I @ Folateantagonistsalsoillustrated;Inadditiontobaker's

[CANCER RESEARCH 39, 293-304, February 1979] 0008-5472/79/0039-0000$02.00 Toward Improved Selectivity in Cancer Chemotherapy: The Richard and Hinda Rosenthal Foundation Award Lecture1

Joseph R. Bertlno2

Yale University School of Medicine, New Haven, Connecticut 06510

dylate synthetase per se or as facilitators of 5-fluorodeox yuridine 5'-monophosphate binding to this enzyme are also discussed. The potential for selectivity using nonclassical @ I folateantagonistsisalsoillustrated;inadditiontoBaker's antifol (triazinate), a triazine folate antagonist, now in clinical trial, several 2,4-diaminoquinazolines were found to be potent exhibitors of DNA synthesis in human leukemic leukocytes. Drug resistance to antineoplastic agents continues to be an important clinical problem. By using resistanceto meth otrexate as an example, exploitation of drug-resistant mu tants by design of specific inhibitors is possible. A strategy h for the prevention of drug resistance is advanced, based on F dataderivedfromexperimentalsystems.Itisconcluded

â€”@-@ that large doses of drugs used in intermittent fashion and @1 sequentially in combination may be the best strategy to avoid this problem. An example of this strategy is Adriamy cin-cyclophosphamide-Oncovin-methotrexate-1 -f3-D-arabi nofuranosylcytosine, a five-drug sequential combination used to treat diffuse histiocytic lymphoma.

Dr. Miller, Mr. and Mrs. Rosenthal, members and guests of the American Association for Cancer Research, I am deeply honored to receive the Rosenthal Award. I have been extremely fortunate over the years I have been at Yale to have many outstanding colleagues, fellows, I and graduate students, and they havereally been responsi ble for whatever success we have had. I had prepared a list of their names to show you but decided not to; it might embarrass those who wish not to admit an association, and it was embarrassing to me to realize I have accomplished so little with so much talent. I will acknowledge their respective contributions during the course of this lecture. Abstract The history of chemotherapy has not been characterized by smooth sailing. There have been many Bermuda trian Approaches to improving the selectivity of drugs for the gles along the way, and the success achieved has surprised treatment of patients with cancer are discussed, with em many scientists who doubted that progress was possible phasis on folate antagonists as prototype compounds. The with the deficiencies in our basic knowledge of the neoplas useof metabolites (leucovorin, 5-methyltetrahydrofolate)as tic process. What will the role of chemotherapy be in the â€œrescue' â€˜ agents following methotrexate treatment with next decade? I was going to indicate in Table 1 that the appropriate monitoring of blood levels of methotrexate has 1980's would establish chemotherapy as an alternative to led to the safe use of this antimetabolite in high doses and surgery or radiation therapy for the treatment of some an improved therapeutic index for the use of this drug in human cancers, but since some of my best friends are some human cancers. The use of methotrexate in conjunc surgeons and radiation therapists, I decided not to strain tion with fluorouracil administration is sequence dependent our relationships. in experimental tumor models and is being tested in human I had the good fortune to have Frank Huennekens and tumors. New folate antagonists, designed to inhibit thymi Clem Finch as my mentors while a postdoctoral fellow quite a few years ago, and then to work with Arnold Welch and

I Presented at the 1978 Meeting of the American Association for Cancer the â€œgangoffourâ€•(Dr.Evangelo Canellakis, Dr. Robert Research in Washington, D. C. Supported in part by Grants CAO8O1Oand Handschumacher, Dr. William Prusoff, and Dr. Alan C. CA08341 and Contract NO-1-CM-33711. 2 American Cancer Society Professor of Medicine and Pharmacology. Sartorelli) at Yale for several more years; therefore it's Received October 16, 1978; accepted October 26, 1978. probably not surprising that my approach to chemotherapy

