WO2021121068A1 - 促抗癌药摄入的大分子、组合物及其制备抗癌药物的用途 - Google Patents

促抗癌药摄入的大分子、组合物及其制备抗癌药物的用途 Download PDF

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WO2021121068A1
WO2021121068A1 PCT/CN2020/134363 CN2020134363W WO2021121068A1 WO 2021121068 A1 WO2021121068 A1 WO 2021121068A1 CN 2020134363 W CN2020134363 W CN 2020134363W WO 2021121068 A1 WO2021121068 A1 WO 2021121068A1
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macromolecule
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amine
guanidine
cancer
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French (fr)
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阎虎生
黄鑫
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南开大学
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F283/00—Macromolecular compounds obtained by polymerising monomers on to polymers provided for in subclass C08G
    • C08F283/002—Macromolecular compounds obtained by polymerising monomers on to polymers provided for in subclass C08G on to polymers modified by after-treatment
    • 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
    • A61K31/74—Synthetic polymeric materials
    • A61K31/785—Polymers containing nitrogen
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • 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
    • A61K47/51—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 non-active ingredient being a modifying agent
    • A61K47/56—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 non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—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 non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0215—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing natural amino acids, forming a peptide bond via their side chain functional group, e.g. epsilon-Lys, gamma-Glu
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F222/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a carboxyl radical and containing at least one other carboxyl radical in the molecule; Salts, anhydrides, esters, amides, imides, or nitriles thereof
    • C08F222/04—Anhydrides, e.g. cyclic anhydrides
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F283/00—Macromolecular compounds obtained by polymerising monomers on to polymers provided for in subclass C08G
    • 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
    • C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
    • C08G73/02—Polyamines

Definitions

  • the invention relates to the field of anti-cancer drug synergists, in particular to macromolecular substances that promote the aggregation and penetration of anti-cancer drugs in tumor tissues.
  • Cancer chemotherapy is currently one of the main methods of treating cancer.
  • the drugs used in traditional chemotherapy usually have disadvantages such as high toxicity and large side effects.
  • chemotherapy drugs are not selectively distributed in the human body. Killing cancer cells will also cause serious damage to normal tissue cells, causing adverse reactions such as bone marrow suppression, gastrointestinal toxicity, skin toxicity, and allergies.
  • the initial chemotherapy often has a good effect, but this effect is often not sustainable.
  • the tumor is prone to recurrence after chemotherapy, and the effect of chemotherapy after recurrence is often poor. The reason is that chemotherapeutic drugs are not easy to accumulate and penetrate in tumor tissues, and are not easily taken up by cancer cells.
  • drugs that are not easy to accumulate and penetrate deep into tumor tissues are the most critical factors hindering the efficacy of anti-cancer drugs.
  • the accumulation of anticancer drugs in tumor tissues through blood circulation is much less than that in normal tissues (R. Nandigama, et al., Restriction of drug transport by the tumor environment, Histochemistry and Cell Biology 2018, 150, 631-648), Even a small amount of anticancer drugs that gather in tumor tissues are mainly distributed around the capillaries, while the drugs that penetrate deep away from the capillaries are few, that is, the distribution of drugs in the tumor tissue is extremely uneven, which is mainly due to the solid body
  • the high density of extracellular matrix and increased interstitial fluid pressure in tumor tissue hinders drug penetration (AI Minchinton, et al., Drug penetration in solid tumors, Nature Reviews Cancer 2006, 6,583-592).
  • the concentration of the drug that enters the tumor tissue away from the capillary vessels is often very small, and it does not reach the minimum drug concentration required to kill the cancer cells.
  • cancer cells that receive low concentrations of anticancer drugs often develop drug resistance. This is cancer.
  • One of the main mechanisms of cancer cell resistance is to prevent chemotherapeutics from entering the cancer cells and pumping the chemotherapeutics that enter the cancer cells out of the cell, so that the concentration of the chemotherapeutics in the cancer cells is not enough to kill the cancer cells. The lowest concentration.
  • chemotherapeutic drugs can increase the drug concentration in tumor tissues far away from capillaries, and the concentration of chemotherapeutic drugs in drug-resistant cancer cells will also increase. However, due to the severe side effects of chemotherapeutics, increasing the dose may be fatal.
  • nanocarriers loaded with anticancer drugs have been developed as a delivery system for anticancer drugs.
  • the basic principle is that nanoparticles cannot easily penetrate the dense normal capillary wall to enter normal tissues, but easily penetrate the porous tumor tissue capillary wall to enter tumor tissues, improving the anti-cancer efficiency of anti-cancer drugs (A. Wicki ,et al., Nanomedicine in cancer therapy: Challenges, opportunities, and clinical applications, Journal of Controlled Release 2015, 200, 138–157).
  • nano-carriers loaded anti-cancer drugs Although more than 10 nano-carriers loaded anti-cancer drugs have been used in clinical applications, the efficacy of these nano-drugs has not increased much compared with the corresponding small molecule drugs, but the side effects have been reduced (YSYoun, et al., Perspectives on the past, present, and future of cancer nanomedicine, Advanced Drug Delivery Reviews 2018, 130, 3-11).
  • the high proportion of aggregation in tumor tissues but the lack of improvement in efficacy is due to the fact that nanoparticles entering tumor tissues are mainly concentrated around capillaries, and because of their larger (compared to small molecules) size it is more difficult to penetrate far away The depths of the capillaries (D.
  • nano-carrier drug-loading system involves issues such as drug loading, loading, and controlled release, and is a very complex system. If any step in the complex system does not work well, the final effect is definitely poor, and the complex system is not easy to scale production, the repeatability between batches is poor, and the production cost is high.
  • amine-containing polymers can increase the entry of small molecule chemotherapeutics into cancer cells.
  • the polymers promote chemotherapy More drugs enter the cancer cells, so its anti-cancer effect is better than that of small molecule drugs alone.
  • the amine-containing polymer enhances the effectiveness of chemotherapeutic drugs. Because the drug is not loaded on the polymer, it is simpler than the nanocarrier loaded drug system.
  • tumor penetrating peptide a 9-amino acid cyclic peptide called tumor penetrating peptide can promote the accumulation of anticancer drugs in tumor tissues and the penetration of anticancer drugs in tumor tissues (KNSugahara, et al., Coadministration of a tumor-penetrating peptide enhances the efficiency of cancer drugs, Science 2010, 328, 1031-1035).
  • tumor-bearing nude mice were injected with cyclic peptides and anti-cancer drugs at the same time, so that the anti-cancer drugs were more concentrated in the tumor tissues and penetrated deeper into the tumor tissues, thereby enhancing the anti-tumor effect of the anti-cancer drugs and increasing The role of potentiator.
  • the synergist and anticancer drug combination system is simpler, does not involve drug loading, loading, controlled release and other issues, and can be combined with a variety of anticancer drugs.
  • peptide drugs have poor stability and are easily inactivated during storage and use. For example, peptide drugs are easily hydrolyzed by peptide hydrolase or protease after entering the blood and become invalid. Moreover, the synthesis cost of peptide drugs is expensive, which limits its practical application.
  • the main purpose of the present invention is to provide a substance that can promote the accumulation of anti-cancer drugs at tumor sites and penetrate deeper into tumor tissues, thereby improving the treatment of anti-cancer drugs, especially chemotherapeutics. effect.
  • the guanidine groups in the macromolecules of the present invention and the number of guanidine groups contained in each macromolecule play a key role in promoting the aggregation of anticancer drugs in tumor tissues and the penetration of anticancer drugs in tumor tissues by the macromolecules.
  • the first aspect of the present invention provides a macromolecule having multiple guanidine groups and optionally amine groups, wherein at least part of the amine groups and/or guanidine groups are substituted by the following formula I Is substituted to form an amide bond, wherein the macromolecule has an average molecular weight less than or equal to 50,000Da, and the zeta potential of the macromolecule is -15mV ⁇ +5mV under normal physiological conditions, and the macromolecule is in slightly acidic conditions After being hydrolyzed, it has 5-100 guanidine groups,
  • R 1 and R 2 may be independently selected from substituted or unsubstituted C1-C6 alkyl groups, or R 1 and R 2 may be connected to each other and form a five-membered or six-membered ring together with the carbon atoms to which they are connected, wherein The substitution refers to substitution by 1 to 2 carboxyl groups.
  • R 1 and R 2 may be independently selected from substituted or unsubstituted C1-C3 alkyl groups, or R 1 and R 2 may be connected to each other and form a carbon atom to which they are connected.
  • substituent of formula I may be selected from the group consisting of the following substituents:
  • the guanidine group exists in the form of protonation under normal physiological conditions, that is, the macromolecules containing the guanidine group are positively charged under normal physiological conditions.
  • the positively charged macromolecules are easily eliminated in the body, and the circulation time in the blood is short, which cannot produce a satisfactory effect of promoting the intake of anti-cancer drugs.
  • a certain amount of carboxyl group is further introduced into the macromolecule, so that the zeta potential of the macromolecule is -15mV ⁇ +5mV under normal physiological conditions (pH ⁇ 7.4). At this time, the net charge of the molecule is close to 0 or slightly negative.
  • Macromolecules with a net charge close to 0 or slightly negatively charged are not easily cleared in the body, and their blood circulation time is long (E. Blanco, et al., Principles of nanoparticle design for overcoming biological barriers to drug delivery, Nature Biotechnology 2015, 33,941 -951).
  • the amide bond formed by the substituent of formula I in the macromolecule of the present invention is hydrolyzed, and the carboxyl group carried by the substituent is separated from the macromolecule.
  • the amine groups and guanidine groups in the hydrolyzed macromolecules are released, so that they can help the anticancer drug to penetrate into the cells and diffuse into the tissues.
  • the "release” mentioned here means that the acylated amine groups and guanidine groups before hydrolysis become free amine groups and guanidine groups after being hydrolyzed and released, as well as the unacylated amine groups and guanidine groups in the macromolecule before hydrolysis.
  • the carboxyl group and the carboxyl group of the macromolecule form an ion pair so that the amine group and the guanidine group are protected. After the hydrolysis, the amine group and the guanidine group protected by the ion pair are also released due to the departure of the carboxyl part.
  • the amide bond formed by the substituent with the structure shown in formula I and the amine group (primary amine group or secondary amine group) and/or guanidine group in the macromolecule can exist stably in a slightly alkaline environment under normal physiological conditions. Normal tissues and normal tissues will not be hydrolyzed in the blood circulation, but in the slightly acidic environment of tumor tissue, the amide bond is easily hydrolyzed, so that it can smoothly reach the tumor tissue and exert its effect.
  • the average number of guanidine groups contained in the macromolecule is at least 5, it has a more significant functional effect, because the number of guanidine groups and molecular weight have opposite effects on its functional effect.
  • the molecular weight is as small as possible and the number of guanidine groups is as high as possible, a better effect can be achieved, and these two factors have the best balance range.
  • the macromolecule may have 10-60 guanidine groups, preferably 15-40 guanidine groups after being hydrolyzed under slightly acidic conditions.
  • the macromolecule may have an average molecular weight of 1,500 to 40,000 Da, preferably an average molecular weight of 2,500 to 25,000 Da, and more preferably 3,000 to 15,000 Da.
  • the macromolecule has a molecular weight of 3,000 to 15,000 Da, and may have 15 to 40 guanidine groups after being hydrolyzed under slightly acidic conditions.
  • the net charge number of the macromolecule of the present invention is close to zero.
  • the net charge number is characterized by the zeta potential of the macromolecule. Under normal physiological conditions (pH ⁇ 7.4), when the zeta potential is between -5mv and +5mv, it can be considered that its net charge is close to 0, and when it is between -15mv and -5mv, it is slightly negative.
  • the zeta potential of the macromolecule of the present invention is between -15mv and +5mv, preferably between -10mv and +2mv, and more preferably between -8mv and 0mv.
  • the guanidine group can be introduced into the macromolecule by guanidinating at least part of the amino group in the macromolecule containing the amine group or reacting with a compound having a guanidine group.
  • the amine-containing macromolecule contains multiple primary and/or secondary amine groups.
  • the amine group-containing macromolecule may further contain a tertiary amine group.
  • the amine group-containing macromolecule may be an amine group-containing linear macromolecule or an amine group-containing branched macromolecule.
  • Such linear or branched macromolecules can be polyolefin molecules with amine side chains, polyamino acid molecules with amine groups on their side chains, and aminated hyperbranched polyglycerols.
  • the amine group-containing linear macromolecule can be selected from polyvinylamine, polyallylamine and linear polyethyleneimine.
  • the amine-containing branched macromolecule can be selected from branched polyethyleneimine, hyperbranched polylysine, dendrimer-amine, dendritic polypropyleneimine, dendritic polylysine and amination Of hyperbranched polyglycerol. Among them, the most preferred are branched macromolecules containing amine groups.
  • amine-containing macromolecules have low price or low synthesis cost, are easy to synthesize, have stable structure, and have low toxic and side effects, and are target macromolecules with good biocompatibility.
  • the reacting at least part of the amine groups in the amine group-containing macromolecule with the guanidine group-containing compound includes reacting at least part of the amine groups in the amine group-containing macromolecule with the guanidine group and carboxyl group.
  • the compound undergoes an amidation reaction.
