Application of deoxycholic acid compound in treatment of diabetic kidney diseases

Abstract

The invention belongs to the field of treatment of diabetic kidney diseases (DKD), and particularly relates to application of deoxycholic acid compounds in treatment of diabetic kidney diseases, the deoxycholic acid compounds have the structure shown in the formula I, and n and R1 are defined as in the specification.

Description

Technical Field

[0001] This invention belongs to the field of diabetic kidney disease (DKD) treatment, and specifically relates to the application of a deoxycholic acid compound in the treatment of diabetic kidney disease. Background Technology

[0002] Diabetic kidney disease (DKD) is a serious complication of diabetes mellitus (DM). Renal fibrosis is a key pathological process in DKD, and its progression ultimately leads to the loss of kidney function. Currently, approximately 700 million people worldwide suffer from end-stage renal disease (ESRD) due to DKD. Despite the increasing understanding and research progress regarding DKD, and the emergence of drugs that benefit DKD, such as nonsteroidal mineralocorticoid receptor antagonists and glucagon-like peptide-1 receptor agonists, these drugs can only slow, not halt, the progression of the disease. Therefore, there is an urgent need to find other means to benefit the kidneys.

[0003] Bile acids (BAs) are amphiphilic steroid molecules. As important endogenous metabolites, they are converted into secondary bile acids after being modified by gut microbiota, playing a crucial role in the metabolic homeostasis of various organs throughout the body. Changes in BA levels or signaling pathways are closely related to metabolic diseases, including obesity, type 2 diabetes mellitus (T2DM), non-alcoholic steatohepatitis (NAH), and atherosclerosis. Therefore, regulation of the bile acid pool may be an effective treatment method in preclinical and clinical models. Previous articles have reported that changes in the circulating BA profile are associated with kidney disease, and that the lower the level of unconjugated bile acids and their glycine conjugates in the serum of patients with type 2 diabetes, the higher the risk of CKD (paper: Nutr J. 2024 Mar 2; 23(1):28. doi:10.1186 / s12937-024-00928-2). Summary of the Invention

[0004] The purpose of this invention is to provide an application of deoxycholic acid compounds in the treatment of diabetic kidney disease.

[0005] A first aspect of the present invention provides the use of a deoxycholic acid compound, its stereoisomers, enantiomers, diastereomers, geometric isomers, solvates, hydrates, isotopic substitutes, various crystal forms, prodrugs, or pharmaceutically acceptable salts thereof, in the preparation of a medicament for treating and / or preventing diabetic kidney disease, said deoxycholic acid compound having the structure shown in Formula I:

[0006]

[0007] in:

[0008] (CH2) n Each CH2 group may be optionally substituted by 1, 2, 3 or 4 substituents selected from C1-C6 alkyl, C1-C6 alkoxy, deuterium, halogen, hydroxyl and amino groups;

[0009] R1 is selected from optionally substituted hydroxyl groups and optionally substituted -NR groups. a R b R a and R b Each is independently selected from H and C1-C6 alkyl groups;

[0010] n can be 1, 2, 3, 4, 5, 6, 7 or 8.

[0011] A second aspect of the present invention provides a pharmaceutical composition comprising (i) the deoxycholic acid compound described in the first aspect of the present invention, its stereoisomers, enantiomers, diastereomers, geometric isomers, solvates, hydrates, isotopic substitutes, various crystal forms, prodrugs or pharmaceutically acceptable salts thereof; and (ii) a pharmaceutically acceptable carrier.

[0012] The present invention has the following beneficial effects:

[0013] The present invention relates to deoxycholic acid compounds, such as glycocodeoxycholic acid (GDCA), which can be used for the prevention and / or treatment of disseminated kidney disease (DKD). Specifically, the inventors have found that gavage administration of the present invention's deoxycholic acid compounds (such as GDCA) to two DKD mouse models can delay the progression of DKD disease, providing a new potential therapeutic agent for DKD treatment (the present invention's deoxycholic acid compounds, such as GDCA). Furthermore, comprehensive results from population cohort studies and drug intervention experiments fully demonstrate that the present invention's deoxycholic acid compounds (such as secondary bile acids, such as GDCA) play a key regulatory role in the progression of DKD disease. Therefore, the present invention's deoxycholic acid compounds (such as GDCA) have potential value in treating DKD, providing a new direction for the treatment and drug development of DKD. Attached Figure Description

[0014] Figure 1 The concentration of DCA-type bile acids in the clinical cohort was negatively correlated with the occurrence and development of DKD. DCA stands for deoxycholic acid; CA for cholic acid; CDCA for chenodeoxycholic acid; LCA for lithocholic acid; and UDCA for ursodeoxycholic acid. *, P < 0.05; **, P < 0.01. Figure 1In the diagram, A represents a comparison of serum bile acid levels of different categories between the DM and DKD groups; B is a heatmap showing the correlation between different categories of serum bile acids and clinical indicators; C is a heatmap showing the distribution changes of DCA type bile acid levels as DM progresses to DKD; and D is a cloud and rain map showing the changes in serum bile acid levels of different categories as the disease progresses.

