title: 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.affiliation: Laboratory of Medicinal Chemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: R
authors.initials: R
authors.lastname: Agbaria
authors.forename: H
authors.initials: H
authors.lastname: Ford
authors.forename: J A
authors.initials: JA
authors.lastname: Kelley
authors.forename: F
authors.initials: F
authors.lastname: Xie
authors.forename: P
authors.initials: P
authors.lastname: Politi
authors.forename: J L
authors.initials: JL
authors.lastname: Grem
authors.forename: D A
authors.initials: DA
authors.lastname: Cooney
authors.forename: V E
authors.initials: VE
authors.lastname: Marquez
authors.forename: C J
authors.initials: CJ
authors.lastname: Allegra
authors.forename: D G
authors.initials: DG
authors.lastname: Johns
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000097:blood*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003562:Cytidine / Q000031:analogs & derivatives* / Q000097:blood; D003570:Cytidine Triphosphate / Q000031:analogs & derivatives* / Q000097:blood; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000737:chemistry / Q000378:metabolism; D010710:Phosphates / Q000097:blood*; D012015:Reference Standards; D015203:Reproducibility of Results; D013601:T-Lymphocytes / Q000737:chemistry / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D010710:Phosphates; C058208:cyclopentenylcytosine 6-triphosphate; D003562:Cytidine; D003570:Cytidine Triphosphate; C045911:cyclopentenyl cytosine