METHODS FOR TREATING OR PREVENTING NEURODEGENERATIVE DISEASE USING A COMBINATION OF AMINOSTEROLS AND AN INSULIN COMPOUND

CROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application the benefit of U.S. Provisional Application No. 63/302,796, filed January 25, 2022, the contents of which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

[0001] The present application relates generally to methods of treatment and/or prevention using a combination of at least one aminosterol compound and at least one insulin compound.

BACKGROUND OF THE INVENTION

[0002] Squalamine is the most abundant member of a larger aminosterol family comprising at least 12 related compounds (Rao et al., 2000). squalamine

[0003] The discovery of squalamine, the structure of which is shown above, was reported by Michael Zasloff in 1993 (U.S. Patent No. 5,192,756).

[0004] Aminosterol 1436 is an aminosterol isolated from the dogfish shark, which is structurally related to squalamine (U.S. Patent No. 5,840,936; Rao, Shinnar et al. 2000). It is also known as MSI-1436, trodusquemine and produlestan.

[0005] Aminosterols also have potential therapeutic use in the areas of neurodegenerative disease. Age-related neurodegeneration is a significant unsolved problem and challenge. The number of people over 60 years is expected to rise from 841 million in 2013 to more than 2 billion in 2050 (United Nations. World population ageing 2013. United Nations Department of Economic and Social Affairs Population Division; Available online at: http://www.un.org/en/development/desa/population/publication s/pdf/ageing/WorldPopulationAg eingReport2013.pdf). As populations get older, age-related neurodegenerative diseases such as Alzheimer’s Disease (AD) and Parkinson’s Disease (PD) have become more common.

[0006] The use of aminosterol compounds for the treatment of neurodegenerative conditions such as Alzheimer’s disease and Parkinson’s disease was previously described. See e.g., WO 2019/241503, US 2020-0038412, US 2018-0319837, and US 2020-0038413. Diseases such as Alzheimer’s disease (AD) are prevalent and place great burden on the healthcare system.

[0007] In addition, the use of aminosterol 1436 in delaying the maturation process of mice was previously described. See US 2020-0038412 and US 2018-0319837.

[0008] The present disclosure is directed to the discovery that a combination of an aminosterol and an insulin compound is effective in treating various conditions.

SUMMARY OF THE INVENTION

[0009] In one aspect, a method of treating, preventing, and/or delaying the progression and/or onset of neurodegeneration in a subject in need is provided, comprising administering to the subject a therapeutically effective amount of a combination of: (a) an aminosterol comprising a spermine moiety, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof, and (b) insulin, an immunologically active insulin fragment, an insulin derivative, or a functional equivalent of insulin, or any combination thereof.

[0010] For all of the method described herein requiring administration of at least one aminosterol and at least one insulin compound, the aminosterol and insulin compound can be administered (i) concomitantly, (ii) as an admixture, (iii) separately and simultaneously or concurrently, or (iv) separately and sequentially.

[0011 ] In some embodiments, the functional equivalent of insulin comprises an insulin-like growth factor (IGF). In some embodiments, the IGF comprises IGF-1, IGF-2, or a combination thereof. In some embodiments, the IGF comprises an IGF-binding protein (IGFBP), optionally bound thereto. In some embodiments, the IGF-binding protein comprises one or more of IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP6, and IGFBP7.

[0012] In some embodiments, the insulin derivative comprises lispro, prandial insulin, aspart, glulisine, novolin, velosulin, isophane (neutral protamine hagedorn), insulin glargine, detemir, degludec, humulin, novolog, or a combination thereof.

[0013] In some embodiments, the neurodegeneration is age-related. In some embodiments, the neurodegeneration is correlated with one or more conditions or diseases selected from the group consisting of age-related dementia, Alzheimer’s disease, Parkinson’s disease, Lewy Body dementia, frontotemporal dementia, vascular dementia, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), multiple system atrophy (MSA), progressive supranuclear palsy (PSP), olivo-ponto-cerebellar degeneration, peripheral sensory neuropathy, cerebral palsy, and age related cognitive decline without a specific diagnosis from the group above.

[0014] In some embodiments, the progression or onset of the neurodegeneration is slowed, halted, or reversed over a defined time period following administration of (a) and (b). In some embodiments, the neurodegeneration is positively impacted by administration of (a) and (b). In some embodiments, the positive impact and/or progression of neurodegeneration is measured quantitatively or qualitatively by one or more techniques selected from the group consisting of electroencephalogram (EEG), neuroimaging, functional MRI, structural MRI, diffusion tensor imaging (DTI), [18F]fluorodeoxy glucose (FDG) PET, agents that label amyloid, [18F]F-dopa PET, radiotracer imaging, volumetric analysis of regional tissue loss, specific imaging markers of abnormal protein deposition, multimodal imaging, and biomarker analysis. In some embodiments, the progression or onset of neurodegeneration is slowed, halted, or reversed by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

|0015| In another aspect, a method of reversibly slowing the growth, maturation, and/or aging of subject, and/or extending the potential lifespan of the subject is provided, comprising administering to the subject a therapeutically effective amount of an aminosterol, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof. Optionally, the method comprises additionally administering insulin, an immunologically active insulin fragment, an insulin derivative, or a functional equivalent of insulin. In one aspect, the aminosterol has the formula: wherein:

R ¹ is H or D; and

R ² is H or D; provided that all of R ¹ are H, all of R ² are H, or all of R ¹ and R ² are H, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof.

|00l6| In some embodiments, the method is directed to retarding the aging process of a subject. In some embodiments, the method is directed to extending the potential lifespan of a subject. In some embodiments, the method is directed to delaying maturation and/or slowing growth of a subject. In some embodiments, the aminosterol, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof, and optionally insulin, an immunologically active insulin fragment, an insulin derivative, or a functional equivalent of insulin, are administered during a critical “developmental window” of the subject. In some embodiments, the critical “developmental window” is prior to the onset of maturity of the subject.

[0017] In some embodiments: (a) the delayed maturation and/or slowed growth is measured by height and/or weight, as compared to a subject the same age and sex, who is not administered the aminosterol, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof; and/or (b) the subject administered the aminosterol, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof, has delayed growth and/or maturation, as measured by height and/or weight, as compared to a subject the same age and sex and who is not administered aminosterol, by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%; and/or (c) the delayed maturation is measured by a delay in skeletal maturation; (d) the subject administered the aminosterol, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof, has delayed growth and/or maturation, as measured by skeletal maturation, as compared to a subject not administered aminosterol which is the same age and sex, by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

[0018] In some embodiments, characteristics of aging impacted are selected from the group consisting of muscle endurance, coordination, social behavior and cognitive ability. In some embodiments, the method: (a) improves impaired muscle endurance, as compared to a subject not administered aminosterol, which is the same sex and age, by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%; and/or (b) improves impaired coordination, as compared to a subject not administered aminosterol, which is the same sex and age, by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%; and/or (c) improves impaired cognitive ability, as compared to a subject not administered aminosterol, which is the same sex and age, by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

[0019] In some embodiments, a method of treating, preventing, or delaying the onset of age- related diseases, conditions or health problems in a subject, comprising administering to the subject a therapeutically effective amount of an aminosterol, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof, and optionally insulin, an immunologically active insulin fragment, an insulin derivative, or a functional equivalent of insulin, wherein the aminosterol has the formula: wherein:

R ¹ is H or D; and

R ² is H or D; provided that all of R ¹ are H, all of R ² are H, or all of R ¹ and R ² are H, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof.

[0020] In some embodiments, the age-related disease, condition, or health problem is selected from the group consisting of atherosclerosis and cardiovascular disease, cancer, arthritis, cataracts, osteoporosis, diabetes, hypertension, Alzheimer’s disease, arthritis, and osteoporosis.

[0021 ] In some embodiments: (a) the aminosterol comprising a spermine moiety, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof, and/or (b) the insulin, an immunologically active insulin fragment, a derivative thereof, or a functional equivalent thereof, are administered in combination with at least one additional active agent to achieve either an additive or synergistic effect.

[0022] In some embodiments, the additional active agent is administered via a method selected from the group consisting of (a) concomitantly; (b) as an admixture; (c) separately and simultaneously or concurrently; and (d) separately and sequentially.

[0023] In some embodiments, the neurodegeneration comprises: (a) Parkinson’s disease (PD) and the additional active agent comprises a drug selected from the group consisting of levodopa optionally combined with a dopa decarboxylase inhibitor such as carbidopa and benserazide, or combined with a COMT inhibitor such as tolcapone and entacapone; dopamine agonists such as bromocriptine, pergolide, pramipexole, ropinirole, piribedil, cabergoline, apomorphine, lisuride, and rotigotine; MAO-B inhibitors such as selegiline and rasagiline; amantadine; anticholinergics; clozapine; cholinesterase inhibitors for dementia; and modafinil for daytime sleepiness; (b) Alzheimer’s disease (AD) and the additional active agent comprises a drug selected from the group consisting of glutamate; antipsychotic drugs; huperzine A; acetylcholinesterase inhibitors such as donepezil (Aricept®), galantamine (Razadyne®), and rivastigmine (Exelon®); and NMDA receptor antagonists such as memantine (Akatinol®, Axura®, Ebixa®/Abixa®, Memox® and Namenda®); (c) Huntington’s chorea or disease (HD) and the additional active agent comprises a drug selected from the group consisting of antipsychotic drugs, such as haloperidol and clonazepam; drugs used to treat dystonia, such as acetylcholine regulating drugs (z.e., trihexyphenidyl, benztropine (Cogentin®), and procyclidine HC1); GABA-regulating drugs (z.e., diazepam (Valium®), lorazepam (Ativan®), clonazepam (Klonopin®), and baclofen (Lioresal®)); dopamine-regulators (z.e., levodopa/carbidopa (Sinemet®), bromocriptine (parlodel), reserpine, and tetrabenazine); anticonvulsants (z.e., carbamazepine (Tegretol®) and botulinum toxin (Botox®)); drugs used to treat depression (z.e., fluoxetine, sertraline, and nortriptyline); amantadine; tetrabenazine; dopamine blockers; and co-enzyme Qio; (d) peripheral sensory neuropathy and the additional active agent comprises a drug selected from the group consisting of neurotrophin-3; tricyclic antidepressants (e.g., amitriptyline); antiepileptic therapies (e.g., gabapentin or sodium valproate); synthetic cannabinoids (Nabilone) and inhaled cannabis; opiates; opioids; and pregabalin (Lyrica®); (c) Amyotrophic lateral sclerosis (ALS) and the additional active agent comprises a drug selected from the group consisting of riluzole (Rilutek®); KNS-760704 (an enantiomer of pramipexole); olesoxime (TRO19622); talampanel; and arimoclomol; (d) multiple sclerosis and the additional active agent comprises a drug selected from the group consisting of corticosteroids (e.g., methylprednisolone); plasmapheresis; fmgolimod (Gilenya®); interferon beta- la (Avonex®, CinnoVex®, Reci Gen® and Rebif®); interferon beta- lb (Betaseron® and Betaferon®); glatiramer acetate (Copaxone®); mitoxantrone; natalizumab (Tysabri®); alemtuzumab (Campath®); daclizumab (Zenapax®); rituximab; dirucotide; BHT-3009; cladribine; dimethyl fumarate; estriol; fmgolimod; laquinimod; minocycline; statins; temsirolimus teriflunomide; naltrexone; and vitamin D analogs; (e) cerebral palsy and the additional active agent comprises a drug selected from the group consisting of botulinum toxin and A injections; (f) multiple system atrophy (MSA) and the additional active agent comprises a drug selected from the group consisting of antiparkinson agents; COMT inhibitors; dopamine agonists; anticholinergics; urinary antispasmodic agents; prokinetic agents; agents for erectile dysfunction; corticosteroids; and alpha 1 agonists; or (g) progressive supranuclear palsy (PSP) and the additional active agent comprises a drug selected from the group consisting of levodopa and amantadine.

[0024] In some embodiments, the aminosterol, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof, is of pharmaceutically acceptable grade. In some embodiments, the aminosterol is in the form of a phosphate. [0025] In some embodiments: (a) the subject is a common pet, such as a dog or cat; or (b) the subject is a livestock, such as a horse, cattle, goat, sheep, pig or any farm animal. In some embodiments, the subject is a human.

10026] In some embodiments, the aminosterol or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof, and the insulin or fragment thereof, derivative thereof, or functional equivalent thereof, are administered via a method selected from the group consisting of (a) concomitantly; (b) as an admixture; (c) separately and simultaneously or concurrently; and (d) separately and sequentially.

[0027] In some embodiments, the aminosterol, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof, and the insulin, an immunologically active insulin fragment, an insulin derivative, or a functional equivalent of insulin, are administered via any pharmaceutically acceptable method.

[0028] In some embodiments: (a) the aminosterol or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof, and (b) the insulin or the fragment thereof, derivative thereof, or functional equivalent thereof, are administered from a method independently selected from the group consisting of oral, nasal, pulmonary, inhalation, sublingual, buccal, rectal, vaginal, intravenous, intra-arterial, intradermal, intraperitoneal, intrathecal, intramuscular, epidural, intracerebral, intracerebroventricular, transdermal, and any combination thereof.

[0029] In some embodiments, the insulin or the fragment thereof, derivative thereof, or functional equivalent thereof is administered via an insulin pump. In some embodiments, the method of administration for each of (a) and/or (b) is independently selected from nasal administration, oral administration, or a combination thereof. In some embodiments, the aminosterol, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof is administered intranasally. In some embodiments, the insulin, an immunologically active insulin fragment, an insulin derivative, or a functional equivalent of insulin is administered intranasally.

[0030] In some embodiments, (a) the aminosterol comprising a spermine moiety, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof, and (b) the insulin, an immunologically active insulin fragment, an insulin derivative, or a functional equivalent of insulin, are formulated into the same dosage form or separate dosage forms, each independently selected from the group consisting of liquid dispersions, gels, aerosols, lyophilized formulations, tablets, and capsules.

[0031] In some embodiments, (a) the aminosterol comprising a spermine moiety, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof, and (b) the insulin, an immunologically active insulin fragment, an insulin derivative, or a functional equivalent of insulin, are formulated into the same dosage form or separate dosage forms, each independently selected from the group consisting of controlled release formulations, fast melt formulations, delayed release formulations, extended release formulations, pulsatile release formulations, and mixed immediate release and controlled release formulations.

[0032] In some embodiments, the amount of the aminosterol comprising a spermine moiety, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof administered to the subject comprises: (a) about 0.1 to about 20 mg/kg body weight of the subject; (b) about 0.1 to about 15 mg/kg body weight of the subject; (c) about 0.1 to about 10 mg/kg body weight of the subject; (d) about 0.1 to about 5 mg/kg body weight of the subject; or (e) about 0.1 to about 2.5 mg/kg body weight of the subject.

[0033] In some embodiments, the amount of the aminosterol comprising a spermine moiety, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof administered to the subject comprises: (a) about 0.001 to about 500 mg/day; (b) about 0.001 to about 250 mg/day; (c) about 0.001 to about 125 mg/day; (d) about 0.001 to about 50 mg/day; (e) about 0.001 to about 25 mg/day; (f) about 0.001 to about 10 mg/day; (g) about 0.001 to about 6 mg/day; (h) about 0.001 to about 4 mg/day; or (i) about 0.001 to about 2 mg/day.

(0034] In some embodiments, the amount of insulin, an immunologically active insulin fragment, a derivative thereof, or a functional equivalent thereof can range from about 0.1 to about 2 units/kg/day. In some embodiments, the method of administration of the aminosterol comprising a spermine moiety, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof comprises oral administration and wherein the therapeutically effective amount comprises: (a) about 1 to about 300 mg/day; or (b) about 25 to about 500 mg/day.

[0035] In some embodiments, the method of administration of the aminosterol comprising a spermine moiety, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof comprises intranasal administration and wherein the therapeutically effective amount comprises 0.001 mg/day up to about 6 mg/day.

[0036] In some embodiments, administration of: (a) the aminosterol comprising a spermine moiety, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof, and/or (b) insulin, an immunologically active insulin fragment, a derivative thereof, or a functional equivalent thereof, comprises administration of (a) and/or (b) on an empty stomach, optionally within two hours of the subject waking.

[0037] In some embodiments, no food is consumed by the subject after about 60 to about 90 minutes from administration of the aminosterol comprising a spermine moiety, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof.

]0038[ In some embodiments, the aminosterol has the formula:

Compound III (ENT-03), or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof.

[0039] In some embodiments, the aminosterol has the formula:

C25 (7?) Compound III (ENT-03), or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof.

[0040] In some embodiments, the aminosterol has the formula of Compound III-N:

C25 (S) ENT-03 (Compound III-S), or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof.

[0041] In some embodiments, the aminosterol has the formula:

Compound III-d4 (ENT-03 -d4), or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof. In some embodiments, the C25 configuration is R. In some embodiments, the C25 configuration is S.

[0042] In some embodiments, the aminosterol has the formula:

Compound III (ENT-03-d3), or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof. In some embodiments, the C25 configuration is R. In some embodiments, the C25 configuration is S.

10043] In some embodiments, the aminosterol has the formula:

Compound IV (A5 ENT-03), or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof. In some embodiments, the C25 configuration is R. In some embodiments, the C25 configuration is S.

[0044] In some embodiments, the aminosterol has the formula:

C25 (R) Compound IV (A5 ENT-03), or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof.

[0045] In some embodiments, the aminosterol has the formula:

Compound V (A4 ENT-03), or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof. In some embodiments, the C25 configuration is R. In some embodiments, the C25 configuration is S.

[0046] In some embodiments, the aminosterol has the formula:

C25 (R) Compound V (A4 ENT-03), or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof.

[0047] In another aspect of the disclosure, the aminosterol is selected from the group consisting of the following, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof:

C25 (R) Compound V,

Compound D,

Compound H.

[0048] In some embodiments, the insulin, an immunologically active insulin fragment, an insulin derivative, or a functional equivalent of insulin is selected from the group consisting of: insulin, inhaled insulin, synthetic insulin, human insulin, synthetic human insulin, porcine insulin, bovine insulin, shark insulin, rapid acting insulin analogs, long acting insulin analogs, intermediate-acting insulin analogs, mixed insulin, mixed insulin analogs, aspart insulin, glulisine insulin, lispro (lyspro) insulin, detemir insulin, glargine insulin, degludec insulin, NPH (Neutral Protamine Hagedorn) insulin, NPA insulin, insulin aspart protamine, and any combination thereof.

[0049] Both the foregoing summary and the following description of the drawings and detailed description are exemplary and explanatory. They are intended to provide further details of the disclosure, but are not to be construed as limiting. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following detailed description of the disclosure.

DESCRIPTION OF THE FIGURES

[0050] Figure 1 shows the accumulation of aminosterol 1436 within the centers of the brain that control growth, maturation, and senescence following intravenous administration to a rat via a peripheral vein, or injected directly into the 3 ^rd ventricle of the brain.

[0051] Figure 2 shows weight (g) (y axis) vs age (days) (x axis) for three groups of mice administered 10 mg/kg or 5 mg/kg aminosterol 1436 (MSI-1436), and a control group. While all animals reached the mature weight of about 40 grams, the control animals reached maturity at 120 days, and the animals that received 5 and 10 mg/kg of aminosterol 1436 reached maturity at 150 and 255 days, respectively.

[0052] Figure 3 shows a graph of weight (g) (y axis) vs time for animals given either vehicle or 10 mg/kg (/./?.) of aminosterol 1436 every 3 days for two doses, for a total of 20 mg/kg over a 6 day period. Animals were then weighed and body length measured once weekly for a period of 40 days. At Day 0 animals in the control group had a starting weight (g) of 16 g, while animals in the aminosterol 1436 group had a weight of 12 g. At day 40, the control group had a weight of 24 g, or an increase of 50%. In contrast, at Day 40 the aminosterol 1436 group had a weight of 11 g, or a decrease of 8.3%.

(0053] Figure 4 shows a crystal structure of compound R-(24).

[0054] Figures 5A and 5B show percent weight loss over time in mice administered ENT-02 (MSI-1436) (circles) or ENT-03 (Compound III; squares). Fig. 5C shows intraperitoneal administration of ENT-03 to C57bl/6 male mice once weekly over 6 weeks caused a dose dependent weight loss.

[0055] Figure 6 shows the results of administration of ENT-03 (Compound III) and ENT-02 (MSI-1436) on growing mice. While both compounds affected weight gain, ENT-02 had a more profound effect, having suppressed growth as well as having induced consumption of body fat. In contrast, the animals treated with ENT-03 continued normal growth, although they “slimmed down”, suggesting that ENT-03 re-established a new optimal body weight “set point.”

[0056] Figure 7 shows images of a mucosal layer of the stomach showing a reduced mucosal layer in the 78 week old stomach (Fig. 7B) vs. the younger 20 week old stomach (Fig. 7A).

[0057] Figure 8 shows ICso curves for PTP1B inhibition by three aminosterols tested ENT-02 (MSI-1436), ENT-03 (Compound III), and D-1436; Fig. 8A) and a control PTP1B inhibitor (Fig. 8B) according to Example 1.

[0058] Figures 9 A-H show that PTP1B inhibition by ENT-03 ameliorates the cognitive impairment of hAPP-J20 (Figs. 9A-9D) and PS 19 (Figs. 9E-9H) mice.

[0059] Figures 10A-10C show volcano plots showing significance (as negative logio- transformed FDR-adjusted p-values) against the magnitude (log2-transformed fold change) of differentially expressed genes in the following three contrasts: (Fig. 10A) aged mice compared to young mice, (Fig. 10C) ENT-03 -treated aged mice compared to vehicle-treated aged mice, (Fig. 10B) ENT-03 -treated young mice compared to vehicle-treated young mice. Genes identified as having different levels between groups are represented as red (up-regulated) or blue (down-regulated) dots, and non-significant genes are represented as black dots. The horizontal red lines represent the applied p-value threshold.

[0060] Figures 11 A and 1 IB show chromatograms for the LC/MS/MS analysis of brain extract from elderly humans for ENT-03. Fig. 11 A is the chromatogram for the brain extract and Fig.

1 IB is a quality control sample of synthetic ENT-03.

[0061] Figures 12A and 12B show chromatograms for the LC/MS/MS analysis of mouse pup brain extract (Fig. 12A) and liver extract (Fig. 12B).

[0062] Figure 13 shows approximate concentrations of ENT-03 measured in the brain and liver of neonatal mice over the first 3 weeks of life.

[0063] Figure 14 shows gene expression profiles of the jejunum (Figs. 14A-14D) or ileum (Figs. 14D-14F) of young and aged mice treated with ENT-03 or control.

[0064] Figure 15 shows a set of heat maps investigating the overlap of differentially expressed genes between pairs of contrasts. The values in the plot represent the number of intersecting differentially expressed genes (adjusted p-value < 0.05) between specific pairs of contrasts. The colors of the squares represent the Jaccard index (a quotient of the intersection and the union of genes) for the contrasts on the x- and y-axis: (Fig. 15 A) differentially expressed genes in both directions, (Fig. 15B) up-regulated genes, (Fig. 15C) down-regulated genes, and (Fig.

15D) genes up-regulated in one contrast and down-regulated in the other contrast. Note that the numbers in grey squares represent the total number of selected differentially expressed genes of a specific contrast.

[0065] Figure 16 shows the results on mouse weight gain following administration of ENT-03 as compared to administration of a deuterated form of ENT-03, ENT-03D3.

[0066] Figures 17A and 17B show representative chromatograms of ENT-03 in 4 day old mouse brain extracts. Figure 17A: MRM 619.6/545.5, upper tracing: endogenous ENT-03 in extract; MRM 623.6/549.5 lower tracing: extract + 2.2 ng ENT-03-t/v/gram brain tissue; Figure 17B: MRM 619.6/474.5, upper tracing: endogenous ENT-03 in extract; MRM 623.6/478.5 lower tracing: extract +2.2 ng ENT-03 -t/v/gram brain tissue.

[0067] Figure 18 shows a plot representing the weight loss results of male C57bl6/j (N=5/group) mice administered ENT-03 at varying dosages or vehicle by oral gavage every 3 ^rd day.

[0068] Figure 19 shows a Venn diagram of transcripts down-regulated in ageing and up- regulated by ENT-03. A plot showing the numbers of overlapping and non-overlapping differentially expressed genes between the two sets of transcripts that were down-regulated in old versus young mice and up-regulated upon treatment compared to control. Numbers of features are shown from treatment with ENT-02 (MSI-1436) and ENT-03.

[0069] Figure 20 shows a scatter plot comparing significant genes in ENT-02 (MSI-1436) vs control (young) against ENT-03 vs untreated (young). Genes are represented by points. The color of the point indicates which set the gene is assigned to. For each gene the log2(fold change) in the ENT-02 (MSI-1436) vs control (young) contrast (y-axis) and the log2(fold change) in the ENT-03 vs untreated (young) contrast (x-axis) are shown.

[0070] Figure 21 shows an upset plot of significant genes according to Example 12. A plot showing the interaction between sets of up- and down-regulated genes. The leftmost bar chart shows the size of each set used as input. The top bar chart shows the exclusive size of each set (z.e., each gene is only counted once in this bar chart). The dot-plot in the centre shows the sets interacting in each case.

[0071] Figures 22A-18E: Fig. 22A: A Venn diagram of overlapping genes in MSI-1436 (aminosterol 1436) vs control (young) against ENT-03 vs untreated (young) — all vs all Fig. 22B shows a Venn diagram of overlapping genes in MSI-1436 vs control (young) against ENT- 03 vs untreated (young) — up vs up. Fig. 22C shows a Venn diagram of overlapping genes in MSI-1436 vs control (young) against ENT-03 vs untreated (young) — down vs down. Fig. 22D shows a Venn diagram of overlapping genes in MSI-1436 vs control (young) against ENT-03 vs untreated (young) — up vs down. Fig. 22E shows a Venn diagram of overlapping genes in MSI- 1436 vs control (young) against ENT-03 vs untreated (young) — down vs up.

[0072] Figure 23 shows a scatter plot comparing significant genes in MSI-1436 ( aminosterol 1436) vs control (old) against ENT-03 vs untreated (old). Genes are represented by points. The colour of the point indicates which set the gene is assigned to. For each gene the log2(fold change) in the MSI-1436 vs control (old) contrast (y-axis) and the log2(fold change) in the ENT- 03 vs untreated (old) contrast (x-axis) are shown.

[0073] Figure 24 shows an Upset plot of significant genes according to Example 12. A plot showing the interaction between sets of up- and down-regulated genes. The leftmost bar chart shows the size of each set used as input. The top bar chart shows the exclusive size of each set (z.e., each gene is only counted once in this bar chart). The dot-plot in the centre shows the sets interacting in each case.

[0074] Figures 25A-25E: Fig. 25A shows a Venn diagram of overlapping genes in ENT-02 (MSI-1436) vs control (old) against ENT-03 vs untreated (old) — all vs all. Fig. 25B shows a Venn diagram of overlapping genes in ENT-02 (MSI-1436) vs control (old) against ENT-03 vs untreated (old) — up vs up. Fig. 25C shows a Venn diagram of overlapping genes in ENT-02 (MSI-1436) vs control (old) against ENT-03 vs untreated (old) — down vs down. Fig. 25D shows a Venn diagram of overlapping genes in ENT-02 (MSI-1436) vs control (old) against ENT-03 vs untreated (old) — up vs down. Fig. 25E shows a Venn diagram of overlapping genes in ENT-02 (MSI-1436) vs control (old) against ENT-03 vs untreated (old) — down vs up.

10075] Figure 26 shows a scatter plot comparing significant genes in Old vs young (control) against Old vs young (untreated). Genes are represented by points. The colour of the point indicates which set the gene is assigned to. For each gene the log2(fold change) in the Old vs young (control) contrast (y-axis) and the log2(fold change) in the Old vs young (untreated) contrast (x-axis) are shown.

[0076] Figure 27 shows an Upset plot of significant genes according to Example 12. A plot showing the interaction between sets of up- and down-regulated genes. The leftmost bar chart shows the size of each set used as input. The top bar chart shows the exclusive size of each set (z.e., each gene is only counted once in this bar chart). The dot-plot in the centre shows the sets interacting in each case.

