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Title:
MOLECULAR PROBES FOR DETECTING GRAM-NEGATIVE BACTERIA IN VITRO AND IN VIVO
Document Type and Number:
WIPO Patent Application WO/2016/075483
Kind Code:
A1
Abstract:
A probe is provided comprising a label and a binding moiety, wherein the binding moiety is adapted to bind to gram-negative bacteria, and to substantially not bind to animal cells or gram-positive bacteria. A method of detecting the presence of bacteria in a target area is also provided, which allows the detection of bacteria generally, and the determination of whether that bacteria is gram negative or gram positive.

Inventors:
BRADLEY MARK (GB)
LILIENKAMPF ANNAMARIA HELENA (GB)
AVLONITIS NIKOLAOS (GR)
ESCOBAR MARC VENDRELL (GB)
MCDONALD NEIL ALEXANDER (GB)
HASLETT CHRISTOPHER (GB)
DHALIWAL KANWALDEEP (GB)
WALSH TIMOTHY SIMON (GB)
Application Number:
PCT/GB2015/053454
Publication Date:
May 19, 2016
Filing Date:
November 13, 2015
Export Citation:
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Assignee:
UNIV EDINBURGH (GB)
International Classes:
A61K49/00; C12Q1/22; C12Q1/37; G01N33/52
Domestic Patent References:
WO2014093031A12014-06-19
WO2003079015A12003-09-25
WO2012136958A22012-10-11
Foreign References:
US20120252137A12012-10-04
US6051395A2000-04-18
Other References:
DERIS ZAKUAN Z ET AL: "Probing the Penetration of Antimicrobial Polymyxin Lipopeptides into Gram-Negative Bacteria", BIOCONJUGATE CHEMISTRY, vol. 25, no. 4, April 2014 (2014-04-01), pages 750 - 760, XP002754088, ISSN: 1043-1802
WARSEN A E ET AL: "Simultaneous discrimination between 15 fish pathogens by using 16S ribosomal DNA PCR and DNA microarrays", APPLIED AND ENVIRONMENTAL MICROBIOLOGY JULY 2004 AMERICAN SOCIETY FOR MICROBIOLOGY US, vol. 70, no. 7, July 2004 (2004-07-01), pages 4216 - 4221, XP002754089, DOI: 10.1128/AEM.70.7.4216-4221.2004
LIU C ET AL: "Noninvasive optical imaging of staphylococcus aureus infection in vivo using an antimicrobial peptide fragment based near-infrared fluorescent probes", JOURNAL OF INNOVATIVE OPTICAL HEALTH SCIENCES JULY 2013 WORLD SCIENTIFIC PUBLISHING CO. PTE. LTD SGP, vol. 6, no. 3, 1350026, July 2013 (2013-07-01), pages 1 - 8, XP002753983, DOI: 10.1142/S1793545813500260
LEEVY W MATTHEW ET AL: "Quantum dot probes for bacteria distinguish Escherichia coli mutants and permit in vivo imaging.", CHEMICAL COMMUNICATIONS (CAMBRIDGE, ENGLAND) 28 MAY 2008, no. 20, 28 May 2008 (2008-05-28), pages 2331 - 2333, XP002754090, ISSN: 1359-7345
EL-KOSASY A M ET AL: "Screening and optimization of the reaction of polymyxin B sulphate with NBD-Cl for the synchronous spectrofluorimetric determination of polymyxin B sulphate in human plasma", JOURNAL OF FLUORESCENCE 20150501 SPRINGER NEW YORK LLC USA, vol. 25, no. 3, 1 May 2015 (2015-05-01), pages 695 - 705, XP002753986, DOI: 10.1007/S10895-015-1555-8
AHSAN R. AKRAM ET AL: "A labelled-ubiquicidin antimicrobial peptide for immediate in situ optical detection of live bacteria in human alveolar lung tissue", CHEMICAL SCIENCE, vol. 6, no. 12, 1 January 2015 (2015-01-01), United Kingdom, pages 6971 - 6979, XP055247541, ISSN: 2041-6520, DOI: 10.1039/C5SC00960J
ROGER Y. TSIEN, J.AM. CHEM. SOC, vol. 2014, pages 874
CHASTRE J ET AL., AM J RESPIR CRIT CARE MED., vol. 165, no. 7, 1 April 2002 (2002-04-01), pages 867 - 903
M. TERNON; J. J. DIAZ-MOCHON; A. BELSOM; M. BRADLEY, TETRAHEDRON, vol. 60, 2004, pages 8721
H. J. KNOLKER; T. BRAXMEIER; G. SCHLECHTINGEN, ANGEW. CHEM. INT. ED., vol. 34, 1995, pages 2497
E. KAISER; R. L. COLESCOTT; C. D. BOSSINGER; P. I. COOK, ANALYTICAL BIOCHEMISTRY, vol. 34, 1970, pages 595 - 598
Attorney, Agent or Firm:
HGF LIMITED (GLASGOW) (Belgrave Hall Belgrave Street,Leeds, Yorkshire LS2 8DD, GB)
Download PDF:
Claims:
Claims

1. A probe comprising a label and a binding moiety, wherein the binding moiety is adapted to bind to gram-negative bacteria, and to substantially not bind to animal cells or gram- positive bacteria.

2. The probe according to any claim 1 , wherein the binding moiety is a polymyxin moiety, such as full length polymyxin (SEQ ID NO.4) or a fragment or variant thereof.

3. The probe according to either one of claim 1 or claim 2, wherein the label is a fluorophore.

4. The probe according to either one of claim 1 or claim 2, wherein the label is a radioactive label, or a magnetic resonance label.

5. The probe according to any one preceding claim, wherein the probe comprises a secondary label.

6. The probe according to claim 3, wherein the fluorophore is an environmentally sensitive fluorophore.

7. The probe according to claim 6, wherein the fluorophore is NBD.

8. The probe according to claim 7, wherein the probe comprises a polymyxin moiety and NBD.

9. A method of detecting bacteria and/or fungi in a target area, the method comprising the steps:

(1) providing a first probe adapted to label bacteria and/or fungi;

(2) delivering the first probe to the target area;

(3) illuminating the target area with an appropriate wavelength of light to excite the first probe;

(4) determining whether bacteria and/or fungi has been labelled by the first probe in the target area;

wherein if bacteria is determined to have been labelled by the first probe in the target area the method further comprises the steps: (5) providing a second probe adapted to label gram-negative or gram- positive bacteria only;

(6) delivering the second probe to the target area;

(7) illuminating the target area with an appropriate wavelength of light to excite the second probe;

(8) determining whether the second probe has labelled bacteria in the target area;

wherein, bacteria labelled with the second probe in the target are identified as gram- negative or gram-positive bacteria.

The method according to claim 9, wherein the target area is a portion of tissue within a patient, and the method is carried out in vivo.

The method according to claim 10, wherein the target area is a portion of the lung, on the skin of a patient, in joints, in the circulatory system, epithelial linings such as but not limited to the digestive system or the reproductive system.

The method according to claim 9, wherein the target area is a portion of a cell culture, a tissue sample such as a biopsy sample, a liquid sample such as a bodily fluid sample, or a portion of a medical device such as a catheter or implant, and the method may be carried out in vitro.

The method according to any one of claims 9 to 12, wherein the method of the invention may be carried out by a clinician in situ, at the point of care, and determines whether bacteria and/or fungi is present in a target area, and whether any bacteria is gram- negative or gram-positive.

The method according to any one of claims 9 to 13, wherein, if bacteria is detected in the target area, and an appropriate antibiotic is given to the patient, the method may be carried out to determine the efficacy of the antibiotic.

The method according to any one preceding claim, wherein, if bacteria is detected in the target area, and an appropriate surgical procedure is given to the patient, the method may be carried out to determine the efficacy of the procedure.

16. The method according to any one of claims 9 to 13, wherein, if fungi is detected in the target area and an appropriate antifungal agent is given to the patient, the method may be carried out to determine the efficacy of the antifungal. 17. The method according to any one of claims 9 to 16, wherein the first probe comprises at least one first probe element.

18. The method according to claim 16, wherein the or each first probe element comprise a first label and a first binding moiety.

19. The method according to any one of claims 9 to 18, wherein the second probe comprises at least one second probe element.

20. The method according to claim 19, wherein the or each second probe element comprises a second label and a second binding moiety.

21. The method according to claim 20, wherein the first and/or second label are a fluorophore, and the first and/or second probes are adapted to fluorescently label bacteria and/or fungi.

22. The method according to claim 20, wherein the first and/or second label are a radioactive label.

23. The method according to claim 22, wherein the first and/or second label are a magnetic resonance label.

24. The method according to any one of claims 9 to 23, wherein the first and/or second probe comprise a secondary label. 25. The method according to claim 21 , wherein the first probe comprises a first fluorophore and the second probe comprises a second fluorophore.

26. The method according to claim 25, wherein the first and/or second fluorophore emit light in the near infrared or infrared range of the spectrum.

27. The method according to claim 26, wherein the fluorophore is detectable via photoacoustic imaging.

28. The method according to any one of claims 25 to 27, wherein the first and/or second fluorophore is an environmentally sensitive fluorophore, such that the intensity/quantum yield of fluorescence of the first and/or second fluorophore depends on the surroundings of the first and/or second fluorophore.

29. The method according to claim 28, wherein the quantum yield or intensity of fluorescence of the first and/or second fluorophore is higher in a hydrophobic environment, such as within a cell membrane.

30. The method according to any one of claims 25 to 29, wherein the first and/or second fluorophore are independently 7-nitrobenz-2-oxa-1 ,3-diazole (NBD), malachite green, a styryl-based dye, Cascade Yellow, prodan (aka 1-Propanone, 1-(6-(dimethylamino)- 2-naphthalenyl), Dansyl (aka. 5-(dimethylamino)naphthalene-1-sulfonyl), Dapoxyl, PyMPO (aka. 1-(3-(Succinimidyloxycarbonyl)Benzyl)-4-(5-(4-Methoxyphenyl)Oxazol- 2-yl)Pyridinium, pyrene and diethylaminocumarin, or derivatives or variants thereof.

31. The method according to claim 30, wherein the first and/or second fluorophore is a NBD moiety.

32. The method according to any one of claim 21 and claims 25 to 31 , wherein the fluorophore of one or more of the probe elements within the plurality of probe elements has a longer fluorescent lifetime and the longer fluorescent lifetime of the fluorophore allows the detection of the probe over background autofluorescence.

33. The method according to claim 32, wherein the fluorophore is azadioxatnangulene (ADOTA) or diazaoxatriangulene (DAOTA), or a derivative thereof.

34. The method according to any one of claims 9 to 33, wherein the method comprises the step of observing the target area under fluorescent light to determine the morphology of any infective agent (bacteria or fungi) identified in the target area.

35. The method according to claim 34 comprising the step of observing the target area under fluorescent light to identify microbes within the target area, and the first and second probes are used to determine the identity of those microbes.

36. The method according to any one of claims 9 to 35, wherein the first binding moiety is a first bacteria binding moiety.

37. The method according to claim 36, wherein the first bacteria binding moiety binds to substantially all bacteria but not to animal cells, or does so weakly.

38. The method according to any one of claims 9 to 37, wherein the second binding moiety is a second bacteria binding moiety.

39. The method according to claim 38, wherein the second bacteria binding moiety selectively binds to substantially gram-negative bacteria, but not bind to gram-positive bacteria, or animal cells such as mammalian cells, or does so weakly.

40. The method according to claim 39, wherein the second probe is a probe according to any one of claims 1 to 8.

41. The method according to claim 38, wherein the second bacteria binding moiety selectively binds to substantially gram-positive bacteria, but not bind to gram-negative bacteria, or animal cells such as mammalian cells, or does so weakly.

42. The method according to any one of claims 9 to 41 , wherein the first and/or second binding moiety is a first and/or second fungi binding moiety and may bind to fungi hyphae.

43. The method according to any one of claims 9 to 42, wherein the first binding moiety is a ubiquicidin moiety, such as the full length ubiquicidin (SEQ ID N0.1) or a fragment or variant thereof.

44. The method according to claim 43, wherein the first binding moiety is the ubiquicidin fragment of amino acids 29 to 41 (UBI29-41 , SEQ ID NO.2).

45. The method according to claim 43, wherein the binding moiety is UBI29-41 comprising a substitution of a norleucine amino acid for the original methionine amino acid (UBI29- 41 Nle, SEQ ID NO.3). The method according to any one of claims 9 to 45, wherein the second binding moiety is a polymyxin moiety, such as full length polymyxin (SEQ ID NO.4), or a fragment or variant thereof.

The method according to any one of claims 9 to 46, wherein the first fluorophore and the second fluorophore are different.

The method according to claim 47, wherein the first fluorophore has an emission peak that is significantly different than the emission peak of the second fluorophore.

The method according to either one of claims 47 and 48, wherein the step of delivering the second probe to the target area may be carried out at the same time as the step of delivering the first probe to the target area, and the target area may be illuminated with light of a suitable wavelength or suitable wavelengths to excite the first and second fluorophores, such that it is possible to determine whether bacteria has been labelled by the first probe and whether that bacteria has been labelled by the second probe at the same time.

The method according to any one of claims 9 to 49, wherein the first and second probe comprise a first and second probe element respectively, the first probe element comprising a first fluorophore and a first binding moiety, and the second probe element comprising a second fluorophore and a second binding moiety, wherein the first and/or second probe comprise a quencher connected to the at least one first and/or second probe element by a cleavable linker.

The method according to claim 50, wherein the cleavable linker comprises an enzyme cleavable peptide sequence, and the linker is cleaved when a cleaving enzyme cleaves the enzyme cleavable peptide sequence.

The method according to either one of claims 50 or 51 , wherein the method may determine whether:

(a) a cleaving agent is present, indicated by an increase in fluorescence of the first probe, or a change in colour of fluorescence of the first probe;

(b) whether bacteria and/or fungi is present, indicated by the labelling of the bacteria and/or fungi with the first probe; and if bacteria is present

(c) whether that bacteria is gram-negative or gram-positive depending on whether or not it is labelled by the second probe. The method according to either one of claims 51 or 52, wherein the cleaving agent i a cleaving enzyme.

The method according to claim 53, wherein the cleaving enzyme is elastase and cleavage of the enzyme cleavable peptide sequence is indicative of the presence of elastase.

Description:
MOLECULAR PROBES FOR DETECTING GRAM-NEGATIVE BACTERIA IN

VITRO AND IN VIVO

Field of the Invention The invention relates to the field of molecular probes, more specifically to molecular probes for the detection of micro-organisms such as bacteria and fungi.

Background of the Invention There is a growing burden of infections worldwide and accurate diagnosis remains a cornerstone to providing accurate treatment.

Patients within hospitals and in healthcare generally, are at risk of infections. Hospital acquired infections (HAI) are becoming more common, and the ability to respond to such infections rapidly and accurately, is increasingly important. The immune system of patients that are seriously ill are often at least partially compromised, making the patient especially vulnerable to HAIs.

