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This study addresses the question whether inductively coupled plasma mass spectrometry (ICP-MS) can be used as a method for the in vitro and in vivo characterization of non-radioactive metal conjugates to predict the properties of analogous radiopharmaceuticals. In a "proof-of-concept" study, the prostate-specific membrane antigen (PSMA)-targeting [ 175 Lu]Lu-PSMA-617 and [ 159 Tb]Tb-PSMA-617 were compared with their respective radiolabeled analogues, [ 177 Lu]Lu-PSMA-617 (PLUVICTO, Novartis) and [ 161 Tb]Tb-PSMA-617. ICP-MS and conventional γ-counting of the cell samples revealed almost identical results (<6% absolute difference between the two technologies) for the in vitro uptake and internalization of the (radio)metal conjugates, irrespective of the employed methodology. In vivo, an equal uptake in PSMA-positive PC-3 PIP tumor xenografts was determined 1 h after the injection of [ 175 Lu]Lu-/[ 177 Lu]Lu-PSMA-617 (41 ± 6% ID/g and 44 ± 12% IA/g, respectively) and [ 159 Tb]Tb-/[ 161 Tb]Tb-PSMA-617 (44 ± 5% ID/g and 44 ± 5% IA/g, respectively). It was further revealed that it is crucial to use the same ratios of the (radio)metal-labeled and unlabeled ligands for both methodologies to obtain equal data in organs in which receptor saturation was reached such as the kidneys (12 ± 2% ID/g vs 10 ± 1% IA/g, 1 h after injection). The data of this study demonstrate that the use of high-sensitivity ICP-MS allows reliable and predictive quantification of compounds labeled with stable metal isotopes in cell and tissue samples obtained in preclinical studies. It can, hence, be employed as a valid alternative to the state-of-the-art γ-counting methodology to detect radioactive ligands.

[ 177 Lu]Lu-Ibu-DAB-PSMA, a radioligand modified with ibuprofen as the albumin binder, showed higher accumulation in PSMA-positive tumors of mice than the clinically used [ 177 Lu]Lu-PSMA-617 but lower retention in non-targeted tissues than previously developed albumin-binding PSMA radioligands. The aim of this study was to investigate whether the stereochemistry of the incorporated ibuprofen affects the radioligand's in vitro and in vivo properties and to select the more favorable radioligand for further development. For this purpose, SibuDAB and RibuDAB containing ( S )- and ( R )-ibuprofen, respectively, were synthesized and labeled with lutetium-177. In vitro , the two isomers had similar properties; however, [ 177 Lu]Lu-SibuDAB showed increased binding to mouse and human plasma proteins (91 ± 1 and 88 ± 2%, respectively) compared to [ 177 Lu]Lu-RibuDAB (75 ± 2 and 79 ± 2%, respectively). In vivo , [ 177 Lu]Lu-SibuDAB was metabolically more stable than [ 177 Lu]Lu-RibuDAB with ∼90 vs ∼67% intact radioligand detected in the blood at 4 h post injection (p.i.). In line with the lower albumin-binding affinity, the blood clearance of [ 177 Lu]Lu-RibuDAB in mice was considerably faster [27% of injected activity (% IA), 1 h p.i.] than for [ 177 Lu]Lu-SibuDAB (50% IA, 1 h p.i.). Time-dependent biodistribution studies performed in tumor-bearing athymic nude mice showed high PSMA-specific tumor uptake for both isomers. A twofold increased area under the curve (AUC 0→8d ) of the blood retention was determined for [ 177 Lu]Lu-SibuDAB as compared to [ 177 Lu]Lu-RibuDAB, whereas the kidney AUC 0→8d value of [ 177 Lu]Lu-SibuDAB was only half as high as for [ 177 Lu]Lu-RibuDAB. As a result, a more favorable tumor-to-kidney AUC 0→8d ratio was obtained for [ 177 Lu]Lu-SibuDAB, which was also visualized on SPECT/CT images. Based on its improved kidney clearance and higher metabolic stability, [ 177 Lu]Lu-SibuDAB was selected as the more favorable radioligand. Therapy studies performed with [ 177 Lu]Lu-SibuDAB (5 MBq/mouse) demonstrated the anticipated therapeutic superiority over the current gold-standard [ 177 Lu]Lu-PSMA-617 (5 MBq/mouse). The significantly increased survival time of mice treated with [ 177 Lu]Lu-SibuDAB as compared to those injected with [ 177 Lu]Lu-PSMA-617 justifies further development of this novel radioligand toward clinical application.

