Potent topoisomerase I inhibition by novel silatecans eliminates glioma proliferation in vitro and in vivo.

Although topoisomerase inhibitors, such as camptothecin and topotecan, have been widely used in the treatment of nonglial tumors, their application for gliomas has been limited by poor efficacy relative to toxicity that may in part reflect limited bioavailability and blood stability of these agents. However, the potential promise of this class of agents has fostered efforts to develop new, more potent, and less toxic inhibitors that may be clinically relevant. Using a cascade radical annulation route to the camptothecin family, we developed a series of novel camptothecin analogues, 7-silylcamptothecins ("silatecans"), that exhibited potent inhibition of topoisomerase I, dramatically improved blood stability, and sufficient lipophilicity to favor blood-brain barrier transit. We explored the efficacy of a series of these agents against a panel of five high-grade glioma cell lines to identify a promising compound for further preclinical testing. One of the most active agents in our systems (DB67) inhibited tumor growth in vitro with an ED50 ranging between 2 and 40 ng/ml, at least 10-fold more potent than the effects observed with topotecan, and at least comparable with those of SN-38, the active metabolite of CPT-11. Because DB67 also exhibited the highest human blood stability of any of the agents examined, this agent was then selected for in vivo studies. A dose-escalation study of this agent in a nude mouse U87 glioma model system demonstrated a concentration-dependent effect, with tumor growth inhibition at day 28 postimplantation (the day control animals began to require sacrifice because of large tumor size) of 61 +/- 7% and 73 +/- 3% after administration of DB67 doses of 3 and 10 mg/kg/day, respectively, for 5 days beginning on postimplantation day 7. Animals that continued treatment with 10 mg/kg/day in three additional 21-day cycles all remained progression free after >90 days of follow-up but later developed enlarging tumors after treatment was stopped. However, a slightly higher dose (30 mg/kg/day) induced complete tumor regression after only two cycles in all study animals and was effective even if treatment was delayed until large, bulky tumors had developed. Application of the 30 mg/kg/day dose to treat established intracranial glioma xenografts led to long-term (>90 day) survival in six of six animals, whereas all controls died of progressive disease (P < 0.00001). No apparent toxicity was encountered in any of the treated animals. In summary, the present studies indicate that silatecans may hold significant promise for the treatment of high-grade gliomas and provide a rationale for proceeding with further preclinical evaluation of their efficacy and safety versus commercially available camptothecin derivatives.

Feasibility of lovastatin analysis by packed column supercritical fluid chromatography with ultraviolet detection.

A reliable supercritical fluid chromatography (SFC) method was developed for the analysis of lovastatin, a hypocholesterolaemic drug, from MEVACOR. Methanol-modified carbon dioxide was shown to elute the drug, and its dehydrolovastatin and hydroxy acid lovastatin degradation products from a Hypersil silica column. However, the hydroxy acid lovastatin was found to tail in this mobile phase. The phenomena was eliminated by the addition of trifluoroacetic acid [Haouck, S. Thomas, D. K. Ellison, Talanta 40 (1993) 491] to the mobile phase which permitted the drug and its two main degradation products to all elute from the Hypersil silica column in under 6 min with symmetrical peak shape. Chromatographic limit of detection (LOD) and limit of quantification (LOQ), linear dynamic range (LDR), and injection precision were obtained in order to assess the chromatographic performance of the SFC system for the lovastatin separation.

Investigation of primary and secondary modifiers for the subcritical extraction of lovastatin from MEVACOR tablets with carbon dioxide.

The subcritical fluid extraction of lovastatin from tablet powder mixtures prepared in this laboratory and MEVACOR tablets is successfully demonstrated. Methanol modifier percentage, additive type (acidic, basic, or neutral), and additive concentration on the extraction efficiency are examined. The extraction recoveries of lovastatin from MEVACOR tablets are shown to be highly dependent on methanol concentration and additive type. Isopropylamine is shown to be the most successful additive investigated. An optimized extraction method is developed, and lovastatin recoveries of 99.5% were achieved with a relative standard deviation of 1.2% from MEVACOR tablets with 15% (v/v) (1.0% [v/v] isopropylamine) methanol-modified CO2.

Effect of helium in helium headspace carbon dioxide cylinders on packed-column supercritical fluid chromatography.

Supercritical fluid chromatography of PAHs was performed with pure carbon dioxide and helium headspace carbon dioxide at various cylinder fill levels. The retention times of the PAHs increased when helium headspace carbon dioxide was used as a carrier fluid relative to pure carbon dioxide. The increased retention times were affected by the level of the liquid phase present in the helium headspace carbon dioxide cylinder. As more liquid phase was removed from the cylinder, the effect of helium on the solvating power of CO(2) was reduced because the relative amount of helium dissolved in the liquid phase decreased. Furthermore, the effect of helium headspace carbon dioxide was investigated with methanol-modified carbon dioxide mobile phases for the analysis of steroids. We observed that the relative solubility of helium in carbon dioxide resulted in longer retention times when compared to pure carbon dioxide as the liquid level of carbon dioxide decreased.

Use of supercritical fluid extraction for sample preparation of sustained-release felodipine tablets.

Supercritical fluid extraction (SFE) was shown to be an accurate and precise alternative to liquid extraction for sample preparation of sustained-release felodipine tablets (5 mg potency) while realizing an 80% reduction in solvent consumption. Extractions of felodipine spiked on an inert support were used to evaluate the solubility of felodipine in CO2 as well as analyte trapping after SFE. Even though the pure drug was found to be soluble in pure CO2, extractions of felodipine from the tablet matrix required moderate modifier concentrations [8.7% (v/v) methanol in CO2] in order to overcome strong matrix-drug interactions. Sequential static/dynamic extraction steps were also required to quantitatively recover the drug from the tablet matrix, indicating that the drug extraction was diffusion-limited. Average recoveries (n = 5) for the optimized SFE method were determined to be 4.93 mg felodipine tablet (98.6% claim) with an RSD of 1.2% versus those for the liquid extraction procedure (n = 5, 4.98 mg/tablet, 99.6% claim, 2.4% RSD). Similar levels of drug degradation (0.12% expressed as felodipine) were also obtained with both the traditional liquid extraction and with the SFE method.

Analysis of felodipine by packed column supercritical fluid chromatography with electron capture and ultraviolet absorbance detection.

A reproducible and selective supercritical fluid chromatography (SFC) method was developed for the analysis of felodipine, a drug indicated for the treatment of hypertension. Methanol-modified carbon dioxide was employed as the SFC mobile phase with both electron capture detection (ECD) and multi-wavelength detection (MWD) being used simultaneously for analyte determination. Chromatography limit of detection (LOD) and limit of quantitation (LOQ), linear dynamic range (LDR) and injection precision were obtained in order to assess chromatographic and detector performance for both the SFC/MWD and SFC/ECD/MWD systems. The method was shown to be stability indicating since felodipine could be separated from its potential oxidative degradation product, H152/37, in under 6 min (felodipine k' = 2.44). Sample throughput was increased by 60% with the SFC assay vs LC. The optimized SFC method was shown to be equivalent to an existing LC/UV procedure for the analysis of a sustained-release tablet while realizing a 92% saving in disposable solvent waste. In order to achieve further solvent savings overall, supercritical fluid extraction (SFE) with 8% methanol-modified carbon dioxide as the extraction fluid was used to extract felodipine from a sustained-release tablet (as opposed to traditional solvent extraction). Comparable drug recoveries were obtained with SFE sample preparation technique when either SFC or LC extract analysis was utilized.