Classifications

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Definitions

• Alzheimer's disease As the human population is aging, the prevalence of age-related conditions, including, but not limited to, cancers and neurodegenerative diseases increases, yet interventions to prevent or treat these conditions are lacking or have undesirable side effects.
• BCa Breast cancer
• Treatments of malignant diseases or disorders are also limited and suffer from numerous drawbacks.
• Treatments generally include local therapy (for instance: surgery with or without radiation in breast cancer, surgery or radiation in prostate cancer) and adjuvant systemic therapy (hormonal therapy, chemotherapy, and biologic agents) for cancer cells that may have spread. Radiation and chemotherapy often cause substantial side-effects including, but not limited to, nausea and hair loss.
• Hormone therapy for prostate cancer includes anti- androgens.
• SERM selective estrogen receptor modulators
• tamoxifen and raloxifene such as tamoxifen and raloxifene
• aromatase inhibitors such as exemestane and anastrozole
• Monoclonal antibodies such as trastuzumab and pertuzumab, are approved for the treatment of HER2 positive cancer.
• HER2 positive cancer For patients with triple-negative breast cancer (absence of estrogen, progesterone, and the Her2/neu receptor), treatment options are limited.
• Lysosomal dysfunction is implicated in a diverse range of disorders and diseases, including genetic diseases.
• high levels of endocytosis have been suggested as a pleiotropic factor in the etiology underlying malignant and neurodegenerative processes, including, but not limited to, a role of endocytosis in cancer, and "deranged" endocytosis in Alzheimer's disease, where endocytic pathway abnormalities precede ⁇ deposition and inhibition of endocytosis reduces amyloid precursor protein ("APP”) internalization and immediately lowers ⁇ levels in vivo.
• APP amyloid precursor protein
• endocytosis While sufficient levels of endocytosis are necessary in early life, high levels of endocytosis may become detrimental with age. Enlarged macrophages have been identified in atherosclerosis, steatohepatitis, and cystic fibrosis, and high levels of endocytosis can increase accumulation of lipids in macrophages. Activation of the early endocytic pathway has been observed in autoimmune-diseases, such as systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), and arthritis. Attenuation of phosphoinositides (PIPs), which regulate endocytosis, restrains autoimmune disease and phosphoinositides have also been linked to several genetic diseases.
• SLE systemic lupus erythematosus
• IBD inflammatory bowel disease
• PIPs phosphoinositides
• Impaired phospholipid (PL) efflux increases accumulation of lipids in macrophages (MOs) and enlarged macrophages have been identified in atherosclerosis, steatohepatitis, and cystic fibrosis (CF).
• PI3K phosphoinositide-3 kinase
• Activation of the early endocytic pathway can lead to accumulation of lipids in macrophages and enlarged macrophages have been identified in atherosclerosis, steatohepatitis, and cystic fibrosis.
• Activation of the early endocytic pathway has been observed in autoimmune-diseases, such as systemic lupus erythematosus (SLE), inflammatory bowel disease, and arthritis. Attenuation of phosphoinositides restrains autoimmune disease.
• cyclin-dependent kinase (CDK)] inhibitors yielded largely disappointing results, and only a small number of patients benefit from phosphoinositide 3-kinase (PI3K) inhibitors.
• PI3K phosphoinositide 3-kinase
• ⁇ -CD The lipid-carrier molecule ⁇ -cyclodextrin ( ⁇ -CD) is "generally recognized as safe” (GRAS) as food additives and the more water-soluble derivative 2-hydroxypropyl-P- cyclodextrin ( ⁇ - ⁇ -CD, HPpCD) is frequently used as an excipient to form water soluble compounds with lipophilic drugs.
• GRAS generally recognized as safe
• ⁇ - ⁇ -CD 2-hydroxypropyl-P- cyclodextrin
• HPpCD 2-hydroxypropyl-P- cyclodextrin
• ⁇ - ⁇ -CD has been shown to exert some of its effect by reducing lysosomal dysfunction (LYD) and improving autophagy ( ⁇ ), another common factor in the etiology of age-related conditions.
• ⁇ - ⁇ -CD has been used for the treatment of Niemann-Pick disease type C (NPC), where its function is to reduce the aggregation of cholesterol, a lipid molecule, in lysosomes (LYs). Again, the mechanism of action is unknown. Clinical applications of ⁇ - ⁇ -CD have been limited, however, because depriving the ear's outer hair cells of cholesterol may cause permanent hearing loss.
• NPC Niemann-Pick disease type C
• LYs lysosomes
• a-CD a-cyclodextrin
• HP-a-CD hydroxypropyl- a-cyclodextrin
• provided herein are methods of targeting the PI cycle as a means for treating diseases or conditions causing a dysfunctional lysosomal pathway and/or elevated endocytosis, phagocytosis, or pinocytosis in a subject, in which the diseases or condition may be caused by e.g., aberrant expression of a gene or combination of genes as identified e.g., by the novel GWAS approach (see e.g., Example 1) described herein.
• Such diseases or conditions include e.g., breast cancer (BCa), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), coronary artery disease (CAD), and Niemann-Pick type C disease (NPC). See e.g., Examples 2, 3, 6, and 7.
• beta-cyclodextrins include e.g., methyl-P-cyclodextrin ( ⁇ - ⁇ -CD) and ⁇ - ⁇ -CD. See e.g., J Membr Biol. 2011 May, 241(1): 1-10; The Journal of Experimental Medicine, 209 (13), 2501-13, and the discussion set forth in Example 5.
• beta-cyclodextrins were believed to remove and to extract cholesterol (Choi) from cell membranes and/or organelles.
• ⁇ -CDs have therapeutic potential, they are ototoxic, i.e., cause damage to the ear which can result in permanent hearing loss. See J Assoc Res Otolaryngol, 16 (5), 599-611.
• a-CD restores what has been called “derailed” endocytosis in breast cancer, “deranged” endocytosis in AD and “defective” endocytosis in PD. See e.g., Example 4.
• a-CD is more efficient than ⁇ -CD in solubilizing phospholipids (see Example 8). Therefore, in one aspect, provided herein are methods of using a-cyclodextrins (e.g., a-CD or HP-a-CD) for treating a subject having a disease, disorder, or condition that involves impaired lysosomal function.
• Such diseases and disorders include e.g., breast cancer (BCa), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), coronary artery disease (CAD), and Niemann-Pick type C disease (NPC)
• BCa breast cancer
• AD Alzheimer's disease
• PD Parkinson's disease
• HD Huntington's disease
• MS multiple sclerosis
• ALS amyotrophic lateral sclerosis
• CAD coronary artery disease
• NPC Niemann-Pick type C disease
• alpha- cyclodextrins e.g., HP-a-CD
• a composition comprising a clathrate of HP-a-CD and medium chain fatty acids substantially improved absorption.
• pharmaceutical compositions comprising an alpha- cyclodextrin (e.g., HP-a-CD) and a medium chain fatty acid (e.g., capric acid), as well as and their use for treating a disease or disorder that involves impaired lysosomal function and autophagy.
• Such diseases and disorders include e.g., breast cancer (BCa), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), coronary artery disease (CAD), and Niemann-Pick type C disease (NPC).
• BCa breast cancer
• AD Alzheimer's disease
• PD Parkinson's disease
• HD Huntington's disease
• MS multiple sclerosis
• ALS amyotrophic lateral sclerosis
• CAD coronary artery disease
• NPC Niemann-Pick type C disease
• compositions comprising an alpha- cyclodextrin (e.g., HP-a-CD) and a medium chain fatty acid (e.g., capric acid) for use in treating inflammatory diseases.
• an alpha- cyclodextrin e.g., HP-a-CD
• a medium chain fatty acid e.g., capric acid
• the present disclosure provides a method of treating a malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition in a subject, the method comprising administering to the subject an effective amount of a cyclic oligosaccharide (e.g., a- cyclodextrin (a-CD)), or an analogue or derivative thereof (e.g., HP-a-CD), either alone or in combination with one or more additional active agents.
• a cyclic oligosaccharide e.g., a- cyclodextrin (a-CD)
• an analogue or derivative thereof e.g., HP-a-CD
• the present disclosure provides a composition comprising a-CD, or an analogue or derivative thereof (e.g., HP-a-CD), for use in the treatment of an epithelial cancer (carcinoma) or the treatment of Parkinson's, Alzheimer's, or Huntington's disease.
• the composition comprising HP-a-CD further comprises a medium-length chain fatty acid (MCFA), e.g., fatty acids having an aliphatic tail of 6-12 carbon atoms.
• MCFA medium-length chain fatty acid
• the HP-a-CD and medium- length chain fatty acid (MCFA) in the composition form a clathrate. In such clathrates, the MCFAs are guests of HP-a-CD.
• the MCFA in the composition is caprate or caprylate or is a salt thereof such as sodium caprate.
• the present disclosure provides a method of improving one or more indicators or symptoms of a malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition in a subject, the method comprising administering to a subject exhibiting one or more indicators or symptoms of a malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition, an effective amount of a-CD, or an analogue or derivative thereof (e.g., HP-a-CD), either alone or in combination with one or more additional active agents.
• a-CD or an analogue or derivative thereof (e.g., HP-a-CD)
• Suitable indicators include, but are not limited to, results of a blood test (including, but not limited to circulating tumor DNA and/or prostate-specific antigen), an x-ray evaluation, the result of a physical examination (including, but not limited to a palpable tumor), a psychiatric evaluation, or a tissue biopsy for histological evaluation and/or determination of hormone receptor status.
• a symptom or indicator is selected from the group consisting of survival, disease-free survival, distant metastasis-free survival, results of a blood test (including, but not limited to circulating tumor DNA and prostate- specific antigen), an x-ray evaluation, the result of a physical examination (including, but not limited to a palpable tumor), or a tissue biopsy for histological evaluation.
• the "improving" comprises an increase of at least 1% in a measurement of the one or more indicators or symptoms
• the present disclosure provides a composition comprising a-CD, or an analogue or derivative thereof (e.g., HP-a-CD), for use in the treatment of an epithelial cancer (carcinoma) or the treatment of Parkinson's, Alzheimer's, or Huntington's disease.
• the composition comprising HP-a-CD further comprises a medium-length chain fatty acid (MCFA), e.g., fatty acids having an aliphatic tail of 6-12 carbon atoms.
• MCFA medium-length chain fatty acid
• the HP-a-CD and medium-length chain fatty acid (MCFA) in the composition form a clathrate. In such clathrates, the MCFAs are guests of HP-a-CD.
• the MCFA in the composition is caprate or salt thereof such as sodium caprate.
• the disclosure relates to a method of restoring the synthesis of sphingomyelin in a subject in need thereof, the method comprising administering a cyclodextrin (e.g., a-CD), or an analogue or derivative thereof (e.g., HP-a-CD), either alone or in combination with one or more additional active agents.
• a cyclodextrin e.g., a-CD
• an analogue or derivative thereof e.g., HP-a-CD
• the present disclosure provides a method of improving one or more indicators or symptoms of a malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition in a subject in need thereof, the method comprising administering to a subject exhibiting one or more indicators or symptoms of a malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition, an effective amount of a-CD, or a salt, analogue or derivative thereof (e.g., HP-a-CD), either alone or in combination with one or more additional active agents.
• a-CD or a salt, analogue or derivative thereof (e.g., HP-a-CD)
• Suitable indicators include, but are not limited to, results of a blood test (including, but not limited to circulating tumor DNA and/or pro state- specific antigen), an x-ray evaluation, the result of a physical examination (including, but not limited to a palpable tumor), a psychiatric evaluation, or a tissue biopsy for histological evaluation and/or determination of hormone receptor status.
• a symptom or indicator is selected from the group consisting of survival, disease-free survival, distant metastasis-free survival, results of a blood test (including, but not limited to circulating tumor DNA and prostate-specific antigen), an x-ray evaluation, the result of a physical examination (including, but not limited to a palpable tumor), or a tissue biopsy for histological evaluation.
• the "improving" comprises an increase of at least 1% in a measurement of the one or more indicators or symptoms.
• the present disclosure provides a method of treating and/or preventing focal segmental glomerulosclerosis (FSGS) and nephrotic swelling in a subject in need thereof the method comprising administering to the subject an effective amount of a-CD, a salt or an analogue or derivative thereof (e.g., HP-a-CD), either alone or in combination with one or more additional active agents.
• FSGS focal segmental glomerulosclerosis
• HP-a-CD an analogue or derivative thereof
• the disclosure relates to a method of treating and/or preventing kidney damage caused by excess or dysfunctional sphingolipid catabolism in a subject in need thereof, the method comprising administering to the subject an effective amount of a-CD, or a salt, an analogue or derivative thereof (e.g., HP- a-CD), either alone or in combination with one or more additional active agents.
• a-CD a salt, an analogue or derivative thereof
• HP-a-CD an analogue or derivative thereof
• the disclosure relates to a method of restoring the synthesis of sphingomyelin in a subject in need thereof, the method comprising administering a-CD, or an analogue or derivative thereof (e.g., HP-a-CD or salt thereof), either alone or in combination with one or more additional active agents.
• compositions described herein comprise resorption enhancers known in the art including, but not limited to bile salts (sodium glycocholate, sodium deoxycholate, sodium taurocholate, sodium fusidate, sodium glycodeoxycholate, sodium taurodihydrofusidate), surfactants (sodium lauryl sulfate, lysophosphatidylcholine (LPC), dioctyl sodium sulfosuccinate, laurenth-9, polysorbate, polyethyleneglycol-8-laurate, glyceryl monolaurate, glyceryl monocaprylate/caprate, glyceryl dicaprylate/caprate, saponin), fatty acids and derivatives (sorbitan laurate, sodium caprate, sucrose palmitate, lauroyl choline, sodium myristate, palmitoyl carnitine), glycerides (phospholipids, monohexanoin, medium chain g
• the cyclodextrin is administered to the subject at a dose of about 2500 mg/kg a-CD bi-weekly (-700 mg/kg/d), the same dose used for ⁇ - ⁇ -cyclodextrin in two children with NPC for "over a year, with no discernable side effects" for a "targeted concentration of 0.1-1.0 mM.” (INDs 104,114 and 104,116, approval date: 2009-04-13).
• the dose will be adjusted over time to the highest dose not causing renal adverse events or hemolysis in the patient.
• the cyclodextrin such as a- CD
• the cyclodextrin such as a-CD
• each of the dosages described above is mg/kg/day. Additional dosages that may be used are provided in the Detailed Description section of this patent application.
