JP2672303B2 - Infection prevention and treatment for horses - Google Patents

Description

TECHNICAL FIELD OF THE INVENTION The present invention relates to highly purified equine immunoglobulin (Ig
The present invention relates to a preventive / therapeutic agent for foal, which contains G) and has a preventive or curative effect on the infection of foals. Conventional technology and its problems In the horse, the maternal immune antibody does not transfer to the foal through the placenta, and the antibody contained in the colostrum after birth is orally ingested to establish the maternal-fetal immunity. This maternal and child immunity is about 24 after birth
Although it is established within the time, the foal needs to ingest a sufficient amount of colostrum containing sufficient antibody during this period. If there is little or no antibody in the colostrum of the mare, or
Foals may be exposed to a variety of illnesses due to inadequate breastfeeding due to maternal accidents (eg, dystocia, mastitis, amyloidosis) or inability of foals to receive colostrum. become. McGuine et al. (J.Am.Vet.Med.Assoc., 170: 1302-1304 (1
977)) considers that the blood IgG concentration in the foal after feeding is 4 g / L or less as a criterion for colostrum immunoglobulin-passed deficiency, and 2-4 g / L is partially deficient. . More than 20% of foals with passive pass-through are present in Thoroughbreds, and about 10 foals with a blood IgG concentration of 2 g / L or less are closely associated with the occurrence and death of "foal disease". There is also around%. Among foals with severe combined immunodeficiency, those with colostral immunoglobulin-passed impaired deficiency develop onset at 4-8 days of age and die at 21-25 days, but those with normal passive imputation , An average of 23 days old and alive up to 59 days old. At present, colostrum bank or serum bank is considered as a preventive or therapeutic method for infections due to poor immunity antibody transfer, but there is a possibility of serotype atypical transfusion, and it is recommended that the serotype be transferred from the mother horse. Since it is limited, its range of application is limited. Means for Solving the Problems, Effects of the Invention Under such circumstances, the present inventors would like to have the above-mentioned problems if immunoglobulin (IgG) derived from equine blood itself could be administered to the foal. As a result of repeated studies, it was possible to complete the present invention by purifying highly purified immunoglobulin IgG derived from equine blood without specific immunization. Thus, according to the present invention, a prophylactic treatment for foals with follicular colostrum immunoglobulin-passed deficiency, which can be orally or intravenously administered, containing immunoglobulin G (IgG) derived from horse blood without specific immunization. An agent is provided. Thus, a preparation in which horse IgG was purified to a high degree of purity has never existed before. In the present invention, as a raw material, serum or plasma aseptically collected from a horse without specific immunization can be used, and this raw material may be obtained from a single horse or serums of a plurality of horses or It may be a pool of plasma. According to the present invention, immunoglobulin is highly purified from horse serum or horse plasma as follows. After appropriately diluting the raw material horse serum or horse plasma to adjust the protein concentration, add alcohol while cooling, and repeat the precipitation fraction to repeat the fibrinogen, albumin fraction, α-globulin fraction and β-globulin fraction. Remove minutes.
The alcohol used in the precipitation fraction fractionation step is removed from the purified immunoglobulin fraction. At this time, a mild method such as dialysis is preferred to prevent denaturation of the immunoglobulin. The purified immunoglobulin fraction is dissolved in physiological saline to a desired protein concentration, preferably 4 to 15%. The immunoglobulin solution is like a human body preparation,
A stabilizer, such as amino acids, saccharides, or protein components, may be added alone or as a mixture to give a liquid preparation or a lyophilized preparation. Further, in preparing the prophylactic / therapeutic agent for foal infection of the present invention, it is preferable to heat-treat the raw material (horse blood, horse plasma), the purified immunoglobulin-containing liquid or the lyophilized immunoglobulin. Such heat treatment can inactivate viruses that may get lost. The heat treatment condition is 50 to 60 ° C when liquid is used.
At 0.5 to 10 hours, preferably at 56 to 58 ° C for 1 to 3 hours, and when performed in a dry state at 60 to 70 ° C for 70 to 120 hours,
It is preferably at 65 to 68 ° C. for 90 to 100 hours. The immunoglobulin IgG obtained according to the present invention has a purity of 95.
In addition, the IgG content is 95% by weight or more as a monomer. Such a formulation also passed various standards such as an exothermic test. The administration method of the preparation according to the present invention is not particularly limited. Oral administration may be carried out within 24 hours after birth when the ability of the foal to absorb immunoglobulin in the intestinal tract is maintained, and thereafter, intravenous administration is preferable. In this way, according to the present invention, by administering the preparation of the present invention to a foal having an early immunoglobulin passive impaired deficiency, infection of various diseases can be prevented, or such an infection can be prevented. The symptoms caused by can be reduced and cured. Examples are shown below, but the present invention is not limited thereto. Example 1 40 L of horse blood collected aseptically in the presence of an anticoagulant was centrifuged to obtain about 20 L of plasma. This was diluted three times with purified water, adjusted to an ionic strength of 0.03 and pH 7.0, and ethanol was added to a final concentration of 8% to remove the fibrinogen fraction. -5 to -6 in the supernatant after centrifugation
