WO2024109724A1 - 一种卟硒啉环糊精包合物、药物组合物及其制备方法和应用 - Google Patents

一种卟硒啉环糊精包合物、药物组合物及其制备方法和应用 Download PDF

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WO2024109724A1
WO2024109724A1 PCT/CN2023/132850 CN2023132850W WO2024109724A1 WO 2024109724 A1 WO2024109724 A1 WO 2024109724A1 CN 2023132850 W CN2023132850 W CN 2023132850W WO 2024109724 A1 WO2024109724 A1 WO 2024109724A1
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cyclodextrin
preparation
solution
pharmaceutical composition
tmz
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French (fr)
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曾慧慧
尹汉维
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Shanghai Yuanxi Medicine Corp
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Shanghai Yuanxi Medicine Corp
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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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4188—1,3-Diazoles condensed with other heterocyclic ring systems, e.g. biotin, sorbinil
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • 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
    • A61K33/04—Sulfur, selenium or tellurium; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6949—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
    • A61K47/6951—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes using cyclodextrin
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
    • C08B37/0012—Cyclodextrin [CD], e.g. cycle with 6 units (alpha), with 7 units (beta) and with 8 units (gamma), large-ring cyclodextrin or cycloamylose with 9 units or more; Derivatives thereof
    • C08B37/0015—Inclusion compounds, i.e. host-guest compounds, e.g. polyrotaxanes
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C08L5/16—Cyclodextrin; Derivatives thereof

Definitions

  • the invention belongs to the field of medicine and relates to a porphyrin cyclodextrin inclusion compound, a pharmaceutical composition and a preparation method and application thereof.
  • Pulmonary fibrosis is a type of lung disease characterized by fibroblast proliferation and accumulation of a large amount of extracellular matrix (ECM), accompanied by inflammatory damage and tissue structure destruction. Once it occurs, it is irreversible. Impaired lung function will seriously affect the patient's quality of life and even endanger his life.
  • ECM extracellular matrix
  • the spinal cord belongs to the central nervous system, and its glioma incidence is relatively low, accounting for 8-10% of all primary spinal cord tumors and 2-4% of all central nervous system tumors. According to its cell source, it can be divided into ependymomas (60-70%) and astrocytomas of various levels (30-40%). Since the transverse diameter of the spinal cord is limited and it has important physiological functions, it is quite difficult to treat.
  • various treatment guidelines for gliomas are mostly aimed at intracranial diseases, and there are no good diagnosis and treatment standards for spinal gliomas as a basis, resulting in large differences among treatment centers, thus affecting the quality of life of patients.
  • the treatment options for spinal gliomas are also diverse, including microsurgery, radiotherapy, chemotherapy, immunotherapy, etc.
  • Porselen is a chemical small molecule drug designed to target thioredoxin reductase (TR).
  • TR thioredoxin reductase
  • Porphyrin tablets that have been approved for clinical use have a therapeutic effect on pulmonary fibrosis. However, due to the first-pass effect in the liver, the drug efficiency is reduced, and due to the influence of the blood-brain barrier, it has no effect on intracranial gliomas. More effective porphyrin preparations targeting intracranial gliomas are urgently needed.
  • porphyrin According to the research and analysis of the properties of porphyrin, it is a poorly soluble drug, and its solubility in water is about 150ng/mL, so its bioavailability in the body is low, which greatly limits its clinical application.
  • the figure below is the chemical structure of porphyrin.
  • the present invention provides an inclusion compound, which is a complex comprising porphyrin or its derivatives and cyclodextrin substances.
  • the cyclodextrin substance is selected from ⁇ -cyclodextrin substances and/or ⁇ -cyclodextrin substances, for example, it can be selected from one or more of hydroxypropyl- ⁇ -cyclodextrin, sulfobutyl- ⁇ -cyclodextrin, hydroxypropyl- ⁇ -cyclodextrin, methyl- ⁇ -cyclodextrin and 2,6-dimethyl- ⁇ -cyclodextrin; preferably hydroxypropyl- ⁇ -cyclodextrin.
  • the inclusion compound is formed by inclusion of porphyrin or its derivative by a cyclodextrin substance.
  • the mass ratio of porphyrin or its derivative to cyclodextrin is 1:(10-480), for example 1:(10-100), exemplified by 1:10, 1:20, 1:30, 1:40, 1:45, 1:49, 1:50, 1:60.
  • the inclusion ratio of the cyclodextrin substance to porphyrin or its derivative is (1-15):1, for example (2-10):1, such as 1.5:1, 2.5:1, 3:1, 4:1.
  • the inclusion compound may be any one of the following inclusion compounds:
  • the present invention also provides a method for preparing the inclusion compound, comprising: using cyclodextrin substances to include porphyrin or its derivatives.
  • the selection and mass ratio of the porphyrin or its derivative and the cyclodextrin substance are as shown above.
  • the inclusion is carried out in water or Tween 80 solution.
  • the water is water for injection.
  • the Tween 80 solution is a Tween 80 aqueous solution.
  • the concentration of the Tween 80 solution is 0.01-0.1%, such as 0.05%.
  • the preparation method comprises the following steps:
  • step 1) After step 1) is completed, stirring, centrifuging, and optionally filtering or not filtering.
  • the mass ratio of the cyclodextrin substance to the water in the system is (10-55):100, for example (20-50):100, exemplified by 30:100, 40:100, 48:100; the mass of water in the system is the sum of the mass of water used to dissolve the cyclodextrin substance and the mass of quantitatively added water.
  • the mass ratio of the cyclodextrin substance to the Tween 80 solution in the system is (10-55):100, for example (20-50):100, exemplified by 30:100, 40:100, 48:100; the mass of the Tween 80 solution in the system is the sum of the mass of the dissolved cyclodextrin substance and the quantitatively added Tween 80 solution.
  • the porphyrin or its derivative is added in the form of micro powder.
  • the stirring temperature is 25-80°C, preferably 40-70°C, for example 30°C, 40°C, 50°C, 60°C, 70°C.
  • the stirring time is 0.5 to 5 hours, preferably 3 hours.
  • the centrifugal rotation speed is 4000-10000 rpm, preferably 7000-9000 rpm, for example 9000 rpm.
  • the centrifugation time is at least 5 min, such as 5 to 30 min, and exemplarily 20 min.
  • the preparation method further comprises step 3), maintaining the transparent solution obtained in step 2) at a temperature of 2 to 8° C. for 2 to 10 days, for example, for 3 to 8 days.
  • the preparation method further comprises step 4), wherein the solution obtained in step 3) is filtered to obtain the inclusion compound.
  • the filtration is microfiltration, for example passing the solution through a 0.45 micron filter.
