WO2023109563A1 - 一种卡巴他赛前药抗肿瘤制剂 - Google Patents

一种卡巴他赛前药抗肿瘤制剂 Download PDF

Info

Publication number
WO2023109563A1
WO2023109563A1 PCT/CN2022/136743 CN2022136743W WO2023109563A1 WO 2023109563 A1 WO2023109563 A1 WO 2023109563A1 CN 2022136743 W CN2022136743 W CN 2022136743W WO 2023109563 A1 WO2023109563 A1 WO 2023109563A1
Authority
WO
WIPO (PCT)
Prior art keywords
cabazitaxel
fatty alcohol
prodrug
small molecule
acid
Prior art date
Application number
PCT/CN2022/136743
Other languages
English (en)
French (fr)
Inventor
孙进
孙丙军
何仲贵
张宇
王丹平
罗聪
左诗意
李凌霄
杜超颖
Original Assignee
沈阳药科大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 沈阳药科大学 filed Critical 沈阳药科大学
Publication of WO2023109563A1 publication Critical patent/WO2023109563A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • 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
    • A61K47/54—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 compound
    • 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
    • A61K47/54—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 compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • 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
    • 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
    • A61K49/00—Preparations for testing in vivo
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D305/00—Heterocyclic compounds containing four-membered rings having one oxygen atom as the only ring hetero atoms
    • C07D305/14—Heterocyclic compounds containing four-membered rings having one oxygen atom as the only ring hetero atoms condensed with carbocyclic rings or ring systems
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention belongs to the field of new excipients and new dosage forms of pharmaceutical preparations, and relates to a cabazitaxel prodrug antitumor preparation, in particular to the construction of cabazitaxel-branched fatty alcohol prodrug and its self-assembled nanoparticles, and its application in drug delivery applications in the system.
  • Cabazitaxel belongs to taxane antineoplastic drugs, which has strong cytotoxicity and antitumor effect.
  • CX cytotoxicity and antitumor effect.
  • cabazitaxel has been associated with serious adverse effects, including gastrointestinal disturbances, anaphylaxis, renal failure, and neutropenia.
  • Cabazitaxel has very poor solubility in water, and the commercially available solution of cabazitaxel Tween 80 and ethanol must be used to aid dissolution.
  • Prodrug strategy is an effective way to improve the delivery efficiency of chemotherapeutic drugs. Structural modification of cabazitaxel through prodrug strategy can effectively improve the problems of poor solubility and severe side effects of cabazitaxel. Nano-drug delivery system can effectively prolong the circulation time of drugs in the body and enhance the anti-tumor effect. Therefore, self-assembled nano-drug delivery systems based on prodrug strategies combine the advantages of nanotechnology and prodrug strategies, and have the advantages of high drug loading and no need for solubilizers, and have been extensively studied in recent years.
  • Prodrugs usually consist of three parts: parent drug, connecting chain and side chain.
  • the parent drug and side chains are linked together by linking chains.
  • most of the existing cabazitaxel prodrugs use straight-chain fatty acids or fatty alcohols as side chains.
  • Aliphatic side chains can increase the structural flexibility of prodrug molecules, balance the intermolecular forces, and promote the self-assembly of prodrugs.
  • branched chain fatty alcohols can effectively disrupt the tight packing of prodrug molecules, which is expected to further enhance the self-assembly ability of prodrugs.
  • the carbon chain length of branched fatty alcohols may affect the formulation properties, in vivo fate, and antitumor effects of prodrug self-assembled nanoparticles.
  • the carbon chain length of branched fatty alcohols may affect the formulation properties, in vivo fate, and antitumor effects of prodrug self-assembled nanoparticles.
  • there is no study comparing the effect of the carbon chain length of branched fatty alcohols on the self-assembled nanoparticles of prodrugs and no research has reported the effects of branched fatty alcohols and linear fatty alcohols as side chains on the self-assembled nanoparticles of prodrugs.
  • the tumor microenvironment is significantly different from that of normal tissue cells.
  • Tumor cells produce a large amount of reactive oxygen species and glutathione, resulting in a tumor microenvironment with an imbalanced redox state.
  • Monosulfide bonds, disulfide bonds, and monoselenium bonds are redox-sensitive, which can intelligently respond to the high redox state in tumor cells and release drugs.
  • the elemental composition of the different linking chains differs, as do the redox sensitivities. Therefore, cabazitaxel prodrugs modified with different linker chains will have different formulation properties, fate in vivo and antitumor effects.
  • the chain length of the linking chain will also affect the redox sensitivity of the prodrug self-assembled nanoparticles, which in turn affects the antitumor activity of the prodrug self-assembled nanoparticles.
  • the purpose of the present invention is to overcome the deficiencies in the above-mentioned prior art, and provide a kind of cabazitaxel prodrug antitumor preparation, specifically cabazitaxel-branched fatty alcohol small molecule prodrug and its nanoparticle and its preparation and application,
  • the nanoparticle is a self-assembled nanoparticle and has the advantages of small particle size, uniform distribution, high drug loading, good stability, good antitumor effect and good safety.
  • the purpose of the present invention is to design and synthesize cabazitaxel prodrugs containing branched fatty alcohol side chains of different lengths, straight chain fatty alcohol side chains and different linking chains, and to prepare self-assembled nanoparticles thereof.
  • the experimental results show that the length of the branched fatty alcohol side chain, the structure of the fatty alcohol side chain (branched or straight chain), the elemental composition of the linking chain, and the length of the linking chain all affect the antitumor effect of the prodrug self-assembled nanoparticles. and security impact.
  • the invention provides more options for the development of new prodrug self-assembled nanometer drug delivery system, and meets the clinical urgent demand for high-efficiency-low toxicity chemotherapy preparations.
  • the present invention adopts the following technical solutions:
  • Cabazitaxel-branched chain fatty alcohol small molecule prodrug or its pharmaceutically acceptable salt, described cabazitaxel-branched chain fatty alcohol small molecule prodrug structure is as follows general formula (I), (II), (III ) as shown:
  • n 1 ⁇ 3;
  • R is a saturated or unsaturated C 3 -C 30 hydrocarbon group, said R is a hydrocarbon group containing a branched chain structure, and the branched chain is C 1 -C 18 alkyl, C 2 -C 18 alkenyl or C 2 -C One or more of 18 alkynyl groups.
  • R is a saturated or unsaturated C 3 -C 24 hydrocarbon group
  • the R is a hydrocarbon group with a branched chain structure
  • the branched chain is a straight chain C 6 -C 10 alkyl group, C 6 -C 10 alkenyl group or one or more of C 6 -C 10 alkynyl groups.
  • R is a saturated or unsaturated C 10 -C 24 hydrocarbon group
  • the R is a hydrocarbon group with a branched chain structure
  • the branched chain is a straight chain C 6 -C 10 alkyl group, C 6 -C 10 alkenyl group or one or more of C 6 -C 10 alkynyl groups.
  • R is a C 10 -C 24 alkyl group, said R is an alkyl group containing a branched chain structure, and said branched chain is a straight chain C 6 -C 10 alkyl group.
  • R is a saturated or unsaturated C 16 -C 24 hydrocarbon group, said R is a hydrocarbon group containing a branched chain structure, and said branched chain is a straight chain C 6 -C 10 alkyl group.
  • R is a C 16 -C 24 alkyl group, said R is an alkyl group containing a branched chain structure, and said branched chain is a straight chain C 6 -C 10 alkyl group.
  • R is an unsaturated hydrocarbon group
  • the sum of the number of alkenyl groups, alkynyl groups or alkenyl and alkynyl groups contained in the unsaturated hydrocarbon group is 1-5.
  • Described branched chain fatty alcohol is 2-hexyl-octanol, 1-heptyl-octanol, 2-hexyl-decyl alcohol, 1-butyl-dodecanol, 1-heptyl-nonanol, 1-octyl alcohol 1-nonyl-nonanol, 2-octyl-decanol, 2-heptyl-undecanol, 1-nonyl-decanol, 2-octyl-dodecanol, 2-decyl-tetradecanol or 2- A type of dodecyl-tetradecyl alcohol.
  • the branched chain fatty alcohol is 2-hexyl-decyl alcohol, 2-heptyl-undecyl alcohol, 2-octyl-dodecanol or 2-decyl-tetradecyl alcohol.
  • the cabazitaxel in the described cabazitaxel-branched fatty alcohol small molecule prodrug is connected with the branched fatty alcohol through a dibasic acid as a connecting chain, and the dibasic acid is a monothiodibasic acid, monoselenium substituted dibasic acid or dithiodibasic acid, wherein, the monothiodibasic acid is monothiodiacetic acid, monothiodipropionic acid or monothiodibutyric acid;
  • the dibasic acid is monoselenodiacetic acid, monoselenodipropionic acid or monoselenodibutyric acid;
  • the dithiodibasic acid is 2,2'-dithiodiacetic acid, 3,3'- Dithiodipropionic acid or 4,4'-dithiodibutyric acid.
  • the present invention provides cabazitaxel-2-hexyl-decanol prodrug, cabazitaxel-2-heptyl-undecyl alcohol prodrug, cabazitaxel-2-octyl-dodecanol prodrug and carbazitaxel Taxa-2-decyl-tetradecyl alcohol prodrug, choose 2,2'-dithiodiacetic acid as the connecting chain, and name the corresponding prodrugs as CTX-SS-HD, CTX-SS-HU, CTX -SS-OD and CTX-SS-DT, its structural formula is:
  • the present invention provides a small molecule prodrug of straight-chain fatty alcohol-cabazitaxel-arachiditol prodrug, choose 2,2'-dithiodiacetic acid as the connecting chain, and name the corresponding prodrug CTX-SS- AA, its structural formula is:
  • the present invention also provides 4,4'-dithiodibutyric acid as the cabazitaxel-2-octyl-dodecanol prodrug of the connecting chain, and the corresponding prodrug is named ⁇ -CTX-SS-OD, Its structural formula is:
  • the present invention also provides cabazitaxel-2-octyl-dodecanol prodrugs using monothiodiacetic acid and monoselenodiacetic acid as connecting chains, and the corresponding prodrugs are respectively named CTX-S-OD, CTX-Se-OD, its structural formula is:
  • the synthetic method of described cabazitaxel-branched fatty alcohol small molecule prodrug comprises the steps:
  • Step 1 after dibasic acid is dissolved into dibasic acid anhydride, carry out esterification reaction with branched chain fatty alcohol, obtain branched chain fatty alcohol-dibasic acid unilateral ester intermediate product, described branched chain fatty alcohol: dibasic acid anhydride
  • the molar ratio is (1-10):(5-15)
  • the dibasic acid is a monothio dibasic acid, a monoseleno dibasic acid or a dithio dibasic acid;
  • n 1 ⁇ 3;
  • R is a saturated or unsaturated C 3 -C 30 hydrocarbon group, said R is a hydrocarbon group containing a branched chain structure, and the branched chain is C 1 -C 18 alkyl, C 2 -C 18 alkenyl or C 2 -C One or more of 18 alkynyl groups.
  • the synthetic method of above-mentioned cabazitaxel-branched fatty alcohol small molecule prodrug specifically comprises the following steps:
  • the dibasic acid is monothiodiacetic acid, monothiodipropionic acid, monothiodibutyric acid, monoselenodiacetic acid, monoselenodipropionic acid, monoselenodi Butyric acid, 2,2'-dithiodiacetic acid, 3,3'-dithiodipropionic acid, or 4,4'-dithiodibutyric acid.
  • the branched fatty alcohol is C 3 -C 30 saturated or unsaturated fatty alcohol, and the branched chain is C 1 -C 18 alkyl, C 2 -C 18 alkenyl or C 2 one or more of -C 18 alkynyl groups.
  • the purity of the prepared cabazitaxel-branched fatty alcohol small molecule prodrug is above 99%.
  • the present invention simultaneously provides the synthetic method of monoselenodiacetic acid, comprises the steps:
  • the present invention also provides self-assembled nanoparticles of cabazitaxel-fatty alcohol small-molecule prodrug, wherein the prodrug self-assembled nanoparticles are non-PEGylated prodrug self-assembled nanoparticles, PEG modified/active targeting modified Prodrug self-assembled nanoparticles or prodrug self-assembled nanoparticles loaded with hydrophobic fluorescent substances/drugs.
  • the preparation method of the described cabazitaxel-branched fatty alcohol small molecule prodrug self-assembled nanoparticles comprises the following steps:
  • the preparation method dissolve a certain amount of prodrug into an appropriate amount of organic solvent, and slowly drop the solution into water under stirring , the prodrug spontaneously forms uniform nanoparticles; the organic solvent in the preparation is removed by vacuum rotary evaporation to obtain a nanocolloid solution without any organic solvent, which is the non-PEGylated cabazitaxel-fatty alcohol small molecule prodrug .
  • PEG modified/active targeting group modified cabazitaxel-fatty alcohol small molecule prodrug self-assembled nanoparticle preparation method a certain amount of PEG modifying agent/active targeting modifying agent and prodrug are dissolved into an appropriate amount In an organic solvent, under stirring, the solution is slowly added dropwise to water, and the prodrug spontaneously forms uniform nanoparticles; the organic solvent in the preparation is removed by a reduced-pressure rotary evaporation method, and a nano-colloid solution without any organic solvent is obtained.
  • PEG-modified/active targeting group modified cabazitaxel-fatty alcohol small molecule prodrug self-assembled nanoparticles, wherein, cabazitaxel-branched chain fatty alcohol small molecule prodrug and PEG modifier/active targeting modification
  • the mass ratio of the agent is 1:(0.1-1)
  • the PEG modifier is an amphiphilic polymer such as DSPE-PEG, TPGS, PLGA-PEG, PE-PEG or DSPE-PEG-FA or a targeting group
  • the active target Targeting modifiers are substances that can target specific tissues, such as antibodies, sugar residues, hormones, receptors, or ligands.
  • the preparation method dissolve a certain amount of PEG modifier, hydrophobic fluorescent substance/drug and prodrug to an appropriate amount In an organic solvent, the solution is slowly added dropwise into water under stirring, and the prodrug spontaneously forms uniform nanoparticles; the organic solvent in the preparation is removed by a reduced-pressure rotary evaporation method, and a nano-colloid solution without an organic solvent is obtained, namely Cabazitaxel-fatty alcohol small-molecule prodrug self-assembled nanoparticles loaded with hydrophobic fluorescent substance/drug, wherein, cabazitaxel-branched-chain fatty alcohol small-molecule prodrug and PEG modifier and hydrophobic fluorescent substance/drug
  • the mass ratio is 1:(0.1-1):(0.1-1).
  • cabazitaxel-branched fatty alcohol small molecule prodrug or the self-assembled nanoparticle in the preparation of injection, oral or local drug delivery systems.
  • the lyophilized powder injection of cabazitaxel-branched fatty alcohol small molecule prodrug self-assembled nanoparticle comprises cabazitaxel-branched fatty alcohol small molecule prodrug self-assembled nanoparticle solution and a freeze-drying protective agent
  • the concentration of the self-assembled nanoparticle solution is 0.1mg/mL-20mg/mL
  • the described freeze-drying protective agent is monosaccharides such as glucose and galactose, disaccharides such as trehalose and sucrose, and mannose Alcohol, sorbitol, xylitol and other polyols, one or more of polymers such as polyethylene glycol, hydroxyethyl starch or dextran
  • the amount of the freeze-drying protectant is 1%-20% (W/V) (that is, the mass concentration of the lyoprotectant is 10 g/L-200 g/L), preferably 5%-10% (W/V).
  • the preparation method of the described cabazitaxel-branched fatty alcohol small molecule prodrug self-assembled nanoparticle freeze-dried powder injection comprises the following steps:
  • Cabazitaxel-branched fatty alcohol small molecule prodrug self-assembled nanoparticle solution was placed in a vial, added with a lyoprotectant, and after the lyoprotectant was completely dissolved, it was pre-frozen at -80°C for 8-12 hours, and then placed in a lyophilizer to freeze-dry for 24-72 hours to obtain a white cake, which is the self-assembled nanoparticle freeze-dried powder injection of cabazitaxel-branched fatty alcohol small molecule prodrug.
  • the present invention has designed and synthesized cabazitaxel-fatty alcohol small molecule prodrugs containing different fatty alcohol side chains and different connecting chains, and the synthesis method is simple and easy; and prepared cabazitaxel with smaller particle size and uniform particle size distribution Sai-fatty alcohol small molecule prodrug self-assembled nanoparticles, the preparation method is simple and easy; The effects of the linking chains on the formulation properties, in vivo fate and antitumor activity of prodrug self-assembled nanoparticles.