• the present disclosure provides a method of treating and/or preventing Huntingdon's disease, Alzheimer's disease, Parkinson's disease, multiple sclerosis, in a subject in need thereof, the method comprising administering to the subject an effective amount of a-CD, or an analogue or derivative thereof (e.g., HP-a-CD), either alone or in combination with one or more additional active agents.
• a-CD or an analogue or derivative thereof (e.g., HP-a-CD)
• the present disclosure provides a method of treating and/or preventing a malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition in a subject not exhibiting disease-specific indicators or symptoms, except indicators of the subject to belong to an at- risk subpopulation.
• the indicator will be age. In some embodiments, the indicator will be more than 30, 40, 50, or 60 years of age.
• the present disclosure provides a method of treating or preventing a malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition in a subject in need of treatment, the method comprising administering to the subject an effective amount of a drug reducing extracellular phospholipid.
• the malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition will be an epithelial cancer (carcinoma).
• the malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition will be breast cancer.
• the malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition will be Alzheimer's disease.
• the malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition will be Parkinson's disease.
• the malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition will be Huntington's disease.
• malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition will be non-alcoholic steatohepatitis (NASH).
• NASH non-alcoholic steatohepatitis
• the present disclosure provides various combinations of treatments, including pharmaceutical compositions.
• cyclodextrins are used in combination with established pharmaceutical, radiological, or surgical interventions comprising cytotoxic interventions, receptor antagonists, monoclonal antibodies, radiation therapy, removal of tumor tissue, and the like.
• the subject is a human. In another embodiment the subject is an adult human. In some embodiments, the subject is in need of the treatment and/or has been identified as having a risk of developing a disease or disorder resulting from one or a plurality of cellular defects caused by lysosomal dysfunction.
• the disclosure relates to a composition comprising an effective amount of a clathrate of HP-a-CD and a salt, such as sodium caprate.
• the molar ratio of HP-a-CD to the fatty acid is from about 0.5 to about 5. In some embodiments, the molar ratio of HP-a-CD to the fatty acid is from about 2.0 to about 1.0. In some embodiments, the molar ratio of HP-a-CD to the fatty acid is from about 1.5 to about 1.0. In some embodiments, the molar ratio of HP-a-CD to the fatty acid is from about 1.0 to about 1.0.
• the disclosure relates to a pharmaceutical composition
• a pharmaceutical composition comprising an effective amount of a clathrate of HP-a-CD and a salt, such as sodium caprate, wherein the composition is free or substantially free of beta-cyclodextrin and/or a-CD.
• the disclosure relates to a method of treating any disorder disclosed herein by administering an effective amount of a pharmaceutical composition comprising (i) one or a plurality of a-cyclodextrin, derivative thereof, or a salt thereof and (ii) a fatty acid molecule, each of (i) and (ii) at an amount in weight to form a clathrate of the one or a plurality of a- cyclodextrin, derivative thereof, or a salt thereof.
• the clathrate is from about 80 to 90% in weight of one or a plurality of a-cyclodextrin, derivative thereof, or a salt thereof, and from about 10% to about 20% in weight of the fatty acid.
• the clathrate is from about 85% in weight of one or a plurality of ⁇ -cyclodextrin, derivative thereof, or a salt thereof, and about 15% in weight of the fatty acid.
• the fatty acid is capric acid or a salt thereof.
• the disclosure relates to a method of treating and/or preventing Alzheimer's disease in a subject in need thereof, the method comprising administering an effective amount of a pharmaceutical composition comprising one or a plurality of alpha-cyclodextrins, a derivatives, salt thereof or combinations thereof to the subject.
• the disclosure relates to a method of treating and/or preventing Parkinson's disease in a subject in need thereof, the method comprising administering an effective amount of a pharmaceutical composition comprising one or a plurality of alpha-cyclodextrins, a derivatives, salt thereof or combinations thereof to the subject.
• the disclosure relates to a method of treating and/or preventing Huntington's disease in a subject in need thereof, the method comprising administering an effective amount of a pharmaceutical composition comprising one or a plurality of alpha-cyclodextrins, a derivatives, salt thereof or combinations thereof to the subject.
• the disclosure relates to a method of treating and/or preventing amyotrophic lateral sclerosis (ALS) disease in a subject in need thereof, the method comprising administering an effective amount of a pharmaceutical composition comprising one or a plurality of alpha- cyclodextrins, a derivatives, salt thereof or combinations thereof to the subject.
• ALS amyotrophic lateral sclerosis
• the disclosure relates to a method of treating and/or preventing multiple sclerosis (MS) disease in a subject in need thereof, the method comprising administering an effective amount of a pharmaceutical composition comprising one or a plurality of alpha-cyclodextrins, a derivatives, salt thereof or combinations thereof to the subject.
• the pharmaceutical composition comprises an effective amount of a clathrate of the one or a plurality of alpha-cyclodextrins, a derivatives, salt thereof or combinations thereof, wherein the clathrate comprises a medium-length chain of fatty acid or salt thereof and the one or a plurality of alpha-cyclodextrins, a derivatives, salt thereof or combinations thereof.
• the one or a plurality of alpha-cyclodextrins, a derivatives, salt thereof or combinations thereof and the fatty acid are in a weight/weight ratio from about 15% weight fatty acid and about 85% weight of the one or a plurality of alpha- cyclodextrins, a derivatives, salt thereof or combinations thereof in the clathrate.
• the weight:weight ratio of fatty acid to a-cyclodextrin is sufficient for absorption into the blood.
• the disclosure relates to a method of treating and/or preventing inflammation in a subject in need thereof, the method comprising administering an effective amount of a pharmaceutical composition comprising one or a plurality of alpha-cyclodextrins, a derivatives, salt thereof or combinations thereof to the subject.
• the disclosure also relates to a method of treating and/or preventing local metastases of a breast cancer or prostate cancer characterized by dysfunctional or deficient endocytosis caused by dysfunction or deficiency of any genes identified in the disclosure related to endocytosis.
• the disclosure also relates to a method of treating and/or preventing local metastases of a breast cancer or prostate cancer characterized by dysfunctional or deficient lysosomal function caused by dysfunction or deficiency of any genes identified in the disclosure related to lysosomal function. in a subject in need thereof, the method comprising administering an effective amount of a pharmaceutical composition comprising one or a plurality of alpha-cyclodextrins, a derivatives, salt thereof or combinations thereof to the subject.
• the disclosure relates to a method of reducing phospholipid levels in the blood of a subject, the method comprising administering an effective amount of a pharmaceutical composition comprising one or a plurality of alpha-cyclodextrins, a derivatives, salt thereof or combinations thereof to the subject.
• the method of reducing phospholipid levels in the blood is accomplished without scavenging lysophopholipids.
• the disclosure also relates to a method of activating autophagy, the method comprising administering an effective amount of a pharmaceutical composition comprising one or a plurality of alpha-cyclodextrins, a derivatives, salt thereof or combinations thereof to the subject.
• the method of improving or activating autophagy is accomplished without causing lysosomal dysfunction.
• FIG. 1 Cyclodextrins, Including Hydroxypropyl Cyclodextrins. Up to nx3 degrees of substitution may be realized, with numerous positional and regio-isomers possible. Substitutions for R include, but are not limited to, H (parent), methyl (including randomly methylated), butyl, 2-hydroxypropyl (HP) (shown), acetyl, succinyl, glucosyl, maltoseyl, carboxymethyl ether, phosphate ester, simple polymers, or carboxymethyl. Typical cyclodextrins contain 6 (a-CD), 7 ( ⁇ -CD) or 8 ( ⁇ -CD) D-glucose monomers in a ring, creating a cone shape that can accommodate guest molecules into their hydrophobic cavity.
• FIG. 2 Clustering analysis of cholesterol interaction with a-CD and ⁇ - ⁇ -CD.
• CD molecules are depicted using the solid surface representation method with a probe radius of 0.4 A, density isovalue of 0.7 and grid spacing of 0.5 to visualize the binding cavity. Hydrogen atoms are omitted to enhance clarity. Modified from (Shityakov S, Salmas RE, et al. (2016) J Toxicol Sci 41: 175-84)
• FIG. 3A through FIG. 3C Specificity of Lipid Release I. Release of phospholipids
• FIG. 4A-FIG. 4B Specificity of Lipid Release II.
• 4A Cholesterol released from brain capillary endothelial cell (BCECs) after 2 h of incubation in the presence of various concentrations of a- and ⁇ -CDs. Results are expressed as a percentage of cholesterol released from BCECs compared with the control. Each percentage is the mean of three different filters and representative of two series of independent experiments.
• 4B Phosphatidylcholine (PC) (light columns) and sphingomyelin (SM, dark columns) released from BCECs after 2 h of incubation in the presence of a- and ⁇ -CDs at 0.5, 5, and 50 mM, respectively.
• PC Phosphatidylcholine
• SM sphingomyelin
• Results are expressed as a percentage of phospholipids released from BCECs compared with the control. Each percentage is the mean of three different filters and representative of two series of independent experiments. Modified from: (Monnaert V, Tilloy S, et al. (2004) Journal of Pharmacology and Experimental Therapeutics 310:745-51)
• FIG. 5 MpCD, but not aCD protect A2E against oxidation.
• the bisretinoid A2E was the first compound identified in extracts of ocular lipofuscin. Protection of A2E against oxidation was monitored by changes in the UV-visible absorption spectra of 5 ⁇ A2E solutions.
• FIG. 6 QR-Plot of ssGWAS results for CGEM.
• the "null" projection ends at the median among the endpoints of the convex projections for individual chromosomes. Genes to the right of the vertical line are above the cut-off for study- specific genome-wide significance. Regions below (to the left) of the cut-off are given for descriptive purposes only.
• FIG. 7 QR-Plot of muGWAS results for CGEM. (see FIG. 6 for legend)
• FIG. 8 QR-Plot of ssGWAS results for EPIC, (see FIG. 6 for legend)
• FIG. 9 QR-Plot of muGWAS results for EPIC, (see FIG. 6 for legend)
• FIG. 10 QR-Plot of ssGWAS results for PBCS. (see FIG. 6 for legend)
• FIG. 11 QR-Plot of muGWAS results for PBCS. (see FIG. 6 for legend)
• FIG. 12 Phosphatidyl-inositol (PI) cycle.
• Phosphatidyl-inositol (PI) is synthesized from myo-inositol (imported by HMIT) and phosphatidic acid (PA) (via CDP-DAG), which can be synthesized from lysophosphatic acid (lyso-PA (LPA), PC, or (cytosolic) phosphatidyl-serine PS), or salvaged from IP 3 and DAG. It can also be synthesized from 1- acyl GPI.
• phosphoinositides are phosphorylated at a 3-, 4-, or 5- position by PI-kinases (left to right) and hydrolyzed by a plethora of phosphatases (right-to- left).
• Genes associated with breast cancer in this GWAS are highlighted as inverted (bold: aGWS).
• Wide arrows in the center indicate the sequence of phosphoinositides (shown as hexagons) involved in EEC. Hexagons: PI/phosphoinositides, PM: plasma membrane, CCV: clathrin-coated vesicle, UCV: uncoated vesicle, EE: early endosome, LE: late endosome LY: lysosome.
• Inverted gene names indicate genes associated with phosphatidylinositol signaling and/or endocytosis.
• Bold arrows indicate phosphoinositides associated with endocytosis.
• FIG. 13A and 13B Known relationship of genes implicated in muGWAS with stages in the process of endocytosis (13A) and exocytosis / lysosomal function (13B).
• CMVs clathrin-coated vesicles
• E3 ubiquitination separation of inactive integrin (fast recycling) from active integrins (slow recycling), sorting between secretory, lysosomal, and (slow) recycling pathway, and lysosomal degradation.
• Underlined genes are known breast cancer promoters and suppressors, respectively.
• Clathrin-mediated endocytosis (CME) begins with co-assembly of the heterotetrameric clathrin adaptor complex AP-2 with clathrin at PI(4,5)P2-rich plasma membrane (PM) sites.
• AP-2 in its open conformation recruits clathrin and additional endocytic proteins, many of which also bind to PI(4,5)P 2 .
• Clathrin-coated pit (CCP) maturation may be accompanied by SHIP-2-mediated dephosphorylation of PI(4,5)P 2 to PI(4)P.
• Synthesis of PI(3,4)P 2 is required for assembly of the PX-BAR domain protein SNX9 at constricting CCPs and may occur in parallel with PI(4,5)P 2 hydrolysis to PI(4)P via synaptojanin, thereby facilitating auxilin-dependent vesicle uncoating by the clathrin- dependent recruitment and activation of PI3KC2a, a class II PI3-kinase.
• PI(3,4)P 2 may finally be converted to PI(3)P en route to endosomes by the 4-phosphatases INPP4A/B, effectors of the endosomal GTPase Rab5.
• FIG. 14 Endocytic mechanisms underlying tumor cell migration and invasion through tissue barriers.
• the diagram presents a motile cell, the advancing lamellipodium of which moves directionally (arrow).
• Focal adhesions (FAs) are schematically shown, and integrin heterodimers are present at these.
• Cell migration necessitates polarized endocytosis and trafficking of FA complexes.
• Integrin internalization is controlled by dynamin, which is activated by microtubules (not shown), and protein kinases, such as FAK and protein kinase Ca (PKCa). Both clathrin- and caveolin 1 (CAVl)-coated domains of the plasma membrane are involved in internalization of integrin.
• CAVl caveolin 1
• integrins are sorted for degradation in lysosomes (LYs), recycled to the plasma membrane through a RAB4- dependent route, or transported to the perinuclear recycling compartment (PNRC). Recycling from the PNRC requires Rabl l family members, such as RAB25, and for some integrin heterodimers, also the protein kinase B (PKB) - GSK3P (glycogen synthase kinase- ⁇ ) axis, ARF6 or certain isoforms of PKC.
• PPKB protein kinase B
• GSK3P glycose kinase- ⁇
• FIG. 15 EEC in Alzheimer's disease (AD).
• AD Alzheimer's disease
• APP is synthesized in the endoplasmic reticulum (ER), transported to the TGN, and inserted into the plasma membrane via secretory vesicles.
• Cell-surface APP can be internalized to endosomes from which it can either be recycled back to the cell surface or delivered to lysosomes for degradation.
• the acidic environment favors production of amyloid-beta, which can be degraded in lysosomes by cathepsins, accumulated in EEs, or released to extracellular spaces via exocytosis (modified from Chen X, Hui L, et al. (2014) J Parkinsons Dis Alzheimers Dis 1).
