Therapy of Clostridium Difficile-Associated Diarrheas
Development of Laboratory Sample of Complex Preparation Based on Enzymes of Specific Bacterial Phages, Anti-Toxic Antibodies and Recombinant Human Alfa-Fetoprotein and Interferon 2 for Therapy of Clostridium Difficile-Associated Diarrheas
Tech Area / Field
- BIO-SFS/Biosafety and BioSecurity/Biotechnology
- MED-DRG/Drug Discovery/Medicine
3 Approved without Funding
Institute of Immunological Engineering, Russia, Moscow reg., Lyubuchany
- McGill University / Sir Mortimer B. Davis Jewish General Hospital, Canada, QC, Montreal\nUniversity of Turku / Finnish-Russian Joint Biotechnology Laboratory, Finland, Turku
Project summaryClostridium difficile-associated diarrheas (CDADs; clostridiosis) are presently one of the most serious clinical problems with high infectivity and lethality rates. CDADs origin from often-used antimicrobial therapies which weaken the natural resistance of the human organism against the pathogens. CDADs vary from self-terminated diarrheas to heavy forms accompanied by the expressed inflammatory-hemorrhagic changes in the form of pseudomembranous and fulminant colitis with signs of systemic inflammatory reactions. C. difficile is an anaerobic, spore forming bacillus, widely spread, long-viable, highly resistant to standard disinfectants and to the majority of antibiotics. The main origin of the pathogenicity is the exotoxins damaging the wall of intestines and causing the clinical symptoms. In the US alone, up to 300 000 cases are annually registered. The costs for treatment of one patient are up to 10 000 USD. In the European Union, costs of the infections have been estimated to be 3 billion euro a year. These amounts are expected to be doubled during the next decade. Starting from the year 2003, outbreaks of infections caused by a new, earlier unknown highly pathogenic form of C. difficile have occurred in the US, Canada and in certain areas of Europe. High lethality by the new form is related to a more extensive toxin formation. The possibility of hospitals to be widely inhabited by the new form makes it extremely dangerous to the public health.
Despite certain recent progress in the prevention of clostridial infections, there remains several problems. Metronidazole and vancomycine are commonly appliedv to patients with C. difficile infection. Their application has, however, a constant risk of generation of resistant forms of these microbes towards the antibiotics followed by losing the only ways to combat against the disease. The risk is highly aggravated by the need of using very long curing times with the antibiotics. The antibiotics have also non-desired side action including harmful allergic reactions. Furthermore, none of the presently applied therapies guarantee full sanitation of intestine from clostridium spores. Due to the spores, relapse of the disease often occurs with plural form of the disease. Treatment of such patients is even more complex. The existing situation is, in fact, that there are no effective and safe drugs against CDADs.
Because of the high probability of relapses of clostridiosis, due to the antibiote-resistant spores, and because of the long times of antibiotic therapies with unpredictable risks formation oif resistant strains, development of more sophisticated innovative therapies avoiding the use of antibiotics is highly wanted. The present Project Proposal aims at combining pieces of existing knowledge and practical experience from different Russian research teams and to integrate them to obtain a novel effective way to combat against clostridiosis. We propose to approach the problem through three apparently independent research lines:
- applying specific bacteriophages against C. difficile,
- increasing the resistivity of cells in the intestinal wall by the anti-apoptotic protein, alfafetoprotein.
- decreasing active concentration of toxins in intestine by specific antibodies,
These sub-tasks can be studied separately and subsequently integrated to make a drug formulation releasing all the active agents in intestine. The participating research groups have know-how on formulation of bacteriophages. Because C. difficile is highly dangerous microbe, it can be expected that fastened drug registration can be permitted to effective preparations.
