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March 2000 Addendum |
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The article Phage Therapy: Bacteriophages as Antibiotics found on our web site was written and posted in the fall of 1997. A few corrections and additions have been made, but it seems better to do the major updating in the form of this addendum. We strongly welcome additions and comments.
Industry and Government Involvement in the US The general awareness and attitude in the US has been changing substantially over the last few years in both scientific and popular circles. At NIH, both Carl Merrill and Sankar Adhya have had substantial interest in phage therapy for a number of years. Merrill in turn inspired Dr. Richard Carlton, who in 1993 formed Exponential Biotherapies -- the first American company devoted exclusively to phage therapy work. Its first employees worked in the labs of Merrill and Adhya under a CRADA agreement with NIH, leading to several patents and a very interesting PNAS paper on the selection for long-circulating bacteriophages for therapeutic purposes. In 1993, Exponential rented its own lab space in Rockvillle, MD and hired several additional employees. The company focussed initially on vancomycin-resistant enterococcus (VRE), and now has phages in preparation for initial clinical trials. They have also been doing exploratory work related to several other interesting phage therapy applications. Intralytix, a company started in Baltimore in 1998 by Glenn Morris and Alexander Sulakvelidze of the University of Maryland and VA Medical Center, also has a phage approach to VRE ready for initial clinical testing, as well as a treatment for Salmonella in chicken flocks that has the potential to substantially decrease food poisoning problems. Sulakvelidze came to Baltimore from the Georgian Ministry of Health in 1993, and he was instrumental in establishing connections between the University of Maryland and scientists at the Eliava Institute beginning in 1995. These collaborations have been further enhanced through the founding of Intralytix, which is currently funding projects at the Eliava Institute on phage treatments for Listeria and for Campylobacter. It is also performing the important function of providing dependable electricity for the whole Institute as part of the indirect costs. Intralytix also is working on developing an American version of PhageBioDerm in collaboration with the two scientists who invented it in Tbilisi, and with the help of several visiting Georgian scientists. The one publicly-listed US company focusing totally on phage therapy is Phage Therapeutics International, started by Canadian venture capitalist Caisey Harlingten in response to the 1996 Scientific American article on phage therapy in Tbilisi. Based in Seattle, they have concentrated on antibiotic-resistant staphylococcus, collecting strains from all over the world. They also have phage ready for phase 1 clinical trials - phage that made headlines Sept. 16 1999 with news of a successful compassionate use case in Toronto, involving a woman with Marfan syndrome who had contracted a deadly case of staph during heart surgery. The clinical trials will be a very expensive hurdle, with the need to prepare each phage in a cocktail separately in the special GMP (Good Manufacturing Practice) facilities required for FDA approval and most manufacturers with GMP capability may be nervous about phage "contamination" of their production facilities. This will, in turn, mean that phage therapy in the West will at least initially be relatively expensive and restricted in scope of applications, without the ability to respond within days with new phages for new pathogens that has been so useful in Tbilisi. Interest is also growing at NIH. Several years ago, a good deal of educating of an NIH small-business study section was needed to get them to even consider phage therapy proposals as representing a potentially viable alternative to antibiotics. However, those running the study section were at least interested enough in the concept to include someone with extensive phage experience on the panel. By Feb. 10 2000, NIH released a challenge-grant announcement that specifically called for proposals using phage therapies for emerging and resistant infections, with special focus on vancomycin-resistant enterococci (VRE) and multi-drug-resistant staphylococcus. This is being given a very high priority and rapid response; the application receipt date for phase I proposals was March 9, with an April 10 award date and a June application date for phase II proposals, with a September award date. Intramurally, a "Phage-Tech Interest Group (PhTIG)" has formed, building on the early interest of Merrill and Adhya and the extensive fundamental phage research going on in a number of labs at NIH. Recent Western Phage Therapy-Related Articles Paul Barrow et al. (1998) of the Institute for Animal Health, Compton Laboratory, Berkshire, United Kingdom published an excellent study on using lytic bacteriophage for control of experimental Escherichia coli septicemia and meningitis in chickens and calves. Abstract: A lytic bacteriophage which was previously isolated from sewage and which attaches to the K1 capsular antigen has been used to prevent septicemia and a meningitis-like infection in chickens