Efficient Synthesis and HPLC-Based Characterization for Developing Vanadium-48-Labeled Vanadyl Acetylacetonate as a Novel Cancer Radiotracer for PET Imaging.

Bis(acetylacetonato)oxidovanadium(IV) [(VO(acac)2], generally known as vanadyl acetylacetonate, has been shown to be preferentially sequestered in malignant tissue. Vanadium-48 (48V) generated with a compact medical cyclotron has been used to label VO(acac)2 as a potential radiotracer in positron emission tomography (PET) imaging for the detection of cancer, but requires lengthy synthesis. Current literature protocols for the characterization of VO(acac)2 require macroscale quantities of reactants and solvents to identify products by color and to enable crystallization that are not readily adaptable to the needs of radiotracer synthesis. We present an improved method to produce vanadium-48-labeled VO(acac)2, [48V]VO(acac)2, and characterize it using high-performance liquid chromatography (HPLC) with radiation detection in combination with UV detection. The approach is suitable for radiotracer-level quantities of material. These methods are readily applicable for production of [48V]VO(acac)2. Preliminary results of preclinical, small-animal PET studies are presented.

Accelerator-Based Production of Scandium Radioisotopes for Applications in Prostate Cancer: Toward Building a Pipeline for Rapid Development of Novel Theranostics.

In the field of nuclear medicine, the ß+ -emitting 43Sc and ß- -emitting 47Sc are promising candidates in cancer diagnosis and targeted radionuclide therapy (TRT) due to their favorable decay schema and shared pharmacokinetics as a true theranostic pair. Additionally, scandium is a group-3 transition metal (like 177Lu) and exhibits affinity for DOTA-based chelators, which have been studied in depth, making the barrier to implementation lower for 43/47Sc than for other proposed true theranostics. Before 43/47Sc can see widespread pre-clinical evaluation, however, an accessible production methodology must be established and each isotope's radiolabeling and animal imaging capabilities studied with a widely utilized tracer. As such, a simple means of converting an 18 MeV biomedical cyclotron to support solid targets and produce 43Sc via the 42Ca(d,n)43Sc reaction has been devised, exhibiting reasonable yields. The NatTi(γ,p)47Sc reaction is also investigated along with the successful implementation of chemical separation and purification methods for 43/47Sc. The conjugation of 43/47Sc with PSMA-617 at specific activities of up to 8.94 MBq/nmol and the subsequent imaging of LNCaP-ENZaR tumor xenografts in mouse models with both 43/47Sc-PSMA-617 are also presented.

Stapled peptides as scaffolds for developing radiotracers for intracellular targets: Preliminary evaluation of a radioiodinated MDM2-binding stapled peptide in the SJSA-1 osteosarcoma model.

Stapled peptides are promising scaffolds for inhibiting protein-protein interactions in cells, including between the intracellular oncoprotein MDM2 and p53. Herein, we have investigated the potential utility of a stapled peptide, VIP116, for developing radiolabeled agents targeting MDM2. VIP116 was radioiodinated using the prosthetic agent N-succinimidyl-3-[*I]iodobenzoate ([*I]SIB). The resulting labeled peptide [*I]SIB-VIP116 exhibited high uptake (165.3 ± 27.7%/mg protein) and specificity in SJSA-1 tumor cells. Tissue distribution studies of [*I]SIB-VIP116 revealed a peak tumor uptake of 2.19 ± 0.56 percent injected dose per gram (%ID/g) in SJSA-1 xenografts at 2 h post-injection, which was stable until 6 h. [*I]SIB-VIP116 exhibited high activity (8.33 ± 1.18%ID/g) in the blood pool but had high tumor-to-muscle ratios (12.0 ± 5.7), at 30 min. Metabolic stability studies in mice indicated that about 80% of the activity in plasma was intact [*I]SIB-VIP116 at 4 h. Our results confirm the cell permeability and specific binding of [*I]SIB-VIP116 to MDM2 and the suitability of the VIP116 scaffold for radiolabeled probe development.

Fluorine-18 Labeling of the MDM2 Inhibitor RG7388 for PET Imaging: Chemistry and Preliminary Evaluation.