  • the compound containing a guanidino group and a carboxyl group is selected from the group consisting of guanidinoacetic acid hydrochloride, 3-guanidinopropionate hydrochloride, ⁇ -amino and guanidino double-protected arginine.
  • the compound containing a guanidine group and a carboxyl group is selected from ⁇ -amino and guanidine group double-protected arginine
  • the resulting guanidine group-containing macromolecule after removal of the protective group also introduces a primary amino group (arginine ⁇ -amino).
  • the aminoguanidine in the amino-containing macromolecule is reacted by a guanidine reagent.
  • the guanidine reagent can be selected from the group consisting of cyanamide, O-methylisourea, thiourea, S-methylisothiourea, N,N-bis(tert-butoxycarbonyl)thiourea, 1H- Pyrazol-1-formamidine hydrochloride and 3,5-dimethyl-1-pyrazole formamidine nitrate.
  • the hydrolyzed macromolecules with a certain number of guanidine groups of the present invention can not only promote the entry of anti-cancer drugs into cells to play a role, but also can effectively promote the aggregation of anti-cancer drugs in solid tumors. Helps the anti-cancer drugs penetrate from the blood vessel to the deeper part of the tumor tissue. Therefore, under the same dose of anticancer drugs, the use of the macromolecules of the present invention significantly increases the anticancer effect; or at a lower dose of anticancer drugs, the use of the macromolecules of the present invention greatly reduces the cost of anticancer drugs. toxic side effect.
  • the second aspect of the present invention provides a method for preparing the above-mentioned macromolecule.
  • the method includes:
  • R 1 and R 2 may be independently selected from the group consisting of carboxyl, substituted or unsubstituted C1-C6 alkyl groups, or R 1 and R 2 may be connected to each other to form a substituted or unsubstituted alkylene group.
  • the connected carbon atoms together form a group consisting of a five-membered or six-membered ring, wherein the substitution is by 1 to 2 carboxyl groups.
  • R 1 and R 2 may be each independently selected from the group consisting of carboxyl, substituted or unsubstituted C1-C3 alkyl, or R 1 and R 2 are connected to each other as a butylene group and the carbon atom to which it is connected Together to form a six-membered ring, wherein the substitution is substitution by 1 carboxyl group.
  • the compound of formula II may specifically be the following compounds:
  • the compound of the formula II is most preferably 2,3-dimethylmaleic anhydride, cyclohexene-1,2-dicarboxylic anhydride, cis-aconitic anhydride and 2-(2-carboxyethyl)-3-methyl One of maleic anhydride.
  • the macromolecule containing an amine group is as defined above.
  • the introduction of a guanidine group into a macromolecule containing an amine group may include forming an amide bond between the macromolecule containing an amine group and a carboxyl group in a compound containing a guanidine group and a carboxyl group.
  • the compound containing a guanidino group and a carboxyl group is selected from the group consisting of guanidinoacetic acid hydrochloride, 3-guanidinopropionate hydrochloride, ⁇ -amino and guanidine double-protected arginine. If the compound containing a guanidine group and a carboxyl group is selected from arginine double-protected with ⁇ -amino group and guanidine group, the protective group needs to be removed at the end.
  • the introduction of a guanidine group into a macromolecule containing an amine group may include: reacting the macromolecule containing an amine group with a guanidine reagent.
  • the guanidine reagent is selected from the group consisting of cyanamide, O-methylisourea, thiourea, S-methylisothiourea, N,N-bis(tert-butoxycarbonyl)thiourea, 1H- Pyrazol-1-formamidine hydrochloride and 3,5-dimethyl-1-pyrazole formamidine nitrate.
  • reaction of introducing guanidine groups in the present invention can also adopt any method conventionally used in the art, and the present invention has no particular limitation on the method of introducing guanidine groups.
  • the third aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned macromolecule.
  • the pharmaceutical composition may further include at least one anti-cancer drug.
  • the anti-cancer drug may be selected from antimetabolites, alkylating agents, anti-microtubule drugs, anti-cancer antibiotics, and combinations thereof.
  • the anti-metabolite can be selected from 5-fluorouracil, methotrexate and gemcitabine; the alkylating agent is selected from cyclophosphamide, phenylbutyrate and platinum anticancer drugs; the anti-microtubule The medicine is selected from paclitaxel, vinblastine, vincristine, camptothecin and irinotecan; the anticancer antibiotic is selected from doxorubicin, pirarubicin and epirubicin.
  • the pharmaceutical composition may further include pharmaceutically acceptable excipients and/or carriers.
  • excipients and carriers that can be used in the pharmaceutical composition of the present invention can be selected according to the specific mode of administration and route of administration, and the present invention is not particularly limited thereto.
  • the fourth aspect of the present invention provides the use of the above-mentioned macromolecules to promote the penetration of anticancer drugs into tumor tissues.
  • the anticancer drugs are small molecule anticancer drugs, especially those defined above.
  • the present invention also provides the use of the above-mentioned pharmaceutical composition in the preparation of a medicine for treating cancer.
  • the present invention also provides a method for treating cancer, which includes the step of administering a drug containing the above-mentioned macromolecule or the above-mentioned pharmaceutical composition to a subject suffering from cancer.
  • the present invention further provides a pharmaceutical composition as described above for the treatment of cancer.
  • the treatment includes the macromolecule according to the present invention to promote the accumulation of the anticancer drug in the tumor tissue and the penetration into the tumor tissue.
  • the macromolecules of the present invention mainly promote the penetration and uptake of anticancer drugs in solid tumors. Therefore, the cancer is a malignant solid tumor.
  • the malignant solid tumor may be selected from liver cancer, lung cancer, stomach cancer, bowel cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, and cervical cancer.
  • the macromolecules of the present invention can significantly improve the curative effect of anti-cancer drugs, and thus can make the anti-cancer drugs be used at a lower dosage, thereby reducing their side effects.
  • the macromolecules of the present invention can promote the anticancer drug to penetrate deeper into the solid tumor tissue, it can also prolong the time for the emergence of drug resistance or prevent the emergence of drug resistance.
  • the macromolecule preparation method of the present invention is simple, has low preparation cost, high stability, is easier to store, and is convenient for transportation.
  • BPEI 0.6k -G-DMA, BPEI 1.8k -G-DMA, BPEI 3.5k -G-DMA, BPEI 5k -G-DMA and BPEI 1.8k -Lys-DMA promote adriamycin in MCF-7 cells Confocal laser photo of penetration in cultured cell spheres (DOX is doxorubicin, scale is 100 ⁇ m);
  • BPEI 1.8k -G, BPEI 1.8k -G-DMA, BPEI 1.8k -Lys-DMA, bPEI 0.6k -G-DMA and G4DPLys-G-DMA promote the penetration of doxorubicin in tumor tissues (DOX It is adriamycin, the scale is 50 ⁇ m);
  • FIG. 7 Tumor volume and time changes in xenograft MCF-7 nude mice after injection of different formulations on day 0 and day 7 (DOX is adriamycin).
  • the terms "including”, “including” or any other variants thereof are intended to cover non-exclusive inclusion, so that a method or device including a series of elements not only includes what is clearly stated Elements, but also include other elements not explicitly listed, or elements inherent to the implementation of the method or device. Without more restrictions, the element defined by the sentence “including a" does not exclude the presence of other related elements (such as steps or components in the method) in the method or device that includes the element.
  • normal physiological conditions refers to a homeostasis environment in mammals, especially humans, which maintains normal physiological activities of cells.
  • the pH of the internal environment is referred to. Under normal physiological conditions, the pH of the internal environment is about 7.4.
  • lightly acidic conditions refers specifically to the acid-base environment in tumor tissues.
  • the acid-base environment in tumor tissues is slightly acidic with a pH of 6.5 to 7.0.
  • the macromolecule of the present invention can be prepared by the following method: firstly, a macromolecule containing multiple guanidine groups is prepared, and then a carboxyl group is introduced into the prepared macromolecule containing guanidine groups.
  • Macromolecules containing multiple guanidine groups can be prepared in two ways.
  • the first method is to introduce a guanidine group by forming an amide bond between a macromolecule containing a primary and/or secondary amino group and a compound containing both a guanidine group and a carboxyl group. That is, the primary and/or secondary amino groups in the macromolecules condense with the carboxyl groups in the compound containing the guanidine group and the carboxyl group to form an amide bond.
  • the second method is to introduce the guanidine group by carrying out the guanidine reaction of the amine group from the macromolecule containing the primary and/or secondary amine group.
  • the macromolecules containing primary and (or) secondary amino groups include linear macromolecules and branched macromolecules, and linear macromolecules containing primary and (or) secondary amino groups include polyvinylamine, polyallyl Amines, linear polyethyleneimine, etc.; branched macromolecules containing primary and/or secondary amine groups include branched polyethyleneimine, hyperbranched polylysine, aminated hyperbranched polyglycerol, etc., Dendritic polyamide-amine, aminated hyperbranched polyglycerol, dendritic polypropylene imine and dendritic polylysine, etc.
  • branched macromolecules containing primary and/or secondary amine groups are used.
  • branched polyethyleneimine, hyperbranched polylysine, aminated hyperbranched polyglycerol and dendritic polyglycerol are preferred. Lysine.
  • the guanidine group and carboxyl group-containing compound used for introducing the guanidine group by forming an amide bond between a macromolecule containing a primary and/or secondary amino group and a compound containing both a guanidine group and a carboxyl group include: guanidino acetate Salt, 3-guanidinopropionate hydrochloride, protected arginine (Boc-Arg(Pbf)-OH, Boc-Arg-OH ⁇ HCl, Boc-Arg(Boc)-OH, Fmoc-Arg-OH ⁇ HCl).
  • the condensing agent in which the carboxyl group and the amine group condense to form an amide bond includes carbodiimide condensing agents (such as dicyclohexylcarbodiimide, diisopropylcarbodiimide), onium salt condensing agents (such as TATU, TBTU, BOP, PyBOP, PyAOP, etc.). If a protected arginine is used, after the amide bond is formed, the protecting group can be removed to obtain a macromolecule containing a guanidine group.
  • carbodiimide condensing agents such as dicyclohexylcarbodiimide, diisopropylcarbodiimide
  • onium salt condensing agents such as TATU, TBTU, BOP, PyBOP, PyAOP, etc.
  • Said macromolecules containing primary and (or) secondary amine groups are introduced into the guanidine group by the guanidine reaction of the amine group.
  • the guanidine reagent used in the reaction includes cyanamide, O-methylisourea, sulfur Urea, S-methylisothiourea, N,N'-bis(tert-butoxycarbonyl)thiourea, 1H-pyrazole-1-carboxamidine hydrochloride, etc. and 3,5-dimethyl-1- Pyrazole formamidine nitrate. If the guanidinating reagent contains a protecting group, the protecting group can be removed after the guanidation reaction to obtain a macromolecule containing a guanidin group.
  • the amine group or the guanidine group can be protected, and the protecting group can be removed after the reaction.
  • the protecting group may be any commonly used amino protecting agent, such as tert-butoxycarbonyl (Boc), etc., but it is not limited thereto.
  • the macromolecules of the present invention can promote the accumulation of anticancer drugs in tumor tissues.
  • the macromolecules and anticancer drugs are injected into the tumor-bearing animals, and the concentration distribution of the anticancer drugs in the tumor, liver, kidney, spleen and lung is analyzed after a certain period of time.
  • the results show that compared with only injecting anticancer drugs, the macromolecules of the present invention significantly promote the accumulation of anticancer drugs in tumor tissues.
  • macromolecules with similar structures that do not contain guanidine groups but contain amine groups have little effect on promoting the aggregation of anticancer drugs in tumor tissues.
  • the macromolecules of the present invention can promote the entry of anticancer drugs into cancer cells, that is, when the cancer cells are incubated with the macromolecules and anticancer drugs in a slightly acidic environment that mimics the tumor, they enter the cancer cells.
  • the concentration of the anticancer drug is higher than the concentration of the anticancer drug that enters the cancer cells when the same dose of the single anticancer drug is incubated.
  • Cell flow cytometry experiments show that compared with the use of anticancer drugs alone (taking doxorubicin as an example), simultaneous use of the macromolecules and anticancer drugs of the present invention can significantly increase the concentration of anticancer drugs that enter cancer cells.
  • the macromolecules of the present invention can promote the entry of anticancer drugs into cancer cells, the efficiency of the anticancer drugs in killing cancer cells can be improved.
  • the cancer cells are incubated with a single anticancer drug under the same conditions. In the incubation experiment where macromolecules exist, The survival rate of cancer cells is significantly reduced.
  • the three-dimensional cancer cell spheres are incubated with the macromolecules of the present invention and the anticancer drug (take doxorubicin as an example) in a slightly acidic environment that mimics the tumor, and then confocal laser imaging is used to detect the cross-sections of the cancer cells at different depths.
  • the fluorescence intensity is proportional to the concentration of doxorubicin.
  • the macromolecule of the present invention can promote a deeper penetration of doxorubicin in the cancer cell sphere and a more even distribution in the cancer cell sphere.
  • macromolecules that do not contain guanidine groups but contain amine groups with similar structures have little effect on the penetration of doxorubicin in cancer cell spheres.
  • Further animal experiments have also shown that the macromolecules of the present invention can promote the penetration of anticancer drugs into deep tumor tissues far away from blood vessels, while macromolecules with similar structures that only contain amine groups have an impact on the penetration of adriamycin in tumor tissues. The promotion is obviously smaller.
