WO2024041532A1 - 含四价铂的配合物、前药及其制备方法和应用 - Google Patents

含四价铂的配合物、前药及其制备方法和应用 Download PDF

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WO2024041532A1
WO2024041532A1 PCT/CN2023/114257 CN2023114257W WO2024041532A1 WO 2024041532 A1 WO2024041532 A1 WO 2024041532A1 CN 2023114257 W CN2023114257 W CN 2023114257W WO 2024041532 A1 WO2024041532 A1 WO 2024041532A1
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cancer
platinum
complex
self
tryptophan
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PCT/CN2023/114257
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English (en)
French (fr)
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石凯
毕洪书
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赣州和美药业股份有限公司
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Priority claimed from CN202211006334.0A external-priority patent/CN115554413A/zh
Priority claimed from CN202211005108.0A external-priority patent/CN115368436A/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • 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
    • A61K47/50—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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0819—Tripeptides with the first amino acid being acidic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0827—Tripeptides containing heteroatoms different from O, S, or N
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present disclosure relates to the field of biomedicine technology, and in particular to a complex containing tetravalent platinum, a prodrug assembled from the precursor, and preparation methods and applications of the complex and prodrug, such as a peptidyl platinum-based self-containing complex. Assembled nanoprodrugs and their preparation methods and applications.
  • Bivalent platinum drugs such as cisplatin, carboplatin, oxaliplatin
  • Bivalent platinum drugs have strong broad-spectrum anti-cancer activity. They mainly exert anti-tumor effects by destroying the DNA of tumor cells, inhibiting replication and inducing apoptosis. It has been widely used in the clinical treatment of various malignant tumors.
  • this class of drugs has the physical and chemical characteristics of low solubility and poor stability, and lacks selectivity for tumor tissues. It can easily lead to serious side effects such as nephrotoxicity, neurotoxicity, and bone marrow suppression, as well as the risk of innate and acquired drug resistance. impact, seriously limiting its clinical efficacy and application.
  • the tetravalent platinum precursor complex Pt(IV) formed by adding two axial ligands has lower reactivity and toxic side effects. After entering the tumor cells, it will Under the action of internal reducing mediators such as ascorbic acid, methionine, glutathione, etc., the axial ligand can be shed and converted into a cytotoxic Pt(II) structure, thereby producing cell killing effect.
  • platinum-based small molecule prodrugs also have limitations in the human body such as fast metabolism, poor accumulation ability in the lesion site, and easy development of drug resistance.
  • Nano-drug-carrying systems constructed based on various carrier materials show great potential application value and development prospects in realizing effective transportation of chemotherapy drugs in the body. They can carry drugs to overcome physiological and pathological barriers, and realize the delivery of drugs to tumor tissues and cells. Effective enrichment to achieve sustained, controllable and targeted delivery, thereby achieving the purpose of enhancing therapeutic effects and reducing adverse reactions.
  • platinum-based small molecules are metal drugs that are poorly soluble in oil and water. They have weak affinity for most drug carriers and have limited payload capacity. Therefore, it is necessary to structurally modify existing platinum drugs and develop multifunctional platinum prodrugs with one or more of the following: high anti-tumor activity, low toxic and side effects, strong selectivity for tumor tissue and controllable release. Thereby improving the clinical application potential of this type of drugs.
  • a complex comprising a tetravalent platinum moiety and an oligopeptide as an axial ligand chemically linked to a tetravalent platinum moiety, wherein the oligopeptide can be dipeptide Xaa1-Xaa4, tripeptide Xaa1-Xaa2-Xaa4 or tetrapeptide Xaa1-Xaa2-Xaa3-Xaa4, wherein Xaa1, Xaa2 and Xaa3 each represent any natural amino acid, and Xaa4 represents tryptophan, phenylalanine, histidine, tyrosine or arginine.
  • the tetravalent platinum moiety in the complex is represented by the following structural formula (a):
  • R 1 , R 2 , R 3 , R 4 and R 5 can be independently selected from: Cl, Br, I, OH, NH 3 ; R 1 and R 2 can form a ring with Pt, and R 3 and R 4 can form a ring with Pt. Pt forms a ring.
  • R 1 and R 2 or R 3 and R 4 can be selected in combination from
  • the tetravalent platinum moiety in the complex is represented by the following structural formulas (a-1) to (a-6):
  • Xaa1 can represent glutamic acid, aspartic acid, tryptophan, proline, tyrosine, or phenylalanine.
  • Xaa1 in the dipeptide can represent glutamic acid, aspartic acid, tryptophan, or tyrosine.
  • Xaa1 in the tripeptide can represent aspartic acid, tryptophan, proline, phenylalanine, or glutamic acid.
  • Xaa1 in the tetrapeptide may represent glutamic acid, tryptophan, or aspartic acid.
  • Xaa2 can represent tryptophan, glycine, alanine, valine, leucine, phenylalanine, proline, serine, threonine, cysteine, tyrosine , methionine, asparagine, glutamine, aspartic acid, glutamic acid, histidine or arginine.
  • Xaa2 in the tripeptide can represent tryptophan, glycine, alanine, valine, leucine, phenylalanine, proline, serine, threonine, cysteine Acid, tyrosine, methionine, asparagine, glutamine, Aspartic acid, glutamic acid or histidine.
  • Xaa2 in the tetrapeptide can represent tryptophan, glycine, valine, leucine, glutamic acid, phenylalanine, proline, aspartic acid, threonine, Histidine, tyrosine or arginine.
  • Xaa3 can represent tryptophan, glycine, leucine, phenylalanine, proline, histidine, aspartic acid, lysine, tyrosine, or arginine.
  • two or three of Xaa2, Xaa3, and Xaa4 in the tetrapeptide are the same.
  • Xaa2 or Xaa3, when present, is the same as Xaa4.
  • Xaa2 and Xaa4 can both be tryptophan
  • Xaa3 and Xaa4 can both be tryptophan.
  • the tetravalent platinum moiety is chemically linked to any one of Xaa1 to Xaa3, for example, to Xaa1, Xaa2, or Xaa3.
  • the tetravalent platinum moiety is chemically linked to Xaa1.
  • the oligopeptide is represented by Xaa1-Xaa2-Xaa4, where Alanine, tryptophan or tyrosine, Xaa4 represents tryptophan.
  • the oligopeptide is represented by .
  • the oligopeptides in the complex can be selected from the compounds shown in Table 1 below:
  • the complex is represented by Formula (I):
  • R 1 , R 2 , R 3 , R 4 and R 5 can be independently selected from: Cl, Br, I, OH, NH 3 ; R 1 and R 2 can form a ring with Pt, and R 3 and R 4 Can form a ring with Pt.
  • R 1 and R 2 or R 3 and R 4 can be selected in combination from
  • the complex may be selected from any of the following:
  • the complex may be selected from any of the following:
  • a peptidyl platinum prodrug comprising a tetravalent platinum complex having an axial ligand containing a tryptophan dipeptide.
  • This embodiment provides a peptide-based platinum self-assembled nanoprodrug, characterized in that the axial ligand contains a tetravalent platinum complex containing tryptophan dipeptide.
  • the structure of the self-assembled nanoprodrug is as shown in Formula I:
  • R 1 , R 2 , R 3 , R 4 and R 5 can be independently selected from: Cl, Br, I, OH, NH 3 ; R 1 and R 2 can form a ring with Pt, and R 3 and R 4 Can form a ring with Pt.
  • R 1 and R 2 or R 3 and R 4 can be selected in combination from
  • the self-assembled nanoprodrug has the structure shown in any one of Formulas II-XIII;
  • the prodrug can self-assemble into nanostructures with a particle size of 10-1000 nm, preferably 20-200 nm, in an aqueous medium.
  • the aqueous medium in which the prodrug is capable of self-assembly has a pH of 7.0-7.8.
  • the aqueous medium in which the prodrug can self-assemble is preferably purified water, water for injection, 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer, tris (Tris) buffer or PBS Buffer.
  • HEPES 4-hydroxyethylpiperazineethanesulfonic acid
  • Tris tris buffer or PBS Buffer.
  • the preparation method of peptidyl platinum prodrugs includes the following steps: oxidizing the divalent platinum complex Pt(II) with chlorosuccinimide to the monochloromonohydroxytetravalent platinum complex Pt ( IV), then in benzo Under the catalysis of triazoletetramethyltetrafluoroborate (TBTU) and triethylamine, it is condensed with oligopeptides to obtain peptidyl platinum prodrugs.
  • TBTU triazoletetramethyltetrafluoroborate
  • a method for preparing self-assembled nanoprodrugs includes: dissolving peptide platinum prodrugs in a first solvent to obtain organic phase A; mixing organic phase A and aqueous phase B to obtain mixed solution C; removing Mix the first solvent in solution C to obtain monodispersed nanoprodrugs.
  • the first solvent is one or a mixture of at least two of acetone, ethanol, methanol, tetrahydrofuran, acetonitrile, dimethyl sulfoxide and N,N-dimethylformamide; preferably, the The first solvent is acetone and/or ethanol; the water phase B is purified water, water for injection, 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer, tris (Tris) buffer or PBS Buffer; preferably, the water phase B is water for injection or HEPES buffer; preferably, the pH value of the water phase B is 7.0-7.8.
  • HEPES 4-hydroxyethylpiperazineethanesulfonic acid
  • Tris Tris
  • the volume ratio of aqueous phase B and organic phase A when mixed is 1-100:1, preferably 1-40:1.
  • the solvent removal method in the mixed solution C is a vacuum evaporation method, a high-speed centrifugation method, a dialysis method or an ultrafiltration method; preferably, a vacuum evaporation method is used to remove the solvent in the mixed solution C.
  • the application of peptide-based platinum self-assembled nanoprodrugs in the preparation of anti-tumor drugs is provided.
  • the anti-tumor drugs can be used for lung cancer, breast cancer, gastric cancer, esophageal cancer, adrenocortical cancer, skin squamous cell carcinoma, head and neck cancer, thyroid cancer, liver cancer, pancreatic cancer, bile duct cancer, colorectal cancer, ovarian cancer, Cervical cancer, endometrial cancer, vaginal squamous cell carcinoma, testicular cancer, prostate cancer, bladder cancer, urothelial cancer, melanoma, osteosarcoma, malignant lymphoma, neuroblastoma, cisplatin-acquired resistance to lung cancer , cisplatin-acquired resistance to ovarian cancer, cisplatin-acquired resistance to breast cancer, cisplatin-acquired resistance to cervical cancer, cisplatin-acquired
  • a self-assembly body is provided, which self-assembly body is self-assembled from the above-described complex or the above-described peptidyl platinum prodrug.
  • the self-assembly is self-assembled from the complex described above or the peptidyl platinum prodrug described above in an aqueous medium.
  • the self-assembly has a particle size of 10 nm to 10,000 nm.
  • the self-assembly has a particle size of 20 nm to 200 nm.
  • a method for preparing the complex described above comprising: oxidizing divalent platinum Pt(II) to hydroxytetravalent platinum Pt(IV), and then reacting with it in the presence of a catalyst. Oligopeptides are condensed to obtain the complex.
  • the oxidizing agent can be chlorosuccinimide.
  • the catalyst may be TBTU, triethylamine, or a combination thereof.
  • a method for preparing the self-assembly described above comprising dissolving the complex described above in a polar solvent to obtain an organic phase; and mixing the organic phase with an aqueous phase. A solution is obtained; the polar solvent in the solution is removed to obtain the self-assembly.
  • the polar solvent may be one or a mixture of at least two of acetone, ethanol, methanol, tetrahydrofuran, acetonitrile, dimethyl sulfoxide and N,N-dimethylformamide.
  • the polar solvent is acetone and/or ethanol.
  • the aqueous phase can be one or a combination of one or more of purified water, water for injection, HEPES buffer, Tris buffer, or PBS buffer. Zaiyou In selected embodiments, the aqueous phase is water for injection or HEPES buffer.
  • the pH of the aqueous phase is 7.0-7.8.
  • the aqueous phase and the organic phase are mixed in a volume ratio of 1-100:1. In other embodiments, the aqueous phase and the organic phase are mixed in a volume ratio of 1-40:1.
  • the solvent in the solution is removed by evaporation under reduced pressure, high-speed centrifugation, dialysis or ultrafiltration. In other embodiments, the solvent in the solution is removed by evaporation under reduced pressure.
  • some embodiments provide pharmaceutical compositions comprising the complexes, peptidyl platinum prodrugs or self-assemblies described above.
  • the pharmaceutical composition includes a therapeutically effective amount of a pharmaceutical composition of the complex, peptidyl platinum prodrug or self-assembly described above.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.
  • some embodiments provide the complexes, peptidyl platinum prodrugs, or self-assemblies described above for use in the treatment of tumors.
  • Other embodiments provide the use of the complexes, peptidyl platinum prodrugs or self-assemblies described above in the preparation of anti-tumor drugs.
  • Still other embodiments provide methods of treating tumors, comprising administering to a subject in need thereof a therapeutically effective amount of a complex, peptidyl platinum prodrug, or self-assembly as described above.
