WO2019096096A1 - Conjugué de ciblage à plusieurs bras - Google Patents

Conjugué de ciblage à plusieurs bras Download PDF

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WO2019096096A1
WO2019096096A1 PCT/CN2018/115059 CN2018115059W WO2019096096A1 WO 2019096096 A1 WO2019096096 A1 WO 2019096096A1 CN 2018115059 W CN2018115059 W CN 2018115059W WO 2019096096 A1 WO2019096096 A1 WO 2019096096A1
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compound
pharmaceutically acceptable
cancer
acceptable salt
salt
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Chinese (zh)
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袁建栋
黄仰青
宋云松
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Brightgene Bio Medical Technology Co Ltd
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Brightgene Bio Medical Technology Co Ltd
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Priority claimed from CN201711119266.8A external-priority patent/CN109771658B/zh
Priority claimed from CN201711119279.5A external-priority patent/CN109776788B/zh
Priority claimed from CN201711119216.XA external-priority patent/CN109776787B/zh
Application filed by Brightgene Bio Medical Technology Co Ltd filed Critical Brightgene Bio Medical Technology Co Ltd
Publication of WO2019096096A1 publication Critical patent/WO2019096096A1/fr
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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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • 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
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/334—Polymers modified by chemical after-treatment with organic compounds containing sulfur

Definitions

  • the present invention generally relates to multi-arm targeting conjugates.
  • the present invention relates to multi-arm PEG-modified targeted anti-cancer conjugates, and more particularly, the present invention is a conjugate of a brain-targeting molecule linked to an anti-cancer drug by a multi-arm PEG.
  • WO2005028539, WO2010019233, WO2011063156, WO2011063158 disclose a clinical phase III drug nktr 102, which is mainly used for metastatic breast cancer, developed by Nektar Therapeutics.
  • the drug is a water-soluble multi-branched polymer prodrug to increase the drug loading, and the structure is as follows:
  • the compound is linked to irinotecan using a multi-arm PEG to increase water solubility, increase drug loading, and reduce side effects without changing the anticancer effect.
  • the drug still has disadvantages, for example, poor targeting, can not act on specific cancer cells, while killing cancer cells, it also affects the performance of normal cells, and the incidence of adverse reactions is still relatively high.
  • Apolipoprotein E (APOE gene, apoE protein) is a 34 kD glycoprotein composed of a 10 kD lipid-binding domain and a 22 kD receptor domain (binding to the low-density lipoprotein family of receptors). Endogenous apoE or intraventricular injection of apoE whole protein plays a neuroprotective role by down-regulating the central nervous system inflammation, reducing oxidative stress, anti-excitatory amino acid toxicity, and directly increasing neurotrophic nutrition.
  • the synthetic apoE peptidomim retains most of the natural three-dimensional structure of the receptor binding domain, retains most of the properties of the whole protein, competes with the apoE whole protein for receptor binding sites on the surface of macrophages, and initiates a signaling cascade. To play its neuroprotective role.
  • LRKLRKRLLLRKLRKRLL is an apoE peptidomimetic that acts as a brain targeting peptide through the blood-brain barrier. Its structure is as follows:
  • the folate receptor (FR) is a glycosylated phosphatidylinositol-linked membrane glycoprotein, and folate (FA) can specifically bind to folate receptors.
  • the addition of folic acid to the complex allows folate to bind to the folate receptor on the tumor cells, so that the complex can be taken up into the cell by cell receptor-mediated endocytosis. This provides a specific targeted drug delivery route for folate receptor-positive tumor cells. Due to this stable binding of folic acid to folate receptors, folic acid is now widely used as a targeting system for various targeted drug delivery.
  • the present invention discloses three novel targeted multi-arm drug conjugates, which are compound a, compound b, and compound c represented by formula (I), formula (II), and formula (III), respectively:
  • R is the organic center, ie in the structure of the conjugate Represents the junction of atoms.
  • R is the organic center, ie in the structure of the conjugate Represents the junction of atoms.
  • branch consists of a polymer POLY, a multivalent linker L, a targeting molecule T, and an active agent D.
  • the polymer POLY is polyethylene glycol, and in the present invention, it is specifically:
  • n 113, Representing the junction of an atom, the oxygen atom labeled "&” is an atom attached to the organic center "R".
