Novel treatments of glaucoma

Abstract

To provide an ophthalmic formulation for treating or preventing glaucoma or pathogenic intraocular pressure.SOLUTION: The ophthalmic formulation comprises a Wnt receptor inhibitor that specifically inhibits expression of a Wnt receptor selected from among frizzled-2 (FZD2), frizzled-5 (FZD5), and receptor tyrosine kinase like orphan receptor 1 (ROR1) through genetic manipulation.SELECTED DRAWING: None

Description

[Background technology]

[0001] This invention was made with United States Government support under Grant No. EY028995 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.

[0002] Introduction Glaucoma is a major health problem affecting over 3 million people in the United States and 60 million people worldwide. It is estimated that by 2040, 111.8 million people will suffer from glaucoma worldwide. One of the major risk factors for glaucoma is elevated intraocular pressure (IOP), which can damage the optic nerve and, if untreated, can lead to permanent blindness. Currently, there is no cure for glaucoma. Existing eye drops and oral medications have limited efficacy and are associated with numerous side effects. Additionally, surgery is often ineffective due to scarring and fibrosis.

[0003] Aqueous humor is a clear, colorless liquid that fills the anterior and posterior chambers of the eye. It is produced in the ciliary body in the posterior chamber and drains from the anterior chamber angle via the normal route through the trabecular meshwork and Schlemm's canal, as well as via an accessory route called the uveoscleral outflow. In a normal eye, a dynamic balance between the production and outflow of aqueous humor is maintained, keeping IOP within the normal range.

[0004] Schlemm's canal (SC) is a ring-shaped canal located at the iridocorneal angle of the anterior chamber of the eye. It is part of the normal aqueous humor outflow system, and in humans, 70-90% of the aqueous humor that leaves the eye passes through it. The endothelial lining of Schlemm's canal is one of the major sites of resistance to aqueous humor outflow and is a major determinant of IOP. Increased resistance in Schlemm's canal with age or under pathological conditions leads to elevated IOP and glaucoma with irreversible optic nerve damage and vision loss. Therefore, Schlemm's canal is an important target for glaucoma treatment. We recently demonstrated for the first time that Schlemm's canal expresses Prox-1, a gene that is a master regulator of lymphangiogenesis (Truong TN, Li H, Hong YK, Chen L. Novel characterization and live imaging of Schlemm's canal expressing Prox-1. PLoS One. 2014; 9(5): e98245).

[0005] Previously, we reported that Wnt5a is expressed in Schlemm's canal and that its expression level is regulated in response to changes in shear stress. Furthermore, we reported that inhibition of Wnt5a effectively lowered IOP in vivo. Wnt5a acts through multiple alternative signaling pathways depending on cell type, microenvironment, stimuli, etc. To identify other drug targets for glaucoma, we attempted to determine the downstream effectors using glaucoma-related models and confirm the druggability of the effectors in the treatment of glaucoma.

[0006] Here, we report that FZD2 (frizzled-2), FZD5, and ROR1 (receptor tyrosine kinase-like orphan receptor 1) are specifically expressed in SCs, and that their expression is regulated by changes in shear stress, stimulation or intervention with Wnt5a. For example, regulation of FZD2, FZD5, and ROR1 controls SC functions such as tube formation. In SC cells cultured under pressure (high shear stress), an increase in Wnt5a, Fzd2, Fzd5, and ROR1 was observed, and when Wnt5a was downregulated, Fzd2, Fzd5, and ROR1 were also downregulated, and when human SC cells were stimulated with Wnt5a, Fzd5 and ROR1 were increased accordingly.

[0007] Furthermore, we also disclose that calcium signaling system is involved in the function of SCs, and that several related molecules, such as PLCB1 (phospholipase C, β1), PPP3R1 (protein phosphatase 3 regulatory subunit B, α), NFATC3 (nuclear factor of activated T cells 3), and CAMK2D (calcium-calmodulin-dependent protein kinase II δ), are regulated by changes in shear stress, stimulation or intervention of Wnt5a. We disclose that such Wnt receptors (FZD2, FZD5, ROR1, etc.) and related molecules of calcium signaling pathway (PLCB1, PPP3R1, NFATC3, CAMK2D, etc.) regulate the function of SCs and are targets for treating glaucoma.

[0008] Although it is known that Wnt5a acts through multiple alternative signaling pathways depending on cell type, microenvironment, and stimuli, it has remained unclear whether its downstream effectors are operative in glaucoma-related models and, if so, which effectors could be targeted for drug discovery to control intraocular pressure. Summary of the Invention [Means for solving the problem]

[0009] The present invention provides methods and compositions for the localized treatment of glaucoma or pathological intraocular pressure.

