Synthesis and biological evaluation of EGFR binding peptides for near-infrared photoimmunotherapy.

Near-infrared photoimmunotherapy (NIR-PIT) is a new cancer treatment that involves photoimmunotherapy drug injection and NIR light exposure. In NIR-PIT, antibodies are commonly used as target-directed molecules carrying IRDye700DX (IR700). However, antibodies have disadvantages, such as high cost, complex development strategies, and poor tumor penetration. In contrast, peptides have lower production costs, can be easy to chemically synthesize and modify, and can also be used for tumor-targeting like antibodies. In this study, we developed a novel PIT drug using a peptide as the target-directed molecule. Epidermal growth factor receptor (EGFR) was selected as the target, and monovalent and bivalent EGFR-binding peptides were synthesized. The bivalent peptide showed sufficient binding to EGFR-positive cells, and a bivalent peptide-IR700 conjugate with a long linker induced morphological changes in EGFR-positive cells. Additionally, the drug significantly reduced cell viability in vitro in an NIR light-dose- and drug-concentration-dependent manner. These results indicate the feasibility of NIR-PIT in treating cancer using peptide-based drugs.

Comparative evaluation of radionuclide therapy using 90Y and 177Lu.

OBJECTIVE: Both 90Y and 177Lu are attractive ß-emitters for radionuclide therapy and have been used in clinical practice. Nevertheless, comparative evaluation between 90Y- and 177Lu-labeled molecules has not been fully conducted. Thus, in this study, the features of 90Y and 177Lu for radionuclide therapy were assessed in tumor-bearing mice. METHODS: Two tumor cell lines with different growth rates were used. Biodistribution studies of 177Lu-labeled antibodies (177Lu-Abs) were conducted in each tumor-bearing mouse model. Subsequently, the therapeutic effect of 90Y- and 177Lu-Ab were assessed in tumor-bearing mice. The absorbed radiation dose for the tumor was estimated using the Monte Carlo simulation. RESULTS: 177Lu-Abs demonstrated high tumor accumulation in both tumor-xerograph. In the fast-growing tumor model, 90Y-Ab showed a better therapeutic effect than 177Lu-Ab, reflecting a higher absorbed radiation dose of 90Y-Ab than that of 177Lu-Ab. In the slow-growing tumor model, both 90Y- and 177Lu-Ab showed an excellent therapeutic effect; however, 177Lu-Ab had a longer efficacy period than 90Y-Ab, which could be attributed to the longer half-life and better dose uniformity of 177Lu than those of 90Y. CONCLUSIONS: To accomplish a maximum therapeutic effect, selecting 90Y or 177Lu, to depend on the growth rate of individual cancer, would be helpful.

Enhancing the accumulation level of 3-[18F]fluoro-L-α-methyltyrosine in tumors by preloading probenecid.

INTRODUCTION: 3-[18F]fluoro-α-methyl-L-tyrosine ([18F]FAMT) is a promising amino acid tracer targeting L-type amino acid transporter 1 (LAT1). One concern regarding the diagnosis using [18F]FAMT is the possibility of false-negative findings because of its relatively low accumulation level even in malignant tumors. Moreover, preloading probenecid, an organic anion transporter inhibitor, markedly increased the tumor accumulation level of radioiodine-labeled α-methyltyrosine. In this study, we evaluated the usefulness of preloading probenecid in improving the tumor-imaging capability of [18F]FAMT. METHODS: Three biodistribution studies of [18F]FAMT were conducted in normal mice to elucidate the usefulness of probenecid preloading. Later, a biodistribution study and positron emission tomography (PET) imaging of [18F]FAMT were conducted with or without probenecid injection in tumor-bearing mice. RESULTS: Probenecid preloading significantly delayed blood clearance and consequently enhanced the accumulation of [18F]FAMT in the pancreas, a LAT1-positive organ. The effects of probenecid preloading were independent of the administration route. Tumor accumulation level in the biodistribution study and the maximum standardized uptake value in tumors on PET imaging of the probenecid preloading group were significantly higher than those of the control (without probenecid injection) group in tumor-bearing mice. CONCLUSIONS: Preloading probenecid significantly delayed blood clearance and consequently enhanced the accumulation of [18F]FAMT in tumors. These results indicate that preloading probenecid could improve the diagnostic accuracy of [18F]FAMT.

Nonclinical study and applicability of the absorbed dose conversion method with a single biodistribution measurement for targeted alpha-nuclide therapy.