The folate receptor (FR) is an interesting target for radiotheranostics due to its overexpression in several tumor types. The progress in developing novel folate radioconjugates is, however, slow due to the synthetic challenges that folate chemistry presents. The goal of this study was, thus, to establish versatile solid-phase synthetic strategies for a convenient preparation of novel folate conjugates. Two approaches were established based on an orthogonal fluorenylmethyloxycarbonyl (Fmoc)-protection strategy to enable a modular buildup of an albumin-binding DOTA conjugate (known as OxFol-1) using folic acid (oxidized folate version) as a targeting agent. The main difference between the two approaches was the sequence of conjugating the single structural units. The approach that introduced the folate entity as the last unit appeared particularly useful for the preparation of conjugates based on 6 R - or 6 S -5-methyltetrahydrofolic acid (5-MTHF; a reduced folate version) as targeting entity. Three types of folate conjugates were synthesized either with a p -iodophenyl-based albumin binder (OxFol-1, 6 R -RedFol-1, and 6 S -RedFol-1) or without an albumin-binding entity (OxFol-14, 6 R -RedFol-14, and 6 S -RedFol-14). All six conjugates were obtained with high chemical purity (>98%) after 9-13 synthesis steps and a single final HPLC purification. Radiolabeling with lutetium-177 was feasible at high molar activity, and the resulting radioconjugates were stable over at least 24 h. Biodistribution and SPECT/CT imaging studies confirmed the favorable effect of an albumin-binding entity to increase the tumor uptake and reduce kidney retention of folate radioconjugates. The increased tumor-to-kidney ratios obtained with [ 177 Lu]Lu-6 R -RedFol-1 and [ 177 Lu]Lu-6 S -RedFol-1 as compared to [ 177 Lu]Lu-OxFol-1 indicated that 5-MTHF is the preferred FR-targeting agent for albumin-binding radioconjugates. This was, however, not the case for folate radioconjugates without an albumin binder. Thanks to the established synthesis strategy, the preparation of further folate radioconjugates will be facilitated, potentially enabling the optimization of the tissue distribution characteristics even more.

The strategy of using radioligands for targeting the prostate-specific membrane antigen (PSMA) revealed to be promising for the treatment of metastatic castration-resistant prostate cancer (mCRPC). Recently developed albumin-binding PSMA radioligands showed a remarkably increased tumor uptake because of the enhanced blood circulation, but higher accumulation of activity was also observed in off-target organs and tissues. The aim of this study was to investigate the option of using fast-cleared, small-molecular-weight PSMA inhibitors (PSMA-11, 2-PMPA, and ZJ-43) to reduce the kidney uptake of [ 177 Lu]Lu-PSMA-ALB-56, a previously developed albumin-binding PSMA radioligand. Dual-isotope SPECT/CT imaging was performed with tumor-bearing mice coinjected with [ 177 Lu]Lu-PSMA-ALB-56 and a 2.5-fold molar excess of [ 67 Ga]Ga-PSMA-11. At early timepoints after injection, the high renal uptake of [ 67 Ga]Ga-PSMA-11 reduced the accumulation of [ 177 Lu]Lu-PSMA-ALB-56 in the kidneys substantially, whereas the tumor uptake of [ 177 Lu]Lu-PSMA-ALB-56 was only slightly affected. These findings were confirmed in biodistribution studies, which revealed reduced uptake of [ 177 Lu]Lu-PSMA-ALB-56 in the kidneys due to coadministered unlabeled PSMA-11 (9.1 ± 0.8% IA/g vs 46 ± 11% IA/g; 1 h p.i.). The tumor uptake of [ 177 Lu]Lu-PSMA-ALB-56 was almost the same at 1 h p.i., irrespective of whether or not PSMA-11 was coinjected (24 ± 6% IA/g vs 27 ± 7% IA/g). The application of [ 177 Lu]Lu-PSMA-ALB-56 with 2-PMPA or ZJ-43, respectively, showed similar results in biodistribution studies. Among all three tested PSMA inhibitors, 2-PMPA, applied at a 2.5-fold molar excess relative to [ 177 Lu]Lu-PSMA-ALB-56, was most effective to improve the tumor-to-kidney ratios over the first hours after injection of [ 177 Lu]Lu-PSMA-ALB-56. The concept of using a PSMA inhibitor together with [ 177 Lu]Lu-PSMA-ALB-56 appears promising in view of a clinical translation of this and possibly other long-circulating PSMA radioligands.

Recently, we developed an albumin-binding radioligand ( 177 Lu-PSMA-ALB-56), which showed higher PSMA-specific tumor uptake in mice than the previously developed 177 Lu-PSMA-617 under the same experimental conditions. Such a radioligand may be of interest also for PET imaging, possibly enabling better visualization of even small metastases at late time-points after injection. The aim of this study was, therefore, to modify PSMA-ALB-56 by exchanging the DOTA chelator with a NODAGA chelator for stable coordination of 64 Cu ( T 1/2 = 12.7 h; Eβ + av = 278 keV). The resulting NODAGA-functionalized PSMA-ALB-89 ligand, and the previously establish DOTA-functionalized PSMA-ALB-56 ligand were labeled with 64 Cu and evaluated in vitro and in vivo. Both radioligands showed plasma protein-binding properties in vitro and PSMA-specific uptake in PC-3 PIP cells. Biodistribution studies, performed in tumor-bearing mice, revealed high accumulation of 64 Cu-PSMA-ALB-89 in PSMA-positive PC-3 PIP tumor xenografts (25.9 ± 3.41% IA/g at 1 h p.i.), which was further increased at later time-points (65.1 ± 7.82% IA/g at 4 h p.i. and 97.1 ± 7.01% IA/g at 24 h p.i.). High uptake of 64 Cu-PSMA-ALB-89 was also seen in the kidneys, however, 64 Cu-PSMA-ALB-89 was efficiently excreted over time. Mice injected with 64 Cu-PSMA-ALB-56 showed increased accumulation of radioactivity in the liver (25.3 ± 4.20% IA/g) when compared to the liver uptake of 64 Cu-PSMA-ALB-89 (4.88 ± 0.21% IA/g, at 4 h p.i.). This was most probably due to in vivo instability of the 64 Cu-DOTA complex, which was also the reason for lower tumor uptake (49.7 ± 16.1% IA/g at 4 h p.i. and 28.3 ± 3.59% IA/g at 24 h p.i.). PET/CT imaging studies confirmed these findings and enabled excellent visualization of the PSMA-positive tumor xenografts in vivo after injection of 64 Cu-PSMA-ALB-89. These data indicate that 64 Cu-PSMA-ALB-89 is favorable over 64 Cu-PSMA-ALB-56 with regard to the in vivo stability and tissue distribution profile. Moreover, 64 Cu-PSMA-ALB-89 outperformed previously developed 64 Cu-labeled PSMA ligands. Further optimization of long-circulating PSMA-targeting PET radioligands will be necessary before translating this concept to the clinics.

