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 proliferation of endometrial tissue (glandular and stromal) outside the uterine cavity, primarily in the pelvic compartment. The present inventors have demonstrated that the use of neuropilin / VEGF binding inhibitors, so-called neuropilin antagonists (NRPa), offers new perspectives for the treatment and cure of women suffering from endometriosis. NRPa alone can suppress endometrial cell proliferation and inhibit the apoptotic / necrotic program of target cell death. The effective concentration of NRPa required is very low (NRPa-48 IC 50 =10 -7 M), depending on the structure of NRPa. NRPa can also enhance its antiproliferative effect by binding to progestins. Therefore, the present invention relates to the use of neuropilin antagonists for the treatment of endometriosis.

Description

Technical Field

[0001] This invention Ming relates to medicine, particularly gynecology relates to the field of .

[0002] Background of the Invention Endometriosis is a chronic inflammatory systemic sex hormone-dependent gynecological disease medicine, and mainly, although not exclusively, to the presence and growth outside the uterine cavity in the pelvic compartment characterized by the presence of endometrial tissue (glands and stroma) characterized by . This proliferation 、 is called a so-called lesion and also occurs in the genital organs, but there is a rare form of endometriosis the form (20% of cases). This rare form, so-called extra-pelvic the form is classified, as classified in the Orphanet rare disease classification (ORPHA: 137820), of the lungs in the pleura, kidney, bladder, abdominal wall, umbilicus, and cesarean section scar scar (skin uterus endometriosis morphology ) etc 、 and is characterized by the presence of functioning endometrial glands morphological uterus and stroma in several locations and .

[0003] This disease affects at least 10% of women of reproductive age (about 300 million people worldwide in women ) is affected, and the annual incidence of endometriosis in women aged 15 - 49 years is 0.1%, with a peak between 25 - 35 years of age. [Parazzini, F., Vercellini, P. & Pelucchi, C. in Endometriosis science and practice (eds Giudice, L. C., Johannes, L. H. & Healy,D. L.) 19 - 26 (Wiley - Blackwell Publishing, 2012)][Vigano, P., Parazzini, F., Somigliana, E. & Vercellini, P. Endometriosis: epidemiology and aetiological factors. Best Pract. Res. Clin. Obstet. Gynaecol. 18, 177 - 200 (2004)] 。 This disease is pelvic pain and and is associated with infertility. [L. C. Giudice, “Clinical practice. Endometriosis,” New England Journal of Medicine, vol. 362, no. 25, pp. 2389 - 2398, 2010][[S. E. Bulun, “Endometriosis,” New England Journal of Medicine, vol. 360, no. 3, pp. 268 - 279, 2009].

[0004] The clinical symptoms are related to menstruation and are at the sites of ectopic tissue dependent but are generally related sites in pain, tumors / small nodules, swelling and / or bleeding including .

[0005] superficial, ovarian uterus endometriomas and deep infiltrating endometriosis including 3 two different phenotypes have been recognized. This disease affects approximately 30% of patients to with adenomyosis accompanied by . This disease has an impact on the quality of women's liveshaving a major effect along with, over a long period of time considerable should be considered as an important public health issue that imposes a long-term economic burden. Pelvic pain having , for patients who do not wish to become pregnant immediately, medical treatment is the first choice of treatment options . For infertility patients, before undergoing endometriosis surgery, it is necessary to carefully consider providing whether or not to perform assisted reproductive medicine The drug is Analgesia the drug and Combined use doing is typical no treatment In the case of these previous endometriosis hormones treatment the focus has been on stopping estradiol production from the ovaries focus . The failure of this direction of treatment is or the fact that estrone from the adrenal glands heterogeneity environmental toxins leaking from adipose tissue ( not suppressing estrogen)

[0006] Among the hormones that have been used for treatment endometriosis, there are progest - gen oral contraceptives, etc. Modern endometriosis management should be multiple modes and interdisciplinary such Integrated approach and Individualized being .

[0007] therapeutically For for treatment endometriosis medical treatment , surgical surgery , and Assisted reproductive medicine (ART) including the main options is existing .

[0008] Widely used used hormones treatment are effective for treatment symptoms, but not radical, that is, without removing endometriosis lesions pain to relieving . Hormones treatment For patients who do not respond, new drugs substance are urgently needed. GnRH antagonist , selective estrogen or progesterone receptor modulators, anti-angiogenic drugs, antioxidants, immune modulator , and newly emerging drugs such as certain epigenetic agents substance are all new and promising indicating treatment but all require more thorough experimental and clinical evaluation.

[0009] Summary of the Invention As defined by the claims, the present invention relates to the use of neuropilin treatment for endometriosis. antagonist

[0010] Detailed Description of the Invention The inventors have shown that the use of neuropilin / VEGF binding inhibitor , so-called neuropilin antagonist (NRPa), brings new prospects for the suffering from it treatment of women in the treatment of endometriosis and . NRPa alone primary inhibits the proliferation of endometrial cells and and the apoptosis / necrosis program mucous of target cells efficiently at will. Certainly effective concentrations of NRPa that are 、 very low (NRPa-48 IC 50 = 10 -7 M), and depending on the structure of NRPa. further , NRPa and and in combination with progestative drugs by reproductive effect enhances the anti-proliferative

[0011] Therefore, a first one object of the present invention is related to a method for endometriosis in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a neuropilin antagonist (NRPa). treating

[0012] As used herein, the term "endometriosis" has its ordinary meaning in the art (see "Background" section). In particular, this term refers to peritoneal uterus endometriosis, ovarian uterus endometriosis, deep uterus endometriosis and extra-pelvic uterus endometriosis, and the like.

