阅读说明：本技术 青光眼的新疗法 (Novel treatment for glaucoma ) 是由 陈璐 施萌 于 2020-01-29 设计创作，主要内容包括：通过向有其需要的眼睛局部给予Wnt5a受体抑制剂的制剂来治疗青光眼或致病性眼内压。(Glaucoma or pathogenic intraocular pressure is treated by topically administering to an eye in need thereof a formulation of a Wnt5a receptor inhibitor.)

1. A method of treating glaucoma or pathogenic intraocular pressure comprising administering to a human in need thereof an inhibitor of an ocular Wnt5a effector selected from the group consisting of:

FZD 2(frizzled-2), FZD5 (frizzled-5), and ROR1 (receptor tyrosine kinase-like orphan receptor 1); or

PLCB1 (phospholipase C, β 1), PPP3R1 (protein phosphatase 3 regulatory subunit B, α), NFATC3 (nuclear factor 3 of activated T) and CAMK2D (calcium/calmodulin-dependent protein kinase II δ).

2. The method of claim 1, wherein the inhibitor inhibits effector expression by gene manipulation, such as CRISPR gene editing or siRNA.

3. The method of claim 1, wherein the inhibitor directly inhibits the effector and is selected from the group consisting of an antibody, a small interfering peptide, and a small molecule inhibitor.

4. The method of claim 1, 2, or 3, wherein the administering step comprises topically administering the inhibitor to an eye in need thereof.

5. The method of claim 1, 2, 3, or 4, wherein the administering step comprises delivery by eye drops or by intracameral administration or injection, subconjunctival administration or injection, or intravitreal administration or injection.

6. The method of claim 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 claim 1, 2, 3, 4, 5 or 6, wherein the inhibitor is a ROR1 inhibitor, e.g. selected from the group consisting of cetuzumab and KAN0439834, or a FZD5 inhibitor, e.g. selected from the group consisting of anti-FZD 5 antibodies IgG-2919 and IgG-2921, or a FZD2 inhibitor, e.g. selected from the group consisting of dFz7-21, a selective peptide, or FZD2 antibody, or a siRNA, e.g. as disclosed herein.

8. The method of claim 1, 2, 3, 4, 5, 6, or 7 further comprising topically administering or coadministering to the eye a second, different inhibitor that is an inhibitor of an ocular Wnt5a effector.

9. An ophthalmic formulation of an inhibitor of an ocular Wnt5a effector for use in the treatment of glaucoma or pathogenic intraocular pressure, in unit dosage form, the effector being selected from the group consisting of:

FZD 2(frizzled-2), FZD5 (frizzled-5), and ROR1 (receptor tyrosine kinase-like orphan receptor 1); or

PLCB1 (phospholipase C, β 1), PPP3R1 (protein phosphatase 3 regulatory subunit B, α), NFATC3 (nuclear factor 3 of activated T) and CAMK2D (calcium/calmodulin-dependent protein kinase II δ).

10. The formulation of claim 9, wherein the inhibitor inhibits effector expression by gene manipulation, such as CRISPR gene editing or siRNA.

11. The formulation of claim 9, wherein the inhibitor directly inhibits the effector and is selected from the group consisting of an antibody, a small interfering peptide, and a small molecule inhibitor.

12. The formulation of claim 9, 10 or 11 in the form of a topical ophthalmic gel, ointment, suspension or solution.

13. The formulation of claim 9, 10, 11 or 12, wherein the dosage form is an inhibitor-loaded contact lens, eye drops, a depot, or a single bolus dosage form.

14. The formulation of claim 9, 10, 11, 12 or 13 packaged in an eye drop dispenser.

15. The formulation of claim 9, 10, 11, 12, 13, or 14 loaded in a syringe configured for intracameral, subconjunctival, or intravitreal administration or injection.

16. The formulation of claim 9, 10, 11, 12, 13, 14 or 15 further comprising excipients and features suitable for direct, topical delivery to the eye selected from the group consisting of ophthalmically suitable clarity, pH buffers, tonicity, viscosity, stability and sterility.

