Folate-based Thymidylate Synthase Inhibitors Research Articles

Use of the combination of raltitrexed (R) and oxaliplatin (O) as a salvage treatment for patients with advanced colorectal cancer (ACRC) refractory to 5-fluorouracil (5-FU) based chemotherapy and with hyperexpression of thymidylate synthase (TS): Final report

3727 Background: TS is an important target for fluoropyrimidine based TS inhibitors and its hiperexpression has been correlated with resistance to 5-FU, but not necessarily to R, a folate-based TS inhibitor, which appears to be more correlated to FPGS activity (van Triest et al, 1999; Cheradame et al, 1999). O is a novel diaminocyclohexane platinum agent also active in ACRC and obviously not related to TS status. Methods: 21 patients with ACRC not responsive to a first line 5-FU based chemotherapy: 14(67%) to IFL(Saltz) regimen, 4(19%) to 5-FU/LV- (Mayo Clinic) regimen and 3(14%) to capecitabine) and high expression of TS (determined by IHC technique-grades 2+ and 3+) and age less than 70 Y/O, measurable disease, WHO PS 0–2, life expectancy ≥ 2 months, adequate renal and liver functions and informed consent were accrued to receive: T – 3.0 mg/m2 and O – 120mg/m2 every 21 days until progression. Results: Thirteen were male and 8 female; median age: 58(39–67); median PS: 1 (1–2). Number of cycles administered: 98 (median per patient: 4). There were 7 PR (33%), 3 SD(14%) and 11(53%) PD. Clinical benefit based upon weight gain, PSand analgesic consumption was observed in all 7 responders (33%). The median PFS was 6.5 months (1–12) and the median overall SV was 8 months (2–12+). The 1 year SV was 17%. The regimen was well tolerated. WHO grade 4 diarrhea occurred in one patient, grade 3 in 12(12%) cycles; grade 2 vomiting in 10(10%) cycles; grades 3 and 4 neutropenia in 16(16%) cycles; reversible febrile neutropenia in 2 patients: (onedied due to sepsis); grade 3 thrombocitopenia in 6(6%) cycles; anemia grades 2 and 3 in 21(21%) cycles and grade 2 oral mucositis in 3 (3%) of the cycles. Grades 1 and 2 neuropathy was observed in 61% of the cycles. Conclusions: These favorable results suggest that this combination might be effective in the treatment of patients with ACRC with high expression of TS and should be also explored as a first line regimen for this subset of patients with predictable resistance to fluoropyrimidine based chemotherapy. No significant financial relationships to disclose.

Pemetrexed (alimta, MTA, multitargeted antifolate, LY231514) for malignant pleural mesothelioma

Regimens with antimetabolites such as gemcitabine administered alone or in combination with cisplatin have shown promising phase II study data. Accordingly, antifolate agents are of considerable interest. Pemetrexed (Alimta; Eli Lilly and Company, Indianapolis, IN), a folate-based inhibitor of thymidylate synthase, is currently under phase III investigation. The Evaluation of Mesothelioma in a Phase III Trial of Pemetrexed with Cisplatin (EMPHACIS) trial is the first phase III randomized study in mesothelioma with survival as the primary end point; it showed statistically significant superiority of the pemetrexed/cisplatin combination over cisplatin in survival, response, and patient quality-of-life parameters. Moreover, this trial reported that consequent low-dose folic acid and vitamin B 12 supplementation significantly reduced the toxicity observed with the use of pemetrexed. In light of these data, it is likely that pemetrexed/cisplatin will soon be recommended for malignant pleural mesothelioma as first-line standard chemotherapy.

Chromosomal imbalance maps of human 5FU-resistant colorectal cancer cell lines: implications in the analysis of 5FU-acquired resistance mechanisms.

