A pharmaceutical composition for treating refractory aplastic anemia and application thereof

Abstract

The application provides a kind of drug composition for treating refractory aplastic anemia, the drug composition per unit dose includes the following components: cyclosporine A 50~100 mg, metformin hydrochloride 250~375 mg, stanozolol 2~5 mg and nutritional supplement 30~65 mg.The drug composition of the application takes cyclosporine A, metformin and stanozolol as core, and combines nutritional supplement, can simultaneously regulate key links such as immune suppression, metabolic adaptation and hematopoietic output etc.The research results show that the drug composition applied to RAA patient has clinical implementation feasibility, can realize the gradual improvement of patient hematology index, has higher clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and relates to a pharmaceutical composition for treating refractory aplastic anemia and its application. Background Technology

[0002] Aplastic anemia (AA) is caused by immune-mediated damage to hematopoietic stem cells (HSCs), with the core pathology being bone marrow failure and pancytopenia. Patients often experience fatigue, bleeding, and a tendency to infection. The choice of treatment strategy depends on the patient's age, disease subtype, donor availability, and the body's tolerance. Allogeneic hematopoietic stem cell transplantation (HSCT) is the first choice for eligible patients due to its potential for cure; while for patients unsuitable for transplantation, immunosuppressive therapy (IST) with equine anti-thymocyte globulin and cyclosporine A is the core standard treatment. However, despite the significant efficacy of IST, some patients with severe aplastic anemia still experience poor treatment response or relapse. These patients who do not respond to IST or relapse are defined as having refractory aplastic anemia (RAA). Given the limited options for subsequent treatment of RAA patients, optimizing their clinical management remains a major challenge for the hematology community.

[0003] For refractory aplastic anemia (RAA), various second-line regimens, including alemtuzumab, androgens, cyclophosphamide, and glucocorticoids, have been explored in clinical practice. However, current evidence has not yet confirmed that these regimens consistently outperform standard immunosuppressive therapy in terms of overall efficacy. These therapies benefit a limited population and face practical challenges such as slow onset of action, high treatment costs, and limited drug accessibility, failing to fundamentally overcome the treatment bottleneck. Given the risks of disease relapse, treatment non-response, and progression, there is an urgent need to develop new, more effective, accessible, and long-term efficacy-maintaining treatment strategies to optimize the clinical management of RAA. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art. This invention provides a pharmaceutical composition for treating refractory aplastic anemia and its application. This invention uses multi-omics and systems biology methods to identify potential therapeutic targets and reusable candidate drugs, prioritizes potential candidate drugs, and screens out drug combinations with therapeutic translational value.

[0005] The technical solution adopted in this invention is as follows: According to one aspect of this application, the present invention provides a pharmaceutical composition for treating refractory aplastic anemia, wherein each unit dose of the pharmaceutical composition comprises the following components: cyclosporine A 50-100 mg, metformin hydrochloride 250-375 mg, stanozolol 2-5 mg, and a nutritional supplement 30-65 mg.

[0006] In one specific embodiment, the nutritional supplement comprises the following components: folic acid 10-20 mg, vitamin B2 10-20 mg, vitamin B12 0.5-1.0 mg and coenzyme Q10 10-20 mg.

[0007] In one specific embodiment, each unit dose of the pharmaceutical composition comprises the following components: cyclosporine A 50-100 mg, metformin hydrochloride 250-375 mg, stanozolol 2-5 mg, folic acid 10-20 mg, vitamin B2 10-20 mg, vitamin B12 0.5-1.0 mg, and coenzyme Q10 10-20 mg.

[0008] In one specific embodiment, each unit dose of the pharmaceutical composition comprises the following components: cyclosporine A 50 mg, metformin hydrochloride 250 mg, stanozolol 3 mg, folic acid 15 mg, vitamin B2 15 mg, vitamin B12 0.75 mg, and coenzyme Q10 15 mg.

[0009] In one specific embodiment, the vitamin B12 is selected from adenosylcobalamin or methylcobalamin. In a preferred embodiment, the vitamin B12 is adenosylcobalamin.

[0010] In one specific embodiment, the components of the pharmaceutical composition are mixed to form a formulation, and the dosage form of the formulation is a pharmaceutically acceptable dosage form. In a preferred embodiment, the dosage form of the formulation is selected from tablets, capsules, granules, and suppositories.

