JP4629964B2 - Cattle digestive disease treatment - Google Patents

Description

  The present invention relates to a bovine digestive tract disease therapeutic agent comprising, as an active ingredient, animal medicine for cattle, particularly human interferon α (hereinafter sometimes abbreviated as “HuIFN-α”).

US Pat. No. 5,910,304 JP-A-6-340549 JP 2002-201141 A Livestock Diagnosis, No.272, Lines 5-14 (issued in 1986) “Clinics and Microorganisms”, Joseph, M, Cummins and William, E, Stewart II (Volume 18, No. 63, 635-1991 (1991), J. Jeff M. Cummins and William E. Stewart II).

  Cattle are widely bred for livestock or dairy cattle, and a variety of breeds have been created through breeding improvements over many years, and now there are surprisingly expensive breeds. Cattle are nervous and easily develop gastrointestinal diseases mainly due to diarrhea due to transportation by means of transportation such as trucks, changes in feed and breeding environment, infection with pathogenic microorganisms, and the like. In particular, viral digestive organ diseases such as rotavirus and coronavirus are very damaging due to the pathogenicity and infectiousness of these viruses. In the case of cattle, symptom manifestation is severe, and even after recovering from the disease, there are many cases in which later growth is delayed or even death occurs, so economic loss is a big problem. Currently, there is no such treatment for bovine viral diseases, and in the case of gastrointestinal diseases, nutritional therapy with a replacement fluid is also given to relieve dehydration and restore physical strength, so that complex infections and secondary infections do not occur. In fact, only symptomatic treatment such as administration of antibiotics is performed (see, for example, Patent Document 1). For these reasons, there is a demand for the development of methods that can easily and effectively treat bovine gastrointestinal diseases and have less economic burden on the owner.

  Recently, it has been reported that oral administration of relatively small amounts of interferon of the same species or different species is effective for diseases of mammals such as cattle, cats, pigs, rats, mice and chickens and birds. (Patent Document 1, Non-Patent Document 2). In addition, the present applicant also discloses a therapeutic agent and a method for treating a respiratory disease of a cat having a small amount of HuIFN-α as an active ingredient in Patent Document 2, and Patent Document 3 discloses a subtype of HuIFN-α. It has been disclosed that the method of combining α2 and subtype α8 is excellent in terms of expression of the action effect of HuIFN-α. However, these documents show that oral administration of interferon can improve diarrhea symptoms caused by infections such as bovine rotavirus and coronavirus, and HuIFN-α preparations composed of specific subtypes have diarrhea. It has not been described at all as being effective as a therapeutic agent for digestive diseases of cattle infected with these main viruses.

  The present invention provides a therapeutic agent for digestive tract disease of cattle that shows remarkable effects even when administered by a method other than oral or nasal injection, and that requires less labor and burden on the person in charge of treatment and less economic burden on the owner. It is a first problem to provide a method for treating bovine digestive tract diseases using the therapeutic agent.

  In order to solve the above-mentioned problems, the present inventors have been conducting research for many years on a method for treating bovine digestive diseases using human interferon (hereinafter, sometimes abbreviated as “HuIFN”). As a result, HuIFN-α, particularly its subtype α8, is 10% by mass or more in the total HuIFN-α mass (hereinafter referred to as “% by mass” unless otherwise specified). We found that the preparation containing it shows a significant effect on digestive tract diseases mainly caused by diarrhea in cattle, including calves, and established therapeutic agents for bovine digestive tract diseases, as well as bovine digestive tract diseases using the same. Thus, the present invention has been completed.

  The therapeutic agent for bovine gastrointestinal diseases comprising HuIFN as an active ingredient according to the present invention and the method for treating bovine gastrointestinal diseases using the therapeutic agent are mainly characterized by diarrhea associated with bovine viral gastrointestinal diseases. Effective in improving clinical symptoms or complete cure.

  HuIFN as referred to in the present invention has a high therapeutic effect on bovine viral digestive tract diseases, and usually a very small dose of about 0.005 to 5,000 international units per kg body weight of bovine is sufficient.

  HuIFN is highly effective in treating bovine gastrointestinal diseases, whether administered orally or parenterally.

