Aleksandr Shavrin

Molecular Imaging of EGFR Kinase Activity in Tumors with 124I-Labeled Small Molecular Tracer and Positron Emission Tomography

Positron emission tomography (PET) with epidermal growth factor receptor (EGFR) kinase-specific radiolabeled tracers could provide the means for noninvasive and repetitive imaging of heterogeneity of EGFR expression and signaling activity in tumors in individual patients before and during therapy with EGFR signaling inhibitors. We developed the synthesis and 124I-radiolabeling of the (E)-But-2-enedioic acid [4-(3-[124I]iodoanilino)-quinazolin-6-yl]-amide-(3-morpholin-4-yl-propyl)-amide (morpholino-[124I]-IPQA), which selectively, irreversibly, and covalently binds the adenosine-triphosphate-binding site to the activated (phosphorylated) EGFR kinase, but not to the inactive EGFR kinase. The latter was demonstrated using in silico modeling with crystal structures of the wild type and different gain-of-function mutants of EGFR kinases. Also, this was demonstrated by selective radiolabeling of the EGFR kinase domain with morpholino-[131I]-IPQA in A431 human epidermoid carcinoma cells and Western blot autoradiography. In vitro radiotracer accumulation and washout studies demonstrated a rapid accumulation and progressive retention postwashout of morpholino-[131I]-IPQA in A431 epidermoid carcinoma and in U87 human glioma cells genetically modified to express the EGFRvIII mutant receptor, but not in the wild-type U87MG glioma cells under serum-starved conditions. Using morpholino-[124I]-IPQA, we obtained noninvasive PET images of EGFR activity in A431 subcutaneous tumor xenografts, but not in subcutaneous tumor xenografts grown from K562 human chronic myeloid leukemia cells in immunocompromised rats and mice. Based on these observations, we suggest that PET imaging with morpholino-[124I]-IPQA should allow for identification of tumors with high EGFR kinase signaling activity, including brain tumors expressing EGFRvIII mutants and nonsmall-cell lung cancer expressing gain-of-function EGFR kinase mutants. Because of significant hepatobiliary clearance and intestinal reuptake of the morpholino-[124I]-IPQA, additional [124I]-IPQA derivatives with improved water solubility may be required to optimize the pharmacokinetics of this class of molecular imaging agents.

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-([18F]fluoroethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}-ethoxy]-quinazoline-6-yl-acrylamide ([18F]F-PEG6-IPQA), a radiotracer with increased selectivity and irreversible binding to the active mutant L858R EGFR kinase. We show that PET with [18F]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 [18F]F-PEG6-IPQA from irreversible binding to EGFR. These results suggest that PET with [18F]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).

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 (∼2–3 mm) early pancreatic carcinoma lesions. Therefore, we have assessed the efficacy of positron emission tomography and computer tomography (PET/CT) imaging with β-O-D-galactopyranosyl-(1,4′)-2′-deoxy-2′-[18F]fluoroethyl-D-glucopyranose ([18F]FEDL) for detection of less than 3 mm orthotopic xenografts of L3.6pl pancreatic carcinomas in mice. [18F]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 [18F]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 [18F]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 [18F]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 [18F]FEDL PET/CT of tumor biomarker HIP/PAP over-expressed in peritumoral pancreatic tissue. Non-invasive non-invasive detection of early pancreatic carcinomas with [18F]FEDL PET/CT imaging should aid the guidance of biopsies and additional imaging procedures, facilitate the resectability and improve the overall prognosis.

Synthesis of N3-substituted thymidine analogues for measurement of cellular kinase activity.

