CS265962B1 - Therapeutical means against infective gastro-enterites - Google Patents

Abstract

Therapeutic agent against infectious contains gastroenteritis as effective bovine colostral antibody component from prepared colostrum against enteropathogenic serotypes of Escherichia coli, for example 055, 086, 0 111, 0 119, 0 126, 0 127, samonellam for example salmonella typhimurium and human rotaviruses, for example serotypes 1 and 2 in the direct titers agglutination reaction of at least 640 with bacterial antigens and at least 16 in antisense immunoelectrophoresis with human antigens rotaviruses. Process for preparing the composition it is that they milk the dairy cows intramuscularly 6 to 2 weeks before term inactivated heat adjuvant composition intestinal microorganisms, in particular enteropathogenic serotypes of Escherichia 055, 086, 0 111, 0 119, 0 126, 0 127 a salmonell typhimurium having the ability to to induce morphological changes in cellular cultures and mutants of human rotaviruses serotypes 1 and 2, selected in cellular cultures capable of propagation to titers reaching at least 10 ® FU / ml infectious media and colostrum taken 3 to 45 h after calving is freed from fat after cooling, by acidification and the precipitation of casein, whey is filtered or centrifuged and lyophilized.

Description

The invention solves the problem of inducing resistance and treatment of newborns and children exposed to infections or indicated in particular by enteropathogenic stones of Escherichia, salmonella and rotaviruses.

In addition to respiratory tract infections, foodborne infections are one of the most serious causes of the disease, especially in neonates and children. The etiology of these infections can be very diverse and depends on factors of epidemiological, climatic or seasonal, hygienic habits of the individual and his immunobiological condition.

In our country, the most common causes of gastroenteritis in childhood are enteropathogenic Escherichia coli, rotaviruses and salmonella. Compared to these, other, laboratory-proven infectious agents play - outside the epidemic period - a much smaller role.

Long-term statistics during the years 1982 to 1985, which monitored the etiology of foodborne infections under 12 years of age hospitalized at infectious disease clinics, Faculty of General Medicine, Charles University and Faculty of Pediatrics in Prague, based on laboratory verification of antibody dynamics showed a separate rotavirus etiology in 16.6% and together with pathogenic or potentially pathogenic agents in 14.4 8, ie in total in 31 8.

Havlík et al. found in a group of 664 children, 50% of whom were infants, bacterial etiology in 35.4 8, of which enteropathogenic strains of E. coli were detected in 8.4 8. They most often occurred in children under 6 months, predominant types of Escherichia coli 0 111 026 and 0 119.

From a statistical survey from 1984, it is known that from the proven etiology of gastrointestinal infections, 43% of enteropathogenic E. coli strains in children under one year of age. The frequent occurrence of foodborne infections, sometimes severe or fatal, has led to the search for effective preventive and curative measures.

Active immunization against foodborne infections is still an unsolved problem, especially for the short-term persistence of antibodies locally acting in the intestinal lumen and for the unresolved issue of preparation of active antigen, suitable route of administration and immunization schedule. In addition to active immunization, another option for protecting the alimentary tract is to prevent the infectious agent from binding to susceptible cells covering its lumen. In this regard, antibodies can be used that bind to active sites on the microbial surface or substances that block receptors on the cell surface for binding to the microorganism.

Individual methods of prevention of foodborne infections are applied by different mechanisms and this itself defines the possibilities of their use. Parenteral immunization against foodborne infections does not affect susceptibility to enteral infection. Active oral immunization, leading to local immunity against battery alimentary infections with inactivated bacterial strains, has not been successful. The effect of vaccination, even using suitably prepared vaccines with conserved microbial surface antigens, is short-lived. For this reason, a solution was sought in the preparation of live vaccines (salmonella, thiezelly), using strains with altered biological properties and preserved antigenicity. Active immunization with live vaccines has not yet found general use in human medicine.

Currently developed live rotavirus vaccines for oral administration-RIT 44 237 live attenuated bovine strain and MMU 18 006 - attenuated monkey rotavirus strain and in recent years developed cold mutants of human rotavirus are candidates for vaccine against rotavirus infections. Another possibility is to obtain a suitable strain by recombination between human rotavirus and rotavirus of another animal species, which is able to achieve a high titer when propagated in cell cultures.

