A canine canine parvovirus specific transfer factor injection for dogs and a preparation method thereof

Abstract

The application discloses a canine parvovirus specific transfer factor injection for dogs and a preparation method thereof, and belongs to the field of veterinary biotechnology. The method comprises the following steps: preparation of a multi-type canine parvovirus inactivated vaccine, immunization of a mother dog, collection of a placenta, soaking in physiological saline, stirring and crushing, four times of gradient cryogenic grinding, centrifugation, microfiltration, inactivation, multi-stage membrane separation and purification, sterilization and sub-packaging. The mother dog is immunized with the multi-type canine parvovirus vaccine, the transfer factor is extracted from the placenta as a specific raw material, the four times of gradient cryogenic grinding is carried out at-5 DEG C, -10 DEG C, -15 DEG C and -20 DEG C, and the multi-stage membrane separation technology is used, so that the transfer factor activity is maximally retained in the whole low-temperature preparation process. The product realizes the double functions of precise targeted treatment of canine parvovirus multi-subtype infection and broad-spectrum enhancement of basic immunity, and has no pyrogen, no abnormal toxicity and good safety.

Description

Technical Field

[0001] This invention belongs to the field of veterinary biotechnology, specifically relating to a canine parvovirus-specific transfer factor injection and its preparation method. Background Technology

[0002] The prevalence of canine parvovirus in China is characterized by a high incidence in puppies, minimal urban-rural disparities, and rapid subtype iteration. Since its introduction to my country in the 1990s, the virus has become the leading infectious disease in dogs, especially among unvaccinated puppies under 6 months of age, where the infection rate can reach 30% to 50%. In rural areas, due to weaker immunization awareness, the incidence rate is 15% to 20% higher than in urban areas. Currently, the prevalent CPV-2a, CPV-2b, and CPV-2c subtypes have similar pathogenicity, but the newer CPV-2a, CPV-2b, and CPV-2c subtypes have wider spread due to mutations. Domestic vaccines are mostly based on traditional subtypes and have limited coverage, resulting in insufficient protective efficacy.

[0003] Transfer factor is a low-molecular-weight polynucleotide-peptide complex found in animal immune lymphocytes. As a novel immunomodulator, it can enhance the animal's immune function in multiple dimensions by specifically delivering immune information and stimulating immune cell activity. Transfer factor can carry specific immune information from donor-sensitized T lymphocytes, transferring the cellular immune capacity against pathogens such as bacteria, viruses, and fungi to recipient lymphocytes, transforming inactive recipient lymphocytes into specifically sensitized cells and rapidly initiating an immune response against specific pathogens. Simultaneously, transfer factor enhances the body's broad-spectrum defense against unknown pathogens by activating macrophage phagocytic activity and promoting interferon release.

[0004] Transfer factor has a small molecular weight, is non-toxic and non-antigenic, does not cause allergic reactions or antagonistic antibodies, can transmit immune information across species, transcends species boundaries, and has strong oral stability. It requires small doses, has a rapid onset of action, and has a long-lasting effect, making it a highly efficient and safe preparation in the field of animal immune regulation.

[0005] However, most veterinary transfer factor products currently on the market are non-specific transfer factors, lacking the ability to target specific pathogens and thus failing to achieve precise targeted treatment of canine parvovirus multi-subtype infections. To date, there are no veterinary drug products in China that simultaneously possess the dual functions of precise targeted treatment of canine parvovirus multi-subtypes and broad-spectrum enhancement of basic immunity; therefore, there is an urgent need for such a veterinary drug product. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a canine parvovirus-specific transfer factor injection and its preparation method, so as to achieve the dual functions of precise targeted treatment of canine parvovirus multi-subtype infection and broad-spectrum enhancement of basic immunity.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing a canine parvovirus-specific transfer factor injection includes the following steps: preparation of a multi-type canine parvovirus inactivated vaccine, collection of placenta from immunized mother dogs, soaking in physiological saline, crushing, four-stage gradient freeze-grinding, centrifugation, microfiltration, inactivation, multi-stage membrane separation purification, and sterilization and packaging. By selectively immunizing mother dogs with a multi-type canine parvovirus vaccine, transfer factor is extracted from the placenta as a specific raw material. The entire process, using four-stage gradient freeze-grinding and multi-stage membrane separation technology, is prepared at low temperatures to maximize the retention of the effective active components of the transfer factor.

