EA044938B1 - COMPOSITIONS FOR TREATMENT OF EYE DISEASES - Google Patents

Description

Related applications

This application claims benefit from U.S. Provisional Patent Application Nos. 62/971002 and 63/010212, filed February 6, 2020 and April 15, 2020, respectively; the entire contents of which are incorporated herein by reference for all purposes.

Field of technology

The present invention relates to the field of medicine and the treatment of eye diseases. More specifically, the present invention relates to the use of certain compositions containing crystalline forms of edonentane for the treatment or improvement of glaucoma, diabetic retinopathy (DR), retinal vein occlusion (RVO) and retinopathy of prematurity (ROP).

BACKGROUND OF THE INVENTION

Eye diseases have a huge impact on a person's quality of life and yet still have no effective treatment. In the United States, it is estimated that vision loss, eye diseases, and visual impairment are associated with an annual economic burden of more than $100 billion. Examples of debilitating eye diseases include glaucoma, diabetic retinopathy (DR), retinal vein occlusion (RVO), and retinopathy of prematurity (ROP).

Glaucoma is an eye disease characterized by visual field defects and cupping of the optic disc. It is well known that abnormally high intraocular pressure is harmful to the eye, and there are clear indications that in patients with glaucoma it is probably the most important physical change causing retinal degeneration. Ultimately, if left untreated, gradual vision loss occurs over time. However, the pathophysiological mechanism of glaucoma is still unknown.

There are three main types of glaucoma: primary, secondary and congenital. Primary glaucoma is the most common type and can be divided into open-angle and closed-angle glaucoma. Primary open-angle glaucoma (POAG; eg, normal tension glaucoma (NTG)) is the most common type of glaucoma seen in the United States. POAG is usually detected in its early stages during routine eye examinations. Primary angle-closure glaucoma, also called acute glaucoma, usually has a sudden onset and is characterized by eye pain and blurred vision. Secondary glaucoma occurs as a complication of a variety of other conditions, such as pseudoexfoliation syndrome, trauma, inflammation, generalized vascular disease, and diabetes. Congenital glaucoma occurs due to a defect in the development of the drainage mechanism of the eye.

Diabetic retinopathy (DR) is the most common complication of diabetes mellitus and a leading cause of decreased visual acuity and blindness in the working-age population in developed countries. The incidence of DR increases as diabetes progresses. Thus, 90% of patients with type 1 diabetes and 60% of patients with type 2 DR have some degree of DR after 20 years of developing diabetes. The prevalence of DR in Western countries is very similar at about 30%, and in 10% of cases DR is in advanced stages that seriously threaten vision.

DR occurs when changes in blood glucose levels cause changes in the blood vessels in the retina. In some cases, these vessels swell (macular edema) and leak fluid into the back of the eye. In other cases, abnormal blood vessels grow on the surface of the retina. If left untreated, DR can gradually become more severe and progress from underlying retinopathy to severe visual impairment and may lead to blindness.

Retinal vein occlusion (RVO) is a retinal vascular disease that is one of the most common causes of vision loss worldwide. In particular, it is the second most common cause of blindness due to retinal vascular disease after diabetic retinopathy. RVO is often the result of underlying health problems (such as high blood pressure, high cholesterol, diabetes and other health problems). There are two types of retinal vein occlusion: central retinal vein occlusion (CRVO) is a blockage in the main retinal vein, and branch retinal vein occlusion (BRVO) is a blockage in one of the smaller branch veins.

There is currently no way to unblock retinal veins, and accepted treatments are aimed at addressing the health problems associated with retinal vein occlusion. In some eyes that have had retinal vein occlusion, vision may return. About 1/3 have some improvement, about 1/3 stay the same, and about 1/3 gradually improve, but it may take a year or more to determine the final result. In some cases, blockage of blood vessels leads to fluid accumulation in the retina. In other cases, the occurrence of ischemia causes the formation of new blood vessels. Currently, RVO is treated with intravitreal injections of anti-VEGF drugs.

Retinopathy of prematurity (ROP) can occur due to premature birth. Abnormal leaky growth of blood vessels (neovascularization) in the retina occurs secondary to other treatments for prematurity and can often lead to neonatal blindness. During pregnancy, blood vessels grow from the center of the developing baby's retina during the mother's 16th week of pregnancy and then branch outward to reach the edges of the retina at 8 months.

- 1 044938 pregnancy. In babies born prematurely, normal retinal vascular growth is incomplete and can therefore be more easily disrupted.

Endothelins (ETs) are a family of naturally occurring peptides identified as ET-1, ET-2 and ET-3. Produced primarily in endothelial cells and playing a key role in vascular homeostasis, ETs constrict blood vessels and increase blood pressure in the kidney, affecting glomerular hemodynamics, sodium and water homeostasis. When overexpressed, ETs contribute to high blood pressure (hypertension), heart disease, kidney problems, and possibly other diseases such as eye disease. See, for example, Salvatore et al., J Ophthalmol. 2010, 2010: 354645.

An endothelin receptor antagonist is a pharmacological agent that inhibits endothelin receptor(s). Some agents inhibit both the endothelin A receptor and the endothelin B receptor, while others selectively inhibit only one of the two receptors (eg, endothelin A receptor antagonist). Endothelin receptor antagonists have been shown to reduce mortality and improve hemodynamics in experimental models (eg, heart failure).

Edonentan (BMS-207940) is a highly selective and very potent endothelin A receptor antagonist. Edonentan was developed as a second-generation analog following the discontinuation of the first clinical candidate, BMS-193884, which was being developed for the treatment of congestive heart failure (CHF). By April 2002, edonentane was being studied in phase I trials, but its development was discontinued.

There remains a need to more effectively reduce the incidence of, treat, or otherwise improve conditions in glaucoma, DR, RVO, and ROP.

The essence of the invention

The present invention relates to a method of using certain compositions containing crystalline forms of edonentane for the treatment of eye diseases selected from glaucoma, diabetic retinopathy (DR), retinal vein occlusion (RVO) and retinopathy of prematurity (ROP).

In one aspect, this invention relates to a solid form of a compound of formula (I) “AA' Jo G J ^HN to (I), wherein said solid form is an anhydrous crystalline form (Form 4) having an X-ray powder diffraction pattern containing at least three characteristic peaks in terms of 2θ selected from peaks at 5.6±0.2°, 11.4±0.2°, 17.7±0.2°, 19.3±0.2°, 21.1± 0.2° and 21.9±0.2°.

In certain solid form embodiments, anhydrous crystalline Form 4 has the following X-ray powder diffraction pattern expressed in terms of diffraction angles (2θ): 5.6±0.2°, 11.4±0.2°, 17.7±0.2°, 19 .3±0.2° and 21.9±0.2°.

In certain solid form embodiments, anhydrous crystalline Form 4 has the following X-ray powder diffraction pattern expressed in terms of diffraction angles (2θ): 11.4±0.2°, 17.7±0.2° and 19.3±0.2°.

In some solid form embodiments, anhydrous crystalline Form 4 exhibits a T _m of about 163°C by DSC analysis.

In another aspect, the present invention includes a composition containing a compound of formula (I)

wherein said compound is an anhydrous crystalline form (Form 4) having an X-ray powder diffraction pattern containing at least three characteristic peaks in terms of 2θ selected from peaks at 5.6±0.2°, 11.4±0.2° , 17.7±0.2°, 19.3±0.2°, 21.1±0.2° and 21.9±0.2°; and 90% by weight or more of said compound is in the form of crystalline Form 4, based on the total weight of the compound present in the composition.

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In some embodiments, the compound of Formula (I) is 95% by weight or more of crystalline Form 4, based on the total weight of the compound present in the composition.

In some embodiments, the compound of Formula (I) is 96% by weight or more of crystalline Form 4, based on the total weight of the compound present in the composition.

In some embodiments, the compound of formula (I) is 97% by weight or more of crystalline Form 4, based on the total weight of the compound present in the composition.

In some embodiments, the compound of Formula (I) is 98% by weight or more in crystalline Form 4, based on the total weight of the compound present in the composition.

In some embodiments, the compound of formula (I) is 99% by weight or more in crystalline Form 4, based on the total weight of the compound present in the composition.

