WO2016078616A1 - Cellule progénitrice mésenchymateuse adipeuse et composition de fraction vasculaire stromale adipeuse utilisée pour le traitement de l'hépatite b - Google Patents

Cellule progénitrice mésenchymateuse adipeuse et composition de fraction vasculaire stromale adipeuse utilisée pour le traitement de l'hépatite b Download PDF

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WO2016078616A1
WO2016078616A1 PCT/CN2015/095204 CN2015095204W WO2016078616A1 WO 2016078616 A1 WO2016078616 A1 WO 2016078616A1 CN 2015095204 W CN2015095204 W CN 2015095204W WO 2016078616 A1 WO2016078616 A1 WO 2016078616A1
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cells
hepatitis
surface antigen
mesenchymal progenitor
adipose
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PCT/CN2015/095204
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English (en)
Chinese (zh)
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曹卫
张丽
戴成祥
刘佳
蔡松柏
郑成小
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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
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Priority claimed from CN201410668435.3A external-priority patent/CN105663163A/zh
Priority claimed from CN201410668392.9A external-priority patent/CN105663165A/zh
Application filed by 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 filed Critical 西比曼生物科技（上海）有限公司
Publication of WO2016078616A1 publication Critical patent/WO2016078616A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/35—Fat tissue; Adipocytes; Stromal cells; Connective tissues
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]

Definitions

  • the present invention relates to the field of application of adipose stem cells, and in particular, the present invention provides a use of a combination of adipose mesenchymal progenitor cells and a fat matrix vascular component for the treatment of hepatitis B.
  • Liver diseases are classified into viral liver disease and non-viral hepatitis.
  • Viral hepatitis mainly includes hepatitis A, B, C, D, E virus
  • non-viral liver diseases mainly include alcoholic liver disease, drugs or toxic liver disease, metabolic abnormal liver disease, fatty liver disease.
  • Chronic hepatitis B is globally distributed and may lead to diseases such as liver decompensation, cirrhosis, and hepatocellular carcinoma.
  • Active hepatitis B virus (HBV) replication is a major driver of liver damage and disease progression.
  • Severe hepatitis B is a severe hepatitis caused by HBV infection. Its incidence is dangerous and rapid progress. Most patients have poor prognosis. It can rapidly cause large-scale hepatocyte necrosis and severe liver damage, liver metabolism, excretion and interpretation. Decrease. As a result, the body's immunity is reduced, resulting in a vicious circle.
  • the traditional drug interferon alpha can effectively inhibit viral replication, and its efficacy is relatively long-lasting, but its range of applicable population is narrow and the incidence of adverse reactions is high.
  • Lafmidine is the first nucleoside analogue, a drug that can rapidly and permanently inhibit viral replication, improve liver function, and delay the progression of hepatitis B disease, but its HBeAg seroconversion rate is low.
  • the vaginal rate of HBeAg smear for lamivudine for 1 year is about 16%. It is easy to relapse after stopping the drug, the virus can not be completely removed, and the incidence of virus-resistant mutations increases with the treatment time (1st, 2nd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd, 3rd 4 years were 14%, 38%, 49%, 66%).
  • Adefovir dipivoxil has a low incidence of long-term treatment and is still effective against lamivudine-resistant people, but its antiviral effect is weak, slow onset, and potentially nephrotoxic.
  • Entecavir has strong antiviral effect and low drug resistance rate. It is carcinogenic in animal experiments and is more expensive. Telbivudine has strong antiviral effect and high HBeAg conversion rate, but its mutation rate is high, side effects such as creatine kinase increase, short time to market, antiviral effect, long-term efficacy and safety have to be confirmed. Nucleoside (acid) similar drugs can not be stopped at any time, long-term use of hepatitis B virus easily mutated resistance, there will be side effects such as flu-like, anorexia, nausea, diarrhea, depression, angina. Moreover, antiviral therapy can only inhibit HBV replication, can not completely eliminate the virus, and can not repair liver cell necrosis caused by hepatitis.
  • hepatitis B is mainly antiviral treatment.
