An Intra-Individual Comparison of MRI, [18F]-FET and [18F]-FLT PET in Patients with High-Grade Gliomas

Objectives

Intra-individual spatial overlap analysis of tumor volumes assessed by MRI, the amino acid PET tracer [¹⁸F]-FET and the nucleoside PET tracer [¹⁸F]-FLT in high-grade gliomas (HGG).

Methods

MRI, [¹⁸F]-FET and [¹⁸F]-FLT PET data sets were retrospectively analyzed in 23 HGG patients. Morphologic tumor volumes on MRI (post-contrast T1 (cT1) and T2 images) were calculated using a semi-automatic image segmentation method. Metabolic tumor volumes for [¹⁸F]-FET and [¹⁸F]-FLT PETs were determined by image segmentation using a threshold-based volume of interest analysis. After co-registration with MRI the morphologic and metabolic tumor volumes were compared on an intra-individual basis in order to estimate spatial overlaps using the Spearman''s rank correlation coefficient and the Mann-Whitney U test.

Results

[¹⁸F]-FLT uptake was negative in tumors with no or only moderate contrast enhancement on MRI, detecting only 21 of 23 (91%) HGG. In addition, [¹⁸F]-FLT uptake was mainly restricted to cT1 tumor areas on MRI and [¹⁸F]-FLT volumes strongly correlated with cT1 volumes (r = 0.841, p<0.001). In contrast, [¹⁸F]-FET PET detected 22 of 23 (96%) HGG. [¹⁸F]-FET uptake beyond areas of cT1 was found in 61% of cases and [¹⁸F]-FET volumes showed only a moderate correlation with cT1 volumes (r = 0.573, p<0.001). Metabolic tumor volumes beyond cT1 tumor areas were significantly larger for [¹⁸F]-FET compared to [¹⁸F]-FLT tracer uptake (8.3 vs. 2.7 cm³, p<0.001).

Conclusion

In HGG [¹⁸F]-FET but not [¹⁸F]-FLT PET was able to detect metabolic active tumor tissue beyond contrast enhancing tumor on MRI. In contrast to [¹⁸F]-FET, blood-brain barrier breakdown seems to be a prerequisite for [¹⁸F]-FLT tracer uptake.     

3′-Deoxy-3′-[18F]-Fluorothymidine PET Imaging Reflects PI3K-mTOR-Mediated Pro-Survival Response to Targeted Therapy in Colorectal Cancer

Biomarkers that predict response to targeted therapy in oncology are an essential component of personalized medicine. In preclinical treatment response studies that featured models of wild-type KRAS or mutant BRAF colorectal cancer treated with either cetuximab or vemurafenib, respectively, we illustrate that [¹⁸F]-FLT PET, a non-invasive molecular imaging readout of thymidine salvage, closely reflects pro-survival responses to targeted therapy that are mediated by PI3K-mTOR activity. Activation of pro-survival mechanisms forms the basis of numerous modes of resistance. Therefore, we conclude that [¹⁸F]-FLT PET may serve a novel and potentially critical role to predict tumors that exhibit molecular features that tend to reflect recalcitrance to MAPK-targeted therapy. Though these studies focused on colorectal cancer, we envision that the results may be applicable to other solid tumors as well.     

