USE OF NEUROPILIN ANTAGONISTS FOR THE TREATMENT OF ENDOMETRIOSIS

Abstract

Endometriosis is a chronic inflammatory systemic sex hormone-dependent gynecological disease, characterized by the presence and growth of endometrial tissue (glands and stroma) outside the uterine cavity, predominantly, but not exclusively, in the pelvic compartment. Here, the inventors show that the use of Neuropilin/VEGF binding inhibitors, so called, Neuropilin antagonist (NRPa), bring new perspective to treat and cure endometriosis in women suffering thereof. NRPa alone is efficient to inhibit primary endometrial cell proliferation and apoptosis/necrosis program cell death of targeted cells. The effective NRPa concentration needed is very low (NRPa-48 IC.sub.50=10.sup.−7M) and is dependent of the NRPa structure. Moreover, the association of NRPa with progestogen drug increases the anti-proliferative effect. Therefore, the present invention relates to the use of neuropilin antagonists for the treatment of endometriosis.

Claims

1. A method of treating endometriosis in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a neuropilin antagonist (NRPa).

2. The method of claim 1 wherein the patient suffers form peritoneal endometriosis, ovarian endometriosis, deep endometriosis or extrapelvic endometriosis.

3. The method of claim 1 wherein the subject is a human.

4. The method of claim 1 wherein the subject is a non-human mammal.

5. The method of claim 1 wherein the neuropilin antagonist is selected from the group consisting of antisense polynucleotides, interfering RNAs, catalytic RNAs, RNA-DNA chimeras, neuropilin-specific aptamers, anti-neuropilin antibodies, neuropilin-binding fragments of anti-neuropilin antibodies, neuropilin-binding small molecules, neuropilin-binding peptides, and other polypeptides that specifically bind neuropilin (including, but not limited to, neuropilin-binding fragments of one or more neuropilin ligands, optionally fused to one or more additional domains), such that the interaction between the neuropilin antagonist and neuropilin results in a reduction or cessation of neuropilin activity or expression.

6. The method of claim 1 wherein the neuropilin antagonist inhibits the interaction between a neuropilin protein (e.g. NRP-1) and its partners, in particular VEGF-A.sub.165.

7. The method of claim 1 wherein the neuropilin antagonist is an antibody that specifically binds to a neuropilin (e.g. NRP-1 or NRP-2) and neutralizes its activity to activate neuropilin signalling pathway, and in particular inhibits the binding neuropilin and VEGF-A.sub.165.

8. The method of claim 1 wherein the neuropilin antagonist is NRPa-47 or NRPa-48.

9. The method of claim 1 wherein the neuropilin antagonist is administered to the patient in combination with a progestogen.

10. The method of claim 1 wherein the progestogen is selected from chlormadinone acetate, cyproterone acetate, desogestrel, dienogest, 5α-dihydroprogesterone, drospirenone (Yasmit®), ethanediol acetate, ethynodiol diacetate, etonouestrel (Nexplanon®), gestodene, 17-hydroxyprogesterone, levonorgestrel (Alesse®), medroxyprogesterone acetate (17α-hydroxy-6α-methylprogesterone acetate; Provera®), megestrol, megestrol acetate (17αacetoxy-6-dehydro-6-methylprogesterone), nestorone, nomegestrol acetate, norethindrone, norethindrone acetate (also known as norethindrone acetate), norethynodrel Enovid®), norgestimate, norgestrel, progesterone, tanaproget, trimegestone, pharmaceutically acceptable salts of any of the foregoing, and any combination thereof.

Description

FIGURES

[0048] FIG. 1: Primary endometrial cell line (TH-EM1) express Neuropilin-1 and is sensitive to Neuropilin-1 inhibitor. [0049] A—Histogram shows the expression and the high amount of Neuropilin-1 at the cell surface of the primary endometrial cell line. [0050] B—Neuropilin antagonist (NRPa) such as NPRa-47 at 10.sup.−5M is significantly efficient to decrease 80% of cell survival in culture after 48 h of exposure (D2) and remains consistent at 72 h (D3). Data represent means±SD of 3 separate experiments, each. (***p<0.001).