  • the macromolecules can promote the accumulation of anticancer drugs in tumor tissues, promote the penetration of anticancer drugs in tumor tissues, and promote the entry of anticancer drugs into cancer cells, when such macromolecules are used in combination with anticancer drugs to treat cancer Can significantly increase the effectiveness of anticancer drugs. Compared with a single injection of an anticancer drug, the weight of the tumor of the tumor-bearing animal is significantly reduced after the injection of the macromolecule and the same dose of the anticancer drug.
  • the product was dissolved in dichloromethane, and trifluoroacetic acid was added with stirring, so that the final ratio of trifluoroacetic acid/dichloromethane was 9/1 (volume ratio), the solution was stirred at room temperature for 12 hours, and the dichloromethane was removed by rotary evaporation , The residue was precipitated with ether, and the obtained product was expressed as BPEI 0.4k -G.
  • the zeta potential of BPEI 0.4k -G-DMA at pH 7.4 was determined by dynamic light scattering method (Malvern's Zetasizer Nano ZS90) to be -0.8mV, measured by MALDI-TOF-MS (Bruker Daltonics AutoflexIII LRF200-CID) The average molecular weight is 2020Da.
  • BPEI 0.6k The branched polyethyleneimine (BPEI 0.6k ) with an average molecular weight of 600Da was used to replace the BPEI 0.4k in Example 1, and the other steps were the same as in Example 1.
  • the resulting macromolecule with guanidine and carboxyl groups used BPEI 0.6k -G -DMA means that the average number of guanidine groups introduced per BPEI 0.6k -G-DMA macromolecule is 10.2, the zeta potential at pH 7.4 is -3.5mV, and the average molecular weight is 3450Da.
  • Trifluoroacetic acid was added with stirring to make the final trifluoroacetic acid/dichloromethane The ratio is 9/1 (volume ratio), the solution is stirred at room temperature for 12 hours, and the dichloromethane is removed by rotary evaporation. The pH of the resulting solution is adjusted to 6-7 with 1mol/L NaHCO 3 , and the molecular weight cut-off is 100-500Da The dialysis bag is dialyzed, and the product obtained by freeze-drying the dialysate is expressed as BPEI 1.8k -G.
  • BPEI 1.8k -G the average molecular weight
  • BPEI 1.8k -G and 2,3-dimethylmaleic anhydride were carried out with reference to the method of Example 1, and the obtained macromolecule was represented by BPEI 1.8k -G-DMA.
  • the zeta potential of BPEI 1.8k -G-DMA at pH 7.4 is -4.2mV, and the average molecular weight is 8080Da.
  • BPEI 3.5k 3500kDa average molecular weight branched polyethyleneimine (BPEI 3.5k) in place of Example 3 BPEI 1.8k embodiments, additional process steps as in Example 3, to give the macromer represented by BPEI 3.5k -G-DMA.
  • the average number of guanidine groups contained in each BPEI 3.5k-G-DMA macromolecule is 39.7, the zeta potential at pH 7.4 is -2.5mV, and the average molecular weight is 14.2kDa.
  • Example 3 Refer to the method of Example 3 to treat the obtained precipitate with dichloromethane/trifluoroacetic acid, and the obtained product is represented by BPEI 5k -G. It is estimated that 55.8 guanidine groups are introduced into each BPEI 5k -G macromolecule by the change of average molecular weight.
  • BPEI 5k -G and 2,3-dimethylmaleic anhydride were carried out according to the method of Example 1, and the obtained product was represented by BPEI 5k -G-DMA.
  • the zeta potential of BPEI 5k -G-DMA at pH 7.4 is -1.6mV, and the average molecular weight is 20.1kDa.
  • BPEI 10k branched polyethyleneimine with an average molecular weight of 10kDa was reacted with Boc-Arg(Pbf)-OH, and the product obtained was represented by BPEI 10k -G. Based on the change of the average molecular weight before and after the reaction, it is estimated that 97.2 guanidine groups are introduced into each BPEI 10k -G macromolecule on average.
  • the linear polyethyleneimine (LPEI 1.8k ) with an average molecular weight of 1800 Da was used to replace the BPEI 1.8k in Example 3.
  • the other operation steps were the same as those in Example 3.
  • the resulting macromolecules with guanidine and carboxyl groups used LPEI 1.8k -G- According to DMA, the average number of guanidine groups introduced per LPEI 1.8k -G-DMA macromolecule is 22.5, the zeta potential at pH 7.4 is -3.1 mV, and the average molecular weight is 9032 Da.
  • the average molecular weight determined by MALDI-TOF-MS are 928Da, 2442Da, 4609Da and 25.6kDa, respectively. They are represented by HBPL 0.9k , HBPL 2.4k , HBPL 4.6k and HBPL 26k , respectively.
  • Example 1 for the reaction of HBPL 0.9k -G (0.5g) with 2,3-dimethylmaleic anhydride (1.1g), and the other steps are the same as in Example 1.
  • the macromolecule obtained is HBPL 0.9k -G -DMA said.
  • the zeta potential of HBPL 0.9k -G-DMA at pH 7.4 is -1.5 mV, and the average molecular weight is 1528 Da.
  • Example 9 Replace HBPL 0.9k in Example 9 with HBPL 2.4k , and the other steps are the same as in Example 9.
  • the resulting macromolecules with guanidine and carboxyl groups introduced are represented by HBPL 2.4k -G-DMA, and each HBPL 2.4k -G-DMA
  • the average number of guanidine groups introduced into the macromolecule is 16.5, the zeta potential at pH 7.4 is -3.8mV, and the average molecular weight is 4256Da.
  • Example 9 Replace HBPL 0.9k in Example 9 with HBPL 4.6k .
  • the other steps are the same as in Example 9.
  • the resulting macromolecules with guanidine and carboxyl groups introduced are represented by HBPL 4.6k -G-DMA, each HBPL 4.6k -G-DMA
  • the average number of guanidine groups introduced into the macromolecule is 28.7
  • the zeta potential at pH 7.4 is +0.8 mV
  • the average molecular weight is 7809 Da.
  • the product was subsequently deprotected in 90% trifluoroacetic acid and precipitated in cold ether.
  • the resulting product was denoted by G1Lys.
  • the guanidine group was introduced according to the method of Example 1, and the obtained product was represented by G1Lys-G. Through the average molecular weight change before and after the introduction of guanidine groups, 6 guanidine groups are introduced into each G1Lys-G molecule.
  • Example 1 Refer to the method of Example 1 for the reaction of G1Lys-G (1g) with 2,3-dimethylmaleic anhydride (1.2g). The other steps are the same as in Example 1.
  • the macromolecule obtained is represented by G1Lys-G-DMA .
  • the zeta potential of G1Lys-G-DMA at pH 7.4 is -4.5mV and the molecular weight is 2116Da.
  • Example 3 Refer to the method of Example 3 to treat the obtained precipitate with dichloromethane/trifluoroacetic acid, and the obtained product is represented by G4DPLys-G.
  • the average molecular weight changes before and after the introduction of guanidine groups estimated that on average 29.3 guanidine groups were introduced per G4DPLys-G macromolecule.
  • BPEI 1.8k -G (from Example 3) was reacted with 0.72g cyclohexene-1,2-dicarboxylic anhydride, and the other steps were the same as the guanidine group-containing macromolecule and 2,3-dicarboxylic acid in Example 1.
  • the reaction of methyl maleic anhydride, the macromolecule obtained is represented by BPEI 1.8k -G-DCA.
  • the zeta potential of BPEI 1.8k -G-DCA at pH 7.4 is -2.2 mV, and the average molecular weight is 8565 Da.
  • Example 15 The cyclohexene-1,2-dicarboxylic anhydride in Example 15 was replaced with cis-aconitic anhydride (0.36g), and the other operation steps were the same as those in Example 15.
  • the obtained macromolecule was represented by BPEI 1.8k -G-CAA.
  • the zeta potential of BPEI 1.8k -G-CAA at pH 7.4 is -8.0mV, and the average molecular weight is 7125Da.
  • Example 15 Use 2-(2-carboxyethyl)-3-methylmaleic anhydride (0.42g) to replace the cyclohexene-1,2-dicarboxylic anhydride in Example 15.
  • the other operation steps are the same as those in Example 15.
  • the macromolecule is represented by BPEI 1.8k -G-CDM.
  • the zeta potential of BPEI 1.8k -G-CDM at pH 7.4 is -4.8mV, and the average molecular weight is 7409Da.
  • the solution was stirred at room temperature for 12 hours, and the dichloromethane was removed by rotary evaporation.
  • the resulting solution was adjusted to pH 6-7 with 1mol/L NaHCO 3 and dialyzed with a dialysis bag with a molecular weight cut-off of 100-500Da.
  • the product obtained by freeze-drying the dialysate was expressed as BPEI 1.8k- Lys.
  • BPEI 1.8k- Lys and 2,3-dimethylmaleic anhydride were carried out according to the method of Example 1, and the obtained macromolecule was represented by BPEI 1.8k- Lys-DMA.
  • the zeta potential of BPEI 1.8k- Lys-DMA at pH 7.4 is -3.8mV, and the average molecular weight is 7428Da.
  • the macromolecules prepared above promote the aggregation experiment of anticancer drugs in tumor tissues.
  • Female BALA/c nude mice purchased from Beijing Huafukang Biotechnology Co., Ltd.), aged 6-8 weeks and weighing 18-20 g, were used as experimental animals. 200 ⁇ L of physiological saline solution containing 5 ⁇ 10 6 MCF-7 cells was injected subcutaneously into the armpit of the right forelimb of nude mice.
  • the dose of doxorubicin and macromolecules are both 10 mg/kg body weight.
  • the mice were sacrificed 0.5 hours after the administration, and the autopsy was completed within 10 minutes.
  • the tumor, heart, liver, spleen, lung and kidney were taken respectively, and the tissue homogenate (20mmol/containing 1% SDS) was added at a rate of 0.1g/mL.
  • HPLC instrument is Shimadzu's model Nexera HPLC System
  • the chromatographic column is Shim-pack XR-ODSIII (150mm ⁇ 2mm particle size 2.2 ⁇ m), and the fluorescence detector (RF-20A).
  • Table 1 The distribution of adriamycin in tumors and different organs is shown in Table 1.
  • the difference is that the anticancer drug used is paclitaxel, the macromolecule is G4DPLys-G-DMA, and the distribution of paclitaxel in tumors and different organs is shown in Table 2.
  • the anticancer drug used is cisplatin
  • the macromolecule is G3PAMAM-G-DMA
  • the tumor tissues and organs obtained by dissection are decomposed with hot nitric acid
  • the inductively coupled plasma mass spectrometry The model of Thermo Elemental is IRIS Intrepid II XSP ICP-MS) analyzes the platinum content, and then converts it to the cisplatin content.
  • the distribution of cisplatin in tumors and different organs is shown in Table 3.
  • a laser confocal microscope (Nikon's model is A1R+microscope) is used to scan the 3D cell sphere layer by layer to study the penetration of macromolecule anticancer drugs in the cell sphere (simulating tumor tissue).
  • complete medium RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin
  • MCF-7 suspension of human breast cancer cells at a cell concentration of 1 ⁇ 10 4 /mL.
  • the cell sphere was aspirated and transferred to a glass-bottomed culture dish for laser confocal.
  • the cells were washed twice with PBS solution, and then 2mL of pH 6.5 serum-free medium diluted (1) Adriamycin (4 ⁇ g/mL), ( 2) Doxorubicin (4 ⁇ g/mL) + BPEI 0.6k -G-DMA (25 ⁇ g/mL), (3) Doxorubicin (4 ⁇ g/mL) + BPEI 1.8k -G-DMA (25 ⁇ g/mL), ( 4) Doxorubicin (4 ⁇ g/mL) + BPEI 3.5k -G-DMA (25 ⁇ g/mL), (5) Doxorubicin (4 ⁇ g/mL) + BPEI 5k -G-DMA (25 ⁇ g/mL) or (6 ) Doxorubicin (4 ⁇ g/mL)+BPEI 1.8k- Lys-DMA, continue to incubate for 2 hours.
  • the group with BPEI 1.8k -G-DMA added showed the strongest and most uniform fluorescence, while the group containing BPEI 1.8k -Lys-DMA with similar structure showed the weakest fluorescence (compared to the group with only adriamycin added) Fluorescence intensity is similar), and more distributed on the surface of the cell sphere.
  • the fluorescence intensity of BPEI 0.6k -G-DMA and BPEI 3.5k -G-DMA group was slightly lower than that of BPEI 1.8k -G-DMA group, but significantly higher than that of BPEI 5k -G-DMA group.
  • Example 22 colorectal cancer cell SW480 was used to culture cell spheroids, and the penetration of HBPL 2.4k -G-DMA on doxorubicin in the cell spheroids was studied. The results are shown in Figure 2. Similarly, in the group containing the macromolecule HBPL 2.4k -G-DMA of the present invention, compared with the group containing only doxorubicin, the spheroids showed significantly stronger and more uniform fluorescence intensity.
  • the cell spheroids were cultured with BxPC-3 cells to study the penetration of adriamycin in the cell spheroids by G4DPLys-G-DMA.
  • the results are shown in Figure 3.
