Combination for the treatment and / or prevention of diabetic nephropathy

Abstract

The application provides a combined medicine, which comprises Dapagliflozin or pharmaceutically acceptable salts, esters, solvates or prodrugs thereof, and a recombinant adeno-associated virus AAV carrying a nucleotide sequence encoding shRNA or miRNA targeting the Acsf2 gene, and can be prepared into a medicine for treating and / or preventing diabetic nephropathy. The application has the following advantages: firstly, the two have a synergistic effect and a double blocking mechanism, and the drug efficacy is significantly better than that of a single group; secondly, the treatment effect is long-lasting; thirdly, the limitation of single-drug treatment is overcome; fourthly, the "small molecule drug + gene therapy" opens up a new direction for the precise treatment of diabetic nephropathy and other Acsf2-related kidney diseases, and has extremely high clinical conversion value and commercial potential.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically to combination drugs for the treatment and / or prevention of diabetic nephropathy. Background Technology

[0002] Diabetic nephropathy is one of the most common and serious microvascular complications of diabetes, and has become a leading cause of end-stage renal disease. Its pathological features include glomerular hyperfiltration, thickening of the glomerular basement membrane, and expansion of the mesangial matrix, ultimately leading to glomerular sclerosis and progressive decline in renal function. For a long time, the standard treatment for diabetic nephropathy has focused primarily on strict glycemic control and renin-angiotensin-aldosterone system blockade. However, these treatments can only partially slow disease progression and cannot completely prevent its deterioration; a significant unmet clinical need remains.

[0003] In recent years, sodium-glucose cotransporter 2 (SGLT2) inhibitors, as a novel type of hypoglycemic drug, have demonstrated renal protective effects beyond simply lowering blood glucose in the treatment of diabetic nephropathy. Dapagliflozin is one of the representative SGLT2 inhibitors. Current technology generally recognizes and confirms that dapagliflozin reduces glucose and sodium reabsorption by inhibiting SGLT2 in the proximal convoluted tubules of the kidney, thereby lowering blood glucose and urinary glucose levels. Its renal protective effect is primarily attributed to this "glucose-dependent" pathway and the resulting series of physiological effects: improved intraglomerular hypertension (tubule-glomerular feedback mechanism), improved metabolic disorders, weight loss, and blood pressure control.

[0004] like Figure 1 As shown, dapagliflozin primarily acts on the SGLT2 target site on the proximal convoluted tubule epithelial cell membrane of the kidney, inhibiting its reabsorption of glucose and sodium. This action directly leads to a decrease in blood glucose and activates tubuloglomerular feedback by increasing distal sodium transport, causing constriction of the afferent arteriole, ultimately reducing intraglomerular hypertension and hyperfiltration, thus protecting the kidneys. Current technology suggests that its renal benefits are an indirect or downstream consequence of this "blood glucose lowering-SGLT2 inhibition-hemodynamic improvement" axis.

[0005] However, existing technologies have the following significant problems and limitations: First, there is a disconnect between clinical efficacy and blood glucose-lowering effects. Large-scale clinical trial data show that the renal protective effect of dapagliflozin can appear early in treatment, and its benefit is not entirely parallel to the improvement in blood glucose control; even in patients with kidney disease but without diabetes, a clear benefit has been observed. This strongly suggests that there may be a direct renal protective pathway independent of blood glucose-lowering effects, and existing theories cannot fully explain this phenomenon. Second, the mechanism of direct renal cell protection is insufficiently elucidated. Existing technologies focus too much on glomerular hemodynamics, lacking in-depth and direct elucidation of whether and how dapagliflozin directly acts on renal parenchymal cells such as renal tubular epithelial cells, affecting key pathological processes such as apoptosis, fibrosis, and inflammation. Acsf2 (Acyl-CoA synthase family member 2) is a key molecule involved in lipid metabolism and cellular energy homeostasis. Recent basic research shows that it plays an important role in renal cell stress and injury, but existing technologies have never revealed any regulatory relationship between dapagliflozin and Acsf2 targets in renal cells. Third, existing technologies have blind spots, limiting the development of more precise treatment strategies. Because the renal effects of dapagliflozin are mainly attributed to SGLT2 inhibition and its subsequent systemic and hemodynamic effects, current technologies have failed to identify cell-specific, hemodynamically independent direct targets such as Acsf2.

