Modulation of wnt5a to treat glaucoma

Abstract

To provide compositions for locally treating glaucoma or pathogenic intraocular pressure.SOLUTION: The present invention provides an ophthalmic formulation that comprises a Wnt5a-specific inhibitor, is in a unit dosage form, and treats glaucoma. The Wnt5a-specific inhibitor is a small interfering peptide, a small molecule inhibitor, or a gene editing composition. Preferably, the small interfering peptide is t-butyloxycarbonyl-modified Wnt5a-derived hexapeptide (Box5), the small molecule inhibitor is 6,7-dihydro-10α-hydroxy radicicol, and the gene editing composition is a CRISPR gene editing composition.SELECTED DRAWING: None

Description

Background Art

[0001] The present invention was made with government support under Grant Numbers EY017392 and EY028995 awarded by the National Institutes of Health, USA. The US government has certain rights in this invention.

[0002] Introduction Glaucoma is one of the major health problems that affects over 3 million people in the United States and 60 million people worldwide. It is estimated that 118 million people will suffer from glaucoma worldwide by 2040. One of the major risk factors for glaucoma is elevated intraocular pressure (IOP). Elevated IOP can damage the optic nerve and, if left untreated, can lead to permanent blindness. Currently, there is no cure for glaucoma. Existing eye drops or oral medications have limited efficacy and many side effects. Surgery also often fails due to scarring and fibrosis.

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

[0004] The Schlemm's canal (SC) is an annular canal that exists at the iridocorneal angle of the anterior chamber of the eye. The Schlemm's canal forms part of the normal aqueous humor outflow system, and in humans, 70 - 90% of the aqueous humor flowing out of the eye passes through the Schlemm's canal. The inner layer consisting of endothelial cells of the Schlemm's canal is one of the main sites showing resistance to aqueous humor outflow and is a major determinant of intraocular pressure (IOP). With age or under pathological conditions, when the resistance of the Schlemm's canal increases, the IOP rises, leading to glaucoma accompanied by irreversible optic nerve damage and vision loss. Therefore, the Schlemm's canal is an important target for glaucoma treatment. We recently demonstrated for the first time that the Schlemm's canal expresses Prox-1, a major regulatory gene for 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] Wnt5a belongs to the Wnt family, which includes multiple ligands and multiple receptors identified in mammals.

[0006] Herein, we disclose that Wnt5a is expressed in the Schlemm's canal and its expression level is regulated in response to changes in shear stress.

[0007] Furthermore, we also disclose that it is possible to effectively lower the IOP in vivo by inhibiting Wnt5a. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0008] The present invention provides a method and a composition for locally treating glaucoma or causative intraocular pressure.

[0009] In one aspect, the present invention provides a method for treating glaucoma or pathogenic intraocular pressure, the method comprising the step of topically administering a Wnt5a inhibitor to an eye in need of treatment.

[0010] In some embodiments,

[0011] - the administration step includes delivery by eye drops, or delivery by intracameral injection, subconjunctival injection or intravitreal injection,

[0012] - the inhibitor is selected from antibodies, siRNAs, small interfering peptides and small molecule inhibitors,

[0013] - the inhibitor is delivered by a viral vector such as an adeno-associated virus (AAV) or a lentivirus, and / or

[0014] - the administration is topical, and the inhibitor is administered in the form of a topical ophthalmic gel, ointment, suspension or solution.

[0015] In another aspect, the present invention provides an ophthalmic formulation of a Wnt5a-specific inhibitor in the form of a unit dosage form for treating glaucoma or pathogenic intraocular pressure, wherein the Wnt5a-specific inhibitor is selected from antibodies, siRNAs, small interfering peptides and small molecule inhibitors.

[0016] In some embodiments,

[0017] - the formulation is in the form of a topical ophthalmic gel, ointment, suspension or solution such as an ophthalmic lubricant,

[0018] - the dosage form is a contact lens, eye drops, depot or bolus containing the inhibitor,

[0019] - the formulation is filled in an eye drop dispenser,

[0020] - The preparation is filled in a syringe for intracameral injection, subconjunctival injection or intravitreal injection, and / or

[0021] - The preparation further contains additives and / or properties suitable for delivery by direct topical application to the eye, and the additives and / or the properties are selected from the group consisting of transparency, pH buffering, isotonicity, viscosity, stability and sterility suitable for ophthalmic use.

