Multi-Aspect Optoacoustic Imaging of Breast Tumors under Chemotherapy with Exogenous and Endogenous Contrasts: Focus on Apoptosis and Hypoxia.

Breast cancer is a complex tumor type involving many biological processes. Most chemotherapeutic agents exert their antitumoral effects by rapid induction of apoptosis. Another main feature of breast cancer is hypoxia, which may drive malignant progression and confer resistance to various forms of therapy. Thus, multi-aspect imaging of both tumor apoptosis and oxygenation in vivo would be of enormous value for the effective evaluation of therapy response. Herein, we demonstrate the capability of a hybrid imaging modality known as multispectral optoacoustic tomography (MSOT) to provide high-resolution, simultaneous imaging of tumor apoptosis and oxygenation, based on both the exogenous contrast of an apoptosis-targeting dye and the endogenous contrast of hemoglobin. MSOT imaging was applied on mice bearing orthotopic 4T1 breast tumors before and following treatment with doxorubicin. Apoptosis was monitored over time by imaging the distribution of xPLORE-APOFL750©, a highly sensitive poly-caspase binding apoptotic probe, within the tumors. Oxygenation was monitored by tracking the distribution of oxy- and deoxygenated hemoglobin within the same tumor areas. Doxorubicin treatment induced an increase in apoptosis-depending optoacoustic signal of xPLORE-APOFL750© at 24 h after treatment. Furthermore, our results showed spatial correspondence between xPLORE-APO750© and deoxygenated hemoglobin. In vivo apoptotic status of the tumor tissue was independently verified by ex vivo fluorescence analysis. Overall, our results provide a rationale for the use of MSOT as an effective tool for simultaneously investigating various aspects of tumor pathophysiology and potential effects of therapeutic regimes based on both endogenous and exogenous molecular contrasts.

Resolution of Spatial and Temporal Heterogeneity in Bevacizumab-Treated Breast Tumors by Eigenspectra Multispectral Optoacoustic Tomography.

Understanding temporal and spatial hemodynamic heterogeneity as a function of tumor growth or therapy affects the development of novel therapeutic strategies. In this study, we employed eigenspectra multispectral optoacoustic tomography (eMSOT) as a next-generation optoacoustic method to impart high accuracy in resolving tumor hemodynamics during bevacizumab therapy in two types of breast cancer xenografts (KPL-4 and MDA-MB-468). Patterns of tumor total hemoglobin concentration (THb) and oxygen saturation (sO2) were imaged in control and bevacizumab-treated tumors over the course of 58 days (KPL-4) and 16 days (MDA-MB-468), and the evolution of functional vasculature "normalization" was resolved macroscopically. An initial sharp drop in tumor sO2 and THb content shortly after the initiation of bevacizumab treatment was followed by a recovery in oxygenation levels. Rim-core subregion analysis revealed steep spatial oxygenation gradients in growing tumors that were reduced after bevacizumab treatment. Critically, eMSOT imaging findings were validated directly by histopathologic assessment of hypoxia (pimonidazole) and vascularity (CD31). These data demonstrate how eMSOT brings new abilities for accurate observation of entire tumor responses to challenges at spatial and temporal dimensions not available by other techniques today. SIGNIFICANCE: Accurate assessment of hypoxia and vascularization over space and time is critical for understanding tumor development and the role of spatial heterogeneity in tumor aggressiveness, metastasis, and response to treatment.

Comparative Studies of Three Pairs of α- and γ-Conjugated Folic Acid Derivatives Labeled with Fluorine-18.

