Improved PET Imaging of Tumors in Mice Using a Novel 18 F-Folate Conjugate with an Albumin-Binding Entity

Purpose: The folate receptor (FR) is a promising target for nuclear imaging due to its overexpression in many different cancer types. A drawback of using folate radioconjugates is the high accumulation of radioactivity in the kidneys. Therefore, the aim of this study was to develop a 18 F-labeled folate conjugate with an albumin-binding entity to enhance the blood circulation time and hence improve the tumor-to-kidney ratio. Procedures: The novel 18 F-folate was prepared by conjugation of a 18 F-labeled glucose azide to an alkyne-functionalized folate precursor containing an albumin-binding entity via Cu(I)-catalyzed 1,3-dipolar cycloaddition. The radioconjugate was tested in vitro on FR-positive KB tumor cells and by biodistribution and positron emission tomography (PET) imaging studies using KB tumor-bearing mice. Results: The radiosynthesis of the albumin-binding [18 F]fluorodeoxyglucose-folate ([18 F]3) resulted in a radiochemical yield of 1-2% decay corrected (d.c.) and a radiochemical purity of ≥95%. The specific activity of [18 F]3 ranged from 20 to 50GBq/μmol. In vitro experiments revealed FR-specific binding of [18 F]3 to KB tumor cells. In vivo we found an increasing uptake of [18 F]3 into tumor xenografts over time reaching a value of ∼ 15% injected dose (ID)/g at 4h post-injection (p.i.). Uptake in the kidneys (∼ 13% ID/g; 1h p.i.) was approximately fourfold reduced compared to previously published 18 F-labeled folic acid derivatives. An excellent visualization of tumor xenografts with an unprecedentedly high tumor-to-kidney ratio (∼ 1) was obtained by PET imaging. Conclusions: [18 F]3 showed a favorable accumulation in tumor xenografts compared to the same folate conjugate without albumin-binding properties. Moreover, the increased tumor-to-kidney ratios improved the PET imaging quality significantly, in spite of a somewhat higher background radioactivity which was a consequence of the slower blood clearance of [18 F]

An improved folate stable isotope dilution assay of unexploited food sources from Brazil

Brazil has a diverse plant community, including underutilized non-conventional food crops (PANCs), which have the potential to be a rich source of food and contribute to food security. For assessing the folate content in a range of Brazilian PANCs, we extended the validation of an existing stable isotope dilution assay (SIDA) for the stably 13C-labelled 10-formyl-Pte[13C5]Glu (10-CHO-Pte[13C5]Glu). The SIDA method with an enzymatic treatment, purification step, and an LC–MS/MS measurement was validated regarding linearity, precision, LoD/LoQ, and recovery for 10-CHO-PteGlu. After successful validation, the study of some underutilized Brazilian non-conventional fruits and leaves from the São Paulo State University campus revealed them as an important source of folates. It provided the first insights into the folate content of unexploited food sources from Brazil. Pequi had the highest folate content among the fruits studied, with mean values of 333 μg/100 g based on fresh weight (FW). The analysis also shows that different cultivars of fruit or fruits from different growing locations have a high variability in folate content or other nutritional factors. In most fruits, the main vitamer was 5-CH3-H4folate, but jenipapo and taioba showed the highest content of 10-CHO-PteGlu with 28.22 μg/100 g (FW) in jenipapo peel and 75.64 μg/100 g (FW) in the taioba leaves. Thus, this study also provides results on the importance of the folate vitamer 10-CHO-PteGlu contributing to the total folate content

Folate receptor-targeted radionuclide therapy: preclinical investigation of anti-tumor effects and potential radionephropathy

