Stabilization Strategies for Linear Minigastrin Analogues: Further Improvements via the Inclusion of Proline into the Peptide Sequence.

Minigastrin (MG) analogues, known for their high potential to target cholecystokinin-2 receptor (CCK2R) expressing tumors, have limited clinical applicability due to low enzymatic stability. By introducing site-specific substitutions within the C-terminal receptor-binding sequence, reduced metabolization and improved tumor targeting can be achieved. In this work, the influence of additional modification within the N-terminal sequence has been explored. Three novel 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated CCK2R ligands with proline substitution at different positions were synthesized. Substitution did not affect CCK2R affinity, and the conjugates labeled with indium-111 and lutetium-177 showed a high enzymatic stability in different incubation media as well as in vivo (57-79% intact radiopeptide in blood of BALB/c mice at 1 h p.i.) combined with enhanced tumor uptake (29-46% IA/g at 4 h in xenografted BALB/c nude mice). The inclusion of Pro contributes significantly to the development of CCK2R ligands with optimal targeting properties for application in targeted radiotherapy.

Design of Radiolabeled Analogs of Minigastrin by Multiple Amide-to-Triazole Substitutions.

The insertion of single 1,4-disubstituted 1,2,3-triazoles as metabolically stable bioisosteres of trans-amide bonds (triazole scan) was recently applied to the 177Lu-labeled tumor-targeting analog of minigastrin, [Nle15]MG11. The reported novel mono-triazolo-peptidomimetics of [Nle15]MG11 showed either improved resistance against enzymatic degradation or a significantly increased affinity toward the target receptor but never both. To enhance further the tumor-targeting properties of the minigastrin analogs, we studied conjugates with multiple amide-to-triazole substitutions for additive or synergistic effects. Promising candidates were identified by modification of two or three amide bonds, which yielded both improved stability and increased receptor affinity of the peptidomimetics in vitro. Biodistribution studies of radiolabeled multi-triazolo-peptidomimetics in mice bearing receptor-positive tumor xenografts revealed up to 4-fold increased tumor uptake in comparison to the all-amide reference compound [Nle15]MG11. In addition, we report here for the first time a linear peptidomimetic with three triazole insertions in its backbone and maintained biological activity.

Triazolo-Peptidomimetics: Novel Radiolabeled Minigastrin Analogs for Improved Tumor Targeting.

MG11 is a truncated analog of minigastrin, a peptide with high affinity and specificity toward the cholecystokinin-2 receptor (CCK2R), which is overexpressed by different tumors. Thus, radiolabeled MG11 derivatives have great potential for use in cancer diagnosis and therapy. A drawback of MG11 is its fast degradation by proteases, leading to moderate tumor uptake in vivo. We introduced 1,4-disubstituted 1,2,3-triazoles as metabolically stable bioisosteres to replace labile amide bonds of the peptide. The "triazole scan" yielded peptidomimetics with improved resistance to enzymatic degradation and/or enhanced affinity toward the CCK2R. Remarkably, our lead compound achieved a 10-fold increase in receptor affinity, resulting in a 2.6-fold improved tumor uptake in vivo. Modeling of the ligand-CCK2R complex suggests that an additional cation-π interaction of the aromatic triazole moiety with the Arg356 residue of the receptor is accountable for these observations. We show for the first time that the amide-to-triazole substitution strategy offers new opportunities in drug development that go beyond the metabolic stabilization of bioactive peptides.

Elucidating the Structure-Activity Relationship of the Pentaglutamic Acid Sequence of Minigastrin with Cholecystokinin Receptor Subtype 2.

