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WO2018108862A1 - Antagoniste du récepteur 2 de la prokinéticine destiné à être utilisé en tant que médicament pour le traitement d'un cancer associé à un vegf - Google Patents

Antagoniste du récepteur 2 de la prokinéticine destiné à être utilisé en tant que médicament pour le traitement d'un cancer associé à un vegf Download PDF

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WO2018108862A1
WO2018108862A1 PCT/EP2017/082321 EP2017082321W WO2018108862A1 WO 2018108862 A1 WO2018108862 A1 WO 2018108862A1 EP 2017082321 W EP2017082321 W EP 2017082321W WO 2018108862 A1 WO2018108862 A1 WO 2018108862A1
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antagonist
vegf
jeg3
prokr2
cells
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PCT/EP2017/082321
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Nadia ALFAIDY-BENHAROUGA
Wael TRABOULSI
Frédéric SERGENT
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Commissariat à l'énergie atomique et aux énergies alternatives
Université Grenoble Alpes
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Publication of WO2018108862A1 publication Critical patent/WO2018108862A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • Gestational choriocarcinoma is a malignant trophoblastic tumor that develops upon normal or abnormal pregnancy.
  • the latter includes complete (CHM) or partial hydatidiform moles (PHM), spontaneous abortions, or ectopic pregnancies.
  • CHM develops when one or two spermatozoa fertilized an oocyte in the absence of the maternal nucleus, while PHM results from dispermic fertilization of a normal oocyte.
  • CHM and PHM patients are at high risk of developing choriocarcinoma; nevertheless, this risk is much higher in CHM (20%) as compared to PHM (1 .5%).
  • Choriocarcinoma is a rare disease with an estimated incidence of 2 to 7 in 100,000 pregnancies in North America and Europe. This number reaches 5 to 202 in 100,000 pregnancies in Asia. Choriocarcinoma is highly metastatic due to the intrinsic invasive property of trophoblast cells. Most patients with non-metastatic gestational choriocarcinoma are successfully treated with single-agent chemotherapy. Metastatic cases are only curable using multiagent chemotherapy, known to induce considerable adverse effects. Choriocarcinoma diagnosis and progression is based on the measurement of the human chorionic gonadotropin (hCG), released by the syncytiotrophoblast.
  • hCG human chorionic gonadotropin
  • hCG measurements have recently been reported to be associated to false positive diagnoses and unnecessary invasive therapeutic procedures including chemotherapy, hysterectomy, and other surgeries. Thus, it is still necessary to prevent the occurrence of this cancer and develop novel and more specific diagnostic markers as well as less toxic therapeutic approaches.
  • EG-VEGF Endocrine Gland-Derived Vascular Endothelial Growth Factor
  • prokineticin-1 represents the canonical member of the prokineticin family.
  • EG-VEGF expression is grossly restricted to endocrine tissues, including the placenta (Brouillet, Hoffmann, Feige, et ai, 2012).
  • EG-VEGF i is abundantly produced by the endocrine unit of the placenta, the syncytiotrophoblast during the first trimester of human pregnancy with a peak of expression just before the establishment of the feto-maternal circulation, ii) its expression is up-regulated by hypoxia, a key parameter of tumor development and by hCG and that iii) its receptors, PROKR1 and PROKR2 are highly expressed in trophoblast and placental endothelial cells (Brouillet, Hoffmann, Feige, etal., 2012; Hoffmann et al., 2009). EG-VEGF circulating levels are around 50 pg/ml in non-pregnant women and increase 5-fold during the first trimester of pregnancy
  • trophoblast cells are excessively proliferative, a phenomenon that ultimately results in an increased pool of cells acquiring migratory and invasive phenotype.
  • Brouillet et al. demonstrated that EG-VEGF increases trophoblast proliferation and ensures their survival under deprived conditions (Brouillet et al., 2013).
  • EG-VEGF has recently been reported to be associated with tumor development of multiple reproductive organs such as ovary (Zhang et al., 2003), testis (Samson et al., 2004) and prostate (Monnier & Samson, 2010).
  • EG-VEGF could be a potential actor in choriocarcinoma development and progression.
  • No previous study has investigated the role of EG-VEGF in the development and/or progression of this pathology.
  • the inventors conducted a clinical study to determine EG-VEGF association to CHM, choriocarcinoma development and progression (Example 1 ) and an in vitro study, using a human choriocarcinoma cell line (JEG3 cells) to characterize EG-VEGF effects on their proliferation, migration and invasion (Example 2).
  • JEG3 cells human choriocarcinoma cell line
  • the inventors used a new animal model of choriocarcinoma obtained by orthotropic injection of JEG3 cells in immunodeficient gravid mice, to characterize choriocarcinoma progression and test the effects of EG-VEGF receptors antagonists on tumor development and progression (Example 3).
  • mice treated with a PROKR1 antagonist (PC1 ) and with a PROKR2 antagonist (PKRA-505) showed a significant reduction (compared to control mice) in the size of the tumor development, as well as a reduction in the tumor metastasis.
  • PKRA-505 had a stronger inhibitory effect on tumor progression, compared to PC1 .
  • the present invention hence pertains to the use of an antagonist of prokineticin receptor 2 (PROKR2), as a medicament for treating an EG-VEGF-related cancer.
  • PROKR2 prokineticin receptor 2
  • EG-VEGF-related cancer any cancer in which this factor may be involved.