  • the spheroids of the group containing the macromolecule G4DPLys-G-DMA of the present invention showed significantly stronger and more uniform fluorescence intensity.
  • the cell spheroids were cultured with PANC-1 cells to study the penetration of G1Lys-G-DMA in the cell spheroids.
  • the results are shown in Figure 4.
  • the spheroids of the group containing the macromolecule G1Lys-G-DMA of the present invention showed significantly stronger and more uniform fluorescence intensity.
  • Example 19 In the animal experiment that macromolecules promote the penetration of anticancer drugs in tumor tissues, refer to Example 19 to create xenograft tumors in nude mice.
  • the tumor volume grows to about 400 mm 3 , it is injected through the tail vein (1) doxorubicin, (2) Adriamycin + bPEI 1.8k -G, (3) Adriamycin + BPEI 1.8k -G-DMA, (4) Adriamycin + BPEI 1.8k -Lys-DMA, (5) Adriamycin + bPEI 0.6k -G-DMA, (6)
  • Doxorubicin + G4DPLys-G-DMA the dose of doxorubicin is 1 mg/kg
  • the dose of macromolecules is 10 mg/kg
  • the experimental mice are sacrificed 0.5 hours later, and the tumors are dissected and removed.
  • the obtained tumor was frozen sectioned, the blood vessels were stained with CoraLite 594-labeled CD34 antibody, and the fluorescence signals of CD34 and adriamycin were observed under a laser confocal microscope.
  • the results are shown in Figure 5. It can be seen from Figure 5 that when only doxorubicin is injected, the doxorubicin in the tumor tissue is mainly distributed around the blood vessel, while when doxorubicin and the macromolecule of the present invention are injected at the same time, the doxorubicin is more easily penetrated to the part far away from the blood vessel and distributed More uniform.
  • the macromolecule bPEI 1.8k -G which contains only guanidine groups but no carboxyl groups, has little promotion effect on the penetration of adriamycin, but the macromolecule (BPEI 1.8k -G-DMA) of the present invention after introducing carboxyl groups has little effect on adriamycin The penetration promotion effect is very significant.
  • the macromolecules (BPEI 1.8k -Lys-DMA) with similar structures that do not contain guanidine groups and only contain amine and carboxyl groups have little effect on the penetration of adriamycin.
  • the cancer cells were cultured with doxorubicin (2 ⁇ g/mL) or doxorubicin (2 ⁇ g/mL) + macromolecules (10 ⁇ g/mL) in a serum-free medium at pH 6.5 for 2 hours, washed with PBS and flow cytometry The analysis was performed on a cytometer (FACS Calibur flow cytometer of Dickinson and Company). The specific types of cancer cells and macromolecules, and the results are shown in Table 4.
  • HepG2 human liver cancer cells
  • Hela human cervical cancer cells
  • BxPC-3 human pancreatic cancer cells
  • SW480 colorectal cancer
  • MCF-7 human breast cancer cells
  • PANC-1 human pancreatic cancer cells
  • the tumor-inhibiting animal experiment of macromolecule promoting anticancer drugs refer to Example 19 to create xenograft tumors in nude mice.
  • the tumor-bearing mice are randomly divided into 6 groups (5 in each group), respectively Intraperitoneal injection: (1) normal saline, (2) BPEI 1.8k -G-DMA (10mg/kg), (3) doxorubicin (1mg/kg), (4) doxorubicin (5mg/kg), ( 5) Adriamycin (1mg/kg) + BPEI 1.8k -G-DMA (10mg/kg), (6) Adriamycin (5mg/kg) + BPEI 1.8k -G-DMA (10mg/kg), or Doxorubicin (5mg/kg) + BPEI 1.8k -Lys-DMA (10mg/kg), macromolecules were injected first, then doxorubicin was injected 30 minutes later, once on day 0 and day 7, tumor
  • Example 29 for tumor suppression animal experiments.
  • PC-3 is used for xenograft tumors
  • the macromolecule used is BPEI 0.6k -G-DMA
  • the dose is 10 mg/kg
  • the anticancer drug used is cis Platinum
  • the dose was 5 mg/kg
  • the average tumor weight on the 15th day after injection is shown in Table 6.
  • Example 29 for tumor suppression animal experiments.
  • BxPC-3 is used for xenograft tumors
  • the macromolecule used is HBPL 0.9k -G-DMA
  • the dose is 20 mg/kg
  • the anticancer drug used is gemcitabine.
  • the dose is 25 mg/kg
  • the average tumor weight on the 15th day after injection is shown in Table 6.
  • Example 29 for tumor suppression animal experiments.
  • human ovarian cancer cell SKOV3 was used for xenograft tumors
  • the macromolecule used was G4DPLys-G-DMA
  • the dose was 10 mg/kg
  • the anticancer drug used was paclitaxel.
  • the dose is 10 mg/kg
  • the average tumor weight on the 15th day after injection is shown in Table 6.
  • Example 29 for tumor suppression animal experiments.
  • human lung cancer cells A549 were used for xenograft tumors
  • the macromolecule used was G3PAMAM-G-DMA
  • the dose was 10 mg/kg
  • the anticancer drug used was irinote Kang
  • the dose is 10mg/kg
  • the average weight of the tumor on the 15th day after injection is shown in Table 6.

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Abstract

一种促抗癌药摄入的大分子、组合物及其制备抗癌药物的用途，所述大分子具有多个胍基且可选地具有胺基，其中至少部分胺基和/或胍基被式I所示的取代基所取代而形成酰胺键，其中所述大分子具有小于等于50,000Da的平均分子量，并且在正常生理条件下所述大分子的ζ电位为-15mV～+5mV，以及所述大分子在微酸性条件下水解后具有5～100个胍基。所述大分子可以促进抗癌药在肿瘤组织的聚集、渗透和促进抗癌药进入癌细胞内，此类大分子与抗癌药联用时可以增加抗癌药的抗癌效果，因此在相同的抗癌药剂量下，与所述的大分子联用显著增大抗癌的效果，或者在较低的抗癌药剂量下，与所述的大分子联用大大减小抗癌药的毒副作用。

Description

促抗癌药摄入的大分子、组合物及其制备抗癌药物的用途 技术领域
本发明涉及抗肿瘤药物增效剂领域，具体涉及促进抗癌药在肿瘤组织的聚集和渗透的大分子物质。
背景技术