  • the present invention also provides the use of the inclusion compound in preparing a therapeutic pharmaceutical composition or preparation.
  • the present invention also provides a pharmaceutical composition or preparation containing the inclusion compound.
  • the pharmaceutical composition or preparation may further comprise a pharmaceutically acceptable excipient, for example, the excipient includes but is not limited to one or more of an excipient, a lubricant, a binder, a disintegrant, a solvent, a dissolution aid, a suspending agent, an isotonic agent, a pH regulator, a buffer, a preservative, an antioxidant, a colorant, a foaming agent, and a flavoring agent, preferably containing one or more of a solvent, an isotonic agent, and a pH regulator.
  • the pharmaceutical composition or preparation contains NaCl.
  • the pharmaceutical composition or preparation contains the following raw materials: porphyrin or its derivatives, cyclodextrin substances, water for injection, Tween and pH regulator.
  • the pharmaceutical composition or preparation contains the following raw materials: porphyrin or its derivatives, cyclodextrin Essence, water for injection and pH adjuster.
  • the mass percentage of the porphyrin or its derivative is 0.4-5%, such as 1-4%.
  • the mass percentage of the cyclodextrin substance is 30-50%, such as 35-45%.
  • the preparation is administered by injection, such as intrathecal, intraperitoneal, subcutaneous, intravenous, acupuncture point, intramuscular injection, etc.
  • the preparation may be an injection, such as an injection solution or a lyophilized powder injection.
  • the injection solution can be prepared by filtering and sterilizing the inclusion compound in the form of a solution prepared above.
  • the filtration is microfiltration, for example, passing the solution through a 0.22 micron filter.
  • the lyophilized powder injection can be prepared by filtering, sterilizing, and aseptically lyophilizing the inclusion compound in the form of a solution prepared above.
  • the filtration is microfiltration, for example, passing the solution through a 0.22 micron filter.
  • the pharmaceutical composition or preparation contains a second active substance, for example, the second active substance is an anti-tumor drug, preferably a glioma therapeutic agent, and as an example, it may be temozolomide (TMZ).
  • the second active substance is an anti-tumor drug, preferably a glioma therapeutic agent, and as an example, it may be temozolomide (TMZ).
  • the mass ratio of the porphyrin to the second active substance is (2-20):1, for example 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 15:1.
  • the present invention also provides the pharmaceutical composition or preparation for preventing and/or treating at least one of the following diseases: brain tumor disease or glioma, including but not limited to one or more of intracranial glioma (i.e., brain glioma), spinal cord glioma (e.g., spinal cord astrocytoma, preferably high-grade spinal cord astrocytoma), medulloblastoma and brain tumor formed by brain metastasis.
  • intracranial glioma i.e., brain glioma
  • spinal cord glioma e.g., spinal cord astrocytoma, preferably high-grade spinal cord astrocytoma
  • medulloblastoma medulloblastoma
  • brain tumor formed by brain metastasis a tumor formed by brain metastasis.
  • the invention provides a cyclodextrin inclusion compound of porphyrin and its derivatives, which greatly increases the solubility of poorly soluble porphyrin and has a simple preparation process and is easy to industrialize.
  • the injection containing the cyclodextrin inclusion compound has good stability and safety, and has a good therapeutic effect on pulmonary fibrosis and glioma.
  • the injection can still achieve good therapeutic effects on brain glioma at a low dosage; it is also found that the injection improves the drug distribution of active ingredients into the brain, which is of great value for improving the distribution of drugs in brain tissue.
  • the inventors also unexpectedly found that the injection containing the cyclodextrin inclusion complex of porphyrin and its derivatives can be combined with TMZ Combined use in the treatment of brain glioma can effectively reduce the dosage of TMZ, mitigate the drug's toxic side effects on the treated subjects, and improve the safety of medication.
  • the injection also has a good therapeutic effect on high-grade spinal astrocytoma.
  • Figure 1 is a comparison of the oral and intraperitoneal efficacy of BS alone (red frame) and BS + TMZ (blue frame).
  • Figure 2 shows the changes in body weight and tumor in the nude mouse U87 glioma model treated with BS, TMZ, and the combination of BS and TMZ.
  • A Body weight of mice in each group during the experiment;
  • B Tumor weight of mice in each group at the end of the experiment;
  • C Tumor volume of mice in each group during the experiment;
  • D Photos of tumors in each group at the end of the experiment.
  • FIG4 is a bar graph showing the body weight change rate of NPG mice after administration.
  • FIG5 is a graph showing the mean tumor volume of NPG mice at D24.
  • 100 mg of porselenide was added to the transparent light yellow solution under magnetic stirring, and the final weight was made up to 100 g with water.
  • the porselenide concentration was found to be 1869.30 ⁇ g/g by HPLC analysis.
  • the solution thus obtained was kept for 8 days at 4° C. A white precipitate formed under these conditions and was removed from the solution by filtration through a 0.45 micron filter.
  • the solution so obtained was kept at 4° C. for 3-8 days.
  • a white precipitate formed under these conditions and was removed from the solution by filtration through a 0.45 micron filter.
  • the solution so obtained was kept at 4° C. for 3-8 days.
  • a white precipitate formed under these conditions and was removed from the solution by filtration through a 0.45 micron filter.
  • the solution so obtained was kept at 4° C. for 3-8 days.
  • a white precipitate formed under these conditions and was removed from the solution by filtration through a 0.45 micron filter.
  • Test Examples 1-3 contain BS injection groups, in which BS exists in the form of cyclodextrin inclusion complexes.
  • BS group of Test Example 1 its prescription raw materials include: an inclusion complex formed by porphyrin and hydroxypropyl- ⁇ -cyclodextrin, the mass ratio of the two is 1:40, and the concentration of Tween 80 solution is 0.05%; the other groups are prepared with reference to the BS group of Test Example 1.
  • BS and TMZ solution for oral administration in subcutaneous Balb/c nude mouse U87 glioma model
  • mice After being prepared into a solution or suspension, the dosage for mice was 5 mL/kg.
  • BS and TMZ solution for intraperitoneal injection in the subcutaneous U87 glioma model of Balb/c nude mice.
  • mice After being prepared into a solution or suspension, the dosage for mice was 5 mL/kg.
  • the drug treatment regimen was as follows: blank control group (5 ⁇ CMC-Na, i.g., q.d.); TMZ group (30 mg/kg, i.g., q.d.); BSL group (10 mg/kg, i.g., q.d.); BSM group (90 mg/kg, i.g., q.d.); BSH group (180 mg/kg, i.g., q.d.); BSM+TMZ group (TMZ 30 mg/kg, BS 90 mg/kg, i.g., q.d.).
  • the qualified animals were randomly divided into 5 groups, control group, TMZ group, BS group, 6 animals in each group, BS+TMZ (treatment group) and BS+TMZ (prevention) group, 5 animals in each group.