RG7388 (Idasanutlin) is a potent inhibitor of oncoprotein murine double minute 2 (MDM2). Herein we investigated the feasibility of developing 18F-labeled RG7388 as a radiotracer for imaging MDM2 expression in tumors with positron emission tomography (PET). Two fluorinated analogues of RG7388, 6 and 7, were synthesized by attaching a fluoronicotinyl moiety to RG7388 via a polyethylene glycol (PEG3) or a propyl linker. The inhibitory potency (IC50) of 6 and 7 against MDM2 was determined by a fluorescence polarization (FP)-based assay. Next, compound 6 was labeled with 18F using a trimethylammonium triflate precursor to obtain [18F]FN-PEG3-RG7388 ([18F]6), and its properties were evaluated in MDM2 expressing wild-type p53 tumor cell lines (SJSA-1 and HepG2) in vitro and in tumor xenografts in vivo. The FP assays revealed an IC50 against MDM2 of 119 nM and 160 nM for 6 and 7, respectively. 18F-labeling of 6 was achieved in 50.3 ± 7.5% radiochemical yield. [18F]6 exhibited a high uptake (∼70% of input dose) and specificity in SJSA-1 and HepG2 cell lines. Saturation binding assays revealed a binding affinity (Kd) of 128 nM for [18F]6 on SJSA-1 cells. In mice, [18F]6 showed fast clearance from blood with a maximum tumor uptake of 3.80 ± 0.85% injected dose per gram (ID/g) in HepG2 xenografts at 30 min postinjection (p.i.) and 1.32 ± 0.32% ID/g in SJSA-1 xenografts at 1 h p.i. Specificity of [18F]6 uptake in tumors was demonstrated by pretreatment of mice with SJSA-xenografts with a blocking dose of RG7388 (35 mg/kg body weight, i.p.). In vivo stability studies in mice using HPLC showed ∼60% and ∼30% intact [18F]6 remaining in plasma at 30 min and 1 h p.i., respectively, with the remaining activity attributed to polar peaks. Our results suggest that RG7388 is a promising molecular scaffold for 18F-labeled probe development for MDM2. Additional labeling strategies and functionalizing locations on RG7388 are under development to improve binding affinity and in vivo stability of the 18F-labeled compound to make it more amenable for PET imaging of MDM2 in vivo.

Observations on the Effects of Residualization and Dehalogenation on the Utility of N-Succinimidyl Ester Acylation Agents for Radioiodination of the Internalizing Antibody Trastuzumab.

Trastuzumab is an antibody used for the treatment of human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancers. Since trastuzumab is an internalizing antibody, two factors could play an important role in achieving high uptake and prolonged retention of radioactivity in HER2-positive tumors after radioiodination-residualizing capacity after receptor-mediated internalization and susceptibility to dehalogenation. To evaluate the contribution of these two factors, trastuzumab was radiolabeled using the residualizing reagent N-succinimidyl 4-guanidinomethyl-3-[*I]iodobenzoate ([*I]SGMIB) and the nonresidualizing reagent N-succinimidyl-3-[*I]iodobenzoate ([*I]SIB), both of which are highly dehalogenation-resistant. Paired-label uptake and intracellular retention of [125I]SGMIB-trastuzumab and [131I]SIB-trastuzumab was compared on HER2-expressing BT474 human breast carcinoma cells. Tumor uptake and normal tissue distribution characteristics for the two labeled conjugates were assessed in mice bearing BT474M1 xenografts. The internalization and intracellular retention of initially-bound radioactivity in BT474 cells was similar for the two labeled conjugates up to 4 h, but were significantly higher for [125I]SGMIB-trastuzumab at 6 and 24 h. Similarly, [*I]SGMIB labeling resulted in significantly higher uptake and retention of radioactivity in BT474M1 xenografts at all studied time points. Moreover, tumor-to-tissue ratios for [125I]SGMIB-trastuzumab were consistently higher than those for [131I]SIB-trastuzumab starting at 12 h postinjection. Thus, optimal targeting of HER2-positive breast cancers with a radioiodinated trastuzumab conjugate requires an acylation agent that imparts residualizing capacity in addition to high stability towards dehalogenation in vivo.

Synthesis and evaluation of radiolabeled AGI-5198 analogues as candidate radiotracers for imaging mutant IDH1 expression in tumors.

