Imaging probes for the non-invasive detection of infection sites

FIELD OF THE INVENTION

The present invention relates to imaging probes, and in preferred embodiments to radioactive compounds, also referred to in the present invention as radiotracers, wherein said imaging probes are based on proteins, such as collagen, fibronectin, and fibrinogen, binding to bacterial cell wall-anchored proteins. These imaging probes are used for the non-invasive detection of infection sites caused by Gram+ bacteria by means of imaging techniques, such as nuclear imaging.

Therefore, the present invention is comprised in the field of medicine, and more specifically in the field of diagnosis or detection of bacterial infections.

BACKGROUND OF THE INVENTION

The imaging techniques used to date in the diagnosis of infection sites are based either on conventional structural imaging techniques, such as magnetic resonance (MR), or on computed tomography (CT). The low sensitivity of these techniques, i.e., the inability to detect infection sites at initial stages, stands out as their main limitation. Moreover, in recent years, molecular imaging, and particularly nuclear imaging, began to be used in the development of more selective techniques due to the high sensitivity of the technique. Despite the improvement in the diagnosis of infections by means of the image provided by this methodology, the use of known and commercially available radiotracers, such as 18F-FDG (a radiopharmaceutical made up of a glucose analog 2- [18F]fluoro-2-deoxy-D-glucose bonded to the radioactive isotope fluorine-18), that are not only specific for bacteria, but also other cell lines such as macrophages or tumor cell lines, gives rise to false positives in many cases. Thus, there is a need of providing an imaging probe with the ability to discriminate between infection and inflammation and/or cancer.

Moreover, the use of conventional diagnostic techniques such as cell or bacterial cultures require long times for result analysis (several days of incubation), as well as for biopsy or blood sampling.

One object of the present invention is to solve current clinical problems by allowing an early, selective, and non-invasive diagnosis of infectious processes, preferably with high sensitivity and/or specificity. It further provides a non-invasive method of monitoring the evolution of an infection.

Antimicrobial peptide-based probes have been described as potential infection imaging PET radiotracers such as [68Ga]DOTA-GF-17 (Chopra S. et al., Appl Radiat Isot. 2019 Jul;149:9-21) or Ga-NOTA-UBI 29-4 (Vilche M. et al., J Nucl Med. 2016 Apr;57(4):622- 7). However, these do not distinguish between Gram + and Gram - bacteria.

Infections caused by Gram+ bacteria such as methicillin-resistant staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE) and Clostridium difficile are amongst the most common multidrug-resistant infections.

Still a further object of the present invention is the provision of an imaging probe which discriminates between Gram+ and Gram- bacteria from an early stage of infection. Having such a tool enables to treat the patient from the moment the infection is detected with a specific antibiotic, thus avoiding the use of an empirical anti-infective treatment and contributing to a more rational use of antibiotics and to prevent the appearance of antibiotic resistances.

SUMMARY OF THE INVENTION

The inventors have shown that the imaging probes of the invention are characterized by their high sensibility, specificity and selectivity for Gram positive bacteria. Thus, providing an imaging agent for the early non-invasive detection of infectious foci of Gram-positive bacteria.

As shown in Example 2, an in vitro study was conducted to assess the collagen-based probe binding capacity to the target G+ bacteria. A linear relationship between the concentration of bacteria and the percentage of binding of the tracer was observed (Figure 2A). In addition, the observed difference between the specific radiotracer and the free isotope was of at least 7-fold, reaching differences of up to 26-fold higher when incubated for 2 h and with 10 ⁸ CFUs (Figure 2C). Thus, confirming the specificity of the radiotracer detection.

Moreover, the probes of the invention were also shown to have selectivity for G+ bacteria, specially G+ cocci, with respect to other infectious pathogens, such as G- bacteria or fungi (Fig. 2 D). In particular, a high percentage of specific binding was observed for S. aureus, S. epidermidis and S. faecalis strains for bacterial concentrations of 10 ³ CFU or higher. Surprisingly, for lower bacterial concentrations, a high percentage of specific binding was only maintained for Staphylococcus strains, especially for S. aureus. Thus, a collagen-based probe of the invention was shown to have particularly high sensitivity for Staphylococcus strains, detecting in vitro staphylococcal concentrations below 10 ³ CFU, even of 10 CFUs or below. This unexpectedly high sensitivity makes it particularly suitable for detecting staphylococcal infections at a very early stage.

Besides, the molecular probe of the invention showed an increased sensitivity with respect to 18F-FDG which is a glucose analog and the standard radiotracer used in clinical setting for detection of infection foci. In particular, the in vitro assay evidenced the higher capacity of the collagen-based probe to detect staphylococcal cultures, with a sensitivity 3.5-fold greater than that of the commercial tracer (Figure 2B).

As shown in the ex vivo biodistribution studies, contrary to 18F-FDG, the imaging probes of the invention do not accumulate in the brain or heart (see Table 1), which make them ideal for detecting brain infections, such as meningitis or encephalitis or for infective endocarditis (IE) diagnosis purposes.

The imaging probe of the invention also showed its ability to distinguish between infection and inflammation. In Example 3, a local infection-inflammation model was used to demonstrate binding capacity of the imaging probe only in active infection sites. More specifically, in this model, the left hind leg of the rat was infected intramuscularly with a live S. aureus inoculum and the right hind leg was infected with the same amount of an inactive strain of the bacterium. The tracer demonstrated its ability to bind mainly to regions where the active (live) strain was found, with little or no binding to tissues inoculated with the inactivated (heat-killed) strain (Figure 3A - left). The results observed by means of in vivo imaging were confirmed by means of ex vivo measurements of biodistribution and autoradiography (Figure 3B). Conversely, as expected on the basis of previous works, 18F-FDG was not able to discriminate between inflammation and infection, showing intense uptake of the tracer in both regions (Figure 3A - right).

Moreover, the ability of the imaging probe of the invention to distinguish between infection and inflammation was further evidenced by the in vivo evaluation in an IE model, where no tracer signaling was observed on the control sham model (Fig. 4A), which presented surgery but not infection, thereby demonstrating the selectivity of the tracer only for infection sites.

Based on these findings, in a first aspect the present invention relates to a compound labelled with a marker suitable for detection by imaging techniques (also referred herein as image probe) comprising a protein which is a substrate of bacterial cell wall- anchored proteins as described herein.

A second aspect of the invention relates to the preparation of imaging probes as described herein, which comprises contacting the compound comprising a protein which is a substrate of bacterial cell wall-anchored proteins as described herein with the labelling agent under conditions suitable for achieving the labelling of the compound.

A third aspect of the invention relates to a composition comprising the compound defined in the first aspect of the invention.

