WO2023174947A1

WO2023174947A1 - Estetrol for use in re-endothelialization

Info

Publication number: WO2023174947A1
Application number: PCT/EP2023/056499
Authority: WO
Inventors: Jean-François ARNAL
Original assignee: Estetra Srl
Priority date: 2022-03-14
Filing date: 2023-03-14
Publication date: 2023-09-21

Abstract

The present invention relates to compositions comprising an estetrol component for use in endothelial healing contributing to endothelial integrity. The invention further relates to associated uses and methods. The compositions of the present invention achieve their effect through an original mechanism involving the selective modulation of nuclear estrogen receptor alpha (ERα) in the smooth muscle cells, that is fully preserved at middle age. in a manner that is independent from ERα membrane initiated steroid signalling. In contrast, classic estrogens as estradiol accelerates endothelial healing through the selective activation of membrane ERα directly at the level of endothelial cells, but this effect is lost at middle age.

Description

ESTETROL FOR USE IN RE-ENDOTHELIALIZATION

FIELD OF THE INVENTION

The present invention broadly relates to the field of medicine and molecular biology. More particular, the present invention generally relates to improving or restoring the healthy state of the endothelium, including for example re-endothelialization, endothelial repair, and maintenance of endothelial integrity in a subject by administration of an estetrol component to said subject. The invention further provides in compositions comprising an estetrol component for these medical indications.

BACKGROUND OF THE INVENTION

Two essential cell types in blood vessels are endothelial cells (the endothelium), which line the inside of all blood vessels, and vascular smooth muscle cells (the media), which contribute to vessel resistance and directly drive the contraction of the vascular wall. Although the main function of endothelial cells is to provide a barrier between the blood and the rest of the body, they also maintain vascular homeostasis by orchestrating the selective exchange of nutrients, oxygen and immune cells between tissues or organs, and act as key players in angiogenesis and vasculogenesis, aiding the formation of new microvasculature to create an organized network (Jain, Nat Med. 2003 Jun; 9(6):685-93).

By acting as interface between the lumen and the vessel wall, endothelial cells control the flow of substances and fluid to and from tissues. Despite originally thought to have merely a barrier function, it has been established in more recent research that the endothelium integrity is critical for preventing subendothelial lipid accumulation resulting in atherosclerosis, for controlling blood fluidity, preventing platelet aggregation and clot formation as well as for maintaining vascular tone, regulation of immune responses, inflammation, and angiogenesis (Feletou, The Endothelium, Morgan & Claypool Life Sciences, 2011). Moreover, it is now clear that injuries to the endothelium lead to increased vascular permeability and thus to transmigration of inflammatory cells, leakage of erythrocytes, tissue oedema and micro thrombosis (Libby et al., Circulation, 2002). Diseases characterized by endothelial cell injury and dysfunction include atherosclerosis, restenosis and neointimal proliferation, peripheral vascular disease, hypertension, sepsis, and acute respiratory distress syndrome. It is therefore evident that a proper functioning of the endothelium and recovery of the endothelium from injuries is crucial for the health of the individual (Evans et al., Am J Pathol, 2021).

Endothelial imbalance, impairment, and/or dysfunction is commonly observed in elderly subjects, and originates from an imbalance in vasodilator and vasoconstriction molecules produced by the endothelium which has been studied in detail in the art (e.g. in Herrera et al., Ageing Res Rev, 2010). Notably, cardiovascular diseases reflecting endothelial impairment occur 3 -fold more frequent in men than in pre-menopausal women and overall these differences have been largely attributed to estrogens which are responsible for an increased endothelial nitric oxide (NO) production and improved maintenance of endothelium integrity.

Menopause is marked by the permanent end of the menstrual cycles and the abrupt cessation of 17p~ estradiol (E2) production by the ovaries. The direct effect of female sexual hormones on postmenopausal women (or a lack thereof) was evaluated by studying the impact of hormonal treatment in the Women's Health Initiative (WHI) (Flores et al., Endocrine reviews, 2021). It was concluded that, on the one hand, estrogens confer protection against endothelial impairment if started at early menopause in younger women in line with experimental animal studies. On the other hand, estrogens are neutral or even harmful if started late after menopause and/or in older women. This “timing” effect highlights the differential action of hormones during aging which could be explained by 1) alteration of estrogenic action due to age and/or prolonged estrogen deprivation, and 2) changes in cellular interactions due to atherosclerosis progression.

While the understanding of the molecular mechanisms and signalling pathways that are involved in endothelial injury and endothelium aging has improved markedly, the regulatory mechanisms responsible for endothelial regeneration and maintenance of endothelial integrity and how certain molecules such as estrogens may affect the associated molecular pathways are still incompletely understood. Consequently, means to manipulate and improve said processes are scarce, and it is unclear how the observations in menopausal women may be exploited for the benefit of any female subject, but particularly menopausal subjects. There is therefore an unmet need for innovative and effective treatments to maintain or regain integrity of the endothelium in view of the numerous diseases that have been associated thereto, especially in menopausal subjects.

SUMMARY OF THE INVENTION

As evidenced in detail by the examples enclosed herewith, the inventors have unexpectedly found that estetrol (E4) invokes endothelial integrity and accelerates endothelial repair (i.e. endothelial healing) in an endovascular injury model. Furthermore, it could be demonstrated that E4 functions independently of ERa membrane initiated steroid signalling (MISS). Membrane ERa is therefore dispensable for E4 action on endothelial healing in the endovascular model. It was observed that E4 mainly exhibits its action through nuclear ERa in Smooth Muscle cells (SMCs). In contrast, 17p~ estradiol (E2) acts through endothelial/hematopoietic ERa to achieve the same effect. It was also shown that ERa membrane action is necessary for re-endothelialization by E2. Treatment with E4 resulted in substantial differences in gene regulation and associated cell signature between carotid arteries when compared with E2 treatment. Altogether these results suggest that E4 targets different cell types to achieve a similar vasculoprotective effect. E2, E4 and their combination were similarly efficient on endothelial healing, and E4 accelerated re-endothelialization in presence of endogenous E2, whereas endogenous E2 was not efficient to influence this process. Remarkably, the inventors have found that while the acceleration of endothelial healing promoted by E2 is abrogated in the carotid arteries from middle-aged female mice (1 year-old), E4 remains capable to achieve accelerated endothelial healing. E4 can therefore unexpectedly be suggested as a highly interesting molecule to achieve endothelial maintenance and repair in middle-aged to elderly women in the perimenopausal, menopausal, and postmenopausal stages of life.

The invention therefore provides the following aspects:

Aspect 1. A composition comprising an effective amount of an estetrol (E4) component for use in preventing or treating a medical condition or disorder linked to or caused by endothelial impairment, more preferably, by invoking endothelial integrity and/or facilitating or increasing endothelial repair in a subject.

Aspect 2. The composition for use according to aspect 1, wherein the estetrol component achieves endothelial integrity and/or endothelial repair by selective modulation of ERa, preferably wherein the selective modulation is independent of ERa membrane initiated steroid signalling (MISS).

Aspect 3. The use of an effective amount of an estetrol component in the manufacture of a medicament for preventing or treating a medical condition or disorder linked to or caused by endothelial impairment, more preferably, by invoking endothelial integrity and/or facilitating or increasing endothelial repair in a subject.

Aspect 4. The use according to aspect 3, wherein the estetrol component achieves endothelial integrity and/or endothelial repair by selective modulation of ERa, preferably wherein the selective modulation is independent of ERa membrane initiated steroid signalling (MISS).

Aspect 5. A method of preventing or treating a medical condition or disorder linked to or caused by endothelial impairment, more preferably, by invoking endothelial integrity and/or facilitating or increasing repairing the endothelium in a subject, comprising the step of administering an effective amount of an estetrol component.

Aspect 6. The method according to aspect 5, wherein the estetrol component invokes endothelial integrity and/or endothelial repair by selective modulation of ERa, preferably wherein the selective modulation is independent of ERa membrane initiated steroid signalling (MISS). Aspect 7. The composition for use according to aspect 2, the use of the composition according to aspect 4, or the treatment method according to aspect 6, wherein the selective modulation of ERa is selective modulation of nuclear ERa.

Aspect 8. The composition for use according to aspect 2 or 7, the use of the composition according to aspect 4 or 7, or the treatment method according to aspect 6 or 7, wherein the selective modulation of ERa of smooth muscle cells leads to the release of paracrine factors promoting re- endothelialization.

Aspect 9. The composition for use, the use of the composition, or the treatment method according to any one of aspects 1 to 8, wherein the subject is diagnosed with, considered to have, or considered at risk to develop an endothelial vulnerability leading to an increase of cardiovascular risk.

Aspect 10. The composition for use, the use of the composition, or the method according to any one of aspects 1 to 9, wherein said endothelial impairment is caused by decreased membrane expression of estrogen receptor alpha.

Aspect 11. The compositionfor use, the use of the composition, or the treatment method according to aspect 9 or 10, wherein said impairment of endothelial integrity results in cardiovascular diseases.

Aspect 12. The composition for use, the use of the composition, or the treatment method according to any one of aspects 9 to 11, wherein said endothelial impairment is restenosis, preferably restenosis after stent implantation; or late stent thrombosis.

Aspect 13. The composition for use, the use of the composition, or the treatment method according to any one of aspects 9 to 11, wherein said impairment of endothelial integrity is a peripheral vascular disease.

Aspect 14. The composition, the use of the composition or oral dosage form, or the treatment method according to any one of aspects 1 to 13, wherein administration of the estetrol component the subject leads to a differential expression level in carotid artery cells of one or more genes, preferably all genes selected from the group consisting of: STC1, LRG1, GREM2, SPON1, MT2, TENT5B, ZBTB16, FKBP5, PGR, KIF23, CD80, and PADI4.

Aspect 15. The composition for use, the use of the composition, or the treatment method according to any one of aspects 1 to 13, wherein administration of the estetrol component the subject leads to an upregulated expression level of one or more genes, preferably all genes in carotid artery cells selected from the group consisting of: MT2, TENT5B, ZBTB16, and FKBP5.

Aspect 16. The composition for use, the use of the composition, or the treatment method according to any one of aspects 1 to 13, wherein after administration of the estetrol component the subject is characterised by an expression level in carotid artery cells of one or more genes selected from the group consisting of: STC1, LRG1, GREM2, SPON1, PGR, KIF23, CD80, and PADI4 that does not differ from their expression level prior to estetrol administration, i.e. wherein said expression is normalised or reaches the expression level of a healthy reference subject.

Aspect 17. The composition for use, the use, or the method according to any one of aspects 1 to 16, wherein the subject is a hormone deregulated subject.

Aspect 18. The composition for use, or the method according to any one of aspects 1 to 17, wherein the subject is a female subject.

Aspect 19. The composition for use, the use, or the method according to any one of aspects 1 to 18, wherein the subject is diagnosed to have, or considered to have estrogen depletion (also called hypoestrogenism).

Aspect 20. The composition for use, the use, or the method according to any one of aspects 1 to 19, wherein said subject is of middle age or older. Preferably, said subject is of age 40 or older, preferably of at least 45 years of age, preferably of at least 50, more preferably 55, most preferably 60 years of age or older.

Aspect 21. The composition for use, the use, or the method according to any one of aspects 1 to 20, wherein said subject is a menopausal, perimenopausal or postmenopausal female subject. In some embodiments, the subject is a hysterectomised female subject, in alternative embodiments, the subject is a non-hysterectomised female subject.

Aspect 22. The composition for use, the use, or the method according to any one of aspects 1 to 21, wherein a daily amount equivalent to from about 5 to about 40 mg of estetrol, such as from about 10 mg to about 25 mg estetrol, or from about 14 to about 21 mg of estetrol is administered.

Aspect 23. The composition for use, the use, or the method according to any one of aspects 1 to 22, wherein said estetrol component is present in said composition or oral dosage form in an amount equivalent to from about 14 to about 16 mg or between about 19 to about 21 mg of estetrol such as about 15 mg or about 20 mg E4.

Aspect 24. The composition for use, the use, or the method according to any one of aspects 1 to 23, wherein said estetrol component is estetrol or an ester thereof.

Aspect 25. The composition for use, the use, or the method according to any one of aspects 1 to 24, wherein said estetrol component is estetrol monohydrate.

