The Skp2-SCF E3 ligase regulates Akt ubiquitination, glycolysis, herceptin sensitivity, and tumorigenesis.

Akt kinase plays a central role in cell growth, metabolism, and tumorigenesis. The TRAF6 E3 ligase orchestrates IGF-1-mediated Akt ubiquitination and activation. Here, we show that Akt ubiquitination is also induced by activation of ErbB receptors; unexpectedly, and in contrast to IGF-1 induced activation, the Skp2 SCF complex, not TRAF6, is a critical E3 ligase for ErbB-receptor-mediated Akt ubiquitination and membrane recruitment in response to EGF. Skp2 deficiency impairs Akt activation, Glut1 expression, glucose uptake and glycolysis, and breast cancer progression in various tumor models. Moreover, Skp2 overexpression correlates with Akt activation and breast cancer metastasis and serves as a marker for poor prognosis in Her2-positive patients. Finally, Skp2 silencing sensitizes Her2-overexpressing tumors to Herceptin treatment. Our study suggests that distinct E3 ligases are utilized by diverse growth factors for Akt activation and that targeting glycolysis sensitizes Her2-positive tumors to Herceptin treatment.

Amitriptyline Accelerates SERT Binding Recovery in a Rat 3,4-Methylenedioxymethamphetamine (MDMA) Model: In Vivo 4-[18F]-ADAM PET Imaging.

Numerous studies have confirmed that 3,4-Methylenedioxymethamphetamine (MDMA) produces long-lasting changes to the density of the serotonin reuptake transporter (SERT). Amitriptyline (AMI) has been shown to exert neuroprotective properties in neuropathologic injury. Here, we used a SERT-specific radionuclide, 4-[18F]-ADAM, to assess the longitudinal alterations in SERT binding and evaluate the synergistic neuroprotective effect of AMI in a rat MDMA model. In response to MDMA treatment regimens, SERT binding was significantly reduced in rat brains. Region-specific recovery rate (normalized to baseline) in the MDMA group at day 14 was 71.29% ± 3.21%, and progressively increased to 90.90% ± 7.63% at day 35. AMI dramatically increased SERT binding in all brain regions, enhancing average ~18% recovery rate at day 14 when compared with the MDMA group. The immunochemical staining revealed that AMI markedly increased the serotonergic fiber density in the cingulate and thalamus after MDMA-induction, and confirmed the PET findings. Using in vivo longitudinal PET imaging, we demonstrated that SERT recovery was positively correlated with the duration of MDMA abstinence, implying that lower SERT densities in MDMA-induced rats reflected neurotoxic effects and were (varied) region-specific and reversible. AMI globally accelerated the recovery rate of SERT binding and increased SERT fiber density with possible neuroprotective effects.

Targeted molecular-genetic imaging and ligand-directed therapy in aggressive variant prostate cancer.

Aggressive variant prostate cancers (AVPC) are a clinically defined group of tumors of heterogeneous morphologies, characterized by poor patient survival and for which limited diagnostic and treatment options are currently available. We show that the cell surface 78-kDa glucose-regulated protein (GRP78), a receptor that binds to phage-display-selected ligands, such as the SNTRVAP motif, is a candidate target in AVPC. We report the presence and accessibility of this receptor in clinical specimens from index patients. We also demonstrate that human AVPC cells displaying GRP78 on their surface could be effectively targeted both in vitro and in vivo by SNTRVAP, which also enabled specific delivery of siRNA species to tumor xenografts in mice. Finally, we evaluated ligand-directed strategies based on SNTRVAP-displaying adeno-associated virus/phage (AAVP) particles in mice bearing MDA-PCa-118b, a patient-derived xenograft (PDX) of castration-resistant prostate cancer bone metastasis that we exploited as a model of AVPC. For theranostic (a merging of the terms therapeutic and diagnostic) studies, GRP78-targeting AAVP particles served to deliver the human Herpes simplex virus thymidine kinase type-1 (HSVtk) gene, which has a dual function as a molecular-genetic sensor/reporter and a cell suicide-inducing transgene. We observed specific and simultaneous PET imaging and treatment of tumors in this preclinical model of AVPC. Our findings demonstrate the feasibility of GPR78-targeting, ligand-directed theranostics for translational applications in AVPC.

Molecular imaging of active mutant L858R EGF receptor (EGFR) kinase-expressing nonsmall cell lung carcinomas using PET/CT.