[0011] In one specific embodiment, each component of the pharmaceutical composition is formulated separately, and each separate formulation is packaged in the same box; the dosage form of the formulation is a pharmaceutically acceptable dosage form.

[0012] According to another aspect of this application, the present invention also provides the use of the pharmaceutical composition in the preparation of a drug for treating refractory aplastic anemia.

[0013] In one specific embodiment, the refractory aplastic anemia is refractory severe aplastic anemia or refractory chronic aplastic anemia.

[0014] This invention proposes a multi-target combination therapy for refractory aplastic anemia. By analyzing publicly available expression datasets, differentially expressed genes between healthy subjects and aplastic anemia patients are screened. Furthermore, messenger RNA and miRNA expression profiles are integrated to construct a miRNA-mRNA regulatory network. Simultaneously, an integrative omics inference platform is used to prioritize potential candidate drugs to screen drug combinations with potential therapeutic translational value, and the rationality of the treatment plan is validated.

[0015] The beneficial effects of the present invention include, but are not limited to: This invention provides a pharmaceutical composition for treating refractory aplastic anemia (RAA). The composition, with cyclosporine A, metformin, and stanozolol as its core components, combined with nutritional supplements, can simultaneously regulate key aspects such as immunosuppression, metabolic adaptation, and hematopoietic output. Research results show that this pharmaceutical composition is clinically feasible for RAA patients, achieving gradual improvement in hematological indicators and possessing high clinical application value. Attached Figure Description

[0016] Figure 1 This is a schematic diagram illustrating the potential application value of the multi-target treatment strategy proposed in this invention in the diagnosis and treatment of refractory aplastic anemia. Figure 2 This is a study of the different characteristics between AA patients and healthy controls according to an embodiment of the present invention; A: Cell distribution characteristics of AA patients and healthy controls; B: miRNA expression profiles of AA patients and healthy controls; Figure 3 This invention provides an embodiment for the study of different functions of AA-related phenotypes. Figure 4 This is a 2SMR analysis result of a candidate drug according to an embodiment of the present invention; Figure 5 This invention provides an example of an age-stratified advantage analysis of AA risk. Figure 6 This is an example of clinical cumulative efficacy evaluation in one embodiment of the present invention; Figure 7 This is a clinical cumulative efficacy assessment in another embodiment of the present invention. Detailed Implementation

[0017] The present invention will be described in detail below with reference to specific embodiments. The following embodiments are only for the purpose of enabling those skilled in the art to understand the technical solutions of the present invention, implement or use the present invention, and are not intended to limit the scope of protection of the present invention.

[0018] In this invention, unless otherwise specified, all raw materials and equipment used can be purchased from the market or are commonly used in the field. Unless otherwise specified, the methods in the embodiments are conventional methods in the field.

[0019] This invention utilizes multi-omics and systems biology methods to identify potential therapeutic targets and reusable drug candidates. By analyzing publicly expressed datasets, it screens differentially expressed genes (DEGs) between healthy subjects and patients with aplastic anemia. Considering the crucial roles of miRNAs in immune regulation, hematopoietic function maintenance, and bone marrow microenvironment regulation, it further integrates messenger RNA (mRNA) and miRNA expression profiles to construct a miRNA-mRNA regulatory network. Simultaneously, this invention employs an integrative omics inference platform to prioritize potential drug candidates, thereby screening for drug combinations with potential therapeutic translational value.

[0020] Given that refractory aplastic anemia is a complex systemic disease involving immune dysfunction, impaired hematopoietic function, abnormal bone marrow microenvironment, and altered energy metabolism, this invention also draws upon the dynamic balance framework of Traditional Chinese Medicine as a conceptual integration tool, corresponding key physiological domains related to energy metabolism, growth and repair, immune regulation, nutritional support, and structural homeostasis. Combining this framework with a Phenotypic Association Analysis (PheWAS) based on data from the UK Biobank, this invention identifies a group of candidate drugs targeting complementary physiological domains, aiming to jointly improve the multidimensional functional deficiencies present in aplastic anemia.