caused by a K1+ bacteremic strain of Escherichia coli. Protection was obtained even when administration of the phage was delayed until signs of disease appeared. The phage was able to multiply in the blood. In newly borne colostrum-deprived calves given the E. coli orally, intramuscular inoculation of phage delayed appearance of the bacterium in the blood and lengthened life span. With some provisos there is considerable potential for this approach to bacterial-disease therapy. There is also interesting research on phage therapy going on in Sweden. J. Cao et al. (2000), from the Department of Biomedicine and Surgery of Linkoping University, just published a BBA article entitled Helicobacter pylori-antigen-binding fragments expressed on the filamentous M13 phage prevent bacterial growth. Abstract: "Colonization of the human stomach by Helicobacter pylori is associated with the development of gastritis, duodenal ulcer, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer. H. pylori-antigen-binding single-chain variable fragments (ScFv) were derived from murine hybridomas producing monoclonal antibodies and expressed as a g3p-fusion protein on a filamentous M13 phage. The recombinant ScFv-phage reacted specifically with a 30-kDa monomeric protein of a H. pylori surface antigen preparation and by means of immunofluorescence microscopy the phage was shown to bind to both the spiral and coccoid forms of the bacterium. In vitro, the recombinant phage exhibited a bacteriocidal effect and inhibited specifically the growth of all the six strains of H. pylori tested. When H. pylori was pretreated with the phage 10 min before oral inoculation of mice, the colonization of the mouse stomachs by the bacterium was significantly reduced (P<0.01). The results suggest that genetic engineering may be used to generate therapy-effective phages." The most exciting new phage-therapy product of recent years is Phage Bioderm. This is a perforated biodegradable nontoxic polymer composite containing bacteriophages and other therapeutic compounds. It is designed to treat and prevent infection and maintain appropriate moisture levels, with maximum mobility and minimum need for materials and time involvement from therapists. A special version called "PhageDent" has been formulated for periodontal applications; its use in treatment situations was described by Shishniashvili, (1999). The Bioderm material was developed by Dr. Ramaz Katsarava, a professor at the Georgian Technical University (GTU), to provide an effective covering for wounds and burns that would breath while helping to prevent infection and dessication.. He drew Zemphira Alavidze into helping him develop a version which incorporated bacteriophage and could be used both as a surface covering and as a way of providing localized, metered release at internal sites. An early major success of the latter type involved osteomyelitis, as reported at the 1998 Evergreen International Phage Biology meetings. The first paper involving a bioactive composite containing bacteriophage was "Amino Acid Based Bioanalogous Polymers. Some biological studies of regular poly(ester amide)s and bio-active composites based on them" presented at the International Symposium "Biodegradable Materials", Hamburg, Germany, Oct 1996; the authors were G.Tsitlanadze, T. Khosruashvili, N. Nadirashvili, A. Meipariani, Z. Alavidze, M. Goderdzishvili, N. Kvatadze, Sh. Dgebuadze, and R. Katsarava. A short clinical paper has recently been published by K. Markoishvili, N. Djavakhishvili, M. Goderdzishvili, A. Meipariani, Z. Chavchanidze, G. Tsitlanadze and R. Katsarava, "PhageBioDerm - new prospects for treatment of wounds and trophic ulcers", Experimental and Clinical Medicine, #2, 83-84 (1999). Katsarava had a joint appointment at the Institute of Molecular Biology and Biophysics of the Georgian Academy of Sciences until 1998, but ironically the Director there explicitly discouraged his interest in developing bio-active compounds for medical applications and he had to do that work on his own using the more limited facilities at GTU. In 1998, the Georgian Ministry of Health opened a special Research Center for Medical Polymers and Biomaterials which Katsarava directs, along with his ongoing teaching at GTU. The clinical trials of PhagoBioDerm have now been completed and official governmental approval given on Jan. 24, 2000 for this Research Center for Medical Polymers to move ahead with its commercial production and distribution. As mentioned above, under a cooperative, exclusive agreement with Katsarava and Alavidze, Intralytix Corporation is now developing an American version of Phage Bioderm (PhagoBioDerm), which exclusively uses phages that were isolated in the US by a team of visiting Georgian scientists headed by Alavidze. Recent Phage Therapy News from Tbilisi The power of the Western press seems to be felt even in Tbilisi. On Feb. 23, after the publicity of a 6-page NY Times Magazine article on phage therapy that focussed largely on the Eliava Institute, Georgian President Shevarnadze visited the Eliava Institute with a strong delegation, including Jorbenadze, the Director of the Ministry of Health, and the president of the Academy of Sciences. Shevarnadze spent several hours listening to presentations by Director Teimuraz Chanishvili and a number of Institute scientists (Nino