  • n represents the degree of polymerization of the polymer, that is, the average number of repeating units contained in the polymer macromolecular chain, depending on the molecular weight of the polymer. For example, when n is 113, it means that the average value is 113.
  • the multivalent linker L of compound a is:
  • the multivalent linker L of compound b is:
  • the multivalent linker L of compound c is:
  • the targeting molecule T of compound a is apoE peptidomimetic LRKLRKRLLLRKLRKRLL;
  • the target molecule T of compound b and compound c is folic acid, and the folic acid structure is as follows:
  • the active agent D of compound a, compound b and compound c are all irinotecan, and the structure of irinotecan is as follows:
  • the present invention is a multi-arm polymer modified targeted anti-cancer conjugate, wherein the water-soluble polymer modification enhances the water solubility of the conjugate and increases the drug loading; the targeting molecule LRKLRKRLLLRKLRKRLL increases brain targeting
  • the conjugate is more likely to pass the blood-brain barrier and exert the function of brain-targeting peptide to make it higher in the target tissue; the target molecule is folic acid, and folic acid acts as a targeting molecule to actively target the expression of folate receptor. Rich tumor cells, better anti-tumor efficacy, increased targeting, so that the concentration of the conjugate in the target tissue is higher.
  • L is an arbitrary linker, which firstly connects the targeting molecule with the anticancer drug, and then connects the "targeting molecule, the anticancer drug and the polymer arm, so that the whole conjugate forms an organic whole.
  • the conjugate of the present invention is a typical prodrug, and the active agent D is released by hydrolysis or enzymatic action, and is separated from the mother to exert physiological activity.
  • the conjugates of the present invention exhibit high loading capacity so that the total dose can be lowered to treat a particular disease, such as cancer. That is, the conjugate active agent carrier of the present invention is capable of covalently bonding to the active agent molecule, allowing a greater amount of therapeutic dosage form (i.e., active agent moiety) to be administered per certain amount of conjugate.
  • the modification of the conjugate of the present invention by the water-soluble polymer is essentially that the conjugate is also hydrophilic, and in particular, when the active agent is a water-insoluble drug, the bioavailability of the conjugate is improved.
  • the conjugates of the invention are capable of exhibiting a stronger effect than tissues that are not coupled, and are more enriched in the human or other animal tissues.
  • the conjugate drug precursors of the present invention contain a number of unique properties, especially where the active agent is an anti-cancer compound.
  • This prodrug inhibits tumor growth with higher efficiency.
  • the small molecule we use is a small molecule known to have anticancer properties. However, by combining with a multi-branched polymer as described above, its efficacy and pharmacokinetics are greatly improved compared to the small molecule (e.g., the anticancer compound itself).
  • the conjugates of the present invention include pharmaceutically acceptable salts including inorganic salts and organic salts, and typical salts include nitrates, sulfates, phosphates, hydrofluoric acid salts, hydrochlorides, hydrobromides, hydroiodides. , formate, lactate, benzoate, acetate, trifluoroacetate, dichloroacetate, trichloroacetate, mixed chlorofluoroacetate, citrate, oxalic acid Salts, sulfonates, methanesulfonates, triflate, heptanesulfonate and the like, of which trifluoroacetate and heptanesulfonate are preferred.
  • Compound a typical trifluoroacetate salt comprises from one to forty molecules of trifluoroacetate.
  • each branch is bound to a conjugate of twelve molecules of trifluoroacetate, the preferred conjugate being forty-eight molecules of trifluoroacetate:
  • Typical heptane sulfonates include from one to forty molecules of heptane sulfonate. Preferably, each branch is bound to a conjugate of twelve molecules of heptane sulfonate, respectively. The preferred conjugate is forty-eight molecules of heptane sulfonate:
  • Typical trifluoroacetate salts include from one to twelve molecules of trifluoroacetate. Preferably, each branch is bound to a conjugate of three molecules of trifluoroacetate, respectively. The preferred conjugate is twelve molecules of trifluoroacetate:
  • Typical heptane sulfonates include from one to twelve molecules of heptane sulfonate.
  • each branch is bound to a conjugate of three molecules of heptane sulfonate, respectively.
