RADIOLABELED PEPTIDES FOR NON-INVASIVE DIAGNOSIS AND TREATMENT OF CXCR4 EXPRESSING TUMORS
20230099200 · 2023-03-30
Assignee
Inventors
Cpc classification
A61K51/088
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K38/12
HUMAN NECESSITIES
International classification
A61K51/08
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
Abstract
The present invention concerns radiolabeled peptides that are suitable tracers for specific targeting and imaging of human CXCR4 in vivo, in particular for the detection of human primary and secondary CXCR4 overexpressing tumors. In addition, the radiolabeled peptides according to the present invention can be advantageously used in the treatment of human primary and secondary CXCR4 overexpressing tumors.
Claims
1. A radiopharmaceutical compound targeting CXCR4 receptor, said compound comprising: a) a cyclic peptide, as monomer or multimer, having the following formula I:
Arg-Ala-[D-Cy s-Arg-X-Y-Z]—COOH wherein X is L-2-Nal or L-Phe; Y is L-Phe or L-His; Z is L-Cys or L-Pen; b) a linker, said linker being bound to the N-terminus of said cyclic peptide; c) a chelator, said chelator being bound to said linker; and d) a radioisotope, said radioisotope being bound to said chelator.
2. The radiopharmaceutical compound according to claim 1, wherein the linker is a combination of at least two subunits A and B, wherein A is bound to said chelator, whereas B is bound to the N-terminus of said cyclic peptide, subunits A and B being connected to each other by amide bond or by a subunit C, preferably by amide bond, wherein subunit A is chosen from the group consisting of 2-aminobenzoic acid, 3-aminobenzoic acid, 4-aminobenzoic acid, 2-aminomethylbenzoic acid, 3-aminomethylbenzoic acid, 4-aminomethylbenzoic acid, amino-(PEG)n-COOH wherein n is from 2 to 10, 8-amino octanoic acid, 7-amino heptanoic acid, 6-aminohexanoic acid, 5-aminovaleric acid, 4-aminobutyric acid, 3-aminopropionic acid, when subunits A and B are connected to each other by amide bond; or (4-Ethynylphenyl)methanamine, (3-Ethynylphenyl)methanamine, 3-Ethynylaniline, 4-Ethynylaniline, 4-Pentyn-1-amine, But-3-yn-1-amine, propargylamine, 3-Azido-1-propanamine, 4-azido-1-butylamine, 5-azido-1-pentylamine, 6-azido-1-hexylamine, when subunits A and B are connected to each other by a subunit C; subunit B is chosen from the group consisting of 2-aminobenzoic acid, 3-aminobenzoic acid, 4-aminobenzoic acid, 2-aminomethylbenzoic acid, 3-aminomethylbenzoic acid, 4-aminomethylbenzoic acid, amino-(PEG)n-COOH wherein n is from 2 to 10, 8-amino octanoic acid, 7-amino heptanoic acid, 6-aminohexanoic acid, 5-aminovaleric acid, 4-aminobutyric acid, 3-aminopropionic acid, when subunits A and B are connected to each other by amide bond; or 2-azidobenzoic acid, 3-azidobenzoic acid, 4-azidobenzoic acid, 2-azidomethyl benzoic acid, 3-azidomethylbenzoic acid; 4-azidomethylbenzoic acid, Azido-(PEG)n-COOH wherein n is from 2 to 10, 8-azidooctanoic acid, 7-azidoheptanoic acid, 6-azidohexanoic acid, 5-azidovaleric acid, 4-azidobutyric acid, 3-butynoic acid, 4-pentynoic acid and 6-hexynoic acid, when subunits A and B are connected to each other by a subunit C; and subunit C is chosen from the group consisting of 1,4 substituted 1,2,3 triazole or 1,5 substituted 1,2,3 triazole linkers.
3. The radiopharmaceutical compound according to claim 1, wherein the chelator is chosen from the group consisting of 1,4,7-triazacyclononane-triacetic acid (NOTA), 1,4,7, 10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), {4-[2-(bis-carboxymethyl-amino)-ethyl]-7-carboxymethyl-[1,4, 7]triazonan-1-yl}-acetic acid (NETA), 1,4,8,11-tetraaza-cyclotetradecane 1,4,8,11-tetraacetic acid (TETA), p-SCN-Bn-NOTA(C-NOTA), NODASA, NODAGA, C-DOTA, DOTAGA, TRAP(Azide).sub.1, TRAP(Azide).sub.2, TRAP(Azide).sub.3.