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Table 1 MIX@ Historyof chemotherapy:the pessimist'sview 1940's Chemotherapycan't workâ€”wedon't understandthe dif @,MTX(G)N ferencebetweennormal and cancercells. @MTX@ @ â€˜ Purines(C2,C1) 1950's Chemotherapyis effective, but patientsalwaysrelapse. DHFRMTx(G)N 1960's Chemotherapy can cure choriocarcinoma and Burkitt's FH2(G).@- u.FH4(G)Nâ€• lymphoma,but theseare unusualtumors. 1970's Chemotherapycancure severaltumors, but the drugs are @ dTMP FH4(G @N too toxic, i.e., lack selectivity. â€˜I 1980's ? DNA dUMP Eztrocslhilor TT@ Compartment Cell Membrane Chart 1. Mechanism of MTx action and folate metabolism. 5CH3FH4, has been to try to understand why drugs work or don't MeFH4;5-CHOFH4, leucovorin. work. The following quote from GeorgeHitchings, who has done this as well as anyone fairly well summarizes this especially in the presence of NADPH and at a slightly acidic approach (64). pH (15). Under these conditions, the binding is one to one and has been referred to by the late Dr. William Werkheiser , â€˜Historically most chernotherapeutic agents have been used without an understandingof their mechanismsof action. However, as â€œstoichiometricâ€•(111,112). the potential rewardsof such understandingareenormous,for the Since thymidylate synthetase action, rather than FH4 practical application of the drug, the discovery of more selective formation via DHFR activity, is rate limiting, only when agents,and for fundamentalknowledge.â€• DHFR is completely inhibited and when excess or â€œfreeâ€• MTX is present in the cell, as emphasized by Goldman, With that preamble, I would like to review for you some of Sirotnak, and coworkers, does FH4 synthesis stop and, our studies and indicate the approaches we are taking in consequently, thymidylate biosynthesis (32, 52, 100). Cells our current research. rapidly synthesizing DNA will of course be depleted of FR, During the past 20 years, a relatively complete description most rapidly. Since FR, is also required for purine synthe of how one clinically useful drug, MTX,3 exerts its effects sis, purine deficiency may also occur; as shown by Hryniuk on cells has become available. I would like to review this et a!. (66), this may be an important mechanism of cell information briefly for those who are not working in this death in some cell types. field (Chart 1). Therefore, some of the factors that relate to cell death In order to prevent a cell from replicating, MTX must first due to MTX are the level of excess or free MTX in cells, the cross the cell membrane. Work from several laboratories rate of dissociation of MTX from the inhibited enzyme when has shown that MTX uptake is an active process and that intracellular levels decrease as a function of decreasing this drug enters cells predominantly by utilizing the carrier extracellular levels, the rate of synthesis of DNA, and the system responsible for transport of reduced folates (10, 11, rate of synthesis of the target enzyme DHFR (9, 19, 21, 65, 17, 39, 50, 51, 53, 56, 67, 85, 99). Since MTX competes 104). effectively with the naturally occurring folates for uptake, it In recent years, as a result of studies in several laborato follows that high extracellular concentrations of this drug ries, it has become clear that the folate coenzymes in cells will inhibit uptake of MeFH4, the predominant form of folate exist as polyglutamates and that these compounds, rather in plasma. Thus an additional site of action of MTX, espe than being inactive storage forms, are the natural cofactors cially as used in the high-dose regimens, may be to starve for one-carbon enzyme reactions (33, 34). Recent studies cells of reduced folates. When extracellular concentrations have indicated some conversion of MTX to a diglutamate of MeFH4 or 5-formyltetrahydrofolate (leucovorin) are in form in vivo (8, 113) and in vitro (83). The consequences of creased by administration of these compounds, effective this conversion are not clear; however, this derivative is as inhibition of MTX transport may result. In addition, as potent an inhibitor of DHFR as is MTX (70). A mutant line of leucovorin enters the cell, because of countertransport, Chinese hamster ovary cells has recently been shown to be more MTX leaves the cell (85). Thus leucovorin may amelio unable to synthesize folate polyglutamates (80, 106). These rate MTX toxicity not only by providing cells with a reduced cells cannot survive without the addition of the end prod folate coenzyme, thus bypassing the MTX block, but also ucts of folate metabolism, namely, a purine and thymidine, by decreasing the intracellular concentration of MTX as a indicating that the formation of folate polyglutamates is result of these effects on its transport (51, 85). Obviously, essential for cell growth, perhaps because these forms are the higher the concentration of leucovorin relative to MTX, retained by the cell, while folate monoglutamates are not. the more important are these effects. Clearly, we need to learn a great deal more about folate Once inside the cell, MTX rapidly binds to the enzyme polyglutamate forms and cell regulation. DHFR. This binding, although reversible, is extremely tight, How can we use what information we have on folate metabolism to improve the selectivity of folate antagonist chemotherapy? Since stem cells from normal renewal tis 3 The abbreviations used are: MTX, methotrexate; MeFH4, 5-methyltetra hydrofolate; DHFR, dihydrofolate reductase; FH4, tetrahydrofolate; ALL, sues also utilize the same pathways to synthesize DNA as acute lymphatic leukemia; 5-FU, 5-fluorouracil; CNS, central nervous system; do tumor cells, how can we get selective antitumor effects? i.t., intrathecal; dThd, thymidine; CPG,, carboxypeptidase G, TZT, triazin The realization that MTX, like other antimetabolites, is a ate; FdUMP, fluorodeoxyuridine 5'-monophosphate; CH,FH4, 5,10- methylenetetrahydrofolate; FdUrd, fluorodeoxyuridine; cDNA, complemen schedule-dependent drug, i.e. , most effective when used tary DNA. intermittently when the tumor cell burden is low and when

294 CANCER RESEARCH VOL. 39

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1979 American Association for Cancer Research. Improved Selectivity in Cancer Chemotherapy most cells are in cycle, has already led to improved results prophylaxis of CNS leukemia with high-dose MTX-Ieucovo in the treatment of ALL (1). nfl regimens have given rise to the hope that this technique During this talk I will discuss our attempts to improve with or without i.t. MTX may replace brain irradiation in the selectivity with folate antagonists. These are the use of MTX management of patients with ALL and possibly result in a

