Preparation method of nystatin fatty acid ester vaginal suppository for inflammation response and product and application thereof

Abstract

The application discloses a preparation method of a nystatin fatty acid ester vaginal suppository responding to inflammation, a product and application thereof, and belongs to the field of medicine. The preparation method comprises the following steps: dissolving fatty acid in a solvent, adding a catalyst, uniformly mixing, and activating a carboxyl group of the fatty acid, adding nystatin, and stirring to obtain nystatin fatty acid ester. The fatty acid is saturated or unsaturated fatty acid with a carbon chain length of 4-17 carbons. The application effectively solves the problems that nystatin is unevenly distributed in the suppository matrix and is prone to suppository sedimentation and stratification. Meanwhile, the nystatin fatty acid ester disclosed by the application can be hydrolyzed under the action of leukocyte esterase at an inflammation site of a vagina, release nystatin raw medicine, and play a bactericidal role responding to inflammation. In the non-inflammation area, the nystatin fatty acid ester is hardly hydrolyzed into nystatin, and thus the local adverse reaction of the vagina caused by nystatin can be effectively reduced.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmacy, and particularly relates to a preparation method of an inflammation-responsive nystatin fatty acid ester vaginal suppository, a product thereof and application. BACKGROUND

[0002] The nystatin vaginal suppository is a suppository formed by dispersing nystatin in a fatty acid glyceride to form a yellow or light yellow oily base, which is often used for treating candidal vulvovaginitis. Since the solubility of nystatin in the fatty acid glyceride is low, the nystatin is dispersed in the form of drug microparticles in the suppository base. During long-term storage, the dispersed nystatin microparticles are prone to form aggregates under the action of van der Waals force, especially under the condition of high temperature in summer, the oily base of the suppository softens, and the nystatin microparticles and aggregates are prone to settle in the base, resulting in the occurrence of stratification of the suppository, which finally has a great impact on the quality of the suppository.

[0003] At the same time, the local use of nystatin can cause allergic reactions, which can cause local itching and red rash, and the use of nystatin vaginal suppository can easily lead to an increase in vaginal secretion, affecting the quality of life of patients. SUMMARY

[0004] This section aims to summarize some aspects of the embodiments of the present application and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of the specification to avoid obscuring the purpose of this section, abstract and title, and such simplifications or omissions cannot be used to limit the scope of the present application.

[0005] As one aspect of the present application, the present application provides a preparation method of an inflammation-responsive nystatin fatty acid ester vaginal suppository, which comprises,

[0006] dissolving the fatty acid in a solvent, adding a catalyst, mixing and activating the carboxyl group of the fatty acid, adding nystatin, stirring, and obtaining nystatin fatty acid ester;

[0007] The fatty acid is a saturated or unsaturated fatty acid with a carbon chain length of 4-17 carbons.

[0008] As a preferred scheme of the preparation method of the inflammation-responsive nystatin fatty acid ester vaginal suppository, the fatty acid is a saturated or unsaturated fatty acid with a carbon chain length of 7-17 carbons.

[0009] As a preferred scheme of the preparation method of the inflammation-responsive nystatin fatty acid ester vaginal suppository, the equivalent ratio of nystatin to fatty acid in the nystatin fatty acid ester is 1:1-3.

[0010] As a preferred embodiment of the preparation method of the nystatin fatty acid ester vaginal suppository for inflammation response of the present application: after the oil base matrix is heated and melted, the nystatin fatty acid ester is added, and after stirring and fully dissolving, the suppository mold is filled, cooled, scraped flat, and demolded.

[0011] As a preferred embodiment of the preparation method of the nystatin fatty acid ester vaginal suppository for inflammation response of the present application: the fatty acid includes one or more of valeric acid, octanoic acid, lauric acid, stearic acid, and oleic acid.

[0012] As a preferred embodiment of the preparation method of the nystatin fatty acid ester vaginal suppository for inflammation response of the present application: the oil base matrix includes fatty acid glyceride.

[0013] As a preferred embodiment of the preparation method of the nystatin fatty acid ester vaginal suppository for inflammation response of the present application: the solvent includes dimethyl sulfoxide, and the catalyst includes 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride and 4-dimethylaminopyridine.

[0014] As a preferred embodiment of the preparation method of the nystatin fatty acid ester vaginal suppository for inflammation response of the present application: the feeding equivalent of the catalyst is 0.01-0.5 times the feeding equivalent of the fatty acid.