  • these include choriocarcinoma, ovary cancer and testis cancer, but increasing evidence suggests that EG-VEGF may play a role in the development of other cancers.
  • EG-VEGF has been proposed as a potential prognostic factor in colorectal, gastrointestinal, and neuroblastoma cancer (Goi et al., 2004; E. S. Ngan et al., 2007).
  • EG-VEGF has been reported as an indicator of ovarian cancer progression and as a good marker of its unfavorable prognosis (Zhang et al., 2003).
  • the present invention pertains to the use of EG-VEGF receptors antagonists, especially PKOKR2 antagonists, as therapeutics to treat other cancers such as colorectal cancer, prostate cancer, gastrointestinal cancer, neuroblastoma cancer, breast cancer and lung cancers.
  • treat refers to any reduction or amelioration of the progression, severity, and/or duration of cancer, particularly a solid tumor; for example in a breast cancer, reduction of one or more symptoms thereof that results from the administration of one or more therapies.
  • the PROKR2 antagonist is used for treating a solid tumor (leading to its reduction or at least impairing its development) and/or preventing the development of metastasis.
  • antagonist a natural or synthetic compound that has a biological effect opposite to that of an agonist.
  • An antagonist binds the receptor and blocks the action of a receptor agonist by competing with the agonist for receptor.
  • An antagonist is defined by its ability to block the actions of an agonist.
  • PROKR2 antagonist has its general meaning in the art and refers to any compound, natural or synthetic, that blocks, suppresses, or reduces (including significantly) the biological activity of PROKR2 or to any compound that inhibit PROKR2 gene expression.
  • PROKR2 antagonist to a compound which causes mobilization of calcium or/and stimulation of phosphoinositide turnover or/and activation of p44/p42 mitogen-activated protein kinase and/or cell migration and/or proteases secretion (see for instance tests described in the experimental part of the present application, pages 20-22, example 2).
  • PROKR2 antagonist includes but is not limited to: small organic molecule, antibody or antibody fragment, a polypeptide or an inhibitor of PROKR2 expression.
  • PROKR2 antagonist Any technique suitable for determining the functionality of PROKR2 antagonist may be used.
  • the man skilled in the art may use techniques describes in Brouillet S et al (Molecular characterization of EG-VEGF-mediated angiogenesis: differential effects on microvascular and macrovascular endothelial cells. Mol Biol Cell. 2010 Aug 15;21 (16):2832-43) or Hoffmann P et al (Role of EG-VEGF in human placentation: Physiological and pathological implications. J Cell Mol Med. 2009 Aug;13(8B):2224-35).
  • a PROKR2 antagonist can be identified by carrying out the following steps: i) providing a plurality of test substances ii) determining whether the test substances are PROKR2 antagonists and iii) positively selecting the test substances that are PROKR2 antagonists.
  • Such cells include cells from mammals, yeast, Drosophila or E. coli.
  • a polynucleotide encoding PROKR2 is used to transfect cells to express the receptor.
  • the expressed receptor is then contacted with a test substance and a PROKR2 ligand, as appropriate, to observe activation of a functional response.
  • comparison steps may involve to compare the activity induced by the test substance and the activity induced by a well- known PROKR2 antagonist.
  • substances capable of having an activity similar or even better than a well-known PROKR2 antagonist are positively selected.
  • test substances capable of binding to PROKR2 present at the cell surface.
  • test substance is labelled (e.g. with a radioactive label) and the binding is compared to a well known PROKR2 antagonist.
  • the candidate compound is selected from the group consisting of small organic molecules, peptides, polypeptides or oligonucleotides.
  • test substances that have been positively selected may be subjected to further selection steps in view of further assaying its properties for the treatment of EG-VEGF-related cancer.
  • candidate compounds that have been positively selected may be subjected to further selection steps in view of further assaying its properties on animal models.
  • the above assays may be performed using high throughput screening techniques for identifying test substances for developing drugs that may be useful to the treatment of EG-VEGF-related cancer.
  • High throughput screening techniques may be carried out using multi-well plates (e.g., 96-, 389-, or 1536- well plates), in order to carry out multiple assays using an automated robotic system.
  • multi-well plates e.g., 96-, 389-, or 1536- well plates
  • large libraries of test substances may be assayed in a highly efficient manner.
  • stably-transfected cells growing in wells of micro-titer plates 96 well or 384 well
  • Compounds in the library will be applied one at a time in an automated fashion to the wells of the microtitre dishes containing the transgenic cells described above.
  • test substances which induce the activity of PROKR2 are identified, they can be positively selected for further characterization.
  • These assays offer several advantages.
  • the exposure of the test substance to a whole cell allows for the evaluation of its activity in the natural context in which the test substance may act. Because this assay can readily be performed in a microtitre plate format, the assays described can be performed by an automated robotic system, allowing for testing of large numbers of test samples within a reasonably short time frame.
  • the assays of the invention can be used as a screen to assess the activity of a previously untested compound or extract, in which case a single concentration is tested and compared to controls.
  • These assays can also be used to assess the relative potency of a compound by testing a range of concentrations, in a range of 100 ⁇ to 1 ⁇ , for example, and computing the more efficient concentration.
  • any antagonist of prokineticin receptor 2 can be used according to the present invention, including antibodies and fragments thereof, antisense nucleic acids and non-protein small molecules.
  • the antagonist is a small molecule.
