WO2020124495A1 - 含有亚铁氨基酸粒子的组合物及其用于制造治疗或改善胰脏相关疾病的医药品的用途 - Google Patents

含有亚铁氨基酸粒子的组合物及其用于制造治疗或改善胰脏相关疾病的医药品的用途 Download PDF

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WO2020124495A1
WO2020124495A1 PCT/CN2018/122406 CN2018122406W WO2020124495A1 WO 2020124495 A1 WO2020124495 A1 WO 2020124495A1 CN 2018122406 W CN2018122406 W CN 2018122406W WO 2020124495 A1 WO2020124495 A1 WO 2020124495A1
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composition
pancreatic cancer
present
gemcitabine
group
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PCT/CN2018/122406
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English (en)
French (fr)
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林村源
陈木桂
陈沧泽
詹勋锦
傅嘉慧
王开鼎
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普惠德生技股份有限公司
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Priority to CN201880086420.2A priority Critical patent/CN111655252B/zh
Priority to EP18943815.3A priority patent/EP3900719A4/en
Priority to US16/965,181 priority patent/US20220031651A1/en
Priority to JP2020540748A priority patent/JP6998087B2/ja
Priority to PCT/CN2018/122406 priority patent/WO2020124495A1/zh
Priority to CA3087911A priority patent/CA3087911A1/en
Priority to AU2018454589A priority patent/AU2018454589A1/en
Publication of WO2020124495A1 publication Critical patent/WO2020124495A1/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
    • A61K31/28—Compounds containing heavy metals
    • A61K31/295—Iron group metal compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/26—Iron; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis

Definitions

  • the invention relates to a composition containing ferrous amino acid chelate particles sintered from a ferrous amino acid chelate, and also relates to the use of the composition in the preparation of a medicinal product for treating or alleviating pancreas-related diseases.
  • pancreatic cancer Common diseases of the pancreas include pancreatic cancer and pancreatitis.
  • pancreatic cancer and pancreatitis.
  • the difficulty in the treatment of pancreas-related diseases lies in the location, function and limited treatment of the organs of the pancreas.
  • pancreatic cancer is the fourth leading cause of cancer death
  • pancreatic cancer is the eighth leading cause of cancer death. Every year, 8.5 people per 100,000 people die from this disease. The reason for the slow progress is due to the limited availability of drugs. Therefore, the development of new drugs for pancreatic cancer is extremely valuable.
  • Malignant ascites is a common symptom of clinical patients with advanced pancreatic cancer. It is mostly caused by peritoneal infiltration of pancreatic cancer. The phenomenon of malignant ascites may be bloody or serous. As the amount of ascites increases, it will press against the organs in the abdominal cavity.
  • pancreatitis is the digestive enzymes secreted by the pancreas that begin to digest the pancreas itself and surrounding tissues, which leads to inflammation, which is not related
  • Corresponding treatment drugs usually use supportive therapy to restore the pancreas on its own. Therefore, searching for a drug that can effectively treat or slow down pancreatic diseases is an urgently needed subject.
  • One of the objects of the present invention is to provide a medicine for effectively treating or alleviating pancreatic diseases.
  • the present invention provides a composition, and the composition can be used to treat or slow down pancreas-related diseases.
  • the composition contains ferrous amino acid chelate particles sintered from ferrous amino acid chelate, and the average particle diameter of the ferrous amino acid chelate particles is 500 nm to 2600 nm, and the average molecular weight is 1,500 Dalton to 600,000 Dalton.
  • the average molecular weight of the ferrous amino acid chelate particles is 1,500 Daltons to 15,000 Daltons; in another embodiment, preferably, the The average molecular weight of the iron amino acid chelate particles is from 400,000 Daltons to 550,000 Daltons, more preferably, the average molecular weight of the ferrous amino acid chelate particles is 550,000 Daltons.
  • the chelating ratio of the ferrous amino acid chelate of the ferrous amino acid chelate in the composition is between 1:1 and 1:4.
  • the chelating ratio of the ferrous amino acid chelate of the ferrous amino acid chelate in the composition is between 1:1.5 and 1:2.5.
  • the ferrous amino acid chelate in the composition is a combination of ferrous amino acid chelate prepared by mixing inorganic iron and amino acid and heating at 60°C to 90°C for 8 hours to 48 hours
  • the weight ratio of inorganic iron to amino acids is between 1:1.2 and 1:1.5.
  • the inorganic iron is ferrous sulfate, ferrous chloride, ferrous pyrophosphate, or a combination thereof; the amino acid is glycine.
  • Effective dose in the present invention refers to the effective amount in terms of dose and for the required period of time to achieve the desired treatment or alleviation of pancreas-related diseases; according to the present invention, it refers to the amount by administering a specific range
  • the composition containing ferrous amino acid chelate particles sintered by ferrous amino acid chelate can reduce the survival rate of human or mouse pancreatic cancer cells, induce the death of pancreatic cancer cells, and inhibit the migration of human pancreatic cancer cells And the ability to invade, inhibit the growth of orthotopic transplanted pancreatic cancer tumors, reduce or slow down the malignant ascites of orthotopic transplanted pancreatic cancer, treat or slow down pancreatitis.
  • the subject of administration of the composition of the present invention may be human, mouse, dog or cat, etc.
  • the effective dose of the composition may be 0.1 mg/kg/day to 120 mg/kg/day.
  • the effective dose of the composition is between 1 milligram per kilogram (mg/kg/day) and 120 mg/kg/day per day for mice; more preferably, between 10 mg/kg/day and 120mg/kg/day, more preferably, between 24mg/kg/day and 72mg/kg/day.
  • the effective dose of the composition is only between 0.1 mg/kg/day and 20 mg/kg/day per kilogram per day for dogs and cats; more preferably, between 1 mg/kg/day day to 5mg/kg/day.
  • the effective dose of the composition is between 1 mg/day and 7000 mg/day in humans; more preferably, between 10 mg/day and 700 mg/day.
  • the above doses are calculated according to the initial estimation method published by the US Food and Drug Administration in 2005 (Estimating the maximum safety in starting initial clinical trials for therapeutics in adulthealthyvolunteers).
  • the "pharmaceutical acceptable carrier” described in the present invention includes, but is not limited to, reducing agent (solving agent), solvent (solvent), emulsifier (emulsifier), suspending agent (suspending agent), decomposer (decomposer) , Binding agent, excipient, stabilizer, stabilizing agent, cheating agent, diluent, gelling agent, preservative, lubrication Lubricant, surfactant, and other similar or applicable carriers of the present invention.
  • the reducing agent includes, but is not limited to, ascorbic acid, citric acid, acetic acid, propionic acid, butyric acid, and lactic acid ), hydroxysuccinic acid (malic acid), sulfonic acid (sulfonic acid), succinic acid (succinic acid) or a combination thereof.
  • the "pharmaceutical products” described in the present invention may exist in various forms, including, but not limited to, liquid, semi-solid, and solid pharmaceutical forms, such as solutions, emulsions, suspensions, and powders ( powder, tablet, pill, lozenge, troche, chewing gum, capsule, liposome, suppository and other similar or applicable The dosage form of the present invention.
  • the pharmaceutical product is a parenteral or parenteral dosage form.
  • the enteral dosage form is an oral dosage form
  • the oral dosage form is a solution, an emulsion, a suspension, a powder, a lozenge, a pill, a lozenge, a tablet, a chewing gum, or a capsule.
  • the pancreas-related diseases include, but are not limited to, pancreatic cancer, pancreatic cancer metastasis, ascites due to pancreatic cancer, and pancreatitis.
  • the administration method of the composition is simultaneous administration with gemcitabine (gemcitabine). More preferably, the administration method of gemcitabine is to perform more than one administration cycle of gemcitabine, and the administration period of gemcitabine is Gemcitabine was administered twice a week, and after three weeks of continuous administration, Gemcitabine was stopped in the fourth week.
  • composition of the present invention can treat or slow down pancreas-related diseases without obvious side effects.
  • Simultaneous administration of gemcitabine to treat or slow down pancreatic carcinoma in situ has better efficacy and less Hepatotoxic side effects, such as: jaundice.
  • Figure 1 Cell migration ability and invasion ability of PANC-1 cells treated with the composition of the present invention in a dose-dependent manner for 24 hours to analyze the inhibition of the mobility of PANC-1 cells by the composition of the present invention (error bar is at least three The average ⁇ standard deviation of independent experiments; ns is no significant difference, *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001; Student tests).
  • Figure 2 The cell migration ability and invasion ability of the SUIT-2 cells treated with the composition of the present invention in a dose-dependent manner for 24 hours to analyze the inhibition of the mobility of the SUIT-2 cells by the composition of the present invention (error bar is at least three The average ⁇ standard deviation of three independent experiments; ns is no significant difference, **p ⁇ 0.05, ***p ⁇ 0.001; Student test).
  • Figure 3 Cell migration and invasion ability of BxPC-3 cells treated with the composition of the present invention in a dose-dependent manner for 24 hours to analyze the inhibition of the mobility of BxPC-3 cells by the composition of the present invention (error bar is at least three The average ⁇ standard deviation of three independent experiments; ns is no significant difference, ***p ⁇ 0.001; Student test).
  • Fig. 4 The cell migration ability and invasion ability of AsPC-1 cells treated with the composition of the present invention in a dose-dependent manner for 24 hours to analyze the inhibition of AsPC-1 cell mobility by the composition of the present invention (error bar is at least three Mean ⁇ standard deviation of independent experiments; ns is no significant difference, *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001; Student tests).
  • Figure 5-1 Ten-day baseline (10D) bioluminescence image of orthotopic transplanted pancreatic cancer tumors treated with the composition of the present invention.
  • Figure 5-2 The bioluminescent image of the composition of the present invention on the orthotopic transplantation of pancreatic cancer tumors on the 13th day.
  • Figure 5-3 The bioluminescent image of the composition of the present invention on the orthotopic transplantation of pancreatic cancer tumor on the 17th day.
  • Figure 5-4 Bioluminescent image of the composition of the present invention on the orthotopic transplantation of pancreatic cancer tumors on the 20th day.
  • Figure 5-5 Bioluminescent image of the composition of the present invention on the orthotopic transplantation of pancreatic cancer tumor on day 24.
  • Figure 5-6 Bioluminescent image of the composition of the present invention on an orthotopic transplantation of pancreatic cancer tumors on day 31.
  • Figure 5-7 Bioluminescent image of the composition of the present invention on the orthotopic transplantation of pancreatic cancer tumor on day 38.
  • Figure 6 Tumor growth trend graph of the composition of the present invention on orthotopically transplanted pancreatic cancer mice.