Chanishvili, Marina Tediashvili, Amiran Mepariani, Nana B., Dato, Inga, Rezo Adamia). His detailed comments on the importance of the Institute work, requests for information on precise needs and pledges of support seemed more meaningful in view of the fact that the officials who would have to come up with the funds were present there in the room at the time. It is interesting that this appears to have been Shevarnadze's first official visit to the Institute and open statement of support, even though Rezo Adamia, a senior Institute scientist, has been a Member of the Georgian Parliament, Chairman of the Defense Committee and a close associate of Shevarnadze's for several years. Other Western Companies Involved in Phage Research PhageTech Inc. is in Montreal, Canada, reachable through saritab@phagetech.com. They have a variety of therapy-related phage interests and have donated $5000 to help support the Year 2000 "Evergreen" International Phage Biology Meeting in Montreal, in addition to providing computer support for the web site, registration and abstract submission. Biophage, Inc., also in Montreal, is a small knowledge-based biotechnology company with a specific focus on infection and inflammation, along with immune modulation and cancer. The president, Rosemonde Mandeville, is an immunologist. Their web site is http://www.biophage.com/. PhaGen AB in Berzelius Science Park, Linkoeping, Sweden is a company with patent rights to phage therapy-related technology trying to raise sufficient funds to develop them appropriately. They are also interested in alliances and partners in phage therapy and phage technologies (Fax number: +46 13 22 42 54). Russian Production of Therapeutic Phages While the Eliava Institute in Tbilisi was the central facility for developing therapeutic phages and for collecting pathogenic bacteria from all over the Soviet Union for this purpose, there were also three major Russian production centers. One of these was in Gorky (Nizhniy Novgorod), associated with the Gorky Research Institute of Epidemiology and Microbiology. It has now become a private company, Imbio, producing a variety of preparations in liquid, aerosol and tablet form, which are detailed in an extensive website (in Russian). They are reported to have just signed an agreement with a foreign company (neither British or US) and the details of their product composition and manufacturing processes are now confidential for commercial reasons, but their products and quality control standards had to be approved by the Russian Ministry of Health. A third center was in Ufa, where a.company called Immunopreparat has evolved. Their bacteriophages are actually produced by a smaller daughter company called Biophage. The Ufa factory is the biggest for phage production and they make tablets and 'candles'. It appears that none of the companies produce products tested for IV use. Their web site contains much historical information about their work (in the section that is only in Russian), though again, no manufacturing or commercial information. Another large Russian company currently producing phages is Biomed, in the city of Perm in the Urals. Biomed is a major Russian producer of pharmaceuticals and biological preparations, including vaccines, diagnostic and therapeutic sera, anatoxins and variousdiagnostic reagents. The company developed from a bacteriological laboratory that was opened in 1898. Biomed currently contains both a research institute employing biologists, chemists, pharmacists and physicians and a production facility that manufactures over 100 products. They first manufactured bacteriophage against dysentery in 1940-1950. They began producing bacteriophage with other specificities in 1994 - at the time when obtaining them from Tbilisi became much more difficult. Their first commercial batches of staphylococcal phages were released in March, 1994, and they now have a substantial variety of products available. It appears that all of the Russian manufacturers have now realized that there is a western commercial potential for their products and they are trying to establish contacts which can result in making a direct profit. This is a commercial sector after all. The web sites for the Russian phage companies: Imbio (Nizhniy Novgorod) http://www.sinn.ru/~imbio/Bakteriofag.htm Soviet Research on Phage Therapy Some progress has been made in making the available literature more available in the West. Alisky et al. (1998) published a review of most of the 27 articles related to the therapeutic use of phage that they found in Medline between 1966 and 1996. In addition to the articles in English discussed in our original review, they found 8 articles in Russian and summarized 7 of them. (Clearly, far more articles were written in Russian during this period; the low number of citations primarily reflects the low rate of inclusion of Russian journals in Medline.) The most detailed and quantitative study they found was by Kochetkova et al. (1989) of the Cancer Research Center in Moscow. They looked at a total of 131 cancer patients with postoperative infections; their cancers were in the larynx, pharynx, esophagus, lung and breast. Of these, 65 received phage with or without antibiotics while sixty six acted as a control, receiving only antibiotics; the two groups were matched for age, location and stage of