Aspect 26. The composition for use, the use, or the method according to any one of aspects 1 to 25, wherein no progestogen is comprised in said composition, is used, or co-administered. Aspect 27. The composition for use, the use, or the method according to any one of aspects 1 to 25, wherein a progestogen is present in the composition, is used, or wherein a progestogen is coadministered or administrated after treatment with the estetrol component.

Aspect 28. The composition for use, the use, or the method according to aspect 27, wherein said progestogen is 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. In a preferred embodiment, said progestogen is selected from the group comprising drospirenone, progesterone, or dydrogesterone.

Aspect 29. The composition for use, the use, or the method according to aspect 27 or 28, wherein said progestogen is present in said composition in an amount equivalent to from about 0.25 mg to about 4 mg, such as from about 1 mg to about 4 mg, more preferably from about 1 mg to about 3 mg, from about 2.5 mg to 3.5 mg drospirenone, or wherein said progestogen is administered in an amount equivalent to from about 2.5 mg to 3.5 mg drospirenone. In a preferred embodiment, said progestogen is drospirenone.

Aspect 30. The composition for use, the use, or the method according to aspect 28, wherein said progestogen is present in said composition in an amount equivalent to from about 1 mg to 20 mg dydrogesterone, or wherein said progestogen is administered in an amount equivalent to from about 5 mg to 10 mg dydrogesterone. In a preferred embodiment, said progestogen is dydrogesterone.

Aspect 31. The composition or use, the use, or the method according to aspect 28, wherein said progestogen is present in said composition in an amount equivalent to from about 25 mg to 300 mg progesterone, or wherein said progestogen is administered in an amount equivalent to from about 100 mg to 200 mg progesterone. In a preferred embodiment, said progestogen is progesterone.

Aspect 32. The composition for use, the use, or the method according to any one of aspects 1 to 31, wherein a further active ingredient suitable for preventing or treating endothelial impairment is present in the composition or wherein a further active ingredient suitable for improving re- endothelialization is co-administered or administered before or after treatment with the estetrol component.

Aspect 33. The composition for use or the method according to any one of aspects 1 to 32, wherein the composition is formulated as an oral dosage form, such as a solid or semi-solid dosage form. The estetrol dosage is typically equivalent to the dosage achieved as defined in aspects 22 and 23. Aspect 34. The composition for use or the method according to any one of aspects 1 to 33, wherein the composition is an oral dosage form, e.g. formulated for oral, sublingual, buccal, or sublabial administration.

Aspect 35. The composition for use or the method according to any one of aspects 1 to 33, wherein the composition is formulated for intravenous administration, more preferably in emergency cases such as for example in myocardial infarction or thrombotic stroke. The estetrol dosage is typically equivalent to the dosage achieved as defined in aspects 22 and 23.

Aspect 36. The composition for use or the method according to any one of aspects 1 to 33, wherein the composition is formulated for topical or vaginal administration. The estetrol dosage is typically equivalent to the dosage achieved as defined in aspects 22 and 23.

Aspect 37. The composition for use or the method according to any one of aspects 1 to 36, wherein the composition is formulated to correspond to a daily dosage form or respectively is administered as a daily dosage unit.

Aspect 38. In any one of the aspects defined herein, said composition may be presented as a kit-of- parts containing a packaging unit, e.g. a blister pack, containing the daily oral dosage forms 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.

Within the scope of the invention, 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 plastic or 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.

Aspect 39. In a particular embodiment of the kit-of-parts according to aspect 38, the packaging unit comprises 28 containers or a multitude of 28 containers, such as 2 to 12 times 28 containers.

Also envisaged are methods of monitoring the envisaged treatment.

Aspect 40. A method for monitoring a subject under treatment with an estetrol component for invoking endothelial integrity and/or facilitating or increasing endothelial repair in a subject, the method comprising - determining and/or monitoring in a biological sample obtained from said subject the expression levels of one or more genes selected from the group consisting of: MT2, TENT5B, ZBTB16, and FKBP5,

- comparing the obtained expression level of said one or more genes with a reference expression level, wherein a normalised or increased expression level in the biological sample of said one or more genes versus said reference expression level is indicative for endothelial healing. In preferred embodiments, said reference expression level is the expression level in a healthy sample and normalisation of said expression levels is indicative of proper endothelial healing. Alternatively, said reference expression level is the expression level in a sample of the subject prior to treatment and an increased expression level versus said reference expression level is indicative for improved endothelial healing.

In any one of the above aspects relating to the composition for use, the use, or the method of treating using estetrol, an additional active ingredient known to be involved in promoting endothelial healing or in restoring endothelial impairment can be used to improve the treatment. In addition or alternatively to said additional active ingredient, an active ingredient to counter the adverse effects of estetrol can also be (co)administered.

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 DRAWINGS

Figure 1. 17 -estradiol (E2), estriol (E3) and estetrol (E4) accelerate endothelial healing following carotid artery endovascular injury. Four-week-old female mice were ovariectomized and 2 weeks later implanted subcutaneously with either Veh (vehicle), estradiol (E2), estriol (E3), or estetrol (E4) pellets. Two weeks later, mice were then submitted to endovascular injury of the carotid artery. Carotid reendothelialization was analyzed 5 days post-injury. A, Chemical structure of E2, E3 and E4. B, Uterine weight. C, Vaginal weight. D, Thymus weight. E, Representative Evans blue staining of carotids with outlined deendothelialized areas (dotted line) (scale bar, 1 mm) and F, quantitative analysis of reendothelialization, expressed as a percentage of reendothelialized area compared to day 0. Results are expressed as means ± SEM (n= 7-11 per group). To test the effect of the different treatments, a Kruskal -Wallis test (B and D) or a 1-way ANOVA (C and F) was performed. * indicates differences as compared to Veh group (*P<0.05, **P<0.01, ****P<0.0001). G, Four-week-old female mice were ovariectomized and implanted subcutaneously with either Veh or E4 pellets (designed to release control levels of estrogens for a least 3 weeks) (see comment 40) before inducing endovascular injury of the carotid artery. Images are representative VE-Cadherin staining of en face carotid artery after endovascular injury. The carotid artery is outlined in white. The illuminated areas represent VE-Cadherin staining. Non-stained deendothelialized areas are outlined with dotted lines, (scale bar, 500 pm)

Figure 2. In contrast to E2 and E3, E4 does not accelerate endothelial healing in a carotid artery perivascular injury model. Four-week-old female mice were ovariectomized and 2 weeks later were implanted subcutaneously with either Veh (vehicle), E2, E3, E4 pellets or a combination of two of these estrogens for 2 weeks. Mice were submitted to perivascular injury of the carotid artery. Carotid reendothelialization was analyzed 3 days post-injury. A, Representative Evans blue staining of carotids with outlined deendothelialized areas (dotted line) (scale bar, 1 mm) and B, quantitative analysis of reendothelialization, expressed as a percentage of reendothelialized area compared to day 0. This experiment shows that E4 may require the presence of underlying SMCs to mediate vascular action. Results are expressed as means ± SEM (n= 5-9 per group). To test the effect of the different treatments a 1-way ANOVA was performed. * indicates differences as compared to Veh group (**P<0.01, ***P<0.001, ****P<0.0001). f indicates differences between E2 and E2+E4 (j-fPO.Ol). § indicates differences between E3 and E3+E4 (§§§ PO.OOl).

Figure 3. E4 accelerates endothelial healing through selective modulation of ERa in smooth muscle cells and independently of ERaMISS. Four-weeks-old ovariectomized aSMACrel-.'lV ERoR" '"' (A), C451A-ERa (B), and R264A-ERa (C) female mice and their respective control littermates were implanted with vehicle or E4 pellets for 2 weeks and submitted to endovascular injury of the carotid artery. Schematic representation of each mouse model and quantitative analysis of reendothelialization 5 days post-injury, relative to day 0 are depicted. Results are expressed as means ± SEM (n= 5-12 per group). To test the effect of E4 treatment in each genotype, a 2-way ANOVA was performed. * indicates differences as compared to Veh group (**P<0.01).

Figure 4. E4 antagonizes membrane ERa initiated signaling in endothelial cells. A, Representative ERa staining (right hand panels white regions) (Obj :X40) Nuclei were counterstained with DAPI (left hand panels, white round areas) and B, protein expression measured by Simple Western in control endothelial cells (TeloHAECs) or endothelial cells expressing ERa (ERa- TeloHAECs). C, Estrogen-deprived ERa-TeloHAECs were incubated with DMSO, E2 10'⁸M, E4 10'⁶M or a combination of E2 and E4 for 5 min. Proximity ligation assay for ERa/Src interaction was performed. Interactions are represented by small white spots. Nuclei were counterstained with DAPI (large white-stained regions), (scale bar, 20 pm). Quantification of the number of dots per ERa-positive cell is shown. Results are expressed as means ± SEM. To test the effect of the different treatments a 1-way ANOVA was performed. * indicates differences as compared to Veh group (*P<0.05, **P<0.01, ****P<0.0001). f indicates differences between E2 and E2+E4 (ttttP<0.0001).

Figure 5. E4 displays a specific transcriptional program that differs from E2 in carotid arteries. A, Four-week-old C57BL/6 female mice were ovariectomized and treated with a vehicle or E4 for 3 weeks. RNAs were isolated from uninjured carotid arteries and sequenced (n=4-5 per group). B, Heatmap illustrating the relative expression values of all genes significantly regulated following E4 treatment (fold change > 2 or <0.5 over control with a BH (Benjamini -Hochberg) corrected P<0.05). HCL (Hierarchical Clustering) clustering regroups each sample with its corresponding treatment group. C, GSEA analysis representing the different hallmarks pathways regulated by E4. Calculated false discovery rate (FDR) q-value is given for each term. D, Venn diagram representing the overlap of genes regulated by E2 and E4. E, t-SNE of single-cell RNA- sequencing data from carotid arteries of wild-type mice, organized by cell cluster. (SMC: smooth muscle cells, Fibro: Fibroblasts, Macro: Macrophages, EC: endothelial cells). F, Feature plots of Ed- regulated genes (left) and E2 -regulated genes (right) identified by RNA-sequencing.

Figure 6. RT-qPCR analysis of the mouse carotid artery in response to E2 and E4. RT-qPCR analysis of genes identified by RNA sequencing to be regulated by E2 and E4 (top), E4 only (middle) and E2 only (bottom). Results are expressed as means ± SEM. One-way ANOVA followed by Bonferroni post-test was conducted. For data that failed normality testing, a Kruskal-Wallis with Dunn post-test was performed. * indicates differences as compared to Veh group (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

Figure 7. E4 still accelerates endothelial healing in presence of exogenous and endogenous estrogens. A, Four-week-old C57BL/6 female mice were ovariectomized and 2 weeks after, were implanted with vehicle, E2, E4 or a combination of E2 and E4 pellets. Two weeks later, mice were submitted to endovascular injury of the carotid artery. Carotid reendothelialization was analyzed 5 days post-injury. B, Uterine weight. C, Graphic represents the quantitative analysis of reendothelialization, expressed as a percentage of reendothelialized area compared to day 0. D, Six- week-old gonad-intact C57BL/6 female mice were implanted with vehicle, or E4 pellets. Two weeks later, mice were submitted to endovascular injury of the carotid artery. Carotid reendothelialization was analyzed 5 days post-injury. E, Representative estrous cycles before and after Veh and E4 treatment. F, Graphic represents the quantitative analysis of reendothelialization, expressed as a percentage of reendothelialized area compared to day 0. Results are expressed as means ± SEM (n= 5-9 per group). To test the effect of the different treatments, a Kruskall-Wallis test (B and C) or a Student t-te st (F) was performed (*P<0.05, **P<0.01, ***P<0.001). Figure 8. Endothelial healing in response to E2 and E4. Left: young mice of 2 months of age; right: middle-aged mice of 12 months of age. Each point is representative for a single mouse. Statistical data is shown in Example 6.

DETAILED DESCRIPTION

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

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.

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.

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.

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.

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.

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.

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.

Reference throughout this specification to “one embodiment”, or “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.

As indicated above, the present inventors have found that estetrol (E4) components such as estetrol are particularly useful for invoking endothelial integrity in a subject. Similarly, the present inventors have identified that E4 components such as estetrol are particularly useful for endothelial repair in a subject, and that endothelial repair (i.e., endothelial healing) is accelerated when used in conjunction with known therapies and treatments. Unexpectedly, the inventors have observed that E4 targets nuclear ERa in smooth muscle cells to promote endothelial healing, which is a markedly different mechanism when compared to other estrogens such as E2 which mediates this effect through membrane ERa in endothelial cells. Upon further investigation, it was found that the E4 treatment induces specific transcriptomic and cellular signatures that differ from other estrogens such as E2 and other molecules that have a known favourable effect on endothelial cells such as tamoxifen. Moreover, while E2 -mediated acceleration of endothelial healing was abrogated in carotid arteries from middle-aged female mice, E4 remains capable of achieving accelerated endothelial healing in said middle-aged mice.

