KR20040028336A - Novel composite of thermosensitive antifungal gel for vaginal administration - Google Patents

Abstract

PURPOSE: Provided is a novel antimicrobial thermal sensitive gel composition for the through vagina administration, which is in a liquid state at ambient temperature while forming a gel in the vagina to be adhered to the vaginal mucosa, and easily makes the administration thereof while continuously exerting excellent medicinal effect. CONSTITUTION: An antimicrobial thermal sensitive gel composition for the through vagina administration comprises 30-35wt.% of poloxamer, 0.2-1wt.% of polycarbophil, and 0.9-1.1wt.% of clotrimazole. The pH of the composition is 3.5-4.5. The poloxamer is a mixture of poloxamer 188 and poloxamer 407 in a weight ratio of 1:1-1.5. The gelation temperature of the poloxamer is 25-35 deg.C.

Description

Novel composite of thermosensitive antifungal gel for vaginal administration

The present invention relates to a novel antimicrobial thermosensitive gel composition for intravaginal administration, and more particularly, to a composition that exhibits a continuous antimicrobial activity while maintaining a certain time at a site such as vagina for a temperature-sensitive gel having appropriate mucoadhesion and ease of administration. It is about.

Conventional intravaginal formulations have the disadvantage of discomfort during administration, and frequent administration by reducing the retention time of the drug administered by the physiological state according to the protective mechanism of the vagina to reduce the therapeutic effect.

Existing vaginal preparations used polyethyleneglycol or witepsol as bases, which are solid at room temperature and are solid solidifying agents that are dissolved or softened in the body to release active ingredients. Since solid vaginal suppositories can reduce side effects that may occur during systemic administration such as oral and injection administration, drugs such as antibacterial agents have been used for the purpose of topical administration for the treatment of vaginitis. However, since the solid vaginal preparation is solid at room temperature, the patient feels a foreign object and a sense of rejection during administration, and has a disadvantage in that it can be taken out of the body after administration. The situation is limited.

Therefore, in order to solve this problem, it has been expected to develop a new type of preparation composition called a temperature sensitive gel that is liquid at room temperature and forms a gel in the body.

Thermosensitive gels have been developed as a new type of suppository composition called liquid suppositories for rectal administration. This liquid suppository uses gels of 2 or more types of poloxamer as the base to have a gelation temperature of 30 to 36 ° C., and has appropriate gel strength and bioadhesive power by adding bioadhesive polymers such as carbopol, polycarbophil or sodium alginate. Intravitreal administration is easy, and after administration, it adheres to the rectal mucosa so that it does not come out of the body and does not migrate to the end of the colon (Choi et al., Int. J. Pharm., 165, 33-44 (1998); Choi et al., Int. J. Pharm., 190, 13-19 (1999); Choi et al., Int. J. Pharm., 165, 23-32 (1998). In addition, the liquid suppositories are easier to manufacture than conventional suppositories, such as not undergoing a melting step during preparation, and the bioavailability of these active ingredients is increased by applying active ingredients such as acetaminophen, propranolol, insulin and prostaglandin E1 to the liquid suppositories. (Korean Patent Application Publication Nos. 97-61240, 97-64596, PCT / KR97 / 0032, PCT / KR97 / 0042).

However, there is a problem in applying pH control as it is to apply the above technique to a vaginal suppository. In other words, if the pH is not controlled, infection is facilitated by increasing adhesion to the vaginal mucosa of Candida, and physiological pH may be disturbed considering that the physiological pH in the vagina is acidic.

Accordingly, the present invention improves the problems of conventional vaginal suppositories, and is liquid at room temperature and forms a gel in the vagina to adhere to the vaginal mucosa, which is easy to administer and adheres to the vaginal mucosa while maintaining the gel state after administration. Without feeling the discomfort in the state It is an object of the present invention to provide a novel antimicrobial thermosensitive gel composition for intravaginal administration that shows excellent efficacy by not leaving the body continuously and is safe from pH disturbances.

1 is a flow curve of the temperature sensitive gel composition of the present invention using a cone and plate viscometer.

Figure 2 shows the results for cytotoxicity according to the increase in the concentration of clotrimazole against vaginal epithelial cells.

Figure 3 shows the microscopic observation of vaginal mucosal tissue damage after intravaginal administration of the temperature sensitive gel composition of Example 4 according to the present invention and the commercial preparation of Comparative Example 1 and the control group.

The present invention relates to an antimicrobial thermosensitive gel composition for vaginal administration containing 30 to 35 parts by weight of poloxamer, 0.2 to 1 part by weight of polycarbophil, and 0.9 to 1.1 parts by weight of clotrimazole, and having a pH of 3.5 to 4.5.