Increased vascular permeability is an important hallmark of many diseases, including cancer, cerebral ischemia, and severe inflammatory disorders. In this regard, the noninvasive assessment of pathologically increased vascular permeability in vivo is of great interest. In this study, the potential of albumin- and transthyretin-binding radioligands was evaluated for imaging of vascular hyperpermeability. For this purpose, the bleomycin-induced lung injury model was used as a model of inflammation-associated vascular leakage. The plasma protein-binding ligands, which bind to albumin (DOTA-PPB-01) and transthyretin (DOTA-PPB-03), were radiolabeled and used for nuclear imaging and biodistribution studies. In this regard, 177 Lu was employed as a surrogate nuclide for detailed preclinical investigations, including single-photon emission computed tomography (SPECT) studies, whereas 44 Sc was proposed as a radionuclide for positron emission tomography (PET), which may be relevant for future clinical translation. Mice were administered with these radioligands 6-9 days after intratracheal instillation of bleomycin or saline. Bleomycin-treated mice developed pronounced lung inflammation with enhanced vascular permeability that was reflected in significantly increased lung size and weight due to edema and infiltration with inflammatory cells. Biodistribution studies revealed significantly higher accumulation of 177 Lu-DOTA-PPB-01 in injured lungs as compared to lungs of control animals at all investigated time points (4-48 h p.i.). The best contrast was achieved at late time points (16.1 ± 2.91% IA/g vs 2.03 ± 1.22% IA/g, 48 h p.i.) when the blood activity levels were ∼7.5% IA/g. Injection of 177 Lu-DOTA-PPB-03 also resulted in increased lung accumulation in bleomycin-treated mice at all investigated time points (2-8 h p.i.). The pharmacokinetics was significantly faster, however, resulting in good contrast already at 8 h p.i. (4.32 ± 0.85% IA/g vs 1.06 ± 0.10% IA/g) when blood activity levels were ∼2% IA/g. The absolute lung accumulation of 177 Lu-DOTA-PPB-03 was significantly lower than that of 177 Lu-DOTA-PPB-01. PET/CT scans performed with 44 Sc-DOTA-PPB-01 distinguished injured from healthy lungs only at late time points (20 h p.i.), whereas 44 Sc-DOTA-PPB-03 already allowed the differentiation at 4 h p.i. due to its faster clearance. The investigated radioligands, 44 Sc/ 177 Lu-DOTA-PPB-01 and 44 Sc/ 177 Lu-DOTA-PPB-03, hold promise for the visualization of vascular leakage in a variety of pathological conditions. 44 Sc would be the radionuclide of choice for clinical application as it can be stably coordinated with a DOTA chelator and enables PET imaging over extended periods.

The treatment of metastatic castration-resistant prostate cancer (mCRPC) remains challenging with current treatment options. The development of more effective therapies is, therefore, urgently needed. Targeted radionuclide therapy with prostate-specific membrane antigen (PSMA)-targeting ligands has revealed promising clinical results. In an effort to optimize this concept, it was the aim of this study to design and investigate PSMA ligands comprising different types of albumin binders. PSMA-ALB-53 and PSMA-ALB-56 were designed by combining the glutamate-urea-based PSMA-binding entity, a DOTA chelator and an albumin binder based on the 4-( p-iodophenyl)-moiety or p-(tolyl)-moiety. The compounds were labeled with 177 Lu (50 MBq/nmol) resulting in radioligands of high radiochemical purity (≥98%). Both radioligands were stable (≥98%) over 24 h in the presence of l-ascorbic acid. The uptake into PSMA-positive PC-3 PIP tumor cells in vitro was in the same range (54-58%) for both radioligands; however, 177 Lu-PSMA-ALB-53 showed a 15-fold enhanced binding to human plasma proteins. Biodistribution studies performed in PC-3 PIP/flu tumor-bearing mice revealed high tumor uptake of 177 Lu-PSMA-ALB-53 and 177 Lu-PSMA-ALB-56, respectively, demonstrated by equal areas under the curves (AUCs) for both radioligands. The increased retention of 177 Lu-PSMA-ALB-53 in the blood resulted in almost 5-fold lower tumor-to-blood AUC ratios when compared to 177 Lu-PSMA-ALB-56. Kidney clearance of 177 Lu-PSMA-ALB-56 was faster, and hence, the tumor-to-kidney AUC ratio was 3-fold higher than in the case of 177 Lu-PSMA-ALB-53. Due to the more favorable tissue distribution profile, 177 Lu-PSMA-ALB-56 was selected for a preclinical therapy study in PC-3 PIP tumor-bearing mice. The tumor growth delay after application of 177 Lu-PSMA-ALB-56 and 177 Lu-PSMA-617 applied at the same activities (2 or 5 MBq per mouse) revealed better antitumor effects in the case of 177 Lu-PSMA-ALB-56. As a consequence, the survival of mice treated with 177 Lu-PSMA-ALB-56 was prolonged when compared to the mice, which received the same activity of 177 Lu-PSMA-617. Our results demonstrated the superiority of 177 Lu-PSMA-ALB-56 over 177 Lu-PSMA-ALB-53 indicating that the p-(tolyl)-moiety was more suited as an albumin binder to optimize the tissue distribution profile. 177 Lu-PSMA-ALB-56 was more effective to treat tumors than 177 Lu-PSMA-617 resulting in complete tumor remission in four out of six mice. This promising results warrant further investigations to assess the potential for clinical application of 177 Lu-PSMA-ALB-56.