BACKGROUND: We recently reported a new absorbed dose conversion method, RAP (RAtio of Pharmacokinetics), for 211At-meta-astatobenzylguanidine (211At-MABG) using a single biodistribution measurement, the percent injected dose/g. However, there were some mathematical ambiguities in determining the optimal timing of a single measurement of the percent injected dose/g. Thus, we aimed to mathematically reconstruct the RAP method and to examine the optimal timing of a single measurement. METHODS: We derived a new formalism of the RAP dose conversion method at time t. In addition, we acquired a formula to determine the optimal timing of a single measurement of the percent injected dose/g, assuming the one-compartment model for biological clearance. RESULTS: We investigated the new formalism's performance using a representative RAP coefficient with radioactive decay weighting. Dose conversions by representative RAP coefficients predicted the true [211At]MABG absorbed doses with an error of 10% or less. The inverses of the representative RAP coefficients plotted at 4 h post-injection, which was the optimal timing reported in the previous work, were very close to the new inverses of the RAP coefficients 4 h post-injection. Next, the behavior of the optimal timing was analyzed by radiolabeled compounds with physical half-lives of 7.2 h and 10 d on various biological clearance half-lives. Behavior maps of optimal timing showed a tendency to converge to a constant value as the biological clearance half-life of a target increased. The areas of optimal timing for both compounds within a 5% or 10% prediction error were distributed around the optimal timing when the biological clearance half-life of a target was equal to that of the reference. Finally, an example of RAP dose conversion was demonstrated for [211At]MABG. CONCLUSIONS: The RAP dose conversion method renovated by the new formalism was able to estimate the [211At]MABG absorbed dose using a similar pharmacokinetics, such as [131I]MIBG. The present formalism revealed optimizing imaging time points on absorbed dose conversion between two radiopharmaceuticals. Further analysis and clinical data will be needed to elucidate the validity of a behavior map of the optimal timing of a single measurement for targeted alpha-nuclide therapy.

Reduced Fatty Acid Use from CD36 Deficiency Deteriorates Streptozotocin-Induced Diabetic Cardiomyopathy in Mice.

Cardiac dysfunction is induced by multifactorial mechanisms in diabetes. Deranged fatty acid (FA) utilization, known as lipotoxicity, has long been postulated as one of the upstream events in the development of diabetic cardiomyopathy. CD36, a transmembrane glycoprotein, plays a major role in FA uptake in the heart. CD36 knockout (CD36KO) hearts exhibit reduced rates of FA transport with marked enhancement of glucose use. In this study, we explore whether reduced FA use by CD36 ablation suppresses the development of streptozotocin (STZ)-induced diabetic cardiomyopathy. We found that cardiac contractile dysfunction had deteriorated 16 weeks after STZ treatment in CD36KO mice. Although accelerated glucose uptake was not reduced in CD36KO-STZ hearts, the total energy supply, estimated by the pool size in the TCA cycle, was significantly reduced. The isotopomer analysis with 13C6-glucose revealed that accelerated glycolysis, estimated by enrichment of 13C2-citrate and 13C2-malate, was markedly suppressed in CD36KO-STZ hearts. Levels of ceramides, which are cardiotoxic lipids, were not elevated in CD36KO-STZ hearts compared to wild-type-STZ ones. Furthermore, increased energy demand by transverse aortic constriction resulted in synergistic exacerbation of contractile dysfunction in CD36KO-STZ mice. These findings suggest that CD36KO-STZ hearts are energetically compromised by reduced FA use and suppressed glycolysis; therefore, the limitation of FA utilization is detrimental to cardiac energetics in this model of diabetic cardiomyopathy.

The Role of Ferrous Ion in the Effect of the Gadolinium-Based Contrast Agents (GBCA) on the Purkinje Cells Arborization: An In Vitro Study.