  • BS was inoculated with U87 cells subcutaneously in the right armpit of mice, 2 ⁇ 10 6 /mouse.
  • Drug administration began 14 days after inoculation, which was the 0th day of treatment. The drug administration lasted for 18 days in total.
  • the drug treatment scheme is as follows: blank control group (0.9% NaCl, i.p., q.d.); TMZ group (10 mg/kg, i.p., qd.); BS group (30 mg/kg, i.p., q.d.); BS+TMZ treatment group (TMZ 10 mg/kg, i.p., qd., BS 90 mg/kg, i.g., q.d.); BS+TMZ prevention group (TMZ 10 mg/kg, i.p., qd., BS 90 mg/kg, i.g., q.d.).
  • TMZ cells were injected by tail vein.
  • the general condition of the animals and the time of tumor occurrence were recorded daily.
  • the long and short diameters of the mouse tumors were recorded daily, and the tumor volume was calculated according to the formula: long diameter ⁇ short diameter 2 ⁇ 0.5236.
  • the weight of the animals was measured every 3 days. After 18 days of administration, the mice were anesthetized with chloral hydrate and the eyeballs were removed for blood collection. The mice were killed, and the tumors, hearts, livers, spleens, lungs, kidneys, thymuses, whole brains, and intestines were quickly separated. The blood stains were washed off with ice saline, and the animals were wiped dry with filter paper.
  • the internal organs of the animals were observed for lesions and weighed.
  • a portion of the tumor tissue was stored in liquid nitrogen for RNA and protein extraction.
  • the other part of the tumor tissue was fixed in tissue fixative.
  • the distribution of Se content in brain tissue was detected by ICP-MS.
  • the efficacy of BS single-drug oral gavage/injection was compared, as shown in Figure 1 and Table 1.
  • the comparison of the efficacy of BS oral gavage (90 mg/kg) and intraperitoneal injection (30 mg/kg) showed that the tumor volume inhibition rate of BS oral gavage (90 mg/kg) was 78.87%, and the tumor volume inhibition rate of BS intraperitoneal injection (30 mg/kg) was 81.12%.
  • This result shows that the intraperitoneal injection method can inhibit tumor growth more significantly under the premise of reducing the amount of BS.
  • BS single-drug injection can significantly inhibit the proliferation of human glioma cells U87, and the inhibitory effect is far better than TMZ.
  • the efficacy of BS+TMZ combined oral gavage/intraperitoneal injection was compared.
  • the tumor volume inhibition rate of BS+TMZ oral administration (90+30mg/kg) was 78.48%, and the tumor volume inhibition rate of BS+TMZ intraperitoneal injection (90+10mg/kg) was 84.29%.
  • the intraperitoneal injection method can inhibit tumor growth more significantly under the premise of reducing the dosage of TMZ. It is suggested that BS alone and BS+TMZ combined intraperitoneal injection have a more obvious effect of inhibiting tumor growth than oral administration, and can achieve BS or TMZ drug reduction and reduce TMZ toxicity.
  • mice The brain tissues of mice after the BS+TMZ gavage (90+10 mg/kg) and BS intraperitoneal injection (30 mg/kg) experiments were taken for ICP-MS detection.
  • the BS content in each gram of brain tissue was calculated by detecting the selenium content in each gram of brain tissue.
  • the distribution of BS in brain tissue is shown in Table 2.
  • BS is distributed in brain tissue.
  • the BS content in brain tissue of BS intraperitoneally injected at a dose of 30 mg/kg was significantly higher than that of BS gavage at a dose of 90 mg/kg. This shows that the administration of BS by intraperitoneal injection is more conducive to BS entering the brain tissue and achieving the effect of BS reduction.
  • mice Six-week-old female Balb/c nude mice were selected to establish an animal model, and 2 ⁇ 10 6 U87 human glioma cells were subcutaneously inoculated. Drug administration began on the 14th day after inoculation and continued for 18 days.
  • control group TMZ group (10 mg/Kg) and BS group (30 mg/Kg) were intraperitoneally injected once a day in this round of experiments.
  • the mice in the control group were given 0.9% NaCl.
  • this round of experiments set up a combined administration group: BS+TMZ treatment group (BS 90mg/Kg & TMZ 10mg/Kg) and BS+TMZ prevention group (BS 90mg/Kg & TMZ 10mg/Kg).
  • the BS+TMZ treatment group is referred to as the BS+TMZ (A) group.
  • the BS+TMZ prevention group was referred to as the BS+TMZ (B) group.
  • mice On the 14th day after U87 inoculation, the tumor volume of mice was less than 20 mm 3 and the drug was started to observe the efficacy of the combination in preventing tumor growth.
  • the combination group was administered by oral gavage once a day. The start time of drug administration was Day 1, the second day of drug administration was Day 2, and so on.
  • mice in each group during the administration process were shown in Figure 2 (A).
  • the weight of mice in the TMZ group (30 mg/Kg) decreased significantly, and the TMZ group experiment was terminated on the 12th day of administration due to the extremely poor animal condition.
  • the mice in the BS group, BS+TMZ (A) group, and BS+TMZ (B) group had normal diet and water intake, normal activities, good mental state, and smooth and shiny hair, indicating that BS had no obvious toxic side effects on tumor-bearing nude mice.
  • mice in the TMZ group were in extremely poor condition on the 12th day of administration, so the experiment was terminated early, so the data are missing. * indicates a significant difference compared with the control group, *p ⁇ 0.05; **p ⁇ 0.05, ***p ⁇ 0.001.
  • mice in the TMZ group were in extremely poor condition on the 12th day of administration, and the experiment was terminated early, so their data were not compared.
  • Gliomas are located in the brain and have a special lesion location. Therefore, it is crucial whether the drug can pass through the blood-brain barrier and work at the lesion.
  • the study selected female six-week-old ICR mice, injected 90 mg/kg BS intraperitoneally, and brain tissues were taken before, 0.5 h, 1 h, 2 h, and 3 h after administration.
  • ICP-MS detection By detecting the selenium content in each gram of brain tissue, the BS content in each gram of brain tissue can be calculated.
  • BS The distribution of BS in brain tissue is shown in Table 4.
  • BS is distributed in brain tissue.
  • the BS content in brain tissue reached 438.75 ng/g.
  • the BS content in brain tissue reached a peak of 528.50 ng/g and then decreased. This result shows that BS can break through the blood-brain barrier and be distributed in the brain tissue of mice.
  • Detection indicators mouse body weight and subcutaneous tumor volume.
  • BS injection drug content 30mg and 90mg BS; weigh 1mg TMZ, dissolve it in 1ml 5 ⁇ CMC-Na, assist with ultrasound to evenly disperse TMZ to obtain TMZ drug solution, prepare it before each use.