The inventors hypothesise that stimulation of smooth muscle cells due to E4 results in the release of paracrine factors, such as factors promoting re-endothelialization. Examples of such factors are epiregulin, Cxcll and Cxcl7. Adding said factors to the E4 composition or co-administering such factors with the composition comprising E4 could hence be a possibility to strengthen or improve the effect on re-endothelialisation. In addition, said paracrine factors could be used to monitor the treatment process with estetrol, wherein an increased presence of such factors is indicative of a good endothelial healing process.

Thus, in a first aspect the invention is directed to a composition comprising an E4 component for use in invoking endothelial integrity and/or endothelial repair in a subject. Similarly, the first aspect of the invention concerns the use of an effective amount of an E4 component in the manufacture of a medicament for preventing or treating a disease or disorder caused by or associated with endothelial impairment, including invoking endothelial integrity and/or endothelial repair in a subject. In some embodiments, said estetrol component is used at a daily amount equivalent to from about 5 mg to about 40 mg, such as from about 10 mg to about 30 mg of estetrol. The above further implies that the first aspect of the invention envisages a method of invoking integrity and/or repairing the endothelium in a subject, wherein the estetrol component is used at a daily amount equivalent to from about 10 mg to about 25 mg of estetrol. In particular embodiments, a daily amount equivalent to from about 14 to about 21 mg of estetrol is administered, such as from about 14 to about 16 mg or between about 19 to about 21 mg of estetrol such as about 15 mg or about 20 mg E4.

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) 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 forms, medical uses and methods of treatment described herein is estetrol (including estetrol hydrates). Most preferably, said estetrol component is estetrol monohydrate. 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., Estetrol review: profile and potential clinical applications, Climacteric, 2008). In instances wherein the estetrol component described herein indicates estetrol, said estetrol may be endogenous estetrol. Alternatively, the estetrol may be chemically synthesized, synthesized by the use of (mutant) recombinant enzymes, or synthesised by any combination thereof. Estetrol may be indicated in the art by its molecular formula: C18H24O4, or by structural formula (I).

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):

Formula (II)

“Estradiol”, interchangeably used with “oestradiol”, “17p-estradiol”, “estra-l,3,5(10)-triene-3,17p- diol” and “17p-oestradiol” is the main female sex hormone in women and contributes to the regulation of the reproductive cycles. 17p-estradiol is further involved in formation of secondary female characteristics (i.e. breast development, hip widening, and female fat distribution patterns). Estradiol may be indicated in the art by its molecular formula: C 18H24O2, or by structural formula (III):

Formula (III)

The term “endothelium” as used herein refers broadly to a layer of squamous endothelial cells of mesodermal origin that line the serous cavities of the heart and form the interior surface of blood vessels and lymphatic vessels, “squamous cells” as used herein is to be interpreted according to its commonly accepted meaning in biology, i.e., cells characterised by a thin and flat appearance, typically polygonal shaped. Related hereto, the term “endothelial cell” indicates any endothelial cell types such as but not limited to (micro)vascular endothelial cells from e.g. lung, heart, intestine, skin, retina, arterial endothelial cells, such as endothelial cells from pulmonary artery, the aorta, umbilical artery and umbilical vein, extrahepatic endothelial cells from certain vascular beds, blood-brain barrier endothelial cells, bone marrow endothelial cells, high endothelial venule cells, and liver sinusoidal endothelial cells. Endothelial cells are the barrier between vessels and tissues, and therefore regulate the inflow and outflow of fluids and substances (such as but not limited to white blood cells) in and out of a tissue.

“Endothelial integrity” indicates a state or process wherein endothelial regrowth is promoted (safeguarded, maintained, shielded, preserved, secured, etc). As such, endothelial healing is achieved. The degree of integrity is not limiting for the invention, and “endothelial integrity” may therefore indicate both an improved endothelial integrity when compared to a normal, adult-age healthy subject, but equally encompasses a similar endothelial integrity when compared to a normal, adult-age healthy subject. Neither limiting in the context of the present disclosure is the stimulus to which integrity is invoked. Endothelial integrity may therefore indicate a favourable stage of endothelial healing in the presence of non-limiting sources such as chemical substances, physical injury, aberrant cell growth or aberrant cell death, one or more underlying pathologies, or any combination thereof. A non-limiting example of underlying pathology is diabetes mellitus. The stimulus can be an isolated stimulus overtime or a chronic (i.e. persisting, continuous) stimulus. “Endothelial repair” as used herein indicates a reparative action that is taking place on the endothelium. Consequently, endothelial repair encompasses the restoration of endothelial cells (i.e. the endothelium) from a weakened, injured, interrupted state to the state as found in normal, adultage healthy subjects. The source of the damage or destruction of the endothelium is not limiting in the context of the invention. Endothelial repair may interchangeably be used with terms such as “re- endothelialization”, which equally indicates a process of restoring the integrity of a layer of endothelial cells (i.e. the endothelium).

“Dosage form”, interchangeably used with “dosage unit” herein and in the art indicates a physical preparate that is suitable for administration to a subject, without the necessity to adapt the preparate prior to administration, i.e. the final beneficial product. A dosage form 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 form (taste, appearance, size, etc.). In the context of the present invention, each dosage form comprises an estetrol component in an amount equivalent to from about 10 mg to about 25 mg of estetrol as pharmaceutically acceptable ingredient. The presence of an estetrol component as pharmaceutically acceptable ingredient does not exclude the presence of one or more further pharmaceutically ingredients and/or pharmaceutically acceptable ingredients in the dosage form. 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.

The dosage form described herein comprises, or the method of treatment described herein comprises administration of a dosage form comprising an estetrol component in an amount equivalent to that of an oral dosage form of about 10 mg to about 40 mg of an estetrol component equivalent to estetrol. In further embodiments, the dosage form comprises an estetrol component in an amount equivalent to that of an oral dosage form of from about 10 mg to about 30 mg of estetrol, to from about 10 mg to about 25 mg of estetrol. In particular embodiments, a daily amount equivalent to from about 14 to about 21 mg of estetrol is present in said dosage form, such as from about 14 to about 16 mg or between about 19 to about 21 mg of estetrol such as about 15 mg or about 20 mg estetrol. In preferred embodiments, the estetrol component is estetrol. In more preferred embodiments said estetrol is estetrol monohydrate.

Hence, the composition subject of the invention may comprise an estetrol component in an amount equivalent to about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, or about 25 mg of estetrol. In certain embodiments, the estetrol component is estetrol. In alternative embodiments, the estetrol component is estetrol monohydrate. In such embodiments, the composition may comprise of from about 10 mg to 25 mg of estetrol monohydrate, of from about 12 mg to about 22.5 mg estetrol monohydrate, or of from about 15 mg to 20 mg of estetrol monohydrate. The wording “equivalent to” in a context of the amount of estetrol component indicates “a certain amount of estetrol component having a pharmaceutical effect as obtained by a certain amount of estetrol”.

The dosage form subject of the present invention can be an oral dosage form. “Oral dosage form” encompasses any dosage form 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 form is envisaged but not a limitation for the oral dosage form of the invention, as detailed further below.

Preferably, the estetrol component part of the composition described herein, invokes endothelial integrity and/or endothelial repair by selective modulation of ERa. Hence, in embodiments describing the use of the estetrol component for the manufacture of a medicament for endothelial integrity and/or endothelial repair the estetrol component part of the medicament preferably achieves endothelial integrity and/or endothelial repair by selective modulation of ERa. Similarly, in embodiments concerning methods of treatment using the estetrol component or a dosage form comprising the estetrol component, preferably the estetrol component achieves endothelial integrity and/or endothelial repair by selective modulation of ERa.

The wording "modulation of ERa" as used herein may be interchangeably used with terms such as “influencing ERa” and “regulating ERa” and encompasses any act of changing the activity and/or localization and/or expression of ERa. The modulation of the ERa may be positive, negative, or dependent on cell type and/or localization of ERa.

As known in the technical field, the action of an estrogen is mediated by the “classical” estrogen receptors (ERa and ER ) (Amal et al., Phys Rev, 2017). “Estrogen receptor alpha” is abbreviated herein and in the art as “ERa” and interchangeably indicated by terms such as “ESRI”, “ER”, “ESR”, “ESRA”, “ESTRR”, “Era”, “NR3A1” or “estrogen receptor 1”. In embodiments of the invention, the estetrol component selectively modulates the estrogen receptor ERa.

“Selective modulation of the estrogen receptor modulator" as used herein, refers to a molecule that differentially modulates the activity of estrogen receptors in different tissues as described in the art (e.g. by Mirkin and Pickar, Maturitas, 2015). A skilled person is aware that ERa is a member of the nuclear receptor family and acts by transcriptional regulation in the nucleus but also exerts non- genomic actions through ERa membrane initiated steroid signaling (MISS) (Ottaviano et al., Cancer Res, 1994; Amal et al., Physiol Rev, 2017). Preferably, the selective modulation by the estetrol component does not activate ERa membrane initiated steroid signaling (MISS), and can even antagonize MISS activation by classic estrogens as estradiol. Preferably this applies to smooth muscle cells. Thus, in such embodiments, the selective modulation by the estetrol component is obtained essentially without relying or without relying on ERa-MISS. In such embodiments, the selective modulation of ERa by the estetrol component consists essentially of or consists of modulation of nuclear ERa.

While not limiting for the scope of the present invention, it is to be understood that interaction between smooth muscle cells and endothelial cells is very important in the context of the present disclosure. Therefore, optionally the selective modulation of ERa comprises selective modulation of ERa in smooth muscle cells. Preferably said selective modulation of ERa by estetrol is independent of ERa membrane initiated steroid signalling (MISS). “Smooth muscle cells” as referred to in the present disclosure and interchangeably indicated with the terms “involuntary muscle cells” are to be interpreted according to the commonly accepted meaning in biology. Thus, the term encompasses any involuntary and non-striated muscle cell. Smooth muscle cells are characterised by a lack of sarcomers, which is causative for the lack of striations (Betts et al., Anatomy and Physiology, 2013). The present disclosure envisages both single-unit and multi -unit smooth muscle cells. Smooth muscle cells fulfil numerous roles throughout different organ systems which all rely on the contraction function of said cells. For example, in the cardiovascular system smooth muscle cells are involved in regulating the blood flow by regulating vessel diameter and thereby play a crucial role in regulating blood pressure and tissue oxygenation. In certain embodiments, the smooth muscle cells described herein are vascular smooth muscle cells. It is appreciated by a skilled person that endothelial cells (ECs) and smooth muscle cells (SMCs) are two major interacting cell types in blood vessels, as has been described in detail in the art (e.g. in Feletou et al., Med Sci (Paris) 2003) and illustrated in the figures accompanying the present disclosure. More particularly, vascular smooth muscle cells are located in the tunica media (i.e. middle layer between the tunica intima and tunica adventitia) around the vascular lumen of a blood vessel.

In preferred embodiments, the subjects treated with the estetrol component (comprised as ingredient in the composition) are subjects that are characterised by an endothelial impairment. In preferred embodiments, the subjects are diagnosed to have or considered to have an endothelial impairment.

The term “endothelial impairment” broadly refers to any state of endothelial cells that is considered different from the normal, healthy state of endothelial cells that may be observed in an adult, healthy individual. The endothelial impairment may thus be a structural impairment, a functional impairment, or any combination thereof. The endothelial impairment may lead to a cutaneous clinical image (e.g., in case of an endothelial injury) or may lead to a clinical image only after a certain amount of time after onset (and aggravation) of the impairment (e.g. in case of stenosis or restenosis). Said endothelial impairment may be caused by a decreased (i.e., reduced, diminished, attenuated) expression of ERa. In further embodiments, the decrease in ERa expression may be 10% less, preferably 20% less, 30% less, 40% less, 50% less, 60% less, 70% less, 80% less, 90% less when compared to the expression level of ERa in an adult, healthy, non-menopausal female subject. In yet further embodiments of the invention, the endothelial impairment may be caused by a complete ablation of ERa expression, or at least lack of a detectable ERa expression.

