HK1212271B - Inhibitors of alpha-crystallin aggregation for the treatment for cataract - Google Patents

Description

Non-surgical methods for treating cataracts

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 61/672,569, filed July 17, 2012, which is incorporated herein by reference in its entirety.

Statement of Government Interest

This invention was made with government support under Grant No. RR024986 awarded by the National Institutes of Health. The government has certain rights in this invention.

Field of the Invention

The present disclosure generally relates to inhibitors of α-crystallin aggregation, uses thereof, and methods of screening for therapeutically effective modulators of protein aggregation.

Background of the Invention

Cataracts, or clouding of the eye's lens, are a condition that affects more than half of all adults over the age of 80, affecting approximately 25 million people in the U.S. Furthermore, cataracts are considered the leading cause of blindness worldwide. αA-crystallin (cryAA) and αB-crystallin (cryAB) comprise 30% of the protein content of the eye lens, where they are responsible for maintaining the transparency of the lens (Haslbeck et al., Nat Struct Mol Biol 12, 842 (2005). cryAA and cryAB belong to a family of small heat shock proteins (sHSPs) that contain a conserved crystallin domain (Bloemendal et al., Prog Biophys Mol Biol 86, 407 (2004); Haslbeck, supra ). Once synthesized, these lens sHSPs are never degraded, so any damage accumulates throughout life and ultimately leads to age-related cataracts (Haslbeck, supra; Perng et al. , J Biol Chem 274, 33235 (1999); Meehan et al. , J Biol Chem 279, 3413 (2004); Meehan et al., J Mol Biol 372, 470 (2007)). Similarly, destabilizing mutations in cryAB, such as R120G, lead to an inherited early-onset form of cataract (Vicart et al ., Nat Genet 20, 92 (1998)). In cataracts, cryAB tends to aggregate and form amyloid-like fibrils in vitro (Andley et al., PLoS One 6, e17671 (2011)).

Currently, the treatment of cataracts involves surgical removal of the cloudy lens and insertion of an artificial replacement. Surgery can be expensive and unsuitable for all patients. Therapies targeting the underlying mechanisms of protein aggregation would benefit these patients. Therefore, there is still a need in the art for therapeutic agents and methods for screening therapeutic agents that block aggregation-prone proteins, such as cryAB, from forming pathological aggregates (e.g., aggregates relevant to cataracts).

SUMMARY OF THE INVENTION

The present invention provides a method for treating or preventing cataracts, comprising administering to a subject in need thereof an effective amount of a composition comprising a compound of formula I or a prodrug or pharmaceutically acceptable salt thereof:

in:

Both R ¹ are H or both R ¹ are Me;

^R2 is H or OH;

The dashed line between carbons 5 and 6 represents an optional double bond;

^R3 is H or Me;

^R4 is H or Me;

n is 0 or 1;

(a) R ⁶ and each R ⁵ are independently H or Me or (b) R ⁶ and one R ⁵ are taken together to form an optionally substituted 6-membered ring, and the other R ⁵ is Me;

The dashed line between carbons 12 and 13 is an optional double bond, provided that when the double bond between carbons 12 and 13 is present, R ⁷ is absent, and when the double bond between carbons 12 and 13 is absent, R ⁷ is H or Me;

^R8 is H or OH;

Two R ⁹ together form oxo (=O) or two R ⁹ are hydrogen; and

R ¹⁰ is CO ₂ H or a linear or branched C ₁ -C ₆ alkyl group.

The present invention also provides an ophthalmic pharmaceutical composition comprising a pharmaceutically acceptable ophthalmic carrier and a compound of formula I.

In various aspects of the methods and/or compositions, the compound of Formula I has the structure of Formula IA or Formula IB:

or

wherein each R ¹¹ is independently alkyl, CO ₂ H or CO ₂ alkyl.

In more particular aspects of the methods and/or compositions, the compound has the structure of Formula II:

wherein R ¹² is H or OH and R ¹³ is H or OH. In one aspect of the methods and/or compositions, the compound is 5-cholestene-3b,25-diol.

In various aspects of the method, the composition is administered topically, subconjunctivally, retrobulbarly, periocularly, subretinaly, suprachoroidally, or intraocularly. In various aspects of the method, the cataract is age-related cataract or diabetic cataract. In certain aspects of the method, the individual has a hereditary early-onset form of cataract. In certain specific aspects of the method, the individual has an R120G mutation and/or a D109H mutation in cryAB.

In certain aspects of the composition, the pharmaceutically acceptable ophthalmic carrier is a cyclodextrin. In a specific aspect, the cyclodextrin is (2-hydroxypropyl)-β-cyclodextrin.

Additionally, the invention includes a high throughput method for screening for compounds that modulate the thermal stability of a protein, the method comprising: (a) contacting a protein with each of a plurality of test compounds; and (b) measuring the melting transition ( _Tm ) of the protein in the presence of each of the plurality of test compounds, wherein a compound that decreases or increases the apparent _Tm by at least 2 standard deviations is a pharmaceutical protein chaperone.

In various aspects of the method, the protein is an amyloid-forming protein or a protein that causes a loss of function disease. In certain aspects, the amyloid-forming protein is selected from Hsp27, αA-crystallin, αB-crystallin, βB2-crystallin, βB1-crystallin, γD-crystallin, Hsp22, Hsp20, τ, α-synuclein, IAPP, β-amyloid, PrP, huntingtin, calcitonin, atrial natriuretic factor, apolipoprotein AI, serum amyloid A, Medin, prolactin, transthyretin, lysozyme, β2 microglobulin, gelsolin, corneal epithelial protein, cystatin, immunoglobulin light chain AL and S-IBM. In other aspects, the protein that causes a loss of function disease is selected from mutant β-glucosidase, cystic fibrosis transmembrane receptor, hexosaminidase A, hexosaminidase B, β-galactosidase and α-glucosidase.

In certain aspects of the method, the _Tm is determined using a high-throughput differential scanning fluorimetry apparatus.