[0021] To further validate the rationale for the proposed therapeutic composition, this invention utilizes large-scale human genetic data and employs Mendelian randomization (MR) analysis to assess the potential causal association between genetic proxy indicators related to the priority screening of candidate compounds and aplastic anemia-related phenotypes. Subsequently, an integrative omics inference platform is used to further evaluate the association between candidate drug components and the aplastic anemia disease network. Furthermore, this invention designs and conducts a prospective single-arm clinical study to preliminarily evaluate the clinical feasibility and hematological efficacy of this multi-target combination therapy strategy in patients with refractory aplastic anemia. Through the aforementioned complementary systems biology analysis, genetic causal inference, and clinical translation validation steps, this invention systematically evaluates the potential application value of the proposed multi-target therapy strategy in the management of refractory aplastic anemia, see [link to relevant documentation]. Figure 1 .

[0022] Example 1: Screening of potential drugs for the treatment of RAA This invention employs an integrative multi-omics and clinical translational research framework to systematically construct a multi-target combination therapy regimen for recurrent acute exacerbations (RAA). The overall research process specifically includes: (1) Differential expression analysis and construction of miRNA-mRNA regulatory network This invention focuses on differentially expressed genes between patients with aplastic anemia and healthy controls to explore potential disease-related pathways and therapeutic targets. First, the single-cell RNA sequencing (scRNA-seq) dataset GSE181989 was downloaded from the Gene Expression Comprehensive Database (GEO). This dataset, generated by the Illumina NovaSeq 6000 platform, includes samples from two patients with refractory aplastic anemia and two healthy controls, containing a total of 33,538 cells and 20,632 gene expression feature maps. Further analysis was conducted on differentially expressed miRNAs (DE-miRNAs) between patients with aplastic anemia and healthy controls. miRNA expression data were obtained from the GEO dataset GSE82095, generated by the Affymetrix GeneChipmicroRNA 2.0 chip platform. The samples were bone marrow T cells from three patients with aplastic anemia and three healthy controls (see Table 1).

[0023] Table 1

[0024] Subsequently, the Seurat package in R was used for quality control, standardization, normalization, dimensionality reduction, and cluster analysis of the single-cell sequencing data. Low-quality cells were filtered based on indicators such as gene detection count, UMI count, and mitochondrial gene proportion. After quality control, the Unified Manifold Approximation and Projection Algorithm (UMAP) was used for visual clustering of cells, such as... Figure 2 As shown in Figure A, the results revealed different cell distribution characteristics between AA patients and healthy controls. Further analysis of gene expression differences between AA patients and healthy controls was conducted using the FindMarkers function in Seurat. Differentially expressed mRNAs (DE-mRNAs) were obtained using the adjusted p-value or false discovery rate (FDR) as statistical screening criteria. Figure 2 As shown in B and Table 2, further differential expression analysis identified 1,261 differentially expressed mRNAs, of which 471 were upregulated and 790 were downregulated. Simultaneously, miRNA expression profiling identified 78 differentially expressed miRNAs, of which 45 were upregulated and 33 were downregulated. These results indicate differences in cellular composition and gene expression patterns between AA patients and healthy controls.

[0025] Table 2

[0026] As shown in Table 3, a total of 26 miRNAs and 168 target mRNAs were included, forming an AA-related miRNA-mRNA regulatory network. Some differentially expressed miRNAs may participate in the disease process of AA by regulating hematopoietic, immune, and metabolic-related genes.

[0027] Table 3

[0028] The raw microarray data underwent background correction using the affy package in R and normalization was performed using robust multi-chip averaging (RMA). Missing values ​​were estimated using the K-nearest neighbor (KNN) algorithm. Subsequently, differential expression analysis was conducted using the limma package in Bioconductor, with p < 0.05 and |log2 fold change| > 0 as the selection thresholds to obtain differentially expressed miRNAs.

[0029] To construct a miRNA-mRNA regulatory network, this invention further utilizes the miRNet platform to predict potential target genes of differentially expressed miRNAs. Subsequently, the intersection of the target mRNAs predicted by the miRNet platform and the differentially expressed mRNAs obtained from the aforementioned scRNA-seq analysis is taken, retaining genes that are both predicted as miRNA target genes and show differential expression in patients with aplastic anemia, thereby improving the biological relevance and disease specificity of the constructed regulatory network.

