Ex vivo imaging and analysis of ROS generation correlated with microglial activation in rat model with acute neuroinflammation induced by intrastriatal injection of LPS.

Neuroinflammation and oxidative stress are hallmarks of neurodegenerative diseases. Microglia, the major important regulators of neuroinflammation, are activated in response to excessive generation of reactive oxygen species (ROS) from damaged cells and resulting in elevated and sustained damages. However, the relationship between microglia and ROS-regulatory system in the early stages of neuroinflammation prior to the appearance of neuronal damages have not been elucidated in detail. In this study, we analyzed the time-dependent changes in ROS generation during acute neuroinflammation in rats that were given an intrastriatal injection of lipopolysaccharide (LPS). We evaluated the effects of minocycline, an anti-inflammatory antibiotic, and N,N'-dimethylthiourea (DMTU), a radical scavenger, to understand the correlation between activated microglia and ROS generation. Ex vivo fluorescence imaging using dihydroethidium (DHE) clearly demonstrated an increased ROS level in the infused side of striatum in the rats treated with LPS. The level of ROS was changed in time-dependent manner, and the highest level of ROS was observed on day 3 after the infusion of LPS. Immunohistochemical studies revealed that time-dependent changes in ROS generation were well correlated to the presence of activated microglia. The inhibition of microglial activation by minocycline remarkably reduced ROS levels in the LPS-injected striatum, which indicated that the increased ROS generation caused by LPS was induced by activated microglia. DMTU decreased ROS generation and resulted in remarkable inhibitory effect on microglial activation. This study demonstrated that ROS generation during acute neuroinflammation induced by LPS was considerably associated with microglial activation, in an intact rat brain. The results provides a basis for understanding the interaction of ROS-regulatory system and activated microglia during neuroinflammation underlying neurodegenerative diseases.

Influence of Linker Molecules in Hexavalent RGD Peptides on Their Multivalent Interactions with Integrin αvß3.

Multivalent RGD peptides have been used as an excellent targeting vector to integrin αvß3-positive tumors. However, little attention has been paid to the influence of linker molecules in multivalent RGD peptides on their dissociation kinetics from tumor cells. In this study, we evaluated the dissociation kinetics of 99mTc-labeled hexavalent RGD peptides which have (CH2-CH2-O)n (n = 4, [99mTc][Tc(L1)6]+ and n = 12, [99mTc][Tc(L2)6]+) or (DPro-Gly)n (n = 1, [99mTc][Tc(L3)6]+; n = 6, [99mTc][Tc(L4)6]+; and n = 9, [99mTc][Tc(L5)6]+) as a linker molecule. The results showed that [99mTc][Tc(L4)6]+ and [99mTc][Tc(L5)6]+ displayed slower dissociation kinetics and [99mTc][Tc(L4)6]+ showed exceptionally high in vitro cellular uptake (203.1 ± 16.7% dose/mg protein) and the highest tumor to blood ratio (138.1 ± 26.3 at 4 h p.i.) in tumor bearing nude mice. These findings indicate that the use of appropriate length of (DPro-Gly)n would maximize the binding of multivalent RGD peptides to clustered integrin αvß3.

Aryl isocyanide derivative for one-pot synthesis of purification-free 99mTc-labeled hexavalent targeting probe.

INTRODUCTION: 99mTc-labeled hexavalent probes can be readily synthesized by the coordination of six equivalent isocyanide ligands towards TcI, and alkyl isocyanide ligands have been extensively used for preparing such probes. However, high ligand concentration (>1 mM) is generally required due to their insufficient coordination ability to TcI. METHODS AND RESULTS: In this study, we revealed that aryl isocyanide ligands, which have greater π-accepting ability compared with alkyl ones, provided 99mTc-labeled hexavalent probes in high radiochemical yields (>95%) even at low ligand concentration (50 µM). We applied this finding to the synthesis of a 99mTc-labeled hexavalent RGD probe, targeting integrin αvß3. This 99mTc-labeled probe was prepared in a 5 min reaction at ligand concentration of 50 µM, and exhibited high tumor localization in vivo without post-labeling purification. CONCLUSION: The present findings indicate that aryl isocyanide ligands would be a useful precursor to a variety of 99mTc-labeled hexavalent targeting probes for molecular imaging of saturable systems. ADVANCES IN KNOWLEDGE: Aryl isocyanide is a better precursor than alkyl isocyanide for preparing 99mTc-labeled hexavalent targeting probe. IMPLICATION FOR PATIENT CARE: This work provides a straightforward method to prepare molecular imaging agents of high target uptake, which would facilitate nuclear medicine imaging in clinical settings.

The synthesis of a 99mTc-labeled tetravalent targeting probe upon isonitrile coordination to 99mTcI for enhanced target uptake in saturable systems.

