OSU-03012, a 3-phosphoinositide-dependent kinase-1 (PDK1) inhibitor, destabilizes MYCN and MYC proteins in neuroblastoma cells. However, AKT phosphorylation is barely detectable in neuroblastoma cells under normal culture conditions whether treated with OSU-03012 or not. This observation suggests that PDK1 is not the main target of OSU-03012 to destabilize MYC and MYCN in neuroblastoma cells. In the present study, we explored one of the possible mechanisms by which OSU-03012 destabilizes MYC and MYCN. Since Aurora kinase A is reported to phosphorylate GSK3β, leading to its inactivation, we hypothesized that one of the targets of OSU-03012 is Aurora kinase A. Comparative analysis of OSU-03012 and VX-680, a potent and specific inhibitor of Aurora kinases, showed that both inhibitors destabilized MYC and MYCN and were significantly growth suppressive to neuroblastoma cell lines. In silico molecular docking analysis further showed that the calculated interaction energy between Aurora kinase A and OSU-03012 was −109.901 kcal/mol, which was lower than that (−89.273 kcal/mol) between Aurora kinase A and FXG, an Aurora kinase-specific inhibitor. Finally, an in vitro Aurora kinase A inhibition assay using a recombinant Aurora kinase A showed that OSU-03012 significantly inhibited Aurora kinase A, although it was weaker in potency than that of VX-680. Thus, OSU-03012 has a likelihood of binding to and inhibiting Aurora kinase A in vivo. These results suggest that OSU-03012 affects multiple cellular targets, including Aurora kinase A, to exhibit its growth suppressive and MYC and MYCN-destabilizing effects on neuroblastoma and other cancer cells.

3,4,3-LI(1,2-HOPO), 1,5,10,14-tetra(1-hydroxy-2-pyridon-6-oyl)-1,5,10,14-tetraazatetradecane), is a potent octadentate chelator of actinides. It is being developed as a decorporation treatment for internal contamination with radionuclides. Conventional HPLC methods exhibited speciation peaks and bridging, likely attributable to the agent’s complexation with residual metallic ions in the HPLC system. Derivatization of the target ligand in situ with Fe(III) chloride, however, provided a single homogeneous iron-complex that can readily be detected and analyzed by HPLC. The HPLC method used an Agilent Eclipse XDB-C18 column (150 mm × 4.6 mm, 5 μm) at 25 °C with UV detection at 280 nm. A gradient elution, with acetonitrile (11% to 100%)/buffer mobile phase, was developed for impurity profiling. The buffer consisted of 0.02% formic acid and 10 mM ammonium formate at pH 4.6. An Agilent 1200 LC-6530 Q-TOF/MS system was employed to characterize the [Fe(III)-3,4,3-LI(1,2-HOPO)] derivative and impurities. The proposed HPLC method was validated for specificity, linearity (concentration range 0.13–0.35 mg/mL, r = 0.9999), accuracy (recovery 98.3–103.3%), precision (RSD ≤ 1.6%) and sensitivity (LOD 0.08 μg/mL). The LC/HRMS revealed that the derivative was a complex consisting of one 3,4,3-LI(1,2-HOPO) molecule, one hydroxide ligand, and two iron atoms. Impurities were also identified with LC/HRMS. The validated HPLC method was used in shelf-life evaluation studies which showed that the API remained unchanged for one year at 25°C/60% RH.

No abstract

An HPLC method for the assay of an anticancer nucleoside, 4’-thio-2’-deoxycytidine (T–dCyd, NSC 764276), has been developed and validated. The stress testing of T-dCyd was carried out in accordance with ICH guidelines Q1A (R2) under acidic, alkaline, oxidative, thermolytic, and photolytic conditions. The separation of T-dCyd from its impurities and degradation products was achieved in 40 min on a Luna® Phenyl-Hexyl column (150 mm × 4.6 mm i.d., 3 µm) with a gradient elution using ammonium phosphate buffer (pH 3.85) and methanol as the mobile phase. The gradient starts from 2% and ends at 80% of methanol. Detection is by UV at 282 nm. LC-QTOF/MS was used to obtain mass data for characterization of impurities and degradation products. The proposed HPLC assay method was validated for specificity, linearity (concentration range 0.25–0.75 mg/mL, r ≥ 0.9998), accuracy (recovery 98.1–102.0%), precision (RSD ≤ 1.5%), and sensitivity (LOD 0.1 µg/mL). The developed method was suitable for the quality control and stability monitoring of the T-dCyd drug substance.

No abstract

An HPLC method for the assay of the heat shock protein 90 inhibitor, PU-H71 (NSC 750424), has been developed and validated. The stress testing of PU-H71 was carried out in accordance with ICH guidelines Q1A (R2) under aqueous, acidic, alkaline, oxidative, thermolytic and photolytic conditions. The separation of PU-H71 from its impurities and degradation products was achieved within 50 min on a Mac-Mod ACE 3 C18 column (150 mm × 4.6 mm i.d., 3 μm) with a gradient mobile phase comprising 20–95% acetonitrile in water, with 0.1% trifluroacetic acid in both phases. LC - Quadruple TOF/MS was used to obtain accurate mass data on various components as well as on their fragments for characterization of impurities and degradation products. The proposed HPLC assay method was validated for specificity, linearity (concentration range 0.1–0.3 mg/mL, r ≥ 0.9998), accuracy (recovery 99.7–101.1%), precision (intra-lab RSD ≤ 1.39%, inter-lab RSD ≤ 0.91%), sensitivity (LOD 0.08 μg/mL), and ruggedness. The developed method was suitable for the assay and stability monitoring of PU-H71 drug substance.

An HPLC method for the assay of a DNA topoisomerase inhibitor, LMP776 (NSC 725776), has been developed and validated. The stress testing of LMP776 was carried out in accordance with International Conference on Harmonization (ICH) guidelines Q1A (R2) under acidic, alkaline, oxidative, thermolytic, and photolytic conditions. The separation of LMP776 from its impurities and degradation products was achieved within 40 min on a Supelco Discovery HS F5 column (150 mm × 4.6 mm i.d., 5 μm) with a gradient mobile phase comprising 38–80% acetonitrile in water, with 0.1% trifluoroacetic acid in both phases. LC/MS was used to obtain mass data for characterization of impurities and degradation products. One major impurity was isolated through chloroform extraction and identified by NMR. The proposed HPLC assay method was validated for specificity, linearity (concentration range 0.25–0.75 mg/mL, r = 0.9999), accuracy (recovery 98.6–100.4%), precision (RSD ≤ 1.4%), and sensitivity (LOD 0.13 μg/mL). The validated method was used in the stability study of the LMP776 drug substance in conformance with the ICH Q1A (R2) guideline.

No abstract