MXPA97001683A - Therapeutic treatment of diseases associated with clostridium difficile - Google Patents

Abstract

The treatment of diseases associated with CLOSTRIDIUM DIFFICILE, such as CLOSTRIDIUM DIFFICILE COLITIS, Pseudomembranous colitis and diarrhea associated with antibiotics by administering an antibody having specific activity against CLOSTRIDIUM DIFFICICLE COLITIS and toxins thereof alone or combination with vancomycin, bacitracin or metronidazole is described in the present, as well as pharmaceutical compositions for the same

Description

TRAT- ^ IIENTQ THERAPEUTIC OF C OSTRIDim DIFFICILE This invention relates to the treatment of Clostridium difficile-associated diseases such as Clostridium difficile colitis, pseudomembranous colitis and diarrhea associated with antibiotics, by administration of specific antibodies in the form of oral and / or pharmaceutical compositions.

AWGJCJ¿DEWGES AND THE BWBMCIQN Clostridium difficile was first described in 1935 as a gram-positive anaerobic bacillus. The bacterium is called the "clostridium of difficult growth" because it resisted the first attempts at isolation and grows very slowly in culture. There was no news of the bacteria until the 1960s and 1970s when there was an increase in pseudomembranous colitis associated with antibiotics due to the wide use of broad spectrum antibiotic agents such as lincomycin and clindamycin. It was found that these antibiotics cause diarrhea in approximately REF: 24129 10% of patients, and produce pseudomembranous colitis in approximately 1%. It is now evident that C. difficile is responsible for colitis in humans, diarrhea associated with antibiotics and virtually all cases of pseudomembranous colitis. The disease develops as a result of the production of two major toxins, toxin A (Mr, 308,000) and toxin B (Mr, 279,000), by the organism in the colon (Barroso, et al., Nucleic Acids Res. 18: 4004; Dove, et al., Infect, Immu., 58: 480-488; Lyerly, et al., Clin.

Microbe !, Rev,, 1: 1-18). It is considered that toxin A causes most of the gastrointestinal symptoms due to its enterotoxic activity in experimental animals (Borriello, et al., Microecol. Ther .. 15: 213-236; Lyerly, et al., Infect. Immun. 35: 1147-1150; Lyerly, et al., Infect. Irr., 47: 349-352). There is some evidence to suggest that the toxins act synergistically during the development of the disease and that the initial tissue damage caused by toxin A allows toxin B to exert its toxic effect (Lyerly, et al., Immun., Immun., 47: 349-352). Most patients with C. difficile disease are treated effectively with vancomycin, bacitracin, or metronium. However, relapse occurs in approximately 10-20% of cases, indicating that antibiotic therapy is not always completely effective. As a result, research on alternative types of therapy continues. A yeast used to treat gastrointestinal diseases, Saccharomyces boulardii, has shown promising results as a probiotic for the treatment of the disease in humans and in experimental animals (Cort'hier, et al., Can. J. Microbio! .. 32: 894-896; Elmer, et al., Antimicrob. Aaents Chemo her .. 31: 129-131; Kimmey, et al. , Day. Dis. Sci. . 35: 897-901; Surawicz, et al., Gastroenterolosy, 96: 981-988; Toothaker, et al. , Antimicrob. Agents Chemother .. 26: 552-556). The actual manner in which the yeast confers protection is not clear, although it has been reported that the yeast must be viable in order to provide protection (Lucas, et al., Presented at the 15th International Congress of Microbial Ecology of the Disease, September 7 to 9, 1990, Ioannina, Greece). Immunoprophylaxis has also been suggested as a type of treatment. It is known that vaccination against toxins A and B stimulates active immunity against C. difficile disease in experimental animals (Fernie, et al., Dev. Biol. Stand .. 53: 325-332; Libby, et al. , Tnfsc, Tmmun .. 36: 822-829). However, until now, adequate vaccines against the organism and its toxins have not been developed for individuals at high risk, and it is not clear if active immunization is appropriate. Alternatively, treatment by passive immunization has been suggested. In preliminary studies, serum antibodies against a toxigenic isolate of C. difficile protect Hamster against C. difficile disease when administered orally to animals. Thus, passive immunity can be beneficial for a prophylactic treatment. Passive immunization with bovine antibodies has been examined as a possible alternative therapy in the treatment of other infectious diseases of the gastrointestinal tract, including diseases caused by rotavirus, Escherichia coli, enteropathogenic and enterotoxigenic, Vibrio clolerae and Crystosporidium parvum,? the results indicate that the antibodies administered in this manner provide protection (Boesman-Finkelstein, et al., Infect. Trnmun .. 57: 1227-1234; Brussow, et al., J. Clin. Microbiol. , 25: 982-986; Fayer, et al. , Infec. Tn-im-in. , 58: 2962-2965; Hilpert, et al., J. Infec. Dis .. 156: 158-166; Mietens, et al., Eur. J. Pediatr. 132: 239-252; Tacket, et al., N. Engl. J. Med .. 318: 1240-1243; Yoshiyama, et al., Immunology. 61: 543-547). In the passive immunity from bovine antibodies offers the advantages that most animals and humans tolerate the material when administered orally and that the predominant antibody species present, the immunoglobulin Gl (IgGl), is relatively resistant to proteolysis . A recent study has reported the ability of a bovine immunoglobulin G (IgG) concentrate (BIC) colostrum from cows vaccinated with C. difficile toxoid to protect hamsters against experimental antibiotic-associated cecitis. The results indicate that hamsters have been passively immunized against the disease if they are treated before the onset of diarrhea. The authors indicate that they were not able to effectively treat hamsters with hyperimmune BIC once the diarrhea had developed Lyerly, et al. , Infection and Im unity, Vol. 59, No. 6, pages 2215-2218 (1991). Therefore, there is a strong need for a method for an effective treatment of antibiotic-associated diarrhea, pseudomembranous colitis and Clostridium difficile colitis in humans without the risk of relapses in a significant amount of the patient population.