The folate receptor (FR) is upregulated in various epithelial cancer types (FR α-isoform), while healthy tissues show only restricted expression. FR-targeted imaging using folate radiopharmaceuticals is therefore a promising approach for the detection of FR-positive cancer tissue. Almost all folate-based radiopharmaceuticals have been prepared by conjugation at the γ-carboxylic functionality of the glutamate moiety of folic acid. In this work, three pairs of fluorinated α- and γ-conjugated folate derivatives were synthesized and their in vitro and in vivo properties compared. The syntheses of all six regioisomers were obtained in good chemical yields using a multistep synthetic approach including the highly selective Cu(I)-catalyzed 1,3-dipolar cycloaddition. The radiosyntheses of the α- and γ-conjugated (18)F-labeled folate derivatives were accomplished in moderate to good radiochemical yields, high radiochemical purities (>95%), and specific activities ranging from 25 to 196 GBq/µmol. In vitro, all folate derivatives showed high binding affinity to the FR-α (IC50 = 1.4-2.2 nM). In vivo PET imaging and biodistribution studies in FR-positive KB tumor-bearing mice demonstrated similar FR-specific tumor uptake for both regioisomers of each pair of compounds. However, FR-unspecific liver uptake was significantly lower for the α-regioisomers compared to the corresponding γ-regioisomers. In contrast, kidney uptake was up to 50% lower for the γ-regioisomers than for the α-regioisomers. These results show that the site of conjugation in the glutamyl moiety of folic acid has a significant impact on the in vivo behavior of (18)F-based radiofolates, but not on their in vitro FR-binding affinity. These findings may potentially stimulate new directions for the design of novel (18)F-labeled folate-based radiotracers.

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 (10-30 MBq/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 ≥23 Gy 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.

Violacein as a genetically-controlled, enzymatically amplified and photobleaching-resistant chromophore for optoacoustic bacterial imaging.

There is growing interest in genetically expressed reporters for in vivo studies of bacterial colonization in the context of infectious disease research, studies of the bacterial microbiome or cancer imaging and treatment. To empower non-invasive high-resolution bacterial tracking with deep tissue penetration, we herein use the genetically controlled biosynthesis of the deep-purple pigment Violacein as a photobleaching-resistant chromophore label for in vivo optoacoustic (photoacoustic) imaging in the near-infrared range. We demonstrate that Violacein-producing bacteria can be imaged with high contrast-to-noise in strongly vascularized xenografted murine tumors and further observe that Violacein shows anti-tumoral activity. Our experiments thus identify Violacein as a robust bacterial label for non-invasive optoacoustic imaging with high potential for basic research and future theranostic applications in bacterial tumor targeting.

Folate receptor targeted alpha-therapy using terbium-149.

Terbium-149 is among the most interesting therapeutic nuclides for medical applications. It decays by emission of short-range α-particles (Eα = 3.967 MeV) with a half-life of 4.12 h. The goal of this study was to investigate the anticancer efficacy of a 149Tb-labeled DOTA-folate conjugate (cm09) using folate receptor (FR)-positive cancer cells in vitro and in tumor-bearing mice. 149Tb was produced at the ISOLDE facility at CERN. Radiolabeling of cm09 with purified 149Tb resulted in a specific activity of ~1.2 MBq/nmol. In vitro assays performed with 149Tb-cm09 revealed a reduced KB cell viability in a FR-specific and activity concentration-dependent manner. Tumor-bearing mice were injected with saline only (group A) or with 149Tb-cm09 (group B: 2.2 MBq; group C: 3.0 MBq). A significant tumor growth delay was found in treated animals resulting in an increased average survival time of mice which received 149Tb-cm09 (B: 30.5 d; C: 43 d) compared to untreated controls (A: 21 d). Analysis of blood parameters revealed no signs of acute toxicity to the kidneys or liver in treated mice over the time of investigation. These results demonstrated the potential of folate-based α-radionuclide therapy in tumor-bearing mice.

Direct in vitro and in vivo comparison of (161)Tb and (177)Lu using a tumour-targeting folate conjugate.

