Molecular Imaging of Sirtuin1 Expression-Activity in Rat Brain Using Positron-Emission Tomography-Magnetic-Resonance Imaging with [18F]-2-Fluorobenzoylaminohexanoicanilide.

Sirtuin 1 (SIRT1) is a class III histone deacetylase that plays significant roles in the regulation of lifespan, metabolism, memory, and circadian rhythms and in the mechanisms of many diseases. However, methods of monitoring the pharmacodynamics of SIRT1-targeted drugs are limited to blood sampling because of the invasive nature of biopsies. For the noninvasive monitoring of the spatial and temporal dynamics of SIRT1 expression-activity in vivo by PET-CT-MRI, we developed a novel substrate-type radiotracer, [18F]-2-fluorobenzoylaminohexanoicanilide (2-[18F]BzAHA). PET-CT-MRI studies in rats demonstrated increased accumulation of 2-[18F]BzAHA-derived radioactivity in the hypothalamus, hippocampus, nucleus accumbens, and locus coeruleus, consistent with autoradiographic and immunofluorescent (IMF) analyses of brain-tissue sections. Pretreatment with the SIRT1 specific inhibitor, EX-527 (5 mg/kg, ip), resulted in about a 20% reduction of 2-[18F]BzAHA-derived-radioactivity accumulation in these structures. In vivo imaging of SIRT1 expression-activity should facilitate studies that improve the understanding of SIRT1-mediated regulation in the brain and aid in the development and clinical translation of SIRT1-targeted therapies.

A Novel Substrate Radiotracer for Molecular Imaging of SIRT2 Expression and Activity with Positron Emission Tomography.

PURPOSE: The purpose of this study was to develop a SIRT2-specific substrate-type radiotracer for non-invasive PET imaging of epigenetic regulatory processes mediated by SIRT2 in normal and disease tissues. PROCEDURES: A library of compounds containing tert-butyloxycarbonyl-lysine-aminomethylcoumarin backbone was derivatized with fluoroalkyl chains 3-16 carbons in length. SIRT2 most efficiently cleaved the myristoyl, followed by 12-fluorododecanoic and 10-fluorodecanoic groups (Kcat/Km 716.5 ± 72.8, 615.4 ± 50.5, 269.5 ± 52.1/s mol, respectively). Radiosynthesis of 12- [18F]fluorododecanoic aminohexanoicanilide (12-[18F]DDAHA) was achieved by nucleophilic radiofluorination of 12-iododecanoic-AHA precursor. RESULTS: A significantly higher accumulation of 12-[18F]DDAHA was observed in MCF-7 and MDA-MB-435 cells in vitro as compared to U87, MiaPaCa, and MCF10A, which was consistent with levels of SIRT2 expression. Initial in vivo studies using 12-[18F]DDAHA conducted in a 9L glioma-bearing rats were discouraging, due to rapid defluorination of this radiotracer upon intravenous administration, as evidenced by significant accumulation of F-18 radioactivity in the skull and other bones, which confounded the interpretation of images of radiotracer accumulation within the tumor and other regions of the brain. CONCLUSIONS: The next generation of SIRT2-specific radiotracers resistant to systemic defluorination should be developed using alternative sites of radiofluorination on the aliphatic chain of DDAHA. A SIRT2-selective radiotracer may provide information about SIRT2 expression and activity in tumors and normal organs and tissues, which may help to better understand the roles of SIRT2 in different diseases.

Novel Histone Deacetylase Class IIa Selective Substrate Radiotracers for PET Imaging of Epigenetic Regulation in the Brain.

Histone deacetylases (HDAC's) became increasingly important targets for therapy of various diseases, resulting in a pressing need to develop HDAC class- and isoform-selective inhibitors. Class IIa deacetylases possess only minimal deacetylase activity against acetylated histones, but have several other client proteins as substrates through which they participate in epigenetic regulation. Herein, we report the radiosyntheses of the second generation of HDAC class IIa-specific radiotracers: 6-(di-fluoroacetamido)-1-hexanoicanilide (DFAHA) and 6-(tri-fluoroacetamido)-1-hexanoicanilide ([18F]-TFAHA). The selectivity of these radiotracer substrates to HDAC class IIa enzymes was assessed in vitro, in a panel of recombinant HDACs, and in vivo using PET/CT imaging in rats. [18F]TFAHA showed significantly higher selectivity for HDAC class IIa enzymes, as compared to [18F]DFAHA and previously reported [18F]FAHA. PET imaging with [18F]TFAHA can be used to visualize and quantify spatial distribution and magnitude of HDAC class IIa expression-activity in different organs and tissues in vivo. Furthermore, PET imaging with [18F]TFAHA may advance the understanding of HDACs class IIa mediated epigenetic regulation of normal and pathophysiological processes, and facilitate the development of novel HDAC class IIa-specific inhibitors for therapy of different diseases.

