Comparison of Estetrol Exposure between Women and Mice to Model Preclinical Experiments and Anticipate Human Treatment.

Estetrol (E4) is a natural estrogen with promising therapeutic applications in humans. The European Medicines Agency and the Food and Drug Administration have approved the use of 15 mg E4/3 mg drospirenone for contraceptive indication. Phase III clinical trials with 15-20 mg E4 for the relief of climacteric complaints are currently running. Relevant data from preclinical animal models are needed to characterize the molecular mechanisms and the pharmacological effects of E4 and possibly to reveal new therapeutic applications and to anticipate potential adverse effects. Therefore, it is important to design experimental procedures in rodents that closely mimic or anticipate human E4 exposure. In this study, we compared the effects of E4 exposure after acute or chronic administration in women and mice. Women who received chronic E4 treatment per os at a dose of 15 mg once daily reached a steady state within 6 to 8 days, with a mean plasma concentration of 3.20 ng/mL. Importantly, with subcutaneous, intraperitoneal or oral administration of E4 in mice, a stable concentration over time that would mimic human pharmacokinetics could not be achieved. The use of osmotic minipumps continuously releasing E4 for several weeks provided an exposure profile mimicking chronic oral administration in women. Measurements of the circulating concentration of E4 in mice revealed that the mouse equivalent dose necessary to mimic human treatment does not fit with the allometric prediction. In conclusion, this study highlights the importance of precise definition of the most appropriate dose and route of administration to utilize when developing predictive preclinical animal models to mimic or anticipate specific human treatment.

Sex-Based Differences in the Tumor Microenvironment.

Cancers are heterogeneous multifactorial diseases consisting of a major public health issue worldwide. Sex disparities are evidenced in cancer incidence, mortality, expression of prognosis factor, response to treatment, and survival. For both sexes, an interplay of intrinsic and environmental factors influences cancer cells and tumor microenvironment (TME) components. The TME cumulates both supportive and communicative functions, contributing to cancer development, progression, and metastasis dissemination. The frontline topics of this chapter are focused on the contribution of sex, via steroid hormones, such as estrogens and androgens, on the following components of the TME: cancer-associated fibroblasts (CAFs), extracellular matrix (ECM), blood and lymphatic endothelial cells, and immunity/inflammatory system.

Estetrol and Mammary Gland: Friends or Foes?

Estrogens have pleiotropic effects on many reproductive and non-reproductive tissues and organs including the mammary gland, uterus, ovaries, vagina, and endothelium. Estrogen receptor α functions as the principal mediator of estrogenic action in most of these tissues. Estetrol (E4) is a native fetal estrogen with selective tissue actions that is currently approved for use as the estrogen component in a combined oral contraceptive and is being developed as a menopause hormone therapy (MHT, also known as hormone replacement therapy). However, exogenous hormonal treatments, in particular MHTs, have been shown to promote the growth of preexisting breast cancers and are associated with a variable risk of breast cancer depending on the treatment modality. Therefore, evaluating the safety of E4-based formulations on the breast forms a crucial part of the clinical development process. This review highlights preclinical and clinical studies that have assessed the effects of E4 and E4-progestogen combinations on the mammary gland and breast cancer, focusing in particular on the estrogenic and anti-estrogenic properties of E4. We discuss the potential advantages of E4 over current available estrogen-formulations as a contraceptive and for the treatment of symptoms due to menopause. We also consider the potential of E4 for the treatment of endocrine-resistant breast cancer.

Estetrol Combined to Progestogen for Menopause or Contraception Indication Is Neutral on Breast Cancer.

Given the unequivocal benefits of menopause hormone therapies (MHT) and combined oral contraceptives (COC), there is a clinical need for new formulations devoid of any risk of breast cancer promotion. Accumulating data from preclinical and clinical studies support that estetrol (E4) is a promising natural estrogen for MHT and COC. Nevertheless, we report here that E4 remains active on the endometrium, even under a dose that is neutral on breast cancer growth and lung metastasis dissemination. This implies that a progestogen should be combined with E4 to protect the endometrium of non-hysterectomized women from hyperplasia and cancer. Through in vivo observations and transcriptomic analyses, our work provides evidence that combining a progestogen to E4 is neutral on breast cancer growth and dissemination, with very limited transcriptional impact. The assessment of breast cancer risk in patients during the development of new MHT or COC is not possible given the requirement of long-term studies in large populations. This translational preclinical research provides new evidence that a therapeutic dose of E4 for MHT or COC, combined with progesterone or drospirenone, may provide a better benefit/risk profile towards breast cancer risk compared to hormonal treatments currently available for patients.

Liposomes and drug-in-cyclodextrin-in-liposomes formulations encapsulating 17ß-estradiol: An innovative drug delivery system that prevents the activation of the membrane-initiated steroid signaling (MISS) of estrogen receptor α.

The encapsulation into liposomes of several types of molecules presents the advantages to protect the activity of these molecules and to target specific tissues. Nevertheless, a major obstacle remains the incomplete understanding of nano-bio interactions. Specifically, the impact that inclusion of drug into liposomes or of drug-in-cyclodextrin-in liposomes (DCL) could have on the molecular and cellular mechanism of drug action is largely unknown. As a proof of concept, we evaluated the impact of 17ß-estradiol (E2) included into liposomes or DCL on estrogen receptor (ER)α signaling pathways. Indeed, ERα relays the pleiotropic actions of E2 in physiology and pathophysiology through two major pathways: (1) the genomic/nuclear effects associated to the transcriptional activity of the ERα and (2) the rapid/nongenomic/membrane-initiated steroid signaling (MISS) effects related to the induction of fast signaling pathways occurring when ERα is anchored to the plasma membrane. We evidenced that the inclusion of E2 into liposomes (Lipo-E2) or into DCL (DCL-E2) prevented the activation of the rapid/nongenomic/extranuclear/MISS pathway of ERα, while the activation of the genomic/nuclear pathway was maintained. These results support that Lipo-E2 and DCL-E2 could be a useful tool to delineate the complex molecular mechanisms associated to ERα. In conclusion, this study supports the notion that inclusion of drugs into liposomes or DCL could modify some specific pathways of their molecular and cellular mechanisms of action. These results emphasized that attention should be paid to nano-bio interactions induced by the use of nanovectors in medicine.