Gadolinium deposition in the brain has been observed in areas rich in iron, such as the dentate nucleus of the cerebellum. We investigated the role of Fe2+ in the effect of gadolinium-based contrast agents (GBCA) on thyroid hormone-mediated Purkinje cell dendritogenesis in a cerebellar primary culture. The study comprises the control group, Fe2+ group, GBCA groups (gadopentetate group or gadobutrol group), and GBCA+Fe2+ groups. Immunocytochemistry was performed with an anti-calbindin-28K (anti-CaBP28k) antibody, and the nucleus was stained with 4',6-diamidino-2-phenylindole (DAPI). The number of Purkinje cells and their arborization were evaluated with an analysis of variance with a post-hoc test. The number of Purkinje cells was similar to the control groups among all treated groups. There were no significant differences in dendrite arborization between the Fe2+ group and the control groups. The dendrite arborization was augmented in the gadopentetate and the gadobutrol groups when compared to the control group (p < 0.01, respectively). Fe2+ significantly increased the effect of gadopentetate on dendrite arborization (p < 0.01) but did not increase the effect of gadobutrol. These findings suggested that the chelate thermodynamic stability and Fe2+ may play important roles in attenuating the effect of GBCAs on the thyroid hormone-mediated dendritogenesis of Purkinje cells in in vitro settings.

64Cu-ATSM Predicts Efficacy of Carbon Ion Radiotherapy Associated with Cellular Antioxidant Capacity.

Carbon ion radiotherapy is an emerging cancer treatment modality that has a greater therapeutic window than conventional photon radiotherapy. To maximize the efficacy of this extremely scarce medical resource, it is important to identify predictive biomarkers of higher carbon ion relative biological effectiveness (RBE) over photons. We addressed this issue by focusing on cellular antioxidant capacity and investigated 64Cu(II)-diacetyl-bis(N4-methylthiosemicarbazone) (64Cu-ATSM), a potential radioligand that reflects an over-reduced intracellular environment. We found that the carbon ion RBE correlated with 64Cu-ATSM uptake both in vitro and in vivo. High RBE/64Cu-ATSM cells showed greater steady-state levels of antioxidant proteins and increased capacity to scavenge reactive oxygen species in response to X-rays than low RBE/64Cu-ATSM counterparts; this upregulation of antioxidant systems was associated with downregulation of TCA cycle intermediates. Furthermore, inhibition of nuclear factor erythroid 2-related factor 2 (Nrf2) sensitized high RBE/64Cu-ATSM cells to X-rays, thereby reducing RBE values to levels comparable to those in low RBE/64Cu-ATSM cells. These data suggest that the cellular activity of Nrf2-driven antioxidant systems is a possible determinant of carbon ion RBE predictable by 64Cu-ATSM uptake. These new findings highlight the potential clinical utility of 64Cu-ATSM imaging to identify high RBE tumors that will benefit from carbon ion radiotherapy.

Enhancing the Therapeutic Effect of 2-211At-astato-α-methyl-L-phenylalanine with Probenecid Loading.

L-type amino acid transporter 1 (LAT1) might be a useful target for tumor therapy since it is highly expressed in various types of cancers. We previously developed an astatine-211 (211At)-labeled amino acid derivative, 2-211At-astato-α-methyl-L-phenylalanine (2-211At-AAMP), and demonstrated its therapeutic potential for LAT1-positive cancers. However, the therapeutic effect of 2-211At-AAMP was insufficient, probably due to its low tumor retention. The preloading of probenecid, an organic anion transporter inhibitor, can delay the clearance of some amino acid tracers from the blood and consequently increase their accumulation in tumors. In this study, we evaluated the effect of probenecid preloading on the biodistribution and therapeutic effect of 2-211At-AAMP in mice. In biodistribution studies, the blood radioactivity of 2-211At-AAMP significantly increased with probenecid preloading. Consequently, the accumulation of 2-211At-AAMP in tumors was significantly higher with probenecid than without probenecid loading. In a therapeutic study, tumor growth was suppressed by 2-211At-AAMP with probenecid, and the tumor volume was significantly lower in the treatment group than in the untreated control group from day 2 to day 30 (end of the follow-up period) after treatment. These results indicate that probenecid loading could improve the therapeutic effect of 2-211At-AAMP by increasing its accumulation in tumors.

Urinary FABP1 is a biomarker for impaired proximal tubular protein reabsorption and is synergistically enhanced by concurrent liver injury.

