[CANCER RESEARCH 42, 1707-1 715, May 1982]0008-5472/82/0042-OOOOS02.00

Toxicity and Anticancer Activity of a New Triazine Antifolate(NSC127755)1

Thomas H. Corbett,2 Wilbur R. Leopold, Donald J. Dykes, Billy J. Roberts, Daniel P. Griswold, Jr., and

Frank M. Schabel, Jr.

Southern Research Institute, Birmingham, Alabama 35255

ABSTRACT

A new triazine folate antagonist, 3-chloro-4-{{4-{2-chloro-4-[4,6-diamino-2,2-dimethyl-s-triazin-1 (2H)-yl]phenyl) butyl}}

benzenesulfonyl fluoride compounded with ethanesulfonic acid(1:1) (NSC 127755), was highly active against four transplant-able colon adenocarcinomas (36, 38, 10/A, 12/A) and theDunning murine ovarian tumor M5076. Treatment schedulestudies indicated that a prolonged time of exposure providedoptimum antitumor activity for the compound. The combinationof NSC 127755 plus 4-amino-1-[5-O-(1-oxohexadecyl)-/3-D-arabinofuranosyl]-2(1H)-pyrimidinone (palmO-ara-C, NSC

135962) was found to have therapeutic synergism againstgrossly evident colon adenocarcinoma 36.

INTRODUCTION

The triazine antifolate, 3-chloro-4-{{4-{2-chloro-4-[4,6-dia-mino-2,2-dimethyl-s-triazin-1 (2H)- yl]phenyl} butyl}} benzene

sulfonyl fluoride compounded with ethanesulfonic acid (1:1)(NSC 127755), was originally synthesized by Baker and Ver-meulen (3) and is an irreversible inhibitor of dihydrofolatereducíase (specifically alkylating the enzyme through the sul-fonyl group) (11 ). That NSC 127755 acts as a folate antagonistis indicated by the abolition of its antitumor activity againstovarian tumor M5076 by administration of leucovorin factor(the end product of the dihydrofolate reducíase reaction).3 Theconcept of active site-directed, irreversible inhibitors of dihy

drofolate reductase has been reviewed by Baker (2).In preliminary studies, NSC 127755 was highly active against

the Dunning murine ovarian tumor M5076.4 This finding was

especially interesting since no other folate antagonist [includingamethopterin (NSC 740), aminopterin (NSC 739), and Baker's

antifol (NSC 139105)] had more than marginal activity againstmurine solid tumors (6, 7, 19). The work reported hereinextends the testing of this new agent to include a number ofadditional solid tumors of mice to determine the broadness ofthe activity and to determine the pharmacodynamics of theantitumor action. The structures of NSC 127755 and Baker's

antifol, another triazine antifolate that has received a clinicaltrial, are shown in Chart 1.

' The work reported herein was supported by Contract NO1-CM-97309 with

the Division of Cancer Treatment. National Cancer Institute, NIH, Department ofHealth and Human Services; by Grant CA 17303 from the National Large BowelCancer Project, the University of Texas System Cancer Center, M. D. AndersonHospital and Tumor Institute; and by grant RO1-CA-26485 from the NationalCancer Institute, NIH, Department of Health and Human Services.

2 To whom requests for reprints should be addressed.3 Dr. R. Johnson. A. D. Little, Inc., personal communication.4 Drug Evaluation Branch, Division of Cancer Treatment, National Cancer

Institute, unpublished screening data.Received September 25. 1981 ; accepted January 20, 1982.

MATERIALS AND METHODS

All mice and drugs were obtained through the Drug EvaluationBranch of the Division of Cancer Treatment, National Cancer Institute.The Dunning ovarian tumor M5076 was obtained from the frozen tumorbank at Mason Research Institute, Worcester, Mass. The 239Pu-induced

osteogenic sarcoma was obtained from Dr. Lowell Glasgow, Department of Pediatrics, University of Utah College of Medicine, Salt LakeCity, Utah. The MXT, hormone-dependent mammary ductal carcinoma

was obtained from Dr. Dan Medina, Department of Cell Biology, BaylorCollege of Medicine, Texas Medical Center, Houston, Texas. Thesetumors have subsequently been maintained in serial passage in thislaboratory. All other tumors used in this study originated and havebeen maintained in serial passage in this laboratory. The biological anddrug response characteristics of the tumors used in these studies arelisted in Tables 1 and 2. The mice were fed a 6% fat diet (Wayne LabBlox F-6; Allied Mills, Inc., Chicago, III.) and tap water ad libitum. Eachmouse was weighed at 7-day intervals beginning on the first day of

drug treatment. The mice were checked daily for deaths.The new triazine antifolate, NSC 127755, was prepared in 0.9%

NaCI solution containing 0.05% Tween 80 for injection as a suspension(0.5 ml/mouse, ¡.p.,or 0.1 ml/mouse, s.c.). For injection as a solution,the agent was first dissolved in DMA5 and diluted with distilled water.

The final percentage of DMA and the injection volume are specified inthe tables for each experiment. All solutions of NSC 127755 wereinjected within 24 hr of preparation. Treatment schedules includedsingle doses, daily doses (for 9 or more consecutive days), and multipledaily injections (3 to 4 times/day for 9 or more consecutive days).

The techniques of chemotherapy and data analysis have been presented in detail elsewhere (6, 8, 13, 14). A summary of these methodsis as follows. Chemotherapy was started within 1 to 5 days of tumorimplantation while the number of cells was relatively small (107 to 108

cells, early stage disease), or alternatively, chemotherapy was delayeduntil the tumors became large (-200 mg, advanced stage disease). A200 mg tumor contains approximately 2 x 108 total cells. Ten normal

mice (without tumors) were often matched to each tumor-bearing

treatment group and given injections of the same agents at the samedosages, schedules, days of administration, and drug route as thetumor-bearing mice. In this way, drug-induced toxicity (lethal and

weight loss) could be clearly separated from the effects of the tumors.The animals necessary to begin an experiment were pooled, and the

normal (tumor-free) toxicity controls were then removed. The remainingmice were implanted with the tumors and again pooled before unselec-

tive distribution to the various treatment groups (early stage tumortreatment). If the experiment was designed for advanced stage treatment, the tumors were allowed to grow to the desired range. Animalswith tumors that were not in the desired range were removed; theremaining mice were pooled and unselectively distributed to the varioustreatment and control groups. Tumors were measured with a caliper

5 The abbreviations used are: DMA, dimethylacetamide; MOD, median day of

death; Td, tumor volume doubling time; LD., the dose level at which x percentageof the treated animals die; CTI, combination toxicity index; HNTD, highestnontoxic dosage; palmO-ara-C, 4-amino-1-{5-O-(1-oxohexadecyl)-/?-D-arabino-furanosyl]-2(1 HVpyrimidinone; ara-C, 4-amino-1 -/3-o-arabinofuranosyl-2(1 H>-pyrimidinone.

