HU197846B - Process for producing therapeutic composition suitable for treating virus infections - Google Patents

Abstract

A biological preparation for the treatment of virus infections, and the procedure of its production. The principle of the method is to combine attenuated Gumboro virus with at least one registered carrier. The preparation which is the object of the invention is characterized by comprising attenuated Gumboro virus.

Description

The present invention relates to a method of treating viral infections.

Most of the compositions of the present invention are useful in the treatment of viral infections, namely hepatitis, cancerous conditions, and loss.

Hepatitis is a viral infection that causes parenchymal liver damage. There are three forms of occurrence; hepatitis epidemica caused by Hepatitis A virus and serum hepatitis caused by Hepatitis B virus Infections not belonging to the above two types that are suspected to be caused by the virus are again classified as "would grow A - grow B" or "C" .

“The infection is relatively mild. The incubation period is usually 10 to 28 days, while the disease causes 30 to 45 days to lie down and further incapacity for work. The outbreak of infection leaves you protected. No vaccine is known to prevent virus A infection.

The infection caused by “B virus is more severe; incubation time is longer, 50-160 days. The resistance of virus "B" is significantly higher, not only against virus "A" but also other viruses in general, against heat and chemicals. Hepatocellular cancer often occurs as an aftermath of the 'B virus. Infection with virus "B" generally has no immune consequences. A vaccine known as "B" virus, which is made from the blood of people who are infected, is known, but may not be considered because of its side effects (AIDS) and its high cost.

To date, no therapeutic agent for hepatitis has been known, and it is therefore an object of the present invention to provide a therapeutic agent for treating and controlling viral hepatitis.

FIELD OF THE INVENTION The present invention relates to a therapeutic composition for viral infection, or to the preparation thereof, which comprises attenuated Gumboro virus (Avian bursa, Avian bronchitis).

It is another object of the present invention to provide a therapeutic composition for viral infections comprising attenuated Gumboro virus.

The vaccine against Gumboro disease is known and used.

Gumboro disease is primarily an acute viral disease of chickens aged 3 to 6 weeks, associated with severe watery diarrhea, Fabricius hose swelling, and inflammation of the lymphoid organs. Gumboro disease was otherwise first described by Cosgrove in the United States as infectious bursitis in chicks. 1957. Outside the United States, it occurs in European countries, including Hungary. The disease-causing enterovirus is highly resistant. The disease starts abruptly after a 2-4 day incubation period, and most deaths occur within 3-5 days and usually occur within one week. In the cases examined, the morbidity ranged from 1 to 30% and the mortality ranged from 4 to 5%.

In medical practice, vaccines are used to immunize against certain diseases. The vaccine usually contains the disease-causing virus in attenuated or killed form. In some cases, vaccines are also used for therapeutic purposes, but in all cases the pathogen virus is present.

The indications of the compositions of the present invention differ substantially from the disease caused by the virus used in the composition. The use of this virus in this area was unpredictable at all.

The preparation of the Gumboro vaccine and of the attenuated Gumboro virus itself is accomplished by methods well known in the art and practiced. For example, primary or secondary fibroblast cultures prepared from pre-incubated chicken embryos for 10 to 11 days are used and the lyophilized vaccine is prepared from virus of reduced virulence grown in tissue culture. Alternatively, Avian bursa or Avian bronchitis virus is introduced into pre-incubated eggs and incubated at 37 ° C. The chicken embryos are then processed and the lyophilized vaccine is prepared from the virus of reduced virulence obtained. For example, the preparation of the vaccine is described in Section VI. A sterile viral material of at least 10 ⁶ TCID ₅ O / 0.1 ml, tested according to the Pharmacopoeia, is used. Sterile viral material is mixed with 50% sterile skimmed milk and lyophilized in 10 ml lyophilisation bottles every 2 ml.

The vaccine is available in lyophilized doses of 100, 200, 500 and 1000 doses and is used to prevent disease caused by the Gumboro virus. The vaccine is administered orally in the drinking water.

The above vaccine is useful as an active ingredient in the composition of the invention. Of course, the attenuated virus itself, or any solution thereof, such as physiological saline, can be used.

The Gumboro virus, even when virulent, is apathogenic to humans. However, the use of the attenuated form in the formulation provides double safety in human terms.

In the case of human vaccines, it usually contains killed viruses. The killed virus cannot be used in the composition of the invention, only the attenuated form of the virus. Viral interference occurs when using this product; that is, the dosed virus tries to displace the infectious virus.