  • Figure 1 is a diagram of the tumor volume change in the in vivo anti-tumor experiment of the PEG-modified small molecule prodrug self-assembled nanoparticles of Example 13 of the present invention.
  • Fig. 2 is a diagram of the body weight change of mice in the in vivo anti-tumor experiment of the PEG-modified small molecule prodrug self-assembled nanoparticles of Example 13 of the present invention.
  • Fig. 3 is a graph of blood routine indicators in the in vivo anti-tumor experiment of the PEG-modified small molecule prodrug self-assembled nanoparticles of Example 13 of the present invention.
  • Fig. 4 is a graph showing the change in tumor volume in the in vivo anti-tumor experiment of the PEG-modified small molecule prodrug self-assembled nanoparticles of Example 14 of the present invention.
  • Fig. 5 is a diagram of the body weight change of mice in the in vivo anti-tumor experiment of the PEG-modified small molecule prodrug self-assembled nanoparticles of Example 14 of the present invention.
  • Fig. 6 is a graph of tumor burden in an in vivo anti-tumor experiment of the PEG-modified small molecule prodrug self-assembled nanoparticles of Example 14 of the present invention.
  • Fig. 7 is a graph of blood routine indicators in the in vivo anti-tumor experiment of the PEG-modified small molecule prodrug self-assembled nanoparticles of Example 14 of the present invention.
  • Fig. 8 is a diagram of biochemical routine indicators in the in vivo anti-tumor experiment of the PEG-modified small molecule prodrug self-assembled nanoparticles of Example 14 of the present invention.
  • Fig. 9 is a graph showing the change in tumor volume in the in vivo anti-tumor experiment of the PEG-modified small molecule prodrug self-assembled nanoparticles of Example 15 of the present invention.
  • Fig. 10 is a diagram of the body weight change of mice in the in vivo anti-tumor experiment of the PEG-modified small molecule prodrug self-assembled nanoparticles of Example 15 of the present invention.
  • Fig. 11 is a graph of tumor burden in an in vivo anti-tumor experiment of the PEG-modified small molecule prodrug self-assembled nanoparticles of Example 15 of the present invention.
  • Fig. 12 is a graph showing the change in tumor volume in the in vivo anti-tumor experiment of the PEG-modified small molecule prodrug self-assembled nanoparticles of Example 16 of the present invention.
  • Fig. 13 is a graph showing the body weight changes of mice in the in vivo anti-tumor experiment of the PEG-modified small molecule prodrug self-assembled nanoparticles of Example 16 of the present invention.
  • Fig. 14 is a graph of tumor burden in an in vivo anti-tumor experiment of the PEG-modified small molecule prodrug self-assembled nanoparticles of Example 16 of the present invention.
  • Fig. 15 is a graph showing the change in tumor volume in the in vivo anti-tumor experiment of the PEG-modified small molecule prodrug self-assembled nanoparticles of Example 17 of the present invention.
  • Fig. 16 is a graph showing the body weight changes of mice in the in vivo anti-tumor experiment of the PEG-modified small molecule prodrug self-assembled nanoparticles of Example 17 of the present invention.
  • the product is separated using a preparation liquid to prepare the prodrug of cabazitaxel-2-hexyl-decanol with 2,2'dithiodiacetic acid as the connecting chain.
  • the product is separated using a preparation solution to prepare the prodrug of cabazitaxel-2-octyl-dodecanol with 2,2'dithiodiacetic acid as the connecting chain.
  • the preparation solution is used to separate the product to prepare the cabazitaxel-arachiditol prodrug with 2,2'dithiodiacetic acid as the connecting chain.
  • the product is separated using a preparation solution to prepare a prodrug of cabazitaxel-2-octyl-dodecanol with 4,4'-dithiodibutyric acid as the connecting chain.
  • the preparation solution is used to separate the product to prepare the cabazitaxel-2-octyl-dodecanol prodrug with the monosulfide bond as the linking chain.
  • Example 8 Synthesis of the Cabazitaxel-2-octyl-dodecanol prodrug with a single selenium bond as the connecting chain
  • the preparation solution is used to separate the product to prepare the cabazitaxel-2-octyl-dodecanol prodrug with the monoselenium bond as the linking chain.
  • cabazitaxel prodrugs with different linking chains and fatty alcohol side chains could form self-assembled nanoparticles
  • cabazitaxel-branched-chain fatty alcohol small molecule prodrug self-assembled nanoparticles had higher Smaller particle size and size distribution of fatty alcohol small molecule prodrug self-assembled nanoparticles.
  • the particle size of cabazitaxel-branched fatty alcohol small molecule prodrug self-assembled nanoparticles is about 100nm, and the particle size distribution is very uniform, and the particle size distribution is about 0.1, which is helpful for the nanoparticles to pass through the high-throughput of solid tumors. Permeability and retention effects enable tumor-targeted accumulation.
  • the surface charge of nanoparticles is about -20mV, which is beneficial to prevent the aggregation of nanoparticles through charge repulsion.
  • Example 10 Preparation of lyophilized powder of small molecule prodrug self-assembled nanoparticles
  • Example 9 Take the PEGylated monosulfide bond prepared in Example 9 as the cabazitaxel-2-octyl-dodecanol prodrug self-assembled nanoparticle of the connecting chain in a vial, and use 5% sucrose, 10% Sucrose, 5% lactose, 10% lactose, 5% trehalose, 10% trehalose, 5% mannitol, 10% mannitol, 5% glucose, 10% glucose were used as lyoprotectants, and then placed in a lyophilizer Freeze drying for 24 hours yielded a white cake. The resulting lyophilized powder was reconstituted with deionized water and the particle size and surface charge were measured. The results are shown in Table 2. The results showed that the particle size and particle size distribution of the prodrug self-assembled nanoparticle lyophilized powder prepared with a single lyoprotectant were larger after reconstitution.
  • Example 11 Preparation of small molecule prodrug self-assembled nanoparticles lyophilized powder
  • Example 9 Take the PEGylated monosulfide bond prepared in Example 9 as the cabazitaxel-2-octyl-dodecanol prodrug self-assembled nanoparticle of the connecting chain in a vial, and use 5% lactose and 5% Mannitol, 5% lactose and 5% maltose, 5% lactose and 5% sucrose, 5% lactose and 5% trehalose were used as lyoprotectant, and the lyoprotectant was completely dissolved and placed in -80°C for 12 hours , and then freeze-dried in a freeze dryer for 24 hours to obtain a white cake.
  • the resulting lyophilized powder was reconstituted with deionized water and the particle size and surface charge were measured.
  • the results are shown in Table 3. The results showed that: compared with the lyophilized powder prepared with a single protective agent, the particle size of the lyophilized powder prepared with the compound protective agent was smaller after reconstitution.
  • Example 12 Pharmacokinetic study of small molecule prodrug self-assembled nanoparticles
  • CTX-SS-OD nanoparticles In contrast, the circulation time of PEG-modified small molecule prodrug self-assembled nanoparticles was significantly prolonged, the bioavailability was significantly improved, and the pharmacokinetic parameters were significantly improved.
  • the overall AUC 0-24h (sum of prodrug and parent drug) of CTX-SS-OD nanoparticles, CTX-Se-OD nanoparticles and CTX-S-OD nanoparticles is 395.47 times that of cabazitaxel solution, respectively. 277.37 times, 260.19 times; t 1/2 were 19.72 times, 10.16 times, 11.51 times of cabazitaxel solution; C max were 76.35 times, 51.80 times, 51.18 times of cabazitaxel solution.
  • the improvement of pharmacokinetic parameters of CTX-SS-OD nanoparticles is the most obvious, indicating that disulfide bonds are more advantageous as linking chains.
  • Example 13 In vivo anti-tumor experiments of PEGylated small molecule prodrug self-assembled nanoparticles
  • Mouse breast cancer cell suspension (4T1, 5x10 6 cells/100 ⁇ L) was inoculated subcutaneously on the dorsal side of female BALB/c mice.
  • the tumor-bearing mice were randomly divided into 6 groups, 8 in each group: saline group (Saline), cabazitaxel solution group, CTX-SS-HU nanoparticle group, CTX-SS-HD nanoparticle group, CTX-SS-OD nanoparticle group and CTX-SS-DT nanoparticle group.
  • the nanoparticles used for administration were the PEG-modified small molecule prodrug self-assembled nanoparticles prepared in Example 9, and the administration dose was 10 mg/kg (calculated based on the concentration of cabazitaxel). Dosing once every other day for 5 consecutive doses. After administration, the survival status and body weight changes of the mice were detected every day, and the tumor volume was measured. One day after the last administration, the mice were sacrificed, and the organs and tumors were obtained for further analysis and evaluation. The results are shown in Figures 1-3. Figure 1 shows that in the saline group, the tumor volume grew rapidly, reaching around 1000 mm at day 10.
  • Example 14 In vivo anti-tumor experiments of PEGylated small molecule prodrug self-assembled nanoparticles
  • Mouse breast cancer cell suspension (4T1, 5x10 6 cells/100 ⁇ L) was inoculated subcutaneously on the dorsal side of female BALB/c mice.
  • the tumor-bearing mice were randomly divided into 7 groups, 8 in each group: saline group (Saline), CTX-SS-OD nanoparticles 2mg/kg, 10mg/kg group, 20mg/kg group and CTX-SS-AA nanoparticles 2mg/kg, 10mg/kg group, 20mg/kg group.
  • the nanoparticle used for administration is the PEG-modified small molecule prodrug self-assembled nanoparticle prepared in Example 9, and the administration dose is calculated based on the concentration of cabazitaxel. Dosing once every other day for 5 consecutive doses. After administration, the survival status and body weight changes of the mice were detected every day, and the tumor volume was measured. The mice were sacrificed one day after the last administration, and the organs and tumors were obtained for further analysis and evaluation. The results are shown in Figures 4-8. Figure 4 shows that in the normal saline group, the tumor volume increased rapidly, reaching about 1000 mm 3 on the 10th day, while the tumor growth in the prodrug self-assembled nanoparticles group was inhibited.
  • the tumor volume of the CTX-SS-OD nanoparticle group is smaller than that of the CTX-SS-AA nanoparticle group;
  • Figure 5 shows that when the dosage is the same, the body weight ratio of the CTX-SS-AA nanoparticle group The CTX-SS-OD nanoparticle group decreased more seriously;
  • Figure 6 shows that the prodrug self-assembled nanoparticle group has a lower tumor burden than the normal saline group, and at the same concentration, the tumor burden of the CTX-SS-OD nanoparticle is higher than that of the CTX -SS-AA is lower;
  • Figure 7 shows that when the dosage is equivalent, the number of leukocytes in the CTX-SS-OD nanoparticle group decreases less;
  • Figure 8 shows that when the dosage is 20mg/kg, the CTX-SS-OD The urea nitrogen rise was less in the nanoparticle group.
  • Example 15 In vivo anti-tumor experiments of PEGylated small molecule prodrug self-assembled nanoparticles
  • Mouse breast cancer cell suspension (4T1, 5x10 6 cells/100 ⁇ L) was inoculated subcutaneously on the dorsal side of female BALB/c mice.
  • the tumor-bearing mice were randomly divided into 5 groups, 8 mice in each group: saline group (Saline), cabazitaxel solution group, CTX-S-OD nanoparticle group, CTX-SS-OD nanoparticle group, CTX-Se-OD nanoparticle group.
  • the nanoparticles used for administration were the PEG-modified small molecule prodrug self-assembled nanoparticles prepared in Example 9, and the administration dose was 4 mg/kg (calculated based on the concentration of cabazitaxel).
  • Figure 9 shows that in the normal saline group, the tumor volume increased rapidly, reaching about 700mm 3 on the 10th day, and the tumor volume of the CTX-S-OD nanoparticle group was comparable to that of the CTX-Se-OD nanoparticle group ( ⁇ 400mm 3 ) , the tumor volume of the CTX-SS-OD nanoparticle group and the cabazitaxel solution group was small ( ⁇ 200mm 3 );
  • Figure 10 shows that the body weights of the three nanoparticle groups had no significant change, and the body weight of the cabazitaxel solution group The body weight decreased significantly;
  • Figure 11 shows that there is no statistical difference between the tumor burden of the CTX-SS-OD nanoparticle group and the cabazitaxel solution group.
  • CTX-SS-OD nanoparticles have a good anti-tumor effect, but also have better safety than cabazitaxel solution; disulfide bonds have more advantages than monosulfide bonds and monoselenium bonds as connecting chains .
  • Example 16 In vivo anti-tumor experiments of PEGylated small molecule prodrug self-assembled nanoparticles
  • Mouse breast cancer cell suspension (4T1, 5x10 6 cells/100 ⁇ L) was inoculated subcutaneously on the dorsal side of female BALB/c mice.
  • the tumor-bearing mice are randomly divided into 8 groups, 8 in each group: saline group (Saline), cabazitaxel solution 2mg/kg group, and cabazitaxel solution 10mg /kg group, cabazitaxel solution 15mg/kg group, CTX-SS-OD nanoparticle 2mg/kg group, CTX-SS-OD nanoparticle 10mg/kg group, CTX-SS-OD nanoparticle 15mg/kg group, CTX-SS-OD nanoparticles 20mg/kg group.
  • the nanoparticle used for administration is the PEG-modified small molecule prodrug self-assembled nanoparticle prepared in Example 9, and the administration dose is calculated based on the concentration of cabazitaxel. Dosing once every other day for 5 consecutive administrations. The survival status and body weight changes of the mice were detected every day, and the tumor volume was measured. The mice were sacrificed one day after the last administration, and the organs and tumors were obtained for further analysis and evaluation. The results are shown in Figures 12-14. Figures 12 and 14 show that in the blank control group, the tumor volume increased rapidly and reached about 800 mm 3 on the 10th day.
  • CTX-SS-OD nanoparticles have the same anti-tumor effect as the cabazitaxel solution, and their toxicity is lower than that of the cabazitaxel solution.
  • Example 17 In vivo anti-tumor experiments of PEGylated small molecule prodrug self-assembled nanoparticles
  • Mouse breast cancer cell suspension (4T1, 5x10 6 cells/100 ⁇ L) was inoculated subcutaneously on the dorsal side of female BALB/c mice.
  • the tumor-bearing mice were randomly divided into 7 groups, 8 mice in each group: saline group (Saline), CTX-SS-OD nanoparticle 20mg/kg group, CTX-SS- OD nanoparticle 30mg/kg group, CTX-SS-DT nanoparticle 20mg/kg group, CTX-SS-DT nanoparticle 30mg/kg group, ⁇ -CTX-SS-OD nanoparticle 20mg/kg group, ⁇ -CTX- SS-OD nanoparticles 30mg/kg group.
  • the nanoparticle used for administration is the PEG-modified small molecule prodrug self-assembled nanoparticle prepared in Example 9, and the administration dose is calculated based on the concentration of cabazitaxel. Dosing once every other day for 5 consecutive administrations. The survival status and body weight changes of the mice were detected every day, and the tumor volume was measured. The mice were sacrificed one day after the last administration, and the organs and tumors were obtained for further analysis and evaluation. The results are shown in Figures 15-16. Figure 15 shows that three kinds of nanoparticles all have good anti-tumor effect, and significantly inhibit the growth of tumor; -OD nanoparticles, CTX-SS-DT nanoparticles, ⁇ -CTX-SS-OD nanoparticles have better security.
  • Example 18 Tolerance experiment of PEGylated small molecule prodrug self-assembled nanoparticles
  • mice Female BALB/c mice were divided into 11 groups with 3 mice in each group. Among them, the four groups were injected with 30 mg/kg, 40 mg/kg, 50 mg/kg, and 60 mg/kg of cabazitaxel solution through tail vein respectively. The other seven groups were given tail vein injections of CTX-SS-HD nanoparticles, CTX-SS-HU nanoparticles, CTX-SS-OD nanoparticles, CTX-SS-DT nanoparticles, CTX-S-OD nanoparticles, CTX - SS-AA nanoparticles, ⁇ -CTX-SS-OD nanoparticles, administered once every 8 hours, 200 mg/kg once administered, the dosage is calculated by the concentration of cabazitaxel, until all the mice died.
  • CTX-SS-HD nanoparticles CTX-SS-HU nanoparticles
  • CTX-SS-OD nanoparticles CTX-SS-DT nanoparticles containing different branched fatty alcohol side chains
  • CTX-SS-OD The safety of nanoparticles is the best, indicating that 2-octyl-dodecyl alcohol has more advantages in safety as a side chain
  • CTX-SS-OD nanoparticles are safer than CTX-SS-AA nanoparticles containing straight-chain fatty alcohols, indicating that branched-chain fatty alcohols have more advantages in safety than straight-chain fatty alcohols
  • ⁇ -CTX-SS-OD nanoparticles containing longer linker chains have better safety than CTX-SS-OD nanoparticles when compared with link

