Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
  • A61K31/716—Glucans
  • A61K31/724—Cyclodextrins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents

Definitions

• the human eye is designed so that light traverses the anterior portions (cornea, aqueous humor and lens), the vitreous and the anterior layers of the retina before reaching the outer segments of light-sensitive cells or photoreceptors (PROS), i.e., the rods and cones.
• PROS light-sensitive cells or photoreceptors
• RPE retinal pigment epithelial cells
• RPE transporters e.g., glucose, amino acids
• maintaining the ionic composition of the subretinal space space between RPE and PR
• phagocytosis i.e., “eating up and digestion of’
• RPE In the visual cycle, light impinges on cis-retinal and converts it into trans-retinal, which is quickly converted into an alcohol derivative (cis-retinol), which is transported to the RPE for regeneration of cis-retinal. All of these RPE functions are essential for normal vision.
• RPE cells do not divide, and therefore, are more susceptible to accumulation of materials as they age.
• One RPE cell disposes the debris generated by 30-50 adjacent PR cells. This debris is produced by circadian shedding of PR tips, the oldest part of PROS.
• Lipofuscin is a fine yellow-brown pigment composed of indigestible material that is believed to be remnants after lysosomal digestion.
• Ocular lipofuscin is mostly composed of dimers of retinaldehydes known as lipid bisretinoids, and small amounts of carbohydrates, oxidized proteins and metals. Accumulation of lipofuscin in retinal cells causes retinal toxicity, which is associated with conditions like macular degeneration, a degenerative disease of the eye, and Stargardt disease.
• the lipofuscin deposits in RPE cells contain almost no protein ( ⁇ 2%); rather, they are constituted of lipidic pigment derivatives of trans -retinal, generated by the visual cycle.
• the most abundant and toxic lipidic pigments found are the bisretinoids, primarily A2E, followed by A2E isomers, oxidized derivatives of A2E, A2-dihydropyridine-phosphatidylethanolamine (A2-DHP- PE), and smaller quantities of other Vitamin A conjugates belonging to the all-trans-retinal dimer series.
• A2-DHP- PE A2-dihydropyridine-phosphatidylethanolamine
• A2E-lipofuscin accumulates linearly with age, and beyond a certain threshold, A2E- lipofuscin becomes toxic to RPE cells, which eventually results in their malfunction and death. This deterioration process results in the decrease or loss of the ability of RPE cells to support adjacent PR cells. Loss of PR cells resulting from the toxic effects of lipofuscin on RPE cells is considered a central pathogenetic mechanism in genetic and age-related retinal degenerations.
• Age-related macular degeneration is the most common cause of blindness, affecting 36% of Americans in their eighth decade of life, with a devastating decrease in their quality of life.
• clinical evidence shows that photoreceptors overlying bisretinoid- loaded RPE areas (containing mostly A2E) are the most prone to degeneration.
• A2E reaches pathogenic levels typically by about 30 years of age, typically resulting in blindness in the fourth decade of life.
• A2E and its derivatives have intrinsic fluorescence and account for most of RPE- lipofuscin (RPE-LF) autofluorescence. Moreover, it is known that, with aging, the RPE-LF fluorescence shifts even more toward blue, suggesting that with time A2E deposits may adopt stiffer organization inside these granules.
• RPE-LF RPE- lipofuscin
• A2E deposits are housed within the interior of discrete membrane-bound organelles that are uniformly dense, roughly spherical, and approximately 1 micrometer in diameter.
• Data obtained using atomic force microscopy and purified granules has revealed that the bulk of A2E deposits in RPE cells reside in the lumen of these post-lysosomal bodies, forming an orderly aggregated structure. Because of their ultrastructural appearance, A2E-containing formations are sometimes also referred to as “lipofuscin granules.”
• embodiments of the present invention provide compounds and methods of treating an ocular disease or disorder associated with retinal lipofuscin accumulation.
• the invention provides a method of treating an ocular disease or disorder associated with retinal lipofuscin accumulation, wherein said treatment comprises administering to a subject in need thereof a therapeutically effective amount of a compound of formula I:
• X is O or S
• R 1 is independently selected in each instance from hydrogen, -(Ci-8)hydrocarbon, -(Ci- s)hydroxyalkyl, R 2 , and -C(O)R 2 ;
• R 2 is an acid moiety with a pKa of between 2.5 and 6.7; and n is 2 or 3; wherein between one and twelve R 1 are R 2 or -C(O)R 2 and, when more than one of R 1 is R 2 or -C(O)R 2 , each R 1 is independently selected in each instance; and wherein the compound of formula I is not P-cyclodextrin substituted only with between 2.5 and 7 succinyl groups.
• the invention provides a method of removing lipofuscin from retinal pigment epithelial cells, comprising contacting the cells with a compound of formula I:
• X is O or S
• R 1 is independently selected in each instance from hydrogen, -(Ci-8)hydrocarbon, -(Ci- s)hydroxyalkyl, R 2 , and -C(O)R 2 ;
• R 2 is an acid moiety with a pKa of between 2.5 and 6.7; and n is 2, or 3; wherein between one and twelve R 1 are R 2 or -C(O)R 2 and, when more than one of R 1 is R 2 or -C(O)R 2 , each R 1 is independently selected in each instance; and wherein the compound of formula I is not P-cyclodextrin substituted only with between 2.5 and 7 succinyl groups.
• the invention provides a compound of formula IA:
• X is O or S
• R 1 is independently selected in each instance from hydrogen, -(Ci-8)hydrocarbon, -(Ci- s)hydroxyalkyl, and -C(O)R 2 ;
• R 2 is an acid moiety comprising 2, 3, 4, 5, 6, 7, or 8 carbon atoms, having a pKa of between 2.5 and 6.7; and n is 2 or 3; wherein between one and twelve R 1 is independently in each instance -C(O)R 2 and, when more than one of R 1 is -C(O)R 2 , each R 1 is independently selected in each instance; and wherein the compound of formula IA is not 0-cyclodextrin substituted only with between 2.5 and 7 succinyl groups, y-cyclodextrin substituted only with 3 to 5 succinyl groups, 0- cyclodextrin substituted only with 3 to 5 succinyl groups and 3 hydroxypropyl groups, 0- cyclodextrin substituted only with 3 to 5 carboxymethyl groups, or 0-cyclodextrin substituted only with 3 to 5 carboxyethyl groups.
• the invention provides a pharmaceutical composition
• a pharmaceutical composition comprising a compound of formula IA disclosed herein, and a pharmaceutically acceptable excipient or carrier.