[0013] As used herein, the term "patient" refers to a female mammal to whom the present invention may be applied. Typically, the mammal is animal a human (i.e., a woman), but may also relate to primates, 、 ,,,, dog ,,,, cat ,,,, pig ,,,, sheep ,,,, such as cattle or other mammals.

[0014] As used herein, terms " "treatment" " or "treatment "treating" means ,,,, no treatment prophylactic or preventive no treatment as well as curative or disease-modifying patients at risk of suffering from a disease or patients suspected of suffering from a disease ,,,, treatment of patients diagnosed with a disease or medical condition and includes not only suppressing clinical recurrence. treatment is administered to a patient having a medical disorder or a patient who may ultimately acquire a disorder to prevent, cure, delay onset, reduce severity, or improve one or more symptoms of the disorder or the disorder that recurs, or to extend the patient's survival period beyond what would be expected if such treatment were not performed. "Treatment regimen" means a treatment pattern of the disease, e.g., a dosing pattern used during treatment. A treatment regimen can include an induction regimen and and a maintenance regimen. The phrase "induction regimen" or "induction period" means an initial used for treatment treatment regimen (or a part of a treatment regimen) of a disease. The general goal of an induction regimen is to treatment administer a high level of drug to the patient during the initial period of the regimen providing is to be done. The induction regimen may (partially or wholly) adopt a "loading regimen", which may include administering a drug in an amount greater than that adopted by the physician during the maintenance regimen, administering the drug more frequently than the physician administers the drug during the maintenance regimen, or both. The term "maintenance" or "maintenance period" refers to a treatment regimen (or a part of the treatment regimen) used for maintaining the patient during the course of the disease, for example, used to keep the patient in a remission state for a long period (several months or years). Maintenance can adopt continuous therapy (for example, administering the drug at regular intervals such as weekly, monthly, annually, etc.) or intermittent therapy (for example, treatment at the time of interruption, intermittent, recurrence, achievement of specific criteria [such as pain, disease, etc.]). In particular, in the case of AML, minimal residual disease that is clinically invisible can be eradicated by maintenance therapy. many doses than the physician administers during the maintenance regimen, administering the drug more frequently than the physician administers the drug during the maintenance regimen, or both may be included. "Maintenance" regimen or the phrase "maintenance period" refers to the treatment regimen (or part of the treatment regimen) used for maintaining the patient during the treatment course of the disease, for example, used to keep the patient in a remission state for a long period (several months or years). Maintenance regimen can adopt continuous therapy (for example, administering the drug at regular intervals such as weekly, monthly, annually, etc.) or intermittent therapy (for example, treatment at the time of substance interruption, intermittent treatment , recurrence treatment , at the time of treatment specific predetermined criteria [such as pain, disease symptoms etc.])

[0015] More specifically, terms "endometriosis "treatment" means to reduce (or remove) the amount of endometrial tissue present inside and / or outside the uterine cavity treatment (for example, shrinking or removing endometriosis lesions); and / or reduce one or more symptoms associated with endometriosis and / or improve treatment (for example, reducing and / or improving the symptoms of dysmenorrhea treatment ; reducing and / or or improving the symptoms of dyspareunia treatment , and / or bone pelvic pain pain improvement and / or relief alleviating treatment ) including 。The American Society for Reproductive Medicine (ASRM) has defined classification systems for various stages of endometriosis, which are divided into four stages (stage IV being the most severe and stage I being the least severe). American Society for Reproductive Medicine. Revised American Society for Reproductive Medicine classification of endometriosis: 1996. Fertil Steril 1997; 67,817 821.] Therefore, the term " treatment of endometriosis" refers to the severity of the condition as measured by the ASRM classification, relief such as treatment reducing the severity of endometriosis from stage IV, III, II, or I more to a lower stage relief or treatment until the symptoms are completely alleviated treatment .

[0016] As used herein when treated, the term "neuropilin" or "NRP " in the art in its generally refers to having a transmembrane glycoprotein typically consisting of 3 one extracellular domain (a1 a2, b1, b2, and c), a transmembrane domain, and two domains of the cytoplasmic domain 5 . There are two types of neuropilin, neuropilin-1 (NRP-1) referring to and neuropilin-2 (NRP-2), which have 44% sequence homology and . Neuropilin is a member of the VEGF (Vascular Endothelial Growth Factor) family, including VEGFA doing , 165 a class 3 semaphorin, TGF-β (transforming growth factor beta-1), HGF (hepatocyte growth factor), FGF (fibroblast growth factor) and other ligands, for example, but not limited to, PDGF (platelet-derived growth factor and ), ) to Neuropilin-1 is known as a multifunctional non-tyrosine kinase receptor for malignant melanoma [Neuropilin-1 as therapeutic target for malignant melanoma. Front. Oncol., 03 June 2015|https: / / doi.org / 10.3389 / fonc.2015.00125].

