Chie Suzuki

Synthesis and in vitro cellular uptake of 11C-labeled 5-aminolevulinic acid derivative to estimate the induced cellular accumulation of protoporphyrin IX.

Protoporphyrin IX (PpIX) accumulation induced by exogenous 5-aminolevulinic acid (ALA) in tumors affects the therapeutic efficacy of ALA-based photodynamic and sonodynamic therapies. To develop a new imaging probe to estimate the ALA-induced PpIX accumulation, (11)C-labeled ALA analog (4), an ALA-dehydratase inhibitor, was radiosynthesized via (11)C-methylation of a Schiff-base-activated precursor in the presence of tetrabutylammonium fluoride, followed by the hydrolysis of ester and imine groups. The cellular uptake of 4 linearly increased with time and was inhibited by ALA and other transporter competitors. Monitoring analog 4 with positron emission tomography might be useful to estimate the ALA-induced PpIX accumulation in tumors.

Benzyl [11C]Hippurate as an Agent for Measuring the Activities of Organic Anion Transporter 3 in the Brain and Multidrug Resistance-Associated Protein 4 in the Heart of Mice

Multidrug resistance-associated protein 4 (MRP4) and organic anion transporter 3 (OAT3) mediate the efflux of organic anions from the brain and heart. In this study, we have developed a probe for estimating the activity of these transporters in these tissues using positron emission tomography. Several (11)C-labeled hippuric acid ester derivatives were screened with the expectation that they would be hydrolyzed in situ to form the corresponding (11)C-labeled organic acids in target tissues. Among the compounds screened, benzyl [(11)C]hippurate showed favorable hydrolysis rates and uptake properties in the target tissues of mice. Subsequent evaluation using transporter knockout mice revealed that radioactivity was retained in the brain and heart of Oat3(-/-) and Mrp4(-/-) mice, respectively, compared with that of control mice after the intravenous administration of benzyl [(11)C]hippurate. Benzyl [(11)C]hippurate could therefore be used as a probe for estimating the activities of OAT3 and MRP4 in mouse brain and heart, respectively.

Preclinical Characterization of 5-Amino-4-Oxo-[6-11C]Hexanoic Acid as an Imaging Probe to Estimate Protoporphyrin IX Accumulation Induced by Exogenous Aminolevulinic Acid

Preoperative noninvasive imaging to estimate the quantity and spatial distribution of protoporphyrin IX (PpIX) accumulation in tumors induced by 5-aminolevulinic acid (ALA) administration is expected to improve the efficacy of ALA-based fluorescence-guided resection and photo- and sonodynamic therapies. PpIX synthesis from exogenous ALA has been reported to be regulated by ALA influx or ALA dehydratase (ALAD) activity, which catalyzes the first step of the synthesis. In this study, we characterized the properties of a 11C-labeled ALA analog, 5-amino-4-oxo-[6-11C]hexanoic acid (11C-MALA), as a PET tracer to estimate PpIX accumulation. Methods: In vitro uptake of 11C-MALA and 3H-ALA was determined in 5 tumor cell lines after 10-min incubation with each tracer at 37°C. The expression levels of ALAD were determined by Western blot analysis. In vivo distribution and dynamic PET studies were conducted in tumor-bearing mice. In vitro and in vivo accumulation of ALA-induced PpIX was determined by measuring fluorescence in extracts of cells or tumors. Results: In vitro uptake of 11C-MALA in 5 tumor cell lines was correlated with ALAD expression levels and PpIX accumulation. In vivo biodistribution and dynamic PET studies showed that 11C-MALA was rapidly incorporated into tumors, and the tumor-to-muscle ratio of 11C-MALA at 1 min after injection was significantly correlated with that of 3H-ALA. 11C-MALA in tumors was continuously decreased thereafter, and the elimination rate of 11C-MALA from AsPC-1 tumors with the highest ALAD expression level was slower than from other tumors with lower expression levels. These results suggest that the influx and intracellular retention of 11C-MALA reflect ALA influx and ALAD expression levels, respectively. Tumor accumulation of 11C-MALA at 60 min after injection was strongly correlated with PpIX accumulation in tumor tissues. Conclusion: 11C-MALA PET has the potential to noninvasively estimate the quantitative and spatial accumulation of exogenous ALA-induced PpIX.

