Bile acid complex

Abstract

The present invention relates to compounds that have a wide range of beneficial therapeutic properties and effects, including neuroprotective effects, anti-inflammatory activity, antioxidant properties, and epithelial cell protective effects.These properties allow the compounds to be useful for treating or preventing a wide range of disorders and conditions.These disorders and conditions include neurological disorders (including Alzheimer's disease, dementia caused by Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (motor neuron disease), multiple sclerosis, and neurodegenerative conditions such as brain injury), cognitive decline (such as memory loss) associated with these neurological disorders, and disorders caused by stress-induced cell damage in the inner or middle ear of subjects (such as vestibular disorders, hearing disorders, and conditions associated with hair cell degeneration or hair cell death).

Description

[Technical field]

[0001] This application claims priority from Australian Provisional Patent Application No. 2021904008, filed on December 10, 2021. The entire contents of Australian Provisional Patent Application No. 2021904008 are incorporated herein by reference.

[0002] Technical Field The present invention relates to compounds that have a wide range of beneficial therapeutic properties and effects, including neuroprotective effects, anti-inflammatory activity, antioxidant properties, and epithelial cell protective effects.These properties allow the compounds to be useful for treating or preventing a wide range of disorders and conditions.These disorders and conditions include neurological disorders (including Alzheimer's disease, dementia caused by Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (motor neuron disease), multiple sclerosis, and neurodegenerative conditions such as brain injury), cognitive decline (such as memory loss) associated with these neurological disorders, and disorders caused by stress-induced cell damage in the inner or middle ear of subjects (such as vestibular disorders, hearing disorders, and conditions associated with hair cell degeneration or hair cell death). [Background technology]

[0003] background Probucol (2,6-ditert-butyl-4-[2-(3,5-ditert-butyl-4-hydroxyphenyl)sulfanylpropan-2-ylsulfanyl]phenol) is a hydrophobic compound that is widely prescribed as an orally taken lipid-lowering or antihypercholesterolemic drug. Probucol is a potent antioxidant that has been in clinical use for the past several decades for the treatment and prevention of cardiovascular disease. Common reported side effects of probucol include bloating, diarrhea, nausea and vomiting, abdominal pain, dizziness or fainting, and fast or irregular heartbeat. Headache and numbness or tingling in the fingers, toes, or face have also been reported as side effects of probucol. Probucol is no longer available in many countries due to concerns regarding its efficacy and adverse and undesirable effects.

[0004] Succinobucol is a monosuccinic acid ester derivative of probucol and has anti-inflammatory and antioxidant properties. Succinobucol has been studied for use in the treatment of atherosclerosis, coronary artery disease, diabetes (type 2 diabetes), and in-stent restenosis. Compared to placebo treatment, succinobucol has been reported to increase LDL (bad) cholesterol and systolic blood pressure, and decrease HDL (good) cholesterol.

[0005] It would be advantageous to provide new derivatives of probucol and succinobucol that retain the useful properties and beneficial therapeutic effects of these drugs, but that result in fewer or reduced side effects typically associated with these drugs.

[0006] All references, including any patents or patent applications cited in this specification are hereby incorporated by reference.

[0007] A number of prior art publications are referenced herein, but it will be understood that this reference does not constitute an admission that any of these publications form part of the common general knowledge in the art in Australia, or in any other country. Summary of the Invention

[0008] overview In various embodiments, the following are provided:

[0009] In one embodiment, the compound of formula (I):

[0010] [ka]

[0011] (In the formula: R 1 teeth:

[0012] [ka]

[0013] and; R 2 , R 3 , R 4 , R 5 , R 7 , R 8 , R 9 , R 10 and R 11 each independently represents H, substituted or unsubstituted C 1-30 Acyloxy, substituted or unsubstituted benzoyloxy, substituted or unsubstituted C 1-30 Alkyl, substituted or unsubstituted C 2-30 Alkenyl, substituted or unsubstituted C 2-30 Alkynyl, substituted or unsubstituted C 3-30cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, an amino acid moiety, or GL, where GL is independently selected from OL, SL, PL2, CL3, or NL2; 2 , R 3 , R 4 , R 5 , R 7 , R 8 , R 9 , R 10 or R 11 When substituted, the substituents are independently OH, F, SH, =O, =S, Cl, Br, SC 1-6 Alkyl, C 1-6 Alkyl or C 1-6 alkoxy; Each L is independently H, a metal ion, or a substituted or unsubstituted C. 1-30 Alkyl, substituted or unsubstituted C 2-30 Alkenyl, substituted or unsubstituted C 2-30 Alkynyl, substituted or unsubstituted C 3-30 cycloalkyl, a substituted or unsubstituted benzyl radical, -CH2CO2H, or -(CH2)2SO3H; where when L is substituted, the substituents are independently selected from OH, SH, =O, =S, F, Cl, Br, SC 1-6 Alkyl, C 1-6 Alkyl or C 1-6 alkoxy; R 6 is -(CH2) n - (wherein n is 0 to 12), -CH2C(=O)NHCH2-, or -CH2C(=O)NHCH2CH2-; Y is -C- or -S(=O)-; and R a is -H or -C(=O)CH2CH2COOH; or a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof is provided.

[0014] The compound of formula (I) has neuroprotective effect, anti-inflammatory activity, antioxidant properties, and epithelial cell protective effect.It is hypothesized that these useful and beneficial properties may be due to the phenolic structure of the compound.The compound of formula (I) is therefore useful in treating or preventing neurological disorders (including Alzheimer's disease, dementia caused by Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (motor neuron disease), multiple sclerosis, and neurodegenerative conditions such as brain injury), cognitive decline (such as memory loss) associated with these neurological disorders, and disorders caused by stress-induced cell damage in the inner or middle ear of a subject (such as vestibular disorders, hearing impairment, and conditions associated with hair cell degeneration or hair cell death).

[0015] In a further aspect, there is provided a pharmaceutical composition comprising a compound of formula (I) or a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier, adjuvant or diluent.

[0016] In a further aspect, there is provided a method for treating or preventing neurological disorder or cognitive decline associated with neurological disorder in a subject, said method comprising administering to said subject an effective amount of the compound of formula (I) or its stereoisomer, or its pharmacologic acceptable salt.Neurological disorder can be Alzheimer's disease, dementia caused by Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (motor neuron disease), multiple sclerosis, or brain injury.Cognitive decline includes memory loss.

[0017] In a further aspect, there is provided a method of preventing, reducing or treating the occurrence and / or severity of a disorder caused by stress-induced cell damage in the middle or inner ear of a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of formula (I) or its stereoisomer, or a pharma- ceutically acceptable salt thereof. The disorder caused by stress-induced cell damage in the inner or middle ear can be a vestibular disorder, a hearing disorder, or a condition associated with hair cell degeneration or hair cell death.

[0018] In a further aspect, there is provided a method for preparing a compound of formula (I) comprising coupling probucol or succinobucol to C17 of a bile acid or derivative thereof. [Brief description of the drawings]

[0019] Embodiments of the present invention will now be further described, by way of example only, with reference to the accompanying drawings in which: * p<0.05, ** p<0.001, *** p<0.0001, and ns=not significant. The accompanying figures are explained below: [Figure 1] Figure 1 shows cytotoxicity studies by MTT cell viability assay. AM-MSCs were incubated with various concentrations of (A) probucol, (B) SPQ, (C) HA-1, and (D) DMSO for 24 hours. D1=DMSO control for 10 μM, D2=DMSO control for 20 μM (n=3). [Diagram 2] Figure 2 shows the estimation of LDH release. Neurodegenerative cells were incubated with probucol (20 μM), SPQ (10 μM), HA-1 (10 μM), and rivastigmine (2 μM) for 24 h (n=3). [Diagram 3] Figure 3 shows the effects of probucol, SPQ, HA-1 and rivastigmine on (A) acetylcholinesterase (AChE) activity and (B) acetylcholine release in an Alzheimer's disease model using an Amplex Red reagent-based assay. Neurodegenerative cells were incubated with probucol (20 μM), SPQ (10 μM), HA-1 (10 μM) and rivastigmine (2 μM) for 24 hours (n=3). [Figure 4] Figure 4 shows the measurement of mitochondrial membrane potential by TMRE assay. Neurodegenerative cells were incubated with probucol (20 μM), SPQ (10 μM), HA-1 (10 μM) and rivastigmine (2 μM) for 24 hours (n=3). [Diagram 5] FIG. 5 shows gene expression analysis by qRT-PCR for neuronal, cholinergic and AD-related markers (A) BACE1, (B) ChAT, (C) synapsin 1, and (D) neuropilin (n=3). [Figure 6] FIG. 6 shows the effects of probucol, SPQ, HA-1 and rivastigmine on (A) intracellular reactive oxidative stress and (B) nitric oxide. [Figure 7] FIG. 7 shows the effect of probucol, SPQ, HA-1 and rivastigmine on glutathione activity. [Figure 8] Figure 8 shows the results of a cytotoxicity study by MTT cell viability assay. AM-MSCs were incubated with various concentrations of (A) probucol, (B) SPQ, (C) HA-1, and (D) DMSO for 24 hours. D1=DMSO control for 10 μM, D2=DMSO control for 20 μM (n=3). [Figure 9] Figure 9 shows the measurement of mitochondrial membrane potential by TMRE assay. Neurodegenerative cells were incubated with probucol (20 μM), SPQ (10 μM), HA-1 (10 μM) and L-dopa (50 μM) for 24 hours (n=3). [Figure 10] Figure 10 shows the evaluation of ATP levels. Neurodegenerative cells were incubated with probucol (20 μM), SPQ (10 μM), HA-1 (10 μM) and L-dopa (50 μM) for 24 hours (n=3). [Figure 11] Figure 11 shows gene expression analysis by qRT-PCR for neuronal, dopaminergic and PD-related markers: (A) Synapsin I, (B) Nurr1, (C) DAT, (D) TH and (E) α-synuclein. (n=3). [Figure 12] FIG. 12 shows the effects of probucol, SPQ, HA-1 and L-dopa on (A) intracellular reactive oxidative stress and (B) nitric oxide. [Figure 13]FIG. 13 shows the effect of probucol, SPQ, HA-1 and L-dopa on glutathione activity. [Figure 14] FIG. 14 shows rotarod latency (mean) in seconds 12 weeks following a sham procedure, subconcussion with administration of AIN93M ("SC"), subconcussion with administration of probucol, subconcussion with administration of AGE, and subconcussion with administration of HA-1. [Figure 15] FIG. 15 shows the number of slips (Y-axis) in a beam-walk test on a 2 cm beam 6 and 12 weeks after the sham procedure, subconcussion with administration of AIN93M, subconcussion with administration of probucol, subconcussion with administration of AGE, and subconcussion with administration of HA-1. [Figure 16] FIG. 16 shows NIT-1 cell viability at 5.5 mM glucose: under normoglycemic conditions, none of the treatment groups exerted any effect, and this was expected since under physiological conditions NIT-1 cells are already at optimal function. [Figure 17] FIG. 17 shows NIT-1 cell viability at 25.5 mM glucose: In hyperglycemic conditions, NIT-1 cells are under stress due to glucose-mediated toxicity, mitochondrial dysfunction and oxidative stress. [Figure 18] FIG. 18 shows the results of the passive avoidance test at 14 weeks for the negative control (non-diabetic mice), positive control (diabetic db / db mice), probucol, HA-1 and ApocIII. [Figure 19] FIG. 19 shows the results of the novel object recognition test at 14 weeks for negative control (non-diabetic mice), positive control (diabetic db / db mice), probucol, HA-1 and ApocIII. [Figure 20]FIG. 20 shows the effect of HA-1 on repeated subconcussion in rats as measured by rotarod and beam-walk tests: (A) shows the mean rotarod latency in seconds 12 weeks after sham procedure, subconcussion, subconcussion with administration of probucol, and subconcussion with administration of HA-1; and (B) shows the number of slips (Y-axis) in the beam-walk test on a 2 cm beam 12 weeks after sham procedure, subconcussion, subconcussion with administration of probucol, and subconcussion with administration of HA-1 (n=12). [Figure 21] FIG. 21 shows an increase in IgG extravasation in (A) the hippocampal formation (HPF) and (B) cortical (CTX) regions of the brain 12 weeks after repeated subconcussion. [Figure 22] FIG. 22 shows changes in expression of 8dOHG, a marker of oxidative stress, in (A) the hippocampal formation (HPF) and (B) cortical (CTX) regions of the brain after 12 weeks of repeated subconcussion. [Diagram 23] FIG. 23 shows plasma glucose (A and B), triglyceride (C and D), insulin (E and F), and cholesterol (G and H) levels measured in 14- and 28-week-old diabetic db / db mice (n=12). [Figure 24] FIG. 24 shows levels of apoB (a marker for lipid-bearing particles) in the small intestine of 14- and 28-week-old diabetic db / db mice (A and B) and levels of small intestinal Aβ in 14- and 28-week-old diabetic db / db mice (C and D). [Diagram 25] Figure 25 shows plasma Aβ40 levels in 14- and 28-week-old diabetic db / db mice (A and B), plasma Aβ42 levels in 14- and 28-week-old diabetic db / db mice (C and D), plasma concentrations of Aβ oligomers in 14- and 28-week-old diabetic db / db mice (E and F), and Aβ42 / 40 ratios in 14- and 28-week-old diabetic db / db mice (G and H) (n=12). [Figure 26]FIG. 26 shows the results (A and B) of the passive avoidance latency test in 14- and 28-week-old diabetic db / db mice (n=12). [Figure 27] FIG. 27 shows the levels of IgG extravasation in the hippocampal formation (HPF) in 14- and 28-week-old diabetic db / db mice (A and B) and the levels of IgG extravasation in the cortex (CTX) in 14- and 28-week-old diabetic db / db mice (C and D) (n=12). [Figure 28] FIG. 28 shows the expression levels of 8dOHG in HPF and CTX (A and B), the expression levels of GFAP in HPF and CTX (C and D), and the expression levels of Iba-1 in HPF and CTX (E and F) (n=12). [Figure 29] FIG. 29 shows the oxygen consumption rate (OCR) of OC-1 cells after 24 hours of exposure to cisplatin. [Diagram 30] FIG. 30 shows OCR data for OC-1 cells following 48 hour exposure to cisplatin followed by treatment with HA-1 and HA-2. [Diagram 31] FIG. 31 shows the results of a caspase assay using analogs HA-1 and HA-2. [Diagram 32] FIG. 32 shows shws cell morphology and viability following a cobalt chloride assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Detailed Description of the Preferred Embodiments Preferred embodiments of the invention are now described, by way of example only.

[0021] 1.Definition Unless otherwise defined herein, the following terms shall be understood to have the following general meanings. The terms referred to below shall have the following general meanings when used alone and when used in combination with other terms, unless otherwise indicated. Thus, for example, the definition of "alkyl" applies to "alkyl" as well as the "alkyl" portions of "haloalkyl", "arylalkyl", etc.

[0022] As used herein, the term "amphiphile" or "amphiphilic compound" is any compound having a hydrophilic / lipophilic balance (HLB) value between about 2 and about 20, inclusive. Preferred amphiphilic compounds have an HLB value between about 6 and about 16.

[0023] The term "alkyl" refers to linear or branched saturated hydrocarbyl groups. Unless otherwise indicated, preferred are 1-6 Alkyl groups and C 1-4 The term "C" is an alkyl group. x-y "Alkyl" (where x and y are integers) refers to an alkyl group having from x to y carbon atoms. For example, the term "C 1-6 "Alkyl" refers to an alkyl group having from 1 to 6 carbon atoms. 1-6 Examples of alkyl include methyl (Me), ethyl (Et), propyl (Pr), isopropyl (i-Pr), butyl (Bu), isobutyl (i-Bu), sec-butyl (s-Bu), tert-butyl (t-Bu), pentyl, neopentyl, hexyl, etc. Unless the context requires otherwise, the term "alkyl" also includes alkyl groups containing one less hydrogen atom, such that the group is attached through two positions, i.e., divalent.

[0024] The term "alkenyl" refers to straight or branched chain hydrocarbyl groups, where applicable, having at least one double bond of either E- or Z-stereochemistry. Unless otherwise indicated, preferred are 2-6 Alkenyl groups and C 2-3 The term "C" is an alkenyl group. x-yAlkenyl" (wherein x and y are integers) refers to an alkenyl group having from x to y carbon atoms. For example, the term "C 2-6 "Alkenyl" refers to an alkenyl group having from 2 to 6 carbon atoms. 2-6 Examples of alkenyl include vinyl, 1-propenyl, 1- and 2-butenyl, and 2-methyl-2-propenyl. Unless the context requires otherwise, the term "alkenyl" also includes alkenyl groups containing one less hydrogen atom, such that the group is attached through two positions, i.e., divalent.

[0025] The term "alkynyl" refers to a straight or branched chain hydrocarbyl group having at least one triple bond. Unless otherwise indicated, preferred are 2-6 Alkynyl groups and C 2-3 The term "C" is an alkynyl group. x-y "Alkynyl" (wherein x and y are integers) refers to an alkynyl group having from x to y carbon atoms. For example, the term "C 2-6 "Alkynyl" refers to an alkynyl group having from 2 to 6 carbon atoms. 2-6 Examples of alkynyl include ethynyl, 1-propynyl, 1- and 2-butynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl and 5-hexynyl, etc. Unless the context indicates otherwise, the term "alkynyl" also includes alkynyl groups containing one less hydrogen atom, such that the group is attached through two positions, i.e., divalent.

[0026] The term "cycloalkyl" refers to a non-aromatic cyclic hydrocarbyl group having three or more carbon atoms in the ring. 3-30 "Cycloalkyl" refers to a non-aromatic cyclic hydrocarbyl group having from 3 to 30 carbon atoms. Such groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. The term "Cycloalkyl" refers to a non-aromatic cyclic hydrocarbyl group having from 3 to 30 carbon atoms. Such groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. 3-30"Cycloalkyl" includes groups where the cyclic hydrocarbyl group is saturated, such as cyclohexyl, or unsaturated, such as cyclohexenyl. In some embodiments, C 3-30 Cycloalkyl is C 3-8 Cycloalkyl. C such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. 3-6 Cycloalkyl is preferred.

[0027] The term "acyloxy" refers to R m C 1-30 The alkyl group -OC(=O)R m Refers to...

[0028] The terms "hydroxy" and "hydroxyl" refer to the group --OH.

[0029] The term "oxo" refers to the group ═O.

[0030] The term "alkoxy" refers to an alkyl group as defined above covalently bonded through an O linkage, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy and pentoxy. Unless otherwise indicated, preferred are 1-6 Alkoxy group, C 1-4 Alkoxy groups and C 1-3 It is an alkoxy group.

[0031] The term "aryl" refers to a carbocyclic (non-heterocyclic) aromatic ring or monocyclic, bicyclic, or tricyclic ring system. The aromatic ring or ring system is generally composed of 6 to 10 carbon atoms. Examples of aryl groups include, but are not limited to, phenyl, biphenyl, naphthyl, and tetrahydronaphthyl. Six-membered aryls, such as phenyl, are preferred.

[0032] The term "arylalkyl" or "aralkyl" refers to an aryl C 1-6 Refers to alkyl-.

[0033] The term "heteroaryl" is used herein to refer to heterocyclic groups having aromatic character and includes aromatic monocyclic ring systems and polycyclic (e.g., bicyclic) ring systems containing one or more aromatic rings. The term aromatic heterocyclyl also includes pseudoaromatic heterocyclyl. The term "pseudoaromatic" refers to a ring system that is not strictly aromatic but is stabilized by electron delocalization and behaves similarly to an aromatic ring. The term aromatic heterocyclyl thus includes polycyclic ring systems in which all of the fused rings are aromatic as well as ring systems in which one or more rings are non-aromatic, provided that at least one ring is aromatic. In polycyclic ring systems that contain both aromatic and non-aromatic rings fused together, the group may be attached to another moiety by the aromatic ring or by the non-aromatic ring.

