Patent Application
20240082221
The present technology relates generally to compositions and methods for treating eye diseases (e.g., retinopathies), and more particularly, eye diseases associated with cytotoxic lipofuscin-associated cytotoxicity in retinal cells.
The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.
Retinal pigment epithelium (RPE) cell-death is the primary cause of geographic atrophy (GA) in retinas with Stargardt and dry-AMD, the most prevalent and incurable genetic and age-related blinding disorders among young and old, respectively. Lipofuscin (LF) is a fine yellow-brown pigment composed of indigestible material that is believed to be remnants after lysosomal digestion. LF is mostly composed of dimers of retinaldehydes known as lipid bisretinoids, and small amounts of carbohydrates, oxidized proteins and metals. Accumulation of LF in retinal cells causes retinal toxicity, which is associated with conditions like macular degeneration, a degenerative disease of the eye, and Stargardt disease. Yet, the mechanisms and extent by which LF contributes to the degeneration is unclear in part because all attempts at targeting its cytotoxic effects have failed to maintain the RPE's viability and stop the retina's decay. Accordingly, there is an urgent need for novel molecular targets to treat GA secondary to Stargardt and dry-AMD.
In one aspect, the present disclosure provides a method for preventing or treating an eye disease associated with retinal cell lipofuscin-associated cytotoxicity in a subject in need thereof comprising administering to the subject an effective amount of at least one therapeutic agent selected from the group consisting of dabrafenib, necrosulfonamide (NSA), arimoclomol, a Kinase Inhibiting RNase Attenuator (KIRA) compound, salubrinal, SAL003 and any pharmaceutically acceptable salt thereof, wherein the eye disease associated with retinal cell lipofuscin-associated cytotoxicity is autosomal recessive retinitis pigmentosa (RP), Stargardt disease (STGD), Best disease (BD), cone-rod dystrophy, or ABCA4 mutant Age-Related Macular Degeneration (AMD). Examples of KIRA compounds include, but are not limited to, KIRA3, KIRA6, KIRA7, or KIRA8. In some embodiments, the subject comprises a mutation in ABCA4 and/or RDH12. The mutation in ABCA4 and/or RDH12 may be homozygous or heterozygous. Additionally or alternatively, in some embodiments of the methods disclosed herein, administration of the effective amount of the at least one therapeutic agent prevents exacerbation of lipofuscin-associated cytotoxicity in retinal cells in the subject.
In another aspect, the present disclosure provides a method for preventing or treating an ABCA4 mutant eye disease associated with retinal cell lipofuscin-associated cytotoxicity in a subject in need thereof comprising administering to the subject an effective amount of Necrostatin 7 (Nec7) or a pharmaceutically acceptable salt thereof, wherein the ABCA4 mutant eye disease associated with retinal cell lipofuscin-associated cytotoxicity is autosomal recessive retinitis pigmentosa (RP), cone-rod dystrophy, or Age-Related Macular Degeneration (AMD). In some embodiments, administration of the effective amount of Nec7 or pharmaceutically acceptable salt thereof prevents exacerbation of lipofuscin-associated cytotoxicity in retinal cells in the subject.
In any and all embodiments of the methods disclosed herein, the eye disease is genetic, non-genetic, or associated with aging. In some embodiments of the methods disclosed herein, the AMD is dry AMD. In other embodiments of the methods disclosed herein, the cone-rod dystrophy is autosomal recessive cone-rod dystrophy.
Additionally or alternatively, in some embodiments of the methods disclosed herein, the subject harbors at least one ABCA4 mutation selected from the group consisting of ABCA4 D2177N, ABCA4 G1961E, ABCA4 G863A, ABCA4 1847delA, ABCA4 L541P, ABCA4 T2028I, ABCA4 N247I, ABCA4 E1122K, ABCA4 W499*, ABCA4 A1773V, ABCA4 H55R, ABCA4 A1038V, ABCA4 IVS30+1G→T, ABCA4 IVS40+5G→A, ABCA4 IVS14+1G→C, and ABCA4 F1440del1cT. In any and all embodiments of the methods disclosed herein, the subject harbors at least one RDH12 mutation selected from the group consisting of RDH12 G127*, RDH12 Q189*, RDH12 Y226C, RDH12 A269Gfs*, RDH12 L274P, RDH12 R65*, RDH12 H151D, RDH12 T155I, RDH12 V41L, RDH12 R314W and RDH12 V146D. cone-rod dystrophy.
Additionally or alternatively, in some embodiments of the methods disclosed herein, the at least one therapeutic agent of the present technology (e.g., dabrafenib, NSA, arimoclomol, the KIRA compound, salubrinal, SAL003, Nec7, or the pharmaceutically acceptable salt thereof) reduces or eliminates lipofuscin bisretinoid (LB) lipid-induced phosphorylation and/or polymerization of MLKL. In any and all embodiments of the methods disclosed herein, the at least one therapeutic agent of the present technology (e.g., dabrafenib, NSA, arimoclomol, the KIRA compound, salubrinal, SAL003, Nec7, or the pharmaceutically acceptable salt thereof) reverses LB lipid-induced translocation of phosphorylated MLKL (pMLKL) to plasma membraned in retinal pigment epithelium cells. In certain embodiments, the LB lipids are selected from the group consisting of N-retinylidene-N-retinylethanolamine (A2E), an A2E isomer, an oxidized derivative of A2E, and all-trans-retinal dimers (ATRD).
In any of the preceding embodiments of the methods disclosed herein, the at least one therapeutic agent of the present technology (e.g., dabrafenib, NSA, arimoclomol, the KIRA compound, salubrinal, SAL003, Nec7, or the pharmaceutically acceptable salt thereof) reduces mRNA or protein levels of one or more genes associated with retinal degeneration, inflammation/angiogenesis, ER-stress and/or necroptosis. Examples of genes associated with retinal degeneration, inflammation/angiogenesis, ER-stress and/or necroptosis include, but are not limited to, EDN2, FGF2, GFAP, SERP, VEGF, CXCL15, XBP1s, SCAND1, CEBPA and HMGA. Additionally or alternatively, in some embodiments of the methods disclosed herein, the at least one therapeutic agent of the present technology (e.g., dabrafenib, NSA, arimoclomol, the KIRA compound, salubrinal, SAL003, Nec7, or the pharmaceutically acceptable salt thereof) inhibits or mitigates lipofuscin-induced necroptosis and/or reduces infiltration of activated microglia/macrophage in retinal pigment epithelium cells.
In any and all embodiments of the methods disclosed herein, the at least one therapeutic agent of the present technology (e.g., dabrafenib, NSA, arimoclomol, the KIRA compound, salubrinal, SAL003, Nec7, or the pharmaceutically acceptable salt thereof) is administered via topical, intravitreous, intraocular, subretinal, or subscleral administration. Additionally or alternatively, in some embodiments of the methods disclosed herein, the at least one therapeutic agent of the present technology (e.g., dabrafenib, NSA, arimoclomol, the KIRA compound, salubrinal, SAL003, Nec7, or the pharmaceutically acceptable salt thereof) is conjugated to an agent that targets retinal pigment epithelium cells. Examples of agents that target retinal pigment epithelium cells include, but are not limited to, tamoxifen, chloroquine (CQ)/hydroxychloroquine (HCQ), ethambutol (EMB), or sodium iodate (NaIO3). Other examples of RPE targeting agents are described in Crisóstomo S, Vieira L, Cardigos J (2019) Retina:23-28; Michaelides M (2011) Arch Ophthalmol 129(1):30; Tsai R K, He M S, Chen Z Y, Wu W C, Wu W S (2011) Mol Vis 17(June):1564-1576; MacHalińska A, et al. (2010) Neurochem Res 35(11):1819-1827; Tsang S H, Sharma T (2018) Drug-Induced Retinal Toxicity. Atlas of Inherited Retinal Diseases, eds Tsang S H, Sharma T (Springer International Publishing, Cham), pp 227-232.
It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology.
In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir's Handbook of Experimental Immunology. Methods to detect and measure levels of polypeptide gene expression products (i.e., gene translation level) are well-known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also, Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)).
Provided are methods and compositions for maintaining the viability of the RPE layer in subjects with Stargardt's disease (STGR1 and STGR3); vitelliform macular degeneration (Best's macular dystrophy or Best's disease); autosomal recessive cone-rod dystrophy (ar-CRD) and autosomal recessive retinitis pigmentosa (ar-RP), secondary to mutations in either the ABCA4 or RDH12 genes; and finally individuals with dry age-related macular degeneration (dry-AMD); choroidal melanoma; or severe ocular trauma associated with increased fundus autofluorescence (FAF) (C. J. Kennedy et al., Eye (Lond). 9 (Pt 6) (1995) 763-71; S. K. Verbakel et al., Prog. Retin. Eye Res. 66 (2018) 157-186; M. a van Driel et al., Ophthalmic Genet. 19 (1998) 117-122; A. Maugeri et al., Am. J. Hum. Genet. 67 (2000) 960-966; A. V. Cideciyan et al., Hum. Mol. Genet. 13 (2004) 525-534).
Excessive ER-stress in photoreceptors has been only associated with autosomal dominant forms of retinitis pigmentosa (adRP) but never with autosomal recessive retinitis pigmentosa (arRP). Comitato et al., Human Molecular Genetics, Vol. 25, No. 13 2801-2812 (2016). Chemical chaperones, i.e. drugs that increase the folding capacity in the cell and reduce the activity of all (IRE1α, PERK and ATF) UPR sensors were beneficial for adRP (S. X. Zhang et al., Exp. Eye Res. 125 (2014) 30-40; M. S. Gorbatyuk et al., Prog. Retin. Eye Res. (2020) 100860), but had no effect on ER-stress provoked by lipofuscin. In addition, treatment with Salubrinal, actually increased IRE1α activity in adRP retinas (Comitato et al., Human Molecular Genetics, Vol. 25, No. 13 2801-2812 (2016)), whereas the Examples herein demonstrate that Salubrinal reduced the same activity in cells with ER-stress due to lipofuscin.
In another model of retinal degeneration due to ER-stress induced by administration of oxidative stress causing agents, suppression of IRE1α resulted detrimental (S. X. Zhang et al., Exp. Eye Res. 125 (2014) 30-40; T. McLaughlin et al., Mol. Neurodegener. 13 (2018) 1-15). While KIRA inhibitors of IRE1α have been shown useful to protect photoreceptors, with massive amounts of misfolded proteins, from apoptosis (most cases of adRP) (R. Ghosh et al., Cell. (2014) 1-15; H. C. Feldman et al., ACS Chem. Biol. 11 (2016) 2195-2205) but were never used to protect RPE from lipid cytotoxicity.
The methods of the present disclosure are based on the following unexpected discoveries, that challenge current dogmas in the field of lipofuscin pathogenesis: 1) lipid-bisretinoids render the lysosomes in which they are trapped, leaky (increased lysosomal membrane permeabilization (LMP)); 2) cytosolic lipofuscin triggers the unfolded protein response (UPR); 3) lipofuscin elicited UPR induces via the ER-stress sensor IRE1α, the formation of an atypical necrosome that phosphorylates MLKL. Phospho-MLKL subsequently self-assembles into pores that damage the ER, lysosomal and plasma membranes, creating an amplification loop “ER-stress↔phospho-HLKL” that culminates with the necrosis of the lipofuscin occupied cells. The lipofuscin-elicited cell death pathway is fundamentally different from previously reported mechanisms of cell death because it does not involve oxidative stress, apoptosis or classical necrosomes containing RIPK1 and RIPK3 kinases.