The preparation of live rotavirus vaccines and their potential use are based on the finding that rotavirus infection early in birth will affect the severity of the clinical picture of rotavirus infection at a later age. Both RIT 4,237 and MMU 18,006 attenuated vaccines have been validated in clinical controlled studies. The evaluation of their effect is not uniform - from very favorable to denying their effect at all.

Depending on the dosage, side effects are also present, which can be marked. In any case, active immunization does not prevent rotavirus infection and virus shedding. The term of protection of the vaccinated individual also remains a question, because local immunity in the alimentary tract persists for a short time. Repeated rotavirus infections in one child are not uncommon. Verification of efficacy in a clinical trial may be affected even by random selection of the kit in certain circumstances, in particular low numbers of vaccinees or an epidemiological situation in general.

The mechanism of immunity in rotavirus infections remains a question. Antibodies persist for a short time, cellular immunity is unlikely to apply, so other mechanisms involved in limiting the development of rotavirus alimentary tract infection need to be considered. Such a possibility could be a latent infection of the small intestinal epithelium and the presence of incomplete virus particles that would interfere with virus replication.

The possibility of protecting the alimentary tract from bacterial infection with orally administered serum or colostrum antibodies was experimentally demonstrated in 1968 by Rejnek et al. Folia Microbiol. 1968, 13:36 on the module of microbial piglets and the authors Otnalss et al. in Acta pathol. microbiol. scand. Seet. C. 1980, 88: 15-21. The efficacy of orally administered antibodies on alimentary tract infection was confirmed in a series of later studies.

The effect of breastfeeding on the development and course of rotavirus infections in children has been described in a number of communications. The vast majority of work agrees on the beneficial effect of breastfeeding in rotavirus infections, doubts about this effect were expressed sporadically.

Antibodies against rotavirus surface glycoproteins, which are the neutralizing antigen, are of protective importance. It has been shown experimentally that monoclonal antibodies against VP3 surface glycoproteins VP7 can protect against rotavirus disease.

Specific and cross-reactive antibodies may be used in protection. Human rotavirus serotype 2 carries specific antigens that do not induce the production of antibodies cross-reactive with other rotavirus serotypes. Serotype 3 can induce antibodies reactive with seriotypes 1 and 4.

Enteritis therapy is based primarily on rehydration in mild oral cases, in more severe cases parenteral, dietary adjustment and antibiotics. Although the efficacy of antimetabolites on rotavirus infection has been published in a model experiment, this experience is not used in current medical practice.

The physiological possibility of preventing foodborne illness is the oral administration of specific antibodies, which has been widely tested experimentally in animal experiments and whose beneficial effect has been observed in human practice in breastfed infants exposed to foodborne bovine serum antibody infection by Lodinova R. et al. (Cs. Pediatr. 33: 186-188) and bovine colostrum antibodies by Mietens C. et al. (Eur. J. Pediatr. 1979, 132: 239-252) and Lodinova et al. (Ann. N. Y. Acad. Sci. 1983, 409: 841-844). Oral gamma globulins administered orally in rotavirus enteritis of premature infants also had a therapeutic effect, as reported by Offit. P. A., et al. (J. Virol. 1986, 58: 700-703) and Barnes G. L. et al. (Lancet 1982/1: 1 371-1 373). The use of antibodies specifically targeted to infection by individual bacterial or viral species did not take into account a wide range of etiological factors involved in the induction of enteritis. The need to initiate specific therapy for enteritis at the time of initial symptoms, prior to laboratory-determined etiology, requires a preparation containing polyvalent antibodies capable of specifically targeting the vast majority of infectious agents causing foodborne infections. A preparation of a similar type has not yet been developed. At the same time, commonly used antibiotic therapy is not able to treat viral infections, in addition, it has side effects, especially in newborns and young children.

The above-mentioned disadvantages are eliminated by the therapeutic agent against infectious gastroenteritis according to the invention, which consists in containing as active ingredient bovine colostrum antibodies from prepared colostrum against enteropathogenic serotypes of Escherichia coli, for example 055, 086, 0 111, 0 119, 0 126, 0 127 and salmonellae, for example salmonella typhimurium, and human rotaviruses, for example serotypes 1 and 2 in titers reaching at least 640 in direct agglutination with bacterial antigens and at least 16 in reverse immunoelectrophoresis with human rotavirus antigens.