[0051] FIG. 2: Low concentration of NRPa is required to suppress primary endometrial cell proliferation. [0052] A—A large concentration range of NRPa-48 is tested alone on primary endometrial cell proliferation. Calculated NRPa-48 IC.sub.50 is closed to 10.sup.−7 M. Curves are representative of at least 3 separate experiments. [0053] B—A large concentration range of NRPa-47 is tested alone on primary endometrial cell proliferation. Calculated NRPa-47 IC.sub.50 is comprise between 10.sup.−6 to 10.sup.−7 M. Curves are representative of at least 3 separate experiments. [0054] Data represent means±SD. (***p<0.001).

[0055] FIG. 3: NRPa induces apoptosis and necrosis of primary endometrial cell. [0056] A—Dot plots show the induction of apoptosis (Annexin-V.sup.+/7-AAD.sup.+) and necrosis (Annexin-V.sup.−/7-AAD.sup.+) of endometrial cells induced by NRPa-47 and NRPa-48 at 10.sup.−6 M at 72 h compare to untreated and DMSO treated cells as control. [0057] B—Histogram compared the ratio of live, apoptotic and necrotic endometrial cells under NRPa treatment.

[0058] FIG. 4: NRPa and Progestatif drug association increases the inhibition of endometrial cell proliferation. [0059] A—Histogram shows the percentage of growth inhibition of primary endometrial cells induced by Lutenyl® (Nomegestrol acetate) at 3.Math.10.sup.−5 M, NRPa-47 (5.Math.10.sup.−7M) alone or in association. Additif effect of the association is observed. [0060] B—Histogram shows the percentage of growth inhibition of primary endometrial cells induced by Lutenyl® (Nomegestrol acetate) at 3.Math.10.sup.−s M, NRPa-48 (5.Math.10.sup.−8M) alone or in association. Synergistic effect of the association is observed. [0061] Data represent means±SD. (**p<0.01, ***p<0.001).

EXAMPLE

[0062] Methods

[0063] Cell Culture

[0064] The human primary unmodified endometrial cell line (TH-EM1) describes by Gogusev j et al. 2019 is cultured with Dulbecco modified Eagle medium supplemented with 10% of fetal calf serum (FCS), L-glutamine, and antibiotics (Invitrogen-Thermo Fisher, Cergy-Pontoise, France). Cells were plated in 96-well plates or in 12-well plates to study cell proliferation or apoptosis, respectively.

[0065] Flow Cytometry

[0066] Untreated proliferating TH-EM1 cells (2.Math.10.sup.5 cells) were harvested and washed with PBS 1× with ca.sup.2+/mg.sup.2+ containing 2% FCS buffer. Then, cells were directly stained with phycoerythrin (PE)-Neuropilin-1 or irrelevant appropriate monoclonal antibodies as controlled using PBS 1×2% FCS (Miltenyi biotec, France). Then, stained cells were analyzed on a fluorescence activated cell sorter (FACS) Calibur flow cytometer (Becton Dickinson Co, Mountain View, Calif.) and data analysis was performed with Flowjo software.

[0067] Cell Proliferation Assay

[0068] Cells were plated in 200 μL/well in 96-well plates at 10.sup.4 cells/well and were treated or not with Neuropilin antagonists (NRPa) such as NRPa-47, NRPa-48 at several concentration (10.sup.−5 to 5.Math.10.sup.−8M) alone or in association with different concentrations of progestatif drugs such as Lutenyl® (Nomegestrol acetate) at 3.Math.10.sup.−5 M during 48 h, 72 h or 120 h. WST-1 (Roche®, France) was added for 2 h, then Optical Density was analyzed with a microplate reader (Microplate Manager 5.2, Bio-Rad) at 490 nm to determine the cell viability.

[0069] Apoptosis Assay

[0070] Cells were plated in 2000 μL/well in 12-well plates at 5.Math.10.sup.5 cells/well and were treated or not with Neuropilin antagonists (NRPa) such as NRPa-47, NRPa-48 during 48 h to 72 h at 10.sup.−6M. Then, cells are harvested and stained using Annexin-V/7AAD kit protocol following manufacturer's recommendation. Then, stained cells were analyzed on a fluorescence activated cell sorter (FACS) Calibur flow cytometer (Becton Dickinson Co, Mountain View, Calif.) and data analysis was performed with Flowjo software.

[0071] Statistical Analysis:

[0072] Data are expressed as the arithmetic mean+/−SD of at least three different experiments. The statistical significance of results was evaluated by ANOVA, with probability values **p<0.01, ***p<0.001, being considered as significant.