Ventilated patients in critical care, and all immunocompromised patients, are especially vulnerable to HAI, and one of the most devastating HAI remains ventilator associated pneumonia (VAP). VAP remains notoriously difficult to accurately diagnose and inappropriate treatment has been shown to be harmful to patients. Accordingly, VAP has a high mortality rate, significant morbidity and remains a burden on healthcare resources. In terms of diagnosis, clinical signs of fever, increased oxygen dependence and tachycardia remain as non-specific means of detecting inflammation or acute lung injury, while the gold standard remains pulmonary biopsy, which is an invasive and rarely utilised investigation owing to the intrinsic invasive nature of the test. Other methods such as bronchoalveolar lavage remain controversial, and while non-culture methods have not had any significant impact or robust validation. Therefore, alternative approaches are required that will allow the accurate and timely diagnosis of VAP, which will allow immediate healthcare decisions to be made and, with appropriate therapy, improve patient outcomes.

Currently clinicians are faced with significant uncertainty in relation to when and if to commence antimicrobial treatment, the choice of agents to use, and if treatment begins, when to de-escalate therapy. These issues are barriers to effective antibiotic stewardship because of the proven association between delayed and inadequate antibiotic therapy and adverse clinical outcomes. Molecular imaging technologies can allow the use of microbial specific tracers and when combined with imaging modalities such as positron emission tomography (PET), have the ability to delineate infective from sterile sites. This approach, however is not be applicable for some patient groups, such as intensive care cohorts where a point of care diagnostic test would be required, and the administration of radioactive agents can be problematic and restrictive. These also involve radiation and are not readily applicable to imaging outside hospital settings. An alternative approach in the art is the application of optical probes to allow direct visualisation of a target area of a patient, through the use of an endoscope. WO 2003/079015 in the name of Visen Medical, Inc., discloses optical imaging probes for identifying and characterising normal and diseased tissues with regards to altered metabolic activity. WO 2012/136958 in the name of the present applicant discloses branched dyes to allow visualisation of cells in vivo by an increase in fluorescence when the dye is internalised by specific cell types.

However, there remains a need for improved methods of determining the cause of inflammation that will allow in situ, point of care determination of whether a patient's condition is due to an infection, and if so, whether that infection is bacterial or fungal.

Therefore, an object of the present invention is to provide improved imaging/sensing methods suitable for rapid and accurate point of care diagnosis.

Statements of the Invention

According to a first aspect of the invention there is provided a method of detecting bacteria and/or fungi in a target area, the method comprising the steps:

(1) providing a first probe adapted to label bacteria and/or fungi;

(2) delivering the first probe to the target area;

(3) illuminating the target area with an appropriate wavelength of light to excite the first probe;

(4) determining whether bacteria and/or fungi has been labelled by the first probe in the target area;

if bacteria is determined to have been labelled by the first probe in the target area the method further comprises the steps: (5) providing a second probe adapted to label gram-negative or gram-positive bacteria only;

(6) delivering the second probe to the target area;

(7) illuminating the target area with an appropriate wavelength of light to excite the second probe;

(8) determining whether the second probe has labelled bacteria in the target area; wherein, bacteria labelled with the second probe in the target are identified as gram- negative or gram-positive bacteria. If bacteria are not labelled by the first probe, there is no bacteria within the target area, and it is not necessary to carry our steps (5) to (8). If bacteria is labelled by the first probe in the target area, carrying out steps (5) to (8) determines whether that bacteria is gram-negative or gram-positive. For example, if the second probe is adapted to label gram-negative bacteria, bacteria that has been determined to be labelled by the first probe in step (4) and is then determined to have been labelled by the second probe in step (8) is gram-negative bacteria. Bacteria that has been determined to be labelled by the first probe in step (4) and is then determined not to have been labelled by the second probe (step (8)) is gram-positive bacteria.

Therefore, the method of the invention allows the determination of the presence of bacteria in the target area, and if bacteria is present in the target area, whether that bacteria is gram- negative or gram-positive. In addition, the method allows the determination of the presence of fungi in the target area.

In embodiments where the target area comprises gram-negative bacteria and gram-positive bacteria, the first probe will label all bacteria in the target area, and the second probe will label either the gram-positive or gram-negative bacteria, thereby identifying both gram of bacteria either directly (by labelling them) or indirectly (by not labelling them).

Preferably, the target area may be a portion of tissue within a patient, and the method may be carried out in vivo. For example, the target area may be a portion of the lung of a patient, and the method may be carried out using the working channel of a bronchoscope to both deliver the probe to the target area, to deliver light to the target area, and to detect fluorescence from the target area. Alternatively, individual instruments may be used to deliver the probe to the target area, to deliver light to the target area and to detect fluorescence from the target area. For example, fluorescence may be detected from the tissue of a patient using optical emission microscopy (OEM), such as fibered confocal fluorescence microscopy (FCFM).

The target area could be on the skin of a patient, in joints, in the circulatory system, epithelial linings such as but not limited to the digestive system or the reproductive system, or any area accessible by intraoperative procedure in a human, and the probes may be delivered and/or observed via an endoscope, or by direct spraying, for example.

Alternatively, the target area may be a portion of a cell culture, a tissue sample such as a biopsy sample, or a liquid sample such as a bodily fluid sample, and the method may be carried out in vitro.

Preferably, in embodiments where the target area is a portion of tissue of a patient, the patient is a human patient. However, the patient may be a non-human animal such as equine, ovine, bovine, or rodent, for example.

In many areas of healthcare, such as critical or intensive care, it is necessary to identify whether inflammation of the lungs, for example, is sterile, bacterial or fungal, and if bacterial, whether that bacteria is gram-positive or gram-negative, or a mixture of both. However, it is not typically possible to use standard methods such as PET scans to investigate the inflammation for patients that cannot be safely moved, especially patients that are being ventilated. Accordingly, clinicians often do not have sufficient information to confidently diagnose the cause of the inflammation and to prescribe a suitable corrective course of action. For example, if the inflammation is sterile, giving the patient antibiotics will not help, and may have adverse side effects. Again, if the bacteria is gram-negative and antibiotics are given to the patient that are only effective against gram-positive bacteria or vice versa, the antibiotics will not clear the bacteria, and may have adverse and harmful side effects.

The method of the invention may be carried out by a clinician in situ, at the point of care, and determines whether bacteria and/or fungi is present in a target area, such as within the lungs, or a portion of the lungs of the patient, and whether any bacteria is gram-negative or gram- positive. Accordingly, the method of the invention advantageously provides the clinician with the information they need to confidently determine the cause of inflammation, and to determine the best course of action, such as giving an appropriate antibiotic or antifungal to the patient, for example. If bacteria is detected in the target area, and an appropriate antibiotic is given to the patient, the method may be carried out to determine the efficacy of the antibiotic. For example, a reduction in the number of bacteria, or the absence of bacteria in the target area typically indicates that the course of treatment is effectively clearing the bacterial infection. Similarly, if fungi is detected in the target area and an appropriate antifungal agent is given to the patient, the method may be carried out to determine the efficacy of the antifungal.

Preferably, the first probe comprises at least one first probe element. The first probe may comprise at least two first probe elements. The first probe may comprise at least three first probe elements.

The or each first probe element may comprise a first label and a first binding moiety.

Preferably, the second probe comprises at least one second probe element. The second probe may comprise at least two second probe elements. The second probe may comprise at least three second probe elements.

The or each second probe element may comprise a second label and a second binding moiety. The first and/or second label may be a fluorophore, and the first and/or second probes may be adapted to fluorescently label bacteria and/or fungi.

The first and/or second label may be a radioactive label. The radioactive label may comprise a radionucleotide. The radioactive label may comprise one or more of 18 F, 64 Cu, 68 Ga, "Tc, 111 In, 123 l, 124 l, 90 Y, 177 Lu, 11 C, 14 C, 3 H, 32 P, 33 P, 186 Re, 188 Re, or 86 Zr.

The first and/or second label may be a magnetic resonance label or core. The magnetic resonance label or core may be Fe, Mn, or Gd, for example. The first and or second probe may comprise a secondary label. For example, the first probe may comprise a fluorophore and radioactive label, or the first probe may comprise a fluorophore and a secondary fluorophore etc. Further variations and combinations will be appreciated by the person skilled in the art.

Preferably, the first probe comprises a first fluorophore and the second probe comprises a second fluorophore. The first and/or second fluorophore may be any suitable fluorophore, such as fluorescein, or a derivative thereof such as fluorescein, a cyanine based fluorophore, a rhodamine based fluorophore, or a boron-dipyrromethene (BODIPY) based fluorophore.

The first and/or second fluorophore may emit light in the red, near-infrared or infrared range of the spectrum. For example, the first and/or second fluorophore may be Cy7 or similar fluorophore. Infrared light may pass through the majority of tissue to allow the whole body, or a large portion of the body, to be imaged. In addition, absorption of the infrared light may lead to thermal expansion that is detectable via photoacoustic imaging, thereby allowing infective agents present to be detected indirectly.

Preferably, the first and/or second fluorophore is an environmentally sensitive fluorophore, such that the intensity/quantum yield of fluorescence of the first and/or second fluorophore depends on the surroundings of the first and/or second fluorophore. For example, the quantum yield or intensity of the first and/or second fluorophore may be different in a free aqueous environment than when the first and/or second fluorophore is in a hydrophobic environment, such as within a cell membrane. Preferably, the quantum yield or intensity of fluorescence of the first and/or second fluorophore is higher in a hydrophobic environment, such as within a cell membrane. Accordingly, the intensity of fluorescence emitted by the first and/or second fluorophore increases when the first and/or second fluorophore is within a cell membrane, such as when the first and/or second probe element is bound within a cell membrane of bacteria, for example. Therefore, the first and/or second fluorophore may be particularly effective when used to fluorescently label cells via their cell membranes in the method of the invention. For example, the first and/or second fluorophore may be 7-nitrobenz-2-oxa-1 ,3-diazole (NBD), malachite green, a styryl-based dye, Cascade Yellow, prodan (aka 1-Propanone, 1-(6- (dimethylamino)-2-naphthalenyl), Dansyl (aka. 5-(dimethylamino)naphthalene-1-sulfonyl), Dapoxyl, PyMPO (aka. 1-(3-(Succinimidyloxycarbonyl)Benzyl)-4-(5-(4-

Methoxyphenyl)Oxazol-2-yl)Pyridinium, pyrene and diethylaminocumarin, or derivatives or variants thereof.

Preferably, the first and/or second fluorophore is a NBD moiety. NBD has been found by the inventors to be a particularly suitable fluorophore for the probe of the invention, providing strong fluorescence when the probe is bound to bacteria or at least some fungi, and a low background fluorescence, allowing clear and reliable labelling of bacteria and at least some fungi compared to other fluorophores tested. The fluorophore of one or more of the probe elements within the plurality of probe elements may have a long fluorescent lifetime and the long fluorescent lifetime of the fluorophore allows the detection of the probe over background autofluorescence. For example, during use, the fluorescence of the fluorophore may have a lifetime significantly longer than the autofluorescence of the background of a target area such that the fluorescence from the fluorophore is readily distinguishable from the fluorescence of the background.

For example, the fluorophore is azadioxatriangulene (ADOTA) dye or diazaoxatriangulene (DA OTA), or derivatives thereof.

Fluorescence from the first and/or second fluorophore may be imaged directly by the fluorescence emitted from the first and/or second fluorophore being directed onto a detecting device, such as a charge-coupled device (CCD) or a complementary metal-oxide- semiconductor (CMOS) device, for example. Fluorescence from the first and/or second fluorophore may be imaged indirectly. For example, the fluorescence may be converted into acoustic waves by using photoacoustic imaging. Photoacoustic imaging may allow high- resolution images of the target area to be generated. In embodiments where the first and/or second fluorophore emit light in the near infrared or infrared range of wavelengths (~700nm - 1 mm), the whole or substantially the whole, of the body of the patient may be imaged. Alternatively, in embodiments where the the first and/or second fluorophore emit light in the visible range of wavelengths (~390nm - 700nm), a specific target area of the patient may be imaged, and the light may be delivered to and received from the target area via a fibre optic, for example. The method may comprise the step of observing the target area using photoaccoustic ultrasound to determine the identity of the infective agent (bacteria or fungi) detected in the target area. The method may comprise the step of observing the target area using photoaccoustic instruments to identify microbes within the target area, and the first and second probes may be used to determine the identity of those microbes using direct fluorescent detection.

The method may comprise the step of observing the target area under white or fluorescent light to determine the morphology of any infective agent (bacteria or fungi) identified in the target area. The method may comprise the step of observing the target area under white or fluorescent light to identify microbes within the target area, and the first and second probes may be used to determine the identity of those microbes. For example, it is additionally envisaged that if bacteria is identified by white or fluorescent light or auto-fluorescence, carrying out steps (5) to (8) determines whether that bacteria is gram-negative or gram-positive. The first binding moiety may selectively bind to at least some bacteria, and not bind to animal cells, such as mammalian cells, for example, or does so weakly. Accordingly, the first binding moiety may be a first bacteria binding moiety. The first bacteria binding moiety may bind to substantially all bacteria but not to animal cells, or does so weakly. The second binding moiety may selectively bind to at least some bacteria, and not bind to animal cells, such as mammalian cells, for example, or does so weakly. Accordingly, the second binding moiety may be a second bacteria binding moiety. The second bacteria binding moiety may selectively bind to substantially gram-negative bacteria, but not bind to gram- positive bacteria, or animal cells such as mammalian cells, or does so weakly. The second bacteria binding moiety may selectively bind to substantially gram-positive bacteria, but not bind to gram-negative bacteria, or animal cells such as mammalian cells, or does so weakly.

The first and/or second binding moiety may selectively bind to fungi, and not bind to animal cells, such as mammalian cells, for example. Accordingly, the first and/or second binding moiety may be a first and/or second fungi binding moiety and may bind to fungi hyphae.

The first and/or second binding moiety may selectively bind to at least some bacteria and to at least some fungi, and not bind to animal cells such as mammalian cells. Accordingly, the first and/or second probe may allow the detection of fungi and/or at least some bacteria in a target area. For example, the binding moiety may be adapted to bind to fungal hyphae of A. fumigatus.

The first binding moiety may be a ubiquicidin moiety, such as the full length ubiquicidin (SEQ ID N0.1) or a fragment or variant thereof. Preferably, the ubiquicidin moiety is capable of selectively binding to bacteria over mammalian cells. The first binding moiety may be a fragment of ubiquicidin comprising at least 10 consecutive amino acids, or at least 12 consecutive amino acids of ubiquicidin. For example, the first binding moiety may be the ubiquicidin fragment of amino acids 29 to 41 (UBI29-41 , SEQ ID NO.2). The first binding moiety may be a ubiquicidin moiety comprising one or more substitutions. The or each substitution may be a conservative substitution, and have little or preferably, no effect on the bacteria binding properties of the ubiquicidin moiety. The or each substitution may provide increased stability to the ubiquicidin moiety against degradation or oxidation. For example, the binding moiety may be UBI29-41 comprising a substitution of a norleucine amino acid for the original methionine amino acid (UBl29-4i Nie, SEQ ID NO.3).

Ubiquicidin is a mammalian anti-microbial peptide present in airway epithelial cells, gut mucosa and in macrophages. Ubiquicidin binds specifically to the cell membrane of prokaryotes such as bacteria and does not bind to mammalian cells.

