DOTA conjugate with an albumin-binding entity enables the first folic acid-targeted 177Lu-radionuclide tumor therapy in mice.

UNLABELLED: The folate receptor (FR) has proven a valuable target for nuclear imaging using folic acid radioconjugates. However, using folate-based radiopharmaceuticals for therapy has long been regarded as an unattainable goal because of their considerable renal accumulation. Herein, we present a novel strategy in which a DOTA-folate conjugate with an albumin-binding entity (cm09) was designed with the aim of prolonging circulation in the blood and therewith potentially improving tumor-to-kidney ratios. METHODS: The folate conjugate cm09 was radiolabeled with (177)LuCl(3), and stability experiments were performed in plasma. Cell uptake studies were performed on FR-positive KB tumor cells, and an ultrafiltration assay was used to determine the plasma protein-binding properties of (177)Lu-cm09. In vivo, (177)Lu-cm09 was tested in KB tumor-bearing mice using SPECT/CT. The therapeutic anticancer effect of (177)Lu-cm09 (20 MBq) applied as a single injection or as fractionated injections was investigated in different groups of mice (n = 5) by monitoring tumor size and the survival time of treated mice, compared with untreated controls. RESULTS: Compound cm09 was radiolabeled at a specific activity of 40 MBq/nmol, a radiochemical yield of more than 98%, and a stability of more than 99% over 5 d in plasma. Ultrafiltration revealed significant binding of (177)Lu-cm09 to serum proteins (∼91%) in plasma, compared with folate radioconjugate without an albumin-binding entity. Cell uptake and internalization of (177)Lu-cm09 was FR-specific and comparable to other folate radioconjugates. In vivo studies resulted in high tumor uptake (17.56 percentage injected dose per gram [%ID/g] at 4 h after injection), which was almost completely retained for at least 72 h. Renal accumulation was significantly reduced (28 %ID/g at 4 h after injection), compared with folate conjugates that lack an albumin-binding entity (∼70 %ID/g at 4 h after injection). These circumstances enabled SPECT imaging of excellent quality. Radionuclide therapy (1 × 20 MBq) revealed complete remission of tumors in 4 of 5 cases and a significantly prolonged survival time, compared with untreated controls. CONCLUSION: The modification of a folate radioconjugate with an albumin-binding entity resulted in a significant increase of the tumor-to-kidney ratio of radioactivity, enabling for the first time, to our knowledge, the preclinical application of folic acid-targeted radionuclide therapy in mice.

Radioiodinated folic acid conjugates: evaluation of a valuable concept to improve tumor-to-background contrast.

Folic acid radioconjugates can be used for targeting folate receptor positive (FR(+)) tumors. However, the high renal uptake of radiofolates is a drawback of this strategy, particularly with respect to a therapeutic application due to the risk of damage to the kidneys by particle radiation. The goal of this study was to develop and evaluate radioiodinated folate conjugates as a novel class of folate-based radiopharmaceuticals potentially suitable for therapeutic application. Two different folic acid conjugates, tyrosine-folate (1) and tyrosine-click-folate (3), were synthesized and radioiodinated using the Iodogen method resulting in [(125)I]-2 and [(125/131)I]-4. Both radiofolates were highly stable in mouse and human plasma. Determination of FR binding affinities using (3)H-folic acid and FR(+) KB tumor cells revealed affinities in the nanomolar range for 2 and 4. The cell uptake of [(125)I]-2 and [(125/131)I]-4 proved to be FR specific as it was blocked by the coincubation of folic acid. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in vitro assays were employed for the determination of tumor cell viability upon exposure to [(131)I]-4. Compared to untreated control cells, significantly reduced cell viability was observed for FR(+) cancer cells (KB, IGROV-1, SKOV-3), while FR(-) cells (PC-3) were not affected. Biodistribution studies performed in tumor bearing nude mice showed the specific accumulation of both radiofolates in KB tumor xenografts ([(125)I]-2: 3.43 ± 0.28% ID/g; [(125)I]-4: 2.28 ± 0.46% ID/g, 4 h p.i.) and increasing tumor-to-kidney ratios over time. The further improvement of the tumor-to-background contrast was achieved by preinjection of the mice with pemetrexed allowing excellent imaging via single-photon emission computed tomography (SPECT/CT). These findings confirmed the hypothesis that the application of radioiodinated folate conjugates may be a valuable concept to improve tumor-to-background contrast. The inhibitory effect of [(131)I]-4 on FR(+) cancer cells in vitro indicates the potential of this class of radiofolates for therapeutic application.

