Satish K. Chitneni

Molecular Imaging of Hypoxia

A wide variety of imaging approaches have been developed in the past few decades for monitoring tumor oxygenation and hypoxia in vivo. In particular, nuclear medicine has seen the development of several radiolabeled hypoxia markers and is the preferred method for imaging of tumor hypoxia. Hypoxia imaging is increasingly being used in the clinical setting and is progressing from a mere detection method to application in individualization of chemoradiotherapy.

Synthesis and biological evaluation of a 99mTc-labelled sulfonamide conjugate for in vivo visualization of carbonic anhydrase IX expression in tumor hypoxia

INTRODUCTION Carbonic anhydrase (CA) IX is a transmembrane protein overexpressed in many frequently occurring tumors associated with tumor hypoxia. Sulfonamides and their bioisosteres are known to inhibit CA IX activity. In this study, 4-(2-aminoethyl)benzenesulfonamide was conjugated to a tridentate ligand, N-2-picolyl-N-acetic acid and labeled with a (99m)Tc(I)-tricarbonyl moiety resulting in [(99m)Tc(CO)(3) (L)] (L=N-(pyridin-2-yl-methyl)-N[2-(4-sulfamoylphenyl)-ethyl]aminoethyl acetate) complex, [(99m)Tc]-5. Similarly the corresponding rhenium congener (Re-4) was synthesized. The in vitro CA IX affinity and inhibitory activity of Re-4 were determined and [(99m)Tc]-5 was evaluated as a tracer for in vivo visualisation of CA IX expression. METHODS Evaluation of the in vitro affinity (inhibition constant, K(i)) of Re-4 for CA isozymes I, II, IX and XII was carried out by assaying the CA catalyzed CO(2) hydration activity and efficacy studies were performed in HT 29 cell lines expressing CA IX under normoxia or hypoxia. Biodistribution studies of [(99m)Tc]-5 were performed in xenograft mice bearing CA IX expressing tumors. RESULTS The in vitro affinity of Re-4 for CA IX was 58 nM and CA IX induced acidification of extracellular medium was efficiently reduced (P<.05) in the presence of 1 mM Re-4. Biodistribution studies indicated a maximal tumor uptake of [(99m)Tc]-5 of 0.1% ID/g at 30 min post injection. CONCLUSION [(99m)Tc]-5 and its rhenium congener were synthesized and characterized. In vitro studies showed that the rhenium compound has a high affinity for CA IX and effectively inhibits CA IX activity. In vivo studies revealed a limited tracer accumulation in a CA IX expressing tumor but with increasing tumor-to-blood activity ratios as a function of time.

Preclinical Evaluation of 18F-JNJ41510417 as a Radioligand for PET Imaging of Phosphodiesterase-10A in the Brain

Phosphodiesterases are enzymes that inactivate the intracellular second messengers 3′,5′-cyclic adenosine-monophosphate and/or cyclic guanosine-monophosphate. Of all 11 known phosphodiesterase families, phosphodiesterase-10A (PDE10A) has the most restricted distribution, with high expression in the striatum. PDE10A inhibitors are pursued as drugs for treatment of neuropsychiatric disorders. We have synthesized and evaluated 18F-JNJ41510417 as a selective and high-affinity radioligand for in vivo brain imaging of PDE10A using PET. Methods: The biodistribution of 18F-JNJ41510417 was evaluated in rats. Rat plasma and perfused brain homogenates were analyzed by high-performance liquid chromatography to quantify radiometabolites. Dynamic small-animal PET was performed in rats and in wild-type and PDE10A knock-out mice and compared with ex vivo autoradiography. Blocking and displacement experiments were performed using the nonradioactive analog and other selective PDE10A inhibitors. Results: Tissue distribution studies showed predominant hepatobiliary excretion, sufficient brain uptake (0.56 ± 0.00 percentage injected dose at 2 min after tracer injection), and continuous accumulation of the tracer in the striatum over time; rapid washout of nonspecific binding from other brain regions was observed. Polar radiometabolites were detected in plasma and brain tissue. Dynamic small-animal PET showed continuous tracer accumulation in the striatum, with rapid decline in the cortex and cerebellum. Pretreatment and chase experiments with PDE10A inhibitors showed that the tracer binding to PDE10A was specific and reversible. Imaging in PDE10A knock-out and wild-type mice further confirmed that binding in the striatum was specific for PDE10A. Conclusion: Experiments in rats and PDE10A knock-out mice indicate that 18F-JNJ41510417 binds specifically and reversibly to PDE10A in the striatum, suggesting that this new fluorinated quinoline derivative is a promising candidate for in vivo imaging of PDE10A using PET.

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.