• FIG. 16 EEC in Parkinson's disease (PD). Following endocytic entry, cargo is transported to early endosomes. From there, cargo can recycle back to the plasma membrane, either directly or via recycling endosomes. Alternatively, cargo can be retained in the EEs, which will form LEs/MVBs, and fuse with lysosomes for degradation. In parallel, cargo are transported between EEs and the trans-Golgi network (TGN). Alterations in these processes lead to lysosomal dysfunction and accumulation of undegraded macromolecules, toxic to the cell, (adopted from (Schreij AM, Fon EA, et al. (2015) Cell Mol Life Sci))
• FIG. 17 Macropinocytosis in Atherosclerosis (CAD) Following endocytic entry, low-density lipoprotein (LDL) is transported to the lysosome for degradation. Deficiencies in the lysosomal process lead to accumulation of LDL and. macrophages turn into foam cells, which accumulate to form atherosclerotic plaques.
• CAD Atherosclerosis
• LDL low-density lipoprotein
• FIG. 18A-C Lysosomal Dysfunction in Cancer (18A), AD/PD (18B, and CAD (18C). Published results show overlapping genetic risk factors for lysosomal dysfunction across diseases, leading to reduced lysosomal clearance (M) and, in cancer, increases in recovery of integrins from the lysosome (1)
• FIG. 19 Viruses "Hijacking" Endocytosis. Viral particles depend on clathrin- mediated endocytosis for entry into cells.
• CCV clathrin-coated vesicle. Modified from Fig. 2 in (Blaising J, Polyak SJ, et al. (2014) Antiviral research 107:84-94)
• FIG. 20 MpCD in AD Mouse Cells.
• MpCD ⁇ - ⁇ -CD
• APPsa levels were increased in the medium from ⁇ - ⁇ -CD-treated cells, while APPsP levels did not change (C and D).
• Fig. 21 ⁇ - ⁇ -CD Clearance of a-Syn. ⁇ - ⁇ -CD-mediated clearance of a-syn aggregates does not depend on the ability of ⁇ - ⁇ -CD to alter cholesterol levels. H4/a-syn- GFP cells untreated or treated with ⁇ - ⁇ -CD (1 mM) or ⁇ - ⁇ -CD-cholesterol complex (1 mM) for 24 h. Representative images are reported. 21A) Immunofluorescense microscopy analyses of TFEB subcellular localization using a FLAG-specific anti-body. Scale bar represents 10 ⁇ . 21B) a-syn-GFP fluorescense microscopy analyses. Scale bar represents 20 ⁇ .
• FIG. 22 Cholesterol Accumulation in Mouse Brain.
• ⁇ - ⁇ -CD shows highly effective reduction in UC storage.
• FIG. 23 CDs in Lysosomal Storage Diseases. Effect of cyclodextrins using lysotracker red staining to indicate enlarged lysosomes in ML111 fibroblasts.
• Wolman LAL
• TSD Fabry
• GLA Farber
• AC Farber
• MPSIIIB mucopolysaccharidose II type B
• NPA Niemann-Pick type A
• HBCD ⁇ - ⁇ -CD (Kleptose)
• the other cyclodextrins did not have that profound an effect",(McKew J, Zheng WEI, et al.
• FIG. 24 Pino-/Phago-/Endocytosis: Macropinocytosis, phagocytosis, and endocytosis are regulated in the same fashion by the PI cycle and lysosomal dysfunction in all three processing pathways causes substrates with unwanted function to be excreted (modified from FIG. 1 in (Sole-Domenech S, Cruz DL, et al. (2016) Ageing Res Rev 32:89- 103)).
• FIG. 25 HPpCD activates autophagy.
• Administration of ⁇ - ⁇ -CD results in activation of transcription factor EB TFEB.
• TFEB regulates the expression of genes involved in biogenesis and fusion of lyso- and autophago-somes.
• ⁇ - ⁇ -CD administration results in enhanced clearance of the autophagy substrate ceroid lipo-pigment.
• FIG. 26 Activation of TFEB by HPbCD: ⁇ - ⁇ -CD has been shown to activate transcription factor EB (TFEB), yet the mechanism of action is unknown (FIG. 25).
• the unexpected genetics results confirmed by the urinalysis results (Example 16) showed that the mechanism of action of ⁇ - ⁇ -CD is (a) reduction of serum phospholipids, (b) reduction of ⁇ -cycle activity, (c) reduction of endocytosis, (d) prevention of lysosomal stress, (e) prevention of MTORC1 activation, (f) continued dephosphorylation and translocation of TFEB, (g) continued activation of autophagy.
• Adapted from Kim S, Choi KJ, et al. (2016) Sci Rep 6:24933; Medina DL, Ballabio A (2015) Autophagy 11:970-1)
• FIG. 27 Phago-/Endocytosis in Multiple Sclerosis (MS).
• MS Multiple Sclerosis
• Fig. 28 MpCD Restores Surface Tension. "Minimum surface tension during dynamic cycle 20 with [Bovine Lipid Extract Surfactant] BLES containing 27 mg/ml BLES in control CBS buffer (checkers bars), 50% w/w FFA (oleic acid [, OA]) or LPC in control CBS buffer (horizontal bars) or buffer containing 40 mg/ml ⁇ - ⁇ -CD (solid black bars). (** p ⁇ 0.01, *** p ⁇ 0.001). BLES with FFA or LPC shows marked impairment, which is repaired to normal functionality in the presence of ⁇ - ⁇ -CD.
• FIG. 29 HaCaT Proliferation. Influence of cyclodextrins on the proliferation of spontaneously immortalized aneuploid human (HaCaT) keratinocytes. Mean values after 48 h incubation, normalized to the control, of at least six independent measurements. Top: ATP- Assay, bottom: PicoGreen-Assay). W6: a-CD, W7: ⁇ -CD, W7 HP: ⁇ - ⁇ -CD. Modified from (Hipler UC, Schonfelder U, et al. (2007) J Biomed Mater Res A 83:70-9)
• FIG. 30 HaCaT Caspase 3/7 Activity. Influence of cyclodextrins on Caspase 3/7 activity of HaCaT keratinocytes. Mean values after incubation (24 h), normalized on the control and the protein content, of at least six independent measurements.
• W6 a-CD
• W6 ⁇ - CD
• W7 HP ⁇ - ⁇ -CD. Modified from (Hipler UC, Schonfelder U, et al. (2007) J Biomed Mater Res A 83:70-9)
• FIG. 31 HaCaT Lactose Dehydrogenase. Influence of cyclodextrins on lactose- dehydrogenase (LDH) of HaCaT keratinocytes. Mean values after incubation (48 h), normalized on the control and the protein content, of at least six independent measurements.
• W6 a-CD
• W6 ⁇ -CD
• W7 HP ⁇ - ⁇ -CD. Modified from (Hipler UC, Schonfelder U, et al. (2007) J Biomed Mater Res A 83:70-9)
• FIG. 32 Solubility Enhancements by Hydroxypropyl-cyclodextrins (HP-CD).
• FIG. 33 Nephrotoxicity of a-CD and ⁇ -CD.
• the 2017 assessment by the EMA (and several other authors) of similar risks associated with parenteral administration of a-CD and ⁇ -CD in rats is based on a single study (Frank DW, Gray JE, et al. (1976) Am J Pathol 83:367-82).
• FIG. 34 Ototoxicity of CDs. Ototoxicity of different CDs as assessed by auditory brainstem response (ABR) recordings at 12 weeks of age. Plot of hearing thresholds for individual mice reveals minute variability across mice treated with a particular CD with the exception of HPyCD, in which hearing thresholds were more variable. Modified from Figure 5B in (Davidson CD, Fishman YI, et al. (2016) Ann Clin Transl Neurol 3:366-80)
• FIG. 35 Wound healing by cyclodextrins in breast cancer cell lines. Dashed horizontal line indicates inhibition of wound healing in HPaCD at 1 and 4 mM respectively. ANOVA P values are shown for HPaCD v. HP ⁇ CD with MCF-7 and MDA-MB-231 as (fixed) blocks:
• FIG. 36 Wound Healing Assay: Modified from Cell BioLabs Inc., Assay CBA-120
• FIG. 37 Impact of PL selectivity: (upper left corner of FIG. 12, see FIG. 12 for legends) HP-a-CD only scavenged PC (and, potentially PI), but not lysophospholipids including, but not limited to, LPC and LPA, upstream of sPLA2, which splits PC into LPC and arachidonic acid (AA).
• FIG. 39 BCa Lung and Liver Metastases. P-values are from pairwise comparisons of bivariate (lung/liver) data via u-statistics for multivariate data (Wittkowski KM, Lee E, et al. (2004) Stat Med 23: 1579-92)
• FIG. 40 BCa Plasma Cytokines. Individual P-values are derived from two sample t- tests p(b): ⁇ - ⁇ -CD v. Vehicle, p(a): HP-a-CD v. Vehicle, p(a:b): HP-a-CD v. ⁇ - ⁇ -CD. Overall P-value for all six cytokines is calculated via u-statistics for multivariate data (Wittkowski KM, Lee E, et al. (2004) Stat Med 23: 1579-92)
• FIG. 41 HTT Mice: Body weight. Estimates are means + SD, P-values for Treatment*Day interaction (P(T*D)) overall and by sex (F: female, M: male) are derived from mixed model ANOVA with mice as random factor.
• FIG. 42 SOD1 Mice: Bodyweight. Estimates are means + SD, P-values for Treatment*Day interaction (P(T*D)) overall and by sex (F: female, M: male) are derived from mixed effects ANOVA with mice as a random factor.
• FIG. 43A and 43 FPLC Standard curves for the type of FPLC columns used.
• 43A) HP-a-CD (HPaCD, 1180 Da) is confirmed to be measured mostly in Fraction 2 (nominally 1060-1267 Da).
• 43B) Phosphatidylcholine (PC) is confirmed to be measured mostly in Fraction 5 (nominally 734-795 Da).
• FIG. 44 HPaCD, FA mixtures (&) v. clathrate.
• HPaCD/FA&PE The clathrate mixed with the above penetration enhancer.
• Axes as in Fig. 43.
• Y-axis with labels added: aCD, PC, and LPC indicate the median amount of substrate identified in the HP-a-CD fraction 2 (1060-1267 Da), the phosphatidylcholine fraction 5 (724-795 Da), and the lyso-phosphatidyl choline fractions 9 and 10 (450-534 Da).
• the fractions referred to on the y-axis are indicated by the same labels on the x-axis.
• the upward open arrows indicate the difference between the corresponding median for HP-a-CD (horizontal line) and the median for each of the four mixtures and/or clathrates, respectively, (short horizontal dotted lines).
• FIG. 45 In vivo Evidence for Clinical Efficacy of pCDs. Efficacy of ⁇ -CD in animal models of breast cancer, Parkinson's disease, Alzheimer's disease, CAD, systemic lupus erythematosus/ALS/MS, cystic fibrosis/IPF, NAFLD/NASH, and T2DM was consistently attributed to the ability of ⁇ -CD to scavenge cholesterol (Choi, dashed arrows), which carries the now well-known risk of cholesterol-mediated ototoxicity. The mechanism by which depletion of cholesterol should improve the various phenotypes, however, was rarely explained. Clinical results also showed phospholipid upregulation in several of these diseases. ⁇ -CDs, however, also scavenge phospholipids (phospholipids) and, thus, also downregulate the PI cycle (center), which directly benefits the various disease phenotypes (solid arrows).
• FIG. 46 HP-a-CD Restores LY Function: (update of FIG. 26).
• ⁇ - ⁇ -CD has been shown to activate transcription factor EB (TFEB), yet the mechanism of action is unknown (Kilpatrick K, Zeng Y, et al. (2015) PLoS One 10:e0120819; Song W, Wang F, et al. (2014) J Biol Chem 289:10211-22; Sardiello M (2016) Ann N Y Acad Sci 1371:3-14; Moors TE, Hoozemans JJ, et al. (2017) Mol Neurodegener 12: 11).
• the unexpected genetics results confirmed by the urinalysis results (Example 16 ) identified the mechanism.
• FIG. 47 HP-a-CD Reduces Inflammation: Effects of a-CD on arachidonic acid (AA) and known drugs with similar effects downstream of arachidonic acid.
• AA arachidonic acid
• sPLA2 the effect of a-CD is better tolerated than the effect of corticosteroids.
• the combination of ibuprofen and cromolyn is tested as the dry-powder inhaler ALZT-OP1 (Elmaleh D (2017)).
• ALZT-OP1 the dry-powder inhaler ALZT-OP1
• FIG. 48 a-CD/Pemetrexed Compounds: To target the compound to the neighborhood of cancer cells, which have folate-receptors, a-CD (or a derivative, such as HP- a-CD, with hydroxypropyl) can be linked, as a non-limiting example, to pemetrexed with a disulfide linker that is bound to both the a-CD and pemetrexed via an ester.
• the disulfide linker could be formed by either cysteine or 3-maercapto-propionic acid and 2-mercapto- ethanol.
• ester or disulfide links can be formed by methods well established, such as those described in (Michel D, Chitanda JM, et al. (2012) Eur J Pharm Biopharm 81:548-56) and (Hunter R, Whyboom N, et al. (2008) Synlett 252-4) (removing one H 2 0, each to form an O from two OH), and (Butt AM, Mohd Amin MC, et al. (2015) Int J Nanomedicine 10: 1321-34) (removing H 2 to form an S-S from two SH).
• FIG. 49A, 49B, 49C Genetic Analysis: Table 4 (FIGS. 49A through 49C) depicts the genetic analysis performed through IPV6/IPV1 : -log 10(p- value) in muGWAS/ssGWAS. DETAILED DESCRIPTION OF EMBODIMENTS
• the present disclosure is based, in part, on the discovery of certain disease-relevant collections of genes based on a reanalysis of three independent sets of breast cancer genetic data, which are available for analysis from the National Institutes of Health's dbGaP collection.
• This reanalysis differed from the earlier analyses by using a novel computational biostatistics method, which incorporates knowledge about genetics into the method itself. (Wittkowski KM, Sonakya V, et al.
• this method addresses the following four points, which prior analyses of the same data using conventional bioinformatics approaches failed to consider: (i) non-additive relationships between risk alleles and incidence, (ii) cis-epistatic interaction, (iii) correlation between significance and minor allele frequency or "MAF" and (iv) non-randomization bias. It also addresses multiplicity adjustment for diplotype length, a problem arising from the use of a wide-locus approach. By addressing these points, the same strategy previously identified two novel collections of autism- specific genes now identified another novel collection of genes related to endocytosis and lysosomal function.. (Wittkowski KM, Sonakya V, et al. (2014) Transl Psychiatry 4:e354)
• genes presented herein comprises eight genes (AGPAT3/4 ATP8A1/B1, ABCA1, AN04, LPPR1, DGKQ) whose roles include providing the phosphatidyl inositol (PI) cycle with its substrate, PI.