In one aspect of the method, the measuring step comprises: (b1) heating the protein from 50°C to 80°C in the presence of each of a plurality of test compounds, (b2) cooling the protein to 25°C, (b3) maintaining the protein at 25°C for 10 seconds, and (b4) measuring the fluorescence of the protein.

In various aspects, the method further comprises repeating steps (b1)-(b4) 2-30 times, wherein each repetition of step (b1) is performed at an increasingly higher temperature. In particular aspects, the amyloid-forming protein is heated from 65°C to 80°C in increments of 1°C. In other aspects, (b1) further comprises, after heating, equilibrating the amyloid-forming protein and the test compound for 60-180 seconds. In various aspects of the method, the equilibration step is 130 seconds.

Additionally, the present invention includes a high-throughput screening system comprising: (a) an amyloid-forming protein; (b) a device capable of measuring the melting transition (T _m ) of the amyloid-forming protein; and (c) a plurality of test compounds.

In various aspects of the screening system, the protein is selected from Hsp27, αA-crystallin, αB-crystallin, βB2-crystallin, βB1-crystallin, γD-crystallin, Hsp22, Hsp20, τ, α-synuclein, IAPP, β-amyloid, PrP, huntingtin, calcitonin, atrial natriuretic factor, apolipoprotein AI, serum amyloid A, Medin, prolactin, transthyretin, lysozyme, β2-microglobulin, gelsolin, corneal epithelial protein, cystatin, immunoglobulin light chain AL and S-IBM.

In certain aspects of the screening system, the device is a high-throughput differential scanning fluorimeter device.

Specifically contemplated are the use of any of the compounds of Formula I, IA, IB, or II in any of the methods disclosed herein or in the preparation of a medicament for administration according to any of the methods disclosed herein. In this regard, the present invention provides any of the compounds of Formula I, IA, IB, or II for use in a method of treating or preventing cataracts, wherein the method comprises administering to a subject in need thereof an effective amount of the compound.

Other features and advantages of the present disclosure will become apparent from the detailed description that follows. However, it will be understood that the detailed description and specific examples, while indicating specific embodiments of the present disclosure, are given by way of illustration only, as various changes and modifications within the spirit and scope of the present disclosure may become apparent to those skilled in the art from the detailed description. It is intended that the entire document be related as a unified disclosure, and it will be understood that all combinations of features described herein are contemplated, even if the combination of features is not found together in the same sentence or paragraph or section of this document. In addition to the foregoing, the present invention also includes, as a further aspect, all embodiments of the present invention that are narrower in scope in any respect than the variations specifically mentioned above. For example, if an aspect of the invention is described as "comprising" a feature, it is also contemplated that the embodiment "consists of" or "consists essentially of" that feature.

Detailed Description of the Invention

The present invention provides inhibitors of α-crystallin aggregation and methods of using α-crystallin aggregation inhibitors to treat or prevent cataracts, for example, in subjects with cataracts or at risk of developing cataracts. The inhibitors of α-crystallin aggregation of the present invention are, for example, sterols represented by Formula I, Formula IA, Formula IB, and Formula II and can be formulated as ophthalmic pharmaceutical compositions comprising a pharmaceutically acceptable ophthalmic carrier. Since cataracts affect such a large portion of the population (more than half of all adults over the age of 80) and the primary treatment is surgical intervention, the findings described herein represent a significant advance in non-surgical methods that can be used to treat cataracts. Moreover, many non-surgical treatments currently in use tend to further inhibit the aggregation of α-crystallin. Significantly, the compounds of the present invention are able to reverse the aggregation of α-crystallin and also inhibit the aggregation of α-crystallin.

The present invention also provides a high-throughput method for screening compounds for modulating the thermal stability of a protein, the method comprising contacting a protein with each of a plurality of test compounds; and measuring the melting transition ( _Tm ) of the protein in the presence of each of the plurality of test compounds, wherein compounds that decrease or increase the apparent _Tm by at least 2 standard deviations are identified as pharmaceutical protein chaperones.

Methods of treating or preventing cataracts

In some embodiments, the present invention provides a method of treating or preventing cataracts, comprising administering to a subject in need thereof an effective amount of a composition comprising a compound of any one of Formulas I, IA, IB, or II, or, for example, a compound in Table 1. In some embodiments, the cataract is age-related cataract, diabetic cataract, cataract associated with surgery, cataract caused by radiation exposure, cataract caused by a genetic disease, cataract caused by infection, or cataract caused by a drug. In some embodiments, the subject has a hereditary early-onset form of cataract. For example, hereditary forms of cataract include subjects with an R120G mutation and/or a D109H mutation in cryAB.

An individual who is "in need" of treatment according to the present invention is an individual who suffers from cataracts. For example, the individual may suffer from age-related cataracts or cataracts caused by diabetes. Similarly, an individual who is "in need" of treatment according to the present invention is an individual who is at risk of developing cataracts. Individuals at risk of developing cataracts include, but are not limited to, individuals with a family history of developing cataracts, individuals with mutations associated with early-onset cataracts, such as individuals with R120G mutations and/or D109H mutations in cryAB, individuals exposed to radiation, diabetes, etc. For example, in one aspect, the individual is diagnosed with cataracts in one eye and a compound is administered to prevent or slow cataract formation in the fellow eye.

"Treating" cataracts does not require 100% elimination of cataracts. Similarly, "prevention" does not require 100% inhibition of cataract formation. Any reduction in opacity or deceleration of cataract progression constitutes a beneficial biological effect in a subject. In this regard, compared to levels observed without the methods of the present invention (e.g., in biologically matched control subjects or in samples not exposed to the compounds of the methods of the present invention), the present invention reduces cataracts, e.g., by at least about 5%, at least about 10%, or at least about 20%. In some embodiments, cataracts are reduced by at least about 30%, at least about 40%, at least about 50%, or at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more (about 100%).