  • mice Animal species: NPG mice, animal grade: SPF grade, gender and number: female, 32
  • Animal weight range 20-25 g
  • animal age range 5-6 weeks old
  • animal source Beijing Weitongda Biotechnology Co., Ltd.
  • mice were randomly divided into 4 groups: control group, BSL group, BSH group and TMZ group, with 8 mice in each group.
  • mice Prepare human spinal cord high-grade astrocytoma tumors (2-3 mm 3 ) and inoculate them in the right axilla of NPG mice. The mice were observed and the tumor size was measured. The animals were screened according to the tumor volume, and the animals with a tumor volume of 450 mm3 were selected for the experiment. Those with a tumor volume that was too large or without a tumor were not selected. A total of 32 suitable tumor-bearing animals were screened for the experiment and divided into 4 experimental groups, with 8 mice in each group. The day of the first administration was recorded as D1, the second day of administration was recorded as D2, and so on. All animals were administered a single intraperitoneal injection per day for 24 consecutive days.
  • this injection dosage form experiment selected 90 mg/kg as the maximum dose for intraperitoneal injection.
  • tumor volume 0.5236 ⁇ long diameter ⁇ short diameter ⁇ short diameter.
  • Figure 3 is the tumor volume change curve of NPG mice during the treatment of BS and TMZ after grouping.
  • Figure 4 shows the weight changes of mice in each group during the administration process.
  • the BS group (30, 90 mg/kg) did not show obvious weight loss at this study dose, indicating that intraperitoneal injection of BS had no obvious toxic side effects on NPG mice.
  • the mice were in good mental state, indicating that the drug toxicity was low and conducive to long-term maintenance.
  • BS had significant tumor inhibition in the 90 mg/kg dose group and the TMZ group.
  • the tumor sizes of the Control group, BS (30, 90 mg/kg) and TMZ group were 4155.70 ⁇ 1809.90, 4593.83 ⁇ 2725.69, 2999.76 ⁇ 2191.03, 2693.29 ⁇ 1682.23, respectively.
  • the tumor volume inhibition rates of the low and high dose groups of BS at the end of the experiment were -10.54%, 35.19%, and the tumor volume inhibition rate of the TMZ group (5 mg/kg) was 27.82%, indicating that BS (90 mg/kg) and TMZ can inhibit the growth of spinal cord glioma.

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Abstract

本发明公开了一种卟硒啉环糊精包合物、药物组合物及其制备方法和应用。所述包合物为包含卟硒啉或其衍生物与环糊精类物质的复合物，所述药物组合物包含所述包合物及药学上可以接受的辅料。含有该环糊精包合物的注射剂具有良好的稳定性、安全性，对脑肿瘤疾病或胶质瘤具有良好的治疗效果。

Description

一种卟硒啉环糊精包合物、药物组合物及其制备方法和应用
本申请要求2022年11月21日向中国国家知识产权局提交的专利申请号为202211459427.9、发明名称为“一种卟硒啉环糊精包合物、药物组合物及其制备方法和应用”的在先申请的优先权。所述在先申请的全文通过引用的方式结合于本申请中。
技术领域
本发明属于医药领域，涉及一种卟硒啉环糊精包合物、药物组合物及其制备方法和应用。
背景技术