A fourth aspect of the invention relates to the use of the compound of the invention or of the composition comprising same as an imaging agent for the visualization of infections or infection sites caused by Gram-positive bacteria in a subject.

A fifth aspect of the invention relates to a method for providing an image of infection sites caused by Gram-positive bacteria in vivo in a subject, which comprises (i) administering the compound of the invention to the subject, and (ii) scanning the subject using an imaging technique, such as scintigraphy, PET or SPECT imaging, optionally combined with a structural technique such as computed tomography or magnetic resonance imaging to obtain a visible image.

A sixth aspect of the invention relates to a method for detecting infection sites or infections, preferably caused by Gram-positive bacteria, in vivo in a subject comprising the following steps:

- administering an effective amount of the image probe (e.g. radioactive compound) of the invention to a subject,

- obtaining an image after the administration of the image probe (e.g. radioactive compound), providing a suitable time for the administered image probe (e.g. radioactive compound) to bind to the target infection site,

- optionally, reconstructing and analyzing the acquired image by computer means.

DESCRIPTION OF THE INVENTION

The present specification describes imaging probes, preferably radioactive compounds (also referred to as radiotracers) based on a protein, such as collagen or other similar proteins binding to proteins present in the bacterial wall, for the non-invasive detection of infection sites caused by Gram-positive bacteria by means of imaging techniques, such as nuclear imaging (e.g., SPECT (single photon emission computed tomography) or PET(positron emission tomography)).

In a first aspect, the present invention provides a compound labelled with a marker suitable for detection by imaging techniques (also referred herein as image probe) comprising a protein which is a substrate of bacterial cell wall-anchored proteins.

These bacterial cell wall-anchored proteins can be the microbial surface components recognizing adhesive matrix molecules (M SC RAM Ms) which are a family of proteins that are defined by the presence of two adjacent IgG-like folded subdomains. These promote binding to ligands by mechanisms that involve major conformational changes exemplified by the binding to fibrinogen by the 'dock-lock-latch' mechanism or to collagen by the 'collagen hug'. Clumping factors A and B are two such MSCRAMMs that have been described to have several important roles in the pathogenesis of Staphylococcus aureus infections. Timothy J. Foster has described MSCRAMM architecture, ligand binding, and roles in infection and colonization (Trends Microbiol. 2019 Nov;27(11):927-941).

Examples of bacterial cell wall-anchored MSCRAMMs are collagen binding proteins (e.g. Collagen adhesin (Cna)), fibronectin binding proteins (e.g., Fibronectin-binding proteins A (FnBPA) and B (FnBPB)) and fibrinogen binding proteins (e.g., FnBPA, Bone sialoprotein-binding protein (isoform of SdrE), Clumping factor A (ClfA) or Clumping factor B (ClfB)). For illustrative purposes, these and other MSCRAMMs found at the surface of Staphylococcus aureus are described at Table 1 of Foster T.J. et al. Nat Rev Microbiol. 2014 Jan; 12(1): 49-62. Accordingly, in a particular embodiment, said protein is a substrate of the MSCRAMMs, such as collagen, fibronectin or fibrinogen, or derivatives thereof which maintain the respective binding properties. These proteins can be collectively referred as adhesive matrix molecules.

These molecules are extracellular matrix molecules naturally present in a mammalian subject, thus these have the advantage of having low toxicity. It was surprisingly found by the inventors that there was a selective binding in vivo of the imaging probes described herein at the site of infection. Moreover, it was unexpectedly found that the half-life of the collagen-based radiotracer was appropriate for imaging detection purposes, despite the constitutive expression of collagenases which expression occurs at several sites during normal and abnormal tissue remodeling.

In the development of the new imaging probe, the protein, preferably collagen, is labelled with a marker suitable for detection by imaging techniques. Protein marking can be conducted by any method known in the art, such as optical labelling with fluorophore for optical image detection or by the conjugation with paramagnetic and/or superparamagnetic nanoparticles based on iron oxide nanoparticles for detection using MR.

In a particular embodiment, the protein is radioactively labeled with a radioisotope. Labelling can be performed by the covalent attachment or by passive absorption (non- covalent) of the radionuclide in the biomolecule. Covalent binding is conducted by conjugating the protein with a chelating agent which will coordinate with the radionuclide. Covalent binding is preferred, since it provides higher yields and a stable linkage of the nuclide to the biomolecule, which will prevent the release of the free isotope that may give rise to false positives.. In Example 1 is shown that the emission of the ^99m Tc-DTPA-Collagen radiotracer was maintained constant until 120 minutes.

Different radioisotopes such as ^99m Tc, ⁶⁸ Ga, ¹⁸ F, ⁸⁹ Zr, ⁶⁴ Cu, ¹³¹ l, ¹²⁴ l or ¹¹¹ ln may be used without modifying the protein native structure. The ability to radiolabel the protein of high molecular weight as described herein without modifying its native structure is an advantage over the use of small peptide-based radiotracers described in the prior art wherein the coupling of the peptide with the chelating agent oftentimes leads to a modification of the peptide structure and binding properties. After its injection, the imaging probe (e.g., radioactive probe) will act by binding specifically to the wall of Gram-positive bacteria, through the binding receptors or MSCRAMMs of the protein in question, allowing the specific and non-invasive detection of infection sites caused by said bacteria by means of imaging techniques (e.g. nuclear imaging).

In a particular embodiment, the present invention relates to a compound comprising a protein as described herein covalently bonded to a chelating agent and a radioisotope coordinated to the chelating agent, wherein preferably the protein is selected from the list consisting of collagen, fibronectin, and fibrinogen, all of them being molecules related to bacterial wall-anchored proteins.

The chelating agent is a compound or molecule comprising at least a first group selected from: amine, maleimide, and carboxyl configured for being covalently bonded to proteins, and at least a second group selected from carboxyl, amino, and phosphine configured for being coordinated to the radioisotope, such as a metal isotope.

In a preferred embodiment, the chelating agent is selected from the list comprising:

- pentetic acid, also referred to as diethylenetriamine pentaacetate (DTPA);

- 1 ,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and its derivatives: DOTA-NHS ester, maleimide-DOTA, and NH2-DOTA-GA;

- 1 ,4,7-triazacyclononane-1 ,4,7-triacetic acid (NOTA) and its derivatives: NOTA-NHS ester, NODAGA-NHS ester, maleimide-NOTA, and NH2-NODA-GA; deferoxamine (DFO) and its derivative p-SCN-Bn-deferoxamine (1-(4- isothiocyanatophenyl)-3-[6, 17-dihydroxy-7, 10, 18,21 -tetraoxo-27-(N- acetylhydroxylamino)-6,11,17,22-tetraazaheptaeicosine]thiour ea)]

- diethylenetriamine-N,N,N”,N”-tetra-tert-butyl acetate-N’-acetic acid, also known as DTPA-tetra (t-Bu ester).