  • Figure 7 Life and survival graph of high-low dose experimental group of the composition of the present invention on orthotopically transplanted pancreatic cancer mice.
  • Figure 8 Quantitative test of malignant ascites in orthotopic transplantation of pancreatic cancer mice in the experimental group of high and low dose of the composition of the present invention.
  • Figure 9 Anatomical anterior ventral and dorsal appearance of experimental mice treated with the low-dose composition of the present invention.
  • Figure 10-1 Baseline ten-day bioluminescence image of the composition of the present invention and Gemcitabine (GEM) alone or in combination for the treatment of pancreatic cancer tumors.
  • GEM Gemcitabine
  • Figure 10-2 The experiment of treating the pancreatic cancer tumor with the composition of the present invention and gemcitabine alone or in combination is carried out on the 17th day of bioluminescence imaging.
  • Figure 10-3 The experiment of treating the pancreatic cancer tumor with the composition of the present invention and gemcitabine alone or in combination is carried out on the 24th day of bioluminescence imaging.
  • Figure 10-4 The experiment of treating the pancreatic cancer tumor with the composition of the present invention and gemcitabine alone or in combination is performed on the 31st day of bioluminescent imaging.
  • Figure 10-5 The experiment of treating the pancreatic cancer tumor with the composition of the present invention and Gecmitabine alone or in combination was carried out on the 38th day of bioluminescence imaging.
  • Fig. 11 Trend graph of pancreatic cancer tumor growth treated with the composition of the present invention and gemcitabine alone or in combination.
  • Figure 12 Anatomical diagram of jaundice in mice at 9 weeks after administration of the composition of the present invention and gemcitabine for the treatment of pancreatic cancer tumors.
  • Figure 13-1 The total blood bilirubin value of each group of blood collected from experimental animals treated with the composition of the present invention and gemcitabine alone or in combination with pancreatic cancer tumors.
  • Figure 13-2 GOT/AST values of blood of each group of blood collected from experimental animals treated with the composition of the present invention and gemcitabine alone or in combination with pancreatic cancer tumors.
  • Figure 13-3 GPT/ALT values of blood of each group of blood collected from experimental animals treated with the composition of the present invention and Gemcitine alone or in combination with pancreatic cancer tumors.
  • Fig. 14 Life-survival diagram of experimental animals treated with the composition of the present invention and gemcitabine alone or in combination to treat pancreatic cancer tumors.
  • Figure 15-1 Baseline ten-day bioluminescence image of the composition of the present invention and gemcitabine alone or in combination to treat pancreatic cancer tumors (II).
  • FIG 15-2 The experiment of treating the pancreatic cancer tumor (II) with the composition of the present invention and gemcitabine alone or in combination is carried out on the 17th day of bioluminescence imaging.
  • FIG. 15-3 The experiment of treating the pancreatic cancer tumor (II) alone or in combination with the composition of the present invention and gemcitabine was carried out on the 24th day of bioluminescence imaging.
  • FIG. 15-4 The experiment of treating the pancreatic cancer tumor (II) alone or in combination with the composition of the present invention and Gemcitabine was performed on the 31st day of bioluminescence image.
  • FIG. 15-5 The experiment of treating the pancreatic cancer tumor (II) alone or in combination with the composition of the present invention and Gemcitabine was performed on the 38th day of bioluminescence imaging.
  • FIG. 15-6 The experiment of treating the pancreatic cancer tumor (II) alone or in combination with the composition of the present invention and Gemcitabine was carried out on the 45th day of bioluminescence imaging.
  • Figure 15-7 The experiment of treating the pancreatic cancer tumor (II) alone or in combination with the composition of the present invention and Gemcitabine was performed on the 59th day of bioluminescence imaging.
  • Figure 16 A graph showing the growth trend of pancreatic cancer tumors treated with the composition of the present invention and gemcitabine alone or in combination (II).
  • Figure 17-1 The average trend graph of total bilirubin, GOT/AST and GPT/ALT in blood of experimental animals of each group in the treatment of pancreatic cancer tumors (II) by the composition of the present invention and gemcitabine alone or in combination.
  • Figure 17-2 Gemcitabine alone treatment of pancreatic cancer tumors (II) Gemcitabine group experimental animal blood total bilirubin, GOT/AST and GPT/ALT trend analysis chart.
  • Figure 17-3 Trend analysis chart of total bilirubin, GOT/AST and GPT/ALT in blood of experimental animals in the composition group of the present invention for the treatment of pancreatic cancer tumor (II) by the composition of the present invention alone.
  • Figure 17-4 Trend analysis of the total blood bilirubin, GOT/AST and GPT/ALT of experimental animals in the combined group of the composition of the present invention and Gecmitabine for the treatment of pancreatic cancer tumor (II).
  • Fig. 18 Lifetime charts of experimental mice in the combined group for treating pancreatic cancer tumor (II) alone or in combination with the composition of the present invention and gemcitabine.
  • the composition containing ferrous amino acid chelate particles of the present invention is made by China Taiwan Ligand Co., Ltd. (batch number: F171001; manufacturing date: October 5, 2017), and the composition is freeze-dried Powder, which is prepared in the following manner.
  • ferrous sulfate and glycine purity 98% or more
  • ferrous amino acid chelate in which the ferrous amino acid chelate
  • the chelating ratio of ferrous iron to amino acid is between 1:1 and 1:4, and then the ferrous amino acid chelate is sintered at 200-240°C to obtain ferrous amino acid chelate particles.
  • the average particle size of the ferrous amino acid chelate particles was measured to be 1465.90 ⁇ 132.29 nm by dynamic light scattering in water using a laser particle size analyzer (Beckman Coulter, N5, Submicron Particle Size Analyzer). Using Waters Alliance 2695 System for gel penetration chromatography (GPC) to determine the number average molecular weight (Mn), weight average molecular weight (Mw), peak average molecular weight (MP) and polydispersity (PDI), respectively, 68188 Dalton, 525538 Dalton, 286426 Dalton and 7.707205.
  • GPC Waters Alliance 2695 System for gel penetration chromatography
  • Mn number average molecular weight
  • Mw weight average molecular weight
  • MP peak average molecular weight
  • PDI polydispersity
  • the human pancreatic cancer cells contain 10% fetal bovine serum (Fetal bovine serum, FBS, GIBCO, Invitrogen), penicillin [100 units/ml (U/mL)], streptomycin [100 ⁇ g/ Ml ( ⁇ g/mL)] of Dulbecco's Modified Medium (GIBCO, Invitrogen) cultured in a 37°C, humidified 5% carbon dioxide incubator; human pancreatic cancer cells (BxPC-3, SUIT-2 And AsPC-1) containing 10% fetal bovine serum (Fetal bovine serum, FBS, GIBCO, Invitrogen), penicillin [100 units/ml (U/mL)], streptomycin [100 ⁇ g/ml ( ⁇ g/mL) ] RMPI-1640) was cultured in a 37°C, humidified 5% carbon dioxide incubator.
  • PANC-1, BxPC-3 and AsPC-1 were purchased from the Biological Resources Conservation and Research Center (Institute of Food Industry Development
  • HPDE-E6E7 Human pancreatic duct epithelial cells HPDE-E6E7 (purchased from Expasy No. CVCL_S972) with KSF supplemented with epidermal growth factor (epidermal growth factor) and bovine pituitary extract (Life Technologies, Inc., Grand Island, NY) The culture medium was cultured in an incubator with humidified 5% carbon dioxide at 37°C.
  • the MTT test was used to test the half maximal inhibitory concentration (IC 50 ) of the composition of the present invention. Take the normal pancreatic cells (human pancreatic duct epithelial cells) and pancreatic cancer cells (pancreatic cancer cells) prepared in Preparation Example 2-3 and plant them on a 96-well plate with 4 ⁇ 10 3 cells per well, and use the preparation examples 1
  • the composition of the present invention is treated with a dose-dependent method (10 0 , 10 1 , 10 2 , 10 3 , 10 4 ⁇ g/mL), cultured for 24, 48, and 72 hours, and MTT reagent is added to each well before continuing After incubation for four hours (37°C and 5% carbon dioxide), the absorbance at 570 nm was measured using a microplate analyzer (BioTek).
  • the composition of the present invention has a half-maximal inhibitory concentration on human pancreatic duct epithelial cells and human pancreatic cancer cells after 24, 48, and 72 hours of treatment.
  • the composition of the present invention inhibits the cell proliferation rate of pancreatic cancer cells (human pancreatic cancer cells) more significantly than normal pancreatic cells.
  • Example 2 The experiment of the composition of the present invention inducing death of pancreatic cancer cells
  • the composition of the present invention was analyzed for the induction of cell death.
  • Human pancreatic cancer cells were planted in 6-well dishes and treated in a dose-dependent manner (0, 100, 250, 500, 750, 1000 ⁇ g/mL).
  • the composition of the invention of Preparation Example 1 was treated with PBS for 48 and 72 hours. After rinsing the buffer solution, adding trypsin, and fixing it with 70% ethanol at -20°C for one hour, the cells were resuspended in PBS containing RNase and propidium iodide (propidium iodide) while staining, using flow cytometry FACSCalibur Flow (cytometer, Becton Dickinson) represents the accumulation of sub-G1 cells in cell death.
  • Example 3-1 Effect of the composition of the present invention on migration of human pancreatic cancer cells
  • Transwell cells with 8 micron holes were placed in 24-well cell culture plates for cell migration testing.
  • the human pancreatic cancer cells of Preparation Example 2 were used for cell migration test, and the cells of the control group were not treated by the composition of the present invention; the cells of the experimental group were treated by the composition of the invention of Preparation Example 1 in a dose-dependent manner for 24 hours.
  • the cells of the experimental group and the control group (2 ⁇ 10 4 cells/well) were planted in the serum-free medium in the upper chamber, and the medium supplemented with 10% fetal bovine serum as the chemical attraction in the lower chamber After incubating for 24 hours at 37°C and 5% carbon dioxide, the cells on the lower surface of the porous membrane of the transwell chamber were fixed with methanol, stained with crystal violet (0.05% by weight), and light microscope (40 times, random 3 fields/ Well) Count the number of migrated cells that penetrate the porous membrane.
  • Example 3-2 Effect of the composition of the present invention on the invasion of human pancreatic cancer cells
  • a transwell cell with 8 micron holes (Corning Costar; Lowell, MA, USA) was placed in a 24-well cell culture dish for cell invasion test, and the porous membrane of the transwell cell was coated with matrigel (matrigel, 60 ⁇ g; BD) Bioscience).
  • the pancreatic cancer cells of Preparation Example 2 were used for cell migration test.
