ES2894201B2 - RADIOACTIVE COMPOUNDS BASED ON SELECTIVE BACTERIA PROTEINS FOR THE NON-INVASIVE DETECTION OF INFECTIOUS FOCUSES - Google Patents

Description

DESCRIPTION

Radioactive compounds based on selective bacterial proteins for the non-invasive detection of infectious foci

The present invention relates to radioactive compounds, also referred to herein as radiotracers, based on proteins, such as collagen, fibronectin and fibrinogen, for the non-invasive detection of infectious foci caused by Gram+ bacteria by nuclear imaging.

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

BACKGROUND OF THE INVENTION

To date, the imaging techniques used in the diagnosis of infectious foci are based on classical techniques such as magnetic resonance imaging (MRI) or computed tomography (CT). The main limitation of these techniques is their low sensitivity, incapable of detecting infectious foci in the initial stages. On the other hand, in the development of more selective techniques in recent years, molecular imaging has begun to be used, especially nuclear imaging, due to the high sensitivity of the technique. Despite the improvement in infectious diagnosis through imaging provided by this methodology, the use of known and commercially available radiotracers, such as 18FDG (a radiopharmaceutical consisting of a glucose analog 2-[18F]fluoro-2-deoxy-D- glucose linked to the radioactive isotope fluorine-18), specific not only for bacteria, but also for other cell lines such as macrophages or tumors, in many cases gives rise to false positives.

On the other hand, the use of classic diagnostic techniques such as cell or bacterial cultures require long times in the analysis of the results (several days of incubation), as well as taking a biopsy or blood.

The present invention aims to solve current clinical problems, allowing an early, selective and non-invasive diagnosis of infectious processes.

DESCRIPTION OF THE INVENTION

The present specification describes radioactive compounds (also called radiotracers, imaging probes or simply probes) based on a protein, such as collagen or other similar proteins related to the binding proteins present in the bacterial wall, for the non-invasive detection of Infectious foci caused by Gram-positive bacteria by nuclear imaging (SPECT/PET, single photon emission computed tomography/positron emission tomography).

In the development of the new imaging probe, the protein, preferably collagen, is radioactively labeled with different radioisotopes such as 99mTc, 68Ga, 18F, 89Zr, 64Cu or 111 ln without modifying its native structure. After the injection of the radioactive probe, it will act by binding specifically to the wall of Gram-positive bacteria through the binding receptors to the protein in question, allowing the non-invasive and specific detection of infectious foci produced by said bacteria by means of core image.

In a first aspect, the present invention relates to a compound comprising a protein covalently linked to a chelating agent and a radioisotope coordinated to the chelating agent, where the protein is selected from the list comprising: collagen, fibronectin and fibrinogen, all of them molecules related to proteins anchored to the bacterial wall.

The chelating agent is a compound or molecule that comprises at least one first group selected from: amine, maleimide and carboxyl configured to covalently bind to proteins and at least one second group selected from carboxyl, amino and phosphine configured to coordinate with the metal isotope

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

- Pentetic Acid, also called 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]thiourea)] and

- 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 or linking agent is pentenic 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 to through oxygens of carboxyl groups and nitrogens that pentenic acid comprises in its structure.

In another preferred embodiment, the protein is collagen, more preferably type I or type IV collagen.

The radioisotope is preferably selected from the list comprising: 99mTc, 68Ga, 18F, 89Zr, 64Cu or 111ln. In a more preferred embodiment, the radioisotope is 99mTc and in an even more preferred embodiment, the radioisotope R is 99mTc (IV):

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

The preparation of radiotracers requires two synthetic stages: a first stage of covalent bond formation between the protein with a suitable chelator, thus forming a protein-chelator conjugate, and a second radiochemical stage based on the radioactive labeling of the protein-chelator conjugate with the corresponding radioisotope.

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

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

Another aspect of the invention refers to the use of the compound of the invention, or of the composition that comprises it, as an imaging agent for the visualization of infections or infectious foci caused by Gram-positive bacteria in a subject (animal or human).

