Measurement of cyclopentenyl cytosine 5'-triphosphate in vitro and in vivo by multidimensional high-performance liquid chromatography.
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.
Agbaria
R
R
Laboratory of Medicinal Chemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Ford
H
H
Kelley
J A
JA
Xie
F
F
Politi
P
P
Grem
J L
JL
Cooney
D A
DA
Marquez
V E
VE
Allegra
C J
CJ
Johns
D G
DG
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
D000970:Antineoplastic Agents; D010710:Phosphates; C058208:cyclopentenylcytosine 6-triphosphate; D003562:Cytidine; D003570:Cytidine Triphosphate; C045911:cyclopentenyl cytosine