Thymidylate synthase (TS), a critical enzyme in the de novo synthesis of thymidylate, is an important target for fluoropyrimidines and folate-based TS inhibitors. Overexpression of TS has been correlated to 5-fluorouracil (5FU)-resistance. Because 5FU still remains a basic component of the treatment of colorectal cancer, circumvention of resistance is of vital importance. A panel of sensitive (HT29 and LoVo) and 5FU-resistant colorectal cancer cell lines (HT29-5FUR and LoVo-5FUR) were subjected to comparative genomic hybridization (CGH) analysis to identify possible amplified/deleted regions associated with 5FU-resistance in colon tumours. We have identified chromosomal gains at 5p, 6, 7p, 7q and 8q and one loss at 3q in 5FU-resistant cells as compared to corresponding sensitive cell lines. Neither chromosomal gains at 18p nor gene amplification of TS were observed in our resistant cell lines although an overexpression of TS gene exists (at mRNA level) in these cell lines as compared with corresponding parental cells. Most of the chromosomal gains identified in this study occur frequently in sporadic colorectal tumours and has been associated to a poor prognosis and a greater progression of the tumour and could be related to a worse chemotherapy response. The chromosomal imbalance profile detected in 5FU-resistant cell lines should provide a basis for interpreting mechanisms of 5FU-resistance in colorectal cancer and also possibly in other tumours treated with this agent. This study also identified new genes potentially implicated in 5FU-resistance and suggests new targets that could be useful for the chemotherapy treatment of colorectal cancer.

Experimental and Clinical Pharmacology: New drugs for colorectal cancer - mechanisms of action

SYNOPSIS Several new drugs have recently been approved for the treatment of advanced colorectal cancer. Raltitrexed is a folate-based inhibitor of thymidylate synthase. Capecitabine is an orally active prodrug of 5-fluorouracil which undergoes some tumour selective activation. Irinotecan is the first registered topoisomerase I poison with activity against this tumour, whereas oxaliplatin is a platinum analogue. The lack of cross-resistance between these drugs has sparked preclinical and clinical studies of a multitude of combination regimens. These regimens may improve the outcomes for patients in the near future.

Pemetrexed for diffuse malignant pleural mesothelioma

The treatment of diffuse malignant pleural mesothelioma (MPM) is currently less than satisfactory. Survival statistics are best for the epithelial subtype and for tumors that can be completely removed by surgery. Radiotherapy provides no survival benefit and is used only to palliate symptoms or to prevent growth along needle tracks and incisions. The response to chemotherapeutic agents is poor, and to date there is no standard cytotoxic treatment. Antimetabolites may have some activity, and one trial of high-dose methotrexate has suggested a moderate response rate. There is also no evidence that any chemotherapy regimen given as a part of multimodality treatment, including radiotherapy and surgery, can improve survival. Recently, a renewed interest in MPM led to clinical trials with promising preliminary results for several agents alone or in combination, including gemcitabine, raltitrexed, vinorelbine, and pemetrexed. Pemetrexed, a folate-based inhibitor of thymidylate synthase and other enzymes, is currently under investigation in multiple malignancies. A phase I study of pemetrexed in combination with cisplatin showed a remarkable response rate of approximately 50% in patients with the epithelial subtype of MPM. This led to a large international study of the combination of pemetrexed plus cisplatin. Semin Oncol 29 (suppl 5):30-35. Copyright 2002, Elsevier Science (USA). All rights reserved.

Pemetrexed for diffuse malignant pleural mesothelioma.

The treatment of diffuse malignant pleural mesothelioma (MPM) is currently less than satisfactory. Survival statistics are best for the epithelial subtype and for tumors that can be completely removed by surgery. Radiotherapy provides no survival benefit and is used only to palliate symptoms or to prevent growth along needle tracks and incisions. The response to chemotherapeutic agents is poor, and to date there is no standard cytotoxic treatment. Antimetabolites may have some activity, and one trial of high-dose methotrexate has suggested a moderate response rate. There is also no evidence that any chemotherapy regimen given as a part of multimodality treatment, including radiotherapy and surgery, can improve survival. Recently, a renewed interest in MPM led to clinical trials with promising preliminary results for several agents alone or in combination, including gemcitabine, raltitrexed, vinorelbine, and pemetrexed. Pemetrexed, a folate-based inhibitor of thymidylate synthase and other enzymes, is currently under investigation in multiple malignancies. A phase I study of pemetrexed in combination with cisplatin showed a remarkable response rate of approximately 50% in patients with the epithelial subtype of MPM. This led to a large international study of the combination of pemetrexed plus cisplatin.