BRIEF DBSCRIPTION OF THE I VBJECTION Accordingly, the present invention includes a method for treating diseases associated with C. difficile colitis in a mammal, comprising: administering to the mammal an effective amount of an antibody having specific activity against C. difficile toxins thereof that are found in the colon. Such diseases include, for example, Clostridium difficile coli tis, pseudomembranous colitis and diarrhea associated with antibiotics. A second aspect of the present invention is the production of a pharmaceutical composition for use in the treatment of C. difficile-associated diseases including, for example, pseudomembranous colitis or antibiotic-associated diarrhea, in a patient comprising: an effective amount of an antibody in combination with a pharmaceutically acceptable carrier adapted for oral or rectal administration, or enteric delivery, the antibody has specific activity against C. difficile and toxins thereof. A third aspect of the present invention is a pharmaceutical composition for use in the treatment of pseudomembranous colitis or antibiotic-associated diarrhea, comprising: a combination of (a) an effective amount of an antibody having specific activity against C. difficile and toxins thereof, and (b) an effective amount of vacomycin, bacitracin or metronidazole, in combination with a pharmaceutically acceptable carrier. Finally, a fourth aspect of the present invention is a method for treating pseudomembranous colitis or diarrhea associated with antibiotics in a patient, comprising: administering to the patient an effective amount of an antibody having specific activity against C. difficile and toxins thereof. concurrently or following the administration of an effective amount of, vancomycin, bacitracin or metronidazole.

