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Bacteriophages in clinical practice: follow the guide! From provision to administration.

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The English version of the French phage therapy guide to help clinicians and pharmacist to learn about and conduct treatments with bacteriophages.
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Jan
Bacteriophages in clinical
practice: follow the guide!
From provision to administration
Version January 2021
Anthia MONRIBOT1, Raphaëlle DELATTRE2,3, Nicolas DUFOUR3,4, Camille d’HUMIERES5, Nathalie
PONS-KERJEAN1, Julie BATAILLE1
1 In-house Pharmacy (IHP), Hôpital Beaujon AP-HP, 100 boulevard du Général Leclerc, 92110 Clichy
2 Anaesthesia-intensive care department, Hôpital Beaujon AP-HP, 100 boulevard du Général Leclerc, 92110 Clichy
3 Institut Pasteur, Department of Microbiology, Molecular Biology of Gene in Extremophiles, 75015 Paris
4 Medical-surgical resuscitation department, Centre Hospitalier René Dubos, 95300 Pontoise
5 Bacteriology laboratory, Hôpital Bichat-Claude Bernard AP-HP, 46 Rue Henri Huchard, 75877 Paris
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Index
SUMMARY ....................................................................................................................................................4
INTRODUCTION .............................................................................................................................................7
SOME GENERAL DATA ...................................................................................................................................8
BACTERIOPHAGES: DEFINITION ........................................................................................................................... 8
THE REGULATORY STATUS OF BACTERIOPHAGES ................................................................................................... 10
THE MODALITIES FOR PROVISIONING BACTERIOPHAGE LYOPHILISATES/SUSPENSIONS ................................................... 11
THE REGULATION ON THE PRODUCTION AND QUALITY CONTROL OF BACTERIOPHAGE LYOPHILISATES/SUSPENSIONS ........... 13
CLINICAL EFFICACY DATA ................................................................................................................................. 14
SAFETY OF THE THERAPEUTIC USE OF BACTERIOPHAGES ......................................................................................... 16
THE DIFFERENT SOURCES OF THERAPEUTIC BACTERIOPHAGES IN FRANCE AND EUROPE ................................................ 19
THE TERMS OF ACCESS TO PHAGOTHERAPY IN FRANCE .............................................................................. 22
THE CLINICAL SITUATIONS POTENTIALLY ELIGIBLE FOR A TREATMENT BY BACTERIOPHAGE ............................................. 22
INFORMATION FROM ANSM ON THE PROJECT TO USE BACTERIOPHAGES FOR THERAPY ............................................... 23
DETERMINING THE SENSITIVITY OF THE BACTERIAL STRAINS TO BACTERIOPHAGES AND THE EFFICACY IN FORMING LYSIS
PLAQUES 24
ASSESSMENT OF THE PHARMACEUTICAL QUALITY OF BACTERIOPHAGE RAW MATERIALS AND THE STAGES OF PREPARATION
PHARMACEUTICAL QUALITY CONTROL OF FINISHED PREPARATIONS......................................................................................... 28
ASSESSMENT OF THE ADMINISTRATION PLAN FOR THERAPEUTIC BACTERIOPHAGES ..................................................... 36
ASSOCIATING THERAPEUTIC BACTERIOPHAGES AND ANTIBIOTICS ............................................................................. 36
THE PRACTICAL CONDITIONS OF USE OF BACTERIOPHAGES ........................................................................ 38
TRANSPORTING AND STORING THE DIFFERENT FORMS OF BACTERIOPHAGES .............................................................. 38
HANDLING PROCEDURES FOR BACTERIOPHAGES................................................................................................... 38
THE HYGIENE AND PROTECTION MEASURES FOR HANDLING BACTERIOPHAGE PREPARATIONS......................................... 41
BACTERIOPHAGE ADMINISTRATION PROCEDURES ................................................................................................. 42
FUNDING BACTERIOPHAGE PREPARATIONS ......................................................................................................... 42
CONCLUSION ............................................................................................................................................... 43
LIST OF ABBREVIATIONS ............................................................................................................................. 45
DEFINITIONS OF TERMS USED ..................................................................................................................... 46
BIBLIOGRAPHY ............................................................................................................................................ 49
APPENDIX 1 .......................................................................................................................................................... 55
APPENDIX 2 .......................................................................................................................................................... 60
1. CLINICAL TRIAL IN SKIN INFECTIONS............................................................................................................ 60
2. CLINICAL TRIAL IN CHRONIC ENT INFECTIONS ............................................................................................. 63
3. CLINICAL TRIAL IN URINARY INFECTIONS .................................................................................................... 65
4. CLINICAL TRAIL IN HEALTHY VOLUNTEERS .................................................................................................. 66
5. CLINICAL TRIAL IN DIGESTIVE INFECTIONS .................................................................................................. 67
APPENDIX 3 .......................................................................................................................................................... 71
APPENDIX 4 .......................................................................................................................................................... 72
APPENDIX 5 .......................................................................................................................................................... 73
LIST OF TABLES ..................................................................................................................................................... 76
LIST OF FIGURES ................................................................................................................................................... 76
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We would like to thank the Products Management service responsible for vaccinations and anti-infective
drugs, in hepato-gastroenterology, dermatology, gene therapy and rare metabolic disorders (INFHEP), the
MALINF centre at the National Agency for Drug and Health Product Safety (ANSM) for their help in
producing this document.
We sincerely thank Professor Jean-Hugues Trouvin for his expertise in the fields of pharmaceutical
regulation and legislation, and for repeatedly rereading the text.
We also thank Doctor Gilles Leboucher for rereading the document.
WARNING
Please note that this document contains data related to drug regulation and administration of
unmarketed substances (here bacteriophages). These data cannot be interpreted and used outside
the French (and subsequent European when applicable) regulatory framework.
This guide does not aim to substitute the contents of the MA file, nor the case by case assessments by
ANSM. It is subject to modification in function to the legislation, European and French recommendations,
feedback from practitioners and results of pre-clinical and clinical studies.
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Summary
Phagotherapy is the medical use of bacteriophages, these viruses that have the particular characteristic of
only infecting bacteria. Bacteriophages have small spectra: they are specific to one bacterial species, or
even to a few strains, and most often to a limited number of strains within this species (14). Bacteriophages
are capable of presenting two main types of infectious cycle:
- the lytic cycle, specific to virulent bacteriophages, which destroys the bacteria by diverting
the bacterial machinery to benefit the viral protein and genome synthesis;
- the lysogenic cycle, specific to temperate bacteriophages. These viruses have the property of
incorporating their genome into the bacterial chromosome, without systematically lysing the
bacteria. They can thus confer new properties to the bacteria, both beneficial and non-
beneficial, such as virulence factors for example (14).
When producing bacteriophages for therapeutic use, it is thus recommended to verify the absence of
temperate bacteriophages in the final product, in other words in the raw material/active principle, since
these are potential vectors of undesirable genes (virulence or antibiotic-resistance genes) (14).
There exist different sources of bacteriophages in France and Europe (cf. III.7, non-exhaustive list).
Bacteriophages are available the form of suspensions or lyophilisates.
Currently, the use of bacteriophages is empirical, and mainly based on a rational of pharmacodynamic
efficacy. Despite widespread use in Eastern Europe, the clinical data are limited. Notably, experimental
studies on animals have demonstrated that bacteriophages can be effective in treating mainly respiratory,
skin and digestive bacterial infections, induced by P. aeruginosa, E. coli or S. aureus (5). The safety of
phagotherapy has also been demonstrated in preclinical and clinical studies(6,7). However, to date, only a
limited number of clinical trials using bacteriophages have been carried out in compliance with the current
standards in biomedical research: 1 with healthy subjects, 4 with ill subjects (812). In France, the National
Agency for the Safety of Medicines and Health Products (ANSM) has supervised the provision of
bacteriophages for fifteen patients since 2016. The majority of these were cases of joint infections where
in situ local administrations were carried out (13).
In 2015, the European Medicines Agency (EMA) agreed to include treatment by bacteriophage in the
European regulatory framework applicable to biological drugs, as defined in the 2001/83/EC
directive of the European Parliament and Council of 6 November 2001, modified by the 2004/27/EC
directive on 31 March 2004 on the Community code relating to medicinal products for human use (1416).
In the same vein, during a meeting of its temporary specialised scientific committee (CSST) on
phagotherapy on 24/03/2016, ANSM decided that bacteriophages should be included in the regulation
applicable to medicinal products (17). They will thus be made available according to the statutes defined
in article L.5111-1 of the French Public Health Code (CSP) (18).
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Given that no industrial developer has applied for a marketing authorisation (MA) in France or in
Europe for bacteriophage lyophilisates/suspensions and in a context of growing demand from the scientific
and medical communities, ANSM has supervised and authorised the provision of bacteriophage
lyophilisates/suspensions for compassionate use. Currently, bacteriophage lyophilisates/suspensions
are not manufactured following a standardised industrial process capable of guaranteeing the quality,
safety and efficacy of these products. The bacteriophage lyophilisates/suspensions produced (made to
order or “as a batch”) to treat bacterial infections in humans, can thus not be considered as drugs in the
sense of specialty pharmaceuticals. In this context, lyophilisates/suspensions can only be provisioned
in the context of clinical trials or for compassionate use.
In this last case, the product is provisioned:
under a nominative Authorisation for Temporary Use (ATU);
depending on the status of the pharmaceutical raw materials, used to create a magistral
preparation (Appendix 1). The in-house pharmacies (IHP) are thus responsible for the
preparation, meaning producing the pharmaceutical form (reconstitution, dilution) of these raw
materials.
At the CSST meeting on phagotherapy organised by ANSM in 2016, it was agreed that the prescription,
dispensing and administration of bacteriophage suspensions should be carried out within a health
establishment (17).
Thus, a request for bacteriophages can only be initiated by a hospital doctor and bacteriophage
lyophilisates/suspensions can only be provisioned by a IHP at a health establishment. Bacteriophages
are administrated under the responsibility of the patient’s prescribing doctor and the hospital pharmacist.
ANSM supports the hospital teams in the process of provisioning the bacteriophages. A collegiate
discussion of the case should also be held in order to analyse the patient’s clinical and therapeutic history,
and the risk/benefit balance of a therapy using bacteriophages.
In 2016, the CSST reached a consensus on the criteria justifying a situation of need, applicable to all
types of infection and justifying access to bacteriophages:
- a prognosis that threatens the patient’s life or functional prognosis
- a situation of a therapeutic "dead end" (“salvage situation”);
- a monomicrobial infection.
Additionally, considering the current limitations in data able to underpin the use of bacteriophages, it was
agreed that in vitro efficacy data would be required, notably a phagogram, before deciding to initiate
the treatment. A phagogram aims to test the sensitivity of the bacterial strain vis-a-vis one or several
bacteriophage candidates that are kept in a phage library (17).
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However, three factors have influenced the evolution of some of these criteria: the clinical experience in
bacteriophages acquired over the last 2 years, the limited availability of bacteriophage cocktails and the
remaining uncertainties about this therapy. Thus, the practical cases that could justify early access to
bacteriophages have been defined:
- a bacterial resistance to antibiotics;
- an infection where the bacteria is still sensitive to some antibiotics but the morbidity linked to the
treatment is not acceptable (nephrotoxicity, allergy, etc.);
- a clinical inefficacy: an infection that is hard to eradicate which is evolving towards chronicity.
Additionally, discussions should be held in order to determine the route of administration, the
composition of the solution in active principle and excipients (concentration, number of different
bacteriophages, etc.), the galenic form and the medical devices (MD) to be used for the administration
(taking into account the physico-chemical characteristics and techniques of the MDs and the potential
interactions between these MDs and the bacteriophages). The route of administration should make it
possible to concentrate the bacteriophages in the infected site while remaining compatible with the
pharmaceutical characteristics of the bacteriophage cocktails. Thus, local administration of bacteriophages
could be considered as the safest method according to the available data. The advantage is that the quantity
of bacteriophages delivered to the infection site is known. During the CSST meeting in 2019, all of the
experts agreed to exclude oral administration, as well as, a priori, intrathecal administration (13).
The hospital pharmacist is responsible for assessing the pharmaceutical quality of the raw material.
It should be recalled that the main tests on the pharmaceutical quality of the raw materials/active substances
of the bacteriophages should be undertaken by the industrial developer/manufacturer, in compliance with
the regulation in force. The IHP pharmacist should critically analyse the control report of the raw
material provided by the manufacturer, to decide whether he/she can take the responsibility to use this
active substance in Humans. This guide lists the information and controls required to assess the
pharmaceutical quality of raw materials/active substances, which should be detailed in the control report.
After having validated the pharmaceutical quality of the raw material, the hospital pharmacist then
carries out the preparation stages (reconstitution, galenic formulation adapted to the route of
administration) in compliance with theGood Preparation Practices (GPP) of the IHP. He/she should also
undertake a pharmaceutical quality control of the final magistral (or hospital) preparation of
bacteriophages, so as to allow administration to Humans (19).
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Introduction
Phagotherapy is the medical use of bacteriophages, these viruses that have the particular characteristic of
only infecting bacteria. This therapy has been in used since the 1930s, but was abandoned in Western
countries in favour of antibiotic therapies. However, it continued to be practiced in Eastern Europe.
Bacteriophages are once again appearing in the therapeutic arsenal as a potential alternative to antibiotic
therapy (or to complement the latter), in situations reaching a therapeutic dead end, considering the growing
multi-resistance, pressures in the supply chain and a non-satisfied demand for new antibiotics.
