THERAPY FOR ENTERIC INFECTIONS

Abstract

A method and composition for treating enteric pathogen infections in animals suffering from such infections or displaying diseases or conditions consistent with such infections or for preventing or reducing the likelihood of enteric pathogen infections in animals at risk for developing such infections.

Claims

1. A method for treating an enteric pathogen infection in a subject suffering from the enteric infection or displaying a disease or condition consistent with the enteric infection, or for preventing or reducing the likelihood of a subject acquiring an enteric pathogen infection, wherein the subject is at risk of developing the enteric infection, the method comprising administering to the subject: (1) an antibody or a mixture of antibodies directed against said enteric pathogen, and (2) a probiotic or mixture of probiotics directed against, or capable of eradicating or suppressing the growth of, the enteric pathogen or a group of enteric pathogens, wherein the antibody or the mixture of antibodies and the probiotic or the mixture of probiotics are contained in and administered in a delivery unit, and the antibody or the mixture of antibodies and the probiotic or the mixture of probiotics are formulated so they are not capable of contacting each other until a substantial amount of the antibody or the mixture of antibodies has bound the enteric pathogen or the group of enteric pathogens in the subject, and the antibody or the mixture of antibodies is coated or encapsulated, or is in an outside or outer layer of a capsule, such that the antibody or the mixture of antibodies are not in physical contact with the probiotic or mixture of probiotics in the delivery unit, and the probiotic or the mixture of probiotics is coated or encapsulated, or is in an inner part of the capsule, such that the probiotic or the mixture of probiotics are released in the subject after the antibody or the mixture of antibodies are released in the subject.

2. (canceled)

3. The method of claim 1, wherein the subject is a human.

4. The method of claim 1, wherein the pathogen or group of pathogens are selected from the group consisting of Aeromonas hydrophilia, Bacillus cereus, Vibrio parahemolyticus, Vibrio cholerae 01, Vibrio cholera non-01, Vibrio vulnificus, Salmonella enteric, Salmonella typhi, Salmonella paratyphi, Salmonella entertidis, Salmonella cholerasuis, Salmonella typhimurium, Clostridium difficile, Clostridium botulinum, Clostridium perfringens, Staphylococcus aureus, Escherichia coli, Campylobacter jejuni, Campylobacter coli, Campylobacter lari, Campylobacter fetus, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Plesiomonas Shigelloides, Listeria monocytogenes, an enteric virus, a parasite, fungi, a luminal or a tissue mycobacteria, Helicobacter pylori, Giardia lamblia, Dientamoeba fragilis, Blastocystis hominis and Entamoeba histolytica.

5. The method or the composition of of claim 4, wherein the enteric virus is a non-enveloped enterovirus, or is selected from the group consisting of a rotavirus, a Norwalk-like virus, an enteric adenovirus, and a coronavirus, wherein the fungi is selected from the group consisting of Cryptosporidium and Cyclospora, and wherein the mycobacteria is selected from the group consisting of Mycobacterium avium avium, Mycobacterium avium paratuberculosis, and Mycobacterium avium silvaticum and other components of the Mycobacterium avium complex.

6. The method of claim 1, wherein the enteric pathogen or group of enteric pathogens is selected from the group consisting of an enteric pathogen or a group of enteric pathogens related to Clostridium difficile infection (CDI) and fragments, components, and products of those enteric pathogens.

7. The method of claim 1, wherein the antibody or group of antibodies are selected from the croup consisting of polyclonal antibodies, monoclonal antibodies, mixtures of polyclonal antibodies and monoclonal antibodies, Fab, Fab, F (ab).sub.2, Fv, dAb, complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, humanized or human antibodies, diabodies and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding.

8. The method of claim 7, wherein the antibodies are selected from the group consisting the groups of antibodies directed to Toxin A, Toxin B, binary Toxin, a supernatant toxin, vegetative forms of the bacterium fimbriae, glycocalyces, pilli, spores, capsules, secreted enzymes proteins, lipids isolated from the cell membranes, the lipopolysaccharide fraction spore, and fractions of spores.

