Pharmaceutical composition comprising TRPV4 inhibitor for preventing or treating cerebral cavernous malformation (CCM)

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cerebral cavernous malformation (CCM), comprising a transient receptor potential vanilloid 4 (TRPV4) inhibitor.

Description

Technical Field

[0001] This invention relates to the field of therapeutic agents for the prevention or treatment of cerebral cavernous malformations (CCM). Background Technology

[0002] Cavernous malformations (CCMs) occur in 0.5% of the world's population. They account for a large proportion (8-15%) of all cerebral and spinal vascular malformations. People with familial CCM (20% of cases) typically develop multiple lesions that progress over time. Familial CCM is associated with mutations in any of the specific genes, among which the most recent (but not limited to) are the KRIT1 gene (also known as CCM1), the MGC4607 gene (also known as CCM2), and the PDCD10 gene (also known as CCM3).

[0003] The most common clinical manifestations are epilepsy, migraine, bleeding, and many other related neurological deficits, such as paralysis of the arm or leg, hearing or visual impairment, or cognitive deficits (such as speech, memory, and attention difficulties).

[0004] CCM (cavernous malformation) lesions are formed by the stacking of tortuous, dilated, and hemorrhaging capillaries in the brain. These cerebral capillaries lack wall cells and are formed by a single layer of weakly connected endothelial cells (ECs). 3D reconstructions of human CCM lesions have demonstrated the presence of numerous endothelial buds expanding into spongy structures that extend from innervated vessels into the brain parenchyma. The reason for this spongy growth, which occurs only in low-flow venous capillaries, remains unclear.

[0005] The main steps in the development of CCM disease are known to be: 1) dedifferentiation / senescence, 2) loss of blood-brain barrier (BBB) ​​(leading to cell-cell junctions / destruction of actin stress fibers and increased BBB permeability), 3) degradation of extracellular matrix (ECM) by secreted matrix metalloproteinases (MMPs) and 4) invasion of surrounding tissues.

[0006] Over the past 20 years, research on CCM signaling has increased exponentially with the identification of downstream deregulated pathways of various CCM gene mutations. The most extensively studied pathways include RhoA / ROCK, VEGF, MEKK3-KLF2 / 4, PI3K / AKT / mTOR, and reactive oxygen species.

[0007] Drugs targeting the RhoA / ROCK-related signaling pathway (ROCK inhibitors and statins) are among the most promising. However, the adverse side effects of these treatments have prompted the search for other, more specific targets.

[0008] Currently, there is no proven effective and safe treatment for CCM, and surgical resection is not always feasible, depending on the location of the lesion in the brain.

[0009] Therefore, it is indeed necessary to understand the pathophysiological mechanisms involved in the formation of these vascular malformations in order to identify therapeutic targets.

[0010] Transient receptor potential vanilloid 4 (TRPV4) is a member of the TRP superfamily and is a Ca2+-transmissible channel. In the central nervous system, TRPV4 is expressed in various cell types, including neurons, glial cells, and vascular endothelial cells. TRPV4 is known to play a crucial role in central nervous system disorders such as ischemic stroke, traumatic brain injury, and Alzheimer's disease. TRPV4 is also known for its role in restoring the blood-brain barrier (BBB) ​​in a model of intracerebral hemorrhage (H. Zhao et al., TRPV4 Blockade Preserves the Blood–Brain Barrier by Inhibiting Stress Fiber Formation in a Rat Model of Intracerebral Hemorrhage Front. Mol. Neurosci. 11 (2018), doi:10.3389 / fnmol.2018.00097). Summary of the Invention

[0011] In their research, the inventors surprisingly discovered that transient receptor potential vanillin 4 (TRPV4) has an upstream effect on deregulation pathways in CCM gene-deficient cells. More precisely, the inventors have determined that TRPV4 is abnormally overexpressed at both the mRNA and protein levels in these endothelial cells and is involved in many steps of CCM disease, including steps 1) dedifferentiation / senescence, 2) loss of the blood-brain barrier (BBB), and 3) degradation of the extracellular matrix (ECM) by secreted matrix metalloproteinases (MMPs).

[0012] Therefore, the present invention relates to a pharmaceutical composition for the prevention or treatment of cerebral cavernous malformation (CCM) comprising a transient receptor potential vanillin 4 (TRPV4) inhibitor.