MAY 1982 1707

on May 16, 2020. © 1982 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

T. H. Corbeff ef al.

twice weekly until the death of the animal or until cure was assured.Tumor weights were calculated from 2-dimensional measurements.

Tumor weight (mg) =axo2

where a and b are the tumor length and width (mm), respectively.End Points for Assessing Antitumor Activity. The following quan

titative end points were used to assess antitumor activity, (a)

NH2

NSC 127755NEW TRIAZINE ANTIFOLATE

NSC 139105BAKER'S ANTIFOL

Chart 1. Chemical structures of NSC 127755, 3-chlorc~4-{{4-{2-chloro-4-[4,6-diamino-2,2-dimethyl-s-triazin-1 (2H)-yl]phenyl} butyl}} benzenesulfonyl fluoride with ethanesulfonic acid (1:1), and NSC 139105. 3-{[2-chloro-4-<4,6-diamino-2,2-dimethyl-1,3,5-triazin-1 (2H)-yl)phenoxy]methyl} -A/,/v-dimethylben-zamide compounded with ethanesulfonic acid (1:1) (Baker's antifol).

Percentage increase in host life span

MOD (treated tumor-bearing mice) —_ MOD (tumor-bearing control mice)

MOD (tumor-bearing control mice)x 100

(b) Tumor growth delay (T - C value), where T and C are the median

times (days) required for the treatment group and the control grouptumors, respectively, to reach a predetermined size (usually 750 or1250 mg). Tumor-free survivors were excluded from these calculations.

In our judgment, this value is the single most important criterion ofantitumor effectiveness because it allows the quantitation of tumor cellkill, (c) Regressions (advanced stage primary site tumor): completeregression = regression below the limit of palpation; partial regression= greater than 50% reduction in tumor mass (complete regressions

were included in the partial regressions).Calculation of Tumor Cell Kill. For s.c. growing tumors, the logio

cell kill/dose was calculated from the following formula.

Log,o kill/doser - C value

(3.32) (Td) (no. of doses)

where 7 —C is the tumor growth delay as described above, and Td(days) is estimated from the best-fit straight line from a log-lineargrowth plot of the control group tumors in the 100- to 1000-mg range.

Table 1

Biological characteristics of 16 transplantable mouse tumors

~. abTumorColon

26Colon

36Colon38Colon51Colon

06/AColon07/AColon08/AColon10/AColon

11/AColon12/AMammary16/CMammary

14M

XTOvarian

M5076Pancreas

02Osteogenic

sarcomaHistological

typeUndifferentiated

coloncarcinomaColon

adenocarcinomaColonadenocarcinomaMucinous

colon adenocarcinomaColonadenocarcinomaColonadenocarcinomaColonadenocarcinomaColonadenocarcinomaColonadenocarcinomaColonadenocarcinomaMammary

adenocarcinomaMammary

adenocarcinomaMammary

ductal carcinoma (hormone de

pendent)UndifferentiatedovariantumorModerately

differentiatedpancreascarcinomaUndifferentiated

sarcomaTrans

plantgenera

tion(range)9789-9860-898020221712-151616-2347114414-512872Metastasis

from s.c.implant (-1 gsize)>90%

tolungs<5%

tolungs<30%tolungs>80%to lungsandlymph

nodes>70%tolungs<15%to

lungs<20%tolungs<40%tolungs<3%

tolungs<5%tolungs>80%

tolungs<30%tolymphnodes<

17% tolungsNone>80%

to liver, kidneys, andovaries>80%

tolungs<15%

to lungsInvasivenessVery

highLowModerateModerate

—»highModerateVery

lowModerateLowModestLowHighLow

-»moderateLow

—»moderateModerate

—»highHighModerateApproxi

mate Tdin days

(60-1000mg)1.73.5-4.32.5-4.04.32.03.74.24.6-9.12.74.0-4.92.01.92.52.0-3.24.21.9

Colon tumor 26 was induced in female BALB/c mice by repeated intrarectal injections of N-methyl-W-nitrosourea.Colon tumors 36, 38, 51, 06/A, 07/A, 08/A, 10/A, 11/A, and 12/A were induced with s.c. injections of 1,2-dimethylhydrazine in female BALB/c mice (except for colon tumor 38 which was induced in female C57BL/6 mice).Mammary adenocarcinomas 16/C and 14 originated as spontaneous (virus-associated) mammary adenocarcinomas infemale C3H/He mice. The MXT tumor was induced with ethyl carbamate in female B6D2Fi mice (18). The ovariantumor M5076 arose spontaneously in a female C57BL/6 mouse. Pancreas tumor 02 was induced by implantation of acotton thread saturated with 3-methylcholanthrene into the pancreas of a male C57BL/6 mouse. The osteogenicsarcoma was induced by 239Puin a C57BL/6 mouse (10).

b Host for tumor passage of colon 38, pancreas 02, and osteogenic sarcoma was C57BL/6; host for chemotherapy

studies was B6D2F, or C57BL/6 X C3H (hereafter called B6C3Fi). Host for tumor passage of colon 26, 36, 51, 06/A, 07/A, 08/A, 10/A, 11/A, and 12/A was BALB/c; host for chemotherapy studies was CD2F,. Host for tumorpassage of mammary 16/C and 14 was C3H/He; host for chemotherapy studies was B6C3F,. Host for tumor passageas well as host for chemotherapy studies of the MXT tumor was B6D2F,. Host for tumor passage of ovarian M5076 wasfemale C57BL/6; host for chemotherapy studies was B6D2F, or B6C3F,.

1708 CANCER RESEARCH VOL. 42

on May 16, 2020. © 1982 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

A New Thazine Antifolate

Table 2

Drug response characteristics of 16 transplantable mouse tumors used for theevaluation of NSC 127755, a triazine antifolate'

Selected agents moderately to highly active {+ + +-»Tumor + + + + ) against the tumors listed: early stage disease

Table 3

Logic */// value conversion to arbitrary activity rating

Colon 26

Colon 36

Colon 38

Colon 51Colon 06/A

Colon 07/A

Colon 08/A

Colon 10/A

Colon 11 /A

Colon 12/A

Mammary 16/C

Mammary 14

MXT

Ovarian M5076

Pancreas 02Osteogenic sarcoma

Aziridinylbenzoquinone, BCNU, c/s-DDPt, dianhydro-galactitol, MeCCNU, 6-MeMPR, L-PAM, PCNU. pi-

perazinedioneAAFC. ara-C, carminomycin, cyclocytidine, cyclophos-

phamide, phosphoramide mustard, procarbazine,triazine antifol

AAFC, anguidine, anthracenedione acetate, 5-FU, ho-moharringtonine, phosphoramide mustard, triazineantifol

ACNU, PCNUAnthracenedione acetate, dihydroxyanthracenedione,

ara-C, chlorozotocin, cyclophosphamide, neocarzi-nostatin, L-PAM

Anguidine. anthracenedione acetate, chlorozotocin, di-

bromodulcitol, DTIC, procarbazineAdriamycin, anguidine, ara-C, c/s-DDPt, cyclophospha

mide, DTIC, hydroxyurea, indicine-N-oxide. mitomy-cin C, L-PAM. procarbazine

Adriamycin, anguidine. cyclophosphamide, indicine-N-

oxide, mitomycin C, neocarzinostatin, triazine antifol,vincristine

Adriamycin, dihydroxyanthracenedione, BCNU, cyclophosphamide, DTIC, neocarzinostatin

Dihydroxyanthracenedione, DTIC, MeCCNU, triazineantifol

Adriamycin, cyclophosphamide, daunomycin, dibromo-dulcitol, 5-FU, isophosphoramide mustard, vincristine

Adriamycin, anthracenedione acetate, dihydroxyanthracenedione, cyclophosphamide. c/s-DDPt, dibro-modulcitol, 5-FU, L-PAM

Adriamycin, anthracenedione acetate, aziridinylbenzo-quinone, carminomycin, diethylstilbesterol, L-PAM.