[0008] The present invention also provides a canine parvovirus-specific transfer factor injection prepared by the above preparation method.

[0009] Compared with the prior art, the beneficial effects of the present invention are: This invention overcomes the limitations of traditional transfer factor methods, which employ a combined process of targeted immunization of maternal dogs with a multi-type canine parvovirus vaccine, placental-specific extraction, and end-to-end low-temperature gradient purification. This achieves the dual function of precisely targeting multiple canine parvovirus subtypes and broadly enhancing basic immunity. Using the placenta of immunized maternal dogs as the targeted raw material, this invention utilizes a four-stage gradient cryo-grinding and multi-level membrane separation technology to preserve activity throughout the process, resolving the contradiction between virus inactivation and immune activity preservation. Clinically, this fills the gap in the prevention and treatment of acute canine parvovirus infection, reducing the cost of disease onset and treatment; industry-wide, it promotes the upgrading of veterinary immunomodulators from broad-spectrum regulation to precise targeting. Detailed Implementation

[0010] Example 1 This embodiment provides a method for preparing a canine parvovirus-specific transfer factor injection, the specific steps of which are as follows: Step 1: Preparation of multi-type canine parvovirus inactivated vaccine. Six to eight different strains of canine parvovirus with good immunogenicity and antigenicity were selected and inoculated into cells. After cytopathic effects reached 80%, the viral fluids were collected, mixed, and inactivated. Vaccine adjuvants were added to prepare the inactivated vaccine, which was stored at 2 to 5°C for later use. The multi-type canine parvoviruses used included CPV-2 classical type, CPV-2a, CPV-2b, CPV-2c, novel CPV-2a, novel CPV-2b, and novel CPV-2c.

[0011] Step Two: Immunization of the Maternal Dog and Placental Collection. The prepared inactivated vaccine is administered once during the mother dog's estrus cycle, followed by three more immunizations during pregnancy, with each immunization spaced 12 days apart. Immunization is discontinued three weeks before the expected delivery date. The placenta is collected at 2-5°C during the mother dog's delivery.

[0012] Step 3, soaking in physiological saline. Weigh the placenta and add it to physiological saline solution at a ratio of 1:1 by mass, with a concentration of 0.05% and a temperature of 2 to 5°C. Soak for 15 minutes.

[0013] Step 4, Grinding. The soaked placenta and soaking liquid are fed into a meat grinder and ground twice.

[0014] Step 5, four-stage gradient freeze-milling. Place the crushed mixture into a freeze mill with an output particle size of 1 to 10 μm, set to -5℃ for the first freeze-milling, and after it is completely thawed, put it into the freeze mill and set to -10℃ for the second freeze-milling, and after it is completely thawed, put it into the freeze mill and set to -15℃ for the third freeze-milling, and after it is completely thawed, put it into the freeze mill and set to -20℃ for the fourth freeze-milling.

[0015] Step 6, centrifugation. Place the fourth freeze-milled thawed liquid into a low-temperature refrigerated centrifuge and centrifuge at 2 to 5°C and 8500 r / min for 15 minutes. After centrifugation, remove the precipitate and retain the supernatant.

[0016] Step 7, microfiltration. The supernatant from centrifugation is microfiltered in a tank containing a 0.45μm hollow fiber column to obtain microfiltrate.

[0017] Step 8, virus inactivation. Add 0.001% β-propiolactone to the microfiltrate, inactivate at 1 to 4°C for 20 hours, then hydrolyze at 37°C for 2 hours to obtain the final inactivated solution, and store at 2 to 5°C.

[0018] Step 9, multi-stage membrane separation and purification. The inactivation solution is subjected to a first microfiltration using a 10K membrane; the collected first microfiltrate is then subjected to a second microfiltration using a 0.5K membrane, retaining the permeate from the second microfiltration and storing it temporarily at 2 to 5°C; the reflux liquid from the second microfiltration is then subjected to a third microfiltration using a 1K membrane, removing the permeate from the third microfiltration and retaining the reflux liquid; finally, the collected reflux liquid from the third microfiltration is subjected to a fourth microfiltration using a 5K membrane, retaining the permeate from the fourth microfiltration and storing it temporarily at 2 to 5°C.