[0010] In one aspect, the invention provides a method of treating glaucoma or pathological intraocular pressure, comprising administering to a human in need of such treatment an inhibitor of an ocular Wnt5a effector, wherein the effector is selected from FZD2 (frizzled-2), FZD5 (frizzled-5), and ROR1 (receptor tyrosine kinase-like orphan receptor 1); and PLCB1 (phospholipase C, beta 1), PPP3R1 (protein phosphatase 3 regulatory subunit B, alpha), NFATC3 (nuclear factor of activated T cells 3), and CAMK2D (calcium-calmodulin-dependent protein kinase II delta).

[0011] In some embodiments,

[0012] -the inhibitor inhibits expression of the effector by genetic manipulation, such as CRISPR gene editing or siRNA;

[0013] - the inhibitor directly inhibits the effector and is selected from an antibody, a small interfering peptide and a small molecule inhibitor;

[0014] - the administering step comprises administering the inhibitor topically to an eye in need thereof;

[0015] - the administering step comprises delivery by eye drops, or by intracameral, subconjunctival, or intravitreal injection;

[0016] - the administration is topical and the inhibitor is administered in the form of a topical ophthalmic gel, ointment, suspension or solution, or contact lens;

[0017] said inhibitor is a ROR1 inhibitor, e.g. selected from cirumutuzumab and KAN0439834, a FZD5 inhibitor, e.g. selected from the anti-FZD5 antibodies IgG-2919 and IgG-2921 (Steinhardt et al., Nat. Med. 2017; 23: 60-68), a FZD2 inhibitor, e.g. selected from the selective peptide dFz7-21 (Nile et al., Nat. Chem. Biol. 2018; 14: 582-590) and an anti-FZD2 antibody, or a siRNA as disclosed herein; and / or

[0018] - the method further comprises locally administering to the eye a second, different inhibitor that is an inhibitor of an ocular Wnt5a effector, or locally administering to the eye together with the inhibitor a second, different inhibitor.

[0019] In another aspect, the present invention provides an ophthalmic formulation of an inhibitor of an ocular Wnt5a effector in the form of a unit dosage form for treating glaucoma or pathological intraocular pressure, characterized in that the effector is selected from FZD2 (frizzled-2), FZD5 (frizzled-5), and ROR1 (receptor tyrosine kinase-like orphan receptor 1); and PLCB1 (phospholipase C, beta 1), PPP3R1 (protein phosphatase 3 regulatory subunit B, alpha), NFATC3 (nuclear factor of activated T cells 3), and CAMK2D (calcium-calmodulin-dependent protein kinase II delta).

[0020] In some embodiments,

[0021] -the inhibitor inhibits expression of the effector by genetic manipulation, such as CRISPR gene editing or siRNA;

[0022] - the inhibitor directly inhibits the effector and is selected from an antibody, a small interfering peptide and a small molecule inhibitor;

[0023] the formulation is in the form of a topical ophthalmic gel, ointment, suspension or solution,

[0024] - the dosage form is a contact lens, eye drop, depot or bolus containing the inhibitor;

[0025] - the formulation is packaged in an eye drop dispenser;

[0026] the formulation is packaged in a syringe for intracameral administration or injection, subconjunctival administration or injection, or intravitreal administration or injection,

[0027] - the formulation further comprises additives and properties suitable for direct topical delivery to the eye, the additives and properties being selected from the group consisting of ophthalmic transparency, pH buffering, isotonicity, viscosity, stability and sterility; and / or

[0028] said inhibitor is a ROR1 inhibitor, e.g. selected from cirumutuzumab and KAN0439834, a FZD5 inhibitor, e.g. selected from anti-FZD5 antibodies IgG-2919 and IgG-2921, a FZD2 inhibitor, e.g. selected from selective peptide dFz7-21 and an anti-FZD2 antibody, or a siRNA as disclosed herein.

[0029] The present invention includes all combinations of the individual embodiments described herein. The methods of the present invention may be practiced with any of the disclosed compositions, including specific embodiments. [Brief description of the drawings]

[0030] [Fig. 1A-1G](A-C) Real-time quantitative PCR data showing that the expression levels of FZD5, ROR1, and FZD2 in human Schlemm's canal cells are regulated by shear stress (A), Wnt5a intervention with small interfering RNA (siRNA) (B), or Wnt5a stimulation (C). (D and E) Summary of data showing that human Schlemm's canal cell tube formation is inhibited by FZD5 siRNA (D) or ROR1 siRNA (E). Scrambled siRNA was used as a negative control. *P<0.05. (F and G) Representative images showing that human Schlemm's canal cell tube formation is regulated by siRNA intervention of FZD5 (F) or ROR1 (G). *P<0.05.