PCNUChlorozotocin, cyclophosphamide, MeCCNU, L-PAM,

triazine antifolNone of 31 agents testedc/s-DDPt, cyclophosphamide

" See Appendix for complete chemical identfication.

The logio cell kill (gross) was the log,0 kill/dose multiplied by thenumber of doses or

T - C value

(3.32) (Td)

The log,o cell kill (net)

_ (T —C value in days) - (duration of treatment in days)

(3.32) (Td)

If the logto cell kill (net) value was positive, there were fewer cellspresent at the end of therapy than at the start. If, on the other hand, thevalue was negative, the tumor grew under treatment. A positive grossvalue with a negative net value indicated inhibition of growth of thetumor cell population during drug treatment. The log,0 kill values wereconverted to an arbitrary activity rating (Table 3). No agent received a+ + + + activity rating unless a 40% or greater increase in life spanvalue was also obtained.

Determination of Therapeutic Synergism. The methods used forthe determination of therapeutic synergism have been described indetail elsewhere (13). Briefly, a combination is considered to havetherapeutic synergism if it is therapeutically superior to the optimumuse of either agent alone (5, 17). In all trials listed, comparisonsbetween the single agents and the combination were made at equitoxicdosages, the maximum tolerated dose levels (LD,: or less, except inone case an LD2o was used). Dosages approximately 1.5 times those

Duration oftreatmentDuration

oftreatment

5-20daysLog,o

KillDuration

oftreatment>20daysLog.o

Kill

Activity rating Log,0 Kill Net Net Gross Net Gross

Highly+activeInactive++ +>2.6+

+ +1.6-2.6++0.9-1.5+

0.5-0.8<0.5>2.00.8-2.0>2.82.0-2.81.3-1.90.7-1.2<0.7aO.8

>3.42.5-3.41.7-2.41.0-1.6<1.0

listed were excessively toxic within the same experiment, inducingeither 4 g or more weight loss per mouse (-20% of body weight) or

death in 20% or more of the mice.Definition of CTI. Skipper (16) has reported a method for quantita

tively determining the lethal toxicity of 2-drug combinations for mice.

Briefly, his procedure is as follows: (a) the dosages of individual drugsused in a combination toxicity study are tabulated as decimal fractionsof the LDio's determined previously for the same treatment schedule;and (6) the decimal fractions of the LD,0's of the individual drugs used

in the drug combination are added, and the sums are plotted againstobserved mortality on log-probit paper. The LDio (sum) of the combi

nation is read from a line of best fit. He has identified the drugcombination LDto determined in this manner as the CTI. A CTI of about1 indicates additive toxicity for vital normal cells, a CTI less than 1indicates greater than additive toxicity, and a CTI greater than 1indicates less than additive toxicity. A CTI of 2 (an exceptionally rareevent) indicates completely nonadditive toxicity; i.e., both agents canbe used in simultaneous combination at full maximum tolerated dosageswithout additional toxicity.

RESULTS

Mice treated ¡.p.or s.c. at maximum tolerated dosages(<LDio) with a suspension of NSC 127755 had minimum weightloss (less than 5% of body weight), had rapid recovery, andhad no apparent residual ill effects as judged by rapid weightgain and general health observations. Ten consecutive dailys.c. injections of a suspension of NSC 127755 resulted inneither local damage nor other untoward effects; the associated lethalities were >156 mg/kg/dose (LD,oo), 97 mg/kg/dose (LD20), and <60 mg/kg/dose (LD0). When NSC 127755was administered P.O., its potency was reduced approximately10-fold; i.e., the LDio (daily for 10 days) for i.p. administrationis about 10-fold lower than that for p.o. administration (480mg/kg/dose). At that p.o. dosage, the agent was only marginally active or inactive against the early stage ovarian tumorM5076 (data not shown). Potential causes of this inactivity(e.g., poor absorption, low pH inactivation, etc.) were notevaluated.

NSC 127755 was 3 to 5 times more toxic if administered asa solution in 6% DMA than as a suspension in 0.9% NaCIsolution containing 0.05% Tween 80 (injection schedule, i.p.daily for 9 days) (Tables 4 and 5). The increased toxicity of theaqueous DMA formulation was not due to the DMA, sinceadministration of a 10% DMA solution 30 min prior to eachdaily injection of a suspension of NSC 127755 in 0.9%NaCI:0.05% Tween 80 did not increase lethality over that seenwith the agent in 0.9% NaCI:0.05% Tween 80 only. Thus, the

MAY 1982 1709

on May 16, 2020. © 1982 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

T. H. Corbe»et al.

Table 4

Treatment of early stage colon adenocarcinoma 38 withNSCNSC

127755 single-agenttreatment0As

a suspension in 0,9% NaCIsolution:0.05%Tween 80, oncedaily135

mg/kg/dose, i.p.. Days3-1184mg/kg/dose, i.p., Days 3-1152mg/kg/dose, i.p., Days 3-11As

a solution in 6% DMA, oncedaily22mg/kg/dose, ¡.p.,Days 3-11(0.3

ml/injection)14mg/kg/dose. ¡.p.,Days3-11(0.3

ml/injection)9mg/kg/dose, i.p.. Days 3-1 1(0.3ml/injection)5.7

mg/kg/dose, i.p., Days 3-11(0.3

ml/injection)Total

dose(mg/kg)12157564681981268151.3Days

oftumorgrowthDrug

deaths in % in- delaytumor-bearing crease in (T —Cmice

life spanvalue)7/10(70)c3/10(30)1/8

(13) 5911.45/10(50)0/10

(0) 02.91/10(10)

-82.30/10

(0) -3 0.3127755:

diluent and schedulestudyTotal

logkillLog

kill/doseGrossNet0.1

1.30.40.03

0.3-0.60.03

0.3-0.60.0

0.0 -0.8ActivityCures

ratingToxicToxic0/8

++Toxic0/100/100/10CommentLD70LDjoHNTDLDMHNTD"

As a solution in 6% DMA, 3 times daily14 mg/kg/dose, i.p.. every 6 hr X 378 8/10(80) Toxic LD80