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Nanotechnology (AREA)
  • Optics & Photonics (AREA)
  • Biomedical Technology (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Physics & Mathematics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

涉及一种卡巴他赛前药抗肿瘤制剂，设计合成了涉及通式(I)(II)(III)的含有不同脂肪醇侧链与不同连接链的卡巴他赛-脂肪醇小分子前药，并制备了自组装纳米粒。结果表明：卡巴他赛-脂肪醇小分子前药自组装纳米粒能有效提高卡巴他赛疗效，降低其毒副作用。支链脂肪醇侧链的长度、脂肪醇侧链的结构、连接链元素组成以及连接链长度会显著影响卡巴他赛前药自组装纳米粒的制剂学性质及抗肿瘤活性，该前药自组装纳米粒具有比卡巴他赛-直链脂肪醇小分子前药自组装纳米粒更高的抗肿瘤活性与更低的毒性。

Description

一种卡巴他赛前药抗肿瘤制剂 技术领域：
本发明属于药物制剂新辅料和新剂型领域，涉及一种卡巴他赛前药抗肿瘤制剂，具体涉及卡巴他赛-支链脂肪醇前药及其自组装纳米粒的构建，以及其在药物递送系统中的应用。
背景技术：
近年来恶性肿瘤的发病率越来越高，严重威胁着人类的健康。化疗是癌症治疗中最有效的策略之一。卡巴他赛(Cabazitaxel,CTX)属于紫杉烷类抗肿瘤药物，具有很强的细胞毒性和抗肿瘤作用。然而，卡巴他赛会引发严重的不良反应，包括胃肠道功能紊乱、过敏反应、肾功能衰竭以及中性粒细胞减少。卡巴他赛在水中溶解性极差，市售的卡巴他赛溶液剂
Figure PCTCN2022136743-appb-000001
必须使用吐温80和乙醇助溶。即使是在增溶剂的帮助下，卡巴他赛溶液剂的稳定性也很差，稀释后容易沉淀，药动学性质较差。这些缺点限制了卡巴他赛的临床应用。
前药策略是提高化疗药物递送效率的有效方法。通过前药策略对卡巴他赛进行结构修饰可以有效的改善卡巴他赛溶解性差、毒副作用大等问题。纳米药物递送系统可以有效的延长药物在体内的循环时间，增强抗肿瘤效果。因此，基于前药策略的自组装纳米药物递送系统结合了纳米技术和前药策略的优点，并且具有载药量高、不需要增溶剂等优势，近年来得到广泛研究。
前药通常由母药、连接链和侧链三部分组成。通过连接链将母药和侧链连接到一起。为了构建具有自组装能力的前药，现有的卡巴他赛前药大多使用直链结构的脂肪酸或脂肪醇作为侧链。脂肪族侧链能够增加前药分子的结构灵活性，平衡分子间作用力，促进前药自组装。我们推测支链脂肪醇能够有效打乱前药分子的紧密堆积，有望进一步增强前药的自组装能力。此外，支链脂肪醇的碳链长度可能会影响前药自组装纳米粒的制剂学性质、体内命运和抗肿瘤效果。目前尚无研究对比支链脂肪醇碳链的长度对前药自组装纳米粒的影响，也没有研究报道过支链脂肪醇和直链脂肪醇作为侧链对前药自组装纳米粒的影响。
肿瘤微环境与正常组织细胞的微环境具有显著差异。肿瘤细胞内产生大量的活性氧和谷胱甘肽，造成氧化还原状态失衡的肿瘤微环境。单硫键、二硫键以及单硒键均具有氧化还原双敏感的特性，可以智能响应肿瘤细胞内的高氧化还原状态并释放药物。不同连接链的元素组成不同，氧化还原敏感性也不同。因此，不同连接链修饰的卡巴他赛前药会具有不同的制剂学性质、体内命运以及抗肿瘤效果。此外，连接链的链长也会影响前药自组装纳米粒的氧化还原敏感性，进而影响前药自组装纳米粒的抗肿瘤活性。
发明内容：
本发明的目的是克服上述现有技术存在的不足，提供一种卡巴他赛前药抗肿瘤制剂，具体为卡巴他赛-支链脂肪醇小分子前药及其纳米粒及其制备与应用，该纳米粒为自组装纳米粒，具有粒径较小且分布均一、载药量高、稳定性好、抗肿瘤效果好和安全性好的优点。
本发明的目的是设计和合成含有不同长度支链脂肪醇侧链、直链脂肪醇侧链以及不同连接链的卡巴他赛前药，并制备其自组装纳米粒。实验结果表明，支链脂肪醇侧链的长度、脂肪醇侧链的结构(支链或直链)、连接链的元素组成，以及连接链的长度都会对前药自组装纳米粒的抗肿瘤效果和安全性产生影响。本发明为开发新的前药自组装纳米药物递送系统提供更多的选择，满足了临床对于高效-低毒化疗制剂的迫切需求。
为实现上述目的，本发明采用以下技术方案：
卡巴他赛-支链脂肪醇小分子前药或其药学上可接受的盐，所述的卡巴他赛-支链脂肪醇小分子前药结构如下通式(I)、(II)、(III)所示：
Figure PCTCN2022136743-appb-000002
Figure PCTCN2022136743-appb-000003
其中，n＝1～3；
R为饱和或不饱和的C 3-C 30烃基，所述R为含支链结构的烃基，所述支链为C 1-C 18烷基、C 2-C 18烯基或C 2-C 18炔基中的一种或多种。
进一步地，R为饱和或不饱和的C 3-C 24烃基，所述R为含支链结构的烃基，所述支链为直链C 6-C 10烷基、C 6-C 10烯基或C 6-C 10炔基中的一种或多种。
进一步地，R为饱和或不饱和的C 10-C 24烃基，所述R为含支链结构的烃基，所述支链为直链C 6-C 10烷基、C 6-C 10烯基或C 6-C 10炔基中的一种或多种。
进一步地，R为C 10-C 24烷基，所述R为含支链结构的烷基，所述支链为直链C 6-C 10烷基。
进一步地，R为饱和或不饱和的C 16-C 24烃基，所述R为含支链结构的烃基，所述支链为直链C 6-C 10烷基。
进一步地，R为C 16-C 24烷基，所述R为含支链结构的烷基，所述支链为直链C 6-C 10烷基。
所述的R为不饱和烃基时，不饱和烃基中含有的烯基、炔基或烯基与炔基数目之和为1-5个。
所述的支链脂肪醇为2-己基-辛醇、1-庚基-辛醇、2-己基-癸醇、1-丁基-十二醇、1-庚基-壬醇、1-辛基-壬醇、2-辛基-癸醇、2-庚基-十一醇、1-壬基-癸醇、2-辛基-十二醇、2-癸基-十四醇或2-十二烷基-十四醇中的一种。
优选地，所述的支链脂肪醇为2-己基-癸醇、2-庚基-十一醇、2-辛基-十二醇或2-癸基-十四醇。
所述的卡巴他赛-支链脂肪醇小分子前药中的卡巴他赛与支链脂肪醇通过二元酸为连接链连接，所述的二元酸为单硫代二元酸、单硒代二元酸或二硫代二元酸，其中，所述的单硫代二元酸为单硫代二乙酸、单硫代二丙酸或单硫代二丁酸；所述的单硒代二元酸为单硒代二 乙酸、单硒代二丙酸或单硒代二丁酸；所述的二硫代二元酸为2,2'-二硫代二乙酸、3,3'-二硫代二丙酸或4,4'-二硫代二丁酸。
具体地，本发明提供卡巴他赛-2-己基-癸醇前药、卡巴他赛-2-庚基-十一醇前药、卡巴他赛-2-辛基-十二醇前药以及卡巴他赛-2-癸基-十四醇前药，选择2,2'-二硫代二乙酸为连接链，将对应的前药分别命名为CTX-SS-HD、CTX-SS-HU、CTX-SS-OD和CTX-SS-DT，其结构式为：
Figure PCTCN2022136743-appb-000004
Figure PCTCN2022136743-appb-000005
本发明提供了一种直链脂肪醇小分子前药-卡巴他赛-花生醇前药，选择2,2'-二硫代二乙酸为连接链，将对应的前药命名为CTX-SS-AA，其结构式为：
Figure PCTCN2022136743-appb-000006
以2,2'-二硫代二乙酸为连接链的卡巴他赛-花生醇前药(CTX-SS-AA)
本发明还提供了4,4'-二硫代二丁酸作为连接链的卡巴他赛-2-辛基-十二醇前药，将对应的前药命名为γ-CTX-SS-OD，其结构式为：
Figure PCTCN2022136743-appb-000007
以4,4'-二硫代二丁酸作为连接链的卡巴他赛-2-辛基-十二醇前药(γ-CTX-SS-OD)
本发明还提供了使用单硫代二乙酸、单硒代二乙酸作为连接链的卡巴他赛-2-辛基-十二醇前药，将对应的前药分别命名为CTX-S-OD、CTX-Se-OD，其结构式为：
Figure PCTCN2022136743-appb-000008
所述的卡巴他赛-支链脂肪醇小分子前药的合成方法，包括如下步骤：
步骤1：将二元酸溶解成二元酸酐后，与支链脂肪醇进行酯化反应，获得支链脂肪醇-二元酸单边酯中间产物，所述的支链脂肪醇：二元酸酐摩尔比为(1-10):(5-15)，所述的二元酸为单硫代二元酸、单硒代二元酸或二硫代二元酸；
步骤2：支链脂肪醇-二元酸单边酯与卡巴他赛发生成酯反应，得到终产物卡巴他赛-支链脂肪醇小分子前药，其中，按摩尔比，支链脂肪醇-二元酸单边酯：卡巴他赛＝1:(0.5-10)，反应方程式如下：
Figure PCTCN2022136743-appb-000009
Figure PCTCN2022136743-appb-000010
其中，n＝1～3；
R为饱和或不饱和的C 3-C 30烃基，所述R为含支链结构的烃基，所述支链为C 1-C 18烷基、C 2-C 18烯基或C 2-C 18炔基中的一种或多种。
上述卡巴他赛-支链脂肪醇小分子前药的合成方法，具体包括如下步骤：
(1)将二元酸溶于乙酸酐中，室温下搅拌2-4小时，使二元酸成二元酸酐，反应完毕后，加入甲苯，减压旋转蒸发除去甲苯和乙酸酐；
(2)取支链脂肪醇和4-二甲氨基吡啶(DMAP)，与步骤(1)所得二元酸酐一并溶于二氯甲烷中，室温条件下搅拌12-18小时，通过层析柱分离得到中间产物：支链脂肪醇-二元酸单边酯；
(3)取1-(3-二甲氨基丙基)-3-乙基碳化二亚胺盐酸盐(EDCI)、1-羟基苯并三唑(HOBt)和4-二甲氨基吡啶(DMAP)，与中间产物支链脂肪醇-二元酸单边酯一并溶于无水二氯甲烷中，冰浴搅拌2-4小时，然后加入卡巴他赛，室温条件下搅拌24-48小时，再经制备液相分离纯化得终产物：卡巴他赛-支链脂肪醇小分子前药。
所述的卡巴他赛-支链脂肪醇小分子前药的合成方法反应全程在氮气保护下进行。
所述的步骤(1)中，二元酸为单硫代二乙酸、单硫代二丙酸、单硫代二丁酸、单硒代二乙酸、单硒代二丙酸、单硒代二丁酸、2,2'-二硫代二乙酸、3,3'-二硫代二丙酸或4,4'-二硫代二丁酸。
所述的步骤(1)中，按比例，二元酸：乙酸酐＝1:(1-10)，优选为1:(1-2)，单位mmol:ml。所述的步骤(2)中，支链脂肪醇为C 3-C 30饱和或不饱和脂肪醇，所述的支链为C 1-C 18烷基、C 2-C 18烯基或C 2-C 18炔基中的一种或多种。
所述的步骤(2)中，按摩尔比，DMAP：支链脂肪醇：二元酸酐＝1:(1-10):(5-15)，优选为1:(2-5):(10-15)。
所述的步骤(3)中，按摩尔比，中间产物支链脂肪醇-二元酸单边酯：HOBt：EDCI：DMAP：卡巴他赛＝1:(1-10):(2-6):(0.2-5):(0.5-10)，优选为1:(1-2):(2-4):(0.5-2):(0.8-2)。
所述的步骤(3)中，制备的卡巴他赛-支链脂肪醇小分子前药纯度在99％以上。
本发明同时提供单硒代二乙酸的合成方法，包括如下步骤：
将硒粉与少量水加入茄形瓶中，茄形瓶置于冰水浴中，缓缓滴加硼氢化钠水溶液，搅拌至溶液澄清透明，升温至100-110℃，搅拌30-45分钟，降至室温后，滴加溴乙酸水溶液，反应10-12小时后，将反应液过滤，加乙酸乙酯萃取直至水层无色，将乙酸乙酯层干燥后减压旋转蒸发除去乙酸乙酯，得到产物。上述反应全程都在氮气保护下进行。
本发明还提供了卡巴他赛-脂肪醇小分子前药的自组装纳米粒，所述的前药自组装纳米粒为非PEG化的前药自组装纳米粒、PEG修饰/主动靶向修饰的前药自组装纳米粒或包载疏水性荧光物质/药物的前药自组装纳米粒。
所述的卡巴他赛-支链脂肪醇小分子前药自组装纳米粒的制备方法，包括如下步骤：
当为非PEG化的卡巴他赛-脂肪醇小分子前药自组装纳米粒时，制备方法：将一定量的前药溶解到适量的有机溶剂中，搅拌下将该溶液缓缓滴加到水中，前药自发形成均匀的纳米粒；采用减压旋转蒸发法除去制剂中的有机溶剂，得到不含任何有机溶剂的纳米胶体溶液，即为非PEG化的卡巴他赛-脂肪醇小分子前药。
当为PEG修饰/主动靶向基团修饰的卡巴他赛-脂肪醇小分子前药自组装纳米粒的制备方法：将一定量的PEG修饰剂/主动靶向修饰剂和前药溶解到适量的有机溶剂中，搅拌下，将该溶液缓缓滴加到水中，前药自发形成均匀的纳米粒；采用减压旋转蒸发法除去制剂中的有机溶剂，得到不含任何有机溶剂的纳米胶体溶液，即为PEG修饰/主动靶向基团修饰的卡巴他赛-脂肪醇小分子前药自组装纳米粒，其中，卡巴他赛-支链脂肪醇小分子前药与PEG修饰剂/主动靶向修饰剂的质量比为1:(0.1-1)，PEG修饰剂为DSPE-PEG、TPGS、PLGA-PEG、PE-PEG或DSPE-PEG-FA等两亲性聚合物或靶向基团，主动靶向修饰剂为抗体、糖残基、激素、受体或配体等能够靶向到特定组织的物质。
当为包载疏水性荧光物质/药物的卡巴他赛-脂肪醇小分子前药自组装纳米粒时，制备方法：将一定量的PEG修饰剂、疏水性荧光物质/药物以及前药溶解到适量的有机溶剂中，搅拌下将该溶液缓缓滴加到水中，前药自发形成均匀的纳米粒；采用减压旋转蒸发法除去制剂中的有机溶剂，得到不含有机溶剂的纳米胶体溶液，即为包载疏水性荧光物质/药物的卡巴他赛-脂肪醇小分子前药自组装纳米粒，其中，卡巴他赛-支链脂肪醇小分子前药与PEG修饰剂以及疏水性荧光物质/药物的质量比为1:(0.1-1):(0.1-1)。
所述的卡巴他赛-支链脂肪醇小分子前药或所述的自组装纳米粒在制备抗肿瘤药物中的 应用。