17. The formulation of claim 9, 10, 11, 12, 13, 14, 15 or 16, wherein the inhibitor is a ROR1 inhibitor, e.g. selected from the group consisting of cetuzumab and KAN0439834, or a FZD5 inhibitor, e.g. selected from the group consisting of anti-FZD 5 antibodies IgG-2919 and IgG-2921, or a FZD2 inhibitor, e.g. selected from the group consisting of dFz7-21, a selective peptide, or FZD2 antibody, or a siRNA, e.g. as disclosed herein.

Summary of The Invention

The present invention provides methods and compositions for the topical treatment of glaucoma or pathogenic intraocular pressure.

In one aspect, the invention provides a method of treating glaucoma or pathogenic intraocular pressure comprising administering to a human in need thereof an inhibitor of an ocular Wnt5a effector selected from the group consisting of: FZD 2(frizzled-2), FZD5 (frizzled-5), and ROR1 (receptor tyrosine kinase-like orphan receptor 1); or PLCB1 (phospholipase C, β 1), PPP3R1 (protein phosphatase 3 regulatory subunit B, α), NFATC3 (nuclear factor 3 of activated T cells), and CAMK2D (calcium/calmodulin-dependent protein kinase II δ).

In an embodiment:

inhibitors inhibit effector expression by gene manipulation, such as CRISPR gene editing or siRNA;

-said inhibitor directly inhibits said effector and is selected from the group consisting of an antibody, a small interfering peptide and a small molecule inhibitor;

-said administering step comprises topically administering said inhibitor to an eye in need thereof;

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

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

-the inhibitor is a ROR1 inhibitor, e.g. selected from the group consisting of cetuzumab (cirmtuzumab) and KAN0439834, or a FZD5 inhibitor, e.g. selected from the group consisting of anti-FZD 5 antibodies IgG-2919 and IgG-2921(Steinhardt et al, nat. med.2017; 23: 60-68), or and a FZD2 inhibitor, e.g. selected from the group consisting of dFz7-21, a selective peptide (Nile et al, nat. chem.biol.2018; 14: 582-590), or a FZD2 antibody, or a siRNA, e.g. as disclosed herein; and/or

-said method further comprises administering locally or coadministering to the eye a second, different inhibitor which is an inhibitor of the ocular Wnt5a effector.

In another aspect, the invention provides an ophthalmic formulation of an inhibitor of the ocular Wnt5a effector for use in the treatment of glaucoma or pathogenic intraocular pressure, in unit dosage form, the effector being selected from the group consisting of: FZD 2(frizzled-2), FZD5 (frizzled-5), and ROR1 (receptor tyrosine kinase-like orphan receptor 1); or PLCB1 (phospholipase C, β 1), PPP3R1 (protein phosphatase 3 regulatory subunit B, α), NFATC3 (nuclear factor 3 of activated T cells), and CAMK2D (calcium/calmodulin-dependent protein kinase II δ).

In an embodiment:

-the inhibitor inhibits effector expression by gene manipulation, such as CRISPR gene editing or siRNA;

-said inhibitor directly inhibits said effector and is selected from the group consisting of an antibody, a small interfering peptide and a small molecule inhibitor;

-the formulation is in the form of a topical ophthalmic gel, ointment, suspension or solution;

-the dosage form is a contact lens loaded with inhibitor, eye drops, depot or single bolus dosage form;

-the formulation is packaged in an eye drop dispenser;

-the formulation is loaded in a syringe configured for intracameral, subconjunctival, or intravitreal administration or injection;

-the formulation further comprises excipients and features suitable for direct, topical delivery to the eye selected from the group consisting of ophthalmically suitable clarity, pH buffering agents, tonicity, viscosity, stability and sterility; and/or

-said inhibitor is a ROR1 inhibitor, e.g. selected from the group consisting of cetuzumab and KAN0439834, or a FZD5 inhibitor, e.g. selected from the group consisting of anti-FZD 5 antibodies IgG-2919 and IgG-2921, or an FZD2 inhibitor, e.g. selected from the group consisting of dFz7-21, a selective peptide, or an FZD2 antibody, or an siRNA, e.g. as disclosed herein.

The present invention includes all combinations of the specific embodiments described herein. The methods may be practiced with all disclosed compositions, including the specific embodiments.

