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WO2018033117A1 - 一种全反式维甲酸注射剂与应用 - Google Patents

一种全反式维甲酸注射剂与应用 Download PDF

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Publication number
WO2018033117A1
WO2018033117A1 PCT/CN2017/097868 CN2017097868W WO2018033117A1 WO 2018033117 A1 WO2018033117 A1 WO 2018033117A1 CN 2017097868 W CN2017097868 W CN 2017097868W WO 2018033117 A1 WO2018033117 A1 WO 2018033117A1
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retinoic acid
trans retinoic
tumor
cells
injection
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French (fr)
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徐宇虹
陈晓龙
郑安杰
吴烈宜
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Highfield Biopharmaceutical Corp
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Highfield Biopharmaceutical Corp
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Priority to ES17841089T priority Critical patent/ES2966738T3/es
Priority to US16/326,216 priority patent/US10952984B2/en
Priority to EP17841089.0A priority patent/EP3501517B1/en
Priority to JP2019530536A priority patent/JP6942183B2/ja
Publication of WO2018033117A1 publication Critical patent/WO2018033117A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/203—Retinoic acids ; Salts thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid

Definitions

  • the invention belongs to the field of biopharmaceutical technology, and particularly relates to an all-trans retinoic acid injection and application.
  • Retinoic acid is a metabolite of vitamin A in the body. All-trans retinoic acid (ATRA) is used as a drug to treat acne. It is also an important drug for the clinical treatment of acute promyelocytic leukemia (APL). The current clinical application is through oral preparation.
  • ATRA All-trans retinoic acid
  • All-trans retinoic acid affects gene expression by binding to specific receptors (RARs, RXRs and RORs) in cells, and promotes APL cell differentiation and PML/RAR ⁇ gene degradation in acute promyelocytic leukemia therapy To achieve therapeutic effects (Effectiveness and Pharmacokinetics of Low-Dose All-trans Retinoic Acid (25 mg/m2) in Acute Promyelocytic Leukemia. Blood 82, 12, 1993. P. 3560).
  • CD34+CD33+CD13+ in humans and, like myeloid precursor cells, can form clones on agar.
  • myeloid suppressor cells inhibit the maturation of DC cells by secreting arginase, ROS and IL-6, down-regulating the killing activity of effector T cells.
  • ATRA has also been reported to have a role in promoting tumor stem cell differentiation (Targeting cancer stem cells in glioblastoma multiforme using mTOR inhibitors and the differentiating agent all-trans retinoic acid. ONCOLOGY REPORTS 30: 1645-1650, 2013.
  • all-trans retinoic acid drugs is limited by the following aspects: 1 all-trans retinoic acid has very low water solubility (4.77e-03g/l); 2 all-trans retinoic acid has a short half-life plasma To exert its efficacy, it is necessary to maintain a certain blood concentration for a long period of time, and the concentration of the drug in the target organ. At present, almost all studies use oral preparations, and their bioavailability is only 30% or less. The study found that ATRA has the ability to induce differentiation only when it is maintained above a certain plasma concentration. Therefore, it has been found that a liquid preparation having a higher all-trans retinoic acid apparent concentration is particularly important.
  • an all-trans retinoic acid injection comprising all-trans retinoic acid and a solubilizing molecule.
  • the solubilizing molecule is selected from a combination of any one or more of a lipid, polyoxyethylene castor oil, PVP, HPMC, Pluronic block copolymer, cyclodextrin or PEG.
  • the mass ratio between the solubilizing molecule and all-trans retinoic acid ranges from (10 to 80):1.
  • the lipid is selected from a combination of any one or more of phospholipids, cholesterol or pegylated phospholipids.
  • the mass ratio between the lipid and all-trans retinoic acid ranges from (20 to 80):1.
  • the phospholipid is selected from PC phospholipids. Further preferably, the phospholipid is selected from a combination of any one or more of EPC, HSPC or DPPC.
  • the PEGylated phospholipid has a molecular weight in the range of 50 to 10,000.
  • the all-trans retinoic acid injection is a solution injection, a suspension injection, an emulsion injection, or a sterile powder injection for injection.
  • the all-trans retinoic acid injection is a solution injection, a suspension injection or an emulsion injection
  • the all-trans retinoic acid injection contains a solvent containing an isotonicity adjusting agent.
  • the isotonicity adjusting agent is: sodium chloride.
  • the mass percent concentration of the sodium chloride in the solvent is from 0.5 to 0.9%.
  • the solvent further contains a protective agent.
  • the protective agent is sucrose.
  • the mass percent by volume of the sucrose in the solvent is from 2 to 5%.
  • the mass percent by volume refers to the mass g of the solute contained per 100 ml of the solvent.
  • the concentration of the all-trans retinoic acid is 0.1 mg/ml or more. Further preferably, in the all-trans retinoic acid injection, the concentration of the all-trans retinoic acid is greater than 1.0 mg/ml. More preferably, in the all-trans retinoic acid injection, the concentration of the all-trans retinoic acid ranges from 1 to 5 mg/ml.
  • the administration route of the injection preparation is selected from the group consisting of intradermal injection, subcutaneous injection, intramuscular injection, and intravenous injection.
  • the tumor therapeutic agent is a drug for abnormal myeloid suppressor cells, differentiation of myeloid suppressor cells, inhibition of tumor proliferation and recurrence in a tumor patient.
  • the myeloid suppressor cells are breast cancer, colon cancer, ovarian cancer, lung cancer, kidney cancer, stomach cancer, liver cancer, cervical cancer, endometrial cancer, bladder cancer, prostate cancer, pancreatic cancer, colorectal cancer, Myeloid suppressor cells of basal cell carcinoma, melanoma, follicular lymphoma or small lymphoma.
  • MDSCs myeloid suppressor cells
  • PBMC peripheral blood mononuclear cells
  • a method of treating a tumor comprising the step of administering to the patient the all-trans retinoic acid injection described above.
  • the particular dosage administered will be within the purview of those skilled in the art.
  • the present invention has the following beneficial effects:
  • the all-trans retinoic acid injection prepared by the present invention increases the solubility of all-trans retinoic acid by 0.01 mg/ml in the prior art, and at least 0.1 mg/ml or more, thereby improving at least 10 times.
  • the application of the preparation can effectively regulate the microenvironment of tumor development, and can effectively induce differentiation of tumor-associated macrophages.
  • the plasma terminal half-life of the invention can be as long as 8-12 h when administered by intravenous or intravenous drip.
  • 40% to 70% of myeloid suppressor cell differentiation or apoptosis in tumors can be promoted, and the formation of mature dendritic cells (DCs) can be induced.
  • the level of interleukin-6 (IL-6) secreted by myeloid suppressor cells is significantly decreased by the treatment of the present invention.
  • the effects of immunotherapy alone or in combination can be applied to inhibit tumor cell proliferation and tumor recurrence, and thus have great medical utility.
  • Figure 1 Blood concentration curve of EPC liposomes in mice.
  • Figure 2 MDSC inhibition assay of all-trans retinoic acid injection on mouse tumor site.
  • Figure 3 All-trans retinoic acid injection promotes the differentiation of MDSC in the blood of patients with head and neck mucosal squamous cell carcinoma.
  • Figure 4 All-trans retinoic acid injection significantly reduced iNOS expression in MDSCs in neoplastic patients.
  • Figure 5 All-trans retinoic acid injections on tumor tissues in tumor patients can significantly reduce the number of MDSC phenotype cells in bladder cancer cells infiltrating myeloid cells.
  • Figure 7 All-trans retinoic acid injection promotes apoptosis of human breast cancer cell MCF-7.
  • Figure 8 All-trans retinoic acid injection promotes apoptosis of human peripheral blood leukemia T cell Jukat.
  • Figure 10 All-trans retinoic acid injection increases the number of tumor infiltrating lymphocytes (CD4+ and CD8+ cells).
  • Figure 11 All-trans retinoic acid injection reduced liver tissue infiltration and metastasis of 4T1 tumor-bearing mouse tumor cells (white arrows indicate tumor cell infiltration).
  • Figure 12 All-trans retinoic acid injection reduces renal tissue infiltration and metastasis of 4T1 tumor-bearing mouse tumor cells (white arrows indicate tumor cell infiltration).
