Positron emitting radionuclide labeled peptides for human uPAR PET imaging

Abstract

There is provided a positron-emitting radionuclide labelled peptide for non-invasive PET imaging of the Urokinase-type Plasminogen Activator Receptor (uPAR) in humans. More specifically the invention relates to human uPAR PET imaging of any solid cancer disease for diagnosis, staging, treatment monitoring and especially as an imaging biomarker for predicting prognosis, progression and recurrence.

Claims

1. A method of generating images of uPAR expression in a human by diagnostic imaging of uPAR expressing tumors involving administering an imaging agent to said human, and generating an image of at least a part of said body to which said imaging agent is administered; wherein the imaging agent is a positron-emitting radionuclide labelled peptide conjugate, said conjugate comprising a uPAR binding peptide coupled via the chelating agent DOTA to a .sup.64Cu radionuclide; wherein the conjugate is to be administered in a dose of 100-500 MBq followed by PET scanning ½-24h after the conjugate has been administered, and quantification through SUVmax and/or SUVmean. wherein the peptide is selected from the group consisting of: TABLE-US-00004 (D-Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)- (D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (Ser)-(Leu)-([beta]-cyclohexyl-L-alanine)-(Phe)- (D-Ser)-(Gln)-(Tyr)(Leu)-(Trp)-(Ser), (D-Glu)-([beta]-cyclohexyl-L-alanine)-(Phe)- (D-Ser)-(D-Tyr)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)- (D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(Ser)- (D-Arg)-(Tyr)-Leu)-(Trp)-(Ser), (D-Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(Ser)- (D-Arg)-(Tyr)-Leu)-(Trp)-(Ser), (D-Thr)-([beta]-cyclohexyl-L-alanine)-(Phe)- (D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (D-Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)- (D-Ser)-(D-Arg)-(Tyr)-(Leu)-([beta]-2-naphthyl- L-alanine)-(Ser), (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)- (Arg)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)- (D-Arg)-(Tyr)-(Leu)([beta]-1-naphthyl-L-alanine)- (Ser), (D-Glu)-([beta]-cyclohexyl-L-alanine)-(Phe)- (D-Ser)-(Tyr)-(Tyr)-(Leu)-(Trp)-(Ser), (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)- (D-Arg)-(Leu)-(Leu)-(Trp)-(D-His), (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)- (D-Arg)-([beta]-cyclohexyl-L-alanine)-(Leu)-(Trp)- (Ile), (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)- (D-Arg)-(Tyr)-(Leu)([beta]-1-naphthyl-L-alanine)- (D-His), (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)- (D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)- (N-(3-indolylethyl)glycine)-(N- (2-methoxyethyl)glycine), (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)- (D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)-(D-Phe)- (N-benzylglycine)-(N-(2[beta]thoxyethyl)glycine), (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)- (D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)- (D-Phe)-(N-(methylnaphthalyl)glycine)-(N- (2-methoxyethyl)glycine), and (Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)- (D-Ser)-(D-Arg)-(N-(2,3-dimethoxybenzyl)glycine)- (D-Phe)-(N-(2,3-dimethoxybenzyl)glycine)-(Ile),    wherein the C-terminal is either a carboxylic acid or an amide.

2. The method according to claim 1, wherein the peptide is (D-Asp)-([beta]- cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Trp)-(Ser).

3. The method according to claim 1, having the formula: ##STR00005##

4. The method according to claim 1, wherein the cancer is selected from prostate, breast, pancreatic, lung, brain and colorectal cancer.

5. The method according to claim 1, wherein the conjugate is administered in a dose of 200-400 MBq.

6. The method according to claim 1, wherein the imaging agent is provided in a pharmaceutical composition comprising the imaging agent, together with one or more pharmaceutical acceptable adjuvants, excipients or diluents.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A shows In vitro competitive inhibition of the uPA:uPAR binding for AE105 and AE152 using surface Plasmon resonance.