5-Methyltetrahydrofolate (5-MTHF), a reduced folate form, is the biologically active folate involved in many different metabolic processes. To date, there are no studies available in the literature on 18 F-labeled 6 S- and 6 R-5-MTHF radiotracers for imaging folate receptor (FR)-α-positive tissues. Therefore, the goal of this study was to synthesize four 18 F-labeled 5-MTHF derivatives conjugated at either the α- or γ-carboxylic functionality of glutamate and to assess their suitability for FR-targeting. Organic syntheses of the precursors and the four reference compounds, namely, 6 S-α, 6 S-γ, 6 R-α, and 6 R-γ-click-fluoroethyl-5-MTHF, were carried out in low to moderate overall chemical yields. The radiosyntheses of the α- and γ-conjugated 18 F-labeled folate derivatives were accomplished in approximately 100 min, low radiochemical yields (1-7% d.c.) and high molar activities (139-245 GBq/μmol). Radiochemically pure tracers were obtained after the addition of a mixture of antioxidants consisting of sodium ascorbate and l-cysteine. In vitro, all four 5-MTHF conjugates showed similar binding affinities to FR-α (IC 50 = 17.7-24.0 nM), whereas folic acid showed a significantly higher binding affinity to the FR-α. Cell uptake and internalization experiments with KB cells demonstrated specific uptake and internalization of the radiofolate conjugates. Metabolite studies in mice revealed high in vivo stability of the radiotracers in mice. Biodistribution and positron emission tomography (PET) imaging studies in FR-positive KB tumor-bearing mice demonstrated that the 6 S- and 6 R-5-MTHF conjugates exhibited a different accumulation pattern in various organs including the kidneys and the liver, whereas no significant differences in radioactivity accumulation in the kidneys and the liver were found for both the α- and γ-conjugated diastereoisomers. Despite the considerably lower binding affinities of the 5-MTHF derivatives compared to the corresponding folic acid conjugates similar high KB tumor uptake was observed for all the folate conjugates investigated (8-11% IA/g). Based on these results, we conclude that 18 F-labeled 5-MTHF conjugates are a promising new class of radiotracers for targeting FR-positive tumor tissues.

The prostate-specific membrane antigen (PSMA) has emerged as an attractive prostate cancer associated target for radiotheragnostic application using PSMA-specific radioligands. The aim of this study was to design new PSMA ligands modified with an albumin-binding moiety in order to optimize their tissue distribution profile. The compounds were prepared by conjugation of a urea-based PSMA-binding entity, a DOTA chelator, and 4-( p-iodophenyl)butyric acid using multistep solid phase synthesis. The three ligands (PSMA-ALB-02, PSMA-ALB-05, and PSMA-ALB-07) were designed with varying linker entities. Radiolabeling with 177 Lu was performed at a specific activity of up to 50 MBq/nmol resulting in radioligands of >98% radiochemical purity and high stability. In vitro investigations revealed high binding of all three PSMA radioligands to mouse (>64%) and human plasma proteins (>94%). Uptake and internalization into PSMA-positive PC-3 PIP tumor cells was equally high for all radioligands. Negligible accumulation was found in PSMA-negative PC-3 flu cells, indicating PSMA-specific binding of all radioligands. Biodistribution and imaging studies performed in PC-3 PIP/flu tumor-bearing mice showed enhanced blood circulation of the new radioligands when compared to the clinically employed 177 Lu-PSMA-617. The PC-3 PIP tumor uptake of all three radioligands was very high (76.4 ± 2.5% IA/g, 79.4 ± 11.1% IA/g, and 84.6 ± 14.2% IA/g, respectively) at 24 h post injection (p.i.) resulting in tumor-to-blood ratios of ∼176, ∼48, and ∼107, respectively, whereas uptake into PC-3 flu tumors was negligible. Kidney uptake at 24 h p.i. was lowest for 177 Lu-PSMA-ALB-02 (10.7 ± 0.92% IA/g), while 177 Lu-PSMA-ALB-05 and 177 Lu-PSMA-ALB-07 showed higher renal retention (23.9 ± 4.02% IA/g and 51.9 ± 6.34% IA/g, respectively). Tumor-to-background ratios calculated from values of the area under the curve (AUC) of time-dependent biodistribution data were in favor of 177 Lu-PSMA-ALB-02 (tumor-to-blood, 46; tumor-to-kidney, 5.9) when compared to 177 Lu-PSMA-ALB-05 (17 and 3.7, respectively) and 177 Lu-PSMA-ALB-07 (39 and 2.1, respectively). The high accumulation of the radioligands in PC-3 PIP tumors was visualized on SPECT/CT images demonstrating increasing tumor-to-kidney ratios over time. Taking all of the characteristics into account, 177 Lu-PSMA-ALB-02 emerged as the most promising candidate. The applied concept may be attractive for future clinical translation potentially enabling more potent and convenient prostate cancer radionuclide therapy.

