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).

Algorithmic guided screening of drug combinations of arbitrary size for activity against cancer cells.

The standard treatment for most advanced cancers is multidrug therapy. Unfortunately, combinations in the clinic often do not perform as predicted. Therefore, to complement identifying rational drug combinations based on biological assumptions, we hypothesized that a functional screen of drug combinations, without limits on combination sizes, will aid the identification of effective drug cocktails. Given the myriad possible cocktails and inspired by examples of search algorithms in diverse fields outside of medicine, we developed a novel, efficient search strategy called Medicinal Algorithmic Combinatorial Screen (MACS). Such algorithms work by enriching for the fitness of cocktails, as defined by specific attributes through successive generations. Because assessment of synergy was not feasible, we developed a novel alternative fitness function based on the level of inhibition and the number of drugs. Using a WST-1 assay on the A549 cell line, through MACS, we screened 72 combinations of arbitrary size formed from a 19-drug pool across four generations. Fenretinide, suberoylanilide hydroxamic acid, and bortezomib (FSB) was the fittest. FSB performed up to 4.18 SD above the mean of a random set of cocktails or "too well" to have been found by chance, supporting the utility of the MACS strategy. Validation studies showed FSB was inhibitory in all 7 other NSCLC cell lines tested. It was also synergistic in A549, the one cell line in which this was evaluated. These results suggest that when guided by MACS, screening larger drug combinations may be feasible as a first step in combination drug discovery in a relatively small number of experiments.

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.

Targeting SRC family kinases inhibits growth and lymph node metastases of prostate cancer in an orthotopic nude mouse model.

Aberrant expression and/or activity of members of the Src family of nonreceptor protein tyrosine kinases (SFK) are commonly observed in progressive stages of human tumors. In prostate cancer, two SFKs (Src and Lyn) have been specifically implicated in tumor growth and progression. However, there are no data in preclinical models demonstrating potential efficacy of Src inhibitors against prostate cancer growth and/or metastasis. In this study, we used the small molecule SFK/Abl kinase inhibitor dasatinib, currently in clinical trials for solid tumors, to examine in vitro and in vivo effects of inhibiting SFKs in prostate tumor cells. In vitro, dasatinib inhibits both Src and Lyn activity, resulting in decreased cellular proliferation, migration, and invasion. In orthotopic nude mouse models, dasatinib treatment effectively inhibits expression of activated SFKs, resulting in inhibition of both tumor growth and development of lymph node metastases in both androgen-sensitive and androgen-resistant tumors. In primary tumors, SFK inhibition leads to decreased cellular proliferation (determined by immunohistochemistry for proliferating cell nuclear antigen). In vitro, small interfering RNA (siRNA)-mediated inhibition of Lyn affects cellular proliferation; siRNA inhibition of Src affects primarily cellular migration. Therefore, we conclude that SFKs are promising therapeutic targets for treatment of human prostate cancer and that Src and Lyn activities affect different cellular functions required for prostate tumor growth and progression.

Evaluation of 2'-deoxy-2'-[18F]fluoro-5-methyl-1-beta-L: -arabinofuranosyluracil ([18F]-L: -FMAU) as a PET imaging agent for cellular proliferation: comparison with [18F]-D: -FMAU and [18F]FLT.

PURPOSE: Clevudine (L: -FMAU) an un-natural analogue of thymidine, is in clinical trials for the treatment of hepatitis B virus (HBV). L: -FMAU is phosphorylated by cellular kinases such as thymidine kinase 1 and deoxycytidine kinase, and its triphosphate form inhibits HBV deoxyribonucleic acid synthesis. Thus, L: -FMAU, radiolabeled with an appropriate isotope, may be useful for positron emission tomography (PET) imaging of tumor proliferation. We evaluated [18F]-L-FMAU as a PET imaging agent in tumor-bearing mice and compared the results with those of two other radiotracers, [18F]-d-FMAU and [18F]-FLT. METHODS: Subcutaneous xenografts of the human lung cancer cell lines H441 and H3255 were established in mice. A micro-PET scanner was used to obtain images of the tumor-bearing animals with [18F]-L-FMAU, [18F]-D-FMAU, and [18F]-FLT. RESULTS: At 2 h postinjection, the tumor uptake (% ID/g) of 18F]-L: -FMAU, 18F]-D: -FMAU, and [18F]-FLT in the faster-growing H441 cells was 3.13 +/- 1.11, 7.74 +/- 1.39, and 5.10 +/- 1.45, respectively. The corresponding values for the slower-growing H3255 cells were 1.38 +/- 0.81, 4.49 +/- 1.08, and 0.57 +/- 0.33. Tumor/muscle ratios of accumulation for [18F]-L: -FMAU, [18F]-D: -FMAU, and [18F]-FLT in H441 cells were 4.15 +/- 1.82, 3.37 +/- 1.19, and 12.94 +/- 4.38, respectively, and the corresponding values in H3255 cells were 1.62 +/- 0.50, 1.96 +/- 0.74, and 1.50 +/- 0.90. CONCLUSIONS: [18F]-L: -FMAU may be a useful agent for imaging tumor proliferation in fast-growing human lung cancers by PET.