Effects of halogenation on tyrosine phosphorylation and peptide binding to the SRC homology 2 domain of lymphocyte-specific protein tyrosine kinase.

Phosphorylation of tyrosine residues by protein tyrosine kinases (PTK) and phosphotyrosine/Src homology 2 (SH2) domain interactions are crucial not only for signal transduction but also for regulation of PTK activity. Tyrosine residues also receive nitration and halogenation under oxidative conditions. It has been reported that nitration of tyrosine residue caused peptides to be a poor substrate for PTK and that nitrotyrosine residues could bind to SH2 domains as a phosphotyrosine mimic to activate Src family kinase. However, the effect of halogenation on tyrosine phosphorylation or SH2 domain binding is not well understood. We examined the phosphorylation of model peptides containing 3-halotyrosine or 3-nitrotyrosine using typical receptor tyrosine kinase, epidermal growth factor receptor (EGFR), and nonreceptor tyrosine kinase, lymphocyte-specific protein tyrosine kinase (Lck). The EGFR- and Lck-mediated phosphorylation was markedly inhibited by tyrosine halogenation. Iodination showed the strongest inhibition of the phosphorylation among four types of halogenation, and its inhibitory effect was stronger than that of nitration. We also examined the effect of iodination and nitration of tyrosine residues on binding to the SH2 domain of Lck, using a model peptide containing the phosphoTyr-Glu-Glu-Ile motif, which has a high affinity for the SH2 domain. The relative affinities of the modified peptides whose phosphotyrosine was substituted with unphosphorylated tyrosine, 3-nitrotyrosine, and 3-iodotyrosine, and of the model peptide were 0.024, 0.26, 1, and 16, respectively. These results suggest that tyrosine iodination may have an effect on the phosphorylation or binding to the SH2 domain similar to nitration. Tyrosine iodination possibly modulates signal transduction, with the potential impairment of cell function.

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.

In vivo tracking of transplanted mononuclear cells using manganese-enhanced magnetic resonance imaging (MEMRI).

BACKGROUND: Transplantation of mononuclear cells (MNCs) has previously been tested as a method to induce therapeutic angiogenesis to treat limb ischemia in clinical trials. Non-invasive high resolution imaging is required to track the cells and evaluate clinical relevance after cell transplantation. The hypothesis that MRI can provide in vivo detection and long-term observation of MNCs labeled with manganese contrast-agent was investigated in ischemic rat legs. METHODS AND FINDINGS: The Mn-labeled MNCs were evaluated using 7-tesla high-field magnetic resonance imaging (MRI). Intramuscular transplanted Mn-labeled MNCs were visualized with MRI for at least 7 and up to 21 days after transplantation in the ischemic leg. The distribution of Mn-labeled MNCs was similar to that of ¹¹¹In-labeled MNCs measured with single-photon emission computed tomography (SPECT) and DiI-dyed MNCs with fluorescence microscopy. In addition, at 1-2 days after transplantation the volume of the site injected with intact Mn-labeled MNCs was significantly larger than that injected with dead MNCs, although the dead Mn-labeled MNCs were also found for approximately 2 weeks in the ischemic legs. The area covered by CD31-positive cells (as a marker of capillary endothelial cells) in the intact Mn-MNCs implanted site at 43 days was significantly larger than that at a site implanted with dead Mn-MNCs. CONCLUSIONS: The present Mn-enhanced MRI method enabled visualization of the transplanted area with a 150-175 µm in-plane spatial resolution and allowed the migration of labeled-MNCs to be observed for long periods in the same subject. After further optimization, MRI-based Mn-enhanced cell-tracking could be a useful technique for evaluation of cell therapy both in research and clinical applications.

Whole-body distribution and brain tumor imaging with (11)C-4DST: a pilot study.