BRIEF DESCRIPTION OF IOS DRAWINGS Figure 1 is a graphic illustration of the concentrations of antibody against Clostridium difficile toxin A by the ELISA method. Figure 2 is a graphic illustration of the concentrations of antibody against Clostridium difficile toxin B by the ELISA method. Figure 3 is a photomicrograph of monolayer of IMR-90 fibroblasts after exposure to a culture filtrate of C. difficile (xlO minimum cytotoxic dose) for 24 hours. There is a remarkable cytopathic effect which surrounds all the cells. Figure 4 is a photomicrograph of an IMR-90 fibroblast monolayer exposed to a culture filtrate of C. difficile for 24 hours (xlO minimum cytotoxic dose) preincubated with BIC antifiltration (100 mg / ml). The BIC antifiltration effectively inhibits the itopático effect and the monolayer shows a normal morphology. Figure 5 is a graphic illustration of BIC against clostridium difficile that inhibits the binding of toxin A to its receptor in enterocyte. Figure 6 is a block graphic illustration showing the effect of the bovine immunoglobulin concentrate (BIC) on a clostridium difficile culture filtrate and induces fluid secretion in rat ileal loops. Figure 7 is a graphic block illustration showing the effect of BIC on the culture filtrate of C. difficile which induces mannitol permeability in rat ileal loops. Figure 8 is a block graphic illustration showing the effect of BIC on the culture filtrate of C. difficile that induces enteritis in rat ileal loops. Figure 9 is a photomicrograph of tissue of the ileum exposed to culture filtrate of C. difficile. Figure 10 is a photomicrograph of ileum tissue exposed to C. difficile culture filtrate preincubated with BIC against C. difficile filtering.

DETAILED DESCRIPTION OF IA TMVENCTÓW Current therapies to treat C. difficile infection, diarrhea and colitis associated with antibiotics, use antimicrobials such as metronidazole, bacitracin or vancomycin. These agents result in an additional breakdown of colon flora and are associated with a 10-20% incidence of diarrhea due to relapse. The present invention provides an effective treatment which would not alter the flora of the colon. A concentrate of bovine immunoglobulin (BIC) against C. difficile is prepared from the colostrum milk of Holstein cows previously immunized with C. difficile toxoid (as described in more detail below). This concentrate contains high concentrations of bovine IgG specific for toxins A and B of C. difficile, evaluated by ELISA. BIC against C. difficile neutralizes the cytotoxic effects of both toxin A and toxin B of C. difficile, while non-immune BIC, control, does not do so. BIC against C. difficile also inhibits the enterotoxic effects of C. difficile toxins in rat ileum measured by increasing the weight / length ratio of the rat ileal loop (64% P &l inhibition).0.01), increased permeability of mannitol (89% inhibition, P <0.01) and histological grading of enteritis (P <0.01 versus non-immune BIC). Therefore, the BIC against C. di ffici neutralizes the cytotoxic effects of C. difficile toxins in vitro and inhibits its enterotoxic effects in vivo. As a result of the above experiments, (described in more detail in the examples) the inventors have discovered that the administration of an immunoglobulin product containing specific antibodies against C. difficile results in the elimination of C. difficile toxins and also destroys bacteria. Within the colon and provides a realistic approach to effectively treat C. difficile-associated diseases such as colitis, pseudomembranous colitis and antibiotic-associated diarrhea, and especially for patients experiencing multiple relapses. For purposes of the present invention, the term "antibodies having specific activity against clostridium difficile and toxins thereof" is synonymous with "bovine immunoglobulin concentrate against C. difficile (BIC)". The antibodies can be used alone as a liquid or solid, preferably in the form of a solid powder, and preferably in admixture with a carrier to form a pharmaceutical composition such as a tablet, capsule or suppository. Since the preferred methods of administration are oral and rectal, or enteric, most preferred oral, tablet and capsule are especially preferred, or enteric. Of course, these are prepared according to conventional methods known in the art. The antibodies can also be coed with other pharmaceutically acceptable carriers such as various liquids, proteins or oils which also provide additional nutritional and / or pharmaceutical benefits. Since the effect of the antibody depends on its reaching the colon, the preferred tablets or capsules must be enteric coated. Alternatively, the active antibodies themselves may be microencapsulated prior to formulation. The preparation of antibody microcapsules as well as the preparation of enteric coated tablets or capsules can be obtained by conventional methods known in the art. Because the antibodies present first remove toxins from C. difficile, it is also advantageous to administer to patients suffering from diseases associated with C. difficile a coation of the antibodies of the present invention with antibiotics known to treat pseudomembranous colitis. and / or diarrhea associated with antibiotics. Such antibiotics are, for example, vancomycin, bacitracin and metronidazole. Due to the rapid and rapid elimination of C. difficile toxins, the coation of antibody and antibiotic may require, synergistically much less antibiotic than is usually used to treat such diseases, which results in a reduced development of symptoms, faster symptomatic relief and a lower relapse rate. The doses recognized for administration of metronidazole, for example, are 250 mg four times a day and oral vancomycin is 125 mg four times a day. Administration of these antibiotics with the antibody of the present invention would result in the use of a substantially reduced dosage of the antibiotics. The administration of such coation may be in the form of a single dosage in which both active ingredients are coed and mixed with a pharmaceutically acceptable carrier. Preferred compositions are those adapted for oral or rectal administration and may include enteric coated tablets, capsules or suppositories. The administration of the coation concurrently or subsequently to another in separate dosage forms can still be formulated together in divided tablets or in capsules. This is also known in the pharmaceutical art.

