WO2024067600A1 - 糖响应性复合物及其制备方法和应用 - Google Patents

糖响应性复合物及其制备方法和应用 Download PDF

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WO2024067600A1
WO2024067600A1 PCT/CN2023/121610 CN2023121610W WO2024067600A1 WO 2024067600 A1 WO2024067600 A1 WO 2024067600A1 CN 2023121610 W CN2023121610 W CN 2023121610W WO 2024067600 A1 WO2024067600 A1 WO 2024067600A1
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insulin
sugar
polylysine
phenylboronic acid
responsive
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PCT/CN2023/121610
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English (en)
French (fr)
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顾臻
王金强
张娟
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浙江大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • 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
    • A61K47/50—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
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

  • the invention relates to a sugar-responsive complex and a preparation method and application thereof.
  • Glucose-responsive insulin can simulate the function of human ⁇ cells, release insulin quickly when blood sugar is high, and release insulin slowly when blood sugar is low. Therefore, glucose-responsive insulin can actively track the fluctuation of blood sugar, dynamically adjust the insulin release rate, meet the dynamic demand of insulin in real time, improve the therapeutic index of insulin, and improve the blood sugar control effect of insulin.
  • glucose-responsive mechanisms including phenylboronic acid derivatives, glucose-binding molecules, and glucose oxidase. Phenylboronic acid-based glucose-responsive insulin has been verified to have rapid and powerful in vitro and in vivo glucose-responsive insulin release performance, which is closer to clinical application.
  • glucose-responsive insulin it is still difficult for glucose-responsive insulin to control blood sugar within a normal range for a long period of time after a single injection. How to achieve rapid insulin release after a meal and continuous and slow basal insulin release during fasting for a long period of time after a single injection is still a considerable challenge for glucose-responsive insulin.
  • the present invention provides a sugar-responsive complex and its preparation method and application.
  • the sugar-responsive complex of the present invention has good glucose-responsive release performance, and can release insulin quickly at high blood sugar concentrations and continuously and slowly at normal blood sugar concentrations. It is worth noting that the complex The formation of subcutaneous fibrous capsules can be avoided, which is essential for the release of insulin from the complex reservoir.
  • the present invention adopts the following technical solutions.
  • the present invention provides a sugar-responsive complex, which includes mutually complexed phenylboronic acid-based polylysine and insulin with a diol structure, wherein the complexing force includes: (1) dynamic electrostatic attraction, and (2) covalent force between the phenylboronic acid structure of the phenylboronic acid-based polylysine and the diol structure of the insulin with a diol structure.
  • the phenylboronic acid-based polylysine is obtained by grafting polylysine with a carboxyl-modified phenylboronic acid compound. That is, in the phenylboronic acid-based polylysine, the phenylboronic acid group is grafted onto the side chain of the polylysine via an amide bond.
  • the polylysine may be E-polylysine and/or L-polylysine, preferably phenylboronic acid-based L-polylysine.
  • the molecular weight of the polylysine can be 2k-1000k, preferably 30k-70k.
  • the phenylboronic acid group may be a phenylboronic acid group substituted or unsubstituted with a substituent, wherein the substituent may be one or more of a halogen and a nitro group.
  • the phenylboronic acid group is selected from one or more of the following groups:
  • the phenylboronic acid grafting rate of the phenylboronic acid-based polylysine is preferably 25% to 75%, such as 30%, 35%, 40%, 45%, 50% or 60%.
  • the phenylboronic acid grafting rate refers to the percentage of amino groups covalently modified by phenylboronic acid groups on polylysine to the total number of amino groups on polylysine before modification.
  • the phenylboronic acid groups in the phenylboronic acid-based polylysine are randomly distributed.
  • R represents a hydrogen atom or one or more substituents, and the substituents are as described above; and the grafting rate of phenylboronic acid is y/(x+y).
  • the insulin having a diol structure can be prepared from insulin and a compound containing an ortho- or meta-hydroxyl group.
  • the compound containing an ortho- or meta-hydroxyl group can be one or more of gluconic acid, dopamine, fructose, lactose, ribose, deoxyribose, ⁇ -D-pyranoside, trehalose and maltose; preferably gluconic acid, more preferably D-gluconic acid.
  • the insulin having a diol structure is called gluconyl insulin (Glu-insulin).
  • the insulin loading rate in the sugar-responsive complex can be 5%-80%, preferably 40%-60%, more preferably 40%-50%, and further preferably 45%-48%, wherein the percentage is the insulin loading rate in the sugar-responsive complex.
  • R represents a hydrogen atom or one or more substituents, and the substituents are as described above; and the grafting rate of phenylboronic acid is y+z/(x+y+z).
  • the present invention provides a method for preparing the sugar-responsive complex, which comprises the following steps: mixing the aqueous solution of the phenylboronic acid-based polylysine with the aqueous solution of the insulin having a diol structure, and adjusting the pH to 6.5-8.0 to obtain the complex.
  • the mass ratio of the insulin having a diol structure to the phenylboronic acid-based polylysine may be 1:(0.5-10), preferably 1:(1-2), such as 1:1.5 or 1:1.
  • the concentration of the aqueous solution of phenylboronic acid-based polylysine may be 1 to 200 mg/mL, for example, 10 mg/mL.
  • the preparation method of the aqueous solution of phenylboronic acid-based polylysine can be: dissolving the phenylboronic acid-based polylysine in weakly acidic water; wherein the pH range of the weakly acidic water can be 2.0 to 7.0, preferably 2.0 to 3.0; the weakly acidic water can be phosphate buffer, deionized water or pure water.
  • the phenylboronic acid-based polylysine can be prepared by conventional methods in the art.
  • the method for preparing phenylboronic acid-modified polylysine comprises the following steps: mixing a polylysine solution and a solution of a carboxyl-modified phenylboronic acid compound and then performing a grafting reaction.
  • the molar ratio of the structural unit of polylysine to the carboxyl-modified phenylboronic acid compound is preferably 4:(3-1).
  • the solvent in the polylysine solution can be a conventional aqueous solvent in the art that can dissolve polylysine, preferably deionized water or pure water.
  • the concentration of the polylysine solution is preferably 1-200 mg/mL, such as 10 mg/mL.
  • the solvent in the solution of the carboxyl-modified phenylboronic acid compound can be a conventional water-miscible organic solvent in the art that can dissolve the carboxyl-modified phenylboronic acid compound, preferably dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF).
  • DMSO dimethyl sulfoxide
  • DMF N,N-dimethylformamide
  • the carboxyl-modified phenylboronic acid compound may be a substituent-substituted or unsubstituted p-carboxylphenylboronic acid or o-carboxylphenylboronic acid, wherein the substituent may be one or more of a halogen and a nitro group.
  • the carboxyl-modified phenylboronic acid compound is selected from one or more of the following compounds:
  • the carboxyl-modified phenylboronic acid compound is 4-carboxy-3-fluorophenylboronic acid.
  • the concentration of the solution of the carboxyl-modified phenylboronic acid compound is preferably 1-500 mg/mL, for example 24 mg/mL.
  • the mixing method includes dropping the solution of the carboxyl-modified phenylboronic acid compound into the polylysine solution and then stirring.
  • the stirring time is preferably 5 min to 24 h, for example 30 min.
  • the grafting reaction further includes a dialysis step.
  • the dialysis can be performed in deionized water using a conventional dialysis bag in the art.
  • the molecular weight cutoff of the dialysis bag is preferably 1k to 10k.
  • the purpose of the dialysis is to remove free carboxyl-modified phenylboronic acid compounds.
  • the grafting reaction further includes a freeze-drying step.
  • the freeze-drying step is performed after the dialysis. A white solid is obtained after the freeze-drying step.
  • the concentration of the aqueous solution of insulin having a diol structure may be 1 to 200 mg/mL, preferably 1 to 100 mg/mL, such as 10 mg/mL.
  • the preparation method of the aqueous solution of insulin with a diol structure can be: dissolving the insulin with a diol structure in weakly acidic water; wherein the pH range of the weakly acidic water can be 2.0 to 7.0, preferably 2.0 to 3.0; the weakly acidic water can be phosphate buffer, deionized water or pure water.
  • the insulin having a diol structure is preferably gluconic acid insulin.
  • Gluconate-based insulin can be prepared by conventional methods in the art.