  The bovine referred to in the present invention is an animal belonging to the so-called Bovidae family, and when the therapeutic agent of the present invention is used prophylactically, rotavirus and coronavirus, which are one of the main causes of digestive diseases in cattle. In general, cattle affected by viral diarrhea virus and other gastrointestinal illnesses are targeted, and for treatment purposes, they are infected with these viruses, and other viruses, mycoplasma, It means animals that have complex or secondary infection with bacteria, parasites, etc., and have symptoms that are mainly diarrhea. General clinical symptoms such as loss of appetite, loss of vitality, loss of hair, hair growth, and dehydration. This includes the animals shown.

The therapeutic agent for bovine digestive tract diseases according to the present invention comprises HuIFN produced by human-derived cells, or HuIFN produced by microorganisms or animal cells introduced with HuIFN-related genes collected from human-derived cells. is there. Today, HuIFN is mainly classified into three types, HuIFN-α, HuIFN-β, and HuIFN-γ, based on the difference in antigenicity, all of which have therapeutic effects on digestive diseases in cattle. Can be used in the present invention, although there is a slight difference. At that time, only one type of HuIFN may be used, or two or more types of HuIFNs having different types may be used in combination. Moreover, existence of a plurality of subtypes is known for HuIFN-α, and these can be used alone or in combination of two or more. In any case of HuIFN, it is desirable to use the one with the highest specific activity. For oral administration, a purified product having a specific activity of about 10 ⁵ international units / mg protein or more, and for parenteral administration, A purified product of about 10 ⁷ international units / mg protein or more is desirable.

  As a result of testing by the present inventors, among these HuIFNs, HuIFN-α, particularly so-called “natural HuIFN-α” produced by human lymphoblastoid cell lines such as BALL-1 cells and Namalva cells, is bovine. It has a high therapeutic effect on gastrointestinal diseases and has few side effects. In particular, in terms of effect, a preparation containing HuIFN-α subtype α8 (hereinafter referred to as “subtype α8”) of 10% or more of the total mass of HuIFN-α is desirable. A preparation containing 10 to 80% of the total mass and containing HuIFN-α subtype α2 (hereinafter referred to as “subtype α2”) of 20 to 90% or less of the total mass of HuIFN-α has a strong therapeutic effect. From the above, it is more desirable that a preparation containing 20 to 40% of the total mass of HuIFN-α and subtype α2 and 60 to 80% of the total mass of HuIFN-α is highly effective. This is particularly desirable. HuIFN-α derived from BALL-1 cells contains subtype α2 of about 75% of the total mass of HuIFN-α and subtype α8 of about 25% of the total mass of HuIFN-α, and the balance is extremely high Well, it was used in this invention and brought the best effect. Moreover, it was confirmed that when HuIFN-α and HuIFN-β are used in combination, the therapeutic effect of HuIFN-α is synergistically enhanced.

  The therapeutic agent for bovine digestive tract diseases according to the present invention is not only in the form of HuIFN alone, but also HuIFN and other physiologically and pharmaceutically acceptable, for example, carriers, excipients, diluents, immune aids, It includes a stabilizer and, if necessary, a form as a composition with an antipyretic agent, an anti-inflammatory agent, an antibacterial agent, a digestive agent, a nutrient, a feed and the like. Furthermore, the therapeutic agent of the present invention also includes a drug in a dosage unit form. The drug in the dosage unit form is, for example, a daily dose of HuIFN, which is the active ingredient of the present invention, or an integral multiple thereof (four times). Or a divisor (up to ¼) thereof and means a drug in a physically separable dosage form suitable for administration. When two or more types and / or HuIFN-α different subtypes are used in combination, a single agent of each type of HuIFN and / or HuIFN-α subtype may be used in combination. Two or more types of HuIFN may be mixed and used as a single agent, or these formulations may be used in combination. Examples of such dosage forms include powders, gel oral preparations, granules, solutions, tablets, capsules, sublinguals, injections, and film oral preparations. In addition, when using powders or granules, it is optional to use them by dissolving them in distilled water, physiological saline, milk, milk substitute or the like according to the administration route.

