Exploiting the Concept of Multivalency with 68Ga- and 89Zr-Labelled Fusarinine C-Minigastrin Bioconjugates for Targeting CCK2R Expression.

Cholecystokinin-2 receptors (CCK2R) are overexpressed in a variety of malignant diseases and therefore have gained certain attention for peptide receptor radionuclide imaging. Among extensive approaches to improve pharmacokinetics and metabolic stability of minigastrin (MG) based radioligands, the concept of multivalency for enhanced tumour targeting has not been investigated extensively. We therefore utilized fusarinine C (FSC) as chelating scaffold for novel mono-, di-, and trimeric bioconjugates for targeting CCK2R expression. FSC-based imaging probes were radiolabelled with positron emitting radionuclides (gallium-68 and zirconium-89) and characterized in vitro (log⁡D, IC50, and cell uptake) and in vivo (metabolic stability in BALB/c mice, biodistribution profile, and microPET/CT imaging in A431-CCK2R/A431-mock tumour xenografted BALB/c nude mice). Improved targeting did not fully correlate with the grade of multimerization. The divalent probe showed higher receptor affinity and increased CCK2R mediated cell uptake while the trimer remained comparable to the monomer. In vivo biodistribution studies 1 h after administration of the 68Ga-labelled radioligands confirmed this trend, but imaging at late time point (24 h) with 89Zr-labelled counterparts showed a clearly enhanced imaging contrast of the trimeric probe compared to the mono- and dimer. Furthermore, in vivo stability studies showed a higher metabolic stability for multimeric probes compared to the monomeric bioconjugate. In summary, we could show that FSC can be utilized as suitable scaffold for novel mono- and multivalent imaging probes for CCK2R-related malignancies with partly improved targeting properties for multivalent conjugates. The increased tumour accumulation of the trimer 24 h postinjection (p.i.) can be explained by slower clearance and increased metabolic stability of multimeric conjugates.

Medullary thyroid carcinoma - PET/CT imaging with 68Ga-labelled gastrin and somatostatin analogues.

CASE PRESENTATION: a 75-year-old man with a 10-year history of nodular goitre was referred for clinical evaluation. The ultrasound scan revealed enlarged thyroid right lobe almost fully filled with a heterogeneous nodule with numerous calcifications. Fine-needle aspiration biopsy suggested medullary thyroid carcinoma (MTC). Before the surgery the patient was referred to the nuclear medicine department and somatostatin receptor imaging (SRS; 68Ga-DOTATATE) with PET/CT was performed. The scan demonstrated an increased uptake within the right thyroid mass. Subsequent PET/CT with 68Ga-gastrin analogue (MG48) revealed the same indications as the SRS: an increased alveolar uptake in the right thyroid mass without the signs of lymph node metastases. The patient underwent total thyroidectomy and central lymph nodes dissection. Histopathology examination confirmed the presence of MTC with vascular invasion, but without lymph node metastases (pT3NoMx according to the 7th edition of the AJCC Cancer Staging Manual). Immunohistochemical staining revealed positive reaction to calcitonin and CD56, whereas the reaction to thyroglobulin remained negative. The Ki-67 was 1%. Staining for SSTR2 and CCK2 showed high cytoplasmic expression in both cases. Knowledge of the presence of CCK2 receptor in MTC patients may be an important indication for the choice of diagnostic and therapeutic procedures. The presence of both the receptor types, cholecystokinin-2/gastrin and somatostatin, is possibly an interesting combination as far as the therapeutic target is concerned.

Cholecystokinin receptor antagonist halts progression of pancreatic cancer precursor lesions and fibrosis in mice.