Molecular assembly of multifunctional 99(m)Tc radiopharmaceuticals using "clickable" amino acid derivatives.

Synthetic strategies that enable the efficient and selective combination of different biologically active entities hold great promise for the development of multifunctional hybrid conjugates useful for biochemical and medical applications. Starting from side-chain-functionalized N(α)-propargyl lysine derivatives, conjugates containing a 99(m)Tc-based imaging probe for SPECT and two different moieties (e.g., tumor-targeting vectors, pharmacological modifiers, affinity tags, or second imaging probes) can be assembled using the Cu(I)-catalyzed alkyne-azide cycloaddition in efficient one-pot protocols. This strategy was successfully applied to the preparation of a 99(m)Tc-labeled conjugate comprising a tumor-targeting peptide sequence (bombesin(7-14)) and a low-molecular-weight albumin binder, a pharmacological modifier that prolongs the blood circulation time of the conjugate. Evaluation of the conjugate in vitro and in vivo provided promising results for its use as an imaging agent for the visualization of tumors positive for the gastrin-releasing peptide receptor. The methodology presented herein provides an attractive synthetic tool for the preparation of multifunctional 99(m)Tc-based radiopharmaceuticals with significant potential for a multitude of applications.

Synthesis and evaluation of bombesin analogues conjugated to two different triazolyl-derived chelators for (99m)Tc labeling.

Overexpression of the gastrin-releasing peptide receptor (GRPR) in a variety of human carcinomas has provided a means of diagnosis and treatment. Previously we reported a metabolically stable (N(α)His)Ac-ßAla-ßAla-[Cha(13),Nle(14)]BBS(7-14) analogue with high affinity for the GRPR. We have also shown that the biodistribution pattern of this fairly lipophilic, radiolabeled peptide can be enhanced by glycation, which is easily carried out by Cu(I)-catalyzed cycloaddition. Herein, we further elaborate this "click approach" in the synthesis of a new series of triazole-based chelating systems as alternatives to the (N(α)His)Ac chelator for labeling with the (99m)Tc(CO)(3) core. The bombesin analogues, containing these new chelating systems, were evaluated with regard to their synthesis and in vitro and in vivo properties, and were compared with their (N(α)His)Ac counterparts. The influence of the chelator on biodistribution properties was less than that of glycation, which clearly improved the tumor-to-background ratios.

Charge dependent substrate activity of C3' and N3 functionalized, organometallic technetium and rhenium-labeled thymidine derivatives toward human thymidine kinase 1.

Human cytosolic thymidine kinase (hTK1) has proven to be a suitable target for the noninvasive imaging of cancer cell proliferation using radiolabeled thymidine analogues such as [(18)F]3'-fluoro-3'-deoxythymidine ([(18)F]FLT). A thymidine analogue for single photon emission computed tomography (SPECT), which incorporates the readily available and inexpensive nuclide technetium-99m, would be of considerable practical interest. hTK1 is known to accommodate modification of the structure of the natural substrate thymidine at the positions N3 and C3' and, to a lesser extent, C5. In this work, we used the copper-catalyzed azide-alkyne cycloaddition to synthesize two series of derivatives in which thymidine is functionalized at either the C3' or N3 position with chelating systems suitable for the M(CO)(3) core (M = (99m)Tc, Re). The click chemistry approach enabled complexes with different structures and overall charges to be synthesized from a common precursor. Using this strategy, the first organometallic hTK1 substrates in which thymidine is modified at the C3' position were identified. Phosphorylation of the organometallic derivatives was measured relative to thymidine. We have shown that the influence of the overall charge of the derivatives is dependent on the position of functionalization. In the case of the C3'-functionalized derivatives, neutral and anionic substrates were most readily phosphorylated (20-28% of the value for the parent ligand thymidine), whereas for the N3-functionalized derivatives, cationic and neutral complexes were apparently better substrates for the enzyme (14-18%) than anionic derivatives (9%).

