Biological and biochemical properties of new anticancer folate antagonists

Cancer and Metastasis Reviews 5: 251-270, 1987 © Martinus Nijhoff Publishers, Boston - Printed in the Netherlands.

Biological and biochemical properties of new anticancer folate antagonists

David W. Fry & Robert C. Jackson Warner-Lambert/Parke-Davis Pharmaceutical Research, Ann Arbor, Michigan 48105, USA

Keywords: antifolates, 10-ethyl-10-deazaaminopterin, N10-propargyl-5,8-dideazafolic acid, 5,10-dideazatetrahydrofolic acid, piritrexim, trimetrexate

Summary

We review the biology and biochemical pharmacology of four antifolates that were recently introduced into clinical trial as anticancer agents, and one compound in preclinical development. Toxicology and clinical data are not discussed. 10-Ethyl-10-deazaaminopterin (10-EdAM) is a classical antifolate, structurally related to methotrexate (MTX) but with greater activity against murine tumors. 10-EdAM has more efficient membrane transport, and relatively greater polyglutamylation in murine tumors than in normal mouse tissues, and these differential effects are greater for 10-EdAM than for other 10-deaza antifolates or for MTX. Trimetrexate and piritrexim are nonclassical antifolates, lacking a glutamate substitution. They are lipophilic, cross cell membranes more rapidly than does MTX, and retain activity against tumors resistant to MTX because of impaired drug transport. These nonclassical antifolates are active against several MTX- insensitive murine tumors, and both have demonstrated clinical anticancer activity. 10-EdAM, trimetrexate and piritrexim all inhibit dihydrofolate reductase (DHFR) as their primary site of action. As such, they deplete cellular thymidylate and purine pools, and inhibit DNA replication. N10-Propargyl-5,8-dideazafolic acid (CB3717) differs from the first three compounds in acting primarily on thymidylate synthase. Like DHFR inhibitors, it blocks DNA replication through depletion of dTTP, but it does not exert an antipurine effect. CB3717 retains activity against transport-defective MTX-resistant cells, and also against cells that overproduce DHFR. 5,10-Dideazatetrahydrofolic acid (DDATHF) is a selective inhibitor of glycinamide ribotide transformylase, and its biochemical pharmacology may differ appreciably from that of the other antifolates under study. DDATHF has strong antitumor activity in several murine systems.

Introduction

Since the introduction of methotrexate (MTX) into clinical cancer chemotherapy in 1949, its utility has been demonstrated in treatment both of leukemias and a range of solid tumors. It has been shown to be therapeutically superior to the earlier agent, aminopterin, against experimental and human tumors. The limitations of MTX are its incomplete

tumor spectrum, the rather frequent development of resistance, and its acute and chronic toxicity. These limitations are shared, in greater or lesser degree, by all anticancer drugs, but they raise the possibility that better analogues, or at least analogues with a different antitumor spectrum, could be discovered. Many newer antifolates have been synthesized in attempts to produce agents that are more effective and less toxic than MTX. Recent

Address for offprints: D.W. Fry, Warner-Lambert/Parke-Davis Pharmaceutical Research, P.O. Box 1047, Ann Arbor, M148106-1047, USA

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reviews of these synthetic efforts have been published by Roth and Cheng [1], Montgomery and Piper [2] and Werbel [3]. Some of the more inten- sively studied antifolates produced during the 1950s and 1960s were metoprine (DDMP), methasqin, triazinate, and 5-methyltetrahydrohomofolate. Properties of these agents have been summarized in references [2] and [3], and in articles by Hill and Price [4], and Hutchison and Schmid [5]. Despite the large number of classical (glutamate-containing) and nonclassical (lipophilic, non-glutamate- containing) antifolates produced during this period, and the fact that occasional clinical responses were seen to some of these compounds, none of them appeared to have clear advantages over MTX.

Over the last fifteen years much detailed infor- mation on the mechanism of action of antifolates has made MTX one of the most studied and best understood of all anticancer drugs. The wealth of new data includes biochemical and kinetic studies on MTX and polyglutamylation (reviewed by Sir- otnak and DeGraw, 6), detailed X-ray crys- tallographic and NMR studies of the interaction of DHFR with MTX (reviewed by Freisheim and Matthews, 7), cell cycle effects studied by flow cytometry [8] and measurements of the MTX-induced perturbations in cellular pools of ribonucle- otides and deoxyribonucleotides [9]. The new biological and biochemical data suggest ways of screening novel antifolates for greater therapeutic selectivity, activity against MTX-resistant tumor cells, or against sites of action other than DHFR. The high resolution X-ray crystallography studies [7] have already suggested convincing explanations for the tight-binding inhibition of DHFR by 2,4- diamino analogues of folic acid, and for the anti- bacterial selectivity of trimethoprim. Crystallogra- phy studies open up the possibility of computer- assisted drug design of more potent and more selective inhibitors of folate enzymes.

The structures of MTX and of the five novel folate antagonists discussed in this review are shown in Figure 1.10-Ethyl-10-deazaaminopterin is a classical antifolate synthesized by DeGraw et al. [10]. Trimetrexate is one of a series of quinazoline antifolates synthesized in the Parke-Davis labora-

,OH2 A COOH

NH2 CH3CHz N.,-J~-.-N- Cl-I~ ~ QOOH ~,~y, rNy H ~ - C O - N H - C H

CO~H B

"'"AVAV-J bc. c

NH~ CH~ .OC H~

OCH~ 0 HC--C-CH2 J[ ---- -CH~ I ~ ,COOH

HN" Y ~ "~l--(( I~'CO-NH-CH

H2N" "N" Y/"~ ~H~) H E

0 I

0 NH C~OHm

Fig. 1. Structures of methotrexate and of new antifolate anticancer drugs. A. methotrexate (MTX); B. 10-ethyl-10-de- azaaminoptcrin (10-EdAM); C. trimetrexate (TMQ or JB-11); D. piritrexim (BW 301U or PTX); E. Nl°-propargyl-5,8-di - deazafolic acid (CB3717); G. 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF).

tories [11, 12]. Another lipophilic antifolate is the 2,4-diaminopyridopyrimidine, piritrexim, described by the Burroughs-Wellcome group [13]. Trimetrexate and piritrexim have some close simi- larities, but also interesting differences, as described below. CB3717 [14] differs from the previous compounds in having an oxygen substitution at position 4. This is also true of 5,10-dideazatetrahydrofolic acid [15], which also differs from most other antifolates studied in having a tetrahydro structure.

lO-Ethyl-lO-deazaaminopterin

lO-Ethyl-lO-deaza-aminopterin (IO-EdAM) is one

of several derivatives synthesized by DeGraw et al. [10] in which the Nl°-amine of aminopterin is re- placed by a methylene group. The initial compound produced in this series, 10-deaza-aminopterin (10-dAM) [16], is clearly superior to methotrexate in several murine tumor models [17, 18] and eventually went to clinical trial [19]. The rationale for synthesis of the 10-ethyl derivative is the concept that membrane transport is an important determinant of cytotoxicity [20, 21]. Previous work [20] shows that the Nl°-alkyl derivatives of aminopterin are transported much less efficiently in normal tissues, such as gut, than in tumor tissue and therefore have the potential for greater selectivity and perhaps an enhanced therapeutic index. The results of an initial study [10] comparing in vivo antitumor properties in L1210 leukemia, inhibition of dihydrofolate reductase, and the transport characteristics in L1210, of methotrexate, aminopterin 10-dAM and 10-EdAM are consistent with these predictions. Although there were only minor differences in K i for inhibition of dihydrofolate reductase between these analogs, 10-EdAM extended the life span of tumor-bearing mice to a greater extent. An analysis of the kinetic parameters for transport of these analogs in and out of L1210 cells shows that the influx Vma x and effiux rate constant for 10-EdAM are identical to that obtained for the other agents. The influx Km, however, is 4-fold lower than that for methotrexate and is similar or perhaps slightly lower than aminopterin and 10-dAM. These preliminary observations indicate that alkylation of the N ~° position of aminopterin reduces the affinity for this transport carrier in tumor cells whereas alkylation of the C ~° position in 10-dAM did not produce this effect. Substitution of either position does not alter binding affinity to dihydrofolate reductase.