[0017] As used herein, the term "neuropilin antagonist" refers to an agent that inhibits, partially or completely, the biological activity or expression of a neuropilin protein. block Neuropilin refers to a molecule that inhibits or neutralizes antagonist A neuropilin can be any type of molecule that interferes with signal transduction associated with at least one or more neuropilin family members (e.g., NRP-1 or NRP-2) in a cell, for example, by reducing the transcription or translation of a nucleic acid encoding a neuropilin, or by inhibiting or blocking neuropilin polypeptide activity, or both. antagonist Examples of neuropilin-specific aptamers include, but are not limited to, antisense polynucleotides, interfering RNA, catalytic RNA, 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 to neuropilins (e.g., in some cases, Neuropilin binding of one or more neuropilin ligands fused to one or more additional domains fragment (including, but not limited to, a neuropilin antagonist and a neuropilin and By the interaction between 、 Polypeptides that result in the reduction or abolishment of neuropilin activity or expression. including In particular, neuropilin antagonists inhibit the activity of neuropilin proteins (e.g., NRP-1) and one of their partners, particularly VEGF-A. 165 It inhibits the interaction between

[0018] Neuropilin antagonist is well-known in the art and typically includes those described below: - Liu WQ, Megale V, Borriello L, Leforban B, Montes M, Goldwaser E, Gresh N,Piquemal JP, Hadj-Slimane R, Hermine O, Garbay C, Raynaud F, Lepelletier Y,Demange L. Synthesis and structure-activity relationship of non-peptidic antagonists of neuropilin-1 receptor. Bioorg Med Chem Lett. 2014 Sep 1;24(17):4254-9. - Tymecka D, Puszko AK, Lipinski PFJ, Fedorczyk B, Wilenska B, Sura K, Perret GY, Misicka A. Branched pentapeptides as potent inhibitors of the vascular endothelial growth factor 165 binding to Neuropilin-1: Design, synthesis and biological activity. Eur J Med Chem. 2018 Oct 5;158:453-462. - Liu WQ, Lepelletier Y, Montes M, Borriello L, Jarray R, Grepin R, Leforban B, Loukaci A, Benhida R, Hermine O, Dufour S, Pages G, Garbay C, Raynaud F, Hadj-Slimane R, Demange L. NRPa-308, a new neuropilin-1 antagonist, exerts in vitro anti-angiogenic and anti-proliferative effects and in vivo anti-cancer effects in a mouse xenograft model. Cancer Lett. 2018 Feb 1;414:88-98. - Borriello L, Montes M, Lepelletier Y, Leforban B, Liu WQ, Demange L, Delhomme B, Pavoni S, Jarray R, Boucher JL, Dufour S, Hermine O, Garbay C, Hadj-Slimane R, Raynaud F. Structure-based discovery of a small non-peptidic Neuropilins antagonist exerting in vitro and in vivo anti-tumor activity on breast cancer model. Cancer Lett. 2014 Jul 28;349(2):120-7. - Getz JA, Cheneval O, Craik DJ, Daugherty PS. Design of a cyclotide antagonist of neuropilin-1 and -2 that potently inhibits endothelial cell migration. ACS Chem Biol. 2013;8(6):1147-54. - Jia H, Bagherzadeh A, Hartzoulakis B, Jarvis A, Lohr M, Shaikh S, Aqil R, Cheng L, Tickner M, Esposito D, Harris R, Driscoll PC, Selwood DL, Zachary IC. Characterization of a bicyclic peptide neuropilin-1 (NP-1) antagonist (EG3287) reveals importance of vascular endothelial growth factor exon 8 for NP-1 binding and role of NP-1 in KDR signaling. J Biol Chem. 2006 May 12;281(19):13493-502. -A. Starzec, P. Ladam, R. Vassy, S. Badache, N. Bouchemal, A. Navaza, C.H. du Penhoat and G.Y. Perret, Structure-function analysis of the antiangiogenic ATWLPPR peptide inhibiting VEGF(165) binding to neuropilin-1 and molecular dynamics simulations of the ATWLPPR / neuropilin-1 complex, Peptides 28 (2007) 2397-402. - A. Novoa, N. Pellegrini-Moise, D. Bechet, M. Barberi-Heyob and Y. Chapleur, Sugar-based peptidomimetics as potential inhibitors of the vascular endothelium growth factor binding to neuropilin-1, Bioorg. Med. Chem 18. (2010) 3285-98. - C. Nasarre, M. Roth, L. Jacob, L. Roth, E. Koncina, A. Thien, G. Labourdette, P. Poulet, P. Hubert, G. Cremel, G. Roussel, D. Aunis, D. Bagnard, Peptide-based interference of the transmembrane domain of neuropilin-1 inhibits glioma growth in vivo, Oncogene 29 (2010) 2381-92.

[0019] Some embodiment instances, neuropilin antagonists are antibodies that specifically bind to neuropilin (e.g., NRP-1 or NRP-2), neutralize its activity to activate the neuropilin signal transduction pathway, and particularly inhibit the binding of neuropilin to VEGF-A 165 Some embodiment instances, the antibody binds to the extracellular domain of neuropilin. Some embodiment instances, the antibody binds to domain c of NRP-1. Examples of antibodies antagonist that are neuropilin include those described in WO2011 / 143408, which particularly describes the anti-NRP-1 antibody MNRP1685A including .

