76 research outputs found
To serve and protect: Enzyme inhibitors as radiopeptide escorts promote tumor targeting
Radiolabeled octreotide analogs are most successfully being applied today in clinical cancer imaging and treatment. Propagation of this paradigm to other radiopeptide families has been greatly hampered by the inherent poor metabolic stability of systemically administered peptide analogs. We hypothesized that the in vivo coadministration of specific enzyme inhibitors would improve peptide bioavailability and hence tumor uptake. Through single coinjection of the neutral endopeptidase inhibitor phosphoramidon (PA), we were able to provoke remarkable rises in the percentages of circulating intact somatostatin, gastrin, and bombesin radiopeptides in mouse models, resulting in a remarkable increase in uptake in tumor xenografts in mice. Methods: The peptide conjugates [DOTA-Ala1]SS14 (DOTA-Ala-Gly-c[Cys-Lys- Asn-Phe-Phe-Trp-Lys-Thr-Phe- Thr-Ser-Cys]-OH), PanSB1 (DOTAPEG2- DTyr-Gln-Trp-Ala-Val-ÎČAla- His-Phe-Nle-NH2), and DOTA-MG11 (DOTA-DGlu-Al
Side-Chain Modified [<sup>99m</sup>Tc]Tc-DT1 Mimics:A Comparative Study in NTS<sub>1</sub>R-Positive Models
Radiolabeled neurotensin analogs have been developed as candidates for theranostic use against neurotensin subtype 1 receptor (NTS1R)-expressing cancer. However, their fast degradation by two major peptidases, neprilysin (NEP) and angiotensin-converting enzyme (ACE), has hitherto limited clinical success. We have recently shown that palmitoylation at the Δ-amine of Lys7 in [99mTc]Tc-[Lys7]DT1 (DT1, N4-Gly-Arg-Arg-Pro-Tyr-Ile-Leu-OH, N4 = 6-(carboxy)-1,4,8,11-tetraazaundecane) led to the fully stabilized [99mTc]Tc-DT9 analog, displaying high uptake in human pancreatic cancer AsPC-1 xenografts but unfavorable pharmacokinetics in mice. Aiming to improve the in vivo stability of [99mTc]Tc-DT1 without compromising pharmacokinetics, we now introduce three new [99mTc]Tc-DT1 mimics, carrying different pendant groups at the Δ-amine of Lys7: MPBA (4-(4-methylphenyl)butyric acid)â[99mTc]Tc-DT10; MPBA via a PEG4-linkerâ[99mTc]Tc-DT11; or a hydrophilic PEG6 chainâ[99mTc]Tc-DT12. The impact of these modifications on receptor affinity and internalization was studied in NTS1R-positive cells. The effects on stability and AsPC-1 tumor uptake were assessed in mice without or during NEP/ACE inhibition. Unlike [99mTc]Tc-DT10, the longer-chain modified [99mTc]Tc-DT11 and [99mTc]Tc-DT12 were significantly stabilized in vivo, resulting in markedly improved tumor uptake compared to [99mTc]Tc-DT1. [99mTc]Tc-DT11 was found to achieve the highest AsPC-1 tumor values and good pharmacokinetics, either without or during NEP inhibition, qualifying for further validation in patients with NTS1R-positive tumors using SPECT/CT.</p
Optimizing the Profile of Tc-99m Tc-NT(7-13) Tracers in Pancreatic Cancer Models by Means of Protease Inhibitors
Background: The overexpression of neurotensin subtype 1 receptors (NTS1Rs) in human
tumors may be elegantly exploited for directing neurotensin (NT)-based radionuclide carriers
specifically to cancer sites for theranostic purposes. We have recently shown that [99mTc]TcâDT1
([99mTc]Tcâ[N4âGly7
]NT(7â13)) and [99mTc]TcâDT5 ([99mTc]Tcâ[N4âÎČAla7
,Dab9
]NT(7â13)) show
notably improved uptake in human colon adenocarcinoma WiDr xenografts in mice treated with
neprilysin (NEP) inhibitors and/or angiotensin-converting enzyme (ACE) inhibitors compared with
untreated controls. Aiming toward translation of this promising approach in NTS1R-positive