4. The radiopharmaceutical compound according to claim 1, wherein the radioisotope is chosen from the group consisting of .sup.68Ga.sup.3+, .sup.67Ga.sup.3+, .sup.64Cu.sup.2+, .sup.44Sc.sup.3+, .sup.47Sc.sup.3+, .sup.111In.sup.3+, .sup.177Lu.sup.3+, .sup.86Y.sup.3+, .sup.90Y.sup.3+, .sup.225Ac.sup.3+, .sup.213Bi.sup.3+, .sup.212Pb.sup.2+ or .sup.18F.sup.−.
5. A precursor compound of the radiopharmaceutical compound as defined by claim 1, comprising a) a cyclic peptide, as monomer or multimer, having the following formula I:
Arg-Ala-[D-Cys-Arg-X-Y-Z]—COOH wherein X is L-2-Nal or L-Phe; Y is L-Phe or L-His; Z is L-Cys or L-Pen: b) a linker, said linker being bound to the N-terminus of said cyclic peptide; and c) a chelator, said chelator being bound to said linker.
6. The precursor compound according to claim 5, wherein the linker is a combination of at least two subunits A and B, wherein A is bound to said chelator, whereas B is bound to the N-terminus of said cyclic peptide, subunits A and B being connected to each other by amide bond or by a subunit C, wherein subunit A is chosen from the group consisting of 2-aminobenzoic acid, 3-aminobenzoic acid, 4-aminobenzoic acid, 2-aminomethylbenzoic acid, 3-aminomethylbenzoic acid, 4-aminomethylbenzoic acid, amino-(PEG)n-COOH wherein n is from 2 to 10, 8-amino octanoic acid, 7-amino heptanoic acid, 6-aminohexanoic acid, 5-aminovaleric acid, 4-aminobutyric acid, 3-aminopropionic acid, when subunits A and B are connected each other by amide bond; or (4-Ethynylphenyl)methanamine, (3-Ethynylphenyl)methanamine, 3-Ethynyl aniline, 4-Ethynyl aniline, 4-Pentyn-1-amine, But-3-yn-1-amine, propargyl amine, 3-Azido-1-propanamine, 4-azido-1-butylamine, 5-azido-1-pentylamine, 6-azido-1-hexylamine, when subunits A and B are connected to each other by a subunit C; subunit B is chosen from the group consisting of 2-aminobenzoic acid, 3-aminobenzoic acid, 4-aminobenzoic acid, 2-aminomethylbenzoic acid, 3-aminomethylbenzoic acid, 4-aminomethylbenzoic acid, amino-(PEG)n-COOH wherein n is from 2 to 10, 8-amino octanoic acid, 7-amino heptanoic acid, 6-aminohexanoic acid, 5-aminovaleric acid, 4-aminobutyric acid, 3-aminopropionic acid, when subunits A and B are connected to each other by amide bond; or 2-azidobenzoic acid, 3-azidobenzoic acid, 4-azidobenzoic acid, 2-azidomethyl benzoic acid, 3-azidomethylbenzoic acid; 4-azidomethylbenzoic acid, Azido-(PEG)n-COOH wherein n is from 2 to 10, 8-azidooctanoic acid, 7-azidoheptanoic acid, 6-azidohexanoic acid, 5-azidovaleric acid, 4-azidobutyric acid, 3-butynoic acid, 4-pentynoic acid and 6-hexynoic acid, when subunits A and B are connected to each other by a subunit C; preferably 6 aminohexanoic acid; and subunit C is chosen from the group consisting of 1,4 substituted 1,2,3 triazole or 1,5 substituted 1,2,3 triazole linkers.
7. The precursor compound according to claim 5, wherein the chelator is chosen from the group consisting of 1,4,7-triazacyclononane-triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), {4-[2-(bis-carboxymethylamino)-ethyl]-7-carboxymethyl-[1,4, 7]triazonan-1-yl}-acetic acid (NETA), 1,4,8, 11-tetraazacyclotetradecane1,4, 8,11-tetraacetic acid (TETA), p-SCN-Bn-NOTA(C-NOTA), NODASA, NODAGA, C-DOTA, DOTAGA, TRAP(Azide).sub.1, TRAP(Azide).sub.2, TRAP(Azide).sub.3.
8. A radiopharmaceutical composition comprising the radiopharmaceutical compound as defined in claim 1, in association with one or more excipients and/or adjuvants.
9. A pharmaceutical composition comprising the precursor compound as defined in claim 5, in association with one or more excipients and/or adjuvants.