PURPOSE: The radiolanthanide (161)Tb (T 1/2 = 6.90 days, Eß(-) av = 154 keV) was recently proposed as a potential alternative to (177)Lu (T 1/2 = 6.71 days, Eß(-) av = 134 keV) due to similar physical decay characteristics but additional conversion and Auger electrons that may enhance the therapeutic efficacy. The goal of this study was to compare (161)Tb and (177)Lu in vitro and in vivo using a tumour-targeted DOTA-folate conjugate (cm09). METHODS: (161)Tb-cm09 and (177)Lu-cm09 were tested in vitro on folate receptor (FR)-positive KB and IGROV-1 cancer cells using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay. In vivo (161)Tb-cm09 and (177)Lu-cm09 (10 MBq, 0.5 nmol) were investigated in two different tumour mouse models with regard to the biodistribution, the possibility for single photon emission computed tomography (SPECT) imaging and the antitumour efficacy. Potentially undesired side effects were monitored over 6 months by determination of plasma parameters and examination of kidney function with quantitative SPECT using (99m)Tc-dimercaptosuccinic acid (DMSA). RESULTS: To obtain half-maximal inhibition of tumour cell viability a 4.5-fold (KB) and 1.7-fold (IGROV-1) lower radioactivity concentration was required for (161)Tb-cm09 (IC50 ~0.014 MBq/ml and ~2.53 MBq/ml) compared to (177)Lu-cm09 (IC50 ~0.063 MBq/ml and ~4.52 MBq/ml). SPECT imaging visualized tumours of mice with both radioconjugates. However, in therapy studies (161)Tb-cm09 reduced tumour growth more efficiently than (177)Lu-cm09. These findings were in line with the higher absorbed tumour dose for (161)Tb-cm09 (3.3 Gy/MBq) compared to (177)Lu-cm09 (2.4 Gy/MBq). None of the monitored parameters indicated signs of impaired kidney function over the whole time period of investigation after injection of the radiofolates. CONCLUSION: Compared to (177)Lu-cm09 we demonstrated equal imaging features for (161)Tb-cm09 but an increased therapeutic efficacy for (161)Tb-cm09 in both tumour cell lines in vitro and in vivo. Further preclinical studies using other tumour-targeting radioconjugates are clearly necessary to draw final conclusions about the future clinical perspectives of (161)Tb.

Future prospects for SPECT imaging using the radiolanthanide terbium-155 - production and preclinical evaluation in tumor-bearing mice.

INTRODUCTION: We assessed the suitability of the radiolanthanide (155)Tb (t1/2=5.32 days, Eγ=87 keV (32%), 105keV (25%)) in combination with variable tumor targeted biomolecules using preclinical SPECT imaging. METHODS: (155)Tb was produced at ISOLDE (CERN, Geneva, Switzerland) by high-energy (~1.4 GeV) proton irradiation of a tantalum target followed by ionization and on-line mass separation. (155)Tb was separated from isobar and pseudo-isobar impurities by cation exchange chromatography. Four tumor targeting molecules - a somatostatin analog (DOTATATE), a minigastrin analog (MD), a folate derivative (cm09) and an anti-L1-CAM antibody (chCE7) - were radiolabeled with (155)Tb. Imaging studies were performed in nude mice bearing AR42J, cholecystokinin-2 receptor expressing A431, KB, IGROV-1 and SKOV-3ip tumor xenografts using a dedicated small-animal SPECT/CT scanner. RESULTS: The total yield of the two-step separation process of (155)Tb was 86%. (155)Tb was obtained in a physiological l-lactate solution suitable for direct labeling processes. The (155)Tb-labeled tumor targeted biomolecules were obtained at a reasonable specific activity and high purity (>95%). (155)Tb gave high quality, high resolution tomographic images. SPECT/CT experiments allowed excellent visualization of AR42J and CCK-2 receptor-expressing A431 tumors xenografts in mice after injection of (155)Tb-DOTATATE and (155)Tb-MD, respectively. The relatively long physical half-life of (155)Tb matched in particular the biological half-lives of (155)Tb-cm09 and (155)Tb-DTPA-chCE7 allowing SPECT imaging of KB tumors, IGROV-1 and SKOV-3ip tumors even several days after administration. CONCLUSIONS: The radiolanthanide (155)Tb may be of particular interest for low-dose SPECT prior to therapy with a therapeutic match such as the ß(-)-emitting radiolanthanides (177)Lu, (161)Tb, (166)Ho, and the pseudo-radiolanthanide (90)Y.

Promises of cyclotron-produced 44Sc as a diagnostic match for trivalent ß--emitters: in vitro and in vivo study of a 44Sc-DOTA-folate conjugate.