9. The method of claim 8, wherein the secreted enzymes are selected from the group consisting of collagenase, hyaluronidase, coagulase and immunoglobulin A protease.

10. The method of claim 1, wherein the antibody or group of antibodies are IgY antibodies, optionally IgY antibodies raised in chickens.

11. The method of claim 1, wherein the probiotic or mixture of probiotics are selected from the group consisting of Lactobacilli, Bifidobacteria, Escherichia coli, Eubacteria, Saccharomyces, Enterococci, Bacteroides and non-pathogenic Clostridia.

12. The method of claim 11, wherein the non-pathogenic Clostridia is selected from the group consisting of Clostridium butyricum and non-pathogenic C. difficile.

13. The method of claim 1, wherein the probiotic or mixture of probiotics are capable of eradicating or suppressing the growth of the enteric pathogen or group of enteric pathogens in vitro or in vivo.

14. The method of claim 1, wherein the infection or syndrome is, or a symptom or condition caused by the infection is, selected from the group consisting of Irritable Bowel Syndrome, bloating, small bowel bacterial overgrowth, a diverticular disease, a colitis, Crohn's Disease, idiopathic ileitis, constipation, flatulence, and halitosis, a dysmotility condition, reflux disease, pseudo-obstruction, bloating and traveler's diarrhea and Parkinson's disease constipation.

15. The method of claim 14, wherein the Irritable Bowel Syndrome is caused by or results in a symptom selected from the group consisting of diarrhea, pain, and constipation, wherein the diverticular disease is diverticulitis, wherein the colitis is or is associated with a disease or condition selected from the group consisting of ulcerative, Crohn's disease, lymphocytic colitis, microscopic colitis, indeterminate pseudo membranous colitis, proctitis and post infective colitis, and wherein the dysmotility condition is gastroparesis.

16. (canceled)

17. The method of claim 1, wherein the delivery unit is manufactured or formulated as a pharmaceutical composition, or in the form of a capsule, a sachet, a tablet, a granule, a pill, a suppository, an enema, or a formulation or suspension capable of being infused trans-endoscopically or trans-colonoscopically into a duodenum, terminal ileum or via an enteric tube into a jejunum.

18. The method of claim 1, wherein the delivery unit is manufactured or formulated in the form of a food or a drink.

19. The method of claim 1, wherein the probiotic Lactobacillus rhamnosus in the delivery unit is at a dose of about 10.sup.10 probiotic bacteria per delivery unit.

20. The method of claim 1, wherein the antibody or group of antibodies comprises a C. difficile immune egg powder preparation, optionally at 10 gm per unit composition.

21. The method of claim 1, wherein the antibody or the mixture of antibodies is in an outside or outer layer of the capsule, such that the antibody or the mixture of antibodies are not in physical contact with the probiotic or mixture of probiotics in the delivery unit.

22. The method of claim 1, wherein the probiotic or the mixture of probiotics is in an inner part of the capsule, such that the probiotic or the mixture of probiotics are released in the subject about four to twelve hours after the antibody or the mixture of antibodies are released in the subject.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0015] This invention relates to methods and compositions for the treatment and prophylaxis of enteric pathogen infections, diseases and conditions in animals, including humans.

[0016] The methods of the invention comprise the step of administering, serially or separately, (1) antibodies directed against a pathogen or group of pathogens that are related to enteric infections and (2) probiotics directed against at least some of said pathogen or group of pathogens to humans or other animals suffering from infections by the pathogens, or displaying diseases or conditions consistent with such infections, or at risk of developing such infections.

[0017] The compositions of the invention comprise antibodies directed against a pathogen or group of pathogens that are related to enteric infections and probiotics directed against at least some of those pathogen. The compositions of the invention are formulated or administered so as to prevent the antibodies and the probiotics coming into functional contact with each other before the antibodies have substantially bound to the pathogen(s) in the human or other animal.

[0018] As used herein enteric pathogens or pathogens that are related to or consistent with enteric infections refer to organisms capable of causing an infection in the gastrointestinal tract of an animal, including a human.

