CN115414387A - Medical kit for treating pulmonary fibrosis - Google Patents
Medical kit for treating pulmonary fibrosis Download PDFInfo
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/007—Pulmonary tract; Aromatherapy
- A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
- A61K9/0078—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy for inhalation via a nebulizer such as a jet nebulizer, ultrasonic nebulizer, e.g. in the form of aqueous drug solutions or dispersions
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Abstract
The invention provides a medical kit for treating pulmonary fibrosis, and belongs to the field of medicine. The medical kit comprises an aerosol inhalation device and a drug solution; the concentration of microvesicles in the drug solution is 1X 10 7 ‑1×10 10 particle/ml; the temperature of the drug solution was 18-37 deg.C. The invention controls the specific concentration and the specific temperature of the drug solution in the medical package at the same timeSpecific administration dosage is controlled by controlling atomization speed and duration of atomization inhalation of a treatment object, and the medical kit disclosed by the invention is found to be capable of obviously improving pulmonary inflammation infiltration and fibrosis of a pulmonary interstitial fibrosis model mouse and relieving excessive deposition of pulmonary interstitial collagen induced by bleomycin. The medical kit has the characteristics of quick response, small dosage, safety, effectiveness and good patient compliance. The medical kit has wide application prospect in the treatment of pulmonary fibrosis.
Description
Technical Field
The invention belongs to the field of medicine, and particularly relates to a medical kit for treating pulmonary fibrosis.
Background
Pulmonary fibrosis is a group of diseases characterized by progressive destruction of the alveolar structure, massive deposition of extracellular matrix, and scarring of the lungs. It is clinically manifested as impaired lung function and impaired alveolar gas exchange, leading to hypoxemia, dyspnea, and even respiratory failure and death. The incidence rate and death rate of pulmonary fibrosis are high, and the pulmonary fibrosis is a public problem threatening the health of human beings. At present, the clinical therapeutic drugs for pulmonary fibrosis mainly comprise glucocorticoid, immunosuppressant and anti-fibrosis drugs (including pirfenidone and nintedanib), but the glucocorticoid can cause diabetes, hypertension, infection, osteoporosis and the like after long-term use, the excessive use of the immunosuppressant can cause infection, tumor and the like, and the anti-fibrosis drugs not only have high cost, but also have poor therapeutic effect on part of patients, thereby bringing heavy burden to families, society and economy.
Mesenchymal Stem Cells (MSCs) are non-hematopoietic multipotent stem cells derived from mesoderm and widely present in connective tissues and organ mesenchyme, have the ability to self-renew and differentiate into mesoderm tissues, and have anti-inflammatory, immunosuppressive, anti-apoptotic, anti-fibrotic, pro-angiogenic functions. Meanwhile, preclinical research and clinical trial exploratory research on the treatment of pulmonary fibrosis by MSCs also show a satisfactory effect. However, at present, MSCs still have the defects of easy death after transplantation, low transplantation rate, easy blockage, easy tumor formation for a long time and the like, and are feared by many doctors and patients, thereby limiting the clinical application of MSCs. Recent studies have found that Extracellular Vesicles (EVs) can avoid these drawbacks very well.
The literature (research progress of mesenchymal stem cell-derived extracellular vesicles in pulmonary fibrosis treatment, journal of medical research, 12 months at 2021, volume 50, stage 12) discloses that mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) are a natural and efficient transport carrier, and have angiogenesis promoting, anti-apoptosis, anti-fibrosis, immunoregulation and certain proliferation and regeneration functions. And as a membrane structure, the MSC-EVs has the advantages of low immunogenicity, long half-life, good in-vivo stability, high transfer efficiency and the like, and is a novel non-cell biological treatment method which is safer and more effective than stem cell treatment. However, most of the existing methods for treating pulmonary fibrosis by using MSC-EVs are intravenous injection administration, and on one hand, the intravenous injection administration has poor targeting on a focus part and is easy to cause systemic adverse reaction; on the other hand, the drug has slow effect after intravenous injection administration; more importantly, for some patients who do not engage intravenous injections (e.g., children), patient compliance is poor.
Therefore, the method for treating pulmonary fibrosis, which has better patient compliance, quicker response time and less systemic adverse reaction, has important clinical significance.
Disclosure of Invention
The invention aims to provide a medical kit for treating pulmonary fibrosis and application thereof.
The invention provides a medical kit for treating pulmonary fibrosis, which comprises an aerosol inhalation device and a medicinal solution; the concentration of microvesicles in the drug solution was 1X 10 7 -1×10 10 particle/ml; the temperature of the drug solution was 18-37 ℃.
