INTRA-OPERATIVE IMAGING

Abstract

The present invention relates to the use of a NIR fluorescent probe comprising an aza-bicycloalkane based cyclic peptide labelled with a Cy5.5 dye moiety in the fluorescence- guided surgery of pathologic regions and to an optical imaging method that comprises using this fluorescent probe for the identification and demarcation of tumor margins during the surgical resection.

Claims

1. A method of using a contrast agent of formula ##STR00007## comprising administering an effective amount of the contrast agent in a patient, wherein the contrast agent is used as a fluorescent probe for a detection and demarcation of a tumor margin of a tumor in a surgery of the patient.

2. The method according to claim 1, wherein the contrast agent is used for a real-time identification of a surgical border of a pathologic area under the surgery.

3. The method according to claim 1, wherein the detection and demarcation of the tumor margin is carried out under a fluorescent light that is capable of exciting the contrast agent.

4. The method according to claim 3, wherein the fluorescent light has a wavelength of from 630 to 690 nm.

5. The method according to claim 1, wherein said tumor is a tumor showing a reduced expression of a α.sub.vβ.sub.3 integrin receptor.

6. The method according to claim 1, wherein said tumor is a tumor showing a variable expression of a α.sub.vβ.sub.3 integrin receptor.

7. The method according to claim 1, wherein the detection and demarcation comprises: illuminating, with a fluorescent light capable of exciting the contrast agent, a region comprising the tumor in the patient administered with the contrast agent; and identifying the tumor margin of the tumor under the fluorescent light.

8. The method according to claim 7, wherein the effective amount of the contrast agent is from 0.05 to 20 mg.

9. The method according to claim 8, wherein the patient is administered with the contrast agent within 48 hours before the surgery.

10. A method of using a pharmaceutical diagnostic composition comprising a contrast agent of formula ##STR00008## comprising administering the pharmaceutical diagnostic composition in a patient, wherein the pharmaceutical diagnostic composition is used in an intraoperative or a pre-operative optical imaging to provide a real-time detection and demarcation of a tumor margin.

11. The method according to claim 10, wherein the intraoperative or the pre-operative optical imaging is carried out in a surgery of a tumor of patient to provide the real-time detection and demarcation of the tumor margin of the tumor undergoing a curative resection.

12-17. (canceled)

18. The method according to claim 1, wherein the tumor is selected from the group consisting of melanoma, glioma, glioblastoma, neuroblastoma, sarcoma, ovarian cancer, breast cancer, lung cancer, liver cancer, colon cancer, head-neck cancer, and prostate cancer.

19. The method according to claim 18, wherein the tumor is a prostate cancer.

20. (canceled)

21. A method of using a contrast agent of formula ##STR00009## comprising: illuminating a region in a patient with a fluorescent light capable of exciting the contrast agent, wherein the patient is pre-administered an effective amount of the contrast agent, and detecting a tumor margin of a tumor under the fluorescent light.

22. The method according to claim 21, wherein the patient is pre-administered with from 0.05 to 20 mg of the contrast agent within one week before the step of detecting the tumor margin under the fluorescent light.

23. The method according to claim 21, wherein the step of detecting the tumor margin under the fluorescent light is performed in real-time, during a surgical inspection and a curative resection of the tumor.

24. The method according to claim 21 further comprising operating a curative resection of the tumor.

25. The method according to claim 21 comprising: administering the effective amount of the contrast agent to the patient, exposing the region of the patient to a surgeon's visual inspection, illuminating the region of the patient with the fluorescent light capable of exciting the contrast agent, generating an intraoperative image of the region of the patient, detecting the tumor margin of the tumor under the fluorescent light, operating a curative resection of the tumor by following the tumor margin, and assessing the region under the fluorescent light and verifying the tumor including the tumor margin has been excised.

26. The method according to claim 25 further comprising repeating the step of operating the curative resection of the tumor by following the tumor margin.

27. The method according to claim 21, wherein the tumor is selected from the group consisting of melanoma, glioma, glioblastoma, neuroblastoma, sarcoma, ovarian cancer, breast cancer, lung cancer, liver cancer, colon cancer, head-neck cancer, and prostate cancer.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0131] FIG. 1. Linear regression for the evaluation of the molar extinction coefficient of the fluorescent probe DA364.

[0132] FIG. 2 shows excitation and emission spectra of DA364 in PBS (left plot; ex/em: 674/694; SS:20 nm) and in Seronorm (right plot; 694/704, SS:10 nm).

[0133] FIG. 3 shows fluorescent images (Panels A, B and C) acquired during the guided surgical procedure using DA364 and the corresponding images acquired under white light (Panels D, E and F). In particular: Panels A and D show intact prostate, in Panels B and E the prostate where the capsule of the right ventral lobe was removed, and Panels C and F show the surgical field at the end of the procedure.

[0134] FIG. 4 shows excised tissue samples. In Panel A the tissue samples are imaged under white light and Panel B show the corresponding NIR fluorescent images.

[0135] FIG. 5 shows the results of the histological validation of the florescence-based visual classification. In particular: Panel A shows an example of Hematoxylin/Eosin stained sections of prostate cancer showing healthy (LOWER density and LOWER FLUORESCENCE) and pathological (HIGH Density and higher FLUORESCENCE) areas; Paned C shows the fluorescence staining with DAPI, and the NON EMPTY AREA automatically detected in white is shown in panel D; Panel B reports the plot of the correspondence between histological and visual results for the parameter TISSUE DENSITY measured in sections from LOW and FLUORESCENT samples (pvalue <0.001, Mann Whitney U Test).

[0136] FIG. 6 shows the DA364 plasma concentration (concentration of free agent in circulating blood) time curve recorded in the Experimental test of Example 6. The fast elimination of the circulating DA364 probe from the plasma compartment is clearly shown in the plot.

[0137] FIG. 7 Black and white images of Mat-Ly/Lu tumor cells in tissue slides obtained overlapping the signals from DA364 and the plasma membrane stain WGA.