(2) FIG. 1B shows Radiolabeling method for .sup.18F-AIF-NOTAAE105.

(3) FIG. 2A shows representative HPLC UV chromatograms of NOTA-AE105.

(4) FIG. 2B shows cold standard AIF-NOTA-AE105.

(5) FIG. 2C shows radio chromatograms for the final product .sup.18F-AIFNOTA-AE105.

(6) FIG. 2D shows radio chromatograms for the final product .sup.18F-AIFNOTA-AE105 and after 30 min in PBS.

(7) FIG. 3A shows Representative PET images after 0.5 h, 1.0 h and 2.0 h p.i of .sup.18F-AIF-NOTA-AE105 (top) and .sup.18F-AIF-NOTA-AE105 with a blocking dose of AE152. White arrows indicate tumor.

(8) FIG. 3B shows quantitative ROI analysis with tumor uptake values (% ID/g). A significant higher tumor uptake was found at all three time points. Results are shown as % ID/g±SEM (n=4 mice/group). ** p<0.01, *** p<0.001 vs blocking group at same time point.

(9) FIG. 4 shows biodistribution results for .sup.18F-AIF-NOTA-AE105 (normal) and .sup.18F-AIFNOTA-AE105+blocking dose of AE152 (Blocking) in nude mice bearing PC-3 tumors at 2.5 h p.i. Results are shown as % ID/g±SEM (n=4 mice/group). *p<0.05 vs blocking group.

(10) FIG. 5A shows uPAR expression level found using ELISA in PC-3 cells.

(11) FIG. 5B shows uPAR expression level found using ELISA in PC-3 cells in resected PC-3 tumors.

(12) FIG. 5C shows a significant correlation between uPAR expression and tumor uptake was found in the four mice injected with 18F-AIF-NOTA-AE105 (p<0.05, r=0.93, n=4 tumors).

(13) FIG. 6 shows in vivo uPAR PET imaging with [.sup.64Cu]NOTA-A.E105 in a orthotropic human glioblastoma mouse model

(14) FIG. 7 shows in vivo uPAR PET imaging with [.sup.68Ga]NOTA-AE105 in a orthotropic 5 human glioblastoma mouse model.

(15) FIG. 8 shows a flow diagram summarizing the steps carried out in accordance with the present invention.

DETAILED DESCRIPTION OF THE INVENTION

(16) Surprisingly, a radiolabeled peptide of the present invention is very useful in the prediction of cancer metastasis of uPAR expressing tumors.

(17) The peptides selected for use in the conjugates of the present invention are typically radiolabeled by coupling a chelating agent to the peptide. The chelating agent is capable of binding a selected radionuclide thereto. The chelating agent and radionuclide is coupled to the peptide in a manner that does not interfere or adversely affect the binding properties or specificity of the peptide. The use of various chelating agents for radio labelling of peptides is well known in the art. The chelating agent is coupled to the peptide by standard methodology known in the field of the invention and may be added at any location on the peptide provided that the biological activity of the peptide is not adversely affected. Preferably, the chelating group is covalently coupled to the amino terminal amino acid of the peptide. The chelating group may advantageously be attached to the peptide during solid phase peptide synthesis or added by solution phase chemistry after the peptide has been obtained. Preferred chelating groups include DOTA, NOTA, NODAGA or CB-TE2A.

(18) Concerning the synthesis of the peptides used in the present invention reference is made to U.S. Pat. No. 7,026,282.

(19) The peptide/chelate conjugates of the invention are labeled by reacting the conjugate with radionuclide, e.g. as a metal salt, preferably water soluble. The reaction is carried out by known methods in the art.

(20) The conjugates of the present invention are prepared to provide a radioactive dose of 35 between about 100-500 MBq (in humans), preferably about 200-400 MBq, to the individual. As used herein, “a diagnostically effective amount” means an amount of the conjugate sufficient to permit its detection by PET. The conjugates may be administered intravenously in any conventional medium for intravenous injection. Imaging of the biological site may be effected within about 30-60 minutes post-injection, but may also take place several hours post-injection. Any conventional method of imaging for diagnostic purposes may be utilized.