[0013] The inventors have discovered that the use of one or more TRPV4 inhibitors, or pharmaceutical compositions containing such TRPV4 inhibitors, in endothelial cells with CCM gene defects has a reversing effect on the early stages of said CCM disease. The pharmaceutical compositions according to the invention can block or delay cell dedifferentiation, reverse BBB permeability, and block or reduce ECM degradation. Furthermore, the TRPV4 channel has the advantage of direct access from the bloodstream, thus bypassing the problem of BBB penetration. In this way, TRPV4 inhibitors (especially chemical compounds) can directly reach the TRPV4 channel without having to cross the BBB, and without the risk of being destroyed or blocked by the BBB.

[0014] In the context of this invention:

[0015] – “Treatment” refers to a method or process aimed at (1) delaying or preventing the onset of a disease or condition (in this case, CCM); (2) slowing or stopping the progression, aggravation, or worsening of the symptoms of a disease or condition; (3) causing improvement in the symptoms of a disease or condition; or (4) curing a disease or condition. Treatment may be administered after the onset of a disease or condition to have a therapeutic effect. Alternatively, treatment may be administered before the onset of a disease or condition to have a prophylactic or preventive effect. In this case, the term “prevention” is used.

[0016] – The terms “transient receptor potential vanillin 4 (TRPV4) inhibitor,” “TRPV4 inhibitor,” “transient receptor potential vanillin 4 (TRPV4) blocker,” “TRPV4 blocker,” “transient receptor potential vanillin 4 (TRPV4) repressor,” or “TRPV4 repressor” are used interchangeably to refer to any therapeutic agent suitable for the therapeutic application of this invention, which reduces or even completely inhibits / represses the expression of a normally expressed gene or protein in cells. TRPV4 inhibitors include therapeutic agents that block TRPV4 activity, inhibit TRPV4 expression, disrupt, degrade, or cleave TRPV4 mRNA or protein, block TRPV4 mRNA translation, or target the therapeutic portion to TRPV4-overexpressing cells. The above definitions also apply to TRPV2, PIEZO1, and PIEZO2 inhibitors, respectively.

[0017] – The terms “TRPV4 RNA interference agent” and “TRPV4 RNAi agent” are used interchangeably and refer to any RNA molecule that can specifically inhibit or downregulate the expression of a target gene (in this case, the TRPV4 gene). “Silencing, inhibiting, or downregulating the expression of a target gene” means that the expression of the target gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits is reduced to a level or activity observed in the absence of an RNAi agent.

[0018] RNA interference (or RNAi) refers to any biological process by which RNA molecules silence, suppress, or downregulate gene expression by causing the destruction, degradation, and / or cleavage of specific mRNA molecules or by blocking their translation. As is known in the art, RNA interference is now being developed and utilized in therapies. In fact, RNAi can be artificially initiated, for example, for silencing the expression of target genes.

[0019] – The terms “TRPV4 siRNA” or “TRPV4 small interfering RNA” are used interchangeably herein and refer to any RNA (or RNA analog) containing about 10-50 nucleotides (or nucleotide analogs) capable of directing or mediating TRPV4 RNAi. Preferably, the siRNA contains about 15-30 nucleotides or nucleotide analogs, more preferably about 16-25 nucleotides (or nucleotide analogs), even more preferably about 18-23 nucleotides (or nucleotide analogs), and even more preferably about 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21, or 22 nucleotides or nucleotide analogs).

[0020] – “TRPV4 aiRNA” or “TRPV4 asymmetric interfering RNA” are used interchangeably in this article to refer to any TRPV4 siRNA characterized by asymmetric lengths of its two RNA strands.

[0021] – The terms “TRPV4 shRNA” or “TRPV4 short hairpin RNA” are used interchangeably herein to refer to an RNA sequence having one or more loop structures and a stem containing self-complementary sense and antisense regions, wherein the antisense regions contain sequences complementary to the target TRPV4 mRNA region. The short hairpin RNA is cleaved into siRNA by cellular mechanisms. The stem length can be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 base pairs, for example, 19 to 25 base pairs or 19 to 21 base pairs. The loop length can vary. For example, the loop length can be 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides. The hairpin structure can also include a 3' or 5' overhang. For example, the overhang is a 3' or 5' overhang of 1, 2, 3, 4, or 5 nucleotides in length.

[0022] – The terms “microRNA” or “miRNA” are used interchangeably in this article to refer to a major group of non-coding RNAs known to regulate almost one-third of all coding genes. They are small (approximately 20-25 nucleotides long) endogenously formed gene expression repressors. miRNAs typically bind to the 3' untranslated region (3'UTR) of target RNA transcripts (mRNA or circRNA) and can induce post-transcriptional gene regulation by blocking translation, degrading the target RNA, or both. miRNAs can also be chemically synthesized. Unlike siRNAs, which are completely complementary to target RNA transcripts, miRNAs bind incompletely to their target RNA transcripts.