[0030] (2) Screening of candidate drugs using an epigenetic precision treatment prediction platform developed based on the principles of logistic omics. This invention uses differentially expressed mRNA as input data and employs the EpiMed platform for epigenetic precision treatment prediction to screen drugs. Within the EpiMed analysis framework, if the molecular expression characteristics induced by a compound show a significant negative correlation with the disease expression profile, it suggests that the compound may have the potential to reverse disease-related molecular abnormalities and could be considered a candidate therapeutic drug. As shown in Table 4, the analysis results indicate that metformin and cyclosporine A rank highly among the candidate compounds, suggesting a possible reversal relationship with AA-related abnormal expression profiles. Furthermore, based on clinical experience, stanozolol was also selected as a candidate therapeutic drug.

[0031] Table 4

[0032] (3) Mendelian randomization (MR) and multigene risk score analysis were used to validate the genetic proxy indicators related to the candidate drugs. This invention employs two-sample Mendelian randomization analysis (2SMR). Pooled statistical data for exposure and outcome factors were derived from independent genome-wide association studies (GWAS). Genetic instrumental variables for cyclosporine A were constructed based on cis-eQTL data, focusing on single nucleotide polymorphisms (SNPs) located near its drug metabolism and transport-related genes ABCB1 (also known as MDR1) and CYP3A5. Genetic proxy indicators for metformin were derived from GWAS data related to glycated hemoglobin in the OpenGWAS database (ebi-a-GCST90014006). Genetic proxy indicators for stanozolol were derived from GWAS datasets related to hemoglobin levels and telomere length in the GWAS Catalog (GCST90469136, GCST90002310, GCST90435144).

[0033] All exposure-related instrumental variables were evaluated based on the GWAS significance threshold of P < 5 × 10⁻⁶. -8 Preliminary screening was conducted to ensure sufficient correlation between instrumental variables and exposure factors. Outcome factors, including aplastic anemia, were pooled from the GWAS Catalog (GCST90018794) and the FinnGen study (phenotype code: D3_OTHERAPLASTICANAEMIA).

[0034] All GWAS data included in the analysis underwent genotype-level quality control, with key criteria including: minor allele frequency (MAF) ≥ 0.0001, minor allele count (MAC) ≥ 10, genotype deletion rate ≤ 0.1, and Hardy–Weinberg equilibrium test P ≥ 1 × 10⁻⁶. -15 To ensure the independence of instrumental variables, further linkage disequilibrium (LD) clumping is performed, excluding variables within a 10,000 kb window. 2 SNPs > 0.001.

[0035] This invention employs the TwoSampleMR package in R language for MR analysis. The primary analytical method used is inverse variance weighting (IVW), and based on the number of available instrumental variables, further sensitivity analyses are conducted using MR-Egger, weighted median, and weighted pattern methods. For exposures with multiple instrumental variables, heterogeneity and potential pleiotropy are further assessed to improve the robustness of causal inference results. Genetic instrumental variables, as proxy indicators of natural random assignment, can, to some extent, reduce bias caused by confounding factors and reverse causality.

[0036] Two-segment morphology (SMR) analysis showed a significant association between genetic proxies related to ABCB1 and CYP3A5 expression and the risk of AA (OR ≈ 1.7, p = 0.0181). Combined with previous pharmacokinetic studies, a negative correlation may exist between cyclosporine A plasma concentration and ABCB1 and CYP3A5 expression levels. Therefore, these results suggest that genetic differences related to drug transport and metabolism may affect cyclosporine A exposure levels and further influence AA-related immune regulation processes.

[0037] 2SMR analysis showed a significant association between genetically predicted HbA1c levels and the risk of atrial fibrillation (AA) (OR ≈ 1.5, p = 0.010). Given that metformin treatment is usually associated with decreased HbA1c levels, this result suggests that glucose metabolism-related pathways may be linked to the risk of AA, and also provides an indirect genetic reference for metformin as a candidate therapeutic component.

[0038] As shown in Figure 4, the 2SMR analysis revealed a protective association between genetically predicted hemoglobin levels and the risk of aplastic anemia (AA) (OR ≈ 0.3, p = 0.0073), consistent with the direction of promoting erythroid formation. Telomere length also showed a protective association trend (OR ≈ 0.3, p = 0.050), suggesting that telomere maintenance-related mechanisms may be involved in the regulation of AA risk. In contrast, genetically predicted EPO levels were associated with an increased risk of AA (OR ≈ 1.7, p = 0.021), which may reflect the body's compensatory response to anemia in bone marrow failure rather than the pathogenic effect of EPO itself.