OBJECTIVES: Exogenous administration of cholecystokinin (CCK) induces hypertrophy and hyperplasia of the pancreas with an increase in DNA content. We hypothesized that endogenous CCK is involved in the malignant progression of pancreatic intraepithelial neoplasia (PanIN) lesions and the fibrosis associated with pancreatic cancer. METHODS: The presence of CCK receptors in early PanIN lesions was examined by immunohistochemistry in mouse and human pancreas. Pdx1-Cre/LSL-Kras transgenic mice were randomized to receive either untreated drinking water or water supplemented with a CCK receptor antagonist (proglumide, 0.1 mg/mL). Pancreas from the mice were removed and examined histologically for number and grade of PanINs after 1, 2, or 4 months of antagonist therapy. RESULTS: Both CCK-A and CCK-B receptors were identified in early stage PanINs from mouse and human pancreas. The grade of PanIN lesions was reversed, and progression to advanced lesions arrested in mice treated with proglumide compared with the controls (P = 0.004). Furthermore, pancreatic fibrosis was significantly reduced in antagonist-treated animals compared with vehicle (P < 0.001). CONCLUSIONS: These findings demonstrate that endogenous CCK is in part responsible for the development and progression of pancreatic cancer. The use of CCK receptor antagonists may have a role in cancer prophylaxis in high-risk subjects and may reduce fibrosis in the microenvironment.

Characterization of gastrins and their receptor in solid human gastric adenocarcinomas.

OBJECTIVE: The gastrin and the gastrin/CCK-B receptor genes are co-expressed in several carcinomas. The primary translational product, progastrin, however, is processed to several peptides of which only those that are α-amidated at their C-terminus are receptor ligands. So far, characterization of the progastrin-derived peptides in gastric cancer has not been reported. The authors therefore examined the molecular nature of gastrin and its receptor in human gastric carcinomas. MATERIALS AND METHODS: Twenty patients with adenocarcinoma underwent partial or total gastrectomy. In samples from each carcinoma, gastrin peptides were characterized, using a library of sequence-specific immunoassays. Expression was also demonstrated by immunohistochemistry. In addition, the gastrin and gastrin/CCK-B receptor gene expression was quantitated using real-time PCR, and the receptor protein demonstrated by western blotting. RESULTS: α-Amidated gastrins were detectable in 16 of 20 carcinomas (median concentration 2.1 pmol/g tissue; range 0-386 pmol/g tissue). The tissue concentrations correlated closely to the gastrin mRNA contents (r = 0.75, p < 0.0001). Moreover, progastrin and non-amidated processing intermediates, including glycine-extended gastrins, were detected in 19 carcinomas. Immunohistochemistry corroborated gastrin expression in carcinoma cells. Chromatography revealed extensive progastrin processing with α-amidated gastrin-34 and -17 (tyrosyl-sulfated as well as non-sulfated) as major products. Finally, gastrin/CCK-B receptor mRNA and protein were detected in all tumors. CONCLUSIONS: The results show that the elements for a local loop of α-amidated gastrins and their receptor are detectable in 80% of human gastric adenocarcinomas. Therefore, the results support the contention that locally expressed gastrin may be involved in the tumorigenesis of gastric adenocarcinomas.

In vitro and in vivo characterization of three 68Ga- and 111In-labeled peptides for cholecystokinin receptor imaging.

Cholecystokinin (CCK) receptors are overexpressed in several human tumor types, such as medullary thyroid carcinomas and small cell lung cancers. Several ligands for the CCK2 receptor (CCK2R) have been developed for radionuclide targeting of these tumors. In this study, we evaluated whether radiolabeled DOTA-sCCK8 and its stabilized derivative, DOTA-sCCK8[Phe(2)(p-CH2SO3H), Nle(3,6)], are suitable for imaging of CCK2R-positive tumors, using DOTA-MG0 as a reference. In vivo targeting of CCK2R-positive tumors with DOTA-sCCK8, DOTA-sCCK8[Phe(2)(p-CH2SO3H), Nle(3,6)], and DOTA-MG0, labeled with (111)In or (68)Ga, was evaluated in BALB/c nude mice with a subcutaneous A431-CCK2R tumor. Biodistribution studies and single-photon emission computed tomography (SPECT) and positron emission tomography (PET) were performed at 1 hour postinjection. All peptides specifically accreted in the CCK2R-expressing tumors. Both (111)In-DOTA-sCCK8 and (111)In-DOTA-sCCK8[Phe(2)(p-CH2SO3H), Nle(3,6)] showed good tumor retention (4.65% ID/g and 5.44% ID/g, respectively, at 4 hours postinjection). On PET/computed tomographic (CT) and SPECT/CT scans, subcutaneous A431-CCK2R tumors were clearly visualized with low uptake of sCCK8 peptides in the intestines. Whereas radiolabeled DOTA-MG0 showed high kidney uptake (70% ID/g), the sCCK8 peptides showed low uptake in the kidneys. Sulfated CCK8 analogues combined high tumor uptake with low retention in the kidney and are therefore promising tracers for imaging of CCK2R-positive tumors.