  The dose and usage of the bovine digestive tract disease therapeutic agent according to the present invention will be described. The therapeutic agent of the present invention can be administered orally or parenterally to achieve the desired preventive and / or therapeutic effect. Is obtained. Specifically, although it depends on the type of cattle, the type of causative virus, symptoms, dose, usage, type of HuIFN used, etc. 0.005 to 5,000 international units / time, preferably about 0.05 to 100 international units / time, more preferably about 0.1 to 50 international units / time HuIFN 1 to 3 times a day or It may be administered 1 to 5 times a week for about 1 day to 6 months.

  In order to orally or nasally administer the therapeutic agent of the present invention, the therapeutic agent is prepared in an orally administrable form such as powder, troche, granule, solution, tablet, sublingual agent, feed, etc. Or, if necessary, mix and dissolve in food, milk, milk substitute, etc., or use appropriate administration aids such as oral administration device, spray, gastric sonde, etc. Insert into the esophagus or stomach. For parenteral administration, the injection according to the present invention may be injected intradermally, subcutaneously, intramuscularly, intravascularly (referring to veins or arteries) or intraperitoneally. When the present inventors tested, it was difficult to obtain the desired therapeutic effect when the dose was below the above range, and conversely, when the dose was exceeded, antibody production and side effects became prominent, and the owner compared with the therapeutic effect. The economic burden may be increased, and the above range was taken as the best.

  Comparing individual administration routes from the viewpoint of therapeutic effect and ease of administration, there is an advantage that oral administration can be easily carried out at general breeding farms or cattle farms. When a predetermined amount of HuIFN is accurately administered, it is also optional to administer it intravascularly, intradermally, subcutaneously or intramuscularly.

  The therapeutic agent of the present invention has effects such as prevention and treatment of bovine respiratory diseases, suppression of weight loss during transportation, and promotion of calf fattening in addition to the treatment and prevention effects of bovine digestive diseases. Have. In addition, it has the same effects as those of cattle on animals such as horses, pigs, cats, dogs, mice, chickens, and other mammals and birds. Therefore, the therapeutic agent of the present invention is used not only as a therapeutic agent for digestive diseases of cattle, but also as a prophylactic agent or therapeutic agent for various diseases of these animals in general, a weight loss inhibitor during transportation, a fattening promoter, etc. can do. The usage and dose in this case may be the same as those in the treatment of bovine digestive diseases.

  Throughout this specification, the activity of HuIFN is determined by measuring the degree to which HuIFN suppresses cytopathic degeneration of FL cells by Sindbis virus by the microtiter method, and the measured value is measured by the international standard for HuIFN “Ga23” “−901-532” is converted into “international units” determined as a standard and displayed.

<Experiment 1>
<Preparation of HuIFN-α preparation and HuIFN-α preparation>
Based on the method described in Example 1 described later, a purified product of HuIFN-α derived from BALL-1 cells (hereinafter sometimes abbreviated as “BALL-1 interferon”) (specific activity 2 × 10 ⁸ international units / mg protein), and based on the method of Example 2, recombinant subtype α8 (specific activity 2 × 10 ⁸ international units / mg protein) and recombinant subtype α2 (specific activity 7 × 10 ⁷ international units). / Mg protein) was prepared. Separately from these, HuIFN-α (hereinafter sometimes abbreviated as “leukocyte interferon”) induced by causing Sendai virus to act on leukocytes isolated from human peripheral blood in a conventional manner. Purified according to the method described in K. Cantel et al., “The Journal of General Virology”, Vol. 39, pp. 541-543 (1978), with a specific activity of about Partially purified leukocyte interferon of 2 × 10 ⁶ international units / mg protein was obtained. Subsequently, this partially purified HuIFN was further purified by antibody chromatography using a monoclonal anti-HuIFN-α antibody in the same manner as in Example 1 described later, and then a phosphate buffer (pH 7) containing about 0.1 mg / ml of human albumin. 0.0) equilibrated with gel filtration chromatography to obtain a purified leukocyte interferon (specific activity about 2 × 10 ⁸ international units / mg protein) solution. Using these two types of HuIFN and recombinant subtype α8 and recombinant subtype α2, according to the method of Example 5 described later, anhydrous crystalline maltose (trade name “Finetose” sold by Hayashibara Corporation) ]) As excipients to prepare powdered formulations each containing 200 international units / g HuIFN-α. For the recombinant subtype α8 and the recombinant subtype α2, the mass ratio of the subtype α8 and the subtype α2 is 10: 0, 9: 1, 8: 2, 4: 6, 2: Seven powdered formulations containing 8, 1: 9, 0:10 were prepared.