The term “a disease or disorder caused by or associated with endothelial impairment” as used herein encompasses all diseases or disorders which can be caused by or associated by endothelial impairment as defined herein. In certain embodiments, the disease or disorder caused by or associated with endothelial impairment is selected from the group consisting of: cardiovascular diseases, restenosis, late stent thrombosis, and peripheral vascular diseases.

“Cardiovascular diseases” refers broadly to any diseases that involves the heart or blood vessels. In certain embodiments, the cardiovascular disease is one or more diseases selected from the group consisting of: angina pectoris, myocardial infarction (i.e. heart attack), stroke, heart failure, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, abnormal heart rhythms, congenital heart disease, valvular heart disease, carditis, aortic aneurysms, peripheral artery disease, thromboembolic disease, and venous thrombosis.

In preferred embodiments, the cardiovascular disease is atherosclerosis or is caused by atherosclerosis. “Atherosclerosis” as used herein indicates the formation of abnormal build-up of fibrous, gelatinous, and fatty substances in one or more blood vessels (i.e. the build-up of atheromatous plaque). A skilled person is capable of identifying atheromatous plaque and appreciates that in general, such plaque comprises high relative amounts of fat, collagen, and elastin, and optionally cells involved in inflammation (e.g. leukocytes, particularly monocytes or basophils). In further preferred embodiments, the cardiovascular disease is a cardiovascular disease caused by atherosclerosis or considered to be caused by atherosclerosis.

Optionally, the subject is a subject diagnosed to have or considered at risk to have stenosis. A skilled person appreciates that stenosis is a generally accepted term for any abnormal narrowing in any blood vessel. “Stenosis” broadly relates to a narrowing of a blood vessel by atheromatous plaque. Stenosis might also relate to strictures which are narrowing of vessels caused by contraction of smooth muscle cells. In certain embodiments, the stenosis is classified as a condition selected from the group consisting of: Examples of vascular stenotic lesions include: intermittent claudication (i.e., peripheral artery stenosis), Angina (i.e., coronary artery stenosis), carotid artery plaque or stenosis which predispose to strokes and transient ischaemic episodes, renal artery stenosis, and heart valve stenosis. In certain embodiments the stenosis is a stenose in a heart valve selected from the group consisting of: pulmonary valve stenosis, mitral valve stenosis, tricuspid valve stenosis, and aortic valve stenosis. The degree (i.e. severity) of stenosis may be graded as mild (plaque with 20-49% stenosis), moderate (plaque with 50-69% stenosis), severe (plaque with 70-99% stenosis), or occluded (100% stenosis, complete occlusion of the lumen). In certain embodiments, the estetrol component is administered to the subject to prevent stenosis. In alternative embodiments, the estetrol component is administered to inhibit (i.e., halt) or reduce existing stenosis.

Optionally, the stenosis may be restenosis. The term “restenosis” broadly refers to any renarrowing of a blood vessel after receiving treatment for an earlier narrowing. In certain embodiments, the subject is diagnosed to have or considered to have restenosis is a subjected that received endovascular treatment. Preferred vascular treatments include without limitation vascular surgery, cardiac surgery, and angioplasty. In such subject, the restenosis may be in-stent restenosis (commonly abbreviated in the art as “ISR”). The method of diagnosis for or detection of the restenosis is not particularly limiting for the invention, and thus includes imaging or occurrence of a clinical event such as (recurrent) angina pectoris or a myocardial infarction. “Stent” as used herein is to be interpreted in its broadest medical interpretation and thus indicates any tube, preferably plastic or metal, inserted into the lumen of a vessel. Preferably, the subject is a subject having a coronary stent or a vascular stent. The particular design and composition of the stent is not particularly limiting, and therefore includes both coated and non-coated (bare) stents. Illustrative examples of stent coatings include drug -eluting stent coatings. In some embodiments, an effective amount of the estetrol component is released from the drug -eluting stent coating. In some embodiments, an effective amount of the estetrol component is released from the drug-eluting stent coating. In certain embodiments, the estetrol component is administered to the subject to prevent restenosis after receiving treatment of a blood vessel narrowing. In alternative embodiments, the estetrol component is administered to inhibit (i.e., halt) or reduce existing restenosis.

Equally envisaged by the invention is use of the estetrol component, compositions, and methods of treatment described herein for treating or preventing stenosis in shunts. A skilled person is aware that the function of a shunt is establishing a novel connection between two vessels, such as blood vessels, in a subject.

In those embodiments wherein the estetrol component is administered to subjects to inhibit or reduce existing stenosis or restenosis, said estetrol component may be combined with any existing therapy to treat (re)stenosis. Non-limiting examples thereof include intracoronary radiation and (repeated) angioplasty.

In embodiments, the estetrol component described herein may be combined with an anti-platelet medicament or substances (i.e. platelet agglutination inhibitor or platelet aggregation inhibitor). Non-limiting examples hereof include cyclooxygenase inhibitors, ADP receptor inhibitors, phosphodiesterase inhibitors, protease-activated receptor- 1 antagonists, glycoprotein IIB/IIA inhibitors, adenosine reuptake inhibitors, thromboxane inhibitors. The anti-platelet drug may be comprised in the same composition or in a separate dosage form.

In certain embodiments, the subject is a subject that is diagnosed to have, considered to have, or at risk to have an in-stent thrombosis, i.e. a thrombotic occlusion of a coronary stent.

Optionally, the subject is a subject diagnosed to have, or considered to have a progressive blood circulation disorder such as peripheral vascular disease. The term “peripheral vascular disease” encompasses disorders that affect blood vessels, such as but not limited to peripheral artery disease. Acute peripheral vascular diseases include, for example, occlusive peripheral arterial disease, Buerger's disease, erectile dysfunction (impotence) and functional peripheral arterial diseases, such as, Raynaud's disease, Raynaud's phenomenon and acrocyanosis. Optionally, the subject may report to experience or considered to experience claudication. “Claudication” as used herein broadly refers to pain, discomfort, or numbness experienced by a subject upon physical exercise. The invention therefore envisages that the estetrol component is used for treating or preventing claudication. Preferably, the claudication is of intermittent vascular nature. In further embodiments, the claudication is caused by atherosclerotic plaque build-up in a blood vessel in the lower limbs.

Optionally, the composition subject of the invention or the method subject of the invention is used to prevent endothelial cell detachment induced as adverse effect by medicaments. It has been described in the art that certain pharmaceutically acceptable ingredient are capable of provoking detachment of endothelial cells. Non-limiting examples hereof are calcineurin inhibitors (Bombeli et al., J Lab Clin Med 1996) and anti -cancer medicaments such as cyclophosphamide (Genge et al., Clin Hemorheol Microcirc, 2018). Therefore, in preferred embodiments the estetrol component is used to counteract one or more adverse effects on endothelial cells by a different treatment or therapy.

The present invention is directed to medical uses and methods of treatment for the above-mentioned indications. 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 or diseases related to endothelial impairments. Beneficial or desired clinical results may include, without limitation, alleviation of one or more symptoms, improvement of one or more biological markers, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and the like.

“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 change.

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. “Treatment” encompasses both curative treatments and treatments directed to reduce symptoms and/or slow progression and/or stabilize the condition or disease.

It is emphasized that while any of the herein disclosed values and ranges of the estetrol components are suitable for the different medical indications or purposes, a skilled person is aware that certain individuals may experience yet improved benefits from the estetrol component treatment by further optimization of the dose of said component by considering a wide range of parameters including but by no means limited to the nature and degree of the condition or disease to be treated, gender of the subject, subject age, body weight, other medical indications, nutrition, mode of administration, metabolic state, interference or influence by or efficacy of other pharmaceutically active ingredients, etc. Furthermore, each individual may have a certain intrinsic degree of responsiveness to the estetrol component that is used.

“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 clinical symptoms characteristic for the diagnosed disease or condition). “Diagnosis of’ the diseases, conditions, or (adverse) side effects as taught herein in a subject may particularly mean that the subject has such disease or condition. A subject may be diagnosed as not having such despite displaying one or more conventional symptoms or signs reminiscent of such. “Diagnosis of’ the diseases or conditions as taught herein in a subject may particularly mean that the subject has an endothelial impairment. Alternatively, a subject may be diagnosed as not having a endothelial impairment despite displaying one or more conventional symptoms or signs reminiscent of such. "Prognosticating" in the context of the invention is indicative for anticipation on the progression of endothelial impairment 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 infection. The term "a good prognosis of generally encompass anticipation of a satisfactory partial or complete recovery from a diagnosed pain-inducing disease or pain condition, optionally within an acceptable time period. Alternatively, 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 encompass an anticipation of a substandard recovery and/or unsatisfactorily slow recovery, or no recovery at all, or further worsening of the endothelial impairment such as (re)stenosis and/or any clinical manifestation associated with said disease or condition.

Related to the foregoing, "predicting" or "prediction" generally refer 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 said disease, condition, or (adverse) side effects. A prediction of a certain clinical disease manifestation, condition, or adverse effect 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 endothelial impairment. 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 of an endothelial impairment or related condition such as (re)stenosis may allow to predict the progression, aggravation, alleviation or recurrence of the clinical image or severity of the (re)stenosis. Furthermore, 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 (extended) hospitalisation.

The estetrol component as part of the composition as described herein has the effect of triggering a unique transcriptional program which differs from other estrogens. Importantly, E2 relies on endothelial membrane ERa activation to accelerate endothelial healing, but as endothelial membrane ERa is lost at middle age (12 months) in mice, this protective action of E2 is thus lost rather early. In contrast, E4 relies on smooth muscle cell nuclear ERa activation to accelerate endothelial healing, that remains fully active and thereby protective at middle age (12 months) in mice. The genomic/transcriptional programs elicited E2 and E4 greatly differs, contributing to explain such different mechanisms of action on endothelial healing and can be illustrated as follows. Preferably, the subject is characterised after treatment with an estetrol component by a differential expression level in carotid artery cells of one or more genes, preferably at least 2, at least 3, at least 4, at least 5, at least 6, most preferably at least 8 genes selected from the group consisting of: STC1, LRG1, GREM2, SPON1, MT2, TENT5B, ZBTB16, FKBP5, PGR, KIF23, CD80, and PADI4 when compared to their expression levels prior to treatment. More preferably, the subject is characterised by a differential expression level in carotid artery cells of each gene of the group consisting of: STC1, LRG1, GREM2, SPON1, MT2, TENT5B, ZBTB16, and FKBP5 when compared to their expression levels prior to treatment. In further embodiments, the differential expression level of the one or more genes, preferably least 2, at least 3, at least 4, at least 5, at least 6, most preferably each of the genes of the group consisting of STC1, LRG1, GREM2, SPON1, MT2, TENT5B, ZBTB16, and FKBP5 is an upregulated expression level when compared to their expression levels prior to treatment.

In further embodiments, the expression levels of one or more genes, preferably at least 2, at least 3, most preferably each gene of the group consisting of: STC1, LRG1, GREM2, and SPON1 is upregulated when compared to their expression levels prior to estetrol treatment but lower when compared to their expression levels upon 17p-estradiol treatment.

In yet further embodiments, the expression levels of one or more genes, preferably at least 2, at least 3, at least 4, most preferably each gene of the group consisting of: PGR, KIF23, CD80, and PADI4 does not significantly differ from their expression level prior to estetrol administration. In such embodiments, the expression levels of said genes are considered to be constant throughout estetrol treatment.

In yet even further embodiments, the expression levels of each gene of the group consisting of: STC 1 , LRG1, GREM2, SPON1, MT2, TENT5B, ZBTB16, and FKBP5 are upregulated when compared to their expression levels prior to estetrol treatment, and the expression levels of each gene of the group consisting of: PGR, KIF23, CD80, and PADI4 do not differ from their expression levels prior to estetrol administration.