Mutations in the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) are commonly found in gliomas. AGI-5198, a potent and selective inhibitor of the mutant IDH1 enzyme, was radiolabeled with radioiodine and fluorine-18. These radiotracers were evaluated as potential probes for imaging mutant IDH1 expression in tumors with positron emission tomography (PET). Radioiodination of AGI-5198 was achieved using a tin precursor in 79 ±â€¯6% yield (n = 9), and 18F-labeling was accomplished by the Ugi reaction in a decay-corrected radiochemical yield of 2.6 ±â€¯1.6% (n = 5). The inhibitory potency of the analogous nonradioactive compounds against mutant IDH1 (IDH1-R132H) was determined in enzymatic assays. Cell uptake studies using radiolabeled AGI-5198 analogues revealed somewhat higher uptake in IDH1-mutated cells than that in wild-type IDH1 cells. The radiolabeled compounds displayed favorable tissue distribution characteristics in vivo, and good initial uptake in IDH1-mutated tumor xenografts; however, tumor uptake decreased with time. Radioiodinated AGI-5198 exhibited higher tumor-to-background ratios compared with 18F-labeled AGI-5198; unfortunately, similar results were observed in wild-type IDH1 tumor xenografts as well, indicating lack of selectivity for mutant IDH1 for this tracer. These results suggest that AGI-5198 analogues are not a promising platform for radiotracer development. Nonetheless, insights gained from this study may help in design and optimization of novel chemical scaffolds for developing radiotracers for imaging the mutant IDH1 enzyme.

Fluorine-18 labeling of an anti-HER2 VHH using a residualizing prosthetic group via a strain-promoted click reaction: Chemistry and preliminary evaluation.

In a previous study, we evaluated a HER2-specific single domain antibody fragment (sdAb) 2Rs15d labeled with 18F via conjugation of a residualizing prosthetic agent that was synthesized by copper-catalyzed azide-alkyne cycloaddition (CuAAC). In order to potentially increase overall efficiency and decrease the time required for labeling, we now investigate the use of a strain-promoted azide-alkyne cycloaddition (SPAAC) between the 2Rs15d sdAb, which had been pre-derivatized with an azide-containing residualizing moiety, and an 18F-labeled aza-dibenzocyclooctyne derivative. The HER2-targeted sdAb 2Rs15d and a nonspecific sdAb R3B23 were pre-conjugated with a moiety containing both azide- and guanidine functionalities. The thus derivatized sdAbs were radiolabeled with 18F using an 18F-labeled aza-dibenzocyclooctyne derivative ([18F]F-ADIBO) via SPAAC, generating the desired conjugate ([18F]RL-II-sdAb). For comparison, unmodified 2Rs15d was labeled with N-succinimidyl 4-guanidinomethyl-3-[125I]iodobenzoate ([125I]SGMIB), the prototypical residualizing agent for radioiodination. Radiochemical purity (RCP), immunoreactive fraction (IRF), HER2-binding affinity and cellular uptake of [18F]RL-II-2Rs15d were assessed in vitro. Paired label biodistribution of [18F]RL-II-2Rs15d and [125I]SGMIB-2Rs15d, and microPET/CT imaging of [18F]RL-II-2Rs15d and the [18F]RL-II-R3B23 control sdAb were performed in nude mice bearing HER2-expressing SKOV-3 xenografts. A radiochemical yield of 23.9 ±â€¯6.9% (n = 8) was achieved for the SPAAC reaction between [18F]F-ADIBO and azide-modified 2Rs15d and the RCP of the labeled sdAb was >95%. The affinity (Kd) and IRF for the binding of [18F]RL-II-2Rs15d to HER2 were 5.6 ±â€¯1.3 nM and 73.1 ±â€¯22.5% (n = 3), respectively. The specific uptake of [18F]RL-II-2Rs15d by HER2-expressing BT474M1 breast carcinoma cells in vitro was 14-17% of the input dose at 1, 2, and 4 h, slightly higher than seen for co-incubated [125I]SGMIB-2Rs15d. The uptake of [18F]RL-II-2Rs15d in SKOV-3 xenografts at 1 h and 2 h p.i. were 5.54 ±â€¯0.77% ID/g and 6.42 ±â€¯1.70% ID/g, respectively, slightly higher than those for co-administered [125I]SGMIB-2Rs15d (4.80 ±â€¯0.78% ID/g and 4.78 ±â€¯1.39% ID/g). MicroPET/CT imaging with [18F]RL-II-2Rs15d at 1-3 h p.i. clearly delineated SKOV-3 tumors while no significant accumulation of activity in tumor was seen for [18F]RL-II-R3B23. With the exception of kidneys, normal tissue levels for [18F]RL-II-2Rs15d were low and cleared rapidly. To our knowledge, this is the first time SPAAC method has been used to label an sdAb with 18F, especially with residualizing functionality.

Feasibility of Developing Radiotracers for MDM2: Synthesis and Preliminary Evaluation of an 18F-Labeled Analogue of the MDM2 Inhibitor SP-141.