When explaining in detail the respective components contained in the antimicrobial temperature-sensitive gel composition for vaginal administration according to the present invention.

Poloxamers contained as bases in the novel antimicrobial thermosensitive gel compositions for vaginal administration according to the present invention are known to not damage mucosa (Choi et al., Int. J. Pharm., 165, 23-32 ( Yun et al., IInt. J. Pharm., 189, 137-145 (1999). In the present invention, it is selected from commercially available poloxamers, and preferably a mixture of two kinds of poloxamer 188 and poloxamer 407 is used. It is preferable to contain 30-35 weight part of said poloxamer bases in the whole liquid suppository composition, and it is preferable that the gelatinization temperature of a poloxamer base maintains 25-35 degreeC range.

In order to maintain this gelation temperature, it is preferable to add poloxamer 188 and poloxamer 407 in a ratio of 1: 1 to 1.5.

If the gelation temperature of the poloxamer base is less than 25 ° C., the thermosensitive gel for vaginal administration at room temperature becomes a solid phase and is difficult to administer to the body, and if it exceeds 35 ° C., there is a possibility that the gel becomes liquefied from the body, so it may come out of the body.

The clotrimazole is preferably contained 0.9 to 1.1 parts by weight in the total temperature-sensitive gel composition, and can be expected to have an effective effect at the same composition ratio as commercial creams, and polycarbophil, which is a mucoadhesive polymer, is less than 0.2 parts by weight. If it contains, there is a problem that the mucoadhesiveness does not appear significantly, and if it exceeds 1 part by weight, it may show a very strong mucoadhesive force, which may cause damage to the vaginal mucosa.

The pH of the composition of the present invention is preferably 3.5 to 4.5, and when the pH is higher than 4.5, the solubility of clotrimazole is reduced to decrease the antimicrobial activity, so the effect is very decreased, and when the pH is lower than 3.5, It may cause damage.

The composition of the present invention may further comprise a pH adjuster to adjust the pH. Preferred pH adjusters include citric acid or sodium hydrogen phosphate, and when 0.7 to 0.8 parts by weight of citric acid and 0.8 to 0.9 parts by weight of sodium hydrogen phosphate are added, a preferred composition can be prepared.

The method for formulating an intravaginal thermosensitive gel composition according to the present invention may be prepared in a pharmaceutical form including conventional excipients, diluents, and the like by methods well known to those skilled in the art.

As described above, the liquid suppository composition for intravaginal administration according to the present invention has a gelation temperature of 25 to 35 ° C. of poloxamer base, and has a suitable gel strength and mucoadhesion strength using polycarbophil as a mucoadhesive polymer. Convenience and good efficacy can be shown continuously.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited by Examples.

Examples 1-6 and Comparative Example 1

Next, polycarbophil was dissolved and cooled in pH 4.0 buffer at room temperature with a composition as shown in Table 1, and then poloxamer was added at 4 ° C. The solution was left in the refrigerator for one day, and clotrimazole was dissolved in a mixture of ethanol and polyethylene glycol, and then dissolved in the poloxamer solution at 4 ° C. to prepare an antibacterial thermosensitive gel for intravaginal administration.

division Example Comparative example One 2 3 4 5 6 One Clotrimazole One One One One One One One Poloxamer P 188 15 15 15 15 15 15 - P 407 15 15 15 20 20 20 - Bio-Adhesive Polymer Polycarbophil 0.2 0.4 1.0 0.2 0.4 1.0 - ethanol 3 3 3 3 3 3 - PEG 4000 5 5 5 5 5 5 99 Citric acid 0.78 0.78 0.77 0.78 0.78 0.77 0 Sodium hydrogen phosphate 0.88 0.88 0.87 0.88 0.88 0.87 0 Purified water q.s q.s q.s q.s q.s q.s q.s Sum 100 100 100 100 100 100 100

(Unit: g)

Test Example 1: Antimicrobial Activity Test

Clotrimazole was dissolved in ethanol and then diluted with 0.1 M citric acid-phosphate buffer at various pHs. An antibacterial activity test plate, SDA medium (sabouraud dextrose agar medium) was prepared and inoculated with 5 ml of SDA medium containing Candida albicans ATCC 10231 and then solidified. The results of measuring the diameter of the inhibitory zone (Inhibition zone), which is obtained by dispensing and incubating various concentrations of clotrimazole solution in each medium, are shown in the following table.