[0015] The present application effectively solves the problem that nystatin is unevenly distributed in the suppository matrix and prone to suppository sedimentation and stratification, and the nystatin fatty acid ester disclosed in the present application can be hydrolyzed under the action of leukocyte esterase at the inflammation site of the vagina, releasing the nystatin raw drug to play a bactericidal role in response to inflammation, and the nystatin fatty acid ester is hardly hydrolyzed to nystatin in the non-inflammation area, which can effectively reduce the local adverse reactions of the vagina caused by nystatin. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced as follows:

[0017] Figure 1 Reaction equation for the reaction of nystatin and fatty acid to form nystatin fatty acid ester.

[0018] Figure 2 NMR spectrum of nystatin oleate.

[0019] Figure 3 Solubility of nystatin oleate in different oil base matrices.

[0020] Figure 4 Effect of the type of fatty acid on the solubility of nystatin fatty acid ester in fatty acid glyceride.

[0021] Figure 5 Effect of the equivalent ratio of nystatin to oleic acid on the solubility of nystatin oleate in fatty acid glyceride.

[0022] Figure 6 Effect of the fatty acid species on the hydrolysis rate of nystatin fatty acid ester under the action of esterase.

[0023] Figure 7 DSC pattern of nystatin oleate vaginal suppository.

[0024] Figure 8 Microscopic observation results (x40) of the thermal stability of nystatin oleate vaginal suppository. DETAILED DESCRIPTION

[0025] In order to make the above-mentioned objectives, features and advantages of the present application more apparent and comprehensible, the specific embodiments of the present application will be described in detail below with specific examples.

[0026] Example 1:

[0027] Preparation and identification of nystatin oleate:

[0028] Figure 1 Equation for the reaction of nystatin with fatty acid. 0.5 g of oleic acid was dissolved in 5 mL of dimethyl sulfoxide (DMSO) to obtain a DMSO solution of oleic acid with a concentration of 1.77 mM, 0.07 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (0.35 mM) and 0.04 g of 4-dimethylaminopyridine (DMAP) (0.35 mM) were added, and the carboxyl group of the fatty acid was activated by stirring at room temperature for 4 h; 0.4 g of nystatin (0.7 mM) was added to the reaction system and stirred overnight; a large amount of deionized water was added to the reaction system, the filter residue was collected by filtration, and was freeze-dried; the freeze-dried product was dissolved in dichloromethane, and the product was separated by silica gel column (dichloromethane:methanol 50:50 as eluent), and the obtained product was dried by rotary evaporator. Nuclear magnetic resonance and infrared spectroscopy were used for structure identification.

[0029] Figure 2 NMR spectrum of nystatin oleate obtained in Example 1. The results show that the product has both the characteristic NMR peaks of nystatin and the characteristic NMR peaks of oleic acid, and the carboxyl peak of oleic acid with a shift of 11 disappears, indicating that the synthesis of nystatin oleate is successful. At the same time, the peak area ratio of the vibration peak of the carbon-hydrogen bond in the carbon-carbon double bond in nystatin (6.5 ppm) to the vibration peak of the carbon-hydrogen bond in the carbon-carbon double bond in nystatin (5.5 ppm) is calculated, and the results show that the ratio of nystatin to oleic acid in the product is 1:2.8, i.e., an average of 1 mole of nystatin molecule is connected to 2.8 moles of oleic acid molecule.

[0030] Screening of reaction solvent: DMSO in Example 1 was replaced by dichloromethane, trichloromethane, N, N-dimethylformamide, acetone, ethyl acetate respectively, it was found that the raw materials of each reaction could not be dissolved at the same time, while DMSO could dissolve the reactants in Example 1 at the same time, so the solvent of the reaction system was selected as DMSO.

[0031] Example 2:

[0032] Solubility of nystatin oleate in oleaginous suppository base:

[0033] 1 g of nystatin oleate obtained in Example 1 was added into 1 g of cocoa butter, Litsea cubeba oil ester (36 type), Litsea cubeba oil ester (38 type), and mixed fatty acid glyceride (from Shaanxi Zhengyi Pharmaceutical Auxiliary Co., Ltd.) respectively, the mixed system was incubated at 80℃ to melt the oil, and stirred at 80℃ for 2 h to completely dissolve the nystatin oleate, an 80℃ preheated syringe and a 0.22 μm microporous filter membrane were used to filter the oil system, 0.01 g of the filtered oil was cooled, added into 1 mL of acetonitrile, vortexed for 3 min, filtered with a 0.22 μm microporous filter membrane, and the content of nystatin oleate was determined by HPLC with a C18 column, the solubility was calculated. The mobile phase was methanol: water (75:35, V / V), the detection wavelength was 224 nm, and the column temperature was 30℃.