PI3K Inhibition Enhances Doxorubicin-Induced Apoptosis in Sarcoma Cells

We searched for a drug capable of sensitization of sarcoma cells to doxorubicin (DOX). We report that the dual PI3K/mTOR inhibitor PI103 enhances the efficacy of DOX in several sarcoma cell lines and interacts with DOX in the induction of apoptosis. PI103 decreased the expression of MDR1 and MRP1, which resulted in DOX accumulation. However, the enhancement of DOX-induced apoptosis was unrelated to DOX accumulation. Neither did it involve inhibition of mTOR. Instead, the combination treatment of DOX plus PI103 activated Bax, the mitochondrial apoptosis pathway, and caspase 3. Caspase 3 activation was also observed in xenografts of sarcoma cells in nude mice upon combination of DOX with the specific PI3K inhibitor GDC-0941. Although the increase in apoptosis did not further impact on tumor growth when compared to the efficient growth inhibition by GDC-0941 alone, these findings suggest that inhibition of PI3K may improve DOX-induced proapoptotic effects in sarcoma. Taken together with similar recent studies of neuroblastoma- and glioblastoma-derived cells, PI3K inhibition seems to be a more general option to sensitize tumor cells to anthracyclines.     

Limits of [18F]-FLT PET as a Biomarker of Proliferation in Oncology

Background

Non-invasive imaging biomarkers of cellular proliferation hold great promise for quantifying response to personalized medicine in oncology. An emerging approach to assess tumor proliferation utilizes the positron emission tomography (PET) tracer 3’-deoxy-3’[¹⁸F]-fluorothymidine, [¹⁸F]-FLT. Though several studies have associated serial changes in [¹⁸F]-FLT-PET with elements of therapeutic response, the degree to which [¹⁸F]-FLT-PET quantitatively reflects proliferative index has been continuously debated for more that a decade. The goal of this study was to elucidate quantitative relationships between [¹⁸F]-FLT-PET and cellular metrics of proliferation in treatment naïve human cell line xenografts commonly employed in cancer research.

Methods and Findings

[¹⁸F]-FLT-PET was conducted in human cancer xenograft-bearing mice. Quantitative relationships between PET, thymidine kinase 1 (TK1) protein levels and immunostaining for proliferation markers (Ki67, TK1, PCNA) were evaluated using imaging-matched tumor specimens. Overall, we determined that [¹⁸F]-FLT-PET reflects TK1 protein levels, yet the cell cycle specificity of TK1 expression and the extent to which tumors utilize thymidine salvage for DNA synthesis decouple [¹⁸F]-FLT-PET data from standard estimates of proliferative index.

Conclusions

Our findings illustrate that [¹⁸F]-FLT-PET reflects tumor proliferation as a function of thymidine salvage pathway utilization. Unlike more general proliferation markers, such as Ki67, [¹⁸F]-FLT PET reflects proliferative indices to variable and potentially unreliable extents. [¹⁸F]-FLT-PET cannot discriminate moderately proliferative, thymidine salvage-driven tumors from those of high proliferative index that rely primarily upon de novo thymidine synthesis. Accordingly, the magnitude of [¹⁸F]-FLT uptake should not be considered a surrogate of proliferative index. These data rationalize the diversity of [¹⁸F]-FLT-PET correlative results previously reported and suggest future best-practices when [¹⁸F]-FLT-PET is employed in oncology.     

Synergistic Effects of Concurrent Blockade of PI3K and MEK Pathways in Pancreatic Cancer Preclinical Models

Patients with pancreatic cancer have dismal prognoses, and novel therapies are urgently needed. Mutations of the KRAS oncogene occur frequently in pancreatic cancer and represent an attractive target. Direct targeting of the predominant KRAS pathways have been challenging and research into therapeutic strategies have been now refocused on pathways downstream of KRAS, phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK [MEK]). We hypothesized that concurrent inhibition of the PI3K and MEK pathways would result in synergistic antitumor activity, as it would circumvent the compensatory feedback loop between the two pathways. We investigated the combined effect of the PI3K inhibitor, GDC0941, and the MEK inhibitor, AZD6244, on cell viability, apoptosis and cell signaling in a panel of pancreatic cancer cell lines. An in vivo analysis was conducted on pancreatic cancer xenografts. While BxPC-3 (KRAS wild type) and MIA PaCa-2 (KRAS mutated) cell lines were sensitive to GDC0941 and AZD6244 as single agents, synergistic inhibition of tumor cell growth and induction of apoptosis were observed in both cell lines when the two drugs were combined. Interestingly, phosphorylation of the cap-dependent translational components, 4E-binding protein (p-4E-BP1) and S6 was found to be closely associated with sensitivity to GDC0941 and AZD6244. In BxPC-3 cell xenografts, survival differences were observed between the control and the AZD6244, GDC0941, and combination groups. Our study provides the rationale for concurrent targeting of the PI3K and MEK pathways, regardless of KRAS status, and suggests that phosphorylation of 4E-BP1and S6 can serve as a predictive biomarker for response to treatment.     