The presence of excess unlabeled ligands in the injectate hinders the target uptake of 99mTc-labeled targeting vectors. To address the issue, we previously developed a chemical design which provides a 99mTc-labeled trivalent RGD probe upon CN-ßAla-Gly-Gly-c(RGDfK) (Lß) coordination to [99mTc][Tc(CO)3]+ core at pH 6.0. In this study, we extended our coordination mediated synthesis of the trivalent RGD probe to that of a tetravalent one. Our initial attempts reacting Lß with [99mTc][Tc(CO)3]+ core at pH 8.0 failed to provide [99mTc][Tc(CO)2(Lß)4]+ due to the formation of multiple side products. A γ-aminobutylic acid (GABA) based isonitrile ligand CN-GABA-Gly-Gly-c(RGDfK) (LG), on the other hand, avoided the side reaction and selectively provided [99mTc][Tc(CO)2(LG)4]+ (99mTc-[LG]4) at pH 8.0. 99mTc-[LG]4 exhibited higher binding affinity to integrin αvß3 than its unlabeled ligand, and visualized U87MG tumor without tedious post-labeling purification. These results indicate that the metal coordination-mediated syntheses of 99mTc-labeled multivalent probes have been successfully applied to a tetravalent one, which would allow a wider range of choices for designing novel 99mTc-labeled multivalent probes of high in vivo target uptake.

Coordination-Mediated Synthesis of Purification-Free Bivalent 99mTc-Labeled Probes for in Vivo Imaging of Saturable System.

In the synthesis of technetium-99m (99mTc) labeled target-specific ligands, the presence of a large excess of unlabeled ligands over 99mTc in the injectate hinders target accumulation of 99mTc-labeled ligands by competing for target molecules. To circumvent the problem, we recently developed a concept of the metal coordination-mediated multivalency, and proved the concept with a 99mTc-labeled trivalent compound [99mTc(CO)3(CN-RGD)3]+. In this study, D-penicillamine (Pen) was selected as a chelating molecule and a cyclic RGDfK peptide was conjugated to Pen via a hexanoic linkage (Pen-Ahx-c(RGDfK)). 99mTc complexation reaction, and the stability, integrin αvß3 binding affinity, and biodistribution of the 99mTc-labeled probe were investigated to evaluate the applicability of the concept to bivalent probes. 99mTc-[Pen-Ahx-c(RGDfK)]2 was obtained over 95% radiochemical yields under low Pen-Ahx-c(RGDfK) concentration (50 µM). 99mTc-[Pen-Ahx-c(RGDfK)]2 showed approximately 10-times higher integrin αvß3 binding affinity than the monovalent compounds, Pen-Ahx-c(RGDfK) and c(RGDyV). In biodistribution studies, the tumor accumulation of 99mTc-[Pen-Ahx-c(RGDfK)]2 was decreased to 77% and 43% of HPLC-purified (Pen-Ahx-c(RGDfK)-free) 99mTc-[Pen-Ahx-c(RGDfK)]2 by the presence of 5 nmol of unlabeled Pen-Ahx-c(RGDfK) and Re-[Pen-Ahx-c(RGDfK)]2, respectively. 99mTc-[Pen-Ahx-c(RGDfK)]2 provided tumor image without removing unlabeled ligand, while a 99mTc-labeled monovalent probe prepared from a monovalent ligand could not. These findings indicate the availability of the design concept to prepare 99mTc-labeled bivalent probes with a variety of 99mTc core and other metallic radionuclides of clinical relevance.

111In-DTPA-d-Phe-1-Asp0-d-Phe1-octreotide exhibits higher tumor accumulation and lower renal radioactivity than 111In-DTPA-d-Phe1-octreotide.