10.-15. (canceled)
16. A method for obtaining a radiopharmaceutical compound comprising: a) radiolabeling a precursor compound as defined in claim 5 with a radioisotope chosen from the group consisting of .sup.68Ga.sup.3+, .sup.67Ga.sup.3+, .sup.64Cu.sup.2+, .sup.44Sc.sup.3+, .sup.47Sc.sup.3+, .sup.111In.sup.3+, .sup.177Lu.sup.3+, .sup.86Y.sup.3+, .sup.90Y.sup.3+, .sup.225Ac.sup.3+, .sup.213Bi.sup.3+, .sup.212Pb.sup.2+ or .sup.18F.sup.−.
17. A kit for the preparation of a radiopharmaceutical compound comprising: a precursor compound as defined in claim 5.
18. A method for the treatment of CXCR4 expressing disorders comprising administering a cyclic peptide, monomer or multimer, or a pharmaceutical composition comprising said cyclic peptide wherein said cyclic peptide has the following formula: ##STR00005## ##STR00006## to a human in need of such treatment.
19. The method according to claim 18, wherein the CXCR4 expressing disorders are CXCR4 expressing tumours selected from the group consisting of Non-small-cell lung carcinoma, pancreatic cancer, prostate cancer, breast cancer, glioblastoma, sarcoma, colon cancer, melanoma, lung cancer, neuroendocrine tumors, and renal cancer.
20. The method according to claim 18 wherein said peptide is administered in association with an immunomodulating therapy.
21. A cyclic peptide or a pharmaceutical composition comprising said cyclic peptide, said cyclic peptide having formula II:
W-Arg-Ala-[D-Cys-Arg-X-Y-Z]—COOH wherein X is L-2-Nal or L-Phe; Y is L-Phe or L-His; Z is L-Cys or L-Pen, and W is CH.sub.3—(CH.sub.2).sub.n—CO, wherein n is from 1 to 30 when said cyclic peptide is chosen from the group consisting of: TABLE-US-00007 (SEQ ID NO: 10) CH.sub.3(CH.sub.2).sub.nCO-Arg-Ala-[D-Cys-Arg-2-Nal-His-Pen]-COOH (SEQ ID NO: 11) CH.sub.3(CH.sub.2).sub.nCO-Arg-Ala-[D-Cys-Arg-Phe-Phe-Cys]-COOH (SEQ ID NO: 12) CH.sub.3(CH.sub.2).sub.nCO-Arg-Ala-[D-Cys-Arg-Phe-His-Pen]-COOH (SEQ ID NO: 13) CH.sub.3(CH.sub.2).sub.nCO-Arg-Ala-[D-Cys-Arg-Phe-Phe-Pen]-COOH and (SEQ ID NO: 14) CH.sub.3(CH.sub.2).sub.nCO-Arg-Ala-[D-Cys-Arg-2-Nal-Phe-Pen]- COOH; and, wherein n is from 0 to 30 when said cyclic peptide is chosen from the group consisting of: TABLE-US-00008 (SEQ ID NO: 15) CH.sub.3(CH.sub.2).sub.nCO-Arg-Ala-[D-Cys-Arg-2-Nal-Phe-Cys]-COOH (SEQ ID NO: 16) CH.sub.3(CH.sub.2).sub.nCO-Arg-Ala-[D-Cys-Arg-2-Nal-His-Cys]-COOH and (SEQ ID NO: 17) CH.sub.3(CH.sub.2).sub.nCO-Arg-Ala-[D-Cys-Arg-Phe-His-Cys]-COOH.
22. A method for the treatment of CXCR4 expressing disorders comprising administering a compound of claim 21 to a human in need of such treatment.
23. The method of claim 22, wherein the CXCR4 expressing disorders are selected from the group consisting of Non-small-cell lung carcinoma, pancreatic cancer, prostate cancer, breast cancer, glioblastoma, sarcoma, colon cancer, melanoma, lung cancer, neuroendocrine tumors, and renal cancer.
24. The method according to claim 22 wherein said peptide is administered in association with an immunomodulating therapy.
Description
[0082]
[0083]
[0084]
[0085]
[0086] EXAMPLE 1: Synthesis of the radiolabeled peptides of the present invention and study concerning their CXCR4 affinities, in vivo biodistribution, in vivo CXCR4 PET imaging and uptake in CXCR4 expressing tumors
MATERIALS AND METHODS
[0087] Synthesis of NOTA-AMBHA-R54, DOTA-AMBHA-R54, [.sup.natGa]NOTA-and [.sup.natga]DOTA-AMBRA-R54
[0088] The synthesis of the peptides was accomplished using 2-C1-TrtCl resin by applying an ultrasonic-assisted Fmoc/tBu solid phase peptide synthesis (US-SPPS) previously described [33].