â€œrescueâ€• techniques; the use of MTX in combination with decreased incidence of CNS toxicity (110). other drugs, in particular 5-FU; new folate antagonists of The mechanism of the selectivity of leucovorin rescue for the â€œnonciassicalâ€•variety;and, finally, the MTX-resistant certain tumors is not clear, and the pharmacology of leu cell as a chemotherapeutic target. covorin is of interest in this regard. When leucovorin is administered p.o. , it is converted almost quantitatively to Leucovorin â€œRescueâ€• MeFH4 (89). Even when administered parenterally, leucovo rin is rapidly converted to the methyl compound. Therefore, Goldin et al. (46-49), who first pointed out the importance reports that MeFH4 may selectively rescue normal cells are of dose schedule on the effects of MTX against early Li 210 of interest and worthy of further study (59, 60). leukemia, in the 1950's also indicated another way to During recent years, other â€œrescueâ€•techniques have improve selectivity with MTX. In the L1210 system, use of been used to attempt to improve MTX selectivity; asparagi leucovorin allowed larger doses of MTX to be administered nase following MTX is a synergistic drug combination (27, safely, with improved therapeutic results. Our group at Yale 28, 109) and appears to be a very effective selective combi and Dr. Isaac Djerassi in Philadelphia began using this nation treatment for ALL (27). The use of concomitant MTX technique in the late 1960's (reviewed in Ref. 13). and dThd selectively to rescue normal cells and to kill In the treatment of cancer of the head and neck, it is clear neoplastic cells that are sensitive to the purineless state that 2 constant infusion regimens, either 240 mg/sq m over induced by MTX is also of great interest. This technique, as 24 hr every4 days,or 1 g/sq m over36 to 42 hr every2 well as that of using dThd to rescue normal cells after an weeks, produce substantial but transient benefit in those interval of exposure to MTX, has been studied by Tattersall, patients (reviewed in Ref. 16) with advanced disease (Table Frei, and their coworkers (37, 104) and Semon and Grindey 2). Dr. Pitman and her colleagues have introduced another (98). Whether selectivity with MTX will be improved by effective schedule, using a push of MTX (3 g/cu m) with concomitant MTX and dThd, or dThd rescue, remains to be leucovorin rescue starting at 24 hr that also is at least as determined. effective as other MTX-leucovorin regimens (90). Although more conventional â€œlow-dose'â€˜regimens, such as weekly Carboxypeptidase G administration, may give response rates also approaching 50% (16),theuseofMTX withleucovorinrescue,especially Chart 2 shows the reaction carried out by an enzyme we in patients not previously treated by X-ray or surgery, has have called CPG1 (82). This enzyme has been investigated been safe and well tolerated and should allow the addition as a method to produce enzymic folate deficiency in man of other drugs in an attempt to improve the magnitude of (29), since it hydrolyzes folate cofactors and since dietary the responses and the response duration (13, 16). This folate deficiency has been difficult to achieve and patient approach is being explored by several groups as preopera compliance has been poor (44). The enzyme has good tive or preirradiation therapy, as well as adjuvant treatment antitumor activity against the Walker 256 carcinosarcoma, postoperative and post-X-ray. a transplanted rat tumor shown previously by Rosen and In ALL and in lymphomas, high-dose MTX regimens with Nichol to be relatively insensitive to MTX treatment but leucovorin appear also to be safe and effective (13, 35, 36, sensitive to dietary folate depletion (87, 92). CPGI has been 38, 65, 97, 107, 116). An additional unanticipated bonus has purified to homogeneity and characterized (82), and after been the good cerebrospinal fluid levels of MTX obtained initial clinical trials (22) large-scale production of a similar with these programs, important in those diseases that often enzyme for clinical trial is being carried out by the New disseminate to the meninges. Successful treatment of me England Enzyme Center. ningeal leukemia and lymphoma as well as studies of Inasmuch as CPG, also hydrolyzes MTX to the corre

Table 2 Useof MTXinfusions with leucovorin in headand neckcancerResponsesNo.

ofMTX schedulepatients>50%>25%24-hrdosage scheduleLeucovorin doses184120iv. infusions every 2 wk, 40-6 mg i.m. every 12 hr for 6 m24-hr mg/sq doses1963mg/sqi.v. infusions every5 days,806 mg i.m. every6 hr for 4 m24-hr then2113240iv. infusions every 4 days,75 mg in 12-hr iv. infusion, mg/sq m12 mg i.m. every 6 hr for 4 doses36â€”42-hr infusion,1713720-1080iv. infusion every 2 wk,40 mg/sq m in 6-hr iv. mg/sq mthen 25 mg p.o. every 6 hr for dosesTotal75363 4