Cell-specific targeting by heterobivalent ligands.

Current cancer therapies exploit either differential metabolism or targeting to specific individual gene products that are overexpressed in aberrant cells. The work described herein proposes an alternative approach--to specifically target combinations of cell-surface receptors using heteromultivalent ligands ("receptor combination approach"). As a proof-of-concept that functionally unrelated receptors can be noncovalently cross-linked with high avidity and specificity, a series of heterobivalent ligands (htBVLs) were constructed from analogues of the melanocortin peptide ligand ([Nle(4), dPhe(7)]-α-MSH) and the cholecystokinin peptide ligand (CCK-8). Binding of these ligands to cells expressing the human Melanocortin-4 receptor and the Cholecystokinin-2 receptor was analyzed. The MSH(7) and CCK(6) were tethered with linkers of varying rigidity and length, constructed from natural and/or synthetic building blocks. Modeling data suggest that a linker length of 20-50 Å is needed to simultaneously bind these two different G-protein coupled receptors (GPCRs). These ligands exhibited up to 24-fold enhancement in binding affinity to cells that expressed both (bivalent binding), compared to cells with only one (monovalent binding) of the cognate receptors. The htBVLs had up to 50-fold higher affinity than that of a monomeric CCK ligand, i.e., Ac-CCK(6)-NH(2). Cell-surface targeting of these two cell types with labeled heteromultivalent ligand demonstrated high avidity and specificity, thereby validating the receptor combination approach. This ability to noncovalently cross-link heterologous receptors and target individual cells using a receptor combination approach opens up new possibilities for specific cell targeting in vivo for therapy or imaging.

Stabilized (111)in-labeled sCCK8 analogues for targeting CCK2-receptor positive tumors: synthesis and evaluation.

Radiolabeled cholecystokinin-8 (CCK8) peptide analogues can be used for peptide receptor radionuclide imaging and therapy for tumors expressing CCK2/gastrin receptors. Earlier findings indicated that sulfated CCK8 (sCCK8, Asp-Tyr(OSO(3)H)-Met-Gly-Trp-Met-Asp-Phe-NH(2)) may have better characteristics for peptide receptor radionuclide therapy (PRRT) than gastrin analogues. However, sCCK8 contains an easily hydrolyzable sulfated tyrosine residue and two methionine residues which are prone to oxidation. Here, we describe the synthesis of stabilized sCCK8 analogues, resistant to hydrolysis and oxidation. Hydrolytic stability was achieved by replacement of the Tyr(OSO(3)H) moiety by a robust isosteric sulfonate, Phe(p-CH(2)SO(3)H). Replacement of methionine by norleucine (Nle) or homopropargylglycine (HPG) avoided undesired oxidation side-reactions. The phenylalanine analogue Phe(p-CH(2)SO(3)H) of l-tyrosine, synthesized by a modification of known synthetic routes, was incorporated in three peptides: sCCK8[Phe(2)(p-CH(2)SO(3)H),Met(3,6)], sCCK8[Phe(2)(p-CH(2)SO(3)H),Nle(3,6)], and sCCK8[Phe(2)(p-CH(2)SO(3)H),HPG(3,6)]. All peptides were N-terminally conjugated with the macrocyclic chelator DOTA (1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) and radiolabeled with In-111. In vitro binding assays on CCK2R-expressing HEK293 cells revealed that all three peptides showed specific binding and receptor-mediated internalization, with binding affinity values (IC(50)) in the nanomolar range. In vitro oxidation studies demonstrated that peptides with Nle or HPG indeed were resistant to oxidation. In vivo targeting studies in mice with AR42J tumors showed that tumor uptake was highest for (111)In-DOTA-sCCK8 and (111)In-DOTA-sCCK8[Phe(2)(p-CH(2)SO(3)H),Nle(3,6)] (4.78 +/- 0.64 and 4.54 +/- 1.15%ID/g, respectively, 2 h p.i.). The peptide with the methionine residues replaced by norleucine ((111)In-DOTA-sCCK8[Phe(2)(p-CH(2)SO(3)H), Nle(3,6)]) showed promising in vivo characteristics and will be further investigated for radionuclide imaging and therapy of CCK2R-expressing tumors.

