Novel 4-nitroimidazole analogues: synthesis, in vitro biological evaluation, in silico studies, and molecular dynamics simulation.

A new series of 4-nitroimidazole bearing aryl piperazines 7-16, tetrazole 17 and 1,3,4-thiadiazole 18 derivatives was synthesized. All derivatives were screened for their anticancer activity against eight diverse human cancer cell lines (Capan-1, HCT-116, LN229, NCI-H460, DND-41, HL-60, K562, and Z138). Compound 17 proved the most potent compound of the series inhibiting proliferation of most of the selected human cancer cell lines with IC50 values in the low micromolar range. In addition, compound 11 exhibited IC50 values ranging 8.60-64.0 µM against a selection of cancer cell lines. These findings suggest that derivative 17 can potentially be a new lead compound for further development of novel antiproliferative agents. Additionally, 17-18 were assessed for their antibacterial and antituberculosis activity. Derivatives 17 and 18 were the most potent compounds of this series against both Staphylococcus aureus strain Wichita and a methicillin resistant strain of S. aureus (MRSA), as well as against Mycobacterium tuberculosis strain mc26230. The antiviral activity of 7-18 was also evaluated against diverse viruses, but no activity was detected. The docking study of compound 17 with putative protein targets in acute myeloid leukemia had been studied. Furthermore, the molecular dynamics simulation of 17 and 18 had been investigated.

Preparation and bioevaluation of a novel 99mTc-labelled propylene amine oxime (PnAO) containing two 4-methyl-2-nitroimidazole groups as a promising tumor hypoxia imaging agent.

Hypoxia is a common phenomenon in solid tumors, and its presence inhibits the efficacy of tumor chemotherapy and radiotherapy. Accurate measurement of hypoxia before tumor treatment is essential. Three propylene amine oxime (PnAO) derivatives with different substituents attached to 2-nitroimidazole were synthesized in the work, they are 3,3,9,9-tetramethyl-1,11-bis(4-bromo-2-nitro-1H-imidazol-1-yl)-4,8-diazaundecane-2,10-dione dioxime (Br2P2), 3,3,9,9-tetramethyl-1,11-bis(4-methyl-2-nitro-1H-imidazol-1-yl)-4,8-diazaundecane-2,10-dione dioxime (Me2P2) and 3,3,9,9-tetramethyl-1,11-bis(4,5-dimethyl-2-nitro-1H-imidazol-1-yl)-4,8-diazaundecane-2,10-dione dioxime (2Me2P2). The three compounds were radiolabeled with 99mTc to give three complexes([99mTc]Tc-Br2P2, [99mTc]Tc-Me2P2 and [99mTc]Tc-2Me2P2) with good in vitro stability. [99mTc]Tc-Me2P2 with a more suitable reduction potential had the highest hypoxic cellular uptake, compared with [99mTc]Tc-2P2 that have been previously reported, [99mTc]Tc-Br2P2 and [99mTc]Tc-2Me2P2. Biodistribution results in S180 tumor-bearing mice demonstrated that [99mTc]Tc-Me2P2 had the highest tumor-to-muscle (T/M) ratio (12.37 ± 1.16) at 2 h in the four complexes. Autoradiography and immunohistochemical staining results revealed that [99mTc]Tc-Me2P2 specifically targeted tumor hypoxic regions. The SPECT/CT imaging results showed that [99mTc]Tc-Me2P2 could target the tumor site. [99mTc]Tc-Me2P2 may become a potential hypoxia imaging agent.

Pimonidazole-alkyne conjugate for sensitive detection of hypoxia by Cu-catalyzed click reaction.