3, Days 3-11 (0.1 ml/injection)9 mg/kg/dose. i.p.. every 6 hr X 3, 243 1/10(10) 42 20.3 0.1 2.2 1.3 2/10 + + + HNTD0

Days 3-11 (0.1 ml/injection)5.7 mg/kg/dose, i.p., every 6 hr x 153.9 1/9 (11) 26 6.4 0.03 0.8 -0.2 0/9 +

3, Days 3-11 (0.1 ml/injection)3.6 mg/kg/dose, i.p.. every 6 hr X 97.2 0/10 (0) 9 3.1 0.01 0.3 -0.5 0/10

3, Days 3-11 (0.1 ml/injection)

a Control: MOD = 38 days; time for median tumor to reach 750 mg = 20.4 days; there were no tumor-free survivors among the 19 control mice. Td = 2.8 days;

62nd transplant generation. Experiment 1734M2.6 See "Materials and Methods."c Numbers in parentheses, percentage.'' Assignment of this HNTD is problematic due to the occurrence of several acute toxicity deaths at the higher dosages. It is clear that the total dosage could be

increased with more frequent administration of smaller doses. See text for further discussion.

increased toxicity of the solubilized compound was apparentlydue to an increased peak plasma level. The intolerance of miceto a high plasma level of the solubilized agent was furtherestablished by single bolus i.v. injections into C57BL/6 xDBA/2 F! (hereafter called B6D2Fi) mice. The highest nontoxicsingle dosage of NSC 127755 (administered i.v. in 10% DMA,0.1 ml/mouse) was 3.7 mg/kg; this schedule was inactiveagainst small s.c. implants of the ovarian tumor M5076. Higherdosages caused deaths within sec of injection: 7.5 mg/kg =LD20; 15 mg/kg = LDeo! and 30 mg/kg = LD10o-Theseimmediate deaths were not due to fluoride toxicity, since asingle i.v. dose of sodium fluoride equivalent to 10 times theamount of fluoride that could be released by a single LDBOdose(i.v.) of NSC 127755 was not lethal to the mice. The lack ofantitumor activity of the solubilized NSC 127755 given as asingle bolus i.v. injection might be due to a reduced time ofexposure at an effective plasma concentration. This type ofproblem (toxic peak plasma levels followed by a rapid declinein serum drug concentrations to therapeutically ineffectivelevels) has been frequently encountered in the evaluation ofpotentially useful antitumor agents. The usual solution hasbeen to reduce the dosage per injection and to administer theagent more frequently (2 to 4 times/day) for a greater numberof days (5 to 15 days) until meaningful, dose-limiting, subacutetoxicity (leukopenia, thrombocytopenia, gastrointestinal tracttoxicity, etc.) is obtained. A detailed explanation of the influence of the schedule of drug administration on host toxicity isprovided in the "Discussion."

The antitumor activity of NSC 127755 is listed in Table 6 and

summarized in Table 7. As can be seen in Table 7, NSC127755 showed marked antitumor activity against 4 of 10transplantable colon adenocarcinomas and against the Dunning murine ovarian tumor M5076.

The results of schedule and diluent studies with NSC 127755against colon adenocarcinoma 38 and the ovarian tumorM5076 are compiled in Tables 4 and 5. In both experiments,solutions of NSC 127755 (in 6% DMA) were less toxic (highertotal HNTD) and showed greater antitumor activity when administered as multiple (3 or 4) i.p. injections per day (every dayfor 9 days) as compared to 9 single daily injections. Despitethe observed beneficial effect of dosage fractionation for thesolubilized agent, treatment with 9 single daily i.p. injections ofsuspensions of NSC 127755 was less toxic than treatment withthe solubilized drug with either of the tested schedules. Thesedata suggest that still more frequent injections or infusion ofsolubilized NSC 127755 would further alleviate peak plasmalevel toxicity and provide optimum antitumor activity.

Although the triazine antifolate is an alkylating agent by virtueof the sulfonyl fluoride group (11), it does not appear to functionas the more classical alkylating agents (e.g., bis-2-chloroethylagents, nitrosoureas, epoxides, or c/s-diamminedichloroplat-inum), since the tumors most sensitive to these agents (colontumors 26 and 06/A and the 239Pu-inducedosteogenic sar

coma) were nearly totally unresponsive to NSC 127755 (Tables2 and 7). An examination of the agents most effective againstthe tumors tested (Table 2) or of the biological characteristicsof the tumors (Table 1) did not, in general, reveal any obviouspattern that would allow the prediction of those tumors likely to

1710 CANCER RESEARCH VOL. 42

on May 16, 2020. © 1982 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

A New Triazine Antifolate

Table 5

Treatment of advanced stage (75 to 300 mg) ovarian tumor M5076 with NSC 127755: diluent and schedule study

NSC 127755 single-agent treatment8Totaldose

(mg/kg)Drug

deaths intumor-bearing

miceAs

a suspension in 0.9% NaCI solution: 0.05% Tween 80, oncedaily133

mg/kg/dose,i.p89mg/kg/dose.¡.p..60mg/kg/dose,i.p.,40mg/kg/dose,i.p..27mg/kg/dose. ¡.p...,

Days 10-18Days10-18Days

10-18Days10-18Days

10-1811978015403602436/100/100/100/100/10(60)"(0)(0)(0)(0)%

increase inlifespan1241036267Days

oftumorgrowthdelay

(T- C

value)56.146.721.724.4Total

logkillLog

kill/dose0.70.60.30.3Gross6.25.22.42.7Net5.24.21.41.7CuresActivityratingToxic0/10

+ + ++0/10+ ++0/10+ ++0/10+ ++CommentLDeoHNTDAs

a solution in 6% DMA, oncedaily27mg/kg/dose,i.p..(0.4

ml/injection)18mg/kg/dose,i.p.,(0.4

ml/injection)12mg/kg/dose,i.p..(0.4

ml/injection)Days

10-18Days

10-18Days

10-188

mg/kg/dose, ¡.p..Days10-18(0.4ml/injection)5.4

mg/kg/dose,i.p.(0.4ml/injection),

Days 10-1 82431621087248.68/102/100/100/100/10(80)(20)(0)(0)(0)41515116.615.716.10.20.20.21.81.81.80.80.70.8ToxicToxic0/10

++ -»+ ++0/10

++0/10

++-»+ ++LO«,LD20HNTDCAs

a solution in 6% DMA, 4timesdaily18

mg/kg/dose,¡.p..4.Days10-18(0.112

mg/kg/dose,i.p.,4,Days 10-18(0.1every

6 hrxml/injection)every

6 hrxml/injection)8

mg/kg/dose, ¡.p..every 6 hrx4,Days10-18(0.15.4mg/kg/dose.¡.p.4.Days10-18(0.13.6

mg/kg/dose,i.p.4.Days 10- 18(0.12.4

mg/kg/dose,i.p.4.Days 10-18(0.1ml/Injection),

every 6 hrxml/injection),

every 6 hrxml/injection),

every 6 hrxml/injection)648432288194.4129.686.410/107/100/100/100/100/10(100)(70)(0)(0)(0)(0)6658515028.429.822.316.80.090.090.070.053.23.32.51.92.22.31.50.9ToxicToxic0/10

+ + ++0/10

+ + ++0/10

+ ++0/10

++ -»+ + +LD,ooLD,oHNTDC

Control: MOD = 39 days; time for median tumor to reach 1250 mg = 20.8 days; there were no tumor-free survivors among the 20 control mice. Td = 2.7 days;14th transplant generation. Experiment M5-114.