Cyclic minigastrin analogues for gastrin receptor scintigraphy with technetium-99m: preclinical evaluation.

Two cyclized minigastrin analogues for gastrin receptor scintigraphy were synthesized and derivatized with HYNIC at the N-terminus for labeling with 99mTc. Radiolabeling efficiency, stability, cell internalization, and receptor binding on CCK-2 receptor expressing AR42J cells were studied and the biodistribution evaluated in tumor bearing nude mice, including NanoSPECT/CT imaging. Metabolites in urine, liver, and kidneys were analyzed by radio-HPLC. Radiolabeled cyclic MG showed high stability in vitro and receptor mediated uptake in AR42J cells. In the animal tumor model, fast renal clearance and low nonspecific uptake in most organs were observed. A tumor uptake >3% was calculated ex vivo 1 h p.i. for both 99mTc-EDDA-HYNIC-cyclo-MG1 and 99mTc-EDDA-HYNIC-cyclo-MG2. In an imaging study with 99mTc-EDDA-HYNIC-cyclo-MG1, the tumor was clearly visualized. The metabolite analysis indicated rapid enzymatic degradation in vivo.

Optimised labeling, preclinical and initial clinical aspects of CCK-2 receptor-targeting with 3 radiolabeled peptides.

Medullary thyroid carcinoma (MTC) expresses CCK-2 receptors. (111)In-labeled DOTA-DGlu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH(2) (DOTA-MG11), DOTA-DAsp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH(2) (DOTA-CCK), and (99m)Tc-labeled N(4)-Gly-DGlu-(Glu)(5)-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH(2) ((99m)Tc-Demogastrin 2) are analogs developed for CCK-2 receptor-targeted scintigraphy. All 3 radiolabeled analogs were selected on the basis of their high CCK-2 receptor affinity and their good in vitro serum stability, with in vitro serum t(1/2) values of several hours. Radiolabeling of DOTA-peptides with (111)In requires a heating procedure, typically in the range of 80 degrees -100 degrees C up to 30 min. Following this procedure with DOTA-MG11 resulted in a >98 % incorporation of (111)In, however, with a radiochemical purity (RCP) of <50 %. The decrease in RCP was found to be due to oxidation of the methionine residue in the molecule. Moreover, this oxidized compound lost its CCK-2 receptor affinity. Therefore, conditions during radiolabeling were optimised: labeling of DOTA-MG11 and DOTA-CCK with (111)In involved 5 min heating at 80 degrees C and led to an incorporation of (111)In of >98 %. In addition, all analogs were radiolabeled in the presence of quenchers to prevent radiolysis and oxidation resulting in a RCP of >90 %. All 3 radiolabeled analogs were i.v. administered to 6 MTC patients: radioactivity cleared rapidly by the kidneys, with no significant differences in the excretion pattern of the 3 radiotracers. All 3 radiolabeled analogs exhibited a low in vivo stability in patients, as revealed during analysis of blood samples, with the respective t(1/2) found in the order of minutes. In patient blood, the rank of radiopeptide in vivo stability was: (99m)Tc-Demogastrin 2 (t(1/2) 10-15 min)>(111)In-DOTA-CCK (t(1/2) approximately 5-10 min)>(111)In-DOTA-MG11 (t(1/2)<5 min).

The [Tc(N)(PNP)]2+ metal fragment labeled cholecystokinin-8 (CCK8) peptide for CCK-2 receptors imaging: in vitro and in vivo studies.