In some embodiments, cataracts are "treated" according to the methods of the invention by inhibiting cataract formation by, for example, at least about 5%, at least about 10%, or at least about 20%, compared to levels observed without the methods of the invention (e.g., in a biologically-matched control subject or a sample not exposed to a compound of the methods of the invention). In some embodiments, cataract formation is inhibited by at least about 30%, at least about 40%, at least about 50%, or at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more (about 100%), compared to cataract formation in the absence of the compounds of the methods of the invention. Cataracts are typically detected using any of a number of optical tests, including, but not limited to, visual acuity testing, fundus examination, slit lamp examination, keratometer examination, tonometry, contrast testing, glare sensitivity, and wavefront mapping.

The " effective amount " of the composition comprising any one of the compound of structural formula I, IA, IB or II, or the compound in Table 1 is the amount that suppresses or reduces the aggregation of amyloid-forming proteins such as cryAB in an individual. Suppression of aggregation does not require 100% inhibition of aggregation. Any inhibition of aggregation constitutes a beneficial biological effect in a subject. In this regard, compared with the observed level without the inventive method (e.g., in a sample of a biologically paired control subject or a compound not exposed to the inventive method), the present invention suppresses, for example, at least about 5%, at least about 10% or at least about 20% of the amyloid-forming protein. In some embodiments, the formation of amyloid aggregates is suppressed by at least about 30%, at least about 40%, at least about 50%, or at least about 60%. In some embodiments, compared with amyloid formation in the absence of the compound of the inventive method, the inventive method suppresses amyloid formation by at least about 70%, at least about 80%, at least about 90% or more (about 100%).

In some embodiments, the amyloid aggregates are reduced by at least about 30%, at least about 40%, at least about 50% or at least about 60%. In some embodiments, the amyloid aggregates are reduced by at least about 70%, at least about 80%, at least about 90% or more (about 100%) compared to the amyloid aggregates in the absence of the compound of the present invention.

Route of administration

As will be appreciated by those skilled in the art, the most appropriate method of administering a compound to a subject depends on a variety of factors. In various embodiments, the compounds according to the present invention are administered topically to the eye, e.g., topically, subconjunctivally, retrobulbarly, periocularly, subretinally, suprachoroidally, or intraocularly. For example, in various embodiments, the composition is delivered topically to the eye by injection. The injection solution can be injected directly into the cornea, crystallin lens, and vitreous body, or adjacent tissues thereof, using a fine needle. The composition can also be administered as an intraocular irrigant.

Additional desirable routes of administration include, but are not limited to, one or more of the following: oral (e.g., as a tablet, capsule, or as an ingestible solution), mucosal (e.g., as a nasal spray or aerosol inhalation), nasal, parenteral (e.g., by injection), gastrointestinal, intraspinal, intraperitoneal, intramuscular, intravenous, intrauterine, intradermal, intracranial, intratracheal, intravaginal, intracerebroventricular, intracerebral, subcutaneous, transdermal, rectal, subbuccal, epidural, and sublingual.

In some embodiments, the composition of the present invention is delivered to the eye via a contact lens. The lenses can be provided pre-treated with the desired compound. Alternatively, the lenses are provided in a kit with the ingredients for preparing the coated lenses, either as a lyophilized powder for reconstitution or as a concentrate or ready-to-use solution. The composition can be provided as a kit for single or multiple use.

In some embodiments, the compositions of the present invention are delivered to the eye via the ophthalmic rods (Gwon et al., Ophthalmology . 1986 Sep;93(9 Suppl):82-5). In some embodiments, the compositions of the present invention are delivered to the eye via the intraocular lens-hydrogel assembly (Garty et al., Invest Ophthalmol Vis Sci, 2011 Aug 3;52(9):6109-16).

dose

The composition comprising the compound is provided in a therapeutically effective amount to obtain the desired biological effect at a medically acceptable toxicity level. The dosage of the composition may vary depending on the route of administration and the severity of the disease. The dosage may also be adjusted according to the weight, age, sex and/or degree of symptoms of each patient to be treated. The precise dosage and route of administration will ultimately be determined by the on-site physician or veterinarian. It should be appreciated that, depending on the age and weight of the patient and the severity of the condition to be treated, it may be necessary to make routine changes to the dosage. The frequency of administration depends on the formulation and the above-mentioned parameters. For example, it may be necessary to administer eye drops at least once a day, including 2, 3, 4 or 5 times a day.

An exemplary dosage of the compound administered to humans (about 70 kg body weight) via the ocular route is 0.1 mg to 1 g, e.g., 1 mg to 500 mg of compound per unit dose. In various embodiments, the dosage is about 1 μg/kg body weight to 15 mg/kg body weight. For example, the dosage may be 1 μg/kg, 5 μg/kg, 10 μg/kg, 20 μg/kg, 30 μg/kg, 40 μg/kg, 50 μg/kg, 60 μg/kg, 70 μg/kg, 80 μg/kg, 90 μg/kg, 100 μg/kg, 120 μg/kg、140μg/kg、160μg/kg、180μg/kg、200μg/kg、300μg/kg、400μg/kg、500μg/kg、600μg/kg、700μg/kg、800μg/kg、900μg/kg、1 mg/kg, 2mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 An exemplary dosage of the compound administered by systemic route to a human (approximately 70 kg body weight) is 0.1 mg to 5 g, e.g., 1 mg to 2.5 g, of the compound per unit dose.

The preferred concentration range of the compound of Formula I, IA, IB or II, or the compound listed in Table 1 is from about 1 μg/ml to 500 μg/ml, for example, about 1 μg/ml, about 2 μg/ml, about 3 μg/ml, about 4 μg/ml, about 5 μg/ml, about 10 μg/ml, about 20 μg/ml, about 30 μg/ml, about 40 μg/ml, about 50 μg/ml, about 60 μg/ml , about 70 μg/ml, about 80 μg/ml, about 90 μg/ml, about 100 μg/ml, about 120 μg/ml, about 140 μg/ml, about 160 μg/ml, about 180 μg/ml, about 200 μg/ml, about 250 μg/ml, about 300 μg/ml, about 350 μg/ml, about 400 μg/ml, about 450 μg/ml or about 500 μg/ml. The inhibitor can be provided in combination with other pharmaceutically active agents.

The compositions according to the present invention are provided in containers, as concentrates to be diluted with a suitable diluent before use, or in ready-to-use concentrations. Preferably, single doses are provided in sterile vials.