Numbers in parentheses, percentage.'" Assignment of this HNTD is problematic due to the occurrence of several acute toxicity deaths at the higher doses. It ¡sclear that the total dosage level could

be increased with more frequent administration of smaller doses. See text for further discussion.

respond or those likely to be insensitive. Potential causes fordifferences in innate sensitivities among the various tumorstested (e.g., intracellular uptake and retention times, intracel-

lular metabolism, etc.) were not evaluated.Because of its broad spectrum of activity against mouse

tumors and because of its possible clinical utility, we evaluatedNSC 127755 in combination with several clinically useful anti-

tumor drugs. The therapeutic synergisms of 2 of these combinations, NSC 127755 plus vincristine and NSC 127755 pluspalmO-ara-C, are shown in Table 8 and Chart 2, respectively.

The therapeutic synergism of NSC 127755 with palmO-ara-C was particularly marked on a simultaneous administrationschedule; the CTI was a highly favorable 1.6. In the sameexperiment, the 2 drugs (palmO-ara-C and NSC 127755) were

also tested in combination at a variety of dosage levels inalternating cycles (9-day course of palmO-ara-C, 7-day rest,9-day course of NSC 127755, 7-day rest, return to palmO-ara-

C, etc., for 3 courses of each agent). The results of thealternating cycles were far inferior (data not shown) to thesimultaneous schedule (Chart 2) because of the more favorableCTI (1.6) of the simultaneous schedule, which allowed a largertotal dose of each drug to be administered at equal toxicity.

DISCUSSION

The anticancer activity against 16 transplantable solid tumors of mice and the associated host toxicities of NSC 127755have been examined. Assessment of the anticancer activity ofany chemotherapeutic agent requires that meaningful dose-

limiting toxicities be reached.In the selection of optimum therapeutic dose levels for an

agent, dosage limitations are usually imposed by leucopenia,thrombocytopenia, or damage to the gastrointestinal epithelium. These subacute toxicities from anticancer drugs usuallydo not start to cause deaths until 3 or 4 days after the firstinjection (even with dosages 3 or 4 times an LD,0). Injection ofa drug by a schedule or route that causes acute deaths (withinmin or hr of the injection) will provide no relevant informationas to the true dose-limiting toxicity and may result in antitumor

evaluation at a suboptimal dose regimen. The following are themost common causes of immediate deaths, (a) Intolerance toa high peak plasma level (causing central nervous system,cardiac, or perhaps other problems) is a common cause ofimmediate death. In these cases, it is necessary to reduce thedosage per injection, increase the number of injections per

MAY 1982 1711

on May 16, 2020. © 1982 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

T. H. Corbett et al.

inCN.

CNJi§i

10)V)"oI1

§a<D

Orsza

coooo)

ai|ilM6OMZì§'

s0Oa2

S^IIrii¿inoUntreated

controloHighest

nontoxictreatment3Ì2o

co; «E £IoK"~ °'í!fi§1

s|ó¿»x

A Ogh-*"IlsIPI>.cl11o

«1°°1t

(0HI!11LÃœt||O)

Ci 1 CM

OCO1

1f-»0

0 |COo

'óo

a>o^ co~00o0a>O»

0*15S

"iSN;

COCOO

OOCM-r-CVJ0^0in

»"o+:+++ì+++

,î++iCM1

III1cMOcocoeMcocoooocoinincooocMCOCMCM

—CMO*-OOOOCOOOO§00

co at CMOCM —CM—CM—O —"-COOOOoooooooooooooooOOOOOOOOOOOOOOOO)OO

CMOOOOOCOI^OOo

o o o ooO

•- CM O *-COo

o o ooc-CM-^coœmcoocoi-oxnnœCOCMCO^CMi-.-^

in^-CM1"S

8 ? S ~ ñCMœœco7 o o *?mcoœcocooincoor-CMcDco^i--COCOCOCMCM^CM^CMCOftCOOlCMOOO)O>O>OOOO)

O~O> O O O 0>CMCM-- — .-CMCM — — .-.- — CMCM—OOOOOOOOOOCMOOOOCMCSiin^^CÛ

^COQO'-COCM'-^'Ol+í

Õ + ++++CM-fl-M^CMCO^inr-^in^COCMlili

IIcoocM^cor-minco^cococM^1CM

r- CM»-oooooooooooooo1O1OO)OOOOO)OOO>OOOO

OOOO OO O O^00

0«-

ooo

oov-

OOOUll-TftvOUÌ^tvCO-»^0)^oinœ.ocoococo.jçocMO77

•-•-•-Vo>cocoocooo>inocM*-r^cMO>•«••^•«••»•«•CM^CgCMCOCOCM-»^Ot-r-OOOOOOOO

O~O O

»-CMOJCMCMCMCMi-COCOWCMCMCMOOOOOOOOOOOOCMOco

o o""inX cococo^f coo"ìx>in•-Õ82

SSSSgSsîiSScoScogSSScScowStwÎïtS-01q

o o^- ^t CMCMincoo

oo

oo1—

co—a

a aooooomoooininininoo5

%5 %%çS 5 S 5 C g S 2Sco

oOO

OOOOOOOOOOOOO.-—

O — —OOO--OOO1

1»5» 8IECM

•—oo

ñ7mo —in 0)cotoCOCOI

1 ICOCMI 1 1 1ICOQOIó.

°.ááá.cÕQ.ci¿a.á.á.Q.ci¿oo>o>mcMinmoinoinooin5

? f s ^ °s 8 S S S S 5So

oO

OOOO OO OOOOli-O)*-*-»-T-C)»-»-0)'-^^OOOOOO'-OOO'-O'-'-18h-

^coIllOiOltllllOIIdd9ddddddddddd_

_ _ _ ._ ._ . ._ ._ ._ W, ._ ._ . ._ ._ ._ ._ . ._ ._._o

ooCOCOfcocoinco

cocoCMCO COooooooooooooooor-OCOCMO)l~COOOCOOOOJOJOcococoaocococo-coicoôcîo^O

O O O 0) O O O^O^OOOOOCOCOCD(O^iniD(D^-<DCDCOCo5tf>

Iß IO IO o O—22 2 2 »e |

eo eo (0 «£5?o^^\\\\£««««£•£$SSS

8S8888888a^S5SSssSS^ìSSsSSSÌlis.

Tf-.GDlu(fi,-Q_ T-^.^.^.CMCM,-^.