Subsequent to these initial data more extensive work was completed on the transport as well as the metabolism of 10-EdAM [22]. These studies show that influx is saturable for 10-EdAM with a Km of approximately l uM in L1210 leukemia, Ehrlich carcinoma and Sarcoma 180. In all cell lines 10-EdAM exhibits a lower K m for influx than methotrexate and reinforces the conclusion that 10-EdAM and other analogs of this series, as well

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as aminopterin, are better substrates for this carrier system than methotrexate. Consistant with these data is the observation that the exchangeable steady-state concentrations of these analogs are 3--4 times higher than methotrexate. Secondly, even though there are large differences in the K m of influx for methotrexate in different cell lines, the parameter remains constant for 10-EdAM in L1210, Ehrlich and S180 cells. Since the limiting toxicity in mice is considered to be associated with antifolate effects in crypt cell epithelium of the small intestine [23], kinetic parameters for the transport of 10-EdAM were also determined in isolated murine intestinal epithelial cells. Several relevant observations are notable from this study. First, the K m values for influx for all compounds both N ~° and 10-deaza are much higher than in tumor cells. Sec- ond, chemical modification from N 1° to 10-deaza as well as alkylation at the C l° position contribute to a significantly lower affinity for the carrier in the mucosal cells and, since this lowered affinity did not occur in tumor cells, these characteristics provide a basis for selectivity. The study shows that compounds demonstrating the greatest ratio for influx K m Of gut epithelial cells to tumor cells are the most effective in prolonging survival in tumor bearing mice [24]. Similar relationships between methotrexate, aminopterin, 10-EdAM and 10-dAM with regard to transport kinetic constants and cellular accumulation are found in the estab- lished human tissue culture cell lines Manca (human B-cell Leukemia) and SKBRlll (human mammary carcinoma) and human ovarian tumor cells obtained from patients as a suspension from malignant effusions [25].

An area of intense research in recent years is the metabolism of classical antifolates to their poly- glutamyl derivatives and the relationship between the formation of these metabolites and their selectivity and antitumor efficacy. The general concepts emerging from this work have been recently reviewed [21, 26, 27].

Initial studies examine the formation of poly- glutamyl derivatives of 10-EdAM as well as 10-dAM in L1210 cells, S180 cells and the small intestine four hours after administration to tumor bearing mice [22]. All analogs are substrates for

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folypolyglutamate synthetase but net accumulation of the metabolites derived from all analogs is consistently lower in the small intestine than in the tumor cells. In the small intestine the extent of polyglutamylation is in the order aminopterin > 10-EdAM >methotrexate >10-dAM (2.8: 0.9: 0.5: 0.3) and consists mainly of the triglutamate with lower levels of di- and heptaglutamates. The same relative order of polyglutamylation is found in L1210 cells. In S180 the order of 10-EdAM and aminopterin is switched. In tumor cells the major metabolites are the tri- and heptaglutamates.

Further studies [28] examine the relationship between transport, polyglutamylation and cytotoxicity of 10-EdAM in 3 cell lines in tissue culture. The kinetics of the formation of each derivative is determined in each cell line and the accumulation of these metabolites reaches 85-95% of the total cellular drug in 24 hours in L1210 and Manca cells, however $180 cells require only 6 hours to attain these levels. Polyglutamates of 10-EdAM consist- ing of 1, 2 and 3 or more additional glutamyl residues appear in sequential order and by 24 hours the predominate forms contain 3 or more polyglutamates. The extent of polyglutamation for 10-EdAM in all cell types is consistently greater than methotrexate and 10-dAM. The retention of these metabolites is similar to earlier findings for methotrexate in that no efflux of higher polyglutamates is observed and, although there is some loss of lower derivatives, it is slower than the loss of monoglutamate. The significance of these observations is reflected in the good correlation between the cytotoxicity of the various analogs and their potential for polyglutamylation.

The fluorometric high-performance liquid chromatographic assay [29, 30] takes advantage of the high native fluorescence exhibited by pteridine- containing compounds which have carbon in the 10 position. This technique was shown to be suitable for analysis of 10-EdAM, its analogs, and poly- glutamyl derivatives in plasma, urine and tissue. The HPLC assay is therefore quite useful for pharmacokinetic studies in patients treated with 10-EdAM.

In summary, the structural modifications and the resulting apparent benefits of 10-EdAM as corn-

pared to methotrexate are as follows: (1) The re- placement of the N ~° with methylene (a) increases its affinity for the folate carrier system in tumor cells (b) decreases the affinity in gut epithelial cells. (2) The substitution of ethyl for methyl at the C 1° position (a) retains affinity for the transport system in tumor cells (alkyl substitution at the N 1° position reduces affinity) (b) further reduces affinity for the influx carrier in gut, and (c) increases polyglutamylation in tumor cells with little increase of polyglutamylation in gut cells.

10-EdAM is a tight-binding competitive inhibitor of dihydrofolate reductase, with K~ of 2 pM, similar to that of aminopterin and 10-deaza-aminopterin, and slightly tighter than methotrexate [22]. 10-EdAM is only a weak inhibitor of thymidylate synthase. Ueda et al. [31], studying thymidyla~e synthase from human leukemia cells, reports that when 5,10-methylenetetrahydrofolate is used as the folate cofactor, 10-EdAM inhibition is competitive with this cofactor, with K~ of 410/zM. Poly- glutamates of 10-EdAM are also competitive, with K i values 50-138-fold tighter than the parent compound. However, when 5,10-methylenetetrahydrofolate pentaglutamate is used as substrate, inhibition by 10-EdAM and its diglutamate is noncompetitive, and the 10-EdAM pentaglutamate is a mixed-type inhibitor. Kisliuk et al. [32] studied inhibition of thymidylate synthase from Lac-

tobacillum casei. The IC50 concentration for 10-EdAM is greater than 300/xM. Addition of glutamate residues to 10-EdAM increases the inhibitory potency, with an ICs0 value for the pentaglutamate of 7.4/xM. These values are considerably weaker than for methotrexate and its polyglutamates, and suggest that inhibition of thymidylate synthase does not contribute to the pharmacological effect of 10-EdAM.

At the time of writing, no published data are available concerning effects of 10-EdAM on cellular nucleotide pools, nor on cell-cycle phase dependence. It seems likely that 10-EdAM will generally resemble methotrexate in these respects.

