Biomarker-based treatment of focal segmental glomerulosclerosis and diabetic kidney disease

Abstract

To provide means of selecting and treating subjects suffering from a kidney disease.SOLUTION: Disclosed are compounds having structural formulas (I)-(XI), and related pharmaceutical compositions. Also disclosed are methods of selecting and treating human subjects suffering from a kidney disease, using the compounds of formulas (I)-(XI), and methods of determining the efficacy of TRPC5 inhibitor therapies using the same.SELECTED DRAWING: None

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 910,758, filed October 4, 2019. [Background technology]

[0002] Mammalian TRP channel proteins form six-spanning, cation-permeable channels that can be classified into six subfamilies (TRPC, TRPV, TRPM, TRPA, TRPP, and TRPML) based on amino acid sequence homology. Recent studies of TRP channels indicate that they are involved in numerous fundamental cellular functions and are thought to play important roles in the pathophysiology of many diseases. Many TRPs are expressed along various parts of the nephron in the kidney, and growing evidence suggests that these channels are involved in inherited and acquired renal disorders. TRPC6, TRPM6, and TRPP2 are involved in hereditary focal segmental glomerulosclerosis (FSGS), hypomagnesemia with secondary hypocalcemia (HSH), and polycystic kidney disease (PKD), respectively.

[0003] Podocyte damage and loss are central components of the pathogenesis of FSGS and diabetic kidney disease (DKD) (Jefferson et al. 2014; Weil et al. 2012; Lin et al. 2016). FSGS is considered a primary podocyte pathology, indicating that the pathogenesis is the result of podocyte dysfunction and damage. While several mechanisms have been hypothesized to induce podocyte damage, a recently described genetic cause of the disease is mutation of Rho-GTPase, a regulator of actin cytoskeleton dynamics (Weiner et al. 2018). Loss of properly functioning Rho-GTPases (e.g., mutations in ARHGAP24 and ARHGDIA) leads to unopposed activation of Rac1 in podocytes, which promotes cytoskeletal remodeling and podocyte death via elevation of cytosolic calcium and reactive oxygen species (Akilesh et al. 2011; Gee et al. 2013; Greka et al. 2011). Many familial and sporadic forms of FSGS have been associated with genetic dysregulation of Rac1, highlighting its importance as a driver in this disease (Lovric et al. 2015).

[0004] Both in vitro and in vivo experimental data support the role of Rac1 activation in the pathogenesis of DKD. In a diabetic environment, cultured podocytes undergo cytoskeletal remodeling and epithelial-mesenchymal transition (e.g., epithelial-mesenchymal transition), both of which are abrogated by knockdown of Rac1 (Liu et al. 2013). Furthermore, podocyte-specific Rac1-deficient mice are protected from diabetic nephropathy (Liu et al. 2018). Activation of the Rac1 pathway is also directly mediated by activation of TRPC5 channels via either epidermal growth factor receptor, toll-like receptor (TLR), or AT1R (Liu et al. 2018), all of which are involved in the pathogenesis of DKD (Greka et al. 2011). Synaptopodin, an actin-associated podocyte protein that is degraded after TRPC5 activation, has been detected in the urine of DKD patients (Zheng et al. 2011), further supporting the relevance of Rac1 activation in DKD. Given the role of TRPC5-Rac1 signaling in mediating damage in DKD, inhibition of TRPC5 represents a viable therapeutic option in this area of ​​high unmet medical need.

[0005] Based on United States (US) Renal Data System estimates, FSGS accounts for 4% of adult ESKD cases and 12% of pediatric ESKD cases (USRDS 2018a; USRDS 2018b). The overall incidence of FSGS is estimated to be 0.2 / 100,000 / year and 1.1 / 100,000 / year, with this range attributed to geographic variations in biopsy rates and likely genetic differences between populations (McGrogan et al. 2011; Rosenberg 2017). In a study analyzing renal biopsy diagnoses by glomerular disease subtype in the United States and Canada, 19.1% were FSGS (O'Shaughnessy et al. 2018). FSGS accounts for an estimated 40% of nephrotic syndrome cases in adults and 20% of nephrotic syndrome cases in children (Kitiyakara et al. 2003).

[0006] Currently, there are no approved therapies specifically indicated for the treatment of FSGS in the United States. Generally, for both pediatric and adult patients, initial treatment involves renin-angiotensin-aldosterone system (RAAS) blockade and corticosteroids (KDIGO 2012; D'Agati et al. 2011). In FSGS, the response to corticosteroids is often incomplete, or if remission is achieved, patients frequently relapse when treatment is discontinued, thus potentially becoming dependent on long-term corticosteroid administration. Patients with FSGS who do not respond to corticosteroids are administered calcineurin inhibitors (CNIs) or, in some cases, other immunomodulatory agents to achieve a reduction in proteinuria (D'Agati et al. 2011; Gipson et al. 2011a; Ochi et al. 2012). Overall, remission of proteinuria can be achieved with corticosteroids or CNIs, but the toxicity associated with long-term use limits the chronic use of these agents at effective dose levels (Gipson et al. 2011a; Gipson et al. 2007). When the need for treatment outweighs the associated toxicity, patients, particularly children, suffer long-term consequences to their health. Given the importance of achieving meaningful reduction in proteinuria, the limited effectiveness of currently available treatments, and the significant side effect profile associated with these agents, there is a need to develop novel therapies to treat patients of all ages with TR-MCD or FSGS.

[0007] Diabetes, with an estimated global prevalence of 415 million patients, is a leading cause of morbidity and mortality both in the United States and worldwide (Ogurtsova et al. 2017). Diabetic kidney disease (DKD) is a major long-term consequence of diabetes, estimated to develop in approximately 40% of diabetic patients and associated with significantly increased rates of ESKD, cardiovascular complications, and premature death (Alicic et al. 2017). Despite overall improvements in the control of hyperglycemia and hypertension, patients continue to experience progressive loss of kidney function. Compared with individuals with diabetes but without kidney disease, patients with DKD have an elevated risk of death, with an adjusted 10-year cumulative mortality rate reaching 47% (Afkarian et al. 2013). These data support the need for a renewed focus on targeted drug development for patients with diabetes and kidney disease. Summary of the Invention [Means for solving the problem]

[0008] One aspect of the invention is a method of selecting and treating a human subject suffering from a kidney disease. In some embodiments, the method comprises: a. selecting a subject if the subject has a urinary level of one or more biomarkers selected from Rac1, Rac1-GTP, phospho-LIM kinase 1, and phospho-cofilin above a predetermined threshold; and b. administering to the selected subject a pharmaceutical composition comprising a TRPC5 inhibitor or a calcineurin inhibitor and a pharmaceutically acceptable carrier; Includes.

[0009] In one aspect, the present invention provides a method of treating a human subject suffering from a kidney disease, comprising: only if the subject is determined to have a pre-treatment urinary level of one or more biomarkers selected from Rac1, Rac1-GTP, phospho-LIM kinase 1, and phospho-cofilin above a predetermined threshold, TRPC5 inhibitors or calcineurin inhibitors; and a pharmaceutically acceptable carrier; The present invention relates to a method comprising administering to a subject a pharmaceutical composition comprising:

[0010] In some embodiments, the renal disease is diabetic nephropathy, focal segmental glomerulosclerosis, minimal change disease, membranoproliferative glomerulonephritis, membranous nephropathy, other hepatitis C virus-associated glomerulopathies, or Alport syndrome.

[0011] In one aspect, the invention provides a method for determining the efficacy of TRPC5 inhibitor treatment in a human subject suffering from kidney disease, wherein prior to initiating treatment, the subject is determined to have a pre-treatment urinary level of one or more biomarkers selected from Rac1, Rac1-GTP, phospho-LIM kinase 1, and phospho-cofilin above a predetermined threshold; a. Obtaining urinary levels of selected biomarkers in human subjects at time points after initiation of TRPC5 treatment; b. comparing the level of the selected biomarker in step a. with the urinary level of the selected biomarker before treatment; c. determining that the TRPC5 inhibitor treatment is efficacious if the level of the selected biomarker in step a. is lower than the urinary level of the selected biomarker before treatment; The present invention relates to a method comprising:

[0012] In one aspect, the invention provides a method for determining the efficacy of TRPC5 inhibitor treatment in a human subject suffering from kidney disease, wherein prior to initiating treatment, the subject is determined to have a pre-treatment urinary level of one or more biomarkers selected from Rac1, Rac1-GTP, phospho-LIM kinase 1, and phospho-cofilin above a predetermined threshold; a. Obtaining urinary levels of selected biomarkers in human subjects at time points after initiation of TRPC5 treatment; and b. determining that the TRPC5 inhibitor treatment is efficacious if the level of the selected biomarker in step a. is lower than a predetermined threshold value for the selected biomarker; The present invention relates to a method comprising:

[0013] The method is effective in a variety of subjects, including mammals, e.g., humans, and other animals, such as laboratory animals (e.g., mice, rats, rabbits, or monkeys), or domestic and livestock animals (e.g., cats, dogs, goats, sheep, pigs, cows, or horses). In some embodiments, the subject is a human.

[0014] The present invention provides several advantages.The preventive and therapeutic methods described herein are effective for treating kidney disease (e.g., proteinuria), and have minimal side effects, if any.In addition, the methods described herein are effective for identifying compounds that treat kidney disease, anxiety, depression, or cancer, or reduce the risk of developing them.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention belongs.Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below.All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, will prevail.In addition, the materials, methods, and examples are merely illustrative and are not intended to be limiting.

[0016] Other features, objects, and advantages of the invention will become apparent from the detailed description and claims. [Brief explanation of the drawings]

[0017] [Figure 1A]Scatter plots showing urinary Rac1 levels in healthy individuals (circles) and human patients with DN (squares), FSGS (diamonds) and Alport syndrome (triangles). [Figure 1B] Scatter plots showing urinary Rac1 levels in a larger number of healthy individuals (circles), a larger number of DN patients (squares), a larger number of FSGS patients (diamonds), PKD patients (hexagons) and an equal number of Alport syndrome patients (triangles) are shown. [Figure 2] Shown is the Rac1:creatinine ratio in urine of naive rats over time after treatment with 10 mg / kg Compound 1 or vehicle control. [Figure 3] 1 shows the Rac1:creatinine ratio in urine of DOCA-treated DOCA-salt hypertensive rats over time after treatment with 10 mg / kg Compound 1 or vehicle control. [Figure 4A] 1 shows the change in urinary Rac1:creatinine ratio in healthy subjects over time after treatment with a single oral dose of 20 mg of Compound 1 or placebo, expressed as a percentage of the pre-treatment Rac1:creatinine ratio. [Figure 4B] 1 shows the change in Rac1 in the urine of healthy subjects over time after treatment with a single oral dose of placebo, 5 mg of Compound 1 as a liquid suspension, or 20, 40, or 80 mg of Compound 1 as tablets, expressed as a percentage of the pre-treatment Rac1 concentration. [Figure 5] The figure shows the comparison of the amount of Rac1 in the urinary extracellular vesicle fraction and the supernatant in healthy individuals. [Figure 6] 1 shows the daily amount of albumin excreted in the urine of ZDSD rats over time after treatment with different doses of Compound 1 (3 mg / kg or 10 mg / kg) or control vehicle. [Figure 7] 1 shows the daily amount of albumin excreted in the urine of DOCA-treated DOCA-salt hypertensive rats over time after treatment with different doses of Compound 1 (3 mg / kg or 10 mg / kg) or control vehicle. [Figure 8]1 shows the urinary protein:creatinine ratio ("UPCR") over time in COL4A4 knockout mice after treatment with different doses of Compound 1 (3 mg / kg or 10 mg / kg) or control vehicle. [Figure 9] 1 shows the daily amount of albumin excreted in the urine of DOCA-treated DOCA-salt hypertensive rats over time after treatment with different doses of Compound 2 (10 mg / kg, or 60 mg / kg, increased to 100 mg / kg after 1 week) or control vehicle. [Figure 10] 1 shows the daily amount of albumin excreted in the urine of DOCA-treated DOCA-salt hypertensive rats over time after treatment with different doses of Compound 3 (30 mg / kg), eplerenone (50 mg / kg BID), or control vehicle. [Figure 11] 1 shows the daily amount of protein excreted in the urine of DOCA-treated DOCA-salt hypertensive rats over time after treatment with different doses of Compound 4 (20 mg / kg, 50 mg / kg, or 100 mg / kg) or control vehicle. [Figure 12] 1 shows the daily amount of albumin excreted in the urine of DOCA-treated DOCA-salt hypertensive rats over time after treatment with cyclosporine A (3 mg / kg), tacrolimus (0.3 mg / kg reduced to 0.1 mg / kg after 14 days), or control vehicle. [Figure 13-1] 1 shows urinary Rac1 levels in six human subjects diagnosed with active acute kidney injury after a positive PCR test for COVID-19. [Figure 13-2] Continuation of Figure 13-1. DETAILED DESCRIPTION OF THE INVENTION

[0018] definition The term "acyl" is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.

[0019] The term "acylamino" is art-recognized and refers to an amino group substituted with an acyl group and may be represented, for example, by the formula hydrocarbylC(O)NH-.

[0020] The term "acyloxy" is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)O-, preferably alkylC(O)O-.

[0021] The term "alkoxy" refers to an alkyl group, preferably a lower alkyl group, having an oxygen attached thereto. Representative alkoxy groups include methoxy, trifluoromethoxy, ethoxy, propoxy, tert-butoxy, and the like.

[0022] The term "alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group and can be represented by the general formula alkyl-O-alkyl.

[0023] The term "alkenyl," as used herein, refers to an aliphatic group containing at least one double bond and is intended to include both "unsubstituted alkenyl" and "substituted alkenyl," the latter of which refers to an alkenyl moiety having substituents replacing hydrogen on one or more carbons of the alkenyl group. Such substituents may be present on one or more carbons that are included or not included in one or more double bonds. Furthermore, such substituents include all of the substituents contemplated for alkyl groups discussed below, except where stability would be impaired. For example, substitution of alkenyl groups with one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated.

[0024] An "alkyl" group or "alkane" is a straight-chain or branched non-aromatic hydrocarbon that is completely saturated. Typically, a straight-chain or branched alkyl group has from 1 to about 20 carbon atoms, preferably from 1 to about 10 carbon atoms, unless otherwise defined. Examples of straight-chain and branched alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl, and octyl. C1-C6 straight-chain or branched alkyl groups are also referred to as "lower alkyl" groups.

[0025] Furthermore, the term "alkyl" (or "lower alkyl"), as used throughout the specification, examples, and claims, is intended to include both "unsubstituted alkyl" and "substituted alkyl," the latter of which refers to an alkyl moiety having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents include, unless otherwise specified, for example, halogen (e.g., fluoro), hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (e.g., thioester, thioacetate, or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moieties. In a preferred embodiment, the substituent on a substituted alkyl is C 1-6 Alkyl, C 3-6The substituents on the substituted alkyl are selected from cycloalkyl, halogen, carbonyl, cyano, or hydroxyl. In a more preferred embodiment, the substituents on the substituted alkyl are selected from fluoro, carbonyl, cyano, or hydroxyl. It will be understood by those skilled in the art that the substituted moieties on the hydrocarbon chain can themselves be substituted, if appropriate. For example, the substituents on the substituted alkyl can include substituted and unsubstituted amino, azido, imino, amido, phosphoryl (including phosphonates and phosphinates), sulfonyl (including sulfates, sulfonamides, sulfamoyl, and sulfonates), and silyl groups, as well as ethers, alkylthio, carbonyl (including ketones, aldehydes, carboxylates, and esters), -CF3, -CN, and the like. Exemplary substituted alkyls are described below. The cycloalkyl can be further substituted with alkyl, alkenyl, alkoxy, alkylthio, aminoalkyl, carbonyl-substituted alkyl, -CF3, -CN, and the like.

[0026] Unless otherwise specified, "alkylene," alone or as part of another substituent, refers to a saturated, straight-chain or branched divalent radical having the specified number of carbon atoms and derived from the corresponding alkane by removing two hydrogen atoms. Examples of straight-chain and branched alkylene radicals include -CH- (methylene), -CH-CH- (ethylene), -CH-CH-CH- (propylene), -C(CH)-, -CH-CH(CH)-, -CH-CH-CH-CH-, -CH-CH-CH-CH- (pentylene), -CH-CH(CH)-CH-, and -CH-C(CH)-CH-.

[0027] The term “C x-y " when used in conjunction with a chemical moiety such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, is meant to include groups containing x to y carbons in the chain. For example, "C x-yThe term "alkyl" refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain alkyl and branched-chain alkyl groups containing x to y carbons in the chain, including haloalkyl groups. Preferred haloalkyl groups include trifluoromethyl, difluoromethyl, 2,2,2-trifluoroethyl, and pentafluoroethyl. CO alkyl refers to a hydrogen atom if the group is in a terminal position, or a bond if internal. "C 2-y alkenyl" and "C 2-y The term "alkynyl" refers to substituted or unsubstituted unsaturated aliphatic groups similar in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.

[0028] The term "alkylamino," as used herein, refers to an amino group substituted with at least one alkyl group.

[0029] The term "alkylthio," as used herein, refers to a thiol group substituted with an alkyl group and can be represented by the general formula alkylS-.

[0030] As used herein, the term "alkynyl" refers to an aliphatic group containing at least one triple bond and is intended to include both "unsubstituted alkynyl" and "substituted alkynyl," the latter of which refers to an alkynyl moiety having substituents replacing hydrogen on one or more carbons of the alkynyl group. Such substituents may be present on one or more carbons included or not included in one or more triple bonds. Furthermore, such substituents include all of the substituents contemplated for alkyl groups discussed above, except where stability would be impaired. For example, substitution of alkynyl groups with one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated.

[0031] The term "amide," as used herein, refers to the following group: [ka] In the formula, each RA independently represent hydrogen or a hydrocarbyl group, or two R A together with the N atom to which they are attached complete a heterocycle with 4 to 8 atoms in the ring structure.

[0032] The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines and salts thereof, for example, a moiety that can be represented by the formula: [ka] In the formula, each R A independently represent hydrogen or a hydrocarbyl group, or two R A together with the N atom to which they are attached complete a heterocycle with 4 to 8 atoms in the ring structure.

[0033] The term "aminoalkyl," as used herein, refers to an alkyl group substituted with an amino group.

[0034] The term "aralkyl," as used herein, refers to an alkyl group substituted with an aryl group.

[0035] The term "aryl" as used herein includes substituted or unsubstituted monocyclic aromatic groups in which each atom of the ring is carbon. Preferably, the ring is a 6-membered or 10-membered ring, more preferably a 6-membered ring. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings, where two or more carbon atoms are common to two adjacent rings, and at least one of the rings is aromatic, for example, the other cyclic rings can be cycloalkyl, cycloalkenyl, aryl, heteroaryl, and / or heterocyclyl. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, etc.

[0036] The term "carbamate" is art-recognized and refers to the following group: [ka] In the formula, each R A independently represent hydrogen or a hydrocarbyl group (e.g., an alkyl group), or both R A together with the intervening atoms complete a heterocyclic ring having 4 to 8 atoms in the ring structure.

