Pharmaceutical composition based on mesenchymal stem cells and application thereof

Abstract

The invention relates to the technical field of medicine preparation, in particular to a medicine composition based on mesenchymal stem cells and application of the medicine composition. And a pharmaceutically acceptable carrier. The pharmaceutical composition based on the mesenchymal stem cells has the following beneficial effects that the inflammatory response and fibrosis process of the focus of alveolar coccosis can be remarkably inhibited through the immune regulation function of the mesenchymal stem cells, and meanwhile, the cytokines secreted by the mesenchymal stem cells can directly inhibit the vitality of the protoscolex and the cell proliferation of the hair growth layer; the synergistic treatment effect of parasite killing, immunoregulation and tissue repair is realized; when the traditional Chinese medicine composition is combined with the existing anti-parasitic medicine, the dosage can be reduced, the hepatotoxicity can be reduced, the traditional Chinese medicine composition has remarkable advantages especially for patients with advanced diffuse lesions and postoperative recurrence, and a novel treatment strategy with high efficiency and low toxicity is provided for alveolar coccosis.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to a mesenchymal stem cell-based pharmaceutical composition and application thereof. Background Art

[0002] Alveolar Echinococcosis (AE) is caused by Echinococcus multilocularis ( Echinococcus multilocularis ) larvae infection, a serious zoonotic parasitic disease caused by AE, which mainly invades the liver and grows invasively, with biological behavior similar to that of malignant tumors. The 10-year mortality rate of untreated AE patients can exceed 90%, and even after treatment, advanced patients still face high recurrence rates and risks of liver failure. Currently, the standard treatment for AE includes surgical resection of the lesions combined with long-term chemotherapy with benzimidazole drugs such as albendazole, but this type of drug has problems such as hepatotoxicity, drug resistance, and limited efficacy in late-stage diffuse lesions. Therefore, the development of new, efficient, and low-toxic treatment strategies is of great clinical significance.

[0003] Prior art treatments for AE primarily focus on the development of antiparasitic drugs (e.g., CN110934876A discloses an anti-echinococcosis drug composition) or vaccine research (e.g., WO2019086327A1 relates to an Echinococcus multilocularis vaccine). However, these approaches struggle to effectively address the immune escape and microenvironmental regulation issues associated with late-stage AE. Furthermore, while MSCs have been used in certain infectious diseases (e.g., CN112007136A describes their use in sepsis), strategies for their combined use with antiparasitic drugs and mechanisms of direct inhibition of the germinal layer cells and protoscolices of alveolar cysts remain largely unresolved. Summary of the Invention

[0004] In response to the problems existing in the prior art, the present invention develops a novel MSCs pharmaceutical composition based on the immunomodulatory and anti-fibrotic properties of MSCs for AE, which provides a breakthrough solution for the treatment of AE through the synergistic effect of multiple mechanisms of inhibiting parasite activity, regulating host immune response and promoting tissue repair.

[0005] The present invention provides a mesenchymal stem cell-based pharmaceutical composition comprising: a therapeutically effective amount of mesenchymal stem cells; Pharmaceutically acceptable carrier.

[0006] Furthermore, mesenchymal stem cells are derived from umbilical cord, bone marrow or adipose tissue.

[0007] Furthermore, the dosage of mesenchymal stem cells is 1×10^6 to 5×10^6 cells / kg body weight.

[0008] Furthermore, mesenchymal stem cells were expanded and cultured in vitro and retained the characteristics of positive expression of CD73, CD90 and CD105 and negative expression of CD34 and CD45.

[0009] Furthermore, it also contains one or more antiparasitic drugs, preferably albendazole, mebendazole or praziquantel.

[0010] Furthermore, the antiparasitic drug is combined with the mesenchymal stem cells in a synergistically effective amount.

[0011] Furthermore, the pharmaceutically acceptable carrier comprises physiological saline, human serum albumin or glucose solution.

[0012] Furthermore, the composition is formulated as an intravenous injection preparation.

[0013] Furthermore, mesenchymal stem cells are genetically modified to enhance their antiparasitic or immunomodulatory functions.

[0014] The present invention also provides an application of a mesenchymal stem cell-based pharmaceutical composition, wherein the pharmaceutical composition is applied to inhibit the growth of key parts of alveolar larvae.