Derivatized minigastrin analogues make up a promising class of candidates for targeting cholecystokinin receptor subtype 2 (CCK2R), which is overexpressed on cancer cells of various neuroendocrine tumors. The pentaglutamic acid sequence of minigastrin influences its biological properties. In particular, it plays a crucial role in the kidney reuptake mechanism. However, the importance of the binding affinity and interaction of this region with the receptor on a molecular level remains unclear. To elucidate its structure-activity relationship with CCK2R, we replaced this sequence with various linkers differing in their amount of anionic charge, structural characteristics, and flexibility. Specifically, a flexible aliphatic linker, a linker with only three d-Glu residues, and a structured linker with four adjacent ß3-glutamic acid residues were evaluated and compared to the lead compound PP-F11N (DOTA-[d-Glu1-6,Nle11]gastrin-13). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to the minigastrin derivatives, which allowed radiolabeling with Lutetium-177. The levels of In vitro internalization into MZ-CRC1 cells and in vivo tumor uptake as well as human blood plasma stability increased in the following order: aliphatic linker < three d-Glu < (ß3-Glu)4 < (d-Glu)6. The in vitro and in vivo behavior was therefore significantly improved with anionic charges. Computational modeling of a CCK2 receptor-ligand complex revealed ionic interactions between cationic residues (Arg and His) of the receptor and anionic residues of the ligand in the linker.

Targeting of the Cholecystokinin-2 Receptor with the Minigastrin Analog 177Lu-DOTA-PP-F11N: Does the Use of Protease Inhibitors Further Improve In Vivo Distribution?

Patients with metastatic medullary thyroid cancer (MTC) have limited systemic treatment options. The use of radiolabeled gastrin analogs targeting the cholecystokinin-2 receptor (CCK2R) is an attractive approach. However, their therapeutic efficacy is presumably decreased by their enzymatic degradation in vivo. We aimed to investigate whether the chemically stabilized analog 177Lu-DOTA-PP-F11N (177Lu-DOTA-(dGlu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2) performs better than reference analogs with varying in vivo stability, namely 177Lu-DOTA-MG11 (177Lu-DOTA-dGlu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2) and 177Lu-DOTA-PP-F11 (177Lu-DOTA-(dGlu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2), and whether the use of protease inhibitors further improves CCKR2 targeting. First human data on 177Lu-DOTA-PP-F11N are also reported. Methods: In vitro stability of all analogs was assessed against a panel of extra- and intracellular endoproteases, whereas their in vitro evaluation was performed using the human MTC MZ-CRC-1 and the transfected A431-CCK2R(+) cell lines. Biodistribution without and with the protease inhibitors phosphoramidon and thiorphan was assessed 4 h after injection in MZ-CRC-1 and A431-CCK2R(+) dual xenografts. Autoradiography of 177Lu-DOTA-PP-F11N (without and with phosphoramidon) and NanoSPECT/CT were performed. SPECT/CT images of 177Lu-DOTA-PP-F11N in a metastatic MTC patient were also acquired. Results:natLu-DOTA-PP-F11N is less of a substrate for neprilysins than the other analogs, whereas intracellular cysteine proteases, such as cathepsin-L, might be involved in the degradation of gastrin analogs. The uptake of all radiotracers was higher in MZ-CRC-1 tumors than in A431-CCK2R(+), apparently because of the higher number of binding sites on MZ-CRC-1 cells. 177Lu-DOTA-PP-F11N had the same biodistribution as 177Lu-DOTA-PP-F11; however, uptake in the MZ-CRC-1 tumors was almost double (20.7 ± 1.71 vs. 11.2 ± 2.94 %IA [percentage injected activity]/g, P = 0.0002). Coadministration of phosphoramidon or thiorphan increases 177Lu-DOTA-MG11 uptake significantly in the CCK2R(+) tumors and stomach. Less profound was the effect on 177Lu-DOTA-PP-F11, whereas no influence or even reduction was observed for 177Lu-DOTA-PP-F11N (20.7 ± 1.71 vs. 15.6 ± 3.80 [with phosphoramidon] %IA/g, P < 0.05 in MZ-CRC-1 tumors). The first clinical data show high 177Lu-DOTA-PP-F11N accumulation in tumors, stomach, kidneys, and colon. Conclusion: The performance of 177Lu-DOTA-PP-F11N without protease inhibitors is as good as the performance of 177Lu-DOTA-MG11 in the presence of inhibitors. The human application of single compounds without unessential additives is preferable. Preliminary clinical data spotlight the stomach as a potential dose-limiting organ besides the kidneys.

