Future directions for antifolate drug development

FUTURE DIRECTIONS FOR ANTIFOLATE DRUG DEVELOPMENT CLEMENTB. KNIGHT*,PATRICKC. ELWOODand BRUCE A. CHABNER Medicine Branch, Divisionof CancerTreatment, NationalCancer Institute, 9~XX)RockvillePike, Bethesda,Maryland20892 INIRODUCTION The folic acid antagonists have earned an important role in cancer therapeutics as part of the curative treatment of childhood acute lymphocytic leukemia and non-Hodgkin's lymphoma. It is logical that attempts should be made to improve on antifolate chemotherapy as knowledge progresses regarding the mechanism of action of the drug and the determinants of response. MECHANISM OF ACTION The mechanism of cytotoxicity of methotrexate was originally thought to be the intracellular depletion of reduced folate cofactors, as the result of its stoichiometric inhibition ofdihydrofolate reductase (DHFR, EC 1.5.1.3) (Fig. 1). More recently, it has become clear that under conditions of drug cxposure sufficient to produce cytotoxicity in tissue culture, depletion of reduced folatc cofactors for the important reactions mediating synthesis of thymidylate (5-10 methylene tctrahydrofolate, 5-10 CH 2 THF) and purines (10-formyl THF) is only partial and insufficient to account for cytotoxicity (1). However, the accumulation of toxic oxidized folates, such as dihydrofolate (DHF) (2) and 10-formyl DHF (3), as well as methotrexate polyglutamates, may produce direct inhibition of these folate-dependcnt reactions. These actions are illustrated in Figure 1. DHF strongly inhibits both thymidylate synthetase (TS, EC 2.1.1.45) and amino-imidazole carboxamide ribonucleotide transformylase (AICAR T'ase, EC 2.1.2.3) (4), and 10-formyl DHF blocks TS as well (5). Thus, the action of methotrexate may be self-limiting in that inhibition of TS by DHF, 10-formyl DHF, and the methotrexate polyglutamates will block the further depletion of reduced folates and the generation of higher levels of toxic

*To whomall correspondenceshouldbe addressed. 3

C. B. KNIGHT,

et al.

~AMP -

-

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~ IMP < ~

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(GAR and AICAR transformylases)

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ThymidyiateV'~'" Synthase ]~,.,~.~.

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{ Thymidylate synthase AICAR transformylase

10"CHO'FH2 (GlUn) { Thymtdylate synthase GAR transformylase

FIG. 1.

DHFs. These considerations lead to the following conclusions regarding the development of new folate antagonists: (1) New inhibitors of DHFR are unlikely to be more effective than methotrexate unless they incorporate features that enhance drug transport or polyglutamation. Drug design based simply on attempts to enhance DHFR inhibition is unlikely to be more effective because of the potency of methotrexate inhibition and the self-limited nature of DHFR inhibitors, as described above. (2) Alternative folate dependent enzymes provide attractive targets for design of new inhibitors. These targets include AICAR T'ase and glycineamide-ribonucleotide (GAR) T'ase, two early enzymes in purine

ANTIFOLATE DRUG DEVELOPMENT biosynthesis that utilize 10-formyl THF as their cofactor. Both enzymes show enhanced inhibition by polyglutamate forms of methotrexate, but these compounds are relatively modest inhibitors when facing a polyglutamate folate substrate (4). Examples of other inhibitory compounds of the basic folate structure include 5,10 dideazatetrahydrofolate (6) and tctrahydrohomofolate (7), both of which inhibit G A R T'ase. However, little work has been done to design inhibitors of these enzymes, as key structure-activity relationships or molecular interactions of substrate or inhibitor with purified enzyme have not been examined. (3) Improved antifolates might be designed if the processes of transport and polyglutamation were better understood. This information could be helpful in two ways. lnhibitors of these processes could be designed if the proteins responsible for these reactions were better characterized. Secondly, inhibitory compounds for intracellular folate-dependent enzymes require both intracellular uptake and polyglutamation for optimal activity, but optimization of these reactions will require a more detailed knowledge of the responsible proteins (8).