Urinary fatty acid binding protein 1 (FABP1, also known as liver-type FABP) has been implicated as a biomarker of acute kidney injury (AKI) in humans. However, the precise biological mechanisms underlying its elevation remain elusive. Here, we show that urinary FABP1 primarily reflects impaired protein reabsorption in proximal tubule epithelial cells (PTECs). Bilateral nephrectomy resulted in a marked increase in serum FABP1 levels, suggesting that the kidney is an essential organ for removing serum FABP1. Injected recombinant FABP1 was filtered through the glomeruli and robustly reabsorbed via the apical membrane of PTECs. Urinary FABP1 was significantly elevated in mice devoid of megalin, a giant endocytic receptor for protein reabsorption. Elevation of urinary FABP1 was also observed in patients with Dent disease, a rare genetic disease characterized by defective megalin function in PTECs. Urinary FABP1 levels were exponentially increased following acetaminophen overdose, with both nephrotoxicity and hepatotoxicity observed. FABP1-deficient mice with liver-specific overexpression of FABP1 showed a massive increase in urinary FABP1 levels upon acetaminophen injection, indicating that urinary FABP1 is liver-derived. Lastly, we employed transgenic mice expressing diphtheria toxin receptor (DT-R) either in a hepatocyte- or in a PTEC-specific manner, or both. Upon administration of diphtheria toxin (DT), massive excretion of urinary FABP1 was induced in mice with both kidney and liver injury, while mice with either injury type showed marginal excretion. Collectively, our data demonstrated that intact PTECs have a considerable capacity to reabsorb liver-derived FABP1 through a megalin-mediated mechanism. Thus, urinary FABP1, which is synergistically enhanced by concurrent liver injury, is a biomarker for impaired protein reabsorption in AKI. These findings address the use of urinary FABP1 as a biomarker of histologically injured PTECs that secrete FABP1 into primary urine, and suggest the use of this biomarker to simultaneously monitor impaired tubular reabsorption and liver function. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Potential of three-step pretargeting radioimmunotherapy using biotinylated bevacizumab and succinylated streptavidin in triple-negative breast cancer xenograft.

OBJECTIVE: Pretargeting radioimmunotherapy (PRIT) is a promising approach that can reduce long-time retention of blood radioactivity and consequently reduce hematotoxicity. Among the PRIT strategies, the combination of biotin-conjugated mAb and radiolabeled streptavidin (StAv) is a simple and convenient method because of its ease of preparation. This study performed three-step (3-step) PRIT using the sequential injection of (1) biotinylated bevacizumab (Bt-BV), (2) avidin, and (3) radiolabeled StAv for the treatment of triple-negative breast cancer (TNBC). METHODS: Four biodistribution studies were performed using 111In in tumor-bearing mice to optimize each step of our PRIT methods. Further, a therapeutic study was performed with optimized 3-step PRIT using 90Y-labeled StAv. RESULTS: Based on the biodistribution studies, the protein dose of Bt-BV and avidin was optimized to 100 µg and 10 molar equivalent of BV, respectively. Succinylation of StAv significantly decreased the kidney accumulation level (with succinylation (6.96 ± 0.91) vs without succinylation (20.60 ± 1.47) at 1 h after injection, p < 0.0001) with little effect on the tumor accumulation level. In the therapeutic study, tumor growth was significantly suppressed in treatment groups with optimized 3-step PRIT using 90Y-labeled succinylated StAv compared to that of the no-treatment group (p < 0.05). CONCLUSIONS: The 3-step PRIT strategy of this study achieved fast blood clearance and low kidney uptake with little effect on the tumor accumulation level, and a certain degree of therapeutic effect was consequently observed. These results indicated that the pretargeting treatment of the current study may be effective for human TNBC treatment.

DPP-4 inhibitor induces FGF21 expression via sirtuin 1 signaling and improves myocardial energy metabolism.

Dipeptidyl peptidase-4 (DPP-4) inhibitors are widely used incretin-based therapy for the treatment of type 2 diabetes. We investigated the cardioprotective effect of a DPP-4 inhibitor, vildagliptin (vilda), on myocardial metabolism and cardiac performance under pressure overload. Mice were treated with either vehicle or vilda, followed by transverse aortic constriction (TAC). After 3 weeks of TAC, cardiac hypertrophy and impairment of systolic function were attenuated in vilda-treated mice. Pressure-volume analysis showed that vilda treatment significantly improved left-ventricular contractile efficiency in TAC heart. Myocardial energy substrate analysis showed that vilda treatment significantly increased glucose uptake as well as fatty acid uptake. Fibroblast growth factor 21 (FGF21), a peptide involved in the regulation of energy metabolism, increased in TAC heart and was further increased by vilda treatment. FGF21 was strongly expressed in cardiac fibroblasts than in cardiomyocytes in mouse heart after TAC with vilda treatment. Vilda treatment markedly induced FGF21 expression in human cardiac fibroblasts through a sirtuin (Sirt) 1-mediated pathway, suggesting that fibroblast-mediated FGF21 expression may regulate energy metabolism and exert vilda-mediated beneficial effects in stressed heart. Vilda induced a metabolic regulator, FGF21 expression in cardiac fibroblasts via Sirt1, and increased contractile efficiency in murine pressure-overloaded heart.

