WO2016107563A1

WO2016107563A1 - Fatty mesenchymal progenitor cell complex for treating upper respiratory tract illnesses

Info

Publication number: WO2016107563A1
Application number: PCT/CN2015/099568
Authority: WO
Inventors: 刘佳; 曹卫; 戴成祥; 蔡松柏; 郑成小; 张丽; 王立群; 汪文; 王飞; 孙中伟
Original assignee: 西比曼生物科技（上海）有限公司; 西比曼生物科技（无锡）有限公司
Priority date: 2014-12-29
Filing date: 2015-12-29
Publication date: 2016-07-07

Abstract

Provided in the present invention is a use of fatty mesenchymal progenitor cells in the prevention or treatment of upper respiratory tract illnesses (e.g., chronic obstructive pulmonary diseases or asthma). The fatty mesenchymal progenitor cells can be used for preparing a pharmaceutical composition for treating upper respiratory tract illnesses. The pharmaceutical composition is administrated to the subject in need, and can improve the indexes thereof (pulmonary function, airway inflammation, quality of life score, airway hyperreactivity, etc.), thereby promoting the repair of pathological changes in the upper respiratory tract.

Description

Adipose mesenchymal progenitor cell complex for treating upper respiratory tract diseases

Technical field

The present invention relates to the field of application of adipose stem cells, and in particular to the use of adipose mesenchymal progenitor cells for the treatment of chronic obstructive pulmonary diseases.

Background technique

Chronic obstructive pulmonary disease (COPD) is a common and frequently-occurring disease that seriously endangers human health, seriously affects the quality of life of patients, has a high mortality rate, and brings heavy burdens to patients, their families and society. economic burden. According to the World Bank and the World Health Organization, by 2020, COPD will rank fifth in the world's disease economy.

The pathogenesis of COPD is not fully understood. Inhalation of harmful particles or gases can cause oxidative stress in the lungs, protease and anti-protease imbalance, and pulmonary inflammation. Chronic obstructive pulmonary disease is currently not cured, and its stabilization period is different from that in acute exacerbation. The goal of COPD stabilization is to reduce current symptoms and reduce future risks. Patients in the stable phase of COPD can perform abdominal breathing and lip-reducing exercises and take drugs. Bronchodilators (β2 receptor agonists, anticholinergic drugs, theophylline drugs) are the main treatment measures for controlling chronic obstructive pulmonary disease. Short-term on-demand medications can alleviate symptoms, and long-term regular use can prevent and alleviate symptoms and increase exercise tolerance, but not all patients with improved FEV1. Most of the existing methods belong to symptomatic treatment. According to the guidelines for diagnosis and treatment of COPD, the existing feasible treatment methods include drug treatment, oxygen therapy, rehabilitation therapy and surgical treatment, but these treatments can only alleviate symptoms, prevent the development of the disease and improve the quality of life. There is still a lack of ways to fundamentally reverse the disease. Therefore, both patients and physicians expect a new type of treatment with reliable efficacy, unreasonable adverse reactions and economics.

Bronchial asthma (referred to as asthma) is one of the common chronic respiratory diseases. In recent years, its prevalence has increased year by year in the world. Asthma is currently not cured, and standardized treatments that inhibit inflammation can only control the clinical symptoms of asthma.

The drugs currently used to treat asthma can be divided into control drugs and relief drugs. Most patients with bronchial asthma (abbreviated as asthma) have effective control, but about 5% to 8% of them are difficult to control despite standardized treatment, and these patients are refractory asthma. Its clinical manifestations are divided into: (1) hormone-dependent/resistance asthma, which responds poorly to hormone therapy and exhibits varying degrees of hormonal resistance, requiring long-term dependence on large doses of inhaled hormones, or even oral hormones. (2) Brittle asthma, divided into two types. Type I is characterized by a large fluctuation in peak breathing and repeated asthma attacks despite high-dose inhaled steroid therapy; type II is characterized by a sudden onset of lethal asthma in the case of well-controlled asthma. (3) Lethal asthma, despite Such patients are using "appropriate" treatments, but still have fatal or sudden deaths. At present, there is still no effective treatment for refractory asthma.

In addition, long-term high-dose inhaled hormone may cause some systemic adverse reactions in patients, including skin ecchymosis, adrenal function inhibition, bone density reduction, cataract, glaucoma and so on. Other treatments also have the disadvantages of poor efficacy and large adverse reactions. Therefore, both patients and physicians expect a new type of treatment with reliable efficacy, unreasonable adverse reactions and economics.

Stem cells are a class of cells with self-renewal and differentiation potential that can differentiate into multiple functional cells under certain circumstances. Bone marrow has long been used as the gold standard for the origin of hematopoietic progenitor cells, but with the increasing number of research and application, the insufficiency of bone marrow-derived stem cells in clinical use has become more prominent, such as from collection to clinical use. The process takes too long, collecting bone marrow damage to the donor itself, higher culture cost, lower HLA matching success rate, etc., which restricts its wide application in clinical treatment.

In summary, there is an urgent need in the art for a method for effectively treating upper respiratory tract diseases, particularly chronic obstructive pulmonary disease and asthma.

Summary of the invention

It is an object of the present invention to provide a method for effectively treating upper respiratory tract diseases such as chronic obstructive pulmonary disease and asthma.

In a first aspect of the invention, there is provided the use of a fat mesenchymal progenitor cell for the preparation of a pharmaceutical composition for the treatment of chronic obstructive pulmonary disease and asthma.

In another preferred embodiment, the concentration of the adipose mesenchymal progenitor cells in the pharmaceutical composition is a concentration of adipose mesenchymal progenitor cells of 0.1 to 10 × 10 ⁶ /ml, preferably 0.2 to 5 × 10 ⁶ /ml, more preferably 0.5 to 2 × 10 ⁶ /ml.

In another preferred embodiment, the adipose mesenchymal progenitor cells are subcultured adipose mesenchymal progenitor cells.

