WO2020140237A1 - 类肽化合物及其制备方法、纳米载体以及药物组合物 - Google Patents

类肽化合物及其制备方法、纳米载体以及药物组合物 Download PDF

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WO2020140237A1
WO2020140237A1 PCT/CN2019/070261 CN2019070261W WO2020140237A1 WO 2020140237 A1 WO2020140237 A1 WO 2020140237A1 CN 2019070261 W CN2019070261 W CN 2019070261W WO 2020140237 A1 WO2020140237 A1 WO 2020140237A1
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subunit
peptoid compound
peptoid
nanocarrier
compound
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PCT/CN2019/070261
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English (en)
French (fr)
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赵子健
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京东方科技集团股份有限公司
北京京东方技术开发有限公司
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Priority to CN201980000007.4A priority Critical patent/CN111868072B/zh
Priority to US16/633,321 priority patent/US11352390B2/en
Priority to PCT/CN2019/070261 priority patent/WO2020140237A1/zh
Publication of WO2020140237A1 publication Critical patent/WO2020140237A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • C07K17/08—Peptides being immobilised on, or in, an organic carrier the carrier being a synthetic polymer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • 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
    • 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
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • 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
    • 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

Definitions

  • the embodiments of the present disclosure relate to a peptoid compound and its preparation method, nanocarrier, pharmaceutical composition, and use of the pharmaceutical composition in the preparation of a medicament for treating diseases related to human epidermal growth factor receptor 1 (EGFR).
  • EGFR human epidermal growth factor receptor 1
  • gliomas Tumors derived from neuroepithelium are collectively called gliomas, and account for 40% to 50% of craniocerebral tumors. They are the most common intracranial malignant tumors, with an annual incidence of 3 to 8 people per 100,000 population. Lung cancer is the cancer with the highest morbidity and mortality rate. Lung cancer brain metastasis and lung cancer due to blood-brain barrier factors make it more difficult to be cured.
  • Cancer targeted therapy and in vivo imaging have become one of the hottest topics in academia and clinical medicine today.
  • the clinical anti-tumor drugs are mainly cytotoxic chemotherapy drugs, but these drugs have poor selectivity, large toxic and side effects, and are likely to cause adverse reactions.
  • protein drugs such as antibodies have high specificity and low toxic and side effects, due to their large molecular mass, complex structure, easy to cause immune reactions and drug resistance, and the preparation process is so complicated that the price is high, it is difficult for ordinary cancer patients Suffered.
  • At least one embodiment of the present disclosure provides a peptoid compound including: a cysteine (Cys) subunit, 1,4-butanediamine (Nlys) subunit, piperonylamine subunit, propionate amine Subunits and 1-naphthylamine subunits.
  • the order of the subunits included in the peptoid compound is a cysteine (Cys) subunit—1,4-butanediamine (Nlys) subunit —1,4-butanediamine (Nlys) subunit—piperonylamine subunit—1,4-butanediamine (Nlys) subunit—propionate subunit—1-naphthylamine subunit.
  • the peptoid compound has the structure shown in Formula I:
  • At least one embodiment of the present disclosure also provides a method for preparing the peptoid compound according to any one of the above, wherein the method includes a solid phase synthesis method to connect subunits.
  • the method includes the following steps: (1) Connect the first subunit of the peptoid compound according to the connection order of the subunits of the peptoid compound Onto a solid support; (2) reacting bromoacetic acid or bromoacetyl chloride with the amino group of the first subunit attached to the solid support to form an amide bond; (3) placing the first of the peptoid compound The donors of the two subunits react with the product obtained in step (2) to replace the bromine atom and complete the connection of the second subunit; (4) Repeat steps (2) and (3) until all subunits are completed Unit connection; (5) Cleavage of the synthetic peptoid compound from the solid support to obtain the peptoid compound.
  • At least one embodiment of the present disclosure further provides a nanocarrier including the peptoid compound, polyamide-amine dendrimer (PAMAM) and polyethylene glycol (PEG) as described in any one of the above, wherein, the polyethylene glycol (PEG) connects the peptoid compound and the polyamide-amine dendrimer (PAMAM).
  • PAMAM polyamide-amine dendrimer
  • PEG polyethylene glycol
  • the molar ratio of the peptoid compound to the polyamide-amine dendrimer (PAMAM) is 1:5 to 1:1, and the PEG The molar ratio to the PAMAM is 1:25 to 1:35.
  • the generation number of the polyamide-amine dendrimer (PAMAM) is more than 4 generations.
  • At least one embodiment of the present disclosure also provides a pharmaceutical composition, including: the nanocarrier as described in any one of the above; and pharmaceutically acceptable auxiliary materials.
  • the auxiliary material is any one or a combination of at least two of excipients, diluents, carriers, flavoring agents, binders, and fillers.
  • At least one embodiment of the present disclosure also provides a use of the pharmaceutical composition according to any one of the above in the preparation of a medicament for treating a disease related to human epidermal growth factor receptor 1 (EGFR).
  • EGFR human epidermal growth factor receptor 1
  • the disease includes glioma and brain metastasis from lung cancer.
  • FIG. 1 is a flowchart of a method for preparing a peptoid compound provided by an embodiment of the present disclosure
  • FIG. 2 is a schematic structural diagram of a nanocarrier provided by an embodiment of the present disclosure
  • FIG. 3 is a scanning electron microscope image of a nanocarrier provided by an embodiment of the present disclosure.
  • FIG. 4 is a schematic diagram of the ability of a nanocarrier to penetrate the blood-brain barrier provided by an embodiment of the present disclosure
  • FIG. 5 is a schematic diagram of the nanocarrier penetrating the blood-brain barrier over time provided by an embodiment of the present disclosure.
  • FIG. 6 is a graph showing the results of surface plasmon resonance detection of a nanocarrier combined with EGFR proteins at concentrations of 55.6 nM, 22.8 nM, 11.4 nM, 5.70 nM, and 2.85 nM, according to an embodiment of the present disclosure.
  • the sub-unit (monomer) of the present disclosure refers to a raw material amine added to a solid-phase synthetic peptoid compound.
  • the subunit of the present disclosure refers to a structural unit constituting a peptoid compound.
  • EGFR human epidermal growth factor receptor 1.
  • nM refers to “nmol/L”
  • ⁇ M refers to “ ⁇ mol/L”
  • mM refers to "mol/L”.
  • the new drug carrier can carry drugs to the tumor site through passive targeting.
  • Specific targeted drug delivery requires the use of specific recognition elements to enable selective active enrichment of the drug at the tumor site, which increases the concentration of the drug and enhances the penetration, thereby significantly improving the therapeutic effect.
  • In vivo imaging takes the identification element as the main body, and combines fluorescence, nuclear magnetic imaging and other imaging methods to achieve the goal. Therefore, the recognition element is the top priority of the entire targeted drug and in vivo imaging.
  • the recognition element includes antibodies, peptides, peptoids, nucleic acid aptamers, and other targeting molecules of specific receptor proteins in tumor sites.
  • aptamer small molecules have a series of unique advantages, such as low immunogenicity, good tissue permeability, small molecular weight, high stability, easy modification and economical.
  • At least one embodiment of the present disclosure provides a peptoid compound including: a cysteine (Cys) subunit, 1,4-butanediamine (Nlys) subunit, piperonylamine subunit, propionate amine Subunits and 1-naphthylamine subunits.
  • the peptoid compound is a peptide mimetic that uses N-substituted glycine as a structural unit. Compared with polypeptide compounds, the side chain of peptoid compounds is transferred from ⁇ -carbon to N. Unlike traditional polypeptide compounds, which have only 20 amino acids, peptoid compounds are synthesized by subunit synthesis. The constituent units are determined by different amines. There are thousands of amines, so the sequence of peptoid compounds is extremely rich. Different chemical sequence structures can be developed for different targets, and because peptoid compounds are not recognized by enzymes, peptoid compounds can effectively resist proteolysis in vivo, which makes peptoid compounds more obvious as molecular probes The advantages.