UNLABELLED: In recent years, implementation of (68)Ga-radiometalated peptides for PET imaging of cancer has attracted the attention of clinicians. Herein, we propose the use of (44)Sc (half-life = 3.97 h, average ß(+) energy [Eß(+)av] = 632 keV) as a valuable alternative to (68)Ga (half-life = 68 min, Eß(+)av = 830 keV) for imaging and dosimetry before (177)Lu-based radionuclide therapy. The aim of the study was the preclinical evaluation of a folate conjugate labeled with cyclotron-produced (44)Sc and its in vitro and in vivo comparison with the (177)Lu-labeled pendant. METHODS: (44)Sc was produced via the (44)Ca(p,n)(44)Sc nuclear reaction at a cyclotron (17.6 ± 1.8 MeV, 50 µA, 30 min) using an enriched (44)Ca target (10 mg (44)CaCO3, 97.00%). Separation from the target material was performed by a semiautomated process using extraction chromatography and cation exchange chromatography. Radiolabeling of a DOTA-folate conjugate (cm09) was performed at 95°C within 10 min. The stability of (44)Sc-cm09 was tested in human plasma. (44)Sc-cm09 was investigated in vitro using folate receptor-positive KB tumor cells and in vivo by PET/CT imaging of tumor-bearing mice RESULTS: Under the given irradiation conditions, (44)Sc was obtained in a maximum yield of 350 MBq at high radionuclide purity (>99%). Semiautomated isolation of (44)Sc from (44)Ca targets allowed formulation of up to 300 MBq of (44)Sc in a volume of 200-400 µL of ammonium acetate/HCl solution (1 M, pH 3.5-4.0) within 10 min. Radiolabeling of cm09 was achieved with a radiochemical yield of greater than 96% at a specific activity of 5.2 MBq/nmol. In vitro, (44)Sc-cm09 was stable in human plasma over the whole time of investigation and showed folate receptor-specific binding to KB tumor cells. PET/CT images of mice injected with (44)Sc-cm09 allowed excellent visualization of tumor xenografts. Comparison of cm09 labeled with (44)Sc and (177)Lu revealed almost identical pharmacokinetics. CONCLUSION: This study presents a high-yield production and efficient separation method of (44)Sc at a quality suitable for radiolabeling of DOTA-functionalized biomolecules. An in vivo proof-of-concept study using a DOTA-folate conjugate demonstrated the excellent features of (44)Sc for PET imaging. Thus, (44)Sc is a valid alternative to (68)Ga for imaging and dosimetry before (177)Lu-radionuclide tumor therapy.

177Lu-EC0800 combined with the antifolate pemetrexed: preclinical pilot study of folate receptor targeted radionuclide tumor therapy.

Targeted radionuclide therapy has shown impressive results for the palliative treatment of several types of cancer diseases. The folate receptor has been identified as specifically associated with a variety of frequent tumor types. Therefore, it is an attractive target for the development of new radionuclide therapies using folate-based radioconjugates. Previously, we found that pemetrexed (PMX) has a favorable effect in reducing undesired renal uptake of radiofolates. Moreover, PMX also acts as a chemotherapeutic and radiosensitizing agent on tumors. Thus, the aim of our study was to investigate the combined application of PMX and the therapeutic radiofolate (177)Lu-EC0800. Determination of the combination index (CI) revealed a synergistic inhibitory effect of (177)Lu-EC0800 and PMX on the viability of folate receptor-positive cervical (KB) and ovarian (IGROV-1) cancer cells in vitro (CI < 0.8). In an in vivo study, tumor-bearing mice were treated with (177)Lu-EC0800 (20 MBq) and a subtherapeutic (0.4 mg) or therapeutic amount (1.6 mg) of PMX. Application of (177)Lu-EC0800 with PMXther resulted in a two- to four-fold enhanced tumor growth delay and a prolonged survival of KB and IGROV-1 tumor-bearing mice, as compared to the combination with PMXsubther or untreated control mice. PMXsubther protected the kidneys from undesired side effects of (177)Lu-EC0800 (20 MBq) by reducing the absorbed radiation dose. Intact kidney function was shown by determination of plasma parameters and quantitative single-photon emission computed tomography using (99m)Tc-DMSA. Our results confirmed the anticipated dual role of PMX. Its unique features resulted in an improved antitumor effect of folate-based radionuclide therapy and prevented undesired radio-nephrotoxicity.

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 50 GBq/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 4 h post-injection (p.i.). Uptake in the kidneys (∼ 13 % ID/g; 1 h 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]3.

In vitro and in vivo evaluation of an innocuous drug cocktail to improve the quality of folic acid targeted nuclear imaging in preclinical research.

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.

Radiosynthesis and preclinical evaluation of 3'-Aza-2'-[(18)F]fluorofolic acid: a novel PET radiotracer for folate receptor targeting.

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.

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.

[18F]fluoro-deoxy-glucose folate: a novel PET radiotracer with improved in vivo properties for folate receptor targeting.

The folate receptor (FR) is upregulated in various cancer types (FR-α isoform) and in activated macrophages (FR-ß 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.