Hypoxia is involved in various diseases, such as cancers. Pimonidazole has often been used as the gold-standard marker to visualize hypoxic regions. Pimonidazole labels hypoxic regions by forming a covalent bond with a neighboring protein under hypoxic conditions in the body, which is detected by immunohistochemistry performed on tissue sections. To date, some pimonidazole-fluorophore conjugates have been reported as fluorescent probes for hypoxia imaging that do not require immunostaining. They are superior to pimonidazole because immunostaining can produce high background signals. However, large fluorophores in the conjugates may alter the original biodistribution and reactivity. Here, we report a new hypoxia marker, Pimo-yne, as a pimonidazole-alkyne conjugate. Pimo-yne has a similar hypoxia detection capability as pimonidazole because the alkyne tag is small and can be detected by Cu-catalyzed click reaction with azide-tagged fluorescent dyes. We successfully visualized hypoxic regions in tumor tissue sections using Pimo-yne with reduced background signals. The detected regions overlapped well with those detected by pimonidazole immunohistochemistry. To further reduce the background, we employed a turn-on azide-tagged fluorescent dye.

Heterobifunctional Cross-Linker with Dinitroimidazole and N-Hydroxysuccinimide Ester Motifs for Protein Functionalization and Cysteine-Lysine Peptide Stapling.

A heterobifunctional cross-linker with one sulfhydryl-reactive dinitroimidazole end and another amine-reactive N-hydroxysuccinimide (NHS) ester end was designed and synthesized. The two motifs of this cross-linker, dinitroimidazole and NHS ester, proved to react with thiol and amine, respectively, in an orthogonal way. The cross-linker was further applied to construct stapled peptides of different sizes and mono- and dual functionalization (including biotinylation, PEGylation, and fluorescence labeling) of protein.

Comparison of three 18F-labeled 2-nitroimidazoles for imaging hypoxia in breast cancer xenografts: [18F]FBNA, [18F]FAZA and [18F]FMISO.

BACKGROUND: Tumour hypoxia is associated with increased metastasis, invasion, poor therapy response and prognosis. Most PET radiotracers developed and used for clinical hypoxia imaging belong to the 2-nitroimidazole family. Recently we have developed novel 2-nitroimidazole-derived PET radiotracer [18F]FBNA (N-(4-[18F]fluoro-benzyl)-2-(2-nitro-1H-imidazol-1-yl)-acet-amide), an 18F-labeled analogue of antiparasitic drug benznidazole. The present study aimed to analyze its radio-pharmacological properties and systematically compare its PET imaging profiles with [18F]FMISO and [18F]FAZA in preclinical triple-negative (MDA-MB231) and estrogen receptor-positive (MCF-7) breast cancer models. METHODS: In vitro cellular uptake experiments were carried out in MDA-MB321 and MCF-7 cells under normoxic and hypoxic conditions. Metabolic stability in vivo was determined in BALB/c mice using radio-TLC analysis. Dynamic PET experiments over 3 h post-injection were performed in MDA-MB231 and MCF-7 tumour-bearing mice. Those PET data were used for kinetic modelling analysis utilizing the reversible two-tissue-compartment model. Autoradiography was carried out in tumour tissue slices and compared to HIF-1α immunohistochemistry. Detailed ex vivo biodistribution was accomplished in BALB/c mice, and this biodistribution data were used for dosimetry calculation. RESULTS: Under hypoxic conditions in vitro cellular uptake was elevated in both cell lines, MCF-7 and MDA-MB231, for all three radiotracers. After intravenous injection, [18F]FBNA formed two radiometabolites, resulting in a final fraction of 65 ± 9 % intact [18F]FBNA after 60 min p.i. After 3 h p.i., [18F]FBNA tumour uptake reached SUV values of 0.78 ± 0.01 in MCF-7 and 0.61 ± 0.04 in MDA-MB231 tumours (both n = 3), representing tumour-to-muscle ratios of 2.19 ± 0.04 and 1.98 ± 0.15, respectively. [18F]FMISO resulted in higher tumour uptakes (SUV 1.36 ± 0.04 in MCF-7 and 1.23 ± 0.08 in MDA-MB231 (both n = 4; p < 0.05) than [18F]FAZA (0.66 ± 0.11 in MCF-7 and 0.63 ± 0.14 in MDA-MB231 (both n = 4; n.s.)), representing tumour-to-muscle ratios of 3.24 ± 0.30 and 3.32 ± 0.50 for [18F]FMISO, and 2.92 ± 0.74 and 3.00 ± 0.42 for [18F]FAZA, respectively. While the fraction per time of radiotracer entering the second compartment (k3) was similar within uncertainties for all three radiotracers in MDA-MB231 tumours, it was different in MCF-7 tumours. The ratios k3/(k3 + k2) and K1*k3/(k3 + k2) in MCF-7 tumours were also significantly different, indicating dissimilar fractions of radiotracer bound and trapped intracellularly: K1*k3/(k2 + k3) [18F]FMISO (0.0088 ± 0.001)/min, n = 4; p < 0.001) > [18F]FAZA (0.0052 ± 0.002)/min, n = 4; p < 0.01) > [18F]FBNA (0.003 ± 0.001)/min, n = 3). In contrast, in MDA-MB231 tumours, only K1 was significantly elevated for [18F]FMISO. However, this did not result in significant differences for K1*k3/(k2 + k3) for all three 2-nitroimidazoles in MDA-MB231 tumours. CONCLUSION: Novel 2-nitroimidazole PET radiotracer [18F]FBNA showed uptake into hypoxic breast cancer cells and tumour tissue presumably associated with elevated HIF1-α expression. Systematic comparison of PET imaging performance with [18F]FMISO and [18F]FAZA in different types of preclinical breast cancer models revealed a similar tumour uptake profile for [18F]FBNA with [18F]FAZA and, despite its higher lipophilicity, still a slightly higher muscle tissue clearance compared to [18F]FMISO.

