CN1110323C - Rotavirus resisting immune globulin composite and its preparation and use - Google Patents

Abstract

The invention relates to an anti-rotavirus immunoglobulin composition, a preparation method thereof and an application for treating rotavirus diarrhea. The composition is an enteric solution, and contains effective doses of anti-rotavirus specific immunoglobulins and a pharmaceutically acceptable carrier, wherein the immunoglobulins are obtained from specific pathogen-free chickens after booster immunization. The composition can be used for preventing or treating rotavirus diarrhea, and has the advantages of high efficiency, safety, and convenience for children to take.

Description

Anti-rotavirus immunoglobulin composition and its preparation method and application

The invention relates to an immunoglobulin composition, a preparation method thereof and an application for preventing and treating rotavirus diarrhea.

Rotavirus diarrhea in infants and young children is a worldwide disease. According to the statistics of the United Nations Health Organization, in developing countries (not including China), the annual incidence of rotavirus diarrhea in children under the age of 5 is 125 million, of which 18 million are severe cases, and the death cases are as high as 8. 170,000. In the United States, there are 3 million infants with rotavirus diarrhea and 82,000 hospitalizations each year. In our country, there are about 350 million infant diarrhea cases every year, and about 40-50% of the cases are caused by rotavirus. What is more noteworthy is that there is currently no specific drug for the treatment of rotavirus infection other than supportive therapy.

In recent years, studies have found that oral immunoglobulin can effectively prevent and treat infantile rotavirus diarrhea. The results of field protective trials in the United States, Australia, Bangladesh and other countries have shown that oral administration of specific immunoglobulins can achieve complete protection against rotavirus diarrhea in infants and young children. The Phase II clinical trial report of the European Santos Pharmaceutical Company (SANDOZ) in Italy showed that in a randomized double-blind controlled trial, 98 children with rotavirus diarrhea in group A took oral immunoglobulin 300mg/Kg at one time, and the diarrhea time of the children It was shortened from an average of 131 hours to 76 hours, and the intestinal detoxification time was shortened from an average of 180 hours to 114 hours. The clinical effect was remarkable, and no toxic side effects were found (Guarino A, Canani RB, Russo S, et al. Oral immunoglobulins for treatment of acute rotaviral gastroenteritis. Pediatrics, 1994, 93(1), 12-6.). The results of a clinical study in Bangladesh showed that 35 children with rotavirus diarrhea were given oral antibody therapy in a randomized double-blind controlled trial. It was found that there was a significant difference between the treatment group and the placebo group in terms of shortening the course of the disease or reducing diarrhea. Differences (Mitra AK, Mahalanabis D, Ashraf H, et al. Hyperimmune cow colostrum reduces diarrhea due to rotavirus: a doubie-blind, controlled clinical trail. Acta Poediatr 1995, 84: 996-1001.). British patients with severe rotavirus diarrhea after bone marrow transplantation were treated with oral immunoglobulin. As a result, all children were cured within 72 hours (Kanfer EJ, Abrahamson G, Taylor J. et al. Severe rotavirus-associated diarrhea fol lowingmarrow transplantation : treatment with oral immunoglobul in. BoneMarrow Transplant, 1994, 14: (4) 651-2.). A Swedish study showed that oral immunoglobulin can significantly shorten the shedding period of rotavirus patients (Bogstedt AK, Johansen K, Hatta H, et al. Passive immunity against diabetes. 1996, 85: (2) 125-8. ). In addition, oral immunoglobulin can significantly shorten the clinical course of severe Crohn's enteritis (Jjellstr B, Stenhammar L, Magnusson KE. et al. Oral immunoglobulin treatment in Crohn's disease. Pcediatr, 1997, 86: (2) 221-3.). Clinical trials in the United States also found that for patients with advanced AIDS complicated by severe cryptosporidium diarrhea, oral immunoglobulin can significantly reduce the degree of diarrhea and improve clinical symptoms (Greenberg PD, Cello JP. Treatment of severe diarrhea caused by Cryptosporidium parvum with oral Bovine immunoglobulin concentrate in patients with AIDS. J AcquirImmune Defic Syndr Hum Retrovirol, 1996, 13: 4 348-54.). However, the oral immunoglobulin used in these studies has the following problems: ①The preparation cost of serum immunoglobulin and its monoclonal antibody and other products is high, and the yield is low, which cannot meet the high-dose requirements of oral administration. ②The use of immunoglobulin without booster immunization requires a large amount of antibody and the treatment effect is not good. ③ The non-protective immunoglobulin is degraded by gastric acid and digestive enzymes in the gastrointestinal tract after oral administration, resulting in a large dosage of immunoglobulin and a short half-life of the antibody. These disadvantages increase the production and treatment costs of immunoglobulins, limiting their clinical application.

