Carbonic anhydrase v inhibitors and methods of use for treatment of neurological and psychiatric disorders

Abstract

The invention is directed to the use of carbonic anhydrase V inhibitors for treatment of neurological and psychiatric disorders. In one aspect, the disclosure provides compositions for treating or preventing a neurological or psychiatric disease or disorder comprising a mitochondrial carbonic anhydrase V (CA-V) inhibitor. In various embodiments, the mitochondrial CA-V inhibitor selectively inhibits at least one or more of CA-VA and CA-VB. In some embodiments, the chemical compound is 4-phenylacetamidomethyl-benzenesulfonamide (4ITP), derivative, and analogs thereof.

Description

[0001] CARBONIC ANHYDRASE V INHIBITORS AND METHODS OF USE FOR TREATMENT

[0002] OF NEUROLOGICAL AND PSYCHIATRIC DISORDERS

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to U.S. Provisional Application No. 63 / 695,477, filed September 17, 2024, and U.S. Provisional Application No. 63 / 850,721, filed July 25, 2025, the contents of each of which are incorporated by reference herein in their entireties.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0006] This invention was made with government support under R01AG062572 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] BACKGROUND OF THE INVENTION

[0008] Alzheimer’s Disease (AD), the most common form of dementia, is a devastating neurodegenerative disease, currently afflicting over 55 million people worldwide (2023 Alzheimer's disease facts and figures. Alzheimers & Dementia, 2023. 19(4): 1598-1695). The most common risk factor for AD and other forms of dementia is age.

[0009] AD is characterized by the accumulation of extracellular Amyloid-beta (A0) aggregates (or plaques) and intraneuronal hyperphosphorylated tau tangles in multiple brain areas, including the hippocampus and cortex (Lemon, N., et al., Front Aging Neurosci, 2021. 13:772278). Recently, therapies employing antibodies targeting Ap to facilitate its clearance have been approved by the FDA for the treatment of AD. However, due to their concerning side effects, such as amyloid related imaging abnormalities (ARIA) (Beshir, S.A., et al., Int J Alzheimers Dis, 2022. 2022:9343514; Wu, W., et al., Eur J Med Res, 2023. 28(1):544; Hampel, H., et al., Brain, 2023; Kelly, L., et al., Alzheimers Dement, 2023), and only partial efficacy in decreasing cognitive impairment, there is still no effective cure for AD. As the aging population is growing rapidly, it is evident that novel therapies against this devastating disorder are vital.

[0010] Thus, there remains a need in the art for improved compositions to treat AD. The present invention satisfies this unmet need. SUMMARY OF THE INVENTION

[0011] In one aspect, the disclosure provides compositions for treating or preventing a neurological or psychiatric disease or disorder comprising a mitochondrial carbonic anhydrase V (CA-V) inhibitor. In various embodiments, the mitochondrial CA-V inhibitor selectively inhibits at least one or more of CA-VA and CA-VB.

[0012] In various embodiments, the mitochondrial CA-V inhibitor is selected from the group consisting of a chemical compound, a protein, a peptide, a peptidomimetic, an antibody, a ribozyme, a small molecule chemical compound, a nucleic acid, a vector, an antisense molecule, an siRNA, an shRNA, and a guide RNA. In various embodiments, the mitochondrial CA-V inhibitor is a chemical compound. In some embodiments, the chemical compound is 4- phenylacetamidomethyl-benzenesulfonamide (4ITP), derivative, and analogs thereof.

[0013] In various embodiments, the neurological or psychiatric disease or disorder is selected from the group consisting of: Alzheimer's disease (AD), Mild Cognitive Impairment (MCI), Parkinson's disease (PD), Huntington's disease (HD), prion-caused diseases, frontotemporal dementia (FTD), Lewy body dementia, vascular dementias, white matter disease, traumatic brain injury, post-traumatic stress, stroke, tauopathies, Down Syndrome, Amyotrophic Later Sclerosis (ALS), chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), multiple system atrophy (MSA), corticobasal degeneration (CBGD), Pick's disease, olivopontocerebellar atrophy (OPCA), senile dementia of the Alzheimer type, progressive supranuclear palsy (Steel-Richardson-Olszewski), corticodentatonigral degeneration, Hallervorden-Spatz disease, striatonigral degeneration, torsion dystonia, spasmodic torticollis and other dyskinesis, familial tremor, Gilles de la Tourette syndrome, cerebellar cortical degeneration, spinocerebellar degeneration, Shy-Drager syndrome, spinal muscular atrophy, primary lateral sclerosis, hereditary spastic paraplegia, peroneal muscular atrophy (Charcot- Marie-Tooth), hypertrophic interstitial polyneuropathy (Dejerine-Sottas), chronic progressive neuropathy, pigmentary degeneration of the retina (retinitis pigmentosa), hereditary optic atrophy (Leber's disease), Cognitive Dysfunction Syndrome, White dog shaker syndrome, degenerative myelopathy, neuroaxonal dystrophy, cerebellar degeneration, cerebellar abiotrophy, cerebral amyloid angiopathy (CAA), and amyloid related imaging abnormalities (ARIA). In various embodiments, the neurological or psychiatric disease or disorder is a neurodegenerative disease. In various embodiments, the neurological or psychiatric disease or disorder is Alzheimer’s disease.

[0014] In one aspect, the disclosure provides methods of treating a neurological or psychiatric disease or disorder in a subject in need thereof comprising administering to the subject a composition comprising a mitochondrial carbonic anhydrase V (CA-V) inhibitor. In various embodiments, the mitochondrial CA-V inhibitor selectively inhibits at least one or more of CA-VA and CA-VB.

[0015] In various embodiments, the mitochondrial CA-V inhibitor is selected from the group consisting of a chemical compound, a protein, a peptide, a peptidomimetic, an antibody, a ribozyme, a small molecule chemical compound, a nucleic acid, a vector, an antisense molecule, an siRNA, an shRNA, and a guide RNA. In various, embodiments, the mitochondrial CA-V inhibitor is a chemical compound. In some embodiments, the chemical compound is 4- phenylacetamidomethyl -benzenesulfonamide (4ITP).

[0016] In various embodiments, the neurological or psychiatric disease or disorder is selected from the group consisting of: Alzheimer's disease (AD), Mild Cognitive Impairment (MCI), Parkinson's disease (PD), Huntington's disease (HD), prion-caused diseases, frontotemporal dementia (FTD), Lewy body dementia, vascular dementias, white matter disease, traumatic brain injury, post-traumatic stress, stroke, tauopathies, Down Syndrome, Amyotrophic Later Sclerosis (ALS), chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), multiple system atrophy (MSA), corticobasal degeneration (CBGD), Pick's disease, olivopontocerebellar atrophy (OPCA), senile dementia of the Alzheimer type, progressive supranuclear palsy (Steel-Richardson-Olszewski), corticodentatonigral degeneration, Hallervorden-Spatz disease, striatonigral degeneration, torsion dystonia, spasmodic torticollis and other dyskinesis, familial tremor, Gilles de la Tourette syndrome, cerebellar cortical degeneration, spinocerebellar degeneration, Shy-Drager syndrome, spinal muscular atrophy, primary lateral sclerosis, hereditary spastic paraplegia, peroneal muscular atrophy (Charcot- Marie-Tooth), hypertrophic interstitial polyneuropathy (Dejerine-Sottas), chronic progressive neuropathy, pigmentary degeneration of the retina (retinitis pigmentosa), hereditary optic atrophy (Leber's disease), Cognitive Dysfunction Syndrome, White dog shaker syndrome, degenerative myelopathy, neuroaxonal dystrophy, cerebellar degeneration, cerebellar abiotrophy, cerebral amyloid angiopathy (CAA), and amyloid related imaging abnormalities (ARIA). In some embodiments, the neurological or psychiatric disease or disorder is a neurodegenerative disease. In some embodiments, the neurological or psychiatric disease or disorder is Alzheimer’s disease.

[0017] In one aspect, the disclosure provides methods for inhibiting blood-brain barrier permeability in a subject in need thereof comprising administering to the subject a composition comprising a mitochondrial carbonic anhydrase V (CA-V) inhibitor. In various embodiments, the mitochondrial CA-V inhibitor selectively inhibits at least one or more of CA-VA and CA-VB.

[0018] In various embodiments, the mitochondrial CA-V inhibitor is selected from the group consisting of a chemical compound, a protein, a peptide, a peptidomimetic, an antibody, a ribozyme, a small molecule chemical compound, a nucleic acid, a vector, an antisense molecule, an siRNA, an shRNA, and a guide RNA. In various embodiments, the mitochondrial CA-V inhibitor is a chemical compound. In some embodiments, the chemical compound is 4- phenylacetamidomethyl-benzenesulfonamide (4ITP).

[0019] In one aspect, the disclosure provides methods for inhibiting one or more of neuronal degeneration, glial degeneration, and vascular degeneration in a subject in need comprising administering to the subject a composition comprising a mitochondrial carbonic anhydrase V (CA-V) inhibitor. In various embodiments, the mitochondrial CA-V inhibitor selectively inhibits at least one or more of CA-VA and CA-VB.

[0020] In various embodiments, the mitochondrial CA-V inhibitor is selected from the group consisting of a chemical compound, a protein, a peptide, a peptidomimetic, an antibody, a ribozyme, a small molecule chemical compound, a nucleic acid, a vector, an antisense molecule, an siRNA, an shRNA, and a guide RNA. In various embodiments, the mitochondrial CA-V inhibitor is a chemical compound. In some embodiments, the chemical compound is 4- phenylacetamidomethyl-benzenesulfonamide (4ITP).

[0021] In one aspect, the disclosure provides methods for inhibiting one or more of mitochondrial dysfunction in a cell, caspase activation in a cell, Cytochrome C (CytC) release in a cell, inhibiting insoluble protein accumulation in a cell, and cell death in a subject in need thereof comprising administering to the subject a composition comprising a mitochondrial carbonic anhydrase V (CA-V) inhibitor. In various embodiments, the mitochondrial CA-V inhibitor selectively inhibits at least one or more of CA-VA and CA-VB.

[0022] In various embodiments, the mitochondrial CA-V inhibitor is selected from the group consisting of a chemical compound, a protein, a peptide, a peptidomimetic, an antibody, a ribozyme, a small molecule chemical compound, a nucleic acid, a vector, an antisense molecule, an siRNA, an shRNA, and a guide RNA. In various embodiments, the mitochondrial CA-V inhibitor is a chemical compound. In some embodiments, the chemical compound is 4- phenylacetamidomethyl-benzenesulfonamide (4ITP).

[0023] In one aspect, the disclosure provides methods for one or more of inhibiting gliosis, improving microglia clearance, or increasing CD68 expression in microglia comprising administering a composition comprising a mitochondrial carbonic anhydrase V (CA-V) inhibitor.

[0024] In various embodiments, the mitochondrial CA-V inhibitor is selected from the group consisting of a chemical compound, a protein, a peptide, a peptidomimetic, an antibody, a ribozyme, a small molecule chemical compound, a nucleic acid, a vector, an antisense molecule, an siRNA, an shRNA, and a guide RNA. In various embodiments, the mitochondrial CA-V inhibitor is a chemical compound. In some embodiments, the chemical compound is 4- phenylacetamidomethyl -benzenesulfonamide (4ITP).

[0025] In various embodiments, the cell is selected from the group consisting of neuronal cells, glial cells, endothelial cells, and smooth muscle cells. In some embodiments, the neuronal cell is a dopaminergic neuron. In some embodiments, the endothelial cell is a microvascular endothelial cell. In some embodiments, the cell is in a subject.

[0026] In various embodiments, the caspase is one or more of caspase 3, caspase 7, and caspase 9.

[0027] In various embodiments, the mitochondrial dysfunction is a loss of mitochondrial membrane potential.

[0028] In various embodiments, the mitochondrial dysfunction is an increase in mitochondrial H2O2 production.

[0029] The present invention further provides, in part, a compound represented by

[0030] General Formula I: wherein ring A represents a ring selected from the group consisting of substituted or unsubstituted: aryl, cycloalkyl, cycloalkenyl, heteroaryl, and heterocyclyl; L represents a single bond or a linking group selected from the group consisting of alkylene, cycloalkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, silyl, amine, amide, ester, ether, carbonyl, carbamate, sulfamate, thioether, thioester, disulfide, hydrazine, urea, thiourea, phosphate, phosphonate ester, poly(alkyl ether), heteroatom, and combinations thereof; and any two adjacent substituents may optionally join to form a ring.

[0031] In one embodiment, the compound is represented by General Formula la: wherein R1, R2, R3, R4, and R5are each independently selected from the group consisting of hydrogen, deuterium, Ci-6 alkyl, C2-6 alkenyl, alkynyl, hydroxy, thio, C1-6 alkoxy, halo, haloCi-6 alkyl, haloCi-6 alkoxy, C3-8 cycloalkyl, cyano, amino, nitro, carbonyl, carboxylate, C1-6 alkyl ester, and combinations thereof.

[0032] In one embodiment, L represents a single bond. In one embodiment, L is selected from the group consisting of CRL1RL2, NRL1, O, and combinations thereof; wherein RL1and RL2are each independently selected from the group consisting of hydrogen, deuterium, C1-6 alkyl, C2- 6 alkenyl, alkynyl, hydroxy, thio, C1-6 alkoxy, halo, haloCi-6 alkyl, haloCi-6 alkoxy, C3- 8 cycloalkyl, cyano, amino, and combinations thereof. In one embodiment, at least one of R1, R2, R3, R4, and R5are each independently selected from the group consisting of carboxyl, amino, methoxy, cyano, fluoro, chloro, bromo, methyl, trifluoro, methoxycarbonyl, and nitro. In one embodiment, R3is not hydrogen.

[0033] In one embodiment, the compound is represented by General Formula lb: wherein RL1and RL2are each independently selected from the group consisting of hydrogen, deuterium, Ci-6 alkyl, C2-6 alkenyl, alkynyl, hydroxy, thio, C1-6 alkoxy, halo, haloCi- 6 alkyl, haloCi-6 alkoxy, C3-8 cycloalkyl, cyano, amino, and combinations thereof. In one embodiment, at least one of RL1and RL2is methyl.

[0034] In one embodiment, the compound is selected from the group consisting of the following structures:

[0035] The present invention further provides, in part, a method of treating or preventing a neurological or psychiatric disease or disorder in a subject in need thereof comprising administering to the subject a composition comprising a compound represented by General Formula I: wherein ring A represents a ring selected from the group consisting of substituted or unsubstituted: aryl, cycloalkyl, cycloalkenyl, heteroaryl, and heterocyclyl; L represents a single bond or a linking group selected from the group consisting of alkylene, cycloalkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, silyl, amine, amide, ester, ether, carbonyl, carbamate, sulfamate, thioether, thioester, disulfide, hydrazine, urea, thiourea, phosphate, phosphonate ester, poly(alkyl ether), heteroatom, and combinations thereof; and any two adjacent substituents may optionally join to form a ring.

[0036] The present invention further provides, in part, a method for inhibiting bloodbrain barrier permeability in a subject in need thereof comprising administering to the subject a composition comprising Q a compound represented by General Formula I: wherein ring A represents a ring selected from the group consisting of substituted or unsubstituted: aryl, cycloalkyl, cycloalkenyl, heteroaryl, and heterocyclyl; L represents a single bond or a linking group selected from the group consisting of alkylene, cycloalkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, silyl, amine, amide, ester, ether, carbonyl, carbamate, sulfamate, thioether, thioester, disulfide, hydrazine, urea, thiourea, phosphate, phosphonate ester, poly(alkyl ether), heteroatom, and combinations thereof; and any two adjacent substituents may optionally join to form a ring.

[0037] The present invention further provides, in part, a method for inhibiting one or more of neuronal degeneration, glial degeneration, and vascular degeneration in a subject in need thereof comprising administering to the subject a composition comprising a compound represented by General Formula I: wherein ring A represents a ring selected from the group consisting of substituted or unsubstituted: aryl, cycloalkyl, cycloalkenyl, heteroaryl, and heterocyclyl; L represents a single bond or a linking group selected from the group consisting of alkylene, cycloalkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, silyl, amine, amide, ester, ether, carbonyl, carbamate, sulfamate, thioether, thioester, disulfide, hydrazine, urea, thiourea, phosphate, phosphonate ester, poly(alkyl ether), heteroatom, and combinations thereof; and any two adjacent substituents may optionally join to form a ring.

[0038] The present invention further provides, in part, a method for inhibiting one or more of mitochondrial dysfunction in a cell, caspase activation in a cell, Cytochrome C (CytC) release in a cell, inhibiting insoluble protein accumulation in a cell, and cell death in a subject in need thereof comprising administering to the subject a composition comprising a compound represented by General Formula I: wherein ring A represents a ring selected from the group consisting of substituted or unsubstituted: aryl, cycloalkyl, cycloalkenyl, heteroaryl, and heterocyclyl; L represents a single bond or a linking group selected from the group consisting of alkylene, cycloalkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, silyl, amine, amide, ester, ether, carbonyl, carbamate, sulfamate, thioether, thioester, disulfide, hydrazine, urea, thiourea, phosphate, phosphonate ester, poly(alkyl ether), heteroatom, and combinations thereof; and any two adjacent substituents may optionally join to form a ring.

[0039] The present invention further provides, in part, a method for one or more of inhibiting gliosis, improving microglia clearance, or increasing CD68 expression in microglia comprising administering a composition comprising a mitochondrial carbonic anhydrase V (CA-V) inhibitor. wherein ring A represents a ring selected from the group consisting of substituted or unsubstituted: aryl, cycloalkyl, cycloalkenyl, heteroaryl, and heterocyclyl; L represents a single bond or a linking group selected from the group consisting of alkylene, cycloalkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, silyl, amine, amide, ester, ether, carbonyl, carbamate, sulfamate, thioether, thioester, disulfide, hydrazine, urea, thiourea, phosphate, phosphonate ester, poly(alkyl ether), heteroatom, and combinations thereof; and any two adjacent substituents may optionally join to form a ring

[0040] BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0042] Figure 1, comprising Figure 1A through Figure ID, depicts representative experimental results demonstrating that CA-V inhibition protects against Ap40-Q22-induced apoptosis in hCMEC. Figure 1A depicts a quantification of 25pM AP40-Q22 (Q22) 24 h treatment inducing DNA fragmentation in human CMEC, expressed in fold of change (FOC) of untreated control (Ctrl), which is reduced by 4ITP in a dose dependent manner (1,10 and 30pM). Figure IB depicts a quantification of 4ITP preventing cell-viability loss triggered by 24h Q22 treatment, expressed as % of Ctrl. Figure 1C depicts a western blot of CMEC lysates showing that 10 and 30pM 4ITP reduces the significant increase in cleaved (activated) caspase-9 (cCas9) observed after 24h of treatment with Q22, in comparison to Ctrl; cCas9 normalized to actin on the right. Figure ID depicts representative immunofluorescence (IF) images of hCMEC, challenged with Q22 alone or in combination with 10 and 30pM 4ITP. Both 4ITP concentrations significantly reduced the percentage of active caspase 3 / 7-positive cells induced by Q22, as plotted on the right. Original magnification: 1 Ox. These data represent the combination of at least three experiments, each with 2 replicates, graphed as mean + SEM. Statistical significance was evaluated by One-way ANOVA, followed by Tukey post-hoc test. **** p<0.0001, *** p<0.001, ** p<0.01, * p<0.05

[0043] Figure 2, comprising Figure 2A through Figure 2D, depicts representative experimental results demonstrating that 4ITP protects against A04O-Q22 induced mitochondrial apoptotic mechanisms and oxidative stress in hCMEC. HCMEC were treated for 16h with 25pM AP40-Q22 (Q22), in the presence or absence of 10 or 30pM 4ITP. Figure 2A depicts representative immunofluorescent images depicting the loss of Mito-tracker signal observed in CMEC challenged with Q22 indicating a loss of mitochondrial membrane potential. The release of cytochrome C is visualized as a diffused, cytosolic signal (indicated with arrows) in Q22- treated cells, compared to untreated cells (ctrl). Both loss of mitochondrial membrane potential and release of cytochrome C were prevented by lOpM 4ITP. Original magnification: lOOx. Figure 2B depicts representative western blot images of mitochondrial apoptotic mediators BCL- 2, BIM, BAX, and BAK, following Q22 treatment, and relative quantification below (loading control, actin). Both 10 and 30pM 4ITP significantly prevent Q22-induced BAX increase, and reduce BIM. Figure 2C depicts a quantification of the Q22-induced increase in mitochondrial H2O2 production, measured by Amplex Red, prevented by 4ITP. Figure 2D depicts a quantification of 4-HNE, a marker of lipid peroxidation, increasing in Q22 treated CMEC, and rescued by 30pM 4ITP. Data represent the combination of at least three experiments each with 2 replicates, graphed as mean + SEM. Statistical significance was evaluated by One-way ANOVA followed by Tukey post-hoc test. *** p<0.001, ** p<0.01, * p<0.05

[0044] Figure 3, comprising Figure 3A through Figure 3C, depicts representative experimental results demonstrating that 4ITP prevents A[340-Q22 induced loss in endothelial barrier integrity. Figure 3 A depicts a quantification of hCMEC barrier resistance assessed by ECIS Z0 to evaluate barrier integrity. Human CMEC were treated with lOpM AP40-Q22 (Q22), alone or in combination with 10 or 30pM 4ITP. Q22 treatment (line) significantly decreases CMEC resistance, compared to control (Ctrl, black straight line). 4ITP prevents Q22 induced decrease in resistance in a dose-dependent manner. Figure 3B and Figure 3C depict representative western blot analysis of occludin and oligomeric claudin-5 (normalized to actin, and plotted as % change of Ctrl), following 24h (Figure 3B) and 48h (Figure 3C) Q22 treatment. Figure 3B demonstrates that a 24 hour-Q22 challenge resulted in no significant changes in occludin and claudin-5 expression in CMEC. 4ITP, with or without Q22 induced claudin-5 upregulation, compared to Ctrl. Figure 3C demonstrates that after 48 hours, Q22 treatment, but not Q22 + 4ITP, significantly decreased occludin expression. 30pM 4ITP alone increased Occludin expression, compared to Ctrl. Data represents 3 individual experiments with 2 replicates per group, graphed as mean ± SEM. Statistical significance was evaluated by One-way ANOVA followed by Tukey post-hoc test. *P< 0.05, **P<0.01, ***P<0.001, ****P<0.0001

[0045] Figure 4, comprising Figure 4A through Figure 4E, depicts representative experimental results demonstrating that 4ITP mitigates A04O-Q22 induced brain endothelial activation. Human CMEC were treated with 10 pM AP40-Q22 (Q22), alone or in combination with 10 or 30pM of 4ITP. Figure 4A depicts a representative western blot demonstrating that compared to untreated control, (Ctrl), Q22 upregulates ICAM-1 expression at 6 hours, with no significant changes in VCAM1. At 24h (Figure 4B) and 48h (Figure 4C), Q22 induces a significant increase in VCAM-1, which is rescued by 30pM 4ITP. No significant changes were observed in ICAM-1 expression at 24h or 48h. Figure 4D and Figure 4E depict quantification of cytokine release evaluated after 6h and 48h Q22 treatment using a multiplex MSD proinflammatory panel. Figure 4D demonstrates that after 6h, Q22 significantly reduces antiinflammatory cytokine IL- 10 and increases pro-inflammatory IL-6 release, compared to Ctrl. The levels of IL-10 are rescued by 4ITP co-treatment. Figure 4E demonstrates that after 48 hours, Q22 induces a significant increase in the release of proinflammatory cytokines IL-6, IL-4, and IL-12p70, and a reduction in IL10 (p=0.0585). 4ITP reverts the increases in IL-6, IL-4, and IL-12p70 , and increases the release of IL-10 compared to Q22-tretated CMEC. Data represent 3 individual experiments with 2 replicates per group, graphed as mean ± SEM. Statistical significance was evaluated by One-way ANOVA followed by Tukey ’s or Dunnett’s T3 (D and E) post-hoc test. *P< 0.05, **P<0.01, ***P<0.001, ****P<0.0001

[0046] Figure 5, comprising Figure 5A through Figure 5F, depicts representative experimental results demonstrating that CA-VB KO in hCMEC protects from Ap-induced apoptosis and BBB permeability. Figure 5A depicts a representative western blot confirming CA-VB absence in KO cells. Figure 5B depicts a quantification of DNA fragmentation, plotted as fold of change (FOC) of untreated control (ctrl) cells, measured by cell death ELISA. CMEC treated with 25pM AP40-Q22 (Q22) for 24 hours induced apoptosis, which was prevented in CA-VB KO hCMEC. Figure 5C depicts representative immunofluorescent images of active caspase-3 / 7 and quantification to the right in CMEC treated with 25 pM Q22 for 24 hours. Caspase 3 / 7 activation is prevented in CA-VB KO hCMEC, as plotted below in % cells with active caspase 3 / 7 versus total cells. Original magnification: lOx. Figure 5D depicts representative immunofluorescent images demonstrating CA-VB KO prevented the release of cytochrome C (Cyto C) as well as the loss of mitochondrial membrane potential (Mito-tracker) in hCMEC challenged with Q22 for 16 hours. Zoom images of the merge signal at the bottom depict altered mitochondrial network with perinuclear mitochondria in Q22-treated hCMEC, but not in CA-VB KO cells. Original magnification: lOOx. Figure 5E depicts representative experimental results demonstrating that CA-VB KO prevented Q22-induced mitochondrial H2O2 production. Figure 5F depicts a quantification measuring barrier resistance over time with ECIS- Z0 in WT and CA-VB KO hCMEC treated or not with 10pM Q22. CA-VB KO did not affect barrier properties compared to the WT control. CA-VB KO hCMEC monolayers treated with A04O-Q22 presented a significantly preserved barrier Resistance compared to treated WT cells. Data represents 3 individual experiments with 2 replicates each, graphed as mean ± SEM. Statistical significance was evaluated by One-way ANOVA followed by Tukey post-hoc test. *P< 0.05, **P<0.01 ****P<0.0001

[0047] Figure 6, comprising Figure 6A and Figure 6B, depicts representative experimental results demonstrating that 4ITP protects from apoptosis and preserves BBB integrity in 3xTG mice. Cleaved caspase-3 (cCAS3), VCAM-1, ICAM-1 and occludin expression were evaluated in the cortex (Figure 6A) and (Figure 6B) hippocampus of WT, untreated 3xTG mice and 3xTG mice treated with 4ITP (6-16M, in the diet). N= 6 / group, males and female. Figure 6A depicts representative western blot analysis demonstrating that in the cortex, occludin expression is significantly increased by 4ITP treatment. Additionally, a significant increase in cCAS3 was observed in 3xTG mice, which is mitigated by 4ITP treatment. Both VCAM-1 and ICAM1 are upregulated in 3xTG mice, and the increase is significantly prevented by 4ITP treatment. Figure 6B depicts representative western blot analysis demonstrating that in the hippocampus, cCAS3 is significantly increased in 3xTG mice, and then reverted with 4ITP -treated 3xTG mice, compared to WT mice. VCAM1 and ICAM are significantly upregulated in untreated 3xTg, but not in 4ITP -treated 3xTg mice, compared to WT animals. Occludin levels are significantly downregulated in 3xTg mice, but not in 4ITP -treated 3xTG mice, compared to WT animals. Data represents N=6 mice per group, 2 replicates per mouse, graphed as mean ± SEM. Statistical significance was evaluated by Two-way ANOVA followed by Tukey’s post-hoc test. *P< 0.05, **P < 0.01, ***P <0.001, ****P<0.0001.