Still included within the scope of the present invention is a method for treating an ocular disease, comprising: contacting optical tissue of a subject with a composition containing a therapeutically effective amount of a compound of formula (I)

wherein the eye disease is selected from the group consisting of glaucoma, diabetic retinopathy (DR), retinal vein occlusion (RVO), and retinopathy of prematurity (ROP); said compound is in a crystalline form (Form 4), wherein Crystalline Form 4 has an X-ray powder diffraction pattern containing at least three characteristic 2θ peaks selected from peaks at 5.6±0.2°, 11.4±0, 2°, 17.7±0.2°, 19.3±0.2°, 21.1±0.2° and 21.9±0.2°; and 90% by weight or more of the compound is in crystalline form 4, based on the total weight of the compound present in the composition.

The present invention further covers a process for preparing an anhydrous crystalline form (Form 4) of the compound of formula (I)

where the method includes:

(a) stirring the compound of formula (I) in an aqueous solution, an organic solvent, or a mixture thereof at a temperature ranging from about 40 to about 120°C;

(b) cooling the resulting solution to a temperature in the range of about 0°C to room temperature (for example, about 25°C) and, when the compound is in an alkaline aqueous solution, adjusting the pH value in the range of about 3 to about 7 (for example, by adding HCl); and (c) filtering the sample thus obtained to obtain crystalline form 4, wherein said crystalline form 4 has an X-ray powder diffraction pattern containing at least three 2θ characteristic peaks selected from peaks at 5.6±0.2°, 11.4±0.2°, 17.7±0.2°, 19.3±0.2°, 21.1±0.2° and 21.9±0.2°.

In some embodiments of the production method, the aqueous solution is water.

In some embodiments of the production method, the aqueous solution is an alkaline aqueous solution. In some embodiments, the pH of the alkaline aqueous solution is 8 or higher.

In some embodiments of the production method, the aqueous solution of the base is an aqueous solution of potassium hydroxide or potassium carbonate.

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In some embodiments of the production method, the organic solvent is a water-soluble organic solvent. In some embodiments, the organic solvent is tetrahydrofuran or isopropanol.

In some embodiments of the production method, the stirring temperature is in the range of about 80 to about 120°C (e.g., from about 80 to about 90°C, from about 90 to about 100°C, from about 100 to about 110°C, or from about 110 to about 120°C).

In some embodiments of the production method, the stirring temperature is in the range of about 40 to about 80°C (e.g., from about 40 to about 50°C, from about 50 to about 60°C, from about 60 to about 70°C, or from about 70 to about 80°C).

In some embodiments of the production method, mixing is carried out for about 20 to about 50 hours (eg, about 20 to about 30 hours, about 30 to about 40 hours, or about 40 to about 50 hours).

In some embodiments of the production process, a slurry of the compound of formula (I) is formed during the mixing step.

In some embodiments of the preparation method, the method further includes seeding with crystalline form 4 and incubating the resulting solution for about 2 hours.

In some embodiments of the production method, cooling is carried out at a temperature of about 5°C for about 15 hours.

In some embodiments of the production method, the pH of the alkaline aqueous solution is adjusted to a range of from about 3 to about 4. In some embodiments of the production method, the pH of the alkaline aqueous solution is adjusted to a range of from about 3 to about 5. In some embodiments of the production method, the pH value the alkaline aqueous solution is adjusted to a range of from about 3 to about 6. In some embodiments of the production method, the pH of the alkaline aqueous solution is adjusted to a range of from about 5 to about 6. In some embodiments of the production method, the pH of the alkaline aqueous solution is adjusted to from about 5 5 to about 6. In some embodiments of the production method, the pH of the alkaline aqueous solution is adjusted to from about 6 to about 7.

Also included within the scope of the present invention is a method of treating an ocular disease, comprising: contacting optical tissue of a subject with a composition containing a therapeutically effective amount of a compound of formula (I), i.e. edonentana:

wherein the eye disease is selected from the group consisting of glaucoma, diabetic retinopathy (DR), retinal vein occlusion (RVO), and retinopathy of prematurity (ROP); said compound is selected from the group consisting of anhydrous crystalline form (Form 1); monohydrate crystalline form (Form 2); and an anhydrous crystalline form (Form 3), where:

(i) anhydrous crystalline Form 1 has an X-ray powder diffraction pattern containing at least three characteristic 2θ peaks selected from peaks at 6.3±0.2°, 7.5±0.2°, 11.7±0, 2°, 15.1±0.2° and 17.3±0.2°; and 90% by weight or more of said compound is in the form of crystalline Form 1, based on the total weight of the compound present in the composition;

(ii) monohydrate crystalline Form 2 has an X-ray powder diffraction pattern containing at least three characteristic 2θ peaks selected from peaks at 9.6±0.2°, 10.4±0.2°, 19.6±0, 2°, 19.7±0.2°, 22.0±0.2°, 22.9±0.2° and 23.7±0.2°; and 90% by weight or more of said compound is in the form of crystalline Form 2 based on the total weight of the compound present in the composition;

(iii) anhydrous crystalline Form 3 has an X-ray powder diffraction pattern containing at least three characteristic 2θ peaks selected from peaks at 7.8±0.2°, 9.0±0.2°, 11.6±0, 2°, 15.8±0.2° and 19.1±0.2°; and 90% by weight or more of said compound is in the form of crystalline Form 3, based on the total weight of the compound present in the composition.

In some embodiments, contacting includes administering the topical composition to the surface of the eye or portion thereof. In other embodiments, contacting involves injecting compositions containing crystalline forms of edonentane into the eye as a whole or into a specific area thereof.

In some embodiments, the eye disease is glaucoma. In additional embodiments, therapeutic efficacy in the treatment of glaucoma is determined by detecting a decrease in intraocular pressure or a decrease in the rate of optic nerve damage/retinal nerve fiber layer thinning, loss of contrast sensitivity, or loss of visual field in an amount sufficient to alleviate or prevent optic nerve damage. In other embodiments, the therapeutic efficacy of a glaucoma treatment is determined by measuring the improvement in retinal, optic disc, or tissue perfusion.

In some embodiments, for treating glaucoma, the regimen further includes adding a therapeutically effective amount of an intraocular pressure (IOP) lowering agent or a neuroprotective agent, or a pharmaceutically acceptable salt of any of the foregoing. In some embodiments, the IOP lowering agent is selected from the group consisting of prostaglandins (such as latanoprost or travoprost), beta blockers (such as timolol or betaxolol), alpha adrenergic agonists (such as brimonidine, apraclonidine), carbonic anhydrase inhibitors (such as dorzolamide or brinzolamide), Rho kinase inhibitors (such as netarsudil), and miotic or cholinergic agents (such as pilocarpine). In some embodiments, the neuroprotective agent is selected from the group consisting of anti-apoptotic agents (such as a caspase-2 inhibitor) and neurotrophic factors (such as ciliary neurotrophic factor).

In some embodiments, the eye disease is diabetic retinopathy (DR). In additional embodiments, the therapeutic efficacy of treatment for DR is determined by a reduction in retinal neovascularization, an assessment of the severity of diabetic retinopathy, and diabetes-induced neurodegeneration. In other embodiments, the therapeutic efficacy of treatment for DR is determined by measuring the improvement in retinal or choroidal perfusion.

In some embodiments, the disease is retinal vein occlusion (RVO). In additional embodiments, the therapeutic efficacy of treatment for RVO is determined by measuring improvement in tissue perfusion, reduction in inflammation, or a combination of the above.

In some embodiments, the eye disease is retinopathy of prematurity (ROP). In additional embodiments, the therapeutic efficacy of treatment for ROP is determined by measuring the improvement in retinal perfusion and the reduction in abnormal neovascularization.