[0034] Figure 3 The solubility of nystatin oleate obtained in Example 1 in different oils was shown in the results. The results showed that the nystatin oleate obtained in Example 1 had high solubility in different oils, among which the solubility in cocoa butter and mixed fatty acid glyceride could reach 513 mg / g and 589 mg / g, while the solubility of nystatin in the above two oils was only 2.1 mg / g and 1.6 mg / g, indicating that the nystatin oleate synthesized in Example 1 significantly improved the solubility of nystatin in oleaginous matrix.

[0035] Example 3:

[0036] Synthesis of nystatin fatty acid esters of different fatty acids:

[0037] A certain amount of acetic acid, pentanoic acid, octanoic acid, lauric acid, stearic acid, lignoceric acid, and octacosanoic acid was dissolved in 5 mL of dimethyl sulfoxide (DMSO) to obtain a DMSO solution of fatty acid with a concentration of 1.77 mM, 0.07 g of EDC (0.35 mM) and 0.04 g of DMAP (0.35 mM) were added, and the fatty acid carboxyl group was activated by stirring at room temperature for 4 h; 0.4 g of nystatin (0.7 mM) was added to the reaction system and stirred overnight; a large amount of deionized water was added to the reaction system, the filtrate was collected by filtration, and the freeze-dried; the freeze-dried product was dissolved in dichloromethane, and the product was separated by silica gel column (dichloromethane:methanol 50:50 as eluent), and the obtained product was dried by rotary evaporator.

[0038] 1 g of the nystatin fatty acid ester prepared above was added to 1 g of mixed fatty acid glycerol, the mixed system was incubated at 80°C to melt the oil, and the nystatin fatty acid ester was completely dissolved by stirring at 80°C for 2 h; the oil system was filtered by using a preheated syringe at 80°C and a 0.22 μm microporous filter membrane; 0.01 g of the filtered oil was taken, added to 1 mL of acetonitrile, vortexed for 3 min, filtered by a 0.22 μm microporous filter membrane, and the content of nystatin fatty acid ester was determined by HPLC with a C18 column; the mobile phase was methanol:water (75:35, V / V), the detection wavelength was 224 nm, and the column temperature was 30°C.

[0039] Figure 4 The effect of the type of fatty acid on the solubility of nystatin fatty acid ester in mixed fatty acid glycerol was studied. It was found that the solubility of nystatin fatty acid ester in mixed fatty acid glycerol gradually increased with the increase of the carbon chain length of fatty acid. When the carbon chain length of fatty acid was less than 4, such as acetic acid and nystatin to form nystatin acetate, the solubility of the product in mixed fatty acid glycerol was only 11 mg / g, while the solubility of nystatin pentanoate could be increased to 231 mg / g; when the carbon chain length of fatty acid was greater than 17, such as nystatin lignocerate and nystatin tetracosanoate, the solubility in mixed fatty acid glycerol was similar to that of nystatin stearate and nystatin oleate, indicating that when the carbon chain length of fatty acid was greater than 17, the extension of the carbon chain had no significant effect on the improvement of the solubility of nystatin.

[0040] Example 4:

[0041] The effect of the equivalent ratio of nystatin to oleic acid on the solubility of nystatin oleate:

[0042] By adjusting the equivalent ratio of nystatin to oleic acid in the reaction, nystatin oleate with different degrees of esterification can be obtained (see Table 1). Figure 5The effect of the equivalent ratio of natamycin to oleic acid in the synthesized natamycin oleate on the solubility of the natamycin oleate in the mixed fatty acid glyceride was determined. The results show that the solubility of the natamycin oleate in the mixed fatty acid glyceride gradually increases as the equivalent ratio of natamycin to oleic acid in the natamycin oleate increases, but the solubility no longer increases when the equivalent ratio of the two increases from 1:3 to 1:3.7. The above results show that the equivalent ratio of natamycin to oleic acid in the natamycin oleate is preferably 1:1-1:3.