[0048] Figure 7, comprising Figure 7A through Figure 7C, depicts representative experimental results demonstrating that 4ITP prevents loss of memory in 3xTG mice. (WT, N=20 3XTG, N=21 3XTG+4ITP N=21, males, and females 15-16 months) (Figure 7A and Figure 7B). Figure 7A depicts an experiment where spatial memory was assessed using Barnes Maze over a 6-day period. 3xTG mice presented a significantly impaired ability (latency, s) to find the escape hole compared to WT mice. 4ITP treatment resulted in a significant amelioration in the latency (s) to find the escape hole, compared to non-treated 3xTG mice on day 1, 2 and 6 of the maze. Figure 7B depicts a quantification demonstrating that on the probe day (day 6), untreated 3xTG animals made significant higher number of mistakes, compared to WT mice, and 4ITP treatment rescued this deficit. Similarly, the average latency (s) to find the escape hole, significantly increased in untreated 3xTG, compared to age-matched WT, and was significantly lowered by 4ITP treatment. Figure 7C depicts a quantification demonstrating that the rotarod test displayed no locomotor differences between groups. For Figure 7A, statistical significance was evaluated by Two-way ANOVA followed by Tukey’s multiple comparison test. For Figure 7B and Figure 7C, statistical significance was evaluated by One-way ANOVA followed by Tukey post-hoc test. *P< 0.05, **P<0.01 ****P<0.0001

[0049] Figure 8 depicts representative experimental results demonstrating that 4ITP is not toxic at concentrations of 10 or 30pM in hCMEC. There is no significant change in cellviability, plotted as % of Ctrl, after 24-hour 4ITP treatment.

[0050] Figure 9 depicts representative experimental results demonstrating that 4ITP increases expression of claudin-5 in hCMEC after 6 hours of treatment. Human CMEC were treated with 10 pM A04O-Q22, alone or in combination with 10 or 30, pM of 4ITP. Western blot analysis of tight junction proteins occludin and claudin-5 were evaluated at 6 hours. At 6 hours claudin-5 was significantly increased in the presence of Q22 + 4ITP in addition to 4ITP alone when compared to the control. Data represents 3 individual experiments of 2 replicates per group and graphed as mean ± SEM. Statistical significance was evaluated by One-way ANOVA followed by Tukey post-hoc test. *P< 0.05, **P< 0.01, ****p<o.0001.

[0051] Figure 10 depicts representative experimental results demonstrating that expression of CA-VA and CA-II isoforms does not change in hCMEC / D3 CA-VB KO cells. WB analysis confirmed CA-VA and CA-II normalized to loading control actin are expressed in hCMEC CA-VB KO cells.

[0052] Figure 11, comprising Figure 11 A and Figure 1 IB, depicts representative experimental results demonstrating that long-term treatment with 4ITP in WT mice did not alter spatial memory. Figure 11 A depicts a quantification of results from a Barnes maze (WT, N=20 WT+4ITP N=11, males and females 15-16 months of age). Latency to find the escape hole was recorded two sessions / day and averaged and then graphed per day. There are no significant differences between treated and non-treated WT mice in the latency to find the escape hole. Figure 1 IB depicts a quantification of motor function assessed with Rotarod. There were no significant changes between treated and non-treated 16 month wild-type mice.

[0053] Figure 12, comprising Figure 12A through Figure 12F, depicts representative experimental results demonstrating inhibition of CA-V is not toxic and prevents cognitive decline in 3xTG mice. Figure 12A depicts a schematic explaining time-points of behavior and biochemistry analysis of 3xTG mice treated and not treated with CA-V inhibitor at 20mg / kg / day. Figure 12B depicts a graph demonstrating there are no significant differences between 3xTG treated and not treated in survival. Figure 12C depicts a graph demonstrating there is a significant reduction in body weight in 3xTG mice compared to wild-type which is slightly reverted with CA-V inhibition, indicating a healthier phenotype. Figure 12D depicts a graph illustrating results from experiments where, over a 6-day period, wild-type mice, 3xTG and 4ITP -treated 3xTG mice, underwent the barnes maze to assess spatial memory as well as memory consolidation. CA-V inhibition prevented cognitive decline in 3xTG mice, already apparent on day 1 of the maze. Figure 12E depicts a graph demonstrating the number of mistakes as well as the latency to find the escape hole on the probe day was significantly reduced in CA-V treated mice, returning down to wild-type levels. Figure 12F depicts a graph illustrating results from a behavioral test, rotarod, which also confirmed there are no motor deficits in these in mice attributing to the changes seen in the barnes maze. Statistical significance was evaluated by Oneway ANOVA followed by Tukey’s multiple comparison test *p<0.05 **p< 0.01 ***p< 0.001.

[0054] Figure 13, comprising Figure 13 A through Figure 13C, depicts representative results demonstrating CA-V inhibition lessens caspase activation and vascular stress in 3xTG mice. Figure 13A depicts a representative immunohistochemical analysis of cleaved-caspase3 in the cortex (top) and hippocampus (bottom) of WT, 3xTG, and 3xTG + CA-Vi treated mice. Figure 13B and Figure 13C depicts a representative western blot analysis in cortex and hippocampus of occludin, cCASp3, VCAM-1, and ICAM-1 all normalized to loading control actin. Number of dots indicates number of mice (N). Dots indicating male and dots indicating female are presented. Statistical significance was evaluated by One-way ANOVA followed by Tukey’s multiple comparison test *p<0.05 **p< 0.01 (Figure 13A). Statistical significance was evaluated by Two-way ANOVA followed by Tukey’s multiple comparison test *p<0.05 **p< 0.01 ***p< 0.001 (Figure 13B and Figure 13C).

[0055] Figure 14, comprising Figure 14A through Figure 14C, depicts representative results demonstrating CA-V inhibition mitigates gliosis, and insoluble protein accumulation in 3xTG mice. Immunohistochemical analysis of gliosis in wild-type, 3xTG treated and non-treated mice. Figure 13A and Figure 13B depicts representative images of GFAP and IBA1, respectively, in the hippocampus and cortex. There is an evident decrease in the treated 3xTG mice that there is a reduction in % area of both GFAP and IBA1. Figure 13C depicts a quantitative analysis of amyloid-beta soluble and insoluble 40 and 42 as well as soluble and insoluble pTau231 in the hippocampus. Dots indicate number of mice (N). Statistical significance was evaluated by T test followed by Welch’s correction. *p<0.05.

[0056] Figure 15, comprising Figure 15A and Figure 15B, depicts representative results demonstrating CA-VB protein expression increases with amyloid after 6-hours of treatment. Western blot analysis with quantification on the left (Figure 15 A) and WB images on the right (Figure 15B) of mitochondrial protein CA-VB normalized to ATP5a in wild-type and CA-VB KO HMC3 cells. Wild-type and CA-VB KO cells were treated with AP42 or Q22 alone or in combination with CA-Vi for 6 hours. Statistical significance was evaluated by Two-Way ANOVA followed by Tukey’s multiple comparison test *p<0.05 **p< 0.01 ***p< 0.001.

[0057] Figure 16, comprising Figure 16A through Figure 16C, depicts representative results demonstrating CD68 expression is increased following 6 hours of CA-V inhibition as well as in CA-VB KO HMC3 compared to wild-type. A western blot analysis with quantification on the left (Figure 16A) and WB images on the right (Figure 16B) of lysosomal protein CD68 normalized to loading control actin in wild-type and CA-VB KO HMC3 cells is depicted. Figure 16C depicts representative ICC images of WT and CA-VB KO cells treated for 48 hours with the %area of amyloid quantified on the right. Statistical significance was evaluated by Two-Way ANOVA followed by Tukey’s multiple comparison test *p<0.05 **p< 0.01 ***p< 0.001.

[0058] DETAILED DESCRIPTION

[0059] The invention is based, in part on the finding that a selective inhibitor of carbonic anhydrase V (CA-V) ameliorates symptoms of Alzheimer’s disease. Thus, the invention is based on the discovery of a previously unknown mechanism of carbonic anhydrase V in regulating blood-brain barrier permeability, neuronal degeneration, glial degeneration, vascular degeneration, mitochondrial dysfunction in a cell, caspase activation in a cell, Cytochrome C (CytC) release in a cell, cell death, gliosis, insoluble protein accumulation, and CD68 expression and provides a novel treatment strategy for treatment of neurodegenerative diseases including, but not limited to, Alzheimer’s Disease (AD).

[0060] Therefore in various embodiments, the invention provides compositions and methods for inhibiting CA-V. In some embodiments, the CA-V is a mitochondrial CA-V. Thus, in some embodiments, the invention provides compositions comprising a mitochondrial CA-V inhibitor. In some embodiments, the CA-V inhibitor selectively inhibits at least one or more of carbonic anhydrase VA (CA-V A) and carbonic anhydrase VB (CA-VB). In some embodiments, the CA-V is CA-VA. In some embodiments, the CA-V is CA-VB. In some embodiments, the CA-V is mitochondrial CA-VA. In some embodiments, the CA-V is mitochondrial CA-VB. In some embodiments, the invention provides 4-phenylacetamidomethyl-benzenesulfonamide (4ITP) for inhibition of CA-V.

[0061] Definitions

[0062] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

[0063] As used herein, each of the following terms has the meaning associated with it in this section.

[0064] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. “About” as used herein when referring to a measurable value, for example numerical values and / or ranges, such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. For example, “about 40 [units]” may mean within ± 25% of 40 (e.g., from 30 to 50), within ± 20%, ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, ± 1%, less than ± 1%, or any other value or range of values therein or therebelow. Furthermore, the phrases “less than about [a value]” or “greater than about [a value]” should be understood in view of the definition of the term “about” provided herein.

[0065] As used herein, the term “organic” includes polymeric materials as well as small molecule organic materials. “Small molecule” refers to any organic material that is not a polymer, and “small molecules” may actually be quite large. Small molecules may include repeat units in some circumstances. For example, using a long chain alkyl group as a substituent does not remove a molecule from the “small molecule” class. Small molecules may also be incorporated into polymers, for example as a pendent group on a polymer backbone or as a part of the backbone. Small molecules may also serve as the core moiety of a dendrimer, which consists of a series of chemical shells built on the core moiety.

[0066] As used herein, the term “alkyl,” by itself or as part of another substituent means, unless otherwise stated, a straight or branched chain hydrocarbon having the number of carbon atoms designated (i.e. Ci-6 means one to six carbon atoms) and including straight, branched chain, or cyclic substituent groups. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, and cyclopropylmethyl.

[0067] As used herein, the term “substituted alkyl” means alkyl as defined above, substituted by one, two or three substituents selected from the group consisting of halogen, -OH, alkoxy, -NH2, amino, azido, -N(CH3)2, -C(=O)OH, trifluoromethyl, -C=N, -C(=O)O(Ci-C4)alkyl, -C(=O)NH2, -SO2NH2, -C(=NH)NH2, and -NO2. Examples of substituted alkyls include, but are not limited to, 2,2-difluoropropyl, 2-carboxycyclopentyl and 3-chloropropyl.

[0068] As used herein, the term “alkenyl” encompasses compounds having a C=C bond. As used herein, the term “alkynyl” encompasses compounds having a carbon-carbon triple bond.

[0069] As used herein, the term “heteroalkyl” by itself or in combination with another term means, unless otherwise stated, a stable straight or branched chain alkyl group consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, and S, and wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heteroatom(s) may be placed at any position of the heteroalkyl group, including between the rest of the heteroalkyl group and the fragment to which it is attached, as well as attached to the most distal carbon atom in the heteroalkyl group. Examples include -O-CH2-CH2-CH3, -CH2-CH2-CH2-OH, -CH2-CH2-NH-CH3, -CH2-S-CH2-CH3, and -CH2CH2-S(=O)-CH3. Up to two heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3, or -CH2-CH2-S-S-CH3.

[0070] As used herein, the term “alkoxy” employed alone or in combination with other terms means, unless otherwise stated, an alkyl group having the designated number of carbon atoms, as defined above, connected to the rest of the molecule via an oxygen atom, such as, for example, methoxy, ethoxy, 1 -propoxy, 2-propoxy (isopropoxy) and the higher homologs and isomers.

[0071] As used herein, the term “halo” or “halogen” alone or as part of another substituent means, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.

[0072] As used herein, the term “cycloalkyl” refers to a mono cyclic or polycyclic nonaromatic radical, wherein each of the atoms forming the ring (i.e. skeletal atoms) is a carbon atom. In one embodiment, the cycloalkyl group is saturated or partially unsaturated. In another embodiment, the cycloalkyl group is fused with an aromatic ring. Cycloalkyl groups include groups having from 3 to 10 ring atoms. Illustrative examples of cycloalkyl groups include, but are not limited to, the following moi eties:

[0073] Monocyclic cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Dicyclic cycloalkyls include, but are not limited to, tetrahydronaphthyl, indanyl, and tetrahydropentalene. Polycyclic cycloalkyls include adamantine and norbornane. The term cycloalkyl includes “unsaturated nonaromatic carbocyclyl” or “nonaromatic unsaturated carbocyclyl” groups, both of which refer to a nonaromatic carbocycle as defined herein, which contains at least one carbon double bond or one carbon triple bond.

[0074] As used herein, the term “heterocycloalkyl” or “heterocyclyl” or “heterocyclic” refers to a cyclic group containing one to four ring heteroatoms each selected from O, S, and N. In one embodiment, each heterocycloalkyl group has from 4 to 10 atoms in its ring system, with the proviso that the ring of said group does not contain two adjacent O atoms. In another embodiment, the heterocycloalkyl group is fused with an aromatic ring. In one embodiment, the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen atom may be optionally quatemized. The heterocyclic system may be attached, unless otherwise stated, at any heteroatom or carbon atom that affords a stable structure. A heterocycle may be aromatic or nonaromatic in nature. In one embodiment, the heterocycle is a heteroaryl.

[0075] An example of a 3-membered heterocycloalkyl group includes, and is not limited to, aziridine. Examples of 4-membered heterocycloalkyl groups include, and are not limited to, azetidine and a beta lactam. Examples of 5-membered heterocycloalkyl groups include, and are not limited to, pyrrolidine, oxazolidine and thiazolidinedione. Examples of 6-membered heterocycloalkyl groups include, and are not limited to, piperidine, morpholine and piperazine.

[0076] Examples of non-aromatic heterocycles include monocyclic groups such as aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, pyrroline, pyrazolidine, imidazoline, dioxolane, sulfolane, 2, 3 -dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane, piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine, morpholine, thiomorpholine, pyran, 2, 3 -dihydropyran, tetrahydropyran, 1,4-dioxane, 1,3-dioxane, homopiperazine, homopiperidine, 1,3-dioxepane, 4,7-dihydro-l,3-dioxepin, and hexamethyl eneoxi de .

[0077] As used herein, the term “aromatic” refers to a carbocycle or heterocycle with one or more polyunsaturated rings and having aromatic character, i.e. having (4n + 2) delocalized TI (pi) electrons, where n is an integer.

[0078] As used herein, the term “aryl,” employed alone or in combination with other terms, means, unless otherwise stated, a carbocyclic aromatic system containing one or more rings (typically one, two or three rings), wherein such rings may be attached together in a pendent manner, such as a biphenyl, or may be fused, such as naphthalene. Examples of aryl groups include phenyl, anthracyl, and naphthyl.

[0079] As used herein, the term “aryl-(Ci-C3)alkyl” means a functional group wherein a one- to three-carbon alkylene chain is attached to an aryl group, e.g., -CH2CH2- phenyl, -CH2- phenyl (benzyl), aryl-CHz- and aryl-CH(CH3)-. The term “substituted aryl-(Ci-C3)alkyl” means an aryl-(Ci-C3)alkyl functional group in which the aryl group is substituted. Similarly, the term “heteroaryl-(Ci-C3)alkyl” means a functional group wherein a one to three carbon alkylene chain is attached to a heteroaryl group, e.g., -CEECHz-pyridyl. The term “substituted heteroaryl-(Ci-C3)alkyl” means a heteroaryl -(Ci-C3)alkyl functional group in which the heteroaryl group is substituted.

[0080] The term “cycloalkenyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms and containing at least one carbon-carbon double bound, i.e., C=C. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, norbornenyl, and the like. The term “heterocycloalkenyl” is a type of cycloalkenyl group as defined above, and is included within the meaning of the term “cycloalkenyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkenyl group and heterocycloalkenyl group can be substituted or unsubstituted. The cycloalkenyl group and heterocycloalkenyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein.

[0081] The term “cycloalkynyl” as used herein is a non-aromatic carbon-based ring composed of at least seven carbon atoms and containing at least one carbon-carbon triple bound. Examples of cycloalkynyl groups include, but are not limited to, cycloheptynyl, cyclooctynyl, cyclononynyl, and the like. The term “heterocycloalkynyl” is a type of cycloalkenyl group as defined above, and is included within the meaning of the term “cycloalkynyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkynyl group and heterocycloalkynyl group can be substituted or un substituted. The cycloalkynyl group and heterocycloalkynyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein.

[0082] As used herein, the term “heteroaryl” or “heteroaromatic” refers to aryl groups which contain at least one heteroatom selected from N, O, Si, P, and S; wherein the nitrogen and sulfur atoms may be optionally oxidized, and the nitrogen atom(s) may be optionally quaternized. Heteroaryl groups may be substituted or unsubstituted. A heteroaryl group may be attached to the remainder of the molecule through a heteroatom. A polycyclic heteroaryl may include one or more rings that are partially saturated. Examples include tetrahydroquinoline, 2,3-dihydrobenzofuryl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4- imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4- isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3- thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2- benzimidazolyl, 5-indolyl, 1 -isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3- quinolyl, and 6-quinolyl.

[0083] The term “heterocyclyl,” as used herein refers to single and multi-cyclic nonaromatic ring systems and “heteroaryl as used herein refers to single and multi-cyclic aromatic ring systems: in which at least one of the ring members is other than carbon. The terms includes azetidine, dioxane, furan, imidazole, isothiazole, isoxazole, morpholine, oxazole, oxazole, including, 1,2,3-oxadiazole, 1,2,5-oxadiazole and 1,3,4-oxadiazole, piperazine, piperidine, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolidine, tetrahydrofuran, tetrahydropyran, tetrazine, including 1,2,4,5-tetrazine, tetrazole, including 1,2,3,4-tetrazole and 1, 2, 4, 5-tetrazole, thiadiazole, including, 1,2, 3 -thiadiazole, 1,2,5-thiadiazole, and 1,3,4- thiadiazole, thiazole, thiophene, triazine, including 1,3,5-triazine and 1,2,4-triazine, triazole, including, 1,2,3-triazole, 1,3,4-triazole, and the like.

[0084] Examples of non-aromatic heterocycles include monocyclic groups such as aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, pyrroline, imidazoline, pyrazolidine, dioxolane, sulfolane, 2,3 -dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane, piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine, morpholine, thiomorpholine, pyran, 2, 3 -dihydropyran, tetrahydropyran, 1,4-dioxane, 1,3-dioxane, homopiperazine, homopiperidine, 1,3-dioxepane, 4,7-dihydro-l,3-dioxepin and hexamethyleneoxide. Examples of heteroaryl groups also include pyridyl, pyrazinyl, pyrimidinyl (particularly 2- and 4-pyrimidinyl), pyridazinyl, thienyl, furyl, pyrrolyl (particularly 2-pyrrolyl), imidazolyl, thiazolyl, oxazolyl, pyrazolyl (particularly 3- and 5-pyrazolyl), isothiazolyl,

[0085] 1.2.3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl,

[0086] 1.3.4-thiadiazolyl and 1,3,4-oxadiazolyl.

[0087] Examples of polycyclic heterocycles and heteroaryls include indolyl (particularly 3-, 4-, 5-, 6- and 7-indolyl), indolinyl, quinolyl, tetrahydroquinolyl, isoquinolyl (particularly 1- and 5 -isoquinolyl), 1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (particularly 2- and 5-quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl, 1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl, benzofuryl (particularly 3-, 4-, 5-, 6- and 7-benzofuryl), 2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl (particularly 3-, 4-, 5-, 6-, and 7-benzothienyl), benzoxazolyl, benzothiazolyl (particularly 2-benzothiazolyl and 5-benzothiazolyl), purinyl, benzimidazolyl (particularly 2-benzimidazolyl), benzotriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, and quinolizidinyl.

[0088] As used herein, the term “substituted” means that an atom or group of atoms has replaced hydrogen as the substituent attached to another group. The term “substituted” further refers to any level of substitution, namely mono-, di-, tri-, tetra-, or penta-substitution, where such substitution is permitted. The substituents are independently selected, and substitution may be at any chemically accessible position. In one embodiment, the substituents vary in number between one and four. In another embodiment, the substituents vary in number between one and three. In yet another embodiment, the substituents vary in number between one and two.

[0089] As used herein, the term “optionally substituted” means that the referenced group may be substituted or unsubstituted. In one embodiment, the referenced group is optionally substituted with zero substituents, i.e., the referenced group is unsubstituted. In another embodiment, the referenced group is optionally substituted with one or more additional group(s) individually and independently selected from groups described herein.

[0090] In one embodiment, the substituents are independently selected from the group consisting of oxo, halogen, -CN, -NEE, -OH, -NH(CHs), -N(CHs)2, alkyl (including straight chain, branched and / or unsaturated alkyl), substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, fluoro alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkoxy, fluoroalkoxy, -S-alkyl, S(=O)2alkyl, S(=O)2N[H, alkyl, or aryl], - C(=O)NH[substituted or unsubstituted alkyl, or substituted or unsubstituted phenyl], -C(=O)N[H or substituted or unsubstituted alkyl or aryl]2, -OC(=O)N[substituted or unsubstituted alkyl]2, -NHC(=O)NH[substituted or unsubstituted alkyl, or substituted or unsubstituted phenyl], -NHC(=O)alkyl, -N[substituted or unsubstituted alkyl]C(=O)[substituted or unsubstituted alkyl], -NHC(=O) [substituted or unsubstituted alkyl], -C(OH)[substituted or unsubstituted alkyl]2, and - C(NH2)[substituted or unsubstituted alkyl]2. In another embodiment, by way of example, an optional substituent is selected from oxo, fluorine, chlorine, bromine, iodine, -CN, -NH2, -OH, - NH(CH3), -N(CH3)2, -CH3, -CH2CH3, -CH(CH3)2, -CF3, -CH2CF3, -OCH3, -OCH2CH3, - OCH(CH3)2, -OCF3, - OCH2CF3, -S(=O)2-CH3, -C(=O)NH2, -C(=O)-NHCH3, - NHC(=O)NHCH3, -C(=O)CH3, -ON(O)2, and -C(=O)OH. In yet one embodiment, the substituents are independently selected from the group consisting of C1-6 alkyl, -OH, C1-6 alkoxy, halo, amino, acetamido, oxo and nitro. As used herein, where a substituent is an alkyl or alkoxy group, the carbon chain may be branched, straight or cyclic.

[0091] As used herein, the term “protected,” as used herein, refers to the presence of a “protecting group” or moiety that prevents reaction of the chemically reactive functional group under certain reaction conditions. The protecting group will vary depending on the type of chemically reactive group being protected. By way of example only, (i) if the chemically reactive group is an amine or a hydrazide, the protecting group may be selected from tertbutyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc); (ii) if the chemically reactive group is a thiol, the protecting group may be orthopyridyldisulfide; and (iii) if the chemically reactive group is a carboxylic acid, such as butanoic or propionic acid, or a hydroxyl group, the protecting group may be benzyl or an alkyl group such as methyl, ethyl, or tert-butyl. Additionally, protecting groups include, but are not limited to, photolabile groups, such as Nvoc and MeNvoc, and other protecting groups known in the art. Other protecting groups are described in Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, N.Y., 1999.

[0092] The term “derivative” refers to a small molecule that differs in structure from the reference molecule but retains the essential properties of the reference molecule. A derivative may change its interaction with certain other molecules relative to the reference molecule. A derivative molecule may also include a salt, an adduct, tautomer, isomer, or other variant of the reference molecule. The term “tautomers” are constitutional isomers of organic compounds that readily interconvert by a chemical process (tautomerization).

[0093] The term “isomers” or “stereoisomers” refer to compounds, which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.

[0094] The term “dysregulation” when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells or components thereof that display the normally “regulated” (expected) respective characteristic. Characteristics which are regulated or expected for one cell or tissue type, might be dysregulated for a different cell or tissue type.

[0095] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.

[0096] In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

[0097] A disease or disorder is “alleviated” if the severity of a sign or symptom of the disease or disorder, the frequency with which such a sign or symptom is experienced by a patient, or both, is reduced.

[0098] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In some embodiments, the patient, subject or individual is a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) and a primate (e.g., monkey and human), most preferably a human. In certain non-limiting embodiments, the patient, subject or individual is a human.

[0099] A “therapeutic” treatment is a treatment administered to a subject who exhibits signs or symptoms of a disease or disorder, for the purpose of diminishing or eliminating those signs or symptoms. As used herein, “treating a disease or disorder” means reducing the severity and / or frequency with which a sign or symptom of the disease or disorder is experienced by a patient.

[0100] An “effective amount” or “therapeutically effective amount” of a compound is that amount of a compound which is sufficient to provide a beneficial effect to the subject to which the compound is administered. An “effective amount” of a delivery vehicle is that amount sufficient to effectively bind or deliver a compound.

[0101] As used herein, the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material may be administered to an individual without causing an undesirable biological effect or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0102] As used herein, the term “pharmaceutical composition” refers to a mixture of at least one compound useful within the invention with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient or subject. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary, and topical administration.

[0103] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the invention within or to the patient such that it may perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the invention, and not injurious to the patient. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the invention and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions. The “pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compound useful within the invention. Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the invention are known in the art and described, for example in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

[0104] As used herein, the term “potency” refers to the dose needed to produce half the maximal response (ED50).

[0105] As used herein, the term “efficacy” refers to the maximal effect (Emax) achieved within an assay.

[0106] By the term “modulating,” as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and / or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.

[0107] The term “activate,” as used herein, means to induce or increase an activity or function, for example, about ten percent relative to a control value. Preferably, the activity is induced or increased by 50% compared to a control value, more preferably by 75%, and even more preferably by 95%. “Activate,” as used herein, also means to increase a molecule, a reaction, an interaction, a gene, an mRNA, and / or a protein’s expression, stability, function or activity by a measurable amount or to increase entirely. Activators are compounds that, e.g., bind to, partially or totally induce stimulation, increase, promote, induce activation, activate, sensitize, or up regulate a protein, a gene, and an mRNA stability, expression, function and activity, e.g., agonists.

[0108] The phrase “inhibit,” as used herein, means to reduce a molecule, a reaction, an interaction, a gene, an mRNA, and / or a protein’s expression, stability, function or activity by a measurable amount or to prevent entirely. Inhibitors are compounds that, e.g., bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate a protein, a gene, and an mRNA stability, expression, function and activity, e.g., antagonists. In one embodiment, the activity is suppressed or blocked by 10% compared to a control value. In one embodiment, the activity is suppressed or blocked by 50% compared to a control value. In one embodiment, the activity is suppressed or blocked by 75%. In one embodiment, the activity is suppressed or blocked by 95%.

[0109] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in its normal context in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural context is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0110] An “isolated nucleic acid” refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, i.e., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, i.e., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, i.e., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence. In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[0111] The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.

[0112] A “coding region” of a gene consists of the nucleotide residues of the coding strand of the gene and the nucleotides of the non-coding strand of the gene which are homologous with or complementary to, respectively, the coding region of an mRNA molecule which is produced by transcription of the gene.