• FIG. 1 shows the comparative toxicity and acidity of cells exposed to acid cyclodextrins and to free acids.
• FIG. 2 shows confocal fluorescence images of the removal of Ocular Lipofuscin from ARPE19 cultures by various cyclodextrins.
• FIG. 3A shows a comparison of the speed of removal of lipofuscin with acid cyclodextrins versus the speed with conventional cyclodextrins.
• FIG. 3B demonstrates the removal of lipofuscin from cells using acid cyclodextrins versus conventional cyclodextrins.
• FIG. 4 demonstrates the expulsion of lipofuscin in the soluble fraction of the supernatant using conventional cyclodextrins and acid cyclodextrins.
• FIG. 5 shows flat mounted RPE eyecups showing the autofluorescence lipofuscin in green in (A) unextracted and (B) acid cyclodextrin-treated eyes.
• FIG. 6 shows a comparison of the removal of lipofuscin and preservation of cell number in untreated cells and in those treated with an acid cyclodextrin.
• FIG. 7 shows the reduction of lipofuscin after single IVT administration of various acid cyclodextrins.
• FIG. 8 shows levels of visual acuity determined by OKT, before and after treatment with acid cyclodextrins.
• FIG. 9 shows a comparison of the levels of ocular lipofuscin in the RPE of control and acid cyclodextrin treated eyes.
• FIG. 10 shows a comparison of the levels of ocular lipofuscin in control and acid cyclodextrin treated eyes.
• FIG. 11 shows a comparison of the in vivo removal of ocular lipofuscin in control cells and in those treated with a non-cyclodextrin acid.
• FIG. 12 shows a comparison of the in vivo removal of lipofuscin in control cells and in those treated with an acid cyclodextrin.
• FIG. 13 shows a comparison of the in vivo removal of lipofuscin in control cells and in those treated with an acid alpha-cyclodextrin.
• FIG. 14 shows a comparison of the in vivo removal of lipofuscin in control cells and in those treated with a non-acid cyclodextrin.
• FIG. 15 shows a comparison of the in vivo removal of lipofuscin in control cells and in those treated with a non-acid cyclodextrin.
• FIG. 16 demonstrates the ability of acid cyclodextrins, in comparison to non-acid cyclodextrin molecules, to clear beta-hexosaminidase from ARPE19 cells.
• FIG. 17 shows a comparison of various cyclodextrins in ocular lipofuscin removal.
• FIG. 18 demonstrates the ability of an acid cyclodextrin to rescue RPE cells from toxicity.
• Embodiments of the inventive method are distinguished from the disclosures references disclosed herein, each of which is hereby incorporated herein by reference.
• Cyclodextrins (CDs) with one or more acidic group(s) and with affinity for lipid bisretinoids can be used effectively to remove ocular lipofuscin from RPE and, therefore, for treating eye diseases associated with retinal lipofuscin accumulation, such as Stargardt Disease and Age-Related Macular degeneration (AMD), as well as cone-rod dystrophies or retinitis pigmentosa, in elderly individuals or patients with defects in the metabolism of retinaldehydes.
• AMD Age-Related Macular degeneration
• Cyclodextrins are well known enhancers of solubility, stability and permeability of guest molecules due, in part, to their ability to dissolve lipids and transiently destabilize biological membranes. Although they are cell membrane impermeant molecules, they are avidly taken up, through clathrin-mediated endocytosis. Without being held to any one theory, it is hypothesized that endocytic uptake gives cyclodextrins prompt access to lipofuscin buildups in secondary lysosomes.
• cyclodextrins can then dissolve lipophilic materials from previously considered unremovable lysosomal deposits.
• Their ability to perturb, without damaging, cellular membranes favors their leakage into the cytosol; this is an important step in which only acid cyclodextrins will generate cytoplasmic protons that will activate multiple acid-removal pathways.
• These include: (1) increased proton uptake into lysosomes which, coupled with (2) increased lysosomal exocytosis, allows the cells to efficiently promote cytosolic alkalization and extracellular acidification.
• Free acids in contrast, cannot do the same. To percolate into cells, free acids require higher concentrations, which are detrimental to cell membranes and, most importantly, they lack the capacity to dissolve lipofuscin.
• canonical cyclodextrins such as methyl -beta-CD, hydroxypropyl-beta-CD, and sulfobutyl-beta-CD dissolve lipofuscin well, they cannot trigger the exocytic response and, therefore, they are less efficient in eliminating lysosomal aggregates from cells.
• the current application discloses acid cyclodextrins as a method to induce cellular emesis to remove solubilized lipofuscin; this is used to treat ocular disorders.
• the invention provides a method of treating an ocular disease or disorder associated with retinal lipofuscin accumulation, wherein said treatment comprises administering to a subject in need thereof a therapeutically effective amount of a compound of formula I:
• X is O or S
• R 1 is independently selected in each instance from hydrogen, -(Ci-s)hydrocarbon, -(Ci- sjhydroxyalkyl, R 2 , and -C(O)R 2 ;
• R 2 is an acid moiety with a pKa of between 2.5 and 6.7; and n is 2 or 3; wherein between one and twelve R 1 are R 2 or -C(O)R 2 and, when more than one of R 1 is R 2 or -C(O)R 2 , each R 1 is independently selected in each instance; and wherein the compound of formula I is not P-cyclodextrin substituted only with between 2.5 and 7 succinyl groups.
• the invention provides a method of removing lipofuscin from retinal pigment epithelial cells, comprising contacting the cells with a compound of formula I: wherein:
• X is O or S
• R 1 is independently selected in each instance from hydrogen, -(Ci-s)hydrocarbon, -(Ci- s)hydroxyalkyl, R 2 , and -C(O)R 2 ;
• R 2 is an acid moiety with a pKa of between 2.5 and 6.7; and n is 2 or 3; wherein between one and twelve R 1 are R 2 or -C(O)R 2 and, when more than one of R 1 is R 2 or -C(O)R 2 , each R 1 is independently selected in each instance; and wherein the compound of formula I is not P-cyclodextrin substituted only with between 2.5 and 7 succinyl groups.
• the invention provides a method of removing lipofuscin from retinal pigment epithelial cells, comprising contacting the cells with a compound of formula I, as shown supra.