Metal chelating systems synthesized using the copper(I) catalyzed azide-alkyne cycloaddition.

The copper(I) catalyzed azide-alkyne cycloaddition (CuAAC) is the premier example of a click reaction. The reaction is modular, reliable and easy to perform, providing easy access to molecular diversity. The majority of reported applications of the reaction employ the 1,2,3-triazole as a stable linkage to connect two chemical/biological components, while the potential for metal coordination of the heterocycle itself has received much less attention. In fact, 1,4-functionalized 1,2,3-triazoles are versatile ligands offering several donor sites for metal coordination, including N3, N2 and C5. In this article, we summarize the areas in which the CuAAC has been applied to the synthesis of novel triazole-containing ligands for transition metals.

Organometallic [Re(CO)3]+ and [Re(CO)2(NO)]2+ labeled substrates for human thymidine kinase 1.

Thymidine was functionalized at position N3 with a tridentate iminodiacetic acid chelating system and a potentially tetradentate mercaptoethyliminodiacetic acid chelating system. Spacers of different lengths (ethyl and butyl) were introduced between the chelators and thymidine. The derivatives were labeled with the [Re(CO)(2)(NO)](2+) and [Re(CO)(3)](+) cores to give isostructural complexes with different overall charges. All complexes were analyzed by NMR, MS, and IR, and in addition, the X-ray structure of a [Re(CO)(2)(NO)](2+) labeled thymidine derivative functionalized at the N3 position was solved. The ligands incorporating the potentially tetradentate mercaptoethyliminodiacetic acid chelating system coordinated tridentately through iminodiacetic acid to both the [Re(CO)(2)(NO)](2+) core and the [Re(CO)(3)](+) core. This was surprising given that the reaction of [NEt(4)][Re(CO)(2)(NO)Br(3)] with the model ligand ethylmercaptoethyliminodiacetic acid led to dissociation of a carbonyl ligand and formation of a monocarbonyl-mononitrosyl complex, as confirmed by X-ray structure analysis. All of the organometallic thymidine derivatives were substrates for human thymidine kinase 1, a key enzyme in (cancer) cell proliferation. Neutral [Re(CO)(2)(NO)](2+) labeled thymidine derivatives revealed substrate activity ranging from 24 to 40%, and the structurally analogous anionic [Re(CO)(3)](+) labeled thymidine derivatives from 20 to 38% compared with the natural substrate thymidine.

Synthesis, in vitro, and in silico evaluation of organometallic technetium and rhenium thymidine complexes with retained substrate activity toward human thymidine kinase type 1.

Human cytosolic thymidine kinase (hTK1) has proven to be a suitable target for noninvasive imaging of cancer cell proliferation using radiolabeled substrates such as [ (18)F]fluorothymidine ([ (18)F]FLT). However, a thymidine tracer useful for single photon emission tomography (SPECT) based on the inexpensive radionuclide technetium-99m would be of significant interest. In this work, a series of thymidine derivatives labeled with the organometallic [M(CO) 3] (+) core (M = (99m)Tc, Re) were synthesized. Neutral, cationic, and anionic complexes were readily formed in aqueous media, and all were substrates of recombinant hTK1 when incubated with ATP. The neutral complexes were phosphorylated to a greater extent than the charged complexes. The extent of phosphorylation was further improved by increasing the spacer length separating thymidine and the organometallic core. A molecular dynamics simulation study performed with a modified hTK1 structure supported the experimental findings. In vitro cell internalization experiments performed in a human neuroblastoma cell line (SKNMC) showed low uptake of the charged complexes but significant uptake for the neutral, lipophilic complexes with a log P value >1.