Improving tumor/muscle and tumor/blood ratios of 99mTc-labeled nitroimidazole propylene amine oxime (PnAO) complexes with ethylene glycol linkers.

Three nitroimidazole propylene amine oxime (PnAO) derivatives with different lengths of ethylene glycol chain were synthesized and radiolabeled with 99mTc. The radiochemical purities of three 99mTc-labeled complexes, oxo[[6,6,12,12-tetramethyl-1,17-bis(2-nitro-1H-imidazol-1-yl)-3,15-dioxa-7,11-diazaheptadecane-5, 13-dione dioximato] (3-)-N,N',N'',N''']-technetium-99m (99mTc-2P2O1), oxo[[9,9,15,15-tetramethyl-1,23-bis(2-nitro-1H-imidazol-1-yl)-3,6,18,21-tetraoxa-10, 14-diazatricosane-8,16-dione dioximato] (3-)-N,N',N'',N''']-technetium-99m (99mTc-2P2O2) and oxo[[15,15,21,21-tetramethyl-1,35-bis(2-nitro-1H-imidazol-1-yl)-3,6,9,12,24,27,30,33-octaoxa-16,20-diazapentatriacontane-14,22-dione dioximato] (3-)-N,N',N'',N''']-technetium-99m (99mTc-2P2O4), were above 90%, and they were all stable both in vitro and in vivo. The hypoxia/normoxia uptake ratios of the three complexes were 2.92 ± 0.61, 2.63 ± 0.64 and 2.29 ± 0.67 in S180 cellular uptake assay (4 h). All of these complexes presented good hypoxia selectivity. The results of biodistribution studies in S180 tumor-bearing mice revealed that the tumor/muscle (T/M) ratios (7.20 ± 2.37, 7.19 ± 1.75, 5.56 ± 1.10) and tumor/blood (T/B) ratios (1.66 ± 0.34, 1.73 ± 0.25, 2.13 ± 0.19) at 4 h of three complexes were significantly higher than those of 99mTc-2P2 (3.24 ± 0.65, 0.81 ± 0.34) without the ethylene glycol chains. Among them, 99mTc-2P2O4 had the best T/B ratio. The new complexes have higher tumor/blood and tumor/muscle ratios by adding suitable length of ethylene glycol chain. It is helpful for the design and optimization of hypoxic imaging agents.

Reactive Molecular Dynamics Simulation of the Structural Damages of the B-DNA Induced by the Oxidation/Nitration of Guanine.