Further, the microvesicles are stem cell-derived microvesicles, preferably the stem cells are mesenchymal stem cells, more preferably umbilical cord mesenchymal stem cells.
Further, the solvent in the drug solution is physiological saline or buffer, and the temperature of the drug solution is room temperature.
In the present invention, room temperature means 20. + -. 5 ℃.
Further, the concentration of microvesicles in the drug solution is 2 × 10 9 particle/ml。
Further, in the medical kit, the atomization speed of the drug solution is set to be 0.05-0.5ml/min, and the duration of each atomization is set to be 8-30min;
preferably, in the medical kit, the atomization speed of the drug solution is set to be 0.1ml/min, and the duration of each atomization is set to be 10min.
Further, the amount of microvesicles in the drug solution is 0.5 × 10 10 -2×10 11 particle, preferably 1.1X 10 10 -1.0×10 11 particle。
Further, the atomization inhalation device comprises a medicine storage bottle for containing medicine solution, an atomization generator, a face mask, a conduit and a connecting piece; the medicine storage bottle is connected with the atomization generator through a conduit, and the medicine storage bottle is connected with the face mask through a connecting piece.
The invention also provides application of the medical kit in preparation of a medical-mechanical combination product for treating pulmonary fibrosis.
Further, the pulmonary fibrosis is pulmonary interstitial fibrosis.
Further, the pharmaceutical-mechanical combination product is capable of improving inflammatory infiltration of the lung, and/or reducing interstitial collagen deposition in the lung.
Compared with the prior art, the medical kit for treating pulmonary fibrosis provided by the invention has the following beneficial effects:
1. the medicine administration mode of the medical kit provided by the invention is local medicine administration, the atomized medicine can directly reach the focus, the effect is very direct, and the side effect is less compared with that of the whole body. Compared with oral drug therapy or intravenous drug therapy, the medical kit for treating pulmonary fibrosis has the characteristics of quick response, small dosage, safety, effectiveness and good patient compliance.
2. According to the invention, the specific concentration and the specific temperature of the microvesicle solution in the medical package are controlled, and the specific administration dosage is controlled by controlling the atomization speed and the atomization inhalation duration of a treatment object, so that the medical package disclosed by the invention can obviously improve the pulmonary inflammation infiltration and fibrosis of a pulmonary interstitial fibrosis model mouse, reduce the infiltration of proinflammatory mononuclear cells and macrophages in lung and peripheral blood, and reduce the excessive deposition of the pulmonary interstitial collagen induced by bleomycin. The medical kit provided by the invention has a wide application prospect in the treatment of pulmonary fibrosis.
It will be apparent that various other modifications, substitutions and alterations can be made in the present invention without departing from the basic technical concept of the invention as described above, according to the common technical knowledge and common practice in the field.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
Figure 1 is a photograph of an aerosol inhalation device of the present invention.
FIG. 2 is a schematic flow chart of the experiment of example 2.
FIG. 3 is the results of in vivo tracking of MSC-MVs.
FIG. 4 is a general view of the lungs of each group of mice.
FIG. 5 is a pathological analysis of inflammatory infiltration and fibrosis in the lungs of each group of mice. * Denotes p <0.001; scale bar =100nm.
Detailed Description
The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.
Example 1: the invention relates to a medical kit for treating pulmonary interstitial fibrosis
The medical kit for treating pulmonary interstitial fibrosis of the invention comprises liquid medicine and an aerosol inhalation device, and the picture of the aerosol inhalation device is shown in figure 1. The atomization inhalation device comprises an air compression type atomization generator, a medicine storage bottle, an atomization catheter and a mask. The liquid medicine is microvesicle (MSC-MVs) solution from human umbilical cord mesenchymal stem cells, and is filled in a medicine storage bottle.
The air compression type atomization generator atomizes the liquid medicine into tiny fogdrops with the grain diameter less than 5 mu m through compressed air. The atomization conduit is a transparent hose and is connected with an atomization generator and a medicine storage bottle. The medicine storage bottle is connected with the face mask through a porous structure, the bottle body is transparent, and the internal liquid medicine and the atomization condition can be observed.
The solvent of the MSC-MVs solution is PBS buffer solution, and the concentration of the MSC-MVs in the MSC-MVs solution is 2 multiplied by 10 9 particle/ml, temperature of MSC-MVs solution was room temperature.