  • Drug delivery systems and molecular imaging systems developed based on peptoid compound molecular probes can enhance the stability of drugs, improve the interaction of drugs with tumor cells and tissues, and can increase the circulating metabolic cycle in the body. Therefore, in vivo imaging diagnosis and enhancement of drugs It has advantages in terms of effectiveness, overcoming drug resistance and reducing toxic and side effects.
  • the order of the subunits included in the peptoid compound is the cysteine (Cys) subunit—1,4-butanediamine (Nlys) subunit— 1,4-Butanediamine (Nlys) subunit—piperonylamine subunit—1,4-butanediamine (Nlys) subunit—propionic acid amine subunit—1-naphthylamine subunit.
  • the peptoid compound provided in at least one embodiment of the present disclosure is a compound of formula I or a pharmaceutically acceptable salt thereof:
  • the peptoid compound is a compound represented by Formula I.
  • stereoisomer belongs to a type of isomers, which refers to isomers caused by the same order in which the atoms or atomic groups in the molecule are connected to each other, but with different spatial arrangements.
  • the compound of formula I has one asymmetric carbon atom, which may exist as a single enantiomer or as a mixture of enantiomers such as racemic mixtures or enantiomers The enriched mixture exists.
  • Embodiments of the present disclosure encompass any stereoisomers including compounds of formula I or pharmaceutically acceptable salts thereof and mixtures of various forms thereof.
  • the peptoid compound may exist in the form of a salt.
  • the salt can be prepared by reacting a peptoid compound with an inorganic acid or an organic acid.
  • the inorganic acid is, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like.
  • the organic acid is, for example, formic acid, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, almond Acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc.
  • the salt is a pharmaceutically acceptable salt.
  • the pharmaceutically acceptable salt may be hydrochloride, hydrobromide, sulfate, nitrate, phosphate, formate, acetate, propionate, fumarate, glycolate, pyruvate, Malate, malonate, benzoate, cinnamate, mandelate, salicylate, maleate, citrate, succinate, tartrate, methanesulfonate, ethanesulfonate Acid salt, or p-toluenesulfonate.
  • pharmaceutically acceptable salts such as hydrochloride, nitrate, sulfate, phosphate, formate, acetate, fumarate, maleate, citrate , Succinate, tartrate, mesylate, or p-toluenesulfonate.
  • the above-mentioned peptoid compounds can specifically recognize the full-length human epidermal growth factor receptor 1 (EGFR protein), and can achieve the targeting of brain metastases from glioma or lung cancer with high expression of EGFR at a lower cost, more accurately, and more efficiently. Drug loaded treatment.
  • EGFR protein human epidermal growth factor receptor 1
  • At least one embodiment of the present disclosure also provides a method for preparing the above-mentioned peptoid compound, wherein the method includes a solid-phase synthesis method to connect subunits.
  • FIG. 1 is a flowchart of a method for preparing a peptoid compound according to an embodiment of the present disclosure.
  • the preparation method includes the following steps:
  • S101 Connect the first subunit of the peptoid compound to the solid phase carrier according to the connection order of the subunits of the peptoid compound;
  • the input order of subunits is: cysteine, mono-protected 1,4-butanediamine, mono-protected 1,4-butanediamine, piperonylamine, mono-protected 1,4-butanediamine , Propionate amine and 1-naphthylamine, single protection means that one amino group in the diamine is protected by an amino protecting group.
  • the protecting group of the amino group a group known in the art for protecting an amino group during the synthesis of a protein, polypeptide, or peptoid compound can be used without limitation, for example, the amino protecting group is 9- Fluorene methoxycarbonyl (Fmoc) or tert-butoxycarbonyl (Boc).
  • the amino protecting group is tert-butoxycarbonyl.
  • the peptoid compounds of the present disclosure can be prepared by selecting raw materials to prepare individual enantiomers or racemic mixtures.
  • the cysteine raw material may be selected from a mixture of D-cysteine and L-cysteine.
  • peptoid compounds in the examples of the present disclosure can also be resolved by chiral separation, for example, by chiral HPLC to obtain individual enantiomers.
  • the reaction conditions of the amidation reaction in the above step S102 are not particularly limited, and conventional conditions for carrying out the amidation reaction of protein, polypeptide or peptoid compound synthesis in the art can be adopted as long as the amino group can be acylated and not destroyed The function of the peptoid is sufficient.
  • the amidation reaction can be carried out in the presence of a condensing agent.
  • the condensing agent can be used without limitation for the condensing agent known in the art for protein, polypeptide or peptoid compound synthesis.
  • the condensing agent may be a carbodiimide-based condensing agent, for example, N,N'-diisopropylcarbodiimide (DIC), N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC), etc.; benzotriazole condensing agent, such as 1-hydroxy-benzo-triazole (HOBt); benzene Sulfonyl chloride-based condensing agents, such as triisopropylbenzenesulfonyl chloride (TPS), etc.; succinimide-based condensing agents, such as disuccinimide carbonyl ester (DSC), succinimide diphenyl phosphate (SDPP), etc.; 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ); 3-(diethoxyphosphoryl)-
  • the reaction conditions of the nucleophilic substitution reaction in the above step S103 are not particularly limited, and conventional conditions for carrying out the nucleophilic substitution reaction of protein, polypeptide or peptoid compound synthesis in the art can be adopted, as long as the bromine atom can be replaced and not destroyed
  • the function of the peptoid compound may be sufficient. For example, it can be reacted at a temperature of 35 to 40°C for 30 minutes or more, 60 minutes or more, or 90 minutes or more.
  • the removal of the side-chain amino protecting group and the cleavage of the peptoid compound from the resin can be performed simultaneously or successively. For example, first cleave the peptoid compound from the resin and then remove the side chain amino protecting group; you can also remove the side chain amino protecting group and then cleave the peptoid compound from the resin; or remove The side chain amino protecting group cleaves the peptoid compound from the resin at the same time.
  • the removal of the side-chain amino protecting group and the cleavage of the peptoid compound from the resin can use conventional conditions in the art for protein, polypeptide or peptoid compound synthesis, as long as the purpose can be achieved and the peptoid compound is not destroyed Function.
  • a cleavage solution containing 95% trifluoroacetic acid, 2.5% ultrapure water, and 2.5% triisopropylsilane in volume ratio can be used to remove the side chain amino protecting group while the peptoid compound Cleavage from the resin.
  • the peptoid compound represented by formula I is synthesized by solid phase synthesis, and the specific preparation process includes:
  • a step of purifying the obtained product may also be included as necessary.
  • the purification method is not particularly limited, and methods known in the art for purifying corresponding similar products can be used, such as precipitation, filtration, dialysis, gel permeation chromatography, or HPLC.
  • FIG. 2 is a schematic structural diagram of a nanocarrier provided by an embodiment of the present disclosure.
  • the nanocarrier 10 includes: any of the above-mentioned peptides Compound 11, a polyamide-amine dendrimer (PAMAM) 12 and polyethylene glycol (PEG) 13, wherein the polyethylene glycol (PEG) 13 connects the peptide compound 11 and the polyamide-amine type Dendrimer (PAMAM) 12.
  • the molar ratio of the peptoid compound 11 and the polyamide-amine dendrimer (PAMAM) 12 in the nanocarrier 10 is 1:5 to 1:1.
  • the molar ratio of PEG13 to PAMAM12 is 1:25 to 1:35, for example, the molar ratio of PEG to PAMAM is 1:30.
