A Nintedanib composition and its application

Abstract

The present invention belongs to the technical field of pharmaceutical preparations, and specifically relates to a nintedanib composition and its application. In the treatment of age-related macular degeneration, the anti-VEGF drugs used in the prior art have a short duration of action, require multiple intraocular injections, and are more likely to be accompanied by serious eye complications. The present invention provides a novel ophthalmic preparation and a preparation method thereof, which utilizes hemoglobin and nintedanib ethanesulfonate to combine, and uses dopamine polymerization on the surface to form a nano preparation that can be used to consume active oxygen in the eye, inhibit angiogenesis, and treat age-related macular degeneration.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and in particular relates to a nintedanib composition and an application thereof. Background Art

[0002] Age-related macular degeneration (AMD), also known as age-related macular degeneration, is a degenerative and irreversible disease that will lead to progressive vision loss. AMD is the most common visual disease in the West and the most common cause of blindness. About 1-3% of the population is in the late stage of the disease. In this posterior segment disease, excessive angiogenesis can cause intraocular hemorrhage, macular edema and retinal detachment, and ultimately lead to loss of visual function. In the early stage, photodynamic therapy mediated by the photosensitizer Verteporfin and laser photocoagulation reduced the rate of vision loss in patients to a certain extent. As the core role of vascular endothelial growth factor (VEGF) in angiogenesis has been confirmed, common anti-VEGF drugs are divided into two categories: synthetic monoclonal antibodies and receptor fusion proteins. The former is represented by ranibizumab and bevacizumab, and the latter is represented by Conbercept and Aflibercept. Although the two types of drugs have different mechanisms of action, they can effectively inhibit the progression of neovascularization in the short term. However, long-term follow-up data show that anti-VEGF drugs have a short duration of action, decompose quickly, are prone to relapse after discontinuation of the drug, require multiple intraocular injections, and have strict requirements on the doctor's skills. They are more likely to be accompanied by serious ocular complications (such as endophthalmitis, intraocular hemorrhage, retinal tears and detachment), which bring huge life and economic burdens to patients. Ordinary local eye drops and systemic administration are hindered by the blood-retinal barrier, resulting in the inability of the drug to reach the posterior segment of the eye to take effect. Therefore, it is urgent to develop a minimally invasive / non-invasive and safe alternative treatment strategy for this type of blinding eye disease.

[0003] Studies have found that oxidative stress has been reported to be one of the important mechanisms of AMD, because the macula is a structural site that is particularly susceptible to damage by reactive oxygen species (ROS). The retina is one of the most oxygen-consuming tissues in the human body, with the highest oxygen content in the choroid, but the oxygen content drops sharply in the outermost retina, which contains a higher content of polyunsaturated fatty acids. Intense oxygen metabolism, prolonged light exposure, high concentrations of polyunsaturated fatty acids, and the presence of photosensitizers increase the production of reactive oxygen species (ROS) in the retina. ROS are produced when light interacts with endogenous chromophores in ocular tissues. In addition, retinal pigment epithelial (RPE) cells produce reactive (ROS) protein adducts such as advanced glycation end products (AGES) malondialdehyde and ethylpyrrole when exposed to light, which accumulate in the degenerating retina, causing RPE cell senescence and / or death, and then causing photoreceptor degeneration and vision loss. Excessive ROS will further upregulate VEGF to accelerate the development of AMD, so methods to rescue RPE cells through antioxidant and / or cell protection will likely contribute to interventional treatment of AMD.

[0004] Nintedanib esylate (NDNB) is an oral tri-linked ligand kinase vascular inhibitor developed by Boehringer Ingelheim. It targets platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR) and vascular endothelial growth factor receptor (VEGFR). It is clinically used for the treatment of idiopathic pulmonary fibrosis and lung cancer. Oral nintedanib can easily cause liver damage in patients, and may also cause adverse gastrointestinal reactions such as diarrhea and nausea. Given the excellent effect of nintedanib in inhibiting vascular endothelial growth factor, it is possible to develop it into an ocular preparation, but oral administration is difficult to reach the posterior segment of the eye. Therefore, the development of new nintedanib preparations will help provide an option for the treatment of AMD. Summary of the invention

[0005] In view of the shortcomings of the prior art, the inventors provide a composition of nintedanib and a preparation method thereof, which uses hemoglobin and nintedanib ethanesulfonate to form a nanostructure, which does not require the addition of cross-linking agents such as glutaraldehyde, and has a uniform particle size. After external adsorption of dopamine hydrochloride, it is further polymerized into nanoparticles to form a nanoparticle that can consume active oxygen in the eye and inhibit new blood vessels. It is further wrapped with a macrophage membrane to form a nanoparticle that can target new blood vessels in the posterior segment of the eye and treat age-related macular degeneration. The preparation is simple to prepare, has a uniform particle size, and is simple in composition.