[0039] (4) Conduct a full phenotypic association analysis (PheWAS) based on data from the UK Biobank. This invention employs the PHESANT tool for PheWAS analysis. PHESANT is a large-scale automated full-phenotype scanning tool applicable to UK biobanks, capable of systematically analyzing the association between specified exposure factors and continuous, integer, and categorical variable phenotypes in the UK biobank. Using this method, this invention assesses a broad range of phenotypic features associated with aplastic anemia or its genetic risk, and, in conjunction with the concept of dynamic equilibrium, systematically integrates and interprets candidate treatment directions.

[0040] A polygenic risk score (PRS) was constructed based on genetic proxy indicators associated with cyclosporine A, metformin, and stanozolol, and age-stratified analysis was performed by age 60. The model was adjusted for sex and the top 10 genetic principal components (PC1–PC10) to reduce the influence of residual population stratification.

[0041] like Figure 5 As shown, no significant association was observed between PRS and AA risk in individuals aged 60 years and older (p = 0.979). In contrast, in individuals under 60 years of age, each 1 standard deviation increase in PRS was associated with a decrease in AA risk (OR ≈ 0.93, p = 0.038). These results suggest that the relationship between candidate drug-related genetic proxy indices and AA risk may be age-dependent, with related genetic signals potentially being more readily observed in younger individuals.

[0042] PheWAS results showed that phenotypes associated with autoimmune atrophy (AA) could be summarized into five interrelated functional aspects, including energy metabolism, tissue support, immune regulation, nutritional status, and hematopoietic growth. Referring to the concept of interrelationship and dynamic balance among different functional systems in Traditional Chinese Medicine, these five functional aspects were respectively mapped to energy metabolism, tissue support, immune regulation, nutritional status, and hematopoietic growth. (See...) Figure 3 This correspondence is mainly used to help summarize multi-dimensional phenotypic results and serve as a conceptual framework for constructing multi-target combined treatment plans. Based on this framework, this invention believes that the treatment strategy for RAA should not be limited to immunosuppression, but can further consider covering multiple directions such as metabolic support, hematopoietic promotion, nutritional supplementation, and tissue microenvironment regulation, as shown in Table 5.

[0043] Table 5

[0044] Example 2 This embodiment provides a pharmaceutical composition for refractory aplastic anemia, with each unit dose consisting of the following components: cyclosporine A 75 mg, metformin hydrochloride 250 mg, stanozolol 2 mg, folic acid 10 mg, vitamin B2 20 mg, adenosylcobalamin 0.5 mg, and coenzyme Q10 10 mg. The cyclosporine A, metformin hydrochloride, stanozolol, folic acid, vitamin B2, adenosylcobalamin, and coenzyme Q10 are each formulated separately, and these seven separate formulations are packaged in the same box. The dosage form of the formulation is any pharmaceutically acceptable dosage form.

[0045] Typical Case: Patient: Guan, age: 31, gender: male; diagnosed with refractory chronic aplastic anemia; after 2.5 years of treatment with the above-mentioned drug combination (twice daily), the patient's blood routine returned to normal (hemoglobin 113 (normal is above 120), platelets 55,000 (normal is above 100,000), white blood cells 5,300 (normal is 3,500-10,000)), successfully getting rid of dependence on red blood cell and apheresis platelet transfusions, and his quality of life returned to normal.

[0046] Example 3 This embodiment provides a pharmaceutical composition for refractory aplastic anemia, with each unit dose consisting of the following components: cyclosporine A 50 mg, metformin hydrochloride 250 mg, stanozolol 3 mg, folic acid 15 mg, vitamin B2 15 mg, adenosylcobalamin 0.75 mg, and coenzyme Q10 15 mg. The cyclosporine A, metformin hydrochloride, stanozolol, folic acid, vitamin B2, adenosylcobalamin, and coenzyme Q10 are each formulated separately, and these seven separate formulations are packaged in the same box. The dosage form of the formulation is any pharmaceutically acceptable dosage form.