The treatment of patients suffering from diseases associated with C. difficile with the combination of two active ingredients can take place not only concurrently in a single or separate dosage form but also after the administration of one ingredient with another. Preferably, the administration of the antibody followed by the administration of an antibiotic results in an effective treatment of the diseases. The antibody of the present invention is contained in an immunoglobulin fraction provided to the patient. In such form, the amount of immunoglobulin provided to the patient is approximately 1 gram per day. Typically, amounts from about 1 to 20 grams per day will be used. For example, about 1 to 2 grams of immunoglobulin can be administered to a patient, 3 to 4 times per day. The dosage of the antibody formulation to be administered will depend on the patient and the medical history of the patient. Dosages of the specific antibody for adult humans considered by the present invention are considered to be therapeutically effective and range from about 0.1 to 500 mg. However, it should be understood that these doses can be easily adjusted to provide adequate amounts of the antibody to any patient, including children. The invention is further described with reference to the following detailed examples, in which the methodologies are as described in the following. These examples do not mean that they limit the scope of the invention as set forth in the preceding description. The variation within the concepts of the invention is evident to those skilled in the art. The descriptions of the references mentioned are incorporated herein by reference.

EXAMPLE 1 Production of antibodies in Cows Toxoid is prepared from culture filtrate of C. di fficile VPI 10463, which contains high concentrations of toxin A and B. The strain is grown in dialysis flasks by brain heart infusion at 37 ° C for 72 hours, as it has been previously described (Sullivan, et al., Infect. Immun .. 35: 1032-1040) for the production of the culture filtrate. The culture filtrate is subsequently converted to the toxoid by adding 1/100 volumes of formalin and incubating the mixture at 37 ° C for 1 hour. Analysis of the toxoid by test in tissue culture and assay in mice shows that it retains < 1% of its original cytotoxicity and deadly activity. The exposure of the strain used in the animal model is C. difficile VPI 7698, which was isolated from a patient with pseudomembranous colitis and which produces intermediate concentrations of toxin A and B. Both bacterial strains are obtained from a collection of hosted anaerobes in the Department of Anaerobic Microbiology at the Polytechnic Institute and State University of Virginia (Blacksburg, Va) (Department of Anaerobic Microbiology at Virginia Polytechnic Institute and State University). A total of 14 pregnant Holstein cows were maintained in accordance with generally accepted dairy management practices, at the Land O'Lakes Answer Farm (Webster City, Iowa) or the research farm at the Polytechnic Institute and Virginia State University. The cows were injected simultaneously with 5 ml of toxoid (ca. 5 mg of protein) multiplied in 5 ml of incomplete Freund's adjuvant. The cows received multiple immunizations which began at least 60 days before the expected calving date. Blood samples were taken for testing at the time of injection. Colostrum from the first six milkings of hyperimmunized cows was collected and frozen within 2 to 3 hours of collection. The colostrum was stored frozen until it was ready for processing. Briefly, the colostrum of all 14 cows was reheated, I accumulate and centrifuge to reduce the fat component. The pH was lowered to precipitate casein. The pH of the supernatant was then re-adjusted and the material treated by heat to reduce bioburden. Ultrafiltration was used to reduce lactose and mineral content. The material was heat treated again and spray dried to produce the bulk immunoglobulin concentrate to a shelf stable powder. The non-immune IgG concentrate of colostrum from unvaccinated cows is prepared in a similar manner.

EXAMPLE 2 To confirm that immunization of cows with C. difficile antigens is associated with a subsequent increase in specific C. difficile IgG in colostrum milk.