  • part or all of the amino groups in the periphery of PAMAM12 are connected to one end of a PEG13 molecule, and the other end of part or all of PEG13 is connected to peptoid compound 11.
  • part or all of the other end of PEG13 is connected to peptoid compound 11 by a chemical bond.
  • PAMAM dendrimers are flexible, have a quasi-spherical structure in aqueous solution, and have a dense inner and outer structure, that is, as the distance between the crystal phases increases, the density decreases.
  • PAMAM dendrimers are used in drug carriers, nanocomposites, nanoreactors, capillary gas chromatography stationary phases, wastewater treatment, emulsion explosive stabilizers, catalysts, rheology modifiers for polymer materials, photoelectric sensing, liquid crystal, Monomolecular membranes and gene carriers have broad application prospects in many aspects.
  • the polyamide-amine dendrimer (PAMAM) coupled peptoid compound as a stable drug carrier has the advantages of easy design synthesis and modification, good biocompatibility, high drug loading, low toxicity and drug delivery
  • the advantages of high rate and so on, have a wide range of application prospects.
  • High-generation (eg, 4th generation or more) polyamide-amine dendrimers change the charge distribution by adjusting the pH in an aqueous solution, so that the hydrophobic drug is encapsulated in the core as a drug carrier.
  • Encapsulation or chemical bonding are combined or bonded to the nanocarrier formed by the polyamide-amine dendrimer (PAMAM) coupling peptoid compound.
  • the drug is degraded by the nanocarrier itself or broken from the nanocarrier To achieve the effect of sustained release, this can increase the utilization rate of drugs, improve the targeting of drugs, and reduce the toxic and side effects of drugs.
  • PAMAM dendrimers have different charges at different pHs. Under acidic conditions, the outermost amine and the internal tertiary amine of PAMAM are all protonated and have a total of 126 positive charges. Under neutral conditions, only the outermost amine of PAMAM is protonated. Such a fourth-generation dendrimer bears 64 positive charges. The drug enclosed in the PAMAM dendrimer and the peptoid compound coupled to the outside are Neither acid nor neutral carry static charge.
  • the pH value is low, and the acidic condition is such that the surface of the PAMAM dendrimer has more charges, and the charged nanoparticles can sneak into the cell membrane.
  • the ability to induce the formation of voids in the membrane is also related to the size of the nanoparticles.
  • the peptide compound 11 has high affinity for EGFR protein, strong targeting effect on EGFR protein, strong selectivity, high drug loading efficiency, no toxicity, and improved safety, so it can recognize EGFR by
  • the protein achieves targeted drug loading and localized imaging, which provides new options for the diagnosis and treatment of EGFR highly expressed gliomas, lung cancer brain metastases, and other EGFR positive tumors. It also provides in vivo data for sequencing and digital PCR results. Pharmaceutically acceptable excipients or adjuvants make new and more effective targeted anticancer drugs.
  • the generation number of the polyamide-amine dendrimer is 4 or more generations, for example, the generation number of the polyamide-amine dendrimer (PAMAM) is 5th generation, 6th generation, and the like.
  • the synthesis process of the nanocarrier is:
  • S201 Synthetic polyamide-amine dendrimer (PAMAM) (purchased from sigma)-polyethylene glycol (PEG) (purchased from sigma).
  • PAMAM polyamide-amine dendrimer
  • PEG polyethylene glycol
  • the methanol (MeOH) and dimethyl sulfoxide (DMSO) in a molar ratio of 1:1 are mixed to form a mixed solvent.
  • MeOH methanol
  • DMSO dimethyl sulfoxide
  • N-Hydroxysuccinimide (NHS) activates PEG, mixes PAMAM dendrimer and activated PEG, adds to the above mixed solvent, reacts at room temperature for 15 minutes to synthesize PAMAM-PEG, and uses Sephadex G-50 Column purification to remove unreacted PEG.
  • step S203 PAMAM-PEG formed in step S201 is activated using maleamide (purchased from sigma), and reacted with a certain proportion of peptoid compounds at room temperature to obtain a nanocarrier.
  • reaction mechanism diagram of PAMAM-PEG activated with maleamide and peptoid compounds is:
  • PAMAM-PEG activated by maleamide (Mal) and peptoid compound in a molar ratio of 1:15 are mixed, and continuously stirred at room temperature for 30 hours, and the reaction mixture is dialyzed and lyophilized to obtain a nanocarrier.
  • FIG. 3 is a scanning electron microscope image of a nanocarrier provided by an embodiment of the present disclosure.
  • the nanocarrier is spherical and has an average particle size of about 4.2 nm. Can easily enter the cell membrane.
  • FIG. 4 is a schematic diagram of the ability of a carrier to penetrate the blood-brain barrier provided by an embodiment of the present disclosure. As shown in FIG. 4, The barrier includes the medial and lateral sides. Cells and labeled nanocarriers are formed on the medial side of the blood-brain barrier. The blood-brain barrier, cells, and labeled nanocarriers are placed in the culture medium by measuring the labeled The number of nanocarriers calculates the penetration efficiency of the nanocarriers.
  • the cell insert with 12 holes was soaked with a gelatin solution with a mass percentage of 2% for 30 minutes for pretreatment, and then washed twice with D-Hank’s solution and sucked dry.
  • epithelial vascular cells in the brain are relatively dense, and the pretreatment of the gelatin solution can be used to make the growing cells more dense; D-Hank’s solution is a commonly used cleaning solution.
  • FITC fluorescein isothiocyanate
  • the process of labeling nanocarriers with fluorescein isothiocyanate is as follows: add an aqueous solution (concentration of 1 mg/mL) formed by 1 mL of nanocarriers to 50 ⁇ LFITC (5 mg/mL), mix well and put Incubate in a 37°C incubator in the dark for 2h. After the labeling is completed, add tribenzol to the ultrafiltration tube, and the volume of tribenzyl blue added accounts for 1/10 of the total volume of the ultrafiltration tube at a speed of 15000rpm.
  • FITC fluorescein isothiocyanate
  • the process of obtaining penetration efficiency using fluorescence spectroscopy is to fluorescently label all nanocarriers and the number of labeled nanocarriers is n, and the number of nanocarriers observed through the blood-brain barrier is m, so that m/n is Nanocarrier penetration efficiency.
  • MALDI-TOF can be used to identify whether nanocarriers penetrate the blood-brain barrier.
  • Fig. 5 is a schematic diagram of the carrier penetrating the blood-brain barrier with time.
  • the nano-carrier has a strong ability to penetrate the blood-brain barrier.
  • the incubation time is 1h
  • the amount of penetration of the nano-carrier is 0.8%
  • the penetration of the nanocarrier is 1.8% when the incubation time is 4h
  • the penetration of the nanocarrier is 3.4% when the incubation time is 8h
  • the penetration of the nanocarrier can reach 5 when the incubation time is 12h %.
  • surface plasmon resonance imaging technology can be used to test the binding ability between the nanocarrier with peptoid compound and EGFR protein, the specific steps are as follows:
  • S302 Spot the samples on the surface of a 3D chip. Repeat 3 spots for each sample. After incubating at 4°C for 12 hours, wash with 10XPBS, 1XPBS, and ultrapure water. Then, the chip was blocked with 1M hydrochloric acid aminoethanol for 30 minutes, then washed 5 times with ultrapure water, and finally dried with clean nitrogen;
  • S303 Install the chip on the SPRi instrument, measure the SPRi angle and adjust to the best optical position, select the relevant detection points in the detection area, including sample points and blank points, and set the experimental flow rate to 2 ⁇ L/s;
  • S304 Select PBS as the buffer solution and pass it into the flow cell until the baseline is stable, and then pass the EGFR protein concentration of 55.6nM, 22.8nM, 11.4nM, 5.70nM and 2.85nM for detection.