(21) The following example focuses on the specific conjugate denoted .sup.18F-AIF-NOTA-AE105. Other conjugates within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein.

(22) The following chemistry applies to the Examples:

(23) TABLE-US-00002 AE105 (Asp-Cha-Phe-Ser-Arg-Tyr-Leu-Trp-Ser—OH) (1)

(24) The peptide according to the above mentioned sequence was synthesized by standard solid-phase peptide chemistry.

(25) TABLE-US-00003 NOTA-AE105 (NOTA-Asp-Cha-Phe-Ser-Arg-Tyr-Leu-Trp-Ser—OH)

(26) ##STR00004##

(27) The product is purified by RP-HPLC and analysed by RP-HPLC (retension time: 11.5 min, purity >98%) and electrospray-MS (1510.8 m.u.).

Example 1

(28) The aim of the present study was to synthesize a NOTA-conjugated peptide and use the Al.sup.18F method for development for the first .sup.18F-labeled PET ligand for uPAR PET imaging and to perform a biological evaluation in human prostate cancer xenograft tumors. To achieve this, the present inventors synthesized high-affinity uPAR binding peptide denoted AE105 and conjugated NOTA in the N-terminal. .sup.18F-labeling was done according to a recently optimized protocol.sup.26. The final product (.sup.18F-AIF-NOTA-AE105) was finally evaluated in vivo using both microPET imaging in human prostate tumor bearing animals and after collection of organs for biodistribution study.

(29) Chemical Reagents

(30) All chemicals obtained commercially were of analytical grade and used without further purification. No-carrier-added .sup.18F-fluoride was obtained from an in-house PETtrace cyclotron (GE Healthcare). Reverse-phase extraction C18 Sep-Pak cartridges were obtained from Waters (Milford, Mass., USA) and were pretreated with ethanol and water before use. The syringe filter and polyethersulfone membranes (pore size 0.22 μm, diameter 13 mm) were obtained from Nalge Nunc International (Rochester, N.Y., USA). The reverse-phase HPLC using a Vydac protein and peptide column (218TP510; 5 μm, 250×10 mm) was performed as previously described.sup.21.

(31) MicroPET scans were performed on a microPET R4 rodent model scanner (Siemens Medical Solutions USA, Inc., Knoxville, Tenn., USA). The scanner has a computer-controlled bed and 10.8-cm transaxial and 8-cm axial fields of view (FOVs). It has no septa and operates exclusively in the three-dimensional (3-D) list mode. Animals were placed near the center of the FOV of the scanner.

(32) Peptide Synthesis, Conjugation and Radiolabeling

(33) NOTA-conjugated AE105 (NOTA-Asp-Cha-Phe-(D)Ser-(D)Arg-Tyr-Leu-Trp-Ser-COOH) was purchased from ABX GmbH. The purity was characterized using HPLC analysis and the mass was confirmed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) (Se Suppl. FIG. 1A). The radiolabeling of NOTAAE105 with .sup.18F-AIF is shown in FIG. 1 and was done according to a recently published protocol with minor modifications.sup.26.

(34) In brief, a QMA Sep-Pak Light cartridge (Waters, Milford, Ma, USA) was fixed with approximately 3 GBq of 18F-fluoride and then washed with 2.5 ml of metal free water. Na18F was then eluted from the cartridge with 1 ml saline, from which 100 μl fraction was taken. Then amounts of 50μ1 0.1 M Na-Acetate buffer (pH=4), 3 μl 0.1 M AlCl.sub.3 and 100 μl of Na.sup.18F in 0.9% saline (300 MBq) were first reacted in a 1 ml centrifuge tube (sealed) at 100° c. for 15 min. The reaction mixture was cooled. 50μ1 ethanol and 30 nmol NOTA-AE105 in 3 μl DMSO were added and the reaction mixture were heated to 95° C. for 5 min. The crude mixture was purified with a semi-preparative HPLC. The fractions containing .sup.18F-AIF-NOTA-AE105 were collected and combined in a sterile vial. The product was diluted in phosphate-buffered saline (PBS, pH=7.4) so any organic solvents were below 5% (v/v) and used for in vivo studies.