[0037] The terms "carbocycle" and "carbocyclic," as used herein, refer to saturated or unsaturated rings in which each atom of the ring is carbon. The term carbocycle includes both aromatic and non-aromatic carbocycles. Non-aromatic carbocycles include both cycloalkane rings, in which all carbon atoms are saturated, and cycloalkene rings, which contain at least one double bond. "Carbocycle" includes 5- to 7-membered monocyclic rings and 8- to 12-membered bicyclic rings. Each ring of a bicyclic carbocycle may be selected from saturated, unsaturated, and aromatic rings. Carbocycles include bicyclic molecules in which one, two, or three or more atoms are shared between the two rings. The term "fused carbocycle" refers to a bicyclic carbocycle in which each ring shares two adjacent atoms with the other ring. Each ring of a fused carbocycle may be selected from saturated, unsaturated, and aromatic rings. In an exemplary embodiment, an aromatic ring, such as phenyl, may be fused to a saturated or unsaturated ring, such as cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated, unsaturated, and aromatic bicyclic rings is included in the definition of carbocycle, as long as valence permits. Exemplary "carbocycles" include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene, and adamantane. Exemplary fused carbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-1H-indene, and bicyclo[4.1.0]hept-3-ene. A "carbocycle" can be substituted at any one or more positions that can have a hydrogen atom.

[0038] A "cycloalkyl" group is a fully saturated cyclic hydrocarbon. "Cycloalkyl" includes monocyclic and bicyclic rings. Typically, a monocyclic cycloalkyl group has 3 to about 10 carbon atoms, more typically 3 to 8 carbon atoms, unless otherwise defined. The second ring of a bicyclic cycloalkyl can be selected from saturated, unsaturated, and aromatic rings. Cycloalkyl includes bicyclic molecules in which one, two, or three or more atoms are shared between the two rings. The term "fused cycloalkyl" refers to a bicyclic cycloalkyl in which each ring shares two adjacent atoms with the other ring. The second ring of a fused bicyclic cycloalkyl can be selected from saturated, unsaturated, and aromatic rings. A "cycloalkenyl" group is a cyclic hydrocarbon containing one or more double bonds.

[0039] The term "carbocyclylalkyl," as used herein, refers to an alkyl group substituted with a carbocyclic group.

[0040] The term "carbonate" is art-recognized and refers to a group -OCO-R A refers to R A represents a hydrocarbyl group.

[0041] The term "carboxy," as used herein, refers to a group represented by the formula -CO2H.

[0042] The term "ester" as used herein refers to the group -C(O)OR A refers to R A represents a hydrocarbyl group.

[0043] The term "ether" as used herein refers to a hydrocarbyl group bonded to another hydrocarbyl group via an oxygen atom. Thus, the ether substituent of a hydrocarbyl group can be hydrocarbyl-O-. Ethers can be symmetrical or asymmetrical. Examples of ethers include, but are not limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethers include "alkoxyalkyl" groups, which can be represented by the general formula alkyl-O-alkyl.

[0044] The terms "halo" and "halogen," as used herein, mean halogen and include chloro, fluoro, bromo, and iodo.

[0045] The terms "hetaralkyl" and "heteroaralkyl," as used herein, refer to an alkyl group substituted with a heteroaryl group.

[0046] The term "heteroalkyl," as used herein, refers to a saturated or unsaturated chain of carbon atoms and at least one heteroatom, wherein no two heteroatoms are adjacent.

[0047] The terms "heteroaryl" and "hetaryl" include substituted or unsubstituted aromatic monocyclic ring structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered rings, in which the ring structure contains at least one heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "heteroaryl" and "hetaryl" also include polycyclic ring systems having two or more cyclic rings, in which two or more carbons are common to two adjacent rings, and at least one of the rings is heteroaromatic; for example, the other cyclic rings may be cycloalkyl, cycloalkenyl, aryl, heteroaryl, and / or heterocyclyl. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine.

[0048] The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

[0049] The terms "heterocyclyl," "heterocycle," and "heterocyclic" refer to a substituted or unsubstituted non-aromatic ring structure, preferably a 3- to 10-membered ring, more preferably a 3- to 7-membered ring, which ring structure contains at least one heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "heterocyclyl" and "heterocyclic" also include polycyclic ring systems having two or more cyclic rings, where two or more carbons are common to two adjacent rings and at least one of the rings is heterocyclic, e.g., the other cyclic rings can be cycloalkyl, cycloalkenyl, aryl, heteroaryl, and / or heterocyclyl. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, tetrahydropyran, tetrahydrofuran, morpholine, lactones, lactams, and the like.

[0050] The terms "heterocyclylalkyl" or "heterocycloalkyl," as used herein, refers to an alkyl group substituted with a heterocycle group.

[0051] The term "hydrocarbyl," as used herein, refers to a group that has no =0 or =5 substituents and is typically bonded through at least one carbon-hydrogen bond and a carbon atom having a primarily carbon backbone, although it may optionally contain heteroatoms. Thus, groups such as methyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered hydrocarbyl for purposes of this application, while substituents such as acetyl (which has an =0 substituent on the linking carbon) and ethoxy (which is linked through an oxygen rather than a carbon) are not. Hydrocarbyl groups include, but are not limited to, aryl, heteroaryl, carbocycle, heterocyclyl, alkyl, alkenyl, alkynyl, and combinations thereof.

[0052] The term "hydroxyalkyl," as used herein, refers to an alkyl group substituted with a hydroxy group.

[0053] The term "lower," when used in conjunction with chemical moieties such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, is meant to include groups in which there are 10 or fewer, preferably 6 or fewer, non-hydrogen atoms in the substituent. "Lower alkyl," for example, refers to an alkyl group containing 10 or fewer, preferably 6 or fewer, carbon atoms. In certain embodiments, each of the acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents defined herein, whether occurring alone or in combination with other substituents, such as those described for hydroxyalkyl and aralkyl (where, for example, atoms in an aryl group are not counted when counting carbon atoms in an alkyl substituent), is lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy.

[0054] The terms "polycyclyl," "polycycle," and "polycyclic" refer to two or more rings (e.g., cycloalkyl, cycloalkenyl, aryl, heteroaryl, and / or heterocyclyl) in which two or more atoms are common to two adjacent rings, e.g., the rings are "fused rings." Each of the rings of a polycycle can be substituted or unsubstituted. In certain embodiments, each ring of a polycycle contains 3 to 10, preferably 5 to 7, atoms in the ring.

[0055] The term "silyl" refers to a silicon moiety having three hydrocarbyl moieties attached thereto.

[0056] The term "substituted" refers to moieties having substituents replacing a hydrogen on one or more backbone carbons. It will be understood that "substituted" or "substituted with" includes the implicit condition that such substitution is in accordance with the allowed valencies of the substituted atom and substituent, as well as the implicit condition that the substitution results in a stable compound that does not spontaneously undergo transformation, for example, by rearrangement, cyclization, elimination, and the like. As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, heteroatoms, such as nitrogen, can have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valencies of the heteroatoms. The substituents can include any of the substituents described herein, for example, halogen, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (e.g., thioester, thioacetate, or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moiety. In a preferred embodiment, the substituent on the substituted alkyl is C 1-6 Alkyl, C 3-6 In a more preferred embodiment, the substituent on the substituted alkyl is selected from fluoro, carbonyl, cyano, or hydroxyl. Those skilled in the art will understand that the substituent itself can be substituted, if appropriate. Unless specifically stated as "unsubstituted," reference to a chemical moiety herein is understood to include substituted variants. For example, reference to an "aryl" group or moiety implicitly includes both substituted and unsubstituted variants.

[0057] The term "sulfate" is art-recognized and refers to the group -OSO3H or a pharmaceutically acceptable salt thereof.

[0058] The term "sulfonamide" is art-recognized and can be represented by the general formula: [ka] In the formula, each R A independently represent hydrogen or hydrocarbyl (e.g., alkyl), or R A together with the intervening atoms complete a heterocycle having 4 to 8 atoms in the ring structure.

[0059] The term "sulfoxide" is art-recognized and refers to the group -S(O)-R A refers to R A represents a hydrocarbyl.

[0060] The term "sulfonate" is art-recognized and refers to the group SO3H, or a pharmaceutically acceptable salt thereof.

[0061] The term "sulfone" is art-recognized and refers to the group S(O)-R A refers to R A represents a hydrocarbyl.

[0062] The term "thioalkyl," as used herein, refers to an alkyl group substituted with a thiol group.

[0063] The term "thioester" as used herein refers to the group -C(O)SR A or -SC(O)R A refers to R A represents a hydrocarbyl.

[0064] The term "thioether" as used herein corresponds to an ether, where the oxygen is replaced by a sulfur.

[0065] The term "urea" is art-recognized and may be represented by the general formula: [ka] In the formula, each R A independently represent hydrogen or hydrocarbyl (e.g., alkyl), or R A Any occurrence of together with another atom and intervening atoms completes a heterocycle having from 4 to 8 atoms in the ring structure.

[0066] "Protecting group" refers to a group of atoms that, when attached to a reactive functional group in a molecule, masks, reduces, or prevents the reactivity of the functional group. Typically, a protecting group can be selectively removed as desired during synthesis. Examples of protecting groups are found in Greene and Wuts, Protective Groups in Organic Chemistry, 3 rdEd., 1999, John Wiley & Sons, NY and Harrison et al., Compendium of Synthetic Organic Methods, Vols. 1-8, 1971-1996, John Wiley & Sons, NY. Representative nitrogen protecting groups include, but are not limited to, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl ("Cbz"), tert-butoxycarbonyl ("Boc"), trimethylsilyl ("TMS"), 2-trimethylsilyl-ethanesulfonyl ("TES"), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl ("FMOC"), nitro-veratryloxycarbonyl ("NVOC"), and the like. Representative hydroxyl protecting groups include, but are not limited to, those in which the hydroxyl group is acylated (esterified) or alkylated, such as, for example, benzyl and trityl ethers, as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers (e.g., TMS or TIPS groups), glycol ethers (e.g., ethylene glycol and propylene glycol derivatives), and allyl ethers.

[0067] As used herein, a therapeutic agent that "prevents" or "reduces the risk of developing" a disease, disorder, or condition refers to a compound that reduces the occurrence of the disease, disorder, or condition in a statistical sample in a treated sample compared to an untreated control sample, or delays the onset of or reduces the severity of one or more symptoms of the disorder or condition compared to an untreated control sample.

[0068] The term "treating" includes prophylactic and / or therapeutic treatment. The term "prophylactic or therapeutic" treatment is art-recognized and includes administration of one or more subject compositions to a host. If administered prior to the manifestation of clinical symptoms of an undesirable condition (e.g., a disease or other undesirable condition in a host animal), the treatment is prophylactic (i.e., protects the host from developing the undesirable condition), and if administered after the manifestation of symptoms of the undesirable condition, the treatment is therapeutic (i.e., intended to reduce, ameliorate, or stabilize an existing undesirable condition or its side effects).

[0069] The phrases "conjoint administration" and "co-administered" refer to any form of administration of two or more different therapeutic compounds, such that a second compound is administered while a previously administered therapeutic compound is still effective in the body (e.g., the two compounds are effective in a patient simultaneously, which may involve a synergistic effect of the two compounds). For example, the different therapeutic compounds can be administered simultaneously or sequentially, either in the same formulation or in separate formulations. In certain embodiments, the different therapeutic compounds can be administered within 1 hour, 12 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours, or 1 week of each other. Thus, individuals receiving such treatment can benefit from the combined effects of the different therapeutic compounds.

[0070] The term "prodrug" is intended to encompass compounds that are converted into the therapeutically active substances of the present invention under physiological conditions. A common method for making a prodrug is to include one or more selected moieties that are hydrolyzed under physiological conditions to reveal the desired molecule. In other embodiments, the prodrug is converted by the enzymatic activity of the host animal. For example, esters or carbonates (e.g., esters or carbonates of alcohols or carboxylic acids) are preferred prodrugs of the present invention. In certain embodiments, some or all of the compounds of the present invention in the formulations represented above can be replaced with corresponding suitable prodrugs; for example, hydroxyls in the parent compound are presented as esters or carbonates, or carboxylic acids present in the parent compound are presented as esters.

[0071] As used herein, "small molecule" refers to a small organic or inorganic molecule with a molecular weight of less than about 3,000 Daltons. Generally, small molecules useful in the present invention have a molecular weight of less than 3,000 Daltons (Da). Small molecules can be, for example, at least about 100 Da to about 3,000 Da (e.g., about 100 to about 3,000 Da, about 100 to about 2,500 Da, about 100 to about 2,000 Da, about 100 to about 1,750 Da, about 100 to about 1,500 Da, about 100 to about 1,250 Da, about 100 to about 1,000 Da, about 100 to about 750 Da, about 100 to about 500 Da, about 200 to about 1,500, about 500 to about 1,000, about 300 to about 1,000 Da, or about 100 to about 250 Da).

[0072] In some embodiments, "small molecule" refers to an organic, inorganic, or organometallic compound that typically has a molecular weight of less than about 1000. In some embodiments, small molecules are organic compounds with sizes on the order of 1 nm. In some embodiments, small molecule drugs of the present invention include oligopeptides and other biomolecules with a molecular weight of less than about 1000.

[0073] An "effective amount" is an amount sufficient to produce a beneficial or desired result. For example, a therapeutic amount is an amount that achieves a desired therapeutic effect. This amount may be the same as or different from a prophylactically effective amount, which is the amount necessary to prevent the onset of a disease or disease symptoms. An effective amount can be administered in one or more administrations, applications, or dosages. The therapeutically effective amount of a composition will depend on the composition selected. The composition can be administered one or more times daily to one or more times weekly, including once every other day. Those skilled in the art will understand that certain factors, including, but not limited to, the severity of the disease or disorder, previous treatments, the subject's overall health and / or age, and other diseases present, can affect the dosage and timing required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective amount of a composition described herein can include a single treatment or a series of treatments.

[0074] Compounds of the Invention One aspect of the invention is a method for selecting and treating a human subject suffering from a kidney disease, comprising: a. selecting a subject if the subject has a urinary level of one or more biomarkers selected from Rac1, Rac1-GTP, phospho-LIM kinase 1, and phospho-cofilin above a predetermined threshold; and b. administering to the selected subject a pharmaceutical composition comprising a TRPC5 inhibitor or a calcineurin inhibitor and a pharmaceutically acceptable carrier; The present invention provides a method comprising:

[0075] In one aspect, the present invention provides a method of treating a human subject suffering from a kidney disease, comprising: only if the subject is determined to have a pre-treatment urinary level of one or more biomarkers selected from Rac1, Rac1-GTP, phospho-LIM kinase 1, and phospho-cofilin above a predetermined threshold, TRPC5 inhibitors or calcineurin inhibitors, and Pharmaceutically acceptable carrier The method comprises administering to the subject a pharmaceutical composition comprising:

[0076] In some embodiments, the TRPC5 inhibitor is a small molecule inhibitor of TRPC5. a. A compound of formula (I) or formula (II): [ka] or a pharmaceutically acceptable salt of any of the above, During the ceremony, X is CH, C(R 3 ) or N; R 1 is selected from the group consisting of H; alkyl; cycloalkyl; heterocycloalkyl; alkenyl; aryl; heteroaryl; alkylene-aryl; alkylene-heteroaryl; -CH2(O)N(R)-heteroaryl; -CH2(O)N(R)-alkyl; alkylene-N(alkyl)2; heterocycloalkyl; alkylene-O-alkyl; alkylene-O-aryl; alkylene-N(R)-C(O)-aryl; alkylene-N(R)-C(O)-alkyl; alkylene-C(O)-N(R)-alkyl; alkylene-C(O)-N(R)-aryl; alkylene-C(O)-cycloalkyl; and alkylene-C(O)-N(R)-heteroaryl; R 2is selected from the group consisting of H; NH2, alkyl; cycloalkyl; aryl; heteroaryl; alkylene-aryl, alkylene-N(alkyl)2; alkylene-heterocycloalkyl; alkylene-cycloalkyl; -N(R)-alkyl; -N(R)-aryl; -N(R)-alkylene-aryl; -N(R)-cycloalkyl; -N(R)-heterocycloalkyl; -O-aryl; alkylene-O-aryl; heterocycloalkyl; -N=C(R)-aryl; -N(R)-alkylene-heteroaryl; -N(R)-alkylene-OH; -S-alkylene-C(O)N(R)-aryl; -S-alkylene-C(O)N(R)-heteroaryl; alkylene-C(O)-heterocycloalkyl; alkylene-N(R)-alkyl; alkylene-N(R)-aryl; and -S-alkyl; R 3 is independently selected from alkyl, halogen, —CN, —OMe, —OH, —NO 2 , —NH 2 , N(Me) 2 , —CF 3 , —OCF 3 , —CHF 2 , —OCHF 2 , and —O-alkylene-OH; R is H, or Me; and n is 0, 1, 2, 3, or 4; b. A compound of formula (III), (IV), or (V): [ka] or a tautomer or pharmaceutically acceptable salt of any of the above, During the ceremony, R 11 and R 13 are independently H, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, heteroaryl, halogen, -OH, -CN, -cycloalkyl, -O-alkyl, -O-cycloalkyl, O-aryl, -aryl-O-aryl-CF3, -C(H)F2, alkylene-CF3, alkylene-C(H)F2, -SO2-alkyl, and O-alkylene-O-alkyl, -heterocyclyl-LR 4 , and -heteroaryl-LR 4 selected from the group consisting of: R12 is -heterocyclyl-LR 14 and; R 14 is absent or is selected from alkyl, cycloalkyl, aryl, alkylene-aryl, alkylene-heteroaryl, heteroaryl, heterocyclyl, -C(O)N(R 15 CF3; R 15 is independently H or alkyl; R 16 is an alkyl, cycloalkyl, aryl, heterocyclyl, heteroaryl, alkylene-aryl, -C(O)N(R 15 )2, and CF3; L is absent or is methylene, -C(O)-, -SO2-, CH2N(Me)-, -N(R 15 )(R 16 )-, -C(R 15 )(R 16 )-, and -OR 16 selected from the group consisting of: R 11 , R 12 and R 13 one and only one of -heterocyclyl-LR 14 or -heteroaryl-LR 14 is; c. A compound of formula (VI) or (VII): [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, R 21 is selected from the group consisting of alkyl; cycloalkyl; heterocycloalkyl; aryl; heteroaryl; alkylene-aryl; alkylene-heteroaryl; alkylene-O-aryl; alkylene-N(alkyl)2; alkylene-heterocycloalkyl; alkylene-cycloalkyl; N(alkyl)2; and -C(O)-aryl; R 22is selected from the group consisting of alkyl; cycloalkyl; heterocycloalkyl; aryl; heteroaryl; alkylene-N(alkyl)2; alkylene-heterocycloalkyl; alkylene-cycloalkyl; alkylene-heterocycloalkyl; and alkylene-OR'; R 23 is independently selected from alkyl, halogen, OMe, OH, N(Me)2, CF3, or OCF3, -O-, and alkylene-OH; R is H or Me; R' is H, methyl, ethyl, or isopropyl; and n is 0, 1, 2, 3, or 4; or d. A compound of formula (VIII) or (IX): [ka] or a pharmaceutically acceptable salt of any of the above, A and A' are CR a and N are independently selected; R a is LR 31 and; L is absent or is CH2, O, SO2, or NR 32 and; R 31 is selected from optionally substituted alkyl, optionally substituted aryl, and optionally substituted heteroaryl; Each R 32 is independently H or alkyl; R 33 is an optionally substituted alkyl, an optionally substituted alkylene -OR 32 , optionally substituted cycloalkylene-OR 32 , optionally substituted alkylene-N(R 37 )2, optionally substituted cycloalkylene-N(R 37 )2, optionally substituted alkylene -C(O)N(R 32 )2, optionally substituted cycloalkylene-C(O)N(R32 )2, optionally substituted alkylene -S(O)2N(R 32 )2, and optionally substituted cycloalkylene -S(O)2N(R 32 )2 is selected; R 34 is selected from alkyl, optionally substituted alkylene-aryl, and optionally substituted alkylene-heteroaryl; Each R 35 are independently H, N(R 32 )2, OR 32 Selected from; Each R 37 is independently selected from H, alkyl, (alkyl)C(O)—, (aryl)C(O)—, (alkyl)S(O)2—, and (aryl)S(O)2—; Y is -C(O)-, CH2, CHR 36 , C(R 36 )2; Each R 36 is independently selected from H, alkyl, and optionally substituted alkylene-OH; Y' is -C(O)-, CH2, CHR 33 ', C(R 33 ')2 or Y' is R 33 together to form a five- or six-membered ring; Each R 33 ' are independently an optionally substituted alkyl, an optionally substituted alkylene -OR 32 , optionally substituted cycloalkylene-OR 32 , optionally substituted alkylene-N(R 37 )2, optionally substituted cycloalkylene-N(R 37 )2, optionally substituted alkylene -C(O)N(R 32 )2, optionally substituted cycloalkylene-C(O)N(R 32 )2, optionally substituted alkylene -S(O)2N(R 32 )2, and optionally substituted cycloalkylene -S(O)2N(R 32 )2; and Z is absent or CH2, CHR 35 , O, -NR 32 -, or -SO2-; However, both Y and Y' are not -C(O)-.