[0022] The present invention encompasses all combinations of the individual embodiments described herein. The methods of the present invention may be practiced using any of the disclosed compositions, including particular embodiments.

Mode for Carrying Out the Invention

[0023] The examples and embodiments described herein are merely for illustrative purposes of the present invention, and it will be apparent to those skilled in the art that various modifications or changes can be made based on these examples and embodiments, and such modifications or changes are included in the present invention. Those skilled in the art will understand that for various non-essential parameters, similar results can be obtained even if they are changed or modified. The present invention may not include compounds, components, elements or processes that are not disclosed as essential components herein, and may be practiced in the absence of these. Throughout the following description and the entire specification, unless otherwise interpreted or stated otherwise, the terms "a" and "an" mean one or more. All publications, patents and patent applications cited herein, and the cited references described therein, are hereby incorporated by reference in their entirety for all purposes.

[0024] The method of inhibiting Wnt5a of the present disclosure can be genetic manipulation and / or administration of small interfering RNA (siRNA), antibody, small molecule, etc. Many of the small interfering RNA (siRNA), antibody, small molecule, etc. are commercially available from suppliers such as Applied Biological Materials (ABM, Richmond, British Columbia), Life Technologies (ThermoFisher Scientific), Sigma-Aldrich, etc. The above method may be used alone for the purpose of reducing intraocular pressure and preventing or treating glaucoma, and / or in combination with other therapies such as eye drops, medication, laser, implant devices and surgery for the purpose of preventing or treating glaucoma.

[0025] Typical examples

[0026] Wnt5a has been confirmed to be expressed in cultured human primary SC cells and also in mouse SCs in vivo. Analysis by quantitative real-time PCR assay has revealed that the expression of Wnt5a is regulated in response to changes in shear stress. In addition, we have confirmed that siRNA specific to Wnt5a has the effect of down-regulating Wnt5a expression in human SC cells and also affects the function of SC cells. In conditional knockout mice with specifically deleted Wnt5a gene in SCs, the increase in IOP induced in a glaucoma model is significantly suppressed compared to their littermate control mice. There was no significant difference in baseline IOP between these knockout mice and their littermate control mice. In the littermate control mice, an increase in IOP was observed at all measurement time points, but in the Wnt5a knockout mice, an increase in IOP was observed only at the initial time point (within 24 hours), and no increase in IOP was observed at subsequent time points. This suggests that Wnt5a intervention makes it impossible for the increase in IOP to persist. In addition, we have also confirmed that in the control of ocular hypertension, Wnt5a intervention is effective in protecting the retinal nerve fiber layer and increasing SC permeability (the target for promoting water movement by the normal outflow system described above) (for example, Tam et al., Scientific Reports 7:40717, DOI: 10.1038 / srep40717). These experiments indicate that Wnt5a is an effective therapeutic target for controlling glaucoma. Furthermore, such results are also shown by the selective inhibition of Wnt5a by CRISPR gene editing performed using the method of Huang et al. (Nature Communications, 2017; 8 (1) DOI: 10.1038 / s41467-017-00140-3).

[0027] Next, we developed an experimental protocol to confirm the IOP-lowering effect by administration of Wnt5a siRNA as a Wnt5a inhibitor. In carrying out this protocol, commercially available Wnt5a-specific siRNA (human WNT5A siRNA, Life Technologies; Anastas et al., J. Clin. Investig. 2014, 124, 2877-2890) was obtained. In one protocol, siRNA was subconjunctivally injected according to the method described by Yuen et al. (2014, Invest Ophthalmol Vis Sci. 2014;55:3320-3327). Mice to receive siRNA or control were randomly selected, and a dose of 5 μL (0.2 μg / μL) was subconjunctivally injected twice a week for 2 weeks. In another protocol, siRNA was intravitreally injected according to the method described by Tam et al. (2017, Scientific Reports 7, 40717). Mice were anesthetized by intraperitoneal injection to dilate the pupils. First, a flat-tipped pulled glass micro-needle was used to make a hole in the cornea to drain aqueous humor. Immediately after perforation, the flat-tipped pulled glass micro-needle was attached to a 10 μL syringe and inserted into the perforated hole, and 1.5 μL of PBS containing 1 μg of siRNA was injected into the anterior chamber. In the contralateral eye, 1.5 μL of PBS containing scrambled siRNA at the same concentration was similarly injected. From these experiments, it was confirmed that locally delivered Wnt5a-specific siRNA by subconjunctival injection or intravitreal injection as a Wnt5a inhibitor is effective for the treatment of pathogenic IOP.

