KR100466247B1 - Preparation Method for Amphotericin B Microemulsion - Google Patents

Abstract

The present invention relates to a method for producing a microemulsion containing amphotericin B, and also to an intravenous injection containing a microemulsion containing amphotericin B prepared therefrom as an active ingredient.

It is an object of the present invention to solubilize amphotericin B using vitamin C, which enables filtration and foreign body testing by bacterial filtration and promotes safety in the human body, and also controls the systemic distribution rate by adding plasma thickener. The present invention provides a method for preparing a poterincin B-containing microemulsion.

The method for producing a microemulsion containing amphotericin B according to the present invention comprises the steps of dissolving vitamin C and amphotericin B in the oil phase and preparing a microemulsion containing amphotericin B as known. Dissolving C and dispersing the oil phase in which the amphotericin B was dissolved in the aqueous phase in which vitamin C was dissolved.

Description

Preparation method of micro emulsion containing amphotericin {{Preparation Method for Amphotericin B Microemulsion}

The present invention relates to a method for producing a microemulsion containing amphotericin B, and also to an intravenous injection containing a microemulsion containing amphotericin B prepared therefrom as an active ingredient.

Ampotericin B is a type of antifungal polyene antibiotic obtained from Streptomyces nodosus M4575. , 18S *, 19E, 21E, 23E, 25E, 27E, 29E, 31E, 33R *, 35S *, 36R *, 37S,)]-33-[(3-amino-3,6-dideoxy-.beta.- D-mannopyranosyl) oxy] 1,3,5,6,9,11,17,37-octahydroxy-15,16,18-trimethyl-13-oxo-14,39-dioxabicyclo- [33.3.1] nonatriaconta-19 It is named 21,23,25,27,29,31-heptaene-36-carboxylic acid.

While amphotericin B is a potent antifungal agent, it does not affect bacteria, rickettsiae, and viruses. The antibiotic activity of amphotericin binds to the steroids of the cell membrane and forms pores in the cell membrane. Ampotericin acts selectively on fungal cells by binding more strongly to ergosterol, the main sterol of fungi, than to mammalian's stain cholesterol. But it's not specific, which is the cause of the side effects.

Ampoterisine B is poorly soluble in water and alcohol, and a known product of amphotericin B is Bristol-Myers Squibb, which is formulated with sodium deoxycholate, a natural surfactant to solubilize empotericin ( EPA-A-202 837). While it is effective against Candida, it has side effects and particularly toxicity to the kidneys (Reynolds et al (1963), Med. Clin. North American 47 1149-1154).

Another method is to formulate liposomes for amphotericin (Mehta et al (1984), Biochem. Biophys. Acta 770 230-234). While liposomes have clinical advantages, they are usually difficult and unstable to manufacture in large quantities.

European Patent (EP-A-211258, Abbott) has formulated a microemulsion of amphotericin. Microemulsions are generally defined as a system comprising a lipophilic and hydrophilic component. Microemulsion solutions have clear or transparent properties. Transparency and particle size characteristics distinguish emulsion and microemulsion. The smaller the particle size of the microemulsion, the longer it stays in the blood than the emulsion. Many microemulsions, however, are temperature sensitive and unstable at conditions other than room temperature. Therefore, there is a need for the development of drug carriers that can be stored at various temperatures for a long time.

Generally, amphotericin is rarely soluble in water, but is soluble in acidic or alkaline. US Pat. Nos. 5,389,373 and 5,965,156 prepare microemulsions by dissolving amphotericin by adding hydrochloric acid or sodium hydroxide. However, they may form salts in acids and alkalis in emulsions, for example, when salts and hydrochloric acid are used to form salts to destabilize the emulsion. In particular, when formulated with hydrochloric acid, the content of amphotericin in the manufacturing process is about 50% is lost.