INTRODUCTION: 111In-DTPA-d-Phe1-octreotide scintigraphy is an important method of detecting neuroendocrine tumors. We previously reported that a new derivative of 111In-DTPA-d-Phe1-octreotide, 111In-DTPA-d-Phe-1-Asp0-d-Phe1-octreotide, accomplished the reduction of prolonged renal accumulation of radioactivity. The aim of this study was to evaluate the tumor accumulation of 111In-DTPA-d-Phe-1-Asp0-d-Phe1-octreotide in vitro and in vivo by comparing it with 111In-DTPA-d-Phe1-octreotide. METHODS: The tumor accumulation of this octreotide derivative was determined by measuring its uptake using cultured AR42J cells in vitro and biodistribution studies in vivo. The distribution of the radiotracer and the extent of somatostatin receptor-specific uptake in the tumor were estimated by a counting method using AR42J tumor-bearing mice. The radioactive metabolite species in the tumor and kidney were identified by HPLC analyses at 3 and 24h post-injection of the 111In-DTPA-conjugated peptide. RESULTS: In both cases, in vitro and in vivo, the tumor radioactivity levels of 111In-DTPA-d-Phe-1-Asp0-d-Phe1-octreotide were approximately 2-4 times higher than those of 111In-DTPA-d-Phe1-octreotide. On in vitro cellular uptake inhibition and radioreceptor assay, 111In-DTPA-d-Phe-1-Asp0-d-Phe1-octreotide exhibited a binding affinity to somatostatin receptor highly similar to that of 111In-DTPA-d-Phe1-octreotide. As the additional cellular uptake of 111In-DTPA-d-Phe-1-Asp0-d-Phe1-octreotide was significantly lower at low temperature than at 37°C, it was considered that a cellular uptake pathway is involved in energy-dependent endocytotic processes. In the radiometabolite analysis of 111In-DTPA-d-Phe-1-Asp0-d-Phe1-octreotide, 111In-DTPA-d-Phe-Asp-OH was a major metabolite in the tumor at 24h post-injection. CONCLUSION: 111In-DTPA-d-Phe-1-Asp0-d-Phe1-octreotide exhibited higher tumor accumulation and persistence of tumor radioactivity than 111In-DTPA-d-Phe1-octreotide. We reasoned that this higher tumor accumulation would not be based on the receptor affinity but on a receptor-mediated endocytotic process involved in temperature-dependent cellular uptake. The present study demonstrated the great potential of the pharmaceutical development of a new radiolabeled peptide with high tumor accumulation and low renal radioactivity by the chemical modification of 111In-DTPA-d-Phe1-octreotide.

Redesign of negatively charged 111In-DTPA-octreotide derivative to reduce renal radioactivity.

INTRODUCTION: Radiolabeled octreotide derivatives have been studied as diagnostic and therapeutic agents for somatostatin receptor-positive tumors. To prevent unnecessary radiation exposure during their clinical application, the present study aimed to develop radiolabeled peptides which could reduce radioactivity levels in the kidney at both early and late post-injection time points by introducing a negative charge with an acidic amino acid such as L-aspartic acid (Asp) at a suitable position in 111In-DTPA-conjugated octreotide derivatives. METHODS: Biodistribution of the radioactivity was evaluated in normal mice after administration of a novel radiolabeled peptide by a counting method. The radiolabeled species remaining in the kidney were identified by comparing their HPLC data with those obtained by alternative synthesis. RESULTS: The designed and synthesized radiolabeled peptide 111In-DTPA-d-Phe-1-Asp0-d-Phe1-octreotide exhibited significantly lower renal radioactivity levels than those of the known 111In-DTPA-d-Phe1-octreotide at 3 and 24h post-injection. The radiolabeled species in the kidney at 24h after the injection of new octreotide derivative represented 111In-DTPA-d-Phe-OH and 111In-DTPA-d-Phe-Asp-OH as the metabolites. Their radiometabolites and intact 111In-DTPA-conjugated octreotide derivative were observed in urine within 24h post-injection. CONCLUSION: The present study provided a new example of an 111In-DTPA-conjugated octreotide derivative having the characteristics of both reduced renal uptake and shortened residence time of radioactivity in the kidney. It is considered that this kinetic control was achieved by introducing a negative charge on the octreotide derivative thereby suppressing the reabsorption in the renal tubules and affording the radiometabolites with appropriate lipophilicity.

The Thymidine Phosphorylase Imaging Agent 123I-IIMU Predicts the Efficacy of Capecitabine.

UNLABELLED: Recently, companion diagnostics with nuclear medicine techniques have been anticipated as more suitable means than biopsy for predicting treatment efficacy. The anticancer effect of capecitabine, an orally administered chemotherapeutic agent activated by thymidine phosphorylase (TP), is positively associated with tumor TP expression levels. This study aimed to assess whether TP imaging using a radiolabeled uracil derivative, (123)I-5-iodo-6-[(2-iminoimidazolidinyl)methyl]uracil ((123)I-IIMU), could predict the efficacy of capecitabine treatment. METHODS: Sensitivity to doxifluridine, a metabolite of capecitabine and direct substrate for TP, was assessed by water-soluble tetrazolium salt assays in vitro for 3 human colon cancer cell lines with different TP expression profiles. The intracellular uptake and retention of (123)I-IIMU were evaluated. Mice inoculated with each cell line were treated with capecitabine for 2 wk, and tumor growth was compared. In vivo distribution studies and SPECT/CT imaging of (123)I-IIMU were performed in inoculated mice. RESULTS: In vitro experiments showed a positive relation between TP expression levels and doxifluridine sensitivity. In vitro studies revealed that intracellular uptake and retention of (123)I-IIMU were dependent on TP expression levels. In vivo experiments in inoculated mice showed that (123)I-IIMU accumulation in tumor tissue was in line with TP expression levels and susceptibility to capecitabine treatment. Moreover, SPECT/CT imaging of (123)I-IIMU in tumor-inoculated mice showed that (123)I-IIMU reflects TP expression levels in tumor tissues. CONCLUSION: (123)I-IIMU could be used as an in vivo companion diagnostic for predicting the efficacy of capecitabine treatment.