The second binding moiety may selectively bind to gram-positive bacteria and may not substantially bind to gram-negative bacteria. Preferably, the second binding moiety selectively binds to gram-negative bacteria and does not substantially bind to gram-positive bacteria. Accordingly, in embodiments where the second binding moiety selectively binds to gram- negative bacteria, the second probe may be randomly distributed in a target area that does not comprise gram-negative bacteria, and be localised to the cell membranes of any gram- negative bacteria present in the target area, thereby labelling the gram-negative bacteria. Therefore, the second probe may be adapted to selectively indicate the presence, or absence, of gram-negative bacteria in a target area.

For example, the second binding moiety may be a polymyxin moiety, such as full length polymyxin (SEQ I D NO.4), or a fragment or variant thereof. Polymyxin binds selectively to gram-negative bacteria, and therefore, a bacterial binding moiety comprising a polymyxin moiety will selectively bind the respective probe element to gram negative bacteria only, and thereby allow detection of any gram negative bacteria within the target area. The second binding moiety may be a fragment of polymyxin comprising at least 6 consecutive amino acids, or at least 8 consecutive amino acids of polymyxin. The second binding moiety may be a polymyxin moiety comprising one or more substitutions. The or each substitution may be a conservative substitution, and have little or preferably, no effect on the gram-negative bacteria binding properties of the polymyxin moiety. The or each substitution may provide increased stability to the polymyxin moiety against degradation or oxidation. In embodiments where the first and second fluorophores are the same (for example, the first fluorophore is N BD and the second fluorophore is NBD), the method may comprise the step of washing the target area or otherwise removing the first probe from the target area before the second probe is contacted to the target area. Therefore, contamination of target area by the first probe when determining whether bacteria in the target area is gram-negative or gram- positive is prevented or at least minimised. The first fluorophore and the second fluorophore may be different. In embodiments where the first and second fluorophores are different, preferably, the first fluorophore has an emission peak that is significantly different than the emission peak of the second fluorophore. (i.e. the fluorescence of the first fluorophore is at a different wavelength to the fluorescence of the second fluorophore and therefore, it is possible to selectively detect fluorescence from the first fluorophore and fluorescence from the second fluorophore). For example, the first fluorophore may be NBD and the second fluorophore may be TAMRA.

In embodiments where the first fluorophore is different to the second fluorophore, the step of delivering the second probe to the target area may be carried out at the same time as the step of delivering the first probe to the target area, and the target area may be illuminated with light of a suitable wavelength or suitable wavelengths to excite the first and second fluorophores, such that it is possible to determine whether bacteria has been labelled by the first probe and whether that bacteria has been labelled by the second probe at the same time. Therefore, it is possible to detect bacteria and/or fungi and determine whether any detected bacteria is gram-negative or gram-positive in a single procedure.

Preferably, the fluorescence of the fluorophore is detectable directly or indirectly above the background or, where present, auto-fluorescence in the target area. The autofluorescence of the indigenous cells or tissue within the target area may have a shorter fluorescent lifetime than the fluorophore of the first probe. The autofluorescence of the indigenous cells or tissue within the target area may reduce over time at a faster rate than that of the fluorophore of the probe. Accordingly, fluorescence observed in the target area that reduces more slowly over time may be indicative of the first probe, and fluorescence observed in the target area that reduces more quickly over time may be indicative of autofluorescence. For example, the fluorophore may be azadioxatriangulene (ADOTA) or diazaoxatriangulene (DAOTA), or a derivative thereof.

In embodiments where the first and/or second probe comprise at least two probe elements, the first and/or second probe may comprise a core and each of the at least two probe elements may be connected to the core.

By the term "core" we refer to a common moiety that joins the plurality of probe elements to form a single unit. Accordingly, the core could be a single atom, or comprise a functional group, a saturated or unsaturated hydrocarbon chain or a polyglygol (linear, branched, or cyclical), a peptide sequence, or a polymer. Typically, each probe element of the at least two probe elements comprises a fluorophore and a binding moiety. For example, a first probe comprising three first probe elements comprises three first fluorophores and three first binding moieties. A first and/or second probe comprising at least two fluorophores may be expected to be brighter per probe, thereby allowing the same signal to be obtained from fewer probes. Accordingly, a lower dose of first and/or second probes may be expected to be required to be applied to the target area to allow bacteria to be reliably detected. However, in at least some embodiments, the at least two fluorophores may self-quench, such as a first fluorophore dissipating the energy absorbed by a second fluorophore non-radiatively as heat. Therefore, a first or second probe comprising at least two fluorophores may or may not be fluorescent, and indeed may be expected by the person skilled in the art to have poor fluorescence due to self-quenching.

Surprisingly, the inventors have found that at least some fluorophores that self-quench in probes comprising a plurality of fluorophores are unable to self-quench when in a hydrophobic environment. For example, a first probe comprising a plurality of NBD fluorophores shows little fluorescence in an aqueous environment, but fluoresces brightly when at least partially embedded within a cell membrane.

Optical probes known in the art are typically poorly retained in a target area in vivo where challenging conditions can lead to degradation or oxidation of the probes, and/or protease breakdown, and as a result, the probes are unable to remain bound to their target to allow reliable detection of the target. For example, optical probes used for imaging within the lung where there are high surfactant concentrations, typically do not allow their targets to be detected. Further challenges within the lung include "washing off" of probes by the circulating fluid therein. Surprisingly, the inventors have found that first and/or second probes comprising a plurality of are more stable to oxidation, degradation and protease activity than probes known in the art, and that such probes allow reliable detection of bacteria in vivo, even in challenging conditions such as those found in the lung using the method of the invention. The first and/or second probe may comprise a quencher connected to the at least one first/and or second probe element by a cleavable linker. The or each first and/or second fluorophore may be substantially fluorescently quenched by the quencher when the at least one first and/or second probe element is connected to the quencher by the cleavable linker; wherein the at least one first and/or second probe element is separated from the quencher when the cleavable linker is cleaved. Preferably, the cleavable linker comprises an enzyme cleavable peptide sequence, and the linker is cleaved when a cleaving enzyme cleaves the enzyme cleavable peptide sequence.

Accordingly, cleavage of the enzyme cleavable peptide sequence typically corresponds to cleavage of the linker, and thereby cleavage of the quencher from the at least one probe element of the first and/or second probe. Accordingly, in embodiments where the cleavable linker comprises an enzyme cleavable peptide sequence, the term "cleavage of the linker" refers to cleavage of the enzyme cleavable peptide sequence unless stated otherwise.

Optical probes known in the art typically are either fluorescent at all times (so-called "always on" fluorophores), and it is the location of these probes that is determined, or the probes change their fluorescence when their environment is changed, be that the removal of or separation from a quencher (for fluorescence resonance energy transfer, or FRET, based probes, for example), or being internalised by a cell. However, in embodiments where the first and/or second probes comprise one fluorophore, or comprise at least two fluorophores that do not self-quench, the first and/or second probes comprising a quencher and a cleavable linker advantageously change their fluorescence due to whether the cleavable linker has been cleaved by a cleaving agent, such as a cleaving enzyme, and the location of the first and/or second probes when observed can indicate the presence of bacteria and/or fungi. Accordingly, the method of the invention may provide more specific and more detailed information than methods using optical probes known in the art.

In embodiments where the first probe comprises a quencher and a cleavable linker, the method of the invention may determine whether (a) the cleaving agent is present, indicated by an increase in fluorescence of the first probe, or a change in colour of fluorescence of the first probe, (b) whether bacteria and/or fungi is present, indicated by the labelling of the bacteria and/or fungi with the first probe, and if bacteria is present, (c) whether that bacteria is gram-negative or gram-positive depending on whether or not it is labelled by the second probe. The cleaving agent may be produced or expressed by the indigenous cells within the target area. The cleaving agent may be produced or expressed by additional cells produced by the patient that have migrated to the target area, such as neutrophils, for example. The cleaving agent may be produced or expressed by an infective cell in the target area, such as bacteria or fungi, for example.

Preferably, the cleaving agent is a cleaving enzyme. The cleaving enzyme is elastase and cleavage of the enzyme cleavable peptide sequence is indicative of the presence of elastase. Typically, the elastase is neutrophil elastase and the elastase is produced or expressed by neutrophils. Typically, the elastase is the active form of the enzyme capable of proteolytic cleavage. Neutrophils typically target sites within the body that are undergoing an inflammatory response, either pathophysiological or part of normal function. Therefore, the presence of neutrophils in a target area within a patient is indicative that the tissue or a portion of the tissue within the target area is inflamed. In embodiments where the patient is a human, preferably the cleaving enzyme is human neutrophil elastase (HNE). Therefore, fluorescence, or an increase in fluorescence of the first and/or second fluorophore resulting from cleavage of the linker is indicative of the presence of neutrophil elastase, thereby indicative of the presence of neutrophils in the target area, and therefore is indicative of inflammation of the tissue within the target area. Accordingly, the method of the invention is adapted to determine whether the tissue within a target area is inflamed.

Therefore, advantageously, the method of the invention is a method of determining whether (a) tissue is inflamed, (b) whether bacteria and/or fungi is present, and (c) whether any bacteria present is gram-positive or gram-negative, that may be carried out in vivo or in vitro. Accordingly, the method may provide information to allow a health care professional to determine the appropriate treatment to clear the inflammation. Preferably, the cleavable linker is dimensioned such that the quencher is sufficiently close to the or each first and/or second fluorophore to quench the or each first and/or second fluorophore. Typically, the quencher is less than 10nm away from the or each first and/or second fluorophore. Preferably, the quencher is less than 5nm away from the or each first and/or second fluorophore.

Typically, the enzyme cleavable peptide linker is a peptide sequence that is a or the cleavage site of the cleaving enzyme. Preferably, the or each cleavage site of the cleaving enzyme comprises multiple amino acids. In embodiments where the cleaving enzyme is elastase, preferably, the enzyme cleavable peptide sequence comprises the amino acid sequence A-A- P-V (i.e. alanine-alanine-proline-valine, or Ala-Ala-Pro-Val) or E-E-I-Nle-R-R. The enzyme cleavable peptide sequence may comprise one or more additional amino acids either side of the sequence AAPV in positions x and/or y, such as xAAPVy, xAAPV, or AAPVy, for example. Alternatively, the cleaving enzyme may be a matrix metalloproteinase (MMP), such as MMP- 9 and the enzyme cleavable peptide sequence may comprise G-P-K-G-L-K-G. The cleaving enzyme may be proteinase 3 and the enzyme cleavable peptide sequence may comprise V- A-D-C-A-D-Y. The cleaving enzyme may be cathepsin G and the enzyme cleavable peptide sequence may comprise A-A-P-F, or F-V-T-Gnf-S-W (where Gnf= 4-guanidine-L- phenylalanine). The cleaving enzyme may be a caspase and the enzyme cleavable peptide sequence may comprise D-E-V-D. In alternative embodiments, the cleavable linker may be cleaved by a reactive oxygen species, such as superoxide (Of) or hydrogen peroxide (H2O2), generated by the presence of microbes, or inflammation processes such as activated neutrophils. Accordingly, a reactive oxygen species may be a cleaving agent. For example, the linker may be a modified boronic acid based linker, such as that described in J.Am. Chem. Soc, 2014, 874, Roger Y. Tsien.

In embodiments where the first and/or second probe comprises a quencher, the or each first and/or second fluorophore of the or each first and/or second probe element may be any fluorophore that may form a FRET pair with a suitable quencher. Typically, the fluorophore and the quencher are chosen as a pair to ensure that they have appropriate excitation and emission spectra for the transfer of energy from the fluorophore to the quencher (i.e. they form a FRET pair). For example, typical fluorophore/quencher pairs include Cy3/Cy5, Cy3/QSY21 , fluorescein/tetramethylrhodamine, fluorescein/methyl red, cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP), etc. Further examples of FRET pairs may be readily identified by the skilled person.

The quencher may be a dark quencher. A dark quencher is a moiety that is able to accept energy from an excited fluorophore and dissipate that energy non-radiatively, typically as heat or acoustic energy, for example. Therefore, when the fluorophore/quencher pair are sufficiently close together and irradiated with a wavelength of light that is within the excitation spectra of the fluorophore, the quencher dissipates the energy absorbed from the light by the fluorophore in a non-radiative manner and no fluorescence is observed. In this way, the dark quencher suppresses the fluorescence of the fluorophore.

In embodiments where the quencher is a dark quencher, the quencher may be methyl red, dimethylaminoazobenzenesulfonic acid (DABSYL), Iowa black FQ or Iowa black RQ (Integrated DNA Technologies, Inc. Iowa, USA), BHQ1 , BHQ2 or BHQ3, for example. The quencher may be a fluorescent quencher. A fluorescent quencher is a moiety that is able to accept energy from an excited fluorophore and to radiate that energy. Accordingly, fluorescent quenchers are typically fluorophores, and emit light at a different wavelength to that emitted by the fluorophore from which they have accepted energy. In this way, the change in colour of fluorescence is indicative of the relative proximity of the quencher to the fluorophore.

In embodiments where the quencher is a fluorescent quencher, the quencher may be rhodamine or a derivative thereof, such as carboxytetramethylrhodamine (TAMRA) for example, fluorescein, or a derivative thereof, a cyanine fluorophore or BODIPY fluorophores.

In embodiments where the quencher is a fluorescent quencher, the quencher may be adapted to label neutrophils. Accordingly, in embodiments where the cleaving enzyme is produced by neutrophils, the method of the invention may be adapted to determine the presence of a cleaving enzyme, such as elastase, to determine the presence of neutrophils, to label any bacteria and/or fungi that may be present, and to determine whether the bacteria is gram- positive or gram-negative.

For example, in embodiments where the cleaving enzyme is HNE, the fluorescent quencher may be taken up by endocytosis by neutrophils that are activated by the presence of bacteria or by stimuli such as calcium ionophore, for example. Accordingly, the fluorescent quencher may selectively label neutrophils within the target area. The inventors have observed that fluorescent quenchers, such as TAMRA, are selectively taken up by neutrophils and are not taken up, or are taken up to a much lesser extent, by bacteria and by other inflammatory cells, such as monocytes. Without wishing to be bound by theory, the inventors speculate that this observed difference in uptake of the fluorescent quencher may be due to the higher endocytic and pinocytic activity in highly phagocytic cells such as neutrophils.

In embodiments where the or each first and/or second fluorophore of the or each first and/or second probe element is NBD, the quencher is preferably a moiety that is able to accept energy from NBD to form a FRET pair with the NBD. Preferably, the quencher is methyl red or tetramethylrhodamine or a derivative thereof. For example, the quencher may be carboxytetramethylrhodamine (TAMRA). Further suitable fluorophore/quencher pairs may be determined by the person skilled in the art.

Preferably, in embodiments where the method is a method of determining whether tissue in the target area is inflamed, and whether inflammation of the tissue is sterile or infective, the first probe comprises a quencher and a cleavable linker. Accordingly, the method may detect inflammation and whether that inflammation is infective/caused by bacteria and/or fungi present in the target area, before the second probe is delivered to the target area. In embodiments where the first and/or second probes comprise a quencher and a cleavable linker, the binding moiety of the first and/or second probe may be at the distal end of the probe element from the linker. Alternatively, the bacterial binding moiety may be at the proximal end of the probe element, adjacent to the linker. The fluorophore may be connected to the linker via the binding moiety. The fluorophore may be directly connected to the linker.