The compositions of the invention comprise the attenuated virus strain together with at least one solid or liquid carrier.

For oral use, tablets or gelatin capsules are used which contain the active ingredient in diluents such as lactose,

-2197846 containing dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine and lubricating agents such as diatomaceous earth, talc, stearic acid or salts thereof such as magnesium or calcium stearate and / or polyethylene glycol. The tablets also contain excipients such as magnesium aluminum silicate, starches such as corn, wheat, rice or Maranta starch, gelatin, tragacanth, methyl-target cellulose, sodium carboxymethyl-cellulose and / or polyvinyl. pyrrolidone and, if desired, disintegrating agents such as starches, agar, alginic acid or salts thereof such as sodium alginate and / or effervescent agents, colorants, flavors or sweeteners.

For parenteral administration, solutions for infusion are particularly suitable, and are preferably isotonic aqueous solutions or suspensions, or they may be prepared, for example, from lyophilized preparations, immediately prior to use, containing the active ingredient alone or together with a carrier such as mannitol. Such formulations may be pre-sterilized and / or contain auxiliaries, such as preserving, stabilizing, potentiating, solubilizing and / or emulsifying agents, salts and / or buffers for controlling osmotic pressure.

The effect of attenuated Gumboro virus can be potentiated primarily by certain sedatives (many tranquilizers). Fentazin derivatives, which are generally substituted at the 2- and 10-positions, are of particular interest in this regard. Chlorpromazine, 10- (3'-dimethylaminopropyl) -2-chlorophthiazine, promethazine, metophenazine, aminopromazine and the like are particularly preferred.

Preferably, the composition is a <RTI ID = 0.0> 1L </RTI> of the virus in which the virus and vehicle are present. The composition may also be in the form of a solution, suppository, capsule, emulsion, suspension, etc. in shape.

The dosage of the compositions of the invention will depend on the subject being treated, the composition of the composition, the stage of disease and the strain of virus. The dose will generally be 1000-5000, preferably 3000-4000 U / day, relative to the attenuated virus, as a single dose or possibly 2 to 5 times a day in divided doses. Generally, administration for 6 consecutive days will provide the full effect. The administration may conveniently be oral or rectal, however, total treatment may be expedient, for example in the form of a suspension, solution, ointment.

The compositions of the present invention have been tested in a number of animal and human studies and have demonstrated a virtually 100% therapeutic effect against both A and B viral infections without any toxic side effects. In addition to the symptomatic treatment, the compositions of the present invention are also useful in preventive protection: for example, during the therapeutic treatment of virus A virus infection, no further infection has occurred after 5 weeks of incubation.

The effect of the composition of the invention was demonstrated in the following experiments:

Example 1

Fecal samples were taken from infected chimpanzees during the acute phase of hepatitis A (type MS-I). The inoculum for intramuscular administration was a 5% faecal suspension, diluted with pH 7.2 phosphate buffered saline, filtered (300 g, 30 min) after filtration (0.45 mm filter, Hank's saline containing 1% aibumin) after washing), then diluted 500-fold and used 0.5 ml of inoculum per animal. This dose of virus usually induces hepatitis in animals within 5 to 6 weeks.

White silk monkeys were inoculated with Bursa-vac G-603 vaccine (Sterwin Laboratories, Inc., Millsboro, Delaware). The vaccine is used to prevent infection with Avian bursa. Monkeys were inoculated with 50 units orally and 50 units intranasally.

During the experiment, the effect of Avian bursa virus was investigated on 4 adult, wild-born and / or colony-born red-breasted silkworm (Saguinus labiatus labiatus). 4 monkeys were also used as untreated controls.

During the experiment, the monkeys were divided into 5 groups of 4 animals each. All animals were assayed for normal enzyme activities prior to treatment for three consecutive weeks. Serum glutamic oxalacetic acid transaminase (SGOT) and serum glutamine pyruvate transaminase (SGPT) were tested. After the third week, a percutaneous liver biopsy was performed. Each group was treated as follows:

Group 1: inoculated with saline.

Group 2: Inoculated with hepatitis A virus (HAV).

Group 3: Inoculated with HAV and treated with Bursa vaccine after one week.

Group 4: Inoculated with HAV and treated with Bursa vaccine after 3 weeks.

Group 5: Inoculated with HAV and treated with Bursa vaccine after 5 weeks.

