Bacteriophage therapy: a regulatory perspective

J Antimicrob Chemother 2016; 71: 2071 – 2074 doi:10.1093/jac/dkw083 Advance Access publication 10 April 2016 Bacteriophage therapy: a regulatory perspective Eric Pelfrene1*, Elsa Willebrand1, Ana Cavaleiro Sanches2, Zigmars Sebris3 and Marco Cavaleri1 1 *Corresponding author. Tel: +44-20-3660-8593; Fax: +44-20-3660-5515; E-mail: eric.pelfrene@ema.europa.eu Despite the recognized problem of antibiotic multidrug resistance, very few antibacterial agents with new mechanisms of action are under development. Bacteriophage therapy could offer one alternative strategy to mitigate this challenge. Although widely used throughout the 20th century in Eastern Europe and the former Soviet Union, this potential therapy has not yet been investigated according to rigorous scientific standards. This paper reports on a multistakeholder meeting held at the EMA, which outlined the existing regulatory framework to which such therapy should adhere and reviewed the current obstacles and shortcomings in scientific development for bacteriophage therapy. Introduction Lytic bacteriophage (phage) therapy may be a promising intervention for the treatment of bacterial infection, either in addition to or as an alternative to conventional antibacterial treatments. The investigation of phage therapy is particularly pertinent considering the emerging global threat of antimicrobial resistance. Co-discovered by Twort1 and d’Hérelle,2 phages have been used in medicine since 1919, a decade before the discovery of antibiotics. The first scientific article describing bacteriophage therapy was published in 1921.3 Although bacteriophage therapy was largely replaced by antibiotics in Western countries after the Second World War, it remained a popular treatment throughout the 20th century in Eastern Europe (Poland) and in the former Soviet Union (Georgia, Russia). As a result, extensive observational data have been gathered for various infection types.4 More recently, some data have been emerging from human trials conducted to modern standards5,6; however, there is still a lack of data to prove their efficacy and safety. Phage therapy has been conceived either as a ‘personalized’ treatment option administered to an individual patient (as a specific, tailored bacteriophage following isolation and identification of the causative pathogen) or as a ‘ready to use’ cocktail that comprises several different strains of phages targeting one or more pathogenic bacterial species. Potential advantages and drawbacks for the use of natural phages and genetically modified phages have been described.7 Some investigators have claimed that the available regulatory framework potentially hampers successful commercialization of phage therapy and access by patients, particularly for the personalized treatment option.8,9 Aim of the EMA workshop In June 2015, the EMA invited representatives from industry, academia, regulatory authorities and European legislators for a workshop on the therapeutic use of bacteriophages. This workshop was part of the EMA’s commitment to explore new therapeutic options for difficult-to-treat infections, including those due to MDR bacteria. The aim of the meeting was to facilitate the development of bacteriophage therapy by reviewing the scientific and regulatory aspects related to this potential therapeutic alternative. It covered general regulatory considerations and quality aspects of phage therapy as well as the state of the art of clinical development to date. Participants explored how regulators could ease the path towards mainstream clinical use without compromising on standards as laid down in current legislation on requirements for proof of quality, safety and efficacy. The recording of the workshop and various presentations have been made available on the EMA web site event page (http://www.ema. europa.eu/ema/index.jsp?curl=pages/news_and_events/events/ 2015/05/event_detail_001155.jsp&mid=WC0b01ac058004d5c3). Regulatory considerations At the workshop, it was clarified that bacteriophage therapy falls under the scope of the existing European regulatory framework on biological medicinal products, as outlined in Directive 2001/83/ EC.10 Hence, in general, a marketing authorization is required before these products can be used in patients, based on detailed pharmaceutical, preclinical and clinical documentation, in accordance with the directive. However, the legislation foresees some exceptions that would not require a marketing authorization. Amongst others, these exceptions include new medicines that are intended for research and development trials, named-patient use programmes and compassionate use programmes. Further, natural phages are not considered advanced therapy medicinal products (ATMPs), and there are no precedents of bacteriophages expressing recombinant nucleic acid being classified as a gene therapy product (a subcategory of ATMPs). However, if bacteriophages expressing recombinant nucleic acid were to be # The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com 2071 Downloaded from https://academic.oup.com/jac/article-abstract/71/8/2071/2237822 by guest on 14 June 2020 Office of Anti-infectives and Vaccines, Human Medicines Evaluation Division, European Medicines Agency, London, UK; 2Quality Office, Human Medicines Evaluation