  • the all-trans retinoic acid injection of the present invention includes all-trans retinoic acid and a solubilizing molecule.
  • the solubilizing molecule is selected from a combination of any one or more of a lipid, polyoxyethylene castor oil, PVP, HPMC, Pluronic block copolymer, cyclodextrin or PEG.
  • the mass ratio between the solubilizing molecule and all-trans retinoic acid ranges from (10 to 80):1.
  • the lipid is selected from a combination of any one or more of phospholipids, cholesterol or pegylated phospholipids.
  • the mass ratio between the lipid and all-trans retinoic acid ranges from (20 to 80):1.
  • the mass ratio between the lipid and the all-trans is also in the range of (20 to 50):1.
  • the phospholipid may be selected from PC phospholipids. Further, the phospholipid is selected from a combination of any one or more of EPC, HSPC or DPPC.
  • the PEGylated phospholipid has a molecular weight in the range of 50 to 10,000.
  • the PEGylated phospholipid is 2,000.
  • the polyoxyethylene castor oil is Cremophor RH40.
  • the all-trans retinoic acid injection is a solution injection, a suspension injection, an emulsion injection, or a sterile powder injection for injection.
  • the all-trans retinoic acid injection is a solution injection, a suspension injection or an emulsion injection
  • the all-trans retinoic acid injection contains a solvent containing an isotonicity adjusting agent.
  • the isotonicity adjusting agent may be selected from sodium chloride.
  • the mass percent concentration of the sodium chloride in the solvent is from 0.5 to 0.9%.
  • the solvent may also contain a protective agent.
  • the protective agent is sucrose.
  • the mass percent by volume of the sucrose in the solvent is from 2 to 5%.
  • the mass percent by volume refers to the mass g of the solute contained per 100 ml of the solvent.
  • the concentration of the all-trans retinoic acid is 0.1 mg/ml or more. Further, in the all-trans retinoic acid injection, the concentration of the all-trans retinoic acid is greater than 1.0 mg/ml. Further, in the all-trans retinoic acid injection, the concentration of the all-trans retinoic acid ranges from 1 to 5 mg/ml.
  • the administration route of the injection preparation is selected from the group consisting of intradermal injection, subcutaneous injection, intramuscular injection, and intravenous injection.
  • the use may be: the use of an all-trans retinoic acid injection for the preparation of a medicament for treating a tumor.
  • the tumor therapeutic agent is a drug for abnormal myeloid suppressor cells, differentiation of myeloid suppressor cells, inhibition of tumor proliferation and recurrence in a tumor patient.
  • the myeloid suppressor cells are breast cancer, colon cancer, ovarian cancer, lung cancer, kidney cancer, gastric cancer, liver cancer, cervical cancer, endometrial cancer, bladder cancer, prostate cancer, pancreatic cancer, colon cancer, basal cell carcinoma, Myeloid suppressor cells of melanoma, follicular lymphoma or small lymphoma.
  • MDSCs myeloid suppressor cells
  • PBMC peripheral blood mononuclear cells
  • the method for treating a tumor of the present invention comprises the step of administering to the patient the aforementioned all-trans retinoic acid injection.
  • the particular dosage administered will be within the purview of those skilled in the art.
  • the experimental methods, detection methods, and preparation methods disclosed in the present invention employ conventional techniques of molecular biology, biochemistry, analytical chemistry, cell culture, recombinant DNA techniques, and related art in the art. These techniques are well described in the prior literature, see Sambrook et al.
  • MOLECULAR CLONING A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (PBBecker, ed.) Humana Press, Totowa, 1999, and the like.
  • (1) emulsification method dissolving an appropriate amount of the solubilizing molecule in 1 ml of ultrapure water as an aqueous phase; dissolving 1 mg of the drug in 1 ml of an organic solvent as an organic phase; adding the organic phase to the aqueous phase under stirring After stirring overnight, the organic solvent is volatilized or the organic solvent is removed by rotary evaporation to obtain a drug-containing solution.
  • Dialysis method The drug is dissolved in an organic solvent together with the solubilizing molecule, and then mixed with 1 ml of ultrapure water, and the resulting solution is dialyzed in pure water to obtain a drug-containing solution.
  • EPC, HSPC and DPPC were selected as the main lipid materials.
  • Mixing in a molar ratio of PC:Chol:DSPE-PEG2000 2:1:0.125, adding ATRA (wherein the lipid/ATRA mass ratio is 20:1, 40:1, 50:1, respectively), and adding 5-7 when hydrating A small glass bead that is hydrated for 30 minutes.
  • the polycarbonate film having a pore diameter of 400 nm, 200 nm, and 100 nm was sequentially extruded 15 times.
  • the average particle size of the all-trans retinoic liposome was found to be in the range of 100 nm ⁇ 30 nm, and the PDI was about 0.1.
  • ATRA free all-trans retinoic acid
  • the all-trans retinoic acid liposome prepared above is administered by intravenous injection, and the half-life of ATRA in mice is between 4 and 12 hours.
  • the plasma concentration curve of EPC liposome in mice is shown by the red curve in Figure 1, and the calculated half-life is 247 min.
  • HSPC hydrogenated soybean phosphatidylcholine
  • DSPE-PEG2000 pegylated phospholipid 1,2-disuccinoylphosphatidylethanolamine-polyethylene glycol 2000
  • cholesterol 0.031 g
  • the water bath in a Celsius water bath is used to dissolve and mix.
  • the ethanol mixture was added to 6.4 ml of calcium acetate buffer (pH 9.0). And placed in a water bath at 70 degrees Celsius for 30 minutes.
  • the obtained liposome vesicles were sequentially extruded through a polycarbonate membrane having a pore diameter of 400 nm, 200 nm, 100 nm, and 50 nm for 8 times to finally obtain a liposome having an average particle diameter of about 90 nm.
  • the liposome prepared in the previous step was dialyzed through a 10000 pore size dialysis membrane, and the aqueous phase was replaced with a 10% mass fraction, a sucrose solution having a pH of 6-7, and a suspension of 4 mg/ml all-trans retinoic acid was added. And incubating at 60 degrees Celsius for 45 minutes. After the incubation, the free all-trans retinoic acid not loaded into the liposome was removed again with a 10000 pore size dialysis membrane, and finally the whole trans-retinoic acid liposome injection was obtained, wherein ATRA The concentration is at 2.0 mg/ml.
  • the plasma terminal half-life of ATRA in mice can reach 8-12 h.
  • CT-26 cells were cultured to logarithmic growth phase using trypsin digestion, and the digested cells were collected and centrifuged at 300 g for 5 min in a centrifuge, the supernatant was decanted, and the cells were resuspended in sterile PBS. Count the cells and adjust the cell concentration to 1*10 7 cells/ml;
  • mice Purchase a 6-week-old Balb/c mouse, and shave the side of the skin inoculated beforehand.
  • the mice were anesthetized with 4% chloral hydrate 200 ⁇ l. After anesthesia, the mice were subcutaneously injected into the right lower arm.
  • the CT-26 suspension has a cell inoculation amount of 5*10 5 -1*10 6 /piece, and continues to be reared after inoculation;
  • mice were sacrificed by cervical dislocation, and the tumor was removed from the skin using tweezers and scissors, and cut on a 40 ⁇ m cell strainer. Care should be taken to avoid damage to the tumor cells by shearing the tumor tissue while cutting the tissue. Rinse the tissue continuously with 5% PBS;
  • the digested cells were again passed through a 40 ⁇ m cell strainer, and the cells were washed with PBS to remove residual tumor tissue digestive juice and cell debris and dead cells (conditions for centrifugation after washing: rotation speed: 1000 rpm centrifugation time: 5 min), washing 2-3 times.
  • the cells were finally resuspended in PBS, at which time a single cell suspension of the tumor was obtained.
  • the tumor single cell suspension of the incubated magnetic beads is added from above the MS column, and the MS column is washed with 1 ml of Buffer to wash the cells of the unbound magnetic beads to the column;
  • Example 2 For the other all-trans retinoic acid injections of Example 1 and Example 2, the same experimental conclusion was also obtained, that is, it was possible to induce differentiation of the MDSC at the tumor site to cause a decrease in the number of MDSCs.