  • the inventors obtained a strong anti-cancer effect with ( ⁇ )-4-Benzyl- N-(3,4-dihydro-2H-1 ,5-benzodioxyepin-7-ylmethyl)-N-isobutylmorpholine-2- carboxabide (Formula I), also designated "PKRA” or "PKRA-505" in the present text:
  • the compound used as a medicament to treat or prevent an EG-VEGF-related cancer is thus PKRA-505 or a derivative thereof still being an antagonist of PROKR2.
  • PROKR2 inhibitors are disclosed in US 8,722,896.
  • Non-limiting examples of molecules which can be advantageously used in the frame of the present invention are illustrated below:
  • the antagonist used to treat an EG-VEGF-related cancer is specific for PROKR2.
  • its IC50 for PROKR2 is at least 3, 5, 10 or 20-fold inferior to its IC50 for PROKR1 .
  • the present invention is particularly useful for treating human patients suffering from an EG-VEGF-related cancer.
  • the PROKR2 antagonist can be administered alone or in combination with another antineoplastic agent.
  • antineoplastic agents that can be used in combination with a PROKR2 antagonist include chemotherapy (such as single-agent chemotherapy, for example with methotrexate, but also multiagent chemotherapy), hormonal and biological therapies, and radiotherapy.
  • the PROKR2 antagonist can also be administered before or after a surgery.
  • PKRA-505 has been used in mice at concentrations of 100mg/kg, four times during gestation (every three days). A simple extrapolation would suppose treating patients at a corresponding frequency until their recovery, which comes to inject them 6g/injection, assuming their average weight is about 60 kg.
  • PKRA-505 has been reported in the literature to alleviate pain and to exhibit strong analgesic effects (Hu et al., 2006).
  • PKRA is hence used to treat an EG- VEGF-related cancer at a posology between 3 to 10 g per administration. Administrations can be done by intravenous injections or by oral administration, every 1 to 10 days, for example every 3 days until recovery.
  • the present invention also pertains to a therapeutic composition
  • a therapeutic composition comprising a PROKR2 antagonist as above-described and an antineoplastic agent such as a chemotherapeutic agent.
  • antineoplastic agent such as a chemotherapeutic agent.
  • the skilled artisan can chose, in the anti-neoplastic armamentarium, the agent(s) that will best interact with the PROKR2 antagonist to treat an EG-VEGF-related cancer.
  • a kit of parts comprising a PROKR2 antagonist as above-described and an antineoplastic agent is also part of the present invention.
  • Figure 1 EG-VEGF expression in control, CHM and choriocarcinoma patients during the first trimester of pregnancy.
  • Panel B reports representative photographs of EG-VEGF immunoreactivity (Ir) in placental villous tissues from women CTL (a), CHM (b) and Choricarcinoma patient (c).
  • Cytotrophoblast (Ct), Hobfauer cells (Ho), Extravillous trophoblast (EVT),
  • FIG. 2 EG-VEGF effect on JEG3 proliferation in the absence or presence of PROKR1 or PROKR2 antagonist.
  • Panel A reports a graph that summarizes EG-VEGF effect on proliferation/viability of JEG3. Cell proliferation was determined using WST-1 assay after incubation with increasing concentrations of EG-VEGF (0, 5, 10, 25 ng/ml) in the absence or presence of PROKR1 and PROKR2 antagonists (1 ⁇ ). * p ⁇ 0.01 and ** p ⁇ 0.01 ).
  • Panel B reports Western blot analyses of protein extracted from JEG3 cells treated with EG-VEGF (10 ng/ml). Membranes were incubated with antibodies to detect phosphorylation of Akt, p44/42 MAPK and Src. The total (T)- AKT, -p42/44 MAPK and -Src were used as a loading control.
  • Figure 3 EG-VEGF effect on JEG3 migration in the absence or presence of PROKR1 or PROKR2 antagonists.
  • Panel A shows representative images of wounded JEG3 monolayers at 0 and 24 h post-wounding in presence of increasing concentrations of EG-VEGF (0, 5, 10, 25 ng/ml).
  • Panel B shows representative images of wounded JEG3 monolayers at 0 and 24 h post- wounding in the presence of increasing concentrations of EG-VEGF (0, 5, 10, 25 ng/ml) and the PROKR1 antagonist (1 ⁇ ).
  • Panel C shows representative images of wounded JEG3 monolayers at 0 and 24h post-wounding in the presence of increasing concentrations of EG-VEGF (0, 5, 10, 25 ng/ml) and the PROKR2 antagonist (1 ⁇ ).
  • Panel D reports a graph that reports percentages of wound closures after 24 h of treatment. * p ⁇ 0.05 and ** p ⁇ 0.001 .
  • FIG. 4 EG-VEGF effect on JEG3 invasion in the absence or presence of PROKR1 or PROKR2 antagonists.
  • Panel A shows representative images of pre-labeled JEG3 cells that invaded the filter under control or treated conditions. Cells were treated with EG-VEGF (0, 5, 10 ng/ml), in the absence or presence of PROKR1 antagonist (1 ⁇ ) or PROKR2 antagonist (1 ⁇ ).
  • Panel B Quantification of JEG3 invasion is reported.
  • FIG. 5 EG-VEGF effect on JEG3 invasion in 3D culture system.
  • Panel A shows representative images of JEG3 cells spheroid at to (a, b, c, d, e, f) and 24 h (g, h, i, j, k, I) after their treatment with EG-VEGF in absence or presence of PROKR1 and PROKR2 antagonists.