  • antiviral drugs can only inhibit HBV replication, and can not completely eliminate the virus, nor can it repair hepatocyte necrosis caused by hepatitis. Therefore, it is easy to form chronic hepatitis B after infection with hepatitis B virus, which may lead to liver cirrhosis and liver cancer.
  • a composition of adipose mesenchymal progenitor cells and a fat matrix vascular component for the preparation of a pharmaceutical composition for treating hepatitis B.
  • the adipose mesenchymal progenitor cells are subcultured adipose mesenchymal progenitor cells.
  • the adipose mesenchymal progenitor cells are present in the composition in an amount of from 1 ⁇ 10 6 /ml to 1 ⁇ 10 8 /ml.
  • the volume ratio of the adipose mesenchymal progenitor cells to the fat matrix vascular component is 1:9 to 2:8.
  • the main active ingredient is only adipose mesenchymal progenitor cells and adipose-derived stromal vascular components.
  • the pharmaceutical composition comprises: adipose mesenchymal progenitor cells, an adipose-derived stromal vascular component, and a pharmaceutically acceptable carrier.
  • the carrier is an infusion carrier and/or an injection carrier.
  • the treatment comprises one or more indicator improvements selected from the group consisting of:
  • Hepatitis B surface antigen hepatitis B surface antibody, hepatitis B e antigen, hepatitis B e antibody, hepatitis B core antibody, hepatitis B virus DNA, and ALT.
  • the adipose mesenchymal progenitor cells have any one or more of the characteristics selected from the group consisting of:
  • more than 90% of the cells have the surface antigen CD29.
  • more than 80% of the cells have the surface antigen CD73.
  • more than 70% of the cells have the surface antigen CD105.
  • more than 80% of the cells have the surface antigen CD90.
  • the adipose mesenchymal progenitor cells have any one or more of the characteristics selected from the group consisting of:
  • less than 5% of the cells have the surface antigen CD34.
  • less than 5% of the cells have the surface antigen CD45.
  • less than 1% of the cells have the surface antigen CD34.
  • less than 1% of the cells have the surface antigen CD45.
  • the stromal vascular component comprises a cytokine selected from the group consisting of stem cell growth factor (HGF), vascular endothelial growth factor (VEGF), platelet-derived factor (PDGF), human transforming growth factor beta. (TGF- ⁇ ), macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-10 (IL-10), or any combination thereof.
  • HGF stem cell growth factor
  • VEGF vascular endothelial growth factor
  • PDGF platelet-derived factor
  • TGF- ⁇ human transforming growth factor beta.
  • GM-CSF macrophage colony-stimulating factor
  • IL-2 interleukin-2
  • IL-10 interleukin-10
  • the concentration of stem cell growth factor (HGF) in the stromal vascular fraction is > 0.5 ng/ml.
  • the concentration of vascular endothelial growth factor (VEGF) in the stromal vascular component is ⁇ 35 pg/ml, preferably ⁇ 40 pg/ml.
  • the concentration of human transforming growth factor beta (TGF-[beta] in the stromal vascular fraction is > 150 pg/ml, preferably > 180 pg/ml.
  • the concentration of interleukin-2 (IL-2) in the stromal vascular component is ⁇ 15 pg/ml, preferably ⁇ 20 pg/ml, more preferably ⁇ 30 pg/ml.
  • the concentration of interleukin-10 (IL-10) in the stromal vascular component is ⁇ 15 pg/ml, preferably ⁇ 20 pg/ml, more preferably ⁇ 30 pg/ml, optimally ⁇ 40 pg/ Ml.
  • the stromal vascular component has any one or more of the characteristics selected from the group consisting of:
  • more than 30% of the cells have the surface antigen CD29.
  • more than 60% of the cells have the surface antigen CD73.
  • more than 90% of the cells have the surface antigen CD49d.
  • more than 60% of the cells have the surface antigen CD90.
  • more than 70% of the cells have the surface antigen CD34.
  • more than 35% of the cells have the surface antigen HLA-DR.
  • less than 5% of the cells have the surface antigen CD14.