[0015] Figure 2 In clinical cohorts, GDCA concentrations were negatively correlated with the occurrence and progression of DKD. GDCA stands for glycodeoxycholic acid; UDCA for ursodeoxycholic acid; TCA for taurocholic acid; CDCA for chenodeoxycholic acid; DCA for deoxycholic acid; CA for cholic acid; GCA for glycodeoxycholic acid; TCDCA for taurochenodeoxycholic acid; TUDCA for taurochenodeoxycholic acid; GCDCA for glycodeoxycholic acid; TDCA for taurochenodeoxycholic acid; TLCA for taurolithocholic acid; GLCA for glycolithocholic acid; and GUDCA for glycodeoxycholic acid. Figure 2 In the table, A represents the association analysis between individual bile acid levels and DKD. The dashed line extends from the box to the maximum value not exceeding 1.5 times the interquartile range of the box, and the error bars represent the 95% confidence interval of the odds ratio (OR); B represents the OPLSDA-VIP analysis of individual bile acids in DKD; C represents the distribution of individual bile acids in the DM and DKD groups.

[0016] Figure 3 The results of renal function tests in db / db mice showed that, compared with the model group, the 24-hour urine volume and the urinary albumin-to-cretinine ratio (UACR) levels in the GDCA gavage group were significantly lower. *, P<0.05. Figure 3 In the table, A represents a comparison of 24-hour urine output after 4 weeks of GDCA supplementation; B represents a comparison of UACR levels after 4 weeks of GDCA supplementation.

[0017] Figure 4 Pathological staining of the renal cortex in db / db mice showed that, compared with the model group, the GDCA gavage group significantly improved collagen area, glomerular size, and the level of neutrophil gelatinase-associated lipocalin (NGAL), a marker of renal tubular injury. **,*,P<0.05;**,P<0.01. Figure 4In the middle, A shows representative images of the results of hematoxylin-eosin staining (H&E), MASSON staining, periodic acid-Schiff staining (PAS), and NGAL immunohistochemical staining of mouse renal cortex after 4 weeks of GDCA reinjection; B shows quantitative analysis of MASSON collagen fiber area, glomerular mesangial fractional area, glomerular size, and NGAL positive signal marker area.

[0018] Figure 5 Transmission electron microscopy results of the renal cortex of db / db mice showed that, compared with the model group, the podocytes and mitochondria in the GDCA gavage group were significantly improved. *, P<0.05; **, P<0.01; ***, P<0.001. Figure 5 Figure A shows representative TEM images of glomerular basement membrane (GBM) thickness and podocyte morphology in the renal cortex of mice 4 weeks after GDCA inoculation; Figure B shows representative TEM images of mitochondria in the renal cortex of mice 4 weeks after GDCA inoculation; Figure C shows quantitative analysis of GBM thickness, number of foot processes, and number of mitochondria.

[0019] Figure 6 The results of renal function index detection in HFD combined with STZ-induced DKD mice showed that, compared with the model group, the UACR and Scr levels in the GDCA gavage group were significantly reduced. ***, P<0.001. Figure 6 In the table, A represents the comparison of UACR levels after 4 weeks of GDCA supplementation; B represents the comparison of Scr levels after 4 weeks of GDCA supplementation.

[0020] Figure 7 The pathological staining results of renal cortex in DKD mice induced by HFD combined with STZ showed that, compared with the model group, the collagen area, glomerular size, and NGAL level were significantly improved in the GDCA gavage group. *, P<0.05; **, P<0.01; ***, P<0.001. Figure 7 In the figure, A represents the H&E, MASSON, PAS, and NGAL staining results of the renal cortex of mice in the three groups after 4 weeks of GDCA supplementation; B is the quantitative analysis of MASSON collagen fiber area, glomerular mesangial fractional area, glomerular size, and NGAL positive signal marker area.

[0021] Figure 8 Transmission electron microscopy results of the renal cortex of DKD mice induced by HFD combined with STZ showed that, compared with the model group, the podocytes and mitochondria in the GDCA gavage group were significantly improved. *, P<0.05; **, P<0.01; ***, P<0.001. Figure 8 In the table, A represents a representative TEM image of GBM thickness and podocyte morphology in the renal cortex of mice 4 weeks after GDCA inoculation; B represents a representative TEM image of mitochondria in the renal cortex of mice 4 weeks after GDCA inoculation; and C represents a quantitative analysis of GBM thickness, number of foot processes, and number of mitochondria. Detailed Implementation

[0022] To enable those skilled in the art to understand the features and effects of the present invention, the terms and expressions used in the specification and claims are explained and defined in general below. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0023] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0024] In this document, the terms “contains,” “includes,” “containing,” and similar terms encompass the meanings of “basically composed of” and “composed of.” For example, when this document discloses “A contains B and C,” “A is basically composed of B and C” and “A is composed of B and C” should be considered as having been disclosed in this document.