Tumor targeting with folic acid radioconjugates has been proposed as a promising strategy for radionuclide therapy of folate receptor α (FR)-positive cancer. Recently, it was shown that modification of radiofolates with an albumin-binding entity increased the tumor-to-kidney ratios of accumulated radioactivity in mice. The goal of this study was to evaluate the lead compound cm10 and compare it with new albumin-binding folate conjugates. Compound cm12 was designed with a long spacer consisting of a PEG-11 entity, and compound cm13 contained a short alkane chain between the albumin-binding moiety and folic acid. All of the derivatives were labeled with 177 Lu (t 1/2 = 6.65 days, E β - ,average = 134 keV; E γ = 113 keV, 208 keV), a clinically established radionuclide for therapeutic purposes. The evaluation revealed that all of the albumin-binding radiofolates exhibited increased in vitro stability compared with the reference compound ( 177 Lu-cm14) without albumin binder. Serum protein binding, determined with an ultrafiltration assay, was high (>88%) for the derivatives with albumin-binding entities. The FR-binding affinity was in the same range (K D = 4.0-7.5 nM) for all of the radiofolates, independent of the albumin-binding entity and spacer length. FR-specific uptake was proven in vitro using FR-positive KB tumor cells. In vivo studies with KB-tumor-bearing mice were performed in order to assess the tissue distribution profile of the novel radiofolates. 177 Lu-cm13 showed high tumor uptake at late time points (13.3 ± 2.94% IA/g, 48 h p.i.) and tumor-to-kidney ratios (0.59 ± 0.03, 48 h p.i.) in the same range as 177 Lu-cm10 (0.55 ± 0.07, 48 h p.i.). However, the tumor-to-kidney ratio of 177 Lu-cm12 (0.28 ± 0.07, 48 h p.i.) was reduced compared with 177 Lu-cm10 and 177 Lu-cm13. The results of this study indicate that the spacer entity between folic acid and the albumin binder is of critical importance with regard to the tissue distribution profile of the radiofolate. The PEG spacer compromised the beneficial effects of the lead compound, but the design with a short alkane spacer appeared to be promising. Future studies will focus on the design of radiofolates with lipophilic and more rigid spacer entities, which may allow a further improvement of their tissue distribution profiles.

A number of folate-based radioconjugates have been synthesized and evaluated for nuclear imaging purposes of folate receptor (FR)-positive tumors and potential therapeutic application. A common shortcoming of radiofolates is, however, a significant accumulation of radioactivity in the kidneys. This situation has been faced by modifying the folate conjugate with an albumin-binding entity to increase the circulation time of the radiofolate, which led to significantly improved tumor-to-kidney ratios. The aim of this study was to develop an albumin-binding folate conjugate with a NODAGA-chelator (rf42) for labeling with (64)Cu and (68)Ga, allowing application for PET imaging. The folate conjugate rf42 was synthesized in 8 steps, with an overall yield of 5%. Radiolabeling with (64)Cu and (68)Ga was carried out at room temperature within 10 min resulting in (64)Cu-rf42 and (68)Ga-rf42 with >95% radiochemical purity. (64)Cu-rf42 and (68)Ga-rf42 were stable (>95% intact) in phosphate-buffered saline over more than 4 half-lives of the corresponding radionuclide. In vitro, the plasma protein-bound fraction of (64)Cu-rf42 and (68)Ga-rf42 was determined to be >96%. Cell experiments proved FR-specific uptake of both radiofolates, as it was reduced to <1% when KB tumor cells were coincubated with excess folic acid. In vivo, high accumulation of (64)Cu-rf42 and (68)Ga-rf42 was found in KB tumors of mice (14.52 ± 0.99% IA/g and 11.92 ± 1.68% IA/g, respectively) at 4 h after injection. The tumor-to-kidney ratios were in the range of 0.43-0.55 over the first 4 h of investigation. At later time points (up to 72 h p.i. of (64)Cu-rf42) the tumor-to-kidney ratio increased to 0.73. High-quality PET/CT images were obtained 2 h after injection of (64)Cu-rf42 and (68)Ga-rf42, respectively, allowing distinct visualization of tumors and kidneys. Comparison of PET/CT images obtained with (64)Cu-rf42 and a (64)Cu-labeled DOTA-folate conjugate (cm10) clearly proved the superiority of NODAGA for stable coordination of (64)Cu. (64)Cu-cm10 showed high liver uptake, most probably as a consequence of released (64)Cu(2+). The data reported in this study clearly proved the promising features of (64)Cu-rf42, particularly in terms of favorable tumor-to-kidney ratios. The relatively long half-life of (64)Cu (T1/2 = 12.7 h) matches well with the enhanced circulation time of the albumin-binding NODAGA-folate, allowing PET imaging at longer time points after injection than is possible when using (68)Ga (T1/2 = 68 min).