<Experiment 2>
To administer the HuIFN-α preparation prepared in Experiment 1 orally once a day as a therapeutic agent for digestive diseases to 100 cows infected with rotavirus and exhibiting diarrhea, and observe its progress. Thus, the therapeutic effect and side effects of the therapeutic agent according to the present invention were evaluated.

  In other words, 50 bulls and 50 cows estimated to be 1 to 6 years old who had symptoms of diarrhea at the first visit and were found to have rotavirus antigens in feces as a result of routine virus tests were targeted. , Randomly selected 5 males and 5 females as a group, and any of the above HuIFN-α preparations were orally administered at a dose of about 0.5 international units / kg body weight / dose in Experiment 1. The prepared preparation was administered once a day for 5 days from the first administration day (Day 1). The course of 12 days from the start of administration was carefully observed, and in addition to diarrhea symptoms and stool color, general clinical findings of 6 items of vitality, appetite, hair coat, and dehydration were recorded along with the dose. As a control, 0.1 g of anhydrous crystalline maltose was orally administered to cattle once a day. Cattle were fed with feed containing no antibacterial agent and water was freely consumed.

  The experiment was mainly conducted by a veterinarian, and the dose of the HuIFN preparation was adjusted based on the body weight of each individual while observing the state of the patient, symptoms and side effects. Then, the first day of administration was defined as the first day, and the effectiveness of the therapeutic agent was determined based on the observation results on the 12th day from the day of administration. That is, the six general clinical findings were scored based on the criteria shown in Table 1, and the total clinical score of each cow was determined, and the average total clinical score of each administration group was determined. The percentage obtained by subtracting the average total clinical score from the first day of administration from the average total clinical score on the first day of administration to the average total clinical score on the first day of administration and dividing by the average total clinical score on the first day of administration is 100 ) The experimental results show that the improvement rate of 85-100% is “effective”, the improvement rate is less than 70-85% “effective”, and the improvement rate is less than 50-70% “slightly effective” The improvement rate was less than 50% and the improvement of diarrhea was not observed on the 3rd day after the start of administration, and it was predicted that it would be worsened. ”And“ effective ”were evaluated as having a therapeutic effect. In each HuIFN preparation administration group, the number of cattle determined to be highly effective and effective was divided by the total number of cattle used in the test group and multiplied by 100 to obtain the response rate (%). Furthermore, of the overall clinical score, only the stool score was separately compiled to obtain an average stool property score for each group, which was used as an index for improving stool properties. The overall clinical score in this experiment was calculated in accordance with the clinical trial implementation standard for antibacterial agents against bovine Escherichia coli diarrhea in the veterinary drug manufacturing approval application to the Ministry of Agriculture, Forestry and Fisheries of Japan. The results are shown in Table 2.

  As is clear from Table 2, when any of the HuIFN preparations were administered, a significant clinical score was improved as compared to the control group, and the digestive system in cattle infected with rotavirus was obtained by oral administration of HuIFN. It was confirmed that clinical symptoms such as diarrhea associated with the disease were improved. This improvement effect is most prominent when BALL-1 interferon (containing about 25% of subtype α8 in the total HuIFN-α mass and about 75% of subtype α2 in the total HuIFN-α mass) is administered. The success rate reached 80%. In the administration group of only leukocyte interferon or recombinant subtype α2, the response rate was as low as 40%, whereas BALL-1 interferon and recombinant subtype α8 were combined with total HuIFN- In the group administered with HuIFN preparation (subtype α8: subtype α2 ratio of 1: 9 to 10: 0) containing 10% or more of α mass, either leukocyte interferon or recombinant subtype α2 A higher response rate was obtained than that of the group administered only. Among them, BALL-1 interferon and recombinant subtype α8 contain 10 to 80% HuIFN-α in the total HuIFN-α mass, and recombinant subtype α2 contains 20 in the total HuIFN-α mass. In the group administered with a HuIFN preparation containing 90% to 90% (subtype α8: subtype α2 ratio is 1: 9 to 8: 2), a high response rate was obtained, and BALL-1 interferon and recombinant subtype A group to which a HuIFN preparation containing 20 to 40% of type α8 in the total HuIFN-α mass and 60 to 80% of recombinant subtype α2 in the total HuIFN-α mass (subtype α8: A particularly high response rate was obtained when the ratio of the subtype α2 was 1: 9 to 4: 6). The fecal property score data is not specifically shown, but the improvement of the fecal property score also showed the same result as the success rate. In addition, it was confirmed that the HuIFN preparation of the present invention showed a therapeutic effect equivalent to that of bovine gastrointestinal diseases infected with rotavirus against digestive diseases in cattle infected with coronavirus.