  • Figure 1 is a transmission electron microscope morphological photograph of the nanoprodrug prepared in Example 5;
  • Figure 2 is a particle size distribution diagram of the nanoprodrug prepared in Example 5;
  • Figure 3 is a particle size distribution diagram of the nanoprodrug prepared in Example 6;
  • Figure 4 is a particle size distribution diagram of the nanoprodrug prepared in Example 7;
  • Figure 5 is the active ingredient release curve of the nanoprodrug prepared in Example 5.
  • Figure 6 is a particle size distribution change diagram of the nanoprodrug prepared in Example 5.
  • Figure 7 is the in vitro toxicity curve based on human ovarian cancer (SKOV3) of the nanoprodrug and cisplatin prepared in Example 5;
  • Figure 8 is the in vitro toxicity curve of the nanoprodrug and cisplatin prepared in Example 5 based on the human ovarian cancer cisplatin-resistant strain (SKOV3/DDP);
  • Figure 9 is the in vivo anti-tumor efficacy evaluation of the nanoprodrug and cisplatin prepared in Example 5;
  • Figure 10 is the in vivo anti-tumor efficacy evaluation of the nanoprodrug and cisplatin prepared in Example 5;
  • Figure 11 is the pharmacokinetic curve of the nanoprodrug and cisplatin prepared in Example 5.
  • Figure 12 is the tissue distribution of the nanoprodrug and cisplatin prepared in Example 5;
  • Figure 13 is the body weight change of the in vivo safety evaluation of the nanoprodrug prepared in Example 5;
  • Figure 14 is H&E staining of tissue sections for in vivo safety evaluation of the nanoprodrug prepared in Example 5. color.
  • Example embodiments will now be described more fully below; however, they may be embodied in different forms and should not be construed as limited to the manner shown herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the exemplary embodiments to those skilled in the art.
  • prodrug is intended to mean a substance that can be converted to a biologically active agent under physiological conditions or by solvolysis.
  • the prodrug may be inactive when administered to a patient in need thereof, but is converted to an active agent in the body.
  • Prodrugs are often rapidly transformed in the body, for example by hydrolysis in the blood.
  • subject may include humans and domestic animals, such as laboratory animals and household pets (eg, cats, dogs, pigs, cattle, sheep, goats, horses, rabbits), as well as non-domesticated animals, such as wild animals, and the like.
  • laboratory animals and household pets eg, cats, dogs, pigs, cattle, sheep, goats, horses, rabbits
  • non-domesticated animals such as wild animals, and the like.
  • a “therapeutically effective amount” means an amount that, when administered to a mammal, preferably a human, is sufficient to effect treatment of a tumor in the mammal, as defined below. What constitutes a “therapeutically effective amount” varies depending on the substance administered, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by one of ordinary skill in the art based on his or her own knowledge and this disclosure. And decide.
  • “Pharmaceutically acceptable excipients” as used herein refer to add-ons other than the main drug in pharmaceutical preparations that are approved by the national drug regulatory agency as acceptable for use in humans or livestock, and may also be called excipients.
  • excipients for example, binders, fillers, disintegrants, and lubricants in tablets; matrix parts in semi-solid preparations; solvents, preservatives, antioxidants, flavorings, aromatics, etc. in liquid preparations.
  • treating encompasses the treatment of a tumor in a mammal, preferably a human, and includes: (i) preventing the occurrence of tumors in the mammal, particularly when such mammals are susceptible to When you have a tumor but have not been diagnosed as having a tumor; (ii) inhibit the tumor, that is, prevent its progression; (iii) alleviate the tumor, that is, cause the tumor to return; or (iv) alleviate the symptoms caused by the tumor.
  • tetravalent platinum complex means the tetravalent platinum moiety represented by the following structural formula (a), and therefore cannot be interchanged with peptidyl platinum prodrugs, etc., because the substances they refer to different.
  • natural amino acids refer to 20 amino acids that exist in nature, including alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, and histamine Acid, different Leucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Unless expressly stated otherwise, when referring to these amino acids, the L- and D-forms may be included.
  • the present disclosure provides a complex comprising a tetravalent platinum moiety represented by the following structural formula (a) and an oligopeptide as an axial ligand chemically linked to the tetravalent platinum moiety,
  • R 1 , R 2 , R 3 , R 4 and R 5 can be independently selected from: Cl, Br, I, OH, NH 3 ; R 1 and R 2 can form a ring with Pt, and R 3 and R 4 can form a ring with Pt. Pt forms a ring.
  • R 1 and R 2 or R 3 and R 4 can be selected in combination from
  • those skilled in the art can reasonably select R 1 , R 2 , R 3 , R 4 and R 5 according to the tetravalent platinum coordination principle.
  • R 1 , R 2 , R 3 , R 4 and R 5 may independently be Cl, Br, I, OH or NH 3 , or both of R 1 , R 2 , R 3 and R 4 can also be combined with each other to form
  • the tetravalent platinum moiety of the present disclosure is represented by the following structural formulas (a-1) to (a-6):
  • Oligopeptides of the present disclosure may be dipeptides Xaa1-Xaa4, tripeptides Xaa1-Xaa2-Xaa4, or tetrapeptides Xaa1-Xaa2-Xaa3-Xaa4.
  • the above-mentioned tetravalent platinum moiety can be chemically connected to each part of the oligopeptide.
  • the divalent platinum complex can be oxidized into a monochloromonohydroxytetravalent platinum moiety, and the hydroxyl group of the tetravalent platinum moiety is condensed with an amino acid to form a connection.
  • Xaa4 represents tryptophan, phenylalanine, histidine, tyrosine or arginine, preferably tryptophan, phenylalanine, histidine or tyrosine.
  • These amino acids can drive platinum through ⁇ - ⁇ stacking Parts self-assemble in aqueous media to form stable monodisperse nanostructures.
  • the tetravalent platinum moiety is chemically linked to Xaa1, where Xaa1 can be glutamic acid, aspartic acid, tryptophan, proline, tyrosine, or phenylalanine.
  • Xaa1 can be glutamic acid, aspartic acid, tryptophan, proline, tyrosine, or phenylalanine.
  • the tetravalent platinum moiety can also be chemically connected to Xaa2 or Xaa3, as long as its position does not hinder ⁇ - ⁇ stacking.
  • Xaa1 when the oligopeptide is a dipeptide, Xaa1 can be glutamic acid, aspartic acid, tryptophan, or tyrosine, and Xaa4 can be tryptophan, phenylalanine, histidine, Tyrosine or arginine.
  • Xaa1 can be aspartic acid, tryptophan, proline, phenylalanine or glutamic acid
  • Xaa2 can be tryptophan
  • Xaa4 can be tryptophan, phenylalanine, histidine or tyrosine.
  • Xaa1 may represent aspartic acid, tryptophan, proline, phenylalanine or glutamic acid
  • Xaa2 may represent histidine
  • Xaa4 may represent Tryptophan.
  • Xaa1 may represent aspartic acid, tryptophan, proline, phenylalanine or glutamic acid
  • Xaa2 and Xaa4 both represent tryptophan.
  • Xaa1 can be glutamic acid, tryptophan or aspartic acid
  • Xaa2 can be tryptophan
  • Xaa3 can be tryptophan, glycine, leucine, phenylalanine, proline, histidine , aspartic acid, lysine, tyrosine or arginine
  • Xaa4 can be tryptophan, phenylalanine, histidine or tyrosine.
  • Xaa2 can be the same as Xaa4, or Xaa1 can be the same as Xaa4.
  • Xaa2 and Xaa4 can both be tryptophan.
  • two or three of Xaa2, Xaa3 and Xaa4 are the same, for example, Xaa2 and Xaa3 are the same, or Xaa3 and Xaa4 are the same.
  • Xaa3 and Xaa4 can both be tryptophan.
  • Complexes of the present disclosure may be selected from any of the following:
  • the complex may be selected from any of the following:
  • the oligopeptide (such as tryptophan dipeptide) motif in the prodrug molecular structure can provide an aromatic indole ring as an assembly unit, driving the platinum complex in aqueous media through ⁇ - ⁇ stacking. Self-assembly forms stable monodisperse nanostructures.
  • the preparation method of peptidyl platinum prodrugs is characterized by comprising the following steps: oxidizing the divalent platinum complex Pt(II) with chlorosuccinimide into the monochloromonohydroxytetravalent platinum complex Pt(IV), Then, under the catalysis of TBTU and triethylamine, it is condensed with an oligopeptide containing a tryptophan dipeptide moiety to obtain a peptide-based platinum prodrug.
  • the prepared peptide-based platinum prodrug self-assembles into a nanostructure; the prodrug can self-assemble into a nanostructure with a particle size of 10-1000 nm, preferably 20-200 nm, in an aqueous medium.
  • the aqueous medium for self-assembly of prodrugs has a pH of 7.0-7.8.
  • the aqueous medium for self-assembly of prodrugs can be purified water, water for injection, HEPES buffer, Tris buffer or PBS buffer.
  • the self-assembly method is as follows:
  • the first solvent is acetone, ethanol, methanol, tetrahydrofuran, acetonitrile, dimethyl sulfoxide and N, N-dimethylmethane
  • the first solvent is acetone and/or ethanol
  • aqueous phase B is purified water, water for injection, HEPES buffer, Tris buffer or PBS buffer; preferably, the aqueous phase B is water for injection or HEPES buffer liquid; preferably, the pH value of the aqueous phase B is adjusted to 7.0-7.8; when the aqueous phase B and the organic phase A are mixed, the volume ratio is 1-100:1, preferably 1-40:1.
  • the solvent removal method in the mixed solution C is a reduced pressure evaporation method, a high-speed centrifugation method, a dialysis method or an ultrafiltration method; preferably, a reduced pressure evaporation method is used to remove the solvent in the mixed solution C. , monodisperse nanoprodrugs are obtained after removing the solvent;
  • Nano-form drugs have good stability and can avoid premature release of active divalent platinum complexes in the blood circulation, thereby reducing toxic side effects and Promote the selective accumulation of platinum drugs in tumor tissues and the controlled release within tumor cells, thereby improving the inhibitory effect on the growth of drug-resistant tumors.
  • the degradation products of the assembly units in the prodrug are all endogenous amino acids in the human body, and their application in tumor treatment can significantly reduce the toxic and side effects of platinum drugs.
  • the preparation method of the complex of the present disclosure may include oxidizing divalent platinum Pt(II) to hydroxytetravalent platinum Pt(IV), and then condensing it with an oligopeptide in the presence of a catalyst to obtain the complex.
  • the oxidizing agent may be chlorosuccinimide or the like
  • the catalyst may be TBTU, triethylamine or a combination thereof, and a combination of the two is preferably used.
  • the formation method of the self-assembly body of the present disclosure may include dissolving the complex in a polar solvent to obtain an organic phase; mixing the organic phase and the aqueous phase to obtain a solution; and removing the polar solvent in the solution to obtain the self-assembly body.
  • the polar solvent can be one or a mixture of at least two of acetone, ethanol, methanol, tetrahydrofuran, acetonitrile, dimethyl sulfoxide and N,N-dimethylformamide, preferably acetone and/or ethanol.
  • the aqueous phase can be one or a combination of purified water, water for injection, HEPES buffer, Tris buffer or PBS buffer, preferably water for injection or HEPES buffer.
  • the pH value of the aqueous phase is 7.0-7.8.
  • the volume ratio of the aqueous phase and the organic phase when mixed can be 1-100:1, preferably 1-40:1.
  • the removal method may be a reduced pressure evaporation method, a high speed centrifugation method, a dialysis method or an ultrafiltration method, with the reduced pressure evaporation method being preferred.
  • the self-assembly of the present disclosure can be used to prepare anti-tumor drugs suitable for, for example, treating the following conditions: Lung cancer, breast cancer, gastric cancer, esophageal cancer, adrenocortical cancer, cutaneous squamous cell carcinoma, head and neck cancer, thyroid cancer, liver cancer, pancreatic cancer, bile duct cancer, colorectal cancer, ovarian cancer, cervical cancer, endometrial cancer, vaginal cancer Squamous epithelial cancer, testicular cancer, prostate cancer, bladder cancer, urothelial cancer, melanoma, osteosarcoma, malignant lymphoma, neuroblastoma, cisplatin-acquired resistance to lung cancer, cisplatin-acquired resistance to ovarian cancer , cisplatin-acquired resistance to breast cancer, cisplatin-acquired resistance to cervical cancer, cisplatin-acquired resistance to gastric cancer, c
  • compositions of the present disclosure may comprise a complex, prodrug or self-assembly as described above, for example, a therapeutically effective amount of the complex, prodrug or self-assembly, and a pharmaceutically acceptable excipient.
  • Typical routes of administration of pharmaceutical compositions of the present disclosure include, but are not limited to, oral, topical, inhalation, parenteral, intranasal, intraocular, intramuscular, subcutaneous, intravenous administration.
  • the pharmaceutical composition of the present disclosure can be manufactured using methods well known in the art, such as conventional mixing methods, dissolving methods, granulation methods, sugar-coated pill making methods, grinding methods, emulsification methods, freeze-drying methods, etc.
