Immunopotentiator comprising peptide and hyaluronic acid

Abstract: An immunopotentiator containing peptide and hyaluronic acid extracted from chicken comb or mammalian skin or organ is provided, the immunopotentiator having a great immunopotentiation effect on biological organisms; additionally, a method for preparing the immunopotentiator is provided, comprising immersing and swelling chicken comb or mammalian skin or mammalian organ in a minced state and hydrolyzing the protein in the mincemeat to peptide.

Description: The present invention relates to a method for preparing an immunopotentiator enhancing the immunopotency of biological organisms. Biological organisms essentially have immunopotency, but the intensity of the immunopotency varies, depending on individual differences and conditions at a certain time. The present inventors have made investigations about hyaluronic acid for a long time and have found that the simultaneous administration of hyaluronic acid and peptide can enhance the immunopotency of biological organisms. Uhlin et al., Int. J. Immunopharmac. 1982, Vol. 4, No. 6, 529-532 disclose compositions comprising complexes of hyaluronic acid and egg albumin, dog albumin or birch pollen extract proteins. CN-A-1063692 discloses a method for extracting hyaluronic acid from pork skin, which method comprises mincing the pork skin, mixing it with water and neutral protease, carrying out enzymolysis at 30 to 50°C at pH 8.5 and then filtering the obtained enzymatic liquid. JP-A-59116204 discloses a method, wherein cock's crest or naval cord is decomposed with an enzyme and the resulting decomposition product is extracted. JP-A-58037001 discloses the extraction of hyaluronic acid from the crest of birds, joint, vitreous body, cartilage of cow, horse, or goat by heating said material in water to 70 - 100°C and then mincing the material, disintegrating the minced material by the action of a proteolytic enzyme and then extracting to obtain hyaluronic acid. WO-A-98/52605 discloses an immunopotentiating substance comprising an antigen, which may be a peptide, and a carrier comprising a biocompatible material, which may be hyaluronic acid, and a method for producing antibodies using said substance. WO-A-00/12122 discloses systems containing an antigen or peptide, optionally together with a carrier system, and coupled thereto a low molecular hyaluronic acid-fragment, wherein the fragments of hyaluronic acid are generated by treating hyaluronic acid with hyaluronidase. In view of the above it is an object of the present invention to provide a method for preparing an immunopotentiator, which can enhance the immunopotency of biological organisms. The above object is achieved by the method of Claim 1. The immunopotentiator prepared according to the present invention exerts a great immunopotentiation effect in biological organisms. Additionally, because chicken comb contains protein and hyaluronic acid abundantly, both the protein and hyaluronic acid are simultaneously extracted from the same source of chicken comb, satisfactorily. Compared with a process of separately extracting peptide and hyaluronic acid and mixing them together, the process of the present invention does not include a useless step but is highly safe because chicken comb is used as a raw material. Fig. 1 shows bar graphs depicting test results; Fig. 2 shows bar graphs depicting test results; Fig. 3 (a) and (b) show plain views depicting tumor-transplanted sites and the immunopotentiator-dosed sites in mice; and Fig. 4 shows graphs depicting test results. The method for preparing the immunopotentiator from a raw material for example chicken comb according to the present invention is now described specifically. A. Chicken comb is generally in a frozen state, thus chicken comb is firstly thawed. The thawed comb is then rinsed with distilled water dissolving therein 0.1 % chlorine. Then, the rinsed comb is minced with a mincing machine. B. The mincemeat is placed in a reaction oven, followed by addition of caustic soda; and the resulting mixture is left to stand for about 15 hours. In this manner, the mincemeat is sufficiently swollen. C. Hydrochloric acid is added to the swollen mincemeat to a final pH of 7-8; protease as the enzyme for decomposing proteins is added at 0.5 % to the resulting mincemeat for enzymatic decomposition (hydrolysis) for 2.5 hours; then, the mixture is preliminarily filtered through a 50-mesh screen; to the filtrate is additionally added celite as a filtration auxiliary agent for prevention of clogging, followed by a second filtration through a 10-micron filter paper. D. Active charcoal is added at 0.5 % to the filtrate. The filtrate is deodorized and decolored with the active charcoal, which is then filtered through a 1-micron filter paper to remove the active charcoal. After the completion of the process D, the resulting filtrate corresponds to the immunopotentiator containing peptide and hyaluronic acid as the effective components. The immunopotentiator can be given orally or parenterally in appropriate dosage forms (for example, liquids, granules, tablets, capsules, etc.) suitable for individual dosage recipes. An immunopotentiation test using the immunopotentiator prepared according to the present invention is now described below. [Cellular immunopotentiation test 1] 40 µl of 2.5 × 10 8 cells/ml sheep red blood cell (SRBC) was subcutaneously injected to left pads of ICR mice (age 6 to 8 weeks; male; 7 animals per group) by using a microsyringe, for antigen sensitization. Six days after sensitization, 40 µl of 2.5 × 10 9 cells/ml SRBC was subcutaneously injected to the right pads; 24 hours later, the size of edema on the right pads was measured with a dial thickness gauge (0.01 mm). The immunopotentiator was intraperitoneally dosed at 100 mg/kg one hour prior to the antigen SRBC sensitization. To a control group, physiological saline was dosed at the same dose. The results are shown in the graphs of Fig. 1. [Test results] In the control mouse group, the swelling degree was at 16.2 ± 2.4 %, while in the immunopotentiator-dosed group, the swelling degree was at 30.1 ± 3.5 %. The degree of swelling was increased with a significant difference (p < 0.05). In the test, a delayed dermal reaction was examined; T lymphocyte sensitized with the preliminarily injected red blood cell was allowed to react with the antigen (red blood cell boosted at a second time) 6 days later, to consequently release lymphokine, so that macrophage and white blood cell were activated for infiltration, leading to the emergence of edema. In the test, strong swelling was apparently observed in the immunopotentiator-dosed group. Thus, it is indicated that cellular immunity was enhanced. [Cellular immunopotentiation test 2: oral dosing] 40 µl of 4.0 × 10 8 cells/ml sheep red blood cell (SRBC) was subcutaneously injected to left pads of ICR mice (age 6 to 8 weeks; male; 7 animals per group) by using a microsyringe, for antigen sensitization. Four days after the sensitization, 40 µl of 4.0 × 10 9 cells/ml SRBC was subcutaneously injected to the right pads; 24 hours later, the size of edema on the right pads was measured with a dial thickness gauge (0.01 mm). The immunopotentiator was orally dosed at 500 mg/kg continuously six times, prior to the sensitization. To a control group, alternatively, physiological saline was dosed at the same dose. The results are shown in the graphs of Fig. 2. [Test results] In the control mouse group, the swelling degree was at 29.3 ± 4.5 %, while in the immunopotentiator-dosed group, the swelling degree was at 65.5 ± 10.3 %. The degree of swelling was increased with a significant difference (p < 0.05). Thus, immunopotentiation via oral dosing was observed at the test. [Somatic immunopotentiation test] So as to examine somatic immunity, the enhancement of the generation of immunoglobulin (IgM, IgG) was confirmed. [Test method] The immunopotentiator was intraperitoneally dosed at 300 mg/kg in ICR mice (of body weight about 30 g). One hour later, the mice were sensitized with % SRBC at 0.4 ml/kg body weight. Six days later, blood was drawn, from which serum was separated to recover anti-serum; aggregation was confirmed on a 96-well plate to determine the antibody titer. To the mice in a control group, physiological saline in place of the immunopotentiator was dosed, to recover the sera similarly. The results are shown in Table 1. [Test results] Compared with the control group, both the antibody titers of IgM and IgG in the mice in the immunopotentiator-dosed group were significantly enhanced (p < 0.05). In the test, the antibody titer was distinctively elevated in the immunopotentiator-dosed group, which indicates enhancement of antibody generation potency. [Anti-tumor test] Meth-A fibreosarcoma was transplanted subcutaneously at 1 × 10 6 cells and 2 × 10 5 cells in the right lower abdominal part and left lower abdominal part, respectively, of BALB/c mouse (age 7 weeks), as shown in Fig. 3 (a). The immunopotentiator was dissolved in physiological saline and then administered at a dose of 1 mg/kg/0.1 ml/mouse into only the right tumor transplanted, three times continuously on days 3, 4 and 5 after tumor transplantation. The long diameter and short diameter of the right and left tumors were measured over time; the size of the tumors was compared with the size in the control group dosed with physiological saline. The results are shown in Table 2 and the graphs of Fig. 4. Test results of the dosing of the immunopotentiator into Meth-A fibreosarcoma (solid tumor) in BALB/c mouse Dose in tumor (mg/animal) × days Dosing period (days) Animal number (animals) Mean tumor weight (g ± SE) Tumor suppression ratio (%) Dosing group 1 × 3 3 to 5 9 0.589 ± 0.231 52.5 Control group 0 × 3 3 to 5 10 1.240 ± 0.329 - * Tumor cell (1 × 10 6 ) was subcutaneously transplanted (on day 0). * The immunopotentiator was dosed into the tumor on the right side on days 3, 4 and 5. Although the immunopotentiator was dosed into only the tumor on the right side, it was observed that the tumor growth was suppressed on the tumors on both the right and left sides. This indicates that the anti-tumor action of the immunopotentiator includes a direct suppressive action and an action suppressing remote tumor. Thus, it is indicated that host immune function is possibly activated in a certain mode. The foregoing description of the preferred embodiments of the invention has been presented for purposes of illustration and description only. It is not intended to be exhaustive or to limit the invention to the precise form disclosed, and modifications and variations are possible in the light of the above teachings or may be acquired from practice of the invention. The embodiments are chosen and described in order to explain the principles of the invention and its practical application to enable one skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated.