[0138] FIG. 8 Flow cytometry of α.sub.vβ.sub.3 integrin expression in rat prostatic tumor Mat-Ly-Lu cells and in human prostatic tumor PC3 cells, used as internal reference. Expression of α.sub.vβ.sub.3 integrin is shown in the gray histograms, and the background of mouse IgG negative control is shown by the marker. Numbers show percentage of positive cells, calculated by subtracting the value obtained with isotype-matched control Ig from that detected with the specific mAb.

EXPERIMENTAL PART

Example 1

Preparation of the NIR agent DA364

[0139] The preparation of the NIR agent DA364 was performed substantially as described in the formerly quoted reference Contrast Media Mol. Imaging 2011, 6, 449-458, by using the synthetic procedure schematized above

##STR00002##

[0140] including:

[0141] 1) Preparation of the Integrin Targeted Cyclic Pentapeptide cRGD-NH.sub.2

[0142] The preparation of the integrin targeted peptidomimetic moiety cRGD-NH.sub.2 was carried out substantially as described in WO2006/095234 and, in better details, respectively in Angew. Chem. Int. Ed., 2010, 49, 7111-7115, J. Org. Chem. 2005, 70, 4124-4132 and Chem Med. Chem. 2009, 4, 615-632 describing, respectively, the preparations of the individual intermediates and the cyclic cRGD-NH.sub.2 integrin targeted scaffold according to the following schemes:

[0143] a. Synthesis of Intermediate 5

##STR00003##

[0144] b. Synthesis of Intermediate 10

##STR00004##

[0145] c. Synthesis of cRGD-NH.sub.2

##STR00005## ##STR00006##

[0146] 2) Preparation of the NIR Agent DA364

[0147] A solution of Cy5.5-NHS ester (13 mg, 0.011 mmol) (purchased from GE Healthcare, Product Code PA15602) in borate buffer pH 9 (4 mL) was added with a solution of cyclic cRGD-NH.sub.2 scaffold (8.8 mg, 0.011 mmol) in borate buffer pH 9 (3.5 mL). The reaction mixture was stirred for 24 h at room temperature and protected from light (resulting pH 8.1). At the end of the reaction (assessed by HPLC-MS) the crude reaction is diluted with a 5% CH3COOH aqueous solution (1.5 mL) and the residual reaction solvent was then removed by freeze-drying.

[0148] The crude product was purified by preparative HPLC (eluent A: H.sub.2O +0.1% TFA, eluent B: CH.sub.3CN+0.1 TFA, flow: 15 mL/min, stationary phase: Waters Atlantis Prep T3 OBD 5 μm, 19×100 mm, detection: UV, λ.sub.1: 210 nm, λ.sub.2: 254 nm) to give the desired product as a dark blue solid (11.8 mg, 0.0082 mmol) after freeze-drying. Yield: 74.6%. HPLC purity: 92.96% (as area %)

[0149] Analytical Characterization

[0150] Analytical characterization of the obtained NIR Agent was performed by HPLC-MS and .sup.1H-NMR. [0151] Nuclear Magnetic Resonance [0152] .sup.1H-NMR spectra was recorded at 298 K in DMSO-d6 or D.sub.2O, with a Bruker Avance-III (.sup.1H, 600.13 MHz) with deuterium as lock channel. [0153] Recorded .sup.1H-NMR was consistent with the expected structure. [0154] HPLC-UV-MS Preparative Method [0155] LC column: Agilent Zorbax SB-PHENYL—250 mm×4.6 mm×5 μm (P/N:880975-312) [0156] HPLC parameters [0157] Instrument: Triple quadrupole Thermo Fisher LC Accela equipped with Accela Pump, Accela Autosampler, Accela PDA Detector and TSQ Quantum Access. [0158] Injection volume: 5 μl (with a sample concentration of 200 μg/ml) [0159] Column oven: 40° C. [0160] Tray temperature: 5° C. [0161] Wash needle program: inside needle washing occurs to prevent memory effects. [0162] Washing solution: 40:50:10—water:methanol:i-propanol [0163] Uv-vis acquisition range: 200-790 nm (Tungsten lamp needing:max abs. 776 nm) [0164] MSD acquisition range: 50-1500 a.m.u. [0165] MSD mode: ESI both polarities (positive and negative, negative is preferred) [0166] The compound under analysis was diluted with milli-Q water. [0167] Pump flow:0.5 ml/min; Max pressure: 400 bar [0168] Solvent A:Water+ammonium acetate (1 g/l); Solvent B:Acetonitrile 100% [0169] Stop time: 42 mins. [0170] Gradient:

TABLE-US-00001 Time (min) A (%) B (%) 0 98 2 2 98 2 10 80 20 25 70 30 30 64 40 35 0 100 36 98 2 42 98 2

[0171] Fluorimetric Characterization of DA364

Example 2

Determination of the Extinction Coefficient

[0172] Material and Methods

[0173] A dual-beam Perkin Elmer Lambda 40 UV-VIS spectrophotometer was used to determine the extinction molar coefficient of the NIR agent DA364.

[0174] Two standard stock solutions containing the NIR agent in PBS at 1000 μM (sol I: 1.27 mg, 0.884mL) and 1250 μM (sol II: 7.03 mg, 3.915 mL) were prepared. Diluted standard solutions were then prepared as reported in Table A by diluting the stock solutions in an appropriate volumetric flask using PBS. The absorbance of each standard diluted solution was measured without any further dilution.

TABLE-US-00002 TABLE A Standard solutions dilution table Stock V Standard Concentration, μM Sol. I, mL Stock Sol. II, mL tot, mL Blank 0 0 0 — STD 1 1.25 0.050 50 STD 2 2 0.040 25 STD 3 3 0.060 25 STD 4 1 0.040 50 STD 5 0.5 0.050 100 STD 6 1.5 0.030 20 STD 7 2.5 0.050 20 STD 8 3.5 0.035 10

[0175] Results

[0176] The absorbance of each diluted standard solutions was measured and the molar extinction coefficient of DA364 was calculated from the slope of the linear regression reported in FIG. 1. The measured extinction coefficient in this condition was 208500 M.sup.−1.Math.cm.sup.−1 (corresponding to 83% of the theoretical value reported for Cy5.5 dye: 250000 M.sup.−1.Math.cm.sup.−1).

[0177] Statistical analysis was also carried out (data not reported) to verify the linear regression reliability.