ELISA method The concentrations of bovine IgG for C. difficile were measured by ELISA using a modification of the previously described method (CP Kelly et al., Astroenterolosy 1992; 102: 35-40; DYM Leung et al., J. Pediatr. 1991; 118: 633-637). The coating antigens used to measure the IgG titers include purified C. difficile toxin A. and purified C. difficile toxin. Toxigenic Clostridium difficile was cultured for 72 hours in brain heart infusion broth (Beckton Dickinson, Cockeysville, MD). The conditioned medium is centrifuged and the supernatant is sterilized by filtration by passing it through a 45 μm filter (Nalgene). C. difficile toxins A and B are purified from the supernatant of the culture broth as previously described (C. Pothoulakis et al., J. Clin.Invest 1991, 88: 119-125). Microtiter plates (Immunol II, Dynatech) are coated with toxin A or C. difficile toxin B (each at 10 μg protein per ml in carbonate buffer pH 9.6, 100 μl per well) by incubation for 2 hours at 37 ° C. ° C followed by incubation overnight at 4 ° C. Plates are washed between each incubation step using phosphate buffered saline with 0.05% Tween 20 (PBS-T). The plates are then blocked with 2% human serum albumin (ICN, 100 μl / pczo) in PBS incubated for 1 hour at room temperature. The following preparations of BIC i) immune filtrates from C. difficile culture, lot # 900918 ("# 18") and lot # 921019 ("# 19"), ii) C. difficile immune toxin A, lot # 900927 ("# 27"), and iii) control, "non-immune" BIC lot # 910311 ("# 11"). All BIC samples are diluted in PBS-T (serial serial dilutions) and incubated 100 μl per sample well for 1 hour at 37 ° C. All tests were performed in triplicate. Goat antibody against bovine IgG, labeled with horseradish peroxidase (KPL Laboratories) is used as the secondary antibody (0.2 μg / ml in PBS with 2% human serum albumin) incubated for 1 hour at 37 ° C. The TMB microwell peroxidase substrate (KPL Laboratories) was used as substrate (100 μl / well) and stopped after 2 to 5 minutes with an equal volume of 1 M phosphoric acid. The optical density is then read at 450 nm with 630 nm as reference using an automated photometer (Dynatech). Controls include the replacement of the secondary antibody with peroxidase-labeled anti-human IgG and the suppression of the peroxidase substrate solution. The results are expressed as the optical density of the test wells minus the average optical density of the bottom wells (coated with human serum albumin alone). Both the BIC versus anti-toxin A preparations have substantially higher IgG antibody concentrations compared to purified toxin A. In this case, the highest antibody concentrations are found in colostrum from animals immunized for toxin A [Figure 1]. Increased concentrations of purified B-toxin IgG antibody are evident in the counter-filtered BIC. A BIC against toxin A shows a marginal increase in the concentrations of antibody to toxin B, compared to the control when tested on a weight-to-weight basis [Figure 2].

Bovine Antibody Binding of C. difficile Complete Cell: C. intact toxigenic difficile is washed to remove extracellular toxins, diluted in carbonate buffer pH 9.6 and incubated overnight at 4 ° C in a microtiter plate to attach bacteria to the plate. Bovine immunoglobulin concentrate (BIC) powder, non-immune and immune to C. difficile antigens, is incubated with bound C. difficile intact. Purified affinity-purified anti-peroxidase goat antibody is used for bovine immunoglobulin (heavy and light chain) and tetramethylbenzidine to detect the amount of bovine immunoglobulin bound to C. difficile intact. Antibody binding was quantified by reading the absorbance at 480 nm from the wells in the microtiter plate.

Results: The BIC immune to C. difficile toxin A lot # 900927 has eight times more specific activity for antibodies with respect to C. toxigenic cell difficile compared to non-immune BIC from lot # 910311.

Immune BIC of the culture filtrate of C. difficile, lot # 921019 has four to eight times more antibody activity specific for the entire C. difficile toxigenic whole cell compared to non-immune BIC, lot # 910311.