The binding moiety may bind to bacteria and/or fungi prior to cleavage of the cleavable linker. Alternatively, the binding moiety may bind to bacteria and/or fungi after cleavage of the cleavable linker. In embodiments where the first and/or second probe comprises at least two probe elements, a core, a quencher and a cleavable linker, the core connects each probe element to the linker. Accordingly, when the cleavable linker is cleaved (e.g. the enzyme cleavable peptide sequence of the linker is cleaved by a cleaving enzyme) the probe is split into the quencher, and the at least two probe elements, and the core, and the at least two probe elements remain connected to the core.

Typically, the provision of a first and/or second probe comprising at least two probe elements, corresponds to a probe comprising at least two fluorophores and at least two binding moieties. According to a second aspect of the invention there is provided a probe for use in the method of the first aspect of the invention comprising a label and a binding moiety.

The label may be a fluorophore, and the probe may be adapted to fluorescently label bacteria and/or fungi.

The label may be a radioactive label. The radioactive label may comprise a radionucleotide. The radioactive label may comprise one or more of 18 F, 64 Cu, 68 Ga, "Tc, 111 In, 123 l, 124 l, 90 Y, 177 Lu, 11 C, 14 C, 3 H, 32 P, 33 P, 186 Re, 188 Re, or 86 Zr. The label may be a magnetic resonance label or core. The magnetic resonance label or core may be Fe, Mn, or Gd, for example. The probe may comprise a secondary label. For example, the probe may comprise a fluorophore and radioactive label, or the probe may comprise a fluorophore and a secondary fluorophore etc. Further variations and combinations will be appreciated by the person skilled in the art.

Preferably, the fluorophore is an environmentally sensitive fluorophore. Preferably, the fluorophore is NBD.

Preferably, the binding moiety is a ubiquicidin moiety, such as full length ubiquicidin (SEQ ID N0.1) or a fragment or variant thereof. Preferably, the ubiquicidin moiety is a ubiquicidin fragment comprising amino acids 29 to 41 of full length ubiquicidin (UBI29-41 , SEQ ID NO.2). Preferably, the ubiquicidin moiety comprises an amino acid substitution. Preferably, the ubiquicidin moiety comprises the amino acid substitution of the methionine residue of the ubiquicidin moiety to norleucine (ΙΙ ΒΙΝΙΘ, SEQ ID NO.3).

The fluorophore may be connected to the binding moiety directly. Alternatively, the fluorophore may be connected to the binding moiety by a spacer. The spacer may be a hydrocarbon chain, an ether, a polymer, a polyethylglycol (PEG), a poly glycol or similar. The spacer may be a peptide. In embodiments where the spacer is a peptide, the peptide may be 1-10 amino acids in length, 1-20 amino acids in length, or 1-30 amino acids in length.

Preferably, the probe comprises a core and a plurality of binding moieties connected to the core. Preferably, the probe comprises at least three binding moieties. For example, in a preferred embodiment, the probe comprises a core, three fluorophores and three binding moieties, and each of the three binding moieties comprises ΙΙ ΒΙΝΙ Θ and each of the three fluorophores is NBD.

According to a third aspect of the invention there is provided a probe for use in the method of the first aspect of the invention comprising a label and a binding moiety adapted to bind to gram-negative bacteria or gram-positive bacteria, and to substantially not bind to animal cells or gram-positive bacteria or gram-negative bacteria respectively.

The label may be a fluorophore, and the probe may be adapted to fluorescently label bacteria and/or fungi. The label may be a radioactive label. The radioactive label may comprise a radionucleotide. The radioactive label may comprise one or more of 18 F, 64 Cu, 68 Ga, "Tc, 111 In, 123 l, 124 l, 90 Y, 177 Lu, 11 C, 14 C, 3 H, 32 P, 33 P, 186 Re, 188 Re, or 86 Zr. The label may be a magnetic resonance label or core. The magnetic resonance label or core may be Fe, Mn, or Gd, for example.

The probe may comprise a secondary label. For example, the probe may comprise a fluorophore and radioactive label, or the probe may comprise a fluorophore and a secondary fluorophore etc. Further variations and combinations will be appreciated by the person skilled in the art.

Preferably, the fluorophore is an environmentally sensitive fluorophore. Preferably, the fluorophore is NBD.

Preferably, the binding moiety is adapted to bind to gram-negative bacteria, and to substantially not bind to animal cells or gram-positive bacteria.

Preferably, the binding moiety is a polymyxin moiety, such as full length polymyxin (SEQ ID NO.4) or a fragment or variant thereof. The polymyxin moiety is typically a truncated polymyxin where at least a portion of the hydrocarbon tail of the polymyxin has been removed. For example, the polymyxin moiety may be derived from polymyxin B1 and the 6- methyloctanoic acid group may have been removed, or the polymyxin may be derived from polymyxin B2 and the 6-methylheptanoic acid may have been removed.

The fluorophore may be connected to the binding moiety directly. Alternatively, the fluorophore may be connected to the binding moiety by a linker. The linker may be a saturated or unsaturated hydrocarbon chain, an ether, a polymer, a polyethylglycol (PEG), a poly glycol, a poly ether or similar. The linker may be a peptide. In embodiments where the linker is a peptide, the peptide may be 1-10 amino acids in length, 1-20 amino acids in length, or 1-30 amino acids in length.

In a preferred embodiment, the probe comprises a polymyxin moiety and NBD. Preferably, the probe of the second aspect is suitable to be used as the first probe of the method of the first aspect, and the probe of the third aspect is suitable to be used as the second probe of the method of the first aspect. Accordingly, the invention extends in a fourth aspect to the use of the probes of the second aspect of the invention and the probes of the third aspect in the method of the first aspect of the invention.

In a preferred embodiment, the probe of the second aspect comprises a core and three binding moieties and three NBD fluorophores, and each of the three binding moieties comprises UBlNie, and the probe of the third aspect comprises a polymyxin moiety and NBD. Preferred and optional features of the first aspect of the invention are preferred and optional features of the probes of the second, third and fourth aspects.

Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the accompanying drawings.

Brief Description of the Figures

Figure 1 : A flow diagram showing an example method of the invention

Figure 2A: Elastase dependant bacterial labelling scheme. The fluorophore on the bacterial binding component is quenched by a dark quencher meaning that no fluorescence is seen upon binding to bacteria. Cleavage of the elastase specific sequence AAPV liberates the bacterial binding component (PV-NBD-UBI) from the dark quencher methyl red (MR). Figure 2B: Elastase dependant bacterial labelling in vitro. Methicillin sensitive staphylococcus aureus (MSSA), counterstained with FM-464(red), co-incubated with isolated human neutrophils demonstrating bacterial labelling in the presence of neutrophils (green), which is inhibited in the presence of an elastase inhibitor.

Figure 3A: Elastase dependant bacterial labelling scheme. The fluorophore on the bacterial binding component is quenched by a different fluorophore of a longer wavelength meaning that no fluorescence is seen upon binding to bacteria. Cleavage of the elastase specific sequence AAPV liberates the bacterial binding component (PV-NBD-UBI) from the quencher (TAMRA). TAMRA reports the presence of activated neutrophils by endocytic uptake.

Figure 3B: Elastase dependant bacterial labelling in vitro. Pseudomonas aueriginosa (PA) counterstained with PKH 660 (purple) was incubated with T AM R A- A A P V- N B D- P MX and demonstrated no labelling in the presence of unactivated neutrophils (top panel), bacterial labelling (shown in green) in an elastase dependent manner and labelling of activated neutrophils (shown in red) in an elastase independent manner. Figure 3C: Elastase dependent fluorescence increase of TAMRA following cleavage.

Spectrophotometer data to demonstrate the unexpected observation of an increase in fluorescence of TAMRA-AA (cleaved compound) compared to TAMRA-AAPV-NBD-PMX in an elastase dependent manner, and inhibited with an elastase inhibitor (ex 525, Em 580). Figure 4: Fluorophore choice impacts on ability to image labelled bacteria with Improved signal to noise for NBD constructs over Fluorescein (FAM). A) Bacteria (MSSA), counterstained with FM-464 (shown in red), and incubated with FAM-UBI or NBD- UBI demonstrating an improved signal to noise ratio with NBD which allowed the majority of bacteria to be detected (shown in green). This was compared with FAM-UBI which detected a minor subset of clumped bacteria. B) NBD-UBI also exhibits specificity for bacteria (Pseudomonas aeruginosa) over mammalian cells (white arrow).

Figure 5: FAM-PMX does not allow sufficient signal to noise to detect bacteria in vitro.

A) Pseudomonas aeruginosa (PA), counterstained with FM-464 (red) and incubated with A549 cells (human lung epitheial cells in purple) demonstrate bacterial labeling is not seen above background but also there is no labeling of the epithelial calls. B) NBD-PMX (green) with a nuclear counterstain Syto 82 (red) allows bacterial labelling and retains selectivity of labeling over mammalian cells (purple).

Figure 6: Emission spectra of probes confirms increase in fluorescence in hydrophobic environments. A) Emission spectra shown for NBD-U BIdend (5μΜ) and NBD-U BlNie (15μΜ) in the presence of PBS or DMSO (hydrophobic environment) when excited at 488nm wavelength. B) Emission spectra shown for NBD-PMX (10μΜ) in the presence of PBS or DMSO (hydrophobic environment) when excited at 488nm wavelength. The fluorescence of both probes greatly increases under hydrophobic conditions as would be present upon binding to the bacterial membrane target.

Figure 7: NBD-UBIdend labels bacteria selectively over mammalian cells in vitro: A) Example images of a clinically relevant bacterial panel with NBD-U BIdend (5μΜ; green) counterstained with the fluorescent generic cellular DNA dye Syto-82 (red) imaged by laser scanning confocal microscopy. B) Quantification of bacterial panel labelling NBD-U BIdend 5μΜ (optimal concentration) where every bacteria in the panel is brighter when compared with MSSA and NBD-U BInie 10μΜ (optimal concentration) C) Flow cytometry data with unstained bacteria (red), NBD-U BI N ie 15μΜ (blue) and NBD-U BI de nd 5μΜ labelled bacteria (orange). D) MSSA and NBD-U BIdend (5μΜ; green), counterstained with Syto-82 and merge showing human neutrophils (red arrow) and E) A549 cells (red arrow) demonstrating no labelling of mammalian cells. Representative images shown, n≥3 for all experiments.

Figure 8: NBD-PMX selectively labels Gram-negative bacteria in vitro. Example images of the bacterial panel with NBD-PMX imaged by laser scanning confocal microscopy. A) Bacterial panel with NBD-PMX 1 μΜ (green) and counterstain with syto-82 (red). Gram-positive bacteria (bounded by red box) display minimal/no labelling compared with Gram-negative bacteria. B) Quantification of bacterial panel with NBD-PMX 1 μΜ with Gram-positive bacteria in white bars and Gram-negative in black bars, showing high intensity selective labelling of Gram-negative bacteria compared with Gram-positive. All Gram-negatives showed a statistically significant increase over all Gram-positives. C) Flow cytometric evaluation of bacterial labelling with NBD-PMX showing unstained bacteria (red) and NBD-PMX (blue) labelled bacteria. No significant labelling of Gram-positive bacteria (bounded by red box). D) MSSA and NBD-PMX 1 μΜ (green), counterstained with Syto-82 and merge showing human neutrophils (red arrow) and E) A549 cells (red arrow) demonstrating no labelling of mammalian cells (reproduced from figure 4). Representative images shown, n≥3 for all experiments. Figure 9: Ex vivo model of bacterial infection. A) Ovine lungs were harvested, ventilated and placed in a neonatal incubator with ambient temperature of 37°C. Pulmonary segments were instilled with PBS (control) or bacteria via bronchoscopy. Probes were then instilled and segments imaged with fibered confocal fluorescence microscopy (FCFM) using 488nm Laser Scanning Unit (LSU). B) Ex vivo ovine model demonstrates viable bacteria 5 hours following instillation. Bacteria (MSSA) instilled into segments of ovine lung and lavaged at 1 , 3 and 5 hours (n=3), plated for CFU/ml and counted following 16 hour incubation.

Figure 10: Representative FCFM images of bacteria in the distal lung demonstrating a distinctive punctate pattern. A) Instillation of PKH labelled S. aureus, Calcein labelled S. aureus or GFP-expressing strain of S. aureus, generate a punctate pattern of fluorescence when segments are imaged using FCFM. Images used to generate positive control for subsequent in situ labelling experiments. Representative images shown, n=3 for all experiments. B) PKH dyes do not stain by-stander cells. Confocal images showing epithelial cells in co-culture with bacteria (S. aureus) that have been counterstained with Syto-82 (red) as well as PKH660 (purple). Bystander mammalian cells (red arrows) are labelled with Syto- 82 that has leached from the labelled bacteria but there is no transfer of PKH660.

Figure 11 : NBD-UBI fails to label bacteria in the ovine lung. There was no consistent labelling of bacteria in the ovine lung confirmed by counterstaining bacterial and imaging on a spectrally distinct imaging system. A) Lung segment with PKH660 labelled MSSA at 1 hour post-instillation. B) Control segment (instilled with PBS and no bacteria) imaged at 660nm. C) The same segment imaged at 488nm following 10μΜ NBD-UBI instillation. However, the same experiment with counterstained PKH660 labelled K. pneumoniae (Gram negative) shown in panel D in purple, with NBD-PMX added demonstrating the same punctate signal.

Figure 12. NBD-UBIdend detects bacteria in situ in the distal ovine lung. Representative FCFM images of NBD-UBIdend showing that no punctate signal is seen in control segments whereas the distinctive punctate signal described in Figure 10 above is seen above background fluorescence in segments instilled with bacteria. Furthermore, agarose beads alone show no fluorescence (control beads) but when beads are coated with bacteria a signal is seen when labelled in situ in the ovine lung. Representative images shown, n≥3 for all experiments.

Figure 13: NBD-PMX selectively labels Gram-negative bacteria in situ in the ovine lung.

A) Control segments and Gram-positive segments show no punctate signal, whereas Gram- negative instilled segments show signal identical to positive control. B) Lavage counts from segments demonstrating no significant difference in counts between segments confirming the presence of bacteria. C) Agarose beads alone show no fluorescence but when beads are coated with bacteria a signal is seen when labelled in situ in the ovine lung. Representative images shown, n≥3 for all experiments.

Figure 14: NBD-UBIdend and NBD-PMX are resistant to 'wash off allowing detection of bacteria in the ovine lung. Bacteria pre-labelled with either NBD-U BIdend or NBD-PMX were readily visualised by FCFM when instilled into the ovine lung. However, when bacteria pre- labelled with linear NBD-U BInie are instilled, no punctate signal is seen. Labelling was confirmed by imaging bacterial suspensions (left hand panels) before instillation into the lung and imaging by FCFM (right hand panels). Note the lower SNR for NBD-U BlNie labelled bacteria in suspension. Representative images shown, n=3 for each experiment.