N3-Substitued thymidine analogues that carry a carboranylalkyl moiety at the N3-position with various spacer lengths have been reported to be good substrates for thymidine kinase (TK1). As part of our continuing effort towards the development of new TK1 substrates for imaging tumor proliferative activity, we have synthesized a series of new N3-substituted analogues of thymidine that carry an aromatic ring with different spacer lengths. The overall yields for 6 and 7 were 13% and 39% in four steps and three steps, respectively, and those for 14, 16 and 18 were in the range of 13%-15% in six steps. The overall yield for 24 was 33% in three steps, and those for 25 and 26 were 64% and 58%, respectively, in one step. Most of these compounds have been tested for TK1 activity by enzymatic assay to identify a good substrate that can be radiolabeled for imaging. The phosphorylation rates of these compounds were 2%-6% compared with that of thymidine. The results from the in vitro enzymatic assays suggest that these N3-substituted thymidine analogues have some potential for imaging TK1 activity if radiolabeled with a suitable isotope.

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.

Erratum: Molecular Imaging of EGFR Kinase Activity in Tumors with 124I-Labeled Small Molecular Tracer and Positron Emission Tomography (Mol Imaging Biol (2006) vol. 8 (5) (262-277) 10.1007/s11307-006-0049-0))

Molecular Imaging of EGFR Kinase Activity in Tumors with I-Labeled Small Molecular Tracer and Positron Emission Tomography A. Pal, A. Glekas, M. Doubrovin, J. Balatoni, M. Namavari, T. Beresten, D. Maxwell, S. Soghomonyan, A. Shavrin, L. Ageyeva, R. Finn, S. M. Larson, W. Bornmann, J. G. Gelovani Department of Experimental Diagnostic Imaging, MD Anderson Cancer Center, Unit 057, 1515 Holcombe Blvd., Houston, TX 77030, USA Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA

Molecular imaging of Sirtuin1 expression-activity in the rat brain using Positron Emission Tomography/Magnetic Resonance Imaging (PET/MRI) 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.

Abstract 4210: Non-invasive molecular imaging of SIRT1-mediated epigenetic regulation in cancer using PET/CT with a novel substrate-type radiotracer 2-[18F]PhAHA

SIRT1 is involved in a wide variety of cellular processes and functions and is known to mediate cleavage of the acetyl moiety from the acetylated lysine residues of several proteins, including p53, PPARγ, members of the FOXO family and NF-KB. Therefore, SIRT1 has emerged as an important target for therapy of cancer. However, currently there are no methods for non-invasive monitoring of pharmacodynamics of novel SIRT1 isoform-selective inhibitors in norm and disease. Therefore, there is a need for development of agents for non-invasive imaging of expression and activity of various SIRT1 in vivo. Previous studies demonstrated that SIRT1 can dephenylacetylate a lysine residue at a rate of ∼56% of its natural deacetylation rate, while other SIRT isoforms cannot cleave an aromatic leaving group. This provided an opportunity to develop a SIRT1-selective imaging agent for PET imaging, which would allow for quantitative measurement of the SIRT1 expression-activity product in vivo. We developed a focused library of compounds to elucidate the structure-activity relationship of SIRT1 and to identify the most selective and efficient imaging substrate for SIRT1. This preliminary screening utilized high-throughput fluorogenic assay with the Carbazol-L-Lysine-Aminomethylcoumarin as a backbone [1]. Subsequently, this backbone was exchanged for 6-aminohexanoicanilide, AHA [2] to develop PET radiotracer. The lead compound, 2-fluorophenylaminohexanoicanilide, was developed in both F-19 (2-FPhAHA) and F-18 (2-[18F]PhAHA) versions. 2-[18F]PhAHA was injected i.v. into Sprague-Dawley (SD) rats followed by dynamic imaging using MicroPET R4 (Siemens, TN), followed by CT imaging on INVEON microCT (Siemens, TN). The kinetics of radiotracer uptake was quantified using Logan graphical analysis [3], which demonstrated differential accumulation of 2-[18F]PhAHA-derived radioactivity in specific areas of the rat brain with high expression of SIRT1 (i.e., the dentate gyrus, CA1, nucleus accumbens, and caudate putamen). Following characterization of 2-[18F]PhAHA-derived radioactivity in the normal rat brain, 2-[18F]PhAHA was administered to SD rats bearing intracerebral 9L gliomas. PET/CT demonstrated significantly increased uptake of 2-[18F]PhAHA-derived radioactivity in the 9L tumors vs. normal brain tissue (P In summary, SIRT1 activity is upregulated in intracerebral 9L gliomas, as evidenced by increased accumulation of 2-[18F]PhAHA on PET/CT images, which demonstrates the feasibility of this method for visualization and quantification of SIRT1 activity in brain tumors. Also, 2-[18F]PhAHA on PET/CT should allow for future monitoring of pharmacodynamics of SIRT1 inhibitors during anti-cancer therapy. Citation Format: Robin E. Bonomi, Maxwell Laws, Aleksandr Shavrin, Thomas Mangner, Juri G. Gelovani. Non-invasive molecular imaging of SIRT1-mediated epigenetic regulation in cancer using PET/CT with a novel substrate-type radiotracer 2-[18F]PhAHA. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4210.