  • the control group cells were not treated with the composition of the invention; the experimental group cells were treated with the composition of Preparation Example 1 of the invention in a dose-dependent manner for 24 hours.
  • the cells of the group and the control group (1 ⁇ 10 5 cells/well) were planted in the serum-free medium in the upper chamber, and the medium in the lower chamber was supplemented with 10% fetal bovine serum as the chemical attraction and cultured.
  • the cells on the lower surface of the porous membrane of the transwell chamber were fixed with methanol, stained with crystal violet (0.05% by weight), and the cells that penetrated the porous membrane were calculated using an optical microscope (40 times, random 3 fields/well) number.
  • FIGS. 1 to 4 show that the composition of the present invention inhibits the cell migration ability and cell invasion ability of the PANC-1 cell line in a dose-dependent manner of. Moreover, the mobility of SUIT-2 cells shown in FIG. 2, BxPC-3 cells shown in FIG. 3, and AsPC-1 cells shown in FIG. 4 is also inhibited by the composition of the present invention. The above data results show that the composition of the present invention plays an important role in inhibiting the mobility of cancer cells.
  • Example 4 Inhibition of pancreatic carcinoma tumor in situ transplanted by the composition of the present invention
  • the cold light fluorescent labeled cells were implanted into experimental mice in situ at an amount of 5 ⁇ 10 5 cells by surgery (60 NOD-SCID experimental mice purchased from Lesco). After the cells are implanted into the pancreas of the animal for 10 days, the baseline signal of the non-invasive in vivo imaging system (IVIS) for measuring pancreatic cancer is started. The baseline signal measurement value of the cold light in vivo is obtained by random average grouping.
  • IVIS non-invasive in vivo imaging system
  • mice All experimental mice were grouped [the total average of the measured values of IVIS signal plus or minus three standard deviations, and the animals within the standard of the measured values of IVIS signal were selected and divided into a control group (control, control group). 24 mg per kg (24 mg/kg) of the low-dose composition group and 72 mg (72 mg/kg) per kg of the high-dose group of the composition of the present invention].
  • drug administration was started, and two concentrations of the composition of the present invention dissolved in physiological saline and physiological saline were directly tube-fed into the stomach of experimental mice via a feeding tube. Mice continue to receive drug toxicity observation, body weight measurement, and IVIS signal measurement for four weeks after receiving treatment with the composition of the present invention.
  • each group in the control group, high-dose group and low-dose group can be divided into 13 orthotopic transplanted pancreas Dental cancer experimental mice (including three experimental mice in each group as a quantitative test for pancreatic cancer malignant ascites)
  • the measurement time points of each IVIS signal are 10 days baseline (Baseline 10D), 13 days (13D), 17 days (17D), 20 days (20D), 24 days (24D), 31 days ( 31D) and 38 days (38D), a total of 7 IVIS signal value measurement time points.
  • IVIS is measured twice a week for the first two weeks after dosing, mainly to observe changes in the initial therapeutic effect of the composition of the present invention on pancreatic cancer, and the weekly IVIS measurement record is restored for the next two weeks.
  • the composition of the present invention is continuously administered for 90 days without stopping.
  • the administration method and time are mainly in combination with the control group implanting PANC-1 (5 ⁇ 10 5 ) Under the number of cells, the survival time of mice implanted in situ with pancreatic cancer is about 90 days. The life span of each mouse was recorded during the experiment.
  • the two groups of experimental groups (high dose and low dose) treated with the composition of the present invention for orthotopic transplantation of pancreatic cancer are compared with the control group After the 24th day of the experiment (24D), there was a significant effect of inhibiting the growth of pancreatic carcinoma in situ.
  • the difference of the detection signal reaches the maximum with the 38th day.
  • each IVIS measurement point can be observed on the third day after the treatment of the composition of the present invention (D13, which is the 13th day of the experiment).
  • D13 which is the 13th day of the experiment.
  • the growth of squamous cancer tumors is slow, so the composition of the present invention has an effect of inhibiting tumor growth for pancreatic cancer.
  • the two experimental groups of the composition of the present invention have about 50% to 60% effect on inhibiting the growth of pancreatic cancer for pancreatic cancer.
  • FIG. 7 for the difference in life span between the two experimental groups and the control group for the treatment of high and low doses of the composition of the present invention.
  • the first dead experimental mouse appeared on the 65th day of the experiment. After 80 days of the experiment, the control group experienced large-scale deaths, and the last one on the 91st day.
  • the experimental mice in the group died, and the average number of life span of the control group was 85.7 days.
  • the life-survival period of ten experimental mice in each of the two experimental groups that treated the composition of the present invention at high and low doses the first experimental mice that died in the two groups also occurred on the 86th day of the experiment, and the two groups survived in life.
  • the trend graph of the period almost showed a very consistent slow decline, indicating that the composition of the present invention, regardless of the high or low dose, after 90 days of administration, the experimental mice transplanted with orthotopic pancreatic cancer had the same life extension of the experimental mice The therapeutic effect of survival period.
  • the longest number of days of life survival of the two groups of experimental mice treated with the composition of the present invention also appeared on the 137th day.
  • the average number of life survival periods of the entire group in the low-dose group was 108.7 days;
  • the average life expectancy is 107 days, and the survival time of the high and low dose groups is about 22 days higher than the average of the control group.
  • Example 5 Effect of the composition of the present invention on ascites accompanying pancreatic cancer
  • mice reserved for the quantitative test of pancreatic cancer malignant ascites in Example 4 two experimental groups (the composition of Preparation Example 1 in the high-dose group and the low-dose group), a control group, and three in each of the three groups were used to observe the malignancy
  • the body weight of the mice was measured daily, and the mice were dissected for 90 days during the experiment to quantify the malignant ascites of pancreatic cancer, and the appearance and activity of the mice were observed daily.
  • the weight change of each group As shown in FIG. 8, on the 80th day of the treatment of the composition of the present invention (that is, the experiment was carried out for 90 days), the weight change of each group. During the 80-day treatment of the composition of the present invention, the average daily weight of the two groups of high and low doses Change, almost no ups and downs. In the control group, on the 70th day of the treatment (80 days of the experiment), the average weight change began to increase significantly. By the 80th day of the treatment of the composition of the present invention (90 days of the experiment), the average weight gain was nearly 3 grams. Please further refer to FIG. 9, it can be seen that the control group and the low-dose group treated with the composition of the present invention were processed for 90 days before the experiment.
  • mice in the control group mice had an average of 6 ml of ascites; while the composition of the present invention treated the low-dose group with an average of about 1 ml of ascites.
  • mice in the control group and the low-dose treatment experimental group of the composition of the present invention have a great gap in the quantification of malignant ascites of orthotopic transplantation of pancreatic cancer, indicating that the treatment of the composition of the present invention has a smaller
  • the development of malignant ascites in the late stage of rats has the effect of slowing down or reducing.
  • Example 6 Therapeutic effect of combining gemcitabine and the composition of the present invention on orthotopic transplantation of pancreatic cancer (1)
  • the dosage and administration method of the composition of the present invention in this experiment are based on the high (72mg/kg) and low dose (24mg/kg) of Example 4 with no significant difference, so the dose of 24mg/kg was chosen for this time Experiment; and the dosage and administration of first-line drugs for pancreatic cancer are based on Cook, Natalie, et al. "Gamma secretase inhibition promotes hypoxic necrosis in mouse pancreatic ductal adenocarcinoma.” Journal of Experimental Medicine 209.3 (2012): 437-444. And Olive, Kenneth P., et al. "Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer.” Science (2009).
  • the composition group of the present invention has a significant effect of inhibiting the growth of pancreatic cancer tumors, and is higher than that of Example 5.
  • (72mg/kg) is similar to the low-dose (24mg/kg) experimental group, with about 50% of the effect of inhibiting the growth of pancreatic cancer tumors;
  • Gemcitabine group inhibits the growth of orthotopically transplanted pancreatic tumors after 28 days of administration
  • the effect is similar to the composition group of the present invention, and the trend of IVIS signal measurement results is also consistent during the overall experiment, about 50% of the effect of inhibiting pancreatic cancer tumor growth; the combined group 28 days after administration, compared with the original
  • the composition group of the invention and the Gemcitabine group have a better effect of inhibiting the growth of pancreatic cancer tumors in situ transplantation.
  • the growth inhibition of pancreatic cancer tumors is as high as 80%.
  • mice in the Gemcitabine group and the combined group all had jaundice, which means that the mice were observed to be thin, abnormally swollen bile, dark yellow fur, limbs and tail also showed abnormal yellow and accompanied by malignant ascites , And soon after discovery, two groups of mice began to die. Immediately after the event (9 weeks of drug administration), blood was collected from all mice throughout the experiment, and the total bilirubin, GOT, and GPT in the blood were measured. The results are shown in Figures 13-1 to 13-3.
  • the total bilirubin, GOT, and GPT of the Gemcitabine group and the combined group were significantly higher than those of other groups not given Gemcitabine, so it is speculated that continuous uninterrupted administration of Gemcitabine may cause severe jaundice.
  • FIG. 14 Of the 8 experimental mice in the control group, the first experimental mouse that died on the 60th day of the experiment was dead. After the 91st day of the experiment, the last experimental mouse in the control group died. The average life span of the control group was 80.1 days. In the combined group and Gemcitabine group, jaundice appeared in the experimental mice after 9 weeks of administration, and large-scale experimental mouse death began at the 10th week of administration (the 80th day of the experiment).
  • the first experimental mouse that died in the Gemcitabine group occurred on the 77th day of the experiment, and the last experimental mouse died on the 100th day of the experiment.
  • the average number of life spans of the entire group was 89.4 days.
  • the first dead mouse of the combined group appeared on the 84th day of the experiment, and the last experimental mouse died on the 108th day of the experiment.
  • the average number of life spans of the entire group was 94.1 days. According to the above survival data, the experimental mice in these two groups will die completely after about two weeks if they appear jaundice.
  • the first experimental mouse died in the composition group of the present invention occurred on the 88th day of the experiment, and the last experimental mouse died on the 138th day of the experiment.
  • the average life span of the entire group of the composition group of the present invention was The number is 106 days, which is about 26 days higher than the average survival time of the control group, which has the effect of prolonging the life span of experimental mice transplanted with orthotopic pancreatic cancer.
  • Example 7 The therapeutic effect of combining gemcitabine and the composition of the present invention on orthotopic transplantation of pancreatic cancer (2)
  • gemcitabine administration cycle a total of three gemcitabine administration cycles were performed and the combined group (gemcitabine was given twice a week at 100 mg/kg, After three consecutive weeks, the application of gemcitabine was stopped in the fourth week and the composition of the invention of Preparation Example 1 (24 mg/kg) was administered daily.
