Use of finerenone for the manufacture of a medicament for the treatment of frasier syndrome

Abstract

The application discloses a use of finerenone in preparation of a medicine for treating Frasier syndrome and belongs to the technical field of biological medicines. The end-stage FS model pig is subjected to 30-day standard oral intervention by using the finerenone, so that the kidney injury can be effectively relieved, the urine protein content is reduced by 70%, is reduced from 365 mg / dL to the minimum 42 mg / dL, the urine protein creatinine ratio is reduced from 10.12 to 1.09, and the serum albumin is increased from 3.33 g / dL to 3.86 g / dL. Meanwhile, the end-stage clinical symptoms, such as anorexia, tremor and bed rest, of the animal can be effectively improved, the self-feeding and activity ability is restored, and it is confirmed that the MR antagonist can effectively relieve the FS kidney inflammation and fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the use of fenelone in the preparation of drugs for treating Frasier syndrome. Background Technology

[0002] Frasier syndrome (FS) is a fatal childhood disease characterized by steroid-resistant nephrotic syndrome, progressive glomerulosclerosis, gonadal dysgenesis, and premature renal failure. It is caused by a mutation at the splicing donor site of the 9th intron of the WT1 gene. Unlike the more common Denys-Drash syndrome, FS does not originate from a significant loss of WT1 function, but rather from alternative splicing. Between the 3rd and 4th zinc finger domains of the 9th intron, two isoforms, +KTS and -KTS, are formed by the insertion or deletion of three amino acids (lysine, threonine, and serine). Normally, the +KTS / -KTS expression ratio is constant; disruption of this balance leads to disease caused by the disruption of the +KTS / –KTS splicing equilibrium. Although the genetic basis of FS is well understood, the lack of large animal models has long limited the elucidation of its mechanisms and the development of treatments. While rodent models have some value, they cannot fully reproduce the complete picture of human FS due to significant interspecies differences in kidney anatomy, hemodynamics, and extracellular matrix composition.

[0003] Current technologies have only constructed a mouse Frasier syndrome (FS) model. This method mainly uses homologous recombination targeting technology from mouse embryonic stem cells to introduce a point mutation at the Intron 9+2T>C site of the WT1 gene in vitro, simulating the loss of the +KTS isoform in humans due to alternative splicing imbalance. However, current research on Frasier syndrome (FS) mouse models is generally at a very basic level and cannot meet the actual needs of clinical translation and pharmacological evaluation. First, there are significant physiological and anatomical barriers between species. Mice have a single-papillary kidney structure, while humans and pigs have complex two-tiered multi-papillary kidney structures. This structural difference means that mice cannot accurately replicate the disease progression and slowly developing end-stage renal disease pathology in human patients when simulating progressive focal segmental nephrosclerosis (FSGS) caused by WT1 gene mutations. Second, the preclinical evaluation value is severely insufficient, leading to a gap in subsequent research. Due to the small size and short lifespan of mice, researchers cannot perform continuous blood biochemical tracking, multi-time renal biopsies, and dynamic imaging monitoring at the clinical level. Meanwhile, the pharmacokinetics of rodents differ significantly from those of humans, which may lead to clinical translation failures in drug screening based on mouse models.

[0004] Currently, all medications used to treat Frasier syndrome are symptomatic relief or complication management, such as cyclosporine A and glucocorticoids. Traditional pediatric non-remission syndrome (NS) is 80%-90% sensitive to glucocorticoids, but Frasier syndrome (FS) is classified as a secondary non-remission syndrome (SRNS). Prednisone, methylprednisolone pulse therapy, cyclophosphamide, mycophenolate mofetil (MMF), and tacrolimus monotherapy have all been shown to fail to induce sustained remission, with multiple case series documenting "persistent proteinuria despite multiple drug rotations." Mechanistically, podocyte damage in FS stems from transcription factor imbalance rather than immune complex deposition; therefore, immunosuppression should not be effective—this understanding constitutes the first stage conclusion in the evolution of FS medications.

[0005] ACEIs (enalapril, etc.) and ARBs (losartan, etc.) have become the foundational medications for managing proteinuria in Frasier syndrome (FS) by reducing intraglomerular pressure and improving filtration barrier selectivity. A case report in 2013 indicated that a combination of RAS inhibitors and indomethacin produced a clear protein-lowering response. However, the evidence level for this combination is limited to case studies / small-scale observations and cannot alter the progression of ESKD. Therefore, exploring new therapeutic targets for Frasier syndrome and modifying +KTS / Imbalance in the KTS isomer ratio is a new research direction for the treatment of Frasier syndrome. Summary of the Invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention provides the use of fenelone in the preparation of a medicament for treating Frasier syndrome.

[0007] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows: The purpose of this invention is to provide the use of nonsteroidal MR antagonists in the preparation of medicaments for the treatment of Frasier syndrome.

[0008] Furthermore, the nonsteroidal MR antagonist is phenelzine.