• PI phosphatidyl inositol
• the second gene cluster comprises genes directly associated with endocytosis, a process controlled by phosphoinositides (Table 4, column PI/EC).
• the stages include, but are not limited to, invagination and forming of CCVs and EEs (FIG. 13).
• the present disclosure provides, in part, a shift in the focus for cancer treatments from controlling cell growth, a process involved in many vital functions even in older subjects, to controlling spread, migration and invasion of cells, a process of relevance primarily during pre- and early postnatal development.
• the present disclosure provides compositions and methods for treating malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition by modulating the PI cycle and its activity.
• the present disclosure provides compositions and methods that may be useful for the treatment of malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition during periods, such as adulthood, where cell migration, including neuronal growth, has mostly ceased, while cell growth, such as growth of hair, skin, and the like, continues and where other cellular mechanism may decline in an age-dependent manner.
• the present disclosure is based on the re-evaluation of interpretations of published findings, which are commonly believed to assert that a-CD is as nephrotoxic as ⁇ - CD, although nephrotoxicity was primarily demonstrated for ⁇ -CD, which has substantially lower aqueous solubility than a-CD, and, thus, a higher risk of forming the long cytoplasmic crystals seen in the kidneys of rats.
• a-CD v. ⁇ -CD - typically at the level of the safer ⁇ - ⁇ -CD - was shown in HaCaT cells proliferation (FIG. 29), caspase activity (FIG. 30), and lactose dehydrogenase (FIG. 31).
• nephrotoxicity is further reduced by reducing the rate of delivery, the method comprising repeated doses per day, administering the drug over several hours via a peristaltic pump or administering the drug continuously via an implanted drug delivery system.
• aCD alpha-cyclodextrin
• AD Alzheimer's disease
• AKT protein kinase B
• ALS amyotrophic lateral sclerosis
• APP amyloid precursor protein
• BCa breast cancer
• CAD coronary artery disease
• CD refers to cyclodextrin
• CDK cyclin-dependent kinase
• CF cystic fibrosis
• CGEM Cancer Genetic Markers of Susceptibility.
• Chr refers to chromosome.
• dbGaP refers to database of Genotypes and Phenotypes.
• DNA refers to deoxyribonucleic acid
• EPIC European Prospective Investigation into Cancer
• ER refers to endoplasmic reticulum
• FAK focal adhesion kinase
• FDA Food and Drug Administration
• FSGS focal segmental glomerulosclerosis
• gCD gamma-cyclodextrin
• GPCR G-protein coupled receptor
• GRAS refers to generally recognized as safe.
• GWAS refers to genome-wide association study.
• GRAS refers to generally recognized as safe.
• HA Hyaluronic acid
• HER2/neu refers to receptor tyrosine-protein kinase erbB-2.
• HLA refers to human leukocyte antigen.
• HP refers to hydroxypropyl.
• HPaCD 2-hydroxypropyl- alpha-cyclodextrin.
• HPbCD ⁇ - ⁇ -CD
• HPpCD hydroxypropyl- beta-cyclodextrin
• IND refers to investigational new drug.
• IPV refers to inverse p-value.
• the abbreviation "i.v.” refers to intravenous. ⁇ 01271
• the abbreviation “LD” refers to linkage disequilibrium.
• LD 50 refers to median lethal dose.
• LPC lysophosphatidylcholine.
• LY refers ot lysosome.
• LYD refers to lysosomal dysfunction
• MAF minor allele frequency.
• MAP refers to mitogen-activated protein.
• ⁇ - ⁇ -CD methyl- ⁇ - cyclodextrin
• PCa refers to prostate cancer.
• PD Parkinson's disease
• PI refers to phosphatidylinositol
• P3R phosphoinositide-3 kinase
• PIP phosphoinositide
• PIP1 refers to PI(4)P.
• PIP2 refers to PI(4,5)P 2 .
• PIP3 refers to PI(3,4,5)P 3 .
• PKT protein kinase B
• PLC protein kinase C
• PL refers to phospholipid
• QQ refers to "quantile-quantile”.
• RTK receptor tyrosine kinase
• SLE systemic lupus erythematosus
• SNP single nucleotide polymorphism
• ssGWAS single-SNP genome-wide association study
• TFEB transcription factor EB
• TSC tuberous sclerosis
• U.S refers to the United States.
• references to "A and/or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
• carrier means a diluent, adjuvant, or excipient with which a compound is administered.
• Pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
• the pharmaceutical carriers can also be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like.
• auxiliary, stabilizing, thickening, lubricating and coloring agents can be used.
• the terms “comprising” (and any form of comprising, such as “comprise”, “comprises”, and “comprised”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”), are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
• the phrase "in need thereof means that the animal or mammal has been identified as having a need for the particular method or treatment. In some embodiments, the identification can be by any means of diagnosis. In any of the methods and treatments described herein, the animal or mammal can be in need thereof. In some embodiments, the animal or mammal is in an environment or will be traveling to an environment in which a particular disease, disorder, or condition is prevalent.
• the terms “treat,” “treating,” and “treatment” encompass a variety of activities aiming at desirable changes in clinical outcomes.
• the term “treat”, as used herein encompasses any detectable improvement in one or more clinical indicators or symptom of malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition - such as carcinomas, including, but not limited to, breast and prostate cancer or neurodegenerative diseases, including, but not limited to, Parkinson's and Alzheimer's Disease, or coronary artery disease, including, but not limited to atherosclerosis, CAD and stroke, or a digestive disorder, including, but not limited to T2DM.
• such terms encompass alleviating, abating, ameliorating, relieving, reducing, inhibiting or slowing at least one clinical indicator or symptom, preventing additional clinical indicators or symptoms, reducing or slowing the progression of one or more clinical indicators or symptoms, causing regression of one or more clinical indicators or symptoms, relieving a condition caused by the disease or disorder, and the like.
• the methods and compositions provided herein can be used in subjects that already exhibit one or more clinical indicators or symptoms of the disease or disorder, such as malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition.
• various clinical indicators and symptoms are known to medical practitioners and those of skill in the art.
• prevent encompasses stopping a disease, disorder, or symptom from starting, as well as reducing or slowing the progression or worsening of a disease or disorder.
• preventing breast cancer or prostate cancer includes, but is not limited to, inhibiting the formation of cancerous cells, growth of tumors, local spread of cancerous spread, or inhibiting the metastasis of malignant growths, preventing the onset of symptoms of Alzheimer's, Parkinson's, Huntington's, ALS and/or MS.
• the methods and compositions provided herein can be used to prevent progression or clinical exhibiting of symptoms in subjects that do not yet exhibit any clear or detectable clinical indicators or symptoms of the disease or disorder but that are believed to be at risk of developing the disease or disorder, such as malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition.
• malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition is used herein in accordance with it usual usage in the art and includes, but is not limited to malignant disorders, such as carcinomas, breast cancer, prostate cancer, malignancies of the breast, and malignancies of the prostate, as well as neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and "a range of disorders including brain overgrowth syndromes, [and] Charcot-Marie-Tooth neuropathies"(Waugh MG (2015) Biochim Biophys Acta 1851:1066-82).
• cancer or “hyperproliferative disease” is meant to refer to those diseases and disorders characterized by hyperproliferation of cells.
• hyperproliferative disease includes all forms of cancer, psoriasis, neoplasia, and hyperplasia.
• subject or “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, cows, pigs, goats, sheep, horses, dogs, sport animals, and pets.
• Tissues, cells and their progeny obtained in vivo or cultured in vitro are also encompassed by the definition of the term subject.
• subject is also used throughout the specification in some embodiments to describe an animal from which a cell sample is taken or an animal to which a disclosed cell or nucleic acid sequences have been administered. In some embodiment, the animal is a human.
• the term "patient” may be interchangeably used. In some instances in the description of the present disclosure, the term “patient” will refer to human patients suffering from a particular disease or disorder. In some embodiments, the subject may be a non-human animal from which an endothelial cell sample is isolated or provided.
• the term “mammal” encompasses both humans and non- humans and includes but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, caprines, and porcines.
• an effective amount refers to an amount of an active agent as described herein that is sufficient to achieve, or contribute towards achieving, one or more desirable clinical outcomes, such as those described in the "treatment” description above.
• An appropriate “effective” amount in any individual case may be determined using standard techniques known in the art, such as a dose escalation study.
• the term “therapeutically effective amount” is meant to refer to an amount of an active agent or combination of agents effective to ameliorate, delay, or prevent the symptoms, shrink tumor size, prevent progression of cancer from non-metastatic to metastatic disease, or prolong the survival of the patient being treated for cancer or neurodegenerative disease. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.
• cyclodextrin or derivatives thereof or pharmaceutically acceptable salts thereof can be co-administered with other therapeutics and/or part of a treatment regimen that includes radiation therapy.
• chemo therapeutics include common cytotoxic or cytostatic drugs such as for example: methotrexate (amethopterin), doxorubicin (adrimycin), daunorubicin, cytosinarabinoside, etoposide, 5-4 fluorouracil, melphalan, chlorambucil, and other nitrogen mustards (e.g. cyclophosphamide), cis-platin, vindesine (and other vinca alkaloids), mitomycin and bleomycin.
• cytotoxic or cytostatic drugs such as for example: methotrexate (amethopterin), doxorubicin (adrimycin), daunorubicin, cytosinarabinoside, etoposide, 5-4 fluorouracil, melphalan, chlorambucil, and other nitrogen mustards (e.g. cyclophosphamide), cis-platin, vindesine (and other vinca alkaloids), mito
• chemotherapeutics include: purothionin (barley flour oligopeptide), macromomycin. 1,4-benzoquinone derivatives and trenimon.
• Anti-cancer antibodies, such as herceptin, and toxins are also examples of other additional therapeutics.
• the therapeutic regimens can include sequential administration of cyclodextrin or derivatives thereof or pharmaceutically acceptable salts thereof and initiation of radiation therapy in either order or simultaneously.
• Those skilled in the art can readily formulate an appropriate radiotherapeutic regimen. Carlos A Perez & Luther W Brady: Principles and Practice of Radiation Oncology, 2nd Ed. JB Lippincott Co, Phila, 1992, which is incorporated herein by reference in its entirety describes radiation therapy protocols and parameters which can be used in the present disclosure.
• the therapeutically effective amount of the inhibitor may be adjusted such that the amount is less than the dosage required to be effective if used without other therapeutic procedures.
• treatment with pharmaceutical compositions described herein is preceded by surgical intervention.
• the disclosure also relates to methods of reducing the number of exosomes in a cancer cell by contacting said cancer cell with a therapeutically effective amount of a cyclodextrin.
• composition refers to a composition comprising at least one active agent as described herein (such as, for example, an (a-CD), and one or more other components suitable for use in pharmaceutical delivery such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, excipients, and the like.
• the disclosure relates to a pharmaceutical composition comprising an effective amount of any alpha-cyclodextrin molecule or derivative or salt disclosed herein and a pharmaceutically acceptable carrier.
• the pharmaceutical composition comprises any one or plurality of fatty acid molecules disclosed herein or a slat thereof.
• the pharmaceutical composition is free of beta or gamma cyclodextrin.
• active agent refers to a molecule that is intended to be used in the compositions and methods described herein and that is intended to be biologically active, for example for the purpose of treating malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition.
• active agent is intended to include molecules that either are, or can be converted to a form that is, biologically active.
• active agent includes pro-drugs and/or molecules that are inactive or lack the intended biological activity but that can be converted to a form that is active or has the intended biological activity.
• sample refers generally to a limited quantity of something which is intended to be similar to and represent a larger amount of that thing.
• a sample is a collection, swab, brushing, scraping, biopsy, removed tissue, or surgical resection that is to be tested for clinical indicators of a disease or disorder.
• samples are taken from a patient or subject that is believed to have malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition.
• a sample believed to contain clinical indicators of a disease or disorder such as malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition
• a control sample that is known not to contain one or a plurality of clinical indicators of a disease or disorder, such as malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition.
• a sample believed to contain a clinical indicator of a disease or disorder such as malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition
• a control sample that is known to not contain a clinical indicator of a disease or disorder, such as malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition.
• a sample believed to contain a clinical indicator of a disease or disorder such as malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition
• a control sample that contains the same clinical indicators of a disease or disorder, such as malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition.
• scavenge means uptake or chemically combine with and transport to including, but not limited to, the kidney for excretion.
• salt refers to acidic salts formed with inorganic and/or organic acids, as well as basic salts formed with inorganic and/or organic bases. Examples of these acids and bases are well known to those of ordinary skill in the art. Such acid addition salts will normally be pharmaceutically acceptable although salts of non-pharmaceutically acceptable acids may be of utility in the preparation and purification of the compound in question. Salts of the embodiments include those formed from hydrochloric, hydrobromic, sulphuric, phosphoric, citric, tartaric, lactic, pyruvic, acetic, succinic, fumaric, maleic, methanesulphonic and benzenesulphonic acids.
• salts of the compositions comprising one or may be formed by reacting the free base, or a salt, enantiomer or racemate thereof, with one or more equivalents of the appropriate acid.
• pharmaceutical acceptable salts of the present disclosure refer to amino acids having at least one basic group or at least one basic radical.
• pharmaceutical acceptable salts of the present disclosure comprise a free amino group, a free guanidino group, a pyrazinyl radical, or a pyridyl radical that forms acid addition salts.
• the pharmaceutical acceptable salts of the present disclosure refer to amino acids that are acid addition salts of the subject compounds with (for example) inorganic acids, such as hydrochloric acid, sulfuric acid or a phosphoric acid, or with suitable organic carboxylic or sulfonic acids, for example aliphatic mono- or di- carboxylic acids, such as trifluoroacetic acid, acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, fumaric acid, hydroxymaleic acid, malic acid, tartaric acid, citric acid or oxalic acid, or amino acids such as arginine or lysine, aromatic carboxylic acids, such as benzoic acid, 2-phenoxy-benzoic acid, 2-acetoxybenzoic acid, salicylic acid, 4- aminosalicylic acid, aromaticaliphatic carboxylic acids, such as mandelic acid or cinnamic acid, hetero aromatic carboxylic acids, such as nicotinic acid or isonico
• salts may be formed.