The importance of the EGF receptor (EGFR) signaling pathway in the development and progression of nonsmall cell lung carcinomas (NSCLC) is widely recognized. Gene sequencing studies revealed that a majority of tumors responding to EGFR kinase inhibitors harbor activating mutations in the EGFR kinase domain. This underscores the need for novel biomarkers and diagnostic imaging approaches to identify patients who may benefit from particular therapeutic agents and approaches with improved efficacy and safety profiles. To this goal, we developed 4-[(3-iodophenyl)amino]-7-{2-[2-{2-(2-[2-{2-([(18)F]fluoroethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}-ethoxy]-quinazoline-6-yl-acrylamide ([(18)F]F-PEG6-IPQA), a radiotracer with increased selectivity and irreversible binding to the active mutant L858R EGFR kinase. We show that PET with [(18)F]F-PEG6-IPQA in tumor-bearing mice discriminates H3255 NSCLC xenografts expressing L858R mutant EGFR from H441 and PC14 xenografts expressing EGFR or H1975 xenografts with L858R/T790M dual mutation in EGFR kinase domain, which confers resistance to EGFR inhibitors (i.e., gefitinib). The T790M mutation precludes the [(18)F]F-PEG6-IPQA from irreversible binding to EGFR. These results suggest that PET with [(18)F]F-PEG6-IPQA could be used for the selection of NSCLC patients for individualized therapy with small molecular inhibitors of EGFR kinase that are currently used in the clinic and have a similar structure (i.e., iressa, gefitinib, and erlotinib).

Challenges in the maintenance of an open hospital-based cancer registry system in a low-to-middle-income country (LMIC): 2017-2022 experience.

Hospital-based cancer registries (HBCRs) record data on all patients diagnosed and/or treated for cancer at healthcare facilities and evaluate the burden of the disease and the quality of healthcare services at that hospital, helping improve patient care, and providing an assessment of healthcare quality. The CARE PH app was created as a tool to facilitate a system of hospital-based cancer registries in the Philippines, a lower middle-income country. From 2017 to 2022, a total of 60,021 cancer registrants from 44 CARE PH hospitals were entered into the database. Breast cancer was the most common primary site, accounting for 17,660 cases (29.4%). This was followed by colorectal cancer at 11.1%, cervical cancer at 6.2%, head and neck cancer at 5.9%, and prostate and other male genital cancer at 5.1%.Among the 30 data fields collected, 17 exhibited 0-20% missing data, eight displayed 21%-90% missing data, while five depicted 91%-100% missing data. Most of the data fields with missing data are in the treatment and follow-up modules, which are stored in separate forms in a patient's record. Digital transformation of hospitals from paper-based charts to electronic medical records, and the integration of the HBCR to the EMR and hospital information system, will likely be the best solution for these limitations. It is recommended that the creation and maintenance of HBCRs nationwide must be harmonized, and embedded in all relevant national programs and legislations. The development of an information technology process that is based on a cancer patient's journey, should be built on an open system embedded in a well designed enterprise architecture, functioning under the guidance of a strong leadership and governance team. All these must be present in order to create and maintain a robust HBCR that is useful for furthering cancer registry and research in the country.

Imaging epigenetic regulation by histone deacetylases in the brain using PET/MRI with ¹8F-FAHA.

Epigenetic modifications mediated by histone deacetylases (HDACs) play important roles in the mechanisms of different neurologic diseases and HDAC inhibitors (HDACIs) have shown promise in therapy. However, pharmacodynamic profiles of many HDACIs in the brain remain largely unknown due to the lack of validated methods for noninvasive imaging of HDAC expression-activity. In this study, dynamic PET/CT imaging was performed in 4 rhesus macaques using [(18)F]FAHA, a novel HDAC substrate, and [(18)F]fluoroacetate, the major radio-metabolite of [(18)F]FAHA, and fused with corresponding MR images of the brain. Quantification of [(18)F]FAHA accumulation in the brain was performed using a customized dual-tracer pharmacokinetic model. Immunohistochemical analyses of brain tissue revealed the heterogeneity of expression of individual HDACs in different brain structures and cell types and confirmed that PET/CT/MRI with [(18)F]FAHA reflects the level of expression-activity of HDAC class IIa enzymes. Furthermore, PET/CT/MRI with [(18)F]FAHA enabled non-invasive, quantitative assessment of pharmacodynamics of HDAC inhibitor SAHA in the brain.

Very rapid cloning, expression and identifying specificity of T-cell receptors for T-cell engineering.

Neoantigens can be predicted and in some cases identified using the data obtained from the whole exome sequencing and transcriptome sequencing of tumor cells. These sequencing data can be coupled with single-cell RNA sequencing for the direct interrogation of the transcriptome, surfaceome, and pairing of αß T-cell receptors (TCRαß) from hundreds of single T cells. Using these 2 large datasets, we established a platform for identifying antigens recognized by TCRαßs obtained from single T cells. Our approach is based on the rapid expression of cloned TCRαß genes as Sleeping Beauty transposons and the determination of the introduced TCRαßs' antigen specificity and avidity using a reporter cell line. The platform enables the very rapid identification of tumor-reactive TCRs for the bioengineering of T cells with redirected specificity.

The pharmacological properties of 3-arm or 4-arm DOTA constructs for conjugation to α-melanocyte-stimulating hormone analogues for melanoma imaging.