Mandelbaum-Shavit [33] studied the effect of 10-EdAM on MCF-7 human breast cancer cells in vitro. The IC50 concentration is 6 nM, compared to 20 nM for methotrexate. Potencies of the two com-

pounds as inhibitors of dihydrofolate reductase from MCF-7 cells are similar, with ICs0 of 4.2 nM for methotrexate and 3.9nM for 10-EdAM. It should be noted that this kind of analysis would not distinguish between quite large differences in K i values when the Ki is in the picomolar range. Based upon inhibition of uptake of radiolabelled methotrexate, Mandelbaum-Shavit finds a K i for transport of 2.4/xM, for 10-EdAM, compared t o K m

values for methotrexate and 5-methyltetra hydrofolate of 7.8 ~M and 3.2/xM, respectively. These results suggest that the greater cytotoxicity to MCF-7 cells of 10-EdAM in comparison with methotrexate is attributable to its higher affinity for the transport carrier.

In vivo studies by Sirotnak et al. [24, 34] show that 10-EdAM is superior to methotrexate in four murine ascites tumors (L1210 leukemia, sarcoma 180, Ehrlich ascites carcinoma, and Tapper carcinosarcoma) and has similar activity to MTX in two other ascites tumors (P288 and C1498 leukemias). 10-EdAM gives a proportion of long-term survivors in the L1210 and sarcoma 180 systems. Against subcutaneous solid tumor implants, 10-EdAM is superior to MTX in four of six systems (sarcoma 180, Tapper, E0771 mammary adenocarcinoma, and T241 fibrosarcoma). 10-EdAM and MTX have similar, modest activity against Lewis lung carcinoma, and both agents are inactive against the B16 melanoma.

Schmid et al. [35] compare the activity of 10-EdAM and MTX against subcutaneous implants of three human tumor xenograft lines in nude mice. In all cases drugs are administered by intraperitoneal injection daily for five days, beginning two days after implantation. Tumor size is evaluated on day 11. Against the MX-1 breast carcinoma, MTX is inactive, and on day 11 tumor mass relative to control (T/C) is 8% for animals treated with 10-EdAM at dose of 2.2 mg/kg, with 2 of 9 animals tumor-free. In the more refractory LX-1 lung carcinoma, MTX at its optimal dose gives T/C of 51%, and 10-EdAM at dose of 4.5 mg/kg gives T/C of 12%. In the highly refactory CX-1 colon carcinoma xenograft, MTX is totally inactive at all tolerated doses, and 10-EdAM at dose of 3.0 mg/kg gives T/C of 33%. Higher doses of 10-EdAM are toxic. In all

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three xenograft systems the tumor regresses to some extent, i.e. its size is smaller on the evaluation day than at the start of treatment. Sirotnak [31] reports that 10-EdAM has activity superior to that of MTX in two other human tumor xenografts (SK melanoma 109 and MBA 9812 non-small cell lung cancer). MTX and 10-EdAM have similar activity against two other xenografts. It has been suggested that the marked therapeutic superiority of 10-EdAM relative to MTX against murine tumors and human tumor xenografts may be attributable to its more efficient transport into tumor cells (but not into mouse intestinal epithelium cells) and to the more extensive polyglutamylation of 10-EdAM in tumor cells.

The optimal doses for 10-EdAM are very similar to those for MTX. Kinahan et al. [36] report preliminary toxicology findings and pharmacokinetics for 10-EdAM. The terminal plasma half-life in dogs is 9 hours. In rats, 50% of the drug is excreted in the bile, much of it as the 7-hydroxy derivative. Poly- glutamates of 10-EdAM are found in liver, small intestine, and kidney of treated rats soon after drug administration, but decline rapidly thereafter.

Trimetrexate

Trimetrexate (2,4-diamino-5-methyl-6 [(3,4,5-trimethoxyanilino) methyl] quinazoline), also referred to in some literature as TMQ or JB-11 is one of a series of 2,4-diaminoquinazolines synthesized in an effort to circumvent certain types of resistance associated with classical antifolates including transport impairment and poor penetration of the blood-brain barrier [3, 11, 12].

A number of studies have demonstrated, at least in vitro, that trimetrexate is active against methotrexate-resistant cells having a defective folate transport system. This has been shown in sublines of several cell types including a human acute lymphocytic T-cell line, MOLT-3 [37, 38], a human T-lymphoblast, CCRF-CEM [39, 40], a human osteosarcoma line, SAOS-2 [39], and a human squamous cell carcinoma (SCC15) [41] as well as murine L1210 leukemia [42-44].

Although the above data repeatedly demon-

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strate that trimetrexate is active against methotrexate transport impaired cells and therefore must enter by a mechanism distinct from the reduced folate carrier system, few studies have appeared that attempt actually to define what that mechanism might be. The first account describes some properties of trimetrexate transport in L1210 cells [42]. Uptake was found to be 30-fold faster than methotrexate and proportional to the extracellular concentration of trimetrexate between the concentrations of 10 -9 and 10 -4 M. Uptake is not inhibited by 20 uM p-chloromecuribenzoate (PCMB), 10 mM sodium azide, the natural folates folic acid or 5-methyltetrahydrofolate or the folate analogs 2,4-diaminoquinazoline, trimethoxyaniline and methotrexate at concentrations of 100/~M each. Uptake of trimetrexate is temperature-sensitive and a Q~0 is calculated to be 2.71. Effiux of trimetrexate is rapid and not affected by 20/~M PCMB or 10mM sodium azide. After cells are incubated with 4/~M trimetrexate for 30 minutes and then washed out, a nonexchangeable fraction is retained which is 3-5 times higher than the dihydrofolate reductase level. No metabolites or trimetrexate are detected under the conditions of these experiments.

A more extensive characterization of trimetrexate transport has been conducted in a human lymphoblastoid cell line, WI-L2 [45, 46, D.W. Fry, unpublished results]. A plot of cell-associated trimetrexate for these cells versus time at an extracellular concentration of 1/~M shows a very high intercept on the y axis implying a component of very rapid association with the cell which may represent some form of surface binding. Uptake is rapid and attains a steady-state concentration of 120 pmol/107 cells within 30 minutes. Undirectional influx studies show that influx rates are 25-fold faster than methotrexate influx. At an extracellular concentration of 1 ~M the influx rate for trimetrexate in 107 cells is 3.11pmol/second whereas that for methotrexate in 107 cells is 0.13 pmol/second. The difference in efflux rate constants between trimetrexate and methotrexate is not nearly as great. Trimetrexate effiux is about 2-3 times faster than methotrexate. Efflux studies show a large nonexchangeable fraction of trimetrexate that is well

above the dihydrofolate reductase binding capacity. This nonexchangeable fraction is not constant and is dependent on the level of drug originally associated with the cells. Influx of trimetrexate does not appear to be saturable and a plot of influx rate versus extracellular trimetrexate concentration is linear up to 500 p~M. At higher concentrations the response deviates from linearity but even at concentrations as high as 5 mM the system does not fully saturate. Furthermore, influx cannot be inhibited to any significant extent with lmM concentrations of methotrexate, folic acid, folinic acid, or a wide variety of natural substrates and metabolites including purine and pyrimidine bases and nucleosides, sugars and amino acids. Although the above data are consistant with a mechanism for transport of passive diffusion other data are somewhat compatible with a carrier mediated process or at least a process more complex than simple diffusion. This possibility is supported by the fact that the infux and exchangeable cellular steady-state levels of trimetrexate are reduced by 50% in cells preincubated 30 minutes with 10 mM sodium azide. Glucose, however, does not reverse this effect and indeed reduces the steady-state levels of trimetrexate by 50% itself, indicating that these effects on

trimetrexate transport may not be strictly a func- tion of altered energy metabolism. Influx can be completely abolished with 50~M p-(chloromer- curi)benzene-sulfonic acid (PCMBS) or by lower- ing the temperature to 4 °C. The difference between influx rates at 37 ° and 27 ° C is about 2.5 fold. Efflux of trimetrexate is also sensitive to metabolic inhibitors in that 10 mM sodium azide stimulated efflux by 2- to 3-fold and this effect could be partially reversed by 10 mM glucose. Finally, the exchangeable intracellular concentration of trimetrexate at the steady-state is always 4 to 5 times that of the extracellular concentration.