[0034] Examples of heteroaryl groups are monocyclic and bicyclic groups containing 5 to 10 ring members. Heteroaryl groups can be, for example, 5- or 6-membered monocyclic rings or fused 5- and 6-membered rings or bicyclic structures formed from two fused 6-membered rings or two fused 5-membered rings. Each ring can contain up to 4 heteroatoms selected from nitrogen, sulfur and oxygen. Heteroaryl groups can contain up to 4 heteroatoms, more typically up to 3 heteroatoms, more usually up to 2 heteroatoms. In one embodiment, heteroaryl groups contain at least one ring nitrogen atom. The nitrogen atom in a heteroaryl group can be basic, as in the case of imidazole or pyridine, or essentially non-basic, as in the case of indole or pyrrole nitrogen. Generally, the number of basic nitrogen atoms present in a heteroaryl group will be less than 5, including any amino group substituents on the ring.

[0035] Examples of heteroaryl groups containing aromatic and non-aromatic rings include tetrahydroisoquinoline, tetrahydroquinoline, dihydrobenzothiophene, dihydrobenzofuran, 2,3-dihydro-benzo[1,4]dioxine, benzo[1,3]dioxole, 4,5,6,7-tetrahydrobenzofuran, indoline and isoindoline groups.

[0036] The term "chiral" refers to molecules that have the property of not being superimposable on their mirror image counterparts, while the term "achiral" refers to molecules that are superimposable on their mirror image counterparts.

[0037] The term "diastereomer" refers to a stereoisomer with two or more centers of dissymmetry and whose molecules are not mirror images of one another.

[0038] The term "enantiomers" refers to two stereoisomers of a compound that are non-superimposable mirror images of one another. An equimolar mixture of two enantiomers is called a "racemic mixture" or "racemate."

[0039] The term "stereoisomers" refers to compounds that have identical chemical constitution, with atoms bonded in the same order, but differ with regard to the arrangement of the atoms in space.

[0040] Throughout this specification, including the claims, the word "include" or variations thereof, such as "includes" or "including," will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0041] Throughout this specification, including the claims, unless the context requires otherwise by express predicate or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" are used in the inclusive sense, i.e. to specify the presence of a stated element, integer or step, or group of elements, integers or steps, but do not preclude the presence or addition of further elements, integers or steps, or further groups of elements, integers or steps, in various embodiments of the invention.

[0042] As used herein, the term "pharmaceutical acceptable carrier" refers to a carrier or excipient or diluent suitable for use in humans and / or animals, without undue adverse side effects (such as toxicity, irritation, and allergic reactions), commensurate with a reasonable benefit / risk ratio.It may be a pharmaceutical acceptable solvent, suspending agent, or vehicle for delivering an active ingredient to a subject.

[0043] As used herein, the term "controlled release" refers to controlling the rate and / or amount of an active ingredient delivered according to the pharmaceutical compositions described herein. The controlled release kinetics can be prolonged or sustained release, fast or immediate release, delayed release or pulsatile drug delivery systems.

[0044] The terms "individual," "subject," and "patient" are used interchangeably herein. In certain embodiments, the subject is a mammal. Mammals include, but are not limited to, primates (including humans and non-human primates). In a preferred embodiment, the subject is a human.

[0045] The term "neuropathy" as used herein is defined as any disorder that affects the brain and nerves found throughout the human body and spinal cord.Neuropathy includes neurodegenerative diseases such as Alzheimer's disease, dementia caused by Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (motor neuron disease), multiple sclerosis, and brain injury, and may lead to cognitive decline (such as memory loss).

[0046] The term "disorder caused by stress-induced cell damage in the middle or inner ear" as used herein is defined as any disorder caused by stress-induced cell damage in the middle or inner ear. Cell damage in the middle or inner ear can be caused by any form of insult that can increase cell stress in the middle or inner ear through molecular disturbance, including, for example, by physical or acoustic trauma, bacterial, viral or other types of infection, or chemical ototoxic insult, which can lead to high levels of free radicals, oxidative stress, proinflammatory cytokines, which can affect normal cell function in the middle or inner ear, and can cause inflamed or damaged specialized middle or inner ear epithelium, such as the stria vascularis, spiral ligament, organ of Corti, endolymphatic sac, crista ampulla, otoliths, spiral ganglion, auditory nerve, along with loss of neural signaling and general apoptosis throughout the middle or inner ear. Changes in the middle ear can affect the inner ear. For example, otitis media, which is primarily a middle ear problem, can lead to sensorineural hearing loss and imbalance, both of which are inner ear problems.

[0047] The term "hearing disorder" as used herein is defined as any disorder caused by molecular disturbances that affect the inner or middle ear and their associated neural connections to the brain.

[0048] The term "hearing impairment" as used herein is defined as any impairment of hearing caused by molecular disturbances that affect the inner or middle ear and their associated neural connections to the brain.

[0049] The term "hearing loss" as used herein is defined as a decreased ability to perceive sound relative to a normal level. This can be caused by either conductive hearing loss, sensorineural hearing loss, or a combination of both. Hearing loss includes presbycusis (age-related hearing loss).

[0050] As used herein, the term "conductive hearing loss" is one in which the transmission of sound pressure from the outer ear to the inner ear is diminished due to, for example, excess earwax buildup, glue ear, ear infections with inflammation and fluid buildup, perforated or defective eardrums, skin growths in the middle ear (cholesteatoma), or poor functioning of the ossicles (bones in the middle ear).

[0051] As used herein, the term "sensorineural hearing loss" is caused by dysfunction of the sensory and / or nerve cells of the cochlea and / or specialized epithelium in the inner ear, such as the streak and supporting cells of the cochlea, and associated neural connections from the ear to the brain.

[0052] As used herein, the term "vestibular dysfunction" is any impairment in the stabilization of balance or vision caused by molecular disturbances affecting the inner ear.

[0053] As used herein, the term "hair cell degeneration" or "hair cell loss" refers to the gradual loss of hair cell function and integrity and / or ultimately leading to hair cell death.

[0054] As used herein, the term "hair cell death" refers to apoptosis of hair cells in the middle or inner ear.

[0055] The terms "identification of hair cell damage" and "detection of hair cell damage" are used interchangeably herein and refer to methods that can determine the extent of hair cell damage in the middle or inner ear. Such methods are known in the art and include, for example, fluorescent imaging of hair cells. An audiogram that demonstrates loss of hearing sensitivity at mid to high frequencies also indicates hair cell damage. A decrease in the likelihood of hearing without any subsequent recovery also aids in the diagnosis of hair cell damage.

[0056] As used herein, the terms "chemically induced hearing loss" and "chemically induced hearing loss" refer to hearing loss that is induced and / or caused by chemicals, such as solvents, gases, paints, heavy metals, and / or drugs that are ototoxic.

[0057] An "effective amount" or "therapeutically effective amount" is an amount sufficient to produce a beneficial or desired therapeutic effect. This amount may be the same as or different from a "prophylactically effective amount," which is the amount necessary to prevent the onset of a disorder or symptoms of a disorder. An effective amount may be administered in one or more administrations, applications, or dosages. The therapeutically effective amount (i.e., effective dosage) of a therapeutic compound will depend on the therapeutic compound selected.

[0058] As used herein, the term "treating" refers to affecting a subject, tissue or cell to obtain a desired pharmacological and / or physiological effect, and includes inhibiting a condition, i.e., arresting its progression; or reducing or ameliorating the effects of a condition, i.e., causing a reversal or regression of the effects of a condition.

[0059] As used herein, the term "preventing" refers to preventing a condition from occurring in a cell, tissue, or subject that may be at risk of having the condition, but does not necessarily mean that the condition will not eventually develop or that the subject will not eventually develop the condition. Preventing includes delaying the onset of a condition in a cell, tissue, or subject.

[0060] The singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below.

[0061] The abbreviation "eg" is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example, i.e., the abbreviation "eg" is synonymous with the term "for example."

[0062] The use of any and all examples or exemplary language provided herein with respect to specific embodiments (e.g., "such as") is intended merely to better illuminate the application and does not pose limitations on the scope of the application as otherwise claimed. No language herein should be construed as indicating any non-claimed element essential to the practice of the application.

[0063] As used herein, the term "about" refers to ± up to 20% of a given measurement. For example, the term "about" refers to a value that is within plus or minus 10% or within plus or minus 5% of the recited value.

[0064] The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and unless otherwise indicated herein, each individual value is incorporated herein as if it were individually recited herein.

[0065] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0066] 2. Compounds of formula (I) The present invention broadly relates to compounds of formula (I), including stereoisomers of compounds of formula (I) and pharma- ceutically acceptable salts of compounds of formula (I). Compounds of formula (I) are formed by conjugating probucol or succinobucol to a bile acid, or by conjugating probucol or succinobucol to a derivative of a bile acid. Conjugates of formula (I) are also referred to herein as "bile acid conjugates." References herein to "compounds of formula (I)" include stereoisomers of formula (I) and any mixture of stereoisomers of formula (I).

[0067] Bile acids are steroids whose structure is related to cholane or cholestane. Bile acids may be called "cholanoids" when it is convenient to have a name for this subclass of steroids. The term "bile acid" is a general term for such molecules that have a carboxyl group and does not indicate any state of ionization. The numbering of the carbon atoms of the steroid ring and side chain is shown below in the structure of the cholestane skeleton.

[0068] [ka]

[0069] Bile acids are facial amphiphiles, i.e., they contain both hydrophobic and hydrophilic faces. The cholesterol-derived portion of the bile acids has one face that is hydrophobic (bearing a methyl group) and one that is hydrophilic (bearing a hydroxyl group).

[0070] Examples of bile acids that may be used to prepare compounds of formula (I) or their stereoisomers or pharma- ceutically acceptable salts are: JPEG2025503406000004.jpg242139JPEG2025503406000005.jpg108158 Includes.

[0071] Probucol (2,6-ditert-butyl-4-[2-(3,5-ditert-butyl-4-hydroxyphenyl)sulfanylpropan-2-ylsulfanyl]phenol) is a hydrophobic compound with antioxidant properties, which may be due to its phenolic structure. In addition to being a cholesterol-lowering and antiatherogenic drug, probucol is a potent oxygen radical scavenger that may serve as a potent anti-inflammatory drug to inhibit oxidant-induced tissue damage. Because glutathione peroxidase (GPx) plays a pivotal role in preventing oxidative stress, the pharmacological use of its mimetics has been proposed as a strategy to treat oxidative stress-related pathologies. In some studies, the protective effect of probucol was paralleled by a significant increase in GPx activity.

[0072] The inventors have discovered that conjugating probucol to bile acids results in conjugates (referred to herein as "bile acid conjugates") that have useful and beneficial properties and effects, such as neuroprotective effects, anti-inflammatory activity, antioxidant properties, and epithelial cell protective effects.

[0073] Advantageously, bile acid conjugates can be used in amounts that are effective to produce the desired therapeutic effect, but with reduced toxicity and reduced or no adverse side effects that are normally associated with probucol and succinobucol.Because of their stability, lipophilicity and tissue uptake, bile acid conjugates are expected to provide greater tissue penetration and enhanced pharmacodynamic response with improved pharmacokinetic profile.That is, a more predictable dose-response effect is achieved, which in turn will minimize side effects.

[0074] Without wishing to be bound by theory, the inventors believe that the compounds of formula (I), the conjugates, may act as neuroprotective agents, anti-inflammatory agents, antioxidants, and / or epithelial cytoprotective agents and may provide cytoprotective effects.

[0075] In one embodiment, the present invention provides a compound of formula (I):

[0076] [ka]

[0077] (In the formula: R 1 teeth:

[0078] [ka]

[0079] and; R 2 , R 3 , R 4 , R 5 , R 7 , R 8 , R 9 , R 10 and R 11 each independently represents H, substituted or unsubstituted C 1-30 Acyloxy, substituted or unsubstituted benzoyloxy, substituted or unsubstituted C 1-30 Alkyl, substituted or unsubstituted C 2-30 Alkenyl, substituted or unsubstituted C 2-30 Alkynyl, substituted or unsubstituted C 3-30 cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, an amino acid moiety, or GL, where GL is independently selected from OL, SL, PL2, CL3, or NL2; 2 , R 3 , R 4 , R 5 , R 7 , R 8 , R 9 , R 10 or R 11 When substituted, the substituents are independently OH, F, SH, =O, =S, Cl, Br, SC 1-6 Alkyl, C 1-6 Alkyl or C 1-6 alkoxy; Each L is independently H, a metal ion, or a substituted or unsubstituted C. 1-30 Alkyl, substituted or unsubstituted C 2-30 Alkenyl, substituted or unsubstituted C 2-30 Alkynyl, substituted or unsubstituted C 3-30 cycloalkyl, a substituted or unsubstituted benzyl radical, -CH2CO2H, or -(CH2)2SO3H; where when L is substituted, the substituents are independently selected from OH, SH, =O, =S, F, Cl, Br, SC 1-6 Alkyl, C 1-6 Alkyl or C 1-6 alkoxy; R 6 is -(CH2) n - (wherein n is 0 to 12), -CH2C(=O)NHCH2-, or -CH2C(=O)NHCH2CH2-; Y is -C- or -S(=O)-; and R a is -H or -C(=O)CH2CH2COOH; or a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof.

[0080] In some embodiments, R a is -H. That is, in some embodiments, R 1 teeth:

[0081] [ka]

[0082] It is.

[0083] In some embodiments, R a is -C(=O)CH2CH2COOH. That is, in some embodiments, R 1 teeth:

[0084] [ka]

[0085] It is.

[0086] In some embodiments, each R 2 , R 3 , R 4 , R 5 , R 7 , R 8 , R 9 , R 10 and R 11 are independently H; substituted or unsubstituted C 1-30 Acyloxy (e.g., substituted or unsubstituted C 1-6 Acyloxy or substituted or unsubstituted C 1-4 acyloxy; substituted or unsubstituted benzoyloxy; substituted or unsubstituted C 1-12 Alkyl (e.g., substituted or unsubstituted C 1-6 Alkyl or substituted or unsubstituted C 1-4 alkyl); substituted or unsubstituted C 2-12 Alkenyl (e.g., substituted or unsubstituted C 2-6 Alkenyl or substituted or unsubstituted C 2-4 alkenyl); substituted or unsubstituted C 2-12 Alkynyl (e.g., substituted or unsubstituted C 2-6 Alkynyl or substituted or unsubstituted C 2-4 alkynyl); substituted or unsubstituted C 3-8 cycloalkyl; substituted or unsubstituted C6 aryl (e.g., substituted or unsubstituted phenyl); substituted or unsubstituted heteroaryl (e.g., substituted or unsubstituted pyridyl), or an amino acid moiety. In some embodiments, R 2 , R 3 , R 4 , R 5 , R 7 , R 8 , R 9 , R 10 or R 11 When substituted, the substituents are independently F, Cl, OH, SH, Br, SC 1-4 Alkyl, C 1-4Alkyl or C 1-4 In some embodiments, R 2 , R 3 , R 4 , R 5 , R 7 , R 8 , R 9 , R 10 or R 11 When substituted, it is F, Cl, Br, OH, SH, =O, =S, SC 1-4 Alkyl, C 1-4 Alkyl or C 1-4 Substituted with 1, 2, 3 or 4 substituents independently selected from alkoxy.

[0087] In some embodiments, GL is selected from OL, SL, PL2, CL3, or NL2, where each L is independently H, a metal ion, substituted or unsubstituted C. 1-30 Alkyl, substituted or unsubstituted C 2-30 Alkenyl, substituted or unsubstituted C 2-30 Alkynyl, substituted or unsubstituted C 3-30 cycloalkyl, a substituted or unsubstituted benzyl radical, -CH2CO2H, or -(CH2)2SO3H; where when L is substituted, the substituents are independently selected from OH, SH, =O, =S, F, Cl, Br, SC 1-6 Alkyl, C 1-6 Alkyl or C 1-6 alkoxy.

[0088] In some embodiments, L is H; substituted or unsubstituted C 1-12 Alkyl (e.g., substituted or unsubstituted C 1-6 Alkyl or substituted or unsubstituted C 1-4 alkyl); substituted or unsubstituted C 2-12 Alkenyl (e.g., substituted or unsubstituted C 2-6 Alkenyl or substituted or unsubstituted C 2-4 alkenyl); substituted or unsubstituted C 2-12 Alkynyl (e.g., substituted or unsubstituted C2-6 Alkynyl or substituted or unsubstituted C 2-4 alkynyl); substituted or unsubstituted C 3-8 cycloalkyl; a substituted or unsubstituted benzyl radical; -CH2CO2H; or -(CH2)2SO3H. In some embodiments, when L is substituted, the substituents are independently F, Cl, OH, SH, =O, =S, Br, SC 1-4 Alkyl, C 1-4 Alkyl or C 1-4 In some embodiments, when L is substituted, it is selected from F, Cl, OH, SH, ═O, ═S, Br, SC 1-4 Alkyl, C 1-4 Alkyl or C 1-4 Substituted with 1, 2, 3 or 4 substituents independently selected from alkoxy.

[0089] In some embodiments, GL is OH.

[0090] In some embodiments, L is, for example, a metal(I) ion (e.g., Na + , K + , and Li + ), and metal (II) ions (e.g., cadmium (II), iron (II), lead (II), zinc (II), copper (II), magnesium (II), and manganese (II) ions). That is, in some embodiments, the metal cation (e.g., Na + , K + , Li + , Cd 2+ , Fe 2+ , Pb 2+ , Zn 2+ , Cu 2+ , Mg 2+ , or Mn 2+ ) is attached to an organic moiety.

[0091] In some embodiments, R 2 , R 3 , R 4 , R5 , R 7 , R 8 , R 9 , R 10 or R 11 can be an amino acid moiety. The amino acid moiety can be formed from any amino acid, such as, for example, any of the major types of amino acids available. For example, the amino acid can be selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and taurine. In some embodiments, the amino acid is glycine, taurine, or alanine. In some embodiments, the amino acid is glycine. In some embodiments, the amino acid is taurine.

[0092] In some embodiments, R 2 , R 3 , R 4 , R 5 , R 7 , R 8 , R 9 , R 10 and R 11 each independently represents H, -OH, or -OC(=O)R f (In the formula, R f is C 1-6 alkyl).

[0093] In some embodiments, R 6 is -(CH2) n -, where n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, n is 0. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, n is 1, 2, 3, 4, or 5. Preferably, n is 1. In some embodiments, R 6 is -CH2-.

[0094] In some embodiments, R 6 is -CHC(=O)NHCH-. In some embodiments, R 6 is -CH2C(=O)NHCH2CH2-.

[0095] In some embodiments, Y is -C-. In some embodiments, Y is -S(=O)-.

[0096] In some embodiments, the compound of formula (I) has two or more hydroxy (-OH) groups attached to two different rings of formula (I), e.g., two different rings of a carboxylated steroid compound of formula (I).

[0097] R 2 , R 3 , R 4 , R 5 , R 7 , R 8 , R 9 , R 10 and R 11 Each of the -CH groups at C10 and C13, the hydrogen atoms at C5, C8, C9 and C14, and the group on C17 may be attached in an α or β configuration on the respective ring. For example, R 5 The group may be located at 3α or 3β.

[0098] In one embodiment, the compound of formula (I) has the formula (Ia):

[0099] [ka]

[0100] (In the formula: R a is -H or -C(=O)CH2CH2COOH; R b are -H, -OH and -OC(=O)R f (In the formula, R f is C 1-6 alkyl; R c are -H, -OH and -OC(=O)R f (In the formula, R f is C 1-6 alkyl; R d are -H, -OH and -OC(=O)R f (In the formula, R f is C 1-6 alkyl; R e -C 1-6 alkyl; R 6 is -CH2-, -CH2C(=O)NHCH2-, or -CH2C(=O)NHCH2CH2-; and Y is C or S(=O); or a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof.