[0031] [Figure 2A-2C] (A-C, top) Real-time quantitative PCR data showing that the expression levels of PLCB1, PPP3R1, and NFATC3 in human Schlemm's canal cells are regulated by shear stress (A), Wnt5a intervention with siRNA (B), or Wnt5a stimulation (C). (A-C, bottom) Representative images and data of Fluo-4 staining showing that intracellular calcium signals are regulated by shear stress (A), Wnt5a intervention with siRNA (B), or Wnt5a stimulation (C). Green: Fluo-4, blue: DAPI nuclear staining. *P<0.05.

[0032] [Figure 3A-3B] Real-time quantitative PCR data showing that expression of CAMK2D in human Schlemm's canal cells is regulated by shear stress (A) or wnt5a stimulation (B). *P<0.05.

[0033] [Figure 4A-4I]Data are summarized showing that human Schlemm's canal cell tube formation is inhibited in vitro by intervention with anti-ROR1 antibody (A), ROR1 small molecule inhibitor DB03208 (0.07% ethanol solution) (B), FZD2 siRNA (C), anti-FZD2 antibody (D), anti-FZD5 antibody (E), CAMK2D siRNA (F), PLCB1 siRNA (G), PPP3R1 siRNA (H), or NFATC3 siRNA (I). Isotype control, vehicle control ethanol (0.07%), or scrambled siRNA were used as negative controls in the antibody, small molecule inhibitor DB03208, and siRNA treatment assays, respectively. *P<0.05.

[0034] [Figure 5A-5B] (A) ROR1 small molecule inhibitor. (B) ROR1 antibody inhibitor.

[0035] [Figure 6A-6B] (A) FZD5 antibody inhibitor. (B) FZD2 antibody inhibitor. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] The examples and embodiments described herein are merely for the purpose of illustrating the present invention, and it is clear to those skilled in the art that various modifications or variations are possible based on these examples and embodiments, and such modifications or variations are included in the present invention. Those skilled in the art will understand that various non-critical parameters can be modified or varied to obtain essentially similar results. The present invention may not include or may be practiced in the absence of compounds, ingredients, elements, or steps that are not disclosed herein as essential components. In the following description and throughout the present specification, unless otherwise understood or otherwise stated, the terms "a" and "an" mean one or more. All publications, patents, and patent applications cited herein, as well as the references cited therein, are incorporated herein by reference in their entirety for all purposes.

[0037] The disclosed method of inhibiting Wnt5a receptor / effector can be genetic engineering and / or administration of small interfering RNA (siRNA), antibodies, small molecules, etc. Many of the small interfering RNA (siRNA), antibodies, small molecules, etc. are commercially available from suppliers such as Applied Biological Materials (ABM, Richmond, BC), Life Technologies (ThermoFisher Scientific), Sigma-Aldrich, etc. The above method can be used alone to reduce intraocular pressure and prevent or treat glaucoma, and / or can be used in combination with other therapies such as eye drops, medications, lasers, implanted devices and surgery to prevent or treat glaucoma.

[0038] Exemplary embodiment

[0039] Identification and targeting of Wnt5a

[0040] In WO2019 / 040311, we disclose the identification and targeting of Wnt5a.

[0041] Wnt5a has been expressed in cultured human primary SC cells and in mouse SCs in vivo. Wnt5a expression was regulated in response to changes in shear stress, as determined by quantitative real-time PCR assays. We also confirmed that Wnt5a-specific siRNA down-regulates Wnt5a expression in human SC cells and also affects SC cell function. Conditional knockout mice with SC-specific deletion of the Wnt5a gene significantly suppressed IOP elevation induced in a glaucoma model compared with their littermate control mice. There was no significant difference in baseline IOP between the knockout mice and their littermate control mice. While littermate control mice showed elevated IOP at all time points, Wnt5a knockout mice showed elevated IOP only at early time points (within 24 hours) and not at later time points. This suggests that Wnt5a intervention results in an unsustainable elevated IOP. We also confirmed that wnt5a intervention is effective in protecting the retinal nerve fiber layer and increasing SC permeability (a target for promoting water movement by the normal outflow system mentioned above) in controlling ocular hypertension (e.g., Tam et al., Scientific Reports 7:40717, DOI: 10.1038 / srep40717). These experiments indicate that Wnt5a is a valid therapeutic target for controlling glaucoma. Furthermore, such results are also shown by selective inhibition of Wnt5a by CRISPR gene editing using the method of Huang et al. (Nature Communications, 2017; 8 (1) DOI: 10.1038 / s41467-017-00140-3).