FEBRUARY 1979 295

@OH z H2N (I,

U U. ,!:@:IIIIiT,:I:I:@J_CH2-NHâ€”3â€”â€”C Nâ€”@ii -J z OH @OH 0 FOLIC ACID U 2 1Zn@ z I CARBOXYPEPTIDASE G1 COON U (I) 4 H3N 1H20 CH3 U I) crIz z @@IIIitI1II)_cH2-NH-1@@â€”Câ€”OH+ NCâ€”NH2 OH COOH 20 50 100 250 500 PTEROICACID GLUTAMICACID DOSEMTX(mg/kg) Chart 2. Hydrolysis of folates by CPGI. Chart 3. Effect of CPG, on the antitumor effects of MTx. CPG, (800 g.tg/ kg) was administered 24 and 48 hr after MTX (from Ref. 30). sponding pteroate and glutamic acid, thereby inactivating it, administration of this enzyme after MTX administration dUMP â€” @-@-dTMP can limit the effects of the antifolate. Evidence that this (Fd UM7,@#â€•@NS@ enzyme might be useful as a rescue agent is shown in Chart 3. In this seriesof experiments, carried out in collaboration with Dr. DavidJohns and Dr. Bruce Chabner, CPGIadmin istered 24 and 48 hr after MTXto L1210tumor-bearing mice CH2FH4 FH2 was able to protect animals from MTX toxicity, and the (MTX) higher doses of MTX that could be administered safely resulted in an increase in life span of the animals (30). Dr. K. Kalghatgi reported last year at these meetings that CPGI, in combination with the â€œnonclassicalâ€•folateantagonists @ 2,4-diamino-5(3' ,4'-dichlorophenyl)-6-methylpyrimidine and EFdUMP E + FdUMP Baker's antifol (TZT)(see below) potentiated the effects of these drugs (71). We hope to initiate a clinical trial of these EFdUMPCH2FH4 if combinations in the near future as well as to test CPGI as a MTX rescue agent. @ E'FdUMPMTX @7 Fluoropyrimidines and Folate Antagonists Chart 4. Ternary complex formation between CH,FH4 or MTX, FdUMP, and thymidylate synthetase. E, enzyme. Although MTX has been used extensively in drug combi nations with 5-FU, especially in the CMF regimen (Cytoxan, binding of FdUMP to TMP synthetase could result. The methotrexate, 5-fluorouracil) for breast cancer (24), its results of such an experiment are shown in Chart 5. MTX contribution to such a combination has not been estab pretreatment enhanced 5-FU antitumor activity in the Sar lished. In experimental models, the combination of 5-FU coma 180 system, but concomitant MTX and 5-FU or the and MTX has been reported to be antagonistic (105)as well reverse sequence resulted in less than additive effects and as synergistic (7, 20, 76). even antagonism (20).The importance of the MTX-+ 5-FU Our interest in this combination was stimulated by the sequence in obtaining optimum antitumor effects with work of Santi et a!. (93), who showed that the binding of these drugs has also been reported by Martin et a!. (79), FdUMPto TMP synthetase appeared to be covalent in the who used an experimental breast tumor, and by Bareham presence of the folate coenzyme, CH2FH4.It follows, there et a!. (7), who studied the effects of these drugs on an fore, that when insufficient levels of CH2FH4are present in immune response. Whether the mechanism proposed, i.e., cells, 5-FU and FdUrd cell kill may be lessened. In fact, increased binding of FdUMP in ternary complex with MTX decreased cell kill by FdUrd in folate-depleted cells has and the enzyme TMP synthetase, accounts for the se beendescribed recently by Ullmanetal. (108). quence-dependent synergy is not yet established. Other The formation of this ternary complex was measured effects of MTX, i.e., an enhancement of 5-FU uptake and using nitrocellulose filter assay,since the ternary complex incorporation into RNA, also result when cells are pre is retained by the filter. Using this assay, Santi et al. (93) treated with high levels of MTX (26). D. Fernandes in our found that folate analogs such as MTX and 2-amino-4- laboratory has been studying the formation of the MTX hydroxy-N'Â°-methylquinazolinecould also substitute for FdUMP-enzymecomplexas a function of sequenceof drug CH2FH4in the ternary complex. We attempted to increase 5- administration. FU effectiveness by pretreating cells with high doses of Willa similarsequenceof treatmentleadto improved MTX. We reasoned that high intracellular levels of MTX results in human cancers sensitive to 5-FU? We are cur could provide the cell with an analog of CH2FH4 when rently studying this possibility in patients with breast and intracellular levelsof MTXexceededthe binding capacity of gastrointestinal cancer. DHFR (Chart 4). Under these circumstances, enhanced Since MTX may not be the best CH2FH4antagonist to use

296 CANCER RESEARCH VOL. 39

w in Table 5. The Walker 256 carcinosarcoma is only moder ately sensitive to MTX treatment, presumably because of I.- the limited uptake of this drug into the tumor (103). How -j 20 4 ever, TZT or Baker's antifol (NSC139105)is curative when > animals bearing this tumor are treated even in doses as > U) a: >- CONTROL small as @/iootheminimal toxic dose (6). In contrast, this :D 4 U) 0 drug is essentially without effect against the L1210 tumor. I0 2 We haveshown that the reasonfor the tremendous selectiv 4 ity of this drug against the Walker 256 tumor, in contrast to