[0020] As used herein The term "antibody" polyclonal, monoclonal, humanized, chimeric, Fab fragment , Fv fragment , F(ab’) fragment and F(ab’)2 fragment , as well as single single-chain antibodies (scFv), fusion proteins and antigen-binding sites of antibodies including include other synthetic proteins 、 but not limited . Antibodies can be obtained by those skilled in the art using methods and commercially available services the known in the art and kitsusing to It can be prepared. Methods for preparing monoclonal antibodies are 、 well the known in the art and include hybridoma technology and phage display technology to including aim . Further antibodies suitable for use in the present disclosure are described, for example, in the following publications: Antibodies A Laboratory Manual, Second edition. Edward A. Greenfield. Cold Spring Harbor Laboratory Press (Sep. 30, 2013); Making and Using Antibodies: A Practical Handbook, Second Edition. Eds. Gary C. Howard and Matthew R. Kaser. CRC Press (Jul. 29, 2013); Antibody Engineering: Methods and Protocols, Second Edition (Methods in Molecular Biology). Patrick Chames. Humana Press (Aug. 21, 2012); Monoclonal Antibodies: Methods and Protocols (Methods in Molecular Biology). Eds. Vincent Ossipow and Nicolas Fischer. Humana Press (Feb. 12, 2014); and Human Monoclonal Antibodies: Methods and Protocols (Methods in Molecular Biology). Michael Steinitz. Humana Press (Sep. 30, 2013)).

[0021]

[0022] In some embodiment cases, neuropilin antagonist is small A molecule, for example, an organic small molecule, and typically has a molecular weight of less than 5,000 kDa.

[0023] Neuropilin antagonist Examples of small molecules that are :

[0024] - N-[5-(1H-benzimidazol-2-yl)-2-methylphenyl]-N'-(2,3-dihydro-1,4-benzodioxin-6-ylcarbonyl)thiourea (also referred to as NRPa-47)

[0025]

Chemical formula

[0026] - N-[3-(1H-benzimidazol-2-yl)phenyl]-N'-(2,3-dihydro-1,4-benzodioxin-6-ylcarbonyl)thiourea (also referred to as NRPa-48)

[0027]

Chemical formula

[0028] And / or - N-[3-(1H-benzimidazol-2-yl)phenyl]-N'-(1,3-benzodioxol-5-ylcarbonyl)thiourea

[0029]

Chemical formula

[0030] Or a salt thereof and ester, and a mixture thereof including 。

[0031] Another example is described in WO2015004212 and has the following formula : N-(2-Ethoxyphenyl)-4-methyl-3-(N-(p-tolyl)sulfamoyl)benzamide domain

[0032]

Chem.

[0033] Some embodiment neuronectin antagonists are those that inhibit the expression of neuronectin inhibitor are.

[0034] "Expression inhibitor " refers to natural or synthetic compounds having the biological effect of inhibiting gene expression referring to . Some embodiment in which the gene expression inhibitor is siRNA, antisense oligonucleotide or ribozyme. For example, antisense oligonucleotides containing antisense RNA molecules and antisense DNA molecules directly inhibit the translation of NRP-1 mRNA by binding to it effectively blocking and thus 、 inhibit protein translation preventing either or increase mRNA degradation, and thus the level of NRP-1 in the cell and and thus 、 activity Reduce will act to reduce. For example, antisense oligonucleotides complementary to the region of the mRNA transcript sequence encoding NRP-1 of at least about 15 bases and can be synthesized by conventional phosphodiester technology. Antisense technology is used to specifically inhibit the gene expression of genes with known sequences unique region can be synthesized, for example, by conventional phosphodiester technology. Antisense technology is used to specifically inhibit the gene expression of genes with known sequences forThe methods are well known in the art (see, for example, U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). (zhao) Small interfering RNAs (siRNAs) can also function as an expression inhibitor for use in the present invention. Expression of the NRP-1 gene can be no inhibited specifically by contacting a patient or cell with a small double-stranded RNA (dsRNA), or a vector or construct agent that causes the production of small double-stranded RNA Reduce (i.e., RNA interference or RNAi). The antisense oligonucleotides, siRNAs, shRNAs and ribozymes of the present invention can be delivered in vivo alone or with a vector. In its broadest sense, a "vector" along with, yi is any vehicle that can facilitate the transfer of an antisense oligonucleotide, siRNA, shRNA and or ribozyme nucleic acid or into a cell to . Typically, a vector transports nucleic acid into a cell in a form that is and typically to cells expressing NRP-1 degraded to a lesser extent compared to the degree of degradation that would occur in the absence of the vector lu . Generally, vectors useful in the present invention are reduce engineered by the insertion or incorporation of an antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence not and include, but are not limited to, vectors derived from viruses of or other bacterial sources. Viral vectors are plasmids, phagemids, viruses and preferred vectors and include nucleic acid sequences from the following viruses ta wu Moloney murine leukemia virus, Harvey murine sarcoma virus, mouse mammary tumor virus, and Ra derived and others 、 are preferred type vectors and include nucleic acid sequences from the following viruses : Moloney murine leukemia virus, Harvey murine sarcoma virus, mouse mammary tumor virus, and Ra wuRetroviruses such as sarcoma virus; adenovirus, adeno-associated virus; SV40 virus; polyomavirus; Epstein-Barr virus; papilloma virus; herpes virus; vacc xi nia virus; poliovirus; and RNA viruses such as retroviruses including, but not limited to . name Although not listed, other vectors known in the art can be easily employed. In some the cases, the expression embodiment is inhibitor an endonuclease. In certain 、 cases, the endonuclease is CRISPR embodiment cas. In some - cases, the endonuclease is CRISPR-cas9 derived from Streptococcus pyogenes. The CRISPR / Cas9 system is described in US 8697359 B1 embodiment and US 2014 / 0068797. In some and cases, the endonuclease is CRISPR-Cpf1, which was more recently characterized by Zetsche et al.( embodiment ) and is a CRISPR derived from Francisella 1 (Cpf1). "Cpf1 is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13 、 Prevotella and