Murine double minute 2 (MDM2), a negative regulator of the p53 tumor suppressor protein, is overexpressed in several human cancers. Herein we investigate the feasibility of developing 18F-labeled compounds based on the small molecule inhibitor SP-141 for imaging tumor MDM2 expression levels with positron emission tomography (PET). Three nonradioactive fluorinated SP-141 analogues, 1-3, were synthesized, and their binding to the MDM2 protein was analyzed by surface plasmon resonance (SPR). One of these, a fluoroethoxy analogue, was labeled with fluorine-18 (18F) using 18F-fluorethyl bromide to provide [18F]1 and evaluated in vitro and in vivo. SPR analysis confirmed the binding of the fluorinated analogues to MDM2 at 1.25-20 µM concentrations. Cell uptake studies revealed high uptake (67.5-71.4%/mg protein) and specificity of [18F]1 in MCF7 and HepG2 cells. The uptake of [18F]1 in these cells could be modulated using 100 µM SP-141, potentially reflecting changes in MDM2 expression because of p53 activation by SP-141. [18F]1 exhibited stable uptake and retention in HepG2 tumor xenografts (~3 %ID/g) in vivo, but poor clearance from blood and other normal tissues, yielding low tumor-to-background ratios (<2) at 2 h post injection. Our results suggest that [18F]1 has suboptimal characteristics for in vivo evaluation as a PET tracer for MDM2, but warrant radiolabeling and assessment of the other fluorinated analogues synthesized in this work, 2 and 3, and potentially other molecular scaffolds for developing MDM2 targeted radiotracers.

Increased Glutaminolysis Marks Active Scarring in Nonalcoholic Steatohepatitis Progression.

BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH) occurs in the context of aberrant metabolism. Glutaminolysis is required for metabolic reprograming of hepatic stellate cells (HSCs) and liver fibrogenesis in mice. However, it is unclear how changes in HSC glutamine metabolism contribute to net changes in hepatic glutaminolytic activity during fibrosis progression, or whether this could be used to track fibrogenic activity in NASH. We postulated that increased HSC glutaminolysis marks active scarring in NASH. METHODS: Glutaminolysis was assessed in mouse NASH fibrosis models and in NASH patients. Serum and liver levels of glutamine and glutamate and hepatic expression of glutamine transporter/metabolic enzymes were correlated with each other and with fibrosis severity. Glutaminolysis was disrupted in HSCs to examine if this directly influenced fibrogenesis. 18F-fluoroglutamine positron emission tomography was used to determine how liver glutamine assimilation tracked with hepatic fibrogenic activity in situ. RESULTS: The serum glutamate/glutamine ratio increased and correlated with its hepatic ratio, myofibroblast content, and fibrosis severity. Healthy livers almost exclusively expressed liver-type glutaminase (Gls2); Gls2 protein localized in zone 1 hepatocytes, whereas glutamine synthase was restricted to zone 3 hepatocytes. In fibrotic livers, Gls2 levels reduced and glutamine synthase zonality was lost, but both Slc1a5 (glutamine transporter) and kidney-type Gls1 were up-regulated; Gls1 protein was restricted to stromal cells and accumulated in fibrotic septa. Hepatocytes did not compensate for decreased Gls2 by inducing Gls1. Limiting glutamine or directly inhibiting GLS1 inhibited growth and fibrogenic activity in cultured human HSCs. Compared with healthy livers, fibrotic livers were 18F-fluoroglutamine-avid by positron emission tomography, suggesting that glutamine-addicted myofibroblasts drive increased hepatic utilization of glutamine as fibrosis progresses. CONCLUSIONS: Glutaminolysis is a potential diagnostic marker and therapeutic target during NASH fibrosis progression.

Improved synthesis and metabolic stability analysis of the dopamine transporter ligand [(18)F]FECT.

INTRODUCTION: [2'-[(18)F]Fluoroethyl (lR-2-exo-3-exe)-8-methyl-3-(4-chlorophenyl)-8-azabicyclo[3.2.1]-octane-2-carboxylate] ([(18)F]FECT) is a positron emission tomography (PET) tracer for imaging the dopamine transporter (DAT) in vivo. We report an improved radiosynthesis procedure and affinity data and have analyzed both brain tissue and plasma samples for the presence of radiometabolites as a function of time post intravenous injection of [(18)F]FECT to rats. METHODS: The radiosynthesis of [(18)F]FECT was carried out using [(18)F]fluoroethyltriflate ([(18)F]FEtOTf) as a labeling agent. The affinity of FECT for DAT was determined in vitro by binding experiments on rat striatal membranes. Three rats were injected with [(18)F]FECT and blood samples were collected at 1 or 3 h post injection (p.i.). Plasma was separated and analyzed using reversed-phase high-performance liquid chromatography (RP-HPLC). Similarly, cerebrum and cerebellum were isolated after sacrifice of the animals at 3 h p.i. of the tracer and homogenized. HPLC analysis was performed on extracts of both samples to examine the presence of metabolites. RESULTS: The radiochemical yield for [(18)F]FECT was 85% relative to the starting activity of [(18)F]FEtOTf. The inhibitory constant (K(i)) of FECT for DAT was found to be 6 nM. The fraction of radioactivity corresponding to intact [(18)F]FECT was 93% in plasma at both 1 and 3 h p.i. and 96% in cerebrum as well as cerebellum samples at 3 h p.i. CONCLUSIONS: FECT has a high affinity for the dopamine transporter. [(18)F]FECT was found to be stable in vivo and the amount of radiolabeled metabolites in plasma and brain at 3 h p.i. is negligible. Hence, [(18)F]FECT can be used for the in vivo quantification of DAT using PET.