  • Panel C reports representative zymograms of matrix- metalloproteinases, MMP2 (72 kDa) and MMP9 (92 kDa) activity in conditioned medium collected from JEG3 cells treated or not with EG-VEGF.
  • FIG. 6 Bioluminescence images of SCID mice injected with JEG3-luc cells in the absence or presence of PROKR1 and PROKR2 antagonists.
  • Panels A and B show representative images that illustrate the bioluminescence of gravid mice injected by JEG3-luc in their placenta (a) or in their uterine horn (b).
  • Photographs in (c) and (d) report images of the same mice after their surgery.
  • Panel C reports representative images of gravid mice injected in their placenta by either JEG3-luc cells (a), or JEG3-luc plus vehicle for the antagonist's treatments (b); or JEG3-luc plus PROKR1 antagonist (500 g/kg) (c); or by JEG3-luc cells plus PROKR2 antagonist (100mg/Kg) (d). All mice were injected with JEG3-luc within the placenta at 7.5 dpc and imaged at 19.5 dpc.
  • Panel D reports quantification of the values of photon flux (p/sec/cm2/sr) for each group of mice.
  • Figure 7 Effects of PROKR1 and PROKR2 antagonists on placental development and on circulating angiogenic factors in JEG3- injected mice.
  • Panel A reports histology of placentas collected from, Matrigel, JEG3-luc, Jeg3-luc+ PROKR1 antagonist and from JEG3-luc+PROKR2 antagonist-injected placentas.
  • Photographs (a) (b) and (c) on histology of placentas collected from mice that were injected by Matrigel alone.
  • Photographs (d) (e) and (f) report photographs of placentas that were injected with JEG3 cells.
  • Photographs in (g) (h) and (i) report histology of placentas collected from mice that were injected by JEG3 cells and treated by PROKR1 antagonist.
  • Photographs in (j) (k) and (I) report histology of placentas collected from mice that were injected by JEG3 cells and treated by PROKR1 antagonist.
  • Photographs on (b, c); (e,f); (h,i) and (k,e) are higher magnifications of photographs in a, d, g, and j, respectively.
  • Panel B reports Ki67 staining of placentas collected from mice that were injected with, Matrigel alone, JEG3-luc, JEG3-luc+ PROKR1 antagonist and from JEG3-luc+PROKR2 antagonist- injected placentas.
  • Photographs (c) (d) report photographs of placentas that were injected with JEG3 cells.
  • Photographs in (e) (f) report Ki67 staining of placentas collected from mice that were injected by JEG3 cells and treated by PROKR1 antagonist.
  • Photographs in (g) (h) report histology report Ki67 staining of placentas collected from mice that were injected by JEG3 cells and treated by PROKR1 antagonist. Photographs on (b); (d); (f) and (h) are higher magnifications of photographs in a, c, e, and g, respectively.
  • Panel C reports representative antibody microarray blots of key angiogenic factors assessed in sera collected from SCID mice injected with JEG3-luc cells and treated or not with PROKR1 or PROKR2 antagonists.
  • the graph on Panel D below reports the quantification of the intensity of the bands of three angiogenic factors that showed significant difference between the groups. Data are presented as mean ⁇ SEM. * p ⁇ 0.05.
  • FIG. 8 EG-VEGF effect on PROKR1 and PROKR2 expression.
  • Panel A and B report Western blot analysis of protein extracted from JEG3 cells treated with increasing concentrations of EG-VEGF. Membranes were incubated with PROKR1 (A) and PROKR2 (B) antibodies, respectively. Standardization of protein signals was performed using antibodies against ⁇ -actin. Quantification of the intensity of the bands is illustrated in panels C and D, respectively below. Values overwritten with different letters are significantly different from each other. * P ⁇ 0.05.
  • FIG. 9 EG-VEGF effect on JEG3 proliferation in the absence or presence of siRNA for PROKR1 or PROKR2. JEG3 proliferation was determined using WST-1 assay after their incubation with increasing concentrations of EG-VEGF (0, 5, 10, 25 ng/ml) in the absence or presence of siRNA for PROKR1 and PROKR2 * p ⁇ 0.05 and ** p ⁇ 0.01 ).
  • FIG 10 Experimental procedures. The flow chart shows the experimental procedures performed at certain time-points during the study. The gravid mice were randomly assigned to be injected either with Matrigel alone, JEG3 cells in Matrigel, JEG3-luc, JEG3-luc+ PROKR1 antagonist or JEG3- luc+PROKR2 antagonist. Mice were imaged using MS imaging system and sacrificed at 19.5dpc. Placenta and maternal blood were collected.
  • Figure 11 Comparison of analogues of PROKR2 antagonists on JEG3 cells proliferation. Note that PROKR2 antagonist was more potent for the inhibition of JEG3 cells proliferation compared to all other analogues. M-7 antagonist was toxic to JEG3 cells.
  • PROKR2 antagonist was more potent for the inhibition of JEG3 cells migration compared to all other analogues.
  • M-6 and M-7 antagonists were toxic to JEG3 cells.
  • JEG3 (ATCC® HTB-36TM) is one of six clonally derived lines isolated from the Woods strain of the Erwin-Turner tumor by Kohler and associates (Kohler et al., 1971 ). JEG3 cells were used as a choriocarcinoma cell line model. Cells were used between 4 and 10 passages and grown in Dulbecco's modified Eagle's medium DMEM-F12 supplemented with 10% fetal bovine serum (FBS), penicillin-streptomycin, and amphotericin B (Invitrogen, Cergy Pontoise, France). Cells were maintained at 37°C under normoxic (20% O2) or hypoxic (1 .5% O2) conditions.