Currently, the therapeutic use of bacteriophages is empirical. Despite widespread use in Eastern Europe,
the clinical data are limited. Very few clinical trials compliant with the current standards on biomedical
research have been carried out (randomisation, controlled trials, double blind…).
Currently, there are no market authorisations (MA) in France or Europe for bacteriophages to be used as
drugs (in the sense of a specialty pharmaceuticals under French legislation) for indications in infectiology.
There are very few specific European recommendations on the development of bacteriophages in terms of
pharmaceutical production and quality (quality control). The current regulatory framework must therefore
adapt to specific clinical situations (emergency situations, patients reaching a therapeutic dead end, a threat
to the patient’s life or functional prognosis) and to the specificities of ‘made to order’ bacteriophage
cocktails (customised approach).
In this context and in response to the growing number of questions and requests from the scientific and
medical communities, we have produced a practical guide on the therapeutic use of bacteriophages. There
are two objectives for this work:
- providing information on the current state of knowledge for this therapy and the regulation it is
subject to;
- supporting the use of bacteriophages by describing each of the stages, from production to
administration, with the aim of guaranteeing the quality, efficacy and safety of this therapy.
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Some general data
Bacteriophages: definition
Bacteriophages are viruses that specifically infect bacteria and do not have the capacity to infect the
eukaryotic cells (1,3,4,20). Bacteriophages have small spectra: they are specific to one bacterial species, or
even a few strains, and most often to a limited number of strains within this species. They are present in
large quantities wherever bacteria can be found. Bacteriophages are thus present in the whole biosphere: in
waters, soils, plants, food, on the skin,and in our digestive tract and mucous (2123). The vast majority
(96%) of bacteriophages belong to the Caudovirales order (tailed viruses) and are most often viruses with
double stranded, non-enveloped DNA. The vast majority of bacteriophages of interest in phagotherapy
belong to three families: Myoviridae, Podoviridae and Siphoviridae (1,20,24).
As illustrated in Figure 1, they are composed of:
a protein capsid protecting their genome (DNA-RNA);
a tail of variable length;
tail fibres allowing the virus to interact with the specific host receptors.
They measure on average between 24 and 200 nanometres (24).
Figure 1. Structure of bacteriophage T4 (1)
Bacteriophages are capable of presenting two main types of infectious cycle depending on the information
coded in their genome and the metabolic status of the infected cells as illustrated in Figure 2. (1,3,4,20).
Virulent bacteriophages destroy the bacteria by diverting the bacterial machinery to benefit the viral
protein and genome synthesis. Viral structural proteins and the viral genome are produced in large
quantities, respectively assembled and encapsidated, thus resulting in the formation of mature virions. At
1: head
2: tail
3: nucleic acid
4: capsid
5: collar
6: sheath
7: long tail fibres
8: short tail fibres
9: baseplate
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the end of this process of lysis, the bacterial lysis liberates several dozen virions. This phenomenon of
amplification at the infectious site is a completely new property of phagotherapy compared to antibiotic
therapy (14).
The lysogenic cycle is specific to temperate bacteriophages. These have the property of incorporating
their genome in the bacterial chromosome, without altering the bacterial viability. The viral genome is
replicated at each cell division and transmitted to the descendance. These bacteriophages can thus confer
new properties to the bacteria, both beneficial and non-beneficial, such as virulence factors for example.
They can remain inactive for a very long time, but in cases of physiological stress (UV irradiation,
mytomycin, energy depletion of the medium) they can excise from the bacterial genome and begin a lytic
infectious cycle (14).
Figure 2. The main infectious cycles of bacteriophages (2)
Thus, phagotherapy, by its expected mode of action, requires the exclusive use of virulent bacteriophages
to treat bacterial infections in humans. When producing bacteriophages for therapeutic use, it is thus
recommended to verify that there are no temperate bacteriophages in the bacteriophage
lyophilisate/suspension produced, i.e. the active principle/raw material (by analysing the genome by
sequencing the DNA-RNA of the bacteriophages of the suspension), since they are potential vectors of
genes that could harm the patient (virulence or antibiotic resistant genes). In particular, this elimination of
the temperate bacteriophages is done by verifying the absence of possibilities for the bacteriophage genome
to incorporate in the bacterial genome (the working method is detailed in chapter IV.4.b).
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The regulatory status of bacteriophages
The European Medicines Agency (EMA) held a conference on bacteriophages on 8 June 2015 (“Workshop
on the therapeutic use of bacteriophages”) which aimed to discuss the issues related to developing
bacteriophage treatments. At this event they discussed the regulatory framework, the quality considerations
and the state of clinical development (16).
During this discussion, it agreed to include treatment by bacteriophage in the European regulatory
framework applicable to biologic drugs, as defined in the 2001/83/EC directive of the European
Parliament and Council of 6 November 2001, modified by the 2004/27/EC directive of 31 March 2004 on
the Community code relating to medicinal products for human use. This directive requires that a MA is
awarded (14,15). The present directive applies to medicinal products for human use, more precisely to
specialty pharmaceuticals, intended for marketing in the Member States and prepared industrially or
manufactured following a method which includes an industrial process (25). In conformity with this, the
MA application should include detailed preclinical and clinical pharmaceutical data. Additionally, it was
confirmed that non-genetically modified bacteriophages do not meet the classification for an advanced
therapy medicinal product (ATMP) because of their mode of action and origins (16).
In the same vein, on 24/03/2016, the French National Agency for Drug and Health Product Safety (ANSM)
created a temporary specialised scientific committee on phagotherapy (CSST)
1
. At this CSST, it was agreed
that bacteriophages should be included in the regulation applicable to drugs (14,15,17).
Given that no industrial developer has applied for a marketing authorisation (MA) in France or in
Europe for bacteriophage lyophilisates/suspensions and in a context of growing demand from the scientific
and medical communities, ANSM has supervised and authorised the provision of bacteriophage
lyophilisates/suspensions for compassionate use, according to the derogation procedures, classed as raw
materials (active principle), for use as components of a magistral preparation. Currently, bacteriophage
lyophilisates/suspensions are not manufactured following a standardised industrial process able to
guarantee the quality, safety and efficacy of these products. This type of production must therefore be
qualified as “experimental”. The bacteriophage lyophilisates/suspensions produced (made to order or “as a
batch”) to treat bacterial infections in humans, can thus not be considered as drugs in the sense of specialty
pharmaceuticals, but can be provisioned depending on the status of the raw material(25). The in-house
pharmacies (IHP) are thus responsible for the preparation, meaning producing the pharmaceutical
form: reconstitution, dilution of these raw materials.
1http://ansm.sante.fr/content/download/91159/1144681/version/1/file/CR_CSST_Phagotherapie_CSST20161101
3_24-03-2016.pdf.
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The modalities for provisioning bacteriophage lyophilisates/suspensions
Bacteriophage lyophilisates/suspensions must thus be provisioned according to the statutes defined by
article L.5111-1 of the French Public Health Code (CSP)(18).
At the CSST meeting on phagotherapy organised by ANSM in 2016, it was agreed that the prescription,
dispensing and administration of bacteriophage suspensions should be carried out within a health
establishment (17).
Thus, a request for bacteriophages can only be initiated by a hospital doctor and bacteriophage
lyophilisates/suspensions can only be provisioned by a IHP at a health establishment. The bacteriophage
lyophilisate-suspension, produced based on the initial prescription, is received by the IHP who should then
produce the pharmaceutical form in order to ‘facilitate’ the administration of the suspension.
Bacteriophages are administrated under the responsibility of the patient’s prescribing doctor and the
hospital pharmacist.
Currently, no developer has applied for a marketing authorisation in France or in Europe for a suspension
of bacteriophages. Therefore, there are no ‘specialty pharmaceuticals’, or finished products, based on
bacteriophages on the market.
In view of these points, the regulatory considerations should be understood in terms of the clinical context.
Indeed, requests for bacteriophages are made in the context of emergencies since they are used for patients
who have reached a therapeutic dead end due to a multi-resistance to antibiotics causing the treatments
available on the market to fail, resulting in a threat to the patient’s life and/or functional prognosis. The
regulatory system will also depend on:
the data documenting the intrinsic pharmaceutical quality of the product guaranteed by a in-
depth scientific development which could eventually be based on monographs or other reference
texts such as guidelines that make it possible to identify the critical elements that contribute to the
intrinsic quality of the bacteriophage lyophilisate-suspension (identity, purity, biological activity
and dose);
quality standards applied during the production and control stages before release (Good
manufacturing practices (GMP), Monograph, etc.);
the geographical location of the establishment ensuring the production/control and release of
bacteriophage lyophilisates/suspensions: France, countries in the European Union, outside the EU,
etc.
In this context, lyophilisates/suspensions can only be provisioned in the context of clinical trials or for
compassionate use. In this last case, the product is provisioned:
under a nominative Authorisation for Temporary Use (ATU);
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depending on the status of the raw pharmaceutical materials, used to create a magistral
preparation (Appendix 1).
Bacteriophage lyophilisates/suspensions can be provisioned in the context of a nominative ATU for
preparations for which the intrinsic quality is guaranteed by an appropriate pharmaceutical
development (if need be supported by monographs and other guidelines) and a quality control
strategy that makes it possible to ensure the final quality of the bacteriophage lyophilisate/suspension
in terms of identity, purity and activity, and for which the industrial production is carried out in a
GMP-certified establishment. As such, the application for an ATU, made by the holder of the exploitation
rights or its proxy at ANSM, must include:
a file presenting the data relating to the pharmaceutical quality of the drug as documented in the
MA application (module 3 of the Common Technical Document);
a copy of the MA application or, if this has not be filed, a letter from the body requesting the
authorisation for temporary use committing to filing such an application within a set timeframe
(26).
Thus the ATU exclusively applies to a “specialty pharmaceutical candidate”. At present, ANSM can not be
led to award any ATUs in the absence of bacteriophage lyophilisates/suspensions that meet the quality and
industrial production standards.
For the bacteriophage lyophilisates/suspensions that do not have the status of specialty
pharmaceutical/finished product, and cannot obtain an ATU, provision for compassionate use
depending on the status of the raw pharmaceutical materials can be considered. The Queen Astrid
Military Hospital in Brussels (QAMH) produces bacteriophages suspensions or lyophilisates, or more
precisely the active pharmaceutical ingredients” (API) that can be used as components or “raw materials”
of a magistral or hospital preparation (27). These are characterised, lytic bacteriophages, active on a
bacterial strain.
The preparation stages (reconstitution, galenic formulation adapted to the route of administration) and the
control of the pharmaceutical quality of the magistral preparation of bacteriophages should be carried out
by the IHP hospital pharmacist, considering that the main controls of the API’s quality will be carried out
by the QAMH team, in compliance with the regulation in force that requires all suppliers to provide a
control report of the material supplied. The IHP pharmacist should critically analyse this control report to
decide whether he/she can take the responsibility to use this active substance in Humans. The controls
carried out on the APIs by QAMH are detailed in the Belgian National General Monograph of
Bacteriophages (2731). It should be noted that Pherecydes Pharma® produces bacteriophage suspensions
that can be provisioned depending on the status of the raw materials. It should be recalled that if the
substance is considered as a raw material, the manufacturer of the Raw Material for Pharmaceutical Use
(RMPU) must have authorisation from ANSM (or the supervisory authority of the country in which the
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manufacturing establishment is located) to carry out manufacturing, import and distribution activities for
active substances.
Bacteriophage lyophilisates/suspensions should, as much as possible, be used in priority in the context of
a clinical trial in relation to a compassionate use.
ANSM supports the hospital teams in the process of provisioning the bacteriophages. A collegiate
discussion of the case should also be held in order to analyse the patient’s clinical and therapeutic history,
the risk/benefit balance of a therapy using bacteriophages and to assess the pharmaceutical quality of the
substance in terms of the information provided on the product.
The regulation on the production and quality control of bacteriophage
lyophilisates/suspensions
Currently, no specific European recommendations exist on the production or the requirements regarding
pharmaceutical quality of bacteriophage lyophilisates/suspensions, i.e. the bacteriophage raw materials. It
is therefore not possible to refer to specific “bacteriophage” recommendations. However, it is possible to
refer to the existing texts and publications on the production and pharmaceutical quality control of
bacteriophage lyophilisates/suspensions such as:
an international group of 29 “bacteriophage experts” from ten countries met in 2015 to define the
requirements in terms of quality, safety and efficacy for bacteriophage preparations (32);
the European Directive on tissues and cells (EUTCD) (3335);
the CHMP/ICH/294/95 Q5D recommendations (36) on cell substrates and their use in the
production of biological substances;
the text of the Food and Drug Administration (FDA): “Characterization and Qualification of Cell
Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious
Disease Indications” (37);
the Belgian National General Monograph of Bacteriophages written by the QAMH team and
validated by the Belgian Federal Agency for Medicines and Health Products (AFMPS) in January
2018 (27,30);
many other publications (28,31,3841).
Considering all of these initial items on ensuring quality for a biological product, the developers of
bacteriophage lyophilisates/suspensions thus possess an already significant arsenal to establish the terms
of reference for their own procedures and control strategies for the bacteriophage lyophilisates/suspensions
they are planning to produce. Also, for the production and control stages, the acceptability criteria will thus
be adapted to and justified for their specific product.
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Clinical efficacy data
In the current state of knowledge, very little data exists on the clinical efficacy of bacteriophages in
function to the route or administration and the dosage. During the EMA’s workshop on bacteriophages, the
considerable lack of scientific evidence to prove the efficacy and safety of bacteriophages was emphasised
(16). The clinical experience acquired on bacteriophages over many years in the Eastern European countries
does not in itself constitute a sufficient level of efficacy and safety, simply based on an anteriority principle.