pancreatic ductal adenocarcinoma (PDAC) patients, we now report on the impact of registered
NEP/ACE inhibitors on the performance of [99mTc]TcâDT1 and [99mTc]TcâDT5 in pancreatic cancer
models. Methods: The cellular uptake of [99mTc]TcâDT1 and [99mTc]TcâDT5 was tested in a panel
of pancreatic cell lines, and their stability was assessed in mice treated or not treated with Entresto,
lisinopril, or their combinations. Biodistribution was conducted in severe combined immunodeficiency
(SCID) mice bearing pancreatic AsPC-1 xenografts. Results: The Entresto + lisinopril combination
maximized the metabolic stability of the fast-internalizing [99mTc]TcâDT1 in mice, resulting in notably
enhanced tumor uptake (7.05 ± 0.80% injected activity (IA)/g vs. 1.25 ± 0.80% IA/g in non-treated
controls at 4 h post-injection; p < 0.0001). Conclusions: This study has shown the feasibility of
optimizing the uptake of [99mTc]TcâDT1 in pancreatic cancer models with the aid of clinically
established NEP/ACE inhibitors, in favor of clinical translation prospects
Key-protease inhibition regimens promote tumor targeting of neurotensin radioligands
Neurotensin subtype 1 receptors (NTS1R) represent attractive molecular targets for directing radiolabeled neurotensin (NT) analogs to tumor lesions for diagnostic and therapeutic purposes. This approach has been largely undermined by the rapid in vivo degradation of linear NT-based radioligands. Herein, we aim to increase the tumor targeting of three99mTc-labeled NT analogs by the in-situ inhibition of two key proteases involved in their catabolism. DT1 ([N4- Gly7]NT(7-13)), DT5 ([N4-ÎČAla7,Dab9]NT(7-13)), and DT6 ([N4-ÎČAla7,Dab9,Tle12]]NT(7-13)) were labeled with99mTc. Their profiles were investigated in NTS1R-positive colon adenocarcinoma WiDr cells and mice treated or not with the neprilysin (NEP)-inhibitor phosphoramidon (PA) and/or the angiotensin converting enzyme (ACE)-inhibitor lisinopril (Lis). Structural modifications led to the partial stabilization of99mTc-DT6 in peripheral mice blood (55.1 ± 3.9% intact), whereas99mTc-D
Bis(Disulfide)-Bridged Somatostatin-14 Analogs and Their [<sup>111</sup>In]In-Radioligands:Synthesis and Preclinical Profile
The overexpression of one or more somatostatin receptors (SST1â5R) in human tumors has provided an opportunity for diagnosis and therapy with somatostatin-like radionuclide carriers. The application of âpansomatostatinâ analogs is expected to broaden the clinical indications and upgrade the diagnostic/therapeutic efficacy of currently applied SST2R-prefering radioligands. In pursuit of this goal, we now introduce two bicyclic somatostatin-14 (SS14) analogs, AT5S (DOTA-Ala1-Gly2-c[Cys3-Lys4-Asn5-c[Cys6-Phe7-DTrp8-Lys9-Thr10-Cys11]-Thr12-Ser13-Cys14]) and AT6S (DOTA-Ala1-Gly2-c[Cys3-Lys4-c[Cys5-Phe6-Phe7-DTrp8-Lys9-Thr10-Phe11-Cys12]-Ser13-Cys14]), suitable for labeling with trivalent radiometals and designed to sustain in vivo degradation. Both AT5S and AT6S and the respective [111In]In-AT5S and [111In]In-AT6S were evaluated in a series of in vitro assays, while radioligand stability and biodistribution were studied in mice. The 8/12-mer bicyclic AT6S showed expanded affinity for all SST1â5R and agonistic properties at the SST2R, whereas AT5S lost all affinity to SST1â5R. Both [111In]In-AT5S and [111In]In-AT6S remained stable in the peripheral blood of mice, while [111In]In-AT6S displayed low, but specific uptake in AR4-2J tumors and higher uptake in HEK293-SST3R tumors in mice. In summary, high radioligand stability was acquired by the two disulfide bridges introduced into the SS14 motif, but only the 8/12-mer ring AT6S retained a pansomatostatin profile. In consequence, [111In]In-AT6S targeted SST2R-/SST3R-positive xenografts in mice. These results call for further research on pansomatostatin-like radioligands for cancer theranostics.</p