[0035] "Therapeutically effective amount" means the amount necessary to achieve the desired therapeutic results effect achieve over a administration period and of time. The amount of a therapeutically effective drug can vary depending on factors such as the medical condition of the individual, age, sex, weight, refers to an effective amount in and the desired response in the individual, as well as the potency of the drug. A therapeutically effective amount is one in which the toxicity or harmful and effects of the antibody or antibody portion are induce outweighed by the therapeutically beneficial any effects effect and effect which superseded is also the case. drug The efficient dosage and dosing regimen of treatment will be ru ji ​​​depending on the disease or condition, and can be determined by one of ordinary skill in the art. in the art A physician having ordinary skill can readily determine and prescribe an effective amount of the pharmaceutical composition required. For example, the physician starts with a dose of the drug employed in the pharmaceutical composition at a level lower than the level required to achieve the desired therapeutic effect and gradually dosage increases it until the desired effect is achieved. Generally of the appropriate dosage of the composition of the present invention will be the amount of the compound that is the lowest dosage effective to produce a therapeutic effect according to a particular dosing regimen. Such effective amount generally of depends on the factors described above. For example, for use in treatment treatment an effective amount can be measured by the ability to stabilize the progression of the disease. of a therapeutic compound A therapeutically effective amount is amount can reduce the size tumor or otherwise, subject or improve the symptoms of size the subject. One of ordinary skill in the art will be able to determine such amount based on factors such as the and subject, the severity of the subject's symptoms, or the particular composition selected of a drug and the route of administration. example Exemplary non-limiting ranges of therapeutically effective amounts are from about 0.1 to 100 mg / kg, such as from about 0.1 to 50 mg / kg, such as from about 0.1 to 20 mg / kg, such as from about 0.1 to 10 mg / kg, For example, about 0.5, such as about 0.3, about 1, about 3 mg / kg, about 5 mg / kg or about 8 mg / k g are. Exemplary non-limiting ranges of therapeutically effective amounts of the antibodies of the present invention are from 0.02 to 100 mg / kg, such as from about 0.02 to 30 mg / kg, such as from about 0.05 to 10 mg / kg or 0.1 to 3 mg / kg, such as from about 0.5 to 2 mg / k above effective no are. Administration can be, for example, intravenous, intramuscular, intraperitoneal, or subcutaneous, and can be administered, for example, in proximity to the target site. The above g is treatmentThe dosing regimens in the methods and methods of use are adjusted to provide an optimal desired response (e.g., a therapeutic response). For example, a single times bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally decreased or increased as indicated by the urgency of the treatment situation. In some embodiment cases, treatment of efficacy is monitor during treatment, for example, at predefined time points such treatment is Single or divided doses every 24, 12, 8, 6, 4 or 2 hours, or any combination thereof, are used at least 1 day out of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days, or instead, at least 1 week out of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks after the start of treatment, or a combination thereof, per day, about 0.1 to 100 mg / kg 、 For example, 0.2, 0.5, 0.9, 1.0, 1.1 、1 .5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or in an amount of 100 mg / kg or, as the daily dose of the agent of the present invention, can be provided.

[0036] The selected mode of administration will depend on the acuity and severity of the condition being treatment treated. Any mode of administration that provides the desired therapeutic effect without unacceptable side effects is relevant to the practice of the present invention. Such modes of administration include oral, rectal, topical, transdermal, sublingual, intramuscular, parenteral, intravenous, intracavity, intravaginal, and those used during surgery adhesion matrix including . Intravaginal administration of neuropilin antagonist is also possible, for example, by means of a vaginal pessary or tablet, or a vaginal ring.