  • less than 15% of the cells have the surface antigen CD45.
  • less than 5% of the cells have the surface antigen Actin.
  • a pharmaceutical composition for preventing or treating hepatitis comprising: an effective amount of adipose mesenchymal progenitor cells and a fat matrix vascular component, and pharmaceutically acceptable a.
  • the pharmaceutically acceptable carrier includes, but is not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical composition is an intravenous injection.
  • the concentration of the adipose mesenchymal progenitor cells in the intravenous injection is 0.1-100 ⁇ 10 7 /ml, preferably 1-10 ⁇ 10 7 /ml, more preferably It is 2-5 x 10 8 /ml.
  • the concentration of the stromal vascular component in the intravenous injection is from 10% to 20% by volume.
  • the volume of the injection reagent is 30-70 ml.
  • a pharmaceutical combination kit for preventing or treating hepatitis comprising: adipose mesenchymal progenitor cells, a fat matrix vascular component, a pharmaceutically acceptable carrier, and The specification; and the described specification describes the method of use.
  • the method of use comprises: mixing adipose mesenchymal progenitor cells, a fat matrix vascular component, and a pharmaceutically acceptable carrier into a formulation and administering to the subject.
  • the preparation is an injection.
  • a method for preventing or treating hepatitis comprising the steps of: administering to a subject in need thereof (a) adipose mesenchymal progenitor cells or a population of adipose mesenchymal progenitor cells, or containing fat a pharmaceutical composition of a mesenchymal progenitor cell or a population of adipose mesenchymal progenitor cells; and (b) a fatty matrix vascular component.
  • the subject is a human or non-human mammal.
  • the non-human mammal is a mouse, a rabbit, a cow, a sheep, a pig, or the like.
  • the method of administration is intravenous reinfusion.
  • a fatty mesenchymal progenitor cell which is characterized in that it is used for the preparation of a pharmaceutical composition for treating hepatitis B.
  • the adipose mesenchymal progenitor cells are subcultured adipose mesenchymal progenitor cells.
  • the main active ingredient is only adipose mesenchymal progenitor cells.
  • the adipose mesenchymal progenitor cells are present in the composition in an amount of from 1 ⁇ 10 5 /ml to 1 ⁇ 10 8 /ml.
  • the pharmaceutical composition comprises: adipose mesenchymal progenitor cells, and a pharmaceutically acceptable carrier.
  • the carrier is an infusion carrier and/or an injection carrier.
  • the treatment comprises one or more indicator improvements selected from the group consisting of:
  • Hepatitis B surface antigen hepatitis B surface antibody, hepatitis B e antigen, hepatitis B e antibody, hepatitis B core antibody, hepatitis B virus DNA, and ALT.
  • the adipose mesenchymal progenitor cells have any one or more of the characteristics selected from the group consisting of:
  • more than 90% of the cells have the surface antigen CD29.
  • more than 80% of the cells have the surface antigen CD73.
  • more than 70% of the cells have the surface antigen CD105.
  • more than 80% of the cells have the surface antigen CD90.
  • the adipose mesenchymal progenitor cells have any one or more of the characteristics selected from the group consisting of:
  • less than 5% of the cells have the surface antigen CD34.
  • less than 5% of the cells have the surface antigen CD45.
  • less than 1% of the cells have the surface antigen CD34.
  • less than 1% of the cells have the surface antigen CD45.
  • a pharmaceutical composition for preventing or treating hepatitis comprising: an effective amount of adipose mesenchymal progenitor cells, and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier includes, but is not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical composition is an intravenous injection.
  • the concentration of the adipose mesenchymal progenitor cells in the intravenous injection is 0.1-100 ⁇ 10 4 /ml, preferably 1-10 ⁇ 10 4 /ml, more preferably It is 2-5 x 10 5 /ml.
  • the volume of the injection reagent is 30-70 ml.
  • a method for preventing or treating hepatitis comprising the steps of: administering to a subject in need thereof a fat mesenchymal progenitor cell or a population of adipose mesenchymal progenitor cells, or containing adipose mesenchymal progenitor cells or A pharmaceutical composition of a population of adipose mesenchymal progenitor cells.
