JPS6116248B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a solid preparation that maintains the utilization of cytochrome c and a method for producing the same. Cytochrome c is an enzyme that is present in animals, higher plants, yeast, molds, etc., and is involved in energy production, and plays important roles such as activating cell movement. Cytochrome c is a protein and is therefore susceptible to denaturation and inactivation due to heat, oxidation by air, pressure, or contact with organic solvents. For this reason, various methods have been devised to formulate pharmaceuticals that stably retain their specific pharmacological actions. Conventional cytochrome c preparations have been formulated as injections, and are made by adding reducing agents such as sulfites and ascorbic acid to an aqueous solution containing cytochrome c, or adding amino acids such as glycine and monosodium glutamate, and proteins such as albumin. Furthermore, it is supplied as an ampoule agent in which the existing air is replaced with nitrogen gas and sealed. The usage of this cytochrome c injection is limited to intravenous or intramuscular administration, and in this case, it is difficult to maintain an effective concentration due to the rapid drop in blood concentration after administration, and its excretion rate is low. The drawback is that it is extremely fast. In recent years, attention has been focused on the fact that when cytochrome c is absorbed through the intestinal wall, its effective blood concentration lasts for a long time, and many studies have been conducted on methods of formulating it into oral preparations such as granules and tablets. However, for example, when making tablets, cytochrome c is required to be stable in each step of kneading with an organic solvent, granulation, drying, tabletting, film coating, and drying, and in the tablets obtained. Cytochrome c is also degraded by contact with air, moisture content, etc.
There is a risk of degeneration and deactivation. The stability in the process under such conditions is completely incomparable with that of the conventional injection preparations. In order to manufacture oral preparations such as granules and tablets in this manner, the raw material cytochrome c bulk powder itself must not only be stable, but also sufficiently stable to form solid preparations. In addition, as a conventional example of solid formulation of cytochrome c, there is Patent Application Publication No. 8716 of 1972, which describes a solid formulation using gelatin as a stabilizer and its manufacturing method, but the manufacturing process It has the drawbacks that the process is long and therefore requires a long time, and the manufacturing operation is difficult and requires special equipment. An object of the present invention is to eliminate the drawbacks of the conventional methods described above and to provide a solid preparation that effectively maintains the activity of cytochrome c, and a manufacturing method with easy-to-operate steps for obtaining this solid preparation. In order to achieve this object, the present invention provides a stable solid cytochrome c preparation, which is characterized in that cytochrome c molecules are dispersed and encapsulated in dry solid dextran. The above solid cytochrome c preparation according to the present invention can be obtained by the following manufacturing method. The first manufacturing method is by freeze-drying a mixed aqueous solution of cytochrome c and dextran. For example, 10-30% (by weight) of cytochrome c
A 20 to 40% (by weight) aqueous polymer dextran solution is added to the aqueous solution, and a small amount of a disintegrant that can be mixed with water is added to improve the disintegration properties.The mixture is then freeze-dried to obtain a cytochrome c solid. This solid product is granulated or tableted by a known method. During the above manufacturing process, cytochrome c molecules are not heated, and even during the drying process, they are effectively encapsulated and protected by dextran, so the activity of cytochrome c is almost maintained. This activity maintenance effect is shown in Table 2 below. Moreover, the second manufacturing method is based on spray drying a mixed aqueous solution of cytochrome c and dextran. For example, a 5-20% dextran aqueous solution is added to a 5-20% cytochrome c aqueous solution, mixed, sprayed and dried while maintaining the hot air flow temperature at the nozzle side at 100°C or less to obtain a cytochrome c solid. . This solid product is granulated or tableted by a known method. In this method, by maintaining the hot air flow temperature at the nozzle side of the spray dryer below 100℃,
The product temperature is maintained below 40℃. In addition,
The temperature of this hot air stream is preferably around 80°C in terms of product temperature and drying state. As a result, the activity of cytochrome c is almost maintained. This activity maintenance effect is shown in Table 3 below. By composing the cytochrome c solid preparation in this way, it is possible to effectively maintain 90% or more of the cytochrome c activity for a long period of 100 days or more, as shown by the results of the abuse test. In the present invention, the above-mentioned effects can be observed with a wide range of molecular weights of dextran, and the amount of cytochrome c included in dextran is preferably 10 to 20% by weight. Table 1 below shows the residual activity percentage of cytochrome c in composition examples of the solid cytochrome c preparation according to the present invention in comparison with other composition examples.

