An erythromycin micellar type ointment overcoming erythromycin multidrug resistance and a preparation method and application thereof

Abstract

The application discloses erythromycin micellar type ointment for overcoming erythromycin multidrug resistance and a preparation method and application thereof, and the erythromycin micellar type ointment is composed of liposoluble acid modified dextran, erythromycin, hydrophilic gel matrix material and water; wherein the mass ratio of the liposoluble acid modified dextran and the erythromycin is 1:1-1:10. The application effectively solves the killing of erythromycin resistant bacteria by erythromycin, and can also effectively reduce the occurrence of the drug resistance phenomenon of non-drug resistant bacteria.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmacy, and particularly relates to erythromycin micellar ointment for overcoming erythromycin multidrug resistance and a preparation method and application thereof. BACKGROUND

[0002] Erythromycin is a kind of macrolide antibiotic. Erythromycin ointment is often used for treating mild to moderate skin and soft tissue infections caused by bacteria. With the increase of the use frequency of drugs, the problem of erythromycin resistance is becoming increasingly serious. In order to solve the problem of erythromycin resistance, researchers have developed second-generation macrolide antibiotics, such as roxithromycin, azithromycin, and third-generation macrolide antibiotics, such as telithromycin and quinolone.

[0003] However, considering that the development speed of new antibiotics is far slower than the generation speed of drug-resistant bacteria, more effective use of existing antibiotics against drug-resistant strains may be a good way to solve the problem of antibiotic resistance. SUMMARY

[0004] This section aims to summarize some aspects of the embodiments of the present application and briefly introduce some preferred embodiments.

[0005] The present application provides the following technical scheme: a preparation of a new erythromycin micellar ointment for overcoming erythromycin multidrug resistance, which comprises,

[0006] Preparation of hydrophobically modified dextran micelles: dissolve the liposoluble acid in dimethyl sulfoxide (DMSO), add dicyclohexyl carbodiimide (DCC) and 4-dimethylaminopyridine (DMAP), stir overnight, and the molar ratio of DCC, DMAP and liposoluble acid is liposoluble acid:DCC:DMAP=1:1.5:1.5; add dextran to the reaction system, stir at room temperature for 24 hours; after the reaction is completed, the reaction product is dialyzed for 72 hours, the supernatant is centrifuged and freeze-dried, and hydrophobically modified dextran is obtained.

[0007] Preparation of erythromycin-loaded hydrophobically modified dextran micellar ointment: weigh an appropriate amount of hydrophobically modified dextran and dissolve it in water, add a hot ethanol solution dissolving erythromycin dropwise into the above solution, stir uniformly, and then add sodium carboxymethyl cellulose to swell and dissolve, and erythromycin-loaded hydrophobically modified dextran micellar ointment is obtained.

[0008] As a preferred method for preparing a new erythromycin micellar ointment for overcoming erythromycin multidrug resistance, the liposoluble acid includes dodecanoic acid, stearic acid, and tretinoin, and tretinoin is preferred.

[0009] As a preferred method for preparing the novel erythromycin micellar ointment for overcoming erythromycin multidrug resistance according to the present application, the dextran has a molecular weight range of 1000-20000 Da, wherein the molecular weight is preferably 1000 Da.

[0010] As a preferred method for preparing the novel erythromycin micellar ointment for overcoming erythromycin multidrug resistance according to the present application, the linking molar ratio of the liposoluble acid to the dextran is 1:18.3-1:4.3, and the linking ratio is preferably 1:1.

[0011] As a preferred method for preparing the novel erythromycin micellar ointment for overcoming erythromycin multidrug resistance according to the present application, the mass ratio of the erythromycin to the hydrophobically modified dextran is 0.01-0.1, and the mass ratio is preferably 0.02.

[0012] The present application has the beneficial effect of effectively killing erythromycin-resistant bacteria and effectively reducing the occurrence of drug resistance of non-drug-resistant bacteria. BRIEF DESCRIPTION OF DRAWINGS

[0013] 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 below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor. Among them:

[0014] Figure 1 Critical micelle concentration of various hydrophobically modified dextrans.