[0077] Figures 28A-24E: Fig. 28A shows a Venn diagram of overlapping genes in Old vs young (control) against Old vs young (untreated) — all vs all. Fig. 28B shows a Venn diagram of overlapping genes in Old vs young (control) against Old vs young (untreated) — up vs up. Fig. 28C shows a Venn diagram of overlapping genes in Old vs young (control) against Old vs young (untreated) — down vs down. Fig. 28D shows a Venn diagram of overlapping genes in Old vs young (control) against Old vs young (untreated) — up vs down. Fig. 28E shows a Venn diagram of overlapping genes in Old vs young (control) against Old vs young (untreated) — down vs up.

]0078J Figure 29 shows a scatter plot comparing significant genes in Old vs young (ENT-02; MSI-1436) against Old vs young (ENT-03). Genes are represented by points. The colour of the point indicates which set the gene is assigned to. For each gene the log2(fold change) in the Old vs young (ENT-02; MSI- 1436) contrast (y-axis) and the log2(fold change) in the Old vs young (ENT-03) contrast (x-axis) are shown.

]0079[ Figure 30 shows an Upset plot of significant genes according to Example 12. A plot showing the interaction between sets of up- and down-regulated genes. The leftmost bar chart shows the size of each set used as input. The top bar chart shows the exclusive size of each set (z.e., each gene is only counted once in this bar chart). The dot-plot in the centre shows the sets interacting in each case. [0080] Figures 31 A-27E: Fig. 31 A shows a Venn diagram of overlapping genes in Old vs young (MSI- 1436) against Old vs young (ENT-03) — all vs all. Fig. 3 IB shows a Venn diagram of overlapping genes in Old vs young (MSI- 1436) against Old vs young (ENT-03) — up vs up. Fig. 31C shows a Venn diagram of overlapping genes in Old vs young (MSI- 1436) against Old vs young (ENT-03) — down vs down. Fig. 3 ID shows a Venn diagram of overlapping genes in Old vs young (MSI- 1436) against Old vs young (ENT-03) — up vs down. Fig. 3 IE shows a Venn diagram of overlapping genes in Old vs young (MSI- 1436) against Old vs young (ENT-03) — down vs up.

[0081] Figure 32: Heat maps of overlaps between contrasts: A plot showing the number of overlapping selected genes between the contrasts performed. Note that the numbers on the diagonal represent the total number of selected genes found for each contrast. The colours of the squares represent the Jaccard index (the intersection over the union) for the contrasts on the x- axis with those on the y-axis. Fig. 32A: Heat map of overlaps of up- and down-regulated (y- axis) vs. up- and down-regulated (x-axis) selected genes for each contrast. Fig. 32B: Heat map of overlaps of up-regulated (y-axis) vs. up-regulated (x-axis) selected genes for each contrast. Fig. 32C: Heat map of overlaps of down-regulated (y-axis) vs. down-regulated (x-axis) selected genes for each contrast. Fig. 32D: Heat map of overlaps of up-regulated (y-axis) vs. down- regulated (x-axis) selected genes for each contrast.

[0082] Figure 33 shows HPLC overlay of ENT-037?, ENT-03S, and ENT-03 (Injection volume: 1 pL).

[0083] Figure 34 shows a picture depicting the elimination of neuroinflammation following intranasal administration of ENT-03 to PS 19 mice, which are engineered to produce large amounts of tau-protein and develop a very aggressive form of neurodegeneration/ Alzheimer’s disease.

[0084] Figure 35A and 35B show improved performance by the PS19 mice following ENT-03 treatment in a Morris water maze, which is a test of spatial learning for rodents that relies on distal cues to navigate from start locations around the perimeter of an open swimming arena to locate a submerged escape platform. Spatial learning is assessed across repeated trials and reference memory is determined by preference for the platform area when the platform is absent. Trial-dependent, latent and discrimination learning can be assessed using modifications of the basic protocol. Fig. 35A shows the number of training days on the X axis vs escape latency in seconds (e.g., time it takes to find the platform) for four groups of tested animals: wild type (WT) vehicle, WT ENT-03, PS19 vehicle, and PS19 ENT-03. Escape latency significantly declined for the PS19 ENT-03 group as compared to the PS19 untreated group. Fig. 35B shows platform area (Y axis) vs each of the four groups of animals: WT vehicle, WT ENT-03, PS19 vehicle, and PS 19 ENT-03.

[0085] Figure 36 shows localization to the arcuate nucleus, proximity to NPY producing cells, and action via pstat3 stimulation in the arcuate and subventricular zone (which is the neurogenic zone). The four groups of tested animals are shown on the X axis, e.g., WT vehicle, WT ENT- 03, PS19 vehicle, and PS19 ENT-03. Measurement of P-STAT3, NPY, P-STAT3/NPY, and P- STAT3/NPY/DAPI is shown on the X axis.

[0086] Figure 37 shows a graph of ENT-03 concentration in brain of mice vs age, with the X axis showing days after birth and Y axis showing the quantity of ENT-03. As body weight increases (open triangle), the amount of brain ENT-03 sharply decreases.

[0087] Figure 38 shows a graph of time (days) vs body weight for ENT-03 treated animals and vehicle-treated animals. ENT-03 treated animals exhibited significantly slower weight gain, and overall less weight gain, indicating an ENT-03 impact on metabolicy function.

[0088] Figure 39 shows the results of a glucose tolerance test (GTT), which measures normalized blood glucose (%) vs minutes for WT vehicle treated animals (6) and WT ENT-03 treated animals (8). The figure shows that ENT-03 treated animlas has a much quicker attainment of normalized blood glucose.

DETAILED DESCRIPTION OF THE INVENTION

I. Overview of the Invention

[0089] This invention relates to methods of treating and/or preventing neurodegeneration to a subject in need. The neurodegeneration may be age-related, and/or may be correlated with a condition such as age-related dementia, Alzheimer’s disease, Parkinson’s disease, Lewy Body dementia, frontotemporal dementia, vascular dementia, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), multiple system atrophy (MSA), progressive supranuclear palsy (PSP)), olivo-ponto-cerebellar degeneration, or age related cognitive decline without a specific diagnosis from the group above. The method comprises administering a therapeutically effective amount of a combination of: (a) an aminosterol comprising a spermine moiety, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof, and (b) insulin, an immunologically active insulin fragment, an insulin derivative, or a functional equivalent of insulin, or a combination thereof.

[0090] The present invention is also directed to methods of delaying maturation, retarding the aging process, and/or increasing the potential lifespan of subject, which can be an animal or human. The invention is also directed to methods of preventing, treating, and/or delaying onset of age-related diseases or conditions in a subject. The methods comprise administering to the subject an aminosterol, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof, and optionally insulin, an immunologically active insulin fragment, an insulin derivative, or a functional equivalent of insulin.

[00911 Aminosterols such as ENT-03, particularly when combined with insulin or an insulin analog, are expected to be useful in treating neurodegenerative diseases such as for example Alzheimer’s disease and Parkinson’s disease, as well as other neurodegenerative diseases described herein. 85% of AD patients have insulin resistance or Type 2 diabetes, and insulin is known to be important in nerve cell and blood vessel health. Further, neuroinflammation (amyloid and tau) is secondary to the metabolic abnormalities associated with neurodegenerative diseases. ENT-03 is expected to be useful in treating neurodegenerative diseases, particularly when combined with insulin or an insulin analog, as aminosterols such as ENT-03 (i) are a centrally active inhibitor of phosphatases, including PTP1B, (ii) reverse insulin resistance in the brain, (iii) promote dose dependent weight loss, (iv) eliminate inflammation in the brain of AD models, and (v) reverse memory loss and normalize behavior in animal models of AD and increase lifespan by more than 50%. See Figs. 34 and 35 and the corresponding examples. Fig. 34 shows elimination of hypothalamic elimination, and elimination of hippocampal inflammation has also been demonstrated (data not shown). Fig. 36 shows localization to the arcuate nucleus, proximity to NPY producing cells, and action via pstat3 stimulation in the arcuate and subventricular zone (which is the neurogenic zone). See also Figs. 37, 38, and 39.

[0092] In terms of treating metabolic diseases, such as diabetes, aminosterols such as ENT-03 act on hypothalamic centers involved in energy, reduce body weight, and normalize glycemia. In particular, Figure 39 shows the results of a glucose tolerance test (GTT), which measures normalized blood glucose (%) vs minutes for WT vehicle treated animals (6) and WT ENT-03 treated animals (8). The figure shows that ENT-03 treated animlas has a much quicker attainment of normalized blood glucose. In addition, Figure 38 shows a graph of time (days) vs body weight for ENT-03 treated animals and vehicle-treated animals. ENT-03 treated animals exhibited significantly slower weight gain, and overall less weight gain, indicating an ENT-03 impact on metabolicy function.

A. Summary of Experimental Results i. ENT-03 and insulin for the treatment of Alzheimer’s disease

[0093] The present invention is based on the discovery of the unexpected and unprecedented activity of a combination of an aminosterol, including but not limited to Compound III (ENT- 03), and insulin in effectively treating mice induced to form high levels of tau protein and develop Alzheimer’s disease. See Example 1. It was observed that the diseased mice treated with this regimen experienced increase in life-span relative to untreated mice, and improvements in mobility and exploratory behavior which rendered the treated mice indistinguishable from healthy mice in these behaviors.

[0094] More particularly, ENT-03 treated PS19 mice showed the elimination of neuroinflammation following intranasal administration of ENT-03. This is highly surprising, as PS 19 mice are engineered to produce large amounts of tau-protein and develop a very aggressive form of neurodegeneration/ Alzheimer’s disease. The elimination of neuroinflammation was correlated with an improvement in spatial learning for the treated mice. This is shown in Figs. 35 A and 35B, which show the results for wild type and PS 19 mice following a Morris water maze test, which is a test of spatial learning for rodents that relies on distal cues to navigate from start locations around the perimeter of an open swimming arena to locate a submerged escape platform. The time it took to find the platform significantly declined for the PS19 ENT-03 group as compared to the PS 19 untreated group.

[0095] The exact mechanism by which an aminosterol such as Compound III (ENT-03) promotes effects against a neurodegenerative disease such as AD is not yet known. However, without being bound by theory, it is theorized that the effect may be similar to that of aminosterol 1436 ’which has effects on the hypothalamus within the brain of the animal. As seen in Fig. 1, when radioactive aminosterol 1436 is administered to a rat intravenously via a peripheral vein (IV), or injected directly into the 3 ^rd ventricle of the brain (ICV), the compound accumulates within the centers of the brain that control growth, maturation and senescence.

10096] The hypothalamus is a portion of the brain that contains a number of small nuclei with a variety of functions. One of the most important functions of the hypothalamus is to link the nervous system to the endocrine system via the pituitary gland. The hypothalamus is responsible for the regulation of certain metabolic processes and other activities of the autonomic nervous system.

[0097] Example 9 also details data supporting the use of ENT-03 (Compound III) in treating neurological diseases such as Alzheimer’s disease. PTP1B dependent mechanisms have been utilized for reversal of memory impairment and normalization of behavior and reduction in neuronal loss in beta amyloid and tau mouse models of Alzheimer’s disease (Rieke, Cruz et al. 2020). Other studies have shown reduction in the toxicity of beta amyloid aggregates by ENT- 02 in vitro and in a C. elegans model of Alzheimer’s disease (Limbocker, Chia et al. 2019).

[0098] ENT-02 (MSI- 1436) reverses several conditions (in mice) that are associated with ageing, such as metabolic syndrome, Alzheimer’s disease, atherosclerosis, cancer and a reduced capacity for regenerative repair. The data in Example 6 demonstrates that ENT-03 can treat Alzheimer’s disease in murine models, which are acceptable animal models for human Alzheimer’s disease.

[0099] The Morris water maze was used to test the effect of ENT-03 (Compound III) on spatial learning and memory deficits in 2 mouse models of familial Alzheimer’s disease, hAPP-J20 mice that express a double mutant of the human amyloid precursor protein (Mucke et al., 2000), and PS19 mice that express the P301S mutant of the human microtubule associated protein tau (Yoshiyama et al., 2007). In both disease models, one with amyloidopathy and the other with a tauopathy, learning during the training phase (Figs. 9A and 9E) and memory on the probe day (Figs. 9B, 9C, 9F and 9G) were improved with ENT-03 treatment as compared to vehicle treated hAPP-J20 or PS 19 littermate control mice (Fig. 9). The effect on preventing cognitive decline is similar to what has been observed with the related compound ENT-02 and hAPP-J20 and PS 19 mice (Rieke et al., 2020). ii. ENT-03 and longevity [0100] The disclosed methods can be used to treat a range of subjects, including human and non-human animals, including mammals, as well as immature and mature animals, including human children and adults. Examples of livestock that can be treated with the methods of the invention include but are not limited to goats, sheep, horses, rabbits, cattle, chickens and other poultry, pigs, camel, alpaca, llama, etc. Examples of zoo animals that can be treated with the methods of the invention include but are not limited to elephants, lions, tigers, giraffes, etc. Examples of pets that can be treated with methods of the invention include but are not limited to dogs, cats, pigs, ferrets, rabbits, rodents (e.g., gerbils, hamsters, chinchillas, rats, and guinea pigs), and avian pets (e.g., parrots, passerines, and fowl).

[0101 ] Methods of slowing maturation and aging, and extending potential lifespan, are useful for example, in animal husbandry, to extend the potential lifespan of, for example, livestock animals such as horses, cattle, sheep, pigs and goats. The methods of the invention can also be used to slow the growth of common pets such as dogs or cats, maintaining them in a smaller, younger state for a longer period of time than would normally occur. In addition, methods of delaying aging could result in extending the potential lifespan of an animal such as a pet.

[0102] The methods of the invention can be administered to animals or humans either prior to maturity or after maturity. The methods of the invention administered to a subject prior to maturity can result in slowed growth, slowed maturation, as well as other results described herein. The methods of the invention administered to a subject after maturity can result in (1) delayed aging; (2) treating, preventing, or delaying onset of age-related diseases and/or conditions; and (3) extending potential lifespan, as well as other results described herein.

[0103] In another embodiment, the invention can be used to slow the maturation and aging process in subjects, and additionally extend potential lifespan. In one embodiment, a subject can be treated with a pharmaceutical composition comprising a therapeutically effective amount of an aminosterol (i.e., ENT-03) or a derivative or salt thereof prior to maturity and would thereby grow more slowly than an untreated subject, potentially resulting in an extended lifespan, barring unforeseen consequences such as infection, accidents, or organic disease. The method can also comprise administering a combination of at least one aminosterol and an insulin compound described herein.

[0104] The invention can also be used to slow the aging process. The subject would be treated at maturity and beyond with a pharmaceutical composition comprising a therapeutically effective amount of an aminosterol (i.e., ENT-03) or a derivative or salt thereof, which can result in a slowed aging process for the subject as compared to an untreated subject, resulting in the treated subject remaining more youthful for a longer period of time. Characteristics of aging that may be impacted by the methods of the invention are described herein. The method can also comprise administering a combination of at least one aminosterol and an insulin compound described herein.

|0105| In another embodiment, encompassed are methods of treating, preventing, or delaying the onset of age-related diseases, conditions, or health problems. The method comprises administering to a subject, such as an animal or human, a pharmaceutical composition comprising a therapeutically effective amount of an aminosterol (i.e., ENT-03) or a derivative or salt thereof for a desirable period of time. The age-related disease, condition, or health problem can be, for example, selected from the group consisting of atherosclerosis and cardiovascular disease, cancer, arthritis, cataracts, osteoporosis, diabetes, hypertension, Alzheimer’s disease, arthritis, and osteoporosis. The method can also comprise administering a combination of at least one aminosterol and an insulin compound described herein.

II. Aminosterols

[0106] Recently, a human derived derivative of aminosterol 1436, Compound III (ENT-03 or Hu-1436) was discovered, as shown below. See PCT/US2020/044390, filed July 31, 2020, the entire disclosure of which is hereby incorporated by reference in its entirety.

Compound III (ENT-03).

[0107] As described in PCT/US2020/044390, it was discovered that Compound III is found in subdural hematoma fluid in humans and in mouse pup brains. Compound III-7? (Compound III having a C25 stereo center of configuration R) may synthesized in the brain through the condensation of spermine and Compound I, as shown below.

Compound III -R

[0108] Compound I is believed to arise from the metabolism of 27-hydroxycholesterol (also called (25R)26-dihydroxycholesterol), a biologically active oxysterol released into the circulation from many tissues, including vascular endothelium and macrophages (Griffiths et al., 2019; Bjorkhem et al., 2002; Javitt et al., 2002). Within the brain, 27-hydroxycholesterol undergoes successive metabolism by CYP27A1, CYP7B1, and HSD-3B7, possibly in that sequence (Meaney et al., 2007). The synthesis of Compound III-7? would require three additional biosynthetic steps: the reduction of the double bond in the cholesterol A ring to create the 5 alpha hydrogen; the condensation of spermine with the 3 -oxo group to form the imine; and the subsequent reduction of the imine. The first step is likely catalyzed by a brain steroid 5 alpha-reductase. The missing biosynthetic link is the enzyme that couples the spermine to the bile acid.

[0109] ENT-03 (Compound III) is the preferred compound, although any derivative or salt thereof that improves the pharmacological characteristics of Compound III can be used in the methods of the invention. A derivative of Compound III may have one or more chemical modifications which do not modify, or drastically diminish, or may improve, the activity of Compound III. Such “activity” may include pharmacological targets and affinity therefore, including changes in affinities for different subtypes of a particular receptor target. A “derivative” of an aminosterol or Compound III in which modifications well known in the art of medicinal chemistry to “mimic” the original spatial and charge characteristics of a portion of the original structure can be introduced to improve the therapeutic characteristics of the aminosterol. In general, such modifications are introduced to influence metabolism, ease of administration, biodistribution, or any combination thereof. Examples of such variants or derivatives include, but are not limited to, (1) substitutions of the sulfate or carboxylic acid by a sulfonate, sulfate, phosphate, carboxylate, or other anionic moiety chosen to circumvent metabolic removal of the sulfate moiety and oxidation of the cholesterol side chain; (2) replacement of a hydroxyl group by a non-metabolizable polar substituent, such as a fluorine atom, to prevent its metabolic oxidation or conjugation; and (3) substitution of various ring hydrogen atoms to prevent oxidative or reductive metabolism of the steroid ring system. Other derivatives include replacement of one or more hydrogens of the aminosterol with deuterium or the unsaturation of any one or more C-C single bonds of the aminosterol. The pharmaceutical composition can comprise one or more pharmaceutically acceptable carriers or excipients.

[0110] In some embodiments, the methods of the invention can employ a formulation of Compound III as an insoluble salt of phosphate, polyphosphate, hydrochloride or an organic phosphate ester. Compound III is shown below:

(Compound III, ENT-03)

[0111] In some embodiments, the aminosterol has the formula: wherein: R ¹ is H or D; and

R ² is H or D; provided that all of R ¹ are H, all of R ² are H, or all of R ¹ and R ² are H, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof.

[0112] In some embodiments, the aminosterol has the formula:

Compound III (ENT-03), or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof.

101131 In some embodiments, the aminosterol has the formula:

C25 (R) Compound III-7? (ENT-03), or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof.

[0114] In some embodiments, the aminosterol has the formula of Compound III-N:

C25 (S) ENT-03 (Compound III-S), or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof.

[0115] In some embodiments, the aminosterol has the formula:

Compound III (ENT-03 -d4), or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof.

[0116] In some embodiments, the aminosterol has the formula:

Compound III (ENT-03-d3), or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof.

101171 In some embodiments, the aminosterol has the formula:

Compound IV (A5 ENT-03), or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof.

[0118] In some embodiments, the aminosterol has the formula:

C ₂₅ (R) Compound IV (A5 ENT-03), or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof.

[0119] In some embodiments, the aminosterol has the formula:

Compound V (A4 ENT-03), or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof.

[0120] In some embodiments, the aminosterol has the formula:

C25 (R) Compound V (A4 ENT-03), or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof.

]0121[ Aminosterol 1436 or its derivatives or salts thereof can be administered via any pharmaceutically acceptable method. For example, the pharmaceutical composition in the methods of the invention can be administered intravenously, intradermally, subcutaneously, orally, rectally, sublingually, intrathecally, intranasally, or by inhalation. Pharmaceutical compositions appropriate for each of the specific routes are utilized.

(0122] Other exemplary aminosterols include but are not limited to: Compound B, Compound F,

Compound H, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof.f

III. Insulin

[0123] Insulin is a peptide hormone produced in the pancreas by the islets of Langerhans, which regulates the amount of glucose in the blood. Insulin is produced in the pancreas and the Brockmann body (in some fish), and released when any of several stimuli are detected. Insulin is produced commercially for use in medicine using recombinant technology. Human insulin is mainly produced either in E. coli or Saccharomyces cerevisiae and purified for medicinal use. (Baeshen et al. 2014). Insulin functions to maintain normal blood glucose levels by facilitating cellular glucose uptake, regulating carbohydrate, lipid and protein metabolism and promoting cell division and growth through its mitogenic effects. Insulin is an anabolic hormone that promotes glucose uptake, glycogenesis, lipogenesis, and protein synthesis of skeletal muscle and fat tissue through the tyrosine kinase receptor pathway. The most important hormone that the pancreas produces is insulin. Insulin is released by the 'beta cells' in the islets of Langerhans in response to food. Its role is to lower glucose levels in the bloodstream and promote the storage of glucose in fat, muscle, liver and other body tissues. The function of insulin is to promote the uptake of glucose by muscle cells that use it for energy and by fat cells that store it as triglycerides, or fats, and by liver cells. It does this by upregulating GLUT4 in muscle, fat, and liver cells.

10124] Recently, it has been shown that neurodegenerative diseases such as AD can be regarded as a brain form of diabetes in which insulin resistance and deficiency develop either primarily in the brain, or due to systemic insulin resistance disease with secondary involvement of the brain, (de la Monte 2017). Moreover, insulin signaling and autophagy impairment are a common factor in both metabolic disease (e.g., diabetes) and neurodegenerative disorders, (de Mello et al. 2019.) Although the exact link between brain insulin and neurodegenerative diseases remains still unknown, a plethora of studies have demonstrated that an optimal insulin signaling homeostasis is important to the maintenance of brain health. Id.

[0125] Various forms of insulin including, an immunologically active insulin fragment, an insulin derivative, or a functional equivalent of insulin are within the scope of the present invention.

|0126| An insulin “fragment” may include a small part derived, cut off, or broken from a larger peptide, polypeptide or protein, which retains the desired biological activity of the larger peptide, polypeptide or protein, for example, any biological activity (i.e., immunologically) known in the art to belong to insulin. The terms “functional equivalent” or “functionally equivalent” to described different types of insulin are used interchangeably herein to refer to proteins, peptides, or polypeptides having similar or identical activity (i.e., immunologically) known in the art to belong to wild-type or native insulin. In some embodiments, the functional equivalent of insulin comprises an insulin-like growth factor (IGF). In some embodiments, the IGF comprises IGF-1, IGF-2, or a combination thereof. In some embodiments, the IGF comprises an IGF-binding protein (IGFBP), optionally bound thereto. In some embodiments, the IGF-binding protein comprises one or more of IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP6, and IGFBP7. Insulin-like growth factors (IGFs) are proteins with high sequence similarity to insulin. The similarity may comprise at least 80% sequence identity, at least 90% sequence identity, or at least 100% sequence identity to insulin.

10.127] “Derivatives” of insulin are used herein to refer to sequences with substantial identity to a reference insulin sequence (i.e., wild type human insulin). A skilled artisan can produce polypeptide variants having single or multiple amino acid substitutions, deletions, additions or replacements. These variants may include inter alia: (a) variants in which one or more amino acid residues are substituted with conservative or non-conservative amino acids; (b) variants in which one or more amino acids are added; (c) variants in which at least one amino acid includes a substituent group; (d) variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at conserved or non-conserved positions; and (d) variants in which a target protein is fused with another peptide or polypeptide such as a fusion partner, a protein tag or other chemical moiety, that may confer useful properties to the target protein, such as, for example, an epitope for an antibody. The techniques for obtaining such variants, including genetic (suppressions, deletions, mutations, etc.), chemical, and enzymatic techniques are known. In some embodiments, the insulin derivative comprises a commercially available or known insulin derivative, for example, lispro, prandial insulin, aspart, glulisine, novolin, velosulin, isophane (neutral protamine hagedorn), insulin glargine, detemir, degludec, humulin, novolog, or a combination thereof.

IV. Developmental Window

[0128] In one embodiment, the methods of the invention are administered to a subject, including a human, during a “developmental window” in the life of the subject. For example, administration of the method during a developmental window of the animal can result in reversible slowing of the growth rate and maturation process of the animal. The “developmental window” is from birth to the animal or human reaches maturity as evidenced by ceased growth. In mice, this window extends from weaning to just prior to maturity, e.g., about 4-5 months of age. Comparable windows for other animals would correspond to the periods of growth specific for those animals, as described below.

V. Treatment and/or Prevention of Neurodegeneration

[0129] As noted above, this invention relates to methods of treating and/or preventing neurodegeneration to a subject in need, including but not limited to age-related neurodegeneration. The neurodegeneration may also be correlated with age-related dementia, Alzheimer’s disease, Parkinson’s disease, Lewy Body dementia, frontotemporal dementia, vascular dementia, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), multiple system atrophy (MSA), progressive supranuclear palsy (PSP)), olivo-ponto-cerebellar degeneration, or age related cognitive decline without a specific diagnosis from the group above. The method comprises comprising administering a pharmaceutical composition comprising a therapeutically effective amount of (a) an aminosterol comprising a spermine moiety, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof, and (b) insulin, an immunologically active insulin fragment, an insulin derivative, or a functional equivalent of insulin, or a combination thereof, to the subject. The subject can be an animal or human.

[0130] In an exemplary method, the aminosterol is administered intranasally, although any pharmaceutically acceptable delivery method can be used, as detailed herein, including but not limited to oral administration. In addition, the insulin compound can be administered using any pharmaceutically acceptable method.

[01 1 ] Research to date regarding neurodegenerative diseases has resulted in only modest success. For AD, PD, and ALS, researchers have looked at everything from misfolded proteins to infectious agents. As a result there are acetyl cholinesterase inhibitors that transiently improve cognition in the early stages of AD (Bond et al., “The effectiveness and cost-effectiveness of donepezil, galantamine, rivastigmine and memantine for the treatment of Alzheimer’s disease (review of Technology Appraisal No. I l l): a systematic review and economic model,” Health Technol. Assess., 76: 1-470 (2012)), dopamine modifying drugs for the temporary amelioration of motor symptoms in the early stages of PD (Muller, T., “Drug therapy in patients with Parkinson’s disease,” Transl. Neurodegener ., 7: 10 (2012)), and an NMDA antagonist which prolongs life for around 3 months in ALS (Gibson and Bromberg, “Amyotrophic lateral sclerosis: drug therapy from the bench to the bedside,” Semin. Neurol., 32: 173-178 (2012)). However, none of these treatments alters the course of these age-related diseases. They remain incurable.

[0132] Examples of neurodegenerative diseases that can be treated with methods of the invention include but are not limited to Parkinson’s disease (PD), Alzheimer’s disease (AD), Lewy body dementia (LBD), Multiple sclerosis (MS), multiple system atrophy (MSA), Progressive supranuclear palsy, Olivo-ponto-cerebellar degeneration, Amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD), frontotemporal dementia (FTD), vascular dementia, Friedreich’s ataxia and/or a related symptom in a subject in need is provided, comprising administering to the subject a therapeutically effective amount of a combination of: (a) an aminosterol comprising a spermine moiety, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof, and (b) insulin, an immunologically active insulin fragment, an insulin derivative, or a functional equivalent of insulin, or a combination thereof.

[0133] In another embodiment, administration of an effective amount of a combination of: (a) an aminosterol comprising a spermine moiety, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof, and (b) insulin, an immunologically active insulin fragment, an insulin derivative, or a functional equivalent of insulin, or a combination thereof, to a neurodegenerative disease patient results in improvement of one or more symptoms of the neurodegenerative disease or on one or more clinically accepted scoring metrics, by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. The improvement can be measured using any clinically recognized tool or assessment.

[0134] The period of time over which the progression or onset of a neurodegenerative disorder discussed above is measured can be for example, one or more months or one or more years, e.g., about 6 months, about 1 year, about 18 months, about 2 years, about 36 months, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 years, or any amount of months or years in between the values of about 6 months to about 20 years or more.