Bactericidal Activity of Bovine Antibody for C. difficile: The toxigenic log phase of C. difficile is combined with either BIC powder immune to C. difficile toxins or medium alone which serves as a 100% growth control. After four hours of incubation at room temperature in an anaerobic chamber, the bacteria + BIC and the bacteria + medium were serially diluted in medium and then plated on plates of Tryptic Soy Blood (TSBA). The TSBA plates were incubated for 16 hours at 37 ° C in an anaerobic chamber and then colonies of individual bacteria were counted.

Results: BIC immune to C. difficile toxin A, lot # 900927 reconstituted at 10% (bp / volume) and subsequently diluted 1: 2 (final) destroys -90% C. difficile in the reaction culture. - "2 - BIC immune to the culture filtrate of C. difficile, batch # 921019 reconstituted at 10% (w / v) and further diluted 1: 2 (final) destroys 90-95% C. difficile in the reaction culture See also table 1.

I? ELA_1 Bactericidal effects of bovine immunoglobulin against C. difficile toxigenica EXAMPLE 4 BIC against C. difficile is able to inhibit the cytotoxic effects of C. difficile toxins.

Cytotoxicity method Cytotoxicity is determined by rounding cells of confluent monolayers of IMR-90 fibroblasts in tissue culture. The cytotoxic effects of a culture filtrate of C. difficile and purified C. difficile toxins A and B were examined. The minimum cytotoxime dose of each preparation is defined as the minimum dose resulting in 100% cell rounding at 24 hours (0.1-1 ng protein / ml for culture filtrate, 10-25 ng / ml for toxin A and 0.003 ng / ml for toxin B, the intervals represent the variable potency of the different toxin preparations used in these studies). The neutralizing activity of the various preparations of BIC was quantified by adding semidilutions of BIC both at the minimum cytotoxic dose of each toxin preparation or at 10 times the minimum cytotoxime dose and upon determining cell rounding after 24 hours. In each case the results are expressed as the lowest concentration of BIC that is required to avoid 50% rounding of the IMR-90 cells.

Results: The results for the cytotoxicity inhibition experiments are summarized in Tables 2 and 3. Figure 3 is a photomicrograph of an IMR-90 monolayer which has been exposed to xlO the minimum cytotoxic dose of C. difficile culture filtrate during 24 hours. All cells demonstrate cellular rounding typical of the cytopathic effects of C. difficile toxins. Figure 4 illustrates a similar IMR-90 monolayer which has been exposed to the same concentration of C. difficile filtrate. However, in this case the filtrate is incubated for 1 hour at room temperature with BIC against C. difficile (# 18, 100 μg / ml) before its addition to the monolayer. BIC filtering against C. difficile effectively prevents the cytotoxic effects of C. difficile toxins.

TABLE 2 Icirca xlO minimum cytotoxic dose) TABLE 3 (circa xl minimum cytotoxin dose) The culture filtering BIC against C. difficile (# 18) effectively blocks the cytotoxic effects of the culture filtrate of C. difficile and purified toxin A and toxin B. The BIC against toxin A is less effective in inhibiting cytotoxicity.

EXAMPLE 5 BIC against C. difficile is able to block the binding of C. difficile toxin A to its receptor at the rough edge of the entity.