Discovery of thiazolobenzoxepin PI3-kinase inhibitors that spare the PI3-kinase β isoform

A series of suitable five-membered heterocyclic alternatives to thiophenes within a thienobenzoxepin class of PI3-kinase (PI3K) inhibitors was discovered. Specific thiazolobenzoxepin 8-substitution was identified that increased selectivity over PI3Kβ. PI3Kβ-sparing compound 27 (PI3Kβ K_i,app/PI3Kα K_i,app = 57) demonstrated dose-dependent knockdown of pAKT, pPRAS40 and pS6RP in vivo as well as differential effects in an in vitro proliferation cell line screen compared to pan PI3K inhibitor GDC-0941. A new structure-based hypothesis for reducing inhibition of the PI3K β isoform while maintaining activity against α, δ and γ isoforms is presented.     

Lactate and Choline Metabolites Detected In Vitro by Nuclear Magnetic Resonance Spectroscopy Are Potential Metabolic Biomarkers for PI3K Inhibition in Pediatric Glioblastoma

The phosphoinositide 3-kinase (PI3K) pathway is believed to be of key importance in pediatric glioblastoma. Novel inhibitors of the PI3K pathway are being developed and are entering clinical trials. Our aim is to identify potential non-invasive biomarkers of PI3K signaling pathway inhibition in pediatric glioblastoma using in vitro nuclear magnetic resonance (NMR) spectroscopy, to aid identification of target inhibition and therapeutic response in early phase clinical trials of PI3K inhibitors in childhood cancer. Treatment of SF188 and KNS42 human pediatric glioblastoma cell lines with the dual pan-Class I PI3K/mTOR inhibitor PI-103, inhibited the PI3K signaling pathway and resulted in a decrease in phosphocholine (PC), total choline (tCho) and lactate levels (p<0.02) as detected by phosphorus (³¹P)- and proton (¹H)-NMR. Similar changes were also detected using the pan–Class I PI3K inhibitor GDC-0941 which lacks significant mTOR activity and is entering Phase II clinical trials. In contrast, the DNA damaging agent temozolomide (TMZ), which is used as current frontline therapy in the treatment of glioblastoma postoperatively (in combination with radiotherapy), increased PC, glycerophosphocholine (GPC) and tCho levels (p<0.04). PI-103-induced NMR changes were associated with alterations in protein expression levels of regulatory enzymes involved in glucose and choline metabolism including GLUT1, HK2, LDHA and CHKA. Our results show that by using NMR we can detect distinct biomarkers following PI3K pathway inhibition compared to treatment with the DNA-damaging anti-cancer agent TMZ. This is the first study reporting that lactate and choline metabolites are potential non-invasive biomarkers for monitoring response to PI3K pathway inhibitors in pediatric glioblastoma.     

