Efficiency of succinylated gelatin and amino acid infusions for kidney uptake reduction of radiolabeled αvß6-integrin targeting peptides: considerations on clinical safety profiles.

PURPOSE: 68Ga-Trivehexin is an investigational PET radiopharmaceutical (NCT05799274) targeting αvß6-integrin for PET imaging of carcinomas. 177Lu-D0301 is a structurally related therapeutic peptide tetramer. However, it showed considerable kidney uptake in rodents, impeding clinical applicability. We therefore evaluated the impact of different kidney protection strategies on the biodistribution of both agents in normal and tumor-bearing mice. METHODS: Ex-vivo biodistribution of 68Ga-Trivehexin (90 min p.i.) and 177Lu-D0301 (90 min and 24 h p.i.) was determined in healthy C57BL/6N and H2009 (human lung adenocarcinoma) xenografted CB17-SCID mice without and with co-infusion of 100 µL of solutions containing 2.5% arginine + 2.5% lysine (Arg/Lys), 4% succinylated gelatin (gelofusine, gelo), or combinations thereof. Arg/Lys was injected either i.p. 30 min before and after the radiopharmaceutical, or i.v. 2 min before the radiopharmaceutical. Gelo was administered either i.v. 2 min prior activity, or pre-mixed and injected together with the radiopharmaceutical (n = 5 per group). C57BL/6N mice were furthermore imaged by PET (90 min p.i.) and SPECT (24 h p.i.). RESULTS: Kidney uptake of 68Ga-Trivehexin in C57BL/6N mice was reduced by 15% (Arg/Lys i.p.), 25% (Arg/Lys i.v.), and 70% (gelo i.v.), 90 min p.i., relative to control. 177Lu-D0301 kidney uptake was reduced by 2% (Arg/Lys i.p.), 41% (Arg/Lys i.v.), 61% (gelo i.v.) and 66% (gelo + Arg/Lys i.v.) 24 h p.i., compared to control. Combination of Arg/Lys and gelo provided no substantial benefit. Gelo furthermore reduced kidney uptake of 177Lu-D0301 by 76% (90 min p.i.) and 85% (24 h p.i.) in H2009 bearing SCID mice. Since tumor uptake was not (90 min p.i.) or only slightly reduced (15%, 24 h p.i.), the tumor/kidney ratio was improved by factors of 3.3 (90 min p.i.) and 2.6 (24 h p.i.). Reduction of kidney uptake was demonstrated by SPECT, which also showed that the remaining activity was located in the cortex. CONCLUSIONS: The kidney uptake of both investigated radiopharmaceuticals was more efficiently reduced by gelofusine (61-85%) than Arg/Lys (25-41%). Gelofusine appears particularly suitable for reducing renal uptake of αvß6-integrin targeted 177Lu-labeled peptide multimers because its application led to approximately three times higher tumor-to-kidney ratios. Since the incidence of severe adverse events (anaphylaxis) with succinylated gelatin products (reportedly 0.0062-0.038%) is comparable to that of gadolinium-based MRI or iodinated CT contrast agents (0.008% and 0.04%, respectively), clinical use of gelofusine during radioligand therapy appears feasible if similar risk management strategies as for contrast agents are applied.

The influence of the combination of carboxylate and phosphinate pendant arms in 1,4,7-triazacyclononane-based chelators on their 68Ga labelling properties.

In order to compare the coordination properties of 1,4,7-triazacyclononane (tacn) derivatives bearing varying numbers of phosphinic/carboxylic acid pendant groups towards 68Ga, 1,4,7-triazacyclononane-7-acetic-1,4-bis(methylenephosphinic) acid (NOPA) and 1,4,7- triazacyclononane-4,7-diacetic-1-[methylene(2-carboxyethyl)phosphinic] acid (NO2AP) were synthesized using Mannich reactions with trivalent or pentavalent forms of H-phosphinic acids as phosphorus components. Stepwise protonation constants logK1-3 12.06, 3.90 and 1.95, and stability constants with GaIII and CuII, logKGaL 24.01 and logKCuL 16.66, were potentiometrically determined for NOPA. Both ligands were labelled with 68Ga and compared with NOTA (tacn-N,N',N″-triacetic acid) and NOPO, a TRAP-type [tacn-N,N',N″- tris(methylenephosphinic acid)] chelator. At pH 3, NOPO and NOPA showed higher labelling efficiency (binding with lower ligand excess) at both room temperature and 95 °C, compared to NO2AP and NOTA. Labelling efficiency at pH = 0-3 correlated with a number of phosphinic acid pendants: NOPO >> NOPA > NO2AP >> NOTA; however, it was more apparent at 95 °C than at room temperature. By contrast, NOTA was found to be labelled more efficiently at pH > 4 compared to the ligands with phosphinic acids. Overall, replacement of a single phosphinate donor with a carboxylate does not challenge 68Ga labelling of TRAP-type chelators. However, the presence of carboxylates facilitates labelling at neutral or weakly acidic pH.