  • the binding time is 300 seconds and the dissociation time is 300. In seconds, phosphoric acid is passed between each concentration for regeneration.
  • the SPRi instrument in the embodiment of the present disclosure is Plexera Kx5V2, Plexera Bioscience LLC, USA.
  • the instrument is mainly equipped with a 660 nm LED light source, a CCD image collector, and a sensor chip with a microfluidic channel.
  • the instrument displays each monitoring point The change of the reflected light intensity with time is recorded as an SPR curve.
  • FIG. 6 is a graph showing the results of surface plasmon resonance detection of a nanocarrier provided with an EGFR protein at a concentration of 55.6 nM, 22.8 nM, 11.4 nM, 5.70 nM, and 2.85 nM, according to an embodiment of the present disclosure.
  • the curve is the test result of PlexArray HT.
  • the equilibrium dissociation constant K D is 1.21 ⁇ 10 -9mol /L, which indicates that the nanocarrier has a very high affinity level with EGFR protein and can be used as a target Drug carriers for EGFR tumors are used in related research.
  • the nanocarrier can be prepared by chemical synthesis method, with high purity, small molecular weight, strong specificity, no immunogen, safe and reliable.
  • At least one embodiment of the present disclosure also provides a pharmaceutical composition, including: any nanocarrier as described above; and pharmaceutically acceptable auxiliary materials.
  • the auxiliary material is any one or a combination of at least two of excipients, diluents, carriers, flavoring agents, binders and fillers, which is not limited by the embodiments of the present disclosure.
  • excipients and nanocarriers constitute a new and more effective pharmaceutical composition.
  • excipients refer to other components in the pharmaceutical preparations than the main drug, and are non-therapeutic substances.
  • the carrier included in the excipient usually refers to the drug-carrying system in the drug delivery system, such as liposomes, starch microspheres, and albumin microspheres.
  • the pharmaceutical composition has an obvious targeting effect on the tumor marker EGFR protein, strong selectivity, high drug loading efficiency, and almost no toxicity, which greatly improves the safety of the drug carrier and is high in EGFR Diagnosis and treatment of brain metastases expressing glioma or lung cancer provide new options.
  • At least one embodiment of the present disclosure also provides use of any one of the above pharmaceutical compositions in the preparation of a medicament for treating diseases related to human epidermal growth factor receptor 1 (EGFR).
  • EGFR human epidermal growth factor receptor 1
  • the embodiments of the present disclosure also provide the use of the above pharmaceutical composition for imaging detection or prognosis monitoring of diseases related to EGFR protein.
  • diseases related to human epidermal growth factor receptor 1 include glioma and brain metastases from lung cancer.
  • the embodiments of the present disclosure provide a peptoid compound and a preparation method thereof, a nanocarrier, a pharmaceutical composition, and the use of the pharmaceutical composition in the preparation of a medicament for treating diseases related to human epidermal growth factor receptor 1 (EGFR). At least one beneficial effect:
  • the peptoid compound has high affinity for EGFR protein, strong targeting effect on EGFR protein, strong selectivity, and high drug loading efficiency It has no toxicity and improves safety. Therefore, it can realize targeted drug loading and localized imaging by recognizing EGFR protein, providing new options for the diagnosis and treatment of EGFR highly expressed gliomas, lung cancer brain metastases and other EGFR positive tumors. It also provides in vivo data support for sequencing and digital PCR results, and makes new and more effective targeted anticancer drugs by adding pharmaceutically acceptable excipients or adjuvants;
  • the nanocarrier has a strong binding ability to the EGFR protein, and the kinetic constant of the binding between the nanocarrier and the EGFR protein is obtained by surface plasmon resonance technology
  • the equilibrium dissociation constant KD is 1.21 ⁇ 10 -9mol /L;
  • the synthesis method of the peptoid compound and the nanocarrier provided by at least one embodiment of the present disclosure is simple, the preparation efficiency is high, and the production cost is low.

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Abstract

提供一种类肽化合物，包括：半胱氨酸(Cys)亚单位、1,4-丁二胺(Nlys)亚单位、胡椒基胺亚单位、丙酸胺亚单位和1-萘胺亚单位，所述类肽化合物对EGFR蛋白具有亲和力和选择性，同时具有药物包载效率，没有毒性。还提供了一种包括所述类肽的纳米载体、药物组合物以及该药物组合物在制备治疗与人表皮生长因子受体1(EGFR)相关的疾病的药物中的用途。

Description

类肽化合物及其制备方法、纳米载体以及药物组合物 技术领域
本公开的实施例涉及一种类肽化合物及其制备方法、纳米载体、药物组合物以及该药物组合物在制备治疗与人表皮生长因子受体1(EGFR)相关的疾病的药物中的用途。
背景技术
源自神经上皮的肿瘤统称为脑胶质瘤，占颅脑肿瘤的40％～50％，是最常见的颅内恶性肿瘤，年发病率为3～8人/10万人口。肺癌是目前发病及致死率最高的癌症，肺癌脑转移和由于血脑屏障的因素导致肺癌更加难以被治愈。
癌症的靶向治疗和活体成像已经成为当今学术界、临床医学界最为热门的课题之一。目前临床上应用的抗肿瘤药物主要是细胞毒类化疗药物，但该类药物选择性差，毒副作用大，容易引起不良反应。而抗体等蛋白类药物虽然特异性高、毒副作用小，但由于其分子质量大、结构复杂、容易引起免疫反应和产生耐药性，且其制备工艺复杂以至于价格高昂，难以为普通肿瘤患者所承受。
发明内容
本公开至少一实施例提供一种类肽化合物，该类肽化合物包括：半胱氨酸(Cys)亚单位、1,4-丁二胺(Nlys)亚单位、胡椒基胺亚单位、丙酸胺亚单位和1-萘胺亚单位。
例如，在本公开至少一实施例提供的类肽化合物中，所述类肽化合物包含的亚单位的顺序为半胱氨酸(Cys)亚单位—1,4-丁二胺(Nlys)亚单位—1,4-丁二胺(Nlys)亚单位—胡椒基胺亚单位—1,4-丁二胺(Nlys)亚单位—丙酸胺亚单位—1-萘胺亚单位。
例如，在本公开至少一实施例提供的类肽化合物中，所述类肽化合物具有式I所示的结构：
Figure PCTCN2019070261-appb-000001
本公开至少一实施例还提供一种制备上述任一项所述的类肽化合物的方法，其中，所述方法包括固相合成法连接亚单位。
例如，在本公开至少一实施例提供的方法中，所述方法包括以下步骤：(1)按照所述类肽化合物的亚单位的连接顺序，将所述类肽化合物的第一个亚单位连接至固相载体上；(2)将溴乙酸或溴乙酰氯与连接至所述固相载体上的第一个亚单位的氨基进行反应形成酰胺键；(3)将所述类肽化合物的第二个亚单位的供体与步骤(2)得到的产物进行反应，取代掉溴原子，完成第二个亚单位的连接；(4)重复进行步骤(2)和(3)，直至完成所有亚单位的连接；(5)从所述固相载体上将合成得到的类肽化合物裂解下来得到该类肽化合物。
本公开至少一实施例还提供一种纳米载体，该纳米载体包括：如上任一项所述的类肽化合物、聚酰胺-胺型树枝状高分子(PAMAM)和聚乙二醇(PEG)，其中，所述聚乙二醇(PEG)连接所述类肽化合物和所述聚酰胺-胺型树枝状高分子(PAMAM)。
例如，在本公开至少一实施例提供的纳米载体中，所述类肽化合物与所述聚酰胺-胺型树枝状高分子(PAMAM)的摩尔比为1:5～1:1，所述PEG与所述PAMAM的摩尔比为1:25～1:35。
例如，在本公开至少一实施例提供的纳米载体中，所述聚酰胺-胺型树枝状高分子(PAMAM)的代数为4代以上。
本公开至少一实施例还提供一种药物组合物，包括：如上任一项所述的纳米载体；以及药学上接受的辅料。
例如，在本公开至少一实施例提供的药物组合物中，所述辅料为赋形剂、稀释剂、载体、调味剂、粘合剂和填充剂中的任意一种或至少两种的组合。
本公开至少一实施例还提供一种如上任一项所述的药物组合物在制备治疗与人表皮生长因子受体1(EGFR)相关的疾病的药物中的用途。
例如，在本公开至少一实施例提供的用途中，所述疾病包括胶质瘤和肺癌脑转移。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例的附图作简单地介绍，显而易见地，下面描述中的附图仅仅涉及本发明的一些实施例，而非对本发明的限制。
图1为本公开一实施例提供的一种类肽化合物的制备方法的流程图；
图2为本公开一实施例提供的一种纳米载体的结构示意图；
图3为本公开一实施例提供的一种纳米载体的扫描电子显微镜图；
图4为本公开一实施例提供的一种纳米载体穿透血脑屏障的能力示意图；
图5为本公开一实施例提供的随着时间的变化纳米载体穿透血脑屏障的示意图；以及
图6为本公开一实施例提供的一种纳米载体与浓度为55.6nM、22.8nM、11.4nM、5.70nM和2.85nM的EGFR蛋白结合的表面等离基元共振检测的结果图。
附图标记：
10-纳米载体；11-类肽化合物；12-聚酰胺-胺型树枝状高分子(PAMAM)；13-聚乙二醇(PEG)。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例的附图，对本发明实施例的技术方案进行清楚、完整地描述。显然，所描述的实施例是本发明的一部分实施例，而不是全部的实施例。基于所描述的本发明的实施例，本领域普通技术人员在无需创造性劳动的前提下所获得的所有其他实施例，都属于本发明保护的范围。
除非另外定义，本公开使用的技术术语或者科学术语应当为本发明所属领域内具有一般技能的人士所理解的通常意义。本公开中使用的“包括”或者“包含”等类似的词语意指出现该词前面的要素涵盖出现在该词后面列举的要素及其等同，而不排除其他要素。本公开的亚单元(monomer)是指在固 相合成类肽化合物中加入的原料胺。本公开的亚单位(subunit)是指组成类肽化合物的结构单元。
除非特殊说明，本文中的“EGFR”指的是人表皮生长因子受体1。