[0025] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0026] Unless otherwise specified, percentages refer to mass percentages and proportions refer to mass ratios in this article.

[0027] In this document, when describing embodiments or examples, it should be understood that it is not intended to limit the invention to those embodiments or examples. Rather, all alternatives, modifications, and equivalents of the methods and materials described herein are covered within the scope defined by the claims.

[0028] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0029] the term

[0030] As used herein, "alkyl" refers to a straight-chain or branched monovalent saturated hydrocarbon group having a specified number of carbon atoms. Specifically, alkyl groups are those having 1 to 6 carbon atoms (C1-C6 alkyl, such as C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl), typically containing 1 to 4 carbon atoms (C1-C4 alkyl), and preferably containing 1 to 3 carbon atoms (C1-C3 alkyl). Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, etc.

[0031] As used in this article, "alkoxy" refers to alkyl-O-, and the definition of alkyl is as described above.

[0032] "Optional" or "optionally" means that the event described below may or may not occur, and the description includes instances where said event or situation occurs and instances where it does not occur. For example, "optionally substituted alkyl" means that the alkyl group may or may not be substituted, and the description includes both substituted and unsubstituted alkyl groups.

[0033] As used herein, "solvent complex" refers to a complex formed by the coordination of the compound of the present invention with solvent molecules in a specific ratio.

[0034] As used herein, "hydrate" refers to a complex formed by the coordination of the compound of the present invention with water.

[0035] As used herein, "pharmaceutical composition" refers to a formulation of the compounds of the present invention with a medium generally accepted in the art for delivering a bioactive compound to a mammal (e.g., a human). This medium includes pharmaceutically acceptable carriers. The purpose of a pharmaceutical composition is to facilitate administration to the organism, thereby promoting the absorption of the active ingredient and the exertion of its bioactivity.

[0036] As used herein, “prodrug” includes simple esters of compounds containing carboxylic acids (e.g., esters obtained by condensation with C1-C4 alcohols according to methods known in the art); esters of compounds containing hydroxyl groups (e.g., esters obtained by condensation with C1-C4 carboxylic acids, C3-C6 diacids, or their anhydrides, such as succinic anhydride and fumaric anhydride, according to methods known in the art); imines of compounds containing amino groups (e.g., imines obtained by condensation with C1-C4 aldehydes or ketones according to methods known in the art); carbamates of compounds containing amino groups, such as those esters described by Leu et al. (J.Med.Chem.42:3623-3628 (1999)) and Greenwald et al. (J.Med.Chem.42:3657-3667 (1999)); and aldol acetals or ketal acetals of compounds containing alcohols (e.g., those acetals obtained by condensation with chloromethyl methyl ether or chloromethyl ethyl ether according to methods known in the art).

[0037] As used herein, the term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. A "pharmaceutically acceptable acid addition salt" is a salt formed with an inorganic or organic acid that retains the bioavailability of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobroms, sulfates, nitrates, and phosphates; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetate, trifluoroacetate, propionate, hexanoate, octanoate, decanoate, undecanoate, glycolate, gluconate, lactate, sebate, adipate, glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate, cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, alginate, ascorbate, salicylate, 4-aminosalicylic acid, and naphthalenedisulfonate. These salts can be prepared using methods known in this field. "Pharmaceutically acceptable base addition salts" refer to salts formed with inorganic or organic bases that retain the bioavailability of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, and aluminum salts. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts, and magnesium salts. Salts derived from organic bases include, but are not limited to, the following: primary amines, secondary amines, and tertiary amines; substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, and polyamine resins. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0038] Unless otherwise specified, the structural formulas described in this invention are intended to include all isomers (e.g., enantiomers, diastereomers, geometric isomers, conformational isomers), such as R and S configurations containing an asymmetric center, and (Z) and (E) isomers with double bonds. Therefore, any single stereochemical isomer of the compounds of this invention, or a mixture of its enantiomers, diastereomers, geometric isomers, or conformational isomers, is within the scope of this invention.

[0039] The embodiments of the invention disclosed herein are also intended to encompass pharmaceutically acceptable compounds of all Formula I compounds that are isotopically labeled by replacing one or more atoms with atoms of different atomic masses or mass numbers. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, respectively, as follows: 2 H, 3 H, 11 C 13 C 14 C 13 N、 15 N、 15 O、 17 O、 18 O、 31 P, 32 P, 35 S, 18 F, 36 Cl、 123 I and 125 I. These radiolabeled compounds can be used to help determine or measure the efficacy of compounds by characterizing, for example, the site or pattern of action or binding affinity to pharmacologically important sites of action. Certain isotope-labeled compounds of formula I, such as those doped with radioactive isotopes, can be used for drug and / or matrix tissue distribution studies. Radioactive isotope tritium (i.e., 3 H) and carbon-14 (i.e. 14 C) It is particularly suitable for this purpose due to its ease of binding and the availability of readily available detection methods. In some embodiments, heavier isotopes such as deuterium (i.e., 2 H) substitution can provide certain therapeutic advantages because of its higher metabolic stability, such as a longer half-life in vivo or a reduced dose requirement, and is therefore preferred in some cases.