  • the route of administration is the same as in Example 6, but the mode of administration is adjusted compared to Example 6, because Example 6 found that the continuous administration of gemcitabine for nine consecutive weeks caused jaundice in mice undergoing orthotopic transplantation of pancreatic cancer, and He died successively in the 10th to 12th week of administration.
  • the IVIS measurement time points of this experiment are: ten-day baseline (Baseline 10D), experiment on the 17th day (7 days after administration), experiment on the 24th day (14 days after administration), experiment on the 31st day (21 days after dosing), 38 days (28 days after dosing), 45 days (35 days after dosing), 59 days (49 days after dosing); and fixed Time points [ten-day baseline (week 0 of dosing), fourth week after dosing, fourth week after dosing, tenth week after dosing, tenth week after dosing] blood was collected from the experimental mice and tested The total bilirubin, GOT and GPT in the blood are used to monitor the index factors that cause jaundice, and the presence of jaundice is observed with the naked eye. The indicators of jaundice observed with the naked eye are whether the mouse is thin, abnormally swollen, and the fur is yellowish. 3. The limbs and tail are abnormally yellow. The life span of each mouse was recorded during the experiment.
  • the composition group of the present invention inhibited orthotopic transplantation of pancreatic cancer tumors after 49 days.
  • the growth also has a very good effect (inhibition effect is more than 50%), the effect is similar to the effect of Examples 4 and 6 at 28 days of administration, and the trend line graph of tumor growth also shows the same trend, and Examples 4, 6 7 and 3 experiments consistently showed the effect of inhibiting the growth of pancreatic cancer tumors in orthotopic transplantation by more than 50%; the gemcitabine group inhibited the growth of pancreatic cancer tumors in orthotopic transplantation 49 days after administration and the composition group of the present invention The effect is very similar.
  • the results of the tumor growth trend line at the time point of IVIS measurement are also very consistent, with about 50 to 60% of the effect of inhibiting tumor growth of orthotopically transplanted pancreatic cancer. Therefore, changing the way of gemcitabine administration does not have much effect on the effect of the gemcitabine group.
  • the growth inhibition of pancreatic cancer transplanted in situ between 28 and 49 days after administration still has 50 to 60% of the therapeutic effect; After the drug treatment, the combined group had a better growth inhibition effect on orthotopic transplantation of pancreatic cancer tumors than the two groups administered alone (composition group of the present invention and gemcitabine group).
  • blood is collected at fixed time points to monitor total bilirubin, GOT, and GPT.
  • the average of the three items of blood collection monitoring in the first 12 weeks after administration fell within the range of normal blood biochemical values (normal value: total bilirubin: 0 ⁇ 1mg/dl; GOT/AST: 40 ⁇ 100U/L; GPT/ALT: 30 ⁇ 50U/L).
  • the Gemcitabine group may have an abnormal increase in GPT values due to an experimental mouse, blood hemolysis or machine detection errors in the eighth week after administration, and it will return to normal by the tenth week of administration.
  • the total bilirubin and GOT values were slightly higher than those in the other three experimental groups.
  • the experimental group began to die successively at 10 weeks after administration, so the three blood The gap between the average of the detected values and other groups has not been significantly widened.
  • three groups were administered except for the control group (one mouse left in the experimental mice): the composition group of the present invention, Gemcitabine There were 1 or 2 experimental mice in the group and the combined group who developed thinness, abnormal bile enlargement, dark yellow fur, abnormal yellow in the limbs and tail, etc.
  • Urgent blood collection test, the total bilirubin, GOT and GPT of each group obtained The trend graphs are Figures 17-2, 17-3, and 17-4. From these three graphs, the total bilirubin is observed.
  • the life survival time of the composition group of the present invention is significantly better than that of the control group, which is consistent with the results of Examples 4 and 6.
  • the maximum survival time of the experimental mice of the composition group of the present invention is 140 days, the average ratio About 25.2 days higher in the control group, the composition of the present invention can prolong the life span of experimental mice transplanted with orthotopic pancreatic cancer tumors; and this experiment compared with Example 6 after changing the Gemcitabine administration method, there was Gemcitabine's Gemcitabine group and the combined group of experimental mice have a significantly longer life span than the experimental group of the corresponding group in Example 6; if the experimental mice develop jaundice, they will be concentrated for a period of time (13 weeks and Week 14) died one after another, but if the experimental mice survived this period, they would have a longer survival period.