The methods of the present disclosure preserve visual function of a subject suffering from lipofuscin pathologies such as Stargardt disease (STGD), autosomal recessive retinitis pigmentosa (RP), Age-Related Macular Degeneration (AMD), Best disease (BD), or autosomal recessive cone-rod dystrophy: i) by administering an effective amount of KIRA compounds (e.g., KIRA3, KIRA6, KIRA7, KIRA8); ii) by administering an effective amount of Salubrinal-derivatives; iii) by administering effective amounts of Necrostatin 7, Necrosulfonamide (NSA), Dabrafenib, or Arimoclomol which, inhibit the formation of phospho-MLKL, and so, interrupt the “phospho-MLKL→ER-stress→IRE1α→phospho-MLKL” loop. These strategies can be applied individually or in combination to halt the degenerative process.
The aforementioned approaches differ radically from all previous attempts used to date to protect the retina from lipofuscin cytotoxicity, including the blockage of lipid bisretinoids formation, the use of antiapoptotic agents, antioxidants, or light blocking lenses.
Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a cell” includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.
As used herein, the term “about” in reference to a number is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).
As used herein, the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including but not limited to, orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, or topically. Administration includes self-administration and the administration by another.
As used herein, the term “biological sample” means sample material derived from living cells. Biological samples may include tissues, cells, protein or membrane extracts of cells, and biological fluids (e.g., ascites fluid or cerebrospinal fluid (CSF)) isolated from a subject, as well as tissues, cells and fluids present within a subject. Biological samples of the present technology include, but are not limited to, samples taken from eye, breast tissue, renal tissue, the uterine cervix, the endometrium, the head or neck, the gallbladder, parotid tissue, the prostate, the brain, the pituitary gland, kidney tissue, muscle, the esophagus, the stomach, the small intestine, the colon, the liver, the spleen, the pancreas, thyroid tissue, heart tissue, lung tissue, the bladder, adipose tissue, lymph node tissue, the uterus, ovarian tissue, adrenal tissue, testis tissue, the tonsils, thymus, blood, hair, buccal, skin, serum, plasma, CSF, semen, prostate fluid, seminal fluid, urine, feces, sweat, saliva, sputum, mucus, bone marrow, lymph, and tears. Biological samples can also be obtained from biopsies of internal organs. Biological samples can be obtained from subjects for diagnosis or research or can be obtained from non-diseased individuals, as controls or for basic research. Samples may be obtained by standard methods including, e.g., venous puncture and surgical biopsy. In certain embodiments, the biological sample is a tissue sample obtained by needle biopsy.
As used herein, a “control” is an alternative sample used in an experiment for comparison purpose. A control can be “positive” or “negative.” For example, where the purpose of the experiment is to determine a correlation of the efficacy of a therapeutic agent for the treatment for a particular type of disease, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or a sample that does not receive the therapy or receives a placebo) are typically employed.
As used herein, the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will vary depending on the composition, the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic compositions may be administered to a subject having one or more signs or symptoms of a disease or condition described herein. As used herein, a “therapeutically effective amount” of a composition refers to composition levels in which the physiological effects of a disease or condition are ameliorated or eliminated. A therapeutically effective amount can be given in one or more administrations.
As used herein, “expression” includes one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and/or other modifications of the translation product, if required for proper expression and function.
As used herein, the terms “individual”, “patient”, or “subject” can be an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the individual, patient or subject is a human.
As used herein, the term “pharmaceutically-acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption delaying compounds, and the like, compatible with pharmaceutical administration. Pharmaceutically-acceptable carriers and their formulations are known to one skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20th edition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.). Examples of pharmaceutically-acceptable carriers include 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.
As used herein, “prevention,” “prevent,” or “preventing” of a disorder or condition refers to one or more compounds that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset of one or more symptoms of the disorder or condition relative to the untreated control sample.
As used herein, the term “separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.
As used herein, the term “sequential” therapeutic use refers to administration of at least two active ingredients at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.
As used herein, the term “simultaneous” therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time.
“Treating” or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, i.e., arresting its development; (ii) relieving a disease or disorder, i.e., causing regression of the disorder; (iii) slowing progression of the disorder; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. In some embodiments, treatment means that the symptoms associated with the disease are, e.g., alleviated, reduced, cured, or placed in a state of remission.
It is also to be appreciated that the various modes of treatment of disorders as described herein are intended to mean “substantial,” which includes total but also less than total treatment, and wherein some biologically or medically relevant result is achieved. The treatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.
Eye Diseases Associated with Retinal Cell Lipofuscin Cytotoxicity
Lipofuscin accumulates with age and can increase due to genetic predispositions and certain underlying conditions. See Molday R S, Zhong M, Quazi F, Biochim Biophys Acta 1791(7):573-83 (2009); Zaneveld J, et al. Genet Med 17(4):262-270 (2015); Allikmets R et al., Science 277(5333):1805-7 (1997); van Driel M a, Maugeri a, Klevering B J, Hoyng C B, Cremers F P, Ophthalmic Genet 19(3):117-122 (1998); Fishman G A, Ophthalmic Genet 31(4):183-9 (2010); Lim L S, Mitchell P, Seddon J M, Holz F G, Wong T Y, Lancet 379(9827):1728-1738 (2012); Swaroop A, Chew E Y, Rickman C B, Abecasis G R, Annu Rev Genomics Hum Genet 10:19-43 (2009); Charbel Issa P, Barnard A R, Herrmann P, Washington I, MacLaren RE (2015) Proc Natl Acad Sci 112(27):8415-20 (2017). ABCR mutations may occur in patients with age-related macular degeneration (AMD), Stargardt's disease, fundus flavimaculatus, cone dystrophy (COD, where only the cone cells undergo degeneration), and cone-rod dystrophy (CRD, where both rods and cones are undergo degeneration) and Retinitis pigmentosa. Examples of such ABCR mutations include, but are not limited to, ABCA4 D2177N, ABCA4 G1961E, ABCA4 G863A, ABCA4 1847delA, ABCA4 L541P, ABCA4 T2028I, ABCA4 N247I, ABCA4 E1122K, ABCA4 W499*, ABCA4 A1773V, ABCA4 H55R, ABCA4 A1038V, ABCA4 IVS30+1G→T, ABCA4 IVS40+5G→A, ABCA4 IVS14+1G→C, ABCA4 F1440del1cT, as well as those disclosed in Allikmets R et al., Science 277(5333):1805-7 (1997).
Retinitis pigmentosa (RP) is a group of diseases where photoreceptor cells die. RP is the most common inherited retinal dystrophy (IRD), with a worldwide prevalence of approximately 1:4000 (S. K. Verbakel, et al., Prog. Retin. Eye Res. 66, 157-186 (2018)). RP can be inherited in an autosomal dominant, autosomal recessive or X-linked manner. Over 40 genes have been associated with RP so far, with the majority of them expressed in either the photoreceptors or the retinal pigment epithelium. The tremendous heterogeneity of the disease makes the genetics of RP complicated. Ferrari et al., Current Genomics, 12, 238-249 (2011).
Typical fundus abnormalities include bone spicule pigmentation predominantly in the periphery and/or mid-periphery of the retina, which gives the name to the disease. The typical bone-spicule dark pigmentation, is observable with the ophthalmoscope and represents RPE cells that detached from the Bruch membrane following photoreceptor degeneration and migrated to intra-retinal perivascular sites, where they form melanin pigment deposits around the blood vessels. These bone spicules often arise in the mid-periphery, where the concentration of rod cells is the highest. Precisely what triggers RPE migration is unknown, but the migration is suspected to be facilitated by the reduced distance between the inner retinal vessels and the RPE, due to the degeneration of the photoreceptors. Almost all forms of RP go through a stage where no pigmentary changes exist in the retina. This stage may exist for decades before typical RP signs appear.
There are two autosomal recessive RP subtypes due to mutations in either the ABCA4 or RDH12 genes, which are very severe forms of RP with an early onset in life (R. F. Mullins, et al., Invest. Ophthalmol. Vis. Sci. 53, 1883-94 (2012); A. Schuster, et al., Investig. Ophthalmol. Vis. Sci. 48, 1824-1831 (2007)). A study in an Asian population revealed that they represent at least 3 and 2% of all RP cases, respectively (L. Huang, et al., Sci. Rep. 7, 1-10 (2017)). Examples of such RDH12 mutations include, but are not limited to, RDH12 p.G127X, RDH12 p.Q189X, RDH12 p.Y226C, RDH12 p.A269GfsX1, RDH12 p.L274P, RDH12 p.R65X, RDH12 p.H151D, RDH12 p.T155I, RDH12 p.V41L, RDH12 p.R314W and RDH12 p. V146D. Unlike autosomal dominant RP, autosomal recessive RP is characterized by high content of retinal lipofuscin in the RPE.
The present disclosure provides compositions that protect against lipofuscin cytotoxicity in retinal cells, e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003, or pharmaceutically acceptable salts thereof.
In one aspect, the present disclosure provides a method for preventing or treating an eye disease associated with retinal cell lipofuscin-associated cytotoxicity in a subject in need thereof comprising administering to the subject an effective amount of at least one therapeutic agent selected from the group consisting of dabrafenib, necrosulfonamide (NSA), arimoclomol, a Kinase Inhibiting RNase Attenuator (KIRA) compound, salubrinal, SAL003 and any pharmaceutically acceptable salt thereof, wherein the eye disease associated with retinal cell lipofuscin-associated cytotoxicity is autosomal recessive retinitis pigmentosa (RP), Stargardt disease (STGD), Best disease (BD), cone-rod dystrophy, or ABCA4 mutant Age-Related Macular Degeneration (AMD). Examples of KIRA compounds include, but are not limited to, KIRA3, KIRA6, KIRA7, or KIRA8. In some embodiments, the subject comprises a mutation in ABCA4 and/or RDH12. The mutation in ABCA4 and/or RDH12 may be homozygous or heterozygous. Additionally or alternatively, in some embodiments of the methods disclosed herein, administration of the effective amount of the at least one therapeutic agent prevents exacerbation of lipofuscin-associated cytotoxicity in retinal cells in the subject.
In another aspect, the present disclosure provides a method for preventing or treating an ABCA4 mutant eye disease associated with retinal cell lipofuscin-associated cytotoxicity in a subject in need thereof comprising administering to the subject an effective amount of Necrostatin 7 (Nec7) or a pharmaceutically acceptable salt thereof, wherein the ABCA4 mutant eye disease associated with retinal cell lipofuscin-associated cytotoxicity is autosomal recessive retinitis pigmentosa (RP), cone-rod dystrophy, or Age-Related Macular Degeneration (AMD). In some embodiments, administration of the effective amount of Nec7 or pharmaceutically acceptable salt thereof prevents exacerbation of lipofuscin-associated cytotoxicity in retinal cells in the subject.
