A nanoemulsion preparation containing recombinant human insulin and a method for preparing the same

Abstract

The application relates to the technical field of ophthalmic pharmaceutical preparations, and particularly discloses a nanoemulsion preparation containing recombinant human insulin and a preparation method thereof. The nanoemulsion preparation containing recombinant human insulin comprises the following components: an active ingredient, which is sulfobutyl ether-beta-cyclodextrin-included recombinant human insulin; an oil phase component, which comprises medium-chain triglyceride or vegetable oil and has dispersed aluminum monostearate with a mass ratio of 0.1-0.5%; a functional composition, which is composed of lecithin, cholesterol and poloxamer 407 at a mass ratio of 1:(0.2-0.5):(0.1-0.5) and has a dosage of 10-30% of the mass of the oil phase component; a surfactant; a co-surfactant; and an aqueous phase component, which is used to make up to 100%. The application can protect the recombinant human insulin in a tear enzyme environment for a long time, significantly improves the stability of the recombinant human insulin in a tear environment and the corneal bioavailability, and can realize a long-acting and stable corneal repair effect.

Description

Technical Field

[0001] This application relates to the field of ophthalmic drug formulation technology, and more specifically, it relates to a nanoemulsion formulation containing recombinant human insulin and a method for preparing the same. Background Technology

[0002] The corneal epithelium is a vital barrier structure for maintaining ocular surface homeostasis and visual function. Various pathological conditions can lead to corneal epithelial damage, including diabetic keratosis, neurotrophic keratitis, dry eye-related corneal injury, and epithelial defects caused by trauma or surgery. Among these, diabetic patients, due to long-term hyperglycemia leading to corneal neurodegeneration and decreased epithelial barrier function, are among the most common causes of corneal epithelial damage in clinical practice. If the damage is prolonged and unhealed, it can develop into persistent corneal epithelial defects or even corneal ulcers, seriously threatening vision.

[0003] Insulin, a classic metabolic regulatory hormone, has attracted increasing attention for its pro-repair effects on ocular surface tissues. Studies have shown that both corneal epithelial cells and stromal cells express insulin receptors. Insulin binding to these receptors activates signaling pathways such as PI3K / Akt and MAPK / ERK, significantly promoting corneal epithelial cell proliferation, migration, and adhesion, thus accelerating wound healing. In recent years, multiple clinical studies have confirmed that topical application of recombinant human insulin ophthalmic preparations demonstrates good pro-healing effects on refractory corneal ulcers unresponsive to conventional treatments, showcasing its great potential as a novel ophthalmic drug.

[0004] Despite the significant therapeutic potential of recombinant human insulin in corneal epithelial repair, it still faces the challenge of degradation by tear film enzymes. Tears are a complex liquid film covering the ocular surface, serving not only lubrication and protection but also forming a robust biochemical defense barrier. Tears naturally contain various proteolytic enzymes, primarily including lysozyme, trypsin-like serine proteases, and matrix metalloproteinases. These enzymes collectively constitute the ocular surface's "enzyme barrier," whose main physiological function is to defend against pathogenic microorganisms. However, they also pose a serious challenge to protein-based drugs. Insulin, as a polypeptide hormone, contains multiple cleavage sites in its molecular structure, making it highly susceptible to degradation and inactivation by these proteases. This results in significant degradation before reaching the corneal epithelium, leading to extremely low bioavailability and severely limiting its therapeutic efficacy.

[0005] Therefore, there is an urgent need to propose a solution to address the aforementioned technical problems. Summary of the Invention

[0006] In order to provide long-term protection for recombinant human insulin in the tear fluid enzyme environment and significantly improve the stability and corneal bioavailability of recombinant human insulin in the tear fluid environment, this application provides a nanoemulsion formulation containing recombinant human insulin and its preparation method.

[0007] In a first aspect, this application provides a nanoemulsion formulation containing recombinant human insulin, employing the following technical solution: A nanoemulsion formulation containing recombinant human insulin comprises the following components: The active ingredient is recombinant human insulin encapsulated by sulfobutyl ether-β-cyclodextrin, and the concentration of recombinant human insulin is 0.1-1.0 U / mL; The oil phase component, with a concentration of 5-15% (v / v), contains medium-chain triglycerides or vegetable oils and disperses 0.1-0.5% aluminum monostearate by mass. A functional composition comprising lecithin, cholesterol, and poloxamer 407 in a mass ratio of 1:(0.2-0.5):(0.1-0.5), wherein the amount of the composition is 10-30% of the mass of the oil phase components; Surfactant, concentration 1-5% (v / v); Co-surfactant, concentration 0.5-3% (v / v); The aqueous phase, used to bring the total to 100%, contains sodium hyaluronate, buffer solution, isotonicity regulator, preservative, and water for injection.