Abstract 4234: Elucidating structure-activity relationships of SIRT2 and fluoroalkyl chain length to identify the lead candidate substrate radiotracer for PET imaging

Histone deacetylases (HDACs) play a pivotal role in epigenetic regulation through post translational modifications of histone core and other critical proteins in cellular pathways. The HDACs class III, sirtuins (SIRTs) 1-7, possess limited deacetylase activity. Each of these nicotinamide dinucleotide (NAD+) dependent enzymes have unique structures of catalytic sites that prefer larger lysine-modifying groups as substrates (i.e., glutaryl, succinyl, lipoyl, crotanyl, myristoyl). In particular, SIRT2 is a key regulator of nuclear H4K16 and cytoplasmic alpha-tubulin proteins, thereby acting as a mitotic exit regulator. From the literature, it is clear that SIRT2 plays a pivotal role in the regulation of cancers, so much so that it may switch between tumor suppressor and regulator at different stages of tumor progression. For this reason, SIRT2 has been identified as a possible target for cancer therapy but due to the pleiotropic roles of SIRT2 under normal and pathologic conditions, it is important to assess its activity during pharmacomodulation (i.e. activation or inhibition). Therefore, non-invasive PET imaging using SIRT2-specific substrate-type radiotracers may aid in the development of new SIRT2 inhibitors for therapy of cancer. Herein, we report novel synthesis of a focused library of compounds to assess the structure activity relationship (SAR) of SIRT2 with fluoroalkyl chain length varying between 3 and 16 carbons. The preliminary screening was performed with the tert-butyloxycarbonyl-lysine-aminomethylcoumarin (Boc-lys-AMC) backbone and Fluor-de-lys assay [1] to determine the chain length for optimal SIRT2 catalytic efficiency. Based on the analysis of Michealis-Menten kinetics of each substrate, myristoyl (14-carbon carboxylic acid) was cleaved most efficiently, followed by 12-fluorododecanoic and 10-fluorodecanoic acids (kcat/km = 4E-3, 1E-4, 8.9E-5 s-1uM-1, respectively). For in vivo PET imaging radiotracer development, we linked the 12-fluorododecanoic acid with the aminohexanoicanilide (AHA) backbone [2]. This compound was developed with a terminal bromine (precursor), iodine, fluorine, and F-18 (12-[18F]DDAHA). In vitro cellular uptake studies demonstrated a three-fold increase in accumulation of 12-[18F]DDAHA in U87 glioma cells as compared to MiaPaCa, MDA-MB-231, and MCF10A cell lines. The increased accumulation in the U87 glioma cells indicates that this compound may prove useful for detection of brain gliomas and for monitoring therapies with novel SIRT2 specific inhibitors. This insight into the catalytic activity of SIRT2 provides a basis for the development of a SIRT2-specific PET imaging agent to further probe the role of SIRT2-mediated epigenetic regulation in normal physiology and cancer. Citation Format: Robin E. Bonomi, Aleksandr Shavrin, Vadim Popov, Thomas Mangner, Juri G. Gelovani. Elucidating structure-activity relationships of SIRT2 and fluoroalkyl chain length to identify the lead candidate substrate radiotracer for PET imaging. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4234.