[0019] Step 10, sterilization and aliquoting. Mix the liquid from the second and fourth microfiltration permeates, sterilize and aliquot using a 0.22 μm membrane to obtain canine parvovirus-specific transfer factor injection solution. Store the aliquoted transfer factor injection solution at 2 to 5°C.

[0020] The ambient temperature of the preparation sites in steps three through ten above is controlled between 2 and 5°C.

[0021] Example 2: Efficacy of different doses of canine parvovirus-specific transfer factor injection in treating canine parvovirus disease in dogs weighing less than 5 kg. 1. Materials 1.1 The canine parvovirus-specific transfer factor injection (TF) prepared by the method in Example 1, batch number TF001, was prepared by Paishengte (Fuzhou) Biotechnology Co., Ltd.

[0022] 1.2 Canine Parvovirus Used for Challenge: Canine parvovirus FJFZCPV strain (typed as novel CPV-2C) was isolated and identified by our company in Fuzhou, Fujian Province in 2024 as the 5th generation strain. To determine the virulence of canine parvovirus FJFZCPV strain to dogs, challenge experiments were conducted on dogs using different doses of FJFZCPV strain. The results showed that after challenge with 1 ml / dog of diluted 5th generation FJFZCPV strain, 10... 5.50 TCID 50 / ml group, 5 / 5 shots for experimental dogs; 10 4.50 TCID 50 In the / ml group, 4 / 5 of the experimental dogs developed the disease; 10 3.50 TCID 50 In the / ml challenge group, 3 / 5 of the experimental dogs developed the disease. To ensure the validity of the challenge experiment, the challenge dose of canine parvovirus FJFZCPV strain was set at 1ml / dog, with a viral load of 10. 5.50 TCID 50 / ml, the route of attack was oral administration.

[0023] 1.3 The experimental dogs, healthy and susceptible beagles weighing 5 kg or less and aged 3-4 months, with negative canine parvovirus antibodies, were purchased from Fuzhou Zhenhe Experimental Animal Technology Development Co., Ltd. Information on the experimental animals is shown in Table 1.

[0024] Table 1. Information on the experimental dogs 2 Methods 2.1 Trial of treating canine parvovirus disease in dogs weighing less than 5 kg with different doses of canine parvovirus-specific transfer factor The experimental protocol is detailed in Table 2. The 5th generation of canine parvovirus FJFZCPV strain was used to treat 20 healthy dogs aged 3-12 months of age and of different weights at a dose of 1.0 ml / dog (10 ml / dog). 5.5 TCID 50Dogs were orally challenged with TF (1.0 ml / dog). Once any of the following symptoms appeared in the challenged dogs: fever, lethargy, vomiting, decreased appetite, diarrhea, or bloody stools, the dogs were immediately randomly assigned to one of three treatment options: subcutaneous TF injection alone, conventional treatment, or no treatment. All dogs were divided into four groups according to the treatment plan: Group 1 received subcutaneous TF injection alone (5 dogs each received 1.0 ml / dog / dose); Group 2 received subcutaneous TF injection alone (5 dogs each received 2.0 ml / dog / dose); Group 3 received conventional treatment (5 dogs); and Group 4 was the untreated group (5 dogs). All dogs were treated for no more than 7 days from the onset of symptoms to the end of treatment, regardless of effectiveness. The experimental protocol is shown in Table 2.

[0025] Table 2. Trial protocols for treating canine parvovirus disease in dogs weighing less than 5 kg with different doses of TF 2.2 Conventional Treatment Plan Conventional treatment consists of antiviral and antibacterial drugs, symptomatic treatment, and fluid replacement therapy.

[0026] 2.2.1 Antiviral and antibacterial drug therapy: Ribavirin injection 15mg / kg, cefalexin 35-55mg / kg, intravenous injection, once daily.

[0027] 2.2.2 Symptomatic treatment: For antiemetics, administer 0.15 ml / kg of antiemetic subcutaneously twice daily; for hemostasis, administer 1.5–2.5 ml of tranexamic acid subcutaneously once daily; for gastric protection and antidiarrheal treatment, administer 1–2 mg / kg of omeprazole intravenously once daily.