In vivo antitumor efficacy of CW252053, a folate-based thymidylate synthase inhibitor.

Previous studies have demonstrated that CW252053, a quinazoline antifolate, exhibits potent inhibitory activity against thymidylate synthase (TS) as well as cytotoxic activity against tumor cell lines in vitro. In this study, we evaluated the in vivo antitumor efficacy of CW252053 in the mouse tumor model. Female B6D2F1 mice were injected with LY3.7.2C TK-/- (thymidine kinase deficient mouse lymphoma) cells into the gastrocnemius muscle. Then, CW252053 was administered twice daily by intraperitoneal injection for 10 days, and tumor growth was monitored daily by leg diameter measurement. All animals in the vehicle, 5-FU, and low dose (30 mg/kg) CW252053 treated groups died between days 12 and 23 because of the tumor burden. In contrast, dosing with 60 mg/kg of CW252053 produced a cure rate against tumor growth of 37.5% and a survival rate of 50%. Even more significantly, a higher dose of CW252053 (120 mg/kg) elicited both a 100% cure rate and a 100% survival rate at the termination of the study, confirming that this compound has very potent in vivo antitumor activity against tumor growth. During the experimental period of this study no signs of toxicity were observed even at the high CW252053 dosage rate of 120 mg/kg.

Clinical and preclinical pharmacokinetics of raltitrexed.

Raltitrexed is a specific, folate-based inhibitor of thymidylate synthase with activity in advanced colorectal cancer comparable with that of fluorouracil (5-fluorouracil) plus folinic acid. Its activity is enhanced by rapid cellular entry and polyglutamation, with the polyglutamated derivatives having approximately 100-fold greater inhibitory potency than the parent compound. A number of phase I/pharmacokinetic studies have been performed, including schedules involving a 15-minute infusion every 3 weeks, weekly x 6 every 8 weeks, and every 2 weeks. The maximum tolerated dose (MTD) for the 3-weekly schedule was 3.5 to 4.5 mg/m2 in adults and 6 mg/m2 in a paediatric population. The MTDs for the other schedules have not yet been reported. The disposition of raltitrexed in patients is best described by a 3-compartment model with a terminal half-life (t1/2gamma) of 260 hours, the latter being subject to significant interpatient variability. A similar protracted t1/2gamma has been detected in all of the animal species studied. Together with evidence from the mass-balance studies performed, this delayed elimination suggests considerable sequestration of raltitrexed in tissues, predominantly as polyglutamate forms. Nevertheless, there has been no pharmacokinetic evidence of drug accumulation in plasma following repeated administration. On the basis of animal experiments, the oral bioavailability and penetration of raltitrexed into cerebrospinal fluid are both likely to be limited in the clinical setting. Raltitrexed is over 90% bound to plasma protein over the concentration range of 20 to 100 micromol/L. Apart from poly-glutamation, raltitrexed does not appear to be metabolised to a significant extent, and most of the excreted drug (approximately 20% of the administered dose) is recovered unchanged in the urine within the first 24 hours post-administration. The average clearance of raltitrexed is 2.4 L/h (40 ml/min), and this value is significantly reduced in patients with compromised renal function (glomerular filtration rate of 25 to 65 ml/min). These patients are more likely to experience severe antiproliferative toxicity with raltitrexed. A careful evaluation of renal function, particularly in the elderly, is warranted. It has not been possible to establish strong correlations between the plasma pharmacokinetics of raltitrexed and toxicity, and the cellular pharmacokinetics of raltitrexed may be more predictive. Studies in mice have demonstrated that delayed administration of folinic acid can assist in the recovery of animals from antiproliferative toxicity, possibly by promoting the release of polyglutamated drug from tissues. This approach should be evaluated as a rescue regimen in patients with severe proliferative toxicity.

Potent and selective activity of a combination of thymidine and 1843U89, a folate-based thymidylate synthase inhibitor, against Plasmodium falciparum.