water pH 4.0 pH 5.0 pH 6.0 pH 7.0 Concentration (㎍ / ml) 100 50 100 50 100 50 100 50 100 50 Restraining ring (mm) 15.7 13.4 19.9 16.3 18.3 13.3 15.4 9.4 13.7 12.3 13.8 13.2 20.9 14.6 17.5 13.4 14.4 11.0 14.8 12.4 13.8 9.3 18.9 15.9 17.6 13.4 14.7 10.5 13.9 12.5 13.6 12.2 18.9 15.4 18.4 13.3 15.2 11.2 14.4 12.3 Average 14.23 ± 0.99 12.03 ± 1.89 19.65 ± 0.96 15.55 ± 0.73 17.95 ± 0.47 13.35 ± 0.06 14.93 ± 0.46 10.53 ± 0.81 14.20 ± 0.50 12.38 ± 0.10 Comparative activity with water (%) 100.0 251.1 160.2 87.5 86.1

Comparative activity with water (%) in Table 2 above was calculated by the following equation.

Comparative activity = A x 100%

Log A = IV / W

I = log 100/50

V = (sum of inhibitory rings at concentrations of 100 μg / mL at each pH + sum of inhibitory rings at concentrations of 50 μg / mL at each pH)-(sum of inhibitory rings at concentrations of 100 μg / mL in water) Is the sum of the inhibitory rings when is 50 μg / mL)

W = (sum of inhibitory rings at concentrations of 100 μg / mL at each pH + sum of inhibitory rings at concentrations of 100 μg / mL at water)-(sum of inhibitory rings at concentrations of 50 μg / mL at each pH + concentration in water Sum of inhibitory rings at 50 μg / mL) (antibiotic drug titer 652-653, 1999).

As shown in Table 2, when the clotrimazole concentration was 100 µg / ml, the antimicrobial activity was the highest at pH 4.0 and decreased when the pH value was increased. Similarly, at 50 μg / ml of clotrimazole, the inhibitory ring had the largest diameter at pH 4.0. The lower the pH, the higher the antibacterial activity of clotrimazole, so the pH of the temperature sensitive gel was adjusted to 4.0. Candida albicans also adheres to the vaginal mucosa with a significantly higher affinity at pH 6.0 than at pH 4.0. Therefore, by adjusting the pH of the preparation to 4.0 it is possible to inhibit the growth of the infection site by reducing the adhesion of fungi. In addition, the acid preparation can help to restore the physiological pH in the vagina, so the preparation is prepared at pH 4.0.

Test Example 2: Ease of Administration and Mucoadhesion

The thermosensitive gel composition of Examples 1 to 6 prepared above was prepared in a mucosal membrane in which the formulation was fixed to a texture analyzer probe in an adhesion mode using a TA-XT 2 texture analyzer. The force required to drop was made mucoadhesive, and the force needed to push the agent out of the syringe in compression mode was filled with the formulation in a 1 ml syringe, and the result was made easy. Table 3 shows.

division Example One 2 3 4 5 6 Ease of administration (dynemm) 443.38 ± 6.42 501.76 ± 11.10 593.77 ± 19.24 543.87 ± 7.92 569.71 ± 11.08 702.41 ± 6.75 Mucoadhesiveness (dynemm) 23.8 ± 1.55 27.0 ± 3.15 45.3 ± 4.28 24.9 ± 1.03 45.3 ± 2.26 104.1 ± 13.2

The mucoadhesive strength of the temperature sensitive gel is an important factor in increasing the retention time of the antimicrobial agent in the vaginal mucosa. On the other hand, ease of administration, which is easy to administer the formulation in a syringe, is a factor that must be considered simultaneously for intravaginal administration of the temperature-sensitive gel in the liquid state.

As shown in Table 3, when the amount of poloxamer is constant, the greater the amount of polycarbophil, the greater the amount of poloxamer, when the amount of polycarbophil is constant, the stronger the mucoadhesive but low ease of administration Therefore, the appropriate composition ratio should be determined. Therefore, in view of the above results, polycarbophil 0.2% -containing preparations (Examples 1 and 4) were used in the following experiments.

Test Example 3: Steady Shear Viscosity

The flow curve of the thermosensitive gel was measured in a controlled rate mode using a cone and plate rheometer. The viscosity was measured at a shear rate of 0.1 s ⁻¹ to 100 s ⁻¹ , and the effect of temperature at 20.0 and 37.2 ° C. is shown in FIG. 1.