In the present invention, the term "imaging agent" refers to a chemical compound whose structure contains a radioactive isotope (also called radiotracer, radiopharmaceutical) that is capable of carrying out the monitoring of a biological process, producing a signal that can be detected by imaging techniques in nuclear medicine, such as single photon emission computed tomography and positron emission tomography. Then, the compound acts as a probe for the detection of infections or infectious foci caused by Gram-positive bacteria.

The term "probe" refers to a chemical compound capable of selectively detecting infectious foci caused by gram-positive bacteria.

In a preferred embodiment, the visualization of infections or infectious foci is performed by the PET (Positron Emission Tomography) or SPECT (Single Photon Emission Computed Tomography) technique.

In a preferred embodiment, the Gram positive bacteria are selected from the list comprising: Staphylococcus aureus, Staphylococcus epidermis, Mycobacterium tuberculosis, Enterococcus faecalis, Staphylococcus agalactiae and Clostridioides difficile,

In a preferred embodiment, the infection caused by the Gram positive bacteria is infective endocarditis.

Another aspect of the invention relates to a method for providing an image of infectious foci caused by Gram-positive bacteria in vivo in a subject, comprising (i) administering the compound of the invention to the subject and (ii) scanning the subject using images PET or SPECT combined with a structural technique such as computerized anatomical tomography to obtain a visible image.

A final aspect of the invention relates to a method for detecting infectious foci or infections in a subject comprising the following steps:

- administration of an effective amount of the compound of the invention to a subject, - obtaining an image after the administration of the compound, providing adequate time for the administered compound to bind to the target infectious focus, - reconstruction and analysis of the acquired image.

In the present invention, the term "effective amount" refers to that amount of the compound which, when administered, is sufficient to be detected by nuclear resonance imaging.

The radioactive compound of the invention, which is used for the non-invasive detection of infectious foci of Gram-positive bacteria, will specifically bind to protein-binding receptors (preferably collagen) present on the wall of Gram-positive bacteria, allowing the non-invasive and specific detection of infectious foci produced by said bacteria by means of nuclear imaging.

The absence of collagen binding proteins (collagen receptors) in macrophages or myocytes allows the compound of the invention to detect only infectious foci. This specific detection will make it possible to overcome the main limitations of the 18F-FDG tracer currently used in the clinic. On the one hand, its inability to distinguish between infectious and inflammatory processes, giving rise to false positives. On the other hand, its high natural uptake in the myocardium, which can mask infectious uptake.

The socioeconomic impact of the use of the compound of the invention in the specific and non-invasive detection of infectious diseases 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 in a improvement in patient care quality.

The compound of the invention, as has been shown in the examples, has high stability under physiological conditions, which will prevent the release of the free isotope that could lead to false diagnoses; It presents selectivity towards Gram positive bacteria against other Gram negative bacteria ( E. coli) or fungi ( C. albicans) and high sensitivity as it is capable of detecting concentrations of bacteria below 10CFU.

Throughout the description and claims the word "comprise" and its variants are not intended to exclude other technical characteristics, additives, components or steps. Other objects, advantages and features of the invention will be apparent to those skilled in the art 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

Fig. 1.- Chemical optimization in the synthesis of the radiotracer 99mTc-DTPA-Collagen and physicochemical characterization. (A) Yield of radioactive labeling as a function of the concentration of collagen I used in the radiosynthesis of 99mTc-DTPA-Collagen. (B) Labeling performance of the 99mTc-DTPA-Collagen compound versus the 99mTc-Collagen control in the absence of a chelator. (C) Radiolabeling performance in the synthesis of 99mTc-DTPA-Collagen as a function of various concentrations of SnCE (D) Radioactive measurement by thin plate chromatography (TLC) for the determination of the purity of the compound 99mTc-DTPA-Collagen (left ) versus the chromatographic measurement of the free radioisotope (99mTc). (E) Stability study of 99mTc-DTPA-Collagen in vitro in PBS by TLC chromatography over time

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

Fig.3 In vivo study of the 99mTc-Collagen probe in a local intramuscular model (rat). (A) In vivo PETC/CT image of the 99mTc-DTPA-Collagen tracer in a double model of localized paw infection (left S. aureus dead vs. right S. aureus live). (B) Ex vivo study by autoradiography of inflamed and infected muscle tissue after intravenous administration of the tracer.