18F-EF5 PET Imaging as an Early Response Biomarker for the Hypoxia-Activated Prodrug SN30000 Combined with Radiation Treatment in a Non–Small Cell Lung Cancer Xenograft Model

Hypoxia is a significant therapeutic problem for solid tumors because hypoxic cells are treatment-resistant and more aggressive. Hypoxia-activated prodrugs such as SN30000 use a mechanism of activation in hypoxic cells similar to that of 2-nitroimidazole hypoxia PET tracers. Therefore, we have evaluated the usefulness of 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-18F-pentafluoropropyl)-acetamide (18F-EF5) PET to monitor and predict tumor response to SN30000 plus radiation treatment (RT). Methods: Human non–small cell lung cancer xenografts (H460) in athymic rats were imaged with 18F-EF5 PET before and after treatment with SN30000 (90 mg/kg), with or without 15-Gy RT. The feasibility of imaging early changes in hypoxia in response to SN30000 was examined 24 h after treatment, followed by ex vivo γ-counting and immunohistochemical examination to study drug-induced apoptosis. Subsequently, the therapeutic effects of SN30000 with or without RT were evaluated in tumor growth delay studies and compared with early treatment-induced changes observed by 18F-EF5 PET. Changes in tumor hemoglobin oxygen saturation as a function of time after treatment measured by optical spectroscopy were compared with PET data. Results: The uptake of 18F-EF5 was significantly lower in SN30000-treated tumors than in saline controls 24 h after treatment (mean standardized uptake value, 0.44 ± 0.08 vs. 0.56 ± 0.08 for control group; P < 0.05). Apoptosis was significantly higher in SN30000-treated tumors than in controls. Early treatment-induced changes in 18F-EF5 uptake were indicative of tumor response in growth delay studies at the group level. SN30000 plus RT significantly decreased 18F-EF5 uptake relative to baseline and resulted in complete tumor remission in 5 of 7 animals. SN30000 alone decreased 18F-EF5 uptake, generally in tumors with high initial standardized uptake values, and showed a minor tumor growth delay effect. The changes induced by SN30000 with or without RT in 18F-EF5 uptake correlated with baseline hypoxia levels. RT caused significant increases in tumor oxygen concentration and hemoglobin oxygen saturation. Conclusion: A hypoxia PET imaging agent can measure changes in tumor hypoxic fraction in response to SN30000. These results suggest the utility of 18F-EF5 PET for monitoring early response to tumor treatment with SN30000 plus RT in the clinical development of this novel hypoxia-activated prodrug.

A simplified synthesis of the hypoxia imaging agent 2-(2-Nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-[18F]pentafluoropropyl)-acetamide ([18F]EF5)

INTRODUCTION [(18)F]EF5 is a validated marker for PET imaging of tumor hypoxia. It is prepared by reacting a trifluoroallyl precursor with carrier-added [(18)F]F(2) gas in trifluoroacetic acid (TFA) solvent. We report here an improved radiosynthesis and purification of [(18)F]EF5 by utilizing an electroformed nickel (Ni) target for [(18)F]F(2) production, and Oasis® HLB cartridges for on-line solid phase extraction of [(18)F]EF5 prior to HPLC purification. METHODS [(18)F]F(2) was produced by deuteron bombardment of neon plus F(2) in an Ni target, and bubbled through the radiolabelling precursor solution. Purification was achieved by extracting the contents of the crude reaction mixture onto Oasis HLB cartridges, and subsequently eluted onto a semi-preparative HPLC column for further separation. Purified [(18)F]EF5 was evaluated in small animal PET studies using HCT116 tumor xenografts in nude mice. RESULTS The electroformed Ni target enabled recovery of >75% of the radioactivity from the cyclotron target, resulting in 16.2 ± 2.2 GBq (438 ± 58 mCi) of [(18)F]F(2) available for the synthesis. Use of Oasis cartridges yielded a less complex mixture for purification. On average, 1140 ± 200 MBq (30.8 ± 5.4 mCi) of [(18)F]EF5 were collected at EOS. Small animal PET imaging studies showed specific retention of [(18)F]EF5 in tumors, with tumor-to-muscle ratios of 2.7 ± 0.3 at about 160 min after injection. CONCLUSION A simple procedure has been developed for the routine synthesis of [(18)F]EF5 in amounts and purity required for clinical studies. This new method avoids the need for TFA evaporation and also enables facile automation of the synthesis using commercially available radiosynthesis modules.

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

The availability of 18F-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 18F-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 18F-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 18F-EF5 distribution by autoradiography and EF5 binding by immunohistochemistry. Additionally, the effects of nonradioactive EF5 (30 mg/kg) on the hypoxia-imaging characteristics of 18F-EF5 were evaluated by comparing the PET data for H460 tumors with those from animals injected with 18F-EF5 alone. Results: The uptake of 18F-EF5 in hypoxic tumor regions and the spatial relationship between 18F-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 18F-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 18F-EF5 and EF5 correlated negatively with Hoechst 33342 perfusion marker distribution in the 3 tumor models. Image contrast and heterogeneous uptake of 18F-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 18F-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 18F-EF5 in hypoxic cells in certain tumor types as observed in H460 in this study.