肺纤维化是一类以成纤维细胞增殖及大量细胞外基质(ECM)聚集并伴炎症损伤、组织结构破坏为特征的肺疾病改变，一旦发生就无法逆转，肺功能受损将严重影响患者的生活质量，甚至危及生命。
脊髓属于中枢神经系统，其胶质瘤发生率较低，占所有原发脊髓肿瘤的8～10％，所有中枢神经系统肿瘤的2～4％。根据其细胞源不同，可分为室管膜瘤(60～70％)和各级星形细胞瘤(30～40％)。由于脊髓横径有限且具有重要生理功能，治疗上颇为棘手，目前胶质瘤的各种治疗指南大多针对颅内疾病，而对脊髓胶质瘤无很好的诊疗规范作为依据，造成了各治疗中心存在较大差异，从而影响患者生活质量。目前对于脊髓胶质瘤的治疗方案也呈现多样化，包括显微外科手术、放化疗、免疫治疗等。
目前国际上临床对于肺纤维化及胶质瘤缺乏显著有效的治疗药物，尤其是针对非IPF的间质性肺病和纤维化患者，未被满足的临床需求极为迫切。
卟硒啉是靶向硫氧还蛋白还原酶(TR)设计的化学小分子药物。已完成的系列研究表明，卟硒啉对于器官纤维化疾病全链条均表现出控制效果。通过抑制炎症信号和关键ECM合成通路，卟硒啉可以缓解器官纤维化表现及后续疾病进展。
目前已批准临床使用的卟硒啉片剂，对肺纤维化疾病有治疗效果，由于肝脏首过效应，导致药物效率降低，且由于血脑屏障影响即对颅内胶质瘤并无药效，更为有效针对颅内胶质瘤的卟硒啉制剂被迫切需要。
根据对卟硒啉性质的研究分析，其为难溶性药物，在水中的溶解度经检测分析为150ng/mL左右，因此其在体内生物利用度低，极大地限制了临床应用。下图为卟硒啉化学结构式。
发明内容
为改善上述技术问题，本发明提供一种包合物，所述包合物为包含卟硒啉或其衍生物与环糊精类物质的复合物。
根据本发明的实施方案，所述环糊精类物质选自β-环糊精类物质和/或γ-环糊精类物质，例如可以选自羟丙基-β-环糊精、磺丁基-β-环糊精、羟丙基-γ-环糊精、甲基-β-环糊精和2,6-二甲基-β-环糊精中的一种或两种以上；优选为羟丙基-β-环糊精。
根据本发明的实施方案，所述包合物由环糊精类物质对卟硒啉或其衍生物进行包合形成。
根据本发明的实施方案，所述卟硒啉或其衍生物与环糊精类物质的质量比为1:(10～480)，例如1:(10～100)，示例性为1:10、1:20、1:30、1:40、1:45、1:49、1:50、1:60。
根据本发明的实施方案，所述环糊精类物质与卟硒啉或其衍生物的包合比为(1～15):1，例如(2～10):1，比如为1.5:1、2.5:1、3:1、4:1。
根据本发明的实施方案，所述包合物可以为下述包合物中的任意一种：
由羟丙基-β-环糊精对卟硒啉包合形成的包合物、由磺丁基-β-环糊精对卟硒啉包合形成的包合物、由羟丙基-γ-环糊精对卟硒啉包合形成的包合物、由甲基-β-环糊精对卟硒啉包合形成的包合物、由2,6-二甲基-β-环糊精对卟硒啉包合形成的包合物。
本发明还提供上述包合物的制备方法，包括：使用环糊精类物质对卟硒啉或其衍生物进行包合。
根据本发明的实施方案，所述卟硒啉或其衍生物与环糊精类物质的选择以及质量比如上文所示。
根据本发明的实施方案，所述包合在水中或吐温80溶液中进行。
根据本发明的实施方案，所述水为注射用水。
根据本发明的实施方案，所述吐温80溶液为吐温80水溶液。优选地，所述吐温80溶液的浓度为0.01～0.1％，例如0.05％。
根据本发明的实施方案，所述制备方法包括如下步骤：
1)先将环糊精类物质溶于水中或吐温80溶液中，形成透明溶液，再将卟硒啉或其衍生物 加入到所述透明溶液中，加水或吐温80溶液定量；
2)步骤1)完成后，搅拌，离心，任选过滤或不过滤。
根据本发明的实施方案，所述环糊精类物质与体系中水的质量比为(10～55):100，例如(20～50):100，示例性为30:100、40:100、48:100；所述体系中水的质量为溶解环糊精类物质用水和定量加水的质量总和。
根据本发明的实施方案，所述环糊精类物质与体系中吐温80溶液的质量比为(10～55):100，例如(20～50):100，示例性为30:100、40:100、48:100；所述体系中吐温80溶液的质量为溶解环糊精类物质用量和定量加吐温80溶液的质量总和。
根据本发明的实施方案，所述卟硒啉或其衍生物以微粉形式加入。
根据本发明的实施方案，所述搅拌的温度为25～80℃，优选40～70℃，例如30℃、40℃、50℃、60℃、70℃。
根据本发明的实施方案，所述搅拌的时间为0.5～5小时，优选3小时。
根据本发明的实施方案，所述离心的转速为4000～10000rpm，优选为7000～9000rpm，例如为9000rpm。
根据本发明的实施方案，所述离心的时间为至少5min，例如5～30min，示例性为20min。
根据本发明的实施方案，所述制备方法还包括步骤3)，将步骤2)得到的透明溶液在2～8℃的温度下保持2～10天，例如保持3～8天。
根据本发明的实施方案，所述制备方法还包括步骤4)，步骤3)得到的溶液经过滤，得到所述包合物。
根据本发明的实施方案，所述过滤为微滤，例如将溶液通过0.45微米过滤器。
本发明还提供上述包合物在制备治疗药物组合物或制剂中的应用。
本发明还提供一种药物组合物或制剂，含有上述包合物。
根据本发明的实施方案，所述药物组合物或制剂还可以包含药学上可接受的辅料，例如所述辅料包括但不限于赋形剂、润滑剂、粘合剂、崩解剂、溶剂、溶解助剂、悬浮剂、等渗剂、pH调节剂、缓冲液、防腐剂、抗氧剂、着色剂、起泡剂和调味剂等中的一种或多种，优选含有溶剂、等渗剂、pH调节剂中的一种或多种。在一些实施方案中，所述药物组合物或制剂含有NaCl。
在一种实施方式中，所述药物组合物或制剂含有下述原料：卟硒啉或其衍生物、环糊精类物质、注射用水、吐温和pH调节剂。
在另一种实施方式中，所述药物组合物或制剂含有下述原料：卟硒啉或其衍生物、环糊 精类物质、注射用水和pH调节剂。
优选地，所述药物组合物或制剂中，所述卟硒啉或其衍生物的质量百分比为0.4～5％，例如1～4％。
优选地，所述药物组合物或制剂中，所述环糊精类物质的质量百分比为30～50％，例如35～45％。
根据本发明的实施方案，所述制剂的给药方式为注射给药，例如鞘内、腹腔、皮下、静脉、穴位、肌内注射等给药。
根据本发明的实施方案，所述制剂可以为注射剂，例如注射液或冻干粉针。
根据本发明的实施方案，所述注射液可以由上述制备得到的溶液形式的包合物经过滤、灭菌制备得到。优选地，所述过滤为微滤，例如将溶液通过0.22微米过滤器。
根据本发明的实施方案，所述冻干粉针可以由上述制备得到的溶液形式的包合物经过滤、灭菌、无菌冻干制备得到。优选地，所述过滤为微滤，例如将溶液通过0.22微米过滤器。