[0042] Next, we developed an experimental protocol to confirm the IOP-lowering effect of administering Wnt5a siRNA as a Wnt5a inhibitor. In carrying out this protocol, Wnt5a-specific siRNA was obtained commercially (human WNT5A siRNA, Life Technologies; Anastas et al., J. Clin. Investig. 2014, 124, 2877-2890). In one protocol, siRNA was injected subconjunctivally according to the method described in Yuen et al. (2014, Invest Ophthalmol Vis Sci. 2014;55:3320-3327). Mice administered with siRNA or control were randomly selected and injected subconjunctivally at a dose of 5 μL (0.2 lg / μL) twice a week for 2 weeks. In another protocol, siRNA was injected into the anterior chamber according to the method described in Tam et al. (2017, Scientific Reports 7, 40717). Mice were anesthetized with intraperitoneal injection and their pupils were dilated. A flat-tipped stretched glass microneedle was used to puncture the cornea to drain aqueous humor. Immediately after puncture, a flat-tipped stretched glass microneedle was attached to a 10 μL syringe and inserted into the puncture to inject 1.5 μL of PBS containing 1 μg of siRNA into the anterior chamber. The contralateral eye was similarly injected with 1.5 μL of PBS containing the same concentration of scrambled siRNA. These experiments confirmed that Wnt5a-specific siRNA delivered locally by subconjunctival or intracameral injection as a Wnt5a inhibitor was effective in treating pathological IOP.

[0043] To evaluate the effect of siRNA delivered as eye drops on IOP, we developed a further protocol following that of Martinez et al. (Mol Ther. 2014 Jan;22(1):81-91). New Zealand white rabbits were topically administered 20 nmol / day of siRNA or phosphate-buffered saline (PBS) for 4 consecutive days. Significant reduction in IOP was observed in siRNA-treated eyes compared to vehicle-treated eyes. The effect of siRNA on IOP was observed 2 days after the first administration, and IOP values ​​remained below baseline levels until about 2 days after the final administration. We also performed an oral water overload model in New Zealand white rabbits to evaluate the IOP-lowering effect of Wnt5a siRNA in pathological conditions such as those observed in glaucoma. First, four doses of siRNA (10 nmol, 20 nmol, 40 nmol, and 60 nmol / eye / day) were administered three times, 48 ​​hours, 24 hours, and 2 hours before induction of intraocular hypertension. Each administration was performed on both eyes, and IOP was measured before induction of intraocular hypertension and after oral overload, and after oral overload, it was measured every 20 minutes until 120 minutes later. Analysis of the results showed that Wnt5a siRNA significantly suppressed the increase in IOP at all doses administered.

[0044] To confirm the effect and specificity of Wnt5a siRNA on IOP, the number of animals in one group was increased and the dose was 40 nmol / eye / day for 4 consecutive days. On the 4th day of administration, ocular hypertension was induced by water loading. The control results confirmed that in animals administered with PBS, IOP increased by water loading during the first hour after induction of ocular hypertension. Analysis of the IOP values ​​at each measurement time point showed that administration of siRNA significantly reduced ΔIOP values ​​during the first hour compared to animals administered with PBS. This effect is specific, since administration of scrambled sequence siRNA had no effect on IOP.

[0045] Next, we developed an experimental protocol to confirm the IOP-lowering effect of administering a Wnt5a-specific antibody as a Wnt5a inhibitor. In this protocol, two types of antibodies are used: anti-human WNT5A antibody (buffered aqueous solution) obtained as rabbit purified immunoglobulin (Sigma-Aldrich, SAB1411396) and anti-human WNT5A monoclonal antibody (ascites) produced in mouse clone 6F2 (Sigma-Aldrich, SAB5300183). However, other Wnt5a antibodies, such as those described in Hanaki et al., Mol Cancer Ther 11(2) Feb 2012; and He et al., Oncogene. 2005, 24 (18): 3054-3058, can also be used. From the experiments performed using both the mouse and rabbit models, it was confirmed that the Wnt5a-specific antibody delivered locally in the form of eye drops as a Wnt5a inhibitor is effective in treating pathological IOP.

[0046] To evaluate the therapeutic effect of Wnt5a neutralizing antibody on IOP and other parameters of glaucoma, such as corneal edema, retinal ganglion cell (RGC) death, and RNFL (retinal nerve fiber layer) thinning, in an exemplary model system, high intraocular pressure was induced in the right eye (OD) of wild-type normal mice and Wnt5a neutralizing antibody was administered. The IOP in the right eye of each mouse was significantly increased in the control group, while the IOP in the eyes treated with Wnt5a antibody was significantly lower and maintained at the baseline level. In addition, central corneal thickness was measured in vivo using optical coherence tomography (OCT), and Wnt5a intervention suppressed corneal edema. After IOP was elevated, corneal thickness increased in the control group, but no increase in corneal thickness was observed in the eyes treated with Wnt5a antibody. In addition, Wnt5a intervention suppressed RGC death as well as RNFL thinning in the eyes treated with Wnt5a antibody. The inhibition of RGC death and RNFL thinning was confirmed by immunohistochemistry and OCT, respectively. These results confirmed that local intervention with Wnt5a antibody significantly reduced IOP and protected the cornea and retina in a mouse model of glaucoma.