[0008] By employing the above technical solution, the inclusion complex of recombinant human insulin with sulfobutyl ether-β-cyclodextrin, and then loading it into the nanoemulsion system, allows the cavity structure of sulfobutyl ether-β-cyclodextrin to physically shield the enzyme cleavage sites in the insulin molecule that are easily recognized by proteases, thus reducing the rate of enzyme cleavage. Furthermore, the inclusion of recombinant human insulin by sulfobutyl ether-β-cyclodextrin inhibits the self-aggregation tendency of insulin molecules, improving its chemical stability in the nanoemulsion formulation. Trace amounts of aluminum monostearate are dispersed in the oil phase, forming a thixotropic network structure within the oil phase, significantly increasing the apparent viscosity of the oil phase. This network structure, on the one hand, increases the diffusion resistance of water-soluble enzyme molecules in the oil phase once they cross the interfacial membrane; on the other hand, it further traps potentially invading enzyme molecules through the weak affinity adsorption of proteins by aluminum soap, forming an auxiliary barrier within the oil core. The functional composition can construct a unique three-layer interfacial membrane structure in nanoemulsion formulations. The inner layer consists of a rigid monolayer of lecithin at the oil-water interface, which is tightly adsorbed and provides a basic barrier. The middle layer consists of cholesterol embedded between lecithin molecules, filling the gaps between phospholipid molecules, significantly reducing the fluidity and permeability of the interfacial membrane, and preventing proteases in the aqueous phase from penetrating into the oil core. The outer layer consists of polyoxyethylene segments of poloxamer 407 forming a hydrophilic "brush-like" three-dimensional barrier on the aqueous side, which prevents proteolytic enzyme molecules in tears from approaching the interface through steric hindrance. The synergistic effect of the three interfacial membranes gives the nanoemulsion excellent anti-enzymatic ability in the tear environment. Therefore, in the above-mentioned nanoemulsion formulations containing recombinant human insulin, the synergistic effect of the three-layer interfacial membrane's steric hindrance and densification, the cyclodextrin inclusion's enzyme cleavage site shielding, and the diffusion retardation effect of the oil phase thixotropic network can enable recombinant human insulin to obtain long-term protection in the tear fluid enzyme environment, significantly improving the stability and corneal bioavailability of recombinant human insulin in the tear fluid environment, thereby achieving a long-term and stable corneal repair effect.

[0009] Preferably, in the recombinant human insulin encapsulated with sulfobutyl ether-β-cyclodextrin, the molar ratio of recombinant human insulin to sulfobutyl ether-β-cyclodextrin is 1:(4-7).

[0010] By adopting the above technical solution, the cavity of sulfobutyl ether-β-cyclodextrin can synergistically encapsulate multiple hydrophobic residue segments on the insulin molecule, maintaining a high encapsulation rate even in ophthalmic systems with extremely low guest molecule concentrations. It physically shields the main cleavage sites of trypsin and matrix metalloproteinases. At the same time, it avoids the decrease in receptor affinity or excessive viscosity of the aqueous phase caused by excessive encapsulation, allowing recombinant human insulin to be released from the ocular surface in a "controlled dissociation" manner. It forms a three-level progressive protection at the molecular, interface, and oil phase levels with the three-layer interface membrane and the oil phase thixotropic network, significantly prolonging the active half-life and improving corneal bioavailability.

[0011] Preferably, the functional composition comprises lecithin, cholesterol, and poloxamer 407 in a mass ratio of 1:0.3:0.3.

[0012] By adopting the above technical solution, cholesterol is appropriately embedded between the lecithin monolayers, making the interfacial membrane liquid and ordered, which significantly reduces membrane fluidity and enzyme permeability. The polyoxypropylene segment of poloxamer 407 is anchored at the interface, and the polyoxyethylene segment extends into the aqueous phase to form a hydrophilic brush layer of appropriate density, which not only provides steric hindrance to tear proteases, but also endows the nanoemulsion formulation with suitable ocular surface mucosal adhesion. Thus, the three components synergistically exhibit better corresponding effects.

[0013] Preferably, the component also contains a polyethylene glycol-polylysine block copolymer modified with phenylboronic acid, the amount of which is 8-15% of the mass of the oil phase component, wherein the degree of polymerization of the polylysine block is 20-50, and 30-60% of its side chain amino groups are replaced by phenylboronic acid.