In any and all embodiments of the methods disclosed herein, the eye disease is genetic, non-genetic, or associated with aging. In some embodiments of the methods disclosed herein, the AMD is dry AMD. In other embodiments of the methods disclosed herein, the cone-rod dystrophy is autosomal recessive cone-rod dystrophy.
Additionally or alternatively, in some embodiments of the methods disclosed herein, the subject harbors at least one ABCA4 mutation selected from the group consisting of ABCA4 D2177N, ABCA4 G1961E, ABCA4 G863A, ABCA4 1847delA, ABCA4 L541P, ABCA4 T2028I, ABCA4 N247I, ABCA4 E1122K, ABCA4 W499*, ABCA4 A1773V, ABCA4 H55R, ABCA4 A1038V, ABCA4 IVS30+1G→T, ABCA4 IVS40+5G→A, ABCA4 IVS14+1G→C, and ABCA4 F1440del1cT. In any and all embodiments of the methods disclosed herein, the subject harbors at least one RDH12 mutation selected from the group consisting of RDH12 G127*, RDH12 Q189*, RDH12 Y226C, RDH12 A269Gfs*, RDH12 L274P, RDH12 R65*, RDH12 H151D, RDH12 T155I, RDH12 V41L, RDH12 R314W and RDH12 V146D. cone-rod dystrophy.
Additionally or alternatively, in some embodiments of the methods disclosed herein, the at least one therapeutic agent of the present technology (e.g., dabrafenib, NSA, arimoclomol, the KIRA compound, salubrinal, SAL003, Nec7, or the pharmaceutically acceptable salt thereof) reduces or eliminates lipofuscin bisretinoid (LB) lipid-induced phosphorylation and/or polymerization of MLKL. In any and all embodiments of the methods disclosed herein, the at least one therapeutic agent of the present technology (e.g., dabrafenib, NSA, arimoclomol, the KIRA compound, salubrinal, SAL003, Nec7, or the pharmaceutically acceptable salt thereof) reverses LB lipid-induced translocation of phosphorylated MLKL (pMLKL) to plasma membraned in retinal pigment epithelium cells. In certain embodiments, the LB lipids are selected from the group consisting of N-retinylidene-N-retinylethanolamine (A2E), an A2E isomer, an oxidized derivative of A2E, and all-trans-retinal dimers (ATRD).
In any of the preceding embodiments of the methods disclosed herein, the at least one therapeutic agent of the present technology (e.g., dabrafenib, NSA, arimoclomol, the KIRA compound, salubrinal, SAL003, Nec7, or the pharmaceutically acceptable salt thereof) reduces mRNA or protein levels of one or more genes associated with retinal degeneration, inflammation/angiogenesis, ER-stress and/or necroptosis. Examples of genes associated with retinal degeneration, inflammation/angiogenesis, ER-stress and/or necroptosis include, but are not limited to, EDN2, FGF2, GFAP, SERP, VEGF, CXCL15, XBP1s, SCAND1, CEBPA and HMGA. Additionally or alternatively, in some embodiments of the methods disclosed herein, the at least one therapeutic agent of the present technology (e.g., dabrafenib, NSA, arimoclomol, the KIRA compound, salubrinal, SAL003, Nec7, or the pharmaceutically acceptable salt thereof) inhibits or mitigates lipofuscin-induced necroptosis and/or reduces infiltration of activated microglia/macrophage in retinal pigment epithelium cells.
In any and all embodiments of the methods disclosed herein, the at least one therapeutic agent of the present technology (e.g., dabrafenib, NSA, arimoclomol, the KIRA compound, salubrinal, SAL003, Nec7, or the pharmaceutically acceptable salt thereof) is administered via topical, intravitreous, intraocular, subretinal, or subscleral administration. Additionally or alternatively, in some embodiments of the methods disclosed herein, the at least one therapeutic agent of the present technology (e.g., dabrafenib, NSA, arimoclomol, the KIRA compound, salubrinal, SAL003, Nec7, or the pharmaceutically acceptable salt thereof) is conjugated to an agent that targets retinal pigment epithelium cells. Examples of agents that target retinal pigment epithelium cells include, but are not limited to, tamoxifen, chloroquine (CQ)/hydroxychloroquine (HCQ), ethambutol (EMB), or sodium iodate (NaIO3). Other examples of RPE targeting agents are described in Crisóstomo S, Vieira L, Cardigos J (2019) Retina:23-28; Michaelides M (2011) Arch Ophthalmol 129(1):30; Tsai R K, He M S, Chen Z Y, Wu W C, Wu W S (2011) Mol Vis 17(June):1564-1576; MacHalińska A, et al. (2010) Neurochem Res 35(11):1819-1827; Tsang S H, Sharma T (2018) Drug-Induced Retinal Toxicity. Atlas of Inherited Retinal Diseases, eds Tsang S H, Sharma T (Springer International Publishing, Cham), pp 227-232.
The term “pharmaceutically acceptable salt” means a salt prepared from a base or an acid which is acceptable for administration to a patient, such as a mammal (e.g., salts having acceptable mammalian safety for a given dosage regime). However, it is understood that the salts are not required to be pharmaceutically acceptable salts, such as salts of intermediate compounds that are not intended for administration to a patient. Pharmaceutically acceptable salts can be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids. In addition, when one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) contain both a basic moiety, such as an amine, pyridine or imidazole, and an acidic moiety such as a carboxylic acid or tetrazole, zwitterions may be formed and are included within the term “salt” as used herein.
In one embodiment, the one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) may contain one or more basic functional groups, such as amino or alkylamino, and thereby, can form pharmaceutically-acceptable salts by reaction with a pharmaceutically-acceptable acid. These salts can be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting a purified compound of the present technology in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed during subsequent purification. In another embodiment, the one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) may contain one or more acidic functional groups, and thereby, can form pharmaceutically-acceptable salts by reaction with a pharmaceutically-acceptable base. These salts can likewise be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting the purified compound in its free acid form (e.g., hydroxyl or carboxyl) with a suitable base, and isolating the salt thus formed during subsequent purification.
Salts derived from pharmaceutically acceptable inorganic bases include ammonium, aluminum, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, and zinc salts, and the like. Salts derived from pharmaceutically acceptable organic bases include salts of primary, secondary and tertiary amines, including substituted amines, cyclic amines, naturally-occurring amines and the like, such as arginine, betaine, caffeine, choline, N,N′-dibenzylethylenediamine, ethylamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, diethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperadine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like. Salts derived from pharmaceutically acceptable inorganic acids include salts of boric, carbonic, hydrohalic (hydrobromic, hydrochloric, hydrofluoric or hydroiodic), nitric, phosphoric, sulfamic and sulfuric acids. Salts derived from pharmaceutically acceptable organic acids include salts of aliphatic hydroxyl acids (e.g., citric, gluconic, glycolic, lactic, lactobionic, malic, and tartaric acids), aliphatic monocarboxylic acids (e.g., acetic, butyric, formic, propionic and trifluoroacetic acids), amino acids (e.g., aspartic and glutamic acids), aromatic carboxylic acids (e.g., benzoic, 2-acetoxybenzoic, p-chlorobenzoic, diphenylacetic, gentisic, hippuric, and triphenylacetic acids), aromatic hydroxyl acids (e.g., o-hydroxybenzoic, p-hydroxybenzoic, 1-hydroxynaphthalene-2-carboxylic and 3-hydroxynaphthalene-2-carboxylic acids), ascorbic, dicarboxylic acids (e.g., fumaric, maleic, oxalic and succinic acids), glucuronic, mandelic, mucic, nicotinic, orotic, pamoic, pantothenic, sulfonic acids (e.g., benzenesulfonic, camphosulfonic, edisylic, ethanesulfonic, isethionic, methanesulfonic, naphthalenesulfonic, naphthalene-1,5-disulfonic, naphthalene-2,6-disulfonic and p-toluenesulfonic acids), xinafoic acid, valeric, oleic, palmitic, stearic, lauric, toluenesulfonic, methansulfonic, ethanedisulfonic, citric, ascorbic, maleic, oxalic, fumaric, phenylacetic, isothionic, succinic, tartaric, glutamic, salicylic, sulfanilic, napthylic, lactobionic, gluconic, laurylsulfonic acids, and the like.
Additionally or alternatively, in some embodiments, administration of the effective amount of the one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts thereof prevent exacerbation of lipofuscin-associated retinal cytotoxicity in the subject. In any and all embodiments of the methods disclosed herein, administration of the effective amount of the one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts thereof block, mitigate, or reverse lipofuscin associated cytotoxicity in retinal pigment epithelium cells.
In some embodiments of the methods disclosed herein, administration of the effective amount of the one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts thereof prevent, slow the onset, or lessen the severity of lipofuscin-associated damage or a disease or condition directly or indirectly associated with lipofuscin-associated damage in RPE cells of the subject. The subject can be of any gender (e.g., male or female), and/or can also be any age, such as elderly (generally, at least or above 60, 70, or 80 years of age), elderly-to-adult transition age subjects, adults, adult-to-pre-adult transition age subjects, and pre-adults, including adolescents (e.g., 13 and up to 16, 17, 18, or 19 years of age), children (generally, under 13 or before the onset of puberty), and infants. The subject can also be of any ethnic population or genotype. Some examples of human ethnic populations include Caucasians, Asians, Hispanics, Africans, African Americans, Native Americans, Semites, and Pacific Islanders.
Additionally or alternatively, in some embodiments, the one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts thereof are configured to localize to RPE cells.
Additionally or alternatively, in certain embodiments, the one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts thereof localize to RPE cells by being administered directly at, into, or in the adjacent vicinity of RPE cells, such as by injection or implantation.
In other embodiments, the one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts thereof localize to RPE cells by coupling the one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts thereof with a targeting agent that selectively targets RPE cells, and the one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts thereof may be administered at, into, or in the adjacent vicinity of RPE cells, or remotely from the RPE cells (e.g., by systemic administration). The cell-targeting agent (i.e., “targeting agent”) is any chemical entity that has the ability to bind to (i.e., “target”) a RPE cell. The cell-targeting agent may target any part of the RPE cell, e.g., cell membrane, organelle (e.g., lysosome or endosome), or cytoplasm. In one embodiment, the cell-targeting agent targets a component of a RPE cell in a selective manner. By selectively targeting a component of an RPE cell, the cell-targeting agent can, for example, selectively target certain components of cells over other types of cellular components. In other embodiments, the targeting agent targets cellular components non-selectively, e.g., by targeting cellular components found in most or all cells.