[0047] Next, we designed an experimental protocol to confirm the IOP-lowering effect of administering Wnt5a-specific antagonist peptides and small molecule inhibitors. In this protocol, we used t-butyloxycarbonyl-modified Wnt5a-derived hexapeptide (Box5), which acts as a potent antagonist of Wnt5a (Jenei et al., PNAS USA, 106 (46), 19473-8), and 6,7-dihydro-10α-hydroxyradicicol, a potent inhibitor of WNT-5A expression with relatively low toxicity and good stability (Shinonaga et al., Bioorg Med Chem. 2009 Jul 1;17(13):4622-35). Again, the experiments performed in both the mouse and rabbit models above confirmed that the Wnt5a-specific modified peptides and small molecule inhibitors of Wnt5a expression, delivered locally in the form of eye drops as Wnt5a inhibitors, were effective in treating pathological IOP.

[0048] Identification and targeting of downstream effectors

[0049] Next, we confirmed that FZD5 (frizzled-5), FZD2, and ROR1 (receptor tyrosine kinase-like orphan receptor 1) are expressed in SCs, and that their expression is regulated by changes in shear stress, stimulation or intervention of Wnt5a. Furthermore, we confirmed that these modulations can control SC functions such as tube formation. Specifically, in SC cells cultured under pressure (high shear stress), increases in Wnt5a, Fzd5, Fzd2, and RoR1 were observed, and when Wnt5a was downregulated, Fzd5, Fzd2, and ROR1 were also downregulated, and when human SC cells were stimulated with Wnt5a, Fzd5, Fzd2, and ROR1 were increased accordingly. See Figure 1.

[0050] Furthermore, we confirmed that calcium signaling system is involved in the function of SCs, for example, several related molecules such as PLCB1 (phospholipase C, β1), PPP3R1 (protein phosphatase 3 regulatory subunit B, α), NFATC3 (nuclear factor of activated T cells 3), and CAMK2D (calcium-calmodulin-dependent protein kinase II δ) are regulated by changes in shear stress, stimulation or intervention of Wnt5a (see Figure 2 and Figure 3).

[0051] We disclose that these Wnt receptors (e.g., FZD5, FZD2, ROR1) and related molecules in the calcium signaling pathway (e.g., PLCB1, PPP3R1, NFATC3, CAMK2D) regulate SC function and are targets for treating glaucoma.

[0052] Next, we developed an experimental protocol to confirm the IOP-lowering effect of administering Wnt5a receptor siRNA as an inhibitor. In carrying out this protocol, siRNA specific to FZD5, FZD2 and ROR1 were obtained commercially (e.g., ThermoFisher Scientific). In one protocol, siRNA was injected subconjunctivally according to the method described by Yuen et al. (2014, Invest Ophthalmol Vis Sci. 2014;55:3320-3327). Mice were randomly selected to receive FZD5 siRNA, FZD2 siRNA, ROR1 siRNA or control, and each was injected subconjunctivally at a dose of 5 μL (0.2 lg / μL) twice a week for 2 weeks. In another protocol, FZD5 siRNA, FZD2 siRNA or ROR1 siRNA is injected intracamerally as described by Tam et al. (2017, Scientific Reports 7, 40717). Mice are anesthetized by intraperitoneal injection and the pupils are dilated. First, a flat-tipped stretched glass microneedle is used to puncture the cornea to drain the aqueous humor. Immediately after puncture, a flat-tipped stretched glass microneedle is attached to a 10 μL syringe and inserted into the puncture hole to inject 1.5 μL of PBS containing 1 μg of siRNA into the anterior chamber. The contralateral eye is similarly injected with 1.5 μL of PBS containing the same concentration of scrambled siRNA. These experiments confirmed that FZD5, FZD2 and ROR1 specific siRNAs delivered locally by subconjunctival or intracameral injection as inhibitors are effective in treating pathological IOP.

[0053] To evaluate the effect of siRNA delivered as eye drops on IOP, we developed a further protocol following the method of Martinez et al. (Mol Ther. 2014 Jan;22(1):81-91). New Zealand white rabbits were topically administered 20 nmol / day of FZD5 siRNA, FZD2 siRNA, ROR1 siRNA or phosphate buffered saline (PBS) for 4 consecutive days. Significant reduction in IOP was observed in siRNA-treated eyes compared to the vehicle-treated group. The effect of FZD5 siRNA, FZD2 siRNA and ROR1 siRNA on IOP could be confirmed at 2 days after the first administration, and IOP values ​​remained below baseline levels until about 2 days after the final administration. We also created an oral water overload model in New Zealand white rabbits to evaluate the IOP-lowering effect of FZD5 siRNA, FZD2 siRNA and ROR1 siRNA in pathological conditions such as those observed in glaucoma. First, four doses of siRNA (10 nmol, 20 nmol, 40 nmol, and 60 nmol / eye / day) were administered three times, 48 ​​hours, 24 hours, and 2 hours before induction of intraocular hypertension. Each administration was performed on both eyes, and IOP was measured before induction of intraocular hypertension and after oral overload, and after oral overload, it was measured every 20 minutes until 120 minutes later. Analysis of the results showed that FZD5 siRNA, FZD2 siRNA, and ROR1 siRNA significantly suppressed the increase in IOP at each dose administered.

