BGC 945, a novel tumor-selective thymidylate synthase inhibitor targeted to alpha-folate receptor-overexpressing tumors.

BGC 945 is a cyclopenta[g]quinazoline-based, thymidylate synthase inhibitor specifically transported into alpha-folate receptor (alpha-FR)-overexpressing tumors. Affinity of BGC 945 for the alpha-FR is 70% of the high-affinity ligand folic acid. In contrast to conventional antifolates, BGC 945 has low affinity for the widely expressed reduced-folate carrier (RFC). The K(i) for isolated thymidylate synthase is 1.2 nmol/L and the IC(50) for inhibition of the growth of alpha-FR-negative mouse L1210 or human A431 cells is approximately 7 micromol/L. In contrast, BGC 945 is highly potent in a range of alpha-FR-overexpressing human tumor cell lines (IC(50) approximately 1-300 nmol/L). Pharmacokinetic variables measured following i.v. injection of 100 mg/kg BGC 945 to KB tumor-bearing mice showed rapid plasma clearance (0.021 L/h) and tissue distribution. The terminal half-lives in plasma, liver, kidney, spleen, and tumor were 2, 0.6, 5, 21, and 28 hours, respectively. Tumor BGC 945 concentration at 24 hours was approximately 1 nmol/g tissue, at least 10-fold higher than that in plasma or normal tissues. Inhibition of thymidylate synthase in tissues leads to increased incorporation of 5-[(125)I]-iodo-2'-deoxyuridine ([(125)I]dUrd) into DNA. Forty-eight hours after injection of 100 mg/kg 6RS-BGC 945 ([(125)I]dUrd injected at 24 hours), tumor was the only tissue with incorporation above control level (6-fold). The RFC-mediated thymidylate synthase inhibitor plevitrexed also increased uptake of [(125)I]dUrd in tumor (10-fold) but, in contrast, also caused increased incorporation in other normal tissues such as spleen and small bowel (4.5- and 4.6-fold, respectively). These data suggest that BGC 945 selectively inhibits thymidylate synthase in alpha-FR-overexpressing tumors and should cause minimal toxicity to humans at therapeutic doses.

A liquid chromatographic-tandem mass spectrometric method for the determination of two selective thymidylate synthase inhibitors, BGC945 and BGC638, in mouse plasma.

A LC-tandem mass spectrometry method to quantify the quinazoline-based thymidylate synthase inhibitors BGC945 and BGC638 in mouse plasma was developed. BGC945 and BGC638 were extracted from mouse plasma using protein precipitation with acetonitrile. Chromatography was performed on a Fluophase RP 5 microm, 100 mmx2.0mm i.d. column using a gradient of ammonium acetate and acetonitrile as a mobile phase with a flow rate of 0.2 mLmin(-1). The injection volume for each sample was 20 microL with a total run time of 7.5 min. This method was validated in the range 25-4000 nM (r2=0.99). The analytical assay performance showed that the method was accurate (mean intra- and inter-day assay R.E. were below 12% and 11%, respectively), reproducible (mean intra- and inter-day R.S.D. were less than 13% and 5% for all quality control levels, respectively) and sensitive (lower limit of quantification was 25 nM) in the range studied. This validated method has been used to define the first pharmacokinetic report of BGC945 and BGC638 in mice.

Antifolates targeted specifically to the folate receptor.

Most antifolate drugs are efficiently transported by the reduced-folate carrier (RFC). However, several also bind with high affinity to the alpha-isoform of the folate receptor (alpha-FR) and there is evidence to suggest that this transport mechanism may contribute to their activity when the receptor is highly overexpressed or when the extracellular folate concentration is very low. In particular, the presence of the alpha-FR on tumour cell lines sensitises them to brief exposures to ZD9331. Nevertheless, it is the ubiquitous expression of the RFC in normal tissues that reduces patient tolerability to antifolate drugs. The overexpression of the alpha-FR in some epithelial tumours and its restricted distribution in normal tissues suggests an opportunity for the development of antifolates specifically targeted at alpha-FR overexpressing tumours. Potent cyclopenta[g]quinazoline-based inhibitors of thymidylate synthase (TS) have been discovered with high and low affinity for the alpha-FR and RFC, respectively. This class of agent is represented by CB300638 (TS Ki=0.24 nM) that displays high potency (IC50 approximately 3 nM) for A431-FBP cells (transfected with the alpha-FR) and KB cells (constitutive overexpression). Importantly, this activity is approximately 300-fold higher than for alpha-FR negative cell lines such as A431. In mice bearing the KB tumour xenograft we have demonstrated localisation of CB300638 to tumour and, more importantly, specific inhibition of TS in tumour and not in normal tissues. Data supports the clinical development of this class of agent with the prediction that toxicity would be reduced compared with conventional antifolate drugs. There are a number of challenges to this development posed by the uniqueness of the compounds and these are discussed.