[0077] In some embodiments, the TRPC5 inhibitor is a compound of structural formula X: [ka] or a pharmaceutically acceptable salt thereof; During the ceremony, "---" is a single or double bond; X 1 is CH or N; If "---" is a double bond, X 2 is CH or N; If "---" is a single bond, X 2 is N(CH3); X 1 If is CH, then X 2 is N or N(CH3); W is -O-, -N(CH3)-, -N(CH2CH2OH)-, cyclopropane-1,1-diyl, or -CH(CH3)-; Q is 2-trifluoromethyl-4-fluorophenyl, 2-difluoromethyl-4-fluorophenyl, 2-trifluoromethylphenyl, 2-methyl-4-fluorophenyl, 2-chloro-4-fluorophenyl, 2-chlorophenyl, 1-(benzyl)-4-methylpiperidin-3-yl, 4-trifluoromethylpyridin-3-yl, 2-trifluoromethyl-6-fluorophenyl, 2-trifluoromethyl-3-cyanophenyl, 2-ethyl-3-fluorophenyl, 2-chloro-3-cyanophenyl, 2-trifluoromethyl-5-fluorophenyl, or 2-difluoromethylphenyl; R 43 is hydrogen, —CHOH, —CH(OH)—CHOH, —NH, —CH(OH)CH, —OCH, or —NH—(CH)OH; and when “---” is a double bond, R44 does not exist; If "---" is a single bond, R 43 and R 44 together form =O; R 45 and R 46 Each of is independently hydrogen or —CH 3 .

[0078] In some embodiments, the TRPC5 inhibitor is a compound of Formula XI: [ka] or a pharmaceutically acceptable salt thereof; During the ceremony, R 41 is chloro, -CF3, -CHF2, or -CH3; R 42 is hydrogen or fluoro; R 43 is hydrogen, -NH2, -CH2OH, or CH(OH)-CH2OH.

[0079] The compounds of Formulas I-XI can be synthesized using methods known to those skilled in the art, for example, the methods disclosed in WO 2019 / 055966, the entire contents of which are incorporated herein by reference.

[0080] In some embodiments, the TRPC5 inhibitor is a compound of formula (HI): [ka] or a pharmaceutically acceptable salt thereof; During the ceremony, R 51 is a C1-C6 alkyl, a C2-C6 alkenyl, or a C2-C6 alkynyl, each of which is selected from 1 to 4 R 55 may be substituted with; R 52is C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, halo, C1-C6 haloalkoxy, hydroxyl, C1-C6 alkoxy, C3-C7 cycloalkyloxy, C6-C 10 Aryl, C6-C 10 Aryloxy, C7-C 16 Arylalkoxy, amino, C1-C6 alkylamino, C2-C 12 dialkylamino, -S-, -S-C1-C6 alkyl, -S(O)-, S(O)2-, heterocycloalkyl, heteroaryl, heteroaryloxy, sulfonamidyl, amido, urea, sulfonylurea, acyl, nitro, cyano; wherein C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, hydroxyl, C1-C6 alkoxy, C3-C7 cycloalkyloxy, C6-C 10 Aryl, C6-C 10 Aryloxy, C7-C16 arylalkoxy, amino, C1-C6 alkylamino, C2-C 12 Dialkylamino, -S-, -S-C1 to C6 alkyl, -S(O)-, -S(O)2-, heterocycloalkyl, heteroaryl, heteroaryloxy, sulfonamidyl, amide, urea, sulfonylurea, and acyl are each substituted with 1 to 3 R 56 may be substituted with; R 53 is C1-C6 alkyl, C1-C6 heteroalkyl, C3-C7 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 hydroxyalkyl, or C1-C6 alkoxy, each of which is selected from 1 to 4 R 57 may be substituted with; R 54 is C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, or C2-C6 alkynyl, each of which is selected from 1 to 4 R 58 may be substituted with; R 55 , R 56 , R 57 , and R58 are each independently C1-C6 alkyl, C1-C6 heteroalkyl, halo, C1-C6 haloalkyl, C1-C6 haloalkoxy, hydroxyl, C1-C6 alkoxy, amino, C1-C6 alkylamino, C2-C 12 Dialkylamino, cyano, nitro, amido, C1-C6 alkylamido, C2-C 12 Dialkylamide, -S-, -S(O)2-, -C(O)O-, -C(O)-, -C(O)O-C1-C6 alkyl, C3-C7 cycloalkyl, C6-C 10 aryl, heterocycloalkyl, or heteroaryl; wherein C1-C6 alkyl, C1-C6 heteroalkyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, hydroxyl, C1-C6 alkoxy, amino, C1-C6 alkylamino, C2-C 12 Dialkylamino, Amido, C1-C6 Alkylamide, C2-C 12 Dialkylamide, -S-, -S(O)2-, -C(O)O-, -C(O)-, -C(O)O-C1-C6 alkyl, C3-C7 cycloalkyl, C6-C 10 An aryl, heterocycloalkyl, or heteroaryl may have 1 to 3 R 59 may be substituted with; Each R 59 are independently C1-C6 alkyl, C1-C6 heteroalkyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, heterocycloalkyl, C6-C 10 Aryl, heteroaryl, C4-C 10 Cycloalkylalkyl, heterocycloalkyl-C1-C6 alkyl, C7-C 16 Aryl alkyl, heteroaryl-C1-C6 alkyl, halo, hydroxyl, C1-C6 alkoxy, C6-C 10 Aryloxy, C7-C 16 Arylalkoxy, C2-C8 alkoxyalkoxyl, amino, C1-C6 alkylamino, C2-C 12 Dialkylamino, C1-C6 alkyl-amino-C1-C6 alkyl, C1-C6 alkyl-amino-C2-C 12Dialkyl, -S-, -S-C1-C6 alkyl, -S(O)2-C1-C6 alkyl, sulfonamidyl, amide, urea, sulfonylurea, acyl, -C(O)-C6-C 10 Aryl, -NHC(O)-C6-C 10 Aryl, -C(O)NH-C6~C 10 aryl, —C(O)OH, —C(O)O—C1-C6 alkyl, —C(O)—C1-C6 alkyl acyl, nitro, or cyano.

[0081] In some embodiments, the TRPC5 inhibitor is a compound of formula H-Ia: [ka] or a pharmaceutically acceptable salt thereof; During the ceremony, R 61 is a C1-C6 alkyl, a C2-C6 alkenyl, or a C2-C6 alkynyl, each of which is selected from 1 to 4 R 65 may be substituted with; R 62 is C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, halo, C1-C6 haloalkoxy, hydroxyl, C1-C6 alkoxy, C3-C7 cycloalkyloxy, C6-C 10 Aryl, C6-C 10 Aryloxy, C7-C 16 Arylalkoxy, amino, C1-C6 alkylamino, C2-C 12 dialkylamino, -S-, -S-C1-C6 alkyl, -S(O)-, S(O)2-, heterocycloalkyl, heteroaryl, heteroaryloxy, sulfonamidyl, amido, urea, sulfonylurea, acyl, nitro, cyano; wherein C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, hydroxyl, C1-C6 alkoxy, C3-C7 cycloalkyloxy, C6-C 10 Aryl, C6-C 10Aryloxy, C7-C 16 Arylalkoxy, amino, C1-C6 alkylamino, C2-C 12 Each of dialkylamino, -S-, -S-C1 to C6 alkyl, -S(O)-, -S(O)2-, heterocycloalkyl, heteroaryl, heteroaryloxy, sulfonamidyl, amido, urea, sulfonylurea, and acyl has 1 to 3 R 66 may be substituted with; R 63 is C2-C6 hydroxyalkyl or C1-C6 heteroalkyl; R 64 is C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, or C2-C6 alkynyl, each of which is selected from 1 to 4 R 68 may be substituted with; R 65 , R 66 , and R 68 are each independently C1-C6 alkyl, C1-C6 heteroalkyl, halo, C1-C6 haloalkyl, C1-C6 haloalkoxy, hydroxyl, C1-C6 alkoxy, amino, C1-C6 alkylamino, C2-C 12 Dialkylamino, cyano, nitro, amido, C1-C6 alkylamido, C2-C 12 Dialkylamide, -S-, -S(O)2-, -C(O)O-, -C(O)-, -C(O)O-C1-C6 alkyl, C3-C7 cycloalkyl, C6-C 10 aryl, heterocycloalkyl, or heteroaryl; wherein C1-C6 alkyl, C1-C6 heteroalkyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, hydroxyl, C1-C6 alkoxy, amino, C1-C6 alkylamino, C2-C 12 Dialkylamino, Amido, C1-C6 Alkylamide, C2-C 12 Dialkylamide, -S-, -S(O)2-, -C(O)O-, -C(O)-, -C(O)O-C1-C6 alkyl, C3-C7 cycloalkyl, C6-C 10 Each aryl, heterocycloalkyl, or heteroaryl may have 1 to 3 R 69may be substituted with; Each R 69 are independently C1-C6 alkyl, C1-C6 heteroalkyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, heterocycloalkyl, C6-C 10 Aryl, heteroaryl, C4-C 10 Cycloalkylalkyl, heterocycloalkyl-C1-C6 alkyl, C7-C 16 Aryl alkyl, heteroaryl-C1-C6 alkyl, halo, hydroxyl, C1-C6 alkoxy, C6-C 10 Aryloxy, C7-C 16 Arylalkoxy, C2-C8 alkoxyalkoxyl, amino, C1-C6 alkylamino, C2-C 12 Dialkylamino, C1-C6 alkyl-amino-C1-C6 alkyl, C1-C6 alkyl-amino-C2-C 12 Dialkyl, -S-, -S-C1-C6 alkyl, -S(O)2-C1-C6 alkyl, sulfonamidyl, amide, urea, sulfonylurea, acyl, -C(O)-C6-C 10 Aryl, -NHC(O)-C6-C 10 Aryl, -C(O)NH-C6~C 10 aryl, —C(O)OH, —C(O)O—C1-C6 alkyl, —C(O)—C1-C6 alkyl acyl, nitro, or cyano.

[0082] In some embodiments, the TRPC5 inhibitor is a compound of formula H-II: [ka] or a pharmaceutically acceptable salt thereof; During the ceremony, Ring D is phenyl, pyridyl, thiazolyl, pyrimidinyl, or oxazolyl; R 72 is C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, halo, C1-C6 haloalkoxy, hydroxyl, C1-C6 alkoxy, C3-C7 cycloalkyloxy, C6-C10 Aryl, C6-C 10 Aryloxy, C7-C 16 Arylalkoxy, amino, C1-C6 alkylamino, C2-C 12 dialkylamino, -S-, -S-C1-C6 alkyl, -S(O)-, S(O)2-, heterocycloalkyl, heteroaryl, heteroaryloxy, sulfonamidyl, amido, urea, sulfonylurea, acyl, nitro, cyano; wherein C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, hydroxyl, C1-C6 alkoxy, C3-C7 cycloalkyloxy, C6-C 10 Aryl, C6-C 10 Aryloxy, C7-C 16 Arylalkoxy, amino, C1-C6 alkylamino, C2-C 12 Each of dialkylamino, -S-, -S-C1 to C6 alkyl, -S(O)-, -S(O)2-, heterocycloalkyl, heteroaryl, heteroaryloxy, sulfonamidyl, amido, urea, sulfonylurea, and acyl has 1 to 3 R 76 may be substituted with; R 73 is C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 hydroxyalkyl, or C1-C6 alkoxy, each of which is selected from 1 to 4 R 77 may be substituted with; R 74 is C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, or C2-C6 alkynyl, each of which is selected from 1 to 4 R 78 may be substituted with; R 76 , R 77 , and R 78 are each independently C1-C6 alkyl, C1-C6 heteroalkyl, halo, C1-C6 haloalkyl, C1-C6 haloalkoxy, hydroxyl, C1-C6 alkoxy, amino, C1-C6 alkylamino, C2-C 12Dialkylamino, cyano, nitro, amido, C1-C6 alkylamido, C2-C 12 Dialkylamide, -S-, -S(O)2-, -C(O)O-, -C(O)-, -C(O)O-C1-C6 alkyl, C3-C7 cycloalkyl, C6-C 10 aryl, heterocycloalkyl, or heteroaryl; wherein C1-C6 alkyl, C1-C6 heteroalkyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, hydroxyl, C1-C6 alkoxy, amino, C1-C6 alkylamino, C2-C 12 Dialkylamino, Amido, C1-C6 Alkylamide, C2-C 12 Dialkylamide, -S-, -S(O)2-, -C(O)O-, -C(O)-, -C(O)O-C1-C6 alkyl, C3-C7 cycloalkyl, C6-C 10 An aryl, heterocycloalkyl, or heteroaryl may have 1 to 3 R 79 may be substituted with; Each R 79 are independently C1-C6 alkyl, C1-C6 heteroalkyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, heterocycloalkyl, C6-C 10 Aryl, heteroaryl, C4-C 10 Cycloalkyl alkyl, heterocycloalkyl C1-C6 alkyl, C7-C 16 Aryl alkyl, heteroaryl-C1-C6 alkyl, halo, hydroxyl, C1-C6 alkoxy, C6-C 10 Aryloxy, C7-C 16 Arylalkoxy, C2-C8 alkoxyalkoxyl, amino, C1-C6 alkylamino, C2-C 12 Dialkylamino, C1-C6 alkyl-amino-C1-C6 alkyl, C1-C6 alkyl-amino-C2-C 12 Dialkyl, -S-, -S-C1-C6 alkyl, -S(O)2-C1-C6 alkyl, sulfonamidyl, amide, urea, sulfonylurea, acyl, -C(O)-C6-C 10 Aryl, -NHC(O)-C6-C 10 Aryl, -C(O)NH-C6~C10 aryl, —C(O)OH, —C(O)O—C1-C6 alkyl, —C(O)—C1-C6 alkyl acyl, nitro, or cyano; Each R 78 is C1-C6 alkyl, C1-C6 haloalkyl, halo; p is 1 or 2; m is 1, 2, or 3.

[0083] In some embodiments, the TRPC5 inhibitor is a compound of formula H-III: [ka] or a pharmaceutically acceptable salt thereof; During the ceremony, R 82 is 1 to 3 R 86 C1-C6 alkoxy or C6-C 10 is aryloxy; R 83 is C1-C6 heteroalkyl or C2-C6 hydroxyalkyl; R 84 is C1-C6 alkyl; R 86 are independently C1-C6 alkyl, halo, C1-C6 haloalkyl, C1-C6 haloalkoxy, or C1-C6 alkoxy; Each R 8a is C1-C6 alkyl, C1-C6 haloalkyl, halo; r is 1 or 2; q is 1, 2, or 3.

[0084] Compounds of formula (HI), (HIa), (HI), and (HI) can be synthesized using methods known to those skilled in the art, for example, the methods disclosed in WO 2014 / 143799, the entire contents of which are incorporated herein by reference.

[0085] In some embodiments, the TRPC5 inhibitor is [ka] or a pharmaceutically acceptable salt thereof.

[0086] In some embodiments, the TRPC5 inhibitor is [ka] [ka] [ka] [ka] or a pharmaceutically acceptable salt thereof.

[0087] These compounds can be synthesized by methods known to those skilled in the art, for example, by the methods disclosed in WO 2019 / 011802 (incorporated by reference), Rubaiy et al., Br. J. Pharmacol. (2019) 176:832-846, and Miller et al., J. Biol. Chem. (2011) 286(38):33436-33446.

[0088] [ka] [ka] [ka] [ka]

[0089] In some embodiments, the TRPC5 inhibitor is [ka] or a pharmaceutically acceptable salt thereof.

[0090] In some embodiments, the calcineurin inhibitor is a small molecule inhibitor of calcineurin. In certain embodiments, the calcineurin inhibitor is cyclosporin A, tacrolimus, or voclosporin, or a pharmaceutically acceptable salt thereof. Cyclosporin A has the following structure: [ka] Tacrolimus has the following structure: [ka] Voclosporin has the following structure: [ka]

[0091] In certain embodiments, the compound of the present invention can be racemic.In certain embodiments, the compound of the present invention can be enriched in one enantiomer (mirror isomer).For example, the compound of the present invention can have an ee of more than 30%, an ee of more than 40%, an ee of more than 50%, an ee of more than 60%, an ee of more than 70%, an ee of more than 80%, an ee of more than 90%, or even an ee of 95% or more.

[0092] The compounds of the present invention have multiple stereocenters. Therefore, the compounds of the present invention can be enriched in one or more diastereomers. For example, the compounds of the present invention can have more than 30% de, more than 40% de, more than 50% de, more than 60% de, more than 70% de, more than 80% de, more than 90% de, or even 95% or more de. In certain embodiments, the compounds of the present invention have substantially one isomeric configuration at one or more stereocenters, and have multiple isomeric configurations at the remaining stereocenters.

[0093] In certain embodiments, the enantiomeric excess of the stereocenter is at least 40% ee, 50% ee, 60% ee, 70% ee, 80% ee, 90% ee, 92% ee, 94% ee, 95% ee, 96% ee, 98% ee or higher ee.

[0094] As used herein, a single bond drawn without stereochemistry does not indicate the stereochemistry of the compound.

[0095] As used herein, hashed or bolded non-wedged bonds indicate relative rather than absolute stereochemical configurations (eg, do not distinguish between enantiomers of a given diastereomer).

[0096] As used herein, a hashed or bold wedge bond denotes absolute stereochemical configuration.

[0097] In some embodiments, the present invention relates to pharmaceutical compositions comprising a compound of the present invention and a pharmaceutically acceptable carrier. In certain embodiments, therapeutic preparations or pharmaceutical compositions of the compounds of the present invention can be enriched to provide primarily one enantiomer of the compound. An enantiomerically enriched mixture can contain, for example, at least 60 mole percent, or more preferably at least 75, 90, 95, or even 99 mole percent of one enantiomer. In certain embodiments, a compound enriched in one enantiomer is substantially free of the other enantiomer, where substantially free means that the material constitutes less than 10%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1% of the amount of the other enantiomer in the composition or compound mixture. For example, if a composition or compound mixture contains 98 grams of a first enantiomer and 2 grams of a second enantiomer, it is said to contain 98 mole percent of the first enantiomer and no more than 2% of the second enantiomer.

[0098] In certain embodiments, therapeutic preparations or pharmaceutical compositions can be enriched to provide predominantly one diastereomer of a compound of the invention. A diastereomerically enriched mixture can contain, for example, at least 60 mole percent, or more preferably at least 75, 90, 95, or even 99 mole percent of one diastereomer.