  • the complexes, peptide-based self-assembled nanoprodrugs and self-assemblies described above are responsive to the tumor microenvironment, and their use in tumor treatment can promote the selective accumulation of platinum drugs in tumor tissues and the controlled release in tumor cells. , thereby improving the inhibitory effect on the growth of drug-resistant tumors;
  • the complexes, peptide-based self-assembled nanoprodrugs and self-assemblies described above can avoid the premature release of active divalent platinum complexes in the blood circulation, thereby reducing toxic and side effects;
  • the above-mentioned complexes, peptide-based self-assembled nanoprodrugs and self-assemblies have good biocompatibility and safety.
  • the assembly unit can contain one of the essential amino acids of the human body (such as tryptophan) and participate in body proteins. Synthesis and metabolism regulation, degradation products are all endogenous substances in the human body, and their application in tumor treatment can significantly reduce the toxic and side effects of platinum drugs;
  • the preparation conditions of the complexes, peptide-based self-assembled nanoprodrugs and self-assemblies described above are mild and simple to operate.
  • the production process does not involve harmful solvents, etc., and is suitable for industrial production.
  • H-Trp-Trp-OMe 243 mg, 0.6 mmol
  • N-methylmorpholine 87 ⁇ L, 0.78 mmol
  • Cbz-Glu(Obzl)-OH 223mg, 0.6mmol
  • HOBt 81mg, 0.6mmol
  • EDCI 150mg, 0.78mmol
  • Cbz-Glu(Obzl)-Trp-Trp-OMe was placed in a 50 mL round-bottomed flask, and 10 mL of methanol was used to dissolve the peptide.
  • the tetravalent platinum intermediate product (176 mg, 0.5 mmol), H-Glu-Trp-Trp-OMe (293 mg, 0.55 mmol), tetramethylurea tetrafluoroborate (TBTU, 264 mg, 0.82 mmol), triethylamine (84 mg, 0.82 mmol) was dissolved in dry dimethylformamide under nitrogen protection, and reacted at room temperature for 24 hours in the dark.
  • Trp-Trp-OMe 243 mg, 0.6 mmol
  • N-methylmorpholine 87 ⁇ L, 0.78 mmol
  • Fmoc-Glu(Obzl)-OH 268mg, 0.6mmol
  • HOBt 882mg, 0.6mmol
  • EDCI 150mg, 0.78mmol
  • dissolve in 10mL DMF activate in ice bath for 40min; add mixture 2, stir After 2 hours, the mixture was naturally raised to room temperature, and the reaction was continued with stirring for 12 hours, dialyzed in distilled water for 4 hours, and freeze-dried to obtain Fmoc-Glu(Obzl)-Trp-Trp-OMe.
  • Fmoc-Glu(Obzl)-Trp-Trp-OMe was placed in a 50 mL round-bottomed flask, and the peptide was dissolved in 10 mL of methanol. Add 180 mg of 5% Pd/C catalyst pre-suspended in 1.2 mL of methanol, add hydrogen while stirring for 6 hours, and filter. The filtrate was concentrated under reduced pressure, dialyzed in distilled water for 4 hours, and freeze-dried to obtain Fmoc-Glu-Trp-Trp-OMe.
  • the tetravalent platinum intermediate product (176 mg, 0.5 mmol), Fmoc-Glu-Trp-Trp-OMe (415 mg, 0.55 mmol), TBTU (264 mg, 0.82 mmol), and triethylamine (84 mg, 0.82 mmol) were dissolved under nitrogen protection.
  • Oxaliplatin (395 mg, 1 mmol) was reacted according to the synthesis method in Example 1 or Example 2 to obtain the product of structural formula (XIV) (yield 71%).
  • the NMR data for this product are shown below.
  • HEPES 4-hydroxyethylpiperazineethanesulfonic acid buffer
  • nanoprodrug The tetravalent peptide platinum (100 mg) prepared in Example 1 or Example 2 was dissolved in 1 mL of ethanol-acetone mixed solvent (1:1), and the resulting solution was injected into 100 mL of HEPES buffer (10 mM, pH 7. 4), continue stirring for 30 minutes after addition, and evaporate the solvent under reduced pressure to obtain a nanocolloid solution, which is a nanoself-assembled prodrug (hereinafter sometimes referred to as nanoprodrug).
  • the appearance of the obtained nanoprodrug is monodisperse spherical, as shown in Figure 1, and the particle size distribution is shown in Figure 2.
  • the average particle size is 118nm, and the PDI (Polymer Dispersibility Index) is 0.12.
  • Example 3 The tetravalent peptide platinum (10 mg) obtained in Example 3 was self-assembled according to the method of Example 5.
  • the particle size distribution of the obtained nano self-loading prodrug is shown in Figure 3, with an average particle size of 132 nm and a PDI of 0.18.
  • Example 4 The tetravalent peptide platinum (10 mg) obtained in Example 4 was self-assembled according to the method of Example 5, and the obtained sodium The particle size distribution of rice prodrug is shown in Figure 4. The average particle size is 121nm and the PDI is 0.09.
  • the nano self-assembled prodrug obtained in Example 5 was dispersed in 20 mL of buffer (10 mM, pH 7.4) containing reduced glutathione (GSH) at different concentrations, and 0.1 mL was sampled at specific time points, at 20,000 rpm high speed. Centrifuge for 30 minutes, draw 20uL supernatant, measure the drug concentration by HPLC, and draw a release curve.
  • the specific steps are as follows, and the results are shown in Figure 5.
  • the nanoprodrug is slowly released in a GSH-free release medium, with a cumulative release rate of less than 20% in 48 hours; in a release medium containing a higher concentration of GSH (5mM), the release of the active ingredient will be significantly accelerated, with a cumulative release rate of 80 in 48 hours. %. It can be seen from the in vitro release results that the nanoprodrug is responsive to the tumor microenvironment and can quickly release active ingredients under the influence of the reducing environment in tumor cells.
  • Mobile phase 0.9% sodium chloride; detection wavelength: 210nm; column temperature: 30°C; injection volume: 20 ⁇ L.
  • Cisplatin has a good linear relationship in the range of 2.5-100 ⁇ g/mL.
  • Figure 6 shows that the particle size distribution of the nanoparticles obtained in Example 5 did not change significantly in 10% FBS within 24 hours, indicating that the nanoparticles obtained in Example 5 will have better stability in blood circulation. .
  • the CCK8 method was used to evaluate the in vitro cytotoxicity of the nanoself-assembled prodrug prepared in Example 5.
  • the specific method is as follows: tumor cells in the logarithmic growth phase are seeded into a 96-well plate (5 ⁇ 10 3 /well). After the cells adhere to the wall, the culture medium is replaced with nano-self-assembly prodrugs, with cisplatin solution as the For the control group, after continuing to incubate at 37°C for 48 hours, add 10 ⁇ L of CCK8 reagent to each well. After incubating for 4 hours, measure the OD value of each well at a wavelength of 450 nm.
  • the half inhibitory concentration of the self-assembled nanoprodrug was equivalent to that of cisplatin solution, indicating that self-assembly of cisplatin into nanoprodrugs does not affect its The ability to inhibit the proliferation of tumor cells, especially after incubation with the platinum-resistant cell line SKOV3/DDP, the half inhibitory concentration of self-assembled nanoprodrugs is significantly lower than that of cisplatin solution, indicating that nanoprodrugs have advantages in alleviating tumor drug resistance. .
  • Example 10 Using the nano self-assembled prodrug prepared in Example 6 and using oxaliplatin solution as the control group, the human colon cancer HCT116 and its drug-resistant cell line HCT116/L-OHPD were tested according to the method in Example 10. The half inhibitory concentration (IC50). The in vitro toxicity results of the prepared self-assembled nanoprodrugs on tumor cells are shown in Table 3.
  • Example 7 Using the nano self-assembled prodrug prepared in Example 7, and using carboplatin solution as the control group, the half inhibition of human ovarian cancer SKOV3 and its platinum-resistant cell line SKOV3/DDP was tested according to the method in Example 11. concentration (IC50). The in vitro toxicity results of the prepared self-assembled nanoprodrugs on tumor cells are shown in Table 4.
  • SKOV3/DDP tumor cell suspension (1 ⁇ 10 7 ) was subcutaneously inoculated into the ventral side of Balb/c nude mice.
  • the tumor-bearing mice were randomly divided into three groups ( 5 animals in each group), 0.2 mL of physiological saline, 0.2 mL of cisplatin solution and 0.2 mL of the nano self-assembled prodrug prepared in Example 5 (calculated as free cisplatin, dose 2 mg/kg) were injected into the tail vein, every 2 Dosing once a day, 5 times in a row. The long and short diameters of the tumors were measured every other day, and the tumor volume and tumor burden were calculated. The results are shown in Figures 9 and 10. In Figure 10, * indicates p ⁇ 0.05, and ** indicates P ⁇ 0.01.
  • the tumor volume in the normal saline group continued to grow and had no inhibitory effect on tumor growth; the cisplatin solution group could not effectively inhibit tumor growth in tumor-bearing mice; the self-assembled nanoprodrug group had a significantly stronger inhibitory effect on tumor growth than cisplatin solution.
  • Platinum solution group
  • Platinum standard working solution (200 ⁇ g/L): Accurately measure 0.8 mL of platinum standard stock solution, and dilute to 10 mL with 0.2% nitric acid.
  • Standard curve good linear relationship within the range of 12.5-200 ⁇ g/L.
  • PDDP nanoprodrug
  • Plasma sample processing and determination Precisely measure 100 ⁇ L of plasma sample, add 2 mL of nitric acid-perchloric acid (9:1), place on an electric hot plate and heat until the nitrate is nearly dry. The residue was dissolved with 0.2% nitric acid and the volume was adjusted to 10 mL, measured with a graphite furnace atomic absorption spectrophotometer, and substituted into the standard curve to calculate the blood drug concentration. Taking the blood drug concentration as the ordinate and time as the abscissa, draw the blood drug concentration-time curve. Kinetica software was used to fit the measured Pt content through a two-compartment model, and various pharmacokinetic parameters were calculated. The results are shown in Figure 11 and Table 5.
  • the elimination half-life of the nanoprodrug prepared in Example 5 is 16.38h, which is significantly higher than the cisplatin group (5.62h), indicating that PDDP can significantly improve the blood circulation time of platinum drugs.
  • SKOV3 tumor cell suspension (1 ⁇ 10 7 ) was subcutaneously inoculated into the ventral side of Balb/c nude mice.
  • cisplatin (CDDP) physiological saline solution and the nanoprodrug (PDDP) physiological saline dilution prepared in Example 5 were injected into the tail vein.
  • the mice were sacrificed 12 hours after injection, and different organs such as the heart, liver, spleen, lungs, kidneys and tumors were removed and weighed.
  • the test results are shown in Figure 12. It can be seen that the nanoprodrug prepared in Example 5 has better tumor targeting ability, and the accumulation amount in the tumor site can reach 6512ng/g tissue, which is significantly better than the cisplatin group (1958ng/g tissue). Verification The targeted delivery efficiency of the nanoprodrug was improved.
  • red blood cells were prepared into a 2% (V/V) suspension with 0.9% sodium chloride solution for testing.
  • Tubes 1-5 are the test tubes, tube 6 is the negative control tube, and tube 7 is the positive control tube.
  • Hemolysis rate % [(OD sample – OD negative)/(OD positive – OD negative)] ⁇ 100%.
  • the hemolysis rate of the nanoprodrug (PDDP) prepared in Example 5 is ⁇ 5% in the concentration range of 12.5 to 200 ⁇ g/mL.
  • mice Healthy KM mice (5-7 weeks), weighing 20-23g, were randomly divided into 3 groups (10 mice in each group), and were injected into the tail vein with physiological saline, cisplatin physiological saline solution and the nanoparticles prepared in Example 5 respectively.
  • Pre-assembled drug (PDDP) physiological saline dilution (calculated as free cisplatin, dose 2 mg/kg) was administered once every 2 days for 5 consecutive administrations. After administration, regular feeding was carried out, and the living conditions of the mice were paid attention to every day, and the body weight was measured. The results are shown in Figure 13.
  • the weight of the mice in the cisplatin solution group decreased significantly after administration, with a weight loss of approximately 25% within 14 days, indicating that the cisplatin solution group had severe systemic toxicity.
  • the weight of the mice in the nanoprodrug group increased slightly, which was similar to the weight change of the normal saline group. The mice were in good health and no death occurred.
  • mice were sacrificed on the 14th day after administration, and the main organs (heart, liver, spleen, lung, and kidney) were collected. After fixing with 4% paraformaldehyde for 24 hours, samples were sent for paraffin embedding, tissue sections were made, and H&E staining was performed. The results are shown in Figure 14.
  • H&E staining was used to evaluate tissue damage caused by PDDP in mice. From the liver tissue sections, the hepatocytes in the normal saline group were aggregated, evenly distributed, with clear membrane boundary structure, uniform eosinophilic staining, round and clear nucleoli, and lighter color in the center. In the CDDP solution group, hepatocytes were dispersed and cavities appeared, the staining became lighter, the liver was edematous and degenerated, and the veins were congested and inflamed, which was consistent with the typical hepatotoxicity of cisplatin. The liver tissue of the PDDP nanopreparation group was more normal than that of the CDDP solution group.
  • the glomeruli in the normal saline group were spherical, with a vascular bulb in the center, a fairly round nucleolus, eosinophilic chromatin, and dark red staining.
  • the degenerated glomeruli in the CDDP solution group had disordered structure, focal cell proliferation, and renal tubular epithelial swelling. No obvious histological damage was observed in the renal tissue sections of the PDDP nanoformulation group.