Example 3

Assessment of the Fluorescence Quantum Yield

[0178] Material and Methods

[0179] Fluorescence quantum yields (0%) measurements were performed on FluoroLog-31IHR-320 (HORIBA-JobinYvon) with integrating sphere accessory.

[0180] The measurements were carried out using an excitation wavelength of 625 nm and using a 450W Xenon Light Source. Detection was performed on TBX-04 detector from 645 to 850 nm (excitation and emission slit were 1.8 nm).

[0181] The fluorescence quantum yield of DA364 solution was measured both in human serum (Seronorm™ Human, NycomedPhama, batch 1109514) and in Phosphate Buffer Saline (PBS).

[0182] Solutions of the NIR agent were prepared in PBS and in human serum with absorbance lower than 0.1. Fluorescence (0%) measurements were carried out after incubation of the NIR agent at Room Temperature (RT) for 15 min.

[0183] Results

[0184] Obtained 0% values, reported in Table B below, show that DA364 exhibits a quantum yield of 36.5% (±2.37) in PBS and 57.5% (±1.26) in Seronorm™.

[0185] The normalized excitation and emission spectra of the NIR agent in PBS (ex/em: 674/694; Stokes Shift: 20 nm) and in Seronorm™ (694/704, Stokes Shift:10 nm) are shown in FIG. 2. As shown in the figure, the interaction of the DA364 contrast agent with serum albumin promotes the shift of the emission maximum from 694 nm to 704 nm (in PBS and in Seronorm™ respectively) and also the reduction of the Stokes shift.

TABLE-US-00003 TABLE B φ% values for DA364 in PBS and Seronorm ™ PBS Seronorm ™ φ% 36.5 (SD 2.37) 57.5 (SD 1.26)

[0186] Biological Characterization of DA364

Example 4

In Vitro Competitive Binding Assay Towards α.SUB.v.β.SUB.3., α.SUB.v.β.SUB.5 .and α.SUB.v.β.SUB.1 .Integrins

[0187] The affinity of DA364 for its specific targeted (the integrin α.sub.vβ.sub.3) and its cross-reactivity for other integrins (e.g. α.sub.vβ.sub.1, α.sub.vβ.sub.5) was assessed by in vitro binding tests.

[0188] Material and Methods

[0189] A 96-well microtiter plate (MediSorp, Nunc) was coated overnight at 4° C., with tested integrins (0.5 μg/mL, diluted in coating/washing buffer (20 mM Tris HCl pH 7.4; 150 mM NaCl; 1 mM MnCl2; 0.5 mM MgCl2; 2 mM CaCl2). After coating, the plate is rinsed with washing buffer and then incubated in blocking buffer (3% BSA in coating buffer) for 2 h at room temperature (RT) (24° C. in Thermomixer, under shaking). The plate was then rinsed in washing buffer and incubated at RT for 3 h with DA364 (concentration range from 5000 to 0.005 nM). DA364 solutions were prepared by serially diluting a stock solution of the NIR agent in 1% BSA in coating buffer in presence of biotinylated vitronectin (1 μg/mL, cod. HVN-U605, Molecular Innovations). The plate is then rinsed again and incubated with Streptavidin-HRP (cod.EG RPN 1051, GE Healthcare) (1:10000 in PBS) 1 h at RT, binding the biotinylated vitronectin. After additional washes in PBS, the plate was incubated with TMB solution (3,3′,5,5′-tetramethylbenzidine) (cod.T-0440, Sigma-Aldrich) for 5-10 min and the colorimetric reaction was then stopped by addition of 2 N sulfuric acid. The quantification of the IC50 for DA364 was performed by measuring the competitor (biotinylated vitronectin) by reading the absorbance at 450 nm. IC50 values were determined by least-square non-linear regression analysis.

[0190] Results

[0191] The competitive binding assays with vitronectin, the natural substrate of α.sub.vβ.sub.3, demonstrated the high affinity of DA364 for its specific target, namely the α.sub.vβ.sub.3, and lower affinity for the analogue α.sub.vβ.sub.5; a crossreactivity is moreover highlighted for α.sub.vβ.sub.1.

[0192] Obtained affinity results for integrins α.sub.vβ.sub.3, α.sub.vβ.sub.5 and α.sub.vβ.sub.1 are provided in Table C below.

TABLE-US-00004 TABLE C Binding affinities of DA364 to integrins expressed as vitronectine IC50 IC50 (nM) SE α.sub.vβ.sub.3* 3.76 0.65 α.sub.vβ.sub.5 280.57 0.97 α.sub.vβ.sub.1 28.03 1.22 *Average value from a triplicate test

Example 5

In Vitro Binding Assay Towards Human Serum Albumin (HSA)

[0193] The affinity of DA364 for the HSA was determined by the spectroscopic measurements of the absorbance, according to the procedure disclosed in, for instance, Connors, K. A. (1987) Binding Constants. The Measurement of Molecular Complex Stability, John Wiley & Sons, New York (see, in particular, chapter 4).

[0194] Material and Methods

[0195] A dual-beam Perkin Elmer Lambda 40 UV-VIS spectrophotometer was used to acquire absorption spectra of DA364 incubated with HSA. Two standard solutions containing HSA were prepared fresh daily in PBS at 75 mM and 500 mM and kept under stirring for about 30 min and 1 h respectively.

[0196] A stock solution of DA364 (845±5 mM) was diluted 1:20 and mixed with HSA (HSA Sigma Aldrich, batch 049K7535, item A1653) to give a ramp of concentrations (0, 1, 10, 20, 40, 60, 30, 80, 100, 200, 400 mM). Final solutions, containing 0.99 mM of the NIR agent and different amount of HSA, were incubated 1 h at room temperature before measurements. The absorbance of each solution was measured against a reference solution of HSA at the same concentration. The absorbance values measured at the wavelength of 689 nm (corresponding to the maximum absorption of DA364 bound to HSA) were fitted using the following equation:

[00001]$\frac{\Delta .Math..Math.A}{b}=\frac{\mathrm{\Delta .Math.}.Math.k_a\left[\mathrm{HSA}\right].Math.C_{\mathrm{DA}.Math..Math.364}}{k_a\left[\mathrm{HSA}\right]+1}$

[0197] where Δε is the difference of molar absorptivities obtained from free and bound DA364, ΔA is the difference in the absorbance of DA364 and reference solutions, b is the length of the optical path through the solutions, [HSA] is the concentration of HSA, C.sub.DA364 is the concentration of the fluorophore and k.sub.a is the association constant.