[0037] Typically, the medicament of the present invention substance is comprising a pharmaceutically acceptable carrier administered to a subject in the form of a pharmaceutical composition can be . Pharmaceutically acceptable can be carriers that can be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, e.g.,Phosphate, glycine, sorbic acid, potassium sorbate, partial glyceride mixture of saturated vegetable fatty acids, water, salts or electrolytes substances, e.g., Protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts colloidal Silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylate wax , polyethylene-polyoxypropylene-block polymer, polyethylene glycol and lanolin including . For use in administration to a subject, the composition is formulated for administration to a subject for and formulated. The sterile injection form of the composition of the present invention possible may be an aqueous suspension or oily a suspension. These suspensions can be formulated according to techniques known in the art with appropriate dispersing or wetting agents and suspending agents use and, according to the techniques the known in the art. The sterile injection possible formulations may also be sterile injection solutions or suspensions in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol possible . Acceptable vehicles and solvents that may be employed include water, Ringer's solution, and isotonic sodium chloride solution is . Furthermore, sterile fixed oils have conventionally been employed as solvents or suspending media. For this purpose, any bland fixed oil containing synthetic mono glyceride or diglycerides can be employed. Fatty acids such as oleic acid and its glyceride derivatives 、 are useful in the preparation of formulation injectables, and pharmaceutically acceptable can be natural oils such as olive oil or castor oil, especially their polyoxyethylated versions, are likewise useful. These oil solutions or suspensions may contain carboxymethyl cellulose such as long-chain alcohol diluents or dispersants, or emulsifying and suspending agents generally used in the formulation of pharmaceutically acceptable can be dosage forms containing emulsions and suspensions in and can be generally used similar for the dispersion agents also contained therein.As other commonly used surfactants, for example, Tweens, Span, and other emulsifiers or bioavailability enhancers, etc., are generally used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms, and can also be used for the purpose of formulation. The composition of the present invention includes capsules, tablets, aqueous suspensions or solutions, but 、 is not limited thereto. 、 Any orally acceptable dosage form can be administered orally. For oral way tablets, generally used carriers include lactose and corn starch including . Lubricants such as magnesium stearate are also typical added thereto. When administered orally in the form of capsules, useful diluents include, for example, lactose including . If an aqueous suspension is required for oral use administration, the active ingredient is 、 combined with emulsifiers and suspending agents. Optionally, certain sweeteners, flavor or colorants can also be added. Alternatively, the composition of the present invention can be administered in the form of suppositories for rectal administration. These are solid at room temperature but liquid at rectal temperature and thus melt in the rectum to release the drug substance and can be prepared by mixing the drug with a suitable non-irritating excipient. Such materials include cocoa butter, and and polyethylene glycol Beeswax . The composition of the present invention can also be administered topically, particularly when the including target includes areas or organs that are easily accessible by topical application, including diseases of the eye, skin, or lower intestinal tract. Appropriate topical formulations can be easily prepared for each of these sites or treatment organs. For topical organ administration, the composition can be a suitable ointment containing the active ingredient suspended or dissolved in one or more carriers apply . Carriers for topical administration of the compounds of the present invention include mineral oil, liquid contain paraffin, white can be formulated paraffin, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax and water petrolatum , but are not limited to petrolatum these. Instead, the composition can be one or more pharmaceutically acceptable including carriers not only. Instead, the composition can be one or more pharmaceutically acceptable able to carriersto the body The suspended or dissolved active ingredient contain can be incorporated into a suitable lotion or cream formulation . appropriate Examples of suitable carriers include mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and containing water, but , this are not limited to these. Local application to the lower intestine can be effected with rectal suppository formulations (see above) or suitable enema formulations. Patches can also be used. The compositions of the invention can also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulations and may employ benzyl alcohol or other suitable preservatives, not absorption promoters for the purpose, fluorocarbons, and / or other conventional solubilizing or dispersing agents and be formulated as solutions in physiological saline. For example, the antibodies present in the pharmaceutical compositions of the invention can be supplied at a concentration of 10 mg / mL in single-use vials of either 100 mg (10 mL) or 500 mg (50 mL). The product is formulated for intravenous administration with 9.0 mg / mL sodium chloride, 7.35 mg / mL sodium citrate dihydrate, 0.7 mg / mL polysorbate 80, and sterile water for injection. The pH is adjusted to 6.5. Exemplary improve bioavailability dosage ranges for the antibodies in the pharmaceutical compositions of the invention can be between about 1 mg / m appropriate ~500 mg / m 2 However, these schedules are exemplary and it will be understood that the optimal schedules and regimens can be adapted taking into account the affinity and tolerability of the particular antibody in the pharmaceutical composition which must be determined in clinical trials. 2 In some

[0038] instances, the neuropyrins of the invention embodiment are administered to the patient in combination with a progestogen. antagonist

[0039] ​As used herein, the term "progest - ogen" or "gestagen" has its general meaning in the art and is an anti-estro n( gen (which cancels the action of estrogen in the body) and an anti-gonadotro n (sex steroid and property gland produce of inhibit ) synthetic hormone compounds that exhibit cover properties.

[0040] In some embodiment cases, progest - ogens are c lorazidinone acetate, s proterone acetate , desogestrel, dienogest, 5α-dihydroprogesterone, drospirenone (Yasmit (registered trademark)) e tangdiol acetate , ethinodiol diacet t ate, ethyno ge stre (nexaplon (registered trademark)), gestoden, 17-hydroxyprogesterone, levonorgestrel (Alesse (registered trademark)) m dydroxyprogesterone acetate (17α-hydroxy-6α-methylprogesterone ace t tate, Provera (registered trademark)) m gestorol m gestorol acetate (17α-acetoxy-6-dehydro-6-methylprogesterone), nestron n megestrol acetate , nor e chindrone n l e chindrone acetate (also known as nor e chindrone acetate), nor e chinodrel ( Enovid (registered trademark)), norgestimat , norgestrel, progesterone, tanapro ge tt, trimegestone, any of the foregoing pharmaceutical permissible able to salts and any combination thereofselected from .