  • the preparation method of the gluconic acid-based insulin comprises: (1) first reacting gluconic acid with N-hydroxysuccinimide or TSTU (2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate) in a solvent; (2) subsequently adding the gluconic acid to an insulin aqueous solution for reaction, adjusting the pH to 7-8, and post-treating the obtained product.
  • the solvent can be any conventional solvent in the art, preferably DMSO.
  • step (1) the reaction is preferably carried out at room temperature.
  • the molar ratio of the gluconic acid to the N-hydroxysuccinimide or TSTU is preferably 1: (1-1.2).
  • the insulin is preferably recombinant human insulin.
  • step (2) the reaction is preferably carried out under ice bath conditions.
  • the concentration of the aqueous insulin solution is preferably 0.2-100 mg/mL.
  • step (2) the pH of the aqueous insulin solution is preferably 7-8.
  • the post-treatment preferably includes dialysis and ion exchange column separation.
  • the dialysis can be performed in deionized water using a conventional dialysis bag in the art, and the molecular weight cutoff of the dialysis bag is preferably 1k to 3.5k.
  • the ion exchange column is preferably an anion exchange column.
  • the pH is adjusted to 6.5-7.4.
  • the white flocculent precipitate formed after adjusting the pH is the sugar-responsive complex.
  • the sugar-responsive complex is soluble or easily soluble in weakly acidic water and insoluble or slightly soluble in weakly alkaline water.
  • the step of centrifugation may be further included after the pH adjustment.
  • the purpose of the centrifugation is to collect the white flocculent precipitate and remove the uncomplexed insulin with a diol structure in the supernatant.
  • the concentration of free insulin in the supernatant obtained by the centrifugation is not more than 10%, preferably not more than 5%, as determined by Coomassie Brilliant Blue.
  • the insulin encapsulation rate of the preparation method can be above 90%, preferably above 95%.
  • the insulin encapsulation rate refers to the percentage of the insulin with a diol structure (complexed amount) compounded with the phenylboronic acid-based polylysine to the total mass (initial feed amount) of the insulin with a diol structure in the aqueous solution of the insulin with a diol structure.
  • the sugar-responsive complex of the present invention is an amorphous flocculent precipitate, which can form an insulin reservoir by subcutaneous injection.
  • the combination of high-concentration glucose and phenylboronic acid groups instantly reduces the positive charge density of the phenylboronic acid polylysine part, and at the same time, the phenylboronic acid ester bond is broken, resulting in a decrease in the interaction between insulin with a diol structure and the polymer, promoting the release of insulin, so that insulin has good high-concentration glucose responsive release performance, and promoting the release of insulin.
  • the body restores normal blood sugar; under normal blood sugar conditions, only a very small amount of insulin is in a free state, reducing the occurrence of hypoglycemia.
  • the present invention also provides an application of the sugar-responsive complex in preparing a drug for treating diabetes.
  • the diabetes may be type 1 diabetes or late stage type 2 diabetes.
  • the sugar-responsive complex can be resuspended in phosphate buffer or physiological saline and administered by subcutaneous injection for treatment.
  • the present invention also provides a method for treating diabetes in a patient, comprising administering the sugar-responsive complex to the patient.
  • the present invention also provides a medicine for treating diabetic patients, comprising the sugar-responsive complex.
  • the present invention also provides a sugar-responsive complex for use in treating diabetes.
  • the reagents and raw materials used in the present invention are commercially available.
  • the sugar-responsive complex of the present invention has good glucose-responsive release performance, and can release insulin quickly at high blood sugar concentrations and continuously and slowly at normal blood sugar concentrations (long-acting release).
  • the sugar-responsive complex of the present invention is an amorphous flocculent precipitate, and subcutaneous injection can form an insulin reservoir.
  • the combination of high concentrations of glucose and phenylboronic acid groups instantly reduces the positive charge density of phenylboronic acid-based polylysine, and is accompanied by the rupture of phenylboronic acid ester bonds, resulting in a reduction in the attraction between the insulin part and the polymer, promoting the release of insulin, so that insulin has good high-concentration glucose-responsive release performance, and promotes the body to restore normal blood sugar; under normal blood sugar conditions, only a very small part of insulin is in a free state, reducing the occurrence of hypoglycemia. It is worth noting that the complex does not cause a strong inflammatory response subcutaneously, and the formation of fibrous capsules can be avoided, which creates favorable conditions for the release of insulin in the sugar-responsive complex.
  • Subcutaneous injection of the sugar-responsive complex of the present invention can form an insulin reservoir, release insulin in a sustained manner, and have a blood sugar lowering effect of up to one week.
  • a higher dose can be given at one time to reduce the injection frequency and improve the compliance of diabetic patients with subcutaneous injection.
  • Figure 1 Schematic diagram of the mechanism of sugar-responsive complex formation and glucose-responsive insulin release.
  • Figure 9 Characterization of AKT signaling phosphorylation by recombinant human insulin or gluconate insulin.
  • Figure 10 Representative fluorescence images of sugar-responsive complexes.
  • FIG. 13 Pulse release curve of insulin from sugar-responsive complexes (sugar-responsive complexes were alternately exposed to 100 and 400 mg/dL glucose solutions).
  • FIG. 14 Blood glucose levels in type 1 diabetic mice treated with Glu-insulin and recombinant human insulin.
  • FIG. 1 Blood glucose levels in type 1 diabetic mice treated with subcutaneous injection of sugar-responsive complexes (20 mg/kg).
  • FIG. 17 Blood glucose levels in type 1 diabetic mice during intraperitoneal glucose tolerance test after treatment with sugar-responsive complexes.
  • FIG. 1 Blood glucose levels in type 1 diabetic mice during intraperitoneal glucose tolerance test after treatment with insulin glargine.
  • FIG. 19 Blood glucose levels and plasma insulin levels in type 1 diabetic mice subjected to intraperitoneal glucose tolerance test after treatment with the sugar-responsive complexes.
  • FIG 20 Blood glucose levels in type 1 diabetic mice injected subcutaneously with multiple sugar-responsive complexes (black arrows indicate three injections of the complexes).
  • Figure 21 Blood glucose levels in miniature pigs with type 1 diabetes treated with sugar-responsive complexes.
  • Figure 22 Levels of major serum biochemical indices in type 1 diabetic mice after subcutaneous injection of sugar-responsive complexes.
  • Figure 23 Representative fluorescence images of PLL-FPBA in the skin and major organs after subcutaneous injection of sugar-responsive complexes.
  • Figure 24 Representative images of H&E and Masson's trichrome staining of the skin at the site of sugar-responsive complex injection.
  • Figure 25 Representative H&E and Masson's trichrome staining images of skin tissue 2 weeks after subcutaneous implantation.
  • Figure 26 Representative H&E and Masson's trichrome staining images of skin tissue 4 weeks after subcutaneous implantation.
  • Figure 27 Representative H&E and Masson's trichrome staining images of skin tissue 12 weeks after subcutaneous implantation.
  • FIG. 28 Immunofluorescence (macrophage biomarker, F4/80, in red; ⁇ -smooth muscle actin, ⁇ -SMA, in green; cell nuclei in blue) and immunohistochemistry (TNF- ⁇ , IL-6, IL-10, IL-12, IL-17) staining.
  • Figure 29 Cumulative optical density statistics of immunohistochemistry (TNF- ⁇ , IL-6, IL-10, IL-12, IL-17) of the skin at the implantation site 2 weeks after subcutaneous implantation.
  • the preparation of the sugar-responsive complex and the glucose-responsive insulin release mechanism are shown in FIG1 .
  • FPBA-NHS 120 mg was dissolved in 5 mL of DMSO and added dropwise to 10 mL of phosphate buffered saline solution containing 100 mg of poly-L-lysine (PLL) (30k-70k), and the pH was controlled at about 7. After adding the FPBA-NHS solution, it was stirred for another 30 min, then dialyzed in deionized water (4 L), and the resulting mixture was lyophilized to obtain a white solid. The product was characterized by 1 H NMR (see Figure 4).
  • the free insulin in the supernatant was determined by Coomassie brilliant blue reagent.
  • Bradford reagent 200 ⁇ L
  • 10 ⁇ L of the supernatant that was resuspended and centrifuged after centrifugation were added to the 96-well plate in sequence.
