gastrins and 1-4-7-10-tetraazacyclododecane--1-4-7-10-tetraacetic-acid

Topics: Amino Acid Sequence; Animals; Cell Line, Tumor; Drug Stability; Female; Gastrins; Heterocyclic Compounds, 1-Ring; Humans; Indium Radioisotopes; Lutetium; Mice, Inbred BALB C; Proline; Protein Binding; Radioisotopes; Radiopharmaceuticals; Rats; Receptor, Cholecystokinin B

The metabolic instability and high kidney retention of minigastrin (MG) analogues hamper their suitability for use in peptide-receptor radionuclide therapy of CCK2/gastrin receptor-expressing tumors. High kidney retention has been related to N-terminal glutamic acids and can be substantially reduced by coinjection of polyglutamic acids or gelofusine. The aim of the present study was to investigate the influence of the stereochemistry of the N-terminal amino acid spacer on the enzymatic stability and pharmacokinetics of (111)In-DOTA-(d-Glu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 ((111)In-PP11-D) and (111)In-DOTA-(l-Glu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 ((111)In-PP11-L). Using circular dichroism measurements, we demonstrate the important role of secondary structure on the pharmacokinetics of the two MG analogues. The higher in vitro serum stability together with the improved tumor-to-kidney ratio of the (d-Glu)6 congener indicates that this MG analogue might be a good candidate for further clinical study.

Topics: Amino Acid Sequence; Animals; Gastrins; Heterocyclic Compounds, 1-Ring; Humans; Indium Radioisotopes; Male; Neoplasms; Positron-Emission Tomography; Rats, Inbred Lew; Receptor, Cholecystokinin B

Radiolabeled gastrin analogs represent attractive candidates for diagnosis and therapy of cholecystokinin subtype-2 receptor (CCK2R)-expressing tumors. Radiolabeled des(Glu)5-gastrins show favorably low renal accumulation, but localize poorly in CCK2R-positive lesions. We introduce herein three truncated [DOTA-DGlu(10)]gastrin(10-17) analogs, with oxidation-susceptible Met(15) replaced by: (1), (2), or (3), and study the profile of [(111)In]1/2/3 during in vivo inhibition of neutral endopeptidase (NEP) in comparison to the non-truncated [ ([(111)In]4) reference.. Blood samples collected from mice 5 min postinjection (pi) of [(111)In]1/2/3/4 without or with phosphoramidon (PA) coinjection were analyzed by RP-HPLC. Biodistribution was conducted in SCID mice bearing A431-CCK2R(+) or AR42J xenografts 4h after administration of [(111)In]1/2/3/4 without or with PA coinjection.. Firstly, we observed remarkable increases in the amount of radiopeptides detected intact in the blood of PA-treated mice at 5 min pi compared to controls. Secondly, we noted impressive enhancement of [(111)In]1/2/3 localization in AR42J and A431-CCK2R(+) tumors in mice after PA coinjection. Specifically, the uptake of [(111)In]1 at 4h pi increased from 2.6 ± 0.3%ID/g to 13.3 ± 3.5%ID/g in the AR42J tumors and from 4.3 ± 0.6%ID/g to 20.4 ± 3.6%ID/g in the A431-CCK2R(+) xenografts, with comparable improvements noted for [(111)In]2 and [(111)In]3 as well. Thirdly, renal uptake remained favorably low and unaffected by PA (<2.5%ID/g). Conversely, although the stability and tumor targeting of [(111)In]4 improved, its high renal uptake (>85%ID/g) increased even further by PA (>140%ID/g).. In situ inhibition of NEP represents a promising new tool to enhance the diagnostic efficacy of biodegradable gastrin radioligands in the visualization of CCK2R-positive lesions in man.