Unlike mammalian cells, malarial parasites are completely dependent on the de novo pyrimidine pathway and lack the enzymes to salvage preformed pyrimidines. In the present study, first, it is shown that 1843U89, even without polyglutamylation, is a potent folate-based inhibitor of purified malarial parasite thymidylate synthase. The binding was noncompetitive with respect to methylenetetrahydrofolate, and 1843U89 had a K(i) of 1 nM. The compound also had potent antimalarial activity in vitro. Plasmodium falciparum cells in culture were inhibited by 1843U89, with a 50% inhibitory concentration of about 70 nM. The compound was effective against drug-sensitive as well as drug-resistant clones of P. falciparum. As predicted by the biochemistry of the parasite, the potent inhibition of parasite proliferation by 1843U89 could not be reversed with 10 microM thymidine. In contrast, in the presence of 10 microM thymidine, mammalian cells were unaffected by 1843U89 even at concentrations as high as 0.1 mM, thus offering a selectivity window of more than 1,000-fold. On this basis, folate-based thymidylate synthase inhibitors may represent a powerful additional tool that can be used to combat drug-resistant malaria.

Thymidine phosphorylase: its role in sensitivity and resistance to anticancer drugs.

Thymidine phosphorylase (TP) is an angiogenic enzyme present in normal tissues. Increased levels are found in many tumors, in stromal cells, tumor cells or both. High tumor TP levels may confer a poor prognosis. Cytokines (including interferons), tissue hypoxia and low pH increase TP levels. The influence of tumor TP on fluoropyrimidine toxicity is variable, but capecitabine is a prodrug of fluorouracil that requires activation by TP and hence may have a higher therapeutic index than other fluoropyrimidines. Folate-based thymidylate synthase inhibitors may also be more effective in tumors with a high TP because of increased degradation of endogenous thymidine. Copyright 1999 Harcourt Publishers Ltd.

Folate-based thymidylate synthase inhibitors in cancer chemotherapy.

Understanding the relationship between chemical structure and biological properties of folate analogs, particularly their interactions with the target enzymes, transport proteins and folate-metabolizing enzyme, folylpolyglutamate synthetase (FPGS), has enabled the rational design and development of the selective thymidylate synthase (TS) inhibitors with folate-based structures for clinical uses. These compounds specifically inhibit TS devoid of concomitant effects at other loci, unlike 5-fluorouracil (5-FU). ZD1694 ('Tomudex') was designed as a non-nephrotoxic and highly active analog of N10-propargyl-5,8-dideazafolic acid (CB3717), which is a potent TS inhibitor but had unacceptable nephrotoxicity caused by its poor water solubility. The potent cytotoxic activity of ZD1694 is dependent upon active uptake into cells via the reduced folate carrier (RFC), and subsequent rapid and extensive metabolism to polyglutamate forms inside cells. Marked enhancement of the TS inhibitory activity has been noted as the glutamate chain is elongated. Polyglutamation is critical to the biological activity of ZD1694 against tumor and normal proliferating tissues. The retentive property of ZD1694 polyglutamates inside cells led to a single, infrequent administration schedule in clinical studies. ZD1694 has completed phase I and phase II evaluation with activity observed in several tumor types, particularly in colorectal cancer with a 26% objective response rate. A recent European phase III study of ZD1694, randomized against a 5-FU plus leucovorin regimen, demonstrated an equivalent response rate for advanced colorectal cancer (complete or partial responses; 20 versus 17%) and less toxicity than seen with the latter regimen. The newer selective TS inhibitors, which retain potency for TS inhibition but are not substrates for RFC and/or FPGS, are currently under clinical evaluation. These classes of compound may have benefits for circumvention of resistance by virtue of alterations in these protein functions and for the management of toxicity.

Carboxylic acid bioisosteres of γ-linked dipeptide analogues of the folate-based thymidylate synthase (TS) inhibitor, 2-desamino-2-methyl- N10-propargyl-5,8-dideazafolic acid (ICI 198583)

Tetrazole carboxylic acid bioisosteres of γ-linked dipeptide derivatives of 2-desamino-2-methyl-N 10-propargyl-5,8-dideazafolic acid (ICI 198583), a folate-based inhibitor of thymidylate synthase (TS), were synthesised by multistep routes starting from the appropriate pteroic acid analogue and Z- d-Ala or d-Glu. They exhibited excellent TS inhibitory activities which, however, were not accompanied by a parallel improvement in the L1210 cell growth inhibition.