  • the preferred conjugate is twelve molecules of heptane sulfonate:
  • the typical trifluoroacetate salt of compound c comprises from one to twelve molecules of trifluoroacetate, preferably a conjugate of three molecules of trifluoroacetate per molecule, respectively.
  • the preferred conjugate is twelve molecules.
  • Typical heptane sulfonates include from one to twelve molecules of heptane sulfonate.
  • each branch is bound to a conjugate of three molecules of heptane sulfonate, respectively.
  • the preferred conjugate is twelve molecules of heptane sulfonate:
  • Solid tumor types suitable for compound a include lymphoid, breast, pancreas, ovary, colon, kidney, bile duct, lung, stomach, brain malignant sarcoma, cancer, and are particularly suitable for treating glioma and breast cancer brain metastasis.
  • Solid tumor types for compounds b and c include ovarian cancer, breast cancer, lung cancer, endometrial cancer, brain tumor, mesothelioma, kidney cancer, stomach cancer, head and neck cancer, lung cancer, colorectal cancer, testicular cancer, Especially suitable for ovarian cancer, breast cancer and lung cancer.
  • 4arm-PEG20K-SCM The structure of 4arm-PEG20K-SCM is as follows:
  • reaction solution was poured into ice-dilute hydrochloric acid solution, extracted with EA was added, EA layer was washed once with dilute hydrochloric acid, saturated sodium bicarbonate, washed with brine, dried over anhydrous Na 2 SO 4, the solvent was distilled off under reduced pressure
  • the silica gel column chromatography obtained 55 g of pure BP103a01.
  • PP04a The synthesis of PP04a is carried out by solid phase synthesis using Fmoc method well known to the skilled person.
  • 2-Cl-Trt Resin is used, Fmoc is removed by using 20% piperidine/DMF, and the coupling reagent is HOBT/DIC, DMF is used as the reaction solvent, and the reaction monitoring is carried out.
  • Compound 11 was purified by reverse-phase HPLC (silica gel: C18, 300A; mobile phase: sodium heptanesulfonate/water, acetonitrile). After the pure product was collected, pH was adjusted to 4 to 5, and then desalted by reverse HPLC (silica gel: C18) , 300A; mobile phase: acetic acid / water, acetonitrile), the pure product was collected, concentrated to remove the organic solvent, and lyophilized to obtain a white powder-like compound 13.
  • Compound 11 MALDI-TOF detected a molecular weight of 34,500.78.
  • the molecular weight of Compound 12 MALDI-TOF was 34,451.86.
  • the synthesis of the polypeptide CDDRD-folic acid is carried out by solid phase synthesis using Fmoc method well known to the skilled person.
  • 2-Cl-Trt Resin is used, Fmoc is removed by using 20% piperidine/DMF, and the coupling reagent is HOBT/DIC, DMF is used for the reaction.
  • the molecular weight of the compound 14 MALDI-TOF was 28,887.03.
  • Compound 15 MALDI-TOF detected a molecular weight of 28920.25.
  • the molecular weight of Compound 16 MALDI-TOF was 28,951.38.
  • Compound 21 was purified by reverse-phase HPLC (silica gel: C18, 300A; mobile phase: sodium heptanesulfonate/water, acetonitrile). After the pure product was collected, pH was adjusted to 4 to 5, and then desalted by reverse HPLC (silica gel: C18) , 300A; mobile phase: acetic acid / water, acetonitrile), the pure product was collected, concentrated to remove the organic solvent, and lyophilized to obtain a white powder-like compound 23.
  • reverse-phase HPLC sica gel: C18, 300A; mobile phase: sodium heptanesulfonate/water, acetonitrile
  • the molecular weight of the compound 21 MALDI-TOF was 27,328.05.
  • Compound 23 MALDI-TOF detected a molecular weight of 27419.56.
  • Test samples irinotecan, nktr-102, compound a.
  • nktr-102 is as follows:
  • Reagents RPMI-1640 medium, trypsin, cyan-chain double antibody, saline.