Previous literature dealing with transport of lipophilic antifolates does little to resolve the poorly understood aspects of trimetrexate transport since a number of discrepancies are evident. Kamen et al. [42] (see above) studied trimetrexate transport in L1210 and while most observations are consistant with those reported in the WI-L2 study, he finds no effect with sodium azide or pCMB.

Duch et al. [47] finds that the rate of uptake of PTX at 4 ° C was the same as at 37 ° degrees which is quite unlike trimetrexate. Hill et al. [48, 49] finds that in L5178Y lymphoblasts, metoprin e transport is highly temperature sensitive, appears to be saturable with a K m for influx of 130/zM and is inhibited by folinic acid. Greco and Hakala [50], in contrast, observe in the human carcinoma cells, KB and Hep-2, that uptake of this drug is not sensitive to temperature nor saturable at least at the concentrations employed (1-200 p.M) and is not inhibited by folinic acid. These studies complicate any attempt to draw firm conclusions concerning a specific mode of transport for these classes of compounds.

Additional data, however, which indicate a more intricate transport system for trimetrexate than passive diffusion may be derived from the fact that cells are capable of altering the rate of transport and thus their sensitivity to trimetrexate. Re- ports [45, 46, D.W. Fry, unpublished results] de- scribing the selection of a trimetrexate-resistant line of human lymphoblastoid cells (WI-L2) by exposing the cells to step-wise increases in drug concentration show that lymphoblastoid cells are 40- to 60-fold resistant to trimetrexate and fully cross-resistant to other lipophilic antifolates including metoprine and BW 301U. However, they are completely sensitive to methotrexate and only 3-fold cross-resistant to methotrexate-gamma- tertbutylmonoester. Furthermore, these cells are completely sensitive to adriamycin and vincristine implying that this is not a form of pleiotropic or multidrug resistance. Although the resistant cells exhibit a slight elevation in dihydrofolate reductase (2-fold), it seems unlikely that this could account for a 60-fold resistance. Nor are there any differences in the affinities of the enzyme for trimetrexate between the two cell lines. Studies investigating the transport of 14C labeled trimetrexate show a 50% decrease in the influx rate of the resistant relative to the sensitive cell line but equal efflux rates. This correlates with a 50% drop in exchangeable trimetrexate observed in the resistant line at the steady-state. Transport differences between the two, cell lines are not due to changes in intracellular water and rates of methotrexate transport are identical in each. Furthermore, analysis of the intra-

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cellular compartment of both cells by high pressure liquid chromatography indicated that no detectable metabolites are formed when cells are incubated for up to six hours with 5/zM trimetrexate.

Resistance to trimetrexate possibly due to impaired transport has also been described in a line of human acute lymphoblastic leukemia, Molt-3 [51]. These cells are 200-fold resistant to trimetrexate and exhibit influx rates 40% of the parent line and steady-state levels of only 23%. Additional abnor- malities are present in these cells, however, including a 6-fold cross-resistance to methotrexate, a 50% reduction in influx of this agent and a 75% decrease in the intracellular levels at the steady- state. Finally, some cell lines appear to take up trimetrexate intrinsically to lesser or greater ex- tents with corresponding effects on sensitivity. Mini et al. [52] reports that CCRF/CEM cells are 10-fold less sensitive to trimetrexate than HCT-8 cells which corresponded to a 10-fold difference in drug accumulation after four hours. Borman et al. [53] finds that levels of trimetrexate in rat colon adenocarcinoma are about 4-fold the level in L1210 after similar incubation conditions and 10- to 100- fold higher than that observed for methotrexate. The enhanced accumulation of trimetrexate correlates with its relative efficacy in experimental colon carcinoma in vivo.

Although multidrug resistance is not associated with the above examples of resistance, recent reports imply that trimetrexate and other lipophilic antifolates have this form of resistance. Klohs et al. [54] find that a pleiotropically resistant line of P388 leukemia is cross-resistant to trimetrexate and BW301U and that resistance can be partially reversed by verapamil and tween-80. Transport studies show that net uptake of trimetrexate is de- pressed by 75% in the resistant line and that intracellular steady-state levels can be restored by the presence of verapamil. These studies indicated that pleiotropic resistance extends to lipophilic antifolates and that the mechanism appears to be alterations in transport similar to what has been observed for adriamycin. Similar results have been observed elsewhere [55]. Multidrug resistance is also found in a line of human acute lymphoblastic leukemia (MOLT-3) made resistant to trimetrex-

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ate [56]. These cells are 800-fold resistant to trimetrexate but are also 280-fold cross-resistant to vincristine, 15-fold to daunorubicin, 14-fold to doxorubicin and 10-fold to mitoxantrone. Cross resistance to these drugs is greatly diminished by 10 -5 M verapamil. Cellular uptake of trimetrexate and vincristine in these cells is diminished 82% and 80% respectively. Uptake of both drugs is enhanced in the presence of verapamil.

These observations imply that although trimetrexate and other lipophilic antifolates are quite capable of circumventing methotrexate resistance due to an impairment of the reduced folate carrier system, the nature of these structures evidently makes them susceptible to at least 2 other forms of resistance related to altered transport. Some studies indicate that since trimetrexate and methotrexate elicite different modes of resistance, alternating use of these drugs may delay onset of resistance to antifolates [57].

Many of the biochemical effects of trimetrexate are typical of most antifolates. Trimetrexate has been shown to be a potent inhibitor of dihydrofolate reductase from several cell types. Earlier studies do not report true K~ values but express inhibition as the concentration of drug necessary to inhibit by 50% (ICs0). Thus the ICs0 values for dihydrofolate reductase from human acute lymphocytic leukemia cells and L1210 cells are 1.9 x 10 -9 and 1.2 x 10 -9 M respectively and are comparable to methotrexate at 9 × 10-~°M for both cells lines [58, 59]. Similar results are obtained for the enzyme from beef liver [60]. A more detailed kinetic analysis of the inhibition of dihydrofolate reductase are conducted with enzyme purified from WI-L2 cells [44]. A Ki.slop e value of 4.3 × 10 -11M are determined for trimetrexate which is about 10-fold greater than that obtained for methotrexate.

Trimetrexate is also a potent inhibitor of deoxyuridine incorporation into DNA [44, 61-63] and, similar to other antifolates, the cytotoxic effects of trimetrexate can be reversed by leucovorin, thymidine or a combination of thymidine and hypoxanthine. Broome et al. [62] finds that the cytotoxic effects of 10txM trimetrexate can be reversed by 100/xM leucovorin in P388 and in the M5076 ovarian tumor, the antitumor effects of trimetrex-

ate are reversed by coadministration of leucovorin in vivo. Complete protection from trimetrexate by leucovorin has also been shown in the human lymphoid cell lines CCRF-CEM and LAZ-007 [65]. Other reports [43] show that protection from both growth inhibition and cytotoxicity of ltxM trimetrexate in L1210 or WI-L2 cells can be attained when 40~M leucovorin is added either simul- taneously or as late as 5 hours after trimetrexate. McCormack et al. [65] show that 4-6txM thymidine alone can prevent the growth inhibition produced in L1210 cells by 2 x 10 -8 M trimetrexate. Further studies [44] show that the protection by thymidine alone is dependent on the concentration of trimetrexate and that if concentrations of drug are 10 -7 M or below, thymidine can fully protect. If, however, concentrations of trimetrexate are greater than 10-7M a combination of thymidine and hypoxanthine is necessary.