A new transplantable ovarian tumor model is presented using a novel folate receptor (FR) positive, murine ovarian cancer cell line that emulates the human disease and induces widespread intraperitoneal (i.p.) tumors in immunocompetent mice within 4-8 weeks of implantation. Tumor development was monitored using a new positron emission tomography (PET) FR-targeting reporter with PET/computerized tomography (PET/CT) and fluorescence molecular tomography (FMT) using a commercial FR-targeting reporter. Conventional structural magnetic resonance imaging (MRI) was also performed. Adult female C57BL/6 mice were injected i.p. with 6 × 10(6) MKP-L FR+ cells. Imaging was performed weekly beginning 2 weeks after tumor induction. The albumin-binding, FR-targeting ligand cm09 was radiolabeled with the positron emitter (68)Ga and used to image the tumors with a small animal PET/CT. The FR-reporter FolateRSense 680 (PerkinElmer) was used for FMT and flow cytometry. Preclinical MRI (7 T) without FR-targeting was compared with the PET and FMT molecular imaging. Tumors were visible by all three imaging modalities. PET/CT had the highest imaging sensitivity at 3-3.5 h postadministration (mean %IA/g mean > 6) and visualized tumors earlier than the other two modalities with lower kidney uptake (mean %IA/g mean < 17) than previously reported FR-targeting agents in late stage disease. FMT showed relatively low FR-targeted agent in the bladder and kidneys, but yielded the lowest anatomical image resolution. MRI produced the highest resolution images, but it was difficult to distinguish tumors from abdominal organs during early progression since a FR-targeting MRI reporter was not used. Nevertheless, there was good correlation of imaging biomarkers between the three modalities. Tumors in the mouse ovarian cancer model could be detected using FR-targeted imaging as early as 2 weeks post i.p. injection of tumor cells. An imaging protocol should combine one or more of the modalities, e.g., PET/CT or PET/MRI for optimal tumor detection and delineation from surrounding tissues.

Folate receptor (FR) targeting is an attractive strategy for nuclear imaging of cancer and activated macrophages through application of folic acid radioconjugates. However, significant renal accumulation of folate radioconjugates and hence exceedingly high emission of radiation from the kidneys may mask uptake of radioactivity at sites of interest such as small metastases in the abdominal region of animal models of ovarian cancer. Recently it was observed that the antifolate pemetrexed (PMX) reduces undesired renal uptake of radiofolates. A disadvantage of this strategy is the fact that pemetrexed is a chemotherapeutic agent which may have toxic side effects. The aims of this study were therefore to investigate whether the desired effect of PMX to reduce renal accumulation of folate radioconjugates would be maintained if it was applied as a cocktail together with its antidote, thymidine, and to explore whether thymidine was an effective rescue agent against PMX's related toxicity in vitro and in vivo. In vitro internalization of (67)Ga-EC0800 was investigated using FR-positive KB tumor cells and embryonic monkey MA104 kidney cells in the absence and presence of PMX alone and in combination with thymidine. Uptake of (67)Ga-EC0800 into KB cells was increased by coincubation of the cells with PMX. In contrast uptake of (67)Ga-EC0800 into MA104 cells was reduced under the same conditions. In both cell lines coincubation of thymidine did not change the results obtained with PMX. Biodistribution and SPECT/CT imaging studies of (67)Ga-EC0800 were performed with KB tumor bearing mice injected with PMX alone or with a cocktail of PMX and thymidine. The radiofolate's kidney uptake reducing effect of PMX in mice was maintained also if PMX was employed together with its antidote thymidine. The tumor uptake of (67)Ga-EC0800 remained unchanged independent of the administration of PMX or a combination of PMX and thymidine. The effect of thymidine to abrogate PMX-induced cytotoxicity was demonstrated in vitro with an MTT assay using KB and MA104 cells. Cell viability was reduced to 50% (KB cells) and 70% (MA104 cells) of untreated controls if PMX (5 μM and 15 μM, respectively) was coincubated. Addition of thymidine (10 μM or 100 μM) compensated PMX's toxic effects in a dose-dependent manner. The effect of thymidine was also investigated in non-tumor bearing mice treated with high-dosed PMX. Rescue of mice with side effects after the third and fourth cycles of PMX application (1 mg/mouse) was achieved by application of thymidine (20 mg/mouse) at five consecutive days starting the day of PMX injection. Application of PMX together with thymidine as a cocktail is effective to improve the tissue distribution of radiofolates while preventing pharmacological and potentially toxic side effects of the chemotherapeutic agent PMX. These findings open new perspectives for folate-based nuclear imaging in preclinical research potentially allowing longitudinal investigations and monitoring therapies in animal models of cancer and inflammatory diseases.

The folate receptor (FR) has been identified as a valuable target for the imaging of cancer and activated macrophages, involved in inflammatory and autoimmune diseases via positron emission tomography (PET). Therefore, conjugates of folic acid have been synthesized by coupling of a radiolabeled prosthetic group to the glutamate part of folic acid (pendent approach). In this work, we present a novel class of folates, where the phenyl ring of folic acid was isosterically replaced by a pyridine moiety for direct labeling with [(18)F]fluoride (integrated approach). 3'-Azafolic acid and its 2'-halogenated derivatives (2'-chloro and 2'-fluoro) were evaluated in vitro to determine their binding affinity. 3'-Aza-2'-[(18)F]fluorofolic acid ([(18)F]6) was obtained, starting from N(2)-acetyl-3'-aza-2'-chlorofolic acid di-tert-butylester (2), in a maximum decay corrected radiochemical yield of about 9% in ≥98% radiochemical purity and high specific activities of 35-127 GBq/μmol. Binding affinity to the FR was high (IC(50) = 0.8 ± 0.2 nM), and the radiotracer was stable in human plasma over 4 h at 37 °C. No degradation or defluorination was detected after incubation of the radiotracer for 1 h at 37 °C with human and murine liver microsomes and human S9-fraction. In vivo PET imaging and biodistribution studies with mice demonstrated a high and specific uptake in FR-positive KB tumor xenografts (12.59 ± 1.77% ID/g, 90 min p.i.). A high and specific accumulation of radioactivity was observed in the kidneys (57.33 ± 8.40% ID/g, 90 min p.i.) and salivary glands (14.09 ± 0.93% ID/g, 90 min p.i.), which are known to express the FR and nonspecific uptake found in the liver (10.31 ± 2.37% ID/g, 90 min p.i.). Preinjection of folic acid resulted in a >85% reduced uptake of [(18)F]6 in FR-positive tissues (xenografts, kidneys, and salivary glands). Furthermore, no radioactive metabolites were detected in the blood, urine, or tumor tissue, 30 min p.i. These characteristics indicate that this new (18)F-labeled 3'-azafolate is an appropriate tool for imaging FR-positive (malignant) tissue.