The radiolabeling of the natural octapeptide CCK8, derivatized with a cysteine residue (Cys-Gly-CCK8), by using the metal fragment [99mTc(N)(PNP3)]2+ (PNP3 = N,N-bis(dimethoxypropylphosphinoethyl)methoxyethylamine) is reported. The [99mTc(N)(NS-Cys-Gly-CCK8)(PNP3)]+ complex was obtained according to two methods (one-step or two-step procedure) that gave the desired compound in high yield. The complex is stable in aqueous solution and in phosphate buffer. In vitro challenge experiments with an excess of cysteine and glutathione indicate that no transchelation reactions occur, confirming the high thermodynamic stability and kinetic inertness of this compound. Stability studies carried out in human and mouse serum, as well as in mouse liver homogenates, show that the radiolabeled compound remains intact for prolonged incubation at 37 degrees C. Binding properties give Kd (19.0 +/- 4.6 nmol/l) and Bmax (approximately 10(6) sites/cell) values in A431 cells overexpressing the CCK2-R. In vivo evaluation of the compound shows rapid and specific targeting to CCK2-R, a fourfold higher accumulation compared to nonreceptor expressing tumors.

Efectos del ayuno del Ramadán sobre la secreción de gastrina en jóvenes musulmanes en edad escolar / No disponible

Fundamento: Los cambios de hábitos durante el Ramadán, obligan a importantes reajustes fisiológicos, para mantener la homeostasis. El objetivo del presente trabajo ha sido analizar el perfil secretor de gastrina durante el ayuno de Ramadán en un grupo de musulmanes voluntarios, en edad escolar (13 a 15 años).Método: La muestra estuvo constituida por17 varones sanos, sin problemas gastrointestinales previos, seleccionados al azar entre todos los alumnos voluntarios de centros de enseñanza secundaria de Ceuta que practican el ayuno del Ramadán. Los niveles hormonales de gastrina se determinaron mediante radioinmunoanálisis. Resultados: Nuestros resultados muestran una disminución significativa de los niveles de gastrina al avanzar el período de ayuno, alcanzando el día 21, cifras de 23,89 pmol/L en comparación con los 28,49 de la semana previa(p=0,006) y de los 28,13 de la primera semana de ayuno (p=0,015). Estos cambios pueden interpretarse como una respuesta homeostática de adaptación a las nuevas pautas de alimentación. Finalizado el tiempo de Ramadán, observamos un incremento en los niveles de gastrina que devuelve los niveles hormonales a 30,15pmol/L, similares a los encontrados las semanas previa y primera del ayuno. Conclusión: Estos resultados sugieren un perfil de secreción para la hormona dependiente de la ingesta, que requiere un tiempo de adaptación a la nueva situación (AU)

Background: The changes in habits during Ramadan result in significant physiological readjustments, in order to maintain homeostasis. The main objective of the present study was to analyze the gastrin secretion during Ramdan Fast in a young muslim group during the school period between 13 - 15 years old. Method: Sample size was integrated by 17healthy men, without digestive personal history, selected by a randomized program between all the muslim students of the High Schools from Ceuta that practise Ramadan fast. Gastrin levels were determined by radioimmuno analysis(RIA).Results: Gastrin levels tend to go down during the fast period and at the 21st the media is23,89 pmol/L comparing with 28,49 pmol/L of the previous week (p=0,006) and the levels of the 1st week (28,13 pmol/L; p=0,015). These changes can be interpreted like an homeostatic answer of adaptation to the new guidelines of feeding. When Ramadan Fast concludes, gastrin levels come up again to basal levels (30,15pmol/L).Conclusion: These results suggest a secretion profile for the dependent hormone of the ingestion, that requires a time from adaptation to the new situation (AU)

Expression of gastrin and its receptor in human gastric cancer tissues.