[0009] Furthermore, fenelazol can alleviate kidney inflammation and fibrosis in patients with FS.

[0010] Furthermore, fenelazol can alleviate kidney damage, reduce urinary protein levels and the urinary protein-to-creatinine ratio, and promote the recovery of serum albumin.

[0011] Another object of the present invention is to provide a medicament for treating Frasier syndrome, which has the above-mentioned nonsteroidal MR antagonist as the active ingredient and includes a pharmaceutically acceptable carrier, excipient or adjuvant thereof.

[0012] Furthermore, the nonsteroidal MR antagonist is phenelzine.

[0013] Furthermore, the dosage form of the drug is tablets, pills, granules, capsules, powders, drops, or injections.

[0014] Another object of the present invention is to provide a pharmaceutical composition for treating Frasier syndrome, comprising the above-mentioned fenelone, or a drug.

[0015] The beneficial effects of this invention are: This invention employs ABE-TadA8e single-base editing technology to replace traditional CRISPR / Cas9 technology, eliminating double-strand breaks in genomic DNA throughout the process. It precisely achieves site-directed single-base mutations (A→G) in the 9th intron of the porcine WT1 gene, avoiding problems such as random insertions and deletions. The editing site is singular and controllable, ensuring a stable supply of mutated positive cells and gene-edited individuals.

[0016] This invention successfully obtained gene-edited piglets for Frasier syndrome (FS) using somatic cell cloning technology. The model pigs stably reproduce the characteristic molecular phenotype of human Frasier syndrome. Compared to wild-type pigs, the model pigs showed significantly reduced expression of the +KTS splice variant of the WT1 gene and significantly increased expression of the -KTS splice variant, accurately replicating the core pathological features of gene splicing disorders in human Frasier syndrome patients. Simultaneously, this model can naturally progress to end-stage renal disease, stably exhibiting typical renal function impairment phenotypes such as severe proteinuria, elevated serum creatinine, and elevated blood urea nitrogen. This overcomes the limitations of small animal models in simulating the renal anatomy, tissue, and clinical lesions of human Frasier syndrome, providing a reliable large-scale animal experimental vehicle for disease research. This invention is the first to construct a large-scale pig disease model that highly replicates human Frasier syndrome in terms of anatomical structure, histopathology, and clinical phenotype, overcoming the technical limitations of traditional small animal models with low phenotypic simulation and inability to reproduce the progression characteristics of human disease. It provides a stable and reliable large-scale animal research vehicle for Frasier syndrome mechanism research and drug development.

[0017] This invention utilizes fenelazol (10 mg / day) for 30 days in a standardized oral intervention in end-stage fibrotic liver disease (FS) model pigs, effectively alleviating kidney damage. Urinary protein levels decreased by 70%, from 365 mg / dL to a minimum of 42 mg / dL; the urinary protein-to-creatinine ratio decreased from 10.12 to 1.09; and serum albumin increased from 3.33 g / dL to 3.86 g / dL. Simultaneously, it effectively improved end-stage clinical symptoms such as anorexia, tremors, and bedriddenness, restoring the animals' ability to eat and move independently. This demonstrates that MR antagonists can effectively alleviate kidney inflammation and fibrosis in FS, providing a valid technical basis for the clinical treatment of FS nephropathy. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of targeted knockout. Figure 2 The proportion of edited cells that are positive for homozygous and heterozygous mutations; Figure 3Flowchart for constructing a pig model of FS disease using somatic cell nuclear transfer and embryo transfer; Figure 4 To quantitatively detect changes in the expression level of the WT1 gene splice isoform in piglet kidney tissue using qPCR; Figure 5 Changes in protein biomarkers in a pig model of FS disease; Figure 6 Transcriptome detection for the analysis of core pathogenic targets and signaling pathways in FS kidney injury; Figure 7 Metabolomics detection for the analysis of core pathogenic targets and signaling pathways in FS kidney injury; Figure 8 Proteomic detection for the analysis of core pathogenic targets and signaling pathways in FS kidney injury; Figure 9 The effects of fenestrone-targeted intervention on FS; Figure 10 This is a schematic diagram illustrating the construction of plasmid functional modules. Detailed Implementation

[0019] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0020] Example 1: Construction of the FS Model 1. Construction of a site-directed ABE single-base editing vector targeting the porcine WT1 gene introns This invention targets the second T site of the 9th intron of the porcine WT1 gene, designs a specific sgRNA based on the antisense strand, and matches the recognition sequence of the ABE-TadA8e single-base editing system; a complete ABE-TadA8e eukaryotic expression editing plasmid is constructed using molecular cloning technology. The plasmid's functional modules include a TadA8e deaminase expression framework, an sgRNA transcription framework, and an resistance selection marker element (see...). Figure 10 ).

[0021] This invention differs from traditional CRISPR / Cas9 double-strand cutting technology in that it does not produce DNA double-strand breaks throughout the process. It only achieves precise A→G single-base substitution at the target site, avoiding random insertion and deletion mutations and ensuring that the mutation site is completely consistent with the pathogenic mutation of human FS.