• the reaction may be carried out in a solvent or medium in which the salt is insoluble or in a solvent in which the salt is soluble, for example, water, dioxane, ethanol, tetrahydrofuran or diethyl ether, or a mixture of solvents, which may be removed in vacuo or by freeze drying.
• the reaction may also be a metathetical process or it may be carried out on an ion exchange resin.
• the salts may be those that are physiologically tolerated by a patient. Salts according to the present disclosure may be found in their anhydrous form or as in hydrated crystalline form (i.e., complexed or crystallized with one or more molecules of water).
• soluble or “water soluble” refers to solubility that is higher than 1/100,000 (mg/ml).
• the solubility of a substance, or solute is the maximum mass of that substance that can be dissolved completely in a specified mass of the solvent, such as water.
• "Practically insoluble” or “insoluble,” on the other hand, refers to an aqueous solubility that is 1/10,000 (mg/ml) or less.
• Water soluble or soluble substances include, for example, polyethylene glycol.
• the polypeptide described herein may be bound by polyethylene glycol to better solubilize the composition comprising the peptide.
• percent “homology” or “sequence identity” is determined by using the stand-alone executable BLAST engine program for blasting two sequences (blZseq), which can be retrieved from the National Center for Biotechnology Information (NCBI) ftp site, using the default parameters (Tatusova TA, Madden TL (1999) FEMS microbiology letters 174:247-50, which is incorporated herein by reference in its entirety).
• derivative as applied to a phosphate containing, monophosphate, diphosphate, or triphosphate group or moiety refers to a chemical modification of such group wherein the modification may include the addition, removal, or substitution of one or more atoms of the phosphate containing, monophosphate, diphosphate, or triphosphate group or moiety.
• such a derivative is a prodrug of the phosphate containing, monophosphate, diphosphate, or triphosphate group or moiety, which is converted to the phosphate containing, monophosphate, diphosphate, or triphosphate group or moiety from the derivative following administration to a subject, patient, cell, biological sample, or following contact with a subject, patient, cell, biological sample, or protein (e.g. enzyme).
• a triphosphate derivative is a gamma-thio triphosphate.
• a derivative is a phosphoramidate.
• the derivative of a phosphate containing, monophosphate, diphosphate, or triphosphate group or moiety is as described in Murakami et al. J.
• Malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, virus, or age-related diseases, disorders, or other conditions are complex conditions in the sense that they involve many genes along common pathways.
• CVDs cardio- vascular diseases
• CAD CAD
• stroke and neurodegenerative diseases
• PI(3,4,5)P 3 and PI(3,4)P 2 are upregulated in malignant and neurodegenerative diseases (Waugh MG (2015) Biochim Biophys Acta 1851: 1066-82) and activate Akt signaling (Ooms Lisa M, Binge Lauren C, et al.
• PI signaling is widely believed to be involved in a broad range of age-related diseases.
• INPP4B is known as a suppressor of some cancers, but as a risk factor for others, including, but not limited to breast or prostate cancer .(Woolley JF, Dzneladze I, et al. (2015) Trends Mol Med 21:530-2; Chew CL, Chen M, et al.
• PI3K signaling has also be implied in aging, cognitive decline, and Alzheimer's disease (O'Neill C (2013) Experimental gerontology 48:647-53). Still, "clinical results with single-agent PI3K inhibitors have been modest to date,"(Mayer IA, Arteaga CL (2016) Annu Rev Med 67: 11- 28) in part because traditional GWAS have largely failed to elucidate the precise mechanism by which PI signaling contributes to complex conditions, such as cancer and neurodegenerative disease.
• the present disclosure provides methods of treatment of breast and prostate cancer, cardiovascular, metabolic, and Parkinson's or Alzheimer's diseases, that comprise administering to a subject one or more therapeutically effective amounts of active agents that downregulate the PI cycle to elicit changes in endo-/exocytosis thereby causing a dampening or decrease of cellular migration and infiltration or processing of proteins, including, but not limited to, LDL, APP, tau, myelin, SOD1, HTT, and a-synuclein.
• active agents that downregulate the PI cycle to elicit changes in endo-/exocytosis thereby causing a dampening or decrease of cellular migration and infiltration or processing of proteins, including, but not limited to, LDL, APP, tau, myelin, SOD1, HTT, and a-synuclein.
• active agents could be, for example, drugs or compounds of drugs that are already being used safely in humans for other indications and could be repurposed for use in the treatment of malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition.
• NCL neural ceroid lipofuscinoses
• FDD frontotemporal dementia
• HD Huntington's disease
• stroke (Vecsernyes M, Fenyvesi F, et al. (2014) Arch Med Res 45:711-29; Rivers JR, Maggo SD, et al. (2012) Neuroreport 23: 134-8), atherosclerosis (Irie T, Fukunaga K, et al.
• Sphingolipids and ceramides are also involved in podocyte dysfunction (including, but not limited to Fabry's disease, Tay-Sachs disease, Alport syndrome, minimal change disease, diabetic kidney disease, focal segmental glomerulosclerosis, end-stage renal disease, kidney failure, podocytopenia, hyperhomocysteinemia, steroid-resistant nephrotic syndrome) and the pathophysiology of cancer, angiogenesis, atherosclerosis, inflammation, and insulin resistance.(Abou Daher A, El Jalkh T, et al. (2017) Int J Mol Sci 18)
• Phosphoinositides which regulate endocytosis, (Posor Y, Eichhorn-Grunig M, et al. (2015) Biochim Biophys Acta 1851:794-804) have been mentioned as drug targets in genetic diseases, including, but not limited to, muscular and corneal dystrophy or myotubular neuropathy, cancers, including, but not limited to, leukemias (ALL, AML, CML) and lymphomas (NHL), epilepsy, and thrombosis. (Viaud J, Mansour R, et al.
• Neurological diseases linked to phosphoinositide dysregulation include, but are not limited to, Friedreich's ataxia, Charcot-Marie-Tooth (CMT) degenerative neuropathy, renal tubulopathies (oculocerebrorenal syndrome of Lowe, Dent's disease), Andersen-Tawil syndrome, mucolipidosis, multiple sclerosis (MS), Yunis-Varon syndrome, ALS FTLD, and ciliopathies.(Waugh MG (2015) Biochim Biophys Acta 1851:1066-82) Additional diseases linked to phosphoinositide dysregulation are osteoporosis, cancers (including, but not limited to, cervical cancer, leiomyosarcoma, gastric cancer, adenocarcinoma, lung cancer), cardiac hypertrophy, and autoimmune diseases (including, but not limited to, rheumatoid arthritis (RA) and systemic lupus erythematosus), and neurological diseases including, but not limited to
• ABCA1 renal disease
• ABCA2 acoustic neuroma
• ABCB9 acute myeloid leukemia (AML)
• ACP2 acid phosphatase deficiency, keratoconus, amyotrophic lateral sclerosis, atrichia, amebiasis
• ACP5 spondyloenchondrodysplasia, bone giant cell tumor, hairy cell leucoma, tooth resorption
• AGA aspartylglucosaminuria, fucosidosis
• AP3B1 Hermansky- Pudlak syndrome (HPS), oculocutaneous albinism, storage pool platelet disease;
• TPP1 NCL, spinocerebellar ataxia, Bielschowsky-Jansky disease.
• cardio- and cerebrovascular diseases including, but not limited to, arteriosclerosis, coronary heart disease, arrhythmia, heart failure, hypertension, orthostatic hypotension, myocardial infarction, angina pectoris, heart failure, atherosclerosis, stroke, renal artery disease or stenosis, peripheral vascular disease, chronic obstructive pulmonary disease, ("COPD”), chronic renal diseases with renal failure, and heart disease.
• exemplary inflammatory diseases and conditions include arthritis, such as rheumatoid arthritis. Additional exemplary age- associated diseases and disorders ulcers, osteopetrosis, progeria, weakness, hearing loss, and type-2 diabetes. It is to be understood that "age-related" refers to diseases frequently associated with aging, however, a given patient need not be of advance age, but rather the subject methods and compositions may he used regardless of the patient's age.
• A) Active Agents refers to diseases frequently associated with aging, however, a given patient need not be of advance age, but rather the subject methods and compositions may
• agents that can be used in the methods of the invention may reduce the concentration of phospholipids available to cells, including, but not limited to, neurons, macrophages, microglia, and tumor cells.
• the agent is a cyclic oligosaccharide.
• the cyclic oligosaccharide is an a-CD optionally modified by an alkyl group from about 1 to about 20 or more carbons in length.
• the cyclodextrin is a-CD optionally modified by a hydroxyl- alkyl group from about 1 to about 20 or more carbons in length.
• the agent is hydroxypropyl- (HP-) a- CD.
• the agent is a clathrate of an a-CD.
• the agent is a clathrate of an a-CD with a fatty acid.
• a "fatty acid” as used herein means a carboxylic acid with a long aliphatic chain, which is either saturated or unsaturated.
• the aliphatic chain is from about 3 to about 70 carbons long.
• the aliphatic chain is from 8 to about 30 carbons long.
• the fatty acid has one or more aliphatic chains branched or unbranched with one or more substituents.
• the fatty acid comprises from about 6 to about 22 carbon atoms in its aliphatic chain. In some embodiments, any one of the bonds between each carbon atom may be in the cis or trans configuration.
• Cyclodextrins are natural compounds formed during bacterial digestion of cellulose. They are not hydrolyzed by common mammalian amylases, but can be fermented by the intestinal microflora. They are poorly resorbed in typical situations and do not interact with cells or tissues.
• a-CD was declared GRAS by the FDA for "use in selected foods, except meat and poultry, for fiber supplementation, as a carrier or stabilizer for flavors (flavor adjuvant), as a carrier or stabilizer for colors, vitamins and fatty acids and to improve mouthfeel in beverages (GRN No. 155, updated Nov 2016 as GRN 678 to reflect an expected 90 th percentile for intake of 420 mg/kg, or -30 g/d at 70 kg).
• the common cyclodextrins are made up of six (a-CD), seven ( ⁇ -CD), or eight ( ⁇ -CD) D-glucose molecules bound together in a toroid (truncated cone) with a lipophilic inner and a hydrophilic outer surface (FIG. 1). This combination of features makes them suitable as an expedient to solubilize lipophilic drugs.
• Common cyclodextrins obtained by the substitution of the R groups on the edge (rim) of the toroid include, but are not limited to, methyl (including randomly methylated) CH 3 , 2- hydroxypropyl (HP): CH 2 CHOHCH 3 , sulfobutylether (CH 2 ) 4 S0 3 Na + , acetyl, succinyl, glucosyl, maltosyl, carboxymethyl ether, phosphate ester, simple polymers, or carboxymethyl.
• each derivative listed should be regarded as a group of closely related cyclodextrin derivatives.
• Phospholipids and cholesterol are all lipids.
• cyclodextrins when cyclodextrins are given parenterally without having their lipophilic cavity filled (or after the lipophilic drug has been delivered), they can potentially extract lipids (phospholipids and cholesterol, depending on the type of cyclodextrin) from plasma membranes.
• cyclodextrins have been demonstrated to cause cell lysis in different types of cells, indicating that the effect is not cell-type specific [(Irie T, Uekama K (1997) J Pharm Sci 86:147-62)]"(Stella VJ, He Q (2008) Toxicol Pathol 36:30-42)
• cyclodextrins are known to induce shape changes of plasma membrane invagination on erythrocytes and, at higher concentrations, induce hemolysis of erythrocytes in the order of ⁇ - CD > a-CD > Y-cyclodextrin.
• HP-a-CD was found to be less cytotoxic than a-CD on heterogeneous human epithelial colorectal adenocarcinoma (Caco-2) cells.
• ⁇ - ⁇ -CD is used as an expedient/solvent for many lipophilic drugs, including the neurosteroid allopregnanolol.
• a-CD has been approved and is used as an expedient of alprostadil (a prostaglandin) for intracavernosal injection in the treatment of erectile dysfunction.
• steroids and sterols including, but not limited to, cholesterol,(Zarzycki PK, Ohta H, et al. (2008) Anal Bioanal Chem 391:2793-801) do not fit its smaller cavity (FIG. 2).
• the specificity of extraction of phospholipids, cholesterol, and proteins are shown in FIG. 3 (Ohtani Y, Irie T, et al. (1989) European Journal of Biochemistry 186: 17-22).
• a-CD is known to scavenge glycerolipids, such as PC, and sphingolipids more specifically than ⁇ -CDs (FIG. 4).
• a-CD is known to form complexes both around both the inositol head or the sn-2 chain of PI.
• a-CDs are expected to downregulate the substrates for the PI cycle and, thereby, downregulate endocytosis with higher specificity than P-CD.
• HP-a-CD retains the preference of a-CD for phospholipids over cholesterol.
• ⁇ -CDs and ⁇ -CDs can bind fats at most at a 1: 1 molar ratio, but a-CD can bind fat 9-times its weight.
• Methylated ⁇ -CDs have a high affinity to common constituents of cell membranes and, thus, an extreme hemolytic effect, while a-CDs have very low effect on human erythrocytes.
• cyclosporin A a mixture of a-CD and ⁇ -CD is used to enhance the drug solubility without causing ocular irritation.
• DIV ⁇ CD and a-CD are potent enhancers, but ⁇ -CD, ⁇ -CD, and HPpCD are not.
• ⁇ - ⁇ -CD is used, for instance,
• the a-CD is a hydroxypropyl, hydroxyethyl, hydroxymethyl, hydroxybutyl, hydroxypentyl- a-CD or salt or derivative thereof.
• the pharmceutical composition of the disclsoure comprises hydroxypropyl, hydroxyethyl, hydroxymethyl, hydroxybutyl, hydroxypentyl- a-CD or salt or derivative thereof but is free of alpha-cycoldextrin or beta-cyclodextrin.
• the pahrmaceutical composition is free of beta or gamma cyclodextrin and or derivatives of salts thereof.
• Embodiments of the present disclosure are particularly useful to treat individuals who have cancer identified as having one or a plurality of cells with an abnormally high rate of endocytosis or exocytosis, or, in some embodiments, a dysfunctional PI cycle or lysosome.
• methods for treating a subject who has cancer comprise the steps of first identifying cancer as having a high rate of endocytosis or exocytosis, or, in some embodiments, a dysregulated PI cycle, and then administering to such a subject a therapeutically effective amount of an a-CD.