  • the subject is a human or non-human mammal.
  • the non-human mammal is a mouse, a rabbit, a cow, a sheep, a pig, or the like.
  • the mode of administration is intravenous reinfusion.
  • Figure 1 Oil red O staining of adipocyte differentiation
  • Figure 2 Oil red O staining control group (negative) for adipocyte differentiation
  • Figure 3 haMPC flow detection test map
  • Figure 4 is a flow cytometric test map of adipose-derived stromal vascular components
  • the present inventors After long-term and intensive research, the present inventors have unexpectedly found that adipose mesenchymal progenitor cells and a fat matrix vascular component have extremely excellent effects of preventing or treating hepatitis (especially hepatitis B).
  • the pharmaceutical composition containing the adipose mesenchymal progenitor cells and the fat matrix vascular component of the present invention is administered to a subject in need thereof, and has a significant preventive or therapeutic effect on hepatitis, which can reduce various surface antigens and significantly reduce viral DNA. And reduce ALT and promote the repair of liver function.
  • the inventors completed the present invention.
  • the terms “above” and “below” include the number, such as “95% or more” means ⁇ 95%, and "0.2% or less” means ⁇ 0.2%.
  • fatty stromal vascular component and "fat-derived stromal vascular component” are inter- Change to use.
  • Hepatitis B is a disease caused by the infection of the body by hepatitis B virus (HBV).
  • HBV hepatitis B virus
  • Hepatitis B virus has a narrow host range and obvious hepatic function, which is difficult to culture in vitro.
  • Hepatitis B model involves hepatitis B virus infection and immune response after infection. Therefore, it is not only necessary to repair damaged liver cells during the treatment process, but also to inhibit viral replication and regulate immune response in the body, which is quite different from non-viral hepatitis.
  • animal models primarily use immunodeficient animals to make human HBV animal models. The requirement is mainly similar to the characteristics of human clinical hepatitis, and the viremia and blood immunological indicators are maintained for a certain period of time to restore the disease state of the human body infected by hepatitis B virus to the greatest extent.
  • Non-viral hepatitis models such as alcoholic liver disease models, drug-induced hepatitis models, etc., directly intervene through diet or drugs, impair liver cell function in animals, and mimic the clinical manifestations of human hepatitis.
  • hepatitis B five items include hepatitis B surface antigen, hepatitis B surface antibody, e antigen, e antibody, core antibody.
  • the surface antigen is the coat protein of hepatitis B virus and is not contagious by itself, but its appearance is often accompanied by the presence of hepatitis B virus, so its positive is a sign that it has been infected with hepatitis B virus.
  • serum is detected in the serum 2-6 months after infection, and serum transaminase has not risen.
  • Most patients with acute hepatitis B can turn negative in the early stage of the disease, but patients with chronic hepatitis B will continue to be positive.
  • Surface antibodies are immunological and protective antibodies against hepatitis B virus in the body, and are mostly positive during the recovery period. At the same time, the majority of those receiving hepatitis B vaccine were also positive.
  • the e antigen is usually positive after the hepatitis B virus infection, while the surface antigen is positive, or a few days later. e antibody positive appeared several months after the antigen turned negative.
  • the core antibody is usually detected in the serum 3-5 weeks after the appearance of the surface antigen.
  • the treatment of hepatitis B is mainly for reversing hepatitis B virus and other related indicators of hepatitis B virus, including (but not limited to): hepatitis B surface antigen, hepatitis B surface antibody, hepatitis B e antigen, hepatitis B e antibody, hepatitis B core antibody, hepatitis B Viral DNA, and ALT.
  • 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.
  • 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.
  • 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.
  • the stromal vascular component or the adipose-derived stromal vascular component, is the most important component of stem cell-assisted fat transplantation.
  • the cell cluster formed by a mixture of various cells separated from adipose tissue by collagenase digestion is called a fat matrix vascular component.
  • Interstitial blood vessel fragments are rich in mesenchymal cells and can be differentiated into cells of various lineages. They are the most ideal seed cells for regenerative medicine and tissue engineering.