[0113] “Complementary” as used herein to refer to a nucleic acid, refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds (“base pairing”) with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. More preferably, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.

[0114] “Antisense” refers particularly to the nucleic acid sequence of the non-coding strand of a double stranded DNA molecule encoding a protein, or to a sequence which is substantially homologous to the non-coding strand. As defined herein, an antisense sequence is complementary to the sequence of a double stranded DNA molecule encoding a protein. It is not necessary that the antisense sequence be complementary solely to the coding portion of the coding strand of the DNA molecule. The antisense sequence may be complementary to regulatory sequences specified on the coding strand of a DNA molecule encoding a protein, which regulatory sequences control expression of the coding sequences.

[0115] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0116] “Variant” as the term is used herein, is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence respectively, but retains essential biological properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide can differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A variant of a nucleic acid or peptide can be a naturally occurring such as an allelic variant, or can be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis.

[0117] The term “antibody,” as used herein, refers to an immunoglobulin molecule which specifically binds with an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The an antibody in the present invention may exist in a variety of forms where the antigen binding portion of the antibody is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv) and a humanized antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0118] The term “antibody fragment” refers to at least one portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, sdAb (either VL or Vn), camelid Vim domains, scFv antibodies, and multi-specific antibodies formed from antibody fragments. The term “scFv” refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it was derived. Unless specified, as used herein an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N- terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-Vn or may comprise Vu-linker-VL.

[0119] As used herein, “conjugated” refers to covalent attachment of one molecule to a second molecule.

[0120] By the term “specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific.

[0121] In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

[0122] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0123] Compounds

[0124] In one embodiment, the composition of the invention comprises a compound represented by General Formula I: wherein ring A represents a ring selected from the group consisting of substituted or unsubstituted: aryl, cycloalkyl, cycloalkenyl, heteroaryl, and heterocyclyl;

[0125] L represents a single bond or a linking group selected from the group consisting of alkylene, cycloalkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, silyl, amine, amide, ester, ether, carbonyl, carbamate, sulfamate, thioether, thioester, disulfide, hydrazine, urea, thiourea, phosphate, phosphonate ester, poly(alkyl ether), heteroatom, and combinations thereof; and any two adjacent substituents may optionally join to form a ring.

[0126] The ring A may comprise any cyclic moiety known in the art, including, but not limited to, 5-10 membered rings and fused rings, such as five-membered rings, six-membered rings, seven-membered rings. The ring A may be partially or completely aliphatic or may comprise at least one, two, three, four, five, or six double bonds. The ring A may represent any 5-10 membered aryl, cycloalkyl, cycloalkenyl, heteroaryl, or heterocyclyl ring, and may comprise heteroatoms, such as N, S, or O. In any example, the ring A may be further substituted, and the substituents can optionally join to form fused rings. Any two substituents on ring A may join to form a ring to provide fused rings, spirocycles, bicycles, or combinations thereof. The fused rings may also be selected from 5-10 membered aryl, cycloalkyl, cycloalkenyl, heteroaryl, or heterocyclyl ring.

[0127] Exemplary rings which may be represented by A include, but are not limited to, phenyl, benzyl, pyridine, piperidine, pyran, thiopyran, diazines, triazines, oxazines, thiazines, pyrimidine, quinoline, purine, chroman, pyridazine, acridine, isoquinoline, phenanthridine, furan, thiophene, pyrrole, pyrrolidine, naphthalene, anthracene, pyrene, and the like.

[0128] In one embodiment, the compound is represented by General Formula I: wherein R1, R2, R3, R4, and R5are each independently selected from the group consisting of hydrogen, deuterium, Ci-6 alkyl, C2-6 alkenyl, alkynyl, hydroxy, thio, C1-6 alkoxy, halo, haloCi-6 alkyl, haloCi-6 alkoxy, C3-8 cycloalkyl, cyano, amino, nitro, carbonyl, carboxylate, C1-6 alkyl ester, and combinations thereof.

[0129] In one embodiment, L represents a single bond, wherein the carbonyl carbon is directly bonded to the phenyl. In one embodiment, L is selected from the group consisting of CRL1RL2, NRL1, O, and combinations thereof, and RL1and RL2are each independently selected from hydrogen, deuterium, C1-6 alkyl, C2-6 alkenyl, alkynyl, hydroxy, thio, C1-6 alkoxy, halo, haloCi-6 alkyl, haloCi-6 alkoxy, C3-8 cycloalkyl, cyano, amino, and combinations thereof. In some examples, L is CRL1RL2. In some examples, L is CRL1RL2, wherein at least one of RL1and RL2is alkyl.

[0130] In one embodiment, R1, R2, R3, R4, and R5are each independently selected from the group consisting of carboxyl, amino, methoxy, cyano, fluoro, chloro, bromo, methyl, trifluoro, methoxycarbonyl, and nitro. In one embodiment, R3is not hydrogen.

[0131] In one embodiment, the compound is represented by General Formula la:

[0132] In one embodiment, at least one of RL1and RL2is alkyl. In one embodiment, at least one of RL1and RL2is methyl. In one embodiment, both RL1and RL2are each methyl.

[0133] In one embodiment, the compound is selected from the group consisting of following structures:

[0134]

[0135] Carbonic Anhydrase Inhibitors In various embodiments, the present invention includes compositions for inhibiting carbonic anhydrases (CA). In some embodiments, the carbonic anhydrase is carbonic anhydrase V (CA-V). In some embodiments, the carbonic anhydrase is mitochondrial carbonic anhydrase. In some embodiments, the carbonic anhydrase is mitochondrial carbonic anhydrase V. In some embodiments, the carbonic anhydrase is one or more of carbonic anhydrase VA (CA- VA) and carbonic anhydrase VB (CA-VB). In some embodiments, the carbonic anhydrase is CA-VA. In some embodiments, the carbonic anhydrase is CA-VB. In some embodiments, the carbonic anhydrase inhibitor is a mitochondrial CA-V inhibitor. In some embodiments, the CA- V inhibitor selectively inhibits at least one or more of CA-VA and CA-VB. In some embodiments, the CA-V inhibitor selectively inhibits CA-VA. In some embodiments, the CA-V inhibitor selectively inhibits CA-VB. In some embodiments, the CA-V inhibitor selectively inhibits at least one or more of mitochondrial CA-VA and mitochondrial CA-VB. In some embodiments, the CA-V inhibitor selectively inhibits mitochondrial CA-VA. In some embodiments, the CA-V inhibitor selectively inhibits mitochondrial CA-VB. In some embodiments, the CA-V inhibitor is 4-phenylacetamidomethyl-benzenesulfonamide (4ITP).

[0136] In various embodiments, the present invention includes compositions comprising at least one compound represented by General Formula I. In some embodiments, the CA-V inhibitor is a compound represented by General Formula I.

[0137] Inhibition of a gene, or gene product, can be assessed using a wide variety of methods, including those disclosed herein, as well as methods known in the art or to be developed in the future. That is, the routineer would appreciate, based upon the disclosure provided herein, that decreasing the level or activity of a gene, or gene product, can be readily assessed using methods that assess the level of a nucleic acid encoding a gene product (e.g., mRNA), the level of polypeptide gene product present in a biological sample, the activity of polypeptide gene product present in a biological sample, or combinations thereof. In some embodiments, the gene or gene product is CA-V. In some embodiments, the gene or gene product is CA-VA and CA-VB. In some embodiments, the gene or gene product is CA-VA. In some embodiments, the gene or gene product is CA-VB.

[0138] The inhibitor compositions and methods of the invention that decrease the level or activity of a gene, or gene product, include, but should not be construed as being limited to, a chemical compound, a protein, a peptide, a peptidomimetic, an antibody, a ribozyme, a small molecule chemical compound, a nucleic acid, a vector, an antisense nucleic acid molecule (e.g., siRNA, miRNA, etc ), a guide RNA, or combinations thereof. One of skill in the art would readily appreciate, based on the disclosure provided herein, that an inhibitor composition encompasses a chemical compound that decreases the level or activity of a gene, or gene product. Additionally, an inhibitor composition encompasses a chemically modified compound, and derivatives, as is well known to one of skill in the chemical arts. In some embodiments, the gene or gene product is CA-V. In some embodiments, the gene or gene product is CA-VA and CA-VB. In some embodiments, the gene or gene product is CA-VA. In some embodiments, the gene or gene product is CA-VB. In some embodiments, the CA V inhibitor is a chemical compound. In some embodiments, the CA-V inhibitor is 4-phenylacetamidomethyl- benzenesulfonamide (4ITP). In some embodiments, the CA-V inhibitor is a compound represented by General Formula I. In some embodiments, the inhibitor composition of the present invention is an antagonist, which decreases the expression, activity, or biological function of a gene or gene product. For example, in certain embodiments, the inhibitor of the present invention is an antagonist of CA-V. In other embodiments, the inhibitor of the present invention is an antagonist of CA-VA and CA-VB. In other embodiments, the inhibitor of the present invention is an antagonist of CA-VA. In other embodiments, the inhibitor of the present invention is an antagonist of CA-VB.

[0139] Further, one of skill in the art would, when equipped with this disclosure and the methods exemplified herein, appreciate that inhibitors include such inhibitors as discovered in the future, as can be identified by well-known criteria in the art of pharmacology, such as the physiological results of modulation of the genes, and gene products, as described in detail herein and / or as known in the art. Therefore, the present invention is not limited in any way to any particular inhibitor composition as exemplified or disclosed herein; rather, the invention encompasses those inhibitor compositions that would be understood by the routineer to be useful as are known in the art and as are discovered in the future.

[0140] Further methods of identifying and producing inhibitor compositions are well known to those of ordinary skill in the art. Alternatively, an inhibitor can be synthesized chemically. Further, the routineer would appreciate, based upon the teachings provided herein, that an inhibitor composition can be obtained from a recombinant organism. Compositions and methods for chemically synthesizing inhibitors and for obtaining them from natural sources are well known in the art and are described in the art.

[0141] One of skill in the art will appreciate that an inhibitor can be administered as a small molecule chemical, a polypeptide, a peptide, an antibody, a nucleic acid construct encoding a protein, an antisense nucleic acid, a nucleic acid construct encoding an antisense nucleic acid, or combinations thereof. Numerous vectors and other compositions and methods are well known for administering a protein or a nucleic acid construct encoding a protein to cells or tissues. Therefore, the invention includes a peptide or a nucleic acid encoding a peptide that is an inhibitor of a gene, or gene product.

[0142] In some embodiments, the inhibitors of the invention results in a decrease in expression of CA-V, CA-VA, CA-VB, or a combination thereof; including transcription, translation, or both. In some embodiments, the inhibitors of the invention results in a decrease in at least one activity of CA-V, CA-VA, CA-VB, or a combination thereof. Thus, decreasing the level or activity of CA-V, CA-VA, CA-VB, or a combination thereof includes, but is not limited to, decreasing the amount of CA-V, CA-VA, CA-VB, or a combination thereof; decreasing transcription, translation, or both, of a nucleic acid encoding CA-V, CA-VA, CA-VB, or a combination thereof; and it also includes decreasing any activity of CA-V, CA-VA, CA-VB, or a combination thereof polypeptide as well.

[0143] Similarly, the expression of a gene may be inhibited by the hybridization of an antisense molecule to a promoter or other regulatory element of a gene, thereby affecting the transcription of the gene. Methods for the identification of a promoter or other regulatory element that interacts with a gene of interest are well known in the art and include such methods as the yeast two hybrid system (Bartel and Fields, eds., In: The Yeast Two Hybrid System, Oxford University Press, Cary, N.C.).

[0144] Alternatively, inhibition of a gene expressing a protein that diminishes the level or activity of CA-V, CA-VA, CA-VB, or a combination thereof can be accomplished through the use of an siRNA, shRNA, guide RNA, antisense oligonucleotide or ribozyme. Given the nucleotide sequence of the molecule, one of ordinary skill in the art could synthesize an antisense oligonucleotide or ribozyme without undue experimentation, provided with the disclosure and references incorporated herein.

[0145] Nucleic Acids

[0146] In one embodiment, the composition of the invention comprises one or more antisense nucleic acid molecules. For example, in one embodiment, the one or more antisense nucleic acid molecules are specific for targeting CA-V, fragment, or a variant thereof. In one embodiment, the one or more antisense nucleic acid molecules are specific for targeting CA-VA, fragment, or a variant thereof. In one embodiment, the one or more antisense nucleic acid molecules are specific for targeting CA-VB, fragment, or a variant thereof. Antisense oligonucleotides are DNA or RNA molecules that are complementary to some portion of a mRNA or IncRNA molecule. When present in a cell, antisense oligonucleotides hybridize to an existing mRNA or IncRNA molecule and inhibit translation into a gene product or promote degradation of the RNA molecule. Inhibiting the expression of a gene using an antisense oligonucleotide is well known in the art (Marcus-Sekura, 1988, Anal. Biochem. 172:289), as are methods of expressing an antisense oligonucleotide in a cell (Inoue, U.S. Pat. No. 5,190,931). The methods of the invention include the use of antisense oligonucleotide to diminish the amount of CA-V activity, CA-VA activity, CA-VB activity, or the activity of a fragment or variant thereof. Contemplated in the present invention are antisense oligonucleotides that are synthesized and provided to the cell by way of methods well known to those of ordinary skill in the art. As an example, an antisense oligonucleotide can be synthesized to be between about 10 and about 100, more preferably between about 15 and about 50 nucleotides long. The synthesis of nucleic acid molecules is well known in the art, as is the synthesis of modified antisense oligonucleotides to improve biological activity in comparison to unmodified antisense oligonucleotides (Tullis, 1991, U.S. Pat. No. 5,023,243).

[0147] Similarly, the expression of a gene may be inhibited by the hybridization of an antisense molecule to a promoter or other regulatory element of a gene, thereby affecting the transcription of the gene. Methods for the identification of a promoter or other regulatory element that interacts with a gene of interest are well known in the art, and include such methods as the yeast two hybrid system (Bartel and Fields, eds., In: The Yeast Two Hybrid System, Oxford University Press, Cary, N.C.).

[0148] Alternatively, inhibition of CA-V, CA-VA, CA-VB, or a fragment or variant thereof can be accomplished through the use of an siRNA, shRNA, antisense oligonucleotide or ribozyme. Given the nucleotide sequence of the molecule, one of ordinary skill in the art could synthesize an antisense oligonucleotide or ribozyme without undue experimentation, provided with the disclosure and references incorporated herein.

[0149] In one embodiment, siRNA is used to decrease the level of at least one of CA-V, CA-VA, CA-VB, a fragment thereof, or a variant thereof. RNA interference (RNAi) is a phenomenon in which the introduction of double-stranded RNA (dsRNA) into a diverse range of organisms and cell types causes degradation of the complementary mRNA. In the cell, long dsRNAs are cleaved into short 21-25 nucleotide small interfering RNAs, or siRNAs, by a ribonuclease known as Dicer. The siRNAs subsequently assemble with protein components into an RNA-induced silencing complex (RISC), unwinding in the process. Activated RISC then binds to complementary transcript by base pairing interactions between the siRNA antisense strand and the mRNA. The bound mRNA is cleaved and sequence specific degradation of mRNA results in gene silencing. See, for example, U.S. Patent No. 6,506,559; Fire et al., 1998, Nature 391 (19):306-311 ; Timmons et al., 1998, Nature 395:854; Montgomery et al., 1998, TIG 14 (7):255-258; David R. Engelke, Ed., RNA Interference (RNAi) Nuts & Bolts of RNAi Technology, DNA Press, Eagleville, PA (2003); and Gregory J. Hannon, Ed., RNAi A Guide to Gene Silencing, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2003). Soutschek et al. (2004, Nature 432:173-178) describe a chemical modification to siRNAs that aids in systemic delivery. Optimizing siRNAs involves consideration of overall G / C content, C / T content at the termini, Tm and the nucleotide content of the 3’ overhang. See, for instance, Schwartz et al., 2003, Cell, 115: 199-208 and Khvorova et al., 2003, Cell 115:209-216.

[0150] Therefore, the present invention also includes methods of decreasing levels of at least one of CA- V, CA-VB, and a variant thereof at the protein level using RNAi technology.

[0151] In certain embodiments, the inhibitors described herein comprise short hairpin RNA (shRNA) molecules. shRNA molecules are well known in the art and are directed against the mRNA of a target, thereby decreasing the expression of the target. In certain embodiments, the encoded shRNA is expressed by a cell, and is then processed into siRNA. For example, in certain instances, the cell possesses native enzymes (e.g., dicer) that cleaves the shRNA to form siRNA.

[0152] In other related aspects, the invention includes an isolated nucleic acid encoding an inhibitor, wherein an inhibitor such as an siRNA, shRNA, GapmeR or antisense molecule, inhibits at least one of CA-V, CA-VA, CA-VB, a fragment thereof, a variant thereof, a derivative thereof, a regulator thereof, and a downstream effector thereof.

[0153] In one embodiment, the antisense molecule is a GapmeR specific for CA-V, CAVA, CA-VB, or a fragment or a variant thereof. GapmeR molecules typically have a central stretch of chemically modified DNA “gap” flanked by locked nucleic acids (LNA), which increase the binding affinity of GapmeR to the target RNA. In addition to specificity and highly efficient gene silencing, GapmeRs can easily internalize into target cells.

[0154] Antisense molecules of the invention may be made synthetically and then provided to the cell. Antisense oligomers of between about 10 to about 30, and more preferably about 15 nucleotides, are preferred, since they are easily synthesized and introduced into a target cell. Synthetic antisense molecules contemplated by the invention include oligonucleotide derivatives known in the art which have improved biological activity compared to unmodified oligonucleotides (see U.S. Patent No. 5,023,243). Ribozymes and their use for inhibiting gene expression are also well known in the art (see, e.g., Cech et al., 1992, J. Biol. Chem. 267: 17479-17482; Hampel et al., 1989, Biochemistry 28:4929-4933; Eckstein et al., International Publication No. WO 92 / 07065; Altman et al., U.S. Patent No. 5,168,053). Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases. Through the modification of nucleotide sequences encoding these RNAs, molecules can be engineered to recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, 1988, J. Amer. Med. Assn. 260:3030). A major advantage of this approach is the fact that ribozymes are sequence-specific.

[0155] There are two basic types of ribozymes, namely, tetrahymena-type (Hasselhoff, 1988, Nature 334:585) and hammerhead-type. Tetrahymena-type ribozymes recognize sequences which are four bases in length, while hammerhead-type ribozymes recognize base sequences I lls bases in length. The longer the sequence, the greater the likelihood that the sequence will occur exclusively in the target mRNA species. Consequently, hammerhead-type ribozymes are preferable to tetrahymena-type ribozymes for inactivating specific mRNA species, and 18-base recognition sequences are preferable to shorter recognition sequences which may occur randomly within various unrelated mRNA molecules.

[0156] In one embodiment of the invention, a ribozyme is used to inhibit at least one of CA-V, CA-VA, CA-VB, and a fragment or a variant thereof. Ribozymes useful for inhibiting the expression of a target molecule may be designed by incorporating target sequences into the basic ribozyme structure which are complementary, for example, to the mRNA sequence of at least one of CA-V, CA-VA, CA-VB, and a fragment or a variant thereof of the present invention. Ribozymes targeting at least one of CA-V, CA-VA, CA-VB, and a fragment or a variant thereof may be synthesized using commercially available reagents (Applied Biosystems, Inc., Foster City, CA) or they may be genetically expressed from DNA encoding them.

[0157] In one embodiment, the inhibitor of at least one of CA-V, CA-VA, CA-VB, and a fragment or a variant thereof may comprise one or more components of a CRISPR-Cas system, where a guide RNA (gRNA) targeted to a gene encoding at least one of CA-V, CA-VA, CA-VB, and a fragment or a variant thereof, and a CRISPR-associated (Cas) peptide form a complex to induce mutations within the targeted gene. In one embodiment, the inhibitor comprises a gRNA or a nucleic acid molecule encoding a gRNA. In one embodiment, the inhibitor comprises a Cas peptide or a nucleic acid molecule encoding a Cas peptide.

[0158] When the inhibitor of the invention is a small molecule, a small molecule antagonist may be obtained using standard methods known to the skilled artisan. Such methods include chemical organic synthesis or biological means. Biological means include purification from a biological source, recombinant synthesis and in vitro translation systems, using methods well known in the art.

[0159] Combinatorial libraries of molecularly diverse chemical compounds potentially useful in treating a variety of diseases and conditions are well known in the art as are method of making the libraries. The method may use a variety of techniques well-known to the skilled artisan including solid phase synthesis, solution methods, parallel synthesis of single compounds, synthesis of chemical mixtures, rigid core structures, flexible linear sequences, deconvolution strategies, tagging techniques, and generating unbiased molecular landscapes for lead discovery vs. biased structures for lead development.

[0160] In a general method for small library synthesis, an activated core molecule is condensed with a number of building blocks, resulting in a combinatorial library of covalently linked, core-building block ensembles. The shape and rigidity of the core determines the orientation of the building blocks in shape space. The libraries can be biased by changing the core, linkage, or building blocks to target a characterized biological structure (“focused libraries”) or synthesized with less structural bias using flexible cores.

[0161] In other related aspects, the invention includes an isolated peptide inhibitor that inhibits at least one of CA-V, CA-VA, CA-VB, a fragment thereof, a variant thereof, or a combination thereof. For example, in one embodiment, the peptide inhibitor of the invention inhibits at least one of CA-V, CA-VA, CA-VB, a fragment thereof, a variant thereof, or a combination thereof directly by binding to at least one of CA-V, CA-VA, CA-VB, a fragment thereof, and a variant thereof thereby preventing the normal functional activity of at least one of CA-V, CA-VA, CA-VB, a fragment thereof, and a variant thereof. In another embodiment, the peptide inhibitor of the invention inhibits at least one of CA-V, CA-VA, CA-VB, a fragment thereof, and a variant thereof by competing with at least one endogenous of CA-V, CA-VA, CA- VB, a fragment thereof, and a variant thereof (e.g., inhibiting the interaction of at least one of CA-V, CA-VA, CA-VB, a fragment thereof, or a variant thereof). In yet another embodiment, the peptide inhibitor of the invention inhibits the activity of at least one of CA-V, CA-VA, CA- VB, a fragment thereof, and a variant thereof by acting as a transdominant negative mutant.

[0162] The inhibitors useful in the methods of the invention can be administered in combination with other known treatments. Such combination treatments may involve the use of pharmaceutical compositions comprising several active ingredients.

[0163] Pharmaceutical Compositions and Formulations

[0164] The invention also encompasses the use of pharmaceutical compositions of the invention or salts thereof to practice the methods of the invention. Such a pharmaceutical composition may consist of at least one inhibitor composition of the invention or a salt thereof in a form suitable for administration to a subject, or the pharmaceutical composition may comprise at least one inhibitor composition of the invention or a salt thereof, and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The compound or conjugate of the invention may be present in the pharmaceutical composition in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

[0165] In some embodiments, the pharmaceutical compositions useful for practicing the methods of the invention may be administered to deliver a dose of between 1 ng / kg / day and 100 mg / kg / day. In another embodiment, the pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of between 1 ng / kg / day and 500 mg / kg / day.

[0166] The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w / w) active ingredient.

[0167] Pharmaceutical compositions that are useful in the methods of the invention may be suitably developed for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ophthalmic, or another route of administration. A composition useful within the methods of the invention may be directly administered to the skin, vagina or any other tissue of a mammal. Other contemplated formulations include liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations. The route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human subject being treated, and the like.

[0168] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

[0169] As used herein, a “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.

[0170] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions that are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist may design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

[0171] In some embodiments, the compositions of the invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In some embodiments, the pharmaceutical compositions of the invention comprise a therapeutically effective amount of a compound or conjugate of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers that are useful, include, but are not limited to, glycerol, water, saline, ethanol and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington’s Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

[0172] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may 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 may 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, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin. In some embodiments, the pharmaceutically acceptable carrier is not DMSO alone.

[0173] Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, vaginal, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and / or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.

[0174] As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fdlers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” that may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Genaro, ed. (1985, Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

[0175] The composition of the invention may comprise a preservative from about 0.005% to 2.0% by total weight of the composition. The preservative is used to prevent spoilage in the case of exposure to contaminants in the environment. Examples of preservatives useful in accordance with the invention included but are not limited to those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea and combinations thereof. A particularly preferred preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.

[0176] The composition preferably includes an anti-oxidant and a chelating agent that inhibits the degradation of the compound. Preferred antioxidants for some compounds are BHT, BHA, alpha-tocopherol and ascorbic acid in the preferred range of about 0.01% to 0.3% and more preferably BHT in the range of 0.03% to 0.1% by weight by total weight of the composition. Preferably, the chelating agent is present in an amount of from 0.01% to 0.5% by weight by total weight of the composition. Particularly preferred chelating agents include edetate salts (e g. disodium edetate) and citric acid in the weight range of about 0.01% to 0.20% and more preferably in the range of 0.02% to 0.10% by weight by total weight of the composition. The chelating agent is useful for chelating metal ions in the composition that may be detrimental to the shelf life of the formulation. While BHT and disodium edetate are the particularly preferred antioxidant and chelating agent respectively for some compounds, other suitable and equivalent antioxidants and chelating agents may be substituted therefore as would be known to those skilled in the art.

[0177] Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water, and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose. Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin, and acacia. Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl-para- hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.

[0178] Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent. As used herein, an “oily” liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water. Liquid solutions of the pharmaceutical composition of the invention may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water, and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.

[0179] Powdered and granular formulations of a pharmaceutical preparation of the invention may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations. A pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.

[0180] Methods for impregnating or coating a material with a chemical composition are known in the art, and include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (i.e., such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying.

[0181] The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the subject either prior to or after a diagnosis of disease. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0182] Administration of the compositions of the present invention to a subject, preferably a mammal, more preferably a human, may be carried out using known procedures, at dosages and for periods of time effective to prevent or treat disease. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular compound employed; the time of administration; the rate of excretion of the compound; the duration of the treatment; other drugs, compounds or materials used in combination with the compound; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A nonlimiting example of an effective dose range for a therapeutic compound of the invention is from about 1 and 5,000 mg / kg of body weight / per day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.

[0183] The compound may be administered to a subject as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc.

[0184] Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.

[0185] A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0186] In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding / formulating such a therapeutic compound for the treatment of a disease in a subject.

[0187] In some embodiments, the compositions of the invention are administered to the subject in dosages that range from one to five times per day or more. In another embodiment, the compositions of the invention are administered to the subject in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. It will be readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the invention will vary from subject to subject depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the invention should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any subject will be determined by the attending physical taking all other factors about the subject into account.

[0188] Compounds of the invention for administration may be in the range of from about 1 mg to about 10,000 mg, about 20 mg to about 9,500 mg, about 40 mg to about 9,000 mg, about 75 mg to about 8,500 mg, about 150 mg to about 7,500 mg, about 200 mg to about 7,000 mg, about 3050 mg to about 6,000 mg, about 500 mg to about 5,000 mg, about 750 mg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 50 mg to about 1,000 mg, about 75 mg to about 900 mg, about 100 mg to about 800 mg, about 250 mg to about 750 mg, about 300 mg to about 600 mg, about 400 mg to about 500 mg, and any and all whole or partial increments therebetween.

[0189] In some embodiments, the dose of a compound of the invention is from about 1 mg and about 2,500 mg. In some embodiments, a dose of a compound of the invention used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound (i.e., a drug used for treating the same or another disease as that treated by the compositions of the invention) as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.

[0190] In some embodiments, the present invention is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound or conjugate of the invention, alone or in combination with a second pharmaceutical agent; and instructions for using the compound or conjugate to treat, prevent, or reduce one or more symptoms of a disease in a subject.