[0014] By employing the above-mentioned technical solution, in the tear film environment, phenylboronic acid forms reversible borate bonds with glycosylation sites or arginine residues on the surface of recombinant human insulin, anchoring insulin to the inner side of the oil-water interface. Meanwhile, the protonated amino groups of the polylysine segment electrostatically bind to the negatively charged phospholipid head, forming a dense "interfacial locking layer" that prevents proteases from contacting recombinant human insulin. On the corneal epithelial surface, the borate bonds dissociate, the polylysine segment protonates, the interfacial locking layer relaxes, and recombinant human insulin is released from the interface and taken up by corneal epithelial cells. Simultaneously, the phenylboronic acid-modified polyethylene glycol-polylysine block copolymer, along with the three-layer interfacial membrane, sulfobutyl ether-β-cyclodextrin inclusion complex, and oil-phase thixotropic network, forms a multi-level synergistic effect in space and time, achieving a smart, targeted delivery mechanism of "locking in the tear film and unlocking on the corneal surface," thereby significantly improving the stability and corneal bioavailability of recombinant human insulin in the tear film environment.

[0015] Preferably, the total molecular weight of the phenylboronic acid-modified polyethylene glycol-polylysine block copolymer is 5000-15000 Da, wherein the molecular weight of the polyethylene glycol segment is 1000-3000 Da.

[0016] By adopting the above technical solution, the above molecular weight parameters are well coupled with the three-layer interface membrane, cyclodextrin inclusion ratio and oil phase thixotropic network, which can bring excellent improvement to the stability of recombinant human insulin in the tear environment and corneal bioavailability.

[0017] Preferably, the nanoemulsion formulation containing recombinant human insulin has an average particle size of 100-200 nm and a zeta potential of -25 to -45 mV.

[0018] By adopting the above technical solutions, the nanoemulsion formulation containing recombinant human insulin avoids both rapid clearance due to its small size and increased enzyme contact cross-section due to its large specific surface area in the ocular tear film, and difficulty in delivery via the corneal epithelial bypass and transcytosis due to its large size. At the same time, it can synergize with the combination mechanism between the three interfacial membranes, cyclodextrin inclusion, and oil phase thixotropic network, so that the nanoemulsion formulation containing recombinant human insulin exhibits excellent tear film enzyme resistance and optimal corneal transmembrane delivery efficiency, thereby ensuring excellent corneal bioavailability.

[0019] Preferably, the buffer solution is a histidine buffer or a citrate-disodium hydrogen phosphate buffer, so that the pH of the nanoemulsion formulation is 6.8-7.4.

[0020] By adopting the above technical solution, the pH range is within the optimal window for the chemical stability of recombinant human insulin, while matching the pH of physiological tears to avoid reflexive tear flushing caused by instillation; in particular, histidine buffer and citrate-disodium hydrogen phosphate buffer can achieve the unity of enzymatic protection, nanoemulsion formulation stability and corneal epithelial repair microenvironment.

[0021] Preferably, the isotonic regulator is one or more of glycerol, mannitol, and sodium chloride, so that the osmotic pressure of the nanoemulsion formulation is 280-320 mOsmol / kg.

[0022] By adopting the above technical solution, the osmotic pressure range matches the physiological osmotic pressure of human tears, and does not cause osmotic impact on the ocular surface or reflexive tear scouring during instillation, thus ensuring the residence time of the nanoemulsion formulation containing recombinant human insulin in front of the cornea; and the selection and combination of glycerol, mannitol and sodium chloride are all suitable for nanoemulsion formulations containing recombinant human insulin, ensuring the stable performance of the excellent synergistic effect among other components.

[0023] Preferably, the surfactant is a mixture of polyoxyethylene hydrogenated castor oil and poloxamer 188, and the co-surfactant is propylene glycol or ethanol.

[0024] By employing the above technical solutions, polyoxyethylene hydrogenated castor oil provides strong emulsifying ability and dense oil core interface shielding. Its hydrogenated structure avoids the destruction of disulfide bonds in recombinant human insulin by the oxidation of unsaturated double bonds. Poloxamer 188 forms a flexible hydrophilic layer at the interface, reducing corneal irritation and inhibiting the adsorption of tear enzymes at the interface. Propylene glycol or ethanol acts as a co-surfactant to fill the interfacial membrane gaps, further reducing the oil-water interfacial tension, and also takes into account microenvironment regulation to ensure the excellent and stable performance of the synergistic effects of other components.