• the invention provides a compound of formula IA: wherein:
• X is O or S
• R 1 is independently selected in each instance from hydrogen, -(Ci-8)hydrocarbon, -(Ci- s)hydroxyalkyl, and -C(O)R 2 ;
• R 2 is an acid moiety comprising 2, 3, 4, 5, 6, 7, or 8 carbon atoms, having a pKa of between 2.5 and 6.7; and n is 2 or 3; wherein between one and twelve R 1 is independently in each instance -C(O)R 2 and, when more than one of R 1 is -C(O)R 2 , each R 1 is independently selected in each instance; and wherein the compound of formula IA is not 0-cyclodextrin substituted only with between 2.5 and 7 succinyl groups, y-cyclodextrin substituted only with 3 to 5 succinyl groups, 0- cyclodextrin substituted only with 3 to 5 succinyl groups and 3 hydroxypropyl groups, 0- cyclodextrin substituted only with 3 to 5 carboxymethyl groups, or 0-cyclodextrin substituted only with 3 to 5 carboxyethyl groups.
• cyclodextrins are typically composed of five or more glucose (i.e., glucopyranoside) units connected in a ring structure, linked as in amylose by alpha 1-4 (i.e., alpha(l — >4)) bonds.
• the cyclodextrins considered herein can conveniently be represented by formula I or formula IA, as shown supra.
• n is 2. In some embodiments, n is 3.
• X is O. In other embodiments, X is S.
• R 1 is independently selected in each instance from hydrogen, -(Ci-8)hydrocarbon, -(Ci-8)hydroxy alkyl, R 2 , and -C(O)R 2 .
• R 1 is independently selected in each instance from hydrogen, -(Ci-8)hydrocarbon, -(Ci-8)hydroxyalkyl, and -C(O)R 2 .
• R 1 between one and twelve R 1 is -C(O)R 2 and, when more than one of R 1 is -C(O)R 2 , each R 1 is independently selected in each instance.
• R 1 is independently selected in each instance, so, as a non-limiting example, some values of R 1 may be hydroxypropyl, some may be hydrogen, and others may be adipic acid in the same compound of formula I or formula IA.
• between one and four of R 1 are independently in each instance R 2 or -C(O)R 2 , between zero and four of R 1 are independently in each instance - (Ci-8)hydroxyalkyl, and the remainder of R 1 are hydrogen.
• between three and four of R 1 are independently in each instance R 2 or -C(O)R 2 , between zero and three of R 1 are independently in each instance -(Ci-8)hydroxyalkyl, and the remainder of R 1 are hydrogen.
• between one and eleven, one and ten, one and nine, one and eight, one and seven, one and six, one and five, one and four, one and three, or one and two of R 1 are independently in each instance R 2 or -C(O)R 2 .
• between two and twelve, two and eleven, two and ten, two and nine, two and eight, two and seven, two and six, two and five, two and four, or two and three of R 1 are independently in each instance R 2 or -C(O)R 2 .
• between three and twelve, three and eleven, three and ten, three and nine, three and eight, three and seven, three and six, three and five, or three and four of R 1 are independently in each instance R 2 or -C(O)R 2 . In some embodiments of formula I, between four and twelve, four and eleven, four and ten, four and nine, four and eight, four and seven, four and six, or four and five of R 1 are independently in each instance R 2 or -C(O)R 2 . In some embodiments of formula I, between five and twelve, five and eleven, five and ten, five and nine, five and eight, five and seven, or five and six of R 1 are independently in each instance R 2 or -C(O)R 2 .
• between six and twelve, six and eleven, six and ten, six and nine, six and eight, or six and seven of R 1 are independently in each instance R 2 or -C(O)R 2 . In some embodiments of formula I, between seven and twelve, seven and eleven, seven and ten, seven and nine, or seven and eight of R 1 are independently in each instance R 2 or -C(O)R 2 . In some embodiments of formula I, between eight and twelve, eight and eleven, eight and ten, or eight and nine of R 1 are independently in each instance R 2 or -C(O)R 2 .
• R 1 between nine and twelve, nine and eleven, or nine and ten of R 1 are independently in each instance -C(O)R 2 . In some embodiments of formula I, one, two, three, four, five, six, seven, eight, nine, or ten of R 1 are independently in each instance R 2 or -C(O)R 2 . In some embodiments of formula I, six of R 1 are independently in each instance R 2 or -C(O)R 2 . In some embodiments of formula I, at least three of R 1 are independently in each instance -(Ci-8)hydroxyalkyl. In some embodiments of formula I, between three and eight of R 1 are independently in each instance -(Ci-8)hydroxyalkyl. In some embodiments of formula I, the -(Ci-s)hydroxyalkyl is hydroxypropyl. In some embodiments of formula I, two R 1 are independently in each instance R 2 or -C(O)R 2 .
• between one and eleven, one and ten, one and nine, one and eight, one and seven, one and six, one and five, one and four, one and three, or one and two of R 1 are independently in each instance -C(O)R 2 .
• between two and twelve, two and eleven, two and ten, two and nine, two and eight, two and seven, two and six, two and five, two and four, or two and three of R 1 are independently in each instance -C(O)R 2 .
• between three and twelve, three and eleven, three and ten, three and nine, three and eight, three and seven, three and six, three and five, or three and four of R 1 are independently in each instance -C(O)R 2 . In some embodiments of formula IA, between four and twelve, four and eleven, four and ten, four and nine, four and eight, four and seven, four and six, or four and five of R 1 are independently in each instance - C(O)R 2 . In some embodiments of formula IA, between five and twelve, five and eleven, five and ten, five and nine, five and eight, five and seven, or five and six of R 1 are independently in each instance -C(O)R 2 .
• between six and twelve, six and eleven, six and ten, six and nine, six and eight, or six and seven of R 1 are independently in each instance -C(O)R 2 . In some embodiments of formula IA, between seven and twelve, seven and eleven, seven and ten, seven and nine, or seven and eight of R 1 are independently in each instance - C(O)R 2 . In some embodiments of formula IA, between eight and twelve, eight and eleven, eight and ten, or eight and nine of R 1 are independently in each instance -C(O)R 2 . In some embodiments of formula IA, between nine and twelve, nine and eleven, or nine and ten of R 1 are independently in each instance -C(O)R 2 .
• one, two, three, four, five, six, seven, eight, nine, or ten of R 1 are independently in each instance -C(O)R 2 .
• six of R 1 are independently in each instance -C(O)R 2 .
• at least three of R 1 are independently in each instance -(Ci- 8)hydroxy alkyl.
• between three and eight of R 1 are independently in each instance -(Ci-8)hydroxyalkyl.