As can be seen in Figure 1 at 20.0 ° C the Newtonian behaviour (Newtonian behaviour) because the temperature sensitive gel composition of Examples 1, 4 did not show a large change in viscosity according to a range of shear rate (shear rate) In contrast to the liquid state shown, but at 37.2 ° C, the shear thinning effect is significant at 37.2 ° C, resulting in gel formation of poloxamers, polycarbophiles, and other components in the formulation as the temperature increases. It can be seen that it forms a non-Newtonian behavior. In this case, the thinning of the layer is shown to be related to the spreadability of the gel, and in the case of Example 4, it is judged to exhibit high spreadability and coating effect in the vagina during intravaginal administration.

Test Example 4: Gelation Temperature

The temperature sensitivity of the gel can be assessed by the sol / gel transition temperature and the sol / gel transition temperature is measured by the rapid change of the elastic modulus. Using a corn and plate rheometer, increase the temperature by 1 ° C per minute while gelling temperature between G '(elastic coefficient) in sol state and G' (elastic coefficient) in gel state It was set as. The gelation temperature was 27.6 ° C. in the formulation containing 20 parts by weight of poloxamer 407 and 32.0 ° C. in the formulation containing 15 parts by weight, and both formulations appeared to exhibit an appropriate gelation temperature, but were tested to maintain the gel structure even in the vagina.

Test Example 5: Gelation Time

In order to measure the gelation time of the temperature sensitive gel at 37.2 ℃, the degree of change of the modulus of elasticity was observed. The gelation time was defined as the time when the elasticity modulus was greater than the viscosity modulus. At this time, the formulation containing 20 parts by weight of poloxamer 407 was 35 seconds, but the formulation containing 15 parts by weight was 72 seconds. It took longer.

Test Example 6 Viscoelasticity of Temperature Sensitive Gel

In vivo, the formulation administered intravaginally is diluted with vaginal secretions. It is known that the dose of vaginal secretion is 0.75 ml and the liquid formulation will be administered in about 1 to 3 ml, so that 3 ml of the temperature sensitive gel of Examples 1 and 4 and 0.75 ml of the vaginal secretion are mixed to make the vaginal situation similar. The change of elastic modulus with dilution was observed. Dilution with vaginal secretions reduced the elasticity of the two formulations to different degrees. Example 4 reduced about 10 times except for the lowest range, but in Example 1 a decrease of more than 1000 times. Depending on the vaginal secretion and dilution, the elastic modulus of Example 1 was lower than the limit that can be measured in the frequency range of 0.1 ~ 2.3 Hz. In Example 1, the dilution with the vaginal secretion showed a large change according to the frequency, but in Example 4, the elastic modulus was kept constant. Dynamic mechanical analysis observes whether the viscoelastic properties of the two formulations are frequency dependent, thus changing the frequency, resulting in elasticity at all frequency ranges except for the lowest frequency (0.1 Hz). The coefficients were higher than the viscous coefficients and appeared almost linear according to the frequency change. This means that Example 4 is gel at 37.2 ° C. The modulus of elasticity of Example 4 was about 10 times higher than that of Example 1, indicating that the elasticity was higher at 37.2 ° C. Compared to Example 4, Example 1 showed a stronger frequency dependence. In other words, in Example 4, it was found that the gel state was maintained even during dilution.

As discussed above, the study of viscoelasticity in preparations based on biopolymers may be basic information for predicting the state and maintenance in vivo through large changes according to vaginal secretion and dilution.

Test Example 7: In vivo ( in vivo) Antimicrobial Activity in

After ovarian ablation, 7 days of recovery period, estradiol benzoate was administered 25 mg / kg every 2 days to maintain psedoestrus. Candida albicans is suspended in physiological saline at 10 ⁸ cfu / ml and then vaginally infected. Two days after infection, Example 4 and Comparative Example 1 were administered at a dose of 1.0 g / kg using an oral solution. At 0, 1, 2, 4, 7, and 10 days after administration, 100 μL of physiological saline is flushed with vagina to obtain vaginal endocrine solution. Vaginal endocrine solution was inoculated in sabouraud dextrose agar and incubated at 37 ° C. for 48 hours to measure the number of bacteria, and the results are shown in Table 4.