[0040] As used in this article, "subject" can be a human, non-human primate, mammal, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc. Subjects may be suspected of having or have DKD disease.

[0041] As used herein, “treatment” encompasses the treatment of the disease or symptom of interest in mammals (preferably humans) suffering from the disease or symptom of interest, including:

[0042] (i) Suppress the disease or symptom, that is, curb its development;

[0043] (ii) Alleviating the disease or symptoms, i.e., causing the disease or symptoms to subside; or

[0044] (iii) To alleviate symptoms caused by the disease or condition, i.e., to relieve pain without resolving the underlying disease or condition.

[0045] The term “prevention” refers to the prevention of the occurrence of a disease or symptom in a mammal, particularly when the mammal is susceptible to the symptom but has not yet been diagnosed with the symptom.

[0046] As used herein, the terms “administer,” “apply,” “dose,” etc., refer to methods that deliver a compound or composition to the desired site for biological action. Administration methods known in the art are applicable to this invention. These methods include, but are not limited to, oral administration, duodenal administration, parenteral administration (including intrapulmonary, intranasal, intrathecal, intravenous, subcutaneous, intraperitoneal, intramuscular, and intra-arterial injection or infusion), local administration, and rectal administration. Those skilled in the art are familiar with administration techniques applicable to the compounds and methods described herein, such as those discussed in Goodman and Gilman, *The Pharmacological Basis of Therapeutics*, current ed.; Pergamon; and Remington's, *Pharmaceutical Sciences* (current edition), Mack Publishing Co., Easton, Pa. In a preferred embodiment, the compounds of the invention, pharmaceutically acceptable salts thereof, or crystalline forms of said compounds or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, are administered orally.

[0047] In this article, diabetic kidney disease (DKD) is a chronic kidney disease caused by diabetes mellitus (DM), clinically characterized by persistently increased albuminuria and / or a progressive decline in glomerular filtration rate, eventually progressing to end-stage renal disease (ESRD). DKD is typically diagnosed using random urinary albumin / creatinine ratio (UACR) or urinary albumin excretion rate (UAER), estimated glomerular filtration rate (eGFR), and renal biopsy. The DKD mouse models used in this article are those recognized in the field for researching DKD treatment methods, including DKD mice induced by a high-fat diet (HFD) combined with streptozotocin (STZ) or db / db induced DKD mice.

[0048] Deoxycholic acid compounds

[0049] The deoxycholic acid compounds of this invention can be used for the prevention and / or treatment of DKD. Therefore, this invention provides a deoxycholic acid compound having the structure shown in Formula I:

[0050]

[0051] (I)

[0052] in:

[0053] (CH2) n Each CH2 group may be optionally substituted by 1, 2, 3 or 4 substituents selected from C1-C6 alkyl, C1-C6 alkoxy, deuterium, halogen, hydroxyl and amino groups;

[0054] R1 is selected from optionally substituted hydroxyl groups and optionally substituted -NR groups. a R b R a and R b Each is independently selected from H and C1-C6 alkyl groups;

[0055] n can be 1, 2, 3, 4, 5, 6, 7 or 8.

[0056] In some implementations, (CH2) n Each CH2 group may be optionally substituted with one, two, three, or four substituents selected from C1-C6 alkyl and C1-C6 alkoxy groups. Preferably, (CH2) n Each CH2 group may optionally be substituted with one C1-C4 alkyl group. Preferably, (CH2) n The CH2 group directly attached to the steroidal ring (a four-membered fused ring) is replaced by a C1-C4 alkyl group.

[0057] When substituted, the number of substituents on R1 can be 1, 2, 3, or 4. The substituents on R1 can be selected from C1-C6 alkyl, carboxyl, and sulfonic acid groups. In some embodiments, R1 is selected from hydroxyl groups and -NR groups optionally substituted with substituents selected from carboxyl and sulfonic acid groups. a R b Preferably, R a For H, R b It can be methyl or ethyl.

[0058] In some implementations, n is 3.

[0059] In some embodiments, the deoxycholic acid compound has the structure shown in Formula II:

[0060]

[0061] R1 is defined as described in any of the embodiments described herein.

[0062] In some embodiments, the deoxycholic acid compound is selected from DCA (deoxycholic acid), GDCA (glycine-deoxycholic acid), and TDCA (taurine-deoxycholic acid); preferably, the deoxycholic acid compound is GDCA. The chemical structures of exemplary deoxycholic acid compounds are shown in the table below:

[0063]

[0064]

[0065] In some embodiments, the deoxycholic acid compounds of the present invention further include stereoisomers, enantiomers, diastereomers, geometric isomers, solvates, hydrates, isotopic substitutes, various crystal forms, prodrugs, or pharmaceutically acceptable salts thereof of the compound of formula I.