[0019] Examples of enteric pathogenic organisms include and are not limited to Aeromonas hydrophilia, Bacillus cereus, Vibrio parahemolyticus, Vibrio cholerae 01, Vibrio cholera non-01, Vibrio vulnificus, Salmonella enteric, Salmonella typhi, Salmonella paratyphi, Salmonella entertidis, Salmonella cholerasuis, Salmonella typhimurium, Clostridium difficile, Clostridium botulinum, Clostridium perfringens, Staphylococcus aureus, Escherichia coli (various subclasses), Campylobacter jejuni, Campylobacter coli, Campylobacter lari, Campylobacter fetus, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Plesiomonas shigelloides, Listeria monocytogenes, enteric viruses, for example, rotavirus, Norwalk-like viruses, enteric adenoviruses, coronavirus and all other non-enveloped enteroviruses, and enteric parasites and fungi, for example, Cryptosporidium, and Cyclospora, luminal and tissue mycobacteria, such as Mycobacterium avium avium, Mycobacterium avium paratuberculosis, Mycobacterium avium silvaticum and other components of the Mycobacterium avium complex, Helicobacter pylori, Giardia lamblia and other parasites including Dientamoeba fragilis, Blastocystis hominis and Entamoeba histolytica.

[0020] Among the diseases and infections, without limitation, that are related to or consistent with enteric pathogens are Irritable Bowel Syndrome in its various forms (diarrhea, pain, constipation, predominant or mixtures thereof), bloating, small bowel bacterial overgrowth, diverticular disease, including diverticulitis, colitis (ulcerative, Crohn's, lymphocytic, microscopic, indeterminate pseudo membranous, proctitis, post infective colitis) among others, as well as Crohn's Disease, idiopathic ileitis, constipation, flatulence, and halitosis, dysmotility conditions, including gastroparesis, reflux disease, pseudo-obstruction, bloating and traveler's diarrhea, as well as Parkinson's disease constipation.

[0021] As described herein, the invention in one embodiment comprises a pharmaceutical composition la comprising antibodies directed against an enteric pathogen or group of enteric pathogens and a probiotic or group of probiotics that are likewise directed against at least some of those pathogens. As used herein, the composition may have the two components, the antibody component and the probiotic component, together in one delivery unit. In this embodiment, the two components are functionally separated. in the delivery unit (e.g. by coating of capsules or microencapsulation). For example, the probiotic component may be encapsulated, such that it is delivered to the human or other animal, more slowly or later than the antibody component of the composition. Alternatively, the antibody component comprises the outside or outer layer of a capsule or other delivery unit or is coated on a capsule and the probiotic component comprises the inner part of the capsule. In either alternative, the probiotic component is delivered to the human or other animal after the antibody component has substantially bound to the pathogen(s).

[0022] A composition as used herein may also have the antibody component and the probiotic component in separate delivery systems or units. For example, two separate capsules, sachets, tablets, granules or pills. Again, the intent is to deliver the probiotic component after the antibody component has substantially bound to the pathogen(s).

[0023] The compositions of this invention can also comprise suppositories, enemas, or can be made into suspensions to be infused trans-endoscopically or trans-colonoscopically into the duodenum, terminal ileum or via an enteric tube into the jejunum. For example, in patients whose access to the GI tract has to be obtained in intensive care situations. The composition may also be administered combined with drinks or foods to be ingested serially, e.g., morning ingestion of the antibody component followed, preferably 4-12 hrs later, by the probiotic component of the composition.

[0024] The probiotic component of the composition of this invention is a micro organism selected, for example, from the group consisting of Lactobacilli, Bifidobacteria, E. coli, Eubacteria, Saccharomyces species, Enterococci, Bacteroides or non pathogenic Clostridia, e.g. Clostridium butyricum and non-pathogenic C. difficile. As will be appreciated by one of skilled in the art, other suitable probiotics known in the art may also be used in the compositions of the invention.