[0006] While existing technologies have recognized that dapagliflozin exerts its renal protective effect by targeting SGLT2, this invention further elucidates its mechanism of action through Acsf2. However, diabetic nephropathy, as a chronic and progressive disease, may face challenges such as compensatory activation of the target, patient compliance, and long-term efficacy attenuation with long-term monotherapy. In particular, Acsf2, as a key molecule mediating renal lipotoxicity and fibrosis, is persistently highly expressed during disease progression, and simply inhibiting its activity with dapagliflozin may not be sufficient to completely control the disease. Therefore, there is an urgent need in the field to develop a novel therapeutic strategy that can block the Acsf2 pathway long-term, stably, and efficiently. Gene therapy (such as AAV-mediated RNA interference) can achieve long-term inhibition of target gene expression, and its combination with small molecule drugs holds promise for producing synergistic and durable control effects. Summary of the Invention

[0007] This invention aims to address the following problems in existing technologies: First, to clarify whether dapagliflozin produces an independent, glucose-independent renal protective effect by regulating the novel target Acsf2. Second, to provide a novel combination therapy strategy that can more effectively block Acsf2-mediated renal lipid metabolism disorders and cell damage pathways. Third, to demonstrate that the combined use of dapagliflozin and AAV-mediated Acsf2 interfering RNA can produce a synergistic renal protective effect beyond monotherapy, achieving dual blockade of "functional inhibition + expression downregulation," thus providing a better treatment option for clinical practice.

[0008] To achieve the above objectives, this application adopts the following technical solution:

[0009] The inventive point of this application is to provide a combination drug comprising dapagliflozin or a pharmaceutically acceptable salt, ester, solvate or prodrug thereof, and a recombinant adeno-associated virus (AAV) carrying a nucleotide sequence encoding shRNA or miRNA targeting the Acsf2 gene.

[0010] Optionally, in the above-mentioned combination drugs, the serotype of the recombinant adeno-associated virus (AAV) is selected from AAV2, AAV5, AAV8, AAV9, or their mutants.

[0011] Optionally, in the above-mentioned combination of drugs, the serotype of the recombinant adeno-associated virus (AAV) is AAV9.

[0012] Optionally, in the above-described combination drug, the nucleotide sequence of the shRNA encoding the Acsf2 gene is shown in SEQ ID No. 1.

[0013] SEQ ID No. 1:

[0014] CAGCCAGACTTCTCCAGTTAT.

[0015] Optionally, in the above-mentioned combination of drugs, dapagliflozin is an oral dosage form, and the recombinant adeno-associated virus (AAV) is an injectable dosage form.

[0016] Optionally, the aforementioned combination therapy involves administering the recombinant adeno-associated virus (AAV) via tail vein injection.

[0017] The second inventive point of this application is to provide the use of the above-mentioned combined drugs in the preparation of a medicament for the treatment and / or prevention of diabetic nephropathy.

[0018] The third inventive point of this application is to provide a pharmaceutical composition comprising a therapeutically effective amount of dapagliflozin or a pharmaceutically acceptable salt, ester, solvate or prodrug thereof, a therapeutically effective amount of recombinant adeno-associated virus (AAV) carrying a nucleotide sequence encoding shRNA or miRNA targeting the Acsf2 gene, and a pharmaceutically acceptable vector or excipient.

[0019] Optionally, in the above-described pharmaceutical composition, the serotype of the recombinant adeno-associated virus (AAV) is AAV9.

[0020] Optionally, in the above-described pharmaceutical composition, the nucleotide sequence encoding the shRNA targeting the Acsf2 gene is shown in SEQ ID No. 1.

[0021] Compared with the prior art, this application has the following advantages:

[0022] First, synergistic effect and dual blocking mechanism: This invention is the first to combine dapagliflozin (inhibiting Acsf2 activity) with AAV-mediated Acsf2 gene knockdown (downregulating Acsf2 expression). The two are complementary in mechanism, achieving dual and synergistic blocking of the Acsf2 pathway in terms of both activity and expression, with significantly better efficacy than the single-drug group.