【table】

[Table] The results shown in Table 1 are based on cytochrome c extracted and purified from horse heart muscle.The aqueous solution was freeze-dried in the form of only cytochrome c (No.
1) Solid products obtained by freeze-drying cytochrome c and low-molecular-weight dextran (No. 2 and No. 3), Solid products obtained by freeze-drying cytochrome c and high-molecular-weight dextran (No. 4 and No. 5) , and also cytochrome c
The results of the stability test for each solid sample (No. 6) obtained by adding acidic sodium sulfite to an aqueous solution and freeze-drying it are shown in terms of the percentage of remaining activity. In this case, the residual activity percentage of cytochrome c was measured after a predetermined period of time had elapsed with each sample filled into an ampoule, sealed, and stored in a thermostat at 45°C. This measurement method was based on a spectroscopic method similar to the cytochrome a concentration measurement method. That is, the sample solution was purified to only cytochrome c using a column of weakly acidic cation exchange resin (Amberlite CG-50), the cytochrome c was reduced to a reduced form with sodium hydrosulfite, and the sample solution was subjected to gel filtration using Sephadex (G-25). After that, a certain amount of cytochrome a is added to a solution of a certain concentration (0.5mM) and reacted, and cytochrome c
This method is based on the method of reading the initial rate of oxidation as the decrease in absorbance at a wavelength of 50 nm and comparing it with the value of a standard cytochrome c product ("Research on Cytochrome", edited by Professor Kazuo Okunuki Retirement Memorial Association, University of Tokyo Press, first edition). be. From the results shown in Table 1, the activity of the solid product containing only cytochrome c (No. 1) was already observed to decrease by nearly 20% after 10 days, and after 150 days, the activity changed by half and the activity decreased by half. In contrast, the cytochrome c solid material (No. 2) according to the present invention cannot be expected to be used effectively.
- No. 5) maintained almost the initial value of activity even after 10 days, and even after 150 days it remained at about 90%.
It was confirmed that the activity of the acidic sodium sulfite additive (No. 6) was maintained and the stability of the activity was excellent.
It turns out that the above is also true. The effect of maintaining cytochrome c activity is achieved by using the above-mentioned cytochrome c composition according to the present invention as a raw material and tableting it using a binder/disintegrant such as low-substituted hydroxypropyl cellulose by a conventional method. You can get excellent tablets. The results of the dissolution test for this tablet are shown in FIG. In this dissolution test, the above tablet was dissolved in constant-temperature distilled water at 37°C in accordance with the disintegration test.
The solubility curve shown in the figure confirms the rapid dissolution of this tablet. Conventionally, low molecular weight sugars such as sucrose, lactose, and glucose have been applied as stabilizers for cytochrome c preparations, but Tables 2 and 3 below show solid preparations according to the present invention and stabilizers. The stability of cytochrome c activity was compared with solid preparations obtained by applying various low molecular weight sugars as a percentage of residual activity.

【table】

[Table] (Residual activity%)
The results shown in Table 2 are based on the knowledge obtained from the results in Table 1, and for the solid preparation according to the present invention, 2 ml of a 20% aqueous solution of cytochrome c extracted and purified from horse heart muscle and 20% dextran (molecular weight: 200000) and 10 ml of an aqueous solution at 4°C, this was put into an ampoule, lyophilized, sealed as a powder, and the dried solid sample was used. and excipients, and tablets obtained by compressing this mixture under the conditions described below (tablet characteristics;
Weight 200mg/tablet, cytochrome c content 5mg/tablet)
The samples were dried solid products and tablets produced under exactly the same conditions as the above-mentioned solid preparations of the present invention for cytochrome c solid preparations containing sucrose, lactose, and glucose as stabilizers, respectively. These results were obtained by storing these specimens in a 45°C incubator.