• the - (Ci-8)hydroxy alkyl is hydroxy propyl.
• two R 1 are independently in each instance -C(O)R 2 .
• R 1 may also be R 2 , i.e., an ether linkage.
• R 2 is an acid moiety with a pKa of between 2.5 and 6.7 (e.g., 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,
• R 2 is an acid moiety with a pKa of between 4.0 and 6.0 (e.g., 4.0, 4. 1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,
• R 2 is an acid moiety with a pKa of between 4.75 and 5.0 (e.g., 4.75, 4.80, 4.85, 4.90, 4.95, or 5.00, including any and all values, ranges, and subranges within).
• R 2 is an acid moiety with a pKa of between 4.0 and 5.0 (e.g., 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, including any and all values, ranges, and subranges within).
• R 2 is an acid moiety with a pKa of between 4.2 and 5.0 (e.g., 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, including any and all values, ranges, and subranges within). In some embodiments, R 2 is an acid moiety with a pKa of between 4.2 and 4.5 (e.g., 4.2, 4.3, 4.4, 4.5, including any and all values, ranges, and subranges within).
• R 2 is an acid moiety with a pKa of between 2.8 and 6.0 (e.g., 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, including any and all values, ranges, and subranges within).
• R 2 is an acid moiety with a pKa of between 2.8 and 4.9 (e.g., 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, including any and all values, ranges, and subranges within).
• R 2 is an acid moiety with a pKa of between 4.3 and 4.9 (e.g., 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, including any and all values, ranges, and subranges within).
• R 2 is an acid moiety with a pKa of greater than or equal to 1.3.
• Acid moi eties with a pKa of between 2.5 and 6.7 include, but are not limited to, formic acid, acetic acid, propionic acid, butyric acid, pentanoic acid, hexanoic acid, heptanoic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, sorbic acid, fumaric acid, malic acid, maleic acid, tartaric acid, citric acid, and propanetricarboxylic acid.
• carboxylic acids, amine-containing moieties, such as imidazole can provide an effective pKa between 2.5 and 6.7 (or any of the other ranges, subranges or values supra).
• the imidazole group provides a pKa within the range between 2.5 and 6.7.
• the imidazole containing carboxylic acids are ester bond attached to cyclodextrin through carboxylic acid functionality, a remaining side group will provide the acidic moiety with a pKa within the range between 2.5 and 6.7.
• so-called “basic amino acids” such as histidine can be attached to cyclodextrin through the acid and nonetheless retain functionality when one of its amines/imines is protonated.
• a preferred counteranion for protonation is one whose conjugate acid is physiologically tolerated, for example, a chloride.
• R 2 is -C(O)(CH2)mCOOH. In other embodiments, R 2 is - (CH 2 )mC00H.
• R 2 is formic acid, acetic acid, propionic acid, butyric acid, pentanoic acid, hexanoic acid, heptanoic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, fumaric acid, malic acid, maleic acid, tartaric acid, citric acid, or propanetricarboxylic acid.
• R 2 is succinic acid.
• R 2 is glutaric acid.
• R 2 is an imidazole containing acid.
• R 2 is histidine.
• m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6. In some embodiments, m is 7. In some embodiments, m is 8. In some embodiments, m is 9. In some embodiments, m is 10.
• the invention is directed to method of treating an ocular disease or disorder associated with retinal lipofuscin accumulation.
• Conditions and diseases treatable by the method described herein include any ophthalmologic or retinal disorder, condition, or disease directly or indirectly caused by the accumulation of lipofuscin in retinal pigment epithelium (RPE) cells, which may be genetic or non-genetic, or is a disease resulting in defects in the metabolism of retinaldehydes.
• the ocular disease or disorder is Stargardt Disease, Age-Related Macular degeneration (AMD), a cone-rod dystrophy, retinitis pigmentosa, Best Disease, or cone-rod dystrophy.
• the acid cyclodextrin has affinity for the lipofuscin, which means it can form complexes with the lipids in the lipofuscin and dissolve it away once it is expelled to the surface of the cell.
• CD is cyclodextrin
• HPBCD is hydroxypropyl beta CD
• HPGCD is hydroxypropyl gamma CD
• HP SB is hydroxypropyl succinyl acid beta
• HPSG is hydroxypropyl succinyl acid gamma
• CD MBCD is methyl beta CD
• SBE is sulfobutyl ether beta
• SUB is succinyl acid beta
• SUB4 is succinyl acid beta CD with a DS of 4
• SUB 10 is succinyl acid beta CD with a DS of 10
• Ci to Cs hydrocarbon includes alkyl, cycloalkyl, poly cycloalkyl, alkenyl, alkynyl, aryl and combinations thereof. Examples include benzyl, phenethyl, cyclohexylmethyl, adamantyl, camphoryl and naphthylethyl. “Hydrocarbyl” or “hydrocarbon” refers to any substituent comprised of hydrogen and carbon as the only elemental constituents. Aliphatic hydrocarbons are hydrocarbons that are not aromatic; they may be saturated or unsaturated, cyclic, linear or branched.
• aliphatic hydrocarbons examples include isopropyl, 2-butenyl, 2-butynyl, cyclopentyl, norbornyl, etc.
• Aromatic hydrocarbons include benzene (phenyl), naphthalene (naphthyl), anthracene, etc.
• alkyl (or alkylene) is intended to include linear or branched saturated hydrocarbon structures and combinations thereof.
• Alkyl refers to alkyl groups from 1 to 20 carbon atoms, or from 1 to 10 carbon atoms, or from 1 to 6 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl, t-butyl and the like.
• Cycloalkyl is a subset of hydrocarbon and includes cyclic hydrocarbon groups of from 3 to 8 carbon atoms. Examples of cycloalkyl groups include cy-propyl, cy-butyl, cy-pentyl, norbomyl and the like.
• the “degree of substitution” (or “ds” or “d.s.” or “DS”) of a cyclodextrin indicates the average number of non-hydrogen substituents at the -R 1 positions of the compounds of formula I and formula IA.
• a P-cyclodextrin for instance, has 21 -R 1 positions and, thus, has the potential for up to 21 substitutions with non-hydrogen moieties, i.e., a degree of substitution of 21.
• a P-cyclodextrin with succinic acid present at ten -R 1 positions would have a ds of ten (or “SUB 10,” for “succinyl beta 10”).
• degree of substitution is meant to represent an average number of non-hydrogen substituents.
• a P-cyclodextrin with succinic acid at a degree of substitution of 3.5 may have molecules with substitutions between 2 and 5, with an average ds of 3.5.