The Mutant KRAS Gene Up-regulates BCL-XL Protein via STAT3 to Confer Apoptosis Resistance That Is Reversed by BIM Protein Induction and BCL-XL Antagonism

In colorectal cancers with oncogenic GTPase Kras (KRAS) mutations, inhibition of downstream MEK/ERK signaling has shown limited efficacy, in part because of failure to induce a robust apoptotic response. We studied the mechanism of apoptosis resistance in mutant KRAS cells and sought to enhance the efficacy of a KRAS-specific MEK/ERK inhibitor, GDC-0623. GDC-0623 was shown to potently up-regulate BIM expression to a greater extent versus other MEK inhibitors in isogenic KRAS HCT116 and mutant KRAS SW620 colon cancer cells. ERK silencing enhanced BIM up-regulation by GDC-0623 that was due to its loss of phosphorylation at Ser⁶⁹, confirmed by a BIM-EL phosphorylation-defective mutant (S69G) that increased protein stability and blocked BIM induction. Despite BIM and BIK induction, the isogenic KRAS mutant versus wild-type cells remained resistant to GDC-0623-induced apoptosis, in part because of up-regulation of BCL-XL. KRAS knockdown by a doxycycline-inducible shRNA attenuated BCL-XL expression. BCL-XL knockdown sensitized KRAS mutant cells to GDC-0623-mediated apoptosis, as did the BH3 mimetic ABT-263. GDC-0623 plus ABT-263 induced a synergistic apoptosis by a mechanism that includes release of BIM from its sequestration by BCL-XL. Furthermore, mutant KRAS activated p-STAT3 (Tyr⁷⁰⁵) in the absence of IL-6 secretion, and STAT3 knockdown reduced BCL-XL mRNA and protein expression. These data suggest that BCL-XL up-regulation by STAT3 contributes to mutant KRAS-mediated apoptosis resistance. Such resistance can be overcome by potent BIM induction and concurrent BCL-XL antagonism to enable a synergistic apoptotic response.     

Development of a Novel PET Tracer [18F]AlF-NOTA-C6 Targeting MMP2 for Tumor Imaging

Background and Objective

The overexpression of gelatinases, that is, matrix metalloproteinase MMP2 and MMP9, has been associated with tumor progression, invasion, and metastasis. To image MMP2 in tumors, we developed a novel ligand termed [¹⁸F]AlF-NOTA-C6, with consideration that: c(KAHWGFTLD)NH₂ (herein, C6) is a selective gelatinase inhibitor; Cy5.5-C6 has been visualized in many in vivo tumor models; positron emission tomography (PET) has a higher detection sensitivity and a wider field of view than optical imaging; fluorine-18 (¹⁸F) is the optimal PET radioisotope, and the creation of a [¹⁸F]AlF-peptide complex is a simple procedure.

Methods

C6 was conjugated to the bifunctional chelator NOTA (1, 4, 7-triazacyclononanetriacetic acid) for radiolabeling [¹⁸F]AlF conjugation. The MMP2-binding characteristics and tumor-targeting efficacy of [¹⁸F]AlF-NOTA-C6 were tested in vitro and in vivo.

Results

The non-decay corrected yield of [¹⁸F]AlF-NOTA-C6 was 46.2–64.2%, and the radiochemical purity exceeded 95%. [¹⁸F]AlF-NOTA-C6 was favorably retained in SKOV3 and PC3 cells, determined by cell uptake. Using NOTA-C6 as a competitive ligand, the uptake of [¹⁸F]AlF-NOTA-C6 in SKOV3 cells decreased in a dose-dependent manner. In biodistribution and PET imaging studies, higher radioactivity concentrations were observed in tumors. Pre-injection of C6 caused a marked reduction in tumor tissue uptake. Immunohistochemistry showed MMP2 in tumor tissues.

Conclusions

[¹⁸F]AlF-NOTA-C6 was easy to synthesize and has substantial potential as an imaging agent that targets MMP2 in tumors.     