  • the free insulin content in the supernatant was calculated by external standard method (standard curve shown in Figure 7) according to its ultraviolet absorption value at 595nm, thereby calculating the insulin encapsulation rate. According to calculations, the encapsulation rates of insulin in the preparation process of the sugar-responsive complex of Examples 1 to 3 were all higher than 95%.
  • the encapsulation rate of insulin in the preparation process of the sugar-responsive complex of Example 1 was higher than 95%; and the insulin used in Example 4 was relatively more, exceeding the loading efficiency of the polymer, so that there was more free insulin in the supernatant.
  • HepG2 cell line was obtained from the Cell Bank of the Chinese Academy of Sciences. HepG2 cells were cultured in a medium containing 10% fetal bovine serum and 1% penicillin/ Cultured in DMEM with streptomycin. When the cell density reached 80-90%, blank medium without serum was used for 12 h. Then, the cells were washed with PBS and medium containing recombinant human insulin or Glu-insulin was added and cultured for 10 minutes (recombinant human insulin or Glu-insulin concentrations were 0, 0.1, 1, 5, 10, 50, 100 nM).
  • the cells were washed with PBS and ice-cold RIPA buffer (Biyuntian) containing protease and phosphatase inhibitors (Meilun Biotechnology) was added for cell lysis.
  • the lysate was centrifuged at 16000g for 10 minutes at 4°C.
  • the protein concentration was determined by the BCA method (Biyuntian), and the samples were boiled in 1 ⁇ Laemmli loading buffer (Bio-Rad) containing 2.5% ⁇ -mercaptoethanol.
  • the proteins were separated by 10% SDS-PAGE gel and transferred to a 0.45 ⁇ m PVDF membrane (Sigma-Aldrich).
  • Blocking was performed with 5% skim milk in PBST (phosphate buffered saline containing 0.1% Tween-20), and antibodies [Anti-phospho-akt (CST, #4060, 1:2000), Anti-akt (CST, #4691, 1:1000) and Anti- ⁇ -Actin (Diagbio, #db7283, 1:1000)] were used in antibody dilution buffer (Biyuntian).
  • PBST phosphate buffered saline containing 0.1% Tween-20
  • Anti-phospho-akt CST, #4060, 1:2000
  • Anti-akt CST, #4691, 1:1000
  • Anti- ⁇ -Actin Diagbio, #db7283, 1:1000
  • Horseradish peroxidase-conjugated secondary antibodies were used to bind primary antibodies according to the manufacturer's instructions, and chemiluminescent substrates (ECL, Thermo Fisher) were used for color development.
  • FITC fluorescein isothiocyanate
  • the sugar-responsive complex was prepared according to step 3 of Example 1 using FITC-labeled Glu-insulin and Cy5-labeled PLL-FPBA.
  • Example 1 Weigh the sugar-responsive complex prepared in Example 1, add water to make the insulin equivalent concentration 1 mg/mL, use an ultrasonic cell disruptor, ultrasonically disrupt for 1 minute at 100 W power, disperse and suspend in 1 mL of water, dilute 10 times and drop onto a copper mesh. Add 10 ⁇ L of 5% uranyl acetate solution to the copper mesh, let it stand for 10 minutes, and remove the solution with filter paper. Observe the sample with a cryo-transmission electron microscope. Place the same sample on a silicon wafer, dry it naturally, and scan it. Scanning electron microscopy observation: Cryo-transmission electron microscopy and scanning electron microscopy confirmed ( FIG. 11 ) that the sugar-responsive complex has a porous and loose microstructure.
  • the glucose-responsive insulin release performance of the sugar-responsive complex prepared in Example 1 was evaluated in a phosphate buffered saline solution at pH 7.4, with four glucose concentrations of 0, 100, 200 and 400 mg/dL, respectively.
  • free Glu-insulin remained at a low level of about 10 ⁇ g/mL (see Figure 12).
  • the free Glu-insulin level increased to 19, 32 and 45 ⁇ g/mL, respectively, at which time the free Glu-insulin level reached equilibrium and remained essentially unchanged.
  • mice C57BL/6 mice (purchased from Hangzhou Medical College) were induced with type 1 diabetes at a dose of 150 mg/kg streptozotocin.
  • Diabetic mice were fed with a standard diet and a circadian cycle of 12 hours of light and 12 hours of darkness.
  • Five mice were subcutaneously injected with Glu-insulin and recombinant human insulin at an insulin equivalent dose of 1.5 mg/kg. Blood glucose was measured using a blood glucose tester.
  • Glu-insulin still has a similar hypoglycemic effect as recombinant human insulin.
  • a type 1 diabetic mouse model was established, and type 1 diabetic mice with blood glucose higher than 300 mg/dL were selected for evaluation of the therapeutic effect.
  • Each group had 5 mice, and each group was subcutaneously injected with glargine insulin (50 U/kg) and sugar-responsive complex (20 mg/kg). Blood glucose was measured using a blood glucose tester.
  • mice treated with the complex can stably maintain blood glucose.
  • diabetic mice treated with glargine insulin could not regulate blood glucose to normal levels 6 and 15 hours after treatment (see Figure 18).
  • Blood glucose regulation in vivo insulin release was further observed by intraperitoneal glucose administration.
  • glucose 3 g/kg was injected intraperitoneally.
  • Plasma was collected, blood glucose was measured, and plasma insulin levels were determined using an enzyme-linked immunosorbent assay (ELISA) kit (see Figure 19).
  • ELISA enzyme-linked immunosorbent assay
  • Injection of the glucose-responsive complex once every 168 hours three injections at doses of 20 mg/kg, 14 mg/kg, and 14 mg/kg, respectively) can control blood sugar to less than 200 mg/dL for at least 22 days.
  • the complex can achieve long-term blood sugar control in diabetic mice through multiple injections, and no obvious hypoglycemia was observed during this period.
  • Bama miniature pigs were intravenously injected with STZ (150 mg/kg) to induce type 1 diabetes.
  • the miniature pigs were fed twice a day and glargine insulin was used to control blood sugar. After the blood sugar was stable for 1 month, they could be used for research.
  • the blood sugar was monitored using a dynamic blood glucose monitoring system (FreeStyle Libre H, Abbott Laboratories, USA), and the blood sugar of the miniature pigs was above 200 mg/dL.
  • the blood sugar of the miniature pigs was usually controlled with glargine insulin. After 48 hours of stopping glargine insulin, the complex treatment was given to test the blood sugar control effect of the sugar-responsive complex.
  • the doses of subcutaneous injection of glargine insulin in miniature pigs were 0.4, 0.5, and 0.6 U/kg, respectively.
  • the doses of subcutaneous injection of the complex in miniature pigs were 0.2 mg/kg, 0.3 mg/kg, and 0.3 mg/kg.
  • a single injection of glargine insulin can reduce blood sugar for no more than 24 hours and will reduce blood sugar to below the detection limit (below 40 mg/dL). After seven consecutive days of injection of insulin glargine, blood sugar fluctuated between normal blood sugar and hyperglycemia, and normal blood sugar could not be maintained for a long time, and blood sugar was less than 40 mg/dL.
  • a type 1 diabetic mouse model was established, with 5 mice in each group. Each group was subcutaneously injected with PBS and sugar-responsive complex (20 mg/kg). Blood was collected after 1 week to measure serum alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin (ALB), blood urea nitrogen (BUN) and creatinine levels to evaluate the toxicity to the liver and kidney (see Figure 22). Compared with the mice in the PBS injection group, the changes in various indicators in the experimental group were not obvious, indicating that PLL-FPBA had little toxicity to the liver and kidney.
  • ALP serum alkaline phosphatase
  • AST aspartate aminotransferase
  • ALT alanine aminotransferase
  • ARB albumin
  • BUN blood urea nitrogen
  • Cy5-labeled PLL-FPBA was subcutaneously injected, and the size of PLL-FPBA was displayed using a live imaging system (IVIS Lumina III, PerkinElmer).
  • IVIS Lumina III PerkinElmer
  • the complex was mainly cleared by the liver in vivo.
  • Hematoxylin-eosin (H&E) staining and Masson's trichrome staining were used to evaluate the immune response of mice to the injected complex. Even four weeks after subcutaneous injection of the complex, no obvious neutrophil infiltration was observed, and no obvious fibrous capsule was observed (see Figure 24).