In some embodiments, the administered composition comprises a crystalline form of edonentane at a dose ranging between about 1 μg and about 5 mg (e.g., between about 1 μg and about 10 μg, between about 10 μg and about 50 μg, between about 50 μg and about 100 mcg, between about 100 mcg and about 500 mcg, between about 500 mcg and about 1 mg, between about 1 mg and about 1.5 mg, between about 1.5 mg and about 2 mg, between about 2 mg and 2, 5 mg, between about 2.5 mg and about 3 mg, between about 3 mg and 3.5 mg, between about 3.5 mg and 4 mg, between about 4 mg and 4.5 mg and between about 4.5 mg and 5 mg). In some embodiments, the administered composition comprises a crystalline form of edonentane at a dose ranging between about 10 μg and about 3 mg (e.g., between about 10 μg and about 100 μg, between about 10 μg and about 500 μg, between about 10 μg and about 1 mg). mg, between about 10 mcg and about 2 mg, about 10 mcg, about 25 mcg, about 50 mcg, about 75 mcg, about 100 mcg, about 125 mcg, about 150 mcg, about 175 mcg, about 200 mcg, about 250 mcg , about 300 mcg, about 350 mcg, about 400 mcg, about 450 mcg, about 500 mcg, about 550 mcg, about 600 mcg, about 650 mcg, about 700 mcg, about 750 mcg, about 800 mcg, about 850 mcg, about 900 mcg, about 950 mcg, about 1 mg, about 1.25 mg, about 1.5 mg, about 1.75 mg, about 2 mg, about 2.25 mg, about 2.5 mg, about 2.75 mg , about 3 mg). In some embodiments, the administered composition contains a crystalline form of edonentane at a dose ranging between about 10 μg and about 1 mg. In some embodiments, the composition described herein is administered in a single dosage form. In some embodiments, the composition described herein is administered in multiple dosage forms.

The present invention also relates to a method for preparing the monohydrate crystalline form (Form 2) of the compound of formula (I)

- 5 044938 where the method includes:

(a) preparing a suspension of a compound of formula (I) (eg, an amorphous form) in an aqueous medium (eg, water) at a temperature in the range of about 15 to about 35°C for a predetermined period of time (eg, about 15 hours) to obtain sample; and (b) filtering the sample thus obtained to obtain crystalline form 2, wherein said crystalline form 2 has an X-ray powder diffraction pattern containing at least three 2θ characteristic peaks selected from peaks at 9.6±0.2°, 10.4±0.2°, 19.6±0.2°, 19.7±0.2°, 22.0±0.2°, 22.9±0.2° and 23.7±0 ,2°.

In some embodiments of the production method, the aqueous medium is water.

In some embodiments of the production method, the specified length of time is about 15 hours.

Additional features and advantages of the method of the invention will be set forth in the description below and, in part, will be apparent from the description or may be learned by practice of the method of the invention. The advantages of the method that is the subject of the invention will be realized and achieved through the structure specifically indicated in the written description and embodiments herein.

Details of one or more embodiments of the invention are set forth in the description below. Other features, objects and advantages of the invention will be apparent from the following drawings, description and claims.

Brief description of the figures

In fig. Figure 1 shows an example of a powder X-ray diffraction pattern of forms 1-4.

In fig. Figure 2 shows an example of a powder X-ray diffraction pattern of form 1.

In fig. Figure 3 shows an example of a powder X-ray diffraction pattern of form 2.

In fig. Figure 4 shows an example of a powder X-ray diffraction pattern of mold 3.

In fig. Figure 5 shows an example of a powder X-ray diffraction pattern of mold 4.

In fig. Figure 6 shows an example of a DSC shape 1 curve.

In fig. Figure 7 shows an example of a DSC shape 2 curve.

In fig. Figure 8 shows an example of a DSC Shape 3 curve.

In fig. Figure 9 shows an example of a DSC shape 4 curve.

Detailed description

The invention is based on the discovery that certain compositions containing crystalline forms of edonentane can be used to prevent, treat, or otherwise improve eye diseases, including, but not limited to, glaucoma, diabetic retinopathy (DR), and retinal vein occlusion (RVO). and retinopathy of prematurity (ROP). The invention is further described below.

As stated in the summary of the invention, the present invention covers a method of treating an eye disease, comprising: contacting optical tissue of a subject with a composition containing a therapeutically effective amount of a compound of formula (I) as defined above, wherein the eye disease is selected from the group consisting of glaucoma, diabetic retinopathy ( DR), retinal vein occlusion (RVO) and retinopathy of prematurity (ROP); the compound is an anhydrous crystalline form (Form 1), wherein the anhydrous crystalline form 1 has an X-ray powder diffraction pattern containing at least three characteristic 2θ peaks selected from peaks at 6.3±0.2°, 7.5±0, 2°, 11.7±0.2°, 15.1±0.2° and 17.3±0.2°; and 90% by weight or more of the compound is in the form of crystalline Form 1, based on the total weight of the compound present in the composition.

In some embodiments, anhydrous crystalline Form 1 has the following X-ray powder diffraction pattern expressed in terms of diffraction angles (2θ): 6.3±0.2°, 7.5±0.2°, 11.7±0.2° and 15 .1±0.2°.

In some embodiments, anhydrous crystalline Form 1 has the following X-ray powder diffraction pattern expressed in terms of diffraction angles (2θ): 7.5±0.2°, 11.7±0.2° and 15.1±0.2°.

In some embodiments, anhydrous crystalline Form 1 has a T _m of about 151°C by DSC analysis.

In some embodiments, anhydrous crystalline Form 1 has a solubility in phosphate buffer of about 264 μg/mL at about pH 7.

In some embodiments, the compound of formula (I) is 95% by weight or more of crystalline Form 1, based on the total weight of the compound present in the composition.

In some embodiments, the compound of formula (I) is 96% by weight or more of crystalline Form 1, based on the total weight of the compound present in the composition.

In some embodiments, the compound of formula (I) is 97% by weight or more of crystalline Form 1, based on the total weight of the compound present in the composition.

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In some embodiments, the compound of Formula (I) is 98% by weight or more of crystalline Form 1, based on the total weight of the compound present in the composition.

In some embodiments, the compound of Formula (I) is 99% by weight or more of crystalline Form 1, based on the total weight of the compound present in the composition.

This invention also covers a method of treating an eye disease, comprising: contacting the optic tissue of a subject with a composition containing a therapeutically effective amount of a compound of formula (I), wherein the eye disease is selected from the group consisting of glaucoma, diabetic retinopathy (DR), retinal vein occlusion (RVO) and retinopathy of prematurity (ROP); said compound is in the form of a monohydrate crystalline form (Form 2), wherein the monohydrate crystalline form 2 has an X-ray powder diffraction pattern containing at least three characteristic 2θ peaks selected from peaks at 9.6±0.2°, 10.4± 0.2°, 19.6±0.2°, 19.7±0.2°, 22.0±0.2°, 22.9±0.2° and 23.7±0.2°; and the compound of formula (I) is 90% by weight or more in the form of crystalline Form 2, based on the total weight of the compound present in the composition.

In some embodiments, the monohydrate crystalline form, crystalline form 2, has the following X-ray powder diffraction pattern expressed in terms of diffraction angles (2θ): 19.6±0.2°, 19.7±0.2°, and 9.6±0.2° .

In some embodiments, the monohydrate crystalline form, crystalline form 2, has the following X-ray powder diffraction pattern expressed in terms of diffraction angles (2θ): 19.6±0.2°, 19.7±0.2°, 9.6±0.2° , 10.4±0.2°, 22.0±0.2° and 22.9±0.2°.

In some embodiments, the monohydrate crystalline form crystalline form 2 has a Tm of about 122°C by DSC analysis.

In some embodiments, the monohydrate crystalline form, crystalline form 2, has a solubility in phosphate buffer of about 35 μg/mL at about pH 7.

In some embodiments, the compound of Formula (I) is 95% by weight or more of crystalline Form 2, based on the total weight of the compound present in the composition.

In some embodiments, the compound of formula (I) is 96% by weight or more of crystalline Form 2, based on the total weight of the compound present in the composition.

In some embodiments, the compound of Formula (I) is 97% by weight or more in crystalline Form 2, based on the total weight of the compound present in the composition.

In some embodiments, the compound of Formula (I) is 98% by weight or more in crystalline Form 2, based on the total weight of the compound present in the composition.

In some embodiments, the compound of Formula (I) is 99% by weight or more in crystalline Form 2, based on the total weight of the compound present in the composition.

The present invention further covers a method of treating an eye disease, comprising: contacting the optical tissue of a subject with a composition containing a therapeutically effective amount of a compound of formula (I), wherein the eye disease is selected from the group consisting of glaucoma, diabetic retinopathy (DR), retinal vein occlusion (RVO) and retinopathy of prematurity (ROP); said compound is an anhydrous crystalline form (Form 3), wherein the anhydrous crystalline form 3 has an X-ray powder diffraction pattern containing at least three characteristic 2θ peaks selected from peaks at 7.8±0.2°, 9.0±0 ,2°, 11.6±0.2°, 15.8±0.2° and 19.1±0.2°; and the compound of formula (I) is 90% by weight or more in the form of crystalline Form 3, based on the total weight of the compound present in the composition.