[0015] In summary, the present invention has the following beneficial effects: The mesenchymal stem cell-based pharmaceutical composition of the present invention has the following beneficial effects: the immunomodulatory function of mesenchymal stem cells can significantly inhibit the inflammatory response and fibrosis process of alveolar coccidioidomycosis lesions. At the same time, the cytokines secreted by the mesenchymal stem cells can directly inhibit the activity of the protoscolices and the proliferation of germinal layer cells, achieving a synergistic therapeutic effect of "parasite killing-immunomodulation-tissue repair"; when used in combination with existing antiparasitic drugs, the drug dosage can be reduced and liver toxicity can be alleviated, especially for patients with late-stage diffuse lesions and postoperative recurrence, providing a new, highly effective and low-toxic treatment strategy for alveolar coccidioidomycosis. DETAILED DESCRIPTION

[0016] To further illustrate the technical means and efficacy of the present invention to achieve the intended purpose, the application of a mesenchymal stem cell-based pharmaceutical composition according to the present invention, its specific implementation methods, characteristics, and efficacy are described in detail below.

[0017] Mesenchymal stem cells (MSCs) are a type of adult stem cell with the potential for self-renewal and multidirectional differentiation. They are widely found in tissues such as bone marrow, adipose tissue, and the umbilical cord. Studies have shown that MSCs not only possess tissue repair capabilities but also regulate the immune microenvironment by secreting multiple cytokines (such as TGF-β and IL-10), inhibiting excessive inflammatory responses and demonstrating potential therapeutic value in areas such as anti-fibrosis and anti-infection. In recent years, the immunomodulatory effects of MSCs in parasitic infections (such as schistosomiasis and malaria) have been gradually revealed, but their application in the treatment of AE has not been systematically studied.

[0018] Current treatments for AE primarily focus on antiparasitic drug development or vaccine research. However, these approaches struggle to effectively address the immune escape and microenvironmental regulation issues associated with late-stage AE. Furthermore, while MSCs have been used in certain infectious diseases, their combined use with antiparasitic drugs and their direct inhibitory mechanisms on the germinal layer cells and protoscolices of alveolar cysts remain largely unresolved.

[0019] Therefore, based on the immunomodulatory and anti-fibrotic properties of MSCs, a new MSCs drug composition for AE is developed, which is expected to provide a breakthrough solution for the treatment of AE through the synergistic effect of multiple mechanisms such as inhibiting parasite activity, regulating host immune response and promoting tissue repair.

[0020] To address the above problems, this embodiment provides a mesenchymal stem cell-based pharmaceutical composition comprising: a therapeutically effective amount of mesenchymal stem cells; Pharmaceutically acceptable carrier.

[0021] It can be understood that the therapeutic principle of the mesenchymal stem cell-based pharmaceutical composition provided in this specific embodiment is that mesenchymal stem cells target the lesion site through the homing effect. On the one hand, they suppress excessive inflammatory responses and improve the immune microenvironment by secreting immunoregulatory factors such as TGF-β and IL-10. On the other hand, they directly inhibit the activity of the protoscolex of the alveolar larvae and the proliferation of the germinal layer cells, while promoting the repair and regeneration of damaged liver tissue. Its advantage is that it breaks through the limitation of traditional antiparasitic drugs that can only kill parasites, and achieves the triple synergistic therapeutic effect of "immunomodulation-parasite inhibition-tissue repair", which has a significant therapeutic effect on late-stage diffuse lesions, and avoids the liver toxicity problem of traditional chemotherapy drugs.

[0022] In some preferred embodiments, the mesenchymal stem cells are derived from umbilical cord, bone marrow, or adipose tissue.

[0023] In some preferred embodiments, the dosage of mesenchymal stem cells is 1×10^6 to 5×10^6 cells / kg body weight.

[0024] It is understood that within the preferred dosage range (1×10^6 to 5×10^6 cells / kg body weight), 1×10^6 cells / kg ensures sufficient therapeutic cells reach the lesion site to exert immunomodulatory functions through paracrine effects, while the upper limit of 5×10^6 cells / kg avoids the risk of excessive cell aggregation. This dosage range has been validated in animal studies to ensure effective enrichment of mesenchymal stem cells in liver lesions, significantly improve liver function indicators, and inhibit lesion growth.

[0025] In some preferred embodiments, mesenchymal stem cells are expanded and cultured in vitro and retain the characteristics of positive expression of CD73, CD90 and CD105 and negative expression of CD34 and CD45.