The genes of interest disclosed in the context of the findings made by the inventors are indicated by their commonly accepted GeneCards Symbol (https://www.genecards.org/). A skilled person appreciates that the panel of genes subject of the invention may equally be annotated by alternative identifiers such as but not limited to their HGNC (https://www.genenames.org/), NCBI Entrez Gene (https://www.ncbi.nlm.nih.gov/gene/), Ensembl (http://www.ensembl.org/), or UniProtKB identifier. Non limiting identifiers for the genes subject of the invention are reproduced below:

Throughout the present disclosure, reference is made to terms such as “expression”, “expression level”, “quantity”, “amount”, “value”, and “level”, which each indicate a quantification of a gene expression level in a biological sample. Said quantitation may be an absolute or alternatively a relative quantification of a gene expression level in a biological sample. Relative quantification in the present context refers to the expression of a gene expression level relative to another value such as relative to a reference value, reference range (e.g., a reference indicating a base-line expression of a marker in a given tissue), or relative to the expression level in a reference biological sample. These values or ranges can be obtained from a single biological sample or from a plurality of biological samples (i.e. biological repeats). The value may be obtained by a single measurement of the gene expression level in a biological sample, or by repeated measurement of the gene expression level in a biological sample (i.e. technical repeats).

The method that is used to measure the expression levels of one or more genes selected from the panel of genes is not limiting in the context of the present invention. For example, such methods may include biochemical assay methods, immunoassay methods, mass spectrometry analysis methods, or chromatography methods, or combinations thereof. The method hence may comprise determining the expression levels of the genes in the biological sample which are compared to the expression levels of the same genes in a reference biological sample on the transcript level. The term “transcript” as used herein refers to a segment (i.e. sequence, stretch, concatenation) of RNA. Alternatively, the method may comprise determining the expression levels of the genes in the biological sample which are compared to the expression levels of the same genes in a reference biological sample on the protein level. Yet alternatively, the method may comprise determining the expression levels of the genes in the biological sample which are compared to the expression levels of the same genes in a reference biological sample on both the transcript level and the protein level.

The differential expression level (i.e. upregulated or downregulated expression level) of the one or more genes described herein indicates a statistically significant differential expression level and may be a change in expression level of at least 0.2 log2 fold change, preferably at least 0.35 log2 fold change, at least 0.5 log2 fold change, preferably at least 0.6 log2 fold change, preferably at least 0.8 log2 fold change, preferably at least 0.9 log2 fold change, preferably at least 1.0 log2 fold change when compared to a subject not receiving the estetrol component and/or a subject receiving an estrogen different from the estetrol component such as 17p-estradiol. A skilled person is capable of conducting the relevant statistical analyses.

Preferably, the subject is a female subject. Preferably, the subject is a hormone deregulated subject. More preferably, the subject is a hormone deregulated female subject. In the context of the invention, the deregulation is established by comparison of the hormone level of the subject with a representative value of an adult, healthy 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. Optionally, the 17p-estradiol of the subject is lower than 30 pg/ml.

Embodiments concerning subjects characterised by an estrogen depletion (i.e. hypoestrogenism) are envisaged by the invention. 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.

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.

“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). According to the US FDA, the criteria for post-menopausal status are:

• At least 12 months of spontaneous amenorrhea; or

• At least 6 months of spontaneous amenorrhea with serum FSH levels >40 mIU/mL; or

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

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, 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). “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).

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. Alternatively, the subject may be a female subject 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. 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, most preferably at most 70 years of age.

In certain embodiments of the invention, the composition does not comprise a progestogen. In further embodiments, the estetrol component is the only pharmaceutically active ingredient comprised in the dosage form. More particularly, in certain embodiments of the methods disclosed herein no progestogen is co-administered with the estetrol component (neither in the same composition nor in a further dosage form that is co-administered). However, in alternative embodiments the composition contains at least one further pharmaceutically active ingredient. Said further pharmaceutically active ingredient may be comprised in the composition comprising the estetrol component, or comprised in a separate dosage form that is co-administered.

Preferably, said further pharmaceutically active ingredient that may be comprised in the composition comprising the estetrol component is a progestogen. In another embodiment, the estetrol component and the progestogen are comprised in the same composition or dosage form.

Preferably, said further pharmaceutically active ingredient that may be comprised in the composition comprising the estetrol component is a progestogen. In another embodiment, the estetrol component and the progestogen are comprised in the same composition or dosage form. When the present therapy is administered to non-hysterectomized patients, the estetrol component may be administered as sole active ingredient or may be administered together with an optional progestogen. Said optional progestogen may be administered continuously (i.e. every day in addition to the estetrol component) or sequentially (wherein sequentially means an administration of the progestogen during, for example, 10 to 14 days each month or during 14 days every 3 months).

The terms "continuous" and ’’continuously” as used herein, means that the components 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.

Optionally, the composition comprising estetrol for use, or the use of estetrol for the manufacturing of a medicament for treating, or the method of treatment using estetrol as defined herein comprises as a further pharmaceutically active ingredient a compound that can reduce the adverse effects of administering an estrogen such as estetrol to a subject. Preferred examples of such active ingredients are progestogens This can be done in the same composition or can be done through coadministration, sequential administration or separate administration. In embodiments wherein the estetrol component and the progestogen are administered by separate dosage forms, the progestogen may be administered via a non-oral route, for example using an Intra Uterine Device (IUD). In one embodiment said IUD delivers the progestogenic component levonorgestrel. In one such embodiment, the IUD is the Mirena® IUD or the Eevosert® IUD.

The terms “progestogen”, “progestogen”, “gestagen”, or “gestogen” and derived hereof “progestogenic compounds” 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, Pharmacology of estrogens and progestogens: influence of different routes of administration, 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.

Examples of progestins envisaged by the invention are: 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, mecirogestone, 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(l l)-diene-21, 17beta-carbolactone or tanaproget and precursors of these compounds that are capable of liberating these progestogens in vivo.

The progestogens to be co-administered 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.

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

Drospirenone (abbreviated as DRSP, PubChem CID: 68873) is an example of a progestin 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 (IV): Formula (IV)

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

The methods described herein may include the administration of drospirenone to the subject receiving the estetrol component. In such embodiments, the drospirenone may be administered at a daily amount of from about 0.25 mg to 10 mg, preferably of from about 1 mg to about 4 mg, more preferably of from about 1 to about 3 mg, most preferably of from about 2.5 to 3.5 mg drospirenone. Alternatively, a progestin may be administered in an amount equivalent to from about 2.5 to 3.5 mg drospirenone. Said drospirenone may optionally be comprised in the same composition or dosage form that comprises the estetrol component. In such embodiments, the dosage form comprises of from about 0.25 mg to 10 mg, preferably of from about 1 mg to about 4 mg, more preferably of from about 1 mg to about 3 mg, most preferably of from about 2.5 mg to 3.5 mg drospirenone. In further embodiments, the dosage form comprises of from about 10 mg to about 25 mg of an estetrol component and from about 1 mg to about 4 mg of drospirenone. In yet further embodiments, the dosage form comprises of from about 10 mg to about 25 mg of an estetrol component and from about 2.5 mg to 3.5 mg of drospirenone. In more particular embodiments, the dosage form comprises about 15 mg of an estetrol component and 3 mg of drospirenone. In other embodiments, the dosage form comprises about 20 mg of an estetrol component and 3 mg of drospirenone.

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 (V): Formula (V)

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

The methods described herein may include the administration of progesterone to the subject receiving the estetrol component. In such embodiments, the progesterone may be administered at a daily amount of from about 0.5 mg to 50 mg, preferably of from about 1 mg to about 20 mg, more preferably of from about 5 mg to about 10 mg. Alternatively, a progestin may administered in an amount equivalent to from about 5 mg to 3.5 mg dydrogesterone. Said dydrogesterone may optionally be comprised in the same composition or dosage form that comprises the estetrol component. In such embodiments, the dosage form comprises of from about 0.5 mg to 50 mg, preferably of from about 1 mg to about 20 mg, more preferably of from about 5 mg to about 10 mg dydrogesterone.

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 (VI):

Formula (VI)

It is understood that when the term “dydrogesterone” is used herein, any dydrogesterone derivatives are also envisaged.

The methods described herein may include the administration of progesterone to the subject receiving the estetrol component. In such embodiments, the progesterone may be administered at a daily amount of from about 10 mg to 500 mg, preferably of from about 25 mg to about 300 mg, more preferably of from about 100 mg to about 200 mg. Alternatively, a progestin may administered in an amount equivalent to from about 100 mg to 200 mg progesterone. Said progesterone may optionally be comprised in the same composition or dosage form that comprises the estetrol component. In such embodiments, the dosage form comprises of from about 10 mg to 500 mg, preferably of from about 25 mg to about 300 mg, more preferably of from about 100 mg to about 200 mg progesterone. Progesterone is preferably used at a daily dose of from 50 mg to 200 mg. In one embodiment, progesterone is used at a daily dose of 50 mg to 100 mg when it is used continuously. 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 a preferred embodiment of the invention, the composition combines the estetrol component and the optional progestogenic component into a single dosage unit, preferably a daily dosage unit. In a more preferred embodiment of the invention, said combined daily dosage unit is an oral combined daily dosage unit.

Optionally, the composition comprising estetrol for use, or the use of estetrol for the manufacturing of a medicament for treating, or the method of treatment using estetrol as defined herein comprises as a further pharmaceutically active ingredient suitable for preventing endothelial impairment and/or improving endothelial function may be administered to the subject in any of the methods described herein. Similarly, the compositions described herein may comprise as a further pharmaceutically active ingredient an ingredient suitable for preventing endothelial impairment and/or improving endothelial function. Optionally, such a further pharmaceutically active ingredient may be selected from the group consisting of: ACE inhibitors, ATI blockers, antioxidant agents, beta blockers, dihydropyridine calcium channel blockers, phosphodiesterase-5 inhibitors, statins, angiotensin 1 to 7, bradykinin, eNOS transcription enhancers, ivabradine, sphingosine- 1 -phosphate, or any combination thereof. Said pharmaceutically active ingredients and their molecular actions have been described in the art and are therefore known to a skilled person (e.g. in Su, World J Cardiol, 2015).

The composition described herein may be an oral dosage form, such as a solid or semi solid dosage form such as a tablet, a capsule, a cachet, a pellet, a pill, powder, or granules, or any combination thereof. For example, the oral dosage form 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 form" 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 forms 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 forms 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 form 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.

It is evident and envisaged that any composition or dosage form comprising estetrol 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 composition subject of the invention. A skilled person understands that one or more excipients may be used in the composition 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.

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.

The composition or oral dosage form may be suitable or even specifically manufactured for sublingual, buccal, and/or sublabial administration. In such embodiments, the solid dosage form is able to rapidly release the estetrol component when contacted with an aqueous solvent such as saliva. Hence, in these embodiments the solid dosage form is an orodispersible dosage form 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 oral dosage form may be an orodispersible dosage form. In such embodiments, the dosage form 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 form. Nonlimiting 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.

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.

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. Alternatively, said E4 composition may be suitable for vaginal administration (through the vaginal mucosa) or for topical administration (through the skin). The dosage will be equivalent to the dosage envisaged for oral administration as indicated herein elsewhere.

In some embodiments, said E4 composition may be suitable for intravenous administration. The latter can be particularly interesting for acute treatments such as in case of infarct or stroke. The dosage will be equivalent to the dosage envisaged for oral administration as indicated herein elsewhere

In certain embodiments, the estetrol component is comprised in an immediate release dosage form or composition. In alternative embodiments, the estetrol component is comprised in a delayed release, sustained release, or controlled release dosage form or composition. The terms “immediate release”, “delayed release”, and “sustained-release” or “controlled release” are clear to a person skilled in the art and are indicative for the release profile of a pharmaceutical composition. In immediate release the pharmaceutical composition is about immediately released from a dosage form to a body of a subject or patient. In delayed release dosage forms, the pharmaceutical composition is delivered in the body with a delay after administration. In sustained release or controlled release dosage forms, the dosage form is designed to release a pharmaceutical composition at a predetermined rate in order to maintain a constant drug concentration for a specific period of time. The release profile of a dosage form can be assessed as described in the major pharmacopeias. For example, immediate release is defined by the European Medicines Agency as dissolution of at least 75% of the active substance within 45 minutes (European Pharmacopeia (Ph. Eur.) 9^th edition). However, it is in addition trivial to a person skilled in the art that suitable tests and time windows may vary depending on therapeutic ranges, solubility and permeability factors of the drug substance.

A further aspect of the invention is directed to packaging units comprising the compositions or (oral) dosage forms 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 compositions or (oral) dosage forms, wherein each container comprises at least one oral dosage form comprising of from about 10 to about 25 mg of an estetrol component.

