Bromfenac prodrug preparation as well as preparation method and application thereof

Abstract

The invention discloses a bromfenac prodrug preparation as well as a preparation method and application thereof. The preparation method comprises the step of forming bromfenac glycerol ester from bromfenac sodium and glycerol through an ester bond. Compared with bromfenac sodium eye drops, the bromfenac prodrug preparation obviously reduces the degree of eye stabbing. Meanwhile, in an inflammation part, the bromfenac prodrug preparation is hydrolyzed under the action of phospholipase, a bromfenac sodium prototype is released, and a powerful anti-inflammatory effect is exerted, so that the bromfenac sodium eye drops effectively solve the symptoms of eye stabbing pain, redness and the like in the use process, and effectively reduce local adverse reactions.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a bromfenac prodrug preparation and a preparation method and application thereof. Background Art

[0002] Bromfenac sodium eye drops, as a nonsteroidal anti-inflammatory drug (NSAIDs), are widely used in clinical practice to treat inflammatory diseases of the external eye and anterior eye, such as blepharitis, conjunctivitis, scleritis, and postoperative inflammation.

[0003] The active ingredient of bromfenac sodium eye drops is bromfenac sodium, which inhibits cyclooxygenase to block the synthesis of inflammatory mediators prostaglandins, thereby exerting a strong anti-inflammatory effect. However, the irritation that bromfenac sodium eye drops may exhibit during use has become an issue that needs to be focused on in research and development and application.

[0004] The irritation of bromfenac sodium eye drops to the eyes mainly comes from its drug ingredients. Some patients may feel temporary stinging, redness or itching in the eyes after using the drops, which reduces the patient's medication compliance.

[0005] Therefore, how to avoid eye stinging caused by the use of bromfenac sodium eye drops is an important issue that needs to be addressed. Summary of the invention

[0006] The purpose of this section is to summarize some aspects of embodiments of the present invention and to briefly introduce some preferred embodiments.

[0007] The present invention provides the following technical scheme: a bromfenac prodrug preparation, comprising forming bromfenac glycerol ester by reacting bromfenac sodium with glycerol via an ester bond.

[0008] As a preferred embodiment of the bromfenac prodrug preparation of the present invention: the bromfenac glycerol ester includes one or more forms of 1-hydroxyl substitution, 2-hydroxyl substitution, a mixture of 1-hydroxyl and 2-hydroxyl substitution, a mixture of 1-hydroxyl and 3-hydroxyl substitution, and a mixture of 1-hydroxyl, 2-hydroxyl and 3-hydroxyl substitution of glycerol;

[0009] As a preferred embodiment of the bromfenac prodrug preparation of the present invention: the bromfenac prodrug preparation includes an emulsion form.

[0010] As a preferred embodiment of the bromfenac prodrug preparation of the present invention, the viscosity of the emulsion is 500-800 Pa·s, and the particle size of the emulsion is 150-300 nm.

[0011] The present invention also provides a method for preparing the bromfenac prodrug preparation: dissolving glycerol in a solvent, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, stirring, adding bromfenac sodium and stirring; adding water to the reaction system, filtering and obtaining the filter residue, and freeze-drying to obtain bromfenac glycerol ester; wherein the molar ratio of bromfenac sodium to glycerol is 2.2 to 3.4:1.

[0012] As a preferred embodiment of the method for preparing the bromfenac prodrug preparation of the present invention, the bromfenac prodrug preparation is prepared into an emulsion, and the preparation method of the emulsion comprises mixing bromfenac glycerol ester, a nonionic surfactant, an osmotic pressure regulator and a thickener, adding water, and homogenizing to obtain the emulsion; the content of bromfenac glycerol ester in the emulsion is 5-5.6 mg / mL, which is equivalent to 1 mg / mL of bromfenac sodium.

[0013] As a preferred embodiment of the method for preparing the bromfenac prodrug preparation of the present invention: the nonionic surfactant includes poloxamer and polyoxyethylene hydrogenated castor oil.

[0014] As a preferred embodiment of the method for preparing the bromfenac prodrug preparation of the present invention: the thickener includes one or more of hydroxypropyl methylcellulose, hydroxypropyl cellulose, methyl cellulose and polyvinyl alcohol.

[0015] As a preferred embodiment of the method for preparing the bromfenac prodrug preparation of the present invention: the osmotic pressure regulator includes sodium chloride and glucose.

[0016] The present invention also provides application of the bromfenac prodrug preparation in ocular medicine: the bromfenac prodrug preparation releases bromfenac sodium under the action of phospholipase.