In some embodiments, anhydrous crystalline Form 3 has the following X-ray powder diffraction pattern expressed in terms of diffraction angles (2θ): 9.0±0.2°, 15.8±0.2°, 7.8±0.2° and 19 .1±0.2°.

In some embodiments, anhydrous crystalline Form 3 has the following X-ray powder diffraction pattern expressed in terms of diffraction angles (2θ): 9.0±0.2°, 15.8±0.2°, and 7.8±0.2°.

In some embodiments, anhydrous crystalline Form 3 has a T _m of about 162°C by DSC analysis.

In some embodiments, anhydrous crystalline Form 3 has a solubility in phosphate buffer of about 251 μg/mL at about pH 7.

In some embodiments, the compound of formula (I) is 95% by weight or more of crystalline Form 3, based on the total weight of the compound present in the composition.

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In some embodiments, the compound of Formula (I) is 96% by weight or more of crystalline Form 3, based on the total weight of the compound present in the composition.

In some embodiments, the compound of Formula (I) is 97% by weight or more of crystalline Form 3, based on the total weight of the compound present in the composition.

In some embodiments, the compound of Formula (I) is 98% by weight or more of crystalline Form 3, based on the total weight of the compound present in the composition.

In some embodiments, the compound of formula (I) is 99% by weight or more in crystalline Form 3, based on the total weight of the compound present in the composition.

Still covered by this invention is a method of treating an ocular disease comprising contacting optical tissue of a subject with a composition containing a therapeutically effective amount of a compound of formula (I), wherein the ocular disease is selected from the group consisting of glaucoma, diabetic retinopathy (DR), retinal vein occlusion (RVO), and retinopathy of prematurity (ROP); said compound is in the form of a crystalline form (Form 4), wherein crystalline Form 4 has an X-ray powder diffraction pattern containing at least three characteristic 2θ peaks selected from peaks at 5.6±0.2°, 11.4±0, 2°, 17.7±0.2°, 19.3±0.2°, 21.1±0.2° and 21.9±0.2°; and the compound of formula (I) is 90% by weight or more in the form of crystalline Form 4, based on the total weight of the compound present in the composition.

In some embodiments, anhydrous crystalline Form 4 has the following X-ray powder diffraction pattern expressed in terms of diffraction angles (2θ): 5.6±0.2°, 11.4±0.2°, 17.7±0.2°, 19 .3±0.2° and 21.9±0.2°.

In some embodiments, anhydrous crystalline Form 4 has the following X-ray powder diffraction pattern expressed in terms of diffraction angles (2θ): 11.4±0.2°, 17.7±0.2° and 19.3±0.2°.

In some embodiments of the invention, crystalline form 4 has the following X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ.

In some embodiments, anhydrous crystalline Form 4 has a Tm of about 163°C by DSC analysis.

In some embodiments, the compound of Formula (I) is 95% by weight or more of crystalline Form 4, based on the total weight of the compound present in the composition.

In some embodiments, the compound of Formula (I) is 96% by weight or more of crystalline Form 4, based on the total weight of the compound present in the composition.

In some embodiments, the compound of formula (I) is 97% by weight or more of crystalline Form 4, based on the total weight of the compound present in the composition.

In some embodiments, the compound of Formula (I) is 98% by weight or more in crystalline Form 4, based on the total weight of the compound present in the composition.

In some embodiments, the compound of formula (I) is 99% by weight or more in crystalline Form 4, based on the total weight of the compound present in the composition.

In some embodiments of the invention, the therapeutic effectiveness of the treatment described above is determined by; (i) detecting a decrease in intraocular pressure or a decrease in the rate of damage to the optic nerve in an amount sufficient to alleviate or prevent damage to the optic nerve; (ii) assessing the degree of improvement in visual acuity or visual field; (iii) measuring the reduction in diabetes-induced retinal neurodegeneration; (iv) measuring improvement in tissue or retinal perfusion; or (v) measuring improvement in tissue or retinal perfusion, reduction in inflammation, or a combination thereof.

The methods of the present invention include contacting ocular tissue (topically or intraocularly) or administering a therapeutically effective amount of compositions containing crystalline forms of edonentane.

Methods for preparing edonentane (eg, amorphous edonentane) are well known to those skilled in the art. Suitable methods are described, for example, in US patent No. 6043265 and international publication No. 2002/32884. Edonentane has the chemical name I-[[2'-[[(4,5-dimethyl-3isoxalyl)amino]sulfonyl]-4-(2-oxazolyl)[1,1'-biphenyl]-2-yl]methyl]- I,3,3-trimethylbutanamide (molecular weight 536.6 g/mol) and the structure of formula (I) shown above.

Crystalline forms of edonentane.

The crystalline forms of edonentane described herein may be significantly more stable compared to the amorphous form of edonentane. For example, the described crystalline form (eg, Form 4) can be stored under practical and economical storage conditions while maintaining physical properties so that it can be manufactured into a dosage form. In another group of examples, the described crystalline form (eg, Form 1, Form 2, Form 3) is converted to the described crystalline form (eg, Form 4). For example, heating a suspension of crystalline form 1 in approximately 20 ml of a mixture of isopropanol and water (1:2) at 80°C for 24 hours, and then cooling and filtering the sample obtained from the solution gives crystalline form 4. In another example, stirring amorphous form of the compound of formula (I) in 20 ml of water at 25°C for 15 hours gives crystalline form 2, and then subsequent heating of crystalline form 2 at 100°C for 40 hours followed by filtration gives crystalline form 4. B In yet another example, heating a suspension of anhydrous crystalline Form 3 in 30 mL of water at 80°C for 40 hours, then cooling and filtering the sample obtained from the solution gives crystalline Form 4. In one embodiment, crystalline Form 4 is more stable (e.g. , thermodynamically) than some other crystalline forms. In one embodiment, crystalline form 4 is more stable (e.g., thermodynamically) than a crystalline form selected from the group consisting of crystalline form 1, crystalline form 2, and crystalline form 3. In one embodiment of the invention, the described crystalline form may have increased chemical and /or physical stability, for example, when included in a pharmaceutical composition, compared to, for example, an amorphous form. In some embodiments, the crystalline form is Crystalline Form 4.

As used herein, the term amorphous refers to a solid that has no long-range order in the arrangement of its molecules. Amorphous solids are typically supercooled liquids in which the molecules are arranged randomly so that there is no well-defined arrangement, such as molecular packing, and there is no long-range order. Amorphous solids are generally isotropic, that is, they exhibit the same properties in all directions and do not have specific melting points. For example, an amorphous substance is a solid that does not have a sharp characteristic crystalline peak(s) in an X-ray powder diffraction (XRPD) pattern (ie, is not crystalline as determined by XRPD). Instead, one or more broad peaks (such as halos) appear on his radiograph.

Hydration forms of crystalline edonentane are contemplated, for example, edonentane(H2O) _m , where m is a fraction or an integer from about 0 to about 4, inclusive. For example, the anhydrate or monohydrate forms of crystalline edonentane are discussed herein. In one embodiment, the disclosed crystalline form of edonentane may have a water content of about 1 to 10% by weight (eg, 3 to 9% or 5 to 8% by weight).

Below is a general method for preparing crystalline form 1 or crystalline form 2: amorphous (crude) edonentane is dissolved in isopropyl alcohol (IPA). The solution is filtered and the filter is washed with a small amount of IPA. The resulting solution is heated to a temperature of 60°C, with vigorous stirring, warm water is added dropwise and the solution is stirred at the same temperature for 1-2 hours. The solution is slowly cooled to room temperature to obtain crystalline form 1 or form 2 depending on the IPA ratio and water. Alternatively, in a preferred method, amorphous (crude) edonentane is suspended in 20 ml of water at 25°C for 15 hours and then filtered to obtain crystalline Form 2.

The following is a general method for preparing crystalline Form 3: amorphous (crude) edonentane is dissolved in ethyl acetate (EA). The resulting solution is filtered and heated to a temperature of 5060°C. With vigorous stirring, hexane is added dropwise. More EA is added to clarify the solution, followed by stirring at the same temperature for 1-2 hours. The solution is slowly cooled to room temperature to obtain crystalline form 3.