Figure 15: NBD-UBIdend retains fluorescence when wash performed. A) The bacterial panel was imaged by laser scanning confocal microscopy in the continued presence of NBD- U BIdend (black) and following PBS wash (white). Quantification demonstrates fluorescence retention above linear NBD-U BI with MSSA in the continued presence of probe (red-line). n≥3, with three random field-of-views assessed for each experiment. B) Bacterial panel imaged with NBD-PMX in the continued presence of probe (black) or following PBS wash (white) demonstrating higher fluorescence retention of all Gram-negative bacteria above NBD-PMX with Gram-positives in the continued presence of probe. n≥3, with three random field-of-views assessed for each experiment.

Figure 16: NBD-UBI de nd is stable in ALI BALF. Mass spectrometry MALDI-TOF analysis demonstrated stability of NBD-U BInie in the presence of saline (arrow indicates correct peak seen at mass of 1949) but degradation in the presence of ALI lavage (no peak seen at mass 1949 but arrows show predictable degradation compounds) when co-incubated for 30 minutes. By contrast NBD-U BIdend remains stable when assessed by FTMS (data shown represents a theoretical plot and an experimental plot demonstrating the peaks correspond indicating presence of compound). NBD-PMX is stable in ALI BALF. MALDI-TOF analysis demonstrated stability of NBD-PMX in the presence of saline or ALI lavage (arrows indicates correct peaks seen at mass of 1291-1293) when co-incubated for 30 minutes. Figure 17: NBD-UBIdend retains higher fluorescence on bacteria in ovine lavage than eqimolar equivalent of linear. A) MSSA imaged by laser scanning confocal microscopy in the presence or absence of ovine lavage with NBD-U BlNie (15 μΜ) and NBD-U BIdend (5 μΜ). Both NBD-U BI compounds have reduced fluorescence in ovine lavage but NBD-U BIdend retains higher fluorescence intensity. B) In contrast, PA3284 imaged in the presence or absence of ovine lavage with NBD-PMX (1 μΜ) demonstrates no significant reduction of fluorescence intensity. Representative images shown, n=3.

Figure 18: NBD-UBIdend and NBD-PMX successfully image bacteria in a surfactant-rich environment. Ratio of bacterial fluorescence intensity: surfactant fluorescent intensity quantified from confocal microscopy images. For equimolar NBD concentrations NBD-U BIdend and NBD-PMX have significantly higher bacterial: surfactant intensity than NBD-U BI n i e . Figure 19: Image Processing Algorithms delineate 'positive' and 'negative' frames from FCFM data A) raw data for a frame that is deemed positive or negative. B) Without preprocessing, spot-detection is ineffective. Following estimation of background noise C), high- pass filtering D) and appropriate thresholding E), the current algorithm detect minimal punctate signal in negative frames F).

Figure 20: Automated Image Processing of entire FCFM videos (up to 3500 frames) demonstrates detection of bacteria. Analysis of NBD-U BIdend videos with thresholding at 80 spots per frame and an arbitrary cut-off of 10% enables objective detection of bacteria using a combination of Laplacian of Gaussian (LoG) and Difference of Gaussians (DoG) positive frames. Analysis of NBD-PMX videos with thresholding at 80 spots per frame and an arbitrary cut-off of 10% enables objective detection of) Gram status using a combination of LoG and DoG positive frames. n=4 for all analyses, except S pneumoniae where n=3. Statistical analysis when compared to control.

Figure 21 : Bacteria detected in the presence of human lung tissue autofluorescence. A) FCFM images of human lung tissue with PA3284 demonstrating elastin autofluorescence and no bacterial signal (left) and human lung tissue with PA3284 co-incubated with NBD-PMX (right) demonstrating bacterial signal. B) Live confocal microscopy images (merge) showing merge images of nucleic acid counterstain (red) with no NBD-PMX labelling. Bacteria in right panel can be clearly seen, labelled with NBD-PMX and above the background fluorescence. C) The fluorescent lifetime of NBD is significantly longer than that of the autofluorescence of the indigenous cells of the lung, measured by laser excitation at 488nm and time resolved single photon counting spectrometer, and this can be used to identify the probes in vivo and increase signal to noise of sensing and imaging.

Figure 22: No haemolysis seen with NBD-UBI d end or NBD-PMX up to 100 μΜ. Haemolysis assay (n=3) demonstrating no haemolysis on concentrations up to 100μΜ. Positive control was 0.2% Triton-X and values corrected to represent 100% haemolysis for Triton-X. Figure 23: A) NBD-PMX shows no organ toxicity in a 2 week single instillation model in mice. Representative histology images (x100) for NBD-PMX compared to PBS control animals at 48 hours and 14 days. No differences from control were seen in any group (blindly scored by a consultant histopathologist).

B) NBD-UBIdend shows no organ toxicity in a 2 week single instillation model in mice. Representative histology images (x100) for NBD-U BIdend compared to PBS control animals at 48 hours and 14 days. No differences from control were seen in any group (blindly scored by a consultant histopathologist).

Figure 24: Fluorescence Life Time Imaging easily distinguishes lung tissue from bacteria. Left panel shows confocal at 488nm excitation of lung and bacteria (dots) in lung tissue. FLIM (right panel) shows clear differences in imaging of lung tissue (green) and bacteria (blue). Imaging performed using NBD-UBIdend and S. aureus on human lung tissue.

Specific Description of Embodiments of the Invention

While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as "a", "an" and "the" are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.

In the following description of example embodiments of the invention, binding moieties comprising a polymyxin moiety are given the code "PMX", and binding moieties comprising a ubiquicidin moiety are given the code "U BI". For example, embodiments of the invention that comprise a plurality of probe elements comprising the NBD and the modified ubiquicidin fragment U BInie ("Dendron Probes") is referred to as NBD-U BIdend. First Example Method

In an example of the invention, a detailed method will now be described. This is an example embodiment of the invention and does not limit the scope of the invention.

A microdose of 500μΙ_ of a first probe (comprising 1 C^g of probe) is delivered to a target area within the lungs of a patient using a bronchoscope. The first probe comprises three ubiquicidin fragments (ΙΙ ΒΙ ΝΙ Θ , acting as binding moieties) and three N BD fluorophores, and therefore is adapted to bind to bacteria and/or fungi specifically, rather than the native cells within the lungs of the patient.

Light of an appropriate wavelength (for example, around the excitation maximum of N BD, 488nm) is delivered to the target area via a fiber-optic within the bronchoscope. Light from the target area is collected by a second fiber-optic within the bronchoscope and incident to a charge-coupled device (CCD) via a confocal optical arrangement or widefield arrangement. Images of the target area produced by the CCD are analysed to determine whether bacteria and/or fungi have been labelled by the first probe.

If bacteria and/or fungi are clearly visible (i.e. labelled by the first probe), it is determined that bacteria and/or fungi is present in the target area and therefore, the clinician may determine that an antibiotic and/or antifungal should be given to the patient.

If bacteria is clearly visible, in order to determine the type of antibiotic required, a further investigation is required.

A bolus of saline buffer is delivered to the target area to wash away the first probe. Once the level of fluorescence in the target area has reached the background level, a microdose of 500μί of a second probe (comprising 1C^g of probe) is delivered to the target area. The second probe comprises a polymyxin moiety (PMX), (acting as a binding moiety) and an N BD fluorophore, and is therefore adapted to selectively bind to gram-negative bacteria specifically, rather than gram-positive bacteria or the native cells within the lungs of the patient.

Again, light of an appropriate wavelength (for example, around the excitation maximum of N BD, 488nm) is delivered to the target area via a fiber-optic within the bronchoscope. Light from the target area is collected by a second fiber-optic within the bronchoscope and incident to a charge-coupled device (CCD) via a confocal optical arrangement or widefield arrangement. Images of the target area produced by the CCD are analysed to determine whether bacteria has been labeled by the second probe. The person skilled in the art will appreciate that any suitable detector may be used, such CMOS detectors, for example, and the method is not limited to the use of CCD detectors. If bacteria are clearly visible (i.e. labelled by the second probe), it is determined that the bacteria is gram-negative bacteria and therefore, the clinician may determine that an antibiotic against gram-negative bacteria should be administered. If no bacteria are clearly visible, it is determined that the bacteria is gram-positive and therefore, the clinician may determine that an antibiotic against gram-positive bacteria should be administered.

Accordingly, the clinician may confidently determine the cause of any inflammation in the lungs of the patient, whether that be sterile inflammation, caused by a single infective agent, or by a mixture of infective agents. Therefore, the clinician may decide the appropriate course of action to take, without any requirement to significantly move the patient, or to remove any ventilator the patient may be using, as a bronchoscope may be fed to the lung of the patient via the ventilator.

Second Example Method In an alternative method, a microdose of 500μΙ_ of a first probe (comprising of 2C^g probe) comprising a TAMRA moiety (acting as a fluorescent quencher), a linker comprising the peptide sequence AAPV, an NBD fluorophore, and a ubuquicidin moiety (ΙΙ ΒΙ ΝΙ Θ , acting as a binding moiety) is delivered to a target area of the lung of a patient and illuminated as per the first example method.

Fluorescence of the TAMRA only is indicative that no HNE is present in the target area, and that therefore the tissue within the target area is not inflamed. Fluorescence of TAMRA and NBD with no labeling of bacteria and/or fungi by the NBD is indicative of the presence of HNE (the linker has been cleaved by HNE to allow the NBD to fluoresce). Fluorescent labelling of human neutrophils by TAMRA is indicative of the presence of human neutrophils within the target area, and fluorescent labelling of bacteria and/or fungi by NBD is indicative of bacteria and/or fungi within the target area.

Accordingly, the method allows inflammation to be detected, including sterile inflammation, bacterial inflammation, and fungal inflammation. If bacteria is detected, the second probe of the first example method may be used to determine whether that bacteria is gram-negative or gram-positive, thereby allowing the determination of whether inflammation is present, whether the inflammation is caused by bacteria and/or fungi, and if the inflammation is caused by bacteria, whether that bacteria is gram-negative or gram- positive.

Third Example Method

In a third example, a microdose of a first probe according to either the first or second example method and a microdose of a second probe comprising a polymyxin moiety and a malachite green fluorophore may be delivered at the same time. Alternatively, the second probe may comprise a cyanine dye such as cy3 or cy5, for example.

Accordingly, fluorescence of the first probe, the second probe, and, if the first probe of the second example method is used, the fluorescent quencher (TAMRA) can be analysed at once, and the presence of bacteria and/or fungi and, if bacteria is detected, whether that bacteria is gram-negative or gram-positive can be determined simultaneously.

Characterisation of appropriate Probes to be used in the Method of the Invention

Example 1

A first example of elastase sensitive probes (shown schematically in Figure 2A) comprises methyl red (acting as a dark quencher) connected via the peptide sequence AAPV (acting as the linker) to 7-nitrobenz-2-oxa-1 ,3-diazole (NBD) and the ubiquicidin fragment UBI29-41 (together acting as the probe element). Accordingly, the NBD fluorescence is quenched by the methyl red and no fluorescent signal will be observed (i.e. the methyl red and NBD are acting as a FRET pair) and therefore, whether or not the probe is bound to any bacteria that may be present in the target area via UBI29-41 , no signal is observed using confocal microscopy.

When the probe is in the presence of human neutrophil elastase (HNE), such as in inflamed tissue, HNE cleaves the peptide sequence of the linker, to thereby free the probe element from the methyl red quencher. Accordingly, the NBD fluorophore is no longer quenched and produces a fluorescent signal. In addition, due to the environmental sensitivity of the NBD, the signal produced is greatly amplified if the NBD is in a hydrophobic environment, such as within a cell membrane. Figure 2B shows confocal images for a sample comprising the probe and that has been incubated with a co-culture of bacteria and neutrophils. These images confirm the ability of the probe to be cleaved by neutrophil derived HNE and to label bacteria. When the elastase inhibitor sivelestat was added to the medium, no labelling was observed as the probe remains uncleaved (and therefore the NBD is quenched by the methyl red).

Example 2 In a second example, the probe comprises carboxytetramethylrhodamine (TAMRA, acting as a fluorescent quencher) connected to NBD and polymyxin (acting as the probe element) via the peptide sequence AAPV (acting as the linker). Accordingly, the NBD fluorescence is quenched by the TAMRA to give rise to a fluorescent signal from TAMRA (i.e. the TAMRA is accepting the energy absorbed by the NBD and is itself fluorescing, TAMRA and NBD are acting as a FRET pair). Accordingly, whether or not the polymyxin has bound to any bacteria that may be present, only a signal from the TAMRA is observed.

Once the probe has been cleaved by elastase, the bacteria are labelled by the probe element due to the fluorescence of NBD (Figure 3A), whereas the neutrophils, once activated, are labelled by the TAMRA moiety (Figure 3B). Furthermore, we demonstrate the fluorescence of the cleaved TAMRA compound increases in an elastase dependent manner and are inhibited with an elastase inhibitor such as sivelestat ("S", Figure 3C).

For the reporting of bacteria we synthesised a probe comprising the bacterial probe element only, and substituting the methionine of norleucine, "NBD-UBlNie". This probe was compared to the same bacterial detecting moiety with another 'always on' fluorophore, fluorescein (FAM) and showed an improved signal-to-noise on live benchtop confocal microscopy for the NBD reported (Figure 4). Furthermore, it was confirmed that the labelling is specific to bacteria and not cell membranes in general. For example, Figure 4 shows that isolated human neutrophils were not labelled by the NBD-UBlNie probe, whilst the bacteria were labelled by the NBD-UBlNie probe.

To confirm the same would be observed for the PMX bacterial detecting moiety we constructed NBD-PMX and FAM-PMX demonstrating an improved signal-to-noise with NBD-PMX over FAM-PMX and confirm this construct is also specific to mammalian cells (Figure 5). "Branched/Dendron" or "Multivalent" Probes

In a further example of the invention, a probe comprising a core and three probe elements connected to the core (a "three branch" probe) was prepared (NBD-UBIdend), each probe element comprising a NBD-UBlNie moiety.

To confirm the fluorescent reporter NBD retains its characteristics when coupled with our peptide moieties we measured the fluorescence of the compounds in conditions to mimic a hydrophobic environment (DMSO). Linear NBD-UBI N ie, NBD-UBI de nd and NBD-PMX were excited at 488 nm (Biotek fluorescent plate reader) and demonstrated significant increase in fluorescence when the probes were in the presence of dimethyl sulfoxide (DMSO) (hydrophobic environment) when compared to phosphate buffered saline (PBS) (Figure 6) confirming environmentally sensitive fluorescent reporting. Surprisingly, NBD-UBIdend demonstrated the same fluorescence increase as linear when using eqimolar concentrations of NBD despite the fact that there were three copies of NBD per probe.

A panel of bacteria which represent >70% of VAP causing pathogens (Chastre J et al. Am J Respir Crit Care Med. 2002 Apr 1 ; 165(7) :867-903) (Gram-negative; P. aeruginosa (two strains), A. baumannii, S. maltophilia, K. pneumoniae, E. coli and H. influenzae. Gram- positive: Methicillin Resistant S. aureus (MRSA), Methicillin Sensitive S. aureus (MSSA) and S. pneumoniae) (strain list in Table 3 below) were labelled with NBD-UBIdend. Labelling was observed with variable intensity (Figures 7A and B). Nevertheless all bacteria were brighter than MSSA labelled with linear NBD-UBI N ie (Figure 7B) and on flow cytometry NBD-UBI de nd demonstrated increased labelling over linear NBD-UBInie (Figure 7C). Furthermore, NBD- UBIdend did not label human neutrophils or A549 cells supporting prokaryotic selectivity. (Figure 7D and E).