In another preferred embodiment, the adipose mesenchymal progenitor cells are present in the composition in an amount of from 1 × 10 ⁶ /ml to 1 × 10 ⁸ /ml.

In another preferred embodiment, the adipose mesenchymal progenitor cells are obtained by purification and expansion of the stromal vascular fraction for 3-10 passages.

In another preferred embodiment, in the composition, the main active ingredient is only adipose mesenchymal progenitor cells.

In another preferred embodiment, the treatment refers to an improvement in pathological symptoms of upper respiratory tract diseases such as chronic obstructive pulmonary disease and asthma.

In another preferred embodiment, the treatment refers to alleviating or reversing pulmonary pathological changes caused by chronic obstructive pulmonary disease.

In another preferred embodiment, the adipose mesenchymal progenitor cells are cells obtained by purification and purification of P3-P10 passages by SVF culture.

In another preferred embodiment, the adipose mesenchymal progenitor cells have the ability to differentiate into bone, cartilage and fat.

In another preferred embodiment, the adipose mesenchymal progenitor cells are adipose mesenchymal progenitor cells expressing a surface marker selected from the group consisting of CD29, CD90, CD73, CD49d, CD44, CD105, SCA-1.

In another preferred embodiment, the adipose mesenchymal progenitor cells are adipose mesenchymal progenitor cells that do not express a surface marker selected from the group consisting of CD34, CD45, CD14, Actin, HLA-DR, CD106, CD80, CD86, CD31.

In another preferred embodiment, the pharmaceutical composition comprises: adipose mesenchymal progenitor cells, and a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutically acceptable carrier is an infusion vehicle carrier and/or an injection vehicle carrier, preferably selected from the group consisting of physiological saline, dextrose saline, or a combination thereof.

In another preferred embodiment, the treatment comprises one or more indicator improvements selected from the group consisting of lung function, airway inflammation, and quality of life score.

In another preferred embodiment, the adipose mesenchymal progenitor cells have any one or more of the characteristics selected from the group consisting of:

(i) more than 90% of the cells have the surface antigen CD29;

(ii) more than 90% of the cells have the surface antigen CD90;

(iii) more than 90% of the cells have the surface antigen CD73;

(iv) more than 90% of the cells have the surface antigen CD49d;

(v) more than 90% of the cells have the surface antigen CD44;

(vi) more than 90% of the cells have the surface antigen CD105;

(vii) More than 90% of the cells have the surface antigen SCA-1.

In another preferred embodiment, more than 95% of the cells have the surface antigen CD29.

In another preferred embodiment, more than 95% of the cells have the surface antigen CD90.

In another preferred embodiment, more than 95% of the cells have the surface antigen CD73.

In another preferred embodiment, more than 95% of the cells have the surface antigen CD49d.

In another preferred embodiment, more than 95% of the cells have the surface antigen CD44.

In another preferred embodiment, more than 95% of the cells have the surface antigen CD105.

In another preferred embodiment, more than 95% of the cells have the surface antigen SCA-1.

In another preferred embodiment, 95-100% (preferably 98-100%, more preferably 99-100%) of the cells have the surface antigen CD29.

In another preferred embodiment, 95-100% (preferably 98-100%, more preferably 99-100%) of the cells have the surface antigen CD90.

In another preferred embodiment, 95-100% (preferably 98-100%, more preferably 99-100%) of the cells have the surface antigen CD73.

In another preferred embodiment, 95-100% (preferably 98-100%, more preferably 99-100%) of the cells have the surface antigen CD49d.

In another preferred embodiment, 95-100% (preferably 98-100%, more preferably 99-100%) of the cells have the surface antigen CD44.

In another preferred embodiment, 95-100% (preferably 98-100%, more preferably 99-100%) of the cells have the surface antigen CD105.

In another preferred embodiment, 95-100% (preferably 98-100%, more preferably 99-100%) of the cells have the surface antigen SCA-1.

(viii) in the cell population, less than 5% of the cells have the surface antigen CD34;

(ix) in the cell population, less than 5% of the cells have the surface antigen CD45;

(x) in the cell population, less than 5% of the cells have a surface antigen Actin;

(xi) in the cell population, less than 5% of the cells have the surface antigen HLA-DR;

(xii) In the cell population, less than 5% of the cells have the surface antigen CD14.

(xiv) In the cell population, less than 5% of cells have the surface antigen CD106

(xv) In the cell population, less than 5% of cells have the surface antigen CD80

(xvi) In the cell population, less than 5% of cells have the surface antigen CD86

(xvii) In the cell population, 5% or less of cells have the surface antigen CD31.

In another preferred embodiment, less than 2% of the cells have the surface antigen CD34.

In another preferred embodiment, less than 2% of the cells have the surface antigen CD45.

In another preferred embodiment, less than 2% of the cells have the surface antigen CD106.

In another preferred embodiment, less than 2% of the cells have the surface antigen CD80.

In another preferred embodiment, less than 1% of the cells have the surface antigen CD34.

In another preferred embodiment, less than 1% of the cells have the surface antigen CD45.

In another preferred embodiment, less than 1% of the cells have the surface antigen CD106.

In another preferred embodiment, less than 1% of the cells have the surface antigen CD80.

In another preferred embodiment, less than 2% of the cells have the surface antigen Actin.

In another preferred embodiment, less than 2% of the cells have the surface antigen HLA-DR.

In another preferred embodiment, less than 2% of the cells have the surface antigen CD14.

In another preferred embodiment, less than 2% of the cells have the surface antigen CD86.

In another preferred embodiment, less than 2% of the cells have the surface antigen CD31.

In another preferred embodiment, less than 1% of the cells have the surface antigen Actin.

In another preferred embodiment, less than 1% of the cells have the surface antigen HLA-DR.

In another preferred embodiment, less than 1% of the cells have the surface antigen CD14.

In another preferred embodiment, less than 1% of the cells have the surface antigen CD86.

In another preferred embodiment, less than 1% of the cells have the surface antigen CD31.