所述的卡巴他赛-支链脂肪醇小分子前药或所述的自组装纳米粒在制备注射给药、口服给药或局部给药系统中的应用。
所述的卡巴他赛-支链脂肪醇小分子前药或所述的自组装纳米粒在制备提高疗效、降低毒性药物传递系统中的应用。
所述的卡巴他赛-支链脂肪醇小分子前药自组装纳米粒的冻干粉针剂，所述的冻干粉针剂包含卡巴他赛-支链脂肪醇小分子前药自组装纳米粒溶液和冻干保护剂，所述自组装纳米粒溶液的浓度为0.1mg/mL-20mg/mL，所述的冻干保护剂为葡萄糖、半乳糖等单糖，海藻糖、蔗糖等二糖，甘露醇、山梨醇、木糖醇等多元醇，聚乙二醇、羟乙基淀粉或葡聚糖等聚合物中的一种或几种，所述的冻干保护剂用量为1％-20％(W/V)(也即冻干保护剂的质量浓度为10g/L-200g/L)，优选为5％-10％(W/V)。
所述的卡巴他赛-支链脂肪醇小分子前药自组装纳米粒冻干粉针剂的制备方法，包括如下步骤：
卡巴他赛-支链脂肪醇小分子前药自组装纳米粒溶液置于西林瓶中，加入冻干保护剂，待冻干保护剂完全溶解后，置于-80℃环境下预冻8-12小时，然后置于冻干机中冷冻干燥24-72小时，得到白色饼块，即为卡巴他赛-支链脂肪醇小分子前药自组装纳米粒冻干粉针剂。
本发明的有益效果：
(1)本发明设计合成了含有不同脂肪醇侧链与不同连接链的卡巴他赛-脂肪醇小分子前药，合成方法简单易行；并制备了粒径较小、粒度分布均一的卡巴他赛-脂肪醇小分子前药自组装纳米粒，制备方法简单易行；(2)考察了四种长度的支链脂肪醇侧链、脂肪醇侧链的结构(支链或直链)、四种连接链对前药自组装纳米粒制剂学性质、体内命运和抗肿瘤活性等方面的影响。结果表明：卡巴他赛-脂肪醇小分子前药自组装纳米粒能有效提高卡巴他赛的疗效，降低其毒副作用；不同的侧链和连接链会对卡巴他赛前药自组装纳米粒的制剂学性质、体内命运与抗肿瘤活性产生显著影响；2-辛基-十二醇作为侧链时前药自组装纳米粒的安全性最佳；卡巴他赛-支链脂肪醇小分子前药自组装纳米粒具有比卡巴他赛-直链脂肪醇小分子前药自组装纳米粒更好的抗肿瘤效果与安全性；二硫键作为连接链的卡巴他赛-脂肪醇小分子前药自组装纳米粒在抗肿瘤效果上更具有优势；当连接链为4,4'-二硫代二丁酸，侧链为2-辛基-十二醇时，化合物的抗肿瘤活性最强，安全性最好，耐受剂量最高。本发明为开发高效-低毒化疗制剂提供了新的策略与选择。
附图说明：
图1为本发明实施例13的PEG修饰的小分子前药自组装纳米粒的在体抗肿瘤实验中肿 瘤体积变化图。
n.s.：P≥0.05  *：P<0.05  **：P<0.01  ***：P<0.001  ****：P<0.0001(均为双侧t检验)
图2为本发明实施例13的PEG修饰的小分子前药自组装纳米粒的在体抗肿瘤实验中小鼠体重变化图。
n.s.：P≥0.05  *：P<0.05  **：P<0.01  ***：P<0.001  ****：P<0.0001(均为双侧t检验)
图3为本发明实施例13的PEG修饰的小分子前药自组装纳米粒的在体抗肿瘤实验中血液常规指标图。
图4为本发明实施例14的PEG修饰的小分子前药自组装纳米粒的在体抗肿瘤实验中肿瘤体积变化图。
n.s.：P≥0.05  *：P<0.05  **：P<0.01  ***：P<0.001  ****：P<0.0001(均为双侧t检验)
图5为本发明实施例14的PEG修饰的小分子前药自组装纳米粒的在体抗肿瘤实验中小鼠体重变化图。
n.s.：P≥0.05  *：P<0.05  **：P<0.01  ***：P<0.001  ****：P<0.0001(均为双侧t检验)
图6为本发明实施例14的PEG修饰的小分子前药自组装纳米粒的在体抗肿瘤实验中肿瘤负荷图。
n.s.：P≥0.05  *：P<0.05  **：P<0.01  ***：P<0.001  ****：P<0.0001(均为双侧t检验)
图7为本发明实施例14的PEG修饰的小分子前药自组装纳米粒的在体抗肿瘤实验中血液常规指标图。
图8为本发明实施例14的PEG修饰的小分子前药自组装纳米粒的在体抗肿瘤实验中生化常规指标图。
图9为本发明实施例15的PEG修饰的小分子前药自组装纳米粒的在体抗肿瘤实验中肿瘤体积变化图。
n.s.：P≥0.05  *：P<0.05  **：P<0.01  ***：P<0.001  ****：P<0.0001(均为双侧t检验)
图10为本发明实施例15的PEG修饰的小分子前药自组装纳米粒的在体抗肿瘤实验中小鼠体重变化图。
n.s.：P≥0.05  *：P<0.05  **：P<0.01  ***：P<0.001  ****：P<0.0001(均为双侧t检验)
图11为本发明实施例15的PEG修饰的小分子前药自组装纳米粒的在体抗肿瘤实验中肿瘤负荷图。
n.s.：P≥0.05  *：P<0.05  **：P<0.01  ***：P<0.001  ****：P<0.0001(均为双侧t检验)
图12为本发明实施例16的PEG修饰的小分子前药自组装纳米粒的在体抗肿瘤实验中肿瘤体积变化图。
n.s.：P≥0.05  *：P<0.05  **：P<0.01  ***：P<0.001  ****：P<0.0001(均为双侧t检验)
图13为本发明实施例16的PEG修饰的小分子前药自组装纳米粒的在体抗肿瘤实验中小鼠体重变化图。
n.s.：P≥0.05  *：P<0.05  **：P<0.01  ***：P<0.001  ****：P<0.0001(均为双侧t检验)
图14为本发明实施例16的PEG修饰的小分子前药自组装纳米粒的在体抗肿瘤实验中肿瘤负荷图。
n.s.：P≥0.05  *：P<0.05  **：P<0.01  ***：P<0.001  ****：P<0.0001(均为双侧t检验)
图15为本发明实施例17的PEG修饰的小分子前药自组装纳米粒的在体抗肿瘤实验中肿瘤体积变化图。
n.s.：P≥0.05  *：P<0.05  **：P<0.01  ***：P<0.001  ****：P<0.0001(均为双侧t检验)
图16为本发明实施例17的PEG修饰的小分子前药自组装纳米粒的在体抗肿瘤实验中小鼠体重变化图。
n.s.：P≥0.05  *：P<0.05  **：P<0.01  ***：P<0.001  ****：P<0.0001(均为双侧t检验)。
具体实施方式：
下面结合实施例对本发明作进一步的详细说明。
实施例1：2,2'二硫代二乙酸作为连接链的卡巴他赛-2-己基-癸醇前药的合成
将适量的2,2'二硫代二乙酸溶于乙酸酐中，置于25mL茄形瓶中，溶解完全后，25℃磁力搅拌2小时后转移到100mL茄形瓶中，加入三倍量甲苯，减压旋转蒸发除去甲苯与乙酸酐，加入适量二氯甲烷溶解所形成的二硫代二乙酸酐，然后加入2-己基-癸醇的二氯甲烷溶液，并 缓缓滴加二氯甲烷溶解的4-二甲氨基吡啶(DMAP)溶液，25℃磁力搅拌12h，得到中间产物2-己基-癸醇-二硫代二乙酸单边酯，层析柱法采用环己烷-丙酮洗脱体系进行分离提纯；得到纯化的上一步产物后加入1-(3-二甲氨基丙基)-3-乙基碳化二亚胺盐酸盐(EDCI)、1-羟基苯并三唑(HOBt)、4-二甲氨基吡啶(DMAP)的二氯甲烷溶液，0℃冰浴活化2h，然后加入卡巴他赛的二氯甲烷溶液，25℃搅拌48h。反应结束后采用制备液相对产物进行分离，制得2,2'二硫代二乙酸作为连接链的卡巴他赛-2-己基-癸醇前药。反应中，按比例，2,2'二硫代二乙酸：乙酸酐＝1:1，单位mmol:ml；按摩尔比，DMAP：2-己基-癸醇：二硫代二乙酸酐＝0.4:2:1，2-己基-癸醇-二硫代二乙酸单边酯：HOBt：EDCI：DMAP：卡巴他赛＝1:1:2:0.4:0.8。
采用质谱和 1H-NMR对产物结构进行确证。波谱解析结果如下：
1H-NMR(600MHz,DMSO-d 6)δ7.966(2H,d),7.840(1H,m,),7.650-7.677(2H,m,),7.348(2H,m),7.174(3H,m),5.812(1H,s,13-H),5.070-5.131(2H,d,2-H,3'-H),4.954(1H,d,2’-H),4.931(3H,m,3-H,-CH＝CH-),4.686(1H,d,4-H),4.459(1H,m,5-H),4.013(1H,d,20α-H),3.996(4H,m,-OCH 2CH 2O-),3.857(1H,d,7-H),3.590(1H,d,8-H),3.291(4H,m,-CH 2-SS-CH 2-),3.207(4H,m,6α-H,s,4-COCHCH 2),2.650(1H,m,14α-H),2.236-2.495(2H,m,14β-H,15α-H),1.791(7H,t,6β-H,-CH 2CH＝CHCH 2-),1.504(5H,s,19-H,10-COCH 3),1.376(6H,m,-CH 2CH 2CH 2CO-),1.245(31H,t,17-H),0.978(7H,s,16-H),0.852(6H,t,-CH 3).
MS(ESI)m/z for C 65H 93NO 17S 2Na[M+Na] +:1246.
实施例2：2,2'二硫代二乙酸作为连接链的卡巴他赛-2-庚基-十一醇前药的合成
将适量的2,2'-二硫代二乙酸溶于乙酸酐中，置于25mL茄形瓶中，溶解完全后，25℃磁力搅拌2小时后转移到100mL茄形瓶中，加入三倍量甲苯，减压旋转蒸发除去甲苯与乙酸酐，加入适量二氯甲烷溶解所形成的二硫代二乙酸酐，然后加入2-庚基-十一醇的二氯甲烷溶液，并缓缓滴加二氯甲烷溶解的4-二甲氨基吡啶(DMAP)溶液，25℃磁力搅拌12h，得到中间产物2-庚基-十一醇-二硫代二乙酸单边酯，层析柱法采用环己烷-丙酮洗脱体系进行分离提纯；得到纯化的上一步产物后加入1-(3-二甲氨基丙基)-3-乙基碳化二亚胺盐酸盐(EDCI)、1-羟基苯并三唑(HOBt)、4-二甲氨基吡啶(DMAP)的二氯甲烷溶液，0℃冰浴活化2h，然后加入卡巴他赛的二氯甲烷溶液，25℃搅拌48h。反应结束后采用制备液相对产物进行分离，制得2,2'二硫代二乙酸作为连接链的卡巴他赛-2-庚基-十一醇前药。反应中，按比例，2,2'二硫代二乙酸：乙酸酐＝1:1，单位mmol:ml；按摩尔比，DMAP：2-庚基-十一醇：二硫代二乙酸酐＝0.4:2:1，2-庚基-十一醇-二硫代二乙酸单边酯：HOBt：EDCI：DMAP：卡巴他赛＝1:1:2:0.4:0.8。
采用质谱和 1H-NMR对产物结构进行确证。波谱解析结果如下：
1H-NMR(600MHz,DMSO-d 6)δ7.967(2H,d),7.817(1H,m),7.737(2H,m),7.658(2H,m), 7.175(3H,m),5.814(1H,s,13-H),5.131-5.358(2H,d,2-H,3'-H),5.071(1H,d,2’-H),4.932(3H,m,3-H,-CH＝CH-),4.687(1H,d,4-H),4.460(1H,m,5-H),4.015(1H,d,20α-H),3.976(4H,m,-OCH 2CH 2O-),3.857(1H,d,7-H),3.574(1H,d,8-H),3.293(4H,m,-CH 2-SS-CH 2-),3.207(4H,m,6α-H,s,4-COCHCH 2),2.651(1H,d,18-H),2.237-2.500(2H,m,14β-H,15α-H),1.792(7H,t,6β-H,-CH 2CH＝CHCH 2-),1.505(5H,s,19-H,10-COCH 3),1.377(6H,m,-CH 2CH 2CH 2CO-),1.243(35H,t,17-H),0.979(7H,s,16-H),0.853(6H,t,-CH 3).
MS(ESI)m/z for C 67H 97NO 17S 2Na[M+Na] +:1276.
实施例3：2,2'二硫代二乙酸作为连接链的卡巴他赛-2-辛基-十二醇前药的合成
将适量的2,2'-二硫代二乙酸溶于乙酸酐后置于25mL茄形瓶中，溶解完全后，25℃磁力搅拌2小时后转移到100mL茄形瓶中，加入三倍量甲苯，减压旋转蒸发除去甲苯与乙酸酐，加入适量二氯甲烷溶解所形成的二硫代二乙酸酐，然后加入2-辛基-十二醇的二氯甲烷溶液，并缓缓滴加4-二甲氨基吡啶(DMAP)的二氯甲烷溶液，25℃磁力搅拌12h，得到中间产物2-辛基-十二醇-二硫代二乙酸单边酯，层析柱法采用环己烷-丙酮洗脱体系进行分离提纯；得到纯化的上一步产物后加入1-(3-二甲氨基丙基)-3-乙基碳化二亚胺盐酸盐(EDCI)、1-羟基苯并三唑(HOBt)、4-二甲氨基吡啶(DMAP)的二氯甲烷溶液，0℃冰浴活化2h，然后加入卡巴他赛的二氯甲烷溶液，25℃搅拌48h。反应结束后采用制备液相对产物进行分离，制得2,2'二硫代二乙酸作为连接链的卡巴他赛-2-辛基-十二醇前药。反应中，按比例，2,2'二硫代二乙酸：乙酸酐＝1:1，单位mmol:ml；按摩尔比，DMAP：2-辛基-十二醇：二硫代二乙酸酐＝0.4:2:1，2-辛基-十二醇-二硫代二乙酸单边酯：HOBt：EDCI：DMAP：卡巴他赛＝1:1:2:0.4:0.8。