Efficient purification and metabolite analysis of radiotracers using high-performance liquid chromatography and on-line solid-phase extraction.

This study describes an efficient method using on-line solid-phase extraction (SPE) (Oasis HLB) for preparative HPLC purification of short-lived radiotracers for positron emission tomography (PET) and for HPLC analysis of radiotracers and their metabolites in cell homogenates, plasma and urine samples. The radiochemical purity of tracers (fluorine-18 labeled) purified using this method (Oasis column) was >99% compared to 90% when no Oasis column was used. Radiometabolites of several fluorine-18 and carbon-11-labeled tracers and one technetium-99m tracer were quantified in cell homogenates, plasma and urine samples. Samples were analyzed using Oasis column and analytical HPLC system without prior precipitation of proteins or removal of other biological matrices. The metabolites observed for the evaluated tracers were all polar relative to the unchanged tracer. The extraction repeatability was found to be good (RSD 2.2%) and recoveries of Oasis column/HPLC-injected radioactivity (plasma) were found to be high (mean recovery >91%). The same Oasis column was used for several times without back pressure build-up or decrease of the HPLC separation characteristics.

Synthesis and Evaluation of a 18F-Labeled Triazinediamine Analogue for Imaging Mutant IDH1 Expression in Gliomas by PET.

Mutations in the isocitrate dehydrogenase gene 1 (IDH1) are common in gliomas. Studies suggest that IDH1 mutations are early events in glioma formation and are important drivers of malignant progression. Herein, we report the synthesis and evaluation of a 18F-labeled triazinediamine analogue, [18F]1, as a candidate radiotracer for noninvasive imaging of IDH1 mutations in gliomas by positron emission tomography (PET). In vitro studies revealed good binding inhibition potency and binding affinity for [18F]1 in IDH1 mutant glioma cell lines, with a half-maximal inhibitory concentration value (IC50) of 54 nM and an equilibrium dissociation constant (Kd) of 40 nM. In vivo studies using mutant IDH1 glioma xenografts showed good tumor uptake of [18F]1 and specific inhibition by the unlabeled 1, but also elevated radioactivity uptake in the bone, suggesting significant defluorination. The results support further optimization of the triazinediamine scaffold to develop a more stable and potent 18F-labeled analogue for PET imaging of IDH1 mutations in gliomas.

A Rationally Designed Fully Human EGFRvIII:CD3-Targeted Bispecific Antibody Redirects Human T Cells to Treat Patient-derived Intracerebral Malignant Glioma.

Purpose: Conventional therapy for malignant glioma fails to specifically target tumor cells. In contrast, substantial evidence indicates that if appropriately redirected, T cells can precisely eradicate tumors. Here we report the rational development of a fully human bispecific antibody (hEGFRvIII-CD3 bi-scFv) that redirects human T cells to lyse malignant glioma expressing a tumor-specific mutation of the EGFR (EGFRvIII).Experimental Design: We generated a panel of bispecific single-chain variable fragments and optimized design through successive rounds of screening and refinement. We tested the ability of our lead construct to redirect naïve T cells and induce target cell-specific lysis. To test for efficacy, we evaluated tumor growth and survival in xenogeneic and syngeneic models of glioma. Tumor penetrance following intravenous drug administration was assessed in highly invasive, orthotopic glioma models.Results: A highly expressed bispecific antibody with specificity to CD3 and EGFRvIII was generated (hEGFRvIII-CD3 bi-scFv). Antibody-induced T-cell activation, secretion of proinflammatory cytokines, and proliferation was robust and occurred exclusively in the presence of target antigen. hEGFRvIII-CD3 bi-scFv was potent and target-specific, mediating significant lysis of multiple malignant glioma cell lines and patient-derived malignant glioma samples that heterogeneously express EGFRvIII. In both subcutaneous and orthotopic models, well-engrafted, patient-derived malignant glioma was effectively treated despite heterogeneity of EGFRvIII expression; intravenous hEGFRvIII-CD3 bi-scFv administration caused significant regression of tumor burden (P < 0.0001) and significantly extended survival (P < 0.0001). Similar efficacy was obtained in highly infiltrative, syngeneic glioma models, and intravenously administered hEGFRvIII-CD3 bi-scFv localized to these orthotopic tumors.Conclusions: We have developed a clinically translatable bispecific antibody that redirects human T cells to safely and effectively treat malignant glioma. On the basis of these results, we have developed a clinical study of hEGFRvIII-CD3 bi-scFv for patients with EGFRvIII-positive malignant glioma. Clin Cancer Res; 24(15); 3611-31. ©2018 AACR.