Ethanol was added at 20 ° C. to a final concentration of 20%, and the centrifugation precipitate was recovered. By this operation, the albumin fraction was mainly removed. The recovered precipitate was suspended in sodium acetate solution, adjusted to pH 5.1-5.2 and ionic strength 0.013, and then ethanol was added to a final concentration of 18%, and the precipitate was removed by centrifugation. The resulting supernatant was ionic strength 0.05,
Adjust the pH to 7.0 and adjust the ethanol concentration to 25%.
Fractionate at 7 ℃ and centrifuge to remove immunoglobulin precipitates.
I got 300g. The immunoglobulin precipitate was dissolved in physiological saline, and the remaining ethanol was removed by dialysis using an artificial kidney. The immunoglobulin solution was heated at 57 ° C for 2 hours. Add glycine and mannitol as stabilizers,
The protein concentration was adjusted to about 5% and sterile filtration was performed. It was dispensed and freeze-dried to obtain 2.5 g of the final product, 25 bottles. This product was dissolved in 50 mL of distilled water for injection, and the purity and monomer content were determined by Separax membrane and sodium diethylbarbitate buffer solution (pH 8.6).
It was examined by using an HPLC composed of a W3000G column (Toyo Soda). The results are shown in the filter paper migration chart of Fig. 1 and the HP of Fig. 2.
As shown in the LC (High Performance Liquid Chromatography) chart, the immunoglobulin purity was 97%, the monomer content was 96%, and it was confirmed that it was highly purified and had no denaturation during the process. Example 2 Using 200 L of horse blood plasma (35 pools for 35 horses) collected in the same manner as in Example 1, fractionation, dealcoholization and aseptic filtration were carried out, followed by dispensing, lyophilization, and final product 2.5 g / I got 260 vial. The same test as in Example 1 was performed and the same test results were obtained. Example 3 For the purpose of confirming the safety of highly purified equine immunoglobulin prepared according to Example 1 above, a fever test (rabbit),
An abnormal toxicity denial test (guinea pig), a sterility test, a mouse acute toxicity test, and a blood pressure reduction test in Beagle dogs were performed. As the results are shown in Table 1, all the tests were satisfied, and the safety of this drug was confirmed. Example 4 The highly purified immunoglobulin prepared according to the above Example 2 was administered to a foal immediately after birth, and its safety was examined.
Two foals were used. On the day of birth and on the day after birth, 2.5 g of each, 2 in total, 4 in total, 10 g of immunoglobulin was administered. Observation begins 1 hour before administration and 6 after administration
By observing every 30 minutes until time, body temperature, respiratory rate, heart rate, etc. were measured. No notable changes were observed in any of the items, confirming the safety of this drug. Example 5 For the purpose of confirming the effectiveness of the equine immunoglobulin preparation prepared in Example 2, an administration test was conducted on a foal in which a cord was infected with a foal. The foal was born by spontaneous birth and had a low colostrum intake and thus had a colostrum-immunoglobulin passive impaired deficiency. It was accompanied by swelling. As a result, he continued to have a high fever, had no appetite, and was in a state of energy. Therefore, 5 g of the equine immunoglobulin preparation of the present invention was intravenously injected. From the first day after the injection, the fever began to fall and my appetite was restored and my energy recovered. Five days after the injection, 5 g of this drug was intravenously administered again to maintain immunoglobulin levels. As a result, from about 7 days after the injection, umbilical cord inflammation, arthritis and swelling were alleviated, and motor function similar to that of a normal foal was recovered. After that, he saw a good recovery and lived a normal life. Therefore, the effectiveness of the equine immunoglobulin preparation of the present invention was confirmed.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the immunoglobulin (Ig
2) is a filter paper migration chart showing the purity of G),
The figure is an HPLC chart showing the content of immunoglobulin (IgG) monomers in the preparation of the present invention.

   ────────────────────────────────────────────────── ─── Continuation of front page    (72) Inventor Kei Taneno               Kumamoto City, Kumamoto Prefecture 6-2-2 Black Hair (72) Inventor Masanobu Eto               Kumamoto City, Kumamoto Prefecture 4-75, Black Hair                (56) References "Infection and Im               unity ”Vol. 47 No. 3                 (1985) PP. 623 ~ 628

Claims (1)

(57) [Claims] A prophylactic / therapeutic agent for a foal having a deficiency of colostrum immunoglobulin-passed deficiency, which can be orally or intravenously administered and contains immunoglobulin G (IgG) derived from horse blood without specific immunization. 2. The prophylactic / therapeutic agent for foal infection according to claim 1, comprising the immunoglobulin G (IgG) in a purity of 95% by weight or more, and the monomer content of the IgG is 95% by weight or more. . 3. The preventive and therapeutic agent for foal infection according to claim 1, wherein the immunoglobulin G (IgG) is contained in a liquid state or a lyophilized state. 4. The foal infection preventive / therapeutic agent according to claim 1, wherein the immunoglobulin G (IgG) is heat-treated in a liquid form or a dried form.