In another preferred embodiment, 0.01 to 5% (preferably 0.1 to 4%, more preferably 1-3%) of the cells have the surface antigen CD34.

In another preferred embodiment, 0.01-1% (preferably 0.05-0.5%, more preferably 0.08-0.2%) of the cells have the surface antigen CD45.

In another preferred embodiment, 0.01 to 3% (preferably 0.05 to 2%, more preferably 0.1 to 1%) of the cells have the surface antigen CD14.

In another preferred embodiment, 0.01-1% (preferably 0.05-0.5%, more preferably 0.08-0.2%) of the cells have the surface antigen HLA-DR.

In another preferred embodiment, 0.01 to 3% (preferably 0.05 to 2%, more preferably 0.1 to 1%) of the cells have the surface antigen Actin.

In another preferred embodiment, 0.01-1% (preferably 0.05-0.5%, more preferably 0.08-0.2%) of the cells have the surface antigen CD80.

In another preferred embodiment, 0.01 to 3% (preferably 0.05 to 2%, more preferably 0.1 to 1%) of the cells have the surface antigen CD86.

In another preferred embodiment, 0.01-1% (preferably 0.05-0.5%, more preferably 0.08-0.2%) of the cells have a table Surface antigen CD31.

According to a second aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating an upper respiratory tract disease such as chronic obstructive pulmonary disease and asthma, the pharmaceutical composition comprising: an effective amount of adipose mesenchymal progenitor cells, And a pharmaceutically acceptable carrier.

In another preferred embodiment, the carrier is an infusion carrier and/or an injection carrier.

In another preferred embodiment, the pharmaceutically acceptable carrier includes, but is not limited to, saline, buffer, dextrose, water, glycerol, ethanol, physiological saline, dextrose saline, and combinations thereof.

In another preferred embodiment, the concentration of the adipose mesenchymal progenitor cells in the pharmaceutical composition is 0.1 to 10 × 10 ⁶ /ml, preferably 0.2 to 5 × 10 ⁶ /ml, more preferably The ground is 0.5 to 2 x 10 ⁶ /ml.

In another preferred embodiment, the volume of the injection reagent is from 10 to 1000 mL, preferably 100 mL.

(i) more than 90% of the cells have the surface antigen CD29;

(ii) more than 90% of the cells have the surface antigen CD90;

(iii) more than 90% of the cells have the surface antigen CD73;

(iv) more than 90% of the cells have the surface antigen CD49d;

(v) More than 90% of the cells have the surface antigen CD44.

(vi) More than 90% of the cells have the surface antigen CD105.

(vii) More than 90% of the cells have the surface antigen SCA-1.

(xiii) In the cell population, 5% or less of cells have the surface antigen CD106.

(xiv) In the cell population, less than 5% of the cells have the surface antigen CD80.

(xv) In the cell population, cells of 5% or less have the surface antigen CD86.

(xvi) In the cell population, cells of 5% or less have the surface antigen CD31.

In another preferred embodiment, the adipose mesenchymal progenitor cells are purified and cultured in a serum-containing medium, or the adipose mesenchymal progenitor cells are purified and cultured in a serum-free medium. cell.

In another preferred embodiment, the adipose mesenchymal progenitor cells further express a cytokine, and the cytokine is selected from the group consisting of TGF-β1, HGF, VEGF, IL6, IL8, IL10, or a combination thereof.

In another preferred embodiment, the adipose mesenchymal progenitor cells express the cytokine TGF-β1 in an amount of 1000-1300 pg/ml/10 ⁶ cells.

In another preferred embodiment, the adipose mesenchymal progenitor cells express cytokine HGF in an amount of 9000-10000 pg/ml/10 ⁶ cells.

In another preferred embodiment, the adipose mesenchymal progenitor cells express the cytokine VEGF in an amount of 700 - 800 pg / ml / 10 ⁶ cells.

In another preferred embodiment, the adipose mesenchymal progenitor cells express the cytokine IL6 in an amount of 5000 - 8000 pg / ml / 10 ⁶ cells.

In another preferred embodiment, the adipose mesenchymal progenitor cells express the cytokine IL8 in an amount of 6000 - 10000 pg / ml / 10 ⁶ cells.

In another preferred embodiment, the adipose mesenchymal progenitor cells express the cytokine IL10 in an amount of 500 to 1000 pg/ml/10 ⁶ cells.

In another preferred embodiment, the mesenchymal progenitor cells have cartilage, osteogenic and adipogenic differentiation capabilities.

In another preferred embodiment, the pharmaceutical composition is an injectable preparation, preferably an intravenous preparation.

In another preferred embodiment, the adipose-derived mesenchymal progenitor cells have the ability to differentiate into bone, cartilage and/or fat.

According to a third aspect of the invention, a method for preventing or treating chronic obstructive pulmonary disease, the method comprising the steps of: administering adipose mesenchymal progenitor cells to a subject in need thereof, or according to the second aspect of the invention Pharmaceutical composition.

In another preferred embodiment, the subject is a human or non-human mammal.

In another preferred embodiment, the non-human mammal is a mouse, a rabbit, a cow, a sheep, a pig, or the like.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

DRAWINGS

Fig. 1 Experimental diagram of oil red O staining, acin blue staining and alizarin red staining for differentiation of adipose mesenchymal progenitor cells;

Figure 2 Oil red O staining control group (negative);

Figure 3haMPC flow detection test map;

Figure 4 is a graph showing the results of airway hyperresponsiveness test in the tail vein injection group in Example 2;

Figure 5 is a diagram showing the results of pathological test of lung tissue in the tail vein injection group in Example 2;

Figure 6 is a diagram showing the results of airway hyperresponsiveness test of the tracheal instillation group in Example 2;

Figure 7 is a diagram showing the results of pathological examination of lung tissue in the tracheal instillation group in Example 2;

Figure 8 is a graph showing the results of lung function test in the tail vein injection group in Example 3;