Relationship between biodistribution of a novel thymidine phosphorylase (TP) imaging probe and TP expression levels in normal mice.

OBJECTIVE: Thymidine phosphorylase (TP) is a key enzyme in the pyrimidine nucleoside salvage pathway and its expression is upregulated in a wide variety of solid tumors. In mice, we previously observed high and specific accumulation levels of our TP imaging probe, radioiodinated 5-iodo-6-[(2-iminoimidazolidinyl)methyl]uracil (IIMU) not only in high-TP-expressing tumors, but also in the liver and small intestine. To clarify the reason for the high accumulation levels of radioiodinated IIMU in the liver and small intestine, we investigated the expression levels of TP in mice in comparison with the biodistribution of radioiodinated IIMU ((123)I-IIMU). METHODS: BALB/cCrSlc mice were injected with (123)I-IIMU, and the radioactivity levels [%ID/g (normalized to a mouse of 25 g body weight)] in the tissues of interest were determined 0.5, 1, 3 and 24 h after the injection (n = 5, each time point). To determine the expression levels of TP, BALB/cCrSlc and ddy mice (n = 3/each strain) were euthanized, and the heart, liver, lung, spleen, kidney, stomach, small intestine, large intestine and brain were collected. The mRNA and protein expression levels of TP in these organs were examined by quantitative reverse transcription-polymerase chain reaction and western blot analyses, respectively. RESULTS: In BALB/cCrSlc mice administered (123)I-IIMU, markedly high radioactivity levels were observed in the liver [1.568 ± 0.237 (%ID/g)] and small intestine [0.506 ± 0.082 (%ID/g)], whereas those in the other tissues were fairly low [<0.010 ± 0.003 (%ID/g)] 30 min after the injection. The highest expression levels of TP mRNA were also observed in the liver and small intestine among the tissues tested. Immunoblotting showed intense immunoreactive bands of the TP protein for the liver and small intestine, whereas no notable bands were detected for other tissues. Similar expression profiles of TP mRNA and protein were observed in ddy mice. CONCLUSION: We confirmed TP expression in various tissues of mice at the mRNA and protein levels: high TP expression levels were observed in the liver and small intestine. These high TP expression levels are consistent with the high accumulation levels of (123)I-IIMU in these tissues. Our results may provide important information about the physiological accumulation of (123)I-IIMU, which may be useful for the clinical diagnostic imaging of TP.

Design, synthesis and biological evaluation of negatively charged ¹¹¹In-DTPA-octreotide derivatives.

Our previous studies indicated that (111)In-diethylenetriaminepentaacetic acid ((111)In-DTPA)-octreotide derivatives with an additional negative charge by replacing N-terminal d-phenylalanine (d-Phe) with an acidic amino acid such as l-aspartic acid (Asp) or its derivative exhibited low renal radioactivity levels when compared with (111)In-DTPA-D-Phe(1)-octreotide. On the basis of the findings, we designed, synthesized and evaluated two Asp-modified (111)In-DTPA-conjugated octreotide derivatives, (111)In-DTPA-Asp(1)-octreotide and (111)In-DTPA-Asp(0)-D-Phe(1)-octreotide. While (111)In-DTPA-Asp(1)-octreotide showed negligible AR42J cell uptake, (111)In-DTPA-Asp(0)-D-Phe(1)-octreotide exhibited AR42J cell uptake similar to that of (111)In-DTPA-D-Phe(1)-octreotide. When administered to AR42J tumor-bearing mice, (111)In-DTPA-Asp(0)-D-Phe(1)-octreotide exhibited renal radioactivity levels significantly lower than did (111)In-DTPA-D-Phe(1)-octreotide at 1 and 3 h post-injection. No significant differences were observed in tumor accumulation between (111)In-DTPA-Asp(0)-D-Phe(1)-octreotide and (111)In-DTPA-D-Phe(1)-octreotide after 1 and 3h injection. The findings in this study suggested that an interposition of an Asp at an appropriate position in (111)In-DTPA-D-Phe(1)-octreotide would constitute a useful strategy to develop (111)In-DTPA-D-Phe(1)-octreotide derivatives of low renal radioactivity levels while preserving tumor accumulation.

Chemical design of (99m)Tc-labeled probes for targeting osteogenic bone region.

