Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0023418 (leukemia)
 93,477 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Six new analogues of 5,8-dideazaisofolic acid and 5,8-dideazaisoaminopterin were synthesized in an effort to obtain enhanced antitumor activity. The modifications included the replacement of the 2-amino group by hydrogen or methyl as well as the inclusion of a methyl substituent at position 9. Based upon activity against L1210 leukemia cells in culture, three of the new analogues together with one compound described previously were evaluated for cytotoxicity in vitro using three human tumor cell lines (Colo 320 DM, Hep G2 and HL-60). The most effective compound was 2-desamino-N9-methyl-5,8-dideazaisoaminopterin (2c) with the HL-60 cells being the most sensitive to its cytotoxic effects. These analogues were evaluated in vitro as inhibitors of dihydrofolate reductase (DHFR) and thymidylate synthase (TS) from human as well as bacterial (Lactobacillus casei) sources. All four of the 4-amino analogues were most effective toward L. casei DHFR compared with human DHFR, with 2-desamino-2-methyl-5,8-dideazaisoaminopterin (2d) and its 9-methyl derivative (2e) having 818- and 430-fold greater selectivity (L. casei/human). Most of the compounds studied were found to be only modest inhibitors of human TS (I50 values = 1.5 to 20 microM) and were therefore at least 40-fold less inhibitory than 10-propargyl-5,8-dideazafolic acid. Nevertheless, reversal of cytotoxicity studies with thymidine, hypoxanthine and folinic acid using the HL-60 cell line suggested that TS is the primary target for these analogues.
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PMID:Studies on the antitumor effects of analogues of 5,8-dideazaisofolic acid and 5,8-dideazaisoaminopterin. 757 41

A series of alkylating phosphoramidate analogs of 5-fluoro-2'-deoxyuridine has been prepared and their growth inhibitory activity evaluated against murine L1210 leukemia and B16 melanoma cells in vitro. These compounds were designed to undergo intracellular release of the phosphoramidate anions, which it was hoped would function as irreversible inhibitors of thymidylate synthase. The expectation was that binding of the nucleoside moiety would be followed by alkylation of the enzyme via the phosphoramidate. The chloride, bromide, iodide, and tosylate analogs were highly potent inhibitors of L1210 cell proliferation, with increased inhibition observed at both higher drug concentrations and longer exposure times. Addition of thymidine completely reversed the inhibition for all compounds, suggesting that these compounds are acting via inhibition of thymidylate synthase. Although the nonalkylating morpholine analog 1f was ca. 50-fold less potent than the methyl(chloroethyl)amino compound, the piperidine analog 1g was only 2-fold less potent, confirming that nitrogen basicity may be as important as the presence of an alkylating group. Addition of thymidine reversed the growth inhibition of the morpholine and piperidine analogs, suggesting that these compounds may also undergo intracellular conversion to 5-fluoro-2'-deoxyuridine 5'monophosphate. The thymidine and deoxyuridine derivatives 2 and 3 showed minimal growth inhibition in the L1210 assay. The alkylating analogs showed modest cytotoxicity against B16 melanoma cells, and the potency of the analogs was more dependent upon the alkylating moiety than on the 5-substituent.
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PMID:Synthesis and biological evaluation of 5-fluoro-2'-deoxyuridine phosphoramidate analogs. 762 6

