Measurement of Cyclopentenyl Cytosine 5′-Triphosphate in Vitro and in Vivo by Multidimensional High-Performance Liquid Chromatography

✍ Scribed by R. Agbaria; H. Ford; J.A. Kelley; F.M. Xie; P. Politi; J.L. Grem; D.A. Cooney; V.E. Marquez; C.J. Allegra; D.G. Johns

• Publisher: Elsevier Science
• Year: 1993
• Tongue: English
• Weight: 478 KB
• Volume: 213
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1993.1390

✦ Synopsis

Cyclopentenyl cytosine 5'-triphosphate (CPEC-TP) is the active metabolite of the investigational drug cyclopentenyl cytosine (CPEC), a nucleoside analogue which exhibits noteworthy antineoplastic activity against several murine and human tumors in tissue culture, and which is now undergoing Phase I clinical trials. This study describes a method to measure the intracellular CPEC-TP levels in peripheral blood mononuclear cells (PBM cells) from patients treated with CPEC, without using radiolabeled drug. The method utilizes on-line multidimensional high-performance liquid chromatography (HPLC) with two columns of different retention mechanisms connected via an automated programmable switching valve. The elution fraction containing CPEC-TP is initially separated from cellular components using a gel sizing column (TSK-G2000-SW) and then rechromatographed by means of a reversed-phase column operated in an ion-pairing mode (YMC-A-312ODS). The limit of quantitation of CPEC-TP by this method is (2.5 \mathrm{pmol}) per injection. When CPEC-TP levels were measured in PBM cells from 12 cancer patients after (20 \mathrm{~h}) continuous infusion of CPEC at doses ranging from 3.5 to (5.9 \mathrm{mg} / \mathrm{m}^{2} / \mathrm{h}), the levels attained ranged from 1.4 to (13.4 \mu \mathrm{M}) ( 3.6 to (33.5 \mathrm{pmol} / 10^{7}) cells). However, wide variability in the concentrations of CPECTP achieved were evident at each dose and did not appear to correlate either with the CPEC dose or with CPEC plasma levels. This method was validated by a comparison of the quantitation of CPEC-TP in cultured