In an even more preferred embodiment, the chelating agent or linker is pentetic acid which would bind to the protein through a covalent bond between an amino group of the protein and a carboxylate of the chelating agent and will bind to the radioisotope by coordination through the oxygen atoms of carboxyl groups and the nitrogen atoms comprised in the structure of pentetic acid. In another preferred embodiment, the protein is collagen, more preferably a type I or type IV collagen.

The radioisotope is preferably selected from the list consisting of ^99m Tc, ⁶⁸ Ga, ¹⁸ F, ⁸⁹ Zr, ⁶⁴ Cu, ¹³¹ l, ¹²⁴ l or ¹¹¹ ln. Radioisotopes which can be used for SPECT are preferred, such as ^99m Tc, ¹³¹ 1, or ¹¹¹ In. SPECT is a less expensive technique than PET and is thus the most common nuclear imaging technique in the clinical setting. In a more preferred embodiment, the radioisotope is ^99m Tc, and in an even more preferred embodiment, the radioisotope R is ^99m Tc (IV). ^99m Tc is a radionuclide of low energy which can be produced in a generator. Thus, an imaging probe as described herein radiolabeled with ^99m Tc has the advantage that can be produced in situ in the hospital without requiring the use of cyclotrons.

A radioisotope (radionuclide, radioactive nuclide, or radioactive isotope) has the conventional meaning, i.e., an atom that has excess nuclear energy and is therefore radioactive.

In a particular embodiment, said radiotracer is ^99m Tc-DTPA-Collagen, in which the protein is collagen, the chelate is DTPA and the radionuclide is metastable Technetium 99 ( ^99m Tc).

The imaging probe of the invention as described herein is preferably suitable for parenteral administration, such as intravenous administration. In particular embodiments, it is characterized by having a LogP value below -2, preferably below -3.

Another aspect of the invention relates to the preparation of imaging probes as described herein, which comprises contacting the compound comprising a protein which is a substrate of bacterial cell wall-anchored proteins as described herein with the labelling agent under conditions suitable for achieving the labelling of the compound. Possible labelling agents are as described herein above.

In a particular embodiment, it provides the preparation of radiotracers as described herein. The preparation of radiotracers requires two synthetic steps: a first step of forming a covalent bond between the protein and a suitable chelating agent, a proteinchelate conjugate thus being formed, and a second radiochemical step based on radioactively labeling the protein-chelate conjugate with the corresponding radioisotope.

Further to creating a protein-chelator conjugate, this may be isolated or purified by any method known in the art, for instance by filtration. Prior to the radiolabeling step, the reduction of the commercial radioisotope may be required, for instance, for the radiolabeling of the proteins with 99mTc (IV) commercial pertechnetate was reduced first with SnCh. The obtained radiolabeled protein-chelate conjugate will generally be further isolated or purified, for instance by filtration.

A further aspect of the invention relates to a composition comprising the compound defined in the first aspect of the invention.

In a particular embodiment, the composition of the invention is a pharmaceutical composition. Said pharmaceutical composition may comprise one or more pharmaceutically acceptable excipient or carrier.

As used herein, "pharmaceutically acceptable excipient" or “pharmaceutically acceptable carrier” means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and, without limiting the scope of the present invention, include: additional buffering agents; preservatives; co-solvents; antioxidants, including ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (e.g., Zn-protein complexes); biodegradable polymers, such as polyesters; salt-forming counterions, such as sodium, polyhydric sugar alcohols; amino acids, such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, such as lactitol, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol; sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, [alpha]-monothioglycerol, and sodium thio sulfate; low molecular weight proteins, such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; and hydrophilic polymers, such as polyvinylpyrrolidone. Other pharmaceutically acceptable carriers, excipients, or stabilizers, such as those described in Remington: The Science and Practice of Pharmacy 22nd edition, Pharmaceutical press (2012), ISBN-13: 9780857110626 may also be included.

In another embodiment of the present invention, the radiotracer or composition as described herein is provided in lyophilized form, for instance in a sterilized container or vial. In this form, the lyophilized composition can be readily reconstituted by adding water or an aqueous solution, preferably having a pH in the range of about 5 to 8, more preferably physiological pH.

The pharmaceutical composition comprises the compound defined in the first aspect of the invention and has at least one application in the detection of infection sites caused by Gram-positive bacteria.

The composition of the invention can be administered by any known methods of administration. In a preferred embodiment, the composition is configured for being able to be administered intravenously, intraperitoneally, or orally. Even more preferably, the composition is configured for being injected intravenously or intraperitoneally.

Another aspect of the invention relates to the use of the compound of the invention or of the composition comprising same as an imaging agent for the visualization of infections or infection sites caused by Gram-positive bacteria in a subject (animal, preferably a mammal or human).

Preferably, the time period between administration of the compound or composition of the invention and image acquisition is in the range of 1 -3h, preferably about 2h.

In the present invention, the term “imaging agent” refers to a chemical compound the structure of which contains a marker suitable for detection by imaging techniques, such as radioactive isotope (also referred to as radiotracer, radiopharmaceutical) when said agent is a “nuclear imaging agent”, which is capable of tracking a biological process, producing a signal that can be detected by means of imaging techniques for instance, those used in nuclear medicine, such as single photon emission computed tomography and positron emission tomography.

The compound therefore acts as a probe for the detection of infections or infection sites caused by Gram-positive bacteria. The term “probe” refers to a chemical compound capable of selectively detecting infection sites caused by Gram-positive bacteria.

In a preferred embodiment, said protein is radiolabeled and the visualization of infections or infection sites is performed by means of the scintigraphy, PET (positron emission tomography) or SPECT (single photon emission computed tomography) technique, optionally combined with a structural technique such as computed tomography or magnetic resonance imaging to obtain a visible image.

Gram-positive bacteria are bacteria classified by the color they turn in the staining method developed by Hans Christian Gram in 1884. The staining method uses crystal violet dye, which is retained by the thick peptidoglycan cell wall found in gram-positive organisms (typically 20 to 80 nm thick peptidoglycan). This reaction gives gram-positive organisms a blue color when viewed under a microscope. Although gram-negative organisms classically have an outer membrane, they have a thinner peptidoglycan layer, which does not hold the blue dye used in the initial dying process. Other information used to differentiate bacteria is the shape. Gram-positive bacteria comprise cocci, bacilli, or branching filaments (Gram Positive Bacteria, Omeed Sizar; Chandrashekhar G. llnakal, StatPearls, NCBI Bookshelf).