  • mice Female BALB/c nude mice (only: 60; weeks of age: 6-8 weeks) purchased from Beijing Vital Lihua Experimental Animal Technology Co., Ltd., raised in SPF animal room, temperature 20 ⁇ 25 ° C, Relative humidity 40% ⁇ 70%, light and dark lighting for 12 hours; animals free access to water and feeding. After about 1 week of normal feeding, veterinary examination, mice with good signs of good condition can be included in this experiment. Before the grouping, use the marker to mark the root of the animal. After grouping, each animal is identified by ear clipping.
  • Transplanted tumor tumor strain glioma cell line U87MG, derived from the Cell Bank of the Typical Culture Collection Committee of the Chinese Academy of Sciences (CAS, laboratory laboratory liquid nitrogen cryopreservation).
  • NCI-N87 cell culture NCI-N87 cells were cultured in RPMI-1640 medium in 5% CO 2 at 37 ° C; digested with 0.25% trypsin; passaged weekly according to cell growth 1 to 2 times, the passage ratio is 1:2 to 1:6.
  • NCI-N87 cells were collected in logarithmic growth phase. After cell counting, resuspend in serum-free RPMI-1640 medium, adjust the cell concentration to 1 ⁇ 10 8 cells/mL; pipette the cells with a pipette. After dispersing evenly, it was placed in a 50 mL centrifuge tube, and the centrifuge tube was placed in an ice box; the cell suspension was aspirated with a 1 mL syringe, and the human glioma cell line U87MG was cultured in vitro by microinjection using the guidance of an animal stereotactic instrument.
  • Formulation of irinotecan preparation Weigh 12.0 mg of irinotecan, add 0.15 mL of 1% lactic acid, vortex to completely dissolve the drug, and then add 2.85 mL of 1% sorbitol aqueous solution separately, vortex and mix. Evenly, the ratio of 1% lactic acid to 1% sorbitol aqueous solution in the solution was about 5:95 (v/v). The free form concentration of irinotecan in the solution was 4.0 mg ⁇ mL -1 .
  • nktr-102 preparation Before each administration, accurately weigh 101.5mg of nktr-102, add 2.5mL of normal saline, vortex to completely dissolve the drug, and the concentration of irinotecan in the solution is 4.0mg. ⁇ mL -1 .
  • Compound a administration preparation preparation accurately weighed, added 2.5 mL of physiological saline, vortexed to completely dissolve the drug, and the concentration of irinotecan was 4.0 mg ⁇ mL -1 .
  • Animal grouping and administration The first administration was started on the day of grouping, and the administration volume was 10 mL ⁇ kg -1 .
  • the first group was the solvent control group, and the blank solvent was administered by tail vein injection once every 4 days for a total of 3 times (Q4D ⁇ 3).
  • Groups 2-4 were administered with irinotecan, nktr-102, and compound a, respectively, at a dose of 40 mg ⁇ kg -1 (calculated as irinotecan), Q4D ⁇ 3, respectively.
  • Test samples irinotecan, nktr-102, compound a.
  • Reagents RPMI-1640 medium, trypsin, cyan-chain double antibody, saline.
  • mice Female BALB/c nude mice (only: 60; weeks of age: 6-8 weeks) purchased from Beijing Vital Lihua Experimental Animal Technology Co., Ltd., raised in SPF animal room, temperature 20 ⁇ 25 ° C, Relative humidity 40% ⁇ 70%, light and dark lighting for 12 hours; animals free access to water and feeding. After about 1 week of normal feeding, veterinary examination, mice with good signs of good condition can be included in this experiment. Before the grouping, use the marker to mark the root of the animal. After grouping, each animal is identified by ear clipping.
  • Transplanted tumor tumor strain Human breast cancer MDA-MB-231-Luc was purchased from the Institute of Cell Biology, Shanghai Institute of Chinese Academy of Sciences.
  • MDA-MB-231-Luc was cultured in DMEM medium (GIBCO, USA) containing 10% fetal bovine serum FBS (GIBCO, USA) and cultured in a 37 ° C incubator containing 5% CO 2 .
  • Brain metastasis MDA-MB-231-Luc cell preparation MDA-MB-231-Luc cells in logarithmic growth phase were collected, cell counted and resuspended in serum-free RPMI-1640 medium, and heart injected with 5 ⁇ 10 5 cells .
  • brain metastatic nude mice were selected by bioimaging and brain metastatic MDA-MB-231-Luc cells were isolated.