[0089] After elongation of linear sequence, the resulting peptidomimetic and acidic sensitive protective groups were simultaneously cleaved from the resin treating with a solution of TFA/TIS for 2 h at room temperature. Oxidation of sulfide side chains was achieved dissolving the crude peptide in H.sub.2O and adding an aqueous solution of N-chlorosuccinimide. The crude mixture was purified by reverse-phase preparative HPLC and the product was characterized by analytical HPLC and MS-ESI spectrometry.
[0090] A detailed description of synthesis, yield, purity, retention times, analytical data is provided below: Synthesis of NOTA-AMBHA-R54 and DOTA-AMBHA-R54
[0091] Standard Na-Fmoc-protected amino acids, O-benzotriazole-N, N, N′, N′-tetra-methyl-uroniumhexafluorophosphate (HBTU, purity 99%), N,N-diisopropylethylamine (DIEA, purity 99%), trifluoroacetic acid (TFA, purity 99%), piperidine, Fmoc-D-Cys(Trt)-OH, Fmoc-L-2Nal-OH, Fmoc-L-Pen(Trt)-OH, 4-(Fmoc-aminomethyl)benzoic acid and Fmoc-6-Ahx-OH were purchased from IRIS Biotech (Marktredwitz, Germany). DOTA-tris(tBu)ester and NOTA-bis(tBu)ester were purchased from CheMatech (Dijon, France). Triisopropylsilane (TIS) (purity 98%), 1-hydroxybenzotriazole hydrate (HOBt) (purity >97% dry weight, water ≈12%) (1H-7-Azabenzotriazol-1-yl-oxy)tris-pyrrolidinophosphonium hexafluorophosphate (PyAOP) (purity 96%) %), 1-hydroxy-7-azabenzotriazole (HOAt, purity 96%), N-chloro succinimide (purity 98%), acetic anhydride (Ac20, purity >98%) %), anhydrous N,N-dimethylformamide (DMF), and anhydrous dichloromethane (DCM) were purchased from Sigma-Aldrich (Milano, Italy).
[0092] Peptide synthesis solvents, water and acetonitrile for HPLC, were reagent grade and were acquired from commercial sources (Sigma-Aldrich, Milano, Italy) and used without any further purification unless otherwise stated. Peptides were purified by preparative HPLC (Shimadzu HPLC system) equipped with a C18-bounded preparative RP-HPLC column (Phenomenex Kinetex 21.2 mm×150 mm, 5 μm). Peptides were analyzed by analytical HPLC (Shimadzu Prominance HPLC system) equipped with a C18-bounded analytical RP-HPLC column (Phenomenex Kinetex, 4.6 mm×150 mm, 5 μM) using a gradient elution (10-90% acetonitrile in water (0.1% TFA) over 20 min; flow rate=1.0 mL/min; diode array UV detector). Molecular weights of compounds were confirmed by HMRS-ESI-mass spectrometry using a Q Exactive Orbitrap LC-MS/MS (Termo Fisher Scientific Walthman, MA, USA).