[0198] Results

[0199] The association constant with HSA (k.sub.a(M.sup.−1)) measured for DA364 was 2.8670.Math.10.sup.4±0.1140.Math.10.sup.4 (R.sup.2=0.9949).

[0200] This result demonstrates the existence of an affinity towards serum albumin displayed by the NIR agent identified by the present invention, which is consistent with the in vivo hybrid behaviour it displays, allowing its accumulation and diffusion towards tumors as a results of both an high specificity for integrins receptors and an non-specific retention, e.g. mediated by a non-specific binding to extracellular components, including extravascular (leaked) albumin.

Example 6

Assessment of the Plasma Kinetic of DA364

[0201] The plasma kinetic of the NIRF probe DA364 was evaluated after a single intravenous administration in CD-1(ICR)BR mice.

[0202] Protocol

[0203] The fluorescent probe is administered at dose of 0.4 μmol/kg, corresponding to about 10 times the effective dose used in imaging studies (1 nmol/mouse, considering an average mouse body weight of 25 g). Animals were sacrificed at different time points up to and including 14 days after treatment and blood samples were collected for the determination of DA364 content, performed by using an HPLC method coupled with a fluorimetric detector.

[0204] Preparation of the Formulation

[0205] DA364 was supplied as a 655 μM bluish solution. Some days before the animal administration, after being thawed, the 655 μM solution of DA364 was diluted up to the final concentration of 0.04 μmol/mL. To this end, 0.8 mL of the 655 μM solution of DA364 was diluted with 12.2 mL of 0.9% NaCl physiological saline. The 0.04 μmol/mL formulation was stored at −80° C. until administration.

[0206] Animals

[0207] The experiment was performed with CD-1(ICR)BR mice (40 males; weight at arrival: 22-30 g; age: 4-5 weeks old), purchased from Charles River Laboratories Italia, Calco (LC), Italy. All procedures involving the animals have been conducted according to national and international laws and policies for the Care and Use of Laboratory Animals (L.D. 116/92; Authorization n.19/2008-A issued Mar. 6, 2008, by the Italian Ministry of Health; EEC Council Directive 86/609CEE, and EEC Council Directive 2010/63/EU).

[0208] Experimental Design

[0209] The plasma kinetic of DA364 was measured in a pharmacokinetic study. A period of at least 3 days was allowed between animal arrival and treatment. On the day of dosing, animals were randomly assigned to the pharmacokinetic time points and to the calibration group. Animals were identified by colored marks made on the tail. Each cage label was uniquely numbered with study number, compound, administration route, species, dose, date of treatment and date of sacrifice and animal numbers.

[0210] The NIR agent DA364, pre-warmed at room temperature, was intravenously injected at a dose of 0.4 μmol/kg, via tail vein, at an injection rate of 1 mL/min with a Harvard infusion pump. The administered dose corresponds to about 10 times the effective dose used in imaging studies. The intravenous administration route was chosen as possible route of exposure for humans.

[0211] Experimental Scheme:

[0212] Animals (three for each time point) were sacrificed before and 5 min, 10 min, 20 min, 30 min, 60 min, 4 h, 24 h, 48 h, 7 days and 14 days after DA364.

[0213] Seven animals were moreover used, to collect blank plasma for the calibration curve.

[0214] The day of sacrifice animals were anesthetized with sevoflurane, at an expiratory concentration of 3-4% in induction and 2-3% in maintenance and then sacrificed by decapitation. Blood for HPLC determinations (at least 0.5 mL) was collected using heparinized tubes. Plasma obtained after centrifugation (2100 g for 10 min) was stored at −80° C. until the day of analysis.

[0215] Assessment of DA364 in Plasma

[0216] Determination of the amount of DA364 in plasma samples was carried out by an HPLC method coupled with a fluorimetric detector previously prompted.

[0217] Preparation of the Acetate Buffered Solution of DA364

[0218] Acetate buffer solution was prepared by dissolving 1 g of Acetate buffer in 1 L of Milli-Q water. The appropriate amount of the NIR agent was then added to the obtained solution.

[0219] Preparation of Plasma “CAL” Samples

[0220] Calibration standards (CAL) were prepared by adding 10 μL of DA364 acetate buffer solution (from 0.04 to 4 nmol/mL), and 100 μL of a Methanol and Acetonitrile solution (50:50) to 90 μL of blank plasma. Tubes were stirred and subsequently centrifuged at 4° C. for 10 minutes at 13000 g. Ten microliters of the clear supernatant were injected into the HPLC equipment.

[0221] Preparation of Plasma “QC” Samples

[0222] Quality controls (QC) were prepared by adding 10 μL of DA364 acetate buffer solution (from 0.06 to 3 nmol/mL), and 100 μL of a Methanol and Acetonitrile solution (50:50) to 90 μL of plasma. Tubes were stirred and subsequently centrifuged at 4° C. for 10 minutes at 13000 g. Ten microliters of the clear supernatant were injected into the HPLC equipment.

[0223] Preparation of Plasma “S” Samples

[0224] Plasma samples (S) were prepared by adding 100 μL of acetate buffer and 100 μL of a Methanol Acetonitrile solution (50:50) to 100 μL of diluted plasma. Tubes will be stirred and subsequently centrifuged at 4° C. for 10 minutes at 13000 g. Ten microliters of the clear supernatant will be injected into the HPLC equipment. Appropriate dilution was performed in case of plasma samples too concentrated.