Gram-positive cocci include inter alia the genus Staphylococcus (catalase-positive), which grows in clusters, and Streptococcus (catalase-negative), which grows in chains. The staphylococci further subdivide into coagulase-positive (S. aureus) and coagulasenegative (S. epidermidis and S. saprophyticus) species. Streptococcus bacteria subdivide into Strep, pyogenes (Group A), Strep. agalactiae (Group B), enterococci (Group D), Strep viridans, and Strep pneumonia.

Gram-positive bacilli (rods) subdivide according to their ability to produce spores. Bacillus and Clostridia are spore-forming rods while Listeria and Corynebacterium are not. Spore-forming rods that produce spores can survive in environments for many years. Also, the branching filament rods encompass Nocardia and actinomyces.

In a preferred embodiment, the Gram-positive bacteria are selected from the genus Staphylococcus, Streptococcus, Enterococcus, Mycobacterium, Bacillus and Clostridioides, preferably from Staphylococcus, Streptococcus, Enterococcus and Bacillus.

In a more preferred embodiment, the Gram-positive bacteria are selected from list consisting of Staphylococcus aureus, Staphylococcus epidermis, Streptococcus mutans, Mycobacterium tuberculosis, Enterococcus faecalis, Staphylococcus agalactiae, Bacillus anthracis and Clostridioides difficile.

In another more preferred embodiment, optionally in combination with the one described above, the Gram-positive bacteria are selected from list consisting of Streptococcus equi, Streptococcus mutans, Streptococcus gallolyticus, Enterococcus faecalis, Staphylococcus aureus and Bacillus anthracis. These Gram + bacteria have been described to comprise M SC RAM Ms in their cell wall which bind collagen (van Wieringen, T., et al., 2010, J. Biol. Chem. 285, 35803-35813; Nomura, R., et al., 2013, Oral Dis. 19, 387-393 ; Nallapareddy, S. R. et al., 2003, Mol. Microbiol. 47, 1733- 1747 ; Ross, C. L., et al., 2012, J. Biol. Chem. 287, 34856-34865 ; Nallapareddy, S. R., et al., 2000, Infect. Immun. 68, 5218-5224 ; Sillanpaa, J., et al., 2009, J. Bacteriol. 191, 6643-6653 ; Sato, Y., et al., 2004, J. Dent. Res. 83, 534-539 ; Xu, Y., et al., 2004a J. Biol. Chem. 279, 51760-51768).

In a further embodiment, said Gram-positive bacteria are Gram-positive cocci, preferably selected from the genus Staphylococcus, Enterococcus or Streptococcus, more preferably from the genus Staphylococcus. In a further preferred embodiment, said Gram-positive bacteria are Staphylococcus aureus.

The infection caused by said Gram-positive bacteria, may occur in any organ or tissue of the subject. In a particular embodiment, the infection caused by said Gram-positive bacteria occurs in bone, cartilage, muscle, soft tissues or blood, for instance in the gastrointestinal tract, heart, cardiovascular system, liver, lung, respiratory tract, kidney, urinary tract, brain, nervous central system, skin, subcutaneous tissues or surgical wounds. In a particular embodiment, said infection is a local infection. In another embodiment, said infection is a blood infection or bacteraemia and the compounds of the invention may enable the visualization of metastatic infection foci.

In a preferred embodiment, the infection caused by Gram-positive bacteria occurs in skeletal or cardiac muscle, see example 3 b) and c), respectively. In an embodiment, said infection is an intramuscular infection. In another embodiment, said infection is infective endocarditis. In a further embodiment, the infection occurs in the bone, also referred as osteomyelitis.

Another aspect of the invention relates to a method for providing an image of infection sites caused by Gram-positive bacteria in vivo in a subject, which comprises (i) administering the compound of the invention to the subject, and (ii) scanning the subject using an imaging technique, such as scintigraphy, PET or SPECT imaging, optionally combined with a structural technique such as computed tomography or magnetic resonance imaging to obtain a visible image.

An additional aspect of the invention relates to a method for detecting infection sites or infections, preferably caused by Gram-positive bacteria, in vivo in a subject comprising the following steps:

- administering an effective amount of the image probe (e.g. radioactive compound) of the invention to a subject,

- obtaining an image after the administration of the image probe (e.g. radioactive compound), providing a suitable time for the administered image probe (e.g. radioactive compound) to bind to the target infection site,

- optionally, reconstructing and analyzing the acquired image by computer means.

A preferred time period between administration of the image probe (e.g. radioactive compound) of the invention and image acquisition is as described herein above.

Said imaging technique may be as defined herein above. Preferably, it is a nuclear imaging technique, such as scintigraphy, PET or SPECT imaging, optionally combined with a structural technique such as computed tomography or magnetic resonance imaging.

Said method for detecting infection sites or infections or for providing an image of infection sites may be useful for diagnostic and/or monitoring purposes. In a particular embodiment, said method is useful for the early diagnosis of a Gram-positive infection. Said Gram-positive infection can be as described herein above.

In the present invention, the expression “effective amount” refers to that amount of the compound which, when administered, is sufficient for being detected by means of nuclear resonance imaging. The image probe (e.g., radioactive compound) of the invention, which is used for the non-invasive detection of infection sites of Gram-positive bacteria, will bind specifically to the protein (preferably collagen) binding receptors or microbial surface component recognizing adhesive matrix molecules (M SC RAM Ms) present in the wall of Grampositive bacteria, allowing the specific and non-invasive detection of infection sites caused by said bacteria by means of imaging, such as nuclear imaging.

The absence of collagen binding proteins (collagen receptors or MSCRAMMs) in macrophages or myocytes allows the compound of the invention to only detect infection sites of Gram+ bacteria. This specific detection will allow solving the main limitations of the tracer 18F-FDG currently used in the clinical setting for the detection of infection foci.

As discussed above, 18F-FDG is a radioactive glucose analog. The accumulation of 18F-FDG by tissues is a marker for the tissue uptake of glucose, which in turn is closely correlated with glucose metabolism involved in a biological process in that specific tissue. Typically, after 18F-FDG is injected into a patient, a PET scanner can form two-dimensional or three-dimensional images of the distribution of 18F-FDG within the body.

18F-FDG presents limitations with respect to the molecular imaging compounds of the invention. On the one hand, the uptake of the 18F-FDG by inflammatory cells such as macrophages and by bacteria does not enable to distinguish between infectious and inflammatory processes, giving rise to false positives when aiming to detect infectious foci. On the other hand, it presents a high natural uptake in organs with a high glucose demand such as the brain or the myocardium, which may mask the infectious uptake.