[0066] Pharmaceutical Composition

[0067] Since the deoxycholic acid compounds of the present invention can be used to prevent and / or treat DKD, pharmaceutical compositions containing the deoxycholic acid compounds of the present invention (compound of formula I), their stereoisomers, enantiomers, diastereomers, geometric isomers, solvates, hydrates, isotope substitutes, various crystal forms, prodrugs or pharmaceutically acceptable salts thereof as the main active ingredient can be used to treat and / or prevent DKD disease.

[0068] In some embodiments, the pharmaceutical compositions of the present invention comprise (i) the deoxycholic acid compounds of the present invention, their stereoisomers, enantiomers, diastereomers, geometric isomers, solvates, hydrates, isotopic substitutes, various crystal forms, prodrugs or pharmaceutically acceptable salts thereof; and (ii) pharmaceutically acceptable carriers.

[0069] In some embodiments, the pharmaceutical compositions of the present invention comprise therapeutic and / or preventative amounts of the deoxycholic acid compounds of the present invention, their stereoisomers, enantiomers, diastereomers, geometric isomers, solvates, hydrates, isotopic substitutes, various crystal forms, prodrugs, or pharmaceutically acceptable salts thereof. As used herein, “effective amount” means the amount of a therapeutic agent used to treat and / or prevent DKD, or the amount that exhibits a detectable therapeutic or preventative effect. The precise effective amount for a given subject depends on the subject’s body size and health status, the nature and severity of the condition, and the choice of the therapeutic agent and / or combination of therapeutic agents administered. Therefore, it is not useful to predetermine an accurate effective amount. However, for a given condition, the effective amount can be determined using routine experiments, and is readily apparent to a clinician.

[0070] As used herein, "pharmaceutically acceptable" means a substance that does not affect the biological activity or properties of the compound of the present invention (compound of Formula I), its stereoisomers, enantiomers, diastereomers, geometric isomers, solvates, hydrates, isotopic substitutes, crystal forms, prodrugs or pharmaceutically acceptable salts thereof, and is relatively non-toxic, i.e., the substance can be administered to an individual without causing an adverse biological reaction or interacting adversely with any component contained in the composition.

[0071] As used herein, a "pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers or gelling substances suitable for human use, and which must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with and with the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc. When selecting a pharmaceutically acceptable carrier, the primary consideration is the route of administration of the drug formulation. This is well known in the art. In some embodiments, the pharmaceutically acceptable carrier is selected from sodium carboxymethyl cellulose and water.

[0072] There are no particular limitations on the administration of the compounds or pharmaceutical compositions of the present invention. Representative administration methods include (but are not limited to): oral, parenteral (intravenous, intramuscular or subcutaneous) or local administration.

[0073] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the compounds of the present invention are mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following components: (a) fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin wax; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. Buffers may also be included in the dosage forms of capsules, tablets, and pills.

[0074] Solid dosage forms such as tablets, sugar pills, capsules, pellets, and granules can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compound from such compositions can be delayed in a portion of the digestive tract. Examples of encapsulating components that can be used are polymeric substances and waxes. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0075] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0076] In addition to these inert diluents, the composition may also contain auxiliaries such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances.

[0077] In addition to the compounds of the present invention, the suspension may contain suspending agents, such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0078] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0079] Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays, and inhalers. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be necessary.

[0080] In some embodiments, the pharmaceutical composition further includes at least one existing drug for treating DKD. Preferably, the existing drug for treating DKD is selected from: insulin, SGLT2 inhibitors (such as dapagliflozin, empagliflozin, canagliflozin, etc.), GLP-1 receptor agonists (such as liraglutide, lixisenatide, exenatide, semaglutide, etc.), DPP-4 inhibitors (sitagliptin, alogliptin, linagliptin, saxagliptin, and vildagliptin, etc.), metformin, sulfonylureas, etc.

[0081] application

[0082] The deoxycholic acid compounds of this invention can be used to treat diabetic kidney disease. Therefore, this invention provides the use of the deoxycholic acid compounds of this invention, their stereoisomers, enantiomers, diastereomers, geometric isomers, solvates, hydrates, isotopic substitutes, various crystal forms, prodrugs, or pharmaceutically acceptable salts thereof in the preparation of medicaments for the treatment and / or prevention of diabetic kidney disease.

[0083] In some implementations, the treatment and / or prevention of diabetic kidney disease includes improving kidney function.

[0084] In some implementations, the treatment and / or prevention of diabetic kidney disease includes improving glomerular and / or tubular damage.

[0085] In some implementations, the treatment and / or prevention of diabetic kidney disease includes regulating and / or improving the progression of diabetic kidney disease.