  • Example 5 All-trans retinoic acid injection promotes phenotypic changes of CD33+ cells in PBMC of patients with head and neck mucosal squamous cell carcinoma
  • 2ml of peripheral blood of patients with head and neck mucosal squamous cell carcinoma was diluted with 2ml of PBS, and 4ml of diluted blood was slowly added to the 3ml human lymphocyte separation solution along the inner wall of the test tube, so that the diluted blood sample was superimposed on the human lymphocyte separation solution. .
  • the room temperature was centrifuged at 300 g per minute for 30 min (acceleration 2, deceleration 1), and the mononuclear cell layer was completely aspirated by a pipette, diluted to 10 ml with PBS solution, and washed twice by centrifugation at 300 g.
  • PBMC peripheral blood mononuclear cell
  • the myeloid cells in the PBMC cells were isolated by magnetic beads labeled with CD33 antibody, referring to the magnetic bead separation apparatus operating manual.
  • the cells were cultured in a RPMI1640 complete medium (addition of 10% FBS) to a 12-well cell plate according to 5 ⁇ 10 5 cells per well, and the whole trans-retinoic acid injection prepared in Example 1 or Example 2 or Example 3 was added for 24 hours.
  • Flow cytometry was used to detect the percentage of HLA-DR + CD11c + phenotypic DC cell population in myeloid cells.
  • the tumor samples of patients with bladder cancer were washed with sterile saline and placed in a cell culture medium containing RPMI 1640 (containing penicillin 100 ⁇ g/ml, streptomycin 100 ⁇ g/ml) containing 10% fetal bovine serum at 4 ° C, and then sent to the laboratory for treatment. .
  • the tumor was placed in 2 ml of medium, and the tissue was minced on ice, and transferred to a 15 ml centrifuge tube containing 2 ml of enzyme digestive solution (collagenase I and IV digest, 0.6 to 1 mg/ml), and gently vortexed and mixed. 37 ° C constant temperature shaker, 200 rpm, 2 h.
  • the myeloid cells in the tumor tissue infiltrating cells were obtained by separation with magnetic beads labeled with CD33 antibody, referring to the magnetic bead separation device operating manual.
  • the cells were cultured in a RPMI1640 complete medium (addition of 10% FBS) to a 12-well cell plate according to 5 ⁇ 10 5 cells per well, and the all-trans retinoic acid injection prepared in Example 1 or Example 2 or Example 3 was added. 24 hours. Flow cytometry was then used to detect the percentage of CD33 + HLA-DR - MDSC cell population in myeloid cells.
  • the all-trans retinoic acid injection prepared in Example 3 of the present invention acts on tumor tissues in tumor patients, and can significantly reduce the CD33+HLA-DR-MDSC phenotype in bladder cancer infiltrating myeloid cells. The number of cells. As shown in Fig. 6, the all-trans retinoic acid injection prepared in Example 3 of the present invention can reduce the inhibitory effect of MDSC on T cells in tumor patients.
  • the tumor tissue acting on the tumor patient can significantly reduce the number of CD33+HLA-DR-MDSC phenotype cells in the bladder cancer infiltrating myeloid cells.
  • the all-trans retinoic acid injection can reduce the inhibitory effect of MDSC on T cells in a tumor patient.
  • 5x10 5 MCF-7 cells were inoculated on a 6-well cell culture plate one day before the experiment, and cultured overnight at 37 ° C in a 5% CO 2 cell incubator. On the next day, after removing the culture medium from the cell culture plate, fresh medium and the all-trans retinoic acid injection drug prepared in Example 1 or Example 2 or Example 3 were added, and the solution was cultured in a 5% CO 2 cell incubator at 37 ° C. Culture for 24h.
  • the cells were collected by centrifugation at 300 g for 5 min at 4 ° C, and then the cells were washed twice with pre-cooled PBS, 300 g each time, and centrifuged at 4 ° C for 5 min to collect 1 to 5 ⁇ 10 5 cells.
  • the PBS was aspirated and the cells were resuspended by adding 100 ⁇ L of 1 ⁇ Binding Buffer. Add 5 ⁇ L of Annexin V-FITC and 10 ⁇ L of PI Staining Solution and mix gently. After 10-15 min in the dark and room temperature, 400 ⁇ L of 1 ⁇ Binding Buffer was added, mixed and placed on ice, and the sample was detected by flow cytometry within 1 hour.
  • the all-trans retinoic acid injection prepared in Example 3 of the present invention can significantly promote human breast cancer cells.
  • MCF-7 apoptosis.
  • each of the all-trans retinoic acid injections prepared in Examples 1 and 2 can significantly promote apoptosis of human breast cancer cell MCF-7.
  • Example 8 all-trans retinoic acid injection promotes apoptosis of human peripheral blood leukemia T cell Jukat
  • the PBS was aspirated and the cells were resuspended by adding 100 ⁇ L of 1 ⁇ Binding Buffer. Add 5 ⁇ L of Annexin V-FITC and 10 ⁇ L of PI Staining Solution and mix gently. After 10-15 min in the dark and room temperature, 400 ⁇ L of 1 ⁇ Binding Buffer was added, mixed and placed on ice, and the sample was detected by flow cytometry within 1 hour.
  • the all-trans retinoic acid injection prepared in Example 3 of the present invention can significantly promote the apoptosis of human peripheral blood leukemia T cell Jukat.
  • each of the all-trans retinoic acid injections prepared in Examples 1 and 2 can significantly promote the apoptosis of human peripheral blood leukemia T cell Jukat.
  • the mouse tumor was grown to about 10 mm 3 , and the all-trans retinoic acid injection (5 mg/kg) prepared in Example 1 or Example 2 or Example 3 was injected into the tail vein, and the mice were administered every other day. The same amount of all-trans retinoic acid injection was injected and administered every other day. The body weight of the mice was observed, and the average tumor volume was measured and counted, and a growth curve was drawn.
  • the all-trans retinoic acid injection prepared in Example 3 of the present invention acts on 4T1 tumor-bearing mice, and the tumor tissue volume of the mice is reduced.
  • Tumor tissue was excised from the 4T1 tumor-bearing mice of Example 9 and fixed in 4% formaldehyde solution for 3-5 days.
  • the tissue was removed from the fixative and trimmed to the appropriate shape and thickness; the tissue block was 80%, 90%. %, 95%, 100% ethanol I, 100% ethanol II, 100% ethanol III were dehydrated, and then treated with xylene I for 30 min, xylene II for 30 min, paraffin I for 1 h, paraffin II for 6 h; According to the principle of taking the material facing down, the tissue was embedded in paraffin.
  • the slice thickness was 4 ⁇ m, and the slice was placed in a 65 ° C incubator for 6-12 h, and the box was stored at room temperature.
  • paraffin immunohistochemistry procedure was as follows: Paraffin sections were deparaffinized to water and incubated at 3% H 2 O 2 for 5-10 minutes at room temperature to eliminate endogenous peroxidase activity. Rinse with distilled water and soak for 5 minutes in PBS and repeat twice. 5-10% normal goat serum (diluted in PBS) was blocked, incubated for 10 minutes at room temperature, and the serum was decanted and not washed. The primary antibody working solution was added dropwise, and incubated at 37 ° C for 1-2 hours or 4 ° C overnight. Rinse with PBS for 5 minutes each time and repeat three times. Add appropriate amount of secondary antibody working solution and incubate at 37 ° C for 10-30 minutes. Rinse with PBS for 5 minutes each time and repeat three times. After 3-15 minutes of color development of the DAB developer, the tap water is thoroughly rinsed, counterstained, dehydrated, transparent, and sealed.
  • the all-trans retinoic acid injection prepared in Example 3 of the present invention acts on 4T1 tumor-bearing mice, and the number of lymphatic infiltrating cells (CD4+ and CD8+ T cells) in the tumor tissues of the mice is increased.
  • each of the all-trans retinoic acid injections prepared in Examples 1 and 2 was applied to 4T1 tumor-bearing mice, and the number of lymphatic infiltrating cells (CD4+ and CD8+ T cells) in the tumor tissues of the mice was increased.
  • Liver tissue and kidney tissue were excised from the 4T1 tumor-bearing mice of Example 9. After fixing for 3-5 days in 4% formaldehyde solution, the tissue was taken out from the fixative and trimmed to an appropriate shape and thickness; %, 90%, 95%, 100% ethanol I, 100% ethanol II, 100% ethanol III were dehydrated, then treated with xylene I for 30 min, xylene II for 30 min, paraffin I for 1 h, paraffin II dip Wax 6h; according to the principle of taking the material facing down, embed the tissue with paraffin, wait for the wax block to cool and solidify and store at -20 °C; slice thickness 4 ⁇ m, slice into 65 °C incubator for 6-12h, box, room temperature save.