• an exclusionary proviso that states, “wherein the compound of formula IA is not P-cyclodextrin substituted only with between 2.5 and 7 succinyl groups” does not exclude a P-cyclodextrin substituted only with a ds of more than 7 succinyl groups.
• P-cyclodextrins substituted with only 7.4, or 7.5, or 8, or 10, or more than 8, or more than 7.4 succinyl groups are included in the claimed subject matter, as is a P-cyclodextrin substituted with only 2, or 2.4, or fewer than 2.5 succinyl groups.
• treatment is intended to encompass any beneficial or ameliorating effect on lipofuscin-associated damage or associated disease or condition directly or indirectly caused by the accumulation of lipofuscin bisretinoid lipid in RPE cells.
• treatment may also include prevention of lipofuscin -associated damage or a disease or condition directly or indirectly associated with lipofuscin-associated damage or the accumulation of lipofuscin in RPE cells in a subject that is at risk of (i.e., not yet suffering from) lipofuscin-associated damage or accumulation, or a disease or condition directly or indirectly associated therewith.
• treatment may also include prophylaxis, therapy, and/or cure.
• the treatment considered herein has the effect of stopping, mitigating, or reversing the accumulation of lipofuscin bisretinoid lipid in RPE cells, and likewise, stopping, mitigating, or reversing the lipofuscin-associated damage or associated disease or condition.
• the method accomplishes this by solubilizing and removing lipofuscin bisretinoid lipid in RPE cells using a compound of formula I or formula IA.
• a method or device, composition, etc. that “comprises”, “has”, “includes” or “contains” one or more steps or elements possesses those one or more steps or elements, but is not limited to possessing only those one or more steps or elements.
• a step of a method or an element of a composition or article that “comprises”, “has”, “includes” or “contains” one or more features possesses those one or more features, but is not limited to possessing only those one or more features.
• the compound of formula I or formula IA can be administered by any route that permits the compound to contact RPE cells.
• the administration can be, for example, ocular, parenteral (e.g., subcutaneous, intramuscular, or intravenous), topical, transdermal, intravitreous, retro-orbital, subretinal, subscleral, oral, sublingual, or buccal modes of administration.
• Some of the foregoing exemplary modes of administration can be achieved by injection. However, in some embodiments, injection is avoided by use of a slow -release implant in the vicinity of the retina (e.g., subscleral route) or by administering drops to the conjuctiva.
• the compound of formula I or formula IA is administered via topical, intravitreous, intraocular, subretinal, or subscleral administration. In some embodiments, the compound is administered via intravitreous administration. In certain embodiments, subscleral administration is achieved by implanting a slow-release subscleral implant in the subject.
• the compound of formula I or formula IA is administered in a therapeutically-effective amount (i.e., therapeutically-effective dosage).
• a therapeutically-effective amount i.e., therapeutically-effective dosage
• therapeutically-effective dosage corresponds to an amount of active agent effective for providing any of the desired therapeutic effects described above, preferably without a substantial toxic effect to the subject.
• the compound of formula I or formula IA is administered, at least initially, at levels lower than that required in order to achieve a desired therapeutic effect, and the dose gradually or suddenly increased until a desired effect is achieved.
• the active compound is administered, at least initially, at levels higher than that required in order to accelerate a desired therapeutic effect, and the dose gradually or suddenly moderated until a desired effect is achieved.
• the selected dosage level will depend upon several factors, as determined by a medical practitioner.
• Some of these factors include the type of disease or condition being treated, the stage or severity of the condition or disease, the efficacy of the active compound being used and its bioavailability profile, as well as the specifics (e.g., genotype and phenotype) of the subject being treated, e.g., age, sex, weight, and overall condition.
• the dosage can be, for example, in the range of about 0.01, 0.1, 0.5, 1, 5, or 10 mg per kg of body weight per day to about 20, 50, 100, 500, or 1000 mg per kilogram of body weight per day, or bi-daily, or twice, three, four, or more times a day.
• the dosage can disregard body weight, and can be in smaller amounts (e.g., 1-1000 pg per dose, or about 1-500 pg per dose, or about 1-100 pg per dose, or about 1-50 pg per dose, or about 1-25 pg per dose, or about 1-10 pg per dose, or about 1-1000 mM per dose, or about 1-500 mM per dose, or about 1-100 mM per dose, or about 1-50 mM per dose, or about 1-25 mM per dose, or about 1-10 mM per dose, or about 1-5 mM per dose).
• the daily dose of the active compound is the lowest dose effective to produce a therapeutic effect.
• the active compound is not administered in discrete dosages, but in a continuous mode, such as provided by a slow release implant or intravenous line.
• the compound of formula I or formula IA in order for the compound of formula I or formula IA to be administrable to a subject, the compound is formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents, as well known in the art of pharmaceutical compositions.
• compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) sublingually; (5) ocularly; (6) transdermally; or (7) nasally.
• oral administration for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses
• phrases “pharmaceutically acceptable” is used herein to refer to those compounds, materials, compositions, and/or dosage forms that are, within the scope of sound medical judgment, suitable for entering a living organism or living biological tissue, preferably without significant toxicity, irritation, or allergic response.
• phrases “pharmaceutically-acceptable carrier,” as used herein, generally refers to a pharmaceutically-acceptable composition, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, useful for introducing the active agent into the body.
• a pharmaceutically-acceptable composition such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, useful for introducing the active agent into the body.
• manufacturing aid e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid
• solvent encapsulating material useful for introducing the active agent into the body.
• Each carrier must be “acceptable” in the sense of being compatible with other ingredients of the
• aqueous and non-aqueous carriers examples include, for example, water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate), and suitable mixtures thereof.
• polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
• vegetable oils such as olive oil
• injectable organic esters such as ethyl oleate
• materials that can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydrox
• the cyclodextrins disclosed herein are administered as a standalone therapy and are not administered with a guest molecule, i.e., they are not administered as a part of a clathrate or complex.
• the cyclodextrin products were dissolved in water, and their experimental pKas were determined by acid-base titration, using a pH meter.
• the beta-hexosaminidase assay measures lysosomal exocytosis in RPE cells.
• This lysosomal enzyme release assay measures accumulatively the extent of lysosomal exocytosis from RPE cells over a period of time. By comparing with the activity of the total cell-associated P-hexosaminidase, this assay was used to estimate the size of the releasable lysosomal pool for specific stimuli and conditions in a given time. In the case of acidic cyclodextrins, it was extremely high (i.e., more than 90% of the lysosomes in RPE cells are releasable).