In various embodiments, the targeting agent can be, or include, for example, a peptide, dipeptide, tripeptide (e.g., glutathione), tetrapeptide, pentapeptide, hexapeptide, higher oligopeptide, protein, monosaccharide, disaccharide, trisaccharide, tetrasaccharide, higher oligosaccharide, polysaccharide (e.g., a carbohydrate), nucleobase, nucleoside (e.g., adenosine, cytidine, uridine, guanosine, thymidine, inosine, and S-Adenosyl methionine), nucleotide (i.e., mono-, di-, or tri-phosphate forms), dinucleotide, trinucleotide, tetranucleotide, higher oligonucleotide, nucleic acid, cofactor (e.g., TPP, FAD, NAD, coenzyme A, biotin, lipoamide, metal ions (e.g., Mg2+), metal-containing clusters (e.g., the iron-sulfur clusters), or a non-biological (i.e., synthetic) targeting group. Some particular types of proteins include enzymes, hormones, antibodies (e.g., monoclonal antibodies), lectins, and steroids.
Antibodies for use as targeting agents are generally specific for one or more cell surface antigens. In a particular embodiment, the antigen is a receptor. The antibody can be a whole antibody, or alternatively, a fragment of an antibody that retains the recognition portion (i.e., hypervariable region) of the antibody. Some examples of antibody fragments include Fab, Fc, and F(ab′)2. In particular embodiments, particularly for the purpose of facilitating crosslinking of the antibody to the one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts thereof described herein, the antibody or antibody fragment can be chemically reduced to derivatize the antibody or antibody fragment with sulfhydryl groups. In certain embodiments, the targeting agent is a ligand of an internalized receptor of the target cell. For example, the targeting agent can be a targeting signal for acid hydrolase precursor proteins that transport various materials to lysosomes. One such targeting agent of particular interest is mannose-6-phosphate (M6P), which is recognized by mannose 6-phosphate receptor (MPR) proteins in the trans-Golgi. Endosomes are known to be involved in transporting M6P-labeled substances to lysosomes.
In other embodiments, the targeting agent is a peptide containing an RGD sequence, or variants thereof, that bind RGD receptors on the surface of many types of cells. Other targeting agents include, for example, transferrin, insulin, amylin, and the like. Receptor internalization may be used to facilitate intracellular delivery of the one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts thereof described herein. In certain embodiments, one cell-targeting molecule or group, or several (e.g., two, three, or more) of the same type of cell-targeting molecule or group are attached to the one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts thereof directly or via a linker. In other embodiments, two or more different types of targeting molecules are attached to the one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts thereof directly or via a linker.
Additionally or alternatively, in some embodiments, a fluorophore may be attached to the one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts thereof. Incorporation of one or more fluorophores can have several purposes. In some embodiments, one or more fluorophores are included in order to quantify cellular uptake and retention of the one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts thereof (e.g., by a fluorescence spectroscopic method).
As used herein, a “fluorophore” refers to any species with the ability to fluoresce (i.e., that possesses a fluorescent property). For example, in one embodiment, the fluorophore is an organic fluorophore. The organic fluorophore can be, for example, a charged (i.e., ionic) molecule (e.g., sulfonate or ammonium groups), uncharged (i.e., neutral) molecule, saturated molecule, unsaturated molecule, cyclic molecule, bicyclic molecule, tricyclic molecule, polycyclic molecule, acyclic molecule, aromatic molecule, and/or heterocyclic molecule (i.e., by being ring-substituted by one or more heteroatoms selected from, for example, nitrogen, oxygen and sulfur). In the particular case of unsaturated fluorophores, the fluorophore contains one, two, three, or more carbon-carbon and/or carbon-nitrogen double and/or triple bonds. In a particular embodiment, the fluorophore contains at least two (e.g., two, three, four, five, or more) conjugated double bonds aside from any aromatic group that may be in the fluorophore. In other embodiments, the fluorophore is a fused polycyclic aromatic hydrocarbon (PAH) containing at least two, three, four, five, or six rings (e.g., naphthalene, pyrene, anthracene, chrysene, triphenylene, tetracene, azulene, and phenanthrene) wherein the PAH can be optionally ring-substituted or derivatized by one, two, three or more heteroatoms or heteroatom-containing groups.
In other embodiments, the organic fluorophore is a xanthene derivative (e.g., fluorescein, rhodamine, Oregon green, eosin, and Texas Red), cyanine or its derivatives or subclasses (e.g., streptocyanines, hemicyanines, closed chain cyanines, phycocyanins, allophycocyanins, indocarbocyanines, oxacarbocyanines, thiacarbocyanines, merocyanins, and phthalocyanines), naphthalene derivatives (e.g., dansyl and prodan derivatives), coumarin and its derivatives, oxadiazole and its derivatives (e.g., pyridyloxazoles, nitrobenzoxadiazoles, and benzoxadiazoles), pyrene and its derivatives, oxazine and its derivatives (e.g., Nile Red, Nile Blue, and cresyl violet), acridine derivatives (e.g., proflavin, acridine orange, and acridine yellow), arylmethine derivatives (e.g., auramine, crystal violet, and malachite green), and tetrapyrrole derivatives (e.g., porphyrins and bilirubins). Some particular families of dyes considered herein are the Cy® family of dyes, the Alexa® family of dyes, the ATTO® family of dyes, and the Dy® family of dyes. The ATTO® dyes, in particular, can have several structural motifs, including, coumarin-based, rhodamine-based, carbopyronin-based, and oxazine-based structural motifs.
The fluorophore can be attached to the one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts thereof by any of the linking methodologies known in the art. For example, a commercial mono-reactive fluorophore (e.g., NHS-Cy5) or bis-reactive fluorophore (e.g., bis-NHS-Cy5 or bis-maleimide-Cy5) can be used to link the fluorophore to one or more molecules containing appropriate reactive groups (e.g., amino, thiol, hydroxy, aldehydic, or ketonic groups). Alternatively, the one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts thereof can be derivatized with one, two, or more such reactive groups, and these reactive portions reacted with a fluorophore containing appropriate reactive groups (e.g., an amino-containing fluorophore).
The one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts thereof can be administered by any route that permits contact with 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 one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts thereof of the present technology may be administered locally, to the eyes of patients suffering from lipofuscin cytotoxicity including Stargardt, carriers of ABCA4 defective genes, dry AMD or at risk for developing retinal degeneration due to lipofuscin cytotoxicity. Local administration includes intravitreal, topical ocular, transdermal patch, subdermal, parenteral, intraocular, subconjunctival, or retrobulbar or subtenon's injection, trans-scleral (including iontophoresis), posterior juxtascleral delivery, or slow release biodegradable polymers or liposomes. The one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts thereof can also be delivered in ocular irrigating solutions. Concentrations may range from about 0.001 μM to about 100 μM, preferably about 0.01 μM to about 5 μM.
In some embodiments, the one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts thereof are administered, at least initially, at levels lower than that required in order to achieve a desired therapeutic effect, and the dose is gradually or suddenly increased until a desired effect is achieved. In other embodiments, the one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts thereof are 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 therapeutic 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.
Particularly for systemic modes of administration, 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. Particularly in embodiments where the one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts thereof are administered non-systemically directly at the retina, the dosage can disregard body weight, and can be in smaller amounts (e.g., 1-1000 μg per dose). In some embodiments, the daily dose of the one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts thereof is the lowest dose effective to produce a therapeutic effect. In some embodiments, the one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts thereof are not administered in discrete dosages, but in a continuous mode, such as provided by a slow release implant or intravenous line.
In one aspect, the present disclosure provides pharmaceutical compositions comprising arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, SAL003, and pharmaceutically acceptable salts thereof.
The one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts thereof may be formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents, known in the art. The pharmaceutical compositions of the present technology 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.
In some embodiments, pharmaceutical compositions of the present technology may contain one or more “pharmaceutically-acceptable carriers,” which 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. Each carrier must be “acceptable” in the sense of being compatible with other ingredients of the formulation and not injurious to the patient. Examples of suitable aqueous and non-aqueous carriers that may be employed in the pharmaceutical compositions of the present technology 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. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
In some embodiments, the formulations may include one or more of sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; alginic acid; buffering agents, such as magnesium hydroxide and aluminum hydroxide; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides; preservatives; glidants; fillers; and other non-toxic compatible substances employed in pharmaceutical formulations.
Various auxiliary agents, such as wetting agents, emulsifiers, lubricants (e.g., sodium lauryl sulfate and magnesium stearate), coloring agents, release agents, coating agents, sweetening agents, flavoring agents, preservative agents, and antioxidants can also be included in the pharmaceutical composition. Some examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. In some embodiments, the pharmaceutical formulation includes an excipient selected from, for example, celluloses, liposomes, micelle-forming agents (e.g., bile acids), and polymeric carriers, e.g., polyesters and polyanhydrides. Suspensions, in addition to the active compounds, may contain suspending agents, such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof. Prevention of the action of microorganisms on the active compounds may be ensured by the inclusion of various antibacterial and antifungal agents, such as, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.
Pharmaceutical formulations of the present technology may be prepared by any of the methods known in the pharmaceutical arts. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound that produces a therapeutic effect. Generally, the amount of active compound will be in the range of about 0.1 to 99 percent, more typically, about 5 to 70 percent, and more typically, about 10 to 30 percent.
The compositions of the present technology may be administered locally, to the eyes of patients suffering from lipofuscin cytotoxicity including Stargardt, carriers of ABCA4 defective genes, dry AMD or at risk for developing retinal degeneration due to lipofuscin cytotoxicity. The one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts thereof can be incorporated into various types of ophthalmic formulations for delivery to the eye (e.g., topically, intracamerally, juxtasclerally, or via an implant). The one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts thereof may be combined with ophthalmologically acceptable preservatives, surfactants, viscosity enhancers, gelling agents, penetration enhancers, buffers, sodium chloride, and water to form aqueous, sterile ophthalmic suspensions or solutions or preformed gels or gels formed in situ.
In some embodiments, the compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, SAL003, and pharmaceutically acceptable salts thereof) is administered 1-10 times a day, once a day, twice, three, four, or more times a day, 1-3 times a day, 2-4 times a day, 3-6 times a day, 4-8 times a day or 5-10 times a day. In some embodiments, the compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, SAL003, and pharmaceutically acceptable salts thereof) is administered every day, every other day, 2-3 times a week, or 3-6 times a week.
In some embodiments, the dose of the compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, SAL003, and pharmaceutically acceptable salts thereof) can be, for example, in the range of about 0.01, 0.1, 0.5, 1, 5, 10, or 100 mg per kg of body weight per day to about 20, 50, 100, 500, or 1000 mg per kilogram of body weight. Particularly in embodiments where the active substance is administered directly at the retina, the dosage administered can be independent of body weight, and can be in smaller amounts (e.g., 1-1000 μg per dose).
If dosed topically, the one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts thereof may be formulated as topical ophthalmic suspensions or solutions, with a pH of about 4 to 8. The one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts thereof will normally be contained in these formulations in an amount 0.001% to 5% by weight, or in an amount of 0.01% to 2% by weight. Thus, for topical presentation, 1 to 2 drops of these formulations would be delivered to the surface of the eye 1 to 4 times per day according to the discretion of a skilled clinician. In some embodiments, the pharmaceutical compositions of the present technology, containing therapeutically effective amounts of at least one composition of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003, or pharmaceutically acceptable salts thereof), are delivered intravitreally either through an injection (perhaps microspheres), an intravitreal device, or placed in the sub-Tenon space by injection, gel, or implant, or by other methods discussed above. If delivered as a solution, the therapeutically effective amount of the one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts thereof in the composition might be about 18-44 μM, of a concentration of about 20-50%. If formulated as a suspension, a therapeutically effective amount of the one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts thereof is about 20-80%.
