In vivo imaging of Bcr-Abl overexpressing tumors with a radiolabeled imatinib analog as an imaging surrogate for imatinib.

UNLABELLED: Imatinib mesylate is a tyrosine kinase inhibitor that was approved by the U.S. Food and Drug Administration in 2001 for treatment of many different stages of chronic myeloid leukemia and in 2002 for treatment of gastrointestinal stromal tumors. Imatinib is known to inhibit the dysregulated proliferation of chronic myeloid leukemia, which is associated with the Bcr-Abl kinase; in gastrointestinal stromal tumors, imatinib is known to act via c-Kit kinase inhibition. The objective of this study was to synthesize an (18)F-labeled analog of imatinib not as a primary imaging agent but rather as a tracer for in vivo drug distribution and tracer concentration that can be used as a PET imaging surrogate for imatinib. METHODS: Molecular modeling studies based on the crystal structure of imatinib bound to the active site of Abl were performed for designing the fluorinated analog. A 2-fluoroethyl analog of imatinib (SKI696) was synthesized using well-established procedures. The selectivity and binding affinity of SKI696 were compared with those of imatinib in vitro. Mice bearing K562 tumor xenografts, which are known to overexpress Bcr-Abl, were imaged with (18)F-SKI696 PET. A biodistribution study was also performed on K562 tumor-bearing mice to which our radiolabeled tracer was administered. RESULTS: The kinase assay verified that imatinib and SKI696 bind to the same targets with similar affinities. The feasibility of using (18)F-SKI696 as a PET agent was examined in vivo, and (18)F-SKI696 PET was shown to visualize K562 tumor xenografts in nude mice. The tumor was visible on PET 1 h after injection, with uptake of 1% of the injected dose. Biodistribution studies in K562-bearing mice were also performed, and the uptake of (18)F-SKI696 (percentage injected dose per gram) for each organ was calculated. CONCLUSION: The results of the binding assay showed that SKI696 has selectivity and binding affinity comparable to imatinib. Small-animal PET of K562 tumor-bearing mice using (18)F-SKI696 as a PET agent displayed promising tumor uptake and tumor-to-nontarget contrast. Because (18)F-SKI696 has been taken up in vivo by tumors that overexpress Bcr-Abl, we are exploring a possible role for identifying tumors that will respond to imatinib before therapy.

Optimizing tumor targeting of the lipophilic EGFR-binding radiotracer SKI 243 using a liposomal nanoparticle delivery system.

Positron emission tomography (PET) of epidermal growth factor receptor (EGFR) kinase-specific radiolabeled tracers could provide a means for non-invasively characterizing EGFR expression and signaling activity in patients' tumors before, during, and after therapy with EGFR inhibitors. Towards this goal, our group has developed PET tracers which irreversibly bind to EGFR. However, tumor uptake is relatively low because of both the lipophilicity of such tracers (e.g. the morpholino-[124I]-IPQA [SKI 212243]), with octanol-to-water partition coefficients of up to 4, and a short dwell time in the blood and significant hepatobiliary clearance and intestinal reuptake. Liposomal nanoparticle delivery systems may favorably alter the pharmacokinetic profile and improve tumor targeting of highly lipophilic but otherwise promising cancer imaging tracers, such as the EGFR inhibitor SKI 243. SKI 243 is therefore an interesting model molecule for incorporation into lipid-based nanoparticles, as it would not only improve their solubility but also increase the circulation time, availability and, potentially, targeting of tumors. In the current study, we compared the pharmacokinetics and tumor targeting of the bare EGFR kinase-targeting radiotracer SKI 212243 (SKI 243) with that of the same tracer embedded in liposomes. SKI 243 and liposomal SKI 243 are both taken up by tumor xenografts but liposomal SKI 243 remained in the blood longer and consequently exhibited a 3- to 6-fold increase in uptake in the tumor among several other organs.

Fidelity of mispair formation and mispair extension is dependent on the interaction between the minor groove of the primer terminus and Arg668 of DNA polymerase I of Escherichia coli.