Division, European Medicines Agency, London, UK; 3Regulatory Affairs Office, Human Medicines Research and Development Support Division, European Medicines Agency, London, UK Review Quality aspects The main quality prerequisites of a pharmaceutical product and particularly a biological product were discussed during the EMA meeting. Manufacturers mentioned that in the absence of specific 2072 quality guidelines for phage products, they generally adhere to existing guidance for biotechnology and biological products. Participants discussed and agreed that the following parameters need careful consideration in the production process of a phage product: bacteriophage identification, potency and biological activity of the preparation, product purity, stability and storage conditions as well as control of sterility. Bacteria and phage bank systems, which typically consist of master cell banks and working cell banks, need to be established. The generation and characterization of the banks should be performed in accordance with the general principles laid down in the Committee for Medicinal Products for Human Use and International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (CHMP/ICH) Q5D Guideline ‘Quality of biotechnological products: derivation and characterization of cell substrates used for production of biotechnological/biological products’.15 The banked phages and bacteria should be characterized for relevant phenotypic and genotypic markers so that amongst other parameters, identity, viability, potency and purity are ensured. With regard to the phages, it is recommended to use only lytic phages (and no temperate phages) unable to transfer host bacterial DNA (potential virulence genes) into non-targeted bacteria [transduction or phage-mediated horizontal gene transfer (HGT)]. Following lysogenic induction, temperate phages may transfer fragments of DNA containing toxin-encoding or antibiotic resistance-mediating genes, and thus temperate phages could produce new pathogenic strains. Therefore, it is important to ensure that therapeutic phages do not carry out generalized transduction and do not possess gene sequences having significant homology with major antibiotic resistance genes, genes for phage-encoded toxins or genes for other bacterial virulence factors. Nevertheless, it was pointed out that currently it is not feasible to exclude the possibility of low levels of generalized transduction by therapeutic phages into any of the infecting and commensal bacteria present in the patient. Moreover, it was mentioned that the use of phages that mediate some random general transduction might be considered in certain circumstances (science- and riskbased decision, taking into consideration the patients’ needs).16 Concerning the host bacteria for manufacturing, it was mentioned that non-lysogenic strains containing few phages or other phage-like elements of genetic exchange should be used. Nevertheless, it was emphasized that most often it will not be possible to find or quickly generate a suitable host bacterium that is free of prophages or phage-like elements. An example was given in which several active substances (phages) can be produced in parallel to be combined into a drug product (phage cocktail), which is prepared according to the aseptic principles described in the EU Guidelines to Good Manufacturing Practices (GMP).17 The preparation needs to be free from host-cell proteins and DNA, pyrogenic exotoxins, endotoxins, residual agents such as solvents, and haemolysins. For the purpose of control, the potency of the active substance can be assessed by methods such as pfu determination or negative-staining transmission electron microscopy (as an orthogonal test). Participants discussed that control of the drug product requires flexibility to accommodate adapting the product to the diversity of bacterial strains. It was reported that the GMP production process should support changes in the composition of a phage preparation and that the criteria for accepting these changes should be pre-established. Downloaded from https://academic.oup.com/jac/article-abstract/71/8/2071/2237822 by guest on 14 June 2020 considered ATMPs, exceptions foreseen for ATMPs as defined in Regulation (EC) number 1394/200711 and in Directive 2001/83/ EC may also apply, e.g. the possibility of a risk-based approach to determine the extent of quality, preclinical and clinical data required for the granting of a marketing authorization. Workshop participants discussed various proposed approaches in developing bacteriophage therapy.12 A ‘library’ approach entails the creation of a phage bank from which one or several bacteriophages are selected for the individual patient, based on the characteristics of the isolated pathogen—such a concept has some similarities with the approach for allergen products that require a large number of substances. It has also been suggested that phages could be cultivated and selected in the presence of the pathogen isolated from the patient. This fully individualized concept may, from a regulatory perspective, have some similarities with autologous cell-based products, in which an inherently variable biological material from the patient affects the final product. However, for autologous ATMPs, the active substance (cells) will have the same predefined