Synthesis and preliminary evaluation of 18F- or 11C-labeled bicyclic nucleoside analogues as potential probes for imaging varicella-zoster virus thymidine kinase gene expression using positron emission tomography.

Two radiolabeled bicyclic nucleoside analogues (BCNAs) were synthesized, namely 3-(2'-deoxy-beta-d-ribofuranosyl)-6-(3-[18F]fluoroethoxyphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one ([18F]-2) and 3-(2'-deoxy-beta-d-ribofuranosyl)-6-(3-[11C]methoxyphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one ([11C]-3), and evaluated as PET reporter probes for varicella-zoster virus thymidine kinase (VZV-tk) gene expression imaging in brain. [18F]-2 and [11C]-3 were synthesized starting from phenol precursor 1. The phenol precursor 1 was converted to stable as well as to radiolabeled compounds 2 and 3 using (19/18)FCH(2)CH(2)Br or (12/11)CH(3)I as alkylating agent. In vitro evaluation of [18F]-2 and [11C]-3 in 293T cells showed a 4.5 and 53-fold higher uptake, respectively, into VZV-tk gene-transduced cells compared to control cells. However, biodistribution studies in mice demonstrated low uptake of these tracers in the brain. RP-HPLC analysis of plasma and urine samples of mice injected with [11C]-3 revealed that this tracer is very stable in vivo. These data warrant further evaluation of these tracers as noninvasive imaging agents for VZV infection and VZV-tk reporter gene expression in vivo.

A p-[18F]fluoroethoxyphenyl bicyclic nucleoside analogue as a potential positron emission tomography imaging agent for varicella-zoster virus thymidine kinase gene expression.

We recently reported a new positron emission tomography (PET) reporter gene, namely, varicella-zoster virus thymidine kinase (VZV-tk) in combination, with carbon-11 or fluorine-18 labeled m-alkoxyphenyl bicyclic nucleoside analogues (BCNAs) as PET reporter probes. We now report the synthesis and evaluation of p-alkoxyphenyl-BCNA tracers ([11C]-4 and [18F]-5), which are found to be superior to the m-alkoxyphenyl-BCNA tracers. In particular, the fluorine-18 labeled tracer ([18F]-5, IC50 of 5 is 4.2 microM) shows a higher accumulation in VZV-tk expressing cells than the previously reported m-methoxyphenyl BCNA. [11C]-4 and [18F]-5 were synthesized by heating the phenol precursor 3 with 11CH 3I and 18FCH 2CH 2Br, respectively, as alkylating agents. In vitro evaluation of [11C]-4 and [18F]-5 in 293T cells showed about 14- and 54-fold higher uptake, respectively, into VZV-tk gene-transduced cells compared to control cells. LC-MS analysis confirmed the formation of monophosphate derivative of 5 upon catalysis by VZV TK. In vivo studies of this new reporter gene/probe system are in progress.

Fetal gene transfer with lentiviral vectors: long-term in vivo follow-up evaluation in a rat model.

OBJECTIVE: The purpose of this study was to evaluate the long-term expression of a transgene and subsequent immune response after the injection of lentiviral vectors in a fetal rats. STUDY DESIGN: Fetal rats were injected in the liver, peritoneal cavity, or lung at E19 (term, E21) with a lentiviral vector expressing enhanced green fluorescent protein and luciferase. Controls received saline solution. After birth, full body bioluminescence was done at weeks 1, 4, 10, and 30 of life; seroconversion for the transgene was assessed. RESULTS: All surviving fetuses that had been injected in the liver (8/9 fetuses), peritoneum (3/3 fetuses), or lung (9/10 fetuses) showed a signal on bioluminescence imaging scan up to 30 weeks. None of the survivors displayed seroconversion against the transgene. CONCLUSION: In the rat model, the administration of lentiviral vectors into the fetal lung and liver resulted in long-term transgene expression without detectable humoral immune response.