[0023] – “TRPV4 antagonist” refers to any synthetic or natural molecule that is described as having TRPV4 blocking activity or properties;

[0024] –GSK2193874 refers to 3-([1,4'-bipiperidin]-1'-ylmethyl)-7-bromo-N-(1-phenylcyclopropyl)-2-[3-(trifluoromethyl)phenyl]-4-quinoline carboxamide and its pharmaceutically acceptable salts;

[0025] –GSK2798745 refers to 1-(((5S,7S)-3-(5-(2-hydroxypropyl-2-yl)pyrazin-2-yl)-7-methyl-2-oxo-1-oxa-3-azaspiro[4.5]dec-7-yl)methyl)-1H-benzo[d]imidazol-6-carboxynitrile and its pharmaceutically acceptable salts;

[0026] –HC 067047 refers to 2-methyl-1-[3-(4-morpholinyl)propyl]-5-phenyl-N-[3-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxamide and its pharmaceutically acceptable salts;

[0027] –RN-1734 refers to 2,4-dichloro-N-isopropyl-N-(2-isopropylaminoethyl)benzenesulfonamide and its pharmaceutically acceptable salts;

[0028] –GSK3395879 refers to 2-[(3R,4S)-4-(4-cyano-3-fluorophenoxy)-3-hydroxy-3-(hydroxymethyl)pyrrolidone-1-yl]sulfonyl-5-(trifluoromethyl)benzyl nitrile and its pharmaceutically acceptable salts;

[0029] –GSK3491943 refers to 3-chloro-4-(((3R,4S)-4-(4-chlorophenoxy)-3-hydroxy-3-(hydroxymethyl)pyrrolidone-1-yl)sulfonyl)benzyl nitrile and its pharmaceutically acceptable salts;

[0030] –GSK3527497 refers to 4-[(3S,4S)-4-(aminomethyl)-1-(5-chloropyridin-2-yl)sulfonyl-4-hydroxypyrrolidine-3-yl]oxy-2-fluorobenzyl nitrile and its pharmaceutically acceptable salts;

[0031] –RN-9893 refers to 2-nitro-N-[4-(4-propyl-2-ylpiperazin-1-yl)sulfonylphenyl]-4-(trifluoromethyl)benzamide and its pharmaceutically acceptable salts;

[0032] –PF-05214030 refers to 4-((1-((2,4-dichlorophenyl)sulfonyl)-3-hydroxyazacyclobutane-3-yl)methoxy)-2-fluorobenzyl nitrile and its pharmaceutically acceptable salts;

[0033] –GSK205 refers to N-[4-[2-[methyl(phenylmethyl)amino]ethyl]phenyl]-5-(3-pyridyl)-2-thiazolamine, hydrobromide (also known as N-[4-[2-[methyl(phenylmethyl)amino]ethyl]phenyl]-5-(3-pyridyl)-2-thiazolamine, hydrobromide) and its pharmaceutically acceptable salts;

[0034] –Tranilast refers to 2-[[3-(3,4-dimethoxyphenyl)-1-oxo-2-propen-1-yl]amino]-benzoic acid and its pharmaceutically acceptable salts;

[0035] –SET2 refers to N-(2-furanylmethyl)-3-[[4-(methylpropylamino)-6-(trifluoromethyl)-2-pyrimidinyl]thio]-propionamide and its pharmaceutically acceptable salts;

[0036] –OB-1 refers to ethyl 5-[(1-methyl-4-nitro-1H-imidazol-5-yl)oxy]-2-phenyl-3-benzofuran carboxylate and its pharmaceutically acceptable salts;

[0037] –GsMTX4 refers to any peptide consisting of the amino acid sequence GCLEFWWKCNPNDDKCCRPKLKCSKLFKLCNFSF or an amino acid sequence that has at least 80% identity with this sequence.

[0038] – “continuous schedule” refers to continuous therapeutic treatment of a patient, including the successive administration of one or more therapeutic compositions (which may or may not be part of the multiple therapy according to the invention), the therapeutic compositions being the same or different, each of which has its own treatment regimen (the number of times and days of daily administration within a given period, such as a week), and thus administered in an unlimited manner and over time, without sequential or intermittent administration, that is, without interruption of treatment;

[0039] – “Daily application” means application once a day or once every 24 hours;

[0040] – A “pharmaceutically acceptable salt” of an active ingredient means any addition salt formed by the active ingredient and an inorganic or organic acid in an organic or aqueous solvent (such as an alcohol, ketone, ether, or chlorinated solvent) through the action of such acid, and that such salt is pharmaceutically acceptable; and

[0041] - Unless otherwise stated, all % values ​​are by weight.