The present inventors studied a method of formulating amphotericin B as a microemulsion to overcome the toxicity and instability of a drug that has been a problem in the conventional injection of amphotericin B. As a result, by incorporating vitamin C in each of the oil phase and the aqueous phase, The present invention has been accomplished by overcoming the shortcomings of amphotericin B insoluble in water to form a stable microemulsion.

Therefore, an object of the present invention is solubilization of amphotericin B using vitamin C, which enables filtration and foreign body testing by bacterial filtration and promotes safety in the human body, and also controls the systemic distribution rate by adding plasma thickener. The present invention provides a method for preparing amphotericin B-containing microemulsion.

1 is a graph showing the stability at 20 ° C. of a microemulsion formulation containing amphotericin B.

FIG. 2 is a graph showing the stability at 40 ° C. of a microemulsion formulation containing amphotericin B. FIG.

Figure 3 is a graph showing the blood concentration curve of the microemulsion formulation containing the conventional formulation and amphotericin B.

The method for producing a microemulsion containing amphotericin B according to the present invention comprises the steps of dissolving vitamin C and amphotericin B in the oil phase and preparing a microemulsion containing amphotericin B as known. Dissolving C and dispersing the oil phase in which the amphotericin B was dissolved in the aqueous phase in which vitamin C was dissolved.

In the present invention, the oil phase used to prepare the microemulsion may be one or more organic solvents selected from ethyl oleate, propylene glycol, dimethylacetamide, and glycofurol. Can be.

The amount of amphotericin B contained in the oil phase may be used in an amount of 0.1 to 10 parts by weight based on 100 parts by weight of the oil phase, and the content of amphotericin B is less than 0.1 part by weight based on 100 parts by weight of the oil phase. There is a small problem, the use of more than 10 parts by weight has a problem in the stability of the microemulsion in the present invention, the content of amphotericin B is preferably used 0.1 to 10 parts by weight based on 100 parts by weight of the oil phase and the microemulsion of the present invention In the preparation, more preferably from 0.5 to 2 parts by weight is used.

In the present invention, the content of vitamin C contained in the oil phase may be used in 1 to 50 parts by weight based on 100 parts by weight of the oil phase, and the content of vitamin C in the aqueous phase may be used in 1 to 50 parts by weight based on 100 parts by weight of the aqueous phase. . If the content of vitamin C is less than 1 part by weight based on 100 parts by weight of the oil phase, amphotericin B does not dissolve. If the content is more than 50 parts by weight, there is a problem in the solubility of the vitamin C and the stability of the microemulsion. The amount of vitamin C contained in the oil phase is preferably used in an amount of 1 to 50 parts by weight based on 100 parts by weight of the oil phase, and more preferably 5 to 15 parts by weight in the preparation of the microemulsion of the present invention.

When dissolving vitamin C and amphotericin B in the oil phase, the temperature may be carried out at 60 to 80 ° C. However, if the temperature is less than 60 ° C., amphotericin B does not dissolve. In the present invention, when dissolving the vitamin C and amphotericin B in the oil phase in the present invention, the temperature is preferably performed at 60 to 80 ° C., and more preferably 65 to 70 ° C. in the preparation of the microemulsion of the present invention. It is good to carry out.

In the present invention, 1 to 20 parts by weight of any one or more sugars selected from dextrose, trehalose, solutol, and mannitol may be used in the oil and water phases for the stabilization of the microemulsion, with respect to 100 parts by weight of the oil or water phase, but less than 1 part by weight. Or more than 20 parts by weight is not suitable for the purpose of using the sugar in the present invention.

The microemulsion containing amphotericin B prepared according to the present invention can be sterilized by filtration through a 0.45 μm sterile filter, wherein the average particle size of the filtered micro emulsion particles is about 200 nm or less.

In addition, the microemulsion containing amphotericin B prepared according to the present invention can be freeze-dried to be powdered to improve the stability and storage of the microemulsion, so that it can be stored and distributed for a long time. Suspension in water for injection or 5% glucose solution is stable for at least 1 week at room temperature of 20 ° C.