Brief Description of Drawings

Figures 1A-G. (a-C) real-time quantitative PCR data showing that expression levels of FZD5, ROR1, and FZD2 in human schlemm's canal cells are modulated by shear stress (a), Wnt5a intervention via small interfering rna (sirna) (B), or Wnt5a stimulation (C). (D and E) summarized data showing that tube formation of human Schlemm's canal cells was inhibited by FZD5 siRNA (D) or ROR1 siRNA (E), respectively. Scrambled siRNA was used as negative control. P < 0.05. (F and G) representative images showing that tube formation of human Schlemm's canal cells is regulated by the intervention of FZD5(F) or ROR1(G) via siRNA. P < 0.05.

Fig. 2A-C (a-C, top panel) real-time quantitative PCR data showing that expression levels of PLCB1, PPP3R1, and NFATC3 in human schlemm's canal cells are modulated by shear stress (a), Wnt5a intervention via siRNA (B), or Wnt5a stimulation (C). (A-C, lower panel) representative images of Fluo-4 staining and summarized data showing that intracellular calcium signaling is modulated by shear stress (A), intervention of Wnt5a via siRNA (B), or stimulation of Wnt5a (C). Green: fluo-4, blue: DAPI nuclear staining. P < 0.05.

Fig. 3A-B real-time quantitative PCR data showing that CAMK2D expression in human schlemm's canal cells is regulated by shear stress (a) or wnt5a stimulation (B), respectively. P < 0.05.

Fig. 4A-i. summarized data showing that tube formation of human schlemm's tube cells was inhibited in vitro by intervention of anti-ROR 1 antibody (a), ROR1 small molecule inhibitor, 0.07% ethanol solution of DB03208 (B), FZD2 sirna (c), anti-FZD 2 antibody (D), anti-FZD 5 antibody (E), CAMK2D sirna (f), PLCB1 sirna (g), PPP3R1 sirna (h), or NFATC3 sirna (i), respectively. Isotype control, ethanol vehicle control (0.07%), or scrambled siRNA served as negative controls for antibody, DB03208 small molecule inhibitor, and siRNA treatment analysis, respectively. P < 0.05.

Fig. 5A-B, (a) ROR1 small molecule inhibitors. (B) ROR1 antibody inhibitors.

Figure 6A-b. (a) FZD5 antibody inhibitors. (B) An FZD2 antibody inhibitor.

Description of specific embodiments of the invention

The examples and embodiments described herein are for illustrative purposes and various modifications or changes in light thereof will be apparent to those skilled in the art and are to be included herein. Those skilled in the art will recognize that various non-critical parameters may be changed or modified to produce substantially similar results. The invention can be practiced without, or in the absence of, any compound, component, element, or step not disclosed or claimed herein. Unless indicated to the contrary or otherwise indicated, the terms "a" and "an" mean one or more in these descriptions and throughout the specification. All publications, patents, and patent applications cited herein, including citations thereof, are hereby incorporated by reference in their entirety for all purposes.

The disclosed Wnt5a receptor/effector inhibition methods may be gene manipulation and/or administration of small interfering rnas (sirnas), antibodies, small molecules, and the like, many of which are commercially available from sources such as Applied Biological Materials Inc (ABM, Richmond BC), Life Technologies (ThermoFisher Scientific), Sigma-Aldrich, and the like. The method can be used alone to reduce intraocular pressure and prevent or treat glaucoma, and/or in combination with other treatments, such as eye drops, drugs, lasers, implant devices, surgery, and the like, to prevent or treat glaucoma.

Exemplary embodiments

Wnt5a determination and targeting

We disclose the determination and targeting of Wnt5a in WO 2019/040311.