[0101] In some embodiments of Formula (Ia), R a is -H. In some embodiments of Formula (Ia), R a is -C(=O)CH2CH2COOH.

[0102] That is, in some embodiments, formula (Ia) is:

[0103] [ka]

[0104] It is.

[0105] In some embodiments of Formula (Ia), R b , R c and R d each independently represents -H, -OH, or -OC(=O)R f (In the formula, R f is C 1-6 In some embodiments, R f is C1-6 Alkyl, C 1-5 Alkyl, C 1-4 Alkyl, or C 1-3 In some embodiments, R f is methyl (Me), ethyl (Et), propyl (Pr), isopropyl (i-Pr), butyl (Bu), isobutyl (i-Bu), sec-butyl (s-Bu), tert-butyl (t-Bu), pentyl, neopentyl, or hexyl.

[0106] In some embodiments of Formula (Ia), R e is C 1-6 Alkyl, C 1-5 Alkyl, C 1-4 Alkyl, or C 1-3 In some embodiments, R e is methyl (Me), ethyl (Et), propyl (Pr), isopropyl (i-Pr), butyl (Bu), isobutyl (i-Bu), sec-butyl (s-Bu), tert-butyl (t-Bu), pentyl, neopentyl, or hexyl.

[0107] In some embodiments of Formula (Ia), R 6 In some embodiments of Formula (Ia), R 6 In some embodiments of Formula (Ia), R 6 is -CH2C(=O)NHCH2CH2-.

[0108] In some embodiments of Formula (Ia), Y is C. In some embodiments of Formula (Ia), Y is S(=O).

[0109] In some embodiments, the compound of formula (Ia) is 2,6-di-tert-butyl-4-((2-((3,5-di-tert-butyl-4-hydroxyphenyl)thio)propan-2-yl)thio)phenyl (4R)-4-[(3R,5R,8R,9S,10S,13R,14S,17R)-3-acetoxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoate (herein referred to as “HA-1”):

[0110] [ka]

[0111] It is.

[0112] In some embodiments, the compound of formula (Ia) is 2,6-di-tert-butyl-4-((2-((3,5-di-tert-butyl-4-hydroxyphenyl)thio)propan-2-yl)thio)phenyl (4R)-4-[(3R,5R,7R,8R,9S,10S,13R,14S,17R)-3,7-diacetoxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoate (herein referred to as “HA-2”):

[0113] [ka]

[0114] It is.

[0115] HA-1 is probucol conjugated to lithocholic acid. HA-2 is probucol conjugated to chenodeoxycholic acid.

[0116] The salts of the compounds of formula (I) are pharmaceutically acceptable. When the compounds of formula (I) contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium salts, potassium salts, calcium salts, ammonium salts, organic amino salts, or magnesium salts, or similar salts. When the compounds contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharma- ceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid, or phosphorous acid, and the like, as well as salts derived from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like. Also included are salts of amino acids, such as arginates, and salts of organic acids, such as glucuronic acid or galactunic acid (see, e.g., Berge et al., Journal of Pharmaceutical Science 66:1-19 (1977)). Certain specific compounds of formula (I) contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts. Other pharma-ceutically acceptable carriers known to those skilled in the art are suitable for the present invention.

[0117] The neutral forms of the compounds can be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent forms of the compounds differ from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent forms of the compounds for purposes of the invention.

[0118] Compounds of formula (I) may be synthesized by methods known in the art of organic synthesis. Methods for optimizing reaction conditions, optionally minimizing competing by-products, are known in the art. Reaction optimization and scale-up may advantageously utilize high speed parallel synthesizers and computer-controlled microreactors (see, for example, Design And Optimization in Organic Synthesis, 2001). nd Edition, Carlson R, Ed, 2005; Elsevier Science Ltd.; Jaehnisch, K et al, Angew. Chem Int. Ed. Engl. 2004 43 : 406; and references therein). Additional reaction schemes and protocols may be determined by one of skill in the art through the use of commercially available structure-searchable database software, such as SciFinder® (CAS division of the American Chemical Society) and CrossFire Beilstein® (Elsevier MDL), or through appropriate keyword searches using Internet search engines such as Google® or keyword databases such as the U.S. Patent and Trademark Office text database. The present invention includes intermediate compounds used in making compounds of the formulae herein, including, without limitation, those specifically described in the Examples herein, as well as methods of making such compounds and intermediates.

[0119] Compounds of formula (I) may also contain linkages (e.g., carbon-carbon bonds) in which bond rotation is restricted for that particular linkage, e.g., restrictions resulting from the presence of a ring or double bond. Thus, all cis / trans and E / Z isomers are expressly included in the present invention. Compounds herein may also be represented in multiple tautomeric forms, and in such cases, the present invention expressly includes all tautomeric forms of the compounds described herein, even if only a single tautomeric form may be represented. All such isomeric forms of such compounds herein are expressly included in the present invention. Also embodied are extracts and fractions containing compounds of formula (I) or stereoisomers thereof. The term "isomer" is intended to include diastereoisomers, enantiomers, positional isomers, rotamers, tautomers, and the like. For compounds containing one or more stereocenters, e.g., chiral compounds, the methods described herein may be carried out using enantiomerically enriched compounds, racemates, or mixtures of diastereomers.

[0120] Preferred enantiomerically enriched compounds have an enantiomeric excess of 50% or greater, more preferably the compounds have an enantiomeric excess of 60%, 70%, 80%, 90%, 95%, 98%, or 99% or greater. In a preferred embodiment, only one enantiomer or diastereoisomer of the chiral compound of formula (I) or a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof, is administered to a cell or subject.

[0121] 3. Methods for preparing compounds of formula (I) The compound of formula (I) can be synthesized by methods known in the art.Various synthesis schemes are described below and in the examples.The examples describe the preparation of various specific compounds of formula (I).Those skilled in the art can modify the synthesis schemes described below and in the examples to prepare other compounds of formula (I) and their salts.

[0122] A typical general procedure for the preparation of compounds of formula (I) from bile acids and probucol is set out below.

[0123] Examples of bile acids that may be used in the preparation of compounds of formula (I) include: deoxycholic acid, cholic acid, taurocholic acid, glycocholic acid, glycodeoxycholic acid, taurodeoxycholic acid, ursodeoxycholic acid, tauchenodeoxycholic acid, lithocholic acid, glycolithocholic acid, chenodeoxycholic acid, taurolithocholic acid, tauroursodeoxycholic acid, obeticholic acid, any of the muricholic acids (e.g. α-muricholic acid, β-muricholic acid, γ-muricholic acid, ω-muricholic acid), glycomuric acid, tauromuricholic acid, or glycochenodeoxycholic acid, or a salt thereof.

[0124] For example, the starting bile acid may be represented by the following formula (B1):

[0125] [ka] It may be a compound of JPEG2025503406000015.jpg84158.

[0126] The compound of formula (B1) may be dissolved in pyridine and then acetic anhydride added to form the O-acyl protected bile acid of formula (B2).

[0127] [ka]

[0128] (Wherein, in formula (B2), R b , R c , R d , R 6 , and Y have the following meaning: JPEG2025503406000017.jpg75158 (having

[0129] The protected bile acid of formula (B2) is then reacted with, for example, thionyl chloride or oxalyl chloride to give the O-acyl bile acid chloride of formula (B3).

[0130] [ka]

[0131] Alternatively, the potassium salt of probucol may be prepared by adding probucol to a stirring mixture of potassium tert-butoxide in anhydrous tetrahydrofuran.

[0132] [ka]

[0133] The O-acetyl bile acid chloride of formula (B3) is then reacted with the potassium salt of probucol to form the bile acid conjugate of formula (B4).

[0134] [ka]

[0135] In the above general procedure, succinobucol may be substituted for probucol to produce bile acid conjugates with succinobucol.

[0136] Any bile acid or bile salt can be used in the preparation of the compound of formula (I), such as those mentioned above.For example, the bile acid or bile salt can be a primary bile acid, or a secondary bile acid, or any bile acid with an amino acid moiety attached.A variety of bile acids and bile salts are commercially available. For example, the bile acid or bile salt may be selected from deoxycholic acid (DCA), ursodeoxycholic acid (UDCA; also known as ursodiol), cholic acid, taurocholic acid, glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholic acid, taurodeoxycholic acid, taurochenodeoxycholic acid, lithocholic acid, glycolithocholic acid, chenodeoxycholic acid, taurolithocholic acid, tauroursodeoxycholic acid, obeticholic acid, all muricholic acids (α-muricholic acid, β-muricholic acid, γ-muricholic acid and ω-muricholic acid), glycomuric acid, tauromuric acid, or salts, derivatives or metabolites thereof. In some embodiments, the bile acid is deoxycholic acid. In some embodiments, the bile acid is ursodeoxycholic acid. In some embodiments, both deoxycholic acid and ursodeoxycholic acid are used. In some embodiments, the bile acid is lithocholic acid. In some embodiments, the bile acid is taurolithocholic acid. In some embodiments, the bile acid is tauroursodeoxycholic acid. In some embodiments, the bile acid is glycocholic acid.

[0137] 4. Application The compound of formula (I) has neuroprotective effects, anti-inflammatory activity, antioxidant properties, and epithelial cell protective effects. It is hypothesized that these useful and beneficial properties may be due to the S-benzene moiety of the cholestane backbone structure of the compound.

[0138] Compounds of formula (I), such as HA-1 or HA-2, are therefore useful in the treatment or prevention of neurological disorders (including neurodegenerative conditions such as Alzheimer's disease, dementia caused by Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (motor neuron disease), multiple sclerosis, and brain injury), cognitive decline (such as memory loss) associated with these neurological disorders, and disorders caused by stress-induced cellular damage in the inner or middle ear of a subject (such as vestibular disorders, hearing disorders, and conditions associated with hair cell degeneration or hair cell death).

[0139] Thus, there is provided a pharmaceutical composition comprising a compound of formula (I), or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier, adjuvant or diluent.

[0140] Also provided is a method of treating or preventing a neurological disorder or cognitive decline associated with a neurological disorder in a subject, the method comprising administering to the subject an effective amount of a compound of formula (I) or a pharma- ceutically acceptable salt thereof.

[0141] There is also provided a compound of formula (I) or a pharma- ceutically acceptable salt thereof for use in treating or preventing a neurological disorder, or cognitive decline associated with a neurological disorder, in a subject.

[0142] There is also provided the use of a compound of formula (I), or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for treating or preventing a neurological disorder, or cognitive decline associated with a neurological disorder, in a subject.

[0143] Also provided is a method of preventing, reducing or treating the occurrence and / or severity of damage caused by stress-induced cellular damage in the middle or inner ear of a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of formula (I) or a pharma- ceutical acceptable salt thereof.

[0144] Also provided is a method of preventing, reducing or treating the incidence and / or severity of a vestibular or hearing disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of formula (I) or a pharma- ceutical acceptable salt thereof.

[0145] Also provided is a method of preventing, reducing or inhibiting hair cell degeneration or hair cell death in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of formula (I) or a pharma- ceutically acceptable salt thereof.

[0146] Also provided is the use of a compound of formula (I) or a pharma- ceutically acceptable salt thereof in the manufacture of a medicament for preventing, reducing or treating the incidence and / or severity of a disorder caused by stress-induced cellular damage in the middle or inner ear of a subject.

[0147] There is also provided the use of a compound of formula (I), or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for preventing, reducing or treating the incidence and / or severity of a vestibular or hearing disorder in a subject.

[0148] Also provided is the use of a compound of formula (I), or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for preventing, reducing or inhibiting hair cell degeneration or hair cell death in a subject.

[0149] Also provided is a compound of formula (I) or a pharma- ceutically acceptable salt thereof for use in preventing, reducing the incidence and / or severity or treating a disorder caused by stress-induced cellular damage in the middle or inner ear of a subject.

[0150] Also provided is a compound of formula (I) or a pharma- ceutically acceptable salt thereof for use in preventing, reducing the incidence and / or severity or treating a vestibular or hearing disorder in a subject.

[0151] Also provided is a compound of formula (I), or a pharma- ceutically acceptable salt thereof, for use in preventing, reducing or inhibiting hair cell degeneration or hair cell death in a subject.

[0152] Cellular damage in the middle or inner ear can be induced or caused by, for example, high levels of free radicals, oxidative stress, proinflammatory cytokines, noise-induced stress, chemically induced stress, infection with a virus or bacteria or other infectious agent, physical trauma, molecular perturbations affecting the cochlea, an inflamed or damaged stria vascularis, stress due to loss of neural signaling and apoptosis throughout the middle or inner ear. Cellular damage in the middle or inner ear can lead, for example, to hair cell degeneration and / or hair cell death.

[0153] The pharmaceutical composition may also comprise additional active ingredients useful for treating or preventing neurological disorders, including Alzheimer's disease, dementia caused by Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (motor neuron disease), multiple sclerosis, memory loss, cognitive decline, or neurodegenerative conditions such as brain damage, or for preventing, reducing or treating the occurrence and / or severity of disorders caused by stress-induced cell damage in the middle ear or inner ear.The pharmaceutical composition may also be used as a medicament for recovery and / or treatment and / or pretreatment for ear-related surgery for outer ear / middle ear / inner ear implantation devices (e.g., cochlear implants and grommets).

[0154] Additional active ingredients that may be usefully administered with the pharmaceutical composition include, for example, corticosteroids, antibiotics, neurotrophic factors, growth factors, antifibrotic agents, and stem cell promoting agents. In some embodiments, the additional active ingredient is probucol or succinobucol.

[0155] In some embodiments, the present invention relates to maintaining, inducing, promoting, or enhancing the viability or regeneration of cells of the middle or inner ear, particularly supporting cells and hair cells of the middle or inner ear. The inventors believe that the compound of formula (I) or a pharma- ceutically acceptable salt thereof has the potential to aid in the restoration of hearing loss and associated cellular functions.

[0156] Whereas mechanical devices merely increase volume, the compounds, methods, uses and compositions described herein may advantageously restore the frequency response of the ear, and unlike surgical intervention, the compounds, methods, uses and compositions described herein are non-invasive.

[0157] 5. Pharmaceutical Compositions The present invention also provides a pharmaceutical composition comprising a compound of formula (I) or a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.

[0158] In some embodiments, the compound of formula (I) or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound of formula (I) or a pharma- ceutically acceptable salt thereof, may be used in combination with one or more other drugs. In some embodiments, the compound of formula (I) or a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof, is the only active ingredient used in the pharmaceutical composition.

[0159] Thus, in some embodiments, the pharmaceutical composition can further comprise or be administered in combination with one or more other agents.For example, the pharmaceutical composition can further comprise or be administered in combination with the agent useful in treating or preventing Alzheimer's disease, dementia caused by Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (motor neuron disease), multiple sclerosis, cognitive decline, memory loss, or brain damage.

[0160] It will be appreciated that the administration of a compound of formula (I) or a pharma- ceutically acceptable salt thereof in combination with one or more other agents may be simultaneous, sequential or separate administration.

[0161] In some embodiments, the compound of formula (I) is present in the pharmaceutical composition in an amount of about 0.1% w / w to about 10% w / w, e.g., about 0.5 to about 5% w / w, about 5 to about 10% w / w, about 1 to about 4% w / w, about 1 to about 8% w / w, about 2 to about 7% w / w, about 3 to about 6% w / w, about 4 to about 8% w / w, or about 6 to about 10% w / w. In some embodiments, the compound of formula (I) is present in the pharmaceutical composition in an amount of 0.1 wt / wt%, 0.2 wt / wt%, 0.3 wt / wt%, 0.4 wt / wt%, 0.5 wt / wt%, 0.6 wt / wt%, 0.7 wt / wt%, 0.8 wt / wt%, 0.9 wt / wt%, 1 wt / wt%, 2 wt / wt%, 3 wt / wt%, 4 wt / wt%, 5 wt / wt%, 6 wt / wt%, 7 wt / wt%, 8 wt / wt%, 9 wt / wt%, or 10 wt / wt%.

[0162] The term "composition" includes formulations that contain the compound of formula (I) or its stereoisomer, or its pharmaceutically acceptable salt with conventional carriers and excipients, and also formulations that have encapsulating materials as carriers to provide capsules in which the compound (with or without other carriers) is surrounded by an encapsulating carrier. In pharmaceutical compositions, the carrier is "pharmaceutically acceptable", meaning that it is compatible with other components of the composition and is not harmful to the subject. Pharmaceutical compositions can contain other agents or additional active agents as described above, and can be formulated according to techniques such as those well known in the art of pharmaceutical formulations (see, for example, Remington: The Science and Practice of Pharmacy, 21st Ed., 2005, Lippincott Williams & Wilkins), for example, by using conventional solid or liquid vehicles or diluents, and pharmaceutical additives (e.g., excipients, binders, preservatives, stabilizers, flavorings, etc.) of the type appropriate for the desired mode of administration.

[0163] Pharmaceutical compositions may be suitable for oral, rectal, nasal, topical (including transdermal, buccal and sublingual), or parenteral (including intramuscular, subcutaneous and intravenous) administration.

[0164] The compound of formula (I) or its stereoisomer, or its pharma- ceutically acceptable salt, may be placed together with conventional adjuvants, carriers, or diluents, i.e., in the form of pharmaceutical compositions and unit doses thereof.The pharmaceutical compositions may be solid, such as tablets or filled capsules, for oral administration, or liquid, such as solutions, suspensions, emulsions, elixirs, or capsules filled therewith.The pharmaceutical compositions may also be in the form of suppositories for rectal administration or in the form of sterile injectable solutions for parenteral (including subcutaneous) use.

[0165] Such pharmaceutical compositions and unit dosage forms thereof may contain conventional ingredients in conventional proportions, with or without additional active compounds or ingredients, and such unit dosage forms may contain any suitable effective amount of the active ingredients consistent with the daily dosage range intended for use.

[0166] For preparing pharmaceutical compositions from the compound of formula (I) or its stereoisomer, or its pharma- ceutically acceptable salt, the pharma-ceutically acceptable carrier can be either solid or liquid.Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispensable granules.Solid carriers can be one or more substances that can also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or encapsulating materials.

[0167] Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, etc. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid forms suitable for oral administration.

[0168] Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water-propylene glycol solutions.For example, parenteral injection liquid preparations can be formulated as solutions in aqueous polyethylene glycol solution.

[0169] The solution or suspension may contain the following components: sterile diluent such as water for injection, saline, fixed oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvent; antibacterial agent such as benzyl alcohol or methylparaben; antioxidant such as ascorbic acid or sodium bisulfite; chelating agent such as ethylenediaminetetraacetic acid; buffer such as acetate, citrate or phosphate and agent for adjusting tonicity such as sodium chloride or dextrose. pH may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be enclosed in glass or plastic ampoules, disposable syringes or multiple dose vials.

[0170] Sterile liquid form compositions include sterile solutions, suspensions, emulsions, syrups and elixirs. The active ingredient can be dissolved or suspended in a pharma- ceutically acceptable carrier, such as sterile water, sterile organic solvent, or a mixture of both.

[0171] That is, the pharmaceutical composition according to the present invention can be formulated for parenteral administration (e.g., by injection, e.g., by bolus injection or continuous infusion) and can be provided in unit dose form in ampoules, prefilled syringes, small injections or in multi-dose containers with added preservatives.The pharmaceutical composition can take the form of suspension, solution or emulsion in oily or aqueous vehicles, and can contain formulation agents such as suspending agents, stabilizing agents and / or dispersing agents.Alternatively, the active ingredient can be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with suitable vehicle, e.g., sterile, pyrogen-free water, before use.

[0172] The pharmaceutical forms suitable for injectable use include sterile injectable solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions. They should be stable under the conditions of manufacture and storage and can be preserved against oxidation and the contaminating action of microorganisms such as bacteria or fungi.

[0173] The solvent or dispersion medium for the injectable solutions or dispersions may contain any of the conventional solvents or carrier systems for injectable solutions or dispersions and may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.