Fig. 4.- In vivo study of the 99mTc-DTPA-Collagen tracer in an animal model (rat) of infective endocarditis. (A) In vivo rat PET/CT image of the radiotracer 99mTc-DTPA-Collagen in a control model (sham) and in an El model (infective endocarditis). (B) Ex vivo study by autoradiography of cardiac tissue after administration of the 99mTc-DTPA-Collagen tracer in control (sham) and El models. (C) Ex vivo quantification of the % of injected dose per gram of tissue (heart) of the radiotracer. (D) Histological study with H&E stain (hematoxylin-eosin stain) of rat heart tissue with El.

EXAMPLES

Next, the synthesis of the radiotracer 99mTc-DTPA-Collagen is described, where the chelating agent used will be DTPA and the radionuclide for its radioactive labeling will be metastable Technetium 99 (99mTc). In addition, its complete physicochemical characterization is presented, as well as its validation ( in vitro and in vivo) as a non-invasive diagnostic tool for infections caused by gram-positive bacteria.

In section (A) the experiments or tests carried out will be indicated and in section (B) the results and conclusions thereof.

(A) REALIZED EXAMPLES

Example 1: Preparation of the radiotracer

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

To a solution of type I collagen (Sigma-Aldrich) of 0.7 mg/mL (1 mL, H2O mQ) was added 100 pl of a 10 mg/ml solution of DTPA-bis-anhydride (Chematech, France) in 0.1 M NaHC03. The reaction was kept at 37 °C for 20 h with stirring. constant. The conjugation product was centrifuged 35 minutes at 3000 g using Amicon Ultra 100 KDa filter units. Pure samples were recovered by centrifugation for 5 minutes at 3000 g and raised to a final volume of 400 ml with 1X PBS.

1.2.- Radioactive labeling of the DTPA-Collagen conjugate with 99m-Technetium, 99mTc (IV) After the conjugation of the collagen molecule with the chelating agent DTPA, the radioactive labeling of the DTPA-Collagen conjugate was carried out with sodium pertechnetate [99mTc-NaTc04 El Commercial sodium pertechnetate [99mTc-NaTc04] was obtained from the technetium99mTcTEKCIS ™ generator (Curium Pharma, Spain). As a first stage in the radiolabeling and for optimal incorporation of the radionuclide into the collagen structure, the commercial radiopharmaceutical 99mTc-NaTc04 (100 pl, 20 mCi) was reduced to 99mTc(IV) in the presence of stannic chloride (60 pl SnCl2 in HAc 10%) for 5 minutes at 37 0C, under N2 atmosphere. The final solution was neutralized using NaOH (10 pl, 2.8N). The DTPA-Collagen conjugate (400 pl) was radioactively labeled with this 99mTc solution (IV) with constant stirring at 370C for 30 minutes. The resulting product was centrifuged 10 minutes at 15,000 rpm using Amicon Ultra 100 KDa filter units. The final radioactive product 99mTc-DTPA-Collagen was recovered by centrifugation (5 min, 7500 rpm) and dissolved in 500 µl 1X PBS.

The yield of the radiochemical reaction and the purity of the radiotracer were estimated by TLC (Instant Thin Layer Chromatography) chromatographic analysis using silica gel as stationary phase and mobile phase = 90:10 MeOH:H20 (Merck, Germany).

Example 2.- In vitro stability studies

The in vitro stability of the radiotracer 99mTc-DTPA-Collagen in PBS 1X was evaluated over time (0h-50h) by incubating 1 mCi at 37°C with constant agitation. Aliquots of 3 pl of the 99mTc-DTPA-collagen probe were analyzed at each time point (1h to 50h) using silica gel plate TLC (Merck, Germany) and mobile phase of 90:10 MeOH:H20.