[0022] 2. Editing, screening, and preparation of positive donor cells from porcine fetal fibroblasts The constructed ABE-TadA8e editing plasmid was electrotransfected into porcine fetal fibroblasts. After transfection, positive edited cells were screened by flow cytometry, and single-cell platelets were obtained to obtain monoclonal cell lines. Sanger sequencing was used to identify the genotypes of all monoclonal cells, and the proportions of homozygous and heterozygous mutant cells were statistically analyzed. Figure 2 ).

[0023] 3. Constructing a pig model of FS disease using somatic cell nuclear transfer and embryo transfer. The positive gene-edited fibroblasts obtained in step 2 were used as nuclear donors, and porcine oocytes were used as recipients. Somatic cell nuclear transfer was used to reconstruct cloned embryos. The successfully developed reconstructed embryos were then transferred into the uterus of surrogate sows. After 114 days of gestation, five gene-edited FS piglets were obtained. Figure 3 ).

[0024] The expression of WT1 gene splice isoforms in piglet kidney tissue was detected by qPCR. The results showed that the +KTS isoform mRNA was significantly downregulated and the -KTS isoform mRNA was significantly upregulated in the model pigs, replicating the characteristic splicing disorder phenotype of human Frasier syndrome, thus completing the construction of a large animal model of Frasier syndrome. Figure 4 ).

[0025] Example 2: Multi-omics integrated analysis to screen core pathogenic targets and signaling pathways in FS renal injury Multi-omics analysis, including transcriptomics, metabolomics, and proteomics, was employed to perform parallel multidimensional omics analysis on kidney tissues from FS pigs and wild-type pigs. Differential expression analysis, pathway enrichment analysis, and protein-protein interaction verification were used to identify core differentially expressed proteins and pathogenic pathways. The results are presented below. Figures 6-8 .

[0026] like Figures 6-8 As shown, FKBP5 protein was found to be significantly downregulated in the kidney tissue of pigs with Frasier syndrome. This indicates that downregulation of FKBP5 can relieve its negative regulatory effect on mineralocorticoid receptor (MR), leading to persistent overactivation of the MR signaling pathway, which in turn induces renal inflammatory infiltration, extracellular matrix deposition, and ultimately drives renal fibrosis and end-stage renal injury.

[0027] Example 3: Feneldrolone-targeted intervention in a terminal FS pig model One disease-end-stage FS model pig was used as the intervention subject. This experimental pig exhibited typical and severe clinicopathological features of FS, including severe proteinuria and severe renal impairment, specifically serum creatinine of 3.58 mg / dL and blood urea nitrogen of 70.39 mg / dL. It also presented with severe clinical symptoms such as anorexia, body tremors, and inability to move independently, resulting in bedridden state. This FS pig was continuously treated with oral fenelazol at a dose of 10 mg / day for 30 days. The intervention effect was evaluated after 30 days of treatment, and the results are shown below. Figure 9 .

[0028] like Figure 9 As shown, the first 4 days of treatment were the drug adaptation period, during which proteinuria in FS pigs showed a transient compensatory increase. From the 5th day of treatment, all renal function indicators showed sustained and significant improvement. Urinary protein (UPRO) decreased from 365 mg / dL to 100 mg / dL, with a minimum value of 42 mg / dL, representing an overall reduction of approximately 70%; the urine protein / creatinine ratio (UPC) decreased from 10.12 to 1.09; and serum albumin increased from 3.33 g / dL to 3.86 g / dL. Clinically, end-stage FS pigs regained their ability to eat and move freely after treatment, and critical clinical symptoms subsided. This confirms that MR antagonists can effectively alleviate renal inflammation and fibrosis in FS, providing a valid technical basis for the clinical treatment of FS nephropathy.

[0029] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Use of nonsteroidal anti-inflammatory drugs (NSAIDs) in the preparation of drugs for the treatment of Frasier syndrome.

2. The use according to claim 1, characterized in that, The nonsteroidal MR antagonist is phenelzine.

3. The use according to claim 2, characterized in that, The fenelone can alleviate kidney inflammation and fibrosis in patients with FS.

4. The use according to claim 2, characterized in that, The fenelazol can alleviate kidney damage, reduce urinary protein levels and the urinary protein-to-creatinine ratio, and promote the recovery of serum albumin.

5. A drug for treating Frasier syndrome, characterized in that, The drug uses the nonsteroidal MR antagonist of claim 1 as its active ingredient and includes a pharmaceutically acceptable carrier, excipient or adjuvant thereof.

6. The drug according to claim 5, characterized in that, The nonsteroidal MR antagonist is phenelzine.

7. The drug according to claim 5 or 6, characterized in that, The dosage form of the drug is tablets, pills, granules, capsules, powders, drops, or injections.

8. A pharmaceutical composition for treating Frasier syndrome, characterized in that, This includes fenelone as described in claim 2, or the drug as described in claim 7.

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