[0017] Beneficial effects of the present invention: The present invention discloses a bromfenac prodrug preparation, a preparation method and an application thereof. The prodrug is formed by ester bonding bromfenac sodium and glycerol. Compared with bromfenac sodium eye drops, the bromfenac prodrug preparation significantly reduces the degree of eye stinging. At the same time, at the site of inflammation, the bromfenac prodrug preparation is hydrolyzed under the action of phospholipase to release the bromfenac sodium prototype, exerting a strong anti-inflammatory effect. The present invention effectively solves the symptoms of eye stinging, redness and the like that occur during the use of bromfenac sodium eye drops, and effectively reduces the occurrence of local adverse reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the following briefly introduces the drawings required for describing the embodiment, wherein:

[0019] Figure 1 It is a synthetic route for bromfenac glycerol ester;

[0020] Figure 2 This is the 1H NMR diagram of bromfenac glycerol ester;

[0021] Figure 3 This is a particle size diagram of bromfenac prodrug emulsion;

[0022] Figure 4 is the ultraviolet absorption wavelength diagram of bromfenac glycerol ester;

[0023] Figure 5 The transdermal release diagram of bromfenac prodrug emulsion with different particle sizes;

[0024] Figure 6 This is the HPLC diagram of bromfenac sodium;

[0025] Figure 7 The release profile of bromfenac prodrug emulsion under different phospholipase concentrations. DETAILED DESCRIPTION

[0026] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0027] Embodiment 1:

[0028] Preparation of bromfenac glycerol ester: Figure 1 As shown, 1 g of glycerol was dissolved in 20 mL of dimethyl sulfoxide (DMSO), 2.5 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 1.9 g of N-hydroxysuccinimide (NHS) were added, and the mixture was stirred at room temperature for 4 h to activate the carboxyl group of glycerol. 4.7 g of bromfenac sodium (C 15 H 11 BnN y ), stirred overnight; added a large amount of deionized water to the reaction system, filtered the residue, and freeze-dried.

[0029] Detection: Dissolve the freeze-dried product in dichloromethane, separate the product with a silica gel column, and use nuclear magnetic resonance for structural identification. Figure 2 As shown, 2-3 ppm in the glycerol spectrum was identified as its characteristic peak, and 5-6 ppm could be determined as the characteristic peak of bromfenac sodium. In the spectrum of bromfenac glycerol ester, 2-3 ppm of glycerol and 5-6 ppm of bromfenac sodium were both present, confirming the synthesis of bromfenac glycerol ester.

[0030] Embodiment 2:

[0031] Determination of bromfenac grafting rate: At room temperature, the JNM-ECZ400S* nuclear magnetic resonance spectrometer was used to 1 H NMR experiment, the bromfenac glycerol ester obtained in Example 1 was dissolved in 0.6 mL D 2O or DMSO-d6, filtered through a 0.22 μm filter, and then transferred to an NMR tube for further analysis.

[0032] Embodiment 3:

[0033] Effect of the feed ratio of bromfenac sodium to glycerol on grafting efficiency: By changing the feed amount of glycerol in Example 1, the ratio of bromfenac sodium to glycerol can be adjusted. As shown in Table 1, when the molar ratio of bromfenac sodium to glycerol in the feed is increased from 1.4:1 to 2.2:1, a grafting rate of 20.13% of bromfenac glycerol ester can be obtained; when the molar ratio of bromfenac sodium to glycerol in the feed is increased from 3:1 to 3.8:1, the grafting rate of bromfenac glycerol ester is reduced to 12.23%. In order to obtain a higher grafting rate of bromfenac glycerol ester, we prefer a feed molar ratio of bromfenac sodium to glycerol of 2.6:1.

[0034] Table 1 Effect of Bromfenac Sodium and Glycerol Feed Ratio on Grafting Rate

[0035]

[0036]

[0037] Embodiment 4:

[0038] Preparation of bromfenac prodrug emulsion: 4.8g of poloxamer 188 and 4.8g of polyoxyethylene castor oil were mixed and stirred continuously at a temperature of 40-50°C until a mixed emulsifier was formed, followed by the addition of 0.45g of bromfenac glycerol ester (bromfenac grafting rate of about 22%) to form an oil phase; 0.5g of cysteine, 5g of mannitol, 0.1g of benzalkonium chloride, and 0.4g of sodium chloride were added to 60mL of water, and stirred continuously at 40-50°C, and the pH value of the solution was adjusted to 6.5 with a pH regulator to obtain an aqueous phase. Then, the oil phase was slowly added dropwise to the aqueous phase and stirred to form an emulsion. Finally, 10mL of 30% hydroxypropyl methylcellulose (model: K15M) swelling solution was added to the emulsion, stirred evenly and fixed to 100mL, and finally a bromfenac prodrug emulsion was obtained. The content of bromfenac prodrug is determined according to the bromfenac grafting rate. The prodrug concentration in the finished emulsion is 5-5.6 mg / mL, which is equivalent to 1 mg / mL of bromfenac sodium. Figure 3 is the particle size distribution of the emulsion.