Bis(Disulfide)-Bridged Somatostatin-14 Analogs and Their [<sup>111</sup>In]In-Radioligands:Synthesis and Preclinical Profile
The overexpression of one or more somatostatin receptors (SST1â5R) in human tumors has provided an opportunity for diagnosis and therapy with somatostatin-like radionuclide carriers. The application of âpansomatostatinâ analogs is expected to broaden the clinical indications and upgrade the diagnostic/therapeutic efficacy of currently applied SST2R-prefering radioligands. In pursuit of this goal, we now introduce two bicyclic somatostatin-14 (SS14) analogs, AT5S (DOTA-Ala1-Gly2-c[Cys3-Lys4-Asn5-c[Cys6-Phe7-DTrp8-Lys9-Thr10-Cys11]-Thr12-Ser13-Cys14]) and AT6S (DOTA-Ala1-Gly2-c[Cys3-Lys4-c[Cys5-Phe6-Phe7-DTrp8-Lys9-Thr10-Phe11-Cys12]-Ser13-Cys14]), suitable for labeling with trivalent radiometals and designed to sustain in vivo degradation. Both AT5S and AT6S and the respective [111In]In-AT5S and [111In]In-AT6S were evaluated in a series of in vitro assays, while radioligand stability and biodistribution were studied in mice. The 8/12-mer bicyclic AT6S showed expanded affinity for all SST1â5R and agonistic properties at the SST2R, whereas AT5S lost all affinity to SST1â5R. Both [111In]In-AT5S and [111In]In-AT6S remained stable in the peripheral blood of mice, while [111In]In-AT6S displayed low, but specific uptake in AR4-2J tumors and higher uptake in HEK293-SST3R tumors in mice. In summary, high radioligand stability was acquired by the two disulfide bridges introduced into the SS14 motif, but only the 8/12-mer ring AT6S retained a pansomatostatin profile. In consequence, [111In]In-AT6S targeted SST2R-/SST3R-positive xenografts in mice. These results call for further research on pansomatostatin-like radioligands for cancer theranostics.</p
Impact of clinically tested NEP/ACE inhibitors on tumor uptake of [111In-DOTA]MG11
Background: We have recently shown that treatment of mice with the neutral endopeptidase (NEP) inhibitor phosphoramidon (PA) improves the bioavailability and tumor uptake of biodegradable radiopeptides. For the truncated gastrin radiotracer [111In-DOTA]MG11 ([(DOTA)DGlu10]gastrin(10â17)), this method led to impressively high tumor-to-kidney ratios. Translation of this concept in the clinic requires the use of certified NEP inhibitors, such as thiorphan (TO) and its orally administered prodrug racecadotril (Race). Besides NEP, angiotensin-converting enzyme (ACE) has also been implicated in the catabolism of gastrin analogs. In the present study, we first compared the effects induced by NEP inhibition (using PA, TO, or Race) and/or by ACE inhibition (using lisinopril, Lis) on the biodistribution profile of [111In-DOTA]MG11 in mice. In addition, we compared the efficacy of PA and TO at different administered doses to enhance tumor uptake. Methods: [111In-DOTA]MG11 was coinjected with (a) vehicle, (b) PA (300 Όg), (c) TO (150 Όg), (d) Lis (100 Όg), (e) PA (300 Όg) plus Lis (100 Όg), or (f) 30â40 min after intraperitoneal (ip) injection of Race (3 mg) in SCID mice bearing AR42J xenografts. In addition, [111In-DOTA]MG11 was coinjected with vehicle, or with progressively increasing amounts of PA (3, 30, or 300 Όg) or TO (1.5, 15, and 150 Όg) in SCID mice bearing twin A431-CCK2R(+/â) tumors. In all above cases, biodistribution was conducted at 4 h postinjection (pi). Results: During NEP inhibition, the uptake of [111In-DOTA]MG11 in the AR42J tumors impressively increased from 1.8 ± 1.0 % ID/g (controls) to 15.3 ± 4.7 % ID/g (PA) and 12.3 ± 3.6 % ID/g (TO), while with Race tumor values reached 6.8 ± 2.8 % ID/g. Conversely, Lis had no effect on tumor uptake and no additive effect when coinjected with PA. During the dose dependence study in mice, PA turned out to be more efficacious in enhancing tumor uptake of [111In-DOTA]MG11 in the CCK2R-positive tumors compared to equimolar amounts of TO. In all cases, renal accumulation remained low, resulting in notable increases of tumor-to-kidney ratios. Conclusions: This study has confirmed NEP as the predominant degrading enzyme of [111In-DOTA]MG11 and ruled out the involvement of ACE in the in vivo catabolism of the radiotracer. NEP inhibition with the clinically tested NEP
From preclinical development to clinical application : kit formulation for radiolabelling the minigastrin analogue CP04 with In-111 for a first-in-human clinical trial
Introduction A variety of radiolabelled minigastrin analogues targeting the cholecystokinin 2 (CCK2) receptor were developed and compared in a concerted preclinical testing to select the most promising radiotracer for diagnosis and treatment of medullary thyroid carcinoma (MTC). DOTA-DGlu-DGlu-DGlu-DGlu-DGlu-DGlu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 (CP04) after labelling with 111In displayed excellent characteristics, such as high stability, receptor affinity, specific and persistent tumour uptake and low kidney retention in animal models. Therefore, it was selected for further clinical evaluation within the ERA-NET project GRAN-T-MTC. Here we report on the development of a pharmaceutical freeze-dried formulation of the precursor CP04 for a first multi-centre clinical trial with 111In-CP04 in MTC patients. Materials and methods The kit formulation was optimised by adjustment of buffer, additives and radiolabelling conditions. Three clinical grade batches of a final kit formulation with two different amounts of peptide (10 or 50 Όg) were prepared and radiolabelled with 111In. Quality control and stability assays of both the kits and the resulting radiolabelled compound were performed by HPLC analysis. Results Use of ascorbic acid buffer (pH 4.5) allowed freeze-drying of the kit formulation with satisfactory pellet-formation. Addition of methionine and gentisic acid as well as careful selection of radiolabelling temperature was required to avoid extensive oxidation of the Met11-residue. Trace metal contamination, in particular Zn, was found to be a major challenge during the pharmaceutical filling process in particular for the 10 Όg formulation. The final formulations contained 10 or 50 Όg CP04, 25 mg ascorbic acid, 0.5 mg gentisic acid and 5 mg l-methionine. The radiolabelling performed by incubation of 200-250 MBq 111InCl3 at 90°C for 15 min resulted in reproducible radiochemical purity (RCP) > 94%. Kit-stability was proven for > 6 months at + 5°C and at + 25°C. The radiolabelled product was stable for > 4 h at + 25°C. Conclusion A kit formulation to prepare 111In-CP04 for clinical application was developed, showing high stability of the kit as well as high RCP of the final product
Comparative biodistribution of 12 ÂčÂčÂčIn-labelled gastrin/CCK2 receptor-targeting peptides.