64Cu-ATSM and 99mTc(CO)3-DCM20 potential in the early detection of rheumatoid arthritis.

OBJECTIVES: Molecular imaging constitutes a promising technique for the early detection of rheumatoid arthritis (RA). Macrophages and hypoxia play significant roles in inflamed synovium. In the present study, we evaluated the efficacy of radiopharmaceuticals that target macrophage mannose receptors (99mTc-labeled mannosylated dextran or 99mTc(CO)3-DCM20) and hypoxia (copper(II) diacetyl-di(N4-methylthiosemicarbazone) or Cu-ATSM) for the early detection of RA in collagen-induced arthritis (CIA) mice models. METHODS: CIA model was developed in DBA/1 mice, and the clinical score for arthritis was visually assessed on a regular basis. Two biodistribution studies were performed in a paired-labeled format using 2-deoxy-2-18F-fluoro-D-glucose (18F-FDG) as a reference: (1) 99mTc(CO)3-DCM20 with 18F-FDG and (2) 67Cu-ATSM with 18F-FDG. RESULTS: The accumulation levels of 99mTc(CO)3-DCM20 and 67Cu-ATSM in forepaws, hindpaws, and knee joints of CIA mice were significantly higher than that of control mice. In contrast, 18F-FDG uptake in hindpaws and knee joints showed no significant difference between CIA and control mice. The radioactivity levels of 99mTc(CO)3-DCM20 and 67Cu-ATSM were significantly correlated with the clinical scores for the paws. CONCLUSION: These results suggest the potential usefulness of 99mTc(CO)3-DCM20 and radiolabeled Cu-ATSM for the imaging and early detection of RA.

Absorbed dose simulation of meta-211At-astato-benzylguanidine using pharmacokinetics of 131I-MIBG and a novel dose conversion method, RAP.

OBJECTIVE: We aimed to estimate in vivo 211At-labeled meta-benzylguanidine (211At-MABG) absorbed doses by the two dose conversion methods, using 131I-MIBG biodistribution data from a previously reported neuroblastoma xenograft model. In addition, we examined the effects of different cell lines and time limitations using data from two other works. METHODS: We used the framework of the Monte Carlo method to create 3200 virtual experimental data sets of activity concentrations (kBq/g) to get the statistical information. Time activity concentration curves were produced using the fitting method of a genetic algorithm. The basic method was that absorbed doses of 211At-MABG were calculated based on the medical internal radiation dose formalism with the conversion of the physical half-life time of 131I to that of 211At. We have further improved the basic method; that is, a novel dose conversion method, RAP (Ratio of Pharmacokinetics), using percent injected dose/g. RESULTS: Virtual experiments showed that 211At-MABG and 131I-MIBG had similar properties of initial activity concentrations and biological components, but the basic method did not simulate the 211At-MABG dose. Simulated 211At-MABG doses from 131I-MIBG using the RAP method were in agreement with those from 211At-MABG, so that their boxes overlapped in the box plots. The RAP method showed applicability to the different cell lines, but it was difficult to predict long-term doses from short-term experimental data. CONCLUSIONS: The present RAP dose conversion method could estimate 211At-MABG absorbed doses from the pharmacokinetics of 131I-MIBG with some limitations. The RAP method would be applicable to a large number of subjects for targeted nuclide therapy.

Reduced fatty acid uptake aggravates cardiac contractile dysfunction in streptozotocin-induced diabetic cardiomyopathy.