[0015] Figure 2 Effect of dextran molecular weight on critical micelle concentration of hydrophobically modified dextran.

[0016] Figure 3 Effect of tretinoin connection ratio to dextran monomer on erythromycin solubility.

[0017] Figure 4 Appearance photo of the novel erythromycin micellar ointment.

[0018] Figure 5 Induction results of erythromycin-resistant Staphylococcus aureus

[0019] Figure 6 Effect of tretinoin connection ratio on MIC of the novel erythromycin micellar ointment on erythromycin-resistant Staphylococcus aureus

[0020] Figure 7 Effect of dextran molecular weight on MIC of the novel erythromycin micellar ointment on erythromycin-resistant Staphylococcus aureus

[0021] Figure 8 Effect of the mass ratio of tretinoin-modified dextran to erythromycin on the MIC of a new erythromycin micellar ointment against erythromycin-resistant Staphylococcus aureus

[0022] Figure 9 Results of the induction of resistance to a new erythromycin micellar ointment against non-resistant Staphylococcus aureus

[0023] Figure 10 Results of the affinity experiment of a new erythromycin ointment against Staphylococcus aureus. DETAILED DESCRIPTION

[0024] In order to make the above 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.

[0025] Example 1:

[0026] Preparation of hydrophobically modified dextran: tretinoin was dissolved in dimethyl sulfoxide (DMSO, 100 mM), dicyclohexyl carbodiimide (DCC, 150 mM) and 4-dimethyl aminopyridine (DMAP, 150 mM) were added, and the mixture was stirred overnight; dextran (80 mg / mL, monomer concentration 500 mM) was added to the reaction system, and the mixture was stirred at room temperature for 24 hours; after the reaction was completed, the reaction product was dialyzed for 72 hours, the supernatant was obtained by centrifugation, and then freeze-drying was performed, thereby obtaining tretinoin-modified dextran.

[0027] In the nuclear magnetic resonance spectrum of tretinoin-modified dextran, the characteristic absorption peaks of dextran are located at 2.5, 4.5, 4.7, 4.9 and 5.2 ppm. The obvious carboxyl characteristic peak of tretinoin is located at 12 ppm. In the spectrum of tretinoin-modified dextran, the characteristic peaks of dextran and tretinoin can be simultaneously observed, and the carboxyl peak of tretinoin disappears, indicating that the ester bond is formed between the carboxyl group of tretinoin and the hydroxyl group of dextran. By calculating the abundance of the methylene carbon-hydrogen stretching vibration peak at 1.2 ppm from the six-membered ring of tretinoin and the methylene carbon-hydrogen stretching vibration peak at 3.3 ppm from dextran in the final product, under the feeding ratio conditions of Example 1, the ratio of tretinoin to dextran monomer is 1:10.4.

[0028] Example 2:

[0029] Determination of the critical micelle concentration:

[0030] An equal volume of 6.0×10 -7A propyl alcohol solution of 1.0 x 10-6 mol / L pyrene was added to a series of 10 ml vials, and the vials were placed in the dark for 24 h to allow the acetone to evaporate completely. 1 mL of different concentrations of retinoic acid modified dextran with a concentration range of 0.006 to 6 mg / mL was added to each vial to make the final concentration of pyrene in each vial 6.0 x 10-6 mol / L. After vortex mixing for 1 min and ultrasonic treatment for 5 min, the excitation spectrum of pyrene was tested using a F-4600 fluorescence spectrophotometer at a temperature of 25°C, a maximum emission wavelength of 200 nm, a slit width of 10 nm, and a scanning speed of 1200 nm / min. -7 The fluorescence intensity of pyrene at different concentrations of retinoic acid modified dextran is shown in Table 1. The results show that the fluorescence intensity of the retinoic acid modified dextran in Example 1 is 0.06 mg / mL. Figure 1 The fluorescence intensity of pyrene at different concentrations of retinoic acid modified dextran is shown in Table 1. The results show that the fluorescence intensity of the retinoic acid modified dextran in Example 1 is 0.06 mg / mL.