[0025] Secondly, this application provides a method for preparing a nanoemulsion formulation containing recombinant human insulin, using the following technical solution: A method for preparing a nanoemulsion formulation containing recombinant human insulin includes the following steps: (1) Take sulfobutyl ether-β-cyclodextrin-encapsulated recombinant human insulin and disperse it in an oil phase containing aluminum monostearate. Stir at 40-50℃ until completely dissolved to obtain an oil phase mixture; (2) After dissolving lecithin and cholesterol in anhydrous ethanol, the ethanol is removed by rotary evaporation to form a lipid film. Then, poloxamer 407 and the oil phase mixture obtained in step (1) are added and treated at 50-60℃ for 25-35 min to form a lipid-oil phase premix. (3) After mixing the surfactant, co-surfactant and aqueous phase components, preheat to 50-55℃, and then add to the lipid-oil phase premix obtained in step (2) under high-speed shearing, and continue to shear to form crude emulsion. After high-pressure microfluidic homogenization and filtration sterilization, nanoemulsion preparation containing recombinant human insulin is obtained.

[0026] By adopting the above technical solution, the above operation is simple, suitable for large-scale industrial production, and can ensure that the components are fully coordinated and exert excellent corresponding effects, thereby ensuring that a high-quality and stable nanoemulsion formulation containing recombinant human insulin is obtained.

[0027] In summary, this application has the following beneficial effects: 1. This application establishes a three-layer interfacial membrane synergistic mechanism by adding a functional composition composed of lecithin, cholesterol and poloxamer 407. It also works synergistically with the enzyme cleavage site shielding effect of cyclodextrin inclusion and the diffusion blocking effect of the oil phase thixotropic network to enable recombinant human insulin to obtain long-term protection in the tear enzyme environment, significantly improve the stability and corneal bioavailability of recombinant human insulin in the tear environment, and thus achieve a long-term and stable corneal repair effect. 2. This application, by adding a polyethylene glycol-polylysine block copolymer modified with phenylboronic acid, not only forms a dense "interfacial locking layer" in the tear environment to prevent proteases from contacting recombinant human insulin, but also regulates the release of recombinant human insulin on the corneal epithelial surface and its uptake by corneal epithelial cells. It also forms a multi-level synergy in space and time with the three-layer interfacial membrane, sulfobutyl ether-β-cyclodextrin inclusion complex and oil phase thixotropic network, realizing an intelligent targeted delivery assistance mechanism of "locking in the tear film and unlocking on the corneal surface", thereby significantly improving the stability of recombinant human insulin in the tear environment and corneal bioavailability. Detailed Implementation

[0028] The present application will be further described in detail below with reference to embodiments and comparative examples.

[0029] Unless otherwise specified, all raw materials used in the embodiments and comparative examples of this application are commercially available.

[0030] The lecithin used is injection-grade egg yolk lecithin EPC-96; The recombinant human insulin encapsulated with sulfobutyl ether-β-cyclodextrin was obtained by dissolving recombinant human insulin in hydrochloric acid solution at pH 3.0, mixing it with an aqueous solution of sulfobutyl ether-β-cyclodextrin, filtering at 0.22 μm, dispensing, pre-freezing at -40℃ for 2 h, and then lyophilizing (first drying at -20℃ / 0.1 mbar, second drying at 25℃) to obtain a lyophilized amorphous powder with an encapsulation rate of 93.5%. The sulfobutyl ether-β-cyclodextrin contained therein is in sodium salt form, while the sodium sulfobutyl ether-β-cyclodextrin is injection grade, with an average degree of substitution of 6.5 and an average molecular weight of 2163 Da. The recombinant human insulin is preferably recombinant human short-acting insulin, meeting pharmacopoeia standards, with a purity ≥98% (HPLC).

[0031] Example Example 1

[0032] A nanoemulsion formulation containing recombinant human insulin, the components of which are shown in Table 1, is prepared by the following steps (based on a total volume of 1000 mL of nanoemulsion formulation): (1) Take sulfobutyl ether-β-cyclodextrin-encapsulated recombinant human insulin and disperse it in an oil phase containing aluminum monostearate. Stir at 40-50°C until completely dissolved (preferably at 45°C in this embodiment) to obtain an oil phase mixture; (2) After dissolving lecithin and cholesterol in anhydrous ethanol, the ethanol is removed by rotary evaporation to form a lipid film. Then, poloxamer 407 and the oil phase mixture obtained in step (1) are added and treated at 50-60℃ for 25-35 min (preferably at 55℃ for 30 min in this embodiment) to form a lipid-oil phase premix. (3) After mixing the surfactant, co-surfactant and aqueous phase components, preheat to 50-55℃ (preferably 55℃ in this embodiment), add it to the lipid-oil phase premix obtained in step (2) under high-speed shear (12000rpm), and continue shearing for 8min to form crude emulsion. After high-pressure microfluidic homogenization (5 cycles at 1000bar pressure) and 0.22μm filtration sterilization, nanoemulsion preparation containing recombinant human insulin is obtained.