Indeed, very little data has been published on this widespread use of bacteriophage therapy. Some
publications say that this lack of communication can be partly explained by the Cold War, where the
government of the ex-USSR only allowed the researchers to communicate their knowledge and discoveries
in the national journals, meaning they are in Georgian or Russian (1,3,4). Additionally, the rare published
works do not adhere to the current standards for clinical research.
The use of bacteriophages is thus empirical, and mainly based on a rational of pharmacodynamic
efficacy. As such, and despite the anteriority, the notion of well-established medical use, as stipulated
in the European regulation, cannot currently serve as a basis for awarding a MA.
ANSM, like the EMA, is strongly encouraging clinical trials to be set up to assess bacteriophages
(16,17). Only randomised clinical trials using relevant assessment criteria will make it possible to obtain
scientific proof of the clinical efficacy and safety of bacteriophages in the different clinical contexts
(indications) studied by the clinical trials. As such, by definition, a clinical trial makes it possible to define
an experimental protocol (ideally randomised) with the aim of reducing bias to a minimum and establishing
a causal link at the end of the experiment.
Many experimental studies have been carried out on animals. A certain number of articles have been
published in the last 20 years demonstrating, for the vast majority, the efficacy of bacteriophages on
different types of infections, notably in treating respiratory, skin and digestive bacterial infections induced
by P. aeruginosa, E. coli or S. aureus (5).
The animal models could make it possible to verify whether the bacteriophages are active in vivo on a panel
of bacterial strains and to propose a dose regimen for clinical use. Thus, the assessment of phagotherapy
can be based on predictive elements (efficacy, resistance) provided by the experimental animal models.
However, the methodology of these studies, and the choice of bacteriophage, animal model and “bacteria-
bacteriophages” pair, is fundamental for the results of these experimental studies to be useable and then
transposable to a clinical trial, which will always be necessary to assess safety and efficacy in Humans and
in the clinical situation demanded.
15
To date, there have been five clinical trials using bacteriophages, conducted in accordance with the
biomedical research standards (double blind, randomised, controlled): 1 with healthy subjects, 4 with ill
subjects (812). These trials are summarised in Appendix 2.
Two clinical research hospital programmes are about to be commenced:
the PHAGOS project: a phase I/II, multi-centre, randomised, open-label, controlled versus
standard treatment clinical trial, with the aim of assessing the tolerance and efficacy of
phagotherapy associated with standard treatment (surgery and antibiotics) in chronic
Staphylococcus infections of hip or knee prostheses (42) ;
the PHAGOPIED project: a phase I/II, multi-centre, randomised, controlled versus
placebo, double blind, superiority clinical trial, aiming to compare the efficacy of a
standard treatment associated with bacteriophages versus a standard treatment associated
with a placebo in the treatment of S. aureus-infected foot ulcers in diabetic patients
(ClinicalTrials.gov Identifier: NCT02664740) (43).
It should be noted that in a context of growing demand, the National Agency for Drug and Health
Product Safety (ANSM) has supervised the use of bacteriophages for around fifteen patients since 2016.
The majority of these were cases of joint infections where in situ local administrations of phages were
carried out (13).
16
Safety of the therapeutic use of bacteriophages
Temperate bacteriophages
The main risk in phagotherapy is the presence of temperate bacteriophages in a therapeutic preparation.
Indeed, temperate bacteriophages present the property of incorporating their genome into the bacterial
chromosome, without killing their host. They are thus potential vectors of virulence or antibiotic-resistant
genes and can confer new properties to the bacteria, which can be beneficial or non-beneficial.
In therapies, it is thus important to use characterised lytic bacteriophages and to verify the absence of
temperate bacteriophages in the lyophilisate/suspension product, i.e. the active principle/raw material
of bacteriophages (28,44). Investigations should be conducted, notably through genome analyses that
sequence the DNA-RNA of the bacteriophages present in the suspension in order to limit the risk linked to
the presence of temperate bacteriophages, which are potentially and indirectly pathogenic to humans.
The risks for the patient
Bacteriophages do not have the capacity to infect the eukaryotic cells and therefore present no reprotoxic
or mutagenic effects. Analysis of the data in the literature provides no evidence that bacteriophages can be
physico-chemically toxic, which would result in tissue damage.
Since bacteriophages are viruses, administering them could provoke an immune response. Such a response
is potentially harmful, and can entail either innate immunity mechanisms with the secretion of cytokines
and the involvement of pro-inflammatory cells, or adaptive immunity mechanisms with antibody
synthesis. The direct immunogenicity of bacteriophages involving innate immunity mechanisms appears
negligible as long as the suspensions are correctly purified, as demonstrated by the animal experiments and
the 2 studies carried out by Nestlé® with the bacteriophage T4 cocktail and the Microgen® cocktail cited
previously in Appendix 2 (8,9,45).
Indeed, the bacterial lysis following the bacteriophage action results in the liberation of bacterial DNA,
endotoxins and bacterial membrane components such as lipoteichoic acid and peptidoglycan. These
proteins are powerful antigens which can be immunogenic in humans. It should be recalled that the
liberation of endotoxins is a key stage in the physiopathology of sepsis and septic shock. The bacterial lysis
could thus be the cause of indirect immune responses, via the liberation of antigenic components. A recent
study carried out by Dufour et al. compared, in vitro, the liberation of endotoxins and the capacity to kill
the bacteria: of therapeutic bacteriophages versus 4 antibiotic treatments (7). The results showed that the
bacteriophages provoke a faster bacterial lysis than that provoked by the action of β-lactamines and that the
concentration in cumulated endotoxins generated by the action of these bacteriophages on the bacterial
strains is less than that generated by the action of β-lactamines (7). The same authors also showed, this time
in vivo in animals (acute pneumonia model), that the administration of two bacteriophages does not result
17
in an increase in the synthesis of the cytokines of the acute inflammation. To the contrary, in an identical
(or even superior) way to that observed in animals treated with reference antibiotics, the phage treatment
reduced the inflammation in the infected animals (6).
The second immunogenic mechanism is the induction of a humoral response with the production of
neutralising antibodies. The development of these anti-phage antibodies could result in an inefficacy of a
future treatment based on bacteriophages as sources of the immunisation. However, M. Żaczek et al.
demonstrated that the production of antibodies did not appear to influence the infective prognosis and the
efficacy of the treatment (46). In another study on 15 healthy volunteers who received a bacteriophage T4
cocktail, none developed antibodies directed against the bacteriophage being tested (8).
The clinical trials cited previously do not reveal any particular adverse side effect. The tolerance profiles
of each of the bacteriophage suspensions administered by various routes were acceptable (cf. Appendix 2).
The tolerance data we now possess mainly come from studies on animals and the 5 clinical trials conducted
following a satisfactory methodology. Additional research is thus needed.
Despite the abundant clinical experience in Eastern Europe, the limited number of publications on this
widespread therapeutic use of bacteriophages means that potential vigilance signals relating to tolerance
and the safety of administering bacteriophages cannot be excluded.
In summary, the potential risks and adverse effects linked to the administration of bacteriophages are:
- an inefficacy of the bacteriophages chosen and selected based on the sensitivity of the bacterial
strain targeted, for the treatment of bacterial infections in connection with the development of a
bacterial resistance to bacteriophages (47);
- a production of specific anti-phage antibodies in connection to a humoral type of adaptive immune
response;
- an acute inflammatory response in connection with an innate immune response;
- an immune response in connection to the liberation of antigenic components during the bacterial
lysis.
The risks associated with handling the bacteriophages
It is important look into the preventive rules and standards required for the handling of bacteriophages.
There exists a classification of natural micro-organisms which covers bacteria, viruses, parasites and fungi
(48). The criteria of this classification are present in Table I.
There are 4 groups depending on the pathogenicity of the micro-organism, and the existence or not of a
prophylaxis or an effective treatment. Knowledge of the biological agents and their classification conditions
the preventative rules, notably the biosafety levels 1, 2, 3 or 4, required to handle them.
18
Table I. Group of risk: from lowest (1) to highest (4) (48)
Therapeutic bacteriophages belong to group 1 of the classification of biological agents (28,44). They are
not directly pathogenic to humans. However, they can transfer virulence factors and toxins to another host:
the bacteria. Through this transfer, they increase the virulence of the bacteria. Thus, bacteriophages could
be vectors of disease, but an intermediary host is required. The main risk of bacteriophages should thus be
considered in relation to the environment.
The level of biological safety is also dependent on the bacterial strain used during the different stages of
producing the bacteriophages (isolation, selection, amplification, purification). A bacteriophage production
with an E. coli O157:H7
2
-type bacteria should be undertaken in a contained laboratory of biosafety level 2,
whereas a production with a non-pathogenic strain of E. coli should be undertaken in a contained laboratory
of biosafety level 1.
2
E. coli O157:H7 is one of the serotypes of enterohaemorrhagic E. coli or EHEC. EHECs colonise the digestive tract then liberate toxins
(Shigatoxins) which can induce intestinal, renal or cerebral vascular lesions. EHECs can thus result in various troubles, ranging from benign
diarrhoea to more serious forms such as haemorrhagic diarrhoea and/or severe renal impairment known as haemolytic uraemic syndrome (HUS)
mainly in young children or thrombotic microangiopathy (TMA) in adults.
19
The different sources of therapeutic bacteriophages in France and
Europe
The bacteriophages currently available do not cover all of the pathogenic bacterial strains likely to be found
in clinical practice in situations of multi-resistance. There is a limited diversity and number of
bacteriophages available in the banks (phage libraries). Yet, it should be recalled that producing
bacteriophages is a long and complex process, broken down into several stages: isolating the phage of
interest (except if the relevant strain already exists in a phage library), then characterising, qualifying and
producing it in sufficient quantities.
The different sources of bacteriophages available in France and Europe are presented below (non-
exhaustive list).
Bacteriophages from the Pherecydes Pharma® company (France)
Pherecydes Pharma® is a French start-up created to develop therapeutic solutions based on bacteriophages
to treat bacterial infections. Pherecydes Pharma® has thus developed technologies to select and produce
bacteriophages that can act against drug-resistant and multidrug-resistant bacteria and now possesses a
limited collection of bacteriophages. The processes they use to isolate, characterise, purify and produce the
bacteriophage suspensions are known to the authorities. Pherecydes Pharma® carries out a certain number
of controls, mainly microbiological, on the bacteriophage suspensions, in compliance with the
microbiological methods described in the European Pharmacopoeia (9th edition), and they provide a batch
analysis certificate.
Since no specific Monographs for bacteriophages currently exist in the European or French Pharmacopoeia,
these controls are not specific to the quality attributes of bacteriophages, but are mainly intended to verify
the general requirements relating to the route of administration chosen.
Pherecydes Pharma® produces characterised bacteriophage suspensions, provisioned depending on the
status of the raw materials. The stages of preparation (reconstitution, galenic formulation adapted to the
route of administration) and control of the pharmaceutical quality of the magistral preparation of
bacteriophages should thus be undertaken by the hospital pharmacist of the IHP in order to enable an
administration in humans.
Pherecydes Pharma® performs the phagogram at its own premises.
Bacteriophages from the Queen Astrid Military Hospital (Belgium)
Researchers at the QAMH in Brussels have been studying bacteriophages since 2006. They took part in the
multi-centre, international clinical trial, Phagoburn®, which assessed the efficacy and tolerance of
bacteriophages in the treatment of E. coli or Pseudomonas aeruginosa infected wounds in serious burn
victims.
20
This organisation possesses a bacteriophage bank of diverse origins: Eastern Europe, Germany, etc. The
bacteriophages are sequenced, characterised and then stored at their premises.
The researchers isolate, purify and characterise the bacteriophages in compliance with the Belgian National
General Monograph of Bacteriophages validated by AFMPS in January 2018 (2731). The Monograph
gives recommendations and details the technical points to take into account when isolating, characterising
and purifying a type of bacteriophage. All these stages are undertaken within a health establishment, in a
controlled environment (microbiological and air particulate quality control) in order to limit any microbial-
particulate contamination and degradation of the bacteriophages. The QAMH guarantees that the
bacteriophages characterised are lytic and undertakes a certain number of controls (2731).
Currently, QAMH is producing bacteriophage active pharmaceutical ingredients (API), in the form of
suspensions or lyophilisates, which can be used as components or “raw materials” of a magistral or hospital
preparation.
Bacteriophages from academic research laboratories (France)
The Pasteur Institute in Paris possesses bacteriophages that are mainly active on strains of E. coli or P.
aeruginosa. They are capable of performing a phagogram at their premises with the bacteriophages in their
bank. The purification is carried out following a specific working method. They guarantee that the
bacteriophages characterised are lytic. The bacteriophages in their possession cannot be used directly in
clinics, since the stages of producing in quantity and controlling the suspensions obtained remain to be
completed.
Bacteriophages from DSMZ (Deutsche Sammlung von Mikroorganismen
und Zellkulturen GmbH, Germany)
The DSMZ centre (a German collection of microorganisms and cell cultures) is part of the Leibniz Institute,
located in Germany. This centre has a large collection of yeasts, bacteria, fungi and bacteriophages. The
research team carries out the stages involved in characterising the bacteriophages in their collection. They
guarantee that the bacteriophages characterised are lytic. They do not however carry out the stages of
producing in large quantities, purification and pharmaceutical quality control of the bacteriophage
suspensions. Thus, their bacteriophages cannot be used directly in the clinic, and they would need to find a
sub-contractor licensed to undertake the production and control stages before these suspensions can be used
in clinical practice.