  • VEGF vascular endothelial growth factor
  • HGF Hepatocyte growth factor
  • TGF-beta The transforming growth factor-beta superfamily is a large family of extracellular ligands involved in many biological processes including development, wound healing and cell proliferation and survival.
  • GM-CSF, PDGF, Bfgf, Factors such as IL-2 and IL-10 also have a repairing effect on cells and promote immune function.
  • the concentration of stem cell growth factor (HGF) secreted by the stromal vascular component is > 0.5 ng/ml.
  • the stromal vascular component secretes a concentration of vascular endothelial growth factor (VEGF) of > 35 pg/ml, preferably > 40 pg/ml.
  • VEGF vascular endothelial growth factor
  • the concentration of human transforming growth factor beta (TGF-[beta]) secreted by the stromal vascular component is > 150 pg/ml, preferably > 180 pg/ml.
  • the stromal vascular component secretes a concentration of interleukin-2 (IL-2) ⁇ 15 pg/ml, preferably ⁇ 20 pg/ml, more preferably ⁇ 30 pg/ml.
  • IL-2 interleukin-2
  • the concentration of interleukin-10 (IL-10) secreted by the stromal vascular component is ⁇ 15 pg/ml, preferably ⁇ 20 pg/ml, more preferably ⁇ 30 pg/ml, optimally ⁇ 40 pg/ Ml.
  • Human adipose derived msenchymal progenitor cells (haMPCs): no CD34+ cells, SVF culture P3-P10 generation purified amplification.
  • 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 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.
  • 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.
  • HSC human hematopoietic stem
  • PC progenitor
  • EC vascular endothelial
  • 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, CD73, CD105, CD90, 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 ⁇ 80%, more preferably ⁇ 90%.
  • the proportion of adipose mesenchymal progenitor cells bearing CD73 in total stem cells is preferably ⁇ 70%, more preferably ⁇ 80%.
  • the proportion of adipose mesenchymal progenitor cells bearing CD105 in total stem cells is preferably ⁇ 60%, more preferably ⁇ 70%.
  • the proportion of adipose mesenchymal progenitor cells bearing CD90 in total stem cells is preferably ⁇ 70%, more preferably ⁇ 80%.
  • 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.
  • 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.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of adipose mesenchymal progenitor cells, a fatty matrix vascular component, and a pharmaceutically acceptable carrier.
  • 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.
  • 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.
  • 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.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.
  • 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.
  • 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.
  • the concentration of the adipose mesenchymal progenitor cells in the subcutaneous or intravenous injection is 1 ⁇ 10 5 - 2 ⁇ 10 9 /ml, preferably 1 ⁇ 10 6 - 1 ⁇ 10 9 / ml, more preferably 1 ⁇ 10 7 - 1 ⁇ 10 8 / ml.
  • hepatitis B especially hepatitis B surface antigen, hepatitis B surface antibody, hepatitis B e antigen, hepatitis B e antibody Reversal of hepatitis B virus related indicators such as hepatitis B core antibody.
  • DMEM serum-free medium
  • SFM serum-free medium
  • Type I collagenase 0.1% collagenase I preparation method: Weigh 0.1 g of collagenase I powder and dissolve it in 100 ml of medium without any factor, and preheat it at 37 ° C before;
  • each T175 flask is divided into 50 ml of adipose tissue. 10ml pipette, remove the tip, first absorb the lower layer of red liquid in the fat collection bottle and discard it. The remaining upper layer of fat is mixed and then dispensed.
  • Collagenase I digestion Add an equal amount of freshly prepared collagenase I solution (preheated in a gas bath shaker at 37 ° C for half an hour), seal the sealing membrane, and shake the flask vigorously for 5 to 10 seconds. In a vibrating air bath, digest for 60 minutes at 37 ° C, 70 rpm, shake the flask vigorously for 5 to 10 seconds every 15 minutes until it looks It is smoother.
  • stromal vascular fraction The digested tissue was dispensed into a 50 ml centrifuge tube using a sterile 40 mesh sieve, and centrifuged at 400 g for 10 minutes at room temperature to obtain a fat-derived matrix vascular component.
