JPS631293B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a solid preparation of lysozyme. More specifically, the present invention relates to a stable solid preparation of lysozyme in which lysozyme is prevented from being digested by pepsin in gastric juice. Lysozyme is a basic protein that exists in animal tissues, secretions, and egg whites.As a mucopolysaccharide-degrading enzyme, it promotes tissue repair, eliminates inflammation, dissolves thick juices and sputum, and
It is widely used for excretion, strengthening capillaries, and improving the body's infection defense function. Chicken egg white lysozyme is currently available for medical use, and the hydrochloride is usually available in tablets, granules, and
It is used in preparations such as syrups. Lysozyme is a relatively stable enzyme, particularly stable in acidic conditions at room temperature, but is known to be degraded by pepsin in gastric juice. By the way, since the optimal pH of pepsin is 1.5 to 2.0, if lysozyme is taken orally after meals, the phlegm in the stomach will be formed by the contents of the meal, and the pH in the stomach will be around 4 to 2.0.
5, so it is expected that there is little risk of lysozyme deactivation due to pepsin. However, lysozyme is not necessarily taken after meals; it can also be taken when the pH in the stomach is low, such as on an empty stomach or fasting, so lysozyme does not have any stability problems when taken at any time. A formulation is desired. Conventionally, enteric-coated preparations have been used for drugs that are unstable in gastric juice. However, because enteric-coated preparations are hard-pressed to increase resistance to gastric juices, the release of the active ingredient may be delayed, resulting in bioavailability problems. Furthermore, in the case of lysozyme, which is a basic protein, it may become insolubilized by binding with the acid groups of enteric bases such as cellulose acetate phthalate and methacrylic acid-methacrylic acid methyl ester copolymer, reducing its medicinal efficacy. . Therefore, we considered the development of a pepsin-resistant lysozyme preparation to replace the enteric-coated preparation, and conducted various studies. As a result, by using phospholipids and sorbitan unsaturated fatty acid esters, if the pH of gastric juice is in the active region of gastric proteolytic enzymes, the release of lysozyme from the preparation can be suppressed and stabilized, and if it is transferred to the intestines. The present invention was completed by successfully designing a stable lysozyme preparation in the stomach that is easily soluble and releases lysozyme. The present invention involves dissolving lysozyme in a hydrophilic solvent,
This is a solid lysozyme preparation made by mixing phospholipids and sorbitan unsaturated fatty acid esters, and adding additives for formulation into a predetermined shape. Lysozyme in the present invention includes both free lysozyme and its acid salt. Examples of phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositic acid, but soybean lecithin and egg yolk lecithin, which are commercially available mixtures of these, are usually used. It will be done.
For the purpose of the present invention, soybean lecithin is most suitable, but egg yolk lecithin further blended with a basic phospholipid such as phosphatidylserine or phosphatidylinosite is also preferred. Sorbitan unsaturated fatty acid esters include sorbitan monooleate, sorbitan dioleate, sorbitan trioleate, and mixtures thereof. The hydrophilic solvent is selected from those in which lysozyme can be dissolved. Usually water is used. Pharmaceutical additives include excipients, binders, disintegrants,
Refers to lubricants and other substances normally used when formulating formulations. For example, starch, lactose, glucose, mannitol, hydroxypropyl starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, hydroxypropyl cellulose,