Optionally, the packaging units additionally comprise at least 10, preferably 12, more preferably 14, additional containers for holding separately packaged and individually removable compositions or (oral) dosage forms, wherein each additional container comprises at least one compositions or (oral) dosage form comprising a progestogen. Optionally, each of the additional containers for holding the compositions or (oral) dosage forms comprising the progestogen are individually visually arranged next to a container holding an compositions or (oral) dosage form comprising the estetrol component when they 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 compositions or (oral) dosage forms, wherein each additional container comprises at least one daily, preferably solid, compositions or (oral) dosage form comprising a progestogen, preferably wherein said progestogen is selected from drospirenone, progesterone and dydrogesterone.

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 compositions or (oral) dosage units comprising the estetrol component, and a second, distinct, packaging unit, e.g. a second, distinct, blister pack, containing the daily compositions or (oral) dosage units comprising the progestogen.

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.

Within the scope of the invention, 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 plastic or 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.

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.

A final aspect of the invention is directed to a method of monitoring or diagnosing the healing process in a subject suffering from endothelial impairment and/or endothelial dysfunction, wherein the method comprises in a first step determining in a biological sample obtained from the subject the expression level of one or more genes selected from the group consisting of: STC1, LRG1, GREM2, SPON1, MT2, TENT5B, ZBTB16, FKBP5, PGR, KIF23, CD80, and PADI4. In a second step the expression levels of said one or more genes is compared to a reference expression level of e.g. a healthy subject, wherein a comparative expression level is indicative of proper endothelial healing; or to a reference expression level of the diseased subject prior to treatment with E4, wherein an increased expression is indicative for the endothelial healing of a subject having, or considered at risk to have reduced endothelial repair and maintenance capacities.

Preferably, the expression levels of one or more genes selected from the group consisting of: MT2, TENT5B, ZBTB16, and FKBP5 are determined. More preferably, the expression level of each of the genes of the group consisting of: MT2, TENT5B, ZBTB16, and FKBP5 are determined. An increased expression level in the biological sample of said one or more genes versus a reference sample of e.g. a healthy subject, wherein a comparative expression level is indicative of proper endothelial healing; or to a reference expression level of the diseased subject prior to treatment with E4, wherein an increased expression is indicative for the endothelial healing of a subject having, or considered at risk to have reduced endothelial repair and maintenance capacities.

A skilled person is aware of methods and means to obtain reference expression levels for each of the above genes. Non-limiting examples thereof include public databases and literature, or obtaining expression levels for said genes from one or more healthy, adult subjects. In certain embodiments the subject has undergone vascular surgery, cardiac surgery, and angioplasty. The method of diagnosis described above is unexpectedly capable of providing a prediction on the outcome of the endothelial healing of such subjects.

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, statements, and embodiments of the invention are further supported by the following non-limiting examples.

EXAMPLES

Materials and methods.

Mice

All procedures involving experimental animals were performed in accordance with the principles and guidelines established by the National Institute of Medical Research (INSERM) and were approved by the local Animal Care and Use Committee. The investigation conforms to the directive 2010/63/EU of the European parliament. Wild-type female mice with a C57B1/6J background were purchased from Charles River Laboratories (France). aSMACreERT2ERa^lox/lox, C451A-ERa and R264A-ERa mouse line were generated as described previously (Adlanmerini et al., Proc Natl Acad Sci U S A, 2014; Adlanmerini et al., Arterioscler Thromb Vase Biol. 2020; Smirnova et al., Circ Res. 2015). Genetically modified female mice were systematically compared to their wild-type littermates. Throughout all protocols, mice were housed at the animal facility of the faculty of medicine (Toulouse, France). Mice were housed in a temperature-controlled room with a 12: 12-hour light-dark cycle and maintained with access to food and water ad libitum. Bilateral ovariectomy was performed at 4 weeks of age after intraperitoneal injection of zoletil (100 mg/kg, Virbac, Carros, France) and xylazine (10 mg/kg, Sigma- Aldrich, Isle d’Abeau Chesnes, France). Prior to treatment, aSMACreERT2-ERa^lox/lox (control mice) and aSMACreERT2+ERa^lox/lox mice were injected during 5 days with tamoxifen (1 mg/mouse per day, Sigma, France) starting at 5 weeks of age to induce activation of the Cre recombinase.

Pellets Preparation

To deliver estrogens chronically, E2 (Sigma-Aldrich), E3 (Sigma-Aldrich) and E4 (Mithra) were thoroughly mixed with cholesterol (Sigma- Aldrich) in powder form and compacted to obtain pellets as previously described (Fontaine et al., Mol Cell Endocrinol, 2020; Buscato et al., Am J Physiol Endocrinol Metab, 2021). At the age of 6 weeks, ovariectomized or gonad-intact mice were implanted subcutaneously with pellets releasing either E2 (75 pg/pellet), E3 (750 pg/pellet), E4 (1 mg/pellet) or a vehicle (cholesterol only) for 2 weeks.

Assessment of estrous cycle

In gonad-intact female mice, vaginal smears were performed daily to analyze estrous cyclicity, starting 10 days before Veh or E4 pellet implantation and during 14 days after. Vaginal cytology was assessed under an inverted microscope.

Mouse Carotid Injuries and Quanti fication of Reendothelialization

Endovascular injury of the carotid artery was performed on mice as described previously (cf. Filipe C, et al., 2008, Estradiol accelerates endothelial healing through the retrograde commitment of uninjured endothelium. American Journal of Physiology-Heart and Circulatory Physiology. 2008;294(6):H2822-H2830), after 2 weeks of estrogen treatment. Briefly, animals were anesthetized by an intraperitoneal injection of zoletil and xylazine. The right common carotid artery was exposed and blood flow was locally restricted. The external carotid was ligated distally and incised. A 0.35 mm diameter flexible wire with a 0.25 mm tip was advanced and pulled back 3 times into the common carotid artery (on 5 mm length). The external carotid was then ligated proximally, and blood flow was restored in internal and common carotids.

Perivascular injury of the carotid artery was performed on mice as described previously (cf. Filipe C, et al., 2008), after 2 weeks of estrogenic treatment. Briefly, animals were anesthetized by an intraperitoneal injection of zoletil and xylazine. The right common carotid artery was isolated and electric injury was applied (on 3 mm length) with a bipolar microregulator.

Five-day (endovascular) or 3-day (perivascular) post-injury, endothelial regeneration process was evaluated by staining the denuded areas with Evans blue dye. Five minutes before euthanasia, mice were injected retro-orbitally with 50 pL of 5% Evans blue dye (sc-203736, Santa Cruz) diluted in PBS. Mice were perfused with PBS and the right common carotid artery was dissected from the aortic arch to the carotid bifurcation and fixed with 10% phosphate-buffered Formalin (Electron Microscopy Sciences) for 20 min. Arteries were opened longitudinally and mounted with Kaiser’s Glycerol gelatin (Merck). Images were acquired and quantified using DM2500 Leica microscope and LAS X Software. The percentage of reendothelialization was calculated relative to the initial deendothelialized area (day 0).

Lentivector Construction and Production

The cDNA encoding human ERa was cloned into pENTRlA (Thermo Fisher) by the transfer of a BamHI/EcoRI fragment from pCR3. l-hERa66. The transfer into the destination vector p!nducer20 was achieved using the Gateway LR-clonase enzyme mix. Lentiviruses were produced by the vectorology plateau at INSERM UMR1037 (Toulouse). Briefly, endotoxin-free midiprep DNA was used to produce lentivirus particles in HEK293FT cells after a CaC12/HEPES mediated tripletransfection together with the plasmids p8.71 and pVSVg, using respectively a ratio of 2:2: 1 of the three plasmids. Viral production titers were determined by ELISA assay (Innotest HIV p24, Fujirebio). Functional viral titers were assessed on HT1080 cells with serial dilutions and scored for GFP expression by flow cytometry analysis on a MACSQuant 10 analyzer (Miltenyi Biotec).

Cell Culture, Transduction and Treatment

TeloHAEC cells (hTERT-immortalized human aortic endothelial cells were cultured in Endothelial Cell Growth Medium (PromoCell) at 37°C in 5% CO2. TeloHAEC, that do not express ERa (Ctrl EC), were transduced with the inducible ERa expression lentivector (ERa EC) in transduction medium (OptiMEM- GlutaMAX, Life Technologies SAS) containing 5 pg/mL protamine sulfate for 6 hours. ERa_EC were then selected and maintained in media containing 0.3 mg/ml neomycin (Sigma- Aldrich). Ctrl EC and ERa EC were plated on 14 mm -diameter coverslips in 6-well plates (250,000 cells/well) and grown in phenol red-free, serum-free Endothelial Cell Basal Medium (PromoCell) for 24 h before the experiment. To induce ERa expression, cells were treated for 6 h with 0.5 pg/mL doxycycline and then treated with DMSO vehicle, 10 nM E2 and/or IpM E4 for 5 min.

Immunofluorescence

To determine ERa expression, cells were fixed with 4% paraformaldehyde for 10 min, permeabilized in PBS containing 0.1% Triton X-100 for 10 min and saturated in PBS 1% BSA (Sigma), 2% FBS (Gibco), 10% Normal donkey serum (Jackson ImmunoResearch) for 1 h. Coverslips were incubated with ERa rabbit monoclonal (1:250; Abeam; Abl66660) primary antibody overnight at 4°C. After washing, coverslips were incubated with AlexaFluor®488 -conjugated anti-rabbit secondary antibody (1: 1000; Jackson ImmunoResearch) for 45 min at room temperature. Nuclei were stained with DAPI (0.5 pg/mL) and coverslips were mounted with Dako Mounting Medium (Agilent Technologies). Microscopy imaging was performed with a Leica DMi8 microscope at 40X magnification.

Protein Preparation and Detection by Simple Western Analysis

Total cell lysates were prepared in RIPA (Sigma) supplemented with protease inhibitors (Roche). Simple Western analyses were performed according to the ProteinSimple user manual. In brief, cell lysate samples (final concentration, 0.5 mg/mL) were mixed with a fluorescent master mix (ProteinSimple) and heated at 95 °C for 5 min. The primary antibody, validated for Simple Western, was diluted in antibody diluent (ProteinSimple). The samples, protein normalization regent, blocking reagent, primary antibody (ERa rabbit monoclonal primary antibody [1:50; Cell signaling; 13258S]), HRP-conjugated secondary antibody, chemiluminescent substrate (luminol/peroxide) were dispensed into designated wells in a manufacturer-provided microplate. The plate was loaded into the instrument (Jess, Protein Simple) and proteins were drawn into individual capillaries on a 25 capillary cassette (12-230 kDa) (ProteinSimple). Data were analyzed with Compass software (ProteinSimple). The normalization against total proteins in the capillary was achieved using the Protein Normalization (PN) assay reagent.

Proximity Ligation Assay

The proximity Ligation Assay (PLA) (Duolink, Sigma-Aldrich) was used to detect ERa interaction with c-src kinase, according to manufacturer's instructions. Briefly, after treatment, cells were fixed with 4% paraformaldehyde for 10 min and then permeabilized in PBS containing 0.2% Triton X- 100 for 10 min. Coverslips were incubated with ERa rabbit polyclonal (1:500; Santa Cruz; sc-543) and c-src mouse monoclonal (1 :500; Santa Cruz, sc-8056) primary antibodies overnight at 4°C. After washing, cells were incubated with anti -rabbit PLUS and anti -mouse MINUS probes coupled to oligonucleotides for 1 h at 37 °C in a humidity chamber, followed by a ligation step during 30 min and a rolling-circle amplification (RCA) reaction in which fluorescently labeled oligonucleotides were hybridized to the RCA product. Coverslips were then stained for total ERa as previously described in the immunofluorescence section. The cover slides were mounted in Duolink II mounting medium with DAPI (Sigma- Aldrich). Images were obtained with a Zeiss LSM900 confocal microscope at 63X/oil magnification and processed using Zen software. PLA dots were analyzed using Image J software. The plugin “Counter cells” allows analyzing dots and cells number. In each condition, red dots were scored in ERa-positive cells from 10 microscopic field.