根据本发明的实施方案，所述药物组合物或制剂含有第二活性物质，例如所述第二活性物质为抗肿瘤药物，优选为胶质瘤治疗剂，作为示例可以为替莫唑胺(TMZ)。
在一些实施方案中，所述卟硒啉与所述第二活性物质的质量比为(2-20):1，例如3:1、4:1、5:1、6:1、7:1、8:1、9:1、10:1、12:1、15:1。
本发明还提供所述药物组合物或制剂用于预防和/或治疗下述疾病中的至少一种：脑肿瘤疾病或胶质瘤，包括但不限于颅内胶质瘤(即脑胶质瘤)、脊髓胶质瘤(例如脊髓星形细胞瘤，优选为高级别脊髓星形细胞瘤)，髓母细胞瘤及脑转移癌形成的脑肿瘤等其中的一种或多种。
本发明的有益效果：
本发明提供了一种卟硒啉及其衍生物的环糊精包合物，极大地增加了难溶性卟硒啉的溶解度，且制备工艺简单、易于工业化。
含有该环糊精包合物的注射剂具有良好的稳定性、安全性，对肺纤维化疾病、胶质瘤具有良好的治疗效果。
相比于口服给药，所述注射剂能够在低给药剂量下依旧对脑胶质瘤治疗取得良好的治疗效果；还发现所述注射剂提高了活性成分进入脑部的药物分布结果，对于改善药物的脑组织分布具有重要价值。
发明人还出人意料地发现，含有卟硒啉及其衍生物的环糊精包合物的注射剂能够与TMZ 联用治疗脑胶质瘤，有效降低TMZ的用药剂量，减缓药物对治疗对象的毒副作用，提升用药安全性。
同时，所述注射剂对于高级别脊髓星形细胞瘤也具有良好的治疗效果。
附图说明
图1为BS单药(红框)及BS+TMZ(蓝框)口服以及腹腔注射药效对比。
图2为BS、TMZ以及二者联合作用于裸鼠U87胶质瘤模型的体重和肿瘤变化。(A)实验过程中各组小鼠体重情况；(B)实验结束时各组小鼠的肿瘤重量；(C)实验过程中各组小鼠肿瘤体积；(D)实验终点各组小鼠肿瘤照片。(A)、(B)、(C)数据表现为均值±SD，n＝5or 6。*表示与对照组相比具有统计学差异，*P<0.05，**P<0.01。
图3为NPG小鼠治疗过程中肿瘤体积变化曲线(n＝8)。
图4为NPG小鼠给药后体重变化率柱形图。
图5为NPG小鼠D24时肿瘤体积均值图。
具体实施方式
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。
实施例1
在磁搅拌下将2,6-二甲基-β-环糊精溶于大约51.9g水中(环糊精：水＝55:100)。在磁搅拌下将100mg卟硒啉加到该透明淡黄色溶液，并用水使最终重量补充至100g。于70℃水浴加热，搅拌3小时，将溶液通过0.45微米过滤器以获得一种透明淡黄色溶液。用HPLC分析发现卟硒啉浓度为1869.30μg/g。
实施例2
在磁搅拌下将4.8g羟丙基-β-环糊精溶于大约4.7g 0.05％吐温80溶液。在磁搅拌下将480mg卟硒啉加到该透明无色溶液，并用0.05％吐温80溶液使最终重量补充至10g。将其搅拌2小时于10000rpm离心10min，将溶液过滤通过0.45微米过滤器以获得一种透明无色溶液。用HPLC分析发现卟硒啉浓度为560μg/g。
将如此得到的溶液在4℃下保持8天。在这些条件下形成白色沉淀，经过滤通过0.45微米过滤器从溶液中除去沉淀。
获得一种透明无色溶液，用HPLC分析发现卟硒啉浓度为430μg/g。
实施例3
在磁搅拌下将9.6g羟基丙基-β-环糊精溶于大约9.4g 0.05％吐温80溶液。在磁搅拌下将196mg卟硒啉加到该透明无色溶液，并用0.05％吐温80溶液使最终重量补充至20g。于70℃将其搅拌5小时后于9000rpm离心10min，将溶液过滤通过0.45微米过滤器以获得一种透明无色溶液。用HPLC分析发现卟硒啉浓度为414μg/g。
将如此得到的溶液在4℃下保持3-8天。在这些条件下形成白色沉淀，经过滤通过0.45微米过滤器从溶液中除去沉淀。
获得一种透明无色溶液，用HPLC分析发现卟硒啉浓度为352μg/g。
实施例4
在磁搅拌下将9.6g羟基丙基-β-环糊精溶于大约9.4g注射用水。在磁搅拌下将192mg卟硒啉加到该透明无色溶液，并用水使最终重量补充至20g。于40℃将其搅拌3小时于9000rpm离心10min。将溶液过滤通过0.45微米过滤器以获得一种透明无色溶液。用HPLC分析发现卟硒啉浓度为366μg/g。
将如此得到的溶液在4℃下保持3-8天。在这些条件下形成白色沉淀，经过滤通过0.45微米过滤器从溶液中除去沉淀。
获得一种透明无色溶液，用HPLC分析发现卟硒啉浓度为310μg/g。
实施例5
在磁搅拌下将4.8g磺丁基-β-环糊精溶于大约4.7g注射用水。在磁搅拌下将480mg卟硒啉加到该透明无色溶液，并用水使最终重量补充至10g。于25℃将其搅拌1小时于10000rpm离心10min。将溶液过滤通过0.45微米过滤器以获得一种透明无色溶液。用HPLC分析发现卟硒啉浓度为133μg/g。
实施例6
在磁搅拌下将9.6g羟基丙基-β-环糊精溶于大约9.4g注射用水溶液。在磁搅拌下将196mg卟硒啉加到该透明无色溶液，并用水使最终重量补充至20g。于70℃将其搅拌3小时于9000rpm离心10min。将溶液过滤通过0.45微米过滤器以获得一种透明无色溶液。用HPLC分析发现卟硒啉浓度为601μg/g。
将如此得到的溶液在4℃下保持3-8天。在这些条件下形成白色沉淀，经过滤通过0.45微米过滤器从溶液中除去沉淀。
获得一种透明无色溶液，用HPLC分析发现卟硒啉浓度为473μg/g。
下述试验例1-3中的BS为卟硒啉的简写；
试验例1-3含有BS的注射组，其中的BS均以环糊精包合物形式存在。以试验例1BS组为例，其处方原料包括：卟硒啉与羟丙基-γ-环糊精对形成的包合物，二者质量比1:40，吐温80溶液的浓度为0.05％；其他各组参照试验例1BS组制备。
试验例1
1.供试品
BS和TMZ溶液(用于Balb/c裸鼠U87脑胶质瘤皮下模型灌胃给药)
制备成溶液或者混悬液后，小鼠的给药剂量均为5mL/kg。
BS和TMZ溶液(用于Balb/c裸鼠U87脑胶质瘤皮下模型腹腔注射给药)
制备成溶液或者混悬液后，小鼠的给药剂量均为5mL/kg。
2.实验方法
2.1 Balb/c裸鼠U87脑胶质瘤皮下模型灌胃给药实验
动物适应性饲养2天后，保留合格动物随机分为6组，每组7只。于小鼠右腋皮下接种U87细胞，2×106/只。接种12天后开始给药，此为治疗第0天。共给药11天。
药物治疗方案如下：空白对照组(5‰CMC-Na,i.g.,q.d.)；TMZ组(30mg/kg,i.g.,q.d.)；BSL组(10mg/kg,i.g.,q.d.)；BSM组(90mg/kg,i.g.,q.d.)；BSH组(180mg/kg,i.g.,q.d.)；BSM+TMZ组(TMZ 30mg/kg,BS 90mg/kg,i.g.,q.d.)。