[0031] Example 3:

[0032] Effect of the molecular weight of dextran on the critical micelle concentration of modified dextran:

[0033] The critical micelle concentration of the retinoic acid modified dextran was determined by changing the molecular weight of the dextran in Example 1. The results are shown in Table 2. Figure 2 The results show that within the molecular weight range of 1000-20000 Da, the critical micelle concentration of the retinoic acid modified dextran increases with the increase of the molecular weight of the dextran. In order to obtain a lower critical micelle concentration, we prefer to use the dextran with the lowest molecular weight (1000 Da) available at present for modification.

[0034] Effect of the ratio of retinoic acid to dextran on the critical micelle concentration of modified dextran: By changing the amount of dextran in Example 1, the ratio of retinoic acid to dextran can be adjusted. As shown in Table 1, when the molar ratio of retinoic acid to dextran (calculated as monomer) in the feed is increased from 1:20 to 5:1, the ratio of the two connected can be between 1:34 and 1:1.2. When the molar ratio of the two is lower than 1:18.3, the product cannot form micelles due to its strong hydrophilicity; when the molar ratio of the two is higher than 1:1.5, the modified dextran cannot be dissolved in water due to the introduction of a large number of hydrophobic groups. Therefore, when retinoic acid is used to modify dextran, the ratio of the two is between 1:18.3 and 1:4.3, and increasing the ratio of retinoic acid within this range can effectively reduce the critical micelle concentration of the product.

[0035] Table 1 Effect of the ratio of retinoic acid to dextran on the critical micelle concentration of modified dextran

[0036]

[0037] Example 4:

[0038] A 20 mg / mL solution of the different retinoid-modified dextran micelles prepared in Example 3 was prepared and 100 mg of erythromycin powder was added. After shaking for 24 hours, the solution was filtered through a 0.22 micron filter and the solubility of erythromycin in the system was determined. Figure 3 It was shown that the solubilization of erythromycin by the modified dextran increased as the proportion of retinoid attached to the modified dextran increased. This is likely to be due to the increased hydrophobic solubilization space provided by the increased proportion of retinoid.

[0039] Example 5:

[0040] Preparation of a new erythromycin micellar ointment:

[0041] A 200 mg solution of the retinoid-modified dextran described in Example 1 was prepared in 5 mL of deionized water. 0.5 mL of a hot ethanolic solution of erythromycin (erythromycin concentration 200 mg / mL, 50°C) was added dropwise to the above solution. After stirring to homogeneity, 0.2 g of sodium carboxymethylcellulose was added and dissolved. Water was added to make up the total weight to 10 g. A uniform, translucent, erythromycin-loaded hydrophobically modified dextran micellar ointment was obtained. Figure 4

[0042] Example 6:

[0043] Induction of erythromycin-resistant Staphylococcus aureus:

[0044] Staphylococcus aureus was cultured on agar medium containing different concentrations of erythromycin (0.09, 0.17, 0.35, 0.75, 1.5, 3, 6, 12, 24, 48 and 72 μg / mL). Several single colonies that grew on the highest concentration of erythromycin were selected and their minimum inhibitory concentration (MIC) of erythromycin was measured. These strains were then cultured on agar medium containing a higher concentration of erythromycin and again, colonies that grew on the highest concentration of erythromycin were selected. This process was repeated until the MIC of erythromycin for Staphylococcus aureus was greater than 16 μg / mL. Erythromycin-resistant Staphylococcus aureus was obtained. Figure 5 It was shown that after 59 days of resistance culture, the MIC of erythromycin increased to 16 μg / mL, indicating that Staphylococcus aureus had developed resistance.

[0045] Example 7:

[0046] Dodecanoic acid-modified dextran and stearic acid-modified dextran were prepared according to the method of Example 1, in which retinoic acid was replaced by dodecanoic acid and stearic acid, respectively. An ointment containing 1% erythromycin was prepared according to Example 5.