[0093] The synthesis of the peptides was accomplished using a 2-Cl-TrtCl resin (58 mg, 0.09 mmol, 1.56 mmol/g), which was first swelled in anhydrous DMF over 30 min and then drained on solid phase peptide manifold without any further treatment. Then a solution of Fmoc-L-Pen(Trt)-OH (18 mg, 0.03 mmol, 0.33 equiv relative to the initial loading of the resin) and DIPEA (16 μL, 0.09 mmol, 1 equiv relative to the initial loading of the resin) in anhydrous DMF (1.5 mL) was added and the so obtained mixture was shaken overnight at room temperature. The residual chloride reactive groups were capped by adding a previously mixed solution of DIPEA (16 μL, 0.09 mmol, 1 equiv relative to the initial loading of the resin) in DCM/MeOH (9:1, 3 mL) and allowing the resin to gently shake for 30 min. The resin was washed with DMF (3×0.5 min), DCM (3×0.5 min) and Et.sub.2O (3×0.5 min), dried exhaustively and its loading level was quantified (approx. 0.03 mmol/g) by measuring the level of the first amino acid attachment as already described in literature. The remaining cycles of peptide bond formation and AP-Fmoc removal reactions required to build the linear sequences were both performed placing the reactors in an ultrasonic bath (SONOREX RK 52 H by BANDELIN electronic, Germany). Specifically, after swelling again the resin in DCM/DMF 1:1, the Fmoc-protecting group was removed by treatment with a 20% piperidine solution in DMF (1×0.5 min and 1×1 min). The so obtained free amine was reacted with subsequent amino acid using 3 equivalents (relative to the 0.03 mmol/g loading) of Fmoc-protected amino acid and coupling reactants. Briefly, to a pre-stirred solution of Fmoc-L-His(Trt)-OH (56 mg, 0.09 mmol, 3 equiv), HBTU (35 mg, 0.09 mmo, 3 equiv) and HOBt (14 mg, 0.09 mmol, 3 equiv) in DMF (1.5 mL), DIPEA (32 μL, 0.18 mmol, 6 equiv) was added portionwise and the so obtained solution poured to the pre-swelled resin. The mixture was placed in the ultrasonic bath for 5 min at room temperature and then washed with DMF (3×1 min) and DCM (3×1 min). Subsequently, to cap the remaining free amines the resin was treated with 1.2 mL of an acetylating solution containing 2 equiv of Ac.sub.2O (relative to the 0.03 mmol/g loading) and 4 equiv of DIPEA (relative to the 0.03 mmol/g loading) in DMF and then placed in the ultrasonic bath for 2 min. Elongation of the linear peptide sequence was obtained by iterative cycles of the aforementioned Fmoc deprotection and coupling reaction steps till 4-(Fmoc-aminomethyl)benzoic acid was anchored to the resin.
[0094] For the synthesis of reference Fmoc-AMBHA-R54, the peptide cleaved from the resin treating with a solution of TFA/TIS (95:5, 1.5 mL) for 1 h at room temperature. The resin was filtered and the crude peptide precipitated from the TFA solution diluting to 15 mL with cold Et.sub.2O and then centrifuged (6000 rpm×15 min). The supernatant was carefully removed and the precipitate washed again with another volume of Et.sub.2O as described above. The resulting wet solid was dried for 1 h under reduced pressure. An aliquot of the crude reduced peptide was dissolved in water/acetonitrile (9:1) and analyzed, before cyclization step, by reverse-phase analytical HPLC (solvent A: water+0.1% TFA; solvent B: acetonitrile+0.1% TFA; from 10 to 60% of solvent B over 30 min, flow rate: 1 mL min-1). Oxidation of sulfide side chains was achieved dissolving the crude peptide in H.sub.2O (30 mL, approx. 0.5 mM) and adding an aqueous solution (5 mL) of N-chlorosuccinimide (5 mg, 0.036 mmol, 1.2 equiv) dropwise. The reaction was allowed to stir at room temperature for 30 minutes and complete conversion of the starting compound ascertained by HPLC profile comparison.
[0095] For the synthesis of DOTA- and NOTA-AMBHA-R54, the 4-(Fmoc-aminomethyl)benzoic acid functionalized resin underwent the last Fmoc deprotection and was functionalized with DOTA-tris(tBu)ester or NOTA-bis(tBu)ester using PyAOP and HOAt as coupling reactants. To a pre-stirred solution of DOTA-tris(tBu)ester (45 mg, 0.075 mmol, 2.5 equiv) (or NOTA-bis(tBu)ester: 31 mg, 0.075 mmol, 2.5 equiv), PyAOP (39 mg, 0.075 mmol, 2.5 equiv) and HOAt (10 mg, 0.075 mmol, 2.5 equiv) in DMF/DCM 2:1 (1.5 mL), DIPEA (26 μL, 0.15 mmol, 5 equiv) was added and the so obtained solution poured to the pre-swelled resin. The mixture was placed in the ultrasonic bath for 10 min at room temperature and the completion of the reaction ascertained by Kaiser ninhydrin and TNBS tests. The resin was washed with DMF (3×1 min), DCM (3×1 min), and Et.sub.2O (3×1 min) and then dried exhaustively. The cleavage of the peptides from the resin and the subsequent oxidation was carried out as above describe for AMBHA-R54.
[0096] At this stage the reaction solutions of AMBHA-R54 and DOTA-and NOTA-R54 were freezed, lyophilized and the crude mixtures purified by reverse-phase preparative HPLC (solvent A: water+0.1% TFA; solvent B: acetonitrile+0.1% TFA; from 0 to 30% of solvent B over 25 min, flow rate: 10 mL min-1). Fractions of interest were evaporated from organic solvents under reduced pressure, freezed and then re-lyophilized. Obtained products were characterized by analytical HPLC and MS-ESI spectrometry. Yield, purity, retention times, and analytical data are reported as follows.