[0006] The present invention specifically provides a nintedanib composition and a preparation method thereof

[0007] The composition comprises hemoglobin, nintedanib and dopamine.

[0008] The nintedanib in the composition is nintedanib ethanesulfonate, and the weight proportions of the components are nintedanib ethanesulfonate: 0.1-5 parts, hemoglobin: 10-100 parts, and dopamine: 10-100 parts.

[0009] The particle size of the composition is less than 400 nm.

[0010] The composition is a nanocomposite formed by hemoglobin and nintedanib ethanesulfonate, and the outer layer of the nanocomposite is wrapped by dopamine.

[0011] The composition is prepared by the following method: hemoglobin is dissolved in water as an aqueous phase, nintedanib ethanesulfonate is dissolved in an organic solvent as an organic phase, the organic phase is added dropwise to the aqueous phase with stirring, dopamine hydrochloride is added with stirring, and then tris (hydroxymethyl)aminomethane (Tris) solution is added and stirring is continued.

[0012] Preferably, the composition is prepared by the following method: dissolving hemoglobin in water as the aqueous phase, dissolving nintedanib ethanesulfonate with a hemoglobin concentration of 5-50 mg / mL in an organic solvent as the organic phase, the volume ratio of the aqueous phase to the organic phase is 1:0.2-8, adding the organic phase dropwise into the aqueous phase and stirring for 0.2-10 hours, removing the organic solvent, adding dopamine hydrochloride and stirring, adding a Tris solution with a pH of 8.5, the volume ratio of the aqueous phase to the Tris solution is 1:1-100, and stirring is continued for 1-48 hours.

[0013] The organic solvent is one or both of methanol and acetone.

[0014] The outer layer of the nanocomposition is coated with a macrophage membrane to form a nanopreparation that can target new blood vessels in the posterior segment of the eye.

[0015] Preferably, the nanocomposition uses a thin film extrusion technique to wrap the outer layer with a macrophage membrane to form a nanoformulation that can target ocular choroidal neovascularization.

[0016] The composition is for intravenous injection.

[0017] The composition further comprises other pharmaceutically acceptable excipients, which are osmotic pressure regulators.

[0018] The present invention also provides a use of the nintedanib composition in the preparation of a drug for treating age-related macular degeneration.

[0019] The nintedanib composition eliminates active oxygen in retinal endothelial cells, inhibits new blood vessels, and achieves the purpose of treating age-related macular degeneration.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] (1) Effectively improve the solubility of nintedanib ethanesulfonate;

[0022] (2) A method for preparing a novel formulation of nintedanib was provided, which had a uniform and stable particle size and a high drug encapsulation efficiency;

[0023] (3) Provides a multifunctional nanoformulation for posterior segment ocular diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : Particle size distribution diagram of the preparation of Example 1;

[0025] Figure 2 : Transmission electron micrograph of the preparation of Example 1 (scale bar = 100 nm);

[0026] Figure 3 : Confocal laser microscopy images of ROS expression in ARPE-19 cells (A is the comparative example 4 group, B is the example 2 group);

[0027] Figure 4 : Cellular uptake diagram of ARPE-19 cells (A is the group in Example 8, B is the group in Example 7). DETAILED DESCRIPTION

[0028] The present invention is further described below by way of examples. It should be correctly understood that the examples of the present invention are provided only for illustrating the present invention, rather than limiting the present invention, and therefore, simple modifications of the present invention under the premise of the method of the present invention are within the scope of protection claimed by the present invention. Example 1

[0029] (1) 0.1 mg of nintedanib esylate was dissolved in 8 mL of acetone as the organic phase, and 10 mg of hemoglobin was dissolved in 2 mL of deionized water as the aqueous phase;