[0054] To confirm the effect and specificity of FZD5 siRNA, FZD2 siRNA and ROR1 siRNA on IOP, the number of animals in each group was increased and the dose was 40 nmol / eye / day for 4 consecutive days. On the 4th day of administration, ocular hypertension was induced by water loading. The control results confirmed that in animals administered with PBS, IOP increased by water loading during the first hour after induction of ocular hypertension. Analysis of the IOP values ​​at each measurement time point showed that administration of FZD5 siRNA, FZD2 siRNA or ROR1 siRNA significantly reduced ΔIOP values ​​during the first hour compared to animals administered with PBS. This effect is specific, since administration of scrambled sequence siRNA had no effect on IOP.

[0055] Next, we developed an experimental protocol to confirm the IOP-lowering effect of administering specific antibodies against FZD5, FZD2, and ROR1 as inhibitors. In this protocol, OMP18R5 (humanized monoclonal antibody that binds to FZD5), Abcam ab52565 (monoclonal antibody that binds to FZD2), and cirumutuzumab (humanized IgG1 anti-ROR1 monoclonal antibody) are used, but polyclonal and monoclonal antibodies against human FZD5, FZD2 siRNA, and ROR1 are commercially available from multiple sources, e.g., ThermoFisher Scientific, Abcam, Sigma-Aldrich, etc. For human FZD5 antibodies, see also US9573998. Experiments performed in both mouse and rabbit models as described above confirmed that FZD5, FZD2 siRNA, and ROR1-specific antibodies, delivered locally in the form of eye drops as inhibitors, were effective in treating pathologic IOP.

[0056] To evaluate the therapeutic effects of neutralizing antibodies against FZD5, FZD2, or ROR1 on IOP and other parameters of glaucoma, such as corneal edema, retinal ganglion cell (RGC) death, and RNFL (retinal nerve fiber layer) thinning, in an exemplary model system, high intraocular pressure was induced in the right eye (OD) of wild-type normal mice, and neutralizing antibodies against FZD5, FZD2, or ROR1 were administered. The IOP of the right eye of each mouse was significantly increased in the control group, whereas the IOP of the eyes treated with FZD5, FZD2, or ROR1 antibodies was significantly lower. In addition, central corneal thickness was measured in vivo using optical coherence tomography (OCT), and interventions against FZD5, FZD2, or ROR1 suppressed corneal edema. After IOP was elevated, the corneal thickness increased in the control group, but not in the eyes treated with FZD5, FZD2, or ROR1 antibodies. In addition, in eyes treated with each antibody, intervention with FZD5, FZD2 or ROR1 suppressed RGC death as well as RNFL thinning. Suppression of RGC death and RNFL thinning could be confirmed by immunostaining and / or OCT, respectively. These results confirmed that local intervention with FZD5, FZD2 or ROR1 antibodies significantly reduced IOP and protected the cornea and retina in a mouse model of glaucoma.

[0057] Next, we designed an experimental protocol to confirm the IOP-lowering effect of administering antagonist peptides and small molecule inhibitors specific for FZD5, FZD2, and ROR1, respectively. In this protocol, we use FZD5 fragment mutants acting as potent antagonists (e.g., Liu et al., Hum Mol Genet. 2016 Apr 1; ​​25(7): 1382-1391) and oral ROR1 small molecule inhibitors (KAN0439834; Hojjat-Farsangi et al., Leukemia 32, p2291-2295, 2018). Again, experiments performed using both the above mouse and rabbit models confirmed that modified peptide and small molecule inhibitors specific for FZD5, FZD2, and ROR1, respectively, delivered locally in the form of eye drops, were effective in treating pathological IOP.

[0058] In vivo data in exemplary models were obtained using an established mouse model of glaucoma in which episcleral veins were occluded with a laser to induce glaucoma (Zhang L et al., Establishment and Characterization of an Acute Model of Ocular Hypertension by Laser-Induced Occlusion of Episcleral Veins. Invest Ophthalmol Vis Sci. 2017 Aug 1; 58(10): 3879-3886). Intraocular hypertension was induced in the right eye (OD) of the mice, and the left eye (OS) served as a control. In these examples, after induction of intraocular hypertension in the right eye of the mice, inhibitors or corresponding controls were administered topically to the right eye by subconjunctival injection daily from day 1. The left eye served as a control. Central corneal thickness and RNFL were measured in vivo using OCT on days 3 or 7. All in vitro data were collected from cultured human Schlemm cells.