Bursa vaccine treatment is repeated for each of the treated animals for four consecutive days.

During the experiment, animals were bled weekly and percutaneous liver biopsies were performed every two weeks except at weeks 5 and 6. Serum samples were assayed for SGOT and SGPT activity. Serum samples of approximately 0.2 ml were stored at −20 ° C for further evaluation. Tissue samples obtained by liver biopsy were subjected to histopathological examination for hepatitis. After 10 weeks, the animals were excised and examined. Tissue samples were 10% 3

It was stored in -3197846 buffered formalin solution. The evaluation of the experiment was evaluated by the increase in SGPT enzyme activity and the histological presence of hepatitis.

Transaminase activity in Group 1 animals remained normal and no histological evidence of hepatitis was found. In Group 2, SGPT activity remained normal for 5 weeks. At week 5, mean SGPT activity increased 6-fold above normal and hepatitis was detected histologically. At week 6, SGPT activity was twice the normal value and hepatitis was still detectable under the microscope, although the inflammation was not as severe as at week 5. At week 7, SGPT activity and liver biopsy were again normal and remained normal during the study (week 10).

In group 3, no hepatitis was observed, SGPT activity remained normal, and no histological lesions were observed for 10 weeks. Group 4 showed the same results as Group 3.

Group 5 SGPT activity was normal until Week 5, but showed a 5-fold increase. The presence of hepatitis could also be detected histologically. At week 6, SGPT activity fell to twice normal levels, while microscopically

The presence of hepatitis similar to Group 2 was observed. At week 7, SGPT activity declined to normal levels, and neither did morphology of liver biopsy by the end of week 10.

After the experiment, the animals were necropsied and no pathological lesions were observed in any of the animals. The liver, lung, kidney, spleen and brain tissues were examined under a microscope.

It should be noted that SGOT enzyme activity showed no significant change in any of the experiments. At the same time, SGPT is considered to be the most sensitive monitoring method for hepatitis.

Example 2

The effect of the composition of the invention against rabies virus in mice was tested. The animals were divided into two groups and one group was treated with 0.5 ml of Gumboro vaccine intraperitoneally; after three days, 20 animals and 20 animals from the untreated group were also inoculated intracerebrally with 0.03 ml single dose as a pathogen with rabies vaccine (RV). Inoculation of the virus in different dilution of 5 (10 ^_1 - 10 ⁵⁾ were carried out, each dilution is added 4-4 animals. Five to five animals in the treated groups served as vaccine controls and 5 animals in the untreated group served as absolute controls. Animals were observed for 25 days and mortality was determined.

In the experiment, RV killed 50% of control animals. This value was taken as the Relative Death Rate (RDF) unit (1.0). During the experiment, ABuV vaccine reduced the RDF values to 0.5, but did not affect the course of infection in unprotected animals.

Example 3 Day-old, specially pathogen-free chickens were inoculated using virulent Rous sarcoma virus (RSV) as pathogen

In a single dose of 0.3 ml, diluted 10 ^-3 ' ^{7 of} the virus, added to the humoral radius of the right wing. The vaccines were administered in a single dose, 7 days prior to RSV infection. Group 1 consisted of 15 non-infected animals (absolute control), 15 animals of Group 2 were inoculated with RSV (virulent virus control). Twenty-four animals in Group 3 were vaccinated intramuscularly with ABuV vaccine, and 12 animals were considered as vaccine controls and 12

After 7 days, an RSV dose was administered. The animals were observed for 5 weeks. In the experiment, we found that the ABuV vaccine showed significant protection against the Rous sarcoma virus. An RSV infection with an RDF value of 1 was 0.25 for ABuV. From the above experiments it can be concluded that the compositions according to the invention show protective and therapeutic effects against infections caused by various viruses.

Claims (2)

PATENT CLAIMS CLAIMS 1. A method for the preparation of a therapeutic composition comprising mixing a reduced amount of Gumboro virus with known pharmaceutically acceptable carriers, preferably from chicken embryos, propagated in fibroblast tissue culture and pre-incubated eggs, and embryo processing, to at least one pharmaceutically acceptable carrier. hepatitis, cancers of viral origin, and anti-rabies. (Priority: July 2, 1984) 2. A method for the preparation of a therapeutic composition comprising mixing attenuated Gumboro virus prepared in a known culture, preferably in primary or secondary fibroblasts made from chicken embryos, and resulting in embryo processing, with at least one pharmaceutically acceptable carrier, and .