The method of preparation of these bovine colostrum antibodies consists in immunizing pregnant dairy cows intramuscularly 6 to 2 weeks before the term of calving with an adjuvanted mixture of inactivated intestinal microorganisms, especially enteropathogenic serotypes Escherichia 055, 086, 0 111, 0 119, 0 126, 0 127 and salmonella. typhimurium having the ability to induce morphological changes in cell cultures and mutants of human rotavirus serotypes 1 and 2, selected in cell cultures, capable of propagation to titers of at least 10® FU / ml of infectious medium and colostrum collected 3 to 45 h after calving after cooling. defatted, acidified and precipitated with casein, the whey is filtered or centrifuged and lyophilized at high speed. .

The advantages of bovine colostrum antibodies for the prevention and treatment of the most common foodborne infections in newborns and children lie primarily in the efficacy of the preparation, because in addition to the specific anti-infective effect, non-immunoglobulin factors having anti-infective activity and nutritive or rehydrating effect apply. in safety - orE the preparation does not lead to any side symptoms when administered orally.

The content of antibodies against a wide range of enteropathogenic batteries and viruses, representing approximately 80% of the etiology of childhood enteritis, guarantees the effectiveness of the preparation in a high percentage of cases. The preparation can be applied with a high probability of a specific effect at the beginning of the disease, before laboratory verification of the etiology. The preparation makes it possible to replace antibiotics which have hitherto been part of the conventional therapy for infant enteritis and which carry the risk of secondary, often severe, symptoms. Compared to standard therapy, oral treatment or preventive administration of the preparation has a higher effectiveness, which is manifested mainly in shortening the treatment time by more than half the time compared to conventional therapy. The effectiveness of the preparation is enhanced by its early application.

Antibodies in the alimentary tract prevent the development of the disease, or will affect its course by accelerating healing. It does not affect the intracellular virus present. In rotavirus infection, even in the presence of antibodies, detectable viral antigens in the faeces are sensitive serological reactions.

Bacterial strains, especially enteropathogenic Escherichia, isolated best from epidemiologically verified cases of the disease, after serological determination of their O serotypes are inoculated into cell culture medium, most often VĚRO line, 24 h old, forming a monolayer, 2x rinsed with maintenance medium without antibiotics. Biologically active strains induce changes in different morphology corresponding to different individual serotypes, within 90 min. from inoculation. Selected identified enteropathogenic Escherichia strains are propagated in an antigen-free medium, for example 0.5% lactalbumin hydrolyzate, at 37 ° C in a water bath for about 5 hours, when the number of O cells reaches a concentration of about 10 bacteria per ml and the log growth phase is not completed. The culture is concentrated by centrifugation sedimentation, resuspended in 1/10 of the original medium and inactivated with formol at a final concentration of 0.2 to 0.4% v / v at 37 ° C for 72 to 48 h. Before mixing with adjuvant (eg 8% aluminum hydroxide). gel) the sterility of the bacterial suspension is verified by aerobic and anaerobic culture, the suspensions of adjuvanted inactivated cultures are mixed together with individual freon (1: 2-4) purified and centrifuged (100,000 g / 90 min) concentrated human rotavirus isolates propagated in cell cultures MA 104. Viral antigen, added to the vaccine at a ratio of 1:10, usually gives a clear precipitation line in a 1:32 to 1:64 dilution in reverse immunoelectrophoresis. Healthy, veterinarily controlled cows are immunized with 10 ml of polyvalent vaccine i.m. divided by two injections (2x5 ml) 6 to 4 weeks before the calving date and revaccinated with 5 ml of adjuvant vaccine 14 to 7 days before the calving date. From the third hour after birth up to 40 hours after birth, colostrum is collected, the individual doses being stored separately at 4 ° C until processing, usually within one week. From the cooled colostrum, stratified into a fat upper and lower liquid phase, the lower liquid layer is sieved by means of a pump, tempered to 40 to 42 ° C, precipitated by acidification with 0.1 N HCl to pH 4.4 to 4.5 with continuous stirring. , poured through a narrow porous sieve with an inert surface, allowed to stand at refrigerator temperature to separate the crumb of casein and finally filtered through a clarifying filter. Samples are taken from the whey of each vessel to orientatively evaluate the amount of antibodies against the individual antigens used for vaccination and, according to the titration results, the prepared whey is mixed into a mixture which is filtered through sterilizing filters. Advantageously, it can be used for the separation of whey pre-filtered by a clarifying filter by a high-speed flow centrifuge. The final product is tested microbiologically and the presence of antibodies is quantified. Immediately after filtration or centrifugation and sampling, the whey is filled in 150 ml portions into NTS 300 ml bottles in which it is lyophilized. The lyophilized preparation is again controlled bacteriologically and the amounts of antibodies against individual serotypes are verified by direct agglutination (enteropathogenic Escherichia, Salmonella typhimurium) and human rotavirus by immunoelectrophoresis. The minimum titers required for the active preparation are 640 for bacterial antigens and 16 for human rotavirus antigens. The moisture of the preparation must not exceed 3% by weight.