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).

Radiosynthesis and initial in vitro evaluation of [18F]F-PEG6-IPQA--a novel PET radiotracer for imaging EGFR expression-activity in lung carcinomas.

INTRODUCTION: Epidermal growth factor receptor (EGFR)-targeted therapies with antibodies and small molecular EGFR kinase inhibitors have shown poor efficacy in unselected populations of patients with advanced non-small cell lung carcinomas (NSCLC). In contrast, patients with overexpression of EGFR and activating mutations in EGFR kinase domain demonstrated improved responses to EGFR kinase inhibitors. Therefore, we have developed a novel radiotracer, [(18)F]F-PEG(6)-IPQA for PET imaging of EGFR expression-activity in NSCLC, and have described its radiosynthesis and in vitro evaluation in two NSCLC cell lines with wild-type and L858R active mutant EGFR. METHODS: A mesylate precursor was synthesized in multiple steps and radiofluorinated using K(18)F/Kryptofix. The fluorinated intermediate compound was reduced to an amino derivative then treated with acryloyl isobutyl carbonate, followed by purification by HPLC to obtain the desired product. RESULTS: Decay-corrected radiochemical yields of [(18)F]F-PEG(6)-IPQA were 3.9-17.6%, with an average of 9.0% (n = 11). Radiochemical purity was >97% with specific activity of 34 GBq/µmol (mean value, n = 10) at the end of synthesis. The accumulation of [(18)F]F-PEG(6)-IPQA in H3255 cells was ten-fold higher than in H441 cells, despite a two-fold lower level of activated phospho-EGFR expression in H3255 cells compared with H441 cells. The accumulation of [(18)F]F-PEG(6)-IPQA in both cell lines was significantly decreased in the presence of a small molecular EGFR kinase inhibitor, Iressa, at 100 µM concentration in culture medium. CONCLUSION: We have synthesized [(18)F]F-PEG(6)-IPQA and demonstrated its highly selective accumulation in active mutant L858R EGFR-expressing NSCLC cells in vitro. Further in vivo studies are warranted to assess the ability of PET imaging with [(18)F]F-PEG(6)-IPQA to discriminate the active mutant L858R EGFR-expressing NSCLC that are sensitive to therapy with EGFR kinase inhibitors vs NSCLC that express wild-type EGFR.

Detection of pancreatic carcinomas by imaging lactose-binding protein expression in peritumoral pancreas using [18F]fluoroethyl-deoxylactose PET/CT.

BACKGROUND: Early diagnosis of pancreatic carcinoma with highly sensitive diagnostic imaging methods could save lives of many thousands of patients, because early detection increases resectability and survival rates. Current non-invasive diagnostic imaging techniques have inadequate resolution and sensitivity for detection of small size ( approximately 2-3 mm) early pancreatic carcinoma lesions. Therefore, we have assessed the efficacy of positron emission tomography and computer tomography (PET/CT) imaging with beta-O-D-galactopyranosyl-(1,4')-2'-deoxy-2'-[(18)F]fluoroethyl-D-glucopyranose ([(18)F]FEDL) for detection of less than 3 mm orthotopic xenografts of L3.6pl pancreatic carcinomas in mice. [(18)F]FEDL is a novel radioligand of hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP), which is overexpressed in peritumoral pancreatic acinar cells. METHODOLOGY/PRINCIPAL FINDINGS: Dynamic PET/CT imaging demonstrated rapid accumulation of [(18)F]FEDL in peritumoral pancreatic tissue (4.04+/-2.06%ID/g), bi-exponential blood clearance with half-lives of 1.65+/-0.50 min and 14.14+/-3.60 min, and rapid elimination from other organs and tissues, predominantly by renal clearance. Using model-independent graphical analysis of dynamic PET data, the average distribution volume ratio (DVR) for [(18)F]FEDL in peritumoral pancreatic tissue was estimated as 3.57+/-0.60 and 0.94+/-0.72 in sham-operated control pancreas. Comparative analysis of quantitative autoradiographic images and densitometry of immunohistochemically stained and co-registered adjacent tissue sections demonstrated a strong linear correlation between the magnitude of [(18)F]FEDL binding and HIP/PAP expression in corresponding regions (r = 0.88). The in situ analysis demonstrated that at least a 2-4 fold apparent lesion size amplification was achieved for submillimeter tumors and to nearly half a murine pancreas for tumors larger than 3 mm. CONCLUSION/SIGNIFICANCE: We have demonstrated the feasibility of detection of early pancreatic tumors by non-invasive imaging with [(18)F]FEDL PET/CT of tumor biomarker HIP/PAP over-expressed in peritumoral pancreatic tissue. Non-invasive non-invasive detection of early pancreatic carcinomas with [(18)F]FEDL PET/CT imaging should aid the guidance of biopsies and additional imaging procedures, facilitate the resectability and improve the overall prognosis.

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.