Figure 9 is a diagram showing the results of pathological examination of lung tissue in the tail vein injection group in Example 3;

Figure 10 is a diagram showing the results of pathological examination of lung tissue in the tail vein injection group in Example 3;

Figure 11 is a graph showing the results of lung function test of the intratracheal instillation group in Example 3;

Figure 12 is a graph showing the results of pathological examination of lung tissue in the intratracheal instillation group in Example 3;

Figure 13 is a graph showing the results of pathological examination of lung tissue in the intratracheal instillation group in Example 3;

Figure 14 is a diagram showing the results of the FISH test in Example 4;

Figure 15 is a graph showing the results of immunomodulatory effects of adipose mesenchymal progenitor cells in Example 1.

detailed description

After long-term and intensive research, the present inventors have unexpectedly found that adipose mesenchymal progenitor cells have an extremely excellent effect of preventing or treating chronic obstructive pulmonary diseases and upper respiratory diseases such as asthma. Specifically, the pharmaceutical composition containing the adipose-derived mesenchymal progenitor cells of the present invention is administered to a subject in need thereof, and has a significant preventive or therapeutic effect on diseases of the upper respiratory tract disease. On this basis, the inventors completed the present invention.

the term

As used herein, the terms "above" and "below" include the number, such as "95% or more" means ≥ 95%, and "0.2% or less" means ≤ 0.2%.

As used herein, the terms "fatty stromal vascular component" and "fat derived stromal vascular component" are used interchangeably.

As used herein, the terms "adipose mesenchymal progenitor cells", "adipose mesenchymal stem cells" and "fat-derived mesenchymal progenitor cells", "fat-derived mesenchymal stem cells" are used interchangeably.

In particular, in the present invention, the term "95% or more" includes 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more.

Fat

Autologous fat is an excellent source of plastic and anti-aging treatments. Adipose tissue materials can be derived from the waist, hips, abdomen, thighs, upper arms and other parts. Those skilled in the art can obtain autologous adipose tissue using general technical methods including, but not limited to, methods of aspiration, surgical separation, and the like.

In the present invention, the adipose tissue or the fat raw material is not particularly limited, and may be an adipose tissue derived from any part of an animal or a human, preferably a human adipose tissue. Preferably, the adipose tissue may be a tissue of a part such as a waist, a buttocks, an abdomen, a thigh, an upper arm or the like.

Human adipose derived mesenchymal progenitor cells

Mesenchymal progenitor cells secrete a broad spectrum of biological factors with immunomodulatory and/or regenerative activity. In the present invention, human adipose derived msenchymal progenitor cells (haMPCs) are preferably used. Among them, one of the most preferred adipose-derived mesenchymal progenitor cells is a CD34+-free cell, which is usually obtained by SVF culture P3-P10 generation purification and amplification.

In the present invention, the method for preparing adipose mesenchymal progenitor cells may include the steps of: washing adipose tissue, then digesting with collagenase, centrifuging the stromal vascular component, removing oil and collagenase, culturing the primary cell, and obtaining the fat after passage. Mesenchymal progenitor cells.

Antigen detection of adipose mesenchymal progenitor cells

The adipose mesenchymal progenitor cells used in the present invention are of high purity and substantially free of other types of cells or stem cells. This can be verified by detection of cell surface antigens.

Adipose mesenchymal progenitor cells have a variety of specific antigens and receptors, mainly CD3, CD13, D29, CD34, CD45, CD49e, CD59, CD73, CD90, CD105, HLA-ABC and so on.

CD34 antigen is a highly glycosylated type I transmembrane protein that is selectively expressed on human hematopoietic stem (HSC), progenitor (PC) and vascular endothelial (EC) surfaces with adipose mesenchyme of CD34. The proportion of progenitor cells in total stem cells is preferably ≤ 0.2%, more preferably ≤ 0.1%.

CD45 is present on the surface of all hematopoietic cells, including hematopoietic stem cells and osteoclasts. The proportion of adipose mesenchymal progenitor cells bearing CD45 in total stem cells is preferably ≤ 0.1%.

CD29, CD44, CD105, CD90, CD106, etc. are mainly present on the surface of adipose mesenchymal progenitor cells.

The proportion of adipose mesenchymal progenitor cells bearing CD29 in total stem cells is preferably ≥ 90%, more preferably ≥ 95%.

The proportion of adipose mesenchymal progenitor cells bearing CD73 in total stem cells is preferably ≥ 80%, more preferably ≥ 85%.

The proportion of adipose mesenchymal progenitor cells bearing CD49d in total stem cells is preferably ≥ 80%, more preferably ≥ 85%.

The proportion of adipose mesenchymal progenitor cells bearing CD105 in total stem cells is preferably ≥ 70%, more preferably ≥ 75%.

The proportion of adipose mesenchymal progenitor cells bearing CD90 in total stem cells is preferably ≥ 70%, more preferably ≥ 75%.

The proportion of adipose mesenchymal progenitor cells bearing SCA-1 in total stem cells is preferably ≥ 70%, more preferably ≥ 75%.

One skilled in the art can use a general method to detect the purity and degree of differentiation of adipose mesenchymal progenitor cells, such as flow cytometry. At the time of detection, different specific and targeted specific antibodies are added, and the antibody may be a complete monoclonal or polyclonal antibody, or may be an immunologically active antibody fragment, such as a Fab' or (Fab) 2 fragment; an antibody heavy chain; An antibody light chain; a genetically engineered single chain Fv molecule (Ladner et al., U.S. Patent No. 4,946,778); or a chimeric antibody, such as an antibody having murine antibody binding specificity but still retaining antibody portions from humans. The antibody is added to the antigen on the cell surface for a certain period of time, and the cells are automatically analyzed and sorted by flow cytometry.

Pharmaceutical composition

The invention also provides a pharmaceutical composition comprising an effective amount of adipose mesenchymal progenitor cells, and a pharmaceutically acceptable carrier.