The requirement that both thymidine and hypoxanthine be present to protect cells from trimetrexate cytotoxicity suggests both an antipyrimidine and antipurine effect. Direct measurement of purine and pyrimidine ribonucleotide pools has been made in L1210 and WI-L2 cells preincubated with 50 nM or 1/xM trimetrexate for 6 hours [44]. Both adenylates and guanylates are reduced to approximately the same extent and the depression in these pools is dependent on drug concentration. In contrast pyrimidine nucleotides, CTP, UTP, and UDP are elevated, most probably due to a reduced rate of RNA synthesis. Qualitatively similar results are obtained in both cell lines although the reduction in purines is much more pronounced in the L1210 cell. In L1210 cells treated 2 hours with 1/xM trimetrexate a strong antithymidylate effect is apparent as dTTP levels drop to 29% of the control. The levels of dATP and dGTP also drop significantly to 41% and 15% of control respectively. No significant change is observed in the dCTP pool.

A recent report has examined the cell cycle phase specificity of trimetrexate [66]. Cultures of Chinese hamster ovary (CHO) cells were syn- chronized by mitotic selection and were exposed periodically throughout the cell cycle to 50/xM trimetrexate for 2 hours. The surviving fractions as determined by a cloning assay show that only those

cells in S phase are sensitive to trimetrexate. Fur- thermore, CHO cells in plateau phase are relatively insensitive to trimetrexate when compared to log phase cells. In other experiments exponen- tionally growing L1210 leukemia cells were con- tinuously exposed to either 3 or 30 nM trimetrexate and their progression through the cell cycle moni- tored by flow cytometry. Cells exposed to 3 nM trimetrexate exhibit a delay in progression through S phase whereas 30 nM produce cell cycle arrest in late G1 or early S phase.

Trimetrexate and methotrexate were evaluated side by side in the human tumor cloning system [67]. A total of 69 (51%) human tumors, obtained from a total of 136 human tumors representing 19 different histological types were prepared and evaluated. A comparison of the relative sensitivity of the two drugs revealed that 15% (8 of 54) of the tumors were sensitive (greater than 50% cell kill) when exposed for 1 hour to 0.1~g/ml of trimetrexate but resistant to 0.3 ~g/ml methotrexate whereas 7% (4 of 54) were sensitive to methotrexate but refractory to trimetrexate.

A number of studies have evaluated the antitumor properties of trimetrexate especially in comparison to methotrexate. In general, trimetrexate shows a much broader spectrum of activity especially against solid tumors. Initial studies [59] show trimetrexate to have moderate to good activity against L1210 and P388 leukemias, colon 26 and 38, and B16 melanoma whereas methotrexate is active against just the leukemias. Subsequent reports confirmed the activity in L1210 [44, 68] and also demonstrate mild activity against the M5076 ovarian [44, 62]. A more recent report [69] com- pares trimetrexate and methotrexate against a wide variety of murine tumor models and human tumor xenografts. In this study trimetrexate is active against B16 melanoma, CD8F1 mammary tumor, colon 26 and 38, and L1210 and P388 leukemias. Trimetrexate has no activity against Lewis lung carcinoma. Methotrexate is active only against the CD8F1 mammary tumor cells and the leukemias. Neither drug is active against CX-1 colon, LX-1 lung or MX-1 mammary human tumor xenografts in nude mice.

In a study to determine the potential for thera-

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peutic synergy between trimetrexate and several clinically proven agents [70, 71] life span assays were conducted in F1 hybrid mice bearing either early or advanced stage P388 leukemia. The mice were treated with each agent singly or in combination. A high degree of therapeutic synergism is observed between trimetrexate and doxorubicin, cytoxan or 6-thioguanine. Combinations of trimetrexate and 5-fluorouracil, vincristine or cisplatin also produce enhanced tumor cell kill although the results are not nearly as dramatic. Over- lapping host toxicities of trimetrexate and methotrexate appeared to negate any therapeutic benefit produced by this combination.

The pharmacokinetics of trimetrexate have been evaluated in several animal systems in addition to a number of clinical studies. In mice the elimination of trimetrexate in the plasma is reported to be a single exponential with a tv2 of 50 minutes [65]. In dogs the elimination is biphasic with an alpha t~/2 of 10-17 minutes and a beta tv2 of 2.5-3.6 hours [72, 73]. These parameters appeared to be identical in normal or lymphoma bearing dogs [74]. In Rhesus monkeys the elimination was triphasic having half lives of 3.3-3.9, 39-45, and 199-248 minutes after an IV bolus of 100mg/m 2 [75]. Further analysis shows that elimination is primarily by biotransfor- mation, that there is substantial plasma protein binding and little penetration into the CFS. Several studies in humans report biexponential plasma elimination with alpha tv_, values ranging from 40 to 78 minutes and beta tv2 values from 10 to 44 hours [76-78]. Other studies indicate a triphasic pattern with halflives of approximately 10 minutes, 2 hours and 16 hours [79, 80].

The major metabolic route for trimetrexate is considered to be oxidative demethylation in the liver and occurs via a conjugated glucuronide [81, 82]. Metabolism of trimetrexate readily occurs in the isolated perfused liver [83] and is inhibited by hypoxic conditions [84]. It also can be observed in rat liver microsomes and is inhibited by clinically used imidazole drugs [851.

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Piritrexim

Piritrexim (PTX), 2,4.diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido-[2,3-d]pyrimidine also referred to in the literature as BW 301U was se- lected out of over 300 heterocyclic structures based on a series of 6-substituted 2,4-diaminopyrido[2,3- d]-pyridines originally synthesized as potential antimicrobial agents [86-88]. In subsequent years the synthetic route to these structures was im- proved and it was found that PTX exhibited significant antitumor activity against Walker 256 carcinoma [13].

One criterion for selecting PTX was its greater affinity for DHFR than a previously developed lipophilic antifolate, metoprine (DDMP). PTX inhibits DHFR by 50% at a concentration of 5 nM which is comparable to MTX at 7 nM [11]. A second advantage PTX has over metoprine is its more favorable plasma pharmacokinetic properties [47]. A third reason for selecting PTX was its lack of inhibition on histamine metabolizing enzymes [90, 91]. Studies with partially purified enzymes show that PTX competitively inhibits histamine meth- yltransferase from bovine cerebral cortex with a K i of 1.8/xM and inhibit diamine oxidase from rat cecum in a noncompetitive manner with a K i of 32/aM. These values, however, represent a much lower affinity for the enzymes than other lipophilic antifolates such as trimetrexate which exhibits K i values for these enzymes of 0.007/aM and 4.1/xM respectively. It was shown that administration of trimetrexate to rats produces a 2-fold elevation of histamine in the brain and a 7-fold elevation in the kidney. In contrast, administration of PTX at the same dose causes no elevation in the brain and a 1.5- to 3-fold elevation in the kidney which is of short duration and returns to normal after 2 hours.