  • the isolated MDA-MB-231-Luc cells were inoculated into the nude mouse heart, brain-transferred nude mice were selected by bioimaging, and brain metastatic MDA-MB-231-Luc cells were isolated. This was repeated 8 times until no MDA-MB-231-Luc cells were transferred in other tissues.
  • Animal grouping and administration Animal hearts were inoculated with previously prepared brain metastasis MDA-MB-231-Luc cells 5 x 10 5 /only. The brain metastasis was confirmed by bioimaging 21 days after the inoculation, and the nude mice which confirmed the brain metastasis were selected and the first administration was started on the day of grouping, and the administration volume was 10 mL ⁇ kg -1 .
  • the first group was the solvent control group, and the blank solvent was administered by tail vein injection once every 4 days for a total of 3 times (Q4D ⁇ 3).
  • Groups 2-4 were administered with irinotecan, nktr-102, and compound a, respectively, at a dose of 40 mg ⁇ kg -1 (calculated as irinotecan), Q4D ⁇ 3, respectively.
  • Test samples irinotecan, nktr-102, compound b.
  • McCoy's 5A medium McCoy's 5A medium, fetal bovine serum (FBS), trypsin, cyan-chain double antibody, physiological saline.
  • FBS fetal bovine serum
  • trypsin trypsin
  • cyan-chain double antibody physiological saline.
  • mice Female BALB/c nude mice (only: 150; weekly age: 6-8 weeks) purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., and raised in SPF animal room of Suzhou Shengsu New Drug Development Co., Ltd. , temperature 20 ⁇ 25 ° C, relative humidity 40% ⁇ 70%, light and dark lighting for 12 hours; animals free access to water and feeding. After about 1 week of normal feeding, veterinary examination, mice with good signs of good condition can be included in this experiment. Before the grouping, use the marker to mark the root of the animal. After grouping, each animal is identified by ear clipping.
  • Transplanted tumor tumor strain human ovarian cancer SK-OV-3, derived from the Cell Bank of the Typical Culture Collection Committee of the Chinese Academy of Sciences (CAS, laboratory laboratory liquid nitrogen cryopreservation).
  • SK-OV-3 was cultured in McCoy's 5A medium (GIBCO, USA) containing 10% fetal bovine serum FBS (GIBCO, USA) and cultured in a 37 ° C incubator containing 5% CO 2 .
  • Animal model preparation cell inoculation method to establish a subcutaneous transplantation model of tumor nude mice: collect tumor cells in logarithmic growth phase, count and resuspend in 1 ⁇ PBS, adjust the cell suspension concentration to 5 ⁇ 10 7 /ml and compare with Matrigel 1:1 Mix well. Tumor cells were inoculated subcutaneously in the right side of nude mice with a 1 ml syringe (4 gauge needle), 5 x 10 6 /mouse, and 50 animals were inoculated. When the tumor volume reached 100-300 mm 3 , the animals were randomly grouped by random block method. After the start of the experiment, the tumor diameter was measured twice a week, the tumor volume was calculated, and the animal weight was weighed and recorded.
  • Solvent preparation Weigh 0.5g of sorbitol into a 50mL centrifuge tube, add 50mL of water for injection to the centrifuge tube, vortex to completely dissolve the solid substance, and prepare a 1% concentration of sorbitol aqueous solution (w/v) Store in a refrigerator at 4 °C.
  • the concentration of irinotecan in the compound b solution was 4.0 mg ⁇ mL -1 .
  • Animal grouping and administration The first administration was started on the day of grouping, and the experiment was terminated after 21 days, and the administration volume was 10 mL ⁇ kg -1 .
  • the first group was the solvent control group, and the blank solvent was administered by tail vein injection once every 4 days for a total of 3 times (Q4D ⁇ 3).
  • Groups 2-4 were administered with irinotecan, nktr-102, and compound b in the tail vein, respectively, at a dose of 40 mg ⁇ kg -1 (calculated as irinotecan).
  • the animals were weighed and the tumor diameter was measured and the animals were euthanized (CO 2 ). Tumor tissue was stripped and weighed to calculate the tumor inhibition rate.
  • the tumor volume (TV) is calculated as follows:
  • the relative tumor volume (RTV) is calculated as:
  • TV initial is the tumor volume measured at the time of group administration
  • TV t is the tumor volume at each measurement during administration.