Quantitation of rat cerebral blood flow using 99mTc-HMPAO

INTRODUCTION Technetium-99m-hexamethylpropyleneamine oxime (99mTc-HMPAO) is potentially useful for the assessment of cerebral blood flow (CBF) in small animals. In this paper, a procedure for quantitation of rat CBF using 99mTc-HMPAO was determined. METHODS Biodistribution of 99mTc-radioactivity in normal rats was determined after intravenous administration of 99mTc-HMPAO. Acetazolamide treated rats were intravenously administered with the mixture of 99mTc-HMPAO and N-isopropyl-[125I]iodoamphetamine ([125I]IMP), and arterial blood was then collected for 5min. After blood sampling, the brain radioactivity concentration was measured with the auto-well γ counter. RESULTS The brain radioactivity concentration after intravenous administration of 99mTc-HMPAO was steady from 14s to 60min post-injection. A double tracer experiment using 99mTc-HMPAO and [125I]IMP showed that 19s was the average of the optimal integration interval of arterial blood 99mTc-radioactivity concentration to obtain CBF values measured by 99mTc-HMPAO identical to those determined by [125I]IMP. The CBF value determined by 99mTc-HMPAO, calculated by dividing the brain radioactivity concentration at 5min post-injection by the integrated arterial blood radioactivity concentration until 19s post-injection, was well correlated with CBF as determined by [125I]IMP. CONCLUSION These results suggest that the CBF quantitation procedure described in this paper could be useful for rat CBF assessment.

Preclinical assessment of early tumor response after irradiation by positron emission tomography with 2-amino-[3-11C]isobutyric acid

The positron emission tomography (PET) probe, 2-amino-[3-¹¹C]isobutyric acid ([3-¹¹C]AIB), is reported to accumulate less in inflammatory lesions than 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]FDG) and has the potential for evaluation of the efficacy of radiotherapy. To determine whether [3-¹¹C]AIB is useful to monitor early metabolic change in tumors after radiotherapy, we evaluated the temporal change in [3-¹¹C]AIB tumor uptake, tumor volume, histological features and expression of amino acid transporters early after radiotherapy in a mouse tumor model. PET with [3-¹¹C]AIB was conducted in mice bearing a subcutaneous tumor (SY, derived from small cell lung cancer) in two schedules: schedule 1, before (day -1) and after (days 1 and 3) 15 Gy of radiation and schedule 2, days -1, 1 and 5. [3-¹¹C]AIB tumor uptake tended to increase on day 1 after irradiation and decreased thereafter. Tumor uptake was not correlated with tumor volume in schedule 1. Although tumor uptake was correlated with tumor volume in schedule 2, this correlation was lost when the day 5 data of greatly reduced tumor volumes were excluded. In a separate group of tumor-bearing mice, excised tumor sections were stained with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) or anti-Ki-67 antibody. There was no correlation between tumor uptake and percentages of TUNEL- or Ki-67-positive cells. Expression of amino acid transporters, SLC38A1, SLC38A2 and SLC38A4, was determined by real-time RT-PCR. SLC38A1 and SLC38A2 were expressed in SY tumors, and a significant correlation was observed between [3-¹¹C]AIB tumor uptake and SLC38A1 expression. In conclusion, early change in [3-¹¹C]AIB tumor uptake after irradiation reflected the temporal change in amino acid transporter expression, while it was independent of change in tumor volume, apoptosis and cell proliferation. PET with [3-¹¹C]AIB has the potential for use in non-invasive evaluation of early metabolic change after irradiation before morphological change of tumors.

Inhibition of radical reactions for an improved potassium tert-butoxide-promoted (11) C-methylation strategy for the synthesis of α-(11) C-methyl amino acids.

α-(11) C-Methyl amino acids are useful tools for biological imaging studies. However, a robust procedure for the labeling of amino acids has not yet been established. In this study, the (11) C-methylation of Schiff-base-activated α-amino acid derivatives has been optimized for the radiosynthesis of various α-(11) C-methyl amino acids. The benzophenone imine analog of methyl 2-amino butyrate was (11) C-methylated with [(11) C]methyl iodide following its initial deprotonation with potassium tert-butoxide (KOtBu). The use of an alternative base such as tetrabutylammonium fluoride, triethylamine, and 1,8-diazabicyclo[5.4.0]undec-7-ene did not result in the (11) C-methylated product. Furthermore, the KOtBu-promoted (11) C-methylation of the Schiff-base-activated amino acid analog was enhanced by the addition of 1,2,4,5-tetramethoxybenzene or 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and inhibited by the addition of 1,10-phenanthroline. These results suggest that inhibition of radical generation induced by KOtBu improves the α-(11) C-methylation of the Schiff-base-activated amino acids. The addition of a mixture of KOtBu and TEMPO to a solution of Schiff-base-activated amino acid ester and [(11) C]methyl iodide provided optimal results, and the tert-butyl ester and benzophenone imine groups could be readily hydrolyzed to give the desired α-(11) C-methyl amino acids with a high radiochemical conversion. This strategy could be readily applied to the synthesis of other α-(11) C-methyl amino acids.