[0097] Fmoc-AMBHA-R54. 36 mg, overall yield: 83%, purity: 95%, tR 28.25 min, (analytical HPLC, 10 to 60% acetonitrile (0.1% TFA) in water (0.1% TFA) over 30 min, flow rate of 1.0 mL/min); HRMS (ESI-MS): Calculated: 1435,63180 for C.sub.71H.sub.89N.sub.17O.sub.12S.sub.2 [M+H]+, found: 1436,64050
[0098] NOTA-AMBHA-R54. 29 mg, overall yield: 64%, purity: 95%, tR 16.25 min, (analytical HPLC, 10 to 60% acetonitrile (0.1% TFA) in water (0.1% TFA) over 30 min, flow rate of 1.0 mL/min); tR 23.48 min, (analytical HPLC, 0 to 30% acetonitrile (0.1% TFA) in water (0.1% TFA) over 30 min, flow rate of 1.0 mL/min); HRMS (ESI-MS): Calculated: 1499,70402 for C.sub.68H.sub.99N.sub.20O.sub.15S.sub.2 [M+H].sup.+, found: 1500,71305
[0099] DOTA-AMBHA-R54. 26 mg, overall yield: 56%, purity: 95%, tR 15.56 min, (analytical HPLC, 10 to 60% acetonitrile (0.1% TFA) in water (0.1% TFA) over 30 min, flow rate of 1.0 mL/min); tR 22.80 min, (analytical HPLC, 0 to 30% acetonitrile (0.1% TFA) in water (0.1% TFA) over 30 min, flow rate of 1.0 mL/min); HRMS (ESI-MS): Calculated: 1600,75170 for C.sub.72H.sub.106N.sub.21O.sub.17S.sub.2 [M+H].sup.+, found: 1601,76010
##STR00004##
[0100] .sup.68Ga-labeling of NOTA- and DOTA-AMBHA-R54 The non-processed fractionated eluate (1.6 mL containing 80% of the total eluted activity) of a .sup.68Ge/.sup.68Ga-generator with TiO.sub.2 matrix (GalliaPharma, Eckert&Ziegler; eluent: 0.1 M aq. HCl, total .sup.68Ga activity: 148-222 MBq) was adjusted to pH 2,5 by adding HEPES buffer (5 mL of a 0.1 M aq. solution) and used for labelling of 50 nmol of NOTA- or DOTA-Ahx-R54 for 10 minutes at 124° C. The labeled product was isolated by passing the reaction mixture through a C18 light solid phase extraction cartridge (SepPak), which was then purged with water (5 mL), followed by elution of the respective .sup.68Ga-labeled peptide using 0.4 mL pure ethanol. The product fraction was diluted with saline and sterilized by filtration through a 0.22 μm membrane filter. Radiochemical yields (before sterile filtration) were 80±5% based on .sup.68Ga-chloride starting activity. Radiochemical purity was determined via radio-TLC using silica coated TLC strips and 0.1 M Na-citrate pH 5 as mobile phase. Strips were analyzed using a Cyclone Plus storage phosphor system (PerkinElmer). Radiochemical purity was always >99%.
[0101] Cell Culture
[0102] Human T-cell Leukemia CCRF-CEM cells were obtained from the NCI 60 cancer cell line collection and grown in RPMI-1640 with 10% FBS and 2 mM glutamine and 100 units/mL of penicillin/streptomycin. Chinese hamster ovary (CHO) and CHO cells expressing human CXCR4 (CHO-hCXCR4) grown in complete medium supplemented with 1 mg/mL G418 (a kind gift of Dr. David McDermott (NIAID, NIH, Bethesda, Md.).
[0103] Flow Cytometry
[0104] CXCR4 levels were evaluated by flow cytometry using PE-conjugated anti-human CXCR4 (clone 12G5) (Catalog #FAB170, R&D Systems). Flow data were acquired using a FACSAria II Cell Sorter cytometer (BD Biosciences) and analyzed using FACS Diva 8 (BD Biosciences).