Before use, the preparation is dissolved in 100 ml of sterile distilled water, in which it is easily soluble. The dissolved preparation is a yellow-green opalescent liquid, somewhat viscous, with a pleasant milky sour aroma. It is administered diluted in the same volume of tea, usually in a volume of 2.5 to 5 ml 6 times a day for 5 days, but it can also be applied as a nutritional solution orally in doses of up to 1,000 ml per child. In the lyophilized state, the preparation is quite stable, stored at 4 ° C and retains its activity for at least 5 years.

The preparation is indicated in children with enteritis (dyspepsia) of bacterial and viral origin.

Anti-bacterial and anti-rotavirus efficacy have been tested in wider clinical trials. The results of these studies are summarized in TAB. 1 to 4.

The efficacy of polyvalent bovine colostrum antibodies has been validated in clinical trials in children with entericitis of bacterial or viral etiology. The colostral antibodies used had titers ranging from 640 to 2,560 in a direct agglutination reaction with antigens of six enteropathogenic serotypes of Escherichia coli and Salmonella typhimurium and 32 in counter-immunoelectrophoresis with antigens of two serotypes (1 and 2) of human rotavirus. One batch of lyophilized prepared colostral antibodies was used for all clinical studies. For reconstitution in sterile distilled water, the preparation was administered orally to sick children in a volume of 3 ml for infants and 2 ml for premature infants in a mixture with the same volume of tea, 6 times daily before feeding for 5 days. Groups of children with enteritis of the same etiology, treated with conventional therapy - diet, rehydration and antibiotics, were used to compare the effectiveness of the preparation. Attention was focused on the effect of the preparation separately in colibacillary, rotavirus, non-colibacillary and rotavirus infections, and combined.

Examples of such clinical trials are provided below and in the tables.

The efficacy of the colostrum antibodies of the invention in foodborne infections of bacterial etiology was tested in a group of 32 premature infants and 24 preterm infants compared to groups of 14 premature infants and 15 infants treated in the usual manner, i.e. diet, rehydration and antibiotics. Individual indicators defining enteritis were monitored, especially the number and quality of stools, weight gain, the need to introduce other therapies, such as parenteral rehydration or total parenteral antibiotics, laboratory findings of pathogenic or potentially pathogenic microbes before and after therapy, other diseases associated with dyspepsia. The results of the comparative study showed that this treatment was safe and effective. Administration of colostrum antibodies in approximately 80% of cases in both groups of children resulted in recovery without any other therapy, the remaining percentage had to be supplemented with parenteral rehydration or antibiotics. In comparison with the control group, colostrum antibody therapy was beneficial in terms of weight gain, number and quality of stools, reduction of persistence of pathogenic or potentially pathogenic microorganisms in the intestine, and finally in the need for perenteral rehydration as an adjunct to treatment.

A comparison of the cumulative number of cured children on individual days during five days of treatment with colostrum antibodies and antibiotics, expressed in%, is shown in the table.

Table 1 Cumulative% to days Number of children' 1 2 3 4 5 premature conventional therapy - - 21 21 57 14 children antibodies - 6 45 69 75 33 delivered conventional therapy - - 6 20 53 15 0 to 6 m. antibodies - 8 33 42 83 24

The etiology of enteritis was bacteriologically confirmed in both groups of children treated with bovine antibodies. In the vast majority of cases, serotypes of enteropateogenic Escherichia corresponding to the specific antibodies present in the vaccine were isolated.

Verification of the effect of bovine polyvalent colostrum antibodies in rotavirus enteritis in newborns and infants.

The effect of polyvalent bovine colostrum antibodies on enteritis of children of rotavirus origin was tested in a group of 30 children in whom rotavirus infection was demonstrated by an increase in specific antibodies in serum or by finding rotavirus antigen in faeces. The results were compared with a group of 38 children with enteritis of the same etiology, conventional treatments. Premature babies and children up to the age of 6 months were assessed separately. The results of this comparative study are shown in tab. 2. The cumulative static data presented in the table represent the percentage of children who experienced clinical recovery, assessed according to the same criteria as in the previous study.

