HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC ASSAY AND STRUCTURE ACTIVITY RELATIONSHIPS OF THE EPIPODOPHYLLOTOXIN ANTICANCER DRUGS VP 16-213 (ETOPOSIDE) AND VM 26 (TENIPOSIDE)
Abstract
A rapid and convenient high-performance liquid chromatographic procedure for the analysis of the clinically useful anticancer agents VP 16-213 and VM 26 is described. The drugs, which are semi-synthetic derivatives of the natural product podophyllotoxin, are extracted from plasma with chloroform. The extracts are evaporated to dryness, reconstituted in methanol, and chromatographed on a reversed-phase microparticle C(,18) column using isocratic elution with a mixture of methanol-water (60:40). Each drug is used as the internal standard for the other. Quantitation to 500 ng/ml (0.85 nmole/ml) plasma is based on peak height ratios using UV detection at 254 nm. Patient plasma concentration versus time data agree well with previously published data obtained using radiolabelled drug. To improve the specificity and sensitivity of the assay, fluorescence detection by means of a dedicated HPLC detector was implemented. While early fluorescence experiments involving insertion of a flow cell into the block of a typical spectrophotofluorometer were limited by instrumental instability, 5 ng of VP 16 were easily detected by the dedicated HPLC detector. The detection limit from plasma was thus lowered to 50 ng VP 16 per ml. A clinical profile from a patient receiving a small dose of VP 16 showed levels of only 100 ng per ml plasma at 24 hours post infusion. The specificity of the method was demonstrated by concurrent UV detection which showed interferences from other drugs which the patient had received along with VP 16. Investigations into the nature of the hydroxy acid metabolite of VP 16-213 carried out using paired-ion chromatography with tetrabutylammonium bromide and fluorescence detection, are described. Also, a unique separation of VP 16-213 and a possible metabolite, the isomer, picro VP 16-213, is described. A study on the structure activity relationships in the lactone ring D of VP 16-213 was also carried out. Lithium aluminum hydride reduction produced the ring D reduced diol, and recyclization by tosyl chloride in pyridine produced the ring D cyclic ether analog of VP 16 (C=O converted to CH(,2)). Stereospecificity and retention of stereochemistry in the reactions were proven by determination of J(,2,3) using 470 Mhz('1)H NMR spectroscopy. J(,2,3) was shown to correlate with cis or trans stereochemistry at the C-2, C-3 ring C-D fusion. Biological testing in L 1210 mouse leukemia showed that VP 16 gave two long-term survivors and that the ether analog gave a 25% ILS, each at doses of 45 mg/kg.
Degree
Ph.D.
Subject Area
Pharmacology
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