The imaging probe of the invention showed its ability to distinguish between infection and inflammation in a local infection-inflammation model (Fig. 3A) and in an infective endocarditis model (Fig. 4A). Moreover, it was shown not to accumulate in brain and heart (Table 1).

Moreover, the imaging probe of the invention as described herein has been shown to have high sensitivity in vitro for the detection of Gram+ bacteria, specially Gram+ cocci (Fig. 2D). In particular embodiments, the compound of the invention detects in vitro bacterial amounts as low as 10 ⁵ CFUs, 10 ⁴ CFUs and even 10 ³ CFUs. For S. aureus bacterial amounts of 10 ² CFUs, and even 10 CFUs or lower may be detected. Thus, the compound of the invention may enable to detect in vivo Gram + bacteria (e.g., Gram+ cocci) infection sites at very early stages of infection when bacterial amount is still very low.

The socio-economic impact of the use of the compound of the invention in the specific and non-invasive detection of infectious diseases, specially Gram-positive infections, will allow optimization of personalized treatment in patients, which will result in a reduction in the costs associated with the treatment of infectious processes, as well as an improvement in patient care quality.

As demonstrated in the examples, the compound of the invention is extremely stable in physiological conditions, which will prevent the release of the free isotope that may give rise to false diagnoses, is selective for Gram-positive bacteria compared to other Gram-negative bacteria (E. coli) or fungi (C. albicans), and is highly sensitive, especially for S. aureus, as it is capable of detecting bacterial concentrations below 10 ³ CFUs, even equal to or below 10 CFUs.

It is contemplated that any features described herein can optionally be combined with any of the embodiments of the compound, composition, uses or methods of the invention; and any embodiment discussed in this specification can be implemented with respect to any of these.

All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The use of the word "a" or "an" may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one". The use of the term “another” may also refer to one or more. The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

Throughout the description and claims, the word “comprises” and variants thereof do not intend to exclude other technical features, additives, components, or steps. For those skilled in the art, other objects, advantages, and features of the invention will become apparent in part from the description and in part from the practice of the invention. The following examples and figures are provided by way of illustration and are not intended to be limiting of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Chemical optimization in the synthesis of radiotracer ^99m Tc-DTPA-Collagen and physicochemical characterization. (A) Radioactive labeling yield based on the concentration of collagen I used in the radiosynthesis of ^99m Tc-DTPA-Collagen. (B) Labeling yield of the compound ^99m Tc-DTPA-Collagen with respect to the control ^99m Tc- Collagen in the absence of chelate. (C) Radiolabeling yield in the synthesis of ^99m Tc- DTPA-Collagen based on several concentrations of SnCh. (D) Radioactive measurement by means of thin layer chromatography (TLC) for determining the purity of the compound ^99m Tc-DTPA-Collagen (left) with respect to the chromatographic measurement of the free radioisotope (99mTc). (E) Study of the stability of ^99m Tc- DTPA-Collagen in vitro in PBS over time by means of TLC chromatography.

Figure 2. In vitro study of the uptake of radioactive probe ^99m Tc-DTPA-Collagen. (A) Yield of the binding of probe ^99m Tc-DTPA-Collagen to the S. aureus strain at different times. (B) Study of the % of binding of radiotracer ^99m Tc-DTPA-Collagen to the bacterium S. aureus vs. commercial tracer 18F-FDG and free isotope 99mTc. (C) Comparison of the % of binding of tracer ^99m Tc-DTPA-Collagen vs. free isotope 99mTC in the presence of S. aureus. (D) In vitro binding study of probe ^99m Tc-DTPA-Collagen in the presence of various bacteria and fungi to determine tracer selectivity.

Figure 3. In vivo study of probe ^99m Tc-Collagen in a local intramuscular model (rat). (A) left: In vivo SPETC/CT imaging of tracer ^99m Tc-DTPA-Collagen in a model with double local infection located in legs (dead S. aureus on the left side vs. live S. aureus on the right side); right: In vivo PET/CT imaging of tracer 18F-FDG in a model with double local infection located in legs (dead S. aureus on the left side vs. live S. aureus on the right side). (B) Ex vivo autoradiography study of inflamed and infected muscle tissue after the intravenous administration of the tracer.

Figure 4. In vivo study of tracer ^99m Tc-DTPA-Collagen in an animal model (rat) of infective endocarditis. (A) Rat in vivo PET/CT imaging of radiotracer ^99m Tc-DTPA- Collagen in a control (sham) model and in an IE (infective endocarditis) model. (B) Ex vivo autoradiography study of heart tissue after the administration of tracer ^99m Tc- DTPA-Collagen in a control (sham) model and in an IE model. (C) Ex vivo quantification of the % of injected dose/gram of (heart) tissue of the radiotracer. (D) Histological study with H&E (hematoxylin-eosin) staining of rat heart tissue with IE.

EXAMPLES

The synthesis of radiotracer ^99m Tc-DTPA-Collagen, in which the chelate used will be DTPA and the radionuclide used for its radioactive labeling will be metastable Technetium 99 ( ^99m Tc), is described below. Furthermore, its complete physicochemical characterization, as well as in vitro and in vivo) validation as a non-invasive diagnostic tool for infections caused by Gram-positive bacteria is provided.

The experiments or assays performed will be indicated in section (A) and the results and conclusions thereof in section (B).

(A) MATERIAL AND METHODS

1 : Preparation of the radiotracer

1.1. Conjugation of type I collagen with DTPA-bis-anhydride

100 pl of a 10 mg/ml solution of DTPA-bis-anhydride (Chematech, France) in 0.1 M NaHCOs were added to a 0.7 mg/mL solution (1 mL, mQ H2O) of type I collagen (Sigma-Aldrich). The reaction was kept at 37°C for 20 h with constant stirring. The conjugation product was centrifuged for 35 minutes at 3000 g using Amicon Ultra 100 KDa filter units. Pure samples were recovered by means of centrifugation for 5 minutes at 3000 g and brought to a final volume of 400 pl with 1X PBS.