[0047] ​The KB paper disc method was used for the inhibition zone experiment. Filter paper discs with a diameter of approximately 5 mm were used. 0.05 g of erythromycin gel was spread onto the paper discs under sterile conditions for the inhibition zone experiment. Using the spread plate method, 0.25 mL of bacterial suspension (OD600 = 0.5) was added to each agar plate, spread evenly, and then the drug-treated and dried paper discs were placed in the plate. After incubation at 37℃ for 24 h, the diameter of the inhibition zone was measured using calipers. Table 2 shows the inhibition zone values ​​of erythromycin ointment prepared with different types of hydrophobic modified dextran. The results show that the inhibition zone diameter of both erythromycin ointment without hydrophobic modified dextran and erythromycin suspension was the same as that of the paper discs, both being 5 mm, indicating that neither had a significant inhibitory effect on erythromycin-resistant Staphylococcus aureus. After adding hydrophobically modified dextran, the inhibition zones of erythromycin ointment were all greater than 7 mm. Among them, the inhibition zone of dodecanoic acid modified dextran erythromycin ointment was 8 mm, the inhibition zone of stearic acid modified dextran erythromycin ointment was 9 mm, and the inhibition zone of retinoic acid modified dextran erythromycin ointment reached 14 mm.

[0048] Table 3 shows the MIC values ​​of erythromycin ointments prepared from different types of hydrophobically modified dextran against erythromycin-resistant Staphylococcus aureus. The results are consistent with the inhibition zone results. The above results also indicate that retinoic acid is the preferred hydrophobic modification for dextran.

[0049] For susceptible bacteria, the addition of retinoic acid-modified dextran erythromycin ointment increased the MIC value of erythromycin from 0.3 μg / mL to 0.6 μg / mL, possibly because the micelle system formed a physical barrier between erythromycin and bacteria.

[0050] Table 2. Inhibition zone values ​​of erythromycin ointments prepared from different types of hydrophobically modified dextran against erythromycin-resistant Staphylococcus aureus.

[0051]

[0052] Table 3. MIC values ​​of erythromycin ointments prepared from different types of hydrophobically modified dextran against erythromycin-resistant Staphylococcus aureus.

[0053]

[0054] Example 8:

[0055] The retinoic acid-modified dextran with different linkage ratios obtained in Example 4 were used to prepare erythromycin ointment according to Example 5, and their MIC values ​​against erythromycin were determined. Figure 6The MIC value of erythromycin-resistant S. aureus is the lowest when the ratio of retinoic acid to dextran monomer is 1:10.4. This is probably because the solubilization of erythromycin is poor when the amount of retinoic acid is too small, and the amount of erythromycin in the micelles is reduced when the amount of retinoic acid is too large, which reduces the interaction between erythromycin and bacteria. This phenomenon is more obvious in sensitive bacteria. The MIC value of the erythromycin gel prepared from the hydrophobically modified dextran with a ratio of 1:4.3 is the highest. The results show that the ratio of retinoic acid to dextran monomer is preferably 1:10.

[0056] Example 9:

[0057] Dextran with different molecular weights was used to prepare retinoic acid-modified dextran. Figure 7 The MIC value of the retinoic acid-modified dextran erythromycin ointment prepared from dextran with different molecular weights was determined. The results show that the MIC value of the retinoic acid-modified dextran erythromycin ointment prepared from dextran with a molecular weight of 1000 Da is the lowest for both sensitive and resistant bacteria. This is probably because the increase in the molecular weight of dextran increases the critical micelle concentration of the modified dextran micelles, which in turn affects the solubilization of erythromycin. The results show that the dextran disclosed in the present application preferably has a molecular weight of 1000 Da.