[0028] 2.2.3 Fluid replacement (supportive therapy): 200-500ml of 5% glucose, 3-4ml of vitamin C, 1-2ml of dexamethasone, 50-200 units of coenzyme A for injection, and 3-4ml of adenosine triphosphate disodium injection, administered intravenously once daily.

[0029] 2.4 Observation and Analysis: The treatment effect was observed and analyzed, including the dogs' body temperature, defecation, mental state, food and water intake, and the effective treatment time, all of which were recorded in detail. The results were then compiled and analyzed, the effectiveness rate was statistically analyzed, and the differences in effective treatment time among the groups were compared.

[0030] 2.5 Criteria for Evaluating Therapeutic Effect 2.5.1 After effective treatment, the patient's mental state, appetite, and body temperature return to normal, and vomiting or diarrhea stops.

[0031] 2.5.2 Ineffective treatment results in lethargy or depression, loss of appetite or complete anorexia, elevated or decreased body temperature below normal, persistent vomiting or diarrhea, or death.

[0032] 2.5.3 Treatment duration is from the start of medication until effective results are observed. Treatment should be discontinued if no improvement is seen after 7 days. Effective treatment time is the period from the start of medication to the observation of effective results.

[0033] 3 Results 3.1 Trial of treating canine parvovirus disease in dogs weighing less than 5 kg with different doses of canine parvovirus-specific transfer factor The results showed that the effective rate of subcutaneous TF treatment alone in Group 1 (1.0 ml / animal / dose) was 80%, with an average effective treatment time of 5.5 days; the effective rate of subcutaneous TF treatment alone in Group 2 (2.0 ml / animal / dose) was 100%, with an average effective treatment time of 4.2 days; and the effective rate of conventional treatment in Group 3 was 40%, with an average effective treatment time of 6.5 days. The mortality rate of the untreated Group 4 was 100%. See Table 5 for detailed results.

[0034] Table 3. Results of trials of canine parvovirus infection in dogs under 5 kg treated with different doses of TF. Note: 1. "●" indicates that the indicator belongs to this column, and " / " indicates that it does not meet the criteria of this indicator.

[0035] 2. Average effective treatment time = Sum of effective treatment times within the group / Number of effective treatments within the group.

[0036] The results showed that the treatment group receiving subcutaneous injection of TF at a dose of 1.0 ml / animal / dose had a significantly better therapeutic effect on canine parvovirus than the conventional treatment group alone. Similarly, the treatment group receiving subcutaneous injection of TF at a dose of 2.0 ml / animal / dose had a significantly better therapeutic effect on canine parvovirus than the conventional treatment group alone. All of these groups showed better therapeutic effects than the untreated group. This indicates that TF treatment is more effective and has a faster cure rate than conventional treatment alone.

[0037] The results showed that TF has a non-specific immunomodulatory effect and can effectively improve the body's immune function. Dogs infected with diseases such as canine parvovirus have weakened immunity; using TF can effectively enhance their immunity and treat these diseases.

[0038] Example 3 Heat Source Inspection This experiment tested the transfer factor injection prepared in Example 1 according to the "Pyrogen Test Method" in the appendix of the current Chinese Veterinary Pharmacopoeia to determine whether the pyrogen limit contained in the transfer factor injection prepared by our company complies with the current Chinese Veterinary Pharmacopoeia.

[0039] 1. Materials 1.1 The experimental rabbits weighing over 1.7 kg were purchased from Jiashan County Jintu Rabbit Industry Professional Cooperative.

[0040] 1.2 The product, canine parvovirus-specific transfer factor injection (TF), with batch number TF001, was prepared by Paishengte (Fuzhou) Biotechnology Co., Ltd.

[0041] 2 Methods 2.1 Preparations before the experiment One to two days before the pyrogen test, the experimental rabbits were kept in an environment with the same temperature, with the temperature difference between the laboratory and the breeding room not exceeding 3°C, and controlled between 17 and 25°C. Throughout the experiment, the laboratory temperature fluctuation was not to exceed 3°C, while preventing animal agitation and avoiding noise interference. The rabbits were not fed at least one hour before the experiment and were placed in a spacious and suitable environment until the experiment was completed. A rectal thermometer (precision ±0.1°C) was inserted into the anus to the same depth and for the same duration for all rabbits, approximately 6 cm deep, for at least 1.5 minutes. Body temperature was measured every 30 minutes, for a total of two measurements. The difference between the two measurements should not exceed 0.2°C, and the average of the two temperatures was taken as the rabbit's normal body temperature. Normal body temperature is within the range of 38–39.6°C, and the difference in normal body temperature between rabbits in the same group should not exceed 1°C.