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Abstract

本公开内容涉及一种配合物，包含四价铂部分和与所述铂化学连接的作为轴向配体的寡肽。

Description

含四价铂的配合物、前药及其制备方法和应用
相关申请的交叉引用
本申请要求于2022年8月22日提交的第202211005108.0号和第202211006334.0号中国专利申请的优先权，上述中国专利申请的公开内容均通过援引加入的方式整体并入本文，用于所有目的。
领域
本公开内容涉及生物医药技术领域，尤其涉及一种含四价铂的配合物、由该前体组装的前药以及该配合物和前药的制备方法和应用，例如一种肽基铂类自组装纳米前药及其制备方法和应用。
背景
二价铂类药物(如顺铂、卡铂、奥沙利铂)具有很强的广谱抗癌活性，主要是通过破坏肿瘤细胞的DNA、抑制复制和诱导细胞凋亡而发挥抗肿瘤作用，目前已被广泛用于多种恶性肿瘤的临床治疗。然而，该类药物具有溶解度低、稳定性差的理化特征，且对肿瘤组织缺乏选择性，极易导致肾毒性、神经毒性和骨髓抑制等严重副作用的发生，以及受到先天和获得性耐药性的影响，严重限制其临床疗效以及应用。
近年来，药物前体技术在改善铂类药物的理化特性以及降低毒副作用等方面提供了新的解决途径。相对于二价铂配合物Pt(II)，增加两个轴向配体所形成的四价铂前体配合物Pt(IV)具有较低的反应活性和毒副作用，进入肿瘤细胞后，在胞内还原介质如抗坏血酸、蛋氨酸、谷胱甘肽等作用下可脱落轴向配体转化成具有细胞毒性的Pt(II)结构，从而产生细胞杀伤作用。然而，铂类小分子前体药物在人体内同样存在代谢速度快、病灶部位蓄积能力差以及易产生耐药性等局限性。
基于各种载体材料所构筑的纳米载药系统在实现化疗药物的体内有效运输方面显示出极具潜力的应用价值和开发前景，其可携载药物克服生理及病理屏障，实现对肿瘤组织、细胞的有效富集，完成持续、可控和靶向递送，进而达到增强治疗效果、降低不良反应的目的。然而，铂类小分子属于油水均难溶性的金属药物，对多数药物载体的亲和性弱，有效负载量有限。因此，需要对现有铂类药物进行结构改造，开发具有以下之一或多种的多功能铂类前体药物：抗肿瘤活性高、毒副作用小、对肿瘤组织选择性强和释放可控，从而提高该类药物的临床应用潜力。
概述
在本公开内容的第一方面，提供了一种配合物，所述配合物包含四价铂部分 和与四价铂部分化学连接的作为轴向配体的寡肽，其中寡肽可以为二肽Xaa1-Xaa4、三肽Xaa1-Xaa2-Xaa4或四肽Xaa1-Xaa2-Xaa3-Xaa4，其中Xaa1、Xaa2和Xaa3各自表示任意天然氨基酸，Xaa4表示色氨酸、苯丙氨酸、组氨酸、酪氨酸或精氨酸。
在一些实施方式中，配合物中的四价铂部分由以下结构式(a)表示：
其中R1、R2、R3、R4和R5可以独立地选自：Cl、Br、I、OH、NH3；R1与R2可与Pt成环，R3与R4可与Pt成环，当成环时R1与R2或R3与R4可以组合地选自
在一些实施方式中，配合物中的四价铂部分由以下结构式(a-1)至(a-6)表示：
在一些实施方式中，Xaa1可以表示谷氨酸、天冬氨酸、色氨酸、脯氨酸、酪氨酸或苯丙氨酸。在另一些实施方式中，二肽中的Xaa1可以表示谷氨酸、天冬氨酸、色氨酸或酪氨酸。在又一些实施方式中，三肽中的Xaa1可以表示天冬氨酸、色氨酸、脯氨酸、苯丙氨酸或谷氨酸。在还一些实施方式中，四肽中的Xaa1可以表示谷氨酸、色氨酸或天冬氨酸。
在一些实施方式中，Xaa2可以表示色氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、苯丙氨酸、脯氨酸、丝氨酸、苏氨酸、半胱氨酸、酪氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、天冬氨酸、谷氨酸、组氨酸或精氨酸。在另一些实施方式中，三肽中的Xaa2可以表示色氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、苯丙氨酸、脯氨酸、丝氨酸、苏氨酸、半胱氨酸、酪氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、 天冬氨酸、谷氨酸或组氨酸。在又一些实施方式中，四肽中的Xaa2可以表示色氨酸、甘氨酸、缬氨酸、亮氨酸、谷氨酸、苯丙氨酸、脯氨酸、天冬氨酸、苏氨酸、组氨酸、酪氨酸或精氨酸。
在一些实施方式中，Xaa3可以表示色氨酸、甘氨酸、亮氨酸、苯丙氨酸、脯氨酸、组氨酸、天冬氨酸、赖氨酸、酪氨酸或精氨酸。在又一些实施方式中，四肽中的Xaa2、Xaa3和Xaa4中的两者或三者是相同的。在还一些实施方式中，Xaa2或Xaa3，在存在时，与Xaa4相同。在此实施方式中，对于三肽而言，Xaa2和Xaa4可以均为色氨酸，对于四肽而言，Xaa3和Xaa4可以均为色氨酸。
在一些实施方式中，四价铂部分与Xaa1至Xaa3中的任一者化学连接，例如，与Xaa1、Xaa2或Xaa3化学连接，优选地，四价铂部分与Xaa1化学连接。在另一些实施方式中，寡肽由Xaa1-Xaa2-Xaa4表示，其中Xaa1可以表示天冬氨酸、色氨酸、脯氨酸、苯丙氨酸或谷氨酸，Xaa2表示组氨酸、苯丙氨酸、色氨酸或酪氨酸，Xaa4表示色氨酸。在又一些实施方式中，寡肽由Xaa1-Xaa2-Xaa4表示，Xaa1可以表示天冬氨酸、色氨酸、脯氨酸、苯丙氨酸或谷氨酸，Xaa2和Xaa4均表示色氨酸。
在一些实施方式中，配合物中的寡肽可以选自以下表1所示的化合物：
表1

















在一些实施方式中，配合物由式(I)表示：
其中，其中R1、R2、R3、R4和R5可以独立地选自：Cl、Br、I、OH、NH3；R1与R2可与Pt成环，R3与R4可与Pt成环，当成环时R1与R2或R3与R4可以组合地选自
在更具体的实施方式中，配合物可以选自以下任一者：
在此实施方式中，配合物可以选自以下任一者：

在本公开内容的第二方面，提供了一种肽基铂类前药，包含四价铂配合物，所述四价铂配合物具有含有色氨酸二肽的轴向配体。此实施方式提供了一种肽基铂类自组装纳米前药，其特征在于轴向配体中含有色氨酸二肽的四价铂配合物。
在一些实施方式中，自组装纳米前药的结构如式I所示：
其中，其中R1、R2、R3、R4和R5可以独立地选自：Cl、Br、I、OH、NH3；R1与R2可与Pt成环，R3与R4可与Pt成环，当成环时R1与R2或R3与R4可以组合地选自
在另一些实施方式中，自组装纳米前药具有式II-XIII中任一所示结构；