BACKGROUND: Although a 3-arm DOTA construct, which has three carboxylic acids, h has been applied for conjugation to many peptides, we investigated if a 4-arm DOTA construct conjugated to peptides improves chemical properties for melanoma imaging of the melanocortin 1 receptor compared to 3-arm DOTA-conjugated peptides. METHODS: Specific activities, radiolabeling efficiencies, and partition coefficients were evaluated using 111In-labeled 3-arm and 4-arm DOTA-α-melanocyte-stimulating hormone (MSH). For assessment of MC1-R affinity and accumulation in tumor cells in vitro, B16-F1 melanoma and/or 4T1 breast cancer cells were incubated with 111In-labeled 3-arm and 4-arm DOTA-α-MSH with and without α-MSH as a substrate. The stability was evaluated using mouse liver homogenates and plasma. Biological distribution and whole-body single photon emission computed tomography imaging of 111In-labeled 3-arm and 4-arm DOTA-α-MSH were obtained using B16-F1 melanoma-bearing mice. RESULTS: Specific activities and radiolabeling efficiencies of both radiotracers were about 1.2 MBq/nM and 90-95%, respectively. The partition coefficients were -0.28 ± 0.03 for 111In-labeled 3-arm DOTA-α-MSH and -0.13 ± 0.04 for 111In-labeled 4-arm DOTA-α-MSH. Although accumulation was significantly inhibited by α-MSH in B16-F1 cells, the inhibition rate of 111In-labeled 4-arm DOTA-α-MSH was lower than that of 111In-labeled 3-arm DOTA-α-MSH. 111In-labeled 4-arm DOTA-α-MSH was taken up early into B16-F1 cells and showed higher accumulation than 111In-labeled 3-arm DOTA-α-MSH after 10 min of incubation. Although these stabilities were relatively high, the stability of 111In-labeled 4-arm DOTA-α-MSH was higher than that of 111In-labeled 3-arm DOTA-α-MSH. Regarding biological distribution, 111In-labeled 4-arm DOTA-α-MSH showed significantly lower average renal accumulation (1.38-fold) and significantly higher average melanoma accumulation (1.32-fold) than 111In-labeled 3-arm DOTA-α-MSH at all acquisition times. 111In-labeled 4-arm DOTA-α-MSH showed significantly higher melanoma-to-kidney, melanoma-to-blood, and melanoma-to-muscle ratios than 111In-labeled 3-arm DOTA-α-MSH. CONCLUSIONS: The 4-arm DOTA construct has better chemical properties for peptide radiotracers than the 3-arm DOTA construct.

N(3)-Substituted thymidine analogues V: synthesis and preliminary PET imaging of N(3)-[(18)F]fluoroethyl thymidine and N(3)-[(18)F]fluoropropyl thymidine.

INTRODUCTION: [(18)F]-Labeled analogues of thymidine have demonstrated efficacy for PET imaging of cellular proliferation. We have synthesized two [(18)F]-labeled N(3)-substituted thymidine analogues, N(3)-[(18)F]fluoroethyl thymidine (N(3)-[(18)F]-FET) and N(3)-[(18)F]fluoropropyl thymidine (N(3)-[(18)F]-FPrT), and performed preliminary PET imaging studies in tumor-bearing mice. METHODS: Thymidine was converted to its 3',5'-O-bis-tetrahydropyranyl ether, which was then converted to the N(3)-ethyl and propyl-substituted mesylate precursors. Reactions of these mesylate precursors with n-Bu(4)N[(18)F] or K[(18)F]/kryptofix followed by acid hydrolysis and HPLC purification yielded N(3)-[(18)F]-FET and N(3)-[(18)F]-FPrT, respectively. Subcutaneous (sc) xenografts of H441 human non-small cell lung cancer were established in two groups of mice (each n=6). Micro-PET images of the tumor-bearing animals were acquired after intravenous injection of N(3)-[(18)F]-FET or N(3)-[(18)F]-FPrT (3700 KBq/animal). RESULTS: The radiochemical yields were 2-12% (d.c.) for N(3)-[(18)F]-FET and 30-38% (d.c.) for N(3)-[(18)F]-FPrT. Radiochemical purity was >99% and calculated specific activity was >74 GBq/mumol at the end of synthesis. The accumulation of N(3)-[(18)F]-FET and N(3)-[(18)F]-FPrT in the tumor tissue at 2 h postinjection was 1.81+/-0.78 and 2.95+/-1.14 percent injected dose per gram (%ID/g), respectively; tumor/muscle ratios were 5.57+/-0.82 and 7.69+/-2.18, respectively; the unidirectional influx rates (K(i)) were 0.013 and 0.018 ml/g per minute, respectively. CONCLUSION: Two novel [(18)F]- N(3)-substituted thymidine analogues have been synthesized in good yields, high purity and high specific activity. Preliminary in vivo studies demonstrated the efficacy of these [(18)F]- N(3)-substituted thymidine analogues for PET imaging of tumors.

Development of radioiodine labeled acetaminophen for specific, high-contrast imaging of malignant melanoma.

