Davinder S. Theti

The Role of α-Folate Receptor-Mediated Transport in the Antitumor Activity of Antifolate Drugs

Purpose: Raltitrexed, pemetrexed, lometrexol, and ZD9331 are antifolate drugs transported into cells via the ubiquitously expressed reduced-folate carrier. They display also high affinity for the α-folate receptor (α-FR), a low capacity folate transporter that is highly overexpressed in some epithelial tumors. The role of α-FR in the activity of the antifolates has been evaluated in two α-FR-overexpressing cell lines grown in a physiological concentration of folate (20 nm R,S-Leucovorin). Experimental Design and Results: A431-FBP cells (transfected with the α-FR) were 3–5-fold more sensitive to the antifolates than A431 cells. KB cells (constitutive α-FR overexpression) were less sensitive to the drugs when coexposed to 1 μm folic acid to competitively inhibit binding to the α-FR. Raltitrexed, pemetrexed, and lometrexol are polyglutamated in cells leading to drug retention, e.g., the raltitrexed 4- and 24-h IC50s in A431 cells were ∼0.6 and 0.008 μm, respectively, compared with 0.003 μm for 72-h continuous exposure. A431-FBP cells were ∼3-fold more sensitive to raltitrexed and pemetrexed at all exposure times. ZD9331 is not polyglutamated, and the 4- and 24-h IC50s in A431 cells were >100 and ∼100 μm, respectively, reducing to 2 and 0.1 μm, respectively, in A431-FBP cells. The ZD9331 4- and 24-h IC50s in KB cells were 20 and 1 μm, respectively, and reversible by coaddition of 1 μm folic acid. An in situ thymidylate synthase assay demonstrated continued thymidylate synthase inhibition after ZD9331-treated A431-FBP and KB, but not A431, cells were placed in drug-free medium for 16 h. A model is proposed in which the antifolates accumulate in the α-FR/endosomal apparatus, leading to slow release into the cytoplasm. In particular, this leads to cellular retention of the nonpolyglutamatable ZD9331. Conclusions: Antifolate drugs, particularly ZD9331, have the potential for increased efficacy in tumors that highly overexpress the α-FR.

A rationale for the clinical development of the thymidylate synthase inhibitor ZD9331 in ovarian and other solid tumours

ZD9331 is an antifolate drug that potently and specifically inhibits thymidylate synthase (TS). In contrast with TS inhibitors such as raltitrexed, it cannot be polyglutamated, leading to antitumour activity independent of folylpolyglutamyl synthetase (FPGS) activity. The growth inhibition IC50 values for ZD9331 and raltitrexed were determined for a panel of 18 human tumour cell lines, that included six colon and six ovarian. The colon lines largely displayed overlapping sensitivities to both drugs with only one of the six lines being drug resistant. In contrast, the ovarian cell lines displayed non-overlapping sensitivities with four being highly resistant to raltitrexed and only one was cross-resistant to ZD9331. Studies were undertaken to explain these results. The colon and ovarian cell lines were characterised for TS activity, and TS and FPGS mRNA expression. TS activity correlated with sensitivity to ZD9331 (r=0.50; p=0.097) and raltitrexed (r=0.74; p=0.0063). Provided the data from the highly drug-resistant cell lines (BE and 41 M) were omitted, TS mRNA expression levels also correlated with ZD9331 (r=0.77; p=0.013) and raltitrexed IC50 (r=0.84; p=0.0031). FPGS mRNA expression correlated with higher sensitivity to raltitrexed relative to ZD9331 (higher ZD9331/raltitrexed IC50 ratios) (r=0.62; p=0.048). Similarly, cell lines with IC50 ratios>median expressed a 1.8-fold higher median level of FPGS mRNA (p=0.0087) compared with those with ratios<or=median. The four ovarian and one colon cell line that were relatively more sensitive to ZD9331 expressed FPGS mRNA<or=median (p=0.061). Thus, ZD9331 overcomes resistance to raltitrexed in ovarian tumour cell lines expressing low levels of FPGS. These data, and others demonstrating a lack of cross-resistance between cisplatin and ZD9331, support the clinical evaluation of ZD9331 in platinum-refractory ovarian cancer.

Cyclopenta[g]quinazoline-based antifolates: the effect of the chirality at the 6-position on the inhibition of thymidylate synthase (TS).

