GB1560344A

GB1560344A - Agent for the prophylaxis and therapy of gastroenterits

Info

Publication number: GB1560344A
Application number: GB15618/77A
Authority: GB
Original assignee: Behringwerke AG
Current assignee: Siemens Healthcare Diagnostics GmbH Germany
Priority date: 1976-04-14
Filing date: 1977-04-14
Publication date: 1980-02-06
Also published as: NO771289L; PT66433A; SE7704245L; IL51863A0; NL7703879A; FR2347934B1; ES457602A1; AU523590B2; AU2420677A; BE853603A; IT1075316B; FI771143A; AR211891A1; JPS52125621A; CA1092974A; DE2616406A1; FR2347934A1; FI61406C; IE44814L; YU96877A

Abstract

The agent for the prophylaxis and therapy of gastroenteritis in primates is prepared by immunising ruminants. The immunisation is effected with Nebraska calf diarrhoea virus and/or serologically related viruses. The antibodies which are produced are preferably isolated from the milk of the ruminants.

Description

(54) AGENT FOR THE PROPHYLAXIS AND THERAPY OF GASTROENTERITIS (71) We, BEHRINGWERKE AKTIEN GESELLSCHAFT, a body corporate organised according to the laws of the Federal Republic of Germany, of D-3550 Marburgl Lahn, Federal Republic of Germany, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: The present invention relates to an agent for the porphylaxis and therapy of gastroenteritis, in particular of infantile gastroenteritis, of humans.

Infantile gastroenteritis (diarrhoea) in humans and animals has always been a serious problem, and an active prophyactic and therapeutic agent has been sought for years.

In 1969 there was isolated the agent causing a great number of cases of diarrhoea of calves. This agent was found to be a virus and was called the Nebraska Calf Diarrhoea Virus (NCDV). In 1975, it was found that the calf diarrhoea could be prevented by the colostrum of animals which were immune to the NCDV and/or antigenetically as well morphologically undifferentiable viruses.

Since 1973, in many cases of human infantile gastroenteritis a virus (Rotavirus) has been isolated from the faeces of the sick children, but it had not been proved that the isolated virus can be considered to be the causative agent of infantile gastroenteritis.

The present invention is based on the observation that orally administrable antibodies against Rotavirus and/or NCDV obtained from the colostrum and/or milk of immunized ruminants give protection against a gastroenteritis in primates induced experimentally with Rotavirus. The clinical manifestation in primates is very similar to that of gastroenteritis in humans for which hitherto no infectious agent could be found, in particular that known as winterdiarrhoea.

Thus, the present invention provides a pharmaceutical preparation, especially for oral administration, for the prophylaxis and therapy of gastroenteritis in primates, especially infantile gastroenteritis, which comprises antibodies against the Nebraska Calf Diarrhoea Virus (NCDV) and/or serologically related viruses, hereinafter referred to as Rotavirus, in admixture or conjunction with a pharmaceutically suitable carrier.

The viruses described in the literature in connection with infantile gastroenteritis and having different names, for example, Rotavirus, Orbivirus, and similar to Reo-virus, Duavirus, seem to ;be synonyms for only one microorganism, according to the present state of our knowledge. However, it is also possible that the viruses that have been described are not identical but are related viruses within a group. As indicated above, we designate this virus or group of viruses as Rotavirus. Since the virus or group of viruses in question have not yet been classified, it will be appreciated that their nomenclature may be changed. Antigenetically related viruses, the relationship of which has not yet been completely clarified but which we include in the category of Rotaviruses, may serve in the same manner or in a very similar manner as an antigen for the production of antibodies for use in the preparation of the invention.

Secretory antibodies of the classes IgA, IgG and IgM are preferred, for use in the preparation of the invention because they have a longer half-life time in the intestine than serum antibodies. Secretory antibodies are found in colostrum and milk, and also in the intestinal juice and in saliva. They may be isolated, for example, from the milk of ruminants immunized with NCDV andi or serologically related viruses (Rotavirus).

However, according to the invention, there may also be used antibodies which are obtained in the usual manner by immunization of animals and isolation of the antiserum.