Site-specific stabilization of minigastrin analogs against enzymatic degradation for enhanced cholecystokinin-2 receptor targeting.

Minigastrin (MG) analogs show high affinity to the cholecystokinin-2 receptor (CCK2R) and have therefore been intensively studied to find a suitable analog for imaging and treatment of CCK2R-expressing tumors. The clinical translation of the radioligands developed thus far has been hampered by high kidney uptake or low enzymatic stability. In this study, we aimed to develop new MG analogs with improved targeting properties stabilized against degradation through site-specific amino acid modifications. Method: Based on the lead structure of a truncated MG analog, four new MG derivatives with substitutions in the C-terminal part of the peptide (Trp-Met-Asp-Phe-NH2) were synthesized and derivatized with DOTA at the N-terminus for radiolabeling with trivalent radiometals. The in vitro properties of the new analogs were characterized by analyzing the lipophilicity, the protein binding, and the stability of the Indium-111 (111In)-labeled analogs in different media. Two different cell lines, AR42J cells physiologically expressing the rat CCK2R and A431 cells transfected with human CCK2R (A431-CCK2R), were used to study the receptor affinity and cell uptake. For the two most promising MG analogs, metabolic studies in normal BALB/c mice were carried out as well as biodistribution and imaging studies in tumor xenografted athymic BALB/c nude mice. Results: Two out of four synthesized peptide analogs (DOTA-MGS1 and DOTA-MGS4) showed retained receptor affinity and cell uptake when radiolabeled with 111In. These two peptide analogs, however, showed a different stability against enzymatic degradation in vitro and in vivo. When injected to normal BALB/c mice, for 111In-DOTA-MGS1 at 10 min post injection (p.i.) no intact radiopeptide was found in the blood, whereas for 111In-DOTA-MGS4 more than 75% was still intact. 111In-DOTA-MGS4 showed a clear increase in injected activity per gram tissue (IA/g) for A431-CCK2R xenografts (10.40±2.21% IA/g 4 h p.i.) when compared to 111In-DOTA-MGS1 (1.23±0.15% IA/g 4 h p.i.). The tumor uptake of 111In-DOTA-MGS4 was also combined with a low uptake in stomach and kidney leading to high-contrast NanoSPECT/CT images. Conclusion: Of the four new MG analogs developed, the best results in terms of enzymatic stability and increased tumor targeting were obtained with 111In-DOTA-MGS4 showing two substitutions with N-methylated amino acids. 111In-DOTA-MGS4 was also superior to other MG analogs reported thus far and seems therefore an extremely promising targeting molecule for theranostic use with alternative radiometals.

(99m)Tc-labeled gastrins of varying peptide chain length: Distinct impact of NEP/ACE-inhibition on stability and tumor uptake in mice.