[0042] Therefore, the present invention relates to pharmaceutical compositions for the prevention or treatment of cerebral cavernous malformations (CCM) comprising transient receptor potential vanillin 4 (TRPV4) inhibitors. Preferably, the present invention relates to pharmaceutical compositions as defined above, wherein the TRPV4 inhibitor is selected as a TRPV4 antagonist, such as GSK2193874, GSK2798745, HC 067047, RN-1734, GSK3395879, GSK3491943, GSK3527497, RN-9893, PF-05214030, or selected as a TRPV4 RNA interfering agent, such as TRPV4 siRNA, TRPV4 shRNA, microRNA, or aiRNA. More preferably, the TRPV4 inhibitor is selected as TRPV4 siRNA. More preferably, the TRPV4 inhibitor is selected from GSK2193874, GSK2798745, HC 067047, RN-1734, GSK3395879, GSK3491943, GSK3527497, RN-9893, and PF-05214030. Even more preferably, the TRPV4 antagonist is selected from GSK2193874. The pharmaceutical composition according to the invention contains a sufficient amount of the active ingredient to ensure the desired therapeutic effect on the endothelial cells of CCM patients.

[0043] Advantageously, the pharmaceutical composition according to the invention further comprises a transient receptor potential vanillin 2 (TRPV2) inhibitor, a piezoelectric mechanosensitive ion channel component 1 (PIEZO 1) inhibitor, and / or a piezoelectric mechanosensitive ion channel component 2 inhibitor. Preferably, the TRPV2 inhibitor is selected from the group consisting of tranilast and SET2, the PIEZO 1 inhibitor is selected from the group consisting of OB-1 and GsMTX4, and the PIEZO 2 inhibitor is GsMTX4. More preferably, the pharmaceutical composition according to the invention comprises GSK2193874 as a TRPV4 inhibitor, OB-1 as a PIEZO 1 inhibitor, and / or tranilast as a TRPV2 inhibitor.

[0044] Preferably, the pharmaceutical composition according to the invention comprises 0.05% to 12% of a TRPV4 inhibitor, more preferably 0.1% to 6% of a TRPV4 inhibitor.

[0045] The pharmaceutical compositions according to the invention can be formulated into any gallon formulation required for their administration. In particular, for topical application, the compositions according to the invention can be formulated into gels or thermogels. For oral administration, the compositions according to the invention can be formulated into coated or uncoated, effervescent, soluble, orally dispersible, gastric-resistant, or modified-release tablets; sugar-coated pills; hard-shell capsules (or gel capsules); soft-shell capsules; granules; microcapsules; pills; lozenges. For nasal administration, the compositions can be formulated into sprays or powders for inhalation. For systemic administration, the compositions according to the invention can be formulated into sterile lyophilized powders for injection.

[0046] Therefore, in addition to the active ingredient, the pharmaceutical compositions of the present invention may contain any adjuvant known to those skilled in the art and essential for the preparation of the desired form of the pharmaceutical composition, which is pharmaceutically acceptable.

[0047] The pharmaceutical composition according to the invention can be administered to patients receiving therapy for the treatment of CCM up to 1 to 4 times daily.

[0048] The pharmaceutical composition according to the invention can be administered at any time of day (before, during, or after a therapy for treating CCM) without affecting the effectiveness of the treatment.

[0049] The pharmaceutical compositions according to the invention can be administered to a patient once or more per week. Therefore, the invention also relates to a pharmaceutical composition, such as the previously defined pharmaceutical composition administered to a patient receiving therapy for the treatment of CCM for 1 to 7 days per week.

[0050] The composition according to the invention can be applied in a continuous manner.

[0051] The present invention also relates to a method for preventing or treating cerebral cavernous malformation (CCM) by applying a pharmaceutical composition as defined above.

[0052] The present invention also relates to a method for preventing or treating cerebral cavernous malformation (CCM) by administering a pharmaceutical composition as defined above 1 to 4 times daily.

[0053] The present invention also relates to a method for preventing or treating cerebral cavernous malformation (CCM), the method being carried out by administering a pharmaceutical composition as defined above daily for 1 to 7 days per week, the administration being carried out according to or not according to a continuous regimen.