癌症的化疗是目前治疗癌症的主要方法之一，但是，传统化疗所使用的药物通常存在毒性大、副作用大等弊端，经由口服或注射给药后，化疗药物在人体内无选择性分布，在杀死癌细胞的同时也会对正常组织细胞造成严重的损害，产生骨髓抑制、胃肠道毒性、皮肤毒性和过敏等不良反应。而且，对于实体瘤患者，最初的化疗往往疗效较好，但这种疗效往往不能持续，化疗后肿瘤容易复发，复发后再进行化疗其疗效往往不佳。其原因是化疗药不易在肿瘤组织聚集和渗透，也不易被癌细胞摄取，特别是药物不易在肿瘤组织聚集和不易渗透到肿瘤组织的深处是阻碍抗癌药物疗效的最为关键的因素。抗癌药物通过血液循环在肿瘤组织的聚集量比在正常组织的聚集量少很多(R.Nandigama,et al.,Restriction of drug transport by the tumor environment,Histochemistry and Cell Biology 2018,150,631–648)，即使是少量聚集于肿瘤组织的抗癌药也主要分布于毛细血管周围，而渗透到远离毛细血管的深处的药物很少，即药物在肿瘤组织内的分布极不均匀，这主要是由于实体瘤组织的高密度的胞外基质和升高的组织间隙液压阻碍了药物的渗透(A.I.Minchinton,et al.,Drug penetration in solid tumours,Nature Reviews Cancer 2006,6,583–592)。进入肿瘤组织中远离毛细血管部位的药物的浓度往往很小，达不到杀死癌细胞所需的最低药物浓度，而且接受低浓度抗癌药的癌细胞往往会产生耐药性，这就是癌症的持续化疗疗效差和化疗后肿瘤易复发的主要原因。癌细胞耐药性的主要机理之一是阻止化疗药进入癌细胞内和将进入癌细胞的化疗药泵出到胞外，使癌细胞内的化疗药的浓度达不到杀死癌细胞所需的最低浓度。理论上，增大化疗药的剂量可以增大远离毛细血管的肿瘤组织的药物浓度，耐药癌细胞内的化疗药的浓度也会增加。但由于化疗药的严重毒副作用，增大剂量可能是致命的。
为了增加抗癌药在肿瘤组织的聚集，近几十年来发展了纳米载体负载抗癌药作为抗癌药的传输体系。其基本原理是，纳米粒子不易透过致密的正常毛细血管壁而进入正常组织，但易透过多孔性的肿瘤组织毛细血管壁而进入肿瘤组织，提高抗癌药物的抗癌效率(A.Wicki,et al.,Nanomedicine in cancer therapy:Challenges,opportunities,and clinical applications,Journal of Controlled Release 2015,200,138–157)。虽然已经有10多个纳米载体负载抗癌药得到临床应用，但这些纳米药物与对应的小分子药物相比疗效并没有增加多少，只是副作用有所减小(Y.S.Youn,et al.,Perspectives on the past,present,and future of cancer nanomedicine,Advanced Drug Delivery Reviews 2018,130,3–11)。在肿瘤组织高 比例的聚集但其疗效并未提高的原因归因于，进入肿瘤组织的纳米粒子主要集中于毛细血管周围，由于其较大(相比于小分子)的尺寸更难渗透到远离毛细血管的深处(D.Rosenblum,et al.,Progress and challenges towards targeted delivery of cancer therapeutics,Nature Communications 2018,9,1410；M.van Elk,et al.,Nanomedicines for advanced cancer treatments:Transitioning towards responsive systems,International Journal of Pharmaceutics 2016,515,132–164)。此外，纳米载体负载药物体系涉及到药物的负载、负载量、可控释放等问题，是一个非常复杂的体系。如果复杂体系中的任何一步效果不佳，则最终的效果肯定很差，而且复杂体系不易规模化生产，批次之间重复性差，生产成本高。
在我们先前发表的研究论文(J.Cao,et al.,European Journal of Pharmaceutics and Biopharmaceutics 2018,127,371–377；X.Huang,et al.,Materials Science&Engineering C 2019,102,558–568)和中国专利申请No.：201710402913.X中发现，含胺基的聚合物可以提高小分子化疗药进入癌细胞内，通过给荷瘤裸鼠共注射或先后注射含胺基聚合物和化疗药，因聚合物促进化疗药更多地进入癌细胞内，因而其抗癌效果比只注射小分子药物的效果好，基含胺基聚合物使化疗药增效。因为药物并没有负载于聚合物上，与纳米载体负载药物体系相比更简单。
Sugahara等发现，称作肿瘤穿透肽的由9个氨基酸组成的环肽可以促进抗癌药在肿瘤组织的聚集和促进抗癌药在肿瘤组织的渗透(K.N.Sugahara,et al.,Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs,Science 2010,328,1031–1035)。该研究中给予荷瘤裸鼠同时注射环肽和抗癌药，使抗癌药更多地聚集于肿瘤组织，并更深地渗透于肿瘤组织，从而提高抗癌药的抗肿瘤效果，起到增效剂的作用。与纳米粒子负载抗癌药物体系相比，增效剂与抗癌药物联用体系更简单，不涉及药物负载、负载量、可控释放等问题，可以与多种抗癌药联用。但多肽药物的稳定性差，在储存和使用过程中都易失活，如多肽药物进入血液后极易被多肽水解酶或蛋白酶水解而失效。而且多肽药物的合成成本昂贵，限制了其实际应用
发明内容
有鉴于此，本发明的主要目的在于提供一种能够促进抗癌药物更多地在肿瘤部位聚集，并更深入地渗透到肿瘤组织内部的物质，从而提高抗癌药物，特别是化疗药物的治疗效果。
本发明人发现，一类含多个胍基的大分子不仅像先前报道的含胺基聚合物一样可以促进化疗药更多地进入癌细胞，而且还可以促进抗癌药在肿瘤组织的聚集和促进抗癌药在肿瘤组织的渗透，从而取得了更好的疗效。本发明的大分子中的胍基以及每一大分子所含的胍基的数量对大分子促进抗癌药在肿瘤组织的聚集和促进抗癌药在肿瘤组织的渗透起关键的作用。
因此，本发明的第一方面提供一种大分子，所述大分子具有多个胍基且可选地具有胺基，其中至少部分胺基和/或胍基被下式I所示的取代基所取代而形成酰胺键，其中所 述大分子具有小于等于50,000Da的平均分子量，并且在正常生理条件下所述大分子的ζ电位为-15mV～+5mV，以及所述大分子在微酸性条件下水解后具有5～100个胍基，
Figure PCTCN2020134363-appb-000001
其中R 1和R 2可各自独立地选自取代或未取代的C1～C6的烷基，或者R 1和R 2可相互连接和与其连接的碳原子共同形成五元或六元环，其中所述取代指被1～2个羧基所取代。
较优地，式I的取代基中R 1和R 2可各自独立地选自取代或未取代的C1～C3的烷基，或者R 1和R 2可相互连接和与其连接的碳原子共同形成六元环，其中所述取代指被1个羧基所取代。
具体地，式I的取代基可选自以下取代基组成的组：
Figure PCTCN2020134363-appb-000002
由于胍基在正常的生理条件下是以质子化的形式存在，即含有胍基的大分子在正常生理条件下带正电荷。然而带正电荷的大分子在体内易被清除，在血液中的循环时间短，无法产生令人满意的促进抗癌药物摄入的作用。为了解决这一问题，进一步在大分子中引入一定量的羧基，使大分子在正常生理条件下(pH～7.4)ζ电位为-15mV～+5mV。此时，分子的净电荷接近于0或略带负电荷。净电荷接近于0或略带负电荷的大分子在体内不易被清除，其血液循环时间长(E.Blanco,et al.,Principles of nanoparticle design for overcoming biological barriers to drug delivery,Nature Biotechnology 2015,33,941–951)。
在肿瘤组织的微酸性的环境中，本发明大分子中由式I取代基形成的酰胺键发生水解，该取代基带有的羧基脱离大分子。水解后的大分子中的胺基和胍基被释放出来，从而能够发挥其帮助抗癌药渗透到细胞中以及扩散至组织内部的作用。此处所述的“释放”是指水解前酰化的胺基和胍基水解后变成自由的胺基和胍基而被释放出来，以及水解前大分子中未酰化的胺基和胍基与大分子所具有的羧基形成离子对而使胺基和胍基被保护起来，水解后由于羧基部分的离去而使由离子对保护的胺基和胍基也被释放出来。
具有式I所示结构的取代基与大分子中的胺基(伯胺基或仲胺基)和/或胍基形成的酰胺键在正常生理条件的微碱性环境中可以稳定存在，因而在正常组织以及正常组织的血液循环中不会水解，而在肿瘤组织的微酸性的环境下，该酰胺键易于水解，从而能够 顺利到达肿瘤组织发挥其作用。
本发明人发现，在模拟肿瘤微酸性的环境中，本发明的大分子在平均分子量相同或相近的情况下，每分子中所含的胍基的平均数愈多则有利于促进抗癌药在肿瘤组织聚集、渗透和进入癌细胞内；当所述大分子的每一分子所含的胍基的平均数相同时，则平均分子量愈小愈有利于促进抗癌药在肿瘤组织聚集、渗透和进入癌细胞内。所述大分子所含的平均胍基数最少为5个时才有较显著的功能作用，由于胍基数和分子量对其功能作用具有相反的影响。本发明中，分子量尽量小，且胍基数量尽量高，则可取得较佳的效果，而这两个因素具有最佳的平衡范围。
根据较佳的实施方式，所述大分子在微酸性条件下水解后可具有10～60个胍基，优选具有15～40个胍基。
根据另一种实施方式，所述大分子可具有1,500～40,000Da的平均分子量，优选具有2,500～25,000Da的平均分子量，更优选具有3,000～15,000Da。
根据更优的实施方式，所述大分子具有3,000～15,000Da的分子量，且在微酸性条件下水解后可具有15～40个胍基。
本发明中，通过控制大分子中式I所示的取代基的取代率，使本发明的大分子的净电荷数接近0。其中净电荷数通过大分子的ζ电位来表征。在正常生理条件下(pH～7.4)的ζ电位在-5mv到+5mv之间时可以认为其净电荷接近于0，在-15mv到-5mv之间时为略带负电荷。本发明的大分子的ζ电位在-15mv到+5mv之间，较优地在-10mv到+2mv之间，更优地在-8mv到0mv之间。
也就是说，整体略带负电荷的大分子是更为优选的。
在本发明的大分子中，所述胍基可通过使含胺基的大分子中的至少部分胺基胍基化或与具有胍基的化合物反应而引入所述大分子中。为了便于引入胍基，所述含胺基的大分子含有多个伯胺基和/或仲胺基。所述含胺基的大分子可进一步含有叔胺基。
所述含胺基的大分子可为含胺基的线性大分子或含胺基的支化大分子。这样的线性或支化的大分子可以是带有胺基侧链的聚烯烃类的分子，也可以是侧链带有胺基的聚氨基酸类的分子，氨基化的超支化聚甘油等。
根据优选的具体实施方式，所述含胺基的线性大分子可选自聚乙烯胺、聚烯丙基胺和线形聚乙烯亚胺。所述含胺基的支化大分子可选自支化聚乙烯亚胺、超支化聚赖氨酸、树枝状聚酰胺-胺、树枝状聚丙烯亚胺、树枝状聚赖氨酸和氨基化的超支化聚甘油。其中最优选的是含胺基的支化大分子。
这些含胺基的大分子价格较低，或合成成本较低，易于合成且结构稳定，毒副作用小，是生物相容性好的目标大分子。
根据一种具体实施方式，所述使含胺基的大分子中的至少部分胺基与具有胍基的化合物反应包括使含胺基的大分子中的至少部分胺基与含有胍基和羧基的化合物进行酰胺化反应。优选地所述含有胍基和羧基的化合物选自胍基乙酸盐酸盐、3-胍基丙酸盐酸盐、α-氨基和胍基双保护的精氨酸。当含有胍基和羧基的化合物选自α-氨基和胍基双保护的精氨酸时，所得到的脱掉保护基后的含胍基大分子中同时也引入了伯胺基(精氨酸 的α-氨基)。
根据另一种具体实施方式，使含胺基的大分子中的至少部分胺基胍基化通过胍基化试剂进行反应。优选地所述胍基化试剂可选自单氰胺、O-甲基异脲、硫脲、S-甲基异硫脲、N,N-二(叔丁氧羰酰基)硫脲、1H-吡唑-1-甲脒盐酸盐和3,5-二甲基-1-吡唑硝酸甲脒。
由以下详述的实施例可知，本发明的水解后带有一定数量胍基的大分子不仅能促进抗癌药物进入细胞内发挥作用，而且能够有效促进抗癌药物在实体肿瘤中聚集，并明显有助于抗癌药物从血管部位渗透到肿瘤组织更深入的部位。因此在相同的抗癌药剂量下，本发明的大分子的使用显著增大抗癌的效果；或者在较低的抗癌药剂量下，本发明的大分子的使用大大减小抗癌药的毒副作用。
本发明的第二方面提供一种制备上述大分子的方法。所述方法包括：
在含胺基的大分子中引入胍基，和
进一步与式II所示的化合物反应得到所述大分子，
Figure PCTCN2020134363-appb-000003
其中R 1和R 2可各自独立地选自由羧基、取代或未取代的C1～C6的烷基所组成的组，或者R 1和R 2相互连接为取代或未取代的亚烷基并和与其连接的碳原子共同形成五元或六元环所组成的组，其中所述取代为被1～2个羧基所取代。
优选地，R 1和R 2可各自独立地选自由羧基、取代或未取代的C1～C3的烷基所组成的组，或者R 1和R 2相互连接为亚丁基并和与其连接的碳原子共同形成六元环，其中所述取代为被1个羧基所取代。
所述式II的化合物可具体为以下化合物：
Figure PCTCN2020134363-appb-000004
所述式II的化合物最优选为2,3-二甲基马来酸酐、环己烯-1,2-二羧酸酐、顺乌头酸酐和2-(2-羧乙基)-3-甲基马来酸酐中的一种。
所述含有胺基的大分子如前述所定义。
根据一种实施方式，所述在含有胺基的大分子中引入胍基可包括：使所述含胺基的大分子与含胍基和羧基的化合物中的羧基形成酰胺键。优选地，所述含胍基和羧基的化合物选自胍基乙酸盐酸盐、3-胍基丙酸盐酸盐、α-氨基和胍基双保护的精氨酸。如果所述含胍基和羧基的化合物选自α-氨基和胍基双保护的精氨酸，最后需要将保护基脱除。