  • the relative tumor growth rate (%T/C) is calculated as:
  • RTV T represents the treatment group RTV
  • RTV C represents the solvent control group RTV.
  • test data was calculated by Microsoft Office Excel 2007 software and related statistical processing. Data were expressed as mean ⁇ standard error (Mean ⁇ SE) unless otherwise specified, and t-test was used for comparison between the two groups.
  • Example 8 Antitumor efficacy test of compound b on human breast cancer MDA-MB-231 xenograft tumor in nude mice
  • Test samples irinotecan, nktr-102, compound b.
  • Reagents DMEM medium, fetal bovine serum (FBS), trypsin, cyan-chain double antibody, saline.
  • mice Female BALB/c nude mice (only: 150; weekly age: 6-8 weeks) purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., and raised in SPF animal room of Suzhou Shengsu New Drug Development Co., Ltd. , temperature 20 ⁇ 25 ° C, relative humidity 40% ⁇ 70%, light and dark lighting for 12 hours; animals free access to water and feeding. After about 1 week of normal feeding, veterinary examination, mice with good signs of good condition can be included in this experiment. Before the grouping, use the marker to mark the root of the animal. After grouping, each animal is identified by ear clipping.
  • Transplanted tumor tumor strain human breast cancer MDA-MB-231, derived from the Cell Bank of the Typical Culture Collection Committee of the Chinese Academy of Sciences (CAS, laboratory laboratory liquid nitrogen cryopreservation).
  • MDA-MB-231 was cultured in DMEM medium (DMEM, USA) containing 10% fetal calf serum FBS (GIBCO, USA) and cultured in a 37 ° C incubator containing 5% CO 2 .
  • Animal model preparation Cell inoculation method To establish a subcutaneous transplantation model of tumor nude mice: tumor cells in logarithmic growth phase were collected, counted, resuspended in 1 ⁇ PBS, and the cell suspension concentration was adjusted to 1 ⁇ 10 7 /ml. Tumor cells were inoculated subcutaneously in the right side of nude mice with a 1 ml syringe (4 gauge needle), 1 x 10 6 /mouse, and 50 animals were inoculated. When the tumor volume reached 100-300 mm 3 , the animals were randomly grouped by random block method. After the start of the experiment, the tumor diameter was measured twice a week, the tumor volume was calculated, and the animal weight was weighed and recorded.
  • Example 4 For the preparation method of the test article for administration, see Example 4, and the concentration of irinotecan in the compound b solution was 4.0 mg ⁇ mL -1 .
  • Animal grouping and administration The first administration was started on the day of grouping, and the experiment was terminated after 21 days, and the administration volume was 10 mL ⁇ kg -1 .
  • the first group was the solvent control group, and the blank solvent was administered by tail vein injection once every 4 days for a total of 3 times (Q4D ⁇ 3).
  • Groups 2-4 were administered with irinotecan, nktr-102, and compound b in the tail vein, respectively, at a dose of 40 mg ⁇ kg -1 (calculated as irinotecan).
  • Test samples irinotecan, nktr-102, compound b.
  • Reagents RPMI-1640 medium, fetal bovine serum (FBS), trypsin, cyan-chain double antibody, saline.
  • mice Female BALB/c nude mice (only: 150; weekly age: 6-8 weeks) purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., and raised in SPF animal room of Suzhou Shengsu New Drug Development Co., Ltd. , temperature 20 ⁇ 25 ° C, relative humidity 40% ⁇ 70%, light and dark lighting for 12 hours; animals free access to water and feeding. After about 1 week of normal feeding, veterinary examination, mice with good signs of good condition can be included in this experiment. Before the grouping, use the marker to mark the root of the animal. After grouping, each animal is identified by ear clipping.
  • Transplanted tumor tumor strain human lung cancer SPC-A-1, derived from the Cell Bank of the Typical Culture Collection Committee of the Chinese Academy of Sciences (CAS, laboratory laboratory liquid nitrogen cryopreservation).
  • SPC-A-1 was cultured in RPMI-1640 medium containing 10% fetal bovine serum FBS (GIBCO, USA) and cultured in a 37 ° C incubator containing 5% CO 2 .
