The measurement of polyglutamate metabolites of the thymidylate synthase inhibitoR, ICI D1694, in mouse and human cultured cells

A method is described for the measurement of the polyglutamates of the quinazoline thymidylate synthase inhibitor, N-(5-[N-(3,4- dihydro-2- methyl-4- oxoquinazolin-6- ylmethyl)-N- methylamino]-2- theonyl)- l - glutamic acid (ICI D1694). This involved incubation of cells with [5- 3H]ICI D1694, extraction of the polyglutamates and their analysis by HPLC using an ion-pairing method. Co-chromatography with ICI D1694 and its synthetic di-hexaglutamate standards (UV detection) aided identification of the [ 3H]polyglutamates in the fractions recovered from the HPLC. Recovery of the polyglutamates at each stage of extraction and analysis was very good (77–84% overall recovery). Polyglutamates readily accumulated as the tri-, tetra and penta forms and occasionally a small amount of hexaglutamate was found. After mouse L1210 leukemia or human W1L2 lymphoblastoid cells were incubated for 30 min with 0.1 μM [ 3H]ICI D1694 there was a ∼6-fold concentration effect intracellularly with most of the 3H associated with polyglutamate forms (∼75% and 96% for the L1210 and W1L2, respectively). Even some of the higher chain length tetra- and pentaglutamates could be detected at this time. After 4 hr incubation the total level of intracellular 3H had risen to 2–3 μM, greater than 96% of which was associated with polyglutamates (mainly tetra- and pentaglutamates). Four other human cell lines, two ovarian (CH1 and 41M), the MCF-7 breast and the HT-29 colon, were examined for their ability to form intracellular polyglutamates. A 4 hr incubation with 0.1 μM [ 3H]ICI D1694 resulted in a substantial intracellular accumulation of the drug (20–100-fold) in its polyglutamate forms with only 2–20% remaining as the parent monoglutamate, depending on the cell line. The major polyglutamate was again cell line dependent, ranging from the tri to the penta form. Prolonging the incubation time to 24 hr allowed a further accumulation of drug with a larger percentage appearing as tri- to hexaglutamates. Although cell lines differed in the total level of polyglutamates formed and the pattern of chain length observed, rapid and extensive polyglutamation of ICI D1694 occurred in all the cell types examined.