[0023] Secondly, the therapeutic effect is long-lasting: AAV-mediated gene interference can achieve long-term and stable inhibition of Acsf2 expression in kidney cells. The effect of a single injection can last for several weeks to several months, which makes up for the shortcomings of small molecule drugs with short half-life and the need for daily dosing. It is expected to improve patients' long-term treatment compliance and efficacy stability.

[0024] Third, it overcomes the limitations of monotherapy: AAV-mediated gene silencing reduces the number of target proteins from the source, making the inhibitory effect of dapagliflozin more efficient, which can effectively address the efficacy bottleneck of monotherapy, given that Acsf2 is persistently highly expressed in diabetic nephropathy.

[0025] Fourth, a new paradigm for precision combination therapy: This invention provides a novel combination strategy of "small molecule drugs + gene therapy", which opens up a new direction for the precision treatment of diabetic nephropathy and other Acsf2-related kidney diseases, and has extremely high clinical translational value and commercial potential. Attached Figure Description

[0026] Figure 1 This diagram illustrates the classic mechanism of dapagliflozin's renal protective effect in the prior art.

[0027] Figure 2The data shows the dynamic changes in random blood glucose levels in mice after STZ combined with high-fat feeding to induce diabetic nephropathy and then administering dapagliflozin via gavage for 4 weeks. The DKD+insulin group and the DKD+Dapa group showed comparable hypoglycemic effects.

[0028] Figure 3 The results show the analysis of kidney tissue samples taken from mice that developed diabetic nephropathy by STZ combined with a high-fat diet and were subsequently administered dapagliflozin via gavage for 4 weeks. Figure 3 A represents transcriptomics and proteomics analysis. The intersection of differentially expressed genes and proteins yielded three differentially expressed molecules: Cybb, Dpysl3, and Acsf2. Figure 3 B and Figure 3 C represents the results of RT-qPCR and WB validation, which showed that the levels of Acsf2 gene and protein were significantly upregulated in the kidney tissue of diabetic nephropathy mice. Compared with the insulin group, the levels of Acsf2 gene and protein were significantly downregulated in the dapagliflozin group, suggesting that Acsf2 is a potential target for dapagliflozin to treat diabetic nephropathy through a non-glucose-dependent pathway.

[0029] Figure 4 The model demonstrates the interaction between Recombinant Human ACSF2 protein and the small molecule compound Dapagliflozin, established using SPR (surface plasmon resonance) technology. Compound determination was performed based on its solubility concentration. Figure 4 A is the interaction fitting plot. Figure 4 B represents the quality control results. The kinetic fitting results of the compound show that the KD dissociation constant is 2.05e-06 (M), indicating that Dapagliflozin has a high affinity for the target ACSF2, and confirming that Dapagliflozin can directly bind to the target ACSF2.

[0030] Figure 5 The image shows the results of the Cellular Thermal Shift Assay (CETSA) experiment, illustrating the changes in the thermal stability of ACSF2 protein in the dapagliflozin-treated group and the control group.

[0031] Figure 6 This study demonstrates the knockdown efficiency of recombinant AAV9-shAcsf2 via tail vein injection in the kidneys of diabetic nephropathy mice. Four weeks after tail vein injection, RT-qPCR was used to detect the mRNA level of Acsf2 in kidney tissue. The results showed that AAV9-shAcsf2 could effectively and specifically knock down Acsf2 expression in the kidneys (knockdown efficiency >70%).

[0032] Figure 7The results show the therapeutic effect of dapagliflozin combined with AAV9-shAcsf2 on diabetic nephropathy mice. Serum creatinine (Scr), blood urea nitrogen (BUN), and urinary albumin / creatinine ratio (UACR) were measured in each group of mice. The results showed that in the diabetic nephropathy mouse model, compared with the single-drug group, the combination of dapagliflozin and AAV9-shAcsf2 significantly improved renal function and reduced proteinuria. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this application clearer, a more detailed description is provided below. However, it should be understood that the description herein is merely for explaining this application and is not intended to limit its scope.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. All reagents and instruments used herein are commercially available, and the characterization methods involved can be found in relevant descriptions in the prior art, and will not be repeated here.