(35) Cell Line and Animal Model

(36) Human prostate cancer cell line PC-3 was obtained from the American Type Culture Collection (Manassas, Va., USA) and culture media DMEM was obtained from Invitrogen Co. (Carlsbad, Calif., USA). The cell line was cultured in DMEM supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin/Streptomycin at 37° C. and 5% CO.sub.2. Xenografts of human PC-3 prostate cancer cells were established by injection of 200 μl cells (1×10.sup.8 cells/ml) suspended in 100 μl Matrigel (BO Biosciences, San Jose, Calif., USA), subcutaneously in the right flank of male nude mice obtained from Charles River Laboratory (Wilmington, Mass. USA), Tumors were allowed to grow to a size of 200-500 mg (3-4 weeks).

(37) MicroPET Imaging

(38) Three min static PET scans were acquired 0.5, 1.0 and 2.0 h post injection (p.i) of .sup.18FAIF-NOTA-AE105 via tail-vein injection of 2-3 MBq (n=4). Similar, the blocking study was performed by injection of the ligand together with 100 μg of AE152 (uPAR antagonist) through the tail vein (n=4) and PET scanned at the same time points. During each three minutes PET scan, mice were anesthetized with isoflurane (5% induction and 2% 30 maintenance in 100% 02). Images were reconstructed using a two-dimensional ordered subsets expectation maximization (OSEM-2D) algorithm. No background correction was performed. All results were analyzed using Inveon software (Siemens Medical Solutions) and PET data was expressed as percent of injected dose per gram tissue (% ID/g) based on manual region-of-interest drawing on PET images and the use of a calibration constant. An assumption of a tissue density of 1 g/ml was used. No attenuation correction was performed.

(39) Biodistribution Studies

(40) After the last PET scan, all PC-3 bearing mice were euthanized. Blood, tumor and major organs were collected (wet-weight) and the radioactivity was measured using a y-counter from Perkin Elmer, Mass., USA (N=4 mice/group).

(41) uPAR ELISA

(42) uPAR ELISA on resected PC-3 tumors was done as described previously in detail.sup.15. All results were performed as duplicate measurements.

(43) Statistical Analysis

(44) All quantitative data are expressed as mean±SEM (standard error of the mean) and means were compared using Student t-test. Correlation statistics was done using linear regression analysis. A P-value of s 0.05 were considered statistically significant.

(45) uPAR Binding Affinity

(46) The uPAR binding affinity of AE105 and AE152.sup.27 (used for blocking studies) was in this 20 study found to be 14.1 nM and 2.9 nM, respectively (FIG. 1A). A high uPAR binding affinity for AE105 with different chelators conjugated in the N-terminal, including the NOTA analogue NODAGA, has been confirmed in our previously studies.sup.15, 16, thus confirming the ability to make modifications in the N-terminal of AE105 without losing affinity towards human uPAR.sup.14, 27, 28.

(47) Radiochemistry

(48) The .sup.18F-labeling of NOTA-AE105 was synthesized based on a recently published procedure with some modification (FIG. 1B). During our labeling optimization, we found that 33% ethanol (v/v) was optimal using 30 nmol NOTA-AE105. We first formed the 18F-AIF complex in buffer at 100° C. for 15 min. Secondly was the NOTA-conjugated peptide added and incubated together with Ethanol at 95° C. for 5 min. By adding ethanol we were able to increase the overall yield to above 92.7% (FIG. 2C), whereas the yield without ethanol was only 30.4%, with otherwise same conditions. No further increase in the overall yield was observed using longer incubation time and/or different ethanol concentrations or using less than 30 nmol conjugated peptide. Two isomers were observed for .sup.18F-AIF-NOTA-AE105.