Preferential cleavage of a tripeptide linkage by enzymes on renal brush border membrane to reduce renal radioactivity levels of radiolabeled antibody fragments

The obstructive renal radioactivity after injection of antibody fragments/constructs labeled with metallic radionuclides would be improved by liberating a radiometal chelate of urinary excretion from the antibody molecules by enzymes on the renal brush border membrane (BBM). A tripeptide GFK sequence was newly evaluated as an enzyme-cleavable linkage and conjugated to a 99mTc chelate of an isonicotinic acid derivative of 2-picolylglycine (99mTc-IPG). 99mTc-IPG-glycine was liberated from 99mTc-IPG-GFK by the enzymes, while 99mTc-IPG-GK (where the tripeptide GFK was substituted with a dipeptide GK) did not. When injected into mice, 99mTc-IPG-GFK-conjugated Fab exhibited lower renal radioactivity levels than directly radioiodinated Fab shortly after injection without reducing the tumor radioactivity levels, due to a release and excretion of 99mTc-IPG-glycine by enzymes present on the renal BBM. These findings would provide insights to develop antibody fragments/constructs labeled with metallic radionuclides of the clinical relevance for improved renal radioactivity levels.

Noninvasive quantitation of rat cerebral blood flow using 99m Tc-HMPAO—assessment of input function with dynamic chest planar imaging

BackgroundCerebral blood flow (CBF) quantitation using technetium-99m hexamethylpropyleneamine oxime (99mTc-HMPAO) generally requires assessment of input function by arterial blood sampling, which would be invasive for small animals. We therefore performed chest dynamic planar imaging, instead of arterial blood sampling, to estimate the input function and establish noninvasive quantitation method of rat CBF using the image-derived input function.ResultsIntegrated radioactivity concentration in the heart-blood pool on planar images (AUCBlood-planar) was identical to that in arterial blood samples (AUCBlood-sampling). Radioactivity concentration in the brain determined by SPECT imaging (CBrain-SPECT) was identical to that using brain sampling (CBrain-sampling). Noninvasively calculated CBF obtained by dividing CBrain-SPECT by AUCBlood-planar was well correlated with conventionally estimated CBF obtained by dividing CBrain-sampling by AUCBlood-sampling.ConclusionRat CBF could be noninvasively quantitated using 99mTc-HMPAO chest dynamic planar imaging and head SPECT imaging without arterial blood sampling.

Preclinical evaluation of 2-amino-2-[11C]methyl-butanoic acid as a potential tumor-imaging agent in a mouse model.

Objective14C-labeled 2-amino-2-methyl-butanoic acid (Iva) was previously reported to provide high tumor uptake; however, the pharmacokinetic properties of 11C-labeled Iva have not been characterized. In the present study, we evaluated the potential of [11C]Iva as a PET probe for tumor imaging. Methods[11C]Iva was incubated in mouse serum for 60 min at 37°C and then analyzed by high-performance liquid chromatography and thin-layer chromatography. In-vitro cellular uptake of [11C]Iva was determined in PBS and sodium-free buffer at 37°C using SY human small-cell lung cancer cells. The effects of inhibitors of amino acid transporters on [11C]Iva uptake were also determined in PBS. In-vivo distribution and dynamic PET studies were conducted in SY tumor-bearing mice. Results[11C]Iva was stable in mouse serum in vitro for 60 min. The cellular uptake of [11C]Iva was linearly increased for 20 min in both PBS and sodium-free buffer and almost completely inhibited by an inhibitor of system L amino acid transporters and another of LAT1, a transporter of system L. In-vivo distribution and dynamic PET studies showed that [11C]Iva was highly accumulated in tumor, but not in normal tissues, except for the pancreas and kidneys. The [11C]Iva uptake ratio of tumor to several normal tissues, such as the lung, muscle, and brain, was high. Conclusion[11C]Iva was stable in mouse serum and transported through system L amino acid transporters including LAT1, which is highly expressed in several tumors. [11C]Iva is a promising PET probe for noninvasive tumor imaging.