Radionuclide bone imaging using polynuclear (99m)Tc complexes of bisphosphonates is the most common clinical practice in nuclear medicine. However, the improvement in the contrast between normal and osteogenic bone regions has been required. Herein we reported a new (99m)Tc-labeled compound considering the increased vascular permeability of osteogenic region. We selected penta-d-Asp as both a targeting motif to hydroxyapatite (HA) and a molecular size modifier, and two penta-d-Asp molecules were conjugated with the two carboxylate residues of ethylene dicysteine (EC) selected as the (99m)Tc chelating moiety to prepare EC-[(d-Asp)5]2. The molecular size, HA binding, and pharmacokinetics of (99m)Tc-EC-[(d-Asp)5]2 in normal mice and model rats bearing osteogenic tumor were compared to those of (99m)Tc-MDP and (99m)Tc-EC with one (d-Asp)5 motif, (99m)Tc-EC-(d-Asp)5. The molecular size of (99m)Tc-EC-[(d-Asp)5]2 was higher than that of (99m)Tc-MDP and (99m)Tc-EC-(d-Asp)5 when determined by permeability of the (99m)Tc-compounds through a membrane filter (10 kDa). The HA binding of (99m)Tc-EC-[(d-Asp)5]2 was higher than and similar to that of (99m)Tc-EC-(d-Asp)5 and (99m)Tc-MDP. (99m)Tc-EC-[(d-Asp)5]2 exhibited significantly lower accumulation in normal bone of mice than did (99m)Tc-MDP. In osteogenic tumor bearing model rats, (99m)Tc-EC-[(d-Asp)5]2 accumulated in the osteogenic and normal bone region similar to and lower than (99m)Tc-MDP, respectively. Although further studies including the chain length of d-Asp are required, these findings indicated that the present chemical design of (99m)Tc-labeled probe would be applicable to develop (99m)Tc-labeled probes for selective imaging of osteogenic bone region as well as develop therapeutic agents using therapeutic radionuclides such as (90)Y, (177)Lu, (186)Re, or (188)Re and cytotoxic agents to osteogenic bone tumor region.

Renal brush border enzyme-cleavable linkages for low renal radioactivity levels of radiolabeled antibody fragments.

We previously demonstrated that Fab fragments labeled with 3'-[(131)I]iodohippuryl N(ε)-maleoyl-l-lysine ([(131)I]HML) showed low renal radioactivity from early postinjection time, due to a liberation of m-[(131)I]iodohippuric acid by the action of renal brush border enzymes. Since there are lots of enzymes on renal brush border membrane, peptide linkages other than the glycyl-l-lysine were evaluated as the cleavable linkages to explore the chemical design. In this study, we evaluated four peptide linkages with a general formula of m-iodobenzoyl-glycyl-X (X: l-tyosine O-methyl, l-asparagine, l-glutamine, and N(ε)-Boc-l-lysine). In vitro studies using renal brush border membrane vesicles (BBMVs) demonstrated that 3'-[(125)I]iodohippuryl O-methyl-l-tyrosine (2c) liberated the highest amount of m-[(125)I]iodohippuric acid among the four substrates and the change in the linkage structure altered enzyme species responsible for the hydrolysis reaction. To further assess the applicability of the linkage, a radioiodination reagent containing a glycyl-tyrosine linkage, 3'-[(125)I]iodohippuryl O-((2-maleimidoethyl)carbamoyl)methyl-l-tyrosine (HMT, 12c), was designed, synthesized, and subsequently conjugated to an Fab fragment. [(125)I]HMT-Fab exhibited renal radioactivity levels similar to and significantly lower than [(125)I]HML-Fab and directly radioiodinated Fab, while the blood clearance rates of the three were similar. The analyses of urine for 24 h postinjection of [(125)I]HMT-Fab showed that m-[(125)I]iodohippuric acid was excreted as the major radiometabolite. The findings indicated that glycyl-tyrosine linkage is also available to reduce renal radioactivity levels of radioiodinated Fab fragments, due to liberation of m-iodohippuric acid by the action of enzymes present on renal brush border membrane. These findings suggest that an appropriate selection of peptide linkages would allow the liberation of a designed radiolabeled compound from covalently conjugated polypeptides to prepare radiolabeled polypeptides of low renal radioactivity levels. For the selection of the most appropriate peptide linkage, the in vitro system using BBMVs would be useful to narrow the candidates to just a few.

Synthesis and evaluation of diastereoisomers of 1,4,7-triazacyclononane-1,4,7-tris-(glutaric acid) (NOTGA) for multimeric radiopharmaceuticals of gallium.