• ARPE19 cells were seeded in a 96-well plate (45,000/well) with DMEM+10%FBS, and treatment occurred 48h after seeding.
• the cells were treated with the cyclodextrin (at the desired time and concentration) in TransfectagroTM media (Coming, a reach and defined media that contains no FBS).
• TransfectagroTM media Coming, a reach and defined media that contains no FBS.
• the top media was removed into a black clear bottom glass plate (96 well), and 25ul of P-Hexosaminidase working substrate solution was added into each well of black plate (so, total became 200ul).
• the + symbols represent a semiquantitative, visual method for evaluating the results (that is, “++” indicates a low level of lysosomal exocytosis over the time period measured, while “+++++++” indicates a very high level of lysosomal exocytosis).
• Lipofuscin affinity was determined using a microplate reader fluorescence assay. The assay was developed considering that ocular lipofuscin is almost exclusively composed of lipid bisretinoids of which A2E is one of the most abundant and best studied. A2E and other lipid bisretinoids’ fluorescence change with their interaction with other molecules in the environment, to detect formation of inclusion complexes. Insertion of lipid bisretinoids into the hydrophobic cavity of cyclodextrins protects their fluorescence from quenching by the water which leads to an increase in intensity and a solvatochromic shift toward the blue. The bigger the shift, the higher the CI) affinity for A2E.
• A2E tends to form aggregates in aqueous environment, so it cannot cross 0.2 um membrane filters, despite having a small molecular weight of 592.
• 200 ul 25 uM A2E dilutions in water or in 25mM cyclodextrins were prepared. After 4 hrs incubation at RT in the dark, fluorescence was determined before (pre-filtration) and after passing it through a 0.2 um syringe filter (pre-filtration fluorescence). In this way, passage of A2E fluorescence through the filter indicated the formation of soluble complexes between A2E and cyclodextrins.
• ARPE19 cells were preloaded with A2E by growing them in serum free media supplemented with 10 pM A2Efor 4 days.
• A2E is one of the most abundant lipid bisretinoids found in the retina and was used as a surrogate of ocular lipofuscin.
• Cells were then treated with 5 mM CDs for 24 hrs in the same serum free media. Lipofuscin content was proportional to the amount of green fluorescence.
• the + gives a semi -quantitative, visual assessment of the percentage of lipofuscin removal (that is, “++” indicates a low level of lysosomal exocytosis over the time period measured, while “++++-1-1-1-” indicates a very high level of lysosomal exocytosis).
• SUB 12 induces significantly high levels of lysosomal exocytosis from RPE cells in the beta-hex test, but shows very low affinity for and solubilization of lipofuscin.
• SUB2 was a moderate inducer of lysosomal exocytosis from RPE cells but exhibited the highest affinity for lipofuscin and showed excellent lipofuscin removal, while the lot with an intermediate degree of substitution (designated SUB 10) was an excellent inducer of exocytosis from RPE cells but had a lower affinity and solubilization activity.
• Example 1 Comparative toxicity and acidity of cells exposed to acid cyclodextrins and free acids.
• Assays were performed to assess the comparison of acid cyclodextrins and free acids on the toxicity and the acidity of cell. For these assays, cells were seeded two days before the assay to have 80% confluency at the moment of the assay. Cells were treated with 1, 5, 10, or 50 mM of free acid (0.4N HC1 or 1.0N HC1) or Succinyl P CD (SUB4), in serum free medium for 1 hour at 37 °C. Viability was assessed in a confocal fluorescence microscope using 0.45 pM DRAQ7, a cell impermeant far-red dye that stains red the nuclei, only when cells are dead.
• Example 2 Removal of Ocular Lipofuscin from ARPE19 cultures after 24 hours.
• ARPE19 cells were preloaded with A2E by growing them in serum free media supplemented with 10 pM A2E for 4 days.
• A2E is one of the most abundant lipid bisretinoids found in the retina and was used as a surrogate of ocular lipofuscin.
• Cells were then treated with 5 mM CDs for 24 hrs in the same serum free media.
• Confocal fluorescence images of ARPE cultures preloaded with ocular lipofuscin green autofluorescence
• Lipofuscin content was proportional to the amount of green fluorescence.
• DRAQ7 was used for viability and Hoechst to show that the reduction in the green was not due to the loss/detachment of cells.
• lipofuscin was partially removed by the conventional CDs, i.e., Methyl Beta CD and Hydroxypropyl Beta CD but was totally removed by the acid Succinyl Beta CD.
• the blue coloring is Hoechst, a cell permeable dye that stains all nuclei in cells irrespective whether they are dead or alive.
• DRAQ7 is a cell impermeant dye that cannot stain the nuclear DNA unless there is a rupture in the plasma membrane that allows it to enter, so it only stains dead cells.
• Example 3 Speed of removal of lipofuscin.
• FIG. 3A and FIG. 3B demonstrate that the removal of lipofuscin with acid CDs is much faster than the removal with conventional CDs. Removal was determined with a fluorescence plate. 4% final concentration of Triton XI 00 was added to supernatants, and adherent cells (as described above) were lysed with 4% Triton XI 00 in serum free media, as well. A2E content was measured using a fluorescence plate reader to determine the stochiometric redistribution of lipofuscin after cyclodextrin treatments.
• FIG. 3A shows the comparison of no treatment with cyclodextrin (top line), lOmM non-acid cyclodextrin (methyl beta CD, middle line), and lOmM acid cyclodextrin (SUB4, bottom line).
• FIG. 3B shows the removal of lipofuscin at 4 hours with classical non-acid versus acid Cyclodextrins.
• nonacid cyclodextrins MBCD is methyl beta CD
• HPBCD is hydroxypropyl beta CD
• SBE is sulfobutyl ether beta CD
• HPGCD is hydroxypropyl gamma CD.
• Example 4 Stoichiometry of the removal of lipofuscin.
• a removal assay was performed as described in Example 3, but the supernatants were collected after 24 hrs of treatment with media alone or media supplemented with 10 mM of the indicated CDs, then centrifuged at the indicated speeds to determine the fraction in which the A2E was released.
• FIG. 4 shows that lipofuscin is expelled in the soluble fraction of the supernatant. The extraction was done for 24 hrs. with lOmM CDs. As can be seen, very little lipofuscin came out with the standard CD as compared with the acid CD at the same concentration during the same period. The results indicate that extraction of A2E by SUB4 neither occurs due to the loss of loaded cells, lysis and release of organelles, nor to exosome production, but rather by promoting the release of solubilized lipid bisretinoid material.