[0025] As used in this invention, the probiotic component is one or a group of probiotic organisms that are directed at the pathogen or group of pathogens being targeted or whose risk of infections is being reduced or prevented. As used herein, a probiotic(s) that is directed to an enteric pathogen or group of those pathogens are those probiotics that are capable in culture of eradicating or suppressing the growth of the targeted pathogen or pathogens. Thus, the probiotics of the compositions of this invention are preferably selected by co-culturing the enteric pathogens with a probiotic groups thereof and selecting the probiotics or group that inhibits or suppresses growth of at least some of the pathogen(s).

[0026] The antibody component of the composition of this invention, may be polyclonal antibodies, monoclonal antibodies, Fab, Fab, F(ab).sub.2, Fv, dAb, and complementarity determining region fragments, single-chain antibodies (scFv), chimeric antibodies, humanized or human antibodies, diabodies and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding. Preferably, they are polyclonal antibodies derived from eggs, including egg yolk and albumin, from poultry immunized with at least one antigen derived from at least one enteric pathogen. When the antibodies are monoclonal antibodies, they are also preferably mixtures of monoclonal antibodies or mixtures of monoclonal antibodies and polyclonal antibodies.

[0027] The antibodies of the compositions of this invention are directed against antigens of the enteric pathogens being targeted. The antigens may comprise whole organisms, spores, fimbriae, pilli capsules, glycocalyces, secreted enzymes, e.g. collagenase, hyaluronidase, coagulase, protease, immunoglobin, proteins isolated from cell membrane, lipopolysaccharide fraction as well as attenuated virus particles, toxins, viral proteins and cell surface proteins as well as fragments and. mixtures thereof. Preferably, mixtures are used.

[0028] For example, it is particularly preferred to use as the antigen multiple strains of the pathogen(s) being targeted, or fragments or products of those strains. In one embodiment, for example, strains 027, 017, and 014 of CDI and fragments and toxins produced by those strains are used.

[0029] The antibody components of the compositions of the invention are preferably produced by immunizing a host, preferably poultry and most preferably chickens, with a diverse collection of antigens or immunogens or a group of antigens and immunogens derived from a group of pathogens. However, other hosts known in the art for antibody production including sheep, horses, and cows, can be used. Preferably, IgY polyclonal antibodies are used in the methods and compositions of the invention.

[0030] An alternate approach of antibody generation is the production of monoclonal antibodies. Again, however, it is preferred to use diverse mixtures of monoclonal antibodies in the methods and compositions of this invention. For example, when treating CDI, a mixture of monoclonal antibodies directed to Toxin A, Toxin B. or the binary toxin, or preferably all these are used. In the more preferred embodiments of a composition for treating CDI, the antibody component would contain a multiplicity of monoclonal antibodies, selected from the groups of antibodies directed to Toxin A, Toxin B, binary Toxin and supernatant toxins yet to be identified but able to be used to immunize from the supernantant, vegetative forms of the bacterium fimbriae, glycocalyces, spores, capsules, secreted enzymes e.g. collagenase, hyaluronidase, coagulase and immunoglobulin A protease, proteins and lipids isolated from the cell membranes and the lipopolysaccharide fraction spore and fractions of spores.

[0031] Monoclonal antibodies can be used alone (singularly) or preferably in combinations (mixtures) and alone or with polyclonal antibodies.

[0032] In a preferred embodiment when antibodies raised in chickens are used, the egg product, egg yolk alone or the whole egg content may be used to produce an antibody powder for administration after e.g. freeze drying spray drying. The whole egg versus yolk alone is preferred because of the increased amount of I Y available and the presence of the albumin as a support vehicle to stimulate the antibody-antigen binding within the GI tract during administration.

[0033] The amount of the antibody component and the probiotic component and the other treatment parameters of the compositions of this invention and in the methods of this invention are easily determined by those of skill in the art taking into account the patient, his or her history, the infection or condition being treated and the effect of various treatments.