TRANSPORT IN NOVEL ANTIFOLATE DEVELOPMENT Since n e t intracellular accumulation of antineoplastic agents is essential for inhibition of sensitive tumor cell growth, perturbation of influx or efflux will affect sensitivity, and thus resistance, of tumor cells to these drugs. Folates and methotrexate are accumulated within eukaryotic cells via an energy dependent specific transport system(s) (9). Efflux mechanisms for methotrexate have been studied in murine leukemia L1210 cells (10) and are characterized by either inhibition (>95%) with N-hydroxysuccinamide ester of methotrexate which covalently attaches to a folate transport protein, inhibition by bromosulfophthalein or resistance to both the ester derivative of methotrexate and bromosulfophthalein. Knowledge of the transport systems may facilitate development of new antifolates which could be transported more efficiently or effect higher intracellular concentrations of drug by other means. Various factors may be considered in the development or potentiation of antifolates including: (1) bypassing the specific folate transport system; (2) exploiting differences in transport of antifolates between normal and malignant tissues; (3) alteration of the K m (Kt) and/or Vma x for antifolates; (4) inhibition of specific folate transport system; and (5) reduction of drug efflux. The various components of the specific folate transport (uptake) system have not been completely characterized, though the initial cell surface binding of ligand is an essential component of the system. A 40-50 kDa membrane-associated folate binding protein (M-FBP) has been isolated

6

¢. B. KNIGHT. et al.

and/or characterized from mammalian tissues (9, 11, 12), cultured cells [human nasopharyngeal epidermoid carcinoma (KB), monkey kidney epithelial (MAI04)] (13, 14) and human reticulocytes (15). M-FBP is an integral membrane protein with an exteriorly oriented ligand binding site (16) such that it is capable of binding significant quantities of 5-methyltetrahydrofolate (5-MTHF) (K~ = 0.3 × 109 i/moi) or methotrexatc (K a = 0.004 × 109 l/mol) with high affinity (13). M-FBP's affinity for 5-MTHF is sufficiently high for M-FBP to serve a transport function in these cells considering that the normal serum level of 5-MTHF is 5-50 nM. Although the K a of methotrexate for M-FBP is lower, it is sufficient to transport methotrexate when methotrexate concentrations are at lower therapeutic levels (10 -~, to 10-7 M). In contrast to the FBPs described in murine L1210 leukemia cells (see below), purified human M-FBP has a high affinity for folic acid (K,, = 1.3 × 109 l/mol). Polyclonal monospecific antiserum towards human FBP blocks the uptake of 5-MTHF (16), and when co-cultured with human erythroid progenitor cells, results in a 70% decrease in the intracellular folatc concentration which is associated with megaloblastic change (17). This data indicatcs that M-FBP serves an important role in the folate uptake process in these tissues. Others (18-21) have demonstrated a particulate FBP in the membrane fraction of murine L1210 leukemia cells by means of affinity labeling techniques; however, attempts to isolate the murine FBP have been unsuccessful due to its low concentration. Studies of the murine FBP in the crude state indicate that it requires detergent for solubilization and has an apparent M r ranging from 36,000 to 46,000 on SDS-PAGE. Based on transport kinetics, the K t of the murine folatc transport protein has ranged from 15 nM (18) to 1 ~M (9). The reason for this discrepancy is not certain, but may be related to the folate status of the cells or culture media, to the fact that the murinc protein has not been studied in an homogeneous form, or to induction of factors which could alter the transport system. For example, M-FBP, the human transport protein, exhibits ligand-mediated regulation such that the concentration of protein is inversely related to the folate concentration (22). There is interspecies variation in the eukaryotic folate binding protein as illustrated by the differences in bovine (23) and human amino acid sequences (24). It is possible that changes in the primary structure of FBPs may result in the differences in their Kas (or Kt). The discrepancy in the affinity (or the Kt) for folic acid, reduced folate derivatives, or methotrexate between human FBP and murine leukemia L1210 cells is uncertain but possibly represents the result of such an interspecies variation or of interspecies differences in processing of the protein. Furthermore, the membrane localization, molecular weights based on SDS-PAGE, apparent high affinities for 5-MTHF, 5-formyltetrahydrofolate, and methotrexate,