INTRODUCTION: Due to its poor prognosis, specific imaging for early detection of malignant melanoma is strongly desired. Although radioiodine labeled 4-hydroxyphenylcysteamine, which we previously developed, has good affinity for tyrosinase, an enzyme in the melanin metabolic pathway, image contrast of the melanoma:organ ratios is not sufficiently high for detection of primary melanoma and metastases at early injection times. In this study, we developed radioiodine labeled acetaminophen (I-AP) for specific, high-contrast imaging of malignant melanoma. METHODS: Radioiodine-125-labeled AP (125I-AP) was prepared using the chloramine-T method under no carrier-added conditions. Accumulation of radioactivity and the mechanism were evaluated in vitro using B16 melanoma cells incubated with 125I-AP or 14C(U)-labeled AP (14C-AP) with and without l-tyrosine as a substrate of tyrosinase, phenylthiourea as an inhibitor of tyrosinase, and thymidine as an inhibitor of DNA polymerase. The biological distribution of radioactivity in B16 melanoma-bearing mice was evaluated to determine the accumulation of 125I-AP. The stability of 125I-AP over time was evaluated in mice. RESULTS: The labeling efficiency and radiochemical purity of 125I-AP were >80% and 95%, respectively. Accumulation of 125I-AP was higher than that of 14C-AP at 60 min of incubation in vitro. The affinity of 14C-AP for tyrosinase and DNA polymerase was higher than that of 125I-AP, whereas the Vmax of 125I-AP was higher than that of 14C-AP. 125I-AP showed the highest accumulation in the gall bladder, and clearance from the blood and kidney was rapid. Melanoma:muscle and melanoma:normal skin ratios of 125I-AP for imaging contrast were the highest at 15 min after injection, whereas the melanoma:blood and melanoma:bone ratios gradually increased over time. 125I-AP remained stable for 60 min after injection in mice. CONCLUSIONS: 125I-AP has affinity for tyrosinase and high image contrast at early time points after injection. Therefore, 123I-AP imaging has great potential for specific, high-contrast detection of malignant melanoma. ADVANCES IN KNOWLEDGE: 123I-AP will provide specific, high-contrast imaging for malignant melanoma at early injection times. IMPLICATIONS FOR PATIENT CARE: 123I-AP has good potential for the diagnosis of malignant melanoma compared with 123I-labeled 4-hydroxyphenylcysteamine, which we previously developed.

Detection of maleate-induced Fanconi syndrome by decreasing accumulation of 125I-3-iodo-alpha-methyl-L-tyrosine in the proximal tubule segment-1 region of renal cortex in mice: a trial of separate evaluation of reabsorption.

OBJECTIVE: Fanconi syndrome is a renal dysfunction characterized by various combinations of renal tubular transport dysfunction involving amino acids, glucose, protein and other substances. Most reabsorption of amino acids occurs in proximal renal tubule segment 1 (S1). The present study evaluated the possibility of early detection of drug-induced Fanconi syndrome, based on decreased renal accumulation of 125I-3-iodo-alpha-methyl-L-tyrosine (125I-IMT), an amino acid transport marker, in the S1 region of renal cortex. The present experimental model used maleate (MAL)-induced Fanconi syndrome in mice. Results were compared between 125I-IMT and 3 other clinical renal radiopharmaceuticals: 99mTc-2,3-dimercaptosuccinic acid (99mTc-DMSA); 99mTc-mercaptoacetylglycylglycylglycine (99mTc-MAG3); and 99mTc-diethylenetriaminepentaacetic acid (99mTc-DTPA). METHODS: Male ddY mice (age, 6 weeks; body weight, 25 g) were used to create a Fanconi model of renal dysfunction. A single dose of maleate disodium salt was administered by intraperitoneal injection (6 mmol/kg). Hematoxylin and eosin (HE) staining of the renal cortex, renal autoradiography and measurement of renal radioactivity of labeled compounds were performed at 30, 60, 90 and 120 min after MAL injection. At 5 min after injection of labeled compounds (18.5 kBq for accumulation experiment, 670 kBq for autoradiography), animals were sacrificed by ether overdose and kidneys were removed. For the accumulation experiment, radioactivity was measured using a well-type scintillation counter. For autoradiography, 20-microm sections of frozen kidney were used with Bio-Imaging Analyzer. RESULTS: At 30 min after MAL injection, HE staining showed pyknosis in some proximal tubule cells. At that time, accumulations of 125I-IMT and 99mTc-DMSA in the S1 region were approximately 67% and 55% of control levels (p < 0.005). MAL increased accumulation of 99mTc-DTPA in the S1 region, but had no effect on accumulation of 99mTc-MAG3 in the S 1 region. CONCLUSIONS: Decreased accumulation of 123I-IMT in the S1 region appears to represent a useful marker for detection of MAL-induced Fanconi syndrome. In future, we plan to assess the efficacy of using 125I-IMT to monitor renal dysfunction induced by nephrotoxic clinical drugs.