除非特殊说明，本文中的“nM”指的是“n mol/L”，“μM”指的是“μ mol/L”，“mM”指的是“m mol/L”。
借助实体瘤的高通透性和滞留效应(Enhanced permeability and retention effect,EPR)，新型药物载体可通过被动靶向的方式将药物载带到肿瘤部位。特异性的靶向给药需要借助采用特定的识别元件，使得药物在肿瘤部位选择性地主动富集，使得药物的浓度增加，渗透作用提高，从而显著提高疗效。活体成像以识别元件作为主体，结合荧光、核磁等成像手段实现目的。因此识别元件是整个靶向药物以及活体成像的重中之重，例如，该识别元件包括抗体、多肽、类肽和核酸适配体等肿瘤部位特定受体蛋白的靶向分子。在各种靶向分子中，适配体小分子具备了一系列特有的优势，例如，免疫原性低、组织渗透性好、分子量小、稳定性高、易于修饰且经济等。
本公开至少一实施例提供一种类肽化合物，该类肽化合物包括：半胱氨酸(Cys)亚单位、1,4-丁二胺(Nlys)亚单位、胡椒基胺亚单位、丙酸胺亚单位和1-萘胺亚单位。
需要说明的是，类肽化合物是以N-取代甘氨酸作为结构单元的多肽模拟物。相较于多肽化合物，类肽化合物的侧链从α-碳上转移到了N上。不同于传统的多肽化合物只有20种氨基酸，类肽化合物由亚单元合成法合成，其组成单元由不同的胺所决定的，胺的种类成千上万，因此类肽化合物的序列种类极为丰富，可以针对不同的靶标开发不同的化学序列结构，并且由于类肽化合物不被酶所识别，类肽化合物能够有效地抵抗体内的蛋白酶解作用，这使得类肽化合物在作为分子探针上具有更加明显的优势。
基于类肽化合物分子探针开发的药物递送系统和分子成像系统可以增强药物的稳定性，提高药物与肿瘤细胞、组织的相互作用，可以增加体内的循环代谢周期，因此在体内成像诊断、增强药效、克服耐药性和降低毒副作用等方面具有优势。
例如，在本公开至少一实施例提供的类肽化合物中，该类肽化合物包含的亚单位的顺序为半胱氨酸(Cys)亚单位—1,4-丁二胺(Nlys)亚单位—1,4-丁二胺(Nlys)亚单位—胡椒基胺亚单位—1,4-丁二胺(Nlys)亚单位—丙酸 胺亚单位—1-萘胺亚单位。
例如，本公开至少一实施例提供的类肽化合物中，是式I的化合物或其可药用盐：
Figure PCTCN2019070261-appb-000002
例如，在本公开的实施例中，类肽化合物是式I所示的化合物。
术语“立体异构体”属于同分异构体的一种，其是指分子中原子或原子团互相连接次序相同，但是空间排列不同而引起的异构体。
关于立体异构体，式I化合物具有1个不对称碳原子，其可作为单独的对映异构体存在，也可作为对映异构体的混合物例如外消旋混合物或对映异构体富集的混合物存在。本公开的实施例涵盖包括式I化合物或其可药用盐的任何立体异构体以及其各种形式的混合物。
例如，在本公开的实施例中，类肽化合物可以以盐的形式存在。例如，该盐可以通过类肽化合物与无机酸或有机酸反应制备形成。该无机酸例如为盐酸，氢溴酸，硫酸，硝酸，磷酸等。该有机酸例如为甲酸，乙酸，丙酸，乙醇酸，丙酮酸，草酸，苹果酸，丙二酸，琥珀酸，马来酸，富马酸，酒石酸，柠檬酸，苯甲酸，肉桂酸，扁桃酸，甲磺酸，乙磺酸，对甲苯磺酸，水杨酸等。
例如，在本公开的实施例中，该盐是可药用盐。例如，药用盐可以是盐酸盐、氢溴酸盐、硫酸盐、硝酸盐、磷酸盐、甲酸盐、乙酸盐、丙酸盐、富马酸盐、乙醇酸盐、丙酮酸盐、苹果酸盐、丙二酸盐、苯甲酸盐、肉桂酸盐、扁桃酸盐、水杨酸盐、马来酸盐、柠檬酸盐、琥珀酸盐、酒石酸盐、甲磺酸盐、乙磺酸盐、或者对甲苯磺酸盐。
例如，在本公开的实施例中，可药用盐例如是盐酸盐、硝酸盐、硫酸盐、磷酸盐、甲酸盐、乙酸盐、富马酸盐、马来酸盐、柠檬酸盐、琥珀酸盐、酒石酸盐、甲磺酸盐、或者对甲苯磺酸盐。
例如，上述类肽化合物可以特异性识别全长的人表皮生长因子受体 1(EGFR蛋白)，可以更低成本、更精准、更高效地实现EGFR高表达胶质瘤或肺癌脑转移的靶向载药的治疗。
本公开至少一实施例还提供一种制备上述类肽化合物的方法，其中，该方法包括固相合成法连接亚单位。
例如，图1为本公开一实施例提供的一种类肽化合物的制备方法的流程图，该制备方法包括以下步骤：
S101：按照类肽化合物的亚单位的连接顺序，将所述类肽化合物的第一个亚单位连接至固相载体上；
S102：将溴乙酸或溴乙酰氯与连接至固相载体上的第一个亚单位的氨基进行反应形成酰胺键；
S103：将类肽化合物的第二个亚单位的供体(亚单元)与步骤S102得到的产物进行反应，取代掉溴原子，完成第二个亚单位的连接；
S104：重复进行步骤S102和S103，直至完成所有亚单位的连接；
其中，亚单元的投入顺序为：半胱氨酸、单保护的1,4-丁二胺、单保护的1,4-丁二胺、胡椒基胺、单保护的1,4-丁二胺、丙酸胺和1-萘胺，单保护指的是二胺中的一个氨基被氨基保护基团保护。
例如，作为氨基的保护基团，可以不受限制地使用本领域中已知的用于蛋白、多肽或类肽化合物的合成过程中保护氨基的基团，例如，该氨基保护基团为9-芴甲氧羰基(Fmoc)或叔丁氧羰基(Boc)。例如，在一些实施方式中，氨基保护基团为叔丁氧羰基。
S105：从固相载体上将合成得到的类肽化合物裂解下来得到该类肽化合物。
本公开的类肽化合物可通过选择原料来制备单独的对映异构体或者外消旋混合物。例如在本公开的一种类肽化合物的制备中，半胱氨酸原料可选自D-半胱氨酸、L-半胱氨酸者二者的混合物。
本公开的实施例中的类肽化合物也可通过手性拆分，例如通过手性HPLC拆分得到单独的对映异构体。
例如，当溴乙酸与连接至固相载体上的第一个亚单位的氨基进行反应形成酰胺键时，上述步骤S102和S103的反应示意图如下所示：
Figure PCTCN2019070261-appb-000003
例如，当溴乙酰氯与连接至固相载体上的第一个亚单位的氨基进行反应形成酰胺键时，上述步骤S102和S103的反应示意图如下所示：
Figure PCTCN2019070261-appb-000004
例如，上述步骤S102中的酰胺化反应的反应条件没有特别限制，可以采用本领域中用于进行蛋白、多肽或类肽化合物合成的酰胺化反应的常规条件，只要能够将氨基酰化并且不破坏类肽的功能即可。例如，上述酰胺化反应可以在缩合剂存在的条件下进行。该缩合剂可以不受限制地使用本领域中已知的用于蛋白、多肽或类肽化合物合成的缩合剂。例如，该缩合剂可以为碳二亚胺类缩合剂，例如，N,N’-二异丙基碳二亚胺(DIC)，N,N’-二环己基碳二亚胺(DCC)，1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(EDC)等；苯并三氮唑类缩合剂，例如1-羟基-苯并-三氮唑(HOBt)；苯磺酰氯类缩合剂，例如三异丙基苯磺酰氯(TPS)等；琥珀亚酰胺类缩合剂，例如二琥珀亚酰胺羰酸酯(DSC)，琥珀亚酰胺磷酸二苯酯(SDPP)等；2-乙氧基-1-乙氧碳酰基-1,2-二氢喹啉(EEDQ)；3-(二乙氧基磷酰基)-氧-1,2,3-苯并三嗪-4(3H)-酮(DEPBT)等。
上述步骤S103中的亲核取代反应的反应条件没有特别限制，可以采用本领域中用于进行蛋白、多肽或类肽化合物合成的亲核取代反应的常规条 件，只要能够替换掉溴原子并且不破坏类肽化合物的功能即可，例如可以在35～40℃的温度下反应30分钟以上、60分钟以上或90分钟以上。
上述步骤S105中，脱去侧链氨基保护基团与将类肽化合物从树脂上裂解下来可以同时或先后进行。例如，先将类肽化合物从树脂上裂解下来，然后脱去侧链氨基保护基团；也可以先脱去侧链氨基保护基团，然后将类肽化合物从树脂上裂解下来；或者在脱去侧链氨基保护基团的同时将类肽化合物从树脂上裂解下来。所述脱去侧链氨基保护基团与将类肽化合物从树脂上裂解下来可以采用本领域中用于进行蛋白、多肽或类肽化合物合成的常规条件，只要能够实现目的并且不破坏类肽化合物的功能即可。在一个实施方式中，可以采用以体积比计含95％三氟乙酸、2.5％超纯水和2.5％三异丙基硅烷的裂解液在脱去侧链氨基保护基团的同时将类肽化合物从树脂上裂解下来。
例如，式I所示的类肽化合物通过固相合成法合成，其具体的制备过程包括：
(1)将Rink amide AM树脂(取代水平0.3mmol/g，200mg)浸泡于N,N-二甲基甲酰胺(DMF)中，充分溶胀15分钟后排出DMF溶液；