  • Cell planting After centrifugation, add 20 ml of medium and mix well. Based on the tissue block method: cell cultivation is performed according to the area of the culture bottle. The amount of fat obtained by inoculating 0.16 ml of liposuction per square centimeter was inoculated, that is, 12 ml of liposuction fat was inoculated into each T75 flask. That is, for every 100 ml of fat tissue, 8 T75 flasks can be inoculated. The amount of untreated fat and the resulting cell suspension were converted to inoculate the cells.
  • Primary cell harvest About 7 days, when the percentage of the area of the primary cultured cell clonal reaches 70% to 80%, it is digested and harvested.
  • the volume was adjusted to 50 ml, and the pipette was suspended and suspended, and filtered through a 100-mesh sterile filter. The filtrate was collected into a 50 ml centrifuge tube and centrifuged at 1000 rpm for 10 minutes.
  • the culture vessel was placed in a carbon dioxide constant temperature and humidity incubator to start the culture.
  • Culture conditions carbon dioxide constant temperature and humidity incubator. Conditions: 37 ⁇ 0.5 ° C, carbon dioxide volume fraction is 5 ⁇ 0.2%. Culture to cell fusion up to 85% to 90%.
  • the fold of P1 generation cells can reach 1-2 times;
  • the fold of P2 generation cells can reach 4-6 times;
  • the fold of P3 generation cells can be multiplied by 10 times.
  • HaMPCs were inoculated into a six-well plate at a density of 1.5 ⁇ 10 5 cells/well, and the samples cultured in the normal medium were negative control groups for adipogenic differentiation experiments.
  • the specific method is: adding 1 ⁇ mol/L of dexamethasone, 10 ⁇ mol/L of insulin, 200 ⁇ mol/L of indomethacin and 0.5 mmol/L of isobutylmethyl to the basal medium (DMEM + 10% fetal bovine serum).
  • Astragalus membranaceus was formulated into a lipid-inducing medium and changed twice a week until the adipogenic staining was observed.
  • Each group of samples after adipogenic induction was stained with 0.5% oil red O.
  • the specific operation method is as follows: firstly, the sample cells are thoroughly washed with D-hanks, and then diluted with oil red for 10 to 15 minutes. A random observation of 5 to 10 fields of each group of samples was performed to observe the effect of adipogenic differentiation of the co-culture method.
  • Figure 1 shows oil red O staining of adipocyte differentiation; Oil red O staining control group (negative); the red color in the figure is a lipid droplet, and it can be seen that a large number of dense lipid droplets appear in the cells after adipogenic differentiation.
  • Osteoblast differentiation cultured to the second generation of cells, trypsinized and digested at a density of 1 ⁇ 10 5 ml, and the medium was cultured for 1 d and then replaced with induction medium: DMEM/10% FBS, 0.1 ⁇ mo/L Rice pine, 50 ⁇ mol/L vitamin C, 10 mmol/ ⁇ -glycerophosphate, 100 U/ml penicillin, 100 U/ml streptomycin; medium changed every 3 days.
  • the cells were washed twice with PBS, fixed with 4% formaldehyde, stained with 1% alizarin red for 10 min, and observed under light microscope.
  • the results of osteoblast differentiation of alizarin red staining are shown in Fig. 5, and the results of cell staining of the control group (negative) are shown in Fig. 6.
  • Adipose mesenchymal progenitor cells were cultured for 2 weeks after osteogenic induction. They have osteogenic differentiation potential and stained with alizarin red. It can be seen that there is a large amount of red calcified matrix deposition in the cytoplasm.
  • Example 3 Flow cytometry of vascular components of haMPC and adipose-derived stromal cells and surface antigen detection of vascular components of adipose-derived matrix
  • 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 ⁇ L of the antibody was added, protected from light, and placed on ice for 30 min.
  • the adipose-derived stromal vascular component was cultured in a specific culture chamber for 48 hours, and the supernatant was taken for testing.
  • Umbilical cord stem cells were taken as the control group.