Benefits of NOPO as chelator in gallium-68 peptides, exemplified by preclinical characterization of (68)Ga-NOPO-c(RGDfK).

The αvß3-integrin addressing cyclic pentapeptide cyclo(RGDfK) was conjugated to NOPO, 1,4,7-triazacyclononane-1,4-bis[methylene(hydroxymethyl)phosphinic acid]-7-[methylene(2-carboxyethyl)phosphinic acid], a bifunctional chelator with exceptional gallium-68 labeling properties. NOPO-c(RGDfK) and its Ga(III) and Cu(II) complexes showed high affinity to αvß3 integrin (IC50 = 0.94 ± 0.06, 1.02 ± 0.09, and 0.51 ± 0.06 nM, respectively). (68)Ga labeling of NOPO-c(RGDfK) in an automated GMP-compliant procedure was performed with near-quantitative radiochemical yield, using precursor amounts as low as 0.5 nmol (approximately 0.6 µg). (68)Ga-NOPO-c(RGDfK) was obtained with high purity (>99% by radio-HPLC/TLC) and, optionally, could be produced with specific activities up to 6 TBq/µmol. M21/M21L (human melanoma with high/low αvß3 integrin expression) xenografted athymic CD-1 nude mice were used for biodistribution, in vivo stability studies, and PET imaging. (68)Ga-NOPO-c(RGDfK) showed rapid and specific uptake in M21 tumor xenografts (2.02 ± 0.34% ID/g at 60 min p.i.) and was found stable in vivo. Its high hydrophilicity is reflected by an octanol-water distribution coefficient (log D = -4.6) which is more than 1 order of magnitude lower compared to respective NOTA or DOTA analogues. As expected, (68)Ga-NOPO-c(RGDfK) thus showed fast renal clearance from nontargeted tissues. We conclude that NOPO might generally prove a useful means to improve renal clearance of corresponding radiopharmaceuticals by increasing the polarity of its bioconjugates. Favorable labeling properties render NOPO conjugates highly recommendable for reliable routine production of (68)Ga-radiopharmaceuticals in a clinical setting.

Tailored Gallium(III) chelator NOPO: synthesis, characterization, bioconjugation, and application in preclinical Ga-68-PET imaging.

The bifunctional chelator NOPO (1,4,7-triazacyclononane-1,4-bis[methylene(hydroxymethyl)phosphinic acid]-7-[methylene(2-carboxyethyl)phosphinic acid]) shows remarkably high Ga(III) complexation efficiency and comprises one carboxylic acid moiety which is not involved into metal ion coordination. An improved synthetic protocol affords NOPO with 45% overall yield. Stepwise protonation constants (log Ka), determined by potentiometry, are 11.96, 5.22, 3.77, and 1.54; the stability constant of the Ga(III) complex is log KGaL = 25.0. Within 5 min, (68)Ga(III) incorporation by NOPO is virtually quantitative at room temperature between pH 3 and 4, and at 95 °C at pH ranging from 0.5 to 7, at NOPO concentrations of 30 µM and 10 µM, respectively. During amide bond formation at the distant carboxylate using the HATU coupling reagent, an intramolecular phosphinic acid ester (phosphilactone) is formed, which is cleaved during (68)Ga complexation or in acidic media, such as trifluoroacetic acid (TFA). Phosphilactone formation can also be suppressed by complexation of Zn(2+) prior to conjugation, the resulting zinc-containing conjugates nevertheless being suitable for direct (68)Ga-labeling. In AR42J (rat pancreatic carcinoma) xenografted CD-1 nude mice, (68)Ga-labeled NOPO-NaI(3)-octreotide conjugate ((68)Ga-NOPO-NOC) showed high and fully blockable tumor uptake (13.9 ± 5% ID/g, 120 min p.i., compared to 0.9 ± 0.4% ID/g with 5 mg/kg of nonlabeled peptide). Uptake in other tissues was generally below 3% ID/g, except appearance of excretion-related activity accumulation in kidneys. NOPO-functionalized compounds tend to be more hydrophilic than the corresponding DOTA- and NODAGA-conjugates, thus promoting fast and extensive renal excretion of (68)Ga-NOPO-radiopharmaceuticals. NOPO-functionalized peptides provide suitable pharmacokinetics in vivo and meet all requirements for efficient (68)Ga-labeling even at room temperature in a kit-like manner.

The importance of tyrosines in multimers of cyclic RGD nonapeptides: towards αvß6-integrin targeted radiotherapeutics.