  • JEG3 cells were serum starved for 12h and incubated in absence or presence of human recombinant EG-VEGF (Peprotech, France) for 24h. Protein extracts were prepared as previously described (Alfaidy, Li, Macintosh, Yang, & Challis, 2003).
  • JEG3-LUC (Luciferase positive JEG3 cells) were prepared using a lentivirus supernatant (pLenti-ll-CMV-Luc-IRES-GFP control vector). The protocol was performed according to the company's instructions (Applied Biological Materials Inc.). Briefly, JEG3 cells were plated in DMEM/F-12 (1/1 ) medium supplemented with 10% fetal bovine serum. Cells were infected for 6h with retroviruses at a ratio of 1 :1 in fresh culture medium. Infected cells were selected with G418 (200 pg/ml) for 7 days.
  • EG-VEGF was measured by ELISA (PeproTech, France) in the collected sera and conditioned media. Two separated standard curves were constructed to allow accurate readings of samples at upper and lower ranges of the assay. All samples were in the linear range of the standard curves. The detection limit of the assay was 16 pg/ml. The intra-assay coefficient of variability (CV) was 6.7%, and the inter assay CV was 8.1 %.
  • PROKR1 antagonist was obtained from Dr F. Balboni (University of Cagliari, Italy) and PROKR2 antagonist was obtained from Dr QY Zhou (University of California Irvine, CA, USA). Both antagonists were used at 1 ⁇ .
  • Wound-healing assay was performed using JEG3 cells. Cells were seeded in complete medium (DMEM-F12 10% FBS) at a density of 2 x 10 5 cells/well into 24-well plates. At confluence, complete medium was replaced by serum-free medium with increased concentrations of recombinant EG-VEGF (Peprotech, France) for 24h, in the absence or presence of PROKR1 or PROKR2 antagonists (1 ⁇ ). Wound healing assay was performed in the presence of mitomycin C (Sigma-Aldrich) to inhibit cell proliferation. Cells were scratched with a sterile tip to create an artificial wound and were allowed to heal for 24h. Photographs were taken at regular time intervals (0 to 24h). The size of the wound closure was measured using Scion Image software (version 4.0.2). The results are presented as percentage of wound closure after 24h of treatment.
  • JEG3 cells were stained with the Vybrant Dil Cell-Labeling Solution (Invitrogen) for 1 h at 37°C.
  • the top chamber of 8 ⁇ pore size FluoroBlok cell culture inserts (BD Biosciences) was precoated with 100 ⁇ of 1 :25 Matrigel (BD Biosciences).
  • 2 x 10 4 Dil-stained JEG3 cells/insert were seeded in 500 ⁇ of DMEM-F12 medium in the presence of 1 % FBS.
  • the inserts were placed into 24- well plates containing 750 ⁇ of DMEM-F12 medium with 15% FBS.
  • Cells were treated, for 24 with increased concentrations of recombinant EG-VEGF (Peprotech, France) in the absence or presence of PROKR1 or PROKR2 antagonists (1 ⁇ ).
  • the chambers were then removed and fixed with paraformaldehyde.
  • the membranes of the chambers were excised and placed on glass slides. The cells that invaded the chamber were visualized under the microscope and counted.
  • JEG3 cells Confluent monolayers of JEG3 cells were trypsinized. 1 .000 cells were suspended in culture medium DMEM-F12 supplemented with 10% fetal bovine serum (FBS), and seeded in; non-adherent round-bottom 96-well plates (Greiner, Frickenhausen, Germany) coated previously with Poly-HEMA (Poly (2-hydroxyethyl methacrylate); sigma Aldrich). Under these conditions, all suspended cells contribute to the formation of a single JEG3 cell spheroid specimen. The spheroids were harvested after 24 h and transferred into a collagen gel (3.54 mg/ml, BD Biosciences).
  • FBS fetal bovine serum
  • Spheroids were treated with different concentrations of recombinant EG-VEGF for 24 h. These experiments were also performed in the absence or presence of PROKRs antagonists. Quantification of JEG3 cells spreading was assessed using ImageJ software (http://rsb.info.nih.gov/ij/) after 24h of culture by image analysis of microphotographs. At least, three replicates were included within each experiment. The spreading was assessed by calculating the ratio of invaded area (the whole spheroid) over the non-invaded area (center of the spheroid).
  • JEG3 cells were incubated in the presence of increased concentrations of recombinant EG-VEGF (Peprotech, France) for 24h.
  • Protein extracts were prepared as previously described (Alfaidy et al., 2003). Protein extracts were electrophoretically separated on 0.1 % sodium dodecyl sulfate-12% polyacrylamide gels and electrically transferred onto 0.45 ⁇ nitrocellulose membranes. The membranes were blotted with antibodies against PROKR1 or PROKR2 (Hoffmann et ai, 2009). Both PROKR1 and PROKR2 antibodies were used at a final concentration of 0.45 g/ml.