UNLABELLED: Recently, we developed [methyl-(11)C]4'-thiothymidine ((11)C-4DST) as an in vivo cell proliferation marker. The present study was performed to determine the safety, distribution, radiation dosimetry, and initial brain tumor imaging of (11)C-4DST in humans. METHODS: Multiorgan biodistribution and radiation dosimetry of (11)C-4DST were assessed in 3 healthy humans, who underwent 2-h whole-body PET scanning. Radiation dosimetry was estimated from the residence times of source organs using the OLINDA program. Six brain tumor patients underwent dynamic (11)C-4DST scans with arterial blood sampling. These patients were also evaluated with (11)C-methionine PET on the same day (n = 4) as, or 3 wk before (n = 2), (11)C-4DST PET studies. Metabolites in plasma and urine samples were analyzed by high-performance liquid chromatography. Breakdown of the blood-brain barrier in tumor tissue was confirmed by gadolinium-enhanced T1-weighted MRI. RESULTS: There were no serious adverse events in any subjects at any time during the study period. (11)C-4DST PET demonstrated selective uptake in the bone marrow, which has a high rate of proliferation. In addition, high-level uptake was also seen in the liver. The highest absorbed organ dose was in the urinary bladder wall (17.6 µGy/MBq). The estimated effective dose for (11)C-4DST was 4.2 µSv/MBq. (11)C-4DST showed little uptake in normal brain tissues, resulting in low background activity for imaging of brain tumors. In contrast, (11)C-4DST PET demonstrated rapid uptake in aggressive tumor masses, whereas no signal of (11)C-4DST was seen in clinically stable disease in which (11)C-methionine uptake was high. The distribution pattern of (11)C-methionine in tumor regions was not always identical to that of (11)C-4DST. Analysis of plasma samples by high-performance liquid chromatography indicated that more than 60% of the radioactivity was present as unchanged (11)C-4DST at 20 min. CONCLUSION: The initial findings of the present study in a small group of patients indicated that (11)C-4DST PET is feasible for imaging of brain tumors. Dosimetry and pharmacologic safety were acceptable at the dose required for adequate PET images.

Reactivity of 6-halopurine analogs with glutathione as a radiotracer for assessing function of multidrug resistance-associated protein 1.

6-Bromo-7-[(11)C]methylpurine is reported to react with glutathione via glutathione S-transferases in the brain and to be converted into a substrate for multidrug resistance-associated protein 1 (MRP1), an efflux pump. The compound with a rapid conversion rate allows quantitative assessment of MRP1 function, but this rate is probably susceptible to interspecies differences. Hence, for application to different species, including humans, it is necessary to adjust the conversion rate by modifying the chemical structure. We therefore designed 6-halo-9-(or 7)-[(14)C]methylpurine (halogen: F, Cl, Br, or I), and evaluated them in vitro with respect to enzymatic reactivity with glutathione using brain homogenates from the mouse, rat, or monkey. There was a marked difference in reactivity between these species. Changes in the position of the methyl group and halogen on N-methyl-6-halopurine provided various compounds possessing wide-ranging reactivity with glutathione. In conclusion, the adjustment of reactivity of 6-bromo-7-[(11)C]methylpurine may allow assessment of MRP1 function in the brain in various species.

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.

In vivo evaluation of P-glycoprotein and breast cancer resistance protein modulation in the brain using [(11)C]gefitinib.

Gefitinib (Iressa) is a selective inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase. Recent studies confirmed that gefitinib interacted with the breast cancer resistance protein (BCRP) at submicromolar concentrations, whereas other multidrug transporters, including P-glycoprotein (P-gp), showed much lower reactivity toward gefitinib. Recently, many tracers for positron emission tomography (PET) have been prepared to study P-gp function in vivo; however, PET tracers had not been evaluated for both P-gp and BCRP modulation in the brain. Therefore, we evaluated in vivo brain penetration-mediated P-gp and BCRP in mice using [(11)C]gefitinib. Co-injection with gefitinib (over 50 mg/kg), a nonspecific P-gp modulator cyclosporin A (50 mg/kg), and the dual P-gp and BCRP modulator GF120918 (over 5 mg/kg) induced an increase in the brain uptake of [(11)C]gefitinib in mice 30 min after injection. In the PET study of mice, the radioactivity level in the brain with co-injection of GF120918 (5 mg/kg) was three- to fourfold higher than that in control after initial uptake. The radioactivity level in the brain in P-gp and Bcrp knockout mice was approximately eightfold higher than that in wild-type mice 60 min after injection. In conclusion, [(11)C]gefitinib is a promising PET tracer to evaluate the penetration of gefitinib into the brain by combined therapy with P-gp or BCRP modulators, and into brain tumors. Furthermore, PET study with GF120918 is a promising approach for evaluating brain penetration-mediated P-gp and BCRP.

Noninvasive and quantitative assessment of the function of multidrug resistance-associated protein 1 in the living brain.

Multidrug resistance-associated protein 1 (MRP1) acts as a defense mechanism by pumping xenobiotics and endogenous metabolites out of the brain. The currently available techniques for studying brain-to-blood efflux have significant limitations related to either their invasiveness or the qualitative assessment. Here, we describe an in vivo method, which overcomes these limitations for assessing MRP1 function, using positron emission tomography (PET) and a PET probe. 6-Bromo-7-[(11)C]methylpurine was designed to readily enter the brain after intravenous administration and to be efficiently converted to its glutathione conjugate (MRP1 substrate) in situ. Dynamic PET scan provided the brain time-activity curve after injection of 6-bromo-7-[(11)C]methylpurine into mice. The efflux rate of the substrate was kinetically estimated to be 1.4 h(-1) with high precision. Moreover, knockout of Mrp1 gene caused approximately a 90% reduction of the efflux rate, compared with wild-type mice. In conclusion, our method allows noninvasive and quantitative assessment for MRP1 function in the living brain.