[0028] To evaluate the effect of siRNA delivered as an eye drop on IOP, we developed an additional protocol according to the method of Martinez et al. (Mol Ther. 2014 Jan;22(1):81-91). New Zealand white rabbits are topically administered 20 nmol / day of siRNA or phosphate-buffered saline (PBS) for 4 consecutive days. In the eyes administered with siRNA, a significant decrease in IOP is observed compared to the vehicle-administered group. The effect of siRNA on IOP can be confirmed at the time point 2 days after the first administration, and IOP values below the baseline level are maintained until about 2 days after the final administration. In addition, to evaluate the IOP-lowering effect of Wnt5a siRNA in the pathological conditions observed in glaucoma, we created an oral water overload model in New Zealand white rabbits. First, 4 doses of siRNA (10 nmol, 20 nmol, 40 nmol, and 60 nmol / eye / day) are administered a total of 3 times, 48 hours before, 24 hours before, and 2 hours before inducing high intraocular pressure. All administrations are performed on both eyes, and IOP is measured before inducing high intraocular pressure and after oral overload, and the measurement after oral overload is performed every 20 minutes until 120 minutes later. Analysis of the results showed that Wnt5a siRNA significantly suppressed the increase in IOP at any dose administered.

[0029] To confirm the effect and specificity of Wnt5a siRNA on IOP, the number of animals in one group was increased and the dose was administered continuously for 4 days at 40 nmol / eye / day. On the 4th day of administration, high intraocular pressure is induced by water load. From the control results, it was confirmed that in the animals administered with PBS, IOP increased due to water load during the first hour after induction of high intraocular pressure. By comparing and analyzing the IOP values at each measurement time point, it was found that the administration of siRNA significantly decreased the ΔIOP value compared to the animals administered with PBS during the first hour. Since the administration of scrambled sequence siRNA had no effect on IOP, this effect is a specific effect.

[0030] Next, we developed an experimental protocol to confirm the IOP-lowering effect by administration of a Wnt5a-specific antibody as a Wnt5a inhibitor. In this protocol, two types of antibodies were used: an anti-human WNT5A antibody (buffer aqueous solution) obtained as rabbit purified immunoglobulin (Sigma-Aldrich SAB1411396) and an anti-human WNT5A monoclonal antibody (ascites) produced by mouse clone 6F2 (Sigma-Aldrich SAB5300183). 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 conducted using both the mouse and rabbit models described above, it was confirmed that Wnt5a-specific antibodies locally delivered in the form of eye drops as a Wnt5a inhibitor are effective in the treatment of pathogenic IOP.

[0031] To evaluate the therapeutic effects of Wnt5a-neutralizing antibodies on IOP and other parameters of glaucoma, such as corneal edema, retinal ganglion cell (RGC) death, and thinning of the RNFL (retinal nerve fiber layer), in an exemplary model system, ocular hypertension was induced in the right eye (OD) of wild-type normal mice, and Wnt5a-neutralizing antibodies were administered. In the control group, the IOP of the right eye of each mouse was significantly increased, but the IOP of the eye administered with the Wnt5a antibody was significantly lower than that and was maintained at the baseline level. Also, when the central corneal thickness was measured in vivo using an optical coherence tomography (OCT), corneal edema was suppressed by Wnt5a intervention. After the increase in IOP, the corneal thickness increased in the control group, but no increase in corneal thickness was observed in the eye administered with the Wnt5a antibody. Also, in the eye administered with the Wnt5a antibody, Wnt5a intervention suppressed both RGC death and RNFL thinning. The suppression of RGC death and RNFL thinning was confirmed by immunostaining and OCT, respectively. From these results, it was confirmed that in the glaucoma mouse model, topical Wnt5a antibody intervention significantly decreased IOP and protected the cornea and retina.