characteristics (i.e. same cell type, same cell surface markers). The potential need for a quick change or update of a bacteriophage preparation in response to developing resistance needs to be reconciled with the provisions of current regulations. Changes to the terms of a marketing authorization are covered by Commission Regulation (EC) number 1234/2008 (‘the Variations Regulation’),13 and require time-consuming processes to ensure that the safety and efficacy of the product is maintained. It should be noted that changes in this context do not include the introduction of an additional active substance (phage), because the outcome of such an alteration would be regarded as a different medicinal product requiring a separate marketing authorization. Examples have been brought up from the veterinary regulatory framework, where some vaccines are allowed more flexibility for change of composition via the inclusion of a multistrain dossier.14 However, no such exceptions currently exist within the regulatory framework for human medicinal products. Annex I of the Variations Regulation lists changes that require an extension of the marketing authorization (for which the assessment follows a timeline as for initial applications). As a relevant example, replacement of a biological active substance with one of a slightly different molecular structure, for which the efficacy/safety characteristics are not significantly different, is cited in Annex I of the Variations Regulation. Precedents of exceptions do exist, as specified in the Variations Regulation. Notably, a shorter assessment time frame is foreseen for strain changes of the (seasonal, prepandemic or pandemic) influenza vaccine, which are considered a major (type II) variation rather than an extension. However, although accelerated, this process is still a complex one, with significant time required. It has been suggested that replacing one strain of bacteriophage with another should follow a similar accelerated route. Further evidence would, however, be needed to establish the data required to ensure that the bacteriophage strain change would not have a negative impact on the efficacy or safety of the product. JAC Review Clinical development issues As described in the scientific literature,7,18 bacteriophage therapy has potential advantages, including activity against Grampositive as well as Gram-negative bacteria. Importantly, bacteriophage therapy also covers MDR strains, exhibits high specificity with narrow therapeutic spectrum (limiting disruption to the microbial ecology) and induces few side effects so far reported from observational studies in patients19 and in healthy volunteers.20 Nevertheless, the high specificity of bacteriophage therapy has been recognized as a potential drawback, since for some of the proposed approaches, culturing of the causative bacteria is required prior to initiating treatment. Nevertheless, the high specificity of bacteriophage therapy has been recognized as a potential drawback, since for some of the proposed approaches, culture and identification of the causative bacterium is required prior to initiating treatment, i.e. matching bacteriophages to the infecting strains, in a time-consuming manner. Also, a number of important issues, such as the population dynamics of the bacteria during phage therapy, optimal duration and modalities of treatment (topical versus systemic, stand-alone versus concomitant use with conventional antibiotic agents), need further exploration. Phage therapy could, however, be envisaged for many different indications, ranging from prevention of infection (e.g. selective decontamination in the ICU) to curative usage, e.g. treatment of skin and soft tissue infections, osteomyelitis or respiratory tract infections. Further issues to consider are potential immune recognition with fast clearance of bacteriophages, impact on immune system, release of endotoxin upon lysis of Gram-negative bacteria triggering a pro-inflammatory cascading effect, as well as development of resistance by the bacterial host. With reference to the latter, the application of a cocktail of lytic bacteriophages attacking the same bacteria via different targets appears to be a logical choice. However, whether this approach would be the most effective has not been established. Alternatively, a sequential application of phages may be a viable alternative, with more efficient eradication of the bacterial pathogen as resistance evolves. There was consensus amongst participants that more robust scientific evidence on the value of bacteriophages from adequately designed clinical randomized studies is urgently needed. To date, few comparative data from small-scale clinical trials with bacteriophages are available.5,6 Following a pilot study,21 a first multicentre, open-label, randomized comparative trial is currently ongoing in burns patients to assess tolerance and efficacy of local bacteriophage treatment of wounds infected with Escherichia coli or Pseudomonas aeruginosa (‘Phagoburn’; ClinicalTrials.gov identifier: NCT02116010). The primary outcome of the study is based on microbiological criteria, measuring the time for bacteria reduction adjusted on antibiotic treatment. Secondary outcomes involve safety/tolerance of treatment and healing improvement. Adult patients with burn wounds