• Example 5 Stoichiometry of the removal of lipofuscin.
• FIG. 5 demonstrates the removal of lipofuscin in unextracted (mock treated) and succinyl beta treated eyes, and shows that the acid CD removes much more lipofuscin than does the vehicle control.
• Example 6 Demonstration of the removal of lipofuscin and preservation of cell number.
• Phalloidin green fluorescence
• a lectin that stains actin fibers allows visualization of RPE cell borders in flat-mounted RPE eyecups collected from 1 month old DKOs, 4 days after intravitreal administration of vehicle-control or acid CD (SUB4).
• FIG. 6 shows that lipofuscin (green) was eliminated and RPE cell number was preserved after treatment with SUB4.
• Example 7 Quantification of in vivo removal with acid cyclodextrins.
• FIG. 7 shows the reduction of lipofuscin in vivo after a single 1.5ul IVT administration of succinylated cyclodextrins or glucose (non-treated control) 250mM in PBS in the left and right eyes, respectively.
• Retinal pigment epithelium (RPE)-ey ecups were flat mounted and subjected to autofluorescence microscopy at 63X magnification. Multiple pictures were stitched together to show the complete eyecups. Stitched images were converted to gray scale and mean fluorescence intensity of the retinas were measured using Image J (NIH). No cataracts irritation nor discomfort were observable after injections with any of the Cyclodextrins. Lipofuscin (%) remaining in the eyes were compared to eyes from animals that received the same volume of vehicle (water) in their eyes. Significance was determined by 2 tails, unpaired T-tests.
• Example 8 Visual acuity before and after acid CD treatment.
• FIG. 8 shows visual acuity determined by opticokinetic tracking (OKT), before and after treatment with succinylated CDs.
• SPATIAL FREQUENCY C/D represents cycle/distance or frequency and is a measurement of how thin the bars can be and still be detected. The higher the frequency, the thinner the bars, and the better is the visual acuity.
• the removal of Lipofuscin with Acid-Cyclodextrins did not impair significantly visual acuity.
• Example 9 Treatment of ocular lipofuscin with succinic acid beta cyclodextrins in DKO mice eyes.
• Ocular lipofuscin is progressively accumulated in the lysosomes of the RPE cells with age. Older animals contain more lipofuscin than young ones, which manifests as wider peaks and larger areas under the curves regarding %RPE versus ocular lipofuscin content. Different animals have different content of ocular lipofuscin, so it is important to compare the treated eye vs. the companion eye (i.e., untreated) in the same animal. Therefore, lipofuscin content was always compared between control (left) and treated (right) eyes from the same animal, allowing for an independent assessment of the removal with respect to the original content of those eyes.
• A shows representative images of the RPE in the eyes after mock (left) or 3mM succinic acid beta CD (right) treatments for 1.5 hrs. The ocular lipofuscin is shown in green. RPE cells were stained with Hoechst (blue nucleus) and Phalloidin (red borders) and imaged using confocal microscopy.
• B shows the quantification of the levels of ocular lipofuscin in 10 random fields of the RPE, done using Image J software.
• the comparison is shown between the areas of the untreated and the treated peaks, i.e., the treated eye and the companion eye in the same animal.
• the cells of the treated eye showed a marked decrease in the ocular lipofuscin content as compared to the untreated eye.
• the ocular lipofuscin removal did not affect the integrity of RPE cells and was completed after 1.5 hrs.
• FIG. 1 shows representative images of the RPE in the eyes after mock (left) or 1.5mM SUB7.4 (right) treatments for 1.5 hrs.
• the ocular lipofuscin is shown in green.
• RPE cells were stained with Hoechst (blue nucleus) and Phalloidin (red borders) and imaged using confocal microscopy.
• (B) shows the quantification of the levels of ocular lipofuscin in 10 random fields of the RPE using Image J software. No significant RPE detachment was observed, indicating that the treatment was well tolerated and that the ocular lipofuscin removal did not affect the integrity of RPE cells. The treatment was completed in 1.5 hrs.
• FIG. 11 shows a comparison of the removal of ocular lipofuscin from the eyes of DKO mice in control cells and in those treated with 10.2mM of a non-cyclodextrin containing acid, succinic acid mono ethyl ester (SUCCA), that is, treated with the same number of acid equivalents as with SUB3.4 but without the cyclodextrin moiety.
• A shows representative images of the RPE in the eyes treated with mock (left) or 10.2mM SUCCA (right) treatments for 1.5 hrs. The ocular lipofuscin is shown in green. RPE cells were stained with Hoechst (blue nucleus) and Phalloidin (red borders) and imaged using confocal microscopy.
• (B) shows the quantification of the levels of ocular lipofuscin in 10 random fields of the RPE using Image J software. As can be seen, the succinic acid without the cyclodextrin ring could not remove the ocular lipofuscin.
• Example 11 Treatment of ocular lipofuscin with oxalic acid beta cyclodextrin in DKO mice eyes.
• FIG. 12 shows a comparison of the removal of ocular lipofuscin from the eyes of DKO mice in control cells and in those treated with 3mM of oxalic acid beta cyclodextrin (DS 3.4).
• A shows representative images of the RPE in the eyes treated with mock (left) or 3mM oxalic acid beta cyclodextrin (right) treatments for 1.5 hrs. The ocular lipofuscin is shown in green.
• RPE cells were stained with Hoechst (blue nucleus) and Phalloidin (red borders) and imaged using confocal microscopy.
• B shows the quantification of the levels of ocular lipofuscin in 10 random fields of the RPE using Image J software. The comparison is between areas of the peaks from untreated and treated eyes in the same animal. As can be seen, the 1.5 hrs of treatment with oxalic acid beta cyclodextrin did not affect the integrity of RPE, but the oxalic acid beta cyclodextrin failed to remove the lipofuscin from the cells.
• Example 12 Treatment of ocular lipofuscin with succinic acid alpha cyclodextrin in DKO mice eyes.
• FIG. 13 shows a comparison of the removal of ocular lipofuscin from the eyes of DKO mice in control cells and in those treated with an acid alpha cyclodextrin (i.e., having a smaller cavity).
• A shows representative images of the RPE in the eyes treated with mock (left) or 3mM succinic acid alpha CD (SUA) (DS 3) (right) treatments for 1.5 hrs. The ocular lipofuscin is shown in green. RPE cells were stained with Hoechst (blue nucleus) and Phalloidin (red borders) and imaged using confocal microscopy.