The following is a general method for preparing crystalline Form 4: amorphous (crude) edonentane is added to a solvent mixture consisting of an organic solvent (eg, tetrahydrofuran) and water. The resulting mixture is stirred at elevated temperature, cooled and

- 9 044938 is filtered to obtain Form 4. Alternatively, amorphous edonentane is dissolved in an alkaline aqueous solution (eg, an aqueous solution of potassium hydroxide or potassium carbonate). The resulting solution is heated to a temperature of 50-60°C, filtered while warm and acidified with an acid (for example, HCl). The resulting mixture is then stirred, cooled and filtered to obtain Form 4. Alternatively, in a preferred method, amorphous (crude) edonentane is dissolved in 8 ml of isopropanol at 60°C. The resulting solution is cooled to 57°C and then a small crystal of Crystal Form 4 is added. After 2 hours, the solution is cooled to 5°C, held for 15 hours and filtered to obtain Crystal Form 4.

Eye diseases.

The methods of the present invention include the use of compositions containing the crystalline forms of edonentane described above for the treatment and improvement of an ocular disease selected from glaucoma, diabetic retinopathy (DR), retinal vein occlusion (RVO) and retinopathy of prematurity (ROP), as described Further.

Glaucoma.

In the treatment of glaucoma using compositions containing the crystalline forms of edonentane described herein, the therapeutically effective amount can be determined by assessing the improvement in retinal blood flow (RBF) compared to what can be achieved with standard treatment (reduction of intraocular pressure (IOP)). Improved blood flow in a healthy rabbit eye model can be used to indicate glaucoma as a predictor of pharmacodynamic (PD) response in humans. To evaluate ocular pharmacokinetic/pharmacokinetic relationships for compounds targeting human ocular diseases, rabbits are commonly used due to the anatomical and functional similarities between rabbit and human eyes. It has previously been shown that intravitreal injection of ET-1 into the rabbit eye causes significant vasoconstriction and damage to the optic nerve (Sasaoka M. et al., Exp Eye Res 2006; Sugiyama T. et al., Arch Ophthalmol 2009). To target pharmacodynamic response in glaucoma patients who have elevated plasma and aqueous humor ET-1 levels, this model can simulate a pharmacodynamic response based on reversing the perfusion impairment caused by intravitreal ET-1 administration (Li S. et. al., Journal of Ophthalmology 2016).

Other examples of relevant animal models of glaucoma are the Morrison rat model of chronically elevated IOP and the laser-induced nonhuman primate (NHP) glaucoma model. Glaucoma in the Morrison rat model is induced by a sustained increase in IOP when a hypertonic solution is administered through the episcleral veins. In the laser-induced glaucoma model of NIP, after a sustained increase in IOP, blood flow in the optic nerve head decreases (Wang L. et al., Invest Ophthalmol Vis Sci 2012). Additionally, decreased optic nerve head blood flow has been shown to correlate with long-term structural changes in the optic nerve (Cull G. et al., Invest Ophthalmol Vis Sci 2013). In a model of laser-induced glaucoma, LNP edonentane was able to improve blood flow in the optic nerve head in a dose-dependent manner.

Effectiveness in the glaucoma models described above is defined as a reduction in IOP, improvement in optic nerve head or retinal blood flow compared with baseline, and prevention or slowing of progression of structural neurodegenerative changes such as retinal nerve fiber layer thickness as measured by optical coherence tomography (OCT) or retinal ganglion cell count on a flat film, as well as functional changes such as electroretinographic (ERG) or contrast sensitivity after treatment with a composition containing crystalline forms of edonentane.

It is believed that the effect of compositions containing crystalline forms of edonentane on retinal blood flow can be assessed by the radius of the blood vessel (r) according to Poiseuille's law. An increase in (r) with an endothelin antagonist will cause a greater increase in blood flow than can be achieved by increasing perfusion pressure by lowering IOP:

blood flow = (perfusion pressure x nr ⁴ )/(8nl), where l is the length of the blood vessel;

r is the radius of the blood vessel;

η - blood viscosity;

perfusion pressure - mean arterial pressure-IOP.

In addition, compositions containing crystalline forms of edonentane may reduce intraocular pressure and/or prevent RGC death through mechanisms independent of improving retinal/optic disc tissue perfusion. Accordingly, when using compositions containing certain crystalline forms of edonentane (eg, Form 4), one (r) and/or more (IOP) of the above parameters can be changed to improve RBF, thereby achieving therapeutic efficacy in the treatment of glaucoma.

In some embodiments of the invention, treatment of patients with glaucoma begins immediately after diagnosis. In some embodiments, a composition containing a crystalline form of edonentane is administered topically to the back of the eye using an intravitreal, suprachoroidal, or implant delivery platform at a frequency of 3 to 12 (e.g., every 3-6 or every 4-6) months. In some embodiments for the treatment of glaucoma, a composition containing a crystalline form of edonentane is administered in a topical form (eg, eye drops).

Diabetic retinopathy (DR).

Diabetes can cause serious late complications, which are classified as microangiopathic (retinopathy, neuropathy and diabetic nephropathy) and macroangiopathic (cardiovascular disease). Diabetic retinopathy occurs as a result of damage to the small blood vessels and neurons of the retina. The earliest changes leading to diabetic retinopathy include narrowing of the retinal arteries associated with decreased retinal blood flow; dysfunction of neurons in the inner retina followed in later stages by changes in the function of the outer retina associated with subtle changes in visual function; dysfunction of the blood-retinal barrier, which protects the retina from many substances contained in the blood (including toxins and immune cells), leading to leakage of blood components into the retinal neuropil. Later, the basement membrane of the retinal vessels thickens, the capillaries degenerate and lose cells, especially pericytes and vascular smooth muscle cells. This results in loss of blood flow and progressive ischemia, as well as microscopic aneurysms that appear as balloon-like structures protruding from the capillary walls that recruit inflammatory cells; and leads to progressive dysfunction and degeneration of retinal neurons and glial cells.

The ischemia and oxidative damage observed in DR impair blood flow and tissue ischemia, which the inventors have discovered can be reversed by formulations containing crystalline forms of edonentane. When DR is indicated, improving retinal perfusion is expected to reduce hypoxia and suppress vascular endothelial growth factor (VEGF) activation, resulting in a reduction in vascular proliferative changes, neovascularization, and/or complications of macular edema.

A preclinical model of retinopathy of prematurity (ROP) can be used as a surrogate model for the changes in ischemic retinopathy observed in DR. Oxygen-induced retinopathy in mice is a reproducible and quantifiable model of proliferative retinal neovascularization suitable for studying the pathogenesis and therapeutic intervention of retinal neovascularization in ROP and other vasculopathologies, including DR. The model is induced by exposing 1-week-old C57BL/6J mice to 75% oxygen for 5 days followed by room air as previously described (Smith LEH et al., Invest Ophthalmol Vis Sci 1994). The effectiveness of this preclinical model of ROP can be assessed by studying retinal hypoxia and neovascularization. The current standard of care for DR includes anti-VEGF therapy, which targets only the progressive vascular complications of the disease. In some embodiments, treatment of patients with DR begins in the nonproliferative stages of the disease. In some embodiments, a composition containing a crystalline form of edonentane is administered topically to the back of the eye using an intravitreal, suprachoroidal, or implant delivery platform at a frequency of 3 to 12 (e.g., every 3 to 6 or every 4 to 6) months. In some embodiments for the treatment of DR, a composition containing a crystalline form of edonentane is administered in a topical form (eg, eye drops).

Retinal vein occlusion (RVO).

Retinal vein occlusions (RVO), a vascular disease of the retina, are currently treated with intravitreal injections of anti-VEGF drugs to suppress the growth factor that causes macular edema and corticosteroids to combat the inflammatory components that lead to edema. For the treatment of RVO, the use of compositions containing crystalline forms of edonentane is highly desirable by improving tissue perfusion and reducing inflammation while preventing the unwanted effects of systemic immunosuppression and/or local side effects of steroids.