In general, the adipose mesenchymal progenitor cells and the adipose matrix vascular component can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, such as physiological saline, wherein the pH is usually about 5-8, preferably. Ground, pH is about 7-8.

As used herein, the term "effective amount" or "effective amount" refers to an amount that can produce a function or activity on a human and/or animal and that can be accepted by a human and/or animal. In a preferred embodiment of the invention, the effective amount is: 10 ± 5 x 10 ⁶ cells. Preferably, the effective amount of cells is injected in one shot.

As used herein, a "pharmaceutically acceptable" ingredient is one that is suitable for use in humans and/or mammals without excessive adverse side effects (eg, toxicity, irritation, and allergies), ie, having a reasonable benefit/risk ratio. Operation The phrase "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent, including. In the present invention, the pharmaceutically acceptable carrier which can be used is not particularly limited and may be one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must have sufficient Purity and low enough toxicity. By "compatibility" it is meant herein that the components of the composition are capable of intermingling with the adipose mesenchymal progenitor cells of the invention without significantly reducing the therapeutic effect thereof. Examples of pharmaceutically acceptable carrier parts of the invention are physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous vehicles, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof. In addition to the conventional vectors described above, optimized vectors can also be designed based on the nature of adipose mesenchymal progenitor cells. The carrier is preferably an infusion carrier and/or an injection carrier.

The pharmaceutical compositions of the present invention comprise a safe and effective amount of adipose mesenchymal progenitor cells, a fatty matrix vascular component, and a pharmaceutically acceptable carrier. Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. Usually, the pharmaceutical preparation should be matched to the mode of administration, and the pharmaceutical composition of the present invention can be prepared into an injection form, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. The pharmaceutical preparation of the present invention can also be formulated into a sustained release preparation.

The effective amount of the adipose mesenchymal progenitor cells and the adipose-derived matrix vascular component of the present invention may vary depending on the mode of administration and the severity of the disease to be treated and the like. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials). Such factors include, but are not limited to, the pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc.; the severity of the condition to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, and the like.

The pharmaceutical composition of the invention is preferably a subcutaneous or intravenous injection. In another preferred embodiment, the concentration of the adipose mesenchymal progenitor cells in the subcutaneous or intravenous injection is 0.1 to 10 × 10 ⁶ /ml, preferably 0.2 to 5 × 10 ⁶ /ml. More preferably, it is 0.5 to 2 x 10 ⁶ /ml. The adipose mesenchymal progenitor cells are preferably used in combination with a cell suspension to form a suspension liquid. In another preferred embodiment, the cell suspension is physiological saline.

The injection method of the pharmaceutical composition is not particularly limited, and may be a single injection preparation or a combination of preparations for multiple injections. In a preferred embodiment of the invention, the pharmaceutical composition is a single injection.

In the present invention, the pharmaceutical composition is preferably an intravenous preparation or a tracheal instillation preparation.

The main advantages of the invention:

(1) A fat mesenchymal progenitor cell complex for treating an upper respiratory tract disease, which can exert a certain therapeutic or reversal effect on an upper respiratory tract disease after administration.

(2) Adipose mesenchymal progenitor cells are rich in source, convenient in obtaining materials and low in immunogenicity, and have broad prospects in clinical application.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually in accordance with conventional conditions or according to the conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

Example 1 Culture of adipose mesenchymal progenitor cells

First, reagents and supplies

1. Sterile surgical instruments and consumables

(1) 5 sterile sterile handles

(2) Sterile 100 mesh filter

(3) Sterilization 40 mesh filter

(4) 50ml centrifuge tube

2. Sterile reagents:

(1) DMEM (basic medium),

Iscove's Modification of DMEM (Corning, basal medium);

Stem Cell Factor (SCF) (Corning, supplement factor)

(2) Type I collagenase (currently used): 0.1% collagenase I preparation method: 0.1 g of collagenase I powder was weighed and dissolved in 100 ml of medium without any factor, and preheated at 37 ° C before.

(3) Sodium chloride injection

2. Implementation plan

1. Wash the adipose tissue and remove the blood cells. Add 20 ml of sodium chloride injection to the centrifuge tube, tighten the lid, shake vigorously for 3 minutes to fully wash the adipose tissue, and then stand still for 3-5 minutes to separate the different phases. Aspirate the lower aqueous phase; repeat the above three times until the lower layer becomes clear.

2. Separation of stromal vascular fraction (SVF): Add an equal amount of freshly prepared pre-warmed collagenase II solution, digest for 40-60 minutes at 37 ° C, 200 rpm, and filter the digested tissue with a sterile 100 mesh filter. After centrifugation at 1500 rpm for 5 minutes at room temperature, the resulting precipitate was SVF.

3. Cell culture: After centrifugation, SVF was deposited on the bottom of the centrifuge tube, and the upper layer of the oil and the lower layer of the collagenase solution were carefully removed from the top with a pipette. The cells were resuspended in an appropriate amount of DMEM, and the cells were cultured in a serum-free, antibiotic-free medium under hypoxia (1-20% by volume of oxygen (v/v)), and passaged cells at a cell fusion of 80%. Cultured to 3-10 passages to harvest adipose mesenchymal progenitor cells.

4. Cell reinfusion: The harvested cells are injected into physiological saline to prepare a cell suspension for use in returning.

III. Conclusion:

1. Cell separation and culture experiment results

Cell yield isolated by the above method: 5 × 10 ⁵ to 1 × 10 ⁶ cells / ml of fat

After the cells were cultured, P3-P7 cells were harvested, and the amount of cells was greater than 5 × 10 ⁹ .

2. Immunostaining analysis of adipose stem cell differentiation

Adipogenic induction control and oil red O staining experiment

The cells cultured in Example 1 were used as the group of the present invention to perform cartilage, osteogenic, and adipogenic differentiation tests. After 3-4 weeks of in vitro culture in the direction of cartilage, acin blue staining showed that the cells cultured in Example 1 had the ability to differentiate into cartilage in vitro (Fig. 1B); in vitro, in vitro cultured in the direction of bone for 3-4 weeks, alizarin red staining It is shown that the cells cultured in Example 1 have the ability to differentiate into bone in vitro (Fig. 1C); the oil red O staining after 3-4 weeks of differentiation into the adipogenic direction in vitro indicates that the cells cultured in Example 1 have a tendency in vitro. The ability to differentiate into fat (Figure 1A).