Claims: A method for preparing an immunopotentiator, wherein the method comprises the steps of rinsing chicken comb with distilled water, wherein 0.1 % chlorine are dissolved; mincing the chicken comb with a mincing machine; placing the minced chicken comb in a reaction oven, adding caustic soda to the minced chicken comb, and leaving the resulting mixture to stand for about 15 hours; adding hydrochloric acid to the swollen minced chicken comb to a final pH of 7 to 8; adding protease to the resulting minced chicken comb at 0.5 % for 2.5 hours to achieve an enzymatic decomposition; filtering the obtained mixture through a 50-mesh screen; adding celite to the obtained filtrate, followed by a filtration through a 10-micron filter paper; adding active charcoal at 0.5 % to the obtained filtrate; and filtering the obtained mixture through a 1-micron filter to remove the active charcoal, wherein the thus obtained filtrate corresponds to the immunopotentiator.

Inventors: HAYASHI SEIJI, HAYASHI, SEIJI, Hayashi, Seiji, c/o Adaputogen Seiyaku K.K.
Applicants: ADAPUTOGEN SEIYAKU K K, ADAPUTOGEN SEIYAKU K.K.
Application year: 2001
Main classification: A61K31
IPC codes: A61P35, A61K38, A61K31, A61P37, A61K35