采用质谱和 1H-NMR对产物结构进行确证。波谱解析结果如下： 1H-NMR(600MHz,DMSO-d 6)δ7.967(2H,t),7.662(3H,d),7.378-7.423(3H,m),7.153-7.189(2H,m),5.807(1H,d,-NH),5.355(1H,d,3'-H),5.124(1H,d,2-H),5.065(4H,m,2'-H,-CH＝CH-,5-H),4.924-4.954(1H,t,7-H),4.685(1H,d,20α-H),4.477(1H,d,20β-H),3.993-4.012(2H,d,15α-H,15β-H),3.861(3H,d,3-H),3.736(4H,m,6α-H,4-COCH 3),2.647(1H,m,13-H),2.501(3H,t,14α-H,-CH 2CO-),1.790(6H,s,-CH 2CH＝CHCH 2-),1.618(4H,s,18-H),1.503(t,5H,6β-H),1.377(10H,s,16-H,19-H,-CH 2CH 2CO-),1.240(24H,t,17-H),0.975(3H,t,-CH 3)，0.850(5H,t,-CH 2CH 3).
MS(ESI)m/z for C 69H 101NO 17S 2Na[M+Na] +:1303.
实施例4：2,2'二硫代二乙酸作为连接链的卡巴他赛-2-癸基-十四醇前药的合成
将适量的2,2'-二硫代二乙酸溶于乙酸酐中，置于25mL茄形瓶中，溶解完全后，25℃磁力搅拌2小时后转移到100mL茄形瓶中，加入三倍量甲苯，减压旋转蒸发除去甲苯与乙酸酐，加入适量二氯甲烷溶解所形成的二硫代二乙酸酐，然后加入2-癸基-十四醇的二氯甲烷溶液， 并缓缓滴加二氯甲烷溶解的4-二甲氨基吡啶(DMAP)溶液，25℃磁力搅拌12h，得到中间产物2-癸基-十四醇-二硫代二乙酸单边酯，层析柱法采用环己烷-丙酮洗脱体系进行分离提纯；得到纯化的上一步产物后加入1-(3-二甲氨基丙基)-3-乙基碳化二亚胺盐酸盐(EDCI)、1-羟基苯并三唑(HOBt)、4-二甲氨基吡啶(DMAP)的二氯甲烷溶液，0℃冰浴活化2h，然后加入卡巴他赛的二氯甲烷溶液，25℃搅拌48h。反应结束后采用制备液相对产物进行分离，制得2,2'二硫代二乙酸作为连接链的卡巴他赛-2-癸基-十四醇前药。反应中，按比例，2,2'二硫代二乙酸：乙酸酐＝1:1，单位mmol:ml；按摩尔比，DMAP：2-癸基-十四醇：二硫代二乙酸酐＝0.4:2:1，2-癸基-十四醇-二硫代二乙酸单边酯：HOBt：EDCI：DMAP：卡巴他赛＝1:1:2:0.4:0.8。
采用质谱和 1H-NMR对产物结构进行确证。波谱解析结果如下：
1H-NMR(600MHz,DMSO-d 6)δ7.967(2H,d),7.817(1H,m),7.737(2H,m),7.658(2H,m),7.175(3H,m),5.814(1H,s,13-H),5.131-5.358(2H,d,2-H,3'-H),5.071(1H,d,2’-H),4.932(3H,m,3-H,-CH＝CH-),4.687(1H,d,4-H),4.460(1H,m,5-H),4.015(1H,d,20α-H),3.976(4H,m,-OCH 2CH 2O-),3.857(1H,d,7-H),3.574(1H,d,8-H),3.293(4H,m,-CH 2-SS-CH 2-),3.207(4H,m,6α-H,s,4-COCHCH 2),2.651(1H,d,18-H),2.237-2.500(2H,m,14β-H,15α-H),1.792(7H,t,6β-H,-CH 2CH＝CHCH 2-),1.505(5H,s,19-H,10-COCH 3),1.377(6H,m,-CH 2CH 2CH 2CO-),1.243(35H,t,17-H),0.979(7H,s,16-H),0.853(6H,t,-CH 3).
MS(ESI)m/z for C 73H 109NO 17S 2Na[M+Na] +:1359.
实施例5：2,2'二硫代二乙酸作为连接链的卡巴他赛-花生醇前药的合成
将适量的2,2'-二硫代二乙酸溶于乙酸酐后置于25mL茄形瓶中，溶解完全后25℃磁力搅拌2小时后转移到100mL茄形瓶中，加入三倍量甲苯，减压旋转蒸发除去甲苯与乙酸酐，加入适量二氯甲烷溶解所形成的二硫代二乙酸酐，然后加入花生醇的二氯甲烷溶液，并缓缓滴加4-二甲氨基吡啶(DMAP)的二氯甲烷溶液，25℃磁力搅拌12h，得到中间产物花生醇-二硫代二乙酸单边酯，层析柱法采用环己烷-丙酮洗脱体系进行分离提纯；得到纯化的上一步产物后加入1-(3-二甲氨基丙基)-3-乙基碳化二亚胺盐酸盐(EDCI)、1-羟基苯并三唑(HOBt)、4-二甲氨基吡啶(DMAP)的二氯甲烷溶液，0℃冰浴活化2h，然后加入卡巴他赛的二氯甲烷溶液，25℃搅拌48h。反应结束后采用制备液相对产物进行分离，制得2,2'二硫代二乙酸作为连接链的卡巴他赛-花生醇前药。反应中，按比例，2,2'二硫代二乙酸：乙酸酐＝1:1，单位mmol:ml；按摩尔比，DMAP：花生醇：二硫代二乙酸酐＝0.4:2:1，花生醇-二硫代二乙酸单边酯：HOBt：EDCI：DMAP：卡巴他赛＝1:1:2:0.4:0.8。
采用质谱和 1H-NMR对产物结构进行确证。波谱解析结果如下： 1H-NMR(600MHz,DMSO-d 6)δ7.967(2H,t),7.662(3H,d),7.378-7.423(3H,m),7.153-7.189(2H,m),5.807(1H,d, -NH),5.355(1H,d,3'-H),5.124(1H,d,2-H),5.065(4H,m,2'-H,-CH＝CH-,5-H),4.924-4.954(1H,t,7-H),4.685(1H,d,20α-H),4.477(1H,d,20β-H),3.993-4.012(2H,d,15α-H,15β-H),3.861(3H,d,3-H),3.736(4H,m,6α-H,4-COCH 3),2.647(1H,m,13-H),2.501(3H,t,14α-H,-CH 2CO-),1.790(6H,s,-CH 2CH＝CHCH 2-),1.618(4H,s,18-H),1.503(t,5H,6β-H),1.377(10H,s,16-H,19-H,-CH 2CH 2CO-),1.240(24H,t,17-H),0.975(3H,t,-CH 3)，0.850(5H,t,-CH 2CH 3).
MS(ESI)m/z for C 69H 101NO 17S 2Na[M+Na] +:1280.
实施例6：4,4'-二硫代二丁酸作为连接链的卡巴他赛-2-辛基-十二醇前药的合成
将适量的4,4'-二硫代二丁酸溶于乙酸酐后置于25mL茄形瓶中，溶解完全后25℃磁力搅拌2小时后转移到100mL茄形瓶中，加入三倍量甲苯，减压旋转蒸发除去甲苯与乙酸酐，加入适量二氯甲烷溶解所形成的二硫代二丁酸酐，然后加入2-辛基-十二醇的二氯甲烷溶液，并缓缓滴加4-二甲氨基吡啶(DMAP)的二氯甲烷溶液，25℃磁力搅拌12h，得到中间产物2-辛基-十二醇-二硫代二丁酸单边酯，层析柱法采用环己烷-丙酮洗脱体系进行分离提纯；得到纯化的上一步产物后加入1-(3-二甲氨基丙基)-3-乙基碳化二亚胺盐酸盐(EDCI)、1-羟基苯并三唑(HOBt)、4-二甲氨基吡啶(DMAP)的二氯甲烷溶液，0℃冰浴活化2h，然后加入卡巴他赛的二氯甲烷溶液，25℃搅拌48h。反应结束后采用制备液相对产物进行分离，制得4,4'-二硫代二丁酸作为连接链的卡巴他赛-2-辛基-十二醇前药。反应中，按比例，4,4'-二硫代二丁酸：乙酸酐＝1:1，单位mmol:ml；按摩尔比，DMAP：2-辛基-十二醇：4,4'-二硫代二丁酸＝0.4:2:1，2-辛基-十二醇-二硫代二丁酸单边酯：HOBt：EDCI：DMAP：卡巴他赛＝1:1:2:0.4:0.8。
采用质谱和 1H-NMR对产物结构进行确证。波谱解析结果如下： 1H-NMR(600MHz,DMSO-d6)δ7.965(2H,t),7.691(3H,d),7.349-7.425(3H,m),7.152-7.189(2H,m),5.753(1H,d,-NH),5.352(1H,d,3'-H),5.120(1H,d,2-H),5.026-5.069(4H,m,2'-H,-CH＝CH-,5-H),4.927-4.951(1H,t,7-H),4.682(1H,d,20α-H),4.474(1H,d,20β-H),3.986-4.011(2H,d,15α-H,15β-H),3.583(3H,d,3-H),3.495(4H,m,6α-H,4-COCH 3),2.670(1H,m,13-H),2.501(3H,t,14α-H,-CH 2CO-),1.780(6H,s,-CH2CH＝CHCH 2-),1.609(4H,s,18-H),1.501(t,5H,6β-H),1.370(10H,s,16-H,19-H,-CH 2CH 2CO-),1.210(24H,t,17-H),0.957(3H,t,-CH 3)，0.831(5H,t,-CH 2CH 3).
MS(ESI)m/z for C 73H 101NO 17S 2Na[M+Na] +:1351.
实施例7：单硫键作为连接链的卡巴他赛-2-辛基-十二醇前药的合成
将适量的单硫代二乙酸溶于乙酸酐后置于25mL茄形瓶中，溶解完全后25℃磁力搅拌2小时后转移到100mL茄形瓶中，加入三倍量甲苯，减压旋转蒸发除去甲苯与乙酸酐，加入适量二氯甲烷溶解所形成的单硫代二乙酸酐，然后加入溶于2-辛基-十二醇的二氯甲烷溶液，并 缓缓滴加4-二甲氨基吡啶(DMAP)的二氯甲烷溶液，25℃磁力搅拌12h，得到中间产物2-辛基-十二醇-单硫代二乙酸单边酯，层析柱法采用环己烷-丙酮洗脱体系进行分离提纯；得到纯化的上一步产物后加入1-(3-二甲氨基丙基)-3-乙基碳化二亚胺盐酸盐(EDCI)、1-羟基苯并三唑(HOBt)、4-二甲氨基吡啶(DMAP)的二氯甲烷溶液，0℃冰浴活化2h，然后加入卡巴他赛的二氯甲烷溶液，25℃搅拌48h。反应结束后采用制备液相对产物进行分离，制得单硫键作为连接链的卡巴他赛-2-辛基-十二醇前药。反应中，按比例，单硫代二乙酸：乙酸酐＝1:1，单位mmol:ml；按摩尔比，DMAP：2-辛基-十二醇：单硫代二乙酸＝0.4:2:1，2-辛基-十二醇-单硫代二乙酸单边酯：HOBt：EDCI：DMAP：卡巴他赛＝1:1:2:0.4:0.8。
采用质谱和 1H-NMR对产物结构进行确证。波谱解析结果如下： 1H-NMR(600MHz,DMSO-d6)δ7.965(2H,t),7.691(3H,d),7.349-7.425(3H,m),7.152-7.189(2H,m),5.753(1H,d,-NH),5.352(1H,d,3'-H),5.120(1H,d,2-H),5.026-5.069(4H,m,2'-H,-CH＝CH-,5-H),4.927-4.951(1H,t,7-H),4.682(1H,d,20α-H),4.474(1H,d,20β-H),3.986-4.011(2H,d,15α-H,15β-H),3.583(3H,d,3-H),3.495(4H,m,6α-H,4-COCH 3),2.670(1H,m,13-H),2.501(3H,t,14α-H,-CH 2CO-),1.780(6H,s,-CH2CH＝CHCH 2-),1.609(4H,s,18-H),1.501(t,5H,6β-H),1.370(10H,s,16-H,19-H,-CH 2CH 2CO-),1.210(24H,t,17-H),0.957(3H,t,-CH 3)，0.831(5H,t,-CH 2CH 3).
MS(ESI)m/z for C 69H 101NO 17SNa[M+Na] +:1271.
实施例8：单硒键作为连接链的卡巴他赛-2-辛基-十二醇前药的合成
将适量的单硒代二乙酸溶于乙酸酐后置于25mL茄形瓶中，溶解完全后25℃磁力搅拌2小时后转移到100mL茄形瓶中，加入三倍量甲苯，减压旋转蒸发除去甲苯与乙酸酐，加入适量二氯甲烷溶解所形成的单硒代二乙酸酐，然后加入2-辛基-十二醇的二氯甲烷溶液，并缓缓滴加4-二甲氨基吡啶(DMAP)的二氯甲烷溶液，25℃磁力搅拌12h，得到中间产物2-辛基-十二醇-单硒代二乙酸单边酯，层析柱法采用环己烷-丙酮洗脱体系进行分离提纯；得到纯化的上一步产物后加入1-(3-二甲氨基丙基)-3-乙基碳化二亚胺盐酸盐(EDCI)、1-羟基苯并三唑(HOBt)、4-二甲氨基吡啶(DMAP)的二氯甲烷溶液，0℃冰浴活化2h，然后加入卡巴他赛的二氯甲烷溶液，25℃搅拌48h。反应结束后采用制备液相对产物进行分离，制得单硒键作为连接链的卡巴他赛-2-辛基-十二醇前药。反应中，按比例，单硒代二乙酸：乙酸酐＝1:1，单位mmol:ml；按摩尔比，DMAP：2-辛基-十二醇：单硒代二乙酸＝0.4:2:1，2-辛基-十二醇-单硒代二乙酸单边酯：HOBt：EDCI：DMAP：卡巴他赛＝1:1:2:0.4:0.8。
采用质谱和 1H-NMR对产物结构进行确证。波谱解析结果如下： 1H-NMR(600MHz,DMSO-d 6)δ7.969(2H,t),7.661(3H,d),7.385-7.428(3H,m),7.175(2H,m),5.807(1H,d,-NH), 5.355(1H,d,3'-H),5.117(1H,d,2-H),4.932-4.958(4H,m,2'-H,-CH＝CH-,5-H),4.685(1H,d,20α-H),4.474(1H,d,20β-H),3.986-4.011(2H,d,15α-H,15β-H),3.571-3.829(3H,d,3-H)3.197(4H,m,6α-H,4-COCH 3),2.670(1H,m,13-H),2.492(3H,t,14α-H,-CH 2CO-),1.795(6H,s,-CH 2CH＝CHCH 2-),1.501(4H,s,18-H),1.379(10H,s,16-H,19-H,-CH 2CH 2CO-),1.239(24H,t,17-H),0.958(3H,t,-CH 3)，0.850(5H,t,-CH 2CH 3).