[0035] To further understand this application, the following detailed description is provided in conjunction with the preferred embodiments.

[0036] Example 1

[0037] Evaluation of the hypoglycemic effect of dapagliflozin in a mouse model of diabetic nephropathy

[0038] 1. Laboratory animals and grouping:

[0039] Eight-week-old male C57BL / 6 mice, weighing 20-25g, were selected. The mice were randomly divided into four groups of 12 mice each: a normal control group (Control group), fed with standard diet; a diabetic nephropathy model group (DKD group), fed a high-fat diet combined with STZ induction; a dapagliflozin treatment group (DKD+Dapa group), treated with dapagliflozin by gavage after model establishment; and an insulin treatment group (DKD+insulin group), treated with insulin after model establishment as a positive control.

[0040] 2. Establishment of a diabetic nephropathy model:

[0041] Except for the normal control group, all other groups of mice were fed a high-fat diet (containing 60% fat) for 4 weeks. After 4 weeks of high-fat feeding, the mice were fasted for 12 hours and then intraperitoneally injected with streptozotocin (STZ, dissolved in 0.1 mol / L citrate buffer, pH 4.5) at a dose of 50 mg / kg body weight for 5 consecutive days. Random blood glucose was monitored weekly 2 weeks after STZ injection. Mice with blood glucose ≥16.7 mmol / L and exhibiting symptoms of polydipsia, polyuria, and polyphagia were considered to have successfully established a diabetic model. The high-fat diet was continued until proteinuria (urine albumin / creatinine ratio ≥300 μg / mg) appeared.

[0042] 3. Drug intervention plan:

[0043] In the DKD+Dapa group, dapagliflozin was dissolved in 0.5% sodium carboxymethyl cellulose solution at a dose of 1 mg / kg / day and administered by gavage between 9:00 and 10:00 AM daily for 4 consecutive weeks; in the DKD+insulin group, insulin was injected subcutaneously at a dose of 0.5 U / kg / day twice daily (8:00 AM and 8:00 PM) for 4 consecutive weeks; and in the Control and DKD groups, an equal volume of 0.5% sodium carboxymethyl cellulose solution was administered by gavage.

[0044] 4. Blood glucose monitoring methods:

[0045] Monitoring time points were before treatment (week 0), week 1, week 2, week 3, and week 4 of treatment. Monitoring method included tail vein blood sampling, and random blood glucose measurements were taken using a Roche blood glucose meter and accompanying test strips. Monitoring time was daily from 9:00 AM to 10:00 AM, consistent with the drug administration time. Results were as follows: Figure 2 As shown.

[0046] Example 2

[0047] Molecular target identification method for dapagliflozin in the treatment of diabetic nephropathy

[0048] After 4 weeks of treatment, the mice in each group were fasted for 12 hours and then their kidneys were removed after anesthesia.

[0049] 1. Multi-omics analysis and target screening:

[0050] 1.1 Transcriptomics analysis:

[0051] Total RNA was extracted from kidney tissue using the TRIzol method. After passing quality control (RIN ≥ 8.0), an mRNA library was constructed. Sequencing was performed using an Illumina NovaSeq 6000 platform with PE 150 and n=5 samples per group. Bioinformatics analysis was performed using Hisat2 for sequence alignment, StringTie for transcript assembly, and DESeq2 for differential expression analysis. The screening criteria were |log2FoldChange| > 1 and adjusted P-value (padj) < 0.05.

[0052] 1.2 Proteomics Analysis:

[0053] Total protein was extracted from kidney tissue using RIPA lysis buffer and quantified using the BCA method. Trypsin digestion, TMT labeling, and high-pH reversed-phase chromatography fractionation were performed. Data acquisition was conducted using LC-MS / MS (Q Exactive HF-X mass spectrometer). MaxQuant software was used for library search and quantitative analysis, with the screening criteria being |log2FoldChange| > 0.5 and P < 0.05.