[0033] Note: In the above operation, the molar ratio of recombinant human insulin to sulfobutyl ether-β-cyclodextrin in the recombinant human insulin containing sulfobutyl ether-β-cyclodextrin is 1:5.5. The functional composition consists of lecithin, cholesterol, and poloxamer 407 in a mass ratio of 1:0.3:0.3. The oil phase component is medium-chain triglycerides or vegetable oil, which can be castor oil. In this embodiment, the preferred oil phase component is medium-chain triglycerides, wherein 0.3% aluminum monostearate is dispersed therein. The surfactant is obtained by mixing polyoxyethylene hydrogenated castor oil RH40 and poloxamer 188 in a weight ratio of 2:1, and the co-surfactant is propylene glycol. The aqueous phase component contains sodium hyaluronate, buffer solution, isotonicity regulator, preservative, and water for injection, wherein the concentration of sodium hyaluronate is 0.1 (w / v) and the molecular weight is 1.2 × 10⁻⁶. 6 The buffer solution was histidine buffer to achieve a pH of 7.1 for the nanoemulsion formulation; the isotonic adjuster was glycerol to achieve an osmotic pressure of 300 mOsmol / kg; and the preservative was benzalkonium chloride at a concentration of 0.005% (v / v). The nanoemulsion formulation containing recombinant human insulin had an average particle size of 150 nm and a zeta potential of -35 mV.

[0034] Example 2-3

[0035] A nanoemulsion formulation containing recombinant human insulin differs from Example 1 in that its components are shown in Table 1.

[0036] Table 1. Composition and corresponding concentrations of Examples 1-3

[0037] Example 4

[0038] A nanoemulsion formulation containing recombinant human insulin differs from Example 1 in that the molar ratio of recombinant human insulin to sulfobutyl ether-β-cyclodextrin in the recombinant human insulin encapsulated with sulfobutyl ether-β-cyclodextrin is 1:4.

[0039] Example 5

[0040] A nanoemulsion formulation containing recombinant human insulin differs from Example 1 in that the molar ratio of recombinant human insulin to sulfobutyl ether-β-cyclodextrin in the recombinant human insulin encapsulated with sulfobutyl ether-β-cyclodextrin is 1:7.

[0041] Example 6

[0042] A nanoemulsion formulation containing recombinant human insulin differs from Example 1 in that the functional composition consists of lecithin, cholesterol, and poloxamer 407 in a mass ratio of 1:0.35:0.3.

[0043] Example 7

[0044] A nanoemulsion formulation containing recombinant human insulin differs from Example 1 in that the functional composition consists of lecithin, cholesterol, and poloxamer 407 in a mass ratio of 1:0.2:0.1.

[0045] Example 8

[0046] A nanoemulsion formulation containing recombinant human insulin differs from Example 1 in that the functional composition consists of lecithin, cholesterol, and poloxamer 407 in a mass ratio of 1:0.5:0.5.

[0047] Example 9

[0048] A nanoemulsion formulation containing recombinant human insulin differs from Example 1 in that the oil phase contains 0.1% aluminum monostearate by mass.

[0049] Example 10

[0050] A nanoemulsion formulation containing recombinant human insulin differs from Example 1 in that the oil phase contains 0.5% aluminum monostearate by mass.

[0051] Example 11

[0052] A nanoemulsion formulation containing recombinant human insulin differs from Example 1 in that the buffer solution is a citrate-disodium hydrogen phosphate buffer solution, making the pH of the nanoemulsion formulation 6.8.

[0053] Example 12

[0054] A nanoemulsion formulation containing recombinant human insulin differs from Example 1 in that the buffer solution is a citrate-disodium hydrogen phosphate buffer solution, making the pH of the nanoemulsion formulation 7.4.

[0055] Example 13

[0056] A nanoemulsion formulation containing recombinant human insulin differs from Example 1 in that the isotonicity regulator is glycerol, resulting in an osmotic pressure of 280 mOsmol / kg for the nanoemulsion formulation.