Bacteriophages at the Eliava Institute (Georgia)
This institute located in Tbilisi, Georgia is entirely dedicated to bacteriophages and their therapeutic
applications. Despite the arrival of antibiotics, the Georgians have never stopped practicing phagotherapy.
However, very little data about this use has been published. This institute has a large collection of
bacteriophages that they use to treat infections due to multidrug-resistant bacteria. Unfortunately, we have
21
no data at all on the characterisation of the bacteriophages in their phage library, nor on the production and
control stages for the suspensions prepared for clinical use.
Bacteriophage from the Ludwik Hirszfeld Institute of Immunology and
Experimental Therapy (Poland)
This institute, founded in 1952 and formerly known as the medical microbiology institute, is a research
centre located in Wrocław, Poland. The main founder was the microbiologist and immunologist Ludwik
Hirszfeld. It is one of the rare research centres in Eastern Europe, alongside the George Eliava Institute, to
have developed the field of phagotherapy. This research team has developed methods of isolating
bacteriophages and producing bacteriophage suspensions for clinical use. The institute has its own
production unit, opened in 2005. However, the therapeutic use of bacteriophages at this institute is
undertaken within the legal framework of the Helsinki declaration. As such, in that it is not yet an approved
therapy, it remains classed as experimental therapy. Little data has been published on the therapeutic use
of isolated and amplified bacteriophages by this institute. Unfortunately, we also have no data on the
characterisation of the bacteriophages, nor on the production and pharmaceutical quality control stages for
the suspensions used at this institute.
Bacteriophages from the AmpliPhi Biosciences Corporation® (United
States)
This is a biotechnology company whose main lines of research and development are to develop therapeutic
solutions based on bacteriophages and peptides to treat multidrug-resistant bacterial infections. The
production site is located in Slovenia. A certain number of bacteriophage cocktails produced by AmpliPhi®
have been authorised for compassionate use in Australia and the United States (4953). However, like for
the other centres presented above, we have no data qualifying the bacteriophages contained in their phage
library, nor describing the production processes for the suspensions for clinical use, nor the quality control
procedures and criteria.
22
The terms of access to phagotherapy
in France
The clinical situations potentially eligible for a treatment by
bacteriophage
The decision to initiate a treatment by bacteriophages should be taken by a multi-disciplinary hospital team:
hospital doctor, hospital pharmacist, a leading infectiologist in the field, and if need be, a surgeon. Each of
the parameters, such as the surgical approach, the dosage and the medical devices (MD) used, should be
discussed beforehand in a multi-disciplinary team board meeting, in order to optimise the treatment efficacy
and ensure the administration is safe.
Considering the growing interest in bacteriophages from the scientific and medical community and from
patients, at its CSST on 24/03/2016, ANSM sought to define its position as to the clinical situations that
justify early access to bacteriophages (17).
A consensus of the criteria justifying a situation of need applicable to all types of infection for early access
to bacteriophages has been arrived at:
- a threat to the patient’s life or functional prognosis;
- a situation of a therapeutic "dead end" (meaning a “salvage situation”);
- a monomicrobial infection.
Additionally, considering the current limitations in data able to underpin the use of bacteriophages, it was
agreed that:
- a collegiality with the clinical team is necessary to decide on the therapeutic approach, with
ANSM to assist the ad-hoc experts;
- it is necessary to submit the results of a phagogram that identifies a bacteriophage compatible
with the infection to be treated, before deciding to undertake the treatment (requiring a phagogram
to be performed in a short timeframe). The phagogram tests the different bacteriophages
“available” to produce an appropriate bacteriophage suspension. In the case where it reveals that
no bacteriophage in the phage libraries analysed is appropriate, then recourse should be taken to
the first stage of identifying and selecting a new strain of bacteriophages.
The criteria defined by the CSST describe the ideal clinical situation. Nevertheless, 3 factors that have
influenced the evolution of some of these criteria: the clinical experience on bacteriophages acquired over
the last 2 years, the limited availability of bacteriophage cocktails and the remaining uncertainties about
this therapy. Thus, the practical cases that could justify early access to bacteriophages have been defined:
23
- A bacterial resistance to antibiotics: a plurimicrobial infection with for example a toto-resistant
strain and a strain that responds to antibiotic treatment.
The notion of a monomicrobial infection should thus be reconsidered. In some cases, the
infection can be polymicrobial, but only one bacteria will be targeted by the bacteriophage
suspension since it is the cause of the infectious morbidity. The other bacteria present in the infected
site can be treated with an appropriate antibiotic. For illustrative purposes, in joint infections,
around 10% of prostheses infections are polymicrobial;
- An infection where the bacteria is still sensitive to some antibiotics, but where the morbidity
linked to the treatment becomes unacceptable (impairment to renal function, allergy, etc.);
- A clinical inefficacy: an infection that is hard to eradicate which is evolving towards chronicity.
This is the case for example of bone infections, where the bacteria can be sensitive to antibiotics.
The pharmacokinetic characteristics of the antibiotic play a key role in eradicating the bacteria.
However, in some situations, it is difficult to reach effective concentrations because of a low
diffusion of the antibiotic, for example, in the site of interest. Additionally, the bacteria can produce
a biofilm (on orthopaedic equipment in particular) in order to protect itself from the environment.
They then become inaccessible to the antibiotics and elude the host’s immune system. Interestingly,
some bacteriophages have the ability to hydrolyse the bacterial polysaccharides that make up these
biofilms (5456).
Information from ANSM on the project to use bacteriophages for
therapy
The hospital doctor in charge of the patient and the hospital pharmacist should inform ANSM of their
intention to administer a bacteriophage suspension to a patient. The medical team should present the
case with:
- a detailed history of the disease
- a detailed and exhaustive history of the antibiotic treatments,
- the identification of the bacteria with the antibiogram,
- the route of administration and dosage envisaged for the bacteriophage suspension,
- the conditions for handling the bacteriophage suspensions: bacteriophage
lyophilisates/suspensions should be handled (dilution, syringe preparation, etc.) in a
controlled environment: microbiological quality and air particulate controls.
Indeed, the pertinence of the request for bacteriophages should be assessed in terms of the clinical
data and the data available in the literature before performing the phagogram. ANSM supports the
hospital teams in the process of assessment, with the possibility to consult external experts on an ad-
hoc basis.
24
Contacts: Products Management service responsible for vaccinations and anti-infective drugs in hepato-
gastroenterology, dermatology, gene therapy and rare metabolic disorders (INFHEP), at the MALINF
centre.
Organization chart of INFHEP centre: https://www.ansm.sante.fr/Les-contacts-utiles-a-l-ANSM -
VACCIN
Director: Nathalie.MORGENSZTEJN@ansm.sante.fr
Determining the sensitivity of the bacterial strains to bacteriophages
and the efficacy in forming lysis plaques
The phagogram
In the same way as antibiograms are performed to select the antibiotics that act on the bacteria responsible
for the infection, it is possible and necessary to perform a phagogram. Since the bacteriophages are specific
to a bacterial species, or even only a few strains of this species, the main risk of administering a
bacteriophage without checking its activity via a phagogram is lack of efficacy.
The phagogram aims to test the sensitivity of the bacterial strain vis-a-vis one or several bacteriophage
candidates that are kept in a phage library. It demonstrates in vitro that some bacteriophages present a
bactericidal effect on the bacterial strain. Analysing the results of this test allows one to identify and select
the bacteriophages that are specifically active against the bacteria in question in order to develop, if judged
useful, a “made to order” therapeutic cocktail of several different bacteriophages. Using several
bacteriophages simultaneously on a single bacterial strain can result in a phenomenon of potentiation
(synergy of action) and a reduction in the risk of the appearance of resistance (2).
Performing a phagogram is relatively similar to the process for an antibiogram (spot test technique): a
bacterial culture of the strain of interest (clinical isolate from the patient) is spread out on a Petri dish
containing a solid nutrient medium. Once the surface is dry, a few microlitres of different bacteriophage
suspensions (1 to 2 µL) are deposited. The results are read after an incubation period (4 to 12 hours at
37°C). The activity of a bacteriophage on the sensitive bacteria leads to a lysis of the latter, characterised
by the appearance of lysis “plaques” (clear zones on the culture medium) in the place where the drop of
bacteriophage was deposited (Figure 3.). The presence of a lysis plaque thus attests to the sensitivity of the
bacterial strain to the bacteriophage tested (57,58).
25
Figure 3. View of the spot test technique, the same bacteriophage is tested on 20 different bacterial strains
(2)
The phagogram makes it possible to roughly identify the bacteriophages active on a given bacterial strain.
It gives an initial indication of the efficacy of the available bacteriophages on the bacterial strain. However,
simply noting a bacterial lysis on the phagogram is not enough to confirm, or, even less so, quantify the
efficacy of the bacteriophage(s). It can only be used to determine whether the bacterial strains are sensitive
to the bacteriophages (58).
It is therefore necessary to then perform a titration and a study of the kinetics of lysis.
Titration and kinetics of lysis
The titration stage serves to:
- demonstrate the individual lysis plaques, the only evidence of the presence of a viral infectious
cycle in the bacteria;
- define the concentration of active bacteriophages causing a bacterial lysis, or the number of active
bacteriophages contained in the suspension, in Plaque-Forming Units (PFU)/mL.
During the titration, different dilutions (factor 10) of a suspension containing a bacteriophage clone to be
titrated are applied and tested on the host bacterial strain if interest (Figure 4.). This technique makes it
possible to visualise the lysis plaques individualised to different concentrations of bacteriophages. The titer
is deduced from the number of lysis plaques (PFU/mL), with each bacteriophage potentially producing a
lysis plaque.
Lysis zone by
the phage
26
Figure 4. Example of the titration of 2 solutions of bacteriophages 536-P1 and 536-P7, on the E. coli 536
strain Serial dilutions of 2 bacteriophages (536-P1 and 536-P7) are deposited on strain 536 (2).
Different methods exist to determine the titer of the bacteriophage suspension:
- Double agar overlay method (59);
- Direct plating plaque assay (60);
- Small drop plaque assay system (61).
The titration makes it possible to calculate the efficiency of plaquing (EOP) (57,58). This is the efficiency
of the bacteriophages in forming lysis plaques in an agar medium, i.e. the bacteriophages’ efficiency in
lysing the bacteria. The EOP is based on a comparison of the activity of a bacteriophage suspension on the
patient’s strain versus the strain that made it possible to isolate the bacteriophage (host-strain). It is obtained
by titrating a suspension of a bacteriophage X:
- on a bacteria A of interest, for example the strain responsible for the infection;
- on the reference bacteria B: the primary host of the bacteriophage, meaning the one that made it
possible to isolate the bacteriophage (57,58).
Successive dilutions of the bacteriophage suspension are spread, on one hand, on a dish containing a
bacterial culture of the patient’s strain and, on the other hand, on a dish containing the primary host. After
an incubation of several hours at 37°C, it is then possible to visualise the number of viral particles for a
given dilution and to then deduce the concentration of active viral particles in the suspension (57,58).
The bacteriophage concentration obtained on the patient’s strain, i.e. the titer, can be lower than that
obtained on the host-strain. This reflects a lesser efficacy of the bacteriophage in destroying the patient’s
strain compared to the host-strain.
Each bacteriophage solution is diluted in a series of factor 10,
starting at the highest and going down (10-1 to 10-7), and 4 µL
of each dilution is deposited in triplicate on an agar that has
been covered beforehand with the 536 strain to be tested.
By counting the lysis plaques individually observed in the
higher concentration dilutions, it is possible to determine the
number of infectious viral particles per mL of solution
(PFU/mL).
27
The EOP is thus the relationship between these two concentrations:
EOP = titer on A / titer on B.
M. Khan Mirzaei and A. S. Nilsson defined the intervals in function to the EOP values:
- value above or equal to 0.5: high efficiency in forming lysis plaques;
- value between 0.1 and 0.5: medium efficiency in forming lysis plaques;
- value between 0.001 and 0.1: low efficiency in forming lysis plaques: the bacteriophage may not
be very effective;
- value below or equal to 0.001: insufficient efficiency: the bacteriophage may be ineffective (58).
Studying the kinetics of lysis makes it possible to assess the bacteriophage(s) activity in liquid media
(Figure 5) (2). A bacterial culture is mixed with a bacteriophage suspension. Readings of the optic density
are made at different times. By monitoring the turbidity of the medium, it is possible to confirm the absence
of bacterial growth.
Figure 5. Kinetics of lysis of a strain X by bacteriophages A, B and C.
Performing a phagogram, and determining the parameters such as the titer of the preparation and
the EOP, constitute the preliminary stages to administering bacteriophages. At the CSST meeting on
phagotherapy held on 24/03/2016 by ANSM, it was agreed that, considering the current limitations in data,
Bacteriophages A and B are effective on the bacterial strain of interest, they have caused a bacterial
lysis, as demonstrated by the absence of a rise in the turbidity of the medium after an initial phase
of bacterial growth at H3.
Bacteriophage C is only temporarily active and does not succeed in counteracting the bacterial
growth over the long term.
28
it is necessary to submit the results of the phagogram before a decision can be taken to proceed to treatment
(17). Therefore, the presumed efficacy of a phagotherapy prior to being administrated should, as an
imperative, take into account the activity of the bacteriophage(s) on the isolated bacterial strains in the
subject.