The object of the present invention is to provide an anti-rotavirus immunoglobulin composition with low preparation cost and not damaged by gastric acid and gastric enzymes.

Another object of the present invention is to provide a method for preparing the anti-rotavirus immunoglobulin composition of the present invention.

Another object of the present invention is to provide the use of the immunoglobulin composition of the present invention in preventing and treating rotavirus diarrhea.

In order to achieve the above object, the inventors of the present invention obtained the following technical solutions through experimental exploration:

The immunoglobulin composition of the present invention comprises an effective amount of anti-rotavirus specific immunoglobulin and a pharmaceutically acceptable dressing.

In order to protect the oral immunoglobulin from the damage of gastric acid and gastric enzymes and to facilitate children's administration, the composition of the present invention is an enteric-coated composition, including enteric-coated granules, enteric-coated pellets and enteric-coated microcapsules.

The dressing in the above-mentioned enteric-coated pellets or enteric-coated granules contains enteric coating materials, plasticizers, anti-sticking agents, protein stabilizers, and binders. The content of anti-rotavirus specific immunoglobulin is 5-50% by weight of the composition; preferably 10-30%; most preferably 20%.

Enteric coating materials include hydroxypropylmethylcellulose phthalate, carboxymethylethylcellulose, acrylic resin No. II, acrylic resin No. III, cellulose acetate phthalate, shellac, Polyvinyl acetate phthalate, hydroxypropyl methylcellulose acetate succinate, etc., preferably acrylic resin No. II or acrylic resin No. III.

Suitable plasticizers include triethyl citrate, glyceryl triacetate, acetylated triethyl citrate, polyethylene glycol, diethyl phthalate, dibutyl phthalate, benzene Dibutyl formate, propylene glycol, etc., preferably dibutyl phthalate or triethyl citrate. Anti-sticking agents include magnesium stearate, talc, silicon dioxide, titanium dioxide, etc., preferably magnesium stearate.

Protein stabilizers include mannitol, lactose, albumin, sorbitol, xylitol, arabitol, etc., preferably using mannitol or albumin as a stabilizer.

The binder includes gelatin, sodium alginate, hydroxypropyl methylcellulose, gum arabic, etc., preferably sodium alginate as the binder.

Fillers include starch, lactose, sucrose, pregelatinized starch, and the like.

The above-mentioned enteric-coated microcapsule dressing includes capsule material. The content of anti-rotavirus specific immunoglobulin is 10-30% by weight of the composition.

The composition of the invention can be used for preventing and treating rotavirus diarrhea. The indication is rotavirus diarrhea in infants and young children.

The administration route of the composition of the present invention is oral administration. The dosage is 30-300 mg immunoglobulin/kg body weight.

The specific immunoglobulin composition against rotavirus of the present invention can be prepared by the following method. Using level II clean breeding conditions, rotavirus-enhanced immunization was carried out on specific pathogen-free (SPF) chickens; the specificity of anti-rotavirus was extracted from the eggs of the enhanced immunization by degreasing, salting out, desalting, concentration and chromatography Immunoglobulin (IgY): The specific immunoglobulin is prepared into an enteric-coated composition by using corresponding enteric-coated materials and technologies.