3. Flow detection of cells

The cells were collected into a centrifuge tube by enzymatic digestion. The cell suspension was adjusted to a density of 1×10 ⁵ cells/mL, centrifuged at 1,800 r/min (120 g) for 5 min, the supernatant was discarded, and washed with cold D-Hanks at 4 °C. The cells were resuspended, and the cell suspension was again centrifuged at 800 r/min for 5 min, after which the supernatant was discarded. The cells were then resuspended to 1 mL with D-Hanks, and 5 to 10 μL of the antibody was added, protected from light, and placed on ice for 30 min. Rinse with D-Hanks, centrifuge, discard the supernatant, and repeat the rinsing process 2 to 3 times to ensure that the unbound antibody is removed. Finally, about 200 to 300 μL of D-Hanks was added to make a suspension, which was detected by flow cytometry.

haMPC flow test results

The results of surface antigen flow identification of P3 cells MSCs isolated and cultured by type I collagenase digestion method are shown in Fig. 3, and the flow detection test pattern is shown in Table 2 below.

Table 2 Flow detection test map

in conclusion:

The results showed that the expression of cell surface antigen marker was detected by flow cytometry on adipose mesenchymal progenitor cells. The purity of the cells was high, most of which were adipose mesenchymal progenitor cells, in which CD34 and CD45 were mesenchymal progenitor cells. Negative mark.

4. Detection of cytokines in adipose mesenchymal progenitor cells

The adipose mesenchymal progenitor cells cultured in Example 1 were resuspended after centrifugation, adjusted for cell density, and the supernatant was collected 48 hours later to detect cytokines TGF-β1, HGF and VEGF, IL6, IL8, IL10. table.

Conclusion: The adipose mesenchymal progenitor cells can express TGF-β1, HGF, VEGF, IL6, IL8, and IL10.

5. Immunomodulatory effects of adipose mesenchymal progenitor cells

The haMPC and activated PBMC in Example 1 were co-cultured in serum-free medium for 5 days, and the BMC only group was used as control. The ratio of different cells of haMPC and PBMC was experimental group (1:120, 1:240, 1:480, 1:960, 1:1920).

PBMC were fluorescently stained and activated by CFSE prior to co-culture. After 5 days of co-culture, the difference in fluorescence between the CFSE cells by flow cytometry can reflect the degree of amplification of PBMC, and thus The inhibitory effect of haMPC on PBMC proliferation in vitro and its relationship with cell ratio.

Conclusion: The fluorescence value of the PBMC only group was 100%, and the ratio of the other groups to the PBMC only group was statistically significant (N=3).

The proliferation inhibitory activity of haMPC on PBMC began to decrease at a ratio of 1:960, and dose-related phenomena began to appear in this proportional group.

Example 2 Therapeutic effect of haMPC intravenous injection/tracheal instillation on asthma

The mice were sensitized by intraperitoneal injection of 0.1% OVA 0.1ml at 0, 7, and 14d, and the mice were placed in a self-contained container at 2nd to 77d, and inhaled by 2.5% OVA for 30min/d, 3 times/week. For 8 weeks, an asthma model was established.

The experimental group (n=8) was administered by intravenous injection of 1×10 ⁶ /100 μl and intratracheal instillation of 1×10 ⁶ /30 μl (both 1×10 ⁶ cells+ cell suspension) on 21 days, respectively. The suspension is physiological saline).

On the 57th day after the injection treatment (78d), all the experimental groups were tested for lung function and airway hyperresponsiveness, inflammatory cell classification and cytokine detection, spleen CD4+CD25+Foxp3+ regulatory T cells; alveolar lavage fluid Inflammatory cell differential count and cytokine detection, lung tissue pathology. The control group (n=8) was treated with PBS sensitization and an equal volume of PBS injection. The main results are as follows:

1. Tail vein injection group

1.1 Airway hyperresponsiveness results are shown in Figure 4. The airway hyperresponsiveness of each group increased with the increase of methacholine concentration. The airway hyperresponsiveness of OVA+haMPCs and PBS+PBS groups under the two methacholine concentrations of M128 and M256. Significantly lower than the OVA+PBS group, P<0.05. There was no significant difference in airway hyperresponsiveness between OVA+PBS and OVA+haMPCs in other methacholine concentrations. This suggests that haMPCs have a mitigating effect on airway hyperresponsiveness in the case of methacholine-induced asthma in mice.

1.2 Pathological results of lung tissue are shown in Figure 5. Among them, the observation results of the PBS+PBS group and the PBS+haMPCs group (normal control group not administered and administered) were similar, and no subepithelial fibrosis occurred. In the model control group treated with OVA, it was found that the deposition of subepithelial collagen fibers was significantly thickened (see the black arrow), suggesting that there was significant subepithelial fibrosis in mice with chronic asthma. In the experimental group treated with intravenous injection of haMPCs, only mild subepithelial fibrosis was observed, indicating that intravenous administration of haMPCs produced a reversal of subepithelial fibrosis symptoms in asthmatic mice.

2. Tracheal instillation group

2.1 airway hyperresponsiveness results

The results are shown in Fig. 6. Among them, the airway hyperresponsiveness values of all groups increased with the increase of methacholine concentration. At M256 concentration, the asthma group and asthma treatment group and control group had statistical data. Difference, P < 0.05. This indicates that haMPCs can alleviate the airway hyperresponsiveness of asthma elimination.