The preparation method of the MSC-MVs comprises the following steps: culturing human umbilical cord Mesenchymal Stem Cells (MSCs) in DMEM medium containing 10% fetal calf serum to passage 5, removing cell supernatant when MSCs proliferate to more than 90% and fuse, washing cells with Phosphate Buffered Saline (PBS) for 3 times, adding serum-free DMEM medium, and culturing at 37 deg.C for 5% CO 2 Culturing in an incubator. After starvation for 48 hours, cell supernatants were collected and centrifuged at 400G for 10 minutes at 4 ℃; the mixture was centrifuged again at 2000G for 20 minutes at 4 ℃ to remove the precipitate, and the supernatant was collected. The supernatant was then centrifuged at 50000G at 4 ℃ for 2.5 hours. Resuspend with PBS to obtain MSC-MVs.
Example 2: in vivo experiments using the kit of the invention for the treatment of interstitial pulmonary fibrosis
1. Experimental method
Experimental animals: male C57BL/6 mice of 6-8 weeks of age, body weight 21-24g, average 20g.
(1) In vivo tracing experiment of atomizing inhalation MSC-MVs
In order to determine the relieving effect of MSC-MVs on pulmonary fibrosis, the invention firstly researches whether the atomizing inhalation type MSC-MVs can reach the lung of a target organ, namely whether the MSC-MVs can be concentrated in the lung tissue of a bleomycin-induced pulmonary fibrosis model mouse, so that the invention carries out an in vivo tracing experiment of the MSC-MVs mouse.
The interstitial pulmonary fibrosis model is constructed by adopting a method of injecting Bleomycin (BLM) into the trachea of a mouse. MSC-MVs were stained with PKH26 fluorochrome and 2 BLM model mice and 1 normal mouse were separately aerosolized into PKH 26-labeled MSC-MVs in the following manner: the medical kit is used for providing PKH26 marked MSC-MVs in an atomizing inhalation mode, the atomizing speed is controlled to be 0.1ml/min in the atomizing process, and meanwhile, the mice are controlled to continuously inhale aerosol for 10 minutes; the BLM model group was imbibed with the same volume of solvent Phosphate Buffered Saline (PBS). After 2 hours, lung tissue of 3 mice was completely removed and fluorescence expression of PKH26 was detected using an animal in vivo imager.
(2) Improving effect on pulmonary interstitial fibrosis
Experimental animals were divided into 3 groups: blank Control group (Control group), bleomycin induced pulmonary fibrosis model group (BLM group), and treatment group (BLM + MVs group). The interstitial pulmonary fibrosis model is constructed by adopting a method of injecting bleomycin into a mouse trachea. After the model is constructed, MSC-MVs treatment is given to the treatment groups on the 1 st day (D1) and the 4 th day (D4) by using the medical kit of the embodiment 1 of the invention in an atomizing inhalation mode, wherein the atomizing speed is controlled to be 0.1ml/min in the atomizing process, and meanwhile, the mice are controlled to continuously inhale aerial fog for 10 minutes in each treatment; the BLM model group was imbibed with the same volume of solvent Phosphate Buffered Saline (PBS).
The dose at which mice continue to inhale MSC-MVs per treatment was calculated as: (0.1 ml/min X10 min X2X 10 9 particle/ml)/0.02kg＝1.0×10 11 particle/kg。
On day 21 (D21), mouse blood and lung specimens were collected for testing (fig. 2). Lung tissue was stained for hematoxylin-eosin (HE) and Masson staining at experimental end-points (D21) and scored.
2. Results of the experiment
(1) In vivo tracing experiment of aerosol inhalation MSC-MVs
It can be seen that MSC-MVs can rapidly reach lung tissue after aerosol inhalation; also, MSC-MVs were more aggregated in lung tissue of BLM model mice compared to normal mouse lung tissue (fig. 3).
The results show that the medical bag disclosed by the invention is quick in treatment effect, atomized medicine can directly reach the focus, the effect is very direct, and the side effect of the whole body is less.
(2) Aerosol inhalation type MSC-MVs improve pulmonary inflammation infiltration and fibrosis of bleomycin-induced pulmonary fibrosis mice
As shown in fig. 4, gross visual inspection of the lungs showed that aerosolized inhalation of MSC-MVs significantly improved lung pathology. As shown in fig. 5A and B, in the Control group, the interval of lung tissues was increased, and inflammatory cell infiltration and fibrosis were evident in the BLM group, compared to the BLM group; after the treatment of atomizing and inhaling MSC-MVs, the lung tissue interval, fibrosis and inflammatory cell infiltration are improved obviously, and the pathological score of the alveolar inflammatory cells is improved obviously (p is less than 0.001). As shown in fig. 5C, D, masson staining also suggested a significant increase in collagen deposition in lung tissue compared to Control in BLM group; after the MSC-MVs are treated by atomization inhalation, the deposition of interstitial collagen of the lung is obviously reduced, and the fibrosis score (p is less than 0.001) is also obviously improved.