ANTIFOLATE DRUG DEVELOPMENT

and receptor modulation by folate levels (22, 25), suggest a close relationship between these folate binding proteins. To circumvent the requirements of the specific folate transport system, lipid soluble antifolates have been developed. The prototypic agent, trimetrexate, is undergoing clinical phase II trials. The drug is rapidly accumulated in cells and should be especially useful in tumors with low de n o v o or low acquired influx capacity for antifolates. Trimetrexate was found to accumulate rapidly in a methotrexate transport resistant strain of murine leukemia L1210 cells and required a 5,000-fold lower drug concentration than methotrexate to achieve 50% growth inhibition (26) in this cell line. The antimetabolic effect, responsible for both tumor cell death and tissue toxicity, may be even more pronounced should there be a concomitant reduction in influx of 5-MTHF. Galivan et al. (27) have described such a synergistic effect between low dose trimetrexate and a folate derivative (5,10-dideazatetrahydrofolate) in H35 hepatoma cells. Based on evidence of a difference in methotrexate transport between normal murine tissues and various murine tumors (28), Sirotnak et al. designed 5-alkyl derivatives of 5-deazaaminopterin and 5-deazamethotrexate. The inhibition of D H F R and the extent of polyglutamation of these drugs were comparable to those for methotrexate. However, these drugs showed an improved therapeutic activity (as determined by IC5o) relative to methotrexate, presumably as a result of altered transport since the influx of these drugs was increased (K~ = 0.91 to 1.12/~M, respectively) relative to methotrexate (K~ 12.5 tzM). Others (29) have reported improved inhibitory activity by a similar mechanism with other methotrexate or aminopterin analogs relative to methotrexate. Tissue to tissue variations in parameters such as transport kinetics (30) suggest that differences in processing of the transporter, e.g., glycosylation (31), may exist. These differences between tissues and/or tumors could involve the binding site which would enable engineering of tissue specific antifolates that would spare normal proliferating tissue. The secondary structure and membrane topography of human M-FBP may be predicted from the amino acid sequence deduced from the M-FBP cDNA nucleotide sequence (24). X-ray crystallography and NMR would be helpful in confirming the predicted secondary structure of M-FBP which may prove useful in elucidating the binding site for folates and antifolates. Comparison of the expression and function (e.g., binding and/or transport) in eukaryotic cells of full-length FBP cDNA and deletion constructs from this cDNA may be helpful in the determination of the folate binding site. This should assist in the development of novel antifolates which have higher affinity for the transport protein and are more efficiently transported than methotrexate. Alternatively, this information could be used to design inhibitors of transport.

Fig.2B a.

b.

c.

d.

e.

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FIG. 2 (B). Southern blot analysis. Genomic DNA from human lymphoblastic leukemia C C R F - C E M cells (lanes a and b), Chinese hamster ovary (CHO) cells (lanes c and d), and human mammary carcinoma (ZR75) cells (lanes e and f) were cleaved with EcoRl (lanes a, c, and e) or Hindlll (lanes b, d, and f). The blot was probed as described above.

a.

b.

c.

d.

Fig.3 Kb

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tA FIG. 3. Northern blot analysis. Poly(A) + mRNA isolated from human KB cells (lane a), CCRF-CEM cells (lane b), ZR75 cells (lane c) and CHO cells (lane d) were resolved on a 1% agarose-formaldehyde gel, transferred to nitrocellulose, and probed with nick translated human folate binding protein cDNA under stringent conditions.

8

C. B. KNIGHT,

et al.