[0135] In another embodiment of the invention, a neurodegenerative disorder may be positively impacted by administering a pharmaceutical composition comprising a therapeutically effective amount of (a) an aminosterol comprising a spermine moiety, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof, and (b) insulin, an immunologically active insulin fragment, an insulin derivative, or a functional equivalent of insulin, or a combination thereof. A “positive impact” includes for example slowing advancement of the condition, improving symptoms, etc.

VI. Treatment of Age-Related Health Conditions/Diseases

[0136] In another embodiment of the invention, encompassed are methods of treating, preventing, or delaying the onset of age-related diseases or health conditions. The method comprising administering to a subject, which can be an animal or human, a pharmaceutical composition comprising a therapeutically effective amount of an aminosterol (i.e., ENT-03 or another aminosterol disclosed herein) or a derivative or salt thereof.

10137] Examples of age-related diseases that can be treated, prevented, or onset can be delayed include, but are not limited to, atherosclerosis and cardiovascular disease, cancer, arthritis, cataracts, osteoporosis, type 2 diabetes, and hypertension. The incidence of all of these diseases increases rapidly with aging (increases exponentially with age, in the case of cancer). Of the roughly 150,000 people who die each day across the globe, about two thirds — 100,000 per day — die of age-related causes. In industrialized nations, the proportion is higher, reaching 90%.

|0138| By age 3 about 30% of rats have had cancer, whereas by age 85 about 30% of humans have had cancer. Humans, dogs and rabbits get Alzheimer’s disease, but rodents do not. Elderly rodents typically die of cancer or kidney disease, but not of cardiovascular disease. In humans, the relative incidence of cancer increases exponentially with age for most cancers, but levels off or may even decline by age 60-75 (although colon/rectal cancer continues to increase).

101.391 Age related health conditions include but are not limited to arthritis, heart disease, atherosclerosis, Cardiovascular disease (CVD), osteoporosis, cataracts, Hypertension (HTN or HT), and diabetes. These age-related health conditions may be positively impacted by administering a pharmaceutical composition comprising a therapeutically effective amount of an aminosterol disclosed herein, or a derivative or salt thereof. A “positive impact” includes for example slowing advancement of the condition, improving symptoms, etc.

[0140] In one embodiment of the invention, the progression or onset of the age related health condition is slowed or prevented over a defined time period as measured by a medically- recognized technique. For example, the progression or onset of the age related health condition can be slowed by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

VII. Measurement of impact on Growth/Maturation

[0141 ] In one embodiment the invention is directed to methods of delaying growth and/or maturation of a subject, which can be a human or animal, comprising administering a pharmaceutical composition comprising a therapeutically effective amount of an aminosterol (i.e., ENT-03 or another aminosterol disclosed herein) or a derivative or salt thereof to the subject. Optionally, the method can additionally comprise administering an insulin compound. Administration of the pharmaceutical composition comprising an aminosterol (i.e., ENT-03 or another aminosterol disclosed herein) or a derivative or salt thereof can be limited to a brief period during development of a subject, sufficient to slow the rate of growth. Alternatively, administration of the pharmaceutical composition comprising an aminosterol (i.e., ENT-03 or another aminosterol disclosed herein) can be as a maintenance protocol.

[0142] It is well established that otherwise healthy animals that grow slowly during early postnatal development live longer than animals that exhibit a rapid rate of early growth. For example, mice with various pituitary mutations that result in small body size, such as the Ames and Snell dwarf mice, live among the longest of any strain of mouse (Blagosklonny, 2013). Mice that grow slowly during the first 2-3 months of age outlive mice that grow more rapidly during that early period of life (Miller et al., 2002). Suppression of the growth hormone/IGF-1 hormonal axis is believed to be, in part, involved in the delay in early growth (Vanhooren and Libert, 2013) though how a delay in early growth translates into longevity is unknown.

[0143] By adjusting the aminosterol dosing regimen, the extent of the growth and/or maturation delay can be controlled, with a greater delay occurring with greater doses and longer duration of administration of aminosterol.

[0144] In particular, by the selection of the appropriate dosing regimen of the aminosterol, the growth rate of a subject, which can be an animal or human, can be slowed in a measured fashion from a few % to over 50% as compared to that of an untreated subject, which is the same sex and age. For example, a subject treated at an early age with a dose that reduced growth to 50% normal would take twice as long to reach the size of an untreated subject. In this example, when an untreated sibling reached maturity at 5 months of age, the treated sibling would resemble a 2.5 month old and not reach maturity until 10 months of age. It is theorized that the treated subject with slowed growth will simultaneously age more slowly than the untreated subject and is anticipated to live longer.

(0145] In other embodiments of the invention, administration of an aminosterol (i.e., ENT-03 or another aminosterol disclosed herein) or a derivative or salt thereof delays growth, as measured by height and/or weight, as compared to a subject who is not administered the same aminosterol or a derivative or salt thereof, which is the same sex and age. The delay can be for example about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. The delay in growth can be measured over any time period, and will vary depending upon the subject being treated. See e.g., Fig. 2.

[0146] Maturation can be measured in a variety of ways. In humans, maturation is simply the process of children growing and obtaining adult stature. Females tend to mature sooner than boys. Maturation as the process from early childhood, to adolescence and then to full adult stature. Childhood is generally regarded as the time until which one reaches adolescence. The start of adolescence begins with the onset of puberty where hormonal and physical changes begin to occur. Initially, rapid changes begin to occur with increases in height, weight, stature and the development of secondary sex characteristics (Lloyd, R. S., and Oliver, J. L. Strength and Conditioning for Young Athletes: Science and Application. Routledge, 2014. https://www.routledge.com/Strength-and-Conditioning-for-Youn g-Athletes-Science-and- application/Lloyd-Oliver/p/book/9780415694896). Human biologists usually apply the term “maturity” to level of maturity; that is, the extent to which an individual, or a group of individuals, has proceeded towards adulthood. Therefore, maturation is a particular type of development: development that proceeds to the same end point in all individuals. In this sense, measures relative to adult size for the same individual, for example, present stature as a percentage of actual or predicted adult stature, are measures of maturity. The measurement of skeletal maturity is based on the recognition of maturity indicators; these are visible changes or stages that occur during maturation. Thus, one measurable factor of maturity is carpal and metacarpal bone maturation, which can be readily measured using x-rays. Mohammed et al., The reliability of Fishman method of skeletal maturation for age estimation in children of South Indian population,” J. Nat. Set. Biol. Med., 5(2):297 -302 (2014); and Pichai et al., “A Comparison of Hand Wrist Bone Analysis with Two Different Cervical Vertebral Analysis in Measuring Skeletal Maturation,” J. Int. Oral Health, 6(5):36-4 l (2014).

|0147| In one embodiment of the invention, administration of an aminosterol (i.e., ENT-03 or another aminosterol disclosed herein) or a derivative or salt thereof delays maturation, as measured by skeletal maturation, as compared to a subject who is not administered an aminosterol (i.e., ENT-03 or another aminosterol disclosed herein) or a derivative or salt thereof, which is the same sex and age. The delay can be for example about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. The delay in maturation can be measured over any time period, and will vary depending upon the subject being treated. For example, a mouse having a typical maturation period of 40 days will have a delay in maturation upon administration of a method according to the invention over a typical period of about 150-300 days, depending upon the dose of an aminosterol (i.e., ENT-03 or another aminosterol disclosed herein) or a derivative or salt thereof administered.

|0148| Measurement of bone maturation: Bone age is the degree of maturation of a child’s bones. As a person grows from fetal life through childhood, puberty, and finishes growth as a young adult, the bones of the skeleton change in size and shape. These changes can be seen by x-ray. The “bone age” of a child is the average age at which children reach this stage of bone maturation. See “Bone age,” Wikipedia (https://en.wikipedia.org/wiki/Bone_age

|0149| At birth, only the metaphyses of the “long bones” are present. The long bones are those that grow primarily by elongation at an epiphysis at one end of the growing bone. The long bones include the femurs, tibias, and fibulas of the lower limb, the humeri, radii, and ulnas of the upper limb (arm + forearm), and the phalanges of the fingers and toes. As a child grows the epiphyses become calcified and appear on the x-rays, as do the carpal and tarsal bones of the hands and feet, separated on the x-rays by a layer of invisible cartilage where most of the growth is occurring. As sex steroid levels rise during puberty, bone maturation accelerates. As growth nears conclusion and attainment of adult height, bones begin to approach the size and shape of adult bones. The remaining cartilaginous portions of the epiphyses become thinner. As these cartilaginous zones become obliterated, the epiphyses are said to be “closed” and no further lengthening of the bones will occur.

VIII. Measurement of Impact on Aging

[0150] In another embodiment, the invention is directed to methods of retarding the aging process of a subject, which can be an animal or human, comprising administering a pharmaceutical composition comprising a therapeutically effective amount of an aminosterol (i.e., ENT-03 or another aminosterol disclosed herein) or a derivative or salt thereof to the subject. The composition can be administered either prior to or after maturity of the subject. Optionally, the method can also comprise administration of at least one insulin compound described herein.

|0151 [ Characteristics of aging that may be impacted by administration of an aminosterol (i.e., ENT-03 or another aminosterol disclosed herein) or a derivative or salt thereof include, but are not limited to, aspects of aging impacted by the hypothalamus. It has been theorized that the endocrine function of the hypothalamus essentially controls the aging process. T. Hicklin (2017). The hypothalamus is known to regulate important processes including growth, development, reproduction and metabolism. It has also been shown that the hypothalamus regulates aging throughout the body. Hypothalamic stem cells appear to exert their anti-aging effects by releasing molecules called microRNAs (miRNAs). Zhang et al. (2017); Zhang et al. (2013).

[0152] Delays in aging can be measured by tissue analysis and behavioral testing to assess changes in a subject’s age-impaired muscle endurance, coordination, social behavior and cognitive ability. Muscular endurance, which is the ability to use muscles for extended periods of time at less than their full strength, can be measured using a variety of methods. For example, ACSM (2000) recommends the partial curl-up test to measure endurance of the abdominal muscles and the push-up test to assess endurance of the upper body. Coordination is evaluated by testing the patient’s ability to perform rapidly alternating and point-to-point movements correctly. Cognitive ability can be measured using cognitive ability tests. Cognitive ability tests assess abilities involved in thinking (e.g., reasoning, perception, memory, verbal and mathematical ability, and problem solving). Examples of cognitive ability tests include but are not limited to the Cognitive Abilities Test (CogAT), Wechsler Adult Intelligence Scale for adults and the Wechsler Intelligence Scale for Children for school-age test-takers, the Stanford-Binet Intelligence Scales, Woodcock-Johnson Tests of Cognitive Abilities, the Kaufman Assessment Battery for Children, the Cognitive Assessment System, and the Differential Ability Scales.

[0153] In one embodiment of the invention, administration of an aminosterol (i.e., ENT-03 or another aminosterol disclosed herein) or a derivative or salt thereof improves impaired muscle endurance, and/or improves impaired coordination, and/or improves impaired cognitive ability, as compared to an untreated subject, which is the same sex and age, by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

IX. Measurement of Impact on Aging

[0154] One embodiment of the invention is directed to methods of extending the potential lifespan of a subject, which can be an animal or human. In one aspect the subject has not yet reached maturity, and in another aspect the subject to be treated has reached maturity. The methods comprise administering a pharmaceutical composition comprising a therapeutically effective amount of an aminosterol (i.e., ENT-03 or another aminosterol disclosed herein) or a derivative or salt thereof to the subject. Optionally the method can also comprise administration of at least one insulin compound.

[0155] Lifespan and life expectancy are not synonymous. Life expectancy is defined statistically as the mean number of years remaining for an individual or a group of people at a given age. Id. Life expectancy increases with age as the individual survives the higher mortality rates associated with childhood. Life expectancy is an average for all people in the population — including those who die shortly after birth, those who die in early adulthood (e.g. childbirth, war), and those who live unimpeded until old age. Lifespan is an individual-specific concept — maximum lifespan is therefore an upper bound rather than an average.

10156 J In the present invention, an increased lifespan is defined as a lifespan greater than life expectancy. For example, a dog administered a composition of an aminosterol (i.e., ENT-03 or another aminosterol disclosed herein) according to the invention, and having a life expectancy of about 7 years, is projected to live longer than a dog having the same life expectancy but which is not treated with a method according to the invention. Life expectancies for different animals, breeds of animals, humans in various countries, etc. are all readily available. In an exemplary aspect of the invention, a subject treated with a pharmaceutical composition comprising a therapeutically effective amount of an aminosterol (i.e., ENT-03 or another aminosterol disclosed herein) formulation has an increased lifespan, as compared to a control, of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%. A “control” is defined as an animal which is the same sex, same age, and same type/breed. For a human, a “control” refers to the same sex, same age, same socioeconomic background, and same geographic residence.

X. Dosage Forms/Methods of Treatment

[0157] Various formulations may be used for administration of aminosterols or derivatives or salts thereof. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Any pharmaceutically acceptable dosage form may be employed in the methods of the invention. For example, the composition can be formulated into a dosage form selected from the group consisting of liquid dispersions, gels, aerosols, lyophilized formulations, tablets, capsules, or an intranasal formulations utilizing a powder or liquid. In some embodiments, the aminosterol or derivatives or salts thereof may be incorporated into a dosage form selected from the group consisting of controlled release formulations, fast melt formulations, delayed release formulations, extended release formulations, pulsatile release formulations, and mixed immediate release and controlled release formulations. In some embodiments, the dosage form may comprise a combination of the forgoing formulation options (e.g., a controlled release tablet). An exemplary dosage form is a nasal spray. A nasal spray is designed to deliver drug to the upper nasal cavity, and can be a liquid or powder formulation, and in a dosage form such as an aerosol, liquid spray, or powder.

[0158] Another exemplary dosage form is an orally administered dosage form, such as a tablet or capsule. These dosage forms can be formulated by any method known in the art. Such methods include the step of bringing into association the aminosterol or derivatives or salts thereof with the carrier that constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. Another example of an exemplary dosage form is a nasal spray, comprising a dry powder, liquid suspension, liquid emulsion, or other suitable nasal dosage form.

[0159] In one embodiment of the invention, an oral dosage form is a liquid, capsule, or tablet designed to disintegrate in either the stomach, upper small intestine, or more distal portions of the intestine with a dissolution rate appropriate to achieve the intended therapeutic benefit.

[0160] Formulations or compositions of the invention may be packaged together with, or included in a kit along with instructions or a package insert. Such instructions or package inserts may address recommended storage conditions, such as time, temperature and light, taking into account the shelf-life of the aminosterol or derivatives or salts thereof. Such instructions or package inserts may also address the particular advantages of the aminosterol or derivatives or salts thereof, such as the ease of storage for formulations that may require use in the field, outside of controlled hospital, clinic or office conditions.

[0161 ] The pharmaceutical composition comprising an aminosterol (i.e., ENT-03 or another aminosterol disclosed herein) or derivatives or salts thereof will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient, the method of administration, the scheduling of administration, and other factors known to practitioners. The “effective amount” for purposes herein is thus determined by such considerations.

[0162] Dosing: In one embodiment, the dosage of an aminosterol (i.e., ENT-03 or another aminosterol disclosed herein) or a derivative or salt thereof is selected from the group consisting of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 mg/kg. In another embodiment, the dosage of an aminosterol (i.e., ENT-03 or another aminosterol disclosed herein) or a derivative or salt thereof is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,

26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,

52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,

78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,

103, 104, 105, 106, 107, 108, 109, or 110 mg/m ² .

(0163] In other embodiments of the invention, an effective oral dose generally falls between about 10 mg to about 400 mg, or about 50 mg to about 350 mg, or about 100 mg to about 300 mg, or about 100 mg to about 200 mg. For instance, an effective dose may be about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195, about 200, about 205, about 210, about 215, about 220, about 225, about 230, about 235, about 240, about 245, about 250, about 255, about 260, about 265, about 270, about 275, about 280, about 285, about 290, about 295, about 300, about 305, about 310, about 315, about 320, about 325, about 330, about 335, about 340, about 345, about 350, about 355, about 360, about 365, about 370, about 375, about 380, about 385, about 390, about 395, or about 400 mg.

[0164] Dosing period: The pharmaceutical composition comprising an aminosterol (i.e., ENT-03 or another aminosterol disclosed herein) or a derivative or salt thereof can be administered for any suitable period of time, including as a maintenance dose for a prolonged period of time. Dosing can be done on an as needed basis using any pharmaceutically acceptable dosing regimen. For example, dosing can be once or twice daily, once every other day, once every three days, once every four days, once every five days, once every six days, once a week, or divided over multiple time periods during a given day (e.g., twice daily). The dosing schedule may include administration during the morning, midday, or during the evening, or a combination thereof.

[0165] In other embodiments, the composition can be administered: (1) as a single dose, or as multiple doses over a period of time; (2) at a maintenance dose for an indefinite period of time; (3) once, twice or multiple times; (4) daily, every other day, every 3 days, weekly, or monthly; (5) for a period of time such as 1, 2, 3, or 4 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, 1 year, 1.5 years, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12,

12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22,

22.5, 23, 23.5, 24, 24.5, or 25 years, or (6) any combination of these parameters, such as daily administration for 6 months, weekly administration for 1 or more years, etc.

[0166] Exemplary dosing regimens include, but are not limited to: Initiating with a “low” initial daily dose, and gradually increasing the daily dose until a dose is reached that elicits evidence of a measurable impact, e.g., slowed growth rate (e.g., height and weight), improved age-related conditions (e.g., muscle endurance, coordination, social behavior and cognitive ability), or other indicia of desirable effects. In some embodiments, a “low” dose is from about 10 to about 100 mg per person, and the final effective daily dose may be between about 25 to about 1000 mg/person.

[01 7] Another exemplary dosing regimen includes: Initiating with a “high” initial dose, and reducing the subsequent daily dosing to that required to elicit a desirable response, with the “high” daily dose being between about 50 to about 1000 mg/person, and the subsequent lower daily oral dose being between about 25 to about 500 mg/person.

[0168] Yet another exemplary dosing regimen includes periodic dosing, where an effective dose can be delivered once every about 1, about 2, about 3, about 4, about 5, about 6 days, or once weekly, with the initial dose determined to be capable of delaying maturation, retarding the aging process, and/or extending the potential lifespan of a subject, which can be an animal or human. [0169] In some embodiments, the first or initial “large” dose of an aminosterol (i.e., ENT-03 or another aminosterol disclosed herein) or a derivative or salt thereof can be selected from the group consisting of about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about 850, about 875, about 900, about 925, about 950, about 975, about 1000, about 1025, about 1050, about 1075, about 1100, about 1125, about 1150, about 1175, about 1200, about 1225, about 1250, about 1275, about 1300, about 1325, about 1350, about 1375, about 1400, about 1425, about 1450, about 1475, about 1500, about 1525, about 1550, about 1575, about 1600, about 1625, about 1650, about 1675, about 1700, about 1725, about 1750, about 1775, about 1800, about 1825, about 1850, about 1875, about 1900, about 1925, about 1950, about 1975, or about 2000 mg. In other embodiments of the invention, the second smaller dose of an aminosterol (i.e., ENT-03 or another aminosterol disclosed herein) or a derivative or salt thereof is less than the first or initial dose and can be selected from the group consisting of about, 10, about 25, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about 850, about 875, about 900, about 925, about 950, about 975, or about 1000 mg. Finally, in other embodiments of the invention, the periodic aminosterol or a derivative or salt thereof dosage (per person) can be selected from the group consisting of about 10, about 25, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about 850, about 875, about 900, about 925, about 950, about 975, and about 1000 mg.

[0170] Any pharmaceutical used for therapeutic administration can be sterile. Sterility is readily accomplished by for example filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Any pharmaceutically acceptable sterility method can be used in the compositions of the invention. [0171 ] The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more pharmaceutical compositions useful in the disclosed methods of treatment. The kits may include, for instance, containers filled with an appropriate amount of a pharmaceutical composition, either as a powder, to be dissolved, or as a sterile solution, in addition to the aminosterol or a derivative or salt thereof. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the aminosterol or a derivative or salt thereof may be employed in conjunction with other therapeutic compounds.

[0172] Excipients: Pharmaceutical compositions according to the invention may also comprise one or more binding agents, filling agents, lubricating agents, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, effervescent agents, and other excipients. Such excipients are known in the art. Examples of filling agents include lactose monohydrate, lactose anhydrous, and various starches; examples of binding agents include various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose, such as Avicel® PHI 01 and Avicel® PHI 02, microcrystalline cellulose, and silicified microcrystalline cellulose (ProSolv SMCC™). Suitable lubricants, including agents that act on the flowability of the powder to be compressed, may include colloidal silicon dioxide, such as Aerosil® 200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel. Examples of sweeteners may include any natural or artificial sweetener, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acesulfame. Examples of flavoring agents are Magnasweet® (trademark of MAFCO), bubble gum flavor, and fruit flavors, and the like. Examples of preservatives include potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride.

[0173] Suitable diluents include pharmaceutically acceptable inert fillers, such as microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and/or mixtures of any of the foregoing. Examples of diluents include microcrystalline cellulose, such as Avicel® PHI 01 and Avicel® PHI 02; lactose such as lactose monohydrate, lactose anhydrous, and Pharmatose® DCL21; dibasic calcium phosphate such as Emcompress®; mannitol; starch; sorbitol; sucrose; and glucose.

[0174] Suitable disintegrants include lightly crosslinked polyvinyl pyrrolidone, corn starch, potato starch, maize starch, and modified starches, croscarmellose sodium, cross-povidone, sodium starch glycolate, and mixtures thereof. Examples of effervescent agents include effervescent couples such as an organic acid and a carbonate or bicarbonate. Suitable organic acids include, for example, citric, tartaric, malic, fumaric, adipic, succinic, and alginic acids and anhydrides and acid salts. Suitable carbonates and bicarbonates include, for example, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium glycine carbonate, L-lysine carbonate, and arginine carbonate. Alternatively, only the sodium bicarbonate component of the effervescent couple may be present.

[0175] Optimal oral dosing appears to be on an empty stomach. An aminosterol (i.e., ENT-03 or another aminosterol disclosed herein) or a derivative or salt thereof, for example, is expected to bind tightly to foodstuff, and be unavailable to interact with the intestinal epithelium. Only as the food material is digested is the aminosterol or a derivative or salt thereof freed.

[0176] In another embodiment, an aminosterol (i.e., ENT-03 or another aminosterol disclosed herein) or a derivative or salt thereof is administered in combination with at least one additional active agent to achieve either an additive or synergistic effect. For example, the additional active agent can be administered via a method selected from the group consisting of (a) concomitantly; (b) as an admixture; (c) separately and simultaneously or concurrently; or (d) separately and sequentially. In another embodiment, the additional active agent is a different aminosterol from that administered in primary method. In yet a further embodiment, the method of the invention comprises administering a first aminosterol which is an aminosterol (i.e., ENT-03 or another aminosterol disclosed herein) or a salt or derivative thereof intranasally and administering a second aminosterol, different from the first aminosterol or a salt or derivative thereof, orally.

[0177] For all of the methods of the invention, in one embodiment each dose of an aminosterol (i.e., ENT-03 or another aminosterol disclosed herein) or a derivative or salt thereof is taken on an empty stomach, optionally within about two hours of the subject waking. In another embodiment for all of the methods of the invention, no food is taken or consumed after about 60 to about 90 minutes of taking the dose of an aminosterol (i.e., ENT-03 or another aminosterol disclosed herein) or a derivative or salt thereof. Further, in yet another embodiment applicable to all of the methods of the invention, the aminosterol or a derivative or salt thereof can be a pharmaceutically acceptable grade of at least one aminosterol or a derivative or salt thereof. For all of the methods of the invention the subject can be a human.

[0178] In another embodiment, the subject to be treated according to the methods of the invention can be a member of a patient population at risk for being diagnosed with neurodegenerati on .

XI. Optional Method of Dose Optimization

|0179| Method of dose optimization: In another embodiment, the invention encompasses a method of treating, preventing and/or slowing the onset or neurodegeneration and/or a related symptom in a subject in need. Optionally, the neurodegeneration is correlated with abnormal a- synuclein (aS) pathology and/or dopaminergic dysfunction. The method comprises (a) determining a dose of an aminosterol or a salt or derivative thereof for the subject, wherein the dose of the aminosterol or a salt or derivative thereof is determined based on the effectiveness of the dose in improving or resolving a neurodegeneration symptom being evaluated, (b) followed by administering the dose to the subject for a period of time, wherein the method comprises (i) identifying a neurodegeneration symptom to be evaluated; (ii) identifying a starting dose of the aminosterol or a salt or derivative thereof for the subject; and (iii) administering an escalating dose of the aminosterol or a salt or derivative thereof to the subject over a period of time until an effective dose for the neurodegeneration symptom being evaluated is identified, wherein the effective dose is the aminosterol dose where improvement or resolution of the neurodegeneration symptom is observed, and fixing the dose at that level for that particular neurodegeneration symptom in that particular subject.

[0180] In one embodiment, starting dosages of the aminosterol or a salt or derivative thereof for oral administration can range, for example, from about 1 mg up to about 175 mg/day, or any amount in-between these two values. An exemplary starting dosage is 25 mg/day. In another embodiment, the composition is administered orally and the dosage is escalated in about 25 mg increments. In yet another embodiment, the composition is administered orally and the dose of aminosterol or a salt or derivative thereof for the subject following dose escalation is fixed at a range of from about 1 mg up to about 500 mg/day, or any amount in-between these two values. In another aspect, the dose of the aminosterol or a salt or derivative thereof for the subject following escalation is fixed at a dose of about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195, about 200, about 205, about 210, about 215, about 220, about 225, about 230, about 235, about 240, about 245, about 250, about 255, about 260, about 265, about 270, about 275, about 280, about 285, about 290, about 295, about 300, about 305, about 310, about 315, about 320, about 325, about 330, about 335, about 340, about 345, about 350, about 355, about 360, about 365, about 370, about 375, about 380, about 385, about 390, about 395, about 400, about 405, about 410, about 415, about 420, about 425, about 430, about 435, about 440, about 445, about 450, about 455, about 460, about 465, about 470, about 475, about 480, about 485, about 490, about 495, or about 500 mg/day. In another aspect, the starting oral dose is about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 60, about 65, about 70, or about 75 mg/day.

[0181] In another embodiment, the composition is administered intranasally (IN) and the starting dosage of the aminosterol or a salt or derivative thereof ranges from about 0.001 mg to about 3 mg/day, or any amount in-between these two values. For example, the starting dosage for IN administration, prior to dose escalation, can be, for example, about 0.001, about 0.005, about 0.01, about 0.02, about 0.03, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.15, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, about 0.5, about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, about 1.0, about 1.1, about 1.25, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.75, about 1.8, about 1.9, about 2.0, about 2.1, about 2.25, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.75, about 2.8, about 2.9, or about 3 mg/day.

[0182] In another embodiment, the composition is administered intranasally and the dosage of the aminosterol or a salt or derivative thereof is escalated in increments of about 0.01, about 0.05, about 0.1, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, about 0.5, about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, about 0.95, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, or about 2 mg. [0183] Finally, in yet another embodiment, the composition is administered intranasally and the dose of the aminosterol or a salt or derivative thereof for the subject following escalation is fixed at a range of from about 0.001 mg up to about 6 mg/day, or any amount in-between these two values. In yet a further embodiment, the composition is administered intranasally and the dose of the aminosterol or a salt or derivative thereof for the subject following dose escalation is a dose which is sub therapeutic when given orally or by injection.

[0184] In one aspect, the aminosterol or a salt or derivative thereof is formulated for intranasal administration in a composition which is a dry powder nasal spray or liquid nasal spray.

10185 [ In one embodiment, the dosage of the aminosterol or a salt or derivative thereof is escalated every about 3 to about 5 days. In another embodiment, the dose of the aminosterol or a salt or derivative thereof is escalated about Ix/week, about 2x/week, about every other week, or about Ix/month. In yet another embodiment, the dose of the aminosterol or a salt or derivative thereof is escalated every about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, or about 14 days.