tumor, type of surgery and type of postoperative infection. The study group was further subdivided into phage only, antibiotics and phage administered simultaneously (from the start of supperative infection) or phage administered only after the antibiotics had failed. All phage were prepared at the Institute in Tbilisi and were applied daily topically or, in the case of osteomyelitis, mediastinitis and ampyema of the pleura, via lavage irrigation. Either anti-pseudomonas or anti-staphylococcus phage or pyophage were used, as appropriate. Overall, phage produced positive clinical results in 81.5% of the patients, in comparison with 60.6% in the antibiotics only control group. Phage was most effective for cutaneous wound infections and for pseudomonas; 86.7% of those treated with phage for pseudomonas showed improvement as compared with 74.4% for staphylococcus and only 57.1% for those needing the pyophage mixture. (They did not give the comparable breakdown for the antibiotic group.) Sakandelidze and Meipariani (1974) reported a 92% success rate in eliminating bacteria using phage to treat supperative infections in a series of 236 patients with antibiotic-resistant osteomyelitis, peritonitis, lung abscesses and postsurgical wound infections at the Tbilisi Institute of Vaccines. Their Staph, Proteus and/or Streptococcus infections were treated daily for 5-10 days either subcutaneously or through surgical drains with a Pyophage mixture active against all 3 or diphage, targeting only staph and Proteus. Sakandelidze (1991) later reported using phage in combination with antibiotics and allergic desensitization to treat a total of 1380 patients aged 1-76 for infections secondary to pharyngitis and allergic rhinitis (515 patients), asthma and allergic rhinitis (342), dermatitis and allergic colitis (305), dermatitis and cholecystitis (166) or allergic conjunctivitis (52). Bacterial cultures and antibiotic sensitivity tests showed that 70.6% had monoinfections with Staphylococcus epidermis or aureus and 29% had mixed infections with E. coli, Proteus, Streptococci and highly pathogenic species of Pseudomonas. All patients were given cutaneous allergic desensitization. They were then given antibiotics (404 patients), phage (360 patients) or a combination of the two (576 patients). Antibiotics alone led to clinical improvement (no relapse of allergic symptoms for 5 years) in only 48% of the patients, while for phage alone this figure was 86.3% and it was 82.5% for phage plus antibiotics. Phage was used to help treat intestinal dysbacteriosis in a study by Litvinova et al. (1981) of the Institute of Epidemiology in Sverdlovsk (now Ekaterinburg), Russia. E. coli-proteus phage were given along with Bifidobacteria to 500 low-birth-weight infants (under 2 kg) who had been administered antibiotics for at least 2-3 weeks to treat sepsis and pneumonia. All 500 infants showed clinical improvement, with cessation of diarrhea, remission of the Proteus infections as determined through culturing, and weight gain. They concluded that the phage served to deplete the pathogenic bacteria while the Bifidobacteria (a kind of acidophilus bacteria) provided a source of new normal flora. Tolkacheva et al (1981), of the Haematology and Blood Transfusion Institute in Moscow, used a similar phage-Bifidobacteria combination to treat dysentary in immunosuppressed leukaemia patients. One group received one or more 3-day courses of orally administered phage (20-30 ml 3 times a day) with no other treatment (15 coli-proteus and12 pseudomonas phages), for 10 more the phage were followed with Bifidobacteria, 9 received only Bifidobacteria and 13 received an oral mixture of kanamycin, polymyxin and ristomycin 5X daily for 10-12 days. With phage alone, control of the dysentery was achieved with 1 such course of pseudomonas phages or 2-3 courses of the coli-proteus phage, respectively; in the latter case, proteus would reappear in the faeces shortly after cessation of treatment. The best clinical results were obtained using both Bifidobacteria and phage. Miliutina and Vorotyntseva (1993) compared various antibiotics with and without phage for treating salmonella and generalized bacterial dysentery in pediatric patients during the period from 1977 to 1989 at the Central Research Institute of Epidemiology in Moscow. Antibiotics compared included rifampin, gentamicin, ampicillin, polymixin and furazolidone. Phage plus antibiotics were found to treat some infections resistant to antibiotics alone. It was reported that phage were involved in only a small part of the study, but no actual figures were cited. Alisky et al. also include an excellent, well-documented discussion of the problems that can be caused by the use of lysogenic phages and their relatives, particularly the issue of phage conversion - the horizontal transfer of toxin or toxin-enhancing or antibiotic resistance genes. For example, Scotland et al. (1979) describe methicillin-resistant S. aureus displaying a triple lysogenic conversion of enterotoxin A, staphylokinase and beta-lysin. Also, Chausce et al. (1996) found prophage-encoded erythrogenic toxin genes A and C in 44% and 34%, respectively, of 117 clinical isolates of Streptococcus pyogenes from patients with diseases including necrotizing fasciitis and toxic shock syndrome, with much phenotypic heterogeneity