RADIOLABELED PROGASTRIN IN CANCER DIAGNOSIS

Abstract

The present invention provides a radiotracer comprising a progastrin moiety, a chelating moiety and a radioisotope. Uses of said biomarker for imaging and detecting cancers in a subject are also provided. In one embodiment, the radiotracer is .sup.68Ga-NODAGA-Progastrin.

Claims

1. A compound, or a pharmaceutically acceptable salt thereof, comprising: a progastrin moiety, and a chelating moiety.

2. The compound of claim 1, wherein the progastrin moiety is the peptide of sequence SEQ ID NO:1.

3. The compound of claim 1, wherein the chelating moiety is a bifunctional chelator.

4. The compound of claim 3, wherein the bifunctional chelator is selected from the group consisting of NODAGA, NOTA, DOTA, DOTA-NHS, p-SCN-Bn-NOTA, p-SCN-Bn-PCTA, p-SCN-Bn-oxo-DO3A, desferrioxamine-p-SCN, DTPA, and TETA.

5. The compound of claim 3, wherein the bifunctional chelator is NODAGA, NOTA, or DOTA.

6. The compound of claim 3, wherein the bifunctional chelator is NODAGA.

7. The compound of claim 1, further comprising a radioisotope selected from the group consisting of .sup.68Ga, .sup.64Cu, .sup.89Zr, .sup.186/188Re, .sup.90Y, .sup.177Lu, .sup.153Sm, .sup.213Bi, .sup.225Ac, .sup.111In, .sup.99mTc, .sup.123I, and .sup.223Ra.

8. The compound of claim 7 any, wherein the radioisotope is .sup.68Ga or .sup.64Cu.

9. The compound of claim 7, wherein the radioisotope is .sup.68Ga.

10. A method of preparing the compound of claim 1, comprising the steps of: a) conjugating an amine-reactive chelating moiety to the progastrin moiety; and b) recovering the conjugate of progastrin and chelator.

11. The method of claim 10, wherein the amine-reactive chelating moiety is DOTA-NHS, NOTA-NHS, or NODAGA-NHS ester.

12. The method of claim 10, wherein the amine-reactive chelating moiety is NODAGA-NHS ester.

13. The method of claim 1, further comprising a step of: c) incubating the conjugate of progastrin and chelator with the complementary radioisotope; thus generating the compound of the invention.

14. A method of imaging one or more cancer cells, organs, or tissues in a subject in recognized need thereof, comprising: a) administering a compound, or a pharmaceutically acceptable salt thereof, to said subject; and b) detecting the compound by in vivo PET or SPECT imaging, wherein the compound comprises: a progastrin moiety, and a chelating moiety.

15. A method of determining the localisation of a cancer in a subject in need thereof, comprising: a) administering a compound, or a pharmaceutically acceptable salt thereof, to said subject; and b) detecting the compound by in vivo PET or SPECT imaging: wherein the compound comprises: a progastrin moiety, and a chelating moiety.

16. The method of claim 15, further comprising a prior step of determining the level of progastrin in sample of said subject.

17. The method of claim 16, wherein the level of progastrin is determined with anti-progastrin antibodies.

18. A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable carrier.

19. A kit comprising a compound of claim 1.

Description

FIGURE LEGENDS

[0102] FIG. 1: 3D representation of quantified ROIs on PET/CT. Are represented the tumor (red), liver (blue), kidneys (green), heart (light blue), muscle (yellow) and brains (pink).

[0103] FIG. 2: Sagittal view of the dynamic PET/CT of the C1S3 mouse at different time point after injection 68Ga-NODAGA-Progastrin.

[0104] FIG. 3: Mean biodiversity of .sup.68Ga-NODAGA-Progastrin in each quantified organ during the 2 hours of PET. The values are expressed in % ID/g+standard deviation. (A) kinetics of all quantified regions, (B) kinetics restricted to muscle, brain and tumour

[0105] FIG. 4: Quantity in % ID/g 68Ga-NODAGA-peptide 1 measured in tumour and muscle (A) and Tumour to Muscle (B) ratio for each mouse after 2 hours of PET/CT acquisition.

EXAMPLES

[0106] Peptide Coupling

[0107] The chelator is prepared at a concentration of 10 mg/mL in a 0.2 M sodium bicarbonate solution at pH=9. Then, 10 equivalents of the chelator are added to an aliquot of progastrin. The conjugation reactions are carried out at 37 C. for 2 hours. The purification of the final product is carried out on AMICON filters. Through these filters, the excess unreacted chelator is removed. we obtain the conjugated peptide that we call NODAGA-Progastrin.