Reactive molecular dynamics simulations (RMD) have been carried out to investigate structural alterations of the dodecamer double-strand B-DNA due to the oxidation/nitration modifications introduced to its guanine bases, including 8-oxoguanine, 8-nitroguanine, and 5-guanidino-4-nitroimidazole, considering two distribution patterns. These modifications may arise in the case of cancer treatment using oxidative/nitrosative reactive nitrogen species as anticancer agents. Results show that these mutations affect structural characteristics of the B-DNA dodecamer in the order 8-nitroguanine > 5-guanidino-4-nitroimidazole ≫ 8-oxoguanine. For instance, the base-pair per turn for these modified B-DNA are changed respectively to 9.79, 10.88 and 10.58 from 10.51 in the native defect-free B-DNA, which is compatible with the experimental value of 10.10. In addition, these mutations allow more water molecules to diffuse into the dodecamer structure and consequently increase the possibility of the penetration of reactive and nonreactive species toward constituting nucleic base-pairs. The largest variation of the B-DNA structure is observed for the mutated B-DNA with 8-nitroguanine modifications applied to its separated CG base-pairs along the dodecamer chain. The structural changes introduced by these nitro-/oxo-modified guanine bases can be considered as a critical step in the damage of the DNA structure and alterations of its function.

Mitochondria-targeting multifunctional nanoplatform for cascade phototherapy and hypoxia-activated chemotherapy.

Despite considerable progress has been achieved in hypoxia-associated anti-tumor therapy, the efficacy of utilizing hypoxia-activated prodrugs alone is not satisfied owing to the inadequate hypoxia within the tumor regions. In this work, a mitochondrial targeted nanoplatform integrating photodynamic therapy, photothermal therapy and hypoxia-activated chemotherapy has been developed to synergistically treat cancer and maximize the therapeutic window. Polydopamine coated hollow copper sulfide nanoparticles were used as the photothermal nanoagents and thermosensitive drug carriers for loading the hypoxia-activated prodrug, TH302, in our study. Chlorin e6 (Ce6) and triphenyl phosphonium (TPP) were conjugated onto the surface of the nanoplatform. Under the action of TPP, the obtained nanoplatform preferentially accumulated in mitochondria to restore the drug activity and avoid drug resistance. Using 660 nm laser to excite Ce6 can generate ROS and simultaneously exacerbate the cellular hypoxia. While under the irradiation of 808 nm laser, the nanoplatform produced local heat which can increase the release of TH302 in tumor cells, ablate cancer cells as well as intensify the tumor hypoxia levels. The aggravated tumor hypoxia then significantly boosted the anti-tumor efficiency of TH302. Both in vitro and in vivo studies demonstrated the greatly improved anti-cancer activity compared to conventional hypoxia-associated chemotherapy. This work highlights the potential of using a combination of hypoxia-activated prodrugs plus phototherapy for synergistic cancer treatment.

Synthesis and bioevaluation of the cyclopentadienyl tricarbonyl technetium-99m 2-nitroimidazole derivatives for tumor hypoxia imaging.