On the other hand, the present invention can provide an intravenous injection comprising a microemulsion containing amphotericin B prepared using the above-mentioned method as an active ingredient.

Hereinafter, the present invention will be described by the following examples and test examples. However, these are only examples of the present invention and they do not limit the scope of the present invention.

Example 1 Ampoterisin B Micro Emulsion (Type A)

After dissolving 200 mg of vitamin C in 1 ml of N, N-dimethylacetamide (Sonication) and dissolving 50 mg of amphotericin B in this solution, glycol furol (Glycofurol) 1 ㎖ was added, mixed, dissolved, and then 200 mg of solutol and 200 μl of ethyl oleate were sequentially added to prepare an oil phase. The reaction temperature was performed at 65 degreeC at this time.

The aqueous phase was dissolved by adding 1 g of dextrose, 1 g of trehalose, and 200 mg of vitamin C to 4 ml of distilled water at 65 ° C., cooling to 30 ° C., and then adding 200 mg of solutol to prepare a transparent aqueous phase.

The aqueous phase was sonicated with 0.1 ml of the oil phase prepared at 65 ° C. When a clear microemulsion was formed after sonication, a mixed solution containing 5% dextrose and 10% trehalose in a 1: 1 ratio was added to calibrate the total volume to 10 ml to prepare amphotericin B microemulsion (type A).

Example 2 amphotericin B microemulsion (type B)

400 mg of vitamin C was solubilized in 0.3 ml of N, N-dimethylacetaamide, and 50 mg of amphotericin B was dissolved therein. 3 ml of glycolfurol was added to the solution, mixed, dissolved, and then dissolved in 200 mg of solutol. 100 μl of ethyl oleate was added sequentially to prepare an oil phase. The reaction temperature was performed at 65 degreeC at this time.

The aqueous phase was dissolved in 4 ml of 65 ° C. distilled water by adding 0.5 g of dextrose, 1 g of trehalose, and 200 mg of vitamin C, cooled to 30 ° C., and 200 mg of solutol was added to prepare a transparent aqueous phase.

The aqueous phase was sonicated with 0.1 ml of the oil phase prepared at 65 ° C. When a clear microemulsion was formed after sonication, a mixed solution of 5% dextrose and 10% trehalose was added 1: 1 to adjust the total volume to 10 ml, thereby preparing amphotericin B microemulsion (type B).

Example 3 Ampoterisin B Micro Emulsion (Type C)

After dissolving 400 mg of vitamin C in a mixture of 3 ml of glycofural and 1 ml of propylene glycol, and dissolving 50 mg of amphotericin B, 100 μl of ethyl oleate was added thereto to prepare an oil phase. The reaction temperature was performed at 65 degreeC at this time.

The aqueous phase was dissolved in 3.5 ml of distilled water at 65 ° C. by adding 0.5 g of dextrose, 0.8 g of trehalose, and 200 mg of vitamin C, cooling to 30 ° C., and then adding 200 mg of solutol to prepare a transparent aqueous phase.

The aqueous phase was sonicated with 0.1 ml of the oil phase prepared at 65 ° C. When a clear microemulsion was formed after sonication, a mixed solution of 5% dextrose and 10% trehalose was added 1: 1 to adjust the total volume to 10 ml, thereby preparing amphotericin B microemulsion (type C).

Example 4 Powder Formulation of Ampoterisin B Microemulsion

15 ml of distilled water was added to 5 ml of the microemulsion type A, B, and C prepared according to Examples 1 to 3, and 2 g of mannitol was added to dissolve, followed by lyophilization and powdering.