[0057] Example 14

[0058] A nanoemulsion formulation containing recombinant human insulin differs from Example 1 in that the isotonicity regulator is glycerol, resulting in an osmotic pressure of 320 mOsmol / kg for the nanoemulsion formulation.

[0059] Example 15

[0060] A nanoemulsion formulation containing recombinant human insulin, which differs from Example 1 in that it has an average particle size of 100 nm and a zeta potential of -45 mV.

[0061] Example 16

[0062] A nanoemulsion formulation containing recombinant human insulin, which differs from Example 1 in that it has an average particle size of 200 nm and a zeta potential of -25 mV.

[0063] Example 17

[0064] A nanoemulsion formulation containing recombinant human insulin differs from Example 1 in that it also contains a phenylboronic acid-modified polyethylene glycol-polylysine block copolymer, which is used together with poloxamer 407 in step (2) and its amount is 11.5% of the mass of the oil phase component. The degree of polymerization of the polylysine segment is 35, and 45% of its side chain amino groups are replaced by phenylboronic acid. At the same time, the total molecular weight of the phenylboronic acid-modified polyethylene glycol-polylysine block copolymer is 10000 Da, and the molecular weight of the polyethylene glycol segment is 2000 Da.

[0065] Example 18

[0066] A nanoemulsion formulation containing recombinant human insulin differs from Example 17 in that the amount of phenylboronic acid-modified polyethylene glycol-polylysine block copolymer is 8% of the mass of the oil phase component, wherein the degree of polymerization of the polylysine segment is 50, and 60% of its side chain amino groups are replaced by phenylboronic acid.

[0067] Example 19

[0068] A nanoemulsion formulation containing recombinant human insulin differs from Example 17 in that the amount of phenylboronic acid-modified polyethylene glycol-polylysine block copolymer is 15% of the mass of the oil phase component, wherein the degree of polymerization of the polylysine segment is 20, and 30% of its side chain amino groups are replaced by phenylboronic acid.

[0069] Example 20

[0070] A nanoemulsion formulation containing recombinant human insulin differs from Example 17 in that the total molecular weight of the phenylboronic acid-modified polyethylene glycol-polylysine block copolymer is 5000 Da, wherein the molecular weight of the polyethylene glycol segment is 1000 Da.

[0071] Example 21

[0072] A nanoemulsion formulation containing recombinant human insulin differs from that in Example 17 in that the total molecular weight of the phenylboronic acid-modified polyethylene glycol-polylysine block copolymer is 15,000 Da, wherein the molecular weight of the polyethylene glycol segment is 3,000 Da.

[0073] Comparative Example Comparative Example 1 A nanoemulsion formulation containing recombinant human insulin differs from Example 1 in that the recombinant human insulin encapsulated with sulfobutyl ether-β-cyclodextrin is replaced with recombinant human insulin, aluminum monostearate is not used in the oil phase, and no functional composition is used in the components.

[0074] Comparative Example 2 A nanoemulsion formulation containing recombinant human insulin, differing from Example 1 in that the recombinant human insulin encapsulated by sulfobutyl ether-β-cyclodextrin is replaced with recombinant human insulin, and aluminum monostearate is not used in the oil phase components.

[0075] Comparative Example 3 A nanoemulsion formulation containing recombinant human insulin, differing from Example 1 in that the recombinant human insulin encapsulated by sulfobutyl ether-β-cyclodextrin is replaced with recombinant human insulin, and no functional composition is used in the components.

[0076] Comparative Example 4 A nanoemulsion formulation containing recombinant human insulin differs from Example 1 in that aluminum monostearate is not used in the oil phase and no functional composition is used in the components.

[0077] Comparative Example 5 A nanoemulsion formulation containing recombinant human insulin differs from Comparative Example 2 in that lecithin and cholesterol are not used in the functional composition.

[0078] Comparative Example 6 A nanoemulsion formulation containing recombinant human insulin, which differs from Comparative Example 2 in that lecithin and poloxamer 407 are not used in the functional composition.

[0079] Comparative Example 7 A nanoemulsion formulation containing recombinant human insulin, which differs from Comparative Example 2 in that the functional composition does not use cholesterol or poloxamer 407.

[0080] Comparative Example 8 A nanoemulsion formulation containing recombinant human insulin, differing from Example 17 in that the recombinant human insulin encapsulated with sulfobutyl ether-β-cyclodextrin is replaced with recombinant human insulin, aluminum monostearate is not used in the oil phase, and no functional composition is used in the components.