Synthesis and preclinical evaluation of [18F]FSL25.1188, a reversible PET radioligand for monoamine oxidase-B

Carbon-11 labeled SL25.1188 is a promising reversible monoamine oxidase-B (MAO-B) radioligand that was recently translated for human positron emission tomography (PET) imaging. Herein, we report the development of a novel fluorinated derivative, namely, [¹⁸F](S)-3-(6-(3-fluoropropoxy)benzo[d]isoxazol-3-yl)-5-(methoxymethyl)oxazolidin-2-one ([¹⁸F]FSL25.1188; [¹⁸F]6), as a candidate ¹⁸F-labeled MAO-B radioligand, and, its subsequent preclinical evaluation in non-human primates (NHP). [¹⁸F]6 was produced and isolated (>6 GBq) with high radiochemical purity (>99%), and molar activity (>100 GBq/µmol at time of injection). Autoradiography studies conducted in post-mortem human brain sections revealed [¹⁸F]6 binding in MAO-B rich regions. PET imaging study of [¹⁸F]6 in NHP showed high brain uptake (SUV > 2.5) as well as a regional brain radioactivity distribution in accordance with MAO-B expression. [¹⁸F]6 displayed favorable in vivo kinetics, with an early peak in the time-activity curve followed by progressive wash-out from the NHP brain. Specificity of [¹⁸F]6 was investigated in a pre-treatment study with l-deprenyl (1.0 mg/kg) wherein reduced radioligand uptake was observed in all MAO-B rich regions. Results from the current preclinical investigation suggests [¹⁸F]6 is a promising MAO-B PET radioligand. Further evaluation of [¹⁸F]6 and structurally related ¹⁸F-analogs are underway to identify an optimized candidate for clinical research studies.     

Dual Pharmacological Targeting of the MAP Kinase and PI3K/mTOR Pathway in Preclinical Models of Colorectal Cancer

Background

The activation of the MAPK and PI3K/AKT/mTOR pathways is implicated in the majority of cancers. Activating mutations in both of these pathways has been described in colorectal cancer (CRC), thus indicating their potential as therapeutic targets. This study evaluated the combination of a PI3K/mTOR inhibitor (PF-04691502/PF-502) in combination with a MEK inhibitor (PD-0325901/PD-901) in CRC cell lines and patient-derived CRC tumor xenograft models (PDTX).

Materials and Methods

The anti-proliferative effects of PF-502 and PD-901 were assessed as single agents and in combination against a panel of CRC cell lines with various molecular backgrounds. Synergy was evaluated using the Bliss Additivity method. In selected cell lines, we investigated the combination effects on downstream effectors by immunoblotting. The combination was then evaluated in several fully genetically annotated CRC PDTX models.

Results

The in vitro experiments demonstrated a wide range of IC₅₀ values for both agents against a cell line panel. The combination of PF-502 and PD-901 demonstrated synergistic anti-proliferative activity with Bliss values in the additive range. As expected, p-AKT and p-ERK were downregulated by PF-502 and PD-901, respectively. In PDTX models, following a 30-day exposure to PF-502, PD-901 or the combination, the combination demonstrated enhanced reduction in tumor growth as compared to either single agent regardless of KRAS or PI3K mutational status.

Conclusions

The combination of a PI3K/mTOR and a MEK inhibitor demonstrated enhanced anti-proliferative effects against CRC cell lines and PDTX models.     

Autophagy Correlates with the Therapeutic Responsiveness of Malignant Pleural Mesothelioma in 3D Models