In the conventional synthesis of 1,4,7-tris-(glutaric acid)-1,4,7-triazacyclononane (NOTGA), four isomeric species are usually generated by the alkylation of 1,4,7-triazacyclononane with α-bromoglutaric acid diester. To estimate their biological efficacies as well as their stability and radiochemistry, the RRR/SSS and RRS/SSR NOTGA-(t)Bu prochelators were isolated and the corresponding cyclic RGDfK (RGD) conjugates with triethylene glycol linkages were prepared. The RRR/SSS and RRS/SSR diastereomers were obtained in 69% and 17% yields, respectively. In the complexation reaction with (67)GaCl(3), both diastereomers provided >98% radiochemical yields at pH 5 within 10 min when the reaction was conducted at room temperature. However, the RRR/SSS diastereomer exhibited more pH-sensitive radiochemical yields between pH 3.5 to 4.5. Despite their diasteromeric nature, both (67)Ga-labeled RGD-NOTGA remained stable during the apo-transferrin challenge, exhibiting similar affinity for integrin α(v)ß(3) and biodistribution with predominant renal excretion. Similar tumor uptake was also observed in mice bearing U87MG tumor xenograft, which resulted in impressively high contrast SPECT/CT images. These findings indicate that the RGD-NOTGA conjugates of both diastereomers presented here possess equivalent biological efficacies and their combined usage would be feasible. It is worth noting that specific properties of a given biomolecule, cell expression levels of the corresponding target molecule, and presence or absence of a pharmacokinetic modifier would affect the structural differences between diastereomers on the ligand-receptor interactions and pharmacokinetics. Thus, the preparation of corresponding conjugates and evaluation of their chemical and biological performances still remains important for applying NOTGA to other biomolecules of interest using the diastereomerically pure NOTGA-(t)Bu prochelator.

Toward in vivo imaging of heart disease using a radiolabeled single-chain Fv fragment targeting tenascin-C.

Antibodies specific to a particular target molecule can be used as analytical reagents, not only for in vitro immunoassays but also for noninvasive in vivo imaging, e.g., immunoscintigraphies. In the latter case, it is important to reduce the size of antibody molecules in order to achieve suitable in vivo "diagnostic kinetics" and generate higher-resolution images. For these purposes, single-chain Fv fragments (scFvs; M(r) < 30 kDa) have greater potential than intact immunoglobulins (~150 kDa) or Fab (or Fab') fragments (~50 kDa). Our recent observation of enhanced tenascin-C (Tnc) expression at sites of cardiac repair after myocardial infarction prompted us to develop a radiolabeled scFv against Tnc for in vivo imaging of heart disease. We cloned the genes encoding the heavy and light chain variable domains of the mouse anti-Tnc monoclonal antibody 4F10, and combined them to create a single gene. The resulting scFv-4F10 gene was expressed in E. coli cells to produce soluble scFv proteins. scFv-4F10 has an affinity for Tnc (K(a) = 3.5 × 10(7) M(-1)), similar to the Fab fragment of antibody 4F10 (K(a) = 1.3 × 10(7) M(-1)) and high enough to be of practical use. A cysteine residue was then added to the C-terminus to achieve site-specific (111)In labeling via a chelating group. The resulting (111)In-labeled scFv was administered to a rat model of acute myocardial infarction. Biodistribution and quantitative autoradiographic studies indicated higher uptake of the radioactivity at the infarcted myocardium than the noninfarcted one. Single photon emission computed tomography (SPECT) provided in vivo cardiac images that coincided with the ex vivo observations. Our results will promote advances in diagnostic strategies for heart disease.

Radiolabeled uracil derivative as a novel SPECT probe for thymidine phosphorylase: suppressed accumulation into tumor cells by target gene knockdown.

OBJECTIVES: We have developed a radiolabeled uracil derivative, 5-iodo-6-[(2-iminoimidazolidinyl)methyl]uracil (IIMU) as a novel single photon emission computed tomography probe for thymidine phosphorylase (TP). This radioiodinated IIMU has a high affinity for TP and highly accumulates in the TP-expressing tumor cell line A431 (human epidermoid carcinoma). To evaluate the specificity of the cellular uptake of IIMU to TP expression, we examined the effects of TP knockdown on the uptake of ¹²5I-labeled IIMU (¹²5I-IIMU) in the tumor cells. METHODS: TP-specific small interfering RNA (siRNA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific siRNA (positive control), and negative control siRNA were transfected into A431 cells, respectively. Target-mRNA and protein expression levels of TP and GAPDH were examined 48 and 72 h after transfection, respectively. The cellular uptake level of ¹²5I-IIMU was also evaluated 72 h after transfection. The results were compared after normalization with the corresponding negative controls. RESULTS: After TP-specific and GAPDH-specific siRNA transfection, the expression levels of TP and GAPDH mRNA decreased significantly to 41 and 29%, respectively, compared with the negative control (P<0.001 for both). The expression levels of TP and GAPDH protein also significantly decreased to 34 and 30%, respectively (P<0.001 for both). After TP-specific siRNA transfection, the cellular uptake level of ¹²5I-IIMU decreased significantly to 66% (P<0.001). In contrast, GAPDH siRNA transfection did not significantly affect the cellular uptake level of ¹²5I-IIMU. CONCLUSION: siRNA-mediated TP knockdown significantly decreased the cellular uptake level of ¹²5I-IIMU. This finding indicates that the uptake of IIMU in tumor cells is TP specific and directly corresponds to TP expression levels.