[0174] Pharmaceutical forms suitable for injectable use may be delivered by any suitable route, including intravenous, intramuscular, intracerebral, intrathecal, or epidural injection or infusion.

[0175] Sterile injectable solutions are prepared by incorporating the active ingredient in the required amount in a suitable solvent with various other ingredients, such as those listed above, as required, and then sterilizing by filtration.Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and the other ingredients required from those listed above.In the case of sterile powders for preparing sterile injectable solutions, the preferred method of preparation is vacuum drying or freeze-drying of a previously sterile filtered solution of the active ingredient plus any additional desired ingredients.

[0176] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0177] The compound of formula (I) or its stereoisomer, or its pharma- ceutically acceptable salt, may be formulated, for example, with an inert diluent or with an assimilable edible carrier into a composition suitable for oral administration, or may be enclosed in a hard or soft shell gelatin capsule, or may be compressed into tablets, or may be incorporated directly with dietary food. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.

[0178] The amount of active compound in therapeutically useful compositions should be sufficient so that a suitable dosage will be obtained.

[0179] The tablets, troches, pills, capsules and the like may also contain ingredients as listed hereinafter: binding agents such as gums, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; disintegrating agents such as corn starch, potato starch, alginic acid and the like; lubricants such as magnesium stearate; and sweetening agents such as sucrose, lactose or saccharin may be added or flavoring agents such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier.

[0180] Various other materials may be present as coating or to otherwise modify the physical form of dosage unit.For example, tablet, pill or capsule may be coated with shellac, sugar or both.Syrup or elixir may contain active compound, sucrose as sweetener, methylparaben and propylparaben as preservatives, dye and flavoring such as cherry flavor or orange flavor.Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amount used.In addition, active ingredient(s) may be incorporated into sustained release preparations and sustained release formulations, including those that allow the active ingredient to be delivered to a specific area of ​​the intestine.

[0181] Aqueous solutions suitable for oral use can be prepared by dissolving the active ingredient in water and adding suitable colorants, flavors, stabilizers and thickeners, if desired. Aqueous suspensions suitable for oral use can be prepared by dispersing the finely divided active ingredient in water with viscous substances, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, or other well-known suspending agents.

[0182] Pharmaceutically acceptable carriers include any and all pharma- ceutically acceptable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.

[0183] Also included are solid form preparations that are intended to be converted immediately before use into liquid form preparations for oral administration.Such liquid forms include solutions, suspensions, and emulsions.These preparations may contain, in addition to the active ingredient, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizers, etc.

[0184] For topical administration to the epidermis, the compound of formula (I) or its stereoisomer, or its pharma- ceutically acceptable salt, can be formulated as an ointment, cream, or lotion, or as a transdermal patch. Ointments and creams can be formulated, for example, with an aqueous or oily base with the addition of suitable thickening and / or gelling agents. Lotions can be formulated with an aqueous or oily base and will generally also contain one or more emulsifiers, stabilizers, dispersants, suspending agents, thickening agents, or coloring agents.

[0185] Compositions suitable for topical administration in the mouth include lozenges comprising the active agent in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

[0186] The solution or suspension for nasal administration can be applied directly to the nasal cavity by conventional means, for example, by using a dropper, pipette or spray. The composition can be provided in a single dose form or in a multi-dose form. In the case of a dropper or pipette, this can be achieved by the patient administering an appropriate, predetermined amount of the solution or suspension. In the case of a spray, this can be achieved, for example, by a metered atomizing spray pump. To improve nasal delivery and retention, the compound of formula (I) or its stereoisomer can be encapsulated with cyclodextrin or formulated with other agents that are expected to enhance delivery and retention in the nasal mucosa.

[0187] Administration to the respiratory tract can also be achieved by aerosol formulation in which the active ingredient is provided in a pressurized pack with a suitable propellant, such as a chlorofluorocarbon (CFC), for example, dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, carbon dioxide or other suitable gas.

[0188] The aerosol may conveniently also contain a surfactant such as lecithin. The dosage of active ingredient may be controlled by providing a metered valve.

[0189] Alternatively, the active ingredient can be provided in the form of a dry powder, for example, a powder mixture of the compound in a suitable powder base, such as lactose, starch, hydroxypropylmethylcellulose and starch derivatives such as polyvinylpyrrolidone (PVP).Conveniently, the powder carrier will form a gel in the nasal cavity.The powder composition can be provided in unit dose form, for example, in capsules or cartridges of, for example, gelatin, or blister packs from which the powder can be administered by inhaler.

[0190] In formulations intended for administration to the respiratory tract, including intranasal formulations, the active ingredient will generally have a small particle size, for example of the order of 5 to 10 microns or less. Such a particle size may be obtained by means known in the art, for example by micronization.

[0191] When desired, compositions adapted to give sustained release of the active ingredient may be employed.

[0192] In some embodiments, a pharmaceutical composition comprising a compound of Formula (I) or a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof, is administered or formulated for administration to the outer ear, middle ear, or inner ear by the methods described below: 1) application of a composition, e.g., ear drops, to the outer ear such that the composition travels through the ear canal and flows into the middle ear and subsequently into the inner ear; 2) transtympanic (TT) syringe delivery of the pharmaceutical composition into the middle ear as a bolus (0.1 to 1 mL); 3) TT syringe delivery of the pharmaceutical composition as a small gel or capsule (e.g., about 0.05 to 1 mL), e.g., a single transtympanic dose injected into the round window, or multiple transtympanic doses injected into the round window, particularly to the round window (RW); 4) TT syringe delivery of the pharmaceutical composition as a gel (e.g., about 0.05 to 1 mL), particularly to the oval window; 5) TT delivery of pharmaceutical compositions to the round window via a microwick or other catheter device;

[0193] 6) TT and trans-RW delivery of pharmaceutical compositions directly into the inner ear fluid via a needle; 7) TT delivery of a pharmaceutical composition through a defect in the tympanic membrane (such as a grommet or perforation) that delivers the pharmaceutical composition to the ear canal; 8) Transmastoid or transantral delivery of the pharmaceutical composition, which then flows into the middle ear and subsequently into the inner ear; 9) Direct delivery into the inner ear fluid, where the pharmaceutical composition is released from a "tube" inserted into the cochlea; 10) systemic delivery via either intravenous, intramuscular, or oral routes or a combination of these (combined with a method to enhance inner ear uptake); 11) direct injection into the inner ear via the round window or cochlear fistula, with or without a catheter or device (such as a cochlear implant); 12) Direct injection into the inner ear, such as injection via the cochlear adnexa into the endolymphatic sac, semicircular canal, or vestibule, with or without a catheter or device; 13) Direct injection into the inner ear through an oval window, such as during a stapedotomy or stapedectomy, or through a middle ear prosthesis (such as a stapes prosthesis or middle ear implant).

[0194] The above mentioned methods are known in the art. For example, drug delivery systems have been described in the literature, e.g. M. Peppi, A. Marie, C. Belline & JT Borenstein (2018), “Intracochlear drug delivery systems: a novel approach whose time has come”, Expert Opinion on Drug Delivery, 15:4, 319-324; J. Wang and JL. Puel, “Presbycusis: An Update on Cochlear Mechanisms and Therapies”, J. Clin. Med. 2020, 9, 218; J. Patel, M. Szczupak, S. Rajguru, C. Balaban and ME Hoffer (2019), “Inner Ear Therapeutics: An Overview of Middle Ear Delivery”, Front. Cell. Neurosci. 13:261; S. Nyberg, NJ Abbott, X. Shi, PS Steyger, A. Dabdoub, “Delivery of therapeutics to the inner ear: The challenge of the “blood-labyrinth barrier” Sci. Transl. Med. 11, eaao0935 (2019); AA McCall, EE Leary Swan, JT Borenstein, WF Sewell, SG Kujawa, and MJ McKenna, “Drug Delivery for Treatment of Inner Ear Disease: Current State of Knowledge”, Ear Hear., 2010 April; 31(2): 156-165; K. Mader, E. Lehner, A. Liebau, S.K.Plontke, “Controlled drug release to the inner ear: Concepts, materials, mechanisms, and performance”, Hearing Research, 368 (2018) 49-66; Yutian Ma, Andrew K. Wise, Robert K. Shepherd, and Rachael T. Richardson, “New molecular therapies for the treatment of hearing loss”, Pharmacology & Therapeutics 200 (2019), 190-209; each of which is incorporated herein by reference.

[0195] In some embodiments, the pharmaceutical composition is administered by one of the modes of delivery listed below: - Incorporated into or used in combination with tympanic membrane graft materials as part of tympanoplasty or tympanoplasty procedures; - Incorporation into a middle ear prosthesis as part of ossicular chain reconstruction surgery; - For use with established middle ear packing materials such as gelfoam or gelfilm; - As part of irrigation solutions during surgical procedures of the middle or inner ear; - Incorporate with suture material; - Built into the ventilation tube (grommet); - Incorporated into a bone-conditioning implant such as the BAHA device, Bonebridge device, Ossia device or Ponto device; or - Incorporated into microsurgical drills.

[0196] In some embodiments, the pharmaceutical composition is administered into the middle ear by applying a liquid or gel formulation by bolus transtympanic injection using a microfluidic device, such as a fine syringe in the milliliter or microliter volume range. In some embodiments, the volume administered for the liquid or gel formulation is about 0.05 mL to about 1 mL. For example, the volume administered can be about 0.1 mL to about 0.8 mL, about 0.2 mL to about 0.7 mL, about 0.3 mL to about 0.6 mL, or about 0.4 mL to about 0.5 mL.

[0197] In some embodiments, the pharmaceutical composition is administered by applying the liquid or gel formulation directly onto the round window membrane(s) or oval window membrane(s). Application to these membranes can be achieved using methods known in the art, for example, intratympanic injection of the liquid or gel formulation using a microfluidic device such as a fine syringe in the milliliter or microliter volume range. In some embodiments, the volume administered for the liquid or gel formulation is about 0.05mL to about 1mL. For example, the volume administered can be about 0.1mL to about 0.8mL, about 0.2mL to about 0.7mL, about 0.3mL to about 0.6mL, or about 0.4mL to about 0.5mL.

[0198] In some embodiments, the pharmaceutical composition is administered by applying the liquid formulation through a catheter or wick delivery system.Catheter or wick delivery systems are known in the art.For example, such systems are described in Silverstein, H., Thompson, J., Rosenberg, SI, Brown, N., Light, J., 2004. "Silverstein MicroWick", Otolaryngol. Clin. North Am., 37, 1019-1034, which is incorporated herein by reference.

[0199] In some embodiments, the pharmaceutical composition is administered directly to the middle or inner ear by a drug delivery system that includes a drug embedded in a silicone carrier, such as a cochlear implant or middle ear implant. Drug delivery systems with active ingredients embedded in a silicone carrier are known in the art. Such systems are discussed in Plontke SK, Goetze G, Rahne T, Liebau A. Intracochlear drug delivery in combination with cochlear implants: Current aspects. HNO. 2017;65(Suppl 1):19-28, which is incorporated herein by reference. Specific examples with animal data are discussed in Farhadi M, Jalessi M, Salehian P, et al. Dexamethasone eluting cochlear implant: Histological study in animal model. Cochlear Implants Int. 2013;14(1):45-50, which is incorporated herein by reference. A more recent example using human subjects is Briggs R, O'Leary S, Birman C, et al. Comparison of electrode impedance measures between a dexamethasone-eluting and standard Cochlear TM Contour Advance(registered trademark) electrode in adult cochlear implant recipients. Discussed in Hear Res. 2020;390:107924.

[0200] In some embodiments, the pharmaceutical composition is administered directly to the middle or inner ear by a drug delivery system that includes the drug being administered in droplet form through a defect in the tympanic membrane, including through a ventilation tube. Drug delivery systems through defects in the tympanic membrane are known in the art.

[0201] Therefore, in a preferred embodiment, the pharmaceutical composition is administered by transtympanic route, for example, using a microfluidic device such as a micro syringe with a volume range of milliliter or microliter.In some embodiments, the volume administered for liquid or gel formulation is about 0.05mL to about 1mL.For example, the volume administered can be about 0.1mL to about 0.8mL, about 0.2mL to about 0.7mL, about 0.3mL to about 0.6mL, or about 0.4mL to about 0.5mL.

[0202] The pharmaceutical preparation is preferably in unit dosage form.In such form, the preparation is subdivided into unit doses containing appropriate amounts of active ingredient.The unit dosage form can be a packaged preparation, the package containing discrete amounts of preparation, such as packaged tablets, capsules, and powders in vials or ampoules.Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.

[0203] Formulating parenteral compositions in dosage unit form is particularly advantageous for ease of administration and uniformity of dosage. The parental compositions may be in the form of physically discrete units suitable as single dosages for the subjects to be treated, each unit containing a predetermined amount of active ingredient calculated to produce the desired therapeutic effect in association with a pharmaceutical carrier.

[0204] The compound may also be administered in the absence of a carrier when the compound is in a unit dosage form.

[0205] Compositions comprising a compound of formula (I) or a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof, formulated for oral delivery, either alone or in combination with another agent, are particularly preferred.

[0206] When the pharmaceutical composition is in the form of a solution, mixture, suspension, microparticles, nanoparticles, or gel, the concentration of the compound of formula (I) or a pharma- ceutically acceptable salt thereof may be, for example, 0.1 to 1000 mg / mL, e.g., 0.1 to 2 mg / mL, 0.2 to 5 mg / mL, 1 to 5 mg / mL, 2 to 8 mg / mL, 5 to 10 mg / mL, 10 to 20 mg / mL, 20 to 30 mg / mL, 30 to 40 mg / mL, 40 to 50 mg / mL, 50 to 60 mg / mL, 60 to 70 mg / mL, 70 to 80 mg / mL, 80 to 90 mg / mL, 90 to 100 mg / mL, 100 to 120 mg / mL, 110 to 140 mg / mL, 120 to 160 mg / mL, 130 to 180 mg / mL, 140 to 180 mg / mL, 150 to 190 mg / mL, 160 to 200 mg / mL, 170 to 220 mg / mL, 180 to 240 mg / mL, 190 to 260 mg / mL, 200 to 280 mg / mL, 210 to 240 mg / mL, 220 to 260 mg / mL, 230 to 280 mg / mL, 240 to 280 mg / mL, 250 to 290 mg / mL, 260 to 300 mg / mL, 270 to 300 mg / mL, 280 to 320 mg / mL, 290 to 320 mg / mL, 320 to 360 mg / mL, 340 to 360 mg / In some embodiments, the concentration of the compound of formula (I) or a pharma- ceutical acceptable salt thereof may be from 80 to 80 mg / mL, 80 to 90 mg / mL, 90 to 100 mg / mL, 10 to 100 mg / mL, 100 to 200 mg / mL, 200 to 300 mg / mL, 300 to 400 mg / mL, 500 to 1000 mg / mL, 400 to 500 mg / mL, 500 to 600 mg / mL, 600 to 700 mg / mL, 700 to 800 mg / mL, 800 to 900 mg / mL, or 900 to 1000 mg / mL of the compound of formula (I) or a pharma- ceutical acceptable salt thereof.

[0207] A pharmaceutical composition containing a compound of formula (I), or a pharma- ceutically acceptable salt thereof, is formulated to be compatible with its intended route of administration.

[0208] In one embodiment, the pharmaceutical composition comprises a cyclodextrin (e.g., (2-hydroxypropyl)-β-cyclodextrin) and a compound of formula (I) or a pharma- ceutically acceptable salt thereof in a form suitable for delivery to the cochlea (inner ear). For example, the composition may be a gel formulation suitable for delivery to the cochlea (inner ear). That is, in some embodiments, the composition is formulated for administration to the middle or inner ear. The compound of formula (I) or a pharma- ceutically acceptable salt thereof may be in the form of microparticles. The gel formulation may be an ultrasound gel suitable for delivery to the cochlea (inner ear).

[0209] In some embodiments, the pharmaceutical composition further comprises a bile acid or bile salt, or a derivative or metabolite thereof. Bile acids are facial amphiphiles, i.e., they contain both hydrophobic and hydrophilic faces. Being facial amphiphiles allows bile acids to be used in drug delivery systems for selective drug targeting to the liver or to enhance drug bioavailability by improving intestinal absorption and metabolic stability. Bile acid-based drug delivery systems, in the form of mixed micelles, bilosomes and drug conjugates, are versatile nanocarriers. It is also believed that the steroid backbone preserves lipophilicity and therefore leads to a permeation enhancement effect, which is advantageous, for example, when the composition is for administration to the middle ear or inner ear.

[0210] Any bile acid or bile salt, or its derivative or metabolite, can be used to enhance the bioavailability of the compound of formula (I) or its stereoisomer, or its pharmaceutically acceptable salt, and can be in the form of bile acid-based nanoparticles or bile acid-based microparticles.For example, bile acid or bile salt can be primary bile acid, or secondary bile acid, or any bile acid with amino acid moiety attached.A variety of bile acids and bile salts are commercially available.

[0211] In some embodiments, the pharmaceutical composition further comprises cholic acid, deoxycholic acid (DCA), chenodeoxycholic acid, ursodeoxycholic acid (UDCA; also known as ursodiol), lithocholic acid, muricholic acid (α-muricholic acid, β-muricholic acid, γ-muricholic acid or ω-muricholic acid), murideoxycholic acid, hyocholic acid (α-hyocholic acid or β-hyocholic acid), hyodeoxycholic acid, ursocholic acid, lagocholic acid, lagodeoxycholic acid, cristocholic acid, bulpecolic acid, glycocholic acid, and chelatecholic acid. The fatty acids may include one or more of nodeoxycholic acid, taurocholic acid, taurochenodeoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, glycolithocholic acid, taurolithocholic acid, tauroursodeoxycholic acid, obeticholic acid, glycomuric acid (α-glycomuric acid, β-glycomuric acid, γ-glycomuric acid, ω-glycomuric acid), or tauromuric acid (α-tauromuric acid, β-tauromuric acid, γ-tauromuric acid, ω-tauromuric acid), or salts, derivatives, or metabolites thereof.

[0212] In some embodiments, the bile acid is deoxycholic acid and / or ursodeoxycholic acid. In some embodiments, the bile acid is deoxycholic acid. In some embodiments, the bile acid is ursodeoxycholic acid. In some embodiments, both deoxycholic acid and ursodeoxycholic acid are used. In some embodiments, the bile acid is lithocholic acid. In some embodiments, the bile acid is taurolithocholic acid. In some embodiments, the bile acid is tauroursodeoxycholic acid. In some embodiments, the bile acid is glycocholic acid. In some embodiments, the bile acid is chenodeoxycholic acid, taurocholic acid, or cholic acid.

[0213] In some embodiments, the bile acid or bile salt, or derivative or metabolite thereof, is present in the pharmaceutical composition in an amount of about 0.1% w / w to about 10% w / w, e.g., about 0.5 to about 5% w / w, about 5 to about 10% w / w, about 1 to about 4% w / w, about 1 to about 8% w / w, about 2 to about 7% w / w, about 3 to about 6% w / w, about 4 to about 8% w / w, or about 6 to about 10% w / w. In some embodiments, the bile acid or bile salt is present in the pharmaceutical composition in an amount of 0.1 wt / wt, 0.2 wt / wt, 0.3 wt / wt, 0.4 wt / wt, 0.5 wt / wt, 0.6 wt / wt, 0.7 wt / wt, 0.8 wt / wt, 0.9 wt / wt, 1 wt / wt, 2 wt / wt, 3 wt / wt, 4 wt / wt, 5 wt / wt, 6 wt / wt, 7 wt / wt, 8 wt / wt, 9 wt / wt, or 10 wt / wt.

[0214] In some embodiments, the pharmaceutical composition in the form of a gel may include the following components: a compound of formula (I) or a pharma- ceutically acceptable salt thereof, one or more cyclodextrins, polysorbate 80 (Tween 80), glycerol, propylene glycol, a water-soluble gel, and water.