The invention accordingly provides a process for the production of a pharmaceutical preparation of the invention, which comprises immunising an animal against Nebraska Calf Diarrhoea Virus and/or against a serologically related virus or viruses, isolating the antibodies, and bringing them into the form of a pharmaceutical preparation, preferably for oral administration.

The antibodies may be isolated as an antiserum from the blood of an immunized animal, but it is preferable to isolate the secretory antibodies from the milk or colostrum of a female mammal. Ruminants and especially cows are particularly useful on account of the large amount of milk they produce, but any other mammal may be used.

The antigen used to immunize the host animal is preferably Rotavirus isolated from the faeces of a human being, especially a child, suffering from gastroenteritis. The isolated Rotavirus may be one virus or several related viruses. NCDV itself may be used, either alone or together with Rotavirus. The viral antigen may be inactivated.

The immunization of an animal may be carried out conventionally. It has been found to be advantageous to immunize a female ruminant by an initial oral dose of antigen during the dry period before parturition. Later during the dry period the antigen is administered into the lymph drainage system of the total udder, into the udder through the teat canal, and intramuscularly, and this procedure is repeated before parturition. During lactation, the antigen is preferably administered into the udder through the teat canal and by intramuscular injection.

The antigen used to immunize a host animal is preferably prepared as follows: Rotavirus is isolated from the faeces of diseased children by centrifugation at low and high speeds in the manner described Melnick and Wenner in Diagnostic Procedures for Viral and Rickettsial Infections, 4th Ed. page 561, New York, 1969. The virus is cultivated in tissue cultures of primary, embryonal or postpartal bovine kidney cells or in cells of a permanent pigs kidney cell line (PK 15). After optimum proliferation of the virus in these cells, the infectious supernatant of these cultures, titre about 106-107 ID;,/ml, is harvested and serves as the starting material for immunization.

To prepare an inactivated antigen, the viruses are concentrated by ultracentrifugation (pelletting at about 100,000 x g) or ultrafiltration, by a factor of 5 to 1000, preferably 10 to 100. The inactivation can be effected with formaldehyde according to Lauffer, Biochemistry of Viruses, Pergamon Press, Oxford 1958, or according to LoGruppo, Ann. N.Y. Acad. Sci. 83 (1960) with beta-propiolactone. Other inactivation methods may be used, for example that according to Wiktor et al., Microbiol. 23 (1972).

To obtain secretory antibodies, the following procedure may be used: Female ruminants are infected orally during their dry period about 8 weeks before parturition, with an effective dose of I0i to 101 , preferably 107 to 10S, infectious dose,,(U),0) with proliferable NCDV and/or Rotavirus. Both 4 weeks and 1 week before parturition, proliferable and/or inactivated NOV and/or Rotaviruses with or without adjuvant are administered in the following manner: each time about 5-50 ml, preferably about 10 ml, of proliferable (titre) 10r-101 ID,,/ml) and/or inactivated viruses are injected subcutaneously into the lymph drainage system of the total udder; each time about 50 to 200 ml, preferably about 100 ml, of the same preparation are instilled into each quarter of the udder or each half of the udder through the teat canal; each time about 2 to 10 ml, preferably about 5 ml, of the same antigen are injected intramuscularly.

During the lactation period, about 50 to 200 ml, preferably about 100 ml, of the above antigen preparation are instilled through the teat canal into each udder quarter or half, at intervals of several weeks, preferably about 4 weeks. In addition, about 5-50 ml, preferably about 10 ml, of the antigen preparation are injected intramuscularly at intervals of several, preferably about 12, weeks.

Other immunization methods may be used, for example that according to Pasieka et awl., Can. J. Microbiol. 21: 655 (1974) or according to Watson et al., Res. Vet. Sci.

18: (1975).

The antibody-containing gammaglobulin fraction may be isolated from the colostrum and milk of the immunized ruminants as follows: The colostrum taken from the day of birth on is degreased and decaseinated, generally in the manner described by Knop et al., Aust. J. Exp. Biol. Med. Sci. 49: 405 (1971).