Ui its ineffectiveness against the L1210, is the marked uptake by the Walker 256 cell and the poor uptake of the drug by I. . . . .I the L1210 cell (101) (Chart 6). This drug is now in Phase II -4 -2 -I 0 I 2 4 clinical trials, and it has shown some activity in patients TIME OF MTX ADMINISTRATION with adenocarcinoma of the lung and colon (81, 91, 103). IN RELATIONSHIP TO FU (HR The lack of outstanding activity of TZT against human Chart 5. The effect of sequence of MTX and 5-FU (FU) on survival of mice tumors was predicted, since uptake of this drug by human bearing Sarcoma 180. The MTX dose was 42 mg/kg, and the 5-FU dose was cells has not been to the degree noted for Walker 256 cells 60 mg/kg. Treatment was given on Day 3 (from Ref. 20). (102). However, these findings have encouraged the hope that enhanced selectivity may be obtained with the use of for this purpose, inasmuch as its uptake into cells is limited other nonclassical folate antagonists that may be accumu and since it also inhibits DHFR,we havebeen investigating lated to a greater degree in human neoplastic cells than in quinazoline analog inhibitors, in collaboration with Dr. human normal cells. John Hynes of the University of South Carolina School of Accordingly, we have examined a series of nonclassical Medicine, in an attempt to find CH2FH4analogsthat may be folate antagonists, synthesized by Elslager et al.5 as poten more effective than MTX in enhancing 5-FU activity. tial antimalarials, for activity as antitumor agents with the Our first approach is to identify inhibitors of mammalian potential for selectivity. TMP synthetase, and data obtained with some of the quin The first criterion that we use to select a compound for azolines studied by K. Scanlon in our laboratory are shown further evaluation is that it is a potent inhibitor of mamma in Table 3. Several 2-amino-4-hydroxyquinazoline com han DHFR (50% inhibition <5 x 10@ M). Table 6 indicates pounds have proven to be moderately potent inhibitors of that several of the compounds tested fulfill this require mammalianthymidylate synthetase.4 ment. We then look for selectivity. Selectivity of enzyme After identifying active compounds, we then look for inhibition is tested by comparing the inhibition produced ternary complex formation between the folate analog, against a â€œnormalâ€•enzyme(from human erythrocytes) FdUMP, and thymidylate synthetase using the Santi assay versus DHFR from human leukemia cells (77). We then (93). Data for one candidate compound are shown in Table examine the potency and selectivity of these compounds as 4. This thio analog resulted in good ternary complex for inhibitors of human neoplastic cell growth and are currently mation and will be tested further in vitro and in vivo for its using several human lines (K-562, a myeloid leukemia line, ability to enhance 5-FU and FdUrd cell kill. and a colon carcinoma line) for this purpose. The drugs are â€œNonclassicalâ€•FolateAntagonists as Selective Inhibitors also screened as inhibitors of DNA synthesis against human leukemia cells and other neoplastic cells obtained from An alternative approach to the use of rescue agents with patients and studied in short-term culture. Studies with one conventional folate antagonists is the design of new agents of these compounds (JB-11, NSC249008),shown in Table which possess greater selectivity for neoplastic cells and 7, indicate that this compound is a more potent inhibitor of which may be effective against tumor cells harbored in the DNAsynthesis in human leukemia cells than is MTX. Since CNS. We have initiated a program to develop new agents this compound equally inhibits DHFR from the ABC enzyme with such properties. We have been stimulated by the work and the leukemia enzyme (50% inhibition = 2 x 10@ M), the of Hitchings et al. (64) and of the late B. R. Baker (5), who basis for this increased potency is clearly at least in part demonstratedthat marked selectivity can be obtained using due to the increased uptake of this drug into neoplastic nonclassical folate antagonists in the treatment of bacterial cells as compared to MTX (18, 77). Studies are in progress and protozoal diseases in humans. with this agent of the mechanism of transport, in neoplastic In this regard, important basic information is lacking on cells as well as with normal marrow, to characterize further the biology of normal and neoplastic cells. If we understood the basis of its potency and attempt to demonstrate selectiv the basis of natural (intrinsic) resistance or sensitivity of ity. This drug has recently been shown to have activity tumor and normal stem cells to MTX and the mechanism of against several transplanted mouse tumors, including the acquired resistance in sufficient detail, the design of more B-16 melanoma, the Lewis lung carcinoma, and the colon selective drugs might be facilitated. 25 carcinoma. This drug should be tested in patients with An exampleof the potential of such an approach is shown cancer if preclinical toxicology goes well. Its use against

4 K. J. Scanlon, J. B. Hynes, B. A. Moroson, and J. R. Bertino. Quinazoline

Antifolates as Inhibitors of Thymidylate Synthetase from Bacterial and S E. F. Elslager, J. Davoll, J. Johnson, L. Newton, and L. M. Werbel, Mammalian Sources, submitted for publication. submitted for publication.

FEBRUARY1979 297

Table 3 Inhibition of Lactobacillus casei and L1210 TMP synthetase and human leukemia DHFR by 2- amino-4-hydroxyquinazolines x Y 0 H COOH [email protected]@@I@ITZ_/@@%s@_(?H2)2 COOH

Thymidylatesynthe (MX10@)DHFR(Mxioe)in Com poundXYZtasecaseiL1210353OHHCH2NH5.00.10355OHHCH2N(CH3)0.41360OHHCH3N(HO)300.1555.0379OHCH,CH2NH12.50.1011.5338OHHNHCH29.00.101.7322OHCH@NHCH2506.80.75375OHHCH2O>10010371OHHCH2S2.60.2512.5leukemiaL. human

4Effect Table biochemically different not only from its parent line but also of quinazolinesontoTMP facilitating the binding of (3HJFdUMP from normal cells (21, 25). coli)Coenzyme synthetase(Escherichia Three mechanisms of acquired resistance to MTX have or analog cpm boundÂ° Relativeefficiency been well documented in experimental tumor systems: an None 700Â±7000.0182,000Â±8,3001.028,800 altered DHFR with a decreased affinity to MTX; impaired CH2FH4 transport to MTX; and an elevated DHFR enzyme activity (2, 353b Â±2,6000.3582,800Â±9,1001.0 23, 40, 41, 55, 56, 62, 63, 69, 75, 78, 86, 88, 96, 101). 371 Altered DHFR.In humans, partly becauseof the difficul a See Ref. 93 for methodology. ties in repeated sampling, documentation of why patients b See Table 3 for structure. with leukemia become resistant to drugs has been difficult (21). In 2 patients with ALL resistant to MTX, Dr. Roland Table 5 Skeel and I have adduced some evidence for an altered â€œCureâ€•ofrats bearing Walker256ascitesbyantifol)(from TZT(Baker's 6)Rats Ref. DHFR, a relatively unusual reason for resistance to MTX in 9with bearing the Walker 256 tumor were treated on Days1to experimental tumors (21). Great specificity of inhibition can thedays.Dose dosesindicated. Controls survivedfor 8 be obtained using nonclassical antifolates between mam schedule(mg/kg)survivors0 32-day malian and bacterial or protozoal DHFR's. Thus it may be possible with the large number of folate antagonists availa 0/612.5 Toxic6.25 ble, especially of the nonclassical type, to find inhibitors 5/61.56 specific for the mutant-altered enzyme. 6/60.39 Transport Mutants. In these meetings, several papers 6/60.10 presented described studies of MTX resistance by virtue of 6/6 decreased permeability to MTX (63, 78). To circumvent resistance due to this alteration, it should be possible to human tumors may be guided by the in vitro sensitivity test find folate antagonists that are accumulated by these cells as described in Table 7. despite the impaired MTX transport system. Selectivity may Design of Selective Agents for the Drug-resistant Cell be obtained if normal stem cells do not transport the compound well. Cells with impaired permeability may also One of the most important problems in the drug treatment have other exploitable defects, i.e. , perhaps they would be of cancer is the development of drug resistance. Although more immunogenic (68). a serious problem, it may be viewed also as an opportunity Elevated DHFR.When animals bearing MIX-responsive for great selectivity. The phenomenon of collateral sensitiv tumors or cells in vitro are treated with continuous expo ity, i.e., unusual sensitivity of a drug-resistant tumor to sure to low levels of MTX, the most common event leading another drug, has been well documented, in particular by to drug resistance is an elevation in the enzyme DHFR (69, Doris Hutchison and coworkers, and many examples exist 88, 96). By exposure to increasing levels of MTX, mutant in experimental tumors (68, 95). lines with extremely high levels of this enzyme have been If we understood the mechanism of resistance to a drug, obtained (4, 56, 78). it should be possible to design inhibitors specific for the Studies from the laboratories of Schimke and Littlefield resistant population of cells in circumstances when resist have clearly demonstrated that the increase in DHFR activity ance is due to a mutation which creates a lineage that is is due to an increased rate of synthesis of this enzyme,