[0043] Table 1: The equivalent ratio of natamycin to oleic acid in the raw material and the esterification degree of the product

[0044] Nystatin to oleic acid feed ratio 1∶2 1∶3 1∶4 1∶5 Nystatin to oleic acid ratio in nystatin oleate 1∶1 1∶1.8 1∶3 1∶3.7

[0045] Example 5:

[0046] Hydrolysis of the natamycin fatty acid ester under the action of esterase:

[0047] 0.5 g of the synthesized natamycin acetate, natamycin valerate, natamycin octanoate, natamycin laurate, natamycin stearate, natamycin oleate, natamycin lignocerate and natamycin montanate was respectively added into 100 mL of normal saline containing 0.2% Tween-80, and was ultrasonically dispersed for 20 min. 10 mg of ester hydrolytic enzyme (from Shangke Biomedicine) was added into the system, and was stirred at 37°C. 1 mL of the reaction system was taken at 0.5 h, 2 h and 4 h, respectively, was diluted with 10 mL of acetonitrile, was filtered through a 0.22 μm microporous filter, and the amount of the generated natamycin in the system was determined by HPLC. Figure 6 It is shown that under the same concentration of ester hydrolytic enzyme, the natamycin acetate and the natamycin valerate have the fastest hydrolysis rate, and the hydrolysis rate of the natamycin fatty acid ester gradually slows down as the carbon chain lengthens. When the carbon chain length is extended from C17 of the natamycin stearate or the natamycin oleate to the natamycin lignocerate and the natamycin montanate, the generation rate of the natamycin appears a cliff-like drop, which is probably due to the steric hindrance of the extended carbon chain to the ester hydrolytic enzyme. The carbon chain length of the natamycin fatty acid ester should be controlled to be below 17.

[0048] Example 6:

[0049] Antibacterial test of the natamycin fatty acid ester:

[0050] According to the "Disinfection Technical Standard" issued by the National Health and Health Committee, the MIC value of nystatin fatty acid ester modified by different fatty acids against Aspergillus fumigatus was determined, and the results (see Table 2) showed that nystatin had the lowest MIC value, and as the carbon chain of fatty acid was extended, the MIC value of the obtained nystatin fatty acid ester against Aspergillus fumigatus increased, and when the carbon chain length exceeded C4, the MIC value increased from 5 μM to 10 μM, which may be due to the incomplete hydrolysis of nystatin fatty acid ester in the system; and when the carbon chain length was extended to C24 and C28, the obtained nystatin fatty acid ester had no obvious bacteriostatic effect, which may be due to the low hydrolysis rate of the two. When nystatin fatty acid ester was in the absence of ester hydrolase, there was no obvious inhibition effect on Aspergillus fumigatus. The above results showed that when the carbon chain length of the fatty acid used was less than 17, the obtained nystatin fatty acid ester still retained the mold inhibition effect comparable to nystatin in the presence of ester hydrolase.

[0051] Table 2 MIC value of different nystatin fatty acid esters against Aspergillus fumigatus

[0052]

[0053] Example 7:

[0054] Preparation of nystatin and nystatin oleate vaginal suppositories:

[0055] After heating and melting 90 g of fatty acid glyceride, 10 g of nystatin oleate was added and stirred to dissolve, then poured into the suppository mold, a total of 50 suppositories were prepared, cooled, scraped flat, and demolded to obtain nystatin oleate vaginal suppositories.

[0056] After heating and melting 95 g of fatty acid glyceride, 5 g of nystatin was added and stirred to dissolve, then poured into the suppository mold, a total of 50 suppositories were prepared, cooled, scraped flat, and demolded to obtain nystatin vaginal suppositories. The molar concentration of nystatin in the suppository is the same as that of nystatin oleate in the nystatin oleate suppository.

[0057] Example 8:

[0058] DSC spectrum of nystatin oleate vaginal suppository:

[0059] Figure 7 The DSC spectrum of nystatin oleate vaginal suppository. It can be seen from the results that the characteristic DSC endothermic peak of nystatin is visible in the nystatin vaginal suppository group, while in the nystatin oleate vaginal suppository, the characteristic DSC endothermic peak of nystatin oleate disappears, indicating that nystatin is dispersed in the form of crystal in the mixed fatty acid glyceride matrix, while nystatin oleate is dispersed in the form of molecule in the mixed fatty acid glyceride, forming a solid solution. This dispersion state can effectively avoid the delamination of nystatin oleate in the suppository matrix, and improve the physical stability of the suppository.