The gammaglobulins are isolated from the whey by ammonsulphate precipitation in the manner described, for example by Pasieka et Z., Can. J. Microbiol. 21: 655 (I 974). They are purified by chromatographic methods, for example, according to the methods described by Knop et al., Aus.

J. Exp. Biol. Med. Sci. 49: 405 (1971) and Knop et al., J. Infect. Dis. 130: 368 (1974), or by the methods described by Hammer et al., Eur. J. Biochem. 6: 443 (1968) and Pasieka et al, Can. J. Microbiol. 21: 655 (1974). They are then filtered under sterile conditions and tested for the absence of proliferable germs.

The gammaglobulin fractions which contain such antibodies against NCDV and/or Rotavirus are the essential component of the preparations of the invention for the prophylaxis and therapy of infantile gastroenteritis.

It has proved advantageous to adjust the preparation to a virus-neutralizing titre of about 1:100 to 1: 1000, for example, from 1:300 to 1: 400/my, for example, 1: 320/ml.

It is particularly advantageous to administer the preparation of the invention orally and to allow it to become active in the gastro-intestinal tract. The dose administered is generally dependent on the virus-neutralizing titre, for example, with medium virus-neutralizing titres of 1: 50Q, about 0.1 to 10 g, preferably 0.5-5 g, of antibody protein per dose is administered.

In general, this corresponds to 1-100 ml, or 5-50 ml of an antibody solution.

The gammaglobulin preparations may also comprise one or more further components, for example, substances which neutralize the gastric juice, for example, sodium bicarbonate or magnesium carbonate, carrier substances, for example, aluminium hydroxide, and protein-stabilizers for example, amino acids and peptides, for example., glycine. The galenical preparation is effected in the manner employed for orally administrable medicaments which are active in the gastro-intestinal tract.

The preparation may be used monospecifically or may also comprise antibodies against other microbial or other viral causative germs of gastroenteritis.

The following Examples illustrate the invention.

Example I Five mqnkeys of the genus macaque which had been delivered by Casarean section and kept without mother were infected with 1000 1D,, Rotavirus. One hour later, 10 ml of antibody preparation having an antiviral activity of 1:320/my were administered orally with a syringe without cannula. A control group of 5 monkeys was given an immuno-globulin preparation of non-immunized ruminants (control globulin) obtained in the same manner.

The monkeys treated with the control globulin were all affected with diarrhoea, whereas none of the animals treated with the specific immuno-globulin fell sick.

Exrrn7pSe 2 A group of 5 monkeys kept in the same manner received, 2 hours prior to the infection with 1000 iD,0 Rotavirus, 10 ml per animal of the antibody preparation. The respective control group of 5 animals received at that time 10 ml of control globulin. Whereas all animals which had received control globulin were affected by diarrhoea, the animals which had been treated with the specific immuno-globulin did not show any symptoms of the disease.

Example 3 10 Animals were infected with 1000 ID,, Rotavirus. After outbreak of the disease, 5 animals were treated orally with 10 ml each of specific immunoglobulin with a titre of 1:320/ml. 5 Animals were given at that time 10 ml each of control globulin. All animals treated with control globulin fell severely ill. Of the animals treated with specific immuno-globulin, 3 were free from symptoms within 24 hours and 1 animal showed a strongly shortened and mitigated course of the disease. With one animal, the course of the disease could not be influenced as in the control group.

Exa,wpsle 4 An antibody preparation was prepared as follows: (i} Rotavirus was isolated from the faeces of diseased children by centrifugation at low and high speeds in the manner described Melnick and Wenner in Diagnostic Procedures for Viral and Rickettsial Infections, 4th Ed. page 561, New York, 1969. The virus was cultivated in tissue cultures of primary, embryonal or postpartal bovine kidney cells or in cells of a permanent pigs kidney cell line (PK 15).

After optimum proliferation of the virus in these cells, the infectious supernatant of the cultures, titre 1068 ID,,/ml, was harvested and served as the starting material for immunization of cows.