Crystalline cellulose, dextrin, methylcellulose, ethylcellulose, hydroxyethylcellulose, gum arabic, tragacanth, gelatin, sodium alginate, polyvinylpyrrolidone, macrogol, precipitated calcium carbonate, calcium lactate, magnesium stearate, calcium stearate, talc, silicic anhydride, etc. can give. The blending ratio of lysozyme, phospholipid, and sorbitan unsaturated fatty acid ester of the present invention is preferably equal or more of phospholipid and half or more of sorbitan unsaturated fatty acid ester, but the ratio may be changed as appropriate. No problem. The preparations of the present invention include all solid preparations for internal use such as powders, granules, tablets, and capsules. The formulation of the present invention is manufactured as follows. Lysozyme is dissolved in a hydrophilic solvent, and phospholipid and sorbitan unsaturated fatty acid ester are mixed. Thereafter, various additives for formulation are added depending on the intended dosage form, and each dosage form is obtained by a conventional method. In the case of a powder, it can be obtained, for example, by adding an oil-absorbing additive such as crystalline cellulose, mixing, and drying. In the case of granules, they can be obtained by further adding a binder and granulating them. In the case of tablets, they can be obtained by adding excipients, disintegrants, and lubricants to granules or powder particles, if necessary, and then compressing them. Figure 1 shows various PHs from the formulation of the present invention shown in Example 1.
This shows the elution curve of lysozyme in an aqueous solution, and it can be seen that the elution of lysozyme is suppressed below about pH 4, but lysozyme rapidly elutes from around pH 5. In this way, at pH4 or lower, lysozyme is confined in a matrix mainly composed of phospholipids, and its release is suppressed except for the portion on the surface, and when the pH approaches 5, lysozyme is trapped in a matrix mainly composed of phospholipids, and when the pH approaches 5, lysozyme is trapped in a matrix mainly composed of phospholipids. The interaction of the esters expands the internal voids of the matrix, allowing lysozyme to be released by diffusion. Therefore, when this preparation is used internally, only some lysozyme is released in the stomach, which has a pH below 4, and pepsin, which has an optimal pH of 1.5 to 2.0,
Most lysozyme is stabilized against proteolytic enzymes such as gastricin, which has an optimal pH of around 3.2.
Lysozyme is easily released in the intestinal tract and exhibits medicinal effects. If the pH in the stomach temporarily rises to around 5, such as after a meal, lysozyme will begin to dissolve, but at that pH, the proteolytic enzymes in the stomach will hardly work, and the medicinal efficacy of lysozyme will be reduced. There seems to be no problem with this. Figure 2 shows the first liquid for disintegration test (artificial gastric juice, PH1.2) of the Ninth revised Japanese Pharmacopoeia Law for the preparation of the present invention.
and the elution curve of lysozyme in the second fluid (artificial intestinal fluid, PH7.5). The experimental method was as follows. Weigh out 1.2 g of the powder of Example 1, add NF (National
The dissolution test was conducted in accordance with the 14th edition of the Formulary. The dissolution testing device described in NF (Model TR-3S manufactured by Toyama Sangyo) was used as the device, two stirring propellers were attached in place of the basket, and the rotation speed was 100 revolutions per minute. After starting the dissolution test, sample 2 ml every 10 to 15 minutes until 60 minutes, with a pH of 6.2.
Make the total volume 20ml with 0.1M phosphate (-EDTA) buffer.
The sample solution was taken up into a container and used as a test solution. Separately, prepare a lysozyme chloride standard solution (10 μg/ml), and use Micrococcus lysodeikticus as a substrate for both the test solution and the standard solution [Analytical Biochemistry, Vol. 39]
113 (1971)], and the dissolution rate after each time was determined. The results shown in FIG. 2 show that lysozyme does not elute much in the stomach under strong acidity, but almost completely elutes in the intestinal tract. Furthermore, the residual rate of lysozyme in the formulation of the present invention in the first solution containing pepsin was determined. 3.2 g of Japanese Pharmacopoeia sugar-containing pepsin was added to the first solution 1 to adjust the pH to 1.6. To this, the powder of Example 1
Add 1.2 g and start the experiment under the same conditions as in the previous elution test, and after 15 minutes, add anhydrous disodium phosphate powder to raise the pH of the test solution to 7.5 to elute lysozyme. Thereafter, 2 ml was sampled and the lysozyme activity was measured in the same manner as before. The residual activity of lysozyme after 30 minutes and 60 minutes was determined in the same manner, and the residual rate was calculated. As a control, a powder prepared by mixing crystalline cellulose with lysozyme chloride crystals was selected. The results are shown in Table 1 below.