A method is described for the measurement of the polyglutamates of the quinazoline thymidylate synthase inhibitor, N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin- 6-ylmethyl)-N-methylamino]-2-theonyl)-L-glutamic acid (ICI D1694). This involved incubation of cells with [5-3H]ICI D1694, extraction of the polyglutamates and their analysis by HPLC using an ion-pairing method. Co-chromatography with ICI D1694 and its synthetic di-hexaglutamate standards (UV detection) aided identification of the [3H]polyglutamates in the fractions recovered from the HPLC. Recovery of the polyglutamates at each stage of extraction and analysis was very good (77-84% overall recovery). Polyglutamates readily accumulated as the tri-, tetra and penta forms and occasionally a small amount of hexaglutamate was found. After mouse L1210 leukemia or human W1L2 lymphoblastoid cells were incubated for 30 min with 0.1 microM [3H]ICI D1694 there was a approximately 6-fold concentration effect intracellularly with most of the 3H associated with polyglutamate forms (approximately 75% and 96% for the L1210 and W1L2, respectively). Even some of the higher chain length tetra- and pentaglutamates could be detected at this time. After 4 hr incubation the total level of intracellular 3H had risen to 2-3 microM, greater than 96% of which was associated with polyglutamates (mainly tetra- and pentaglutamates). Four other human cell lines, two ovarian (CH1 and 41M), the MCF-7 breast and the HT-29 colon, were examined for their ability to form intracellular polyglutamates. A 4 hr incubation with 0.1 microM [3H]ICI D1694 resulted in a substantial intracellular accumulation of the drug (20-100-fold) in its polyglutamate forms with only 2-20% remaining as the parent monoglutamate, depending on the cell line. The major polyglutamate was again cell line dependent, ranging from the tri to the penta form. Prolonging the incubation time to 24 hr allowed a further accumulation of drug with a larger percentage appearing as tri- to hexaglutamates. Although cell lines differed in the total level of polyglutamates formed and the pattern of chain length observed, rapid and extensive polyglutamation of ICI D1694 occurred in all the cell types examined.
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PMID:The measurement of polyglutamate metabolites of the thymidylate synthase inhibitor, ICI D1694, in mouse and human cultured cells. 768 Aug 60

Anti-metabolites are among the most important agents used in cancer chemotherapy. Ara-C, the thiopurines and MTX are active drugs for both induction and maintenance chemotherapy of childhood and adult leukaemia, while the new adenosine analogues are active against hairy cell leukaemia, with promising activity against other malignancies such as malignant lymphomas. Methotrexate and 5FU are being used in the treatment of several solid malignancies. Recent advances in the clinical pharmacology of widely used antimetabolites have shown a relationship among dose, plasma concentrations and clearance with the toxicity and anti-tumour activity. Thus, it has been shown that adaptative control of 5FU administration is possible, limiting the toxicity of this drug. Recent advances in the pharmacogenetics of, for example, 6MP and 5FU will possibly enable researchers to identify patients who may have an increased risk of toxicity. For ara-C, some evidence has been obtained to identify populations at risk of no response. In addition, for most anti-metabolites, convincing evidence of their intracellular (intratumour) metabolism has been obtained, thus making it possible to identify patients who are likely to respond to treatment. These studies (eg accumulation of active metabolites such as ara-CTP, thioguanine nucleotides, FdUMP, MTX-polyglutamates; and inhibition of target enzymes such as thymidylate synthase) have made it possible to develop the basis of biochemical modulation--that is, specific manipulation of intracellular metabolism of the drug. It is anticipated that new technical developments in molecular biology, biochemistry, cell biology and immunology will make it possible to improve the identification of resistant patients in order to modulate specifically drug metabolism in the tumour cells. Biochemical modulation has been successful in achieving significant improvements in treatment and currently is a keystone in cancer chemotherapy. Together with the development of promising new anti-metabolites, biochemical modulation (with other drugs, biologicals) will be a major strategy for the future.
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PMID:Clinical pharmacokinetics of anti-metabolites. 813 39