  • mice Six-week-old healthy C57BL/6 mice were selected, with five mice in each group, and implanted with PCL particles (5 mm in diameter; average molecular weight 45,000, Sigma), silicone discs (5 mm in diameter), PLL-FPBA (0.5 mg, 1.0 mg), and PLL-BA (1.0 mg, modified with 64% benzoic acid). All materials were sterilized, and animal surgery was performed under isoflurane anesthesia and aseptic conditions. The skin was disinfected before and after surgery. A longitudinal incision of about 8 mm was made on the back of the mouse for implantation of PCL particles and silicone discs as positive controls. The incision was far enough from the implantation site to avoid the influence of the wound. After the material was implanted, the mouse skin was sutured.
  • PCL particles 5 mm in diameter; average molecular weight 45,000, Sigma
  • silicone discs 5 mm in diameter
  • PLL-FPBA 0.5 mg, 1.0 mg
  • PLL-BA 1.0 mg, modified with 64% benzoic acid
  • PLL-FPBA or PLL-BA was suspended in 0.1 mL of PBS and injected subcutaneously. After the 2nd, 4th, and 12th week of implantation, the mice were euthanized, and the skin containing the implants was removed, fixed with 4% paraformaldehyde for more than 24 hours, and embedded in paraffin. Untreated mice from the same batch served as blank controls. Each skin tissue section, with a thickness of 3-4 ⁇ m, was stained with hematoxylin-eosin (H&E) and Masson's trichrome (M&T).
  • H&E hematoxylin-eosin
  • M&T Masson's trichrome
  • Immunofluorescence staining was performed for ⁇ -SMA and F4/80, and immunohistochemical staining was performed for cytokines TNF- ⁇ , IL-6, IL-10, IL-12, and IL-17. Immunofluorescence images were recorded on a laser scanning confocal microscope (ECLIPSE Ti2, Nikon). H&E, M&T, and immunohistochemical images were collected using a digital slide scanner (VS200, Olympus). Immunohistochemical images were analyzed for positive staining data on ImageJ (Fiji) (within 100 ⁇ m of the implant interface). As shown in Figure 25, the implants were of similar size under the skin 2 weeks after implantation.
  • the sugar-responsive complex prepared by the present invention that can release insulin for a long time is verified by in vitro glucose response and mouse and mini-pig animal models, which confirms that the sugar-responsive complex of the present invention can release insulin slowly and persistently, and a higher dose can be given at one time to reduce the injection frequency.
  • the PLL-FPBA prepared by the present invention has a lower immune response to the host, avoids the formation of subcutaneous fibrous capsules, and is conducive to long-term release of insulin.

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Abstract

一种糖响应性复合物及其制备方法和应用。所述糖响应性复合物包括相互络合的苯硼酸基聚赖氨酸和具有二醇结构的胰岛素，所述络合的作用力包括：(1)动态静电吸引力，和(2)所述苯硼酸基聚赖氨酸的苯硼酸结构与所述具有二醇结构的胰岛素的二醇结构之间的共价作用力。所述的糖响应性复合物具有良好的葡萄糖响应释放性能，可在高血糖浓度快速释放胰岛素、正常血糖浓度持续缓慢释放胰岛素。

Description

糖响应性复合物及其制备方法和应用
本申请要求申请日为2022/9/29的中国专利申请2022112050858的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及一种糖响应性复合物及其制备方法和应用。
背景技术
全球有约10％的糖尿病患者需要使用胰岛素治疗，以将血糖维持在正常范围。市场上，现已有多种胰岛素药物，可以改善血糖控制效果，减少注射频率。在现有的治疗方法下，糖尿病人仍旧需要每天多次皮下注射胰岛素，以配合日常饮食规律，维持空腹血糖和餐后血糖平稳。这种给药方案给糖尿病患者，尤其是青少年人群带来沉重身心负担。此外，胰岛素的治疗窗狭窄，因此胰岛素用量稍有不适，便可能导致低血糖，尤其是夜间低血糖，带来安全风险。因此，我们需要设计新型胰岛素递送体系，在降低注射频率的同时，提高胰岛素调节血糖能力。