Another approach to antifolate therapy could involve the inhibition or blockade of the transport system. Antony et al. (17) demonstrated the functional significance of M-FBP by observing the effects of antiplacental folate receptor antiserum on cultured human bone marrow cells (see above). It may be possible to target tumors expressing high levels of M-FBP with antibody, antibody conjugates, or to use antibody in parallel with antifolates to effect a greater folate deplete state. This “immunotherapy” would require the production of a suitable monoclonal antibody that could be used in conjunction with a lipid soluble antifolate like trimetrexate. Although the FBPs so far described contain a single binding site, it is possible that discrete portions of the molecular structure will enable selective chemical blockade of the receptor binding site for folates but not for antifolates. The Vinca alkaloids facilitate accumulation of and increase the cytotoxicity of methotrexate in murine leukemia L1210 cells by inhibiting the bromosulfophthalein sensitive efflux pathway of methotrexate (18). Since the nonclassical antifolate, trimetrexate, is not polyglutamated and shows significant efflux in HCT-8 cells (32), the combination of trimetrexate with agents, such as vincristine, could potentially enhance the effectiveness of antifolates. At least two mechanisms by which cells become resistant to methotrexate by virtue of defective transport have been described (33) and involve alteration of the K, and/or V,,, of methotrexate. Flintoff et al. (34) described a mutant of a Chinese hamster ovary (CHO) cell line which was resistant to methotrexate because of an inability to transport drug. The authors stated that these resistant CHO cells exhibited a defective membrane binding component. The V,,, was also markedly reduced and binding of methotrexate, folic acid, and 5-MTHF could not be demonstrated in the resistant cell line relative to the wild type CHO cell line. Schuetz et al. (19) have described a methotrexate-resistant murine L1210 leukemia cell line where methotrexate influx was markedly depressed due to a decrease in V,,,, without a change in K,. Since the resistant cell lines contained similar quantities of the membrane transport protein as determined by ztffmity labeling, it was postulated that the defect in transport was related to reduced mobility of the carrier resulting in a failure to translocate methotrexate across the cell membrane. Sirotnak (35) described several categories of methotrexate transport resistant sublines of L1210 cells. Cells from each category were characterized bxa 3- to 4-fold reduction in apparent influx V,,, for methotrexate, a 2-fold increase in K, for methotrexate, or a 5-fold reduction in influx combined with a 3-fold increase in apparent influx K,. The various mechanisms by which cells exhibit methotrexate resistance by virtue of defective transport in vitro have been phenotypically defined by analysis of transport kinetics (K, and V,,,) and affinity labeling techniques.

ANTIFOLATE DRUG DEVELOPMENT

9

Further characterization of methotrexate sensitive and resistant cell lines with anti-FBP antibodies (22) and with FBP cDNA probes (24) should provide insights into the acquisition of resistant states in vitro and in vivo and provide clues toward the development of drugs which could potentially prevent de n o v o or circumvent acquired drug resistance. For example, transport resistance characterized by a low Vm~Xmay result from decreased synthesis or increased catabolism of the transport components (e.g., M-FBP). On Northern blot analysis, the former would be associated with a decreased or absent hybridization signal, whereas, in the latter, the signal may in fact be increased. In either instance, the total M-FBP determined by immunologic or functional assays would be decreased. Transport resistance due to an altered K~ (Kt) presumably due to a mutational change in the protein would result in an increased signal on Northern analysis and perhaps an alteration on Southern blot analysis if the mutation involved an enzyme restriction site (analogous to RFLP, restriction fragment length polymorphism). In preliminary experiments, Southern blot analysis of normal human genomic DNA (derived from lymphocytes) and tissue culture cell [malignant nasopharyngeal cpidermoid carcinoma (KB) cells, human lymphoblastic CCRF-CEM cells, human mammary carcinoma (ZR75) cells, and Chinese hamster ovary (CHO) cells] DNA were probed with radiolabeled human M-FBP eDNA. The hybridization patterns between normal human gcnomic and KB ccli DNA were identical to each other (Fig. 2A) and to other human DNA (Fig. 2B) irrespective of the phenotypic expression of M-FBP dctermined by PtcGlu binding assays (12), RIA (22), or Western blot analysis. Chinese hamster ovary cells (Fig. 2B) appear to contain closely related DNA although the hybridization pattern is different from that of human DNA, presumably a reflection of interspecies DNA differences. Northern blot analysis (Fig. 3) of poly(A) + mRNA isolated from the same cell lines and poly(A) + mRNA from various normal human tissues (24) indicated that the radiolabeled human FBP cDNA hybridized to a 1100 bp species of mRNA that was expressed to a variable degree. The presence of a signal on Northern blot analysis of these tissue culture cells correlated with the level of phenotypic expression of M-FBP. The mRNA from KB cells, which express M-FBP, contains the 1100 bp mRNA species whereas the mRNA from the other cell lines, which do not express detectable quantities of M-FBP, do not contain this species of mRNA (Fig. 3). It is possible that the tissue culture cells (CCRF-CEM, ZR75 and CHO) propagated in media containing high concentrations of folic acid (2-4 p,M) did not contain a detectable signal because M-FBP was down regulated. These studies illustrate the feasibility and potential usefulness of the molecular characterization of this component of the transport system and should provide a tool for further characterization of