Compounds effective in treating or preventing cataracts

In various embodiments, the compound of the methods or compositions of the present invention is a compound of Formula I:

in:

Two R ^1s are H or two R ^1s are Me;

^R2 is H or OH;

The dashed line between carbons 5 and 6 represents an optional double bond;

^R3 is H or Me;

^R4 is H or Me;

n is 0 or 1;

(a) R ⁶ and each R ⁵ are independently H or Me or (b) R ⁶ and one R ⁵ are taken together to form an optionally substituted 6-membered ring, and the other R ⁵ is Me;

The dashed line between carbons 12 and 13 is an optional double bond, provided that when the double bond between carbons 12 and 13 is present, R ⁷ is absent, and when the double bond between carbons 12 and 13 is absent, R ⁷ is H or Me;

^R8 is H or OH;

Two R ⁹ together form oxo (=O) or two R ⁹ are hydrogen; and

R ¹⁰ is CO ₂ H or a linear or branched C ₁ -C ₆ alkyl group.

For example, the compound of the method or composition of the present invention is a compound of Formula IA or Formula IB:

or

wherein each R ¹¹ is independently alkyl, CO ₂ H or CO ₂ alkyl.

In various embodiments, the compound has the structure of Formula II:

wherein R ¹² is H or OH and R ¹³ is H or OH. In some embodiments, the compound is 5-cholestene-3b,25-diol.

Alternatively, the compound is a compound listed in Table 1.

Any prodrugs or pharmaceutically acceptable salts of the above compounds are encompassed within the scope of the present invention.

Pharmaceutical compositions

The present invention also includes compositions comprising a compound of any one of Formulas I, IA, IB, or II, or a compound listed in Table 1, and a pharmaceutically acceptable ophthalmic carrier, e.g., a pharmaceutically acceptable excipient, carrier, binder, and/or diluent. Optionally, the composition comprises a free acid, free base, salt (e.g., acid or base addition salt), hydrate, or prodrug of a compound of any one of Formulas I, IA, IB, or II, or a compound listed in Table 1. A prodrug is a substance comprising a compound of any one of Formulas I, IA, IB, or II, or a compound listed in Table 1, covalently linked to a carrier moiety. The carrier moiety can be released from a compound of any one of Formulas I, IA, IB, or II, or a compound listed in Table 1, in vitro or in vivo to yield a compound of any one of Formulas I, IA, IB, or II, or a compound listed in Table 1. Prodrug forms are well known in the art as exemplified in Sloan, KB, Prodrugs , M. Dekker, New York, 1992; and Testa, B. and Mayer, JM, Hydrolysis in drug and prodrug metabolism: chemistry, biochemistry, and enzymology , Wiley-VCH, Zurich, 2003.

In various embodiments, compositions include any compound of formula I, IA, IB or II formulated as eye drops, injections or eye ointments or the compounds listed in Table 1. These pharmaceutical compositions can be prepared according to conventional methods by making the compound, optionally mixed, diluted or dissolved in wherein with suitable pharmaceutical additives such as excipients, disintegrants, adhesives, lubricants, diluents, buffers, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and dissolution aids and prepared and prepared in a conventional manner according to dosage form. For example, eye drops can be prepared by dissolving the compound in sterile water, wherein surfactant is dissolved and optionally adds suitable pharmaceutical additives such as preservatives, stabilizers, buffers, antioxidants and viscosity modifiers. In some embodiments, compositions include cyclodextrin. In specific embodiments, cyclodextrin is (2-hydroxypropyl)-β-cyclodextrin.

Physiologically acceptable buffers include, but are not limited to, phosphate buffered saline or Tris-HCl buffer (comprising tris (hydroxymethyl) aminomethane and HCl). For example, a Tris-HCl buffer with a pH of 7.4 comprises 3 g/l tris (hydroxymethyl) aminomethane and 0.76 g/l HCl. In another aspect, the buffer is 10x phosphate buffered saline ("PBS") or 5xPBS solution.

Other buffers include, but are not limited to, HEPES (N-{2-hydroxyethyl}piperazine-N'-{2-ethanesulfonic acid})-based buffers, which have a _pKa of 7.5 at 25°C and a pH in the range of about 6.8-8.2; BES (N,N-bis{2-hydroxyethyl}2-aminoethanesulfonic acid), which has a _pKa of 7.1 at 25°C and a pH in the range of about 6.4-7.8; MOPS (3-{N-morpholino}propanesulfonic acid), which has a _pKa of 7.2 at 25°C and a pH in the range of about 6.7-7.9; TES (N-tris{hydroxymethyl}-methyl-2-aminoethanesulfonic acid), which has a pKa of 7.4 at 25°C and a pH in the range of about 6.8-8.2; MOBS (3-{N-morpholino}propanesulfonic acid), which has a _pKa of 7.4 at 25°C and a pH in the range of about 6.8-8.2; (4-{N-morpholino}butanesulfonic acid), which has a _pKa of 7.6 at 25°C and a pH in the range of about 6.9-8.3; DIPSO (3-(N,N-bis{2-hydroxyethyl}amino)-2-hydroxypropane)), which has a _pKa of 7.52 at 25°C and a pH in the range of about 7-8.2; TAPSO (2-hydroxy-3{tris(hydroxymethyl)methylamino}-1-propanesulfonic acid)), which has a _pKa of 7.61 at 25°C and a pH in the range of about 7-8.2; TAPS ({(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino}-1-propanesulfonic acid)), which has a _pKa of 8.4 at 25°C and a pH in the range of about 7.7-9.1; TABS (N-tris(hydroxymethyl)methyl-4-aminobutanesulfonic acid), which has a _pKa of 8.9 at 25°C and a pH in the range of about 8.2-9.6; AMPSO (N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid), which has a _pKa of 9.0 at 25°C and a pH in the range of about 8.3-9.7; CHES (2-cyclohexylamino)ethanesulfonic acid), which has a _pKa of 9.5 at 25°C and a pH in the range of about 8.6-10.0; CAPSO (3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid), which has a _pKa of 9.6 at 25°C and a pH in the range of about 8.9-10.3; and CAPS (3-(cyclohexylamino)-1-propanesulfonic acid), which has a pKa of 10.4 at 25°C. _a and a pH in the range of about 9.7-11.1.