葡萄糖响应性胰岛素可以模拟人体β细胞功能，在高血糖时快速释放胰岛素，在低血糖时缓慢释放胰岛素。因此，葡萄糖响应性胰岛素可以主动跟踪血糖的波动，动态调节胰岛素释放速率，实时满足胰岛素的动态需求，提高胰岛素的治疗指数，改善胰岛素的血糖控制效果。目前有三种主要的葡萄糖响应机制，包括苯硼酸衍生物、葡萄糖结合分子和葡萄糖氧化酶。基于苯硼酸的葡萄糖响应性胰岛素已被验证具有快速而强大的体外和体内葡萄糖响应性胰岛素释放性能，较为接近临床应用。然而，目前葡萄糖响应性胰岛素在单次注射后，仍较难在一个较长的时间周期内，将血糖控制在正常范围。如何在单针注射后，在一个较长的时期内，同时实现餐后快速的胰岛素释放和空腹时持续而缓慢的基础胰岛素释放，对于葡萄糖响应性胰岛素仍然是一个相当大的挑战。
发明内容
本发明为了解决现有技术中葡萄糖响应性胰岛素递送体系难以在单次注射后在一个较长的时间周期内将血糖控制在正常范围的缺陷，提供了一种糖响应性复合物及其制备方法和应用。本发明所述的糖响应性复合物具有良好的葡萄糖响应释放性能，可在高血糖浓度快速释放胰岛素、正常血糖浓度持续缓慢释放胰岛素。值得注意的是，该复合物 可避免皮下纤维囊的形成，这对于胰岛素从复合物储库的释放至关重要。
为了实现上述目的，本发明采用以下技术方案。
本发明提供一种糖响应性复合物，其包括相互络合的苯硼酸基聚赖氨酸和具有二醇结构的胰岛素，所述络合的作用力包括：(1)动态静电吸引力，和(2)所述苯硼酸基聚赖氨酸的苯硼酸结构与所述具有二醇结构的胰岛素的二醇结构之间的共价作用力。
本发明中，所述苯硼酸基聚赖氨酸是由聚赖氨酸与羧基修饰的苯硼酸类化合物通过接枝反应得到。即，在所述苯硼酸基聚赖氨酸中，苯硼酸基通过酰胺键接枝在聚赖氨酸的侧链上。
本发明中，所述聚赖氨酸可为E-聚赖氨酸和/或L-聚赖氨酸，优选为苯硼酸基L-聚赖氨酸。
本发明中，所述聚赖氨酸的分子量可为2k～1000k，较佳地为30k～70k。
本发明中，所述苯硼酸基可为取代基取代或未取代的苯硼酸基团，其中，所述取代基可为卤素和硝基中的一种或多种。较佳地，所述苯硼酸基选自以下基团中的一种或多种：
本发明中，所述苯硼酸基聚赖氨酸的苯硼酸接枝率较佳地为25％～75％，例如30％、35％、40％、45％、50％或60％。其中，所述苯硼酸接枝率是指聚赖氨酸上被苯硼酸基团共价修饰的氨基占修饰前聚赖氨酸上总氨基数目的百分比。所述苯硼酸基聚赖氨酸中苯硼酸基团随机分布。
本发明中，较佳地，所述苯硼酸基聚赖氨酸的化学结构可由式I所示：
其中，R表示氢原子或一个或多个取代基，所述取代基如前所述；苯硼酸接枝率为y/(x+y)。
本发明中，所述具有二醇结构的胰岛素可由胰岛素与含邻位或间位羟基的化合物制备得到。其中，所述含邻位或间位羟基的化合物可为葡萄糖酸、多巴胺、果糖、乳糖、核糖、脱氧核糖、β-D-吡喃甘露糖苷、海藻糖和麦芽糖中的一种或多种；较佳地为葡萄糖酸，更佳地为D-葡萄糖酸。当所述含邻位或间位羟基的化合物为葡萄糖酸时，所述具有二醇结构的胰岛素称为葡萄糖酸基胰岛素(Glu-insulin)。
本发明中，所述糖响应性复合物中胰岛素负载率可为5％-80％，较佳地为40％～60％，更佳地为40％～50％，进一步更佳地为45％～48％，其中，百分比为所述糖响应性复合物中 所述具有二醇结构的胰岛素占所述糖响应性复合物的质量百分比。
本发明中，所述糖响应性复合物的代表性化学结构由式II所示：
其中，R表示氢原子或一个或多个取代基，所述取代基如前所述；苯硼酸接枝率为y+z/(x+y+z)。
本发明提供一种所述糖响应性复合物的制备方法，其包括以下步骤：将所述苯硼酸基聚赖氨酸的水溶液与所述具有二醇结构的胰岛素的水溶液混合，调节pH至6.5～8.0，即得。
本发明中，所述具有二醇结构的胰岛素和所述苯硼酸基聚赖氨酸的质量比可为1：(0.5-10)，较佳地为1：(1～2)，例如1:1.5或1:1。
本发明中，所述苯硼酸基聚赖氨酸的水溶液的浓度可为1～200mg/mL，例如10mg/mL。
本发明中，所述苯硼酸基聚赖氨酸的水溶液的制备方法可为：将所述苯硼酸基聚赖氨酸溶于弱酸性水中即得；其中，所述弱酸性水的pH范围可为2.0～7.0，优选为2.0～3.0；所述弱酸性水可为磷酸盐缓冲液、去离子水或纯水。
本发明中，所述苯硼酸基聚赖氨酸可采用本领域常规的方法制备。
在某些优选的实施方案中，所述苯硼酸改性聚赖氨酸的制备方法包括以下步骤：将聚赖氨酸溶液和羧基修饰的苯硼酸类化合物的溶液混合后进行接枝反应即可。
其中，所述聚赖氨酸的结构单元与所述羧基修饰的苯硼酸类化合物的摩尔比较佳地为4：(3～1)。
本发明中，所述聚赖氨酸溶液中溶剂可为本领域常规的水性溶剂，可溶解聚赖氨酸即可，优选为去离子水或纯水。
本发明中，所述聚赖氨酸溶液的浓度较佳地为1～200mg/mL，例如10mg/mL。
本发明中，所述羧基修饰的苯硼酸类化合物的溶液中溶剂可为本领域常规的与水互溶的有机溶剂，可溶解羧基修饰的苯硼酸类化合物即可，优选为二甲基亚砜(DMSO)或N,N-二甲基甲酰胺(DMF)。
本发明中，所述羧基修饰的苯硼酸类化合物可为取代基取代或未取代的对羧基苯硼酸或邻羧基苯硼酸，其中，所述取代基可为卤素和硝基中的一种或多种。较佳地，所述羧基修饰的苯硼酸类化合物选自以下化合物中的一种或多种：
更佳地，所述羧基修饰的苯硼酸类化合物为4-羧基-3-氟苯硼酸。
本发明中，所述羧基修饰的苯硼酸类化合物的溶液的浓度较佳地为1～500mg/mL，例如24mg/mL。
本发明中，较佳地，所述混合的方式包括将所述羧基修饰的苯硼酸类化合物的溶液滴加到所述聚赖氨酸溶液中后搅拌。其中，所述搅拌的时间较佳地为5min～24h，例如30min。
本发明中，较佳地，所述接枝反应后还包括透析的步骤。其中，所述透析可采用本领域常规的透析袋在去离子水中进行。所述透析袋的截留分子量优选为1k～10k。所述透析的目的是除去游离的羧基修饰的苯硼酸类化合物。
本发明中，较佳地，所述接枝反应后还包括冻干的步骤。当所述接枝反应后还包括透析的步骤时，所述冻干在所述透析之后。所述冻干后得到白色固体。
本发明中，所述具有二醇结构的胰岛素的水溶液的浓度可为1～200mg/mL，较佳地为1～100mg/mL，例如10mg/mL。
本发明中，所述具有二醇结构的胰岛素的水溶液的制备方法可为：将所述具有二醇结构的胰岛素溶于弱酸性水中即得；其中，所述弱酸性水的pH范围可为2.0～7.0，优选为2.0～3.0；所述弱酸性水可为磷酸盐缓冲液、去离子水或纯水。
本发明中，所述具有二醇结构的胰岛素较佳地为葡萄糖酸基胰岛素。其中，所述葡 萄糖酸基胰岛素可采用本领域常规的方法制备。
具体地，所述葡萄糖酸基胰岛素的制备方法包括：(1)先将葡萄糖酸与N-羟基琥珀酰亚胺或TSTU(2-琥珀酰亚胺基-1,1,3,3-四甲基脲四氟硼酸酯)在溶剂中反应；(2)随后加入胰岛素水溶液中反应，调节pH至7～8，后处理即得。
步骤(1)中，所述溶剂可为本领域常规，较佳地为DMSO。
步骤(1)中，所述反应较佳地在常温条件下进行。
步骤(1)中，所述葡萄糖酸与所述N-羟基琥珀酰亚胺或TSTU的摩尔比较佳地为1：(1～1.2)。
步骤(2)中，所述胰岛素较佳地为重组人胰岛素。
步骤(2)中，所述反应较佳地在冰浴条件下进行。
步骤(2)中，所述胰岛素水溶液的浓度较佳地为0.2～100mg/mL。
步骤(2)中，所述胰岛素水溶液的pH较佳地为7～8。
步骤(2)中，所述后处理较佳地包括透析和离子交换柱分离。
其中，所述透析可采用本领域常规的透析袋在去离子水中进行，所述透析袋的截留分子量优选为1k～3.5k。
其中，所述离子交换柱较佳地为阴离子交换柱。
本发明中，较佳地，将所述苯硼酸基聚赖氨酸的水溶液与所述具有二醇结构的胰岛素的水溶液混合后，调节pH至6.5～7.4。
本发明中，所述调节pH后形成白色絮状沉淀，即为糖响应性复合物。所述糖响应性复合物在弱酸性水中可溶或易溶，在弱碱性水中不溶或微溶。