^. ^^ ^.^rtiiiiiHÃœHiSi''

AlldosageslistedweretheHNTDs(unlessotherwisenoted).Inthesetests,thenexthigherdosage(approximately1.5timesthelisteddosage)testedresultedin20%ormoredrugdeaths.

*Tumorgrowthdelay,treatedminuscontrol(r-Cindays)=thetimerequiredforthetreatmentgrouptumors(medianof10)toreachapredeterminedsize(usually750mg.earlystagetreatment,or1250

•ng,advancedstagetreatment)minusthetimerequiredforthecontrolgrouptumors(medianof10)togrowtothesamesize.Tumor-freesurvivorswereexcludedfromthesecalculations.

'Scheduleofdrugadministrationindayspostimplantationof30-to60-mgtumorfragmentss.c.

aTd=theexponentialtumorvolumedoublingtime(indays)measuredfromabest-fitlineofthecontrolgrouptumorsinthe60-to1000-mgrange.8Percentageincreaseinlifespan,T-Cvalue,andlogkillvaluescouldnotbeevaluatedinthisexperimentduetothelargenumberofcures(8/9).

'Thisvalueisthetimeindaysforthemediantumortoreach1250mg.

9Withthisschedule(3times/day).NSC127755wasinjectedasasolutionin6%DMA.

hHighestdosagetested.

'No-takes;tumorfragmentimplantfailedtoestablishitselfandgrow.

1712 CANCER RESEARCH VOL. 42

on May 16, 2020. © 1982 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

day, and inject for a greater period of time (until true cumulativedose-limiting toxicity causes 20% or more deaths at the highest

dosage level), (b) Incorrectly adjusted pH (pH should be between 5 and 8) and (c) intravascular precipitation of drug withi.v. injections (agent should be checked for solubility at pH 7 ifit is to be injected i.v.) also are common causes of immediatedeath.

Historically, we have placed a new agent into one of 3categories based on its toxicity behavior for different schedules. It should be emphasized that the distinction is not madeon comparisons of antitumor efficacy for various schedules,but rather on toxicity only. These categories refer to relativelyshort durations of treatment (e.g., less than 10 days) in whichthere is only minimal host recovery time from dose-limiting

toxicity at maximum tolerated dosages.Category 1. The schedule will markedly influence the total

dosage that can be administered. This occurs for some anti-metabolites (e.g., 6-thioguanine, ara-C, methotrexate). The

total dosage (at the HNTD) will markedly decrease (often by afactor of 100 or more) as the number of injections increaseswithin this time period (<10 days).

Category 2. The schedule will not influence the total dosagethat can be administered. Most agents fall into this category(e.g., most alkylating agents, mitotic inhibitors, DMA binders,and some antimetabolites such as 5-fluorouracil). The total

dosage (at the HNTD) does not change appreciably as the

Table 7Summary of NSC 127755 activity

TumorColon

26Colon36Colon38Colon51Colon06/AColon07/AColon08/AColon10/AColon

11/AColon12/AMammary16/CMammary14MXTOvarian

M5076Pancreas02Osteogenic

sarcomaEarly

stage Advanced stagetreatment3treatment+

NT6++++

+++++ + +++NTNT+

NTNT+

+ + + H »++NT+

+—•+ + + H > +++

NT+— + +NTNT++++

++++NTNT

Tumor weight at first treatment: 60 mg, early stage; 100 to 500 mg,advanced stage.

b NT. no test.

A New Triazine Antifolate

number of injections increases within the time period.Category 3. The total dosage at the apparent highest non-

toxic level appears to increase as the number of injectionsincreases within this time period (<10 days). This phenomenonis usually caused by an intolerance to a high peak plasma levelof the drug (or route) that results in immediate deaths and theerroneous assignment of the highest nontoxic dose level. Thus,the schedule (or route) producing the immedite deaths is notsatisfactory for the evaluation of antitumor activity. In thesecases, efficacy can only be determined in a schedule with morefrequent injections (at reduced dosage levels) for a more prolonged period of time. The triazine antifolate (NSC 127755)discussed in the present work is a Category 3 agent.

Because of the acute toxicity problems with the triazineantifolate, NSC 127755, in solution, this agent has progressedslowly toward clinical trial. There is the possibility that the 8- to10-fold-lower doses (on a mg/kg basis) that are used in hu

mans (as compared to mice) will eliminate the problem. Otherpossibilities include a slow infusion of the drug or the p.o.administration of an enteric-coated preparation. It is also pos

sible that certain structural changes could retain or improvethe antitumor activity, while reducing the acute toxicity problem. Based on the similar peak plasma level problems ofNSC 127755, Baker's antifol, and 5-(3,4-dichlorophenyl)-6-

methyl-2,4-pyrimidinediamine, each of which contains theR—N=C(NH2)—N=C(NH2)—R' group, modification of one or

both of the amino groups with groups that would slowly hydro-

lyze to the parent compound might be effective. In addition, anappropriate choice of the(se) hydrolyzable group(s) could result in greater water solubility, thus simplifying formulation.

Because 4 of 10 transplantable mouse colon adenocarci-

nomas were highly responsive to NSC 127755, a clinical trialagainst this tumor type may be indicated if the drug formulationand acute toxicity problems can be overcome. Similarly, a trialof NSC 127755 with ara-C may be indicated because of the

marked therapeutic synergism observed on a simultaneousadministration schedule. The therapeutic synergism of anotherantifolate (methotrexate) with ara-C has been reported previ

ously (1, 4, 9, 12, 15).

ACKNOWLEDGMENTS

The authors gratefully acknowledge the technical assistance of T. Ayer, D.Beane, M. Black, L. Dorwin, G. Gamble, P. Holum, C. Howard, J. Seay, and S.Wilson in thécollection of laboratory data, and théassistance of T. Ayer, L.Muglach, and C. Schulman in manuscript preparation.

Table 8WSC 127755 plus vincristine treatment of advanced stage (100 to 300 mg) colon adenocarcinoma 10/A

Highest nontoxic single agenttreatment"NSC

127755, 42.0 mg/kg/dose, ¡.p.,Days16-28

Vincristine, 1.3 mg/kg/dose, i.V., Days 16.22,28Drug

deathsin tumor-

bearingmice0/10

0/10RegressionsT

- C value(days)15.4

15.8Partial

(>50%)1/10

0/10Complete1/100/10Tumor-free

survivors0/10

0/10Log

kill/dose0.06

0.25Total

logkillGross0.8

0.8Net0.2 0.2Activity

rating+

+

NSC 127755, 28 mg/kg/dose, i.p., Days 16- 1/1028 + vincristine, 0.8 mg/kg/dose, i.V.,Days 16, 22, 28

24.2 5/10 4/10 1/10 1.2 0.6

Control: MOD = 84.5; time for median tumor to reach 1250 mg = 36.1 days; there were no tumor-free survivors among the 20 control mice. Td = 6.3 days;13th transplant generation. Experiment 1709L1.

b Highest nontoxic combination.