[0042] Test instruments that come into contact with the sample to be tested are sterile and pyrogen-free.

[0043] 2.3 Inspection Method Three experimental rabbits were selected and numbered 1 to 3. Within 15 minutes of measuring their normal body temperature, the sample to be tested, warmed to approximately 38°C, was slowly injected into the ear vein according to Table 1. Measurements were taken every 30 minutes for a total of 6 times. The highest temperature reading was subtracted from the normal body temperature to determine the rabbit's elevated body temperature (negative temperature readings were recorded as 0°C). If one of the three rabbits showed an elevated body temperature of 0.6°C or higher, or if the total elevated body temperature of the three rabbits reached 1.3°C or higher, five more rabbits should be selected for retesting using the same method.

[0044] 2.4 Result Judgment In the case of three experimental rabbits undergoing initial immunization, the body temperature increase was less than 0.6℃, and the total body temperature increase of the three rabbits was less than 1.3℃; or in the case of five experimental rabbits undergoing retesting, no more than one rabbit had a body temperature increase of 0.6℃ or higher, and the total body temperature increase of the eight experimental rabbits undergoing initial immunization and retesting combined was 3.5℃ or lower, in all cases the pyrogen test of the samples was deemed to meet the requirements.

[0045] If more than one of the three rabbits undergoing initial immunization had a body temperature increase of 0.6℃ or higher; or if more than one of the five rabbits undergoing retesting had a body temperature increase of 0.6℃ or higher; or if the total body temperature increase of the eight rabbits undergoing initial immunization and retesting exceeded 3.5℃, the pyrogen test of the sample was deemed to be non-compliant.

[0046] Table 1 Experimental Design 3 Results Following the experimental procedure, the normal body temperature and elevated body temperature of each experimental rabbit were measured, and the results are shown in Table 2.

[0047] Table 2 Results of pyrogen testing The results showed that after injection of the test samples, the body temperature rise of each experimental rabbit was less than 0.6℃, and the sum of the body temperature rises of the three experimental rabbits in each group was less than 1.3℃. Therefore, it was determined that the pyrogen tests of the three batches of transfer factor injection solution prepared by the method in Example 1 all met the requirements of the current Chinese Veterinary Pharmacopoeia.

[0048] Example 4 Abnormal Toxicity Test To verify the safety of the transfer factor injection, this experiment tested the transfer factor injection prepared by the method in Example 1 according to the "Abnormal Toxicity Test Method" in the appendix of the current Chinese Veterinary Pharmacopoeia.

[0049] 1. Experimental Materials 1.1 Healthy Kunming mice weighing 20g ± 2g were provided by Shanghai Bishengchang Biotechnology Co., Ltd.

[0050] 1.2 The product, canine parvovirus-specific transfer factor injection (TF), with batch number TF001, was prepared by Paishengte (Fuzhou) Biotechnology Co., Ltd.

[0051] 2. Test Methods 2.1 Test Method: Three bottles were randomly selected, mixed thoroughly, and used as test samples. Five mice were intraperitoneally injected with 0.5 ml of the TF001 batch of mixed transfer factor, as shown in Table 1.

[0052] Table 1 Experimental Design 2.2 Result Interpretation: After 48 hours of observation, all mice should be healthy and alive. If any mice die, double the number of mice should be taken for retesting. After 48 hours of observation, all mice should be healthy and alive.

[0053] 3 Results The results showed that all five mice survived within 48 hours after intraperitoneal injection without any abnormalities, as shown in Table 2.

[0054] Table 2 Test Results 4. Conclusion This experiment examined the canine parvovirus-specific transfer factor injection prepared according to the method in Example 1 of the "Abnormal Toxicity Test Method" appendix of the current Chinese Veterinary Pharmacopoeia. The results showed that all mice survived within 48 hours after injection, meeting the requirements of the current Chinese Veterinary Pharmacopoeia. The results indicate that the canine parvovirus-specific transfer factor injection prepared by our company according to the method in Example 1 has no abnormal toxicity.