Its advantages are: ① the extraction technology of the present invention can make the purity of the immunoglobulin reach 90%; ② using SPF chicken as the immune medium can significantly reduce the background antibody in the egg and greatly reduce the irrelevant antibody in the immunoglobulin. Helps to obtain a large amount of uniform specific immunoglobulin against rotavirus. ③ Due to the adoption of SPF breeding technology, the risk of pathogen contamination of immunized chickens and eggs can be effectively reduced. Therefore, the biological safety of the prepared immunoglobulin is greatly improved.

The immunoglobulin in the composition of the present invention significantly improves the virus neutralization titer of the immunoglobulin due to the enhanced immunization; at the same time, the use of SPF technology greatly reduces the irrelevant antibody background and eliminates potential pathogenic microorganisms in the antibody preparation The enteric-coated chemical protective dosage form greatly improves the stability of the specific immunoglobulin dosage form, avoids the degradation of immunoglobulin by gastric acid and gastric enzymes, prolongs the half-life of immunoglobulin in the intestinal tract, and reduces irrelevant degradation in the intestinal tract . These measures have significantly reduced the actual dosage of immunoglobulin, thereby reducing the production cost of immunoglobulin and improving production efficiency.

The present invention will be described in further detail below in conjunction with examples, but these examples do not limit the present invention.

Preparation of immunoglobulin

Embodiment one.

1. Immunogen preparation:

1.1 The ATCC standard human rotavirus Wa strain, monkey SA11 strain, and bovine WC3 strain were used as virus production strains, and the culture host cells were MA-104 cell lines.

1.2 Continuously subculture MA-104 cells into a 2500ml rotary culture flask (DMEM medium containing 10% newborn calf serum, 1% antibiotics and 1.5% sodium bicarbonate), rotate at 37°C for 24 hours, and use 20 μg/ml Treat the cells with trypsin for 30 minutes, then add 5ml of virus solution to absorb for 1 hour, then suck off the virus solution, and continue culturing with maintenance solution (serum-free medium, containing 2 μg/ml trypsin) for 48-72 hours until the cytopathic effect reaches 75% Harvest the virus.

1.3 The virus culture medium was repeatedly frozen and thawed three times, intermittently sonicated for 2 minutes, centrifuged at 6000rpm for 15min, 8% PEG6000 was added to the supernatant, 4°C overnight, centrifuged at 8500rpm4°C for 20min, the precipitate was resuspended in 0.05MPBS, and loaded on Sepharose CL-4B row After gel filtration, the main peak was collected and concentrated by ultrafiltration (PELLICONCASSETTE, 100,000NMWL, MILLIPORE), and the concentrated solution was determined for immunology, protein concentration and CPE.

2. Animal immunity:

2.1 Use six-month-old specific pathogen-free (SPF) laying hens (standard level II clean animal room, three-level air filtration), and inject inactivated bivalent virus intramuscularly at multiple points in the chest, abdomen and inner thigh of each chicken solution + incomplete Freund's adjuvant 1.5-2.0ml (Wa+SA11 or Wa+WC3, CPE2×10 ^-5 each 0.5ml, mixed with an equal amount of Freund's incomplete adjuvant to emulsify). Booster immunizations are given every 2-4 weeks. Serum and yolk immunoglobulin titers were regularly tested.

3. Immunoglobulin Preparation

3.1 Aseptically treat the surface of hyperimmune eggs, homogenize after removing the egg white, add acidified sterile deionized water with a pH of 4.0-5.0 at a ratio of 1:6, let stand overnight at 4°C after homogenization, and add 20-50% to the supernatant Gradient precipitation of saturated ammonium sulfate, let stand at 4°C for 6h, centrifuge at 8000rpm for 20min, resuspend the precipitate with 0.0SM PBS, purify it by Sapharose S-300 column chromatography gel filtration, collect the immunoglobulin peak and concentrate it by ultrafiltration desalting ( PELLICON CASSETTE, 100,000NMWL, MILLIPORE).