Malignant pleural mesothelioma is a highly chemoresistant solid tumor. We have studied this apoptotic resistance using in vitro and ex vivo three-dimensional models, which acquire a high level of chemoresistance that can be reduced by PI3K/mTOR inhibitors. Here, we investigate the activity of GDC-0980, a novel dual PI3K/mTOR inhibitor, which has been proposed to be effective in mesothelioma. In this work, we aimed to identify mechanisms and markers of efficacy for GDC-0980 by utilizing 3D models of mesothelioma, both in vitro multicellular spheroids and ex vivo tumor fragment spheroids grown from patient tumor samples. We found that a subset of mesothelioma spheroids is sensitive to GDC-0980 alone and to its combination with chemotherapy. Unexpectedly, this sensitivity did not correlate with the activation of the Akt/mTOR pathway. Instead, sensitivity to GDC-0980 correlated with the presence of constitutive ATG13 puncta, a feature of autophagy, a cellular program that supports cells under stress. In tumor fragment spheroids grown from 21 tumors, we also found a subset (n = 11) that was sensitive to GDC-0980, a sensitivity that also correlated with the presence of ATG13 puncta. Interference with autophagy by siRNA of ATG7, an essential autophagic protein, increased the response to chemotherapy, but only in the sensitive multicellular spheroids. In the spheroids resistant to GDC-0980, autophagy appeared to play no role. In summary, we show that GDC-0980 is effective in mesothelioma 3D models that display ATG13 puncta, and that blockade of autophagy increases their response to chemotherapy. For the first time, we show a role for autophagy in the response to chemotherapy of 3D models of mesothelioma and propose ATG13 as a potential biomarker of the therapeutic responsiveness of mesothelioma.     

N-[18F]-Fluoroacetylcrizotinib: A potentially potent and selective PET tracer for molecular imaging of non-small cell lung cancer

N-[¹⁸F]fluoroacetylcrizotinib, a fluorine-18 labeled derivative of the first FDA approved tyrosine kinase inhibitor (TKI) for the treatment of Anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC), crizotinib, was successfully synthesized for use in positron emission tomography (PET). Sequential in vitro biological evaluation of fluoracetylcrizotinib and in vivo biodistribution studies of [¹⁸F]fluoroacetylcrizotinib demonstrated that the biological activity of the parent compound remained unchanged, with potent ALK kinase inhibition and effective tumor growth inhibition. These results show that [¹⁸F]fluoroacetylcrizotinib has the potential to be a promising PET ligand for use in NSCLC imaging. The utility of PET in this context provides a non-invasive, quantifiable method to inform on the pharmacokinetics of an ALK-inhibitor such as crizotinib prior to a clinical trial, as well as during a trial in the event of acquired drug resistance.     

Genotype Directed Therapy in Murine Mismatch Repair Deficient Tumors

The PI3K/AKT/mTOR pathway has frequently been found activated in human tumors. We show that in addition to Wnt signaling dysfunction, the PI3K/AKT/mTOR pathway is often upregulated in mouse Msh2^−/− initiated intestinal tumors. NVP-BEZ235 is a dual PI3K/mTOR inhibitor toxic to many cancer cell lines and currently involved in clinical trials. We have treated two mouse models involving Msh2 that develop small intestinal and/or colonic tumors with NVP-BEZ235, and a subset of animals with NVP-BEZ235 and MEK inhibitor ADZ4266. The disease phenotype has been followed with pathology, ¹⁸F FDG PET imaging, and endoscopy. Intestinal adenocarcinomas are significantly decreased in multiplicity by both drug regimens. The majority of tumors treated with combined therapy regress significantly, while a small number of highly progressed tumors persist. We have examined PTEN, AKT, MEK 1&2, MAPK, S6K, mTOR, PDPK1, and Cyclin D1 and find variable alterations that include downregulation of PTEN, upregulation of AKT and changes in its phosphorylated forms, upregulation of pMEK 1&2, p42p44MAPK, pS6K, and Cyclin D1. Apoptosis has been found intact in some tumors and not in others. Our data indicate that NVP-BEZ235 alone and in combination with ADZ4266 are effective in treating a proportion of colorectal cancers, but that highly progressed resistant tumors grow in the presence of the drugs. Pathways upregulated in some resistant tumors also include PDPK1, suggesting that metabolic inhibitors may also be useful in treating these tumors.     