In a recent paper in this journal (RSC Med. Chem., 2023, 14, 2429), we described an unusually strong impact of regiospecific exchange of phenylalanines by tyrosines in 10 gallium-68-labeled trimers of certain cyclic RGD peptides, c[XRGDLAXp(NMe)K] (X = F or Y), on non-specific organ uptakes. We found that there was, in part, no correlation of liver uptake with established polarity proxies, such as the octanol-water distribution coefficient (log D). Since this observation could not be explained straightforwardly, we suggested that the symmetry of the compounds had resulted in a synergistic interaction of certain components of the macromolecules. In the present work, we investigated whether a comparable effect also occurred for a series of 5 tetramers labeled with lutetium-177. We found that in contrast to the trimers, liver uptake of the tetramers was well correlated to their polarity, indicating that the unusual observations along the trimer series indeed was a unique feature, probably related to their particular symmetry. Since the Lu-177 labeled tetramers are also potential agents for treatment of a variety of αvß6-integrin expressing cancers, these were evaluated in mice bearing human lung adenocarcinoma xenografts. Due to their tumor-specific uptake and retention in biodistribution and SPECT imaging experiments, these compounds are considered a step forward on the way to αvß6-integrin-targeted anticancer agents. Furthermore, we noticed that the presence of tyrosines in general had a positive impact on the in vivo performance of our peptide multimers. In view of the fact that a corresponding rule was already proposed in the context of protein engineering, we argue in favor of considering peptide multimers as a special class of small or medium-sized proteins. In summary, we contend that the performance of peptide multimers is less determined by the in vitro characteristics (particularly, affinity and selectivity) of monomers, but rather by the peptides' suitability for the overall macromolecular design concept, and peptides containing tyrosines are preferred.

Potential Efficacy of 68 Ga-Trivehexin PET/CT and Immunohistochemical Validation of αvß6 Integrin Expression in Patients With Head and Neck Squamous Cell Carcinoma and Pancreatic Ductal Adenocarcinoma.

PURPOSE: αvß6 integrin is exclusively expressed in epithelial cells and is upregulated in many carcinomas, such as pancreatic ductal adenocarcinomas (PDACs) and head and neck squamous cell carcinomas (H&NSCCs). Trivehexin is a recently synthesized trimerized αvß6 integrin selective nonapeptide, which can be labeled with a positron emitter like 68 Ga. This is a pilot study to assess the potential role of 68 Ga-Trivehexin PET/CT in patients with H&NSCC and PDAC and their correlation with αvß6 integrin expression by the tumor tissue on immunohistochemistry (IHC). PATIENTS AND METHODS: Thirty-two patients with suspected H&NSCC (n = 20) or PDAC (n = 12) underwent whole-body 68 Ga-Trivehexin PET/CT and 18 F-FDG PET/CT scans on 2 separate days. All 32 patients underwent biopsy from the tumor site for histopathological diagnosis and IHC for αvß6 integrin expression. The degree of αvß6 integrin expression on IHC was scored using the immunoreactive score and modified 4-point immunoreactive score classification. RESULTS: The 68 Ga-Trivehexin PET images demonstrated increased tracer uptake (mean SUV max 5.9 ± 3.3) in the primary and metastatic lesions with good lesion delineation in 8 out of the 9 cases of PDACs. However, FDG PET showed increased tracer uptake in 7 cases (6.2 ± 2.6). Among various cases of H&NSCC, increased uptakes of 68 Ga-Trivehexin (6.6 ± 4.5) and 18 F-FDG (12.7 ± 6.7) were seen in 17 out of the 18 patients. The 2 cases of inflammatory changes with suspected disease recurrence showed increased tracer uptake in 18 F-FDG PET (7.98 ± 3.1) and no significant uptake in 68 Ga-Trivehexin PET (2.2 ± 0.34).IHC showed higher expression of αvß6 integrins in lesions with higher uptake of 68 Ga-Trivehexin. A higher sensitivity, specificity, and accuracy of 68 Ga-Trivehexin PET over 18 F-FDG PET was seen for detection of primary and metastatic lesions. CONCLUSIONS: 68 Ga-Trivehexin is a promising noninvasive molecular imaging agent for tumors expressing αvß6 integrin, especially in cases where 18 F-FDG PET/CT scan may be suboptimal due to its low uptake, or due to its nonspecific uptake around tumor sites.

A cyclen-based tetraphosphinate chelator for the preparation of radiolabeled tetrameric bioconjugates.