[0030] (2) The aqueous phase was added dropwise to the organic phase under stirring conditions and stirred for 0.2 h. The organic solvent was removed by rotary evaporation and 1 mg of dopamine hydrochloride was added and stirred;

[0031] (3) Add 20 ml of Tris solution (pH = 8.5) and continue stirring for 1 hour. Example 2

[0032] (1) 1 mg of nintedanib esylate was dissolved in 8 mL of a mixed solution of methanol and acetone (the volume ratio of methanol to acetone was 1:1) as the organic phase, and 10 mg of hemoglobin was dissolved in 1 mL of deionized water as the aqueous phase;

[0033] (2) The aqueous phase was added dropwise to the organic phase under stirring conditions and stirred for 10 h. The organic solvent was removed by rotary evaporation and 5 mg of dopamine hydrochloride was added and stirred;

[0034] (3) Add 100 ml of Tris solution (pH = 8.5) and continue stirring for 48 hours. Example 3

[0035] (1) 5 mg of nintedanib esylate was dissolved in 5 mL of methanol as the organic phase, and 50 mg of hemoglobin was dissolved in 1 mL of deionized water as the aqueous phase;

[0036] (2) The aqueous phase was added dropwise to the organic phase under stirring conditions and stirred for 4 h. The organic solvent was removed by rotary evaporation and 50 mg of dopamine hydrochloride was added and stirred;

[0037] (3) Add 1 ml of Tris solution (pH 8.5) and continue stirring for 24 hours. Example 4

[0038] (1) 0.2 mg of nintedanib esylate was dissolved in 0.4 mL of acetone as the organic phase, and 40 mg of hemoglobin was dissolved in 2 mL of deionized water as the aqueous phase;

[0039] (2) The aqueous phase was added dropwise to the organic phase under stirring conditions and stirred for 0.5 h. The organic solvent was removed by rotary evaporation and 10 mg of dopamine hydrochloride was added and stirred;

[0040] (3) Add 20 ml of Tris solution (pH = 8.5) and continue stirring for 4 hours. Example 5

[0041] (1) 1 mg of nintedanib esylate was dissolved in 8 mL of acetone as the organic phase, and 20 mg of hemoglobin was dissolved in 1 mL of deionized water as the aqueous phase;

[0042] (2) The aqueous phase was added dropwise to the organic phase under stirring conditions and stirred for 0.5 h. The organic solvent was removed by rotary evaporation and 20 mg of dopamine hydrochloride was added and stirred;

[0043] (3) Add 20 ml of Tris solution (pH = 8.5) and continue stirring for 3 hours. Example 6

[0044] (1) 1 mg of nintedanib esylate was dissolved in 8 mL of a mixed solution of methanol and acetone (the volume ratio of methanol to acetone was 1:3) as the organic phase, and 10 mg of hemoglobin was dissolved in 1 mL of deionized water as the aqueous phase;

[0045] (2) The aqueous phase was added dropwise to the organic phase under stirring conditions and stirred for 10 h. The organic solvent was removed by rotary evaporation and 5 mg of dopamine hydrochloride was added and stirred;

[0046] (3) Add 20 ml of Tris solution (pH = 8.5) and continue stirring for 24 hours.

[0047] (4) The resulting solution was filtered through a 0.45 μm microporous filter membrane, and RAW264.7 macrophage cell membrane with a protein concentration of 1 mg / ml was added thereto, and the cell membrane was wrapped using a thin film extrusion technique. Example 7

[0048] The nintedanib ethanesulfonate in Example 5 was replaced by Nile Red, and the other preparation steps were the same as in Example 5. Example 8

[0049] The nintedanib ethanesulfonate in Example 6 was replaced by Nile Red, and the other preparation steps were the same as in Example 6.

[0050] Comparative Example 1

[0051] (1) A mixed solution of 1 mg of nintedanib esylate dissolved in 8 mL of tetrahydrofuran was used as the organic phase, and 10 mg of hemoglobin was dissolved in 1 mL of deionized water as the aqueous phase;

[0052] (2) The aqueous phase was added dropwise to the organic phase under stirring conditions and stirred for 10 h. The organic solvent was removed by rotary evaporation and 5 mg of dopamine hydrochloride was added and stirred;

[0053] (3) Add 100 ml of Tris solution (pH = 8.5) and continue stirring for 48 hours.