Radiosynthesis and evaluation of N-(3-[18F]fluoropropyl)paroxetine as a radiotracer for in vivo labeling of serotonin uptake sites by PET

To visualize serotonin uptake sites by positron emission tomography (PET), N-(3-[¹⁸F]fluoropropyl)-paroxetine ([¹⁸F]FPP) (Fig. 1), a derivative of the selective serotonin uptake blocker paroxetine, was synthesized from 3-[¹⁸F]fluoropropyltosylate and paroxetine via a one-pot procedure. The rate of formation of [¹⁸F]FPP was a function of the ratio of the initial amount of paroxetine to that of 1,3-propanediol bistosylate with which [¹⁸F]fluoropropyltosylate was synthesized. When the reaction mixture contained an excess amount of paroxetine over that of the propyl-bistosylate, the radiosynthesis followed by HPLC purification, which took approx. 90 min, gave [¹⁸F]FPP in a radiochemical yield of approx. 8%, and in high radiochemical and chemical purity. The specific activity was 2640 ± 360 mCi/μmol.The brain biodistribution of [¹⁸F]FPP showed no distinguishable localization in regions with high density of serotonin uptake sites such as hypothalamus or olfactory tubercles. In vitro binding assays revealed that N-fluoropropylation of paroxetine reduced the affinity for the serotonin uptake site by three orders of magnitude.     

Imatinib analogs as potential agents for PET imaging of Bcr-Abl and c-KIT expression at a kinase level

We synthesized two series of imatinib mesylate (STI-571) analogs to develop a Bcr-Abl and c-KIT receptor-specific labeling agent for positron emission tomography (PET) imaging to measure Bcr-Abl and c-KIT expression levels in a mouse model. The methods of molecular modeling, synthesis of STI-571 and its analogs, in vitro kinase assays, and radiolabeling are described. Molecular modeling revealed that these analogs bind the same Bcr-Abl and c-KIT binding sites as those bound by STI-571. The analogs potently inhibit the tyrosine kinase activity of Bcr-Abl and c-KIT, similarly to STI-571. [¹⁸F]-labeled STI-571 was prepared with high specific activity (75 GBq/μmol) by nucleophilic displacement and an average radiochemical yield of 12%. [¹³¹I]-labeled STI-571 was prepared with high purity (>95%) and an average radiochemical yield of 23%. The uptake rates of [¹⁸F]-STI-571 in K562 cells expressing Abl and in U87WT cells overexpressing c-KIT were significantly higher than those in the U87 cell and could be inhibited by STI-71 (confirming the specificity of uptake). PET scans of K562 and U87WT tumor-bearing mice with [¹⁸F]-STI-571 as a contrast agent showed visible tumor uptake and tumor-to-non-target contrast.     

Radiosynthesis,biological evaluation and preliminary microPET study of 18F-labeled 5-resorcinolic triazolone derivative based on ganetespib targeting HSP90

Heat-shock protein 90 (HSP90) is a molecular chaperone that activates oncogenic transformation in several solid tumors, including lung and breast cancers. Ganetespib, a most promising candidate among several HSP90 inhibitors under clinical trials, has entered Phase III clinical trials for cancer therapy. Despite numerous evidences validating HSP90 as a target of anticancer, there are few studies on PET agents targeting oncogenic HSP90. In this study, we synthesized and biologically evaluated a novel ¹⁸F-labeled 5-resorcinolic triazolone derivative (1, [¹⁸F]PTP-Ganetespib) based on ganetespib. [¹⁸F]PTP-Ganetespib was labeled by click chemistry of Ganetespib-PEG-Alkyne (10) and [¹⁸F]PEG-N₃ (11) with 37.3?±?5.11% of radiochemical yield and 99.7?±?0.09% of radiochemical purity. [¹⁸F]PTP-Ganetespib showed proper LogP (0.96?±?0.06) and good stability in human serum over 97% for 2?h. [¹⁸F]PTP-Ganetespib showed high uptakes in breast cancer cells containing triple negative breast cancer (TNBC) MDA-MB-231 and Her2-negative MCF-7 cells, which are target breast cancer cell lines of HSP90 inhibitor, ganetespib, as an anticancer. Blocking of HSP90 by the pretreatment of ganetespib exhibited significantly decreased accumulation of [¹⁸F]PTP-Ganetespib in MDA-MB-231 and MCF-7 cells, indicating the specific binding of [¹⁸F]PTP-Ganetespib to MDA-MB-231 and MCF-7 cells with high HSP90 expression. In the biodistribution and microPET imaging studies, the initial uptake into tumor was weaker than in other thoracic and abdominal organs, but [¹⁸F]PTP-Ganetespib was retained relatively longer in the tumor than other organs. The uptake of [¹⁸F]PTP-Ganetespib in tumors was not sufficient for further development as a tumor-specific PET imaging agent by itself, but this preliminary PET imaging study of [¹⁸F]PTP-Ganetespib can be basis for developing new PET imaging agents based on HSP90 inhibitor, ganetespib.     