  • the HE staining procedure of paraffin tissue is as follows: first dewaxing rehydration: xylene I 15 min, xylene II 15 min, anhydrous Ethanol I 5 min, absolute ethanol II 5 min, 95% ethanol 5 min, 80% ethanol 5 min, tap water for 1 min; staining: the slice was immersed in hematoxylin staining solution for 5 min at room temperature, tap water for 1 min; the slice was immersed in 1% hydrochloric acid Alcohol solution for a few seconds, tap water until the tissue returns to blue; the slice is immersed in Yihong dye solution for 3-5min, tap water can be washed off the floating color on the slide; dehydrated, transparent, sealed: 80% ethanol 0.5min, 95% ethanol I 0.5 min, 95% ethanol II 0.5 min, absolute ethanol I 0.5 min, absolute ethanol II 0.5 min, xylene I transparent for 3 min, xylene II transparent for 3 min, sealed with neutral gum and observed for staining results
  • the all-trans retinoic acid injection prepared in Example 3 of the present invention acts on 4T1 tumor-bearing mice, and the tumor cells infiltrate and metastasize in the liver of the mice compared with the control group.
  • Fig. 11 Comparison of tumor cell infiltration and metastasis in the kidney The control group had less renal tissue (Fig. 12).

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Abstract

本发明提供一种全反式维甲酸注射剂及其在制备肿瘤治疗药物中的用途。所述全反式维甲酸注射剂包括全反式维甲酸和助溶分子，将全反式维甲酸的表观溶解度由0.01mg/ml提升至0.1mg/ml以上。该制剂可以降低肿瘤病人血液中和肿瘤组织中浸润的免疫抑制细胞群的活性，提高针对肿瘤的免疫清除机制的效果，单独使用或联合其他药物应用可以抑制肿瘤生长和肿瘤复发。

Description

一种全反式维甲酸注射剂与应用 技术领域
本发明属于生物制药技术领域，具体涉及一种全反式维甲酸注射剂与应用。
背景技术
维甲酸为体内维生素A代谢产物。全反式维甲酸(ATRA)作为药物被用来治疗粉刺，同时也是临床上治疗急性早幼粒细胞白血病(acute promyelocytic leukemia，APL)的重要药物，目前临床上的应用均通过口服制剂给药。
全反式维甲酸(ATRA)通过结合至细胞内的特定受体(RARs，RXRs和RORs)从而影响基因表达，在急性早幼粒细胞白血病治疗中，可以促进APL细胞分化和PML/RARαgene的降解，达到治疗的效果(Effectiveness and Pharmacokinetics of Low-Dose All-trans Retinoic Acid(25mg/m2)in Acute Promyelocytic Leukemia。Blood 82，12，1993.P.3560)。也有文献提示，ATRA能够促进肿瘤病人体内的髓样抑制性细胞的分化，下调肿瘤组织中各类髓样抑制性细胞的数量(All-trans-Retinoic Acid Improves Differentiation of Myeloid Cells and Immune Response in Cancer Patients.Cancer Res 2006；66：(18).September 15，2006)。髓样抑制性细胞最初发现于20世纪80年代。其主要由处于不同分化程度的相对未成熟细胞组成。在荷瘤小鼠的骨髓、血液、淋巴器官和肿瘤浸润部位发现有大量标志为CD11b和Gr-1的髓样细胞。在人体中这些细胞为CD34+CD33+CD13+，并且与髓样前体细胞一样，能在琼脂上形成克隆。在肿瘤微环境中，髓样抑制性细胞通过分泌精氨酸酶、ROS和IL-6，下调效应T细胞的杀伤活性，抑制DC细胞成熟。此外，ATRA还被报道具有促进肿瘤干细胞分化的作用(Targeting cancer stem cells in glioblastoma multiforme using mTOR inhibitors and the differentiating agent all-trans retinoic acid.ONCOLOGY REPORTS 30：1645-1650，2013。
然而，全反式维甲酸的药物的临床应用受到以下几个方面的限制：1全反式维甲酸的水溶性极低(4.77e-03g/l)；2全反式维甲酸血浆半衰期较短，而发挥其药效需要在一个较长的时间内维持一定的血药浓度，和在靶器官中的药效浓度。而目前几乎所有研究都使用的是口服制剂，其生物利用度只有30％以下。研究发现ATRA只在维持在一定的血药浓度以上才具有诱导分化的能力。因此，发现一种具有较高全反式维甲酸表观浓度的液体制剂尤为重要。
发明内容
为了克服现有技术中所存在的问题，本发明的目的在于提供一种全反式维甲酸注射剂与应用。
为了实现上述目的以及其他相关目的，本发明采用如下技术方案：
本发明的第一方面，提供一种全反式维甲酸注射剂，所述全反式维甲酸注射剂包括全反式维甲酸和助溶分子。
优选地，所述助溶分子选自脂质、聚氧乙烯蓖麻油、PVP、HPMC、Pluronic嵌段共聚物、环糊精或PEG中的任一种或多种的组合。
优选地，所述助溶分子与全反式维甲酸之间的质量比例范围是(10～80)∶1。
优选地，所述脂质选自磷脂、胆固醇或聚乙二醇化的磷脂中的任一种或多种的组合。
优选地，所述脂质与全反式维甲酸之间的质量比例范围是：(20～80)∶1。
优选地，所述磷脂选自PC磷脂。进一步优选地，所述磷脂选自EPC、HSPC或DPPC中任一种或多种的组合。
优选地，所述聚乙二醇化的磷脂的分子量范围是50～10000。
优选地，所述全反式维甲酸注射剂为溶液型注射剂、混悬型注射剂、乳剂型注射剂、或注射用无菌粉针剂。
进一步优选地，当所述全反式维甲酸注射剂为溶液型注射剂、混悬型注射剂或乳剂型注射剂时，所述全反式维甲酸注射剂中含有溶剂，所述溶剂中含有等渗调节剂。
优选地，所述等渗调节剂为：氯化钠。所述氯化钠在溶剂中的质量体积百分浓度是0.5～0.9％。