[0054] Comparative Example 2

[0055] (1) 1 mg of nintedanib esylate was dissolved in 1 mL of acetone solution as the organic phase, and 10 mg of bovine serum albumin was dissolved in 1 mL of deionized water as the aqueous phase;

[0056] (2) The aqueous phase was added dropwise to the organic phase under stirring conditions and stirred for 10 h. The organic solvent was removed by rotary evaporation and 5 mg of dopamine hydrochloride was added and stirred;

[0057] (3) Add 100 ml of Tris solution (pH = 8.5) and continue stirring for 48 hours.

[0058] Comparative Example 3

[0059] (1) 5 mL of methanol was used as the organic phase, and 5 mg of nintedanib esylate and 50 mg of hemoglobin were dissolved in 1 mL of deionized water as the aqueous phase;

[0060] (2) The aqueous phase was added dropwise to the organic phase under stirring conditions and stirred for 4 h. The organic solvent was removed by rotary evaporation and 50 mg of dopamine hydrochloride was added and stirred;

[0061] (3) Add 1 ml of Tris solution (pH 8.5) and continue stirring for 24 hours.

[0062] Comparative Example 4

[0063] (1) 5 mL of methanol was used as the organic phase, and 5 mg of nintedanib esylate and 50 mg of hemoglobin were dissolved in 1 mL of deionized water as the aqueous phase;

[0064] (2) The aqueous phase was added dropwise to the organic phase under stirring conditions and stirred for 4 h, and the organic solvent was removed by rotary evaporation.

[0065] Comparative Example 5

[0066] (1) 0.2 mg of nintedanib esylate was dissolved in 0.4 mL of acetone as the organic phase, and 40 mg of hemoglobin was dissolved in 2 mL of deionized water as the aqueous phase;

[0067] (2) The aqueous phase was added dropwise to the organic phase under stirring conditions and stirred for 0.5 h. The organic solvent was removed by rotary evaporation, and 10 mg of plant polyphenol hematoxylin was added and stirred;

[0068] (3) Add 20 ml of Tris solution (pH = 8.5) and continue stirring for 4 hours.

[0069] Comparative Example 6

[0070] (1) 1 mg of doxorubicin hydrochloride was dissolved in 8 mL of methanol as the organic phase, and 5 mg of hemoglobin was dissolved in 50 mL of deionized water as the aqueous phase;

[0071] (2) The aqueous phase was added dropwise to the organic phase under stirring conditions and stirred for 10 h. The organic solvent was removed by rotary evaporation and 5 mg of dopamine hydrochloride was added and stirred;

[0072] (3) Add 100 ml of Tris solution (pH = 8.5) and continue stirring for 48 hours.

[0073] Comparative Example 7

[0074] (1) 1 mg of nintedanib esylate was dissolved in 8 mL of acetone as the organic phase, and 20 mg of hemoglobin was dissolved in 1 mL of deionized water as the aqueous phase;

[0075] (2) The aqueous phase was added dropwise to the organic phase under stirring conditions and stirred for 0.5 h. 200 μL of 0.5% glutaraldehyde as a cross-linking agent was added and stirred for another 0.5 h. The organic solvent was removed by rotary evaporation. 20 mg of dopamine hydrochloride was added and stirred.

[0076] (3) Add 20 ml of Tris solution (pH = 8.5) and continue stirring for 3 hours.

[0077] Comparative Example 8

[0078] (1) Weigh 30 mg of dopamine hydrochloride and add 10 ml of deionized water to dissolve it. Stir and react for 1 hour.

[0079] (2) Add 15 ml of Tris solution (pH 8.5) and stir for 12 hours.

[0080] Verification Example

[0081] 1. The solution obtained in Example 1 was filtered using a 0.45 μm microporous filter membrane, the obtained liquid was negatively stained with phosphotungstic acid, and the morphology was observed using a transmission electron microscope.

[0082] The results are as follows Figure 2 The nanoparticles prepared in the examples of the present invention are approximately spherical in shape and have a relatively uniform particle size.

[0083] 2. The solutions obtained in Examples 1-6 and Comparative Examples 1-8 were filtered through a 0.45 μm microporous filter membrane, and the particle size and polydispersity index of the obtained liquid were monitored using a laser particle size analyzer.