[0054] The present invention is illustrated by the following embodiments, but is not limited thereto. Attached Figure Description

[0055] • Figure 1The included graphs show that targeting TRPV4 restores the proliferation defects of CCM2-depleted endothelial cells, and

[0056] • Figure 2 The included charts and images show that targeting TRPV4 restores the permeability barrier of CCM2-depleted endothelial cells.

[0057] • Figure 3 The included charts show that targeting TRPV4 restores ECM degradation in CCM2-depleted endothelial cells. Detailed Implementation

[0058] Example

[0059] 1. Materials and Methods

[0060] Reagents, cell culture, and transfection:

[0061] Hybrid human umbilical vein endothelial cells (HUVECs) were obtained from Pelobiotech. Upon receipt, P0 HUVECs were amplified at least two passages in complete EGM-2 medium (Lonza) supplemented with 100 U / ml penicillin and 100 μg / ml streptomycin in a humidified chamber at 37°C, 5% CO2–3% O2. Third-generation HUVECs were transfected twice at 24-hour intervals in OptiMEM with 30 nM siRNA and Lipofectamine RNAi max (Life Technologies, catalog number 13778-150) according to the manufacturer's instructions. For double transfection, use 30 nM of each of the following siRNA duplexes (Dharmacon smart pool ON-TARGET plus Thermo Scientific): non-targeting siRNA #1 (Catalog No. D-001810-01), CCM2 siRNA (Catalog No. L-014728-01), and TRPV4 siRNA (Catalog No. L-004195-00). Inhibitors and agonists were purchased from Bio-Techne / Tocris (GSK2193874, catalog number 5106; RN1734, catalog number 3746; RN9893, catalog number 5678; HC067047, catalog number 4100; GSK1016790A, catalog number 6433; OB1, catalog number 6545) or MedChemExpress (GSK2798745, catalog number HY-19765; GSK205, catalog number HY-120691A). Water-soluble GSK2193874 hydrochloride was obtained by mixing the commercially available compounds with 0.1M HCl (1:1 molar ratio), sonicating, and freeze-drying.

[0062] Immunofluorescence staining:

[0063] Transfected HUVECs were seeded in 24-well plates at 3 × 10⁴ cells (sparse seeding, for Ki67 staining) or 1.5 × 10⁶ cells (confluence seeding, for VE-cadherin staining) on ​​coverslips coated with 10 μg / ml fibronectin (from human plasma, Sigma-Aldrich), and incubated overnight (Ki67 assay) or for 48 hours (VE-cadherin assay) in complete EBM-2 medium with or without calcium channel inhibitors dissolved in DMSO. For accurate comparison, DMSO was also added to samples without inhibitors at the maximum permitted concentration. Cells were fixed with 4% PFA, permeabilized with 0.2% Triton X-100, and incubated with either anti-Ki67 antibody (AN9260 Millipore, 1 / 200) or anti-VE-cadherin antibody (BV9 Millipore MABT129). After rinsing, coverslips were incubated with a goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor-conjugated AF 488 (Invitrogen, 1 / 1000), and TRITC-conjugated phalloidin (1 / 1000, Sigma-Aldrich). The coverslips were mounted in Mowiol / DAPI solution and imaged at 63x or 20x magnification on an epifluorescence Axiomager microscope (Zeiss) to obtain monolayer images. The percentage of Ki67-positive cells was manually counted from a total of over 130 cells. To analyze cell-cell junction width, the VE-cadherin staining signal was segmented using Ilastik (a machine learning-based segmentation tool). Junction width was determined by multiplying the Euclidean distance map by the skeleton of each segmented image, and the cumulative distribution of cell-cell junction widths was compared.

[0064] SA-B-galactosidase staining:

[0065] Forty-eight hours after the second siRNA transfection, transfected HUVECs were seeded at a density of 3 × 10⁴ in 24-well plates coated with 10 µg / mL fibronectin and incubated overnight in complete EBM-2 medium with or without calcium channel inhibitors. For accurate comparison, DMSO was also added to the sample without inhibitors at the maximum usable concentration. Senescence-associated β-galactosidase activity was assessed using the SA-β-galactosidase staining kit according to the manufacturer's instructions (Cell Signaling). Positive cells were manually counted from over 130 cells in total under each condition.