Topics: Amino Acid Sequence; Animals; Cell Line, Tumor; Gastrins; Heterocyclic Compounds, 1-Ring; Humans; Indium Radioisotopes; Ligands; Male; Mice; Molecular Sequence Data; Neprilysin; Protease Inhibitors; Radiochemistry; Rats; Receptor, Cholecystokinin B; Tissue Distribution

The minigastrin analogue - CP04: DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 has been developed for CCK2R targeting. This analogue can be radiolabelled with 111In or 68Ga for imaging, or with 90Y and 177Lu for therapy. However, affinity of the chelator-peptide conjugates to the cell membrane receptors may vary depending on the metal incorporated into the complex. So far, there are no such studies for the ligands of gastrin/cholecystokinin receptor CCK2R. It is supposed that the reason for the differentiation of receptor affinity to the respective receptors is in the changes of structure of chelating system and their influence on the bioactive conformations of the metal conjugated peptides. Herein, we report on the radiolabeling of CP04 with 90Y, 177Lu and 68Ga and synthesis of cold CP04 complexes with respective stable metals for further structural and physico-chemical and biological studies.. From 200 to 600 MBq of 90Y, 177Lu or 68Ga were used for radiolabelling of 20 μg of CP04 dissolved in ascorbic acid solution (50 mg/mL, pH 4.5). Non-radioactive complexes with Lu and Ga were synthesized in milligram amounts starting from 0.5 mg up to 5 mg of CP04 dissolved in ascorbic acid solution (50 mg/mL, pH 4.5) when using 2-molar excess of the metal ions. Complex formation needed 5 min in microwave oven or 12 min in thermo-block at 95°C. RP-HPLC isocratic method (Kinetex 150/4.6 mm; 25% AcN/0.1% TFA, 1 mL/min) with UV/Vis and radiometric detection was developed for investigation of the radiolabelled and "cold" complexes. For LC-MS investigations, HPLC method was modified replacing TFA by formic acid.. Yields of CP04 radiolabelling were greater than 90% for all three radionuclides. The HPLC method enabled identification of these radio-complexes based on comparison to their non-radioactive equivalents. In all cases, chromatograms revealed peaks that could be attributed to the metal-CP04 complexes and to impurities (including methionine oxidation). LC-MS analysis of Ga and Lu complexes revealed conformity of the observed molecular ions to the predicted formulas (m/z 2116 and 2220 Da for Ga and Lu, respectively). Different chromatographic behaviour observed for Ga-CP04 complex comparing to Lu- and Y- labelled peptide (relative retention to CP04: 1.08, 0.86 and 0.85, respectively) suggest different coordination of the metal ions. Therefore, further studies are planned using the non-radioactive complexes in order to assess their structural conformations.

Topics: Chemical Phenomena; Chemistry Techniques, Synthetic; Drug Stability; Gallium Radioisotopes; Gastrins; Heterocyclic Compounds, 1-Ring; Isotope Labeling; Lutetium; Yttrium Radioisotopes

The means of identifying prostate carcinoma and its metastases are limited. The contrast agents used in magnetic resonance imaging clinical diagnostics are not taken up into the tumor cells, but only accumulate in the interstitial space of the highly vasculated tumor. We examined the gastrin/cholecystokinin-B receptor as a possible target for prostate-specific detection using the C-terminal seven amino acid sequence of the gastrin peptide hormone. The correct sequence and a scrambled control sequence were coupled to the fluorescent dye rhodamine and the magnetic resonance imaging contrast agent gadolinium (Gd)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Expression analysis of the gastrin receptor mRNA was performed by reverse transcriptase polymerase chain reaction on PC3 prostate carcinoma cells, U373 glioma, U2OS osteosarcoma and Colo205 colon carcinoma cells. After having confirmed elevated expression of gastrin receptor in PC3 cells and very low expression of the receptor in Colo205 cells, these two cell lines were used to create tumor xenografts on nude mice for in vivo experiments. Confocal lasers scanning microscopy and magnetic resonance imaging showed a high specificity of the correct conjugate for the PC3 xenografts. Staining of the PC3 xenografts was much weaker with the scrambled conjugate while the Colo205 xenografts showed no marked staining with any of the conjugates. In vitro experiments comparing the correct and scrambled conjugates on PC3 cells by magnetic resonance relaxometry and fluorescence-activated cell sorting confirmed markedly higher specificity of the correct conjugate. The investigations show that the gastrin receptor is a promising tumor cell surface target for future prostate-cancer-specific imaging applications.