In another embodiment, the therapeutically effective amount of the one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts thereof is administered in the form of a mini-tablet, each weighing from about 1 mg to about 40 mg, or about 5 mg. From one to twenty such mini-tablets may be injected [dry] into the sub-Tenon space through a trochar in one dose, so that a total single dose of 50-100 mg [44-88 μM] is injected.
In some embodiments, the compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, SAL003, and pharmaceutically acceptable salts thereof) is administered 1-10 times a day, once a day, twice, three, four, or more times a day, 1-3 times a day, 2-4 times a day, 3-6 times a day, 4-8 times a day or 5-10 times a day. In some embodiments, the compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, SAL003, and pharmaceutically acceptable salts thereof) is administered every day, every other day, 2-3 times a week, or 3-6 times a week.
In some embodiments, the dose of the compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, SAL003, and pharmaceutically acceptable salts thereof) can be, for example, in the range of about 0.01, 0.1, 0.5, 1, 5, 10, or 100 mg per kg of body weight per day to about 20, 50, 100, 500, or 1000 mg per kilogram of body weight. Particularly in embodiments where the active substance is administered directly at the retina, the dosage administered can be independent of body weight, and can be in smaller amounts (e.g., 1-1000 μg per dose).
Formulations of the present technology suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present technology as an active ingredient. The active compound may also be administered as a bolus, electuary, or paste.
Methods of preparing these formulations generally include the step of admixing a composition of the present technology or pharmaceutically acceptable salt thereof, with the carrier, and optionally, one or more auxiliary agents. In the case of a solid dosage form (e.g., capsules, tablets, pills, powders, granules, trouches, and the like), the active compound can be admixed with a finely divided solid carrier, and typically, shaped, such as by pelletizing, tableting, granulating, powderizing, or coating. Generally, the solid carrier may include, for example, sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds and surfactants, such as poloxamer and sodium lauryl sulfate; (7) wetting agents, such as, for example, cetyl alcohol, glycerol monostearate, and non-ionic surfactants; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, zinc stearate, sodium stearate, stearic acid, and mixtures thereof; (10) coloring agents; and/or (11) controlled release agents such as crospovidone or ethyl cellulose. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-shelled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
A tablet may be made by compression or molding, optionally with one or more auxiliary ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. The tablets, and other solid dosage forms of the active agent, such as capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. The dosage form may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. The dosage form may alternatively be formulated for rapid release, e.g., freeze-dried.
Generally, the dosage form is required to be sterile. For this purpose, the dosage form may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. The pharmaceutical compositions may also contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
Liquid dosage forms are typically a pharmaceutically acceptable emulsion, microemulsion, solution, suspension, syrup, or elixir of the active agent. In addition to the active ingredient, the liquid dosage form may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
Dosage forms specifically intended for topical or transdermal administration can be in the form of, for example, a powder, spray, ointment, paste, cream, lotion, gel, solution, or patch. Ophthalmic formulations, such as eye ointments, powders, solutions, and the like, are also contemplated herein. The active compound may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants that may be required. The topical or transdermal dosage form may contain, in addition to an active compound of this present technology, one or more excipients, such as those selected from animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, and mixtures thereof. Sprays may also contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
For purposes of this present technology, transdermal patches may provide the advantage of permitting controlled delivery of a compound of the present technology into the body. Such dosage forms can be made by dissolving or dispersing the compound in a suitable medium. Absorption enhancers can also be included to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate-controlling membrane or dispersing the compound in a polymer matrix or gel.
Pharmaceutical compositions of this present technology suitable for parenteral administration generally include one or more compounds of the present technology in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders that may be reconstituted into sterile injectable solutions or dispersions prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, or solutes that render the formulation isotonic with the blood of the intended recipient.
In some cases, in order to prolong the effect of a drug, it may be desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
Injectable depot forms can be made by forming microencapsule matrices of the active compound in a biodegradable polymer, such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.
The pharmaceutical composition may also be in the form of a microemulsion. In the form of a microemulsion, bioavailability of the active agent may be improved. Reference is made to Dordunoo, S. K., et al., Drug Development and Industrial Pharmacy, 17(12), 1685-1713, 1991, and Sheen, P. C., et al., J. Pharm. Sci., 80(7), 712-714, 1991, the contents of which are herein incorporated by reference in their entirety.
The pharmaceutical composition may also contain micelles formed from a compound of the present technology and at least one amphiphilic carrier, in which the micelles have an average diameter of less than about 100 nm. In some embodiments, the micelles have an average diameter less than about 50 nm, or an average diameter less than about 30 nm, or an average diameter less than about 20 nm.
While any suitable amphiphilic carrier is considered herein, the amphiphilic carrier is generally one that has been granted Generally-Recognized-as-Safe (GRAS) status, and that can both solubilize the compound of the present technology and microemulsify it at a later stage when the solution comes into a contact with a complex water phase (such as one found in the living biological tissue). Usually, amphiphilic ingredients that satisfy these requirements have HLB (hydrophilic to lipophilic balance) values of 2-20, and their structures contain straight chain aliphatic radicals in the range of C-6 to C-20. Some examples of amphiphilic agents include polyethylene-glycolized fatty glycerides and polyethylene glycols.
Some amphiphilic carriers are saturated and monounsaturated polyethyleneglycolyzed fatty acid glycerides, such as those obtained from fully or partially hydrogenated various vegetable oils. Such oils may advantageously consist of tri-. di- and mono-fatty acid glycerides and di- and mono-polyethyleneglycol esters of the corresponding fatty acids, such as a fatty acid composition including capric acid 4-10, capric acid 3-9, lauric acid 40-50, myristic acid 14-24, palmitic acid 4-14 and stearic acid 5-15%. Another useful class of amphiphilic carriers includes partially esterified sorbitan and/or sorbitol, with saturated or mono-unsaturated fatty acids (SPAN-series) or corresponding ethoxylated analogs (TWEEN-series). Commercially available amphiphilic carriers are particularly contemplated, including the Gelucire®-series, Labrafil®, Labrasol®, or Lauroglycol®, PEG-mono-oleate, PEG-di-oleate, PEG-mono-laurate and di-laurate, Lecithin, Polysorbate 80.
Hydrophilic polymers suitable for use in the pharmaceutical composition are generally those that are readily water-soluble, can be covalently attached to a vesicle-forming lipid, and that are tolerated in vivo without substantial toxic effects (i.e., are biocompatible). Suitable polymers include, for example, polyethylene glycol (PEG), polylactic (also termed polylactide), polyglycolic acid (also termed polyglycolide), a polylactic-polyglycolic acid copolymer, and polyvinyl alcohol. Exemplary polymers are those having a molecular weight of from about 100 or 120 daltons up to about 5,000 or 10,000 daltons, and more preferably from about 300 daltons to about 5,000 daltons. In certain embodiments, the polymer is polyethylene glycol having a molecular weight of from about 100 to about 5,000 daltons, or a molecular weight of from about 300 to about 5,000 daltons, or a molecular weight of 750 daltons, i.e., PEG(750). Polymers may also be defined by the number of monomers therein. In some embodiments, the pharmaceutical compositions of the present technology utilize polymers of at least about three monomers, such PEG polymers comprising of at least three monomers, or approximately 150 daltons. Other hydrophilic polymers that may be suitable for use in the present technology include polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.
In certain embodiments, the pharmaceutical composition includes a biocompatible polymer selected from polyamides, polycarbonates, polyalkylenes, polymers of acrylic and methacrylic esters, polyvinyl polymers, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, celluloses, polypropylene, polyethylenes, polystyrene, polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, poly(butic acid), poly(valeric acid), poly(lactide-co-caprolactone), polysaccharides, proteins, polyhyaluronic acids, polycyanoacrylates, and blends, mixtures, and copolymers thereof.
The pharmaceutical composition may also be in liposomal form. Liposomes contain at least one lipid bilayer membrane enclosing an aqueous internal compartment. Liposomes may be characterized by membrane type and by size. Small unilamellar vesicles (SUVs) have a single membrane and typically range from 0.02 to 0.05 μm in diameter; large unilamellar vesicles (LUVS) are typically larger than 0.05 μm Oligolamellar large vesicles and multilamellar vesicles have multiple, usually concentric, membrane layers, and are typically larger than 0.1 μm. The liposomes may also contain several smaller vesicles contained within a larger vesicle, i.e., multivesicular vesicles.
In some embodiments, the pharmaceutical composition includes liposomes containing one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts, where the liposome membrane is formulated to provide an increased carrying capacity. Alternatively or additionally, the one or more compositions of the present technology (e.g., arimoclomol, dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), KIRA compounds (e.g., KIRA3/6/7/8), Salubrinal, or SAL003) or pharmaceutically acceptable salts may be contained within, or adsorbed onto, the liposome bilayer of the liposome. In some embodiments, the active agent may be aggregated with a lipid surfactant and carried within the liposome's internal space. In such cases, the liposome membrane is formulated to resist the disruptive effects of the active agent-surfactant aggregate. In certain embodiments, the lipid bilayer of a liposome contains lipids derivatized with polyethylene glycol (PEG), such that the PEG chains extend from the inner surface of the lipid bilayer into the interior space encapsulated by the liposome, and extend from the exterior of the lipid bilayer into the surrounding environment.
Active agents contained within liposomes are preferably in solubilized form. Aggregates of surfactant and active agent (such as emulsions or micelles containing the active agent of interest) may be entrapped within the interior space of liposomes. A surfactant typically serves to disperse and solubilize the active agent. The surfactant may be selected from any suitable aliphatic, cycloaliphatic or aromatic surfactant, including but not limited to biocompatible lysophosphatidylcholines (LPCs) of varying chain lengths, e.g., from about 14 to 20 carbons. Polymer-derivatized lipids, such as PEG-lipids, may also be utilized for micelle formation as they will act to inhibit micelle/membrane fusion, and as the addition of a polymer to surfactant molecules decreases the critical micelle concentration (CMC) of the surfactant and aids in micelle formation. Preferred are surfactants with CMCs in the micromolar range; higher CMC surfactants may be utilized to prepare micelles entrapped within liposomes of the present technology, however, micelle surfactant monomers could affect liposome bilayer stability and would be a factor in designing a liposome of a desired stability.