A number of effective methods for controlled release of active agents are available. See, for example, Wagh VD, Inamdar B., Samanta MK, Polymers used in ocular dosage form and drug delivery systems. Asian J Pharm 2, 2008, 12-17 and references cited therein, the contents of which are incorporated herein by reference. In particular, polymers (e.g., cellulose derivatives such as hydroxypropyl methylcellulose (HPMC) and hydroxypropyl cellulose (HPC), poly(acrylic acid) (PAA), polyacrylates, cyclodextrins and natural gums, polyorthoesters (POE) and mucoadhesive polymers); semisolids such as gels, films and other inserts; resins such as ion exchange resins; iontophoretic delivery; and colloidal particles such as microspheres and nanoparticles are contemplated.

The compounds of the present invention can also be provided in combination with other therapeutic agents. In some embodiments, the compounds of the present invention can be co-formulated with analgesics, anesthetics, artificial tears, enzyme inhibitors, cytokine inhibitors, anti-inflammatory drugs, or antibiotics. In some embodiments, the antibiotic is an antibacterial agent, an antiviral agent, an antifungal agent, an antiprotozoal agent, or a combination thereof.

In various embodiments, the compounds of the present invention may also be provided in combination with an ophthalmic treatment selected from the group consisting of Anhra (ketorolac tromethamine eye drops) 0.5%, Acuvail (ketorolac tromethamine), AK-Con-A (naphazoline ophthalmic), Akten (lidocaine hydrochloride), Alamast, Alphagen (brimonidine), Alrex, Astepro (azelastine hydrochloride nasal spray), AzaSite (azithromycin), Bepreve (bepotastine besylate eye drops), Besivance (besifloxacin ophthalmic suspension), Betaxon, BSS sterile irrigation solution, Cosopt, Durezol (difluprednate), Eylea (aflibercept), Lotemax, Lucentis (ranibizumab), Lumigan (bimatoprost eye drops), Macugen (pegaptanib), Ocuflox (ofloxacin eye drops) 0.3%, OcuHist, Ozurdex (dexamethasone), Quixin (levofloxacin), Rescula (isopropyl unoprostone eye drops) 0.15%, Restasis (cyclosporine ophthalmic emulsion), Sulezin tablets, Suweitan (travoprost eye drops), Wansaiwei (valganciclovir HCl), Viroptic, Vistide (cidofovir), Visudar (verteporfin for injection), Vitrasert Implant, Vitravene Injection, ZADITOR, Zioptan (tafluprost eye drops), Zirgan (ganciclovir ophthalmic gel), Zymaxid Gatifloxacin eye drops, atropine, flurbiprofen, physostigmine, Azopt, gentamicin, pilocarpine, bacitracin, hypromellose eye drops, polymyxin B, povidone iodine, gramicidin, prednisolone, betaxolol, Humorsol, proparacaine, betase, Hylartin, propine, brinzolamide, hypertonic NaCl, Puralube, BSS, indocyanine green, rose bengal, carbachol, itraconazole, sodium hyaluronate, cefazolin, latanoprost, suprofen, xiaolaiwei, mannitol, oxytetracycline, chloramphenicol, methazolamide, timolol, ciprofloxacin hydrochloride solution, miconazole, tobramycin, ciprofloxacin, Miostat, triamcinolone, Cosopt, Muro 128, trifluridine, Demecarium, neomycin, tropicamide, dexamethasone, nibutarone, Solomon's Seal, dipivefrin, ofloxacin eye drops, vidarabine, dorzolamide, ofloxacin, Vira-A, epinephrine, oxytetracycline, Viroptic, fluorescein, phenylephrine, and Xalatan.

Screening methods and systems

In various embodiments, the present invention includes a high-throughput method for screening compounds for modulating the thermal stability of a protein. The method comprises (a) contacting a protein with each of a plurality of test compounds; and (b) measuring the melting transition ( _Tm ) of the protein in the presence of each of the plurality of test compounds, wherein a compound that decreases or increases the apparent _Tm by at least 2 standard deviations is a pharmaceutical protein chaperone. In various embodiments, a compound that decreases or increases the apparent _Tm by at least 3 standard deviations is a pharmaceutical protein chaperone.

Agents that regulate protein thermal stability can be identified using differential scanning fluorimetry (DSF). In DSF, the melting transition ( _Tm ) of a protein target is measured in the presence of an effective ligand. DSF measures the thermal unfolding of a target protein by intrinsic tryptophan fluorescence, or dyes such as 1,8-anilinonaphthalenesulfonic acid (bis-ANS) or Sypro Orange. The binding of the ligand increases the free energy of the folded state or key intermediates, which limits unfolding and shifts the apparent _Tm . Therefore, in some embodiments, the melting transition is measured using a high-throughput differential scanning fluorimetry device, such as the ThermoFluor® 384-well DSF platform, or a real-time PCT thermal cycler.

In various embodiments of the screening method, the step of measuring the melting transition comprises the following steps: (b1) heating the protein from 50°C to 80°C in the presence of each of a plurality of test compounds, (b2) cooling the protein to 25°C, (b3) maintaining the protein at 25°C for 10 seconds, and (b4) measuring the fluorescence of the protein. In more particular embodiments, the method further comprises repeating steps (b1)-(b4) 2-30 times, wherein each repetition of step (b1) is performed at a gradually increasing higher temperature. For example, during the first repetition, the protein is heated to 65°C and cooled to 25°C in the presence of the test compound, maintained there for about 10 seconds, and then the fluorescence of the protein is measured. During the second repetition, the protein is heated to 66°C and cooled to 25°C in the presence of the test compound, maintained there for about 10 seconds, and then the fluorescence of the protein is measured. This process is repeated (e.g., 2-30 times) while increasing the peak temperature to which the protein is heated, for example, in 1°C increments. In some embodiments, during step (b1), the protein is equilibrated at the peak temperature for 60-180 seconds. In a specific embodiment of the method, the equilibration step is 130 seconds.