  • the blots were washed with PBS- Tween 0.1 % and incubated for 1 h in blocking solution (5% skimmed milk in PBS- T). Subsequently, the membranes were immunoblotted with a rabbit antibody against PROKR1 , PROKR2 (0.45 pg /ml) overnight. The blots were then rinsed with PBS-T and incubated with goat anti-rabbit IgG (1 :3.000) for 1 h. After three PBS-T washes, the antibody-antigen complex was detected using the ECL plus detection system (Amersham Pharmacia Biotech). The intensities of immunoreactive bands were measured by scanning the photographic film and analyzing the images on a desktop computer using ImageJ software.
  • the Chemidoc analyzing system was also used (Image Lab version 4.0.1 ). To standardize for sample loading, the blots were subsequently stripped using a commercially available kit, following the manufacturer's instructions (Re-blot; Millipore), and reprobed with an anti- -actin antibody (Sigma-Aldrich) as an internal control for protein loading.
  • an anti- -actin antibody Sigma-Aldrich
  • membranes were incubated at 4 ° C overnight respectively with phospho-MAPK (1 :5.000) (Promega), phospho-AKT (Ser473) (1 :2.000) (Cell Signaling), phospho-Src (pY418) (1 :1 .000) (Invitrogen).
  • Blots were reprobed respectively with anti-MAPK (1 :40.000) (Sigma), anti-AKT (1 :1 .000) (Cell Signaling) or anti-Src (1 :1000) (Millipore) antibodies to standardize for sample loading.
  • JEG3 cells were incubated in the absence or presence of EG-VEGF for 24h.
  • the protein concentration was determined in the culture medium of all conditions.
  • the same amount of proteins was electrophoresed under non- reducing conditions in a 10% acrylamide gel containing 1 mg/ml gelatin (Sigma Aldrich, France) according to the method of Xu et al. (Xu, Alfaidy, & Challis, 2002).
  • tissue sections were subsequently washed three times with PBS and incubated with biotinylated goat anti-rabbit IgGs (1 :400 dilution in blocking solution; Sigma-Aldrich, Saint-Quentin Fallavier, France) for 1 h in a humid chamber.
  • biotinylated goat anti-rabbit IgGs (1 :400 dilution in blocking solution; Sigma-Aldrich, Saint-Quentin Fallavier, France
  • the slides were incubated with an avidin-biotin complex (Vectastain ABC kit; Vector Laboratories, Burlingame, CA) for 1 h.
  • an avidin-biotin complex Vectastain ABC kit; Vector Laboratories, Burlingame, CA
  • the immunoreactive proteins were visualized after the addition of 3, 3'-diaminobenzidine (Dako) for 2min and then counterstained with hematoxylin.
  • Dako 3, 3'-diaminobenz
  • mice All animal studies were approved by the institutional guidelines and those formulated by the European Community for the Use of Experimental Animals. Two to three month-old SHO SCID female mice were mated in the animal facility. The presence of a vaginal plug was observed at 0.5dpc. The gravid mice were randomly assigned to be injected by JEG3-luc cells (see flowchart for detailed protocol, Fig. 10). Five groups of female mice were established. At least five animals were assigned to each experimental group, which were defined as follows: Mice in Group 1 were injected at 7.5 days post coitus (dpc) in two opposed placenta with JEG3 cells embedded in Matrigel.
  • dpc post coitus
  • mice are non-gravid mice that were injected in their uterine horns with JEG3-luc embedded in Matrigel.
  • Group 3 mice were injected with Matrigel alone in either uterine horns or placentas.
  • Mice in the Group 4 were injected in two opposed placenta with JEG3 cells embedded in Matrigel and at days 8.5, 1 1 .5, 14.5, and 17.5. These mice were injected with PC7, the PROKR1 antagonist (500 g/kg, ip).
  • mice were injected in two opposed placentas with JEG3 cells embedded in Matrigel and at 8.5, 1 1 .5, 14.5, and 17.5 dpc, these mice were injected with PKR-A 505, the PROKR-2 antagonist (100 mg/kg, ip).
  • PKR-A 505 the PROKR-2 antagonist (100 mg/kg, ip).
  • Two other control groups of mice were injected with vehicles of PC7 and PKR505 antagonists.
  • bioluminescence imaging was performed with a highly sensitive, cooled CCD camera, mounted in a light-tight specimen box (MS®. In Vivo Imaging System. PerkinElmer). Before imaging, animals were anesthesized in 2% isoflurane. 10 ⁇ /1 Og of body weight of luciferin (potassium salt, Xenogen, USA), was injected to the mice 15 min before imaging. This dose and route of administration have been shown to be optimal for studies in rodents when images were acquired within 15min post-luciferin administration.
  • mice were placed onto the warmed stage inside the light- tight camera box, with continuous exposure to 1 % to 2% isoflurane. The animals were imaged, and data were acquired for 45s; this imaging time was shown to yield superior results.
  • the low levels of light emitted from the bioluminescent tumors were detected by the MS® camera system and were then integrated, digitized, and displayed.
  • the regions of interest (ROI) from displayed images were designated around the tumor area and were quantified as total photon counts or in photons/s, using Living Image® software (Xenogen, USA).
  • a laparotomy was performed to collect blood and to expose and image the uterine horn containing embryos with their attached placentas, as well as the rest of metastatic organs.
  • a second imaging of the organs was performed and quantified as described above. Placentas and metastatic organs were collected and stored at -80°C or collected in PFA for immunohistochemistry analyses.