These tests can be performed by the companies marketing the bacteriophages. To do this, the patient‘s
strain must be sent following the required transport conditions (cf. V.1). In the case where the laboratory
marketing the bacteriophage preparations does not perform the phagogram, it is possible to do this at the
microbiology laboratory. However, such an undertaking requires a dedicated space and rigorous staff
training. Indeed, performing the phagogram (stages of dilution, pipetting, standardising the bacterial density
at the start, etc.) as well as reading the results (analysing the lysis plaques) should be undertaken by an
experienced person, in order to increase the sensitivity of the test and to limit any interpretation bias and
the risk of errors.
Assessment of the pharmaceutical quality of bacteriophage raw
materials and the stages of preparation pharmaceutical quality
control of finished preparations
Assessment of the pharmaceutical quality of bacteriophage raw materials
It should be recalled that the main tests for pharmaceutical quality of the raw materials/active substances
of the bacteriophages should be undertaken by the industrial developer/manufacturer, in compliance with
the regulation in force that requires all suppliers to submit a control report on the raw material being
supplied. The IHP pharmacist should critically analyse this control report to decide whether he/she can take
the responsibility to use this raw material/active substance in Humans.
According to the specific texts on the active principles of bacteriophages, i.e. the lyophilisates/suspensions,
such as the Belgian National General Monograph of Bacteriophages, and the general texts such as the
European General Monograph of Pharmacopoeia 9th edition: “Substances for pharmaceutical use” (2034)
and the other existing texts, a list of requirement relating to the production and quality control of
bacteriophage raw materials has been drawn up (27,28,31,32,3840).
For the controls carried out on the raw material/active substance, the following items should be detailed:
the references used (example European pharmacopoeia, national monograph), the analytical methods, and
the acceptance criteria. These controls should be carried out in a perfectly controlled environment in terms
of microbial, particulate and pyrogen contamination (controlled atmosphere area CAA, etc.), in compliance
with the regulation in force (European pharmacopoeia, GMP etc.).
29
The information and controls listed below are required to assess the pharmaceutical quality of raw
material/active substances, and should be detailed in the control report:
the references used to characterise the bacteria used to isolate the bacteriophage: European
directive on tissues and cells (EUTCD) (3335), the guidelines CHMP/ICH/294/95 Q5D (36), the
text of the Food and Drug Administration (FDA): Characterization and Qualification of Cell
Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious
Disease Indications” (37);
the manufacturing reference used: GMP for example;
details of the manufacturing process for the active substance/raw material: precise
manufacturing process with a list of the raw materials used and their specifications, including the
bacteriophage banks and bacteria used in the production;
the characterisation of the bacteria used in the production: identity, viability, absence of
pathogenic toxin production, genotype, purity;
the characterisation of the bacteriophages: identity (family, sub-family, genre, species), lytic
character, structure by electronic microscopy, genome sequencing, verification of absence of genes
involved in the process of viral incorporation in the bacterial genome, list of bacteria sensitive to
the bacteriophages.
As explained in the preceding chapters, it is essential to use characterised lytic bacteriophages in
therapy, and to verify the absence of temperate bacteriophages in the lyophilisate/suspension
produced by the manufacturer (28,44). Investigations should be conducted, notably through
genome analyses that sequence the DNA-RNA of the bacteriophages present in the
lyophilisate/suspension (raw material) in order to limit the risk linked to the presence of temperate
bacteriophages which are potentially and indirectly pathogenic to humans.
It should be noted that the administration of bacteriophages whose characterisation is incomplete
(considering the continued uncertainties about some genes of antibiotic resistance or coding for
bacterial toxins) could result in the transfer of virulence factors to the bacterial strains responsible
for the infection.
A comparison should be made of the isolated bacteriophage’s genome with that of an identified
close homologue and a search should be done for genes coding for integrases, transposases or
recombinases, and virulence genes (29,39).
These results will provide information on and confirm:
the identity of the bacteriophage;
its lytic character;
the absence of a possibility for the bacteriophage’s genome to incorporate
in the bacterial genome.
Other methods that make it possible to confirm the absence of temperate bacteriophages: “prophage
induction methods”, such as the mitomycin C or ultraviolet (UV) irradiation tests (31,62,63). It is
30
also necessary to verify the absence of genes coding for bacterial toxins (the toxin for toxic shock
syndrome, the exfoliative toxin and the Panton-Valentine leukocidin for example if looking into
phages targeting Staphylococcus) or the genes for antibiotic resistance;
the purity of the raw material/active substance: any impurities originating in the manufacturing
process should be reduced to a minimum: bacterial DNA, bacterial debris, bacterial proteins,
additives from the manufacturing process, solvents, etc. For some impurities, the maximum
tolerated residual concentrations have been defined, such as for example:
- According to the World Health Organisation (WHO) recommendations on
biotherapies, the concentration of residual DNA from the host cell should be under
10 ng/dose. By analogy, this concentration seems acceptable as the maximum
tolerated value of residual bacterial DNA (40).
- Some additives are already being used as excipients in the formulation of drugs
with an MA and for which we know the tolerance profile. Thus, we simply have
to verify the maximum tolerated concentration of this molecule in a drug and
compare this to that present in the bacteriophage preparations.
the concentration in endotoxins of the raw material/active substance: the limit for endotoxins
depends on the dosage, the route of administration and the patient’s body mass. In the European
Pharmacopoeia 9th edition, two parts deal with bacterial endotoxins: the Monograph 2.6.14
“Bacterial Endotoxins” and the general chapter 5.1.10. “Recommendations for performing bacterial
endotoxin tests”. For some routes of administration, the maximum authorised limit in endotoxins
is specified. For example, the Eur. Ph. requires a bacterial endotoxin test for parenteral formulations
by IV route with a limit of 5 IU (International Units)/kg (patient body weight)/h (if perfusion > 1
hour);
the sterility of the raw material/active substance (non-mandatory): sterility test. Monograph
2.6.1 of the European Pharmacopoeia 9th edition describes the working method. It is possible to
perform a sterilising filtration with a 0.22 µm filter and a SteritestTM of the bacteriophage
preparation upon reception.
in the case of a non-sterile raw material/active substance a microbiological test should be
done. Monographs 2.6.12 and 2.6.13 of the European Pharmacopoeia 9th edition describe the
working method for this test. These are the Total Aerobic Count (TAC) and the Total Yeast and
Mould Count (TYMC) tests. The acceptance criteria in function to the route of administration are
described in the general chapter 5.1.4 of the European Pharmacopoeia 9th edition. In the case of
non-sterile active substances for pharmaceutical use, the acceptance criteria for microbiological
quality are:
o TAC 103 colony-forming unit (CFU)/g or CFU/mL
o TYMC 102 CFU/g or CFU/mL;
31
the titer of the raw material/active substance: this titer should be determined according to a
standardised procedure (cf. IV.3.b);
the stability data for the raw material/active substance;
the extractible volume of the raw material/active substance;
the processing of the raw material/active substance;
the pH of the raw material/active substance: close to neutral pH (6.0 to 8.0).
It should be recalled that it is the manufacturer’s responsibility to perform the enumerated tests cited above
on the substance it supplies. The hospital pharmacist then controls the information present in the quality
file.
It is possible that some of this information is not provided by the manufacturer, or is incomplete or given
with a degree of uncertainty. In this situation, each item of information should be evaluated in terms
of:
- the route of administration chosen,
- the dosage,
- the formulation,
- the situation of the patient taking into account the estimated risk/benefit balance.
If necessary, the hospital pharmacist should request additional information from the manufacturers, such
as for example the residual concentration of impurities, or additional data on the characterisation of the
bacteriophages.
The pharmacist could also request a peer evaluation from external experts on the pharmaceutical quality of
the raw material. Indeed, while some tests, such as ascertaining the pH, the concentration of endotoxins, or
the sterilising filtration, can easily be carried out at an IHP, it is not the pharmacist’s role to sequence the
DNA-RNA of bacteriophages present in the raw material so as to ensure there are no temperate
bacteriophages. ANSM supports the hospital teams in the process of assessing the quality of bacteriophage
lyophilisates/suspensions (cf. ANSM contacts, page 24)
In the case of active substances or bacteriophage preparations manufactured by QAMH: these are produced
at a health establishment in compliance with the Belgian National General Monograph of Bacteriophages.
Thus, the pharmaceutical quality is assessed using the data from the batch analysis certificate provided by
QAMH
Appendix 3 and Appendix 4 summarise the control procedures for RMPUs depending on the supply circuit.
32
Preparation and pharmaceutical quality control
After validating the pharmaceutical quality of the raw material using the control report supplied by the
manufacturer (see below), the hospital pharmacist should carry out the preparation stages
(reconstitution, galenic formulation adapted to the route of administration) in compliance with the GPP of
the IHP, and a pharmaceutical quality control of the magistral (or hospital) preparation of the
prepared bacteriophages, in order to enable administration in humans.
Preparation: optimising-defining the galenic formulation, producing the
pharmaceutical form
The pharmacist is responsible for the decision to make the preparations (19). The IHP pharmacist
should undertake an assessment of the added value and the technical feasibility of the preparation.
The added value is estimated by considering for each preparation:
- the pharmaco-therapeutic benefit;
- the best possible acceptance to strengthen compliance;
- a risk/benefit appraisal.
The technical feasibility is estimated by considering for each preparation:
- the presence of defined general procedures and working methods;
- the presence of compliant material and premises to produce the pharmaceutical form;
- the presence of staff trained to analyse the formula of the preparation (19).
The preparation stage in the pharmaceutical sense of the term, meaning producing the
pharmaceutical form, reconstitution and dilution, using bacteriophage raw materials produced
in sufficient quantities by a manufacturer, should be carried out at an IHP, under the responsibility
of a pharmacist, in compliance with the GPP (19). The specific measures to protect the environment
and workers should be applied (cf. V.2.b).
The preparation stages can include:
- optimising the formula so as to ensure the best bioavailability;
- producing the pharmaceutical form (dilution, reconstitution, mix of several bacteriophage
suspensions, preparation in syringes, pouches) so as to “facilitate” the administration of the
bacteriophage suspension (28,31,6470).
In this case, the pharmacist produces a magistral (or hospital) preparation of bacteriophages using the
raw materials (lyophilisates/suspensions) produced by a manufacturer/developer.
The formulation of a preparation is fundamental. It makes it possible to define the physico-chemical
and galenic characteristics of a preparation in relation to the route of administration chosen, with the
33
aim of optimising the pharmacokinetic parameters of a substance, while guaranteeing the efficacy and
safety of the treatment. In view of the pharmacokinetic characteristics of bacteriophages, one of the
objectives of the formulation is to ensure optimum bioavailability of the bacteriophages at the
infectious site in order to enable the viral amplification.
The stability of the preparation should also be under control. The route of administration should thus
make it possible to concentrate the bacteriophages at the infected site while remaining compatible with
the pharmaceutical characteristics of the bacteriophage cocktails.
Thus, local administration of bacteriophages could be considered as the safest method according to the
available data. The advantage is that the quantity of bacteriophages delivered to the infection site is
known. However, such a route of administration can not be used to treat systemic infections, for which
the intravenous (IV) or oral route could be envisaged. But there is still some uncertainty as to the
quantity of bacteriophages reaching the systemic level by oral administration or through the mucous.
Likewise, since the distribution volume of bacteriophages is badly understood, IV administration would
be the best approach for direct information on the quantity of bacteriophages that reach the blood
circulation, but we still cannot know the quantity of bacteriophages at the infection site.
During the CSST of 21 March 2019, all the experts agreed to exclude the oral route. Indeed, it is difficult
to predict a sufficient level of activity on the bacterial target via this route of administration which
involves intestinal absorption with possible loss/degradation. It is also agreed that the intrathecal route
should a priori be excluded, but it could be considered subject to sufficient guarantees of the quality
(notably microbiological and apyrogenicity) of the bacteriophage preparations (13).
Different risks should be considered during the preparation and administration stages of the
bacteriophage suspension:
- the risk of adsorption of the preparation material and the MDs used for the preparation and
administration of bacteriophages;
- the risk of bacteriophage degradation at the administration site, during the administration, this risk
being specific to the route of administration;
- the problem of bacteriophage inactivation by gastric acidity in the case of oral administration;
- the sensitivity to pH, temperature;
- the concentration in endotoxins, the sterility of the preparation (3,9,27)etc.
Bacteriophages are viruses, and are consequently sensitive to temperature, acidic conditions (pH = 4
and below) and alkaline conditions (pH =11 and above) (44).
Thus, the hospital team should determine the route of administration, the composition of the solution
in active principle and excipients (titer/concentration…), the galenic form and the MDs used during the
administration (taking into account the MDs’ physico-chemical characteristics and techniques). It
34
should be noted that the dose of bacteriophages to be administered will depend on the route of
administration envisaged and the bacterial inoculum.
As an example, in the case of a nebulization of the bacteriophage suspension, one should consider (64
66):
- the nebulization system: pneumatic nebulizer, vibrating plate nebulizer (it is recommended to avoid
ultrasonic nebulizers and to use in preference vibrating plate nebulizers for which the perspective
in terms of tolerance [for the bacteriophages] appears to be more positive);
- the nebulization circuit: the interface with the patient, meaning the type of connector, the physico-
chemical characteristics and techniques of the piping, i.e. the materials, length and diameter;
- the size of the nebulized droplets;
- the yield of the bacteriophage titer on exiting the nebulizer;
- the duration of the nebulization as regards the stability data of the bacteriophage suspension;
- the addition of excipient(s): it is recommended that tensioactive agents are added to the preparation
for nebulization.