[0054] 1.3 Key Target Intersection Screening:

[0055] The intersection of differentially expressed genes screened by transcriptomics and differentially expressed proteins screened by proteomics was analyzed. The expression trends of the intersection molecules were further analyzed in the DKD group, DKD+Dapa group, and DKD+insulin group. Screening results: Three common differentially expressed molecules were obtained: Cybb (cytochrome b-245 β chain), Dpysl3 (dihydropyrimidine enzyme-like 3), and Acsf2 (acyl-CoA synthase family member 2). Among them, Acsf2 expression was significantly upregulated in the DKD group, significantly downregulated after dapagliflozin treatment, and showed no significant change in the insulin group, suggesting that it may be a potential target for dapagliflozin-specific action.

[0056] 2. Target validation experiment:

[0057] 2.1 Real-time quantitative PCR (RT-qPCR) validation results:

[0058] Compared with the Control group, the Acsf2 mRNA level in kidney tissue was significantly upregulated in the DKD group (P<0.01). Compared with the DKD group, the Acsf2 mRNA level was significantly downregulated in the DKD+Dapa group (P<0.01), while the downregulation in the DKD+insulin group was smaller and there was no statistical difference compared with the DKD group (P>0.05).

[0059] 2.2 Western Blot Validation Results:

[0060] The trend of Acsf2 protein expression level changes was consistent with that of mRNA level. Acsf2 protein expression in the DKD group was significantly higher than that in the Control group. Acsf2 protein expression in the DKD+Dapa group was significantly lower than that in the DKD group (P<0.01), while there was no significant difference in Acsf2 protein expression between the DKD+insulin group and the DKD group (P>0.05).

[0061] 3. Results Analysis and Conclusions:

[0062] Through combined transcriptomics and proteomics analysis, Acsf2, a key differentially expressed molecule in the development and progression of diabetic nephropathy and during dapagliflozin treatment, was successfully identified. Compared to insulin therapy with comparable glucose-lowering efficacy, dapagliflozin specifically downregulates Acsf2 expression in the kidneys, an effect independent of its glucose-lowering action. Acsf2, an acyl-CoA synthase related to lipid metabolism, is closely associated with lipotoxic damage in diabetic nephropathy due to its abnormal expression. Dapagliflozin may exert a nephroprotective effect by downregulating Acsf2, thereby correcting abnormal lipid metabolism in the kidneys.

[0063] Example 3

[0064] Verification of the direct binding of dapagliflozin to its target protein ACSF2:

[0065] This embodiment aims to clearly verify the direct interaction between dapagliflozin and the ACSF2 protein in vitro and at the cellular level using surface plasmon resonance (SPR) technology and cellular thermal displacement assay (CETSA) experiments.

[0066] 1. Experimental materials:

[0067] The instruments included a Biacore T200 SPR system (Cytiva), a real-time quantitative PCR instrument, a cell culture incubator, and a low-temperature high-speed centrifuge. Reagents included Recombinant Human ACSF2 protein (His tag), purity >95%; Dapagliflozin standard (purity >99%); a series of H buffers (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% v / v surfactant P20, pH 7.4); and an NTA sensor chip (Ni...). 2+ Chelating chip (Cytiva); HK-2 cell line; cell lysate, protease inhibitor mixture, phosphatase inhibitor; protein concentration assay kit (BCA method); anti-ACSF2 antibody; anti-His tag antibody.

[0068] 2. Experimental methods:

[0069] 2.1 SPR technology to verify the direct binding of dapagliflozin to the ACSF2 protein:

[0070] The NTA sensor chip was mounted in a Biacore T200 instrument, and a series of H buffer solutions were used as the run buffer. A 0.5 mM NiCl2 solution was injected at a flow rate of 10 μL / min for 1 minute to chelate Ni on the chip surface. 2+ The Recombinant Human ACSF2 protein (His tag) was diluted in running buffer to a final concentration of 20 μg / mL and injected into the chip surface at a flow rate of 10 μL / min for 7 minutes to specifically immobilize the protein via the His tag, achieving an immobilization level of approximately 5000 RU. 350 mM EDTA solution was injected for 1 minute to elute unstable protein. A reference flow path (immobilizing only Ni) was treated in the same manner. 2+ Dapagliflozin powder (non-immobilized protein) was used for subtraction of non-specific binding signals. A 10 mM stock solution was prepared by dissolving dapagliflozin powder in 100% DMSO. Serial dilutions were performed using run buffer (containing 1% DMSO) to obtain six concentration gradients. The instrument temperature was set to 25°C. Dapagliflozin solutions of different concentrations were sequentially flowed through the chip surface immobilized with ACSF2 protein and a reference surface at a flow rate of 30 μL / min. Each cycle included: sample injection (binding phase, 120 s), run buffer rinsing (dissociation phase, 180 s), and regeneration (rinsing with buffer containing 10 mM EDTA for 30 s). Analysis was performed using Biacore T200 Evaluation Software. First, the active flow path sensor map was subtracted from the reference flow path signal. A 1:1 Langmuir binding model was used to globally fit the binding and dissociation phase data to calculate kinetic parameters. The dissociation equilibrium constant was calculated using the formula KD = kd / ka.

[0071] 2.2 Cellular thermal shift assay (CETSA) to verify intracellular binding:

[0072] HK-2 cells were cultured, and the experiment was divided into a dapagliflozin treatment group (dapagliflozin added to a final concentration of 10 μM) and a solvent control group (dapagliflozin added to an equal volume, final concentration of 0.1%). Cells were incubated at 37°C in a 5% CO2 incubator for 2 hours. After incubation, cells were washed twice with pre-chilled PBS, digested with trypsin, and collected. Cells were resuspended in PBS containing protease inhibitors and divided into several aliquots. Each aliquot of cell suspension was heated for 3 minutes in a metal bath or PCR instrument at different temperatures. The samples were immediately cooled on ice, and then subjected to three freeze-thaw cycles in liquid nitrogen or sonication. The samples were centrifuged at 20,000 × g for 20 minutes at 4°C, and the supernatant was collected. The total protein concentration of the supernatant at each temperature point was determined using the BCA method and normalized. The content of ACSF2 protein in each sample was detected by Western blotting. The ACSF2 signal in the untreated sample at 37°C was taken as 100%, and the relative percentage of soluble ACSF2 protein remaining at each temperature point was calculated.

[0073] 3. Experimental Results:

[0074] SPR experimental results showed that the binding sensing profile of dapagliflozin to immobilized ACSF2 protein exhibited typical concentration-dependent binding and dissociation curves. The binding rate constant (ka) = (3.25 ± 0.42) × 10⁻⁶. 4 M -1 s -1 The dissociation rate constant (kd) = (6.66 ± 0.85) × 10 -2 s -1 The dissociation equilibrium constant (KD) = kd / ka = 2.05 × 10 -6 The KD value of 2.05 μM indicates that dapagliflozin binds directly to recombinant human ACSF2 protein with high affinity. CETSA assays showed that, compared to the DMSO solvent control group, the thermostability curve of ACSF2 protein in cells treated with 10 μM dapagliflozin shifted significantly to the right. The Tm value of ACSF2 protein in the DMSO control group was approximately 49.2°C, while in the dapagliflozin-treated group, its Tm value significantly increased to approximately 52.8°C, ΔTm ≈ +3.6°C. These results further confirm the direct interaction between the two at the cellular level.

[0075] Example 4

[0076] Construction, packaging, and tail vein injection method of recombinant AAV9-shAcsf2:

[0077] 1. shRNA target design and vector construction:

[0078] The optimal shRNA interference sequence (SEQ ID No. 1: 5'-CAGCCAGACTTCTCCAGTTAT-3') was designed and screened for the CDS region of the mouse Acsf2 gene and constructed into the pAAV-U6-MCS-CMV-EGFP vector. The shRNA expression was driven by the U6 promoter and the EGFP reporter gene expression was driven by the CMV promoter.

[0079] 2. AAV Packaging and Purification:

[0080] HEK293T cells were transfected using a three-plasmid co-transfection method (pAAV-shAcsf2, pHelper, pAAV9-RC). After 72 hours of culture, cells and supernatant were collected and purified by iodixanol density gradient centrifugation and ultrafiltration to obtain high-titer recombinant AAV9-shAcsf2 virus (titer ≥ 1 × 10⁻⁶). 12 (vg / mL).