Imaging of Sleeping Beauty-Modified CD19-Specific T Cells Expressing HSV1-Thymidine Kinase by Positron Emission Tomography.

PURPOSE: We have incorporated a positron emission tomography (PET) functionality in T cells expressing a CD19-specific chimeric antigen receptor (CAR) to non-invasively monitor the adoptively transferred cells. PROCEDURES: We engineered T cells to express CD19-specific CAR, firefly luciferase (ffLuc), and herpes simplex virus type-1 thymidine kinase (TK) using the non-viral-based Sleeping Beauty (SB) transposon/transposase system adapted for human application. Electroporated primary T cells were propagated on CD19+ artificial antigen-presenting cells. RESULTS: After 4 weeks, 90 % of cultured cells exhibited specific killing of CD19+ targets in vitro, could be ablated by ganciclovir, and were detected in vivo by bioluminescent imaging and PET following injection of 2'-deoxy-2'-[18F]fluoro-5-ethyl-1-ß-D-arabinofuranosyl-uracil ([18F]FEAU). CONCLUSION: This is the first report demonstrating the use of SB transposition to generate T cells which may be detected using PET laying the foundation for imaging the distribution and trafficking of T cells in patients treated for B cell malignancies.

Development of radioiodine-labeled 4-hydroxyphenylcysteamine for specific diagnosis of malignant melanoma.

INTRODUCTION: A specific diagnosis for melanoma is strongly desired because malignant melanoma has poor prognosis. In a previous study, although radioiodine-125-labeled 4-hydroxyphenyl-L-cysteine ((125)I-L-PC) was found to have good substrate affinity for tyrosinase enzyme in the melanin metabolic pathway, (123/131)I-L-PC had insufficient substrate affinity for tyrosinase to diagnose melanoma. In this study, we synthesized 4-hydroxyphenylcysteamine (4-PCA) and developed a novel radioiodine-125-labeled 4-hydroxyphenylcysteamine ((125)I-PCA) to increase affinity for the melanin biosynthesis pathway. METHODS: 4-PCA was separated with 2-hydroxyphenylcysteamine (2-PCA), which is an isomer of 4-PCA, and was examined using melting point, proton nuclear magnetic resonance, mass spectrometry and elemental analysis. (125)I-PCA was prepared using the chloramine-T method under no-carrier added conditions. We performed biodistribution experiments using B16 melanoma-bearing mice using (125)I-PCA, (125)I-L-PC, (125)I-α-methyl-L-tyrosine, (123)I-m-iodobenzylguanidine and (67)Ga-citrate. In vitro assay was performed with B16 melanoma cells, and affinity for tyrosinase, DNA polymerase and amino acid transport was evaluated using phenylthiourea, thymidine, ouabine and L-tyrosine inhibitor. In addition, partition coefficients of (125)I-PCA were evaluated. RESULTS: In the synthesis of 4-PCA, analysis values did not differ between calculated and reported values, and 4-PCA was separated from 2-PCA at high purity. In biodistribution experiments, (125)I-PCA was accumulated and retained in B16 melanoma cells when compared with (125)I-L-PC. (125)I-PCA showed the highest values at 60 min after radiotracer injection in melanoma-to-muscle ratios, melanoma-to-blood ratios and melanoma-to-skin ratios. Accumulation of (125)I-PCA was significantly inhibited by phenylthiourea and thymidine. Partition coefficients of (125)I-PCA were lower than those of N-isopropyl-p-[(123)I]iodoamphetamine and were not significantly different from (125)I-L-PC. CONCLUSIONS: (125)I-PCA is a better substrate for tyrosinase and DNA polymerase and has higher uptake and longer retention in B16 melanoma cells when compared with (125)I-L-PC. Therefore, (123/131)I-PCA has good potential for diagnosis for malignant melanoma. ADVANCE IN KNOWLEDGE: (125)I-PCA will be a specific diagnosis tool for malignant melanoma. IMPLICATIONS FOR PATIENT CARE: (123/131)I-PCA has good potential for the diagnosis of malignant melanoma when compared with other SPECT tracers, as well as anti-melanoma chemotherapeutic drugs.

An artificial amino acid, 4-iodo-L-meta-tyrosine: biodistribution and excretion via kidney.