MAY 1982 1713

on May 16, 2020. © 1982 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

T. H. Corbett et al.

APPENDIX

AAFC (NSC 166641 ); [2R,(2d,3/3,3a/3,9ay8))-7-fluoro-2,3,3a,9a-tetrahydro-3-hydroxy-6-imino-6H-furo[2',3':4,5]oxazolo[3,2-a]pyrimidine-2-methanol. CAReg. No. [37717-21-8].

ACNU (NSC 245382); N'-[(4-amino-2-methyl-5-pyrimidinyl)methyl>W-(2-chlo-

roethyl)-/V-nitrosourea, monohydrochloride. CA Reg. No. [55661-38-6].Adriamycin (NSC 123127); (8S-c/sM0-[(3-amino-2,3,6-trideoxy-a-L-/yxo-hex-

opyranosyl)-oxy]-7,8,9,10-tetrahydro-6,8.11 -trihydroxy-8 -(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione. CA Reg. No. [23214-92-8].

Aminopterin (NSC 739); W-{{ 4-{[(2,4-diaminc-6-pteridinyl)methyl]amino}ben-zoyl})-L-glutamic acid. CA Reg. No. [54-62-6].

Anguidine (NSC 141537); (3a,4/8)-12,13-epoxytrichothec-9-ene-3.4,15-triol,4,15-diacetate. CA Reg. No. (2270-40-8).

Anthracenedione acetate (NSC 287513); 1,4-bis {{{2-[(2-hydroxyethyl)ammo]ethyl)amino}}-9,10-anthracenedione, diacciate. CA Reg. No. [70711-40-9].

Ara-C (NSC 63878); 4-amino-1-/3-D-arabinofuranosyl-2(1H)-pyrimidinone. CAReg. No. [147-94-4].

Aziridinylbenzoquinone (NSC 182986); [2,5-bisd -aziridinyl)-3,6-dioxo-1,4-cy-clohexadiene-1.4-diyl]biscarbamic acid, diethyl ester. CA Reg. No. [57998-68-2]

Baker's antifol (NSC 139105); 3-{[2-chloro-4-(4,6-diamino-2,2-dimethyl-

1,3,5-triazin-1 (2H)-yl)phenoxy]methyl) -A/.N-dimethylbenzamide compoundedwith ethanesulfonic acid (1:1). CA Reg. No. [41191-04-2].

BCNU (NSC 409962); N,N'-bis(2-chloroethyl)-N-nitrosourea. CA Reg. No.[154-93-8).

Carminomycin (NSC 180024); (8S-cis)-8-acetyl-10-[(3-amino-2,3,6-trideoxy-a-L-/yxo-hexopyranosyl)oxy]-7,8.9,10-tetrahydro-1,6,8,11-tetrahydroxy-5,12-naphthacenedione. CA Reg. No. [50935-04-1].

Chlorozotocin (NSC 178248); 2-{{([(2-chloroethyl)nitrosoamino]carbonyl)amino}}-2-deoxy-D-glucose. CA Reg. No. [54749-90-5].

cis-DDPt (NSC 119875); (SP-4-2)-diamminedichloroplatinum. CA Reg. No.[15663-27-1].

Cyclocytidine (NSC 145668); [2-W-(2a,3/8,3ai8,9a/8)]-2,3,3a,9a-tetrahydro-3-hydroxy-6-imino-6H-turo[21,3':4,5]oxazolo[3,2-a]pyrimidine-2-methanol. CAReg. No. [31698-14-3].

Cyclophosphamide (NSC 26271); /V,N-bis(2-chloroethyl)tetrahydro-2H-1,3,2-oxazaphosphorin-2-amine, 2-oxide. CA Reg. No. [50-18-0].

Daunomycin (NSC 82151); (8S-c/s)-8-acetyl-10-[(3-amino-2,3,6-trideoxy-a-L-/yxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro- 6,8,11-trihydroxy-1-methoxy-5,12-naphthacenedione. CA Reg. No. [20830-81-3].

DDMP (NSC 19494); 5-{3.4-dichlorophenyl)-6-methyl-2.4-pyrimidinediamine.CA Reg. No. [7761-45-7].

Dianhydrogalactitol (NSC 132313); 1,2:5,6-dianhydrogalactitol. CA Reg. No.[23621-20-3].

Dibromodulcitol (NSC 104800); 1,6-dibromo-1,6-dideoxygalactitol. CA Reg.No. [10318-26-0],

Diethylstilbestrol (NSC 3070); 4,4'-(1,2-diethyl-1,2-ethenediyl)bisphenol. CAReg. No. [56-53-1].

Dihydroxyanthracenedione (NSC 279836); 1,4-dihydroxy-5.8-bis(( {2-[(2-hy-droxyethyl)amino]ethyl}amino}}-9,10-anthracenedione. CAReg.No. [65271-80-

9].DTIC (NSC 45388); 5-(3,3-dimethyl-1-triazenyl)-1H-imidazole-4-carboxa-

mide. CA Reg. No. [4342-03-4].5-FU (NSC 19893); 5-fluoro-2,4(1H,3H)-pyrimidinedione. CA Reg. No. [51-

21-8],Homoharringtonine (NSC 141633); cephalotaxine, 4 methyl-2-hydroxy-2-(4-

hydroxy-4-methylpentyl)butanedioate ester. CA Reg. No. [26833-87-4].Hydroxyurea (NSC 32065); CA Reg. No. [127-07-1].Indicine-N-oxide (NSC 132319); [1fl-[1a,7(2R<,3S>),7a/3]]-2.3-dihydroxy-2-

(1 -methylethyObutanoic acid, (2,3,5,7a-tetrahydro-1 -hydroxy-1 H-pyrrolizin-7-yDmethyl ester, N-oxide. CA Reg. No. [41708-76-3).

Isophosphoramide mustard (NSC 297900); W,N'-bis(2-chloroethyl)phos-phorodiamidic acid. CA Reg. No. [31645-39-3].

MeCCNU (NSC 95441); N-(2-chloroethyl)-/V-(4-methylcyclohexyl)-A/-nitro-sourea. CA Reg. No. [13909-09-6).

6-MeMPR (NSC 40774); 6-{methylthio)-9-/3-D-ribofuranosyl-9H-purine. CAReg. No. [7143-67-1].

Methotrexate (NSC 740); amethopterin; N-({4-{[(2,4-diamino-6-pteridinyl)-methyl]methylamino}benzoyl})-L-glutamic acid. CA Reg. No. [59-05-2].

Mitomycin C (NSC 26980); [1aR-(1aa,8^.8aa,8Da))-6-amino-8-{[(amino-carbonyl)oxy]methyl}-1,1a,2,8,8a,8D-hexahydro-8a-methoxy-5-methylazirino-[2',3':3,4)pyrrolo[1,2-a]indole-4,7-dione. CA Reg. No. [50-07-7].

