INTRODUCTION: BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE The increased incidence of antibiotic resistant 'superbugs' has amplified the use of broad spectrum antibiotics worldwide. An unintended consequence of antimicrobial treatment is disruption of gastrointestinal microbiota, resulting in susceptibility to opportunistic pathogens, such as Clostridium difficile (C. difficile).
C. difficile is a gram-positive, spore-forming, anaerobic bacterium that causes hospital and community-acquired enteric infections resulting in C. difficile-associated diarrhea, pseudo membranous colitis, colitis and death.
Clostridium difficile infection (CDI) remains the predominant cause of health care -associated infectious diarrhea, centered mainly in hospitals but also in the community at a rate of \~100/100,000 population and affecting the elderly disproportionately. Antimicrobial damage to the intestinal microbiome combined with the acquisition of spores of C. difficile is the main cause of pathogenesis. CDI is endemic in hospitals, and although infection control measures have reduced nosocomial rates of CDI in Canada over the past 5 years, community cases now appear to be more apparent .Treatment of CDI utilizes alternate antimicrobials directed at the pathogen, mainly metronidazole, vancomycin, or fidaxomicin given for a recommended 10-14 days. While the majority respond to antibiotic treatment, subsequent recurrences of CDI are observed in 25-40% of patients. Recurrences of CDI are retreated with the same antibiotics and due to the non-selective spectrum of activity of metronidazole and vancomycin, recurrences are increasing in probabilities for subsequent recurrences. Fidaxomicin is relatively more selective and its use is associated with reduced recurrences, but the high cost of fidaxomicin has restricted the usage.
C. difficile infection is a serious diarrheal illness associated with substantial morbidity and mortality and is responsible for hospital and community-acquired diarrhea, pseudo membranous colitis colitis and death..
ImmuniMed Inc. has developed an oral Immunotherapy using egg-derived anti- C. difficile polyclonal antibodies (IM-01), that directly targets the virulence factors of C. difficile. IM-01 is produced from chicken eggs following the immunization of laying hens with C. difficile virulent antigens, C. difficile toxin A, toxin B, C. difficile whole cells and C. difficile spores. IM-01 is in the form of spray-dried whole egg powder (yolk plus egg white containing ovalbumin) and is very stable for functional activity. Upon reconstitution, IM-01 is administered orally, which allows it to reach to the intestinal lumen, where C. difficile toxins exert their toxic effects. In addition, IM-01 antibodies bind to C. difficile toxins and spores and neutralize their biological activities in vitro.
The use of polyclonal antibodies derived from chicken eggs (IgY) to confer passive immunity has gained attention as a novel strategy for oral immunotherapy as an alternative to antibiotics for treatment of intestinal pathogens which cause diarrhea in animals and humans including, enterotoxigenic E. coli, Salmonella spp, and both bovine and human rotaviruses. Compared with mammalian antibodies (IgG), chicken egg-derived antibodies (IgY) have high antigen-specificity and avidity, react more rapidly to the same antigens and differ from mammalian antibodies (IgG) in both structural and immunological properties.. Compared to IgG, IgY is more stable in gut at pH 3.5 -11 and better tolerates enzymatic degradation by trypsin and chymotrypsin; the stability of IgY against pepsin is pH-dependent.
Immunimed has determined that IgY polyclonal antibodies-specific to E. coli in egg powder containing albumin, are functionally stable when stored at 2-8°C for over 10 years.
This makes IM-01 antibodies attractive for oral immunotherapy since eggs are normal dietary components, with no known toxic side effects. The IM-01 antibodies are shown to bind with C. difficile toxins and spores as well as to neutralize their biological activities in vitro.
Compared with active vaccination, passive immunotherapy using IM-01 egg-derived polyclonal anti-C. difficile antibodies has advantages including: (i) rapid and local onset of action, (ii) highly specific activity, (iii) applicability to a broader range of patients from children to adults including immunocompromised patients, (iv) strategically can be applied for a specific event when it occurs, in contrast to a vaccination model where large populations need to be treated in advance and sustain active antibodies for long-durations, and (v) is a normal part of human diet.