One of the more peculiar aspects of PTX as an antifolate is the nucleoside requirement for reversal of cytotoxicity in tissue culture. The growth inhibition of tissue culture cells by most known inhibitors of DHFR such as MTX, trimetrexate and metoprine can be fully reversed by a combination of thymidine and hypoxanthine. In S180 cells, however, full protection from PTX is afforded only by a combination of hypoxanthine, thymidine, ade-

nosine and either cytidine or uridine [47, 89]. Fur- thermore, although leucovorin alone is capable of protecting cells from MTX and other antifolates, it is not sufficient in the presence of PTX. In $180 cells the cytotoxic effects of MTX or metoprine can be completely prevented by the addition of leucovorin at a concentration 10-fold higher than that of the drug. However, when cells are grown in a concentration of PTX sufficient to inhibit cell growth by 90%, leucovorin can reverse this inhibition to no more than 60% of the control cells even at concentrations of leucovorin as high as 250 times that of extracellular drug. If, however, thymidine is included along with leucovorin then complete protection can be achieved [47, 89]. The same results are obtained when 5-methyltetrahydrofolate is substituted for leucovorin and similar results are observed in L1210 and Walker 256 cells. In vivo administration of leucovorin alone (200mg/kg, twice a day for 2 days and starting 24 hours after PTX) allows the survival of rats given a lethal dose (100mg/kg, i.p.) of PTX. Thymidine affords no protection from lethal toxicity.

PTX has been tested for its cytotoxicity against a number of ceils both normal and MTX-resistant in tissue culture. PTX inhibits by 50% the growth of Walker 256, sarcoma 180 and L1210 leukemia cells at similar concentrations to MTX; 0.8, 1.2 and 2.2 × 10-SM respectively [47, 89]. Comparable re- suits were obtained in a study with a human lymphoblastoid cell line, WI-L2, and show a C50 value of 2.5 x 10-8M [92]. Since a major rationale for developing a lipophilic antifolate is the circumven- tion of the more frequently observed forms of resistance to classical antifolates, PTX has been tested against several cell lines resistant to MTX. Sedwick et al. [92] tested PTX against mouse 3T6 cells and a mutant line (3T6R400) which overproduces an altered form of DHFR having a 100-fold lesser affinity for MTX than the enzyme from parent 3T6 cells. These cells can be exposed to 1 mM MTX with little reduction in cloning efficiency [93] but they are sensitive to PTX at levels that can be achieved in vivo [92]. In a more recent study [94] PTX was tested against 3 MTX-resistant cell lines. A mutant line of CCRF-CEM ceils (CCRF-CEM/ MTX R) which shows severely impaired transport

of MTX, and is 100-fold resistant to MTX, is collaterally sensitive to PTX. Another cell line (L5178Y/ MTXR) overproduces DHFR by 600-fold and is approximately 10,000-fold resistant to MTX. These cells were cross-resistant to PTX, and at least an order of magnitude more sensitive to PTX than MTX. The third cell line, L1210/MTXR, is a double mutant having both impaired MTX transport and a 24-fold overproduction of DHFR as the cause of its 5000-fold resistance to MTX. These cells are markedly sensitive to PTX compared to MTX, although not to the degree of the 'sensitive' line and the observed IC50 probably reflects a combination of resistance due to the overproduction of DHFR and collateral sensitivity due to an impaired reduced folate carrier system. In general these studies show that PTX effectively circumvents the folate carrier system and is quite capable of overcoming MTX-resistance due to its impairment. Secondly, because entry of PTX is apparently not limited by this low capacity carrier system, resistance due to an overproduction of DHFR is more easily overcome simply by increasing the extracellular concentration.

A number of studies have looked at the effect of PTX on the incorporation of precursors into the macromolecules. Exposure to PTX is associated with a very rapid suppression of deoxyuridine (dUrd) incorporation into DNA. The ability of PTX to inhibit this parameter was assessed in a variety of ceils including human lymphoblastoid cells in culture and normal leukemic myeloid cells from peripheral blood and myeloid cells from bone marrow [95]. In all cells inhibition of deoxyuridine incorporation into DNA is immediate. The degree of inhibition is similar in most leukemic cell culture lines after a 30 or 90 minute incubation, but a somewhat more variable response is found with bone marrow cells. The reversibility of suppressed dUrd incorporation was studied in the WI-L2 cells exposed to PTX as well as MTX and metoprine [92]. After a 1 hour exposure to 1/,M PTX partial recovery occurs when the extracellular drug is re- moved and the cells are resuspended in drug-free media. The initial rate of recovery is faster than in cells exposed to MTX, however the latter phase of recovery is equal to MTX and recovery from both

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drugs equally augmented by leucovorin. The effects of PTX on the incorporation of 32p into DNA in WI-L2 cells was also assessed in this study [92] as a measure of DNA synthesis. Although this parameter is less sensitive to inhibition by antifdl- ates than dUrd incorporation it is a better indicator of the potential lethal toxicity of these drugs. A drug concentration of 1/xM PTX, MTX or metoprine produces equivalent levels of inhibition. After a 90 minute exposure the inhibition produced by PTX and metoprine can be completely reversed simply by removing the drug and resuspending the cells in drug-free media. Recovery from the effect of MTX however requires leucovorin or thymidine.

PTX also inhibits dUrd incorporation much more rapidly than MTX in sarcoma 180 cells [47] but the reversal patterns differ from WI-L2 cells. No difference is observed in reversal of $180 cells exposed to PTX, MTX or metoprine. When resuspended into drug-free media with no additions the pattern of recovery from all drugs shows an initial period of increased activity followed by a subsequent decline of incorporation. The presence of 200/,M leucovorin does not allow cells to recover from the effects of PTX, however, it allows dUrd incorporation to resume at normal rates after inhibition by MTX. The ability of PTX or MTX to inhibit dUrd incorporation has been studied in bone marrow cells from patients treated with PTX [96]. The results are unusual in that dUrd incorporation into DNA is much less susceptible to inhibition by MTX in cells from pretreated than from untreated patients. The response to PTX remains the same. The same results are also obtained in HL-60 cells exposed to cytostatic concentrations of PTX in tissue culture.

The transport of PTX across the plasma membrane in ceils was initially studied indirectly by a method which utilizes the inhibition of dUrd into DNA as a measure of this parameter [97]. Using this technique reducing the temperature of the cultures from 37 ° to 27 ° C does not significantly affect the kinetics of drug entry into WI-L2, indicating a lack of temperature sensitivity for the uptake process [92, 95]. More direct transport studies for PTX are reported in S180 cells using 14C labeled PTX

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[47]. A time course for net uptake indicates that PTX associates rapidly with cells and reaches equilibrium across the membrane after 30 minutes. A very high intercept on the y axis is always present on plots of cellular drug versus incubation time even when very short time points are taken. The high intercept indicates a component of very rapid drug-cell association which may represent some mode of surface binding. Cellular drug levels achieved at the steady-state are directly and lin- early related to the extracellular concentration of drug. Washout experiments show that when cells are in equilibrium with 10/zM PTX there is a large component of exchangeable drug which effiuxes rapidly from the cell. Extended washout reveals a large nonexchangeable fraction which is most likely well above the DHFR binding capacity. Fi- nally, and similar to the indirectly determined measurements above, uptake of PTX appears to be temperature insensitive. No differences are observed between measurements made at 37 ° and 4 °C. These data are taken as evidence for a non- facilitated, passive, diffusional process as the mechanism of membrane transport for TPX.