UNLABELLED: We evaluated the use of radiolabeled 4-iodo-L-meta-tyrosine as an amino acid transport marker. The pharmacologic features of this compound, particularly the biodistribution and excretion, were examined by conducting in vivo and in vitro studies using 4-(125)I-iodo-L-meta-tyrosine (4-(125)I-mTyr). Results obtained for L-(14)C-Tyr and 3-(125)I-iodo-alpha-methyl-L-tyrosine ((125)I-IMT) were used for comparison. METHODS: In vivo biodistribution studies of 4-(125)I-mTyr were performed in male ddY mice. Urinary excretion of 4-(125)I-mTyr and (125)I-IMT with administration of probenecid was studied. Local distribution of 4-(125)I-mTyr and (125)I-IMT in kidney was visualized by autoradiography. We performed metabolite analysis of 4-(125)I-mTyr in mice. For in vitro studies, reabsorption mechanisms of 4-(125)I-mTyr were compared with those of (125)I-IMT and the parent L-(14)C-Tyr using superconfluent monolayers of the porcine kidney epithelial cell line LLC-PK(1) in medium containing inhibitor (L-Tyr, D-Tyr, and 2,4-dinitrophenol), in Na(+)-free medium, and at 4 degrees C. RESULTS: 4-(125)I-mTyr demonstrated high accumulation in the pancreas and kidney and comparable brain uptake to that of (125)I-IMT. Blood clearance of 4-(125)I-mTyr was faster than that of (125)I-IMT. Three hours after administration, >70% of 4-(125)I-mTyr was excreted via the urine, whereas <5% was found in the feces. Renal autoradiography revealed moderate accumulation of 4-(125)I-mTyr and high accumulation of (125)I-IMT in the renal cortex. Probenecid further reduced accumulation of 4-(125)I-mTyr and (125)I-IMT in the kidney as well as urinary excretion. At 30 min after tracer injection, intact free 4-(125)I-mTyr accounted for >98.1% of the total present in kidney and >96.3% in urine. Protein incorporation was not observed. Uptake of 4-(125)I-mTyr into LLC-PK(1) cell monolayers was remarkably reduced by 5 mmol/L L-Tyr (4.6%) and incubation at 4 degrees C (15.6%) but was reduced by 5 mmol/L D-Tyr (50.0%). L-(14)C-Tyr and (125)I-IMT showed similar results; however, uptake of (125)I-IMT was enhanced by 0.1 mmol/L 2,4-dinitrophenol (165.1%), an inhibitor of generation of energy-rich phosphates. CONCLUSION: The artificial amino acid 4-(125)I-mTyr demonstrated high metabolic stability, rapid blood clearance, rapid urinary excretion, and similar biodistribution to other radiolabeled L-Tyr analogs. 4-(125)I-mTyr can be a competitive substrate of L-Tyr reabsorption. However, 4-(125)I-mTyr demonstrates different pharmacologic features than those of (125)I-IMT, particularly in renal handling. 4-(125)I-mTyr may potentially be applied as a new amino acid transport marker.

Renal accumulation and excretion of radioiodinated 3-iodo-alpha-methyl-L-tyrosine.

OBJECTIVE: We investigated mechanisms of renal accumulation of radioiodinated 3-iodo-alpha-methyl-L-tyrosine (IMT), which has been used clinically for tumor imaging and as an amino acid transport marker in studies of brain and pancreas function. METHODS: In this study, we used 125I- or 123I-labeled IMT ([125I]IMT or [123I]IMT) as the transport marker. Partition coefficients of [125I]IMT were determined for hypothetic urine at pH ranging from 5 to 8. The examination of uptake and inhibition of [125I]IMT was performed using normal human renal proximal tubule epithelial cells (RPTEC), which are characteristic of the proximal convoluted tubule. The plasma protein binding ratio of [125I]IMT was determined using rats. In the in vivo experiments using mice, we examined biodistribution and excretion inhibition, and performed whole body autoradiography. Also, renal SPECT using [123I]IMT was performed using a normal canine. RESULTS: Very low lipophilicity of [125I]IMT in hypothetic urine suggests that a carrier-mediated pathway contributes to its marked kidney accumulation. [125I]IMT uptake into RPTEC was significantly inhibited by 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) in a sodium-dependent manner, suggesting reabsorption mainly via system B0 in apical membrane of proximal tubule. Plasma protein binding ratio of IMT was 45.4 +/- 5.6%. At 6 hr after administration of IMT to mice, excretion via urinary tract was 77.51% of injected dose, and excretion into feces was 0.25%. Furosemide, ethacrynic acid and probenecid inhibited tubular secretion of [125I]IMT in mice. We obtained very clear autoradiographs of mouse renal cortex and a canine renal SPECT image (S2-like region). CONCLUSIONS: We believe that [123I]IM-T is useful for kidney imaging. In future studies, we plan to examine the use of [123I]IMT in diagnosis of disease.

Monitoring therapy with MEK inhibitor U0126 in a novel Wilms tumor model in Wt1 knockout Igf2 transgenic mice using 18F-FDG PET with dual-contrast enhanced CT and MRI: early metabolic response without inhibition of tumor growth.