The compared groups of premature infants and groups of children aged 1 to 6 months responded to the administration of the preparation in approximately the same way. Within two days, half of the children treated with bovine polyvalent antibodies were asymptomatic, after the end of therapy all children were without clinical signs of the disease. In the control group of children treated with conventional methods, the remission of the symptoms of the disease was slower. At day 7, 29% of premature infants and 43% of infants aged 1 to 6 months had clinical signs of enteritis.

The difference in the effectiveness of the two treatments compared is clearly evident from the comparison of the duration of the disease, calculated for the individual groups of the group. In the group of premature infants and children aged 1 to 6 months, the persistence of clinical symptomatology was reduced by more than half compared to both groups treated with conventional therapy.

Table 3 shows another group of children with enteritis, where the etiology of the disease was viral, bacterial or combined, in whom treatment with orally administered antibodies was used.

Administration of the product to children with energy of different etiology reduced the symptoms of the disease (Th +) in all fifteen premature infants, in whom the rotavirus etiology was verified by demonstrating the dynamics of antibody in serum (Ab) and rotavirus antigen in faeces (Ag) regardless of any bacterial microorganisms present ( B +). In other cases, the course of the disease was affected in about half of the patients. In most cases, pathogenic or potentially pathogenic bacterial flora was present at the same time or was the sole cause of the disease. At the same time, there is a clear difference in the effect of colostrum antibodies in premature infants and children up to the age of 6 months. ·

The effect of preventively administered rotavirus antibodies was favorably applied in their prophylactic administration to newly admitted patients to the inpatient part of the pediatric clinic where the epidemic of rotavirus enteritis took place.

The effect of the preparation is higher when applied at the beginning of the disease. In advanced infections accompanied by changes in blood electrolytes, the effect of the preparation is limited. In these cases, the recovery time is conditioned by the physiological ability to regenerate the resorption epithelium of the small intestine.

Table 2

Efficacy of bovine colostrum antibody therapy in children with laboratory-confirmed rotavirus enteritis compared to children treated with conventional therapy

Healing to the day Cumulative% Th- 7 Number of children Duration of illness (0 days) 1 2 3 4 5 6 newborns conventional Th 0 0 10 32 67 72 29 24 6.9 including premature colostrum 0 58 75 83 91 100 0 12 2.9 infants conventional Th 0 0 21 36 43 57 43 14 7.5 1 to 5 months colostrum 11 50 67 78 94 100 0 18 3.0

Table 3

Efficacy of bovine colostrum antibody therapy in relation to laboratory findings

Company Ab + B Route AbAg + B Company Ag + B Total company B Unknown etiology Total Th + Th newborns Th + 1 1 3 3 0 4 12 3 2 17 including premature - 0 0 0 0 0 0 0 0 0 0 infants Th + 1 3 3 6 0 5 18 8 0 26 1 to 6 months - 2 2 0 0 2 0 6 3 1 10 children Th + 0 0 0 0 0 0 0 2 0 2 6 to 12 months - 0 0 0 0 0 0 0 0 0 0 45 10

Claims (2)

. OBJECT OF THE INVENTION Therapeutic composition against infectious riastroenteritis, characterized in that it contains as active ingredient bovine colostrum antibodies from prepared colostrum against enteropathogenic serotypes of Escherichia coli, for example 055, 086, 0 111, 0 119, 0 126, 0 127, salmonella, for example salmonella typhimurium and human rotaviruses, for example, serotypes 1 and 2 in titers achieving at least 640 in direct agglutation with bacterial antigens and at least 16 in reverse immunoelectrophoresis with human rotavirus antigens. 2. A process for preparing a composition according to claim 1, characterized in that the pregnant dairy cows are immunized intramuscularly 6 to 2 weeks before the calving date with an adjuvanted mixture of inactivated intestinal microorganisms, in particular enteropathogenic serotypes Escherichia 055, 086, 0 111,, 0 119, 0 126, 0 127 and salmonella typhimurium having the ability to induce morphological changes in cell cultures and mutants of human rotavirus seretypes 1 and 2, selected in cell cultures, capable of propagation to titers of at least 10 FU / ml of infectious medium and colostrum collected 3 to 45 h after calving after cooling, defatting, acidifying and precipitating the casein, filtering the whey or centrifuging and lyophilizing at high speed.