10

C.B. KNIGHT, et al.

transport resistance mechanisms. Further application of these techniques may include the prediction of tumor sensitivity to classic antifolates and the acquisition of methotrexate resistance. SUMMARY

We have discussed potential ways by which new antifolates could be designed and utilized to effect both sensitive and resistant cell/tumor inhibition. Targeting of alternative folate-dependent enzymes and increasing net intracellular accumulation (transport) and polyglutamation of antifolates should be useful approaches. In addition, antibodies to and cloned cDNAs of the transport components (e.g., folate transport proteins) will allow better characterization and understanding of de n o v o and acquired antifolate resistance states and may provide insights into new drug development. REFERENCES I. 2. 3.

4.

5.

6. 7.

8. 9.

10.

C . J . ALLEGRA, R. L. FINE, J. C. DRAKE and B. A. CHABNER, The effect of methotrexale on intracellular folate pools in human MCF-7 breast cancer cells: evidence for direct inhibition of purine synthesis, J. Biol. Chem. 261, 6478-6485 (1986). C.J. ALLEGRA, J. C. DRAKE, J. JOLIVET and B. A. CHABNER, The inhibition of AICAR transformylase by methotrexate and dihydrofolic acid polyglutamates, Proc. Natl. Acad. Sci. U.S.A. 82, 4881-4885 (1985). J. BARAM, B. A. CHABNER, J. C. DRAKE, A. L. FITZHUGH, P. W. SHOLAR and C. J. ALLEGRA, Identification and biochemical properties of 10-formyl dihydrofolate, a novel folate found in methotrexate-treated cells, J. Biol. Chem. 263, 7105-7111 (1988). C. J. ALLEGRA, K. HOANG, G. C. YEH, J. C. DRAKE and J. BARAM, Evidence for direct inhibition of de novo purine synthesis in human MCF-7 breast cells as principal mode of metabolic inhibition by methotrexate, J. Biol. Chem. 262, 13520-13526 (1987). C.J. ALLEGRA, J. BARAM and B. A. CHABNER, Evidence for direct inhibition of metabolic pathways as a mechanism of action of methotrexate, pp. 981-984 in Chemistry and Biology of Pteridines 1986: Pteridines and Folic Acid Derivatives (B. A. COOPER and B. M. WHITEHEAD, eds.), de Gruyter, New York (1986). R. G. MORAN, E. C. TAYLOR, C. SHIH and G. P. BEARDSLEY, The two diastereomers of 5.10-dideazatetrahydrofolate (DDATHF) are equiactive, Proc. Am. Assoc. Cancer Res. 28, 274 (1987). J. THORNDIKE, Y. GAUMONT, R. L. KISLIUK, F. M. SIROTNAK, B. R. MURTHY, M. G. NAIR and J. R. PIPER, Inhibition of glycinamide ribonucleotide formyltransferase and other folate enzymes by homofolate polyglutamates in human lymphoma and routine leukemia cell extracts, Cancer Res. 49, 158-163 (1989). B. A. CHABNER, Methotrexate, pp. 229-255 in Pharmacologic Principles of Cancer Treatment (B. CHABNER, ed.), W. B. Saunders, Philadelphia (1982). F . M . HUENNEKENS, M. R. SURESH, C. E. GRIMSHAW, D. W. JACOBSEN, E. T. QHACHAS, K. S. VITOLS and G. B. HENDERSON, Transport of folate and pterin compounds, pp. 1-22 in Chemistry and Biology ofPteridines, (J. A. BLAIR, ed.), de Gruyter, New York (1983). G. B. HENDERSON and E. M. ZEVELY, Affinity labeling of the 5-methyltetrahydrofolate/methotrexate transport protein of LI210 cells by treatment with an N-hydroxysuccinimide ester of [3Hlmethotrexate J. Biol. Chem. 259, 4558-4562 (1984).