[0041] As used herein, term "combination 」is of a first drug and and further (second, third, etc.) drugs together with is intended to refer to all dosage forms provided. The drugs can be administered simultaneously, separately, or sequentially, and in any order. The drugs administered in combination have biological activity in the patient to whom the drugs are delivered. In the context of the present invention, a combination thus comprises at least two different drugs, where one drug is at least one neuropilin antagonist and the other drug is at least one progesterone - gene.

[0042] The present invention is further illustrated by the following figures and examples. However, these examples and figures should not be construed in any way as limiting the scope of the present invention.

Brief Description of the Drawings

[0043]

Figure 1A

Figure 1B

Figure 2A

Figure 2B

Figure 3A

Figure 3B

Figure 4A

Figure 4B

[0044] Example Method Cell culture The human primary non - modified endometrial cell line (TH - EM1) described by Gogusev j et al. in 2019 was cultured in Dulbecco's Modified Eagle Medium (Invitrogen - Thermo Fisher, Cergy - Pontoise, France) supplemented with 10% fetal calf serum (FCS), L- glutamine, and and antibiotics. with Cultures performed To study cell proliferation or apoptosis, the cells were plated in 96 - well plates or 12 - well plates, respectively.

[0045] Flow cytometry Untreated proliferating TH - EM1 cells (2.10 5 cells) were recovery harvested and washed with PBS 1x containing 2% FCS. Next, PBS 1x 2% FCS (Miltenyi biotec, France) was contain ca 2+ / mg 2+ added. usingCells were directly stained with either control phycoerythrin (PE)-neuropilin-1 or an appropriate irrelevant monoclonal antibody. The stained cells were then analyzed by a fluorescence-activated cell sorter (FACS) Calibur flow cytometer (Becton Dickinson Co, Mountain View, CA) analysis and the data analysis were analyzed using Flowjo software.

[0046] Cell proliferation assay 10 cells 4 were plated at 200 μL / well per well in a 96-well plate, Neuropilin antagonists (NRPa) such as NRPa-47 and NRPa-48 were some added at concentrations (10 -5 to 5.10 -8 M) alone or 3.10 -5 M of in combination with different concentrations of norgestrel acetate) Lutenyl (registered trademark) progestative drug and in combination with, for 48 hours, 72 hours or 120 hours with and treated or not treated incubated. After adding WST-1 (Roche (registered trademark), France) for 2 hours, a microplate reader (Microplate Manager 5.2, Bio-Rad) was used used to 、 measure the optical density at 490 nm analysis and calculate the cell viability. determination The experiment was then completed.

[0047] Apoptosis assay Cells were 5.10 5 12 wells at 2000 μL / well per cell / well seeded onto in plates 10 -6 M of and neuropilin antagonist treated with with NRPa-47, NRPa-48, etc. treated for 48 to 72 hours andThen, the cells were collected and stained according to the protocol of the Annexin-V / 7AAD kit using recommended by the manufacturer according to . After that, the stained cells were analyzed using a fluorescence-activated cell sorter (FACS) Calibur flow cytometer (Becton Dickinson Co, Mountain View, CA), and data analysis was performed using Flowjo software.

[0048] Statistical analysis analysis The data were 、 expressed as the arithmetic mean + / − SD of at least three different experiments. The statistical significance of the results was evaluated by ANOVA, and p-values ** p < 0.01, *** p < 0.001 were considered significant.

[0049] Results Neuropilin expression in endometrial cells and NRPa sensitivity To investigate the effect of neuropilin antagonist (NRPa) on endometriosis test first, primary human non- modification primary endometrial cells (Gogusev et al. 2019) its expression in levels loop were examined. As shown in Figure 1A, neuropilin (NRP-1) was significantly expressed on the cell surface of these cells 、 . Even though NRP-1 was expressed obtained , it was still unclear whether these cells were sensitive to NRPa assuming . Therefore, first, the effect of high-concentration NRPa (NRPa-47, 10 then μM) on cell viability was examined or . As expected, after 48-72 hours of exposure, the amount of endometrial cells decreased significantly (80% inhibition) (Figure 1B). any To inhibit the proliferation of endometrial cells, low concentrations -5 of RP effect were observed. test

[0050] N of of RP N of RPa is is necessary. To determine the sensitivity of endometrial cells to NRPa determined for, several concentrations (10 -5 , 10 -6 , 10 -7 M) were examined over time ( Day 2 ~ Day 5 including the examination of) with evaluated. As expected, at high concentrations (10 -5 M) of NRPa (NRPa-47 and NRPa-48), rapid endometrial proliferation inhibition was obtained from day 2 of exposure (D2) (Figure 2A and Figure 2B). However while , 10 -6 ~10 -7 M of contained in the period low concentrations of NRPa (NRPa-47 and NRPa-48) reached maximum efficiency with 3 days of exposure (D3) and the effect stabilized at D5 remained (Figure 2A and Figure 2B). From these results, the approximate IC 50 of 、 NRPa-48 was -7 10 approximate IC 50 of 、 NRPa-47 was -6 ~10 -7 M contained in the period as such determined (Figure 2A and Figure 2B).