Pharmaceutical quality control of finished bacteriophages preparations
produced by the IHP
The assessment of the pharmaceutical quality of finished pharmaceutical bacteriophage
preparations (magistral or hospital preparations) is an essential prerequisite to administration.
Indeed, the pharmacist should ensure that the different stages of preparation, and the administration
stage, do not result in a degradation alteration of the active substance such as a reduction in the
titer, an inactivation of the bacteriophages, or any other alteration of the pharmaceutical quality
(II.4.1). Additionally, he/she should demonstrate that the development of the pharmaceutical form
conforms to the indication (dose…).
Based on the data in the literature regarding bacteriophage preparations and notably from the
QAMH team, a list has been drawn up of the quality controls that should be performed on the
(magistral or hospital) preparations of bacteriophages prepared by the IHP (30,31,39). For each
control, the following items should be detailed: the references used (e.g. European Pharmacopoeia,
national monograph), the analytical methods, the acceptance criteria. These controls should be
carried out in a perfectly controlled environment in terms of microbial, particulate and pyrogene
contamination (controlled atmosphere area CAA, etc.), in conformity with the regulation in force
(European pharmacopoeia, GPP etc.).
35
The information and controls listed below are essential in the assessment of the pharmaceutical
quality of the finished bacteriophage preparations prepared by the IHP:
- the titer of the preparation. Empirically, it is agreed that the titer of a bacteriophage
preparation should be at least equal to 108-109 PFU/ml (71). This titer should be determined
following a standardised procedure (cf. IV.3.b);
- the MOI (multiplicity of infection), the relationship between the number of bacteriophages
and the number of bacteria present, is one of the parameters that condition the level of efficacy
of the viral infection. Yet, although this is easily controlled in vitro, it is very difficult to
precisely define it in vivo. It is agreed empirically that the MOI should fall between 10 and 0.1
(10,72);
- the stability data: the stability of the mix if excipient(s) are added, stability in the final
container, during the administration;
- depending on whether the preparation is sterile or not: the requirements relating to the
sterility of the preparation depend on the route of administration and the product (cf. Appendix
5) (66).
- according to Monograph 2.6.1 of the European Pharmacopoeia 9th edition the preparation
should be sterile:
notably for an administration by parenteral, ophthalmic or intra-mammary
route and some preparations for inhalation or intrauterine irrigation,
where there is a high risk of the bacteriophages passing into the system
(for example: integrity of the skin barrier seriously damaged in the case of
serious skin damage or deep wound).
In the case of a sterile preparation: sterility test. Monograph 2.6.1 of the European
Pharmacopoeia 9th edition describes the working method. Performing a sterilising filtration
with a 0.22 µm filter and a SteritestTM of the bacteriophage preparation upon reception can be
envisaged;
In the case of a non-sterile preparation: microbiological control of non-sterile
preparations. Monographs 2.6.12 and 2.6.13 of the European Pharmacopoeia 9th edition
describe the working method for this test. These are the Total Aerobic Count (TAC) and the
Total Yeast and Mould Count (TYMC) tests. The acceptance criteria in function to the route
of administration are described in the general chapter 5.1.4 of the European Pharmacopoeia
9th edition.
- pH: close to neutral (6.0 to 8.0);
- visual appearance;
- extractible volume;
- the processing.
36
Each item of information should be evaluated in terms of:
- the route of administration chosen;
- the dosage;
- the formulation;
- and the situation of the patient taking into account the estimated risk/benefit balance.
The Annexe details the requirements relating to the pharmaceutical quality of finished preparations
depending on the route of administration, in compliance with the European Pharmacopoeia 9th
edition.
The pharmacist could also request a peer evaluation from external experts on the pharmaceutical
quality of the preparation, or could outsource some controls (example: titration, etc.).
ANSM supports the hospital teams in the process of assessing the quality of bacteriophage
lyophilisates/suspensions (cf. ANSM contacts, page 24)
Assessment of the administration plan for therapeutic bacteriophages
A collegial discussion of the case should be held with the clinical team in charge of the patient and ANSM,
with the possibility of requesting advice from ad-hoc external experts. A rigorous assessment:
- of the clinical case;
- of the route of administration of the bacteriophages and the dosage envisaged,
- of the pharmaceutical quality of the bacteriophage preparations available
should be carried out.
To do this, the assessment should be based on data from the literature, the clinical and therapeutic history
of the patient, the microbiological data, the phagogram results (once obtained), the data on the quality of
the bacteriophage preparations. The therapeutic use of bacteriophages should be considered in terms of the
risk/benefit balance. It should only be planned if using bacteriophages is likely to be of benefit to the patient.
It is necessary to obtain the free, enlightened, formal, written consent of the patient, or the person of
confidence, or the holders of parental authority in the case of a minor. The expected benefits and foreseeable
risks should be clearly and truthfully explained to the patient and/or the family.
Associating therapeutic bacteriophages and antibiotics
Currently, the use of bacteriophages is empirical, no study carried out following the required
methodological quality standards has demonstrated the efficacy of bacteriophages used alone to treat
bacterial infections.
37
When used for therapy, bacteriophages should be used to support the action of antibiotics. It is thus essential
to associate a bacteriophage administration with antibiotics (continuation of the antibiotic therapy
previously initiated) when this presents a benefit or efficacy, even one considered minimal in the case of
antibiotic resistance.
It appears that alongside the bacterial lysis directly provoked by the bacteriophages, they could also
contribute to improving the efficacy of the antibiotics. In the literature, some studies have demonstrated a
synergy between bacteriophages and antibiotics (29,54,73).
Finally, because of the small spectrum of action of bacteriophages, the other species present in the infected
site should be covered by an appropriate antibiotic therapy if need be.
Currently the fact remains that the role of bacteriophages in relation to antibiotics has not been defined.
The strategy of using bacteriophages depending on the indications must still be validated:
- as a complement to antibiotic therapy? to surgery?
- should bacteriophages be used alone in the particular case of infections caused by multidrug-
resistant bacteria?
38
The practical conditions of use of
bacteriophages
Transporting and storing the different forms of bacteriophages
As indicated previously (cf.III.6.c), therapeutic bacteriophages belong to group 1 of the classification of
biological agents(28,44). Thus, when transporting them, the rules for transporting dangerous biological
products do not need to be applied (meaning those applicable to biological products classed as 2814, 2900
and 3373) (48).
Transporting bacteriophages should be secure and conform to:
- either the Good Distribution Practices for the active substances of medicinal products for human
use for the units of raw materials produced by the manufacturer;
- or the Good Distribution Practices for bulk medicinal products for human use for pharmaceutical,
magistral or hospital preparations, prepared by an IHP.
As an indication, bacteriophage suspensions should be stored at 2-8 °C (74,75). It is essential to monitor
the temperature during the transportation using a probe to guarantee the quality of the suspensions. The
transporter should be able to provide traceability for the temperature monitoring.
Bacteriophage suspensions (raw materials/ magistral preparations) can thus be stored in a refrigerated area
of the IHP, with no additional specific environmental protection measures.
Handling procedures for bacteriophages
The handling of bacteriophage lyophilisates/suspensions, during the preparation and pharmaceutical
reconstitution stages in IHP or when performing the phagogram, should be done in a controlled
environment: microbiological quality and air particulates controls. The premises and the material should
be designed and maintained to best suit the operations to be carried out and should enable all the potential
risks to the environment and workers to be minimised.
The handling of bacteriophages is covered by the European directive 2000/54/EC of the European
Parliament and Council of 18 September 2000 on the protection of workers from risks related to exposure
to biological agents at work (76).
39
The phagogram
The conditions in which the phagogram is performed depend on the virulence of the bacteria used to
isolate the bacteriophage of interest (used as a positive control) and that of the bacterial strain
(patient’s isolate) to be tested. In the vast majority of cases, this is a class 2 pathogen.
The phagogram should be performed under sterile conditions at a type II microbiological safety work
bench (MSW). The use of cones with filters is recommended to avoid contaminating the micropipettes.
The members of the CSST of 21 March 2019 stressed the importance of systematically performing a
phagogram, with a calculation of the EOP and the bacterial kinetics of lysis. They also discussed the benefit
of testing several isolates (of the same bacterial species) from one or several intraoperative samples to
investigate whether there are different phenotypes of bacterial sensitivity to the phages. The number of
isolates to test is still to be determined (13). However, it does not seem useful to calculate the EOP on a
strain where the phagogram using a spot test technique shows a resistance, the same applies to the kinetics
of lysis.
This stage does not have to be carried out under pharmaceutical responsibility, it can be done in a
microbiology laboratory. A phagogram is performed following a medical decision and by a medical biology
laboratory qualified to make this diagnosis. The biologist assumes the responsibility for this act which
includes taking the sample, performing the analysis, validating the results, and if necessary comparing them
with the patients’ clinical and biological data.
The preparation stages
The conditions under which the preparation stages are performed (optimisation of the formula,
reconstitution, etc.) depend on the requirements in terms of the pharmaceutical quality of the finished
product (sterility requirements, etc.).
Different preparation procedures are possible:
- a reconstitution stage when provisioning the bacteriophage lyophilisates;
- producing the pharmaceutical form, for example: mixing several suspensions in a syringe to obtain
a cocktail of several bacteriophages.
In the case of sterile preparations (application on severely damaged skin or in a joint cavity, preparation
for nebulization, cf. Appendix 5), the preparation stages should be carried out in a CAA, in specific
enclosure, in compliance with the GPP:
- either a closed environment, an isolator,
- or a type II MSW.
40
Following the GPPs, the isolator should meet class A requirements in terms of microbiological and air
particulate quality, the immediate environment should conform to the class D requirements of the BPP
(27,28,31).
If the environment is not an enclosed space, meaning a laminar airflow work bench (type II MSW), then
the immediate environment of the flow should meet the class B requirements in terms of microbiological
and air particulate quality, according to the GPP (27,28,31).
There are three main preparation procedures for sterile drugs:
- - terminal sterilisation;
- - sterilising filtration;
- - aseptic preparation.
However, terminal sterilisation is not possible since it would destroy the bacteriophages. Thus, if sterility
is required for the finished product, two processes could be used during the preparation, according to the
GPP:
- a sterilising filtration, with a filter type that is recognised as satisfactory and compliant with the
requirements of the European Pharmacopoeia 9th edition, i.e. a 0.22 m filter made of poly-ether-
sulfone (PES), choosing the model with the lowest dead space. Crucially, it should be noted that
cellulose ester/acetate filters should not be used since they can massively adsorb the phages;
- aseptic preparation (if and only if the starting material used is declared sterile, backed up by test
results).
The dress precautions are similar to those recommended when making sterile preparations (cf.
Table II).
Table II. Dress precautions when making sterile preparations
Isolator
MSW
Dress
Clean adapted clothing
- Bouffant cap
- Overshoes
- Non-sterile gloves
- Surgical mask
- Sterile shoes or boots
- Sterile gown tightened at the wrists
- Sterile gloves
- Mask FFP2
In practice, when handling bacteriophages, the instruments and surfaces can be cleaned and disinfected
with detergents biocide surface disinfectants, such as those routinely used in healthcare environments, of
Surfa’safe Anios type (cf. V.3).
The waste can be treated through the waste from care activities involving a risk of infection and related
risks channel (DASRI).
41
The hygiene and protection measures for handling bacteriophage
preparations
Considering the atypical nature of bacteriophages, it is strongly recommended that the following
members of the health establishment are informed of the bacteriophages administration: the
management, the Operational Hygiene Team (OHT) and the Director of Nursing (DN).
Bacteriophages are naked viruses, and are consequently sensitive to temperature (a temperature above
80 °C can inactivate the bacteriophages), to acidic conditions (pH = 4 and below) and alkaline conditions
(pH = 11 and above) (44).
In practice, when handling bacteriophages, the instruments and surfaces can be cleaned and disinfected
with detergents classic biocide surface disinfectants, such as those routinely used in healthcare
environments, presenting the following properties (77,78):
- bactericide conforming with the standard NF EN 1040, NF EN 13727
- fungicide or yeasticide conforming with the standard NF EN 1275, NF EN 13624
- sporicide conforming with the standard NF EN 14347,
- virucide conforming with the standard NF EN 14476 + A1.
The products that are active on viruses are detailed in Figure 6.
Figure 6. Estimating the biocidal activities of the main substances (78)
Ewan M. Clark et al. showed that bleach (0.5% [vol/vol]) resulted in a reduction of the bacteriophage titer
of 7-log10 (79).
As an example, Surfa’safe Anios®-type products present the properties previously cited in conformity with
the standards, it is thus active on bacteriophage types of naked viruses. It should be noted that the Oxy’floor
42
Anios detergent-disinfectant, which is active on Clostridium difficile spores provides a gain in naked
virus cover of 1 to 2%.
When handling bacteriophages, it is recommended to wash ones hands with water and mild soap(80).
It is also important to take account of the material used during the administration to the patient. Wherever
possible, it is better to use single-use materials. Otherwise, advice should be sought about the possibility of
sterilizing the material. Briers et al. demonstrated that the activity of anti-P. aeruginosa bacteriophages
FKZ progressively decreases from 100% to 0% when exposed to temperatures going from 25 to 90 °C for
60 min (81).
The conventional moist heat autoclaving cycles routinely used in health establishments (classic cycle of
121°C for 30 minutes or ATNC cycle of 134°C for 18 minutes) are perfectly effective to inactivate the
bacteriophages.
Bacteriophage administration procedures
In the current absence of recommendations and data, standard precautions should be applied during the care
provision.
Clean and/or disinfect the environment around the patient (bedside table, adaptable table, bed, etc.), the
frequently used surfaces (door handles, toilets, etc.) and the premises (floors, surfaces) according to the
procedures set out in chapter V.3 and in compliance with the procedures of the premises.