[0186] In another embodiment, the fixed dose of the aminosterol or a salt or derivative thereof is given once per day, every other day, once per week, twice per week, three times per week, four times per week, five times per week, six times per week, every other week, or every few days. In addition, the fixed dose of the aminosterol or a salt or derivative thereof can be administered for a first defined period of time of administration, followed by a cessation of administration for a second defined period of time, followed by resuming administration upon recurrence of SZ or a symptom of SZ. For example, the fixed dose can be incrementally reduced after the fixed dose of the aminosterol or a salt or derivative thereof has been administered to the subject for a period of time. Alternatively, the fixed dose is varied plus or minus a defined amount to enable a modest reduction or increase in the fixed dose. For example, the fixed dose can be increased or decreased by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20%.

|0187] A “fixed aminosterol dose,” also referred to herein as a “fixed escalated aminosterol dose,” which will be therapeutically effective is determined for each patient by establishing a starting dose of an aminosterol composition and a threshold for improvement of a particular symptom which is used as a tool or marker for evaluating the effectiveness of the aminosterol dosage. Following determining a starting aminosterol dosage for a particular patient, the aminosterol dose is then progressively escalated by a consistent amount over consistent time intervals until the desired improvement is achieved; this aminosterol dosage is the “fixed escalated aminosterol dosage” for that particular patient for that particular symptom. In exemplary embodiments, an orally administered aminosterol dose is escalated every about 3 to about 5 days by about 25 mg until the desired improvement is reached. Symptoms evaluated, along with tools for measuring symptom improvement, may be specifically described below, including but not limited to constipation, hallucinations, sleep disturbances (e.g. REM disturbed sleep or circadian rhythm dysfunction), cognitive impairment, depression, or alpha-synuclein aggregation.

10188] This therapeutically effective “fixed dose” is then maintained throughout treatment and/or prevention. Thus, even if the patient goes “off drug” and ceases taking the aminosterol composition, the same “fixed dose” is taken with no ramp up period following re-initiation of aminosterol treatment. Not to be bound by theory, it is believed that the aminosterol dose is dependent on the severity of nerve damage relating to the symptom establishing the “fixed dose” threshold (e.g. for constipation, the dose may be related to the extent of nervous system damage in the patient’s gut).

[0189] Dose escalation: When determining a “fixed aminosterol dosage” for a particular patient, a patient is started at a lower dose and then the dose is escalated until a positive result is observed for the symptom being evaluated. An exemplary symptom to be evaluated can be constipation, but any symptom associated with the disease or disorder to be treated can be used as a marker for evaluating aminosterol dosage. Aminosterol doses can also be de-escalated (reduced) if any given aminosterol dose induces a persistent undesirable side effect, such as diarrhea, vomiting, or nausea.

(0190] The starting aminosterol dose is dependent on the severity of the symptom - e.g. for a patient experiencing severe constipation, defined as less than one spontaneous bowel movement (SBM) a week, the starting oral aminosterol dose can be about 150 mg/day or greater. In contrast, for a patient having moderate constipation, e.g., defined as having more than one SBM a week, the starting oral aminosterol dose can be about 75 mg/day. Thus, as an example, a patient experiencing moderate constipation can be started at an oral aminosterol dosage of about 75 mg/day, whereas a patient experiencing severe constipation can be started at an oral aminosterol dosage of about 150 mg/day.

101911 In other embodiments, a patient experiencing moderate symptoms (for the symptom being used to calculate a fixed escalated aminosterol dose) can be started at an oral aminosterol dosage of from about 10 mg/day to about 75 mg/day, or any amount in-between these values. For example, the starting oral aminosterol dosage for a moderate symptom can be about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 60, about 65, about 70, or about 75 mg/day.

[0192] In yet further embodiments, when the patient is experiencing severe symptoms (for the symptom being used to calculate the fixed escalated aminosterol dose), the patient can be started at an oral aminosterol dosage ranging from about 75 to about 175 mg/day, or any amount inbetween these two values. For example, the starting oral aminosterol dosage for a severe symptom can be about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150 about 155, about 160, about 165, about 170, or about 175 mg/day.

(0193] In some embodiments, the starting oral aminosterol dose may be about 125 mg or about 175 mg/day; again dependent on the severity of the symptom, such as constipation.

101941 Starting intranasal (IN) aminosterol dosages prior to dose escalation can be, for example, about 0.001 mg to about 3 mg/day, or any amount in-between these two values. For example, the starting aminosterol dosage for IN administration, prior to dose escalation, can be, for example, about 0.001, about 0.005, about 0.01, about 0.02, about 0.03, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.15, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, about 0.5, about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, about 1.0, about 1.1, about 1.25, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.75, about 1.8, about 1.9, about 2.0, about 2.1, about 2.25, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.75, about 2.8, about 2.9, or about 3 mg/day.

[0195] In exemplary embodiments, the aminosterol dose is given periodically as needed. For example, the aminosterol dose can be given once per day. The aminosterol dose can also be given every other day, 2, 3, 4, or 5x per week, once/week, or 2x/week. In another embodiment, the aminosterol dose can be given every other week, or it can be given for a few weeks, followed by skipping a few weeks (as the effects persist following treatment), followed by restarting aminosterol treatment.

[0196] When calculating a fixed escalated aminosterol dose, the dose can be escalated following any suitable time period. In one embodiment, the aminosterol dose is escalated every about 3 to about 7 days by about a defined amount until a desired improvement is reached. For example, when the symptom being treated/measured is constipation, threshold improvement can be an increase of one SBM per week or at least a total of three bowel movements per week. In other embodiments, the aminosterol dose can be escalated every about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, or about 14 days. In other embodiments, the aminosterol dose can be escalated about Ix/week, about 2x/week, about every other week, or about Ix/month.

[0197] During dose escalation, the aminosterol dosage can be increased by a defined amount. For example, when the aminosterol is administered orally, the dose can be escalated in increments of about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or by about 50 mg. When the aminosterol is administered intranasally, then the dosage can be increased in increments of about, for example, about 0.1, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, about 0.5, about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, about 0.95, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, or about 2 mg.

|0198[ Other symptoms that can be used as an endpoint to determine aminosterol dosage for a patient’s fixed escalated aminosterol dosage are any symptom known to be associated with the disease, disorder, or condition intended to be treated. For example, neurodisease symptoms described herein and include, but are not limited to, (a) at least one non-motor aspect of experiences of daily living as defined by Part I of the Unified Parkinson’s Disease Rating Scale (UPDRS), such as for example cognitive impairment, hallucinations and psychosis, depressed mood, anxious mood, apathy, features of dopamine dysregulation syndrome, sleep problems, daytime sleepiness, pain, urinary problems, constipation problems, lightheadedness on standing, and fatigue; (b) at least one motor aspect of experiences of daily living as defined by Part II of the UPDRS, such as for example, speech, saliva and drooling, chewing and swallowing, eating tasks, dressing, hygiene, handwriting, turning in bed, tremors, getting out of a bed, a car, or a deep chair, walking and balance, and freezing; (c) at least one motor symptom identified in Part III of the UPDRS, such as for example, speech, facial expression, rigidity, finger tapping, hand movements, pronation-supination movements of hands, toe tapping, leg agility, arising from chair, gait, freezing of gait, postural stability, posture, body bradykinesia, postural tremor of the hands, kinetic tremor of the hands, rest tremor amplitude, and constancy of rest tremor; (d) at least one motor complication identified in Part IV of the UPDRS, such as for example, dyskinesias, functional impact of dyskinesias, time spent in the off state, functional impact of fluctuations, complexity of motor fluctuations, and painful off-state dystonia; (e) constipation; (f) depression; (g) cognitive impairment; (h) sleep problems or sleep disturbances; (i) circadian rhythm dysfunction; (j) hallucinations; (k) fatigue; (1) REM disturbed sleep; (m) REM behavior disorder; (n) erectile dysfunction; (o) apnea; (p) postural hypotension; (q) correction of blood pressure or orthostatic hypotension; (r) nocturnal hypertension; (s) regulation of temperature; (t) improvement in breathing or apnea; (u) correction of cardiac conduction defect; (v) amelioration of pain; (w) restoration of bladder sensation and urination; (x) urinary incontinence; and/or (y) control of nocturia.

[0199] In another embodiment, the starting aminosterol or a salt or derivative thereof dose is higher if the neurodegeneration symptom being evaluated is severe. For example, the starting dose can be based on a baseline score of a cognitive test or tool, wherein if the baseline score correlates with an assessment of mild cognitive impairment, then the starting dose of aminosterol or a salt or derivative thereof is lower than if the baseline score correlates with an assessment of severe cognitive impairment. In another aspect, a subject experiencing moderate or mild cognitive impairment as determined by a clinical scale or test is administered a starting oral dose of from about 10 to about 75 mg/day; or a subject experiencing severe cognitive impairment as determined by a clinical scale or test is administered a starting oral dose greater than about 75 mg/day.

[0200] In one embodiment, the method results in slowing, halting, or reversing progression or onset of neurodegeneration over a defined period of time following administration of the fixed escalated dose of the aminosterol or a salt or derivative thereof, as measured by a medically- recognized technique. In addition, the method of the invention can result in positively impacting the neurodegeneration, as measured by a medically-recognized technique.

[0201] The positive impact and/or progression of neurodegeneration, and/or improvement or resolution of the neurodegeneration symptom being evaluated, may be measured quantitatively or qualitatively by one or more clinically recognized scales, tools, or techniques). Examples of such techniques include computed tomography (CT), magnetic resonance imaging (MRI), magnetic resonance spectroscopy, functional MRI (fMRI), diffusion tensor imaging, single photon emission computed tomography (SPECT), and positron emission tomography (PET). In addition, the progression or onset of neurodegeneration may be slowed, halted, or reversed by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, as measured by a medically-recognized technique.

[0202] In one embodiment, the fixed escalated dose of the aminosterol or a salt or derivative thereof reverses dysfunction caused by the neurodegeneration and treats, prevents, improves, and/or resolves the neurodegeneration symptom being evaluated. Again, the improvement or resolution of the neurodegeneration-related symptom can be measured using a clinically recognized scale or tool. Examples of such scales or tools include, for example, the Uniformed Parkinson’s Disease Scale (UPDRS), Mini Mental State Examination (MMSE), Mini Mental Parkinson (MMP), Informant Questionnaire on Cognitive Decline in the Elderly (IQCODE), The 7-Minute Screen, Abbreviated Mental Test Score (AMTS), Cambridge Cognitive Examination (CAMCOG), Clock Drawing Test (CDT), General Practitioner Assessment of Cognition (GPCOG), Mini-Cog, Memory Impairment Screen (MIS), Montreal Cognitive Assessment (MoCA), Rowland Universal Dementia Assessment (RUD A), Self- Administered Gerocognitive Examination (SAGE), Short and Sweet Screening Instrument (SAS-SI), Short Blessed Test (SBT), St. Louis Mental Status (SLUMS), Short Portable Mental Status Questionnaire (SPMSQ), Short Test of Mental Status (STMS), Time and Change Test (T&C), Test Your Memory (TYM) test, and Addenbrooke’s Cognitive Examination-Revised (ACER). Further, the improvement in the neurodegeneration-related symptom is at least about 3%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%, as measured using a clinically recognized scale or tool.

10203] In one aspect, the neurodegeneration correlated with abnormal aS pathology and/or dopaminergic dysfunction is related to or correlated with a neurodegenerative disease or neurological disease associated with neural cell death. In another aspect, the neurodegenerative disease or neurological disease or related symptom associated with neural cell death is: (a) selected from the group consisting of septic shock, intracerebral bleeding, subarachnoidal hemorrhage, multi-infarct dementia, inflammatory diseases, neurotrauma, peripheral neuropathies, polyneuropathies, metabolic encephalopathies, and infections of the central nervous system; or(b) selected from the group consisting of synucleopathies, Alzheimer’s disease, Parkinson’s disease, dementia with Lewy bodies, multiple system atrophy, Huntington’s disease, multiple sclerosis, parkinsonism, amyotrophic lateral sclerosis (ALS), schizophrenia, Friedreich’s ataxia, vascular dementia, spinal muscular atrophy, frontotemporal dementia, supranuclear palsy, progressive supranuclear palsy, progressive nuclear palsy, degenerative processes associated with aging, dementia of aging, Guadeloupian parkinsonism, spinocerebellar ataxia, hallucinations, stroke, traumatic brain injury, down syndrome, Gaucher’s disease, Krabbe’s disease (KD), lysosomal conditions affecting glycosphingolipid metabolism, cerebral palsy, and epilepsy.

[0204] In another aspect, the neurodegeneration correlated with abnormal aS pathology and/or dopaminergic dysfunction is related to or correlated with a psychological or behavioral disorder. For example, the psychological or behavioral disorder can be selected from the group consisting of aberrant motor and obsessive-compulsive behaviors, sleep disorders, REM sleep behavior disorder (RBD), depression, major depressive disorder, agitation, anxiety, delirium, irritability, ADHD, apathy, bipolar disorder, disinhibition, addiction, illusion and delusions, amnesia, autism,

(0205] In one embodiment, the neurodegeneration correlated with abnormal aS pathology and/or dopaminergic dysfunction is related to or correlated with a cerebral ischemic disorder or a general ischemic disorder. For example, the cerebral ischemic disorder can be selected from the group consisting of cerebral microangiopathy, intrapartal cerebral ischemia, cerebral ischemia during/after cardiac arrest or resuscitation, cerebral ischemia due to intraoperative problems, cerebral ischemia during carotid surgery, chronic cerebral ischemia due to stenosis of bloodsupplying arteries to the brain, sinus thrombosis or thrombosis of cerebral veins, cerebral vessel malformations, and diabetic retinopathy; or the general ischemic disorder can be selected from the group consisting of high blood pressure, high cholesterol, myocardial infarction, cardiac insufficiency, cardiac failure, congestive heart failure, myocarditis, pericarditis, perimyocarditis, coronary heart disease, angina pectoris, congenital heart disease, shock, ischemia of extremities, stenosis of renal arteries, diabetic retinopathy, thrombosis associated with malaria, artificial heart valves, anemias, hypersplenic syndrome, emphysema, lung fibrosis, and pulmonary edema.

[0206| In another embodiment, the neurodegeneration-related symptom is selected from the group consisting of: cognitive impairment (CI) as determined by an IQ score; CI as determined by a memory or cognitive function test; decline in thinking and reasoning skills; confusion; poor motor coordination; loss of short term memory; loss of long term memory; identity confusion; impaired judgement; forgetfulness; depression; anxiety; irritability; obsessive-compulsive behavior; apathy and/or lack of motivation; emotional imbalance; problem solving ability; impaired language; impaired reasoning; impaired decision-making ability; impaired ability to concentrate; impaired communication; impaired ability to conduct routine tasks such as cooking; self-care, including feeding and dressing; constipation; neurodegeneration; sleep problem, sleep disorder, and/or sleep disturbance; hypertension; hypotension; sexual dysfunction; cardiovascular disease; cardiovascular dysfunction; difficulty with working memory; gastrointestinal (GI) disorders; attention deficit and hyperactivity disorder; seizures; urinary dysfunction; difficulty with mastication; vision problems; and muscle weakness.

102071 In one aspect, the neurodegeneration-related symptom to be evaluated is cognitive impairment (CI) as determined by an IQ score or as determined by a memory or cognitive function test and wherein: (a) progression or onset of the CI is slowed, halted, or reversed over a defined period of time following administration of the fixed escalated dose of the aminosterol or a salt or derivative thereof, as measured by a medically-recognized technique; (b) the CI is positively impacted by the fixed escalated dose of the aminosterol or a salt or derivative thereof, as measured by a medically-recognized technique; (c) the CI is positively impacted by the fixed escalated dose of the aminosterol or a salt or derivative thereof, as measured by a medically- recognized technique and the positive impact on and/or progression of cognitive decline is measured quantitatively or qualitatively by one or more medically-recognized techniques selected from the group consisting of ADASCog, Mini-Mental State Exam(MMSE), Mini-cog test, Woodcock-Johnson Tests of Cognitive Abilities, Leiter International Performance Scale, Miller Analogies Test, Raven’s Progressive Matrices, Wonderlic Personnel Test, IQ tests, or a computerized tested selected from Cantab Mobile, Cognigram, Cognivue, Cognision, and Automated Neuropsychological Assessment Metrics Cognitive Performance Test (CPT); and/or (d) the progression or onset of CI is slowed, halted, or reversed by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, as measured by a medically-recognized technique.

[0208] In one embodiment, the neurodegeneration-related symptom to be evaluated is depression and (a) the method results in improvement in a subject’s depression, as measured by one or more clinically-recognized depression rating scale; (b) the method results in improvement in a subject’s depression, as measured by one or more clinically-recognized depression rating scale and the improvement is in one or more depression characteristics selected from the group consisting of mood, behavior, bodily functions such as eating, sleeping, energy, and sexual activity, and/or episodes of sadness or apathy; and/or (c) the method results in improvement in a subject’s depression, as measured by one or more clinically-recognized depression rating scale, and the improvement a subject experiences following treatment is about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95 or about 100%. For example, the one or more clinically-recognized depression rating scale can be selected from the group consisting of the Patient Health Questionnaire-9 (PHQ-9); the Beck Depression Inventory (BDI); Zung Self-Rating Depression Scale; Center for Epidemiologic Studies-Depression Scale (CES- D); and the Hamilton Rating Scale for Depression (HRSD).

[0209] In one embodiment, the neurodegeneration-related symptom to be evaluated is constipation, and (a) treating the constipation prevents and/or delays the onset and/or progression of the neurodegeneration; (b) the fixed escalated aminosterol dose causes the subject to have a bowel movement; (c) the method results in an increase in the frequency of bowel movement in the subject; (d) the method results in an increase in the frequency of bowel movement in the subject and the increase in the frequency of bowel movement is defined as: (i) an increase in the number of bowel movements per week of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 100%; and/or (ii) a percent decrease in the amount of time between each successive bowel movement selected from the group consisting of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%; (e) as a result of the method the subject has the frequency of bowel movement recommended by a medical authority for the age group of the subject; and/or (f) the starting aminosterol dose is determined by the severity of the constipation, wherein: (i) if the average complete spontaneous bowel movement (CSBM) or spontaneous bowel movement (SBM) is one or less per week, then the starting oral aminosterol dose is at least about 150 mg; and (ii) if the average CSBM or SBM is greater than one per week, then the starting oral aminosterol dose is about 75 mg or less.

102101 In one embodiment, the neurodegeneration-related symptom to be evaluated is neurodegeneration correlated with neurodegeneration, and (a) treating the neurodegeneration prevents and/or delays the onset and/or progression of the neurodegeneration; (b) the method results in treating, preventing, and/or delaying the progression and/or onset of neurodegeneration in the subject; (c) progression or onset of the neurodegeneration is slowed, halted, or reversed over a defined period of time following administration of the fixed escalated dose of the aminosterol or a salt or derivative thereof, as measured by a medically-recognized technique; and/or (d) the neurodegeneration is positively impacted by the fixed escalated dose of the aminosterol or a salt or derivative thereof, as measured by a medically-recognized technique. For example, the positive impact and/or progression of neurodegeneration can be measured quantitatively or qualitatively by one or more techniques selected from the group consisting of electroencephalogram (EEG), neuroimaging, functional MRI, structural MRI, diffusion tensor imaging (DTI), [18F]fluorodeoxy glucose (FDG) PET, agents that label amyloid, [18F]F-dopa PET, radiotracer imaging, volumetric analysis of regional tissue loss, specific imaging markers of abnormal protein deposition, multimodal imaging, and biomarker analysis. In addition, the progression or onset of neurodegeneration can be slowed, halted, or reversed by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, as measured by a medically-recognized technique. [0211 ] In one embodiment, the neurodegeneration-related symptom to be evaluated is a sleep problem, sleep disorder, or sleep disturbance and (a) the sleep problem, sleep disorder, or sleep disturbance comprises a delay in sleep onset, sleep fragmentation, REM-behavior disorder, sleep-disordered breathing including snoring and apnea, day-time sleepiness, micro-sleep episodes, narcolepsy, circadian rhythm dysfunction, REM disturbed sleep, or any combination thereof; (b) the sleep problem, sleep disorder, or sleep disturbance comprises REM-behavior disorder, which comprises vivid dreams, nightmares, and acting out the dreams by speaking or screaming, or fidgeting or thrashing of arms or legs during sleep; (c) treating the sleep problem, sleep disorder, or sleep disturbance prevents or delays the onset and/or progression of the neurodegeneration; (d) the method results in a positive change in the sleeping pattern of the subject; wherein the positive change is defined as: (i) an increase in the total amount of sleep obtained of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 100%; and/or (ii) a percent decrease in the number of awakenings during the night selected from the group consisting of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%; and/or (f) as a result of the method the subject obtains the total number of hours of sleep recommended by a medical authority for the age group of the subject.

10212] For all of the embodiments described herein, each defined period of time is independently selected from the group consisting of about 1 day to about 10 days, about 10 days to about 30 days, about 30 days to about 3 months, about 3 months to about 6 months, about 6 months to about 12 months, and about greater than 12 months.

XII. Combination therapy

[0213] In the methods of the disclosure, the combination of (a) an aminosterol comprising a spermine moiety, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof, and optionally (b) insulin, an immunologically active insulin fragment, an insulin derivative, or a functional equivalent of insulin, or a combination thereof may be administered alone or in combination with one or more other therapeutic agents. An example of an additional therapeutic agent is one known to treat the condition the aminosterol is being administered to treat.

[0214] For example, in methods of treating, preventing, and/or slowing the onset or progression of PD and/or a related symptom, the combination of (a) an aminosterol comprising a spermine moiety, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof, and optionally (b) insulin, an immunologically active insulin fragment, an insulin derivative, or a functional equivalent of insulin, or a combination thereof can be co-administered or combined with drugs commonly prescribed to treat PD or related symptoms, such as levodopa (usually combined with a dopa decarboxylase inhibitor or COMT inhibitor), dopamine agonists and MAO-B inhibitors. Exemplary dopa decarboxylase inhibitors are carbidopa and benserazide. Exemplary COMT inhibitors are tolcapone and entacapone. Dopamine agonists include, for example, bromocriptine, pergolide, pramipexole, ropinirole, piribedil, cabergoline, apomorphine, lisuride, and rotigotine. MAO-B inhibitors include, for example, selegiline and rasagiline. Other drugs commonly used to treat PD include, for example, amantadine, anticholinergics, clozapine for psychosis, cholinesterase inhibitors for dementia, and modafinil for daytime sleepiness.

[0215] In methods of treating, preventing, and/or slowing the onset or progression of AD or related symptoms associated with AD, the combination of (a) an aminosterol comprising a spermine moiety, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof, and optionally (b) insulin, an immunologically active insulin fragment, an insulin derivative, or a functional equivalent of insulin, or a combination thereof can be co-administered or combined with drugs commonly prescribed to treat AD or related symptoms, such as glutamate, antipsychotic drugs, huperzine A, acetylcholinesterase inhibitors and NMDA receptor antagonists such as memantine (Akatinol®, Axura®, Ebixa®/Abixa®, Memox® and Namenda®). Examples of acetylcholinesterase inhibitors are donepezil (Aricept®), galantamine (Razadyne®), and rivastigmine (Exelon®).

[0216] In methods of treating, preventing, and/or slowing the onset or progression of diabetes or related symptoms associated with diabetes and/or diabetes mellitus, including both Type 1 and Type 2 diabetes, or neuropathy of diabetes, the aminosterol composition can be co-administered or combined with drugs commonly prescribed to treat diabetes mellitus or related symptoms, such as insulin (NPH insulin or synthetic insulin analogs) (e.g., Humulin®, Novolin®) and oral antihyperglycemic drugs. Oral antihyperglycemic drugs include but are not limited to (1) biguanides such as metformin (Glucophage®); (2) Sulfonylureas such as acetohexamide, chlorpropamide (Diabinese®), glimepiride (Amaryl®), glipizide (Glucotrol®), tolazamide, Tolbutamide, and glyburide (Diabeta®, Micronase®); (3) Meglitinides such as repaglinide (Prandin®) and nateglinide (Starlix®); (4) Thiazolidinediones such as rosiglitazone (Avandia®) and pioglitazone (Actos®); (5) Alpha-glucosidase inhibitors such as acarbose (Precose®) and miglitol (Glyset®); (6) Dipeptidyl peptidase-4 inhibitors such as Sitagliptin (januvia®); (7) Glucagon-like peptide agonists such as exenatide (Byetta®); and (8) Amylin analogs such as pramlintide (Symlin®).

[0217] In methods of treating, preventing, and/or slowing the onset or progression of HD or related symptoms associated with Huntington’s chorea or disease, the combination of (a) an aminosterol comprising a spermine moiety, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof, and optionally (b) insulin, an immunologically active insulin fragment, an insulin derivative, or a functional equivalent of insulin, or a combination thereof can be co-administered or combined with drugs commonly prescribed to treat Huntington’s chorea or related symptoms, such as medications prescribed to help control emotional and movement problems associated with Huntington’s chorea. Such medications include, but are not limited to, (1) antipsychotic drugs, such as haloperidol and clonazepam; (2) drugs used to treat dystonia, such as acetylcholine regulating drugs (trihexyphenidyl, benztropine (Cogentin®), and procyclidine HC1); GABA-regulating drugs (diazepam (Valium®), lorazepam (Ativan®), clonazepam (Klonopin®), and baclofen (Lioresal®)); dopamine-regulators (levodopa/carbidopa (Sinemet®), bromocriptine (parlodel), reserpine, tetrabenazine) ; anticonvulsants (carbamazepine (Tegretol®) and botulinum toxin (Botox®)); and (3) drugs used to treat depression (fluoxetine, sertraline, and nortriptyline). Other drugs commonly used to treat HD include amantadine, tetrabenazine, dopamine blockers, and co-enzyme Qio.

[0218] In methods of treating, preventing, and/or slowing the onset or progression of peripheral sensory neuropathy or related symptoms associated with peripheral sensory neuropathy, the combination of (a) an aminosterol comprising a spermine moiety, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof, and optionally (b) insulin, an immunologically active insulin fragment, an insulin derivative, or a functional equivalent of insulin, or a combination thereof can be co-administered or combined with drugs commonly prescribed to treat peripheral sensory neuropathy or related symptoms. Peripheral sensory neuropathy refers to damage to nerves of the peripheral nervous system, which may be caused either by diseases of or trauma to the nerve or the side-effects of systemic illness. Drugs commonly used to treat this condition include, but are not limited to, neurotrophin-3, tricyclic antidepressants (e.g., amitriptyline), antiepileptic therapies (e.g., gabapentin or sodium valproate), synthetic cannabinoids (Nabilone) and inhaled cannabis, opiate derivatives, and pregabalin (Lyrica®).

[0219] In methods of treating, preventing, and/or slowing the onset or progression of ALS or related symptoms associated with ALS, the combination of (a) an aminosterol comprising a spermine moiety, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof, and optionally (b) insulin, an immunologically active insulin fragment, an insulin derivative, or a functional equivalent of insulin, or a combination thereof can be co-administered or combined with drugs commonly prescribed to treat Amyotrophic lateral sclerosis or related symptoms, such as riluzole (Rilutek®), KNS-760704 (an enantiomer of pramipexole), olesoxime (TRO 19622), talampanel, arimoclomol, medications to help reduce fatigue, ease muscle cramps, control spasticity, reduce excess saliva and phlegm, control pain, depression, sleep disturbances, dysphagia, and constipation.

[0220] In methods of treating, preventing, and/or slowing the onset or progression of MS or related symptoms associated with multiple sclerosis, the combination of (a) an aminosterol comprising a spermine moiety, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof, and optionally (b) insulin, an immunologically active insulin fragment, an insulin derivative, or a functional equivalent of insulin, or a combination thereof can be coadministered or combined with drugs commonly prescribed to treat multiple sclerosis or related symptoms, such as corticosteroids (e.g., methylprednisolone), plasmapheresis, fmgolimod (Gilenya®), interferon beta-la (Avonex®, CinnoVex®, ReciGen® and Rebif®), interferon beta-lb (Betaseron® and Betaferon®), glatiramer acetate (Copaxone®), mitoxantrone, natalizumab (Tysabri®), alemtuzumab (Campath®), daclizumab (Zenapax®), rituximab, dirucotide, BHT- 3009, cladribine, dimethyl fumarate, estriol, fmgolimod, laquinimod, minocycline, statins, temsirolimus teriflunomide, naltrexone, and vitamin D analogs.

[0221 ] In methods of treating, preventing, and/or slowing the onset or progression of cognitive impairment or related symptoms associated with neurodegenerative disease, the combination of (a) an aminosterol comprising a spermine moiety, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof, and optionally (b) insulin, an immunologically active insulin fragment, an insulin derivative, or a functional equivalent of insulin, or a combination thereof can be co-administered or combined with drugs commonly prescribed to treat cognitive impairment, such as donepezil (Aricept®), galantamine (Razadyne®), rivastigmine (Exelon®); and stimulants such as caffeine, amphetamine (Adderall®), lisdexamfetamine (Vyvanse®), and methylphenidate (Ritalin®); NMDA antagonists such as memantine (Nameda®); supplements such as ginko biloba, L-theanine, piracetam, oxiracitam, aniracetam, tolcapone, atomoxetine, ginseng, and salvia officinalis.

