JP2893901B2 - Animal-based oil-based paste formulation - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an antibody-containing oil-based paste formulation suitable for oral administration for preventing or treating infectious diseases in livestock, especially newborn livestock.

(Prior art) As an antibody-containing preparation for preventing or treating an infectious disease by orally administering it to animals, particularly to newborn livestock, only liquid or powdery preparations are conventionally known.

For example, enterotoxigenic E. coli (enteroto) in piglets and calves
Xigenic E. coli, hereinafter developed to intestinal infection or treatment with abbreviated as ETEC), Immune sera preparations are used, K88 ^+, altitude horses inactivation antigen K99 ⁺ and 987P ⁺ ETEC The immunized and obtained serum is directly filled in a container, and the monoclonal antibody (mouse ascites) corresponding to ETEC's K99 antigen is directly filled in a container. Each of these products is in a liquid form, and is transferred to a syringe or the like at the time of use, and is orally administered to a newborn animal. However, such a liquid preparation has to be transferred to a syringe every time of administration, which complicates the operation at the time of administration. In addition, when it is liquid, its activity as an antibody is not only unstable but also unstable to gastric juice. Further, in the case of a liquid, there is a large loss of the preparation due to breakage of the container.

On the other hand, the present inventors have first shown that a method of preparing an antibody specific to a specific antigen from immunized eggs produced by chickens inoculated with an antigen in advance can easily obtain an antibody-containing material having a high antibody titer. (JP-A-62-215534). This material can be obtained in the form of a powder, but it dissolves in water or artificial milk at the time of administration, so it also takes time, and due to the nature of the preparation, the powder scatters, or changes in quality due to moisture absorption, generation of mold, etc. There is a problem.

(Problems to be solved by the invention) Unlike conventional antibiotics and synthetic antibacterial agents, conventional immunological preparations are very safe because they do not show any problem of remaining in animal products or drug resistance, but the product is liquid. The operation at the time of administration to domestic animals is complicated, and there are also problems in stability of antibody activity and stability against gastric juice. In addition, the powdery antibody-containing material still has problems such as troublesome administration, deterioration due to moisture absorption, generation of mold, and the like.

Therefore, an object of the present invention is to provide an antibody-containing preparation which is simple to administer orally by gavage to an animal, has good antibody titer stability upon storage, and is effective for the prevention and treatment of infectious diseases.

(Means for Solving the Problems) As a result of research on the dosage form of an antibody-containing material, the present inventors have found that a paste preparation based on a purified powdered antibody and vegetable oil can achieve the above object. The present invention has been completed.

The present invention provides a veterinary antibody-containing oil-based paste formulation containing an antibody-containing powder and a vegetable oil as essential components, wherein the ratio of the vegetable oil to the total weight of the paste formulation is 30 to 70% by weight. And

The antibody-containing powder that can be used in the present invention is not particularly limited, but since there is a limit on the amount of a single dose, an antibody having an antibody titer such that the antibody titer of the obtained paste preparation becomes a certain level or more. It is preferred to use a contained powder. Desirable antibody titers vary depending on the purpose of administration and the target animal.For example, in the case of a formulation for oral administration for preventing or treating intestinal infectious disease in a newborn animal, the formulation is 500 times or more in antibody titer due to bacterial agglutination. Is preferred.

The antibody titer varies greatly depending on the production method and lot.
From the viewpoint of obtaining a high antibody titer, a powdered version of the antibody-containing material derived from the immunized hen's egg, which was previously proposed by the present inventors, is preferable. Pig obtained from this immunized chicken egg
ETEC's K88, K99, and 987P specific antibody-containing powders have an antibody titer of 2000
About 5000 times, about 5000 to 20000 times, about 4000 to 60,000 times. In order to obtain a preparation having an antibody titer of 500 times or more by mixing these 1 to 3 types, an antibody-containing powder of 0.8% by weight or more based on the total amount of the paste preparation is used. Further, in order to obtain a preparation having a high antibody titer, for example, about 2,000-fold, unless a substance having a high antibody titer is used, the proportion of the antibody-containing powder becomes too high to form a paste. In the case of a preparation having a low antibody titer, the dosage may be increased, but it is actually difficult to increase the dosage in newborn livestock.

The type of antibody used may be one or more depending on the purpose.

The compounding ratio of the antibody-containing powder depends on the antibody titer of the raw antibody powder,
Although it depends on the target antibody titer of the paste preparation, at least 30% by weight of vegetable oil is required for pasting, so that the amount of antibody powder is 70% by weight or less. In addition, when a coloring agent, a flavor, a sweetener, an excipient, and the like are added, the amount of the antibody powder is reduced accordingly.

In the present invention, a vegetable oil which does not solidify even in winter and is liquid at 0 ° C. to ordinary temperature can be used as the vegetable oil. Vegetable fat that is solid at room temperature, such as coconut oil or palm oil, cannot be used. Also,
What is hard to polymerize when left to stand, such as a drying oil such as linseed oil or tung oil, is also not preferable.