  During the course of this study, there were no particular side effects due to the administration of the HuIFN preparation, confirming that the therapeutic agent according to the present invention is extremely effective and safe for the treatment of bovine gastrointestinal diseases. It was. Furthermore, when a blood test was performed on some of these cows on the 30th and 60th days from the start of administration, no antibody production that was attributed to HuIFN-α was detected. This suggests that the therapeutic agent according to the present invention is safe for repeated administration to the same cow for the purpose of treating digestive diseases of the cow.

<Experiment 3>
Based on the above findings, confirm the therapeutic effect of the therapeutic agent of the present invention on a calf that is most affected by rotavirus infection and is the most economically problematic in the livestock industry. An experiment was conducted. That is, for the 140 calves (70 males and 70 females) within 6 months of age that have been confirmed to be rotavirus-infected and exhibit diarrhea symptoms, the HuIFN preparation ( 200 international units / g) was administered, and the effects and side effects were evaluated by the same evaluation method as in Experiment 2. In this experiment, the BALL-1 interferon preparation that showed the highest response rate in Experiment 2 was used, and the dosage was 0.5 international unit / kg body weight and 20 international unit / kg body weight. As a control group, anhydrous crystalline maltose 0.1 g was orally administered to cows once a day. The BALL-1 interferon preparation administration group used 60 calves (30 males and 30 females) each, and the control group used 20 calves (10 males and 10 females each). Table 3 shows the transition of the average overall clinical score, Table 4 shows the transition of the average fecal property score, and Table 5 shows the results of the therapeutic effect determination.

表３から明らかなように、平均総合臨床スコアは、対照群では、実験期間中変動はあったものの、改善は認められなかった。これに対して、ＢＡＬＬ−１インターフェロンの０．５国際単位／ｋｇ体重投与群では投与開始の日から３日目から、対照群に比して有意の改善を示し、投与開始の日から１２日目でも総合臨床スコアの悪化は認められなかった。また、ＢＡＬＬ−１インターフェロンの２０国際単位／ｋｇ体重投与群では投与開始の日から４日目から、対照群と比して有意の改善を示し、投与を開始の日から１２日目でも総合臨床スコアの悪化は認められなかった。

As can be seen from Table 3, the mean overall clinical score did not improve in the control group, although it varied during the experiment. In contrast, in the 0.5 international unit / kg body weight administration group of BALL-1 interferon, it showed a significant improvement compared to the control group from the third day from the administration start date, and 12 days from the administration start date There was no deterioration in the overall clinical score in the eyes. In addition, the BALL-1 interferon 20 international unit / kg body weight administration group showed a significant improvement compared to the control group from the 4th day from the start date of administration, and the comprehensive clinical study was also performed on the 12th day from the start date. No worsening of the score was observed.

  As is apparent from Table 4, the average fecal property score did not improve in the control group, although it varied during the experiment. In contrast, in both the 0.5 international unit / kg body weight administration group and the 20 international unit / kg body weight administration group of BALL-1 interferon, from the second day from the start of administration, compared to the control group There was a significant improvement, and no deterioration was observed even on the 12th day from the start of administration.