MS(ESI)m/z for C 69H 101NO 17SeNa[M+Na] +:1319.
实施例9：PEG修饰的小分子前药自组装纳米粒的制备
精密称取DSPE-PEG 2K 0.4mg和前药2mg，用200μL丙酮将其溶解，搅拌下将该乙醇溶液缓缓滴加到1.8mL去离子水中，自发形成粒径均匀的PEG修饰的纳米粒。用减压旋转蒸发法去除丙酮，得到不含有机试剂的纳米胶体溶液。如表1所示，除CTX-SS-AA纳米粒外，纳米粒的粒径都在100nm左右，粒径分布小于0.2，表面电荷在-20mV左右。
表1.PEG修饰的前药自组装纳米粒的粒径、粒径分布和表面电荷
Figure PCTCN2022136743-appb-000011
结果表明：不同连接链和不同脂肪醇侧链的卡巴他赛前药均可以形成自组装纳米粒，卡巴他赛-支链脂肪醇小分子前药自组装纳米粒具有比卡巴他赛-直链脂肪醇小分子前药自组装纳米粒更小的粒径与粒径分布。卡巴他赛-支链脂肪醇小分子前药自组装纳米粒的粒径均在100nm左右，同时粒径分布非常均匀，粒径分布均在0.1左右，有助于纳米粒通过实体肿瘤的高通透性和滞留效应实现肿瘤靶向蓄积。纳米粒表面电荷在-20mV左右，有利于通过电荷排斥作用阻止纳米粒的聚集。
实施例10：小分子前药自组装纳米粒的冻干粉制备
取实施例9中制备的PEG化修饰的单硫键作为连接链的卡巴他赛-2-辛基-十二醇前药自组装纳米粒1mL于西林瓶中，分别用5％蔗糖、10％蔗糖、5％乳糖、10％乳糖、5％海藻糖、10％海藻糖、5％甘露醇、10％甘露醇、5％葡萄糖、10％葡萄糖作为冻干保护剂，然后置于冻干机中冷冻干燥24小时，得到白色饼块。所得冻干粉用去离子水复溶后测量粒径与表面电荷， 结果如表2所示。结果表明：使用单一冻干保护剂制备的前药自组装纳米粒冻干粉复溶后粒径与粒径分布较大。
表2.冻干复溶后的前药自组装纳米粒的粒径、粒径分布和表面电荷
Figure PCTCN2022136743-appb-000012
实施例11：小分子前药自组装纳米粒冻干粉制备
取实施例9中制备的PEG化修饰的单硫键作为连接链的卡巴他赛-2-辛基-十二醇前药自组装纳米粒1mL于西林瓶中，分别用5％乳糖与5％甘露醇、5％乳糖与5％麦芽糖、5％乳糖与5％蔗糖、5％乳糖与5％海藻糖作为冻干保护剂，冻干保护剂完全溶解后置于-80℃环境中预冻12h，然后置于冻干机中冷冻干燥24小时，得到白色饼块。所得冻干粉用去离子水复溶后测量粒径与表面电荷，结果如表3所示。结果表明：与使用单一保护剂制备的冻干粉相比，使用复合保护剂制备的冻干粉在复溶后的粒径更小。
表3.冻干复溶后的前药自组装纳米粒的粒径、粒径分布和表面电荷
Figure PCTCN2022136743-appb-000013
实施例12：小分子前药自组装纳米粒的药代动力学研究
取24只健康雄性大鼠，体重180-220g，随机分为4组，给药前禁食12h，自由饮水。4 组分别尾静脉注射卡巴他赛溶液剂和实施例9中制备的PEG修饰的前药自组装纳米粒，其剂量为4mg/kg(以卡巴他赛计)。于规定的时间点取血。通过液相色谱-质谱联用仪测定血中的药物浓度。结果如表4所示。结果表明，卡巴他赛溶液剂循环时间很短，给药后在体内迅速代谢清除。相比之下，PEG修饰的小分子前药自组装纳米粒循环时间明显延长，生物利用度明显提高，药动学参数提升明显。CTX-SS-OD纳米粒、CTX-Se-OD纳米粒和CTX-S-OD纳米粒的总体AUC 0-24h(前药和母药的加和)分别为卡巴他赛溶液剂的395.47倍、277.37倍、260.19倍；t 1/2分别为卡巴他赛溶液剂的19.72倍、10.16倍、11.51倍；C max分别为卡巴他赛溶液剂的76.35倍、51.80倍、51.18倍。CTX-SS-OD纳米粒药动学参数提升最为明显，说明二硫键作为连接链更具有优势。
表4.卡巴他赛溶液剂和前药纳米粒的药代动力学参数
Figure PCTCN2022136743-appb-000014
实施例13：PEG化的小分子前药自组装纳米粒的在体抗肿瘤实验
将小鼠乳腺癌细胞悬液(4T1,5x10 6cells/100μL)接种于雌性BALB/c小鼠背侧皮下。待肿瘤体积生长至100-120mm 3时，将荷瘤小鼠随机分为6组，每组8只：生理盐水组(Saline)，卡巴他赛溶液剂组，CTX-SS-HU纳米粒组，CTX-SS-HD纳米粒组，CTX-SS-OD纳米粒组以及CTX-SS-DT纳米粒组。给药所用的纳米粒为实施例9中制备的PEG修饰的小分子前药自组装纳米粒，给药剂量为10mg/kg(以卡巴他赛浓度计算)。每隔1天给药1次，连续给药5次。给药后，每天检测小鼠的存活状态和体重变化情况，测量肿瘤体积。最后一次给药后一天将小鼠处死，获取器官和肿瘤，进行进一步分析评价，结果如图1-3所示。图1表明，在生理盐水组中，肿瘤体积迅速增长，在第10天达到1000mm 3左右。相比之下，纳米粒与卡巴他赛溶液剂均显著抑制了肿瘤的生长(<200mm 3)；图2表明，卡巴他赛溶液剂组的小鼠体重明显下降，而前药自组装纳米粒组体重无显著变化；图3表明，各组制剂均引起了小鼠血中白细胞数量下降，其中CTX-SS-OD纳米粒组与CTX-SS-HU纳米粒组引发的下降最少。结果表明：二硫键作为化学连接的卡巴他赛-支链脂肪醇小分子前药自组装纳米粒在具有强力抗 肿瘤效果的同时，没有造成明显的系统毒性，是安全有效的化疗药物递送系统。
实施例14：PEG化的小分子前药自组装纳米粒的在体抗肿瘤实验
将小鼠乳腺癌细胞悬液(4T1,5x10 6cells/100μL)接种于雌性BALB/c小鼠背侧皮下。待肿瘤体积生长至100-120mm 3时，将荷瘤小鼠随机分为7组，每组8只：生理盐水组(Saline)，CTX-SS-OD纳米粒2mg/kg、10mg/kg组、20mg/kg组与CTX-SS-AA纳米粒2mg/kg、10mg/kg组、20mg/kg组。给药所用的纳米粒为实施例9中制备的PEG修饰的小分子前药自组装纳米粒，给药剂量以卡巴他赛浓度计算。每隔1天给药1次，连续给药5次。给药后，每天检测小鼠的存活状态和体重变化情况，测量肿瘤体积。最后一次给药后一天将小鼠处死，获取器官和肿瘤，进行进一步分析评价，结果如图4-8所示。图4表明，在生理盐水组中，肿瘤体积迅速增长，在第10天达到1000mm 3左右，而前药自组装纳米粒组的肿瘤生长均受到了抑制。给药剂量相同时，CTX-SS-OD纳米粒组的肿瘤体积比CTX-SS-AA纳米粒组更小；图5表明，给药剂量相同时，CTX-SS-AA纳米粒组的体重比CTX-SS-OD纳米粒组下降更严重；图6表明，前药自组装纳米粒组具有比生理盐水组更低的肿瘤负荷，同浓度时，CTX-SS-OD纳米粒的肿瘤负荷比CTX-SS-AA更低；图7表明，给药剂量相当时，CTX-SS-OD纳米粒组的白细胞数量下降更少；图8表明，给药剂量为20mg/kg时，CTX-SS-OD纳米粒组的尿素氮上升更少。结果表明：卡巴他赛-支链脂肪醇小分子前药自组装纳米粒比卡巴他赛-直链脂肪醇小分子前药自组装纳米粒具有更强的抗肿瘤效果的同时，还具有更好的安全性。
实施例15：PEG化的小分子前药自组装纳米粒的在体抗肿瘤实验
将小鼠乳腺癌细胞悬液(4T1,5x10 6cells/100μL)接种于雌性BALB/c小鼠背侧皮下。待肿瘤体积生长至100-120mm 3时，将荷瘤小鼠随机分为5组，每组8只：生理盐水组(Saline)，卡巴他赛溶液剂组，CTX-S-OD纳米粒组，CTX-SS-OD纳米粒组，CTX-Se-OD纳米粒组。给药所用的纳米粒为实施例9中制备的PEG修饰的小分子前药自组装纳米粒，给药剂量为4mg/kg(以卡巴他赛浓度计算)。每隔1天给药1次，连续给药5次。给药后，每天检测小鼠的存活状态和体重变化情况，测量肿瘤体积。最后一次给药后一天将小鼠处死，获取器官和肿瘤，进行进一步分析评价，结果如图9-11所示。图9表明，在生理盐水组中，肿瘤体积迅速增长，在第10天达到700mm 3左右，CTX-S-OD纳米粒组与CTX-Se-OD纳米粒组的肿瘤体积相当(<400mm 3),CTX-SS-OD纳米粒组与卡巴他赛溶液剂组的肿瘤体积较小(<200mm 3)；图10表明，三种纳米粒组的体重均没有明显变化，卡巴他赛溶液剂组的体重出现明显下降；图11表明，CTX-SS-OD纳米粒组的肿瘤负荷与卡巴他赛溶液剂组没有统计学差异。结果表明：CTX-SS-OD纳米粒具有良好的抗肿瘤效果的同时，还具有比卡巴他赛溶液剂更好的安全性；二硫键作为连接链比单硫键、单硒键更具有优势。
实施例16：PEG化的小分子前药自组装纳米粒的在体抗肿瘤实验
将小鼠乳腺癌细胞悬液(4T1,5x10 6cells/100μL)接种于雌性BALB/c小鼠背侧皮下。待肿瘤体积生长至100-120mm 3时，将荷瘤小鼠随机分为8组，每组8只：生理盐水组(Saline)，卡巴他赛溶液剂2mg/kg组，卡巴他赛溶液剂10mg/kg组，卡巴他赛溶液剂15mg/kg组，CTX-SS-OD纳米粒2mg/kg组，CTX-SS-OD纳米粒10mg/kg组，CTX-SS-OD纳米粒15mg/kg组,CTX-SS-OD纳米粒20mg/kg组。给药所用的纳米粒为实施例9中制备的PEG修饰的小分子前药自组装纳米粒，给药剂量以卡巴他赛浓度计算。每隔一天给药一次，连续给药5次。每天检测小鼠的存活状态和体重变化情况，测量肿瘤体积。最后一次给药后一天将小鼠处死，获取器官和肿瘤，进行进一步分析评价，结果如图12-14所示。图12、14表明，在空白对照组中，肿瘤体积迅速增长，在第10天达到800mm 3左右。当给药剂量为2mg/kg时，CTX-SS-OD纳米粒组的肿瘤体积与肿瘤负荷低于卡巴他赛溶液剂组。当给药剂量为10mg/kg、15mg/kg时，CTX-SS-OD纳米粒组的肿瘤体积与肿瘤负荷与卡巴他赛溶液剂组相比没有统计学差异；图13表明，当给药剂量为2mg/kg时，CTX-SS-OD纳米粒组与卡巴他赛溶液剂组的体重没有明显变化。当给药剂量为10mg/kg时，CTX-SS-OD纳米粒组体重没有变化，卡巴他赛溶液剂组的体重下降。当给药剂量为15mg/kg时，卡巴他赛溶液剂组的小鼠全部死亡，CTX-SS-OD纳米粒组的小鼠没有死亡。结果说明：CTX-SS-OD纳米粒在具有与卡巴他赛溶液剂相当的抗肿瘤效果的同时，毒性比卡巴他赛溶液剂更低。
实施例17：PEG化的小分子前药自组装纳米粒的在体抗肿瘤实验
将小鼠乳腺癌细胞悬液(4T1,5x10 6cells/100μL)接种于雌性BALB/c小鼠背侧皮下。待肿瘤体积生长至100-120mm 3时，将荷瘤小鼠随机分为7组，每组8只：生理盐水组(Saline)，CTX-SS-OD纳米粒20mg/kg组，CTX-SS-OD纳米粒30mg/kg组，CTX-SS-DT纳米粒20mg/kg组，CTX-SS-DT纳米粒30mg/kg组，γ-CTX-SS-OD纳米粒20mg/kg组，γ-CTX-SS-OD纳米粒30mg/kg组。给药所用的纳米粒为实施例9中制备的PEG修饰的小分子前药自组装纳米粒，给药剂量以卡巴他赛浓度计算。每隔一天给药一次，连续给药5次。每天检测小鼠的存活状态和体重变化情况，测量肿瘤体积。最后一次给药后一天将小鼠处死，获取器官和肿瘤，进行进一步分析评价，结果如图15-16所示。图15表明，三种纳米粒均具有良好的抗肿瘤效果，显著抑制了肿瘤的生长；图16表明，给药剂量相当时，γ-CTX-SS-OD组体重下降最少，说明与CTX-SS-OD纳米粒、CTX-SS-DT纳米粒相比，γ-CTX-SS-OD纳米粒具有更好的安全性。
实施例18：PEG化的小分子前药自组装纳米粒的耐受性实验
将雌性BALB/c小鼠分为11组，每组3只。其中四组分别尾静脉注射给药30mg/kg、40 mg/kg、50mg/kg、60mg/kg的卡巴他赛溶液剂。另外七组分别尾静脉注射给药CTX-SS-HD纳米粒、CTX-SS-HU纳米粒、CTX-SS-OD纳米粒、CTX-SS-DT纳米粒、CTX-S-OD纳米粒、CTX-SS-AA纳米粒、γ-CTX-SS-OD纳米粒，每隔8小时给药一次，一次给药200mg/kg，给药剂量以卡巴他赛浓度计算，直到小鼠全部死亡。每次给药后观察小鼠的生存状况，结果如表5所示。结果表明，前药自组装纳米粒的耐受性远高于卡巴他赛溶液剂。四种含有不同支链脂肪醇侧链的CTX-SS-HD纳米粒、CTX-SS-HU纳米粒、CTX-SS-OD纳米粒、CTX-SS-DT纳米粒之中，CTX-SS-OD纳米粒的安全性最好，说明2-辛基-十二醇作为侧链在安全性上更具有优势；不同结构(支链或直链)的侧链相比时，含有支链脂肪醇的CTX-SS-OD纳米粒比含有直链脂肪醇的CTX-SS-AA纳米粒安全性更好，说明支链脂肪醇作为侧链比直链脂肪醇在安全性上更具有优势；不同长度的连接链相比时，含有更长连接链的γ-CTX-SS-OD纳米粒比CTX-SS-OD纳米粒具有更好的安全性，说明4,4'-二硫代二丁酸作为连接链在安全性上更具有优势。所有的纳米粒制剂中，γ-CTX-SS-OD纳米粒具有最高的最大耐受剂量，为卡巴他赛溶液剂的40倍。
表5.卡巴他赛溶液剂和前药纳米粒的耐受剂量
Figure PCTCN2022136743-appb-000015