Specific examples of vegetable oils that can be used include common edible oils such as corn oil, rapeseed oil, cottonseed oil, safflower oil and the like.

The mixing ratio of the vegetable oil is usually 30 to 70% based on the total weight of the preparation in order to paste the preparation and impart appropriate viscosity.
%, Preferably 40 to 60% by weight.

The paste formulation of the present invention may contain excipients for maintaining the dosage form in addition to the antibody powder and the vegetable oil. Excipients are starch powder, casein powder,
Examples thereof include cellulose powder and silica powder. The mixing ratio is such that the ratio of vegetable oil in the entire paste preparation is 30 to 70% by weight in consideration of the amounts of the antibody powder and additives.

If necessary, the paste preparation of the present invention may contain additives such as powdered sugar, an activator, a coloring agent, a flavor, and a preservative. Powdered sucrose or the like can be used in an amount of about 7 to 15% by weight to impart sweetness to the powdered sugar. Examples of the activator include Tween 80 and the like, and 0.5 to 5% by weight can be added to improve the dispersibility of the pasted preparation when it enters the stomach or intestine. As the coloring agent, flavoring agent and preservative, those used in ordinary preparations can be used, and it may be added in a total amount of about 0.1 to 2% by weight. Examples of the preservative include an antioxidant such as tocopherol.

The above-mentioned components are blended to prepare the oily paste preparation of the present invention.

The resulting paste formulation has a moderate viscosity, for example,
It can be in the form of a product filled in a syringe or a tube. Useful for oral gavage. In the case of a product filled in a syringe or a tube, the formulation can be forcibly administered orally by extruding and injecting an appropriate amount of the paste formulation from the syringe or the tube into the back of the subject animal's throat. in this way,
The required amount can be reliably administered. This is particularly advantageous in the case of newborn pigs, which are difficult to administer by mixing the preparation in feed or drinking water because they are fed directly from the parent pig. Of course, gavage administration to other animals can be easily performed.

One dose is preferably about 1-2 g in the case of a newborn pig. The filling amount of the paste preparation into a tube or syringe should be such that several to several tens of doses can be used so that it can be used up at one time, and even if a scale is provided on the outer surface of the container to indicate the single dose. Good.

The paste formulation of the present invention is different from the case of liquid or powder, unlike the case of storage or use, in which the loss of the formulation due to breakage or mistake of the container is small, and is economical. It is also advantageous in that no occurrence is observed.

Further, as is clear from the examples described later, the antibody titer does not decrease even after 6 months of long-term storage, and long-term storage is possible. In addition, the antibody titer after degradation by artificial gastric juice does not decrease much. This is because, in the paste formulation of the present invention, the antibody molecules are adsorbed on the surface of the excipient powder, and the oil layer covers the surface thereof, so that contact with acid or proteolytic enzyme in the stomach or intestine is caused by the antibody aqueous solution. This is thought to be due to slower degradation of the antibody and less decrease in antibody activity.

In addition, the paste preparation of the present invention is compatible with an abnormal toxicity test using mice, and its safety has been confirmed.

(Example) Reference Example (Preparation of Antibody-Containing Powder) GL-16 strain (K88 pilus positive), GL-96 strain (K99 pilus positive) and GL-148 strain (987P) Pilus positive) using each Minka liquid medium
After culturing at 37 ° C. by a conventional method, cells were collected by centrifugation,
K88, K99 and 987P fimbriae were separated with a homogenizer. The obtained pili were appropriately mixed with an adjuvant and injected intramuscularly, and the first immunization was performed. Six weeks later, the second immunization was performed in the same manner as the first immunization. The eggs laid by the chickens are used as immunized chicken eggs. After separating the yolk from the immunized chicken egg and diluting it 25 times with water, an emulsion prepared by adding water to a copolymer of methacrylic acid and ethyl acrylate (Eudrakit L100, manufactured by Rohm Pharma) is added thereto. The concentration of the copolymer in the yolk fluid after mixing was 0.3
%. After stirring at 25 ° C., the mixture was left for 30 minutes, and centrifuged at 5000 g for 20 minutes to obtain a supernatant. The powder was dried at 80 ° C. for 3 minutes with a spray dryer to obtain a specific antibody-containing powder.

Example 1 (Preparation of Paste Formulation of the Present Invention) The antibody titer of a powder containing a specific antibody against K88, K99, 987P fimbrial antigen of ETEC manufactured in Reference Example was measured by a bacterial agglutination reaction according to a conventional method. Are 5120 and 1024 respectively
0, 40,960. Using these bulk powders, an oil-based paste formulation of the present invention was prepared by blending excipients, vegetable oils, active agents and powdered sugar such that the antibody titers against K88, K99 and 987P were all 1000-fold. . Table 1 shows the mixing ratio of each preparation.