Subsequently, the in vitro stability of the radiotracer in mouse serum was evaluated by adding 200 pCi of the radiotracer to 1 ml of murine serum and incubating the mixture at 37 0C with constant agitation. Time points selected for evaluation were 30 and 120 minutes and stability was also analyzed by ¡TLC on silica gel plate and mobile phase of 90:10 MeOH:H20.

Example 3.- Determination of LoqP and hydrophobicity

The procedure selected for the evaluation of the hydrophobicity of the radiotracer was the partition method based on the calculation of LogP. For this, 200 μl of pure 99mTc-DTPA-collagen (1 mCi) were added to an immiscible biphasic mixture of 500 μl of 1-octanol and 500 μl of PBS 1X. The mixture was homogenized for two minutes with vigorous stirring and was subsequently allowed to decant for 30 minutes for the correct separation of the phases. Finally, 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).

Example 4.- Preparation of bacterial strains

For the inoculation of animals, a Staphylococcus aureus ATCC 29213 strain was used, which was seeded on blood agar 24 hours before 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 108 cfu/ml.

Inactivation of the strain: in those cases in which it was required to inject a control solution, the S. aureus strain was inactivated using a thermoblock at 100°C for 5 minutes.

Example 5.- In vitro evaluation

The detection capacity of the radiotracer (probe) was initially evaluated by in vitro studies using a panel of both gram-positive bacteria (S aureus ATCC 29213, S epidermidis (clinical strain provided by the microbiology service of the Ramón y Cajal hospital in Madrid), S faecalis ATCC 33186) as negative (E coli ATCC 25922) and fungi (C albicans ATCC 14058). Among the strains described, the last two categories were used as controls to demonstrate the specificity of the probe towards gram-positive bacteria)

Binding studies were performed on 96-well plates in which the corresponding pathogens were found at various concentrations (from 101 to 108) in the form of a biofilm in order to mimic as much as possible the conditions found in clinical practice. 150uL (10 pCi) of pure 99mTc-NaTc04 probe were added to each well. Parallel controls with free 99mTc with the same activities were carried out at each CFU to demonstrate the specificity of collagen. Uptake studies were carried out at 37°C and at different storage times. incubation (30 min, 2h and 4h) for the evaluation of binding kinetics. After the corresponding incubation times, the samples were transferred to eppendorf and centrifuged in order 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 bacteria based on the activity present in the bacterial pellets.

Finally, in order to demonstrate the improvement of our methodology compared to the techniques currently used in clinical practice, the binding capacity of our 99mTc-NaTc04 probe was compared to the commercial 18F-FDG. For this, 10 pCi of each radiotracer were incubated with 108 CFU of S aureus for 180 min. Supernatants were separated from bacterial colonies by centrifugation and collected pellets and supernatants were measured in a gamma counter to establish binding percentages based on radioactive activity present in each fraction.

Example 6.- Half-life time in blood

The blood circulation time of the radiotracer was calculated by measuring the activity in blood samples taken over time from the tail vein. The radioactive probe 99mTc-DTPA-Collagen (910-960 pCi, 250 pL of 1X PBS) was injected intravenously into healthy SD rats (10 weeks old, weight 210-260 g, n = 3). Rats were previously anesthetized with 2% sevoflurane (O2100%; 200-400 cc/min; Zoetis, Belgium) and blood samples were obtained from the saphenous vein in awake mice at 5, 15, 30, 45, 60, 75 minutes. , 90, 120, 180, 240, 300, 360, 420, 1320, 1440, 1620, 2760, 2880 and 3060 min after injection. For sample normalization, blood samples were weighed and radioactivity measured in a Wallac Wizard 1480-011 automatic gamma ray counter (Perkin Elmer, Waltham, MA). Measurements in counts per minute were calculated as %D/g tissue mean.