本发明中，所述调节pH后还可包括离心的步骤。所述离心的目的是收集白色絮状沉淀，去除上清液中未复合的具有二醇结构的胰岛素。使用考马斯亮蓝测定，所述离心所得的上清液中游离胰岛素的浓度不超过10％，优选不超过5％。
本发明中，所述制备方法的胰岛素包封率可在90％以上，较佳地95％以上。所述胰岛素包封率是指与所述苯硼酸基聚赖氨酸复合的具有二醇结构的胰岛素(复合量)占所述具有二醇结构的胰岛素的水溶液中具有二醇结构的胰岛素的总质量(初始投料量)的百分比。
本发明的糖响应性复合物为无定形絮状沉淀，皮下注射可以形成胰岛素储库。在高血糖情况下，高浓度的葡萄糖与苯硼酸基的结合瞬间降低苯硼酸基聚赖氨酸部分的正电荷密度，同时伴随苯硼酸酯键的断裂，导致具有二醇结构的胰岛素与聚合物之间的作用力降低，促进胰岛素的释放，使胰岛素具有良好的高浓度葡萄糖响应释放性能，促进机 体恢复正常血糖；在血糖正常情况下，只有极少部分胰岛素处于游离状态，减少低血糖的发生。
本发明还提供一种所述糖响应性复合物在制备治疗糖尿病药物中的应用。
本发明中，所述糖尿病可为1型糖尿病或晚期2型糖尿病。
本发明中，所述糖响应性复合物在应用时，可用磷酸盐缓冲液或者生理盐水重悬，通过皮下注射给药进行治疗。
本发明还提供一种治疗患者的糖尿病的方法，包括给患者施用所述糖响应性复合物。
本发明还提供一种用于治疗糖尿病患者的药物，其包括所述的糖响应性复合物。
本发明还提供一种所述的糖响应性复合物，其用于治疗糖尿病。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
本发明的糖响应性复合物具有良好的葡萄糖响应释放性能，可在高血糖浓度快速释放、正常血糖浓度持续缓慢释放(长效释放)胰岛素。本发明的糖响应性复合物为无定形絮状沉淀，皮下注射可以形成胰岛素储库，在高血糖情况下，高浓度的葡萄糖与苯硼酸基团的结合瞬间降低苯硼酸基聚赖氨酸的正电荷密度，同时伴随苯硼酸酯键的断裂，导致胰岛素部分与聚合物之间的吸引力降低，促进胰岛素的释放，使胰岛素具有良好的高浓度葡萄糖响应释放性能，促进机体恢复正常血糖；在血糖正常情况下，只有极少部分胰岛素处于游离状态，减少低血糖的发生。值得注意的是，该复合物在皮下不会引起强烈的炎症反应，可以避免纤维囊的形成，这为糖响应复合物中胰岛素的释放创造了有利条件。
本发明的糖响应性复合物皮下注射可以形成胰岛素储库，持久释放胰岛素，降糖效果可达一周，可以一次性给予较高剂量以减少注射频率，提高糖尿病患者对皮下注射的依从性。
附图说明
图1.糖响应性复合物形成和葡萄糖响应性胰岛素释放机制示意图。
图2.合成的葡萄糖酸基胰岛素(Glu-insulin)的MALDI-TOF质谱。
图3.葡萄糖酸基胰岛素的二级质谱。
图4.合成的聚L-赖氨酸-4-羧基-3-氟苯硼酸(PLL-FPBA)的核磁共振氢谱(氘代试 剂为D2O)。
图5.PLL-FPBA的MALDI-TOF质谱。
图6.PLL-FPBA的核磁共振硼谱。
图7.葡萄糖酸基胰岛素(Glu-insulin)浓度-吸光度标准曲线。
图8.不同投料比例下胰岛素的包封率。
图9.重组人胰岛素或葡萄糖酸基胰岛素对AKT信号磷酸化的表征。
图10.糖响应性复合物的代表性荧光图像。
图11.糖响应性复合物的扫描电子显微镜和冷冻透射电子显微镜图像。
图12.糖响应性复合物的葡萄糖响应性胰岛素释放曲线。
图13.糖响应性复合物中的胰岛素脉冲释放曲线(糖响应性复合物交替暴露于100和400mg/dL葡萄糖溶液)。
图14.葡萄糖酸基胰岛素(Glu-insulin)和重组人胰岛素治疗1型糖尿病小鼠的血糖水平。
图15.皮下注射糖响应性复合物(20mg/kg)治疗1型糖尿病小鼠的血糖水平。
图16.商业化长效胰岛素制剂甘精胰岛素治疗1型糖尿病小鼠的血糖水平。
图17.使用糖响应性复合物治疗后腹腔内葡萄糖耐量试验的1型糖尿病小鼠的血糖水平。
图18.使用甘精胰岛素治疗后腹腔内葡萄糖耐量试验的1型糖尿病小鼠的血糖水平。
图19.使用糖响应性复合物治疗后腹腔内葡萄糖耐量试验的1型糖尿病小鼠的血糖水平和血浆胰岛素水平。
图20.皮下注射多次糖响应性复合物的1型糖尿病小鼠血糖水平(黑色箭头表示复合物的三次注射)。
图21.糖响应性复合物治疗1型糖尿病小型猪的血糖水平。
图22.皮下注射糖响应性复合物后1型糖尿病小鼠的血清主要生化指标水平。
图23.皮下注射糖响应性复合物后皮肤及主要器官中PLL-FPBA的代表性荧光图像。
图24.注射糖响应性复合物部位皮肤的H&E和Masson三色染色代表性图像。
图25.皮下植入2周后皮肤组织的代表性H&E和Masson三色染色图像。
图26.皮下植入4周后皮肤组织的代表性H&E和Masson三色染色图像。
图27.皮下植入12周后皮肤组织的代表性H&E和Masson三色染色图像。
图28.皮下植入2周后植入部位皮肤的免疫荧光(巨噬细胞生物标志物，F4/80，为红色；α-平滑肌肌动蛋白，α-SMA，为绿色；细胞核为蓝色)和免疫组织化学(TNF-α、 IL-6、IL-10、IL-12、IL-17)染色情况。
图29.皮下植入2周后植入部位皮肤的免疫组织化学(TNF-α、IL-6、IL-10、IL-12、IL-17)累积光密度统计。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
以下实施例中的所用的试剂或仪器的厂家或型号如表1所示。
表1

实施例1
糖响应性复合物的制备和葡萄糖响应性胰岛素释放机制如图1所示。
1、葡萄糖酸基胰岛素(Glu-insulin)的制备
将15.69g的葡萄糖酸溶解于40mL的二甲基亚砜(DMSO)中，将9mmol的二环己基碳二亚胺(DCC)和10mmol的N-羟基琥珀酰亚胺(NHS)溶解于5mL的DMSO中，加入到葡萄糖酸溶液中，室温搅拌过夜，将沉淀滤除。将0.8g的胰岛素溶解于10mL的磷酸盐缓冲液(pH7.4)中，将上述滤液加入至胰岛素溶液，冰浴反应2小时。用4L去 离子水透析3次或者用离子交换柱分离，冷冻干燥得到葡萄糖酸基胰岛素(Glu-insulin)。制得的Glu-insulin的MALDI-TOF质谱如图2所示；图3结果可见，葡萄糖酸在重组人胰岛素在上的修饰位点是A1(α-链的N端)。
2、聚L-赖氨酸-4-羧基-3-氟苯硼酸(PLL-FPBA)的制备
将120mg的FPBA-NHS溶于5mL的DMSO中，逐滴加入至含有100mg的聚L-赖氨酸(PLL)(30k～70k)的10mL磷酸缓冲盐溶液中，控制pH在7左右。加入FPBA-NHS溶液后，再搅拌30min，然后在去离子水(4L)中透析，将得到的混合物冻干，得到白色固体。用1H NMR对产物进行了表征(参见图4)，从核磁共振氢谱可知PLL上有60％的氨基被4-羧基-3-氟苯硼酸(FPBA)修饰；PLL-FPBA是多分散的(参见图5)；硼的存在通过核磁共振硼谱(参见图6)得到证实。
3、糖响应性复合物的制备
将1mg的Glu-insulin和1mg的PLL-FPBA分别溶于0.1mL的弱酸性纯水(pH为3.0)中。加入1M的NaOH水溶液，将pH调至7.4，形成白色絮状沉淀，离心收集白色絮状沉淀，加入1mL的磷酸盐缓冲液(PBS，10mM,pH 7.4)，冷藏保存。
实施例2
在“3、糖响应性复合物的制备”中，将PLL-FPBA的用量改为2mg，除此之外，其他步骤和条件同实施例1。
实施例3
在“3、糖响应性复合物的制备”中，将PLL-FPBA的用量改为1.5mg，除此之外，其他步骤和条件同实施例1。
实施例4
在“3、糖响应性复合物的制备”中，将PLL-FPBA的用量改为0.5mg，除此之外，其他步骤和条件同实施例1。