在一些实施方式中，前药能够在水性介质中自组装成粒径为10-1000nm的纳米结构，优选为20-200nm。
在一些实施方式中，前药能够自组装的水性介质pH为7.0-7.8。
在一些实施方式中，前药能够自组装的水性介质优选纯化水、注射用水、4-羟乙基哌嗪乙磺酸(HEPES)缓冲液、三羟甲基氨基甲烷(Tris)缓冲液或PBS缓冲液。
在另一些实施方式中，肽基铂类前药的制备方法包括如下步骤：将二价铂配合物Pt(II)用氯代琥珀酰亚胺氧化为单氯单羟基四价铂配合物Pt(IV)，然后在苯并 三唑四甲基四氟硼酸(TBTU)、三乙胺催化作用下，与寡肽缩合得到肽基铂类前药。
在一些实施方式中，制备自组装纳米前药的方法包括：将肽基铂类前药溶于第一溶剂中，得到有机相A；将有机相A与水相B混合得到混合溶液C；去除混合溶液C中的第一溶剂，得到单分散的纳米前药。
在一些实施方式中，第一溶剂为丙酮、乙醇、甲醇、四氢呋喃、乙腈、二甲基亚砜和N，N-二甲基甲酰胺中的一种或至少两种的混合物；优选地，所述第一溶剂为丙酮和/或乙醇；水相B为纯化水、注射用水、4-羟乙基哌嗪乙磺酸(HEPES)缓冲液、三羟甲基氨基甲烷(Tris)缓冲液或PBS缓冲液；优选地，所述水相B为注射用水或HEPES缓冲液；优选地，所述水相B的pH值为7.0-7.8。
在一些实施方式中，水相B与有机相A混合时体积比为1-100:1，优选为1-40:1。在另一些实施方式中，混合溶液C中的溶剂去除方法为减压蒸发法、高速离心法、透析法或超滤法；优选地，采用减压蒸发法去除混合溶液C中的溶剂。
在一些实施方式中，提供了肽基铂类自组装纳米前药在制备抗肿瘤药物中的应用。所述抗肿瘤药物可以是用于肺癌、乳腺癌、胃癌、食管癌、肾上腺皮质癌、皮肤鳞癌、头颈部癌、甲状腺癌、肝癌、胰腺癌、胆管癌、结直肠癌、卵巢癌、宫颈癌、子宫内膜癌、阴道鳞状上皮癌、睾丸癌、前列腺癌、膀胱癌、尿路上皮癌、黑色素瘤、骨肉瘤、恶性淋巴瘤、神经母细胞瘤、顺铂获得性耐药肺癌、顺铂获得性耐药卵巢癌、顺铂获得性耐药乳腺癌、顺铂获得性耐药宫颈癌、顺铂获得性耐药胃癌、顺铂获得性耐药结直肠癌的药物。
在本公开内容的第三方面，提供了一种自组装体，该自组装体由上文所述的配合物或上文所述的肽基铂类前药自组装而成。在一些实施方式中，该自组装体由上文所述的配合物或上文所述的肽基铂类前药在水性介质中自组装而成。在另一些实施方式中，自组装体具有10nm至10000nm的粒径。在又一些实施方式中，自组装体具有20nm至200nm的粒径。
在本公开内容的第四方面，提供了制备上文所述的配合物的方法，包括：将二价铂Pt(II)氧化为羟基四价铂Pt(IV)，然后在催化剂的存在下与寡肽缩合得到所述配合物。在一些实施方式中，氧化剂可以为氯代琥珀酰亚胺。在另一些实施方式中，催化剂可以为TBTU、三乙胺或其组合。
在本公开内容的第五方面，提供了制备上文所述的自组装体的方法，包括将上文所述的配合物溶于极性溶剂中，得到有机相；将有机相与水相混合得到溶液；去除溶液中的极性溶剂，得到所述自组装体。
在一些实施方式中，极性溶剂可以为丙酮、乙醇、甲醇、四氢呋喃、乙腈、二甲基亚砜和N,N-二甲基甲酰胺中的一种或至少两种的混合物。在优选的实施方式中，极性溶剂为丙酮和/或乙醇。在一些实施方式中，水相可以为纯化水、注射用水、HEPES缓冲液、Tris缓冲液或PBS缓冲液中的一种或多种的组合。在优 选的实施方式中，水相为注射用水或HEPES缓冲液。在一些实施方式中，水相的pH值为7.0-7.8。
在一些实施方式中，水相与有机相混合时体积比为1-100:1。在另一些实施方式中，水相与有机相混合时体积比为1-40:1。在一些实施方式中，溶液中的溶剂的去除方法为减压蒸发法、高速离心法、透析法或超滤法。在另一些实施方式中，溶液中的溶剂的去除方法为减压蒸发法。
在本公开内容的第六方面中，一些实施方式提供了包含上文所述的配合物、肽基铂类前药或者自组装体的药物组合物。在另一些实施方式中，所述药物组合物包含治疗有效量的上文所述的配合物、肽基铂类前药或者自组装体的药物组合物。在又一些实施方案中，所述药物组合物还包含药学上可接受的赋形剂。
在本公开内容的第七方面中，一些实施方式提供了用于治疗肿瘤的上文所述的配合物、肽基铂类前药或者自组装体。另一些实施方式提供了上文所述的配合物、肽基铂类前药或者自组装体在制备抗肿瘤药物中的用途。又一些实施方式提供了治疗肿瘤的方法，所述方法包括向有需要的对象给予治疗有效量的上文所述的配合物、肽基铂类前药或者自组装体。
附图描述
包括附图以提供对本公开内容的进一步理解，以及将附图并入本文以构成本公开内容的一部分，由此附图连同相关描述一起用于解释本公开内容的构思。在附图中：
图1为实施例5中所制备的纳米前药的透射电子显微镜形态照片；
图2为实施例5中所制备的纳米前药的粒度分布图；
图3为实施例6中所制备的纳米前药的粒度分布图；
图4为实施例7中所制备的纳米前药的粒度分布图；
图5为实施例5中所制备的纳米前药的活性成分释放曲线；
图6为实施例5中所制备的纳米前药的粒度分布变化图；
图7为实施例5中所制备的纳米前药和顺铂的基于人卵巢癌(SKOV3)的体外毒性曲线；
图8为实施例5中所制备的纳米前药和顺铂的基于人卵巢癌顺铂耐药株(SKOV3/DDP)的体外毒性曲线；
图9为实施例5中所制备的纳米前药和顺铂的体内抗肿瘤药效评价；
图10为实施例5中所制备的纳米前药和顺铂的体内抗肿瘤药效评价；
图11为实施例5中所制备的纳米前药和顺铂的药代动力学曲线；
图12为实施例5中所制备的纳米前药和顺铂的组织分布；
图13为实施例5中所制备的纳米前药的体内安全性评价的体重变化；
图14为实施例5中所制备的纳米前药的体内安全性评价的组织切片H&E染 色。
发明详述
现将在下文更加全面地描述示例性实施方式；然而，它们可以以不同的形式实施并且不应解释为局限于本文所示的方式。相反，提供这些实施方式使得本公开内容将是透彻和完整的，并且会全面地将示例性实施方式传达给本领域技术人员。
另外，在本文中，关于某一个实施方式所阐述的技术特征可以应用于另一个实施方式，或者可以与另一个实施方式的技术特征相组合，只要这样的应用或组合不违背本文所给出的教导即可。
术语“前药”是为了表示可在生理条件下或通过溶剂分解被转化成生物活性药剂的物质。前药当被给予有需要的患者时可以是无活性的，但在体内被转化成活性药剂。前药通常在体内迅速地转变，例如通过在血液中水解等途径。
术语“对象”可以包括人和家畜如实验室动物与家庭宠物(例如猫、狗、猪、牛、绵羊、山羊、马、家兔)，及非驯养动物，如野生动物等。
本文中使用的“治疗有效量”是指当被给予哺乳动物，优选为人时，足以如下文定义地在哺乳动物中实现对肿瘤的治疗的量。构成“治疗有效量”根据给予的物质、疾病状态及其严重性、给药方式以及要治疗的哺乳动物的年龄而改变，但可常规地由本领域一般技术人员根据其自有知识及本公开内容而决定。
本文中使用的“药学上可接受的赋形剂”是指被国家药品管理机构批准为可接受用于人或家畜的在药物制剂中除主药以外的附加物，也可称为辅料。举例而言，片剂中的粘合剂、填充剂、崩解剂、润滑剂；半固体制剂中的基质部分；液体制剂中的溶剂、防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、增溶剂、渗透压调节剂、着色剂等。
本文中使用的“进行治疗”或“治疗”涵盖对患有肿瘤的哺乳动物，优选为人中的肿瘤的治疗，且包括：(i)防止肿瘤发生于哺乳动物中，尤其是当这些哺乳动物易患肿瘤，但尚未被诊断为患有肿瘤时；(ii)抑制肿瘤，即阻止其发展；(iii)缓解肿瘤，即引起肿瘤的复原；或(iv)缓解由肿瘤引发的症状。
在本文中，配合物、肽基铂类前药、肽基铂类自组装纳米前药、纳米自组装前药可以互换使用，除非明确指出。举例而言，明确指出的“四价铂配合物”意指由以下结构式(a)表示的四价铂部分，因而无法与肽基铂类前药等互换，因为二者所指代的物质不同。
在本文中，自组装体、纳米前药、自组装纳米前药、纳米前药和纳米自组装前药可以在某些语境中互换使用，只要其指代的物质相同即可。
在本文中，天然氨基酸是指自然界存在的20种氨基酸，包括丙氨酸、精氨酸、天冬酰胺、天冬氨酸、半胱氨酸、谷氨酰胺、谷氨酸、甘氨酸、组氨酸、异 亮氨酸、亮氨酸、赖氨酸、蛋氨酸、苯丙氨酸、脯氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸和缬氨酸。除非明确指出，当提及这些氨基酸时，可以包括L型和D型。
本公开内容提供了一种配合物，包含由以下结构式(a)表示的四价铂部分和与该四价铂部分化学连接的作为轴向配体的寡肽，
其中R1、R2、R3、R4和R5可以独立地选自：Cl、Br、I、OH、NH3；R1与R2可与Pt成环，R3与R4可与Pt成环，当成环时R1与R2或R3与R4可以组合地选自此处，本领域技术人员可以根据四价铂配位原则合理地选择R1、R2、R3、R4和R5。
举例而言，R1、R2、R3、R4和R5可以独立地是Cl、Br、I、OH或NH3，或者，R1、R2、R3和R4中的两者也可以相互结合以形成
本公开内容的所述四价铂部分由以下结构式(a-1)至(a-6)表示：
本公开内容的寡肽可以为二肽Xaa1-Xaa4、三肽Xaa1-Xaa2-Xaa4或四肽Xaa1-Xaa2-Xaa3-Xaa4。上述四价铂部分可以与寡肽的各个部分化学连接，例如可以将二价铂配合物氧化成单氯单羟基四价铂部分，通过该四价铂部分的羟基与氨基酸缩合，进而形成连接。
在上述寡肽中，Xaa4表示色氨酸、苯丙氨酸、组氨酸、酪氨酸或精氨酸，优选色氨酸、苯丙氨酸、组氨酸或酪氨酸。这些氨基酸能够通π-π堆积作用驱动铂 部分在水性介质中自组装形成稳定的单分散纳米结构。
通常，四价铂部分与Xaa1化学连接，其中Xaa1可以是谷氨酸、天冬氨酸、色氨酸、脯氨酸、酪氨酸或苯丙氨酸。当然，四价铂部分也可以与Xaa2或Xaa3化学连接，只要其位置不妨碍π-π堆积作用即可。
在本公开内容中，当寡肽是二肽时，Xaa1可以是谷氨酸、天冬氨酸、色氨酸或酪氨酸，Xaa4可以是色氨酸、苯丙氨酸、组氨酸、酪氨酸或精氨酸。
当寡肽是三肽时，Xaa1可以是天冬氨酸、色氨酸、脯氨酸、苯丙氨酸或谷氨酸，Xaa2可以是色氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、苯丙氨酸、脯氨酸、丝氨酸、苏氨酸、半胱氨酸、酪氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、天冬氨酸、谷氨酸或组氨酸，Xaa4可以是色氨酸、苯丙氨酸、组氨酸或酪氨酸。举例而言，Xaa1可以表示天冬氨酸、色氨酸、脯氨酸、苯丙氨酸或谷氨酸，Xaa2表示组氨酸、苯丙氨酸、色氨酸或酪氨酸，Xaa4表示色氨酸。还例如，Xaa1可以表示天冬氨酸、色氨酸、脯氨酸、苯丙氨酸或谷氨酸，Xaa2和Xaa4均表示色氨酸。
当寡肽是四肽时，Xaa1可以是谷氨酸、色氨酸或天冬氨酸，Xaa2可以是色氨酸、甘氨酸、缬氨酸、亮氨酸、谷氨酸、苯丙氨酸、脯氨酸、天冬氨酸、苏氨酸、组氨酸、酪氨酸或精氨酸，Xaa3可以是色氨酸、甘氨酸、亮氨酸、苯丙氨酸、脯氨酸、组氨酸、天冬氨酸、赖氨酸、酪氨酸或精氨酸，Xaa4可以是色氨酸、苯丙氨酸、组氨酸或酪氨酸。
在三肽中，Xaa2可以与Xaa4相同，或者Xaa1可以与Xaa4相同，优选地，Xaa2和Xaa4可以均为色氨酸。在四肽中，Xaa2、Xaa3和Xaa4中的两者或三者是相同的，例如Xaa2和Xaa3相同，或者Xaa3和Xaa4相同，优选地，Xaa3和Xaa4可以均为色氨酸。
本公开内容的配合物可以选自以下任一者：