Radiolabeled inhibitors as probes for imaging mutant IDH1 expression in gliomas: Synthesis and preliminary evaluation of labeled butyl-phenyl sulfonamide analogs.

INTRODUCTION: Malignant gliomas frequently harbor mutations in the isocitrate dehydrogenase 1 (IDH1) gene. Studies suggest that IDH mutation contributes to tumor pathogenesis through mechanisms that are mediated by the neomorphic metabolite of the mutant IDH1 enzyme, 2-hydroxyglutarate (2-HG). The aim of this work was to synthesize and evaluate radiolabeled compounds that bind to the mutant IDH1 enzyme with the goal of enabling noninvasive imaging of mutant IDH1 expression in gliomas by positron emission tomography (PET). METHODS: A small library of nonradioactive analogs were designed and synthesized based on the chemical structure of reported butyl-phenyl sulfonamide inhibitors of mutant IDH1. Enzyme inhibition assays were conducted using purified mutant IDH1 enzyme, IDH1-R132H, to determine the IC50 and the maximal inhibitory efficiency of the synthesized compounds. Selected compounds, 1 and 4, were labeled with radioiodine ((125)I) and/or (18)F using bromo- and phenol precursors, respectively. In vivo behavior of the labeled inhibitors was studied by conducting tissue distribution studies with [(125)I]1 in normal mice. Cell uptake studies were conducted using an isogenic astrocytoma cell line that carried a native IDH1-R132H mutation to evaluate the potential uptake of the labeled inhibitors in IDH1-mutated tumor cells. RESULTS: Enzyme inhibition assays showed good inhibitory potency for compounds that have iodine or a fluoroethoxy substituent at the ortho position of the phenyl ring in compounds 1 and 4 with IC50 values of 1.7 µM and 2.3 µM, respectively. Compounds 1 and 4 inhibited mutant IDH1 activity and decreased the production of 2-HG in an IDH1-mutated astrocytoma cell line. Radiolabeling of 1 and 4 was achieved with an average radiochemical yield of 56.6 ± 20.1% for [(125)I]1 (n = 4) and 67.5 ± 6.6% for [(18)F]4 (n = 3). [(125)I]1 exhibited favorable biodistribution characteristics in normal mice, with rapid clearance from the blood and elimination via the hepatobiliary system by 4 h after injection. The uptake of [(125)I]1 in tumor cells positive for IDH1-R132H was significantly higher compared to isogenic WT-IDH1 controls, with a maximal uptake ratio of 1.67 at 3 h post injection. Co-incubation of the labeled inhibitors with the corresponding nonradioactive analogs, and decreasing the normal concentrations of FBS (10%) in the incubation media substantially increased the uptake of the labeled inhibitors in both the IDH1-mutant and WT-IDH1 tumor cell lines, suggesting significant non-specific binding of the synthesized labeled butyl-phenyl sulfonamide inhibitors. CONCLUSIONS: These data demonstrate the feasibility of developing radiolabeled probes for the mutant IDH1 enzyme based on enzyme inhibitors. Further optimization of the labeled inhibitors by modifying the chemical structure to decrease the lipophilicity and to increase potency may yield compounds with improved characteristics as probes for imaging mutant IDH1 expression in tumors.

Comparison of the Hypoxia PET Tracer (18)F-EF5 to Immunohistochemical Marker EF5 in 3 Different Human Tumor Xenograft Models.