  As is apparent from Table 5, the response rate was 63% in the BALL-1 interferon 0.5 international unit / kg body weight administration group, and the response rate was 57% in the 20 international unit / kg body weight administration group. In addition, no particular side effects were observed during the administration period, confirming that the therapeutic agent according to the present invention is extremely effective and safe for the treatment of bovine digestive system diseases. In addition, as in Experiment 2, for some patients, blood tests were carried out on the 30th and 60th days from the start of administration, and no antibody production that could be attributed to BALL-1 interferon was detected. It was.

  From these experiments, it is confirmed that the HuIFN preparation of the present invention is an excellent diarrhea symptom improving agent that can already improve the diarrhea symptom on the second day from the start of administration by oral administration. In addition, since no worsening of clinical symptoms was observed even on the 12th day from the start of administration, it was confirmed that it has an excellent therapeutic effect as a therapeutic agent for digestive diseases of infected bovines such as rotavirus and coronavirus. It was done.

<Acute toxicity test>
A solution obtained by dissolving bovine serum albumin to a concentration of 0.1% in physiological saline containing 0.01 M phosphate buffer (pH 7.0), and this solution was derived from BALL-1 cells prepared in Example 1. Partially purified HuIFN-α, purified HuIFN-α, partially purified HuIFN-α derived from human leukocytes prepared in Experiment 1, purified HuIFN-α, or purified subtype α2 or subtype α8 prepared in Example 2 described later A solution was prepared by dissolving any one of the above to 1 × 10 ⁶ international units / ml. The seven types of solutions thus obtained are 7 groups consisting of 5 healthy cows / group estimated to be 2 to 3 years of age, and 1 × 10 6 corresponding to 20,000 times the maximum dose in the present invention per kg body weight. An amount of physiological saline equivalent to ⁶ international units of HuIFN was administered subcutaneously or orally, and the course after administration was observed. Furthermore, the same amount of physiological saline prepared as described above was administered to the control group by the same route except that HuIFN was not dissolved.

  As a result, any group administered with HuIFN, a group administered with a solution in which bovine serum albumin was dissolved to 0.1% in physiological saline containing phosphate buffer, and physiological saline were administered. In the group, no clinical symptoms that were recognized as side effects were observed. In addition, these dogs did not show any particular changes in death, general condition, body weight, or food intake even after administration. The results of this acute toxicity test and the above-mentioned Experiment 2 and Experiment 3 suggest that HuIFN is extremely low toxicity when administered to cattle.

  Hereinafter, based on an Example, this invention is demonstrated concretely. However, the present invention is not limited to these examples.

In accordance with the method described in Japanese Patent Publication No. 56-54158, HuIFN-α was induced by causing Sendai virus to act on BALL-1 cells grown in hamsters in a culture medium. The supernatant obtained by centrifuging this culture medium is concentrated, and affinity chromatography using phenyl sepharose is applied to the concentrate to obtain partially purified HuIFN-α having a specific activity of about 10 ⁶ international units / mg protein. Obtained. Next, this partially purified HuIFN-α was further purified by affinity chromatography using “NK-2 Sepharose” manufactured by Celltech, in which a monoclonal anti-HuIFN-α antibody was bound to a water-insoluble carrier. By applying gel filtration chromatography equilibrated with phosphate buffer (pH 7.0) containing about 0.1 mg / ml serum albumin, purified HuIFN-α with a specific activity of about 2 × 10 ⁸ international units / mg BALL-1 interferon solution containing When this product was analyzed by high performance liquid chromatography, it contained about 25% of subtype α8 and about 75% of subtype α2 with respect to the total mass of HuIFN-α.

  The purified HuIFN-α derived from BALL-1 thus prepared was concentrated to about 5 mg protein / ml, and 1% (w / v) bovine serum albumin and 0.01 M phosphate buffer (pH 7.0) were added. The diluted physiological saline was diluted to a HuIFN-α concentration of 150 international units / ml, sterilized by membrane filtration, and aseptically filled into vials 1 ml each, and lyophilized.

  Since this product contains purified HuIFN-α derived from BALL-1 cells as an active ingredient and bovine serum albumin is used as an excipient, it is useful as an injection for treatment of digestive diseases in cattle It is.