Method Toxin A purified from C. difficile is labeled with 'H by using the Bolton-Hunter protocol as previously described (C. Pothoulakis et al., J. Clin Clin., 1991, 88: 119-125). Radiolabeled toxin A migrates identically to the native toxin in both SDS-PAGE and CLAP and retains all biological activity as cytotoxin (IMR-90 fibroblast rounding test) and enterotoxin (rabbit ileal loop assay) . In the first studies using 3H-toxin A, the presence of a single class of receptors for toxin A on the rough edge of the rabbit ileum is demonstrated with a K of 5.4 x 10"8M and a maximum binding capacity of 5.9 toxin pmol A / mg rough-rim protein Rough ileal membranes (BBM) of rabbit ileum are purified by chelation by EDTA, as previously described in the above reference.The purity of BBM is determined by light microscopy and measurement of alkaline phosphatase and sucrose activity, toxin binding experiments are carried out A-BBM in triplicate in 1.8 ml Eppendorf tubes precoated with 5% skimmed milk in 50 mM Tris, 0.2 M NaCl (pH 7.4). BIC is incubated against C. difficile (30 μl) at various concentrations, with 200 ng of 3 H-toxin A (approximately 35,000 dpm / pmol of specific activity) in 145 μl of 50 mM Tris (pH 7.4) for 2 hours at 20 ° C. The samples are then cooled to 4 ° C and 200 μg of purified BBM is added in 25 μl of 50 mM Tris. After 1 hour of incubation at 4 ° C, 1 ml of 50 mM Tris buffer is added and the tubes are centrifuged for 4 minutes at 11,000 g. The BBM pellets are washed twice with 1 ml of 50 mM Tris and then dissolved in 0.4 ml of buffer containing 10% SDS. The membrane-associated radioactivity is measured by liquid scintillation counting. The results are expressed as toxin A specific binding. The specific binding is defined as' H-linked total toxin less than the bound H-toxin A in the presence of a 100-fold excess of native toxin A, unlabeled.

Results The results are illustrated in Figure 5 (expressed as mean + EE). The specific binding of toxin A is expressed as pmoles of bound 3 H-toxin A per gram of rough-edged membrane protein (pmol / g). The antifiltered BIC (# 18) inhibits the specific binding of 3 H-toxin to the rough-edged membrane in a dose-dependent manner. At a concentration of 15 μg / ml, BIC against filtration completely blocks the binding of the toxin A receptor. The BIC against toxin A also substantially reduces the binding of toxin A in a dose-dependent manner. At a concentration of 15 μg / ml, BIC against toxin A blocks the binding of toxin A-receptor by 84% compared to the same concentration of non-immune BIC, control. The bovine immunoglobulin concentrate of cows immunized against C. difficile culture filtrate or C. difficile toxin A is able to reduce the specific binding of toxin A to the rough-rim membrane of purified rabbit. The inhibition of the toxin A-receptor binding probably results from the binding of bovine IgG against toxin to toxin A, thereby blocking the subsequent toxin-receptor interaction. This represents a putative mechanism of action for the protective effects of BIC against C. difficile to inhibit the enterotoxic effects of toxin A in vivo.

EXAMPLE 6 To demonstrate that BIC against C. difficile is able to inhibit the enterotoxic effects of C. difficile toxins.

Enterotoxicity method Male Wistar rats are anesthetized by intraperitoneal injection of sodium pentobarbital. A laparotomy is performed, the renal pedicles are tied and 3H-mannitol is administered intravenously (10 uCi, NEN, Boston, MA). Closed ileal loops (5 cm) are then formed and injected with 400 μl of 50 mM Tris buffer (pH 7.4) or with Tris buffer containing culture filtrate of C. difficile (20 μg of protein). The inhibitory effect of BIC against C. difficile is determined by the addition of BIC (200 μg) to the toxins before injection into the ileum lumen. The following preparations of BIC are tested: i) culture filtrate of C. difficile immune (# 18 and # 19), ii) C. difficile immune toxin A (# 27), and iii) non-immune BIC control (# 11).

The abdominal incision is closed and anesthesia is maintained with sodium pentobarbital. The animals are sacrificed after 4 hours and the ileal loops are harvested immediately. The ratio of weight to length of the loops is determined as a measure of the effect of enterotoxin. The excretion of mannitol, which indicates the intestinal permeability when conducting a radioactivity count in the nominal fluid, is measured. The ileum tissue samples are also fixed in formalin, embedded with paraffin and stained sections with hematoxylin and eosin. The histological severity of enteritis is classified according to the following characteristics: i) migration of neutrophils and infiltration of tissue, ii) hemagic congestion and edema of the mucosa, iii) epithelial cell damage. A classification from 0 to 3 is assigned, which indicates severe abnormality increasing, to each of these parameters by a histopathologist who is unaware of the experiment.