To prepare an inactivated antigen, the viruses were concentrated by ultracentrifugation (pelletting at about 100,000 xg) by a factor of 80. The inactivation was effected with formaldehyde according to Lauffer, Biochemistry of Viruses, Pergamon Press, Oxford 1958.

(ii) Cows were infected orally during their dry period, 8 weeks before parturition, with an effective dose of 1075 infectious dose50(IDs0) of the above proliferable Rotavirus. Both 4 weeks and 1 week before parturition, a preparation of the above inactivated Rotaviruses together with an aluminium hydroxide adjuvant (10% by volume of a 10% w/v suspension) has administered in the following manner: each time 12 ml of inactivated viruses (titre 106 ID,,/ml) were injected subcutaneously into the lymph drainage system of the total udder; each time about 120 ml of the same preparation were instilled into each quarter of the udder through the teat canal; each time 7.5 ml of the same antigen were injected intramuscularly.

During the lactation period, 120 ml of the above antigen preparation were instilled through the teat canal into each udder quarter at intervals of 4 weeks. In addition, 15 ml, of the antigen preparation were injected intramuscularly at intervals of 1G weeks for 6 months.

(iii) The colostrum taken from the day of birth on was degreased and decaseinated in the manner described by Knop et al., Aust. J. Exp. Biol. Med. Sci. 49: 405 (1971).

The gammaglobuhus were isolated from the whey by ammonsulfate precipitation in the manner described by Pasieka et awl., Can. J. Microbiol. 21 :655 (1974). They were purified by chromatographic methods according to the methods described by Knop et al, Aus. J. Exp. Biol. Med. Sci.

49: 405 (1971) and Knop et al., J. Infect.

Dis. 130: 368 (1974) or by Pasieka et al., Can. J. Microbiol. 21: 655 (1974).

They were then sterile filtered and tested for the presence of proliferable germs.

The specific immuneglobulin titre was adjusted to 11320/ml.

WHAT WE CLAIM IS: 1. A pharmaceutical preparation for the prophylaxis and therapy of gastroenteritis in primates, which comprises antibodies against Nebraska Calf Diarrhoea Virus (NCDV) and/or against a serologically related virus or viruses. in admixture or conjunction with a pharmaceutically suitable carrier.

2. A pharmaceutical preparation as claimed in claim 1, wherein the antibodies are secretory antibodies.

3. A pharmaceutical preparation as claimed in claim 2, wherein the antibodies have been isolated from the milk or colostrum of a female mammal which has been immunized with NCDV and/or serologically related viruses.

4. A pharmaceutical preparation as claimed in claim 3, wherein the female mammal is a female ruminant.

5. A pharmaceutical preparation as claimed in claim 4, wherein the female ruminant is a cow.

6. A pharmaceutical preparation as claimed in any one of claims 1 to 5, wherein the virus-neutralizing titre of the preparation is within the range of from 1:100 to 1:1000 per ml.

7. A pharmaceutical preparation as claimed in claim 6, wherein the titre is from 1:300 to 1:400 per ml.

8. A pharmaceutical preparation as claimed in any one of claims 1 to 7, wherein the preparation is in unit dosage form.

9. A pharmaceutical preparation as claimed in claim 8, wherein a unit dose comprises from 0.1 to 10 g of antibody protein.

10. A pharmaceutical preparation as claimed in claim 9, wherein a unit dose comprises from 0.5 to 5 g of antibody protein.

11. A pharmaceutical preparation as claimed in any one of claims 1 to 10, which also comprises antibodies against one or more other causative agents of gastroenteritis.

12. A pharmaceutical preparation as claimed in any one of claims 1 to 11, which also comprises a protein stabilizer.

13. A pharmaceutical preparation as claimed in any one of claims 1 to 12, in a form suitable for oral administration.

14. A pharmaceutical preparation as claimed in claim 13, which also comprises a substance capable of neutralizing acidic gastric juice.

15. A pharmaceutical preparation as claimed in claim 1, substantially as described in Example 4 herein.

16. A process for the production of a pharmaceutical preparation as claimed in claim 1, which comprises immunizing an animal against Nebraska Calf Diarrhoea Virus and/or against a serologically related virus or viruses, isolating the antibodies and bringing them into pharmaceutical preparation form.