[0086] In some embodiments, the treatment and / or prevention of diabetic nephropathy includes downregulating the levels of creatinine and / or uric acid in the subject. Preferably, the creatinine is selected from urinary creatinine and serum creatinine. Preferably, the uric acid is selected from urinary uric acid and serum uric acid. Preferably, the subject suffers from diabetic nephropathy.

[0087] In some implementations, the treatment and / or prevention of diabetic kidney disease includes upregulating the subject's estimated glomerular filtration rate (eGFR). Preferably, the subject has diabetic kidney disease.

[0088] In some embodiments, the treatment and / or prevention of diabetic kidney disease includes downregulating the subject's UACR (urine microalbumin to creatinine ratio). Preferably, the subject suffers from diabetic kidney disease.

[0089] In some embodiments, the treatment and / or prevention of diabetic kidney disease includes reducing the subject's urine output. Preferably, the subject suffers from diabetic kidney disease.

[0090] In some embodiments, the treatment and / or prevention of diabetic kidney disease includes improving kidney histopathology; preferably, the treatment and / or prevention of diabetic kidney disease includes reducing glomerular basement membrane thickening and mesangial expansion, improving collagen fiber area, reducing glomerular size, and reducing neutrophil gelatinase-associated lipotransferase, a marker of renal tubular damage.

[0091] In some implementations, the treatment and / or prevention of diabetic kidney disease includes improving the glomerular basement membrane, reducing foot process fusion and loss, alleviating mitochondrial swelling, and / or restoring mitochondrial numbers.

[0092] method

[0093] The present invention provides a method for treating and / or preventing diabetic kidney disease, the method comprising the steps of: administering to a subject in need a therapeutic and / or preventative amount of the deoxycholic acid compound of the present invention, its stereoisomers, enantiomers, diastereomers, geometric isomers, solvates, hydrates, isotopic substitutes, various crystal forms, prodrugs or pharmaceutically acceptable salts thereof; or the pharmaceutical composition of the present invention.

[0094] In some embodiments, the treatment and / or prevention of diabetic kidney disease, deoxycholic acid compounds, and pharmaceutical compositions are as described in any of the embodiments herein.

[0095] In one or more methods of the present invention, the administration may be carried out orally.

[0096] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

[0097] In this study, Ctrl represents the normal control group, Vehicle represents the model group without GDCA supplementation, and the GDCA group represents the intervention group with GDCA supplementation. For db / db-induced DKD mice, the administration method via gavage was as follows: the GDCA group received 50 mg / kg of GDCA daily, with a gavage dose of 5 mL / kg. GDCA was supplemented in vivo in mice for 4 weeks using a 10 mg / mL suspension prepared with 0.5% carboxymethyl cellulose sodium (CMC-Na) solution via gavage. The Vehicle group received an equal volume of 0.5% CMC-Na solution via gavage once daily. Because the db / db mice showed poor condition after supplementation with 50 mg / kg GDCA, the administration method for high-fat combined with STZ-induced DKD mice was adjusted to: the GDCA group received 30 mg / kg of GDCA daily, with a gavage dose of 5 mL / kg. GDCA was administered to mice via gavage for 4 weeks as a 6 mg / mL suspension prepared with 0.5% sodium carboxymethyl cellulose (CMC-Na). The mice remained in good condition throughout the period. The Vehicle group received an equal volume of 0.5% CMC-Na solution via gavage once daily. The Ctrl group received no treatment.

[0098] Example 1 (obtained) Figure 1 (Experimental results)

[0099] Figure 1 A: The serum bile acid levels of different classes in the DM and DKD groups in the clinical cohort were compared by the "fold change" Log2FC on a logarithmic scale. The difference bubble chart on the CNSknowall website was used for visualization. The size of the bubble represents the p-value, and the color of the bubble represents the expression level of that class of bile acid in the DKD group. The bluer the color, the weaker the expression level, and the yellower the color, the stronger the expression level. Figure 1 B: The relationship between different types of bile acids and DKD-related clinical indicators was visualized using the Spearman rank correlation test and the correlation heatmap on the CNSknowall website. Figure 1 C: Use the R package "ComplexHeatmap" to plot a heatmap of the distribution changes of DCA-type bile acid levels as DM progresses to DKD; Figure 1 D: Using cloud and rain plots from the CNSknowall website, the process of changes in serum bile acid levels across different categories as the disease progresses is visualized.

[0100] Example 2 (obtained) Figure 2 (Experimental results)

[0101] Figure 2 A: The association between bile acids and DKD was validated using a conditional logistic regression model in a clinical cohort, and visualized using a combined differential box plot from the CNSknowall website. Figure 2 B: The contribution of each bile acid in the progression from DM to DKD is assessed by supervised machine learning method orthogonal partial least squares discriminant analysis (OPLS-DA) combined with variable projective importance (VIP). VIP ≥ 1 is usually regarded as the bile acid that can distinguish between different groups. Figure 2 C: Use percentage stacking plots from the CNSknowall website to visualize the distribution of individual bile acids in the DM and DKD groups.

