BENEFICIAL EFFECTS OF BACTERIOPHAGE TREATMENTS

Abstract

The invention elates 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.

Claims

1.-25. (canceled)

26. A method of treating a bacterial infection in a human or an animal, said method comprising: a. administration of a bacteriophage preparation comprising one or more bacteriophages to the human or animal in vivo; b. in vitro monitoring of sensitivity to one or more chemical antibiotic(s) of a sample of bacterial cells from said infection or from another infection wherein the bacterial cells are of the same strain as said infection; and c. administration of said one or more chemical antibiotic(s), when it has been established that said sensitivity to said one or more chemical antibiotic(s) has been induced; and wherein said sensitivity to one or more chemical antibiotic(s) is heritable, is independent of continuing bacteriophage metabolism within those cells, and does not relate to the destruction of a biofilm.

27. The method of claim 26, wherein the ability of said bacteriophage preparation to induce sensitivity of said bacterial cells to said one or more chemical antibiotic(s) is determined by in vitro antibiotic sensitivity testing using samples of bacterial cells from said infection.

28. The method of claim 26, wherein induction of sensitivity has been confirmed for one or more bacterial strains from the individual patient.

29. The method of claim 26, wherein said bacteriophage preparation and said one or more chemical antibiotic(s) are administered at intervals of one day to two months apart.

30. The method of claim 26, wherein said bacteriophage preparation and said one or more chemical antibiotic(s) are administered at intervals of one to four weeks apart.

31. The method of claim 26, wherein said bacteriophage preparation and said one or more chemical antibiotic(s) are administered at intervals of one day to two days apart.

32. The method of claim 26, wherein the bacterial infection is a Pseudomonas aeruginosa bacterial infection.

33. The method of claim 26, wherein said chemical antibiotic comprises or consists of an aminoglycoside antibiotic.

34. The method of claim 26, wherein the antibiotic is selected from the group consisting of amikacin, ceftazadime, ciprofloxacin, gentamicin, meropenem, pipericillin, tazobactam, colistin, aztreonam, imipenem, and tobramycin.

35. The method of claim 26, wherein said bacteriophage preparation is a combined preparation of the six bacteriophages NCIMB 41174, NCIMB 41175, NCIMB 41176, NCIMB 41177, NCIMB 41178 and NCIMB 41179 (as deposited at the National Collection of Industrial And Marine Bacteria, UK on 24 Jun. 2003).

36. The method of claim 26, wherein the animal is a canine.

Description

DETAILED DESCRIPTION

[0013] Thus 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 to individual patient or may be identified in other patients with similar bacterial infections following bacteriophage treatment

[0014] 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 is antibiotics, for sequential use in humans or animals, where this is designed to exert beneficial effects by the induction of sensitivity as noted above.

[0015] 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.

[0016] 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.

EXAMPLES

Induction of Antibiotic Sensitivity in a Veterinary Field Trial:

[0017] 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.

[0018] 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

[0019] BioVet-PA was stored at −80° C. Immediately prior to administration, the product was thawed and warmed in the hand. 0.2 ml (containing 1×10.sup.5 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.

[0020] The procedure was as follows:

[0021] Characterisation (2 to 14 days prior to treatment): [0022] Day 0 Swabs taken from each ear by a veterinary surgeon Laboratory tests carried out using these swabs to confirm presence of Pseudomaonas. aeruginosa,

[0023] If Pseudomonas aeruginosa was not detected, the dog was excluded from the trial [0024] Day 1 If Pseudomonas aeruginosa was detected, the isolates were tested tier sensitivity to BioVet-PA.

[0025] 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.