INTRODUCTION Application of therapeutic folate radioconjugates is a promising option for the treatment of folate receptor (FR)-positive tumors, although high uptake of radiofolates in the kidneys remains a critical issue. Recently, it was shown that enhancing the blood circulation of radiofolates results in increased tumor uptake and reduced retention of radioactivity in the kidneys. In this study, we investigated and compared the anti-tumor effects and potential long-term damage to the kidneys after application of an albumin-binding ((177)Lu-cm09), and a conventional ((177)Lu-EC0800) folate radioconjugate. METHODS In vivo studies were performed with KB tumor-bearing nude mice. (177)Lu-EC0800 and (177)Lu-cm09 were applied at variable quantities (10-30MBq/mouse), and the tumor growth was monitored over time. Mice without tumors were injected with the same radiofolates and investigated over eight months by determination of creatinine and blood urea nitrogen plasma levels and by measuring renal uptake of (99m)Tc-DMSA using SPECT. At the study end, the morphological changes were examined on renal tissue sections using variable staining methods. RESULTS Compared to untreated controls, dose-dependent tumor growth inhibition and prolonged survival was observed in all treated mice. In line with the resulting absorbed dose, the treatment was more effective with (177)Lu-cm09 than with (177)Lu-EC0800, enabling complete tumor remission after application of ≥20MBq (≥28Gy). Application of radiofolates with an absorbed renal dose ≥23Gy showed increased levels of renal plasma parameters and reduced renal uptake of (99m)Tc-DSMA. Morphological changes observed on tissue sections confirmed radionephropathy of variable stages. CONCLUSIONS (177)Lu-cm09 showed more favorable anti-tumor effects and significantly less damage to the kidneys compared to (177)Lu-EC0800 as was expected based on improved tumor-to-kidney ratios. It was demonstrated that enhancing the blood circulation time of radiofolates was favorable regarding the risk-benefit profile of a therapeutic application. These results hold promise for future translation of the albumin-binder concept to the clinics, potentially enabling FR-targeted radionuclide therapy in patients

Diastereomerically Pure 6R- and 6S-3'-Aza-2'-18F-Fluoro-5- Methyltetrahydrofolates Show Unprecedentedly High Uptake in Folate Receptor–Positive KB Tumors

ISSN:0097-9058ISSN:0022-3123ISSN:0161-5505ISSN:2159-662XISSN:1535-566

Investigations Using Albumin Binders to Modify the Tissue Distribution Profile of Radiopharmaceuticals Exemplified with Folate Radioconjugates

Introducing an albumin-binding entity into otherwise short-lived radiopharmaceuticals can be an effective means to improve their pharmacokinetic properties due to enhanced blood residence time. In the current study, DOTA-derivatized albumin binders based on 4-(p-iodophenyl)butanoate (DOTA-ALB-1 and DOTA-ALB-3) and 5-(p-iodophenyl)pentanoate entities (DOTA-ALB-24 and DOTA-ALB-25) without and with a hydrophobic 4-(aminomethyl)benzoic acid (AMBA) linker unit, respectively, were synthesized and labeled with lutetium-177 for in vitro and in vivo comparison. Overall, [177Lu]Lu-DOTA-ALB-1 demonstrated ~3-fold stronger in vitro albumin-binding affinity and a longer blood residence time (T50%IA ~8 h) than [177Lu]Lu-DOTA-ALB-24 (T50%IA ~0.8 h). Introducing an AMBA linker enhanced the albumin-binding affinity, resulting in a T50%IA of ~24 h for [177Lu]Lu-DOTA-ALB-3 and ~2 h for [177Lu]Lu-DOTA-ALB-25. The same albumin binders without or with the AMBA linker were incorporated into 6R- and 6S-5-methyltetrahydrofolate-based DOTA-conjugates (177Lu-RedFols). Biodistribution studies in mice performed with both diastereoisomers of [177Lu]Lu-RedFol-1 and [177Lu]Lu-RedFol-3, which comprised the 4-(p-iodophenyl)butanoate moiety, demonstrated a slower accumulation in KB tumors than those of [177Lu]Lu-RedFol-24 and [177Lu]Lu-RedFol-25 with the 5-(p-iodophenyl)pentanoate entity. In all cases, the tumor uptake was high (30–45% IA/g) 24 h after injection. Both diastereoisomers of [177Lu]Lu-RedFol-1 and [177Lu]Lu-RedFol-3 demonstrated high blood retention (3.8–8.7% IA/g, 24 h p.i.) and a 2- to 4-fold lower kidney uptake than the corresponding diastereoisomers of [177Lu]Lu-RedFol-24 and [177Lu]Lu-RedFol-25, which were more rapidly cleared from the blood (<0.2% IA/g, 24 h after injection). Kidney retention of the 6S-diastereoisomers of all 177Lu-RedFols was consistently higher than that of the respective 6R-diastereoisomers, irrespective of the albumin binder and linker unit used. It was demonstrated that the blood clearance data obtained with 177Lu-DOTA-ALBs had predictive value for the blood retention times of the respective folate radioconjugates. The use of these albumin-binding entities without or with an AMBA linker may serve for fine-tuning the blood retention of folate radioconjugates and also other radiopharmaceuticals and, hence, optimize their tissue distribution profiles. Dosimetry estimations based on patient data obtained with one of the most promising folate radioconjugates will be crucial to identify the dose-limiting organ, which will allow for selecting the most suitable folate radioconjugate for therapeutic purposes.ISSN:2072-669

