Investigation of the Activity of Cytoxan Against Leukemia L1210 in Mice

Investigation of the Activity of Cytoxan against Leukemia L1210 in Mice

JOHN M. VENDITTI, STEWART R. HUMPHREYS, AND ABRAHAM GOLDIN

(Laboratory of Chemical Pharmacology, National Canter Institute,* Bethesda, Md.)

Two prime limiting factors in the treatment of dichloroamethopterin and 3'-bromo-5'-chloro- neoplastic disease are (a) the toxicity of active amethopterin, were capable of eliciting extensive anti-tumor agents for the host, and (b) the origin, survival time and apparently cured some of the during therapy, of resistance to the agent being mice, even when treatment was initiated after the employed. The origin of resistant variant sublines disease was frankly systemic (11). However, to therapy with folic acid antagonists, antipurines, despite the marked effectiveness of the antifolics glutamine antagonists, etc., has been amply in this system, host toxicity and the origin of re- demonstrated (5, 6, 22, 25-31, 86, 37). sistant variants, particularly following very ex- For purposes of overcoming the limitation of tensive treatment, appeared to limit the useful- drug toxicity in therapy, a primary objective of ness of therapy (20). screening programs and of detailed structure- In seeking additional anti-leukemic compounds activity studies within active groups of compounds with differing modes of action for use in conjunc- has been to uncover drugs with increased anti- tion with folic acid antagonists, purine and pyrimi- tumor activity and reduced host toxicity (7, 17, dine antagonists, etc., the nitrogen mustard 21, 35, 39, 41). With respect to the origin of re- derivative, N,N-bis (f~-chloroethyl)-N',O-propyl- sistance to treatment, the effort has been twofold: enephosphoric acid ester diamide (Cytoxan I) (1) (a) To uncover more active congeners within the was selected for more extensive study. This cyclic same structural series (2, 7, 17, 41) and (b) to ob- nitrogen mustard phosphamide ester appeared to tain drugs which inhibit at sites in different be of particular interest because of (a) its marked metabolic pathways or at different loci within the inhibitory action against a variety of experimen- same metabolic pathway (8, 38, 40). Such com- tal tumors, and its apparently greater specificity pounds have been employed in clinical studies of of anti-tumor action as compared with a number concomitant and sequential therapy (9). Combina- of other alkylating agents (1, 8, 4, 19, 23, 24), (b) tions of drugs presumed to act at different sites the postulated mechanism for increased specificity, have been employed in studies of anti-tumor involving splitting off of the phosphamide group synergism, empirically or in attempts at multiple and release of mustard activity at the tumor site biochemical blockade (12). (8), and (c) the apparent dissimilarity of this In this laboratory, assay procedures for the action as compared with that of the folic acid quantitative evaluation of chemotherapeutic antagonists, antipurines, antipyrimidines, etc. agents have been developed with the use of early The current study was conducted to character- and advanced leukemia L1210 in mice (14, 15). ize the effectiveness of Cytoxan against leukemia With these procedures, various purine and pyrimi- L1210 and to determine its effectiveness against dine antagonists, nitrogen mustard, and folic acid an antifolic-resistant subline and an antipurine- antagonists etc. (18), demonstrated definite thera- resistant subline of leukemia L1210. peutic effectiveness. The folic acid antagonists were particularly effective in increasing the sur- MATERIALS AND METHODS vival time of mice with this leukemia (17, 18). Al- The general procedures for the assay of drug effectiveness are similar to those used previously in this laboratory (14, 15, though amethopterin was markedly effective in 18). this system, it was surpassed in therapeutic The experiments were conducted in ~0-28-gm. BDFI activity by various halogenated derivatives of (C57BL/6 X DBA/~)FI hybrid, CDBA (BALB/c An X amethopterin (11, 17). Notably, two of the di- DBA/2J)F1 hybrid, or D~BC (ZBC X DBA/~) hybrid mice. halogenated derivatives of amethopterin, 3'5'- Cytoxan, Mead Johnson brand of cyclophosphamide, was formerly designated as Endoxan ("Summary of data on En- * National Institutes of Health, Public Health Service, U.S. doxan," A. G. Astawerke). The Cytoxan used in these experi- Department of Health, Education, and Welfare. ments was obtained from Mead Johnson through the Cancer Received for publication June ~, 1959. Chemotherapy National Service Center. 986