NBD-PMX incubated with bacteria, demonstrated significantly higher fluorescence on Gram- negative bacteria (P. aeruginosa, A. baumannii, S. maltophilia, K. pneumoniae, E. coli and H. influenzae) than Gram-positive bacteria (MRSA, MSSA and S. pneumoniae) (p<0.05) on confocal analysis (Fig 8A and B), which was confirmed by flow cytometry (Figure 7C). Furthermore, there was no labelling of human neutrophils or A549 cells. (Figure 8D and E).

NBD-UBIdend and NBD-PMX were assessed for in situ specificity and sensitivity in an ex vivo ovine model of bacterial infection (Figure 9). In this model, the instillation of PKH fluorescent dye-labelled bacteria into the distal ovine lung and imaged with FCFM reveals a characteristic and distinctive pattern of punctate fluorescence in each field of view (Figure 10A). We observed identical patterns with Calcein-labelled bacteria and Green Fluorescent Protein- expressing S. aureus which were instilled into the lung and imaged with FCFM (Figure 10A). This pattern is reproduced identically by instilling bacteria "pre-labelled" with NBD-UBIdend and NBD-PMX into lung segments. Following this thorough characterisation of the model and positive controls, we instilled PBS or VAP-relevant bacteria into distinct segments within the ex vivo lung model, followed by the microdosed delivery of the probes. We demonstrated that the linear NBD-UBlNie could not label bacteria in situ despite the ability in vitro (Figure 11). For these experiments the assays were repeated with counterstained gram-negative bacteria (K. pneumoniae) which demonstrated labelling with NBD-PMX (Figure 1 1). However, In segments instilled with bacteria we demonstrated the same signal as seen with the 'positive controls' when NBD-UBIdend is instilled (P. aeruginosa, S. aureus, E. coli and K. pneumonia) but minimal/no signal with PBS control, confirming the ability to label bacteria in situ and image using the FCFM system (Figure 12). In segments instilled with gram-negative bacteria, P. aeruginosa (laboratory strain PA01 and clinical VAP isolate J3284), K. pneumoniae and E. coli, we have demonstrated the same signal as in the 'positive controls' when NBD-PMX is instilled but no signal in segments with PBS or gram-positive bacteria MSSA, MRSA and S. pneumoniae (Figure 13). In these experiments we confirmed the equal density of gram-positive and gram-negative bacteria in all of the segments imaged with NBD-UBIdend and NBD-PMX by bronchoalveolar lavage (demonstrating no difference in CFU/ml between segments) and through counterstained bacteria imaged on a Laser Scanning Unit (LSU) at 660nm.

To further demonstrate in situ bacterial detection and to assess the ability of the probes to image bacterial aggregation, we embedded bacteria in agarose beads which were then instilled into the lung. Microdosed probe instillation and FCFM imaging demonstrated that bacterial beads are clearly and exclusively detected whereas control beads (beads without bacteria) are not (Figures 12 and 13). In the distal lung, there is likely to be significant and rapid dissipation of the probes immediately after delivery. Therefore, it is imperative that probe-bacterial labelling remains persistent under these conditions. NBD-UBIdend-labelled bacteria retain labelling upon probe 'wash-off', as is seen for NBD-PMX. When instilled into the ovine lung, bacteria pre-labelled with NBD-UBInie are undetectable by FCFM whereas bacteria pre-labelled with NBD-UBIdend or NBD-PMX are readily visualised (Figure 14). As such, this resistance to 'wash-off represents a surrogate indicator of probe-bacterial affinity which appears to be an absolute requirement for distal lung in situ labelling. Currently, both NBD-UBIdend and NBD-PMX are resistant to wash-off whilst most of the other structural variants do not. Hence, bacteria labelled with both NBD-UBIdend and NBD-PMX retain sufficient intensity of fluorescence upon probe dissipation that occurs in the distal lung (Figure 15). Secondly we assessed stability of the probes in broncholalveolar lavage fluid (BALF) from patients with Acute Lung Injury (ALI) by Fourier Transform Mass Spectrometry (FTMS) and Matrix-Assisted Laser Desorption/lonization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) analysis. NBD-UBI N ie, NBD-UBI de nd and NBD-PMX were incubated for 30 minutes with BALF (Figure 16). NBD-UBIdend and NBD-PMX demonstrate stability with peaks corresponding exactly to the predicted theoretical spectra of the intact probes readily identified in both saline and BALF incubated samples. Predicted spectra of breakdown products were also not seen. By contrast breakdown products of the NBD-UBlNie were seen indicating instability in lavage fluid. Thus, probe stability and retention of function in the context of the 'inflamed' highly enzymatically-active distal lung environment is a key determinant of in vivo utility. This was confirmed by imagining in the presence of ovine lavage, which demonstrated higher fluorescence intensity of NBD-UBIdend over the linear compound, and no reduction in intensity for NBD-PMX when imaged in ovine lavage (Figure 17).

In vitro experiments were conducted using lung surfactant constituents to investigate the ability of probes to preferentially detect bacteria in the presence of large amounts of surfactant. The nature of the fluorescent reporter (NBD) incorporated in NBD-UBIdend and NBD-PMX, suggested the possibility of fluorescent activation in the hydrophobic 'rich' surfactant environment. A suspension of surfactant constituents in buffered saline was prepared (20mg/ml, 65% dipalmitoylphosphatidylcholine, 30% phosphatidylglycerol, 5% palmitic acid with 1 mg/ml tyloxapol as a spreading agent) and incubated with and without A549 epithelial cell monolayer. Particles of surfactant constituents seen to coat the epithelial cell-surface were fluorescent suggesting the NBD fluorescence increases in this hydrophobic solution. We clearly demonstrate that NBD-UBIdend and NBD-PMX both possess selectivity for bacterial labelling over lung surfactant constituents. At equivalent molarity, NBD-UBIdend has significantly improved bacterial selectivity over linear NBD-UBInie in the presence of surfactant constituents (Figure 18). Despite a mechanism relying on fluorescence increase in hydrophobic environments by NBD, both NBD-UBIdend and NBD-PMX preferentially increase in fluorescence on bacteria over purified surfactant constituents, in keeping with their ability to image bacteria in the distal lung. Although the comparative distribution and relative abundance of surfactant constituents in the lung is unknown it is entirely likely that this contributes to the background fluorescence that is observed upon probe instillation and that retention of labelling in vitro in the presence of surfactant is required for the probe to image successfully, providing it is sufficiently resistant to degradation and retains labelling upon wash-off.

We have begun to develop bespoke image analysis/processing strategies to perform rapid real-time objective analysis of the large datasets generated by the probe/FCFM platform. Unequivocal detection of bacteria and the delineation of their Gram status will be achieved by employing these image processing algorithms in real-time. A processing algorithm based upon single frame analysis has been applied to entire video sequences (up to 3500 frames), and even at this early stage we are able to unequivocally delineate bacterial presence and Gram status. These signal and image processing algorithms will be rapidly iterated in readiness for clinical application, and we expect significant components of machine-learning to be incorporated into further optimisation of NBD-U BIdend and NBD-PMX datasets.

Figure 19 shows the stepwise processing for single positive and negative frames that were initially chosen to develop the model, based on two image processing algorithms (The Laplacian of Gaussian (LoG) and the Difference of Gaussians (DoG)) commonly used to detect bright spots in fluorescence microscopy images in an objective computational manner. Spots are enhanced in an image by convolving with a LoG or DoG filter. Pre-processing is necessary to improve the accuracy by removing the influence of noise in the image. First, background noise is estimated by dividing an image into small windows (100 per frame) and then calculating the standard deviation of the pixel intensities. High-pass filtering is used to decrease low frequency noise. Thresholding the resulting image at 3x standard deviation removes background noise and keeps only the higher pixel intensity values. Finally, spots are detected with LoG or DoG.

We have employed the method described above to analyse entire FCFM image videos (up to 3500 frames). With an initial thresholding limit of 80 spots per frame to indicate a positive frame, we determined the percentage positive frames per video and set an arbitrary 'cut-off of 20% as the threshold for a binary outcome of YES/NO. This shows unequivocal detection of bacteria and Gram status using NBD-U BIdend and NBD-PMX respectively (Figure 20).

To ensure the compounds can label bacteria above the normal human lung autofluorescence, bacteria were incubated with human lung tissue and imaged using FCFM and on benchtop confocal (Figure 21) demonstrating that bacteria can be detected, once labelled, above the level of autofluorescence. We have identified a number of determinants of distal lung in situ labelling with probes. These explain why the promising in vitro data for the linear NBD-UBlNie probe did not translate to reproducible in situ labelling in the ovine lung. Structural variants were synthesised and assessed, exploring the structure-activity-relationship. The initial aims were to improve resistance to degradation and improve signal-to-noise. Certain modifications which greatly improved a single functional aspect such as stability (such as insertion of d-amino acids/n- methyl or variants including exclusive d-amino acid variants) or labelling intensity (cyclicisation of the compound) did not permit reproducible visualisation of bacteria in the distal lung. We comprehensively assessed the UBI analogues for stability in ALI BALF, in vitro labelling of bacteria using live bench-top confocal imaging at 37°C and in situ labelling in the ovine lung.

A number of structural modifications have been undertaken which are broadly divided into two groups: (a) Increase the signal-to-noise ratio by examining different environmentally-sensitive fluorophores or increasing the NBD-UBlNie payload.

We have also assessed the utility of including two NBD fluorophores for each UBI fragment and have also assessed this with an N-methylated amino acid variant designed to enhance stability. We have assessed a series of alternative environmentally-sensitive fluorophores, including malachite green and styryl-based dyes, with the aim of producing a higher signal-to- noise ratio, which may enable lower levels of bacterial detection or detection at lower effective probe concentrations. (b) Improve the resistance to proteolytic degradation.

We have assessed a number of compounds including variants incorporating D-amino acids and N-methylated amino acids at selected positions identified by MALDI analysis of the parent NBD-UBI compound as sites susceptible to proteolytic degradation. To reduce degradation without altering the amino acid constituents we have synthesised variants including PEG units at the amino and/or carboxy termini in order to block degradation from the ends of the peptide sequence. We have also synthesised and assessed variants consisting entirely of D-amino acids and D-amino acids with inversion of sequence with or without blocking PEG units. Furthermore, a cyclic variant of NBD-UBlNie has also been assessed as well as further variants of this compound incorporating N-methylated amino acids at selected positions identified by MALDI analysis of the cyclic variant of NBD-UBlNie as remaining susceptible to proteolytic cleavage. KTGRAKRRN | eQYNRRG

Cyclic variant of NBD-UBlNie

All compounds have undergone biological assessment. For stability we have assessed each compound in the presence of 0.9% NaCI (Saline) or pooled lavage fluid from patients with acute lung injury and analysed by matrix-assisted laser desorption/ionization (MALDI) or fourier transform mass spectrometry (FTMS). In vitro labelling was assessed on benchtop confocal in the presence of compound with bacteria and labelling was compared to NBD- UBlNie which served as a reference control for bacterial labelling. Where appropriate we have also assessed labelling of the compound on isolated human neutrophils and primary human cell lines (A549 human lung adenocarcinoma cell line). For the ex vivo and in vivo ovine lung experiments each compound was assessed in a control lung segment (instilled with 2ml PBS) or a bacterial segment (instilled with 2ml of 2 optical density of bacteria). Following bacterial instillation, the compound was administered to the segment of interest and this was imaged by probe based confocal laser endomicroscopy (FCFM).

Assessment of the different NBD-UBlNie variants has given us a clearer understanding of different mechanistic factors affecting the function of the probe in the lung environment, and specifically clarified the reasons why, out of all the UBI variants, only the NBD-UBIdend is able to image bacteria in the lung, although, the teaching in the art would suggest that NBD-UBIdend would self-quench.

The alternative fluorophores were inferior to NBD for this application. The Malachite Green variant gave a much lower labelling intensity on the bacteria and although the Styryl-dye compounds exhibited an increased intensity of labelling on bacteria these compounds had a decrease in selectivity over mammalian cells, most likely due to their propensity of the dyes themselves to enter lipophilic membranes overcoming the targeting of the ubiquicidin moiety. Consequently, the Styryl-dye variants exhibited greatly increased off-target labelling in the ex vivo lung and no bacterial signal was observed.

The D-amino acid variants as well as the variants with PEG blocking groups at the ends of the peptide sequence exhibited reduced labelling in vitro and no labelling ex vivo. Despite improved stability, of the linear UBI variants which retained function in vitro none of these were able to image bacteria in the lung. We have obtained evidence that the wash-off properties of the probes, most likely related to affinity and/or the nature of subsequent insertion into the bacterial membrane, impact on whether or not they can be used to successfully image in the lung. The retention of labelling upon removal of probe solution was investigated in vitro by confocal. With all of the linear UBI variants labelling was lost completely upon wash-off. This suggests that, in the lung, labelling would be rapidly lost once the probe concentration around the bacteria decreased as a consequence of fluid dissipation. Bacteria were pre-labelled with these linear NBD-UBlNie compounds and successful labelling was confirmed by imaging the bacterial suspension pre-instillation. These suspensions were instilled into the ex vivo lung and when the segment was subsequently imaged by FCFM no bacterial signal was detected (there is an inherent time-delay in change-over from the instillation catheter to passage of the FCFM fibre into the same segment). Howeverwhen bacteria pre-labelled with polymyxin-NBD (NBD-PMX), which didn't lose labelling upon wash-off when assessed by in vitro confocal, were instilled a bacterial signal was detected. The NBD-UBIdend construct, as well as giving an increased bacterial signal at equimolar concentrations, retained labelling upon wash-off. As predicted, when bacteria pre-labelled with these compounds were instilled into the ex vivo lung the bacteria were successfully imaged.

As described previously we assessed the cyclic variant of NBD-UBlNie in a synthesised surfactant and fluorescent bacteria were identifiable above the background of the surfactant labelling (as seen for NBD-UBIdend and NBD-PMX). Therefore, both the cyclic variant of NBD- UBlNie and NBD-UBIdend share similar wash-off behaviour, labelling intensity and retention of labelling in the presence of surfactant constituents but only the NBD-UBIdend is able to image bacteria in the lung. Therefore it was hypothesised that degradation may be the limiting factor for this compound. MALDI analysis identified that the cyclic NBD-UBInie degrades selectively at points distinct from the linear NBD-UBInie compounds. Therefore N-methyl amino acids were incorporated at these sites. However, these variants no longer exhibited the increased labelling intensity seen for the cyclic variant of NBD-UBInie and no longer retained labelling upon wash-off. Variants with single N-methyl arginine insertions at the position that was hypothesised to be the primary site of degradation showed reduced labelling compared with the original cyclic variant of NBD-UBInie but still higher intensity compared with the linear NBD- UBlNie. This variant had improved retention of labelling upon wash-off but the retained labelling was lower than that of the cyclic variant of NBD-UBInie and this compound did not image bacteria in the ex vivo lung.