<Recombinant interferon α2 and α8>
According to the method of Experiment 1-1 (a) and Experiment 1-1 (b) of Patent Document 3, recombinant subtype α8 (specific activity, about 3 × 10 ⁸ international units / mg protein) and recombinant type A purified sample of subtype α2 (specific activity, approximately 8 × 10 ⁷ international units / mg protein) was prepared.

  After the subtype α8 preparation and the subtype α2 preparation thus prepared were concentrated to about 5 mg protein / ml, 1 part by mass of subtype α8 and 4 parts by weight of subtype α2 were mixed. Diluted in physiological saline containing% (w / v) pharmacopoeia purified gelatin and 0.01M phosphate buffer (pH 7.0) to a HuIFN-α concentration of 200 international units / ml, sterilized by membrane filtration After that, the vials were aseptically filled in 1 ml portions and lyophilized.

  Although this product containing recombinant purified subtype α8 and subtype α2 as active ingredients is slightly inferior to the injection of Example 1 in therapeutic effect and side effects, it is useful as an injection for the treatment of digestive diseases of cattle It is.

  Purified BALL-1 interferon prepared by allowing Sendai virus to act on BALL-1 cells in the same manner as in Example 1, 3% (w / v) bovine serum albumin and 0.1 M phosphate buffer (pH 7.0). The diluted physiological saline was diluted to a HuIFN-α concentration of 2,000 international units / ml, sterilized by membrane filtration, filled in 1 ml vials, and lyophilized.

  This product contains purified HuIFN-α derived from BALL-1 cells as an active ingredient and the bovine serum albumin is derived from bovine, as in the case of the injection of Example 1. It is useful as an injection for treatment of digestive system diseases.

  1% (w / v) bovine serum albumin was dissolved in distilled water, and a solution containing BALL-1 cell-derived partially purified HuIFN-α prepared by the method of Example 1 was added to the solution, and HuIFN-α per gram. A HuIFN-α solution containing 300 international units was obtained. To 32.0 parts by mass of this solution, 0.5 part by mass of “Tween 80” was dissolved and mixed in 15.0 parts by mass of ethanol, and 0.5 parts by mass of triethanolamine was further added to the mixture. After dissolving and mixing in 0 parts by mass of glycerin, a small amount of triethanolamine was added to obtain a nearly neutral gel-like preparation containing about 100 international units of HuIFN-α per ml.

  This product containing HuIFN-α derived from BALL-1 cells as an active ingredient is a gel-type oral preparation for treatment of digestive tract diseases that has an appropriate intraoral residence time and is easy to swallow.

2 parts by mass of purified HuIFN-α (approximately 10 ⁸ international units / ml) derived from BALL-1 cells prepared by the method of Example 1 in 100 parts by mass of anhydrous crystalline maltose (trade name “FineTose”, sold by Hayashibara Corporation) Were uniformly sprayed, vacuum dried, pulverized, and sieved to obtain a powdery product having a particle size of 100 to 500 microns. An appropriate amount of “Fine Tose” was further uniformly mixed with the powder to obtain a powder containing about 200 international units of HuIFN-α per gram.

  This product has a moderate oral residence time and excellent storage stability, and is useful as a therapeutic agent for digestive diseases of cattle. This product has moderate sweetness, is a powder that is less irritating even when administered into the mouth of cattle and is easy to swallow.

  An aqueous solution (about 1,000 international units / ml) containing partially purified HuIFN derived from BALL-1 cells prepared by the method of Example 1 in 100 parts by mass of anhydrous crystalline maltose (trade name “Finetose” sold by Hayashibara Corporation) ) After uniformly mixing 15 parts by mass, the mixture was put into a tableting machine to obtain granules.

  This product contains about 125 international units of HuIFN-α per gram, has excellent storage stability, and is useful as a powder for treating digestive tract diseases in cattle. In addition, this product is a granule that has moderate sweetness, is less irritating even when administered into the oral cavity of cattle, and is easy to swallow.

  Anhydrous crystalline maltose (Hayashibara Shoji Co., Ltd., trade name “Finetose”) 100 parts by weight of commercially available HuIFN-β preparation (Mochida Pharmaceutical Co., Ltd., trade name “6 million IU for injection”) distilled water 2 parts by mass dissolved in 1 ml were sprayed uniformly, vacuum dried, pulverized, and sieved to obtain a powdery product having a particle size of 100 to 500 microns. An appropriate amount of “Fine Tose” was further uniformly mixed with the powder to obtain a powder containing about 50 international units of HuIFN-β per gram.