The cyclen-based tetraphosphinate chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis[methylene(2-carboxyethyl)phosphinic acid] (DOTPI) comprises four additional carboxylic acid moieties for bioconjugation. The thermodynamic stability constants (logK(ML)) of metal complexes, as determined by potentiometry, were 23.11 for Cu(II), 20.0 for Lu(III), 19.6 for Y(III), and 21.0 for Gd(III). DOTPI was functionalized with four cyclo(Arg-Gly-Asp-D-Phe-Lys) (RGD) peptides through polyethylene glycol (PEG4) linkers. The resulting tetrameric conjugate DOTPI(RGD)4 was radiolabeled with (177)Lu and (64)Cu and showed improved labeling efficiency compared with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). The labeled compounds were fully stable in transchelation challenges against trisodium diethylenetriaminepentaacetate (DTPA) and disodium ethylenediaminetetraacetic acid (ETDA), in phosphate buffered saline (PBS), and human plasma. Integrin αvß3 affinities of the non-radioactive Lu(III) and Cu(II) complexes of DOTPI(RGD)4 were 18 times higher (both IC50 about 70 picomolar) than that of the c(RGDfK) peptide (IC50 = 1.3 nanomolar). Facile access to tetrameric conjugates and the possibility of radiolabeling with therapeutic and diagnostic radionuclides render DOTPI suitable for application in peptide receptor radionuclide imaging (PRRI) and therapy (PRRT).

Complexation of metal ions with TRAP (1,4,7-triazacyclononane phosphinic acid) ligands and 1,4,7-triazacyclononane-1,4,7-triacetic acid: phosphinate-containing ligands as unique chelators for trivalent gallium.

Three phosphinic acid 1,4,7-triazacyclononane (TACN) derivatives bearing methylphosphinic (TRAP-H), methyl(phenyl)phosphinic (TRAP-Ph), or methyl(hydroxymethyl)phosphinic acid (TRAP-OH) pendant arms were investigated as members of a new family of efficient Ga(3+) chelators, TRAP ligands (triazacyclononane phosphinic acids). Stepwise protonation constants of ligands and stability constants of their complexes with Ga(3+), selected divalent metal, and Ln(3+) ions were determined by potentiometry. For comparison, equilibrium data for the metal ion-NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) systems were redetermined. These ligands exhibit high thermodynamic selectivity for Ga(3+) over the other metal ions (log K(GaL) - log K(ML) = 7-9) and a selective complexation of smaller Mg(2+) over Ca(2+). Stabilities of the Ga(3+) complexes are dependent on the basicity of the donor atoms: [Ga(NOTA)] (log K(GaL) = 29.6) > [Ga(TRAP-OH)] (log K(GaL) = 23.3) > [Ga(TRAP-H)] (log K(GaL) = 21.9). The [Ga(TRAP-OH)] complex exhibits unusual reversible rearrangement of the "in-cage" N(3)O(3) complex to the "out-of-cage" O(6) complex. The in-cage complex is present in acidic solutions, and at neutral pH, Ga(3+) ion binds hydroxide anion, induces deprotonation and coordination of the P-hydroxymethyl group(s), and moves out of the macrocyclic cavity; the hypothesis is supported by a combination of results from potentiometry, multinuclear nuclear magnetic resonance spectrometry, and density functional theory calculations. Isomerism of the phosphinate Ga(3+) complexes caused by a combination of the chelate ring conformation, the helicity of coordinated pendant arms, and the chirality of the coordinated phosphinate groups was observed. All Ga(3+) complexes are kinetically inert in both acidic and alkaline solutions. Complex formation studies in acidic solutions indicate that Ga(3+) complexes of the phosphinate ligands are formed quickly (minutes) and quantitatively even at pH <2. Compared to common Ga(3+) chelators (e.g., 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) derivatives), these novel ligands show fast complexation of Ga(3+) over a broad pH range. The discussed TRAP ligands are suitable alternatives for the development of (68)Ga radiopharmaceuticals.

In-House Preparation and Quality Control of Ac-225 Prostate-Specific Membrane Antigen-617 for the Targeted Alpha Therapy of Castration-Resistant Prostate Carcinoma.

PURPOSE: Ac-225 labeled with prostate-specific membrane antigen (PSMA-617), a transmembrane glycoprotein which is highly expressed in prostate carcinoma cells, is presently being considered a promising agent of targeted alpha therapy for the treatment of patients suffering from metastatic castration-resistant prostate cancer. In the present study, we report an optimized protocol for the preparation of therapeutic dose of Ac-225 PSMA-617 with high yield and radiochemical purity (RCP). METHODS: Ac-225 PSMA-617 was prepared by adding the peptidic precursor-PSMA-617 (molar ratios, Ac-225: PSMA-617 = 30:1) in 1 ml ascorbate buffer to Ac-225 and heating the reaction mixture at 90°C for 25 min to obtain the radiopeptide with high RCP and yield. The radiolabeled peptide was administered in patients who met the eligibility criteria and posttherapy assessment was done. RESULTS: Ten batches of Ac-225 PSMA-617 were prepared following this protocol. The radiopeptide was obtained with an adequate yield of 85%-87% and RCP of 97%-99%. CONCLUSION: The current protocol allows single-step, successful, routine inhouse radiolabeling of Ac-225 with PSMA-617 with high yield and RCP.