Wnt5a was expressed on human primary SC cells in culture and in vivo on mouse SCs. Wnt5a expression was modulated as a function of shear stress as analyzed by quantitative real-time PCR analysis. We also demonstrated that Wnt5 a-specific sirnas can down-regulate Wnt5a expression in human SC cells, which also affects SC cell function. SC-specific Wnt5a gene conditional knockout mice induced in the glaucoma model had significantly reduced IOP elevation compared to control littermates. No significant difference in baseline IOP was found between the knockout mice and the control littermates. Compared to control littermates with IOP elevation at all time points of the study, Wnt5a knockout mice showed IOP elevation only at the early stage (within 24 hours), and not at the later time points, indicating that IOP elevation under Wnt5a intervention is not sustainable. We have also demonstrated that wnt5a intervention is an effective target for protecting the retinal nerve fiber layer and increasing SC permeability, which is a target for enhancing the management of intraocular hypertension through aqueous humor movement of conventional outflow systems (e.g., Tam et al, Scientific Reports 7:40717, DOI:10.1038/srep 40717). These experiments indicate that Wnt5a is an effective therapeutic target for glaucoma management. These results were further demonstrated by selective inhibition of Wnt5a by CRISPR gene editing using the method of Huang et al (Nature Communications, 2017; 8(1) DOI:10.1038/s 41467-017-00140-3).

Next, we developed experimental protocols to demonstrate the efficacy of Wnt5a siRNA inhibitor treatment to reduce IOP. For these protocols, Wnt5 a-specific siRNAs were commercially available (human WNT5A siRNA, Life Technologies; Anastas et al, J.Clin.Investig.2014,124, 2877-2890). In one protocol, subconjunctival injection of siRNA was performed as described by Yuen et al (2014, Invest Ophthalmol Vis Sci.2014; 55: 3320-. Randomly selected mice received subconjunctival injections of 5uL (0.2lg/uL) siRNA or control twice weekly for 2 weeks. In a second protocol, intracameral injection of siRNA was performed as described by Tam et al (2017, Scientific Reports 7,40717). Mice were anesthetized by intraperitoneal injection and the pupils were dilated. The cornea is first punctured with a blunt, blunt micro-glass needle that is pulled to withdraw the aqueous humor. Immediately after puncture, a trailing blunt micro-glass needle connected to a 10 μ l syringe was inserted through the puncture and 1.5 μ l of PBS containing 1 μ g siRNA was administered into the anterior chamber. The contralateral eye received 1.5 μ l of the same injection containing the same concentration of scrambled siRNA. These experiments demonstrate that siRNA, a Wnt5 a-specific inhibitor, delivered locally by subconjunctival or intracameral injection, is an effective therapy for pathogenic IOP.

To evaluate the effect of siRNA delivered by eye drops on IOP, we developed an additional protocol based on the method of Martinez et al (Mol ther. 2014jan; 22(l):81-91) in which new zealand white rabbits received a local administration of 20 nmol/day siRNA or Phosphate Buffered Saline (PBS) over a continuous period of 4 days. The treated eyes showed significant IOP reduction compared to the vehicle-treated group. The effect of siRNA on IOP was detectable 2 days after the first administration, and its value remained below basal levels until about 2 days after the last administration. We also adjusted the oral water overload model of new zealand white rabbits to evaluate the effect of Wnt5a siRNA in lowering IOP in pathological conditions such as those observed in glaucoma. Four different doses of siRNA (10nmol, 20nmol, 40nmol and 60 nmol/eye/day) were administered initially, three times: 48, 24 and 2 hours before induction of high pressure. All treatments were performed in both eyes and IOP was measured every 20 minutes to up to 120 minutes before induction of high pressure and after oral overload. Analysis of the results showed that Wnt5a siRNA provided significant protection against IOP elevation at all doses tested.

To confirm the efficacy and specificity of Wnt5a siRNA on IOP, a larger group of animals was treated at a dose of 40 nmol/eye/day over a continuous 4 day period; on the fourth day, intraocular hypertension was induced by water loading. Control results demonstrate that water loading results in elevated IOP during the first hour after induction of high pressure in animals treated with PBS. The analysis by comparison of IOP values at each time point showed that treatment with siRNA significantly reduced the Δ Γ Ρ value within the first hour compared to PBS treated animals. Since treatment with scrambled siRNA had no effect on IOP, the effect was specific.

We next developed an experimental protocol to demonstrate the efficacy of Wnt5 a-specific antibody inhibitors in the treatment of lowering IOP. These protocols use two different antibodies: anti-human WNT5A antibody (Sigma-Aldrich SAB 1411396) produced in rabbit purified immunoglobulin buffered water and anti-human WNT5A monoclonal antibody (Sigma-Aldrich SAB5300183) produced in ascites of mouse clone 6F2, but other WNT5a antibodies may also be used, for example Hanaki et al, Mol Cancer Ther 11(2) Feb 2012; he et al, oncogene.2005,24(18), 3054-3058. These experiments use mouse and rabbit models (supra) to demonstrate that Wnt5 a-specific antibody inhibitors delivered locally by eye drops are an effective therapy for pathogenic IOP.