[0215] For example, the pharmaceutical composition may include the following ingredients: JPEG2025503406000021.jpg97158

[0216] Two specific composition examples are provided below. JPEG2025503406000022.jpg92158

[0217] A typical method of preparation is as follows: A mixture of (2-hydroxypropyl)-β-cyclodextrin in water is mixed with stirring at about 80° C. for about 1 hour. A compound of formula (I) or a pharma- ceutically acceptable salt thereof is then added, and the resulting mixture is heated to about 80° C. with stirring for about 1 hour. Tween 80 is then added, and the resulting mixture is heated to about 80° C. with stirring for about 1 hour. Ursodeoxycholic acid (UDCA) is then added, and the resulting mixture is heated to about 80° C. with stirring for about 1 hour. Deoxycholic acid is then added, and the resulting mixture is heated to about 80° C. with stirring for about 1 hour. Metron water-soluble gel is then added, and the resulting mixture is heated to about 80° C. with stirring for about 1 hour. Glycerol and propylene glycol are then added, and the resulting mixture is heated to about 80° C. with stirring for about 1 hour. The resulting mixture is then stirred at about 80° C. for an additional hour, and then water is added to make up the volume to the desired amount.

[0218] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0219] In further embodiments, the pharmaceutical composition comprising the compound of formula (I) or a pharma- ceutically acceptable salt thereof may comprise at least one additional active ingredient. The additional active ingredient may be an antibacterial agent, an antiviral agent, an antifungal agent, an anti-inflammatory agent, an osmotically active agent (e.g., mannitol), or other suitable therapeutically or pharmacologically active agent. In some embodiments, the additional active ingredient is a steroid, for example, selected from dexamethasone, methylprednisolone, and prednisolone. In some embodiments, the additional active ingredient is an antibiotic, such as gentamicin. In some embodiments, the additional active ingredient may have an otoprotective effect specific to preventing chemotherapy-related damage to the middle or inner ear, such as that occurring with cisplatin treatment or radiation therapy.

[0220] In further embodiments, the pharmaceutical composition comprising the compound of formula (I) or its pharma- ceutically acceptable salts can be administered simultaneously, separately or sequentially in combination with at least one additional active ingredient, and according to different routes of administration for each active ingredient.The additional active ingredient can be an antibacterial agent, an antiviral agent, an antifungal agent, an anti-inflammatory agent, a chemotherapeutic agent, an osmotically active substance (e.g., mannitol), or other suitable therapeutic or pharmacologically active agent.In particular, the additional active ingredient can be an ototoxic or otoirritant therapeutic agent; commonly used ototoxic agents include aminoglycosides, platinum-based chemotherapeutic agents, loop diuretics, macrolide antibiotics, and antimalarials.In some embodiments, the additional active ingredient is a steroid, for example, selected from dexamethasone, methylprednisolone, and prednisolone.

[0221] In some embodiments, a pharmaceutical composition comprising a compound of formula (I) or a pharma- ceutically acceptable salt thereof may be administered to a subject being treated with gene therapy (e.g., viral vector), or stem cell therapy.

[0222] Following treatment, if appropriate, the subject may be tested for improvements in, for example, hearing or other symptoms related to middle or inner ear disorders. Methods for measuring hearing are well known and include pure tone audiometry, air conduction, and bone conduction testing. These tests measure the loudness (intensity) and pitch (frequency) limits of sounds that a subject can hear. Hearing tests in humans include behavioral observation audiometry (infants to 7 months), visual reinforcement directional hearing tests (children 7 months to 3 years); play audiometry for children over 3 years; and standard hearing tests for older children and adults, such as whispered voice, pure tone audiometry; tuning fork testing; brainstem auditory evoked response (BAER) testing or auditory brainstem evoked potential (ABEP) testing. Otoacoustic emission testing can be used to test the function of hair cells in the cochlea, and cochlear electrostimulation provides information about the function of the cochlea and the initial part of the neural pathway to the brain. In some embodiments, treatment can be continued with or without modification or discontinued.

[0223] In some embodiments, the composition comprises an additional agent that is a permeability enhancer.

[0224] Advantageously, the permeability enhancer enhances the permeation through the middle ear (tympanic membrane) into the round window and into the inner ear (cochlea, organ of Corti, scala vestibuli, scala tympani and scala media). The permeation of sufficient amount of drug deep into the cochlea is one of the main challenges of current commercial gels. In addition, the permeability enhancer can function as a stabilizer or as a component that can provide a sustained or controlled release of active ingredients.

[0225] In some embodiments, pharmaceutical compositions comprising such permeability enhancers will facilitate delivery of the composition across the biological barrier separating the middle ear and the inner ear, such as the round window, thereby efficiently delivering a therapeutically effective amount of the pharmaceutical composition to the inner ear. Efficient delivery to the cochlea, organ of Corti, and / or vestibular organs is desired because these tissues host supporting cells that promote the regeneration of sensory hair cells when treated or contacted with the compositions described herein.

[0226] In some embodiments, the additional permeation enhancer is a polyol such as polyethylene glycol (PEG), glycerol (glycerin), maltitol, sorbitol; diethylene glycol monoethyl ether, azone, benzalkonium chloride (ADBAC), cetylperidium chloride, cetylmethylammonium bromide, dextran sulfate, lauric acid, menthol, methoxy salicylate, oleic acid, phosphatidylcholine, polyoxyethylene, polysorbate 80, sodium glycolate, sodium lauryl sulfate, sodium salicylate, sodium taurocholate, sodium taurodeoxycholate, sulfoxide, sodium deoxycholate, sodium glycodeoxycholate, sodium taurocholate, and surfactants such as sodium lauryl sulfate, laureth-9, cetylpyridinium chloride, and polyoxyethylene monoalkyl ethers, sodium salicylate and methoxy salicylate. salicylate), fatty acids such as benzoic acid, lauric acid, oleic acid, undecanoic acid and methyl oleate, fatty alcohols such as octanol and nonanol, laurocapram, cyclodextrin, thymol, limonene, urea, chitosan and other natural and synthetic polymers.

[0227] In some embodiments, the additional permeation enhancer is present in the pharmaceutical composition in an amount of about 0.1% w / w to about 10% w / w, e.g., about 0.5 to about 5% w / w, about 5 to about 10% w / w, about 1 to about 4% w / w, about 1 to about 8% w / w, about 2 to about 7% w / w, about 3 to about 6% w / w, about 4 to about 8% w / w, or about 6 to about 10% w / w. In some embodiments, the permeation enhancer is present in the pharmaceutical composition in an amount of 0.1 wt / wt%, 0.2 wt / wt%, 0.3 wt / wt%, 0.4 wt / wt%, 0.5 wt / wt%, 0.6 wt / wt%, 0.7% wt / wt%, 0.8 wt / wt%, 0.9 wt / wt%, 1 wt / wt%, 2 wt / wt%, 3 wt / wt%, 4 wt / wt%, 5 wt / wt%, 6 wt / wt%, 7 wt / wt%, 8 wt / wt%, 9 wt / wt%, or 10 wt / wt%.

[0228] In some embodiments, the additional permeability enhancer is a cyclic oligosaccharide consisting of a macrocyclic ring of glucose subunits linked by α-1,4 glycosidic bonds.

[0229] In some embodiments, the cyclic oligosaccharide is a cyclodextrin. Cyclodextrins are a family of cyclic oligosaccharides consisting of a macrocyclic ring composed of five or more α-D-glucopyranoside units linked by α-1,4 glycosidic bonds. Typical cyclodextrins contain many glucose monomers ranging from 6 to 8 units in the ring: α-cyclodextrin (6 glucose subunits), β-cyclodextrin (7 glucose subunits), γ-cyclodextrin (8 glucose subunits). Any cyclodextrin or derivative thereof that enhances permeability may be used.

[0230] In some embodiments, the cyclodextrin can be an α-cyclodextrin, a β-cyclodextrin, or a γ-cyclodextrin, or a larger polymeric cyclodextrin. For example, the cyclodextrin can be (2-hydroxypropyl)-β-cyclodextrin.

[0231] Cyclodextrin has a hydrophobic interior and a hydrophilic exterior and forms a complex with a hydrophobic compound, such as a hydrophobic active ingredient (e.g., probucol). Advantageously, cyclodextrin can provide solubility and stability to the active ingredient, and can allow the complex of cyclodextrin with the hydrophobic active ingredient to penetrate body tissue and release the active ingredient.

[0232] In some embodiments, the cyclodextrin is present in the pharmaceutical composition in an amount of about 1% to about 50% w / w. For example, the cyclodextrin can be present in the pharmaceutical composition in an amount of about 1% to about 25% w / w, such as about 2% to about 20% w / w, about 3% to about 15% w / w, or about 5% to about 10% w / w.

[0233] Pharmaceutically acceptable carriers for otic administration In some embodiments, the pharmaceutical composition includes a pharma- ceutically acceptable carrier. The pharma- ceutically acceptable carrier is selected depending on the mode of administration of the pharmaceutical composition, e.g., transtympanic administration. In preferred embodiments, the pharma- ceutically acceptable carrier is not perceived as a foreign body by the ear.

[0234] In some embodiments, the pharma- ceutically acceptable carrier is water. Typically, water is a component of a pharmaceutical composition. In some embodiments, the water is deionized water or distilled water.

[0235] In some embodiments, the pharma- ceutically acceptable carrier comprises a polymer, e.g., a hydrogel (a water-soluble gel), that provides localized and sustained release of the active ingredient (e.g., a bile acid conjugate of formula (I)). Such polymers and hydrogels are known in the art and are suitable for transtympanic administration. In some embodiments, the viscosity of the gel is 1-100 mPa S.

[0236] Examples of polymers and hydrogels suitable for transtympanic administration include the thermoreversible triblock copolymer poloxamer 407 (see, e.g., Wang et al., Audiol Neurootol. 2009;14(6):393-401. Epub 2009 Nov 16; and Wang et al., Laryngoscope. 2011 Feb;121(2):385-91); poloxamer-based hydrogels; Pluronic F-127 (see, e.g., Escobar-Chavez et al., J Pharm Pharm Sci. 2006;9(3):339-5); Pluronic F68, F88, or F108; polyoxyethylene-polyoxypropylene triblock copolymers (e.g., polymers composed of polyoxypropylene and polyoxyethylene, of the general formula E106 P70 E106; GB2459910, US2010 / 0133662; 110319377 and US20100273864); MPEG PCL diblock copolymers (Hyun et al., Biomacromolecules. 2007 Apr;8(4):1093-100. Epub 2007 Feb 28); hyaluronic acid hydrogels (Borden et al., Audiol Neurootol. 2011;16(1):1-11); gelfoam cubes (see, e.g., Havenith et al., Hearing Research, February 2011; 272(1-2):168-177); and gelatin hydrogels (see, e.g., Inaoka et al., Acta Otolaryngol. 2009 Apr; 129(4):453-7). Other biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.For example, the tunable self-assembly hydrogel made from natural amino acids L and D, such as Ac-LD6-COOH(L), as described in Hauser et al., Biotechnol Adv. 2012 May Jun;30(3):593-603, can also be used.Such compositions can be prepared using standard techniques or can be obtained commercially, for example, from Alza Corporation and Nova Pharmaceuticals, Inc.

[0237] In some embodiments, the pharma- ceutically acceptable carrier is selected from one or more of glycerol, propylene glycol, polysorbate 80 (Tween 80), gel (e.g., water-soluble gel), hyaluronic acid, polyvinyl alcohol (PVA), polylactic-co-glycolic acid (PLGA), and PEG 400. That is, in some embodiments, the pharma- ceutically acceptable carrier comprises any two or more of these carriers in combination. Such carriers are suitable for transtympanic administration.

[0238] In some embodiments, the pharma- ceutically acceptable carrier is selected from glycerol, propylene glycol, and polysorbate 80 (Tween 80). In some embodiments, the pharma- ceutically acceptable carrier is glycerol. In some embodiments, the pharma- ceutically acceptable carrier is propylene glycol. In some embodiments, the pharma- ceutically acceptable carrier is polysorbate 80 (Tween 80). In some embodiments, the pharma- ceutically acceptable carrier is a water-soluble gel. In some embodiments, the pharmaceutical composition comprises glycerol, propylene glycol, polysorbate 80 (Tween 80), and a water-soluble gel.

[0239] In some embodiments, the water-soluble gel is selected from a thermosensitive gel or an adhesive sol-gel transition hydrogel. Commercially available water-soluble gels suitable for use in the pharmaceutical compositions described herein include, for example, Metron water-soluble ultrasound gel, Pluronic F127, and hyaluronic acid (HA).

[0240] Thus, in some embodiments, the pharmaceutical composition comprises an active ingredient (e.g., a bile acid conjugate of formula (I)) and: 1: Polyethylene glycol (of any molecular weight) and any of its derivatives; 2: Polyvinylpyrrolidone (of any molecular weight) and any of its derivatives; 3: Polytetrafluoroethylene (PTFE; Teflon) (of any molecular weight) and any of its derivatives; 4: Poly-L-ornithine (of any molecular weight) and any of its derivatives; 5: Polystyrene sulphonate (of any molecular weight) and any derivatives thereof; 6: Acacia (any molecular weight); 7: Starch (of any molecular weight); 8: Chitosan (any molecular weight); 9: Poly-L-lysine (of any molecular weight) and any derivative thereof; 10: Poly-allyl-amine (of any molecular weight) and any derivatives thereof;

[0241] 11: Gelatin (of any molecular weight) and any derivative thereof; 12: Pectin (of any molecular weight) and any of its derivatives; 13: Poly(vinyl alcohol) (of any molecular weight) and any derivatives thereof; 14: Poloxamer 407; 15: Polysorbates of any number and configuration of polyoxyethylene moieties; 16: Pluronic F127; 17: Hyaluronic acid (of any molecular weight) and any derivatives thereof; 18: Alginate and any derivative thereof of any type / combination of G and M units; 19: Bile acids: including salts with any cation and conjugates with any amino acid (natural, synthetic and their derivatives); 20: Spiroquinone: (chemical name: 2,4,9,11-tetrakis(1,1-dimethylethyl)-14,14-dimethyl-13,15-dithiadispiro[5.0.5.3]pentadeca-1,4,8,11-tetraene-3,10-dione); 21: All cyclodextrins and their derivatives: alpha-, beta-, and gamma-cyclodextrins and any of their derivatives: any cyclodextrin including any member of the family of cyclic oligosaccharides consisting of a macrocyclic ring of glucose subunits linked by α-1,4 glycosidic bonds. These include all cyclodextrins composed of 5 or more α-D-glucopyranoside units linked 1→4, such as in amylose with any number of glucose monomers (for example: α (alpha)-cyclodextrin: 6 glucose subunits, β (beta)-cyclodextrin: 7 glucose subunits and γ (gamma)-cyclodextrin: 8 glucose subunits) and also all their derivatives with any modifications to their hydroxyl groups (including (2-hydroxypropyl)-β-cyclodextrin and all its derivatives); 22: Propylene glycol: propane-1,2-diol; or 23: Triethanolamine may include any one or more of the following.

[0242] Dosage An "effective amount" or "therapeutically effective amount" is an amount sufficient to produce a beneficial or desired therapeutic effect. This amount may be the same as or different from a "prophylactically effective amount," which is the amount necessary to prevent the onset of a disorder or symptoms of a disorder. An effective amount may be administered in one or more administrations, applications, or dosages. The therapeutically effective amount (i.e., effective dosage) of a therapeutic compound will depend on the therapeutic compound selected.

[0243] Suitable dosage levels of the compound of formula (I) or its stereoisomer, or a pharma- ceutically acceptable salt thereof, to be administered to a subject will generally be about 0.01 to 500 mg per kg of the subject's body weight per day, which can be administered in a single dose or multiple doses.

[0244] It will be understood that the specific dose level and frequency of administration for any particular subject may vary and will depend on a variety of factors, including the activity of the particular compound used, the metabolic stability and length of action of that compound, the age, weight, general health, sex, genetics and diet of the subject, the mode and time of administration, rate of excretion, drug combination, and the severity of the particular condition.

[0245] The appropriate dosage of a compound of formula (I) or an additional active agent to be administered in combination with a compound of formula (I) can be readily determined by one of skill in the art taking into account the particular compound of formula (I) or additional active agent selected.

[0246] Furthermore, it will be understood that when the compound of formula (I) or its stereoisomer, or a pharma- ceutically acceptable salt thereof is to be administered in combination with one or more agents, or other active agents, the dosage forms and dosage levels may be formulated for either simultaneous, sequential or separate administration, or a combination thereof.

[0247] The subject may be a human subject, which is intended to include both adult and "pediatric populations" (wherein the term "pediatric population" is understood as part of the population ranging from birth to 18 years of age).

[0248] The following examples are included to increase understanding of the invention without having any limiting effect on the invention. EXAMPLES

[0249] The invention will now be further described with reference to the following non-limiting examples.

[0250] material and method All chemical reagents were purchased from commercial sources (Sigma Aldrich, Scharlab SL and Merck & Co, Inc.) and used without further purification.

[0251] (4R)-4-[(3R,5R,8R,9S,10S,13R,14S,17R)-3-acetoxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoic acid

[0252] [ka]

[0253] Lithocholic acid (5.4 g, 14.5 mmol) was dissolved in anhydrous pyridine (20 mL) and acetic anhydride (2.05 mL, 21.7 mmol) was added dropwise to the above solution. After the reaction mixture was stirred at room temperature overnight, water (30 mL) was added and the pH was adjusted to 2 by adding 10% hydrochloric acid solution. The product was extracted with dichloromethane (50 mL x 3). The combined organic fractions were dried over anhydrous sodium sulfate, filtered and evaporated under vacuum to give 5.8 g of a white solid (95% yield). 1 H NMR(400MHz,CDCl3)δ:6.6(br,1H);4.70(tt,1H,J=5.1,J=16.5Hz);2.40(ddd,1H,J=5.3,J=10.3,J=15.7Hz);2.26(ddd,1H,J=6.4,J=9. 6,J=15.9Hz);2.03(s,3H);2.01-1.92(m,1H);1.91-1.74(m,5H);1.74-0.97(m,20H);0.92(d,3H,J=6.32Hz);0.92(s,3H);0.65(s,3H). 13 C NMR(100MHz,CDCl3)δ:180.0;170.9;74.6;56.6;56.1;42.9;42.0;40.6;40.3;35.9;35.5 ;35.2;34.7;32.4;31.1;30.9;28.3;27.2;26.8;26.5;24.3;23.5;21.6;21.0;18.4;12.2.

[0254] (4R)-4-[(3R,5R,8R,9S,10S,13R,14S,17R)-3-acetoxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl chloride

[0255] [ka]

[0256] Protected lithocholic acid (10 g, 23.9 mmol) was dissolved in anhydrous dichloromethane (67 mL) and thionyl chloride (8.66 mL, 119.4 mmol) was added dropwise to the above solution. After the reaction mixture was stirred at room temperature for 3 h, the solvent and excess thionyl chloride were evaporated under reduced pressure to give O-acetyllithocholic acid chloride. The solid was resuspended in anhydrous tetrahydrofuran (50 mL) before it was used for the next step.