优选地，所述溶剂中还含有保护剂。所述保护剂为蔗糖。所述蔗糖在溶剂中的质量体积百分浓度是2～5％。
质量体积百分浓度是指每100ml溶剂中含有的溶质的质量g。
优选地，所述全反式维甲酸注射剂中，所述全反式维甲酸的浓度大于等于0.1mg/ml。进一步优选地，所述全反式维甲酸注射剂中，所述全反式维甲酸的浓度大于等1.0mg/ml。更优选地，所述全反式维甲酸注射剂中，所述全反式维甲酸的浓度范围是1～5mg/ml。
进一步优选地，所述注射制剂的给药途径选自皮内注射、皮下注射、肌内注射剂、静脉注射。
本发明的第二方面，提供前述全反式维甲酸注射剂在制备肿瘤治疗药物中的用途。
优选地，所述肿瘤治疗药物为针对肿瘤病人中异常的髓系抑制性细胞、诱导髓系抑制性细胞分化、抑制肿瘤增殖和复发的药物。
进一步优选地，所述髓系抑制性细胞为乳腺癌、结肠癌、卵巢癌、肺癌、肾癌、胃癌、肝癌、宫颈癌、子宫内膜癌、膀胱癌、前列腺癌、胰腺癌、大肠癌、基底细胞癌、黑色素瘤、滤泡性淋巴癌或小淋巴细胞瘤的髓系抑制性细胞。
本发明的第三方面，提供前述全反式维甲酸注射剂在制备药物中的用途，所述药物起到以下作用的任一种或多种：
(1)降低肿瘤浸润细胞中的髓系抑制性细胞(MDSC)的数量；
(2)诱导肿瘤浸润CD33+HLA-DR-细胞的分化；
(3)促进肿瘤患者外周血单核细胞(PBMC)中CD33+细胞的表型变化；
(4)降低外周血单核细胞(PBMC)中CD33+HLA-DR-细胞对T细胞的抑制作用；
(5)诱导肿瘤细胞的凋亡；
(6)增加肿瘤组织中浸润淋巴细胞比例；
(7)抑制肿瘤细胞转移；
(8)延缓肿瘤生长。
本发明的第四方面，提供一种治疗肿瘤的方法，包括步骤：给患者施用前述全反式维甲酸注射剂。具体给药剂量在本领技术人员所熟知的范围内。
与现有技术相比，本发明具有如下有益效果：
(1)通过长期实验发现并证明本发明所制备的全反式维甲酸注射剂将全反式维甲酸的溶解度由现有技术中的0.01mg/ml，提升至少0.1mg/ml以上，提高了至少10倍。
(2)应用该制剂可以有效调节肿瘤发展的微环境，并可以有效地诱导肿瘤部位相关巨噬细胞的分化。通过静脉注射或静脉滴注给药时，该发明的血浆终末半衰期可达到8-12h。通过本发明治疗以后，可促进肿瘤中的40％～70％的髓系抑制性细胞分化或凋亡，并诱导成熟树突状细胞(DCs)的形成。通过本发明治疗后髓系抑制性细胞分泌的白细胞介素6(IL-6)发生显著水平下降。单独使用或联合应用均有免疫治疗的效果，可以应用于抑制肿瘤细胞增值和肿瘤复发，因此具有极大的医疗实用性。
附图说明
图1：EPC脂质体在小鼠中的血药浓度曲线。
图2：全反式维甲酸注射剂对小鼠肿瘤部位细胞MDSC抑制实验。
图3：全反式维甲酸注射剂促进头颈部黏膜鳞状细胞癌病人血液中MDSC的分化。
图4：全反式维甲酸注射剂使瘤病人MDSC中的iNOS表达明显降低。
图5：全反式维甲酸注射剂作用于肿瘤病人体内的肿瘤组织，可以明显减少膀胱癌肿瘤浸润髓源细胞中MDSC表型细胞的数量。
图6：全反式维甲酸注射剂使瘤病人MDSC对T细胞的抑制作用降低。
图7：全反式维甲酸注射剂促进人乳腺癌细胞MCF-7的凋亡。
图8：全反式维甲酸注射剂促进人外周血白血病T细胞Jukat的凋亡。
图9：全反式维甲酸注射剂减小4T1荷瘤小鼠肿瘤组织体积。
图10：全反式维甲酸注射剂增加肿瘤浸润淋巴细胞(CD4+和CD8+细胞)的数目。
图11：全反式维甲酸注射剂使4T1荷瘤小鼠肿瘤细胞的肝组织浸润转移降低(白色箭头示肿瘤细胞浸润灶)。
图12：全反式维甲酸注射剂使4T1荷瘤小鼠肿瘤细胞的肾组织浸润转移降低(白色箭头示肿瘤细胞浸润灶)。
具体实施方式
全反式维甲酸注射剂
本发明的全反式维甲酸注射剂包括全反式维甲酸和助溶分子。
所述助溶分子选自脂质、聚氧乙烯蓖麻油、PVP、HPMC、Pluronic嵌段共聚物、环糊精或PEG中的任一种或多种的组合。
所述助溶分子与全反式维甲酸之间的质量比例范围是(10～80)∶1。
所述脂质选自磷脂、胆固醇或聚乙二醇化的磷脂中的任一种或多种的组合。
所述脂质与全反式维甲酸之间的质量比例范围是：(20～80)∶1。所述脂质与全反式之间的质量比例范围也可以是：(20～50)∶1。
所述磷脂可选自PC磷脂。进一步地，所述磷脂选自EPC、HSPC或DPPC中任一种或多种的组合。
所述聚乙二醇化的磷脂的分子量范围是50～10000。
本发明一实施例中，列举了所述聚乙二醇化的磷脂的是2000。
本发明一实施例中，所述聚氧乙烯蓖麻油采用Cremophor RH40。
所述全反式维甲酸注射剂为溶液型注射剂、混悬型注射剂、乳剂型注射剂、或注射用无菌粉针剂。
进一步地，当所述全反式维甲酸注射剂为溶液型注射剂、混悬型注射剂或乳剂型注射剂时，所述全反式维甲酸注射剂中含有溶剂，所述溶剂中含有等渗调节剂。
所述等渗调节剂可选用氯化钠。所述氯化钠在溶剂中的质量体积百分浓度是0.5～0.9％。
所述溶剂中还可含有保护剂。所述保护剂为蔗糖。所述蔗糖在溶剂中的质量体积百分浓度是2～5％。
质量体积百分浓度是指每100ml溶剂中含有的溶质的质量g。
所述全反式维甲酸注射剂中，所述全反式维甲酸的浓度大于等于0.1mg/ml。进一步地，所述全反式维甲酸注射剂中，所述全反式维甲酸的浓度大于等1.0mg/ml。更进一步地，所述全反式维甲酸注射剂中，所述全反式维甲酸的浓度范围是1～5mg/ml。
进一步地，所述注射制剂的给药途径选自皮内注射、皮下注射、肌内注射剂、静脉注射。
全反式维甲酸注射剂的用途
本发明中，所述用途可以是：全反式维甲酸注射剂在制备肿瘤治疗药物中的用途。
所述肿瘤治疗药物为针对肿瘤病人中异常的髓系抑制性细胞、诱导髓系抑制性细胞分化、抑制肿瘤增殖和复发的药物。
所述髓系抑制性细胞为乳腺癌、结肠癌、卵巢癌、肺癌、肾癌、胃癌、肝癌、宫颈癌、子宫内膜癌、膀胱癌、前列腺癌、胰腺癌、大肠癌、基底细胞癌、黑色素瘤、滤泡性淋巴癌或小淋巴细胞瘤的髓系抑制性细胞。
所述用途还可以是在制备药物中的用途，所述药物起到以下作用的任一种或多种：
(1)降低肿瘤浸润细胞中的髓系抑制性细胞(MDSC)的数量；
(2)诱导肿瘤浸润CD33+HLA-DR-细胞的分化；
(3)促进肿瘤患者外周血单核细胞(PBMC)中CD33+细胞的表型变化；
(4)降低外周血单核细胞(PBMC)中CD33+HLA-DR-细胞对T细胞的抑制作用；
(5)诱导肿瘤细胞的凋亡；
(6)增加肿瘤组织中浸润淋巴细胞比例；
(7)抑制肿瘤细胞转移；
(8)延缓肿瘤生长。
治疗肿瘤的方法
本发明治疗肿瘤的方法，包括步骤：给患者施用前述全反式维甲酸注射剂。具体给药剂量在本领技术人员所熟知的范围内。
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。下列实施例中未注明具体条件的试验方法，通常按照常规条件，或者按照各制造商所建议的条件。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明，具体可参见Sambrook等MOLECULAR CLONING：A LABORATORY MANUAL，Second edition，Cold Spring Harbor Laboratory Press，1989 and Third edition，2001；Ausubel等，CURRENT PROTOCOLS IN MOLECULAR BIOLOGY，John Wiley&Sons，New York，1987and periodic updates；the series METHODS IN ENZYMOLOGY，Academic Press，San Diego；Wolffe，CHROMATIN STRUCTURE AND FUNCTION，Third edition，Academic Press，San Diego，1998；METHODS IN ENZYMOLOGY，Vol.304，Chromatin(P.M.Wassarman and A.P.Wolffe，eds.)，Academic Press，San Diego，1999；和METHODS IN MOLECULAR BIOLOGY，Vol.119，Chromatin Protocols(P.B.Becker，ed.)HumanaPress，Totowa，1999等。