NBD-PMX and NBD-UBIdend were assessed for direct red cell toxicity by a haemolysis assay and demonstrated no red cell haemolysis up to 100 μΜ (Figure 22). The compounds were assessed for toxicity in a rodent single dose, intratracheal toxicity assessment. Mice received a single intratracheal administration of 100μg/25g mouse (3000 fold human dose assuming Ι ΟΟμς delivered to a 75kg man) of NBD-UBI de nd or NBD-PMX, or PBS control. Mice were then euthanized at 48 hours and 14 days (n=3 per group). The data shows no toxicity for NBD-PMX (Table 1 and Figure 23) or NBD-UBI de nd (Table 2 and Figure 23).

Table 1

NBD-UBIdend

48 hours 14 days

PBS Control NBD-UBIdend PBS Control NBD-UBIdend

Cytospin (% of 96.5/0.7/0 ± 96.4/3.6/0 ± 100/0/0 ± 100/0/0 ±

ns ns

Mononuclear 0.7/0.67/0 2.0/2.0/0 0/0/0 0/0/0

cells/ Neutrophils/ Red

Blood Cells)

BALF (Cells/ul) 354.9 ± 66.6 477.1 ± 150.6 ns 361.1 ± 38.6 342.7 ± 6.5 ns

PenH value 0.5 ± 0.04 0.5 ± 0.06 ns 0.38± 0.03 0.5 ± 0.04 ns

Creatinine (u/l) 8.0 ± 0.6 8.3 ± 0.7 ns 12.7 ± 1.7 9.0 ± 1.5 ns

Bilirubin (u/l) 2 ± 0 2 ± 0 n/a* 2 ± 0 2 ± 0 n/a*

ALT (u/l) 33.3 ± 3.9 24.0 ± 1.2 ns 39.3 ± 10.8 28.3 ± 1.9 ns

ALP (u/l) 120.0 ± 6.2 222.3 ± 80.3 ns 178.3 ± 27.4 227.0 ± 27.1 ns

Albumin (u/l) 21.0 ± 1.0 22.3 ± 1.8 ns 24.3 ± 0.7 25.0 ± 1.2 ns ns=not significant. * Bilirubin for these time points was below 2 for all animals.

Table 2

Methods of Synthesis of Probes

Synthesis of Ubiquicidin Based Elastase Probes ("Methyl Red (MR)-AAPV-NBD-UBI?a-4 " and "TAM RA-AAPV-N BD-U Bl?a-4 ")

M R-AAPV-K(N BD)- PEG-OH (AL3-74) fragment was synthesised on solid-phase employing Fmoc-strategy, with standard amino acid coupling cycles (2 χ 30 min at rt) with DIC and oxyma in peptide grade DMF at -0.1 mM reagent concentration. Fmoc deprotection steps were done in 20% piperidine in DMF (2 χ 30 min). Between each step, the resin was washed with DMF, DCM and MeOH. 2 g of chlorotrityl polystyrene resin (loading -0.3 mmol/g) was treated with Fmoc-PEG-OH (3 eq) and DIPEA (6 eq) in anhyd. DCM (2 ml_) for 3 h. After washing and Fmoc deprotection, the Fmoc-AAPVK(Dde) sequence was synthesised as described above using, Fmoc- Lys(Dde)-OH, Fmoc-Val-OH, Fmoc-Pro-OH, and Fmoc-Ala-OH. After the sequence was completed, the synthesis was continued with half of the resin (0.3 mmol scale) and Dde protecting group was orthogonally removed with NhbOH/imidazole in NMP/DCM (2 χ 90 min). The resin was treated with NDB-CI (3 eq) and DIPEA (6 eq) in DMF (2 χ 45 min). After Fmoc deprotection, the synthesis was continued in 0.15 mmol scale and Methyl Red was coupled to the /V-terminus as described above. After washing, the fragment was cleaved off the resin with TFA-TIS-H2O (95:2.5:2.5) (30 min) and precipitated with cold ether to give AL3-74 (ESI-MS 1044.4 and 1066.4).

Scheme 1 Synthesis of Methyl Red-AAPV-NBD-UBI 2 9-4i

Ubi29-4i sequence was synthesised on Rink-amide ChemMatrix resin (loading 1 mmol/g) using an Fmoc strategy above. Next, AL3-74 (0.055 mmol) in anhyd. DMF (0.6 ml_) was added to Ubi 2 9-4i on a ChemMatrix resin AL3-68 (0.03 mmol), followed by addition of HBTU (0.055 mmol) and DIPEA (0.22 mmol). The reaction mixture was shaken overnight covered from light. After filtration, the resin was washed with DMF, DCM and MeOH. The resin was swollen with DCM and the probe was deprotected and cleaved off the resin with TFA/thioanisole/EDT/anisole (90:5:3:2) (3 h). The crude precipitated with cold ether and collected by centrifugation. The product AL3-79 was purified by preparative HPLC with detection at 490 nm and gradient of H2O-ACN with 0.1 % formic acid as an eluent. MALDI- TOF MS 2719.4, >95% HPLC purity.

TAMRA-AAPV-NBD-UBI 29-41 AL3-88 (Maldi-TOF MS 2881.5, >95% HPLC purity) was synthesised in similar manner expect fragment TAMRA-AAPV-K(NBD)-PEG-OH (AL3-75) was coupled to the /V-terminus of UBI-based peptide on resin AL3-68.

Scheme 2 Synthesis of TAMRA-AAPV-NBD-UBI29-41 Synthesis of Polymyxin- Based NeBac-Probe (AL3-124)

1. HSPyU, DIPEA, DMF

2. Boc-Polymixin, DIPEA

3. TFA-DCM

Molecular Weight: 2150.43

Scheme 3 Synthesis of TAMRA-AAPV-NBD-PMX Tamra-AAPV-K(MR)-PEG-OH 3A fragment was synthesised on solid-phase employing Fmoc- strategy, with standard amino acid coupling cycles (2 χ 30 min at rt) with DIC and oxyma in peptide grade DMF at -0.1 mM reagent concentration. Fmoc deprotection steps were done in 20% piperidine in DMF (2 χ 30 min). Between each step, the resin was washed with DMF, DCM and MeOH.

500 mg of chlorotrityl polystyrene resin (loading -0.3 mmol/g) was treated with Fmoc-PEG- OH (3 eq) and DIPEA (6 eq) in anhyd. DCM (2 mL) for 3 h. After washing and Fmoc deprotection, the Fmoc-AAPVK(Dde)- sequence was synthesised as described above using, Fmoc-Lys(Dde)-OH, Fmoc-Val-OH, Fmoc-Pro-OH, and Fmoc-Ala-OH. After the sequence was completed, the Dde protecting group was orthogonally removed with NhbOI-l/imidazole in NMP/DCM (2 x 90 min). The resin was treated with NDB-CI (3 eq) and DIPEA (6 eq) in DMF (2 x 45 min). After Fmoc deprotection, 5(6)-carboxyTamra was coupled to the /V-terminus as described above. After washing, the fragment was cleaved off the resin with TFA-TIS-H2O (95:2.5:2.5) (30 min) and precipitated with ether. 3A ESI-MS 1044.4 and 1066.4.

Next, to 3A (0.01 1 mmol) in anhyd. DMF (0.5 mL), HSPyU (0.011 mmol) and DI PEA (0.033 mmol) were added, and the reaction was stirred at rt for 1 h. The truncated and selectively Boc-protected Polymyxin (tetra-Boc polymyxin compound C - see below) (15 mg, 0.012 mmol in 0.5 mL DMF) and DIPEA (0.033 mmol) were added, and the reaction mixture was stirred overnight covered from light. DMF was evaporated, the crude dissolved into 1 mL TFA-DCM (1 : 1), and stirred for 90 min. TFA-DCM was evaporated, the crude precipitated with cold ether, and collected by centrifugation. The product was purified by preparative HPLC with detection at 490 nm and gradient of H2O-ACN with 0.1 % formic acid as an eluent.

MALDI-TOF MS 2151.6 and 2173.6, 100% HPLC purity.

Synthesis of NBD-UBIdend

Synthesis of monomer (5)

Monomer (5) was synthesised in six steps 1 as shown in Scheme 1. Monomer (5) was prepared by the 1 ,4 addition of the hydroxy groups of 1 , 1 ,1- fr/ ' s(hydroxymethyl)amino-methane onto acrylonitrile, followed by amino group protection (Boc). Hydrogenolysis of the nitrile groups with Pt02/H2 gave (3) which was treated with DdeOH to give the tris-Dde protected amine (4). Following removal of the Boc protecting group, the isocyanate (5) was prepared following the procedure of Knolker. 2

DdeOH, DIPEA

MeOH

Scheme 4 Synthesis of monomer (5)

Fmoc-Rink amide ChemMatrix resin (6)

4-[(2,4-Dimethoxyphenyl)-(Fmoc-amino)methyl]phenoxyacetic acid (Rink amide linker) was attached to ChemMatrix resin (LV = 1 mmol/g). Thus the Fmoc-Rink-amide linker (0.2mmol, 1 eq) was dissolved in DMF (4ml_) and ethyl oximinocyanoacetate (Oxyma) (0.2mmol, 1eq) was added and the mixture was stirred for 5min. Λ/,Λ/'-Diisopropylcarbodiimide (DIC) (0.2mmol, 1 eq) was then added and the resulting mixture was stirred for a further 2 min. The solution was added to ChemMatrix resin (0.1 mmol, 1.0mmol/g, 1 eq) and shaken for 0.5 hour. The resulting resin was washed with DMF (3x5ml_), DCM (3x5ml_) and MeOH (3x5ml_). The coupling reaction was monitored by a quantitative ninhydrin test 3 .

LV 0.1 mmol/g

1- 2% hydrazine in DMF

2- a) Fmoc-PEG-OH, Oxyma, DIC, DMF

b) 20% piperidine in DMF

NBD-UBIdend

3- a) Fmoc-AA-OH, Oxyma, DIC, DMF

b) 20% piperidine in DMF

4- NBD-PEG-NHS, DIPEA, DMF

5- TFA:TIS:DCM (90:5:5)

Scheme 5 Synthesis of NBD-UBI dend

The probe was synthesised on a ChemMatrix resin derivatized with an Fmoc-Rink Amide type linker (Scheme 2). The linker (6) was loaded with monomer (5) to give the tri-branched scaffold (7). Following the removal of the Dde groups (2% hydrazine in DMF) the appropriate Fmoc- Amino acids were coupled sequentially followed by the attachment of 4-PEG-7- nitrobenzofurazan /V-hydroxysuccinimide ester (NBD-PEG-NHS) and cleaved from the resin using TFA/TIS/DCM (90/5/5). General procedure for the Fmoc deprotection

To the resin (pre-swollen in DCM) was added 20% piperidine in DMF (5ml_) and the reaction mixture was shaken for 10 min. The solution was drained and the resin was washed with DMF (3x 10ml_), DCM (3x 10ml_) and MeOH (3x 10ml_). This procedure was repeated twice. The coupling reaction was monitored by a quantitative ninhydrin test 3 .

Isocyanate coupling to give (7)

To resin (0.30mmol), pre-swollen in DCM (10ml_), was added a solution of isocyanate (6) (920g, 0.93mmol), DIPEA (0.2ml_, 0.93mmol) and DMAP (22mg, 0.17mmol) in a mixture of DCM/DMF (1 : 1 , 5m L) and the mixture was shaken overnight and the reaction monitored by a quantitative ninhydrin test. The solution was drained and the resin was washed with DMF (3x20ml_), DCM (3x20ml_) and MeOH (3x20ml_) and ether (3x20ml_). (3x20ml_). The coupling reaction was monitored by a quantitative ninhydrin test 3 .

PEG Coupling - 8-(9-Fluorenylmethyloxycarbonyl-amino)-3,6-dioxaoctanoic acid (Fmoc-PEG- OH) coupling

A solution of Fmoc-PEG-OH (3.0mmol, 10eq) in DMF (3ml_) and Oxyma (3.0mmol, 10eq) was added and the mixture was stirred for 5min. DIC (3.0mmol, 10eq) was then added and the resulting mixture was stirred for a further 2 min. The solution was added to pre-swollen resin (7) in DCM and the reaction mixture was shaken for 0.5h. The solution was drained and the resin was washed with DMF (3x 10ml_), DCM (3x 10ml_) and MeOH (3x 10ml_). The coupling reaction was monitored by a quantitative ninhydrin test 3 .

Peptide Synthesis

Peptide Sequence: Thr-Gly-Arg-Ala-Lys-Arg-Arg-Nle-Gln-Tyr-Asn-Arg-Arg

A solution of the appropriate Fmoc-amino acid (3.0mmol, 10eq) (Fmoc-Arg(Boc)-OH, Fmoc- Asn(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Nle-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH) and Oxyma (3.0mmol, 10eq) was added and the mixture was stirred for 5min. DIC (3.0mmol, 10eq) was then added and the resulting mixture was stirred for a further 2 min. The solution was added to pre-swollen resin in DCM and the reaction mixture was shaken for 0.5h. The solution was drained and the resin washed DMF (3x20ml_), DCM (3x20ml_) and MeOH (3x20ml_). The coupling reactions were monitored by a quantitative ninhydrin test 3 . 7-Nitrobenzofurazan (NBD) coupling

To a solution of NBD-PEG-NHS (3.0mmol, 10eq) in DMF (3ml_) was added DIPEA (3.0mmol, 10eq). The resulting solution was added to resin (1eq), pre-swollen in DCM, and the reaction mixture was shaken for 0.5h. The solution was drained and the resin washed with DMF (*3), DCM (x3) and MeOH (*3). The coupling reaction was monitored by a quantitative ninhydrin test 3 .

TFA cleavage and purification of reporter NBD-UBIdend

The resin (45mg), pre-swollen in DCM, was treated with a cleavage cocktail of TFA/ TIS/DCM (90/5/5, 300μΙ_) for 2.5h. The solution was drained and the resin was washed with the cleavage cocktail and the solution was removed in vacuo. The crude material was dissolved in a minimum amount of cleavage cocktail (50μΙ_) and added to ice-cold ether (7.5ml_). The precipitated solid (22mg) was collected by centnfugation and the solvent removed by decantation and the precipitate was washed with cold ether (3x5ml_). The precipitate was then purified by preparative reverse phase HPLC and the required fractions were pooled and lyophilized to afford NBD-UBI dend-

N BD-U BIdend

Synthesis of NBD-PMX The NBD-PMX probe was synthesised from its precursor Polymyxin B sulfate in four steps (Scheme 6). The probe is novel and its intermediates were synthesised using reported methods 1 with moderate modifications. The fluorophore is incorporated as an amide coupling between the NHS ester of the NBD-peg and the tetra-boc polymyxin compound C. NBD-PMX is obtained after the TFA cleavage and HPLC purification.

10 g

Scheme 6 Synthetic route of NBD-PMX probe.