[0058] Example 10:

[0059] Figure 8 The MIC value of the erythromycin ointment prepared from different ratios of retinoic acid-modified dextran and erythromycin was determined. As shown in the figure, when the mass ratio of erythromycin to retinoic acid-modified dextran is in the range of 1:1-1:10, the MIC value of the erythromycin ointment is the smallest when the mass ratio is 1:2. The reason is probably that when the amount of hydrophobically modified dextran is too small, the solubilization of erythromycin is weakened, and when the amount of hydrophobically modified dextran is too large, the total amount of micelles increases, and the amount of erythromycin in a single micelle decreases. When the amount of micelles is constant, the amount of erythromycin in the micelles decreases. Therefore, the mass ratio of erythromycin to retinoic acid-modified dextran is preferably 1:2.

[0060] Example 11:

[0061] The retinoic acid-modified dextran erythromycin ointment prepared in Example 5 was used to induce erythromycin-resistant S. aureus according to the method described in Example 6. Figure 9 The results of the induction are shown in the figure. The results show that the production rate of erythromycin-resistant bacteria treated with the retinoic acid-modified dextran erythromycin ointment is significantly slower than that of the raw erythromycin, indicating that the retinoic acid-modified dextran erythromycin ointment disclosed in the present application can effectively reduce the production rate of erythromycin-resistant bacteria during the use of erythromycin.

[0062] Example 12:

[0063] FITC-labeled erythromycin was prepared by co-stirring FITC and erythromycin overnight. The FITC-labeled erythromycin ointment was prepared according to Example 5 by using retinoic acid-modified dextran. The ointment was added to the erythromycin-resistant S. aureus suspension, and after 2 h, the bacterial bodies were centrifuged and observed under a fluorescence microscope. Figure 10 It can be seen that compared with the FITC-labeled erythromycin ointment without the addition of retinoic acid-modified dextran, the addition of retinoic acid-modified dextran significantly improves the fluorescence signal of erythromycin in the bacteria, which may be due to the unique uptake affinity of bacteria for dextran as a carbon-based substance. Moreover, it is found that compared with dextran modified with dodecanoic acid or stearic acid, the dextran modified with retinoic acid has higher affinity for the bacteria.

[0064] 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, it should be understood by those skilled in the art 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, and they should be covered in the scope of the claims of the present application.

Claims

1. An erythromycin mucoadhesive soft gel for overcoming erythromycin multidrug resistance, characterized by: The erythromycin micelle ointment is composed of lipid-soluble acid-modified dextran, erythromycin, a hydrophilic gel matrix material, and water; wherein the lipid-soluble acid-modified dextran includes one or more of dodecanoic acid-modified dextran, stearic acid-modified dextran, and retinoic acid-modified dextran; and the grafting ratio of lipid-soluble acid to dextran monomer in the lipid-soluble acid-modified dextran is 1:18.3-1:4.

3.

2. The erythromycin micelle ointment for overcoming multidrug resistance of erythromycin according to claim 1, characterized in that: The grafting ratio of fat-soluble acid to dextran monomer is 1:

10.

3. The erythromycin mucoadhesive soft gel of claim 1 or 2, wherein the erythromycin is in a mucoadhesive soft gel form. The mass ratio of fat-soluble acid-modified dextran to erythromycin is 1:

2.

4. The erythromycin mucoadhesive soft gel of claim 1 or 2, wherein the erythromycin is in a mucoadhesive soft gel form. The molecular weight of dextran is 1000-20000 Da.

5. The method for preparing erythromycin micelle ointment to overcome multidrug resistance of erythromycin according to claim 1, characterized in that: The process consists of the following steps: dissolving fat-soluble acid-modified dextran in water, adding hot ethanol solution of erythromycin, stirring until homogeneous, and then adding sodium carboxymethyl cellulose to swell and dissolve.

6. The method for preparing erythromycin micelle ointment to overcome multidrug resistance of erythromycin according to claim 5, characterized in that: The fat-soluble acid-modified dextran is prepared by dissolving the fat-soluble acid in dimethyl sulfoxide, adding DCC and DMAP, stirring overnight, adding dextran, stirring at room temperature for 24 hours, dialyzing the reaction product, centrifuging, and freeze-drying the supernatant.

7. The use of the erythromycin micelle ointment according to claim 1 in the preparation of a drug for inhibiting erythromycin-resistant Staphylococcus aureus.

Images