实施例1
本实施例采用以下三种方法考察不同助溶分子对ATRA增溶后的饱和溶解度。
(1)乳化法：将适量助溶分子溶于1ml超纯水中，作为水相；将1mg药物溶解于1ml有机溶剂中，作为有机相；在搅拌的条件下将有机相加入水相中，搅拌过夜，使有机溶剂挥发或通过旋转蒸发法除去有机溶剂，得到含有药物的溶液。
(2)透析法：将药物与助溶分子一起溶于有机溶剂中，再与1ml超纯水混合，将得到的溶液在纯水中透析，得到含有药物的溶液。
(3)冷冻干燥法：将适量助溶分子溶于1ml纯水中，加入适量冻干保护剂，作为水相；将药物溶解于有机溶剂(叔丁醇，TBA)中，作为有机相；将有机相与水相按一定的比例混合均匀，放入冻干机中冻干，24小时后取出，加入1ml超纯水复溶，得到药物的溶液。
筛选获得以下含有以下助溶分子的注射剂：
将20％CremophorRH40与5％甘露醇溶于1ml纯水中，作为水相；将3mg药物ATRA溶解于1.5ml叔丁醇中，作为有机相；将有机相与水相混合均匀，装入5ml西林瓶中，放入冻干机中冻干，24小时后取出，封盖。给药前，加入1ml注射用水复溶，得到含有药物的混悬液。测定其中全反式维甲酸的饱和溶解度为0.1mg/ml。用动态激光散射方法测量，混悬液中的粒子平均粒径为274nm±42nm，并且可以在室温下放置10小时保持粒径不变，具有特别好的稳定性和分散性。
实施例2
分别选用EPC、HSPC、DPPC作为主要脂质材料。按照PC∶Chol∶DSPE-PEG2000＝2∶1∶0.125的摩尔比混合，加入ATRA(其中脂质/ATRA质量比分别为20∶1、40∶1、50∶1)，水合时加入5-7颗小玻璃珠，旋转水合30min。依次挤压通过孔径为400nm，200nm，100nm聚碳酯膜各15次。得到全反式维甲酸脂质体的平均粒径在100nm±30nm范围内，PDI约为0.1。通过葡聚糖凝胶G-50微柱除去游离全反式维甲酸(ATRA)，在全反式维甲酸的标准曲线线性范围内，通过除去包封到脂质体内的ATRA紫外吸收值峰面积与未除前ATRA紫外吸收峰面积的比值，即得到ATRA脂质体的包封率，分别为EPC脂质体：94％；HSPC脂质体：91％；DPPC脂质体：76％。三种脂质体制剂中ATRA的浓度均在0.3mg/ml以上。
以上制备的全反式维甲酸脂质体通过静脉注射给药，在小鼠体内的ATRA的半衰期在4～12小时之间。其中EPC脂质体在小鼠中的血药浓度曲线如图1中红色曲线所示，经计算半衰期其为247min。
实施例3
称取0.097克氢化豆磷脂(HSPC)、0.031克聚乙二醇化磷脂1，2-一二硬脂酰磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000)及0.031克胆固醇，用1.6毫升的乙醇溶解并于70 摄氏度水浴锅中水浴以溶解混合。将乙醇混合物加入6.4毫升的乙酸钙缓冲液(pH为9.0)。并置于70摄氏度中水浴30分钟。将所得脂质体囊泡依次挤压通过孔径为400nm，200nm，100nm，50nm聚碳酯膜各8次，最终得到平均粒径约为90nm左右的脂质体。
将上一步所制得的脂质体通过10000孔径透析膜透析将水相置换为10％质量分数，pH为6～7的蔗糖溶液中，加入4mg/ml的全反式维甲酸的混悬液，并于60摄氏度中孵育45分钟，孵育结束后再次用10000孔径透析膜将未载入脂质体的游离全反式维甲酸去除，最终得到全反式维甲酸脂质体注射液，其中ATRA的浓度在2.0mg/ml。
通过静脉注射或静脉滴注给药时，在小鼠中ATRA的血浆终末半衰期可达到8-12h。
实施例4、全反式维甲酸注射剂体外诱导肿瘤髓系抑制性细胞分化
1.Balb/c小鼠的肿瘤模型建立
(1)CT-26细胞培养至对数生长期时使用胰酶消化，将消化下的细胞收集并于离心机中以300g转速离心5min，倒去上清液，并用无菌PBS重悬细胞，细胞计数，并调整细胞浓度为1*107个细胞/毫升；
(2)购买6周大小的Balb/c白鼠，将皮下接种的一侧皮毛事先剃干净，用4％的水合氯醛200μl腹腔注射麻醉小鼠，麻醉后于右侧腋下部位皮下注射消化下来的CT-26悬液，细胞接种量为5*105-1*106/只，接种后继续饲养；
(3)大约饲养2-3周后，使用游标卡尺测量肿瘤长径及短径，通过肿瘤体积计算公式计算肿瘤大小：V＝1/2*长径*短径2，肿瘤体积约生长至100mm3时即可进行实验。
2.肿瘤相关淋巴细胞MDSC表征和分选
(1)断颈处死小鼠，使用镊子与剪刀从皮下去除肿瘤，并于40μm细胞滤网上剪碎，注意剪碎肿瘤组织时应小心避免剪切力对肿瘤细胞的损伤，同时在剪碎组织的过程中用含有5％的PBS不断冲洗组织；
(2)剪碎组织后将所有组织及冲洗PBS液一起离心，盗去上清后将所有组织一起转移至含1ml肿瘤组织消化液的15ml离心管中，于37℃、200rpm/min的摇床条件下消化1h；
(3)消化后的细胞再次通过40μm细胞滤网，使用PBS清洗细胞以去除残留的肿瘤组织消化液及细胞碎片与死细胞(清洗后离心时的条件为转速：1000rpm离心时间：5min)，清洗2-3遍。最终使用PBS重悬，此时得到肿瘤单细胞悬液。
(4)以每107个细胞/90μl体积的比例加入分选buffer并以每107个细胞/10μl体积的比 例加入CD11b磁珠；
(5)将磁珠与细胞充分混匀，于4℃避光条件下孵育30min；孵育结束后按每107个细胞/1ml体积的比例加入90μl Buffer，1000rpm条件下离心5min，离心结束后使用Buffer继续清洗2遍；
(6)最终加入500μl Buffer重悬制得孵育磁珠的肿瘤单细胞悬液；
(7)将MS柱放入配套的磁铁中，预先使用Buffer进行润洗，使MS柱得到充分饱和；
(8)润洗结束后将孵育磁珠的肿瘤单细胞悬液从MS柱上方加入，并用1ml Buffer冲洗MS柱以将未结合磁珠的细胞冲洗下柱子；
(9)反复冲洗3-5遍后取下MS柱，放入15ml离心管中，加入1ml Buffer并用MS柱配套仪器将其快速推下，此时收集到的是停留在MS柱上的CD11b阳性细胞。
(10)将上一步得到的CD11b阳性细胞悬液进行细胞计数，将细胞浓度调整至107/ml，制备流式细胞术样品；
(11)流式细胞样品分为：阴性组、Gr-1单阳性组、CD11b单阳性组、待测样品组。每组设置2-3个平行管。其中阴性组不加任何荧光抗体以设置阴性条件，2个单阳性分别加入单一荧光抗体以便后续荧光补偿，待测样品组加入待测的荧光抗体；
(12)所有流式管中各加入100μl体积使用流式Staining Buffer重悬的细胞(约106个)，阴性对照组中不加入任何荧光抗体，Gr-1单阳性组加入Gr-1抗体，CD11b单阳性组加入CD11b抗体，待测样品组同时加入Gr-1与CD11b抗体；
(13)于4℃避光条件下孵育30min，孵育结束后加入1ml Staining Buffer离心以洗去未结合至细胞上的抗体，并接着使用Staining Buffer清洗细胞2次，最后用500μl体积Staining Buffer进行细胞重悬；流式细胞仪检测。
