BENEFICIAL EFFECTS OF BACTERIOPHAGE TREATMENTS
The invention relates to use of one or more bacteriophages in vivo in a human or animal in order to induce sensitivity to chemical antibiotics in bacterial cells, where such susceptibility is heritable, independent of continuing bacteriophage metabolism within those cells, and does not relate to the destruction of a biofilm to induce such sensitivity.
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The present invention relates to the sensitisation of previously resistant bacteria to antibiotics following treatment with bacteriophages. In particular, the invention provides in its preferred aspect for the preparation and administration of therapeutic medicaments that use sequential treatments with bacteriophages and conventional antibiotics, to be used with infections of animals and humans caused by pathogenic bacteria.
BACKGROUND TO THE INVENTIONAntibiotic resistance is now seen as one of the major challenges facing modern medicine. Given the shortage of novel antibiotics, a number of alternative approaches are being investigated, including the use of bacteriophages as therapeutic agents (Barrow & Soothill, Trends in Microbiology (1997), 5, 268-271; Dixon B, The Lancet Infectious Diseases (2004), 4, 186; Hausler T, Viruses vs. Superbugs: A Solution to the Antibiotics Crisis? (2006) MacMillan, New York; Matsuzaki et al, Journal of Infection and Chemotherapy (2005), 11, 211-219.
Bacteriophages (often known simply as “phages”) are viruses that grow within bacteria. The name translates as “eaters of bacteria” and reflects the fact that as they grow most bacteriophages kill the bacterial host as the next generation of bacteriophages is released. Early work with bacteriophages was hindered by many factors, one of which was the widespread belief that there was only one type of bacteriophage, a non-specific virus that killed all bacteria. In fact, the host range of bacteriophages (the spectrum of bacteria they are capable of infecting) is often very specific. This specificity may be considered a therapeutic strength as populations of bacteriophages can be selected to specifically eliminate only the target bacterial species.
Despite the therapeutic advantages afforded by the host specificity of bacteriophages, this characteristic has the disadvantage that it can be difficult to achieve breadth of coverage of target strains. For this reason, there has been interest in finding combinations of bacteriophages having broad targeting capability in relation to particular types of bacterial infection (see for example Pirsi, The Lancet (2000) 355, 1418). This has now been achieved with the development of a mixture of six bacteriophages targeting Pseudomonas aeruginosa, which has completed veterinary field trials and is now in human clinical trials (Soothill et al, Lancet Infectious Diseases (2004) 4, 544-545). The challenge now is to develop regimens which optimise the delivery of such therapies.
Bacteriophages and antibiotic therapy have previously been used together in Eastern Europe (see for example Bradbury, The Lancet (February 2004) 363, 624-625), but without specific reporting of synergistic effects. Indeed, there have been suggestions that antibiotics can have adverse effects on use of bacteriophage therapy since bacteriophages use bacterial metabolism to replicate and this is inhibited by antibiotics (Payne and Janssen, Clinical Pharmacokinetics (2002) 42, 315-325).
More recently, bacteriophages have been shown to produce benefits where mixed pathogenic bacteria grow in a biofilm (Soothill et al, 2005, PCT patent application WO2005009451). In this application benefit was shown with respect to subsequent antibiotic treatment of heterologous bacterial infections, apparently by disruption of the biofilm following bacteriophage treatment.
Biofilm formation is now known to be a characteristic of many important pathogenic bacteria contributing to increased resistance to antibiotics. Such biofilms may comprise more than one type of bacterium supported and surrounded by an excreted extracellular matrix and assist bacteria to colonise surfaces from marine reefs to tooth enamel. Biofilms allow bacteria to attach to surfaces and to attain population densities which would otherwise be unsupportable. They impart increased resistance to not only antibiotics but many environmental stresses including toxins such as heavy metals, bleaches and other cleaning agents. It was previously thought that contribution of biofilm formation to antibiotic resistance was primarily a physical process arising from limitation of diffusion, but more recent evidence has shown that some biofilms appear to have specific abilities to trap antibiotics (Mah et al., Nature (2003) 426, 306-310). It is known that bacteria within biofilms can be 100 to 1000 times more resistant to antibiotics than the same strain of bacteria growing in single-celled (“planktonic”) forms. This increased resistance means that bacteria that are apparently sensitive to antibiotics in a laboratory test may be resistant to therapy in a clinical setting. Even if some are cleared, biofilms may provide resistant reservoirs permitting rapid colonisation once antibiotics are no longer present. It is clear therefore that biofilms are major factors in many human diseases.