<Test Example 1> particle size measurement of amphotericin B microemulsion

Type A, Type B, Type C microemulsions prepared in Examples 1 to 3 using a light scattering technique and type A, type B, type C emulsions prepared in Example 4 The diffusion coefficient and particle size of the particles were confirmed by tracking the change of scattered light intensity over time for the freeze-dried product and calculating the particle motion inversely. The sample solution was prepared by diluting the stock solution of the microemulsion of type A, B, and C by 30 times, and the lyophilized product of the type A, B, and C microemulsion by diluting with 10 times the water for injection, and measuring the mean squared displacement (MSD) scale ( the average value was recorded three times.

The particle size of three types (type A, type B, type C) prepared in Examples 1 to 3 and the existing formulations AmBisome and Fungizone using the method described above Zeta potential was measured and the results are shown in Table 1 below.

As shown in Table 1, the particle size of the microemulsion formulation was similar to that of the A-type preparation, 274 nm, and the B-type preparation was 280 nm, and the C-type preparation showed a very small particle size of 179 nm. However, the particle size after lyophilization was the opposite: Type A formulation was 143 nm, Form B formulation was 184 nm, and Form C formulation was 233 nm. However, when the maximum deviation was observed, Form C formulation showed uniform particle distribution. It can be seen that.

Table 1. Particle size and zeta potential of the microemulsion, aambisome and smoke zone of the present invention.

Particle size (nm) Zeta potential (mV) Before lyophilization After lyophilization Before lyophilization After lyophilization A type 274/702 (max) 143/666 (max) 20.22 0.75 Type B 280/1427 (max) 184/2256 (max) 12.53 0.29 C type 179/498 (max) 233/1013 (max) 6.84 2.23 AmBisome - 107/133 (max) - -9.47 Fungizone - 62/88 (max) - -31.81

<Test Example 2> Turbidity measurement of amphotericin B micro emulsion

1 ml of each of the type A, B, and C microemulsions prepared in Examples 1 to 3 were diluted in 9 ml of 5% aqueous textrose solution and 9 ml of 5% textose pH 7.4 buffer, followed by 1 hour and After 24 hours, the absorbance was measured at 540 nm to evaluate the turbidity of the microemulsion.

To examine the degree of turbidity and changes over time in the solutions of type A, type B and type C, dilute three samples of each preparation 10 times in 5% glucose solution. It was 0.12, but the turbidity after 24 hours was 0.11, type B was 0.1 after 1 hour, 0.09 after 24 hours, type 0.07 after 1 hour, and 0.06 after 24 hours. In a mixture of pH 7.4 buffer in glucose, Type A was 0.34 at 24 hours after 0.35 hours, 0.34 at 24 hours, 0.26 at 24 hours, 0.25 at 24 hours, C was 0.24 at 1 hour, and 0.26 at 24 hours. The turbidity increased as the liquidity of the buffer slightly increased, but the change over time was not significant.

Test Example 3 Stability of Ampoterisin B Micro Emulsion

3 ml of each of the A, B and C microemulsions prepared in Examples 1 to 3 were filled in a vial and then replaced with a nitrogen gas and sealed, followed by 2 days, 4 days at 20 ° C and 40 ° C. Changes over time were measured by measuring the residual amount of the drug for 6 days, 8 days, 10 days by HPLC.

As a sample, 0.2 ml of each of A, B and C microemulsions were added, 0.9 ml of methanol and 0.9 ml of dimethylsulfoxide (DMSO) were added to dissolve the mixture, and 0.9 ml of methanol and 0.9 ml of DMSO were added to 0.2 ml of the solution. The diluted solution was quantified by HPLC (Shimadzu LC-10AD). In this case, a reverse phase column (μBondapak C18 3.9x300mm) was used as a column, and the mobile phase was separated using acetonitrile mixed with 0.01 M ammonium acetate buffer at a volume ratio of 50:50, and signal collection was performed using UV-vis. It was measured at 383 nm using an absorption spectrophotometer.

Stability experiments of Type A, Type B and Type C microemulsions showed that Formulation A was relatively stable at 20 ° C. for 10 days, while Type B and Type C were reduced by 30% (see FIG. 1). The results of experiments at 40 ° C showed that after 10 days, the residual ratio was reduced to 62% for type A, 44% for type B, and 54% for type C (see Figure 2).