[0059] ROR1 inhibitors

[0060] 1) Sirmutuzumab

[0061] 2) KAN0439834; See Hojjat-Farsang et al., Leukemia. 2018 Oct;32(10):2291-2295. doi: 10.1038 / s41375-018-0113-1; for related inhibitors see also US2018 / 0002329, which are incorporated herein by reference.

[0062] 3) ROR1 siRNA, Thermofisher

[0063] 4) ROR1 antibody, R&D Systems, Cat# AF2000

[0064] 5) ROR1 small molecule, DB03208, Medkoo Biosciences, Cat#: 564580 TIFF2025038190000001.tif48140

[0065] 6) ROR1 small molecule, strictinin; See Fultang N. et al., Strictinin, a novel ROR1-inhibitor, represses triple negative breast cancer survival and migration via modulation of PI3K / AKT / GSK3s activity. PLoS One. 2019 May 31;14(5):e0217789.

[0066] 7) ROR1 blocking peptide; See https: / / www.mybiosource.com / blocking-peptide / ror1 / 544396.

[0067] 8) ARI-1, identified as a ROR1 inhibitor; See Liu X. et al., Novel ROR1 inhibitor ARI-1 suppresses the development of non-small cell lung cancer. Cancer Lett. 2019 Aug 28;458:76-85.

[0068] 9) ROR1-cFab (anti-ROR1 chimeric Fab antibody); See Yin Z. et al., Antitumor activity of newly developed monoclonal antibody against ROR1 in ovarian cancer cells. Oncotarget. 2017 Oct 7;8(55):94210-94222.

[0069] FZD2 inhibitors

[0070] FZD2 siRNA, Thermofisher

[0071] FZD2 antibody, Abcam ab52565

[0072] FZD5 inhibitors

[0073] FZD5 siRNA, Thermofisher

[0074] FZD5 antibody, R&D Systems AF1617

[0075] CAMK2D inhibitors

[0076] CAMK2D siRNA, Thermofisher

[0077] PLCB1 inhibitors

[0078] PLCB1 siRNA, Thermofisher

[0079] PPP3R1 inhibitors

[0080] PPP3R1 siRNA, Thermofisher

[0081] NFATC3 inhibitors

[0082] 1) NFATC3 siRNA, Thermofisher

[0083] Recombinant anti-human antibodies and variants:

[0084] 2) Creative Biolabs Recombinant Anti-Human NFATC3 Antibody 10188

[0085] 3) Creative Biolabs Recombinant Anti-Human NFATC3 Antibody Fab Fragment 10189

[0086] 4) Creative Biolabs Recombinant Anti-Human NFATC3 Antibody scFv Fragment 10190

[0087] Human siRNA sequences [Table 1]

[0088] The present invention includes the following inventions. [1] A method of treating glaucoma or pathological intraocular pressure, comprising administering to a human in need of such treatment an inhibitor of an ocular Wnt5a effector, wherein the effector is selected from FZD2 (frizzled-2), FZD5 (frizzled-5), and ROR1 (receptor tyrosine kinase-like orphan receptor 1); and PLCB1 (phospholipase C, beta 1), PPP3R1 (protein phosphatase 3 regulatory subunit B, alpha), NFATC3 (nuclear factor of activated T cells 3), and CAMK2D (calcium-calmodulin-dependent protein kinase II delta). [2] The method according to [1], wherein the inhibitor inhibits expression of the effector by genetic manipulation such as CRISPR gene editing or siRNA. [3] The method according to [1], wherein the inhibitor directly inhibits the effector and is selected from an antibody, a small interfering peptide, and a small molecule inhibitor. [4] The method of any one of [1], [2] or [3], wherein the administering step comprises locally administering the inhibitor to an eye in need thereof. [5] The method of any one of [1], [2], [3], or [4], wherein the administering step comprises delivery by eye drops, or delivery by intracameral administration or injection, subconjunctival administration or injection, or intravitreal administration or injection. [6] The method of any one of [1], [2], [3], [4] or [5], wherein the administration is topical and the inhibitor is administered in the form of a topical ophthalmic gel, ointment, suspension or solution, or contact lens. [7] The method of any one of [1], [2], [3], [4], [5] or [6], wherein the inhibitor is a ROR1 inhibitor selected from, for example, cirumutuzumab and KAN0439834, an FZD5 inhibitor selected from, for example, anti-FZD5 antibodies IgG-2919 and IgG-2921, an FZD2 inhibitor selected from, for example, selective peptide dFz7-21 and an anti-FZD2 antibody, or an siRNA as disclosed herein. [8] The method of any one of [1], [2], [3], [4], [5], [6] or [7], further comprising locally administering to the eye a second, different inhibitor that is an inhibitor of an ocular Wnt5a effector, or locally administering to the eye together with the inhibitor, the second, different inhibitor. [9] An ophthalmic formulation of an inhibitor of an ocular Wnt5a effector in the form of a unit dosage form for treating glaucoma or pathological intraocular pressure, wherein the effector is selected from FZD2 (frizzled-2), FZD5 (frizzled-5), and ROR1 (receptor tyrosine kinase-like orphan receptor 1); and PLCB1 (phospholipase C, beta 1), PPP3R1 (protein phosphatase 3 regulatory subunit B, alpha), NFATC3 (nuclear factor of activated T cells 3), and CAMK2D (calcium-calmodulin-dependent protein kinase II delta).