17. A process as claimed in claim 16, wherein a female mammal is immunized and the antibodies are isolated from the milk or colostrum.

18. A process as claimed in claim 16 or claim 17, wherein the virus or viruses is or are inactivated or a mixture of inactivated and noninactivated viruses are used.

19. A process as claimed in any one of claims 16 to 18, wherein the serologically related virus or viruses is or are isolated from the faeces of a human being suffering from gastroenteritis.

20. A process as claimed in claim 19, wherein the human being is a child.

21. A process as claimed in any one of claims 17 to 20, wherein the female mammal is a female ruminant.

22. A process as claimed in claim 21, wherein the female ruminant is a cow.

23. A process as claimed in claim 21 or claim 23, wherein the female ruminant is immunized as follows: (i) before parturition an oral dose of noninactivated virus(es) is admini

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **. each time 12 ml of inactivated viruses (titre 106 ID,,/ml) were injected subcutaneously into the lymph drainage system of the total udder; each time about 120 ml of the same preparation were instilled into each quarter of the udder through the teat canal; each time 7.5 ml of the same antigen were injected intramuscularly. During the lactation period, 120 ml of the above antigen preparation were instilled through the teat canal into each udder quarter at intervals of 4 weeks. In addition, 15 ml, of the antigen preparation were injected intramuscularly at intervals of 1G weeks for 6 months. (iii) The colostrum taken from the day of birth on was degreased and decaseinated in the manner described by Knop et al., Aust. J. Exp. Biol. Med. Sci. 49: 405 (1971). The gammaglobuhus were isolated from the whey by ammonsulfate precipitation in the manner described by Pasieka et awl., Can. J. Microbiol. 21 :655 (1974). They were purified by chromatographic methods according to the methods described by Knop et al, Aus. J. Exp. Biol. Med. Sci. 49: 405 (1971) and Knop et al., J. Infect. Dis. 130: 368 (1974) or by Pasieka et al., Can. J. Microbiol. 21: 655 (1974). They were then sterile filtered and tested for the presence of proliferable germs. The specific immuneglobulin titre was adjusted to 11320/ml. WHAT WE CLAIM IS:

1. A pharmaceutical preparation for the prophylaxis and therapy of gastroenteritis in primates, which comprises antibodies against Nebraska Calf Diarrhoea Virus (NCDV) and/or against a serologically related virus or viruses. in admixture or conjunction with a pharmaceutically suitable carrier.

20. A process as claimed in claim 19, wherein the human being is a child.

22. A process as claimed in claim 21, wherein the female ruminant is a cow.

stered; (ii) later ;but still before parturition the antigen is administered into the lymph drainage system of the total udder, into the udder through the teat canal and intramuscularly, this procedure being repeated before par tuition; (iii) during lactation the antigen is administered into the udder and intra muscularly.

24. A process as claimed in claim 23, wherein (i) the antigen is admanistered 8 weeks before parturition in a dose of 10' to 1010ID50, (ii) the antigen is administered 4 weeks and 1 week before parturition in the following doses: 5 to 50 ml of antigen, titre 104-101 Dá0/ml into the lymph drainage system, 50 to 200 ml of antigen, titre 104-1010 ID,,/ml into each quarter or each half of the udder.

2 to 10 ml of antigen, titre 104-1010 ID50/ml intramuscularly, (iii) the antigen is administered into the udder at 4 week intervals in a dose of 50 to 200 ml, titre 104-101 IDs0lml and intramuscularly at 10-12 week intervals in a dose of 5 to 50 ml, titra 104101o ID,,/ml.

25. A process as claimed in any one of claims 16 to 24, wherein the virus- neutralizing titre of the resulting preparation is brought into the range of from 1:100 to 1:1000 per ml.

26. A process as claimed in claim 25, wherein the titre is from 1:300 to 1:400 per ml.

27. A process as claimed in claim 16, carried out substantially as described in Example 4 herein.

28. A pharmaceutical preparation as claimed in claim 1, whenever produced by a process as claimed in any one of claims 16 to 27.