[0026] Treatment: [0027] Day 0 Ears examined auroscopically to assess their condition. Swabs taken from each ear for microbiological analysis. Dog's core temperature measured [0028] Dog given dose of 0.2ml BioVet-PA into the ear (treatments administered drop-wise using a sterile I mi-capacity syringe, and ear canals then massaged to promote deep penetration). [0029] Day 2 Ears examined to assess their condition. [0030] Swabs taken from each ear for microbiological analysis. [0031] Dog's core temperature measured. [0032] Only where both ears were infected: [0033] Dog given dose of 0.2 ml BioVet-PA into the second ear (treatments administered drop-wise using a sterile l ml-capacity syringe, and ear canals then massaged to promote deep penetration). [0034] Day 4 Only where both ears were infected: [0035] Ears examined to assess their condition. [0036] Swabs taken from each ear for microbiological analysis. [0037] Dog's core temperature measured.

Results:

[0038] 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.

[0039] 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:

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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:

[0044] 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.

[0045] 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.

[0046] 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 June 2003) are as follows:

TABLE-US-00001 Reference NCIMB Deposit Number BC-BP-01 NCIMB 41174 BC-BP-02 NCIMB 41175 BC-BP-03 NCIMB 41176 BC-BP-04 NCIMB 41177 BC-BP-05 NCIMB 41178 BC-BP-06 NCIMB 41179

[0047] These bacteriophages are effective at killing a broad range of P. aeruginosa isolates.

[0048] The study was carried out in two parallel groups of patients with ear infection caused by Pseudomonas aeruginosa. Patients were randomly allocated to receive a single dose of either

[0049] 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

[0050] 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:

[0051] Group 1:

[0052] Patients received a single 0.2 mL dose of BioPhage-PA (containing 1×10.sup.5 pfu by original titration of each of the 6 therapeutic bacteriophages)

[0053] Group 2:

[0054] Patients received a single 0.2 mL dose of placebo (10% v/v glycerol in PBS)

Design Summary

Pre-Study Visit

[0055] 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.

[0056] Treatment period (Days 0-42 inclusive)

[0057] 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.

[0058] Patients returned on Days 7, 21 and 42 for further safety and efficacy tests.

[0059] 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.

[0060] 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

[0061] Each patient attended the unit for the following visits:

[0062] 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.

[0063] If suitable, the patient was enrolled onto trial within 2 weeks of the time that the diagnostic swab was taken.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] Study Day 21: This involved procedures as described for study day 7

[0068] 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

[0069] 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.

[0070] 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:

[0071] Amikacin—30 μg/ml

[0072] Ceftazadime—30 μg/ml

[0073] Ciprotloxacin—5 μg/ml

[0074] Gentamicin—10 μg/ml

[0075] Meropenem—10 μg/ml

[0076] Pipericillin+Tazobactam (7.5:1)—85 μg/ml

[0077] Colistin—25 μg/ml

[0078] Aztreonam—30 μg/ml

[0079] Imipenem—10 μg/ml

[0080] Tobramycin—10 μg/ml

[0081] The test was conducted using lsosensitest agar.

[0082] 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).

TABLE-US-00002 TABLE 2 Antibiotic sensitivity of Pseudomonas aeruginosa: Data from human otitis trial Pre-treatment Day 0 Antibiotic screening (treatment) Day 7 Day 21 Day 42 Amikacin Partially resistant Partially resistant Sensitive Partially resistant Sensitive Gentamicin Resistant Resistant Resistant Resistant Partially resistant Tobramycin Resistant Sensitive Resistant Partially resistant Resistant Meropenem Sensitive Sensitive Sensitive Sensitive Sensitive Imipenem Sensitive Sensitive Sensitive Sensitive Sensitive Ceftazadime Sensitive Sensitive Sensitive Sensitive Sensitive Pipencillin & Sensitive Sensitive Sensitive Sensitive Sensitive Tazobactam Colistin Sensitive Sensitive Sentitive Sensitive Sensitive Aztreonam Sensitive Sensitive Sensitive Sensitive Sensitive Ciprofloxacin Sensitive Sensitive Sensitive Sensitive Sensitive

Summary of the Above Exemplification

[0083] In the veterinary field trial, over a two t© 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).

[0084] 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 testantibiotics monitored (30%), over the longer monitoring period in this trial.

Further Human Trial Results

[0085] 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 to 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.