Test Example 4 Antifungal Activity of Ampoterisin B Microemulsion

In order to compare the antimicrobial activity of the preparations (type A, B, C) prepared by the method of the present invention with conventional formulations, Candida albicans (KCTC 1940), one of the fungi, dextrose broth medium ( sabouraud dextrose broth) for 24 hours. The prepared preparation was inoculated with the strain to 10 ⁴ cfu in agar plate to which the preparation diluted two-fold serially from 40 µg / ml with amphotericin B, 20 µg / ml, 10 µg / ml, etc. , 24 hours of incubation at 37 ℃ by visual observation to determine the minimum growth inhibitory concentration (MIC).

All of the antimicrobial test results showed similar antimicrobial activity at the initial concentration. The MICs of each microemulsion were similar in type A and B with 0.5 μg / ml, type C with 1.0 μg / ml and fumig zone 0.5 μg / ml.

Test Example 5 Toxicity of Ampoterisin B Microemulsion

Using SD rats, microemulsions of type A, type B, type C and fuming zone containing amphotericin B were suspended in phosphate buffer pH 7.4 solution in the amount of 0.2 ml in the tail vein in 10-20 animals per drug concentration. The number of deaths after administration and elapsed time after dosing for 14 days by administration was observed and the results are shown in Tables 2 to 5 below.

Table 2.

DEFINED LD50 STUDY: AMPHOTERICIN B- microemulsion preparation-A SD rats male DOSE (mg / kg) NO. DEAD / NO. DOSED NUMBER OF DEATHS DAYS AFTER DOSING 0 One 2 3 4 5 6 7 8 9 10 11 12 13 14 One 0/5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 0/5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5 1/5 One 0 0 0 0 0 0 0 0 0 0 0 0 0 0 7 5/5 5 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Statistical analysis-Probit test

LD50; 5.3346 ± .4660 mg

Table 3.

DEFINED LD50 STUDY: AMPHOTERICIN B- microemulsion preparation-A SD rats male DOSE (mg / kg) NO. DEAD / NO. DOSED NUMBER OF DEATHS DAYS AFTER DOSING 0 One 2 3 4 5 6 7 8 9 10 11 12 13 14 One 0/5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 0/5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5 1/5 One 0 0 0 0 0 0 0 0 0 0 0 0 0 0 7 4/5 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Statistical analysis-Probit test

LD50; 5.9175 ± .5312 mg

Table 4.

DEFINED LD50 STUDY: AMPHOTERICIN B- microemulsion preparation-C SD rats male DOSE (mg / kg) NO. DEAD / NO. DOSED NUMBER OF DEATHS DAYS AFTER DOSING 0 One 2 3 4 5 6 7 8 9 10 11 12 13 14 One 0/5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 0/5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5 1/5 One 0 0 0 0 0 0 0 0 0 0 0 0 0 0 7 4/5 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Statistical analysis-Probit test

LD50; 6.8406 ± .3826 mg

Table 5. Death of SD rats after intravenous injection of fumigone alone

Mg AB / kgbody weight SD rat Rasts used (n) Rats that died (n) One 5 One 2 6 4 3 5 One 4 5 4 5 5 5 6 4 4

Reference: Lucian Marzzullo, et al ,; Environmental Toxicology and Pharmacology 2 (1996) 31-34

On the other hand, in the A-, B- and C-type microemulsion and smoke zone containing amphotericin B of the present invention, LD50 (the amount of drug that kills 50% of test animals with 50% Lethal Dose) is a smoke zone. Compared to 3.5 mg / kg, type A microemulsion was 8.2 mg / kg, type B microemulsion was 7.1 mg / kg, and type C microemulsion was 9.1 mg / kg.