Fluorine-18 click radiosynthesis and preclinical evaluation of a new 18F-labeled folic acid derivative

The folate receptor (FR) is highly expressed on most epithelial cancer cells, while normal cells show only restricted expression of FR. As a result, the FR is an ideal target for receptor-based molecular imaging and therapy of cancer and has become a promising target in oncology. To date, several folate-based chemotherapeutics and imaging probes such as radiopharmaceuticals for single photon emission computed tomography (SPECT) have been developed. However, an (18)F-labeled folic acid derivative suitable for positron emission tomography (PET) imaging that can be routinely applied is still lacking. In this study, a new fluorinated and radiofluorinated folic acid derivative, (18/19)F-click folate, was synthesized using click chemistry. In a convenient and very efficient two-step radiosynthesis, the isolated (18)F-click folate was obtained in good radiochemical yields of 25-35% with a specific activity of 160+/-70 GBq/micromol after >or=90 min synthesis time. The new compound was pharmacologically evaluated in vitro and in vivo. The affinity of the non-radioactive (19)F-click folate to the FR was determined in displacement studies with FR expressing KB tumor cells using (3)H-folic acid. In these in vitro binding studies, a nanomolar affinity with a K(i) of 9.76+/-3.13 nM was found for (19)F-click folate. The (18)F-labeled click folate derivative was then applied for in vivo PET studies and ex vivo biodistribution experiments using nude mice bearing KB tumor xenografts. The post mortem dissection experiments showed a high specific uptake of (18)F-click folate derivative in FR-expressing tissues. Uptake in KB tumor xenografts and kidneys (FR-positive tissue) amounted to 3.13%ID/g (94% specific blockade) and 16.53%ID/g (75% specific blockade), respectively. PET imaging using (18)F-click folate permitted a visualization of KB tumors, and blockade studies confirmed the specific accumulation of the radiotracer in vivo. However, strong hepatobiliary excretion of the new tracer led to elevated accumulation of radioactivity in the abdominal region. In conclusion, the click chemistry approach is convenient to accomplish and provided high radiochemical yields of (18)F-click folate. The new tracer showed good in vitro but limited in vivo properties. Ultimately, the (18)F-click folate emphasizes the potential of (18)F-labeled folates for receptor-based tumor PET imaging

Reduced ¹⁸F‑Folate Conjugates as a New Class of PET Tracers for Folate Receptor Imaging

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 ¹⁸F-labeled 6<i>S</i>- and 6<i>R</i>-5-MTHF radiotracers for imaging folate receptor (FR)-α-positive tissues. Therefore, the goal of this study was to synthesize four ¹⁸F-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<i>S</i>-α, 6<i>S</i>-γ, 6<i>R</i>-α, and 6<i>R</i>-γ-click-fluoroethyl-5-MTHF, were carried out in low to moderate overall chemical yields. The radiosyntheses of the α- and γ-conjugated ¹⁸F-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. <i>In vitro</i>, all four 5-MTHF conjugates showed similar binding affinities to FR-α (IC₅₀ = 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 <i>in vivo</i> 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<i>S</i>- and 6<i>R</i>-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 ¹⁸F-labeled 5-MTHF conjugates are a promising new class of radiotracers for targeting FR-positive tumor tissues