RVO is currently treated with intravitreal steroids and anti-VEGF drugs. The inventors hypothesize that improving perfusion of existing vessels will reduce the degree of macular edema and VEGF activation, as well as subsequent maladaptive changes that manifest as RVO. A preclinical mouse model of ischemic retinopathy can be used to test efficacy. Oxygen-induced retinopathy in mice is a reproducible and quantifiable model of proliferative retinal neovascularization suitable for studying the pathogenesis and therapeutic intervention of retinal neovascularization in many ischemic retinopathies, including RVO. The model is induced by exposing one-week-old C57BL/6J mice to 75% oxygen for 5 days followed by room air as previously described (Smith LEH et al., Invest Ophthalmol Vis Sci 1994). The effectiveness of this preclinical model of ischemic retinopathy can be assessed by studying retinal hypoxia and neovascularization. A therapeutically effective amount of a composition containing the crystalline form of edo-nentane described herein may be complementary to the current standard of care by improving tissue perfusion and reducing ET-1-mediated inflammation while avoiding the undesirable effects of topical steroids. In some treatment options for RVO, a composition containing a crystalline form of edonentane is administered topically to the back of the eye using an intravitreal, suprachoroidal, or implant platform. The frequency of administration will vary depending on the patient's disease course and response to therapy. In some embodiments for the treatment of RVO, a composition containing a crystalline form of edonentane is administered in a topical form (eg, eye drops).

Retinopathy of prematurity (ROP).

Retinopathy of prematurity (ROP) is a vasoproliferative disease of the retina that affects premature infants. ROP continues to be the leading preventable cause of blindness and visual impairment worldwide. With improvements in perinatal care, survival of moderately preterm infants has improved, and with limited resources for oxygen delivery and poor monitoring, more mature preterm infants develop severe ROP in developing countries.

The pathophysiology of ROP is characterized by two phases. Phase I ROP is caused by vasoobliteration beginning immediately after birth, secondary to marked reductions in vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1). Phase II begins at approximately 33 weeks postmenstrual age (PMA). During this phase, VEGF levels increase, especially in the presence of retinal hypoxia with increased retinal metabolism and oxygen demand, leading to abnormal vasoproliferation. In advanced stages of ROP, laser ablation of the avascular retina, early treatment protocol for ROP (ETPH), intravitreal anti-VEGF antibodies (eg, bevacizumab), and vitrectomy are used to protect central vision and prevent retinal detachment. Long-term complications such as refractory errors, recurrence of ROP, and risk of retinal detachment require constant monitoring by an ophthalmologist during adolescence and beyond.

ROP is caused by severe ischemia due to underdevelopment of retinal vessels due to preterm birth. Therefore, as an aspect of the invention, it is believed that improving the perfusion of existing vessels using compositions containing crystalline forms of edonentane will reduce the degree of ischemia and activation of VEGF, as well as subsequent maladaptive changes that manifest as ROP. A preclinical mouse model of ROP can be used to test efficacy. Oxygen-induced retinopathy in mice is a reproducible and quantifiable model of proliferative retinal neovascularization suitable for studying the pathogenesis and therapeutic intervention of retinal neovascularization in ROP. The model is induced by exposing 1-week-old C57BL/6J mice to 75% oxygen for 5 days followed by room air as previously described (Smith LEH et al., Invest Ophthalmol Vis Sci 1994). The effectiveness of this preclinical model of ROP can be assessed by studying retinal hypoxia and neovascularization. A therapeutically effective amount of a composition containing a crystalline form of edonentane as described herein would be added to the current standard of care by improving tissue perfusion and reducing pathological neovascularization induced by VEGF. In some embodiments, the drug is administered topically to the back of the eye using an intravitreal, suprachoroidal, or implant delivery platform at a frequency of every 4 to 6 weeks, as needed, depending on the patient's disease course and response to therapy. For example, the drug is administered locally to the back of the eye via intravitreal injection at a frequency of every 5 weeks as needed depending on the patient's disease course and response to therapy. In some embodiments for treating ROP, a composition containing a crystalline form of edonentane is administered in a topical form (eg, eye drops).

Pharmaceutical compositions.

Certain embodiments described herein relate to a pharmaceutical composition that may include (eg, made from) a therapeutically effective amount of a crystalline form of edonentane described herein and a pharmaceutically acceptable carrier, diluent, excipient, or a combination thereof.

The term pharmaceutical composition refers to a mixture of one or both of the compounds described herein with other chemical components, such as diluents or carriers. The pharmaceutical composition facilitates the administration of the compound into the body. Pharmaceutical compositions will generally be tailored to the particular intended route of administration.

The term pharmaceutically acceptable means a carrier, diluent, excipient, salt or composition that is safe and effective for its intended use and has the desired biological and pharmacological activity.

As used herein, the term carrier refers to a compound that facilitates the incorporation of the compound into cells or tissues. For example, without limitation, dimethyl sulfoxide (DMSO) is a commonly used vehicle that facilitates the uptake of many organic compounds into cells or tissues of a subject.

As used herein, the term diluent refers to an ingredient in a pharmaceutical composition that does not have pharmacological activity, but may be pharmaceutically necessary or desirable. For example, a diluent can be used to increase the volume of a potent drug that is too small to produce and/or administer. It may also be a liquid for dissolving a drug administered by injection, ingestion or inhalation. A common form of diluent in the art is a buffered aqueous solution, such as, but not limited to, phosphate buffered saline, which mimics the composition of human blood.

As used herein, the term excipient refers to an inert substance that has been added to a pharmaceutical composition to impart, among other things, volume, consistency, stability, binding ability, lubrication, disintegration ability, delayed dissolution, etc. A diluent is a type of excipient.

As used herein, the term will be understood by those skilled in the art and will vary to some extent depending on the context in which it is used. If there are uses of a term that are not clear to those skilled in the art, given the context in which it is used, about will mean up to 10% plus or minus of the specific term.

The pharmaceutical compositions described herein can be administered to a human patient by themselves or in pharmaceutical compositions where they are admixed with other active ingredients, as in combination therapy, or with carriers, diluents, excipients, or combinations thereof. The correct composition depends on the chosen route of administration. Methods for preparing and administering the compounds described herein are known to those skilled in the art.

The pharmaceutical compositions described herein can be manufactured in a manner that is per se known, for example, by conventional processes of mixing, dissolving, granulating, trituration, emulsifying, encapsulating or entrapping. See, for example, Encapsulation Processes, in: Food Powders, 2005, 199-299. In addition, the active ingredients are contained in an amount effective to achieve the intended purpose. The compounds used in the pharmaceutical combinations described herein may be presented in the form of pharmaceutically acceptable salts.

It is preferable to administer the compounds or pharmaceutical compositions of the present invention topically, either as a topical ophthalmic preparation or by injecting the compounds or pharmaceutical compositions directly into the ocular tissue, often in the form of a depot or sustained release formulation. The route of local administration may be intravitreal, suprachoroidal, periocular or subconjunctival injection of the composition, use of implant technology or topical application. For example, the compound is administered as a liposomal formulation that slowly releases the compound to maintain the desired pharmacological effects. Alternatively, to obtain a slow or extended release formulation for topical or intraocular use, polyvinyl alcohol nanoparticles can be prepared by well known methods.

In some embodiments, the pharmaceutical composition is an ophthalmic preparation containing a therapeutically effective amount of a composition comprising a crystalline form of edonentane described herein. As used herein, an ophthalmic drug refers to a specific dosage form intended for instillation on the outer surface of the eye (topical), administration inside (intraocular) or near the eye (periocular), or for use in combination with an ophthalmic drug. In some embodiments, the ophthalmic drug is in the form of a solution, suspension, or ointment. In other embodiments, the ophthalmic drug is in the form of a gel, gelling solution, ophthalmic insert, micro/nanoparticle preparation for local or preferably intravitreal injection, or implant.

In some embodiments, the ophthalmic preparation contains a preservative. Examples of suitable preservatives include, but are not limited to, cationic wetting agents (eg, benzalkonium chloride), organic mercuric compounds (eg, phenylmercuric nitrate, phenylmercuric acetate), organic mercuric compounds (eg, phenylmercuric nitrate, phenylmercuric acetate), organic acids, or esters (eg sorbic acid, p-hydroxybenzoic acid esters such as methylhydroxybenzoate, propylhydroxybenzoate) and alcohol substitutes (eg chlorobutanol, phenylethanol). The preservative may be present in the ophthalmic preparation in an amount of from about 0.002% w/v. to about 0.5% w/v. (e.g. 0.01-0.25% w/v). The ophthalmic preparation may additionally contain a preservative. Examples of suitable preservatives include, but are not limited to, ethylenediaminetetraacetic acid (EDTA).