In an exemplary model system, intraocular hypertension was induced in the right eye (OD) of wild-type normal mice, and Wnt5a neutralizing antibodies were administered to evaluate its therapeutic effect on IOP and other parameters of glaucoma, including corneal edema, Retinal Ganglion Cell (RGC) death, and RNFL thinning. IOP was significantly lower and maintained at baseline levels in the Wnt5a antibody-treated eyes compared to the control group with significant IOP elevation in the right eye of mice. Wnt5a intervention reduced corneal edema as measured by central corneal thickness in vivo by OCT. After IOP elevation, increased corneal thickness was observed in the control group, but not in the Wnt5a antibody-treated eyes. Wnt5a intervention reduced RGC death and RNFL thinning in the treated eyes. These were detected by immunostaining and OCT, respectively. These results demonstrate that in the mouse model of glaucoma, topical Wnt5a antibody intervention significantly lowers IOP and protects the cornea and retina.

We next devised an experimental protocol to demonstrate the efficacy of Wnt5 a-specific antagonist peptide and small molecule inhibitor treatment to reduce IOP. These protocols employ 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-10 α -hydroxyradicicol, a potent Wnt5A expression inhibitor with relatively low toxicity and excellent stability (Shinonaga et al, Bioorg Med chem.2009jul; 17(13): 4622-35). These experiments again demonstrated, using both mouse and rabbit models (supra), that Wnt5 a-specific modified peptide inhibitors and small molecule inhibitors of Wnt5a expression delivered locally by eye drops are effective therapies for pathogenic IOP.

Downstream effector determination and targeting

We next demonstrated that FZD5 (frizzled-5), FZD2, and ROR1 (receptor tyrosine kinase-like orphan receptor 1) are expressed on SCs, whose expression is modulated in response to shear stress changes, Wnt5a stimulation, or intervention. In addition, its modulation may modulate SC function, such as tube formation. In particular, we observed increases in Wnt5a and Fzd5, Fzd2, RoR1 in SC cells cultured under stress (or increased shear stress), Fzd5, Fzd2 and RoR1 were also down-regulated when Wnt5a was down-regulated, and Fzd5, Fzd2 and RoR1 were correspondingly increased when we stimulated human SC cells with Wnt5 a. See fig. 1.

Furthermore, we demonstrate that calcium signaling is involved in SC function, and several related molecules, such as PLCB1 (phospholipase C, β 1), PPP3R1 (protein phosphatase 3 regulatory subunit B, α), NFATC3 (activated T nuclear factor 3), CAMK2D (calcium/calmodulin-dependent protein kinase II δ) are regulated in response to shear stress changes, Wnt5a stimulation or intervention. See fig. 2 and 3.

We disclose that these Wnt receptors (i.e., FZD5, FZD2, ROR1) and related molecules of the calcium signaling pathway (i.e., PLCB1, PPP3R1, NFATC3, CAMK2D) provide targets for modulating SC function and treating glaucoma.

Next, we developed experimental protocols to demonstrate the efficacy of Wnt5a receptor siRNA inhibitor treatment to lower IOP. For these protocols, FZD5, FZD2, and ROR1 specific sirnas (e.g., ThermoFisher Scientific) are commercially available. In one protocol, subconjunctival injection of siRNA was performed as described by Yuen et al (2014, Invest Ophthalmol Vis Sci.2014; 55: 3320-. Randomly selected mice received subconjunctival injections of 5uL (0.2lg/uL) FZD5 siRNA, FZD2 siRNA or ROR1 siRNA or control, twice weekly for 2 weeks. In a second protocol, an intracameral injection of FZD5 siRNA, FZD2 siRNA or ROR1 siRNA was performed as described by Tam et al (2017, Scientific Reports 7,40717). Mice were anesthetized by intraperitoneal injection and the pupils were dilated. The cornea is first punctured with a blunt, blunt micro-glass needle that is pulled to withdraw the aqueous humor. Immediately after puncture, a trailing blunt micro-glass needle connected to a 10 μ l syringe was inserted through the puncture and 1.5 μ l of PBS containing 1 μ g siRNA was administered into the anterior chamber. The contralateral eye received 1.5 μ l of the same injection containing the same concentration of scrambled siRNA. These experiments demonstrate that siRNA, specific inhibitors of FZD5, FZD2, and ROR1 delivered locally by subconjunctival injection or intracameral injection, are effective therapies for pathogenic IOP.