(49) In order to ensure the formation of the right product, a cold standard of the final product was synthesis (AIF-NOTA-AE105). The HPLC analysis of the precursor (NOTA-AE105, FIG. 2A) confirmed the purity of the NOTA-conjugated precursor (>97%) and MALDI-MS confirmed the mass (1511.7 Da) (See suppl. FIG. 1). The cold standard (AIFNOTA-AE105, FIG. 2B), with the right mass confirmed by MALDI-MS (1573.6 Da) (See suppl. FIG. 1B), corresponded well in regards to retention time with the ‘hot’ product (FIG. 2C), thus confirming the formation of 18F-AIF-NOTA-AE105 (FIG. 2C). No degradation of the final product was found after 30 min in PBS (FIG. 2D). The radioactive peaks were collected and diluted in PBS and used for in vivo studies. The specific activity in the final product was above 25 GBq/μmol.

(50) In Vivo PET Imaging

(51) .sup.18F-AIF-NOTA-AE105 was injected i.v. in four mice bearing PC-3 tumors and PET scan were performed 0.5, 1.0 and 2.0 h post injection (p.i). Tumor lesions were easily identified from the reconstructed PET images (FIG. 3A) and ROI analysis revealed a high tumor uptake, with 5.90±0.35% ID/g after 0.5 h, declining to 4.22±0.13% ID/g and 2.54±0.24% ID/g after 1.0 and 2.0 h, respectively (FIG. 3B).

(52) In order to ensure that the found tumor uptake did indeed reflect specific uPAR mediated uptake, four new PC-3 tumor bearing mice were then injected with a mixed solution containing .sup.18F-AIF-NOTA-AE105 and 100 μg of the high-affinitty uPAR binding peptide denoted AE152, in order to see if the tumor uptake could be inhibited. A significant lower amount of .sup.18F-AIF-NOTA-AE105 tumor uptake was found at all three time points investigate (FIG. 3B) and tumor lesions were not as easily identified in the PET images (FIG. 3A). At 1.0 h p.i a tumor uptake of 1.86±0.14% ID/g was found in the blocking group compared with 4.22±0.13% ID/g found in the group of mice receiving only .sup.18F-AIFNOTA-AE105 (p<0.001, 2.3 fold reduction).

(53) Biodistribution

(54) After the last PET scan, each group of mice where euthanized and selected organs and tissues were collected to investigate the biodistribution profile 2.5 h p.i. (FIG. 4). A significant higher tumor uptake in the group of mice receiving .sup.18F-AIF-NOTA-AE105 was found compared with blocking group (1.02±0.37% ID/g vs. 0.30±0.06% ID/g, p<0.05), thus confirming the specificity of .sup.18F-AIF-NOTA-AE105 for human uPAR found in the PET study. Highest activity was found in the kidneys for both groups of mice, confirming the kidneys to be the primarily route of excretion. Beside kidneys, the bone, well known to accumulate fluoride, also had a relatively high uptake of 3.54±0.32% ID/g and 2.34±0.33% ID/g for normal and blocking group, respectively.

(55) uPAR Expression

(56) Both the PC-3 cells used for tumor inoculation and all PC-3 tumors at the end of the study (n=8) were finally analyzed for confirming expression of human uPAR (FIG. 5). An expression in the cells of 6.53±1.6 ng/mg protein was found (FIG. 5A), whereas the expression level in the resected tumors was 302±129 pg/mg tumor tissue (FIG. 5B). A significant correlation between tumor uptake of 18F-AIF-NOTA-AE105 and uPAR expression was found (p<0.05, r=0.93) (FIG. 5C).