Diabetes is an independent risk factor for the development of heart failure. Increased fatty acid (FA) uptake and deranged utilization leads to reduced cardiac efficiency and accumulation of cardiotoxic lipids, which is suggested to facilitate diabetic cardiomyopathy. We studied whether reduced FA uptake in the heart is protective against streptozotocin (STZ)-induced diabetic cardiomyopathy by using mice doubly deficient in fatty acid binding protein 4 (FABP4) and FABP5 (DKO mice). Cardiac contractile dysfunction was aggravated 8 weeks after STZ treatment in DKO mice. Although compensatory glucose uptake was not reduced in DKO-STZ hearts, total energy supply, estimated by the pool size in the TCA cycle, was significantly reduced. Tracer analysis with 13C6-glucose revealed that accelerated glycolysis in DKO hearts was strongly suppressed by STZ treatment. Levels of ceramides, cardiotoxic lipids, were similarly elevated by STZ treatment. These findings suggest that a reduction in total energy supply by reduced FA uptake and suppressed glycolysis could account for exacerbated contractile dysfunction in DKO-STZ hearts. Thus, enhanced FA uptake in diabetic hearts seems to be a compensatory response to reduced energy supply from glucose, and therefore, limited FA use could be detrimental to cardiac contractile dysfunction due to energy insufficiency.

Renal Handling of 99mTc-Labeled Antibody Fab Fragments with a Linkage Cleavable by Enzymes on Brush Border Membrane.

The high and persistent renal radioactivity levels after injection of radiolabeled low-molecular-weight polypeptides constitute a significant problem for their diagnostic and therapeutic applications, especially when they are labeled with metallic radionuclides. To improve the renal radioactivity levels of technetium-99m (99mTc)-labeled Fab fragments, a mercaptoacetyltriglycine (MAG3)-based new bifunctional chelating agent with a cleavable glycyl-phenylalanyl-lysine (GFK) linkage, MAG3-GFK-suc-TFP, was designed, synthesized, and evaluated. 99mTc-labeled Fab was obtained by reacting MAG3-GFK-Fab conjugate with 99mTc-glucarate. The GFK linkage remained stable in plasma but was cleaved by enzymes on the renal brush border membrane. The comparative biodistribution studies with indium-111 (111In)-labeled Fab using SCN-CHX-A″-DTPA showed that while both radiolabeled Fabs exhibited similar elimination rates from the blood, [99mTc]Tc-MAG3-GFK-Fab registered much lower renal radioactivity levels from 30 min post-injection onward due to the release and subsequent urinary excretion of [99mTc]Tc-MAG3-Gly. However, [99mTc]Tc-MAG3-GFK-Fab showed an increase in the intestinal radioactivity levels with the time that was not observed with 111In-labeled Fab. The analysis of the intestinal contents suggested the redistribution of [99mTc]Tc-MAG3-Gly to the intestine. The retrospective comparison of [99mTc]Tc-MAG3-GFK-Fab with the radiolabeled Fabs so far prepared under the identical concept suggested that some portion of [99mTc]Tc-MAG3-Gly was generated after the coated vesicle formation and they were excreted into the blood, and subsequently redistributed in the intestine via hepatobiliary excretion. In conclusion, MAG3-GFK-suc-TFP provided 99mTc-labeled Fabs that exhibit low renal radioactivity shortly after injection by the post-labeling procedure. The present study indicated that, contrary to our earlier proposal, the generation of the radiometabolites would proceed not only during the internalization process of the parental antibody fragments but also after coated vesicle formation. This study also showed that the intracellular behaviors of radiometabolites played crucial roles in the elimination rates and the routes of the radioactivity from the kidney.

Preclinical evaluation of new α-radionuclide therapy targeting LAT1: 2-[211At]astato-α-methyl-L-phenylalanine in tumor-bearing model.

INTRODUCTION: Targeted α-radionuclide therapy has attracted attention as a promising therapy for refractory cancers. However, the application is limited to certain types of cancer. Since L-type amino acid transporter 1 (LAT1) is highly expressed in various human cancers, we prepared an LAT1-selective α-radionuclide-labeled amino acid analog, 2-[211At]astato-α-methyl-L-phenylalanine (2-[211At]AAMP), and evaluated its potential as a therapeutic agent. METHODS: 2-[211At]AAMP was prepared from the stannyl precursor. Stability of 2-[211At]AAMP was evaluated both in vitro and in vivo. In vitro studies using an LAT1-expressing human ovarian cancer cell line, SKOV3, were performed to evaluate cellular uptake and cytotoxicity of 2-[211At]AAMP. Biodistribution and therapeutic studies in SKOV3-bearing mice were performed after intravenous injection of 2-[211At]AAMP. RESULTS: 2-[211At]AAMP was stable in murine plasma in vitro and excreted intact into urine. Cellular uptake of 2-[211At]AAMP was inhibited by treatment with an LAT1-selective inhibitor. After 24 h incubation, 2-[211At]AAMP suppressed clonogenic growth at 10 kBq/ml, and induced cell death and DNA double-strand breaks at 25 kBq/ml. When injected into mice, 2-[211At]AAMP exhibited peak accumulation in the tumor at 30 min postinjection, and radioactivity levels in the tumor were retained up to 60 min. The majority of the radioactivity was rapidly eliminated from the body into urine in an intact form immediately after injection. 2-[211At]AAMP significantly improved the survival of mice (P < 0.05) without serious side effects. CONCLUSION: 2-[211At]AAMP showed α-radiation-dependent cellular growth inhibition after it was taken up via LAT1. In addition, 2-[211At]AAMP had a beneficial effect on survival in vivo. These findings suggest that 2-[211At]AAMP would be useful for the treatment of LAT1-positive cancer. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: This is the first report of an LAT1-targeting radiopharmaceutical for α-radionuclide therapy; this agent would be applicable for the treatment of various types of cancer.