或者根据另一种实施方式，所述在含有胺基的大分子中引入胍基可包括：使所述含有胺基的大分子与胍基化试剂反应。优选地，所述胍基化试剂选自单氰胺、O-甲基异脲、硫脲、S-甲基异硫脲、N,N-二(叔丁氧羰酰基)硫脲、1H-吡唑-1-甲脒盐酸盐和3,5-二甲基-1-吡唑硝酸甲脒。
除上述方法外，本发明中引入胍基的反应还可采用本领域常规使用的任何方法，本发明对引入胍基的方法并没有特别限制。
本发明的第三方面提供一种包括上述大分子的药物组合物。
根据优选的实施方式，所述药物组合物可进一步包括至少一种抗癌药物。优选地，所述抗癌药物可选自抗代谢药、烷化剂、抗微管药、抗癌抗生素和它们的组合。
具体地，所述抗代谢药可选自5-氟尿嘧啶、甲氨蝶呤和吉西他滨；所述烷化剂选自环磷酰胺、苯丁酸单芥和铂类抗癌药；所述抗微管药选自紫杉醇、长春碱、长春新碱、喜树碱和伊立替康；所述抗癌抗生素选自阿霉素、吡柔比星和表柔比星。
所述药物组合物还可进一步包括药学上可接受的赋形剂和/或载剂。
可用于本发明的药物组合物的赋形剂和载剂可根据具体的给药方式、给药途径选择，本发明对此并无特别限制。
本发明的第四方面提供上述大分子促进抗癌药物渗透到肿瘤组织内部的用途。所述抗癌药物为小分子抗癌药物，特别是如上所限定的那些。
并且本发明还提供上述药物组合物在制备治疗癌症的药物中的用途。
本发明还提供一种治疗癌症的方法，所述方法包括将含有上述大分子或上述药物组合物的药物对罹患癌症的受试者给药的步骤。
本发明进一步提供一种用于治疗癌症的上述药物组合物。
特别地，所述治疗包括根据本发明的大分子促进所述抗癌药物在肿瘤组织聚集并向肿瘤组织内部渗透。
本发明的大分子主要促进抗癌药物在实体瘤中的渗透和摄入。因此，所述癌症为恶性实体瘤。
优选地，所述恶性实体瘤可选自肝癌、肺癌、胃癌、肠癌、卵巢癌、乳腺癌、胰腺癌、前列腺癌和宫颈癌。
本发明的大分子能够显著提高抗癌药物的疗效，并因而能够使抗癌药物以更低的剂量使用，从而降低其副作用。此外，由于本发明的大分子能够促进抗癌药物更深入地渗透到实体肿瘤组织内部，因此还能够延长出现耐药性的时间，或者阻止抗耐药性的出现。 此外，与多肽类促进剂相比，本发明的大分子制备方法简单，制备成本低，稳定性高，更易于保存，便于运输。
附图说明
图1.BPEI 0.6k-G-DMA、BPEI 1.8k-G-DMA、BPEI 3.5k-G-DMA、BPEI 5k-G-DMA和BPEI 1.8k-Lys-DMA促进阿霉素在MCF-7细胞培养的细胞球中的渗透的激光共聚焦照片(DOX为阿霉素，标尺为100μm)；
图2.HBPL 4.6k-G-DMA促进阿霉素在SW480细胞培养的细胞球中的渗透的激光共聚焦照片(DOX为阿霉素，标尺为100μm)；
图3.G4DPLys-G-DMA促进阿霉素在BxPC-3细胞培养的细胞球中的渗透的激光共聚焦照片(DOX为阿霉素，标尺为100μm)；
图4.G1Lys-G-DMA促进阿霉素在PANC-1细胞培养的细胞球中的渗透的激光共聚焦照片(DOX为阿霉素，标尺为100μm)；
图5.BPEI 1.8k-G、BPEI 1.8k-G-DMA、BPEI 1.8k-Lys-DMA、bPEI 0.6k-G-DMA和G4DPLys-G-DMA促进阿霉素在肿瘤组织中的渗透(DOX为阿霉素，标尺为50μm)；
图6.bPEI 1.8k-G-DMA促进阿霉素进入MCF-7细胞的激光共聚焦照片(DOX为阿霉素，标尺为50μm)；和
图7.异种移植MCF-7裸鼠在第0天和第7天注射不同配方后肿瘤体积与时间的变化图(DOX为阿霉素)。
具体实施方式
下面将结合本发明实施方式及附图，对本发明实施方式中的技术方案进行清楚、完整地描述，显然，所描述的实施方式仅仅是本发明的一部分实施方式，而不是全部的实施方式。基于本发明中的实施方式，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施方式，都属于本发明保护的范围。
在整个说明书中，除非另有特别说明，本文使用的术语应理解为如本领域中通常所使用的含义。因此，除非另有定义，本文使用的所有技术和科学术语具有与本发明所属领域技术人员的一般理解相同的含义。若存在矛盾，本说明书优先。
需要说明的是，在本发明实施例中，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的方法或者装置不仅包括所明确记载的要素，而且还包括没有明确列出的其他要素，或者是还包括为实施方法或者装置所固有的要素。在没有更多限制的情况下，由语句“包括一个……”限定的要素，并不排除在包括该要素的方法或者装置中还存在另外的相关要素(例如方法中的步骤或者组分)。
本文所述的术语“正常的生理条件”指哺乳动物，特别是人体内的维持细胞正常的生理活动的稳态内环境。本文中尤其指该内环境的pH值。在正常生理条件下，体内环境的pH值为大约7.4。
本文所述的术语“微酸性条件”是特指肿瘤组织中的酸碱性环境，通常肿瘤组织中的酸碱性环境呈现为pH 6.5～7.0的微酸性。
本发明的大分子可采用以下方法制备：首先制备含多个胍基的大分子，然后在所制备的含胍基大分子中引入羧基。
含多个胍基的大分子可采用两种方法制备。第一种方法是含有伯胺基和(或)仲胺基的大分子与同时含有胍基和羧基的化合物形成酰胺键而引入胍基。即大分子中的伯胺基和(或)仲胺基与含有胍基和羧基的化合物中的羧基进行缩合形成酰胺键。第二种方法是由含有伯胺基和(或)仲胺基的大分子进行胺基的胍基化反应而引入胍基。所述的含有伯胺基和(或)仲胺基的大分子包括线形大分子和支化大分子，含有伯胺基和(或)仲胺基的线形大分子包括聚乙烯胺、聚烯丙基胺、线形聚乙烯亚胺等；含伯胺基和(或)仲胺基的支化大分子包括支化聚乙烯亚胺、超支化聚赖氨酸、氨基化的超支化聚甘油等、树枝状聚酰胺-胺、氨基化的超支化聚甘油、树枝状聚丙烯亚胺和树枝状聚赖氨酸等。优选采用含有伯胺基和(或)仲胺基的支化大分子，支化大分子中优选支化聚乙烯亚胺、超支化聚赖氨酸、氨基化的超支化聚甘油和树枝状聚赖氨酸。
所述的通过含有伯胺基和(或)仲胺基的大分子与同时含有胍基和羧基的化合物形成酰胺键而引入胍基所用的含有胍基和羧基的化合物包括：胍基乙酸盐酸盐、3-胍基丙酸盐酸盐、保护的精氨酸(Boc-Arg(Pbf)-OH、Boc-Arg-OH·HCl、Boc-Arg(Boc)-OH、Fmoc-Arg-OH·HCl)。羧基和胺基缩合形成酰胺键的缩合剂包括碳二亚胺类缩合剂(如二环己基碳二亚胺、二异丙基碳二亚胺)、鎓盐类缩合剂(如TATU、TBTU、BOP、PyBOP、PyAOP等)。如果使用保护的精氨酸，形成酰胺键后再将保护基脱掉后即可得到含有胍基的大分子。
所述的含有伯胺基和(或)仲胺基的大分子通过胺基的胍基化反应引入胍基的反应中采用的胍基化试剂包括单氰胺、O-甲基异脲、硫脲、S-甲基异硫脲、N,N′-二(叔丁氧羰酰基)硫脲、1H-吡唑-1-甲脒盐酸盐等和3,5-二甲基-1-吡唑硝酸甲脒。如果胍基化试剂中含有保护基，胍基化反应后再将保护基脱掉后即可得到含有胍基的大分子。
在反应中，可对胺基或胍基进行保护，在反应后脱去保护基即可。所述保护基可为任何常用的氨基保护剂，如叔丁氧羰酰基(Boc)等，但不限于此。
本发明的大分子可以促进抗癌药更多地聚集于肿瘤组织。如以下将详述的，将所述大分子和抗癌药注射于荷瘤动物体内，在一定的时间后分析抗癌药在肿瘤、肝脏、肾脏、脾脏和肺中的浓度分布。结果表明，与只注射抗癌药相比，本发明的大分子显著地促进抗癌药在肿瘤组织的聚集。而不含胍基但含胺基的具有类似结构的大分子对抗癌药在肿瘤组织中的聚集的促进作用很小。
通过细胞实验证明，本发明的大分子可以促进抗癌药进入癌细胞内，即当癌细胞与所述大分子和抗癌药在模拟肿瘤的微酸性环境下共同孵育时，进入癌细胞内的抗癌药的浓度高于相同剂量的单独抗癌药孵育时进入癌细胞内的抗癌药的浓度。细胞流式实验表明，与单独使用抗癌药(以阿霉素为例)相比，同时使用本发明的大分子和抗癌药可以使进入癌细胞内的抗癌药的浓度明显提高。由于本发明的大分子可以促进抗癌药进入癌 细胞，因此可以提高抗癌药的杀死癌细胞的效率。通过癌细胞与本发明的大分子和抗癌药在模拟肿瘤的微酸性环境下共同孵育，作为比较癌细胞与单独抗癌药在相同的条件下进行孵育，在存在大分子的孵育实验中，癌细胞的存活率明显减小。
将三维癌细胞球体与本发明的大分子和抗癌药(以阿霉素为例)在模拟肿瘤的微酸性环境下共同孵育，然后通过激光共聚焦成像检测癌细胞球体中不同深度的截面上的荧光强度，所述荧光强度与阿霉素的浓度成正比。与不使用大分子但其他条件相同的对照实验对比，本发明的大分子可以促进阿霉素在癌细胞球体中更深地渗透，在癌细胞球体中更均匀地分布。而不含胍基但含胺基的结构类似的大分子几乎没有对阿霉素的癌细胞球体中的渗透的促进作用。进一步作动物实验也表明，本发明的大分子可以促进抗癌药渗透到远离血管的肿瘤组织的深处，而仅含胺基的结构类似的大分子对阿霉素在肿瘤组织中的渗透的促进明显要小。
由于所述大分子可以促进抗癌药在肿瘤组织的聚集、促进抗癌药在肿瘤组织的渗透、并促进抗癌药进入癌细胞内，因此该类大分子与抗癌药联合使用治疗癌症时可以显著地使抗癌药增效。与单独注射抗癌药相比，荷瘤动物注射所述大分子和相同剂量的抗癌药后，其肿瘤的重量显著减小。
下面进一步通过实施例对本发明作进一步说明。
实施例1
称取1g平均分子量为400Da的支化聚乙烯亚胺(BPEI 0.4k)溶于200mL二甲基甲酰胺中，在搅拌下加入17.8g保护的精氨酸(Boc-Arg(Boc)-OH)、4.7g 1-羟基苯并三唑、4.4g N,N′-二异丙基碳二亚胺和6.8g N,N-二异丙基乙胺，反应在室温下进行24小时，过滤，减压浓缩去除大部分溶剂，用乙醚沉淀。沉淀溶于二氯甲烷，通过硅胶柱纯化(二氯甲烷/甲醇＝10/1)，所得产物通过NMR估算出每一聚合物分子中含有胍基的平均数5.8个。产物溶于二氯甲烷中，搅拌下加入三氟乙酸，使最后的三氟乙酸/二氯甲烷的比例为9/1(体积比)，溶液在室温下搅拌12小时，旋转蒸发除去二氯甲烷，残液用乙醚沉淀，得到的产物用BPEI 0.4k-G表示。
称取1g BPEI 0.4k-G溶解在50mL去离子水中，在搅拌下加入1.2g 2,3-二甲基马来酸酐(DMA)，随后滴加氢氧化钠溶液保持反应溶液pH在8.5-9之间，室温下反应18小时，其间调节pH值始终为8.5-9。所得溶液用截留分子量为100-500Da的透析袋透析，冷冻干燥得到的大分子用BPEI 0.4k-G-DMA表示。采用动态光散射方法(Malvern的Zetasizer Nano ZS90)测定BPEI 0.4k-G-DMA在pH 7.4时的ζ电位为-0.8mV，采用MALDI-TOF-MS(Bruker Daltonics的AutoflexIII LRF200-CID)测得的平均分子量为2020Da。
实施例2
用平均分子量为600Da的支化聚乙烯亚胺(BPEI 0.6k)替代实施例1中的BPEI 0.4k，其他操作步骤同实施例1，所得引入胍基和羧基的大分子用BPEI 0.6k-G-DMA表示，每 一BPEI 0.6k-G-DMA大分子中引入胍基的平均数为10.2个，在pH 7.4时的ζ电位为-3.5mV，平均分子量为3450Da。
实施例3
称取1g平均分子量为1800Da的支化聚乙烯亚胺(BPEI 1.8k)溶于200mL二甲基甲酰胺中，在搅拌下加入17.8g保护的精氨酸(Boc-Arg(Pbf)-OH)、4.7g N-羟基琥珀酰亚胺(NHS)、9.3g 1-乙基-3-(3-二甲基氨基丙基)碳二亚胺(EDC)和7.5g N,N-二异丙基乙胺，反应在30℃下进行24小时，减压浓缩去除大部分溶剂，用乙醚沉淀，沉淀溶于二氯甲烷中，搅拌下加入三氟乙酸，使最后的三氟乙酸/二氯甲烷的比例为9/1(体积比)，溶液在室温下搅拌12小时，旋转蒸发除去二氯甲烷，所得溶液用1mol/L NaHCO 3调节pH值到6-7，用截留分子量为100-500Da的透析袋透析，透析液冷冻干燥得到的产物用BPEI 1.8k-G表示。通过引入胍基前后平均分子量(MALDI-TOF-MS)的变化估算出平均每一BPEI 1.8k-G大分子上引入22.1个胍基。
参照实施例1的方法进行BPEI 1.8k-G与2,3-二甲基马来酸酐的反应，得到的大分子用BPEI 1.8k-G-DMA表示。BPEI 1.8k-G-DMA在pH 7.4时的ζ电位为-4.2mV，平均分子量为8080Da。
实施例4
用平均分子量为3500kDa的支化聚乙烯亚胺(BPEI 3.5k)代替实施例3中的BPEI 1.8k，其他操作步骤同实施例3，得到的大分子用BPEI 3.5k-G-DMA表示。每一BPEI 3.5k-G-DMA大分子中含有胍基数的平均值为39.7个，在pH 7.4时的ζ电位为-2.5mV，平均分子量为14.2kDa。
实施例5
称取1g平均分子量为5000Da的支化聚乙烯亚胺(BPEI 5k)溶于200mL二甲基甲酰胺中，在搅拌下加入22.9g保护的精氨酸(Boc-Arg(Pbf)-OH)、2.5g N-羟基琥珀酰亚胺、10g 1-乙基-3-(3-二甲基氨基丙基)碳二亚胺和7.5g N,N-二异丙基乙胺，反应在30℃下进行24小时，减压浓缩去除大部分溶剂，用乙醚沉淀。参照实施例3的方法用二氯甲烷/三氟乙酸处理所得沉淀，得到的产物用BPEI 5k-G表示。通过平均分子量的变化估算出平均每一BPEI 5k-G大分子上引入55.8个胍基。