[0081] 3. Tail vein injection method:

[0082] Dilute recombinant AAV9-shAcsf2 with sterile PBS to an appropriate concentration (recommended injection dose is 1×10⁻⁶). 11 vg / animal, injection volume 100-200µl), slowly injected via tail vein (completed within 10 seconds). The control group was injected with an equal volume of negative control AAV9-shNC (targeting a nonspecific sequence).

[0083] Example 5

[0084] Evaluation of the therapeutic effect of dapagliflozin combined with AAV9-shAcsf2 on diabetic nephropathy mice:

[0085] 1. Laboratory animals and grouping:

[0086] Eight-week-old male C57BL / 6 mice were selected, and a DKD model was established using the original method. After successful modeling, the mice were randomly divided into the following four groups, with 15 mice in each group: model control group (DKD group), which received an equal volume of solvent; dapagliflozin monotherapy group (Dapa group), which received 1 mg / kg / day by gavage for 4 consecutive weeks; and AAV9-shAcsf2 monotherapy group (AAV-shAcsf2 group), which received AAV9-shAcsf2 (1×10⁻⁶ mg / kg / day) via tail vein injection on day 1 of the experiment. 11 vg / animal); combined drug group (Dapa + AAV-shAcsf2 group), tail vein injection of AAV9-shAcsf2 (1×10) 11 While administering vg / animal, dapagliflozin (1 mg / kg / day) by gavage daily for 4 weeks.

[0087] 2. Detection indicators:

[0088] After treatment (week 4), 24-hour urine samples were collected to detect UACR; mice were euthanized, and kidney tissue was collected for pathological staining (HE, Sirius red), Western blotting to detect Acsf2 protein expression, and detection of renal function biochemical indicators.

[0089] 3. Experimental results (e.g.) Figure 7 (as shown)

[0090] The combined drug group showed significantly improved renal function and proteinuria compared to the Dapa monotherapy group and the AAV-shAcsf2 monotherapy group.

[0091] 4. Conclusion:

[0092] The combination of dapagliflozin and AAV9-shAcsf2 showed significantly better synergistic effects than monotherapy in improving renal function and reducing proteinuria.

[0093] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A combination drug, characterized in that, A recombinant adeno-associated virus (AAV) containing dapagliflozin or its pharmaceutically acceptable salts, esters, solvates or prodrugs, and carrying a nucleotide sequence encoding shRNA or miRNA targeting the Acsf2 gene.

2. The combined medication according to claim 1, characterized in that, The serotype of the recombinant adeno-associated virus (AAV) is selected from AAV2, AAV5, AAV8, AAV9 or their mutants.

3. The combined medication according to claim 2, characterized in that, The serotype of the recombinant adeno-associated virus (AAV) is AAV9.

4. The combined medication according to claim 1, characterized in that, The nucleotide sequence of the shRNA encoding the Acsf2 gene is shown in SEQ ID No.

1.

5. The combined medication according to claim 1, characterized in that, The dapagliflozin is an oral dosage form, and the recombinant adeno-associated virus (AAV) is an injectable dosage form.

6. The combination drug according to claim 5, characterized in that, The recombinant adeno-associated virus (AAV) was administered via tail vein injection.

7. The use of the combination of drugs according to any one of claims 1-6 in the preparation of a medicament for treating and / or preventing diabetic nephropathy.

8. A pharmaceutical composition, characterized in that, It contains a therapeutically effective amount of dapagliflozin or its pharmaceutically acceptable salt, ester, solvate or prodrug, a therapeutically effective amount of recombinant adeno-associated virus (AAV) carrying a nucleotide sequence encoding shRNA or miRNA targeting the Acsf2 gene, and a pharmaceutically acceptable vector or excipient.

9. The pharmaceutical composition according to claim 8, characterized in that, The serotype of the recombinant adeno-associated virus (AAV) is AAV9.

10. The pharmaceutical composition according to claim 9, characterized in that, The nucleotide sequence of the shRNA encoding the Acsf2 gene is shown in SEQ ID No. 1.

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