Changes in the number of bacteria in vaginal secretions Administration group (n = 5) 0 days after dosing 1 day after dosing 2 days after dosing 4 days after dosing 7 days after dosing 10 days after dosing Control 5.2117 ± 1.543 4.3633 ± 0.6268 4.5333 ± 0.8976 4.7850 ± 0.8117 4.8167 ± 0.6083 4.6983 ± 0.5886 Example 4 4.3900 ± 0.5577 4.7740 ± 0.4101 3.5660 ± 2.1522 1.2740 * ± 1.7451 1.2320 * ± 1.6871 0.0000 * ± 0.0000 Comparative Example 1 3.8633 ± 0.8677 3.5175 ± 0.3523 3.2950 ± 0.2892 2.3675 ± 1.7962 3.5575 ± 2.3726 4.8175 ± 0.4205

(* P <0.05: significant at 0.05 with water as control)

As shown in Table 4, the temperature-sensitive gel composition of Example 4 according to the present invention continued to decrease the number of bacteria until 10 days, the formulation based on the PEG of Comparative Example 1 was reduced until 4 days 7 From day it was found that the number of bacteria increased and became re-fungal infection. From these results, it can be seen that the composition of the present invention can effectively treat the vaginal candidiasis while reducing the frequency of administration according to the continuous antimicrobial activity.

Test Example 8: Cytotoxicity Test

The temperature sensitive gel composition of Example 4 exhibited higher and lasting antimicrobial activity, confirming safety in cells and vaginal mucosal tissues. Human cervical epithelial cells (ME-180) were added to a 24-well well plate using a 5-volume McCoy 5 a medium supplemented with 10 doses of heat-inactivated fetal bovine serum. Incubated in a 5% CO2 incubator at 0.4 μm thickness. The thermosensitive gel composition of Example 4, a commercial formulation based on PEG of Comparative Example 1, and clotrimazole solution were exposed to the cell surface, and then incubated at 37 ° C. for 4 hours, followed by washing with buffered saline buffer to remove the preparation. It was. 500 μl of fresh medium and 50 μl of 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide solution (5 mg / ml) were added to each well and placed in a 37 ° C. CO ₂ incubator. Insoluble purple formazan produced by time incubation was dissolved with acidic alcohol. The absorbance of each cell was measured at 570 nm and the results for cytotoxicity against the concentration of clotrimazole are shown in FIG. 2.

As shown in FIG. 2, clotrimazole increased cytotoxicity in a dose-dependent manner, while temperature sensitive gel compositions decreased cytotoxicity. Therefore, it is thought that the administration of the thermosensitive gel may reduce the cytotoxicity which may occur due to continuous exposure to high concentrations following the continuous release of clotrimazole.

No damage to the vaginal mucosa was observed in the thermosensitive gel. After intravaginal administration using the liquid suppository composition of Example 4 and the preparation based on the PEG of Comparative Example 1, the mucosal mucosa of the rat was separated and fixed in a 10 parts by weight carbonate buffer-formalin solution. Again embedding in paraffin using an embedding center to thin slices. The flakes were stained with Haematoxylin-eosin and vaginal mucosal cells were observed under a microscope, and the results are shown in FIG. 3.

As shown in FIG. 3, the thermosensitive gel composition (A) of Example 4 showed no damage to the vaginal mucosa cells as in the control group (C) which was not treated with the active ingredient, but based on the PEG of Comparative Example 1 (B) In one formulation, the loss of mucosal cells was observed, and thus the damage of the mucosal cells was observed. Thus, it was confirmed that the thermosensitive gel composition according to the present invention secured safety against the vaginal mucosa.

The present invention is liquid at room temperature and has a property of forming a gel in the vagina to adhere to the vaginal mucosa. It is possible to provide a novel antimicrobial thermosensitive gel composition for vaginal administration that continuously shows excellent drug efficacy and ensures safety in pH perturbation.

Claims (7)

An antimicrobial thermosensitive gel composition for vaginal administration comprising 30 to 35 parts by weight of poloxamer, 0.2 to 1 part by weight of polycarbophil, and 0.9 to 1.1 parts by weight of clotrimazole, and having a pH of 3.5 to 4.5. The antimicrobial thermosensitive gel composition for vaginal administration according to claim 1, wherein the poloxamer is a mixture of poloxamer 188 and poloxamer 407. The antimicrobial thermosensitive gel composition for vaginal administration according to claim 2, wherein the weight ratio of poloxamer 188 and poloxamer 407 is 1: 1 to 1.5. The antimicrobial thermosensitive gel composition for vaginal administration according to claim 1, wherein the poloxamer gelation temperature is 25 to 35 ° C. The antimicrobial thermosensitive gel composition for vaginal administration according to claim 1, further comprising a pH adjusting agent. 6. The antimicrobial thermosensitive gel composition for vaginal administration according to claim 5, wherein the pH adjusting agent is citric acid and sodium hydrogen phosphate. The antimicrobial thermosensitive gel composition for vaginal administration according to claim 6, wherein citric acid and sodium hydrogen phosphate are added in an amount of 0.7 to 0.8 parts by weight and 0.8 to 0.9 parts by weight, respectively, based on the whole composition.