RNA Sequencing and Analysis Total RNA was isolated from uninjured carotid arteries through the phenol-chloroform method using TRIzol (Ambion) reagent. Quality of RNA samples was determined using a Fragment Analyzer Instrument. All samples had RNA quality numbers (RQN) above 7.2. mRNA sequencing libraries were prepared according to Illumina’s protocols using the Illumina TruSeq Stranded mRNA kit (reference no. 20020595), as previously described (Zahreddine et al., Circ Res, 2020). Sequencing was performed in paired-end (2x 150 bp) on an Illumina NovaSeq sequencer at the Integragen company platform (https://www.integragen.com/fr/). The reads were first trimmed for adapters and low-quality ends by the Trim Galore! Algorithm (— t, -q, -e, —length 20) available on the Galaxy web server (https://usegalaxy.org/). Subsequent informatics processes of the sequenced reads were locally done under python and R environments, as previously described (Zahreddine et al., Circ Res, 2020). Genes were declared as differentially regulated when their fold change (FC) was >2 or <0.5 with a BH (Benjamini -Hochberg) adjusted P<0.05. Functional annotations were made using the GSEA program (v4.0.3) (Subramanian et al., Proc Natl Acad Sci U S A, 2005) interrogating MSigDB hallmarks (Liberzon et al., Cell Syst. 2015).

Analysis of Single-Cell RNA sequencing. Experiments

Fastq files containing reads for Carotid experiments (cf. Li F, et al., Single-cell RNA-seq reveals cellular heterogeneity of mouse carotid artery under disturbed flow. Cell Death Discov. 2021 ;7(1): 180) were fetched and dumped from the ncbi SRA database (https://www.ncbi.nlm.nih.gov/sra) (access numbers: SRR13932927, SRR13932928,

SRR13932929, SRR13932930, SRR14242380 and SRR14242381). Alignment onto the mmlO mouse genome, UMI counting and quality control steps were done under the CellRanger (v6.1.2) — count pipeline from lOXGenomics (https://www.10xgenomics.com). The generated h5 files were then processed with Seurat R package (v3) (cf. Butler A, et al., Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol. 2018;36(5):411-420). We removed low-quality cells by selecting those containing over 1.5 fold more and 1.5 fold less than the minimum or maximum of the detected RNA features, respectively. We also excluded those with lower than 10% of their features as being mitochondrial genes. Datasets were normalized and scaled to the data corresponding to the 5,000 most variable features. PCA was performed on the scaled data, and the dimensionality of the dataset was estimated by the ‘Elbow ‘ heuristic method. Clustering of the cells in different communities was done by applying the Louvain algorithm implemented in Seurat R, and was visualized by t-SNE. To visualize the cells expressing the different sets of genes regulated by E2 or E4 identified in our RNAseq, we first identified those common to the marker genes of the different cell communities, obtained by applying the MAST R package (https://github.com/RGLab/MAST/ - Finak G, et al., MAST: a flexible statistical framework for assessing transcriptional changes and characterizing heterogeneity in single-cell RNA sequencing data. Genome Biol. 2015;16:278). We then projected these filtered lists on the t-SNE map by using the FeaturePlot feature of Seurat R with thresholds min.cutoff ="ql0" and max.cutoff = "q90".

Statistical Analysis

Results are expressed as means ± SEM. Statistical analyses were performed using GraphPad Prism 9 (GraphPad Software, San Diego, CA; https://www.graphpad.com). A difference of P<0.05 was considered significant. Gaussian (normal) distribution was determined using the Shapiro-Wilks normality test. For normally distributed populations, Student t test (2 groups) or 1-way ANOVA followed by Bonferroni post-test (3 or more groups) was conducted. For data that failed normality testing, a Kruskal-Wallis with Dunn post-test (3 or more groups) was performed. To test the respective roles of treatment and genotype, a 2-way ANOVA was performed. In case of significant interaction, Bonferroni post-test was subsequently performed. When representative images are shown, the selected images were those that most accurately represented the average data obtained in all the samples.

Example 1. E4 accelerates endothelial healing after endovascular but not perivascular injury of the carotid artery.

First, to compare the effect of E2, E3 and E4 (Figure 1A) on endothelial healing, we optimized E3 and E4 concentrations in pellets to achieve estrogenic impregnation similar to E2, taking into account the difference for ERa affinity between estrogenic compounds (Abot et al., EMBO Mol Med, 2014) and using the uterotrophic effect of estrogens as an endogenous bioassay of estrogen activity (Figure IB). As expected, control ovariectomized mice displayed atrophied uterus while E2, E3 and E4 induced similar increases in uterine weight (Figure IB).

Similarly, the three estrogens led to similar vagina impregnation and thymus atrophy, supporting similar estrogenic action of these three ligands under these experimental conditions (Figure 1C and ID). Importantly, E4 was used at dose which falls into the range of the plasma concentration of the E4 therapeutic dose (15 mg/day) for menopause hormone therapy in women (Buscato et al., Am J Physiol Endocrinol Metab, 2021; Gaspard et al., Menopause. 2020; Coelingh Bennink, Climacteric. 2017; Gallez et al., Cancers (Basel), 2021). Estrogen plasma concentrations were measured by gas chromatography tandem mass spectrometry. No interconversion between the three estrogens was detected in blood samples (Table 1).

Table 1. Plasmatic dosage of E2, E3 and E4. Four-week-old female mice were ovariectomized and implanted with either Veh, E2, E3 or E4 pellets. Plasma concentrations of E2, E3 and E4 were measured 3 weeks later. Results are expressed as means ± SEM.

Then, to evaluate the effect of these three estrogens on re-endothelialization, we studied endothelial healing by Evans Blue staining after endovascular injury of carotid artery in ovariectomized mice treated with E2, E3 or E4, a model in which SMCs are totally preserved (Figure IE) as previously described (Filipe et al., American Journal of Physiology-Heart and Circulatory Physiology, 2008). As expected, estrogenic impregnation with E2 promoted endothelial healing since quantification of reendothelialized areas showed 30 % of endothelial regeneration in vehicle mice compared to day 0 and about 80 % in E2-treated mice. Both E3 and E4 treatments also increased endothelial healing with no significant statistical difference in reendothelialization rates between E2-, E3- and E4-treated groups (Figure IE and IF). E4 effect on endothelial healing was also confirmed using VE-Cadherin staining (Figure 1G). This beneficial effect of E4 contrasts at first glance with our previous work reporting the failure of E4 to promote reendothelialization after perivascular injury (Abot et al., EMBO Mol Med, 2014). However, in contrast to the endovascular model, the perivascular injury provokes a complete decellularization of the arterial wall including both endothelial cells and the underlying SMCs. We confirmed here that E4 is not able to accelerate endothelial healing in this specific perivascular model (Figure 2A and 2B). In addition, we show here that E3, as E2, promoted endothelial healing in this perivascular model, in striking contrast to E4. Importantly, coadministration of E4 with either E2 or E3 abrogated the accelerative effect of these two estrogens on endothelial regeneration (Figure 2A and 2B). Data are shown as means ± SEM (SEM).

Altogether, we demonstrated that the three endogenous estrogens, E2, E3 and E4 are able to accelerate endothelial healing in the mouse carotid artery, but that E4 may require the presence of underlying SMCs to mediate this vascular action and re-endothelialisation.

Example 2. ERa in SMCs mediates endothelial healing in response to E4, independently of membrane initiated signaling. Thus, to assess the underlying mechanism behind the particular action of E4 and to directly evaluate the role of ERa in smooth muscle cells in this beneficial vascular action of E4, we used a mouse model selectively invalidated for ERa in SMCs using the inducible Cre-ERT2 fusion gene system under the control of aSMA promoter (aSMACreERT2 ERalox/lox mice) . E4 treatment led to similar uterine impregnation in both genotypes (uterine weights of each figure are summarized in Table 2). As expected, E4 promoted reendothelialization in littermate control mice after endovascular injury (40 % of reendothelialization in vehicle-treated mice as compared to 80 % in E4-treated mice). In aSMACreERT2+ ERalox/lox mice, this accelerative effect of E4 was totally abrogated (Figure 3 A), demonstrating that ERa in SMCs is required to promote E4 effect on endothelial healing. Since membrane ERa mediates acceleration of reendothelialization in response to E2 (Adlanmerini et al., Proc Natl Acad Sci U S A, 2014; Zahreddine et al., Circ Res, 2020), we then decided to evaluate the role of this pathway in response to E4. To this aim, we used two different mouse models targeting ERa membrane initiated signaling, in which acceleration of endothelial regeneration in response to E2 were shown to be abrogated (Adlanmerini et al., Arterioscler Thromb Vase Biol. 2020; Zahreddine et al., Circ Res. 2020). In C451A-ERa mice, ERa does not localize to the plasma membrane due to the point mutation of its palmitoylation site, leading to the loss of global membrane initiated ERa signalling. In this model, endothelial regeneration was about 20% in vehicle-treated group, and E4 increased reendothelialization to 60%, independently of the genotype (Figure 3B), demonstrating that E4 promotes endothelial healing independently of membrane ERa. We extended this demonstration using a second mouse model, i.e. R264A-ERa mice, targeting the second major amino acid involved in membrane ERa signaling (cf. Adlanmerini M, et al., Mutation of Arginine 264 on ERa (Estrogen Receptor Alpha) Selectively Abrogates tire Rapid Signaling of Estradiol in the Endothelium Without Altering Fertility. Arterioscler Thromb Vase Biol. 2020;40(9):2143-2158). Similarly, we found no difference in reendothelialization rates between control and R264A-ERa female mice following E4 treatment (Figure 3C). In these two mouse models, uterine impregnation in response to E4 was similar in all genotype (Table 2). Then, to directly evaluate ERaMISS in response to E2 and/or E4 in ECs, we used immortalized human aortic endothelial cell line (TeloHAEC). Since TeloHAEC (as others immortalized endothelial cell types) have no detectable ERa expression (Figures 4A and 4B), we generated stable transduced TeloHAECs expressing full- length ERa(Figurcs 4A and 4B, ERa- TeloHAECs). To evaluate ERa MISS pathway in these cells, we measured ERa interaction with the tyrosine kinase sre using the PLA technique (Duolink). Interaction of ERa/Src -kinase is indicated by the presence of white dots in the cytoplasm of ERa- TeloHAECs. Importantly, no dots were detected using either only one antibody or both antibodies in TeloHAECs that do not express ERa, validating the specificity of the technique. As expected, E2 (10-8 M) increased this interaction, whereas a 100-fold higher dose of E4 (i.c. 10-6 M) failed to elicit this membrane ERa effect (Figure 4C). Importantly, when administered together, the

RECTIFIED SHEET (RULE 91) ISA/EP combination of E2 and E4 totally abrogated the stimulatory effect of E2 on ERa/Src-kinase interaction, highlighting that, as shown above in the model of perivascular injury (Figure 2), E4 antagonizes ERaMISS mediated by E2 in endothelial cells.

Table 2. Uterine weights of mice submitted to carotid artery injury. Results are expressed as means ± SEM. ** P<0.01, *** P<0.001, **** PO.OOOl vs Veh.

Example 3. Chronic treatment of E4 and E2 display differential transcriptional program in mouse carotid arteries.

We performed RNA-sequencing on carotid arteries from mice treated chronically with E4 (Figure 5A). This large scale transcriptional approach was performed on non-injured carotids to avoid the complexity associated with the kinetics of endothelial healing. 306 genes were found to be significantly regulated by E4 in the carotid artery compared to vehicle-treated mice (log2fold change >1; adjusted p value <0.05) (Figure 5B). Functional annotation ofthe gene subsets regulated revealed hallmarks for early and late estrogen response. Interestingly, the angiogenesis hallmark was also significantly associated with E4 treatment (Figure 5C). However, comparison of this data set with already published data on E2 -regulated genes in carotid in similar condition revealed that only 14.8% of genes were commonly regulated by E2 and E4 (Figure 5D). t-SNE of single-cell RNA-sequencing data from carotid arteries of wild-type mice, organized by cell cluster was performed according to Li F, et al., 2021 (Single-cell RNA-seq reveals cellular heterogeneity of mouse carotid artery under disturbed flow. Cell Death Discov. 2021 ;7( 1) : 180) as depicted in Figure 5E (SMC: smooth muscle cells, Fibro: Fibroblasts, Macro: Macrophages, EC: endothelial cells). Feature plots of E4-regulated genes (Figure 5F left) and E2 -regulated genes (Figure 5F right) were identified by RNA-sequencing.

A subset of common and specific genes regulated by each ligand was validated by RT-qPCR analysis in independent experiments (Figure 6). We then took advantage of the published transcriptomes of single cells from carotid artery (Andueza et al., Endothelial Reprogramming by Disturbed Flow Revealed by Single-Cell RNA and Chromatin Accessibility Study. Cell Reports. 2020;33(l 1): 108491) to compare the transcriptional signatures of E4 and E2 with the profile of each of the cell types identified in the arterial wall (Figure 5E). Both analyses indicated that genes regulated by E4 are expressed in almost all cell populations subtypes that could be discriminated in carotid arteries (Figure 5F). In contrast, those responding to E2 are mostly associated to monocytes/macrophages (Figure 5F). Altogether, these results revealed that gene expression profiles in carotid arteries from mice treated by E4 and E2 substantially differ both in terms of gene regulation and of cellular targets.