[0222] In the methods of treating, preventing, and/or slowing the onset or progression of depression or related symptoms associated with neurodegenerative disease, the combination of (a) an aminosterol comprising a spermine moiety, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof, and optionally (b) insulin, an immunologically active insulin fragment, an insulin derivative, or a functional equivalent of insulin, or a combination thereof can be co-administered or combined with drugs commonly used to treat depression. These include selective serotonin reuptake inhibitors (SSRIs) such as citalopram (Celexa®, Cipramil®), escitalopram (Lexapro®, Cipralex®), paroxetine (Paxil®, Seroxat®), fluoxetine (Prozac®), fluvoxamine (Luvox®, Faverin®), sertraline (Zoloft®, Lustral®), indalpine (Upstene®), zimelidine (Normud®, Zelmid®); serotonin-norepinephrine reuptake inhibitors (SNRIs) such as desvenlafaxine (Pristiq®), duloxetine (Cymbalta®), levomilnacipran (Fetzima®), milnacipran (Ixel®, Savella®), venlafaxine (Effexor®); serotonin modulators and stimulators (SMSs) such as vilazodone (Viibryd®), vortioxetine (Trintellix®); serotonin antagonists and reuptake inhibitors such as nefazodone (Dutonin®, Nefadar®, Serzone®), trazodone (Desyrel®), etoperidone; norepinephrine reuptake inhibitors (NRIs) such as reboxetine (Edronax®), teniloxazine (Lucelan®, Metatone®), viloxazine (Vivalan®), atomoxetine (Strattera®); norepinephrine-dopamine reuptake inhibitors such as bupropion (Wellbutrin®), amineptine (Survector®, Maneon®), nomifensine (Merital®, Alival®), methylphenidate (Ritalin®, Concerta®), lisdexamfetamine (Vyvanse®); tricyclic antidepressants such asamitriptyline (Elavil®, Endep®), amitriptylinoxide (Amioxid®, Ambivalon®, Equilibrin®), clomipramine (Anafranil®), desipramine (Norpramin®, Pertofrane®), dibenzepin (Noveril®, Victoril®), dimetacrine (Istonil®), dosulepin (Prothiaden®), doxepin (Adapin®, Sinequan®), imipramine (Tofranil®), lofepramine (Lomont®, Gamanil®), melitracen (Dixeran®, Melixeran®, Trausabun®), nitroxazepine (Sintamil®), nortriptyline (Pamelor®, Aventyl®), noxiptiline (Agedal®, Elronon®, Nogedal®), opipramol (Insidon®), pipofezine (Azafen®/ Azaphen®), protriptyline (Vivactil®), trimipramine (Surmontil®), butriptyline (Evadyne®), demexiptiline (Deparon®, Tinoran®), fluacizine (Phtorazisin®), imipraminoxide (Imiprex®, Elepsin®), iprindole (Prondol®, Galatur®, Tertran®), metapramine (Timaxel®), propizepine (Depressin®, Vagran®), quinupramine (Kinupril®, Kevopril®), tiazesim (Altinil®), tofenacin (Elamol®, Tofacine®), amineptine (Survector®, Maneon®), tianeptine (Stabion®, Coaxil®); tetracyclic antidepressants such as amoxapine (Asendin®), maprotiline (Ludiomil®), mianserin (Bolvidon®, Norval®, Tolvon®), mirtazapine (Remeron®), setiptiline (Tecipul®), mianserin, mirtazapine, setiptiline; monoamine oxidase inhibitors (MAOIs) such as isocarboxazid (Marplan®), phenelzine (Nardil®), tranylcypromine (Parnate®), benmoxin (Neuralex®), iproclozide (Sursum®), iproniazid (Marsilid®), mebanazine (Actomol®), nialamide (Niamid®), octamoxin (Ximaol®), pheniprazine (Catron®), phenoxypropazine (Drazine®), pivhydrazine (Tersavid®), safrazine (Safra®), selegiline (Eldepryl®, Zelapar®, Emsam®), caroxazone (Surodil®, Timostenil®), metralindole (Inkazan®), moclobemide (Aurorix®, Manerix®), pirlindole (Pirazidol®), toloxatone (Humoryl®), eprobemide (Befol®), minaprine (Brantur®, Cantor®), bifemelane (Alnert®, Celeport®); atypical antipsychotics such as amisulpride (Solian®), lurasidone (Latuda®), quetiapine (Seroquel®); and N-methyl D- aspartate (NMD A) antagonists such ketamine (Ketalar®).

[0223] Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further includes the separate administration of one of the compounds or agents administered first, followed by the second. The regimen selected can be administered concurrently since activation of the aminosterol induced response does not require the systemic absorption of the aminosterol into the bloodstream and thus eliminates concern over the likelihood systemic of drug-drug interactions between the aminosterol and the administered drug. XIII. Definitions

[0224] The following definitions are provided to facilitate understanding of certain terms used throughout this specification.

10225] Technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise defined. Any suitable materials and/or methodologies known to those of ordinary skill in the art can be utilized in carrying out the methods described herein.

[0226] As used in the description of the invention and the appended claims, the singular forms “a”, “an”, and “the” are used interchangeably and intended to include the plural forms as well and fall within each meaning, unless the context clearly indicates otherwise. Also, as used herein, “and/or” refers to _i and encompasses, any and all possible combinations of one or more of the listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0227] “”As used herein, the phrase “therapeutically effective amount” means a dose of aminosterol, or a salt or derivative thereof that provides the specific pharmacological effect for which the compound or compounds are being administered. It is emphasized that a therapeutically effective amount will not always be effective in achieving the intended effect in a given subject, even though such dose is deemed to be a therapeutically effective amount by those of skill in the art. For convenience only, exemplary dosages are provided herein. Those skilled in the art can adjust such amounts in accordance with standard practices as needed to treat a specific subject. The therapeutically effective amount may vary based on the route of administration and dosage form, the age and weight of the subject, and/or the severity of the subject’s condition. For example one of skill in the art would understand that the therapeutically effective amount for treating a small individual may be different from the therapeutically effective amount for treating a large individual.

[0228] The term “administering” as used herein includes prescribing for administration, as well as actually administering, and includes physically administering by the subject being treated or by another.

[0229] As used herein “subject” or “patient” or “individual” refers to any subject, patient, or individual and the terms are used interchangeably herein. In this regard, the terms “subject,” “patient,” and “individual” includes mammals, and, in particular humans.

[0230] As used herein, the term “comprising” or “comprises” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of’ when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) of the claimed invention. “Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention. When an embodiment is defined by one of these terms (e.g., “comprising”), it should be understood that this disclosure also includes alternative embodiments, such as “consisting essentially of’ and “consisting of’ for said embodiment.

[02311 “Substantially” or “essentially” means nearly totally or completely, for instance, 95%, 96%, 97%, 98%, 99%, or greater of some given quantity.

[0232] The term “about” will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” will mean up to plus or minus 10% of the particular term. For example, in some embodiments, it will mean plus or minus 5% of the particular term. Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number, which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0233] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0234] As used herein, the term “treatment” or “treating” means any treatment of a disease or condition or associated disorder, in a patient, including:

[0235] Inhibiting or preventing the disease or condition, that is, arresting or suppressing the development of clinical symptoms, such as neurological deficits resulting from cerebral ischemia, also included within “treatment” is provision of neuroprotection; and/or relieving the disease or condition that is, causing the regression of clinical symptoms (e.g., increasing neurological performance or reducing neurological deficits).

[0236] In some embodiments, “treatment” encompasses “providing neuroprotection” to the subject. “Treatment” and “providing neuroprotection” may comprise the administration of the therapeutics agent(s) or compositions disclosed herein.

[0237] “Pharmaceutically acceptable salt” refers to salts of a compound, which salts are suitable for pharmaceutical use and are derived from a variety of organic and inorganic counter ions well known in the art. Pharmaceutically acceptable salts include, when the compound contains an acidic functionality, by way of example only, sodium, potassium, calcium, magnesium, ammonium, and tetraalkylammonium. When the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, and oxalate. Stahl and Wermuth, eds., “Handbook of Pharmaceutically Acceptable Salts,” (2002), Verlag Helvetica Chimica Acta, Zurich, Switzerland), which is hereby incorporated by reference for its teachings related to pharmaceutically acceptable salts, discusses a variety of pharmaceutical salts, their selection, preparation, and use.

[0238] “Reductive amination” as used herein, refers to a synthetic procedure whereby a ketone or aldehyde is reacts with an amine to form in imine or iminium which is subsequently reduced to form an amine. Methods and conditions to affect reductive amination are known to those or ordinary skill in the art. See, for example, Dangerfield et al., J. Org. Chem., 2010, 75, 5470- 5477; Taibakhsh et al., Synthesis, 2011, 490-496; and Abdel-Magid et al., J. Org.

Chem., 1996, 61, 3849-3862; the entire disclosures of which are hereby incorporated by reference.

[0239] Any references to the C25 carbon of a steroid or intermediate to the synthesis thereof refers to the carbon marked “25” in Compound III and, for example, its synthetic precursors as shown below:

[0240] It is to be understood, that in any aminosterol or steroid compound disclosed herein, the stereochemical configuration of the 17 and 20 carbons are equally represented as depicted in the two example steroid nuclei shown below, i.e., the configurations of the 17 and 20 carbons, as drawn below, are the same:

X. Examples

[02411 The following examples are provided to illustrate the present invention. It should be understood, however, that the invention is not to be limited to the specific conditions or details described in these examples. Throughout the specification, any and all references to a publicly available document, including a U.S. patent, the contents of those documents are specifically incorporated by reference.

Example 1

[0242] PS 19 mice are engineered to produce large amounts of tau-protein and develop a very aggressive form of neurodegeneration/ Alzheimer’s disease. Average lifespan in these animals is 10-11 months.

[0243] PS-19 animals were intranasally treated with ENT-03, which is an aminosterol comprising a spermine moiety, starting at 10 months (in late stages of disease) and dosed biweekly. The animals improved dramatically, becoming much faster and more agile, venturing further in exploratory behavior, indistinguishable from a young animal and from wild-type controls. Neuroinflammation in the mice was eliminated. See Fig. 34. Further, improved performance was seen in a Morris water maze (MWW) test, which is a test of spatial learning for rodents. See Fig. 35. The MWM has proven to be a robust and reliable test that is strongly correlated with hippocampal synaptic plasticity and NMDA receptor function. Fig. 35 A shows the number of training days on the X axis vs escape latency in seconds (e.g., time it takes to find the platform) for four groups of tested animals: wild type (WT) vehicle, WT ENT-03, PS19 vehicle, and PS19 ENT-03. Escape latency significantly declined for the PS19 ENT-03 group as compared to the PS 19 untreated group. Fig. 35B shows platform area (Y axis) vs each of the four groups of animals: WT vehicle, WT ENT-03, PS19 vehicle, and PS19 ENT-03.

[0244] Figure 36 shows localization to the arcuate nucleus, proximity to NPY producing cells, and action via pstat3 stimulation in the arcuate and subventricular zone (which is the neurogenic zone). The four groups of tested animals are shown on the X axis, e.g., WT vehicle, WT ENT-03, PS19 vehicle, and PS19 ENT-03. Measurement of P-STAT3, NPY, P- STAT3/NPY, and P-STAT3/NPY/DAPI is shown on the X axis. P-STAT3, or phosphorylated STAT3, is the activated form of STAT3. STAT3 is a transcription factor involved in many cellular functions. It helps control cell growth and division (proliferation), cell movement (migration), and the self-destruction of cells (apoptosis). Neuropeptide Y (NPY) is one the most potent orexigenic peptides found in the brain. It stimulates food intake with a preferential effect on carbohydrate intake. It decreases latency to eat, increases motivation to eat and delays satiety by augmenting meal size. [0245]

[0246] PS- 19 mice and normal WT controls were subjected to genomic analysis to confirm that they were in fact the PS-19 mice exhibiting the same behavior as the wild-type controls.

[0247] These treated animals were still alive and well at 15 months, which correlates with a 50% extension of life span).

Example 2

[0248] An experiment was conducted to explore the dose response to a delay in growth of B6D2F1 (BDF1) mice to an aminosterol comprising a spermine moiety, e.g., Aminsoterol 1436.

10249] 30 day old male BDF1 mice received either vehicle or Aminosterol 1436 intraveneously (IV) at 5 mg/kg or 10 mg/kg (n=15 for each treatment arm) every 3 days until 51 days of age. Aminosterol 1436 was administered as an aqueous solution in water. BDF1 mice are available from Charles River Laboratories. The animals were fed standard lab chow, offered ad libitum. The animals were weighed and body length measured every 5 days.

10250] As can be seen in Fig. 2, all animals reached the mature weight of about 40 grams. However, while the control animals reached maturity at 120 days, the animals that received 5 mg/kg of aminosterol 1436 reached maturity at 150 days, corresponding to a 25% delay in reaching maturity, as measured by end weight. In addition, the animals receiving 10 mg/kg reached maturity at 255 days, corresponding to a 112.5% delay in reaching maturity, as measured by end weight. Moreover, as can be seen in Fig. 2, the growth rate of the animals treated at 5 mg/kg was about 30% slower than the controls, while the growth rate of those treated at 10 mg/kg was slowed by 50%, where growth rate is measured by increasing weight on the y axis of Fig. 2. Finally, linear growth was slowed to a corresponding degree (data not shown). Most importantly, both treated and untreated animals reached normal adult dimensions, albeit at different rates.

[0251] These results show that administration of an aminosterol comprising a spermine moiety such as aminosterol 1436 can slow the maturation process of an animal, while the endpoint of maturity, as measured by final weight, remains constant. Moreover, the results also show that the delay in maturation is increased with an increased dose of aminosterol 1436. [0252] In a second part of this experiment, it is proposed that the animals additionally be treated with an insulin compound. The insulin compound is expected to act synergistically with the aminosterol compound comprising a spermine moiety.

Example 3

[0253] An experiment was conducted to determine whether treatment of animals with an aminosterol comprising a spermine moiety such as aminosterol 1436 would delay growth of the animal.

[0254] C57BL/6 males (12-16 grams) were administered either vehicle or 10 mg/kg (z. .) of aminosterol 1436 every 3 days for two doses, for a total of 20 mg/kg over a 6 day period. The mice were about 3 weeks old, and there were 10 mice/arm. The animals were weighed and their body length measured once weekly for a period of 40 days.

[0255] The results shown in Fig. 3 indicate that growth rates of the animals were slowed upon administration of the aminosterol comprising a spermine moiety (aminosterol 1436), which is consistent with the results shown in Fig. 2 and described in Example 2. Specifically, at Day 0 animals in the control group had a starting weight (g) of 16 g, while animals in the aminosterol 1436 group had a weight of 12 g. At day 40, the control group had a weight of 24 g, or an increase of 50%. In contrast, at Day 40 the aminosterol 1436 group had a weight of 11 g, or a decrease of 8.3%.

[0256] These results confirm that administration of an aminosterol comprising a spermine moiety such as aminosterol 1436 slows the growth rate of animals.

[0257] In a second part of this experiment, it is proposed that the animals additionally be treated with an insulin compound. The insulin compound is expected to act synergistically with the aminosterol compound comprising a spermine moiety.

Example 4: Synthesis of ENT-03

[0258] This example describes synthetic methods of making compounds described herein.

[0259] Preparation of BDG-5

[0260| BDG-4 (370.54 g, 916.3 mmol, commercially available from Bridge Organics) was dissolved in di chloromethane (DCM, 3,600 mL) and treated with ethylene glycol (210 mL, 3.77 mol, 4.11 eq.) along with /?-toluenesulfonic acid-hydrate (36.27 mmol, 0.04 eq.). The mixture was refluxed until the reaction was complete (TLC in 70:30 hexane: acetone showed ca 3-5% residual starting material that did not change). After about 4 h reflux, the mixture was cooled, treated with of 10% potassium carbonate (150 mL), and the layers separated. The DCM was reextracted with 800 mL of 5% sodium chloride solution and 10 mL of 10% potassium bicarbonate solution. The two aqueous layers were back-extracted with 500 mL of DCM. The combined DCM extracts were dried over magnesium sulfate, filtered, washed with DCM (2 x 100 mL), and concentrated under vacuum to afford a solid, which was slurried in acetonitrile (4.8 L) containing tri ethylamine (2 mL) at 55 °C for 1 h, then 35 °C for 1 hour, and finally at 12 °C for 2 h. The product was filtered and washed with cold acetonitrile (2 x 100 mL) containing tri ethylamine (0.1 mL). The crystals were dried at 50 °C in the vacuum oven (0.2 mm) overnight to afford BDG-5 (351.0 g, 85%); m.p. was 175.6-177.5° (at 2°/ minute from ca 140°); ^x H NMR (500 MHz, CDCk) 5 3.93 (m, 4H), 3.82 (br s, 1H), 3.66 (s, 3H), 2.4-2.2 (m, 2H),1.9-1.2 (m, 24H), 0.92 (d, 3H, J = 7 Hz), 0.81 (s, 3H), 0.66 (s, 3H); ¹³ C NMR (CDCk) 5 177,109, 67.9, 64.2, 64.1, 55.8, 51.4, 50.5, 45.6, 42.6, 39.5, 39.4, 37.5, 36.3, 36.1, 35.7, 35.5, 35.4, 31.2, 31.0, 30.9, 28.0, 23.6, 20.9, 18.3, 11.8, 10.4.

[02611 Preparation of BDG-6

[0262] Lithium aluminum hydride (7.44 g, 196.05 mmol, pellets) was added to anhydrous THF (435 mL) under nitrogen and stirred overnight at 20 °C to break up the pellets. The suspension was cooled to 10 °C and a solution of BDG-5 (77.57 g, 172.91 mmol) was added dropwise over 160 min. The mixture was stirred 1 h further, and was quenched by adding 2 mL of ethyl acetate, followed by dropwise addition of 20% aqueous potassium hydroxide solution (7.44 mL) over 10 min. The mixture became very thick, then thinned out and became more granular with additional stirring for 15 h at 20 °C. The mixture was filtered through Celite® 545 to remove the aluminum salts. The filter cake was washed with of THF (5 x 90 mL) to ensure that all the product was obtained in the filtrate, which was concentrated to afford crude BDG-6 (82.01 g, theory = 72.7 g). In practice in larger runs, the THF solution was concentrated to dryness, and re-concentrated with DCM to remove traces of water prior to next step; ^X H NMR (500 MHz, CDCh) 5 3.93 (m, 4H), 3.82 (br s, 1H), 3.61 (m, 2H), 1.97-1.84 (m, 4H), 1.7-1.0 (m, 22H), 0.93 (d, 3H, J = 7 Hz), 0.81 (s, 3H), 0.66 (s, 3H); ¹³ C NMR (CDCh) 5 109.3, 67.9, 64.15, 64.13, 63.5, 56.0, 50.6, 45.6, 42.6, 39.51, 39.48, 37.5, 36.3, 36.1, 35.7, 35.6, 35.5, 31.8, 31.2, 29.4, 28.2, 23.6, 20.9, 18.6, 11.8, 10.4.

[0263] Preparation of BDG-7 [0264] BDG-6 (214.55 g, 510.1 mmol) was dissolved in DCM (3.0 L) and treated with 4- dimethylaminopyridine (250.3 g, 2.048 mol) and then benzoyl chloride (180 mL, 217.98 g, 1.551 mol) dropwise. An exotherm to 31 °C was noted after about 40 mL of benzoyl chloride was added. The temperature was held to 25 °C for the remainder of the addition which took a total of 35 min. The solution was stirred overnight at 25 °C, diluted with DCM (200 mL), and treated with 10% aqueous potassium bicarbonate (2 L) generating a small amount of carbon dioxide. The layers were separated and the aqueous layer was re-extracted with DCM (500 mL). The total organic layers were dried over magnesium sulfate and concentrated to afford BDG-7 as a solid (163.6 g, 100%). In practice in larger runs, the dibenzoate solution was concentrated to a suitable volume for the next step. ^X H NMR (500 MHz, CDCk) 5 8.07 (d, 2H, J = 8 Hz), 8.00 (d, 2H, J = 8 Hz), 7.57 (t, 1H, J = 8 Hz), 7.53 (t, 1H, J = 8 Hz), 7.47 (t, 2H, J = 8 Hz), 7.41 (t, 2H, J = 8Hz), 5.16 (br s, 1H), 4.25 (m, 2H), 3.87 (m, 4H), 2.02-1.85 (m, 2H), 1.8-1.1 (m, 22H), 0.95 (d, 3H, J = 7 Hz), 0.88 (s, 3H), 0.68 (s, 3H); ¹³ C NMR (CDCh) 5 166.7, 166.0, 132.8,131.1, 130.5, 129.7, 129.5, 128.4, 128.3, 109.0, 71.9, 65.5, 64.2, 64.1, 55.8, 50.7, 47.2, 42.8, 39.5, 38.6, 37.4, 37.2, 35.7, 35.5, 35.3, 33.3, 32.0, 31.2, 28.0, 25.2, 23.6, 21.1, 18.6, 11.8, 10.5.

[0265] Preparation of BDG-8

[0266] BDG-7 (22.13 kg, 35.2 mol) was dissolved in 1 : 1 tetrahydrofuran: methanol solution (220 L) at 25 °C and treated with a solution of 50% sodium hydroxide (3.7 L, 70.4 mol, 2.0 eq.) in deionized water (11 L) and stirred for 4 h. An aliquot (0.1 mL) of the reaction mixture was partitioned between ethyl acetate (0.5 mL each) and 1 M potassium bicarbonate solution. The organic layer was analyzed by TLC (70:30 hexane: acetone) and the reaction was judged complete. An aqueous solution of potassium bisulfate was prepared by mixing deionized water (23 L), 96% sulfuric acid (3.7 L, 66.6 moles), and 45% potassium hydroxide (5.7 L, 66.7 moles). The resulting solution of potassium bisulfate was added via spray ball to the reaction mixture to a final pH of 8.39 (pH meter). The mixture was vacuum-distilled to about 60 L volume; and treated with of ethyl acetate (100 L) and water (100 L). The 2-phase mixture was agitated for 10 min, and the layers separated over about 20 min. The aqueous phase was re-extracted with 37 L of ethyl acetate. The total ethyl acetate layer (about 160 L) was vacuum concentrated to a volume of 80 L at 50 °C. Another 50 L of ethyl acetate was added, and the solution dried over anhydrous sodium sulfate (15 kg) by stirring overnight. The solution was filtered over Celite® 545 to remove the sodium sulfate. The filter cake and the reactor were rinsed with 2 x 37 L of ethyl acetate. The dried ethyl acetate solution was vacuum concentrated from about 225 L volume to 40 L. Acetonitrile (50 L) was added and the solution was re-distilled to remove ethyl acetate. A second and third batch of acetonitrile (50 and 100L) were added and evaporated. Finally, 100 L of acetonitrile was added and this solution was counter-currently extracted with 3 x 104 L of hexane to remove most of the methyl benzoate. The 3 hexane extracts were reextracted with a second 104 L of acetonitrile.

[0267] The hexane layers were checked by TLC for BDG-8 intermediate and none was present. The acetonitrile (ca. 300 L) was vacuum concentrated to ca. 50 L volume. Isopropanol (57 L) was added and concentrated to ca. 50. L; this operation was repeated 2 more times to remove all the acetonitrile. The volume was adjusted to 100 L with isopropanol and the solution slowly cooled to 0 °C and was seeded with crystalline BDG-8 to initiate crystallization. The mixture was slowly cooled to -20 °C overnight. The crystal slurry was filtered through a jacketed filter at -20 °C and rinsed with 10 L of -20 °C isopropanol. The filter cake was dried by vacuum for several h at -20 °C, followed by letting the filter warm slowly to room temperature (about 18 °C) and then drying with warm nitrogen yielding 11.92 kg of BDG-8. The mother liquors were chromatographed to isolate additional product. A column of silica gel (19 kg) was packed in methylene chloride. The filtrate of the product was vacuum concentrated to dryness and /i was added to the column in methylene chloride. Elution with 100 L of methylene chloride (DCM) gave the product. The column was flushed with 20 L of 85: 15 methylene chloride: ethyl acetate, then 20 L of 60:40 methylene chloride: ethyl acetate, and finally back to methylene chloride (40 L).

[0268] The second half of the mother liquors was similarly chromatographed to give a total of 5.32 kg of pure BDG-8 (Total yield of BDG-8: 17.24 kg, 93.3%). ^X H NMR (500 MHz, CDCk) 5 8.07 (d, 2H, J = 8 Hz), 7.57 (t, 1H, J = 7 Hz), 7.48 (t, 2H, J = 7 Hz), 5.15 (br s, 1H), 3.88 (m, 4H), 3.56 (br s, 2H), 2.02-1.84 (m, 2H), 1.75-0.98 (m, 24 H), 0.92 (d, 3H, J = 6.5 Hz), 0.88 (s, 3H), 0.67 (s, 3H). ¹³ C NMR (CDCh) 5 166.0, 132.7, 131.0, 129.7, 128.4, 109.0, 71.9, 64.2, 64.1, 63.5, 55.8, 50.7, 47.2, 42.7, 39.5, 38.6, 37.3, 37.2 35.7, 35.5, 33.3, 31.7, 31.3, 29.3, 28.0, 23.6, 21.1, 18.6, 11.8, 10.5.

[0270] A 50 L jacketed reactor under nitrogen was charged with potassium bromide (250 g, 2.23 mol), sodium bicarbonate (265 g, 3.15 mol), and water (5 L); agitated and cooled with a -5 °C jacket. To the reactor was added BDG-8 (5.32 kg, 10.14 mol) dissolved in ~13 L of di chloromethane, followed by rinse from 8 L of dichloromethane. When the reaction mixture attained a temperature <5 °C, TEMPO (50 g, 0.286 mole) was added through the manhole. The reactor was then charged via pump with 12.5% bleach (5.5 kg), keeping the mixture < 5 °C. The reaction was sampled to estimate additional amount of bleach needed. Bleach continued to be added in small portions while sampling periodically until reaction was judged to be complete (< 2% BDG-8 remaining; a total of 6.2 kg of bleach was added). A solution of sodium thiosulfate (200 g) in water (I L) was added with vigorous stirring until a negative starch/iodide test was obtained. Hexane (20 L) was added to the reactor with stirring and the layers settled. The lower aqueous phase was drained, and the organic layer was washed with saturated aqueous potassium bicarbonate (5 L). The organic phase was vacuum filtered through a pad of sodium sulfate/silica gel (1 kg of each) in a sintered glass filter funnel and rinsed with hexane/di chloromethane (1/1), di chloromethane, and finally 5% methyl -t-butyl ether (MTBE) in dichloromethane. The combined filtrates were evaporated in two batches in a Buchi apparatus at 40 °C. Hexane was added to each batch and evaporated again until a thick slurry formed. The solids were filtered, washed with hexane, and dried in a vacuum oven to afford 1 (4.58 kg). The filtrates and washings were combined and concentrated to get a second crop, which was filtered and washed with 5% MTBE/hexane, then hexane, and dry to get another 565 g of product for a total yield of 5.145 kg (97% yield) of 1.

[0271] The material contained about 1.5% of residual starting material, but no detectable (NMR) carboxylic acid. ’H NMR (500 MHz, CDCh) 5 9.72 (s, 1H), 8.07 (d, 2H, J = 7 Hz), 7.59 (t, 1H, J = 7 Hz), 7.49 (t, 2H, J = 7 Hz), 5.16 (br s, 1H), 3.88 (m, 4H), 2.45-2.27 (m, 2H), 2.00- 1.85 (m, 2H), 1.78-1.17 (m, 22H), 0.913 (d, 3H, J = Hz), 0.88 (s, 3H), 0.68 (s, 3H). ^U C NMR (CDCh) 5 203, 166, 132.8, 131.0, 129.8, 128.4, 109, 72.0, 64.17, 64.11, 55.7, 50.7, 47.2, 42.8, 40.8, 39.5, 38.6, 37.3, 37.2, 35.7, 35.5, 35.4, 33.3, 31.2, 27.9, 27.8, 23.7, 21.1, 18.3, 11.7, 10.5.