Topics: Animals; Cell Line, Tumor; Contrast Media; Fluorescent Dyes; Gadolinium; Gastrins; Heterocyclic Compounds, 1-Ring; Humans; Magnetic Resonance Imaging; Male; Mice; Mice, Nude; Oligopeptides; Prostate; Prostatic Neoplasms; Receptor, Cholecystokinin B; Rhodamines

Cholecystokinin-2 (CCK-2) receptors, overexpressed in cancer types such as small cell lung cancers (SCLC) and medullary thyroid carcinomas (MTC), may serve as targets for peptide receptor radionuclide imaging. A variety of CCK and gastrin analogues has been developed, but a major drawback is metabolic instability or high kidney uptake. The minigastrin analogue PP-F11 has previously been shown to be a promising peptide for imaging of CCK-2 receptor positive tumors and was therefore further evaluated. The peptide was conjugated with one of the macrocyclic chelators DOTA, NOTA, or NODAGA. The peptide conjugates were then radiolabeled with either (68)Ga, (64)Cu, or (111)In. All (radio)labeled compounds were evaluated in vitro (IC50) and in vivo (biodistribution and PET/CT and SPECT/CT imaging). IC50 values were in the low nanomolar range for all compounds (0.79-1.51 nM). In the biodistribution studies, (68)Ga- and (111)In-labeled peptides showed higher tumor-to-background ratios than the (64)Cu-labeled compounds. All tested radiolabeled compounds clearly visualized the CCK2 receptor positive tumor in PET or SPECT imaging. The chelator did not seem to affect in vivo behavior of the peptide for (111)In- and (68)Ga-labeled peptides. In contrast, the biodistribution of the (64)Cu-labeled peptides showed high uptake in the liver and in other organs, most likely caused by high blood levels, probably due to dissociation of (64)Cu from the chelator and subsequent transchelation to proteins. Based on the present study, (68)Ga-DOTA-PP-F11 might be a promising radiopharmaceutical for PET/CT imaging of CCK2 receptor expressing tumors such as MTC and SCLC. Clinical studies are warranted to investigate the potential of this tracer.

Topics: Acetates; Animals; Cell Line, Tumor; Chelating Agents; Copper Radioisotopes; Female; Gallium Radioisotopes; Gastrins; Heterocyclic Compounds; Heterocyclic Compounds, 1-Ring; Humans; Indium Radioisotopes; Inhibitory Concentration 50; Mice; Mice, SCID; Multimodal Imaging; Neoplasm Transplantation; Peptides; Positron-Emission Tomography; Radiopharmaceuticals; Receptor, Cholecystokinin B; Tomography, Emission-Computed, Single-Photon; Tomography, X-Ray Computed

Targeting of cholecystokinin receptor expressing malignancies such as medullary thyroid carcinoma is currently limited by low in vivo stability of radioligands. To increase the stability, we have developed and preclinically evaluated two cyclic 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-minigastrin analogs radiolabeled with (111)In and (68)Ga.. Radiolabeling efficiency, in vitro characterization, cholecystokinin receptor subtype 2 (CCK-2) binding in human tumor tissues, and cell internalization on CCK-2 receptor expressing AR42J cells, as well as biodistribution and small animal imaging in two different mouse xenograft models were studied.. High receptor affinity and receptor-mediated uptake of the radioligands in AR42J cells was confirmed in vitro. (111)In-labeled cyclic DOTA-peptides showed a specific tumor uptake of ~1% ID/g in vivo, (68)Ga-labeled analogs of ~3% ID/g. Small animal SPECT imaging resulted to be superior with (111)In-DOTA-cyclo-MG2 in comparison with (111)In-DOTA-cyclo-MG1.. Cyclic DOTA-minigastrin analogs are promising candidates for gastrin receptor scintigraphy and targeted radionuclide therapy.