PURPOSE: Gastrin is a growth factor of cancerous and normal cells of the gastrointestinal tract, and its effect is known to be mediated by gastrin/cholecystokinin B (CCKB) receptor. This study was performed to investigate the prognostic significance and the expression profiles of gastrin and gastrin receptor in human gastric carcinoma tissues. METHODS: We analyzed the expressions of gastrin and gastrin receptor by immunohistochemical staining using anti-gastrin Ab (Sigma, St. Louis, MO, USA) and anti-gastrin receptor Ab (Aphton Corp., Woodland, CA, USA) in 279 gastric adenocarcinoma patients. Patients' clinicopathologic features and prognoses were analyzed. RESULTS: The gastrin expression rate in these patients was 47.7% (133/279) and the gastrin receptor expression rate was 56.5% (158/279). Gastrin expression was significantly higher in men than in women (54.3% vs. 34.1%), and higher in differentiated gastric adenocarcinoma than in the undifferentiated type (55.1% vs. 43.0%). The gastrin receptor expression rate was also significantly higher in men than in women (61.2% vs. 47.3%), and was higher in the differentiated type than in the undifferentiated type (72.9% vs. 46.5%), and significantly higher in the intestinal type than in the diffuse type (75.2% vs. 42.9%). Gastrin and gastrin/CCKB receptor expressions were not found to be significant prognostic factors in themselves. When focused on correlation between the co-expression of gastrin and gastrin/CCKB receptor and the survival, the prognosis of patients positive for both gastrin and gastrin receptor was significantly poorer than for those negative for gastrin and gastrin receptor in diffuse-type gastric cancer patients. However, multivariate analysis showed that only TNM stage was an independent prognostic factor of survival in diffuse-type gastric cancer patients. CONCLUSIONS: This study shows that the expression rates of gastrin and gastrin receptor are high (about a half) in gastric carcinoma tissues, and that there is an association between gastrin and gastrin receptor expression. We also found that patients with diffuse-type gastric carcinoma tissues expressing both gastrin and gastrin receptor have a poorer prognosis than those negative for both, which suggests that gastrin acts as an autocrine growth factor in a subgroup of gastric carcinomas.

Immunohistochemical localization of CCK1 cholecystokinin receptors in normal and neoplastic human tissues.

BACKGROUND: The biological effects of cholecystokinin (CCK) are mediated by two distinct G protein-coupled receptors, CCK1 and CCK2. Although it is well established that CCK receptors are widely distributed throughout the normal gastrointestinal tract, little is known about their cellular and subcellular localization in human normal and neoplastic tissues. METHODS: We developed and characterized a novel antipeptide antibody to the carboxyl-terminal region of the human CCK1 receptor. Specificity of the antiserum was demonstrated by 1) detection of a broad band migrating at a relative molecular mass of 85,000-95,000 in Western blots of membranes from CCK1-expressing tumors and CCK1-transfected cells, 2) cell surface staining of CCK1-transfected cells, 3) translocation of CCK1 receptor immunostaining after agonist exposure, and 4) abolition of tissue immunostaining by preadsorption of the antibody with its immunizing peptide. The distribution of CCK1 receptors was investigated in 74 human tumors and their tissues of origin. RESULTS: The presence of CCK1 receptors was rarely detected in human tumors except for carcinoids, insulinomas, pituitary adenomas, and meningiomas. CCK1 receptors were clearly located at the plasma membrane and uniformly present on nearly all tumor cells. In the gastrointestinal tract, CCK1 receptor immunoreactivity was highly abundant in chief cells of the gastric mucosa, in myenteric ganglion cells, and in myenteric nerve fibers. CONCLUSION: This is the first localization of CCK1 receptors in human formalin-fixed, paraffin-embedded tissues at the cellular level. The overexpression of CCK1 receptors in a subset of human neuroendocrine tumors may provide a molecular basis for efficient targeting of these tumors with radiolabeled CCK analogs.

99mTc-labeling and in vitro and in vivo evaluation of HYNIC- and (Nalpha-His)acetic acid-modified [D-Glu1]-minigastrin.