Funding bacteriophage preparations
According to article L. 5123-2 of the French Public Health Code (CSP), nominative ATUs and cohort
ATUs are deemed to feature on the public authority list and are thus covered by the health insurance (82).
When bacteriophages are provisioned in the context of a nominative ATU, these specialties feature on the
list called the “list of drugs billable over and above inpatient services” or “over and above list”. This allows
the establishments to bill the health insurers for bacteriophage preparations over and above the diagnosis-
related group-based fees (DRG) and to be reimbursed at 100%.
If bacteriophage lyophilisates/suspensions are considered as raw materials then their cost becomes part of
the hospitalisation services fee, i.e. the DRG fee.
The cost of carrying out the “pharmaceutical magistral preparation” part should be added and included in
the hospitalisation services fee, i.e. the DRG fee.
The phagogram and the other tests needed to diagnose and prescribe bacteriophages can be, by assimilation
with the antibiograms, billed to the health establishments, their costs are included in the hospitalisation
services fee, i.e. the DRG fee.
43
Conclusion
Today, the early provision of bacteriophage lyophilisates/suspensions is restricted to the context of clinical
trials or for compassionate use. Indeed, no bacteriophage suspension is currently subject to a MA which
would permit routine therapeutic use according to the indications stated in the MAs that could be envisaged.
The only industrial producer of bacteriophages in France is Pherecydes Pharma®, promoter of the
Phagoburn clinical trial. It has bacteriophages that are active against some strains of S. aureus and P.
aeruginosa. Thus, beyond these few bacteriophages, in order to treat infections caused by other bacteria, it
is necessary to take recourse, by way of derogation and in the context of compassionate use, to importing
bacteriophage lyophilisates/suspensions produced by the QAMH in Belgium; these are
lyophilisates/suspensions in unitary form that the IHPs who have made the prescription for compassionate
phagotherapy should then prepare to enable a safe administration.
A brief overview of the production capacities for bacteriophage lyophilisates/suspensions in unitary form
(raw material), and the capacities of the phage libraries to allow a relevant phage to be selected for the
resistant infection, shows that the resources in France are very limited. In this context, it is important to
increase the possibilities for bacteriophage provision in France, particularly regarding the production of
customised bacteriophage lyophilisates/suspensions (to tackle the various resistant infectious bacterial
strains) in the country. ANSM strongly encourages the development of an academic production of
bacteriophages, using the same model as QAMH in Belgium or other European countries. Additionally, the
creation of a “phage library” or “bacteriophage bank” should be discussed. This would considerably
increase the number and diversity of bacteriophages available. It would be possible, once the
recommendations on the requirements for production and pharmaceutical quality control are published, to
make available production sites for lytic, characterised bacteriophages, where the safety levels meet the
standards for use in clinical practice. This phage library would be added to after each isolation-
identification-characterisation of a bacteriophage for a given patient. These bacteriophages stored in the
bank would then be available for use in other patients.
In terms of the perspectives for using phagotherapy in exceptional situations of resistance, it
appears that the customised approach “upon request” and “made to order” is plausibly the most
adapted/effective means of targeting a pathogen that is resistant to other therapeutic approaches. The
different bacteriophage suspensions currently available are not of a standardised quality. However, there
are some texts in existence on the biological products, and publications such as the Belgian National
Monograph could be used to institute a production and control procedure and thus ensure that the
44
pharmaceutical quality of the unitary bacteriophage lyophilisates/suspensions available is assessed
(meaning the raw material/active substances).
This raises the question of demonstrating the clinical efficacy and optimum treatment regimens for
bacteriophages. There are no defined prerequisites in the current assessment standards to act as a base for
the clinical demonstration of phagotherapy. It is essential to define the clinical objectives and methodology
of future trials so as to avoid perpetuating the empiricism of this therapy and to obtain data on the efficacy
and tolerance of bacteriophages. Phagotherapy should be subject to in-depth and rigorous evaluation.
Finally, as stressed in the foreword, this guide does not aim to substitute for the contents of a MA file, nor
for ANSM’s case by case assessments. It is subject to change in function to the legislation, European and
French recommendations, data from feedback from practitioners and the results of pre-clinical and clinical
studies.
45
List of Abbreviations
DNA: DeoxyriboNucleic Acid
AFMPS: Agence fédérale des médicaments et des produits de santé / Belgian Federal Agency for
Medicines and Health Products
MA: Marketing Authorisation
ANSM: Agence nationale de sécurité du médicament et des produits de santé / French National Agency
for Drug and Health Product Safety
API: active pharmaceutical ingredient
RNA: RiboNucleic Acid
ATU: Authorisation for Temporary Use
EHARB: Emerging Highly Antibiotic Resistant Bacteria
AMB: Antibiotic-Multiresistant Bacteria
GMP: Good manufacturing practices
GPP: Good preparation practices
AC: Analysis Certificate
CHMP: Committee for Medicinal Products for Human Use
CEP: Certificate of Suitability to the European Pharmacopoeia monographs
CSP: Code de la santé publique / French Public Health Code
CSST: Comité scientifique spécialisé temporaire / Temporary specialised scientific committee
DASRI: Déchets d'activités de soins à risques infectieux et assimilés / Waste from care activities
involving a risk of infection and related risks
TAC: Total Aerobic Count
MD: Medical device
TYMC: Total Yeast and Mould Count
DN: Director of Nursing
AE: Adverse Effects
Etb: Establishment
EMA: European Medicines Agency
EOP: Efficiency of Plaquing (parameter of phage activity)
OHT: Operational Hygiene Team
FDA: Food and Drug Administration
DRG: Diagnosis-Related Group
QAMH: Queen Astrid Military Hospital
ICH: International Conference on Harmonisation
IV: Intravenous
JORF: Journal officiel de la République française / official gazette of the French Republic
ATMP: Advanced Therapy Medicinal Product
MOI: Multiplicity of infection (relation between the number of phages and the number of bacteria
present)
RMPU: Raw Materials for Pharmaceutical Use
WHO: World Health Organisation
PFU: Plaque-forming units
MSW: Microbiological Safety Work bench
IHP: In-house Pharmacy
TUP: Therapeutic Use and information gathering Protocol
SPC: Summary of Product Characteristics
CFU: Colony-Forming Unit
EU: European Union
UV: Ultraviolet
CAA: Controlled Atmosphere Area
46
Definitions of terms used
Good manufacturing practices (GMP): The Good Manufacturing Practices for medicinal products
constitute one of the elements of managing quality. They guarantee that the products are manufactured and
controlled in a coherent way, in adherence to the quality standards adapted to their use and required for the
marketing authorisation, clinical trial authorisation and product specifications. GMPs apply to both the
production and quality control. The GMP guide is intended to serve as a reference when assessing the
manufacturing authorisation applications and when inspecting drug manufacturers (83).
The principles of the GMPs and the specific guidelines apply to all operations requiring authorisations, as
stipulated in articles 40 of the directive 2001/83/EC and 13 of the directive 2001/20/EC, as amended
(14,84). They also apply to pharmaceutical manufacturing procedures, such as those carried out in health
establishments, for example.
Good Preparation Practices (GPP): The Good Preparation Practices are another of the elements of
managing quality. They guarantee that pharmaceutical preparations are manufactured and controlled in a
coherent way, in adherence to the quality standards adapted to their use and their specifications.
GMPs apply to both the production and quality control of pharmaceutical preparations (19).
Manufacture - production: All operations covering the reception of materials, production, processing,
reprocessing, labelling, relabelling, quality control, market release, storage and distribution of active
substances and the associated controls (83).
In the case of bacteriophages, the developers/manufacturers produce/manufacture the bacteriophage
lyophilisates/suspensions in unitary form, these are raw materials. The in-house pharmacy (IHP)
pharmacists are responsible for the preparation, meaning producing the pharmaceutical form:
reconstitution, dilution of these raw materials. Thus, the magistral (or hospital) preparations are produced
by the IHP for a given patient (or several patients in the case of hospital preparations), to treat a bacterial
infection, using these raw materials.
Raw Material for Pharmaceutical Use: All components used to make a preparation (active substances,
excipients, elements used to produce the pharmaceutical form, intended for use in or administration to
humans) (19).
In the case of bacteriophages, these are suspensions or lyophilisates in unitary form, the “active
pharmaceutical ingredients” (API), produced by a developer/manufacturer using a recognised production
process that is compliant to the Good Manufacturing Practices (GMP) (83). The active substances raw
materials produced will become the starting materials used by the IHPs to make magistral or hospital
preparations.
47
Preparation (act of): Term designating all activities involved in preparing and processing (including
labelling and relabelling) (19). The pharmacist is responsible for the decision to make the preparations (19).
In particular, the IHPs’ work includes the preparation, control and supply of medicinal products, in
compliance with article L.5126-5 of the French public health code (85). The preparation activities should
follow defined instructions and procedures. They adhere to the principles of the Good Preparation Practices
(GPP) so as to obtain preparations of the quality required. The specificities of the preparation are defined
in the preparation file. A preparation is only made if the pharmacist responsible for this activity possesses
the specific, appropriate means (equipment, materials, personnel, premises, etc.) to undertake and control
it.
Pharmaceutical preparation: A drug, generally made of active substance(s) that may be combined with
one or more excipients, which is formulated and produced in pharmaceutical form in a way that is adapted
to the intended use, if necessary after reconstitution, and which is presented in an appropriate recipient and
properly labelled. A pharmaceutical preparation is made for the specific needs of one or more patients in
compliance with the legislation. Pharmaceutical preparations are divided into 2 categories:
Extemporaneous preparations are customised for a specific patient or group of patients and
provisioned immediately after being prepared;
stored preparations are prepared in advance and stored until requested (19).
The French Public Health Code (CSP) describes 3 sorts of preparation: magistral preparation, hospital
preparation and officinal preparation (Article L.5121-1 of CSP) (86).
Finished preparation: Drug which has undergone all the stages involved in making the preparation,
including the final processing (19).
Finished product: Drug which has undergone all the stages involved in the manufacture, including the
processing.
Finished products, manufactured by a pharmaceutical establishment, contain pharmaceutical active
principles compliant with the qualitative and quantitative formula detailed in the marketing authorisation
(MA), or the authorisation for temporary use (ATU) or the authorisation for clinical trial. They have the
required purity, are contained in appropriate packaging and are correctly labelled.
The preparation and production of the pharmaceutical form (reconstitution, etc.) of these finished products
is compliant to the Summary of Product Characteristics (SPC) in the case of a product with a MA, or the
Therapeutic Use Protocol (TUP) for a drug subject to an ATU (83).
Reconstitution: A manipulation making it possible to use or administer a drug. Drugs with a MA, awarded
by a competent drug safety agency, are reconstituted in compliance with the instructions figuring in the
48
Summary of Product Characteristics (SPC) or in the patient information leaflet, or in the experimental drug
protocol. When done in a pharmacy, this should adhere to the GPP in force. The reconstitution of specialty
or experimental drugs is a simple mixing operation, notably of a solution, a powder, a lyophilisate, etc. with
a solvent for parenteral or non-parenteral use(19).
The reconstitution of unitary bacteriophage lyophilisates/suspensions, which have the status of raw
materials, is a simple mixing operation, notably of one or several suspensions, of one or several
lyophilisates, to finish with a cocktail of bacteriophages, with a solvent for parenteral or non-parenteral use.
The reconstitution of bacteriophage lyophilisates/suspensions should be carried out in accordance with the
provisions in the GPPs (19).
49
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rticle=LEGIARTI000006690078&dateTexte=&categorieLien=cid
86. Code de la santé publique. Article L.5121-1 relatif aux médicaments à usage humain.
Version en vigueur au 1er juillet 2017. [Internet]. Disponible sur:
https://www.legifrance.gouv.fr/affichCodeArticle.do?idArticle=LEGIARTI000026499732&cidT
exte=LEGITEXT000006072665
87. McCallin S, Alam Sarker S, Barretto C, Sultana S, Berger B, Huq S, et al. Safety analysis
of a Russian phage cocktail: From MetaGenomic analysis to oral application in healthy human
subjects. Virology. 2013;443(2):18796.
55
APPENDIX 1
Algorithm for bacteriophage provision depending on the status of the preparation
56
No
The clinical use of bacteriophages is carried out under the responsibility of
the prescriber and the hospital pharmacist.
No
Manufacture in France.
Example of the Pherecydes
Pharma ® company.
Collegial discussion of the case.
ANSM supports the hospital
teams in the process of
provisioning bacteriophages.
2. The hospital pharmacist then makes the (magistral or hospital6) preparation and controls the pharmaceutical quality
of the bacteriophage preparation.
Cf. part IV.4.b : Preparation and pharmaceutical quality control
Is there any ongoing biomedical research
involving human beings on the pathology
of the patient using bacteriophages?
Preparation of bacteriophages with a
marketing authorisation (MA)
Prescription compliant:
- to the MA;
- to scientific knowledge, data acquired
from medical knowledge
(recommendations of good practices);
- to the analysis of the individual
risk/benefit balance.
The product can only be provisioned within a
hospital structure.1
If it is a product for which (the 2 criteria must
be present):
1. The manufacturing is industrial,
compliant with Good Manufacturing
Practices (GMP), undertaken by an
ANSM-authorised establishment or
the supervisory authority of the
country where the establishment is
located.
2. The intrinsic pharmaceutical quality is
guaranteed by an appropriate pharmaceutical
development, a quality control strategy that
makes it possible to guarantee the final quality,
in compliance with the applicable references.