[0257] 2,6-di-tert-butyl-4-((2-((3,5-di-tert-butyl-4-hydroxyphenyl)thio)propan-2-yl)thio)phenyl (4R)-4-[(3R,5R,8R,9S,10S,13R,14S,17R)-3-acetoxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentannoate ("HA-1")

[0258] [ka]

[0259] Probucol (18.5 g, 35.8 mmol) was added portionwise to a mixture of potassium tert-butoxide (8.3 g, 74.0 mmol) stirred in anhydrous tetrahydrofuran (166 mL). The mixture was allowed to stir at room temperature for 2 hours, during which time it turned dark brown. The resulting solution containing the potassium salt of probucol was added to the suspension of protected lithocholic acid chloride. After stirring the reaction mixture overnight at room temperature, water (500 mL) was added and the product was extracted with dichloromethane (500 mL x 3). The crude material was purified by column chromatography (silica gel, gradient hexane / dichloromethane) (51% yield, 1 93% purity based on H NMR analysis. 1 H NMR(400MHz,CDCl3)δ:7.62(s,2H);7.45(s,2H);5.36(s,1H);4.71(tt,1H,J=5.0,J=16.4Hz);2.7 3-2.61(ddd,1H,J=5.2,J=11.2,J=16.6Hz);2.59-2.47(ddd,1H,J=6.1,J=10.5,J=16.8Hz);2.03(s ,3H),2.03-1.78(m,6H);1.72-1.65(m,1H);1.63-1.56(m,1H);1.55(s,3H);1.48-1.36(m,30H);1. 34(s,18H);1.31-1.20(m,4H);1.19-1.02(m,5H);0.99(d,3H,J=6.4Hz);0.93(s,3H);0.66(s,3H). 13 C NMR(100MHz,CDCl3)δ:173.9;170.8;155.2;149.1;142.8;136.1(2C);134.8;134 .3(2C),129.3(2C);122.2(2C);74.6;59.6;56.7;56.1;42.9;42.0;40.6;40.3;3 6.0;35.7;35.6;35.3;35.2;34.7;34.5(2C);32.6;32.4;31.61(3C);31.59(3C); 30.7;30.4(6C);30.3;28.3;27.2;26.8;26.5;24.4;23.5;21.6;21.0;18.6;12.2.

[0260] (4R)-4-[(3R,5R,7R,8R,9S,10S,13R,14S,17R)-3,7-diacetoxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoic acid

[0261] [ka]

[0262] Chenodeoxycholic acid (5.0 g, 12.7 mmol) was dissolved in anhydrous pyridine (20 mL) and acetic anhydride (3.60 mL, 38.2 mmol) was added dropwise to the above solution. After the reaction mixture was stirred at room temperature overnight, water (30 mL) was added and the pH was adjusted to 2 by adding 10% hydrochloric acid solution. The product was extracted with dichloromethane (50 mL x 3). The combined organic fractions were dried over anhydrous sodium sulfate, filtered and evaporated under vacuum to give 5.5 g of white solid (91% yield). 1 H NMR(400MHz,CDCl3)δ:4.92-4.83(m,1H);4.59(tt,1H,J=4.4,J=11.4Hz);2.40(ddd,1H,J=5.1,J=10.1,J=15.4Hz);2.26(ddd,1H,J=6.5,J=9.7,J =15.9Hz);2.05(s,3H);2.03(s,3H);2.02-1.75(m,7H);1.75-1.67(m,1H );1.65-1.00(m,16H);0.933(d,3H,J=6.2Hz);0.928(s,3H);0.65(s,3H).

[0263] (4R)-4-[(3R,5R,7R,8R,9S,10S,13R,14S,17R)-3,7-diacetoxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl chloride

[0264] [ka]

[0265] Protected chenodeoxycholic acid (1 g, 2.1 mmol) was dissolved in anhydrous dichloromethane (5 mL) and thionyl chloride (0.45 mL, 6.3 mmol) was added dropwise to the above solution. After the reaction mixture was stirred at room temperature for 3 h, the solvent and excess thionyl chloride were evaporated under reduced pressure to give O-acetylchenodeoxycholic acid chloride. The solid was resuspended in anhydrous tetrahydrofuran (5 mL) before it was used for the next step.

[0266] 2,6-di-tert-butyl-4-((2-((3,5-di-tert-butyl-4-hydroxyphenyl)thio)propan-2-yl)thio)phenyl (4R)-4-[(3R,5R,7R,8R,9S,10S,13R,14S,17R)-3,7-diacetoxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoate ("HA-2")

[0267] [ka]

[0268] Probucol (1.6 g, 3.1 mmol) was added in portions to a mixture of potassium tert-butoxide (0.7 g, 6.2 mmol) stirred in anhydrous tetrahydrofuran (20 mL). The mixture was kept stirring at room temperature for 2 h, during which it turned dark brown. The resulting solution containing the potassium salt of probucol was added to a suspension of the protected lithocholic acid chloride. After stirring the reaction mixture at room temperature overnight, water (60 mL) was added and the product was extracted with dichloromethane (60 mL x 3). The crude material was purified by column chromatography (silica gel, gradient hexane / dichloromethane) (37% yield). 1H NMR(400MHz,CDCl3)δ:7.62(s,2H);7.45(s,2H);5.36(s,1H);4.92-4.85(m,1H);4 .59(tt,1H,J=4.6,J=11.5Hz);2.67(ddd,1H,J=4.9,J=10.7,J=16.0Hz);2.53(ddd ,1H,J=6.1,J=10.3,J=16.6Hz);2.06(s,3H);2.03(s,3H),2.03-1.78(m,6H);1.77 -1.67(m,1H);1.65-1.02(m,59H);1.00(d,3H,J=6.4Hz);0.94(s,3H);0.67(s,3H).

[0269] Abbreviation The following abbreviations are used in the examples and figures:

[0270] HA-1:Formula:

[0271] [ka]

[0272] 2,6-di-tert-butyl-4-((2-((3,5-di-tert-butyl-4-hydroxyphenyl)thio)propan-2-yl)thio)phenyl (4R)-4-[(3R,5R,8R,9S,10S,13R,14S,17R)-3-acetoxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoate,

[0273] HA-2:, formula:

[0274] [ka]

[0275] 2,6-di-tert-butyl-4-((2-((3,5-di-tert-butyl-4-hydroxyphenyl)thio)propan-2-yl)thio)phenyl (4R)-4-[(3R,5R,7R,8R,9S,10S,13R,14S,17R)-3,7-diacetoxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoate,

[0276] SPQ: Formula:

[0277] [ka]

[0278] spiroquinone (a metabolite of probucol), which is 2,4,9,11-tetrakis(1,1-dimethylethyl)-14,14-dimethyl-13,15-dithiadispiro[5.0.5.3]pentadeca-1,4,8,11-tetraene-3,10-dione having the formula

[0279] L-Dopa: Formula:

[0280] [ka]

[0281] Leovdopa, which has

[0282] DMSO: Dimethyl sulfoxide SC: subconcussion AIN93M: refers to control diet (AIN93M diet is a semi-pure maintenance diet based on the formula developed by the American Society of Nutritional Sciences (ASNS, formerly the American Institute of Nutrition). The formula meets the maintenance nutritional requirements of rats and mice. AIN93M diet is a modification of the previous formula AIN76A. This well-studied diet was updated to separate maintenance from growing animals and improve the long-term health of rodents fed semi-pure diets. Prob: Probucol PB: Probucol AGE: Aged garlic extract ApoCIII: apolipoprotein C-III ApoC3: apolipoprotein C-III BSA Bovine Serum Albumin PBS Phosphate Buffered Saline PBST 0.1% Tween 20 in PBS

[0283] Experimental Example 1: Effects of probucol and its derivatives SPQ and HA-1 on an in vitro model of Alzheimer's disease Drug Preparation: The compounds tested were probucol, SPQ and HA-1, as well as rivastigmine, a marketed drug for the treatment of mild to moderate Alzheimer's disease (AD), which was used as a positive control.

[0284] Rivastigmine is a cholinesterase inhibitor and has the following structure:

[0285] [ka]

[0286] has.

[0287] Stock solution (10mM) 5.16 mg of probucol and SPQ, and 10.32 mg of HA-1 were dissolved in 100% DMSO to prepare a 10 mM stock solution. Rivastigmine (10 mM) was prepared in nuclease-free water. For treatment, probucol (20 μM), SPQ (10 μM), HA-1 (10 μM), and rivastigmine (2 μM) were prepared in Dulbecco's Modified Eagle Medium (DMEM) directly from the stock solutions.

[0288] A perinatal tissue-derived human stem cell model for Alzheimer's disease (AD) was prepared using a proprietary protocol to test the effects of new drug entities. Drug treatment was for 24 h in all assays.

[0289] (I) Cytotoxicity studies using MTT cell viability assay Figure 1 shows the results of a cytotoxicity study by MTT cell viability assay. Amniotic membrane mesenchymal stem cells (AM-MSCs) were incubated with various concentrations (10, 15, 20, 25 and 30 uM, as well as DMSO control) of (A) probucol, (B) SPQ, (C) HA-1, and (D) DMSO for 24 hours. D1=DMSO control for 10 μM, D2=DMSO control for 20 μM (p<0.05; * p<0.05, ** p<0.001, *** p<0.0001, ns=not significant, n=3).

[0290] Probucol, SPQ, and HA-1 in the range of 10-30 μM were not toxic to the cells. For further analysis, 20 μM probucol and 10 μM SPQ and HA-1 were used.

[0291] (II) Lactate dehydrogenase (LDH) assay LDH leakage is a well-characterized marker of cell membrane damage and its increase, indicative of lactate deficiency, is a hallmark of AD. Using this model, we measured LDH levels in drug-treated (PB, SPQ, and HA-1 as well as rivastigmine) cells and compared them to neurodegenerative (ND) cells.

[0292] Figure 2 shows the evaluation of LDH release. Neurodegenerative cells were incubated with probucol (20 μM), SPQ (10 μM), HA-1 (10 μM), and rivastigmine (2 μM) for 24 h (p<0.05; * p<0.05, ** p<0.001, *** p<0.0001, ns=not significant, n=3).

[0293] It was clearly seen that probucol, SPQ, HA-1 and rivastigmine significantly reduced LDH activity compared to ND cells, indicating a reversal of AD-like neurodegeneration.

[0294] (III) Functional assays for cholinergic neurons - acetylcholinesterase (AChE) activity and acetylcholine (ACh) release Acetylcholinesterase (AChE), which controls the hydrolysis of acetylcholine (ACh) in the brain, is an important target for the treatment of Alzheimer's disease (AD), which is characterized by ACh deficiency. We assayed AChE activity and ACh release in control, neurodegenerated, and drug-treated AD cells.

[0295] Figure 3 shows the effect of probucol, SPQ, HA-1 and rivastigmine on (A) acetylcholinesterase (AChE) activity and (B) acetylcholine release in an Alzheimer's disease model using an Amplex Red reagent-based assay. Neurodegenerative cells were incubated with probucol (20 μM), SPQ (10 μM), HA-1 (10 μM) and rivastigmine (2 μM) for 24 h (p<0.05; * p<0.05, ** p<0.001, *** p<0.0001, ns=not significant, n=3).

[0296] Inhibition of acetylcholinesterase is one of the targets for the treatment of AD, since there is a decrease in cholinergic activity in the brain of AD patients. Probucol, SPQ and HA-1 have shown inhibition of acetylcholinesterase (AChE), as well as rivastigmine, an AChE inhibitor. Importantly, these compounds can also increase / maintain the level of acetylcholine (ACh).

[0297] (IV) Mitochondrial membrane potential test - TMRE (tetramethylrhodamine ethyl ester) assay Aβ plaques are a source of toxicity leading to severe structural and functional abnormalities in mitochondria in Alzheimer's disease. To assess electrophysiological and functional changes in mitochondrial membrane potential, we fluorimetrically quantified changes in mitochondrial membrane potential in living cells using TMRE (tetramethylrhodamine ethyl ester).

[0298] Figure 4 shows the measurement of mitochondrial membrane potential by TMRE assay. Neurodegenerative cells were incubated with probucol (20 μM), SPQ (10 μM), HA-1 (10 μM) and rivastigmine (2 μM) for 24 h (p<0.05; * p<0.05, ** p<0.001, *** p<0.0001, ns=not significant, n=3).

[0299] Mitochondrial dysfunction due to enhanced oxidative stress is observed in AD conditions. Probucol, SPQ and HA-1 were able to maintain (similar to rivastigmine) the mitochondrial membrane potential, which is disrupted due to neurodegeneration.

[0300] (V) Gene expression analysis for neural, cholinergic and AD-related markers To determine the expression of genes involved in AD-specific neurodegeneration and neuronal markers, we analyzed BACE and chAT (AD and cholinergic markers) as well as synapsin 1 and neuropilin (neuronal markers).

[0301] Figure 5 shows gene expression analysis by qRT-PCR for neuronal, cholinergic, and AD-related markers (A) BACE1, (B) ChAT, (C) synapsin 1, and (D) neuropilin (p<0.05; * p<0.05, ** p<0.001, *** p<0.0001, ns=not significant, n=3).

[0302] Interestingly and supporting a role for amyloid beta accumulation, β-site amyloid precursor protein cleaving enzyme 1 (BACE1), which leads to the accumulation of amyloid beta, was significantly higher in neurodegenerative (ND) cells compared to control cells. Further confirmation of cholinergic neuronal degeneration in ND cells was obtained by downregulation of choline acetyltransferase (ChAT) when compared to control neuronal cells.

[0303] Probucol, SPQ and HA-1, as well as the positive control rivastigmine, were able to reduce levels of beta-secretase 1 (BACE1), the main beta-secretase required for the production of Aβ in neurons, and increase levels of choline acetyltransferase (ChAT), involved in the synthesis of the neurotransmitter acetylcholine.

[0304] Gene expression levels of mature neuronal markers, neuropilin and synapsin 1 (Syn), were also increased in probucol-, SPQ- and HA-1-treated neurodegenerative cells.

[0305] (VI) Immunocytochemical localization of ad-specific and neuronal markers For further confirmation and protein localization of AD-specific neurodegenerative and neuronal markers, we performed immunolocalization for BACE1 and synapsin, respectively.

[0306] Fluorescence microscopy images of BACE1-positive neurons were taken and the effects of probucol, SPQ and HA-1 on the neurodegeneration model were compared. BACE1 protein accumulation was reduced by all drug treatments, with HA-1 and rivastigmine showing the lowest expression. In the AD model, BACE1 accumulation was clearly evident, as expected.

[0307] Fluorescent micrographs of synapsin 1 positive neurons were obtained, and the effects of probucol, SPQ, and HA-1 on the neurodegeneration model were compared with rivastigmine as a positive control. In the case of synapsin 1, a disruption in the expression of synapsin protein in ND cells was observed. However, there was a clear reversal of degeneration by probucol, SPQ, and HA-1.

[0308] (VII) Assessment of oxidative stress: ROS (reactive oxygen species) and Greiss (nitric oxide) assays Increases in oxidative stress are associated with the accumulation of amyloid beta seen in AD models. Increasing oxidative stress and nitric oxide levels lead to apoptosis and evoke cytotoxic effects on neurons. We studied these parameters to check the reversal of oxidative stress in AD cells treated with probucol, SPQ, HA-1 and rivastigmine.

[0309] FIG. 6 shows the effects of probucol, SPQ, HA-1 and rivastigmine on (A) intracellular reactive oxidative stress and (B) nitric oxide.

[0310] Probucol, SPQ and HA-1 were able to prevent the decline in endogenous nitric oxide levels, which reduced the levels of ROS and protected neurons from amyloid beta-related oxidative stress.

[0311] (VIII) Assay of glutathione peroxidase activity The increase in oxidative stress associated with Alzheimer's disease is associated with a decrease in the levels of the brain antioxidant glutathione (GSH). We studied GSH levels to analyze whether AD cells treated with probucol, SPQ, HA-1 and rivastigmine had a restoration of GSH activity.

[0312] FIG. 7 shows the effect of probucol, SPQ, HA-1 and rivastigmine on glutathione activity. (Statistical analysis: Results are expressed as mean ± SD. One-way ANOVA followed by Dunnett's post-hoc test was performed to compare all rows vs. neurodegeneration (p<0.05; * p<0.05, ** p<0.001, *** p<0.0001, ns=not significant, n=3).

[0313] The data observed correlated well and demonstrated that concomitant with the reduction in ROS and NO levels by probucol, SPQ, HA-1 and rivastigmine, as seen in FIG. 6, there was an increase in antioxidant GSH activity in drug-treated neurons, thereby indicating protection against oxidative injury (FIG. 7).

[0314] (IX) Conclusion Using an in vitro model of Alzheimer's disease, we observed that probucol and its derivatives SPQ and HA-1 are effective in reversing certain molecular changes of AD-specific neurodegeneration. These studies demonstrate that these drugs act efficiently like a commonly used drug for AD, i.e., rivastigmine, in the AD-specific parameters we tested. We observed that the drugs reverse the changes occurring in the neurodegenerative model by lactate dehydrogenase assay, mitochondrial membrane potential assay, acetylcholine level and acetylcholinesterase activity, ROS and NO assay, glutathione peroxidase assay, which indicate cell membrane damage. We were also able to demonstrate at gene and protein level that the expression of a characteristic marker for AD, i.e., BACE, which accumulates with the accumulation of amyloid beta, was clearly decreased in AD cells treated with probucol, SPQ, HA-1 and rivastigmine. Furthermore, the restoration of active neuronal markers such as synapsin and neuropilin suggested the neuroprotective effect of the drugs.

[0315] Experimental Example 2: Effects of probucol and its derivatives SPQ and HA-1 on an in vitro model of Parkinson's disease Drug Preparation: The compounds tested were probucol, SPQ and HA-1, and levodopa (L-dopa), a known drug for Parkinson's disease (PD), was used as a positive control.

[0316] Stock solution (10mM) 5.16 mg of probucol and SPQ, and 10.32 mg of HA-1 were dissolved in 100% DMSO to prepare a 10 mM stock solution. L-dopa (1 mM) was prepared in nuclease-free water. For treatments, probucol (20 μM), SPQ (10 μM), HA-1 (10 μM), and L-dopa (50 μM) were prepared in Dulbecco's Modified Eagle Medium (DMEM) directly from the stock solutions.

[0317] A perinatal tissue-derived human stem cell model for Parkinson's disease (PD) was prepared using a proprietary protocol to test the effects of new drug entities. Drug treatment was for 24 h in all assays.

[0318] (I) Cytotoxicity studies using MTT cell viability assay An initial toxicity assay was performed.

[0319] Figure 8 shows the results of a cytotoxicity study by MTT cell viability assay. Amniotic membrane mesenchymal stem cells (AM-MSCs) were incubated with various concentrations (10, 15, 20, 25 and 30 uM, as well as DMSO control) of (A) probucol, (B) SPQ, (C) HA-1 and (D) DMSO for 24 hours. D1=DMSO control for 10 μM, D2=DMSO control for 20 μM (p<0.05; * p<0.05, ** p<0.001, *** p<0.0001, ns=not significant, n=3).

[0320] Probucol, SPQ and HA-1 in the range of 10-30 μM were not toxic to the cells. For further analysis, 20 μM probucol and 10 μM SPQ and HA-1 were used.

[0321] (II) Mitochondrial membrane potential test - TMRE (tetramethylrhodamine ethyl ester) assay Dysfunctional mitochondria and changes in mitochondrial membrane potential promote the neurodegeneration seen in Parkinson's disease. To assess changes in mitochondrial membrane potential, we used TMRE (tetramethylrhodamine ethyl ester) to quantitate the changes in live cells fluorimetrically.

[0322] Figure 9 shows the measurement of mitochondrial membrane potential by TMRE assay. Neurodegenerative cells were incubated with probucol (20 μM), SPQ (10 μM), HA-1 (10 μM) and L-dopa (50 μM) for 24 h (p<0.05; * p<0.05, ** p<0.001, *** p<0.0001, ns=not significant, n=3).

[0323] Mitochondrial dysfunction due to enhanced oxidative stress is observed in PD conditions. Probucol, SPQ and HA-1 could maintain (similar to L-dopa) the mitochondrial membrane potential, which is disrupted due to neurodegeneration. SPQ was observed to have enhanced recovery of mitochondrial membrane potential.

[0324] (III) Adenosine triphosphate (ATP) assay Impaired energy metabolism and reduced ATP levels are common features of PD. Using a stem cell-derived PD model, we measured ATP levels in drug-treated (probucol, SPQ, and HA-1 and L-dopa) cells and compared them with neurodegenerative (ND) cells.