Radiation Exposure to the Nuclear Medicine Personnel During Preparation and Handling of 213Bi-Radiopharmaceuticals.

Because of the excellent ability of α-particles to transfer a high amount of energy over a short tissue range, targeted α-therapy has been attracting rising numbers of nuclear medicine centers. In this study, we estimated the radiation exposure to the occupational workers with pocket dosimeters during handling of the α-emitter 213Bi, used for targeted α-therapy of neuroendocrine tumor and castration-resistant prostate cancer patients. The dose rates from patients at different distances and time points after injection of the therapy were also evaluated. Methods: This prospective study was done in the Department of Nuclear Medicine at Fortis Memorial Research Institute, Gurgaon, India. Twelve patients with neuroendocrine tumors or castration-resistant prostate cancer were enrolled to receive 213Bi-DOTATOC or 213Bi-prostate-specific membrane antigen therapy, respectively. Each patient received 2-3 intravenous injections of 213Bi-peptide, 266-362 MBq (7.2-9.8 mCi) in a single cycle over 2-3 d. The radiation exposure to nuclear medicine personnel at the chest and extremity levels was assessed for tasks such as elution, dispensing, injecting, and collecting blood samples. Radiation levels were measured at distances of 1 cm and 1 m from patients immediately after, and at 1, 2, and 4 h after, the administration of 213Bi-peptide. Results: The external dose incurred at the chest level by radiopharmacists during synthesis, by physicians during injection, by technologists during imaging, and by nurses during sample collection was 2-7 µSv/procedure. The extremity dose was 1-14 µSv/procedure. The dose rate at 1 m from patients immediately after 213Bi-radiopharmaceutical injection was 0.02-0.03 µSv/MBq⋅h. Conclusion: The external radiation doses received by occupational workers involved in various procedures were far below the limit prescribed by the regulatory authority (20 mSv/y).

Efficient formation of inert Bi-213 chelates by tetraphosphorus acid analogues of DOTA: towards improved alpha-therapeutics.

BACKGROUND: The recently growing interest in targeted alpha-therapy (TAT) calls for improvement of the labelling chemistry of the corresponding radionuclides. 213BiIII is a short-lived alpha emitter which emits only one alpha particle in its decay chain. Hence, it might be safer in application than other respective nuclides, such as 223Ra or 225Ac, because no alpha-emitting daughters are released upon recoil. We investigated cyclen derivatives with phosphorus-containing pendant arms regarding their suitability for 213Bi labelling. RESULTS: The concentration dependency of 213Bi labelling at 25 °C and 95 °C was determined for DOTP, DOTPH, DOTPEt, and DOTPI, as well as for DOTA and CHX-A"-DTPA for comparison. The labelling efficiency of the phosphorus-containing ligands was at least comparable to CHX-A"-DTPA and exceeded that of DOTA. DOTP was most efficient, requiring chelator concentrations for labelling which were approx. two orders of magnitude lower than those required for CHX-A"-DTPA, both at 25 °C and 95 °C. The 213Bi complexes of phosphorus ligands furthermore showed a higher stability against demetallation (> 96% of intact complex after 120-min incubation in plasma were found for DOTP, DOTPH, and DOTPEt, compared to 85% for DOTA and 76% for CHX-A"-DTPA). CONCLUSION: Cyclen derivatives bearing four N-methylenephosphonic or -phosphinic acid substituents, e.g., DOTP, are capable of complexing the alpha-emitting radionuclide 213BiIII with higher efficiency and in-vitro stability than the current gold standards DOTA and CHX-A"-DTPA.

Twins in spirit - episode I: comparative preclinical evaluation of [(68)Ga]DOTATATE and [(68)Ga]HA-DOTATATE.