[Table] (Residual activity%)
The results shown in Table 3 are based on the knowledge obtained from the results in Table 1, and the solid preparation according to the present invention contains 50 ml of a 10% aqueous solution of cytochrome c extracted and purified from horse heart muscle and 5% dextran. (molecular weight 176,000) was mixed at room temperature and spray-dried in a spray dryer cooled to 80°C on the nozzle (inlet) side, 50°C on the outlet side, and 30°C on the receiver, and the obtained powder was sealed in an ampoule. A sample of the dry solid was prepared by mixing an excipient with the powder of the dry solid, and tableting the mixture under the following conditions (tablet characteristics: weight 200 mg/ Tablet, cytochrome c content 5 mg/tablet) was used as the sample, and sucrose, lactose, and glucose were used as stabilizers.
Regarding solid preparations, the results obtained were obtained by using dried solid products and tablets produced under exactly the same conditions as the above solid preparations of the present invention as specimens, and storing these specimens in a heat insulator at 45°C. It is. The tableting conditions for each of the above cases are: (a) Tableting die diameter; 7.0 mmφ (b) Tableting pressure Preliminary pressure: 1200 Kg/cm ² pressure; 800 Kg/cm ² (c) Tableting speed; 300 It was a tablet/minute. From the results shown in Tables 2 and 3, it can be confirmed that the solid preparation according to the present invention has excellent stability of cytochrome c activity, especially during tableting, and also stability over time. In this respect, there is a significant difference in action and effect from when low molecular weight sugars are applied. As described above, the present invention provides a solid preparation capable of stably maintaining the activity of cytochrome c over time during manufacturing and storage conditions, and a manufacturing method using a simple and rapid production process. [Example 1] Based on the findings obtained from the results in Table 1, 2 ml of a 20% aqueous solution of cytochrome c extracted and purified from horse heart muscle and 10 ml of a 20% dextran (molecular weight: 200,000) aqueous solution were mixed at a temperature of 4°C. The mixture was mixed in an ampoule, freeze-dried, and sealed as a powder. The remaining activity of cytochrome c when the required number of ampoules were made and stored in a 50°C incubator is shown in Table 4 below.

[Table] (Residual activity%)
[Example 2] Based on the knowledge obtained from the results in Table 1, 20 ml of a 25% aqueous solution of cytochrome c extracted and purified from horse heart muscle and 100 ml of 25% dextran (molecular weight 176,000)
ml at room temperature, further disperse 5 g of low-substituted hydroxypropyl cellulose (LH-31) uniformly, freeze-dry this, then crush or granulate at low temperature, and then add excipients to this. Mix and tablet with mortar diameter
7.0mmφ, tableting pressure; preliminary pressure 1200Kg/cm ² , main pressure 800
Kg/cm ² , tableting speed 300 tablets/min (weight 200 mg/tablet, cytochrome c content 5 mg/min)
Tablets) were obtained. The residual activity of team c when this tablet was stored in a 45°C incubator is shown in Table 5 below.

[Table] (Residual activity%)
[Example 3] Based on the knowledge obtained from the results in Table 1, 50 ml of a 10% aqueous solution of cytochrome c extracted and purified from horse heart muscle and 1000 ml of a 5% dextran (molecular weight 176,000) aqueous solution were mixed at room temperature, ) side 80
A powder was obtained by spray drying in a spray dryer cooled to 50°C on the outlet side and 30°C on the receiver. At this time, the product temperature was 40°C. The residual activity of cytochrome c when this bulk powder was sealed in an ampoule and stored in a 50°C incubator is shown in Table 6 below.

[Table] (Residual activity%)

[Brief explanation of the drawing]

FIG. 1: A graph showing the solubility of cytochrome c tablets according to the present invention. Explanation of drawing symbols: A: Solubility curve.

Claims (1)

[Scope of Claims] 1. A stable solid cytochrome c preparation, characterized in that cytochrome c molecules are dispersed and encapsulated in dry solid dextran. 2. A method for producing a stable cytochrome c solid preparation, which comprises freeze-drying a mixed aqueous solution of cytochrome c and dextran. 3. A method for producing a stable cytochrome c solid preparation, which comprises spray-drying a mixed aqueous solution of cytochrome c and dextran.