Measurement of cyclopentenyl cytosine 5'-triphosphate in vitro and in vivo by multidimensional high-performance liquid chromatography.

Abstract

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-312-ODS). The limit of quantitation of CPEC-TP by this method is 2.5 pmol per injection. When CPEC-TP levels were measured in PBM cells from 12 cancer patients after 20 h continuous infusion of CPEC at doses ranging from 3.5 to 5.9 mg/m2/h, the levels attained ranged from 1.4 to 13.4 microM (3.6 to 33.5 pmol/10(7) cells). However, wide variability in the concentrations of CPEC-TP 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 PBM cells and Molt-4 cells (a human T-cell line adapted for growth in tissue culture) after incubation with both unlabeled and radiolabeled CPEC.

• Authors: R Agbaria, H Ford Jr, J A Kelley, F Xie, P Politi, J L Grem, D A Cooney, V E Marquez, C J Allegra, D G Johns
• Journal: Analytical biochemistry (Anal Biochem) Vol. 213 Issue 1 Pg. 90-6 (Aug 15 1993) ISSN: 0003-2697 [Print] United States
• PMID: 8238887 (Publication Type: Journal Article)

Chemical References

• Antineoplastic Agents
• Phosphates
• cyclopentenylcytosine 6-triphosphate
• Cytidine
• Cytidine Triphosphate
• cyclopentenyl cytosine

Topics

• Animals
• Antineoplastic Agents (blood)
• Cattle
• Chromatography, High Pressure Liquid (methods)
• Cytidine (analogs & derivatives, blood)
• Cytidine Triphosphate (analogs & derivatives, blood)
• Humans
• Intracellular Fluid (metabolism)
• Leukocytes, Mononuclear (chemistry, metabolism)
• Phosphates (blood)
• Reference Standards
• Reproducibility of Results
• T-Lymphocytes (chemistry, metabolism)