Jun Niijima

A novel carbazole topoisomerase II poison, ER-37328: potent tumoricidal activity against human solid tumors in vitro and in vivo

We have discovered a novel topoisomerase II (topo II) poison, ER‐37328 (12,13‐dihydro‐5‐[2‐(dimethylamino)ethyl]‐4H‐benzo[c]py‐rimido[5,6,1‐jk]carbazole‐4,6,10(5H, 11H)‐trione hydrochloride), which shows potent tumor regression activity against Colon 38 cancer inoculated s.c. Here, we describe studies on the cell‐killing activity against a panel of human cancer cell lines and the antitumor activity of ER‐37328 against human tumor xenografts. In a cell‐killing assay involving 1‐h drug treatment, ER‐37328 showed more potent cell‐killing activity (50% lethal concentrations (LC50s) ranging from 2.9 to 20 μM) than etoposide (LC50s>60 μM) against a panel of human cancer cell lines. ER‐37328 induced double‐stranded DNA cleavage, an indicator of topo II‐DNA cleavable complex formation, within 1 h in MX‐1 cells, and the extent of cleavage showed a bell‐shaped relationship to drug concentration, with the maximum at 2.5 μM. After removal of the drug (2.5 μM) at 1 h, incubation was continued in drug‐free medium, and the amount of cleaved DNA decreased. However, at 10 μM, which is close to the LC50 against MX‐1 cells, DNA cleavage was not detected immediately after 1‐h treatment, but appeared and increased after drug removal. This result may explain the potent cell‐killing activity of ER37328 in the 1‐h treatment. In vivo, ER‐37328 showed potent tumor regression activity against MX‐1 and NS‐3 tumors. Moreover, ER‐37328 had a different antitumor spectrum from irinotecan or cisplatin against human tumor xenografts. In conclusion, ER‐37328 is a promising topo II poison with strong cell killing activity in vitro and tumor regression activity in vivo, and is a candidate for the clinical treatment of malignant solid tumors. (Cancer Sci 2003; 94: 119–124)

Selective determination of potential impurities in an active pharmaceutical ingredient using HPLC-SPE-HPLC

The present paper describes the selective determination of two synthetic intermediates (2,4-dichloro-6,7-dimethoxyquinazoline (IMP-1) and its derivative (IMP-2) as potential impurities in the active pharmaceutical ingredient (API)-A using two-dimensional high-performance liquid chromatography (HPLC) hyphenated via on-line solid-phase extraction (SPE) (HPLC-SPE-HPLC). Two synthetic intermediates that are potential impurities in API-A were concentrated on-line on two Shimadzu MAYI-ODS SPE columns (10 mm×4.6 mm I.D.) after heartcutting in 1st dimension HPLC (1st HPLC) using a Shiseido CAPCELL PAK ACR C18 column (250 mm × 10.0 mm I.D.). Each analyte retained on these SPE columns was transferred to 2nd dimension HPLC (2nd HPLC) with a Shiseido CAPCELL PAK MG-II column (150 mm × 3.0 mm I.D.) for further separation and was subsequently detected with high sensitivity UV. The HPLC-SPE-HPLC system achieved a stepwise downsizing in HPLC. The method was validated and found to be accurate and precise with a linear range of 0.25-250 ppm of each intermediate in API-A with respect to a 500 μL injection of 40 mg/mL of API-A in dimethyl sulfoxide. The method was successfully applied for the determination of these impurities in API batches, and the results demonstrated the usefulness of HPLC-SPE-HPLC for the selective determination of trace impurities in APIs.