"Click-to-chelate": design and incorporation of triazole-containing metal-chelating systems into biomolecules of diagnostic and therapeutic interest.

The site-specific conjugation of metal chelating systems to biologically relevant molecules is an important contemporary topic in bioinorganic and bioorganometallic chemistry. In this work, we have used the CuI-catalyzed cycloaddition of azides and terminal alkynes to synthesise novel ligand systems, in which the 1,2,3-triazole is an integral part of the metal chelating system. A diverse set of bidentate alkyne building blocks with different aliphatic and aromatic backbones and various donor groups were prepared. The bidentate alkynes were reacted with benzyl azide in the presence of a catalytic amount of CuI to form tridentate model ligands. The chelators were reacted with [ReBr3(CO)3]2- to form well-defined and stable complexes with different overall charges, structures and hydrophilicities. In all cases tridentate coordination of the ligands, including through N3 of the 1,2,3-triazole ring, was observed. The ligand systems could also be quantitatively radiolabelled with the precursor [99 mTc (H2O)3(CO)3]+ at low ligand concentrations. Similarly the alkynes were reacted with an azido thymidine derivative to form a series of compounds, which could be radiolabelled in situ to form single products. Subsequent incubation of the neutral and cationic organometallic 99 mTc thymidine derivatives with human cytosolic thymidine kinase, a key enzyme in tumour proliferation, revealed that only the neutral compounds maintained substrate activity towards the enzyme. Bioconjugation, radiolabelling and enzymatic reactions were successfully performed in a matter of hours. Thus, click chemistry provides an elegant method for rapidly functionalising a biologically relevant molecule with a variety of efficient metal chelators suitable for (radio)labelling with the M(CO)3 core (M=99 mTc, Re), to offer new potential for technetium-99 m in clinical and preclinical tracer development.

The exocyclic functionalisation of bis(thiosemicarbazonate) complexes of zinc and copper: the synthesis of monomeric and dimeric species.

This paper reports the synthesis of bimetallic zinc thiosemicarbazone complexes with rigid aromatic linkers, using either 1,3- or 1,4- benzenediamines or 1,3- or 1,4- benzenedialdehydes as the basis of the linking groups. Non-rigid aliphatic diamines and dialdehydes were also used to link the zinc chelating units. Reaction of a bis(thiosemicarbazone) with a pendant NHNH(2) group with monoaldehydes or ketones gives a range of monomeric complexes with exocylic imine groups bearing a range of substituents. The zinc complexes can be quantitatively and rapidly transmetallated to the corresponding copper complexes and this route or direct reaction with the free ligand can be used to radiolabel the monomeric species with (64)Cu. In vivo and in vitro studies of one of the (64)Cu imine complexes shows substantial hypoxic selectivity and high tumour uptake in a murine model.

New bimetallic compounds based on the bis(thiosemicarbazonato) motif.

Zinc and copper bis(thiosemicarbazonato) complexes containing more than one metal centre have been prepared with a view to examining their application for molecular imaging. The zinc complexes are fluorescent with excitation and emission at relatively long wavelengths. The dinuclear copper complex undergoes two sequential, quasi-reversible reductions.

"Click to chelate": synthesis and installation of metal chelates into biomolecules in a single step.

Click chemistry has been employed for the assembly of novel and efficient triazole-based multidentate chelating systems while simultaneously attaching them to molecules of biological interest. The "click-to-chelate" approach offers a powerful new tool for the modification of (bio)molecules with metal chelators for potential diagnostic and therapeutic applications.