Hypoxia imaging agents can play an important role in the tumor treatment by avoiding the worse effect of radiotherapy and chemotherapy due to the tumor hypoxia. Due to the small size and easy coordination, tricarbonyl technetium-99m can be used to label a wide range of imaging agents. In this work, the tricarbonyl 99mTc labeled small-sized hypoxia imaging agents containing 2-nitroimidazoles were prepared, which have different carbon chain lengths between cyclopentadienyl and 2-nitroimidazole, and which have one or two 2-nitroimidazole groups. The results of S180 cell experiment and biodistribution indicated that these molecules have different hypoxic selectivity. When contains one 2-nitroimidazole, as the carbon chain lengthens, which means the molecular volume becomes larger, hypoxia cellular uptake and selectivity decrease in S180 cell uptake experiment. In biodistribution study in mice bearing S180 tumor, Tc-2 (1-cyclopentadienyl-5-(2-nitro-1H-imidazol-1-yl)-pentan-1-one tricarbonyl 99mTc complex), which has intermediate carbon chain, is better due to the more complex factors. Its tumor/blood (T/B) ratio is 3.56 ± 0.25, tumor/muscle(T/M) ratio is 1.73 ± 0.29 and tumor uptake is 2.23 ± 0.24%ID/g at 2 h. Comparing to other tricarbonyl technetium complexes containing one 2-nitroimidazole, the complexes in this work have an advantage in tumor/blood ratio and tumor uptake. This suggests that the small-volume cyclopentadienyl may have an advantage when used as a ligand. When contains two 2-nitroimidazole groups, the complex, 1-cyclopentadienyl-5-di(2-(2-nitro-1H-imidazol-1-yl)ethyl)amino-pentan-1-one tricarbonyl 99mTc complex (Tc-4), has the better results in the cell experiment than those which contain one 2-nitroimidazole group. Thus the hypoxia imaging agent contains two 2-nitroimidazole groups is more advantageous, but further modifications of Tc-4 are needed to improve its clearance rate in the blood, because the increased lipophilicity leads to a decrease in the T/B ratio of Tc-4. In conclusion, small volume hypoxia imaging agents with two 2-nitroimidazole groups may be the trend of development.

Noncanonical Amino Acids for Hypoxia-Responsive Peptide Self-Assembly and Fluorescence.

Design of endogenous stimuli-responsive amino acids allows for precisely modulating proteins or peptides under a biological microenvironment and thereby regulating their performance. Herein we report a noncanonical amino acid 2-nitroimidazol-1-yl alanine and explore its functions in creation of the nitroreductase (NTR)-responsive peptide-based supramolecular probes for efficient hypoxia imaging. On the basis of the reduction potential of the nitroimidazole unit, the amino acid was synthesized via the Mitsunobu reaction between 2-nitroimidazole and a serine derivate. We elucidated the relationship between the NTR-responsiveness of the amino acid and the structural feature of peptides involving a series of peptides. This eventually facilitates development of aromatic peptides undergoing NTR-responsive self-assembly by rationally optimizing the sequences. Due to the intrinsic role of 2-nitroimidazole in the fluorescence quench, we created a morphology-transformable supramolecular probe for imaging hypoxic tumor cells based on NTR reduction. We found that the resulting supramolecular probes penetrated into solid tumors, thus allowing for efficient fluorescence imaging of tumor cells in hypoxic regions. Our findings demonstrate development of a readily synthesized and versatile amino acid with exemplified properties in creating fluorescent peptide nanostructures responsive to a biological microenvironment, thus providing a powerful toolkit for synthetic biology and development of novel biomaterials.

Synthesis and evaluation of multivalent nitroimidazole-based near-infrared fluorescent agents for neuroblastoma and colon cancer imaging.

Hypoxia is one of key characteristics of microenvironments of solid tumors, and evaluation of hypoxia status in solid tumors is important to determine cancer stage and appropriate treatment. In the present study, novel, multivalent, near-infrared (NIR) fluorescent imaging agents were developed to measure tumor hypoxia. These agents were synthesized using an amino acid as a backbone to connect mono-, bis-, or tris-2-nitroimidazole as a hypoxia-sensitive moiety to enhance uptake by the tumor and to attach sulfo-Cyanine 5.5 as an NIR fluorophore to visualize tumor accumulation. Studies of physical characteristics demonstrated that the novel NIR imaging agents showed suitable optical properties for in vitro and in vivo imaging and were stable in serum. In vitro cellular uptake studies in SK-N-BE(2) and SW620 cell lines demonstrated that NIR imaging agents bearing 2-nitroimidazole structures showed significantly higher tumor uptake in hypoxic cells than in normoxic cells. Moreover, in vivo optical imaging studies using SK-N-BE(2) and SW620 xenografted mice demonstrated that novel, multivalent, 2-nitroimadazole NIR imaging agents with two or three 2-nitroimidazole moieties showed higher uptake in tumor than the control agents with only one 2-nitroimidazole. These observations suggest that novel, multivalent, NIR agents could serve as potential optical imaging agents for evaluating tumor hypoxia.