In a published literature, hamsters treated orally with C. difficile anti-toxin A and anti-toxin B neutralizing egg-derived polyclonal antibodies were protected following C. difficile challenge. Also, hamsters treated with egg-derived antibodies did not develop relapsing disease months after the treatment with antibodies. .
Furthermore, egg-derived polyclonal antibodies have several advantages over the monoclonal antibodies (MAbs), since MAbs are generally specific for single epitope, when polyclonal antibodies - a new class of biopharmaceuticals, that have higher affinities for multiple target antigens and can recognize multiple epitopes resulting in superior potency; since they can recognize multiple toxin epitopes as it is relevant to polymorphism of the toxin A and toxin B genes in C. difficile.
A piglet study was performed to demonstrate the safety and therapeutic effect of egg-derived polyclonal antibodies -specific to E. coli K-88, in farm animals. It has been established that E. coli K88 that cause diarrhea in piglets by colonizing in the lower GI tract of piglets improve their growth performance following treatment with E. coli K-88-specific antibodies orally.Furthermore, the highest body weight gain was achieved in pigs which were fed with the highest dosage of E. coli K-88 antibodies (4kg/1000 kg of nursery diet) compared to the lowest antibody dosage (1kg /1000 kg of nursery diet).
It was concluded that feeding of piglets with egg-derived polyclonal antibodies-specific to E. coli K-88 results in significantly improved growth performance in an antibody dose-dependent manner. The manufacturing process for egg-derived polyclonal antibodies targeted to enterotoxigenic E. coli has been approved and validated by the Canadian Center for Veterinary Biologics.
In conclusion oral Immunotherapy using polyclonal antibodies is considered to have a high safety profile with a low likelihood of adverse effects. If proven to be effective as an anti-infective approach, antibiotic selection pressure could be reduced, a key strategy to limit antibiotic resistance in C. difficile infections.
IM-01 is spray-dried egg powder product containing polyclonal antibodies -specific to C. difficile toxins A and toxin B, plus antibodies to C, difficile spores. A 20g/day dosage of IM-01 was selected based on a pre-clinical animal study data, using piglets to treat with C. difficile enteritis.disease with 0.6g of IM-01 orally daily for 4 days.
Under compassionate grounds, patients with confirmed CDI clinical diagnosis with unformed diarrheal stools, abdominal cramps and a positive stool test for the presence of toxins and/or C. difficile in culture, 106 patients (ages: 11-93 years) were enrolled for the clinical study. All patients signed informed consent forms to participate in the study. The amount of IM-01 antibody powder product (10g or 20g) orally administered daily by each patient with 150 mL of milk and followed for up to 6-week period following IM-01 immunotherapy. Seventy six patients received an oral dose of 20 g of IM-01 and 30 patients received 10 g of IM-01 powder daily for 10 consecutive days, as drinkable therapy with milk or chocolate milk. The primary endpoint of the study was to eradicate C. difficile in stool samples.
Of the 106 patients treated with IM-01 therapy,101 patients showed significant improvement of clinical symptoms with negative stool test results for CDI.Five patients showed reduced clinical symptoms during IM-01 treatment period. No recurrence was observed with IM-01 therapy during the follow up period. No safety concerns were reported.
Standard of care (SOC) antibiotics for the treatment of CDI have observed responses to treatment i.e. resolution of diarrhea and ancillary symptoms/signs in 72-88% of subjects, and for those subjects who do achieve resolution of CDI, \~16-24% experience a recurrence of CDI within an 8 week follow up. Non-responders are treated with higher dosages of vancomycin. day interval. A post-treatment minimal follow up period of 30 days has been used in recent RCTs of antimicrobials. Other trials have used several time points i.e 8 weeks or 12 weeks post -treatment. Therefore, in absence of a standard of care control arm, the foregoing response rates for resolution of CDI symptoms (\~80%), and a recurrence rate of 20% will be used for as a response template for this study. In the absence of randomization of subjects to an antibiotic treatment control group,