who present with local signs of infection and with wounds showing a microbiologically documented infection (positive culture from a surface swab) with either E. coli or P. aeruginosa, regardless of the resistance profile, are to be included. The investigational product (phage cocktail) consists of 12 (E. coli) or 13 (P. aeruginosa) lytic phages, whilst silver sulfadiazine is designated as control treatment. These treatments are locally applied as a daily dressing for 7 days and microbiological swabbing is carried out twice daily during the study phase. Patients are recruited across 11 centres using the same standardized protocol. The trial aims to recruit 220 patients over 1 year. This trial uses a phage cocktail that is GMP compliant, and as such various difficulties were encountered in its preparation. It was evident during the workshop that this posed a great challenge to the supplying manufacturer. The Phagoburn trial is to be viewed as a ‘proof of concept’, and results will further inform subsequent clinical research. Within the current trial, a dose–response relationship is not being investigated, and the approach towards dosefinding for bacteriophages would be significantly different from that for conventional medicines. In future exploration, in vitro models could possibly be employed in order to properly understand the treatment dynamics. Other issues, such as impact on gut microbiota including the important concern of potential transmission of resistance genes; modulation of immune responses; bacterial replacement with ensuing secondary infections; and ultimate positioning of lytic bacteriophages as part of the therapeutic armamentarium need due consideration. To ensure successful development of bacteriophage therapy so as to reach patients in need at the earliest, a regular dialogue with regulatory authorities is warranted. Drug developers should take advantage of early exchanges with regulators and of subsequent formal assistance offered by the scientific-advice process.22 The latter offers a platform for advice on questions concerning quality, non-clinical and clinical aspects of the drug development plan, as well as on issues relating to interpretation and implementation of (draft) EU guidelines; this process ultimately increases the probability of a positive regulatory outcome.23 Conclusions Bacteriophage therapy is a somewhat unique approach to address the need for alternatives to antibacterial medicines in an era of bacterial multidrug resistance. However, there are regulatory and scientific challenges with phage therapy. The EMA workshop, which was attended by various stakeholders, explored the bottlenecks hindering successful therapeutic innovation with bacteriophages. It was clear from this workshop that there are several manufacturers progressing with the development of investigational bacteriophage cocktails to be tested in clinical trials. It was also 2073 Downloaded from https://academic.oup.com/jac/article-abstract/71/8/2071/2237822 by guest on 14 June 2020 Participants acknowledged that bacteriophage therapy, which by its very nature involves a co-evolutionary dynamic pattern between bacteria and lytic phages, poses a unique challenge for regulators. Directive 2001/83/EC10 states that an application for a marketing authorization should be accompanied by qualitative and quantitative particulars of all the constituents of the medicinal product. Biological active substances have inherent variability; nevertheless, they must be appropriately characterized, including identifying critical parameters relevant to produce a high-quality, well-characterized and safe product. Specifications and appropriate acceptance criteria also need to be set and justified, and the manufacturing process should be adequately described, validated and controlled in order to ensure that the product is of consistent quality. Bacteriophage therapy should adhere to these prerequisites; however, the scientific requirements and necessary flexibility require further discussion and definition to accommodate the need for rapid change in composition of the phage preparation. Review Funding The workshop was supported by EMA institutional funding only. Transparency declarations None to declare. Disclaimer The views expressed in this paper are the personal views of the authors and must not be understood or quoted as being made on behalf of or representing the position of the EMA or one of its committees or working parties. References 1 Twort FW. An investigation on the nature of ultra-microscopic viruses. Lancet 1915; 189: 1241– 3. 2 d’Hérelle F. Sur un microbe invisible antagoniste des bacilles dysentériques. C R Acad Sci Paris 1917; 165: 373–5. 3 Bruynoghe R, Maisin J. Essais de thérapeutique au moyen du bacteriophage du Staphylocoque. Compt Rend Soc Biol 1921; 85: 1120– 1. 4 Sulakvelidze A, Alavidze Z, Morris JG Jr. Bacteriophage therapy. Antimicrob Agents Chemother 2001; 45: 649–59. 5 Rhoads DD, Wolcott RD, Kuskowski MA et al. Bacteriophage therapy of venous leg ulcers in humans: results of a phase I safety trial. J Wound Care 2009; 18: 237–8, 240– 3. 6 Wright A, Hawkins CH, Anggård EE et al. A controlled clinical trial of a therapeutic bacteriophage preparation in chronic otitis due to antibiotic resistant Pseudomonas aeruginosa; a preliminary report of efficacy. Clin Otolaryngol 2009; 34: 349– 57. 