[0225] Chromatographic Conditions [0226] Chromatographic: Waters Alliance 2695XC [0227] HPLC column: Zorbax SB-Phenyl, 5 μm, 250×4.6 mm [0228] Mobile phase A: Dissolve 1 g of Acetate buffer in 1 L of Milli-Q water (0.013 M, pH −7.0) [0229] Mobile phase B: Dissolve 1 g of Acetate buffer in 1 L of Methanol and Acetonitrile (50:50) [0230] Sample Temperature: 10° C. [0231] Column Temperature: 60° C. [0232] Injection volume: 10 μL [0233] Detector: FL detector Waters 2475; excitation wavelength: 670 nm; emission wavelength: 700 nm [0234] Run time: 23 min [0235] Elution: gradient

TABLE-US-00005 Time (min) % A % B Flow 0 91 9 1.0 5 91 9 1.0 11 44 56 1.1 15 44 56 1.1 17 91 9 1.0 23 91 9 1.0

[0236] The HPLC determination was performed by interpolating calibration curves of DA364 in plasma and organs, prepared by assaying calibration standard samples (CAL) (6 concentrations, 3 replicates for each concentration) of the fluorescent agent DA364.

[0237] Data Analysis

[0238] Blood for HPLC determinations (at least 0.5 mL) was collected in heparinized tubes. Plasma concentration of DA364 was measured by an HPLC method coupled with a fluorimetric detector.

[0239] To evaluate the systemic exposure, C.sub.max and the area under the plasma DA364 concentration curve as a function of time (AUC) were calculated by non-parametric methods using the computer program WIN-NONLIN 6.3 (WinNonlin 6.3 SCI Software, Lexington, Ky., USA). For the tested dose, a non-compartmental analysis was performed using the average plasma concentrations from 3 animals for each time point. For C.sub.max and the corresponding t.sub.max the observed values were reported. Area under the plasma concentration-time curve to the last observable plasma concentration (AUC.sub.(0 to t) was calculated from observed data using the logarithmic trapezoidal method. In realta possiamo dire di calcolare anche t.sub.1/2, Vd, e Cl.

[0240] Da 364 content in organs will be reported as μg/mL (blood) or μg/g of tissue. Data will be transformed to percent of injected dose (% ID) in each organ. Mean and standard deviation will be then calculated for each sampling time.

[0241] Results

[0242] Plasma concentration of DA364, measured by fluorescence after HPLC separation are included in the Table D below and, graphically, in FIG. 6. Obtained results confirm the rapid clearance of the free, circulating agent, reaching a negligible concentration in the blood 4 hours after the administration to test animals, which becomes then less than the instrumental detection limits (LOD=0.0012 nmol/mL).

TABLE-US-00006 TABLE D nmol/mL Time (min) (Mean from a triplicate result) s.d. 5 1.629 0.063 10 1.10 0.20 20 0.612 0.035 30 0.518 0.052 60 0.278 0.066 240 0.051 0.013 1440 <LOD <LOD 2880 <LOD <LOD 10080 <LOD <LOD 20160 <LOD <LOD

[0243] C.sub.max of 1.629 nmol/mL was measured at 5 minutes (t.sub.max) after injection. The half life values associated with the terminal elimination phase was 64 min. The AUC.sub.(0 to ∞) value was 77 min*nmol/mL. The total plasma clearance value was 5 mL/min/kg (0.15 mL/min). The clearance value results well lower than mice hepatic and renal blood flow (1.8 and 1.3 mL/min) suggesting accumulation in body (o other tissue). The volume distribution value at a steady state (V.sub.dss) was 329 mL/kg (10 mL), slight lower than mice total body water (14.5 mL), suggesting extravasation of DA364 from the plasma compartment.

Example 7

Fluorescence Guided-Surgery in a Rat Prostate Tumor Model using DA364 as NIR Fluorescent Probe

[0244] This example is aimed at assessing the sensitivity of the NIR RGD cyclic probe DA364 in the recognition of tumor masses using rat orthotopic prostate tumor model Mat Ly-Lu (a tumor with reduced expression of α.sub.vβ.sub.3 integrins, as illustrated below).

[0245] Intraoperative imaging was performed on a Zeiss system, a modified surgical microscope including appropriate filters for illumination and selection of signals emitted from the probe. Rat tumoral MatLyLu prostate cells were inoculated in the right ventral lobe of the prostate. Three-four days after inoculation animals were intravenously injected with a dose ranging from 5 to 20 nmol/rat of NIR fluorescent probe and imaged 24 hours after. Tumors have been then resected through fluorescent signal guide. Tissues surrounding the fluorescent area including tissues from the left prostate lobe was excised for histological comparison. All the samples were analysed through routine histological processing (Hematoxylin and eosin stain) and additional immune-labelling.

[0246] Tumor Cells and their Preparation

[0247] Prostatic tumor Mat-Ly-Lu cell line, a subline of the rat prostatic adenocarcinoma model G-Dunning R-3327, was supplied by European Collection of Cell Culture (ECACC). Cells were grown in RPMI 1640 medium supplemented with 10% Foetal Bovine Serum (FBS), 2 mM glutamine, 100 IU/mL penicillin, 100 μg/mL streptomycin and 250 nM dexamethasone (DXMT). The trypan-blue exclusion test was used to assess cell viability before cells inoculation. Viability was expressed in % (ratio of viable cells to total cells×100). Cell cultures with a viability <70% were discarded. Human prostatic adenocarcinoma PC-3 cell line was supplied by American Type Culture Collection (ATCC). Cells were grown in Ham's F-12 medium supplemented with 10% Foetal Bovine Serum (FBS), 2 mM glutamine, 100 IU/mL penicillin, 100 μg/mL streptomycin. The trypan-blue exclusion test was used to assess cell viability before cells inoculation. Viability was expressed in % (ratio of viable cells to total cells×100). Cell cultures with a viability <70% were discarded. Human melanoma cell line WM-266 was supplied by American Type Culture Collection (ATCC). Cells were grown in Eagle's Minimum Essential Medium supplemented with 10% Foetal Bovine Serum (FBS), 2 mM glutamine, 100 IU/mL penicillin, 100 μg/mL streptomycin.

