ISRCTN14301360 and UMIN000024988.

Abnormal activation of the Hedgehog (Hh) signaling pathway has been linked to several types of human cancers, and the development of small-molecule inhibitors of this pathway represents a promising route toward novel anticancer therapeutics. A cell-based screen performed in our laboratories identified a new class of Hh pathway inhibitors, 1-amino-4-benzylphthalazines, that act via antagonism of the Smoothened receptor. A variety of analogues were synthesized and their structure-activity relationships determined. This optimization resulted in the discovery of high affinity Smoothened antagonists, one of which was further profiled in vivo. This compound displayed a good pharmacokinetic profile and also afforded tumor regression in a genetic mouse model of medulloblastoma.

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The analysis of 8-oxo-7,8-dihydro-2 0 -deoxyguanosine (8-oxodG) represents an important biomarker of oxidative stress. A sensitive method for the detection of 8-oxodG in DNA samples has been developed that utilizes immunoaffinity column purification of 8-oxodG followed by liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) multiple reaction monitoring (MRM) mode analysis. An internal standard of stable-isotopically labelled 8-oxodG containing [ 15 N 5 ] was added prior to the enzymatic digestion of DNA to deoxynucleosides, which was then subjected to immunoaffinity column purification followed by microbore positive ion LC/MS/MS MRM. The introduction of the immunoaffinity column purification step into the method represents a significant improvement for the accurate determination of 8-oxodG since all artefactual peaks that are observed following the direct injection of digested DNA onto the LC/MS/MS system are removed. The identity of these artefactual peaks has been confirmed to be 2 0 -deoxyguanosine (dG), thymidine (dT) and 2 0 -deoxyadenosine (dA). The presence of these artefactual peaks in MRM mode analysis can be explained as a consequence of a concentration effect due to their considerably higher relative abundance in DNA compared to 8-oxodG. The highest signal intensity was observed for the artefactual peak for dA due to the fact that the adenine base formed an adduct with methanol, which is a constituent of the mobile phase. The resulting [MþH] þ ion at m/z 284 (dA m/z 252 þ CH 3 OH m/z 32) gave rise to a product ion at m/z 168 following the loss of deoxyribose in MRM mode analysis. Control calf thymus DNA was digested to deoxynucleosides and unmodfied deoxynucleosides were removed by immunoaffinity column purification; the enriched 8-oxodG was determined by LC/MS/MS MRM. The level of 8-oxodG in control calf thymus DNA was determined to be 28.8 AE 1.2 8-oxodG per 10 6 unmodified nucleotides (n ¼ 5) using 5 mg of digested DNA. The limit of detection of the microbore LC/MS/MS MRM for 8-oxodG was determined to be 25 fmol on-column with a signal-to-noise ratio of 3.5. Copyright # 2002 John Wiley & Sons, Ltd.The most commonly studied DNA adduct biomarker for oxidative stress is 8-oxo-7,8-dihydro-2 0 -deoxyguanosine (8-oxodG), which is formed by the hydroxylation of the C-8 position of guanine. 1 This adduct can be formed from reactive oxygen species (ROS) such as the superoxide radical (O 2 À. ), singlet oxygen ( 1 O 2 ), hydrogen peroxide (H 2 O 2 ) and the hydroxyl radical ( . OH) that result from oxidative stress, although 1 O 2 and . OH are the only ROS capable of directly damaging DNA. 2,3 ROS are generated endogenously from cellular metabolism and inflammatory responses, or by exposure to exogenous agents such as ionizing radiation and chemicals such as methylene blue. ROS are implicated to have roles in cancer and aging as well as a host of degenerative diseases that involve inflammation. 4,5 The formation of 8-oxodG can lead to chromosomal aberrations and the induction of mutations, wh...

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