3.全反式维甲酸注射剂对外周血中淋巴细胞及肿瘤部位淋巴细胞的诱导分化作用
(1)按实施例1，2，3的方法，制备全反式维甲酸注射剂；
(2)分选肿瘤组织的中的MDSC细胞，细胞计数后将细胞浓度调整为调整107/ml，将分选后的收集到的细胞接种到24孔版中，使得每孔最终细胞浓度为106/孔；
(3)向铺好细胞的孔板中依次加入20μl PBS、20μl全反式维甲酸注射剂、50μl全反式维甲酸注射剂，37℃中孵育24h；
(4)孵育1天后使用Gr-1及CD11b、CD11c、CD80、CD86、MHC-II抗体对细胞进行孵育，并通过流式细胞仪以观察细胞中MDSC及DC的变化，在流式样品处理过程中注意加入同型对照及做好荧光补偿。
对于，实施例3所制备的全反式维甲酸注射剂，如图2所示，通过给予不同剂量的药物我们可以观察到，Gr-1hi细胞数发生了显著的下降，在高剂量给药组(+50μl全反式维注射剂)中Gr-1hi细胞比例从对照组中17.5％下降至9.10％，而Gr-1int细胞在比例上变化看起来并不明显，可能是由于高表达的Gr-1在全反式维甲酸的诱导作用下转变为了中低表达。而Gr-1low细胞比例随着给药剂量的增加而上升，进一步说明全反式维甲酸诱导MDSC发生分化，从而导致Gr-1表达量下降。由于肿瘤部位大部分浸润的MDSC具有Gr-1hi或Gr-1int的表征，因此我们可以推断全反式维甲酸注射剂能够诱导肿瘤部位MDSC发生分化从而导致MDSC数量的减少。
对于实施例1和实施例2中的其他全反式维甲酸注射剂，也得到了同样的实验结论，亦即，能够诱导肿瘤部位MDSC发生分化从而导致MDSC数量的减少。
实施例5、全反式维甲酸注射剂促进头颈部黏膜鳞状细胞癌病人体内PBMC中CD33+细胞表型变化
取头颈部黏膜鳞状细胞癌病人外周血2ml加入2ml PBS稀释一倍，将4ml稀释血液沿试管内壁缓缓加入3ml人淋巴细胞分离液内，使稀释血液标本叠加在人淋巴细胞分离液上。室温每分钟300g离心力30min(加速2，减速1)，用吸管将单个核细胞层全部吸出，用PBS液稀释至10ml，300g离心洗涤2次。弃上清后加入少量PBS，得到大量PBMC，4℃备用。参照磁珠分离设备操作手册，用CD33抗体标记的磁珠分离获得PBMC细胞中的髓源性细胞。按照每孔5x105个细胞，用RPMI1640完全培养基(添加10％FBS)培养细胞至12孔细胞板，加入实施1或实施例2或实施例3制备获得的全反式维甲酸注射剂培养24小时，运用流式细胞仪检测HLA-DR+CD11c+表型DC细胞群在髓源性细胞中所占百分比。
如图3所示，对于实施例3所制备的全反式维甲酸注射剂，运用流式细胞仪检测HLA-DR+CD11c+表型DC细胞群在髓源性细胞中所占百分比，发现其数量显著增加。此外，如图4所示，这些细胞的iNOS的表达也显著下降。此外，Arg-1，IL-6等的表达水平也显著下降。
对于实施例1和2中的其他各全反式维甲酸注射剂，也得到了同样的实验结论，亦即，采用了其全反式维甲酸注射剂后，能够使得HLA-DR+CD11c+表型DC细胞群在髓源性细胞中所占百分比显著增加，这些细胞的Arg-1，iNOS，IL-6等的表达水平也显著下降。
实施例6、全反式维甲酸注射剂明显减少膀胱癌肿瘤浸润髓源细胞中的CD33+HLA-DR-MDSC数量
取膀胱癌病人肿瘤样本，无菌盐水冲洗干净后放入4℃含10％胎牛血清的RPMI 1640(含青霉素100μg/ml，链霉素100μg/ml)细胞培养液中，送入实验室处理。将肿瘤置于2ml培养基中冰上切碎组织，转移至含2ml酶消化液(胶原蛋白酶I和IV消化液，0.6～1mg/ml)的15ml离心管中，轻轻的涡旋混匀。37℃恒温摇床，200rpm，2h。然后用无菌PBS缓慢冲洗，过40μm膜至50ml离心管。300g离心10min后用PBS重悬细胞并计数。参照磁珠分离设备操作手册，用CD33抗体标记的磁珠分离获得肿瘤组织浸润细胞中的髓源性细胞。按照每孔5x105个细胞，用RPMI1640完全培养基(添加10％FBS)培养细胞至12孔细胞板，加入实施例1或实施例2或实施例3制备好的全反式维甲酸注射剂药物培养24小时。然后运用流式细胞仪检测CD33+HLA-DR-MDSC细胞群在髓源性细胞中所占百分比。
结果，如图5所示，本发明实施例3制备的全反式维甲酸注射剂作用于肿瘤病人体内的肿瘤组织，可以明显减少膀胱癌肿瘤浸润髓源细胞中CD33+HLA-DR-MDSC表型细胞的数量。如图6所示，本发明实施例3制备的全反式维甲酸注射剂能够使瘤病人MDSC对T细胞的抑制作用降低。
对于实施例1和2所制备的各全反式维甲酸注射剂，也得到了同样的实验结论。亦即，作用于肿瘤病人体内的肿瘤组织，可以明显减少膀胱癌肿瘤浸润髓源细胞中CD33+HLA-DR-MDSC表型细胞的数量。且，所述全反式维甲酸注射剂能够使瘤病人MDSC对T细胞的抑制作用降低。
实施例7、全反式维甲酸注射剂促进人乳腺癌细胞MCF-7凋亡
实验前一天接种5x105个MCF-7细胞于6孔细胞培养板，在37℃、5％CO2细胞培养箱中培养过夜。第二天细胞培养板去除培养液后，加入新鲜培养基和实施例1或实施例2或实施例3制备好的全反式维甲酸注射剂药物，在37℃、5％CO2细胞培养箱中培养24h。用不含EDTA的胰酶消化后，300g，4℃离心5min收集细胞，然后用预冷的PBS洗涤细胞2次，每次均需300g，4℃离心5min，收集1～5×105细胞。吸弃PBS，加入100μL 1×Binding Buffer重悬细胞。加入5μL Annexin V-FITC和10μL PI Staining Solution，轻轻混匀。避光、室温反应10-15min后加入400μL 1×Binding Buffer，混匀后放置于冰上，样品在1小时内用流式细胞仪检测。
结果，如图7，本发明实施例3制备的全反式维甲酸注射剂能显著促进人乳腺癌细胞 MCF-7凋亡。
对于实施例1和2所制备的各全反式维甲酸注射剂，也得到了同样的实验结论。亦即，实施例1和2所制备的各全反式维甲酸注射剂能显著促进人乳腺癌细胞MCF-7凋亡。
实施例8、全反式维甲酸注射剂促进人外周血白血病T细胞Jukat凋亡
实验前一天接种5x105个Jukat细胞于6孔细胞培养板，在37℃、5％CO2细胞培养箱中培养过夜。第二天细胞培养板去除培养液后，加入新鲜培养基和实施例1或实施例2或实施例3所制备的全反式维甲酸注射剂药物，在37℃、5％CO2细胞培养箱中培养24h。接着300g，4℃离心5min收集细胞，然后用预冷的PBS洗涤细胞2次，每次均需300g，4℃离心5min，收集1～5×105细胞。吸弃PBS，加入100μL 1×Binding Buffer重悬细胞。加入5μL Annexin V-FITC和10μL PI Staining Solution，轻轻混匀。避光、室温反应10-15min后加入400μL 1×Binding Buffer，混匀后放置于冰上，样品在1小时内用流式细胞仪检测。
结果，如图8，本发明实施例3制备的全反式维甲酸注射剂能显著促进人外周血白血病T细胞Jukat凋亡。
对于实施例1和2所制备的各全反式维甲酸注射剂，也得到了同样的实验结论。亦即，实施例1和2所制备的各全反式维甲酸注射剂能显著促进人外周血白血病T细胞Jukat凋亡。
实施例9、全反式维甲酸注射剂抑制荷瘤小鼠肿瘤生长