[0272] Preparation of Compound 2:

[0273] The phosphonate (A, 3.69 g, 15 mmol) was added to anhydrous tetrahydrofuran (100 mL) and chilled in a salt ice bath to ~0 °C. Potassium tert-butoxide (1.72 g, 15 mmol) was added with vigorous magnetic stirring under nitrogen and the reaction was allowed to stir for 30 min. Compound 1 (8.0 g, 15 mmol) was added and dissolved in tetrahydrofuran (80 mL), the ice bath was removed and the reaction was allowed to warm to RT overnight. The reaction after approximately 16 h total was worked up by partitioning between hexane/ ethyl acetate (50/50, 400 mL) and water (400 mL). The organic layer was washed with an additional portion of water (100 mL) and the organic layer was dried over Na2SC>4, filtered, and the solvent removed in vacuo. The residue was redissolved in a minimal amount of hexane/ethyl acetate 3/1 and passed through a plug of silica gel ~3 x 9 in.

[0274] The eluant was then roto-evaporated to yield Compound 2 (8.3 g, 12.4 mmol, 83%) of satisfactory purity to utilize in the next step without further purification, ^T H NMR (CDCk, 300 MHz) 6 8.10 - 8.07 (m, 2H), 7.60 - 7.57 (m, 1H), 7.52 - 7.47 (m, 2H), 6.7, 5.9 (t, 1H), 5.17 (m, 1H), 4.21 - 4.12 (m, 2H), 3.92 - 3.88 (m, 4H), 2.06 (s, 3H), 2.2 - 1.0 (m, 29 H), 0.95 (d, 3H, J = 7 Hz), 0.90 (s, 3H), 0.69 (s, 3H); MS (ES+) 485.45 (M-C7H7O2+H).

[0275] Preparation of Compound 3:

[0276] Compound 2 (8.25 g, 13.5 mmol) was dissolved in anhydrous ethanol and 10% Pd on C (400 mg) was added under N2 in a Parr bottle (500 mL). The flask was flushed and filled 2x with vacuum and N2 then hydrogenated at 50 psi for 24 h. The uptake of hydrogen had slowed to a near stop, but TLC showed a possible trace of starting material. An additional portion of catalyst (400 mg) was added, and the reaction was allowed an additional 12 h.

[0277] Filtration of catalyst and removal of the solvent in vacuo gave the saturated product in quantitative yield Compound 3 (8.25 g, 13.5 mmol), ^X H NMR (CDCh, 300 MHz) 6 8.09 - 8.06 (m, 2H), 7.58 - 7.56 (m, 1H), 7.55 - 7.45 (m, 2H), 5.16 (m, 1H), 4.12 - 4.05 (m, 2H), 3.90 - 3.85 (m, 4H), 2.39 - 2.36 (m, 1H), 2.0-1.0 (m, 35 H), 1.11 (d, 3H, J = 7Hz), 0.88 (s, 3H), 0.67 (s, 3H); MS (ES+) 487.46 (M-C7H7O2+H).

[0278] Preparation of Compound 4:

[0279] Compound 3 (8.2 g, 13.5 mmol) was dissolved in 3/1/1 tetrahydrofuran/methanol/lM KOH (-100 mL) and stirred until hydrolysis of the ethyl ester appeared to be complete by TLC. There was no evidence of benzoate hydrolysis under these conditions. The solution was neutralized with 1 M hydrochloric acid solution, evaporated to remove the organic solvent, treated with acetone (-100 mL), and evaporated again to ensure removal of any methanol. Acetone (-250 mL) was added to the flask and 3M HC1 was added to lower the pH to the point where it registered in the 1-2 range by pH paper. The hydrolysis of the ketal was carried out overnight at RT, water was then added to the flask, and the majority of the acetone was removed in vacuo. The material was partitioned between ethyl acetate and water, and then the organic layer washed with brine. The organic layer was dried in vacuo to give Compound 4 (6.36 g, 11.9 mmol, 87%) of satisfactory purity to be utilized without further purification, ’H NMR (CDCh, 300 MHz) S 8.05 - 8.02 (m, 2H), 7.60 - 7.57 (m, 1H), 7.51 - 7.45 (m, 2H), 5.21 (m, 1H), 2.4- 1.0 (m, 32 H), 1.15 (d, 3H, J = 7 Hz), 1.09 (s, 3H), 0.91 (d, 3H, J = 7 Hz), 0.67 (s, 3H); MS (ES+) 415.52 (M-C7H7O2+H).

[0280] Preparation of Compound 5: [0281 ] Compound 4 (3.5 g, 6.5 mmol) was dissolved in methanol (100 mL) and treated with spermine (5 g, 24.8 mmol) in methanol (-10 mL). The mixture was stirred for 2 h at RT after which 2-propanol (100 mL) was added, and the majority of the solvent was removed in vacuo. The residue was redissolved in methanol (200 mL) and stirred overnight. Isopropyl alcohol (200 mL) was added and the mixture was evaporated to a thick residue. The residue was dissolved in anhydrous methanol (200 mL), and the solution chilled in a dry ice acetone bath under N2 with vigorous magnetic stirring. When the internal temperature reached - -74 ⁰ C, NaBHi (1.89 g, 50 mmol) was added. The temperature was maintained with the dry ice acetone bath for -4 h and then allowed to come to RT overnight. The reaction mixture was carefully acidified with 10% trifluoroacetic acid in water until pH paper showed pH 2-3 range. Water was added to the mixture, and the mixture was transferred to an oversized flask (to allow for frothing of the mixture on rotary evaporation) and the majority of the methanol removed in vacuo. The resulting solution was applied directly to amberchrome and eluted with a step gradient of acetonitrile in water with 0.5% TFA (10% increments 500 mL per increment) until aminosterol eluted (-60% acetonitrile). The gradient was held at this point until all of the aminosterol eluted.

[0282] The fractions containing aminosterol were analyzed and the relatively clean fractions pooled and lyophilized to afford Compound 5 as the tetra-TFA salt (-4.5 g, 3.8 mmol) of sufficient purity to carry on without further purification, ^X H NMR (CD3OD, 300 MHz) 6 8.05 - 8.02 (m, 2H), 7.66 - 7.60 (m, 1H), 7.54 - 7.49 (m, 2H), 5.17 (m, 1H), 3.36 - 3.04 (m, 13H), 2.37 (m, 1H), 2.1-1.0 (m, 39 H), 1.10 (d, 3H, J = 7 Hz), 0.95 (m, 6H), 0.74 (s, 3H); MS (ES+) 723.78 (M+H).

[0283 ] Preparation of ENT-03 (Compound III): [0284] Compound 5 (3.0 g, 2.5 mmol) was added to of 5% methanolic potassium hydroxide (40 mL), and the solution was stirred for 2 days under N2 at 110 °C, and monitored with TLC (6:3: 1 Chloroform, Methanol, cone. NH4OH). After 2 days, the reaction was cooled to room temperature, evaporated under vacuum, and dissolved in H2O (40 mL). This solution was then acidified with 6M HC1, and the white precipitate was forced back into solution with gentle heat and stirring. The solution was poured onto a large column of Amberchrome, and washed with H2O (350 mL), increments (500 mL) of 10%, 15%, and 25% acetonitrile/water. Almost as soon as one column volume of 25% solution had passed through, the compound began to elute. The fractions (40 mL) that were collected were analyzed via LC/MS to separate away the 3 -a sideproduct, which came off the column immediately following the desired 3-0 product. Although there was some co-elution, a significant portion of the material came off cleanly.

[0285] These fractions were combined and lyophilized overnight to give (1.31 g, 1.7 mmol, 68%) of ENT-03 (Compound III) as the tetra-HCl salt, ’H NMR (CD3OD, 300 MHz) S 3.80 (br s, 1H), 3.20 - 3.05 (m, 13H), 2.37 (m, 1H), 2.2-1.0 (m, 36 H), 1.13 (d, 3H, J = 7 Hz), 0.93 (d, 3H, J = 7 Hz), 0.87 (s, 3H), 0.69 (s, 3H). ENT-03 was analyzed for purity via HPLC (Waters Acquity ELSD) under the following conditions: Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in acetonitrile; Column: Kinetex XB-C18 (2.1 x 75 mm, 1.7pm); Gradient: 5-95%/8 min, hold 95% B/l min; 0.6 mL/min flowrate; ELSD detector; Retention time: 1.96 min and 99.9% peak area. ENT-03 was analyzed by mass spectrometry (Waters Acquity TQD); Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid acetonitrile; Column: Kinetex XB-C18 (2.1 x 75 mm, 1.7 pm); Gradient: 5-95%/8 min, hold 95% B/l min; Flowrate: 0.6 ml/min; MS (ES+, M+H): Calc’d 619.55; Found: 619.31.

| 0286| Preparation of ENT-035: NaH

|0287| Tetrahydrofuran (THF, 10 mL) was added to 60% sodium hydride (330 mg, 8.25 mmol) in a 50 mL flask under nitrogen while cooling in an ice bath. Triethyl 2- phosphonopropionate (A, 1.31 g, 5.55 mmol) was added neat by syringe over 5 min so that gas evolution was controlled. The solution was stirred another 10 min after the addition ended. The substrate aldehyde 1 (2.613 g, 5.00 mmol) was dissolved in THF (4 mL) and this was added to the flask over 5 minutes, keeping the reaction temperature at 3-8°C and washing in the residue with THF (0.5 mL) twice. The reaction was checked by thin layer chromatography (TLC) using 4: 1 heptane:EtOAc and Hannesians stain. Conversion was complete on the first check 5 min after the addition. At 10 minutes, a saturated NH4Q (10 mL) solution was added at once with rapid stirring. The mixture was diluted with water until the solids dissolved. The aqueous phase was extracted with ethyl acetate (2 x 5 mL), and the combined organic phases were dried over Na2SC>4 and concentrated to afford a gel (2.77 g). The TLC before and after workup shows an increase in the polar impurity spots. The crude was analyzed by LC-MS and ’H-NMR. The E:Z ratio of the product was 5.0: 1.0 by the NMR shifts at 6.68 and 5.84 ppm. The crude was purified by MPLC using a 25 g Biotage Snap column. The crude was loaded onto the column with DCM and the product was eluted with 0 to 35% EtOAc in hexanes over 12 column volumes. The top spot on the TLC was collected. The Rf of this spot was 0.65 in 4: 1 4: 1 heptane: EtOAc. The more polar spots present in the crude were removed by this purification to yield Compound 2 (1.99 g, 66%, E:Z ratio remained 5: 1 after column) as a white foam. ^X H NMR (CDCh, 300 MHz) 6 8.12-8.03 (m, 2H), 7.64-7.53 (m, 1H), 7.53-7.42 (m, 2H), 6.70 (td, J = 7.4, 1.6 Hz, 0.8H), 5.8 (t, 0.2H), 5.16 (m, 1H), 4.16 (q, J = 7.1 Hz, 2H), 3.93-3-83 (m, 4H), 2.2-1.0 (m, 29 H), 1.27 (s, 3H), 0.98 - 0.85 (m, 6H), and 0.68 (s, 3H); MS (ES+, M+Na): Cak’d: 629.38; Found: 629.77.

[0288] Preparation of Compound 22.

Molecular Weight: 606.84 Molecular Weight: 578.79

[0289] Compound 2 (1.43 g, 2.36 mmol) was combined with methanol (12.4 mL), tetrahydrofuran (12.4 mL), and 3 M NaOH in water (5.76 mL, 17.3 mmol) in a 50 mL flask with a septum and stir bar. The homogeneous mixture was stirred at 50°C and monitored by LC-MS at 220 nm. At 90 minutes, the area percent of the desired product was 87 percent. 2M citric acid was added until the pH was 3.5, and the THF and MeOH were removed by rotavap at 30 degrees. The aqueous layer was extracted with ethyl acetate (3 x 20 mL) and the combined organic phase was dried with Na2SC>4 and concentrated under vacuum to afford an amorphous white solid (1.488 g). The crude material was purified by Biotage MPLC with a 50 g Snap Ultra column. The product was loaded with a minimal amount of DCM and eluted with a gradient of 0 to 50% EtOAc in hexanes using 20 column volumes. Peaks were collected with a 254 nm detection threshold of 20 mAU. Partial separation of (E) and (Z)-olefins was seen in the fractions. The earlier fractions were combined and analyzed by 1H-NMR. These were enriched for the (Z) isomer as seen by the peak at 6.0 ppm. This was combined with the other productcontaining fractions to give Compound 22 as a white foam (930 mg, 68%) of 10:1 E:Z ratio. ’H NMR (CDCh, 300 MHz) S 8.12-8.03 (m, 2H), 7.64-7.53 (m, 1H), 7.48 (ddt, J = 8.4, 6.7, 1.3 Hz, 2H), 6.84 (td, J = 7.5, 1.6 Hz, 1H), 5.16 (m, 1H), 4.27-4.11 (m, 1H), 3.98-3.81 (m, 4H), 2.3-1.0 (m, 26H), 1.80 (s, 3H), 0.92 (d, 3H), 0.88 (s, 3H) and 0.68 (s, 3H); MS (ES+, M+Na): Cak’d: 601.35; Found: 601.77. [0290] Preparation of (A)-23

[02911 In the glovebox, (Me-Allyl)2Ru(COD) (93.66 mg, 0.293 mmol) and SL-M004-2 (308.74 mg, 0.293 mmol) were added to a 40 mL vial with a septum cap and stir bar. After adding isopropyl acetate (36.17 mL) with rapid stirring at room temperature, a solution of 48% HBr (72.97 pL, 0.645 mmol) in water was added dropwise and the mixture was stirred for 1 h in the glovebox. Compound 22 (3.965 g, 6.85 mmol) was added to each reactor of the HEL instrument in air as a solid. The reactors were sealed and purged with 200 psi of nitrogen 6 times, then with 200 psi of hydrogen 6 times. The orange catalyst solution was removed from the glovebox, the septum was fitted with a nitrogen balloon, and the catalyst solution (17.13 mL, 0.137 mmol) was injected into each purged reactor using a syringe with a 10 inch needle. The ports were shut, and the reactions were pressurized with hydrogen and heated to the desired temperature and pressure with 1000 RPM stirring. Hydrogen uptake was monitored to judge reaction completion. A blank of 21 mL of iPrOAc was subjected to the same program in another reactor to gauge hydrogen uptake by the solvent. Upon completion, the reactions were analyzed by UPLC (Table 4) and chiral HPLC to determine conversion, area percent (AP) of product S- 23, and de of (A)-23 (Table 5). Excellent conversion and stereocontrol was observed under both conditions studied (Table 2) in delivering Compound (5)-23. ^X H NMR (CDCk, 300 MHz) 6 8.06 (m, 2H), 7.6-7.4 (m, 3H), 5.15 (m, 1H), 3.88 (m, 4H), 2.42 (m, 1H), 2.4-1.0 (m, 31 H), 0.88 (m, 6H) and 0.66 (s, 3H). a) Conditions: 6.85 mmol 22 (> 27.6: 1 E:Z) in 16.9 mL z-PrOAc with catalyst, 1000 rpm, 30 °C for R1 or 50 °C for R2. AP of substrate 22 was 87.8 by the achiral UPLC method, b) Determined by the achiral UPLC method, c) Determined by the chiral UPLC method.

[0292] Preparation of Compound (l?)-23. [0293] In the glovebox, SL-M004-1 (111.2 mg, 0.1056 mmol) was added to a 20 mL screwcap vial with a stir bar, followed by (Me-Allyl)2Ru(COD) (33.7 mg, 0.1056 mmol). In a separate 20 mL vial, 48 wt.% HBr (39.3 mg, 0.233 mmol) in water was combined with isopropyl acetate (14.51 mL), making a solution of 0.016 M HBr solution. The HBr solution (13.2 mL) was added to make a catalyst solution of 0.008 M theoretical concentration. This was stirred in the glovebox for 30 minutes at room temperature. In the Biotage Endeavor reactor, Compound 22 (695 mg, 1.2 mmol) of was added and the reactor was sealed. Using the Endeavor control software, the reaction vessels were purged 10 times with 50 psi of nitrogen, then 10 times with 50 psi hydrogen. The catalyst solution (3 mL) was injected into the reactions vessels that were purged with H2 and that each contained Compound 22. The pre-programmed reaction operation was started, raising each reaction to the set temperature, then pressurizing with hydrogen to the set pressure for 16 h. The glass reaction vials were removed from the reactor and 30 pL of the reaction liquid was diluted with 4 mL of MeCN, filtered and analyzed by UPLC and chiral HPLC by the same methods described in Tables 4 and 5. Excellent conversion and stereocontrol was observed under all conditions studied (Table 3) in delivering Compound (l?)-23. ’H NMR. (CDCh, 300 MHz) S 8.06 (m, 2H), 7.6-7.4 (m, 3H), 5.15 (m, 1H), 3.88 (m, 4H), 2.42 (m, 1H), 2.4-1.0 (m, 31 H), 0.88 (m, 6H), and 0.66 (s, 3H).

a) RH was done on a 10 pmol scale. All others were done on a 1.2 mmol scale, b) Actual readings are shown, c) E:Z ratio was determined by proton NMR. TBD = to be determined, d) APs were measured at 238 nm.

[0294] Generation of Compound (l?)-24 for x-ray.

Exact Mass M+H: 477.36

(f?)-23 (K)' ²⁴

[0295] Compound (l?)-23 (lot 830-050) was hydrolyzed in 5% KOH in methanol for 48 h. After acidified with half saturated citric acid solution in water, the crude product was extracted into ethyl acetate, dried over sodium sulfate, and evaporated. The material was dissolved in hot ethyl acetate, allowed to crystallize at room temperature, and collected by vacuum filtration. Large plate crystals of (l?)-24 suitable for X-ray structure determination were grown by recrystallization of a sample (50 mg) in EtO Ac. MS (ES+, M+H): Calc’d: 477.36; Found: 477.30; ^X H NMR (400 MHz, DMSO) 5 11.98 (s, 1H), 4.06 (d, J = 4.0 Hz, 1H), 3.80 (s, 4H), 3.57 (t, J = 3.2 Hz, 1H), 2.32 - 2.23 (m, 1H), 1.88 (ddq, J = 12.6, 9.4, 3.3 Hz, 2H), 1.74 (ddt, J = 12.9, 6.9, 3.0 Hz, 1H), 1.70 - 1.55 (m, 2H), 1.55 - 1.42 (m, 4H), 1.42 - 1.37 (m, 1H), 1.37 - 1.30 (m, 4H), 1.30 - 1.21 (m, 5H), 1.21 - 1.15 (m, 4H), 1.12 (dd, J = 8.3, 3.8 Hz, 2H), 1.02 (d, J = 6.9 Hz, 6H), 1.00 - 0.91 (m, 2H), 0.86 (d, J = 6.5 Hz, 3H), 0.72 (s, 3H), 0.59 (s, 3H); ¹³ C NMR (101 MHz, DMSO) 5 177.99, 108.96, 66.05, 63.98, 56.13, 50.52, 45.44, 42.40, 39.21, 37.91, 37.03, 35.98, 35.82, 35.76, 35.60, 35.53, 34.21, 31.41, 28.29, 23.54, 23.48, 21.07, 18.99, 17.38, 12.20, 10.67.

[0296] The X-Ray structure of (l?)-24 was determined on a Bruker D8 QUEST Single-crystal X-ray Diffractometer, equipped with high brightness IpS 3.0 microfocus (50kV x 1 mA) for Cu radiation (X = 1.54178 A) and with PHOTON II Charge-Integrating Pixel Array Detector of superior speed, sensitivity, and accuracy, was used for screening/evaluation of crystals and for diffraction data collection, as described below. The analogous S isomer may also be prepared and analyzed similarly from Compound 1-5. Bruker APEX3 software suite including SHELXTL was used for diffraction experiments including data collection and integration, and for solving, refining, displaying, and publishing of structural results. A Cryostream 800 PLUS low temperature device was used. Keeping a crystal in a cold nitrogen gas stream prevents possible decay and reduces thermal motion of atoms and increases scattering power leading to better quality structures. A clear colorless plate-like crystal, approximate dimensions 0.020 mm x 0.120 mm x 0.260 mm, was used for the X-ray crystallographic analysis. The X-ray intensity data were measured (X = 1.54178 A) at 223K. A total of 804 frames were collected. The total exposure time was 1.33 hours. The frames were integrated with the Bruker SAINT software package using a narrow-frame algorithm. The integration of the data using a monoclinic unit cell yielded a total of 34512 reflections to a maximum 9 angle of 70.21° (0.82 A resolution), of which 9503 were independent (average redundancy 3.632, completeness = 96.9%, Rint = 8.34%, Rsig = 8.13%) and 6783 (71.38%) were greater than 2o(F2). The final cell constants of a = 11.2128(3) A, b = 11.1701(3) A, c = 21.0641(6) A, p = 91.458(2)°, volume = 2637.38(13) A ³ , are based upon the refinement of the XYZ-centroids of 9993 reflections above 20 c(I) with 8.398° < 29 < 132.0°. Data were corrected for absorption effects using the Multi-Scan method (SADABS). The ratio of minimum to maximum apparent transmission was 0.639. The calculated minimum and maximum transmission coefficients (based on crystal size) are 0.8530 and 0.9870. The structure was solved and refined using the Bruker SHELXTL Software Package, using the space group P 1 21 1, with Z = 4 for the formula unit, C29H48O5. The final anisotropic full-matrix least-squares refinement on F2 with 630 variables converged at R1 = 5.52%, for the observed data and wR2 = 15.29% for all data. The goodness-of-fit was 1.035. The largest peak in the final difference electron density synthesis was 0.348 e'/A3 and the largest hole was -0.246 e'/A ³ with an RMS deviation of 0.051 e'/A ³ . On the basis of the final model, the calculated density was 1.200 g/cm3 and F(000), 1048 e'. Results confirmed assignment of the C- 25 stereochemistry as the //-configuration. The absolute configuration (as shown below) was determined with single crystal diffraction data collected at 223K (see Fig. 4).

[0297] Compound (5)-23 (lot 830-051, de = 96%) and (l?)-23 (lot 830-050, de = 90%) were converted to ENT-035 and ENT-031?, respectively, by the same means described above of the conversion of Compound 3 to ENT-03. ENT-035 and ENT-031? were analyzed by HPLC on a Kromasil, 100-5-C18, 5 pm, 250 x 4.6 mm, P#M05CLA25 column, using a Thermo Ultimate 3000 UPLC with CAD detector. The samples analyzed were ENT-055, Lot# ASI-ESJ-2020- 052A1 and ENT-031?, Lot#ASI-ESJ-2020-048Al. The ^X H NMR and MS data were identical to those obtained for ENT-03. The samples were differentiated by HPLC as described in method below and depicted above in the image of (R)-24. The injection volumes of 1.0 to 2.0 pL appeared best without significantly sacrificing the separation. As can be seen in Figure 33, baseline separation of the two isomers was achieved. The diastereomeric excess of these lots of ENT-035 and ENT-031? were determined to be 96% and 90%, respectively.

[0298] Synthesis of Compound III-5 and Compound III-l?

{0299] Compound S-(23) and R-(23) was subjected to acetone (-250 mL) was added to the flask and 3M HC1 was added to lower the pH to the point where it registered in the 1-2 range by pH paper. The hydrolysis of the ketal was carried out overnight at RT, water was then added to the flask, and the majority of the acetone was removed in vacuo. The material was partitioned between ethyl acetate and water, and then the organic layer washed with brine. The organic layer was dried in vacuo to give the ketone which was subjected to the same conditions as described above in “Preparation of Compound 5” except spermine (for preparation of C25 stereodefined ENT-03 isomers or spermidine for C25 stereodefined ENT-06 isomers, respectively.

10300] Preparation of ENT-03-d3:

[0301 [ Compound 1 (2.6 g, 5 mmol) was dissolved in tetrahydrofuran (THF, 50 mL) and stirred over a sodium citrate/ citric acid buffer ~0.5 M (100 mL). Sodium chlorite (900 mg, 10 mmol) was dissolved in deionized (DI) water (20 mL) and added to the aldehyde solution with ice bath cooling. A slight yellow color was observed, and TLC (SiCh, 15% EtOAc in hexane) shows a rapid conversion to the acid within 20 min. Ethyl acetate/hexane 50/50 (100 mL) was added, the aqueous removed, and the organic layer was washed with water, 10% thiosulfate solution, and brine to give the desired product 11 of satisfactory purity without further purification (2.7 g, 5 mmol, 100%).

[0302] Compound 11 (2.6 g, 4.9 mmol) was dissolved in N-methylpyrrolidone (NMP, 25 mL) and anhydrous K2CO3 (-2 g) was added and the reaction mixture was stirred under nitrogen for 20 min. lodomethane (2.8 g, 20 mmol) was added in one portion and the reaction was stirred overnight under nitrogen. The reaction showed complete conversion to Compound 12 in nearly quantitative yield (2.6 g, 4.9 mmol).

[0303] Compound 12 (1.3 g, 2.3 mmol) was dissolved in anhydrous THF (30 mL) and anhydrous lithium chloride (~1 g) was added. This solution was warmed to 40 °C and stirred under nitrogen. A solution of NaBD4 in EtOD was prepared in 500 mg batches (2 x 3 mL) by dissolving the NaBD4 in EtOD at room temperature and then chilling to prevent decomposition via the alkoxyborohydrides. The NaBD4 solution was added in portions over time (4 h) and then warmed to 50 °C and stirred overnight under nitrogen. The reaction was diluted with water and extracted with 50/50 hexane/ethyl acetate. Organic layers were pooled dried over Na2SO4 and concentrated in vacuo. The residue was chromatographed on silica gel with 25% ethyl acetate in hexane to give the desired Compound 13 as a white solid (1.1 g, 2.0 mmol, 87%).

[0304] Compound 13 (1.0 g, 1.9 mmol) was dissolved in dichloromethane (50 mL), treated with activated 4A sieves (~1 g) and 4-methylmorpholine N-oxide (500 mg, 4.3 mmol), and stirred 10 min at rt. Tetrapropylammonium perruthenate (~40 mg) was added to the reaction and stirring was continued at rt overnight under nitrogen. This gave a single product matching the unlabeled Compound 1 by TLC. ’H NMR did not show the aldehydic proton as expected.

Molecular Weight: 523.73210 Molecular Weight: 607.85010

[0305] Triethyl phosphonopropionate (500 mg 2.0 mmol) was dissolved in EtOD and catalytic sodium ethoxide was added, stirred for 2 h, and the reaction mixture stripped to exchange the majority of the acidic methylene protons for deuterium. The phosphonate was added to anhydrous THF (30 mL) and treated with potassium t-butoxide (225 mg 2.0 mmol) at 0 °C for 30 min under nitrogen. The aldehyde 14 (1.0 g 1.9 mmol) was added in one portion at 0 °C in THF (~5 mL) with rinses. The ice bath was removed, and the reaction mixture was allowed to run overnight at rt. The reaction mixture became slightly cloudy relatively quickly and is likely done in 1-2 h. The reaction mixture was partitioned between hexane/ethyl acetate 50/50 (-100 mL) and water, and then washed with brine. The organic was dried over Na2SC>4 and the solvent removed in vacuo. The relatively clean material was chromatographed on silica gel with a hexane ethyl acetate gradient to afford Compound 15 (860 mg, 1.4 mmol, 74%).

Molecular VXfeight: 607.85010 Molecular Weight: 611.87831

[0306] The unsaturated ester 15 (860 mg, 1.4 mmol) was dissolved in ethyl acetate containing 20% EtOD and treated with the catalyst (10% Pd on Carbon, -100 mg) under nitrogen. The reaction was purged and backfilled with deuterium gas and then charged to 40 PSI in a 500 mL Parr shaker bottle. Agitation at room temperature overnight yielded a slight decrease in pressure. After an additional 8 h, no additional uptake was observed, so the reaction mixture was purged and backfilled with nitrogen three times and suction filtered through Celite®. The filtrate was evaporated in vacuo to give Compound 16 (850 mg, 1.4 mmol, 100%).