2.2 lung pathology test results

The result is shown in Figure 7. Among them, the observation results of the PBS+PBS group and the PBS+haMPCs group (normal control group not administered and administered) were similar, and no subepithelial fibrosis occurred. In the model control group treated with OVA, it was found that the deposition of subepithelial collagen fibers was significantly thickened (see the white arrow), suggesting that there was significant subepithelial fibrosis in mice with chronic asthma. In the experimental group treated with intra-hap instillation of haMPCs, only mild subepithelial fibrosis was observed, indicating that intravenous administration of haMPCs produced a reversal of subepithelial fibrosis symptoms in asthmatic mice.

Example 3 Therapeutic effect of combined intravenous administration of haMPC/tracheal instillation on chronic obstructive pulmonary disease (COPD)

Ozone exposure established experimental mice similar to human COPD (exposure 3 h/d at 2.5 ppm ozone concentration, 2 times/week for 6 weeks). The control group was exposed to air. After 6 weeks of ozone exposure, the experimental group (n=8) mice received 1×10 ⁶ /100 μl haMPCs tail vein injection or 1×10 ⁶ /30 μl haMPCs intratracheal instillation, and the control group (n=8) mice. Treatment with an equal volume of PBS injection. After d71 (after 4 weeks of treatment), the mice were sacrificed after lung function test. The specimens were collected for inflammatory cell classification and cytokine detection, alveolar lavage fluid for inflammatory cell classification and cytokine detection, and lung histopathology. The main results are as follows:

1. Tail vein injection group

1.1 lung function test

The results are shown in Fig. 8, in which the FEV ₂₅ /FVC% of the COPD model group O ₃ +PBS T=4w and O ₃ +PBS T=6w was significantly lower than the normal group, and there was a statistical difference, P<0.01; Compared with the COPD model group O ₃ +PBS T=6w, the FEV ₂₅ /FVC% of the O ₃ +haMPCs T=6w in the 6-week administration group increased significantly, and there was a statistical difference, P<0.01.

1.2 lung tissue pathology test

The results are shown in Figure 9 and in which the MAN, MAA per unit area per unit of field of the lung tissue of the COPD model group was significantly decreased compared with the normal group ( ₃ + PBS T = 4w, O ₃ + PBS T = 6w). P <0.05); compared with model group COPD O ₃ + PBS T = 4w, basis COPD treatment group O ₃ + haMPCs T = 4w lung tissues / vision unit MAN, MAA increased significantly (P <0.05); Compared with CO ₃ model group O ₃ +PBS T=6w, MAN and MAA per unit area/unit field of view of O ₃ +haMPCs T=6w lung tissue in COPD treatment group were significantly increased (P<0.05).

As shown in Fig. 10, the lung tissue LM was significantly increased in the COPD model group O ₃ +PBS T=4w, O ₃ +PBS T=6w; compared with the COPD model group O ₃ +PBS T=4w, COPD treatment group O ₃ +haMPCs T=4w LM of lung tissue was significantly decreased (P<0.05); compared with CO3 model group O ₃ +PBS T=6w, CO ₃ treatment group O ₃ +haMPCs T=6w lung tissue LM significantly decreased (P <0.05).

2. Intratracheal instillation group

2.1 lung function test

The results are shown in Fig. 11, in which the FEV ₂₅ /FVC% of the COPD model group O ₃ +PBS T=4w and O ₃ +PBS T=6w decreased significantly compared with the normal group, and there was a statistical difference, P<0.01; Compared with the normal group, the FEV ₂₅ /FVC% of O ₃ +PBS T=6w was significantly decreased in the COPD model group, P<0.01; compared with the COPD model group O ₃ +PBS T=4w, the 4-week administration group The FEV ₂₅ /FVC% of O ₃ +haMPCs T=6w increased significantly, there was a statistical difference, P<0.01; compared with the COPD model group O ₃ +PBS T=6w, the 6-week administration group O ₃ +haMPCs T= The FEV ₂₅ /FVC% of 6w increased significantly, and the results were statistically different, P <0.01.

2.2 lung histopathology test

The results are shown in Figures 12 and 13, wherein the COPD model group O ₃ + PBS T = 4 w, O ₃ + PBS T = 6 w lung tissue per unit area / unit field of view MAN, MAA decreased significantly (P <0.05); COPD model group compared with the _{O 3 + PBS T = 4w,} COPD O ₃ + treatment group basis haMPCs T = 4w lung / vision unit MAN, MAA increased significantly (P <0.05) Compared with the COPD model group O ₃ +PBS T=6w, the MAN/MAA per unit area/unit field of view of O ₃ +haMPCs T=6w lung tissue in the COPD treatment group was significantly increased (P<0.05). As shown in Fig. 13, the lung tissue LM was significantly increased in the COPD model group O ₃ +PBS T=4w, O ₃ +PBS T=6w; compared with the COPD model group O ₃ +PBS T=4w, COPD treatment group O ₃ +haMPCs T=4w LM of lung tissue was significantly decreased (P<0.05); compared with CO3 model group O ₃ +PBS T=6w, CO ₃ treatment group O ₃ +haMPCs T=6w lung tissue LM significantly decreased (P <0.05).

Example 4 Tracing of male mice with maMPC intravenously into female mice

experiment procedure:

Female mice were sensitized subcutaneously with 2 mM Al(OH) ₃ coated 40 ug ovalbumin (OVA) on day 1, 3, 5, 7, 9, 11, and 13, respectively.

On day 20, male mouse adipose mesenchymal progenitor cells were injected into the tail vein (cell volume: 1 × 10 ⁶ cells).

On

days

21, 22, 23, 24, 25, 26, and 27, 30% 5% nebulized OVA was administered, and 20ul OVA (40 mg/ml) was administered intranasally to stimulate asthma (experimental group).

Part of the mice were sacrificed by cervical dislocation at four time points on the 20th, 21st, 28th and 35th day.

After the mice were sacrificed on the 20th day (ie, after injection of male mouse adipose mesenchymal progenitor cells in the tail vein), the lungs were taken for FISH detection (detection of the presence of the Y chromosome in the tissue).

