Gibson W, Bisset G M, Marsham P R, Kelland L R, Judson I R, Jackman A L
Drug Development Section, Institute of Cancer Research, Sutton, Surrey, U.K.
Biochem Pharmacol. 1993 Feb 24;45(4):863-9. doi: 10.1016/0006-2952(93)90170-2.
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 microM [3H]ICI D1694 there was a approximately 6-fold concentration effect intracellularly with most of the 3H associated with polyglutamate forms (approximately 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 microM, 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 microM [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.
本文描述了一种用于测定喹唑啉胸苷酸合酶抑制剂N-(5-[N-(3,4-二氢-2-甲基-4-氧代喹唑啉-6-基甲基)-N-甲基氨基]-2-噻吩甲酰基)-L-谷氨酸（ICI D1694）多聚谷氨酸盐的方法。该方法包括用[5-³H]ICI D1694孵育细胞，提取多聚谷氨酸盐，并采用离子对方法通过高效液相色谱（HPLC）对其进行分析。与ICI D1694及其合成的二己谷氨酸标准品进行共色谱分析（紫外检测），有助于鉴定从HPLC回收的各馏分中的[³H]多聚谷氨酸盐。在提取和分析的每个阶段，多聚谷氨酸盐的回收率都非常高（总体回收率为77 - 84%）。多聚谷氨酸盐很容易以三、四和五聚体形式积累，偶尔也会发现少量的六聚谷氨酸盐。用0.1微摩尔[³H]ICI D1694孵育小鼠L1210白血病细胞或人W1L2淋巴母细胞30分钟后，细胞内出现约6倍的浓度效应，大部分³H与多聚谷氨酸盐形式相关（L1210和W1L2细胞分别约为75%和96%）。此时甚至可以检测到一些较高链长的四聚和五聚谷氨酸盐。孵育4小时后，细胞内³H的总水平升至2 - 3微摩尔，其中超过96%与多聚谷氨酸盐相关（主要是四聚和五聚谷氨酸盐）。对另外四种人类细胞系，即两种卵巢细胞系（CH1和41M）、MCF - 7乳腺癌细胞系和HT - 29结肠癌细胞系，检测了它们形成细胞内多聚谷氨酸盐的能力。用0.1微摩尔[³H]ICI D1694孵育4小时后，药物以多聚谷氨酸盐形式在细胞内大量积累（20 - 100倍），仅2 - 20%以母体单谷氨酸盐形式存在，具体取决于细胞系。主要的多聚谷氨酸盐同样因细胞系而异，范围从三聚体到五聚体形式。将孵育时间延长至24小时，药物会进一步积累，更多比例以三聚体到六聚体谷氨酸盐形式出现。尽管不同细胞系在形成的多聚谷氨酸盐总量和观察到的链长模式上存在差异，但在所有检测的细胞类型中，ICI D1694都能快速且广泛地发生多聚谷氨酸化。