[0102] Example 3 (obtained) Figure 3 (Experimental results)

[0103] Figure 3 A: Transfer mice into metabolic cages to collect urine, and collect and record the urine volume over 24 hours; Figure 3 B: UACR is the ratio of urinary albumin to urinary creatinine. The collected urine was tested for urinary albumin and urinary creatinine separately. Urinary albumin was detected using a mouse albumin ELISA kit (Mouse Albumin ELISA Kit, ab108792), while urinary creatinine was detected using a biochemical kit (Nanjing Jiancheng Kit, C011-2-1).

[0104] Example 4 (obtained) Figure 4 (Experimental results)

[0105] Figure 4 A: Kidney tissue was collected, fixed with 4% paraformaldehyde at room temperature, and then prepared into paraffin sections. The sections were stained with H&E, PAS, Masson staining, and immunohistochemical NGAL staining. The morphology of glomeruli and tubular cells, and the area of ​​positively stained matrix were observed under a microscope under different staining conditions. Figure 4 B: MASSON staining areas are considered a marker of collagen deposition. PAS staining positivity can help observe the morphology and thickness of the glomerular basement membrane and the presence of mesangial material deposition, which is convenient for assessing glomerular damage. NGAL staining areas are considered a marker of renal tubular damage. The positive area and glomerular size area are calculated using ImageJ software.

[0106] Example 5 (obtained) Figure 5 (Experimental results)

[0107] Figure 5 A: Fresh kidney tissue samples were immediately placed in a 4°C fixative (2.5% glutaraldehyde) within 1 minute, and then immediately divided into 1mm cross-sections in an ice box. 2 The tissue should be cut into strips no longer than 3 mm. After quickly immersing in a 4°C fixative solution for 15 minutes, the tissue should be cut into 1 mm pieces. 3 Small tissue samples were fixed in a fixative solution at 4°C. Osmium tetroxide, ethanol, and propylene oxide were used for tissue dehydration, followed by embedding in a plastic mixture and baking for 48 hours. After embedding, ultrathin sections were serially sectioned at 60–70 nm using a microtome. The sections were then placed on a copper grid, stained with 4% uranium acetate in methanol, and images were acquired using transmission electron microscopy (TEM). Foot processes and mitochondrial morphology were analyzed. Figure 5 B: Analysis of mitochondrial morphology. Figure 5 C: The thickness of GBM and the number of foot processes and mitochondria per μm GBM were quantitatively calculated using ImageJ software.

[0108] Example 6 (obtained) Figure 6 (Experimental results)

[0109] Figure 6 A: Mice were transferred to metabolic cages to collect urine. UACR is the ratio of urinary albumin to urinary creatinine. Urinary albumin was detected using a mouse albumin ELISA kit (Mouse Albumin ELISA Kit, ab108792), while urinary creatinine was detected using a biochemical kit (Nanjing Jiancheng Kit, C011-2-1). Figure 6B: At the end of the experiment, blood was collected by enucleating the mouse eyeballs, centrifuging the blood, and then the serum was collected. Serum creatinine was measured using a biochemical reagent kit (Nanjing Jiancheng Reagent Kit, C011-2-1).

[0110] Example 7 (obtained) Figure 7 (Experimental results)

[0111] The experimental method is the same as in Example 4.

[0112] Example 8 (obtained) Figure 8 (Experimental results)

[0113] The experimental method is the same as in Example 5.

[0114] Experimental results

[0115] I. Secondary bile acid GDCA levels are closely related to the disease status of DKD.

[0116] High-throughput targeted metabolomics is a technique that simultaneously detects the levels of multiple target metabolites in a sample. It features high throughput and high sensitivity, playing a crucial role in exploring risk factors and pathogenesis of diseases. Bile acids are important metabolites in the human body, playing a vital role in various metabolic diseases. Therefore, based on a population cohort (included from Longhua Hospital affiliated with Shanghai University of Traditional Chinese Medicine; n=131, 58 DM patients and 73 DKD patients), we conducted bile acid metabolomics testing on DM and DKD patients. We observed a significant decrease in deoxycholic acid (DCA) species (including DCA, GDCA, and TDCA) levels in the DKD group. Correlation analysis between bile acid levels and disease progression revealed a negative correlation between DCA bile acid levels and serum creatinine and uric acid levels, and a positive correlation with eGFR in the clinical cohort (DM+DKD patients), indicating that DCA bile acid deficiency is closely related to the progression of DKD. Further, based on eGFR and UACR, the DKD cohort was divided into early DKD (DKDE) and advanced DKD (DKDA). The results showed that patients in the early DKD stage had already developed secondary bile acid metabolism defects. Figure 1 Among the DCA-type bile acids, GDCA showed a significant negative correlation with the severity of DKD. Using the OPLSDA-VIP method to analyze the contribution of each bile acid in the clinical cohort, it was also found that GDCA had the highest contribution to DKD. Figure 2 The above results suggest that reduced GDCA levels may be a risk factor for DKD, and that specific targeting of GDCA may have a protective effect against DKD.