The hybrids employed are designated with the individual ex- RESULTS periments. The transplantable tumors employed were the sensitive The results are summarized in Tables 2-4 and leukemia (LI~10) (32) carried in DBA/~J mice, an amethop- Charts 1-3. terin-resistant subline of LI~10 (M46R) carried in CDBA hy- Table 2 shows summary data of a series of ex- brids (~0), and an 8-azaguanine-resistant variant (33) carried periments comparing the relative effectiveness of in DBA/~ mice. For each experiment the mice were/,dven in- oculations subcutaneously in the right hind leg of 0.1 or 0.~ ml. daily administration of Cytoxan, various other of a uniform saline suspension of leukemic cells from stock mice alkylating agents, and amethopterin in increasing with advanced leukemia. The sensitive leukemia (LI~10) and the survival time of mice with systemic leukemia. the amethopterin-resistant LI~10 (M46R) inocula were pre- At the optimal daily dose (62.5 mg/kg) (Table 2, pared from leukemic spleen tissue. The 8-azaguanine-resistant Exp. 1), Cytoxan increased the median survival LI~10 inoctdum was prepared from ascitic fluid. The concentration of the experimental tumor inoculum ranged from 3 X time of the nfice from 11.0 days (untreated con- 105 to 3 )< 10 e cells per mouse for the various experiments. trois) to 26.5 days. The extension in survival time

TABLE 1 LIST OF COMPOUNDS

Compound name Chemical designation Molecular formula Vehicle Source

Cytoxan (Endoxan) N,N-Bis-(f/-chloroethyl)-N',O-propyl- CrHIsCI2N202P-H20 0.85% saline Mead Johnson ene phosphoric acid ester diamide Nitrogen mustard (HN~) 2,2'-Dichloro-N-methyldiethylamine, CzttHC]2N-HCI 0.85% saline Merck hydrochloride Triethylene melamine 2,4,6-Tris(1-aziridinyl)-s-triazine CgHI~N~ 0.85% saline l~derle (TEM) Degranol 1,6-Bis(2-cnloroethylamino)-l,6-dide- CIoH2.~CIaN~O4-~ 0.85% saline Medimpex oxy-D-mannitol,dihydrochloride Chlorambucil 4- {p-[Bis(~-chloroethyl)amino]phenyl }- C14H~gCI~.NOo. MC* Burroughs-Well- butyric acid come Hydroquinone mustard a~,a6-Bis[bis(~-chloroethyl)amino]-2,5- CI~LI~4CI4N.,O2 9 2HC1 MC C. Weatherbee, xylohydroquinone dihydrochloride Millikin Univ. DL-Sarcolysin 3-{ p-[Bis(2-chloroethyl)amino]phenyl I - Cj~H1sCIo_N.~O2"HC1 H,O J. tI. Burckhal- DL-alanine, hydrochloride ter Univ. of Kansas L-Sarcolysin 3- {p-[Bis(2-chloroethyl)amino]phenyl }- CI.~HlsCI2N202-HCI H.,O Chester Beatty L-alanine, hydrochloride Research Inst. Nitromin 2,2'-Dichloro-N-methyl diethylamine, CsHIIC12NO- HCI H20 Takeda N-oxide hydrochloride Uracil mustard 5-[Bis(2-chloroethyl) amino]uracil CsHI~CI~N~O2 5% EtOH- Up john saline Amethopterin 4-amino-N l~ C20H2..NsO~ ~% NaHCO~ Am. Cyanamid acid Co. 6-Mercaptopurine 6-Purinethiol C~H4N4S Dil. NaOH Burroughs-Well- come

* MC = 0.5 per cent methyl cellulo~ solution. Uracil mustard was kindly provided by Dr. John S. Evans, Research Division, Upjohn Co. Amethopterin was obtained from the Lederle Research Div., American Cyanamid Co. The remaining compounds were obtained from the Cancer Chemotherapy National Service Center, National Cancer Inst.

The treatment of advanced leukemia was initiated 7-11 provided by Cytoxan, in this experiment, was 97 days following tumor implantation (2-4 days prior to the per cent as great as that observed with ameth- median day of death of untreated controls) when all the mice displayed palpable local tumors. In all cases, when tested, the opterin. The antileukemic action of Cytoxan was disease had become systemic. This was evidenced by the death also reflected in an inhibitory effect on the growth from tumor of normal mice given implants subcutaneously of of the local tumor at the site of leukemic inocula- spleens from mice selected at random from those to be treated tion. At the optimal dose, the local tumor, ap- (18). The treatment of early leukemia was initiated two days proximately 7-9 mm. in diameter, was non- following tumor implantation. The compounds employed, their sources, and vehicles are palpable after about 1 week of treatment. Relative listed in Table 1. They were administered subcutaneously in to amethopterin, employed as a standard, Cytoxan the axillary area in the constant volume of 0.01 ml/gm of displayed greater antileukemic activity than did mouse weight. Each compound was administered over a wide any of the other alkylating agents tested. Uracil range of doses. There were usually ten mice at each dose level and twenty untreated controls. The experimental design, in- mustard, in one of two experiments (Table 2, Exp. cluding dosages and schedules of treatment, is indicated in the 2), appeared to be next in effectiveness among the individual experiments. alkylating agents, producing an increase in