2.2 Balb/c裸鼠U87脑胶质瘤皮下模型注射给药(腹腔)实验
动物适应性饲养2天后，保留合格动物随机分为5组，对照组、TMZ组、BS组每组6只。BS+TMZ(治疗组和BS+TMZ(预防)组每组5只。BS于小鼠右腋皮下接种U87细胞，2×106/只。接种14天后开始给药，此为治疗第0天。共给药18天。
药物治疗方案如下：空白对照组(0.9％NaCl,i.p.,q.d.)；TMZ组(10mg/kg,i.p.,qd.)；BS组(30mg/kg,i.p.,q.d.)；BS+TMZ治疗组(TMZ 10mg/kg,i.p.,qd.,BS 90mg/kg,i.g.,q.d.)；BS+TMZ预防组(TMZ 10mg/kg,i.p.,qd.,BS 90mg/kg,i.g.,q.d.)。其中，TMZ细胞采取尾静脉注射。
实验期间每日记录动物一般状况和肿瘤发生时间，每日记录小鼠肿瘤长径、短径，并根据公式：长径×短径2×0.5236计算肿瘤体积，每3日测量动物体重。给药18天后，水合氯醛麻醉，摘眼球取血。处死小鼠，迅速分离肿瘤、心脏、肝脏、脾脏、肺脏、肾脏、胸腺、全脑、肠，冰生理盐水洗去血污，滤纸拭干，观察动物内脏有无病变，称重。组织取一部分肿瘤组织液氮保存，以备提取RNA和蛋白质。另一部分肿瘤组织置于组织固定液进行固定。通过ICP-MS检测脑组织Se含量分布情况。
3.实验结果
3.1灌胃与注射剂型BS对裸鼠胶质瘤U87模型的药效对比
将BS单药灌胃/注射剂型(腹腔)药效进行对比，如图1及表1所示。BS灌胃(90mg/kg)与腹腔注射(30mg/kg)药效对比发现，BS灌胃(90mg/kg)肿瘤体积抑制率为78.87％，BS腹腔注射(30mg/kg)肿瘤体积抑制率为81.12％。该结果说明，采用腹腔注射给药方式可以在BS减量的前提下，抑制肿瘤生长的药效更加明显。并且还显示，BS单药注射剂能够显著抑制人源胶质瘤细胞U87的增殖，抑制作用远优于TMZ。
表1 BS单药及BS+TMZ口服/腹腔注射药效对比
将BS+TMZ联用灌胃/腹腔注射药效进行对比，BS+TMZ口服(90+30mg/kg)肿瘤体积抑制率为78.48％，BS+TMZ腹腔注射(90+10mg/kg)肿瘤体积抑制率为84.29％。采用腹腔注射给药方式可以在TMZ减量的前提下，抑制肿瘤生长的药效更加明显。提示BS单药及BS+TMZ联用腹腔注射给药较口服给药，抑制肿瘤生长的药效更加明显，且可以实现BS或TMZ药物减量，降低TMZ毒性。
3.2灌胃与腹腔注射BS在裸鼠胶质瘤U87模型的脑组织分布对比
分别取BS+TMZ灌胃(90+10mg/kg)与BS腹腔注射(30mg/kg)实验结束后的小鼠脑组织进行ICP-MS检测，通过检测每克脑组织中的硒含量，从而计算出每克脑组织中的BS含量。脑组织BS分布情况如表2所示，BS在脑组织中有分布。BS在30mg/kg剂量下腹腔注射给药的脑组织BS含量明显高于BS在90mg/kg剂量下灌胃给药。说明BS采用腹腔注射的给药方式更有利于BS进入脑组织，实现BS减量的效果。
表2 BS在U87裸鼠给药后的脑组织分布
对比灌胃与注射剂型(腹腔)BS对裸鼠胶质瘤U87模型的药效，发现BS单药及BS+TMZ联用注射(腹腔)给药较口服灌胃给药，抑制肿瘤生长的药效更加明显，且可以实现BS或TMZ药物减量，降低TMZ毒性。ICP-MS检测BS脑组织分布情况发现BS采用腹腔注射的给药方式更有利于BS进入脑组织，实现BS减量的效果。
试验例2 BS与TMZ注射联用对裸鼠胶质瘤U87模型药效学研究
1.BS与TMZ协同抑制U87荷瘤裸鼠肿瘤增殖
选择雌性六周龄Balb/c Nude裸鼠建立动物模型，以2×106/只皮下接种U87人源胶质瘤细胞。接种后第14天开始给药，连续给药18天。
为了对比口服灌胃给药与腹腔注射给药的药效差异，本轮实验中对照组、TMZ组(10mg/Kg)及BS组(30mg/Kg)采用每日单次腹腔注射给药。对照组小鼠给予0.9％NaCl。
为了对比BS与TMZ联合应用的治疗药效与预防药效，本轮实验设置联用给药组：BS+TMZ治疗组(BS 90mg/Kg&TMZ 10mg/Kg)和BS+TMZ预防组(BS 90mg/Kg&TMZ 10mg/Kg)。其中，BS+TMZ治疗组简称BS+TMZ(A)组，U87接种后第14天小鼠正常出瘤体积 大于20mm3并开始给药，以观察联用对抑制肿瘤增殖的药效表现。BS+TMZ预防组简称BS+TMZ(B)组，U87接种后第14天小鼠出瘤体积小于20mm3并开始给药，以观察联用对预防肿瘤出瘤的药效表现。联用组给药方式采取每日单次口服灌胃。开始给药时间为Day 1，给药第2天为Day 2，以此类推。
给药过程中各组小鼠体重变化情况如图2中(A)所示，TMZ组(30mg/Kg)小鼠体重明显下降，于给药第12天因动物状态极差而结束TMZ组实验。BS组、BS+TMZ(A)组、BS+TMZ(B)组小鼠饮食饮水正常，活动正常，精神状态良好，毛发光滑有光泽，提示BS对荷瘤裸鼠没有明显毒副作用。
实验期间，各组小鼠肿瘤体积的变化如图2中(C)所示，BS组、BS+TMZ(A)组、BS+TMZ(B)组小鼠肿瘤体积均显著低于对照组小鼠肿瘤体积(p＜0.01)。实验结束后，各组小鼠肿瘤照片如图2中(D)所示，可见给药组小鼠肿瘤相比对照组明显减小。对肿瘤进行称重统计分析结果如图2中(B)及表3所示。
表3实验结束时各组小鼠肿瘤体积及重量
数据表示为均值±SD,n＝5or 6。TMZ组小鼠由于给药第12天状态极差，提前结束实验，故数据缺失。*表示与对照组相比具有显著性差异，*p＜0.05；**p＜0.05，***p<0.001。
分析采取腹腔注射给药的对照组、TMZ组、BS组裸鼠U87胶质瘤模型的药效学表现，发现BS组(30mg/Kg)肿瘤体积及肿瘤重量均显著低于对照组，可显著抑制肿瘤生长。至实验结束，BS组小鼠肿瘤体积抑制率及重量抑制率分别为81.12％、66.67％(表3)。该结果提示BS在采用腹腔注射给药时，BS降低剂量同样能够显著抑制肿瘤生长。TMZ组小鼠由于给药第12天状态极差，提前结束实验，故不对其数据进行比较。
以下考察BS在小鼠脑组织的分布情况：
脑胶质瘤位于颅内，病灶位置特殊。因此，药物是否能够通过血脑屏障并在病灶处发挥作用至关重要。为了检测BS是否能够通过血脑屏障到达脑组织，研究选择雌性六周龄ICR小鼠，小鼠腹腔注射90mg/kg BS，分别取给药前、给药后0.5h、1h、2h、3h时的脑组织进行 ICP-MS检测。通过检测每克脑组织中的硒含量，从而计算出每克脑组织中的BS含量。