[0099] Pharmaceutical Composition The compositions and methods of the present invention can be used to treat subjects in need thereof. In certain embodiments, the subject is a mammal, such as a human, or a non-human mammal. When administered to a subject, such as a human, the composition or compound is preferably administered as a pharmaceutical composition, for example, comprising the compound of the present invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline, or other solvents or vehicles such as glycols, glycerol, oils (e.g., olive oil), or injectable organic esters. In a preferred embodiment, when such pharmaceutical compositions are intended for human administration, particularly for invasive administration routes (i.e., routes such as injection or implantation that avoid transport or diffusion through epithelial barriers), the aqueous solution is pyrogen-free or substantially pyrogen-free. The excipient can be selected, for example, to provide delayed release of the drug or to selectively target one or more cells, tissues, or organs. The pharmaceutical composition may be in the form of a dosage unit such as a tablet, capsule (including a sprinkle capsule and a gelatin capsule), granule, lyophilized product for reconstitution, powder, solution, syrup, suppository, injection, etc. The composition may also be present in a transdermal delivery system, such as a skin patch. The composition may also be present in a solution suitable for topical administration, such as eye drops.

[0100] Pharmaceutically acceptable carriers can contain physiologically acceptable agents that act, for example, to stabilize, increase the solubility, or enhance the absorption of compounds such as the compounds of the present invention. Such physiologically acceptable agents include, for example, carbohydrates such as glucose, sucrose, or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low-molecular-weight proteins, or other stabilizers or excipients. The selection of a pharmaceutically acceptable carrier containing a physiologically acceptable agent depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying or self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) can also be, for example, a liposome or other polymer matrix into which the compounds of the present invention can be incorporated. For example, liposomes containing phospholipids or other lipids are non-toxic, physiologically acceptable, and metabolizable carriers that are relatively easy to prepare and administer.

[0101] The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0102] "Pharmaceutically acceptable salt" is used herein to refer to an acid or base addition salt that is suitable or compatible with the treatment of patients.

[0103] As used herein, the term "pharmaceutically acceptable acid addition salt" refers to any non-toxic organic or inorganic salt of a disclosed compound. Exemplary inorganic acids that form suitable salts include hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Exemplary organic acids that form suitable salts include mono-, di-, and tri-carboxylic acids such as glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, tartaric acid, bitartrate, citric acid, ascorbic acid, maleic acid, benzoic acid, phenylacetic acid, cinnamic acid, salicylic acid, and sulfosalicylic acid, and sulfonic acids such as p-toluenesulfonic acid and methanesulfonic acid. Either mono- or di-acid salts can be formed, and such salts can exist in either hydrated, solvated, or substantially anhydrous form. Generally, the acid addition salt of the compounds disclosed herein is soluble in water and various hydrophilic organic solvents, and generally shows a higher melting point compared with their free base form.The selection of suitable salt is known to those skilled in the art.Other pharmaceutically unacceptable salts, such as oxalates, can also be used in the isolation of the compounds disclosed herein, for example, for laboratory use or for subsequent conversion into pharmaceutically acceptable acid addition salts.

[0104] As used herein, the term "pharmaceutically acceptable base addition salt" refers to any non-toxic organic or inorganic base addition salt of any acidic compound disclosed herein. Exemplary inorganic bases that form suitable salts include lithium, sodium, potassium, calcium, magnesium, or barium hydroxide. Exemplary organic bases that form suitable salts include aliphatic, alicyclic, or aromatic organic amines, such as methylamine, trimethylamine, and picoline or ammonia. The selection of an appropriate salt is known to those skilled in the art.

[0105] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject. Some examples of substances that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and the like. (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer; and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0106] Pharmaceutical compositions (preparations) can be administered to a subject by any of a number of routes of administration, including, for example, oral (e.g., drenches, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue, such as in aqueous or non-aqueous solutions or suspensions); absorption through the oral mucosa (e.g., sublingual); anal, rectal, or vaginal (e.g., as a pessary, cream, or foam); parenteral (e.g., as a sterile solution or suspension, including intramuscular, intravenous, subcutaneous, or intrathecal); nasal; intraperitoneal; subcutaneous; transdermal (e.g., as a patch applied to the skin); and topical (e.g., as a cream, ointment, or spray applied to the skin, or as eye drops). The compounds may also be formulated for inhalation. In certain embodiments, the compounds may simply be dissolved or suspended in sterile water. Details of suitable routes of administration and compositions suitable therefor can be found, for example, in U.S. Pat. Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970, and 4,172,896, and the patents cited therein.

[0107] The formulations can be conveniently provided in unit dosage form and can be prepared by any method well known in the art of pharmacy. The amount of active ingredient (or active ingredient) that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will usually be the amount of compound that produces a therapeutic effect. Generally, out of 100 percent, this amount will range from about 1 percent to about 99 percent of the active ingredient, preferably from about 5 percent to about 70 percent, and most preferably from about 10 percent to about 30 percent.

[0108] Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the present invention, with a carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0109] Formulations of the present invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), lyophilisate, powder, granules, each containing a predetermined amount of a compound of the present invention as the active ingredient, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a pastille (using an inert base such as gelatin and glycerin, or sucrose and acacia), and / or as a mouthwash, etc. The composition or compound may also be administered as a bolus, electuary, or paste.

[0110] To prepare solid dosage forms for oral administration (such as capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules, etc.), the active ingredient is mixed with sodium citrate or dicalcium phosphate, and / or any one or more of the following pharmaceutically acceptable carriers: (1) a filler or extender, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) a binder, such as, for example, carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) a humectant, such as glycerol; (4) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarders such as paraffin; (6) absorption accelerators such as quaternary ammonium compounds; (7) wetting agents such as cetyl alcohol and glycerol monostearate; (8) adsorbents such as kaolin and bentonite clay; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents such as modified and unmodified cyclodextrins; and (11) coloring agents. For capsules (including sprinkle capsules and gelatin capsules), tablets, and pills, the pharmaceutical compositions may also contain buffering agents. Solid compositions of a similar type may also be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols.

[0111] Tablets can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using binders (e.g., gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surfactants, or dispersants. Molded tablets can be made by molding a mixture of the powdered compound moistened with an inert liquid diluent in a suitable machine.

[0112] Tablets and other solid dosage forms of pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills, and granules, can be optionally scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They can also be formulated to provide sustained or controlled release of the active ingredient therein, for example, using various proportions of hydroxypropylmethylcellulose, other polymer matrices, liposomes, and / or microspheres to provide the desired release profile. They can be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved in sterile water or other sterile injectable medium immediately before use. These compositions can also optionally contain opacifying agents and can be compositions that release the active ingredient only, or preferentially, in a certain part of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can, if appropriate, be in microencapsulated form with one or more of the above-mentioned excipients.

[0113] The liquid dosage form useful for oral administration includes pharmaceutically acceptable emulsion, lyophilized product for reconstitution, microemulsion, solution, suspension, syrup and elixir.In addition to active ingredient, liquid dosage form can contain the inert diluent commonly used in the art, such as water or other solvent, cyclodextrin and its derivatives, solubilizer and emulsifier, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oil (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol and fatty acid ester of sorbitan, and their mixtures.

[0114] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0115] Suspensions may contain, in addition to the active compounds, suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, hydroxyaluminum oxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

[0116] Formulations of pharmaceutical compositions for rectal, vaginal, or urethral administration may be provided as suppositories, which can be prepared by mixing one or more active compounds with one or more suitable non-irritating excipients or carriers including, for example, cocoa butter, polyethylene glycol, a suppository wax, or a salicylate, which are solid at room temperature but liquid at body temperature and will therefore melt in the rectum or vaginal cavity and release the active compound.

[0117] Formulations of the pharmaceutical composition for oral administration may be presented as a mouthwash, or an oral spray, or an oral ointment.

[0118] Alternatively, or in addition, the compositions may be formulated for delivery via a catheter, stent, wire, or other intraluminal device, which may be particularly useful for delivery to the bladder, urethra, ureter, rectum, or intestine.

[0119] Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

[0120] Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and any preservatives, buffers, or propellants that may be required.

[0121] The ointments, pastes, creams and gels may contain, in addition to the active compound, excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0122] Powders and sprays can contain, in addition to the active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0123] Transdermal patch has the additional advantage of providing controlled delivery of the compound of the present invention to the body.This dosage form can be prepared by dissolving or dispersing active compound in suitable medium.Absorption enhancers can also be used to increase the flux of compound across the skin.The rate of this flux can be controlled by providing a rate-controlling membrane or dispersing compound in a polymer matrix or gel.

[0124] Ophthalmic preparations, eye ointments, powders, solutions, etc. are also considered to be within the scope of the present invention. Exemplary ophthalmic preparations are described in U.S. Patent Application Publication Nos. 2005 / 0080056, 2005 / 0059744, 2005 / 0031697, and 2005 / 004074, and U.S. Patent No. 6,583,124, the contents of which are incorporated herein by reference. If desired, liquid ophthalmic preparations have properties similar to those of tears, aqueous humor, or vitreous humor, or are compatible with such fluids. Preferred administration routes are topical administration (e.g., topical administration such as eye drops, or administration via implants).

[0125] As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, intraocular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

[0126] Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions immediately before use, which may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0127] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0128] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of microbial action can be ensured by including various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents such as sugars and sodium chloride in the composition. In addition, prolonged absorption of injectable pharmaceutical forms can be brought about by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0129] In some cases, it is desirable to delay the absorption of drugs from subcutaneous or intramuscular injection in order to prolong the effect of drugs.This can be achieved by using a liquid suspension of crystalline or amorphous material with poor water solubility.The absorption rate of the drug then depends on its dissolution rate, which may depend on crystal size and crystalline form.Alternatively, delayed absorption of parenterally administered drug forms can be achieved by dissolving or suspending the drug in an oil vehicle.

[0130] Injectable depot forms are prepared by forming microencapsulated matrices of the target compound in biodegradable polymers such as polylactide-polyglycolide. The drug release rate can be controlled depending on the drug-to-polymer ratio and the properties of the specific polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Injectable depot formulations can also be prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0131] For use in the methods of the present invention, the active compound can be provided per se or as a pharmaceutical composition containing, for example, about 0.1 to about 99.5% (more preferably, about 0.5 to about 90%) of the active ingredient in combination with a pharmaceutically acceptable carrier.

[0132] The introduction method may also be provided by a rechargeable or biodegradable device. In recent years, various sustained-release polymeric devices for the controlled delivery of drugs, including proteinaceous biologics, have been developed and tested in vivo. Various biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form implants for sustained release of compounds at specific target sites.

[0133] Actual dosage levels of the active ingredient in the pharmaceutical compositions may be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and method of administration without toxicity to the patient.

[0134] The selected dosage level will depend on a variety of factors, including the activity of the particular compound or combination of compounds, or esters, salts, or amides thereof, used, the route of administration, the time of administration, the rate of excretion of the particular compound used, the duration of treatment, other drugs, compounds, and / or substances used in combination with the particular compound used, the age, sex, weight, condition, general health, and prior medical history of the subject being treated, and similar factors well known in the medical arts.

[0135] A physician or veterinarian with ordinary skill in the art can easily determine and prescribe the required therapeutically effective amount of the pharmaceutical composition. For example, a physician or veterinarian can start the dosage of the pharmaceutical composition or compound at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. A "therapeutically effective amount" refers to the concentration of the compound sufficient to induce the desired therapeutic effect. It is generally understood that the effective amount of a compound varies depending on the subject's weight, sex, age, and medical history. Other factors that affect the effective amount may include, but are not limited to, the severity of the subject's condition, the disorder being treated, the stability of the compound, and, if desired, other types of therapeutic agents administered together with the compound of the present invention. A larger total dose can be delivered by multiple administrations of the drug. Methods for determining efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13 ed., 1814-1882, incorporated herein by reference).

[0136] In general, a suitable daily dose of an active compound used in the compositions and methods of the present invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[0137] If desired, the effective daily dose of the active compound may be administered as 1, 2, 3, 4, 5, 6 or more subdoses administered separately at appropriate intervals throughout the day, and may be administered in unit dosage form as needed. In certain embodiments of the present invention, the active compound may be administered two or three times a day. In certain embodiments, the active compound is administered once a day.

[0138] In certain embodiments, the compounds of the present invention can be used alone or can be administered in combination with another type of therapeutic agent.As used herein, the phrase "conjoint administration" refers to any form of administration of two or more different therapeutic compounds, such that a second compound is administered while a previously administered therapeutic compound is still effective in the body (e.g., the two compounds are effective in a subject at the same time, which may include the synergistic effect of the two compounds).For example, different therapeutic compounds can be administered simultaneously or sequentially in either the same formulation or separate formulations.In certain embodiments, different therapeutic compounds can be administered within 1 hour, 12 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours, or 1 week of each other.Therefore, subjects receiving such treatment can benefit from the combined effect of different therapeutic compounds.

[0139] In certain embodiments, co-administration of a compound of the invention with one or more additional therapeutic agents provides improved efficacy compared to administration of either the compound of the invention or the one or more additional therapeutic agents individually. In certain such embodiments, the co-administration provides an additive effect, where additive effect refers to the sum of the effects of each of the individual administrations of the compound of the invention and the one or more additional therapeutic agents.

[0140] The present invention includes the use of pharmaceutically acceptable salts of the compounds of the present invention in the compositions and methods of the present invention. In certain embodiments, contemplated salts of the present invention include, but are not limited to, alkyl, dialkyl, trialkyl, or tetra-alkylammonium salts. In certain embodiments, contemplated salts of the present invention include, but are not limited to, L-arginine, benethamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, contemplated salts of the present invention include, but are not limited to, Na, Ca, K, Mg, Zn, or other metal salts.

[0141] Pharmaceutically acceptable acid addition salts may also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, etc. Mixtures of such solvates may also be prepared. The source of such solvates may be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.

[0142] Wetting agents, emulsifying agents and lubricating agents (such as sodium lauryl sulfate and magnesium stearate), as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition.

[0143] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and α-tocopherol; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0144] Treatment method Non-selective Ca 2+ Transient receptor potential (TRP) channels act as sensors that transduce extracellular cues to the intracellular environment in diverse cellular processes, including actin remodeling and cell migration (Greka et al., Nat Neurosci 6, 837-845, 2003; Ramsey et al., Annu Rev Physiol 68, 619-647, 2006; Montell, Pflugers Arch 451, 19-28, 2005; Clapham, Nature 426, 517-524, 2003). Dynamic reorganization of the actin cytoskeleton is governed by spatiotemporally regulated Ca2+ release. 2+These changes are dependent on the influx (Zheng and Poo, Annu Rev Cell Dev Biol 23, 375-404, 2007; Brandman and Meyer, Science 322, 390-395, 2008; Brandman and Meyer, Science 322, 390-395, 2008); Collins and Meyer, Dev Cell 16, 160-161, 2009), and the small GTPases RhoA and Rac1 act as key modulators of these changes (Etienne-Manneville and Hall, Nature 420, 629-635, 2002; Raftopoulou and Hall, Dev Biol 265, 23-32, 2004). RhoA induces stress fiber and focal adhesion formation, and Rac1 mediates lamellipodia formation (Etienne-Manneville and Hall, Nature 420, 629-635, 2002). Transient receptor potential cation channel, subfamily C, member 5 (TRPC5), acts in concert with TRPC6 to regulate Ca2+ transport in kidney podocytes and fibroblasts. 2+ Regulating TRPC5-mediated Ca influx, actin remodeling, and cell motility 2+ Influx of TRPC6-mediated Ca enhances Rac1 activity, but 2+ Influx promotes RhoA activity. Genetic silencing of TRPC6 channels eliminates stress fibers, reduces focal adhesions, and results in a motile, migratory cell phenotype. In contrast, genetic silencing of TRPC5 channels rescues stress fiber formation and results in a contractile cell phenotype. The results described herein reveal a conserved signaling mechanism whereby TRPC5 and TRPC6 channels control a tightly regulated balance of cytoskeletal dynamics through differential coupling to Rac1 and RhoA.

[0145] Actin cytoskeleton Ca 2+ Ca-dependent remodeling is a dynamic process that drives cell migration (Wei et al., Nature 457, 901-905, 2009). RhoA and Rac1 act as switches involved in cytoskeletal rearrangements in migrating cells (Etienne-Manneville and Hall, Nature 420, 629-635, 2002; Raftopoulou and Hall, Dev Biol 265, 23-32, 2004). Rac1 activation mediates a motile cell phenotype, whereas RhoA activity promotes a contractile phenotype (Etienne-Manneville and Hall, Nature 420, 629-635, 2002). 2+ plays a central role in the regulation of small GTPases (Aspenstrom et al., Biochem J 377, 327-337, 2004). 2+ Flicker is abundant near the leading edge of migrating cells (Wei et al., Nature 457, 901-905, 2009). 2+ Microdomains participate in localized bursts of Rac1 activity as key events at the leading edge (Gardiner et al., Curr Biol 12, 2029-2034, 2002; Machacek et al., Nature 461, 99-103, 2009). To date, no studies have demonstrated that Ca2+ is involved in GTPase regulation. 2+ The source of the influx has been very elusive. TRP (Transient Receptor Potential) channels mediate the temporally and spatially restricted Ca influx associated with cell migration in fibroblasts and neuronal growth cones. 2+Specifically, TRPC5 channels are known regulators of neuronal growth cone guidance, and their activity in neurons depends on PI3K and Rac1 activity (Bezzerides et al., Nat Cell Biol 6, 709-720, 2004).

[0146] Podocytes are neuron-like cells derived from the metanephric mesenchyme of the kidney glomerulus and are essential for the formation of the renal filtration apparatus (Somlo and Mundel, Nat Genet. 24, 333-335, 2000; Fukasawa et al., J Am Soc Nephrol 20, 1491-1503, 2009). Podocytes have an extremely sophisticated repertoire of cytoskeletal adaptations to environmental cues (Somlo and Mundel, Nat Genet 24, 333-335, 2000; Garg et al., Mol Cell Biol 27, 8698-8712, 2007; Verma et al., J Clin Invest 116, 1346-1359, 2006; Verma et al., J Biol Chem 278, 20716-20723, 2003; Barletta et al., J Biol Chem 278, 19266-19271, 2003; Holzman et al., Kidney Int 56, 1481-1491, 1999; Ahola et al., Am J Pathol 155, 907-913, 2009). 1999; Tryggvason and Wartiovaara, N Engl J Med 354, 1387-1401, 2006; Schnabel and Farquhar, J Cell Biol 111, 1255-1263, 1990; Kurihara et al., Proc Natl Acad Sci USA 89, 7075-7079, 1992). Early events in podocyte injury include dysregulation of the actin cytoskeleton (Faul et al., Trends Cell Biol 17, 428-437, 2007; Takeda et al., J Clin Invest 108, 289-301, 2001; Asanuma et al., Nat Cell Biol 8, 485-491, 2006) and Ca 2+Renal nephropathy is characterized by dysregulation of calcium homeostasis (Hunt et al., J Am Soc Nephrol 16, 1593-1602, 2005; Faul et al., Nat Med 14, 931-938, 2008). These changes are associated with the development of proteinuria, loss of albumin into Bowman's space, and ultimately renal failure (Tryggvason and Wartiovaara, N Engl J Med 354, 1387-1401, 2006). The vasoactive hormone angiotensin II induces Ca deficiency in podocytes. 2+ Ca influx is induced, and prolonged treatment leads to the loss of stress fibers (Hsu et al., J Mol Med 86, 1379-1394, 2008). 2+ Although there is a recognized relationship between influx and cytoskeletal reorganization, the mechanisms by which podocytes sense and transduce extracellular cues that regulate cell shape and motility have remained elusive. TRP Canonical 6 (TRPC6) channel mutations have been associated with podocyte injury (Winn et al., Science 308, 1801-1804, 2005; Reiser et al., Nat Genet 37, 739-744, 2005; Moller et al., J Am Soc Nephrol 18, 29-36, 2007; Hsu et al., Biochim Biophys Acta 1772, 928-936, 2007), but little is known about the specific pathways that regulate this process. Furthermore, TRPC6 shares close homology with six other members of the TRPC channel family (Ramsey et al., Annu Rev Physiol 68, 619-647, 2006; Clapham, Nature 426, 517-524, 2003). TRPC5 channels antagonize TRPC6 channel activity to control a tightly regulated balance of cytoskeletal dynamics through differential coupling to distinct small GTPases.