Diagnosis of bladder cancer using 18F-labeled α-methyl-phenylalanine tracers in a mouse model.

OBJECTIVE: Although 2-18F-fluoro-2-deoxy-glucose (18F-FDG) has established roles in the diagnosis of a variety of cancers, it has limited value in the detection of primary/recurrent lesions in the bladder, mainly because of interference by the pooled radioactivity in the urine. Our previous study revealed promising properties of L- and D-2-18F-α-methyl-phenylalanine (2-18F-FAMP) as radiotracers; i.e., their rapid blood clearance and low renal accumulation. In the present study we evaluated the utility of L- and D-2-18F-FAMP for imaging bladder cancer in a mouse model. METHODS: We used the human bladder cancer cell line HT1376 to prepare a bladder cancer xenograft model in mice bearing both orthotopic and subcutaneous tumors. Biodistribution and PET imaging studies were performed at 1 and 3 h after injection of L-2-18F-FAMP or D-2-18F-FAMP. 18F-FDG was used as a control. RESULTS: At 1 h after injection, greater accumulations of both L-2-18F-FAMP and D-2-18F-FAMP were observed in the orthotopic tumors compared to 18F-FDG. The orthotopic tumor-to-muscle ratio of D-2-18F-FAMP was significantly higher than that of 18F-FDG (p < 0.01), because of the rapid blood clearance of D-2-18F-FAMP. L-2-18F-FAMP showed the highest subcutaneous tumor-to-muscle ratio (p < 0.01) due to its high subcutaneous tumor uptake. Compared to L-2-18F-FAMP, D-2-18F-FAMP exhibited faster clearance and lower kidney accumulation. In the PET imaging studies, L- and D-2-18F-FAMP both clearly visualized the orthotopic bladder tumors at 1 h after injection. CONCLUSION: Our study showed that L-2-18F-FAMP and D-2-18F-FAMP have the potential to detect bladder cancer.

Selective synthesis of L-2-[18 F]fluoro-alpha-methylphenylalanine via copper-mediated 18 F-fluorination of (mesityl)(aryl)iodonium salt.

L-2-[18 F]fluoro-alpha-methylphenylalanine (2-[18 F]FAMP) is a promising amino acid tracer for positron emission tomography (PET) imaging, yet the low production yield of direct electrophilic radiofluorination with [18 F]F2 necessitates further optimization of the radiolabeling process. This paper describes a two-step preparation method for L-2-[18 F]fluoro-alpha-methylphenylalanine (2-[18 F]FAMP) starting from [18 F]fluoride. The (Mesityl)(L-alpha-methylphenylalanine)-2-iodonium tetrafluoroborate precursors with various protecting groups were prepared. The copper-mediated 18 F-fluorination of the iodonium salt precursors successfully produced 2-[18 F]FAMP. The highest radio chemical conversion of 57.6% was noted with N-Piv-protected (mesityl)(aryl)iodonium salt in the presence of 5 equivalent of Cu (OTf)2 . Subsequent deprotection with 57% hydrogen iodide produced 2-[18 F]FAMP within 120 min in 21.4 ± 11.7% overall radiochemical yield with >95% radiochemical purity and an enantiomeric excess >99%. The obtained 2-[18 F]FAMP showed comparable biodistribution profiles in normal mice with that of the carrier-added 2-[18 F]FAMP. These results indicate that usefulness of copper mediated 18 F-fluorination for the production of 2-[18 F]FAMP, which would facilitate clinical translation of the promising tumor specific amino acid tracer. Individual facilities could adopt either production method based on radioactivity demand and equipment availability.