(0307) Using the same procedures described for conversion of Compound 3 to ENT-03 (Compound III), Compound 16 was converted to ENT-03-d3. ^X H NMR (D2O, 400 MHz) 6 3.90 (br s, 1H), 3.06-3.01 (m, 13H), 2.0-1.0 (m, 34 H), 1.02 (s, 3H), 0.80 (br s, 3H), 0.73 (s, 3H), and 0.57 (s, 3H). ENT-03-d3 was analyzed for purity via HPLC (Agilent) under the following conditions: Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in acetonitrile; Column: Kinetex XB-C18 (2.1 x 75 mm, 1.7 pm); Gradient: 5-95%/8 min, hold 95% B; 0.6 ml/min flowrate; ELSD detector; Retention time: 4.93 and 95.5% peak area. ENT- 03-d3 was analyzed by LC/MS (Waters Aquity HPLC-ZQ MS); Mobile phase A: 0.1% formic acid in water; Mobile phase B: acetonitrile; Column: Waters XBridge C18 (4.6 x 50 mm, 3.5 pm). Flowrate: 1.1 mL/min. MS (ES+, M+H): Calc’d: 622.57; Found: 622.60.

[0308] Preparation of ENT-03-d4:

[0309] Compound 4 (2.14 g, 4.0 mmol) was dissolved in MeOD (50 mL) and 10% K2CO3 in D2O was added (lOmL). The mixture was stirred under nitrogen at reflux for 16 h. The solvent was removed in vacuo and the residue was resuspended in MeOD (50 mL) and stirred at reflux for 8 h. The only change by TLC was the appearance of a small amount of unprotected C-7 hydroxyl, which was not a concern. The solvent was removed in vacuo and the Compound 21 was resuspended in MeOD (50 mL). Spermine (2.1 g, 10 mmol) was exchanged with MeOD (3x10 mL) by dissolving in the methanol stirring for 5 min. then removing the solvent in vacuo. A final portion of MeOD was added and the spermine solution was added to the exchanged sterol and the mixture was stirred overnight at room temperature. The solvent was removed in vacuo and the residue resuspended in MeOD (50 mL). This solution was stirred at rt for ~9 h before chilling in a dry ice acetone bath for 40 min. Solid NaBEL (740 mg, 20 mmol) was added as a solid in two portions approximately 15 min apart. The bath was charged with dry ice and allowed to warm to rt overnight. Thin layer chromatograpy (TLC, SiO2 6/3/1 CHCE/MeOH/NFLOH) of the reaction at ~14 h reaction time showed no starting material and very little sign of 3 -hydroxI of Compound 20. The reaction was diluted with deionized (DI) water and acidified slowly with trifluoro acetic acid (TFA) until strongly acidic by pH paper (pH<2). This gave a thick white precipitate, which was filtered, washed with 0.5% TFA in water, and then dissolved in 5% KOH in MeOH (100 mL) and refluxed overnight under nitrogen. After overnight reflux, the C7- benzoate hydrolysis was complete by TLC. The majority of the methanol was removed in vacuo and the material diluted with DI H2O. The material was applied directly to an Amberchrome packed column (5x20 cm) and washed with water until the strongly basic portion had eluted. The column was then washed with 1% TFA and 5% acetonitrile in water until the eluant was acidic. A step gradient was run in 5% steps, and fractions containing the cleanest material by TLC pooled and the majority of the solvent removed in vacuo near the end of the solvent removal. [0310] A solution (1 mL) of 6 N HC1 was added and the solvent removed to exchange TFA for HC1. This was repeated three times and 2-propanol (100 mL) was added and quickly stripped off to give a free flowing white solid (700 mg, 0.96 mmol, 24% overall yield) in clean fractions of ENT-03-d4. ^X H NMR (D ₂ O, 400 MHz) S 3.77 (br s, 1H), 3.06-2.99 (m, 13H), 2.36 (m, 1H), 2.0-1.0 (m, 33 H), 1.05 (d, 3H), 0.84 (br s, 3H), 0.73 (s, 3H), and 0.57 (s, 3H). ENT-03-d4 was analyzed for purity via HPLC (Agilent) under the following conditions: Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in acetonitrile; Column: Kinetex XB- C18 (2.1 x 75 mm, 1.7 pm); Gradient: 5-95%/8 min, hold 95% B; 0.6 ml/min flowrate; ELSD detector; Retention time: 4.94 and 100% peak area. ENT-03-d4 was analyzed by LC/MS (Waters Aquity HPLC-ZQ MS); Mobile phase A: 0.1% formic acid in water; Mobile phase B: acetonitrile; Column: Waters XBridge C18 (4.6 x 50 mm, 3.5 pm). Flowrate: 1.1 mL/min. MS (ES+, M+H): Cak’d: 623.58; Found: 623.31.

Example 5: ENT-03 (Compound III) activity as an inhibitor of protein tyrosine phosphatase IB (PTP1B)

[0311 ] This example tested the PTP1B inhibitory activity of ENT-03 (Compound III) and ENT-02 (MSL1436). Also included is comparative data of the inhibitory activity of D?-1436 (D-1436), an aminosterol derivative having the structure:

D ₇ -1436

[0312] ENT-02 (MSI-1436), ENT-03 (Compound III), and D-1436 were dissolved in dimethyl sulfoxide (DMSO) to a stock concentration of 10 mM. A known PTP1B inhibitor, 3-(3,5- Dibromo-4-hydroxy-benzoyl)-2-ethyl-benzofuran-6-sulfonicacid -(4-(thiazol-2-ylsulfamyl)- phenyl)-amide (Wiesmann et al., 2004), served as a control. The compounds were tested in a 10- dose IC50 mode with 3 -fold serial dilution, in singlet, starting at 100 pM. The enzyme was the human truncated form (1-321), recombinantly produced in Escherichia coli. Fluorescence was measured to monitor enzyme activity. The phosphatase activities were monitored as a timecourse measurement of the increase in fluorescence signal from fluorescent substrate, and initial linear portion of slope (signal/min) was analyzed. No compounds exhibited fluorescent background that could interfere with the assay.

10313] IC50 curves were generated for the three aminosterols tested (Fig. 8A) and the control compound (Fig. 8B). Curve fits were performed when the activities at the highest concentration of compounds were less than 65%.

[0314] As seen in Table 6, ENT-02 (MSI-1436), already known to inhibit PTP1B, exhibited an IC50 of 2.89 pM; ENT-03 (Compound III) exhibited an IC50 of 1.03 pM; D-1436 exhibited an IC50 of 2.09 pM; and the control PTP1B compound exhibited an IC50 of 2.47 pM.

10315] The activity of ENT-03 and ENT-06 on a number of other phosphatases was also investigated. Both compounds were assayed in vitro against human phosphatases (Tables 2B and 2C). The two compounds exhibited a very similar phosphatase “fingerprint” with respect to their corresponding IC50. These data strongly support the hypothesis that ENT-03 and trodusquemine are phylogenetic chemical orthologs. The compounds were found to be inactive at PPI A, PP1B, PP2A alpha, PTPN6, and PTPN2.

[0316] These data demonstrate that ENT-03 (Compound III) is a potent inhibitor of PTP1B, and has potential therapeutic utility known to be associated with PTP1B inhibitors.

Example 6: ENT-03 (Compound III) as a weight loss agent in mice

[0317] This example demonstrated the promotion of weight loss by ENT-03 (Compound III) in mice.

[0318] ENT-02 (MSI-1436) is known to induce weight loss through a mechanism that involves certain brain circuits that control appetite. Trodusquemine causes weight loss and a shift to lipid oxidation when administered systemically to mice. The pharmacological target appears to lie within the hypothalamus, including the arcuate nucleus, median eminence, and the paraventricular nucleus, based on localization of radioactive Trodusquemine, and cFos activation following intraventricular administration (Ahima et al., 2002).

[0319] Studies on the structure activity relationship of ENT-02, with respect to weight loss, have demonstrated the high degree of structural specificity required for this pharmacological effect (Zasloff et al., 2001). For example, altering the chirality of the spermine at C-3, the hydroxyl at C-7, or the methyl at C-21 eliminates weight loss. Activity is lost if the spermine is replaced by a spermidine, or if the terminal amino group of spermine is methylated. In contrast, both stereo-isomers of the sulfate at C-24 are equally active, demonstrating that spatial constraints are loosened around the anionic moiety, consistent with the activity observed for ENT-03.

[0320] To determine if ENT-03 (Compound III) exhibited a similar activity, male Swiss Webster mice, previously fed ad lib (about 50 grams, N=3 for each group), were treated i.p. with 5 doses of either ENT-02 (MSI-1436) or ENT-03 (Compound III) every other day at 10 mg/kg (e.g., on days 0, 2, 4, 6, 8 and 10). Food was provided ad lib.

[0321] As can be seen in Figs. 5A, 5B and 6, ENT-03 administration results in weight loss with kinetics similar to that seen following administration of ENT-02. Administration of both compounds resulted in a decrease in weight, with a nadir at about day 16, followed a gradual recovery to the starting weight by about day 45. While the initial decrease in weight was similar for both compounds, by about 10 days the effects diverged, with ENT-02 causing a more severe decline. By 27 days both sets of treated mice had lost about 10% of their starting body weight.

[0322] These data demonstrate that ENT-03 (Compound III) exhibits a pharmacological response with respect to weight similar to ENT-02 (MSI-1436) and suggests that ENT-03 (Compound III) can have utility in all therapeutic applications known to be associated with ENT- 02 (MSI-1436) as well as other aminosterols.

103231 In a second experiment, growing male mice, about 25 grams, were administered either compound following a similar dosing regimen. As in the previous experiment, both compounds affected weight gain. See Fig. 6. However, in the case of growing mice, ENT-02 had a more profound effect, having suppressed growth as well as having induced consumption of body fat. While the animals treated with ENT-03 continued normal growth, they “slimmed down”, suggesting that ENT-03 re-established a new optimal body weight “set point.”

[0324] In a third experiment, as seen in Fig. 5C intraperitoneal administration of ENT-03 to C57bl/6 male mice once weekly over 6 weeks caused a dose dependent weight loss. Other than weight loss the treated animals appeared clinically normal.

[0325] Finally, in a fourth experiment, as seen in Figure 18, intraperitoneal administration of ENT-03 (3 mg/kg, 5 mg/kg, 10 mg/kg, or vehicle) to C57bl/6 male mice once weekly over 2 weeks caused a dose dependent weight loss. Other than weight loss the treated animals appeared clinically normal.

(0326] These studies demonstrate that ENT-03 (Compound III) exhibits potent pharmacological activity in mice, similar to ENT-02.

Example 7: Rejuvenation of RNA Transcriptome in the Gut

[0327] The purpose of this example was to evaluate the impact of oral administration of ENT- 02 and ENT-03 (Compound III) on young vs aged gut tissue.

[0328] Aging involves a depletion of gene expression in the gut. Comparison of the images showing mucosal tissue in the stomach of a young mouse (20 week, Fig. 7A) versus an old mouse (78 week, Fig. 7B) shows a reduced thickness of the mucosal layer in the older specimen. This reduction in mucosa is associated with a reduced RNA transcriptome in the stomach in aged mice (78 weeks) vs. young mice (20 weeks), see Table 7 below. The present Example evaluated the impact of oral dosing of ENT-01 and ENT-02 and on old mice.

103291 The dosing schedule used to determine the effect of orally administered squalamine and ENT-02 on the GI tracts of young and old mice was as follows. Male C57B1/6 mice, aged 20 and 78 weeks, were obtained from Jackson labs. Animals were exposed to 12 hr light dark cycles and provided Teklad standard mouse diet and water ad lib. Animals were assigned to the treatment groups shown in Table 7.

[0330] Animals were dosed once daily by oral gavage in the morning for a total of 14 days. Animals were fasted 3-4 hours prior to dosing and at least 1 hour following dosing, with fasting not to exceed 6 hours. The test article was dissolved in 0.5% hydroxypropylcellulose in water. On day 15 the animals were euthanized by CO2 asphyxiation, necropsied, and tissues prepared for histology and RNAseq analysis.

[0331] The GI tracts of the animals were sectioned into stomach, duodenumjejunum, ileum, caecum, colon, and rectum. The tissues were then sent for histology, and the transcriptomes analyzed by RNAseq. Table 8 shows the respective mRNA amounts in young and old mouse stomach.

[0332] As shown in Table 9A below, mRNA levels for all of the genes in the table showed a significant increase after treatment with squalamine (ENT-01). Equally remarkable, while ENT- 03 stimulates induction of the transcriptome of these segments of the GI tract of older mice, it has minimal effect in the younger animals, corresponding to a slight repression or induction of certain genes not associated with ageing. Perhaps ENT-03 is complementing a deficiency in the older mice that does not exist in the younger animals. This suggests that squalamine (ENT-01), and by extension structurally related aminosterols, such as ENT-03 (Compound III) and derivatives thereof, have a rejuvenating effect in the gut.

103331 Applicant also investigated transcriptome changes within each segment of the GI tract. Upon oral administration to aged mice the abundance of gene transcripts that decrease with ageing in the stomachjejunum, and ileum are increased, in some cases to levels observed in the younger individuals as shown in Figs. 10A-10C.

[0334] Young (20 week) and aged (80 week) C57bl/6 male mice were administered ENT-03 (40 mg/kg) or vehicle (water) by oral gavage daily for 2 weeks. Over this period of time clinical observations were unremarkable as were the gross and microscopic examinations of the GI tract. The transcriptomes of the stomach ejunum, and ileum were analyzed by RNAseq technology. Comparative analyses of the transcripts from untreated animals identified numerous transcripts that differed in abundance between the corresponding tissues from old and young individuals. Differential expression was observed for 86 stomach transcripts (p(adj )<0.05): 70 decreased, and 16 increased with ageing; in the case of the jejunum, over 400 transcripts decreased, 200 increased with ageing; and for the ileum, 700 transcripts decreased, while 400 increased with ageing. Thus, within these three distinct regions of the mouse GI tract, ageing is associated with changes in the transcriptome.

[0335 ] When the effect of oral administration of ENT-03 on the transcriptome of the tissues was examined, in each case it was observed that the transcriptional response in the young was blunted in comparison to that occurring in the aged animals (Figs. 10A-10C). For example, in the stomach of treated young animals, 13 genes were differentially expressed (p(adj)<0.05), while 63 were in the aged group. Similarly, for the jejunum 42 genes were differentially expressed in the young, and 382 genes on treatment in the aged group. In the ileum, 80 genes were differentially expressed in the young, and 1162 genes in the aged animals treated with ENT-03.

[0336] Next, the sets of genes that were differentially expressed (DEGs) with ageing were compared with those that were differentially expressed in the treated aged animals or in the treated young animals. Analysis of the overlapping DEGs between contrasts revealed, for example, in the stomach 37 genes down-regulated in ageing (old versus young) significantly overlapped (P < 0.0001, hypergeometric test) with genes up-regulated by treatment with ENT-03 in old mice. In contrast the only significant overlap between the stomach ageing genes and those in the young ENT-03 treated stomach were 12 genes that were further down regulated in the ageing direction.

[0337] The “ageing” genes that ENT-03 appears to complement most significantly within the stomach are listed in Table 9B. Notably, in the stomach, the “restored” ageing genes include those involved in tissue renewal (fibroblast growth factor 2; zinc finger protein 383; forkhead box C2); neuronal differentiation (neural cell adhesion molecule 2); immunity (toll-like receptors 9 and 12; interleukin 2 receptor, beta chain), neurotransmitter synthesis and uptake (choline and serotonin transporters), and mitochondrial respiration (cytochrome c oxidase subunit 6B2).

103381 First, genes that significantly changed in expression between young and aged mice were identified (FDR-adjusted p-value < 0.05) (Fig. 10A). With ageing, the expression of 75 genes in the stomach was significantly decreased, and the expression of 11 genes was significantly increased (Fig. 10A). Meanwhile, fewer differences were observed between the gene expression profiles of the jejunum (Figs. 10A-10D) or ileum (Figs. 14D-14F) of young and aged mice. In the jejunum, 5 genes decreased in expression and 2 genes increased in expression with ageing. Whereas in the ileum, 19 genes decreased and 9 genes increased in expression. These results are consistent with the recognized reduced regenerative capacity of the ageing rodent stomach (Fukunaga et al., 1998), and the resilience with ageing of the small intestine (Eswaran et al., 2006).

[0339] Following oral administration of ENT-03 to young mice (20 weeks), 13 stomach genes responded and all were transcriptionally repressed by ENT-03 treatment (Fig. 10B). The expression of no more than 2 genes changed significantly in response to ENT-03 exposure in the jejunum and ileum for both young and aged mice.

[0340] In contrast, a more robust effect on gene expression in the stomach was observed in the older animals (78 weeks) treated with ENT-03. 63 genes were transcriptionally induced (FDR- adjusted p < 0.05) (Fig. 10C). Interestingly, 36 of the genes that increased in expression upon ENT-03 treatment of aged mice were the same that decreased in expression with ageing. These genes include those involved in tissue renewal (fibroblast growth factor 2 (Fgf2), zinc finger protein 382 (Zfp382) and forkhead box C2 (Foxc2)); in neuronal differentiation (neural cell adhesion molecule 2 (Ncam2)); in immunity (toll-like receptors 9 and 12 (Tlr9, Tlr 12), interleukin 2 receptor, beta chain (I12rb) and CD300 (Cd3001d)); in neurotransmitter synthesis and uptake (choline and serotonin transporters (Slc5a7, Slc6a4)); and in mitochondrial respiration (cytochrome c oxidase subunit 6B2 (Cox6b2)).

[0341] Statistical analysis of RNA-sequencing data was performed using R programming language. Transcripts with less than one read count per million reads in all samples of each tissue were removed. The raw count data for the samples were then normalised using trimmed mean of M-values normalisation and transformed with voom (Law et al., 2014), resulting in log2- transformed counts per million with associated precision weights. Normalised data provide the input for statistical hypothesis testing, in which genes that are significantly different between sample groups are identified. Statistical comparisons were performed using linear modelling, as implemented in the Bioconductor package limma (Ritchie et al., 2015). Significance values (p- values) were adjusted for multiple testing using Benjamini -Hochberg procedure (Benjamini et al., 1995). For each comparison (e.g. group A versus group B), a positive log2 -transformed fold change indicates up-regulation in group A relative to group B.

[0342[ To investigate the overlap between contrasts, the number of overlapping differentially expressed genes (defined using adjusted p-value < 0.05) between all pairwise combinations of the comparisons performed were counted. For each comparison, the Jaccard index, defined as the intersection over the union, measures the similarity between the two contrasts under consideration. The p-value was calculated using a hypergeometric test taking into account the universe size under the assumption that the contrasts and genes are independent.

[0343] Fig. 15 shows a set of heatmaps investigating the overlap of differentially expressed genes between pairs of contrasts.

[0344] These data support the hypothesis that oral administration of ENT-03 to old mice can reverse some of the changes in gene expression associated with ageing within the GI tract.

[0345] In a second part of this experiment, it is proposed that the animals additionally be treated with an insulin compound. The insulin compound is expected to act synergistically with the aminosterol compound comprising a spermine moiety.

Example 8: Pharmacokinetic Study of ENT-03

(Compound III) via Intravenous and Oral Administration

[0346] The objective of this example was to determine the pharmacokinetic profile of ENT-03 (Compound III) via intravenous and oral administration in male SD Rats.

[0347] The study groups are shown in Table 10 below.

* : The salt factor of 1. 177 is used for the formulation preparation of Compound III.

(0348] Animal feeding control: Animals were food fasted overnight prior to dosing and fed 4 hours after administration having free access to water.

[0349] Dose formulation processing during dosing: The dose formulation was kept stirring at room temperature and was used within 2 hours.

Note: Unscheduled clinical observations (cageside or detailed) were also performed, as needed.

[0350] PK Sample Analyses: Concentrations of ENT-03 (Compound III) in the plasma and dose samples were analyzed using a LC-MS/MS method. WinNonlin (Phoenix™, version 6.1) or other similar software was used for pharmacokinetic calculations. The following pharmacokinetic parameters were calculated, whenever possible from the plasma concentration versus time data.

[0351] IV Bolus administration: T1/2, Co, AUCiast, AUCinf, MRTinf, Cl, Vss, Number of Points for Regression.

[0352] PO administration: F, T1/2, Cmax, Tmax, AUCinf, AUCiast, MRTinf, Number of Points for Regression.

[0353] The pharmacokinetic data was described using descriptive statistics such as mean and standard deviation. All the residual biological samples were retained in the freezer (-75±15 °C) for a period of 6 months after testing. The results are tabulated below in Table 18. Example 9: ENT-03 (Compound III) Reverses Alzheimer’s in Mice

[0354] PTP1B dependent mechanisms have been utilized for reversal of memory impairment and normalization of behavior and reduction in neuronal loss in beta amyloid and tau mouse models of Alzheimer’s disease (Rieke, Cruz et al. 2020). Other studies have shown reduction in the toxicity of beta amyloid aggregates by trodusquemine in vitro and in a C. elegans model of Alzheimer’s disease (Limbocker, Chia et al. 2019).

[0355] ENT-02 (MSI- 1436) reverses several conditions (in mice) that are associated with ageing, such as metabolic syndrome, Alzheimer’s disease, atherosclerosis, cancer and a reduced capacity for regenerative repair. As we have shown in this report, ENT-03 can treat Alzheimer’s disease in murine models.

[0356] The Morris water maze was used to test the effect of ENT-03 on spatial learning and memory deficits in 2 mouse models of familial Alzheimer’s disease, hAPP-J20 mice that express a double mutant of the human amyloid precursor protein (Mucke et al., 2000), and PS19 mice that express the P301S mutant of the human microtubule associated protein tau (Yoshiyama et al., 2007).

[0357] hAPP-J20 mice expressing human APP bearing the Swedish and Indiana familial mutations (B6.Cg-Zbtb20Tg(PDGFB-APPSwInd)20Lms/2Mmjax, (Mucke et al., 2000)) and PS19 mice expressing the P301S mutation of human tau protein (B6;C3-Tg(Prnp- MAPT*P301S)PS19Vle/J (Yoshiyama et al., 2007)) were purchased from Jackson laboratories and maintained as colonies in the animal facility of the University of Ottawa. Heterozygous hAPP-J20 and PS 19 mice were used. Genotypes were verified by PCR using genomic DNA isolated from tail or ear biopsies.

[0358] Clinical grade ENT-03 (provided by Enterin, Inc.) was administered intraperitoneally (z. .) once per week for 6 weeks at a dose of 2.5 mg/kg bodyweight starting at 4.5 months of age. The behavioral experiments were conducted 10 days after the last injection. Vehicle treated controls received sterile saline (0.9% NaCl in water).

[0359] The Morris water maze test to analyze the ability of mice to learn and remember the location of a submerged platform in a pool containing opaque water was conducted in the Faculty of Medicine Behavior Core Laboratory at the University of Ottawa. The 80 cm ² platform surface corresponds to 0.6 % of the total pool area. Mice were habituated to the experimental room and consecutive experiments were performed between noon and 4 PM. Mice were trained for 5 days, (four trials per day with an inter-trial interval of 20 minutes and a random start location in one of four positions) to find the invisible, submerged platform at a fixed location. Cues around the pool were provided as spatial references. Trials lasted 1 minute or until the mouse found the platform. Mice were guided to the platform if they did not find the platform. Mice stayed on the platform for 15 seconds of each trial before being removed to their cages. After the training period, the platform was removed from the pool and the probe trial was executed within 1 minute on the following day. On the probe day, crossings of the platform area and target quadrant were counted and swimming speed was measured using Ethovision automated video tracking software (Noldus).

[0360] In both disease models, one with amyloidopathy and the other with a tauopathy, learning during the training phase (Figs. 9A and 9E) and memory on the probe day (Figs. 9B, 9C, 5F and 9G) were improved with ENT-03 treatment compared to vehicle treated hAPP-J20 or PS 19 littermate control mice (Fig. 9). The effect on preventing cognitive decline is similar to what has been observed with the related compound trodusquemine hAPP-J20 and PS 19 mice (Rieke et al., 2020).

[03611 In particular, Morris water maze test reveals ENT-03 treatment over 6 weeks (2.5 mg/kg/wk, i.p.) decreased the latency to find the hidden platform for hAPP-J20 mice during the training phase (Figs. 9A and 9E) and improved memory (crossings at the area where the platform was located (Figs. 9B and 9F) and time in the platform area (Figs. 9C and 9G) on the probe day. (Figs. 9D and 9H) Swimming speed. Fig. 9A, repeated measures two-way ANOVA: genotype/treatment, F (3, 42)= 13.47, p<0.0001; time, F(4,168)=23.45, p<0.0001; subjects (matching), F(42,168)=4.420, p<0.0001; interaction, F(12,168)=3.458, p=0.0001; *, post hoc pairwise comparisons between Veh WT and Veh hAPP-J20, day 2, 3, 4, 5: p=0.0073, 0.0101, <0.0001, <0.0001, respectively; #, post hoc pairwise comparisons between Veh hAPP-J20 and ENT-03 hAPP-J20, day 4, 5: p=0.0260, 0.0298, respectively. Fig. 9B, two-way ANOVA: genotype, F(l,42)=10.44, p=0.0024; treatment, F(l,42)=4.509, p=0.0396; interaction, F(l,42)=3.625, p=0.0638; *, post hoc comparisons between Veh WT and Veh hAPP-J20, p=0.0020 and between Veh and ENT-03 hAPP-J20, p=0.0487. Fig. 9C, two-way ANOVA: genotype, F(l,41)=9.977, p=0.0030; treatment, F(l,41)=4.149, p=0.0482; interaction, F(l,41)=l .570, p=0.2173). *, post hoc comparisons between Veh WT and Veh hAPP-J20, p=0.0080 and between Veh hAPP-J20 and ENT-03 hAPP-J20, p=0.0032. Fig. 9D, two-way ANOVA, genotype, F(l,42) = 10.95, p=0.0019; treatment, F(l, 42) = 1.352, p=0.2515; interaction, F(l,42) = 0.1378, p=0.7123. Fig. 9E, repeated measures two-way ANOVA: genotype/treatment, F(3,45)=l 1.90, p<0.0001; time, F(4,180)=29.09, p<0.0001; subjects (matching), F(45,180)=2.780, p<0.0001; interaction, F(12,180)=2.325, p=0.0087; *, post hoc pairwise comparisons between Veh WT and Veh hAPP-J20, day 2, 3, 4, 5: p=0.0143, <0.0001, 0.0037, <0.0001, respectively; #, post hoc pairwise comparisons between Veh hAPP-J20 and ENT-03 hAPP-J20, day 4: p=0.0111. Fig. 9F, two-way ANOVA: genotype, F(l,45)=5.413, p=0.0245; treatment, F(l,45)=16.87, p=0.0002; interaction, F(l,45)=3.630, p=0.0631; *, post hoc comparisons between Veh WT and Veh hAPP-J20, p=0.0236 and between Veh hAPP-J20 and ENT-03 hAPP-J20, p=0.0004. Fig. 9G, two-way ANOVA: genotype, F(l,45)=4.829, p=0.0332; treatment, F(l,45)=6,861, p=0.0120; interaction, F(l,45)=4.500, p=0.0394). *, post hoc comparisons between Veh WT and Veh hAPP-J20, p=0.0201 and between Veh hAPP-J20 and ENT-03 hAPP-J20, p=0.0064. Fig. 9H, two-way ANOVA, genotype, F(1 ,45) = 4.572, p=0380; treatment, F(l, 45) = 0.6015, p=0.4421; interaction, F(l,45) = 0.0090, p=0.9249).

103621 In a second part of this experiment, it is proposed that the animals additionally be treated with an insulin compound. The insulin compound is expected to act synergistically with the aminosterol compound comprising a spermine moiety.

Example 10: Detection of ENT-03 in Humans and Mouse Pup Brain

[0363] In 1992 Nagata et al identified high concentrations of the bile acid, 7-alpha hydroxy-3- oxo-4-cholestenoic acid (7-HOCA), in human chronic subdural hematoma fluid, (Fig. 5C) (Nagata et al., 1992) and later in acute subarachnoid hemorrhage (Nagata et al., 1995). In 1997, Zhang et al reported that rat brain cells could metabolize 27-hydroxycholesterol to 7-HOCA (Zhang et al., 1997). Subsequently, Bjbrkhem, Sjbvall, Griffiths and their collaborators identified 7-HOCA as the most abundant bile acid in human cerebrospinal fluid (Ogundare et al., 2010; Meaney et al., 2007; Saeed et al., 2014; Saeed et al., 2014). 7-HOCA did not appear to a be a biologically active bile acid, in that it did not activate either the FXR, LXR, or RXR/NURR1 receptors, for which bile acids and oxysterols are known ligands (Ogundare et al., 2010). Bjbrkhem has proposed that the brain metabolizes 27-hydroxycholesterol that enters from the periphery into 7-HOCA to facilitate the efflux of the oxysterol back into the circulation (Meaney et al., 2007).