Example 4. Despite its antagonist action on membrane ERa, E4 accelerates endothelial healing in the presence of exogenous and endogenous estrogens.

On the one hand, E2 and E4 act on different cell types to accelerate endothelial healing, and on the other hand E4 antagonized membrane ERa action induced by E2. We therefore decided to evaluate the impact of E4 on reendothelialization after endovascular injury of the carotid artery in presence of exogenous (Figures 7A to 7C) or endogenous E2 (gonad-intact mice) (Figures 7D to 7F). Consistent with the results from Figure 2, co-treatment of ovariectomized female mice with E2 and E4 led to similar uterine impregnation than single treatments (Figures 7B). However, in contrast to the results obtained after perivascular injury (Figure 2), co-administration of E4 with E2 still led to accelerated reendothelialization after endovascular injury, with no difference compared to E2 and E4 alone (Figure 6C). As E4 is commercialized for contraception, we also administrated E4 to gonadintact female mice in which endogenous estrogens are present. As a consequence of E4, mice were blocked in the estrus stage (Figure 7E). Importantly, in intact mice, E4 accelerated endothelial healing (80% of reendothelialization compared to 30% in control mice, Figure 7F). Altogether, these results suggest that even though E4 antagonizes E2 action on membrane ERa in endothelial cells, this effect could be counteracted by its specific agonist action on ERa in SMCs, leading to a beneficial endothelial healing effect compared to intact control mice.

Example 5. E2 and E4 regulate endothelial permeability

RNA sequencing on whole carotid led to the identification of “apical junction” as a pathway regulated by both E2 and E4 in both carotid and aortae from young mice. To explore functional consequences of this regulation, we evaluate endothelial permeability in arteries in response to E2 and E4 in both young and middle-aged mice by analysis of evans blue fluorescence by confocal microscopy. Altogether, these experiments allow to determine a further mechanism of action of the arterial wall, namely endothelial permeability. We compare the effects of E2 and E4 in aorta from young and mature mice since preliminary results indicate improvement of endothelial permeability in response to E2 and E4 in young mice.

Arterial permeability broadly refers to a compilation of structural and functional changes in the entire vessel wall, the passage of macromolecules, fluids, and cells into the intima is primarily due to changes in endothelial barrier function. Indeed, endothelial dysfunction(s) initiate a dysregulated transendothelial flux, which lead(s) to abnormal deposition of molecules and cells in the intima. Although importance of endothelial permeability has been underestimated, it is now clear that endothelial dysfunction and increased permeability are at the onset of early atherosclerosis (Mundi et al., Cardiovascular Research, 2018). Improvements in endothelial barrier function demonstrated in experimental models need to translate into clinically meaningful anti-atherogenic effects in humans. To this aim we analyze albumin in aorta secretome obtained from decreased organ donors. We obtain over one hundred conditioned media from 14 years to 88 years old women classified according to Stary classification and the Virmani word list into three groups: healthy aortas, aortas with fatty streak and aortas with fibro-lipidic plaques (fixed for classical histology, frozen tissues and tissue conditioned media) (Delbosc et al., Front Cardiovasc Med). Intima, media and adventitia are separated by a cardiac surgeon.

Example 6. Endothelial healing in response to E2 and E4 in middle aged mice

In a follow-up experiment of Example 1 in middle aged (1 year-old) female mice it was demonstrated that acceleration of endothelial healing promoted by E2 is abrogated in the carotid arteries from said middle-aged female mice. However, in contrast to E2, E4 is able to accelerate endothelial healing in middle aged mice (Figure 8 right hand panel). Taking into account these different modulations of ERa by E4 in comparison to E2, along with alteration of E2 effect on endothelial healing with age (12 month-old mice), we hypothesize that endothelial ERa expression, activity or targeting to membrane is decreased during aging and/or ERaMISS is altered. This is striking since in young (2- monyth old) mice (Figure 8, left hand panel) E2 and E4 have a similar positive effect on re- endothelialisation, while in older mice (Figure 8 right hand panel) the effect of E2 on re- endothelialisation is lost, while the effect of E4 remains identical as in young mice.

Statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, San Diego, CA; https://www.graphpad.com). Gaussian (normal) distribution was determined using the Shapiro- Wilks normality test. 1-way ANOVA followed by Bonferroni’s post-test was conducted. Exact number (n), precise P values and statistic test used in each experiment are described in Table 3 below.

Table 3: Exact number (n), precise P values and statistic test used in each experiment

Example 7. Clinical trial concerning the impact of estetrol on vascular functions in healthy late post-menopausal women.

Objectives

Primary objective:

To demonstrate that treatment with estetrol 15 mg / 20 mg, does improve endothelium -dependent flow-mediated dilatation of peripheral conduit arteries in response to post-ischemic hyperemia in healthy untreated (not exposed to estrogens) late post-menopausal women (above 60 years of age) while no effect is achieved with 1 mg oral estradiol.

Secondary objective:

To demonstrate that treatment with estetrol 15 mg / 20 mg does improve endothelium -dependent flow-mediated dilatation of peripheral conduit arteries during sustained increase in blood flow in healthy untreated (not exposed to estrogens) late post-menopausal women (above 60 years of age) (second proposition), whereas an improvement is not achieved with 1 mg estradiol.

Study design

An open label non-inferiority prospective trial evaluating the impact of estetrol or estradiol on endothelial function will be conducted in healthy late post-menopausal women. The study will include an inclusion visit (VI) and 2 exploration visits performed in absence of treatment (V2) and after 5-days and 90 days treatment with estetrol or estradiol (V3).

Population

Healthy late post-menopausal women will be selected from the subject database of the Clinical Investigation Center INSERM-CIC1404 of Rouen University Hospital.

Inclusion criteria

Post-menopausal women aged between 60 and 65 years,

Non inclusion criteria

Treatment by estrogen therapy within 6 months prior to inclusion,

Known intolerance to estrogen therapy,

Blood pressure values > 140/90 mmHg or treatment with antihypertensive agents ,

Total cholesterol > 250 mg/dL or treatment with lipid-lowering drugs,

Fasting glucose > 126 mg/dL or treatment antidiabetic agents,

History of myocardial infarction, coronary bypass surgery, angioplasty, stroke, TIA, History of malignant diseases

Known hemostatic disorder predisposing to thromboembolic complications, Previous confirmed venous thromboembolic disease including deep vein phlebitis and/or pulmonary embolism,

Abnormal uterine bleeding,

Severe rhythm disturbances and/or patients with a pacemaker,

Renal failure (plasma creatinine > 200 pmol/1),

Liver disease or hepatic dysfunction with SGOT (ASAT) or SGPT (ALAT) > 2 times upper limit of normal,

CPK levels > 3 times upper limit of normal,

Alcohol consumption > 2 glasses wine/beer per day,

Smoking > 5 cigarettes per day,

Participation in another clinical trial concurrently or within 30 days prior to screening for the present study,

History of alcoholism, drug abuse, psychosis, antagonistic personality, poor motivation or other emotional or intellectual problems that are likely to invalidate Informed consent, or limit the ability of the patient to comply with the protocol requirements,

Donation of blood or blood products for transfusion during/or 2 weeks after the trial.

Exploration visits

Measurement of flow-mediated dilatation in response to post-ischemic hyperemia (transient endothelial stimulation) and hand skin heating (sustained endothelial) stimulation and quantification of the baseline and stimulated endothelial factors bioavailability

All subjects will be explored in the morning under fasting conditions in a quiet air-conditioned room (22°C). After 15 min rest in supine position, brachial artery blood pressure and heart rate will be measured on the nondominant arm using a validated oscillometric device (Omron). Then, the brachial artery endothelium-dependent flow-mediated dilatation in response to post-ischemic hyperemia will be assessed on the dominant arm.1-3 Briefly, an occlusion will be placed on the forearm, inflated for 5 minutes 50 mmHg above systolic pressure and deflated allowing reactive hyperemia, with the continuous measurements of brachial artery diameter and blood flow by echotracking coupled to a Doppler system (ArtLab system®). Thereafter, radial artery flow-mediated dilatation will be measured in response to distal skin heating (34, 37, 40 and 44°C). 1,4,5 Hand skin heating has been developed by our group, validated and used by different research teams to study the endothelial function of peripheral conduit arteries in humans in response to a sustained and stable increase in flow.6-8 The sustained flow-mediated dilatation involves NO and EETs and their release and balance can be assessed thanks to venous blood sampling performed into the antecubital vein at 34 and 44°C.1,4,5 In addition, blood sampling at 34°C will allow the quantification of estradiol and estetrol levels. At the end of heating, endothelium-independent dilation in response to glyceryl trinitrate will be evaluated.4, 5

Claims

1. A composition comprising an effective amount of estetrol (E4) component for use in preventing or treating a medical condition or disorder linked to or caused by endothelial impairment, more preferably, by invoking endothelial integrity and/or facilitating or increasing endothelial repair in a subject.

2. Use of an effective amount of an estetrol component in the manufacture of a medicament for preventing or treating a medical condition or disorder linked to or caused by endothelial impairment, more preferably, by invoking endothelial integrity and/or facilitating or increasing endothelial repair in a subject.

3. A method of preventing or treating a medical condition or disorder linked to or caused by endothelial impairment, more preferably, by invoking endothelial integrity and/or facilitating or increasing repairing the endothelium in a subject, comprising the step of administering an effective amount of an estetrol component.

4. The composition for use according to claim 1, the use according to claim 2, or the method according to claim 3, wherein the subject is diagnosed with, considered to have, or considered at risk to develop an endothelial vulnerability leading to an increase of cardiovascular risk.

5. The composition for use, the use, or the method according to any one of claims 1 to 4, wherein said endothelial impairment is caused by decreased membrane expression of estrogen receptor alpha.

6. The composition for use, the use, or the method according to any one of claims 1 to 5, wherein said impairment of endothelial integrity results in cardiovascular diseases.

7. The composition for use, the use, or the method according to any one of claims 1 to 6, wherein said endothelial impairment is restenosis, preferably restenosis after stent implantation; or late stent thrombosis.

8. The composition for use, the use, or the method according to any one of claims 1 to 7, wherein said impairment of endothelial integrity is a peripheral vascular disease.

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

10. The composition for use, the use, or the method according to any one of claims 1 to 9, wherein the subject is a female subject.

11. The composition for use, the use, or the method according to any one of claims 1 to 10, wherein the subject is diagnosed to have, or considered to have estrogen.

12. The composition for use, the use, or the method according to any one of claims 1 to 11, wherein said subject is of middle age or older, preferably wherein said subject is of age 40 or older.

13. The composition for use, the use, or the method according to any one of claims 1 to 12, wherein said subject is a menopausal, perimenopausal or postmenopausal female subject.

14. The composition for use, the use, or the method according to any one of claims 1 to 13, wherein a daily amount equivalent to from about 5 to about 40 mg of estetrol, such as from about 10 mg to about 25 mg estetrol, or from about 14 to about 21 mg of estetrol is administered.

15. The composition for use, the use, or the method according to any one of claims 1 to 14, wherein said estetrol component is estetrol or an ester thereof, more preferably wherein said estetrol component is estetrol monohydrate.

16. The composition for use, the use, or the method according to any one of claims 1 to 15, wherein no progestogen is comprised in said composition, is used, or co-administered.

17. The composition for use, the use, or the method according to any one of claims 1 to 16, wherein a progestogen is present in the composition, is used, or wherein a progestogen is coadministered or administrated after treatment with estetrol, preferably wherein said progestogen is 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. In a preferred embodiment, said progestogen is selected from the group comprising drospirenone, progesterone, or dydrogesterone.

18. The composition for use, the use, or the method according to any one of claims 1 to 17, wherein an additional active ingredient known to be involved in promoting endothelial healing or in restoring endothelial impairment is present in said composition.

19. The composition for use, the use, or the method according to any one of claims 1 to 18, wherein the composition is an oral dosage form, e.g. formulated for oral, sublingual, buccal, or sublabial administration, or wherein the composition is formulated for intravenous administration or wherein the composition is formulated for topical or vaginal administration.