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Selective Delivery of CB300638, a Cyclopenta[g]quinazoline-based Thymidylate Synthase Inhibitor into Human Tumor Cell Lines Overexpressing the -Isoform of the Folate Receptor¹

Section of Medicine [D. S. T., D. G., A. L. J.] and the Cancer Research United Kingdom Centre for Cancer Therapeutics [V. B., L. A. S., J. T.], the Institute of Cancer Research, Sutton, Surrey, SM2 5NG, United Kingdom, and Department of Oncology/Rheumatology, University Hospital Vrije Universiteit, Amsterdam, 1081 HV the Netherlands [G. J.]

The -isoform of the glycosylphosphatidylinositol cell membrane tethered folate receptor (-FR) is overexpressed in some carcinomas (notably ovarian carcinomas) relative to normal tissues. The nonpolyglutamatable folate-based thymidylate synthase (TS) inhibitor, CB300638 (TS K_i = 0.24 nM) displayed an IC₅₀ of 0.0028 µM for the inhibition of the growth of human A431-FBP cells transfected with the -FR. In contrast, the IC₅₀ for the neotransfected A431 cells was 0.81 µM (300-fold higher). Similarly, this compound inhibited the growth of human KB cells that constitutively overexpress the -FR with an IC₅₀ of 0.0036 µM. These data were derived from cells grown in a physiological concentration of folate (20 nM R,S-leucovorin). Incubation of KB cells with a 1 µM excess of folic acid (FA), to selectively block uptake via the -FR, increased the CB300638 IC₅₀ to 0.39 µM. The relatively low potency of CB300638 under these conditions, or in cell lines not expressing the -FR, is ascribed to its low affinity for the ubiquitously expressed folate transporter, the reduced-folate carrier (K_i for inhibition of [³H]methotrexate transport >100 µM). The high potency of CB300638 in -FR-overexpressing cell lines is attributable to high affinity of the -FR (53% of FA) and efficient endosomal trafficking mediated by the -FR. Sixteen-h exposure to CB300638 inhibited the rate of ³H₂O release from 5-[³H]dUrd (in situ TS assay) in A431, A431-FBP, and KB cells with IC₅₀ values of 0.1 µM, 0.005 µM, and 0.002 µM, respectively. The coaddition of 1 µM FA increased the IC₅₀s for A431-FBP and KB cells to 0.1 µM consistent with -FR-mediated transport of CB300638. In conclusion, -FR-mediated uptake of CB300638 leads to TS and growth inhibition that is highly selective for -FR overexpressing tumor cell lines. The low expression of the -FR in normal tissues, particularly those sensitive to TS inhibitors, together with the low affinity of CB300638 for the reduced-folate carrier, suggests that the compound may have potential as an antitumor agent with a high therapeutic index.

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