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.

The folate receptor (FR) is upregulated in various cancer types (FR-α isoform) and in activated macrophages (FR-β isoform) which are involved in inflammatory and autoimmune diseases, but its expression in healthy tissues and organs is highly restricted to only a few sites (e.g kidneys). Therefore, the FR is a promising target for imaging and therapy of cancer and inflammation using folate-based radiopharmaceuticals. Herein, we report the synthesis and evaluation of a novel folic acid conjugate with improved properties suitable for positron emission tomography (PET). [(18)F]-fluoro-deoxy-glucose folate ([(18)F]3) was synthesized based on the click chemistry approach using 2-deoxy-2-[(18)F]fluoroglucopyranosyl azide and a folate alkyne derivative. The novel radiotracer [(18)F]3 was produced in good radiochemical yields (25% d.c.) and high specific radioactivity (90 GBq/μmol). Compared to previously published (18)F-folic acid derivatives, an increase in hydrophilicity was achieved by using a glucose entity as a prosthetic group. Biodistribution and PET imaging studies in KB tumor-bearing mice showed a high and specific uptake of the radiotracer in FR-positive tumors (10.03 ± 1.12%ID/g, 60 min p.i.) and kidneys (42.94 ± 2.04%ID/g, 60 min p.i.). FR-unspecific accumulation of radioactivity was only found in the liver (9.49 ± 1.13%ID/g, 60 min p.i.) and gallbladder (17.59 ± 7.22%ID/g, 60 min p.i.). No radiometabolites were detected in blood, urine, and liver tissue up to 30 min after injection of [(18)F]3. [(18)F]-fluoro-deoxy-glucose-folate ([(18)F]3) is thus a promising PET radioligand for imaging FR-positive tumors., (© 2012 American Chemical Society)

Administration of certain antifolates before radiofolate application has previously proven to have a positive effect on undesired kidney uptake of radiofolates in mice bearing human tumor xenografts. The aims of this study were to (i) test the effects of the antifolates, pemetrexed and CB3717, on tissue distribution of the clinically investigated radiofolate, (99m)Tc-EC20, and (ii) to determine if pemetrexed's kidney-selective blocking effect also functions in mice bearing syngeneic tumors. Relative binding affinities of pemetrexed and CB3717 were determined in folate receptor (FR)-positive KB cells at 0 and 37 degrees C using (3)H-folic acid. In vivo studies were performed in nude mice with KB tumor xenografts (A) and in Balb/c mice bearing FR-positive M109 tumor grafts (B). (99m)Tc-EC20 was prepared via a kit formulation. The antifolates pemetrexed and CB3717 (20 mumol/kg body weight) were administered intravenously 1 h before injection of (99m)Tc-EC20 (67 nmol/kg body weight). Similar to previously published data we found that FR-binding affinities of pemetrexed and CB3717 at 0 degrees C were in the same range as that of folic acid. Interestingly, experiments performed at 37 degrees C showed that pemetrexed has a nearly approximately 700-fold lower FR-affinity than CB3717. Tissue distribution of (99m)Tc-EC20 was largely comparable in both animal models (A and B). Radiofolate accumulation was found in FR-positive tumors (A, 8.92 +/- 2.14% ID/g; B, 15.02 +/- 0.95% ID/g) and FR-positive kidneys (A, 59.10 +/- 8.03% ID/g; B, 69.44 +/- 4.66% ID/g, 4 h p.i.). Preinjection of pemetrexed resulted in a significant decrease of (99m)Tc-EC20 uptake in kidney (A, 18.80 +/- 2.73% ID/g; B, 15.27 +/- 2.64% ID/g; 4 h p.i), whereas uptake in the tumors was unaltered. However, administration of the CB3717 resulted in a reduction of (99m)Tc-EC20 uptake in both the kidney and tumor (<1% ID/g, 4 h p.i.). We have thus demonstrated that pemetrexed effectively reduces kidney uptake of radiofolates not only in xenografted mice but also in a syngeneic tumor mouse model, thereby indicating that the kidney-specific blocking effect is not based on differences between human and murine FRs that are expressed in xenografts and kidneys, respectively. This effect was not observed with the antifolate, CB3717, which targets the FR selectively in contrast to pemetrexed that is predominantly transported into cells through carrier systems.