(57) Data Interpretation

(58) The above experiments provide evidence for the applicability of an 18F-labeled ligand for 15 uPAR PET. The ligand was characterized in a human prostate cancer xenograft mouse model. Based on the obtained results, similar tumor uptake, specificity and tumor-to-background contrast were found compared to our previously published studies using 64Cu- and 68Ga-based ligands for PET.sup.15, 16. Based on the superior physical characteristics of 18F and the high tumor-to-background contrast found already after 1 h p.i, our new 18F-based ligand must be considered the so far most promising uPAR PET candidate for translation into clinical use in order to non-invasively characterize invasive potential of e.g. prostate cancer.

(59) .sup.18F-labeling of peptides using the AIF-approach has previously been described to be performed at 100° c. for 15 min, at pH=4.sup.17-20. This protocol was modified, since degradation of the NOTA-conjugated peptide was observed using these conditions. The present inventors therefore first produced the .sup.18F-AIF complex using the above mentioned conditions and next added the NOTA-conjugated peptide and lowered the temperature to 95° C., and within 5 min obtained a labeling yield of 92.7% and with no degradation of the peptide. Two isomers of .sup.18F-AIF-NOTA-AE105 were produced. Same observations have been reported by others for .sup.18F-AIF-NOTA-Octreotide.sup.18 and all NOTA-conjugated IMP peptide analogues described.sup.19. The ratio of the two peaks were nearly constant for each labeling and both radioactive peaks were collected and used for further in vivo studies. This approach was recently also described by others.sup.26.

(60) Besides optimizing the temperature and time, the present inventors found that the addition of ethanol, to a final concentration of 33% (v/v), resulted in a significant higher labeling yield, compared with radiolabeling without ethanol (30.4% vs 92.7%), using the same amount of NOTA-conjugated peptide. Same observations have recently been described by others.sup.26. Here the effect of lowering the ionic strength was investigated using both acetonitrile, ethanol, dimethylforamide (DMF) and tetrahydrofuran (THF) at different concentrations. A labeling yield of 97% was reported using ethanol at a concentration of 80% (v/v). However, they used between 76-383 nmol NOTA-conjugated peptide, whereas in this study only used 30 nmol was used. The amount needed for optimal labeling yield therefore seems to be dependent on the peptide and on the amount of peptide used for labeling.

(61) The tumor uptake of .sup.18F-AIF-NOTA-AE105 was similarly compared with previously published results pertaining to .sup.64Cu-based ligands.sup.15. The tumor uptake 1 h p.i was 4.79±0.7% ID/g, 3.48±0.8% ID/g and 4.75±0.9% ID/g for .sup.64Cu-DOTA-AE105, .sup.64Cu-CB-TE2A-AE105, .sup.64Cu-CB-TE2A-PA-AE105 compared to 4.22±0.1% ID/g for .sup.18F-AIF-NOTA-AE105. However, all .sup.64Cu-based ligands were investigated using the human glioblastoma cell line U87MG, whereas in this study, the prostate cancer cell line PC-3 was used. Considering that the data show that the level of uPAR in the two tumor types is not similar, with PC-3 having around 300 pg uPAR/mg tumor tissue (FIG. 5B) and U87MG having approximately 1,700 pg/mg tumor tissue (unpublished), the tumor uptake of .sup.18F-AIF-NOTA-AE105 seems to be relatively higher per pg uPAR, However, a direct comparison between the two independent studies is difficult, considering the different cancer cell line used. However, the present inventors have previously shown a significant correlation between uPAR expression and tumor uptake across three tumor types.sup.15, which is confirmed in the present study using PC-3 xenografts (FIG. 5C), further validating the ability of .sup.18F-AIF-NOTA-AE105 to quantify uPAR expression using PET imaging. The uPAR specific binding of .sup.18F-AIF-NOTA-AE105 in the present study was confirmed by a 2.3-fold reduction in tumor uptake of .sup.18F-AIF-NOTA-AE105 1 h p.i. when co-administration of an uPAR antagonist (AE152) was performed for blocking study.