Hypoxia and pH co-triggered oxidative stress amplifier for tumor therapy.

To keep fast proliferation, tumor cells are exposed to higher oxidative stress than normal cells and they upregulate the amount of some antioxidants such as glutathione (GSH) against reactive oxygen species to maintain the balance. This phenomenon is severe in hypoxic tumor cells. Although researchers have proposed a series of treatment strategies based on regulating the intracellular reactive oxygen species level, few of them are related to the hypoxic tumor. Herein, a novel organic compound (PLC) was designed by using lysine as a bridge to connect two functional small molecules, a hypoxia-responsive nitroimidazole derivative (pimonidazole) and a pH-responsive cinnamaldehyde (CA) derivative. Then, the oxidative stress amplifying ability of PLC in hypoxic tumor cells was evaluated. The acidic microenvironment of tumor can trigger the release of CA to produce reactive oxygen species. Meanwhile, large amount of nicotinamide adenine dinucleotide phosphate (NADPH) can be consumed to decrease the synthesis of GSH during the bio-reduction process of the nitro group in PLC under hypoxic conditions. Therefore, the lethal effect of CA can be amplified for the decrease of GSH. Our results prove that this strategy can significantly enhance the therapeutic effect of CA in the hypoxic tumor cells.

Chitosan oligosaccharide decorated liposomes combined with TH302 for photodynamic therapy in triple negative breast cancer.

BACKGROUND: Triple negative breast cancer (TNBC) is an aggressive tumor with extremely high mortality that results from its lack of effective therapeutic targets. As an adhesion molecule related to tumorigenesis and tumor metastasis, cluster of differentiation-44 (also known as CD44) is overexpressed in TNBC. Moreover, CD44 can be effectively targeted by a specific hyaluronic acid analog, namely, chitosan oligosaccharide (CO). In this study, a CO-coated liposome was designed, with Photochlor (HPPH) as the 660 nm light mediated photosensitizer and evofosfamide (also known as TH302) as the hypoxia-activated prodrug. The obtained liposomes can help diagnose TNBC by fluorescence imaging and produce antitumor therapy by synergetic photodynamic therapy (PDT) and chemotherapy. RESULTS: Compared with the nontargeted liposomes, the targeted liposomes exhibited good biocompatibility and targeting capability in vitro; in vivo, the targeted liposomes exhibited much better fluorescence imaging capability. Additionally, liposomes loaded with HPPH and TH302 showed significantly better antitumor effects than the other monotherapy groups both in vitro and in vivo. CONCLUSION: The impressive synergistic antitumor effects, together with the superior fluorescence imaging capability, good biocompatibility and minor side effects confers the liposomes with potential for future translational research in the diagnosis and CD44-overexpressing cancer therapy, especially TNBC.

Development of Novel 18F-PET Agents for Tumor Hypoxia Imaging.

Tumor hypoxia is a major factor responsible for tumor progression, metastasis, invasion, and treatment resistance, leading to low local tumor control and recurrence after radiotherapy in cancers. Here,18F-positron emission tomography (PET) probes are developed for visualizing viable hypoxic cells in biopsies. Pimonidazole derivatives and nitroimidazole-based agents bearing sulfonyl linkers were evaluated. A small-animal PET study showed that the tumor uptake of [18F]-23 [poly(ethylene glycols) (PEG)-sulfonyl linker] of 3.36 ± 0.29%ID/g was significantly higher (P < 0.01) than that of [18F]-20 (piperazine-linker tracer, 2.55 ± 0.49%ID/g) at 2 h postinjection in UPPL tumors. The tumor-to-muscle uptake ratio of [18F]-23 (2.46 ± 0.48 at 2 h pi) was well improved compared with that of [18F]-FMISO (1.25 ± 0.14 at 2 h pi). A comparable distribution pattern was observed between ex vivo autoradiography of [18F]-23 and pimonidazole staining of the neighboring slice, indicating that [18F]-23 is a promising PET agent for hypoxia imaging.