(2)脱保护：配置质量百分含量为20％六氢吡啶的DMF溶液，将树脂浸入过量脱保护液中反应10min，脱去保护基团，露出游离的氨基；
(3)清洗树脂：用二氯甲烷(DCM)、DMF交替清洗各3次；
(4)将2M溴乙酸的DMF溶液(2.5ml)和3.2M N,N’-二异丙基碳二亚胺(DIC)的DMF溶液(2.5ml)加入Rink amide AM树脂中，在37℃下反应30min，将树脂末端的氨基酰化；
(5)再加入2M待加入的亚单元的DMF溶液(5ml)在37℃下反应90min，通过亲核取代反应替换掉溴原子，完成一个亚单位的合成；
(6)清洗树脂：用DCM、DMF交替清洗树脂各3次；
(7)重复步骤(4)-(6)直至完成其余亚单位的合成；其中，亚单元的投入顺序为：半胱氨酸、单保护的1,4-丁二胺、单保护的1,4-丁二胺、胡椒基胺、单保护的1,4-丁二胺、丙酸胺和1-萘胺；
(8)待合成完毕后，侧链保护基团被除去，并通过用95％三氟乙酸，2.5％超纯水，2.5％三异丙基硅烷处理2.5小时将类肽化合物从树脂上裂解下来；
(9)在过滤、稀释和冷冻干燥之后，通过HPLC纯化类肽化合物。
在本公开的类肽化合物的制备方法中，还可以视需要包括将所得产物纯化的步骤。该纯化的方法没有特殊限制，可以采用本领域中已知的纯化相应类似产物的方法，例如沉淀、过滤、透析、凝胶渗透色谱或者HPLC等。
本公开至少一实施例还提供一种纳米载体，例如，图2为本公开一实施例提供的一种纳米载体的结构示意图，如图2所示，该纳米载体10包括：上述任一类肽化合物11，聚酰胺-胺型树枝状高分子(PAMAM)12和聚乙二醇(PEG)13，其中，该聚乙二醇(PEG)13连接该类肽化合物11和该聚酰胺-胺型树枝状高分子(PAMAM)12。
例如，在该纳米载体10中的类肽化合物11与聚酰胺-胺型树枝状高分子(PAMAM)12的摩尔比为1:5～1:1。
例如，PEG13与PAMAM12的摩尔比为1:25～1:35，例如，PEG与PAMAM的摩尔比为1:30。
例如，该PAMAM12外围的部分或全部氨基各自连接一个PEG13分子的一端，部分或全部PEG13的另一端连接类肽化合物11。
例如，部分或全部PEG13的另一端与类肽化合物11通过化学键连接。
例如，PAMAM树状大分子具有柔性，在水溶液中具有类球形的结构，且具有内密外疏的结构，即随着晶相距离增大，其密度降低。PAMAM树状大分子在药物载体、纳米复合材料、纳米反应器、毛细管气相色谱固定相、废水处理、乳化炸药稳定剂、催化剂、高分子材料的流变学改性剂、光电传感、液晶、单分子膜和基因载体等多方面具有广阔的应用前景。
例如，聚酰胺-胺型树枝状大分子(PAMAM)偶联类肽化合物作为一种稳定的药物载体，具有易于设计合成和修饰、生物相容性好、载药量高、毒性低及药物递送率高等优点，具有广泛的应用前景。高代数(例如，4代或者4代以上)的聚酰胺-胺型树枝状大分子通过在水溶液中调控pH而改变电荷分布，从而将疏水性药物包裹入核内作为药物载体，药物可以通过物理包载或化学键合两种方式结合或键合到聚酰胺-胺型树枝状大分子(PAMAM)偶联类肽化合物形成的纳米载体中，药物通过纳米载体自身的降解或从纳米载体上断裂来达到缓释的效果，这样可以增大药物的利用率、提高药物的靶向性、降低药物的毒副作用。
例如，不同pH下PAMAM树枝状大分子所带的电荷不同，在酸性条件 下，PAMAM最外层胺及内部叔胺全部质子化，共有126个正电荷。在中性条件下，PAMAM只有最外层胺被质子化，这样一个第4代树枝状大分子带64个正电荷，PAMAM树枝状大分子内包裹的药物和其外侧偶联的类肽化合物在酸性和中性条件下都不携带静电荷。
例如，在EGFR高表达胶质瘤或肺癌脑转移靶的细胞的周围，pH值较低，偏酸性条件，这样PAMAM树枝状大分子的表面所带的电荷较多，带电纳米粒子可以潜入到细胞膜，且诱导膜形成空洞的能力和纳米粒子的尺寸也相关。
例如，该类肽化合物11对EGFR蛋白具有高的亲和力，对EGFR蛋白的靶向作用强，选择性强，同时具有高的药物包载效率，没有毒性，提高了安全性，因此能够通过识别EGFR蛋白实现靶向载药和定位成像，为EGFR高表达胶质瘤、肺癌脑转移和其它EGFR阳性肿瘤的诊断和治疗提供了新的选择，也为测序及数字PCR结果提供活体数据佐证，通过添加药学上可接受的辅料或佐剂制成新型的更有效的靶向抗癌药物。
例如，该聚酰胺-胺型树枝状高分子(PAMAM)的代数为4代以上，例如，该聚酰胺-胺型树枝状高分子(PAMAM)的代数为5代、6代等。聚酰胺-胺型树枝状高分子(PAMAM)的代数越高，分支越多，其越接近于球状。
例如，该纳米载体的合成过程为：
S201：合成聚酰胺-胺型树枝状高分子(PAMAM)(购自sigma公司)-聚乙二醇(PEG)(购自sigma公司)。
例如，以第四代聚酰胺-胺型(G4PAMAM)树枝状大分子为例加以说明，将摩尔比为1:1的甲醇(MeOH)和二甲基亚砜(DMSO)混合形成混合溶剂，用N-羟基琥珀酰亚胺(NHS)对PEG进行活化，将PAMAM树枝状聚合物和活化后的PEG混合后加入上述混合溶剂中，在室温下反应15分钟合成PAMAM-PEG，并用Sephadex G-50柱纯化以除去未反应的PEG。
S202：合成类肽化合物。
上述类肽化合物的合成过程可以参见上述步骤S101～S105中的相关描述。
S203：使用马来酰胺(购自sigma公司)活化步骤S201中形成的PAMAM-PEG，并与一定比例的类肽化合物在室温下反应，得到纳米载体。
例如，采用马来酰胺活化的PAMAM-PEG与类肽化合物的反应机理图 为：
Figure PCTCN2019070261-appb-000005
例如，将摩尔比为1:15的通过马来酰胺(Mal)活化的PAMAM-PEG和类肽化合物进行混合，且在室温下连续搅拌30小时，将反应混合物透析并冻干，得到纳米载体。
例如，图3为本公开一实施例提供的一种纳米载体的扫描电子显微镜图，从图3中可以看出，该纳米载体成球状，且平均粒径大约为4.2nm，该尺寸的纳米载体可以很容易地进入细胞膜。
例如，可以通过建立血脑屏障模型，验证药物穿透血脑屏障的能力，图4为本公开一实施例提供的一种载体穿透血脑屏障的能力示意图，如图4所示，血脑屏障包括内侧和外侧，在血脑屏障的内侧形成有细胞和被标记的纳米载体，血脑屏障、细胞和被标记的纳米载体均放在培养液中，通过测量血脑屏障外侧的被标记的纳米载体的数量计算纳米载体的穿透效率。
验证药物穿透血脑屏障的能力的具体步骤如下：
(1)在37℃下，用质量百分含量为2％的明胶溶液将具有12孔的细胞插入器浸泡30min以进行前处理，然后用D-Hank’s溶液清洗2次后吸干。
需要说明的是，脑内上皮血管细胞比较密集，可以通过明胶溶液前处理以使得生长的细胞更加密集；D-Hank’s溶液是常用的清洗溶液。
(2)在该具有12孔的细胞插入器的每个孔中放置75000个鼠源BMVECs细胞(来源于北京中日友好医院)，该细胞插入器的平均孔径为3cm，并在细胞插入器外侧加入1mL培养液，培养4d(2天换一次培养液)。
(3)4d后对细胞插入器进行检漏，将细胞插入器外侧的培养液加入内层，若液面高度差能保持至少4小时不变，且上皮细胞电阻高于250Ω/cm 2则模型建立完成。
(4)将异硫氰酸荧光素(FITC)标记的纳米载体加入到细胞插入器的内层，最终浓度为0.2g/mL，细胞插入器的外侧是1mL培养液，在37℃下孵育12h。
(5)分别在孵育时间为1h、4h、8h、12h时从外侧取400μL培养液，并立刻补充400μL新的培养液。
(6)用荧光光谱和MALDI-TOF来检测并计算穿透效率。
例如，采用异硫氰酸荧光素(FITC)对纳米载体进行标记的过程为：将1mL纳米载体形成的水溶液(浓度为1mg/mL)加入到50μLFITC(5mg/mL)中，混合均匀后放入37℃恒温箱中避光温育2h，标记结束以后加入曲利苯蓝至超滤管中，且加入的曲利苯蓝的体积占超滤管总体积的1/10，在转速为15000rpm的离心机中充分离心10min，然后加入一定量的缓冲液(PBS，pH＝7.2)至上述超滤管中，并轻轻捶打混匀，然后在转速为15000rpm的离心机中充分离心10min，重复上述离心操作多次，直至超滤管中的蓝色消失，未标记的FITC被彻底清除干净，这样标记过程完成。