[0034] The methods and compositions of the invention may also include additional steps or components. For example, in one embodiment, the human or other animal is pre-treated with antibiotics targeted against the enteric pathogens to reduce the infective load or numbers of the pathogens in the enteric tract. For example, in the situation where a patient is infected with Clostridium difficile pre-treatment with anti-clostridial agents including metronidazole, vancomycin, rifampicin, rifaximin, nitazoxanide or rifabutin used singly or in combinations, for at least one day and up to 3 monthsreduces the load of the bacteria and spores.

[0035] In a further embodiment of the invention, the effectiveness of the methods and compositions may be enhanced by reducing the acid in the stomach during antibody ingestion to prevent acid damage of the antibody protein [curdling]. This can be achieved with H2 receptor antagonists, but more preferably by proton pump inhibitorsPPI's (e.g. omeprazole, lansoprazole, esomeprazole, pantoprazole, rabeprazole and other PPI's), administered prior to the ingestion of the medications. Ideally, the PPI should he given at least 2-4 hours before and in some situations a PPI could be combined with a H2RA to maximize acid suppression. This will allow the passage both of the probiotic component and especially the antibody component of the compositions of this invention through the gastric space without causing precipitation of the large proteins which make up the antibodies.

[0036] In some embodiments of the invention species other than humans benefit from methods compositions of this invention. In particular, for example, dogs which develop chronic Clostridium perfringens diarrhoea and other such diarrhea conditions caused by specific and non-specific pathogens may be treated in accordance with this invention.

[0037] In the prophylactic embodiment of this invention, the compositions of the invention are administered, often at lower doses than in the situation of active infection, to humans and other animals at risk of enteric pathogen infections.

EXAMPLES

Example 1

[0038] A 48 yr old female with longstanding and recurrent urine infections treated with antibiotics developed chronic diarrhoea. After a number of stool tests toxigenic Clostridium difficile was detected in the stool. This was a non-epidemic strain but nevertheless caused chronic diarrhoea which occurred between 10 and 15 times per day causing occasional incontinence.

[0039] The patient had been treated initially with 20 gm/d of C. difficile immune egg powder preparation for 10 days but her stool continued to be C. difficile-positive and her diarrhoea recurred. She was then given a combination of 10 gm of the same egg powder but this time together with Lactobacillus rhamnosus strain CDD1. This strain was selected because it could inhibit C. difficile in vitro and was added at a dose which was equivalent to 10.sup.10 bacteria for ten days. The antibodies were administered in the morning and the probiotic bacteria eight hours later for 10 days.

[0040] On Completion of the study (at 4 and 8 weeks), the patient was free of C. difficile infection on stool tests. Her diarrhoea settled by day 3 of the combined therapy.

Example 2

[0041] A 9 year old male allergic to penicillin was given prophylactic clindamycin following a cut finger which was then sutured in the emergency room in a San Francisco hospital. 3 to 4 weeks after finishing the clindamycin he developed diarrhoea. This diarrhoea was associated with cramping, urgency, malaise and progressive weight loss of about 2 to 3 kg. He was diagnosed as having the epidemic strain of C. difficile and was given metronidazole. He developed nausea and was then given vancomycin capsules 250 mg tds. His diarrhoea was inhibited quite effectively both by the metronidazole and by the vancomycin. Within 2 to 4 weeks of stopping the medications the diarrhoea would recur with up to 8 or 12 diarrhoeal stools per day. Over the next 18 months there were numerous recurrences of the diarrhoea each time suppressed by vancomycin. Numerous protocols of reducing doses of Vancomycin were tried but he continued to have diarrhoea.

[0042] The patient was then treated with 10 gm daily of the anti-C. difficile antibodies for 10 days. His diarrhoea initially settled but then recurred 3 to 4 weeks after the cessation of the 10 day treatment. He was then given a 10 gm morning dose of the antibody followed by an evening dose of a Bifidobacterium strain CDD2 strain that could inhibit C. difficile as a combination therapy for 10 days. The diarrhoea again ceased by day 3 and has not recurred now for nearly 8 months. His stool remain C. difficile negative. The patient has gained weight, has formed stools, has no pain, no urgency and no incontinence.