参照实施例1的方法进行BPEI 5k-G与2,3-二甲基马来酸酐的反应，得到的产物用BPEI 5k-G-DMA表示。BPEI 5k-G-DMA在pH 7.4时的ζ电位为-1.6mV，平均分子量为20.1kDa。
实施例6
参照实施例5的方法用平均分子量为10kDa的支化聚乙烯亚胺(BPEI 10k)与Boc-Arg(Pbf)-OH反应，得到的产物用BPEI 10k-G表示。通过反应前后平均分子量的变化估算出平均每一BPEI 10k-G大分子上引入97.2个胍基。
参照实施例1的方法进行BPEI 10k-G(0.5g)与2,3-二甲基马来酸酐(0.65g)的反 应，其他操作步骤同实施例1，得到的产物用BPEI 10k-G-DMA表示。BPEI 10k-G-DMA在pH 7.4时的ζ电位为+1.3mV，平均分子量为37.8kDa。
实施例7
用平均分子量为1800Da的线性聚乙烯亚胺(LPEI 1.8k)替代实施例3中的BPEI 1.8k，其他操作步骤同实施例3，所得引入胍基和羧基的大分子用LPEI 1.8k-G-DMA表示，平均每一LPEI 1.8k-G-DMA大分子中引入的胍基数为22.5个，pH7.4时的ζ电位为-3.1mV，平均分子量为9032Da。
实施例8
参考文献(Z.Kadlecova,et al.,Biomacromolecules 2012,13,3127-3137)合成超支化聚赖氨酸：27.5g赖氨酸盐酸盐溶于50mL去离子水中，加入8.4g氢氧化钾，溶液在氮气保护下加热到150℃并保持16小时。所得产物溶于去离子水，用截留分子量为100-500Da的透析袋透析，透析液冷冻干燥，然后通过Sephadex G25凝胶色谱柱分离得到4个级分，采用MALDI-TOF-MS测定的平均分子量分别为928Da、2442Da、4609Da和25.6kDa。分别用HBPL 0.9k、HBPL 2.4k、HBPL 4.6k和HBPL 26k表示。
实施例9
取1g HBPL 0.9k溶于40mL去离子水中，在搅拌下加入1.4g 1H-吡唑-1-甲脒盐酸盐，滴加4mol/L的氢氧化钠溶液控制反应溶液pH值在9.5左右。反应在室温下进行60小时。采用截留分子量为100-500Da的透析袋透析，冷冻干燥得到的产物用HBPL 0.9k-G表示。通过胍基化前后氮元素含量分析计算出平均每一HBPL 0.9k-G大分子上引入6.4个胍基。
参照实施例1进行HBPL 0.9k-G(0.5g)与2,3-二甲基马来酸酐(1.1g)的反应，其他操作步骤同实施例1，得到的大分子用HBPL 0.9k-G-DMA表示。HBPL 0.9k-G-DMA在pH7.4时的ζ电位为-1.5mV，平均分子量为1528Da。
实施例10
用HBPL 2.4k替代实施例9中的HBPL 0.9k，其他操作步骤同实施例9，所得引入胍基和羧基的大分子用HBPL 2.4k-G-DMA表示，每一HBPL 2.4k-G-DMA大分子中引入胍基的平均数16.5个，在pH7.4时的ζ电位为-3.8mV，平均分子量为4256Da。
实施例11
用HBPL 4.6k替代实施例9中的HBPL 0.9k，其他操作步骤同实施例9，所得引入胍基和羧基的大分子用HBPL 4.6k-G-DMA表示，每一HBPL 4.6k-G-DMA大分子中引入胍基的平均数28.7个，在pH7.4时的ζ电位为+0.8mV，平均分子量为7809Da。
实施例12
称取5.5g Boc-Lys(Boc)-OH、2g DIC(N,N'-二异丙基碳二亚胺)、2g HOBT(1- 羟基苯并三氮唑)和4.2g DIPEA(N,N-二异丙基乙胺)溶于50mL二氯甲烷中，在搅拌下加入1g三(2-氨基乙基)胺，在氩气保护下室温反应24小时。反应溶液过滤后依次用饱和NaHCO 3溶液、NaHSO 4溶液和盐水洗涤。溶液经过MgSO 4干燥后进行旋蒸得到粗产物，随后通过硅胶柱(二氯甲烷：甲醇＝15：1)得到白色产物。随后将产物在90％三氟乙酸中进行脱保护，并在冷乙醚中进行沉淀，得到的产物用G1Lys表示。参照实施例1的方法引入胍基，得到的产物用G1Lys-G表示。通过引入胍基前后的平均分子量变化得出每一个G1Lys-G分子上引入6个胍基。
参照实施例1的方法进行G1Lys-G(1g)与2,3-二甲基马来酸酐(1.2g)的反应，其他操作步骤同实施例1，得到的大分子用G1Lys-G-DMA表示。G1Lys-G-DMA在pH 7.4时的ζ电位为-4.5mV，分子量为2116Da。
实施例13
根据文献(M.Ohsaki,et al.,Bioconjugate Chem.2002,13,510-517)合成4代树枝状聚赖氨酸G4DPLys。称取1g G4DPLys溶于200mL二甲基甲酰胺中，在搅拌下加入8.4g Boc-Arg(Pbf)-OH、1g N-羟基琥珀酰亚胺、3.4g 1-乙基-3-(3-二甲基氨基丙基)碳二亚胺和3g N,N-二异丙基乙胺，反应在室温下反应24小时，所得溶液用乙醚沉淀。参照实施例3的方法用二氯甲烷/三氟乙酸处理所得沉淀，得到的产物用G4DPLys-G表示。通过引入胍基前后的平均分子量的变化估算出平均每一G4DPLys-G大分子上引入29.3个胍基。
参照实施例1的方法进行G4DPLys-G(0.5g)与2,3-二甲基马来酸酐(0.48g)的反应，其他操作步骤同实施例1，得到的大分子用G4DPLys-G-DMA表示。G4DPLys-G-DMA在pH 7.4时的ζ电位为-6.6mV，平均分子量为13.1kDa。
实施例14
称取1g 3代聚酰胺-胺树枝状大分子(G3PAMAM)溶于40mL去离子水中，在搅拌下加入0.78g 1H-吡唑-1-甲脒盐酸盐，滴加4mol/L的氢氧化钠溶液控制反应溶液pH值在9.5左右。反应在室温下进行60小时。经过透析、冷冻干燥，得到胍基化3代聚酰胺-胺树枝状大分子，用G3PAMAM-G表示。通过胍基化前后的氮元素含量分析计算出平均每一G3PAMAM-G大分子上引入28.6个胍基。
参照实施例1的方法进行G3PAMAM-G(0.5g)与2,3-二甲基马来酸酐(0.67g)的反应，其他操作步骤同实施例1，得到的大分子用G3PAMAM-G-DMA表示。G3PAMAM-G-DMA在pH7.4时的ζ电位为-1.8mV，平均分子量为10.2kDa。
实施例15
0.5g BPEI 1.8k-G(来自实施例3)与0.72g环己烯-1,2-二羧酸酐进行反应，其他操作步骤同实施例1中的含胍基大分子与2,3-二甲基马来酸酐的反应，得到的大分子用BPEI 1.8k-G-DCA表示。BPEI 1.8k-G-DCA在pH7.4时的ζ电位为-2.2mV，平均分子量为8565Da。
实施例16
用顺式乌头酸酐(0.36g)替代实施例15中的环己烯-1,2-二羧酸酐，其他操作步骤同实施例15，得到的大分子用BPEI 1.8k-G-CAA表示。BPEI 1.8k-G-CAA在pH 7.4时的ζ电位为-8.0mV，平均分子量为7125Da。
实施例17
用2-(2-羧乙基)-3-甲基马来酸酐(0.42g)替代实施例15中的环己烯-1,2-二羧酸酐，其他操作步骤同实施例15，得到的大分子用BPEI 1.8k-G-CDM表示。BPEI 1.8k-G-CDM在pH 7.4时的ζ电位为-4.8mV，平均分子量为7409Da。
实施例18
称取1g平均分子量为1800Da的支化聚乙烯亚胺(BPEI 1.8k)溶于200mL二氯甲烷中，在搅拌下加入11.7g Boc-Lys(Boc)-OH(11.7/346.4＝33.8mmol)、6.4g二异丙基碳二亚胺、6.8g 1-羟基苯并三唑和13.0g N,N-二异丙基乙胺，反应在30℃下进行24小时，过滤，滤液依次用1mol/L NaHCO 3、1mol/L NaHSO 4和饱和NaCl各洗2次，旋转蒸发除去大部分溶剂，用乙醚沉淀。沉淀溶于二氯甲烷中，搅拌下加入三氟乙酸，使最后的三氟乙酸/二氯甲烷的比例为9/1(体积比)，溶液在室温下搅拌12小时，旋转蒸发除去二氯甲烷，所得溶液用1mol/L NaHCO 3调节pH值到6-7，用截留分子量为100-500Da的透析袋透析，透析液冷冻干燥得到的产物用BPEI 1.8k-Lys表示。通过反应前后平均分子量(MALDI-TOF-MS)的变化估算出平均每一BPEI 1.8k-Lys大分子上引入22.7个赖氨酸。
参照实施例1的方法进行BPEI 1.8k-Lys与2,3-二甲基马来酸酐的反应，得到的大分子用BPEI 1.8k-Lys-DMA表示。BPEI 1.8k-Lys-DMA在pH 7.4时的ζ电位为-3.8mV，平均分子量为7428Da。
实施例19
以上制备的大分子促进抗癌药在肿瘤组织的聚集实验。采用6-8周龄、体重18-20g的雌性BALA/c裸鼠(购买自北京华阜康生物科技股份有限公司)作为实验动物。将200μL含有5×10 6个MCF-7细胞的生理盐水溶液皮下注射在裸鼠右前肢腋下。当肿瘤体积长到大约400mm 3时进行尾静脉注射(每组5只)：(1)阿霉素、(2)阿霉素+BPEI 0.4k-G-DMA、(3)阿霉素+BPEI 0.6k-G-DMA、(4)阿霉素+BPEI 1.8k-G-DMA、(5)阿霉素+BPEI 3.5k-G-DMA、(6)阿霉素+BPEI 5k-G-DMA、(7)阿霉素+BPEI 10k-G-DMA、(8)阿霉素+BPEI 1.8k-G-DCA、(9)阿霉素+BPEI 1.8k-G-CAA、(10)阿霉素+BPEI 1.8k-G-CDM、(11)阿霉素+LPEI 1.8k-G-DMA、(12)阿霉素+BPEI 1.8k-Lys-DMA、(13)阿霉素+G1Lys-G-DMA或(14)阿霉素+HBPL 2.4k-G-DMA。阿霉素和大分子的剂量都为10mg/kg体重。给药0.5小时后将小鼠处死，在10min内完成解剖，分别取其肿瘤、心脏、肝脏、脾、肺和肾，以0.1g/mL的比例加入组织匀浆 液(含1％SDS的20mmol/L的KH 2PO 4溶液，pH＝3.8)，将全部组织进行研磨，匀浆。取100μL匀浆液，加入250μL丙酮和100μL饱和ZnSO 4溶液，于37℃下培育15min，离心，取上层有机相并将其用氩气流吹干，用HPLC流动相溶解并用HPLC分析阿霉素的浓度。HPLC仪器为岛津的型号为Nexera HPLC System，色谱柱为Shim-pack XR-ODSIII(150mm×2mm粒径2.2μm)，荧光检测器(RF-20A)。流动相为乙腈(含0.1％TFA)：水(含0.1％TFA)＝36：64，洗脱速度为1mL/min，检测器激发/发射波长为498/593nm。阿霉素在肿瘤和不同器官中的分布见表1。
表1.阿霉素在肿瘤和器官中的分布
Figure PCTCN2020134363-appb-000005
(单独注射阿霉素时肿瘤和各器官中的值定义为1.00)
实施例20
参照实施例19的操作步骤，所不同的是采用的抗癌药为紫杉醇，大分子为G4DPLys-G-DMA，紫杉醇在肿瘤和不同器官中的分布见表2。
表2.紫杉醇在肿瘤和器官中的分布
Figure PCTCN2020134363-appb-000006
(单独注射紫杉醇时肿瘤和各器官中的值定义为1.00)
实施例21
参照实施例19的操作步骤，所不同的是采用的抗癌药为顺铂，大分子为G3PAMAM-G-DMA，解剖得到肿瘤组织和器官用热硝酸进行分解，用电感耦合等离子体质谱(Thermo Elemental的型号为IRIS intrepid II XSP的ICP-MS)分析铂的含量，然后换算成顺铂的含量，得到的顺铂在肿瘤和不同器官中的分布见表3。
表3.顺铂在肿瘤和器官中的分布
Figure PCTCN2020134363-appb-000007
(单独注射顺铂时肿瘤和各器官中的值定义为1.00)
实施例22
通过激光共聚焦显微镜(Nikon的型号为A1R+显微镜)逐层扫描3D细胞球的方法研究大分子对抗癌药在细胞球(模拟肿瘤组织)中的渗透性能。将100μL完全培养基(含有10％胎牛血清和1％盘尼西林/链霉素的RPMI-1640培养基)稀释的人乳腺癌细胞MCF-7悬液按照1×10 4/mL的细胞浓度加入到琼脂涂层的细胞孔板中，在37摄氏度下培养24小时以形成3D细胞球。随后将细胞球吸出转移至激光共聚焦用玻底培养皿中，用PBS溶液洗涤细胞两次，然后加入2mLpH为6.5的无血清培养基稀释的(1)阿霉素(4μg/mL)、(2)阿霉素(4μg/mL)+BPEI 0.6k-G-DMA(25μg/mL)、(3)阿霉素(4μg/mL)+BPEI 1.8k-G-DMA(25μg/mL)、(4)阿霉素(4μg/mL)+BPEI 3.5k-G-DMA(25μg/mL)、(5)阿霉素(4μg/mL)+BPEI 5k-G-DMA(25μg/mL)或(6)阿霉素(4μg/mL)+BPEI 1.8k-Lys-DMA，继续培养2小时。随后吸出溶液，用PBS溶液清洗3次后，加入1mL PBS，在激光共聚焦显微镜下逐层扫描3D细胞球，拍摄其在不同层面的照片，如图1所示。由图可知，在模拟肿瘤微酸性环境(pH 6.5)下，聚合物可促进阿霉素在细胞球内的渗透。由图1可见，阿霉素与大分子共孵育的情况下，细胞球均显示出比单独用阿霉素孵育时更强和更均匀的荧光。其中加入BPEI 1.8k-G-DMA的组显示最强、且最均匀的荧光，而结构类似的含BPEI 1.8k-Lys-DMA的组则显示出最弱的荧光(与只加阿霉素显示的荧光强度相近)，且更多分布在细胞球表面。BPEI 0.6k-G-DMA和BPEI 3.5k-G-DMA组的荧光强度略低于BPEI 1.8k-G-DMA组，但显著高于BPEI 5k-G-DMA组。