Cyclopenta[g]quinazoline-based inhibitors of thymidylate synthase (TS) possess a chiral centre at the 6-position of the molecule. The effect of this chirality on the inhibition of TS was investigated by synthesising compounds 6S-1a-c, 6R-1a-c. It was shown, in particular with the diastereoisomers 6S-1c, 6R-1c, that the inhibitory activity against TS is mainly due to the 6S diastereoisomer rather than the 6R diastereoisomer, which is virtually inactive.

The impact of arteriovenous fistula creation in pulmonary hypertension: measurement of pulmonary pressures by right heart catheterization in a patient with respiratory failure following arteriovenous fistula creation.

Pulmonary hypertension (PHT) is frequent in patients receiving hemodialysis (HD) and carries a high mortality. While it has been suggested that arteriovenous fistulae (AVF) may exacerbate PHT in HD patients, it has also been observed that creating AVF in patients with chronic lung disease and normal renal function may lead to improved exercise tolerance. Most of the observations regarding HD patients using echocardiography demonstrated that temporary closure of AVF improved pulmonary pressures. We present the case of a 45‐year‐old patient with chronic obstructive pulmonary disease on HD who experienced respiratory failure following AVF formation and underwent right heart catheterization. Severe PHT was diagnosed but transient occlusion of the fistula failed to improve the PHT. This case supports the theory that fistula creation does not exacerbate pre‐existing PHT and that AVF can be the access of choice in patients with known chronic lung disease and pulmonary hypertension.

Abstract 2544: Preclinical pharmacodynamics (PD) of ONX 0801, a folate receptor-α (FRα) and tumor-targeted thymidylate synthase (TS) inhibitor

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL ONX 0801, a first-in-class tumor-targeted antifolate, selectively targets TS in tumor cells due to transport by FRα, thus differentiating it from other antifolates. FRα expression is restricted to the apical membrane of specialised tissues generally inaccessible to drugs in circulation; however, it is highly expressed in several cancers including ovarian. FRα is a high affinity, low capacity transporter and the rate of drug uptake and cell line sensitivity to ONX 0801 is related to the number of FRαs tethered to the cell surface (72h IC50 range: 0.003-0.3µM). Non-FRα mediated uptake becomes relevant at relatively high concentrations (>1µM). The purpose of the study was to investigate the relationship between drug exposure and FRα- or non-FRα mediated TS inhibition in both tumors and normal proliferating tissues. PD endpoints of TS inhibition included an intact TS assay for cultured cells, and tumor and plasma dUrd in mice bearing FRα positive IGROV-1 ovarian tumors. In IGROV-1 cells, the 72h IC50 was 0.003µM; however, blocking FRα-mediated uptake with excess folic acid increased it to 2µM. In FRα negative A431 cells the IC50 was 7µM. Drug washout experiments demonstrated that exposure to high concentrations for 30µM for 4h exposure (72h endpoint) but was reduced to 8µM and 5µM for a 24h and 48h exposure, respectively. These data suggest that this level of exposure in vivo could produce effects in normal proliferating tissues (NPT). We have tested this in mice bearing IGROV-1 xenografts by injecting ONX 0801, as either a bolus to achieve temporary high exposure, or as a 3 day continuous infusion (CI) with osmotic minipumps. During the 4hr following a bolus of 100-200mg/kg of ONX 0801 the plasma drug levels were high (∼500-1µM) and plasma dUrd was elevated consistent with non FR-mediated TS inhibition in NPT. However, plasma dUrd had normalised by 24h. Consistent with tumor-targeting, tumor dUrd was still elevated 24h after 15mg/kg ONX 0801. CI of ONX 0801 of 5mg/kg/24h produced a steady-state plasma drug level of 0.05µM and increased tumour dUrd for 72h. No increases in plasma dUrd were observed up to a dose of 300mg/kg/24h where a small increase was seen (plasma ONX 0801 ∼4µM). Taken together, these data are consistent with cell line data and predict that increasing exposure in patients to >5µM for >24h is unnecessary and could be associated with toxicity. In conclusion, ONX 0801 has a high therapeutic index in model systems using PD markers of TS inhibition. Further, we have defined guiding pharmacokinetic and PD parameters for the ongoing Phase 1 clinical trial to aid the determination of the biologically effective dose. Supported by grants from Onyx Pharmaceuticals, BTG PLC and Cancer Research UK Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2544. doi:10.1158/1538-7445.AM2011-2544