In various embodiments of the screening method, wherein the step of measuring the melting transition comprises gradually heating the protein to 80° C. in the presence of each of a plurality of test compounds while continuously measuring the fluorescence of the protein.

In various aspects of the screening method, the protein is an amyloid-forming protein. Generally, when the protein is an amyloid-forming protein, the required modulator reduces the melting transition of the protein. Such a modulator stabilizes the non-amyloid form of the protein. In some embodiments, the amyloid-forming protein is selected from Hsp27, αA-crystallin (cataract), αB-crystallin (cataract), βB2-crystallin (cataract), βB1-crystallin (cataract), γD-crystallin (cataract), Hsp22, Hsp20, τ, α-synuclein (Parkinson's disease), IAPP (type 2 diabetes), β-amyloid (Alzheimer's disease), PrP (transmissible spongiform encephalopathy), Huntington's protein (Huntington's disease), calcitonin (thyroid duct carcinoma), atrial natriuretic factor (isolated atrial amyloid degeneration), apolipoprotein AI (atherosclerosis), serum amyloid A (rheumatoid arthritis), Medin (intra-aortic amyloid), prolactin (prolactinoma), transthyretin (familial amyloid polyneuropathy), lysozyme (hereditary non-neuropathic systemic amyloidosis), beta-2 microglobulin (dialysis-related amyloidosis), gelsolin (Finnish amyloidosis), keratoepitheliin (lattice corneal dystrophy), cystatin (cerebral amyloid angiopathy: Icelandic type), immunoglobulin light chain AL (systemic AL amyloidosis), actin (glaucoma), and S-IBM (sporadic inclusion body myositis).

In some embodiments of the screening methods, the protein is a protein that causes a loss-of-function disease. Generally, when the protein is a protein that causes a loss-of-function disease, the desired modulator increases the melting transition of the protein. Such a modulator stabilizes the mutant form of the protein so that it remains active (i.e., degradation of the protein is reduced). Proteins that cause loss-of-function diseases can be, for example, mutant β-glucosidase, mutant glucosylceramidase (Gaucher's disease), mutant cystic fibrosis transmembrane receptor (cystic fibrosis), mutant hexosaminidase A (Tay-Sachs disease), mutant hexosaminidase B (Sandhoff disease), mutant β-galactosidase (Morquio syndrome), and mutant α-glucosidase (i.e., Pompe disease).

In some embodiments, the present invention includes a high-throughput screening system comprising: (a) an amyloid-forming protein; (b) a device capable of measuring the melting transition (T _m ) of the amyloid-forming protein; and (c) a plurality of test compounds. The device capable of measuring the T _m of the amyloid-forming protein can be any device known in the art. As noted above, in some embodiments, the device is a differential scanning fluorimeter device, such as the ThermoFluor® 384-well DSF platform. In various aspects of the screening system, the protein is selected from Hsp27, αA-crystallin, αB-crystallin, βB2-crystallin, βB1-crystallin, γD-crystallin, Hsp22, Hsp20, τ, α-synuclein, IAPP, β-amyloid, PrP, huntingtin, calcitonin, atrial natriuretic factor, apolipoprotein AI, serum amyloid A, Medin, prolactin, transthyretin, lysozyme, β2-microglobulin, gelsolin, corneal epithelial protein, cystatin, immunoglobulin light chain AL and S-IBM.

Example

The following are non-limiting examples of various aspects of the methods described herein. The examples are given for illustrative purposes only and are not to be construed as limiting the present disclosure, as many variations thereof are possible.

Example 1

This example describes a high-throughput method for screening compounds that stabilize the exemplary aggregation-prone protein Hsp27.

Differential scanning fluorimetry (DSF) was used to identify compounds that stabilize cryAB. In a DSF experiment, the melting transition (T _m ) of a protein target is measured in the presence of an effective ligand (Cummings et al., J Biomol Screen 11, 854 (2006). DSF measures the thermal unfolding of a target protein via intrinsic tryptophan fluorescence or (more commonly) dyes such as 1,8-anilinonaphthalenesulfonic acid (bis-ANS) or SyproOrange. Binding of the ligand increases the free energy of the folded state or a key intermediate, which restricts unfolding and shifts the apparent T _m . These values are used to calculate the unfolding transition. When compounds bind to the target before heating, they stabilize against unfolding, shifting the ΔT m . In the case of cryAB and the cryAB R120G mutant, compounds that reduce the apparent T _m are sought, as amyloidogenic proteins generally have a higher apparent stability in this platform. For example, the T _m of wild-type cryAB is 64.1 ± 0.5°C, while the more amyloid-prone R120G cryAB mutant has an apparent T _m of 68.3 ± 0.2°C. (3) This difference correlates with the increased amyloidogenicity of R120G. Therefore, it is reasonable to suggest that screening for “hits” that reduce the apparent T _m may inhibit aggregation.

Using a small 384-well DSF platform (ThermoFluor®), 2,446 compounds were screened for their ability to block sHSP aggregation. As a model, human Hsp27 was used for HTS because it easily forms amyloid and it retains the highly conserved crystallin domain found in all sHSPs. Hsp27 is soluble at room temperature, but it is thermally unfolded and rapidly forms stable amyloid when heated.

Screening was performed in a 7 µL volume of Abgene black 384-well PCR plates containing 10 µM Hsp27 in 50 mM NaPO₄ (pH 7.4), 700 mM NaCl, 50 mM LiCl, and 100 µM bis-ANS. Reactions were covered with silicone oil to limit evaporation. Control experiments indicated that the assay tolerated up to 4% DMSO, but 1% was used as the final concentration. Plates were measured using up/down mode, empirically determined to provide a better signal-to-noise ratio than continuous ramp mode. Plates were heated from 65°C to 80°C in 1°C increments, equilibrated at each high temperature for 130 seconds, cooled to 25°C, held at 25°C for 10 seconds, and then imaged with a single 10-second exposure for each temperature reading. Plate uniformity testing determined the T _m of Hsp27 to be 72.3 ± 0.16°C. The calculated Z-factors varied between ~0.59 and 0.71 and the CV was 8%. Most of the variation appeared to be due to some "marginal effect."