Six novel antifolates with 2,4-diaminopyrimidine-fused five-membered rings containing either pyrrole or cyclopentene rings were characterized at the cellular and biochemical level. Five of these antifolates were more growth inhibitory to the CCRF-CEM human leukemia cell line than methotrexate [MTX; drug concentration effective at inhibiting cell growth by 50% relative to untreated control (EC50), 12 nM], the antifolate used in the clinic, and two were more potent than 10-ethyl-10-deazaaminopterin (EC50, 2.7 nM); similar patterns of response were obtained in the FaDu and A253 squamous carcinoma cell lines. In addition, the growth inhibitory potency of these antifolates was generally less dependent on exposure time than was MTX. Growth inhibitory effects could be reversed by leucovorin, indicating an antifolate mechanism. These antifolates targeted dihydrofolate reductase (DHFR) based on direct human DHFR inhibition assays [drug concentration inhibiting enzyme activity by 50% (IC50), 0.6-28 nM; MTX IC50, 0.8 nM] and the cross-resistance of MTX-resistant CCRF-CEM cells containing elevated DHFR. Inhibition of human thymidylate synthase was generally weak. These 6,5-fused ring heterocyclic antifolates utilized the reduced folate/MTX transporter for uptake, based on the cross-resistance of MTX uptake-impaired CCRF-CEM cells, and were efficient substrates for this uptake system, based on inhibition of [3H]MTX uptake (IC50, 0.3-5.8 microM; aminopterin IC50, 2.6 microM). These analogues were substrates for CCRF-CEM folylpolyglutamate synthetase, with several being among the most active substrates now known (highest Vrel/Km 0.73; MTX and 10-ethyl-10-deazaaminopterin, 0.013 and 0.24, respectively). Substrate activity for murine intestinal folylpolyglutamate synthetase was also assayed, and a different specificity pattern was observed. These new antifolates are apparently not substrates for aldehyde oxidase. Analogues containing the fused cyclopentene ring are preferred to those containing the fused pyrrole ring based on growth inhibitory potency, effectiveness against decreased uptake mutants and apparent affinity for transport, and inhibition of DHFR. In addition, fused cyclopentene-containing analogues are efficiently polyglutamylated. The data indicate that antifolates with 2,4-diaminopyrimidine-fused five-membered rings, especially those containing the fused cyclopentene ring, are an important new class of antifolates which warrant further exploration at the synthetic and preclinical levels.
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PMID:Novel 6,5-fused ring heterocyclic antifolates: biochemical and biological characterization. 816 96

CCRF-CEM human leukemia sublines resistant to short-term methotrexate (MTX) exposure as a result of decreased folylpolyglutamate synthetase (FPGS) activity were examined for their response to other cytotoxic agents. The R3/7 and R30dm sublines display 25 and 1%, respectively, of the FPGS activity of CCRF-CEM cells as measured with MTX in vitro. Response to agents in outgrowth experiments was examined under both continuous exposure (120 h, where MTX resistance is not observed) and short-term (6-14.5 h) exposure. During continuous exposure to various classes of agents, cross-resistance of R3/7 and R30dm that correlated with FPGS level was not observed, although some minor (< or = 3-fold) stochastic variations in sensitivity were noted. These agents included actinomycin D, Adriamycin, etoposide, vincristine, cisplatin, cytosine arabinoside, 5-fluorouracil, and some other antifolates. Cross-resistance during continuous exposure that did correlate with FPGS level was noted, however, to glutamate-containing thymidylate synthase inhibitors (including ICI D1694) and, to a minor extent, to 6-mercaptopurine and 5-fluorodeoxyuridine. Slight collateral sensitivity during continuous exposure that apparently correlated with FPGS level was noted to the lipid-soluble antifolate trimetrexate and to 5,8-dideazapteroyl-L-ornithine, an FPGS-specific inhibitor. In short-term exposures (where MTX resistance of the sublines is observed), the resistant sublines displayed sensitivity or cross-resistance to each agent that was qualitatively similar to that observed for the same agent in continuous exposure. Because of the requirement for reduced folates in the anti-DNA mechanism of action of fluoropyrimidines and the current clinical use of leucovorin (LV) to enhance their effects, the interaction of LV and fluoropyrimidines was examined. The results suggest that even highly FPGS-deficient cells are as sensitive to the effects of LV modulation as are wild-type cells even at fluoropyrimidine exposure times as short as 4 h.
Leukemia 1993 Dec
PMID:Cross-resistance studies of folylpolyglutamate synthetase-deficient, methotrexate-resistant CCRF-CEM human leukemia sublines. 825 99