A speculative but very appealing theory about the mechanism of antifolate-induced cytotoxicity is that uracil misincorporation into DNA results in accumulation of apyrimidinic sites. Since the nucleotide pool imbalance caused by antifolates is characterized by depletion of dTI'P and elevated dUTP, repair synthesis will simply cause further misincorporation, and lethal DNA damage will eventually result. Richards et al. [98] show that piritrexim causes uracil misincorporation, and that addition of deoxyuridine to the growth medium slightly increases the cytotoxicity of piritrexim to HL-60 cells. In MOLT-4 and SB cells, piritrexim causes DNA fragmentation, and in WI-L2 ceils, chromatin maturation is inhibited. It is proposed that these effects are caused by uracil misincorporation, followed by excision of uracil to leave apyrimidinic sites [98].

Piritrexim has in vivo activity against leukemias P388 and L1210, sarcoma 180, Ehrlich ascites carcinoma and the Walker 256 carcinosarcoma [99, 100]. Drug distribution studies in rats indicate a plasma half-life of 38 minutes following intra-

venous administration (in dogs, the half-lif~ is 130 minutes). Piritrexim extensively penetrates rat tissues, including the brain. The major pathway of metabolism is O-demethylation. The demethyl- ated metabolite is an effective inhibitor of dihydrofolate reductase.

Nl°-Propargyl-5,8-dideazafolic acid

This quinazoline antifolate, generally referred to in the literature as CB3717, was synthesized by Jones et al. [14]. The biochemistry and pharmacology of CB3717 have been reviewed recently [101]. The original antitumor studies of this compound were carried out using the tetraploid ICR subline of L1210 leukemia [14]; in this system CB3717 is far more active than MTX, giving 90% cures against intraperitoneal tumor and 40% cures against intravenous tumors at optimal doses. In the diploid NCI line of L1210, CB3717 is less active. CB3717 also gives 80% inhibition of tumor growth when tested against subcutaneous implants of the ADJ/PC6 plasmacytoma. CB3717 has no activity or only mar- ginal activity against TLX-5 lymphoma, P388 leukemia, and colon carcinoma 38 [101]. Jackman et al. caution that the interpretation of data obtained in rodents is complicated by their high circulating thymidine concentration. Thymidine not only an- tagonizes antitumor activity, but may also prevent toxicity to normal tissues. Obviously, using xenografts does not help this situation, since the source of the thymidine is the host animal.

In vitro, CB3717 is only moderately cytotoxic, with IC50 values for continuous exposure failing between 1/zM and 5/zM for most cell lines [14,101- 104]. Cytotoxicity of CB3717 to murine and human cells is largely prevented by 10/xM thymidine [14, 102, 104] even when thymidine addition is delayed for many hours after drug administration [102]. In the case of MTX or trimetrexate, protection from cytotoxicity generally requires a purine, such as hypoxanthine, in addition to thymidine. Leuco- vorin does not protect cells very effectively from CB3717, and hypoxanthine not at all [14, 104].

Supporting evidence that the primary site of CB3717 action is thymidylate synthase comes from

studies with drug-resistant cells. Cells with overproduction of dihydrofolate reductase have little or no cross-resistance to CB3717 [103, 104]. Con- versely, cells with overproduction of thymidylate synthase are highly resistant to CB3717 [105].

A number of kinetic studies have examined the inhibition of thymidylate synthase by CB3717 and its polyglutamate derivatives. In purified enzyme preparations of L1210 mouse leukemia cells, and WI-L2 human lymphoblastoid cells, CB3717 is competitive with 5,10-methylenetetrahydrofotate; kinetic analysis using a zone B procedure for tight- binding inhibition indicates a Ki,slop e of 4 to 5 nM [102,106]. Cheng et al., studying enzyme from KB cells, report noncompetitive inhibition with K i of 20 nM [104]. There are two likely reasons for this discrepancy. First, the analysis of Cheng et al. did not use an appropriate method for tight-binding inhibitors, and competitive tight-binding inhibitors may show apparent noncompetitive kinetics if a zone B technique is not used. (Methotrexate was originally reported to be a noncompetitive inhibitor of dihydrofolate reductase for that reason.) Second, Santi (cited in 50) has reported that CB3717 interacts with thymidylate synthase from Lactobacillus casei in a two-stage process. The first stage is competitive with 5,10-methylenetetrahydrofolate. The second stage, which occurs with a half-time of 50 minutes, produces a ternary complex in which dUMP is covalently bound. This ternary complex dissociates with tv2 of 27 hours, If a similar interaction occurs with the human enzyme, long incubation times could result in non- steady-state conditions and apparently anomalous kinetics.

Polyglutamates of CB3717 are more potent inhibitors of thymidylate synthase than CB3717 itself [104, 1071. Sikora et al. report that CB3717 di- and triglutamates are 30 and 84-fold more potent, respectively, as inhibitors of the enzyme from L1210 cells. Cheng et al. report that inhibitory potency increases with addition of successive glutamate residues up to the tetraglutamate, but that the pentaglutamate is weaker. The tri- and tetraglutamates of CB3717 are thus the most potent known inhibitors of thymidylate synthase.

CB3717 is a moderately potent inhibitor of di-

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hydrofolate reductase. It is competitive with dihydrofolate, and has a Ki,slop e estimated at 23-250 nM in various preparations of the enzyme. This inhibitory effect is probably not of great pharmacological importance in vivo; however, in tissue culture cells growing on folic acid-containing medium in presence of high concentrations of CB3717 plus thymidine, growth inhibition is seen which may be reversed by hypoxanthine or leucovorin [108]. Since the predominant physiological folate is 5-methyltetrahydrofotate rather than folic acid, this effect would probably not be seen in vivo.

In WI-L2 cells, CB3717 treatment (20/xM, 4 hours) causes marked depletion of dTTP, with minor decreases in dCTP and dTTP. dATP increases, as do ATP, GTP, UTP and CTP [102]. These results are consistent with the reversal of CB3717 effects by thymidine. Under normal conditions, CB3717 has no antipurine effect. The minor decrease in dGTP may be attributed to the fact that CB3717 causes extensive depletion of DTTP, and dTTP is a required cofactor for the GDP reductase reaction. Unlike MTX, CB3717 does not cause accumulation of dihydrofolates, dUMP accumul- ates in CB3717-treated cells, and significant amounts of dUTP are also seen [102].

The fact that CB3717 polyglutamates are effective thymidylate synthase inhibitors prompted a search for polyglutamate formation in tumor cells and normal tissues. Manteuffel-Cymborowska et al. [109] report formation of di-, tri- and higher glutamates in murine liver and kidney 4 and 24 hours after the drug is administered to mice. Trig- lutamate is predominant. Sikora et al. [107] treated L1210 cells in vitro with radiolabelled CB3717. Twenty four hours after treatment, two-thirds of the extractable radioactivity is unchanged drug, but the remainder is mainly diglutamate. As yet, there is no evidence that CB3717 is selectively polyglutamylated in malignant cells. Nevertheless, it is clear that polyglutamates are formed in both tumor and normal cells.

At present the transport mechanism for CB3717 is poorly understood. CB3717 appears to penetrate cell membranes very slowly. One study suggested that six hours after the start of incubation, at an extracellular concentration of 50/xM CB3717, the

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cellular drug concentration does not exceed 2/xM, despite the fact that tritiated deoxyuridine incorporation is markedly decreased within one hour [101]. The uptake of CB3717 appears to be non- saturable [110], but it is temperature-dependent [101]. Several cell lines with defective membrane transport of MTX have normal sensitivity to CB3717, suggesting that uptake of the latter cannot be through the carrier responsible for methotrexate transport [103].

At present no flow cytometry studies have been reported for CB3717, nor any data concerning cytotoxicity toward nonproliferating cells. On mechanistic grounds it seems likely that CB3717 will be highly S-phase selective in its effect, but experimental confirmation of this would be valuable.