  Using this product, using 30 cows estimated to be 1 to 6 years old infected with rotavirus according to the method of Experiment 3 and exhibiting diarrhea symptoms, 20 international (IU) units / When kg body weight was administered for 14 days, the response rate was 43%, and this product showed a significantly higher response rate compared to the non-administration group (20 animals, response rate 30%). It was judged that there is a therapeutic effect on gastrointestinal diseases including improvement.

  In addition, using 30 cattle estimated to be 1 to 6 years old infected with rotavirus and exhibiting diarrheal symptoms according to the method of Experiment 3, this product was 10 international (I.U.) units / kg body weight. And the subtype α8 preparation prepared in Experiment 1 was administered at 10 international units / kg body weight for 10 days. The response rate was 52%, and the combination of this product and BALL-1 interferon was applied to 30 cattle. Since the α8 preparation showed a significantly higher response rate compared to the group administered 20 international units / kg body weight (response rate 45%) and the non-administration group (20 animals, response rate 30%), It was judged to have a therapeutic effect on digestive system diseases including improvement of diarrhea.

  This product has moderate oral residence time and excellent storage stability. Moreover, the above test results show that this product is useful as a treatment agent for bovine digestive tract diseases, and that this product can enhance the therapeutic effect of the HuIFN-α formulation when used in combination with the HuIFN-α formulation. It turns out that you can. In addition, this product has moderate sweetness, is a powder that is less irritating even when administered to the mouth of cattle and is easy to drink to livestock.

  The HuIFN contained in the therapeutic agent of the present invention as an active ingredient exerts a remarkable effect even in small doses to livestock. Therefore, in the case of small dose administration, the economic burden on the owner is greatly reduced. For the same reason, when using the therapeutic agent of the present invention, it is not always necessary to administer interferon into the blood vessel of a patient, and it is administered parenterally by routes other than blood vessels such as oral administration, intradermal, subcutaneous, intramuscular. In this way, the desired effect can be obtained. As a result, a predetermined amount of HuIFN can be reliably and easily administered to a cattle that is nervous and vigilant, and the labor and burden of treatment personnel such as veterinarians are greatly reduced. In addition, since the therapeutic agent of the present invention exerts a remarkable therapeutic effect even in calves, the economic loss due to calf diarrhea can be greatly reduced. The present invention having such excellent effects contributes to the livestock field, and its industrial significance is extremely large.

Claims (5)

  The active ingredient consists essentially of human interferon α derived from BALL-1 cells, which is a human lymphoblastoid cell line. The human interferon α is a human interferon α induced by acting Sendai virus on BALL-1 cells. Human interferon α obtained by purification by affinity chromatography using a monoclonal anti-human interferon α antibody, comprising about 75% by mass of subtype α2 and about 25% by mass of subtype α8 in the total mass of interferon α protein. The mean fecal property score of cattle with diarrhea associated with rotavirus infection in cattle, wherein the excipient is mainly maltose and contains about 200 international units / g of interferon α, is less than 1.0 An oral preparation used to improve stool properties.   The oral preparation according to claim 1, wherein the dosage form is 0.1 to 50 international units / kg bovine body weight / dose.   The oral preparation according to claim 2, comprising about 1 ng / g of interferon α protein.   The oral preparation according to any one of claims 1 to 3, which is in the form of a powder, a troche, a granule or a tablet. The active ingredient consists essentially of human interferon α derived from BALL-1 cells, which is a human lymphoblastoid cell line. The human interferon α is a human interferon α induced by acting Sendai virus on BALL-1 cells. Human interferon α obtained by purification by affinity chromatography using a monoclonal anti-human interferon α antibody, comprising about 75% by mass of subtype α2 and about 25% by mass of subtype α8 in the total mass of interferon α protein. , excipient mainly maltose, and bovine who developed diarrhea with oral preparation containing about 200 international units / g interferon α rotavirus infection of cattle characterized by oral administration to cattle How to improve fecal stool properties with an average stool property score of less than 1.0 .