As noted above, greater beneficial effects have been observed with the subsequent use of antibiotics against mixed infections following the use of a therapeutic bacteriophage preparation against Pseudomonas aeruginosa, and it has been proposed that this is due to the destruction of Pseudomonas aeruginosa as the key species maintaining the biofilm (Soothill et al, 2005, PCT patent application WO2005009451), which results in loss of biofilm integrity and thus exposure of bacteria to conventional antibiotics.
The teaching of PCT patent application WO2005009451 is against the use of antibiotics that are specifically active against the same bacterial species as that targeted by bacteriophage. The examples cited refer to the use of Synulox (amoxicillin and clavulanic acid) and/or Canaural ear drops (containing diethanolamine fusidate, framycetin sulphate, nystatin and prednisolone). Both of these preparations contain only antibiotics that are not effective against Pseudomonas aeruginosa (Krogh et al, Nordisk Veterinaer Medicin (1975) 27, 285-295; Kucers A, in Kucers et al (eds), The Use of Antibiotics: A Clinical Review of Antibacterial, Antifungal and Antiviral Drugs, Fifth edition (1997), Butterworth-Heinemann, Oxford; Rawal, Journal of Antimicrobial Chemotherapy (1987) 20, 537-540). In particular, while aminoglycoside antibiotics as a class are effective against Pseudomonas aeruginosa, Framycetin is of very limited efficacy. Kucers notes that “Nearly all the medically important Gram-negative aerobic bacteria are sensitive” (to Neomycin, Framycetin and Paromomycin) “with the exception of Pseudomonas aeruginosa”, while the same author states that “Pseudomonas aeruginosa is co-amoxiclav resistant, citing the work of Comber et al, in Rolinson & Watson Augmentin (eds) (1980), Excerpta Medica, Amsterdam, p.19. Co-amoxiclav is defined in the online 52nd edition of the British National Formulary (www.bnf.org) as “a mixture of amoxicillin (as the trihydrate or as the sodium salt) and clavulanic acid (as potassium clavulanate), equating to the veterinary drug Synulox. Thus, PCT patent application WO2005009451 would not indicate the use of antibiotics targeting Pseudomonas in any combination with bacteriophages but rather the use of antibiotics specifically targeting co-infecting bacteria.
Another mechanism has been identified recently by which bacteriophages can increase the sensitivity of bacteria to antibiotics to which they are resistant (Hagens et al, Microbial Drug Resistance (2006), 12, 164-168). This involves active bacteriophage metabolism, and is suggested to involve the formation of pores in the bacterial membrane. However, this teaches that “resensitization of pathogens resistant to a particular antibiotic can be achieved in the presence of phage in vivo” based around the use of “a combination treatment with antibiotics and filamentous phage”. Thus this relates to a non-heritable characteristic which is exerted only in the presence of bacteriophage, which relies on the simultaneous use of both bacteriophages and antibiotics, and which appears to be specific to filamentous to bacteriophages which form pores in the bacterial membrane. This is thus distinct from the inventions claimed herein, which induce heritable changes that persist even when actively replicating bacteriophage is not present.
SUMMARY OF THE INVENTIONThe present invention is based on the induction of sensitivity to chemical antibiotics by the use of bacteriophage treatment in vivo in humans or in animals, where such sensitivity is heritable, does not rely on active bacteriophage metabolism and does not relate to the destruction of biofilm to induce such sensitivity, along with the preparation of medicaments to permit the sequential use of bacteriophages and antibiotics so as to take advantage of such induction in sensitivity in the control of bacterial disease, especially for example a Pseudomonas aeruginosa infection. Induction of sensitivity in this context will be understood to include improvement of sensivity.