[0047] Example 4 This embodiment provides a pharmaceutical composition for refractory aplastic anemia, with each unit dose consisting of the following components: cyclosporine A 100 mg, metformin hydrochloride 375 mg, stanozolol 3 mg, folic acid 20 mg, vitamin B2 10 mg, adenosylcobalamin 0.75 mg, and coenzyme Q10 15 mg. The cyclosporine A, metformin hydrochloride, stanozolol, folic acid, vitamin B2, adenosylcobalamin, and coenzyme Q10 are each formulated separately, and these seven separate formulations are packaged in the same box. The dosage form of the formulation is any pharmaceutically acceptable dosage form.

[0048] Typical Case: Patient: Ms. Yuan, age 63, gender: female; diagnosed with refractory chronic aplastic anemia. After diagnosis, due to the poor response to conventional treatment, the treatment plan was adjusted to systemic therapy using the aforementioned drug combination (twice daily). During the two-year systemic treatment period, the patient maintained good compliance. Through long-term follow-up observation and regular blood routine monitoring, the drug combination demonstrated a significant effect on bone marrow hematopoietic function repair. To date, the patient's clinical symptoms of anemia have achieved comprehensive and substantial improvement, with a significant increase in peripheral blood erythrocyte and hemoglobin levels. She has not only eliminated or significantly reduced her dependence on blood transfusion products, but her overall quality of life has also fundamentally improved.

[0049] Example 5 This embodiment provides a pharmaceutical composition for refractory aplastic anemia, with each unit dose consisting of the following components: cyclosporine A 100 mg, metformin hydrochloride 250 mg, stanozolol 5 mg, folic acid 15 mg, vitamin B2 15 mg, adenosylcobalamin 1.0 mg, and coenzyme Q10 20 mg. The cyclosporine A, metformin hydrochloride, stanozolol, folic acid, vitamin B2, adenosylcobalamin, and coenzyme Q10 are each formulated separately, and these seven separate formulations are packaged in the same box. The dosage form of the formulation is any pharmaceutically acceptable dosage form.

[0050] Typical Case: Patient: Ms. Li, age 58, gender: female, diagnosed with refractory severe aplastic anemia. After 55 days of treatment with the above-mentioned drug combination (twice daily), the patient's hematopoietic function significantly recovered. A comparison of blood routine indicators before and after treatment showed that the white blood cell count decreased from 1.9 × 10⁻⁶ to 1.9 × 10⁻⁶. 9 / L increased to 2.9 ×10 9 / L; hemoglobin increased from 76 g / L to 113 g / L; platelets increased from 9 × 10 / L. 9 / L increased to 22×10 9 / L. Clinical monitoring has confirmed that this treatment regimen has a clear therapeutic effect on relapsed and refractory aplastic anemia.

[0051] Example 6 This invention conducted a prospective single-arm clinical intervention study, enrolling 65 patients with refractory aplastic anemia, including 45 patients with refractory chronic aplastic anemia (CAA) and 20 patients with refractory severe aplastic anemia (SAA). The patients' clinical information is shown in Table 6. All patients received the drug composition of Example 3 orally twice daily.

[0052] After treatment with the drug combination, peripheral blood was collected from patients regularly to monitor hemoglobin (HGB), platelets (PLT), absolute neutrophil count (ANC), white blood cells (WBC), lymphocytes (Lym), monocytes (Mon), hematocrit (HCT), red blood cell count (RBC), and three mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC)). Subjects were followed up regularly at 1, 3, 6, and 12 months after treatment, and the treatment effect was evaluated according to the efficacy grading criteria. The efficacy grades were divided into hematologic remission (HR), complete remission (CR), partial remission (PR), and no remission (NR); HR = CR + PR.

[0053] Table 6

[0054] As shown in Table 7, after 12 months of treatment with the drug combination, the median HGB, ANC, and PLT levels in all 65 patients increased compared to baseline. Stratified by disease severity, patients with refractory severe aplastic anemia showed significant improvements in ANC, Hb, PLT, and red blood cell count compared to baseline. In patients with refractory chronic aplastic anemia, hematological response, HGB, PLT, and red blood cell count also showed significant improvements compared to baseline (p < 0.05). During follow-up, one patient died at month 7 of treatment due to pulmonary infection and multiple organ failure. At 12 months of treatment, of the remaining 64 patients, 51 met the criteria for hematological response; the remaining 13 patients, although not meeting the criteria for hematological response, still showed improvement in HGB or PLT compared to baseline. During the study observation period, no new or amplified clones of paroxysmal nocturnal hemoglobinuria (PNH) were observed, nor were any cases of progression to myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) observed.