  • the harvested cells were injected into 30 ml of physiological saline to prepare a cell suspension at a concentration of 2 ⁇ 10 5 cells/ml for use in returning.
  • Hepatitis B surface antigen 1805 1646 1255 Hepatitis B surface antibody ⁇ 2.0 ⁇ 2.0 ⁇ 2.0 Hepatitis B e antigen 1284 1143 983 Hepatitis B e antibody 5.38 5.39 3.11 Hepatitis B core antibody 0.005 0.005 0.005 Hepatitis B virus DNA 4.73*10 ⁇ 4 3.73*10 ⁇ 4 6.27*10 ⁇ 3 ALT 63 54 28
  • the cell mixture of haMPC has a therapeutic effect on hepatitis B, which can reduce surface antigen, reduce viral DNA, and reduce ALT, thereby promoting liver function repair.
  • Example 5 Therapeutic effect of combination of adipose-derived stromal vascular components and haMPC on hepatitis B
  • the harvested haMPC and the adipose-derived matrix vascular component were injected into 30 ml of physiological saline to prepare a cell suspension for use in returning.
  • Intravenous injection the amount of cells > 10 8 , according to SVF: haMPCs 1:1 ratio, once a week for four weeks.
  • Two patients with hepatitis B were evaluated for clinical efficacy before cell reinfusion and at 3 and 6 months after reinfusion, respectively.
  • the efficacy of adipose-derived progenitor cells and adipose-matrix vascular component complex in the treatment of hepatitis B was evaluated. .
  • the cell mixture of adipose-derived stromal vascular components and haMPC has a therapeutic effect on hepatitis B, which can reduce surface antigen, reduce viral DNA, and reduce ALT, thereby promoting liver function repair.

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Abstract

L'invention concerne une utilisation d'une composition de cellules progénitrices mésenchymateuses adipeuses et une composition de fraction vasculaire stromale adipeuse optionnelle dans la prévention ou le traitement de l'hépatite. Spécifiquement, la cellule progénitrice mésenchymateuse adipeuse et la fraction vasculaire stromale adipeuse peuvent être utilisées pour préparer une composition pharmaceutique pour le traitement de l'hépatite B.
PCT/CN2015/095204 2014-11-20 2015-11-20 Cellule progénitrice mésenchymateuse adipeuse et composition de fraction vasculaire stromale adipeuse utilisée pour le traitement de l'hépatite b WO2016078616A1 (fr)

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CN201410668392.9 2014-11-20
CN201410668435.3 2014-11-20
CN201410668435.3A CN105663163A (zh) 2014-11-20 2014-11-20 用于治疗乙肝的脂肪间充质祖细胞
CN201410668392.9A CN105663165A (zh) 2014-11-20 2014-11-20 用于治疗乙肝的脂肪间充质祖细胞和脂肪基质血管成分组合物

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013017699A1 (fr) * 2011-08-04 2013-02-07 Ethianum Betriebsgesellschaft Mbh & Co. Kg Moyens et procédés de régénération hépatique
CN103436555A (zh) * 2013-08-30 2013-12-11 浙江大学 携带miR-122脂肪间充质干细胞的构建方法及其应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013017699A1 (fr) * 2011-08-04 2013-02-07 Ethianum Betriebsgesellschaft Mbh & Co. Kg Moyens et procédés de régénération hépatique
CN103436555A (zh) * 2013-08-30 2013-12-11 浙江大学 携带miR-122脂肪间充质干细胞的构建方法及其应用

Non-Patent Citations (2)

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Title
NUNES SS. ET AL.: "Generation of a functional liver tissue mimic using adipose stromal vascular fraction cell -derived vasculatures", SCIENTIFIC REPORTS, vol. 3, no. 2141, 5 July 2013 (2013-07-05) *
WANG Y ET AL.: "Human Adipose-Derived Mesenchymal Stem Cells Are Resistant to HBV Infection during Differentiation into Hepatocytes in Vitro", INT.J.MOL.SCI., vol. 15, no. 4, 10 April 2014 (2014-04-10) *

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