[0039] Embodiment 5:

[0040] Effect of particle size on corneal penetration: Using the modified Franz diffusion cell method, the intact porcine cornea was fixed between the supply cell and the receiving cell, and the receiving cell was filled with 15 ml of phosphate buffered saline solution (pH=6.8) containing 20% ​​ethanol. 2 ml of the bromfenac prodrug emulsion with different particle sizes obtained in Example 4 was weighed and added to the exposed surface of the porcine cornea in the supply cell, and magnetic stirring was performed at 350 rpm. The water bath temperature was controlled at 37°C. 1 ml of the receiving solution was taken at 15 min, 30 min, 60 min, and 120 min, and 1 ml of the same amount of isothermal receiving solution was added. 1 ml of methanol was added to the sample and vortexed and mixed, and centrifuged at 10000 rpm for 10 min. It was filtered with a 0.22 μm microporous filter membrane, and the amount of drug transmitted was detected by an ultraviolet spectrophotometer at 400 nanometers. Figure 4 is the UV scanning spectrum of bromfenac glycerol ester. Figure 5 As shown in the figure, when the particle size is 352nm, the drug penetration to the cornea is lower than that of 149nm and 231nm. Therefore, the preferred particle size is 200nm.

[0041] Embodiment 6:

[0042] Thickening experiment: By changing the concentrations of hydroxypropyl methylcellulose, hydroxypropyl cellulose, methylcellulose and polyvinyl alcohol in Example 4, the viscosity, particle size and ease of dropping of the bromfenac prodrug emulsion can be adjusted. As shown in Tables 2, 3, 4, and 5, when the concentrations of hydroxypropyl methylcellulose, hydroxypropyl cellulose, methylcellulose and polyvinyl alcohol increase, the viscosity increases accordingly, and the ease of dropping also decreases accordingly; while when the viscosity decreases, the particle size is too large. Compared with hydroxypropyl cellulose, methylcellulose and polyvinyl alcohol, when the concentration of hydroxypropyl methylcellulose is 3%-4%, the viscosity is between 500-800pa s, and the particle size ranges from about 200. The preferred thickener is hydroxypropyl methylcellulose.

[0043] Table 2 Effect of Hydroxypropyl Methylcellulose Concentration on Viscosity, Particle Size and Ease of Addition

[0044] concentration Viscosity Particle size Convenience of adding drops 1％ 274.36 692 Convenient 2％ 428 494 Convenient 3％ 543 237 Convenient 4％ 729 212 Convenient 5％ 923 176 Inconvenient

[0045] Table 3 Effect of hydroxypropyl cellulose concentration on viscosity, particle size and ease of dropping

[0046] concentration Viscosity Particle size Convenience of adding drops 1％ 449 609 Convenient 2％ 498 528 Convenient 3％ 553 298 Convenient 4％ 849 283 Inconvenient 5％ 899 230 Inconvenient

[0047] Table 4 Effect of methylcellulose concentration on viscosity, particle size and ease of dropping

[0048] concentration Viscosity Particle size Convenience of adding drops 1％ 199 794 Convenient 2％ 384 550 Convenient 3％ 689 302 Inconvenient 4％ 790 247 Inconvenient 5％ 1038 200 Inconvenient

[0049] Table 5 Effect of polyvinyl alcohol concentration on viscosity, particle size and ease of dropping

[0050]

[0051]

[0052] Embodiment 7:

[0053] Effect of bromfenac grafting rate on enzymatic release: 2 ml of bromfenac glycerol ester emulsion in Example 1 and 2 ml of phospholipase (concentrations of 1, 10, and 50 μg / mL) were placed in a dialysis bag, and 100 ml of 0.25% Tween 80-artificial tear solution was used as the release medium (pH = 7.4), and placed in a 37°C constant temperature oscillator with a speed of 100 rpm. Samples were taken at 0h, 0.5h, 1h, 2h, 4h, 6h, 8h, 12h, and 24h, and 0.5 ml of the solution outside the dialysis bag was taken out each time, and an equal amount of fresh release medium at the same temperature was added at the same time. The taken release medium was ultrafiltered and centrifuged, the supernatant was taken, and it was diluted with methanol at a ratio of 1:1. The diluted sample was taken and the content of bromfenac sodium in the release medium was determined by HPLC injection. Figure 6 This is a typical HPLC spectrum of bromfenac.