上清液中游离的胰岛素用考马斯亮蓝试剂测定，在96孔板依次加入Bradford试剂(200μL)和离心后重悬再离心的上清液10μL，根据其在595nm处紫外吸收值，采用外标法(标准曲线见图7)计算上清液中游离的胰岛素含量，从而计算得到胰岛素包封率。经计算，实施例1～3的糖响应复合物的制备过程中，胰岛素的包封率均高于95％。由图8可知，实施例1的糖响应复合物的制备过程中，胰岛素的包封率高于95％；而实施例4中采用的胰岛素相对较多，超过聚合物的负载效能，使得上清液中有较多游离胰岛素。
效果实施例1：Glu-insulin的体外活性研究
HepG2细胞系来自中国科学院细胞库。HepG2细胞在含10％胎牛血清和1％青霉素/ 链霉素的DMEM中培养。培养达到细胞密度80-90％，使用无血清的空白培养基培养12h。然后，将细胞用PBS洗涤并加入含有重组人胰岛素或Glu-insulin的培养基，培养10分钟(重组人胰岛素或Glu-insulin浓度为0、0.1、1、5、10、50、100nM)。用PBS洗涤细胞，加入含有蛋白酶和磷酸酶抑制剂(美仑生物)的冰冷RIPA缓冲液(碧云天)进行细胞裂解。裂解液在4℃下，16000g，离心10分钟。采用BCA法(碧云天)测定蛋白质浓度，样品在含有2.5％β-巯基乙醇的1×Laemmli上样缓冲液(Bio-Rad)中煮沸。蛋白质用10％SDS-PAGE凝胶进行分离，转移到0.45μm PVDF膜(Sigma-Aldrich)上。用5％脱脂牛奶在PBST(含0.1％Tween-20的磷酸盐缓冲盐水)中进行封闭，并在抗体稀释缓冲液(碧云天)中使用抗体[Anti-phospho-akt(CST，#4060，1：2000)，Anti-akt(CST，#4691，1：1000)和Anti-β-Actin(Diagbio，#db7283，1：1000)]进行结合。根据制造商的说明，使用辣根过氧化物酶偶联二抗结合一抗，使用化学发光底物(ECL，赛默飞)进行显色。
结果参见图9，在HepG2细胞中，Glu-insulin显示出与未修饰的重组人胰岛素相似的活性。
效果实施例2：糖响应性复合物形态研究
2.1、荧光显微镜观察糖响应性复合物形态
将0.7mg异硫氰酸荧光素(FITC)溶解于0.3mL的DMSO中，将10mg的Glu-insulin(按照实施例1的步骤1制备)加入到5mL的NaHCO3(0.1M)中。室温搅拌过夜，在4L去离子水中避光透析三次，冷冻干燥后得到FITC标记的Glu-insulin。
将0.1mg的水溶性Cy5-NHS酯溶解于0.02mL的DMSO中，将10mg的PLL-FPBA(按照实施例1的步骤2制备)加入到5mL的NaHCO3(0.1M)中，冰浴搅拌过夜，在4L去离子水中避光透析三次，冷冻干燥后得到Cy5标记的PLL-FPBA。
采用FITC标记的Glu-insulin与Cy5标记的PLL-FPBA按照实施例1的步骤3制备糖响应性复合物。
用荧光显微镜(型号T1，日本尼康)观察，结果如图10。可见，通过FITC标记的Glu-insulin与Cy5标记的PLL-FPBA的荧光重叠，进一步验证了Glu-insulin和PLL-FPBA的络合作用。
2.2、扫描电子显微镜和冷冻透射电子显微镜观察糖响应性复合物形态
称取实施例1制得的糖响应性复合物，加水使得胰岛素当量浓度为1mg/mL，使用超声波细胞破碎仪，在100W功率下超声破碎1分钟，分散悬浮于1mL水中，稀释10倍后滴到铜网上。在铜网中加入5％醋酸铀酰溶液10μL，静置10分钟，用滤纸除去溶液。用冷冻透射电子显微镜对样品进行观察。同样的样品放在硅片上，自然干燥，并通过扫 描电子显微镜观察。通过冷冻透射电子显微镜和扫描电子显微镜证实(图11)，糖响应性复合物具有多孔和松散的微观结构。
效果实施例3：糖响应性复合物的体外糖响应验证
实施例1制得的糖响应性复合物的葡萄糖响应胰岛素释放性能在pH7.4的磷酸缓冲盐溶液中评估，四种葡萄糖浓度分别为0，100，200和400mg/dL。在没有葡萄糖的情况下，游离Glu-insulin保持在10μg/mL左右的低水平(参见图12)。当葡萄糖浓度增加到100，200和400mg/dL，孵育2小时后，游离Glu-insulin水平分别增加到19，32和45μg/mL，此时游离Glu-insulin水平达到平衡并保持基本不变。在400mg/dL葡萄糖溶液中孵育0.2h后，游离Glu-insulin水平达到21μg/mL，几乎是在100mg/mL葡萄糖溶液中的3倍(参见图12)。证明该糖响应性复合物可以以葡萄糖依赖的方式释放胰岛素。将复合物交替暴露于100和400mg/dL葡萄糖溶液中，观察到胰岛素脉冲释放(参见图13)。
效果实施例4：动物药效研究
4.1、Glu-insulin的动物药效研究
将C57BL/6小鼠(购自杭州医学院)以150mg/kg链脲佐菌素剂量诱导1型糖尿病。糖尿病小鼠以标准饮食和12小时光照、12小时黑暗的昼夜节律周期饲养。取各5只小鼠以胰岛素当量剂量为1.5mg/kg皮下注射Glu-insulin和重组人胰岛素。使用血糖检测仪测量血糖。图14结果可见，Glu-insulin仍有与重组人胰岛素类似的降糖效果。
4.2、糖尿病1型小鼠的体内降糖研究
按照上述方法建立1型糖尿病小鼠模型，选取血糖高于300mg/dL的1型糖尿病小鼠进行治疗效果评价。每组5只，各组皮下注射甘精胰岛素(50U/kg)和糖响应性复合物(20mg/kg)。使用血糖检测仪测量血糖。
图15结果可见，小鼠血糖维持在200mg/dL以下超过一周，比使用商业化长效胰岛素制剂甘精胰岛素治疗有效的时间长(参见图16)，且几乎未发生严重的低血糖。通过腹腔内葡萄糖耐量试验(IPGTT)进一步评估复合物对血糖的调节能力。在给予复合物皮下注射15小时、48小时、6天、12天后，按1.5g/kg注射葡萄糖。图17结果可见，给予复合物治疗的小鼠可以稳定维持血糖。作为对照，甘精胰岛素治疗的糖尿病小鼠，在治疗后6和15小时不能将血糖调节到正常水平(参见图18)。通过腹腔内葡萄糖给药，进一步观察血糖调节的体内胰岛素释放。给予糖尿病小鼠复合物(20mg/kg)预处理3d后，腹腔注射葡萄糖(3g/kg)。收集血浆，测量血糖，用酶联免疫吸附试验(ELISA)试剂盒测定血浆胰岛素水平(参见图19)。每168小时注射一次糖响应性复合物(三次注射剂量分别为20mg/kg，14mg/kg，14mg/kg)，在至少22天内可以将血糖控制在200mg/dL以 下(参见图20)。该复合物通过多次注射可以实现对糖尿病小鼠的长期血糖控制能力，且在此期间未观察到明显的低血糖。
4.3、糖尿病1型小型猪的体内降糖研究
给予6个月年龄的巴马小型猪静脉注射STZ(150mg/kg)诱导1型糖尿病。小型猪每天喂食两次，用甘精胰岛素控制血糖，待血糖稳定1个月后可用于研究。使用动态血糖监测系统(FreeStyle Libre H，美国雅培制药有限公司)监测血糖，小型猪血糖均在200mg/dL以上。小型猪的血糖平常使用甘精胰岛素控制。停用甘精胰岛素48h后给予复合物治疗，测试糖响应性复合物血糖控制效果。小型猪皮下注射甘精胰岛素剂量分别为0.4，0.5，0.6U/kg。小型猪皮下注射复合物剂量为0.2mg/kg，0.3mg/kg，0.3mg/kg。图21结果可见，甘精胰岛素注射一次降低血糖的时间不超过24小时，且会将血糖降低至检测限以下(低于40mg/dL)。连续七天注射甘精胰岛素，血糖在正常血糖和高血糖范围内波动，无法长期维持正常血糖，且出现血糖低于40mg/dL。与之相比，所有糖响应性复合物均能将血糖降低至200mg/mL以下，且几乎没有血糖低于40mg/dL。复合物可以在2只小型猪体内调节血糖使其低于200mg/dL超过120小时，并发挥一周以上的治疗效果。由于小型猪比小鼠更具有临床相关性，在小型猪模型中验证的这种长效血糖控制能力和餐后瞬时高血糖调节能力表明，该复合物极有可能以同样方式调控人类血糖。
效果实施例5：复合物的生物组织相容性研究
5.1糖尿病小鼠体内毒性评估