  • the longest survival time of the experimental mice in the combined group was more than 150 days, and the average survival time was about 36 days higher than that of the control group.
  • the combined group treatment was significantly better for the experimental mice with orthotopic transplantation of pancreatic cancer.
  • the composition group and gemcitabine group of the present invention The data of only 7 mice in each group in the table is due to the dissection of one mouse in each group.
  • Example 8 Effect of the composition of the present invention on pancreatitis
  • pancreatitis index amylase value [normal value 500-1500 units per liter (U/L)] in the blood were administered the composition of the present invention of Preparation Example 1 in the manner of Tube feeding once a day, 10 mg per 10 kg (10 mg/10 kg/day) per day, after continuous administration for one week, the amylase value was measured, and a dog with abnormal amylase value was used as the control group, and only saline was administered Supportive treatment was performed as a control. And track the amylase value in the blood of each group of experimental animals to determine whether pancreatitis has improved.
  • the results of this experiment show that the abnormal values of amylase in the blood of dogs administered with the composition of the present invention decreased compared to the control group administered with physiological saline. Therefore, the present invention
  • the composition has an improved effect on pancreatitis.
  • the cat's pancreatitis can be improved by administering the composition of the present invention, and the pancreatitis index-amylase value was reduced to a normal value.
  • the composition of the present invention containing ferrous amino acid particles can treat or slow down pancreas-related diseases, specifically, the composition of the present invention can inhibit the growth and induction of pancreatic cancer cells Pancreatic cancer cell death, inhibit pancreatic cancer cell migration and invasion ability, inhibit orthotopic transplantation of pancreatic cancer tumor growth, slow down the spread of orthotopic transplantation of pancreatic cancer tumor, slow or reduce the late stage of orthotopic transplantation of pancreatic cancer Malignant ascites can have fewer hepatotoxic side effects, and the first-line pancreatic cancer drug Gemcitabine has a better effect of inhibiting pancreatic tumor growth; in addition, the composition of the present invention can treat or slow down pancreatitis.

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Abstract

本发明提供一种组合物，其中该组合物中含有由亚铁氨基酸螯合物烧结而成的亚铁氨基酸螯合物粒子，且所述亚铁氨基酸螯合物粒子的平均粒径为500纳米至2600纳米、平均分子量为1,500道尔顿(Dalton)至600,000道尔顿。且本发明的组合物可用于制备治疗或改善胰脏相关疾病的医药品的用途，其中所述医药品含有有效剂量的所述组合物以及药学上可接受的载剂。

Description

含有亚铁氨基酸粒子的组合物及其用于制造治疗或改善胰脏相关疾病的医药品的用途 技术领域
本发明涉及含有由亚铁氨基酸螯合物烧结而成的亚铁氨基酸螯合物粒子的组合物，还涉及该组合物在制备治疗或减缓胰脏相关疾病的医药品的用途。
背景技术
胰脏常见疾病有胰脏癌及胰脏炎，胰脏相关疾病的治疗难度在于胰脏的脏器位置、功能及有限的治疗方法。
胰脏癌的治疗进展在众多癌症类别中呈现较为缓慢的进步，至今整体的五年存活率只有9％。胰脏癌是第四大癌症致死原因，而在中国台湾，胰脏癌为第八大癌症死亡原因，每年每十万人口中就有8.5人因此病而丧生。归咎缓慢进展的原因，则是因为可用药物比较有限。因此针对胰脏癌发展新药物极为有价值。而恶性腹水是晚期胰脏癌临床患者常见的症状，多为胰脏癌的腹膜浸润所致，恶性腹水的现象可能为血性或浆液性，随着腹水量的增加、压迫到腹腔内的器官，会产生食欲下降、胃口不好等情形，有些患者会因为恶性腹水造成肠子蠕动变差，甚至出现麻痹性肠阻塞，而产生呕吐症状。腹水的形成不仅影响了患者的生活品质，亦不利于患者的后期治疗及康复；胰脏炎是胰脏分泌的消化酶开始消化胰脏自身与周围的组织，而导致发炎的现象，并无相对应的治疗药物，通常使用支持性疗法使胰脏自行恢复。因此寻找一种有效治疗或减缓胰脏疾病的药物是目前亟需的课题。
发明内容
本发明的目的之一在于提供一种有效治疗或减缓胰脏疾病的药物。
为达上述目的，本发明提供一种组合物，且该组合物可用于治疗或减缓胰脏相关疾病。其中该组合物中含有由亚铁氨基酸螯合物烧结而成的亚铁氨基酸螯合物粒子，且所述亚铁氨基酸螯合物粒子的平均粒径为500纳米至2600纳米、平均分子量为1,500道尔顿(Dalton)至600,000道尔顿。
于其中一实施态样中，较佳地，所述亚铁氨基酸螯合物粒子的平均分子量为1,500道尔顿至15,000道尔顿；于另一实施态样中，较佳地，所述亚铁氨基酸螯合物粒子的平均分子量为400,000道尔顿至550,000道尔顿，更佳地，所述亚铁氨基酸螯合物粒子的平均分子量为550,000道尔顿。
较佳地，所述的组合物中的亚铁氨基酸螯合物的亚铁与氨基酸的螯合比例介于1：1至1：4之间。
较佳地，所述的组合物中的亚铁氨基酸螯合物的亚铁与氨基酸的螯合比例介于1：1.5至1：2.5之间。
较佳地，所述的组合物中的亚铁氨基酸螯合物是由无机铁与氨基酸混合并历经60℃至90℃加热8小时至48小时所制得的含有亚铁氨基酸螯合物的组合物，其中无机铁与氨基酸的重量比例介于1：1.2至1：1.5之间。
更佳地，所述的无机铁为硫酸亚铁、氯化亚铁、焦磷酸亚铁或其组合；所述氨基酸为甘氨酸。
本发明所述的「有效剂量」是指在剂量上及对于所需要的时间段而言对达成所要治疗或减缓胰脏相关疾病的有效的量；依据本发明，是指通过施予特定范围量的含有亚铁氨基酸螯合物烧结而成的亚铁氨基酸螯合物粒子的组合物，能够降低人类或小鼠胰脏癌细胞存活率、诱导胰脏癌细胞死亡、抑制人类胰脏癌细胞迁移及侵袭的能力、抑制原位移植胰脏癌肿瘤的生长、降低或减缓原位移植胰脏癌晚期恶性腹水、治疗或减缓胰脏炎。
较佳地，本发明的组合物的给药对象可为人类、小鼠、犬或猫等，所述的组合物的有效剂量可为0.1mg/kg/day至120mg/kg/day。
较佳地，所述的组合物的有效剂量于小鼠是介于每日每公斤1毫克(mg/kg/day)至120mg/kg/day；更佳地，介于10mg/kg/day至120mg/kg/day，更佳地，介于24mg/kg/day至72mg/kg/day。
较佳地，所述的组合物的有效剂量于犬只、猫只是介于每日每公斤0.1毫克(mg/kg/day)至20mg/kg/day；更佳地，介于1mg/kg/day至5mg/kg/day。
较佳地，所述的组合物的有效剂量于人是介于1mg/day至7000mg/day；更佳地，介于10mg/day至700mg/day。以上剂量是根据2005年美国食品药物管理局所公告的实验初期估算方法(Estimating the maximum safe starting dose in initial clinical trials for therapeutics in adult healthy volunteers)计算而得。
本发明所述的「医药学上可接受的载剂」包含，但不限于还原剂(reducing agent)、溶剂(solvent)、乳化剂(emulsifier)、悬浮剂(suspending agent)、分解剂(decomposer)、黏结剂(binding agent)、赋形剂(excipient)、安定剂(stabilizing agent)、螯合剂(chelating agent)、稀释剂(diluent)、胶凝剂(gelling agent)、防腐剂(preservative)、润滑剂(lubricant)、表面活性剂(surfactant)，及其他类似或适用本发明的载剂。
较佳地，所述还原剂包含，但不限抗坏血酸(ascorbic acid)、柠檬酸(citric acid)、乙酸(acetic acid)、丙酸(propionic acid)、丁酸(butyric acid)、乳酸(lactic acid)、羟琥珀酸(malic  acid)、磺酸(sulfonic acid)、丁二酸(succinic acid)或其组合。
本发明所述的「医药品」可以多种形式存在，这些形式包含，但不限于液体、半固体及固体药剂形式，诸如溶液(solution)、乳剂(emulsion)、悬浮液(suspension)、粉末(powder)、锭剂(tablet)、丸剂(pill)、口含锭(lozenge)、片剂(troche)、口嚼胶(chewing gum)、胶囊(capsule)、脂质体、栓剂以及其他类似或适用本发明的剂型。
较佳地，所述的医药品为经肠道的或非经肠道的剂型。
更佳地，所述的经肠道的剂型为口服剂型，其口服剂型为溶液、乳剂、悬浮液、粉末、锭剂、丸剂、口含锭、片剂、口嚼胶或胶囊。
较佳地，所述胰脏相关疾病包括，但不限于胰脏癌、胰脏癌转移、胰脏癌产生的腹水、胰脏炎。
较佳地，所述的组合物给药方式是搭配吉西他滨(Gemcitabine)同时给予，更佳地，所述Gemcitabine的给药方式是进行一次以上的Gemcitabine给药周期，所述Gemcitabine给药周期为每周给药Gemcitabine两次，连续三周给药后于第四周停止给药Gemcitabine。