  • Antibody-array was used to compare the expression profile and levels of angiogenesis-related proteins in sera collected from the five different groups of female mice. Antibody array experiment was assessed using Mouse Angiogenesis Array kit (R&D Systems; USA). Briefly, nitrocellulose membranes were incubated in blocking solution for 1 hour at RT on a rocking platform shaker. Membranes were then incubated with 10 ⁇ of serum and 15 ⁇ of detection antibody cocktail overnight at 4 ° C. After three washes using the washing buffer, membranes were incubated with streptavidin-HRP for 30min at room temperature. After three washes the antibody-antigen complex was detected using the ECL plus detection system (Amersham Pharmacia Biotech). The intensities of immunoreactive bands were measured by scanning the photographic film and analyzing the images on a desktop computer using ImageJ software. The Chemidoc analyzing system was also used (Image Lab version 4.0.1 ).
  • Example 1 Clinical study to determine EG-VEGF association to CHM, choriocarcinoma development and progression
  • EG-VEGF circulating levels and placental expression are increased in CHM and choriocarcinoma
  • EG-VEGF is secreted by the syncytiotrophoblast and that its levels are elevated during the first trimester of pregnancy.
  • Fig. 1A shows that EG-VEGF levels were slightly but significantly higher in the CHM group than the normal group (P ⁇ 0.05). The mean value was increased by almost 1 .5-fold in CHM patients. More importantly, mean value in patients who were diagnosed for choriocarcinoma were significantly elevated compared to normal individuals and CHM patients (Fig 1A).
  • Fig. 1A shows that EG-VEGF levels were slightly but significantly higher in the CHM group than the normal group (P ⁇ 0.05). The mean value was increased by almost 1 .5-fold in CHM patients. More importantly, mean value in patients who were diagnosed for choriocarcinoma were significantly elevated compared to normal individuals and CHM patients.
  • Fig. 1A mean value in patients who were diagnosed for choriocarcinoma were significantly elevated compared to normal individuals and CHM patients.
  • FIG. 1 B shows representative control, CHM and choriocarcinoma placental sections stained for EG-VEGF.
  • EG-VEGF protein expression was markedly increased in CHM placentas and was mostly localized to the syncytiotrophoblast layer.
  • Choriocarcinoma exhibited stronger staining for EG-VEGF as compared to normal and CMH tissues.
  • Example 2 in vitro study, using JEG3 cells, to characterize EG- VEGF effects on their proliferation, migration and invasion
  • EG-VEGF activates key tumor signaling pathways and increases choricarcinoma cell proliferation
  • EG-VEGF increases normal trophoblast proliferation suggesting its potential involvement in choriocarcinoma cell proliferation.
  • JEG3 expresses both PROKR1 and PROKR2 receptors and that their expression is up-regulated by EG-VEGF (Fig. 8).
  • Fig. 2A shows that EG-VEGF significantly increases JEG3 cell proliferation.
  • EG-VEGF effect was abolished in the presence of PROKR1 or PROKR2 antagonists, suggesting that both receptors are involved in the EG-VEGF mediated-proliferative effect of JEG3 cells.
  • EG-VEGF induces the proliferation and survival of choriocarcinoma cells.
  • Fig. 2B shows that EG- VEGF induces the phosphorylation of AKT and MAPKs upon 10 min of treatment. Because Src phosphorylation is associated to tumor cell aggressiveness, we investigated EG-VEGF effect on its phosphorylation in JEG3 cells.
  • Fig. 2B shows that EG-VEGF induces Src phosphorylation after 10min of treatment. Effects of EG-VEGF on choriocarcinoma
  • FIG. 3A shows representative photographs of JEG3 at (TO) and (T24) after wounding and subsequent incubation in the absence or the presence of EG-VEGF (5, 10 and 25 ng/ml). Following 24 h incubation, EG-VEGF induced JEG3 cell migration at all concentrations tested. The closure of the wound reached 80% in cells treated with 10ng/ml of EG-VEGF as compared to 60% of closure in the controls.
  • Fig. 3B and 3C show that EG-VEGF effect on JEG3 migration was abolished in the presence of PROKR1 and PROKR2 antagonists. The quantification is reported in Fig.3D
  • Fig. 4A shows that EG-VEGF significantly increased JEG3 invasion and that this effect was reversed in the presence of PROKR1 and PROKR2 antagonists, suggesting that E G-VE G F med iates its effect on J EG3 invasion throug h PROKR 1 and PROKR2 activation . Quantification of the number of invading cells in each condition is reported in Fig. 4B. Effect of EG-VEGF on tumor trophoblastic cells invasion using
  • EG-VEGF effect on JEG3 differentiation and invasion in a system in which the tumor architecture is maintained.
  • Tumor cells cultured as spheroids form a topology similar to the one observed in vivo (Nath & Devi, 2016).
  • Fig. 5A shows representative photographs of JEG3 spheroids at the time of their incubation with EG-VEGF (a- f) and 24h later (g-l).
  • EG-VEGF treatment resulted in a significant increase in JEG3 spreading within the collagen.
  • EG-VEGF effect was abolished in the presence of PROKR1 and PROKR2 antagonists.
  • a critical element of tumor progression is remodeling of extracellular matrix (ECM) by matrix metalloproteinases (MMPs).
  • Trophoblast cells are known to express high levels of MMP-2 and MMP-9.
  • MMP-2 and MMP-9 production in culture media collected from JEG3 cultures after treatment with EG- VEGF (10 and 25ng/ml).
  • Zymography was used to assess the MMP-2 and MMP- 9 activity.