按照上述方法建立1型糖尿病小鼠模型，每组5只，各组皮下注射PBS和糖响应性复合物(20mg/kg)，1周后采血，测定血清碱性磷酸酶(ALP)、天冬氨酸转氨酶(AST)、丙氨酸转氨酶(ALT)、白蛋白(ALB)、血尿素氮(BUN)和肌酐水平，评价其对肝脏和肾脏的毒性(参见图22)。与PBS注射组小鼠相比，实验组各项指标变化均不明显，表明PLL-FPBA对肝脏和肾脏的毒性很小。此外，皮下注射Cy5标记的PLL-FPBA，并用活体成像系统(IVIS Lumina III，PerkinElmer)显示PLL-FPBA的大小。图23结果可见，复合物在体内主要由肝脏清除。采用苏木精-伊红(H&E)染色和马松三色染色评价小鼠对注射复合物的免疫反应。即使皮下注射复合物四周后，也未观察到明显的中性粒细胞浸润，未见明显的纤维囊(参见图24)。
5.2、PLL-FPBA在小鼠体内的宿主反应评估
选取6周龄健康C57BL/6小鼠，每组五只，植入PCL颗粒(直径5mm；平均分子量45000，Sigma)，硅胶圆片(直径，5mm)，PLL-FPBA(0.5mg，1.0mg)，PLL-BA(1.0mg，64％苯甲酸修饰)。所有材料均经灭菌处理，动物手术在异氟醚麻醉和无菌条件下进行， 手术前后对皮肤进行消毒。在小鼠背部做约8mm的纵向切口，用于植入阳性对照的PCL颗粒和硅胶圆片。切口离植入部位足够远，以避免伤口的影响。材料植入后，缝合小鼠皮肤。将PLL-FPBA或PLL-BA悬浮于0.1mL的PBS中进行皮下注射。在植入第2、4、12周后，对小鼠实施安乐死，取下包含植入物的皮肤，使用4％多聚甲醛固定24小时以上，使用石蜡包埋。同一批次未处理小鼠作为空白对照。每个皮肤组织切片，厚度为3-4μm，进行苏木精-伊红(H&E)染色和马松三色(M&T)染色。对α-SMA、F4/80进行免疫荧光染色，对细胞因子TNF-α、IL-6、IL-10、IL-12、IL-17进行免疫组织化学染色。在激光扫描共聚焦显微镜(ECLIPSE Ti2，Nikon)上记录免疫荧光图像。采用数字切片扫描仪(VS200，Olympus)采集H&E、M&T和免疫组织化学图像。免疫组织化学图像在ImageJ(Fiji)上(距植入物界面100μm范围内)进行阳性染色数据分析。图25结果可见，植入2周后，这些植入物在皮肤下具有相似的大小。皮下注射后2周、4周和12周，PLL-FPBA周围未发现明显的纤维囊(参见图25-27)。相比之下，PCL颗粒和硅胶圆片在植入物周围引发了厚而致密的胶原蛋白或纤维层。植入后2周和4周，在PCL颗粒和硅胶圆片表面有一层较厚的免疫细胞层。与之相比，PLL-FPBA和PLL-BA植入物周围的免疫细胞可忽略不计。通过免疫荧光染色将巨噬细胞标志物F4/80标记为红色。与PCL颗粒和硅胶圆片周围相比，可以观察到PLL-FPBA植入物周围的红色荧光密度较低。同时研究了植入物周围的细胞因子水平(参见图28-29)。PCL颗粒植入物周围的TNF-α、IL-6、IL-10、IL-12、IL-17的积累最为严重，硅胶圆片也引起了明显的细胞因子积累。与之相比，在PLL-FPBA和PLL-BA周围以上所有细胞因子都呈现较低水平，表明FPBA在降低宿主免疫应答中的作用较小。由于PLL骨架具有生物降解性，PLL-FPBA和PLL-BA植入物的尺寸随着时间的推移逐渐减小(参见图25-27)。PLL-FPBA缓慢去除，导致胶原纤维在植入物表面粘附性较弱，从而不容易形成纤维囊。此外，在硅胶圆片组和PLL-FPBA组中观察到α-SMA，它们具有刺激血管生成的潜力。
本发明所制备的可长效释放胰岛素的糖响应性复合物，通过体外葡萄糖响应验证，以及小鼠和小型猪动物模型验证，证实了本发明的糖响应性复合物可以缓慢而持久地释放胰岛素，并且可一次性给予较高剂量以减少注射频率。另外，本发明所制备的PLL-FPBA对于宿主具有较低的免疫反应，避免了皮下纤维囊的形成，有利于长期释放胰岛素。

Claims (13)

  1. 一种糖响应性复合物，其特征在于，其包括相互络合的苯硼酸基聚赖氨酸和具有二醇结构的胰岛素，所述络合的作用力包括：(1)动态静电吸引力，和(2)所述苯硼酸基聚赖氨酸的苯硼酸结构与所述具有二醇结构的胰岛素的二醇结构之间的共价作用力。
  2. 根据权利要求1所述的糖响应性复合物，其特征在于，在所述苯硼酸基聚赖氨酸中，苯硼酸基通过酰胺键接枝在聚赖氨酸的侧链上；
    和/或，所述苯硼酸基聚赖氨酸的苯硼酸接枝率为25％～75％，例如30％、35％、40％、45％、50％或60％。
  3. 根据权利要求1或2所述的糖响应性复合物，其特征在于，所述苯硼酸基聚赖氨酸中，所述聚赖氨酸为E-聚赖氨酸和/或L-聚赖氨酸，优选为苯硼酸基L-聚赖氨酸；
    和/或，所述聚赖氨酸的分子量为2k～1000k，较佳地为30k～70k。
  4. 根据权利要求1～3中至少一项所述的糖响应性复合物，其特征在于，所述苯硼酸基聚赖氨酸中，苯硼酸基为取代基取代或未取代的苯硼酸基团，其中，所述取代基较佳地为卤素和硝基中的一种或多种；
    较佳地，所述苯硼酸基选自以下基团中的一种或多种：
  5. 根据权利要求1～4中至少一项所述的糖响应性复合物，其特征在于，所述具有二醇结构的胰岛素由胰岛素与含邻位或间位羟基的化合物制备得到；
    其中，所述含邻位或间位羟基的化合物较佳地为葡萄糖酸、多巴胺、果糖、乳糖、核糖、脱氧核糖、β-D-吡喃甘露糖苷、海藻糖和麦芽糖中的一种或多种；更佳地为葡萄糖酸，例如D-葡萄糖酸；
    和/或，所述糖响应性复合物中胰岛素负载率为5％-80％，较佳地为40％～60％，更佳地为40％～50％，进一步更佳地为45％～48％，其中，百分比为所述糖响应性复合物中所述 具有二醇结构的胰岛素占所述糖响应性复合物的质量百分比。
  6. 一种如权利要求1～5中至少一项所述的糖响应性复合物的制备方法，其特征在于，其包括以下步骤：将所述苯硼酸基聚赖氨酸的水溶液与所述具有二醇结构的胰岛素的水溶液混合，调节pH至6.5～8.0，即得。
  7. 根据权利要求6所述的糖响应性复合物的制备方法，其特征在于，所述具有二醇结构的胰岛素和所述苯硼酸基聚赖氨酸的质量比为1：(0.5-10)，较佳地为1：(1～2)，例如1:1.5或1:1；
    和/或，所述苯硼酸基聚赖氨酸的水溶液的浓度为1～200mg/mL，例如10mg/mL；
    和/或，所述苯硼酸基聚赖氨酸的水溶液的制备方法为：将所述苯硼酸基聚赖氨酸溶于弱酸性水中即得；其中，所述弱酸性水的pH范围为2.0～7.0，优选为2.0～3.0；所述弱酸性水较佳地为磷酸盐缓冲液、去离子水或纯水。
  8. 根据权利要求6或7所述的糖响应性复合物的制备方法，其特征在于，所述具有二醇结构的胰岛素的水溶液的浓度为1～200mg/mL，较佳地为1～100mg/mL，例如10mg/mL；
    和/或，所述具有二醇结构的胰岛素的水溶液的制备方法为：将所述具有二醇结构的胰岛素溶于弱酸性水中即得；其中，所述弱酸性水的pH范围为2.0～7.0，优选为2.0～3.0；所述弱酸性水较佳地为磷酸盐缓冲液、去离子水或纯水。
  9. 根据权利要求6～8中至少一项所述的糖响应性复合物的制备方法，其特征在于，所述调节pH为调节pH至6.5～7.4；
    和/或，所述调节pH后还包括离心的步骤；
    和/或，所述制备方法的胰岛素包封率在90％以上，较佳地95％以上。
  10. 一种如权利要求1～5中至少一项所述的糖响应性复合物在制备治疗糖尿病药物中的应用。
  11. 一种治疗患者的糖尿病的方法，包括给患者施用如权利要求1～5中至少一项所述的糖响应性复合物。
  12. 一种用于治疗糖尿病患者的药物，其包括如权利要求1～5中至少一项所述的糖响应性复合物。
  13. 一种如权利要求1～5中至少一项所述的糖响应性复合物，其用于治疗糖尿病。
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