Example 7.- Single photon emission computed tomography/computerized tomography (SPECT I CT) in vivo image

In vivo evaluation by SPECT/CT imaging of 99mTc-collagen as a diagnostic agent was carried out in two SD rat models (10 weeks old, weight 230-280 g, n = 17 per animal model). In both cases, the radiotracer 99mTc-DTPA-Collagen was administered by 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 was carried out at different times. after injection (1h, 4h and 24h) to determine optimal uptake times a) Local infection-inflammation model (n=6): Initially, the radiotracer was studied in vivo in a double local model of hind legs to demonstrate the ability binding only in active infectious foci. In this model, the left hind paw of the rat was infected intramuscularly with an inoculum of live S. aureus (0.4 ml, 108 CFU) and the right hind paw another with the inactive strain of the bacterium (0.4 ml, 108 CFU). To evaluate the effect of the extension of the infectious process on the diagnostic capacity of the radiotracer, the bacterial dose was inoculated at different times before the administration of the radiotracer (4 h, 24 h, 48 h) and the Microbiology Service of the Hospital Gregorio Marañón (Madrid, Spain) performed ex vivo confirmation of positive infection.

b) Infective endocarditis model: Once the ability of the 99mTc-DTPA-Collagen radiotracer to detect activated foci by preventing its binding to inflammatory foci was demonstrated, its in vivo evaluation was carried out in a localized cardiac model of infective endocarditis. In order to develop a model similar to clinical reality, the Durack method was applied to generate infective endocarditis in the aortic valve and left ventricle of rats, whose weight was controlled before the intervention and 24 h after bacterial inoculation. Briefly, a polyethylene catheter (PE-50) was introduced through the right common carotid artery. The catheter was then inserted, fixed, and maintained throughout the evaluation in the left ventricle, and its location was monitored by CT (using iodine contrast), ultrasound, and MRI. After 24 h of the surgical intervention, the active strain of S. aureus was inoculated into the rats by intravenous administration (0.4 ml, 108 CFU). The Microbiology Service of Hospital Gregorio Marañón (Madrid, Spain) confirmed the infection by ex vivo evaluation of histological tissue from the catheter (n = 11) and from the heart (n = 8) and kidneys (n = 6). ) **.

In both models, in vivo image acquisition was carried out using the SPECT scanner (MiLabs USPECT II, Boston, MA) and CT (PET/CT SuperArgus, SEDECAL, Spain). The rats were placed in the prone position and the field of view was adjusted to the area of interest. Once the radiotracer was administered, a scan of 2 frames of 30 minutes per animal was acquired. A 1.0 multipinhole collimator was used in the acquisition of SPECT images and, for this radionuclide, an energy window of 126 to 154 KeV was selected. OS-EM reconstruction was performed with a voxel size of 0.75 mm3, 16 subsets, and 1 iteration, using proprietary software (MiLabs, Boston, MA). To correct for scattered events, applied two 20% windows to the left and right of the radioactive technetium peak, and reconstruction was completed under a Gaussian blur post-filter with a FWHM between 0.8 and 0.9 mm. For anatomical image acquisition, the selected CT parameters were 40 KeV, 340 pA, 360 projections, and 2 x 2 binning and 0.12 mm voxel size images were reconstructed using the embedded PET/CT software (SEDECAL, Spain). .

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

Example 8.- Ex vivo biodistribution studies

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

Example 9.- Ex vivo analysis by autoradiography

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

Example 10.-Microbiological Post-Mortem Analysis

a) Culture of Samples

All tissue samples were obtained by surgical excision and sent to the microbiology laboratory in a dry sterile container and stored in a refrigerator at 2-8°C until processing. Once the samples were tempered, they were processed, introducing the tissue in a sterile mortar with 0.5 ml of saline solution. 100 p were obtained from the crushed sample, and it was spread qualitatively with a loop on the blood agar plate.

b) Catheter Culture

All catheter samples were obtained by surgical excision and sent to the microbiology laboratory in a dry sterile container and stored in a refrigerator at 2-8°C until processed. Once the samples were tempered, they were processed using Maki's semiquantitative method, which consists of rolling the distal end of the catheter on a sterile blood agar plate.