[0147] Proteinuria Proteinuria is a pathological condition in which protein is present in the urine. Albuminuria is one type of proteinuria. Microalbuminuria occurs when the kidneys leak small amounts of albumin into the urine. In a normally functioning body, albumin is normally retained in the bloodstream by the kidneys and therefore not present in the urine. Microalbuminuria is diagnosed by a 24-hour urine collection (20-200 μg / min) or, more commonly, by high levels (30-300 mg / L) on at least two occasions. Microalbuminuria may be a precursor to diabetic nephropathy. Albumin levels above these values ​​are called macroalbuminuria. Subjects with certain conditions, such as diabetic nephropathy, progress from microalbuminuria to macroalbuminuria, reaching the nephrotic range (>3.5 g / 24 hours) as kidney disease reaches advanced stages.

[0148] Causes of Proteinuria Proteinuria can be associated with several conditions, including focal segmental glomerulosclerosis, IgA nephropathy, diabetic nephropathy, lupus nephritis, membranoproliferative glomerulonephritis, progressive (crescentic) glomerulonephritis, and membranous glomerulonephritis. Each of these conditions can be treated by the patient stratification methods described herein.

[0149] Some of the renal disorders that can be treated by the methods described herein are detailed below.

[0150] A. Focal segmental glomerulosclerosis (FSGS) Focal segmental glomerulosclerosis (FSGS) is a disease that attacks the kidney's filtration system (glomeruli), causing severe scarring. FSGS is one of many causes of a condition known as nephrotic syndrome, which occurs when blood proteins leak into the urine (proteinuria). Primary FSGS usually presents as nephrotic syndrome when no underlying cause is found. Secondary FSGS, when an underlying cause is identified, usually presents with kidney failure and proteinuria. FSGS can be hereditary; currently, there are several known genetic causes for hereditary forms of FSGS.

[0151] There are very few treatments available for patients with FSGS. Many patients are treated with steroid regimens, most of which have severe side effects. Some patients have been shown to respond positively to immunosuppressant drugs as well as blood pressure medications that have been shown to reduce urinary protein levels. To date, there is no generally accepted, effective treatment or cure, and no FDA-approved drug for treating FSGS. Therefore, more effective methods for reducing or inhibiting proteinuria are desirable.

[0152] B. Diabetic nephropathy Diabetic nephropathy, also known as Kimmelstiel-Wilson syndrome and intercapillary glomerulonephritis, is a progressive kidney disease caused by capillary vascular damage in the renal glomeruli. It is characterized by nephrotic syndrome and diffuse glomerulosclerosis. This is due to long-standing diabetes and is the primary cause of dialysis. The earliest detectable change in the course of diabetic nephropathy is thickening of the glomeruli. At this stage, the kidneys begin to allow more serum albumin into the urine than normal. As diabetic nephropathy progresses, an increasing number of glomeruli are destroyed by nodular glomerulosclerosis, resulting in increased amounts of albumin excreted in the urine.

[0153] C. Membranoproliferative glomerulonephritis I / II / III Membranoproliferative glomerulonephritis is a type of glomerulonephritis caused by deposits and basement membrane thickening in the glomerular mesangium, complement activation, and glomerular damage. There are three types of membranoproliferative glomerulonephritis. Type I is caused by immune complex deposition in the kidney and is thought to be related to the classical complement pathway. Type II is similar to type I but is thought to be related to the alternative complement pathway. Type III is very rare and is characterized by a mixture of subepithelial deposits and the typical pathological findings of type I disease.

[0154] Based on immunofluorescence microscopy, there are two major types of MPGN: immune complex-mediated and complement-mediated. Hypocomplementemia is common in all types of MPGN. In immune complex-mediated MPGN, complement activation occurs via the classical pathway and is typically manifested by normal or mildly decreased serum C3 and low serum C4 concentrations. In complement-mediated MPGN, low serum C3 and normal C4 levels are usually present due to activation of the alternative pathway. However, complement-mediated MPGN is not excluded by normal serum C3 concentrations, and it is not uncommon to find normal C3 concentrations in adults with dense deposit disease (DDD) or C3 glomerulonephritis (C3GN).

[0155] C3 glomerulonephritis (C3GN) demonstrates glomerulonephritis on light microscopy (LM), bright C3 staining on immunofluorescence microscopy (IF), and the absence of C1q, C4, and immunoglobulins (Ig), as well as mesangial and / or subendothelial electron-dense deposits on electron microscopy (EM). Occasional intramembranous and subepithelial deposits are also frequently present. The term "C3 glomerulopathy" is often used to encompass C3GN and dense deposit disease (DDD), both of which result from dysregulation of the alternative complement pathway (AP). C3GN and DDD can be difficult to distinguish from each other in LM and IF studies. However, EM demonstrates mesangial and / or subendothelial, intramembranous, and subepithelial deposits in C3GN, whereas in DDD, dense osmiophilic deposits are present along the glomerular basement membrane (GBM) and in the mesangium. Both C3GN and DDD are distinguished from immune complex-mediated glomerulonephritis by the lack of immunoglobulin staining on IF (Sethi et al., Kidney Int. (2012) 82(4):465-473).

[0156] D. Membranous glomerulonephritis Membranous glomerulonephritis (MGN) is a slowly progressive disease of the kidneys that primarily affects patients aged 30 to 50, usually Caucasians (or Caucasians). It can develop into nephrotic syndrome. MGN is caused by circulating immune complexes. Current research indicates that the majority of immune complexes are formed via in situ binding of antibodies to antigens on the glomerular basement membrane. The antigens can be endogenous to the basement membrane or deposited from the systemic circulation.

[0157] E. Alport syndrome Alport syndrome is a genetic disorder affecting approximately 1 in 5,000 to 10,000 people and characterized by glomerulonephritis, end-stage kidney disease, and hearing loss. Alport syndrome can also affect the eyes, but the changes usually do not affect vision except in later life when changes to the lens occur. Blood in the urine is common. Proteinuria is a hallmark as kidney disease progresses.

[0158] F. Minimal Change Disease Minimal change disease (also known as MCD, minimal change glomerulopathy, and Nil disease, among others) is a disease affecting the kidneys that causes nephrotic syndrome. Clinical signs of minimal change disease are proteinuria (abnormal excretion of protein, primarily albumin, in the urine), edema (swelling of soft tissues as a result of fluid retention), weight gain, and hypoalbuminemia (low serum albumin). These signs are collectively referred to as nephrotic syndrome. The primary clinical sign of minimal change disease is usually edema, accompanied by associated weight gain. While swelling can be mild, patients may present with edema in the lower half of the body, periorbital edema, swelling in the scrotal / labial area, and, in more severe cases, generalized edema. In elderly individuals, patients may also present with acute kidney injury (20–25% of affected adults) and hypertension. Due to the disease process, patients with minimal change disease are also at risk for blood clots and infection.

[0159] G. Membranous nephropathy Membranous nephropathy refers to the deposition of immune complexes in the glomerular basement membrane (GBM), resulting in GBM thickening. The cause is usually unknown (idiopathic), but secondary causes include drugs, infections, autoimmune diseases, and cancer. Symptoms include the insidious onset of edema and heavy proteinuria with benign urinary sediment, normal renal function, and normal or elevated blood pressure. Membranous nephropathy is diagnosed by kidney biopsy. Spontaneous remission is common. Treatment for patients at high risk of progression is usually with corticosteroids and cyclophosphamide or chlorambucil.

[0160] H. Postinfectious glomerulonephritis Acute proliferative glomerulonephritis is a disorder of the glomeruli (glomerulonephritis) or the kidney's microvasculature. It is a common complication of bacterial infection, typically skin infections (impetigo) caused by types 12, 4, and 1 Streptococcus bacteria, but also after streptococcal pharyngitis, which is also known as postinfectious or poststreptococcal glomerulonephritis. This may be a risk factor for future albuminuria. In adults, signs and symptoms of infection may still be present at the time kidney problems develop, and the terms infection-associated glomerulonephritis or bacterial infection-associated glomerulonephritis are also used. Acute glomerulonephritis resulted in 19,000 deaths worldwide in 2013, down from 24,000 deaths in 1990. Acute proliferative glomerulonephritis (poststreptococcal glomerulonephritis) is caused by streptococcal infection, usually 3 weeks after a pharyngeal or skin infection, given the time required to produce antibodies and complement proteins. The infection causes inflammation in the kidney's blood vessels, which interferes with the kidney's ability to filter urine [Eison et al., "Post-streptococcal acute glomerulonephritis in children: clinical features and pathogenesis," Pediatr. Nephrol. 2011, 26:165-180]. Acute proliferative glomerulonephritis occurs most commonly in children. Additionally, glomerulopathies, including glomerulonephritis, are also associated with bacterial endocarditis, hepatitis C infection, and HIV infection.

[0161] I. Goodpasture's syndrome Goodpasture syndrome, also known as anti-glomerular basement membrane disease, is a rare autoimmune disorder in which antibodies attack the basement membranes of the lungs and kidneys, resulting in pulmonary bleeding and kidney failure. It is thought to attack the alpha-3 subunit of type IV collagen, hence the name Goodpasture antigen. Goodpasture syndrome rapidly leads to permanent lung and kidney damage and often death.

[0162] J. IgA nephropathy IgA nephropathy (also known as IgA nephritis, IgAN, Burger's disease, and pharyngitis glomerulonephritis) is a form of glomerulonephritis (inflammation of the kidney's glomeruli). IgA nephropathy is the most common form of glomerulonephritis worldwide. Primary IgA nephropathy is characterized by the deposition of IgA antibodies in the glomeruli. There are other diseases associated with glomerular IgA deposition, the most common of which is Henoch-Schönlein purpura (HSP), which is considered a systemic form of IgA nephropathy. Henoch-Schönlein purpura presents with a characteristic purpuric skin rash, arthritis, and abdominal pain and occurs more commonly in young adults (16–35 years of age). HSP is associated with a more favorable prognosis than IgA nephropathy. IgA nephropathy progresses slowly to chronic renal failure over a 20-year period in 25–30% of cases.

[0163] K. Lupus nephritis Lupus nephritis is a kidney disorder that is a complication of systemic lupus erythematosus (SLE). It occurs when antibodies and complement build up in the kidneys, causing inflammation. This often leads to proteinuria and can rapidly progress to kidney failure. Nitrogenous waste products build up in the bloodstream. SLE causes various disorders of the kidney's internal structures, including interstitial nephritis. Lupus nephritis affects approximately 3 in 10,000 people.

[0164] L. Polycystic kidney disease Polycystic kidney disease (PKD, also known as polycystic kidney syndrome) is a genetic disorder in which renal tubules become structurally abnormal, leading to the development and growth of numerous cysts within the kidneys. These cysts can begin to develop in utero, during infancy, childhood, or adulthood. Cysts, which are nonfunctioning tubules filled with fluid pumped through them, range in size from microscopic to giant, and can crush adjacent normal tubules, eventually rendering them nonfunctional. PKD is caused by an abnormal gene that produces a specific abnormal protein; this protein adversely affects the development of renal tubules. PKD is a general term for two types: autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD), each with its own unique pathology and genetic causes. PKD affects approximately 500,000 people in the United States.

[0165] Measurement of urine protein levels Urine protein levels can be measured using methods known in the art. Until recently, accurate protein measurement required a 24-hour urine collection. In a 24-hour collection, the patient urinates into a container, which is refrigerated during the trip to the bathroom. The patient is instructed to begin collecting urine after the first trip to the bathroom in the morning. All remaining urine drops for the day are collected in the container. The next morning, the patient adds the first urination after waking up, completing the collection.

[0166] More recently, researchers have found that a single urine sample can provide the necessary information. Newer techniques compare the amount of albumin in a urine sample with the amount of creatinine, a waste product of normal muscle breakdown. This measurement is called the urinary albumin-to-creatinine ratio (UACR). A urine sample containing more than 30 milligrams of albumin per gram of creatinine (30 mg / g) is a warning sign that there may be a problem. If the laboratory test shows a reading above 30 mg / g, another UACR test should be performed in 1 to 2 weeks. If the second test also shows high levels of protein, the person has persistent proteinuria, a sign of declining kidney function, and should have further testing to evaluate kidney function.

[0167] Tests that measure the amount of creatinine in the blood also indicate whether a subject's kidneys are efficiently removing waste products. Too much creatinine in the blood is a sign that a person has kidney damage. Doctors can use creatinine measurements to estimate how efficiently the kidneys are filtering blood. This calculation is called the estimated glomerular filtration rate, or eGFR. Chronic kidney disease exists when the eGFR is less than 60 milliliters per minute (mL / min).

[0168] TRPC5 TRPC is a family of transient receptor potential cation channels in animals. TRPC5 is a subtype of the TRPC family of mammalian transient receptor potential ion channels. Three examples of TRPC5 are highlighted in Table 1 below. [Table 1]

[0169] Transient receptor potential channel 5 (TRPC5) is a calcium-permeable nonspecific cation channel primarily expressed in the brain. It can form a heterotetrameric complex with TRPC1 and TRPC4 channel subunits. TRPC5 is also expressed in the kidney, more specifically in podocytes, where it is involved in regulating the podocyte actin cytoskeleton.

[0170] Thus, in certain embodiments, the present invention provides a method for treating diseases including diabetic nephropathy, focal segmental glomerulosclerosis, minimal change disease, membranoproliferative glomerulonephritis (including poststreptococcal glomerulonephritis and bacterial endocarditis-associated glomerulonephritis), membranous nephropathy, other hepatitis C virus-associated glomerulopathies, HIV-associated glomerulopathies, COVID-19-associated acute kidney injury, Alport syndrome, polycystic kidney disease (both autosomal dominant and autosomal recessive), IgA nephropathy, other inherited nephropathies or ciliopathy (e.g., HNF1β, nephronophthisis, autosomal dominant cystic / tubular kidney disease), lupus nephritis, Goodpasture's syndrome (anti-GBM disease), and

[0013] Methods for treating or reducing the risk of developing a renal disease selected from other complement-mediated or immune-mediated renal diseases are provided, wherein the subject has a urinary level of one or more biomarkers selected from Rac1, Rac1-GTP, phospho-LIM kinase 1, and phospho-cofilin above a predetermined threshold, comprising administering to the subject in need thereof a therapeutically effective amount of a compound of the present invention (e.g., a compound of structural formula I, II, III, IV, V, VI, VII, VIII, IX, X, or XI, or a calcineurin inhibitor) or a pharmaceutical composition comprising said compound. In some aspects of these embodiments, the renal disease is selected from diabetic nephropathy, focal segmental glomerulosclerosis, minimal change disease, membranoproliferative glomerulonephritis, membranous nephropathy, other hepatitis C virus-associated glomerulonephropathy, and Alport syndrome.

[0171] In some embodiments, the kidney disease is diabetic nephropathy or focal segmental glomerulosclerosis.

[0172] Treatment target In one aspect of the invention, the subject has a urinary level of one or more biomarkers selected from Rac1, Rac1-GTP, phospho-LIM kinase 1, and phospho-cofilin above a predetermined threshold; and is also diagnosed with diabetic nephropathy, focal segmental glomerulosclerosis, minimal change disease, membranoproliferative glomerulonephritis (including poststreptococcal glomerulonephritis and bacterial endocarditis-associated glomerulonephritis), membranous nephropathy, other hepatitis C virus-associated glomerulopathies, HIV-associated glomerulopathies, and / or urinary tract infections. Patients are selected based on having or being at risk of developing kidney diseases such as globulopathy, COVID-19 associated acute kidney injury, Alport syndrome, polycystic kidney disease (both autosomal dominant and autosomal recessive), IgA nephropathy, other inherited nephropathies or ciliopathies (e.g., HNF1β, nephronophthisis, autosomal dominant cystic / tubular kidney disease), lupus nephritis, Goodpasture syndrome (anti-GBM disease), and other complement-mediated or immune-mediated kidney diseases.

[0173] In some specific embodiments, the subject to be treated has or is at risk of developing diabetic nephropathy, focal segmental glomerulosclerosis, minimal change disease, membranoproliferative glomerulonephritis, membranous nephropathy, other hepatitis C virus-associated glomerulopathies, or Alport syndrome. Subjects with or at risk of developing proteinuria include those with diabetes, hypertension, or certain family backgrounds. In the United States, diabetes is the leading cause of end-stage renal disease (ESRD). In both type 1 and type 2 diabetes, albumin in the urine is one of the first signs of deteriorating kidney function. As kidney function declines, the amount of albumin in the urine increases. Another risk factor for developing proteinuria is hypertension. Proteinuria in hypertensive individuals is an indicator of declining kidney function. If hypertension is not controlled, it can progress to complete kidney failure. African Americans are more likely than Caucasians to have high blood pressure and develop kidney problems from it, even if their blood pressure is only mildly elevated. Other groups at risk for proteinuria are American Indians, Hispanics / Latin Americans, Pacific Islanders, the elderly, and overweight subjects.

[0174] In one embodiment of the present invention, a subject is selected based on having a urinary level of one or more biomarkers selected from Rac1, Rac1-GTP, phospho-LIM kinase 1, and phospho-cofilin that exceeds a predetermined threshold; and having or being at risk for developing proteinuria. A subject with or at risk for developing proteinuria is a subject with one or more symptoms of the condition. Symptoms of proteinuria are known to those skilled in the art and include, but are not limited to, a large amount of protein in the urine, which appears as foaming in the toilet. A large amount of protein loss can lead to edema, which can cause swelling of the hands, feet, abdomen, or face. These are signs of significant protein loss and indicate that kidney disease has progressed. Laboratory testing is the only way to determine whether protein is present in a subject's urine before extensive kidney damage occurs.

[0175] The method is effective for a variety of subjects, including humans and other animals, such as mammals, e.g., laboratory animals (e.g., mice, rats, rabbits, or monkeys), or domestic and livestock animals (e.g., cats, dogs, goats, sheep, pigs, cows, or horses). In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0176] In one aspect, the present invention provides a method for selecting and treating a human subject suffering from a kidney disease, comprising: a. selecting a subject if the subject has a urinary level of one or more biomarkers selected from Rac1, Rac1-GTP, phospho-LIM kinase 1, and phospho-cofilin above a predetermined threshold; and b. administering to the selected subject a pharmaceutical composition comprising a TRPC5 inhibitor or a calcineurin inhibitor and a pharmaceutically acceptable carrier; The present invention relates to a method comprising:

[0177] In one aspect, the present invention provides a method of treating a human subject suffering from a kidney disease, comprising: only if the subject is determined to have a pre-treatment urinary level of one or more biomarkers selected from Rac1, Rac1-GTP, phospho-LIM kinase 1, and phospho-cofilin above a predetermined threshold, TRPC5 inhibitors or calcineurin inhibitors, and a pharmaceutically acceptable carrier; The present invention relates to a method comprising administering to a subject a pharmaceutical composition comprising:

[0178] According to these embodiments, subjects whose pre-treatment urinary levels of one or more biomarkers selected from Rac1, Rac1-GTP, phospho-LIM kinase 1, and phospho-cofilin are below a predetermined threshold for those biomarkers are not treated with a TRPC5 inhibitor or a calcineurin inhibitor. In some specific embodiments, subjects whose pre-treatment urinary Rac1 levels are below a predetermined threshold are not treated with a TRPC5 inhibitor or a calcineurin inhibitor. In other specific embodiments, subjects whose pre-treatment urinary Rac1-GTP levels are below a predetermined threshold are not treated with a TRPC5 inhibitor or a calcineurin inhibitor. In still other specific embodiments, subjects whose pre-treatment urinary levels of phospho-LIM kinase 1 are below a predetermined threshold are not treated with a TRPC5 inhibitor or a calcineurin inhibitor. In still other specific embodiments, subjects whose pre-treatment urinary levels of phospho-cofilin are below a predetermined threshold are not treated with a TRPC5 inhibitor or a calcineurin inhibitor.