Mechanisms of resistance to N-[5-[ N-(3, 4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)- N-methylamino]-2-thenoyl]-L-glutamic acid (ZD1694), a folate-based thymidylate synthase inhibitor, in the HCT-8 human ileocecal adenocarcinoma cell line

N-[5-[ N-(3,4-Dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)- N-methylamino]-2-thenoyl]-L-glutamic acid (ZD1694) is a folate-based thymidylate synthase (TS; EC 2.1.1.45) inhibitor. Metabolism to higher chain length polyglutamates is essential for its optimal cytotoxic effect. A ZD1694-resistant (300-fold) human ileocecal carcinoma cell line (HCT-8/DW2) was developed, and its mechanism of resistance was evaluated. TS activities in situ and TS protein levels in the HCT-8 parental line and HCT-8/DW2 were similar (168 ± 47 vs 137 ± 25 pmol/hr/10 6 cells and 2.05 ±0.28 vs 2.07 ± 0.19 pmol/mg protein, respectively). The ic 50 values of ZD1694 for TS inhibition in cell-free extracts were similar in both lines, but the ic 50 of ZD1694 for TS inhibition in situ in HCT-8/DW2 cells was 27- and 268-fold higher than that in HCT-8 cells at 0 and 24 hr, respectively, after a 2-hr drug exposure. Folylpolyglutamate synthetase (FPGS; EC 6.3.2.17) activity was significantly lower in resistant HCT-8/DW2 cells as compared with parental HCT-8 cells (88 ± 40 vs 1065 ± 438 pmol/hr/mg protein when ZD1694 was used as substrate). The combined endogenous pool of methylenetetrahydrofolate and tetrahydrofolate in HCT-8/DW2 cells was also decreased. In addition, HCT-8/DW2 cells accumulated lower levels of methotrexate (MTX) in a 2-hr period, although the initial velocity of MTX transport was similar to that in parental HCT-8 cells. The lower level of FPGS activity and the lower level of (anti)folate accumulation in HCT-8/DW2 correlated with drug resistance and with the higher ic 50 of ZD1694 for in situ TS inhibition. In addition, drug resistance was also correlated with the rapid recovery of in situ TS activity after drug treatment. In brief, in this highly ZD1694-resistant HCT-8 cell line, resistance is associated with decreased FPGS activity, which, in turn, affects the metabolism of ZD1694 and consequently the extent and duration of in situ TS inhibition by the drug.

Biochemical modulation of iododeoxyuridine by N6-[4-(morpholinosulfonyl)benzyl]- N6-methyl-2, 6-diaminobenz[ cd]indole glucuronate (AG-331) leading to enhanced cytotoxicity

Inhibition of thymidylate synthase (TS) may increase incorporation of thymidine analogues into DNA, leading to increased inhibition of colony formation in tumor cells. We have reported previously that TS inhibition by N-(5-[ N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6,-ylmethyl)- N-methylamino]-2-thenoyl)-L-glutamic acid (ICI D1694 or Tomudex), a folate-based TS inhibitor, increases the cytotoxicity of iododeoxyuridine (IdUrd), a thymidine analogue, in MGH-U1 human bladder and HCT-8 human colon cancer cells. N 6-[4-(Morpholinosulfonyl)benzyl]- N 6-methyl-2,6-diaminobenz[ cd]-indole glucuronate (AG-331) differs from ICI D1694 in that it is a de novo designed lipophilic TS inhibitor, it does not require a specific carrier for cellular uptake, and it does not undergo intracellular polyglutamation. Exposure of MGH-U1 cells to 5 μM AG-331 for 24 hr decreased clonogenic survival by 30%, but almost completely inhibited TS activity. IdUrd is a cytotoxic thymidine analogue, with IC 50 and IC 90 values after 24-hr exposures in MGH-U1 cells of 13 and 81 μM, respectively. The combination of IdUrd and AG-331 resulted in an enhanced antitumor effect, as compared with the effect of either agent alone. The cytotoxic IC 50 of IdUrd decreased from 13 to 1.5 μM, and the IC 90 decreased from 81 to 5 μM with the addition of 5 μM AG-331. Biochemical studies of the combination revealed that pretreating MGH-U1 cells with 5 μM AG-331 increased IdUrd incorporation into cellular DNA by 3.8-fold. This increased incorporation was associated with a greater proportion of DNA single-strand breaks than observed with either agent alone, and the combination of 5 μM AG-331 plus IdUrd produced up to a 2.5-fold increase in DNA single-strand breaks as compared with IdUrd alone. The effects of AG-331, IdUrd, and the combination of IdUrd and AG-331 on the colony-forming ability of normal human bone marrow CFU-GM cells was determined as a measure of myelosuppression. The combination of IdUrd and AG-331, at the same concentrations as those used in the MGH-U1 cells, produced a wider therapeutic index relative to that of IdUrd alone, and the therapeutic index for the combination was 6.5, as compared with 4.0 for IdUrd plus ICI D1694 in previous studies from this laboratory. These observations suggest that the combination of IdUrd and AG-331 may enhance antitumor effects with minimal myelosuppression in vivo.