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1959 American Association for Cancer Research. 988 Cancer Research Vol. 19, October, 1959 median survival time which was 63 per cent as guanine, 5-fluorouracil, ~-amino-l,3,4-thiadiazole, great as that observed with amethopterin. In a and aminopterin were relatively effective, produc- second experiment, however, uracil mustard was ing increases in median survival time which were only 9.5 per cent as effective as amethopterin 42-62 per cent as great as those produced by (Table 2, Exp. 3). Triethylene melamine, DL- amethopterin. Up to the present, the only com- Sarcolysin, L-Sarcolysin, Degranol, nitrogen mus- pounds which have been shown to provide an tard (HN2), and hydroquinone mustard gave in- antileukemic specificity as great as, or greater than, creases in median survival time ranging from 12 amethopterin have been other congeners of folic to 31 per cent as great as that of amethopterin. acid; notably, the halogenated derivatives of Chlorambucil and nitromin were essentially in- amethopterin and aminopterin, adenopterin and effective. Ni~ (11, 17). Cytoxan, then, is In a previous report from this laboratory (18), the only drug, other than other antifolics, shown 38 compounds, including 23 employed in the to be as effective as amethopterin in increasing Gellhorn-Hirschberg report (10), were compared the survival time of mice with systemic leukemia. with amethopterin with respect to their capacity More detailed studies were therefore undertaken to increase the survival time of mice with ad- against the sensitive line of leukemia LI~10. vanced leukemia. Of the compounds tested, An experiment was conducted (Chart 1) to de- reserpine, 8-azaguanine, 6-mercaptopurine, thio- termine the influence of the schedule of treatment

TABLE 2 RELATIVE EFFECTIVENESS OF CYTOXAN AND OTHER ALKYLATING AGENTS AGAINST ADVANCED LEUKEMIA L1210, WITH AMETHOPTERIN USED AS A STANDARD

Relative Per cent increase in increase in Experi- Day of Median sur- median sur- Dose range, Optimal dose median ment initial Compound name vival time at vival time. daily level, daily survival IlO. treatment optimal dose (Amethop- time over terin in- controls erease=100) , (mg/kg) (mg/kg) (days) 8 Amethopterin 0 39-3.02 1.09 27.0 145 100 Cytoxan 31.3 -1000 62.5 26.5 141 97 Hydroquinone mustard 2.00-63.0 63.0" 13.0" 18 Chlorambucil 5.04-180 14.0 11.5 5 3 Controls 11.0

Amethopterin 0.47-2.16 1.30 205 86 100 Uracil mustard 0.28--6.00 2 16 17.0 55 63 Controls ...... 11.0

Amethopterin 0.47-3.60 1.30 ~5 0 127 100 Uracil mustard 0.78-6.00 3.60 14.5 32 25 Controls ...... 11.0 ...... , ......

Amethopterin 0 39-3 02 0.65 ~5.5 132 100 DL-Sarcolysin 1.09-38.9 23.3 15.5 41 31 Degranol 1.81-108 64.8 14.0 ~7 21 L-Sarcolysin O. 65-38.9 38.9* 13.0" 18 14 Nitromin 3 05-108 38.9 11.5 5 3 Controls ...... , ...... 11.0 ...... , ......

Amethopterin O. 39-3.02 1.09 25.5 155 100 Triethylene melamine (TEM) 0.9.3-3.0s 1.09 14.0 40 26 Nitrogen mustard (HN2) 0 23-1.81 0.65 120 20 13 Controls ...... , ...... 10 0 ......

11 Amethopterin 0.39-3 02 0.65 27.0 108 100 L-Sarcolysin ~3 3-108 38.9 16.5 ~7 ~4 Chlorambucil 14 0-108 None 13 0 0 0 Controls 13.0

* The highest daily dose employed was most effective. The mice succumbed, however, with considerable weight loss, which suggests that higher dose levels would not have produced appreciably greater increases in median survival time. Mice employed in the various experiments: BDF:, male (Exps. 1, 3, 4); D2BC, male (Exp. 5); D2BC, female (Exps. 2, 6). Ten mice per treated group; twenty mice per control group.

