Rosemary Kimbell

ZD1694 (Tomudex): A new thymidylate synthase inhibitor with activity in colorectal cancer

ZD1694 (Tomudex) is a new antifolate which is a specific inhibitor of thymidylate synthase (TS). Evidence suggests that ZD1694 has a spectrum of activity that only partially overlaps with 5-fluorouracil (modulated with leucovorin) against colon tumours in vitro. Potent cytotoxic activity is dependent upon active uptake into cells via the reduced folate/methotrexate cell membrane carrier (RFC) and subsequent metabolism to polyglutamated forms (tri, tetra and pentaglutamates). These polyglutamates are approximately 60-fold more active as TS inhibitors and are not effluxed readily from cells. Extensive polyglutamation also occurs in various mouse tissues (e.g. small intestinal epithelium, liver and kidney), resulting in high tissue/plasma drug ratios which persist for a prolonged period. ZD1694 has antitumour activity in mice, although the high plasma thymidine in this species complicates: (1) the interpretation of therapeutic index; (2) tumour types in which activity is likely to be observed; and (3) translation of doses and schedules for clinical evaluation. ZD1694 entered clinical study and has completed Phase I and II evaluation, with activity observed in several tumour types. Appreciable activity in the Phase II colorectal study (29% objective response rate on interim analysis) led to the current Phase III study, randomised against 5-fluorouracil/leucovorin.

Kinetic characteristics of ICI D1694 : a quinazoline antifolate which inhibits thymidylate synthase

The thymidylate synthase (TS) inhibitor ICI D1694 (N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N -methylamino]-2 - thenoyl)-S-glutamic acid) is a structural analogue of the substrate N5,N10-methylenetetrahydrofolate (5,10-CH2FH4) and is currently under clinical evaluation as a treatment for cancer. The compound is shown here to be a mixed non-competitive inhibitor of TS from murine leukemia (L1210) cells when 5,10-CH2FH4 is varied. This result suggests formation of an inactive complex between TS, 5,10-CH2FH4 and the inhibitor. Thus, binding to only one of the two active sites on the TS homodimer may be sufficient to prevent catalysis fully. Treatment of L1210 cells with ICI D1694 is known to cause intracellular accumulation of the tetraglutamate derivative which is shown here to have a 60-fold higher affinity for TS. The IC50 for inhibition of L1210 cell growth is below the Ki value of ICI D1694 for L1210 TS but above that of the tetraglutamate. The formation of polyglutamates and concentration of drug inside cells, therefore, seem to be responsible for biological activity.

The Role of the Reduced-Folate Carrier and Metabolism to Intracellular Polyglutamates for the Activity of ICI D1694

The uptake of ICI D1694 into L1210 cells is very rapid and evidence strongly suggests that transport is via the reduced-folate/MTX cell membrane carrier (RFC); for example a cell line with a greatly impaired RFC is highly resistant to ICI D1694. Polyglutamates can be found intracellularly within a few minutes, so that experiments initially designed to measure transport were actually measuring transport and polyglutamation. After 30mins, in normal serum-containing tissue culture medium, the concentration of polyglutamates (di, tri and tetra) exceeded that of the parent drug 6-fold. Studies where cells were resuspended in drug-free medium demonstrated that the parent drug and its diglutamate could readily leave the cell. Folinic acid could markedly decrease the polyglutamation of ICI D1694, but had to be given simultaneously with the drug as a 4hr delayed rescue was less effective because substantial polyglutamation had already occurred. This effect was translated into considerable antagonism for cell growth inhibition by simultaneous folinic acid. The importance of the metabolism of ICI D1694 to polyglutamates to its potent cytotoxic activity is demonstrated by compounds related in structure to ICI D1694 but with different properties for the RFC and FPGS. For example, 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolate (ICI 198583) owes its less potent cytotoxic activity to its poorer FPGS substrate activity (Km 40 microM compared with 1.3 microM for ICI D1694). Replacing the 2-methyl of either compound with amino, which appears to prevent use of the RFC, has a deleterious effect on growth inhibitory activity presumably by limiting the transport of the parent compounds into the cells, thereby slowing the rate of polyglutamate formation. Again a single change to another part of the molecule, that is methylation of the 7-position can have serious consequences on cytotoxic potency, particularly for the ICI D1694 molecule. The 7-methylated compounds are apparently poor or non-substrates for FPGS and therefore retain activity against a cell line unable to polyglutamate antifolates. These same compounds are only slightly affected by coincubation with folinic acid in L1210 tissue culture, consistent with the failure of these compounds to form intracellular polyglutamates. The results of short-exposure assays and in situ TS assays confirms that 7-methylation largely prevents the formation of a retained drug-form (polyglutamates), continuous exposure being necessary to maintain TS inhibition and cause a cytotoxic effect after removal of extracellular drug.(ABSTRACT TRUNCATED AT 400 WORDS)