[0248] α.sub.vβ.sub.3 Expression

[0249] The expression of α.sub.vβ.sub.3 integrin in MatLyLu cell line was measured by western blot by comparison with WM266 melanoma cell line, known to have a high integrin over-expression (Anticancer Research 33:871-880 (2013)). In order to extract total proteins, cells pellets were dissolved in Lysis buffer (Tris-HCl pH 8 50 mM, NaCl 150 mM, EDTA 1 mM, NaF 100 mM, glycerol 10%, MgCl2 1 mM, TritonX-100 1%, Protease Inhibitors), subjected to 3 rounds of freeze and thaw (−80 ° C./ 37 ° C.) and left in ice for 20 min. Obtained solution was then sonicated for 10 s and centrifuged at 14000 g at 4° C. for 15 min. The protein content of the supernatant was then quantified with BCA method. To this extent, total extract was loaded in a 10% polyacrylamide gel and electrophoresis was applied to separate the proteins based on their molecular weight in denaturing conditions (20% SDS). Proteins within gel were then transferred onto a nitrocellulose membrane and subsequently blotted with the antibodies anti-β.sub.3 integrin. Specific signal was visualized as a single or multiple band by chemiluminescent detection method. The images of labelled nitrocellulose were acquired by the ChemiDoc MP Imaging system and the area of the band relative to β.sub.3 expression level was quantified with the software Image Lab v.4.1.

[0250] The expression of α.sub.vβ.sub.3 in MatLyLu cells was also measured by Flow cytometry (FACS) method and compared to the expression of the PC3 cell line.

[0251] To this extent, cells were recovered and washed in PBS supplemented with 0.2% BSA and 0.01% sodium azide. Nonspecific sites were blocked with rabbit IgG (Sigma-Aldrich). Cells were then incubated with the anti-α.sub.vβ.sub.3 integrin antiboby (dilution 1:100; clone LM609, Millipore) or an isotype-matched negative control for 30 min at 4 ° C., and subsequently incubated with fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse Ig (DakoCytomation). Samples were collected and analyzed with a CyAn ADP flow cytometer and the Summit 4.3 software (DakoCytomation). Cells were gathered according to their light-scattering properties to exclude cell debris, and according to their propidium iodide (Sigma-Aldrich) negativity to exclude dead cells.

[0252] Western blot analysis resulted in a negligible expression of α.sub.vβ.sub.3 integrin by MatLyLu cells as compared to the expression by WM266 melanoma cells. In the FACS experiment, PC3 cells confirmed a relatively high α.sub.vβ.sub.3 expression (45% of cells showing fluorescent signal), consistent with values quoted in the cited literature, and a negligible expression (0.1%) of α.sub.vβ.sub.3 on Mat-Ly-Lu cells (FIG. 8).

[0253] Tumor Model

[0254] The experiment was performed on 10 Copenhagen rats (males) 11 weeks old, purchased from Harlan Laboratories Srl, S.Pietro al Natisone (UD), Italy. Rat prostate adenocarcinoma was induced in Copenhagen rats by orthotopic injection of the MatLylu tumor cells under isofluorane anesthesia. About 1 hour before tumor implantation, an analgesic (Rymadil) was administered subcutaneously (5 mg/kg) to rats. Then the lower abdomen was shaved and disinfected (Betadinen. A 12 mm incision of the abdominal wall along the linea Alba was performed 3 to 4 cm above the urinary orifice. The prostate was exposed after having pushed away the adipose tissue on each side of the animal.

[0255] A cell suspension (30 μL) containing 0.5×10.sup.6 tumor cells was injected with a 27G needle into the right ventral lobe of the prostate. The muscular and cutaneous plan was sewed up with sterile non-resorbable thread (3-0 black silk, Distrex S.p.a.). The wound was painted with disinfectant solution Betadine®. The analgesic was also administered at least once after surgery (about 24 h after). All procedures involving the animals have been conducted according to national and international laws and policies for the Care and Use of Laboratory Animals (EEC Council Directive 86/609CEE; L.D. 116/92 and EEC Council Directive 2010/63/EU; L.D. 26/2014. Authorization n.149/2006-A issued Oct. 27, 2006 by the Italian Ministry of Health).

[0256] Fluorescence-Guided Surgery Experiment

[0257] The tumor removal was performed using a dedicated surgical microscope (Axio Zoom v16, Carl Zeiss Beteiligungs GmbH, Jena, Germany). The microscope was equipped with a PLANAPO Z 0.5X/0.125 FWD 114 MM objective. During surgery an area of about 2.5×2.5 cm.sup.2 was visualized. The surgical field was white-light illuminated by a cold light source (CL 9000 LED CAN (D)) when the fluorescence signal was not required, and by filtered light during the tumor removal (Illuminator: HXP 200C (D), filter set: 32 Cy 5.5 shift free (E)).

[0258] The day before surgery, corresponding to 3-4 days after tumor implantation, animals were administered with a dose of DA364 test agent ranging from 5 to 20 nmol/rat. Twenty-four hours after probes injection, animals were anaesthetized with isofluorane gas 0.5% (O.sub.2 99.5%), secured on the surgical table in the supine position and prostate tumor will be exposed.

[0259] After the removal of the prostatic capsule, the prostatic tumor was exposed to filtered light and fluorescence images were acquired to outline the primary tumor mass and the margins of the pathology (see FIG. 3, Panels A and D). The removal of the tumor mass was then carried out iteratively, by using fluorescence to guide the excision until absence of the fluorescent signal in the area surrounding tumor region (see FIG. 3, Panels B,E and C,F). Most of the fluorescent signal was confined in implantation area, i.e. the prostate right ventral lobe. Tissue samples excised from the area surrounding the fluorescence signal and left ventral, dorsal and anterior prostate lobes were collected and ex vivo fluorescence images were acquired to investigate the histological pattern underlying the accumulation of the fluorophore (FIG. 4). Of note, in this experimental test the samples of tissue were excised strictly following the tumor margins identified by high intensity of the fluorescence signal, although for a safer surgical strategy, a larger excision volume including at least a minimum non-fluorescent, negative, margin is recommended. All the collected tissue samples were finally processed for the following histological analysis. At the end of the experiments animals were sacrificed by overdose of anesthesia.

[0260] Histological Analysis

[0261] Excised samples were directly embedded in Killik compound (Cryostat embedding medium) and immediately frozen in isopentane cooled down in liquid nitrogen.

[0262] The excised tissue blocks were cut into groups of three consecutive 10 μm thick slices for parallel labelling. Each tissue sample was then treated with the standard procedure for histo-pathological analysis. In particular, a histological staining of collected samples was performed with Hematoxylin and Eosin in order to detect and characterize the anatomical tumor margins in excised samples and to verify the presence of optional tumor cells in surrounding healthy prostate tissue, in order to assess and confirm the radical excision of the tumor with surgery.