[0054] Embodiment 8:

[0055] Eyeball irritation test: Three adult male rabbits without any eye diseases and intact corneal epithelium were prepared for the experiment. The day before the experiment, the ocular health was checked, and a wide light band examination was performed using a slit lamp to confirm the transparency of the cornea and the clarity of the iris. At the same time, the conjunctiva was observed for congestion or secretions to exclude the possibility of inflammation. The eyes of the rabbits were stained with sodium fluorescein by adjusting the slit lamp to cobalt blue light to check the integrity of the corneal epithelium and the presence of pathological damage. The left and right eyes of the same rabbit received different treatments. 0.1 ml of bromfenac prodrug emulsion was injected into the conjunctival sac of the right eye, while the same volume of bromfenac sodium was injected into the left eye as a control. After administration, the eyelids were gently closed for 20 seconds to promote drug absorption. The rabbit eyes were observed at 0h, 1h, 2h, 4h, 8h, 12h, and 24h of administration to check whether there was secretion, conjunctival congestion or edema. Use a slit lamp to perform a detailed examination to observe whether the cornea is cloudy or the iris is abnormal. Evaluate according to the eye irritation evaluation criteria.

[0056] Table 6 Rabbit eye irritation scores of bromfenac prodrug emulsion and bromfenac sodium eye drops

[0057]

[0058]

[0059] In summary, the present invention discloses a bromfenac prodrug preparation and a preparation method and application thereof. The prodrug is formed by ester bonding bromfenac sodium and glycerol. Compared with bromfenac sodium eye drops, the bromfenac prodrug preparation significantly reduces the degree of eye stinging. At the same time, at the site of inflammation, the bromfenac prodrug preparation is hydrolyzed under the action of phospholipase, releasing the bromfenac sodium prototype, exerting a strong anti-inflammatory effect. The present invention effectively solves the symptoms of eye stinging, redness, etc. that occur during the use of bromfenac sodium eye drops, and effectively reduces the occurrence of local adverse reactions.

[0060] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A bromfenac prodrug preparation, characterized in that: The method comprises forming bromfenac sodium and glycerol through an ester bond to form bromfenac glycerol ester.

2. The bromfenac prodrug preparation according to claim 1, characterized in that: The bromfenac glycerol ester includes one or more forms of 1-hydroxyl substitution, 2-hydroxyl substitution, a mixture of 1-hydroxyl and 2-hydroxyl substitution, a mixture of 1-hydroxyl and 3-hydroxyl substitution, and a mixture of 1-hydroxyl, 2-hydroxyl and 3-hydroxyl substitution of glycerol.

3. The bromfenac prodrug preparation according to claim 1 or 2, characterized in that: The bromfenac prodrug formulation includes an emulsion form.

4. The bromfenac prodrug preparation according to claim 3, characterized in that: The viscosity of the emulsion is 500-800 Pa·s, and the particle size of the emulsion is 150-300 nm.

5. The method for preparing the bromfenac prodrug preparation according to claim 1, characterized in that: Dissolve glycerol in a solvent, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, stir, add bromfenac sodium and stir; add water to the reaction system, filter out the residue, and freeze-dry to obtain bromfenac glycerol ester; wherein the molar ratio of bromfenac sodium to glycerol is 2.2-3.4:

1.

6. The method for preparing the bromfenac prodrug preparation according to claim 5, characterized in that: It also includes preparing the bromfenac prodrug preparation into an emulsion, wherein the preparation method of the emulsion comprises mixing bromfenac glycerol ester, a nonionic surfactant, an osmotic pressure regulator and a thickener, adding water, and homogenizing to obtain the emulsion; the content of bromfenac glycerol ester in the emulsion is 5 to 5.6 mg / mL.

7. The method for preparing the bromfenac prodrug preparation according to claim 5 or 6, characterized in that: The nonionic surfactants include poloxamer and polyoxyethylene hydrogenated castor oil.

8. The method for preparing the bromfenac prodrug preparation according to claim 5 or 6, characterized in that: The thickener includes one or more of hydroxypropyl methylcellulose, hydroxypropyl cellulose, methyl cellulose and polyvinyl alcohol.

9. The method for preparing the bromfenac prodrug preparation according to claim 5 or 6, characterized in that: The osmotic pressure regulator includes sodium chloride and / or glucose.

10. Use of the bromfenac prodrug preparation according to claim 1 in ocular medicine, characterized in that: The bromfenac prodrug preparation releases bromfenac sodium under the action of phospholipase.

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