在此实施方式中，配合物可以选自以下任一者：
对于上述化合物而言，前药分子结构中的寡肽(例如色氨酸二肽)基元可提供芳香性吲哚环作为组装基元，通π-π堆积作用驱动铂配合物在水性介质中自组装形成稳定的单分散纳米结构。
肽基铂类前药的制备方法，其特征在于包括如下步骤：将二价铂配合物Pt(II)用氯代琥珀酰亚胺氧化为单氯单羟基四价铂配合物Pt(IV)，然后在TBTU、三乙胺催化作用下，与含有色氨酸二肽基元的寡肽缩合得到肽基铂类前药。
制备得到的肽基铂类前药自组装成纳米结构；前药能够在水性介质中自组装成粒径为10-1000nm的纳米结构,优选为20-200nm。前药自组装的水性介质pH为7.0-7.8。前药自组装的水性介质可以是纯化水、注射用水、HEPES缓冲液、Tris缓冲液或PBS缓冲液。自组装方法如下：
将制得的肽基铂类前药溶于第一溶剂中，得到有机相A；第一溶剂为丙酮、乙醇、甲醇、四氢呋喃、乙腈、二甲基亚砜和N，N-二甲基甲酰胺中的一种或至少两种的混合物；优选地，所述第一溶剂为丙酮和/或乙醇；
将有机相A与水相B混合得到混合溶液C；水相B为纯化水、注射用水、HEPES缓冲液、Tris缓冲液或PBS缓冲液；优选地，所述水相B为注射用水或HEPES缓冲液；优选地，所述水相B的pH值调节为7.0-7.8；水相B与有机相A混合时体积比为1-100:1，优选为1-40:1。
去除混合溶液C中的第一溶剂，混合溶液C中的溶剂去除方法为减压蒸发法、高速离心法、透析法或超滤法；优选地，采用减压蒸发法去除混合溶液C中的溶剂，去除溶剂后得到单分散的纳米前药；
肽基铂类自组装纳米前药能够用于制备抗肿瘤药物，纳米形态的药物具有良好的稳定性，可以避免活性二价铂配合物在血液循环中的过早释放，从而降低毒副作用，并促进铂类药物对肿瘤组织的选择性蓄积以及肿瘤细胞内的可控释放，从而提高对耐药肿瘤生长的抑制效果。前药中组装基元的降解产物均为人体内源性氨基酸，应用于肿瘤治疗可显著减低铂类药物的毒副作用。
本公开内容的配合物的制备方法可以包括将二价铂Pt(II)氧化为羟基四价铂Pt(IV)，然后在催化剂的存在下与寡肽缩合得到所述配合物。如上所示，氧化剂可以为氯代琥珀酰亚胺等，并且催化剂可以为TBTU、三乙胺或其组合，优选使用二者的组合。
本公开内容的自组装体的形成方法可以包括将配合物溶于极性溶剂中，得到有机相；将有机相与水相混合得到溶液；去除溶液中的极性溶剂，得到所述自组装体。对此，极性溶剂可以为丙酮、乙醇、甲醇、四氢呋喃、乙腈、二甲基亚砜和N,N-二甲基甲酰胺中的一种或至少两种的混合物，优选为丙酮和/或乙醇。水相可以为纯化水、注射用水、HEPES缓冲液、Tris缓冲液或PBS缓冲液中的一种或多种的组合，优选为注射用水或HEPES缓冲液。水相的pH值为7.0-7.8。在该方法中，水相与有机相混合时体积比可以为1-100:1，优选1-40:1。在该方法中，去除方法可以为减压蒸发法、高速离心法、透析法或超滤法，优选减压蒸发法。
本公开内容的自组装体可用于制备适用于、例如治疗以下病症的抗肿瘤药物： 肺癌、乳腺癌、胃癌、食管癌、肾上腺皮质癌、皮肤鳞癌、头颈部癌、甲状腺癌、肝癌、胰腺癌、胆管癌、结直肠癌、卵巢癌、宫颈癌、子宫内膜癌、阴道鳞状上皮癌、睾丸癌、前列腺癌、膀胱癌、尿路上皮癌、黑色素瘤、骨肉瘤、恶性淋巴瘤、神经母细胞瘤、顺铂获得性耐药肺癌、顺铂获得性耐药卵巢癌、顺铂获得性耐药乳腺癌、顺铂获得性耐药宫颈癌、顺铂获得性耐药胃癌、顺铂获得性耐药结直肠癌。
本公开内容的药物组合物可以包含上文所述的配合物、前药或自组装体，例如治疗有效量的配合物、前药或自组装体，以及药学上可接受的赋形剂。本公开内容的药物组合物的典型途径包括但不限于口服、局部、吸入、肠胃外、鼻内、眼内、肌内、皮下、静脉内给药。本公开内容的药物组合物可以采用本领域众所周知的方法制造，如常规的混合法、溶解法、制粒法、制糖衣药丸法、磨细法、乳化法、冷冻干燥法等。
本公开内容可以涉及以下优点和效果中的一者或多者：
上文所述的配合物、肽基自组装纳米前药和自组装体具有肿瘤微环境响应性，用于肿瘤治疗能够促进铂类药物对肿瘤组织的选择性蓄积以及肿瘤细胞内的可控释放，从而提高对耐药肿瘤生长的抑制效果；
上文所述的配合物、肽基自组装纳米前药和自组装体可以避免活性二价铂配合物在血液循环中的过早释放，从而降低毒副作用；
上文所述的配合物、肽基自组装纳米前药和自组装体具有良好的生物相容性和安全性，组装基元可以包含人体必需氨基酸之一(例如色氨酸)，参与机体蛋白质合成和代谢调节，降解产物均为人体内源性物质，应用于肿瘤治疗可显著减低铂类药物的毒副作用；
上文所述的配合物、肽基自组装纳米前药和自组装体的制备条件温和、操作简单，生产过程不涉及有害溶剂等，适合工业化生产。
实施例
提供以下实施例以对本公开内容进行说明，其中，未具体说明操作步骤的实验方法，均按照相应商品说明书进行，实施例中所用到的仪器、试剂、耗材如无特殊说明，均可从商业公司购买得到。
缩略语及结构说明：
H-Trp-OMe.HCl：
Cbz-Trp-OH：
Cbz-Glu(Obzl)-OH：
H-Glu-Trp-Trp-OMe：
实施例1
提供了如式(VIII)所示结构的四价肽铂的制备方法，合成路线如下：
(1)称取H-Trp-OMe.HCl(291mg，1mmol)和N-甲基吗啉(145μL，1.3mmol)，溶于5mL二甲基甲酰胺(DMF)，得到混合物1。称取Cbz-Trp-OH(338mg，1mmol)、1-羟基苯并三唑(HOBt，135mg，1mmol)和1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDCI，250mg，1.3mmol)，溶于20mL DMF，于冰浴下活化40min，加入混合物1，搅拌2h后自然升至室温，继续搅拌反应12h。减压浓缩后，于蒸馏水中透析4h，冻干，得Cbz-Trp-Trp-OMe。
将Cbz-Trp-Trp-OMe置于50mL的圆底烧瓶内，用10mL甲醇使该肽溶解。加入150mg预先悬浮于1mL甲醇中的5％Pd/C催化剂，搅拌下通入氢气氢化6h，过滤。将滤液减压浓缩后，于蒸馏水中透析4h，冻干，得H-Trp-Trp-OMe。
称取H-Trp-Trp-OMe(243mg，0.6mmol)和N-甲基吗啉(87μL，0.78mmol)，溶于5mL DMF，得到混合物2。称取Cbz-Glu(Obzl)-OH(223mg，0.6mmol)，HOBt (81mg，0.6mmol)和EDCI(150mg，0.78mmol)，溶于10mL DMF，于冰浴下活化40min，加入混合物2，搅拌2h后自然升至室温，继续搅拌反应12h，于蒸馏水中透析4h，冻干，得Cbz-Glu(Obzl)-Trp-Trp-OMe。
将Cbz-Glu(Obzl)-Trp-Trp-OMe置于50mL的圆底烧瓶内，用10mL甲醇使肽溶解。加入180mg预先悬浮于1.2mL甲醇中的5％Pd/C催化剂，搅拌下通入氢气氢化6h，过滤。滤液减压浓缩后，于蒸馏水中透析4h，冻干，得H-Glu-Trp-Trp-OMe。
(2)将顺铂(300mg，1mmol)、N-氯代琥珀酰亚胺(NCS，150mg,1.1mmol)混悬于60mL蒸馏水中，室温下避光反应12h。将反应液过滤后，滤液减压浓缩至析出淡黄色沉淀。将沉淀依次用丙酮和乙醚洗涤后，得到四价铂中间产物(收率91.2％)。
将四价铂中间产物(176mg,0.5mmol)、H-Glu-Trp-Trp-OMe(293mg，0.55mmol)、四甲基脲四氟硼酸酯(TBTU，264mg，0.82mmol)、三乙胺(84mg，0.82mmol)氮气保护下溶于干燥二甲基甲酰胺中，避光室温下反应24h。加入1M氢氧化钠溶液调至pH 12.8，15℃搅拌6h；加入1M盐酸溶液调至pH 6.0；减压浓缩后加入到10倍体积量冰乙醚中沉淀，离心，取沉淀物加冷乙醚重复上述操作5次，减压干燥得到结构式(II)产物。该产物的质谱和核磁数据如下所示。
ESI-MS[M+H]+:854.64
1HNMR(600MHz,DMSO-d6):δ10.22(s,1H),8.05(d,J＝6.7Hz,2H),7.62-7.58(m,2H),7.36(dd,J＝7.7,1.5Hz,2H),7.21-7.10(m,4H),7.05(d,J＝6.7Hz,1H),6.98-6.91(m,2H),6.76(d,J＝6.7Hz,1H),4.55-4.47(m,2H),3.53(p,J＝5.6Hz,1H),3.26(dd,J＝14.5,6.7Hz,1H),3.20(dd,J＝15.6,5.9Hz,1H),2.98(dd,J＝15.6,5.9Hz,1H),2.92(dd,J＝14.5,6.7Hz,1H),2.42(t,J＝7.5Hz,3H),2.37-2.28(m,1H),2.30-2.16(m,2H),1.96(p,J＝7.6Hz,2H),1.79(dtd,J＝15.3,8.4,5.0Hz,1H),1.68(d,J＝5.9Hz,2H),1.50(s,3H)。
实施例2
提供了如式(VIII)所示结构的四价肽铂的制备方法，合成路线如下：
(1)称取H-Trp-OMe.HCl(291mg，1mmol)和N-甲基吗啉(145μl，1.3mmol)，溶于5mL DMF，得到混合物1。称取Cbz-Trp-OH(338mg，1mmol)，HOBt(135mg，1mmol)，EDCI(250mg，1.3mmol)，溶于20mL DMF，于冰浴下活化40min； 加入①，搅拌2h后自然升至室温，继续搅拌反应12h。减压浓缩后，于蒸馏水中透析4h，冻干，得Cbz-Trp-Trp-OMe。
将Cbz-Trp-Trp-OMe置于50mL的圆底烧瓶内，用10mL甲醇使该肽溶解。加入150mg预先悬浮于1mL甲醇中的5％Pd/C催化剂，搅拌下通入氢气氢化6h，过滤。滤液减压浓缩后，于蒸馏水中透析4h，冻干，得Trp-Trp-OMe。
称取Trp-Trp-OMe(243mg，0.6mmol)和N-甲基吗啉(87μL，0.78mmol)，溶于5mL DMF，得到混合物2。称取Fmoc-Glu(Obzl)-OH(268mg，0.6mmol)，HOBt(81mg，0.6mmol)和EDCI(150mg，0.78mmol)，溶于10mL DMF，于冰浴下活化40min；加入混合物2，搅拌2h后自然升至室温，继续搅拌反应12h，于蒸馏水中透析4h，冻干，得Fmoc-Glu(Obzl)-Trp-Trp-OMe。
将Fmoc-Glu(Obzl)-Trp-Trp-OMe置于50mL的圆底烧瓶内，用10mL甲醇使该肽溶解。加入180mg预先悬浮于1.2mL甲醇中的5％Pd/C催化剂，搅拌下通入氢气氢化6h，过滤。滤液减压浓缩后，于蒸馏水中透析4h，冻干，得Fmoc-Glu-Trp-Trp-OMe。
(2)将顺铂(300mg,1mmol)、NCS(150mg,1.1mmol)混悬于60mL蒸馏水中，室温下避光反应12h。将反应液过滤后，滤液减压浓缩至析出淡黄色沉淀。将沉淀依次用丙酮和乙醚洗涤后，得到四价铂中间产物。
将四价铂中间产物(176mg,0.5mmol)、Fmoc-Glu-Trp-Trp-OMe(415mg,0.55mmol)、TBTU(264mg，0.82mmol)、三乙胺(84mg，0.82mmol)氮气保护下溶于干燥二甲基甲酰胺中，避光室温下反应24h；加入1M氢氧化钠溶液调至pH 12.8，15℃搅拌6h；加入1M盐酸溶液调至pH6.0；减压浓缩后加入到10倍量冰乙醚中沉淀，离心，取沉淀加冷乙醚重复上述操作5次，减压干燥得到结构式(VIII)产物。ESI-MS[M+H]+:854.56
实施例3
提供了如式(XIV)所示结构的四价肽铂的制备方法，合成路线如下：
将奥沙利铂(395mg，1mmol)按实施例1或实施例2中合成方法进行反应，得到结构式(XIV)产物(收率71％)。该产物的核磁数据如下所示。
1HNMR(600MHz,DMSO-d6):δ10.22(s,1H),8.05(d,J＝6.7Hz,2H),7.60(dd,J＝7.2,1.2Hz,2H),7.36(dd,J＝7.7,1.5Hz,2H),7.21-7.10(m,4H),7.05(d,J＝6.7Hz,1H),6.98-6.91(m,2H),6.76(d,J＝6.7Hz,1H),4.55-4.47(m,2H),3.53(p,J＝5.5Hz,1H),3.26(dd,J＝14.5,6.7Hz,1H),3.20(dd,J＝15.6,5.9Hz,1H),2.98(dd,J＝15.6, 5.9Hz,1H),2.92(dd,J＝14.5,6.7Hz,1H),2.56-2.48(m,2H),2.37-2.28(m,1H),2.30-2.16(m,2H),2.08-2.00(m,1H),1.84-1.66(m,7H),1.53-1.47(m,2H),1.38-1.30(m,1H),1.32-1.21(m,2H),1.15(dddd,J＝12.4,8.4,5.9,3.9Hz,1H)。
实施例4
提供了如式(XV)所示结构的四价肽铂的制备方法，合成路线如下：
将卡铂(371mg，1mmol)按实施例1或实施例2中合成方法进行反应，得到结构式(XV)产物(收率68％)。该产物的核磁数据如下所示。
1HNMR(600MHz,DMSO-d6):δ10.22(s,1H),8.05(d,J＝6.7Hz,2H),7.62-7.58(m,2H),7.36(dd,J＝7.7,1.5Hz,2H),7.21-7.10(m,4H),7.05(d,J＝6.7Hz,1H),6.98-6.91(m,2H),6.76(d,J＝6.7Hz,1H),4.55-4.47(m,2H),3.53(p,J＝5.6Hz,1H),3.26(dd,J＝14.5,6.7Hz,1H),3.20(dd,J＝15.6,5.9Hz,1H),2.98(dd,J＝15.6,5.9Hz,1H),2.92(dd,J＝14.5,6.7Hz,1H),2.42(t,J＝7.5Hz,3H),2.37-2.16(m,3H),1.96(p,J＝7.6Hz,2H),1.79(dtd,J＝15.3,8.4,5.0Hz,1H),1.68(d,J＝5.9Hz,2H)。
实施例5
如下配制4-羟乙基哌嗪乙磺酸缓冲液(HEPES，1M)：称取119.15g HEPES溶解在400mL蒸馏水中，加1M的NaOH水溶液调节pH至7.4，然后用蒸馏水定容至500mL，于4℃保存。
将实施例1或实施例2制得的的四价肽铂(100mg)溶于1mL乙醇-丙酮混合溶剂(1:1)中，将所得到的溶液注入到100mL HEPES缓冲液(10mM，pH7.4)中，加毕继续搅拌30min，减压蒸发去除溶剂，得到纳米胶体溶液，即为纳米自组装前药(下文有时也简称为纳米前药)。所得的纳米前药的外观形态为单分散的球形，如图1所示，粒度分布如图2所示，平均粒径为118nm，PDI(聚合物分散性指数)为0.12
实施例6
将实施例3所获得的四价肽铂(10mg)按实施例5的方法进行自组装，所得纳米自助装前药的粒度分布如图3所示，平均粒径为132nm，PDI为0.18。
实施例7
将实施例4所获得的四价肽铂(10mg)按实施例5的方法进行自组装，所得纳 米前药的粒度分布如图4所示，平均粒径为121nm，PDI为0.09。
实施例8
环境响应性释放行为
将实施例5所获得的纳米自组装前药分散于20mL含有不同浓度还原型谷胱甘肽(GSH)的缓冲液中(10mM，pH7.4)，分别在特定时间点取样0.1mL，20000rpm高速离心30min，吸取20uL上清液HPLC测定药物浓度，绘制释放曲线，具体步骤如下，并且结果如图5所示。
纳米前药在不含GSH的释放介质中缓慢释放，48h累积释放率低于20％；在含有较高浓度GSH(5mM)的释放介质中会显著加速活性成分的释放，48h累积释放率达到80％。从体外释放结果可以看出，纳米前药具有肿瘤微环境响应性，可在肿瘤细胞内还原性环境的作用下快速释放活性成分。
(1)顺铂(CDDP)含量测定方法：
色谱条件：Shim-Paok VP-ODS C18(150×4.6mm，粒径5μm)；流速1mL/min；
流动相：0.9％氯化钠；检测波长：210nm；柱温：30℃；进样量：20μL。
(2)标准曲线绘制：
精密称取顺铂(10mg)于10mL容量瓶中，加入0.9％氯化钠稀释至刻度，得到顺铂浓储液。精密量取储备液并将其配置成2.5、5、10、20、40、80、100μg/mL标准供试液。取溶液按上述的色谱条件分别进样，纪录峰面积，以峰面积A对浓度C进行线性回归，得回归方程：A＝24644C+21689，r2＝0.9993。顺铂在2.5-100μg/mL范围内线性关系良好。
实施例9
血清稳定性
将实施例5所获得的纳米自组装前药分散在含10％胎牛血清FBS的缓冲液中(pH＝7.4)中，置于37℃恒温摇床中振荡，分别于0、2、4、8、12、24、48h取样，进行粒度分析。结果如图6所示，表明在24h内实施例5所获得的纳米颗粒在10％FBS中粒度分布没有显著变化，说明实施例5所获得的纳米颗粒在血液循环中将具有较好的稳定性。
实施例10
体外细胞毒性
CCK8法评价实施例5所制备的纳米自组装前药的体外细胞毒性。具体方法如下：将对数生长期的肿瘤细胞接种到96孔板中(5×103/孔)，待细胞贴壁后，将培养液更换为含纳米自组装前药，以顺铂溶液为对照组，37℃继续孵育48h后，向每孔加入10μL CCK8试剂，孵育4h后于450nm波长处测定各孔的OD值。计 算细胞存活率，将细胞存活率对浓度进行非线性回归，得到药物对细胞生长的半数抑制浓度(IC50)。所制备的纳米自组装前药对肿瘤细胞的体外毒性结果见表2、图7和图8，其中CDDP表示顺铂，PDDP表示实施例5获得的纳米自组装前药。
表2
从此次实验结果可以看出，在与人卵巢癌SKOV3细胞孵育48小时后，自组装纳米前药的半数抑制浓度与顺铂溶液相当，说明将顺铂自组装成纳米前药后并不影响其对肿瘤细胞的增殖抑制能力，尤其是与铂耐药细胞系SKOV3/DDP孵育后，自组装纳米前药的半数抑制浓度显著低于顺铂溶液，说明纳米前药在缓解肿瘤耐药方面具有优势。
实施例11
体外细胞毒性。
采用实施例6中所制备的纳米自组装前药，并使用奥沙利铂溶液为对照组，按实施例10中的方法测试了对人结肠癌HCT116及其耐药细胞系HCT116/L-OHPD的半数抑制浓度(IC50)。所制备的自组装纳米前药对肿瘤细胞的体外毒性结果见表3。
表3
实施例12：
体外细胞毒性。
采用实施例7中所制备的纳米自组装前药，并且使用卡铂溶液为对照组，按实施例11中的方法测试了对人卵巢癌SKOV3及其铂耐药细胞系SKOV3/DDP的半数抑制浓度(IC50)。所制备的自组装纳米前药对肿瘤细胞的体外毒性结果见表4。
表4