[0066] xCELLigence proliferation assay:

[0067] Proliferation was measured using the xCELLigence Real-Time Cell Analysis (RTCA) DP instrument in E-plate 16 (ACEA Biosciences) coated with 100 µL of 100 µg / mL collagen 1 (derived from rat tail) at 37°C for 30 min, followed by washing twice with PBS 1X. 50 µL of complete EBM-2 medium was added, and a cell-free baseline was created using RTCA software. 24 hours after the second transfection, 2.5 × 10³ transfected HUVECs (4 wells per condition) were seeded in 50 µL of complete EBM-2 medium. Immediately after seeding, 50 µL of a channel inhibitor or an equivalent concentration of DMSO (3 times the final desired concentration) was added. Impedance measurements were recorded every 5 min over 24 hours. Impedance was normalized 4 hours after seeding to eliminate the influence of cell spreading and adhesion on the signal. Slope measurements were performed between 4 and 24 hours.

[0068] Permeability measurement:

[0069] The xCELLigence Real-Time Cell Analyzer (RTCA) system (Ozyme) was used to measure changes in electrical impedance over time. Impedance changes in confluent endothelial cells reflect changes in barrier function (Twiss et al., 2012). Four × 10⁴ transfected HUVECs were confluently seeded in EGM-2 complete medium in wells of E-plate 16 pre-coated with 10 µg / ml fibronectin and 50 µg / ml collagen I (at least four wells per condition). After 4 h of adhesion, cells were starved in serum and cultured for an additional 24 h in basal EGM-2 containing 0.3% BSA, with or without calcium channel inhibitors. Cell indices were normalized to serum starvation time to eliminate signals due to cell spreading and adhesion to the matrix.

[0070] Gelatin degradation assay:

[0071] As previously described, coverslips in 24-well plates (70) were coated with gelatin-Alexa488 dye. Twenty-four hours after the second siRNA transfection, siCT and siCCM2 HUVECs were seeded at 3 × 10⁴ cells / well in OptiMEM medium with or without calcium channel inhibitors and incubated overnight at 37°C, 5% CO₂. Cells were fixed with 4% PFA and washed twice with PBS. Coverlips were mounted in Mowiol / DAPI solution and imaged at 40x magnification on an epifluorescence Axiomager M2 microscope (Zeiss) equipped with an N&B photosensitive camera (OrcaR2). To quantitatively analyze the degradation capacity of siRNA-transfected cells, at least 10 images were randomly acquired under each condition. Degradation areas (displayed as black areas on fluorescent gelatin) were segmented using the Ilastik model (a machine learning-based segmentation tool), and the total degradation area was measured on ImageJ. DAPI-stained images of cell nuclei were randomly acquired at 10x magnification, and cell density was measured using the Stardist plugin of Fiji after nucleus segmentation. Divide the total degradation area by the cell density to obtain the degradation area of ​​each cell.

[0072] 3D-PEG Intrusion Assay:

[0073] Polyethylene glycol (PEG) hydrogels were prepared on ice in EBM-2 complete medium by combining 1.5% polymer concentration of a PEG precursor modified with an MMP-sensitive peptide (8-arm 40 kDa, (71)), 50 μM Lys-RGD peptide (Pepmic), and 1 μM sphingosine-1-phosphate (S1P, Sigma-Aldrich). The hydrogels were enzymatically crosslinked at 10% of the total hydrogel volume using reconstituted and thrombin-activated factor XIII (Fibrogammin, CSLBehring), prepared as previously described (72). 20 μL of the hydrogel suspension was pipetted into a modified imaging chamber (Secure-Seal™ hybridization sealing system, ThermoFisher Scientific) attached to the bottom of a vertically positioned 24-well plate (62). The hydrogels were allowed to polymerize in this orientation at room temperature for 30 min prior to cell seeding. Depending on the study, confluent monolayers of GFP-HUVECs transfected with 5 × 10⁴ siRNA or a mixture of 1:1 siRNA-transfected GFP-HUVECs and immature RFP-HUVECs were adhered to the PEG meniscus at 37°C and 5% CO₂ for 1 h, and then horizontally returned to 1 ml of EBM-2 complete medium. After 24 h, fixation was performed with 4% PFA in DPBS. Shoots invading the PEG hydrogel were imaged using a Leica SP8 inverted confocal microscope equipped with an HC PL APO 10x, 0.4 numerical aperture dry objective to obtain 1024 × 1024 pixel image stacks with a 50 μm Z-stack and 1–1.5 μm Z-interval. The Z-projection of each image was used for manual quantification of the invasion distance using ImageJ's line measurement tool. Each technique was repeated to analyze more than 100 shoots.