Preparation of compound B

Polymyxin B sulfate (10 g, 7.7 mmol, 1 eq) was dissolved in deionized water (200 mL) at a pH of 6.5 (use HCI aq solution to adjust the pH). Papain (1.5 g) was dissolved in water (25 mL) (same pH). The solutions were combined and toluene (0.5 mL) was added, and the mixture was gently stirred at 65 °C overnight. The mixture was then stirred in boiling water for 5 min and the precipitate formed (denatured papain) was removed by centrifugation and filtration. The filtrate was concentrated in vacuo and freeze dried to give the crude product B in quantitative yield. This step was carried forward to the next step without any further purification. MS m/z 963.2 (100%, [M+H] + ).

Preparation of compound C:

Crude B (5.5 g, 5.7 mmol, 1 eq) was dissolved in a mixture of H2O: Dioxane: EtzN (150 mL, 1 :1 : 1) and Boc-ON (4.52 g, 17.1 mmol, 3eq) was added. The solution was stirred for 20 min at room temperature and then quenched with methanolic ammonia (20 mL, 2M ammonia in MeOH). The reaction was followed up by ELSD. Solvents were evaporated and the resulting mixture was subjected to silica gel chromatography column (MeOH: DCM, 15:85) to afford white solid B (1.7 g, 22%). MS m/z 1363.7 (100%, [M+H] + ). V-(4-Nitrobenz-2-oxa-1,3-diazol-7-yl)amino-3,6-dioxaoctanoic acid (NBD-PEG2-OH): 2 DIEA (850 μΙ, 5.00 mmol) and solid 8-Amino-3,6-dioxaoctanoic acid (NH2-PEG2-OH) (392 mg, 2.40 mmol, 1 eq) were added slowly, over an hour, to a solution of NBD-CI (401 mg, 2.01 mmol) in methanol (20 mL) at 0°C. The reaction mixture was stirred overnight at room temperature. The solvent was evaporated and the remaining material was purified by chromatography on silica with DCM/MeOH (8:2) as the eluent to give NBD-PEG2-OH (400 mg, 1.23 mmol, 51 %) as dark red oil. 1 H NMR (500 MHz, DMSO): δ 10.9 (s, 1 H; COOH), 8.49 (d, J = 8.5 Hz, 1 H; CH NBD), 7.1 (s, 1 H, NH), 6.23 (d, J = 8.5 Hz, 1 H; CH NBD), 4.25 (s, 2H), 3.93 (t, J = 5.3 Hz, 2H; CH 2 ), 3.80 (s, 4H), 3.72 (t, J = 6.8 Hz, 2H; CH 2 ) ppm; MS (ESI-): m/z calcd for Ci 2 Hi 4 N40 7 [M-H]: 325.1 ; found: 325.2. V-(4-Nitrobenz-2-oxa-1,3-diazol-7-yl)amino-3,6-dioxaoctanoic acid, succinimidyl ester (NBD-PEG-NHS):

To a solution of NBD-PEG-OH (2.4 g, 7.4 mmol, 1 eq) in anhydrous DCM (500 mL) was added EDC.HCI (1.56 g, 8.18 mmol, 1.1 eq) and DIPEA (1.36 mL, 10 mmol). After stirring the mixture for 10 min, /V-hydroxysuccinimide (0.94 g, 8.18 mmol) was added and allowed to stir for 16 h. The reaction mixture was diluted with DCM (250 mL) and treated with 5% aqueous citric acid (2 x 200 mL), sat. aqueous NaHCOs and brine. The organic layer was dried over Na2S0 4 , filtered and reduced in vacuo to afford product as dark brown solid (1.0 g, quantitative). The crude was used for next step without further purification.

Preparation of compound F:

A solution of NBD-PEG-NHS (466 mg, 1.1 mmol, 1 eq), DIPEA (384 μΙ_, 2.2 mmol, 2 eq) and amine C (1.5 g, 1.1 mmol, 1 eq) in DMF (150 mL) was stirred at room temperature for 1 h and protected from light. After completion of the reaction (TLC), volatiles are removed under vacuum. The crude mixture was purified by flash chromatography (DCM : MeOH, 90: 10) to afford dark orange/brown solid (1.2 g, 65%). HPLC (254nm & 495nm) Rt = 7.80 min; m/z 1671.7 (25%, [M+H] + ); 1693.9 (65%, [M+Na] + ).

Preparation of NBD-PMX probe:

A solution of Boc-protected polymyxin F (150 mg, 0.09 mmol) in 20% TFA in DCM (2 mL) was vigorously stirred for 45 min at room temperature and protected from light. The reaction mixture was evaporated in vacuo and the resultant was dissolved in ether. Ether layer was decanted after centrifugation (3 x 2 mL). The resultant yellow/brown solid (40 mg, quantitative) was dried under vacuum. The crude product was purified by preparative HPLC in MeOH/H20 as gradient solvent system with 0.1 % formic acid as an additive. The fractions collected from prep-HPLC were freeze dried to afford red/orange solid (30 mg, 26% recovery from HPLC). Characterisation: For analytical HPLC, a Poroshell 120 SB-C18, 2.7 μηι, 4.6 x 50mm column was used with a diode array detector. For prep-HPLC method: Discovery C18 reverse-phase column (5 cm x 4.6 mm, 5 μηι) with a flow rate of 1 mL/min and eluting with H 2 0/MeOH/HCOOH (95/5/0.05) to H20/MeOH/HCOOH (5/95/0.05), over 6 min, holding at 95% MeOH for 4 min, with detection at 254 and 495nm and by ELSD. HPLC (495nm): Rt = 4.1 min; MS mlz 1271.7 (95%, [M+H] + ); 1293.7 (100%, [M+Na] + ); FTMS calc. 636.3282 ([M+2H]/2) + , found 636.3344.

Absorption / Emission: 467 nm / 539 nm.

Solubility: Fully soluble in water.

Stability: stable at room temperature for > than 1 week.

Storage: Stored at -20 °C under inert atmosphere. Protect from light.

Biological Methods Bacterial Growth: Bacteria used in assays include Pseudomonas aeruginosa (PA01- reference strain and J3284-clinical isolate from VAP patient), Acinetobacter baumannii, Stenotrophomonas maltophilia, Staphylococcus aureus (Inc. methicillin-resistant S. aureus (MRSA), methicillin-sensitive S. aureus (MSSA)), Klebsiella pneumoniae, Escherichia coli, Haemophilus influenzae and Streptococcus pneumoniae.

rom ro essor o n ovan, n vers y o n urg . All bacteria were grown on agar broth, chocolate agar or blood agar plates, stored at 4°C. For assays a single colony of bacteria was taken using an inoculating loop and added to 10ml liquid broth in a 50ml Falcon Tube. This was transferred to an incubator at 37 ° C for 16 hours (for Streptococcus pneumoniae supplemented with 5% CO2). Cultures were either used as overnight cultures (stationary phase) or from these cultures a sub-culture was (1 :100) and the sample was grown until they entered mid log phase (reads of 0.5-0.6 optical density (OD) on spectrophotometer at 595nm). The culture was then centrifuged at 4000rpm for 5 minutes and pellet resuspended in phosphate buffered saline (PBS). Following three washes this was reconstituted to 0.5 ODs95nm for confocal assays, 0.1 ODs95nm for flow cytometry or 2 OD595nm for ovine ex vivo lung experiments (unless otherwise stated).

Bacterial Counting: Samples (prepared bacteria or lavage from ovine lung segments) were vortexed briefly then serial dilutions (1 : 10) were performed to dilutions to the 8th dilution. The broth/blood agar plate was divided into quadrants with 5 x 20ul drops in each quadrant. These were incubated at 37°C for 16 hours (for Streptococcus pneumoniae supplemented with 5% CO2) and plates were counted with data reported as colony forming units per millilitre (CFU/ml).

Surfactant Constituent Synthesis: Surfactant 5μg 1 ,2-Dipalmitoyl-sn-glycero-3- phosphocholine (DPPC) and 2^g L-a-Phosphatidyl-DL-glycerol sodium salt (from egg yolk lecithin; PG) were dissolved in 500μΙ chloroform and evaporated under nitrogen to a thin lipid film in a round bottom flask. The lipid film was rehydrated with PBS at 48°C for 1 hour with agitation (750rpm) to generate multilammelar vesicles (MLV). These were diluted 1 :4 for use in confocal experiments.

Agarose Bacterial Beads: Bacteria were grown to midlog phase in 400ml TSB, pelleted by centrifugation and resuspended in 2ml PBS. This was mixed with 18ml molten tryptic-soy agar (50°C) and injected rapidly into vortexing mineral oil +0.01 % Span 80, pre-warmed to 50°C. This was then rapidly cooled to 4°C whilst continuing to vortex to allow the beads to set. Bacterial agar beads were pelleted by centrifugation (20 minutes, 3000g) and washed in 0.5% sodium deoxycholate (SDC) in PBS (20 minutes, 3000g), followed by 0.25% SDC (20 minutes, 3000g) in PBS, washed in PBS (10 minutes, 3000g) and 3X PBS (5 minutes, 200g). Beads were resuspended at 50% v/v in PBS for instillation. Neutrophil Extraction: Neutrophils were isolated from the peripheral blood of healthy human volunteers dextran sedimentation followed by centrifugation through discontinuous plasma- Percoll gradients. A549 cultures: A549 cells were grown in Dulbecco's modified Eagle's medium (DM EM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/mL penicillin G and 100 μg/mL streptomycin to 80% confluence. Cells were dispersed with Trypsin-EDTA and seeded onto glass coverslips or 8 well confocal imaging chambers and grown to confluence in the presence of DMEM.

Confocal Analysis: Bacteria were prepared and counterstained with Syto 82 nucleic acid stain (Invitrogen, CA, USA) in a shaking heat block at 37°C and 350rpm for 20 minutes. They were co-incubated with the probe at required concentration in a sealed POC mini chamber, or 8 well confocal chamber. When required the glass coverslip for the POC chamber was coated in fibronectin (for neutrophil experiments) or poly-d-lysine (for bacteria and cell lines) and incubated with cells with one hour at 37°C to allow adherence prior to bacterial innoculation. Analysis was with ImageJ. Briefly, the Syto channel was automatically thresholded (Huang) and an ROI generated from this. The mean fluorescence intensity on the probe channel within this ROI was quantified. Data presented represents the mean of three separate fields of view.

Flow Cytometry: Bacteria were prepared and counterstained with Syto 82 nucleic acid stain (Invitrogen, CA, USA) in a shaking heat block at 37°C and 350rpm for 20 minutes. Bacteria were washed in PBS x 3, and probe (50μΙ) added in 50ul OD595 1od bacteria. This was diluted to 500uL and analysed using BD FACS Calibur using FL-1 and FL-2 channels, with 10,000 events. Analysis was with FlowJo software following gating on the FL-2 channel.

Lung Harvesting and pCLE procedure: From a cohort of surplus stock ewes which were destined for cull, one ewe was identified and terminally euthanized with an overdose of anaesthetic. Death was confirmed and the trachea was identified and clamped in situ. The thoracic cavity was then accessed and the lungs were freed from surrounding tissues and organs and the heart/lung was removed en block. The right pulmonary artery was identified, cannulated and perfused with 1000ml 0.9% NaCI. Once filling of the left ventricle was confirmed an incision was made to allow free drainage and perfusion continued until the drainage from the left atrium was clear. The lungs were then transferred to the Queen's Medical Research Institute and the trachea was intubated with an 8.0 endotracheal tube immediately following clamp release. The lungs were placed in a neonatal incubator with an ambient temperature of 37°C and humidity of 65% and ventilated using a Pressure Controlled Ventilator (Breas Vivo PV 403). Ventilator setting was adjusted to aid maximal parenchymal recruitment and aiming to achieve tidal volume > 1 litre. Following 1 hour of optimal ventilation, bronchoscopy was undertaken and individual segments were identified and instilled with 2 ml of bacteria or PBS control. Following instillation a separate sheath (ERBE) was introduced and the probe instilled. Then the pCLE fibre was passed down the working channel and the segment was imaged. For BALF, the bronchoscope was wedged and 20ml of 0.9% NaCI instilled and carefully withdrawn with lavage yields of 40-50%. Control segments were anatomically distinct and/or in the contralateral lung. The bronchoscope was decontaminated between each segment imaged.

Haemolysis Assay: Erythrocytes were isolated from freshly drawn, anticoagulated human blood and resuspended to 20 vol % in PBS (pH 7.4). In a 96-well microtiter plate, 100 μΙ of erythrocyte suspension was added to 100 μΙ of NLLP solution in PBS (prepared by 1 :2 serial dilutions) or 100 μΙ of PBS in the case of negative controls. One-hundred percent haemolysis wells contained 100 μΙ of red cell suspension with 100μΙ of 0.2 vol% Triton X-100. The plate was incubated for 1 h at 37°C, and then each well was diluted with 150 μΙ of PBS. The plate was then centrifuged at 1 ,200g for 15 min, 100μΙ of the supernatant from each well was transferred to a fresh microtiter plate, and A350 was measured. Percentage of haemolysis was determined as (A-A0)/(Atotal-A0) X100, where A is the absorbance of the test well, AO the absorbance of the negative controls, and Atotal the absorbance of 100% haemolysis wells, all at 350 nm on a Biotek plate reader. MALDI-TOF: Probe was added to saline or pooled BALF from patients with ALI incubated for 30 minutes. A ZipTip (C-18, 0.2 μΙ_) with 5μΙ_ MeCN (with 0.1 % TFA as an additive) followed by 20 μΙ_ of H20 was washed. The ZipTip was loaded with the sample, washed and eluted into 5 μΙ_ of 80% aq. MeCN (with 0.1 % TFA as an additive). The sample was analysed by MALDI-TOF (PerSeptive Biosystems Voyager DE™STR MALDI-TOF mass spectrometer (Applied Biosystems, Foster City, CA)).

Statistical Analysis: All experiments were performed at least three times unless otherwise stated and results expressed as mean ± SEM. Data was analysed by unpaired t-test or ANOVA, significance was determined as p<0.05 (GraphPad Prism).

References

1. M. Ternon, J. J. Diaz-Mochon, A. Belsom, M. Bradley, Tetrahedron, 2004, 60, 8721

2. H. J. Knolker, T. Braxmeier, G. Schlechtingen, Angew. Chem. Int. Ed., 1995, 34, 2497

3. E. Kaiser, R. L. Colescott, C. D. Bossinger and P. I. Cook, Analytical Biochemistry,

1970, 34, 595-598. Listing of Relevant Sequences

SEQ ID NO.1

Ubiquicidin (full)

KVHGSLARAG KVRGQTPKVA KQEKKKKKTG RAKRRMQYNR RFVNVVPTFG KKKGPNANS

SEQ ID NO.2

Ubiquicidin (UBI29-41)

TGRAKRRMQY NRR

SEQ ID N0.3

Ubiquicidin (UBInie)

TGRAKRRNIeQY NRR

SEQ ID N0.4

Polymyxin

Dab - Thr - Dab - Dab - Dab - D-Phe - Leu - Dab - Dab - Thr

† I

Dab = L-a-Y-Diaminobutyric acid

SEQ ID N0.5

Polymyxin B1

6-methyloctanoic acid - Dab - Thr - Dab - Dab - Dab - D-Phe - Leu - Dab - Dab - Thr

SEQ ID N0.6

Polymyxin B2 -methylheptanoic acid - Dab - Thr - Dab - Dab - Dab - D-Phe - Leu - Dab - Dab - Thr