PURPOSE: Cholecystokinin 2 (CCK-2) receptor overexpression has been demonstrated in various tumours such as medullary thyroid carcinomas and small-cell lung cancers. Due to this high expression, CCK-2 receptors might be suitable targets for radionuclide imaging and/or radionuclide therapy. Several CCK-2 receptor-binding radiopeptides have been developed and some have been tested in patients. Here we aimed to compare the in vivo tumour targeting properties of 12 (111)In-labelled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated gastrin/CCK2 receptor-binding peptides. METHODS: Two CCK8-based peptides and ten gastrin-based peptide analogues were tested. All peptides were conjugated with DOTA and labelled with (111)In. Biodistribution studies were performed in mice with subcutaneous CCK2/gastrin receptor-expressing tumours and with receptor-negative tumours contralaterally. Biodistribution was studied by counting dissected tissues at 1 and 4 h after injection. RESULTS: Both the CCK analogues displayed relatively low tumour uptake (approximately 2.5%ID/g) as compared to minigastrin analogues. Two linear minigastrin peptides (MG0 and sargastrin) displayed moderate tumour uptake at both 1 and 4 h after injection, but also very high kidney uptake (both higher than 48%ID/g). The linear MG11, lacking the penta-Glu sequence, showed lower tumour uptake and also low kidney uptake. Varying the N-terminal Glu residues in the minigastrin analogues led to improved tumour targeting properties, with PP-F11 displaying the optimal biodistribution. Besides the monomeric linear peptides, a cyclized peptide and a divalent peptide were tested. CONCLUSION: Based on these studies, optimal peptides for peptide receptor radionuclide targeting of CCK2/gastrin receptor-expressing tumours were the linear minigastrin analogue with six D-Glu residues (PP-F11), the divalent analogue MGD5 and the cyclic peptide cyclo-MG1. These peptides combined high tumour uptake with low kidney retention, and may therefore be good candidates for future clinical studies
Comparative biodistribution of 12 111In-labelled gastrin/CCK2 receptor-targeting peptides
Contains fulltext :
97745.pdf (publisher's version ) (Closed access)PURPOSE: Cholecystokinin 2 (CCK-2) receptor overexpression has been demonstrated in various tumours such as medullary thyroid carcinomas and small-cell lung cancers. Due to this high expression, CCK-2 receptors might be suitable targets for radionuclide imaging and/or radionuclide therapy. Several CCK-2 receptor-binding radiopeptides have been developed and some have been tested in patients. Here we aimed to compare the in vivo tumour targeting properties of 12 (111)In-labelled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated gastrin/CCK2 receptor-binding peptides. METHODS: Two CCK8-based peptides and ten gastrin-based peptide analogues were tested. All peptides were conjugated with DOTA and labelled with (111)In. Biodistribution studies were performed in mice with subcutaneous CCK2/gastrin receptor-expressing tumours and with receptor-negative tumours contralaterally. Biodistribution was studied by counting dissected tissues at 1 and 4 h after injection. RESULTS: Both the CCK analogues displayed relatively low tumour uptake (approximately 2.5%ID/g) as compared to minigastrin analogues. Two linear minigastrin peptides (MG0 and sargastrin) displayed moderate tumour uptake at both 1 and 4 h after injection, but also very high kidney uptake (both higher than 48%ID/g). The linear MG11, lacking the penta-Glu sequence, showed lower tumour uptake and also low kidney uptake. Varying the N-terminal Glu residues in the minigastrin analogues led to improved tumour targeting properties, with PP-F11 displaying the optimal biodistribution. Besides the monomeric linear peptides, a cyclized peptide and a divalent peptide were tested. CONCLUSION: Based on these studies, optimal peptides for peptide receptor radionuclide targeting of CCK2/gastrin receptor-expressing tumours were the linear minigastrin analogue with six D-Glu residues (PP-F11), the divalent analogue MGD5 and the cyclic peptide cyclo-MG1. These peptides combined high tumour uptake with low kidney retention, and may therefore be good candidates for future clinical studies
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