Bacteriophages are presently well-known alternatives for the use of antibiotics. Their main advantage is that they are highly specific and self-propagating in the presence of their hosts. They are not also limited by the existence of antibiotic resistant host strains. High efficiency of phage therapy is proven at a number of infectious diseases. The preparations based on phages do not substantially interference with the balance of bacterial microflora and does not cause secondary infections as do the antibiotics.. Application of specific bacterial phages, or their enzymes in combination with spore germinants, is perspective for sterilization of soil, equipment and hospital rooms, as well.
2. Prevention of injuries of intestine
Whereas the basic pathogenic factors of C. difficile-infection are exotoxins that cause injuries to the tissues of intestine and toxic complications, the research on the mechanisms of apoptosis regulation, that will allow to effect its separate stages to for correction, is required. Inhibition of synthesis or liberation of cytokines is one of the possible ways of combating septic shock that can become a consequence of heavy forms of C. difficile-associated diarrheas. The Institute of Immunological Engineering is involved in the development and research of the human recombinant alfa-fetoprotein (Recalfin), that is capable of regulating molecular and cellular processes of inflammation, infringement of microcirculation and multiple injury of organs, caused by ischemia of tissues and apoptotic destruction of cellular elements, that constitute the pathogenesis of sepsis. At laboratory conditions Recalfin has shown the anti-inflammatory activity and ability to directly suppress the production of inflammatory cytokines in vitro and in vivo.
3. Neutralization of toxins
Defining the measures to combat toxigenic strains, besides destruction of pathogens, the neutralization of their toxins is required. Specific antibodies, influencing pathogenetic mechanisms, will promote faster and long therapeutic effects. Application of various combinations of bacterial phages and their enzymes, anti-inflammatory and anti-apoptosis preparations, antibacterial and immune stimulating substances, monoclonal antibodies, probiotics gives the basis for development of a complex medical preparation.
While developing a complex therapy as a way to combat clostridiosis, it is necessary to consider real danger of relapses occurrence due to development of spores kept in intestines. In order to prevent returnable forms of a disease it is viable to use the spore germinats in combination with lysines, that will promote the fastest elimination of the pathogen from an organism.
For screening the clostridial phages will be used, as from institute’s collection, and isolated from environment. Specific antibodies will be estimated on protective properties and further used for complex application in a combination with bacterial phages or their enzymes. Known spore germinants will be analyzed and the most acceptable will be chosen. Sensitivity of C. difficile to preparations of human recombinant interferon and γ, influence of human recombinant alfa-fetoprotein on apotosis mechanisms, caused by exotoxines A and B of C. difficile, and on production of proinflammatory citokynes at septic complications will be studied.
The collective of executors has wide experience of work with bacterial phages, owns technology of isolation and cleaning of enzymes, isolation of monoclonal antibodies; many years study the kinetics of spore germination, development and research of recombinant interferon and γ and human alpha-fetoprotein, analysis of molecular mechanisms of apoptosis.
The present project is directed to the decision of one of the major problems of modern medicine - creation of new effective and safe means for protection against microbial infections.
The purpose of the project: To prove experimentally the efficiency of complex influence of high-purity phages’ enzymes in combination with spore germinants and specific antibodies on pathogens of Clostridium difficile-infection, and also recombinant human AFP on correction of apoptosis and sepsis, caused by discussed pathogen and to develop the laboratory sample of a treatment-and-prophylactic preparation.
The primary tasks:
- Studying of cultural-morphological properties and factors of pathogenicity of C. difficile.
- Search and characteristic of bacterial phages able to lyse strains of C. difficile.
- Isolation and cleaning of exo-and endolysisnes from pages species-specific to bacteria of family Clostridium, studying of their physical and chemical and biological properties.
- Isolation of specific monoclonal antibodies against toxins A and B of C. difficile, estimation of their biological activity.
- Studying of influence of recombinant human alfa-fetoprotein on apoptosis mechanisms, caused by exotoxins A and B of C. difficile and on production of proinflammatory cytokines.
- Development of an experimental complex preparation based on phages’ lysisnes, spore germinants, specific antibodies and recombinant human alfa-fetoprotein.