[0364] Applicant suggests that the presence of ENT-03 in subdural hematoma fluid reflects its possible role in the development of this structure. Following head injury, generally in older people and rarely in infants, a highly vascularized sac-like “organ” develops, with one side deriving from the dura(“ outer membrane”), the other from the subarachnoid (“inner membrane”) (Yamashima et al., 2000). High concentrations of numerous growth factors, including VEGF accumulate within the fluid (Edlmann et al., 2017). We suggest that ENT-03 appears within the subdural hematoma in an attempt to repair the intracranial injury. In this context, one might wish to speculate why healthy newborns who (rather commonly) experience intracranial trauma following passage through the birth canal, resulting in subdural and parenchymal hemorrhage, generally recover asymptomatically (Looney et al., 2007).

Human Study

103651 Because of the known high concentrations of Compound la (7-HOCA) in the chronic subdural hematoma fluid, Applicant analyzed the fluid evacuated from 3 elderly patients with chronic subdurals via LC/MS/MS for the presence of ENT-03.

[0366] Patient #1 : 66 yo male with 3 weeks of gait instability, headache, difficulty with fine motor movements. CT with 2.2 cm right sided subacute subdural hematoma (SDH) with significant mass effect/midline shift. Indications for drainage were large, symptomatic SDH. Drainage procedure was right sided craniotomy for evacuation of SDH.

[0367] Patient #2: 75 yo male who sustained fall 3 weeks prior. Presented with 2-3 days of worsening headache, gait instability, and left lower extremity weakness. CT with bilateral subacute SDH (1.7 cm on right, 1.6 cm on left). Indications for drainage were large, symptomatic SDH. Drainage procedure was bedside twist drill hole craniostomy.

103681 Patient #3 : 94 yo male with progressive confusion/altered mental status/gait instability. Work-up demonstrated bilateral subacute on chronic subdural hematomas, both 2.5-3.0 cm in size and with mass effect/brain compression. Indications for drainage were large, symptomatic SDH. Drainage procedure was a bedside twist drill hole craniostomy.

[0369] Brain extracts were analyzed via LC/MS/MS for ENT-03. Fig. 11A shows the presence of ENT-03 in brain extract. Fig. 1 IB shows a reference sample of synthetic ENT-03.

Mouse Pup Study

[0370] Based on these reports Applicant analyzed extracts of brain, liver, and kidneys from newborn mice over the first two postnatal weeks for the presence of ENT-03. 3-oxo-bile acids comprise between 18-40% of the unconjugated bile acids within amniotic fluid during the last trimester of human fetal development (Nakagawa et al. 1990) and are present as a significant percentage of the bile acids in healthy newborn urine, gradually diminishing during the first postnatal month (Wahlen et al., 1989 and Kimura et al., 1999). Since 3-oxo-bile acids are abundant during the newborn period, Applicant focused the search for the putative polyamine- bile acid molecule in neonatal mice. Brain and liver extracts were prepared from mice between day 1 through day 24 of age using a protocol designed to capture ENT-03 based on its physical properties as follows.

[0371] Frozen neonatal mice were obtained from Layne Laboratories, and tissues dissected in the frozen state. Tissues were dissected in the frozen state: “pinky” (Id); “large pinky” (2-5d); “small fuzzy”(6-9d); “large fuzzy”(10-14d); “hopper” (15-18d); “small frozen”(18-24d). Tissues (0.5-7 grams) were placed into 4 volumes of methanol containing 0.12N HC1 and heated at 80°C for 5 hours. The tissues were macerated, followed by a brief centrifugation. The supernatant was collected, the volume reduced under a stream of air, and extracted with 1 volume of chloroform/1 volume of methanol. The upper phase was reduced in volume and the samples further analyzed by LC/MS/MS.

[0372J Synthetic ENT-03 was used as internal standard in LC/MS/MS analysis to permit localization and identification of the corresponding molecule within the chromatographic analysis.

[0373] ENT-03 (Compound III) could be detected in brain and liver of neonatal mice (Figs. 12A and 12B, respectively). The identity of the endogenous ENT-03 was established by its retention time, mass ([M+H ⁺ ] m/z =619.6 ), and MS/MS MRM of the characteristic fragments of mass 545.57 (Fig. 17A) and 474.39 (Fig. 17B). Approximate concentrations of ENT-03 (Compound III) measured in the brain and liver of neonatal mice over the first 3 weeks of life are presented in Fig. 13. In both brain and liver the highest concentrations appear at birth with a gradual reduction over the following 3 weeks. [0374] Analytical procedures: Tissue extracts were spiked with ENT-03 -t/v and analyzed on a Triple Quad 5500 MS/MS system equipped with a ExionLC™ LC (SCIEX, Redwood City, CA) using Analyst® 1.7.1 software for instrumental control.

[0375] Separation of ENT-03 (Compound III) from tissue matrix components was achieved using a Kinetex® 5pm C18 100A 50 * 2.1 mm chromatographic column (Phenomenex, Torrance, CA, USA). The column was maintained at 25 °C, the flow rate was 0.3 mL/min, and the injection volume was 10 pL. The mobile phase (MP) consisted of A: 0.1% formic acid in water and B: 0.1% formic acid in acetonitrile (ACN, v/v). The mobile phase gradient was as follows: after injection, initial conditions with MPA at 80% were held for 0.3 min, decreased to 70% in 1.7 min, to 50% in 0.5 min and held constant for 0.5 min, returning to initial conditions for another 3.5 min of reequilibration time.

[0376] Retention time of ENT-03 (Compound III) was approximately 2.3 min and total run time was 7 min. A turbo ion spray interface was used as the ion source, operating in positive ion mode. Acquisition was performed in multiple reaction monitoring (MRM) mode using m/z

619.6 ([M+H] ⁺ ) —> 545.5 (loss of 74.1 (C3H10N2) and m/z 619.6 ([M+H] ⁺ ) 474.4 (loss of

145.2 (C7H19N3) at unit resolution. The internal standard was ENT-03-d4 using MRM of m/z

623.6 ([M+H] ⁺ ) 549.5 or m/z 623.6 ([M+H] ⁺ ) 478.5. Ion spray voltage was 4500 V and collision energy was 55 ( 619.6/474.5) or 43 (619.5/545.5) V. Declustering potential was 70V. The collision gas was nitrogen and the ion spray temperature was 650 °C. The MRM transitions were confirmed with a mass error of 10 ppm or less using high-resolution MRM on a X500 QTOF System (SCIEX).

[0377] For ENT-03 (Compound III) tissue level estimation, calibration curves were prepared from ENT-03 chromatographic peak areas ratios to the internal standard and using linear regression with a (1/x ² ) weighting factor that was chosen based on goodness-of-fit criteria, including coefficient of determination (r ² ), the back-calculated concentration of individual calibrators, and minimization of the intercept value.

Example 11: ENT-05 and regulatory phosphatase inhibition

[0378] The purpose of this example was to evaluate the inhibition of regulatory phosphatases by ENT-05. [0379] ENT-05 was synthesized and assayed against the bank of regulatory phosphatases.

[0380] ENT-05, which differs from ENT-03 (Compound III) with respect to the polyamine, and the presence of a hydroxyl group on Cl 2, exhibits inhibitory activity with great specificity against the proto-oncogene PTPN11 (E76K), as shown in Tables 15A and 15B below.

[0381 [ As another example, ENT-06 was synthesized.

ENT-06

[0382] ENT-06 differs from ENT-03 in the substitution of a spermidine for a spermine. ENT- 06 exhibits the following inhibitory activity against the human regulatory phosphatases (Tables 16A and 16B):

[0383] As can be seen in this example, the substitution of a spermidine for a spermine does not change the inhibitory profile of ENT-03. This example also teaches that the C-12 moiety on ENT-05 (above) drastically alters the specificity of the molecule towards the human regulatory phosphatases.

10384] Phosphatase assays used: The assays used were from Recation Biology Inc. PTPN11/SHP2-FL: Recombinant human PTPN11 full length (Genbank accession# NM_00133043.1; aa 2- 597, isoform 1 (canonical)) was expressed in E. Coli with N-terminal StrepII-TEV, C- terminal His-tag. Mw=71.93 kDa.

[0385] PTPN11/SHP2 (E76K)-FL: Recombinant human PTPN11 full length (Genbank accession# NM_00133043.1; aa 2- 597, isoform 1 (canonical)) with E76K mutation was expressed in E. Coli with N- terminal StrepII-TEV, C-terminal His-tag. Mw=71.93 kDa.

[0386] Activation peptide: H2N-LN(pY)IDLDLV(dPEG8)LST(pY)ASINFQK-amide (Fortanet et al., 2016) Substrate: DiFMUP [6,8-difluoro-7-hydroxy-4-methylcoumarin] Final concentration in the assay: For wild type: 0.35 pM Activating peptide; 100 pM DiFMUP; for mutant: No Activating peptide; 100 pM DiFMUP Assay buffer: 60 mM HEPES (pH 7.4), 1 mM EDTA, 75 mM KC1, 75mM NaCl, 0.01% Brij-35, 5 mM DTT, and 10% DMSO (final).

]0387[ Other phosphatases assayed: Assay buffer: 25 mM HEPES (pH 7.5), 5 mM MgCh, 0.01% Brij-35, 1 mM DTT, and 1% DMSO. For PP2A Alpha/PPP2R1 A Complex, PPI A, and PP1B 1 mM MnCh was added to the assay buffer. The concentration of DiFMUP varied with the phosphatase chosen: 2 pM for PTPN1/PTP1B-CD; 30 pM for PP1B; 10 pM for all other phosphatases. The phosphatase inhibitors were PTP1B CAS 765317-72-4 (Sigma Aldrich cat# 539741); Cantharidic acid (Santa Cruz Biotech, cat# sc-201323); SHP009 (ChemieTek, cat# CT- SHP099).

[0388] General procedure: Enzyme and substrate are freshly prepared in assay buffer; the enzyme solution is introduced into the reaction well, followed by delivery of the inhibitors in 100% DMSO via acoustic technology (Echo550; nanoliter range); the reaction is incubated for 20 min at room temperature; substrate is then delivered into the reaction well to initiate the reaction; the enzyme activities are monitored (Ex/Em 355/460) by an increase in fluorescence for 120 min at room temperature. The slope (signal/min) of the linear portion of time course is determined and the rates calculated in the presence of inhibitor relative to the DMSO control. Example 12: Assessment of in vivo activity of ENT-05

[0389] The purpose of this example was to evaluate the activity of ENT-05 in vivo.

[0390] To assess the activity of ENT-05 in vivo, the compound was added to 500 ml of distilled water to a concentration of 10 pg/ml. A Xenopus leavis tadpole, premetamorphic, was placed into the solution. Over the course of about 1 hour, the tadpole gradually swelled, stopped moving, and eventually died. A second animal was placed in 500ml of water, containing 10 pg/ml ENT-05, but also 50 mM NaCl. In this case, the animal survived. The animal was then transferred to spring water without either salt or ENT-05 and remained active over the following days. No similar effect could be observed with ENT-06 or ENT-03.

[0391] This example demonstrates that modification of ENT-03 to create ENT-05 yielded a compound with specificity towards PTPN11 (E76K) and likely other as yet undescribed phosphatases. The in vivo pharmacological effect on the tadpole is that of inhibition of sodium reabsorption, most likely in the kidney. The likely target is the sodium-potassium chloride cotransporter type 2 (NKCC2). Very little is currently known about the phosphatases that regulate the activity the NKCC2 of the renal tubule.

[0392] The example teaches that ENT-05 has identified a phosphatase that activates the NKCC2 transporter of the channel that can be inhibited by ENT-05. ENT-05 clearly has a potential utility in the treatment of hypertension, similar to the drug furosemide which inhibits renal NKCC2 by a mechanism not involving inhibition of phosphatase activity as well as intracranial hypertension (raised intracranial pressure) and intraocular hypertension (glaucoma).

Example 13: Evaluation of a deuterated ENT-03 Compound

[0393] ENT-03, a polyamine-bile acid conjugate, is expected to be metabolized through the chain of reactions utilized in the synthesis of bile acids. The following molecule was synthesized ENT-03D ₃ (C24D ₂ , C25D1)

[0394] When administered i.p. to mice the animals gained weight, in contrast to the loss of weight observed when ENT-03 was administered. See Fig. 16. The pharmacological effect is best explained by the presence of the deuterium isotope on the kinetics of the enzymes that successively metabolize the cholesterol side chain, since the deuterated form of ENT-03 should exhibit the same pharmacology as ENT-03. The presence of the deuterium on the side chain likely slows the action of the 2-methylacyl-coenzyme A racemase on C-25, the subsequent action of the dehydrogenase that produces the 24,25-/ra//.s-unsaturated derivative by removing the deuterium atoms at C24 and C25, and the subsequent hydration and oxidation at the C24 bond catalyzed by the D-bifunctional protein. Instead, the metabolism of ENT-03D3 will begin with the oxidation at C24 by CYP46A1, yielding the following compound:

[0395] Since the pharmacological effect is weight gain, one can readily predict that the compound inhibits the PTPN11/SHP2 with highest specificity, since SHP2 is known to enhance the central leptin pathway. Inhibition of SHP2 would increase leptin resistance, which would result in an increase in appetite and accumulation of adipose tissue.

Example 14: ENT-03 stimulates the transcription of genes involved in red blood cell production and immune cell function in the spleen of aged mice

[0396] As in humans, the spleen of the mouse plays a primary immune function (Smith et al., 2019). In the adult mouse, however, the spleen can become a red blood cell forming organ in a variety of experimental settings (Morita et al., 2011), although it normally cedes this function to the bone marrow during the first few weeks of postnatal life (Wolber et al., 2002).

[0397] To explore the effects of ENT-03 (Compound III) on the hematopoietic and immune systems of the spleens of young and aged mice, the transcriptomes of the spleens of the animals described in the previous rejuvenation of gut transcriptome Example were studied.

[0398] The transcriptional response to ENT-03 (Compound III) in the aged spleen (Table 21, below) was more intense than in the spleen of the younger animals (Table 22, below). 109 genes in the aged spleen were significantly upregulated by >1.5 fold, compared with 4 genes in the young. The genes most robustly induced in the old spleen are responsible for erythropoiesis (italicized rows in Table 21) and immune functions (bold rows in Table 21). The genes involved in hemoglobin synthesis are at the top of the list (hemoglobin a and 0 proteins, aminolevulinic acid synthase) along with abundant proteins involved in the structure/function of the red blood cell (the chloride-bicarbonate exchanger, band 4.2 protein, and 2,3 biphosphoglycerate mutase). These data are compatible with the hypothesis that ENT-03 is stimulating a program in the aged mouse normally operational during the first few weeks of postnatal life.

[0399] Generally speaking, the immune genes induced by ENT-03 correspond to those of the innate arm. These include IL-21, secreted by T cells and NKT cells, which has broad stimulatory effects across the breadth of the innate immune system (Spolski et al., 2014); and the IL22 binding protein (IL22ra2), secreted by numerous innate immune cells, that helps curb the action of the proinflammatory cytokine IL22 following resolution of an infection (Huber et al., 2012) reducing the probability of post-inflammatory tumorigenesis (Huber et al., 2012). Other highly induced genes encode for receptors that are expressed by dendritic cells and macrophages, such as DC-SIGN (CD209) which plays a key role in the recognition by dendritic cells of viruses and other pathogens (Svajger et al., 2010); the macrophage chemokine receptor CCR2, which plays a critical role in the normal reparative response to tissue injury (Boniakowski et al., 2018); and the Fc alpha/mu receptor (Fcamr) that mediates endocytosis of IgM coated pathogens by the macrophage (Shibuya et al., 2000).

104001 None of the splenic genes highly induced by ENT-03 in the aged spleen appear in the set of responsive genes in the spleen of young animals. [0401 ] In a second part of this experiment, it is proposed that the animals additionally be treated with an insulin compound. The insulin compound is expected to act synergistically with the aminosterol compound comprising a spermine moiety.

Example 15: Treatment induced gene expression in mouse stomach tissues, comparison of ENT-03 and ENT-02 (MSI-1436)

[0402] The purpose of this example is to identify transcriptional changes between young and old mice and compare the effects of ENT-02 (MSI-1436) treatments on gene expression with those of ENT-03. Mice were treated with ENT-03, ENT-02 (MSI-1436) or a vehicle control. Samples of stomach tissues from the mice were analysed by RNA-sequencing on an Illumina platform.

[0403] The aims of this analysis were to: identify transcriptional changes between young and old mice; determine if ENT-01 or ENT-02 (MSI-1436) treatments could reverse those age- related changes; and compare the effects of ENT-02 (MSI-1436) treatments on gene expression with those of ENT-03.

[0404] To achieve these aims, data were subject to a quality control evaluation, differential expression and functional enrichment analyses, as well as a congruence analysis. Data for ENT- ENT-03 treatments were compared to the results generated for ENT-02 (MSI-1436).

|0405] To identify those genes that were significantly differentially expressed between groups, an arbitrary threshold was applied based on fold changes in expression. A four-fold change in expression between groups was used as a measure of pseudo-significance in the absence of replicate samples. At this threshold, differentially expressed genes were identified in all contrasts. For gene expression changes associated with ageing, or ENT-02 (MSI-1436) treatments in young mice, the proportions of down-regulated genes (72-80%) were higher than the proportions of upregulated genes (20-28%). The trend was opposite for both treatments in old mice, where 82-89% of gene expression changes were up-regulation and 12-18% were downregulation.

[0406] When comparing differentially expressed genes across contrasts, it was apparent that genes downregulated in aged mice overlapped significantly with genes up-regulated in response to ENT-02 treatment in old mice (hypergeometric P < 0.0001). To a lesser extent, there was also significant overlap in genes down-regulated in aged mice and up-regulated in response to treatment in old mice. In young mice, genes that were down-regulated upon ENT-02 treatment significantly overlapped with the down-regulated genes associated with ageing. This result was also true for genes up-regulated in both contrasts.

[0407| For each comparison, the number of sample genes significant at various statistical thresholds and fold change 4 were tallied. As mentioned previously, for statistical robustness, only those with an adjusted p-value < 0.05 should be considered.

[0408] The following statistical significance threshold was chosen to define differentially expressed genes: • fold change > 4

[0409| To investigate the overlap between selected genes from the multiple contrasts performed, the number of overlapping differentially expressed genes were counted (defined using fold change 4) between all pairwise combinations of the comparisons performed. The amount of overlap is represented in Figures 32A-32D. For each comparison, the value in the plot represents the number of intersecting selected genes and the colour represents the Jaccard index (the intersection over the union) for the two contrasts under consideration.

[0410] To investigate the overlap between significant genes from the contrasts performed, a set of scatter plots was generated comparing the fold change between pairs of contrasts. Functional enrichment analysis was performed upon those genes where expression differences were greater than four-fold in individual contrasts. Reactome and GO term databases were interrogated to identify relevant terms that were significantly enriched in differentially expressed genes (enrichment P < 0.05). Pathways related to the immune system such as platelet degranulation, antimicrobial peptides and complement cascade were generally amongst the most enriched pathways across the contrasts. Pathways such as keratinisation and keratinocyte differentiation were enriched in genes changed upon ageing or ENT-02 (MSI- 1436) treatments in old mice. Pathways such as muscle contractions and sarcomere organisation were enriched in genes changed upon ageing, but were also enriched in ENT-02 treatment-affected genes in young mice.

[0411] Significant genes (at fold change 4) from each contrast were analyzed for enrichment of Reactome pathway membership using a hypergeometric test by mapping genes to genes (if appropriate). Enrichment (p-value < 0.05) was assessed for the union of selected genes.

(0412] Genes that were differentially expressed in response to ENT-02 treatment, as identified in this study, were compared to those that were associated with ENT-03 treatments, respectively. ENT-03 treatment-associated gene expression changes were identified previously.

[0413] Across all contrasts at the relevant statistical thresholds used to identify significant features, there were ten or less genes that changed in expression upon both treatments (ENT-02 and ENT-03). Note that this included the gene expression changes associated with ageing. Specifically, the expression of only one gene, Sypl2, was changed significantly between the old and young mice of both studies.

[0414] Comparisons of the genes which were down-regulated in ageing and up-regulated in treatment revealed that genes affected by ENT-02-treatment did not overlap with those affected by ENT-03.

[0415] Congruence analysis was performed. Specifically, the analysis compared the effects of human- or shark-origin compound analogues on murine stomach tissue gene expression profile as follows: ENT-02 (MSI-1436) was compared to ENT-03.

[0416] Scatter plots, upset plots, Venn diagrams, hypergeometric tests, and Spearman rank correlation tests were employed to assess the level of overlap as shown in Figures 19-31.

10417] Note that significantly differentially expressed genes in ENT-03-specific contrasts were determined using a statistical threshold of FDR-adjusted P < 0.05. Whereas the ENT-02 (MSI- 1436)-specific contrasts in this study defined significant genes using a cut-off of greater than four-fold change in expression.

[0418] Genes down-regulated in ageing and up-regulated upon treatment: Transcripts that were significantly down-regulated in ageing and up-regulated by treatment of old mice were identified for each compound. Congruence between these sets of genes was then assessed by comparing ENT-02 (MSI-1436)-affected genes to those of ENT- 03, these results are illustrated in the Venn diagram in Fig. 19.

[0419] Congruence analysis results are shown in Fig. 20 which shows a scatter plot comparing significant genes in ENT-02 (MSI-1436) vs. control (young) against ENT-03 vs untreated (young). Figure 26 shows a scatter plot comparing significant genes in Old vs young (control) against Old vs young (untreated). Figure 29 shows a scatter plot comparing significant genes in Old vs young (ENT-02 (MSI-1436)) against Old vs young (ENT-03).

[0420] Fig. 22 shows Venn diagrams of significant genes in ENT-02 (MSI-1436) vs control (young) against ENT-03 vs untreated (young). Each plot considers a different interaction of sets; either ignoring direction of perturbation, considering only up-regulated genes, considering only down-regulated genes, or examining the over- lap between those genes up-regulated in one contrast and those genes down-regulated in another. The symbol U denotes the universe.

[0421] Congruence analysis results are shown in Fig. 23 which shows a scatter plot comparing significant genes in ENT-02 (MSI-1436) vs. control (old) against ENT-03 vs untreated (old).

Fig. 25 shows Venn diagrams of significant genes in ENT-02 (MSI-1436) vs control (old) against ENT-03 vs untreated (old). Figure 28 shows Venn diagrams of significant genes in Old vs young (control) against Old vs young (untreated). Figure 31 shows Venn diagrams of significant genes in Old vs young (ENT-02 (MSI-1436)) against Old vs young (ENT-03). Venn diagrams of up- and down-regulated genes. Each plot considers a different interaction of sets; either ignoring direction of perturbation, considering only up-regulated genes, considering only down-regulated genes, or examining the overlap between those genes up-regulated in one contrast and those genes down-regulated in another. The symbol U denotes the universe.

Example 16

[0422| This prophetic example describes an exemplary method of retarding the aging process of a subject. The method comprising administering a pharmaceutical composition comprising a therapeutically effective amount of Compound III (ENT-03) or a pharmaceutically acceptable salt or derivative thereof to the subject.

[0423] One or more adult human subjects can be given a suitable dosage of Compound III (ENT-03) via any pharmaceutically acceptable method, such as oral, intranasal, or injectable. An exemplary daily or weekly dosage can be, for example, about 1 to about 20 mg administered intranasally daily.

[0424] The characteristics of aging impacted by administration of Compound III (ENT-03) or a derivative or salt thereof that can be measured include muscle endurance, coordination, social behavior and cognitive ability.

[0425] First, muscle endurance is measured for each subject prior to initial Compound III (ENT-03) dosing to establish a baseline. For example, the partial curl-up test can be used to measure endurance of the abdominal muscles and the push-up test can be used to assess endurance of the upper body. Following initiation of Compound III (ENT-03) dosing, the muscle endurance tests are repeated periodically to measure improvement. It is anticipated that muscle endurance will improve following Compound III (ENT-03) dosing by about 5% or more.

[0426] Coordination can also be evaluated for each subject prior to initial Compound III (ENT- 03) dosing to establish a baseline by testing the patient’s ability to perform rapidly alternating and point-to-point movements correctly. Following initiation of Compound III (ENT-03) dosing, the coordination tests are repeated periodically to measure improvement. It is anticipated that coordination will improve following Compound III (ENT-03) dosing by about 5% or more.

[0427] Cognitive ability can also be evaluated for each subject prior to initial Compound III (ENT-03) dosing to establish a baseline using a conventional cognitive ability test. Following initiation of Compound III (ENT-03) dosing, the cognitive ability test is repeated periodically to measure improvement. It is anticipated that cognitive ability will improve following Compound III (ENT-03) dosing by about 5% or more.

10428] In a second part of this experiment, it is proposed that the animals additionally be treated with an insulin compound. The insulin compound is expected to act synergistically with the aminosterol compound comprising a spermine moiety.

Example 17

[0429] This prophetic example describes an exemplary method of delaying growth and/or maturation of a subject, comprising administering a pharmaceutical composition comprising a therapeutically effective amount of Compound III (ENT-03) or a pharmaceutically acceptable salt or derivative thereof to the subject.

[0430] One or more juvenile dogs can be given a suitable dosage of Compound III (ENT-03) via any pharmaceutically acceptable method, such as oral, intranasal, or injectable. An exemplary daily or weekly dosage can be, for example, about 20 to about 160 mg/m ² /day administered daily via any pharmaceutically acceptable route.

[0431] The rate of growth of each dog can be measured by recording each dog’s height and weight prior to treatment, and then periodically after initiation of treatment. At least one control dog, of the same sex and breed as the tested dogs, does not receive Compound III (ENT-03) treatment.

[0432] Consistent with the results described in Example 3 and Figure 2, the treated dogs are expected to show slower growth in terms of height and weight as compared to the untreated dog(s). However, the end point in terms of height and weight of both the treated and untreated dogs is expected to be the same. It is expected that administration of Compound III (ENT-03) will result in slowing growth, in terms of height and/or weight, by about 5% or more.

[0433] In a second part of this experiment, it is proposed that the animals additionally be treated with an insulin compound. The insulin compound is expected to act synergistically with the aminosterol compound comprising a spermine moiety.

Example 18

[0434] This prophetic example describes an exemplary method of delaying and/or preventing progression and/or onset of age-related neurodegeneration in a subject, comprising administering a pharmaceutical composition comprising a therapeutically effective amount of Compound III (ENT-03) or a pharmaceutically acceptable salt or derivative thereof to the subject.

104351 One or more subjects are given a daily dose of a pharmaceutical composition comprising Aminosterol 1436. The composition can be administered via any pharmaceutically acceptable method, such as oral, injectable, or intranasally. In an exemplary method, the composition is administered daily intranasally at a dosage of about 1 to about 20 mg.

[0436] Neurodegeneration is evaluated prior to treatment to form a baseline, using a medically recognized technique, and then periodically following initiation of treatment. At least one control subject, of the same sex and age as the tested subjects, does not receive Compound III (ENT-03) treatment.

[0437] The treated subjects are expected to show slowed progression and/or onset of neurodegeneration as compared to the untreated control subject. It is expected that administration of Compound III (ENT-03) will result in slowing progression and/or onset of neurodegeneration by about 5% or more.

[0438] In a second part of this experiment, it is proposed that the animals additionally be treated with an insulin compound. The insulin compound is expected to act synergistically with the aminosterol compound comprising a spermine moiety.

Example 19

[0439] This prophetic example describes an exemplary method of extending the potential lifespan of a subject, which can be an animal or human.

(0440] One or more mice can be given a suitable dosage of Compound III (ENT-03) via any pharmaceutically acceptable method, such as oral, intranasal, or injectable. The mice can be juveniles or adults. An exemplary daily or weekly dosage can be, for example, about 1 to about 10 mg/kg every 3 days administered via IPeritoneal or INasal. A control group of mice, of the same sex, are not treated.

]0441 [ The lifespan of each mouse is measured and compared to that of the control group. It is expected that administration of Compound III (ENT-03) will result in extending the lifespan of the mice by about 5% or more as compared to the control. [0442] In a second part of this experiment, it is proposed that the animals additionally be treated with an insulin compound. The insulin compound is expected to act synergistically with the aminosterol compound comprising a spermine moiety.

H 1:

[0443] It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention cover the modifications and variations of this invention, provided they come within the scope of the appended claims and their equivalents.

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