(62) The biodistribution study of 18F-AIF-NOTA-AE105 confirmed the kidneys to be the primary route of excretion and the organ with highest level of activity (FIG. 4). Same excretion profiles have been found for .sup.68Ga-DOTA/NODAGA-AE105.sup.16, .sup.177Lu-DOTAAE105.sup.30. Besides the kidneys and tumor, the bone also had a relatively high accumulation of activity. Bone uptake following injection of 18F-based ligands is a well-described phenomenon and used clinically in NaF bone scans.sup.31. A bone uptake of 3.54% ID/g 2.5 h p.i was found, which is similar to the bone uptake following .sup.18F-FDG injection in mice, where 2.49% ID/g have been reported 1.5 h p.i..sup.17.

(63) The development of the first .sup.18F-based ligand for uPAR PET provides of number of advantages compared to previously published .sup.64Cu-based uPAR PET ligands. Considering the optimal tumor-to-background contrast as early as 1 h p.i. as found in this study and in previously studies using 64Cu, the relatively shorter half-life of .sup.18F (T.sub.1/2=1.83 h) compared with .sup.64Cu (T.sub.1/2=12.7 h) seems to be optimal consider the much lower radiation burden to future patients using .sup.18F-AIF-NOTA-AE105. Moreover, is the production of .sup.18F well established in a number of institutions worldwide, whereas the production of .sup.64Cu still is limited to relatively few places.

Example 2

[.SUP.64.Cu]NOTA-AE105 (NOTA-Asp-Cha-Phe-Ser-Arg-Tyr-Leu-Trp-Ser-OH)

(64) .sup.64CuCl2 dissolved in 50 ul metal-free water was added to a solution containing 10 nmol NOTA-AE105 and 2.5 mg gentisic acid dissolved in 500 ul 0.1 M NH4OAc buffer (pH 5.5) and left at room temperature for 10 minutes resulting in 375 MBq [64Cu]NOTA-AE105 20 with a radiochemical purity above 99%. The radiochemical purity decreased to 94% after 48 hours storage.

Example 3

In Vivo uPAR PET Imaging with [.SUP.64.Cu]NOTA-AE105 in a Orthotropic Human Glioblastoma Mouse Model

(65) A mouse was inoculated with human derived glioblastoma cells in the brain. 3 weeks later a small tumor was visible using microCT scan A microPET images was recorded 1 hr post i.v. injection of approximately 5 MBq [.sup.64Cu]NOTA-AE105. Uptake in the tumor and background brain tissue was quantified. Moreover, was a control mouse (with no tumor inoculated) also PET scanned using the same procedure, to investigate the uptake in normal brain tissue with intact blood brain barrier. See FIG. 6.

Example 4

[68Ga]NOTA-AE105 (NOTA-Asp-Cha-Phe-Ser-Arg-Tyr-Leu-Trp-Ser-OH)

(66) A 1 ml fraction of the eluate form a .sup.68Ge/68Ga generator for added to a solution containing 20 nmol NOTA-AE105 dissolved in 1000 ul 0.7M NaOAc buffer (pH 3.75) and heated to 60° C. for 10 minutes. The corresponding mixture could be purified on a C18 SepPak column resulting in 534MBq [.sup.68Ga]NOTA-AE105 with a radiochemical purity above 98%.

Example 5

In Vivo uPAR PET Imaging with [.SUP.68.Ga]NOTA-AE105 in a Orthotropic Human Glioblastoma Mouse Model

(67) A mouse was inoculated with human derived glioblastoma cells in the brain. 3 weeks 15 later a small tumor was visible using microCT scan A microPET images was recorded 1 hr post i.v. injection of approximately 5 MBq [68Ga]NOTA-AE105. Uptake in the tumor and background brain tissue was quantified. Moreover, was a control mouse (with no tumor inoculated) also PET scanned using the same procedure, to investigate the uptake in normal brain tissue with intact blood brain barrier. See FIG. 7.

REFERENCES

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