Immuno-PET imaging for non-invasive assessment of cetuximab accumulation in non-small cell lung cancer.

BACKGROUNDS: Overexpression of epidermal growth factor receptor (EGFR) has been established as a valid therapeutic target of non-small cell lung cancer (NSCLC). However, the clinical benefit of cetuximab as an EGFR-targeting drug is still controversial, partially due to the lack of effective means to identify suitable patients. This study aimed to investigate the potential of radiolabeled cetuximab as a non-invasive tool to predict cetuximab accumulation in NSCLC tumor xenografts with varying EGFR expression levels. METHODS: The NSCLC tumors in model mice were subjected to in vivo biodistribution study and positron emission tomography (PET) imaging 48 h after injection of either 111In- or 64Cu-labeled cetuximab. The EGFR expression levels of NSCLC tumors were determined by ex vivo immunoblotting. RESULTS: We found that tumors with high EGFR expression had significantly higher [111In]In-DOTA-cetuximab accumulation than tumors with moderate to low EGFR expression (P < 0.05). Strong correlations were found between [111In]In-DOTA-cetuximab tumor uptake and EGFR expression level (r = 0.893), and between [64Cu]Cu-DOTA-cetuximab tumor uptake with EGFR expression level (r = 0.915). PET imaging with [64Cu]Cu-DOTA-cetuximab allowed clear visualization of tumors. CONCLUSION: Our findings suggest that this immuno-PET imaging can be clinically translated as a tool to predict cetuximab accumulation in NSCLC cancer patients prior to cetuximab therapy.

Al18F-labeled alpha-melanocyte-stimulating hormone (α-MSH) peptide derivative for the early detection of melanoma.

OBJECTIVE: Early detection plays a role in the prognosis of melanoma, the most aggressive skin cancer. 64Cu- and 68Ga-labeled alpha-melanocyte-stimulating hormone (α-MSH) analogs targeting the melanocortin-1 receptor are promising positron emission tomography (PET) tracers for detecting melanoma, and the use of 18F-labeling will further contribute to the detectability and availability. However, the high radiochemistry demand related to the conventional 18F-labeling methods has restricted the development of 18F-labeled α-MSH analogs. A recently developed radiofluorination method using aluminum-fluoride (Al18F) offers a simple, efficient, and time-saving labeling procedure compared to the conventional 18F-labeling methods. Herein, we sought to establish a simple preparation method for an 18F-labeled α-MSH analog using Al18F, and we examined its potential for the early detection of melanoma. METHODS: A 1,4,7-triazacyclononane-N,N',N″-triacetic acid (NOTA)-conjugated α-MSH analog (NOTA-GGNle-CycMSHhex) was prepared by the Fmoc solid-phase strategy. NOTA-GGNle-CycMSHhex was labeled with Al18F by heating at 105 °C using a microwave synthesizer for 15 min. Biodistribution study was conducted on B16/F10-luc melanoma-bearing mice at 30 min, 1 h and 3 h after injection of Al18F-NOTA-GGNle-CycMSHhex. PET imaging was conducted on melanoma-bearing mice at 1 h post-injection. One day prior to the PET imaging, bioluminescence imaging was also performed. RESULTS: Al18F-NOTA-GGNle-CycMSHhex was readily prepared with a high radiochemical yield (94.0 ± 2.8%). The biodistribution study showed a high accumulation of Al18F-NOTA-GGNle-CycMSHhex in the tumor at 30 min and 1 h post-injection (6.69 ± 1.49 and 7.70 ± 1.71%ID/g, respectively). The tumor-to-blood ratio increased with time: 3.46 ± 0.89, 12.67 ± 1.29, and 35.27 ± 9.12 at 30 min, 1 h, and 3 h post-injection, respectively. In the PET imaging, Al18F-NOTA-GGNle-CycMSHhex clearly visualized the tumors and depicted very small tumors (< 3 mm). CONCLUSIONS: We successfully prepared Al18F-NOTA-GGNle-CycMSHhex in a simple and efficient manner. Al18F-NOTA-GGNle-CycMSHhex showed high tumor accumulation and clearly visualized very small tumors in melanoma-bearing mice. These findings suggest that Al18F-NOTA-GGNle-CycMSHhex will be a promising PET tracer for melanoma imaging at an earlier stage.