实施例13：
体内抗肿瘤药效
将150μL SKOV3/DDP肿瘤细胞悬液(1×107)皮下接种于Balb/c裸鼠的腹侧皮下，待肿瘤体积达到约100-150mm3时，将荷瘤小鼠随机分为三组(每组5只)，分别尾静脉注射0.2mL生理盐水、0.2mL顺铂溶液和0.2mL实施例5中制备的纳米自组装前药(按游离顺铂计算，剂量2mg/kg)，每隔2日给药1次，连续给药5次。隔日测量肿瘤的长径与短径，计算肿瘤体积和肿瘤负担，结果如图9和10所示。图10中*表示p＜0.05，**表示P＜0.01。
治疗期间内，生理盐水组肿瘤体积持续增长，对肿瘤生长没有抑制效果；顺铂溶液组不能有效抑制荷瘤小鼠的肿瘤生长；自组装纳米前药组对肿瘤生长的抑制效果显著强于顺铂溶液组。
实施例14
药代动力学分析
(1)测定方法的建立
铂标准储备液制备(2.5mg/L)：准确量取2.5mL铂标准液(1mg/mL)，用0.2％硝酸定容至10mL；准确量取100μL，用0.2％硝酸定容至10mL。
铂标准工作液(200μg/L)：准确量取0.8mL铂标准贮备液，用0.2％硝酸定容至10mL。
分析条件：工作波长为265.9nm，带宽0.2nm，进样体积20μL。
标准曲线：12.5-200μg/L范围内线性关系良好。
(2)取样
SD大鼠(体重200-250g)随机分为2组(n＝5)，正式给药前禁食12h，自由饮水。各组尾静脉分别单次注射3mg/kg(以顺铂含量计)的CDDP生理盐水溶液、实施例5中制备的纳米前药(PDDP)生理盐水稀释液，在给药后5min、15min、30min、1h、2h、4h、6h、8h、12h、24h、48h眼眶取血至涂有肝素钠的EP管中混匀，5000rpm离心10min获取血浆，-20℃冰箱保存。
血浆样品处理与测定：精密量取血浆样品100μL，加入2mL硝酸-高氯酸(9:1)，置于电热板上加热直至硝解近干。残渣用0.2％硝酸溶解并定容至10mL，石墨炉原子吸收分光光度计测定，并代入标准曲线计算血药浓度。以血药浓度为纵坐标，时间为横坐标，绘制血药浓度-时间曲线。用Kinetica软件通过双室模型对测得的Pt含量进行拟合，并计算各项药代动力学参数，结果如图11和表5所示。
表5
实施例5中所制备的纳米前药的消除半衰期为16.38h，显著高于顺铂组(5.62h)，表明PDDP可显著提升铂类药物的血液循环时间。
实施例15
组织分布
将150μL SKOV3肿瘤细胞悬液(1×107)皮下接种于Balb/c裸鼠的腹侧皮下，待肿瘤体积达到约150mm3时，将荷瘤小鼠随机分为2组(n＝5)，分别尾静脉注射顺铂(CDDP)生理盐水溶液和实施例5中所制备的纳米前药(PDDP)生理盐水稀释液(按游离顺铂计算，剂量5mg/kg)。注射12h后处死小鼠，并取出不同脏器心、肝、脾、肺、肾及肿瘤称重。将组织样本切碎，加入2mL硝酸-高氯酸(9:1)，置于电热板上加热直至硝解近干。残渣用0.2％硝酸溶解并定容至10mL，石墨炉原子吸收分光光度计测定，并代入标准曲线计算Pt浓度。
测试结果如图12所示。可以看出，实施例5中所制备的纳米前药具有更好的肿瘤靶向能力，在肿瘤部位的蓄积量可达6512ng/g组织，显著优于顺铂组(1958ng/g组织)，验证了该纳米前药的靶向输送效率。
实施例16
体内安全性评价
(1)溶血性试验
取兔血数毫升，放入含玻璃珠的三角烧瓶中振摇10分钟除去纤维蛋白原，使成脱纤血液。加入0.9％氯化钠溶液约10倍量，摇匀，1500rpm离心15分钟，除去上清液，沉淀的红细胞再用0.9％氯化钠溶液按上述方法洗涤2-3次，至上清液不显红色为止。将所得红细胞用0.9％氯化钠溶液配成2％(V/V)的混悬液，供试验用。
取洁净试管7只，进行编号，1-5号管为供试品管，6号管为阴性对照管，7号管为阳性对照管。按下表所示依次加入2％红细胞悬液、0.9％氯化钠溶液或蒸馏水。按表6顺序加入各种溶液：
表6
各组设3个平行，涡旋混匀后，于37℃孵育3小时。离心取上清液，于540nm处测吸光度值。结果如表7所示。
溶血率％＝[(OD样品–OD阴性)/(OD阳性–OD阴性)]×100％。
表7
由上可见，实施例5中所制备的纳米前药(PDDP)在12.5～200μg/mL浓度范围内溶血率<5％。
(2)体重变化
取健康KM小鼠(5-7周)，体重20-23g，随机分为3组(每组10只)，分别尾静脉注射生理盐水、顺铂生理盐水溶液和实施例5中制备的纳米自组装前药(PDDP)生理盐水稀释液(按游离顺铂计算，剂量2mg/kg)，每隔2日给药1次，连续给药5次。给药后常规喂养，每日关注小鼠的生存状况，测量体重，结果如图13所示。
从图13结果可以看出，给药后顺铂溶液组小鼠体重明显下降，14天内体重减轻约25％，表明顺铂溶液组具有较严重的系统毒性。纳米前药组小鼠体重略微上升，与生理盐水组体重变化相似，小鼠健康状况良好，没有出现死亡现象。
(3)组织病理学检查
给药后第14日处死小鼠，收集主要器官(心、肝、脾、肺、肾)，4％多聚甲醛固定24小时后，送样进行石蜡包埋，制作组织切片并进行H&E染色，结果如图14所示。
H&E染色用于评估PDDP对小鼠的组织损伤。从肝组织切片来说生理盐水组肝细胞呈聚集状，分布均匀，膜边界结构清晰，染色均匀嗜酸性，核仁圆形清晰，位于中央颜色较浅。CDDP溶液组肝细胞分散出现空腔、染色变浅肝水肿变性、静脉充血出现炎症，符合顺铂的典型肝毒性。PDDP纳米制剂组肝组织相比CDDP溶液组较为正常。从肾组织切片来说，生理盐水组肾小球呈球形，中心为血管球，核仁相当圆，染色质嗜酸，染色暗红色。CDDP溶液组变性的肾小球结构紊乱，局灶性细胞增生，肾小管上皮肿大。PDDP纳米制剂组肾组织切片未观察到明显的组织学损伤。
由此可见，小鼠主要脏器组织切片H&E染色结果显示顺铂溶液组观察到明显的肝、肾损伤，而自组装纳米前药组和生理盐水组的各脏器组织切片均没有明显的病理性变化，说明自组装纳米前药可显著降低原药的毒性，其应用于肿瘤治疗具有较高的体内安全性。
本公开内容中所引用的所有文献，例如专利、专利申请公开、专利申请及非专利出版物，均以其全文并入本文供参考。
自前文可以理解，虽然为了说明的目的在本文中描述了具体实施方案，但可在不偏离本公开内容的精神与范围的情况下进行各种修正或调整。因此，本公开内容的范围应当仅受所附的权利要求及其等同的限定。