实施例23
参照实施例22的操作步骤，所不同的是：用结直肠癌细胞SW480培养细胞球，研究HBPL 2.4k-G-DMA对阿霉素在细胞球中的渗透，结果见图2。同样的，与仅加入阿霉素的组相比，含有本发明大分子HBPL 2.4k-G-DMA的组，细胞球显示出明显更强和更均匀的荧光强度。
实施例24
参照实施例22的方法，用BxPC-3细胞培养细胞球，研究G4DPLys-G-DMA对阿霉素在细胞球中的渗透，结果见图3。同样的，与仅加入阿霉素的组相比，含有本发明大分子G4DPLys-G-DMA的组，细胞球显示出明显更强和更均匀的荧光强度。
实施例25
参照实施例22的方法，用PANC-1细胞培养细胞球，研究G1Lys-G-DMA对阿霉素在细胞球中的渗透，结果见图4。同样的，与仅加入阿霉素的组相比，含有本发明大分子G1Lys-G-DMA的组，细胞球显示出明显更强和更均匀的荧光强度。
实施例26
大分子促进抗癌药在肿瘤组织中渗透的动物实验，参照实施例19造裸鼠异种移植瘤，当肿瘤体积长到大约400mm 3时，通过尾静脉注射(1)阿霉素、(2)阿霉素+bPEI 1.8k-G、(3)阿霉素+BPEI 1.8k-G-DMA、(4)阿霉素+BPEI 1.8k-Lys-DMA、(5)阿霉素+bPEI 0.6k-G-DMA、(6)阿霉素+G4DPLys-G-DMA，阿霉素的剂量为1mg/kg，大分子的剂量为10mg/kg，0.5小时后将实验鼠处死，解剖取出肿瘤后，对所得的肿瘤进行冷冻切片，血管用CoraLite 594标记的CD34抗体染色，在激光共聚焦显微镜下观察CD34和阿霉素的荧光信号，结果如图5所示。由图5可见，只注射阿霉素时肿瘤组织中的阿霉素主要分布于血管周围，而同时注射阿霉素和本发明的大分子时阿霉素更易渗透到远离血管的部位，而且分布较均匀。只含胍基但不含羧基的大分子bPEI 1.8k-G对阿霉素渗透的促进作用很小，但引入羧基后的本发明的大分子(BPEI 1.8k-G-DMA)对阿霉素渗透的促进作用非常显著。不含胍基只含胺基和羧基的结构类似的大分子(BPEI 1.8k-Lys-DMA)对阿霉素渗透的促进作用并不明显。
实施例27
通过激光共聚焦显微镜照片研究大分子促进抗癌药进入癌细胞的实验，将1mL完全培养基(含有10％胎牛血清和1％盘尼西林/链霉素的RPMI-1640培养基)稀释的MCF-7细胞悬液按照1×10 4/mL的细胞浓度加入到玻底培养皿中，在37摄氏度下培养24小时。随后吸出培养基，用PBS洗涤细胞两次，再分别加入1mL不同pH(6.5，6.8或7.4)无血清培养基稀释的浓度为10μg/mL的BPEI 1.8k-G-DMA和1μg/mL的阿霉素的混合溶液，作为对照1μg/mL的阿霉素分别加入其他培养皿中，培养2小时。随后吸出溶液，用PBS溶液清洗3次后，加入0.5mL PBS，细胞的激光共聚焦显微镜照片如图6所示。结果表明在模拟肿瘤组织微酸性(pH 6.8和6.5)的环境下BPEI 1.8k-G-DMA 可促进阿霉素进入癌细胞。
实施例28
通过细胞流式实验研究大分子促进抗癌药进入癌细胞的实验，将2mL完全培养基(含有10％胎牛血清和1％盘尼西林/链霉素的RPMI-1640培养基)稀释的癌细胞悬液按照每孔2×10 5个细胞的浓度加入到6孔板中，在37摄氏度培养24小时。之后，癌细胞与阿霉素(2μg/mL)或阿霉素(2μg/mL)+大分子(10μg/mL)在pH 6.5的无血清培养基中培养2小时，用PBS清洗后用流式细胞仪(Dickinson and Company的型号为FACS Calibur流式细胞仪)进行分析，具体的癌细胞和大分子的种类，以及结果见表4。
表4.细胞流式实验结果
Figure PCTCN2020134363-appb-000008
*HepG2，人肝癌细胞；Hela，人宫颈癌细胞；BxPC-3，人胰腺癌细胞；SW480，结直肠癌；MCF-7，人乳腺癌细胞；PANC-1，人胰腺癌细胞
实施例29
大分子促进抗癌药的抑瘤动物实验，参照实施例19造裸鼠异种移植瘤，当肿瘤体积长到大约150mm 3时，将荷瘤小鼠随机分成6组(每组5只)，分别腹腔注射：(1)生理盐水、(2)BPEI 1.8k-G-DMA(10mg/kg)、(3)阿霉素(1mg/kg)、(4)阿霉素(5mg/kg)、(5)阿霉素(1mg/kg)+BPEI 1.8k-G-DMA(10mg/kg)、(6)阿霉素(5mg/kg)+BPEI 1.8k-G-DMA(10mg/kg)、或阿霉素(5mg/kg)+BPEI 1.8k-Lys-DMA(10mg/kg)，先注射大分子，30分钟后再注射阿霉素，第0天和第7天各注射一次，肿瘤体积变化见图7。在第15天将裸鼠处死，解剖得到的肿瘤的平均重量见表5。
表5.肿瘤重量
组别 肿瘤重量(g)
生理盐水 1.33±0.05
BPEI 1.8k-G-DMA(10mg/kg) 1.29±0.07
阿霉素(1mg/kg) 0.97±0.03
阿霉素(5mg/kg) 0.74±0.03
阿霉素(1mg/kg)+BPEI 1.8k-G-DMA(10mg/kg) 0.65±0.01
阿霉素(5mg/kg)+BPEI 1.8k-G-DMA(10mg/kg) 0.28±0.01
阿霉素(5mg/kg)+BPEI 1.8k-Lys-DMA(10mg/kg) 0.55±0.10
实施例30
参照实施例29进行抑瘤动物实验，所不同的是，采用PC-3进行异种移植肿瘤，采用的大分子为BPEI 0.6k-G-DMA，剂量为10mg/kg，采用的抗癌药为顺铂，剂量为5mg/kg，注射后的第15天的肿瘤的平均重量见表6。
表6.抑癌作用
Figure PCTCN2020134363-appb-000009
实施例31
参照实施例29进行抑瘤动物实验，所不同的是，采用BxPC-3进行异种移植肿瘤，采用的大分子为HBPL 0.9k-G-DMA，剂量为20mg/kg，采用的抗癌药为吉西他滨，剂量为25mg/kg，注射后的第15天的肿瘤的平均重量见表6。
实施例32
参照实施例29进行抑瘤动物实验，所不同的是，采用人卵巢癌细胞SKOV3进行异种移植肿瘤，采用的大分子为G4DPLys-G-DMA，剂量为10mg/kg，采用的抗癌药为紫杉醇，剂量为10mg/kg，注射后的第15天的肿瘤的平均重量见表6。
实施例33
参照实施例29进行抑瘤动物实验，所不同的是，采用人肺癌细胞A549进行异种移植肿瘤，采用的大分子为G3PAMAM-G-DMA，剂量为10mg/kg，采用的抗癌药为伊立替康，剂量为10mg/kg，注射后的第15天的肿瘤的平均重量见表6。
以上所述仅为本发明的优选实施方式，并非因此限制本发明的专利范围，凡是在本发明的发明构思下，利用本发明说明书及附图内容所作的等效结构变换，或直接/间接运用在其他相关的技术领域均包括在本发明的专利保护范围内。

Claims (20)

  1. 一种大分子，所述大分子具有多个胍基且可选地具有胺基，其中至少部分胺基和/或胍基被下式I所示的取代基所取代而形成酰胺键，其中所述大分子具有小于等于50,000Da的平均分子量，并且在正常生理条件下所述大分子的ζ电位为-15mV～+5mV，以及所述大分子在微酸性条件下水解后具有5～100个胍基，
    Figure PCTCN2020134363-appb-100001
    其中R 1和R 2可各自独立地选自由羧基、取代或未取代的C1～C6的烷基所组成的组，或者R 1和R 2相互连接为取代或未取代的亚烷基并和与其连接的碳原子共同形成五元或六元环所组成的组，其中所述取代为被1～2个羧基所取代。
  2. 根据权利要求1所述的大分子，其中R 1和R 2可各自独立地选自由羧基、取代或未取代的C1～C3的烷基所组成的组，或者R 1和R 2相互连接为亚丁基并和与其连接的碳原子共同形成六元环，其中所述取代为被1个羧基所取代。
  3. 根据权利要求1或2所述的大分子，其中所述大分子在微酸性条件下水解后具有10～60个胍基，优选具有15～40个胍基。
  4. 根据权利要求1或2所述的大分子，其中所述大分子具有1,500～40,000Da的平均分子量，优选具有2,500～25,000Da的平均分子量，更优选具有3,000～15,000Da。
  5. 根据权利要求1或2所述的大分子，其中所述大分子的ζ电位为-10mV～+2mV，更优选-8mV～0mV。
  6. 根据权利要求1或2所述的大分子，其中所述大分子中的胍基通过使含胺基的大分子中的至少部分胺基胍基化或与具有胍基的化合物反应而引入所述大分子中。
  7. 根据权利要求6所述的大分子，其中所述含胺基的大分子为含胺基的线形大分子或含胺基的支化大分子；
    优选地，所述含胺基的大分子选自带有胺基侧链的聚烯烃类、侧链带有胺基的聚氨基酸类和氨基化的超支化聚甘油中的至少一种；更优选地，所述含胺基的线形大分子选自聚乙烯胺、聚烯丙基胺和线形聚乙烯亚胺；所述含胺基的支化大分子选自支化聚乙烯亚胺、超支化聚赖氨酸、树枝状聚酰胺-胺、树枝状聚丙烯亚胺、树枝状聚赖氨酸和氨基化的超支化聚甘油。
  8. 根据权利要求6所述的大分子，其中所述使含胺基的大分子中的至少部分胺基与具有胍基的化合物反应包括使含胺基的大分子中的至少部分胺基与含有胍基和羧基的化合物进行酰胺化反应，优选地所述含有胍基和羧基的化合物选自胍基乙酸盐酸盐、3-胍基丙酸盐酸盐、α-氨基和胍基双保护的精氨酸；和
    使含胺基的大分子中的至少部分胺基胍基化通过胍基化试剂进行反应，优选地所述胍基化试剂选自单氰胺、O-甲基异脲、硫脲、S-甲基异硫脲、N,N-二(叔丁氧羰酰基) 硫脲、1H-吡唑-1-甲脒盐酸盐和3,5-二甲基-1-吡唑硝酸甲脒。
  9. 一种制备根据权利要求1～8中任一项所述的大分子的方法，所述方法包括：
    在含胺基的大分子中引入胍基，和
    进一步与式II所示的化合物反应得到所述大分子，
    Figure PCTCN2020134363-appb-100002
    其中R 1和R 2可各自独立地选自由羧基、取代或未取代的C1～C6的烷基所组成的组，或者R 1和R 2相互连接为取代或未取代的亚烷基并和与其连接的碳原子共同形成五元或六元环所组成的组，其中所述取代为被1～2个羧基所取代。
  10. 根据权利要求9所述的方法，其中，R 1和R 2可各自独立地选自由羧基、取代或未取代的C1～C3的烷基所组成的组，或者R 1和R 2相互连接为亚丁基并和与其连接的碳原子共同形成六元环，其中所述取代为被1个羧基所取代。
  11. 根据权利要求9或10所述的方法，其中所述含胺基的大分子为含胺基的线形大分子或含胺基的支化大分子；
    优选地，所述含胺基的大分子选自带有胺基侧链的聚烯烃类、侧链带有胺基的聚氨基酸类和氨基化的超支化聚甘油中的至少一种；
    更优选地，所述含胺基的线形大分子选自聚乙烯胺、聚烯丙基胺和线形聚乙烯亚胺；所述含胺基的支化大分子选自支化聚乙烯亚胺、超支化聚赖氨酸、树枝状聚酰胺-胺、树枝状聚丙烯亚胺、树枝状聚赖氨酸和氨基化的超支化聚甘油。
  12. 根据权利要求9或10所述的方法，其中所述在含胺基的大分子中引入胍基包括：使所述含胺基的大分子与含有胍基和羧基的化合物反应以与所述含有胺基的大分子中胺基形成酰胺键，或者使所述含有胺基的大分子与胍基化试剂反应。
  13. 根据权利要求12所述的方法，其中所述含有胍基和羧基的化合物选自胍基乙酸盐酸盐、3-胍基丙酸盐酸盐、α胺基和胍基双保护的精氨酸；所述胍基化试剂选自单氰胺、O-甲基异脲、硫脲、S-甲基异硫脲、N,N-二(叔丁氧羰酰基)硫脲、1H-吡唑-1-甲脒盐酸盐和3,5-二甲基-1-吡唑硝酸甲脒。
  14. 根据权利要求9所述的方法，其中所述式II所示的化合物选自2,3-二甲基马来酸酐、环己烯-1,2-二羧酸酐、顺乌头酸酐和2-(2-羧乙基)-3-甲基马来酸酐。
  15. 一种药物组合物，所述药物组合物包括根据权利要求1～8中任一项所述的大分子。
  16. 根据权利要求15所述的药物组合物，其中所述药物组合物进一步包括至少一种抗癌药物，优选地，所述抗癌药物选自抗代谢药、烷化剂、抗微管药、抗癌抗生素和它们的组合，更优选地，所述抗代谢药选自5-氟尿嘧啶、甲氨蝶呤和吉西他滨；所述烷化剂选自环磷酰胺、苯丁酸单芥和铂类抗癌药；所述抗微管药选自紫杉醇、长春碱、长 春新碱、喜树碱和伊立替康；所述抗癌抗生素选自阿霉素、吡柔比星和表柔比星。
  17. 根据权利要求15或16所述的药物组合物，其中所述药物组合物进一步包括药学上可接受的赋形剂和/或载剂。
  18. 根据权利要求15～17中任一项所述的药物组合物在制备治疗癌症的药物中的用途。
  19. 根据权利要求18所述的用途，其中所述治疗包括根据权利要求1～8中任一项所述的大分子促进抗癌药物在肿瘤组织聚集并向肿瘤组织内部渗透。
  20. 根据权利要求18或19所述的用途，其中所述癌症为恶性实体瘤，优选地，所述恶性实体瘤选自肝癌、肺癌、胃癌、肠癌、卵巢癌、乳腺癌、胰腺癌、前列腺癌和宫颈癌。
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