5,10-Dideazatetrahydrofolic acid reduces toxicity and deoxyadenosine triphosphate pool expansion in cultured L1210 cells treated with inhibitors of thymidylate synthase

5,10-Dideazatetrahydrofolic acid (DDATHF) reduces de novo purine biosynthesis by inhibiting glycinamide ribonucleotide transformylase. ICI D1694 and CB3717 are folate-based inhibitors of thymidylate synthase (TS). Fluorodeoxyuridine (FdUrd) following metabolism to FdUMP also inhibits TS. In cultured L1210 cells DDATHF reduced the toxicity of ICI D1694, CB3717 and FdUrd in a concentration-dependent manner. This protection correlated with a prevention of the increase in intracellular dATP pools seen in cells exposed to the TS inhibitors alone. The possibility that DDATHF protection might be due to competition for cell entry is not likely since CB3717 and FdUrd but not ICI D1694 enter the cell by independent transport processes. Exogenous hypoxanthine (HX) had no effect on the toxicity of TS inhibitors. However, HX increased the protective effect of DDATHF from ICI D1694 toxicity, had no effect on the CB3717-DDATHF interaction, and reduced the protective effect of DDATHF on FdUrd toxicity. HX prevented the fall in dATP levels caused by DDATHF addition to cells treated with TS inhibitors. HX had different effects on dTTP levels in cells treated with DDATHF and quinazoline TS inhibitors compared to FdUrd. Together these results support the hypothesis that imbalance in dTTP and dATP pools is an important determinant of cytotoxicity in antifolate-treated cells. In addition, these findings suggest that intracellular reduced folates interconvert to catalyse reactions in metabolically perturbed cells.

Biochemical effects of folate-based inhibitors of thymidylate synthase in MGH-U1 cells

Biochemical effects of folate-based inhibitors of thymidylate synthase in MGH-U1 cells

Comparative cytotoxicity of folate-based inhibitors of thymidylate synthase and 5-fluorouracil � leucovorin in MGH-U1 cells

Comparative cytotoxicity of folate-based inhibitors of thymidylate synthase and 5-fluorouracil � leucovorin in MGH-U1 cells

Benzoquinazoline inhibitors of thymidylate synthase: enzyme inhibitory activity and cytotoxicity of some 3-amino- and 3-methylbenzo[f]quinazolin-1(2H)-ones.

The synthesis and thymidylate synthase (TS) inhibitory activity of a series of simple benzo[f]-quinazolin-1(2H)-ones are described. Fully aromatic 3-amino compounds with compact lipophilic substituents in the 9-position were found to have I50 values as low as 20 nM on the isolated enzyme, and represent the first examples of potent, folate-based TS inhibitors that completely lack any structural feature corresponding to the (p-aminobenzoyl)glutamate moiety of the cofactor. A number of the compounds also showed moderate growth inhibitory activity against a human colon adenocarcinoma cell line (SW480), with IC50 values as low as 2 microM.