[Table] As shown in Table 1, the lysozyme residual rate of this powder is higher than that of the control. This is considered to be because, as shown in FIG. 2, the elution of lysozyme was suppressed in this powder, and as a result, it was avoided from being inactivated by pepsin. As mentioned above, the characteristics of the lysozyme solid preparation of the present invention are that it does not require enteric coating.
It is a stable formulation in the stomach, and unlike conventional enteric-coated formulations, it can also be obtained as a powder. EXAMPLES Next, the present invention will be explained in more detail with reference to Examples. Example 1 Powder lysozyme chloride 10g Distilled water 20ml Purified soybean lecithin (manufactured by Central Soya, USA)
Centrolex F) 20g Sorbitan monooleate (manufactured by Nippon Chemicals)
SO-10) 10g Crystalline Cellulose 80g Lysozyme chloride was dissolved in distilled water and kneaded with purified soybean lecithin and sorbitan monooleate in a mortar. Crystalline cellulose was added to this paste-like mixture, and the mixture was ground and dried at 40 to 45°C. After drying, it was sieved through a 60-mesh sieve to obtain a powder. Example 2 Powder lysozyme chloride 10g Distilled water 20ml Purified soybean lecithin (SLP manufactured by Tsuru Lecithin Industries)
White) 20g Sorbitan Sesquioleate (manufactured by Nippon Chemicals)
SO-15) 10g Crystalline cellulose 80g Lactose 80g Lysozyme chloride was dissolved in distilled water and kneaded with purified soybean lecithin and sorbitan sesquioleate in a mortar. Crystalline cellulose was added to this paste-like mixture and ground. Further, lactose was added and mixed uniformly, followed by drying at 40 to 45°C to remove moisture. After drying, it was sieved through a 60-mesh sieve to prepare a 20% lysozyme chloride powder. Example 3 Lysozyme chloride capsule 10g Distilled water 20ml Purified soybean lecithin (Centrolex F) 20g Sorbitan monooleate (SO-10) 10g Crystalline cellulose 80g Corn starch 49g Calcium stearate 1g Lysozyme chloride was dissolved in distilled water and placed in a mortar. It was mixed with purified soybean lecithin and sorbitan monooleate. Crystalline cellulose was added to this paste-like mixture and ground. This powder was dried at 40 to 45°C, then uniformly mixed with corn starch and calcium stearate through a 60-mesh sieve, and filled into No. 3 hard capsules with 170 mg each. Example 4 Tablet Lysozyme chloride 10g Distilled water 20ml Purified soybean lecithin (Centrolex F) 20g Sorbitan monooleate (SO-10) 10g Crystalline cellulose 100g Spray-dried lactose 23g Corn starch 15g Magnesium stearate 2g Lysozyme chloride was dissolved in distilled water. , and kneaded with purified soybean lecithin and sorbitan monooleate in a mortar. 80 g of crystalline cellulose was added to this paste-like kneaded mixture, which was then ground and dried at 40 to 45°C. Spray-dried lactose, the remaining 20 g of crystalline cellulose, and corn starch were added to this and mixed uniformly, followed by sieving through a 60-mesh sieve. Magnesium stearate was sprinkled onto the mixed powder through an 80-mesh sieve, mixed uniformly, and then compressed into tablets having a diameter of 8 mm and a weight of 180 mg.

[Brief explanation of the drawing]

FIG. 1 shows the elution curves of lysozyme in various PH aqueous solutions from the preparation of the present invention. FIG. 2 shows the elution curve of lysozyme from the preparation of the present invention in the first and second liquids for disintegration test according to the Ninth Edition Japanese Pharmacopoeia.

Claims (1)

[Scope of Claims] 1. A solid preparation of lysozyme, which is prepared by dissolving lysozyme in a hydrophilic solvent, mixing phospholipid and sorbitan unsaturated fatty acid ester, and adding additives for the preparation to form a predetermined shape. 2. The lysozyme solid preparation according to claim 1, wherein the phospholipid is soybean lecithin. 3. The lysozyme solid preparation according to claim 1, wherein the sorbitan unsaturated fatty acid ester is one or more selected from sorbitan monooleate, sorbitan dioleate, and sorbitan trioleate. 4 Claim 1 in which the hydrophilic solvent is water
Lysozyme solid preparation as described in Section 1. 5. The solid preparation of lysozyme according to claim 1, which contains at least an equal amount of phospholipid and at least half an amount of sorbitan unsaturated fatty acid ester relative to lysozyme.