脑组织BS分布情况如表4所示，BS在脑组织中有分布。给药0.5h时脑组织BS即达到438.75ng/g，给药2h前后，脑组织中BS含量达到峰值528.50ng/g，随后降低。该结果说明BS能够突破血脑屏障，分布在小鼠脑组织中。
表4 ICR小鼠腹腔注射BS 90mg/kg后的BS脑组织分布
试验例3 BS注射剂对大体积人脊髓高级别星形细胞瘤PDX小鼠皮下模型的药效学研究
1.方法：
1)动物分组：皮下接种肿瘤组织块，待动物出瘤后，以肿瘤体积均值相近原则分组；
2)给药方式：BS、TMZ腹腔注射(i.p.,q.d.×17天)；
3)检测指标：小鼠体重和皮下肿瘤体积。
2.供试品配制
BS注射剂，药物含量30mg和90mg BS；称取1mg TMZ，溶于1ml 5‰CMC-Na中，辅助超声使TMZ均匀分散，得到TMZ药物溶液，每次现用现配。
3.实验动物
动物种属：NPG小鼠，动物等级：SPF级，性别和数量：雌性，32只
动物体重范围：20-25g，动物年龄范围：5-6周龄，动物来源：北京维通达生物技术有限公司。
4.动物筛选和分组
按表5随机分为4组，对照组、BSL、BSH及TMZ组每组8只。
表5
5.给药方式和频率
准备人源脊髓高级别星形细胞瘤肿瘤块(2-3mm3)，于NPG小鼠右腋下接种，接种后常 规观察小鼠状态并测量肿瘤大小。按肿瘤体积大小进行筛选，挑选肿瘤体积450mm3的动物进行实验，肿瘤体积过大及未成瘤者不予入选。共筛选到32只合适的荷瘤动物用于实验，分为4个实验组，每组8只老鼠。首次给药当天记为D1，给药第2天记为D2，以此类推。所有动物均为每日单次腹腔注射给药，连续给药24天。
6.剂量设计依据
前期实验结果，BS注射剂在90,180,360mg/kg剂量范围内灌胃给药，动物无毒性反应，且肿瘤抑制药效呈剂量依赖性。因此本注射剂型实验选择90mg/kg剂量作为本实验最大给药剂 量进行腹腔注射。
7.检测指标
7.1体重
所有动物实验期间每天观察一次。观察内容包括死亡或濒死、粪便、摄食情况、精神状态以及行为活动等，发现异常情况，及时记录。所有动物分组前称重1次，给药后每周称重3次，安乐死前称重1次。
7.2肿瘤体积
实验过程中，所有动物每天量肿瘤的长径和短径。并计算肿瘤体积(mm3)，公式如下：肿瘤体积＝0.5236×长径×短径×短径。
8.实验结果
8.1肿瘤生长曲线
在本实验中，当实验组小鼠生命体征濒临死亡或体重明显下降(＞20％)即终止实验。实验终止时，各实验组小鼠均未死亡，共有32只小鼠实验数据纳入统计。图3为NPG小鼠在分组后使用BS及TMZ治疗过程中的肿瘤体积变化曲线。
8.2体重
本实验过程中，未见因药物毒性出现的临床异常症状或动物死亡。图4为给药过程中各组小鼠体重变化情况。BS组(30，90mg/kg)在此研究剂量下未呈明显体重下降表现，提示BS腹腔注射对NPG小鼠没有明显毒副作用，用药过程中，小鼠精神状态佳，说明药物毒性低，有利于长期维持。
8.3解剖当日小鼠皮下肿瘤体积
由图5所示，BS在90mg/kg剂量组和TMZ组存在显著的肿瘤抑制。试验结束时，Control组、BS(30，90mg/kg)及TMZ组的肿瘤大小分别为4155.70±1809.90,4593.83±2725.69，2999.76±2191.03，2693.29±1682.23，BS低、高剂量组在实验终止时的肿瘤体积抑制率分别为 -10.54％、35.19％，TMZ组(5mg/kg)肿瘤体积抑制率为27.82％，说明BS(90mg/kg)与TMZ可抑制脊髓胶质瘤生长。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种包合物，其特征在于，所述包合物为包含卟硒啉或其衍生物与环糊精类物质的复合物。
  2. 根据权利要求1所述的包合物，其特征在于，所述环糊精类物质选自β-环糊精类物质和/或γ-环糊精类物质，例如可以选自羟丙基-β-环糊精、磺丁基-β-环糊精、羟丙基-γ-环糊精、甲基-β-环糊精和2,6-二甲基-β-环糊精中的一种或两种以上；
    优选地，所述包合物由环糊精类物质对卟硒啉或其衍生物进行包合形成。
    优选地，所述卟硒啉或其衍生物与环糊精类物质的质量比为1:(10～480)，例如1:(10～100)。
  3. 权利要求1或2所述包合物的制备方法，其特征在于，所述制备方法包括：使用所述环糊精类物质对卟硒啉或其衍生物进行包合。
  4. 根据权利要求3所述的制备方法，其特征在于，所述包合在水中或吐温80溶液中进行。
  5. 根据权利要求3或4所述的制备方法，其特征在于，所述制备方法包括如下步骤：
    1)先将环糊精类物质溶于水中或吐温80溶液中，形成透明溶液，再将卟硒啉或其衍生物加入到所述透明溶液中，加水或吐温80溶液定量；
    2)步骤1)完成后，搅拌，离心，任选过滤或不过滤。
    优选地，所述环糊精类物质与体系中水的质量比为(10～55):100，所述体系中水的质量为溶解环糊精类物质用水和定量加水的质量总和。
    优选地，所述环糊精类物质与体系中吐温80溶液的质量比为(10～55):100，所述体系中吐温80溶液的质量为溶解环糊精类物质用量和定量加吐温80溶液的质量总和。
  6. 权利要求1或2所述的包合物在制备治疗药物组合物或制剂中的应用。
  7. 一种药物组合物或制剂，其特征在于，所述组合物或所述制剂含有权利要求1或2所述的包合物。
  8. 根据权利要求7所述的药物组合物或制剂，其特征在于，所述药物组合物或制剂还包含药学上可接受的辅料。
    优选地，所述制剂的给药方式为注射给药。
    优选地，所述制剂为注射剂，例如注射液或冻干粉针。
  9. 根据权利要求7或8所述的药物组合物或制剂，其特征在于，所述药物组合物或制剂含有第二活性物质，例如所述第二活性物质为抗肿瘤药物，优选为胶质瘤治疗剂，作为示例可以为替莫唑胺(TMZ)。
  10. 权利要求7-9任一项所述的药物组合物或制剂用于预防和/或治疗下述疾病中的至少一种：脑肿瘤疾病或胶质瘤，包括但不限于颅内胶质瘤(即脑胶质瘤)、脊髓胶质瘤(例如脊髓星形细胞瘤，优选为高级别脊髓星形细胞瘤)、髓母细胞瘤及脑转移癌形成的脑肿瘤中的一种或多种。
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