Claims (10)

  1. 卡巴他赛-支链脂肪醇小分子前药或其药学上可接受的盐，其特征在于，所述的卡巴他赛-支链脂肪醇小分子前药结构如下通式(I)、(II)、(III)所示：
    Figure PCTCN2022136743-appb-100001
    其中，n＝1～3；
    R为饱和或不饱和的C 3-C 30烃基，所述R为含支链结构的烃基，所述支链为C 1-C 18烷基、C 2-C 18烯基或C 2-C 18炔基中的一种或多种。
  2. 根据权利要求1所述的卡巴他赛-支链脂肪醇小分子前药或其药学上可接受的盐，其特征在于，当所述的R为不饱和烃基时，不饱和烃基中含有的烯基、炔基或烯基与炔基数目之和为1-5个。
  3. 根据权利要求1所述的卡巴他赛-支链脂肪醇小分子前药或其药学上可接受的盐，其特征在于，所述的支链脂肪醇为2-己基-辛醇、1-庚基-辛醇、2-己基-癸醇、1-丁基-十二醇、1-庚基-壬醇、1-辛基-壬醇、2-辛基-癸醇、2-庚基-十一醇、1-壬基-癸醇、2-辛基-十二醇、2-癸基-十四醇或2-十二烷基-十四醇中的一种。
  4. 根据权利要求1所述的卡巴他赛-支链脂肪醇小分子前药或其药学上可接受的盐，其特征在于，所述的卡巴他赛-支链脂肪醇小分子前药中的卡巴他赛与支链脂肪醇通过二元酸为连接链连接，所述的二元酸为单硫代二元酸、单硒代二元酸或二硫代二元酸，其中，所述的单硫代二元酸为单硫代二乙酸、单硫代二丙酸或单硫代二丁酸；所述的单硒代二元酸为单硒代二乙酸、单硒代二丙酸或单硒代二丁酸；所述的二硫代二元酸为2,2'-二硫代二乙酸、3,3'-二硫代二丙酸或4,4'-二硫代二丁酸。
  5. 根据权利要求3所述的卡巴他赛-支链脂肪醇小分子前药或其药学上可接受的盐，其特征在于，所述的卡巴他赛-支链脂肪醇小分子前药或其药学上可接受的盐为如下结构的卡巴他赛-支链脂肪醇小分子前药或其药学上可接受的盐：
    Figure PCTCN2022136743-appb-100002
    Figure PCTCN2022136743-appb-100003
    Figure PCTCN2022136743-appb-100004
  6. 权利要求1-5所述的卡巴他赛-支链脂肪醇小分子前药的合成方法，其特征在于，包括如下步骤：
    步骤1：将二元酸溶解成二元酸酐后，在4-二甲氨基吡啶(DMAP)的催化下与支链脂肪醇进行酯化反应，获得支链脂肪醇-二元酸单边酯中间产物，所述的DMAP：支链脂肪醇：二元酸酐摩尔比为1:(1-10):(5-15)，所述的二元酸为单硫代二元酸、单硒代二元酸或二硫代二元酸；
    步骤2：支链脂肪醇-二元酸单边酯与卡巴他赛发生成酯反应，得到终产物卡巴他赛-支链脂肪醇小分子前药，其中，按摩尔比，支链脂肪醇-二元酸单边酯：卡巴他赛＝1:(0.5-10)，反应方程式如下：
    Figure PCTCN2022136743-appb-100005
    Figure PCTCN2022136743-appb-100006
    其中，n、R如权利要求1所述。
  7. 权利要求1所述的卡巴他赛-支链脂肪醇小分子前药的自组装纳米粒，其特征在于，所述的卡巴他赛-脂肪醇小分子前药的自组装纳米粒为非PEG化的前药自组装纳米粒、PEG修饰/主动靶向修饰剂修饰的前药自组装纳米粒或包载疏水性荧光物质/药物的前药自组装纳米粒。
  8. 根据权利要求7所述的卡巴他赛-支链脂肪醇小分子前药自组装纳米粒的制备方法，其特征在于，包括如下步骤：
    当为非PEG化的卡巴他赛-支链脂肪醇小分子前药自组装纳米粒时，制备方法：将前药溶解到有机溶剂中，搅拌下将该溶液滴加到水中，前药自发形成均匀的纳米粒；采用减压旋转蒸发法除去制剂中的有机溶剂，得到不含任何有机溶剂的纳米胶体溶液，即为非PEG化的卡巴他赛-脂肪醇小分子前药；
    当为PEG修饰/主动靶向基团修饰的卡巴他赛-支链脂肪醇小分子前药自组装纳米粒时，制备方法：将PEG修饰剂/主动靶向修饰剂和前药溶解到有机溶剂中，搅拌下，将该溶液滴加到水中，前药自发形成均匀的纳米粒；采用减压旋转蒸发法除去制剂中的有机溶剂，得到不含任何有机溶剂的纳米胶体溶液，即为PEG修饰/主动靶向基团修饰的卡巴他赛-支链脂肪醇小分子前药自组装纳米粒，其中，卡巴他赛-支链脂肪醇小分子前药与PEG修饰剂/主动靶向修饰剂的质量比为1:(1-0.1)，PEG修饰剂为DSPE-PEG、TPGS、PLGA-PEG、PE-PEG或DSPE-PEG-FA，主动靶向修饰剂为抗体、糖残基、激素、受体或配体；
    当为包载疏水性荧光物质/药物的卡巴他赛-支链脂肪醇小分子前药自组装纳米粒时，制备方法：将PEG修饰剂、疏水性荧光物质/药物以及前药溶解到有机溶剂中，搅拌下将该溶液滴加到水中，前药自发形成均匀的纳米粒；采用减压旋转蒸发法除去制剂中的有机溶剂，得 到不含有机溶剂的纳米胶体溶液，即为包载疏水性荧光物质/药物的卡巴他赛-支链脂肪醇小分子前药自组装纳米粒，其中，卡巴他赛-支链脂肪醇小分子前药与PEG修饰剂以及疏水性荧光物质/药物的质量比为1:(0.1-1):(0.1-1)。
  9. 权利要求1所述的卡巴他赛-支链脂肪醇小分子前药或权利要求7所述的自组装纳米粒的应用，其特征在于，具体为在制备抗肿瘤药物中的应用，在制备注射给药、口服给药或局部给药系统中的应用或在制备提高疗效、降低毒性药物传递系统中的应用。
  10. 权利要求7所述的卡巴他赛-支链脂肪醇小分子前药的自组装纳米粒的冻干粉针剂，其特征在于，所述的冻干粉针剂包含卡巴他赛-支链脂肪醇小分子前药自组装纳米粒溶液和冻干保护剂，所述自组装纳米粒溶液的浓度为0.1mg/mL-20mg/mL，所述的冻干保护剂为葡萄糖、半乳糖、海藻糖、蔗糖、甘露醇、山梨醇、木糖醇、聚乙二醇、羟乙基淀粉或葡聚糖中的一种或几种，所述的冻干保护剂用量为1％-20％(W/V)。
PCT/CN2022/136743 2021-12-15 2022-12-06 一种卡巴他赛前药抗肿瘤制剂 WO2023109563A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111532693.5 2021-12-15
CN202111532693.5A CN116332879A (zh) 2021-12-15 2021-12-15 一种卡巴他赛前药抗肿瘤制剂