DETAILED DESCRIPTIONThus in one aspect, there is provided a bacteriophage preparation comprising one or more bacteriophages for use in combined bacteriophage and antibiotic therapy to treat a bacterial infection in a human or animal, wherein at least one antibiotic is administered following the start of said bacteriophage treatment at a time period at which susceptibility of bacterial cells of said infection to said antibiotic is induced or improved by the bacteriophage treatment, where such susceptibility is heritable, independent of continuing bacteriophage metabolism within those cells, and does not relate to the destruction of a biofilm to induce such sensitivity. Antibiotic sensitivity may be monitored by established procedures in vitro. Induction of sensitivity may be confirmed for one or more bacterial strains from the individual patient or may be identified in other patients with similar bacterial infections following bacteriophage treatment
In a further aspect, there is provided a two stage medicament where the first stage comprises a bacteriophage-based therapeutic and the second is composed of one or more chemical antibiotics, for sequential use in humans or animals, where this is designed to exert beneficial effects by the induction of sensitivity as noted above.
The bacteriophage therapeutic and one or more chemical antibiotics may be administered for example at an interval of one to two days to two months apart, preferably at an interval of one to four weeks, most preferably at an interval of two weeks apart.
As indicated above, combined phage/ antibiotic therapy according to the invention may be particularly useful for example in targeting bacterial infection comprising or consisting of Pseudomonas aeruginosa. Such infection may be, for example, at the site of a skin burn or other skin wound. It may be in the lung, an ocular infection or an ear infection. In this context, such an infection comprising P. aeruginosa will be understood to include an infection consisting essentially of P. aeruginosa. Thus, phage therapy according to the invention may be applied to an infection composed entirely, predominantly or significantly of P. aeruginosa.
EXAMPLESInduction of Antibiotic Sensitivity in a Veterinary Field Trial:
Canine ear infections caused by Pseudomonas aeruginosa (otitis externa and otitis media) are examples of clinical disease associated with biofilm-based colonization of a body surface. Clinical signs of such infection include pain, irritation (erythema), ulceration and the discharge of increased amounts of material from the ear. This is often purulent in nature and is accompanied by a distinctive odour.
A combined preparation of six bacteriophages was named BioVet-PA, and was authorized for trial in dogs with such infection by the Veterinary Medicines Directorate of the United Kingdom in November 2003.
Conduct of the Trial
BioVet-PA was stored at −80° C. Immediately prior to administration, the product was thawed and warmed in the hand. 0.2 ml (containing 1×105 infectious units of each of the 6 bacteriophages) was administered drop-wise using a sterile 1 ml capacity syringe into the ear. Ear condition and microbiology was assessed at 2 days post-administration.
The procedure was as follows:
Characterisation (2 to 14 days prior to treatment):
If Pseudomonas aeruginosa was not detected, the dog was excluded from the trial
If the Pseudomonas aeruginosa strain(s) with which the dog was infected was/were not sensitive to BioVet-PA, the dog was excluded from the trial.
Treatment:
Studies on ten dogs with severe, antibiotic-resistant Pseudomonas aeruginosa ear infections treated with BioVet-PA showed improvement in clinical symptoms within two days of treatment and reductions in bacterial numbers over the same timescale. Bacteriophage replication was observed in all dogs. Analysis of the improvement in clinical symptoms showed this to be significant at the 95% level of confidence by both the t-test and the Wilcoxon matched-pairs test.
Three dogs were excluded from the trial. The first dog to be tested was excluded because of a subsequent change in the scoring system (to take account of ear discharge purulence) while two treated dogs proved to have infections that were not predominantly with the target bacterium at the time of treatment (due to a change in bacterial flora following pre-entry screening).
Antibiotic Resistance:
All isolates of Pseudomonas aeruginosa from all thirteen dogs that were collected before treatment were screened for their sensitivity to antibiotics. The antibiotic sensitivity profile of each Pseudomonas aeruginosa strain was assessed with a range of 10 antibiotics which may be used clinically by veterinarians to treat Pseudomonas aeruginosa infections. The results were recorded in appropriate data collection sheets.
Since differing colony types were often observed in the same dog and both ears were infected in four dogs, a total of 83 individual isolates of Pseudomonas aeruginosa were tested. Thus, 830 tests were carried out, of which 340 were on swabs taken immediately prior to treatment, 340 two days after treatment, and 150 four days after treatment.