Synthesis of Mitochondria-Anchored Nitroimidazoles with a Versatile NIR Fluorophore for Hypoxic Tumor-Targeting Imaging and Chemoradiotherapy.

Nitroimidazoles are one of the most common radiosensitizers investigated to combat hypoxia-induced resistance to cancer radiotherapy. However, due to poor selectivity distinguishing cancer cells from normal cells, effective doses of radiosensitization are much closer to the doses of toxicity induced by nitroimidazoles, limiting their clinical application. In this work, a tumor-targeting near-infrared (NIR) cyanine dye (IR-808) was utilized as a targeting ligand and an NIR fluorophore tracer to chemically conjugate with different structures of hypoxia-affinic nitroimidazoles. One of the NIR fluorophore-conjugated nitroimidazoles (808-NM2) was identified to preferentially accumulate in hypoxic tumor cells, sensitively outline the tumor contour, and effectively inhibit tumor growth synergistically by chemotherapy and radiotherapy. More importantly, nitroimidazoles were successfully taken into cancer cell mitochondria via 808-NM2 conjugate to exert the synergistic effect of chemoradiotherapy. Regarding the important roles of mitochondria on cancer cell survival and metastasis under hypoxia, 808-NM2 may be hopeful to fight against hypoxic tumors.

Synthesis and evaluation of gallium-68-labeled nitroimidazole-based imaging probes for PET diagnosis of tumor hypoxia.

OBJECTIVE: In this study, we designed and synthesized four novel 68Ga-radiolabeled compounds ([68Ga]DN-3, [68Ga]DN-4, [68Ga]NN-3, and [68Ga]NN-4) composed of a nitroimidazole and two types of bifunctional chelates (DOTA or NOTA) via several alkyl linkers of different length. Then, we evaluated their properties as hypoxia imaging probes for positron emission tomography (PET) compared with conventional compounds ([68Ga]DN-2 and [68Ga]NN-2). METHODS: The precursors of 68Ga-radiolabeled compounds were synthesized through a two-step reaction, and then reacted with 68GaCl3 to be 68Ga-radiolabeled compounds. FaDu cells were treated with 68Ga-radiolabeled compounds and then incubated under normoxic (21% O2) or hypoxic (1% O2) conditions. The radioactivity of these cells was measured 2 h after incubation. The biodistribution and PET/CT imaging of 68Ga-radiolabeled compounds in FaDu-bearing Balb/c nude mice were evaluated 2 h after intravenous injection. RESULTS: The 68Ga-radiolabeled compounds were synthesized with radiochemical purities over 95%. In the in vitro study, the levels of 68Ga-radiolabeled compounds were significantly higher in hypoxic cells than in normoxic cells. In hypoxic cells, the compounds we designed in this study demonstrated higher accumulation than the conventional compounds. In the in vivo biodistribution study, [68Ga]DN-3 exhibited the highest accumulation in tumor. In the in vivo PET/CT imaging study, the tumor tissues of the FaDu-xenografted mice were visualized at 2 h after intravenous administration of 68Ga-radiolabeled compounds. CONCLUSIONS: Our study suggested that the length of the linkers connecting nitroimidazole to a bifunctional chelate affect PET imaging of hypoxic tumors with 68Ga-radiolabeled compounds.

An overview of the developments and potential applications of 68Ga-labelled PET/CT hypoxia imaging.

Non-invasive imaging of hypoxia plays a role in monitoring the body's adaptive response or the development of pathology under hypoxic conditions. Various techniques to image hypoxia have been investigated with a shift towards the use of molecular imaging using PET/CT. The role of hypoxia-specific radiopharmaceuticals such as radiolabelled nitroimidazoles is well documented particularly in the oncologic setting. With the increasing utilisation of in-house labelling with a PET benchtop generator, such as the 68Ge/68Ga generator, the use of 68Ga-labelled hypoxic radiopharmaceuticals in the clinical setting is developing. Since hypoxia plays a role in various pathologic states including infectious disease such as TB, there is a need to explore the potential application of 68Ga-labelled hypoxia seeking radiopharmaceuticals beyond oncology. The purpose of this review is to describe the developments of 68Ga-labelled hypoxic radiopharmaceuticals including the various chelators that have been investigated. Further, the role of hypoxia imaging in various pathologies is discussed with particular emphasis on the potential clinical applications of hypoxia PET/CT in TB.