Liposomes according to the present technology may be prepared by any of a variety of techniques known in the art, such as described in, for example, U.S. Pat. No. 4,235,871 and International Published Application WO 96/14057, the contents of which are incorporated herein by reference in their entirety. For example, liposomes may be prepared by diffusing a lipid derivatized with a hydrophilic polymer into preformed liposomes, such as by exposing preformed liposomes to micelles composed of lipid-grafted polymers, at lipid concentrations corresponding to the final mole percent of derivatized lipid which is desired in the liposome. Liposomes containing a hydrophilic polymer can also be formed by homogenization, lipid-field hydration, or extrusion techniques, as are known in the art. By another methodology, the active agent is first dispersed by sonication in a lysophosphatidylcholine or other low critical micelle concentration (CMC) surfactant (including polymer grafted lipids) that readily solubilizes hydrophobic molecules. The resulting micellar suspension of active agent is then used to rehydrate a dried lipid sample that contains a suitable mole percent of polymer-grafted lipid, or cholesterol. The lipid and active agent suspension is then formed into liposomes using extrusion techniques well known in the art, and the resulting liposomes separated from the unencapsulated solution by standard column separation.
In some embodiments, the liposomes are prepared to have substantially homogeneous sizes in a selected size range. One effective sizing method involves extruding an aqueous suspension of the liposomes through a series of polycarbonate membranes having a selected uniform pore size. The pore size of the membrane will correspond roughly with the largest sizes of liposomes produced by extrusion through the membrane (U.S. Pat. No. 4,737,323, the contents of which are herein incorporated by reference in their entirety).
The release characteristics of a formulation of the present technology depend on several factors, including, for example, the type and thickness of the encapsulating material, the concentration of encapsulated drug, and the presence of release modifiers. If desired, the release can be manipulated to be pH dependent, such as by using a pH-sensitive coating that releases only at a low pH, as in the stomach, or releases at a higher pH, as in the intestine. An enteric coating can be used to prevent release from occurring until after passage through the stomach. Multiple coatings or mixtures of cyanamide encapsulated in different materials can be used to obtain an initial release in the stomach, followed by later release in the intestine. Release can also be manipulated by inclusion of salts or pore-forming agents, which can increase water uptake or release of drug by diffusion from the capsule. Excipients that modify the solubility of the drug can also be used to control the release rate. Agents that enhance degradation of the matrix or release from the matrix can also be incorporated. The agents can be added to the drug, added as a separate phase (i.e., as particulates), or can be co-dissolved in the polymer phase depending on the compound. In all cases, the amount is preferably between 0.1 and thirty percent (w/w polymer). Some types of degradation enhancers include inorganic salts, such as ammonium sulfate and ammonium chloride; organic acids, such as citric acid, benzoic acid, and ascorbic acid; inorganic bases, such as sodium carbonate, potassium carbonate, calcium carbonate, zinc carbonate, and zinc hydroxide; organic bases, such as protamine sulfate, spermine, choline, ethanolamine, diethanolamine, and triethanolamine; and surfactants, such as a Tween™ or Pluronic™ commercial surfactant. Pore-forming agents that add microstructure to the matrices (i.e., water-soluble compounds, such as inorganic salts and sugars) are generally included as particulates.
Uptake can also be manipulated by altering residence time of the particles in the body. This can be achieved by, for example, coating the particle with, or selecting as the encapsulating material, a mucosal adhesive polymer. Examples include most polymers with free carboxyl groups, such as chitosan, celluloses, and especially polyacrylates (as used herein, polyacrylates refers to polymers including acrylate groups and modified acrylate groups such as cyanoacrylates and methacrylates).
The present technology is further illustrated by the following Examples, which should not be construed as limiting in any way.
Cell culture. Human retinal pigment epithelium (ARPE-19) cells were obtained from ATCC and were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were kept in an incubator with 5% C02 and 95% humidified air at 37° C. Human fetal RPE (hfRPE) cells from donors at 16 to 18 weeks gestation were cultured at 37° C., 5% C02 in RPE medium. To grow polarized hfRPE cells, the cells were seeded in 12 well transwell plate in 1% RPE medium with Rock kinase inhibitor for the first week. After the first week, cells were cultured in normal 1% RPE medium for 3 weeks. Cells were used in passage 1.
Blue light treatment. ARPE19 cells were grown at 70% confluence and were treated with or without 20 μM A2E in culture medium for 24 h. Then, HBSS replaced the cell culture medium before blue light treatment. ARPE19 cells with or without A2E intake, were illuminated by 460+20 nm wavelength light for 20 min.
Cell Viability Assay. ARPE19 cells (80% confluent) pre-treated with inhibitors (
Real time monitoring of necrotic and apoptotic cell-death. Cell-death assay to study Lipofuscin's dark toxicity was performed. 90% confluent ARPE-19 or hfRPE were incubated overnight in serum free media supplemented with indicated LB concentration. Viability was assessed at 24 hrs by AlamarBlue®, or microscopy with NUC405/DRAQ7. Real time monitoring of necrotic (red) and apoptotic (blue) cell-death was performed by automated fluorescence microscopy. The exact timing of appearance of far-red (DRAQ7=plasma membrane leakage) and blue (NUC405=caspase 3 activation) fluorescence signals was critical to differentiate apoptosis and necrosis from secondary events, such as membrane damage and generalized proteolytic activation during the late phase of apoptosis and necrosis, respectively.
Chemical inhibitors. Chemical inhibitors used in this study are described in
Kinase activity assay. ARPE19 cells were treated with A2E for 6 h followed by total cell lysates preparation by syringe (20 times) with the use of lysis buffer (20 mM TrisHCl, pH 7.5, 150 mM NaCl, 1% Triton, protease-phosphatase inhibitor 1×). Supernatant was collected after centrifugation at 15000×g for 10 mins. 200 μl (3 mg/ml) of protein were taken and incubated overnight with primary RIP3 (13526,CS) or MLKL antibody at 4° C. with rotation. Next, 20 μl agarose beads A (9863,CS) were added and kept at 4° C. with rotation for 3 h followed by centrifugation at 15000×g for 30 sec. Pellet were washed and dissolved in 20 μl of kinase buffer (40 mM TrisHCl pH7.4, 20 mM MgCl2, 0.1 mg/ml BSA) with substrate and ATP (1 mM) and incubated at 30° C. for 1 h (700 speed) for kinase assay. At the end of the incubation 10 μl of the sample was taken in 384 well plate and 5 μl ADP glo solution was added (V6930, Promega) and kept at room temperature for 40 min in the dark followed by addition of 10 μl kinase detection reagent. After 20 min at room temperature, luminometer readings were taken.
Animals. Pigmented ABCA4−/− RDH8−/− double knock-out mice (DKO), free of rd8 mutation, were purchased from Jackson laboratories and every 10 generations were in house backcrossed to the C57BL6J control strain, to prevent genetic drifts. Genotyping was performed at Transnetyx (Memphis, TN). Only mice with ABCA4, RDH8, RPE65-Leu450 but no crb1 mutations (retinal degeneration slow) were maintained in the colony. Controls C57BL/6J (Rpe65-Leu450, crb1negative) were also Jackson's lab. All mice were housed at Weill Cornell Medicine's animal facility under a 12 h light (˜10 lux)/12 h dark cycle environment or under complete darkness. Experimental manipulations in the dark were done under dim red light transmitted through a Kodak No. 1 safelight filter (transmittance >560 nm). No retinal degeneration or necroptosis markers were appreciably detected in 20 months or older C57BL6/N (Rpe65-Met450, crb1 positive) obtained from the NAI/NIH. All animal procedures and experiments were approved by the Animal Care and Use Committee of Weill Cornell Medical College in agreement with the guidelines established by the NIH Office of Laboratory Animal Welfare and the Association of Research for Vision and Ophthalmology (ARVO) statement for the use of animals in ophthalmic research.
Tissue preparation. Mice were euthanized with C02 and mouse eyes were immediately enucleated. For H&E staining, mouse eyes were immersed in 4% paraformaldehyde (PFA), 16.8% isopropyl alcohol, 2% trichloroacetic acid and 2% ZnCl2 in phosphate buffer directly and sent for paraffin embedding and sectioning. For lipofuscin images, mouse eyes were immersed into 4% PFA for one hour before dissection. RPE layer was dissected out from mouse eyes and carefully flat-mounted on slides for lipofuscin assessment under fluorescence microscope. Immunofluorescence images were taken using Zeiss Spinning Disk Confocal Microscope (Zeiss, Jena, Germany).
RPE and Neural retina flat mounts. Mouse eyes were enucleated and placed in 4% PFA in PBS for 1 h at room temperature. After fixing, a 23G needle was used to make a hole at the limbus area and iris scissors used to cut around the circumference of the limbus, remove the cornea, iris and lens, separate the neuronal retina and sclera by micro-forceps. Neuronal retina and RPE layer were dissected out and incubated with blocking buffer (1% BSA and 0.3% Triton-X-100 in PBS) for at least 20 min. Primary antibodies were added to the blocking buffer and kept at 4° C. overnight. Retina and RPE layer were washed and incubated with secondary antibodies for at least 30 min at room temperature, then washed with PBS three times. Under the dissection microscope, retina or RPE layer was cut into a four-leaf clover shape and mount on the slides in mount medium (EMS glycerol mounting medium with DAPI and DABCO, cat. No. 17989-61). Slides were stored at 4° C. until imaging.
Cryosections and Immunofluorescence staining. Mouse eyes were fixed with 4% PFA and penetrated with 30% sucrose overnight at 4° C., then cryopreserved in Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA, USA) and cut into 15 μm sections. Sections were blocked with 1% BSA and 0.1% Triton-X-100 PBS, and then immunofluorescence staining was performed using standard methods and the appropriate dilutions of primary antibodies against p- p-MLKL (cell signaling), XBP1s (Biogend), Iba-1 (Abcam), CD11b (Millipore), Lamp2 (hybridoma bank), CellRox and DRAQ7 (Invitrogen), Rhodopsin (Abcam), phalloidin-CF660 (Biotium, cat.no. 0052), Hoechst33324, caspase3 (clone9664, Cell signaling). Subsequently, slides were incubated with Alexa-647, Alexa-594 secondary antibodies, and counterstained with Hoechst33324. Negative controls were included in each staining, and slides were mounted with anti-fade mount medium. Slides were stored at 4° C. before analysis on SD microscope using Zen software. For paraffin sections, mouse eyes were embedded in paraffin and cut into 5-μm sections. After deparaffinzation using standard protocol, slides were incubated with blocking buffer for 2 hrs and then primary antibodies overnight. All antibodies used for immunofluorescence staining were listed in
Demelanization. Paraffin slides were removed of paraffin using a standard protocol, as described herein. Slides were washed 4 times in Xylene 4 min/wash, 20 dips in 100% Ethanol and repeat 4 times, then air dried the slides for 10 min. For melanin bleach, slides were immersed into PBS with 10% H2O2 at 55° C. for 5 min or until melanin is bleached. Slides were blocked with blocking buffer and performed immunofluorescence staining as above mentioned.
H&E. H&E staining was performed using standard protocol, as described herein. Slides were removed of paraffin using protocol as above described. Slides were air dried. Slides on the rack were put into Xylene for 2 min (repeated once); then in 100% ethanol for 2 min (repeated once); and then 95% ethanol for 2 min once. Slides then were put in Hematoxylin for 3 min, followed by Eosin for 45 seconds, 95% ethanol for 1 min, 100% ethanol for 1 min twice, then in mounting medium and were ultimately coverslipped.