  • Animal model preparation Cell inoculation method was established to establish a subcutaneous transplantation model of tumor nude mice: tumor cells in logarithmic growth phase were collected, counted, resuspended in 1 ⁇ PBS, and the cell suspension concentration was adjusted to 2 ⁇ 10 7 /ml. Tumor cells were inoculated subcutaneously in the right side of nude mice with a 1 ml syringe (4 gauge needle), 2 x 10 6 /mouse, and 50 animals were inoculated. When the tumor volume reached 100-300 mm 3 , the animals were randomly grouped by random block method. After the start of the experiment, the tumor diameter was measured twice a week, the tumor volume was calculated, and the animal weight was weighed and recorded.
  • Example 4 For the preparation method of the test article for administration, see Example 4, and the concentration of irinotecan in the compound b solution was 4.0 mg ⁇ mL -1 .
  • Animal grouping and administration The first administration was started on the day of grouping, and the experiment was terminated after 21 days, and the administration volume was 10 mL ⁇ kg -1 .
  • the first group was the solvent control group, and the blank solvent was administered by tail vein injection once every 4 days for a total of 3 times (Q4D ⁇ 3).
  • Groups 2-4 were administered with irinotecan, nktr-102, and compound b in the tail vein, respectively, at a dose of 40 mg ⁇ kg -1 (calculated as irinotecan).

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Abstract

L'invention concerne un conjugué de ciblage anticancéreux modifié par un polymère à plusieurs bras, la modification par un polymère hydrosoluble étant à même d'améliorer l'hydrosolubilité du conjugué et d'augmenter une quantité de charge de médicament ; lorsque la molécule de ciblage est LRKLRKRLLLRKRLL, le conjugué peut augmenter le ciblage cérébral de façon à le faire passer plus facilement à travers la barrière hémato-encéphalique et peut jouer le rôle d'un peptide de ciblage cérébral, en permettant à ce dernier d'avoir une concentration plus élevée dans un tissu cible ; et lorsque la molécule de ciblage est l'acide folique, le conjugué peut cibler activement des cellules tumorales dans lesquelles les récepteurs de folate sont abondamment exprimés et mieux exercer des effets antitumoraux et améliorer la propriété de ciblage.
PCT/CN2018/115059 2017-11-14 2018-11-12 Conjugué de ciblage à plusieurs bras Ceased WO2019096096A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
CN201711119266.8A CN109771658B (zh) 2017-11-14 2017-11-14 靶向多臂偶联物
CN201711119279.5 2017-11-14
CN201711119266.8 2017-11-14
CN201711119279.5A CN109776788B (zh) 2017-11-14 2017-11-14 叶酸受体靶向多臂偶联物
CN201711119216.X 2017-11-14
CN201711119216.XA CN109776787B (zh) 2017-11-14 2017-11-14 多臂靶向偶联物

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WO2019096096A1 true WO2019096096A1 (fr) 2019-05-23

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005028539A2 (fr) * 2003-09-17 2005-03-31 Nektar Therapeutics Al, Corporation Promedicaments a base de polymere a branches multiples
WO2011063156A2 (fr) * 2009-11-18 2011-05-26 Nektar Therapeutics Formes de sel d'acide de conjugués polymère-médicament et procédés d'alcoxylation
CN104784699A (zh) * 2014-01-20 2015-07-22 博瑞生物医药技术(苏州)有限公司 叶酸受体结合配体-药物偶联物
CN107375288A (zh) * 2016-05-16 2017-11-24 博瑞生物医药（苏州）股份有限公司 多臂的聚合靶向抗癌偶联物

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005028539A2 (fr) * 2003-09-17 2005-03-31 Nektar Therapeutics Al, Corporation Promedicaments a base de polymere a branches multiples
WO2011063156A2 (fr) * 2009-11-18 2011-05-26 Nektar Therapeutics Formes de sel d'acide de conjugués polymère-médicament et procédés d'alcoxylation
CN104784699A (zh) * 2014-01-20 2015-07-22 博瑞生物医药技术(苏州)有限公司 叶酸受体结合配体-药物偶联物
CN107375288A (zh) * 2016-05-16 2017-11-24 博瑞生物医药（苏州）股份有限公司 多臂的聚合靶向抗癌偶联物

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