298 CANCER RESEARCH VOL. 39

chart 6. Uptake of MTX and TZT in L1210 and Walker 256 -Jp cells. Uptake was determined after incubation of cells with @H1MTXor[â€œCJTZTasdescribed (103). U U- 4 I--.- z

TIME (MIN)

Table6 180- and an L1210-derived line, was answered using a Inhibition of human leukemia (ALL) DHFR and L1210 DHFR by purified cDNA probe, made to the mRNA from the Sarcoma 2,4-diaminoquinazolinecompounds 180 resistant cells (3, 94). Hybridization studies with this Inhibition of DHFR was measured as described (19). The 50% cDNA showed an increased rate of association to the mRNA inhibition value is the concentration of drug necessary to produce 50% inhibition of enzymeactivity under standard conditions (the from the Sarcoma 180 cells resistant to MIX, as well as to final reaction mixture contained: 0.1 mmol Tris-HCI, pH 7.0; 0.15 the DNA from the resistant cells. Revertant cells grown in mmol KCI; 0.05 @molNADPH;0.02 @tmoldihydrofolate;approxi the absence of MIX for generations and with enzyme levels mately3 pmol DHFR;variableamounts of inhibitor; and water to a that had decreased to 7- to 10-fold over normal showed a final reaction volumeof 1 ml. All assayswere performedat 37Â°C. 10-fold increased rate of association. Hybridization studies with the Li210 parent line and the MIX-resistant line showed a similar increased rate of association between the _fII@JII@XNH2 cDNA and mRNAof the resistant line, as compared to the parent line. As with the Sarcoma 180 line, hybridization R1 NH2 studies with the cDNA from the Sarcoma 180 line resistant to MTX and the DNA of the L1210 resistant line showed an (M)ALLL1210JB-23' inhibition increased rate of association, of a magnitude similar to the Com increase in enzyme activity in this line. In contrast to the poundXR,R250% revertant Sarcoma 180 line, cells of the L1210 resistant line 10@JB-13',4'C12HH3.8 x [email protected] x grown in the absence of MTX maintained their DHFR level 10@JB-93',4'C12ClH1 .1 x [email protected] x 10'JB-33' ,4'Cl2CH3H2.6 x and increased rate of DHFR synthesis. This line was similar 1O@JB-143',4'Cl2HNO1 .0 x 10@1 .0 x to the high-enzyme mutant propagated in the presence of 108JB-153',4'C12CH,NO1.0,4'Cl,HC5.0 x 1081 .0 x MIX. 10@JB-223' x The data in Table 8 clearly show that the elevated DHFR 10JB-73' ,4'Cl2HCH5.0 x activity and mRNA activity of the resistant sublines are a 10'JB-113',4',5'OCH3CH,H1.3,4',5'OCH3HH6.0 x 10@JB-134'BrHH2.2 x [email protected] x consequence of a proportionally increased number of gene 1O@JB-124'BrCH3H1.5 x [email protected] x copies for this enzyme. Furthermore, in the revertant Sar x [email protected] x 10@ coma 180 subline that stabilized to levels of DHFR 7 to 10 x the sensitive line, the number of gene copies also Table7 corresponded to the levels of DHFR and mANA found. Inhibition of DNA synthesis in human leukemia cells by MTX and The question of what the nature of the variation of DHFR JB-11 levels was in the population of MIX-resistant cells, as well controlAML as the resistant cells partially reverting to MIX sensitivity when grown in the absenceof MTX,was answeredwith the K.)MTX5DrugConcentration (M)% (F. A.)ALL (W. use of fluorescein-labeled MIX, first synthesized by Gapski x 10@ et al. (45), and the fluorescence-activated cell sorter. Fluo 5JB-11SxlOe2x10@18 6.715 rescein-labeled MIX (Chart 7), while not as potent an inhibitor of DHFR as is MTX, still binds tightly to the enzyme 5x107 8.0 4 (45); and the amount of intracellular fluorescence, under 2x10@12 2.35 2 appropriate conditions, corresponds to the level of DHFR found in the cells (72). associated with an increase in mRNA levels for this enzyme The revertant subline of the Sarcoma 180, grown in the (4, 31, 61 , 73). During the past year, while on sabbatical at absence of MTX for 400 cell generations, containing 7- to Stanford, I was fortunate to be able to participate in studies 10-fold greater amounts of DHFR than the parent line, was of these high-level DHFR mutants with Dr. Schimke and his found to be made up not of a mixture of a small percentage colleagues. of highly resistant cells and a large percentage of sensitive The question as to the mechanism of the increased DHFR cells, but rather of a homogeneous population of cells with and the mRNAin 2 lines with high DHFRactivity, a Sarcoma DHFR levels that cluster tightly around the average, i.e. , a