Pharmacological treatment of mice. KIRA6 (Cayman Chemical, item no. 19151) was injected intravitreously with 1 μL total volume. KIRA6 concentration is 20 μg/ml. The control eye received an equal amount of mock reagent (DMSO). Mouse was weighed and anesthetized with Ketamine cocktail at 10 mg/kg, then mouse eyes were dilated with Tropicamide. The exact volume of Mock reagent or Kira6 was determined by 10 μl Hamilton syringe. Under surgery microscope, mouse eye was placed in the center of the field, 34 gauge of needle was inserted into mouse eye at the Ora serrata and towards ONH. Once the needle was inside the mouse eye, 2 μl of the contents in the Hamilton syringe was injected. The needle was slowly withdrawn to prevent the reflux of the solution. Nec7 (2 μL of 33 mM stock in DMSO) was intravitreally injected in the left eye and an equal amount of vehicle (DMSO) was administered to the right eye. After intravitreal injection, the mouse was placed in a warm place until it completely woke up. Single intravitreal injection of Nec7 decreased pMLKL staining with the respective companion eye was performed. Immunofluorescence staining with anti-p-MLKL (red) and XBP1s (green) and LB (white) of RPE flat mount samples from 700 day old mice is shown (n=3). Bar=20 μm.
Lipofuscin synthesis. A2E was synthesized and purified by HPLC (>97%) according to a published protocol. Quality of the material was assessed by mass-spect and UV absorbance between 250 and 600 nm.
HPLC analysis of bisretinoids content. Bisretinoids were extracted from mouse eyecups under red dim light. Briefly, single mouse eyecup (containing RPE/choroid/sclera, devoid of neural retina) or ARPE-19 cells were washed with phosphate buffer (PBS) and homogenized in 1 mL PBS. Four milliliters chloroform/methanol (2:1, vol/vol) was added, and the samples were extracted with the addition of 4 mL chloroform and 3 mL dH2O, followed by centrifugation at 1000×g for 10 min. Extraction was repeated with the addition of 4 mL chloroform. Organic phases were pooled, filtered, dried under a stream of argon, and redissolved in 100 μL 2-propanol. Bisretinoid extracts were analyzed by normal-phase HPLC with a silica column (Zorbax-Sil 5 μm, 250×4.6 mm; Agilent Technologies, Wilmington, DE) as previously described (Sparrow J R, et al. (2003) J Biol Chem 278(20):18207-13). The mobile phase was hexane/2-propanol/ethanol/25 mM potassium phosphate/glacial acetic acid (485:376:100:45:0.275 vol/vol) that was filtered before use. The flow rate was 1 mL/min. Column and solvent temperatures were maintained at 40° C. Absorption units at 435 nm were converted to picomoles using a calibration curve with an authentic A2E standard and the published molar extinction coefficient for A2E; the identity of each bisretinoid peak was confirmed by online spectral analysis.
RNA isolation and quantitative PCR. Total RNA was extracted from cultured cells or mouse eye RPE layer using the RNeasy Mini kit (QIAGEN). The total RNA was digested with deoxyribonuclease I to prevent amplification of genomic DNA. The total RNA then was reversed transcribed using High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific, cat.no. 4368814) and analyzed gene expression using SYBR Green Master Mix (ThermoFisher Scientific, cat.no. 4472908) in an Applied Biosystems StepOne real time PCR machine. GAPDH was used as a reference gene. Primer sequences are displayed in
RNAseq. Cultured ARPE19 cells were treated with or without 15 uM A2E for 24 hours, then cells were harvested and total RNA extracted. Proteins were prepared for mass spectrometry analysis. RNAseq profiles were analyzed further with Ingenuity Pathway Analysis (IPA, Qiagen).
Quantity of lipofuscin per cell. RPE cells were seeded in DMEM-10% FBS overnight. The following day media was replaced with Opti-MEM supplemented with indicated amounts of A2E (0, 10, 20, 30 μM) for 24 hours. A2E-loaded cells were dissociated with trypsin, counted and resuspended in lysis buffer. For eye RPE, 12-months-old WT and DKO mice were sacrificed, their eyes were enucleated and their RPE isolated from the neural retina and underlying choroid using RNA Protect Cell Reagent from Qiagen (Cat.no. 76526) at 100 uL/eyecup for 10 minutes, as previously described (Xin-Zhao Wang C et al., (2012) Exp Eye Res 102:1-9). Mouse RPE cells were counted and resuspended in lysis buffer. The lysis buffer used was 2% Triton X-100 1% SDS in water as it dissolved efficiently cell membranes and lipid-bisretinoids. The fluorescence of the lysates (arbitrary fluorescent units) was measured using a Spectramax M5s plate reader at 430 nm for excitation and 600 nm for detection.
Detection of lipid bisretinoids aggregation. To demonstrate aggregate formation of lipid bisretinoids, fluorescence of 500 μm A2E or ATRD solutions in PBS were determined before and after passage through 13 mm Nylon syringe disc filters 0.45 μm, and 3 μm (Tisch scientific, Ohio).
Immunoblotting. Total cell lysates were extracted using RIPA lysis buffer (50 mM TrisHCl, Ph 8.0, 150 mM NaCl, 1% NP40, 0.5% Sodium Deoxycholate, 0.1% SDS, protease-phosphatase inhibitor) after the incubation of the specific sample group in a 48 well plate. Sonication or syringing (20 times) was done followed by centrifugation at 12,000×g for 10 mins and the supernatant was collected and stored in −80° C. Proten concentration from homogenates were assessed by Pierce Rapid Gold BCA protein assay kit (ThermoFisher Scientific, cat.no. A53225). Then 30 μg of protein was analyzed by standard SDS-PAGE gel. Samples were mixed 4:1 (v:v) with Nupage loading buffer with or without 8% 0-mercaptoethanol and heated for 10 min at 72° C. SDS-PAGE was done using 4-12% Nupage gel and buffer (Life, NP0335BOX). Next, SDS-PAGE gel was transferred to nitrocellulose membrane (Whatman, PROTAN BA83) overnight at 20V. The following day, 5% milk in TBS was used for 2 h blocking the membrane and then primary antibody was added in TBST (TBS, 0.1% Tween-20) for overnight at 4° C. Next day membranes were washed and incubated for 2 hours at room temperature with HRP conjugated secondary antibody (cat #G21234, Invitrogen) at 1:10,000 dilution. After 3 more washed membranes were probed with enhanced chemiluminescence (ECL) reagent and detected using an X-ray films for chemiluminescence image (GE healthcare, RPN2106). Scanning/imaging and quantitation of the image was done using silverfast 8 application software and Fiji Image J. Antibodies for Western blotting were list in
Retina ONL thinning quantification. Whole eyes were embedded in paraffin and sectioned at a thickness of 5 μm. Sections were counterstained with hematoxylin and eosin (H&E). Light microscopy was used to take digital images and the images were stitched by Zen software. Outer nuclear layer (ONL) thickness was measured at 0.2 mm intervals superior and inferior to the edge of the optic nerve head (ONH) along the vertical meridian. The center of ONH was used as the start of the measurement. The thickness of ONL was measured by Zen software.
Retinal RPE layer nuclei quantification. RPE nuclei was quantified in H&E counterstained cross sectioned eyecups. Images were taken at 40× and stitched by the Zen software. Number of RPE nuclei were counted every 0.1 mm intervals and plotted as a function of distance from ONH in 23 months old DKO (n=10) and 27 months old WT retinas (n=8). Mean values (±SEM) were significantly different at each point (p<0.05).
Retinal RPE cell size quantification. Retinal RPE eyecups were carefully dissected out of mouse eyes and stained with Alexa647-phalloidin for 1 hour at room temperature before flat-mounted on the slides. Using a Spinning Disk confocal microscope with a 63× lens, the RPE layers were imaged from the optic nerve head to the peripheral of the layer. The contours of individual RPE cells were visualized with phalloidin. Cell borders were manually selected using ImageJ software to calculate cell area and areas measured using ImageJ after manually selecting cell borders. Number of RPE nuclei were counted every 0.1 mm intervals and plotted as function of distance from ONH in 23 months old DKO (n=10) and 27 months old WT retinas (n=8). Mean values (±SEM) were significantly different at each point (p<0.05).
Statistics. All data were processed in Prism7.0 software. ANOVA, Student's t test or multiple t-test was used when appropriate. P values less than 0.05 were considered statistically significant.
HPLC and more recently quantitative fundus autofluorescence (qFAF) have become gold standards for measuring the content of LBs in retinas of animal models (Sparrow J R, et al. (2013) Investig Ophthalmol Vis Sci 54(4):2812-2820), yet the amounts reported by each method do not completely match. Experiments in mouse lines with excessive accumulation of LF, e.g Abca4−/− or Abca4−/− RDH8−/− (double KO or DKO mouse) have reported a curious dichotomy: while qFAF indicates that the combined content of LBs in RPE and PRs increases continuously throughout life, HPLC shows a sharp decline in RPE's LBs after the first few months (Sparrow J R, et al. (2013) Investig Ophthalmol Vis Sci 54(4):2812-2820). This dichotomy was attributed to an early, sudden loss of RPE cells containing above threshold levels of LF that would permanently compromise the functionality of the epithelium promoting the formation and impairing the phagocytic removal of new LF in PRs (Flynn E, Ueda K, Auran E, Sullivan J M, Sparrow J R (2014) Invest Ophthalmol Vis Sci 55(9):5643-52).
To elucidate the cytological and pathological aspects of this process, experiments in DKO vs control mice were carried out to compare the accumulation of HPLC-extractable LBs with the accumulation of fluorescent granules in the cytoplasm of RPE cells, measured by confocal microscopy, as a function of retina age. Using confocal microscopy, the number of LF-granules per RPE cell increased to fully occupy the whole cytoplasm in old DKOs (
In order to quantify the extent of this phenomena, the size (area in μm2) of cells in random central locations using flat-mounts of DKO RPE detached from the neural retina between 8 and 23 months and age-matched controls (8 and 27 months) was surveyed (
Furthermore, larger (˜5-10 μm) epithelial-sized fragments filled with LF (
Lipofuscin was quantified in mouse RPE cells in DKO and WT mice of different ages by microscope. Mouse RPE cells were photographed from the center (ONH) to the periphery of mouse eyecup. The lipofuscin of central RPE cells was quantified by Image J and graphed. Over 300 RPE cells were quantified in each group, each dot in the graph represent a single cell. The lipofuscin contain in DKO 8 months, 13 months and 26 months are much higher than DKO 3 months (p<0.01 by unpaired t test). DKO 3 months is higher than WT 8 months and 33 months (p<0.01 by t test). WT 33 months group is higher than WT 8 months group with significance (p<0.01 by unpaired t test).