[0060] Example 9:

[0061] Microscopic observation of the heat stability of nystatin oleate vaginal suppository (observed after 14 days at 40°C):

[0062] Figure 8 For the microscopic observation of the heat stability of nystatin oleate vaginal suppository, it can be seen that there are a large number of needle-shaped drug crystals in the nystatin vaginal suppository, and there are no obvious drug crystals in the nystatin oleate vaginal suppository, indicating that the formation of oleate greatly improves the uniformity of the vaginal suppository.

[0063] Example 10:

[0064] Acute irritation test of rabbit skin:

[0065] The rabbit skin on the back was shaved, and nystatin vaginal suppository and nystatin oleate vaginal suppository were evenly applied to one side of the rabbit skin, and the skin was observed after 4 hours. After 4 hours of sealing, nystatin vaginal suppository caused erythema on the skin of the rabbit, which was caused by the local skin irritation of nystatin; and the skin of the rabbit in the nystatin oleate vaginal suppository group had no obvious change. The above results show that nystatin oleate vaginal suppository can effectively reduce the local adverse reactions caused by nystatin.

[0066] In summary, in order to solve the sedimentation and stratification phenomenon of nystatin in the vaginal suppository, and to a certain extent reduce the occurrence of local adverse reactions caused by nystatin, the present application proposes a new type of inflammation-responsive nystatin oleate suppository. In the vaginal suppository, oleic acid fatty acid and nystatin are used to prepare nystatin fatty acid ester. Compared with nystatin, the solubility of nystatin fatty acid ester in oil and fat matrix is significantly improved, which can effectively solve the stratification phenomenon of the suppository after the softening of the oil and fat matrix. At the same time, at the inflammation site of the vagina, nystatin fatty acid ester can be hydrolyzed under the action of leukocyte esterase, releasing nystatin prototype, and playing a role in killing bacteria; while at non-inflammatory sites, or after the inflammation is effectively treated, the number of white blood cells is significantly reduced compared to the inflammation site, and nystatin still exists in the form of fatty acid ester, which can effectively reduce the occurrence of local adverse reactions caused by nystatin.

[0067] It should be noted that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application 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 application can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present application, which should be covered in the scope of the claims of the present application.

Claims

1. A method of preparing a nystatin fatty acid ester pessary for inflammation response, characterized by: The method comprises the following steps: The fatty acid is dissolved in a solvent, a catalyst is added, the carboxyl of the fatty acid is activated by mixing, nystatin is added, and stirring is performed to obtain a nystatin fatty acid ester; The fatty acid is a saturated or unsaturated fatty acid with a carbon chain length of 4-17 carbons. The equivalent ratio of nystatin to the fatty acid in the nystatin fatty acid ester is 1:1-3. The method further comprises the following steps: after the oil and fat matrix is heated and melted, the nystatin fatty acid ester is added, and stirring is performed until the nystatin fatty acid ester is completely dissolved, then the nystatin fatty acid ester is poured into a mold, cooled, scraped flat, and demolded. The oil and fat matrix comprises a fatty acid glyceride.

2. The method of preparing a nystatin fatty acid ester pessary for inflammation response according to claim 1, characterized by: The fatty acid is a saturated or unsaturated fatty acid with a carbon chain length of 7-17 carbons.

3. Process for the preparation of a nystatin fatty acid ester pessary for the response of inflammation according to claim 1 or 2, characterized in that: The fatty acid comprises one or more of valeric acid, octanoic acid, lauric acid, stearic acid, and oleic acid.

4. The method of preparing a nystatin fatty acid ester pessary for inflammation response according to claim 1 or 2, characterized by: The solvent comprises dimethyl sulfoxide, and the catalyst comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine.

5. The method of preparing a nystatin fatty acid ester pessary for inflammation response according to claim 4, characterized by: The feeding equivalent of the catalyst is 0.01-0.5 times the feeding equivalent of the fatty acid.

6. The inflammation-responsive nystatin fatty acid ester vaginal suppository prepared by the preparation method in claim 1.

7. Use of the inflammation-responsive nystatin fatty acid ester vaginal suppository in claim 6 in the preparation of a medicine for treating vaginitis.

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