Example 2 A storage stability test of the oily paste preparation of the present invention was performed using Sample 4 prepared in Example 1. Storage temperature is 4
The measurement was performed at each temperature of 25 ° C., 25 ° C., and 37 ° C., and the antibody titer was measured and the change in the paste properties was observed. For comparison, a test of an aqueous paste formulation in which carrageenan was added to an aqueous antibody solution to form a gel was also performed. The composition of the aqueous paste formulation is
Anti-K88 antibody containing powder 19.5%, anti-K99 antibody containing powder 9.8%,
Powder containing anti-987P antibody 3.0%, carrageenan 4.5%, water 63.7
%. Table 2 shows the measurement results of the antibody titer for each antigen.

As is evident from Table 2, the antibody titer of the oil-based paste formulation of the present invention is stable at 4 ° C., 25 ° C. and 37 ° C. even after 6 months. There was no change in their properties. On the other hand, the aqueous paste preparation showed a decrease in antibody titer, and markedly deteriorated particularly at 25 ° C and 37 ° C.

Example 3 Using Sample 4 prepared in Example 1, changes in the antibody titer in the absence of an enzyme and in the presence of an enzyme were examined under strongly acidic conditions. After diluting the paste preparation five-fold with an acidic buffer solution of KC1 (0.1 M) -HCl, pH 1.8, if no enzyme was present, the diluent was collected at 37 ° C. over time. When observing the change in the antibody in the presence of the enzyme, pepsin 2 mg /
The solution was added to a volume of 100 ml, and after 0, 30, 60, 90 and 120 minutes, the diluent was collected and immediately neutralized to pH 7.0 with 0.1 N sodium hydroxide solution. Thereafter, the antibody titer was measured.

For comparison, the antibody aqueous solution was also examined. From the raw antibody powder prepared in the reference example, an antibody solution having a 1000-fold antibody titer was prepared using phosphate buffered saline, diluted 5 times with the above acidic buffer, and tested in the same manner. Was. The results are shown in Table 3.

As is clear from Table 4, in the oil-based paste formulation,
Antibody activity decreases more slowly in the absence and presence of the enzyme than in the aqueous antibody solution.

Example 4 Using the samples 2, 4 and 5 prepared in Example 1, a negative toxicity test was carried out in accordance with the standard for biologics for animals. DDY mice, 5 weeks old, were used as experimental animals.
Each sample was diluted 4-fold to give a dispersion, which was injected into experimental animals using a 25 ml syringe and a 26G 1/2 needle. Changes in body weight and physical condition were observed as measurement items. Table 4 shows changes in body weight (average of 10 animals). The injection was performed on day 0, and the control was phosphate buffered saline.

No abnormality was observed in any of the experimental animals from immediately after the sample injection until the lapse of 13 days. As is clear from Table 4, the body weight decreased for a few days after the injection, but then recovered satisfactorily.

Example 5 Using the oil-based paste formulation of the present invention, an efficacy test was performed on a formulation intended for treating newborn pigs infected with enterotoxigenic Escherichia coli. One day old large white species were used as experimental animals. E. coli infection is 2 × 10 ¹¹ cfu / ml
GL-16 strain (K88 pilus positive), GL-96 strain (K99 pilus positive)
And 2 × 10 ⁹ cfu / ml of the GL-148 strain (987P fimbria-positive) bacterial cell dispersion were each administered to the stomach in an amount of 5 ml per animal. This administration was performed by inserting a polyethylene tube connected to a syringe into the stomach of the experimental animal and through the tube. Immediately after diarrhea was observed, a predetermined amount of the paste formulation of the present invention (sample 4) was orally administered by gavage three times a day for three days. The measurement items were the measurement of the bacterial isolation rate (rectum positive number / experimental number) in the rectal sample after infection, and the change in the body weight of the experimental animal (rate of change in body weight before infection). Table 5 shows the bacterial isolation rate and Table 6 shows changes in body weight.

As is clear from Tables 5 and 6, with an increase in the dosage of the preparation, a decrease in the bacterial detection rate after onset and an increase in body weight due to the recovery of diarrhea are observed, and the effect of the preparation is exhibited. .

(Effect of the Invention) According to the present invention, it is possible to provide an antibody-containing oil-based paste formulation that is easy to administer by oral gavage. This preparation, as an antibody-containing preparation, can exhibit excellent effects for preventing and treating infectious diseases, and is more excellent in stability of antibody titer than aqueous preparations, and can be used after long-term storage.

Claims (1)

(57) [Claims] 1. An animal antibody-containing paste formulation comprising an antibody-containing powder and a vegetable oil as essential components, wherein the ratio of vegetable oil to the total weight of the paste formulation is 30 to 70% by weight.