[0191] The term “container” includes any receptacle for holding the pharmaceutical composition. For example, in some embodiments, the container is the packaging that contains the pharmaceutical composition. In other embodiments, the container is not the packaging that contains the pharmaceutical composition, i.e., the container is a receptacle, such as a box or vial that contains the packaged pharmaceutical composition or unpackaged pharmaceutical composition and the instructions for use of the pharmaceutical composition. Moreover, packaging techniques are well known in the art. It should be understood that the instructions for use of the pharmaceutical composition may be contained on the packaging containing the pharmaceutical composition, and as such the instructions form an increased functional relationship to the packaged product. However, it should be understood that the instructions may contain information pertaining to the compound’s ability to perform its intended function, e.g., treating or preventing a disease in a subject, or delivering an imaging or diagnostic agent to a subject.

[0192] Routes of administration of any of the compositions of the invention include oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal, and (trans)rectal), intravesical, intrapulmonary, intracerebral, epidural, intracerebroventricular, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration. In some embodiments, the composition can be administered to the cerebrospinal fluid of a subject.

[0193] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present invention are not limited to the particular formulations and compositions that are described herein.

[0194] Therapeutic Methods

[0195] In one embodiment, the invention provides methods of decreasing one or more of CA-V, CA-VA, and CA-VB activity or expression such that the decrease produces a therapeutic effect in a subject or a group of subjects in need. In one embodiment, the invention provides methods that produce a therapeutic effect in a subject or a group of subjects in need, the method comprising administering a composition comprising at least one compound represented by General Formula I. A therapeutic effect is one that results in an amelioration in the symptoms, or progression of a disease or disorder. In one embodiment, the disease or disorder is a neurological or psychiatric disease or disorder. In one embodiment, the disease or disorder is obesity.

[0196] Non-limiting examples of neurological or psychiatric disorders treatable by the methods of the invention include Alzheimer's disease (AD), Mild Cognitive Impairment (MCI), Parkinson's disease (PD), Huntington's disease (HD), prion-caused diseases, frontotemporal dementia (FTD), Lewy body dementia, vascular dementias, white matter disease, traumatic brain injury, post-traumatic stress, stroke, tauopaties, Down Syndrome, Amyotrophic Later Sclerosis (ALS), chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), multiple system atrophy (MSA), corticobasal degeneration (CBGD), Pick's disease, olivopontocerebellar atrophy (OPCA), senile dementia of the Alzheimer type, progressive supranuclear palsy (Steel- Richardson-Olszewski), corticodentatonigral degeneration, Hallervorden-Spatz disease, striatonigral degeneration, torsion dystonia (e.g., torsion spasm; dystonia musculorum deformans), spasmodic torticollis and other dyskinesis, familial tremor, Gilles de la Tourette syndrome, cerebellar cortical degeneration, spinocerebellar degeneration (e.g., Friedreich's ataxia and related disorders), Shy-Drager syndrome, spinal muscular atrophy, primary lateral sclerosis, hereditary spastic paraplegia, peroneal muscular atrophy (Charcot-Marie-Tooth), hypertrophic interstitial polyneuropathy (Dejerine-Sottas), chronic progressive neuropathy, pigmentary degeneration of the retina (retinitis pigmentosa), hereditary optic atrophy (Leber's disease), Cognitive Dysfunction Syndrome, White dog shaker syndrome, degenerative myelopathy, neuroaxonal dystrophy, cerebellar degeneration, cerebellar abiotrophy, cerebral amyloid angiopathy (CAA), and amyloid related imaging abnormalities (ARIA).

[0197] In one embodiment, the method comprises administering a composition described herein to a subject having, or having symptoms indicative of, a neurological or psychiatric disease or disorder. In one embodiment, the method comprises administering a composition described herein to a subject having or having symptoms indicative of a neurodegenerative disease. In one embodiment, the method comprises administering a composition described herein to a subject having or having symptoms indicative of Alzheimer’s disease.

[0198] It will be appreciated by one of skill in the art, when armed with the present disclosure including the methods detailed herein, that the invention is not limited to treatment of a neurological or psychiatric disease or disorder that is already established. Particularly, the disease or disorder need not have manifested to the point of detriment to the subject; indeed, the disease or disorder need not be detected in a subject before treatment is administered. That is, significant signs or symptoms of the disease or disorder do not have to occur before the present invention may provide benefit. Therefore, the present invention includes a method for preventing a neurological or psychiatric disease or disorder, in that an inhibitor composition, as discussed previously elsewhere herein, can be administered to a subject prior to the onset of the disease or disorder, thereby preventing the disease or disorder.

[0199] One of skill in the art, when armed with the disclosure herein, would appreciate that the prevention of a neurological or psychiatric disease or disorder, encompasses administering to a subject an inhibitor as a preventative measure against the development of, or progression of a neurological or psychiatric disease or disorder. As more fully discussed elsewhere herein, methods of decreasing the level or activity of a gene, or gene product, encompass a wide plethora of techniques for decreasing not only the level and activity of polypeptide gene products, but also for decreasing expression of a nucleic acid, including either transcription, translation, or both.

[0200] Additionally, as disclosed elsewhere herein, one skilled in the art would understand, once armed with the teaching provided herein, that the present invention encompasses methods of treating, or preventing, a wide variety of neurological or psychiatric disease or disorder, where decreasing the level or activity of a gene, or gene product treats or prevents the disease. Various methods for assessing whether a neurological or psychiatric disease or disorder are known in the art. Further, the invention encompasses treatment or prevention of such diseases discovered in the future.

[0201] In one embodiment, the method comprises the use of a carbonic anhydrase V inhibitor, at least one compound represented by General Formula I, or a combination thereof to reduce blood-brain barrier permeability. In one embodiment, the method comprises administering a composition described herein to a subject having, or having symptoms indicative of, a disease or disorder associated with blood-brain barrier permeability defects. In one embodiment, the method comprises administering a composition described herein to a subject having defective blood-brain barrier permeability -induced or defective blood-brain barrier permeability-associated neurological or psychiatric disorders.

[0202] In one embodiment, the method comprises the use of a carbonic anhydrase V inhibitor, at least one compound represented by General Formula I, or a combination thereof to reduce neuronal degeneration, glial degeneration, and vascular degeneration. In one embodiment, the method comprises administering a composition described herein to a subject having or having symptoms indicative of a disease or disorder associated with one or more of neuronal degeneration, glial degeneration, and vascular degeneration. In one embodiment, the method comprises administering a composition described herein to a subject having neuronal degeneration, glial degeneration, or vascular degeneration-induced or neuronal degeneration, glial degeneration, or vascular degeneration-associated neurological or psychiatric disorders. In one embodiment, the method comprises administering a composition described herein.

[0203] In one embodiment, the method comprises the use of a carbonic anhydrase V inhibitor, at least one compound represented by General Formula I, or a combination thereof to inhibiting gliosis, improving microglia clearance, or increasing CD68 expression in microglia. In one embodiment, the method comprises administering a composition described herein to a subject having or having symptoms indicative of a disease or disorder associated with one or more of gliosis, insoluble protein accumulation, or decreased CD68 expression. In one embodiment, the method comprises administering a composition described herein to a subject having gliosis, insoluble protein accumulation, or decreased CD68 expression-induced or gliosis, insoluble protein accumulation, or decreased CD68 expression-associated neurological or psychiatric disorders. In one embodiment, the method comprises administering a composition described herein.

[0204] In one embodiment, the method comprises the use of a carbonic anhydrase V inhibitor, at least one compound represented by General Formula I, or a combination thereof to reduce obesity. In one embodiment, the method comprises administering a composition described herein to a subject having, or having symptoms indicative of, obesity.

[0205] In one embodiment, the method comprises the use of a carbonic anhydrase V inhibitor, at least one compound represented by General Formula I, or a combination thereof to reduce one or more of mitochondrial dysfunction in a cell, caspase activation in a cell, Cytochrome C (CytC) release in a cell, insoluble protein accumulation in a cell, and cell death. In one embodiment, the method comprises administering a composition described herein to a subject having or having symptoms indicative of a disease or disorder associated with one or more of mitochondrial dysfunction in a cell, caspase activation in a cell, Cytochrome C (CytC) release in a cell, inhibiting insoluble protein accumulation in a cell, and cell death. In one embodiment, the cell is one or more selected from the group consisting of neuronal cells, glial cells, astrocyte, microglia, endothelial cells, and smooth muscle cells. In some embodiments, the neuronal cell is a dopaminergic neuron. In some embodiments, the glial cell is a microglia or astrocyte. In some embodiments, the endothelial cell is one or more selected from the group consisting of a cerebral endothelial cell and a microvascular endothelial cell. In one embodiment, the insoluble protein is one or more selected from the group consisting of amyloid-beta 40, amyloid-beta 42, and pTau231. In one embodiment, the caspase is one or more of caspase 3, caspase 7, and caspase 9. In one embodiment, the mitochondrial dysfunction is a loss of mitochondrial membrane potential. In one embodiment, the mitochondrial dysfunction is an increase in mitochondrial H2O2 production. In some embodiments, the cell is in a subject.

[0206] In one embodiment, the method comprises administering a composition described herein to a subject having mitochondrial dysfunction, caspase activation, or Cytochrome C (CytC) release-induced or mitochondrial dysfunction, caspase activation, or Cytochrome C (CytC) release-associated neurological or psychiatric disorders.

[0207] EXPERIMENTAL EXAMPLES

[0208] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0209] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore are not to be construed as limiting in any way the remainder of the disclosure.

[0210] Example 1 : Inhibition of Carbonic Anhydrase- VB rescues amyloid-beta induced cerebral endothelial apoptosis, loss of barrier integrity, and cognitive decline in Alzheimer’s model mice

[0211] The demise of the cerebral vessels, with decreased cerebral blood flow (CBF) and increased blood-brain barrier (BBB) permeability, is one of the earliest causal events in the pathogenesis of AD, which in turn affect other cells composing the neurovascular unit (NVU), such as glial and neuronal cells (Kelly, L., et al., Alzheimers Dement, 2023; Carey, A. and S. Fossati, Alzheimers Dement, 2023. 19(2):671-695; Carare, R.O., et al., Alzheimers Dement (Amst), 2020. 12(l):el2053; Sweeney, M.D., et al., Alzheimers Dement, 2019. 15(1): 158- 167; Parodi-Rullan, R., et al., J Alzheimers Dis, 2019. 72(4): 1019-1039). The NVU is a multi-cellular structure composed of cerebral endothelial cells (CEC), pericytes / smooth muscle cells, astrocytes, microglia, and neurons which all work together to execute many vital functions, including maintenance of the physical barrier between the blood and the brain, nutrient exchange, regulation of CBF, and immunological surveillance (Lemon, N., et al., Front Aging Neurosci, 2021. 13:772278; Canepa, E. et al., Front Neurol, 2020. 11 :573324).

[0212] Cerebral amyloid angiopathy (CAA), the accumulation of aggregated Ap around and within the brain vasculature, is observed in about 90% of AD patients (Malek-Ahmadi, M., et al., J Alzheimers Dis, 2020. 74(1 ): 189-197; Cortes-Canteli, M. et al., J Am Coll Cardiol, 2020. 75(8):942-951). Parallel to NVU dysfunction is the rise of neuroinflammatory processes, which exacerbate a vicious cycle of BBB permeability, glial cell activation, and eventually neurodegeneration (Parodi-Rullan, R.M., et al., Cells, 2021. 10(11)).

[0213] Aβ deposition at the cerebral vessels has been observed to cause and worsen NVU dysfunction, by affecting mechanisms such as mitochondrial metabolism, oxidative stress, cell death and neuroinflammation (Parodi-Rullan, R., et al., J Alzheimers Dis, 2019. 72(4): 1019- 1039; Parodi-Rullan, R.M., et al., Cells, 2021. 10(11)). Thus, improving cerebrovascular function is a crucial target for AD therapies.

[0214] In AD brains, Aβ40 is known to accumulate more on the vasculature, while Aβ42 deposits more in parenchymal plaques. Although most cases of CAA are sporadic, there are known familial mutations, such as the Dutch mutation- characterized by Aβ40 mutated in position 22 (AβQ22). The Dutch mutation, among others within the amyloid precursor protein (APP), clinically presents with severe Aβ deposition at the brain vasculature, causing early onset dementia, strokes, and cerebral hemorrhages (Levy, E., et al., J Alzheimers Dis, 2006. 9(3 Suppl):329-39). APQ22 has also a faster aggregation rate, compared to Ap40 wild-type (WT), correlating with its higher toxicity (Carey, A., et al., Aging Cell, 2024:el4106; Fossati, S., et al., Cell Death Dis, 2012. 3(6):e321; Parodi-Rullan, R., et al., Aging Cell, 2020. 19(1 l):el3258; Fossati, S., et al., FASEB J, 2010. 24(1):229-41).

[0215] It has been demonstrated that the FDA-approved, non-selective, pan-carbonic anhydrase (CA) inhibitors, Acetazolamide (ATZ) and Methazolamide (MTZ), are effective in ameliorating mitochondria-mediated cell death and stress mechanisms in models of amyloidosis (Fossati, S., et al., Neurobiol Dis, 2016. 86:29-40; Solesio, M.E., et al., Aging Cell, 2018. 17(4):el2787; Canepa, E., et al., Alzheimers Dement, 2023. 19(11):5048-5073; Anzovino, A., et al., Cells, 2023. 12(24)). In the TgSwDI model of CAA, ATZ and MTZ preserved vascular fitness and prevented gliosis (Canepa, E., et al., Alzheimers Dement, 2023. 19(11):5048-5073). There is also evidence that CA inhibitors are protective in models of stroke, obesity, and diabetes (Lemon, N., et al., Front Aging Neurosci, 2021. 13:772278; Supuran, C.T., J Enzyme Inhib Med Chem, 2022. 37(l):2478-2488; Salameh, T.S., et al., Fluids Barriers CNS, 2019. 16(1): 1). ATZ and MTZ prevent Ap-induced cell death, through both the extrinsic and intrinsic apoptotic pathways, in many cell types, including CEC, smooth muscle cells, glial cells, and neurons, in addition to diminishing mitochondrial oxidative stress (Fossati, S., et al., Neurobiol Dis, 2016. 86:29-40; Solesio, M.E., et al., Aging Cell, 2018. 17(4):el2787; Anzovino, A., et al., Cells, 2023. 12(24)). The intrinsic apoptotic pathway, also referred to as mitochondria-mediated apoptosis is regulated through signaling molecules such as BIM, BAX and BAK, and BCL-2. When BAX and BAK translocate to the outer mitochondrial membrane they form pores, increasing mitochondrial membrane permeability and promoting loss of mitochondrial membrane potential. The mitochondria then release cytochrome C into the cytosol, leading to the activation of caspase-9, which activates caspase-3, resulting in the execution of apoptosis, through DNA fragmentation.

[0216] CEC are the physical barrier responsible for the inhibition of entry of peripheral molecules, immune cells, or foreign substances into the healthy brain. When needed, they have a direct role in the recruitment of peripheral immune cells to the brain, as well as in the activation of glial cells within the brain during inflammation. Tight junction proteins (TJPs), such as occludin and claudin-5, are important regulators of BBB integrity. Although there are mixed reports on the expression of TJPs in models of AD, it is clear that the BBB becomes leaky due to AD and CAA pathology. ECs can become activated through the Aβ-mediated accumulation of reactive oxygen species (ROS), or other mechanisms (Parodi-Rullan, R.M., et al., Cells, 2021. 10(11)). When EC are activated, they release pro-inflammatory cytokines and increase the expression of immune cell adhesion proteins such as vascular cellular adhesion molecule- 1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), which promote the extravasation of immune cells form the periphery into the brain. When this immune response becomes chronic, it is detrimental to the brain tissue (Parodi-Rullan, R.M., et al., Cells, 2021. 10(11)); Munsterman, D., et al., Alzheimers Dement, 2024).

[0217] Carbonic Anhydrases (CA) are zinc metalloenzymes that catalyze the hydration of carbon dioxide to produce bicarbonate and a proton. This reaction is vital in many processes, such as the regulation of pH, ion, and redox homeostasis, as well as metabolism (Lemon, N., et al., Front Aging Neurosci, 2021. 13:772278; Dettori, I., et al., J Enzyme Inhib Med Chem, 2021. 36(l):964-976; Zamanova, S., et al., Expert Opin Ther Pat, 2019. 29(7): 509-533). There are 15 different CA isoforms expressed by humans, each one with its own tissue expression pattern, activity level, and cellular localization (Lemon, N., et al., Front Aging Neurosci, 2021. 13:772278; Supuran, C.T., Nat Rev Drug Discov, 2008. 7(2): 168-81; Zamanova, S., et al., Expert Opin Ther Pat, 2019. 29(7):509-533), highlighting their different functions. CA isoforms are expressed on the plasma membrane of cells, in the cytosol, and two are expressed in the mitochondria (CA-VA and CA-VB). Importantly, the two mitochondrial isoforms have a differential expression pattern, CA-VB being more abundant in the brain (Lemon, N., et al., Front Aging Neurosci, 2021. 13:772278; Aspatwar, A., et al., J Physiol, 2023. 601(2):257-274; Zamanova, S., et al., Expert Opin Ther Pat, 2019. 29(7):509-533).

[0218] The non-selective CA inhibitors ATZ and MTZ are FDA approved for other indications, such as glaucoma and high-altitude sickness. Although, pan-CA inhibitors are demonstrated to be protective \n vitro and in vivo, chronic long-term use is a concern due to their lack of selectivity and potential undesired effects (Lemon, N., et al., Front Aging Neurosci, 2021. 13:772278; Provensi, G., et al., Int J Mol Sci, 2019. 20(19)). ATZ and MTZ belong to the sulfonamide drug class, with established structures which have been modified and screened to determine their inhibitory activity on multiple CA isoforms (Supuran, C.T., Nat Rev Drug Discov, 2008. 7(2): 168-81). In a study evaluating the activity of benzene sulfonamide CA inhibitors, it was determined that the compound 4-phenylacetamidomethyl-benzenesulfonamide (4ITP) has strong selectivity for CA-V (Guzel-Akdemir, O., et al., Bioorg Med Chem, 2013. 21(21):6674-80; Guzel, O., et al., Bioorg Med Chem, 2009. 17(14):4894-9). 4ITP is considered highly selective for CA-VA and CA-VB, with a Ki value of 8.6 and 8.3 nM respectively. This is 10- fold more effective than ATZ and MTZ. 4ITP was determined to be much less effective on cytosolic isoforms CA-I and CA-II, and even less on plasma membrane isoforms CA-IX and CA-XII, emphasizing its selectivity for CA-V (Guzel-Akdemir, O., et al., Bioorg Med Chem, 2013. 21(21):6674-80).

[0219] Previous findings demonstrate that pan-CA inhibitors prevent CEC death through mitochondria-specific mechanisms and that CA-VB is increased in AD models and human AD brains. Hence, it was hypothesized that the selective inhibition of mitochondrial CA-V would protect from Ap induced CEC mitochondria-mediated apoptosis and NVU stress, therefore reducing cognitive impairment, while also decreasing the off-target undesired effects of pan- CAIs. In this study, this was investigated in CECs as well as the 3xTG mouse model of AD (Oddo, S., et al., Neuron, 2003. 39(3):409-21), which presents both Ap and tau pathology, in addition to vascular dysfunction (Quintana, D.D., et al., Neurobiol Aging, 2021. 105: 115-128). This example demonstrates that a known selective CA-V inhibitor, 4ITP, is effective at reducing Aβ-induced mitochondria-mediated CEC death, BBB dysfunction and cerebrovascular stress in vitro. It was also confirmed the specific role of CA-VB in AP-mediated CEC death and BBB dysfunction, through CRISPR Cas9 CA-VB knockout (KO) in human CEC. Moreover, the employment of this specific compound was pioneered in vivo, showing that it can prevent cognitive impairment in 3xTG mice, therefore bringing light to a novel potential target for AD therapy.

[0220] The materials and methods are described herein.

[0221] Cell Culture

[0222] Immortalized human cerebral microvascular endothelial cells (hCMEC / d3) were grown and maintained in endothelial basal medium (EBM-2) (Lonza CC-3156) supplemented with EGM-2 growth factors (Lonza CC-4176) and 5% fetal bovine serum (FBS).

[0223] CRISPR Cas9 mediated knockout cell pool of CA-VB in hCMEC / D3 cells were generated by EditCo Bio, Inc. (Redwood City, CA, USA). To generate these cells, Ribonucleoproteins containing the Cas9 protein and synthetic chemically modified guide RNA were electroporated into the cells using EditCo's optimized protocol. Editing efficiency is assessed upon recovery, 48 hours post electroporation. Genomic DNA is extracted from a portion of the cells, PCR amplified and sequenced using Sanger sequencing. The resulting chromatograms are processed using EditCo's Inference of CRISPR edits software. The wild-type cell line was generated similarly without gRNA specific for the gene of interest.

[0224] Upon receival of the cells and prior to experiments, CA-VB KO was confirmed via protein expression. Cells were grown and maintained in EBM-2, supplemented with EGM-2 growth factors and 5% FBS.

[0225] Cells were grown in a humidified cell culture incubator, under a 5% CO2 atmosphere at 37°C. WT hCMEC were routinely checked to ensure that they maintained their proper function and TJP expression, as CRISPR / Cas9 CA-VB KO to ensure CA-VB KO stability.

[0226] Synthesis and characterization of the CA V selective inhibitor 4ITP: The following materials were used as received: phenylacetic acid, thionyl chloride were from Sigma-Aldrich (St. Louis, MO), 4-aminomethylbenzenesulfonamide hydrochloride was from Oakwood Chemical (Estill, SC), N-methylmorpholine was from TCI America (Portland, OR). Organic solvents (HPLC quality) were purchased from Fisher Scientific (Pittsburgh, PA), EMD (Gibbstown, NJ), and VWR International (West Chester, PA).

[0227] The purity and the structure identity of the intermediary and final products were assessed by thin-layer chromatography (TLC), HPLC-MS,1H-,13C-NMR. TLC was carried out on SiO2-precoated aluminum plates (Alugram® SIL G / UV25420x20 cm with F254 indicator; layer thickness 0.20 nm; pore size 60 A) from MACHEREY-NAGEL, (Bethlehem, PA). Flash chromatography was performed using an Isco Combiflash RF (Teledyne Isco, Lincoln, NE), using Isco prepacked silica columns. The purity of compounds was also assessed via LC-MS, using an Agilent 1200 HPLC DAD-MS system (Santa Clara, CA) equipped with a G1315A DAD and a 6130 Quadrupole MS via a ZORBAX SB-C18 column eluted with H2O (0.1% HCOOH) / MeCN (0.1% HCOOH) 95 / 5 to 0 / 100 linear gradient. NMR spectra were recorded at T = 300 K with a Bruker Avance III 400 Plus spectrometer equipped with a 5 mm indirect detection probe, operating at 400 MHz for 'H NMR, at 100 MHz for13C NMR. Chemical shifts are reported as 5 values, using tetramethylsilane (TMS) as internal standard for proton spectra and the solvent resonance for carbon.

[0228] 4-(Aminomethyl)benzenesulfonamide hydrochloride (1 eq, 4.5 mmol, 1.00 g) was suspended in dry acetonitrile (18 mL) in a 50 mL round bottom flask and the flask was purged with argon. The mixture was cooled to 0°C using an ice bath, then 4-methylmorpholine (2.1 eq, 9.4 mmol, 0.95 g) was added under stirring. After 30 min, the ice bath was removed, and the reaction mixture was stirred overnight at room temperature. Separately, phenylacetic acid (1.05 eq, 4.73 mmol, 0.73 g) was treated with thionyl chloride (10 eq, 45 mmol, 5.35 g) and 1 drop of dimethylformamide in a 100 mL round bottom flask and the mixture was heated to reflux, under stirring, for Ih. Excess thionyl chloride was removed in vacuo using a rotary evaporator and the acid chloride was dissolved in dry acetonitrile (3 mL) and added dropwise to the previously made solution of 4-(aminomethyl)benzenesulfonamide, at 0°C (ice bath cooling). The reaction was stirred at 0°C for 30 min, then at room temperature overnight, when a precipitate was formed. The suspension was filtered and the precipitate was washed subsequently with acetone and water, dried under vacuum, and absorbed onto SiO2 using MeOH / CHCl3 The product was purified by flash chromatography on silica gel (0-40% McOH / CHCl3 gradients). The pure fractions were combined and evaporated to dryness to yield a while solid (55-71% (multiple batches made).

[0229] 4ITP: 'H-NMR (400 MHz, DMSO-d6, 6, ppm): 8.63 (t, J= 5.8 Hz, 1H, NH), 7.74 (d, J= 8.4 Hz, 2H, H2.6-PI1SO2NH2), 7.3 (d, J= 8.4 Hz, 2H, H3.5-PhSO2NH2), 7.20-7.35 (m, 5H, PhCH2CO), 7.29 (s, 2H, SO2NH2), 4.32 (d, J= 5.9 Hz, 2H, NHCH2). 3.49 (s, 2H, CH2CO).13C- NMR (100.6 MHz, DMSO-d6, 5, ppm): 170.2, 143.5, 142.5, 136.2, 128.9 (2C), 128.1 (2C), 127.3 (2C), 126.3, 125.5, 42.2, 41.7.

[0230] 4ITP: LC-MS (ESI) 305.1 (MH+), tR= 2.47 min, > 96% purity).

[0231] Determination of 4ITP solubility in water and PBS buffer.

[0232] The solubility of 4ITP in water and in PBS buffer was determined by the same HPLC method and instrument described above. A stock solution of 4ITP at a concentration of 10 mM were prepared in DMSO and serial dilutions (5 mM, 2.5 mM, 1.25 mM, 0.625 mM, 0.3125 mM and 0.156 mM) were done with the same solvent. A volume of 1 pL of these 4ITP solutions was injected in the instrument and the resulting peak areas were plotted against the corresponding concentrations to obtain a calibration curve, via the least squares linear regression analysis. 4ITP saturated solutions in deionized water and in PBS buffer were made by suspending 10 mg pure 4ITP in 1 mL water or in 1 mL PBS at pH 7.4 in two Eppendorf tubes, vortexing the suspensions for 1 min and subsequently placing them in a mini rotator (Labnet International, Edison, NJ) overnight. The next day the undissolved compound was centrifuged down using a bench centrifuge for 5 min and 10 pL of each supernatant was collected in an LCMS vial. A volume of 1 pL of these 4ITP solutions was injected in the HPLC instrument and the concentration of 4ITP in each solution was determined from the resulting peak areas via the previously constructed calibration curve. The analysis was done in triplicate and the values were reported as mean ± standard deviation.

[0233] 4ITP solubility in water: 0.057 ± 0.011 mg / mL (0.187 mM)

[0234] 4ITP solubility in PBS (pH = 7.4): 0.0399 ± 0.003 mg / mL (0.13 mM)

[0235] 4ITP lipophilicity (Log P) determination. Log P value of 4ITP was determined using the same HPLC method and instrument described above, using an injection volume of 1 gL and a flow rate of ImL / min in all cases. DMSO solutions (1 mg / mL) of reference compounds of known log P (indomethacin, caffeine, ketoprofen, phenytoin, testosterone) were injected into the HPLC system and the retention time of each compound was determined in the same RP Cl 8 column. A calibration curve was constructed by plotting the log P values of these reference compounds against their retention time (Veith, G.D., et al., Water Res, 1979, 13(l):43-47). Subsequently, 1 mg 4ITP was dissolved in 1 mL of DMSO and was injected in the same system to determine the retention time, which was interpolated within the calibration curve to determine the log P value of 4ITP.