[0084] Table 1. Particle size and polydispersity index (PDI) of various examples

[0085]

[0086] The results are as follows Figure 1 As shown in Table 1, the particle size of the nanoparticle preparation prepared in the embodiment of the present invention conforms to the normal distribution, the particle size is less than 200 nm, and the polydispersity index is less than 0.2, and the particle size is uniform.

[0087] 3. The solutions obtained in Examples 1-6 and Comparative Examples 1-5 and 7 were filtered through a 0.45 μm microporous membrane and dialyzed in water. DMSO was added to the obtained liquid to break the emulsion. The drug content was detected at 390 nm using an ultraviolet spectrophotometer to calculate the drug encapsulation efficiency.

[0088] Table 2. Encapsulation efficiency of various examples

[0089]

[0090] The results are shown in Table 2. The drug preparation prepared by the present invention is easily encapsulated, and the encapsulation rate is relatively high, about 90%.

[0091] 4. Human retinal pigment epithelial cells (ARPE-19) were inoculated into a confocal culture dish, and 10 μM H2O2 complete medium (DMEM / F12) was added to induce oxidative stress. The medium was removed, and the cells were washed three times with the medium. Example 2 and Comparative Example 4 were diluted with complete medium and incubated with ARPE-19 cells for 12 hours. The medium was removed, and a ROS fluorescent probe DCFH-DA solution diluted with serum-free medium was added and placed in an incubator for further incubation for 30 minutes. The cells were washed three times with PBS, and the fluorescence intensity of Example 2 and Comparative Example 4 was observed under a confocal laser microscope. Figure 3 It shows that the ROS fluorescence intensity of Example 2 group is weaker than that of Comparative Example 4 group.

[0092] 5. Human retinal pigment epithelial cells (ARPE-19) were seeded into 6-well plates and cultured with complete medium (DMEM / F12) for 24 hours. After lipopolysaccharide stimulation, the medium was discarded, and Examples 7 and 8 were diluted with complete medium to the same concentration of Nile red, respectively, and incubated with ARPE-19 cells for 2 hours. The medium was removed, and the cells were fixed with 4% paraformaldehyde, washed 3 times with PBS, and the cell uptake intensity was observed under an inverted fluorescence microscope. Figure 4 It was shown that both Examples 7 and 8 had good cellular uptake.

Claims

1. A nintedanib composition, characterized in that The composition comprises hemoglobin, nintedanib ethanesulfonate, and dopamine; the weight proportions of each component are 0.1-5 parts of nintedanib ethanesulfonate, 10-100 parts of hemoglobin, and 10-100 parts of dopamine; the composition is a nanocomposite formed by hemoglobin and nintedanib ethanesulfonate, and the outer layer is then wrapped by dopamine to form a nanocomposite; the composition is prepared by the following method: hemoglobin is dissolved in water as an aqueous phase, nintedanib ethanesulfonate is dissolved in an organic solvent as an organic phase, the organic phase is dripped into the aqueous phase and stirred, dopamine hydrochloride is added and stirred, and then Tris solution is added and stirred continuously; The organic solvent is one or both of methanol and acetone.

2. The nintedanib composition according to claim 1, characterized in that The particle size of the composition is less than 400 nm.

3. The nintedanib composition according to claim 1, characterized in that The composition is prepared by the following method: hemoglobin is dissolved in water as an aqueous phase, nintedanib ethanesulfonate with a hemoglobin concentration of 5-50 mg / mL is dissolved in an organic solvent as an organic phase, the volume ratio of the aqueous phase to the organic phase is 1:0.2-8, the organic phase is added dropwise into the aqueous phase and stirred for 0.2-10 hours, the organic solvent is removed, dopamine hydrochloride is added and stirred, and then a Tris solution with a pH of 8.5 is added, the volume ratio of the aqueous phase to the Tris solution is 1:1-100, and stirring is continued for 1-48 hours.

4. The nintedanib composition according to claim 1, characterized in that The outer layer of the nanocomposition is coated with a macrophage membrane to form a nanopreparation that can target new blood vessels in the posterior segment of the eye.

5. The nintedanib composition according to claim 1, characterized in that The composition is for intravenous injection.

6. Use of the nintedanib composition according to any one of claims 1 to 5 in the preparation of a drug for treating age-related macular degeneration.

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