Experimental results:

The preliminary results of the FISH test are shown in Figure 14:

The FISH test results of the lung tissue of the male mice in the positive control group (untreated male mice) were positive; the FISH test results of the lung tissues of the female mice in the negative control group (untreated female mice) were negative; The experimental group (injected with adipose mesenchymal progenitor cells) was injected into the male mouse maMPC to the female mice for several hours, and the FISH test of the lung tissue was positive. The above experimental results indicate that maMPC reaches lung tissue several hours after the tail vein injection of male mouse maMPC into female mice, demonstrating that the adipose-derived progenitor cells of the invention of the same origin are identical to the adipose-derived progenitor cells of autologous origin. The effect, and will not cause rejection.

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims

Use of a fat mesenchymal progenitor cell for the preparation of a pharmaceutical composition for treating upper respiratory tract diseases.
The use according to claim 1, wherein said upper respiratory tract disease is selected from the group consisting of chronic obstructive pulmonary disease and asthma.
The use according to claim 1, wherein said pharmaceutical composition comprises: adipose mesenchymal progenitor cells, and a pharmaceutically acceptable carrier.
The use of claim 1 wherein said treatment comprises one or more indicators selected from the group consisting of lung function, airway hyperresponsiveness, airway inflammation, quality of life score.
The use according to claim 1 wherein said adipose mesenchymal progenitor cells have any one or more of the characteristics selected from the group consisting of:

(i) more than 90% of the cells have the surface antigen CD29;

(ii) more than 90% of the cells have the surface antigen CD90;

(iii) more than 90% of the cells have the surface antigen CD73;

(iv) more than 90% of the cells have the surface antigen CD49d;

(v) more than 90% of the cells have the surface antigen CD44;

(vi) more than 90% of the cells have the surface antigen CD105;

(vii) More than 90% of the cells have the surface antigen SCA-1.
The use according to any one of claims 1 to 5, wherein the adipose mesenchymal progenitor cells have any one or more of the following characteristics selected from the group consisting of:

(viii) in the cell population, less than 5% of the cells have the surface antigen CD34;

(ix) in the cell population, less than 5% of the cells have the surface antigen CD45;

(x) in the cell population, less than 5% of the cells have a surface antigen Actin;

(xi) in the cell population, less than 5% of the cells have the surface antigen HLA-DR;

(xii) in the cell population, less than 5% of the cells have the surface antigen CD14;

(xiv) in the cell population, less than 5% of the cells have the surface antigen CD106;

(xv) in the cell population, less than 5% of the cells have the surface antigen CD80;

(xvi) in the cell population, less than 5% of the cells have the surface antigen CD86;

(xvii) In the cell population, 5% or less of cells have the surface antigen CD31.
The use according to any one of claims 1 to 5, wherein the pharmaceutical composition is a dosage form selected from the group consisting of an intravenous injection or a tracheal instillation.
The use according to claim 7, wherein the intravenous drip agent comprises: adipose mesenchymal progenitor cells, and a cell suspension; and/or

The tracheal instillation agent includes: adipose mesenchymal progenitor cells, and a cell suspension.
A pharmaceutical composition for preventing or treating an upper respiratory tract disease, characterized in that the pharmaceutical composition comprises an effective amount of adipose mesenchymal progenitor cells, and a pharmaceutically acceptable carrier.
The pharmaceutical composition according to claim 9, wherein said upper respiratory tract disease is selected from the group consisting of chronic obstructive pulmonary disease and asthma.
The pharmaceutical composition according to claim 9, wherein said adipose mesenchymal progenitor cells have one or more characteristics selected from the group consisting of:

(i) more than 90% of the cells have the surface antigen CD29;

(ii) more than 90% of the cells have the surface antigen CD90;

(iii) more than 90% of the cells have the surface antigen CD73;

(iv) more than 90% of the cells have the surface antigen CD49d;

(v) more than 90% of the cells have the surface antigen CD44;

(vi) more than 90% of the cells have the surface antigen CD105;

(vii) More than 90% of the cells have the surface antigen SCA-1.
The pharmaceutical composition according to claim 9, wherein said adipose mesenchymal progenitor cells have one or more characteristics selected from the group consisting of:

(viii) in the cell population, less than 5% of the cells have the surface antigen CD34;

(ix) in the cell population, less than 5% of the cells have the surface antigen CD45;

(x) in the cell population, less than 5% of the cells have a surface antigen Actin;

(xi) in the cell population, less than 5% of the cells have the surface antigen HLA-DR;

(xii) in the cell population, less than 5% of the cells have the surface antigen CD14;

(xiv) in the cell population, less than 5% of the cells have the surface antigen CD106;

(xv) in the cell population, less than 5% of the cells have the surface antigen CD80;

(xvi) in the cell population, less than 5% of the cells have the surface antigen CD86;

(xvii) In the cell population, 5% or less of cells have the surface antigen CD31.
The pharmaceutical composition according to claim 9, wherein said adipose mesenchymal progenitor cells further express cytokines, and said cytokines are selected from the group consisting of TGF-β1, HGF, VEGF, IL6, IL8, IL10, or a combination thereof.
The pharmaceutical composition according to claim 9, wherein said adipose-derived mesenchymal progenitor cells have the ability to differentiate into bone, cartilage and/or fat.
The pharmaceutical composition according to claim 9, wherein said adipose-derived mesenchymal progenitor cells are prepared by the following method: culture containing 0.0001 to 20% by weight of serum or serum substitute and containing no antibiotic In the base, the isolated stromal vascular components were cultured and passaged to obtain P3-P7 generation adipose mesenchymal progenitor cells.
The pharmaceutical composition according to claim 9, wherein said adipose mesenchymal progenitor cells are cultured and passaged in an atmosphere containing 1% to 20% (v/v) of oxygen.
The pharmaceutical composition according to claim 9, wherein said adipose mesenchymal progenitor cells have a capacity of >1:960 in a co-culture experiment with monocytes, preferably in said pharmaceutical composition. It is 1:960-1:120.