Feasibility studies of 4'-[methyl-(11)C]thiothymidine as a tumor proliferation imaging agent in mice.

This study reports on the radiosynthesis and feasibility studies of 4'-[methyl-(11)C]thiothymidine ([methyl-(11)C]S-dThd) as a tumor proliferation imaging agent. [Methyl-(11)C]S-dThd was synthesized by rapid methylation of corresponding 5-trimethylstannyl- or 5-tributylstannyl-precursor via a palladium-promoted Stille cross-coupling reaction with [(11)C]methyl iodide. The decay-corrected radiochemical yields of [methyl-(11)C]S-dThd synthesized by the corresponding 5-trimethylstannyl-precursor and 5-tributylstannyl-precursor based on [(11)C]CO(2) were 18.9% and 14.5%, respectively. The radiochemical purity of [methyl-(11)C]S-dThd was always greater than 99%. The specific activities of [methyl-(11)C]S-dThd synthesized by the corresponding 5-trimethylstannyl-precursor and 5-tributylstannyl-precursor were 47 GBq/mumol and 121 GBq/mumol, respectively, at the end of the synthesis. The total synthesis time was 30 min after the end of bombardment. The comparison between in vivo distribution of [methyl-(14)C]S-dThd and that of [methyl-(3)H]FLT showed that tracer uptake was comparable in nonproliferating tissues. In contrast, [methyl-(14)C]S-dThd showed significantly higher uptake in proliferating tissues than did [methyl-(3)H]FLT. [Methyl-(11)C]S-dThd uptake levels in five different tumor tissues were well correlated with the DNA synthesis levels determined by [2-(14)C]thymidine DNA incorporation. At 30 min after injection, plasma analysis found 95% of the activity in unmetabolized form. The microPET imaging of the C6 glioma xenograft showed significantly high uptake in the tumor and urinary bladder, followed by the intestine and marrow. Our results demonstrated that the tumor uptake of [methyl-(11)C]S-dThd was higher than that of [methyl-(3)H]FLT and was well correlated with the DNA synthesis level. Consequently, 4'-[methyl-(11)C]thiothymidine has promise for the imaging of tumor cell proliferation by positron emission tomography.

A novel noninvasive method for assessing glutathione-conjugate efflux systems in the brain.

Brain efflux systems export such conjugated metabolites as glutathione (GSH) and glucuronate conjugates, generated by the detoxification process, from the brain and serve to protect the brain from harmful metabolites. The intracerebral injection of a radiolabeled conjugate is a useful technique to assess brain efflux systems; however, this technique is not applicable to humans. Hence, we devised a novel noninvasive approach for assessing GSH-conjugate efflux systems using positron emission tomography. Here, we investigated whether or not a designed proprobe can deliver its GSH conjugate into the brain. Radiolabeled 6-chloro-7-methylpurine (7m6CP) was designed as the proprobe, and [(14)C]7m6CP was prepared by the reaction of 6-chloropurine with [(14)C]CH(3)I as a model of [(11)C]CH(3)I. The radiochemical yield and purity of [(14)C]7m6CP were 10-20% and greater than 99%, respectively. High brain uptake (0.8% ID/g) at 1 min was observed, followed by gradual radioactivity clearance from the brain for 5-60 min after the injection of [(14)C]7m6CP into rats. Analysis of metabolites confirmed that the presence of [(14)C]7m6CP was hardly observed, and 80% of the radioactivity was identical to its GSH conjugate for 15-60 min. The brain radioactivity was single-exponentially decreased during the period of 15-60 min post-injection of [(14)C]7m6CP, and the first-order efflux rate constant of the conjugate, estimated from the slope, was 0.0253 min(-1). These results showed that (1) [(14)C]7m6CP readily entered the brain, (2) it efficiently and specifically transformed to the GSH conjugate within the brain, and (3) after [(14)C]7m6CP disappearance, the clearance of radioactivity represented the only efflux of GSH conjugate. We conclude that 7m6CP can deliver the GSH conjugate into the brain and would be useful for assessing GSH-conjugate efflux systems noninvasively.

Evaluation of 4'-[methyl-14C]thiothymidine for in vivo DNA synthesis imaging.