1.2. Radioactive labeling of the DTPA-Collagen conjugate with " ^m -Technetium, " ^m Tc (IV)

After conjugating the collagen molecule with the chelate DTPA, radioactive labeling of the DTPA-Collagen conjugate with sodium pertechnetate [ ^99m Tc-NaTcC>4] was carried out. The commercial sodium pertechnetate [ ^99m Tc- NaTcC ] was obtained from the TEKCIS™ technetium ^99m Tc generator (Curium Pharma, Spain). As a first step in radiolabeling and for the optimal incorporation of the radionuclide in the collagen structure, the commercial radiopharmaceutical ^99m Tc-NaTcC>4 (100 pl, 20 mCi) was reduced to ^99m Tc (IV) in the presence of stannous chloride (60 pl SnCh in 10% HAc) for 5 minutes at 37°C, under N2 atmosphere. The final solution was neutralized using NaOH (10 pl, 2.8 N). The DTPA-Collagen conjugate (400 pl) was radioactively labeled with this solution of ^99m Tc (IV) with constant stirring a 37°C for 30 minutes. The resulting product was centrifuged for 10 minutes at 15000 rpm using Amicon Ultra 100 KDa filter units. The radioactive end product ^99m Tc-DTPA-Collagen was recovered by centrifugation (5 min, 7500 rpm) and dissolved in 500 pl of 1X PBS.

The yield of the radiochemical reaction and the purity of the radiotracer were estimated by means of iTLC (Instant Thin Layer Chromatography) chromatographic analysis using silica gel as the stationary phase and 90:10 MeOH:H2O (Merck, Germany) as the mobile phase.

2. In vitro stability studies

The in vitro stability of radiotracer ^99m Tc-DTPA-Collagen in 1X PBS was evaluated over time (0 h-50 h) by incubating 1 mCi at 37°C with constant stirring. 3 pl aliquots of probe ^99m Tc-DTPA-collagen were analyzed for each time point (from 1 h to 50 h) using iTLC in a silica gel plate (Merck, Germany) and a mobile phase of 90:10 MeOhkFW.

The in vitro stability of the radiotracer in mouse serum was then evaluated by adding 200 pCi of the radiotracer to 1 ml of mouse serum and incubating the mixture at 37°C with constant stirring. The selected time points for evaluation were 30 and 120 minutes and stability was also analyzed by means of iTLC in a silica gel plate and a mobile phase of 90:10 MeOH: H2O.

3. Determination of LogP and hydrophobicity

The method selected for evaluating the hydrophobicity of the radiotracer was the partition method based on the calculation of LogP. To that end, 200 pl of pure ^99m Tc- DTPA-collagen (1 mCi) were added to an immiscible two-phase mixture of 500 pl of 1 - octanol and 500 pl of 1X PBS. The mixture was homogenized for two minutes with vigorous stirring and then left to stand for 30 minutes for proper phase separation. Lastly, 100 pl of each phase (octanol and PBS) were taken and the activity present in each sample was measured using a Genesys gamma counter (Laboratory Technologies Inc., USA). 4. Preparation of bacterial strains

A Staphylococcus aureus ATCC 29213 strain was used for inoculation in animals. This strain was seeded in blood agar 24 hours before its in vivo inoculation. On the day of inoculation, a bacteriological suspension was prepared using the S. aureus strain and adjusting the McFarland turbidity standards to 0.5, obtaining a concentration of 10 ⁸ CFU/ml.

Strain inactivation: in those cases in which a control solution had to be injected, the S. aureus strain was inactivated using a heat block at 100°C for 5 minutes.

5. In vitro evaluation

The detection capacity of the radiotracer (probe) was first evaluated by means of in vitro studies using a panel of Gram-positive bacteria (S. aureus ATCC 29213, S. epidermidis (clinical strain provided by the Microbiology Department of Hospital Ramon y Cajal of Madrid), S. faecalis ATCC 33186), Gram-negative bacteria (E. coli ATCC 25922), and fungi (C. albicans ATCC 14058). Among the strains that are described, the last two categories were used as controls to demonstrate the specificity of the probe to Gram-positive bacteria.

Binding studies were performed on 96-well plates in which the corresponding pathogens were found in the form of a biofilm at several concentrations (from 10 ¹ to 10 ⁸ ) to enable mimicking the conditions found in clinical practice as much as possible. 150 pL (10 pCi) of pure probe ^99m Tc-DTPA-Collagen were added to each well. Controls with free ^99m Tc with the same activities were carried out in parallel in each CFU to demonstrate the specificity of collagen. Uptake studies were carried out at 37°C and at different incubation times (30 min, 2 h, and 4 h) for the evaluation of binding kinetics. After the corresponding incubation times lapsed, the samples were transferred to an Eppendorf tube and centrifuged to separate the supernatant from the bacterial pellet. Each fraction was separated and measured in a gamma counter, quantifying the binding of the probe to the bacterium based on the activity present in the bacterial pellets.

Lastly, in order to demonstrate the improvement of the methodology of the present invention with respect to the techniques currently used in clinical practice, the binding capacity of probe ^99m Tc-DTPA-Collagen of the present invention was compared to that of commercial 18F-FDG. To that end, 10 pCi of each radiotracer were incubated with 10 ⁸ CFUs of S. aureus for 180 min. The supernatants were separated from the bacterial colonies by means of centrifugation, and the collected pellets and supernatants were measured in a gamma counter to establish the percentages of binding based on the radioactive activity present in each fraction.

6. Blood half-life

The radiotracer blood circulation time was calculated by measuring activity in blood samples taken from the tail vein over time. Radioactive probe ^99m Tc-DTPA-Collagen (910-960 pCi, 250 pL of 1X PBS) was injected intravenously into healthy SD rats (10 weeks of age, weighing 210-260 g, n = 3). The rats were first anesthetized with 2% sevoflurane (100% O2; 200-400 cc/min; Zoetis, Belgium) and blood samples were obtained from the saphenous vein of mice that were awake 5, 15, 30, 45, 60, 75, 90, 120, 180, 240, 300, 360, 420, 1320, 1440, 1620, 2760, 2880, and 3060 min after the injection. For sample normalization, the blood samples were weighed and radioactivity was measured in an automatic Wallac Wizard 1480-011 gamma ray counter (Perkin Elmer, Waltham, MA). Measurements in counts/minute were calculated as % of ID/mean g of tissue.

7. In v/ o evaluation of " ^m Tc-collagen as imaging agent

In vivo evaluation of ^99m Tc-collagen as a diagnostic agent by means of SPECT/CT imaging was carried out in two SD rat models (10 weeks of age, weighing 230-280 g, n = 17 per animal model). In both cases, radiotracer ^99m Tc-DTPA-Collagen was administered by means of intravenous injection through the lateral tail vein (250 pL in PBS, 1.08 ± 0.15 mCi) 24 h after generating the infection models. In all cases, a longitudinal study at different times after injection (1 h, 4 h, and 24 h) was carried out to determine optimal uptake times. a) Local infection-inflammation model (n=6): First, the radiotracer was studied in vivo in a hind leg double local model to demonstrate binding capacity only in active infection sites. In this model, the left hind leg of the rat was infected intramuscularly with a live S. aureus inoculum (0.4 ml, 10 ⁸ CFUs) and the right hind leg was infected with the inactive strain of the bacterium (0.4 ml, 10 ⁸ CFUs). To evaluate the effect of the spread of the infection process on the diagnostic capacity of the radiotracer, the bacterial dosage was inoculated at different times before the administration of the radiotracer (4 h, 24 h, 48 h) and the Microbiology Department of Hospital Gregorio Maranon (Madrid, Spain) performed ex vivo confirmation of a positive infection. b) Reference example local-infection inflammation model: As control model in the evaluation of the selectivity of the radioprobe, in vivo, [18F]FDG PET-CT imaging was acquired 72 h after inoculation employing a local infection-inflammation model (n=4). PET/CT images were acquired 2 h post-injection of 37 - 48 MBq of [18F]FDG (300 pl).