To evaluate the effect of siRNA delivered by eye drops on IOP, we developed an additional protocol based on the method of Martinez et al (Mol ther. 2014jan; 22(1):81-91) in which new zealand white rabbits received a local administration of 20 nmol/day FZD5 siRNA or FZD2 siRNA or ROR1 siRNA or Phosphate Buffered Saline (PBS) over a continuous period of 4 days. The treated eyes showed significant IOP reduction compared to the vehicle-treated group. The effects of FZD5 siRNA, FZD2 siRNA and ROR1 siRNA on IOP were detectable 2 days after the first administration, and their values remained below basal levels until about 2 days after the last administration. We also adjusted the oral water overload model of new zealand white rabbits to evaluate the effect of FZD5 siRNA, FZD2 siRNA and ROR1 siRNA in lowering IOP in pathological conditions such as those observed in glaucoma. Four different doses of each siRNA (10nmol, 20nmol, 40nmol and 60 nmol/eye/day) were initially administered, three times: 48, 24 and 2 hours before induction of high pressure. All treatments were performed in both eyes and IOP was measured every 20 minutes to up to 120 minutes before induction of high pressure and after oral overload. Analysis of the results showed that FZD5 siRNA, FZD2 siRNA and ROR1 siRNA provided significant protection against IOP elevation at all doses tested.

To confirm the efficacy and specificity of FZD5 siRNA, FZD2 siRNA and ROR1 siRNA on IOP, a larger group of animals was treated at a dose of 40 nmol/eye/day over a continuous 4 day period; on the fourth day, intraocular hypertension was induced by water load. Control results show that water load leads to elevated IOP during the first hour after induction of high pressure in animals treated with PBS. Analysis by comparing IOP values at each time point showed that treatment with FZD5 siRNA, FZD2 siRNA or ROR1 siRNA significantly reduced Δ IOP values within the first hour compared to PBS treated animals. The effect is specific, as treatment with scrambled siRNA had no effect on IOP.

We next developed experimental protocols to demonstrate the efficacy of FZD5, FZD2 and ROR1 specific antibody inhibitors in the treatment of lowering IOP. These protocols use OMP18R5 (a humanized monoclonal antibody that binds FZD 5), Abcam ab52565 (a monoclonal antibody that binds FZD 2) and cetuzumab (a humanized IgG1 anti-ROR 1 monoclonal antibody), but anti-human FZD5, FZD2 siRNA and ROR1 polyclonal and monoclonal antibodies are commercially available from a variety of sources, e.g., ThermoFisher Scientific, Abcam, sigmaldrich, etc.); for antibodies against human FZD5 see also US 9573998. These experiments use mouse and rabbit models (supra) to demonstrate that FZD5, FZD2 siRNA and ROR1 specific antibody inhibitors delivered locally by eye drops are effective therapies for pathogenic IOP.

In an exemplary model system, intraocular hypertension was induced in the right eye (OD) of wild-type normal mice, and FZD5, FZD2, or ROR1 neutralizing antibodies were administered to evaluate their therapeutic effect on IOP and other parameters of glaucoma, including corneal edema, Retinal Ganglion Cell (RGC) death, and RNFL thinning. IOP was significantly lower in the FZD5, FZD2, and ROR1 antibody-treated eyes compared to the control group with significantly elevated IOP in the right eye of mice. FZD5, FZD2, and ROR1 intervene to reduce corneal edema as measured by central corneal thickness in vivo by OCT. After the increase in IOP, an increase in corneal thickness was observed in the control group, but not in the eyes treated with FZD5, FZD2, and ROR1 antibodies. FZD5, FZD2, and ROR1 interventions reduced RGC death and RNFL thinning in treated eyes. These can be detected by immunostaining and/or OCT, respectively. These results demonstrate that local FZD5, FZD2, and ROR1 antibody intervention significantly lowers IOP and protects cornea and retina in a mouse model of glaucoma.