本发明所述的组合物可治疗或减缓胰脏相关的疾病，且无明显副作用产生，搭配Gemcitabine同时给药以治疗或减缓原位移植胰脏癌时有更佳的疗效，并且有较少的肝毒性副作用，如：黄疸的产生。
附图说明
图1：对PANC-1细胞以剂量依赖方法使用本发明的组合物处理24小时的细胞迁移能力及侵袭能力以分析本发明的组合物对PANC-1细胞移动性的抑制(误差杠为至少三个独立实验的平均值±标准差；ns为没有显著差异，*p<0.05，**p<0.01，***p<0.001；Student t test)。
图2：对SUIT-2细胞以剂量依赖方法使用本发明的组合物处理24小时的细胞迁徙能力及侵袭能力以分析本发明的组合物对SUIT-2细胞移动性的抑制(误差杠为至少三个独立实验的平均值±标准差；ns为没有显著差异，**p<0.05，***p<0.001；Student t test)。
图3：对BxPC-3细胞以剂量依赖方法使用本发明的组合物处理24小时的细胞迁徙能力及侵袭能力以分析本发明的组合物对BxPC-3细胞移动性的抑制(误差杠为至少三个独立实验的平均值±标准差；ns为没有显著差异，***p<0.001；Student t test)。
图4：对AsPC-1细胞以剂量依赖方法使用本发明的组合物处理24小时的细胞迁徙能力及侵袭能力以分析本发明的组合物对AsPC-1细胞移动性的抑制(误差杠为至少三个独立实验的平均值±标准差；ns为没有显著差异，*p<0.05，**p<0.01，***p<0.001； Student t test)。
图5-1：以本发明的组合物处理原位移植胰脏癌肿瘤的十天基线(Baseline 10D)生物冷光影像。
图5-2：本发明的组合物对原位移植胰脏癌肿瘤的实验进行第13天的生物冷光影像。
图5-3：本发明的组合物对原位移植胰脏癌肿瘤的实验进行第17天的生物冷光影像。
图5-4：本发明的组合物对原位移植胰脏癌肿瘤的实验进行第20天的生物冷光影像。
图5-5：本发明的组合物对原位移植胰脏癌肿瘤的实验进行第24天的生物冷光影像。
图5-6：本发明的组合物对原位移植胰脏癌肿瘤的实验进行第31天的生物冷光影像。
图5-7：本发明的组合物对原位移植胰脏癌肿瘤的实验进行第38天的生物冷光影像。
图6：本发明的组合物对原位移植胰脏癌小鼠的肿瘤生长趋势线图。
图7：本发明的组合物高低剂量实验组对原位移植胰脏癌小鼠的生命生存期图。
图8：本发明的组合物高低剂量实验组对原位移植胰脏癌小鼠的恶性腹水定量试验。
图9：处理本发明的组合物低剂量组实验小鼠的解剖前腹侧与背侧外观。
图10-1：本发明的组合物与Gemcitabine(GEM)单独或合并治疗胰脏癌肿瘤的基线十天生物冷光影像。
图10-2：本发明的组合物与Gemcitabine单独或合并治疗胰脏癌肿瘤的实验进行第17天生物冷光影像。
图10-3：本发明的组合物与Gemcitabine单独或合并治疗胰脏癌肿瘤的实验进行第24天生物冷光影像。
图10-4：本发明的组合物与Gemcitabine单独或合并治疗胰脏癌肿瘤的实验进行第31天生物冷光影像。
图10-5：本发明的组合物与Gemcitabine单独或合并治疗胰脏癌肿瘤的实验进行第38天生物冷光影像。
图11：本发明的组合物与Gemcitabine单独或合并治疗的胰脏癌肿瘤生长变化趋势图。
图12：本发明的组合物与Gemcitabine合并治疗胰脏癌肿瘤的给药第9周小鼠黄疸现象解剖图。
图13-1：本发明的组合物与Gemcitabine单独或合并治疗胰脏癌肿瘤的实验动物采血的每组血液总胆红素值。
图13-2：本发明的组合物与Gemcitabine单独或合并治疗胰脏癌肿瘤的实验动物采血的每组血液GOT/AST值。
图13-3：本发明的组合物与Gemcitabine单独或合并治疗胰脏癌肿瘤的实验动物采血的每组血液GPT/ALT值。
图14：本发明的组合物与Gemcitabine单独或合并治疗胰脏癌肿瘤的实验动物生命生存期图。
图15-1：本发明的组合物与Gemcitabine单独或合并治疗胰脏癌肿瘤(II)的基线十天生物冷光影像。
图15-2：本发明的组合物与Gemcitabine单独或合并治疗胰脏癌肿瘤(II)的实验进行第17天生物冷光影像。
图15-3：本发明的组合物与Gemcitabine单独或合并治疗胰脏癌肿瘤(II)的实验进行第24天生物冷光影像。
图15-4：本发明的组合物与Gemcitabine单独或合并治疗胰脏癌肿瘤(II)的实验进行第31天生物冷光影像。
图15-5：本发明的组合物与Gemcitabine单独或合并治疗胰脏癌肿瘤(II)的实验进行第38天生物冷光影像。
图15-6：本发明的组合物与Gemcitabine单独或合并治疗胰脏癌肿瘤(II)的实验进行第45天生物冷光影像。
图15-7：本发明的组合物与Gemcitabine单独或合并治疗胰脏癌肿瘤(II)的实验进行第59天生物冷光影像。
图16：本发明的组合物与Gemcitabine单独或合并治疗(II)的胰脏癌肿瘤生长变化趋势图。
图17-1：本发明的组合物与Gemcitabine单独或合并治疗胰脏癌肿瘤(II)的各组实验动物血液总胆红素、GOT/AST与GPT/ALT平均趋势图。
图17-2：Gemcitabine单独治疗胰脏癌肿瘤(II)的Gemcitabine组实验动物血液总胆红素、GOT/AST与GPT/ALT趋势分析图。
图17-3：本发明的组合物单独治疗胰脏癌肿瘤(II)的本发明的组合物组实验动物血液总胆红素、GOT/AST与GPT/ALT趋势分析图。
图17-4：本发明的组合物与Gemcitabine合并治疗胰脏癌肿瘤(II)的合并组实验动物血液总胆红素、GOT/AST与GPT/ALT趋势分析图。
图18：本发明的组合物与Gemcitabine单独或合并治疗胰脏癌肿瘤(II)的合并组实验小鼠生命生存期图。
具体实施方式
本发明将由下列的实施例作为进一步说明，这些实施例并不限制本发明前面所揭示的内容。本领域技术人员，可以做些许的改良与修饰，但不脱离本发明的范畴。
制备例1 制备含有亚铁氨基酸粒子的组合物
本发明的含有亚铁氨基酸螯合物粒子的组合物是由中国台湾配位体股份有限公司制作(批次号码：F171001；制造日期：2017年10月5日)，且该组合物为冷冻干燥的粉末，其是以下述方式制备。首先，将硫酸亚铁与甘氨酸(纯度98％以上)以重量比1：1.3混合并历经60℃至90℃加热8小时至48小时，以获得亚铁氨基酸螯合物，其中亚铁氨基酸螯合物的亚铁与氨基酸螯合比例介于1：1至1：4之间，接着，亚铁氨基酸螯合物在200-240℃下进行烧结，以获得亚铁氨基酸螯合物粒子。经由激光粒径分析仪(Beckman Coulter,N5,Submicron Particle Size Analyzer)在水中进行动态光散射测得亚铁氨基酸螯合物粒子的平均粒径为1465.90±132.29纳米。利用Waters Alliance 2695System进行凝胶穿透层析仪(GPC)测定数目平均分子量(Mn)、重量平均分子量(Mw)、峰值平均分子量(MP)和多分散性(polydispersity，PDI)，分别为68188道尔顿(Dalton)、525538道尔顿(Dalton)、286426道尔顿(Dalton)及7.707205。
制备例2 人类胰腺癌细胞培养
将人类的胰腺癌细胞(PANC-1)以含有10％胎牛血清(Fetal bovine serum,FBS,GIBCO,Invitrogen)、青霉素[100单位/毫升(U/mL)]、链霉素[100微克/毫升(μg/mL)]的杜氏改良培养基(Dulbecco’s Modified Eagle Medium,GIBCO,Invitrogen)培养于37℃、加湿的5％二氧化碳的培养箱中；人类的胰腺癌细胞(BxPC-3、SUIT-2及AsPC-1)以含有10％胎牛血清(Fetal bovine serum,FBS,GIBCO,Invitrogen)、青霉素[100单位/毫升(U/mL)]、链霉素[100微克/毫升(μg/mL)]的RMPI-1640)培养于37℃、加湿的5％二氧化碳的培养箱中。PANC-1、BxPC-3及AsPC-1购自生物资源保存及研究中心(财团法人食品工业发展研究所)，SUIT-2细胞株经由生物资源保存及研究中心的人类细胞复核(STR profile)鉴定。
制备例3 人类正常胰脏导管细胞培养
人类的胰脏导管上皮细胞HPDE-E6E7(购自Expasy No.CVCL_S972)以添加有上皮生长因子(epidermal growth factor)及牛脑下垂体萃取物(Life Technologies,Inc.,Grand Island,NY)的KSF培养基培养于37℃、加湿的5％二氧化碳的培养箱中。
实施例1 抗胰脏癌--细胞增生实验
使用MTT试验测试本发明的组合物的半最大抑制浓度(half maximal inhibitory concentration,IC 50)。取制备例2-3制备的正常胰脏细胞(人类胰脏导管上皮细胞)及胰脏癌细胞(胰腺癌细胞)种植于96孔盘上，每孔4×10 3个细胞，并使用制备例1的本发明的组合物以剂量依赖方法(10 0、10 1、10 2、10 3、10 4μg/mL)处理后培养24、48、72小时，在每 孔中添加MTT试剂，再继续培养四小时(37℃且5％二氧化碳)后，利用微孔盘分析仪(BioTek)测量570nm下的吸光值。
请参阅如下表1所示，本发明的组合物处理24、48、72小时对人类胰脏导管上皮细胞及人类胰腺癌细胞的半最大抑制浓度。总地来说，本发明的组合物对胰脏癌细胞(人类胰腺癌细胞)的细胞增生率的抑制较正常胰脏细胞更为显著。
表1、本发明的组合物对胰脏癌细胞及正常胰脏细胞的24、48、72小时MTT试验半最大抑制浓度
Figure PCTCN2018122406-appb-000001
ns表示与HPDE-E6E7组没有显著差异，Student t test
*表示P<0.05Student t test
实施例2 本发明的组合物诱导胰脏癌细胞死亡实验
分析本发明的组合物对细胞死亡的诱导。将人类胰腺癌细胞种植于6孔盘中，并以剂量依赖方法(0、100、250、500、750、1000μg/mL)处理制备例1的本发明的组合物48及72小时后，以PBS缓冲溶液润洗后，添加胰蛋白酶作用后，并以70％乙醇于-20℃下固定一小时，细胞以含有RNase及碘化丙啶(propidium iodide)的PBS重新悬浮同时进行染色，利用流式细胞仪侦测(FACSCalibur flow cytometer，Becton Dickinson)代表细胞死亡的亚第一间期(sub-G1)细胞累积。
请参阅下表2为本发明的组合物对这些细胞的半最大抑制浓度分析结果，实验数据显示出本发明的组合物可抑制细胞增生率，本发明的组合物可显著诱导人类胰腺癌细胞死亡。
表2、使用流式细胞仪分析本发明的组合物对胰脏癌细胞的半最大抑制浓度
细胞株 处理本发明组合物48小时的IC 50 处理本发明组合物72小时的IC 50
PANC-1 571.7±140.9 398.7±56.4
SUIT-2 688.1±31.8 611±78.2
AsPC-1 598.4±41.4 466.4±19.4
BxPC-3 1076.6±109.8 660.2±66.9
实施例3-1 本发明的组合物对人类胰脏癌细胞迁移的影响
利用有8微米孔洞的transwell小室(Corning Costar；Lowell,MA,USA)置入24孔细胞培养盘中进行细胞迁移测试。使用制备例2的人类胰脏癌细胞进行细胞迁移测试，对照 组细胞未经本发明的组合物处理；实验组细胞经由制备例1的本发明的组合物以剂量依赖方法处理24小时。将实验组及对照组细胞(2×10 4细胞/孔)种植于上层腔室(upper chamber)无血清的培养基中，下层腔室则为添加有10％胎牛血清的培养基作为化学吸引，于37℃、5％二氧化碳下培养24小时后，将transwell小室的多孔膜下表面的细胞利用甲醇固定、使用结晶紫(0.05重量％)染色，并利用光学显微镜(40倍，随机3视野/孔)计算穿透多孔膜的迁移的细胞数。
实施例3-2 本发明的组合物对人类胰脏癌细胞侵袭的影响