例如，利用荧光光谱获得穿透效率的过程为，将所有纳米载体进行荧光标记且标记的纳米载体的数量为n，观察通过血脑屏障的纳米载体的数量为m，这样通过m/n即为纳米载体的穿透效率。
例如，可以利用MALDI-TOF鉴别纳米载体是否穿透血脑屏障。
图5为随着时间的变化载体穿透血脑屏障的示意图，如图5所示，纳米载体具有较强的穿透血脑屏障的能力，在孵育时间为1h时纳米载体的穿透量为0.8％；在孵育时间为4h时纳米载体的穿透量为1.8％；在孵育时间为8h时纳米载体的穿透量为3.4％；在孵育时间为12h时纳米载体的穿透量可以达到5％。
例如，可以利用表面等离子体共振成像技术测试带有类肽化合物的纳米载体与EGFR蛋白间结合能力，具体步骤如下：
S301：将纳米载体溶解到ddH 2O中，使得形成的纳米载体的摩尔浓度为1-1000μM；
S302：将样品点在一张3D芯片表面上，每种样品重复3个点，4℃孵育12小时后，用10XPBS，1X PBS，超纯水清洗。然后将芯片用1M盐酸氨基乙醇封闭30分钟，然后用超纯水清洗5次，最后用干净氮气吹干；
S303：将芯片安装在SPRi仪器上，测定SPRi角并调节至最佳光学位置，在检测区域选取相关检测点，包括样品点与空白点，设置实验流速为2μL/s；
S304：选择PBS为缓冲液通入流通池至基线稳定后依次通过浓度为55.6nM、22.8nM、11.4nM、5.70nM和2.85nM的EGFR蛋白进行检测，结合时间为300秒，解离时间为300秒，每个浓度间通入磷酸进行重生。
例如，本公开的实施例中的SPRi仪器为Plexera Kx5V2，Plexera  Bioscience LLC,USA，该仪器主要装配有660nmLED光源、CCD图像采集器和带微流通道的传感芯片，仪器显示每个监测点上反射光强度随时间的变化并记录为SPR曲线。
图6为本公开一实施例提供的一种纳米载体与浓度为55.6nM、22.8nM、11.4nM、5.70nM和2.85nM的EGFR蛋白结合的表面等离基元共振检测的结果图，其中，ΔAU代表流动相通过阵列后的结合信号减去初始PBS缓冲液的基线信号，表示结合信号的强度，曲线是PlexArray HT的测试结果。经BIA evaluation version 4.1 software(Biacore,Inc.)拟合，平衡解离常数K D为1.21×10 -9摩尔/升，这表明该纳米载体与EGFR蛋白具有相当高的亲和力水平，可以作为靶向EGFR肿瘤的药物载体用于相关的研究中。
例如，在本公开的实施例提供的制备方法中，纳米载体可以采用化学合成的方法制备得到，纯度高，分子量小，特异性强，无免疫原形，安全可靠。
本公开至少一实施例还提供一种药物组合物，包括：如上任一纳米载体；以及药学上接受的辅料。
例如，该辅料为赋形剂、稀释剂、载体、调味剂、粘合剂和填充剂中的任意一种或至少两种的组合，本公开的实施例对此不作限定。上述辅料与纳米载体构成新型的更有效的药物组合物。
需要说明的是，辅料是指药物制剂中除主药以外的其他成分，为非治疗物质。
辅料所包括的载体通常指给药系统中载带药物的系统，如脂质体、淀粉微球和白蛋白微球等。例如，该药物组合物对肿瘤标志物EGFR蛋白的靶向作用明显，选择性强，同时具有高的药物包载效率，而且几乎没有毒性，极大的提高了药物载体的安全性，为EGFR高表达胶质瘤或肺癌脑转移的诊断和治疗提供了新的选择。
本公开至少一实施例还提供一种上述任一药物组合物在制备治疗与人表皮生长因子受体1(EGFR)相关的疾病的药物中的用途。
例如，本公开的实施例还提供上述药物组合物在用于成像检测或预后监测与EGFR蛋白相关的疾病中的用途。
例如，在本公开至少一实施例提供的用途中，与人表皮生长因子受体1(EGFR)相关的疾病包括胶质瘤和肺癌脑转移。
本公开的实施例提供一种类肽化合物及其制备方法、纳米载体、药物组合物以及该药物组合物在制备治疗与人表皮生长因子受体1(EGFR)相关的疾病的药物中的用途具有以下至少一项有益效果：
(1)在本公开至少一实施例提供的类肽化合物中，该类肽化合物对EGFR蛋白具有高的亲和力，对EGFR蛋白的靶向作用强，选择性强，同时具有高的药物包载效率，没有毒性，提高了安全性，因此能够通过识别EGFR蛋白实现靶向载药和定位成像，为EGFR高表达胶质瘤、肺癌脑转移和其它EGFR阳性肿瘤的诊断和治疗提供了新的选择，也为测序及数字PCR结果提供活体数据佐证，通过添加药学上可接受的辅料或佐剂制成新型的更有效的靶向抗癌药物；
(2)在本公开至少一实施例提供的纳米载体中，该纳米载体与EGFR蛋白的结合能力较强，通过表面等离基元共振技术得到该纳米载体与EGFR蛋白的结合动力学常数中的平衡解离常数KD为1.21×10 -9摩尔/升；
(3)本公开至少一实施例提供的类肽化合物和纳米载体的合成方法简单，制备的效率高，且制作成本低。
需要说明的是：在不冲突的情况下，本发明的实施例及实施例中的特征可以相互组合以得到新的实施例。
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，本发明的保护范围应以所述权利要求的保护范围为准。

Claims (12)

  1. 一种类肽化合物，包括：半胱氨酸(Cys)亚单位、1,4-丁二胺(Nlys)亚单位、胡椒基胺亚单位、丙酸胺亚单位和1-萘胺亚单位。
  2. 根据权利要求1所述的类肽化合物，其中，所述类肽化合物包含的亚单位的顺序为半胱氨酸(Cys)亚单位—1,4-丁二胺(Nlys)亚单位—1,4-丁二胺(Nlys)亚单位—胡椒基胺亚单位—1,4-丁二胺(Nlys)亚单位—丙酸胺亚单位—1-萘胺亚单位。
  3. 根据权利要求1所述的类肽化合物，其中，所述类肽化合物具有式I所示的结构：
    Figure PCTCN2019070261-appb-100001
  4. 一种权利要求1-3中任一项所述的类肽化合物的制备方法，其中，所述方法包括固相合成法连接亚单位。
  5. 根据权利要求4所述的方法，其中，所述方法包括以下步骤：
    (1)按照所述类肽化合物的亚单位的连接顺序，将所述类肽化合物的第一个亚单位连接至固相载体上；
    (2)将溴乙酸或溴乙酰氯与连接至所述固相载体上的第一个亚单位的氨基进行反应形成酰胺键；
    (3)将所述类肽化合物的第二个亚单位的供体与步骤(2)得到的产物进行反应，取代掉溴原子，完成第二个亚单位的连接；
    (4)重复进行步骤(2)和(3)，直至完成所有亚单位的连接；
    (5)从所述固相载体上将合成得到的类肽化合物裂解下来得到该类肽化合物。
  6. 一种纳米载体，包括：
    如权利要求1-3中任一项所述的类肽化合物、聚酰胺-胺型树枝状高分子(PAMAM)和聚乙二醇(PEG)，其中，所述聚乙二醇(PEG)连接所述类 肽化合物和所述聚酰胺-胺型树枝状高分子(PAMAM)。
  7. 根据权利要求6所述的纳米载体，其中，所述类肽化合物与所述聚酰胺-胺型树枝状高分子(PAMAM)的摩尔比为1:5～1:1，所述PEG与所述PAMAM的摩尔比为1:25～1:35。
  8. 根据权利要求6或7所述的纳米载体，其中，所述聚酰胺-胺型树枝状高分子(PAMAM)的代数为4代以上。
  9. 一种药物组合物，包括：
    如权利要求6-8中任一项所述的纳米载体；以及
    药学上接受的辅料。
  10. 根据权利要求9所述的药物组合物，其中，所述辅料为赋形剂、稀释剂、载体、调味剂、粘合剂和填充剂中的任意一种或至少两种的组合。
  11. 一种如权利要求9或10所述的药物组合物在制备治疗与人表皮生长因子受体1(EGFR)相关的疾病的药物中的用途。
  12. 根据权利要求11所述的用途，其中，所述疾病包括胶质瘤和肺癌脑转移。
PCT/CN2019/070261 2019-01-03 2019-01-03 类肽化合物及其制备方法、纳米载体以及药物组合物 WO2020140237A1 (zh)

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