BACKGROUND: Recently, an intra-patient comparison demonstrated that the somatostatin (sst) ligand [(68)Ga]HA-DOTATATE ([(68)Ga]DOTA-3-iodo-Tyr(3)-octreotate) provides PET images comparable to or superior to those obtained with [(68)Ga]DOTATATE. To provide a comprehensive basis for nevertheless observed slight differences in tracer biodistribution and dosimetry, the characteristics of [(68)Ga]HA-DOTATATE were investigated in a detailed preclinical study. METHODS: Affinities of (nat)Ga-HA-DOTATATE and (nat)Ga-DOTATATE to sst1-5 were determined using membrane preparations and [(125)I]SST-28 as radioligand. Internalization into AR42J cells was studied in dual-tracer studies with [(125)I]TOC as internal reference. Biodistribution was investigated using AR42J tumor-bearing CD1 mice, and specificity of tracer uptake was confirmed in competition studies by coinjection of 0.8 mg TOC/kg. RESULTS: Sst2 affinities (IC50) of [(nat)Ga]HA-DOTATATE (1.4 ± 0.8 nM, logP: -3.16) and [(nat)Ga]DOTATATE (1.2 ± 0.6 nM, logP: -3.69) were nearly identical. Both compounds displayed IC50 > 1 µM for sst1,3,4, while sst5 affinity was markedly increased for (nat)Ga-HA-DOTATATE (102 ± 65 nM vs >1 µM for (nat)Ga-DOTATATE). [(nat)Lu]HA-DOTATATE and [(nat)Lu]DOTATATE showed slightly lower, identical sst2 affinities (2.0 ± 1.6 and 2.0 ± 0.8 nM, respectively) and sst3 affinities of 93 ± 1 and 162 ± 16 nM. Internalization of [(68)Ga]HA-DOTATATE was tenfold higher than that of [(125)I]TOC but only sixfold higher for [(68)Ga]DOTATATE and [(177)Lu]HA-DOTATATE. While [(68)Ga]HA-DOTATATE and [(68)Ga]DOTATATE had shown similar target- and non-target uptake in patients, biodistribution studies in mice at 1 h post injection (n = 5) revealed slightly increased non-specific uptake of [(68)Ga]HA-DOTATATE in the blood, liver, and intestines (0.7 ± 0.3, 1.0 ± 0.2, and 4.0 ± 0.7 %iD/g vs 0.3 ± 0.1, 0.5 ± 0.1, and 2.7 ± 0.8 %iD/g for [(68)Ga]DOTATATE). However, sst-mediated accumulation of [(68)Ga]HA-DOTATATE in the pancreas, adrenals, and tumor was significantly enhanced (36.6 ± 4.3, 10.8 ± 3.2, and 33.6 ± 10.9 %iD/g vs 26.1 ± 5.0, 5.1 ± 1.4, and 24.1 ± 4.9 %iD/g, respectively). Consequently, tumor/background ratios for [(68)Ga]HA-DOTATATE in the AR42J model are comparable or slightly increased compared to [(68)Ga]DOTATATE. CONCLUSIONS: The present preclinical data fully confirm the general biodistribution pattern and excellent in vivo sst-targeting characteristics previously observed for [(68)Ga]HA-DOTATATE in patients. The effect of slightly enhanced lipophilicity on background accumulation and normal organ dose is compensated by the high uptake of [(68)Ga]HA-DOTATATE in tumor. Thus, [(68)Ga]HA-DOTATATE represents a fully adequate, freely available substitute for [(68)Ga]DOTATATE and, given the superb sst-targeting characteristics of [(177)Lu]HA-DOTATATE in vitro, potential applicability for sst-targeted PRRT.

MA-NOTMP: A Triazacyclononane Trimethylphosphinate Based Bifunctional Chelator for Gallium Radiolabelling of Biomolecules.

In the past few years, gallium-68 has demonstrated significant potential as a radioisotope for positron emission tomography (PET), and the optimization of chelators for gallium coordination is a major goal in the development of radiopharmaceuticals. Methylaminotriazacyclononane trimethylphosphinate (MA-NOTMP), a new C-functionalized triazacyclononane derivative with phosphinate pendant arms, presents excellent coordination properties for (68) Ga (low ligand concentration, labelling at low pH even at room temperature). A "ready-to-be-grafted" bifunctional chelating agent (p-NCS-Bz-MA-NOTMP) was prepared to allow (68) Ga labelling of sensitive biological vectors. Conjugation to a bombesin(7-14) derivative was performed, and preliminary in vitro experiments demonstrated the potential of MA-NOTMP in the development of radiopharmaceuticals. This new chelator is therefore of major interest for labelling sensitive biomolecules, and further in vivo experiments will soon be performed.

68Ga- and 177Lu-Labeled PSMA I&T: Optimization of a PSMA-Targeted Theranostic Concept and First Proof-of-Concept Human Studies.