The results show that the treatment by using the medical kit can obviously improve the lung inflammation infiltration and the collagen deposition of the mouse with the pulmonary interstitial fibrosis.
In addition, according to pharmacologic experimental methodology compiled by Xutaiyun, the equivalent dose of mice is 9.1 times that of human, and it can be calculated that when the therapeutic package of the present invention is used for treating patients with pulmonary interstitial fibrosis, the dose of continuous MSC-MVs inhalation of patients per treatment is 1.0 × 10 11 particle/kg÷9.1＝1.1×10 10 particle/kg。
In summary, the present invention provides a medical kit for the treatment of pulmonary fibrosis. According to the invention, the specific concentration and the specific temperature of the drug solution in the medical kit are controlled, and the specific administration dosage is controlled by controlling the atomization speed and the atomization inhalation duration of a treatment object, so that the medical kit disclosed by the invention can obviously improve the pulmonary inflammation infiltration and fibrosis of a pulmonary interstitial fibrosis model mouse, and reduce the excessive deposition of the pulmonary interstitial collagen induced by bleomycin. The medical kit has the characteristics of quick response, small dosage, safety, effectiveness and good patient compliance. The medical kit has wide application prospect in the treatment of pulmonary fibrosis.
Claims (10)
1. A kit for treating pulmonary fibrosis, comprising: it comprises an aerosol inhalation device and a drug solution(ii) a The concentration of microvesicles in the drug solution is 1X 10 7 -1×10 10 particle/ml; the temperature of the drug solution was 18-37 ℃.
2. The medical kit of claim 1, wherein: the microvesicle is derived from stem cells, and the stem cells are preferably mesenchymal stem cells, and more preferably umbilical cord mesenchymal stem cells.
3. The medical kit of claim 1, wherein: the solvent in the medicine solution is normal saline or buffer solution, and the temperature of the medicine solution is room temperature.
4. The medical kit of claim 1, wherein: the concentration of the microvesicles in the drug solution is 2X 10 9 particle/ml。
5. The medical kit of any one of claims 1-4, wherein: in the medical kit, the atomization speed of the medicinal solution is set to be 0.05-0.5ml/min, and the continuous time of each atomization is set to be 8-30min;
preferably, in the medical kit, the atomization speed of the drug solution is set to be 0.1ml/min, and the duration of each atomization is set to be 10min.
6. The medical kit of any one of claims 1-5, wherein: the amount of microvesicles in the drug solution is 0.5X 10 10 -2×10 11 particle, preferably 1.1X 10 10 -1.0×10 11 particle。
7. The medical kit of any one of claims 1-6, wherein: the atomization inhalation device comprises a medicine storage bottle for containing medicine solution, an atomization generator, a face mask, a conduit and a connecting piece; the medicine storage bottle is connected with the atomization generator through a conduit, and the medicine storage bottle is connected with the face mask through a connecting piece.
8. Use of a kit according to any of claims 1 to 7 for the manufacture of a pharmaceutical-mechanical combination for the treatment of pulmonary fibrosis.
9. Use according to claim 8, characterized in that: the pulmonary fibrosis is pulmonary interstitial fibrosis.
10. Use according to claim 8 or 9, characterized in that: the pharmaceutical composition is capable of ameliorating inflammatory infiltrates in the lung, and/or reducing interstitial collagen deposition in the lung.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113384597A (en) * | 2020-03-13 | 2021-09-14 | 西比曼生物科技（上海）有限公司 | Aerosol inhalation preparation containing human cell-derived extracellular vesicles, preparation method and application thereof |
| CN114652703A (en) * | 2022-03-14 | 2022-06-24 | 陕西兰医维生物科技有限公司 | Application of aerosol inhalation functional extracellular vesicles in improvement of acute lung injury |
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| CN113384597A (en) * | 2020-03-13 | 2021-09-14 | 西比曼生物科技（上海）有限公司 | Aerosol inhalation preparation containing human cell-derived extracellular vesicles, preparation method and application thereof |
| CN114652703A (en) * | 2022-03-14 | 2022-06-24 | 陕西兰医维生物科技有限公司 | Application of aerosol inhalation functional extracellular vesicles in improvement of acute lung injury |
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