• the identification of cancer as having a high rate of endocytosis or exocytosis, or, in some embodiments, a dysfunctional PI cycle is done by PET imaging, preferably using a fluorescent tag, antibody, or other agent that identifies mutations in the amino acid sequence of Table 5b or an amino acid sequence at least about 70%, 80%, 90%, 95% 96%, 97%, 98%, or 99% homology to the amino acid sequences of Table 5b,
• the a-CD or derivative or salt thereof is effective to scavenge phospholipid in greater than about 50% of cells in an in vitro migration assay at a concentration of less than 4 mM, 3 mM, 2 mM, or 1 mM.
• the a-CD or derivative or salt thereof is effective to inhibit migration of cells in greater than about 50% of cells in an in vitro cell migration assay at a concentration of about 1 mM. In some embodiments, the a-CD or derivative or salt or clathrate thereof is effective to reduce cell migration in greater than 50% of cells in an in vitro cell migration assay at a concentration from about 0.5 to about 1.5 mM.
• methods for treating an individual who has been identified as having cancer comprise administering to such an individual a therapeutically effective amount of the a-CD or derivative or salt thereof which is known to be effective to inhibit cell migration in greater than 50% of cells in an in vitro migration assay at a concentration of less than 1 mM.
• the a-CD or derivative or salt thereof is effective to inhibit cell migration in greater than 50% of cells in an in vitro cell migration assay at a concentration of less than about 1.5 mM.
• the a-CD or derivative or salt thereof is effective to inhibit cell migration in greater than about 50%, 60%, 70%, 80%, or 90% of cells in an in vitro cell migration assay at a concentration of about 1 mM,
• the cancer prior to administration of the a-CD or derivative or salt thereof, is confirmed as being a cancer comprising one or a plurality of cells characterized by an abnormally high rate of endo- or exocytosis, or, in some embodiments, a dysfunctional PI cycle or lysosome.
• the preferred method of doing so is be PET imaging, polymerase chain reaction (PCR) of a sample such as a biopsy.
• Methods are provided for inhibiting, even partially, metastasis of a cancer cell.
• the methods comprise delivering to the cancer cell an amount of a-CD or derivative or salt thereof effective to inhibit cell migration of the cell.
• the a-CD or derivative or salt thereof used is effective to slow migration of a cancer cell in greater than 50% of cells in an in vitro cell migration assay at a concentration of less than about 2 mM or about 1.5 mM.
• the a-CD or derivative or salt thereof is effective to inhibit cell migration in greater than 50% of cells in an in vitro cell migration assay at a concentration of less than 1.5 mM.
• the cyclodextrin or derivative or salt thereof is effective to inhibit cell migration in greater than 50% of cells in an in vitro cell migration assay at a concentration of at or about less than 1.0 mM.
• the treatment simultaneously reduces the ototoxicity of the treatment.
• Embodiments of the present disclosure are particularly useful to treat patients who have cancer with cancer cells that have abnormally high rate of endo- or exocytosis, or, in some embodiments, a dysfunctional PI cycle.
• Such cancers include most cancers and generally exclude those cancers arising from tissues associated with lipid production such as liver cancer, and cancer involving fat cells.
• Cancer cells that have abnormally high rate of endocytosis or exocytosis, or, in some embodiments, a dysfunctional PI cycle are generally limited to epithelial cell derived cancers.
• cancer is from epithelial cells of the breast, colon, lung, or prostate.
• some methods of the invention relate to methods of treating a cancer patient who has cancer that have abnormally high rate of endo- or exocytosis, or, in some embodiments, a dysfunctional PI cycle, wherein such methods comprise the step of administering to such patient or subject a therapeutically effective amount of cyclodextrin.
• the a-CD or derivative or salt thereof is known to be effective to slow cell migration in greater than 50% of cells in an in vitro cell migration assay at a concentration of less than 2 mM.
• the a- CD or derivative or salt thereof is effective to induce apoptosis in greater than about 50%, 60%, 70%, 80%, or 90% of cells in an in vitro cell migration at a concentration of less than 1.5 niM.
• Cancer cells that have abnormally high rate of endo- or exocytosis, or, in some embodiments, a dysfunctional PI cycle generally have dysfunction of enzymes of Table 5b and/or metabolize and/or uptake high levels of phospholipids around their microenvironment.
• the cancer prior to administration of an a-CD or derivative or pharmaceutically acceptable salt thereof, is confirmed as being a cancer characterized by an abnormally high rate of endocytosis or exocytosis, or, in some embodiments, a dysfunctional PI cycle.
• the preferred method of doing so is be PET imaging, PCR or immunohistochemistry of a sample.
• Methods are provided for preventing or inhibiting the rate of metastases of a cancer cell characterized by an abnormally high rate of endo- or exocytosis, or, in some embodiments, a dysfunctional PI cycle.
• the methods comprise delivering to the cancer cell an amount of an a-CD or derivative or pharmaceutically acceptable salt thereof effective to reduce cell migration of the cell.
• the a-CD or derivative or pharmaceutically acceptable salt thereof used is effective to inhibit or reduce the rate of cell migration in greater than about 50%, 60%, 70%, 80%, or 90% of cells in an in who cell migration assay at a concentration of less than 1.5 mM.
• the disclosed treatment herein is effective to inhibit or reduce the rate of cell migration in greater than about 50%, 60%, 70%, 80%, or 90% of cells in an in vitro cell migration assay at a concentration of less than 1.1 mM. In some preferred embodiments, the disclosed treatment herein is effective to inhibit or reduce the rate of cell migration in greater than about 50%, 60%, 70%, 80%, or 90% of cells in an in vitro cell migration assay at a concentration of less than about 1.0 mM.
• any of the methods disclosed herein are free of administration of a cyclodextrin that scavenges cholesterol upon administration to a subject. In some embodiments, any of the methods disclosed herein are free of administration of a beta- cyclodextrin ( ⁇ -CD, PCD) or hydroxy-alkyl beta cyclodextrin.
• a-CDs The production of a-CDs is relatively simple and involves treatment of ordinary starch with a set of easily available enzymes.
• CGTase cyclodextrin glycosyltransferase
• First starch is liquefied either by heat treatment or using a-amylase, then CGTase is added for the enzymatic conversion.
• CGTases can synthesize all forms of cyclodextrins, thus the product of the conversion results in a mixture of the three main types of cyclic molecules, in ratios that are strictly dependent on the enzyme used: each CGTase has its own characteristic ⁇ : ⁇ : ⁇ synthesis ratio.
• ⁇ -CD which is very poorly water-soluble (18.5 g/1 or 16.3 mM) (at 25 °C) can be easily retrieved through crystallization while the more soluble a- and ⁇ -CDs (145 and 232 g/1 respectively) are usually purified by means of chromatography techniques.
• a "complexing agent" can be added during the enzymatic conversion step: such agents (usually organic solvents like toluene, acetone or ethanol) form a complex with the desired cyclodextrin which subsequently precipitates.
• Wacker Chemie AG uses dedicated enzymes, that can produce ⁇ -, ⁇ - or ⁇ -cyclodextrin specifically. This is very valuable especially for the food industry, as only a- and ⁇ -cyclodextrin can be consumed without a daily intake limit.
• the present disclosure provides compositions comprising any one or more of the active agents described herein, either alone or in combination, for example for use in treating malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition.
• the present disclosure provides compositions comprising an a-CD, or an analogue or derivative thereof, for example for use in treating breast or prostate cancer.
• the present disclosure provides compositions comprising an a-CD, or an analogue or derivative thereof, for example for use in treating breast or prostate cancer.
• compositions provided by the present disclosure include compositions wherein the active ingredient (e.g., compounds described herein, including embodiments or examples) is contained in a therapeutically effective amount, i.e., in an amount effective to achieve its intended purpose.
• the actual amount effective for a particular application will depend, inter alia, on the condition being treated.
• such compositions When administered in methods to treat a disease, such compositions will contain an amount of active ingredient effective to achieve the desired result, e.g., modulating the activity of a target molecule (e.g., PIP1, PIP2, PIP3), and/or reducing, eliminating, or slowing the progression of disease symptoms (e.g.
• compositions may be formulated by one having ordinary skill in the art with compositions selected depending upon the chosen mode of administration. Suitable pharmaceutical carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, A. 0501, a standard reference text in this field.
• Administering the pharmaceutical composition can be effected or performed using any of the various methods known to those skilled in the art.
• Systemic formulations include those designed for administration by injection, e. g. subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal, oral or pulmonary administration.
• administration of the effective amount of pharmaceutical composition disclosed herein is not limited to any particular delivery system and includes, without limitation, parenteral (including subcutaneous, intravenous (via injection or infusion), intramedullary, intraarticular, intramuscular, or intraperitoneal injection), rectal, topical, transdermal, mucosal or oral (for example, in capsules, suspensions, or tablets) administration.
• parenteral including subcutaneous, intravenous (via injection or infusion), intramedullary, intraarticular, intramuscular, or intraperitoneal injection
• rectal topical, transdermal, mucosal or oral (for example, in capsules, suspensions, or tablets) administration.
• administration to a subject in need thereof occurs in a single dose or in repeat administrations, and in any of a variety of physiologically acceptable salt forms, or with an acceptable pharmaceutical carrier or additive as part of a pharmaceutical composition.
• any suitable and physiological acceptable salt forms or standard pharmaceutical formulation techniques, dosages, and excipients are utilized.
• the step of administering comprises administering the composition or pharmaceutical composition intravenously, intramuscularly, topically, intradermally, intramucosally, subcutaneously, sublingually, orally, intravaginally, intracavernously, intraocularly, intranasally, intrarectally, gastrointestinally, intraductally, intrathecally, subdurally, extradurally, intraventricular, intrapulmonary, into an abscess, intraarticularly, into a bursa, subpericardially, into an axilla, intrauterine, into the pleural space, intraperitoneally, transmucosally, or transdermally.
• compositions of the invention may be administered by a variety of routes including oral, buccal, sublingual, rectal, transdermal, subcutaneous, intravenous injection, intravenous infusion, intramuscular, intrathecal, intraperitoneal and intranasal.
• the active agents can be formulated as compositions that are, for example, either injectable, topical or oral compositions.
• Liquid forms of compositions may include a suitable aqueous or nonaqueous vehicle with buffers, suspending and dispensing agents, colorants, flavors and other suitable ingredients known in the art.
• Solid forms of compositions may include, for example, binders, excipients, lubricants, coloring agents, flavoring agents and other suitable ingredients known in the art.
• the active agents and pharmaceutical compositions described herein may also be administered in sustained release forms or from sustained release drug delivery systems known in the art.
• the pharmaceutical composition may depend on the disease or condition and on whether the administration is to prevent or to treat the disease or condition. For instance, administration for prevention of several diseases or conditions, including, but not limited to cancer, may be orally, without penetration enhancers, and at a lower dose. Administration for a subject showing symptoms of cancer, such as triple-negative node-positive breast cancer, the administration may be parenteral, regioselective, and at higher dose.
• Methods for targeting may include, but are not limited to combination with folate or antifolates (methotrexate, pemetrexed). Combination includes, but is not limited to esterification with or without linkers.
• the linker would be a disulfide linker (including, but not limited linkers comprising cy stein, 3-mercaptopropionic acid, or 2-mercaptoethanol), linking to both the a-CD and pemetrexed via an ester.
• the a-CD/pemetrexed compound would be co-administered with folate.
• Administration to a subject showing symptoms of a neurodegenerative disease may be intrathecally without penetration enhancers and modifications for targeting.
• compositions of the present disclosure are pharmaceutical compositions comprising one or more active agents, as described herein, together with one or more conventionally employed components suitable for use in pharmaceutical delivery such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, excipients, and the like, may be placed into the form of pharmaceutical formulations.
• Nonlimiting examples of such formulations include solutions, creams, gels, gel emulsions, jellies, pastes, lotions, salves, sprays, ointments, powders, solid admixtures, aerosols, emulsions (e.g., water in oil or oil in water), gel aqueous solutions, aqueous solutions, suspensions, liniments, tinctures, and patches suitable for topical administration.
• the pharmaceutical compositions and formulations described herein may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy.
• Such methods include the step of bringing into association an active agent with liquid carriers, solid matrices, semi- solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, shaping the product into the desired delivery system.
• Unit dosage forms of a pharmaceutical composition or formulation preferably contain a predetermined quantity of active agent and other ingredients calculated to produce a desired therapeutic effect, such as an effective amount of a therapeutically effective amount.
• Typical unit dosage forms include, for example, prefilled, premeasured ampules or syringes of liquid compositions, or pills, tablets, capsules or the like for solid compositions.
• the cyclodextrin or derivative thereof can be, for example, formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle or pharmaceutically acceptable carrier.
• a pharmaceutically acceptable parenteral vehicle or pharmaceutically acceptable carrier examples include water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, Ringers dextrose, dextrose and sodium chloride, lactated Ringers and fixed oils, polyethylene glycol, polyvinyl pyrrolidone, lecithin (glycerophospholipids), arachis oil or sesame oil. Liposomes and nonaqueous vehicles such as fixed oils may also be used.
• the vehicle or lyophilized powder may contain additives that maintain isotonicity (e. g, sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives).
• the formulation is sterilized by commonly used techniques.
• Parenteral dosage forms may be prepared using water or another sterile carrier.
• a parenteral composition suitable for administration by injection is prepared by dissolving 1.5% by weight of active ingredient in 0.9% sodium chloride solution.
• the solution can be lyophilized and then reconstituted with a suitable solvent just prior to administration.
• Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, from about 0.01 to about 0.1 M or about 0.05 M phosphate buffer or about 0.8% saline.
• Intravenous carriers include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like.
• such pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
• Aqueous carriers include water, ethanol, alcoholic/aqueous solutions, glycerol, emulsions or suspensions, including saline and buffered media.
• compositions can be prepared using conventional pharmaceutical excipients and compounding techniques.
• Oral dosage forms may be elixirs, syrups, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
• the typical solid carrier may be an inert substance such as lactose, starch, glucose, cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP); granulating agents; binding agents, magnesium stearate, dicalcium phosphate, mannitol and the like.
• a composition in the form of a capsule can be prepared using routine encapsulation procedures.
• pellets containing the active ingredient can be prepared using standard carrier and then filled into a hard gelatin capsule; alternatively, a dispersion or suspension can be prepared using any suitable pharmaceutical carrier(s), for example, aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatin capsule.
• suitable pharmaceutical carrier(s) for example, aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatin capsule.