Early Detection of Erlotinib Treatment Response in NSCLC by 3′-Deoxy-3′-[18F]-Fluoro-L-Thymidine ([18F]FLT) Positron Emission Tomography (PET)

Background

Inhibition of the epidermal growth factor receptor (EGFR) has shown clinical success in patients with advanced non-small cell lung cancer (NSCLC). Somatic mutations of EGFR were found in lung adenocarcinoma that lead to exquisite dependency on EGFR signaling; thus patients with EGFR-mutant tumors are at high chance of response to EGFR inhibitors. However, imaging approaches affording early identification of tumor response in EGFR-dependent carcinomas have so far been lacking.

Methodology/Principal Findings

We performed a systematic comparison of 3′-Deoxy-3′-[¹⁸F]-fluoro-L-thymidine ([¹⁸F]FLT) and 2-[¹⁸F]-fluoro-2-deoxy-D-glucose ([¹⁸F]FDG) positron emission tomography (PET) for their potential to identify response to EGFR inhibitors in a model of EGFR-dependent lung cancer early after treatment initiation. While erlotinib-sensitive tumors exhibited a striking and reproducible decrease in [¹⁸F]FLT uptake after only two days of treatment, [¹⁸F]FDG PET based imaging revealed no consistent reduction in tumor glucose uptake. In sensitive tumors, a decrease in [¹⁸F]FLT PET but not [¹⁸F]FDG PET uptake correlated with cell cycle arrest and induction of apoptosis. The reduction in [¹⁸F]FLT PET signal at day 2 translated into dramatic tumor shrinkage four days later. Furthermore, the specificity of our results is confirmed by the complete lack of [¹⁸F]FLT PET response of tumors expressing the T790M erlotinib resistance mutation of EGFR.

Conclusions

[¹⁸F]FLT PET enables robust identification of erlotinib response in EGFR-dependent tumors at a very early stage. [¹⁸F]FLT PET imaging may represent an appropriate method for early prediction of response to EGFR TKI treatment in patients with NSCLC.     

Synthesis,radiolabeling and preliminary in vivo evaluation of [18F]FE-PE2I,a new probe for the dopamine transporter

A new dopamine transporter (DAT) ligand, (E)-N-(3-iodoprop-2-enyl)-2β-carbofluoroethoxy-3β-(4′-methyl-phenyl) nortropane (FE-PE2I, 6), derived from PE2I (1), was prepared and found to be a potent inhibitor of rodent DAT in vitro. Compound 6 was radiolabelled with fluorine-18 (t_1/2 = 109.8 min) for PET studies in monkeys. In vivo PET measurements showed a regional distribution in brain that corresponds to the known distribution of DAT. This binding was specific, reversible and the kinetics of [¹⁸F]6 binding in brain were faster than for its lead compound, [¹¹C]1. The possible presence of a hydroxymethyl-radiometabolite formed by oxidation in the 3β-benzylic position of [¹⁸F]6 warrants further detailed evaluation of the metabolism of [¹⁸F]6. [¹⁸F]6 is a potential radioligand for imaging DATs in the human brain with PET.