[0074] Calcium imaging experiment:

[0075] P3 HUVECs were seeded in collagen-coated black 96-well clear glass plates (4 × 10⁴ cells / well) in complete EBM-2 medium (Lonza) supplemented with 100 U / ml penicillin and 100 μg / ml streptomycin, and incubated overnight at 37°C in a humidified chamber of 5% CO₂ - 3% O₂. Cells were loaded for 2 h at 37°C with a calcium-6 probe diluted in HBSS (FLIPR kit, Molecular Devices). 15 min before the end of the loading step, a TRPV4 inhibitor or its mediator (DMSO) was added to the wells. After calcium-6 excitation at 485 nm, fluorescence emission at 525 nm was collected every 2 seconds on a FlexStation3 (Molecular Devices). A baseline of 1 min (F0) was established, followed by the addition of 25 nM GSK1016790A (GSK101; TRPV4 agonist). Two minutes later, 1 µM of the ionocarrier drug iomycin was added to induce Ca2+ influx and cell viability was examined. IC50 was determined by the peak value of ΔF (F-F0) in response to GSK101. The peak value was normalized between 100% (mediator) and 0%, and IC50 was calculated using nonlinear regression curve analysis on Prism.

[0076] Statistical tests:

[0077] Results were evaluated using the following methods: paired t-tests were performed to compare two conditions, or Tukey's multiple comparisons test after ANOVA was used to compare more than two conditions with the control group. Fisher's exact test was used to compare the proportion of positive cells under different conditions, using the counts of positive and negative cells for each pair of test conditions. The 0.5α level was used for all comparisons. Statistical analysis of all data was performed using Prism software. P < 0.05 was considered significant. *P < 0.05, **P < 0.05, ***P < 0.005. n represents biological replicates.

[0078] 2. Results

[0079] The restorative effects of siTRPV4 and GSK2193874 (TRPV4 antagonist):

[0080] Secretory phenotype-associated aging (SASP) in endothelial cells with depleted CCM2.

[0081] refer to Figure 1Following CCM2 depletion, HUVECs undergo reprogramming and tend towards senescence (Vannier et al., Angiogenesis, 2021). Targeting TRPV4 with a specific siRNA (siTRPV4) blocked the senescence of CCM2-depleted ECs (siCCM2), as shown by the restoration of senescence-associated β-galactosidase activity-positive cells to control levels (siCT) (C), and restored their proliferative capacity, as shown by immunostaining of the proliferation marker Ki67 (D), and their proliferation rate (A), as measured over 24 hours by RTCA xCelligence impedance assay. Similarly, the addition of 400 nM of GSK2193874 (named "GSK219" in the figure, a specific TRPV4 antagonist) to siCCM2 ECs resulted in the restoration of the proliferation marker Ki67 (E) and proliferation rate (B). When siCCM2 ECs were treated with a combination of 800 nM or 400 nM GSK2193874 salts prepared as salts and 0.05 µM OB1 (a PIEZO1 inhibitor), aging-related β-galactosidase activity was also restored (F) (Reference: https: / / doi.org / 10.1038 / nn.4454; Wetzel et al., Nature Neuroscience volume 20, pages 209–218 (2017)).

[0082] CCM2 depletion of endothelial cell permeability barrier

[0083] refer to Figure 2CCM2 loss triggers a loss of endothelial junction permeability (Stockton et al., J Exp Med, 2010), which precedes ECM invasion and CCM damage formation, leading to hemorrhage. Barrier permeability was restored by targeting TRPV4 with specific siRNA, as demonstrated by impedance measurements of confluent cells using RTCA xCelligence (A). A similar restoration was observed with 800 nM GSK2193874 (B). Following CCM2 depletion, actin stress fibers form, pulling cells apart and weakening junctions. Staining of junctional proteins such as VE-cadherin showed that cell-cell junctions were thinner in CCM2-depleted cells (C, arrow E). Cortical tissue supporting the cell-cell junction architecture was restored by siRNA treatment via immunofluorescence staining (arrow, C). Cell-cell junction thickness was also restored by targeting TRPV4 with siRNA, as demonstrated by a significant reduction in the difference in cell-cell junction thickness compared to siCT conditions (D). Similarly, targeting TRPV4 alone with 0.2 µm GSK2193874 salt or in combination with 0.1 µm OB1 (a PIEZO1 inhibitor) restored the morphology of EC monolayers and the thickness of cell-cell junctions (arrow, E).