FEBRUARY 1979 299

Sarcoma180andLl2lO lines@' â€˜C RelativeDHFR 2 4@D 22 activ se U) 4 LineSpecific itymRNA -J copiesS-i quencesGene a. 805-3111AT-3000250220180Rev-40010710Li

z 0

@4 U D@ 210S11RR VO 4 â€˜--â€˜O C) (+MTX)3545RR(â€”MTX)3535a z

From Ref. 4.

U C) 4 H@N@ ,, I @ @NyN@ (j ?)H U. I N,@,L@N,eLâ€”(H.N-..( )â€”CNHCH (.1 0 @ I' I NH. (â€˜H, (CIIi), S HOOC 0 0 20 30 ., (@NH(CHZ)@NH@NH_.d._.@ TIME (HOURS) Chart 8. Inhibition of DNA synthesis from cells obtained from a patient with ALL refractory to treatment with MTx. Cells were obtained from the patient before, during, and after a 24-hr infusion of MTX (see text). @HJDeoxyuridine(rH@IUrd) incorporation into DNA and DHFR was mea Chart 7. Structure of fluorescein-labeled MDC. sured as described (65). Dark bars, pH 8.5 activity; open bars, pH 7.0 enzyme activity. 7-fold increase in DHFR. In contrast, the highly resistant Sarcoma180line is madeup of a heterogeneouspopulation of cells, containing an average of 200-fold increase in U fluorescence per cell over the sensitive line, but with a 4 z larger spread of fluorescence. 0 Of what relevance are these studies to treatment of 4 patients with cancer? Does the same mechanism of gene a.0 0 amplification occur in patients resistant to MIX? What can C) be done to prevent or take advantage of drug resistance? z Chart 8 shows the results of some studies of a patient V with lymphatic leukemia reported by William Hryniuk and me several years ago (65). The patient who was clinically IO' 10Â° refractory to conventional MTX was treated with what we MOLARITY OF MIX then considered a high dose of MIX (240mg/sq m) by 24- Chart 9. Inhibition of DNA synthesis (@HJdeoxyuridine (UdA) incorpora hr infusion, followed by leucovorin rescue, 24 hr after the tion into DNA) In cells from a patient with ALL before clinical resistance to infusion was started. Despite good levels of MIX in the MTX (A), after developmentof clinical resistanceto MDC (B). after resistance to 1-@-o-arabinofuranosyIcytosine (C), and after additional treatment with serum (>10@M)the patient had less than the usual inhibi MTx(D). tion of DNA synthesis, as measured by [3H]deoxyuridine incorporation into DNA (65), and the infusion had essen patient relapsed, and the patient's cells showed corre tially no effect on her leukemia. The level of DHFR in the spondingly decreased sensitivity to MTX as measured by cells was 10 x normal and, despite the high extracellular [3H]deoxyuridine incorporation into DNA. After another levelsof MTXachieved, DHFRin the cells was not inhibited short remission induced by 1-f3-D-arabinofuranosylcytosine, significantly. This patient was clearly resistant to MTX and the patient again relapsed and, when studied at this time had less than a 0.5-log fall in blast count. (The averagefall with the same assay, the cells appeared once again to be in blast count expected in a sensitive patient was >2.) Thus sensitive to MIX. However, after a single infusion of MTX, the mechanismof MIX resistancenoted in this patient was the cells that were not killed by the treatment rapidly presumably an elevated DHFR. revertedto the high-resistancepattern. Thesedata illustrate In another patient with ALL, a phenomenonsimilar to the that resistance and reversion to a state of less resistance reversion phenomenon seen in the Sarcoma 180 cells was probably also occur in some patients after development of noted (Chart 9). Thus, before treatment with MIX, the drug resistance. patient's cells were extremely sensitive to MTX, as mea If gene amplification is found to be the basis of resistance sured by the effect of MIX on DNAsynthesis. After a short to MTX in some patients with cancer, and as well the basis (3-month) remission induced by infusions of MTX, the for resistance to other drugs, e.g. , the new pyrimidine