UNLABELLED: We evaluated 4'-[methyl-11C]thiothymidine ([methyl-11C]S-dThd) to obtain a thymidine analog that might prove simpler to use for imaging DNA synthesis and that might follow the same biochemistry as its surrogate. METHODS: [Methyl-14C]S-dThd was synthesized by rapid methylation of 5-trimethylstannyl-4'-thio-2'-deoxyuridine via a palladium-mediated Stille coupling reaction with 14C-methyl iodide. Degradation of [methyl-14C]S-dThd, when incubated in human blood, was analyzed by high-performance liquid chromatography (HPLC). The in vivo potential of [methyl-14C]S-dThd was evaluated by studying its distribution in EMT-6 mammary carcinoma-bearing mice. 2-Fluoro-2'-deoxycytidine, a potent inhibitor of DNA synthesis, was used to modulate cell proliferation. Tissue extraction was also performed to investigate the incorporation of [methyl-14C]S-dThd into DNA. RESULTS: [Methyl-14C]S-dThd was obtained in a 31%-41% radiochemical yield (calculated from 14C-methyl iodide) at 130 degrees C, 5-min reaction in N,N-dimethylformamide followed by semipreparative HPLC purification. The radiochemical purity of [methyl-14C]S-dThd was >99% and the specific activity was 2.04 GBq/mmol (according to the specific activity of 14C-methyl iodide). [2-14C]Thymidine, when incubated with human blood, demonstrated rapid degradation. In contrast, [methyl-14C]S-dThd was stable with <3% degradation at 60 min. An in vivo distribution study showed the accumulation of radioactivity in proliferating tissues (spleen, thymus, duodenum, and tumor). On the other hand, the radioactivity of nonproliferating tissues (lung, liver, kidney, and muscle) was rapidly cleared in parallel with the clearance of blood radioactivity. The tumor uptake of [methyl-14C]S-dThd was high (8.8 percentage injected dose per gram at 60 min) and selective (tumor-to-blood ratio, 12.2 at 60 min). 2-Fluoro-2'-deoxycytidine pretreatment significantly reduced the tumor uptake of [methyl-14C]S-dThd. Relative blood flow as measured by the uptake of 4-[N-methyl-14C]iodoantipyrine was similar between the treated and untreated groups. Tissue extraction studies showed that most of the total tissue radioactivity of rapidly proliferating tissues was recovered in the DNA fraction at 60 min after [methyl-14C]S-dThd injection. CONCLUSION: The labeling procedure is rapid and suitable for 11C labeling. Positron-labeled 4'-thiothymidine should be useful for imaging DNA synthesis by PET.

An approach for measuring in vivo cerebral redox states using the oxidative conversion of dihydropyridine to pyridinium ion and the metabolic trapping principle.

This study was undertaken to develop radiopharmaceuticals for measuring in vivo cerebral redox states. Based on the oxidative conversion of dihydropyridine to pyridinium ion and the metabolic trapping principle, five N-[(14)C]methyl-3 or 3,5-substituted 1,4-dihydropyridines with different oxidation rates were designed, synthesized, and evaluated as a prototype of radiotracers for measuring in vivo cerebral redox states. When these tracers were injected into mice, they crossed the blood-brain barrier (BBB) and became trapped in the brain depending on their oxidation rates, while the corresponding oxidized forms hardly crossed the BBB. Furthermore, a significant increase in the radioactivity trapped in the brain was observed following injection of N-[(14)C]methyl-3-acetyl-1,4-dihydropyridine to mice pretreated with diethylmaleate that depletes glutathione in the brain. These findings suggested that an approach based on the oxidative conversion of dihydropyridine to the pyridinium ion and the metabolic trapping principle would be useful for measuring in vivo cerebral redox states.

Detection of experimental autoimmune myocarditis in rats by 111In monoclonal antibody specific for tenascin-C.

BACKGROUND: Although the identification of inflammatory infiltrates in endomyocardial biopsy specimens is necessary for the definite diagnosis of myocarditis, the biopsy test is invasive and is not sensitive. Therefore, a new diagnostic technique for the early and noninvasive evaluation of myocarditis has been awaited. Expression of tenascin-C (TNC), one of the oligometric extracellular glycoproteins, is induced in various pathological states, including inflammation, suggesting that TNC can be a molecular marker of myocarditis. METHODS AND RESULTS: An 111In anti-TNC monoclonal antibody Fab' fragment was injected intravenously into rats with experimental autoimmune myocarditis (EAM), and the biodistribution of this radiotracer was measured. Rapid clearance of radioactivity from the blood was observed in both EAM and control rats (<1% at 6 hours after injection). Myocardial uptake of the tracer was much higher in EAM rats than in control rats (7.54-, 4.39-, and 3.51-fold at 6, 24, and 48 hours after injection, respectively). By autoradiography, high radioactivities were clearly observed in the regions indicative of inflammation in EAM rats. Single-photon emission CT imaging demonstrated the focal myocardial uptake of 111In anti-TNC Fab' in vivo. CONCLUSIONS: Radiolabeled anti-TNC Fab' may be useful for the noninvasive diagnosis of myocarditis.