• suitable pharmaceutical carrier(s) for example, aqueous gums, celluloses, silicates or oils
• Typical liquid oral excipients include ethanol, glycerol, glycerin, non-aqueous solvent, for example, polyethylene glycol, oils, or water with a suspending agent, preservative, flavoring or coloring agent and the like.
• excipients may be mixed as needed with disintegrants, diluents, lubricants, and the like using conventional techniques known to those skilled in the art of preparing dosage forms.
• disintegrating agents may be added, such as the crosslinked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
• solid dosage forms may be sugarcoated or coated using standard techniques, including, but not limited to the use of chitosan, to target specific regions of the gastrointestinal tract.
• suitable carriers, excipients or diluents include water, glycols, oils, alcohols, etc. Additionally, flavoring agents, preservatives, coloring agents and the like may be added.
• compositions of the disclosure may take the form of tablets, lozenges, and the like formulated in conventional manner.
• the compounds may also be formulated in rectal or vaginal compositions such as suppositories or enemas.
• a typical suppository formulation comprises a binding and/or lubricating agent such as polymeric glycols, glycerides, gelatins or cocoa butter or other low melting vegetable or synthetic waxes or fats.
• the compounds for use according to the present disclosure are conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer, with the use of a suitable propellant, e.
• the dosage unit may be determined by providing a valve to deliver a metered amount.
• Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. Even the larger PCD was seen to be suitable for inhalation.
• the formulations may also be a depot preparation which can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
• the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
• Liposomes and emulsions are well known examples of delivery vehicles that may be used.
• Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.
• the compounds may be delivered using a sustained release system, such as semipermeable matrices of solid polymers containing the therapeutic agent.
• sustained- release materials have been established and are well known by those skilled in the art. Sustained release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.
• the compounds described herein may also be formulated for parenteral administration by bolus injection or continuous infusion and may be presented in unit dose form, for instance as ampoules, vials, small volume infusions or prefilled syringes, or in multi-dose containers with an added preservative.
• Preservatives and other additives can also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like. All carriers can be mixed as needed with disintegrants, diluents, granulating agents, lubricants, binders and the like using conventional techniques known in the art. F) Dosages
• the dose of an active agent described herein may be calculated based on studies in humans or other mammals carried out to determine efficacy and/or effective amounts of the active agent (see section E, Clinical Outcomes, below).
• the dose amount and frequency or timing of administration may be determined by methods known in the art and may depend on factors such as pharmaceutical form of the active agent and route of administration, and patient characteristics including age, body weight or the presence of any medical conditions affecting drug metabolism.
• a dose may be administered as a single dose.
• a dose may be administered as multiple doses over a period of time, for example, at specified intervals, such as, daily, bi-weekly, weekly, monthly, and the like.
• the dose will be 700 mg/kg/d (Table 3, 2010-04-15).
• the dose will be increased over time until early signs of renal or cytotoxicity are observed, in which case the dose level will be decreased to the previous, well-tolerated level.
• the dose of active agent is about at least 10 mg, at least 20 mg, at least 30 mg, at least 40 mg, at least 50 mg, at least 75 mg, at least 100 mg, at least 125 mg, at least 150 mg, at least 175 mg, at least 200 mg, at least 225 mg, at least 250 mg, at least 275 mg, at least 300 mg, at least 325 mg, at least 350 mg, at least 375 mg, at least 400 mg, at least 425 mg, at least 450 mg, at least 475 mg, at least 500 mg, at least 550 mg, at least 600 mg, at least 650 mg, at least 700 mg, at least 750 mg, at least 800 mg, at least 850 mg, at least 900 mg, at least 950 mg, at least 1000 mg, at least 1200 mg, at least 1500 mg, at least 2000 mg, at least 2500 mg, at least 3000 mg, at least 4000 mg, at least 5000 mg, at least 7500 mg, at least 10,000 mg, at least 15,000 mg, at least
• the above dosages are mg/day or mg/kg/day.
• the dose of active agent is in the range of 1 to 10000 mg, 1 to 7500 mg, 1 to 5000 mg, 1 to 2500 mg, 1 to 1000 mg, 1 to 500 mg, 1 to 250 mg, 250 to 10000 mg, 250 to 5000 mg, 250 to 1000 mg, 250 to 500 mg, 500 to 10000 mg, 500 to 5000 mg, 500 to 1000 mg.
• the above dosages are mg/day or mg/kg/day.
• the dose of clathrate or a-cyclodextrin, derivative or salt thereof is from about 1 to about 20 grams per day.
• the dose of clathrate or a-cyclodextrin, derivative or salt thereof is from about 10 to about 20 grams per day. In some embodiments, the dose of clathrate of or a- cyclodextrin, derivative or salt thereof is about 15 grams per day. In some embodiments, the dose of clathrate of or a-cyclodextrin, derivative or salt thereof is about 15 grams per day. In some embodiments, the dose of clathrate of or a-cyclodextrin, derivative or salt thereof is about 20 grams per day. In some embodiments, the dose of clathrate of or a-cyclodextrin, derivative or salt thereof is about 25 grams per day.
• the dose of clathrate of or ⁇ -cyclodextrin, derivative or salt thereof is about 30 grams per day. In some embodiments, the dose of clathrate of or a-cyclodextrin, derivative or salt thereof is about 35 grams per day. In some embodiments, the dose of clathrate of or a-cyclodextrin, derivative or salt thereof is about 40 grams per day. In some embodiments, the dose of clathrate of or a- cyclodextrin, derivative or salt thereof is about 45 grams per day. In some embodiments, the dose of clathrate of or a-cyclodextrin, derivative or salt thereof is about 50 grams per day.
• the dose of clathrate of or ⁇ -cyclodextrin, derivative or salt thereof is about 55 grams per day. In some embodiments, the dose of clathrate of or a-cyclodextrin, derivative or salt thereof is about 10 grams per day. In some embodiments, the dose of clathrate of or ⁇ -cyclodextrin, derivative or salt thereof is about 15 grams per day. In some embodiments, the dose of clathrate or ⁇ -cyclodextrin, derivative or salt thereof is about 60 grams per day. In some embodiments, the dose is about 5 grams three times per day.
• a single dose may be administered.
• multiple doses may be administered over a period of time, for example, at specified intervals, such as, four times per day, three times a day, twice per day, once a day, weekly, monthly, 4 times over 14 days, 2 times over 21 days, twice per month, 4 times over 21 days, 4 times per month, or 5, 6, 7, 8, 9, 10, 11, 12 or more times per month, per 21 days, per 14 days, or per week, and the like.
• the doses administered intravenously or intrathecally may be 5600 mg/kg per week or 400 mg per week (Table 3).
• Week 1 500 mg/kg/d; 8 h x1 + 3-4 d 600 mg/kg/d; 8 h x1
• Week 2 700 mg/kg/d; 8 h x1 + 3-4 d 800 mg/kg/d; 8 h x1
• Week 3 900 mg/kg/d; 8 h x1 + 3-4 d 1000 mg/kg/d; 8 h x1
• Initial infusion 500 mg/kg/d over 8 h at a rate of 20 ml/h
• the methods and compositions described herein may be used to treat or prevent malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition, in any subject in need of such treatment.
• the subject is a human.
• the subjects to be treated are post-menopausal women, in other embodiments the methods of treatment described herein are not intended to be limited to such subjects. Rather, in some embodiments the subjects can be of any age, ranging from newborns to older adults.
• the methods and compositions described herein may be employed as prophylactic treatments or therapeutic treatments.
• the methods and compositions provided herein can be used preventatively in subjects that do not yet exhibit any clear or detectable clinical indicators or symptoms of malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition but that are believed to be at risk of developing malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition, such as MS, prostate cancer or breast cancer, or diseases that are characterized by dysfunctional lysosomes.
• a subject receiving prophylactic treatment for a-CD may not exhibit any clinical indicators or symptoms of MS, prostate cancer or breast cancer, or diseases that are characterized by dysfunctional lysosomes.
• the methods and compositions provided herein can be used in subjects that already exhibit one or more clinical indicators or symptoms of the disease or disorder, such as MS, prostate cancer or breast cancer, or diseases that are characterized by dysfunctional lysosomes.
• a subject receiving therapeutic treatment for MS, prostate cancer or breast cancer, or diseases that are characterized by dysfunctional lysosomes may have been clinically diagnosed with MS, prostate cancer or breast cancer, or diseases that are characterized by dysfunctional lysosomes or may otherwise exhibit one or more clinical indicators or symptoms of MS, prostate cancer or breast cancer, or diseases that are characterized by dysfunctional lysosomes.
• the disclosure relates to methods of treating cancers comprising cells that are deficient or substantially deficient in any of the genes identified in the Figures or specification, such that the limited expression or lack of expression of those genes results in lysosomal dysfunction.
• a subject may have been identified as being at risk of developing malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition.
• the subject has a family history of malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition.
• the subject has one or more genetic risk factors associated with malignant, neurodegenerative, vascular, metabolic, inflammatory, autoimmune, pulmonary, fibrotic, hepatic, lysosomal storage, or viral disease, disorder, or condition, for example, a genetic mutation in a gene associated with PI cycle function.
• the methods of treatment provided herein (which comprise, for example, administering to a subject an effective amount of a composition according to the present disclosure) result in, or are aimed to achieve, a detectable improvement in one or more clinical indicators or symptoms of the age-related diseases.
• the clinical indicators or symptoms include, but not limited to, changes growth, migration, or invasion.
• a symptom or indicator of improvement is selected from the group comprising survival, disease-free survival, distant metastasis-free survival, results of a blood test (including, but not limited to circulating tumor DNA, prostate-specific antigen, ⁇ - galactosidase/p-hexosaminidase (Tiribuzi R, Orlacchio A, et al.
• the serum chemistries may include, but will not be limited to evaluation of electrolytes, bicarbonate, glucose, BUN, creatinine, magnesium, phosphate, hepatic enzymes (AST and ALT), total protein, albumin, bilirubin, and alkaline phosphatase.
• a complete lipid panel may be obtained and shape of erythrocytes may be evaluated microscopically. Bone density may be measured to identify early signs of osteoporosis.
• compositions and methods described herein are illustrative only and not intended to be limiting. Those of skill in the art will appreciate that various combinations or modifications of the specific compositions and methods described above can be made, and all such combinations and modifications of the compositions and methods described herein may be used in carrying out the present disclosure. Furthermore, certain embodiments of the present disclosure are further described in the following non-limiting Examples, and also in the following Claims.
• Example 1 The Pi-Cycle is a Drug Target against Metastases in Breast Cancer
• Example 1 The methods used to obtain the results presented in Example 1 are further described in (Wittkowski KM (2014) US 2016/0206581 Al), which is hereby incorporated by reference in its entirety.
• ssGWAS After eliminating non-informative or low-quality SNPs, a traditional ssGWAS was performed, using u-statistics for univariate data (also known as the Mann- Whitney test (Mann HB, Whitney DR (1947) Ann Math Stat 18:50-60) which is equivalent to the Wilcoxon rank-sum test (Wilcoxon F (1954) Biometrics 1:80-3)). By construction, the results of this analysis are very close to those obtained with the traditional Cochran- Armitage trend test. (Armitage P (1955) Biometrics 11:375-86)
• MAF- significance correlation With any finite sample size, the significance of a u- or rank test is limited. Hence, more significant results can only be obtained for SNPs with sufficiently high MAF.
• Non-randomization bias The reason for this curvature often not being recognized is that GWAS subjects are deterministically categorized based on their outcome (e.g., nonverbal v. verbal), rather than randomly assigned to interventions (as in clinical trials). Any deterministically categorized populations, however, are expected to differ systematically in aspects related to neither the condition of interest nor common ancestry factors (which could potentially be accounted for through stratification). When the downward trend from using a limited test and the upward bias from deterministic selection are similar, the s-values may still appear to follow the diagonal, except for loci suggesting 'true association' (Pearson TA, Manolio TA (2008) JAMA 299:1335-44)
• the highest point of the projected QR curve (apex) for each chromosome can be estimated from a smooth projection of the s-values after truncating as many of the highest values as needed for the projection to have a monotone increase and, conservatively for a limited test, a non-positive second derivative. Fitting against the data also reduces the effect of population stratification (Pearson TA, Manolio TA (2008) JAMA 299:1335-44).
• Quantile-rank (QR) plots As is customary with selection procedures, p-values were used mainly for the purpose of ranking loci. As no particular hypotheses regarding specific loci were to be confirmed, the traditional approach of exploring characteristics of the 'QQ plot' as decision criteria was modified and formalized. For multivariate tests of overlapping diplotypes, the straight line expected in the traditional 'QQ plot' under the univariate WG permutation hypothesis turns into a curve because many tests are performed per SNP. Even though the number of tests performed increases substantially, the increase in s-values shown in the QR curve compared to the QQ line (FIG. 7 v. FIG. 6, FIG. 9 v. FIG. 8, FIG. 11 v. FIG. 10) is limited, because most tests are highly dependent.(Wittkowski KM, Sonakya V, et al. (2014) Transl Psychiatry 4:e354)
• Selective chromosome permutation The proposed use of a selected chromosome permutation approach reduces this bias. While chromosomes may differ with respect to their content of disease related and unrelated risk factors, random errors are expected to have the same distribution across all chromosomes. Hence, the above biases are reduced by excluding chromosomes containing regions of high significance when determining the permutation distribution. In particular, the endpoint of the expected distribution for each chromosome can be estimated from the loess projection to the p-values after truncation to ensure a monotone increase and a non-positive second derivative.
• the endpoint of the expected distribution is estimated from the median of the limited set of, e.g., ten, chromosomes with the lowest maximum deviation of the distribution of s-values from the loess projection.
• ssGWAS results Single-SNP GWAS confirmed these findings at essentially equivalent levels of 6.20 and 5.57, respectively. Many other results had been dismissed in previous published analyses because p-values did not reach the traditional level of "fixed genome-wide significance" (typically, 7.5). From the ssGWAS QR plots (CGEM: FIG. 6, EPIC: FIG. 8, and PBCS: FIG. 10) many of the genes above the cut-off for study- specific genome-wide significance fit the paradigm of being involved in signaling at the membrane (GPCRs, Fc receptors, growth factor receptors, ion channels) or processes in the nucleus (cell cycle control, transcription, splicing) (see Table 4, columns Mbrn and Ncls).
• muGWAS results In muGWAS (CGEM: FIG. 7, EPIC: FIG. 9, and PBCS: FIG. 11), the proportion of genes related to membrane signaling and nuclear processes is even higher than in ssGWAS. In addition, a group of genes known to play a role in either in the PI cycle (FIG. 12) or in endocytosis (FIG. 8A) stands out. Table 4: Genes identified by Study.