[0236] 4ITP Log P Value: 1.69

[0237] 4ITP solubilization and delivery in vitro:

[0238] CA-V inhibitor, 4ITP, was dissolved in DMSO at a concentration of lOmM and stored at -20°C. On the day of treatment, the lOmM stock concentration was diluted to 2mM in EBM2 with 50%FBS. Finally, 4ITP was diluted to its final concentration (1, 10, or 30gM) in treatment medium, EBM2 with 1% FBS.

[0239] Aβ preparation

[0240] Aβ40-Q22 was synthesized by peptide 2.0. To obtain monomeric AP40-Q22, the peptide was weighed and dissolved in 1,1,1,3,3,3-169 hexafluoro-2-propanol (HFIP; Sigma, St. Louis, MI, USA), at a concentration of 1 mM, and incubated overnight at room temperature to dissolve beta-sheet structures. The day after, the peptide was flash-frozen in dry ice-cold 100% ethanol, then lyophilized until the pellet was dry, and stored at -20°C. To maintain the peptide in its monomeric form, the pellet was resuspended at lOmM in DMSO, and then diluted to ImM with sterile dH2O immediately before treatment. AP40-Q22 was brought to a final concentration of either 25 or lOpM in EBM2 with 1% FBS for treatment and added to the cells.

[0241] DNA fragmentation Apoptosis

[0242] Cells were seeded at a density of 20,000 cells / well in a 24-well plate 24 hours (h) prior to treatment in complete cell culture media. Following treatments, cells were centrifuged at 200xg for 10 minutes, and the appropriate lysis buffer was then added (Roche Applied Science). DNA fragmentation was measured using the Cell Death ELISA kit (Roche Applied Science), as recommended by the manufacturer. Absorbance was measured with SpectraMax I3x Multimode micro-plate reader (Molecular Devices) at wavelength 405 nm. Absorbance values were analyzed by determining the relative change to the control through the calculation of fold change.

[0243] Cell Viability Assay

[0244] Cells were seeded at a density of 10,000 cells / well in a 96-well plate in complete cell culture media 24h prior to treatment. Cells were treated in EBM-2 supplemented with 1% FBS plus the desired concentrations of AP40-Q22 and 4ITP. Cell viability was measured after 24h of treatment. WST-1 reagent (Roche, Applied Bioscience) was added directly to the cell culture media (1 :10 ratio), and then incubated for 3 hours at 37°C to allow for the colorimetric reaction. Absorbance was read with SpectraMax I3x at a wavelength of 450 nm.

[0245] Cell Event Caspase 3 '7

[0246] A day before treatment, 10,000 cells / well were seeded in a 96-well plate in complete cell culture media. Cells were treated in EBM-2 supplemented with 1% FBS and the experimental concentrations of AP40-Q22 and 4ITP. After 24h of treatment, the Cell Event Caspase 3 / 7 reagent (Thermo Fisher Scientific) was diluted in Hank’s balanced Salt Solution (HBSS) at a concentration of 5pM, added at a volume of I OOpl / well to the cells and incubated at 37°C for 30 minutes. The reagent was then removed and replaced with HBSS during acquisition. Pictures were acquired on an EVOS M5000 microscope at lOx magnification. Images were acquired with the brightfield and GFP channels to visualize the total number of cells and the caspase 3 / 7 activity, respectively. Three pictures per well were taken and then analyzed using Fiji / ImageJ software. The number of active caspase 3 / 7 positive cells was normalized to the total number of cells in each image and expressed as percentage of caspase 3 / 7 positive cells.

[0247] Western blot

[0248] In hCMEC, one day prior to treatment, cells were counted and seeded in a 6-well plate at a density of 350,000 cells / well. Following treatment, proteins were isolated from cells. Briefly, media was collected, and cells were washed with IX PBS and lysed with RIPA buffer (Invitrogen) + Halt protease / phosphatase inhibitors (Thermo Fisher Scientific). Cell debris were pelleted at 16,000xg for 20 minutes at 4°C. Supernatant was collected, and protein concentration was quantified with BCA method, and normalized to 25 pg in each sample.

[0249] For WB analysis, cell lysates, and homogenates from hippocampus and cortex of WT, 3xTG and 3xTG treated with 4ITP were prepared using BOLT 4x loading buffer and lOx reducing agent or Laemmli Buffer 6x (Invitrogen), depending on which gel was used. To further denature the samples, they were incubated for 5 minutes at 95°C.

[0250] Proteins were separated using 4-12% BOLT bis-tris SDS polyacrylamide gels (Invitrogen) or 10% CRITERION SDS polyacrylamide gels (BioRad) and transferred onto 0.45 pm nitrocellulose membrane using lx Towbin buffer containing 20% methanol, at 110 volts for 70 minutes. After transfer, membranes were blocked with 5% milk in TBS. Primary antibodies, Caspase-9 (1 :500 ab202068), Bax (1 :500 Novus Biologicals NBP1-88682), Bcl-2 (1 :500 abl96495) , Bim (1 :500 ab32158), Bak (1 :250 abl04124), CA-VB (Novus Biologicals), Occludin (1 :250 Invitrogen 71-1500), Claudin-5 (1 :500 Invitrogen 352500), ICAM-1 (1 :500 Invitrogen MA5407), VCAM-1 (1 :500 abl34047), and actin (1 :3000 Millipore MAB1501) were incubated on the membrane O / N at 4°C or for 2h room temperature. Membranes were then washed with TBS-T (0.1% Tween) and incubated with the species-appropriate secondary antibody (Licor, 1 :20,000 in TBS) for Ih at room temperature. Images were acquired using LICOR Odyssey CLx Immunoblot imager and then analyzed with LICOR Image Studio Software.

[0251] Cytochrome C and Mito-tracker Staining

[0252] Prior to seeding, 8-well chamber glass slides were pre-coated with attachment factor (Cell Systems). For immunocytochemistry experiments, 15,000cells / well were seeded 24h prior to treatment. Cells were treated with 25pm A04O-Q22 with or without 4ITP. After 16h of treatment, Mito-tracker CMH2xROS (Invitrogen) was diluted in 1% FBS EBM2 (1 :2000) and added to the cells for 30 minutes at 37°C. This is a dye that only enters mitochondria with a healthy membrane potential. After live-cell staining was complete, cells were washed lx with PBS and fixed with 4% PFA for 15 minutes at 37°C, then washed again 3x with PBS for 5 minutes. Cells were then permeabilized with 0.2% Triton in PBS for lOmin at room temperature, followed by one hour of blocking with 3% BSA in PBS. Then, cells were incubated with CytochromeC-AlexaFlour488 antibody (BD Pharmigen 560263) for Ih at room temperature. Finally, to visualize the nucleus, DAPI mounting media (Southern-Biotech 0100-20) was used. Images were acquired using a Nikon Ti2-E fluorescence deconvolution microscope at lOOx resolution.

[0253] Mitochondrial H2O2 production

[0254] HCMEC were seeded at 2 million cells / well in 10cm dishes 24h prior to treatment, and then treated with AP40-Q22 with or without 4ITP. After 16h of treatment, mitochondria were isolated in an isotonic buffer including 75 mM sucrose, 10 mM Tris-HCl pH = 7.4, 2 mM EGTA and 225 mM Mannitol (STEM) buffer (Solesio, M.E., et al., Aging Cell, 2018. 17(4):e 12787). The mitochondrial fraction was quantified using BCA assay. H2O2 was measured with an Amplex red assay (Invitrogen). Levels of H2O2 were measured by absorbance detected at 560 nm, normalized to protein quantification of the mitochondria fraction, and then expressed as fold of change to the control.

[0255] Lipid peroxidation 4-HNE ELISA

[0256] The levels of 4-Hydroxynonenal (4-HNE) in total cell lysates were measured with a commercially available ELISA kit (Abeam 238538) as per the manufacturer’s instructions. Protein samples were collected after 16h of treatment and then flash frozen with dry ice. The levels of 4-HNE were calculated through a standard curve, detected with Spectra I3x Max at a wavelength 450 nm. Concentrations were further converted to %change of control.

[0257] Measurement of barrier resistance by ECIS Z6

[0258] Trans-endothelial electrical resistance was measured using the ECIS Z0 system (Applied Biophysics). All experiments were performed on 8-well ECIS (8WE10+, Applied Biophysics) 40-electrodes-gold plated arrays, pre-coated per the manufacturer’s instructions. Cells were seeded at a density of 200,000 cells / well, and given 48h to form a monolayer, visualized on the recorded plot as a plateau in Resistance over time. The frequency applied to the cells was 4000 Hz. At this point, cells were treated with lOpM AP40-Q22, with or without 4ITP, or 4ITP alone. The resistance was measured for 48h after treatment. Data were expressed as normalized to the untreated control, over a 48-hour period. Meso-Scale Discovery Cytokine analysis

[0259] Cytokine release was evaluated in conditioned media from untreated hCMEC, and Q22-treated cells with or without 4ITP. Conditioned media (900JJ,1) was collected and then centrifuged at 800 x g for 5 minutes at 4°C. CM was then flash frozen and stored at -80 for no longer than 1 month. MesoScaleDiscovery (MSD) V-PLEX proinflammatory cytokine panel 1 was used to evaluate 10 cytokines (IL-ip, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, TNFα, and IFN-y). The technique employs an electrochemical detection method which produces a specific signal in order to evaluate all 10 cytokines in one sample / well. The amount of cytokine was calculated using a standard curve, normalized to the amount of protein in the cell sample, and then further converted to FOC of the untreated control.

[0260] Mouse model and treatment

[0261] The 3xTG AD mouse model was used to evaluate the protective effects of CA-V inhibition in vivo. The 3xTG mouse model carries human Amyloid Precursor Protein (APP) Swedish, Presenilin 1 (PSEN1 M146V) and tau (MAPT P301L) transgenes under the Thyl.2 neuronal promoter, and thus presents both Ap and tau accumulation within the brain, particularly in the hippocampus and cortex, with reports of Ap accumulation within the vasculature (Fu, L., et al., Brain Pathol, 2020. 30( 1 ) :92- 105). Mice are anticipated to present with mild amyloidosis around 6 months of age, while tau accumulation is reported later, around 12 months of age (Oddo, S., et al., Neuron, 2003. 39(3):409-21). Recent studies have demonstrated that vascular abnormalities (Quintana, D.D., et al., Neurobiol Aging, 2021. 105:115-128), metabolic deficits, and chronic inflammation may occur in these mice prior to protein accumulation, particularly more in females than males, translating more accurately to the human progression of disease (Quintana, D.D., et al., Neurobiol Aging, 2021. 105: 115-128; Javonillo, D.I., et al., Front Neurosci, 2021. 15:785276). WT mice (N=20), WT + 4ITP treated mice (N=l 1) 3xTG mice (N=21), and 3xTG + 4ITP (N=20) treated (males and females) mice were compared. 4ITP treatment in 3xTG and WT mice began at 6 months of age until 16 months. CA-V inhibitor, 4ITP, was provided in the mice’ diet at 20mg / kg / day, (100 ppm) in a grain-based diet. This was done in collaboration with bio-Serv. Food was stored in a cool dry place. Body weight and activity levels were monitored and recorded throughout the study. Following behavioral analysis, mice were anesthetized and perfused with ice-cold phosphate-buffered saline PBS, to obtain the brains, which were dissected and flash frozen.

[0262] For WB, hippocampus, and cortex tissue (WT, 3xTG, 3xTG + 4ITP N=6 / group, males and females) was homogenized in Tissue Homogenization Buffer (20mM Tris-Base, 0.25M sucrose, 5mM EDTA, ImM EGTA). Protein content of the homogenates was quantified with BCA and then normalized to 30pg of protein. WB samples were prepared with 6x Laemmli Buffer (Invitrogen).

[0263] Barnes Maze

[0264] When mice reached 15 months of age, spatial memory was evaluated through the Barnes Maze. The Barnes maze runs over the course of 7 days: day 0, habituation, days 1-5 training and day 6 probe day. On habituation day, mice were put into the middle of the maze with a beaker, a white noise at 80 db for 60 seconds, after which, mice were guided to the escape hole box and left there for 1 minute with the noise off. After habituation day (0), every trial day (1-5), mice underwent 2 trials, 2h apart from each other, in which the mouse had 3 minutes to find the escape hole box. The number of mistakes, in addition to the time to find the escape hole (latency) was recorded on each trial. Between mice, the maze was cleaned, and each day the platform was rotated. On probe day, (6), the mice are put into the maze, with the escape box removed. Number of mistakes and the time to find escape hole (when their nose is placed into the correct hole) was recorded.

[0265] Rotarod

[0266] Locomotor function, coordination and balance were evaluated using an accelerating rotarod procedure across six trials spaced by 20-minute inter-trial interval (ITI). Five mice were run simultaneously on 9.5 cm diameter rods of a land rotarod apparatus. All mice were first given a habituation trial where they were required to stay on the platform for 1 minute without falling. Animals were then tested in experimental trials, during which the rod was accelerating steadily from 4 to 40 rpm over the course of 5 minutes. The latency to fall from the platform was recorded.

[0267] The results are described herein. CA-V inhibition reduces mitochondria-mediated Afi-induced apoptosis in hCMEC.

[0268] To confirm that selective inhibition of mitochondrial CA-V prevents CEC death and mitochondria-mediated apoptotic mechanisms (Fossati, S., et al., Neurobiol Dis, 2016. 86:29-40; Solesio, M.E., et al., Aging Cell, 2018. 17(4):el2787; Canepa, E., et al., Alzheimers Dement, 2023. 19(11):5048-5073; Anzovino, A., et al., Cells, 2023. 12(24); Provensi, G., et al., Int J Mol Sci, 2019. 20(19)), hCMEC was treated with 25pM A0Q22 alone or in combination with the CA-V inhibitor 4ITP (l,10,30pM) for 24h (Figure 1) or 16h (Figure 2). These time points were selected based on previous evidence demonstrating APQ22’s effects on mitochondria-mediated cell death within hCMEC (Solesio, M.E., et al., Aging Cell, 2018. 17(4):el2787). After confirming that APQ22 induced apoptosis, measured as DNA fragmentation (the last step of the apoptotic pathway), it was determined that 10 and 30pM 4ITP significantly protected hCMEC from APQ22-induced apoptosis (Figure 1A), nearly rescuing DNA fragmentation, as well as CEC loss of viability (Figure IB), to the control levels. Since both concentrations (10 and 30pM) of 4ITP were non-toxic to hCMEC (Figure 8) and rescued both APQ22-induced hCMEC DNA fragmentation (Figure 1 A) and viability loss (Figure IB), these concentrations were selected for subsequent experiments. To test whether CA-V inhibition could prevent Ap-induced mitochondria-mediated apoptosis, APQ22-induced caspase-9 activation was measured, which is directly promoted by mitochondrial Cytochrome C release. It was demonstrated that 10 and 30pM 4ITP were protective against APQ22-induced caspase-9 activation (Figure 1C), assessed as increased formation of its cleaved fragment by WB. CA-V inhibition also significantly reduced the percentage of active executioner caspases 3 and 7- positive cells (Figure ID), measured by the specific fluorescent marker Cell-Event caspase 3 / 7 (Invitrogen). These results suggest that selective CA-V inhibition with 10 and 30pM 4ITP has a protective effect against APQ22-induced mitochondria-mediated CEC apoptosis.

[0269] Co-treatment with lOpM 4ITP also prevented APQ22-induced loss of mitochondrial membrane potential measured by the Mito-tracker CMH2xROS dye, which enters only mitochondria with a healthy (Figure 2A). Loss of mitochondrial membrane potential was observed in APQ22-treated CEC as a decreased Mito-tracker CMH2xROS signal. The release of CytC, measured via immunofluorescence (IF), was evidenced by a diffused signal in APQ22-treated cells, indicating CytC release into the cytosol. Both loss of and release of CytC were rescued by 4ITP. Due to these observations, measures were taken to understand which pro-apoptotic mediators could drive the mitochondrial membrane permeability loss, and if 4ITP could impact their expression. Many proteins may modulate the pore formation on the outer mitochondrial membrane, including pro-apoptotic BAX, BAK and Bim, and anti-apoptotic protein BCL-2 (Westphal, D., et al., Cell Death Differ, 2014. 21(2): 196-205; Jurgensmeier, J.M., et al., Proc Natl Acad Sci U S A, 1998. 95(9):4997-5002). The inhibition of CA-V with 4ITP was observed to significantly prevent AβQ22-mediated BAX and Bim increase in hCMEC, (Figure 2B), while there were no significant changes in Bcl-2 or pro-apoptotic protein BAK expression.

[0270] One mechanism that can alter the expression and interactions of mitochondrial pore regulating proteins, as well as affecting is the accumulation of reactive oxygen species (ROS) (Provensi, G., et al., Int J Mol Sci, 2019. 20(19); Marchi, S., et al., J Signal Transduct, 2012. 2012:329635). Indeed, after 16h treatment, APQ22 significantly induced production of H2O2 by isolated mitochondria, compared to untreated hCMEC. Mitochondrial H2O2 production was reduced to levels similar to the control by both concentrations of 4ITP (Figure 2C). When H2O2 is produced within the cell, it can react with lipids, forming toxic lipid peroxidation products such as 4-HNE, a known inducer of cell death and marker of oxidative stress, which has also been observed in the brains of AD patients (McGrath, L.T., et al., QJM, 2001. 94(9):485- 90). 4-HNE was measured in CEC treated with APQ22 and 4ITP. The increase in 4-HNE induced by APQ22 was significantly prevented by 30pM 4ITP (Figure 2D). Overall, these results confirm that inhibition of mitochondrial CA-V prevents Q22-induced mitochondrial ROS production and mitochondria-mediated apoptotic mechanisms.

[0271] CA-V inhibition prevents Aβ driven loss of barrier integrity in hCMEC.

[0272] After confirming the positive effects of the CA-V inhibitor on mitochondria- mediated apoptosis and oxidative stress triggered by AβQ22 in hCMEC, whether CA-V inhibition could also prevent Aβ-mediated loss of endothelial barrier function was determined. To do so, a sublethal concentration (lOpM) of AβQ22 was used to observe changes in vascular barrier integrity not due to cell death (Parodi-Rullan, R., et al., Aging Cell, 2020.

[0273] 19(1 l):el3258). The resistance (R) of the CEC barrier was measured through the ECIS Z0 technology, as an indicator of BBB integrity and health, which is decreased in the presence of different Aβ species (Carey, A., et al., Aging Cell, 2024:el4106; Parodi-Rullan, R., et al., Aging Cell, 2020. 19(1 l):el3258). Interestingly, co-treatment with 10 and 30pM 4ITP protected against A0Q22-induced loss in R over-time, improving R to levels higher than the control in a dose dependent manner (Figure 3A). It was also observed that 4ITP alone increased the resistance compared to untreated control CEC. The observed changes in resistance could be due to changes in tight junction expression. Thus, to further investigate whether 4ITP induced BBB functional changes by modulating APQ22 effects on the expression of tight junction proteins (TJPs), occludin and claudin-5 were measured.

[0274] At 6h, A0Q22 did not significantly affect the expression of occludin and claudin- 5 (Figure 9) (Bhardwaj U, et al., Mol Neurobiol, 2021, 58(12):6290-6303), as it did not at 24h (Figure 3B). However, claudin-5 expression was significantly increased in cells treated with 4ITP, alone or in combination with APQ22, for 6h (Figure 9) or 24h (Figure 9), in line with the increased R of CEC monolayers treated with the CA-V inhibitor.

[0275] After 48h of treatment with lOpM APQ22, a significant reduction in the expression of occludin was observed compared to untreated CEC, which was no longer significant in the presence of 4ITP, confirming its protective effects on barrier properties. A significant increase in occludin expression in cells treated with 30pM 4ITP alone was also observed compared to control CEC (Figure 3C). Overall, these results indicate that the CA-V inhibitor 4ITP increases tight junction expression and prevents APQ22-induced loss of barrier resistance, confirming its therapeutical potential against BBB permeability in cerebrovascular disease.

[0276] CA-V inhibition prevents Aβ-initiated endothelial activation.

[0277] Another important function of hCMEC is their role as mediators of the crosstalk between the peripheral and CNS immune systems (Parodi-Rullan, R.M., et al., Cells, 2021. 10(11)). Due to their bi-directional influence on cerebral and peripheral immune mechanisms, CEC activation state and the production of endothelial inflammatory mediators are critical in AD and other cerebrovascular diseases. To evaluate endothelial activation, the expression of vascular adhesion molecules known to extravasate peripheral immune cells into the brain, VCAM-1 and ICAM-1, were measured following 6, 24 and 48h of treatment. Interestingly, APQ22 increased the expression of both VCAM-1 and ICAM-1 at different time-points: at early time points (6h, Figure 4A), ICAM-1 was significantly increased compared to the control, while there were no significant changes in VCAM-1 expression; at later time points (24 and 48h, Figure 4B and 4C, respectively), Q22 treatment significantly increased VCAM-1 expression, compared to the control hCMEC, while ICAM-1 expression was not significantly changed. Remarkably, cotreatment with 4ITP reduced the AβQ22 -mediated increase in ICAM-1 to levels non- significantly different from the control at 6h and significantly restored VCAM-1 levels at both 24 and 48h, in a dose-dependent manner. The CEC inflammatory mediators released into the conditioned media were also elucidated. Interestingly, after 6h of AβQ22 treatment (Figure 4D), a significant decrease in the release of anti-inflammatory interleukin- 10 (IL- 10) was observed which was reverted by 4ITP. Conversely, a significant increase in the release of pro- inflammatory cytokine IL-6 was observed which was not affected by mitochondrial CA-V inhibition. There were no significant changes in IL-4 or IL-12p70 after 6h of treatment. After 48h (Figure 4E), APQ22 treatment lowered IL-10 levels (p= 0.0585) and increased the release of pro-inflammatory cytokines IL-6 and IL-12p70, and pleiotropic cytokine IL-4, compared to untreated hCMEC. Importantly, 4ITP significantly upregulated anti-inflammatory IL- 10 release, compared to Q22 alone (significantly at lOpM), and diminished IL-6, IL-4 (significantly at 30pM), and IL12p70 levels. These results suggest that CA-V mediates endothelial activation, highlighting the therapeutical potential of its inhibition in AD and other cerebrovascular indications.

[0278] CA-VB KO prevents Aβ-induced apoptosis in hCMEC and loss in barrier resistance

[0279] It was recently reported that the expression of mitochondrial CA-VB is upregulated in multiple models of amyloidosis, including hCMEC challenged with APQ22, TgSwDI mice and human AD / CAA brains (Canepa, E., et al., Alzheimers Dement, 2023. 19(11):5048-5073). It was also demonstrated that silencing CA-VB in hCMEC is protective against AP-induced apoptosis. In contrast, expression of CA-VA, the other mitochondrial isoform, or CA-II, the main cytoplasmic CA present in the CNS, did not change. Due to the mounting evidence that CA-VB is likely the mitochondrial isoform mediating Ap toxicity in AD, to further explore the role of this CA isoform in CEC dysfunction, hCMEC CA-VB Crispr / CAS9 KO cells were used. After confirming almost complete KO of CA-VB (Figure 5A), compared to control cells (electroporated with CAS9 alone), it was also confirmed that other isoforms of CA, such as CA-VA and CA-II were still expressed in these cells (Figure 10). Strikingly, after 24h of treatment with 25 pM Q22, CA-VB KO hCMEC did not show the significant increase in apoptosis, measured as DNA fragmentation, seen in the WT cells (Figure 5B), suggesting that CA-VB is necessary for AβQ22-induced apoptosis in CEC. To further investigate the impact of mitochondrial CA-VB on endothelial apoptosis, Q22-induced caspase-3 / 7 activation in CA-VB KO cells was also measured (Figure 5C). It was found that APQ22-induced caspase 3 / 7 activation was completely prevented in CA-VB KO CEC. In addition, IF analysis demonstrated that, after 16h of APQ22 treatment, APQ22-mediated loss of mitochondrial membrane potential and cytochrome C release were prevented in CA-VB KO CEC (Figure 5D). Notably, it was also observed that CA-VB KO protected from APQ22-induced mitochondrial H2O2 production (Figure 5E). Finally, to determine if CA-VB KO protected against barrier integrity loss, endothelial barrier R over time was measured with ECIS Z0. As expected, lOpM APQ22 triggered loss of barrier R in WT CEC, while, in absence of CA-VB KO, barrier R was significantly preserved also in the presence of APQ22 challenge. A remaining slight loss of R in APQ22-challenged CA-VB KO CEC monolayers was observed, which is likely due to the involvement of other players in Ap-mediated endothelial barrier dysfunction (Figure 5F). Overall, these results indicate that CA-VB is necessary for Ap-induced mitochondrial apoptosis and that its KO significantly facilitates barrier integrity maintenance in CEC monolayers.

[0280] CA-V inhibition decreases apoptosis and loss ofBBB integrity) in vivo, preserving cognition in 3xTG mice.

[0281] To further evaluate the impact of CA-V inhibition in vivo, the effects of 4ITP in a mouse model of AD, the 3xTG mouse model which presents both Ap and tau pathology in the hippocampus and cortex, was determined. It has been recently demonstrated these mice also display cerebrovascular deficits and mitochondrial dysfunction (Quintana, D.D., et al., Neurobiol Aging, 2021. 105: 115-128; Jullienne, A., et al., Biomedicines, 2022. 10(8)); Ghosh, D., et al., J Neurosci, 2012. 32(17):5821-32; Espino de la Fuente-Munoz, C., et al., Int J Mol Sci, 2020. 21(22)); Yao, J., et al., Proc Natl Acad Sci U S A, 2009. 106(34): 14670-5). It was shown that 4ITP treatment in 3xTG mice mitigated brain apoptosis, measured as cleaved caspase-3 expression, in both cortex (Figure 6 A) and hippocampus (Figure 6B). It was also found that VCAM-1 and ICAM-1 expression were increased in the cortex (Figure 6A) and hippocampus (Figure 6B) of 3xTG mice, and 4ITP significantly reduced the expression of both these endothelial activation markers. Finally, no significant changes in the expression of occludin between the WT and 3xTG mice in the cortex were found, although 4ITP induced a significant increase in the expression of occludin, compared to WT and untreated 3xTg mice (Figure 6A). Differently, in the hippocampus, there was a significant loss in the expression of occludin in 3xTG mice compared to WT, which was mitigated in 4ITP-treated 3xTG mice, showing no significant difference from the WT animals (Figure 6B). Importantly, it was determined whether these observed changes in the brains of 4ITP -treated 3xTG mice were correlated with an improvement in cognition. To this end, the mice cognitive ability was evaluated over a 6-day period using the Barnes Maze, a paradigm employed to measure the ability to consolidate and retrieve spatial memories. As expected, 3xTG mice exhibited an increased latency to find the escape hole throughout the learning trials. These results demonstrate that 4ITP -treated 3xTG mice learned to find the escape hole at a much faster rate than untreated 3xTG mice. A significant difference between treated and untreated mice was observed on day 1 and 2 (training) of the maze (Figure 7A). Moreover, 4ITP -treated 3xTg mice did not show significant differences from WT mice at any day. On probe day (day 6), 3xTG mice demonstrated a significant increase in the time to find the escape hole (latency) and number of mistakes, which was significantly reduced to WT levels in 4ITP treated 3xTG animals (Figure 7B). Additionally, cognitive differences were not observed between WT and WT + 4ITP (6-16M) mice- also measured using the Barnes maze (Figure 11 A). No motor function changes were observed in any of the groups with rotarod test (Figure 7C and Figure 1 IB).