UNLABELLED: The availability of (18)F-labeled and unlabeled 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide (EF5) allows for a comparative assessment of tumor hypoxia by PET and immunohistochemistry; however, the combined use of these 2 approaches has not been fully assessed in vivo. The aim of this study was to evaluate (18)F-EF5 tumor uptake versus EF5 binding and hypoxia as determined from immunohistochemistry at both macroscopic and microregional levels. METHODS: Three tumor models-PC3, HCT116, and H460-were evaluated. Tumor-bearing animals were coinjected with (18)F-EF5 and EF5 (30 mg/kg), and PET imaging was performed at 2.5 h after injection. After PET imaging and 2 min after Hoechst 33342 injection, the tumors were excised and evaluated for (18)F-EF5 distribution by autoradiography and EF5 binding by immunohistochemistry. Additionally, the effects of nonradioactive EF5 (30 mg/kg) on the hypoxia-imaging characteristics of (18)F-EF5 were evaluated by comparing the PET data for H460 tumors with those from animals injected with (18)F-EF5 alone. RESULTS: The uptake of (18)F-EF5 in hypoxic tumor regions and the spatial relationship between (18)F-EF5 uptake and EF5 binding varied among tumors. H460 tumors showed higher tumor-to-muscle contrast in PET imaging; however, the distribution and uptake of the tracer was less specific for hypoxia in H460 than in HCT116 and PC3 tumors. Correlation analyses revealed that the highest spatial correlation between (18)F-EF5 uptake and EF5 binding was in PC3 tumors (r = 0.73 ± 0.02) followed by HCT116 (r = 0.60 ± 0.06) and H460 (r = 0.53 ± 0.10). Uptake and binding of (18)F-EF5 and EF5 correlated negatively with Hoechst 33342 perfusion marker distribution in the 3 tumor models. Image contrast and heterogeneous uptake of (18)F-EF5 in H460 tumors was significantly higher when the radiotracer was used alone versus in combination with unlabeled EF5 (tumor-to-muscle ratio of 2.51 ± 0.33 vs. 1.71 ± 0.17, P < 0.001). CONCLUSION: The uptake and hypoxia selectivity of (18)F-EF5 varied among tumor models when animals also received nonradioactive EF5. Combined use of radioactive and nonradioactive EF5 for independent assessment of tumor hypoxia by PET and immunohistochemistry methods is promising; however, the EF5 drug concentrations that are required for immunohistochemistry assays may affect the uptake of (18)F-EF5 in hypoxic cells in certain tumor types as observed in H460 in this study.

Copper signaling axis as a target for prostate cancer therapeutics.

Previously published reports indicate that serum copper levels are elevated in patients with prostate cancer and that increased copper uptake can be used as a means to image prostate tumors. It is unclear, however, to what extent copper is required for prostate cancer cell function as we observed only modest effects of chelation strategies on the growth of these cells in vitro. With the goal of exploiting prostate cancer cell proclivity for copper uptake, we developed a "conditional lethal" screen to identify compounds whose cytotoxic actions were manifested in a copper-dependent manner. Emerging from this screen was a series of dithiocarbamates, which, when complexed with copper, induced reactive oxygen species-dependent apoptosis of malignant, but not normal, prostate cells. One of the dithiocarbamates identified, disulfiram (DSF), is an FDA-approved drug that has previously yielded disappointing results in clinical trials in patients with recurrent prostate cancer. Similarly, in our studies, DSF alone had a minimal effect on the growth of prostate cancer tumors when propagated as xenografts. However, when DSF was coadministered with copper, a very dramatic inhibition of tumor growth in models of hormone-sensitive and of castrate-resistant disease was observed. Furthermore, we determined that prostate cancer cells express high levels of CTR1, the primary copper transporter, and additional chaperones that are required to maintain intracellular copper homeostasis. The expression levels of most of these proteins are increased further upon treatment of androgen receptor (AR)-positive prostate cancer cell lines with androgens. Not surprisingly, robust CTR1-dependent uptake of copper into prostate cancer cells was observed, an activity that was accentuated by activation of AR. Given these data linking AR to intracellular copper uptake, we believe that dithiocarbamate/copper complexes are likely to be effective for the treatment of patients with prostate cancer whose disease is resistant to classical androgen ablation therapies.

Development and biological evaluation of 99mTc-sulfonamide derivatives for in vivo visualization of CA IX as surrogate tumor hypoxia markers.

In vivo visualization of tumor hypoxia related markers, such as the endogenous transmembrane protein CA IX may lead to novel therapeutic and diagnostic applications in the management of solid tumors. In this study 4-(2-aminoethyl)benzene sulfonamide (AEBS, K(i) = 33 nM for CA IX) has been conjugated with bis(aminoethanethiol) (BAT) and mercaptoacetyldiglycine (MAG2) tetradendate ligands and the conjugates radiolabelled with (99m)Tc, to obtain anionic and neutral (99m)Tc-labeled sulfonamide derivatives, respectively. The corresponding rhenium analogues were also prepared and showed good inhibitory activities against hCA IX (K(i) = 59-66 nM). In addition, a second generation bis AEBS was conjugated with MAG2 and labeled with (99m)Tc, and the obtained diastereomers were also evaluated in targeting CA IX. Biodistribution studies in mice bearing HT-29 colorectal xenografts revealed a maximum tumor uptake of <0.5% ID/g at 0.5 h p.i for all the tracers. In vivo radiometabolite analysis indicated that at 1 h p.i. MAG2 tetradendate ligands were more stable in plasma (>50% intact) compared to the neutral complex (28% intact). This preliminary data suggest that negatively charged (99m)Tc-labeled sulfonamide derivatives with modest lipophilicity and longer circulation time could be promising markers to target CA IX.