[0263] The detection of the fluorescent agent DA364 distribution in excised tissue samples was operated on tissue slides, after thawing at room temperature (RT) and washing in PBS to remove Killik. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, cod.BML-AP402, Enzo Life Sciences) (5 μg/mL in PBS, 5 min). The plasma membrane was stained with 2 μg/mL of FITC-conjugated Wheat Germ Agglutinin (WGA) (cod.W11261 Life Technologies), in order to identify the internalized fraction of DA364. To this extent, tissues were visualized both with the Aperio digital scanner ScanScope FL, to acquire the whole stained section, and Leica DM2500 fluorescence microscope, for additional details.

[0264] Internalization in Tumor Cells

[0265] The internalization of DA364 in tumor cells was then observed by fluorescence microscopy. In particular, black and white images of Mat-Ly/Lu tumor cells were obtained, for instance shown in FIG. 7, in which the fluorescent signal generated by DA364 is overimposed to the signal obtained from Mat-Ly/Lu tumor tissue slides stained with 2 μg/mL of FITC-conjugated Wheat Germ Agglutinin (WGA), a plasma membrane stain allowing to identify cells membranes and extracellular interstitial spaces.

[0266] Image Analysis

[0267] The ex vivo fluorescence images acquired just after surgery (FIG. 4) were used to visually classify the excised tissue samples in two fluorescence classes: FLUORESCENT and NON FLUORESCENT. The classification between the two fluorescence classes was based only on the fluorescent signal without considering the anatomic origin of the tissue sample. Tissue samples were assigned to the FLUORESCENT class when they included at least a region characterized by intense and bright fluorescent emission (see, for example, samples 3, 4 and 5 of FIG. 4). All remaining tissue samples, i.e. samples displaying no fluorescence (such as sample 1 of FIG. 4) or a fluorescence consistent with the auto-fluorescence observed in a healthy tissue far from the tumor area (such as sample 2 of FIG. 4) were classified in the NON FLUORESCENT class. For each image the fluorescent signal was mapped in pixels intensity using all the pixels intensity range. This classification procedure was intended to reproduce the fluorescence-based visual selection of ‘Healthy’ (NON FLUORESCENT) and ‘Pathological’ (FLUORESCENT) tissues that the surgeon is required to operate during the tumor removal.

[0268] The results of the visual classification (of tissue samples collected during the guided surgery) were then validated histologically, by H&E staining of 2 histological sections from each tissue sample, allowing to identify sections containing a region of tumor, regardless of its dimension (FIG. 5 Panel A). Tissue samples containing tumor (namely tissues samples of which at least 1 of tested histological sections contained tumor cells) were then classified as POSITIVE, whereas tumor-free samples were classified as NEGATIVE. The histological and visual classifications were then compared using a contingency tables providing a measure the sensitivity of the visual classification enabled by the compound identified by the present invention and its reliability in identifying and separating tumor-free and tumor-containing tissues.

[0269] An additional analysis of collected tissue samples was also performed, based on the fluorescence of the nuclear stain 4′,6-diamidino-2-phenylindole (DAPI). In general terms, this analysis relies on differences existing in the morphology of healthy and tumor tissues, where the normal prostatic morphology is disrupted by tumor invasion, that can be easily distinguished with an automatic threshold-based image analysis procedure (FIG. 5. Panels C,D). Healthy prostate tissue, in fact, is mainly constituted by glands and ducts immersed in a fibrous stroma; the empty regions of the tissue represent the wide lumen of prostatic glands, while epithelial glandular cells and stroma represent the parenchima of prostate. Tumor tissue, instead, is more typically constituted by homogeneously packed cells. This automatic procedure consisted in calculating the TISSUE DENSITY defined as the ratio of the sample area occupied by tissue (NON EMPTY AREA) versus the area of the section. Healthy tissues are characterized by a lower TISSUE DENSITY than pathological tissues.

[0270] Results

[0271] Histological Validation of the Surgical Efficacy.

[0272] The results of the histological and visual classifications were compared by using contingency tables. The results of the comparison, for instance included in the contingency Table E below, indicates that a significant correlation exists between the intensity of the detected fluorescence and the pathological state histologically verified in tested tissue samples. In particular, obtained results indicate that DA634 NIR fluorescent probe enabled a visual classification of tumor tissues with a sensitivity of 87% and a specificity of 83% (Table E).

[0273] FLUORESCENT samples were also characterized by a substantial disruption of the normal tissue morphology, by showing a statistically higher tissue density, essentially due to the absence of prostatic glands and ducts verified in pathologic tissues (Mann Whitney U test, p-value <0.05, FIG. 5, panel B).

TABLE-US-00007 TABLE E Contingency table of the correspondences between visual and histological classification of tissue samples. Histology Surgery Positive Negative FLUORESCENT 20 3 NON FLUORESCENT 3 15

[0274] Note: two of the three false negatives recorded in the test (NON-FLUORESCENT but POSITIVE) were tissue samples from the prostate capsule partially infiltrated by tumor cells, though displaying a low fluorescence at the surgical microscope, probably due to the elastic winding on itself caused by incision of the capsule, which made this particular prostatic region less visible and less properly imaged under fluorescent light. The remaining false negative was a very small piece of tissue removed from a big tumor that, when evaluated only by its fluorescence, showed low light intensity.

Example 8

[0275] Determination of α.sub.vβ.sub.3 Integrin Expression with Respect to Tumor Mass

[0276] The expression of α.sub.vβ.sub.3 integrin has been measured by western blot, for three different types of tumors from animal models: [0277] U-87 MG, a xenograft mouse model of human glioblastoma reported to have relative high expression of α.sub.vβ.sub.3 integrin [0278] MatLyLu, an orthotopic rat model of rat prostate cancer reported to have medium-low expression of α.sub.vβ.sub.3 integrin [0279] A431, a xenograft mouse model of human epidermoid carcinoma reported to have relatively low expression of α.sub.vβ.sub.3 integrin

[0280] Methods

[0281] Frozen tumor tissues were cut in small pieces using a scalpel and then pulverized, using a mortar and pestle, in presence of liquid nitrogen. The powder was weighted and then dissolved in an adequate volume of lysis buffer (Tris-HCl pH 8 50 mM, NaCl 150 mM, EDTA 1 mM, NaF 100 mM, glycerol 10%, MgCl2 1 mM, TritonX-100 1%, Protease Inhibitors), vortexed for 1 min, and put on ice for 45 min. Afterwards, the solution was sonicated 15 s and centrifuged 15 min at 4° C. at 14000 rpm.