Hypoxia-Activated Prodrug Evofosfamide Treatment in Pancreatic Ductal Adenocarcinoma Xenografts Alters the Tumor Redox Status to Potentiate Radiotherapy.

Aims: In hypoxic tumor microenvironments, the strongly reducing redox environment reduces evofosfamide (TH-302) to release a cytotoxic bromo-isophosphoramide (Br-IPM) moiety. This drug therefore preferentially attacks hypoxic regions in tumors where other standard anticancer treatments such as chemotherapy and radiation therapy are often ineffective. Various combination therapies with evofosfamide have been proposed and tested in preclinical and clinical settings. However, the treatment effect of evofosfamide monotherapy on tumor hypoxia has not been fully understood, partly due to the lack of quantitative methods to assess tumor pO2in vivo. Here, we use quantitative pO2 imaging by electron paramagnetic resonance (EPR) to evaluate the change in tumor hypoxia in response to evofosfamide treatment using two pancreatic ductal adenocarcinoma xenograft models: MIA Paca-2 tumors responding to evofosfamide and Su.86.86 tumors that do not respond. Results: EPR imaging showed that oxygenation improved globally after evofosfamide treatment in hypoxic MIA Paca-2 tumors, in agreement with the ex vivo results obtained from hypoxia staining by pimonidazole and in apparent contrast to the decrease in Ktrans observed in dynamic contrast-enhanced magnetic resonance imaging (DCE MRI). Innovations: The observation that evofosfamide not only kills the hypoxic region of the tumor but also improves oxygenation in the residual tumor regions provides a rationale for combination therapies using radiation and antiproliferatives post evofosfamide for improved outcomes. Conclusion: This study suggests that reoxygenation after evofosfamide treatment is due to decreased oxygen demand rather than improved perfusion. Following the change in pO2 after treatment may therefore yield a way of monitoring treatment response. Antioxid. Redox Signal. 35, 904-915.

Development of a novel acetyl glucose-modified gefitinib derivative to enhance the radiosensitizing effect.

Various radiosensitizers are being developed to increase the radiation sensitivity of hypoxic cancer cells, which show resistance to radiation. Previously, we demonstrated that an acetyl glucose-modified nitroimidazole derivative showed a high radiosensitizing effect by inhibiting glucose uptake and glycolysis. Based on this finding, we designed and synthesized novel sugar hybrid radiosensitizers, wherein acetyl glucose was introduced into gefitinib. Among them, UTX-114 had higher autophosphorylation and radiosensitizing activity than gefitinib and inhibited glucose uptake. This result supports our hypothesis that an acetyl glucose moiety improves the radiosensitizing effect of the drug, and UTX-114 can be expected to be a leading compound with a radiosensitizing effect.

Synthesis and evaluation of [99mTcN]2+ core and [99mTcO]3+ core labeled complexes with 4-nitroimidazole xanthate derivative for tumor hypoxia imaging.

A 4-nitroimidazole xanthate ligand (NMXT) was synthesized and radiolabeled with [99mTcN]2+ core and [99mTcO]3+ core to obtain 99mTcN-NMXT and 99mTcO-NMXT, respectively. The two 99mTc-complexes were prepared with high radiochemical purity and had good stability. The partition coefficient results indicated both of them were hydrophilic, and cellular uptake studies showed they exhibited good hypoxic selectivity. From the biodistribution study results, 99mTcO-NMXT showed more favourable tumor uptake (1.73 ± 0.14 ID%/g) and higher tumor/muscle ratio (7.01 ± 0.16) than 99mTcN-NMXT at 4 h post-injection. Single photon emission computed tomography (SPECT) imaging study of 99mTcO-NMXT showed there was a visible accumulation in tumor site, suggesting it would be a promising candidate as a tumor hypoxia imaging agent.