利用有8微米孔洞的transwell小室(Corning Costar；Lowell,MA,USA)置入24孔细胞培养盘中进行细胞侵袭测试，且transwell小室的多孔膜上涂布有基质胶(matrigel，60微克；BD Bioscience)。使用制备例2的胰脏癌细胞进行细胞迁移测试，对照组细胞为未经本发明的组合物处理；实验组细胞经由制备例1的本发明的组合物以剂量依赖方法处理24小时，将实验组及对照组细胞(1×10 5细胞/孔)种植于上层腔室(upper chamber)无血清的培养基中，且下层腔室为添加有10％胎牛血清的培养基作为化学吸引，培养24小时后，将transwell小室的多孔膜下表面的细胞利用甲醇固定、使用结晶紫(0.05重量％)染色，并利用光学显微镜(40倍，随机3视野/孔)计算穿透多孔膜侵袭的细胞数。
实施例3-1及3-2的结果，请参照图1至图4，其中，图1，本发明的组合物对PANC-1细胞株的细胞迁移能力及细胞侵袭能力的抑制为剂量依赖性的。且如图2所示SUIT-2细胞、图3所示BxPC-3细胞、图4所示AsPC-1细胞的移动性亦受到本发明的组合物的抑制。上述数据结果显示了本发明的组合物在抑制癌细胞的移动性上扮演一重要角色。
实施例4 本发明的组合物对原位移植胰脏癌肿瘤的抑制
将制备例2制备的PANC-1人类胰腺癌细胞株以习知技术用冷光荧光载体转殖标记后，利用手术将该冷光荧光标记的细胞以5×10 5细胞数量原位植入实验小鼠(自乐思科公司购入的60只NOD-SCID实验小鼠)的胰脏。在细胞植入动物胰脏间隔10日后，开始进行测量胰脏癌的非侵入式活体影像系统(In Vivo Imaging System，IVIS)基线讯号，在取得活体冷光的基线讯号测量值以随机平均分组方式将所有实验小鼠进行分组[IVIS讯号测量值的总平均数加减三个标准差，选取IVIS讯号测量值标准内的动物，并分成控制组(control，对照组)、制备例1的本发明的组合物低剂量组每公斤24毫克(24mg/kg)及本发明的组合物高剂量组每公斤72毫克(72mg/kg)]。分组后开始进行药物的给予，将生理食盐水与溶解于生理食盐水的两种浓度的本发明的组合物经由喂食管直接管喂至实验小鼠胃中。小鼠在接受本发明的组合物处理后一个月内持续接受药物毒性的观察，体重的 测量，并且持续四周测量IVIS讯号。待小鼠出现死亡、不可逆的严重毒性与严重腹水状况则必须牺牲，因此视为事件点，用以统计生存率的差异。在实验分组方面，扣除实验中死亡、IVIS讯号测量值过高或过低(标准外离群值)后，控制组、高剂量组及低剂量组中每组可分得13只原位移植胰脏癌实验小鼠(包含每组各三只实验小鼠作为胰脏癌恶性腹水定量试验)。而每次IVIS讯号的测量时间点，分别为十天基线(Baseline 10D)、实验进行第13天(13D)、17天(17D)、20天(20D)、24天(24D)、31天(31D)与38天(38D)，总共7个IVIS讯号值测量时间点。给药后前两周每周IVIS测量两次，主要在于观察本发明的组合物对于胰脏癌的初期疗效变化，后两周则恢复每周一次IVIS的测量记录。在本发明的组合物给予实验小鼠方面，以不间断地连续给予本发明的组合物90天后停药，给药方式与时间主要是配合控制组在植入PANC-1(5×10 5)的细胞数目下，原位植入胰脏癌小鼠生命生存期大约90天左右。实验期间记录每只小鼠的生命生存期。
请参阅图5-1至图5-7及图6所示，在给予本发明的组合物处理原位移植胰脏癌的两组实验组(高剂量及低剂量)，与对照组相比较下在实验进行第24天(24D)后，就有明显的抑制原位移植胰脏癌肿瘤生长的效果。侦测讯号的差异随时间到第38天达到最大。高剂量与低剂量两组之间的IVIS的讯号测量结果则没有明显的差异。因此，本发明的组合物的使用可抑制胰脏细胞株PANC-1原位移植实验小鼠胰脏的肿瘤生长。
为了能进一步清楚观察出本发明的组合物治疗胰脏癌的效果，将每一个IVIS测量时间点的数据，两组实验组(高剂量及低剂量)与控制组相比较，制成下表3。如下表3所示，在本发明的组合物处理后的第三天(D13，也就是实验进行第13天)后就可以观察到每一个IVIS测量点，两组实验组均比控制组的胰脏癌肿瘤生长情形来的缓慢，因此本发明的组合物对于胰脏癌具有抑制肿瘤生长的效果。且在最后一个IVIS测量时间点(38D)，可以看到本发明的组合物的两组实验组对于胰脏癌约有50％～60％的抑制胰脏癌生长的疗效。
表3、本发明的组合物实验组与控制组在每个IVIS测量时点的比较
Figure PCTCN2018122406-appb-000002
此外，请进一步参考图7所示，为处理本发明的组合物高低剂量的两实验组与控制组在生命生存期的差异。在控制组10只实验小鼠中，实验进行至第65天就出现的第一只死亡的实验小鼠，等到实验进行80天后控制组陆续出现大规模死亡状况，到第91天后最后一只控制组实验小鼠死亡，控制组整组在生命生存期的平均数为85.7天。对于处理本发明的组合物高低剂量的两实验组各十只实验小鼠的生命生存期，两组在出现第一只死亡的实验小鼠同样发生在实验进行第86天，两组在生命生存期趋势线图几乎呈现非常一致的缓慢下降状态，表示本发明的组合物不论高剂量或者低剂量，在给予90天后，对原位移植胰脏癌的实验小鼠，具有相同延长实验小鼠生命生存期的治疗效果。两组以本发明的组合物处理的实验小鼠生命生存期最长天数同样出现在第137天，在低剂量组整组在生命生存期的平均数为108.7天；在高剂量组整组在生命生存期的平均数为107天，高低剂量组生存期比控制组平均数高出约22天。
实施例5 本发明的组合物对伴随胰脏癌发生的腹水的影响
使用实施例4预留的供胰脏癌恶性腹水定量试验的小鼠，两实验组(高剂量组及低剂量组的制备例1组合物)、一控制组，三组各三只，观察恶性腹水的情况，每日测量小鼠的体重，且在实验进行90天解剖老鼠进行胰脏癌恶性腹水定量，且每日观测小鼠外观与活动力。
请参阅图8所示，在本发明的组合物处理第80天(亦即实验进行90天)，每组体重的变化，本发明的组合物处理80天期间，高低剂量两组每日平均体重变化，几乎都没有太大的起伏。而控制组在处理第70天(实验进行80天)左右，平均体重变化开始呈现大幅增加，至本发明的组合物处理第80天(实验进行90天)平均体重增加将近3克重。请进一步参阅图9，可见实验进行90天的解剖前控制组与处理本发明的组合物低剂量组照片，控制组小鼠腹腔变得异常肿胀，而观察本发明的组合物处理的低剂量组实验动物腹腔则无明显改变。根据观察，解剖后小鼠腹腔腹水的状况，控制组小鼠呈现腹水弥漫整个腹腔器官；本发明之组合物低剂量组的小鼠则无这种情形。控制组及低剂量组小鼠腹水定量之结果，控制组平均有6毫升腹水；而本发明组合物处理低剂量组平均约有1毫升的腹水。控制组与本发明之组合物低剂量处理实验组小鼠在原位移植胰脏癌恶性腹水的定量上有极大的差距，表示本发明之组合物的处理对原位移植胰脏癌的小鼠晚期恶性腹水的产生具有减缓或降低的疗效。此外观察整个实验从一开始到实验进行90天后，实验小鼠对于本发明之组合物处理是否有副作用状况。我们发现实验组的小鼠在体重、外观与活动力上并没有太大的改变，据此作合理推测本发明之组合物对原位移植胰脏癌之实验小鼠，应无明显的副作用。
实施例6 合并Gemcitabine与本发明之组合物对原位移植胰脏癌的治疗效果(一)
将制备例2制备的PANC-1人类胰脏癌细胞株以冷光萤光载体转殖标记后，利用手术将该冷光萤光标记之以5×10 5细胞数量原位植入实验小鼠(自乐思科公司购入之50只NOD-SCID实验小鼠)的胰脏，在细胞植入动物胰脏间隔10日后，开始进行测量胰脏癌的非侵入式活体影像系统(In Vivo Imaging System，IVIS)基线讯号，在取得活体冷光的基线讯号测量值以随机平均分组方式将所有实验小鼠进行分组[IVIS讯号测量值的总平均数加减三个标准差，选取IVIS讯号测量值标准内的动物，并分成控制组(control，使用生理食盐水)、制备例制备之本发明之组合物组(每日给予1次24mg/kg之制备例1之组合物，给予方式为经由喂食管直接管喂至实验小鼠胃中)、Gemcitabine组(每周以静脉注射的方式施打两次100mg/kg Gemcitabine)及合并组(本发明之组合物组每日给予24mg/kg及每周给予Gemcitabine两次100mg/kg)]后，即进行药物给予。本实验中之本发明之组合物剂量、给药方式选择是基于实施例4之高(72mg/kg)、低剂量(24mg/kg)无显著差异，因此选择以24mg/kg之剂量进行本次实验；而胰脏癌一线药物之剂量、给药方式是根据Cook,Natalie,et al."Gamma secretase inhibition promotes hypoxic necrosis in mouse pancreatic ductal adenocarcinoma."Journal of Experimental Medicine 209.3(2012):437-444.及Olive,Kenneth P.,et al."Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer."Science(2009).两篇期刊论文决定Gemcitabine以100mg/kg每周两次的给药方式进行实验。本次实验的IVIS测量时间点分别为：十天基线(Baseline 10D)、实验进行第17天(给药后7天)、实验进行第24天(给药后14天)、实验进行第31天(给药后21天)及实验进行第38天(给药后28天)，并于第九周将所有实验动物采血检测总胆红素(T-Bilirubin)、麸胺酸草乙酸转胺酶/天门冬胺酸转胺酶(GOT/AST)与麸胺酸丙酮酸转胺酶/丙胺酸转胺酶(GPT/ALT)，配合肉眼观察是否有出现黄疸现象，肉眼观察的黄疸现象指标分别为是否小鼠瘦弱、胆异常肿大、毛皮暗黄、四肢与尾巴呈现异常黄色。实验期间记录每只小鼠的生命生存期。
请同时参照图10-1至图10-5及图11所示，本发明之组合物组与控制组相比，有明显的抑制胰脏癌肿瘤生长的效果，且与实施例五的高剂量(72mg/kg)与低剂量(24mg/kg)实验组的效果相近，约有50％抑制胰脏癌肿瘤生长的疗效；Gemcitabine组在给药28天后，抑制原位移植胰脏肿瘤生长的结果与本发明之组合物组效果相近，且在整体实验期间，IVIS讯号测量结果呈现的趋势也很一致，约有50％抑制胰脏癌肿瘤生长的效果；合并组在给药28天后，较本发明之组合物组与Gemcitabine组有更佳的抑制原位移植胰脏癌肿瘤生长效果，与控制组相比抑制胰脏癌肿瘤生长更是高达80％，因此合并本发明之组合物与 胰脏癌临床一线化疗药物Gemcitabine，更具有抑制原位移植胰脏癌肿瘤生长的效果。
请进一步参照图12所示，Gemcitabine组与合并组实验小鼠先后都发生黄疸现象，意即观察到老鼠瘦弱、胆异常肿大、毛皮暗黄、四肢与尾巴也呈现异常黄色并伴随恶性腹水产生，并在发现不久后两组老鼠开始陆续死亡。在事件刚发生时(给药9周)，对整个实验中的所有老鼠进行采血，并检测血液中的总胆红素、GOT与GPT，所得的结果如图13-1至13-3所示，Gemcitabine组与合并组两组的总胆红素、GOT与GPT都明显高于其他没有给予Gemcitabine的组别，因此推测连续不间断给予Gemcitabine可能造成严重的黄疸现象产生。请进一步参阅图14所示，在控制组8只实验小鼠中，实验进行至第60天出现的第一只死亡的实验小鼠，于实验第91天后最后一只控制组实验小鼠死亡，控制组整组生命生存期的平均数为80.1天。合并组及Gemcitabine组，两组在给药9周之后发现实验小鼠出现黄疸现象，且在给药第10周(实验进行第80天)开始出现大规模实验小鼠死亡。Gemcitabine组出现第一只死亡的实验小鼠发生在实验进行第77天，于实验进行第100天最后一只实验小鼠死亡，整组在生命生存期的平均数为89.4天。合并组第一只死亡小鼠则出现在实验进行第84天，于实验进行第108天最后一只实验小鼠死亡，整组在生命生存期的平均数为94.1天。根据上述生存期数据，此两组的实验小鼠一旦出现黄疸现象，约两周后就会完全死亡。本发明之组合物组出现第一只死亡的实验小鼠发生在实验进行第88天，于实验进行第138天最后一只实验小鼠死亡，本发明之组合物组整组生命生存期的平均数为106天，较控制组生存期的平均数高出约26天，有延长原位移植胰脏癌实验小鼠生命生存期的效果。
实施例7 合并Gemcitabine与本发明之组合物对原位移植胰脏癌的治疗效果(二)