[0282] Overall, these data confirms that CA-V inhibition prevents apoptosis and cerebrovascular dysfunction in vivo in the brain of 3xTG AD mice, while significantly rescuing cognitive impairment.

[0283] Discussion

[0284] The main findings of these results demonstrate that mitochondrial CA-V inhibition is a reliable target for the treatment of AD and other cerebrovascular pathologies. This is the first study to evaluate a selective CA-V inhibitor in models of amyloidosis. The novel, selective CA-V inhibitor, 4ITP, more lipophilic than acetazolamide and methazolamide, prevented AβQ22 induced mitochondria-mediated apoptosis, loss in mitochondrial membrane potential, accumulation of oxidative stress, loss in hCMEC barrier resistance, and endothelial activation. Additionally, hCMEC CA-VB KO cells confirmed the involvement of mitochondrial isoform CA-VB in AβQ22 induced mitochondria-mediated apoptosis, oxidative stress, and EC barrier integrity through the prevention of AβQ22 induced toxicity when compared to WT hCMEC. Strikingly, the same CA-V inhibitor, 4ITP, was protective in the 3xTG mouse model through the prevention of cerebrovascular activation and cell death, associated with cognitive improvement. Overall, the development of CA-V inhibitors for AD and other cerebrovascular disorders should be further explored.

[0285] Cerebrovascular and mitochondrial dysfunction are early and causative events observed in many diseases associated with aging (Carey, A. and S. Fossati, Alzheimers Dement, 2023. 19(2):671-695; Sweeney, M.D., et al., Alzheimers Dement, 2019. 15(1): 158-167; Parodi- Rullan, R., et al., J Alzheimers Dis, 2019. 72(4): 1019-1039; Parodi-Rullan, R.M., et al., Cells, 2021. 10(11); Parodi-Rullan, R., et al., Aging Cell, 2020. 19(1 l):el3258; Anzovino, A., et al., Cells, 2023. 12(24); Iturria-Medina, Y , et al., Nat Commun, 2016. 7: 11934; Wang, W., et al., Mol Neurodegener, 2020. 15(l):30; Swerdlow, R.H., J Alzheimers Dis, 2018. 62(3): 1403-1416). In AD, both hallmarks are observed to occur even prior to Ap and tau accumulation, and worsen throughout the progression of AD and CAA, causing a vicious loop of impaired clearance, toxic protein accumulation, neuroinflammation, exacerbated cerebrovascular and mitochondrial / metabolic deficits. Due to the high impact of cerebrovascular and mitochondrial deficits on the development and progression of AD and CAA, it is evident that they are both ideal targets for the development of novel therapeutic strategies against AD and CAA.

[0286] Current FDA-approved therapies for AD do not appear to improve cerebrovascular function. On the contrary, toxic effects of the new anti-Ap antibodies, increasing Aβ deposits on the BBB, are likely to be responsible for the development of ARIA-E (edema) or ARIA-H (hemorrhages) in a substantial percentage of patients treated with these new drugs (Hampel, H., et al., Brain, 2023; Piazza, F., et al., Neurology, 2022. 99(12):el265-el277), emphasizing the importance and need for treatments targeting vascular dysfunction in AD and CAA. Moreover, mitochondrial and metabolic deficits are also frequently discussed among the main pathways to target for the development of new drugs against AD and dementias. However, mitochondria-targeted therapies have not yet reached FDA approval for these diseases (Parodi- Rullan, R.M., et al., Cells, 2021. 10(11); Wang, W„ et al., Mol Neurodegener, 2020. 15(l):30; Swerdlow, R.H., J Alzheimers Dis, 2018. 62(3): 1403-1416). Importantly, a multitude of evidence demonstrates that lifestyle factors such as diet and exercise, as well as cardiovascular risk factors, can influence both BBB and mitochondrial function, resulting in the modulation of risk and progression of symptoms associated with the disease (2023 Alzheimer's disease facts and figures. Alzheimers & Dementia, 2023. 19(4): 1598-1695; Carey, A. and S. Fossati, Alzheimers Dement, 2023. 19(2): 671-695; Saeed, A., et al., J Am Heart Assoc, 2023. 12(21):e030780; Faraco, G., et al., Nature, 2019. 574(7780):686-690). Cardiovascular risk factors, in particular, may increase cerebrovascular complications in AD and CAA patients, through their toxic effects on CEC (Carey, A. and S. Fossati, Alzheimers Dement, 2023. 19(2):671-695; Carey, A., et al., Aging Cell, 2024:el4106).

[0287] CAs catalyze the hydrolysis of carbon dioxide to produce bicarbonate and a proton. This is an essential reaction to maintain physiological cell health, and particularly within the mitochondria, due to the high production of carbon dioxide as a by-product of metabolism. The use of CA inhibitors has been first explored in cardiovascular disease (Friedberg, C.K., et al., N Engl J Med, 1953. 248(21):883-9; Warshaw, L.J., Am J Cardiol, 1959. 3(2): 167-79). Pan- CA inhibitors such as ATZ and MTZ were developed as legacy diuretics, being optimized to modulate the function on the kidney and water reabsorption. They are relatively polar (log P ATZ = -0.26, log P MTZ = 0.13) as they were optimized to work primarily at the level of the kidneys. They have then become FDA-approved for many diseases such as glaucoma, epilepsy, and high-altitude sickness (Lemon, N., et al., Front Aging Neurosci, 2021. 13:772278; Provensi, G., et al., Int J Mol Sci, 2019. 20(19)). More recently, they have also been demonstrated to be effective in models of obesity, cerebral edema, stroke, and diabetes-induced cerebrovascular pathology (Supuran, C.T., J Enzyme Inhib Med Chem, 2022. 37(l):2478-2488; Dettori, I., et al., J Enzyme Inhib Med Chem, 2021. 36(l):964-976; Shah, G.N., et al., J Pharmacol Exp Ther, 2013. 344(3):637-45). Many of the studies, dissecting the involvement of CA in cardiovascular pathology or stroke, have used pan-CA inhibitors, most commonly ATZ and MTZ (Lemon, N., et al., Front Aging Neurosci, 2021. 13:772278; Wang, X., et al., Stroke, 2009. 40(5): 1877-85). As previously mentioned, ATZ and MTZ have also been proved effective against Ap-mediated toxicity in many NVU cells (Fossati, S., et al., Neurobiol Dis, 2016. 86:29-40; Solesio, M.E., et al., Aging Cell, 2018. 17(4):el2787; Anzovino, A., et al., Cells, 2023. 12(24)), and in a CAA mouse model, the TgSwDI (Canepa, E., et al., Alzheimers Dement, 2023. 19(11): 5048-5073). Additionally, topiramate, another CA inhibitor, has been demonstrated to be protective in models of diabetes-induced cerebrovascular pathology (Shah, G.N., et al., J Pharmacol Exp Ther, 2013. 344(3):637-45; Price, T.O., et al., Endocrinology, 2012. 153(l):362-72). Although many pan-CA inhibitors have protective effects, they are not ideal for therapeutic use, as their lack of selectivity require significantly higher doses to achieve efficacy, which cause undesired side effects when used systemically in humans (Mincione, F., et al., Curr Pharm Des, 2008. 14(7):649-54).

[0288] Previous studies have demonstrated that ATZ and MTZ prevent Aβ toxicity, specifically through mitochondria-mediated mechanisms (Fossati, S., et al., FASEB J, 2010. 24(1):229-41; Fossati, S., et al., Neurobiol Dis, 2016. 86:29-40; Solesio, M E., et al., Aging Cell, 2018. 17(4):el2787; Anzovino, A., et al., Cells, 2023. 12(24)). Here, a mitochondria-selective CA-V inhibitor was tested for the first time, and it was shown that it is effective at similar concentrations to ATZ (the most effective of the pan-CAIs tested in vitro and in vivo), against mitochondria-mediated apoptosis in hCMEC (Solesio, M.E., et al., Aging Cell, 2018. 17(4):el2787). Hence, selective CA-V inhibitors will be at least as effective as pan-CA inhibitors against AD / CAA, while avoiding side effects that have been reported in humans for pan-CAIs. One of the possible side-effects to be considered when using high concentrations of pan-CA inhibitors, such as ATZ, is short-term memory impairment. Indeed, a high dose of ATZ has been observed to inhibit long-term potentiation, acutely, in mice (Schmidt, S.D., et al., Proc Natl Acad Sci U S A, 2020. 117(27): 16000-16008; Sanku, R.K.K., et al., Behav Pharmacol, 2018. 29(6):482-492). Additionally, humans have reported memory reduction when taking ATZ at high concentrations (Schmidt, S.D., et al., Proc Natl Acad Sci U S A, 2020. 117(27): 16000- 16008). This effect may be due to the activity of non-selective inhibitors on a specific CA isoform, CA-VII, abundantly expressed in pyramidal neurons of the hippocampus (Ruusuvuori, E., et al., I Neurosci, 2004. 24(11):2699-707). However, the dose of ATZ used in animal models (20mg / kg / day), which is the same dose of 4ITP in the present study, corresponds to a human dose of 2.6 mg / kg / day (182 mg / day for a 70 kg person), and is lower than the FDA-approved dose for CAIs (ATZ: up to Ig / day), which is the dose known to induce short-term memory issues. Nevertheless, confirming the safety and lack of this side effect for 4ITP, it was shown here that, while 4ITP treatment ameliorates memory in the 3xTG AD mouse model, the chronic use of 4ITP on WT animals, at the proposed concentration, does not impair spatial memory.

[0289] Due to the diverse expression pattern and functions of CAs, it is evident that not all 15 CA isoforms are involved in the development of AD pathology. It was shown here that eliminating CA-VB completely prevents mitochondria-mediated apoptosis induced by Ap in hCMEC and prevents Aβ-mediated BBB permeability. Thus, selectively targeting the mitochondrial isoform CA-VB, while avoiding CA isoforms not involved in the progression of AD and CAA, may be of high interest for therapeutic development against these disorders, as well as, likely, against the cerebrovascular complications induced by current treatment with FDA-approved anti-Ap antibodies.

[0290] CA-V, expressed in the mitochondrial matrix, has been demonstrated to influence intermediates of the citric acid cycle through the supply of bicarbonate for enzymes like pyruvate carboxylase and propionyl carboxylase, thereby affecting biosynthetic pathways, such as gluconeogenesis and ureagenesis. However, many of these studies have been done in the liver, where CA-VA is much more abundant. Interestingly, the involvement of CA-VB in metabolism is only evident in the absence of CA-VA (Shah, G.N., et al., Proc Natl Acad Sci U S A, 2013. 110( 18): 7423-8). This is observed in cells where CA-VA is not expressed, as well as in Car5a KO mice. Less is known about the function of CA-VB; however, it is much more abundant throughout the body and in the brain (Lemon, N., et al., Front Aging Neurosci, 2021. 13:772278). Interestingly, Car5B global KO mice, differently from Car5A KO mice, are viable and show normal growth and normal ammonia levels, suggesting that chronic inhibition or brain Car5B KO does not have toxic effects (Shah, G.N., et al., Proc Natl Acad Sci U S A, 2013. 110(18)7423-8).

[0291] Besides CA-V’s role in metabolism, it is evident CA-V activity influences mitochondria-mediated apoptosis and mitochondrial membrane potential, as well as oxidative stress. While CA-VA may function mainly to regulate metabolism, CA-VB may regulate mitochondrial pH or redox homeostasis. Mechanistically, in these models, the inhibition of mitochondrial CA activity may reduce the over-production of hydrogen ions, especially in times of stress, as amyloidosis, maintaining a lower proton level within the mitochondrial matrix compared to the inner mitochondrial membrane. Maintaining a low H+concentration in the matrix will aid in preserving a healthy mitochondrial membrane potential, in addition to an optimal proton motive force, especially in the presence of ROS. This is essential for proper mitochondrial function, cell signaling and, ultimately, cell survival. Indeed, it was demonstrated that 4ITP-treated cells present a preserved membrane potential, reduced mitochondrial H2O2 production, as well as reduced CytC release, reinforcing the mitochondria-specific mechanism of action of the drug, as also supported by the same results in CA-VB KO cells.

[0292] A plethora of evidence indicates that preservation of mitochondrial function and mitigation of ROS prevents downstream neuroinflammatory mechanisms. ROS production and release has been observed to induce pro-inflammatory cytokine release resulting in the activation of glial and vascular cell types, and consequently an increase in CEC activation and BBB permeability. When CEC are activated, they increase their expression of extravasation proteins VCAM-1 and ICAM-1 for the purpose of immune cell adherence / recruitment. In the brain this is still not completely understood, and while necessary for the clearance of toxic substances from the brain, chronic inflammation and CEC activation is damaging to the BBB and thus the brain. The prevention of CEC activation and preservation of endothelial cell function through the inhibition of CA-V is likely due to the prevention of ROS and loss in mitochondrial membrane potential.

[0293] The function of CA-V in other neurovascular cell types is not well studied, however there has been indication of its importance in the regulation of cell death in cerebral pericytes (Shah, GN, et al., I Pharmacol Exp Ther, 2013, 344(3):637-45; Price, TO, et al., Endocrinology, 2012, 153(1):362-72). It is unclear if the mitigation of glial cell activation observed with pan-CA inhibitors in TgSwDI mice is direct or indirect through the prevention of CEC activation. Investigating CA-V function in other neurovascular cell types as well as immune cells (both microglia and peripheral immune cells) is critical to further unfold the functions of CA-V.

[0294] The accumulation of Aβ around the brain vessels, found in AD, sporadic CAA, CAA related inflammation (CAAri), as well as in ARIA complications due to high amounts of Aβ unable to be cleared by the cerebral vasculature, is known to exacerbate BBB dysfunction and induce permeability, enhancing the likelihood of ischemic and hemorrhagic strokes, white matter abnormalities, worsening behavioral symptoms, and causing long-lasting damage (Kelly, L., et al., Alzheimers Dement, 2023; Alber, I., et al., Alzheimers Dement (N Y), 2019. 5:107- 117). Therefore, mitochondrial CA inhibitors could not only be a potential therapy for AD and CAA when used alone, but the combination of CA inhibitors and anti-Ap therapies could also be a very beneficial co-treatment strategy.

[0295] One limitation of this study is that it is currently impossible to have complete pharmacological selectivity for one CA-V isoform. 4ITP has been previously demonstrated to have a Ki value of 8.6 nm and 8.3 nm for CA-VA and -VB respectively, indicating equal selectivity for the mitochondrial isoforms. 4ITP is about 8x less effective on CA-I and -II (Ki = 75 and 54 nm respectively), and even less on plasma membrane proteins CA-IX and CA-XII (136 and 212 nm). Although this study, by comparing the effects of 4ITP with CA-VB KO cells, provides strong evidence 4ITP has CA-V specificity, without being bound by a particular theory, the activity of 4ITP on other CA isoforms relevant to the brain / NVU activity remains to be determined. Of interest, CA-IV is highly expressed on the plasma membrane of CEC and should be considered when studying CA function in CEC. Furthermore, knowledge on the activity of 4ITP on CA-VII would be informative, as this is the isoform anticipated to be involved in memory and highly expressed in neurons. The lack of an effect of 4ITP on WT animals’ behavior, however, suggests low affinity for this isoform. Studies providing more evidence about the selectivity of 4ITP would help understanding of the mechanism of action and to design other selective CA-V inhibitors in future studies.

[0296] Another limitation of this study is that BBB function or CBF were not measured in real time in vivo. The effects of CA-V inhibitors and endothelial-specific CA-VB KO on CBF, BBB permeability and neurovascular coupling in AD mouse models are contemplated.

[0297] It was demonstrated that there is mitigation of caspase activation as well as endothelial inflammatory activation in the hippocampus and cortex of 3xTG mice treated with 4ITP. The degree of pathology in treated vs non-treated 3XTG mice, and possible amelioration of brain clearance, through measurements of Ap and tau pathology, glial function, and neurodegeneration markers, as previously shown for MTZ and ATZ, are contemplated. However, the strong protective effects of 4ITP on CEC function and on cognition in the 3xTG mouse model suggest potential benefits in all those mechanisms.

[0298] Overall, this is the first study to demonstrate the protective effects of a selective CA-V inhibitor in models of amyloidosis or AD. Overall, it was demonstrated that the CA-V inhibitor 4ITP, as well as CA-VB CRISPR KO, protect CEC from Aβ-induced mitochondria toxicity and BBB dysfunction, and that 4ITP is effective to reduce cognitive impairment in 3xTG mice. More specifically, CA-VB has been demonstrated to be necessary for Aβ-induced cell death in CEC, and to have an important role in the loss of BBB resistance. Taken together, it is evident that the development of CA-V inhibitors is an important area of research for the improvement of BBB function, cerebrovascular pathology, and mitochondrial function in AD, CAA, and other dementias. This study has also laid the groundwork for future testing of other CA-V inhibitors for the treatment of AD and CAA and for potential clinical trials employing this compound in AD and related dementias.

[0299] Example 2: Design of chemical compounds

[0300] General Procedure for the Synthesis of 5-Benzoylamido-l ,3,4-thiadiazole-2- sulfonamide Derivatives 6.

[0301] A. Synthesis of 5-amino-l,3,4-thiadiazole-2-sulfonamide 3.

[0302] In a 500 mL round bottom flask 22.2 g (0.1 mol) acetazolamide 1 were dissolved in 100 mL of concentrated HC1 solution (35%) and refluxed for 1 h. The solvent was evaporated under vacuum using a rotary evaporator, when the hydrochloride of 5-amino-l,3,4-thiadiazole-2- sulfonamide 2 was obtained as a white precipitate. The hydrochloride 2 was dissolved in 50 mL of water and neutralized with NaOH IM till pH 7, when neutral 5-amino-l,3,4-thiadiazole-2- sulfonamide 3 precipitated. It was filtered and recrystallized from methanol (yield of 89 %). B. Synthesis of substituted benzamido-sulfonamides 6a-i.

[0303] In a 100 mL round-bottom flask, 5-amino-l,3,4-thiadiazole-2-sulfonamide 3 (0.54 g, 3.6 mmol) was suspended in dry acetonitrile (10 mL), and pyridine (0.29 mL, 3.6 mmol) was added via automatic pipette. The reaction mixture was cooled to 0-5°C using an ice bath, under stirring. The acyl chloride 5 (3.6 mmol) was made separately from the corresponding benzoic acid 4 (3.6 mmol), 2.6 mL of thionyl chloride and 2-3 drops of DMF, under reflux, for Ih. Excess thionyl chloride was removed under vacuum on the rotary evaporator and the crude acyl chloride was dissolved in 5-10 mL of acetonitrile and was added dropwise to the cold suspension of 3, under stirring. Stirring was continued overnight when a white precipitate of was formed. The precipitate was filtered, washed with 5-10 mL of acetonitrile, which was combined with the filtrate. The remaining precipitate was washed several times with 5-10 mL of water and was dried under vacuum. Recrystallization with alcohols (MeOH, EtOH) usually yielded pure product, as evidence of LC-MS (>96%). The acetonitrile filtrate was evaporated to dryness using a rotary evaporator. The residue was treated with water and filtered, then purified as described above. When purity was not satisfactory, the solid compound was purified via flash chromatography on SiCh using MeOH / CHCh gradients. The pure fractions (by TLC, MeOH / CHC13 1 / 4 v / v) were grouped, evaporated to dryness, and crystallized from MeOH or EtOH. Final purity was confirmed by LC-MS.

[0304] C. Synthesis of 5-(4-carboxybenzoylamido)-l,3,4-thiadiazole-2-sulfonamide 6j In a 100 mL round bottom flask equipped with a magnetic stir bar, 5-(4- methoxycarbonylbenzamide)-2-sulfonamido-l,3,4-thiadiazole 6h (0.5 g, 1.45 mmol) was dissolved in THF (20 mL). A 20% aq. KOH solution (6 mmol, 1.7 mL) was added. The mixture was stirred at room temperature until the reaction was shown to be completed by TLC (20% CH3OH / CHCI3 v / v). After completion, the organic solvent was removed in vacuo and the aqueous solution was acidified to pH = 1 with HC1 IM. The precipitated product was extracted using ethyl acetate (5 x 30 mL). The organic layer was washed with water, brine, then dried over Na2SO4 and concentrated in vacuo to yield the title compound 6j, which was crystallized from EtOH, filtered out and dried under vacuum. D. Synthesis of 5-(4-aminobenzoylamido)-l,3,4-thiadiazole-2-sulfonamide 6k In a 250 mL round bottom flask, equipped with a magnetic stir bar, 2-(4- nitrobenzamido)-5-sulfonamido-l,3,4-thiadiazole 6i (0.5 g, 1.5 mmol) was dissolved in EtOH (50 mL) and treated with 100 mg Pd / C. The flask was capped with a rubber septa and the air was purged out using Ar gas. A balloon filled with hydrogen was attached to the rubber septa and the argon was replaced with hydrogen via a needle. Hydrogenation was continued until the reaction was shown to be completed by TLC (20% CH3OH / CHCI3 v / v). After completion, the hydrogen was purged out with argon, the rubber septa was removed and the Pd / C was filtered out and washed with EtOH without allowing its complete drying and ensuring subsequent proper disposal. The EtOH filtrate was concentrated under vacuum when the title product 6k crystallizes, being subsequently filtered out and dried under vacuum.

[0305] 2-(4-Methoxybenzamido)-5-sulfonamido-l,3,4-thiadiazole 6a: mp 306-310 °C; ^-NMR (DMSO-d6, 5, ppm): 13.35 (s, 1H, -CONH), 8.37 (s, 2H, -SO2NH2), 8.17 (d, 3J = 8.9 Hz, 2H, H2 and H6 Ph), 7.12 (d, 3J = 8.9 Hz, 2H, H3 and H5 Ph); 13C-NMR (DMSO-d6, δ, ppm): 164.7 (C5 TDA), 164.5 (CONH), 163.2 (C4 Ph), 162.2 (C2, TDA), 130.7 (2C, C2, C6 Ph), 122.7 (Cl Ph), 114.0 (2C, C3, C5 Ph); LC-MS: exact mass: 314.0; Found: 315.0 (MH+).

[0306] 2-(4-Cyanobenzoylamido)-5-sulfonamido-l,3,4-thiadiazole 6b: mp 310-316 °C; ^-NMR (DMSO-d6, δ, ppm): 13.81 (s, 1H, -CONH), 8.42 (s, 2H, -SO2NH2), 8.28 (d, 3J = 8.9 Hz, 2H, H2 and H6 Ph), 8.08 (d, 3J = 8.9 Hz, 2H, H3 and H5 Ph), 13C-NMR (DMSO-d6, 8, ppm): 164.9 (CONH), 164.7 (C5 TDA), 162.0 (C2, TDA), 135.0 (C4Ph), 132.6 (2C, C2, C6 Ph), 129.3 (2C, C3, C5 Ph), 118.0 (Cl Ph), 115.3 (CN); LC-MS: exact mass: 309.0; Found: 310.0 (MH+).

[0307] 2-(4-Fluorobenzoylamido)-5-sulfonamido-l,3,4-thiadiazole 6c: mp 328-331 °C; 'H-NMR (DMSO-d6, 8, ppm): 13.56 (s, 1H, -CONH), 8.39 (s, 2H, -SO2NH2), 8.24 (dd, 3J = 8.9 Hz, 4J = 5.3 Hz, 2H, H2 and H6 Ph), 7.44 (t, 3J = 8.9 Hz, 1H, H3 and H5 Ph); 13C-NMR (DMSO-d6, 8, ppm): 165.0 (d, JF-Cipso = 253 Hz, C4 Ph), 164.7 (-CONH), 164.5 (C5 TDA), 162.1 (C2, TDA), 131.5 (d, JF-Cmeta = 9.5 Hz, C2, C6 Ph), 127.4 (Cl Ph), 115.8 (d, JF-Cortho = 22.2 Hz, C3, C5 Ph); 19F-NMR (DMSO-d6, 8, ppm): -105.57; LC-MS: exact mass: 302.0; Found: 303 (MH+).

[0308] 2-(4-Chlorobenzoylamido)-5-sulfonamido-l,3,4-thiadiazole 6d: mp 332-336 °C; 1-NMR (DMSO-d6, 8, ppm): 13.62 (s, 1H, -CONH), 8.39 (s, 2H, -SO2NH2), 8.16 (d, 3J = 8.9 Hz, 2H, H2 and H6 Ph), 7.67 (d, 3J = 8.9 Hz, 2H, H3 and H5 Ph); 13C-NMR (DMSO-d6, δ, ppm): 164.8 (-CONH), 164.7 (C5 TDA), 162.1 (C2, TDA), 138.3 (C4Ph), 130.5 (2C, C2, C6 Ph), 129.7 (Cl Ph), 128.9 (2C, C3, C5 Ph); LC-MS: exact mass: 319.0; Found: 320.0 (MH+).

[0309] 2-(4-Bromobenzamido)-5-sulfonamido-l,3,4-thiadiazole 6e: mp 334-338 °C; 'H- NMR (DMSO-d6, 8, ppm): 13.64 (s, 1H, -CONH), 8.36 (s, 2H, -SO2NH2), 8.07 (t, 3J = 8.3 Hz, 2H, H2 and H6 Ph), 7.80 (t, 3J = 8.3 Hz, 2H, H3 and H5 Ph);13C-NMR (DMSO-d6, 8, ppm): 165.1 (-CONH), 164.6 (C5 TDA), 162.5 (C2, TDA), 131.8 (2C, C2, C6 Ph), 130.6 (2C, C3, C5 Ph), 130.4 (Cl Ph), 127.3 (C4 Ph); LC-MS: exact mass: 362.0; Found: 363.0 (MH+).

[0310] 2-(4-Methylbenzamido)-5-sulfonamido-l,3,4-thiadiazole 6f: mp 320-325°C; 'H- NMR (DMSO-d6, 8, ppm): 13.45 (s, 1H, -CONH), 8.38 (s, 2H, -SO2NH2), 8.07 (d, J = 8.30 Hz, 2H, H2 and H6 Ph), 7.40 (d, J = 8.03 Hz, 2H, H3 and H5 Ph), 2.41 (s, 3H, H3C-Ph);13C-NMR (DMSO-d6, 8, ppm): 165.3 (C5, TDA), 164.6 (-CONH), 162.1 (C2, TDA), 143.8 (C4Ph), 129.2 (2C, C2, C6 Ph), 128.5 (2C, C3, C5 Ph), 127.9 (Cl Ph), 21.1 (1C, -CH3); LC-MS: exact mass: 298.0; Found: 299.0 (MH+).

[0311] 2-(4-Trifluorobenzamido)-5-sulfonamido-l,3,4-thiadiazole 6g: mp 310-316°C; 'H-NMR (DMSO-d6, 8, ppm): 13.82 (s, 1H, -CONH), 8.42 (s, 2H, -SO2NH2), 8.34 (d,3J = 8.08 Hz, 2H, H2 and H6 Ph), 7.98 (d,3J = 8.47 Hz, 2H, H3 and H5 Ph);13C-NMR (DMSO-d6, 8, ppm); 164.9 (-CONH), 164.8 (C5 TDA), 162.0 (C2 TDA), 132.7 (t,2J=31.54 Hz, C-CF3, C4 Ph), 129.5 (Cl Ph), 125.6 (d,3J=4.35, C-C-CF3, C3, C5 Ph), 123.63 (q,1J=272.87 Hz, CF3);19F- NMR (DMSO-d6, 8, ppm): -61.6; LC-MS: exact mass: 352.0; Found: 353.0 (MH+).