[0325] Figure 10 shows the assessment of ATP levels. Neurodegenerative cells were incubated with probucol (20 μM), SPQ (10 μM), HA-1 (10 μM) and L-dopa (50 μM) for 24 h (p<0.05; * p<0.05, ** p<0.001, *** p<0.0001, ns=not significant, n=3).

[0326] PD neurodegeneration was clearly seen to exhibit a significant decrease in ATP levels compared to controls. Enhanced increases in ATP levels were observed in cells treated with SPQ and HA-1 along with L-dopa.

[0327] (IV) Gene expression analysis of neural, cholinergic and PD-related markers Gene expression levels of mature neuronal markers (synapsin I), dopaminergic markers (nuclear receptor-related 1 (Nurr1), dopamine transporter (DAT) and tyrosine hydroxylase (TH)), and PD-associated markers (α-synuclein) were analyzed to determine the effects of probucol, SPQ, HA-1 and L-dopa.

[0328] FIG. 11 shows gene expression analysis by qRT-PCR for neuronal, dopaminergic and PD-related markers. (A) Synapsin I, (B) Nurr1, (C) DAT, (D) TH and (E) α-synuclein. (p<0.05; * p<0.05, ** p<0.001, *** p<0.0001, n=3).

[0329] Probucol, SPQ and HA-1 had different effects on the expression of dopaminergic markers, including synapsin I and Nurr1, involved in the maintenance of the dopaminergic system in the brain, and DAT, responsible for the reuptake of dopamine from the synaptic cleft.More importantly, the expression of TH, the rate-limiting enzyme that converts L-tyrosine to L-dopa, was as effective in drug treatment as in the positive control L-dopa.

[0330] In comparison, the efficacy of these compounds differed since HA-1 was able to increase the expression of all dopaminergic markers tested, whereas DAT, TH, along with the neuronal marker, synapsin I, were significantly increased after SPQ treatment.

[0331] Interestingly, decreased expression of α-synuclein, the accumulation of which leads to the formation of Lewy bodies and ultimately to the degeneration of dopaminergic neurons, was observed following treatment with all three drugs probucol, SPQ and HA-1.

[0332] (V) Immunocytochemical localization of PD-specific and neuronal markers For further confirmation and protein localization of neuronal, dopaminergic and PD-specific neurodegeneration markers, we performed immunolocalization for synapsin I, Nurr1, TH and α-synuclein, respectively.

[0333] Fluorescent micrographs of synapsin 1 positive neurons were obtained, and the effects of probucol, SPQ and HA-1 on the neurodegeneration model were compared with L-dopa as a positive control. A decrease in the expression of synapsin protein was observed in ND cells. However, there is a clear restoration of synapsin expression by probucol, SPQ and HA-1.

[0334] Fluorescent micrographs of Nurr1-positive dopaminergic neurons were obtained, and the effects of probucol, SPQ and HA-1 on the neurodegeneration model were compared with L-dopa as a positive control.

[0335] Fluorescent micrographs of TH-positive dopaminergic neurons were obtained, and the effects of probucol, SPQ and HA-1 on the neurodegeneration model were compared with L-dopa as a positive control.

[0336] The appearance of dopaminergic markers, Nurr1 and TH, was seen to be more pronounced after treatment with probucol, SPQ and HA-1, with a clear reduction in neurodegeneration.

[0337] Fluorescence micrographs of α-synuclein immunolocalization were obtained and the effects of probucol, SPQ and HA-1 on the neurodegeneration model were compared with L-dopa as a positive control.

[0338] The accumulation of α-synuclein was reduced by all drug treatments, with HA-1 and L-dopa showing the lowest expression. As expected, the accumulation of α-synuclein was clearly increased in the PD model.

[0339] (VI) Assessment of oxidative stress – ROS and Greiss (nitric oxide) assays Increases in oxidative stress associated with α-synuclein accumulation play a central role in disease progression. Nitric oxide enhances α-synuclein aggregation and accelerates disease progression. Increased oxidative stress and nitric oxide levels lead to apoptosis and evoke cytotoxic effects on neurons.

[0340] FIG. 12 shows the effects of probucol, SPQ, HA-1 and L-dopa on (A) intracellular reactive oxidative stress and (B) nitric oxide.

[0341] Probucol, SPQ and HA-1 could reduce the levels of ROS and prevent the decline in endogenous nitric oxide levels, thereby protecting neurons from α-synuclein aggregation and associated oxidative stress.

[0342] (VII) Assay of glutathione peroxidase activity The increase in oxidative stress associated with Parkinson's disease is associated with mitochondrial dysfunction and a subsequent decrease in levels of the brain antioxidant glutathione (GSH).

[0343] FIG. 13 shows the effect of probucol, SPQ, HA-1 and L-dopa on glutathione activity. (Statistical analysis: Results are expressed as mean ± SD. One-way ANOVA followed by Dunnett's post-hoc test was performed to compare all rows vs. neurodegeneration (p<0.05; * p<0.05, ** p<0.001, *** p<0.0001, ns=not significant, n=3).

[0344] Concomitant with the reduction in ROS and NO levels by probucol, SPQ, HA-1 and L-dopa, there was an increase in neuronal GSH activity, thereby suggesting cellular and mitochondrial protection against oxidative injury (FIG. 13).

[0345] (VIII) Conclusion Using an in vitro stem cell-derived model of Parkinson's disease, we observed that probucol, SPQ and HA-1 were effective in reversing certain molecular changes of PD-specific neurodegeneration. These studies show that these agents act efficiently like a commonly used drug for PD, i.e., L-dopa, in the PD-specific parameters we tested. Using a mitochondrial membrane potential assay, we observed mitochondrial dysfunction in the PD model that was restored by drug treatment. Furthermore, the ATP assay, which measures the impaired energy metabolism that occurs in PD, clearly shows that SPQ and HA-1, derivatives of probucol, were effective in restoring ATP levels. Assays such as ROS, NO and GSH also demonstrated that the drugs reversed the cellular and mitochondrial changes that occur in the neurodegenerative model.

[0346] We were also able to demonstrate at gene and protein levels that the expression of an enzyme involved in dopamine metabolism that is downregulated in PD, i.e. tyrosine hydroxylase, which was suppressed in PD models, was obviously upregulated in PD cells treated with probucol, SPQ, HA-1 and L-dopa. Significantly, the drugs also significantly reduced the accumulation of α-synuclein, a major factor responsible for accelerating neurodegeneration. Moreover, the restoration of active neuronal markers such as synapsin and dopaminergic markers Nurr1, DAT and TH indicated the neuroregenerative effect of the drugs.

[0347] Experimental Case 3: Study of Brain Injury (Subconcussion) Repeated subconcussive head impacts have been reported to impair psychomotor function.Rats were subjected to a sham procedure, subconcussive (SC) with a control diet (AIN93M), SC with probucol, SC with aged garlic extract (AGE), or SC with HA-1 for 12 weeks.

[0348] Both the rotarod and beamwalk tests assess neuromotor function. For the beamwalk, higher slips indicate greater impairment. For the rotarod, lower latencies indicate greater impairment.

[0349] Rotarod test FIG. 14 shows rotarod latency (mean) in seconds 12 weeks following a sham procedure, subconcussion with administration of AIN93M, subconcussion with administration of probucol, subconcussion with administration of AGE, and subconcussion with administration of HA-1.

[0350] Rats with SC did not show any change in latency, indicating that there was no detectable neuromotor deficit.Nevertheless, treatment with HA-1 significantly increased latency, indicating higher neuromotor ability.That is, rats with HA-1 performed best on the rotarod, as shown by higher latency compared with sham, SC with AIN93M, SC with probucol, and SC with AGE.

[0351] Beam walk test FIG. 15 shows the number of slips (Y-axis) in the beam-walk test (2 cm) 6 and 12 weeks after the sham procedure, subconcussion with administration of AIN93M, subconcussion with administration of probucol, subconcussion with administration of AGE, and subconcussion with administration of HA-1.

[0352] After 6 weeks of experimental / drug intervention, SC increased beam walk slips on the 2 cm beam. Probucol or AGE did not show any beneficial effect, whereas HA-1 showed a significant reduction in slips on the 2 cm beam. That is, HA-1 showed a significant prevention of neuromotor dysfunction especially for the 2 cm beam walk after 6 weeks, as shown by the lower number of slips compared to SC with AIN93M, SC with probucol, and SC with AGE.

[0353] After 12 weeks, SC increased the number of slips for all 1 cm, 2 cm and 3 cm beam walks. Provision of probucol, AGE or HA-1 did not show a significant preventative effect in reducing slips. No effect was seen after 12 weeks.

[0354] Experimental Example 4: Viability assay of HA-1 on pancreatic NIT-1 cells (I) Cultivating pancreatic NIT-1β cells Cells were grown and treated in Dulbecco's modified Eagle's medium with 11.5% fetal bovine serum according to the protocol described below.

[0355] Specifically, the cells were cultured at T-75cm 2 They were cultured on tissue culture flasks (Thermo Fisher Scientific®, Australia) and fed with Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Life Technologies, USA) supplemented with either 5.5 mM glucose or 25.5 mM glucose (Sigma Chemical Co, USA), 11.5% fetal bovine serum (Thermo Fisher Scientific, Australia), and 5% penicillin-streptomycin (Thermo Fisher Scientific, Australia). β-Cells were incubated for 72 hours at 37°C in a humidified air environment with 5% CO2 using a Nuaire NU-8500 Water Jacket CO2 Incubator (Nuaire, USA).

[0356] (II) MTT Viability Protocol The MTT assay for cellular mitochondrial activity is a common and important analytical technique for determining the degree of cell viability and biological activity. Briefly, MTT was prepared as a 5 mg / ml stock solution (Sigma Chemical CO, USA) in phosphate buffer (Thermo Fisher Scientific, Australia) at pH 7.4. Undissolved residues were removed by sterile filtration. The stock solution was stored in a sterile environment at 4°C in the dark and used within 7 days of preparation. For the MTT assay protocol, 20 μl of MTT from the stock solution was added into each well of a 96-well plate (Thermo Fisher Scientific, Australia) containing 10 μM probucol and 10 μM HA-1 (dissolved in a propylene glycol-water hydrogel mixture). The conversion of MTT to formazan was removed from the incubator after 4 hours by washing the microcapsules with MilliQ water for 5 minutes to remove spectroscopic interference. Formazan was dissolved in 100 μl of dimethylsulfoxide (DMSO) (Sigma Chemical Co., USA) via reverse pipetting and the resulting purple solution was analyzed photometrically at 550 nm.

[0357] (III) Results and Discussion FIG. 16 shows that under normoglycemic conditions, none of the treatment groups exerted any effect, which was expected since under physiological conditions NIT-1 cells are already at optimal function.

[0358] However, under hyperglycemic conditions, NIT-1 cells are under stress due to glucose-mediated toxicity, mitochondrial dysfunction and oxidative stress (Figure 17). Probucol could partially counteract hyperglycemia-induced pancreatic beta cell death, but HA-1 had a much greater effect on ameliorating cell death and could substantially counteract beta cell damage from glucose toxicity.

[0359] (IV) Conclusion HA-1 confers potent pancreatic beta cell protective properties, ensuring cellular viability and function under hyperglycemic conditions, which has major implications for diabetes treatment and HA-1 may be of significant benefit in terms of diabetes management.

[0360] Experimental Example 5: Cognitive function (memory) test Cognitive function in diabetic db / db mice was tested in the passive avoidance test and the novel object recognition test. For both tests, the higher the score, the better the memory.

[0361] Mice were administered either probucol, HA-1 or apoCIII antisense starting at 5 weeks of age, and cognitive performance was tested at 14 weeks of age.

[0362] HA-1 was shown to fully restore memory in diabetic mice comparable to probucol or apoCIII antisense.

[0363] (I) Passive avoidance test FIG. 18 shows the results of the passive avoidance test at 14 weeks for the negative control (non-diabetic mice), positive control (diabetic db / db mice), probucol, HA-1 and ApocIII.

[0364] At 14 weeks, diabetic db / db (positive control) mice showed significantly reduced latency compared to non-diabetic control mice (negative control), indicating significant short-term memory loss. Provision of probucol completely prevented memory loss, with latency comparable to negative control mice. Similarly, mice given HA-1 showed latency comparable to negative control and probucol group mice. Mice maintained on ApoCIII antisense (ApoCIII) also showed a preventative effect.

[0365] (II) Novel object recognition test FIG. 19 shows the results of the novel object recognition test at 14 weeks for negative control (non-diabetic mice), positive control (diabetic db / db mice), probucol, HA-1 and ApocIII.

[0366] At 14 weeks of age, db / db positive mice showed preference indices comparable to negative control mice and did not show any memory deficits. Treatment with probucol, HA-1 or ApocIII did not show any significant effect at 14 weeks of age.

[0367] Experimental Example 6: Effect of HA-1 on repeated subconcussive trauma in rats It is increasingly recognized that repeated subconcussive head impacts cause brain damage and neuromotor dysfunction. This study investigated the effect of HA-1 on preventing blood-brain barrier (BBB) ​​breakdown and attenuating neuroinflammation and neuromotor dysfunction in an established rat model of repeated subconcussive head impacts.

[0368] Female PVG rats aged 5-7 weeks were used. Under full anesthesia with isoflurane, a 25 g weight was dropped from a height of 1 m onto the lambda of the skull. This was repeated 10 consecutive times per procedure. The procedure was repeated three times per week (i.e., Monday, Wednesday, and Friday) for a period of 12 weeks. Rats in the sham group underwent the same procedure without the weight drop. The probucol treatment group received probucol supplemented in the diet at 0.1% w / w. The HA-1 treatment group also received HA-1 in the diet at 0.2% w / w for the entire duration of the study.

[0369] After completion of the 12-week subconcussion procedure, neuromotor function was tested with the established rotarod and beamwalk tests. For the beamwalk, higher slips indicate greater impairment. For the rotarod, lower latencies indicate greater impairment.

[0370] The rats were then sacrificed and brain tissue obtained for further analysis.

[0371] Rotarod test The rotarod test (FIG. 20A) did not show any significant changes in latency in rats subjected to repeated subconcussive (SC) impacts compared to sham rats, indicating the absence of any substantial neuromotor deficits ( * p<0.05, n=12).

[0372] Supplementation with probucol also showed no changes, however HA-1 treatment showed a significant increase in latency, indicating improved neuromotor ability.

[0373] Beam walk test The beam-walk test (FIG. 20B) showed a substantial increase in foot slips in SC rats compared to sham, suggesting significant neuromotor dysfunction, which was somewhat attenuated by the provision of probucol, whereas HA-1 completely normalized it to levels comparable to the sham group, indicating that treatment with HA-1 improves neuromotor performance.

[0374] Blood-brain barrier (BBB) ​​integrity Following 12 weeks of repeated subconcussion, rats showed an increase in IgG extravasation in the hippocampal formation (HPF) and cortical (CTX) regions of the brain compared to rats that underwent a sham procedure (Figures 21A and 21B). The increase in IgG extravasation in SC rats indicates a disruption of the BBB, allowing blood-borne IgG to leak into the brain. Probucol supplementation did not show any protection against BBB disruption in the HPF, whereas there was some reduction in CTX IgG extravasation. HA-1 was found to be more effective than probucol in attenuating BBB disruption in both the HPF and CTX regions.

[0375] Neurooxidative stress Repeated subconcussive impacts did not alter the expression of 8dOHG, a marker of oxidative stress (Figures 22A and 22B). Interestingly, probucol showed some increase in 8dOHG, especially in CTX. However, administration of HA-1 substantially attenuated brain oxidative stress levels, even lower than in sham rats.

[0376] conclusion Collectively, the data indicate that HA-1 prevents BBB disruption in rats subjected to SC head impact, which leads to attenuation of oxidative stress. These effects were better than those observed with probucol. Thus, administration of HA-1 leads to improvement of neuromotor performance and prevents dysfunction induced by repeated SC.

[0377] Experimental Example 7: Effects of HA-1 on diabetes-induced blood-brain barrier breakdown and cognitive decline It is well established that diabetes increases the risk of developing dementia, especially Alzheimer's disease. Although the exact underlying mechanisms are unclear, a growing body of evidence suggests that BBB disruption may be crucial. Thus, we investigated the effects of HA-1, compared with probucol, on BBB integrity and dementia phenotypes in diabetic db / db mice, a clinically relevant mouse model of type 2 diabetes.

[0378] In this study, 4-5 week-old diabetic db / db mice were fed a diet containing probucol 0.1% (wt / wt) or HA-1 0.2% (wt / wt) for either 10 or 24 weeks, until 14 or 28 weeks of age, respectively. A passive avoidance test was used to assess short-term memory. Subsequently, mice were sacrificed to obtain plasma and brain tissue samples for further analysis.

[0379] Diabetic phenotype Elevations in plasma glucose, triglycerides and insulin, and to a lesser extent cholesterol, are typical signs of diabetes. Levels of plasma glucose, plasma triglycerides, plasma insulin and plasma cholesterol levels were measured at 14 and 28 weeks of age (Figure 23) ( * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, n=12).

[0380] At 14 weeks of age, diabetic db / db mice had significantly higher glucose compared to non-diabetic control mice (Figure 23A). Probucol did not show any significant effect, and HA-1 showed a slight decrease in blood glucose in db / db mice. At 28 weeks of age, probucol and HA-1 significantly reduced blood glucose, with HA-1 providing a greater effect (Figure 23B).

[0381] In 14-week-old diabetic db / db mice, plasma triglyceride levels were not increased compared to control mice (FIG. 23C). Nevertheless, probucol and HA-1 significantly attenuated plasma triglycerides at 14 weeks of age (FIG. 23C). Similarly, at 28 weeks of age, probucol and HA-1 significantly lowered plasma triglycerides (FIG. 23D).

[0382] Plasma insulin levels were increased in diabetic db / db mice at 14 weeks of age (Figure 23E). Although insulin levels were significantly reduced by the provision of probucol or HA-1 at 14 weeks of age (Figure 23E), insulin levels remained significantly higher than in non-diabetic control mice. At 28 weeks of age, probucol or HA-1 lost their improving effect on insulin. Similarly, probucol and HA-1 did not show any beneficial effect on plasma cholesterol (Figures 23G and 23H).

[0383] Alzheimer's amyloid-β Emerging in vivo evidence suggests that circulating amyloid-beta (Aβ) is associated with the risk of Alzheimer's disease and cognitive decline. In particular, soluble Aβ40 is thought to be less harmful, while Aβ42 appears to be more toxic. An increasing number of studies also show that plasma concentrations of Aβ oligomers and the Aβ42 / 40 ratio are also highly associated with Alzheimer's disease and cognitive decline.

[0384] As expected, diabetic db / db mice exhibited significantly higher levels of apoB (a marker of lipid-bearing particles) in the small intestine at 14 and 28 weeks of age (Figures 24A and 24B), which coincided with the increase in plasma triglycerides and cholesterol. The increase in small intestinal apoB was significantly attenuated by HA-1 at 14 weeks of age, whereas probucol did not show any significant reduction and apoB remained significantly higher than in control mice (Figure 24A) ( * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, n=12). The ameliorative effect of HA-1 on intestinal apoB was no longer seen in 28-week-old diabetic db / db mice (FIG. 24B).

[0385] Small intestinal Aβ (FIGS. 24C and 24D) was not increased in 14-week-old diabetic db / db mice, whereas it was significantly increased in 28-week-old db / db mice. Probucol significantly attenuated the production of Aβ in the small intestine in db / db mice at 14 and 28 weeks of age. HA-1 showed comparable or superior effects in attenuating small intestinal Aβ (FIGS. 24C and 24D).