[0282] The protein content of the supernatant was quantified with BCA method. Total extracts was loaded in a 10% polyacrylamide gel and electrophoresis was performed to separate the proteins by their molecular weight in denaturing conditions (20% SDS). Proteins within the gel were then transferred onto a nitrocellulose membrane and subsequently blotted with the antibody anti-β3 integrin. Specific signal was visualized as a single or multiple band by chemiluminescent detection method. The images of the labelled nitrocellulose were acquired by the ChemiDoc MP Imaging system and the area of the band relative to β3 expression level was quantified with the software Image Lab v.4.1

[0283] Results

[0284] The amount of integrin β3 was normalised by tumor mass (mg of tumor). The experiments were performed with at least two samples per tumor type. Results (mean value of integrin expression) are shown in Errore. L'origine riferimento non è stata trovata.

TABLE-US-00008 TABLE F Tumor type Mean (ngβ.sub.3/mg tumor) U-87 MG 16.28 Matlylu 6.8 A431 2.39

Example 9

[0285] Determination of α.sub.vβ.sub.3 Integrin Expression with Respect to Total Amount of Protein in the Tumor Mass

[0286] The expression of α.sub.vβ.sub.3 integrin has been measured by western blot, for the three different animal models previously detailed in Example 8 and for additional human tumors purchased as protein extracts.

[0287] The protein extracts from tumor models were obtained starting from tissues with the extraction method described before, while the human proteins extracts (as detailed below in table G) were purchased by OriGene

TABLE-US-00009 Human Extract OriGene ID Pathological Features CP565523 Malignant melanoma, metastatic IV CP565782 Adenocarcinoma of ovary, papillary serous IV (G2) CP541289 Adenocarcinoma of colon IV (G2)

[0288] The data reported in table H below, further to confirming the respective reduced over-expression of α.sub.vβ.sub.3 integrin in Matlylu type tumor and A431 tumor, also show other human tumor with reduced expression of α.sub.vβ.sub.3 integrin which may benefit from the present invention.

TABLE-US-00010 TABLE H Type of Integrin β3 ng β3/μg prot tot Malignant Melanoma IV 0.241149 U-87 MG tumor (Hu in Ms) 0.221000 Matlylu tumor (Rt in Rt) 0.104000 A431 tumor (Hu in Ms) 0.046500 Ovarian adenocarcinoma IV (G2) 0.027133 Colorectal adenocarcinoma IV 0.020731

Example 10

[0289] In Vivo Visualization of Tumors with Reduced Expression of α.sub.vβ.sub.3 Integrin (A431)

[0290] This example shows the uptake of DA364 in the A431 and U87-MG mouse models of human tumors, characterized respectively by low and high α.sub.vβ.sub.3 over-expression.

[0291] Tumor Model

[0292] A431 cells were collected and washed two times with PBS. Five million cells were resuspended in 0.1 mL of DMEM high glucose medium and subcutaneously injected in the right flank of all mice. Eleven days after A431 inoculation, U-87MG cells were collected and washed two times with PBS. Two million cells were resuspended in 0.1 mL of EMEM medium and subcutaneously injected in the left flank of same animals previously injected with A431 cells.

[0293] Tumors development was followed after inoculation by palpation and calliper measurement once a week. Tumor volume has been calculated according to the formula: (L×W.sup.2)/2, where L and W are the maximum length and width of the tumor. Animals were sacrificed by overdose of anaesthesia at the end of the experimental phase or in case the sum of both tumors reaches a volume of about 2000 mm.sup.3.

[0294] Fluorescent Imaging Experiments

[0295] Three weeks after A431 tumor implantation five mice were administered with 1 nmol of DA364 for a total administration volume of 0.2 mL (inj. rate 1 mL/min). In vivo OI acquisitions have been carried out 5 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 24 h and 48 hours after intravenous administration of the fluorescent probe using the IVIS Spectrum optical imaging system (Perkin Elmer). During OI experiments animals were kept under 3-4% Sevofluorane gas anaesthesia (O.sub.2 97.0-96.0%). During anaesthesia animals body temperature has been stabilized at about 37° C. using a heated bed.

[0296] Following in vivo OI experiments, 48 hours after probes injection, animals were sacrificed. Tumors, liver, kidneys, heart and spleen were excised for ex vivo fluorescent signal measure on the whole excised tumor or organ.

[0297] Results

[0298] Fluorescence intensity from U-87 MG and A431 tumors was measured in vivo together with the signal coming from the normal healthy tissues (background).

[0299] In the time-window from 3 hours to 24 hours after injection, U-87 MG and A431 tumors showed stable signal ratio, with respect to the background. FIG. 9 illustrates the fluorescence imaging of a mice, showing that DA364 allows a clear visualization of the low-integrin expressing tumor A431 under skin.

[0300] The fluorescent signal (expressed as Radiant Efficiency (RE): p/s/(μW/cm.sup.2), where p is the photon count), was then measured on the excised ex vivo samples using the IVIS Spectrum optical imaging system (Perkin Elmer).

[0301] As illustrated in the table I below, the fluorescence signal of DA364 in U-87MG tumor was about twice the intensity of the signal measured in the A431 tumor. The fluorescence intensity measured in A431 tumors was however comparable to that measured in liver and kidney (excretion organs where DA364 accumulates)) while it was about 10 times higher with respect to the signal measured in heart and spleen (representative of heathy tissues, background).

TABLE-US-00011 TABLE I Fluorescence Tumor/Organ Signal U87-MG 6.78E+08 A431 3.47E+08 liver 4.04E+08 kidney 3.55E+08 heart 4.61E+07 spleen 5.79E+07

[0302] These results show that DA364 is a suitable imaging agent for tumors having reduced integrin expression, particularly for the intraoperative visualization thereof.