Combination of raltitrexed with other cytotoxic agents : Rationale and preclinical observations

Agents for use in combination therapy should be effective as monotherapy in the tumour type of interest, have different mechanisms of action or pharmacology, and preferably non-overlapping toxicity profiles. Raltitrexed is effective as monotherapy in a number of tumour types, but it is hoped that combining it with other cytotoxic agents will lead to enhanced efficacy. Raltitrexed and 5-fluorouracil (5-FU) are specific and non-specific inhibitors, respectively, of thymidylate synthase, a critical enzyme in the de novo synthesis of DNA. Preclinical studies have indicated that raltitrexed and 5-FU have an incompletely overlapping spectrum of antitumour activity and may have additive or synergistic effects on colon carcinoma cells. These interactions are schedule-dependent (raltitrexed should precede 5-FU). Pre-treatment of colon carcinoma cells with raltitrexed has also been shown to increase intracellular levels of phosphoribosyl pyrophosphate resulting in increased incorporation of 5-FU nucleotides into RNA. Raltitrexed has a different mechanism of action from two other new agents active in colorectal cancer, irinotecan and oxaliplatin, and tumours are therefore not necessarily cross-resistant. Short pre-exposure of colon carcinoma cells to the irinotecan active metabolite, 7-ethyl-10-hydroxy-camptothecin (SN-38), prior to exposure to raltitrexed has consistently resulted in synergistic cell kill, whereas the reverse sequence is antagonistic. Preliminary results indicate that equitoxic doses of raltitrexed and cisplatin, or oxaliplatin, are antagonistic in two colon carcinoma cell lines. However, because there are major difficulties in translating preclinical drug combination results to the clinical settings, these results should be interpreted with caution.

Relationships between resistance to cisplatin and antifolates in sensitive and resistant tumour cell lines

Possible relationships between tumour resistance to cisplatin and the folate-based thymidylate synthase (TS) inhibitors, CB3717 and ZD1694 (tomudex), have been investigated in vitro using a panel of tumour cell lines (predominantly human ovarian), either parental or possessing acquired resistance to cisplatin or ZD1694. Across eight parent human tumour cell lines, ZD1694 was the most potent drug (mean IC50 of 1.9 x 10(-8) M), being over 250 times as potent as its prototype CB3717 (mean IC50 of 4.8 x 10(-6) M). In five pairs of acquired cisplatin-resistant human tumour cell lines (three ovarian, one cervical and one testicular) which encompass all of the main known mechanisms of platinum drug resistance, ZD1694, CB3717 and the DHFR inhibitor, methotrexate, all exhibited non-cross-resistance. The cervical line, HX/155cisR, showed collateral sensitivity to ZD1694, CB3717, 5-fluorouracil (FUra) and fluorodeoxyuridine (FdUrd). One cell line, A2780cisR, showed a low level of cross-resistance to FUra (resistance factor, RF, of 1.5) and FdUrd (RF of 3.8). A2780cisR, in common with two other cisplatin-resistant lines, did not possess elevated TS activity compared with its parent. Cisplatin retained activity in four acquired ZD1694-resistant cell lines (encompassing reduced folate transport, elevated TS and defective polyglutamation mechanisms of resistance). Furthermore, combinations of ZD1694 with each of the platinum-based drugs, cisplatin, carboplatin and the recently introduced orally administrable, JM216, all showed additive growth inhibitory effects by median effect analysis. These data suggest that the tumour inhibitory properties of the recently introduced highly potent TS inhibitor, ZD1694, and cisplatin, and, moreover, their respective mechanisms of resistance, do not overlap. Therefore, these drugs may be considered for combination in the clinic.

Quinazoline antifolate thymidylate synthase inhibitors : γ-linked L-D, D-D, and D-L dipeptide analogues of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583)

The syntheses of gamma-linked L-D, D-D, and D-L dipeptide analogues of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583) are described. The general methodology for the synthesis of these molecules involved the preparation of the dipeptide derivatives employing solution phase peptide synthesis followed by condensation of the dipeptide free bases with the appropriate pteroic acid analogue via diethyl cyanophosphoridate (DEPC) activation. In the final step, tert-butyl esters were removed by trifluoroacetic acid (TFA) hydrolysis. Z-L-Glu-OBut-gamma-D-Ala-OBut, for example, was prepared from alpha-tert-butyl N-(benzyloxycarbonyl)-L-glutamate and tert-butyl D-alaninate via isobutyl-mixed anhydride coupling. The Z-group was removed by catalytic hydrogenolysis and the resulting dipeptide free base condensed with 2-desamino-2-methyl-N10-propargyl-5,8-dideazapteroic acid via DEPC coupling. Finally, tert-butyl esters were removed by TFA hydrolysis to give ICI 198583-gamma-D-Ala. The compounds were tested as inhibitors of thymidylate synthase and L1210 cell growth. Good enzyme and growth inhibitory activity were found with gamma-linked L-D dipeptides, the best examples being the Glu-gamma-D-Glu derivative 35 (Ki = 0.19 nM, L1210 IC50 = 0.20 +/- 0.017 microM) and the Glu-gamma-D-alpha-aminoadipate derivative 39 (Ki = 0.12 nM, L1210 IC50 = 0.13 +/- 0.063 microM). In addition, ICI 198583 L-gamma-D-linked dipeptides were resistant to enzymatic degradation in mice.