(B) RESULTS OBTAINED

Synthesis and physicochemical characterization

Optimization under radioactive labeling conditions of the DTPA-Collagen complex showed a linear relationship between collagen concentrations and labeling yields (Figure 1A). In addition, in the evaluation under the reduction conditions, lower labeling yields were observed at high (0.2 mM SnCh) and low (0.0002 mM SnCh) concentrations of reductant, probably due to a degradative effect in the presence of high concentrations and to the non-reduction. total commercial pertechnetate (99mTc04) in the presence of low concentrations, is caused by the incomplete incorporation of the radionuclide in the chelator and therefore biomolecule. Based on these results, the optimization of the synthesis and radiolabeling conditions determined that the optimal parameters to obtain the highest radiochemical yields were 2mg/ml of collagen and 0.002 mM of SnCE. Using these conditions, a yield of 42.86 ± 6.35% (Fig 1A and C). The radioactive purity of the compound, determined by thin plate chromatography, ¡TLC, established values of 95.84 ± 1.85% (Figure 1.D), thus confirming the ideal purity for its application in vivo and possible translational jump to clinical practice. Using the partition coefficient, a LogP value of -3.69 ± 0.58 was experimentally determined for these compounds, confirming the high hydrophilicity of the compound. This data is crucial when administering the compound intravenously, since it requires a high solubility in water, confirmed in our probe.

The evaluation of the stability of the compound under conditions similar to the physiological environment (37°C, pH: 7, PBS) confirmed a stability greater than 90% even after 50h of incubation (Figure 1E). Similar results were obtained in the evaluation of the radiotracer in serum by ¡TLC. At 30 min we observed a stability of 92.83 ± 1.48% (n=3), remaining constant up to 120 min (92.23 ± 3.45%). These results support its use in vivo by confirming its high stability under physiological conditions, which will prevent the release of the free isotope that could lead to false diagnoses.

in vitro study

As a first stage in the in vitro study of the probe, its ability to bind to the target bacteria (in this case S. aureus) was evaluated against the free radioisotope to demonstrate its selectivity towards the collagen-binding proteins present in the bacterial wall due to the vehicle biomolecule (Collagen). In order to also be able to determine the kinetics of said union, the study was repeated at various time points; 15min, 30min, 90min, 2h and 3h (Fig.1.A). A linear relationship was observed between the concentration of bacteria and the percentage of binding of the tracer, thus confirming the specificity of the probe. In addition, in all cases the difference between the specific radiotracer and the free isotope was at least 7-fold, reaching differences of even 26-fold higher in the case of the studies at 2h min and 108 CFU (Fig 2.C). , confirming the selectivity due to the collagen vector.

Once the selectivity of the probe towards S. aureus bacteria was demonstrated, a broader study was carried out in which the selectivity and specificity of the probe towards Gram positives was evaluated. The tests carried out in the microbiological panel demonstrated the selectivity of the probe towards Gram positives against other Gram negative bacteria ( E. coli) or fungi ( C. albicans). Among Gram-positive bacteria, the probe showed a higher affinity for staphylococcal strains (both S. aureus and S. epidermidis). At the same time, this study made it possible to determine the sensitivity of the probe, establishing the minimum concentration of bacteria at which the probe is capable of binding (detection limit). The high sensitivity of our radiotracer was demonstrated by being able to detect staphylococcal concentrations below 10CFU (Figure 2D).

As the last stage in the in vitro study of the probe, its detection capacity was evaluated against the standard radiotracer used in the clinic; the trade tracer 18F-FDG. This study demonstrated the greater capacity of our probe to bind to staphylococcal cultures, its sensitivity being 3.5 times higher than that of the commercial tracer (Figure 2B).

In vivo evaluation of the 99mTc-Collagen tracer

a) Pharmacokinetic study: Within the pharmacokinetic evaluation of the tracer, both its half-life in blood and its biodistribution were determined to determine its future applications in infectious diagnosis. These studies determined a mean blood life time of less than 20 min (19 ± 2 min), which points to a rapid metabolism and suggests the use of short uptake times in medical imaging diagnosis. Quantitative analysis of the biodistribution of our probe (1h post injection) determined a main accumulation in liver (6.11 ± 2.80%ID/g), spleen (2.06 ± 0.15%ID/g), confirming a typical hepatobiliary metabolism of this type of protein with large molecular weight (300KDa). The high uptake present also in the kidneys (0.94 ± 0.45 %ID/g) confirms a rapid renal excretion, supporting the short blood circulation times.