Different imaging modalities can provide complementary information on biological processes at the cellular or molecular level in vitro and in vivo. However, specific molecular probes suitable for a comparison of different imaging modalities are often not readily accessible because their preparation is usually accomplished by individually developed and optimized syntheses. Herein, we present a general, modular synthetic approach that provides access to multiple probes derived from a single precursor by application of the same, efficient functionalization strategy, the Cu(I)-catalyzed cycloaddition of terminal alkynes and azides (click chemistry). To demonstrate the viability and efficiency of this approach, folic acid (FA) was selected as a targeting vector because the preparation of FA-based imaging probes used for SPECT, PET, MRI, and NIRF by reported synthetic strategies is usually difficult to achieve and often results in low overall yields. We prepared a versatile γ-azido-FA precursor as well as a set of alkyne functionalized probes and precursors including ligand systems suitable for the chelation of various (radio)metals, an NIR dye and (18)F- and (19)F-derivatives, which enabled the parallel development of new FA-imaging probes. The Cu(I)-mediated coupling of the alkynes with the γ-azido-FA precursor was accomplished in high yields and with minimal use of protective groups. The various probes were fully characterized spectroscopically as well as in vitro and in vivo. In vitro, all new FA-derivatives exhibited high affinity toward the folic acid receptor (FR) and/or were specifically internalized into FR-overexpressing KB cells. In vivo experiments with nude mice showed that all probes (except the MRI probes which have not been tested yet) accumulated specifically in FR-positive organs and human KB-cell xenografts. However, in vivo imaging revealed significant differences between the various FA-derivatives with respect to unspecific, off-target localization. In general, the comparison of different probes proved the superiority of the more hydrophilic, radiometal-based imaging agents, a result which will guide future efforts for the development of FA-based imaging probes and therapeutic agents. In addition, the strategy presented herein should be readily applicable to other molecules of interest for imaging and therapeutic purposes and thus represents a valuable alternative to other synthetic approaches.

A side-by-side comparison of the synthesis, radiolabeling, and in vitro and in vivo characterization of two new and isostructural (99m)Tc-tricarbonyl folic acid radiotracers comprising either a N(tau)-functionalized histidine (His) chelator or a 1,4-bifunctionalized 1,2,3-triazole His analogue is described. The 1,2,3-triazole-containing folic acid derivative was synthesized in approximately 80% yield by a short reaction sequence including application of click chemistry (the Cu(I)-catalyzed cycloaddition of azides and terminal alkynes). The synthesis of the ligand system and the functionalization of the folic acid derivative were accomplished simultaneously, which prompted us to call this approach "click-to-chelate". In comparison, the reported regioselective synthesis of the N(tau)-His compound provided the final product in only very low yields (<1%). While the efficiency of the syntheses differs considerably, the two isostructural folate derivatives exhibit virtually identical properties with respect to Tc-99m radiolabeling and in vitro and in vivo characteristics as shown by experiments performed with FR-positive KB cells and xenografted mice bearing folate receptor overexpressing tumors. We have demonstrated herein for the first time that a ligand system known to be an excellent chelator for the stable complexation of the organometallic core [M(CO)3] (+) (M = Tc-99m, Re) can be replaced by an isostructural 1,2,3-triazole analogue without influencing the characteristics of the radiometal conjugate. The "click-to-chelate" strategy provides a highly efficient and convenient entry to metal conjugates suitable for diagnostic and potentially therapeutic applications. The described procedures should be readily applicable to any azide-functionalized (bio)molecule and, thus, are likely to represent the method of choice for the future development of radiopharmaceuticals radiolabeled with the organometallic precursors [M(CO)3(H2O)3] (+) (M = (99m)Tc, (188)Re).

Novel organometallic 99mTc(I)-folate derivatives have been synthesized and evaluated in vitro and in vivo in order to assess the influence of the overall charge of the radioconjugates and the spacer entity on the affinity and pharmacokinetic profile. Folic acid has been functionalized at the gamma-carboxylate group of the glutamate moiety with (i) a hydrophilic diethoxyethyl spacer bearing a picolylamine monoacetic acid chelate, (ii) a hexyl spacer bearing an iminodiacetic acid chelate, and (iii) a hexyl spacer with a bis(pyridylmethyl)amine chelating system. Coordination of the 99mTc(CO)3-core resulted in neutral complex 21, anionic complex 22, and cationic complex 23 in excellent yields (>90%) at ligand concentrations of 10(-4) M. Complexes 21-23 were HPLC purified for in vitro and in vivo experiments. In the case of 23, separation from the unlabeled folate analogue was incomplete, leading to low specific activity and, hence, significantly inferior in vivo uptake in folate-receptor-positive (FR-positive) organs and tissues (tumors and kidneys). Time dependent in vivo studies were performed in female, athymic nude mice bearing subcutaneous FR-positive human KB cell xenografts at 1, 4, and 24 h post injection (p.i.) of the radiotracers. Tumor uptake ranged between 1.9-2.7% ID/g, 4 h p.i. and 1.6-2.2% ID/g, 24 h p.i. for 21 and 22, and 0.9% ID/g, 4 h p.i. and 1.1% ID/g, 24 h p.i. for 23. Blood clearance was fast for all derivatives (< or =0.2% ID/g 1 h p.i.). Significant fractions of radioactivity were found in nontargeted and FR-negative organs and tissues (particularly in the liver and the intestines/intestinal contents) at early time points p.i. Coadministration of folic acid reduced radioactivity in FR-positive tissues and organs to background levels. In conclusion, overall charge and the nature of the spacer entity seemed to have a relatively minor influence on receptor affinity and the in vivo pharmacokinetic profile of the tested radiofolates.