Enhanced target-specific accumulation of radiolabeled antibodies by conjugating arginine-rich peptides as anchoring molecules.

We have devised and estimated a new strategy to prolong the residence time of radiolabeled antibodies in tumor in which an octaarginine peptide (R8) was used as an anchoring molecule to fix antibodies against CD20 (NuB2; IgG2a) on tumor cells. Conjugation of R8 with antibodies was performed by maleimide-thiol chemistry using thiol groups generated by reducing the disulfide bonds of the antibody. The R8-conjugated NuB2 was then reacted with succinimidyl meta-[¹²5I]iodobenzoate to prepare [¹²5I]SIB-NuB2(I) (0.92 R8/NuB2) and [¹²5I]SIB-NuB2(III) (3.38 R8/NuB2). Both SIB-NuB2(I) and SIB-NuB2(III) exhibited size-exclusion HPLC elution profiles and immunoreactivity to CD20-positive cells similar to those of NuB2. NuB2(I) also possessed isoelectric focusing (IEF) profile similar to NuB2. However, NuB2(III) registered a broad IEF band toward higher pI. When incubated with CD20-positive cells, [¹²5I]SIB-NuB2(I) and [¹²5I]SIB-NuB2(III) exhibited 1.4 and 4.0 times higher cell-associated radioactivity than [¹²5I]SIB-NuB2. After the cells were washed and reincubated in a fresh medium for 3 h, [¹²5I]SIB-NuB2(I) and [¹²5I]SIB-NuB2(III) exhibited significantly higher cell-associated radioactivity than [¹²5I]SIB-NuB2. In biodistribution studies in normal mice, while both [¹²5I]SIB-NuB2(I) and [¹²5I]SIB-NuB2 exhibited similar biodistribution profiles, [¹²5I]SIB-NuB2(III) showed faster clearance from the blood and higher hepatic radioactivity levels than [¹²5I]SIB-NuB2. In SCID mice bearing CD20-positive xenografts, [¹³¹I]SIB-NuB2(I) exhibited significantly higher radioactivity in xenografts than those of [¹²5I]SIB-NuB2 with no significant increase being observed in other tissues. The findings indicate that appropriate R8 modification of antibodies satisfies both specific targeting ability of antibody and strong cell-association property of R8, which was reflected in the increased radioactivity levels in tumor. These findings supported the applicability of this approach to enhance target-specific accumulation of radiolabeled antibodies.

In vitro and in vivo evaluations of a radioiodinated thymidine phosphorylase inhibitor as a tumor diagnostic agent for angiogenic enzyme imaging.

INTRODUCTION: The expression of thymidine phosphorylase (TP) is closely associated with angiogenesis, tumor invasiveness and activation of antitumor agents. We evaluated radioiodinated 5-iodo-6-[(2-iminoimidazolidinyl)methyl]uracil ([(125)I]IIMU) having high TP-inhibitory potency as the new radiotracer for SPECT targeting of TP expression in tumors. METHODS: The characteristics of the radioiodinated TP inhibitor IIMU were determined by evaluating the uptake by tumor cells in vitro and by biodistribution studies in vivo. The distribution of the radiotracer and the extent of TP-specific uptake by tumors were evaluated by a counting method in tumor-bearing mice. RESULTS: The in vitro uptake of radiolabeled IIMU by A431 cells along with high TP expressions was attributed to the binding of the radiotracer to its target enzyme, i.e., TP. In vivo distribution of the radiotracer in A431 tumor-bearing mice revealed tumor/blood and tumor/muscle activity uptake ratios of 36 and 106, respectively, at 3 h after the radiotracer injection. On using low TP-expressing tumors and TP blocking studies as controls, minor TP-specific accumulation of the radiotracer was detected in these studies. CONCLUSION: According to the binding of radioiodinated IIMU to the angiogenic enzyme TP, it can be concluded that radioiodinated IIMU might be suitable as a SPECT tracer for tumor imaging.

gamma-Glutamyl PAMAM dendrimer as versatile precursor for dendrimer-based targeting devices.