[0032] Next, we designed an experimental protocol to confirm the IOP-lowering effect by administration of a Wnt5a-specific antagonist peptide and a small molecule inhibitor. In this protocol, a t-butyloxycarbonyl-modified hexapeptide derived from Wnt5a (Box5) (Jenei et al., PNAS USA, 106 (46), 19473-8), which acts as a potent antagonist of Wnt5a, and 6,7-dihydro-10α-hydroxyjasmonico (Shinonaga et al., Bioorg Med Chem. 2009 Jul 1;17(13):4622-35), a potent inhibitor of WNT-5A expression with relatively low toxicity and excellent stability, were used. Also in this case, from experiments conducted using both of the above mouse and rabbit models, it was confirmed that a Wnt5a-specific modified peptide and a small molecule inhibitor of Wnt5a expression, locally delivered in the form of eye drops as Wnt5a inhibitors, are effective for the treatment of pathogenic IOP.

[0033] The present invention includes the following inventions. [1] A method for treating glaucoma or pathogenic intraocular pressure, the method comprising the step of locally administering a Wnt5a inhibitor to an eye in need of treatment. [2] The method according to [1], wherein the administration step includes delivery by eye drops, or delivery by intracameral injection, subconjunctival injection or intravitreal injection. [3] The method according to [1] or [2], wherein the inhibitor is selected from an antibody, siRNA, a small interfering peptide and a small molecule inhibitor. [4] The method according to [1], [2] or [3], wherein the administration is topical, and the inhibitor is administered in the form of a topical ophthalmic gel, ointment, suspension or solution. [5] An ophthalmic preparation of a Wnt5a-specific inhibitor in the form of a unit dosage form for treating glaucoma or pathogenic intraocular pressure, wherein the Wnt5a-specific inhibitor is selected from an antibody, siRNA, a small interfering peptide and a small molecule inhibitor. [6] The preparation according to [5], which is in the form of a topical ophthalmic gel, ointment, suspension or solution. [7] The pharmaceutical preparation according to [5] or [6], wherein the dosage form is a contact lens, eye drops, depot preparation or bolus preparation containing the inhibitor. [8] The pharmaceutical preparation according to [5], [6] or [7], which is filled in an eye drop dispenser. [9] The pharmaceutical preparation according to [5], [6], [7] or [8], which is filled in a syringe for intracameral injection, subconjunctival injection or intravitreal injection.

[10] The pharmaceutical preparation according to [5], [6], [7], [8] or [9], further comprising additives and properties suitable for delivery by direct topical application to the eye, the additives and the properties being selected from the group consisting of transparency, pH buffering, isotonicity, viscosity, stability and sterility suitable for ophthalmic use.

Claims

1. An ophthalmic preparation for treating glaucoma, which contains a Wnt5a-specific inhibitor and is in the form of a unit dosage form, wherein the Wnt5a-specific inhibitor is a small molecule inhibitor.

2. The ophthalmic preparation according to claim 1, wherein the small molecule inhibitor is 6,7-dihydro-10α-hydroxy radicicol.

3. The ophthalmic preparation according to claim 1 or 2, wherein the Wnt5a-specific inhibitor is delivered by a viral vector such as adeno-associated virus (AAV) or lentivirus.

4. The ophthalmic preparation according to claim 1, 2 or 3, which is in the form of a topical ophthalmic gel, ointment, suspension or solution.

5. The ophthalmic preparation according to claim 1, 2, 3 or 4, wherein the dosage form is a contact lens, eye drop, depot or bolus containing the inhibitor.

6. The ophthalmic preparation according to claim 1, 2, 3, 4 or 5, which is filled in an eye drop dispenser.

7. The ophthalmic preparation according to claim 1, 2, 3, 4, 5 or 6, which is filled in a syringe for intracameral injection, subconjunctival injection or intravitreal injection.

8. The ophthalmic preparation according to claim 1, 2, 3, 4, 5, 6 or 7, further comprising additives and properties suitable for direct topical delivery to the eye, and the additives and the properties are selected from the group consisting of transparency, pH buffering, isotonicity, viscosity, stability and sterility suitable for ophthalmology.