All sensitivity assays were compared to identify shifts in sensitivity to any of the antibiotics tested. No individual isolate showed more than a single shift, and no isolate changed from fully sensitive to fully resistant, or fully resistant to fully sensitive. However, shifts from sensitive to partially resistant, partially resistant to resistant, resistant to partially resistant, or partially resistant to sensitive were seen for 16 isolates. Events were observed as shown in Table 1 below.
A total of 30 alterations in antibiotic sensitivity were seen, with 28 being shifts towards sensitivity and 2 being shifts towards resistance. Thus shifts towards sensitivity outnumbered those towards resistance by a factor of 14:1, illustrating the predominance of such “beneficial” shifts following bacteriophage treatment.
Induction of Antibiotic Sensitivity in a Human Clinical Trial
The trial was a single-centre, double-blind, randomised, parallel group study of the safety and efficacy of a single administration of BioPhage-PA (a mixture of six bacteriophages specific for Pseudomonas aeruginosa) compared with placebo in patients with chronic ear infection caused by Pseudomonas aeruginosa shown to be susceptible to one or more of the bacteriophages present in BioPhage-PA.
This study investigated the efficacy and safety of BioPhage-PA, a mixture of six Pseudomonas aeruginosa bacteriophages of the same types as those tested as BioVet-PA in the veterinary field trial, which formed part of the pre-clinical work for this study.
The six bacteriophage strains (which were deposited at the National Collection of Industrial and Marine Bacteria, 23 St Machar Drive, Aberdeen, AB24 3RY, Scotland, UK on 24 Jun. 2003) are as follows:
These bacteriophages are effective at killing a broad range of P. aeruginosa isolates.
The study was carried out in two parallel groups of patients with ear infection caused by to Pseudomonas aeruginosa. Patients were randomly allocated to receive a single dose of either BioPhage-PA or placebo and were be monitored in a double-blind design over a period of 6 weeks post-dose. Efficacy assessments included questions about adverse events, both patient and investigator assessment of disease severity using visual analogue scales, Pseudomonas aeruginosa and bacteriophage ear swab count, audiogram, photography of the ear, and aural temperature analysis. Change from baseline (pre-dose assessment) in active and placebo groups were compared statistically. Safety data was also compared in the 2 groups.
Study Design
This was a single-centre, double-blind, randomised, parallel group study in patients with chronic Pseudomonas aeruginosa infection of the ear. Patients were randomised to one of two groups:
Group 1:
Patients received a single 0.2 mL dose of BioPhage-PA (containing 1×105 pfu by original titration of each of the 6 therapeutic bacteriophages)
Group 2:
Patients received a single 0.2 mL dose of placebo (10% v/v glycerol in PBS)
Design Summary
Pre-Study Visit
Patients attended the clinic within 2 weeks of treatment Day 0 after being informed of the trial verbally. At this visit, they were provided with a written information sheet and were also provided with study details verbally. Patients were questioned regarding their eligibility to participate and if successful signed a consent form prior to Day 0 of the trial.
Treatment Period (Days 0-42 Inclusive)
Patients attended the clinic on the morning of Day 0 for clinical examination and were questioned about adverse events and study compliance. Upon confirmation of eligibility, patients were randomised to one of the two treatment groups and had baseline assessments performed to determine the severity of the infection. Treatment was then administered by the clinician who instilled the therapy drop-wise into the ear. Patients remained in the clinic for 6 hours post-dose. They were issued with diary cards for recording any adverse events or comments on the condition of the ear on a daily basis whilst away from the unit.
Patients returned on Days 7, 21 and 42 for further safety and efficacy tests.
A patient was eligible for inclusion in this study only if all of the following criteria apply: Aged 18 or over; able and willing to give written informed consent to take part in the study; infection of a the ear shown to be caused predominantly or solely by Pseudomonas aeruginosa; Pseudomonas aeruginosa isolated from the infection and shown to be vulnerable to one or more of the bacteriophages present in BioPhage-PA; infection established for at least 6 weeks and proven unresponsive to conventional anti-bacterial therapy; available to attend all clinic visits and complete all study measurements; female patients to be post-menopausal, surgically sterile or willing to use an acceptable form of contraception.