[0084] ECM degradation caused by endothelial cells depleted of CCM2

[0085] refer to Figure 3 Following CCM2 depletion, HUVECs acquire the ability to degrade the extracellular matrix (ECM) as a means of invading the ECM (Vannier et al., Angiogenesis, 2021). The ability of CCM2-depleted ECs to degrade ECM (such as gelatin (denatured collagen)) was inhibited by targeting TRPV4 with specific siRNA (A) or by using 800 nM GSK2193874 (B). Treatment with 800 nM GSK2193874 formulated as a salt provided the same effect, and better recovery was observed with a combination of 400 nM GSK2193874 salt and 100 µM tranilast salt (a TRPV2 antagonist) (C). This increased matrix degradation capacity leads to greater matrix invading ability in CCM2-depleted endothelial cells (Vannier et al., Angiogenesis, 2021). The combination of GSK2193874 and tranilast salt targeted TRPV4 and TRPV2, inhibiting the ability of CCM2-depleted ECs to invade 3D PEG gel (D).

[0086] Effect of TRPV4 antagonists on the response of HUVEC to GSK101 (TRPV4 agonist)

[0087] refer to Figure 4 In HUVECs, TRPV4 can be inhibited by a variety of drug inhibitors. After incubation with a TRPV4 inhibitor, activation of TRPV4 was reduced using the specific agonist GSK1016790A, consistent with the observed dose-dependent peak of intracellular calcium entry. The inhibitory efficacy of GSK2193874 (A), GSK2798745 (B), GSK205 (C), RN-9893 (D), RN-1734 (E), and HC067047 (F) was calculated. On the graph, the points represent the mean of technical replicates, and the curves represent nonlinear regressions for each biological replicate. Inhibitory effects are all in the micromolar range, even lower. The IC50 values ​​of the TRPV4 inhibitors obtained in HUVECs differed from those previously reported in vitro or in other cell types: GSK2193874 had an IC50 value of 25.9 nM instead of 40 nM (A), GSK2798745 had an IC50 value of 0.614 nM instead of 1.8 nM (B), GSK205 had an IC50 value of 0.66 nM instead of 4.19 nM (C), RN9893 had an IC50 value of 2.47 nM instead of 420 nM (D), RN1734 had an IC50 value of 32.2 nM instead of 2.3 nM (E), and HC067047 had an IC50 value of 284 nM instead of 48 nM (F).

Claims

1. A pharmaceutical composition for the prevention or treatment of cerebral cavernous malformation (CCM) comprising a transient receptor potential vanillin 4 (TRPV4) inhibitor.

2. The pharmaceutical composition for the prevention or treatment of CCM according to claim 1, wherein the TRPV4 inhibitor is selected from TRPV4 antagonists and TRPV4 RNA interference agents.

3. The pharmaceutical composition for the prevention or treatment of CCM according to claim 2, wherein the TRPV4 RNA interference agent is selected from the group consisting of TRPV4 siRNA, TRPV4 shRNA, microRNA, and TRPV4 aiRNA.

4. The pharmaceutical composition for the prevention or treatment of CCM according to claim 3, wherein the TRPV4 RNA interfering agent is TRPV4 siRNA.

5. The pharmaceutical composition for the prevention or treatment of CCM according to claim 2, wherein the TRPV4 antagonist is selected from the group consisting of GSK2193874, GSK2798745, HC 067047, RN-1734, GSK3395879, GSK3491943 or GSK3527497, RN-9893, PF-05214030, and GSK205.

6. The pharmaceutical composition for the prevention or treatment of CCM according to claim 5, wherein the TRPV4 antagonist is GSK2193874.

7. The pharmaceutical composition for the prevention or treatment of CCM according to any one of the preceding claims, further comprising a transient receptor potential vanillin 2 (TRPV2) inhibitor, a piezoelectric mechanosensitive ion channel component 1 (PIEZO 1) inhibitor and / or a piezoelectric mechanosensitive ion channel component 2 (PIEZO 2) inhibitor.

8. The pharmaceutical composition for the prevention or treatment of CCM according to claim 7, wherein the transient receptor potential vanillin 2 (TRPV2) inhibitor is selected from the group consisting of tranilast and SET2, the piezoelectric mechanosensitive ion channel component 1 (PIEZO 1) inhibitor is selected from the group consisting of OB-1 and GsMTX4, and the piezoelectric mechanosensitive ion channel component 2 (PIEZO 2) inhibitor is GsMTX4.

9. The pharmaceutical composition for the prevention or treatment of CCM according to claim 8, wherein the TRPV4 inhibitor is GSK2193874 and the PIEZO 1 inhibitor is OB-1.

10. The pharmaceutical composition for the prevention or treatment of CCM according to claim 8, wherein the TRPV4 inhibitor is GSK2193874 and the TRPV2 inhibitor is tranilast.

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