We next designed experimental protocols to demonstrate the efficacy of FZD5, FZD2, and ROR 1-specific antagonist peptides and small molecule inhibitor treatments to reduce IOP. These protocols employ a mutated FZD5 fragment that acts as a potent antagonist (e.g., Liu et al, Hum Mol Genet.2016Apr 1; 25(7): 1382-1391), and an oral small molecule inhibitor of ROR1 (KAN 0439834; Hojjat-Farsangi et al, Leukemia 32, p 2291-2295,2018). These experiments again demonstrated, using both mouse and rabbit models (supra), that FZD5, FZD2, and ROR 1-specific modified peptide inhibitors and small molecule inhibitors delivered locally by eye drops are effective therapies for pathogenic IOP.

In an exemplary Model, in vivo data was obtained using a well established mouse Model of glaucoma with Laser-Induced Episcleral vein Occlusion (Zhang L, et al, assessment and Characterization of an ace Model of Ocular Hypertension by Laser-Induced Occuplusion of Episcleral vessels. invest Ophthalmol Viss Sci.2017aug 1; 58(10): 3879) 3886). Intraocular hypertension was induced in the right eye (OD) of mice, and the left eye (OS) served as a control. In these examples, the inhibitor or its control was delivered to the right eye daily by local administration of subconjunctival injection starting on day 1 after induction of intraocular hypertension in the right eye of the mice. The left eye was used as a control. Central corneal thickness and RNFL were measured by in vivo OCT on day 3 or day 7, respectively. All in vitro data were collected from human schlemm's cells in culture.

ROR1 inhibitors

1) Cetuzumab ozogamicin

2) KAN 0439834; Hojjat-Farsang et al, Leukemia.2018Oct; 32(10) 2291-2295.doi 10.1038/s 41375-018-0113-1; see also the relevant inhibitor classes: US2018/0002329, incorporated herein by reference.

3)ROR1 siRNAs，Thermofisher

4) ROR1 antibody, R & D Systems, Cat No. AF2000

5) ROR1 small molecule, DB03208, Medkoo Biosciences, inc. catalog No.: 564580

6) ROR1 small molecule, strictinin; see: 10, N. et al, striptin, a novel ROR1-inhibitor, a pressurized tertiary culture subset and differentiation modification of PI3K/AKT/GSK3 β activity, PLoS one.2019, 5, 31 days; 14(5) e 0217789.

7) ROR1 blocking peptides; see: https:// www.mybiosource.com/blocking-peptide/ror 1/544396.

8) ARI-1, defined as ROR1 inhibitor; see: liu X, et al Novel ROR1 inhibitor ARI-1 substitutions of the degree of non-small cell Long cancer, cancer Lett.2019, 8/28 days; 458:76-85.

9) ROR1-cFab (chimeric anti-ROR 1 Fab antibody); see: yin z. et al, anticancer activity of new degraded monoclonal antibody against ROR1 in viral cancer cells, oncotarget.2017, 10 months and 7 days; 8(55):94210-94222).

FZD2 inhibitor

FZD2 siRNA，Thermofisher

FZD2 antibody, Abcam ab52565

FZD5 inhibitor

FZD5 siRNA，Thermofisher

FZD5 antibody, R & D Systems AF1617

CAMK2D inhibitors

CAMK2D siRNA，Thermofisher

PLCB1 inhibitors

PLCB1 siRNA，Thermofisher

PPP3R1 inhibitors

PPP3R1 siRNA，Thermofisher

NFATC3 inhibitors

1)NFATC3 siRNA，Thermofisher

Recombinant anti-human antibodies and variants:

2) creativeliolabs recombinant anti-human NFATC3 antibody 10188

3) Creativeliolabs recombinant anti-human NFATC3 antibody Fab fragment 10189

4) Creativeliolabs recombinant anti-human NFATC3 antibody scFv fragment 10190

Human siRNA sequence

Name of ThermoFisher Scientific siRNA gene