[0081] Performance testing Test samples: The nanoemulsion formulations containing recombinant human insulin in Examples 1-21 were selected as test samples 1-21, and the nanoemulsion formulations containing recombinant human insulin in Comparative Examples 1-8 were selected as control samples 1-8.

[0082] Experimental methods: (1) In vitro simulated tear enzyme degradation test; The simulated tear enzyme system is histidine buffer (pH 7.4) containing: lysozyme (1.0 mg / mL, simulated tear lysozyme concentration), trypsin (0.1 mg / mL, simulated trypsin-like serine protease), MMP-9 (50 ng / mL, simulated concentration under inflammatory conditions); The simulated tear enzyme system is preheated to 37°C, a 1.5 mL volumetric EP tube is taken, 900 μL of the preheated enzyme system is added, and then 100 μL of the nanoemulsion preparation containing recombinant human insulin to be tested is added. After vortexing and mixing, it is placed in a constant temperature shaker (100 rpm) at 37°C. After 4 h, 100 μL of reaction solution is taken out and immediately added to an EP tube containing 10 μL of stop solution (0.1% trifluoroacetic acid aqueous solution), vortexed and mixed, and temporarily stored in an ice bath. Next, the recombinant human insulin content was determined using HPLC: a C18 reversed-phase column (4.6 × 250 mm, 5 μm), mobile phase A: 0.1% TFA / water, mobile phase B: 0.1% TFA / acetonitrile, gradient elution (28-38% B, 20 min), flow rate 1.0 mL / min, injection 20 μL, UV detection at 214 nm. The retention rate of recombinant human insulin after 4 h was calculated, with the insulin content at 0 min as 100%.

[0083] (2) In vitro corneal permeability test: A vertical Franz diffusion cell was used to separate the donor chamber and the recipient chamber, with a fresh isolated porcine cornea sandwiched in between. The recipient chamber was injected with histidine buffer at pH 7.4, and the donor chamber was filled with the nanoemulsion preparation containing recombinant human insulin. The temperature was set to 35±0.5 ℃ in a water bath, and the sample was taken and replenished with isothermal buffer at 200 rpm after magnetic stirring at 0.5, 1, 2, 3, 4, 5, and 6 h. The apparent permeability coefficient was calculated according to Papp (apparent corneal permeability coefficient) = (ΔQ / Δt) / (A×C0) (ΔQ / Δt is the steady-state cumulative permeation rate, which is obtained from the slope of the steady-state segment of the cumulative permeation amount-time curve; A is the effective exposed area of ​​the cornea, and C0 is the initial insulin concentration in the donor chamber).

[0084] After performing the above tests on test samples 1-21 and control samples 1-8, the test results are recorded in Table 2.

[0085] Table 2 Test results of test samples 1-21 and control samples 1-8

[0086] Combining Example 1 and Comparative Examples 1-4 with Table 2, it can be seen that by adding the functional composition composed of lecithin, cholesterol, and poloxamer 407, a three-layer interfacial membrane synergistic mechanism is established. This, along with the synergistic effect of the cyclodextrin inclusion encapsulation on enzyme cleavage sites and the diffusion retardation effect of the oil-phase thixotropic network, enables recombinant human insulin to obtain long-term protection in the tear film enzyme environment, significantly improving the stability and corneal bioavailability of recombinant human insulin in the tear film environment. The test results obtained in the above tests all show significant improvements. If any one of the functional composition, aluminum monostearate, and sulfobutyl ether-β-cyclodextrin encapsulation is used alone, although it can bring about corresponding improvements, the improvement is limited, and the sum of the improvements brought by the individual application of the three is far less than the superior synergistic effect of the three. Combined with Comparative Examples 5-7 and Table 2, it can be seen that for the functional composition consisting of lecithin, cholesterol and poloxamer 407, using any one of the three components alone cannot bring about the excellent effect of the combination of the three. Moreover, the test results show that the combination of lecithin, cholesterol and poloxamer 407 can bring about a significant improvement effect of 1+1+1>3.

[0087] Combining Examples 1 and 17-21 with Table 2, it can be seen that the addition of phenylboronic acid-modified polyethylene glycol-polylysine block copolymer resulted in superior performance in the nanoemulsion formulation containing recombinant human insulin, indicating that it can improve the stability and corneal bioavailability of recombinant human insulin in the tear film environment. Furthermore, combining Comparative Examples 1 and 8 with Table 2, it can be seen that the phenylboronic acid-modified polyethylene glycol-polylysine block copolymer can form excellent complexation and enhancement effects with the three-layer interfacial film, sulfobutyl ether-β-cyclodextrin inclusion complex, and oil-phase thixotropic network. However, without the application of the functional composition, aluminum monostearate, and sulfobutyl ether-β-cyclodextrin inclusion complex, the corresponding enhancement effect brought about by the addition of phenylboronic acid-modified polyethylene glycol-polylysine block copolymer is significantly reduced.