The influence of drug-exposure conditions on the development of resistance to methotrexate or ZD1694 in cultured human leukaemia cells.

The influence of drug‐exposure conditions on the development of resistance to methotrexate (MTX) or ZD1694 was studied by treating MOLT‐3 human lymphoblastic‐leukaemia cells in a continuous or a pulsatile (high‐dose, short‐term) drug‐exposure schedule. Continuous exposure of the cells to MTX with stepwise escalation of the drug concentrations resulted in a MTX‐resistant sub‐line (MOLT‐3/MTX10,000) with impaired reduced‐folate carrier (RFC) and increased dihydrofolate‐reductase (DHFR) activity. Conversely, a MTX‐resistant clone (MOLT‐3/MTX·P‐9) with unaltered RFC and DHFR activity, but with decreased cellular accumulation of antifolates, was selected by high‐dose short‐term treatment of the cells with MTX. MTX resistance in the latter cells was pronounced after short‐term rather than continuous‐exposure incubation with MTX, suggesting defective polyglutamation of the drug. On the other hand, 2 ZD1694‐resistant sub‐lines which were established by continuous (MOLT‐3/ZD1694·C) or by pulsatile drug‐exposure schedule (MOLT‐3/ZD1694·P‐9) demonstrated extremely low accumulation and poor retention of [3H]ZD1694, with no change of initial drug uptake and little or no increase of thymidylate‐synthase (TS) activity, irrespective of drug‐exposure conditions for their establishment. HPLC analysis displayed a virtual absence of ZD1694 polyglutamates in both ZD1694‐resistant sub‐lines and low accumulation in MOLT‐3/MTX·P‐9 as compared with the parent line. However, folylpolyglutamate‐synthetase (FPGS) mRNA was only moderately decreased in the 2 ZD1694‐resistant sub‐lines and to an even lesser extent in MOLT‐3/MTX·P‐9. In addition, γ‐glutamyl‐hydrolase (GGH) activity was not increased, but was slightly down‐regulated in the polyglutamation‐defective sublines. These results indicate that the mechanism(s) of the resistance developed may depend not only on drug‐exposure conditions while raising resistance but also on the biochemical properties of the drug.

The Antitumour Activity of ZD9331, a Non-Polyglutamatable Quinazoline Thymidylate Synthase Inhibitor

Thymidylate synthase (TS) is regarded as a good target for the development of quinazoline (folate-based) anticancer agents. The quinazoline-based TS inhibitor, D1694 or Tomudex (trade mark of Zeneca PLC) demonstrated exciting activity against colorectal tumours in the worldwide Phase II1 clinical studies and is currently in Phase III study for this tumour type. Tomudex is an excellent substrate for folylpolyglutamate synthetase (FPGS) and is almost completely metabolised to polyglutamate forms (mainly tetra and pentaglutamates) that are not readily effluxed from the cell2. Prolonged intracellular drug retention is thus a feature of this drug. This has the advantage of showing antitumour activity by bolus administration and the current clinical protocol is a 15min infusion once every 3 weeks. The polyglutamates of Tomudex are ~ 60-fold more active than the parent drug as TS inhibitors2. Polyglutamation is thus a requirement for antitumour activity and may offer some tumour selectivity. However a mechanism of acquired and possibly intrinsic resistance to Tomudex is the failure of cells to polyglutamate the drug3. In order to broaden the spectrum of tumours responsive to TS inhibition by antifolates we synthesised and evaluated compounds unable to undergo such metabolism. As drug retention is not a general feature of this class of compound, infusion protocols were employed to evaluate their activity in mice4. Since TS inhibition is only achieved during drug administration this offers a high degree of control over the length of time that DNA synthesis is inhibited. Prevention of polyglutamation is possible through modification of the quinazoline ring (7-methylation)5 or the glutamate residue. Compound design also focused on developing TS inhibitors with potency at least equal to the polyglutamates of Tomudex (Ki tetraglu = 1nM). Acid-containing (water-soluble) and highly lipophilic analogues were synthesised but we concentrated on those that had high water-solubility and used the reduced-folate/MTX carrier (RFC) for cell entry. One such compound, ZD9331, was chosen as the most promising compound with activity against a number of experimental tumours (murine and human xenografts) in mice6, 7, 8. The in vitro activity of ZD9331 is summarised below and, where appropriate, is compared with other TS inhibitors of known biochemical profile. Thus N10-propargyl-5,8-dideazafolic acid (CB3717), the prototype quinazoline TS inhibitor9, uses the RFC poorly and is slowly polyglutamated when compared with ICI 19858310 or Tomudex2. AG337, represents a newer class of lipophilic quinazolines discovered by Agouron Pharmaceuticals that neither uses the RFC nor is a substrate for FPGS11.