[0051] The proliferation of endometrial cells inhibition is mediated by apoptosis induced by NRPa. Apoptosis of endometrial cells at 48 hours and 72 hours after NRPa exposure was monitored by Annexin-V / 7-AAD staining using NRPa including NRPa-47 and NRPa-48 induced reaching the maximum endometrial cell death may induce after 72 hours of exposure (Figure 3A). This induced cell death was first induced by apoptosis (Annexin-V+ / 7-AAD+ and Annexin-V+ / 7-AAD- staining) andThe induction of necrosis (Annexin-V- / 7-AAD+) was divided into two stages (Figure 3A and Figure 3B). at low concentration The proportion of each cell death phase induced in endometrial cells h This is clearly shown in the histogram.

[0052] NRPa is a traditional progesterone-gene may act synergistically with endometriosis suffering from disease patients Continuously progesterone-gene is being administered due to , N.R.P.A. and progesterone-gene Surprisingly, the combined effect of low concentrations of NRPa-47 (6.10 -7 M) and progesterone-gene drug ( norgestrel acetate ) in combination with inhibition Additive effect of with recognition for (Figure 4A). More importantly, the IC of NRPa-48 was two-fold lower. 50 Concentration (5.10 -8 M) is used, progesterone-gene drug Synergistic effect in the presence of with observation treated These results raise interest in the use of NRPa in this condition and are indicative of the efficacy of NRPa in patients with pulmonary arterial disease (Figure 4B). progesterone-gene drug With in combination This reinforces the importance of

[0053] Consideration This report is about endometriosis treatment and this disease involved Biological pathways for the findings brought new tools to 、 The vital importance of the development of NRPa emphasis In this report, relevant Two types of NRPa (NRPa-47 and NRPa-48) alone with Or traditional hormones in combination with treatment , rapidly inhibiting the proliferation of endometriosis cell lines inhibition Things to do with confirmation treatedTherefore, NRPa may induce apoptosis in endometriosis cell lines. In the future all it is necessary to in the study study the downstream pathways through the kinase control, death receptor, pro-apoptosis and anti regulation of apoptosis proteins of NRPa. identification That is, NRPa may be used alone or in combination with conventional endometriosis hormones

[0054] This observation has brought new interest to the development of NRPa in this disease state. treatment and in combination with This observation has brought new interest to the development of NRPa in this disease state.

[0055] References Throughout this application, various references describe the state of the art to which the present invention pertains. The disclosures of these documents are hereby incorporated by reference into this disclosure.

[0056] 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.

Claims

**Claim 1** A pharmaceutical composition for treating endometriosis in a subject in need thereof, comprising a therapeutically effective amount of a neuropilin antagonist, wherein the neuropilin antagonist is NRPa-47 or NRPa-48, and the subject has peritoneal endometriosis, ovarian endometriosis, deep endometriosis or extra-pelvic endometriosis. **Claim 2** The pharmaceutical composition according to claim 1, wherein the subject is a human. **Claim 3** The pharmaceutical composition according to claim 1, wherein the subject is a non-human mammal. **Claim 4** The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is administered to the subject in combination with a progestogen. **Claim 5** The progestogen is selected from the group consisting of chlormadinone acetate, cyproterone acetate, desogestrel, dienogest, 5α-dihydroprogesterone, drospirenone, ethinodiol diacetate, ethinylestradiol diacetate, etonogestrel, gestoden, 17-hydroxyprogesterone, levonorgestrel, medroxyprogesterone acetate (17α-hydroxy-6α-methylprogesterone acetate), megestrol, megestrol acetate (17α-acetoxy-6-dehydro-6-methylprogesterone), nestorone, nomegestrol acetate, norethisterone, norethisterone acetate, norethindrone, norgestimate, norgestrel, progesterone, tanaproget, trimegestone, any pharmaceutically acceptable salt thereof, and any combination thereof. The pharmaceutical composition according to claim 4. **Claim 6** A pharmaceutical composition for treating endometriosis in a subject in need thereof, comprising a progestogen, administered to the subject in combination with a neuropilin antagonist, wherein the neuropilin antagonist is NRPa-47 or NRPa-48, and the subject has peritoneal endometriosis, ovarian endometriosis, deep endometriosis or extra-pelvic endometriosis. **Claim 7**: The pharmaceutical composition according to claim 6, wherein the progestogen is selected from the group consisting of chlormadinone acetate, cyproterone acetate, desogestrel, dienogest, 5α-dihydroprogesterone, drospirenone, ethandiol acetate, ethinodiol diacetate, etonogestrel, gestoden, 17-hydroxyprogesterone, levonorgestrel, medroxyprogesterone acetate (17α-hydroxy-6α-methylprogesterone acetate), megestrol, megestrol acetate (17α-acetoxy-6-dehydro-6-methylprogesterone), nestoron, nomegestrol acetate, norethisterone, norethisterone acetate, norethindrel, norgestimate, norgestrel, progesterone, tanaproget, trimegestone, any pharmaceutically acceptable salt thereof, and any combination thereof.

Images