INTRODUCTION: In situ inhibition of neutral endopeptidase (NEP) has been recently shown to impressively increase the bioavailability and tumor uptake of biodegradable gastrin radioligands. Furthermore, angiotensin converting enzyme (ACE) has been previously shown to cleave gastrin analogs in vitro. In the present study, we have assessed the effects induced by single or dual NEP/ACE-inhibition on the pharmacokinetic profile of three (99m)Tc-labeled gastrins of varying peptide chain length: [(99m)Tc]SG6 ([(99m)Tc-N4-Gln(1)]gastrin(1-17)), [(99m)Tc]DG2 ([(99m)Tc-N4-Gly(4),DGlu(5)]gastrin(4-17)) and [(99m)Tc]DG4 ([(99m)Tc-N4-DGlu(10)]gastrin(10-17)). METHODS: Mouse blood samples were collected 5min after injection of each of [(99m)Tc]SG6/DG2/DG4 together with: a) vehicle, b) the NEP-inhibitor phosphoramidon (PA), c) the ACE-inhibitor lisinopril (Lis), or d) PA plus Lis and were analyzed by RP-HPLC for radiometabolite detection. Biodistribution was studied in SCID mice bearing A431-CCK2R(+/-) xenografts at 4h postinjection (pi). [(99m)Tc]SG6 or [(99m)Tc]DG4 was coinjected with either vehicle or the above described NEP/ACE-inhibitor regimens; for [(99m)Tc]DG2 control and PA animal groups were only included. RESULTS: Treatment of mice with PA induced significant stabilization of (99m)Tc-radiotracers in peripheral blood, while treatment with Lis or Lis+PA affected the stability of des(Glu)5 [(99m)Tc]DG4 only. In line with these findings, PA coinjection led to notable amplification of tumor uptake of radiopeptides compared to controls (P<0.01). Only [(99m)Tc]DG4 profited by single Lis (2.06±0.39%ID/g vs 0.99±0.13%ID/g in controls) or combined Lis+PA coinjection (8.91±1.61%ID/g vs 4.89±1.33%ID/g in PA-group). Furthermore, kidney uptake remained favourably low and unaffected by PA and/or Lis coinjection only in the case of [(99m)Tc]DG4 (<1.9%ID/g) resulting in the most optimal tumor-to-kidney ratios. CONCLUSIONS: In situ NEP/ACE-inhibition diversely affected the in vivo profile of (99m)Tc-radioligands based on different-length gastrins. Truncated [(99m)Tc]DG4 exhibited overall the most attractive profile during combined NEP/ACE-inhibition in mouse models, providing new opportunities for CCK2R-expressing tumor imaging in man with SPECT.

Preclinical pharmacokinetics, biodistribution, radiation dosimetry and toxicity studies required for regulatory approval of a phase I clinical trial with (111)In-CP04 in medullary thyroid carcinoma patients.

INTRODUCTION: From a series of radiolabelled cholecystokinin (CCK) and gastrin analogues, (111)In-CP04 ((111)In-DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2) was selected for further translation as a diagnostic radiopharmaceutical towards a first-in-man study in patients with medullary thyroid carcinoma (MTC). A freeze-dried kit formulation for multicentre application has been developed. We herein report on biosafety, in vivo stability, biodistribution and dosimetry aspects of (111)In-CP04 in animal models, essential for the regulatory approval of the clinical trial. MATERIALS AND METHODS: Acute and extended single dose toxicity of CP04 was tested in rodents, while the in vivo stability of (111)In-CP04 was assessed by HPLC analysis of mouse blood samples. The biodistribution of (111)In-CP04 prepared from a freeze-dried kit was studied in SCID mice bearing double A431-CCK2R(±) xenografts at 1, 4 and 24h pi. Further 4-h animal groups were either additionally treated with the plasma expander gelofusine or injected with (111)In-CP04 prepared by wet-labelling. Pharmacokinetics in healthy mice included the 30min, 1, 4, 24, 48 and 72h time points pi. Dosimetric calculations were based on extrapolation of mice data to humans adopting two scaling models. RESULTS: CP04 was well-tolerated by both mice and rats, with an LD50>178.5µg/kg body weight for mice and a NOAEL (no-observed-adverse-effect-level) of 89µg/kg body weight for rats. After labelling, (111)In-CP04 remained >70% intact in peripheral mouse blood at 5min pi. The uptake of (111)In-CP04 prepared from the freeze-dried kit and by wet-labelling were comparable in the A431-CCK2R(+)-xenografts (9.24±1.35%ID/g and 8.49±0.39%ID/g, respectively; P>0.05). Gelofusine-treated mice exhibited significantly reduced kidneys values (1.69±0.15%ID/g vs. 5.55±0.94%ID/g in controls, P<0.001). Dosimetry data revealed very comparable effective tumour doses for the two scaling models applied, of 0.045 and 0.044mSv/MBq. CONCLUSION: The present study has provided convincing toxicology, biodistribution and dosimetry data for prompt implementation of the freeze-dried kit formulation without or with gelofusine administration in a multicentre clinical trial in MTC patients.