Results As expected, the culture filtrate of C. difficile shows enterotoxic effects as evidenced by substantial increases in the weight-to-length ratio [Figure 6] and the cleavage of mannitol [Figure 7] in ileum loops exposed to filtrate. The C. difficile culture filtrate from immune BIC (anti-Fil) substantially inhibits these enterotoxic effects (up to 54% by weight / length ratio [Figure 6] and up to 100% for mannitol permeability [Figure 7], p < 0.01 for both). BIC immune to C. difficile toxin A (anti-Txa) 'produces a similar inhibition of the enterotoxicity induced by the filtrate (64% for the weight / length ratio [Figure 6] and 89% for the permeability to mannitol [Figure 7]. ], P <0.01 for both). BIC not immune, control, does not inhibit or have a significant effect on the enterotoxicity of the culture. Histological examination of the ileal loop tissue shows significant damage, which is reflected in an increase in histological classification, in tissues exposed to culture filtrate of C. difficile [Figure 8]. BIC against filtering and BIC against toxin A, both significantly attenuate the enteritis induced by C. difficile filtration as evidenced by a substantial decrease in histological classifications. BIC not immune to control, does not have a significant protective effect. Figure 9 is a photomicrograph illustrating a representative section of ileal mucosa after exposure to culture filtrate of C. difficile. There is a complete destruction of the normal hairy architecture. There is evident vascular congestion (vessels in the lower central part and in the right part) as well as migration of neutrophils and infiltration of tissue. Figure 10 illustrates the protective effect of BIC against filtering. The architecture is preserved in addition to a slight disruption and edema of the hairy tips. There is minimal inflammatory infiltration or congestion of the vessels of the submucosa. BIC immune to the culture filtrate of C. difficile substantially inhibits enterotoxicity induced by filtrate in rat ileum. A similar degree of inhibition is observed in BIC immune to C. difficile toxin A. These data are consistent with our previous findings that the enterotoxicity induced by C. difficile toxin and rodent intestine is mediated by toxin A. Toxin B has no demonstrable enterotoxic effects in this animal model, although toxins A and B may act synergistically to produce symptoms of disease in humans. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (11)

1. Use of an antibody having specific activity against Clostridium difficile and toxins thereof found in the colon for the manufacture of a medicament for treating diseases associated with Clostridium difficile in a mammal, when administered to said mammal, in a dosage form oral or rectal, an effective dose of the medication.

2. The use according to claim 1, characterized in that the medicament is a tablet, capsule or suppository.

3. The use according to claim 2, characterized in that the tablet or capsule is enteric coated.

4. The use according to claim 1, characterized in that the medicament contains the antibody in microencapsulated form.

5. Use of an antibody having specific activity against Clostridium difficile and toxins thereof for the manufacture of a medicament for treating pseudomembranous colitis in a patient, by orally or rectally administering to the patient an effective amount of the drug.

6. The use according to claim 5, characterized in that the medicament is a capsule or tablet with enteric coating.

7. Use of an antibody having specific activity against Clostridium difficile and toxins thereof found in the colon, for the manufacture of a medicament for treating diarrhea associated with antibiotics in a patient by orally administering to the patient an effective amount of the medicament.

8. The use according to claim 7, characterized in that the medicament is a tablet or capsule with enteric coating.

9. A pharmaceutical composition for use in the treatment of pseudomembranous colitis or diarrhea associated with antibiotics in a patient, the composition is characterized in that it comprises: an effective amount of an antibody in combination with a pharmaceutically acceptable carrier adapted for oral or rectal administration, the antibody has specific activity against Clostridium difficile and toxins thereof.

10. A pharmaceutical composition for use in the treatment of pseudomembranous colitis or diarrhea associated with antibiotics, the composition is characterized in that it comprises: (a) an effective amount of an antibody having specific activity against Clostridium difficile and toxins thereof, and (b) a effective amount of vancomycin, bacitracin or metronidazole, in combination with a pharmaceutically acceptable carrier, the combination is adapted for oral or rectal administration.

11. Use of (a) an antibody having specific activity against Clostridium difficile and toxins thereof, found in the colon and (b) vancomycin, bacitracin or metronidazole, for the manufacture of parts of a kit comprising a first medicament including ( a) and a second medicament including (b) to treat pseudomembranous colitis or diarrhea associated with antibiotic in a patient, by orally or rectally administering to said patient an effective amount of the first medication concurrently or followed by the administration of the second medication.