  • Fig. 5C shows that MMP2 was more abundant than MMP9 in JEG3 cells and that EG-VEGF treatment significantly increased both activities.
  • Example 3 Characterization of EG-VEGF role in choriocarcinoma development and progression in vivo
  • Fig.6A shows that JEG3 cell injection in the gravid placentas lead to more aggressive tumor development as compared to their injection within the uterine horn in Fig.6B.
  • placental environment and vascularization contribute to choriocarcinoma metastasis.
  • gestations of all gravid mice injected with JEG3-luc cells were unsuccessful, as all fetuses were dead at 19.5 dpc with resorption of all placentas (Table 2 and control in Table 3).
  • PROKR1 and PROKR2 antagonists Effects of PROKR1 and PROKR2 antagonists on choriocarcinoma development and progression in vivo
  • PROKR1 or PROKR 2 antagonists at three time points during tumor progression.
  • Fig. 6C shows that mice injected with either antagonist exhibited significant decrease in tumor development and progression. These data suggest a remarkable potential of both antagonists to reduce tumor development and metastasis. Quantification of at least three mice per condition shows that
  • PROKR1 or PROKR2 antagonists significantly decreased the intensity of ROI compared to their vehicle, respectively Fig. 6D. More importantly, both in PROKR1 and PROKR2 antagonists-treated JEG3-luc mice we observed a maintenance of the gestation with few resorbed placentas, (Tables 4 and 5).
  • Fig 7A JEG3-Luc, or JEG3-Luc+PROKR1 antagonist or +PROKR2 antagonist is reported on Fig 7A.
  • Placentas collected from mice injected with JEG3-luc cells exhibited strong histological changes with loss of all placental structures and zones (Fig. 7Ad, Ae, Af), compared to placenta collected from mice injected with matrigel (Fig. 7Aa, Ab, Ac).
  • histological analyses of placentas collected from mice injected with JEG3-Luc +PROKR1 or +PROKR2 antagonists exhibited minor placental changes, as all placental zones and structures were conserved, (Fig. 7Ag, Ah, Ai and Fig. 7Aj, Ak, Ae, respectively).
  • JEG3 cells JEG3-luc
  • PROKR1 antagonist JEG3-luc+PROKR1 a
  • PROKR2 antagonist JEG3- luc+PROKR2a
  • MMP-9 is well established as pro-tumoral protein (Farina & Mackay, 2014), while Nov is known as an inhibitor of cancer cell proliferation (Bleau et ai., 2007).
  • Nov overexpression results in reduced tumor size in glioma cell xenografts (Gupta et ai., 2001 ).
  • the decrease in the angiogenic hormone proliferin is in line with the observation that mice injected by JEG3-luc cells had an arrested gestation. In fact, proliferin is secreted specifically by the trophoblast giant cells and ensures the establishment of the fetomaternal circulation early on during gestation.
  • mice with either antagonist exhibited higher levels of proliferin secretion, confirming the beneficial effect of these antagonists on the pregnancy maintenance and progress.
  • the fine mechanism by which these angiogenic factors are locally regulated within the placenta are still to be investigated.
  • the in vitro data further substantiate the control of tumor trophoblast cell's migration upon their treatment by the EG-VEGF antagonist's, PROKR2-atg.
  • the findings were performed using a 3D culture system that allowed the assessment of the degree of the spreading of tumor cells (Figure 5).
  • Figure 1 1 and Figure 12 compared the effects of 8 other analogues of PROKR2 antagonists on trophoblasts proliferation and migration.
  • the new antagonists were obtained from our collaborator Dr Gianfranco Balboni.
  • EG-VEGF controls placental growth and survival in normal and pathological pregnancies: case of fetal growth restriction (FGR).
  • FGR fetal growth restriction
  • Ngan E. S., Sit, F. Y., Lee, K., Miao, X., Yuan, Z., Wang, W., Tarn, P.
  • MMP matrix metalloproteinase

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Abstract

La présente invention concerne l'utilisation d'un antagoniste du récepteur 2 de la prokinéticine en tant que médicament pour traiter un cancer associé à l'EG-VEGF, en particulier pour traiter le choriocarcinome. Les inventeurs ont effectué une étude clinique pour déterminer l'association EG-VEGF à CHM, le développement et la progression du choriocarcinome et une étude in vitro, à l'aide d'une lignée cellulaire de choriocarcinome humain (cellules JEG3) pour caractériser des effets d'EG-VEGF sur leur prolifération, migration et invasion. Les résultats de ces études cliniques et in vitro ont fortement suggéré que l'EG-VEGF peut contribuer au développement et à la progression globaux du choriocarcinome in vivo. En particulier, la présente invention concerne un antagoniste du récepteur 2 de la prokinéticine (PROKR2) destiné à être utilisé en tant que médicament pour traiter un cancer associé à l'EG-VEGF.
PCT/EP2017/082321 2016-12-12 2017-12-12 Antagoniste du récepteur 2 de la prokinéticine destiné à être utilisé en tant que médicament pour le traitement d'un cancer associé à un vegf WO2018108862A1 (fr)

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CN109943530A (zh) * 2019-03-04 2019-06-28 浙江大学医学院附属妇产科医院 人绒癌耐药细胞株

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CN109943530A (zh) * 2019-03-04 2019-06-28 浙江大学医学院附属妇产科医院 人绒癌耐药细胞株

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