3.2 Purified immunoglobulins were vacuum freeze-dried after adding protective agents for use.

Preparation of enteric-coated immunoglobulin compositions

Example 2: Preparation of enteric-coated granules

Formula and dosage:

  Rotavirus antibody freeze-dried powder                                          

Lactose 3000 grams

Gum Arabic Powder 500g

  Microcrystalline Cellulose                                                                                                           

2.5% Hydroxypropyl Methylcellulose 600g

                                           

5% Polyacrylic Resin No. II 800g

Magnesium stearate 100g

Preparation steps:

1. Preparation of rotavirus antibody particles

Weigh each dressing according to the dosage of the formula, pass through a 100-mesh sieve, then mix well, use an appropriate amount of binder solution to make it into a soft material, pass through a 14-mesh sieve, bake at 50°C for 4 hours, and granulate with a 14-mesh sieve. Pass through a 30-mesh sieve to remove fine powder.

2. Preparation of rotavirus antibody enteric-coated particles

Dissolve enteric coating material polyacrylic acid resin П in ethanol solution, add diethyl phthalate and magnesium stearate, and then coat by air suspension coating method. The rotavirus antibody enteric-coated particles were obtained.

Embodiment three, preparation of enteric-coated granule

Formula and dosage:

  Rotavirus antibody freeze-dried powder 800g

Lactose 800 grams

                                                                 

Mannitol 400g

Starch 500g

1% sodium alginate 440g

                                                         

5% Polyacrylic Resin No. III 630g

Talc powder 63 grams

Preparation steps: with embodiment two

Example 4: Preparation of enteric-coated granules

Formula and dosage;

  Rotavirus antibody freeze-dried powder                                                                                

Starch 120g

                                                                                            

Lactose 400 grams

10% gelatin 560 g

  Hydroxypropyl Methyl Cellulose Phthalate   27g

                                         

Titanium Dioxide 20g

The preparation steps are the same as in Example 2.

Example five: Preparation of enteric-coated granules

Formula and dosage:

  Rotavirus antibody freeze-dried powder 90g

Sorbitol 20 grams

                                                                 

  Microcrystalline Cellulose                                                                          

Lactose 20 grams

10% gelatin 32 g

  Cellulose acetate phthalate             3 g

                                                   

Magnesium stearate 4.3 grams

The preparation steps are the same as in Example 2.

Example 6: Preparation of rotavirus antibody enteric-coated pellets

Formula and consumption: with embodiment two to five;

Preparation:

1. Preparation of rotavirus antibody pellets

After mixing the rotavirus antibody and the dressing (through a 100-mesh sieve) according to the formula dosage for preparing granules, prepare a soft material with an appropriate amount of binder solution, pass through a 14-mesh sieve to obtain wet granules, and then place them in a coating granulator In medium, roll into round pellets.

2. Preparation of rotavirus antibody enteric-coated pellets

The rotavirus antibody pellets are placed in a coating granulator, coated with a coating material or a composite coating material, a plasticizer and an anti-adhesive coating liquid to obtain rotavirus antibody enteric-coated pellets.

Example 7: Preparation of rotavirus antibody enteric-coated microcapsules

Formula and dosage:

  Rotavirus antibody lyophilized powder                                          

  Hydroxypropyl Methyl Cellulose Phthalate   2 g

Magnesium stearate 0.12 g

Acetone 20ml

                                                                       

Method steps: dissolving enteric-coated material hydroxypropyl methylcellulose phthalate in organic solvent acetone to form a capsule material solution, suspending rotavirus antibody freeze-dried powder in it, adding ammonium sulfate solution, and fully stirring Emulsify, then heat under stirring to remove the organic solvent by evaporation to obtain circular microcapsules containing rotavirus antibody.

Example 8: Preparation of enteric-coated microcapsules for rotavirus antibody

Formula and dosage:

  rotavirus antibody freeze-dried powder                                      

  Cellulose Acetate Phthalate                          

Magnesium stearate 0.3 g

Acetone 40ml

                                                                                         

Method steps: the enteric-coated material is dissolved in an appropriate amount of organic solvent cellulose acetate phthalate, and the rotavirus antibody freeze-dried powder is suspended therein. Weigh the liquid paraffin, add magnesium stearate, stir and mix well for later use. Slowly add acetone solution dropwise to liquid paraffin under stirring, stir at room temperature to completely volatilize the acetone, filter, and wash with petroleum ether three times to obtain microcapsules.