[0088] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A nanoemulsion formulation containing recombinant human insulin, characterized in that, It contains the following components: The active ingredient is recombinant human insulin encapsulated by sulfobutyl ether-β-cyclodextrin, and the concentration of recombinant human insulin is 0.1-1.0 U / mL; The oil phase component, with a concentration of 5-15% (v / v), contains medium-chain triglycerides or vegetable oils and disperses 0.1-0.5% aluminum monostearate by mass. A functional composition comprising lecithin, cholesterol, and poloxamer 407 in a mass ratio of 1:(0.2-0.5):(0.1-0.5), wherein the amount of the composition is 10-30% of the mass of the oil phase components; Surfactant, concentration 1-5% (v / v); Co-surfactant, concentration 0.5-3% (v / v); The aqueous phase, used to bring the total to 100%, contains sodium hyaluronate, buffer solution, isotonicity regulator, preservative, and water for injection.

2. The nanoemulsion formulation containing recombinant human insulin according to claim 1, characterized in that: In the recombinant human insulin encapsulated with sulfobutyl ether-β-cyclodextrin, the molar ratio of recombinant human insulin to sulfobutyl ether-β-cyclodextrin is 1:(4-7).

3. The nanoemulsion formulation containing recombinant human insulin according to claim 1, characterized in that: The functional composition consists of lecithin, cholesterol, and poloxamer 407 in a mass ratio of 1:0.3:0.

3.

4. The nanoemulsion formulation containing recombinant human insulin according to claim 1, characterized in that: The composition also includes a polyethylene glycol-polylysine block copolymer modified with phenylboronic acid, which is used at 8-15% of the mass of the oil phase component. The degree of polymerization of the polylysine block is 20-50, and 30-60% of its side chain amino groups are replaced by phenylboronic acid.

5. The nanoemulsion formulation containing recombinant human insulin according to claim 4, characterized in that: The total molecular weight of the phenylboronic acid-modified polyethylene glycol-polylysine block copolymer is 5000-15000 Da, wherein the molecular weight of the polyethylene glycol segment is 1000-3000 Da.

6. The nanoemulsion formulation containing recombinant human insulin according to claim 1, characterized in that: The nanoemulsion formulation containing recombinant human insulin has an average particle size of 100-200 nm and a zeta potential of -25 to -45 mV.

7. The nanoemulsion formulation containing recombinant human insulin according to claim 1, characterized in that: The buffer solution is a histidine buffer or a citrate-disodium hydrogen phosphate buffer, so that the pH of the nanoemulsion preparation is 6.8-7.

4.

8. The nanoemulsion formulation containing recombinant human insulin according to claim 1, characterized in that: The isotonic regulator is one or more of glycerol, mannitol, and sodium chloride, which makes the osmotic pressure of the nanoemulsion preparation 280-320 mOsmol / kg.

9. The nanoemulsion formulation containing recombinant human insulin according to claim 1, characterized in that: The surfactant is a mixture of polyoxyethylene hydrogenated castor oil and poloxamer 188, and the co-surfactant is propylene glycol or ethanol.

10. The method for preparing the nanoemulsion formulation containing recombinant human insulin according to claim 1, characterized in that: Includes the following steps: (1) Take sulfobutyl ether-β-cyclodextrin-encapsulated recombinant human insulin and disperse it in an oil phase containing aluminum monostearate. Stir at 40-50℃ until completely dissolved to obtain an oil phase mixture; (2) After dissolving lecithin and cholesterol in anhydrous ethanol, the ethanol is removed by rotary evaporation to form a lipid film. Then, poloxamer 407 and the oil phase mixture obtained in step (1) are added and treated at 50-60℃ for 25-35 min to form a lipid-oil phase premix. (3) After mixing the surfactant, co-surfactant and aqueous phase components, preheat to 50-55℃, and then add to the lipid-oil phase premix obtained in step (2) under high-speed shearing, and continue to shear to form crude emulsion. After high-pressure microfluidic homogenization and filtration sterilization, nanoemulsion preparation containing recombinant human insulin is obtained.

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