ANT1FOLATE DRUG DEVELOPMENT

11

11. A . C . ANTONY, C. UTLEY, K. C. VANHORN and J. F. KOLHOUSE, Isolation, characterization and comparison of the solubilized particulate and soluble folate binding proteins from human milk, J. Biol. Chem. 257, 10081-10089 (1982). 12. A . C . ANTONY, C. UTLEY, K. C. VANHORN and J. F. KOLHOUSE, Isolation and characterization of a folate receptor from human placenta, J. Biol. Chem. 256, 9684-9692 (1981). 13. P. C. ELWOOD, M. A. KANE, R. M. PORTILLO and J. F. KOLHOUSE, The isolation, characterization, and comparison of the membrane-associated and soluble folate-binding proteins from human KB cells, J. Biol. Chem. 261, 15416-15423 (1986). 14. B. A. KAMEN and A. CAPDEVILA, Receptor-mediated folate accumulation is regulated by the cellular folate content, Proc. Natl. Acad. Sci. U.S.A. 83, 5983-5987 (1986). 15. A. C. ANTONY, R. S. KINCADE, R. S. VERMA and S. R. KRISHNAN, Identification of high affinity folate binding proteins in human erythrocyte membranes, J. Clin. Invest. 80, 711-723 (1987). 16. A. C. ANTONY, M. A. KANE, R. M. PORTILLO, P. C. ELWOOD and J. F. KOLHOUSE, Studies of the role of a particulate folate-binding protein in the uptake of 5-methyltetrahydrofolate by cultured human KB cells, J. Biol. Chem. 260, 14911-14917 (1985). 17. A . C . ANTONY, E. BRUNO, R. A. BRIDDELL, J. E. BRANDT, R. S. VERMA and R. HOFFMAN, Effect of perturbation of specific folate receptors during in vitro erythropoiesis. J. Clin. Invest. 80, 1618-1623 (1987). 18. G. B. HENDERSON, J. M. TSUJI and H. P. KUMAR, Mediated uptake of folate by a high-affinity binding protein in sublines of LI210 cells adapted to nanomolar conccntrations of folatc, J. Membr. Biol. 101,247-258 (1988). 19. J. D. SCHUETZ, L. H. MATHERLY, E. H. WESTIN and I. D. GOLDMAN, Evidencc for a functional defect in the translocation of the methotrexatc transport carrier in a methotrcxate-resistant murine LI210 leukemia cell line, J. Biol. Chem. 263, 9840-9847 (1988). 20. E. M. PRICE and J. H. FREISHEIM, Photoaffinity analogues of methotrexatc as folate antagonist binding probes. 2. Transport studies, photoaffinity labeling, and identification of the membrane carrier protein for methotrexate from routine L1210 cells, Biochemistry 26. 4757-4763 (1987). 21. C.-H. YANG, F. M. SIROTNAK and L. S. MINES, Further studies on a novel class of genetic variants of the L1210 cell with increased folate analogue transport inward, J. Biol. (;hem. 263, 9703-9709 (1988). 22. M . A . KANE, P. C. ELWOOD, R. M. PORTILLO, A. C. ANTONY, V. NAJFELD, A. FINLEY, S. WAXMAN and J. F. KOLHOUSE, Influence on immunoreactive folate-binding proteins of extracellular folate concentration in cultured human cells, J. Clin. Invest. 81, 1398-1406 (1988). 23. I. B. SVENDSEN, S. 1. HANSEN, J. HOLM and J. LYNGBYE, The complete amino acid sequence of the folate-binding protein from cow's milk, Carlsberg Res. Commun. 49, 123--131 (1984). 24. P . C . ELWOOD, C. B. KNIGHT and B. A. CHABNER, Cloning of the eDNA for the human membrane-associated folate binding protein from placental and malignant nasopharyngeal carcinoma cell eDNA libraries, Blood 72, 73A (1988). 25. C. A. LUHRS, E. SADASIVAN, M. DA COSTA and S. P. ROTHENBERG, The isolation and properties of multiple forms of folate binding protein in cultured KB cells, Arch. Biochem. Biophys. 250, 94-105 (1986). 26. B . A . KAMEN° B. EIBL, A. CASHMORE and J. BERTINO, Uptake and efficacy of trimetrexate, a non-classical antifolate in methotrexate-resistant cells in vitro. Biochem. Pharmacol. 33, 1697-1699 (1984). 27. J. GALIVAN, Z. MIMEC, M. RHEE, D. BOSCHELLI, A. L. ORONSKY and S. S. KERWAR, Antifolate drug interactions: enhancement of growth inhibition due to the antipurine 5,10-dideazatetrahydrofolic acid by the lipophilic dihydrofolate reductase inhibitors metoprine and trimetrexate, Cancer Res. 48, 2421-2425 (1988). 28. F. M. SIROTNAK, F. A. SCHMID, G. M. OTTER° J. R. PIPER and J. A.