4T1皮下移植瘤模型的建立方法是：在体外培养4T1肿瘤细胞株计数，制备肿瘤细胞悬液，将制备好的肿瘤细胞悬液吹打均匀，固定好Balb/c小鼠(雌性)，乙醇棉球消毒注射部位的皮肤，按每只动物1×106个细胞，进行皮下接种，注射完毕后，轻轻按压针刺部位以防止倒流。接种后观察肿瘤生长情况，测量移植瘤体积，用游标卡尺测量肿瘤额最长径(a)和最短径(b)，按照V＝1/2×ab×(a+b)计算肿瘤体积(V)。待小鼠肿瘤生长至约10mm3，尾静脉注射实施例1或实施例2或实施例3所制备的全反式维甲酸注射剂(5mg/kg)，隔天给药，对照组小鼠尾静脉注射等量的不含全反式维甲酸注射剂，隔天给药。观察小鼠体重，测量并统计得出肿瘤平均体积，并绘制出生长曲线。
结果，如图9，本发明实施例3制备的全反式维甲酸注射剂作用于4T1荷瘤小鼠，小鼠肿瘤组织体积大小减少。
对于实施例1和2所制备的各全反式维甲酸注射剂，也得到了同样的实验结论。亦即， 实施例1和2所制备的各全反式维甲酸注射剂作用于4T1荷瘤小鼠，小鼠肿瘤组织体积大小减少。
实施10、全反式维甲酸注射剂明显增加荷瘤小鼠模型肿瘤浸润淋巴细胞比例
从实施例9的4T1荷瘤小鼠中剥取肿瘤组织，于4％甲醛溶液中固定3-5days后，从固定液中取出组织，修整为适当的形状及厚度；组织块经过80％、90％、95％、100％乙醇I、100％乙醇II、100％乙醇III进行脱水处理，然后用二甲苯I 30min、二甲苯II 30min透明处理后，石蜡I浸蜡1h、石蜡II浸蜡6h；按照取材面向下的原则，用石蜡包埋起组织，待蜡块冷却凝固后置于-20℃冷藏；切片厚度4μm，切片放入65℃恒温箱中6-12h，装盒，常温保存。
石蜡免疫组化步骤如下：将石蜡切片脱蜡至水，3％H2O2室温孵育5-10分钟，以消除内源性过氧化物酶的活性。蒸馏水冲洗，PBS浸泡5分钟，重复两次。5-10％正常山羊血清(PBS稀释)封闭，室温孵育10分钟，倾去血清，勿洗。滴加一抗工作液，37℃孵育1-2小时或4℃过夜。PBS冲洗，每次5分钟，重复三次。滴加适量二抗工作液，37℃孵育10-30分钟。PBS冲洗，每次5分钟，重复三次。DAB显色剂显色3-15分钟后，自来水充分冲洗，复染，脱水，透明，封片。
结果，如图10中所示，本发明实施例3制备的全反式维甲酸注射剂作用于4T1荷瘤小鼠，小鼠肿瘤组织中淋巴浸润细胞(CD4+和CD8+T细胞)数目增加。
对于实施例1和2所制备的各全反式维甲酸注射剂，也得到了同样的实验结论。亦即，实施例1和2所制备的各全反式维甲酸注射剂，作用于4T1荷瘤小鼠，小鼠肿瘤组织中淋巴浸润细胞(CD4+和CD8+T细胞)数目增加。
实施11、全反式维甲酸注射剂明显抑制荷瘤小鼠模型肾脏和肝脏的肿瘤细胞转移
从实施例9的4T1荷瘤小鼠中剥取肝组织和肾组织，于4％甲醛溶液中固定3-5days后，从固定液中取出组织，修整为适当的形状及厚度；组织块经过80％、90％、95％、100％乙醇I、100％乙醇II、100％乙醇III进行脱水处理，然后用二甲苯I 30min、二甲苯II 30min透明处理后，石蜡I浸蜡1h、石蜡II浸蜡6h；按照取材面向下的原则，用石蜡包埋起组织，待蜡块冷却凝固后置于-20℃冷藏；切片厚度4μm，切片放入65℃恒温箱中6-12h，装盒，常温保存。
石蜡组织HE染色步骤如下：首先脱蜡复水：二甲苯I 15min、二甲苯II 15min、无水 乙醇I 5min、无水乙醇II 5min、95％乙醇5min、80％乙醇5min、自来水浸洗1min；染色：将切片浸入苏木精染液中常温染色5min，自来水洗1min；将切片浸入1％盐酸酒精溶液数秒，自来水至组织返蓝；将切片浸入伊红染液中染色3-5min，自来水洗去玻片上的浮色即可；脱水、透明、封固：80％乙醇0.5min、95％乙醇I 0.5min、95％乙醇II 0.5min、无水乙醇I 0.5min、无水乙醇II 0.5min、二甲苯I透明3min、二甲苯II透明3min、取出后用中性树胶封固并观察评估染色结果。
结果，如图11和图12中所示，本发明实施例3制备的全反式维甲酸注射剂作用于4T1荷瘤小鼠，小鼠肝脏中肿瘤细胞浸润转移比较对照组小鼠肝组织较少(图11)，肾脏中肿瘤细胞浸润转移比较对照组小鼠肾组织较少(图12)。
对于实施例1和2所制备的各全反式维甲酸注射剂，也得到了同样的实验结论。亦即，注射全反式维甲酸注射剂的4T1荷瘤小鼠肝脏中肿瘤细胞浸润转移比较对照组小鼠肝组织较少，肾脏中肿瘤细胞浸润转移比较对照组小鼠肾组织较少。
以上所述，仅为本发明的较佳实施例，并非对本发明任何形式上和实质上的限制，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还将可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。凡熟悉本专业的技术人员，在不脱离本发明的精神和范围的情况下，当可利用以上所揭示的技术内容而做出的些许更动、修饰与演变的等同变化，均为本发明的等效实施例；同时，凡依据本发明的实质技术对上述实施例所作的任何等同变化的更动、修饰与演变，均仍属于本发明的技术方案的范围内。

Claims (13)

  1. 一种全反式维甲酸注射剂，所述全反式维甲酸注射剂包括全反式维甲酸和助溶分子。
  2. 根据权利要求1所述的全反式维甲酸注射剂，其特征在于，所述助溶分子选自脂质、聚氧乙烯蓖麻油、PVP、HPMC、Pluronic嵌段共聚物、环糊精或PEG中的任一种或多种的组合。
  3. 根据权利要求1所述的全反式维甲酸注射剂，其特征在于，所述助溶分子与全反式维甲酸之间的质量比例范围是(10～80)∶1。
  4. 根据权利要求2所述的全反式维甲酸注射剂，其特征在于，所述脂质选自磷脂、胆固醇或聚乙二醇化的磷脂中的任一种或多种的组合。
  5. 根据权利要求2所述的全反式维甲酸注射剂，其特征在于，所述脂质与全反式维甲酸之间的质量比例范围是：(20～80)∶1。
  6. 根据权利要求2所述的全反式维甲酸注射剂，其特征在于，所述磷脂选自EPC、HSPC或DPPC中任一种或多种的组合。
  7. 根据权利要求1所述的全反式维甲酸注射剂，其特征在于，所述全反式维甲酸注射剂为溶液型注射剂、混悬型注射剂、乳剂型注射剂、或注射用无菌粉针剂。
  8. 根据权利要求1～7任一权利要求所述的全反式维甲酸注射剂，其特征在于，所述全反式维甲酸的浓度大于等于0.1mg/ml。
  9. 根据权利要求1～7任一权利要求所述的全反式维甲酸注射剂，其特征在于，所述注射制剂的给药途径选自皮内注射、皮下注射、肌内注射剂、静脉注射。
  10. 如权利要求1～7任一权利要求所述全反式维甲酸脂注射剂在制备肿瘤治疗药物中的用途。
  11. 如权利要求10所述的用途，其特征在于，所述全反式维甲酸制剂可以降低肿瘤病人中异常的髓系抑制性细胞的活性、诱导髓系抑制性细胞分化、抑制肿瘤增殖和复发。
  12. 如权利要求11所述的用途，其特征在于，所述髓系抑制性细胞为乳腺癌、结肠癌、卵巢癌、肺癌、肾癌、胃癌、肝癌、宫颈癌、子宫内膜癌、膀胱癌、前列腺癌、胰腺癌、大肠癌、基底细胞癌、黑色素瘤、滤泡性淋巴癌或小淋巴细胞瘤病人体内的髓系抑制性细胞。
  13. 如权利要求1～7任一权利要求所述全反式维甲酸注射剂在制备药物中的用途，所述药物起到以下作用中的任一种或多种：
    (1)降低肿瘤浸润细胞中的髓系抑制性细胞(MDSC)的数量；
    (2)诱导肿瘤浸润CD33+HLA-DR-细胞的分化；
    (3)促进肿瘤患者外周血单核细胞(PBMC)中CD33+细胞的表型变化；
    (4)降低外周血单核细胞(PBMC)中CD33+HLA-DR-细胞对T细胞的抑制作用；
    (5)诱导肿瘤细胞的凋亡；
    (6)增加肿瘤组织中浸润淋巴细胞比例；
    (7)抑制肿瘤细胞转移；
    (8)延缓肿瘤生长。
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