[0055] As can be seen from the above embodiments, the canine parvovirus-specific transfer factor injection prepared by the preparation method provided by the present invention is pyrogen-free and has no abnormal toxicity. It can treat canine parvovirus infection and enhance the immune function of dogs. When used in combination with vaccines, it can enhance the protective effect of vaccines.

[0056] Although embodiments of the present invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and variations can be made to the above embodiments without departing from the principles and spirit of the present invention, the scope of which is defined by the claims and their equivalents.

Claims

1. A method for preparing a canine parvovirus-specific transfer factor injection, characterized in that, Includes the following steps: 1) Preparation of a multi-type canine parvovirus inactivated vaccine; 2) Vaccinate the female dog once during estrus with the inactivated vaccine, and 2 to 3 times during pregnancy; collect the placenta at 2 to 5°C when the female dog gives birth; 3) Soak the placenta in physiological saline solution at 2 to 5°C and a concentration of 0.01% to 0.1% at a mass ratio of 1:1 for 10 to 30 minutes; 4) Crush the soaked placenta and soaking solution twice; 5) Perform four gradient freeze-grinding processes on the crushed mixture. The first grinding temperature is -5℃, the second grinding temperature is -10℃, the third grinding temperature is -15℃, and the fourth grinding temperature is -20℃. After each grinding, wait until it is completely melted before proceeding to the next grinding. 6) Centrifuge the fourth freeze-milled thawed liquid at 2 to 5°C and 8500 r / min for 10 to 20 minutes, and collect the supernatant; 7) The supernatant from centrifugation is microfiltered through a 0.45 μm hollow fiber column to obtain microfiltrate; 8) Add 0.001% β-propiolactone to the microfiltrate, inactivate it at 1 to 4°C for 18 to 20 hours, and then hydrolyze it at 37°C for 2 hours to obtain the inactivated solution; 9) The inactivation solution is sequentially subjected to a first microfiltration through a 10K membrane and a second microfiltration through a 0.5K membrane, and the liquid permeated from the second microfiltration end is collected; The liquid from the second microfiltration reflux end is passed through a 1K membrane for a third microfiltration, and the liquid from the third microfiltration reflux end is collected. Then it is passed through a 5K membrane for a fourth microfiltration, and the liquid from the fourth microfiltration permeate end is collected. 10) Mix the liquid from the second microfiltration end with the liquid from the fourth microfiltration end, filter through a 0.22μm membrane for sterilization, and dispense to obtain canine parvovirus-specific transfer factor injection.

2. The preparation method according to claim 1, characterized in that, In step 1), the multi-type canine parvovirus includes 6 to 8 strains of CPV-2 classical type, CPV-2a, CPV-2b, CPV-2c, novel CPV-2a, novel CPV-2b, and novel CPV-2c.

3. The preparation method according to claim 1, characterized in that, The preparation method of the inactivated vaccine in step 1) is as follows: select 6 to 8 different types of canine parvovirus with good immunogenicity and antigenicity and inoculate them into cells respectively. After the cytopathic effect reaches 80%, collect the virus fluid separately, mix and inactivate it, add vaccine adjuvant to make inactivated vaccine, and store at 2 to 5°C for later use.

4. The preparation method according to claim 1, characterized in that, In step 2), the interval between each immunization for pregnant bitches is 10 to 14 days, and immunization should be stopped within 3 weeks before the expected delivery date.

5. The preparation method according to claim 1, characterized in that, The ambient temperature of the preparation site in steps 3) to 10) is 2 to 5°C.

6. The preparation method according to claim 1, characterized in that, The transfer factor injection solution dispensed in step 10) should be stored at 2 to 5°C.

7. The preparation method according to claim 1, characterized in that, In step 5), the output particle size of the cryogenic grinder is 1 to 10 μm.

8. The preparation method according to claim 1, characterized in that, The inactivation solution obtained in step 8) is stored at 2 to 5°C.

9. A canine parvovirus-specific transfer factor injection, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 8.

Images