Poly(amidoamine) (PAMAM) dendrimers are highly branched spherical polymers that have a unique surface of primary amine groups and provide a versatile design for targeted delivery of pharmaceuticals and imaging agents. Acetylation or succinylation of surface amine groups of PAMAM dendrimer derivatives is frequently performed to reduce nonspecific uptake. However, since targeting molecules, drugs/imaging agents, and acylating reagents react with the amine groups on dendrimer, such modification may limit the number of targeting molecules and/or drugs or may result in insufficient charge reduction. In this study, a gamma-glutamyl PAMAM dendrimer was designed and synthesized as a new precursor for targeting device. The relationship between surface electrical properties of the PAMAM dendrimer derivatives and pharmacokinetics was also determined. A PAMAM dendrimer (generation 4.0) was modified with a small number of Bolton-Hunter reagent to prepare Phe-P (pI 9.2). The amine residues of Phe-P were gamma-glutamylated to prepare Glu-P (pI 7.1). The alpha-amine residues of Glu-P were then acetylated or succinylated to prepare Ac-Glu-P (pI 5.3) or Suc-Glu-P (pI 3.6). For comparison, Phe-P was acetylated or succinylated to prepare Ac-P (pI 6.0) or Suc-P (pI 5.1). All the PAMAM dendrimer derivatives exhibited similar molecular size (7.2 to 7.8 nm) except for Ac-P (5.1 nm). The biodistribution studies were performed after radioiodination of each PAMAM dendrimer derivative with Na[(125)I]I. When injected intravenously to mice, both [(125)I]Ac-P and [(125)I]Suc-P exhibited prolonged radioactivity levels in the blood and significantly lower hepatic and renal radioactivity levels than those of [(125)I]Phe-P. Both [(125)I]Glu-P and [(125)I]Ac-Glu-P showed residence times in the blood similar to those of [(125)I]Ac-P and [(125)I]Suc-P. However, [(125)I]Glu-P also registered higher radioactivity levels in the kidney. High hepatic and renal radioactivity levels were observed with highly anionic [(125)I]Suc-Glu-P. These results indicate that, while the manipulation of pI between 5 to 6 would be appropriate to enhance blood retention and reduce renal and hepatic uptake, the amount of primary amine residues on dendrimer surface may also play a crucial role in their renal uptake. The findings in this study show that gamma-glutamyl PAMAM dendrimers would constitute versatile precursors to prepare PAMAM dendrimer-based targeting devices due to their neutral molecular charge (pI 7.1) and the presence of a large number of alpha-amine residues available for conjugation of targeting molecules and drugs/imaging agents.

Intracellular reactions affecting 2-amino-4-([(11)C]methylthio)butyric acid ([(11)C]methionine) response to carbon ion radiotherapy in C10 glioma cells.

PURPOSE: The response of 2-amino-4-([(14)C]methylthio)butyric acid ([(14)C]Met) uptake and [(125)I]3-iodo-alpha-methyl-l-tyrosine ([(125)I]IMT) uptake to radiotherapy of C10 glioma cells was compared to elucidate the intracellular reactions that affect the response of 2-amino-4-([(11)C]methylthio)butyric acid ([(11)C]Met) uptake to radiotherapy. METHODS: After irradiation of cultured (3 Gy) or xenografted C10 glioma cells (25 Gy) using a carbon ion beam, the accumulation of [(14)C]Met and [(125)I]IMT in the tumors was investigated. The radiometabolites in xenografted tumors after radiotherapy were analyzed by size-exclusion HPLC. RESULTS: [(14)C]Met provided earlier responses to the carbon ion beam irradiation than [(125)I]IMT in both cultured and xenografted tumors. While [(125)I]IMT remained intact in xenografted tumor before and after irradiation, the radioactivity derived from [(14)C]Met was observed both in high molecular fractions and intact fractions, and the former decreased after irradiation. CONCLUSION: The earlier response of [(11)C]Met uptake to tumor radiotherapy could be attributable to the decline in the intracellular energy-dependent reactions of tumors due to radiotherapy.

Renal uptake and metabolism of radiopharmaceuticals derived from peptides and proteins.

Radiolabeled anti-CD20 antibodies have demonstrated impressive efficacy in the treatment of relapsed non-Hodgkin lymphoma. This encourages the treatment of solid tumor with radiolabeled antibody fragments and peptides. However, both preclinical and clinical studies revealed that persistent localization of radioactivity in the kidney constitutes a major obstacle that compromises therapeutic efficacy. Recent extensive studies show that long residence times of radiolabeled end products from lysosomes are responsible for the renal radioactivity levels. Recent studies have also elucidated the involvement of megalin-cubilin in renal tubular reabsorption of radiolabeled antibody fragments and peptides. In light of these findings, efforts are being made to block tubular reabsorption of radiolabeled antibody fragments and peptides by competitive inhibitors, charge modification, and PEGylation. An interposition of an enzyme-cleavable linkage between antibody fragments and radiolabels would constitute an alternative approach to reduce renal radioactivity levels. Recent findings of these studies will be described.