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 microM ZD1694 at 37 degrees 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 degrees C, 85% of the total drug (2.3 pmol/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 microM or 0.1 microM 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 the 3H 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.
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PMID:Cellular pharmacokinetics of ZD1694 in cultured human leukaemia cells sensitive, or made resistant, to this drug. 857 77

To develop novel lipophilic thymidylate synthase (TS) inhibitors, the X-ray structure of Escherichia coli TS in ternary complex with FdUMP and the inhibitor 10-propargyl-5,8-dideazafolic acid (CB3717) was used as a basis for structure-based design. A total of 31 novel lipophilic TS inhibitors, lacking a glutamate residue, were synthesized; 26 of them had in common a N-((3,4-dihydro-2-methyl-6-quinazolinyl)methyl)-N-prop-2-ynylaniline+ ++ structure in which the aniline was appropriately substituted with simple lipophilic substituents either in position 3 or 4, or in both. Compounds were tested for their inhibition of E. coli TS and human TS and also for their inhibition of the growth in tissue culture of a murine leukemia, a human leukemia, and a thymidine kinase-deficient human adenocarcinoma. The crystal structures of five inhibitors complexed with E. coli TS were determined. Five main conclusions are drawn from this study. (i) A 3-substituent such as CF(3), iodo, or ethynyl enhances binding by up to 1 order of magnitude and in the case of CF(3) was proven to fill a nearby pocket in the enzyme. (ii) A simple strongly electron-withdrawing substituent such as NO(2) or CF(3)SO(2) in the 4-position enhances binding by 2 orders of magnitude; it is hypothesized that the transannular dipole so induced interacts favorably with the protein. (iii) Attempts to combine the enhancements of i and ii in the same molecule were generally unsuccessful (iv) A 4-C(6)H(5)SO(2) substituent provided both electron withdrawal and a van der Waal's interaction of the phenyl group with a hydrophobic surface at the mouth of the active site. The inhibition (K(is) = 12 nM) of human TS by this compound, 7n, showed that C(6)H(5)SO(2) provided virtually as much binding affinity as the CO-glutamate which it had replaced. (v) The series of compounds were poorly water soluble, and also the potent TS inhibition shown by several of them did not translate into good cytotoxicity. Compounds with large cyclic groups linked to position 4 by an SO or SO(2) group did, however, have IC(50)'s in the range 1-5 microM. Of these, 4-(N-((3,4-dihydro-2-methyl-6-quinazolinyl)methyl)-N-prop-2-ynylamino )phenyl phenyl sulfone, 7n, had IC(50)'s of about 1 microM and was chosen for further elaboration.
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PMID:Structure-based design of lipophilic quinazoline inhibitors of thymidylate synthase. 863 14