• B shows the quantification of the levels of ocular lipofuscin in 10 random fields of the RPE using Image J software.
• Example 13 Treatment of ocular lipofuscin with a non-acid cyclodextrin in DKO mice eyes.
• FIG. 14 shows a comparison of the removal of ocular lipofuscin from the eyes of DKO mice in control cells and in those treated with non-acid cyclodextrin.
• A shows representative images of the RPE in the eyes treated with mock (left) or 3mM Sulfo Butyl Ether Beta-CD (SBE) (right) treatments for 1.5 hrs. The ocular lipofuscin is shown in green. RPE cells were stained with Hoechst (blue nucleus) and Phalloidin (red borders) and imaged using confocal microscopy.
• B shows the quantification of the levels of ocular lipofuscin in 10 random fields of the RPE using Image J software.
• the SBE potency was not enough to reduce the content of lipofuscin in the RPE under the current treatment conditions. Further, there appears to be a reduction in the level of lipofuscin, due not to removal of lipofuscin from RPE cells but to the loss of entire sections of the RPE.
• FIG. 15 compares the removal of ocular lipofuscin from the eyes of DKO mice in control cells and in those treated with a different nonacid cyclodextrin.
• A shows representative images of the RPE in the eyes treated with mock (left) or 3mM Methyl Beta-CD with a DS of approximately 12 (MBCD) (right) treatments for 1.5 hrs. The ocular lipofuscin is shown in green. RPE cells were stained with Hoechst (blue nucleus) and Phalloidin (red borders) and imaged using confocal microscopy.
• B shows the quantification of the levels of ocular lipofuscin in 10 random fields of the RPE using Image J software. As can be seen, the MBCD potency was not enough to reduce the content of lipofuscin in the RPE under the current treatment conditions.
• Example 14 Comparison of non-acid cyclodextrins and acid cyclodextrins for beta-hexosaminidase removal.
• FIG. 16 demonstrates the extraordinary potency of acid beta cyclodextrins to remove beta-hexosaminidase.
• ARPE19 human-derived RPE
• SBE sulfobutyl ether beta CD
• MBCD methyl beta CD
• SUB4 succinic acid cyclodextrin
• (B) shows a dose response to acid SUB4, neutralized SUB4 (i.e., pH>6), SBE, and MBCD, and demonstrates that only acid SUB can trigger the removal of the beta-hexosaminidase from the cells.
• the cells were all viable at the end of the 4hrs assay, based on Alamar ® blue viability assay. Clearly, then, both the cyclodextrin and the acidity are needed for removal of beta-hexosaminidase.
• Example 15 Comparison of various cyclodextrins in ocular lipofuscin removal.
• To remove ocular lipofuscin not only does exocytosis need to be triggered, but the lipids also need to be dissolved to make them removable. Although some acid alpha CDs may be able to trigger exocytosis, they cannot dissolve A2E. Clearance of ocular lipofuscin can only be achieved effectively and quickly with an acid beta CD or an acid gamma CD. Acid alone or acid cyclodextrins with inappropriate ring sizes cannot remove ocular lipofuscin. The procedure followed can be found in U.S. Patent No. 10,463,687. FIG.
• A shows the level of complexation of A2E as it relates to the concentration of the cyclodextrin (succinic acid alpha CD (SUA), succinic acid beta CD (SUB), succinyl gamma CD (SUG), or Sulfo Butyl Ether Beta-CD (SBE).
• succinic acid cyclodextrin has an extraordinary ability to induce solvatochromic shifts in the fluorescence of A2E and other lipid bisretinoids present in the ocular lipofuscin. Without being held to any one theory, this is an indirect demonstration that SUB, and other acid beta CDs, can incorporate these lipids into their cavity.
• B Solubilization assay of A2E. A2E tends to form large aggregates in aqueous media that cannot pass 0.3um nylon membranes. The A2E can be detected by fluorescence in an aqueous solution, however, it cannot pass a 0.3um syringe polypropylene filter.
• Example 16 Treatment with SUB rescue RPE cells from toxic doses of A2E.
• RPE cell cultures were pre-incubated for at least 24 hrs with 5uM synthetic A2E. During this period, A2E is known to be incorporated into lysosomes. Fresh media with or without SUB4 was added for 1 hr. Cells were left in fresh media overnight and the following day a viability assay using Alamar blue was performed. As shown in FIG. 18, when RPE cells are dosed with 5uM of A2E, the viability decreases by over 20%. However, when 0. ImM SUB is subsequently given to these cells, the viability is restored.
• cyclodextrins The synthesis of cyclodextrins is well known in the art, and numerous types are commercially available. Chemical modification of cyclodextrins can be made directly on the native (alpha, beta, gamma) cyclodextrin rings by reacting a chemical reagent (nucleophiles or electrophiles) with a properly functionalized cyclodextrin (Adair-Kirk, T. L., et al., Nat.
• a chemical reagent nucleophiles or electrophiles
• Cyclodextrins can also be prepared by de novo synthesis, starting with glucopyranose-linked oligopyranosides. Such a synthesis can be accomplished by using various chemical reagents or biological enzymes, such as cyclodextrin transglycosylase.
• 3,453,259 and 3,459,731 describe electroneutral cyclodextrins, the disclosures of which are herein incorporated by reference in its entirety.
• Other derivatives include cyclodextrins with cationic properties, as disclosed in U.S. Pat. No. 3,453,257; insoluble crosslinked cyclodextrins, as disclosed in U.S. Pat. No. 3,420,788; and cyclodextrins with anionic properties, as disclosed in U.S. Pat. No. 3,426,011, the disclosures of which are all hereby incorporated by reference in their entirety.
• cyclodextrin derivatives with anionic properties carboxylic acids, phosphorous acids, phosphinous acids, phosphonic acids, phosphoric acids, thiophosphonic acids, thiosulphinic acids, and sulfonic acids have been appended to the parent cyclodextrin, as disclosed, for example, in U.S. Pat. No. 3,426,011. Sulfoalkyl ether cyclodextrin derivatives have also been described, e.g., in U.S. Pat. No. 5, 134,127, the disclosure of which is hereby incorporated by reference in its entirety.
• each range is intended to be a shorthand format for presenting information, where the range is understood to encompass each discrete point within the range, and further to encompass any subrange within the range between any discrete point within the range and any other discrete point within the range, as if the same were fully set forth herein.

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