Yes
Research involving human beings:
- Article L1121-1 of the French
public health code
- Decree n° 2016-1537 of 16
November 2016
Inclusion of the patient in biomedical
research compliant with the protocol.
Yes
It is a specialty pharmaceutical (article L.5111-
2 of the French public health code2): provisioned
under an Authorisation for Temporary Use
(ATU)3. This nominative ATU equates to an
import authorisation in the case of
pharmaceutical establishments located abroad.4
In instances where a nominative ATU system is
required, the requests should be dependent on the
use of a Therapeutic Use and information
gathering Protocol (TUP), the aim being, on one
hand, to ensure safety, adequate monitoring and
that the patient is informed, and on the other, to
build on knowledge about bacteriophages.
Yes
No
Manufacture by a European Union member
state.
Example: manufacture by the Queen Astrid
Military Hospital team.
Collegial discussion of the case. ANSM
awards a derogating import authorisation
for diagnostic purposes (if the phagogram
is performed within the health
establishment), then a derogating import
authorisation for therapeutic purposes.
Compassionate use
= status of raw material5
(Article L5138-2 of the French public health code)
1. The hospital pharmacist should assess the pharmaceutical quality of the raw materials.
Cf. part IV.4.a: Assessment of the pharmaceutical quality of bacteriophage raw materials
Is the raw material produced in France?
No
Yes
57
1 Bacteriophage lyophilisates/suspensions can only be supplied by an in-house pharmacy of a hospital establishment, this therefore requires a monitoring within
the hospital service.
2Article L5111-2 of the French public health code: “A specialty pharmaceutical is understood to be any medication prepared in advance, presented in a particular
packaging and characterised by a special denomination.”
3 Concerning the ATUs qualified as nominative, ANSM can, exceptionally, authorise the use of some drugs intended to treat, prevent or diagnose serious or rare
diseases, where no appropriate treatment exists, and where the current state of scientific knowledge makes it possible to presume they are effective and safe.
This type of access to drugs is for a single named patient who cannot participate in biomedical research, it concerns drugs for which the efficacy/safety balance
is presumed to be favourable for these patients in view of the data available, and is awarded upon request and under the responsibility of the prescribing doctor
once the drug is deemed likely to present a benefit for this patient.
4 In compliance with articles L.5124-13 and R.5121-108 of the French public health code, the nominative ATU (ATUn) and cohort ATU (ATUc) equate to an
import authorisation. Consequently, it is not necessary to request an import authorisation from ANSM for drugs subject to an ATUn or an ATUc. However, if a
pharmaceutical establishment or in-house pharmacy are importing a drug under a nominative ATU in order to constitute stocks, they need to obtain a specific
prior authorisation awarded by the ANSM director general.
5 Article L.5138-2 gives the following definition: “We understand the term raw materials for pharmaceutical use to mean all components of medicinal products
in the sense of article L.5111-1, meaning:
- the active substance(s);
- the excipient(s);
- the elements of the pharmaceutical form intended for use in or administration to humans or animals."
58
In the framework of transposing the provisions of directive 2011/62/EU of 8 June 2011 into national law, which intends to combat the falsification of medicinal
products, article L. 5138-1 of the French public health code, modified by decree n° 2012-1427 of 19 December 2012 stipulates:
- an authorisation scheme for the establishments exercising manufacturing, import and distribution activities of active substances.
- a declaratory scheme the establishments exercising the same activities for the excipients
The decree implementing this law defines the procedures for authorisation applications and for declaring these activities (decree n° 2012-1562 of 31 December
2012). The decision of the ANSM general director on 24 February 2015 defines the form and contents of the authorisation application and the declaration of
these activities. It revokes decision DG n° 2013-104 of 21 March 2013 and modifies the procedures for updating online registrations.
The administrative and technical information should be submitted to ANSM via a online registration portal.
The ‘annual’ modifications should now be communicated within a timeframe of 12 months following the date of the previous online registration.
The following should be notified without delay:
- all changes to the administrative information
- all modifications likely to affect the quality or safety of the raw materials for pharmaceutical use.
- any cessation of activity
http://ansm.sante.fr/Activites/Matieres-premieres-a-usage-pharmaceutique-MPUP/Autorisation-et-declaration-des-activites-de-fabrication-d-
importation-et-de-distribution/(offset)/0
6Article L5121-1 (Modified by decree n°2016-1729 of 15 December 2016 - art. 2): “Definitions:
1° Magistral preparation, any drug prepared in accordance with a medical prescription intended for a given patient because of an absence of available specialty
pharmaceuticals with a marketing authorisation, of one of the authorisations mentioned in articles L. 5121-9-1 and L. 5121-12, of a parallel import authorisation
or an import authorisation issued to a pharmaceutical establishment in the context of a stock shortage of a drug, either extemporaneously in a pharmacy, or under
the conditions set out by article L. 5125-1 or article L. 5126-6;
59
2° Hospital preparation, any drug, with the exception of gene or cell therapy products, prepared in accordance with the indications in the pharmacopoeia and in
compliance with the good practices cited in article L. 5121-5, because of the absence of available or appropriate specialty pharmaceuticals with a marketing
authorisation, of one of the authorisations mentioned in articles L. 5121-9-1 and L. 5121-12, of a parallel import authorisation or an import authorisation issued
to a pharmaceutical establishment in the context of a stock shortage of a drug, by an in-house pharmacy of a health establishment, or by a pharmaceutical
establishment of this health establishment with authorisation under article L. 5124-9 or under the conditions defined in article L. 5126-6. Hospital preparations
are dispensed under medical prescription to one or several patients by the in-house pharmacy of said establishment. They are subject to declaration at the National
Agency for Drug and Health Product Safety (ANSM), under the conditions defined by the decree of the health minister;
3° Officinal preparation, any drug prepared in a pharmacy, listed in the pharmacopoeia or in the national formulary and intended to be dispensed directly to the
patients served by this pharmacy […] »
60
APPENDIX 2
Clinical trials using bacteriophages
1. CLINICAL TRIAL IN SKIN INFECTIONS
Table III. Result of the Phagoburn clinical trial (10)
Study title
Phase I/II, randomised (1:1), multi-centre, European, controlled versus standard of care clinical trial, with the aim of assessing the efficacy and tolerance of a
cocktail of bacteriophages in treating skin infections caused by P. aeruginosa in burns patients.
The trial also initially tested a cocktail of bacteriophages active on E. coli. But in January 2016, considering the low recruitment levels of patients presenting an
E. coli skin infection (n=1), they decided to stop the clinical assessment of anti-E. coli bacteriophages
Status of the
clinical trial
Complete
Start: 22 July 2015 End: 2 January 2017 because of insufficient efficacy of treatment PP1131.
Study
population
Patients aged 18 years or over, hospitalised in a burns treatment centre and presenting:
- a skin burn infected with P. aeruginosa, confirmed by microbiological sampling;
Initially, to be included in the trial, the patient should present a monobacterial P. aeruginosa infection. However, following an amendment, a minor
colonisation of other bacteria was accepted.
- one or several clinical signs of infection according to the criteria defined by the Société Française d'Étude et de Traitement des Brûlures (French Society for
the Study and Treatment of Burns).
Following the amendment, any patient presenting an increase of 2 points or more in the SOFA (Sequential Organ Failure Assessment) Score in the 48 hours
preceding the inclusion, was excluded.
Primary
endpoint
Time required to obtain a lasting reduction in the bacterial load of two or more quadrants, following the four quadrant seeding method (semi-quantitative method),
multivariate statistical analysis with adjustment depending on whether or not antibiotics active on P. aeruginosa were taken between D0 and D7. Microbiological
samples were taken daily.
Other
endpoints
Tolerance to the treatment was assessed in all the patients who received at least one dose of treatment (suspension of bacteriophages PP1131/sulphadiazine cream).
An ancillary study (sensitivity to the bacterial strain isolated in the patients vis-à-vis the bacteriophages at D7, stability of the bacteriophage suspension) was
carried out to determine the reasons for the successes or failures of treatment PP1131.
Experimental
treatments
- Standard treatment: silver sulphadiazine cream 1%;
- Suspension PP1131 of 12 lytic anti - P. aeruginosa bacteriophages (1st preparation of bacteriophages where the manufacture complied with GMP).
Study design
The study comprised 2 arms:
- Control arm: silver sulphadiazine cream 1%; local topical application of the cream with no support vector, directly on the skin;
- Treatment arm: liquid PP1131 suspension of 12 lytic anti-P. aeruginosa bacteriophages; impregnation of a neutral EOP by the PP1131 suspension, followed
by the local topical application of this “bacteriophage vector” substrate on the wound to be treated; neutral substrate: Algosteril dressing
Duration and frequency of treatment administration: 1 application per day for 7 days.
61
Table III. Result of the Phagoburn clinical trial (10)
Monitoring period: 14 days
Total study duration: 21 days.
The co-prescription of antibiotics was authorised during the 7 days of treatment studied, at the discretion of the clinician.
The clinician was not blinded because of the different galenic forms of the 2 treatments in the study. The microbiologists and patients were blinded.
Results
Total number of patients included: 27
Number of patients for the assessment of treatment efficacy = 25 (N = 12 PP1131 treatments, N = 13 in the control arm)
Number of patients for the assessment of tolerance to the treatment = 26 (N = 13 PP1131 treatments, N = 13 in the control arm)
Results for the primary endpoint (analysis carried out as modified Intent-to-Treat); Median time difference to obtain a reduction in two or more quadrants.
Whether antibiotics active on P. aeruginosa were taken or not could be a confounder, it is an accessory factor as regards the primary endpoint that could mask or
cause differences. The Cox regression model was thus applied to account for this confounding factor. Thus multivariate analyses were carried out to rebalance the
2 groups by statistical adjustment depending on whether or not antibiotics active on P. aeruginosa were taken between D0 and D7.
- Median timeframe in the bacteriophage treatment arm = 144 hours (95% CI 48 not reached);
- Median timeframe in the control arm = 47 hours (23 122; hazard ratio [HR] 0.29 [95% CI 0.100.79]).
Significantly higher median timeframe in the bacteriophage treatment arm compared to the control arm (p = 0.018).
The co-administration of antibiotics (initiated on D0 or during the study treatment period) active on P. aeruginosa does not modify the treatments’ efficacy
(standard treatment or bacteriophage treatment).
Assessment of tolerance to the treatment administered (per-protocol analysis): 3 patients out of 13 presented adverse effects in the “bacteriophage treatment
arm versus 7 out of 13 in the “standard treatment” arm Non-significant difference.
Study
limitations
- Low number of patients included (overly restrictive inclusion criteria and production problems): low statistical power. Although it was initially sized for up
to one hundred patients per treatment arm, the study ended up recruiting 27 patients of which 25 provided useable data. This low level of inclusion
transformed PhagoBurn into a clinical proof of concept study.
- Non-comparable group on the “age”, “burn severity” and “initial bacterial load” criteria: the patients treated with the PP1131 suspension were older and more
severely burned. Yet, age and burn severity are 2 risk factors for burn severity.
The initial bacterial load was higher in the patients treated with the P1131 suspension. By comparing the sensitivity profiles of the antibiotics to the bacterial
strains of the patients in the 2 arms, it has been shown that on D0, the bacterial strains of the patients in the control arm were sensitive to a greater number of
antibiotics.
- The success of bacteriophage treatment depends on the sensitivity of the bacterial strain to the bacteriophages tested: yet, in this trial, the phagogram was not
performed before initiating treatment. An ancillary study, carried out for the patients for whom a failure of the bacteriophage treatment was observed (per-
protocol analysis), showed that the majority of the bacteria isolated were resistant to the doses of bacteriophages administered.
62
Table III. Result of the Phagoburn clinical trial (10)
In the bacteriophage treatment arm: among the patients for whom no reduction in the bacterial load of 2 or more quadrants was observed, a phagogram was
performed a posteriori (n = 5 patients): 24% of the bacterial strains were sensitive to the PP1131 bacteriophage cocktail, 60% had an intermediate sensitivity
and 16% were resistant.
In the bacteriophage treatment arm: a phagogram was performed a posteriori with the bacterial strains of the patients for whom a reduction in the bacterial
load of 2 or more quadrants was observed (n = 5 patients): 89% of the bacterial strains were sensitive to the PP1131 bacteriophage cocktail, 11% had an
intermediate sensitivity and none were resistant. The is no information available on the types of resistance and their molecular mechanisms.
- The stability studies showed a decrease in the titer of the suspension of 1x105 PFU/mL-1x104 PFU/mL (initial titer of 1x109 PFU/ml). Yet, it was necessary
to dilute the suspension to reduce the concentration in endotoxins, thus the titer of the bacteriophage suspension applied daily was of only approximately
1x102 PFU/ml. In accordance with the data in the literature and from preclinical studies, it was decided to use a multiplicity of infection of 10 (relation of the
number of bacteriophages to the number of bacteria, MOI). Yet, given that the titer of the suspension was lower than that planned and that the bacterial load
was very high, the MOI at the infected site was lower. The main consequences of this were:
o the viral amplification at the infected site was slower;
o a slower bacterial lysis slower reduction in the bacterial load.
Daily wound cleaning probably had a negative effect on the bacteriophage activity.
- Differences in the application of the 2 treatments: direct application of the standard treatment versus use of a vector support for the bacteriophage treatment
- The preclinical studies showed in vitro that, for bacteriophage suspensions with a titer of 109 PFU/mL, using a support had no impact on the liberation of
bacteriophages, no “trapping” effect was observe