[0108] Animal Model

[0109] The colorectal cancer cell line T84 were cultured in T75 flask and passed 4 times after thawing to allow optimal growth rate to resume before xenograft in mice. The culture medium used was DMEM-F12 with Glutamax+10% fetal calf serum and 1% antibiotics (Streptomycin, Penicillin). For mouse xenograft, cell cultures are stopped at a confluence of 80% and the cells are taken up in a DMEM-F12 solution without Serum and Matrigel at a ratio of 1:1 to a concentration of 1.Math.10.sup.9 cells/100 l.

[0110] Mice are rapidly anesthetized with isoflurane and an injection of 100 l of T84 (1.Math.10.sup.9 cells/100 l) is done in the subcutaneous area between the shoulder blades. The animals are put back in their cages as soon as their awakening and placed in a stable room until the tumour growth is sufficient to experimentation.

[0111] Radiolabelling and Imaging

[0112] 100 L of a 2 M ammonium acetate solution are added to an aliquot of NODAGA-Progastrin (10 g solubilized in 50 L of PBS). Then, 500 L of gallium-68 eluate, [.sup.68Ga]GaCl3, from an IRE Elit generator, are added to the previously prepared solution. The whole is incubated at room temperature for 10 minutes. The final pH is 4.8. The radiochemical purity is greater than 90% (n=3) and is determined by thin layer chromatography (mobile phase: 0.1 M sodium citrate at pH=5). 2 L of 10 M sodium hydroxide are added to the final mixture to neutralize the pH. This prepared solution is used as is for biodistribution and PET/CT imaging studies.

[0113] The animals are put to sleep by gas anaesthesia (isoflurane at 3% for induction, and at 1.Math.5-2% for mask maintenance). The caudal vein is catheterized (27G catheter). Mice receive a radiotracer injection in a bolus of 3.50.6 MBq for 2-hour dynamics (Table 2).

[0114] All PET/CT imaging is done with the nanoPET/CTO camera (Mediso, Hungary).

[0115] The animals are imaged 3 by 3. To obtain images of the biodistribution kinetics of the NODAGA-Progastrin, 2-hour dynamic PET images (400-600 keV energy window) combined with a scanner (35 kVp, 450 ms exposure time per projection) are performed over the entire mouse body (10 cm window). The PET acquisition starts 10 seconds before the radiotracer injection starts and allows the injection peak to be obtained. The PET images obtained are then reconstructed by applying an anatomical shift, attenuation correction and time division. The time division is as follows: 10, 1, 1, 5, 10, 20, 40, 1h, 1h20, 1h20, 1h40 and 2h.

[0116] Post-analysis of the PET/CT 3D images was performed with VivoQuant 3.5 software (Invicro, USA). For dynamics, 6 regions of interest (ROIs) are detuned on the scanner, then transferred to the PET images for quantification. The quantified organs are the liver, kidneys, heart, brain, tumour and muscle (FIG. 3). The results of the quantifications are expressed either as a percentage of the dose injected per gram of tissue (% ID/g)* or as a Tumour/Muscle** ratio. * % ID/g=Activity calculated in ROI (MBq)/(Injected Activity (MBq)Volume of tissue (ml))100** The muscle is considered as a control region in the non-specific fixation of the radiotracer.

[0117] Results

[0118] In total, the biodistribution kinetics of NODAGA-Progastrin were monitored and quantified on a total of 5 mice that developed an ectopic tumour T84 between 100 and 600 mm3 (PET/CT acquisition in Table 2).

TABLE-US-00002 TABLE 2 Mouse-injected activity for dynamic 2-hour PET/CT acquisitions and percentage purity of radiosynthesis Radiotracer Acquisition Mice Injected activities MBq % purity NODAGA- 1 C3S 3.78 >95% Progastrin C5S3 4.12 2 C1S2 3.45 88% C1S3 2.46 C4S1 3.31

[0119] Tumour volumes of mice were measured on CT images (Table 3).

TABLE-US-00003 TABLE 3 Tumour volume at the time of imaging calculated by clipping on the scanner Mice C3S C5S3 C1S2 C1S3 C4S1 Volume tumours in mm.sup.3 338 553 320 172 398

[0120] FIG. 1 illustrates the bio-allocation of this tracer during the 2 hours of PET imaging in a mouse. The average quantitation values in % ID/g of each interest region were calculated and are presented in FIG. 2.

[0121] As expected, we observed a high concentration in the elimination organs of the liver and kidneys and a much lower level of activity in the muscle or brain that does not specifically fix the tracer. More interestingly, the level of activity in the tumour is higher than in the muscle in the mice with a ratio Tumour/muscle ranking from 1 to 4 in the 5 mice (FIG. 3).

CONCLUSION

[0122] We can conclude that there is an incorporation of radiolabelled Progastrin peptide into the tumour in this model.