A preliminary report of CB3717 pharmacokinetics has been presented [111]. Plasma decay in humans, following a one hour intravenous infusion is biphasic, with half-lives of 70 minutes and 9 hours. Metabolites are not seen in urine or plasma, but the deglutamylated derivative is detected in the feces. The protein binding of CB3717 is more extensive than for methotrexate,

The clinical studies with CB3717 have been reviewed by Jackman et al. [101]. Renal toxicity and hepatic toxicity have been observed. The hepatic toxicity is not seen with the related compound 5,8- dideaza folic acid, nor is it prevented by thymidine. Thus this effect is not directly attributable to inhibition of thymidylate synthase; possibly it is caused by an unrelated reaction of the propargyl moiety [112]. Clinical responses to CB3717 have been reported in several kinds of tumors, including breast cancer patients resistant to the CMF (cyclophos- phamide + methotrexate + 5-fluorouracil) pro- tocol, demonstrating that the approach of design- ing a folate inhibitor aimed at an enzymatic target other than dihydrofolate reductase can overcome clinical MTX resistance [101].

5,10-Dideaza-5,6,7,8-tetrahydrofolic acid

The structure of this compound, which was synthesized by Taylor et al. [15] is shown in Fig. 1 (F).

DDATHF is highly cytotoxic to L1210 murine leukemia cells in culture, with ICs0 of approximately 20 nM. The cytotoxicity is not prevented by addition of thymidine to the medium but it is prevented by hypoxanthine or by aminoimidazole carboxamide [113]. DDATHF retains activity against a methotrexate-resistant subline of L1210. DDATHF gives no significant inhibition of Lac- tobaciUus casei thymidylate synthase when tested at 1 mM concentration, and is a very weak inhibitor of beef liver dihydrofolate reductase, giving 50% inhibition at 0.6 mM. It gives excellent therapeutic activity against a panel of experimental murine tumors, many of which are insensitive to methotrexate [114]. For example, against the subcutaneous B16 melanoma, daily doses of DDATHF at 6.25 mg/kg for ten days give complete inhibition of tumor growth. Daily doses below 100 mg/kg have no toxic effect on the mice [115]. DDATHF depletes cellular pools of ATP and GTP, and block formation of N-formylglycinamide ribotide, indicating that its site of action is inhibition of glycinamide ribotide transformylase [114, 115]. It does not inhibit the incorporation into DNA of radiolabelled deoxyuridine or thymidine [113]. DDATHF is an excellent substrate for folylpolyglutamate synthetase from mouse liver, with K m of 4.7 tzM, and V~a x similar to that of folic acid; high concentrations of DDATHF give excess substrate inhibition [116]. DDATHF appears to be a highly novel kind of antifolate with promising experimental antitumor activity. It will also be a valuable biochemical probe for the study of folate metabolism.

Conclusions

- 10-EdAM appears to be the most active classical antifolate yet described against murine tumors, based on studies in five ascites systems and five subcutaneous solid tumor transplant lines. In addition, 10-EdAM shows significant activity against three human tumor xenograft lines.

- The increased therapeutic activity of 10-EdAM is achieved by optimization of the membrane

transport differential between tumor cells and normal tissues, and of the degree of polyglutamylation of drug in tumor cells relative to normal cells.

- Nonclassical antifolates, such as trimetrexate and piritrexim have activity against murine tumors unresponsive to MTX. Tumors with ac- quired resistance to MTX because of DHFR overproduction are totally or partially cross- resistant to lipophilic DHFR inhibitors. How- ever. tumors with resistance to MTX, caused by deletion of membrane transport, retain sensitivity to trimetrexate and piritrexim, and may even be collaterally sensitive.

- Resistance to trimetrexate and piritrexim may arise by a mutation that decreases the rate of cellular uptake of these drugs. In addition, tumors with pleiotropic drug resistance to an- thracyclines and vinca alkaloids may be cross- resistant to trimetrexate and piritrexim.

- The cytotoxicity of trimetrexate is highly phase- dependent on the cell cycle and occurs only in S-phase.

- Trimetrexate depletes cellular pools of dTTP and of purine nucleotides. Its cytotoxicity can be completely prevented by thymidine plus hypoxanthine.

- The cytotoxicity of piritrexim in vitro cannot be blocked by thymidine plus hypoxanthine. FuIi protection of S-180 cells requires a mixture of thymidine, hypoxanthine, adenosine and uridine.

- CB3717 has very limited activity against murine tumors, but has shown significant activity against some human tumors. This difference is attributed to the fact that thymidine levels in human plasma are much lower than in mouse plasma.

- CB3717 is active against MTX-resistant tumors with D H F R overproduction and impaired MTX uptake. However, routine and human cells have been described with resistance to CB3717 attributable to overproduction of thymidylate synthase.

- Like inhibitors of dihydrofolate reductase, CB3717 depletes cellular dTTP pools, but unlike dihydrofolate reductase inhibitors, CB3717

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appears to have no antipurine effect. - CB3717 is a substrate for folylpolyglutamate

synthetase, and polyglutamates of CB3717 are detectable in L1210 cells treated in vitro, and in normal tissues of CB3717-treated mice.

- D D A T H F is a potent inhibitor of G A R transformylase. It does not inhibit dihydrofolate reductase or thymidylate synthase. DDATHF is a substrate for folylpolyglutamate synthase.

- DDATHF has broad-spectrum activity against a panel of murine tumors.

K e y u n a n s w e r e d q u e s t i o n s

- Why does MTX have such narrow spectrum activity against murine tumors? If this is because of limited transport, does the four-fold lower influx K m of 10-EdAM partly explain its greater activity in murine tumors7 Does inade- quate transport limit the clinical spectrum of MTX, and if so, will 10-EdAM be more active than MTX against human tumors?

- What are the effects of 10-EdAM on cellular nucleotide pools?

- Since nonclassical antifolates are not transported by the 5-methyltetrahydrofolate membrane carrier, and are not polyglutamylated, to what is their antitumor selectivity attributable?

- is the rapid influx of trimetrexate and piritrexim into tumor cells passive or carrier-mediated? If it is passive, what is the nature of the mutation that confers resistance to these agents by impaired uptake?

- What is the mechanism by which cells with pleiotropic drug resistance become cross-resistant to trimetrexate and piritrexim? Does this resistance mechanism, which appears to be present intrinsically in some colon tumors, explain the low response rate of colon tumors to these agents?

- What is the cellular component to which nonexchangeable trimetrexate (in excess of the cellular dihydrofolate reductase level) is bound? Does this bound drug provide a cellular reser- voir of pharmacologically active drug?

- Why can t'-,e cytotoxicity of piritrexim not be

266

reversed by thymidine plus hypoxanthine? Does piritrexim have cellular sites of action other than dihydrofolate reductase?

- What is the mechanism by which CB3717 enters cells?

- What is the mechanism of the renal and hepatic toxicities of CB3717?

- Is the cytotoxic mechanism of DDATHF mediated through inhibition of synthesis of D N A or RNA or both?

- What is the cell cycle phase-dependence of the cytotoxicity of DDATHF? Is D D A T H F cytotoxic to non-cycling cells?

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10. DeGraw JI, Brown VH, Tagawa H, Kisliuk RL, Gaumont Y, Sirotnak FM: Synthesis and antitumor activity of 10-alkyl-10-deazaaminopterin. A convenient synthesis of 10-deazaaminopterin. J Med Chem 25: 1227-1230, 1982

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Biological and biochemical properties of new anticancer folate antagonists

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