A patient was not eligible for inclusion in this study if any of the following criteria applied: Local surgery within 3 months of the pre-study visit; acute or systemic sepsis; use of systemic or topical antibiotics within one week of the pre-study visit or during the study; use of topical antiseptic or anti-inflammatory agents within one week of the pre-study visit or during the study; bacteriophage therapy in the 6 months prior to the pre-study visit; haemolytic Streptococci of groups A, B, C and G or unusual bacterial or fungal flora on ear swab culture at the pre-study visit; female pregnant or intending to become pregnant; patients who have a past or present disease which, as judged by the investigator, may affect the outcome of the study; any other condition which the investigator feels may prejudice the results of the study; participation in another clinical trial involving a new molecular entity within the previous 4 months or any trial within the previous one month.
Study Assessments and Procedures
Each patient attended the unit for the following visits:
Prior to the official start of the study, swabs (in transport media) were taken from the ears of potential trial candidates. General microbiological analysis was carried out to determine the level of Pseudomonas aeruginosa in the ear. This was followed by a diagnostic ear-swab test. The trial was discussed verbally with patient, and the patient was provided with information sheet/consent form; history was taken and recorded on the case report form; a diagnostic swab was taken and the swab sent for microbiological analysis, where it was analysed for Pseudomonas aeruginosa and for sensitivity of Pseudomonas aeruginosa that was present to the bacteriophages in BioPhage-PA.
If suitable, the patient was enrolled onto trial within 2 weeks of the time that the diagnostic swab was taken.
Study Day 0: The patient assessed the condition of their ear for: discomfort, itchiness, wetness, and smell. Using pre-weighed dry swabs, samples were taken for microbiological analysis. Oral and aural temperature were recorded, the ear was cleaned, and the attending physician assessed the ear for: erythema/inflammation, ulceration/granulation/polyps, discharge type (clear/mucoid/mucopurulent), discharge quantity, and odour (immediately prior to study procedures). Digital otoscopic photography was performed, along with a hearing test (audiogram). BioPhage-PA (0.2 ml) was then administered directly into the ear canal using a 1 ml syringe and soft sterile tubing over a period of approximately 30 seconds. The patient remained at the clinic for 6 hours after therapy for observation and was then sent home with a diary card to record any information they felt relevant to their condition.
Study Day 7: This involved adverse event and compliance questioning, patient assessment of the ear, swab sampling with microbiological analysis, recording of aural and oral temperature, physician assessment of the ear, and ear cleaning.
Study Day 21: This involved procedures as described for study day 7
Study Day 42: This involved procedures as described for study days 7 and 21, except that a hearing test was also be performed and the ear was photographed.
Microbiological Assessment
Microbiological assessment involved counting of the Pseudomonas aeruginosa present on the swab, along with counting of all bacteriophages (both extraneous and therapeutic) on the swab.
Sensitivity to ten antibiotics (as for the veterinary field trial) was also monitored for all isolates. The antibiotic sensitivity test was conducted on each strain of Pseudomonas aeruginosa isolated. The test was conducted according to the standard methods of the British Society for Antimicrobial Chemotherapy (BSAC) Disc Diffusion Method for Antimicrobial Susceptibility Testing (May 2003). The antibiotics to be used were as follows:
- Amikacin—30 μg/ml
- Ceftazadime—30 μg/ml
- Ciprofloxacin—5 μg/ml
- Gentamicin—10 μg/ml
- Meropenem—10 μg/ml
- Pipericillin+Tazobactam (7.5:1)—85 μg/ml
- Colistin—25 μg/ml
- Aztreonam—30 μg/ml
- Imipenem—10 μg/ml
- Tobramycin—10 μg/ml
The test was conducted using Isosensitest agar.
It was found that in the first patient in which bacteriophage replication was seen, there was evidence of a movement towards sensitivity for three of the ten antibiotics monitored (see Table 2).
In the veterinary field trial, over a two to four day monitoring period, evidence was found of a movement towards antibiotic sensitivity in 8.24% of Pseudomonas aeruginosa isolates (against 0.59% where movement towards resistance was seen).