Lymph/angiogenesis contributes to sex differences in lung cancer through oestrogen receptor alpha signalling.

Oestrogen signalling pathways are emerging targets for lung cancer therapy. Unravelling the contribution of oestrogens in lung cancer development is a pre-requisite to support the development of sex-based treatments and identify patients who could potentially benefit from anti-oestrogen treatments. In this study, we highlight the contribution of lymphatic and blood endothelia in the sex-dependent modulation of lung cancer. The orthotopic graft of syngeneic lung cancer cells into immunocompetent mice showed that lung tumours grow faster in female mice than in males. Moreover, oestradiol (E2) promoted tumour development, increased lymph/angiogenesis and VEGFA and bFGF levels in lung tumours of females through an oestrogen receptor (ER) alpha-dependent pathway. Furthermore, while treatment with ERb antagonist was inefficient, ERa antagonist (MPP) and tamoxifen decreased lung tumour volumes, altered blood and lymphatic vasculature and reduced VEGFA and bFGF levels in females, but not in males. Finally, the quantification of lymphatic and blood vasculature of lung adenocarcinoma biopsies from patients aged between 35 and 55 years revealed more extensive lymphangiogenesis and angiogenesis in tumour samples issued from women than from men. In conclusion, our findings highlight an E2/ERa-dependent modulation of lymphatic and blood vascular components of lung tumour microenvironment. Our study has potential clinical implication in a personalised medicine perspective by pointing to the importance of oestrogen status or supplementation on lung cancer development that should be considered to adapt therapeutic strategies.

Whole blood microsampling for the quantitation of estetrol without derivatization by liquid chromatography-tandem mass spectrometry.

Quantitative bioanalysis and especially pharmacokinetic studies are challenging since only low volumes of biological material are available and low concentrations (ng/ml) are often expected. In this context, volumetric absorptive microsampling (VAMS) devices were developed to accurately collect 10 or 20µl of whole blood from tested subjects. In this study, we present the development and validation of ultra-high performance liquid chromatography coupled to tandem mass spectrometry method after VAMS sampling for the quantitation of estetrol (E4), a potentially new medicine for hormone replacement, contraception and osteoporosis therapies. Interestingly, a very simple sample preparation procedure was developed without any derivatization step. Even if lack of sensitivity is a common consideration when using negative ionization mode, we demonstrated in this work that an excellent sensitivity could be reached by carefully optimizing the nature and concentration of the mobile phase additive. After the optimization of every experimental parameter, the stability, selectivity, trueness, precision and accuracy of the final method were successfully demonstrated. In addition, the excellent performances of the method were confirmed by two independent proof-of-concept pharmacokinetic studies of E4 after VAMS collection in a murine model.

Accurate Control of 17ß-Estradiol Long-Term Release Increases Reliability and Reproducibility of Preclinical Animal Studies.

Estrogens are the subject of intensive researches aiming to elucidate their mechanism of action on the various tissues they target and especially on mammary gland and breast cancer. The use of ready-to-use slow releasing devices to administer steroids, especially estrogens, to small experimental animals remains the method of choice in terms of animal well-being and of safety for both the researcher and the animal. In this study, we evaluated and compared, in vitro and in vivo, the release kinetic of estradiol (E2) over sixty days from two different slow-releasing systems: the matrix pellet (MP) and the reservoir implant (RI). We compared the impact of these systems in three E2-sensitive mouse models : mammary gland development, human MCF7 adenocarcinoma xenograft and mouse melanoma progression. The real amount of E2 that is released from both types of devices could differ from manufacturer specifications due to inadequate release for MP and initial burst effect for RI. Compared to MP, the interindividual variability was reduced with RI thanks to a superior control of the E2 release. Depending on the dose-dependent sensitivity of the physiological or pathological readout studied, this could lead to an improvement of the statistical power of in vivo experiments and thus to a reduction of the required animal number. Altogether, our data draw attention on the importance to adequately select the slow-releasing device that is the most appropriated to a specific experiment to better fulfill the 3Rs rule (Replacement, Reduction, Refinement) related to animal welfare and protection.

Quantitative Assessment of Mouse Mammary Gland Morphology Using Automated Digital Image Processing and TEB Detection.

The assessment of rodent mammary gland morphology is largely used to study the molecular mechanisms driving breast development and to analyze the impact of various endocrine disruptors with putative pathological implications. In this work, we propose a methodology relying on fully automated digital image analysis methods including image processing and quantification of the whole ductal tree and of the terminal end buds as well. It allows to accurately and objectively measure both growth parameters and fine morphological glandular structures. Mammary gland elongation was characterized by 2 parameters: the length and the epithelial area of the ductal tree. Ductal tree fine structures were characterized by: 1) branch end-point density, 2) branching density, and 3) branch length distribution. The proposed methodology was compared with quantification methods classically used in the literature. This procedure can be transposed to several software and thus largely used by scientists studying rodent mammary gland morphology.