For each well, a series of fluorescence versus temperature points were obtained, plotted, and fitted to determine the _Tm . An automated method was developed in MatLab that performed the individual fits and then ranked each experimental well based on how closely the three independent fit methods (Savitzky-Golay derivative, sigmoidal, and parallel baseline Hill) agreed.

From this initial screen, 45 compounds were identified that decreased the apparent T _m by more than 3 standard deviations (-3SD; ~0.6°C) and 45 compounds that increased the T _m by +3SD. All 90 compounds were subjected to dose-dependency studies to confirm their activity, and from these studies, 64 compounds were found to have activity below 50 µM. The 45 compounds identified as lowering the apparent T _m by more than 3 standard deviations were tested in a 12-point dose-dependency assay, providing 28 compounds that demonstrated and shifted the ΔT _m at concentrations below 20 µM. Interestingly, 12 of these 28 compounds were part of a single structural class of related sterols. This scaffold was selected for further study.

In summary, these studies demonstrate that the HTS approach is a powerful tool for identifying drug partners for amyloid-forming proteins, such as cryAB. In fact, the studies demonstrate that the HTS approach can be used to discover agents that modulate the Tm of any protein where increased stability (e.g., loss-of-function proteins such as mutant forms of α-glucosidase responsible for Pompe disease) or decreased stability (aggregation-prone proteins such as huntingtin) is _desired .

Example 2

This example describes structure-activity relationship (SAR) studies of the sterol backbone identified in the primary screen of Example 1. As described further below, SAR revealed the general chemical structure (Formula I) of a compound that reduced the _Tm of the amyloid-forming protein, R120G cryAB, by at least 2°C.

Thirty-two sterols with chemical structures related to 17-α-hydroxy-progesterone were pooled and subjected to further testing (see Table 2). DSF experiments on this pooled collection using R120G cryAB identified two compounds that reduced _T by at least 2°C: 5α-cholestan-3β-ol-6-one and 5-cholesten-3β,25-diol. Many other closely related sterols, including cholesterol, were inactive, and the resulting SAR supported a fairly specific molecular interaction.

Table 2

name Activity (Transfer of thermal stability at 100 μM) 5-Cholesterene-3b,25-diol -2.0℃ 5a-cholestane-3b-ol-6-one -3.0℃ 5-cholesten-3b-ol -1.1℃ Bencholan-17b-ol-3-one +1.7℃ 17-a-Hydroxy-progesterone Between +1℃ and -1℃ 5a-Androstane-3b,17b-diol Between +1℃ and -1℃ 17a-Hydroxy-pregnenolone Between +1℃ and -1℃ 5-a-Androstane-3,17-dione Between +1℃ and -1℃ Epiandrosterone Between +1℃ and -1℃ progesterone Between +1℃ and -1℃ Dehydroepiandrosterone Between +1℃ and -1℃ D4-androstene-3,17-dione Between +1℃ and -1℃ b-estradiol Between +1℃ and -1℃ testosterone Between +1℃ and -1℃ corticosterone Between +1℃ and -1℃ cortisone Between +1℃ and -1℃ Estrogen triol Between +1℃ and -1℃ 4-cholesten-3-one Between +1℃ and -1℃ cholesterol Between +1℃ and -1℃ hydrocortisone Between +1℃ and -1℃ Estrogen Between +1℃ and -1℃ D5-pregnen-3b-ol-20-one Between +1℃ and -1℃ Testosterone acetate Between +1℃ and -1℃ Deoxycorticosterone Between +1℃ and -1℃ Androsterone Between +1℃ and -1℃ Corticosterone-21-acetate Between +1℃ and -1℃ Digitonin Between +1℃ and -1℃ 5-Cholesteren-3b-ol-7-one Between +1℃ and -1℃ Ursodeoxycholic acid Between +1℃ and -1℃ 6-Ketocholestanol Between +1℃ and -1℃ 18-a-Glycyrrhetinic acid Between +1℃ and -1℃

Further dose-dependence studies with 5a-cholestane-3b-ol-6-one, 5-cholestene-3b,25-diol, and cholesterol showed that both 5a-cholestane-3b-ol-6-one and 5-cholestene-3b,25-diol were able to partially restore the wild-type _Tm , suggesting their utility as drug chaperones. To confirm direct interaction with cryAB in a unique experimental platform, biolayer interferometry (described in Example 1) was employed to determine that 5-cholestene-3b,25-diol bound to R120G cryAB with a _KD of 10.1 ± 4.4 μM. Measurement of the affinity of the test protein by biolayer interferometry (Octet Red) allowed for the identification of false positives. Briefly, biotinylated cryAB was immobilized on streptavidin-coated pins and equilibrium association data were analyzed over a 100-fold concentration range to generate an apparent _KD . To control for nonspecific binding, the response of the biotin-blocked needles was subtracted from each sensorgram.

In summary, the above results demonstrate that the compound of formula I functions as a drug chaperone for R120G cryAB in vitro and binds with high affinity. Furthermore, the results indicate that the disclosed DSF method represents a powerful high-throughput screening system for identifying new drug chaperones for non-enzymes.

Example 3

This example demonstrates that the compounds identified in Example 1 inhibit amyloid formation. Notably, the compounds are also able to reverse amyloid formation.

DSF studies suggest that sterols inhibit R120G cryAB amyloid formation. To test this concept, R120GcryAB (15 µM) was treated with either 5-cholestene-3b,25-diol or cholesterol (100 µM), and its aggregation capacity was measured by electron microscopy. These studies demonstrated that 5-cholestene-3b,25-diol, but not cholesterol or solvent controls, significantly inhibited amyloid formation. Visual inspection of the solutions supported this conclusion, as only 5-cholestene-3b,25-diol reduced the opacity of the R120G cryAB mixture. Furthermore, 5-cholestene-3b,25-diol reversed the aggregation of preformed R120G cryAB amyloid, suggesting that it shifts the equilibrium toward non-amyloid structures.