[10] The formulation described in [9], wherein the inhibitor inhibits expression of the effector by genetic manipulation such as CRISPR gene editing or siRNA.

[11] The formulation described in [9], wherein the inhibitor directly inhibits the effector and is selected from an antibody, a small interfering peptide, and a small molecule inhibitor.

[12] The formulation according to [9],

[10] or

[11] above, which is in the form of an external ophthalmic gel, ointment, suspension or solution.

[13] The formulation described in [9],

[10] ,

[11] or

[12] above, wherein the dosage form is a contact lens, eye drop, depot or bolus formulation containing the inhibitor.

[14] The formulation according to [9],

[10] ,

[11] ,

[12] or

[13] , which is filled in an eye drop dispenser.

[15] The formulation according to [9],

[10] ,

[11] ,

[12] ,

[13] or

[14] , which is filled in a syringe for intracameral administration or injection, subconjunctival administration or injection, or intravitreal administration or injection.

[16] The formulation of [9],

[10] ,

[11] ,

[12] ,

[13] ,

[14] or

[15] , further comprising additives and properties suitable for direct topical delivery to the eye, the additives and properties being selected from the group consisting of ophthalmic transparency, pH buffering, isotonicity, viscosity, stability and sterility.

[17] The formulation of [9],

[10] ,

[11] ,

[12] ,

[13] ,

[14] ,

[15] or

[16] , wherein the inhibitor is a ROR1 inhibitor, e.g. selected from cirumutuzumab and KAN0439834, an FZD5 inhibitor, e.g. selected from anti-FZD5 antibodies IgG-2919 and IgG-2921, an FZD2 inhibitor, e.g. selected from selective peptide dFz7-21 and an anti-FZD2 antibody, or an siRNA as disclosed herein.

Claims

1. 1. An ophthalmic formulation for preventing or treating glaucoma or pathological intraocular hypertension comprising an inhibitor of a Wnt receptor that is an ocular Wnt5a effector in the form of a unitary dosage form, wherein the Wnt receptor is selected from frizzled-2 (FZD2), frizzled-5 (FZD5), and receptor tyrosine kinase-like orphan receptor 1 (ROR1), and wherein the inhibitor specifically inhibits expression of the Wnt receptor by genetic engineering.

2. 1. An ophthalmic formulation for preventing or treating glaucoma or pathological intraocular hypertension comprising an inhibitor of a Wnt receptor that is an ocular Wnt5a effector in the form of a unitary dosage form, characterized in that the Wnt receptor is selected from frizzled-2 (FZD2), frizzled-5 (FZD5), and receptor tyrosine kinase-like orphan receptor 1 (ROR1), and the inhibitor directly and specifically inhibits the Wnt receptor, and the inhibitor is a small interfering peptide or a small molecule inhibitor.

3. 3. The formulation of claim 1 or 2, in the form of a topical ophthalmic gel, ointment, suspension or solution.

4. 4. The formulation of claim 1, 2 or 3, wherein the dosage form is a contact lens, eye drop, depot or bolus containing the inhibitor.

5. 5. The formulation of claim 1, 2, 3 or 4, which is packaged in an eye drop dispenser.

6. 6. The formulation of claim 1, 2, 3, 4 or 5, which is packaged in a syringe for intracameral administration or injection, subconjunctival administration or injection, or intravitreal administration or injection.

7. 7. The formulation of claim 1, 2, 3, 4, 5 or 6, further comprising additives and properties suitable for direct topical delivery to the eye, said additives and properties being selected from ophthalmic transparency, pH buffering, isotonicity, viscosity, stability and sterility.

8. The formulation of claim 2, 3, 4, 5, 6 or 7, wherein the ROR1 inhibitor is KAN0439834 or the FZD2 inhibitor is the selective peptide dFz7-21.

9. The formulation of claim 1, 3, 4, 5, 6 or 7, wherein the genetic manipulation is a conditional knockout or CRISPR gene editing technology.

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