[0386] In plasma, diabetes did not increase Aβ40, a non-toxic form of amyloid (Figures 25A and 25B). * p<0.05, ** p<0.01, *** p<0.001, ****p<0.0001, n=12). Probucol and HA-1 did not change plasma Aβ40 levels in 14-week-old diabetic db / db mice (FIG. 25A), whereas HA-1 increased plasma Aβ40 levels in 28-week-old diabetic db / db mice (FIG. 25B).

[0387] At 14 weeks of age, plasma levels of toxic Aβ42 were significantly increased in diabetic db / db mice compared to control mice (Figure 25C). This was significantly attenuated by HA-1, whereas probucol did not show any beneficial effect (Figure 25C). Similarly, at 28 weeks of age, diabetic db / db mice showed no increase in plasma Aβ42, whereas only HA-1 significantly reduced it (Figure 25D).

[0388] Plasma concentrations of Aβ oligomers (Figures 25E and 25F) were also significantly reduced with HA-1 at 14 and 28 weeks of age, which was not observed with probucol. Similarly, the Aβ42 / 40 ratio was significantly reduced with HA-1 in db / db mice at 14 and 28 weeks of age (Figures 25G and 25H).

[0389] Collectively, these data suggest that HA-1 may significantly reduce the risk of Alzheimer's disease (dementia caused by Alzheimer's disease) through improving diabetes-induced dyslipidemia and regulating plasma Aβ levels.

[0390] Memory function Consistent with our observations in plasma biomarkers, HA-1 appeared to significantly improve short-term memory in db / db mice using a passive avoidance test (Figures 26A and 26B) ( * p<0.05, n=12). Interestingly, db / db mice showed higher latency in the test compared to control mice, indicating no significant memory deficits at 14 or 28 weeks of age. Nevertheless, HA-1 significantly increased latency compared to control mice, indicating improved short-term memory.

[0391] BBB Integrity Diabetic db / db mice showed substantially higher brain leakage of IgG in the hippocampal formation (HPF) and cortex (CTX) (Figures 27A, 27B, and 27D), indicating a breakdown of the BBB, except in CTX at 14 weeks of age (Figure 27C) ( * p<0.05, ** p<0.01, *** p<0.001, n=12).

[0392] At 14 weeks of age, HPF levels of IgG extravasation were significantly attenuated by provision of probucol or HA-1 (Figure 27A), which completely prevented BBB breakdown. In contrast, probucol showed no BBB protection in HPFs at 28 weeks (Figure 27B), whereas HA-1 significantly attenuated BBB breakdown (Figure 27B). Similarly, in 28-week-old db / db mice, probucol did not show any beneficial effect on CTX IgG extravasation, whereas HA-1 significantly reduced it (Figure 27D).

[0393] Oxidative Stress and Neuroinflammation Diabetic db / db mice showed some increase in HPF expression of 8dOHG and CTX expression (Figures 28A and 28B), indicating increased oxidative stress. This was attenuated by both probucol and HA-1 (Figures 28A and 28B), but HA-1 showed better efficacy especially in HPF (Figure 28A) ( * p<0.05, ** p<0.01, n=12). GFAP is a marker of astrocyte activation that may confer anti-inflammatory effects. GFAP was significantly decreased in HPFs of db / db mice (FIG. 28C), which showed significant recovery by provision of HA-1, whereas probucol showed no benefit. In CTX, GFAP was slightly decreased (FIG. 28D), which was similarly ameliorated by probucol and HA-1.

[0394] Iba-1 expression is indicative of microglial activation, a surrogate marker of neuroinflammation. Iba-1 was significantly higher in HPF and CTX in diabetic db / db mice (Figures 28E and 28F). HA-1 substantially reduced Iba-1 expression in HPF and CTX (Figures 28E and 28F). In contrast, probucol did not show any beneficial effect, and Iba-1 expression remained significantly elevated (Figures 28E and 28F).

[0395] conclusion Collectively, the data indicate that HA-1 ameliorates the diabetic phenotype in db / db mice, a clinically relevant mouse model of diabetes. HA-1 also significantly reduced systemic levels of Aβ, especially toxic Aβ42. The latter resulted in the prevention of BBB breakdown and significantly attenuated oxidative stress and neuroinflammation. Collectively, these led to the prevention of memory deficits. The data indicate that HA-1 may be effective in preventing or treating Alzheimer's disease through various aspects.

[0396] Experimental Example 8: Effect of HA1 and HA2 on ear protection Several experiments were performed to evaluate the potential otoprotective effects of two analogs, HA-1 and HA-2.

[0397] Materials and Methods (all data based on OC-1 cells) Seahorse Experiment Mitochondrial function was analyzed by measuring the oxygen consumption rate (OCR) using our well-established method (Kovacevic, Bozica, et al. “Novel hydrogel comprising non-ionic copolymer with various concentrations of pharmacologically active bile acids for cellular injectable gel.” Colloids and Surfaces B: Biointerfaces (2022): 113014) using a real-time Seahorse Flux Analyzer XF 96 (Seahorse Bioscience, USA). Briefly, the first injection was glucose-free medium, followed by the ATP synthase inhibitors oligomycin and FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) and the last injection was the complex I+II inhibitor rotenone + antimycin A 77. Results were generated automatically and analyzed by Wave software. OC-1 cells were pretreated with three different concentrations (20, 30, and 40 μM) of cisplatin for 24 or 48 h to induce cytotoxicity prior to the seahorse bioenergetics assay.

[0398] Caspase assay Caspase assays were performed according to the manufacturer's instructions (abcam, NSW, Australia) as described below.

[0399] Caspase 3 / 9 protein analysis: Materials needed Collagen-coated coverslips Fixative: 4% paraformaldehyde Permeabilization agent: 0.5% Tween 20 or trition-X 100 diluted in PBS · Blocking: 1% BSA in PBST may also be used. PBST: 0.1% Tween 20 in PBS ·Primary ABS (1:500 in PBS) ·Secondary ABS (1:1000 in PBS) ·Hoechst (nuclear staining)

[0400] procedure: Day 1: Fix, permeabilize, block and add primary antibody to cells as described below.

[0401] A) Fixing cells · Begin by aspirating the medium from each well, followed by fixation of the cells at 4% using 4% paraformaldehyde in PBS pH 7.4 for 10 minutes at room temperature (add 1 ml in each well). Shake the plate for a few seconds.

[0402] B) Cell permeabilization 0.5% Tween 20 or triton-X 100 diluted in PBS. Tween-20 is used for epitopes located in the cytoplasm, while triton-x100 is used to permeabilize the nucleus and mitochondria. 1) Incubate the wells with permeabilization buffer for 10 minutes at room temperature. 2) Aspirate the permeabilization buffer and wash with PBS for 5 min each time.

[0403] blocking: Block non-specific binding sites with blocking buffer for 1 hour at room temperature. During this 1 h, prepare the primary abs by diluting it in blocking buffer. Wash three times with PBST for 5 minutes each.

[0404] Primary ABS:(1:500) · Treat cells with 1 abs and incubate cells overnight at 4 degrees. Aspirate off the primary abs solution and then wash the cells three times with PBST for 5 min each. Treat cells with secondary abs for 1 hour at room temperature. · Cover the plate with foil to protect the light-sensitive dye from degradation. Aspirate off the secondary antibody solution and then wash the cells 3 times with PBST for 5 minutes each in the dark.

[0405] Counterstain: · Incubate cells on 0.1-1ug / ml Hoechst for 3 minutes at room temperature.

[0406] Slide preparation: The coverslip is ready to be mounted onto a microscope slide. Take a clean slide and slowly dispense one drop of anti-fade mounting medium from the pipette onto the slide. Carefully remove the coveslip from the well, allow excess wash solution to drip off and place cells face down on the slide. Clear nail polish can be used to seal the coverslip and prevent it from drying out. · Visualize the slides under a microscope.

[0407] Cobalt chloride hypoxia assay This assay is used to determine whether an intervention can reverse hypoxia-induced cell death brought about by cobalt chloride. For this assay, cells are pretreated with 100 μM CoCl2 for 24 h, followed by microscopic examination of cell morphology and viability.

[0408] Results and Discussion Seahorse bioenergetics experiment FIG. 29 shows the oxygen consumption rate (OCR) of OC-1 cells after 24 hours of exposure to cisplatin (in FIG. 29, "PB" is probucol, "HA1" is HA-1, and "HA2" is HA-2). As can be seen, cisplatin has a direct and concentration-dependent cytotoxic effect on OC-1 cells. Both analogs, HA-1 and HA-2, exerted significant and profound otoprotective effects, with HA-2 being the most potent in reversing cisplatin-induced cytotoxicity. This is evident from the enhanced OCR, which indicates greater respiration, and therefore biological activity, taking place.

[0409] The experiments were extended to evaluate complete bioenergetic parameters following 48 h exposure to the above concentrations of cisplatin.

[0410] FIG. 30 shows OCR data for OC-1 cells after 48 hours of exposure to cisplatin followed by treatment with HA-1 and HA-2. FIG. 30 has three graphs presented as line graphs showing cellular respiration (written down as OCR). The top left line graph shows the effect at a cisplatin level of 30 μM, the top right line graph shows the effect at 20 μM, and the bottom line graph shows the effect at 40 μM. For each line graph, the higher the line sits above the X-axis, the better the effect of the treatment on the cells. For these line graphs, the lines that sit highest above the X-axis are HA-1, HA-2, and probucol, and the lowest lines (sit nearly flat) are those with cisplatin and no treatment. As can be seen from FIG. 30, HA-1 and HA-2 are able to significantly reverse the cytotoxic effects of cisplatin as evidenced by enhanced OCR, which in turn indicates greater respiration, metabolism, and bioenergetics.

[0411] To elucidate the mechanisms and possible biological pathways involved in the cytoprotective effects of HA-1 and HA-2, caspase assays were performed to determine whether the analogs could inhibit apoptosis.

[0412] Figure 31 shows the results of the caspase assay. nd Figure 1 shows the population of cells in three sections (left, center and right) for six subgroups designated as Ab Alexa Fluor® 647 only), C1 (control: no treatment), C2 (control: treatment with 12.5 mM AAPH), HA-1, HA-2 and PB. In each image, every dot represents a cell colony. For each subgroup, the left image has the most dots representing the greatest number of cells as a total population, the center image shows dots representing non-living cells, and the right image shows dots representing the cell population that is a viable proportion of the total cell population (which will be composed of live and dead cells that add up to "total"). So, the presence of more dots in the right image for each subgroup indicates that the drug is working better; HA-2 has the most dots in the right image, and is therefore the best at keeping cells alive under stressful, toxic conditions. This is followed by HA-1 and PB, which show similar effects. All therapeutic interventions had a positive impact, and analogs HA-1 and HA-2 showed significant protection against programmed cell death mediated by the caspase-3 activation pathway, with HA-2 showing the least amount of cell death due to cisplatin-induced oxidative stress and cytotoxicity.

[0413] FIG. 32 shows the morphology and viability of cells following a cobalt chloride assay. This figure shows the population of cells occupying the space in each of the eight rectangles that make up the overall figure. The more oval structures in each rectangle, the more live cells will be represented, i.e., the better the outcome will be, since more live cells equal a better biological outcome. The control (healthy), shown in the top left rectangle, had the largest population of healthy cells (the largest number of oval structures scattered throughout the rectangle) and this was similar for the four rectangles in the overall top row (the top row did not have any stress induced on the cells). The bottom row is where the discrepancy occurs and the effect of the administered substance can be seen. The bottom left rectangle shows that the oval shapes have almost disappeared and been replaced by a mixture of all kinds of shapes representing dead cells; however, the bottom row rectangles, in the top right corner, show that HA-2 has retained all the oval shapes, i.e., has protected the entire cell population from damage. This was followed by the rectangles of PB and HA-1, which next showed the largest population of healthy, oval cells. Cells treated with CoCl2 without any other intervention showed a significant loss of characteristic traits with a viability of less than 40±3%. CoCl2 cells treated with probucol maintained normal morphology with a viability of 70±2.5%, whereas HA-1- and HA-2-treated CoCl2 cells showed optimal morphology with the most prominent viabilities of 82±3.5% and 87±1.6%, respectively.

Claims

1. Formula (I): 【Chemistry 1】 (In the formula: R １ teeth: 【Chemistry 2】 and R ２ , R ３ , R ４ , R ５ , R ７ , R ８ , R ９ , R １０ and R １１ each independently represents H, substituted or unsubstituted C １－３０ acyloxy, substituted or unsubstituted benzoyloxy, substituted or unsubstituted C １－３０ Alkyl, substituted or unsubstituted C ２－３０ Alkenyl, substituted or unsubstituted C ２－３０ Alkynyl, substituted or unsubstituted C ３－３０ cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroaryl, an amino acid moiety, or GL (wherein GL is independently OL, SL, PL, ２ , C.L. ３ , or N.L. ２ wherein R ２ , R ３ , R ４ , R ５ , R ７ , R ８ , R ９ , R １０ or R １１ When is substituted, the substituents are independently OH, F, SH, ═O, ═S, Cl, Br, SC １－６ Alkyl, C １－６ Alkyl or C １－６ alkoxy; Each L is independently H, a metal ion, a substituted or unsubstituted C １－３０ Alkyl, substituted or unsubstituted C ２－３０ Alkenyl, substituted or unsubstituted C ２－３０ Alkynyl, substituted or unsubstituted C ３－３０ cycloalkyl, substituted or unsubstituted benzyl radical, —CH ２ CO ２ H, or -(CH ２ ) ２ SO ３ H; where when L is substituted, the substituents are independently selected from OH, SH, ═O, ═S, F, Cl, Br, SC １－６ Alkyl, C １－６ Alkyl or C １－６ alkoxy; R ６ is -(CH ２ ) ｎ - (wherein n is 0 to 12), -CH ２ C(=O)NHCH ２ - or -CH ２ C(=O)NHCH ２ CH ２ - and; Y is —C— or —S(═O)—; and R ａ is —H or —C(═O)CH ２ CH ２ COOH); or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

2. R ａ The compound of claim 1 , wherein is —H.

3. R ａ -C(=O)CH ２ CH ２ 2. The compound of claim 1, wherein the compound is COOH.

4. R ２ , R ３ , R ４ , R ５ , R ７ , R ８ , R ９ , R １０ and R １１ each independently represents H; substituted or unsubstituted C １－３０ Acyloxy; Substituted or unsubstituted benzoyloxy; Substituted or unsubstituted C １－１２ Alkyl; substituted or unsubstituted C ２－１２ Alkenyl; substituted or unsubstituted C ２－１２ Alkynyl; substituted or unsubstituted C ３－８ Cycloalkyl; substituted or unsubstituted C ６ aryl; substituted or unsubstituted heteroaryl; an amino acid moiety; or GL (wherein GL is independently OL, SL, PL, ２ , C.L. ３ , or N.L. ２ 4. The compound according to claim 1, wherein the compound is selected from the group consisting of:

5. R ２ , R ３ , R ４ , R ５ , R ７ , R ８ , R ９ , R １０ and R １１ are independently H; substituted or unsubstituted C １－６ Acyloxy; Substituted or unsubstituted benzoyloxy; Substituted or unsubstituted C １－６ Alkyl; substituted or unsubstituted C ２－６ Alkenyl; substituted or unsubstituted C ２－６ Alkynyl; substituted or unsubstituted C ３－８ 4. The compound of any one of claims 1 to 3, wherein the group is selected from cycloalkyl; substituted or unsubstituted phenyl; substituted or unsubstituted pyridyl; or OH.

6. R ２ , R ３ , R ４ , R ５ , R ７ , R ８ , R ９ , R １０ or R １１ are independently H, —OH, and —OC(═O)R ｆ (In the formula, R ｆ is C １－６ 4. The compound of claim 1, wherein the aryl group is alkyl.

7. R ６ But -(CH ２ ) ｎ - (wherein n is 1, 2 or 3); -CH ２ C(=O)NHCH ２ -; or -CH ２ C(=O)NHCH ２ CH ２ The compound according to any one of claims 1 to 3, selected from:

8. The compound of any one of claims 1 to 3, wherein Y is -C-.

9. When the compound of formula (I) has formula (Ia): 【Transformation 3】 (In the formula: R ａ is —H or —C(═O)CH ２ CH ２ COOH; R ｂ represents —H, —OH, and —OC(═O)R ｆ (In the formula, R ｆ is C １－６ alkyl); R ｃ represents —H, —OH, and —OC(═O)R ｆ (In the formula, R ｆ is C １－６ alkyl); R ｄ represents —H, —OH, and —OC(═O)R ｆ (In the formula, R ｆ is C １－６ alkyl); R ｅ is -C １－６ alkyl; R ６ is -CH ２ -, -CH ２ C(=O)NHCH ２ - or -CH ２ C(=O)NHCH ２ CH ２ - and; and Y is C or S(=O); or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

10. R ｅ 10. The compound of claim 9, wherein is selected from methyl (Me), ethyl (Et), or propyl (Pr).

11. R ６ Ga-CH ２ The compound according to claim 9 or 10, wherein

12. 11. The compound of claim 9 or 10, wherein Y is C.

13. R ａ The compound of claim 9 or 10, wherein is —H.

14. R ｂ is H; R ｃ is H or OAc; and R ｄ 11. The compound of claim 9 or 10, wherein is H.

15. The compound is 2,6-di-tert-butyl-4-((2-((3,5-di-tert-butyl-4-hydroxyphenyl)thio)propan-2-yl)thio)phenyl (4R)-4-[(3R,5R,8R,9S,10S,13R,14S,17R)-3-acetoxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoate: 【Chemistry 4】 11. The compound according to claim 9 or 10, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

16. 11. A pharmaceutical composition comprising a compound according to any one of claims 1 to 3, 9 and 10, and a pharmaceutically acceptable carrier, adjuvant or diluent.

17. A pharmaceutical composition according to claim 16 for use in treating or preventing a neurological disorder or cognitive decline associated with a neurological disorder in a subject, comprising an effective amount of a compound according to any one of claims 1 to 3, 9 and 10.

18. 11. Use of a compound according to any one of claims 1 to 3, 9 and 10 in the manufacture of a medicament for treating or preventing a neurological disorder or cognitive decline associated with a neurological disorder in a subject.

19. 11. A compound according to any one of claims 1 to 3, 9 and 10 for use in treating or preventing a neurological disorder or cognitive decline associated with a neurological disorder in a subject.

20. 20. The compound of claim 19, wherein the neurological disorder is Alzheimer's disease, dementia caused by Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (motor neuron disease), or multiple sclerosis; and / or the cognitive decline is memory loss.

21. A pharmaceutical composition as described in claim 16 for use in preventing, reducing or treating the occurrence and / or severity of disorders caused by stress-induced cell damage in the middle or inner ear of a subject, comprising a therapeutically effective amount of a compound as described in any one of claims 1 to 3, 9 and 10.

22. Use of a compound described in any one of claims 1 to 3, 9 and 10 in the manufacture of a medicament for preventing, reducing or treating the occurrence and / or severity of a disorder caused by stress-induced cellular damage in the middle or inner ear of a subject.

23. 11. A compound according to any one of claims 1 to 3, 9 and 10 for use in preventing, reducing the incidence and / or severity of or treating a disorder caused by stress-induced cellular damage in the middle or inner ear of a subject.

24. 24. The compound of claim 23, wherein the disorder caused by stress-induced cell damage in the middle or inner ear is a vestibular disorder or a hearing disorder.

25. A pharmaceutical composition as described in claim 16 for use in preventing, reducing or inhibiting hair cell degeneration or hair cell death in a subject, comprising a therapeutically effective amount of a compound as described in any one of claims 1 to 3, 9 and 10.

26. 11. Use of a compound according to any one of claims 1 to 3, 9 and 10 in the manufacture of a medicament for preventing, reducing or inhibiting hair cell degeneration or hair cell death in a subject.

27. 11. A compound according to any one of claims 1 to 3, 9 and 10 for use in preventing, reducing or inhibiting hair cell degeneration or hair cell death in a subject.

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