Improving the In Vivo Profile of Minigastrin Radiotracers: A Comparative Study Involving the Neutral Endopeptidase Inhibitor Phosphoramidon.

Minigastrin radiotracers, such as [(111)In-DOTA]MG0 ([(111)In-DOTA-DGlu(1)]minigastrin), have been considered for diagnostic imaging and radionuclide therapy of CCK2R-positive human tumors, such as medullary thyroid carcinoma. However, the high kidney retention assigned to the pentaGlu(2-6) repeat in the peptide sequence has compromised their clinical applicability. On the other hand, truncated des(Glu)(2-6)-analogs, such as [(111)In-DOTA]MG11 ([(111)In-DOTA-DGlu(10),desGlu(2-6)]minigastrin), despite their low renal uptake, show poor bioavailability and tumor targeting. [(111)In]CP04 ([(111)In-DOTA-DGlu(1-6)]minigastrin) acquired by Glu(2-6)/DGlu(2-6) substitution showed promising tumor-to-kidney ratios in rodents. In the present study, we compare the biological profiles of [(111)In]CP04, [(111)In-DOTA]MG11, and [(111)In-DOTA]MG0 during in situ neutral endopeptidase (NEP) inhibition, recently shown to improve the bioavailability of several peptide radiotracers. After coinjection of the NEP inhibitor, phosphoramidon (PA), the stability of [(111)In]CP04 and [(111)In-DOTA]MG0 in peripheral mouse blood increased, with an exceptional >14-fold improvement monitored for [(111)In-DOTA]MG11. In line with these findings, PA treatment increased the uptake of [(111)In]CP04 (8.5 ± 0.4%ID/g to 16.0 ± 2.3%ID/g) and [(111)In-DOTA]MG0 (11.9 ± 2.2%ID/g to 17.2 ± 0.9%ID/g) in A431-CCK2R(+) tumors at 4 hours postinjection, whereas the respective increase for [(111)In-DOTA]MG11 was >6-fold (2.5 ± 0.9%ID/g to 15.1 ± 1.7%ID/g). Interestingly, kidney uptake remained lowest for [(111)In-DOTA]MG11, but unfavorably increased by PA treatment for [(111)In-DOTA]MG0. Thus, overall, the most favorable in vivo profile was displayed by [(111)In-DOTA]MG11 during NEP inhibition, highlighting the need to validate this promising concept in the clinic.

The in vivo disposition and in vitro transmembrane transport of two model radiometabolites of DOTA-conjugated receptor-specific peptides labelled with (177) Lu.

In vivo metabolism of the radiolabelled receptor-specific peptides has been described; however, information regarding the pharmacokinetic behaviour of the degradation products within the body is very scarce. The present study was designed to obtain new knowledge on the disposition and elimination of low-molecular radiometabolites of receptor-specific peptides in the organism and to reveal the potential involvement of selected membrane transport mechanisms in the cellular uptake of radiometabolites, especially in the kidney. The study compared pharmacokinetics of two radiometabolites: a final metabolite of somatostatin analogues, (177)Lu-DOTA-DPhe, and a tripeptide metabolite of (177)Lu-DOTA-minigastrin 11, (177)Lu-DOTA-DGlu-Ala-Tyr. Their pharmacokinetics was compared with that of respective parent (177)Lu-radiopeptide. Both radiometabolites exhibited relative rapid clearing from most body tissues in rats in vivo along with predominant renal excretion. The long-term renal retention of the smaller radiometabolite (177)Lu-DOTA-DPhe was lower than that of (177)Lu-DOTA-DGlu-Ala-Tyr. An uptake of (177)Lu-DOTA-DPhe by human renal influx transporter organic cation transporter 2 was found in vitro in a cellular model. The study brings the first experimental data on the in vivo pharmacokinetics of radiometabolites of receptor-specific somatostatin and gastrin analogues. The found results may indicate a negative correlation between the degree of decomposition of the parent peptide chain and the renal retention of the metabolite.