The hydrogen bonding interactions between the Klenow fragment of Escherichia coli DNA polymerase I with the proofreading exonuclease inactivated (KF(-)) and the minor groove of DNA were examined with modified oligodeoxynucleotides in which 3-deazaguanine (3DG) replaced guanine. This substitution would prevent a hydrogen bond from forming between the polymerase and that one site on the DNA. If the hydrogen bonding interaction were important, then we should observe a decrease in the rate of reaction. The steady-state and pre-steady-state kinetics of DNA replication were measured with 10 different oligodeoxynucleotide duplexes in which 3DG was placed at different positions. The largest decrease in the rate of replication was observed when 3DG replaced guanine at the 3'-terminus of the primer. The effect of this substitution on mispair extension and formation was then probed. The G to 3DG substitution at the primer terminus decreased the k(pol) for the extension past G/C, G/A, and G/G base pairs but not the G/T base pair. The G to 3DG substitution at the primer terminus also decreased the formation of correct base pairs as well as incorrect base pairs. However, in all but two mispairs, the effect on correct base pairs was much greater than that of mispairs. These results indicate that the hydrogen bond between Arg668 and the minor groove of the primer terminus is important in the fidelity of both formation and extension of mispairs. These experiments support a mechanism in which Arg668 forms a hydrogen bonding fork between the minor groove of the primer terminus and the ring oxygen of the deoxyribose moiety of the incoming dNTP to align the 3'-hydroxyl group with the alpha-phosphate of the dNTP. This is one mechanism by which the polymerase can use the geometry of the base pairs to modulate the rate of formation and extension of mispairs.

Positional isomerism markedly affects the growth inhibition of colon cancer cells by nitric oxide-donating aspirin in vitro and in vivo.

NO-donating aspirin (NO-ASA), a novel pharmacological agent currently undergoing clinical testing, consists of ASA to which a nitrate group is covalently linked via a spacer molecule. We synthesized the three positional isomers of NO-ASA with respect to the -CH(2)ONO(2) group (ortho, meta, and para) and examined whether this isomerism affects the biological activity of NO-ASA on HT-29 human colon cancer cells. The ortho- and para-isomers showed similar IC(50) values (1-5 microM) for cell growth inhibition over 72 h, whereas the IC(50) of the meta-isomer was 200 to 500 microM. The ortho- and para-isomers inhibited cell proliferation more potently than the meta-isomer. All three induced apoptosis but the ortho- and para-isomers also induced atypical cells (they maintain their shape but have diminished or absent nuclear material). Treatment for 3 weeks of Min (Apc(min)(/+)) mice, a model of intestinal cancer, with equimolar amounts of meta- and para-NO-ASA decreased the number of tumors in the small intestine by 36 and 59% (P < 0.01), respectively, compared with vehicle-treated controls, thus confirming their in vitro differences in potency. A structure-activity study of the three isomers revealed that substituting an aliphatic for the aromatic spacer or removing the -ONO(2) group profoundly diminished NO-ASA's ability to inhibit cell growth, whereas removal of the acetyl group on the ASA moiety did not affect cell growth inhibition. Thus, positional isomerism is critical for the pharmacological properties of NO-ASA against colon cancer and it should be taken into consideration in rational drug design.

Caenorhabditis elegans ABL-1 antagonizes p53-mediated germline apoptosis after ionizing irradiation.

c-Abl, a conserved nonreceptor tyrosine kinase, integrates genotoxic stress responses, acting as a transducer of both pro- and antiapoptotic effector pathways. Nuclear c-Abl seems to interact with the p53 homolog p73 to elicit apoptosis. Although several observations suggest that cytoplasmic localization of c-Abl is required for antiapoptotic function, the signals that mediate its antiapoptotic effect are largely unknown. Here we show that worms carrying an abl-1 deletion allele, abl-1(ok171), are specifically hypersensitive to radiation-induced apoptosis in the Caenorhabditis elegans germ line. Our findings delineate an apoptotic pathway antagonized by ABL-1, which requires sequentially the cell cycle checkpoint genes clk-2, hus-1 and mrt-2; the C. elegans p53 homolog, cep-1; and the genes encoding the components of the conserved apoptotic machinery, ced-3, ced-9 and egl-1. ABL-1 does not antagonize germline apoptosis induced by the DNA-alkylating agent ethylnitrosourea. Furthermore, worms treated with the c-Abl inhibitor STI-571 (Gleevec; used in human cancer therapy), two newly synthesized STI-571 variants or PD166326 had a phenotype similar to that generated by abl-1(ok171). These studies indicate that ABL-1 distinguishes proapoptotic signals triggered by two different DNA-damaging agents and suggest that C. elegans might provide tissue models for development of anticancer drugs.

Silanediol-based inhibitor of thermolysin.

The first silanediol inhibitor of thermolysin is reported, prepared by analogy with the Grobelny/Bartlett phosphinate inhibitor. A Cbz group on nitrogen proved to be unstable to the triflic acid mediated silanediol deprotection and was replaced with a dihydrocinnamoyl group. The silanediol was prepared in high purity by hydrolysis of a difluorosilane intermediate and proved to be an effective inhibitor, differing from the phosphinate by a factor of 4 (K(i)=41nM).