[0055] Table 7

[0056] like Figure 6 As shown in Table 8, at 1, 3, 6, and 12 months post-treatment, 40 patients (61.5%), 44 patients (67.7%), 46 patients (70.8%), and 51 patients (79.7%) achieved hematologic remission, respectively. Among patients with refractory chronic aplastic anemia, the rates of hematologic remission were 36 (80%), 36 (80%), 37 (82%), and 39 (87%), respectively. Among patients with refractory severe aplastic anemia, the rates of hematologic remission were 4 (20%), 8 (40%), 9 (45%), and 12 (60%), respectively.

[0057] In summary, the hematological response rate observed after treatment with the drug composition gradually increased over time, indicating that the drug composition has value for further evaluation in refractory patients with limited treatment options.

[0058] Table 8

[0059] Example 7 This invention included a clinical study that enrolled 108 patients with refractory aplastic anemia, including 85 patients with refractory chronic aplastic anemia (CAA) and 23 patients with refractory severe aplastic anemia (SAA). All patients received an oral pharmaceutical composition (the pharmaceutical composition in Example 3). Similar to Example 5 above, follow-up data were obtained at 1 month (n=108), 3 months (n=102), 6 months (n=91), and 12 months (n=78). One patient died in the seventh month of treatment due to pulmonary infection and multiple organ failure; this case was statistically classified as NR. Results are as follows... Figure 7 As shown in Table 9, at 1, 3, 6, and 12 months post-treatment, 61 patients (56.5%), 67 patients (65.7%), 67 patients (73.6%), and 61 patients (78.2%) achieved hematologic remission, respectively. Among patients with refractory chronic aplastic anemia, the rates of hematologic remission were 56 (66%), 58 (72%), 57 (81%), and 49 (86%), respectively. Among patients with refractory severe aplastic anemia, the rates of hematologic remission were 5 (22%), 9 (41%), 10 (48%), and 12 (57%), respectively.

[0060] Table 9

[0061] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

Claims

1. A pharmaceutical composition for treating refractory aplastic anemia, characterized in that, Each unit dose of the pharmaceutical composition comprises the following components: cyclosporine A 50-100 mg, metformin hydrochloride 250-375 mg, stanozolol 2-5 mg, and nutritional supplement 30-65 mg.

2. The pharmaceutical composition according to claim 1, characterized in that, The nutritional supplement contains the following components: folic acid 10-20 mg, vitamin B2 10-20 mg, vitamin B12 0.5-1.0 mg and coenzyme Q10 10-20 mg.

3. The pharmaceutical composition according to claim 1, characterized in that, Each unit dose of the pharmaceutical composition consists of the following components: cyclosporine A 50-100 mg, metformin hydrochloride 250-375 mg, stanozolol 2-5 mg, folic acid 10-20 mg, vitamin B2 10-20 mg, vitamin B12 0.5-1.0 mg, and coenzyme Q10 10-20 mg.

4. The pharmaceutical composition according to claim 1, characterized in that, Each unit dose of the pharmaceutical composition consists of the following components: cyclosporine A 50 mg, metformin hydrochloride 250 mg, stanozolol 3 mg, folic acid 15 mg, vitamin B2 15 mg, vitamin B12 0.75 mg and coenzyme Q10 15 mg.

5. The pharmaceutical composition according to any one of claims 1-4, characterized in that, The vitamin B12 is selected from adenosylcobalamin or methylcobalamin.

6. The pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition is prepared by mixing the components to form a formulation, and the dosage form of the formulation is a pharmaceutically acceptable dosage form.

7. The pharmaceutical composition according to claim 6, characterized in that, The dosage form of the preparation is selected from one of tablets, capsules, granules, and suppositories.

8. The pharmaceutical composition according to claim 1, characterized in that, Each component of the pharmaceutical composition is formulated separately, and each separate formulation is packaged in the same box; the dosage form of the formulation is a pharmaceutically acceptable dosage form.

9. Use of the pharmaceutical composition according to any one of claims 1-8 in the preparation of a drug for treating refractory aplastic anemia.

10. The application according to claim 9, characterized in that, The refractory aplastic anemia referred to is either refractory severe aplastic anemia or refractory chronic aplastic anemia.

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