Example 9: Preparation of rotavirus antibody enteric-coated microcapsules

Formula and dosage:

Freeze-dried powder of rotavirus antibody 0.7g

Cellulose acetate phthalate 4 g

Magnesium Stearate 0.25g

Acetone 40ml

  Ammonium Sulfate Solution                                                                

Method steps: same as embodiment seven

Several examples are listed above, where the data are only for the purpose of clarifying the invention. In fact, the anti-rotavirus-specific immunoglobulin and other carriers in the composition can be adjusted arbitrarily within the pharmaceutically acceptable range, and it is possible to achieve the purpose of the present invention beyond the upper and lower proportions of the embodiment of the present invention. Therefore, those skilled in the art can devise many embodiments according to the present invention.

Analytical results of the immunoglobulin prepared in Example 1 of the present invention

The purified immunoglobulin of the present invention carries out the mensuration of immunology, electrophoresis and protein concentration, and the results are as follows:

ELISA Immunospreading Antibody Purity Protein Concentration Before Purification 6×10 ³ 1:32 60% 10.5mg/ml After Purification 1.2-1.5×10 ⁴ 1:128 90% 120mg/ml

The protective effect of the enteric agent immunoglobulin composition of the present invention

1. Dissolution Experiment of Immunoglobulin Composition in Enteric Solution

Physiological saline with different pH values was taken and mixed with the above-prepared enteric-coated immunoglobulin respectively, and incubated at 37° C. for 30 min. The result is as follows:

  Solubility test of immunoglobulin enteric-coated pellets

PH 3.0 4.0 5.0 6.0 7.0 8.0

Solubility Insoluble Insoluble Insoluble Insoluble Insoluble (+) (++)

The results showed that the prepared enteric immunoglobulin was insoluble in solutions below pH7.0. Effectively protects immunoglobulins against degradation in acidic solutions.

2. Stability test of enteric solvent immunoglobulin composition

Store the prepared enteric-coated immunoglobulin at room temperature (18-22°C) and 4°C, respectively, and measure their ELISA immune titers. The results are as follows:

Stability test of immunoglobulin enteric-coated pellets

Original 60 days 120 days 180 days 240 days

Room temperature 1:12000 1:10240 1:10240 1:10240 1:9600

4℃ 1:12000 1:10240 1:10240 1:10240 1:10240

3. Metabolism experiment of enteric-coated immunoglobulin composition in the intestine

18-20 g of Kunming mice were taken, and the enteric-coated immunoglobulin composition (containing 5 mg of immunoglobulin) was orally administered, and ordinary immunoglobulin without protective agent was used as a control. The whole small intestine of the mice was collected at different times, and the supernatant was recovered by tissue homogenization, rinsing with physiological buffer, and centrifugation for ELISA immunoassay.

Metabolism experiment of enteric-coated immunoglobulin composition in intestine

ELISA detection cost time (point) 60 90 120 150 180 240 enteric solvent type group 1: 1200 1: 3200 1: 2400 1: 1200 1: 1200 1: 800 Normal control level 1: 3200 1: 2400 1: 1200 1: 800 1:200 1:100

Specific Immunoglobulin Safety Test

Acute toxicity test in mice: BALB/C mice of 18-20 grams of grade II were randomly divided into different dose groups, given different doses of immunoglobulin by intragastric administration, and observed for 2 weeks. The results showed that when each mouse was given 6 mg immunoglobulin/g body weight (equivalent to 40 times the effective dose), all the tested mice survived without obvious toxic reaction.