[0105] Determination of CXCR4 Affinity
[0106] Binding affinities to hCXCR4 were assessed with CCRF-CEM cells (1.Math.10.sup.6 cells/sample) in binding buffer (PBS containing 50 mM HEPES, 1 mM CaCl.sub.2, 5 mM MgCl.sub.2, 0.5% BSA, and 0.3 mM NaN.sub.3) and [.sup.125I]CXCL12 (Perkin-Elmer, 2200 Ci/mmol) as radioligand. Cells were incubated with 100.000 cpm of the respective radioligand (approx. 0.1 nM) plus increasing concentrations (10.sup.−11 to 10.sup.−5 M) of the respective peptide of interest (n=3 per concentration, total sample volume 250 μL). After incubation at 4° C. for 60 min (CCRF-CEM cells) at room temperature (RT), samples were centrifuged (5 min, 450 g, Megafuge 1.0, Heraeus Thermo Scientific), the supernatant was carefully removed and pooled with the supernatants of two subsequently performed washing steps. Then, the amount of free (pooled supernatants) and bound radioligand (cell pellet) was quantified using a γ-counter and expressed in percent of total added activity. The half maximal inhibitory concentration (IC.sub.50) values were calculated using GraphPad Prism software (GraphPad Software, Inc., California).
[0107] Mouse Tumor Models
[0108] Female athymic nude mice (6 weeks old from Envigo Laboratories, Indianapolis, Ind., USA) were subcutaneously (s.c.) injected with 10.sup.7 CHO (right flank) and CHO-hCXCR4 (left flank) or CHO-hCXCR4 cells alone (right flank). Once tumors became palpable (100=.sup.3 [volume=0.5×long diameter×(short diameter).sup.2]), approximately 15-21 days post injection (p.i.), the animals were used for the experiments.
[0109] Biodistribution Studies
[0110] 10-15 MBq of the respective .sup.68Ga-labeled peptide in 100 μL of PBS were injected into the tail vein of nude mice bearing CHO-hCXCR4 tumor (groups of n=3-5). For competition studies, 8 mg/kg of unlabeled R54, were co-injected with the radioligands. At 45 min p.i., blood was collected from the retro-orbital plexus (100 μl) and mice were sacrificed and the tissues and organs of interest were dissected, weighed, and counted for radioactivity in a γ-counter. Biodistribution data are given as percent injected dose per gram tissue, % ID/g (means±SD).
[0111] PET/CT-Imaging
[0112] Tumor-bearing CHO/CHO-hCXCR4 mice were i.v. injected with 10-15 MBq of the respective radiotracer in 100 μL of PBS. At 45 min p.i., mice were anaesthetized with Avertin and images were acquired using a Discovery 600 PET/TC scanner (GE healthcare) in 3D mode with measured attenuation correction (1 bed position, 6 min scanning time). Data were decay-corrected to the time of tracer injection. The reconstructed images including PET, CT and fused PET/TC images, were generated on the advantage workstation version (AW4.6 software, GE healthcare). Tissue activity (e.g. tumor, lung, liver, kidney) was quantified considering the volume of a region of interest (ROI) to determine maximum standardized uptake values (SUVmax). Non-specific tracer accumulation was investigated by co-injection of 8 mg/kg unlabeled peptide R54.
[0113] Immunohistochemistry
[0114] CHO and CHO-hCXCR4 tumors were microscopically analyzed (H&E) and CXCR4 expression was evaluated through immunohistochemistry (IHC) (mab172, clone 44716, diluition 1:1000R&D system). The percentage of positive cells was evaluated in at least 5 areas (HPF 200× magnification) (Zeiss AxioScope light microscope).
[0115] Statistical Analysis
[0116] Statistical analysis was performed using the MedCalc 9.3.7.0 and Excel software. Unpaired Student t test was used for statistical analysis of ex vivo biodistribution and PET/TC images. The Spearman's correlation was used to measure the degree of association between SUV(max) and % CXCR4 IHC staining. All statistical tests were performed two-sided and a p-value <0.05 was considered to indicate statistical significance.
RESULTS
[0117] CXCR4 Affinities
[0118] To assess the influence of modifications and chelator conjugation on R54, the CXCR4 affinity of Fmoc-AMBHA-R54 (see above the structure), NOTA-AMBHA-R54 and DOTA-AMBHA-R54 peptides as well as unmodified R54 were tested in competitive binding with [.sup.125I]CXCL12 as radioligand on CXCR4-expressing CCRF-CEM cells. Binding affinity data, in particular hCXCR4 binding affinities (IC.sub.50 in nM), are summarized in Table 1. Each experiment was performed in triplicate, and results are means ±SD from a minimum of three separate experiments.