[0179] Rac1 and Rac1-GTP Rac1, also known as Ras-related C3 botulinum toxin substrate 1, is a small (approximately 21 kDa) signaling G protein (more specifically, a GTPase) found in human cells. It is a member of the Rac subfamily of the Rho family of GTPases. Members of this superfamily appear to regulate diverse cellular events, including control of GLUT4 translocation for glucose uptake, cell proliferation, cytoskeletal reorganization, antimicrobial cytotoxicity, and activation of protein kinases. Rac1 is expressed at significant levels in insulin-sensitive tissues such as adipose tissue and skeletal muscle. Here, Rac1 regulates the translocation of glucose-transporting GLUT4 vesicles from intracellular compartments to the plasma membrane. In response to insulin, this allows blood glucose to enter cells and lower blood glucose levels. In obesity and type 2 diabetes, Rac1 signaling in skeletal muscle is dysfunctional, suggesting that Rac1 contributes to disease progression. Rac1 protein is also required for glucose uptake in skeletal muscle, which is activated by exercise and muscle stretching. RAC1 has two conformations: an active form (RAC1-GTP) and an inactive form (RAC1-GDP) [Laboratory Investigation (2018) 98:989-998; Cell Mol Life Sci. (2009) 66:370-4].

[0180] Phospho-LIM kinase 1 LIM kinase-1 (LIMK1) and LIM kinase-2 (LIMK2) are actin-binding kinases that phosphorylate members of the ADF / cofilin family of actin-binding and filament-severing proteins. ADF / cofilin is the only substrate identified to date for LIM kinases. LIM kinases directly phosphorylate and inactivate cofilin family members, resulting in the stabilization of filament (F)-actin. LIM kinases are activated by signaling through the Rho family of small GTPases. The LIM domain is a highly conserved cysteine-rich structure containing two zinc fingers. While zinc fingers typically function by binding to DNA or RNA, the LIM motif likely mediates protein-protein interactions. LIM kinase-1 and LIM kinase-2 belong to a small subfamily of kinases with a unique combination of two N-terminal LIM motifs and a C-terminal protein kinase domain.

[0181] Phospho-cofilin Cofilin and actin-depolymerizing factor (ADF) are members of a family of essentially conserved small actin-binding proteins that play crucial roles in cytokinesis, endocytosis, embryonic development, stress response, and tissue regeneration (Carlier, M.F. et al. (1999) J. Biol. Chem. 274, 33827-30.). In response to stimuli, cofilin promotes the regeneration of actin filaments by severing existing filaments (Condeelis, J. (2001) Trends Cell. Biol. 11, 288-93.). The cleavage activity of cofilin is inhibited by LIMK or TESK phosphorylation at Ser3 of cofilin (Arber, S. et al. (1998) Nature 393, 805-9; Yang, N. et al. (1998) Nature 393, 809-12; Toshima, J. et al. (2001) J Biol Chem 276, 31449-58.). Phosphorylation at Ser3 also regulates cofilin translocation from the nucleus to the cytoplasm (Nebl, G. et al. (1996) J Biol Chem 271, 26276-80.) [https: / / www.cellsignal.com / products / primary-antibodies / phospho-cofilin-ser3-77g2-rabbit-mab / 3313].

[0182] Measuring urinary levels of biomarkers Protein levels in urine can be measured using methods known in the art, such as those described above, and biomarkers can be measured in concentrated urine samples. Antibodies specific to Rac1, Rac1-GTP, phospho-LIM kinase 1, and phospho-cofilin are commercially available. Urine samples can be treated with one or more of these antibodies according to methods known to those skilled in the art.

[0183] In some embodiments, subjects are selected based on having urinary Rac1 levels above a predetermined threshold. The threshold can be adjusted based on the Rac1 (or other metabolite) levels in subjects with specific kidney diseases (e.g., for FSGS vs. membranous nephropathy) and / or based on clinical trial results.

[0184] In some embodiments, the urinary Rac1 level in a subject is measured in the urine fraction containing extracellular vesicles.When active Rac1 is localized in the plasma membrane, microvesicles are a class of extracellular vesicles formed from the plasma membrane budding.The release of microvesicles increases with calcium increase and cytoskeleton disruption, which are the central events in podocyte damage in FSGS and DN.According to some embodiments, extracellular vesicles can be fractionated and isolated from urine using ultracentrifugation.

[0185] In some embodiments, the predetermined threshold level is established by determining the range of urinary levels of a selected biomarker in a population of healthy individuals; and further establishing a predetermined threshold level for the selected biomarker at a level above the 75th percentile in the population. As used herein, "n percentile" refers to a value on a scale of 100 that indicates the percent of the distribution below it. For example, a biomarker level above the 75th percentile in a population refers to a level of a biomarker that is present at a higher concentration than that value in the bottom 75% of the population. As used herein, a "population" is a group or cohort of subjects (e.g., mammals, cats, dogs, primates, or humans); for example, in some embodiments, the predetermined threshold level is established by determining the range of urinary levels of a selected biomarker in a population of healthy individuals (i.e., a group or cohort of healthy individuals); and further establishing a predetermined threshold level for the selected biomarker at a level above the 75th percentile in the population.

[0186] In some embodiments, the predetermined threshold level is greater than the 90th percentile of the population. In some embodiments, the predetermined threshold level is greater than the 95th percentile of the population.

[0187] In some embodiments, the predetermined threshold level of urinary Rac1 is 100 to 500 pg / mL.

[0188] Methods for determining the efficacy of treatment In one aspect, the invention provides a method for determining the efficacy of TRPC5 inhibitor treatment in a human subject suffering from kidney disease, wherein prior to initiating treatment, the subject is determined to have a pre-treatment urinary level of one or more biomarkers selected from Rac1, Rac1-GTP, phospho-LIM kinase 1, and phospho-cofilin above a predetermined threshold; a. Obtaining urinary levels of selected biomarkers in human subjects at time points after initiation of TRPC5 treatment; b. comparing the level of the selected biomarker in step a. with the pre-treatment urinary level of the selected biomarker; c. determining that the TRPC5 inhibitor treatment is efficacious if the level of the selected biomarker in step a. is lower than the pre-treatment urinary level of the selected biomarker; The present invention relates to a method comprising:

[0189] In one aspect, the invention provides a method for determining the efficacy of TRPC5 inhibitor treatment in a human subject suffering from kidney disease, wherein prior to initiating treatment, the subject is determined to have a pre-treatment urinary level of one or more biomarkers selected from Rac1, Rac1-GTP, phospho-LIM kinase 1, and phospho-cofilin above a predetermined threshold; a. Obtaining urinary levels of selected biomarkers in human subjects at time points after initiation of TRPC5 treatment; and b. determining that the TRPC5 inhibitor treatment is efficacious if the level of the selected biomarker in step a. is lower than a predetermined threshold value for the selected biomarker; The present invention relates to a method comprising: [Example]

[0190] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0191] Example 1. TRPC5 activity assay TRPC5-expressing ICLN-1633 cells (HEK-TREx hTRPC5) were generated as follows: 24 hours before transfection, commercially available HekTrex-293 cells were cultured at 0.7 × 10 ng / ml in 2 mL of antibiotic-free cell growth medium (1 × DMEM / high glucose (Hyclone #SH30022.02); 10% fetal bovine serum (Sigma), 2 mM sodium pyruvate, 10 mM HEPES). 6 Cells were seeded at 1x6-well plate per well. The human TRPC5 coding sequence (NM_012471 with a silent T478C mutation) was cloned into pcDNA5 / TO (Invitrogen; Catalog No.: V103320) using hygromycin as a resistance gene, and the plasmid (SEQ ID NO: 2) was propagated in T-Rex-293 cells (Invitrogen; Catalog No.: R71007) according to the manufacturer's instructions. On day 2, 2 μg of plasmid DNA plus 6 μl of Xtreme-GENE HP Reagent in Optimem (total volume 200 μl) was prepared and incubated at room temperature for 15 minutes. The plasmid solution was then gently added dropwise to each well, and the plate was gently swirled for approximately 30 seconds to mix the complex with the medium. The transfected cells were incubated at 37°C in a 10% CO2 incubator for 24 hours. Transfected cells were harvested and transferred to 2 x 150 mm dishes containing cell growth medium without antibiotics at 37°C.

[0192] The next day, selection was initiated to generate stable pools by adding cell growth medium containing 150 μg / mL hygromycin and 5 μg / mL blasticidin, and the cells were allowed to grow. The medium containing the selection agents was changed every 1–2 days as needed to remove dead cells. After 7 days, the hygromycin concentration was reduced to 75 μg / mL, and cell growth was continued.

[0193] Single clones were selected as follows: stable pools were diluted to 10 cells / mL, seeded (100 μl / well) into 24 x 96-well plates (approximately 1 cell / well), and grown in cell growth medium for 7 days. Fresh medium (100 μl) was added, and cells were grown for an additional 1-2 weeks, then either cryopreserved or used immediately.

[0194] Compounds were typically made up to 10 mM stock solutions or supplied as 10 mM stock solutions using DMSO as the vehicle. Ten-point dose-response curves were generated using an Echo-550 acoustic dispenser. Compound source plates were created by serially diluting compound stocks to create 10 mM, 1 mM, and 0.1 mM DMSO solutions in Echo-certified LDV plates. Echo then serially spotted 100% DMSO stock solutions onto the source dose-response plate, creating a 4-fold dilution scheme. 100% DMSO was added to the spotted dose-response plate to a final volume of 5 μl. 300 nl of the dose-response stock plate was then spotted onto the preincubation and stimulation assay plates. 50 μl of preincubation buffer and 100 μl of stimulation buffer were then added to the plate, resulting in a final assay test concentration range of 30 μM to 0.0001 μM with a final DMSO concentration of 0.3%.

[0195] Human ICLN-1633 cells expressing TRPC5 were plated into 384-well black PDL-coated microplates the day before use and maintained in TRPC5 growth medium. TRPC5 expression was induced by application of 1 μg / mL tetracycline at the time of plating. The medium was removed from the plates, and 10 μl of 4 μM Fluo-4AM in EBSS (mixed with an equal volume of Pluronic F-127) was added to the cells. The cells were incubated at room temperature, protected from light, for 60-90 minutes. After the incubation period, the dye was removed and replaced with 10 μl of EBSS. The cell, preincubation, and stimulation plates were loaded onto the FLIPR-II and the assay was initiated. The FLIPR measured a 10-second baseline, followed by the addition of 10 μl of 2X compound (or control). The change in fluorescence was monitored for an additional 5 minutes. After 5 minutes of pre-incubation, 20 μl of 2× Riluzole (containing 1× compound or control) is added to the cell plate. The final Riluzole stimulation concentration in the assay is 30 μM. After adding Riluzole, the change in fluorescence is monitored for an additional 5 minutes.

[0196] Compound modulation of the TRPC5 calcium response was determined as follows: Fluorescence was monitored for 5 minutes after Englerin A. The maximum relative fluorescence response (minus "baseline inhibition" in the following equation, which is the control response of 1 μM of an internal control compound known to maximally block the TRPC5 calcium response) was captured and exported from FLIPR.

[0197] The effect of the compound is calculated as % inhibition using the following formula:

number

[0198] The results of these assays are shown in Table 2 below, where "A" indicates an IC50 of 50 nM or less; "B" indicates an IC50 of greater than 50 nM and less than 500 nM; "C" indicates an IC50 of greater than 500 nM and less than 1 μM; "D" indicates an IC50 of 1 μM or greater; and "NT" indicates that the compound was not tested.

[0199] Example 2. Urinary Rac1 analysis in healthy volunteers, patients with DN, FSGS, PKD and Alport syndrome The purpose of this study was to measure the amount of Rac1 protein in the urine of healthy volunteers and patients with diabetic nephropathy ("DN"), FSGS, polycystic kidney disease ("PKD"), and Alport syndrome.

[0200] Urine samples were obtained from healthy volunteers and patients with DN, FSGS, PKD, and Alport disease. Samples were concentrated using Pierce protein concentrators with a 10 kDa MWCO (catalog number 88516, Thermo Scientific, USA) and centrifuged at 6000 × g for 30 minutes at 4 °C in an AVANTI-JE centrifuge (Beckman Coulter) using rotor JA 14.50 (Beckman Coulter, USA). Samples concentrated to a volume of >1 mL were centrifuged a second time at 6000 × g for 30 minutes at 4 °C to achieve a volume of <1 mL for all samples. Concentrated urine was collected and stored at -80 °C. Samples were then analyzed for urinary Rac1 by ELISA (catalog number abx253084, Abbexa Ltd, UK) using standard procedures according to the manufacturer's instructions.

[0201] As shown in Figure 1A, the Rac1 levels in urine from healthy subjects were 56.4 ± 15.7 pg / mL, those from DN patients were 6600.0 ± 3677.1 pg / mL, those from FSGS patients were 19,610.4 ± 30,070.6 pg / mL, and those from Alport syndrome patients were 20.6 ± 71.4 pg / mL (all results are means ± standard deviations). The lower limit of quantification of the assay is approximately 10 pg / mL, assuming a starting urine volume (before concentration) of >3 mL. Because the available starting volume of Alport patient urine samples was limited, the lower limit of quantification was approximately 100 pg / mL, and most samples were below the limit of quantification (BLQ). On an input-volume-adjusted basis, Alport patients have Rac1 levels comparable to those of healthy subjects.

[0202] When Rac1 levels in additional healthy individuals, DN and FSGS patients, and PKD patients were included in the assay, the urinary Rac1 levels in healthy subjects were 107.0 ± 44.6 pg / mL, the urinary Rac1 levels in DN patients were 1,692.9 ± 3,365.8 pg / mL, the urinary Rac1 levels in FSGS patients were 24,525.9 ± 39,369.2 pg / mL, and the urinary Rac1 levels in PKD patients were 2379.0 ± 654.4 pg / mL (see Figure 1B).

[0203] Example 3. Urinary Rac1 analysis in naive rats after treatment with Compound 1 The purpose of this study was to measure the amount of Rac1 protein in the urine of healthy rats treated with Compound 1.

[0204] Six- to seven-week-old Sprague-Dawley rats were placed in metabolic cage housing for urine collection. After two 24-hour pre-dose urine collections, Compound 1 was administered by oral gavage at 10 mg / kg once daily for 7 days; control animals received vehicle. Urine was collected over a 24-hour period beginning on the first day of dosing and on days 3 and 6 of dosing. No adverse effects were observed in animals treated with Compound 1.

[0205] Urine samples were concentrated as follows: 20 mL of urine was centrifuged at 1500 × g for 5 minutes to remove cellular debris. Samples were concentrated using Pierce protein concentrators with a 10 kDa MWCO (catalog number 88516, Thermo Scientific, USA) and centrifuged at 6000 × g for 30 minutes at 4 °C in an AVANTI-JE centrifuge (Beckman Coulter) using rotor JA14.50 (Beckman Coulter, USA). Samples concentrated to a volume of >1 mL were centrifuged a second time at 6000 × g for 30 minutes at 4 °C to achieve a volume of <1 mL for all samples. The concentrated urine was collected and stored at -80 °C. Samples were then analyzed for urinary Rac1 by ELISA (cat. no. abx253084, Abbexa Ltd, UK) and for urinary creatinine by ELISA (cat. no. ab65340, Abcam, USA) using standard procedures according to the manufacturer's instructions. The amount of urinary Rac1 was normalized to the amount of urinary creatinine to adjust for the amount of urine produced.

[0206] As shown in FIG. 2, Compound 1 reduced the urinary Rac1 level compared with the urinary Rac1 level before administration, and the reduction reached significance on day 4 (p value < 0.01).

[0207] Example 4. Urinary Rac1 analysis in DOCA-salt hypertensive rats after treatment with Compound 1 The purpose of this study was to measure the amount of Rac1 protein in the urine of DOCA-salt hypertensive rats treated with Compound 1.

[0208] The DOCA-salt hypertensive rat model is an established model of mineralocorticoid hypertension with renal dysfunction leading to the FSGS phenotype and is characterized by increased levels of urinary protein and albumin excretion [Schenk et al., "The pathogenesis of DOCA-salt hypertension," J. Pharmacol. Toxicol. Methods (May 1992) 27(3):161-170; Gomez-Sanchez et al., "Mineralocorticoids, salt, and high blood pressure," Steroids (1996) 61:184-188.]

[0209] Six- to seven-week-old Sprague-Dawley rats underwent unilateral nephrectomy. After one week of recovery, rats were implanted with a 45 mg DOCA pellet and provided with tap water containing 0.9% NaCl and 0.2% KCl for 4 weeks of treatment (Day 1). On Day 21, rats were administered Compound 1 by oral gavage at 10 mg / kg once daily for 7 days. Body weights were recorded daily throughout the study. No adverse effects were observed in animals treated with Compound 1. Urine was collected over 24 hours starting on Days 17, 20, 24, and 27, and urinary protein and albumin were measured using standard methods.

[0210] For Rac1 analysis, urine samples were concentrated as follows: 20 mL of urine was centrifuged at 1500 × g for 5 minutes to remove cellular debris. Samples were concentrated using Pierce protein concentrators with a 10 kDa MWCO (catalog no. 88516, Thermo Scientific, USA) and centrifuged at 6000 × g for 30 minutes at 4 °C in an AVANTI-JE centrifuge (Beckman Coulter) using rotor JA14.50 (Beckman Coulter, USA). Samples concentrated to a volume of >1 mL were centrifuged a second time at 6000 × g for 30 minutes at 4 °C to achieve a volume of <1 mL for all samples. The concentrated urine was collected and stored at -80 °C. Samples were then analyzed for urinary Rac1 by ELISA (cat. no. abx253084, Abbexa Ltd, UK) and for urinary creatinine by ELISA (cat. no. ab65340, Abcam, USA) by standard procedures according to the manufacturer's instructions. The amount of Rac1 in urine was normalized to the amount of creatinine in urine to adjust for the amount of urine produced.

[0211] As shown in Figure 3, after the start of administration on day 21, Compound 1 reduced urinary Rac1 levels compared with pre-administration levels, and the reduction reached significance on day 25 (p value < 0.01).

[0212] Example 5. Urinary Rac1 analysis in healthy human subjects after treatment with Compound 1 The purpose of this study was to measure the amount of Rac1 protein in the urine of healthy human subjects treated with Compound 1.

[0213] Healthy human subjects enrolled in a Phase 1 clinical trial ("A First-In-Human, Phase 1, Placebo-Controlled Study to Evaluate the Safety, Tolerability, and Pharmacokinetics of Compound 1, a TRPC5 Channel Inhibitor, in Healthy Subjects and Subjects With Renal Impairment" (NCT03970122)) received a single oral dose of placebo or 20 mg of Compound 1 as a tablet. Urine samples were collected before drug administration, followed by pooled urine collections at 0–4 h, 4–8 h, 8–12 h, 12–24 h, 24–48 h, and 48–72 h after administration.