• IPV6/IPV1 -log 10(p- value) in muGWAS/ssGWAS; Mbrn: membrane-associated genes (GPCR, Fc-Receptor, HA, RTK, Ion channels), PI/EC: PI signaling/endocytosis, MPK: MAP kinases, Ncls: nucleus (cell cycle control, transcription, splicing).
• center block of rows within each study other genes with diplotypes among the top 41 in muGWAS; bottom block of rows within each study: genes with SNPs above the study- specific level of genome-wide significance in ssGWAS (CGEM: 4.03, FIG. 6; EPIC: 4.00, FIG. 8; PBCS: 3.84, FIG. 10); ssGWAS results for genes also implicated in muGWAS are shown next to the muGWAS results); black squares: known breast cancer genes (from http://www.
• MEGF11 (CGEM, PBCS)
• AGPAT4 (CGEM) - AGPAT3 (EPIC)
• PI(3)P (NLRP4, EEAl, RAB32), as well as
• Endocytosis as a critical component of migration and invasion. Endocytosis is known to be controlled by PI signaling, which is consistent with the genes identified in the results presented:
• the subject has a disease or disorder that is characterized by a dysfunctional lysosomal pathway by deficient expression of any one or combination of genes disclosed above.
• the approach used here differs from traditional GWAS in both the statistical method being used and the decision strategy.
• the novel approach (a) avoids making assumptions about a particular degree of dominance, (b) draws for the fact that both SNPs neighboring a disease locus should be in LD, unless they are separated by a recombination hotspot, (c) can distinguish between SNPs belonging to the same tag sets, but differ in their order along the chromosome, (d) accounts for different disease loci within the same region having similar effects and for compound heterozygosity within the statistical method (rather than through visual inspection looking for several SNPs within a region having high significance), and (e) provides additional information (“information content”) that can be used to prioritize results.
• the PI cycle is critical for many cellular functions in eukaryotic cells, including developmental functions such as oocyte maturation, fertilization, embryo genesis, but also cell growth, cytoskeleton dynamics, membrane trafficking, and nuclear events.
• developmental functions such as oocyte maturation, fertilization, embryo genesis, but also cell growth, cytoskeleton dynamics, membrane trafficking, and nuclear events.
• PI(4.5)P2, PI(4)P, PI(3,4)P2, and PI(3)P are tightly regulated by both three kinases and three groups of phosphatases.
• PIPs phosphatidylinositides
• Example 2 The genetic risk factors in the PI cycle and along the endocytosis pathway are known as shared risk factors for "derailed endocytosis” in breast cancer and "defective/deranged endocytosis” Parkinson's disease/Alzheimer's disease.
• Alzheimer's disease and Parkinson's disease are known to share risk factors: ⁇ and a-synuclein have been hypothesized to interact, (Crews L, Tsigelny I, et al. (2009) Neurotox Res 16:306-17; Tsigelny IF, Crews L, et al. (2008) PLoS One 3:e3135) and "moderate association" of AD with PD was found in a meta analysis of 14 studies conducted 1986-2010,(Feldman AL, Johansson AL, et al.
• breast cancer has high co-occurrence with Parkinson's disease.
• Dermatol Surg 42:141-6 Earlier reports that cancers reduce Alzheimer's disease risk were linked to statistical models not accounting for competing risks (Hanson HA, Horn KP, et al. (2016) J Gerontol B Psychol Sci Soc Sci) and/or treatment effects of cancer drugs.
• Table 5 Pi-Cycle overlap between Breast Cancer, PD, and AD.
• Gene Genes identified in breast cancer GWAS.
• EEC function Known function in Pl-cycle and/or EEC.
• KEGG KEGG pathway (http://www.genome.jp/kegg/pathway.html)
• EC epithelial cancer (carcinoma).
• ND Neurodegenerative disease.
• AD Alzheimers Dement 10:45-52
• AGPAT3 converts lyso-PI (LPI) (Bradley RM, Marvyn PM, et al. (2015) Biochimica AGPAT4 into et Biophysica Acta (BBA) - Molecular and Cell phosphatidylinositol Biology of Lipids 1851 :1566-76)
• AD Alzheimers Dis 23:349-59
• DAG diacylglycerol
• PTENP1 PI3K/PTEN and hsa04070 PTEN (Erneux C, Ghosh S, et al. (2016) Curr PI(3,4,5)P3 are Pharm Des 22:2309-14)
• ASTN2 regulates trafficking of hsa04144 (Wilson PM, Fryer RH, et al. (2010) The
• Neural Transm (Vienna) 122:701-8) ID ASTN1: (Anazi S, Maddirevula S, et al.
• TNS1 controls cell polarization, (Rainero E, Howe JD, et al. (2015) Cell Rep migration, and invasion 10:398-413; McCleverty CJ, Lin DC, et al. binds a5b1 integrin during (2007) Protein Science : A Publication of the endocytosis Protein Society 16:1223-9; Burghel GJ, Lin
• ⁇ DNM1 depends on the hsa04721 Development 140:3230-43)
• AD MEGF10 (Sherva R, Tripodis Y, et al.
• SDCBP2 Syndecans bind PI(4,5)P2 (Baietti MF, Zhang Z, et al. (2012) Nat Cell and are involved in both Biol 14:677-85; Hurley JH, Odorizzi G endo- and exocytosis. (2012) Nat Cell Biol 14:654-5)
• N4BP3 NEDD4 controls growth factor hsa04144 (Persaud A, Alberts P, et al. (2011) EMBO J receptor endocytosis 30:3259-73; Jung S, Li C, et al. (2013) Int J ⁇ NEDD9 expression is Oncol 43:1587-95)
• BECN1 a component of the PI3K Immunol 186:1646-55; Zhang Y, Sauler M, complex that mediates vesicle
• AD (Antonell A, Llado A, et al. (2015) Mol Neurobiol ; Swaminathan G, Zhu W, et al.
• AD Woodon BL, Cramer PE, et al. (2012) Neurobiology of Aging 33:197.e21 -.e32
• RAB32 RAB32/RAB38 interact AP-3 hsa05012 (Hesketh GG, Perez-Dorado I, et al. (2014) and with LRRK2 (PARKS) Dev Cell 29:591 -606; Waschbusch D,
• RAPGEF4 GEF for RABIA/I B/2A (Parnell E, Palmer TM, et al. (2015) Trends involved in excocytosis Pharmacol Sci 36:203-14; Almahariq M, through RIMS2 Tsalkova T, et al. (2013) Mol Pharmacol
• AD (Puthiyedth N, Riveras C, et al. (2016) PLoS One 11 :e0152342; Bereczki E, Francis PT, et al. (2016) Alzheimers Dement)
• STXBP4* Prevents interaction hsa04130, (Zhang QY, Tan MS, et al. (2015) Mol between STX4 and VAMP2, hsa04721 Neurobiol)
• ANXA4 Forms exocytotic complexes hsa04721 (Lizarbe MA, Barrasa Jl, et al. (2013) Int J with SYT1 and the RAB3A Mol Sci 14:2652-83; Willshaw A, Grant K, et effector RPH3A. al. (2004) FEBS Lett 559:13-21 )
• AD (Kuzuya A, Zoltowska KM, et al. (2016) BMC Biol 14:25; Tan MG, Lee C, et al. (2014) Neurochem lnt 64:29-36)
• PD DNAJC6/13 (Seaman M, Freeman CL
• Example 3 Elevated endocytosis combined with age-related decline in lysosomal function is a common epistatic risk factor in age-related diseases
• endocytosis is a known component of the etiology of many age-related diseases.
• cancers Mosesson Y, Mills GB, et al. (2008) Nat Rev Cancer 8:835-50
• Alzheimer's disease Schott al. (2015) Cell Mol Life Sci ; Wang X, Huang T, et al. (2014) Molecular Neurodegeneration 9:1-9; Kim S, Sato Y, et al. (2016) Mol Psychiatry 21:707-16
• PD Ravero-Rios P, Gomez-Suaga P, et al. (2015) Biochem Soc Trans 43:390-5
• Alzheimer's disease risk factors [and] endocytosis-related genes are the earliest known disease-specific neuronal response in Alzheimer's disease. They develop early in Down syndrome, a cause of early-onset Alzheimer's disease linked to an extra copy of APP.”(Kim S, Sato Y, et al. (2016) Mol Psychiatry 21:707-16)
• HBV hepatitis B virus
• Example 4 pCDs exert their effects on "derailed”, “deranged”, or “defective” endocytosis not through a mechanism independent of cholesterol.
• the present disclosure provides a-CD or HP-a-CD and analogues and derivatives thereof as non-limiting examples of compounds that may be useful for treating age-related conditions including but not limited to conditions involving "derailed endocytosis," a "hallmark of cancer” (Mitra S, Cheng KW, et al. (2012) Biochem Soc Trans 40:1404-8) also seen in neurodevelopmental diseases. (Rivero-Rios P, Gomez-Suaga P, et al. (2015) Biochem Soc Trans 43:390-5; Van Dooren T, Princen K, et al. (2014) Biomed Res Int 2014: 167024).
• a-CDs are used for treating age-related conditions, such as cancers (including, but not limited to, prostate and breast cancer) and neurodegenerative diseases (including, but not limited to, PD or AD).
• CDs lower the amount of PIPs available without directly interfering with their distribution.
• ⁇ - ⁇ -CD suppressed invasion activity in three H7 Lewis lung cancer cell lines and highly metastatic cell lines had more ⁇ integrin (Zhang Q, Furukawa K, et al. (2006) J Biol Chem 281:18145-55) and breast cancer and prostate cancer cell lines were more sensitive to ⁇ - ⁇ -CD-induced cell death than their normal counterparts.
• ⁇ - ⁇ -CD treatment induced a substantial decrease (40%) in activity of breast cancer resistance protein (BCRP),(Storch CH, Ehehalt R, et al.
• ⁇ - ⁇ -CD was more toxic for invasive than for non-invasive urothelial cancer cells,(Resnik N, Repnik U, et al. (2015) PLoS One 10:e0137878) interfered with RTK-[PIP2]-PI3K-[PIP3]-AKT signaling in HeLa cells,(Yamaguchi R, Perkins G, et al. (2015) FEBS Lett 589:4097-105) and inhibited the growth of leukemia cell lines.(Yokoo M, Kubota Y, et al. (2015) PLoS One 10:e0141946)
• AD ⁇ - ⁇ -CD inhibits secretion of amyloid-beta from hippocampal neurons of rats infected with recombinant Semliki Forest virus (SFV) carrying APP,(Simons M, Keller P, et al. (1998) Proc Natl Acad Sci U S A 95:6460-4) promotes the non-amylogenic a-secretase pathway,(Kojro E, Gimpl G, et al. (2001) Proc Natl Acad Sci U S A 98:5815-20) and increased activity of ⁇ -secretase while decreasing activity of ⁇ -secretase, reducing the level of cell-associated APPsP (FIG.
• SFV Semliki Forest virus
• PD In HeLa cells transfected with a-synuclein, a-synuclein was shown to colocalize with the lipid raft. Incubation with 20 mM ⁇ - ⁇ -CD for 1 hr dramatically reduced this co- location of ⁇ -synuclein with the cell membrane. (Fortin DL, Troyer MD, et al. (2004) J Neurosci 24:6715-23)
• ⁇ - ⁇ -CD had higher concentration in tumor than in other cells (except kidney and liver) and was effective in a mouse model of breast cancer,(Grosse PY, Bressolle F, et al. (1998) Br J Cancer 78: 1165-9) reduced the number of lung metastases in mice implanted with H7-0 Lewis lung cancer cells,(Zhang Q, Furukawa K, et al. (2006) J Biol Chem 281: 18145-55) and inhibited growth of primary effusion lymphoma (PEL) in mice.(Gotoh K, Kariya R, et al.
• AD Scavenging of cholesterol and/or binding directly to amyloid-beta or a-synuclein was also believed to be the mode of action for ⁇ -CD in Alzheimer's disease and Parkinson's disease: "HPpCD, which diminishes the pool of both cholesterol and PLs, had "neuroprotective effects section in a transgenic mouse model of AD [by] enhancing clearance mechanisms".
• HPpCD which diminishes the pool of both cholesterol and PLs
• Tgl9959 mice are transgenic mice with 2 mutations in the amyloid precursor protein (APP) gene which have been associated with human Alzheimer's disease and beneficial effects were attributed to a reduction in cholesterol.
• APP amyloid precursor protein
• cyclodextrins The direct action of cyclodextrins consists in extracting lipids (cholesterol and phospholipids) from membranes and serum. After parenteral application, the lipids are excreted into urine. From the mechanism of ⁇ -CD in NPC and elevated cholesterol levels seen in several cancers, including breast cancer,(Yokoo M, Kubota Y, et al. (2015) PLoS One 10:e0141946) ⁇ -CDs are thought to reduce cancer growth by lowering cholesterol levels. In Alzheimer's disease, ⁇ - ⁇ -CD is believed to act on hydrophobic residues of another large lipid, amyloid-beta.
• ⁇ - ⁇ -CD was the most effective cyclodextrin against various LYD diseases (FIG. 23) and the proposed combination with 10 ⁇ delta- toxopherol (vitamin E) reduced the concentrations needed and, thus, reduced (but not eliminated) the risk of oto- and nephrotoxicity. While a-CD (and ⁇ - ⁇ -CD) were less effective than ⁇ - ⁇ -CD, it was not appreciated that the fact that a-CD (which cannot fit cholesterol) was similarly effective as ⁇ - ⁇ -CD (which can fit cholesterol) implies that the effects cannot be due to scavenging of cholesterol.
• Example 5 HPpCD activates formation of autophagolysosomes through a mechanism involving reduction of serum phospholipids
• TFEB transcription factor EB
• CLAR Coordinated Lysosomal Expression and Regulation
• ⁇ - ⁇ -CD treatment resulted in transcriptional up- regulation of all autosomal (LC3, SQSTM1, BECN1) lysosomal genes tested (GBA, HEX A, LAMP1) through activation of TFEB.
• ⁇ - ⁇ -CD treatment results in coordinated up- regulation of lysosome biogenesis and autophagy and enhanced autophagic clearance through TFEB-induced activation of the autophagy system, but blockage of downstream steps of the autophagy flux (e.g. blockage of ATG7 expression) prevents clearance of ceroid lipopigment.
• ⁇ - ⁇ -CD activates the pro-survival autophagy pathway, but not apoptosis.

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