7 Wittebole X, De Roock S, Opal SM. A historical overview of bacteriophage therapy as an alternative to antibiotics for the treatment of bacterial pathogens. Virulence 2014; 5: 226– 35. 8 Verbeken G, Pirnay JP, De Vos D et al. Optimising the European regulatory framework for sustainable bacteriophage therapy in human medicine. Arch Immunol Ther Exp 2012; 60: 161– 72. Pharmaceutical Legislation: Medicinal Products For Human Use. European Commission. http://ec.europa.eu/health/files/eudralex/vol-1/dir_2001_ 83_consol_2012/dir_2001_83_cons_2012_en.pdf. 11 European Commission. Regulation (EC) no 1394/2007 of the European Parliament and of the council of 13 November 2007 on advanced therapy medicinal products and amending directive 2001/83/EC and regulation (EC) no 726/2004 (consolidated version: 02/07/2012). In: EudraLex—The Rules Governing Medicinal Products in the European Union, Volume 1: Pharmaceutical Legislation: Medicinal Products For Human Use. European Commission. http://ec.europa.eu/health/files/eudralex/vol-1/reg_2007_ 1394/reg_2007_1394_en.pdf. 12 Chan BK, Abedon ST, Loc-Carrillo C. Phage cocktails and the future of phage therapy. Future Microbiol 2013; 8: 769– 83. 13 European Commission. Regulation (EC) no 1234/2008 concerning the examination of variations to the terms of marketing authorisations for medicinal products for human use and veterinary medicinal products (consolidated version: 02/11/2012). In: EudraLex—The Rules Governing Medicinal Products in the European Union, Volume 1: Pharmaceutical Legislation: Medicinal Products For Human Use. European Commission. http://ec.europa.eu/health/files/eudralex/vol-1/reg_2008_1234_cons_ 2012-11-02/reg_2008_1234_cons_2012-11-02_en.pdf. 14 Committee for Medicinal Products for Veterinary Use. Guideline on Data Requirements for Multi-strain Dossiers for Inactivated Vaccines against Avian Influenza (AI), Bluetongue (BT) and Foot-and-Mouth Disease (FMD). European Medicines Agency. http://www.ema.europa.eu/ema/pages/ includes/document/open_document.jsp?webContentId=WC500077950. 15 International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, Committee for Proprietary Medicinal Products. CHMP/ICH Q5D Note for Guidance on Quality of Biotechnology Products: Derivation and Characterisation of Cell Substrates Used for Production of Biotechnological/Biological Products (CPMP/ICH/294/95). European Medicines Agency. http://www.ema. europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/ WC500003280.pdf. 16 Pirnay JP, Blasdel BG, Bretaudeau L et al. Quality and safety requirements for sustainable phage therapy products. Pharm Res 2015; 32: 2173– 9. 17 European Commission. Annex 1: manufacture of sterile medicinal products. In: EudraLex—The Rules Governing Medicinal Products in the European Union, Volume 4: EU Guidelines to Good Manufacturing Practice, Medicinal Products for Human and Veterinary Use. European Commission. http://www.ec.europa.eu/health/files/eudralex/vol-4/2008_11_25_gmpan1_en.pdf. 18 Parracho HMRT, Burrowes BH, Enright MC et al. The role of regulated clinical trials in the development of bacteriophage therapeutics. J Mol Genet Med 2012; 6: 279–86. 19 Mie˛dzybrodzki R, Borysowski J, Weber-Da˛browska B et al. Clinical aspects of phage therapy. Adv Virus Res 2012; 83: 73 –121. 20 Bruttin A, Brüssow H. Human volunteers receiving Escherichia coli phage T4 orally: a safety test for phage therapy. Antimicrobiol Agents Chemother 2005; 49: 2558– 69. 21 Rose T, Verbeken G, de Vos D et al. Experimental phage therapy of burn wound infection: difficult first steps. Int J Burns Trauma 2014; 4: 66 –73. 9 Verbeken G, Pirnay JP, Lavigne R et al. Call for a dedicated European legal framework for bacteriophage therapy. Arch Immunol Ther Exp 2014; 62: 117–29. 22 European Medicines Agency. Scientific Advice and Protocol Assistance. http://www.ema.europa.eu/ema/index.jsp?curl=pages/regulation/general/ general_content_000049.jsp&mid=WC0b01ac05800229b9. 10 Directive 2001/83/EC of the European Parliament and of the council of 6 November 2001 on the community code relating to medicinal products for human use (consolidated version: 16/11/2012). In: EudraLex—The Rules Governing Medicinal Products in the European Union, Volume 1: 23 Regnstrom J, Koenig F, Aronsson B et al. Factors associated with success of market authorisation applications for pharmaceutical drugs submitted to the European Medicines Agency. Eur J Clin Pharmacol 2010; 66: 39– 48. 2074 Downloaded from https://academic.oup.com/jac/article-abstract/71/8/2071/2237822 by guest on 14 June 2020 evident that physicians within the EU are already treating patients with this therapy on a compassionate use basis. However, there is currently a considerable lack of robust evidence to prove the efficacy and safety of these products. More data are urgently needed on the subject; it is anticipated that ongoing research efforts will inform follow-up research and debate. An ongoing dialogue between the drug developers and regulatory authorities on how the regulatory framework might support and offer appropriate flexibility in delineating the tests and studies to be undertaken is highly encouraged. This approach would facilitate the introduction of bacteriophage therapy in clinical practice if such therapy is proven to be safe and effective.