TABLE-US-00005 TABLE 1 Peptide IC.sub.50 (nM) R54 20.0 ± 2.0 Fmoc-AMBHA-R54 38.5 ± 3.5 DOTA-AMBHA-R54 107.0 ± 39.6 NOTA-AMBHA-R54 36.0 ± 11.5
[0119] Compared to R54, Fmoc-AMBHA-R54 and NOTA-AMBHA-R54 displayed slightly decreased CXCR4 affinity, whereas DOTA conjugation clearly reduced CXCR4 affinity. In vivo biodistribution studies
[0120] The biodistribution of [.sup.68Ga]DOTA-AMBHA-R54 and [.sup.68Ga]NOTA-AMBHA-R54 was investigated in CHO-hCXCR4 bearing CD1 mice (45 min p.i.). Table 2 shows the biodistribution of [.sup.68Ga]DOTA-AMBHA-R54 and [.sup.68Ga]NOTA-AMBHA-R54 in CHO hCXCR4 xenograft bearing nude mice at 45 min p.i. Data are given in % ID/g and are means±SD.
TABLE-US-00006 TABLE 2 [.sup.68Ga]DOTA-AMBHA-R54 [.sup.68Ga]NOTA-AMBHA-R54 competition competition tracer only (+8 mg/kg R54) tracer only (+8 mg/kg R54) organ (n = 4) (n = 3) (n = 5) (n = 4) blood 0.41 ± 0.08 0.37 ± 0.11 1.80 ± 1.02 1.10 ± 0.18 lung 0.43 ± 0.10 0.34 ± 0.10 1.41 ± 0.90 0.85 ± 0.11 liver 0.35 ± 0.09 0.26 ± 0.11 0.90 ± 0.40 0.56 ± 0.09 spleen 0.26 ± 0.05 0.14 ± 0.06 0.97 ± 0.40 0.70 ± 0.16 digestive 0.19 ± 0.03 0.20 ± 0.03 0.77 ± 0.33 0.56 ± 0.14 system kidneys 1.79 ± 0.57 2.13 ± 0.9 5.83 ± 2.60 6.04 ± 3.20 muscle 0.24 ± 0.18 0.21 ± 0.06 0.41 ± 0.20 0.41 ± 0.28 CHO-hCXCR4 1.63 ± 1.02 0.37 ± 0.17 4.44 ± 0.84 0.95 ± 0.31 tumor
[0121] Both [.sup.68Ga]DOTA-AMBHA-R54 and [.sup.68Ga]NOTA-AMBHA-R54 showed rapid background clearance and very low accumulation in all non-target tissues except the kidney. [.sup.68Ga]NOTA-AMBHA-R54 displayed higher CXCR4-mediated tumor accumulation, nicely competed with cold R54, as compared to [.sup.68Ga]DOTA-AMBHA-R54, consistent with the higher CXCR4 affinity of the NOTA analog.
[0122] Co-injection of an excess of unlabeled competitor (unlabeled R54) efficiently reduced the tracer uptake to background levels, indicating high CXCR4 specificity of tracer uptake in the tumor xenografts, with the [.sup.68Ga]DOTA- and [.sup.68Ga]NOTA-AMBHA-R54 accumulation in all other tissues remaining essentially unaffected by the competition conditions. High tumor/background ratios was observed for both analogs with values for [.sup.68Ga]NOTA-AMBHA-R54 being substantially higher (
[0123] In Vivo CXCR4 PET Imaging
[0124] Representative PET/CT images of [.sup.68Ga]DOTA-AMBHA-R54 and [.sup.68Ga]NOTA-AMBHA-R54 in CHO-hCXCR4 bearing CD1 mice is shown in
Upon co-injection of unlabeled competitor (R54) tumor accumulation was reduced to background levels (
[0125] [.sup.68Ga]NOTA-AMBHA-R54 Uptake (SUVmax) in the Tumors Correlates with CXCR4 Expression
[0126] To exemplarily correlate [.sup.68Ga]NOTA-AMBHA-R54 uptake with the CXCR4 expression level in tumor tissue, immunohistochemical staining for CXCR4 was performed in CHO-hCXCR4 and CHO tumors. In
CONCLUSION
[0127] This study has conclusively demonstrated that the development of CXCR4 targeted PET tracers based on the well characterized, and structurally as well as physicochemical, optimized R54 peptide scaffold is feasible and successful. Of the two compounds in this study, [.sup.68Ga]NOTA-AMBHA-R54 showed superior overall tracer characteristics as compared to DOTA counterpart, i.e. faster clearance from non-target tissues and higher accumulation in CXCR4-expressing tumor xenografts. Thus, [.sup.68Ga]NOTA-AMBHA-R54 can be considered a suitable candidate for rapid translation into a clinical context.
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