among isolates. As he says, the totality of the evidence in the Soviet and Polish papers suggests that using antibiotics along with phage is more likely to inhibit the phage's effectiveness than enhance it (particularly in external applications), and the antibiotics may well tend to select for virulence genes transferred simultaneously with the antibiotic-resistance genes, as described by Coleman et al. (1989) and others. In the work of Smith and Huggins where phage were isolated that specifically used surface virulence factors as receptors, virulence was largely eliminated in resistant bacterial strains. However, Coleman et al. find that virulence is not impaired in phage-resistant strains with drug-resistant plasmids selected for through antibiotic use. Solid basic knowledge of the properties of proposed therapeutic phage, including the demonstration that they are not related to any of the known lysogenic phage families, will be crucial in ensuring that no toxin-producing genes are inadvertently introduced into non-pathogenic indigenous GI or skin flora. Alisky et al. conclude by calling for extensive East-West collaboration that helps preserve the scientific infrustructure in this area in Russia and Poland (and here he should definitely add Georgia), shares the effective methodology developed over many decades, and supports double-blind studies to confirm the many exciting results that have been reported. As they say, "Further international research on phage therapy should be part of an ongoing multimodality effort as we seek to cope with antibiotic-resistant pathogens in the years to come." Several volunteers have been very thoughtfully translating articles from Soviet journals that were not available on Medline. While most of these are only extended abstracts from meetings or thesis summaries, some are quite extensive and detailed. I summarize several of them here to give a sense of the kind of work that was being carried out. Solodovnikov et al. (1970) published a study entitled "Prophylactic Application of Dry Polyvalent Dysentery Bacteriophage with Pectin in Children's Preschool". This is a very extensive controlled trial of using bacteriophages to try and reduce the incidence of diarrheal disease in nurseries and kindergartens where it had caused rather serious problems for the several preceding years. The test group contained 3212 total children, the control group 3310 children. The two groups were well matched in terms of age, sex, history of disease, number of other children in the family, and living conditions-separate flat with full amenities, communal flat, or private house lacking full amenities. Once a week during the peak problem months (June-September), the test group received a dry pectin-coated tablet containing phage produced at the Gorky Institute of Epidemiology and Microbiology, while the control groups received similar tablets without phage. The specific phages included in the cocktail were directed against Shigella flexner, Zonne, Newcastle, Schmitz-Shtuzer and Grigoryev-Siga, with precise titers on each determined on 3 batches by the State Control Institute of Medical and Biological Preparations. The pectin was demonstrated to make them more resistant to the action of stomach enzymes in 0.1M HCl (no loss after 2-hour treatment, vs a 10-fold drop without the pectin, as measured by the Appelman-Gracia test. The treated group showed a 2.5-fold lower incidence of bacteriologically confirmed dysentery, a 2.3-fold lower rate of clinically and bacteriologically confirmed dysentery, and a 2.1-fold lower total incidence of acute intestinal diseases. Solodovnikov et al. (1971) report an equally well controlled follow-up study looking at administering the same phage preparations every 3 days rather than once a week during the seasonal rise of the incidence of dysentery (June-October) to children' in Yaroslavl pre-school institutions (with 4948 children in the test group and 5094 in the control group). This worked even better; the group receiving phage had 3.9 times less cases of bacteriologically confirmed dysentery, 3.4 fold less clinically and bacteriologically confirmed dysentery, and 3.3 fold less total acute intestinal diseases than the group receiving the placebo. Acknowledgements I would like to express special thanks to Dr. Elena Jones, a graduate of the Gorky Research Institute of Epidemiology and Microbiology, now living and working in England, for tracking down much of the information about what is going on currently in Russia and for translating several of the Russian articles. Thanks also to Natasha Lebedeva of Olympia for her assistance in translating work written in Russian. Added Early History Over 800 articles appeared in the scientific press discussing phage therapy during this period, including work by such prominent microbiologists as M. F. Burnet and R. Dubos. Burnet, F. M., M. McKie and I. J. Wood, 1930. Investigations on bacillary dysentery in infants, with special reference to the bacteriophage phenomena. Medical Journal of Australia 2:71-78. Dubos, R. J., J. H. Straus and C. Pierce, 1943. The multiplication of bacteriophage in vivo and its protective effects against an experimental infection with Shigella dysenteriae. J. Experimental Medicine 78:161-168. Added References Popular press:
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