[0312] 2-(4-Methoxycarbonylbenzamide)-5-sulfonamido-l,3,4-thiadiazole 6h: mp 325-

[0313] 330 °C; ‘H-NMR (DMSO-d6, 8, ppm): 13.77 (s, 1H, -CONH), 8.41 (s, 2H, -SO2NH2), 8.26 (d, J = 8.52 Hz, 2H, H2 and H6 Ph), 8.13 (d,3J = 8.47 Hz,4J = 3.91 Hz, 2H, H3 and H5 Ph);13C- NMR (DMS0-d6, δ, ppm): 165.4 (-COOH), 165.0 (-CONH), 164.8 (C5, TDA), 162.0 (C2, TDA), 134.9 (C4 Ph), 129.3 (2C, C3, C5 Ph), 128.9 (2C, C2, C6 Ph), 133.3 (Cl Ph), 52.5 (- OCH3); LC-MS: exact mass: 342.0; Found: 343.0 (MH+).

[0314] 2-(4-Nitrobenzamido)-5-sulfonamido-l,3,4-thiadiazole 6i: mp 341-344 °C; 'H- NMR (DMSO-d6, δ, ppm): 13.91 (s, 1H, -CONH), 8.90 (s, 2H, -SO2NH2), 8.42 (d,3J=9.08 Hz, 2H, H3 and H5 Ph), 8.34 (d,3J=9.24 Hz, 2H, H2 and H6 Ph);13C-NMR (DMSO-d6, 5, ppm): 163.8 (-CONH), 163.4 (C5 TDA), 160.9 (C2 TDA), 148.9 (C4 Ph), 135.5 (Cl Ph), 129.0 (2C, C3, C5 Ph), 122.6 (2C, C2, C6 Ph); LC-MS: exact mass: 329.0; Found: 330.0 (MH+).

[0315] 2-(4-Carboxybenzamido)-5-sulfonamide-l,3,4-thiadiazole 6j: mp 324-328 °C; 'H-NMR (DMSO-d6, 5, ppm): 13.71 (s, 1H, -CONH), 8.43 (s, 2H, -SO2NH2), 8.23(d,3J=8.55 Hz, 2H, H3 and H5 Ph), 8.12 (d,3J=8.52 Hz, 2H, H2 and H6 Ph ),13C-NMR (DMSO-d6, δ, ppm): 166.5 (s, 1H, C4, CO2H), 165.1 (-CONH), 164.8 (C5, TDA), 162.0 (C2, TDA), 134.7 (C4 Ph), 134.5 (Cl Ph), 129.4 (2C, C3, C5 Ph), 128.8 (2C, C2, C6 Ph); LC-MS: exact mass 330.0; Found: 331.0 (MH+).

[0316] 2-(5-Aminobenzamide)-5-sulfonamido-l,3,4-thiadiazole 6k: mp 300-305 °C; 'H- NMR (DMSO-d6, 8, ppm): 12.93 (s, 1H, -CONH), 8.30 (s, 2H, -SO2NH2), 7.90 (d,3J 8.35 Hz, 2H, H3 and H5 Ph), 6.61 (d,3J=8.75 Hz, 2H, H2 and H6 Ph), 6.16 (s, 2H, -NH2);13C-NMR (DMSO-d6, 8, ppm): 164.5 (C5, TDA), 164.1 (-CONH), 162.39 (C2, TDA), 153.9 (C4 Ph), 130.6 (2C, C2, C6 Ph), 116.1 (Cl Ph), 112.5 (2C, C3, C5 Ph); LC-MS: exact mass: 299.0; Found: 300.0 (MH+).

[0317] General Procedure for the Synthesis of 5-(2-(4-Rl-Phenyl)-2-R2-2-R3- acetamido)-], 3, 4-thiadiazole-2-sulfonaniide Derivatives 9.

[0318]

[0319] The procedure was similar with the above procedure used to generate compounds 6. Thus, in a 100 mL round-bottom flask, 5-amino-l,3,4-thiadiazole-2-sulfonamide 3 (0.54 g, 3.6 mmol) was suspended in dry acetonitrile (10 mL), and pyridine (0.29 mL, 3.6 mmol) was added via automatic pipette. The reaction mixture was cooled to 0-5°C using an ice bath, under stirring. The acyl chloride 8 (3.6 mmol) was made separately from the corresponding phenylacetic acid 7 (3.6 mmol), 2.6 mL of thionyl chloride and 2-3 drops of DMF, under reflux, for Ih. Excess thionyl chloride was removed under vacuum on the rotary evaporator and the crude acyl chloride was dissolved in 5-10 mL of acetonitrile and was added dropwise to the cold suspension of 3, under stirring. Stirring was continued overnight when a white precipitate of was formed. The precipitate was filtered, washed with 5-10 mL of acetonitrile, which was combined with the filtrate. The remaining precipitate was washed several times with 5-10 mL of water and was dried under vacuum. Recrystallization with alcohols (MeOH, EtOH) usually yielded pure product, as evidence of LC-MS (>96%). The acetonitrile filtrate was evaporated to dryness using a rotary evaporator. The residue was treated with water and filtered, then purified as described above. When purity was not satisfactory, the solid compound was purified via flash chromatography on SiO2using MeOH / CHCL gradients. The pure fractions (by TLC, MeOH / CHC13 1 / 4 v / v) were grouped, evaporated to dryness, and crystallized from MeOH or EtOH. Final purity was confirmed by LC-MS.

[0320] 5-(2-(4-methoxyphenyl)acetamido)-l,3,4-thiadiazole-2-sulfonamide 9Aa 'H- NMR (DMSO-d6, 5, ppm): 13.24 (s, 1H, CONH), 8.34 (s, 2H, SO2NH2), 7.25 (d, J= 8.7 Hz, 2H, Ar), 6.89 (d, J = 8.7 Hz, 2H, Ar), 3.81 (s, 2H, CH2), 3.73 (s, 3H, OMe);13C-NMR (DMSO-d6, δ, ppm): 170.5 (CO), 164.3 (CSO2NH2), 161.1 (COCH3), 158.2, 130.3, 125.9, 113.6, 55.0 (OCH3), 40.5 (CH2CO). LC-MS: exact mass: 328.03; Found: 329.1 (MH+). 5-(2-(4-methoxyphenyl)-2-methylacetamido)-l,3,4-thiadiazole-2-sulfonamide 9Ba 'H-NMR (DMSO-d6, 5, ppm): 13.18 (s, 1H, CONH), 8.31 (s, 2H, SO2NH2), 7.29 (d, J= 5.5 Hz, 2H, Ar), 6.91 (d, J= 5.5 Hz, 2H, Ar), 4.00 (q, J = 4.4 Hz, 1H, CH), 3.72 (s, 3H, OMe), 8 = 1.44 (d, J = 4.4, 3H, Me);13C-NMR (DMSO-d6, 8, ppm): 173.5 (CO), 164.4 (CSO2NH2), 161.1 (COCH3), 158.4, 132.0 (CCH), 128.5 (CHCOCH3), 114.0 (CHCCH), 55.1 (OCH3), 44.0 (CHCO), 18.2 (CH3). LC-MS: exact mass: 342.05; Found: 343.1 (MH+).

[0321] 5-(2-(4-methoxyphenyl)-2,2-dimethylacetamido)-l,3,4-thiadiazole-2-sulfonamide 9Ca 'H-NMR (DMSO-d6, 8, ppm): 12.59 (s, 1H, CONH), 8.30 (s, 2H, SO2NH2), 7.22 (d, J= 5.6, 2H, Ar), 6.92 (d, J= 5.6, 2H, Ar), 3.73 (s, 3H, OMe), 1.60 (s, 6H, Me);13C-NMR (DMSO-d6, 8, ppm): 176.1 (CO), 164.5 (CSO2NH2), 161.9 (COCH3), 158.1 (CONHC), 135.6 (CC(CH3)2), 127.1 (CHCOCH3), 113.8 (CHCHCOCH3), 55.1 (OCH3), 46.3 (C(CH3)2), 8 = 25.9 (CH3). LC-MS: exact mass: 356.06; Found: 357.1 (MH+).

[0322] Methyl 2-(4-methoxyphenyl)propionate 11 (Wang, D., et al., J Am Chem Soc, 2017. 139, (44): 15632-15635; Cheng, XF., et al., J Am Chem Soc, 2013. 135, (4): 1236-1239; Clericuzio, M., et al., Synthesis 2002, 2002. (07):0921-0927)

[0323] In a flame-dried flask, methyl (4-methoxyphenyl)acetate 10 (2.0 g, 11 mmol) was dissolved in THF (50 mL). Lithium diisopropylamide (LDA, 1.1 eq, 12.1 mmol) was added dropwise at -78°C under argon atmosphere. The reaction mixture was stirred at 0°C for Ih, then iodomethane (1.5 eq, 16.5 mmol, 2.3 g) was added at -78°C. The reaction was stirred overnight at room temperature. The mixture was acidified to pH = 2 using 10% aq HCl, and was extracted twice with dichloromethane (2 x 50 ml) and the combined organic layers were washed successively with water (10 mL) and brine (10 mL), then dried over anhydrous MgSO4 and concentrated in vacuo to yield the crude product. Subsequent chromatography on silica gel (0- 30% EtOAc / hexanes) yielded the pure product as a yellow oil (2.04 g, 96%).

[0324] 1H NMR (CDCl3) (Clericuzio, M., et al., Synthesis 2002, 2002. (07):0921-0927): δ = 7.45 (d, J= 7.5 Hz, 2H, Ar), 6.86 (d, J= 5.2 Hz, 2H, Ar), 3.79 (s, 3H, OMe), 3.67 (m, 1H, CH), 3.65 (s, 3H, COOMe), 1.47 (d, J= 7.9 Hz, 3H, Me).

[0325] 2-(4-Methoxyphenyl)propionic acid 7Ba (Clericuzio, M., et al., Synthesis 2002, 2002. (07):0921-0927; Page, D„ et al., Bioorg Med Chem Let, 2008. 18 (13):3695-3700)

[0326] Methyl 2-(4-methoxyphenyl)propi onate 11 (6.7 mmol, 1.3 g) was dissolved in THF (16 mL) and was treated with a 20% aqueous KOH solution (4 eq, 1.6 g, 7.8 mL). The mixture was stirred at room temperature until the reaction was shown to be completed by TLC (20% EtOAc / hexanes). After completion, the solvent was removed in vacuo, and the remaining aqueous solution was acidified to pH = 1 using HC1 IM. The precipitated product was extracted using EtOAc (3 x 50 mL). The organic layer was separated, washed with water, dried over Na2SO4 and subsequently concentrated in vacuo to yield the title compound as a yellow oil (1.2 g, 99.5%). No further purification was necessary.

[0327] 1H NMR (CDCh) (Clericuzio, M., et al., Synthesis 2002, 2002. (07):0921-0927): 8 = 7.24 (d, J= 7.5 Hz, 2H, Ar), 6.86 (d, J= 5.2 Hz, 2H, Ar), 3.80 (s, 3H, OMe), 3.69 (q, J= 2.1, 1H, CH), 1.49 (d, J= 4.3 Hz, 3H, Me).

[0328] Methyl 2-(4-methoxyphenyl)-2,2-dimethylacetate 12 (Wang, D., et al., J Am Chem Soc, 2017. 139, (44):15632-15635; Cheng, XF., et al., J Am Chem Soc, 2013. 135, (4): 1236-1239; Palkowitz, AD., et al., J Med Chem, 1994. 37(26):4508-4521)

[0329] Methyl 2-(4-methoxyphenyl)propi onate 11 (3.8 mmol, 0.74 g) were dissolved in THF (20 mL). LDA (2 eq, 7.7 mmol) was added dropwise at -78°C under argon. The reaction was stirred at 0°C for Ih, then cooled down at -78°C and iodomethane (1.5 eq, 5.7 mmol, 0.81 g) was added via syringe. The reaction was stirred at room temperature overnight, then the mixture was acidified to pH = 1 using IM aq HC1. The aqueous layer was extracted twice using dichloromethane (2 x 50 mL) and once using ethyl acetate (50 mL). The organic layers were combined and washed with water (20 mL) and brine (20 mL), then dried over MgSCU. The solvent was removed in vacuo, yielding the crude product, which was purified by chromatography on silica gel (0-30% EtOAc / hexanes) to generate the title compound as a yellow oil (514 mg, 65%).

[0330] 1H NMR (CDCl3) (Palkowitz, AD., et al., J Med Chem, 1994. 37(26):4508-4521): δ = 7.26 (d, J= 5.8, 2H, Ar), 6.86 (d, J= 5.6, 2H, Ar), 3.79 (s,3H, OMe), 3.64 (s, 3H, COOMe), 1.56 (s, 6H, Me).

[0331] 2-(4-Methoxyphenyl)-2,2-dimethylacetic acid 13 (Palkowitz, AD., et al., J Med Chem, 1994. 37(26):4508-4521; Bell, VL„ et al., J Chem Soc, 1986. (0): 1515-1522)

[0332] Methyl 2-(4-methoxyphenyl)-2,2-dimethylacetate 12 (2.5 mmol, 514 mg) was dissolved in EtOH (5 mL) and a 20% aqueous KOH (6.5 eq, 912 mg, 5 mL) was added slowly, under stirring. The reaction was stirred at room temperature for 36 h. The solvent was removed in vacuo. The remaining aqueous solution was acidified to pH = 1 using IM aqueous HC1, the aqueous layer was extracted with EtOAc (3 x 50 mL). The organic layer was separated and the combined EtOAc extracts were washed with water (15 mL), brine (15 mL) and died over MgSO4. The solvent was removed in vacuo and the crude product was purified by chromatography on silica gel (0-40% EtOAc / hexanes) to yield the title compound as a yellow solid (342 mg, 70%). H NMR (CDCl3) (Palkowitz, AD., et al., J Med Chem, 1994. 37(26):4508-4521): δ = 7.32 (d, J= 5.8, 2H, Ar), 6.86 (d, J= 5.6, 2H, Ar), 3.77 (s,3H, OMe), 1.57 (s, 6H, Me).

[0333] General Procedure for the synthesis of 4ITP (4-

[0334] (phenylacetamidomethyl)benzenesulfonamide) 4-(Aminomethyl)benzenesulfonamide hydrochloride 15 (1 eq, 4.5 mmol, 1.00 g) was suspended in dry acetonitrile (18 mL) in a 50 mL round bottom flask and the flask was purged with argon. The mixture was cooled to 0°C using an ice bath, then 4-methylmorpholine (2.1 eq, 9.4 mmol, 0.95 g) was added under stirring. After 30 min, the ice bath was removed, and the reaction mixture was stirred overnight at room temperature. Separately, phenylacetic acid 13 (1.05 eq, 4.73 mmol, 0.73 g) was treated with thionyl chloride (10 eq, 45 mmol, 5.35 g) and 1 drop of dimethylformamide in a 100 mL round bottom flask and the mixture was heated to reflux, under stirring, for Ih. Excess thionyl chloride was removed in vacuo using a rotary evaporator and the acid chloride 14 was dissolved in dry acetonitrile (3 mL) and added dropwise to the previously made solution of 4-(aminomethyl)benzenesulfonamide, at 0°C (ice bath cooling). The reaction was stirred at 0°C for 30 min, then at room temperature overnight, when a precipitate was formed. The suspension was fdtered and the precipitate was washed subsequently with acetone and water, dried under vacuum, and absorbed onto SiO2 using MeOH / CHCl3. The product was purified by flash chromatography on silica gel (0-40% MeOH / CHCh gradients). The pure fractions were combined and evaporated to dryness to yield 4ITP 16 as a while solid (55-71% (multiple batches made).

[0335] 4ITP 16: 'H-NMR (400 MHz, DMSO-d6, 5, ppm): 8.63 (t, J= 5.8 Hz, IH, NH), 7.74 (d, J= 8.4 Hz, 2H, H2,6-PhSO2NH2), 7.3 (d, J= 8.4 Hz, 2H, H3,5-PhSO2NH2), 7.20-7.35 (m, 5H, PhCH2CO), 7.29 (s, 2H, SO2NH2), 4.32 (d, J= 5.9 Hz, 2H, NHCH2), 3.49 (s, 2H, CH2CO).13C-NMR (100.6 MHz, DMSO-d6, δ, ppm): 170.2, 143.5, 142.5, 136.2, 128.9 (2C), 128.1 (2C), 127.3 (2C), 126.3, 125.5, 42.2, 41.7.

[0336] 4ITP 16: LC-MS (ESI) 305.1 (MH+), tR= 2.47 min, > 96% purity).

[0337] Determination of 4ITP solubility in water and PBS buffer.

[0338] The solubility of 4ITP in water and in PBS buffer was determined by the same HPLC method and instrument described above. A stock solution of 4ITP at a concentration of 10 mM were prepared in DMSO and serial dilutions (5 mM, 2.5 mM, 1.25 mM, 0.625 mM, 0.3125 mM and 0.156 mM) were done with the same solvent. A volume of 1 pL of these 4ITP solutions was injected in the instrument and the resulting peak areas were plotted against the corresponding concentrations to obtain a calibration curve, via the least squares linear regression analysis. 4ITP saturated solutions in deionized water and in PBS buffer were made by suspending 10 mg pure 4ITP in 1 mL water or in 1 mL PBS at pH 7.4 in two Eppendorf tubes, vortexing the suspensions for 1 min and subsequently placing them in a mini rotator (Labnet International, Edison, NJ) overnight. The next day the undissolved compound was centrifuged down using a bench centrifuge for 5 min and 10 pL of each supernatant was collected in an LCMS vial. A volume of 1 pL of these 4ITP solutions was injected in the HPLC instrument and the concentration of 4ITP in each solution was determined from the resulting peak areas via the previously constructed calibration curve. The analysis was done in triplicate and the values were reported as mean ± standard deviation.

[0339] 4ITP solubility in water: 0.057 ± 0.011 mg / mL (0.187 mM)

[0340] 4ITP solubility in PBS (pH = 7.4): 0.0399 ± 0.003 mg / mL (0.13 mM)

[0341] 4ITP lipophilicity (Log P) determination.

[0342] Log P value of 4ITP was determined using the same HPLC method and instrument described above, using an injection volume of 1 pL and a flow rate of ImL / min in all cases. DMSO solutions (1 mg / mL) of reference compounds of known log P (indomethacin, caffeine, ketoprofen, phenytoin, testosterone) were injected into the HPLC system and the retention time of each compound was determined in the same RP Cl 8 column. A calibration curve was constructed by plotting the log P values of these reference compounds against their retention time. Subsequently, 1 mg 4ITP was dissolved in 1 mL of DMSO and was injected in the same system to determine the retention time, which was interpolated within the calibration curve to determine the log P value of 4ITP.

[0343] 4ITP Log P Value: 1.69

[0344] Synthesis of 2,4-Diphenyl-N-(5-sulfamoyl-l,3,4-thiadiazole-2-yl) butaneamide 2,4-Diphenylbutanenitrile (He et al., 2015, J. Med. Chem., 58, 7341; Wu et al.,

[0345] 2014, Angew. Chem. Int. Ed., 53, 10510) KOH pellets (2.2 eq, 14.97 mmol, 0.84 g) were weighed and crushed to increase the surface area and dimethl sulfoxide DMSO (5 mL) was added over. In a separate flask, 2-(4- methoxyphenyl)acetonitrile (1 eq, 6.79 mmol, 0.92 mL) and bromoethylbenzene (1.1 eq, 7.47 mmol, 1.02 mL) were dissolved in 1 mL DMSO. This solution was added dropwise to the stirred KOH suspension at room temperature. Afterwards, the mixture was stirred at room temperature overnight. The reaction was quenched with water (20 mL). The layers were separated. The aqueous layer was extracted twice with EtOAc (10 mL). The organic layers were combined, then washed successively with water and brine. The organic layer was dried over MgSO4, filtered and concentrated in vacuo. The crude product was purified by chromatography on silica gel (0-50% EtOAc / Hexanes).

[0346] Yield: clear oil, 729 mg, 43%.

[0347] 1H NMR (CDCl3): 5 = 2.16 (m, 2H, CH2), 2.62 (m, 2H, CH2), 3.68 (t, 1H, CH), 3.80 (s, 3H, OCH3), 6.89 (m, 2H, Ar), 7.22 (m, 7H, Ar).

[0348] 2-(4-Methoxyphenyl)-4-phenylbutanoic acid (lmp32) (Vaccaro et al., 1996, J.

[0349] Med. Chem., 39, 1704)

[0350] 2,4-Diphenylbutanenitrile (1 eq, 2.9 mmol, 0.729 g) and KOH pellets (10 eq, 29 mmol, 1.628 g) were suspended in ethylene glycol (7 mL). The mixture was stirred and heated under reflux for 2h. Afterwards, EtOAc (15 mL) and water (10 mL) were added. The layers were separated. The aqueous layer was acidified to pH 4 using 1 M HC1 in water. Then the aqueous layer was extracted twice with EtOAc. The organic layers were combined and washed with diluted aqueous HC1 (< 0.01 M). The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The crude product was purified by chromatography on silica gel (0- 60% EtOAc / Hexanes). The collected product fractions were subjected to a second chromatographic separation, performed on silica gel, eluted with 0-60% EtOAc / Hexanes. Pure fractions were combined and evaporated to dryness to yield the pure acid.

[0351] Yield: yellow wax, 0.698 g, 89%.

[0352] ‘HNMR (CDCI3): δ = 2.09 (m, 1H, CH2), 2.39 (m, 1H, CH2), 2.57 (t, 2H, CH2), 3.51 (t, 1H, CH), 3.79 (s, 3H, OCH3), 6.87 (d, 2H, Ar), 7.21 (m, 7H, Ar).

[0353] 2,4-Diphenyl-N-(5-sulfamoyl-l,3,4-thiadiazole-2-yl) butaneamide

[0354] 2-(4-Methoxyphenyl)-4-phenylbutanoic acid (1.1 eq, 2.58 mmol, 0.698 g) was dissolved in acetonitrile. One drop of DMF was added. Thionyl chloride (11 eq, 25.8 mmol, 1.87 mL) was added slowly to the resulting suspension. The reaction mixture was stirred and heated under reflux for Ih. Afterwards, the solvent and excess thionyl chloride were removed in vacuo.

[0355] 5-amino-l,3,4-thiadiazole-2-sulfonamide (1 eq, 2.35 mmol, 0.423 g) was dispersed in acetonitrile (6 mL) and pyridine (1.2 eq, 2.82 mmol, 0.23 mL) was added. The acid chloride was redissolved in acetonitrile (5 mL), then added dropwise to the stirred aminosulfonamide suspension at 0°C. The reaction was stirred at room temperature overnight. The crude product was concentrated in vacuo and purified by chromatography on silica gel (0- 50% Methanol / Chloroform). The product fractions were combined and dried in vacuo. The solid residue was subjected to a third chromatographic separation on silica gel (0-60% EtOAc / hexanes) to yield the pure product. Analysis by NMR confirmed the identity and purity of the desired product.

[0356] Yield: yellow gel, 578 mg, 57%.

[0357] 'H NMR (DMSO): 8 = 2.10 (IH, m, CH2), 2.45 (m, IH, CH2), 2.51 (m, CH2; obscured by DMSO solvent peak), 3.78 (s, 3H, OCH3), 3.87 (t, IH, CH), 6.93 (d, 2H, Ar), 7.25 (m, 7H, Ar), 8.32 (s, 2H, NH2), 13.21 (s, IH, NH).

[0358] 13C NMR (DMSO): 8 = 32.82 (CHCH2CH2), 33.07 (CHCH2), 49.41 (CH), 55.03 (OCH3), 114.11 (H3COCCH), 125.88 (CH2CCHCHCH), 128.30 (CH2CCH), 128.92 (CH2CCHCH, H3COCCHCH), 130.31 (CHCCHCO), 140.98 (CH2CCH), 158.55 (NHCS), 161.06 (H3COC), 164.42 (SCSO2NH2), 172.75 (CONH).

[0359] Synthesis of 2-(4-Methoxyphenyl)-3-phenyl-N-(5-sulfamoyl-l,3,4-thiadiazol-2- yl)propenamide

[0360] 2-(4-Methoxyphenyl)-3-phenylpropanenitrile

[0361] KOH pellets (1.9 eq, 15.3 mmol, 0.86 g) were weighed and crushed. Afterwards, DMSO (5 mL) was added. In a separate flask, 2-(4-methoxyphenyl)acetonitrile (1 eq, 8.15 mmol, 1.11 mL) and benzyl bromide (1.1 eq, 8.97 mmol, 1.06 mL) were dissolved in 2 mL DMSO. This solution was added dropwise to the stirred KOH suspension at room temperature. Afterwards, the mixture was stirred at room temperature overnight. The reaction was quenched with water (20 mL), the layers were separated and the aqueous layer was extracted three times with ethyl acetate (10 mL). The organic layers were combined, washed with water and brine, dried over MgSO4 and concentrated in vacuo. The crude product was purified by chromatography on silica gel (0-40% EtOAc / Hexanes), then the useful fractions were subjected to HPLC on a RP-C18 column, 0-100% acetonitrile (0.1% TFA) / water (0.1% TFA)).

[0362] Yield: white wax, 269 mg, 14%.

[0363] ‘HNMR (CDCl3): 5 = 3.15 (m, 2H, CH2), 3.79 (s, 3H, OCH3), 3.90 (t, 1H, CH), 6.90 (m, 2H, Ar), 7.17 (m, 7H, Ar).

[0364] Alternative procedure (Wang et al., 2017, J. Am. Chem. Soc., 139, 15632; Cheng et al., 2013, J. Am. Chem. Soc., 135, 1236) 2-(4-Methoxyphenyl)acetonitrile (1 eq, 8.15 mmol, 1.11 mL) was dissolved in dry THF (25 mL). LDA (1.2 eq, 9.78 mmol, 4.89 mL) was added dropwise at -78°C under argon. The reaction was stirred at room temperature for 30 min. Benzyl bromide (1 eq, 8.15 mmol, 0.97 mL) was added at -78°C. The reaction was stirred at room temperature overnight. The reaction was quenched by acidifying the mixture to pH 1 using 10% aqueous HC1. Afterwards, the layers were separated. The aqueous layer was extracted twice using diethyl ether. The organic layers were combined and washed with water and brine, then dried over MgSCL. The solvent was removed in vacuo. The crude product was purified by chromatography on silica gel (0-40% EtOAc / hexanes), the useful fractions were combined and subjected to a second chromatography on silica gel (0-40% EtOAc / hexanes), which afforded the pure product.

[0365] Yield: white wax, 262 mg, 13%.

[0366] ‘H NMR (CDCI3): 5 = 3.14 (m, 2H, CH2), 3.81 (s, 3H, OCH3), 3.95 (t, 1H, CH), 6.87 (m, 2H, Ar), 7.25 (m, 7H, Ar).

[0367] 2-(4-Methoxyphenyl)-3-phenylpropanoic acid (Vaccaro et al., 1996, J. Med.

[0368] Chem., 39, 1704)

[0369] 2-(4-Methoxyphenyl)-3-phenylpropanenitrile (1 eq, 1.34 mmol, 0.319 g) and KOH pellets (10 eq, 13.4 mmol, 0.754 g) were suspended in ethylene glycol (3 mL). The mixture was stirred and heated under reflux for 2h. Afterwards, EtOAc (6 mL) and water (5 mL) were added. The layers were separated. The aqueous layer was acidified to pH 1.5 using 10% aqueous HC1. Then the aqueous layer was extracted twice with EtOAc. The organic layers were combined and washed with diluted aqueous HC1 (< 0.01 M) and brine. The organic layer was dried over MgSO4, filtered and concentrated in vacuo. The crude product was purified by chromatography on silica gel (0-50% EtOAc / Hexanes).