[0026] Understandably, the expression of these specific surface markers ensures the purity and functional integrity of the stem cells (CD73 / CD90 / CD105 are internationally recognized markers for identifying MSCs), while the negativity of CD34 / CD45 eliminates contamination by hematopoietic cells. This standardized culture of stem cells maintains stable multidirectional differentiation potential and tissue repair capacity, while also increasing the levels of immunomodulatory factors such as TGF-β and IL-10 in their exosomes by 2-3 times compared to unpurified cells.

[0027] In some preferred embodiments, the drug further comprises one or more antiparasitic drugs, preferably albendazole, mebendazole or praziquantel.

[0028] It's understandable that mesenchymal stem cells, by improving the immune microenvironment in lesions, not only enhance the penetration of antiparasitic drugs but also repair drug-induced liver damage. Furthermore, after antiparasitic drugs directly kill protoscolex, mesenchymal stem cells accelerate the removal of parasite debris and inhibit secondary fibrosis. This combination significantly increases the apoptosis rate of protoscolex, accelerates lesion reduction, and allows for lower drug dosage without compromising efficacy. More importantly, it effectively combats drug-resistant strains, providing a breakthrough solution for the clinical treatment of advanced and recurrent AEs.

[0029] In some preferred embodiments, the antiparasitic drug is combined with mesenchymal stem cells in a synergistically effective amount.

[0030] More preferably, when albendazole 10 mg / kg is combined with MSCs 3×10^6 cells / kg, the drug can be enhanced in the lesion through MSCs, and the IL-10 secreted by MSCs can reduce the drug's hepatotoxicity; when praziquantel 25 mg / kg is combined with MSCs 2×10^6 cells / kg, MSCs promote drug-induced calcium overload in the protoscolecus and improve blood supply around the lesion through VEGF secretion; when mebendazole 15 mg / kg is combined with MSCs 4×10^6 cells / kg, MSCs upregulate the expression of the drug target β-tubulin, thereby increasing the inhibition rate of drug-resistant strains.

[0031] In some preferred embodiments, the pharmaceutically acceptable carrier comprises physiological saline, human serum albumin or glucose solution.

[0032] In some preferred embodiments, the composition is formulated as an intravenous injection.

[0033] In some preferred embodiments, the mesenchymal stem cells are genetically modified to enhance their antiparasitic or immunomodulatory functions.

[0034] This specific embodiment also provides an application of a mesenchymal stem cell-based pharmaceutical composition, wherein the pharmaceutical composition is used to inhibit the growth of key parts of alveolar cysts.

[0035] It is understood that the pharmaceutical composition of the present invention can specifically inhibit the growth of multiple key structures of the alveolar cyst: in the germinal layer cells, it inhibits their proliferation activity by downregulating the EGF / EGFR signaling pathway; in the protoscoleces, it promotes apoptosis by inducing calcium ion influx and mitochondrial membrane potential collapse; in the vesicle structure, it inhibits its invasive growth by regulating the secretion of MMP-2 / 9; and in the lesion microenvironment, it reverses the immunosuppressive state by regulating the PD-1 / PD-L1 pathway. This multi-target synergistic action enables the drug to simultaneously block the three key pathological processes of parasite proliferation, differentiation, and spread.

[0036] The present invention will be further described below with reference to specific embodiments.

[0037] Example 1: Pharmaceutical composition based on umbilical cord mesenchymal stem cells Components of the drug composition: Active ingredient: umbilical cord-derived mesenchymal stem cells (UC-MSCs): 3×10^6 cells / kg, in line with international standards (CD73+ / CD90+ / CD105+>95%, CD34- / CD45-<2%); after three generations of expansion culture, the viability is >98%; albendazole: 10 mg / kg; vehicle: physiological saline solution containing 5% human serum albumin.

[0038] Preparation method: S1. Preparation of UC-MSCs: Umbilical cords from healthy newborns were aseptically collected, and primary cells were isolated by collagenase digestion. Cells were expanded to passage 3 in α-MEM medium (containing 10% FBS). Surface markers (CD73: 98.2%, CD90: 97.5%, CD105: 96.8%) were detected by flow cytometry. Cells were digested with 0.25% trypsin and washed twice with PBS before preparation.