PET/CT studies were acquired using a small-animal PET/CT scanner (PET/CT SuperArgus, SEDECAL Molecular Imaging, Madrid, Spain). PET data were collected for 40 min in two bed positions (20 min per bed) 60 min and reconstructed using OSEM-2D with 16 subsets and 1 iteration (voxel size: 0.388 x 0.388 x 0.775 mm). CT parameters were acquired using an X-ray beam current of 340 pA and tube voltage of 40 kVp, and studies were reconstructed using an FDK algorithm. PET and CT images were co-registered using dedicated software (Multimodality Workstation for small animal visualization and analysis (MMWKS). Regions of interest (ROIs) were manually drawn on the CT over hind legs (deleting skin, bone marrow and bone sections) muscles and afterwards applied to the co-registered PET images. c) Infective endocarditis model: Once the capacity of radiotracer ^99m Tc-DTPA- Collagen for detecting active sites, preventing the binding thereof to inflammatory sites, was demonstrated, in vivo evaluation was carried out in a local heart infective endocarditis model. To enable developing a model similar to clinical reality, the Durack method for generating infective endocarditis in the aortic valve and the left ventricle of rats, the weight of which was controlled before the intervention and 24 h after the inoculation of bacteria, was applied. Briefly, a polyethylene (PE-50) catheter was introduced through the right common carotid artery. The catheter was then inserted, fixed, and maintained in the left ventricle throughout the entire evaluation, and its location was monitored by means of TC (using iodine contrast), ultrasound, and magnetic resonance. The active S. aureus strain was inoculated in rats by means of intravenous administration (0.4 ml, 10 ⁸ CFUs) 24 after surgical intervention. The Microbiology Department of Hospital Gregorio Maranon (Madrid, Spain) carried out the confirmation of infection by means of the ex vivo evaluation of the histological tissue of the catheter (n = 11), heart (n = 8), and kidneys (n = 6) **.

In both models, the in vivo image was acquired by means of using SPECT (MiLabs LISPECT II, Boston, MA) and CT (SuperArgus PET/CT, SEDECAL, Spain) scanners. The rats were placed in the prone position and the field of view was set to the area of interest. Once the radiotracer was administered, a 2-frame scan lasting for 30 minutes per animal was acquired. A multi-pinhole collimator 1.0 was used in SPECT image acquisition and an energy window of 126 to 154 KeV was selected for this radionuclide. OS-EM reconstruction was performed with a voxel size of 0.75 mm ³ , 16 subsets, and 1 iteration, using patented software (MiLabs, Boston, MA). To correct scattered events, two windows 20% to the left and to the right of the radioactive technetium peak were applied, and reconstruction was completed under a post-Gaussian blur filter with a FWHM between 0.8 and 0.9 mm. For the acquisition of anatomical images, the selected CT parameters were 40 KeV, 340 pA, 360 projections, and 2 x 2 binning, and 0.12 mm ³ of voxel size images were reconstructed using built-in PET/CT software (SEDECAL, Spain).

SPECT and CT images were recorded together using 3 markers previously loaded with contrast and radioactive agents and using MMWKS software marketed by the company Sedecal.

8. Ex vivo biodistribution studies

The ex vivo biodistribution study was performed in healthy SD rats (10 weeks of age, weighing 210-190 g, n = 3) to quantify probe uptake in the main organs. To that end, the radioactive collagen molecule (250 pL in 1X PBS, 350 pCi, n = 3) was administered by means of intravenous injection in the lateral tail vein and left to circulate for 1 hour. The animals were then sacrificed and the organs of interest, i.e., brain, trachea, lungs, heart, liver, spleen, pancreas, stomach, kidneys, intestines, feces, skin, blood, and urine, removed. The activity of each tissue was measured in an automatic Wallac Wizard 1480-011 gamma counter (Perkin Elmer, Waltham, MA), and expressed as mean % of I D/g tissue.

9. Ex vivo autoradiography analysis

The histological confirmation of the results obtained in vivo by means of PET/CT imaging was carried out by means of autoradiography. To that end, the radiotracer (X pCi in 250 pL of 1X PBS) was intravenously inoculated in SD rats with infective endocarditis model (10 weeks of age, 250-300 g, n = 3). Likewise, as control tests, the same method was carried out on the Sham model (n=4) with ventricular surgery but without infection. The animals were sacrificed 1 h after the circulation of the radiotracer (uptake) and spleen, kidneys, and heart were harvested. These organs were cut into sections, mounted on plastic slides, and placed in a phosphor plate (BAS-MP 2025, Fujifilm) for 1 hour at room temperature. Radiotracer accumulation in the tissues was measured by reading the plate at a resolution of 200 p with a Bio-Imaging Analyzer BAS-500 plate reader (Fujifilm, Japan).

10. Post-mortem microbiological analysis a) Sample culture

All the tissue samples were obtained by surgical excision, sent to the microbiology laboratory in a dry sterile container, and kept in a refrigerator at 2-8°C until the processing thereof. Once the samples reached suitable temperature, the processing thereof commenced, introducing the tissue in a sterile mortar with 0.5 ml of saline. From the crushed sample, 100 p were obtained and qualitatively spread with a loop on the blood agar plate. b) Catheter culture

All the catheter samples were obtained by surgical excision, sent to the microbiology laboratory in a dry sterile container, and kept in a refrigerator at 2-8°C until the processing thereof. Once the samples reached room temperature, the processing thereof commenced, using the semi-quantitative Maki method consisting of rolling the distal end of the catheter across a sterile blood agar plate.