[0370] Yield: yellow solid, 243 mg, 71%. ‘H NMR (CDCh): 3 = 2.97 (dd, 1H, CH2), 3.37 (dd, 1H, CH2), 3.77 (s, 3H, OCH3), 4.10 (q, 1H, CH), 6.82 (d, 2H, Ar), 7.22 (m, 7H, Ar).

[0371] 2-(4-Methoxyphenyl)-3-phenyl-N-(5-sulfamoyl-l,3,4-thiadiazol-2- yl)propenamide

[0372] 2-(4-Methoxyphenyl)-3-phenylpropanoic acid (1.3 eq, 0.88 mmol, 0.225 g) was dissolved in acetonitrile (1 mL). One drop of DMF was added. Thionyl chloride (13 eq, 8.8 mmol, 0.64 mL) was added slowly to the suspension and the reaction mixture was stirred and heated under reflux for Ih. Afterwards, the solvent and excess thionyl chloride were removed in vacuo.

[0373] 5-amino-l,3,4-thiadiazole-2-sulfonamide (1 eq, 0.68 mmol, 0.123 g) was dispersed in acetonitrile (5 mL). Pyridine (1.3 eq, 0.88 mmol, 0.07 mL) was added. The acid chloride was redissolved in acetonitrile (2 mL), then added dropwise to the stirred aminosulfonamide suspension at 0°C. The reaction was stirred at room temperature overnight. Afterwards, the crude product was concentrated in vacuo, partitioned between EtOAc and water, with a small amount of acetonitrile to aid solubility. The desired product was obtained by chromatographic separation of the crude on silica gel (0-30% MeOH / CHCh). The identity of the product was confirmed by1H and13C NMR.

[0374] Yield: yellow crystalline solid, 129 mg, 45%.

[0375] 1H NMR (DMSO): δ = 2.56 (m, IH, CH2, obscured by DMSO peak), 3.10 (m, IH, CH2), 3.77 (s, 3H, OCH3, partially obscured by water peak), 4.25 (m, IH, CH), 6.93 (d, 2H, NH2), 7.37 (m, 9H, Ar), 13.26 (s, IH, NH).

[0376] 13C NMR (DMSO): δ = 51.79 (CHCH2), 55.02 (CHCO), 79.11 (OCH3), 113.99 (H3COCCH), 126.27 (CCHCHCH), 128.22 (CCHCHCH), 128.73 (CCHCHCH), 129.02 (H3COCCHCH), 129.93 (CCHCO), 138.81 (CH2CCH), 158.56 (NHCS), 160.86 (CSO2NH2), 164.44 (H3COCCH), 172.47 (CONH). l-(4-Methoxyphenyl)-N-(5-sulfamoyl-l ,3,4-thiadiazol)cyclobutane-l- carboxamide

[0377] Synthesis of l-(4-Methoxyphenyl)cyclobutane-l-carbomtrile (Barbasiewicz et al.,

[0378] 2006, Tetrahedron Lett., 47, 3871)

[0379] 2-(4-Methoxyphenyl)acetonitrile (1.0 eq, 13.6 mmol, 1.84 mL) and 1,3- dibromopropane (1.05 eq, 14.3 mmol, 1.45 mL) were dissolved in dry THF (50 mL). LDA (3.5 eq, 47.6 mmol, 23.8 mL) was added dropwise at -78°C under argon. The reaction was stirred at -78°C for 2h, then allowed to warm to room temperature and stirred for 2 days. Afterwards, the reaction was quenched by acidifying to pH = 1.5 with 10% aq HC1. The layers were separated, and the aqueous layer was extracted three times with DCM (10 mL). The combined organic layers were washed with water and brine, then dried over Na2SO4, filtered and concentrated in vacuo. The crude product was purified by chromatography on silica gel (0-30% EtOAc / hexanes). Both the cyclized product and the monoalkylated products could be isolated, yet the desired cyclized product remained impure. The monoalkylated product (2.2 mmol, 0.578 g) was dried in vacuo, then redissolved in dry THF (15 mL). LDA (1.3 eq, 2.8 mmol, 5.6 mL) was again added at -78°C under argon. The reaction was stirred at room temperature overnight. The reaction was quenched by acidifying to pH = 1.5 with 10% aq HC1. The layers were separated. The aqueous layer was extracted three times with DCM (5 mL). The combined organic layers were washed with water and brine. The organic layer was dried over Na2SO4, filtered and concentrated in vacuo. The new product was combined with the previously obtained cyclized product and purified by chromatography on silica gel (0-40% EtOAc / hexanes). The product identity and purity were confirmed by NMR.

[0380] Yield: clear oil, 302 mg, 12%.

[0381] 'H NMR (CDCl3): 5 = 2.06 (m, 1H, CH2), 2.40 (m, 1H, CH2), 2.56 (m, 2H, CH2), 2.62 (m, 2H, CH2), 3.81 (s, 3H, OCH3), 6.92 (d, 2H, Ar), 7.34 (d, 2H, Ar). l-(4-Methoxyphenyl)cyclobutane-l -carboxylic acid (lmp56) (Dubois et al., 2020,

[0382] Org. Lett., 22, 5279) l-(4-Methoxyphenyl)cyclobutane-l -carbonitrile (1.0 eq, 1.61 mmol, 0.302 g) was dissolved in ethylene glycol (2 mL). KOH pellets (10 eq, 16.1 mmol, 0.905 g) were crushed and added to the solution. The reaction was stirred and heated under reflux for 2h. Afterwards, EtOAc (15 mL) and water (10 mL) were added and the layers were separated. The aqueous layer was extracted three times with EtOAc (5 mL). The aqueous layer was then acidified to pH = 1 using 10% aq HC1. The aqueous layer was again extracted three times with EtOAc (5 mL). The second organic extract was washed with water and brine, dried over Na2SO4 and concentrated in vacuo. The crude product was purified by chromatography on silica gel (0-40% 1% AcOH in EtOAc / hexanes). To remove the acetic acid from the product, the product crystals were washed with cold diethyl ether, and dried under vacuum.

[0383] Yield: white crystals, 111 mg, 33%.

[0384] 1H NMR (CDCl3): 5 = 1.88 (m, 1H, CH2), 2.06 (m, 1H, CH2), 2.50 (m, 2H, CH2), 2.85 (m, 2H, CH2), 3.80 (s, 3H, OCH3), 6.87 (d, 2H, Ar), 7.26 (d, 2H, Ar). l-(4-Methoxyphenyl)-A-(5-sulfamoyl-l,3,4-thiadiazol)cyclobutane-l- carboxamide (lmp59) l-(4-Methoxyphenyl)cyclobutane-l -carboxylic acid (1.2 eq, 0.538 mmol, 0.111 g) was dissolved in acetonitrile (0.5 mL). DMF (1 drop) and thionyl chloride (12 eq, 5.38 eq, 0.39 mL) were added. The mixture was stirred and heated under reflux for Ih. Afterwards, acetonitrile and excess thionyl chloride were removed in vacuo. 5-amino-l,3,4-thiadiazole-2- sulfonamide (1 eq, 0.448 mmol, 81 mg) and pyridine (1.2 eq, 0.538 mmol, 0.04 mL) were combined and acetonitrile (2 mL) was added. The acid chloride was dissolved in acetonitrile (1 mL), then added dropwise under stirring to the thiadiazol mixture at 0°C. The mixture was stirred at room temperature overnight. The solvent was removed in vacuo, then the crude product was partitioned between ethyl acetate and water. A small amount of acetonitrile was added to aid solubility. The layers were separated, and the organic layer was concentrated in vacuo. The crude product was purified by chromatography on silica gel (0-30% MeOH / chloroform). The product was confirmed by NMR.

[0385] Yield: light-brown crystals, 39 mg, 24%.

[0386] ‘HNMR (DMSO): 5 = 1.89 (m, 2H, CH2), 2.56 (m, 2H, CH2, obscured by DMSO peak), 2.89 (m, 2H, CH2), 3.78 (s, 3H, OCH3), 6.97 (m, 2H, Ar), 7.40 (m, 2H, Ar), 8.32 (m, 2H, NH2, obscured by chloroform peak), 12.96 (bs, 1H, NH).

[0387] 13C NMR (DMSO): 5 = 15.65 (CCH2CH2), 31.67 (CCH2CH2), 52.13 (CCH2CH2), 55.07 (OCH3), 1 13.91 (H3COCCH), 127.40 (H3COCCH CH), 133.99 (CHCCCH2), 158.19 (NHCS), 161.50 (H3COCCH), 164.43 (SCSO2NH2), 175.22 (CONH).

[0388] Example 3: Mitochondrial CA-V inhibition mitigates Alzheimer’s Disease progression via modulation of neurovascular inflammation and microglia function

[0389] The neurovascular unit is known to become dysfunctional in Alzheimer’s Disease (AD). Amyloidosis disrupts the function of neurovascular cells via extrinsic and intrinsic apoptosis, and induction of blood brain barrier (BBB) permeability. Example 1 demonstrated that pan-Carbonic Anhydrase (CA) inhibitors prevent mitochondria-mediated apoptosis and inflammatory activation in multiple neurovascular cell types. Therefore, without being bound by a particular theory, it was hypothesized that the mitochondrial CA isoforms, CA-VA and -VB mediate AP-induced toxicity. Importantly, mitochondrial isoform CA-VB is increased in models of amyloidosis. Example 1 demonstrated that selective CA-V inhibition and -VB KO are effective to rescue cerebrovascular cell death and BBB dysfunction. In the example herein, without being bound by a particular theory, it was hypothesized that CA-V inhibition would prevent apoptosis in 3xTG mice brains. Furthermore, it was hypothesized that CA-V inhibition would ameliorate neuroinflammation and microglia function, preventing the progression of AD pathology and cognitive impairment.

[0390] 3xTG mice were treated with a CA-V inhibitor at 20mg / kg in their diet from 6-16 months of age. Caspase-3 activation, GFAP and IB Al were evaluated through immunohistochemistry. Additionally, vascular stress markers such as VCAM-1 and ICAM-1 through western blot were evaluated. To measure the extent of amyloid and tau accumulation, soluble and insoluble fractions were extracted from the brain, and the expected species were quantified using ELISA sandwich assay. To assess microglia function in vitro, the human microglia cell line (HMC3) WT and CA-VB KO, in the presence or absence of the same CA-V inhibitor, were used.

[0391] Strikingly, CA-V inhibition prevented cognitive decline (Figure 12). Additionally, apoptosis and gliosis was prevented in the brains of treated 3xTG mice. Furthermore, mitigation of insoluble amyloid and tau accumulation was observed (Figure 14). It was also observed that, when HMC3 cells were treated with amyloid in the presence of CA-V inhibitor there was an enhancement in lysosomal maker CD68 (Figure 16). Strikingly, in CA-VB KO cells, the CA-V inhibitor did not have the same effect, implying a CA-VB specific mechanism.

[0392] These findings improve understanding of the function of CA-V in the development of AD pathology and highlight the potential of CA-VB inhibition to improve microglial clearance and reduce brain neuroinflammation. Overall, it was demonstrated that mitochondrial CA-V is emerging as a promising target against AD and other cerebrovascular indications.

[0393] In conclusion, the experiments herein demonstrated (1) CA-V inhibition is not toxic, and prevents cognitive decline in 3xTG mice, (2) CA-V inhibition prevents caspase-3 activation, vascular stress and glial cell activation correlating with a reduction in insoluble amyloid and tau accumulation in 3xTG mice, (3) CA-VB protein expression is increased in human microglia cells treated with amyloid and further mitigated with CA-V inhibitor, (4) CD68 expression is increased in cells treated with CA-V inhibitor as well as CA-VB KO cells, indicating increased clearance and function when CA-V is reduced, and (5) CA-V inhibition and -VB KO reduce neuroinflammation and improve microglia phenotype resulting in a prevention of AD pathology in 3xTG mice.

[0394] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMSWhat is claimed is:

1. A composition for treating or preventing a neurological or psychiatric disease or disorder comprising a mitochondrial carbonic anhydrase V (CA-V) inhibitor.

2. The composition of claim 1, wherein the mitochondrial CA-V inhibitor selectively inhibits at least one or more of CA-VA and CA-VB.

3. The composition of claim 1, wherein the mitochondrial CA-V inhibitor is selected from the group consisting of a chemical compound, a protein, a peptide, a peptidomimetic, an antibody, a ribozyme, a small molecule chemical compound, a nucleic acid, a vector, an antisense molecule, an siRNA, an shRNA, and a guide RNA.

4. The composition of claim 1, wherein the mitochondrial CA-V inhibitor is a chemical compound.

5. The composition of claim 5, wherein the chemical compound is 4- phenylacetamidomethyl-benzenesulfonamide (4ITP), derivative, and analogs thereof.

6. The composition of claim 1, wherein the neurological or psychiatric disease or disorder is selected from the group consisting of: Alzheimer's disease (AD), Mild Cognitive Impairment (MCI), Parkinson's disease (PD), Huntington's disease (HD), prion-caused diseases, frontotemporal dementia (FTD), Lewy body dementia, vascular dementias, white matter disease, traumatic brain injury, post-traumatic stress, stroke, tauopathies, Down Syndrome, Amyotrophic Later Sclerosis (ALS), chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), multiple system atrophy (MSA), corticobasal degeneration (CBGD), Pick's disease, olivopontocerebellar atrophy (OPCA), senile dementia of the Alzheimer type, progressive supranuclear palsy (Steel-Richardson-Olszewski), corticodentatonigral degeneration, Hallervorden-Spatz disease, striatonigral degeneration, torsion dystonia, spasmodic torticollis and other dyskinesis, familial tremor, Gilles de la Tourette syndrome, cerebellar cortical degeneration, spinocerebellar degeneration, Shy-Drager syndrome, spinal muscular atrophy, primary lateral sclerosis, hereditary spastic paraplegia, peroneal muscular atrophy (Charcot-Marie-Tooth), hypertrophic interstitial polyneuropathy (Dejerine-Sottas), chronic progressive neuropathy, pigmentary degeneration of the retina (retinitis pigmentosa), hereditary optic atrophy (Leber's disease), Cognitive Dysfunction Syndrome, White dog shaker syndrome, degenerative myelopathy, neuroaxonal dystrophy, cerebellar degeneration, cerebellar abiotrophy, cerebral amyloid angiopathy (CAA), and amyloid related imaging abnormalities (ARIA).

7. The composition of claim 1, wherein the neurological or psychiatric disease or disorder is a neurodegenerative disease.

8. The composition of claim 1, wherein the neurological or psychiatric disease or disorder is Alzheimer’s disease.

9. A method of treating a neurological or psychiatric disease or disorder in a subject in need thereof comprising administering to the subject a composition comprising a mitochondrial carbonic anhydrase V (CA-V) inhibitor.

10. The method of claim 9, wherein the mitochondrial CA-V inhibitor selectively inhibits at least one or more of CA-VA and CA-VB.11 . The method of claim 9, wherein the mitochondrial CA-V inhibitor is selected from the group consisting of a chemical compound, a protein, a peptide, a peptidomimetic, an antibody, a ribozyme, a small molecule chemical compound, a nucleic acid, a vector, an antisense molecule, an siRNA, an shRNA, and a guide RNA.

12. The method of claim 9, wherein the mitochondrial CA-V inhibitor is a chemical compound.

13. The method of claim 12, wherein the chemical compound is 4- phenylacetamidomethyl-benzenesulfonamide (4ITP).

14. The method of claim 9, wherein the neurological or psychiatric disease or disorder is selected from the group consisting of: Alzheimer's disease (AD), Mild Cognitive Impairment (MCI), Parkinson's disease (PD), Huntington's disease (HD), prion-caused diseases, frontotemporal dementia (FTD), Lewy body dementia, vascular dementias, white matter disease, traumatic brain injury, post-traumatic stress, stroke, tauopathies, Down Syndrome, AmyotrophicLater Sclerosis (ALS), chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), multiple system atrophy (MSA), corticobasal degeneration (CBGD), Pick's disease, olivopontocerebellar atrophy (OPCA), senile dementia of the Alzheimer type, progressive supranuclear palsy (Steel-Richardson-Olszewski), corticodentatonigral degeneration, Hallervorden-Spatz disease, striatonigral degeneration, torsion dystonia, spasmodic torticollis and other dyskinesis, familial tremor, Gilles de la Tourette syndrome, cerebellar cortical degeneration, spinocerebellar degeneration, Shy-Drager syndrome, spinal muscular atrophy, primary lateral sclerosis, hereditary spastic paraplegia, peroneal muscular atrophy (Charcot- Marie-Tooth), hypertrophic interstitial polyneuropathy (Dejerine-Sottas), chronic progressive neuropathy, pigmentary degeneration of the retina (retinitis pigmentosa), hereditary optic atrophy (Leber's disease), Cognitive Dysfunction Syndrome, White dog shaker syndrome, degenerative myelopathy, neuroaxonal dystrophy, cerebellar degeneration, cerebellar abiotrophy, cerebral amyloid angiopathy (CAA), and amyloid related imaging abnormalities (ARIA).

15. The method of claim 9, wherein the neurological or psychiatric disease or disorder is a neurodegenerative disease.

16. The method of claim 9, wherein the neurological or psychiatric disease or disorder is Alzheimer’s disease.

17. A method for inhibiting blood-brain barrier permeability in a subject in need thereof comprising administering to the subject a composition comprising a mitochondrial carbonic anhydrase V (CA-V) inhibitor.

18. The method of claim 17, wherein the mitochondrial CA-V inhibitor selectively inhibits at least one or more of CA-VA and CA-VB.

19. The method of claim 17, wherein the mitochondrial CA-V inhibitor is selected from the group consisting of a chemical compound, a protein, a peptide, a peptidomimetic, an antibody, a ribozyme, a small molecule chemical compound, a nucleic acid, a vector, an antisense molecule, an siRNA, an shRNA, and a guide RNA.

20. The method of claim 17, wherein the mitochondrial CA-V inhibitor is a chemical compound.21 . The method of claim 20, wherein the chemical compound is 4- phenylacetamidomethyl-benzenesulfonamide (4ITP).

22. A method for inhibiting one or more of neuronal degeneration, glial degeneration, and vascular degeneration in a subject in need comprising administering to the subject a composition comprising a mitochondrial carbonic anhydrase V (CA-V) inhibitor.

23. The method of claim 22, wherein the mitochondrial CA-V inhibitor selectively inhibits at least one or more of CA-VA and CA-VB.

24. The method of claim 22, wherein the mitochondrial CA-V is selected from the group consisting of a chemical compound, a protein, a peptide, a peptidomimetic, an antibody, a ribozyme, a small molecule chemical compound, a nucleic acid, a vector, an antisense molecule, an siRNA, an shRNA, and a guide RNA.

25. The method of claim 22, wherein the mitochondrial CA-V inhibitor is a chemical compound.

26. The method of claim 25, wherein the chemical compound is 4- phenylacetamidomethyl -benzenesulfonamide (4ITP).

27. A method for inhibiting one or more of mitochondrial dysfunction in a cell, caspase activation in a cell, Cytochrome C (CytC) release in a cell, insoluble protein accumulation in a cell, and cell death in a subject in need thereof comprising administering to the subject a composition comprising a mitochondrial carbonic anhydrase V (CA-V) inhibitor.

28. The method of claim 27, wherein the mitochondrial CA-V inhibitor selectively inhibits at least one or more of CA-VA and CA-VB.

29. The method of claim 27, wherein the mitochondrial CA-V inhibitor is selected from the group consisting of a chemical compound, a protein, a peptide, a peptidomimetic, an antibody, a ribozyme, a small molecule chemical compound, a nucleic acid, a vector, an antisense molecule, an siRNA, an shRNA, and a guide RNA.

30. The method of claim 27, wherein the mitochondrial CA-V inhibitor is a chemicalcompound.

31. The method of claim 30, wherein the chemical compound is 4- phenylacetamidomethyl -benzenesulfonamide (4ITP).

32. The method of claim 27, wherein the cell is selected from the group consisting of neuronal cells, glial cells, astrocyte, or microglia, endothelial cells, and smooth muscle cells.

33. The method of claim 32, wherein the neuronal cell is a dopaminergic neuron.

34. The method of claim 32, wherein the endothelial cell is one or more selected from the group consisting of a microvascular endothelial cell and a cerebral endothelial cell.

35. The method of claim 32, wherein the cell is in a subject.

36. The method of claim 27, wherein the caspase is one or more of caspase 3, caspase7, and caspase 9.

37. The method of claim 27, wherein the mitochondrial dysfunction is a loss of mitochondrial membrane potential.

38. The method of claim 27, wherein the mitochondrial dysfunction is an increase in mitochondrial H2O2 production.

39. A compound represented by General Formula I:whereinA represents a ring selected from the group consisting of substituted or unsubstituted: aryl, cycloalkyl, cycloalkenyl, heteroaryl, and heterocyclyl;L represents a single bond or a linking group selected from the group consisting of alkylene, cycloalkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene,heteroarylene, silyl, amine, amide, ester, ether, carbonyl, carbamate, sulfamate, thioether, thioester, disulfide, hydrazine, urea, thiourea, phosphate, phosphonate ester, poly(alkyl ether), heteroatom, and combinations thereof; and any two adjacent substituents may optionally join to form a ring.

40. The compound of claim 39, wherein the compound is represented by General Formula la:wherein R1, R2, R3, R4, and R5are each independently selected from the group consisting of hydrogen, deuterium, Ci-6 alkyl, C2-6 alkenyl, alkynyl, hydroxy, thio, C1-6 alkoxy, halo, haloCi-6 alkyl, haloCi-6 alkoxy, C3-8 cycloalkyl, cyano, amino, nitro, carbonyl, carboxylate, C1-6 alkyl ester, and combinations thereof.

41. The compound of claim 39, wherein L represents a single bond.

42. The compound of claim 39, wherein L is selected from the group consisting of CRL1RL2, NRL1, O, and combinations thereof; wherein RL1and RL2are each independently selected from the group consisting of hydrogen, deuterium, C1-6 alkyl, C2-6 alkenyl, alkynyl, hydroxy, thio, C1-6 alkoxy, halo, haloCi-6 alkyl, haloCi-6 alkoxy, C3-8 cycloalkyl, cyano, amino, and combinations thereof.

43. The compound of claim 39, wherein at least one of R1, R2, R3, R4, and R5are each independently selected from the group consisting of carboxyl, amino, methoxy, cyano, fluoro, chloro, bromo, methyl, trifluoro, methoxycarbonyl, and nitro.

44. The compound of claim 39, wherein R3is not hydrogen.

45. The compound of claim 39, wherein the compound is represented by GeneralFormula lb:wherein RL1and RL2are each independently selected from the group consisting of hydrogen, deuterium, Ci-6 alkyl, C2-6 alkenyl, alkynyl, hydroxy, thio, C1-6 alkoxy, halo, haloCi- 6 alkyl, haloCi-6 alkoxy, C3-8 cycloalkyl, cyano, amino, and combinations thereof46. The compound of claim 45, wherein at least one of RL1and RL2is methyl.

47. The compound of claim 39, wherein the compound is selected from the group consisting of the following structures:

47. A method of treating or preventing a neurological or psychiatric disease or disorder in a subject in need thereof comprising administering to the subject a compositioncomprising a compound represented by General Formula T:whereinA represents a ring selected from the group consisting of substituted or unsubstituted: aryl, cycloalkyl, cycloalkenyl, heteroaryl, and heterocyclyl;L represents a single bond or a linking group selected from the group consisting of alkylene, cycloalkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, silyl, amine, amide, ester, ether, carbonyl, carbamate, sulfamate, thioether, thioester, disulfide, hydrazine, urea, thiourea, phosphate, phosphonate ester, poly(alkyl ether), heteroatom, and combinations thereof; and any two adjacent substituents may optionally join to form a ring.

48. A method for inhibiting blood-brain barrier permeability in a subject in need thereof comprising administering to the subject a composition comprising a compound represented by General Formula I:whereinA represents a ring selected from the group consisting of substituted or unsubstituted: aryl, cycloalkyl, cycloalkenyl, heteroaryl, and heterocyclyl;L represents a single bond or a linking group selected from the group consisting of alkylene, cycloalkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, silyl, amine, amide, ester, ether, carbonyl, carbamate, sulfamate, thioether, thioester, disulfide, hydrazine, urea, thiourea, phosphate, phosphonate ester, poly(alkyl ether), heteroatom, and combinations thereof; andany two adjacent substituents may optionally join to form a ring.

49. A method for inhibiting one or more of neuronal degeneration, glial degeneration, and vascular degeneration in a subject in need thereof comprising administering to the subject a composition comprising a compound represented by General Formula I:whereinA represents a ring selected from the group consisting of substituted or unsubstituted: aryl, cycloalkyl, cycloalkenyl, heteroaryl, and heterocyclyl;L represents a single bond or a linking group selected from the group consisting of alkylene, cycloalkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, silyl, amine, amide, ester, ether, carbonyl, carbamate, sulfamate, thioether, thioester, disulfide, hydrazine, urea, thiourea, phosphate, phosphonate ester, poly(alkyl ether), heteroatom, and combinations thereof; and any two adjacent substituents may optionally join to form a ring.

50. A method for inhibiting one or more of mitochondrial dysfunction in a cell, caspase activation in a cell, Cytochrome C (CytC) release in a cell, insoluble protein accumulation in a cell, and cell death in a subject in need thereof comprising administering to the subject a composition comprising a compound represented by General Formula I:whereinA represents a ring selected from the group consisting of substituted or unsubstituted: aryl, cycloalkyl, cycloalkenyl, heteroaryl, and heterocyclyl;L represents a single bond or a linking group selected from the group consisting of alkylene, cycloalkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, silyl, amine, amide, ester, ether, carbonyl, carbamate, sulfamate, thioether, thioester, disulfide, hydrazine, urea, thiourea, phosphate, phosphonate ester, poly(alkyl ether), heteroatom, and combinations thereof; and any two adjacent substituents may optionally join to form a ring.

51. A method for one or more of inhibiting gliosis, improving microglia clearance, or increasing CD68 expression in microglia comprising administering a composition comprising a mitochondrial carbonic anhydrase V (CA-V) inhibitor.

52. The method of claim 51, wherein the mitochondrial CA-V inhibitor selectively inhibits at least one or more of CA-VA and CA-VB.

53. The method of claim 51, wherein the mitochondrial CA-V is selected from the group consisting of a chemical compound, a protein, a peptide, a peptidomimetic, an antibody, a ribozyme, a small molecule chemical compound, a nucleic acid, a vector, an antisense molecule, an siRNA, an shRNA, and a guide RNA.

54. The method of claim 51, wherein the mitochondrial CA-V inhibitor is a chemical compound.

55. The method of claim 51, wherein the chemical compound is 4- phenylacetamidomethyl-benzenesulfonamide (4ITP).

56. A method for one or more of inhibiting gliosis, improving microglia clearance, or increasing CD68 expression in microglia comprising administering a composition comprising a compound represented by General Formula I:whereinA represents a ring selected from the group consisting of substituted or unsubstituted: aryl, cycloalkyl, cycloalkenyl, heteroaryl, and heterocyclyl;L represents a single bond or a linking group selected from the group consisting of alkylene, cycloalkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, silyl, amine, amide, ester, ether, carbonyl, carbamate, sulfamate, thioether, thioester, disulfide, hydrazine, urea, thiourea, phosphate, phosphonate ester, poly(alkyl ether), heteroatom, and combinations thereof; and any two adjacent substituents may optionally join to form a ring.

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