[0039] S2. Final preparation: Resuspend UC-MSCs in the carrier solution to a cell density of 2×10^6 cells / mL; grind albendazole and add it to the carrier, then ultrasonically disperse it until completely dissolved; sterile filter it through a 0.22μm filter membrane; aliquot into 10mL sterile vials and store at 4°C (shelf life 8h).

[0040] Example 2: Pharmaceutical composition based on adipose-derived mesenchymal stem cells Components of the drug composition: Active ingredient: adipose-derived mesenchymal stem cells (AD-MSCs): 4×10^6 cells / kg, meeting ISCT standards (CD73+ / CD90+ / CD105+>96%, CD31- / CD45- / HLA-DR-<1.5%); after four generations of culture under hypoxic conditions (5% O2), the viability is >97%; pre-treatment with IFN-γ (50ng / mL, 24h) enhances the immune regulatory function; praziquantel: 25mg / kg; vehicle: glucose (5%) solution containing 2.5% hydroxyethyl starch.

[0041] Preparation method: S1. Preparation of AD-MSCs: Adipose tissue was obtained by liposuction and digested with type I collagenase to isolate the vascular stromal fraction. The cells were hypoxically expanded to passage 4 in DMEM / F12 medium (containing 5% platelet lysate). Stemness markers (CD73: 97.8%, CD90: 98.1%, CD105: 96.3%) were verified by flow cytometry. IDO activity was measured after IFN-γ activation (>15 μM / 10^6 cells / 24 h).

[0042] S2. Final preparation: Activated AD-MSCs were washed three times with serum-free medium and resuspended in pre-chilled carrier solution to a final concentration of 3 × 10^6 cells / mL. Praziquantel nanocrystals were prepared (average particle size 220 nm, PDI < 0.2). After aseptic mixing, they were dispensed into prefilled syringes and stored at 2–8°C (stability 12 h).

[0043] Example 3: Pharmaceutical composition based on bone marrow mesenchymal stem cells Pharmaceutical composition components: Active ingredient: bone marrow-derived mesenchymal stem cells (BM-MSCs): 5×10 6 cells / kg, meeting ISCT criteria (CD73 + / CD90 + / CD105 + >97%, CD34 - / CD45 - / HLA-DR - <1%), after five generations of normoxia culture, the vitality was >99%; Pretreatment with TGF-β1 (10 ng / mL, 48 h) enhanced anti-fibrotic function and overexpressed the HGF gene (copy number >15 / cell); mebendazole: 15 mg / kg; β-cyclodextrin inclusion technology was used (encapsulation efficiency >92%); carrier: lactated Ringer's solution containing 10% autologous serum.

[0044] Preparation method: S1. Preparation of BM-MSCs: Bone marrow fluid was obtained by iliac puncture, and mononuclear cells were isolated by density gradient centrifugation. The cells were expanded to passage 5 in low-glucose DMEM medium (containing 10% autologous serum and 1 ng / mL bFGF). Flow cytometry confirmed the expression of positive markers (CD73: 98.5%, CD90: 99.2%, CD105: 97.8%) and negative markers (CD34: 0.8%, CD45: 0.5%). Lentivirus was transfected with the HGF gene (MOI = 20), and PCR confirmed the transfection efficiency was >80%. Collagenase secretion was measured after TGF-β1 stimulation (>200 ng / mL). 6 cells / 24h).

[0045] S2. Final preparation: Gene-modified BM-MSCs were washed twice with heparinized PBS and resuspended in carrier solution preheated at 37°C to a final concentration of 4×10 6 cells / mL; preparation of mebendazole-β-cyclodextrin inclusion complex (drug loading 18.7%); aseptic mixing and aliquoting through a cell infusion device (40μm filter membrane); storage at room temperature (shelf life 6h).

[0046] Comparative Example 1: Traditional pharmaceutical composition Pharmaceutical composition components: active ingredient: albendazole (20 mg / kg); carrier: 0.5% sodium carboxymethylcellulose solution.

[0047] Preparation method: S1, albendazole API is ground to a particle size of <50 μm; mixed with carrier solution and then homogenized under high pressure; S2. Divide into capsules (each capsule contains 200 mg).

[0048] Comparative Example 2: Unmodified stem cell pharmaceutical composition Pharmaceutical composition: Active ingredient: Common BM-MSCs (5×10 6 cells / kg; not genetically modified, cultured in conventional normoxia; vehicle: normal saline.

[0049] Preparation method: S1. Conventional bone marrow mesenchymal stem cell isolation and culture (P3 generation), flow cytometry identification meets basic standards (CD73+ / CD90+>90%).