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In some embodiments, the ophthalmic preparation contains one or more additional excipients or agents to impart viscosity or lubrication, stabilize the active ingredients from degradation, increase the solubility of the active or inactive ingredient, control tonicity, or act as a solvent. Examples of excipients or agents for imparting viscosity or lubrication include hypromellose, carbomer 974P, hydroxyethylcellulose (HEC), polyvinyl alcohol, sodium hyaluronate, sodium carboxymethylcellulose, carbopol 940, hydroxypropyl methylcellulose (HPMC), poloxamer, xyloglucan, alginic acid, sodium alginate, gellan gum, cellulose acetate phthalate and xanthan gum. Examples of excipients or agents as stabilizers include sodium bisulfite, sodium metabisulfite, sodium thiosulfate and sodium sulfate/sulfuric acid, which can act as antioxidants. Examples of excipients or solubilizing agents include providone, glycerin, polyethylene glycol (PEG), polypropylene glycol (PPG), PEG stearate, poloxamer 407, tyloxapol, polysorbate 80, creatinine, cyclodextrin and castor oil. Examples of excipients or agents for adjusting tonicity include sodium chloride, potassium chloride, calcium chloride dehydrate, magnesium chloride hexahydrate, sugars (eg, sucrose, maltose, dextrose, etc.), glycerin, propylene glycol, mannitol, ascorbic acid and acetylcysteine.

In some embodiments, the ophthalmic preparation contains one or more buffers to adjust the pH. Examples of buffers for pH adjustment include, but are not limited to, sodium citrate, monobasic sodium phosphate, dibasic sodium phosphate, boric acid, heptahydrate, sodium acetate trihydrate, sodium citrate dihydrate, histidine, and phosphate-buffered saline (PBS) buffers. The resulting composition may have a pH value of 5.0-8.5 (for example, 5.0-6.0, 5.2-5.8, 6.0-8.0, 6.6-7.8, 6. 2-8.2 and 6.2-7.5).

In some embodiments, the phthalmic preparation contains one or more surfactants. Examples of surfactants include sorbitan oleic acid esters (eg polysorbate or Tween 20 and 80) and tyloxapol.

The volume that can be injected into the human eye at one time is about 50-90 μl through the intravitreal route, up to 450 μl through the subretinal route, and up to 200 μl through the suprachoroidal route. The needle used for these routes is typically 27 to 30 G. The dose is based on concentration, which can be adjusted to match this volume, potency, target efficacy, and pharmacokinetic profile for each indication. Typically, injections into the eye will be given at a frequency no more than once per month for each eye. In some embodiments, when a composition of the invention is administered in a topical form (eg, as eye drops), the volume administered to the human eye at a time may be about 50 μL (the concentration of the eye drops may be about 5 mg /ml).

In some embodiments, the intravitreal formulation will contain a dose of the composition comprising the crystalline form of edonentane in the range of about 1 μg to about 1 mg. A first exemplary formulation contains from about 1 μg to about 1 mg of a composition comprising the crystalline form of edonentane described above, about 10 mM histidine HCl, about 10% α,α-trehalose dihydrate, and about 0.01% polysorbate 20. A second exemplary formulation contains about 1 μg to about 1 mg of a composition containing a crystalline form of edonentane, about 10 mM sodium phosphate, about 40 mM sodium chloride, about 0.03% polysorbate 20, and about 5% sucrose.

Without further elaboration, it is believed that one skilled in the art can, based on the above description, use the present invention to its fullest extent. Therefore, the following specific examples should be considered only as illustrative and in no way limiting the remainder of the invention.

Examples

To provide a more complete understanding of the invention described herein, the following examples are provided. The examples described in this application are offered to illustrate aspects of the invention and their embodiments presented herein and should in no way be construed as limiting their scope.

The compounds to be crystallized as provided herein can be prepared from readily available starting materials using known methods. The compounds can be crystallized using the methods described herein.

Abbreviations:

DSC - differential scanning calorimetry,

EA - ethyl acetate,

IPA - isopropyl alcohol,

PBS - phosphate buffered saline,

XRPD - X-ray powder diffractometry.

Example 1 An example of a method for obtaining crystalline form 1.

Amorphous edonentane (840 mg) was dissolved in 12 ml IPA. The resulting solution was filtered and the filter was washed with an additional 2.5 ml of IPA. The filtrate was concentrated to dryness, dissolved in

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11.8 ml IPA and heated with stirring to a temperature of 60°C. Then 18 ml of warm water at 60°C was added dropwise with vigorous stirring and the solution was stirred at 60°C for 1 hour. The solution was slowly cooled to 25°C, filtered and dried under vacuum at 25°C to obtain 660 mg of crystalline form 1 (XRPD and DSC in Fig. 2 and Fig. 6, respectively).

Example 2 An example of a method for obtaining crystalline form 2.

Amorphous edonentane (250 mg) was dissolved in 3.5 ml IPA. The resulting solution was filtered and the filter was washed with an additional 0.25 ml of IPA. The solution was then heated to a temperature of 60°C, after which 7.5 ml of warm water at a temperature of 60°C was added dropwise with vigorous stirring, and then stirred at a temperature of 60°C for 1 hour. After slowly cooling to a temperature of 25°C the mixture was filtered to obtain crystalline form 2 (XRPD and DSC in Fig. 3 and Fig. 7, respectively).

Alternatively, a preferred method for preparing crystalline Form 2 was as follows. Amorphous edonentane (1 g) was suspended in 20 ml water at 25°C for 15 hours. The solution was then filtered to obtain crystalline form 2 (XRPD and DSC in Fig. 3 and Fig. 7, respectively).

Example 3 An example of a method for obtaining crystalline form 3.

Amorphous edonentane (250 mg) was dissolved in 0.5 ethyl acetate. The resulting solution was filtered and heated to a temperature of 60°C; with vigorous stirring, 1.5 ml of hexane was added dropwise at a temperature of 60°C. To the resulting slightly cloudy solution, 0.1 ml of ethyl acetate was added to obtain a clear solution, which was then stirred at 60°C for 1 hour. The solution was slowly cooled to 25°C and the resulting precipitate was filtered to obtain crystalline form 3 (XRPD and DSC in Fig. 4 and Fig. 8, respectively).

Example 4. Examples of methods for obtaining crystalline form 4.

Amorphous edonentane (100 mg) was added to 2 ml of water containing 0.2 ml of tetrahydrofuran (THF). The resulting mixture was stirred at 50°C for 24 hours, cooled and filtered to obtain Form 4, which was confirmed to be different from Forms 1, 2 and 3 by XRPD (Figure 5) and DSC (Figure 9).

In an alternative method, 107 mg of amorphous edonentane was added to 1 ml of water, followed by the addition of an equivalent of KOH in 1 ml of water. The resulting solution was heated to a temperature of 60°C for 20 minutes, filtered while warm and acidified with 1 ml of 0.2 N. HCl. The resulting mixture was stirred for 5 hours at 60°C, cooled and filtered to obtain Form 4, which was confirmed by XRPD.

In an alternative method, 150 mg of edonentane (Form 3) was added to a mixture of isopropanol and water (1 ml and 2 ml, respectively). The resulting suspension was stirred at 15°C for 48 hours and then filtered. XRPD analysis confirmed that the sample was Form 4, demonstrating that under these conditions Form 4 was more thermodynamically stable than Form 3.

In an alternative method, 200 mg of edonentane (Form 1) was added to a mixture of isopropanol and water (1.3 ml and 2.6 ml, respectively). The resulting solution was heated to a temperature of 80°C and stirred for 24 hours, then cooled and filtered. XRPD analysis confirmed that the sample thus obtained was Form 4, demonstrating that under these conditions Form 4 was more thermodynamically stable than Form 1.

In an alternative method, 100 mg of edonentane (amorphous) was dissolved in 10 ml of water and heated to 100°C for 40 hours. The resulting solution was cooled to ambient temperature and filtered to obtain Form 4.

In an alternative method, amorphous (crude) edonentane was dissolved in 8 volumes of isopropanol at 60°C. The resulting solution was cooled to 57°C and then a small crystal of Crystal Form 4 was added. After 2 hours, the solution was cooled to 5°C, held for 15 hours and filtered to obtain Crystal Form 4.

Example 5: Powder X-ray diffraction of crystalline forms.

X-ray powder diffraction patterns of crystalline forms 1-4 are shown in FIG. 1-5. The X-ray powder diffraction pattern of the crystalline form described herein was recorded using a polycrystalline X-ray diffractometer (Bruker, D8 ADVANCE). The CuKa radiation was operated at a voltage of 40 kV and a current of 40 mA with a transmission slot of 1.0 mm and a cable slot of 0.4°. The sample was placed in the center of the groove of the sample holder and the surface of the sample holder was aligned with the surface of the sample holder. Data were collected by continuous scanning at 0.02° increments and 8°/min using a LYNXEYE detector.