PURPOSE: The understanding of the role of genetic alterations in Wilms tumor development could be greatly advanced using a genetically engineered mouse models that can replicate the development and progression of this disease in human patients and can be monitored using non-invasive structural and molecular imaging optimized for renal tumors. PROCEDURES: Repetitive dual-contrast computed tomography (CT; intravenous and intraperitoneal contrast), T2-weighted magnetic resonance imaging (MRI), and delayed 2-deoxy-2-[(18)F]fluoro-D-glucose ((18)F-FDG) positron emission tomography (PET) were utilized for characterization of Igf2 biallelic expression/Wt1 knockout mouse model of Wilms tumor. For CT imaging, Ioversol 678 mg/ml in 200 µl was administered i.p. followed by 100 µl injected intravenously at 20 and 15 min prior to imaging, respectively. Static PET imaging studies were acquired at 1, 2, and 3 h after i.v. administration of (18)F-FDG (400 µCi). Coronal and sagittal T1-weighted images (TE/TR 8.5/620 ms) were acquired before and immediately after i.v. injection of 0.4 ml/kg gadopentetate dimeglumine followed by T2-weighted images (TE/TR 60/300 ms). Tumor tissue samples were characterized by histopathology and immunohistochemistry for Glut1, FASN, Ki67, and CD34. In addition, six Wt1-Igf2 mice were treated with a mitogen-activated protein kinase (MEK) inhibitor U0126 (50 µmol/kg i.p.) every 4 days for 6 weeks. (18)F-FDG PET/CT imaging was repeated at different days after initiation of therapy with U0126. The percent change of initial tumor volume and SUV was compared to non-treated historic control animals. RESULTS: Overall, the best tumor-to-adjacent kidney contrast as well as soft tissue contrast for other abdominal organs was achieved using T2-weighted MRI. Delayed (18)F-FDG PET (3-h post (18)F-FDG administration) and dual-contrast CT (intravenous and intraperitoneal contrast) provided a more accurate anatomic and metabolic characterization of Wilms tumors in Wt1-Igf2 mice during early development and progression of renal tumors. Over the 8-month period, 46 Wt1-Igf2 mice and 8 littermate control mice were studied. Renal tumors were identified in 54.3 % of Wt1-Igf2 mice between post-natal 50-100 days. In 35.6 % of Wt1-Igf2 mice, tumors were localized in the right kidney; in 24 %, in the left kidney, while 40.4 % of Wt1-Igf2 mice had bilateral kidney tumors. Metastatic lesions were identified in 15.4 % of Wt1-Igf2 mice. Increased levels of Glut1 and IGF1R expression, high Ki67 labeling index, and a dense network of CD34+ microvessels in renal tumors was consistent with increased (18)F-FDG accumulation. Treatment with a MEK 1/2 inhibitor U0126 did not cause the inhibition of tumor growth as compared to untreated animals. However, after the first three to four doses (~2 weeks of treatment), a decrease in (18)F-FDG SUV was observed, as compared to pre-treatment levels (p < 0.05, paired Student t test), which constitutes a metabolic response. Six weeks later, despite continuing therapy, the (18)F-FDG SUV increased again to previous levels. CONCLUSIONS: The optimized dual contrast PET/CT imaging with early post i.v. and i.p. contrast CT and 3 h delayed PET imaging after (18)F-FDG administration provides a sensitive and reliable method for detecting early tumor lesions in this endogenous mouse model of Wilms tumor and for monitoring their growth in response to targeted therapies. Therapy with MEK inhibitor U0126 produces only a transient inhibition of tumor glycolytic activity but does not inhibit tumor growth, which is due to continuing IGF2-induced signaling from IGF1R through the PI3K-AKT-mTOR pathway.

Synthesis and preliminary evaluation of [18F]-labeled 2-oxoquinoline derivatives for PET imaging of cannabinoid CB2 receptor.

INTRODUCTION: The cannabinoid receptor type 2 (CB(2)) is an important target for development of drugs and imaging agents for diseases, such as neuroinflammation, neurodegeneration and cancer. Recently, we reported synthesis and results of in vitro receptor binding of a focused library of fluorinated 2-oxoquinoline derivatives as CB(2) receptor ligands. Some of the compounds demonstrated to be good CB(2)-specific ligands with Ki values in the nanomolar to subnanomolar concentrations; therefore, we pursued the development of their (18)F-labeled analogues that should be useful for positron emission tomography (PET) imaging of CB(2) receptor expression. Here, we report the radiosynthesis of two (18)F-labeled 2-oxoquinoline derivatives and the preliminary in vitro and ex vivo evaluation of one compound as a CB(2)-specific radioligand. METHODS: 4-[(18)F]fluorobenzyl amine [(18)F]-3 was prepared by radiofluorination of 4-cyano-N,N,N-trimethylanilinium triflate salt followed by reduction with LiAlH(4) and then coupled with acid chlorides 11 and 12 to afford [(18)F]-13 and [(18)F]-14. In vitro CB(2) receptor binding assay was performed using U87 cells transduced with CB(2) and CB(1) receptor. Ex vivo autoradiography was performed with [(18)F]-14 on spleen and on CB(2)- and CB(1)-expressing and wild-type U87 subcutaneous tumors grown in mice. RESULTS: The radiochemical yields of [(18)F]-13 and [(18)F]-14 were 10%-15.0% with an average of 12% (n=10); radiochemical purity was >99% with specific activity 1200 mCi/µmol. The dissociation constant Kd for [(18)F]-14 was 3.4 nM. Ex vivo autoradiography showed accumulation of [(18)F]-14 in the CB(2)-expressing tumor. CONCLUSION: Two new [(18)F]-labeled CB(2) ligands have been synthesized. Compound [(18)F]-14 appears to be a potential PET imaging agent for the assessment of CB(2) receptor expression; however, poor solubility restrain its use in vivo.