In human trial, evidence was seen of a movement towards sensitivity to chemical antibiotics following the use of a bacteriophage therapeutic in the first patient where bacteriophage replication was observed. Such movement was seen for three of ten antibiotics monitored (30%), over the longer monitoring period in this trial.
Further Human Trial Results
Subsequent analysis of all twenty four participants in the human trial confirmed the above findings as follows with reference to Tables 3 and 4 below. It can be seen that cessation of antibiotic treatment (required for trial entry) itself produced a drift towards antibiotic sensitivity, but that this was more marked for both numbers of patients and for individual antibiotics assayed in the test (bacteriophage-treated) group, with the majority of patients (7/12) showing at least one change towards sensitivity during the monitoring period. Shifts to sensitivity appear particularly marked for aminoglycoside antibiotics with, for example, five of twelve bacteriophage-treated patients showing increased sensitivity to gentamicin. Taken together, the above data exemplifies the present invention.
Claims
1. A method to induce sensitivity to chemical antibiotics in bacterial cells,
- comprising administering one or more bacteriophages in vivo in a human or animal; and
- monitoring induction of the sensitivity by antibiotic sensitivity testing in vitro, both before and after the administering of the one or more bacteriophages;
- wherein the sensitivity is heritable, independent of continuing bacteriophage metabolism within those cells, and does not relate to the destruction of a biofilm to induce sensitivity.
2. (canceled)
3. The method of claim 1 wherein testing is used to select chemical antibiotics for therapeutic use in patients.
4. (canceled)
5. The method of claim 1 wherein there is induction of sensitivity to an aminoglycoside antibiotic.
6-12. (canceled)
13. A method to induce or improve susceptibility to an antibiotic in bacterial cells, comprising:
- administering to a human or animal with a bacterial infection, a bacteriophage preparation comprising one or more bacteriophages, and
- determining the ability of said preparation to increase susceptibility of said cells to said antibiotic is by in vitro antibiotic sensitivity testing in the absence of bacteriophages using samples of bacterial cells from said infection or from another infection by the same or a comparable bacterial strain of the same species subject to identical exposure to said bacteriophage preparation,
- wherein said in vitro antibiotic testing is carried out both before and after the exposure to said bacteriophage preparation.
14. (canceled)
15. The method according to claim 13 wherein said one or more bacteriophages induce or improve susceptibility to an aminoglycoside antibiotic.
16-25. (canceled)
26. The method according to claim 1 further comprising administering at least one antibiotic.
27. The method according to claim 13 wherein the testing is used to select chemical antibiotics for therapeutic use in a patient.
28. The method according to claim 13 further comprising administering at least one antibiotic.
29. A method to treat a bacterial infection in a human or animal, comprising:
- testing bacteria from the bacterial infection for susceptibility to an antibiotic, before the bacteria are exposed to a bacteriophage preparation,
- administering the bacteriophage preparation to the human or animal, to expose the bacteria in the bacterial infection, to the bacteriophage preparation,
- testing the bacteria from the bacterial infection for susceptibility to the antibiotic, after the bacteria are exposed to the bacteriophage preparation, and
- administering the antibiotic to the human or animal, after administering the bacteriophage preparation,
- wherein the bacteriophage preparation comprises one or more bacteriophages, and
- susceptibility of the bacteria to the antibiotic after the bacteria are exposed to the bacteriophage preparation, is greater than susceptibility of the bacteria to the antibiotic before the bacteria are exposed to the bacteriophage preparation.
30. The method according to claim 29 wherein a time period between administering the bacteriophage preparation and administering the antibiotic is one to two days.
31. The method according to claim 29 wherein a time period between administering the bacteriophage preparation and administering the antibiotic is one day to two months.
32. The method according to claim 29, wherein the antibiotic is an aminoglycoside antibiotic.
Type: Application
Filed: May 29, 2013
Publication Date: Feb 6, 2014
Applicant: BIOCONTROL LIMITED (NOTTINGHAM)
Inventor: David Harper (Southampton)
Application Number: 13/904,713
International Classification: A61K 35/76 (20060101); A61K 31/7036 (20060101);