Gastrin/CCK-2 receptors are overexpressed in a number of tumors such as medullary thyroid cancer (MTC) and small cell lung cancer (SCLC). Recently [D-Glu1]-minigastrin (MG) has been radiolabeled with 131I, 111In, and 90Y and evaluated in patients. This study describes the labeling and evaluation of MG with technetium-99m using two different labeling approaches: HYNIC as bifunctional coupling agent and (Nalpha-His)Ac as tridentate ligand for 99mTc(CO3) labeling. Labeling was perfomed at high specific activities using Tricine and EDDA as coligands for HYNIC-MG and [99mTc(OH2)3(CO)3]+ for (Nalpha-His)Ac-MG. Stability experiments were carried out by reversed phase HPLC analysis in PBS, serum, histidine, and cysteine solutions, as well as rat liver and kidney homogenates. Receptor binding and internalization experiments were performed using CCK-2 receptor positive AR42J rat pancreatic tumor cells. Biodistribution experiments were carried out in nude mice carrying AR42J tumors by injection of 99mTc-labeled peptide with or without coinjection of 50 microg of minigastrin I human (MGh). HYNIC-MG and (Nalpha-His)Ac-MG could be radiolabeled at high specific activities (>1 Ci/micromol). For HYNIC-MG, high labeling yields (>95%) were achieved using Tricine and EDDA as coligands. Stability experiments of all 99mTc-labeled conjugates revealed a high stability of the label in PBS and serum as well as toward challenge with histidine and cysteine. Incubation in kidney homogenates resulted in a rapid degradation of all conjugates with <10% intact peptide after 60 min at 37 degrees C, with no considerable differences between the radiolabeled conjugates; a somewhat lower degradation rate was seen in liver homogenates. Protein binding varied considerably with lowest levels for 99mTc-EDDA/HYNIC-MG. Competition experiments of unlabeled conjugates on AR42J membranes versus [125I-Tyr12]-gastrin I showed high CCK-2 receptor affinity for all conjugates under study. Internalization behavior was very rapid for all radiolabeled conjugates in the order of 99mTc-(Nalpha-His)Ac-MG > 99mTc-EDDA/HYNIC-MG > 99mTc-Tricine/HYNIC-MG. In tumor-bearing nude mice the highest tumor-uptake was observed with 99mTc-EDDA/HYNIC-MG (8.1%ID/g) followed by 99mTc-Tricine/HYNIC-MG (2.2%ID/g) and 99mTc-(Nalpha-His)Ac-MG (1.2%ID/g) which correlated with kidney uptake (101.0%ID/g, 53.8%ID/g, 1.8%ID/g respectively). In this series of compounds 99mTc-EDDA/HYNIC-MG with its very high tumor/organ ratios except for kidneys seems to be the most promising agent to target CCK-2 receptors. Despite promising properties concerning receptor binding, internalization, and in vitro stability, 99mTc-(Nalpha-His)Ac-MG showed low tumor uptake in vivo.

In vitro and in vivo characterization of Indium-111 and Technetium-99m labeled CCK-8 derivatives for CCK-B receptor imaging.

Cholecystokinin (CCK) receptors of the subtype B (CCK-BR) have been shown to be overexpressed in certain neuroendocrine tumors including medullary thyroid cancer. Our recent work has focused on new methods to radiolabel the CCK8 peptide with 111In or 99mTc for CCK-B receptor imaging. Derivatives of CCK8 were obtained by addition at the N-terminus in solid phase of a DTPA derivative (DTPAGlu) linked through a glycine spacer (DTPAGlu-G-CCK8) or cysteine, glycine and a diphenylphosphinopropionyl moiety (PhosGC-CCK8) for labeling with 111In and 99mTc, respectively. CCK-BR overexpressing A431 cancer cell lines were utilized to characterize in vitro properties of the two compounds as well as for generating xenografts in nude mice for in vivo characterization. Both 111In-DTPAGlu-G-CCK8 and 99mTcPhosGC-CCK8 showed similar binding affinities for CCK-BR with dissociation constants of 20-40 nM, were internalized after interaction with the receptor and displayed prolonged cellular retention times. Specific in vivo interaction with the receptor of both CCK8 analogs was observed in our animal model. 111In-DTPAGlu-G-CCK8 showed better target to non-target ratios, although it appeared to be rapidly metabolized after injection and activity cleared through the kidneys. 99mTc-PhosGC-CCK8 was more stable in vivo but showed marked hepatobiliary clearance with resulting high background activity in the bowel. The rapid clearance and lower background obtained with 111In-DTPAGlu-G-CCK8 make this a better candidate for further development.