[0117] II. Gavage administration of secondary bile acid GDCA can safely and effectively delay the progression of disease in db / db and high-fat combined with STZ-induced DKD model mice.

[0118] Based on the correlation between GDCA levels and the development of DKD found in Example 1, and considering that animal experiments can more directly study the relationship between GDCA and DKD, we used a DKD mouse model induced by a high-fat diet (HFD) combined with streptozotocin (STZ) to determine the effect of GDCA on the progression of DKD in mice. The results showed that, compared with the model group, the GDCA gavage group effectively improved DKD renal function-related indicators such as the urinary albumin-to-cretinine ratio (UACR) and serum creatinine (Scr) levels. Figure 6 Furthermore, it can effectively improve renal tissue pathology, mainly by effectively reducing glomerular basement membrane thickening and mesangial expansion, improving collagen fiber area, reducing glomerular size, and reducing renal tubular injury markers such as neutrophil gelatinase-associated lipocalin (NGAL). Figure 7 Transmission electron microscopy (TEM) results further indicated that GDCA gavage effectively improved the glomerular basement membrane in DKD nephropathy, reduced foot process fusion and loss, alleviated mitochondrial swelling, and restored mitochondrial numbers. Figure 8 GDCA reinjection in the db / db mouse-induced DKD model also showed the same improvement. Figure 3-5 These results indicate that GDCA can significantly delay the progression of DKD disease in mice.

Claims

1. The use of deoxycholic acid compounds, their stereoisomers, enantiomers, diastereomers, geometric isomers, solvates, hydrates, isotope-substituted derivatives, various crystal forms, prodrugs, or pharmaceutically acceptable salts thereof in the preparation of medicaments for the treatment and / or prevention of diabetic kidney disease, characterized in that, The deoxycholic acid compounds have the structure shown in Formula I: in: (CH2) n Each CH2 group may be optionally substituted by 1, 2, 3 or 4 substituents selected from C1-C6 alkyl, C1-C6 alkoxy, deuterium, halogen, hydroxyl and amino groups; R1 is selected from optionally substituted hydroxyl groups and optionally substituted -NR groups. a R b R a and R b Each is independently selected from H and C1-C6 alkyl groups; n can be 1, 2, 3, 4, 5, 6, 7 or 8.

2. The application as described in claim 1, characterized in that, When substituted, the number of substituents on R1 is 1, 2, 3 or 4; and / or, the substituents on R1 are selected from C1-C6 alkyl, carboxyl and sulfonic acid groups; Preferably, R1 is selected from hydroxyl groups and -NR groups optionally substituted with substituents selected from carboxyl and sulfonic acid groups. a R b ; Preferably, R a For H, R b It can be methyl or ethyl.

3. The application as described in claim 1, characterized in that, The deoxycholic acid compounds are selected from deoxycholic acid, glycodeoxycholic acid, and taurideoxycholic acid.

4. The application as described in claim 1, characterized in that, The treatment and / or prevention of diabetic kidney disease includes improving kidney function; and / or, the treatment and / or prevention of diabetic kidney disease includes regulating and / or improving the progression of diabetic kidney disease.

5. The application as described in claim 1, characterized in that, The treatment and / or prevention of diabetic kidney disease includes downregulating the levels of creatinine and / or uric acid in the subjects.

6. The application as described in claim 1, characterized in that, The treatment and / or prevention of diabetic kidney disease includes upregulating the subject's estimated glomerular filtration rate (eGFR); and / or, the treatment and / or prevention of diabetic kidney disease includes downregulating the subject's UACR (urine microalbumin to urinary creatinine ratio); the treatment and / or prevention of diabetic kidney disease includes downregulating the subject's urine output.

7. The application as described in claim 1, characterized in that, The treatment and / or prevention of diabetic kidney disease includes improving glomerular and / or tubular damage.

8. The application as described in claim 1, characterized in that, The treatment and / or prevention of diabetic kidney disease includes improving kidney tissue pathology.

9. The application as described in claim 1, characterized in that, The treatment and / or prevention of diabetic kidney disease includes reducing glomerular basement membrane thickening and mesangial expansion, improving collagen fiber area, reducing glomerular size, and reducing neutrophil gelatinase-associated lipotransferase, a marker of renal tubular damage; and / or, the treatment and / or prevention of diabetic kidney disease includes improving glomerular basement membrane, reducing foot process fusion and loss, alleviating mitochondrial swelling, and / or restoring mitochondrial numbers.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises (i) any one of the deoxycholic acid compounds of claims 1-3, their stereoisomers, enantiomers, diastereomers, geometric isomers, solvates, hydrates, isotopic substitutes, various crystal forms, prodrugs or pharmaceutically acceptable salts thereof. (ii) pharmaceutically acceptable carriers.

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