Table 1. Values of % injected dose / gram of tissue for ex vivo biodistribution

b) In vivo evaluation in a paw infection model: As an initial evaluation of the ability of our probe to detect infectious foci by imaging, the 99mTc-Collagen tracer was analyzed in a local infection-inflammation model with intramuscular infection. In this model, the tracer demonstrated through SPECT-CT imaging, its ability to bind mainly in the regions where the active (alive) strain was found, with little or no binding to the tissues inoculated with the inactivated strain (heat-killed). In this way, the tracer of the present invention demonstrated its ability to detect active versus inactive infectious foci. The results observed by in vivo imaging were confirmed by ex vivo measurements of biodistribution and autoradiography (Figure 3B).

c) In vivo evaluation in a model of infective endocarditis (El): Once the effectiveness of the tracer was confirmed in a simple local model (intramuscular), the detection capacity of the probe was evaluated in a more complex model of infective endocarditis similar to the clinical pathology . As in previous tests after intravenous administration of the radioactive compound, our tracer was able to bind specifically to the focus of the infection located in the heart valve. To confirm the ability of the tracer to distinguish between infectious and inflammatory processes and thus avoid false positives, the image was carried out in a sham control model, which presented surgery but no infection. In this case, tracer uptake was not observed in the damaged area, thus demonstrating its selectivity only towards infectious foci. These observations were confirmed ex vivo by autoradiography (FIG. 4B) where high cardiac uptake can be clearly seen in the El model but not in the control. Histological validation (Fig. 4D) confirmed damage and bacterial colonization. Microbiological confirmation of the model was carried out through bacterial seeding studies, confirming the presence of the microorganism S. aureus in infected hearts and catheters but not in controls.

Claims (12)

1.- Compound comprising a protein covalently bound to a chelating agent and a radioisotope coordinated to the chelating agent, where the protein is collagen, the chelating agent is selected from the list comprising: - Pentetic acid, - 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 and - Diethylenetriamine-N,N,N”,N”-tetra-tert-butyl acetate-N’-acetic acid and the radioisotope is selected from the list comprising: 99mTc, 68Ga, 18F, 89Zr, 64Cu or 111In. 2. - Compound according to claim 1 wherein the collagen is type I or type IV collagen. 3. - Compound according to claim 1 or 2 where the chelating agent is pentetic acid. 4. - Compound according to any of the previous claims where the radioisotope is 99mTc. 5. Procedure for the preparation of the compound defined in any of claims 1 to 4 comprising a first stage of covalent bond formation between the protein with the chelator, thus forming a protein-chelator conjugate, and a second stage based on the radioactive labeling of the chelating protein conjugate with the corresponding radioisotope. 6. - Composition comprising the compound described in any of the preceding claims 1 to 4. 7. Composition according to claim 6, characterized in that it is configured to be able to administered intravenously, intraperitoneally, or orally 8. - Use of the compound described in any of claims 1 to 4, or of the composition described in claim 6 or 7, as an imaging agent for visualizing infections or infectious foci caused by Gram-positive bacteria in a subject. 9. - Use according to claim 8 where the Gram positive bacteria are selected from the list comprising: Staphylococcus aureus, Staphylococcus epidermis, Mycobacterium tuberculosis, Enterococcus faecalis, Staphylococcus agalactiae and Clostridioides difficile, 10. - Use according to claim 9 where the infection caused by Gram positive bacteria is infective endocarditis. 11. - Use according to any of claims 8 to 10 where the visualization of infections or infectious foci is performed by the PET or SPECT technique. 12.

- Method for providing an image of infectious foci caused by Gram-positive bacteria in vivo in a subject, comprising (i) administering to the subject the compound defined in any of claims 1 to 7 and (ii) scanning the subject using PET images or SPECT combined with structural technique such as anatomical tomography