Topics: Animals; Carcinoma, Neuroendocrine; Cell Line, Tumor; Drug Evaluation, Preclinical; Female; Gallium Radioisotopes; Gastrins; Heterocyclic Compounds, 1-Ring; Indium Radioisotopes; Mice; Mice, Inbred BALB C; Mice, Nude; Mice, SCID; Receptors, Cholecystokinin; Thyroid Neoplasms; Tomography, Emission-Computed, Single-Photon

Lack of correlation between in vitro and in vivo stability is a general problem for the development of radiopeptides especially in the case of minigastrin derivatives for therapeutic applications. In this study, we compared the influence of experimental conditions on radiopeptide stability results in vitro using a model Minigastrin (MG) analogue labelled with Lu-177. Additionally, we attempted to characterize the main serum enzymatic cleavage sites by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) analysis.. In vitro stability of a DOTA-minigastrin derivative ((177)Lu-DOTA-His-His-Glu-Ala-Tyr-Gly-Trp-NIe-Asp-Phe-NH(2)) was tested in serum, rat tissue homogenates and two different standardised enzymatic mixtures. Quantification of the metabolised radiopeptides at different time intervals was performed using reversed-phase high-performance liquid chromatography (RP-HPLC). Metabolites were characterised by MALDI-TOF-MS. Urine was collected after 15 min p.i. into the mice and compared with in vitro metabolites by RP-HPLC.. Faster degradation of the radiopeptide was found in blood in comparison with plasma and serum incubation and in components from rats faster than from human origin. Fast degradation was observed in kidney and liver homogenates as well as in standardised enzymatic mixtures, also revealing variations in the metabolic profile. In urine, no intact peptide was detected already 5 min post injection. MALDI-TOF-MS revealed major cleavage sites at the carboxy terminus of the peptide.. Very variable results may be found when different kind of incubation media for testing radiopeptide stabilities is used. Serum incubation studies may overestimate stability; therefore, results should be interpreted with care and combined with alternative in vitro and in vivo investigations.

Topics: Amino Acid Sequence; Animals; Binding Sites; Biological Assay; Chromatography, High Pressure Liquid; Chromatography, Reverse-Phase; Female; Gastrins; Heterocyclic Compounds, 1-Ring; Humans; Isotope Labeling; Lutetium; Mice; Mice, Inbred BALB C; Peptide Hydrolases; Protein Stability; Radioisotopes; Rats; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tissue Extracts

Gastrin/cholecystokinin subtype 2 receptors (CCK-2Rs) are overexpressed in several tumor types and are, thus, a potential target for peptide receptor radionuclide therapy (PRRT) of cancer. To improve the in vivo performance of CCK-2R binding peptides, we have previously synthesized and screened a series of divalent gastrin peptides for improved biochemical and biologic characteristics. In this study, we explore in more detail the most promising of these compounds and compare its performance with a previously described monomeric peptide.. From six (111)In-labeled 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-conjugated divalent gastrin peptides based on the C-terminal sequence of minigastrin, the maleimide-linked compound DOTA-GSC(succinimidopropionyl-EAYGWNleDF-NH(2))-EAYGWNleDF-NH(2) (MGD5) was selected. The in vitro stability, receptor binding, and internalization of (111)In-MGD5 were studied and compared with those of monomer compound (111)In-APH070. In vivo biodistribution and imaging using a SPECT/CT camera were also performed.. More than 90% of the labeled divalent peptide remained intact after 20 h of incubation in plasma. The inhibitory concentration of 50% of the divalent peptide was 1.0 versus 5.6 nM for the monomer, and the dissociation constant was 0.7 versus 2.9 nM. The rate of internalization of the divalent peptide was twice that of the monomer. Tumor uptake of the divalent peptide in vivo was about 6 times that of the monomer. The rate of washout of the divalent peptide from the tumor was lower than that of the monomer.. Dimerization of the CCK-2R binding site results in an increase in binding affinity and an increase in tumor uptake both in vitro and in vivo. It is likely that these increases would result in improved tumor-targeting efficiency in patients with CCK-2R-positive tumors.

Topics: Amino Acid Sequence; Animals; Cell Line, Tumor; Female; Gastrins; Gene Expression Regulation, Neoplastic; Heterocyclic Compounds, 1-Ring; Isotope Labeling; Mice; Neoplasms; Protein Stability; Protein Transport; Rats; Receptor, Cholecystokinin B; Tissue Distribution; Tomography, Emission-Computed, Single-Photon; Tomography, X-Ray Computed