[0214] For Rac1 analysis, urine samples were concentrated as follows: 20 mL of urine was centrifuged at 1500 × g for 5 minutes to remove cellular debris. Samples were concentrated using Pierce protein concentrators with a 10 kDa MWCO (catalog no. 88516, Thermo Scientific, USA) and centrifuged at 6000 × g for 30 minutes at 4 °C in an AVANTI-JE centrifuge (Beckman Coulter) using rotor JA14.50 (Beckman Coulter, USA). Samples concentrated to a volume of >1 mL were centrifuged a second time at 6000 × g for 30 minutes at 4 °C to achieve a volume of <1 mL for all samples. The concentrated urine was collected and stored at -80 °C. Samples were then analyzed for urinary Rac1 by ELISA (cat. no. abx253084, Abbexa Ltd, UK) and for urinary creatinine by ELISA (cat. no. ab65340, Abcam, USA) using standard procedures according to the manufacturer's instructions. The amount of urinary Rac1 was normalized to the amount of urinary creatinine to adjust for the amount of urine produced.

[0215] As shown in Figure 4, compared with the pre-administration level of urinary Rac1, Compound 1 reduced the urinary Rac1 level, and the reduction reached significance by 8 to 12 hours after administration (p value < 0.05).

[0216] Additional data were obtained from human subjects who received either a single oral dose of placebo, 5 mg of Compound 1 as a liquid suspension, or 20, 40, or 80 mg of Compound 1 as tablets. Urine samples were collected prior to drug administration, followed by 24-hour urine pools at 0-4 hours, 4-8 hours, 8-12 hours, 12-24 hours, and on days 2 through 7 after administration. Each dose level included two placebo subjects and eight treated subjects. These results are shown in Figure 4B.

[0217] As shown in Figure 4B, compared to pre-dose levels, Compound 1 reduced urinary Rac1 levels, reaching significance by 8-12 hours post-dose at the 40 mg and 80 mg doses (p<0.05). Urinary Rac1 levels remained reduced for up to 4 days at the single 40 mg dose and for at least 7 days at the single 80 mg dose, consistent with the maintenance of plasma concentrations based on pharmacokinetic analysis.

[0218] Example 6. Rac1 is found in extracellular vesicles in the urine of healthy human subjects The purpose of this study was to determine whether Rac1 protein is found in urine as a soluble protein or contained in extracellular vesicles.

[0219] Extracellular vesicles (EVs) are cell-derived membrane-bound particles that play an important role in intercellular communication [Stahl et al., “Exosomes and microvesicles in normal physiology, pathophysiology, and renal diseases,” Pediatr. Nephrol. (2019) 34: 11-30].

[0220] Healthy subjects enrolled in a Phase 1 clinical trial ("First-in-Human, Phase 1, Placebo-Controlled Study to Evaluate the Safety, Tolerability, and Pharmacokinetics of Compound 1 (a TRPC5 Channel Inhibitor) in Healthy Subjects and Subjects with Renal Impairment" (NCT03970122)) were administered a single oral dose of placebo or 20 mg of Compound 1 as a tablet. Urine samples were collected before drug administration, and then urine pools were collected at 0–4 h, 4–8 h, 8–12 h, 12–24 h, 24–48 h, and 48–72 h after administration.

[0221] For Rac1 analysis, urine samples were concentrated as follows: 20 mL of urine was centrifuged at 1500 × g for 5 min to remove cellular debris. Samples were concentrated using Pierce protein concentrators with a 10 kDa MWCO (catalog no. 88516, Thermo Scientific, USA) and centrifuged at 6000 × g for 30 min at 4 °C using rotor JA14.50 (Beckman Coulter, USA) in an AVANTI-JE centrifuge (Beckman Coulter, USA) to achieve a volume of 1–1.5 mL for all samples. 1 mL of concentrated urine was centrifuged at 120,000 × g for 16 h at 4 °C (Sorvall mx120+ ultracentrifuge, rotor type S120-AT2, ThermoFisher, USA) to pellet extracellular vesicles. A fixed-angle rotor was chosen for its lower K factor and better pelleting efficiency. Supernatants and pellets were collected and analyzed for Rac1 by ELISA (catalogue no. abx253084, Abbexa Ltd, UK) using standard procedures according to the manufacturer's instructions.

[0222] As shown in Figure 5, the majority of urinary Rac1 was found in the extracellular vesicle pellet, which had significantly higher Rac1 levels than in the supernatant (p-value < 0.005).

[0223] Example 7. Urinary Rac1-GTP analysis in human subjects The purpose of this study was to measure the amount of active Rac1 protein (Rac1-GTP) in the urine of healthy human subjects and patients with renal disease.

[0224] Rac1-GTP is the active form of Rac1, and upon activation, Rac1 localizes to the plasma membrane [Garcia-Mata et al., "The invisible hand: regulation of RHO GTPases by RHOGDIs," Nat. Rev. Mol. Cell Biol. (2011) 12: 493-504]. The membrane localization of Rac1 is consistent with its presence in extracellular vesicles.

[0225] Urine samples are concentrated as follows: 20 mL of urine is centrifuged at 1500 × g for 5 minutes to remove cellular debris. Samples are concentrated using Pierce protein concentrators with a 10 kDa MWCO (catalog number 88516, Thermo Scientific, USA) and centrifuged at 6000 × g for 30 minutes at 4 °C in an AVANTI-JE centrifuge (Beckman Coulter) using rotor JA14.50 (Beckman Coulter, USA). Samples concentrated to a volume of > 1 mL are centrifuged a second time at 6000 × g for 30 minutes at 4 °C to achieve a volume of < 1 mL for all samples. The concentrated urine is collected and stored at -80 °C. Samples were then analyzed for urinary Rac1-GTP by G-LISA (Cat. No. BK128, Cytoskeleton, USA) and for urinary creatinine by ELISA (Cat. No. ab65340, Abcam, USA) according to standard procedures, following the manufacturer's instructions. The amount of urinary Rac1-GTP was normalized to the amount of urinary creatinine, adjusting for the amount of urine produced.

[0226] Example 8. Urinary phospho-LIMK1 analysis in human subjects The purpose of this study was to measure the amount of phospho-LIMK1 in the urine of healthy human subjects and patients with renal disease.

[0227] Urine samples were concentrated as follows: 20 mL of urine was centrifuged at 1500 × g for 5 minutes to remove cellular debris. Samples were concentrated using Pierce protein concentrators with a 10 kDa MWCO (catalog no. 88516, Thermo Scientific, USA) and centrifuged at 6000 × g for 30 minutes at 4 °C in an AVANTI-JE centrifuge (Beckman Coulter) using rotor JA14.50 (Beckman Coulter, USA). Samples concentrated to a volume of >1 mL were centrifuged a second time at 6000 × g for 30 minutes at 4 °C to achieve a volume of <1 mL for all samples. The concentrated urine was collected and stored at -80 °C. Samples were then analyzed for urinary phospho-LIMK1 by ELISA (catalog no. 3842S, Cell Signaling Technologies) and for urinary creatinine by ELISA (catalog no. ab65340, Abcam, USA) using standard procedures according to the manufacturer's instructions. The amount of phospho-LIMK1 in urine is normalized to the amount of creatinine in urine to adjust for the amount of urine produced.

[0228] Phospho-LIMK1 was also assessed by immunoblotting. Concentrated urine was lysed in 1x RIPA lysis buffer (Cat. No. 20-188, EMD Millipore, USA) containing a protease inhibitor cocktail (Cat. No. P8340, Sigma, USA), run on an SDS-polyacrylamide gel, transferred to a polyvinylidene difluoride membrane, and immunoblotted with a primary antibody against phospho-LIMK1 (Cat. No. 3842S, Cell Signaling Technologies) according to standard procedures.

[0229] Example 9. Urinary phospho-cofilin analysis in human subjects The aim of this study was to measure the amount of phospho-cofilin in the urine of healthy subjects and patients with renal disease.

[0230] Urine samples were concentrated as follows: 20 mL of urine was centrifuged at 1500 × g for 5 minutes to remove cellular debris. Samples were concentrated using Pierce protein concentrators with a 10 kDa MWCO (catalog no. 88516, Thermo Scientific, USA) and centrifuged at 6000 × g for 30 minutes at 4 °C in an AVANTI-JE centrifuge (Beckman Coulter) using rotor JA14.50 (Beckman Coulter, USA). Samples concentrated to a volume of >1 mL were centrifuged a second time at 6000 × g for 30 minutes at 4 °C to achieve a volume of <1 mL for all samples. The concentrated urine was collected and stored at -80 °C. Samples were then analyzed for urinary phospho-cofilin by ELISA (catalog no. 3318S, Cell Signaling Technologies) and for urinary creatinine by ELISA (catalog no. ab65340, Abcam, USA) using standard procedures according to the manufacturer's instructions. The amount of phospho-cofilin in urine is normalized to the amount of creatinine in urine to adjust for the amount of urine produced.

[0231] Phospho-cofilin was also evaluated by immunoblotting. Concentrated urine was dissolved in 1x RIPA lysis buffer (Cat. No. 20-188, EMD, Millipore, USA) containing a protease inhibitor cocktail (Cat. No. P8340, Sigma, USA), run on an SDS-polyacrylamide gel, transferred to a polyvinylidene difluoride membrane, and immunoblotted with a primary antibody against phospho-cofilin (Cat. No. 3318S, Cell Signaling Technologies) according to standard procedures.

[0232] Example 10. Effect of Compound 1 in the ZDSD model of diabetic nephropathy The objective of this study was to evaluate the efficacy of compound 1, a TRCP5 inhibitor, in attenuating the development and / or progression of albuminuria in the ZDSD model of diabetic nephropathy (DN).

[0233] The ZDSD model is an established model that recapitulates key features of diabetes, including impaired glucose metabolism, neuropathy, retinopathy, and nephropathy [Peterson et al., “Characterization of the ZDSD Rat: A Translational Model for the Study of Metabolic Syndrome and Type 2 Diabetes,” J. Diabetes. Res. (2015), Article ID 487816, 10 pages; Peterson et al., “The ZDSD rat: a novel model of diabetic nephropathy,” Am. J. Transl. Res. (2017) 9: 4236-4249].

[0234] Male ZDSD rats (Crown Bioscience, Indianapolis, IN; n = 79) were maintained on standard rodent chow (Purina 5008) from weaning until 15 weeks of age. A diabetogenic diet (Research Diet D124668) was initiated and maintained for 3 weeks to synchronize the onset of hyperglycemia. The diabetogenic diet was replaced with Purina 5008 for the remainder of the study. Animals were housed two per cage and maintained on a 12-hour light cycle (06:00-18:00). Room temperature was monitored daily and maintained at 70-74°F. Food and water were available ad libitum throughout the study.

[0235] Hyperglycemic ZDSD rats were selected for the study and randomized into groups of 10 rats based on body weight to receive either vehicle or Compound 1 (3 or 10 mg / kg / day). All compounds were administered by oral gavage daily (6–8 AM) for 12 weeks. The administration volume was maintained at 5 mL / kg.

[0236] Body weights were recorded weekly. Food consumption was recorded weekly during the treatment phase from week 0 to week 12. Blood samples were collected from the tail vein 3 hours after dosing and every 2 weeks until week 6, then weekly from week 8 to week 11. Whole blood was processed to serum for measurement of BUN, creatinine, albumin, and total protein (AU480).

[0237] Twenty-four-hour urine samples were collected at baseline, then every two weeks until week 6, and then weekly thereafter. Samples were collected at room temperature without additives. Food and water were provided ad libitum during the collection period. Urinary total protein (AU480) and albumin (ICL kit #E-25AL) were assayed. After 12 weeks of treatment, animals were terminated using CO2 asphyxiation and cervical dislocation.

[0238] Animals administered Compound 1 at 3 mg / kg and 10 mg / kg showed increased body weight compared to animals in the vehicle group during the final two weeks of the study.

[0239] As shown in Figure 6, Compound 1 reduced urinary albumin excretion from week 6 to week 12 compared to vehicle control rats, with the reduction reaching significance at weeks 10–12 ( p -value <0.001).

[0240] Example 11. Effect of Compound 1 in DOCA-salt hypertensive rats The objective of this study was to evaluate the efficacy of compound 1, a TRCP5 inhibitor, in attenuating the development and / or progression of albuminuria in deoxycorticosterone acetate (DOCA)-salt hypertensive rats.

[0241] The DOCA-salt hypertensive rat model is an established model of mineralocorticoid hypertension with renal dysfunction resulting in an FSGS phenotype and is characterized by increased levels of urinary protein and albumin excretion [Schenk et al., "The pathogenesis of DOCA-salt hypertension," J. Pharmacol. Toxicol. Methods (May 1992) 27(3):161-170; Gomez-Sanchez et al., "Mineralocorticoids, salt, and high blood pressure," Steroids (1996) 61:184-188.]

[0242] Six- to seven-week-old Sprague-Dawley rats underwent unilateral nephrectomy; after one week of recovery, rats were implanted with a DOCA pellet (45 mg) and provided with tap water containing 0.9% NaCl and 0.2% KCl for 4 weeks (Day 1). On Day 1, DOCA-salt rats were administered Compound 1 by oral gavage at 3 mg / kg or 10 mg / kg once daily for 4 weeks; DOCA-treated control animals received vehicle. Sham-treated animals implanted with a silicone-water pellet received tap water and oral administration of vehicle. Body weight was recorded daily, and proteinuria, albuminuria, and arterial blood pressure were recorded weekly.

[0243] No adverse effects were observed in animals administered Compound 1. There were no significant differences in body weight and urinary creatinine excretion in rats treated with DOCA, or DOCA and Compound 1. Animals receiving DOCA, or DOCA and Compound 1, had elevated mean arterial blood pressure (BP), diastolic, and systolic BP compared with sham animals from weeks 1 to 4.

[0244] Water intake and the amount of urine produced per day also increased in animals receiving vehicle or Compound 1 after DOCA-saline treatment.

[0245] As shown in Figure 7, Compound 1 at 10 mg / kg reduced urinary albumin excretion from weeks 2 to 4 compared to DOCA-vehicle control rats, with the reduction reaching significance at week 2 (p-value < 0.05) and p-values ​​< 0.001 at weeks 3 and 4. Compound 1 at 3 mg / kg reduced urinary albumin excretion from weeks 2 to 4 compared to DOCA-vehicle control rats, with the reduction reaching significance at week 3 (p-value < 0.05).

[0246] Example 12. Effect of Compound 1 in COL4A4 knockout mice The objective of this study was to evaluate the efficacy of compound 1, a TRCP5 inhibitor, in attenuating the development and / or progression of albuminuria in COL4A3 knockout mice.

[0247] The COL4A4 knockout mouse model is an established model of Alport disease and is characterized by increased levels of urinary protein and albumin excretion [Korstanje et al., "A mouse Col4a4 mutation causing Alport glomerulosclerosis with abnormal collagen α3α4α5(IV) trimers," Kidney Int. (2014) 85:1461-1468].

[0248] Four- to five-week-old COL4A4 knockout mice were administered Compound 1 by oral gavage at 3 mg / kg or 10 mg / kg once daily for 4 weeks; control animals received vehicle. Body weight was recorded daily, and urinary protein and creatinine were recorded weekly, and the urinary protein / creatinine ratio (UPCR) was calculated.

[0249] As shown in Figure 8, Compound 1 at 3 mg / kg or 10 mg / kg had no effect on the urinary protein / creatinine ratio.

[0250] Example 13. Effect of Compound 2 in DOCA-salt hypertensive rats The objective of this study was to evaluate the efficacy of compound 2, a TRCP5 inhibitor, in attenuating the development and / or progression of albuminuria in deoxycorticosterone acetate (DOCA)-salt hypertensive rats.

[0251] The DOCA-salt hypertensive rat model is an established model of mineralocorticoid hypertension with renal dysfunction resulting in an FSGS phenotype and is characterized by increased levels of urinary protein and albumin excretion [Schenk et al., "The pathogenesis of DOCA-salt hypertension," J. Pharmacol. Toxicol. Methods (May 1992) 27(3):161-170; Gomez-Sanchez et al., "Mineralocorticoids, salt, and high blood pressure," Steroids (1996) 61:184-188.]

[0252] Six- to seven-week-old Sprague-Dawley rats underwent unilateral nephrectomy; after one week of recovery, rats were implanted with a DOCA pellet (45 mg) and provided with tap water containing 0.9% NaCl and 0.2% KCl for 4 weeks (Day 1). On Day 1, DOCA-saline rats were administered Compound 2 by subcutaneous (SC) injection once daily at 10 mg / kg for 4 weeks or 60 mg / kg for 1 week, followed by 100 mg / kg for 3 weeks; DOCA-treated control animals were administered vehicle. Sham animals implanted with silicone-water pellets were provided with tap water and SC administration of vehicle. Body weight was recorded daily, and proteinuria, albuminuria, and arterial blood pressure were recorded weekly.

[0253] No adverse effects were observed in animals administered Compound 2. There were no significant differences in body weight and urinary creatinine excretion in rats treated with DOCA, or DOCA and Compound 2. Animals administered DOCA, or DOCA and Compound 2, had elevated mean arterial blood pressure (BP), diastolic, and systolic BP compared with sham animals from weeks 1 to 4.

[0254] Water intake and the amount of urine produced per day also increased in animals receiving DOCA saline treatment with vehicle or Compound 2.

[0255] As shown in Figure 9, compared with DOCA-vehicle control rats, Compound 2 at 10 mg / kg and 60 / 100 mg / kg reduced urinary albumin excretion from week 2 to week 4, with the reduction reaching significance at week 4 (p-value < 0.05).

[0256] Example 14. Effect of Compound 3 in DOCA-salt hypertensive rats The objective of this study was to evaluate the efficacy of compound 3, a TRCP5 inhibitor, in attenuating the development and / or progression of albuminuria in deoxycorticosterone acetate (DOCA)-salt hypertensive rats.

[0257] The DOCA-salt hypertensive rat model is an established model of mineralocorticoid hypertension with renal dysfunction resulting in an FSGS phenotype and is characterized by increased levels of urinary protein and albumin excretion [Schenk et al., "The pathogenesis of DOCA-salt hypertension," J. Pharmacol. Toxicol. Methods (May 1992) 27(3):161-170; Gomez-Sanchez et al., "Mineralocorticoids, salt, and high blood pressure," Steroids (1996) 61:184-188.]

[0258] Six- to seven-week-old Sprague-Dawley rats underwent unilateral nephrectomy; after 1 week of recovery, rats were implanted with a DOCA pellet (45 mg) and provided with tap water containing 0.9% NaCl and 0.2% KCl for 4 weeks of treatment (Day 1). On Day 1, DOCA-salt rats received Compound 3 by oral gavage at 30 mg / kg once daily for 4 weeks; DOCA-treated control animals received vehicle or the mineralocorticoid receptor antagonist (MCRA) eplerenone by oral gavage at 50 mg / kg twice daily. Sham animals implanted with silicone-water pellets received tap water and SC administration of vehicle. Body weight was recorded daily, and proteinuria, albuminuria, and arterial blood pressure were recorded weekly.

[0259] No adverse effects were observed in animals administered Compound 3. There were no significant differences in body weight and urinary creatinine excretion in rats treated with DOCA, or DOCA plus Compound 3 or eplerenone. Animals administered DOCA, DOCA and Compound 3, or DOCA and eplerenone had elevated mean arterial pressure (BP), diastolic, and systolic BP compared with sham animals from weeks 1 through 4. Water intake and the amount of urine produced per day also increased in animals treated with DOCA saline followed by vehicle, Compound 3, or eplerenone.

[0260] As shown in Figure 10, 30 mg / kg of Compound 3 significantly reduced urinary albumin excretion at week 4 compared to DOCA-vehicle control rats (p value < 0.05). Eplerenone also significantly reduced urinary albumin excretion at week 4 compared to DOCA-vehicle control rats (p value < 0.05).