In vivo inhibition of neutral endopeptidase enhances the diagnostic potential of truncated gastrin (111)In-radioligands.

INTRODUCTION: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Stereochemistry of amino acid spacers determines the pharmacokinetics of (111)In-DOTA-minigastrin analogues for targeting the CCK2/gastrin receptor.

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.

Preclinical evaluation of gastrin derivatives labelled with 111In: radiolabelling, affinity profile and pharmacokinetics in rats.

BACKGROUND: Cholecystokinin receptor subtype 2 (CCK-2) is overexpressed in various tumours like medullary thyroid carcinomas and small cell lung cancer. Radiolabelled peptides that bind with high affinity and specificity to CCK-2 receptors, thus hold great potential for visualizing such tumours. METHODS: We compared four 111In labelled gastrin analogues, called minigastrins (MG), namely MG11, MG45, MG47 and MG48 linked to metal chelating DOTA in preclinical experiments. The radiolabelled peptides were tested for peptide binding in CCK-2 receptor-bearing cell line AR42J and for their pharmacokinetics in normal rats. RESULTS: The experiments suggest that all gastrin analogues had similar and relatively rapid internalization into AR42J cells. Binding to CCK-2 receptors in AR42J cells was saturable for all agents but there were some differences in receptor affinity. This biodistribution study in rats showed a rapid decrease in blood radioactivity, predominantly renal clearance and saturable uptake of the radiopharmaceutical and/or its metabolites in the CCK-2 receptor-positive stomach. Higher kidney accumulation of radioactivity was only found for 111In-DOTA-minigastrin 48. CONCLUSIONS: The data suggest that the 111In-DOTA-minigastrin analogues studied are promising candidates for the scintigraphy of CCK-2 receptor-expressing tumours; 111In-DOTA-MG47 and 111In-DOTA-MG11 are the most promising.

Omeprazole and PGC-formulated heparin binding epidermal growth factor normalizes fasting blood glucose and suppresses insulitis in multiple low dose streptozotocin diabetes model.

PURPOSE: Our objective was to develop novel nanocarriers (protected graft copolymer, PGC) that improve the stability of heparin binding EGF (HBEGF) and gastrin and then to use PGC-formulated HBEGF (PGC-HBEGF) and Omeprazole (+/- PGC-gastrin) for normalizing fasting blood glucose (FBG) and improving islet function in diabetic mice. METHODS: HBEGF, PGC-HBEGF, Omeprazole, Omeprazole + PGC-HBEGF, Omeprazole + PGC-gastrin + PGC-HBEGF and epidermal growth factor (EGF) + gastrin were tested in multiple low dose streptozotocin diabetic mice. RESULTS: Omeprazole + PGC-HBEGF normalized FBG and is better than EGF + gastrin at improving islet function and decreasing insulitis. Groups treated with Omeprazole, Omeprazole + PGC-HBEGF, or EGF + gastrin have significantly improved islet function versus saline control. All animals that received PGC-HBEGF had significantly reduced islet insulitis versus saline control. Non-FBG was lower for Omeprazole + PGC-gastrin + PGC-HBEGF but Omeprazole + PGC-HBEGF alone showed better FBG and glucose tolerance. CONCLUSIONS: Omeprazole + PGC-HBEGF provides a sustained exposure to both EGFRA and gastrin, improves islet function, and decreases insulitis in multiple low dose streptozotocin diabetic mice. Although HBEGF or EGF elevates non-FBG, it facilitates a reduction of insulitis and, in the presence of Omeprazole, provides normalization of FBG at the end of treatment. The study demonstrates Omeprazole and PGC-HBEGF is a viable treatment for diabetes.

Distribution, elimination, and renal handling of (99m)technetium-Demogastrin 1.