Preliminary validation of varicella zoster virus thymidine kinase as a novel reporter gene for PET.

INTRODUCTION Imaging of gene expression with positron emission tomography (PET) has emerged as a powerful tool for biomedical research during the last decade. The prototypical herpes simplex virus type 1 thymidine kinase (HSV1-TK) PET reporter gene (PRG) is widely used and many other PRGs have also been validated. We investigated varicella zoster virus thymidine kinase (VZV-tk) as new PRG with radiolabeled bicyclic nucleoside analogues (BCNAs) as PET tracers. METHODS The uptake and washout of four different radiolabeled BCNAs was evaluated in cells expressing VZV-tk after lentiviral vector (LV) transduction and in control cells. Metabolism of the tracers was assayed by high pressure liquid chromatography (HPLC). Mice bearing VZV-TK expressing xenografts were imaged with PET. RESULTS High uptake in VZV-tk expressing cells was seen for 3 of the 4 tracers tested. The uptake of the tracers could be blocked by the presence of excess thymidine in the incubation solution. Cellular retention was variable, with one tracer showing an acceptable half-life of ~1 hour. The amount of intracellular tracer correlated with the titer of LV used to transduce the cells. VZV-TK dependent conversion into metabolites was shown by HPLC. No specific accumulation was observed in cells expressing a fusion protein containing an HSV1-TK moiety. VZV-tk expression in xenografts resulted in a 60% increase in uptake in vivo as measured with PET. CONCLUSIONS We have validated the combination of VZV-tk and radiolabeled BCNAs as new PRG/PRP system. Further optimization of the PRPs and the PRG are warranted to increase the signal.

D-Amino acid peptide residualizing agents bearing N-hydroxysuccinimido- and maleimido-functional groups and their application for trastuzumab radioiodination.

INTRODUCTION Proteins that undergo receptor-mediated endocytosis are subject to lysosomal degradation, requiring radioiodination methods that minimize loss of radioactivity from tumor cells after this process occurs. To accomplish this, we developed the residualizing radioiodination agent N(ϵ)-(3-[(*)I]iodobenzoyl)-Lys(5)-N(α)-maleimido-Gly(1)-D-GEEEK (Mal-D-GEEEK-[(*)I]IB), which enhanced tumor uptake but also increased kidney activity and necessitates generation of sulfhydryl moieties on the protein. The purpose of the current study was to synthesize and evaluate a new D-amino acid based agent that might avoid these potential problems. METHODS N(α)-(3-iodobenzoyl)-(5-succinimidyloxycarbonyl)-D-EEEG (NHS-IB-D-EEEG), which contains 3 D-glutamates to provide negative charge and a N-hydroxysuccinimide function to permit conjugation to unmodified proteins, and the corresponding tin precursor were produced by solid phase peptide synthesis and subsequent conjugation with appropriate reagents. Radioiodination of the anti-HER2 antibody trastuzumab using NHS-IB-D-EEEG and Mal-D-GEEEK-IB was compared. Paired-label internalization assays on BT474 breast carcinoma cells and biodistribution studies in athymic mice bearing BT474M1 xenografts were performed to evaluate the two radioiodinated D-peptide trastuzumab conjugates. RESULTS NHS-[(131)I]IB-D-EEEG was produced in 53.8%±13.4% and conjugated to trastuzumab in 39.5%±7.6% yield. Paired-label internalization assays with trastuzumab-NHS-[(131)I]IB-D-EEEG and trastuzumab-Mal-D-GEEEK-[(125)I]IB demonstrated similar intracellular trapping for both conjugates at 1h ((131)I, 84.4%±6.1%; (125)I, 88.6%±5.2%) through 24h ((131)I, 60.7%±6.8%; (125)I, 64.9%±6.9%). In the biodistribution experiment, tumor uptake peaked at 48 h (trastuzumab-NHS-[(131)I]IB-D-EEEG, 29.8%±3.6%ID/g; trastuzumab-Mal-D-GEEEK-[(125)I]IB, 45.3%±5.3%ID/g) and was significantly higher for (125)I at all time points. In general, normal tissue levels were lower for trastuzumab-NHS-[(131)I]IB-D-EEEG, with the differences being greatest in kidneys ((131)I, 2.2%±0.4%ID/g; (125)I, 16.9%±2.8%ID/g at 144 h). CONCLUSION NHS-[(131)I]IB-D-EEEG warrants further evaluation as a residualizing radioiodination agent for labeling internalizing antibodies/fragments, particularly for applications where excessive renal accumulation could be problematic.

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 ± 6% yield (n = 9), and 18F-labeling was accomplished by the Ugi reaction in a decay-corrected radiochemical yield of 2.6 ± 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.