[0050] S2. Direct resuspension and infusion.

[0051] Comparative Example 3: Pure Nanomedicine Group Components of the pharmaceutical composition: active ingredient: mebendazole nanocrystals (15 mg / kg), average particle size 200 nm, PDI < 0.25; carrier: 5% glucose solution.

[0052] Preparation method: S1, preparation of nanosuspension by high pressure homogenization method; S2. Terminally sterilize by γ-ray and store lyophilized (reconstitute before use).

[0053] Performance Testing 1. Protoscolecus Killing Experiment Test method: Calcein AM / PI double staining was used to determine the survival rate of the protoscolex; laser confocal microscopy was used to observe the structural integrity of the integument; and the ATP content (an indicator of the metabolic activity of the insect body) in the culture supernatant was detected. The test results are shown in the table below.

[0054]

[0055] 2. Sheep AE model test Animal Model: Sixty adult sheep (half male and half female) were intrahepatically inoculated with alveolar cyst tissue (0.5 g / sheep) and randomly divided into six groups (n=10). Treatment lasted for 12 weeks. Evaluation criteria included ultrasound measurement of lesion volume changes, histopathological scores (0-10), and serum EM18 antibody levels (ELISA). The test results are shown in the table below.

[0056]

[0057] Based on the above results, comprehensive analysis of in vitro and in vivo pharmacodynamic data demonstrated that all three treatment regimens significantly outperformed conventional treatment (p < 0.01). In the in vitro protoscolectomy assay, the mortality rates of groups 1-3 in Examples 1-3 reached 91.2%, 93.5%, and 95.8%, respectively, significantly higher than those in groups 1-3 (42.6%-65.3%). Group 3 demonstrated the strongest inhibition of parasite metabolism (ATP reduction rate of 94.3%). This superiority was further validated in an in vivo sheep model, with group 3 demonstrating a 71.4±6.8% reduction in lesion volume, a 79.2±5.7% improvement in pathological scores, and a 6.5±0.4 log2 decrease in EM18 antibody levels, all significantly superior to those in the other groups. Particularly noteworthy is that the therapeutic efficacy of Example 3 was more than three times greater than that of traditional chemotherapy (Comparative Example 1). Furthermore, the targeted liver enrichment rate of the gene-modified stem cells (Example 3) (58.3%) was nearly four times higher than that of the unmodified stem cells (Comparative Example 2), demonstrating the superiority of the synergistic treatment strategy of "immunomodulation-precision insecticide-tissue repair." All data were statistically significant (p < 0.05) using analysis of variance (ANOVA) and Tukey's post hoc test.

[0058] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been presented as a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with the present profession can make some changes or modifications to equivalent embodiments of equivalent changes using the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A pharmaceutical composition based on mesenchymal stem cells, characterized in that: Include: a therapeutically effective amount of mesenchymal stem cells; Pharmaceutically acceptable carrier.

2. The pharmaceutical composition according to claim 1, characterized in that The mesenchymal stem cells are derived from umbilical cord, bone marrow or adipose tissue.

3. The pharmaceutical composition according to claim 1, characterized in that The dosage of the mesenchymal stem cells is 1×10^6 to 5×10^6 cells / kg body weight.

4. The pharmaceutical composition according to claim 1, characterized in that The mesenchymal stem cells are expanded and cultured in vitro and retain the characteristics of positive expression of CD73, CD90 and CD105 and negative expression of CD34 and CD45.

5. The pharmaceutical composition according to claim 1, characterized in that It also contains one or more antiparasitic drugs, preferably albendazole, mebendazole or praziquantel.

6. The pharmaceutical composition according to claim 5, characterized in that The antiparasitic drug is combined with mesenchymal stem cells in a synergistically effective amount.

7. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutically acceptable carrier comprises physiological saline, human serum albumin or glucose solution.

8. The pharmaceutical composition according to claim 1, characterized in that The composition is formulated for intravenous injection.

9. The pharmaceutical composition according to claim 1, characterized in that The mesenchymal stem cells are genetically modified to enhance their anti-parasitic or immunomodulatory functions.

10. An application of a pharmaceutical composition based on mesenchymal stem cells, characterized in that: The pharmaceutical composition according to any one of claims 1 to 9 is used to inhibit the growth of key parts of alveolar larvae.

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