Radiolabeled cholecystokinin/gastrin (CCK) receptor-targeting peptides are promising compounds for radiodiagnosis and radiotherapy of certain malignancies. This study evaluated the pharmacokinetic profile of a CCK-2 receptor-specific peptide, Demogastrin 1, labeled with technetium-99m ((99m)Tc-Demogastrin 1), in rats. To investigate the fate of (99m)Tc-Demogastrin 1 in the rat, biodistribution and elimination studies in vivo were performed, and elimination parameters in perfused rat liver and kidney were determined. Biodistribution studies showed that (99m)Tc-Demogastrin 1 was rapidly cleared from the blood and most organs. A significant amount of radioactivity was detected in the CCK-2 receptor-rich organs, such as the stomach. Low radioactivity was found in the CCK-1 receptor-rich organs. Radioactivity in bowels and stomach declined relatively slowly. High and long-term retention of radioactivity in the kidneys was observed. Elimination of (99m)Tc-Demogastrin 1 via the bile was negligible. A high and rapid renal excretion was observed in elimination experiments in vivo. In the perfused kidney, glomerular filtration was found to be the main renal excretion mechanism of (99m)Tc-Demogastrin 1. Demogastrin 1 was distributed preferentially to the organs expressing CCK-2 receptors. The decisive elimination route of (99m)Tc-Demogastrin 1 in rats was urinary excretion. A high and prolonged renal retention may limit potential clinical use of the compound.

Biodistribution of 177Lu-octreotate and 111In-minigastrin in female nude mice transplanted with human medullary thyroid carcinoma GOT2.

To be able to evaluate new radiopharmaceuticals and optimize diagnostic and therapeutic procedures, relevant animal models are required. The aim of this study was to evaluate the medullary thyroid carcinoma GOT2 animal model by analyzing the biodistribution of 177Lu-octreotate and 111In-minigastrin (MG0). BALB/c nude mice, subcutaneously transplanted with GOT2, were intravenously injected with either 177Lu-octreotate or 111In-MG0, with or without excess of unlabeled human minigastrin simultaneously with 111In-MG0. Animals were sacrificed 1-7 days after injection in the 177Lu-octreotate study and 1 h after injection of 111In-MG0. The activity concentrations in organs and tissues were determined and mean absorbed doses from 177Lu were calculated. There was a specific tumor uptake of either 177Lu-octreotate or 111In-MG0. 177Lu-octreotate samples showed high activity concentrations in tissues expressing somatostatin receptors (SSTR). For both radiopharmaceuticals the highest activity concentrations were found in the kidneys. Compared to results from similar studies in mice with another MTC cell line (TT) the biodistribution was favorable (higher tumor uptake) for the GOT2 model, while compared to other animal models expressing SSTR, the tumor uptake of 177Lu-octreotate was modest. In conclusion, the GOT2 animal model is a valuable model for evaluation and optimization of diagnostic and therapeutic procedures using radiolabeled somatostatin, CCK2 and gastrin analogues prior to clinical studies.

Preclinical evaluation of radiolabeled DOTA-derivatized cyclic minigastrin analogs for targeting cholecystokinin receptor expressing malignancies.

PURPOSE: 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. PROCEDURES: 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. RESULTS: 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. CONCLUSIONS: Cyclic DOTA-minigastrin analogs are promising candidates for gastrin receptor scintigraphy and targeted radionuclide therapy.

Radiopeptide internalisation and externalization assays: cell viability and radioligand integrity.

Various aspects of radiopeptide receptor-mediated cell internalisation and externalization assays were assessed, including the integrity of externalized peptides and the effect of varying the pH and incubation time of the acid wash step (to remove surface receptor-bound ligand) on efficacy and cell viability. The observed intact proportion of externalized peptide was 5-10%, and acid wash buffers with pH 2.8 or below were found to be detrimental to cell viability and integrity, particularly following prolonged incubation times.

Targeting of CCK-2 receptor-expressing tumors using a radiolabeled divalent gastrin peptide.

UNLABELLED: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

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.