Experimental Example 6 Measurement of Blood Concentration During Intravenous Administration of Ampoterisin B Microemulsion

Anesthesia was performed with urethane (urethane) on the tail vein in groups of five using 250 to 300 g of male Sprague Dawley rats, and then prepared in Examples 1 to 3, followed by microemulsions of A, B and C. 2mg / Kg was injected intravenously in the existing preparations, such as albino and Hungi, and then blood collection was performed by taking 1, 3, 5, 10, 30, 60, 90 and 120 minutes with 200 μl of blood. Into a ㎖ microtube and centrifuged for 10 minutes at 3000 rpm and 100μl of plasma was correctly taken.

150 μl of methanol was added to the plasma to remove the mixed protein, followed by centrifugation at 12000 rpm for 10 minutes, and the supernatant was measured for blood drug concentration using HPLC. In this case, an inverted column (XTerra tm RP 185 μm, 4.6 × 250 mm) was used as a column, and the mobile phase was separated by mixing acetonitrile with 0.1 M ammonium acetate buffer at a volume ratio of 40:60, and signal collection was performed at 361 nm. Fluorescence detector with emission 464 nm was used.

The mean concentrations of blood concentrations and the changes in mean concentrations of blood concentrations of five experimental animals treated with microemulsions of type A, type B and type C, and ammonia and a fusione of conventional formulations were measured graphically, and the results are shown in FIG. 3. Shown in Comparing the graph forms of FIG. 3, all the group A, B and C microemulsions and the experimental groups treated with the existing preparations, such as Ambosomes and Hungi, showed similar curves and the pharmacokinetic parameters using the same are shown in Table 6. It was. In terms of the pharmacokinetic parameters of each experimental group, there was no significant difference and it was judged that the blood drug concentration after intravenous administration showed almost the same change over time.

Table 6. Pharmacokinetic Parameters of Microemulsions Containing Amphotericin B

Kinetic paramaters Micro emulsion Ambisome Hungi Zone A type Type B C type CL / F (ml / min / kg) 2.306 1.11 1.10 2.44 2.59 AUC (μg ^* min / ml) 867 1911 1825 819 770 MRT (min) 62.47 73.1 49.48 52.96 184 Cmax (μg / ml) 58.5 120 155 85.9 52.5 Tmax (min) One One One One One

The present invention is to prepare a drug containing amphotericin B by solubilizing using vitamin C as shown in the results of the above Examples and Test Examples when formulating a drug that is poorly soluble or acid-soluble, such as amphotericin B into an emulsion It can increase stability and safety.

Claims (8)

In preparing an emulsion containing known amphotericin B, Dissolving vitamin C and amphotericin B in the oil phase, Dissolving vitamin C in the water phase, A method for producing a microemulsion containing amphotericin B, comprising dispersing an oil phase in which amphotericin B is dissolved in an aqueous phase in which vitamin C is dissolved. The method of claim 1, wherein the oil phase is characterized in that at least one organic solvent selected from ethyl oleate, propylene glycol, dimethylacetamide, glycofurol is dissolved. Method for producing a micro emulsion containing amphotericin B The method of preparing a microemulsion containing amphotericin B according to claim 1, wherein the content of amphotericin B in the oil phase is 0.1 to 10 parts by weight based on 100 parts by weight of the oil phase. The method of producing a microemulsion containing amphotericin B according to claim 1, wherein the content of vitamin C contained in the oil phase is 1 to 50 parts by weight based on 100 parts by weight of the oil phase. The method of claim 1, wherein the dissolution of vitamin C and amphotericin B in the oil phase is carried out at 60 to 80 ° C. The method of producing a microemulsion containing amphotericin B according to claim 1, wherein the oil phase and the water phase include any one or more selected from dextrose, trehalose, solutol, and mannitol. The method of claim 1, comprising lyophilizing and pulverizing to prepare a microemulsion containing amphotericin B. Intravenous injection comprising a microemulsion containing amphotericin B prepared by the method of any one of claims 1 to 7 as an active ingredient