The biological activity and cellular metabolism of ZD1694, a novel folate-based thymidylate synthase (TS) inhibitor, were analyzed in a human leukemia cell line, MOLT-3, and its antifolate-resistant sublines with different mechanisms of resistance to methotrexate (MTX), trimetrexate (TMQ) and N10-propargyl-5,8-dideazafolic acid (CB3717). MOLT-3/CB3717(40), which was selected for CB3717 resistance, demonstrated impaired membrane drug transport via reduced folate carrier (RFC) and lower accumulation of [3H]ZD1694-polyglutamates in the cells with a shift in the polyglutamate distribution profile to shorter chain length polyglutamates, indicating an alteration in polyglutamation capacity in this subline. Impaired RFC and reduced rate of polyglutamation could explain the cross-resistance (12-fold) of this subline to ZD1694. On the other hand, there was little or no cross-resistance to this drug in a subline (MOLT-3/TMQ800) reportedly resistant to TMQ through impaired membrane transport for TMQ and an increase in dihydrofolate reductase (DHFR) activity. Total amount of ZD1694 polyglutamated to a level higher than diglutamate was approximately 1.7-fold higher in the TMQ-resistant cells than that in the parent cells, but a low degree of increase in TS activity in the cells counteracted the supposed increase in sensitivity to ZD1694. MOLT-3/TMQ800-MTX10000 cells, which were established by sequential exposure of the TMQ-resistant cells to MTX and were previously shown to amplify mutated DHFR with low affinity for MTX, showed a decreased accumulation of polyglutamated ZD1694 as compared with the parent line and this was consistent with cross-resistance to ZD1694 in this subline. Overproduction of variant DHFR scarcely influenced the sensitivity to this drug. These results indicate that ZD1694 could overcome antifolate resistance through a mechanism such as amplified DHFR activity, and the biological activity of this drug against the cells paralleled the amount of polyglutamated drug inside the cells. Determination of polyglutamation capacity in tumor cells may allow prediction of sensitivity to this drug.
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PMID:Biological activity and intracellular metabolism of ZD1694 in human leukemia cell lines with different resistance mechanisms to antifolate drugs. 869 29

The inhibition of thymidylate synthase (TS) as a drug development target has received much attention in recent years, and several compounds have reached clinical evaluation. During drug development, the effectiveness of target inhibition can be assessed by determination of the perturbations of deoxythymidine 5-triphosphate (TTP) and deoxyuridine 5'-monophosphate (dUMP) pools in drug-treated cells. Rapid, sensitive, and reproducible radioimmunoassays for TTP pools and immunoreactive dUMP pools have been developed to meet our requirement for the rapid assessment of TS inhibition by quinazoline antifolates. The assays can be carried out on 1-2 million cells, and require minimal sample preparation. The limit of detection for TTP is 1 pmole/10(6) cells and for immunoreactive dUMP ("dUMP"), 3.0 pmole/10(6) cells, both assays being performed on the same cell extract. TTP and "dUMP" pools have been measured in mouse L1210 leukaemia cells treated with the quinazoline antifolates ZD1694 (N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl )-N-methylamine]-2-thenoyl)-L-glutamic acid) and CB30900 (N-[N-[4-[N-[(3,4-dihydro-2,7-dimethyl-4-oxo-6-quinazolinyl)methyl ]-N-prop-2- ynylamino]-2-fluorobenzoyl]-L-gamma-glutamyl]-D-glutamic acid). Unlike ZD1694, CB30900 is a TS inhibitor that does not rely on polyglutamation for activity. In L1210 cells, both compounds caused a rapid inhibition of TTP pools in a dose- and time-related manner. Greater than 90% TS inhibition was achieved following a 4-hr exposure to each compound at equitoxic doses (up to 100 times the IC50 determine by a 48-hr growth inhibition assay). For both compounds, this was accompanied by a 5-10-fold increase in "dUMP" pools. For ZD1694, neither the TTP pool or "dUMP" levels were normalised when cells were resuspended in a drug-free medium for 4 hr and, at the higher doses studied, TS was still inhibited after a 16-hr period in the absence of drug. This is consistent with the formation and intracellular retention of potent polyglutamated forms of ZD1694. In contrast, TS activity as determined by repletion of the TTP pools and normalisation of "dUMP" levels were demonstrated for CB30900. However, at a high dose (50 microM, equivalent to 250 times the IC50), retention of TS inhibition was observed following 4 hr, but not 16 hr in the absence of drug. The radioimmunoassays described will prove useful to further define the extent and time-course of TS inhibition by novel antifolate compounds, and will also provide valuable in vitro and in vivo pharmacodynamic information on established antimetabolites when used alone or in combination with other drugs and modulators.
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PMID:Immunoreactive dUMP and TTP pools as an index of thymidylate synthase inhibition; effect of tomudex (ZD1694) and a nonpolyglutamated quinazoline antifolate (CB30900) in L1210 mouse leukaemia cells. 878 44


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