将制备例2制备的PANC-1人类胰脏癌细胞株以冷光萤光载体转殖标记后，利用手术将该冷光萤光标记之以5×10 5细胞数量原位植入实验小鼠(自乐思科公司购入之40只NOD-SCID实验小鼠)的胰脏，在细胞植入动物胰脏间隔10日后，开始进行测量胰脏癌的非侵入式活体影像系统(In Vivo Imaging System，IVIS)基线讯号，在取得活体冷光的基线讯号测量值以随机平均分组方式将所有实验小鼠进行分组[IVIS讯号测量值的总平均数加减三个标准差，选取IVIS讯号测量值标准内的动物，并分成控制组(control，使用生理食盐水)、本发明的组合物组(每日给予24mg/kg制备例1的组合物)、Gemcitabine组(每周给予两次100mg/kg，连续三周后，于第四周停止Gemcitabine的施打，此四周称为一个Gemcitabine给药周期，本实施例中总共执行了3个Gemcitabine给药周期)及合并组(每周给予Gemcitabine两次100mg/kg，连续三周后，于第四周停止Gemcitabine的施打及每日给予制备例1本发明的组合物24mg/kg)。给药途径与实施例6相同，但给药方式相较于实 施例6作调整，是由于实施例6发现连续九周不间断给予Gemcitabine使原位移植胰脏癌实验小鼠产生黄疸现象，且于给药第10～12周接连死亡。本次实验的IVIS测量时间点分别为：十天基线(Baseline 10D)、实验进行第17天(给药后7天)、实验进行第24天(给药后14天)、实验进行第31天(给药后21天)、实验进行第38天(给药后28天)、实验进行第45天(给药后35天)、实验进行第59天(给药后49天)；并在固定时间点[十天基线(给药第0周)、给药后第四周、给药后第四周、给药后第十周、给药后第十周]对实验小鼠采血，并检测血中的总胆红素、GOT与GPT以监测造成黄疸原因的指标因素，配合肉眼观察是否有出现黄疸现象，肉眼观察的黄疸现象指标分别为是否小鼠瘦弱、胆异常肿大、毛皮暗黄、四肢与尾巴呈现异常黄色。实验期间记录每只小鼠的生命生存期。
实验结果请参阅图15-1至图15-7及图16所示，在给药49天，与控制组相比，本发明的组合物组于49天后，对于抑制原位移植胰脏癌肿瘤生长亦有非常好的效果(抑制效果达50％以上)，效果与实施例4及6在给药28天的效果接近，肿瘤生长的趋势线图也呈现同样的趋势，且实施例4、6及7三次实验一致呈现出有50％以上抑制原位移植胰脏癌肿瘤生长的疗效；Gemcitabine组在给药后49天，抑制原位移植胰脏癌肿瘤生长的效果与本发明的组合物组的效果呈现很相近的趋势。在49天实验期间，IVIS测量时间点的肿瘤生长趋势线所呈现的结果也很一致，约有50～60％抑制原位移植胰脏癌肿瘤生长的效果。因此，改变Gemcitabine给药方式，对于Gemcitabine组的效果没有太大影响，在给药后28天至49天期间原位移植胰脏癌生长抑制还是有50～60％的治疗效果；在改变Gemcitabine给药方式后，合并组比单独给药的两组(本发明的组合物组及Gemcitabine组)对原位移植胰脏癌肿瘤具有更好的抑制生长的效果，本次实验结果相较于控制组，呈现抑制胰脏癌肿瘤生长高达80％以上(给药28天)的效果，与实施例6几乎相同。且合并组的IVIS时间点讯号测量值相较于同一时间点的控制组讯号测量值，在给药28天与给药49天两个时间点上，可看出对于抑制原位移植胰脏癌肿瘤生长，没有太大的差异(抑制肿瘤生长效果分别为84％与77％)。因此，改变Gemcitabine给药方式，对于合并组的后期治疗效果应该没有太大影响。因此从本实施例重复的动物试验更可以确定合并本发明的组合物与胰脏癌临床一线化疗药物Gemcitabine，对于原位移植胰脏癌肿瘤的生长有更好的抑制效果。
请进一步参阅图17-1至图17-4，本实施例固定时间点采血监测总胆红素、GOT与GPT。分别有十天基线、给药后第四周、给药后第八周(给药后第四周与第八周分别跟第一与第二次Gemcitabine给药周期结束时间点一致)、给药后第十周与给药后第十二周(给药后第十周与第十二周系与第三次Gemcitabine给药周期进行与结束时间点一致)共五个时间点，而给药后第十三周的采血时间点系因发现小鼠异常，出现黄疸现象，所以在预定的五 个时间点额外增加采血监测时间点。如图17-1所示，采血监测的三个项目在给药后的前12周各组平均数都落在正常血液生化值范围内(正常值:总胆红素：0～1mg/dl；GOT/AST:40～100U/L；GPT/ALT:30～50U/L)。Gemcitabine组在给药后第八周可能因为有一只实验小鼠，血液溶血或机器检测误差，导致整组在GPT数值异常往上提高，至给药第十周则恢复正常。控制组在给药后第十周以后总胆红素与GOT两项数值都有略高于其他3组实验组，然控制组在给药后10周实验小鼠开始陆续死亡，所以3项血液检测数值的平均数与其他组的差距并未有明显拉开。进行给药后第十三周(第三次Gemcitabine给药周期结束后一周)，发现除控制组(实验小鼠剩一只)外，其他给药的三组：本发明的组合物组、Gemcitabine组与合并组都有1至2只实验小鼠产生瘦弱、胆异常肿大、毛皮暗黄、四肢与尾巴呈现异常黄色等黄疸现象，紧急采血检测，所得各组总红胆素、GOT与GPT的趋势图为图17-2、图17-3、图17-4，从这3个图观察出总红胆素部分，本发明的组合物组与Gemcitabine组各有一只；合并组有三只实验小鼠明显高于标准值(0.1-0.9mg/mL)，且总胆红素高于标准值的实验小鼠也在10天内陆续死亡。Gemcitabine组在给药后第十三周采血后，发现一开始没有产生黄疸现象的实验小鼠也接连出现黄疸现象，并在出现黄疸后不久实验小鼠陆续死亡。关于本实施例的实验小鼠生命生存期请参照图18，及下表4。
表4、控制组及给药实验组的小鼠生命生存期
Figure PCTCN2018122406-appb-000003
可观察出，本发明的组合物组的生命生存期明显优于控制组，与实施例4、6的结果一致，本发明的组合物组实验小鼠最长生存期为140天，平均数比控制组高出约25.2天，本发明的组合物能延长原位移植胰脏癌肿瘤实验小鼠的生命生存期；且本次实验相较于实施例6改变了Gemcitabine给药方式后，有给予Gemcitabine的Gemcitabine组及合并组实验小鼠生命生存期明显比实施例6的对应组别的实验小鼠要长；实验小鼠若发生黄疸现象，将集中在一段时间(给药后第13周及第14周)内陆续死亡，但是如果实验小鼠撑过此期间就会有较长的生存期。此外，合并组实验小鼠生命生存期最长为150天以上，存活期平均数比控制组高出约36天，合并组治疗对原位移植胰脏癌的实验小鼠的生命生存期明显优于本发明的组合物组及Gemcitabine组。表格中每组仅有7只小鼠的数据，是由于每一组分别均取一只小鼠解剖。
实施例8 本发明的组合物对胰脏炎的影响
对血液中的胰脏炎指标淀粉酶amylase数值[正常值500-1500单位每公升(U/L)]过高的三犬只投予制备例1的本发明的组合物，给药方式为每日管喂一次，每日每十公斤10毫克(10mg/10kg/day)，持续给予一周后，测量淀粉酶数值，并以一淀粉酶数值异常的犬只作为控制组，仅投予生理食盐水进行支持性治疗作为对照。并追踪各组实验动物的血液中淀粉酶数值，判断胰脏炎是否改善。
请参阅下表5所示，本实验结果显示，相较于投予生理食盐水的对照组，投予本发明的组合物进行的犬只血液中的淀粉酶异常值均下降，因此，本发明的组合物对胰脏炎有改善的效果。虽未记载于表格中，然猫只的胰脏炎亦观察到可经由投予本发明组合物改善，使胰脏炎指标—淀粉酶数值降至正常值。
表5、投予本发明的组合物一周对犬只的胰脏炎影响
Figure PCTCN2018122406-appb-000004
综上所述，本发明含有亚铁氨基酸粒子的组合物(本发明的组合物)可治疗或减缓胰脏相关疾病，具体而言，本发明的组合物可抑制胰脏癌细胞的生长、诱导胰脏癌细胞死亡、抑制胰脏癌细胞的迁徙及侵袭能力、抑制原位移植胰脏癌肿瘤生长、减缓原位移植胰脏癌肿瘤往外扩散的情形、减缓或降低原位移植胰脏癌晚期恶性腹水、能有较少的肝毒性副作用，且搭配胰脏癌一线药物Gemcitabine有更佳的抑制胰脏肿瘤生长的效果；此外本发明的组合物可治疗或减缓胰脏炎。
根据本发明可作的不同修正及变化对于本领域技术人员而言均显然不会偏离本发明的范围与精神。虽然本发明已叙述特定的较佳具体事实，必须了解的是本发明不应被不当地限制于这些特定具体事实上。事实上，在实施本发明的已述模式方面，对于本领域技术人员而言显而易知的不同修正亦被涵盖于请求保护范围之内。

Claims (12)

  1. 一种组合物，所述组合物中含有由亚铁氨基酸螯合物烧结而成的亚铁氨基酸螯合物粒子，且所述亚铁氨基酸螯合物粒子的平均粒径为500纳米至2600纳米、平均分子量为1,500道尔顿(Dalton)至600,000道尔顿。
  2. 如权利要求1所述的组合物，其中所述的亚铁氨基酸螯合物的亚铁与氨基酸的螯合比例为1：1至1：4之间。
  3. 如权利要求1所述的组合物，其中所述的亚铁氨基酸螯合物为亚铁甘氨酸螯合物。
  4. 一种如权利要求1至3任一项所述的组合物用于制备治疗或改善胰脏相关疾病的医药品的用途，其中所述医药品含有有效剂量的所述组合物以及药学上可接受的载剂。
  5. 如权利要求4所述的用途，其中所述胰脏相关疾病为胰脏癌。
  6. 如权利要求5所述的用途，其中所述胰脏相关疾病为胰脏癌转移。
  7. 如权利要求5所述的用途，其中所述胰脏相关疾病为胰脏癌产生的腹水。
  8. 如权利要求4所述的用途，其中所述胰脏相关疾病为胰脏炎。
  9. 如权利要求4所述的用途，其中所述组合物的给药对象为人类。
  10. 如权利要求4所述的用途，其中所述组合物的有效剂量介于0.1mg/kg/day至120mg/kg/day。
  11. 如权利要求4所述的用途，其中所述组合物的给药方式是搭配Gemcitabine同时给予。
  12. 如权利要求11所述的用途，所述Gemcitabine的给药方式是进行一次以上的Gemcitabine给药周期，所述Gemcitabine给药周期为每周给药Gemcitabine两次，连续三周给药后于第四周停止给药Gemcitabine。
PCT/CN2018/122406 2018-12-20 2018-12-20 含有亚铁氨基酸粒子的组合物及其用于制造治疗或改善胰脏相关疾病的医药品的用途 WO2020124495A1 (zh)

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CN201880086420.2A CN111655252B (zh) 2018-12-20 2018-12-20 含有亚铁氨基酸粒子的组合物用于制备减缓胰脏癌产生的腹水及治疗胰脏炎的医药品的用途
EP18943815.3A EP3900719A4 (en) 2018-12-20 2018-12-20 COMPOSITION CONTAINING AN AMINO ACID FERROUS PARTICLE AND ITS USE IN THE PREPARATION OF A PHARMACEUTICAL PRODUCT FOR THE TREATMENT OR IMPROVEMENT OF A PANCREAS-RELATED DISEASE
US16/965,181 US20220031651A1 (en) 2018-12-20 2018-12-20 Composition comprising ferrous amino acid particles and method for treating or ameliorating pancres-related disease using the same
JP2020540748A JP6998087B2 (ja) 2018-12-20 2018-12-20 第一鉄アミノ酸粒子を含む組成物、およびその組成物を含む、膵臓関連疾患の治療または改善のための薬剤
PCT/CN2018/122406 WO2020124495A1 (zh) 2018-12-20 2018-12-20 含有亚铁氨基酸粒子的组合物及其用于制造治疗或改善胰脏相关疾病的医药品的用途
CA3087911A CA3087911A1 (en) 2018-12-20 2018-12-20 Composition comprising ferrous amino acid particles and use thereof in manufacture of medicament for treating or ameliorating pancreas-related disase
AU2018454589A AU2018454589A1 (en) 2018-12-20 2018-12-20 Composition comprising ferrous amino acid particles and method for treating or ameliorating pancreas-related disease using the same

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