Cellular pharmacokinetics of ZD1694 in cultured human leukaemia cells sensitive, or made resistant, to this drug

We have analysed the cellular metabolism of a novel thymidylate synthase (TS) inhibitor, ZD1694, in MOLT-3 and K562 human leukaemia cell lines sensitive to or made resistant to ZD1694 by continuous exposure of the cells to ZD1694 with stepwise escalation of the drug concentration. The initial cellular uptake of [3H]ZD1694 was greater in K562 cells than in MOLT-3 cells and the drug accumulated approximately 3-fold more in the former cells following incubation with 0.1 μM ZD1694 at 37°C for 24 h. TS and dihydrofolate reductase activities were not significantly different between the two cell lines. After a 30-min incubation with the drug at 37°C, 85% of the total drug (2.3pmol/mg protein) in K562 cells was found as tri-to pentaglutamates, whereas MOLT-3 cells accumulated less drug in this time (0.83 pmol/mg protein) and polyglutamates of chain length greater than triglutamate were not found to a significant extent. When the incubation time was extended to 24 h, the polyglutamate profile in K562 cells was progressively shifted towards those of long glutamate chain length and 59% of the total cellular drug (204 pmol/mg protein) was identified as the penta form. In contrast, even distribution between tri-and pentaglutamate was observed in MOLT-3 cells. Total cellular polyglutamates were approximately 3-fold higher in K562 cells than in MOLT-3 cells, and this may explain the 2.5-fold difference in the sensitivity to ZD1694 between the two cell lines. Continuous exposure of MOLT-3 and K562 cells to ZD1694 up to 1 μM or 0.1μM resulted in 1600-and 4200-fold resistant sublines, respectively (MOLT-3/ZD1694·C and K562/ZD1694·C). The resistant MOLT-3 cells showed a markedly lower cellular accumulation and poor retention of [3H]ZD1694 with no significant change of initial drug uptake by 10 min and with a little increase of TS activity. HPLC analysis demonstrated that more than 90% of the3H co-eluted with the monoglutamate (parent drug) in the resistant MOLT-3 cells, indicating extremely diminished polyglutamation in the cells. On the other hand, cellular uptake of [3H]ZD1694 was extensively impaired in K562/ZD1694·C cells and cellular accumulation of the drug was only 2.5% of that in the parent cells following 24 h incubation with the drug. Neither an increase of TS or dihydrofolate reductase activity nor a change in the polyglutamate formation profile was observed in the resistant K562 cells. These results indicate that the cellular ability to produce the polyglutamate metabolites of ZD1694 must influence the sensitivity of the tumour cells to this drug, and development of mechanisms involved in the ZD1694 resistance may relate to the intrinsic biochemical properties of the cells.

Synthesis of novel quinazoline-based antifolates with modified glutamate side chains as potential inhibitors of thymidylate synthase and antitumour agents

Abstract Several novel antifolates, derivatives of 2-desamino-2-methyl- N 10 -propargyl-5,8-dideazafolic acid, were synthesised as inhibitors of thymidylate synthase (TS) and antitumour agents. This was accomplished by first developing routes to the key intermediates Glu-Ome-γ-ψ[CSNH]Glu(OEt)-OEt ( 8 ), Glu-OBu t -γ-ψ[CH 2 NH]Glu(OBu t )-OBu t ( 16 ), Glu-OMe-γ-ψ[CN 4 ]Gly-OMe ( 23 ) and its 2,5-disubstituted regioisomer ( 22 ), followed by DEPC coupling to 4-[ N -[3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)-methyl]- N -prop-2-ynylamino]benzoic acid ( 9 ) or 4-[ N -[(3,4-dihydro-2,7-dimethyl-4-oxo-6-quinazolinyl)-methyl]- N -prop-2-ynylamino]-2-fluorobenzoic acid ( 24 ), and finally removal of the protecting groups. The resulting quinazoline-based antifolates with modified glutamate side chains, and in particular, the tetrazole derivatives 26 and 29 displayed potent TS and L1210 cell growth inhibitory activities ( e.g. , for 26 : TS IC 50 = 2.4 nM, L1210 IC 50 = 1.3 μM).