In the following tables. 1-4 list some characteristic X-ray powder diffraction peaks for crystalline forms 1-4, respectively.

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Table 1

Example of X-ray powder diffraction pattern of crystal form 1

2Θ Intensity (number of counts) 6.3 1250 7.5 2750 11.7 1400 15.1 2200 17.3 900

Table 2

Example of X-ray powder diffraction pattern of crystal form 2

Angle [2Θ] Intensity (number of counts) 9.6 2250 10.4 1500 ID 600 12.3 750 14.6 1000 15.1 800 17.2 1000 19.6 3000 19.7 3000 22.0 1500 22.9 1500 23.7 2000

Table 3

Example of X-ray powder diffraction pattern of crystal form 3

2Θ Intensity (number of counts) 7.8 2000 9.0 2750 11.6 750 15.8 2500 19.1 900

Table 4

Example of X-ray powder diffraction pattern of crystal form 4

Angle [2Θ] Intensity (number of counts) 5.6 1800 I,4 12600 14.4 1400 15.7 1200 16.8 1400 17.7 4800 19.3 6700 21.1 2900 21.9 2400 23.9 2400 24.6 1900

In table 5 below lists the characteristic X-ray powder diffraction peaks for crystalline form 4 shown in FIG. 5.

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Table 5

Characteristic X-ray powder diffraction peaks for crystal form 4

SCAN: 3.0/44.9933/0.01973/24.78(sec). Si(40kV,40mA), 1(max)=6860, 04/10/20 10:20

PIC: 35-pts/ ^personal Threshold=3.0, ^”y^ _aniia= 0.1%, VO=3/1.0. Top of the peak=Top

NOTE: 35-рз1/parabolic filter, threshold = 3.0. cutoff = 0-1%.

BG=3/1.0. peak-top = top_______________________________________________________________

# 2- Theta d(A) Height 1% Square l% FWHM 1 5.641 15.6528 1393 20.3 13553 24.5 0.187 2 8.803 10.0363 563 8.2 3809 6.9 0.180 3 9.256 9.5470 504 7.3 2976 5.4 0.169 4 10.494 8.4231 818 11.9 4742 8.6 0.134 5 11.367 7.7780 6860 100.0 55374 100.0 0.140 6 12.198 7.2497 434 6.3 1350 2.4 0.117 7 14.408 6.1424 951 13.9 6234 11.3 0.141 8 15.072 5.8734 484 7.1 1747 3.2 0.110 9 15.710 5.6362 764 11.1 4042 7.3 0.124 10 16.773 5.2814 839 12.2 3958 7.1 0.113 eleven 17.739 4.9959 2150 31.3 20631 37.3 0.183 12 18.629 4.7591 595 8.7 1999 3.6 0.109 13 19.280 4.5999 3173 46.3 23625 42.7 0.137 14 19.733 4.4952 608 8.9 3384 6.1 0.180 15 20.295 4.3721 536 7.8 2106 3.8 0.151 16 21.132 4.2006 1439 21.0 8311 15.0 0.124 17 21.907 4.0538 1206 17.6 11523 20.8 0.222 18 22.495 3.9493 795 11.6 3003 5.4 0.112 19 23.365 3.8041 903 13.2 4778 8.6 0.141 20 23.938 3.7143 1304 19.0 7676 13.9 0.133 21 24.624 3.6123 995 14.5 7730 14.0 0.189 22 26.073 3.4148 595 8.7 2572 4.6 0.146 23 26.718 3.3338 722 10.5 7595 13.7 0.294 24 28.674 3.1107 412 6.0 1069 1.9 0.142 25 29.141 3.0619 398 5.8 754 1.4 0.098 26 30.115 2.9650 440 6.4 3479 6.3 0.309 27 30.841 2.8969 437 6.4 1605 2.9 0.152 28 33.588 2.6660 345 5.0 2082 3.8 0.259

Example 6. Physico-chemical properties of crystalline forms.

Approximate physicochemical properties of the crystalline forms are presented herein. The melting temperatures described herein can be measured using the following method.

Melting point protocol.

The peak maximum melting point ( _Tm ) of each crystalline form was determined by DSC. The DSC of the crystalline form described herein was measured using a TA DSC Q2000 instrument. A sample (1.3010 mg) was weighed in an aluminum crucible and heated from a temperature of 30 to a temperature of 300°C at a heating rate of 10°C/min. The temperatures of the onset of the crystal melting peak, the onset of the peak, the maximum of the peak, and the end of the peak were collected.

The solubility described herein can be measured using the following method:

Solubility analysis protocol.

1. Samples weighing at least 2.0 mg were weighed into the lower chamber of Whatman Mini-UniPrep vials (GE Healthcare). 450 μl of buffer was added to each chamber.

2. The filter pistons of the Mini-UniPrep vials were placed and compressed to the liquid level position to ensure buffer and connection contact with the filter during incubation.

3. Samples were shaken for 2 min, followed by incubation at room temperature (about 25 ± 2°C) for 24 h with shaking at 500 rpm.

4. Mini-UniPrep was pressed to prepare the filtrates for injection into the HPLC system. All vials were checked for visible undissolved material before filtration and for leakage after filtration.

5. The supernatant was diluted 100-fold with buffer to obtain diluents, which were analyzed by HPLC.

In table 6 below shows typical physicochemical properties of crystalline forms 1-4. Physicochemical properties can be obtained using the methods described above.

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Claims (7)

Table 6 Examples of physicochemical properties of crystalline forms 1-4 Polymorph Solvation T _t (°C) Solubility Phosphate buffer pH 7.0 [112] (µg/ml) Form 1 anhydrate 151 264 Form 2 monohydrate 122 35 Form 3 anhydrate 162 251 Form 4 anhydrate 163 138 Among the four crystalline forms, Form 4 had a higher melting point (Tm) than Form 1 and Form 2. Form 4 had lower solubility than Form 1 and Form 3. Form 4 showed higher thermodynamic stability compared to Form 1, Form 2 and form 3 as shown above. In preferred embodiments, Form 4 is preferred for pharmaceutical development. Other embodiments of the invention All features described herein may be combined in any combination. Each function described herein may be replaced by an alternative function serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature described is merely an example of a general series of equivalent or similar features. Moreover, from the foregoing description, one skilled in the art can easily ascertain the essential characteristics of the present invention and, without departing from its spirit and scope, can make various changes and modifications to the invention to adapt it to various applications and conditions. Thus, other embodiments are also included in the claims. CLAIM 1. Solid form of the compound of formula (I) wherein said compound is an anhydrous crystalline form having an X-ray powder diffraction pattern containing at least three characteristic peaks in terms of 2θ selected from peaks at 5.6±0.2°, 11.4±0.2°, 17.7 ±0.2°, 19.3±0.2°, 21.1±0.2° and 21.9±0.2°. 2. The solid form according to claim 1, wherein the anhydrous crystalline form has the following characteristic peaks expressed in terms of diffraction angles (2θ): 11.4±0.2°, 17.7±0.2° and 19.3±0, 2°. 3. The solid form according to claim 1, wherein the anhydrous crystalline form has the following characteristic peaks expressed in terms of diffraction angles (2θ): 11.4±0.2°, 17.7±0.2°, 19.3±0, 2° and 21.1±0.2°. 4. The solid form according to claim 1, wherein the anhydrous crystalline form has the following characteristic peaks expressed in terms of diffraction angles (2θ): 5.6±0.2°, 11.4±0.2°, 17.7±0, 2°, 19.3±0.2°, 21.1±0.2° and 21.9±0.2°. 5. The solid form according to claim 1, wherein the anhydrous crystalline form has the following characteristic peaks expressed in terms of diffraction angles (2θ): 5.6±0.2°, 11.4±0.2°, 14.4±0, 2°, 15.7±0.2°, 16.8°±0.2°, 17.7±0.2°, 19.3±0.2°, 21.1±0.2°, 21 .9±0.2°, 23.9±0.2° and 24.6±0.2°. 6. The solid form of claim 1, wherein the anhydrous crystalline form has a Tm of about 163° C. as determined by differential scanning calorimetry (DSC) analysis. 7. The solid form of claim 1, wherein the anhydrous crystalline form has a solubility in phosphate buffer of about 138 μg/ml at about pH 7. -