[0261] Example 15. Effect of Compound 4 in DOCA-salt hypertensive rats The objective of this study was to evaluate the efficacy of compound 4, a TRCP5 inhibitor, in attenuating the development and / or progression of albuminuria in deoxycorticosterone acetate (DOCA)-salt hypertensive rats.

[0262] The DOCA-salt hypertensive rat model is an established model of mineralocorticoid hypertension with renal dysfunction resulting in an FSGS phenotype and is characterized by increased levels of urinary protein and albumin excretion [Schenk et al., "The pathogenesis of DOCA-salt hypertension," J. Pharmacol. Toxicol. Methods (May 1992) 27(3):161-170; Gomez-Sanchez et al., "Mineralocorticoids, salt, and high blood pressure," Steroids (1996) 61:184-188.]

[0263] Six- to seven-week-old Sprague-Dawley rats underwent unilateral nephrectomy; after one week of recovery, rats were implanted with a DOCA pellet (45 mg) and provided with tap water containing 0.9% NaCl and 0.2% KCl for two weeks (Day 1). On Day 1, DOCA-salt rats were administered Compound 4 via intraperitoneal (IP) injection once daily for two weeks at 20 mg / kg, 50 mg / kg, or 100 mg / kg; DOCA-treated control animals received vehicle. Sham animals implanted with silicone-water pellets received tap water and IP administration of vehicle. Body weight was recorded daily, and proteinuria, albuminuria, and arterial blood pressure were recorded weekly.

[0264] No adverse effects were observed in animals administered Compound 4. There were no significant differences in body weight and urinary creatinine excretion in rats treated with DOCA, or DOCA and Compound 4. Animals administered DOCA, or DOCA and Compound 4, had elevated mean arterial blood pressure (BP), diastolic, and systolic BP compared with sham animals from week 1 to week 2.

[0265] Water intake and the amount of urine produced per day also increased in animals receiving vehicle or DOCA saline treatment with Compound 4.

[0266] As shown in Figure 11, Compound 4 at 20 mg / kg, 50 mg / kg and 100 mg / kg significantly reduced urinary protein excretion at week 2 compared to DOCA-vehicle control rats (p-value < 0.05).

[0267] Example 16. Effects of cyclosporin A and tacrolimus in DOCA-salt hypertensive rats The objective of this study was to evaluate the efficacy of the calcineurin inhibitors cyclosporine A and tacrolimus in attenuating the development and / or progression of albuminuria in deoxycorticosterone acetate (DOCA)-salt hypertensive rats.

[0268] The DOCA-salt hypertensive rat model is an established model of mineralocorticoid hypertension with renal dysfunction resulting in an FSGS phenotype and is characterized by increased levels of urinary protein and albumin excretion [Schenk et al., "The pathogenesis of DOCA-salt hypertension," J. Pharmacol. Toxicol. Methods (May 1992) 27(3):161-170; Gomez-Sanchez et al., "Mineralocorticoids, salt, and high blood pressure," Steroids (1996) 61:184-188.]

[0269] Six- to seven-week-old Sprague-Dawley rats underwent unilateral nephrectomy. After one week of recovery, rats were implanted with DOCA pellets (45 mg) and provided with tap water containing 0.9% NaCl and 0.2% KCl for three weeks (Day 1). On Day 1, DOCA-salt rats received either cyclosporine A at 3 mg / kg once daily by gavage for three weeks or tacrolimus at 0.3 mg / kg for two weeks, followed by 0.1 mg / kg once daily by gavage for one week; DOCA-treated control animals received vehicle. Sham animals implanted with silicone-water pellets received tap water and vehicle. Proteinuria and albuminuria were recorded weekly, and body weights were recorded daily.

[0270] No adverse effects were observed in mice administered DOCA or DOCA and cyclosporine A. Rats treated with DOCA and tacrolimus experienced significant loss of body weight, and 2 weeks after administration, the dose of tacrolimus was reduced from 0.3 mg / kg to 0.1 mg / kg, which reversed the weight loss.

[0271] Water intake and the amount of urine produced per day also increased in animals receiving DOCA saline treatment with vehicle, cyclosporine A, or tacrolimus.

[0272] As shown in Figure 12, 3 mg / kg cyclosporine A significantly reduced urinary albumin excretion at week 3 compared to DOCA-vehicle control rats (p-value < 0.05), and 0.3 / 0.1 mg / mg tacrolimus significantly reduced urinary albumin excretion at weeks 2 and 3 compared to DOCA-vehicle control rats (p-value < 0.05).

[0273] Example 17. Urinary Rac1 analysis in COVID-19 positive patients with acute kidney injury The aim of this study was to measure the amount of Rac1 protein in the urine of patients with acute kidney injury ("AKI") who tested positive for COVID-19 by PCR.

[0274] Urine samples from six patients with active AKI after a positive COVID-19 test were obtained, processed, and analyzed as described in Example 2. The mean Rac1 value for the six patients was 4221.13 ± 5825.17 pg / ml (compared to 107.0 ± 44.6 pg / ml in normal patients). Figure 13 shows that three of the six patients had Rac1 levels that were at least eight-fold elevated, exceeding the upper limit of approximately 300 pg / ml in normal patients. This suggests that a subset of COVID-19 patients with AKI have sufficiently high urinary Rac1 concentrations (e.g., above a predetermined threshold) that they can be treated by the methods of the present invention.

[0275] Incorporation by Reference All US patents and US and PCT published patent applications cited herein are hereby incorporated by reference.

[0276] equivalent The foregoing specification is sufficient to enable one skilled in the art to practice the present invention. The examples are intended as merely illustrative of one aspect of the invention; other functionally equivalent embodiments are within the scope of the invention, and the present invention is not limited in scope by the examples provided. Various modifications of the present invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objectives of the present invention are not necessarily encompassed by each embodiment of the present invention. Finally, preferred embodiments of the present invention are described in sections.

[0277] [Embodiment 1] 1. A method for selecting and treating a human subject suffering from a renal disease, comprising: a. selecting a subject if the subject has a urinary level of one or more biomarkers selected from Rac1, Rac1-GTP, phospho-LIM kinase 1, and phospho-cofilin above a predetermined threshold; and b. administering to the selected subject a pharmaceutical composition comprising a TRPC5 inhibitor or a calcineurin inhibitor and a pharmaceutically acceptable carrier; A method comprising:

[0278] [Embodiment 2] 1. A method of treating a human subject suffering from a renal disease, comprising: only if the subject is determined to have a pre-treatment urinary level of one or more biomarkers selected from Rac1, Rac1-GTP, phospho-LIM kinase 1, and phospho-cofilin above a predetermined threshold, TRPC5 inhibitors or calcineurin inhibitors, and Pharmaceutically acceptable carrier administering to said subject a pharmaceutical composition comprising:

[0279] [Embodiment 3] The method according to embodiment 1 or 2, wherein the TRPC5 inhibitor is any one of the following a to d: a. A compound of formula (I) or formula (II), or a pharmaceutically acceptable salt thereof: [ka] [In the formula, X is CH, C(R 3 ) or N; R 1 is selected from the group consisting of H; alkyl; cycloalkyl; heterocycloalkyl; alkenyl; aryl; heteroaryl; alkylene-aryl; alkylene-heteroaryl; -CH2(O)N(R)-heteroaryl; -CH2(O)N(R)-alkyl; alkylene-N(alkyl)2; heterocycloalkyl; alkylene-O-alkyl; alkylene-O-aryl; alkylene-N(R)-C(O)-aryl; alkylene-N(R)-C(O)-alkyl; alkylene-C(O)-N(R)-alkyl; alkylene-C(O)-N(R)-aryl; alkylene-C(O)-cycloalkyl; and alkylene-C(O)-N(R)-heteroaryl; R 2 is selected from the group consisting of H; NH2, alkyl; cycloalkyl; aryl; heteroaryl; alkylene-aryl, alkylene-N(alkyl)2; alkylene-heterocycloalkyl; alkylene-cycloalkyl; -N(R)-alkyl; -N(R)-aryl; -N(R)-alkylene-aryl; -N(R)-cycloalkyl; -N(R)-heterocycloalkyl; -O-aryl; alkylene-O-aryl; heterocycloalkyl; -N=C(R)-aryl; -N(R)-alkylene-heteroaryl; -N(R)-alkylene-OH; -S-alkylene-C(O)N(R)-aryl; -S-alkylene-C(O)N(R)-heteroaryl; alkylene-C(O)-heterocycloalkyl; alkylene-N(R)-alkyl; alkylene-N(R)-aryl; and -S-alkyl; R 3is independently selected from alkyl, halogen, —CN, —OMe, —OH, —NO 2 , —NH 2 , N(Me) 2 , —CF 3 , —OCF 3 , —CHF 2 , —OCHF 2 , and —O-alkylene-OH; R is H or Me; n is 0, 1, 2, 3, or 4]; b. A compound of formula (III), (IV), or (V), or a tautomer or pharmaceutically acceptable salt thereof: [ka] [In the formula, R 11 and R 13 are independently H, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, heteroaryl, halogen, -OH, -CN, -cycloalkyl, -O-alkyl, -O-cycloalkyl, O-aryl, -aryl-O-aryl-CF3, -C(H)F2, alkylene-CF3, alkylene-C(H)F2, -SO2-alkyl, and O-alkylene-O-alkyl, -heterocyclyl-LR 4 , and -heteroaryl-LR 4 selected from the group consisting of: R 12 is -heterocyclyl-LR 14 and; R 14 is absent or is selected from alkyl, cycloalkyl, aryl, alkylene-aryl, alkylene-heteroaryl, heteroaryl, heterocyclyl, -C(O)N(R 15 CF3; R 15 is independently H or alkyl; R 16 is an alkyl, cycloalkyl, aryl, heterocyclyl, heteroaryl, alkylene-aryl, -C(O)N(R 15 )2, and CF3; L is absent or is methylene, -C(O)-, -SO2-, CH2N(Me)-, -N(R 15)(R 16 )-, -C(R 15 )(R 16 )-, and -OR 16 selected from the group consisting of: R 11 , R 12 and R 13 one and only one of -heterocyclyl-LR 14 or -heteroaryl-LR 14 is]; c. A compound of formula (VI) or (VII), or a pharmaceutically acceptable salt thereof: [ka] [In the formula, R 21 is selected from the group consisting of alkyl; cycloalkyl; heterocycloalkyl; aryl; heteroaryl; alkylene-aryl; alkylene-heteroaryl; alkylene-O-aryl; alkylene-N(alkyl)2; alkylene-heterocycloalkyl; alkylene-cycloalkyl; N(alkyl)2; and -C(O)-aryl; R 22 is selected from the group consisting of alkyl; cycloalkyl; heterocycloalkyl; aryl; heteroaryl; alkylene-N(alkyl)2; alkylene-heterocycloalkyl; alkylene-cycloalkyl; alkylene-heterocycloalkyl; and alkylene-OR'; R 23 is independently selected from alkyl, halogen, OMe, OH, N(Me)2, CF3, or OCF3, -O-, and alkylene-OH; R is H or Me; R' is H, methyl, ethyl, or isopropyl; n is 0, 1, 2, 3, or 4; or d. A compound of formula (VIII) or (IX), or a pharmaceutically acceptable salt thereof: [ka] [In the formula, A and A' are CR a and N are independently selected; R a is LR 31 and; L is absent, CH2, O, SO2, or NR 32 and; R 31 is selected from optionally substituted alkyl, optionally substituted aryl, and optionally substituted heteroaryl; Each R 32 is independently H or alkyl; R 33 is an optionally substituted alkyl, an optionally substituted alkylene -OR 32 , optionally substituted cycloalkylene-OR 32 , optionally substituted alkylene-N(R 37 )2, optionally substituted cycloalkylene-N(R 37 )2, optionally substituted alkylene -C(O)N(R 32 )2, optionally substituted cycloalkylene-C(O)N(R 32 )2, optionally substituted alkylene -S(O)2N(R 32 )2, and optionally substituted cycloalkylene -S(O)2N(R 32 )2 is selected; R 34 is selected from alkyl, optionally substituted alkylene-aryl, and optionally substituted alkylene-heteroaryl; Each R 35 are independently H, N(R 32 )2, OR 32 Selected from; Each R 37 is independently selected from H, alkyl, (alkyl)C(O)—, (aryl)C(O)—, (alkyl)S(O)2—, and (aryl)S(O)2—; Y is -C(O)-, CH2, CHR 36 , C(R 36 )2; Each R 36is independently selected from H, alkyl, and optionally substituted alkylene-OH; Y' is -C(O)-, CH2, CHR 33 ', C(R 33 ')2 or Y' is R 33 together to form a five- or six-membered ring; Each R 33 ' are independently an optionally substituted alkyl, an optionally substituted alkylene -OR 32 , optionally substituted cycloalkylene-OR 32 , optionally substituted alkylene-N(R 37 )2, optionally substituted cycloalkylene-N(R 37 )2, optionally substituted alkylene -C(O)N(R 32 )2, optionally substituted cycloalkylene-C(O)N(R 32 )2, optionally substituted alkylene -S(O)2N(R 32 )2, and optionally substituted cycloalkylene -S(O)2N(R 32 )2 is selected; Z is absent or CH2, CHR 35 , O, -NR 32 -, or -SO2-; provided that both Y and Y' are not -C(O)-.

[0280] [Embodiment 4] 4. The method of embodiment 3, wherein the TRPC5 inhibitor is a compound of structural formula X, or a pharmaceutically acceptable salt thereof: [ka] [In the formula, "---" is a single or double bond; X 1 is CH or N; If "---" is a double bond, X 2 is CH or N; If "---" is a single bond, X 2 is N(CH3); X 1 If is CH, then X 2 is N or N(CH3); W is -O-, -N(CH3)-, -N(CH2CH2OH)-, cyclopropane-1,1-diyl, or -CH(CH3)-; Q is 2-trifluoromethyl-4-fluorophenyl, 2-difluoromethyl-4-fluorophenyl, 2-trifluoromethylphenyl, 2-methyl-4-fluorophenyl, 2-chloro-4-fluorophenyl, 2-chlorophenyl, 1-(benzyl)-4-methylpiperidin-3-yl, 4-trifluoromethylpyridin-3-yl, 2-trifluoromethyl-6-fluorophenyl, 2-trifluoromethyl-3-cyanophenyl, 2-ethyl-3-fluorophenyl, 2-chloro-3-cyanophenyl, 2-trifluoromethyl-5-fluorophenyl, or 2-difluoromethylphenyl; R 43 is hydrogen, —CHOH, —CH(OH)—CHOH, —NH, —CH(OH)CH, —OCH, or —NH—(CH)OH; and when “---” is a double bond, R 44 does not exist; If "---" is a single bond, R 43 and R 44 together form =O; R 45 and R 46 each is independently hydrogen or -CH3.

[0281] [Embodiment 5] 5. The method of embodiment 4, wherein the TRPC5 inhibitor is a compound of formula XI, or a pharmaceutically acceptable salt thereof: [ka] [In the formula, R 41 is chloro, -CF3, -CHF2, or -CH3; R 42 is hydrogen or fluoro; R 43is hydrogen, -NH2, -CH2OH, or CH(OH)-CH2OH].

[0282] [Embodiment 6] The TRPC5 inhibitor is [ka] or a pharmaceutically acceptable salt thereof.

[0283] [Embodiment 7] The TRPC5 inhibitor is [ka] 3. The method of embodiment 1 or 2, wherein the compound is a pharmaceutically acceptable salt thereof.

[0284] [Embodiment 8] 3. The method of embodiment 1 or 2, wherein the calcineurin inhibitor is cyclosporin A, tacrolimus, or voclosporin, or a pharmaceutically acceptable salt thereof.

[0285] [Embodiment 9] 9. The method of any of embodiments 1-8, wherein the renal disease is diabetic nephropathy, focal segmental glomerulosclerosis, minimal change disease, membranoproliferative glomerulonephritis (including poststreptococcal glomerulonephritis and bacterial endocarditis-associated glomerulonephritis), membranous nephropathy, other hepatitis C virus-associated glomerulopathies, HIV-associated glomerulopathies, COVID-19-associated acute kidney injury, Alport syndrome, polycystic kidney disease (both autosomal dominant and autosomal recessive), IgA nephropathy, other inherited nephropathies or ciliopathies (e.g., HNF1β, nephronophthisis, autosomal dominant cystic / tubular kidney diseases), lupus nephritis, Goodpasture's syndrome (anti-GBM disease), and other complement-mediated or immune-mediated renal diseases.

[0286] [Embodiment 10] 10. The method of embodiment 9, wherein the kidney disease is diabetic nephropathy or focal segmental glomerulosclerosis.

[0287] [Embodiment 11] 10. The method of embodiment 9, wherein the kidney disease is autosomal dominant polycystic kidney disease or autosomal recessive polycystic kidney disease.

[0288] [Embodiment 12] 12. The method of any of embodiments 1 to 11, wherein the subject is selected on the basis of having a urinary Rac1 level above a predetermined threshold.

[0289] [Embodiment 13] The method of embodiment 12, wherein the urinary Rac1 level in the subject is measured in a urine fraction containing extracellular vesicles.

[0290] [Embodiment 14] 14. The method of embodiment 12 or 13, wherein the predetermined threshold level is established by: determining a range of urinary levels of the selected biomarker in a population of healthy individuals; and further establishing the predetermined threshold level of the selected biomarker at a level above the 75th percentile in the population.

[0291] [Embodiment 15] 15. The method of embodiment 14, wherein the predetermined threshold level is a level above the 90th percentile in the population.

[0292] [Embodiment 16] 16. The method of embodiment 15, wherein the predetermined threshold level is a level above the 95th percentile in the population.

[0293] [Embodiment 17] 14. The method of embodiment 12 or 13, wherein the predetermined threshold level of urinary Rac1 is between 100 and 500 pg / mL.

[0294] [Embodiment 18] 1. A method for determining the efficacy of TRPC5 inhibitor treatment in a human subject suffering from kidney disease, wherein, prior to initiating treatment, the subject is determined to have a pre-treatment urinary level of one or more biomarkers selected from Rac1, Rac1-GTP, phospho-LIM kinase 1, and phospho-cofilin above a predetermined threshold; a. Obtaining urinary levels of selected biomarkers in human subjects at time points after initiation of TRPC5 treatment; b. comparing the level of the selected biomarker in step a. with the pre-treatment urinary level of said selected biomarker; c. determining that the TRPC5 inhibitor treatment is efficacious if the level of the selected biomarker in step a. is lower than the pre-treatment urinary level of said selected biomarker; A method comprising:

[0295] [Embodiment 19] 1. A method for determining the efficacy of TRPC5 inhibitor treatment in a human subject suffering from kidney disease, wherein, prior to initiating treatment, the subject is determined to have a pre-treatment urinary level of one or more biomarkers selected from Rac1, Rac1-GTP, phospho-LIM kinase 1, and phospho-cofilin above a predetermined threshold; a. obtaining urinary levels of selected biomarkers in said human subject at a time point after initiation of TRPC5 treatment; b. determining that the TRPC5 inhibitor treatment is efficacious if the level of the selected biomarker in step a. is lower than a predetermined threshold value for the selected biomarker; A method comprising:

Claims

[Claim 1] 1. A method for selecting and treating a human subject suffering from a renal disease, comprising: a. selecting a subject if the subject's urinary level of one or more biomarkers selected from Rac1, Rac1-GTP, phospho-LIM kinase 1, and phospho-cofilin is above a predetermined threshold; and b. Administering to the selected subject a pharmaceutical composition comprising a TRPC5 inhibitor or a calcineurin inhibitor and a pharmaceutically acceptable carrier; A method comprising:

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