In vivo small-animal PET/CT of EphB4 receptors using 64Cu-labeled peptide.

UNLABELLED: Many solid tumors overexpress EphB4 receptor, a member of the ephrin receptor tyrosine kinase family. Noninvasive imaging of EphB4 could potentially increase early detection rates, monitor response to therapy directed against EphB4, and improve patient outcomes. The purpose of this study was to evaluate a novel (64)Cu-labeled peptide with high receptor binding affinity for PET of EphB4 receptors. METHODS: The EphB4-binding peptide TNYLFSPNGPIARAW (TNYL-RAW) was conjugated with fluorescein isothiocyanate (FITC) and DOTA. DOTA-TNYL-RAW was labeled with (64)Cu with high labeling efficiency. The binding affinity of TNYL-RAW and its derivatives to purified recombinant EphB4 was determined using surface plasmon resonance technology. In vitro binding of both FITC-TNYL-RAW and (64)Cu-DOTA-TNYL-RAW to cancer cells was assessed by fluorescent microscopy and a radioactivity count method. In vivo biodistribution and small-animal PET/CT were performed in mice bearing EphB4-expressing CT26 and PC-3M tumors as well as EphB4-negative A549 tumors. RESULTS: TNYL-RAW and its derivatives displayed high binding affinity to EphB4, with equilibrium dissociation constant of 1.98-23 nM. In vitro, both FITC-TNYL-RAW and (64)Cu-DOTA-TNYL-RAW were selectively taken up by CT26 and PC-3M cells but not by A549 cells. Binding of FITC-TNYL-RAW and (64)Cu-DOTA-TNYL-RAW to CT26 and PC-3M cells could be blocked by an excess amount of TNYL-RAW. In vivo, (64)Cu-DOTA-TNYL-RAW showed significantly higher uptake in PC-3M tumors than in A549 tumors, with percentages of injected dose per gram of tumor of 0.84 ± 0.09 and 0.44 ± 0.09 at 24 h after radiotracer injection, respectively. Small-animal PET/CT clearly revealed deposition of (64)Cu-DOTA-TNYL-RAW in CT26 and PC-3M tumors but not in A549 tumors. Furthermore, uptake of (64)Cu-DOTA-TNYL-RAW in both CT26 and PC-3M tumors could be blocked by cold TNYL-RAW. CONCLUSION: The expression of EphB4 receptors can be noninvasively interrogated by small-animal PET/CT using (64)Cu-DOTA-TNYL-RAW.

Small-animal PET of tumor damage induced by photothermal ablation with 64Cu-bis-DOTA-hypericin.

UNLABELLED: The purpose of this study was to investigate the potential application of small-molecular-weight (64)Cu-labeled bis-DOTA-hypericin in the noninvasive assessment of response to photothermal ablation therapy. METHODS: Bis-DOTA-hypericin was labeled with (64)Cu with high efficiency (>95% without purification). Nine mice bearing subcutaneous human mammary BT474 tumors were used. Five mice were injected intratumorally with semiconductor CuS nanoparticles, followed by near-infrared laser irradiation 24 h later (12 W/cm(2) for 3 min), and 4 mice were not treated (control group). All mice were intravenously injected with (64)Cu-bis-DOTA-hypericin (24 h after laser treatment in treated mice). Small-animal PET images were acquired at 2, 6, and 24 h after radiotracer injection. All mice were killed immediately after the imaging session for biodistribution and histology study. In vitro cell uptake and surface plasmon resonance studies were performed to validate the small-animal PET results. RESULTS: (64)Cu-bis-DOTA-hypericin uptake was significantly higher in the treatment group than in the control group. The percentage injected dose per gram of tissue in the treated and control groups was 1.72 ± 0.43 and 0.76 ± 0.19, respectively (P = 0.017), at 24 h after injection. Autoradiography and histology results were consistent with selective uptake of the radiotracer in the necrotic zone of the tumor induced by photothermal ablation therapy. In vitro results showed that treated BT474 cells had a higher uptake of (64)Cu-bis-DOTA-hypericin than nontreated cells. Surface plasmon resonance study showed that bis-DOTA-hypericin had higher binding affinity to phosphatidylserine and phosphatidylethanolamine than to phosphatidylcholine. CONCLUSION: (64)Cu-bis-DOTA-hypericin has a potential to image thermal therapy-induced tumor cell damage. The affinity of (64)Cu-bis-DOTA-hypericin for injured tissues may be attributed to the breakdown of the cell membrane and exposure of phosphatidylserine or phosphatidylethanolamine to the radiotracer, which binds selectively to these phospholipids.