Taken together, confocal images of RPE from DKO eyecups showed that the number of LF granules per cell increased uninterruptedly with age (
Photooxidative degradation of LBs contributes to retinal degeneration in albino animals, but it remains unclear whether photooxidation takes place at significant levels in pigmented retinas. Since photooxidation can be stopped by rearing animals in complete darkness (Ueda K et al., (2016) Proc Natl Acad Sci USA. 113(25):6904-9, Boyer N P et al., (2012) J Biol Chem 287(26):22276-22286) without affecting the accumulation of new LBs, studies were performed to determine whether RPE and PRs would still degenerate in DKO retinas never exposed to light. Accordingly, WT and DKO pigmented mice were housed from birth in either continuous darkness or under 12 h cyclic light conditions. The change in the thickness of their ONL as well as the number of RPE nuclei were evaluated for up to one year. As shown in
Taken together, the comparable loss of PRs and RPE cells between dark- and light-reared DKO retinas (
To study the mechanisms involved in light-independent cell death, existing protocols of incorporating LBs into the lysosomes of RPE cells were adapted (Sparrow J R et al., (1999) Invest Ophthalmol Vis Sci 40(12):2988-95, Sparrow J R, Kim S R, Wu Y Chapter 18 Experimental Approaches to the Study of A2E, a Bisretinoid Lipofuscin Chromophore of Retinal Pigment Epithelium. 315-327, Boulton M E (2014) Exp Eye Res 126:61-7) and dose dependent RPE cell-death under no light condition was reproducibly provoked (
The susceptibility to LF depended heavily on cell confluency (
LF-induced apoptosis and necrosis at single-cell level was investigated by adding NucView®405 (a non-fluorescent cell-permeant substrate that stains nuclear DNA blue when cleaved by caspase-3 during the executioner phase of apoptosis) and DRAQ7 (a dye that stains DNA red only if cells have compromised membrane integrity) to the cultures. LF induced necrosis in the absence of light, but if exposed to blue-light, apoptosis was induced (
To determine whether A2E induces necrosis by intercalating into membranes, methyl beta-cyclodextrins (MβCD) was used. MβCD is a cyclic sugar that protects against detergent effects by forming soluble complexes with amphipathic molecules. Remarkably, although MβCD complexed with both A2E and Triton-X100, it did not protect against A2E but fully shielded against lethality caused by Triton-X100 (
Pretreatment with the pan-caspase inhibitor z-VAD(OMe)-FMK and the gasdermin-D inhibitor disulfiram provided no protection. GSK'872, a selective inhibitor of RIPK3 (the only known kinase to phosphorylate human MLKL at Ser358 (pMLKL)) did not protect, either.
However, dabrafenib an ATP competitive inhibitor of B-Raf and RIPK3 or necrosulfonamide (NSA), a drug that prevents the spontaneous assembly of pMILKL into oligomeric pores that insert into membranes that eventually kill, increased RPE survival in a dose dependent manner. For its phosphorylation, MLKL and its kinase need to be recruited into multiprotein complexes, known as necrosomes (
To rule out that the low expression of these proteins in ARPE19 precluded their detection, experiments were performed in human intestinal HT29 cells after treatment with LBs or TNFα/caspase inhibitors (
In contrast, Nec1 did not block phosphorylation and polymerization of MLKL (
These results demonstrate that the light-independent cytotoxicity of A2E or ATRD loaded LF-granules elicited necroptosis, which is relevant to the pathobiology of GA. Necroptosis is a type of programmed cell-death that leads to cell membrane disruption causing atrophic areas and the release of cellular constituents known that elicit local inflammation. Evidence for light-independent LF mediated necroptosis is as follows: (i) real-time monitoring of cell death showed early impairment in membrane integrity without caspase-3 activation; (ii) LF induced a dose-dependent Ser358-phosphorylation, polymerization and plasma membrane translocation of the pseudokinase mixed-lineage kinase domain-like (MLKL), in light-free conditions; (iii) cell-death was preventable with dabrafenib and NSA; (iv) Immunoprecipitation experiments revealed that LF promoted the association of MLKL with a kinase, indicative of necrosome formation; (v) dark cell-death could not be prevented by the pan-caspase inhibitor z-VAD(OMe)-FMK nor anti-oxidants; and (vi) Nec7, potently prevented MLKL phosphorylation, polymerization, plasma membrane localization and cell-death by LF. See
LF cell-death and MLKL phosphorylation/polymerization were not affected by GSK'872 (
Finally, since it has been suggested that apart from the adverse effects of lipid-bisretinoids, the retina of ABCA4−/−RDH8−/− mice succumbs from the toxicity of the all-trans-retinal (ATR) released upon illumination, the link between ATR and necroptosis was investigated. Using the cell-death assay, ATR was found to trigger a light-independent cell-death in ARPE19 cells (
These results demonstrate that Dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), and IRE1α inhibitors that block IRE1α dimerization are useful in methods for preventing or treating an eye disease associated with retinal cell lipofuscin-associated cytotoxicity in a subject in need thereof.
LF deposits are thought to induce oxidative stress (Ueda K, et al. (2018) Proc Natl Acad Sci USA 115(19):4963-4968). Thus, the protective effects of a variety of antioxidants: N-acetyl cysteine (NAC), Trolox, L-cysteine, vitamin C, BHA and TMB against LF cytotoxicity was investigated. None of the tested antioxidants had any effect on RPE survival relative to LF accumulation, although NAC effectively prevented oxidative damage by H2O2 (
Since accumulation of crystalline materials in cells can elicit necroptosis and atomic force microscopy revealed that the core of lipofuscin granules comprises solid aggregates, the ability of lipid bisretinoids to form crystals that kill cells was investigated. A2E (MW 592 Da) in aqueous milieu formed aggregates the size of a bacteria, that could not move across 450 nm filter pores (
To investigate the cellular processes leading to necroptosis, Ingenuity Pathway Analysis (IPA) was used to compare the transcriptomes of cells suffering of dark LF cytotoxicity with cells rescued by Nec7 treatment. As shown in
The causal link between LF accumulation and ER-stress without light assistance was analyzed (
Regular PCR coupled with agarose gel revealed splicing of XBP1 in ARPE19 and hfRPE (
To determine how UPR was implicated in LF induced necroptosis, each of the three endogenous ER-stress sensors/effectors, PERK, ATF6 and IRE1α, were individually knocked out (KO) in ARPE-19 cells, and their susceptibility to LBs was tested with our light-free cell-death assay. Remarkably, IRE1α-KO showed significant increase in survival to toxic doses of LBs (
The critical role of IRE1α in the light-independent cell-death process was evaluated using small-molecules that selectively blocked IRE1α at the various stages of activation. A set of inhibitors selective for the kinase (APY29, sunitinib) or RNAse (STF-0831, 4μ8C, MKC-3946) functions were tested. Surprisingly, individual or combined inhibition of the kinase, RNAse or both activities did not translate into survival suggesting that LF-mediated necroptosis does not rely on the IRE1α canonical signaling pathway as the UPR response (
These results demonstrate that Dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), Arimoclomol, and IRE1α inhibitors that block IRE1α dimerization are useful in methods for preventing or treating an eye disease associated with retinal cell lipofuscin-associated cytotoxicity in a subject in need thereof.
To investigate whether pigmented retinas affected with LF experience progressive IRE1α mediated deposition of pMLKL oligomers, eyes from 2, 12 and 27 months old ABCA4−/− RDH8−/− DKO and WT C57BL6 mice were dissected and their whole RPE, flat mounted and stained with anti-XBP1s (
The atypical necroptosis observed in cell cultures was blocked by Nec7. To verify the role of Nec7 in in vivo detected necroptosis, 1 of vehicle and 1 of Nec7 was intraocularly injected in the right eyes and left eyes, respectively, of 12 month old DKOs and their pMLKL levels were analyzed one week later. As shown in
The phospho-MLKL staining was particularly evident for microglia/macrophages infiltrated into subretinal space that appeared attached to RPE in flat mounts from 20 months old DKO (
XBP1s was also positive around the bodies, inner segments and large parts of the outer segments of PRs. To further confirm the activated status of the microglia, the phenotype of Iba-1+ cells infiltrated in the subretinal space was analyzed. RPE flat-mounts from 25 to 27 month DKOs were prepared and dual stained with Iba-1/XBP1s or Iba-1/pMLKL (
Very impressive was the discovery of a vast phospho-MLKL staining around the zones of the neural retina, where RPE cells had migrated (
To establish the therapeutic potential of inhibiting the atypical necroptosis pathway, 2 μl of necrostatins, or vehicle were injected intraocularly in the eyes of 26-month-old DKO mice. A week later, the status of their retinas was evaluated by staining RPE- and neuroretina-flat-mounts with anti phospho-MLKL antibody. It was verified that Nec7 but not Nec1, reduced membrane and cytosolic phospho-MLKL staining to undetectable levels (
These results demonstrate that Dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), Arimoclomol, and IRE1α inhibitors that block IRE1α dimerization are useful in methods for preventing or treating an eye disease associated with retinal cell lipofuscin-associated cytotoxicity in a subject in need thereof.
To establish the therapeutic potential of inhibiting IRE1α-driven cell-death for pigmented retinas with LF accumulation, the IRE1α inhibitor, KIRA6, or vehicle was injected intraocularly in the eyes of 26 month old DKO mice. A week later, their retinas status was evaluated by staining whole RPE flat mounts with specific antibodies. XBP1s staining was decreased from the center to the periphery by the KIRA6 treatment compared to mock treated eyes. These results demonstrate that KIRA6 was effective at blocking IRE1α signals in vivo and that the XBP1s detection was specific (
Furthermore, KIRA6 abrogated necroptosis, as depicted by the center to periphery disappearance of intracellular and plasma membrane labelling by pMLKL antibody (
Collectively, these results demonstrate that ER-stress and necroptosis were exacerbated in the retinas with LF. Treatment with Nec7 reduced pMLKL staining indicating the labeling was specific and that necroptosis in the retinas was inhibited with Nec7, as in RPE cultures. Histology of retinas with high content of LF revealed that IRE1α and pMLKL membrane deposition were increasingly present in RPE and invading microglia and that both IRE1α and pMLKL tend to colocalize in the same cells as LF-driven degeneration proceeded. The type of necroptosis found in the retinas was susceptible to inhibition with Nec7 suggesting it represented the same type of atypical necroptosis observed in cultured cells. Treatment with KIRA6 normalized the levels of IRE1α activation, pMLKL oligomerization and Iba1+ microglia infiltration as well as multiple markers of ongoing retinal degeneration detected by qPCR (
Only Salubrinal (SAL) and SAL003, that targeted both cellular eIF2a phosphatases comprised of PP1 bound to either GADD34 or CreP, catalytic subunits, protected against lipofuscin. In contrast, Guanabenz, that only disrupts PP1-GADD34 association or eIF4 and mTOR activators, did not confer significant protection (see
These results demonstrate that Dabrafenib, necrosulfonamide (NSA), Necrostatin 7 (Nec7), Salubrinal, SAL003, Arimoclomol, and IRE1α inhibitors that block IRE1α dimerization are useful in methods for preventing or treating an eye disease associated with retinal cell lipofuscin-associated cytotoxicity in a subject in need thereof.
The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.
As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.
All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all FIG.s and tables, to the extent they are not inconsistent with the explicit teachings of this specification.
This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63/140,533 filed Jan. 22, 2021, the entire contents of which is incorporated herein by reference.
This invention was made with government support under EY027422-04 awarded by the National Institutes of Health/National Eye Institute. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/013276 | 1/21/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63140533 | Jan 2021 | US |