Publications (1)

Publication Number Publication Date
WO2023109563A1 true WO2023109563A1 (zh) 2023-06-22

Family

ID=86774860

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/136743 WO2023109563A1 (zh) 2021-12-15 2022-12-06 一种卡巴他赛前药抗肿瘤制剂

Country Status (2)

Country Link
CN (1) CN116332879A (zh)
WO (1) WO2023109563A1 (zh)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105884719A (zh) * 2016-04-14 2016-08-24 沈阳药科大学 紫杉烷类前药的制备和应用
CN108478803A (zh) * 2018-04-08 2018-09-04 沈阳药科大学 氧化还原超敏感二硫键桥连前药自组装纳米粒的构建
CN109350748A (zh) * 2018-10-24 2019-02-19 沈阳药科大学 氧化还原双敏感键桥连小分子前药及其自组装纳米粒
CN111116521A (zh) * 2019-12-11 2020-05-08 河南大学 茄尼醇修饰的紫杉醇前药及其制备方法和应用
CN113398277A (zh) * 2021-06-18 2021-09-17 沈阳药科大学 脂肪酸/脂肪醇-抗肿瘤物质前药及其自组装纳米粒的制备方法
CN115252805A (zh) * 2021-04-30 2022-11-01 苏州裕泰医药科技有限公司 卡巴他赛-脂肪醇小分子前药及其自组装纳米粒的构建

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105884719A (zh) * 2016-04-14 2016-08-24 沈阳药科大学 紫杉烷类前药的制备和应用
CN108478803A (zh) * 2018-04-08 2018-09-04 沈阳药科大学 氧化还原超敏感二硫键桥连前药自组装纳米粒的构建
CN109350748A (zh) * 2018-10-24 2019-02-19 沈阳药科大学 氧化还原双敏感键桥连小分子前药及其自组装纳米粒
CN111116521A (zh) * 2019-12-11 2020-05-08 河南大学 茄尼醇修饰的紫杉醇前药及其制备方法和应用
CN115252805A (zh) * 2021-04-30 2022-11-01 苏州裕泰医药科技有限公司 卡巴他赛-脂肪醇小分子前药及其自组装纳米粒的构建
CN113398277A (zh) * 2021-06-18 2021-09-17 沈阳药科大学 脂肪酸/脂肪醇-抗肿瘤物质前药及其自组装纳米粒的制备方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JIANG YAN, WANG XIUZHEN, LIU XIN, LV WEI, ZHANG HONGJUAN, ZHANG MINGWAN, LI XINRUI, XIN HONGLIANG, XU QUNWEI: "Enhanced Antiglioma Efficacy of Ultrahigh Loading Capacity Paclitaxel Prodrug Conjugate Self-Assembled Targeted Nanoparticles", APPLIED MATERIALS & INTERFACES, AMERICAN CHEMICAL SOCIETY, US, vol. 9, no. 1, 11 January 2017 (2017-01-11), US , pages 211 - 217, XP055981508, ISSN: 1944-8244, DOI: 10.1021/acsami.6b13805 *
XUE PENG, LIU DAN, WANG JING, ZHANG NA, ZHOU JIAHUA, LI LIN, GUO WEILING, SUN MENGCHI, HAN XIANGFEI, WANG YONGJUN: "Redox-Sensitive Citronellol–Cabazitaxel Conjugate: Maintained in Vitro Cytotoxicity and Self-Assembled as Multifunctional Nanomedicine", BIOCONJUGATE CHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 27, no. 5, 18 May 2016 (2016-05-18), US , pages 1360 - 1372, XP055981503, ISSN: 1043-1802, DOI: 10.1021/acs.bioconjchem.6b00155 *

Also Published As

Publication number Publication date
CN116332879A (zh) 2023-06-27

Similar Documents

Publication Publication Date Title
He et al. Biomimetic thiamine-and niacin-decorated liposomes for enhanced oral delivery of insulin
CN111494640B (zh) 氧化还原双敏感三硫键桥连二聚体前药及其自组装纳米粒
CN113398277B (zh) 脂肪酸/脂肪醇-抗肿瘤物质前药及其自组装纳米粒的制备方法
CN103804472B (zh) 一种紫杉烷类药物前体
JP2011517683A (ja) 疎水性タキサン誘導体の組成物およびその使用
CN112604002A (zh) 二硫键桥连的多西他赛-脂肪酸前药及其自组装纳米粒
CN106083769A (zh) 一种还原响应的紫杉醇前药及制备纳米胶束载体方法
CN113264906B (zh) 多西他赛二聚体小分子前药及其自组装纳米粒的构建
WO2021110004A1 (zh) 紫杉醇弱酸性衍生物主动载药脂质体及其制备与应用
CN105777770A (zh) 一种饱和长链脂肪酸修饰的7-乙基-10-羟基喜树碱化合物及其长循环脂质体
US20110059180A1 (en) Method for Preparing Nanoparticles Based on Functional Amphiphilic Molecules or Macromolecules, and the Use Thereof
CN114796513B (zh) 二硒键桥连多西他赛二聚体前药及其自组装纳米粒
WO2021190495A1 (zh) 负载羧酸抗肿瘤药物的peg化肝素纳米胶束及其制备方法
WO2023109563A1 (zh) 一种卡巴他赛前药抗肿瘤制剂
Zhang et al. Preparation, characterization and in vivo antitumor evaluation of a micellar formulation of camptothecin prodrug
WO2022227555A1 (zh) 卡巴他赛-脂肪醇小分子前药及其自组装纳米粒的构建
CN115572302A (zh) 鬼臼毒素修饰的多金属氧酸盐杂化化合物及其制备方法和应用
WO2023109562A1 (zh) 一种多西他赛前药抗肿瘤制剂
KR101332001B1 (ko) 양친성 저분자량 히알루론산 복합체를 포함하는 나노 입자 및 그의 제조 방법
Zhao et al. Self-assembled thermosensitive nanoparticles based on oligoethylene glycol dendron conjugated doxorubicin: preparation, and efficient delivery of free doxorubicin
JP2023526707A (ja) カバジタキセル弱塩基性誘導体およびその製剤
CN116983421A (zh) 阿霉素-脂肪醇小分子前药及其自组装纳米粒的构建
CN111116614A (zh) 免疫调节因子与紫杉烷的共价链接物及其白蛋白纳米制剂及制备方法
CN117244074A (zh) 一种紫杉醇前药抗肿瘤制剂
CN107998403B (zh) Peg修饰的三十烷醇水溶性前药

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22906313

Country of ref document: EP

Kind code of ref document: A1