[0073] Results

[0074] Neuropilin Expression and NRPa Sensitivity of Endometrial Cells

[0075] To test the efficiency of Neuropilin antagonist (NRPa) on endometriosis, we first examined its level expression on the human unmodified primary endometrial cell (Gogusev et al. 2019). As we show in FIG. 1A, Neuropilin (NRP-1) is significantly express at the cell surface of these cells. Even if NRP-1 is expressed, these might be or not sensitive to NRPa, thus we initially tested at high level concentration of NRPa (NRPa-47, 10.sup.−5M) if any effect is observed on cells. As expected, after 48 to 72 hours of exposure, quantity of endometrial cells is significantly decreased (80% of inhibition) (FIG. 1B).

[0076] Low Concentration of NRPa is Needed to Inhibit Endometrial Cell Growth.

[0077] To determine the sensitivity of endometrial cell to NRPa, several concentrations (10.sup.−5, 10.sup.−6, 10.sup.−7 M) were evaluated during a time course (including day-2 to day-5 examination). As expected, a rapid endometrial growth inhibition is obtained at high level concentration (10.sup.−5M) of NRPa (NRPa-47 and NRPa-48) since 2 days (D2) of exposure (FIGS. 2A and 2B). However, Low concentration of NRPa (NRPa-47 and NRPa-48) comprise between 10.sup.−6 and 10.sup.−7 M reached a maximum efficiency after 3 days (D3) of exposure since at D5 the effect stayed stable (FIGS. 2A and 2B). These results allowed us to determine the approximative IC.sub.50 of NRPa-48 at 10.sup.−7 M (100 nM) and of NRPa-47 comprise between 10.sup.−6 to 10.sup.−7 M (FIGS. 2A and 2B).

[0078] Endometrial Cell Growth Inhibition is Mediated by the Apoptosis Induced by NRPa.

[0079] The endometrial cell apoptosis was followed under the NRPa exposure after 48 and 72 hours using Annexin-V/7-AAD staining. NRPa including NRPa-47 and NRPa-48, may induced endometrial cell death reaching a maximum at 72 hours of exposure (FIG. 3A). This induced cell death is divided in two steps, first the induction of apoptosis (Annexin-V.sup.−/7-AAD.sup.+ and Annexin-V.sup.−/7-AAD.sup.− staining) and also the induction of necrosis (Annexin-V.sup.−/7-AAD.sup.+) (FIGS. 3A and 3B). Histogram clearly showed the percentage of each cell death phase induced by NRPa on endometrial cells at low concentration.

[0080] NRPa May Act Synergistically with Conventional Progestogen.

[0081] As patient suffering of endometriosis continuously received progestogen drugs, we investigated the effect of NRPa associated to it. Surprisingly, the association of low NRPa-47 concentration (6.Math.10.sup.−7 M) with progestogen drug (Nomegestrol acetate) procured additive effect on cell growth inhibition (FIG. 4A). More importantly, using 2-fold less of NRPa-48 IC.sub.50 concentration (5.Math.10.sup.−8M) a synergistic effect is observed in presence of progestogen drug (FIG. 4B). These results reinforced the interest of the NRPa use in this pathology and strengthened the importance of its association of conventional progestogen drugs.

[0082] Discussion:

[0083] Taken together, this report highlighted the pivotal importance of the development of NRPa, which brought new tools for endometriosis treatment and for the knowledge of biological pathways involved in this disease. In this report, we observed that two structurally-related NRPa (NRPa-47 and NRPa-48) rapidly inhibit endometriosis cell line growth alone or in association with conventional hormonal treatment. In this context, NRPa may induce endometriosis cell line apoptosis. Future investigations are needed to identify the downstream pathway mediated by NRPa on kinase regulation as well as on the modulation of death receptors, pro-apoptotic and anti-apoptotic proteins.

[0084] In summary, NRPa might be used alone or in association with a conventional endometriosis hormonal treatment. This observation brought newest interest for the development of NRPa in this pathology.

REFERENCES

[0085] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure. [0086] Gogusev J., Lepelletier Y., El Khattabi L., Grigoroiu M., and Validire P. Establishment and Characterization of a Stromal Cell Line Derived From a Patient With Thoracic Endometriosis. Reproductive Sciences, 2019.