(B) RESULTS

Example 1 Synthesis and physicochemical characterization

Optimization under DTPA-Collagen complex radioactive labeling conditions showed a linear relationship between concentrations of collagen and labeling yields (Figure 1A). Furthermore, in the evaluation of reduction conditions, lower labeling yields were observed at high reducer concentration (0.2 mM SnCh) and low reducer concentration (0.0002 mM SnCh) probably due to a degradative effect in the presence of high concentrations and the incomplete reduction of commercial pertechnetate ( ^99m TcC>4) in the presence of low concentrations, entailing the incomplete incorporation of the radionuclide in the chelate, and therefore the biomolecule. Based on these results, the optimization of synthetic conditions and radiolabeling determined that the optimal parameters for obtaining the highest radiochemical yields were 2 mg/ml of collagen and 0.002 mM of SnCh. Using said conditions, a yield of 42.86 ± 6.35% was obtained (Figures 1A and C). The radioactive purity of the compound, determined by means of thin layer chromatography, iTLC, established values of 95.84 ± 1.85% (Figure 1 D), thereby confirming the ideal purity for in vivo application and possible translational leap to clinical practice. By means of the partition coefficient, a LogP value for these compounds of -3.69 ± 0.58 was experimentally determined, confirming the high hydrophilicity of the compound. This datum is a determinant in the intravenous administration of the compound, as it requires high water solubility, confirmed in the probe of the present invention.

The evaluation of the stability of the compound in conditions similar to the physiological environment (37°C, pH 7, PBS) confirmed a stability above 90% even after 50 h of incubation (Figure 1 E). Similar results were obtained in the evaluation of the radiotracer in serum by means of iTLC. A stability of 92.83 ± 1.48% (n=3) was observed after 30 min, remaining constant until 120 min (92.23 ± 3.45%). These results support the in vivo use thereof by confirming the high stability in physiological conditions, which will prevent the release of the free isotope that may give rise to false diagnoses.

Example 2.- In vitro study

As a first step in the in vitro study of the probe, its capacity to bind to the target bacteria (in this case S. aureus) was evaluated in comparison to the free radioisotope to demonstrate the selectivity thereof for collagen binding proteins present in the bacterial wall due to the vehicle biomolecule (collagen). For the purpose of being able to also determine the kinetics of said binding, the study was repeated at various time points, i.e. , 15 min, 30 min, 90 min, 2 h, and 3 h (Figure 2A). A linear relationship between the concentration of bacteria and the percentage of binding of the tracer was observed, the specificity of the probe thus being confirmed. Furthermore, in all cases the difference between the specific radiotracer and the free isotope was at least 7-fold, reaching differences of up to 26-fold higher in the case of studies performed at 2 h and with 10 ⁸ CFUs (Figure 2C), confirming selectivity due to the collagen vector.

Once the selectivity of the probe for the bacterium S. aureus was demonstrated, a more extensive study was carried out in which the selectivity and specificity of the probe for Gram-positive bacteria was evaluated. The tests performed on the microbiological panel demonstrated the selectivity of the probe for Gram-positive bacteria in comparison with other Gram-negative bacteria (E. coli) or fungi (C. albicans). Among Gram-positive bacteria, the probe showed higher affinity for staphylococcal strains (both S. aureus and S. epidermidis). This study allowed the sensitivity of the probe to be determined at the same time, establishing the minimal concentration of bacterium to which the probe is capable of binding (detection limit). The high sensitivity of the radiotracer of the present invention was demonstrated as it is capable of detecting staphylococcal concentrations below 10 CFUs (Figure 2D).

As the last step in the in vitro study of the probe, its detection capacity was evaluated in comparison with the standard radiotracer used in clinical setting for detection of infection foci, i.e., commercial tracer 18F-FDG. Said study demonstrated the higher capacity of the probe of the present invention to detect staphylococcal cultures, the sensitivity thereof being 3.5-fold greater than that of the commercial tracer (Figure 2B).

Example 3.- In vivo evaluation of tracer " ^m Tc-Collagen a) Pharmacokinetic study: Both the blood half-life of the tracer and its biodistribution were determined in the pharmacokinetic evaluation of the tracer in order to determine its future applications in infection diagnosis. Said studies determined a mean blood half-life below 20 min (19 ± 2 min), suggesting a quick metabolism and the use of short uptake times in diagnosis by means of medical imaging. The quantitative analysis of the biodistribution of the probe of the present invention (1 h post-injection) determined a major accumulation in the liver (6.11 ± 2.80% ID/g) and spleen (2.06 ± 0.15% ID/g), confirming a hepatobiliary metabolism typical of proteins with a large weight molecular (300 KDa) of this type. The high uptake that is also present in kidneys (0.94 ± 0.45% ID/g) confirms rapid renal excretion, supporting short blood circulation times.

Table 1. Values of the % of injected dose/gram tissue for ex vivo biodistribution b) In vivo evaluation in a leg infection model: As an initial evaluation of the capacity of the probe of the present invention to detect infection sites by means of imaging, tracer ^99m Tc-Collagen was analyzed in a local infection-inflammation model with intramuscular infection. In said model, by means of SPECT-CT imaging, the tracer demonstrated its ability to bind mainly to regions where the active (live) strain was found, with little or no binding to tissues inoculated with the inactivated (heat-killed) strain. Therefore, the tracer of the present invention clearly showed its capacity to detect active infection sites in comparison to inactive infection sites (Figure 3A - left). As a reference example, we also provide the results of in vivo PET-CT imaging obtained 72 h after the inoculation of [18F]FDG. The bacteria showed similar uptake values in both left and right leg muscles (Log10-infected vs. Log10-inflamed ratio of 1.08 ± 0.01. This result confirms the well-known limitations of FDG in distinguishing between infection and the inflammatory processes (Figure 3A - right). The results observed by means of in vivo imaging were confirmed by means of ex vivo measurements of biodistribution and autoradiography (Figure 3B). c) In vivo evaluation in an infective endocarditis (IE) model: Once the effectiveness of the tracer was confirmed in a simple local (intramuscular) model, the detection capacity of the probe was evaluated in a more complex infective endocarditis model similar to the clinical pathology. Like in the preceding tests, after the intravenous administration of the radioactive compound, the tracer of the present invention was capable of binding specifically to the infection site located in the heart valve. To confirm the ability of the tracer to distinguish between infectious and inflammatory processes and to thereby prevent false positives, imaging was carried out in a control sham model, which presented surgery but not infection. In this case, no tracer uptake was observed in the damaged area, thereby demonstrating the selectivity of the tracer only for infection sites. These observations were confirmed ex vivo by means of autoradiography (Figure 4B) where high cardiac uptake can be clearly observed in the IE model but not in the control. The histological validation (Figure 4D) confirmed bacterial colonization and damage. The microbiological confirmation of the model was carried out by means of bacterial seeding studies, confirming the presence of the microorganism S. aureus in infected hearts and catheters but not in controls.