UNLABELLED: On the basis of the high and consistent expression of prostate-specific membrane antigen (PSMA) in metastatic prostate cancer (PC), the goal of this study was the development, preclinical evaluation, and first proof-of-concept investigation of a PSMA inhibitor for imaging and therapy (PSMA I&T) for (68)Ga-based PET and (177)Lu-based endoradiotherapeutic treatment in patients with metastatic and castration-resistant disease. METHODS: PSMA I&T was synthesized in a combined solid phase and solution chemistry strategy. The PSMA affinity of (nat)Ga-/(nat)Lu-PSMA I&T was determined in a competitive binding assay using LNCaP cells. Internalization kinetics of (68)Ga- and (177)Lu-PSMA I&T were investigated using the same cell line, and biodistribution studies were performed in LNCaP tumor-bearing CD-1 nu/nu mice. Initial human PET imaging studies using (68)Ga-PSMA I&T, as well as endoradiotherapeutic treatment of 2 patients with metastatic PC using (177)Lu-PSMA I&T, were performed. RESULTS: PSMA I&T and its cold gallium and lutetium analog revealed nanomolar affinity toward PSMA. The DOTAGA (1,4,7,10-tetraazacyclododecane-1-(glutamic acid)-4,7,10-triacetic acid) conjugate PSMA I&T allowed fast and high-yield labeling with (68)Ga(III) and (177)Lu(III). Uptake of (68)Ga-/(177)Lu-PSMA I&T in LNCaP tumor cells is highly efficient and PSMA-specific, as demonstrated by competition studies both in vitro and in vivo. Tumor targeting and tracer kinetics in vivo were fast, with the highest uptake in tumor xenografts and kidneys (both PSMA-specific). First-in-human (68)Ga-PSMA I&T PET imaging allowed high-contrast detection of bone lesions, lymph node, and liver metastases. Endoradiotherapy with (177)Lu-PSMA I&T in 2 patients was found to be effective and safe with no detectable side effects. CONCLUSION: (68)Ga-PSMA I&T shows potential for high-contrast PET imaging of metastatic PC, whereas its (177)Lu-labeled counterpart exhibits suitable targeting and retention characteristics for successful endoradiotherapeutic treatment. Prospective studies on larger cohorts of patients are warranted and planned.

Disclosing the CXCR4 expression in lymphoproliferative diseases by targeted molecular imaging.

Chemokine ligand-receptor interactions play a pivotal role in cell attraction and cellular trafficking, both in normal tissue homeostasis and in disease. In cancer, chemokine receptor-4 (CXCR4) expression is an adverse prognostic factor. Early clinical studies suggest that targeting CXCR4 with suitable high-affinity antagonists might be a novel means for therapy. In addition to the preclinical evaluation of [(68)Ga]Pentixafor in mice bearing human lymphoma xenografts as an exemplary CXCR4-expressing tumor entity, we report on the first clinical applications of [(68)Ga]Pentixafor-Positron Emission Tomography as a powerful method for CXCR4 imaging in cancer patients. [(68)Ga]Pentixafor binds with high affinity and selectivity to human CXCR4 and exhibits a favorable dosimetry. [(68)Ga]Pentixafor-PET provides images with excellent specificity and contrast. This non-invasive imaging technology for quantitative assessment of CXCR4 expression allows to further elucidate the role of CXCR4/CXCL12 ligand interaction in the pathogenesis and treatment of cancer, cardiovascular diseases and autoimmune and inflammatory disorders.

Phosphinic acid functionalized polyazacycloalkane chelators for radiodiagnostics and radiotherapeutics: unique characteristics and applications.

Given the wide application of positron emission tomography (PET), positron-emitting metal radionuclides have received much attention recently. Of these, gallium-68 has become particularly popular, as it is the only PET nuclide commercially available from radionuclide generators, therefore allowing local production of PET radiotracers independent of an on-site cyclotron. Hence, interest in optimized bifunctional chelators for the elaboration of (68) Ga-labeled bioconjugates has been rekindled as well, resulting in the development of improved triazacyclononane-triphosphinate (TRAP) ligand structures. The most remarkable features of these ligands are unparalleled selectivity for Ga(III) , rapid Ga(III) complexation kinetics, extraordinarily high thermodynamic stability, and kinetic inertness of the respective Ga(III) chelates. As a result, TRAP chelators exhibit very favorable (68) Ga-labeling properties. Based on the scaffolds NOPO (1,4,7-triazacyclononane-1,4-bis[methylene(hydroxymethyl)phosphinic acid]-7-[methylene(2-carboxyethyl)phosphinic acid]) and TRAP-Pr, tailored for convenient preparation of (68) Ga-labeled monomeric and multimeric bioconjugates, a variety of novel (68) Ga radiopharmaceuticals have been synthesized. These include bisphosphonates, somatostatin receptor ligands, prostate-specific membrane antigen (PSMA)-targeting peptides, and cyclic RGD pentapeptides, for in vivo PET imaging of bone, neuroendocrine tumors, prostate cancer, and integrin expression, respectively. Furthermore, TRAP-based (68) Ga-labeled gadolinium(III) complexes have been proposed as bimodal probes for PET/MRI, and a cyclen-based analogue of TRAP-Pr has been suggested for the elaboration of targeted radiotherapeutics comprising radiolanthanide ions. Thus, polyazacycloalkane-based polyphosphinic acid chelators are a powerful toolbox for pharmaceutical research, particularly for the development of (68) Ga radiopharmaceuticals.