To explore the mechanism of action of 5-cholestene-3b,25-diol, the binding site of R120G cryAB was probed by ¹⁵ NHSQC NMR, and analysis of the resulting chemical shifts suggested that it binds to an exposed surface of a conserved crystallin domain. Specifically, 5-cholestene-3b,25-diol binds to a groove spanning the cryAB dimer interface and contacts residues near R120 and D109.

These results support a model in which sterols stabilize sHSPs and reduce amyloid formation.

Example 4

This example demonstrates that the compounds identified herein reverse the cataract phenotype in vivo .

The R120G cryAB knock-in mouse is a clinically accepted model for age-related and hereditary cataracts. This animal model develops severe cataracts within 20 weeks (Andley et al., PLoS One 6, e17671 (2011)). Eyes removed from these mice were treated with 5-cholestene-3b,25-diol (100 µM) or saline control. Treatment with 5-cholestene-3b,25-diol significantly reduced cryAB aggregation and improved cryAB solubility.

5-Cholestene-3b,25-diol was then formulated in a saline-cyclodextrin solution (5 mM cyclodextrin; 5 mM 5-Cholestene-3b,25-diol) and delivered via eye drops to live animals (n = 15) three times a week for two weeks. cryAB solubility and lens transparency were measured in live animals using slit lens illumination and gel permeation chromatography/light scattering from lens homogenates. This treatment significantly increased cryAB solubility, improved lens transparency, and rescued the cataract phenotype in 10 of 15 treated R120G cryAB knock-in mice. Therefore, 5-Cholestene-3b,25-diol, and more generally, compounds of Formula I, are promising therapeutic agents for the treatment of cataracts. More broadly, these studies suggest that DSF-based HTS activities can be used to identify non-enzymatic drug partners, such as cryAB.

Example 5

This example demonstrates that the compounds identified herein increase the levels of soluble α-crystallin in the eye.

5-Cholesterene-3b,25-diol formulated in a saline-cyclodextrin solution (5 mM cyclodextrin; 5 mM 5-cholestene-3b,25-diol) was delivered to live mice (n = 15) via eye drops three times a week for two weeks. Gel filtration analysis of α-crystallin solubility was performed by treating samples as previously described by Andley et al. ( PLoS ONE , 6:3, 1-13 (2011)). Briefly, the degree of aggregation of α-crystallin was determined using gel permeation chromatography (GPC) with light scattering and refractive index (RI) measurements. All data were analyzed using SAS version 9.3 (SAS Institute; Cary, NC). To control for differences in analytical sensitivity among animals, the area under the curve for α-crystallin in drug-treated eyes was compared to the area under the curve generated from untreated eyes of the same animals and expressed as the area "percentage" difference using the untreated eye as the denominator. The Wilcoxon signed rank test for the null hypothesis of no difference was used to compare the percentage difference between treated and untreated eyes. The relationship between age and percentage difference was calculated using the Spearman rank correlation coefficient. A nonparametric statistical model was used to prevent deviations from normality.

The area under the curve for α-crystallin was found to be statistically significantly higher in drug-treated eyes compared to untreated eyes of the same animals (n = 17, mean percentage difference = 381.2 ± 974.6, median = 63.3, Wilcoxon signed-rank p-value = 0.001). Older mice exhibited a higher percentage increase in α-crystallin in treated eyes relative to untreated eyes (Spearman rank correlation = 0.44, p = 0.075). The data in Table 1 show that the median percentage difference increased significantly when the age was greater than 200 days.

The data reveal that the compound of Formula I, 5-cholestene-3b,25-diol, significantly increases the solubility of α-crystallin in vivo, particularly in older subjects, thereby targeting the underlying mechanism of cataract formation.

Each document cited herein, including any cross-referenced or related patents or applications, is incorporated herein by reference in its entirety unless specifically excluded or otherwise limited. The citation of any document does not constitute an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone or in any combination with any other reference teaches, suggests, or discloses any such invention. In addition, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition of the term in this document shall prevail.

While particular embodiments of the disclosed subject matter have been illustrated and described, it will be apparent to those skilled in the art that various other changes and modifications may be made without departing from the spirit and scope of the claimed subject matter. It is therefore intended to cover all such changes and modifications in the appended claims.

Claims (12)

1. Use of a compound of formula II, or a prodrug or pharmaceutically acceptable salt thereof, in the preparation of a medicament for the treatment or prevention of cataracts in an individual of need: ^R12 is H or OH and ^R13 is H or OH. 2. The use of claim 1, wherein the compound of formula II is, or a prodrug thereof, or a pharmaceutically acceptable salt thereof. 3. Use according to any one of claims 1-2, wherein the drug is formulated for administration topically, subconjunctivally, retroocularly, periocularly, subretinally, suprachoroidally, or intraocularly. 4. Use according to any one of claims 1-3, wherein the cataract is age-related cataract, diabetic cataract, surgery-related cataract, radiation-induced cataract, cataract caused by hereditary disease, infection-induced cataract, or drug-induced cataract. 5. Use according to any one of claims 1-4, wherein the cataract is a hereditary early-onset form of cataract. 6. The use of claim 5, wherein the cataract comprises an R120G mutation and/or a D109H mutation in cryAB. 7. Use according to any one of claims 1-6, wherein the drug interacts with αA-crystal protein or αB-crystal protein, optionally wherein the interaction is to stabilize αA-crystal protein or αB-crystal protein. 8. An ophthalmic pharmaceutical composition comprising a pharmaceutically acceptable ophthalmic carrier and a compound of formula II or a prodrug or a pharmaceutically acceptable salt thereof: ^R12 is H or OH and ^R13 is OH. 9. The composition of claim 8, wherein the compound of formula II is: Or, or its prodrug or a pharmaceutically acceptable salt. 10. The composition of any one of claims 8-9, wherein the pharmaceutically acceptable ophthalmic carrier is cyclodextrin. 11. The composition of claim 10, wherein the cyclodextrin is (2-hydroxypropyl)-β-cyclodextrin. 12. The composition of any one of claims 8-11, further comprising an antioxidant.