Pharmacodynamic experiment of specific immunoglobulin composition

1. In vitro virus neutralization titers of specific immunoglobulins

1.1 MA-104 cytopathic inhibition experiment

MA-104 cells are routinely spread on 96-well cell culture plates, and the rotavirus to be infected is mixed with doubly diluted immunoglobulin, and reacted at 37°C for 1 hour. The maintenance solution of 2% calf serum was 100 μl, and the concentration of trypsin was 2 μg/ml. After 72 hours, observe the test results under the microscope. The results showed that the neutralizing titer of the prepared specific immunoglobulin against Wa strain was 6.5×10 ⁴ (TCID ₅₀ ), and that against SA ₁₁ strain was 8.5×10 ⁴ (TCID ₅₀ )

2. The protective effect of specific anti-rotavirus immunoglobulin on suckling mouse rotavirus diarrhea model

2.1 Establishment of rotavirus diarrhea model in suckling mice

Thirty 5-day-old Bab/c suckling mice of grade II cleanliness were randomly divided into groups, and challenged with different virus doses by intragastric administration to determine the minimum 100% diarrhea-causing virus dose (DD ₁₀₀ ).

2.2 The protective effect of specific anti-rotavirus immunoglobulin on suckling mouse infection model

2.2.1 The anti-rotavirus immunoglobulin was diluted in multiples, mixed with the virus to be infected respectively, and reacted at 37°C for 1 hour, and then challenged the intestinal tract of 5-day-old suckling mice by gavage, within 96 hours, whether diarrhea, Diarrhea duration, body weight gain index analysis results. The results show that the administration of 0.15 mg/g body weight to newborn suckling mice can prevent 100% of the suckling mice in the protection group from diarrhea. There was a significant difference from the control group.

2.2.2 Newborn 5-day-old suckling mice were administered intragastrically with double-diluted anti-rotavirus immunoglobulin, and then intragastrically infected with rotavirus 1 hour later, and the protective effect was determined within 96 hours. The results showed that administration of 0.2 mg immunoglobulin per gram of body weight of suckling mice could protect 100% of infected suckling mice from diarrhea. Significantly different from the control group.

2.2.3 Newborn suckling mice were infected with rotavirus by gavage, and 12 hours later, they were gavaged with double-diluted immunoglobulin, and the protective effect was judged after 96 hours. The results showed that administration of 0.2 mg per gram of body weight of suckling mice could significantly reduce the diarrhea time and amount of diarrhea in suckling mice in the treatment group, and there were significant differences in diarrhea index and weight gain compared with the control group.

Claims (15)

1. An anti-rotavirus immunoglobulin composition, which comprises an effective amount of anti-rotavirus specific immunoglobulin and a pharmaceutically acceptable dressing, wherein the dressing includes an enteric coating material, a plasticizer, Anti-adhesive agent, protein stabilizer, adhesive, the effective amount of the anti-rotavirus specific immunoglobulin is 5-50% by weight of the composition. 2. The composition according to claim 1, characterized in that it is in the form of an enteric solution. 3. The composition according to claim 2, wherein the enteric-coated type is enteric-coated pellets. 4. The composition according to claim 2, characterized in that the enteric-coated type is enteric-coated granules. 5. The composition according to claim 1, characterized in that the content of the specific anti-rotavirus immune protein is 10-30% by weight of the composition. 6. The composition according to claim 5, wherein the content of the specific anti-rotavirus immune protein is 20% by weight of the composition. 7. The composition according to claim 1, wherein said enteric coating material is acrylic resin No. II or acrylic resin No. III. 8. The composition according to claim 1, wherein the plasticizer is dibutyl phthalate or triethyl citrate. 9. The composition of claim 1 wherein said anti-tack agent is magnesium stearate. 10. The composition according to claim 1, wherein said protein stabilizer is mannitol or albumin. 11. The composition of claim 1, wherein said binder is sodium alginate. 12. The composition according to claim 2, wherein the enteric-coated type is enteric-coated microcapsules. 13. The composition according to claim 12, wherein said enteric microcapsules comprise dressing capsules. 14. The composition according to claim 12, characterized in that the content of said anti-rotavirus specific immunoglobulin is 10-30% by weight of the composition. 15. A method for preparing the anti-rotavirus specific immunoglobulin composition of claim 1, comprising the following steps: 1) Carry out rotavirus booster immunization with specific pathogen-free chickens; 2) Extract the specific immunoglobulin against rotavirus from the eggs with enhanced immunity by means of degreasing, salting out, desalting, concentration and chromatography; 3) Prepare the specific immunoglobulin described in 2) into an enteric-coated composition by using corresponding enteric-coated materials and techniques.