Synthesis and preclinical evaluation of DOTAGA-conjugated PSMA ligands for functional imaging and endoradiotherapy of prostate cancer.

BACKGROUND: Due to its high expression in prostate cancer, PSMA (prostate-specific membrane antigen) represents an ideal target for both diagnostic imaging and endoradiotherapeutic approaches. Based on a previously published highly specific PSMA ligand ([(68)Ga]DOTA-FFK(Sub-KuE)), we developed a corresponding metabolically stable 1,4,7,10-tetraazacyclododececane,1-(glutaric acid)-4,7,10-triacetic acid (DOTAGA) construct for theranostic treatment of prostate cancer. METHODS: All ligands were synthesized by a combined solid phase and solution phase synthesis strategy. The affinity of the (nat)gallium and lutetium complexes to PSMA and the internalization efficiency of the radiotracers were determined on PSMA-expressing LNCaP cells. The (68)Ga- and (177)Lu-labelled ligands were further investigated for lipophilicity, binding specificity, metabolic stability, as well as biodistribution and µPET in LNCaP-tumour-bearing mice. RESULTS: Radiochemical yields for (68)Ga (3 nmol, 5.0 M NaCl/2.7 M HEPES (approximately 5/1), pH 3.5 to 4.5, 5 min, 95°C) and (177)Lu labelling (0.7 nmol, 0.1 M NH4OAc, pH 5.5, 30 min, 95°C) were almost quantitative, resulting in specific activities of 250 to 300 GBq/µmol for the (68)Ga analogues and 38 GBq/µmol for (177)Lu complexes. Due to metabolic instability of L-amino acid spacers, D-amino acids were implemented resulting in a metabolically stable DOTAGA ligand. Compared to the DOTA ligand, the DOTAGA derivatives showed higher hydrophilicity (logP = -3.6 ± 0.1 and -3.9 ± 0.1 for (68)Ga and (177)Lu, respectively) and improved affinity to PSMA resulting in an about twofold increased specific internalization of the (68)Ga- and (177)Lu-labelled DOTAGA analogue. Especially, [(68)Ga]DOTAGA-ffk(Sub-KuE) exhibits favourable pharmacokinetics, low unspecific uptake and high tumour accumulation in LNCaP-tumour-bearing mice. CONCLUSIONS: The pair of diagnostic/therapeutic PSMA-ligands [(68)Ga/(177)Lu]DOTAGA-ffk(Sub-KuE) possess remarkable potential for the management of prostate cancer.

How is (68)Ga labeling of macrocyclic chelators influenced by metal ion contaminants in (68)Ge/(68)Ga generator eluates?

To assess the influence of Zn(2+) , Cu(2+) , Fe(3+) , Al(3+) , Ti(IV) , and Sn(IV) on incorporation of (68) Ga(3+) into pendant-arm macrocyclic chelators, the (68) Ga labeling of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-tris[methyl(2-carboxyethyl)phosphinic acid]) (TRAP), and 1,4,7-triazacyclononane-1-[methyl(2-carboxyethyl)phosphinic acid]-4,7-bis[methyl(2-hydroxymethyl)phosphinic acid] (NOPO), as well as their peptide conjugates, was investigated in the presence of varying concentrations of these metal ions. The (68) Ga labeling yield for carboxylate-type chelators NOTA and DOTA is decreased at lower metal ion contaminant concentrations compared with phosphinate-type chelators TRAP and NOPO. The latter are able to rapidly exchange coordinated Zn(II) with (68) Ga(3+) , as confirmed by mass spectrometry and (31) P NMR spectroscopy. (68) Ga labeling of Zn(II) complexes of TRAP and NOPO proceeds as efficient as labeling of neat NOTA; this applies also to the corresponding peptide conjugates of these chelators. This behavior results in substantially improved selectivity for Ga(3+) and, therefore, in more robust and reliable (68) Ga labeling procedures. In addition, none of the investigated chelators binds (68) Ge, rendering post-labeling purification protocols, for example, solid-phase extraction, a reliable means of removal of (68) Ge contamination from (68) Ga radiopharmaceuticals.

Copper-64 labelling of triazacyclononane-triphosphinate chelators.

The 1,4,7-triazacyclononane-1,4,7-tris(methylenephosphinic acid) chelators TRAP and NOPO are complexing copper-64 with similar efficiency as 1,4,7-triazacyclononane-triacetic acid (NOTA). The kinetic stability of Cu-64-labelled TRAP-peptides is sufficient for PET imaging at early time points (1-2 h post injection). For labelling of TRAP conjugates, Cu-64 can be recommended as an alternative to Ga-68 to achieve higher resolution of PET images.