12

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3(1. 31. 32. 33. 34. 35.

c . B . KNIGHT, etal. MONTGOMERY, Structural design, biochemical properties, and evidence for improved therapeutic activity of 5-alkyl derivatives of 5-deazaaminopterin and 5-deazamethotrexate compared to methotrexate in murine tumor models, Cancer Res. 48, 5686-5691 (1988). A. ROSOWSKY, H. BADER, C. A. CUCCHI, R. G. MORAN. W. KOHLER and J. H. FREISHEIM, Methotrexate analogues. 33. N delta-acyI-N alpha-(4-amino-4deoxypteroyl)-L-ornithine derivatives: synthesis and in vitro antitumor activity, J. Med. Chem. 31, 1332-1337 (1988). F. M. SIROTNAK. Obligate genetic expression in tumor cells of a fetal membrane property mediating "'folate" transport: biological significance and implications for improved therapy of human cancer, Cancer Res. 45, 3992-400() (1985). E. SADASIVAN, M. DA COSTA, S. P. ROTHENBERG and L. BRINK, Puritication, properties, and immunologic characterization of folate-binding proteins from human leukemia cells, Biochim. Biophys. Acta 925, 36-47 (19871. J . R . BERTINO, A. SOBRERO, E. MINI, B. A. MOROSON and A. CASHMORE, Design and rationale for novel antifolates, NCI Monogr. 5, 87-91 (19871. Y. G. ASSARAF and R. T. SCHIMKE, Identification of methotrexate transport deficiency in mammalian cells using fluoresceinated methotrexate and flow cytometry, Proc. Natl. Acad. Sci. U.S.A. 1t4, 7154-7158 (1987). W. F. FLINTOFF and C. R. NAGAINIS, Transport of methotrexate in Chinese hamster ovary cells: a mutant defective in methotrexate uptake and cell binding, Arch. Biochem. Biophys. 223. 433--44(I (1983). F. M. SIROTNAK, D. M. MOCCIO, L. E. KELLEHER and L. J. GOUTAS, Relative frequency and kinetic properties of transport-defective phenotypes among methotrexate-resistant LI210 clonal cell lines derived in vivo, Cancer Res. 41, 4447-4452 ( 1981 ).