Neocarzinostatin (NSC 157365); polypeptide antibiotic isolated from Sfrep-tomyces carcinostaticus var. F41.CA Reg. No. [9014-02-2].

PalmO-ara-C (NSC 135962); 4-amino-1-(5-O-(1-oxohexadecyl)-/ì-o-arabino-furanosyl}-2(1H)-pyrimidinone. CA Reg. No. [31088-06-9].

L-PAM (NSC 8806); L-sarcolysin; melphalan; 4-{bis(2-chloroethyl)aminoH-

phenylalanine. CA Reg. No. [148-82-3].PCNU (NSC 95466); N-(2-chloroethyl)-N'-(2,6-dioxo-3-piperdinyl)-W-nitrosou-

rea. CA Reg. No. [13909-02-9].Phosphoramide mustard (NSC 69945);W,N-bis(2-chloroethyl)phosphoro-

diamidic acid. CA Reg. No. [10159-53-2].Piperazinedione (NSC 135758); 3,6-bis-(5-chloro-2-piperidinyl)-2,5-pipera-

zinedione, dihydrochloride. CA Reg. No. [41109-80-2).Procarbazine (NSC 77213); W-(1-methylethyl)-4-[(2-methylhydrazino)meth-

yl]benzamide. CA Reg. No. [671-16-9).6-Thioguanine (NSC 752); 2-amino-1,7-dihydro-6H-purine-6-thione. CA Reg.

No. [154-42-7]. vVincristine (NSC 67574); 22-oxovincaleukoblastine. CA Reg. No. [57-22-7].

REFERENCES

1. Avery, T. L., and Roberts, D. Dose-related synergism of cytosine arabinosideand methotrexate against murine leukemia L1210. Eur. J. Cancer, 10: 425-429, 1974.

2. Baker, B. R. Design of Active-Site-Directed Irreversible Enzyme Inhibitors.The Organic Chemistry of the Enzymic Active-Site. New York: John Wiley& Sons, Inc., 1967.

3. Baker, B. R., and Vermeulen, N. M. J. Irreversible enzyme inhibitors.CLXXVII. Active-site-directed irreversible inhibitors of dihydrofolate reducíase derived from 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(phenylalkyl-phenyl)-S-tnazines. II. J. Med. Chem., »3.1154-1160, 1970.

4. Cadman, E., and Eiferman, F. Mechanism of synergistic cell killing whenmethotrexate precedes cytosine arabinoside. Study of L1210 and humanleukemic cells. J. Clin. Invest., 64: 788-797, 1979.

5. Carter, S. K. Planning combined therapy—The interaction of experimentaland clinical studies. Cancer Chemother. Rep., 4. 3-11, 1974.

6. Corbett, T. H., Griswold, D. P., Jr., Roberts, B. J., Peckham, J. C., andSchabel, F. M., Jr. Evaluation of single agents and combinations of chemo-therapeutic agents in mouse colon carcinomas. Cancer (Phila.). 40: 2660-2680, 1977.

7. Corbett, T. H., Griswold, D. P., Jr., Roberts, B. J., Peckham, J. C., andSchabel, F. M., Jr. Biology and therapeutic response of a mouse mammaryadenocarcinoma (16/C) and its potential as a model for surgical adjuvantchemotherapy. Cancer Treat. Rep., 62. 1471-1488, 1978.

8. Corbett, T. H., Griswold, 0. P., Jr., Wolpert. M. K., Venditti, J. M.. andSchabel, F. M., Jr. Design and evaluation of combination chemotherapytrials in experimental animal tumor systems. Cancer Treat. Rep., 63. 799-801, 1979.

9. Edelstein, M., Vietti, T., and Valeriote, F. The enhanced cytotoxicity ofcombinations of 1-/S-o-arabinofuranosylcytosine and methotrexate. CancerRes., 35. 1555-1558. 1975.

10. Glasgow, L. A., Crane, J. L., Jr., and Kern, E. R. Antitumor activity ofinterferon against murine osteogenic sarcoma cells in vitro. J. Nati. CancerInst., 60: 659-666. 1978.

11. Kumar, A. A., Mangum, J. H., Blankenship, D. T., and Freisheim, J. H.Affinity labeling of chicken liver dihydrofolate reducíase by a substituted4,6-diaminodihydrotriazine bearing a terminal sulfonyl fluoride. J. Biol.Chem., 256. 8970-8976. 1981.

12. Roberts. D., and Milliard, S. L. Combination chemotherapy in sequence forthe treatment of L1210 leukemia. Cancer Res., 38. 2317-2320, 1978.

13. Schabel, F. M., Jr., Griswold, D. P., Jr., Corbett, T. H., Laster, W. R., Jr.,Mayo, J. G., and Lloyd, H. H. Testing therapeutic hypotheses in mice andman: observations on the therapeutic activity against advanced solid tumorsof mice treated with anticancer drugs that have demonstrated or potentialclinical utility for treatment of advanced solid tumors of man. Cancer DrugDevelopment, Part B. Methods Cancer Res., 17: 3-51, 1979.

14. Schabel, F. M., Jr., Griswold. D. P., Jr., Laster, W. R., Jr., Corbett. T. H.,and Lloyd, H. H. Quantitative evaluation of anticancer agent activity inexperimental animals. Pharmacol. Ther. A, ). 411-435, 1977.

15. Schabel, F. M., Jr., Trader, M. W., Laster, W. R., Jr., and Witt, M. W.Therapeutic synergism (TS) of ara-C plus methotrexate (MTX) against murineleukemia L1210. Proc. Am. Assoc. Cancer Res., 22: 214. 1981.

16. Skipper, H. E. Combination therapy: Some concepts and results. CancerChemother. Rep. Part II, 4: 137-145, 1974.

17. Venditti, J. M., and Goldin, A. Drug Synergism in Antineoplastic Chemotherapy. Adv. Chemother., Õ.397-498, 1964.

18. Watson, C., Medina, D., and Clark. J. H. Estrogen receptor characterizationin a transplantable mouse mammary tumor. Cancer Res., 37. 3344-3348,

1977.19. Wodinsky, I., and Kensler, C. J. The biology, kinetics, and chemotherapy of

a slow growing tumor, the B16 melanoma. In: M. Semonsky. M. Hejzlar, andS. Masak (eds.). Advances in Antimicrobial and Antineoplastic Chemotherapy, Vol. 2, pp. 427-429. Baltimore: University Park Press, 1972.

1714 CANCER RESEARCH VOL. 42

on May 16, 2020. © 1982 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

A New Triazine Antifolate

"2

©W)1H9I3M

MAY 1982 1715

on May 16, 2020. © 1982 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

1982;42:1707-1715. Cancer Res   Thomas H. Corbett, Wilbur R. Leopold, Donald J. Dykes, et al.   (NSC 127755)Toxicity and Anticancer Activity of a New Triazine Antifolate

  Updated version

  http://cancerres.aacrjournals.org/content/42/5/1707

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/42/5/1707To request permission to re-use all or part of this article, use this link

on May 16, 2020. © 1982 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from