Claims (18)

  1. 一种配合物，所述配合物包含四价铂部分和与所述四价铂部分化学连接的作为轴向配体的寡肽，所述寡肽为二肽Xaa1-Xaa4、三肽Xaa1-Xaa2-Xaa4或四肽Xaa1-Xaa2-Xaa3-Xaa4，其中Xaa1、Xaa2和Xaa3各自表示任意天然氨基酸，Xaa4表示色氨酸、苯丙氨酸、组氨酸、酪氨酸或精氨酸。
  2. 如权利要求1所述的配合物，其中所述四价铂部分由以下结构式(a)表示：
    其中R1、R2、R3、R4和R5独立地选自：Cl、Br、I、OH、NH3；或者，R1与R2可与Pt成环，R3与R4可与Pt成环，当成环时R1与R2或R3与R4组合地选自
    任选地，其中所述四价铂部分由以下结构式(a-1)至(a-6)表示：
  3. 如权利要求1或2所述的配合物，其中Xaa1表示谷氨酸、天冬氨酸、色氨酸、脯氨酸、酪氨酸或苯丙氨酸；任选地，在二肽中，Xaa1表示谷氨酸、天冬氨酸、色氨酸或酪氨酸；任选地，在三肽中，Xaa1表示天冬氨酸、色氨酸、脯氨酸、苯丙氨酸或谷氨酸；任选地，在四肽中，Xaa1表示谷氨酸、色氨酸或天冬氨酸；
    任选地，其中Xaa2表示色氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、苯丙 氨酸、脯氨酸、丝氨酸、苏氨酸、半胱氨酸、酪氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、天冬氨酸、谷氨酸、组氨酸或精氨酸；任选地，在三肽中，Xaa2表示色氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、苯丙氨酸、脯氨酸、丝氨酸、苏氨酸、半胱氨酸、酪氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、天冬氨酸、谷氨酸或组氨酸；任选地，在四肽中，Xaa2表示色氨酸、甘氨酸、缬氨酸、亮氨酸、谷氨酸、苯丙氨酸、脯氨酸、天冬氨酸、苏氨酸、组氨酸、酪氨酸或精氨酸；
    任选地，其中Xaa3表示色氨酸、甘氨酸、亮氨酸、苯丙氨酸、脯氨酸、组氨酸、天冬氨酸、赖氨酸、酪氨酸或精氨酸；
    任选地，在四肽中Xaa2、Xaa3和Xaa4中的两者或三者是相同的；其中Xaa2或Xaa3，在存在时，与Xaa4相同，优选地，在三肽中，Xaa2和Xaa4均为色氨酸，在四肽中，Xaa3和Xaa4均为色氨酸，
    任选地，所述四价铂部分与Xaa1至Xaa3中的任一者化学连接，优选地，所述四价铂部分与Xaa1化学连接。
  4. 如权利要求1至3中任一项所述的配合物，其中所述寡肽由Xaa1-Xaa2-Xaa4表示，其中Xaa1表示天冬氨酸、色氨酸、脯氨酸、苯丙氨酸或谷氨酸，Xaa2表示组氨酸、苯丙氨酸、色氨酸或酪氨酸，Xaa4表示色氨酸，优选地，Xaa2和Xaa4均表示色氨酸。
  5. 如权利要求1至4中任一项所述的配合物，其中所述寡肽选自：









  6. 如权利要求1至5中任一项所述的配合物，其中所述配合物由式(I)表示：
    其中，R1、R2、R3、R4和R5独立地选自：Cl、Br、I、OH、NH3；或者，R1与R2可与Pt成环，R3与R4可与Pt成环，当成环时R1与R2或R3与R4组合地选自
  7. 如权利要求1至6中任一项所述的配合物，其中所述配合物选自以下任一者：
    任选地，选自以下任一者：

  8. 一种肽基铂类前药，包含四价铂配合物，所述四价铂配合物具有含有色氨酸二肽的轴向配体。
  9. 一种自组装体，所述自组装体由权利要求1至7中任一项所述的配合物或权利要求8所述的肽基铂类前药自组装而成，任选地，所述自组装体由权利要求1至7中任一项所述的配合物或权利要求8所述的肽基铂类前药在水性介质中自组装而成，优选地，所述水性介质的pH为7.0-7.8，优选地，所述水性介质为纯化水、注射用水、HEPES缓冲液、Tris缓冲液或PBS缓冲液；优选地，所述自组装体具有10nm至10000nm的粒径，更优选地，所述自组装体具有20nm至200nm的粒径。
  10. 制备权利要求1至7中任一项所述的配合物的方法，所述方法包括：将二价铂Pt(II)氧化为羟基四价铂Pt(IV)，然后在催化剂的存在下与寡肽缩合得到所述配合物。
  11. 制备权利要求8所述的肽基铂类前药的方法，其中将二价铂配合物Pt(II)用氯代琥珀酰亚胺氧化为羟基四价铂配合物Pt(IV)，然后在TBTU、三乙胺催化作用下，与含有色氨酸二肽基元的寡肽缩合得到肽基铂类前药。
  12. 制备权利要求9所述的自组装体的方法，所述方法包括将权利要求1至7中任一项所述的配合物或权利要求8所述的肽基铂类前药溶于极性溶剂中，得到有机相；将所述有机相与水相混合得到溶液；去除所述溶液中的极性溶剂，得到所述自组装体。
  13. 如权利要求12所述的方法，其中所述极性溶剂为丙酮、乙醇、甲醇、四氢呋喃、乙腈、二甲基亚砜和N,N-二甲基甲酰胺中的一种或至少两种的混合物；优选地，所述极性溶剂为丙酮和/或乙醇；或者
    所述水相为纯化水、注射用水、HEPES缓冲液、Tris缓冲液或PBS缓冲液中的一种或多种的组合；优选地，所述水相为注射用水或HEPES缓冲液；更优 选地，所述水相的pH值为7.0-7.8；或者
    所述水相与所述有机相混合时体积比为1-100:1，优选为1-40:1；或者
    所述溶液中的溶剂的去除方法为减压蒸发法、高速离心法、透析法或超滤法；优选地，减压蒸发法。
  14. 药物组合物，包含权利要求1至7中任一项所述的配合物、权利要求8所述的肽基铂类前药或者权利要求9所述的自组装体，以及药学上可接受的赋形剂。
  15. 用于治疗肿瘤的权利要求1至7中任一项所述的配合物、权利要求8所述的肽基铂类前药、权利要求9所述的自组装体或者权利要求14所述的药物组合物。
  16. 权利要求1至7中任一项所述的配合物、权利要求8所述的肽基铂类前药、权利要求9所述的自组装体或者权利要求14所述的药物组合物在制备抗肿瘤药物中的用途。
  17. 治疗肿瘤的方法，所述方法包括向有需要的对象给予包含治疗有效量的权利要求9所述的自组装体的药物或者权利要求14所述的药物组合物。
  18. 如权利要求15所述的配合物、肽基铂类前药、自组装体或药物组合物，如权利要求16所述的用途，或者如权利要求17所述的方法，其中所述肿瘤选自肺癌、乳腺癌、胃癌、食管癌、肾上腺皮质癌、皮肤鳞癌、头颈部癌、甲状腺癌、肝癌、胰腺癌、胆管癌、结直肠癌、卵巢癌、宫颈癌、子宫内膜癌、阴道鳞状上皮癌、睾丸癌、前列腺癌、膀胱癌、尿路上皮癌、黑色素瘤、骨肉瘤、恶性淋巴瘤、神经母细胞瘤、顺铂获得性耐药肺癌、顺铂获得性耐药卵巢癌、顺铂获得性耐药乳腺癌、顺铂获得性耐药宫颈癌、顺铂获得性耐药胃癌、顺铂获得性耐药结直肠癌。
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