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1979 American Association for Cancer Research. Improved Selectivity in Cancer Chemotherapy analog N-(phosphoacetyl)-L-aspartate (74), these are impor Concluding Remarks tant implications for drug use for prevention of resistance. Some of these principles derived to prevent drug resist It should be apparent from this discussion that despite a ance have been empirically developed in the treatment of large amount of new information there are gaps in our patients with cancer but now have a firm experimental knowledge of not only the mechanisms of natural and basis. Resistance occurs when cells are exposed to low acquired resistance of human tumors to folate antagonists concentrations of drugs for long periods of time, and drug but also the basic biology of human tumors and normal dosage is slowly escalated.Therefore it follows that patients stem cells. with cancer should be treated with intermittent exposure to Some 15 years ago, in a review article (14), I closed with large doses of drug, especially two or more with differing the following statement: mechanisms of action to decrease the probability of resist â€œAlthough it may be pointed out that no new significantly ance. Sequential use of other agents, used in the same improved folate antagonist has been found since the discovery of fashion to avoid continued exposure to the same drugs, aminopterin, this does not justify pessimism, since we are just should then be instituted. Thus, if cells survive the first beginning to appreciatehow thesedrugs should be usedand what treatment and are partially resistant to the first set of drugs, wearelookingfor indevelopingnewdrugs.â€• they may be eliminated by the second set of drugs. Ob viously, this program requires that 4 or more drugs with During this past 15 years, there has been a great deal of marked effectiveness be available for treatment of a cancer. progress in chemotherapy. Improved dosage schedules of It is of interest that, when 3 or 4 effective drugs are available MTXand the useof this drug in combination regimens have and used according to these principles, cures of dissemi played important roles in this progress. Although no addi nated cancers, e.g. , ALL, Hodgkin's disease,diffuse histio tional new clinically useful folate antagonists have been cytic lymphoma, and testicular cancer, have been reported added to the chemotherapy pharmacopoeia, I believe the (reviewed in Refs. 114 and 115). Mutagenic drugs should possibilities of more selective folate analogs are still an be avoided, since they may increase the probability that exciting prospect. Newer techniques of tissue culture and drug-resistant mutants will develop, in addition to having immunodepressed mice to grow human cells may allow the the potential to cause cancer several years later. Of course, gaps in our knowledge to be filled that could be translated since many of the effective drugs available for the treatment into design of drugs that are what we are looking for of cancer are mutagens and carcinogens, especially alkyl selective agents. ating agents, alternatives may not be available. A second generation regimen, based on our previous experience (12), Acknowledgments which we have recently developed for the treatment of This review contains information to be published in more detail at a later diffuse histiocytic leukemia that embodies these principles date. The author expresses his gratitude to the following persons who is given in Table 9. Preliminary results with this program participated in the research and permitted the use of unpublished data: C. Lindquist, J. McGuire, W. Rode, B. Dolnick, D. Fernandes, K. Kalgathi, K. have been very exciting in that 12 of 13 patients treated Scanlon, and A. Cashmore. have achieved complete remission. P. Hsieh, B. Moroson, W. L. Sawicki, and J. Uhoch provided excellent How can we design selective inhibitors of cells resistant assistance in various phases of this work. I am also indebted to P. Kerley for to MIX by virtue of high levels of DHFR? M. Friedkin, her secretarial assistance. several years ago, suggested a strategy that would be lethal to these cells (43). If a dihydrofolate analog could be found References that is a substrate for DHFR, it would be reduced more 1. Acute Leukemia Group B. New Treatment Schedule with Improved effectively by resistant cells as a result of the increase in Survival in Childhood Leukemia. Intermittent Parenteral versus Daily DHFR activity present. The resulting reduced analog, if an Oral Administration of Methotrexate for Maintenance of Induced Re inhibitor of an essential one-carbon enzyme, e.g. , IMP mission. J. Am. Med. Assoc., 194: 75-81 , 1965. 2. Albrecht, A. M., Biedler, J. L., and Hutchison, D. J. Two Different synthetase, would therefore inhibit growth of the resistant Species of Dihydrofolate Reductase in Mammalian Cells Differentially cells. One such analog developed by Goodman and his Resistant to Amethopterin and Methasquin. Cancer Res., 32: 1539- 1546, 1972. colleagues, dihydrohomofolate, has been found to be effec 3. Alt, F. W., Kellems, R. E., Bertino, J. R., and Schimke, A. T. Multiplica tive against a MTX-resistant mouse lymphoma line with tion of Dihydrofolate Reductase Genes in Methotrexate-resistant Var elevated levels of DHFR (42, 54, 84). Whether this com iants of Cultured Murine Cells. J. Biol. Chem., 253: 1357-1370, 1978. 4. Alt, F. N., Kellems, R. E., and Schimke, R. T. Synthesis and Degradation pound and its oxidized and reduced forms, which are also of Folate Reductase in Sensitive and Methotrexate-resistant Lines of S effective, work in the manner hypothesized is not yet clear 180 Cells. J. BioI. Chem., 251: 3063-3074, 1976. (57). 5. Baker, B. R. Design of Active-Site Directed Irreversible Enzyme Inhibi tors, pp. 192-263. New York: John Wiley and Sons, Inc., 1967. 6. Baker, B. R. Active-5ite Directed Irreversible Inhibitors of Dihydrofolate Table 9 Reductase. Ann. N. V. Acad. Sci., 186: 214â€”226,1971. TheACOMAregimen 7. Bareham, C. A., Griswold, D. E., and Calabresi, P. Synergism of Methotrexate with Imuran and with 5-Fluorouracil and Their Effects on Adriamycin401Cyclophosphamide1000 mg/sq mDay Hemolysin Plaque-forming Cell Production in the Mouse. Cancer Res., mOncovin2mgDays2,9,16Methotrexat&'500mg/sq 34: 571-575, 1974. 8. Baugh, C. M., Kramdiek, C. L., and Nair, M. G. Polygammaglutamate wk1-f3-D-Arabinofuranosylcyto mg/sq mEvery wk for 8 Metabolites of Methotrexate. Biochem. Biophys. Res. Commun., 52: 23sine 300 mg/sq mBegin Day 27-34, 1973. 9. Bender, R. A. Antifolate Resistance in Leukemia: Treatment with â€œHigh Doseâ€•Methotrexate and Citrovorum Factor. Cancer Treat. Rev., 2: a With leucovorin rescue (25 mg every 6 hr for 8 doses beginning 215â€”224,1975. 24 hr later). 10. Bender, R. A. Membrane Transport of Methotrexate in Human Neoplas

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