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1959 American Association for Cancer Research. TABLE 3 EFFECT OF CYTOXAN, ALONE AND COMBINED WITH NICOTINAMIDE, ON EARLY LEUKEMIA L1210

SINGLE TREATMENT, DAY DAILY TREATMENT BEGINNING DAY

No. No. Av. survival No. of Median sur- tu mor- Median Treatment vival time of toxic tumor time of free sur. Treatment survival total group deaths deaths mice dying vivors time of tumor (day 60

(mg/kg) (days) (days) (mg/kg/day) (days) 500 Nieotin-+1000 Cytoxan 3.0 10 0 ...... 0 500 Nicotin-+'360 Cyt~xan 7.0 amide+ 600 " 5.5 7 0 i ...... 8 amide+~216 ' 8.5 " + 360 " > 60.0 3 0 ...... 7 " + 180 " 9.5 " + 216 " 34.0 8 0 ...... `2 " + 78.0 " 19.0 " + 180 " >60.0 1 1 `20.0 8 " + 46,8 " `25.5 " + 78.0 " 18.0 0 10 17.9 0 " + `28.1 " `27.5

250 Nicotin-+1000 Cy~xan 8.5 `250 Nicotin-+860 Cytoxan 8.0 amide+ 600 6.5 amide+~216 " 8.0 " + 360 " 38.5 " + 180 " 9.5 " + `216 " >60.0 " + 780 " 17.5 " + 180 " > 60.0 `25.0 " + 46.8 " 19.0 " + 78.0 " 18.0 17.4 " + `28.1 " `27.0

i 1000 Cytoxan 3.0 10 0 ...... 130 Cytoxan 10.0 600 8.5 10 0 78.0 " "20,0 360 " >60.0 3 0 iiiiiiiiii 46.8 " `29.0 216 >60.0 `2 0 `28.1 " `26.0 130 `22.5 5 3 `26.0 16,9 " 14.5 78.0 " 16.0 1 8 16.8 `2.16 Amethopterin 18.0 360 Amethopterin 7.5 `2 `24 .o 1.30 " 59.0 216 "21.0 10 ~.7 0.78 " 83.5 180 " 18.0 18.8 0.47 " `27.5 78.0 " 17.5 17.1 O . `28 " `25.5 I~ ntreated controls 10.0

BDF, male mice. Nicotinamide was administered intraperitoneally 15 minutes prior to the subcutaneous injection of Cytoxan (Exp. 7). Ten mice per group.

TABLE 4 RELATIVE EFFECTIVENESS OF CYTOXAN, AMETHOPTERIN, AND 6-MERCAPTOPURINE AGAINST AN 8-AZAGUANINE-RESISTANT SUBLINE OF Ll`210

Per cent increase in Median sur- Optimal dose median Compound Dose range, daily vival time at level, daily survival optimal dose time over controls

(mg/kg) (mg/kg) (days) Cytoxan 14.0-180 64.8 `25.5 118 Amethopterin O. 14-8. O0 1.08 >50.0 >,'317 6-Mereaptopurine 14.0-800 38.9, 108 18.0 8 Controls 1`2.0

CDBA female mice. Ten per treated group, twenty untreated controls. Exp. 8. Daily treatment was begun 8 days after tumor inoculation.

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1959 American Association for Cancer Research. 990 Caucer Research Vol. 19, October, 1959 on the effectiveness of Cytoxan in increasing the (23, 24). It is of interest to note that amethopterin survival time of mice with systemic leukemia. Cy- has been shown to be relatively ineffective against toxan was given as a single dose, daily, every 2d- advanced leukemia L1210 when given as a single day, and every 4th day, starting at 7 days follow- treatment or when treatments are spaced 4 days ing leukemic inoculation. Comparison is made apart (15). with daily treatment with amethopterin as the With amethopterin daily, the last animal standard. The median survival time of the con- succumbed on the 30th day following leukemic in- trol mice was 10 days. In this experiment, on oculation, while on the four schedules of treatment daily treatment, Cytoxan was slightly more effec- with Cytoxan, 21 mice survived beyond this tive than amethopterin. The alkylating agent gave point, succumbing between the 30th day and the a maximum median survival time of 27 days, as 48th day. There were two such mice on single contrasted with 24.5 days for amethopterin. treatment with Cytoxan and three on daily treat- On two other schedules of treatment, namely, ment. The remaining sixteen long-term survivors every 2d day and every 4th day, Cytoxan was still were in the groups treated with Cytoxan every 2 more effective, giving maximum median survival and every 4 days. times of 31 days and 30.5 days. A single treatment The results of treatment of early leukemia with Cytoxan given at 7 days following leukemic L1210 with Cytoxan are summarized in Table 3. inoculation was also effective, increasing the Two schedules of treatment were employed, median survival time to 23 days. namely, a single treatment given 2 days following Thus, Cytoxan appears to be quite effective in tumor inoculation and daily treatment beginning increasing the survival time of mice with systemic on the same day. Mice receiving a single treat- leukemia over a wide range of schedules of treatment could be separated into three categories; ment. This is in agreement with previous reports those succumbing to drug toxicity, those succumb-

l'l'i'l'lll']' ']'1'I I "I ' I' " I' I' 35-- TREATMENT TREATMENT EVERY EVERY SINGLE -- TREATMENT DAILY SECOND DAY FOURTH DAY TREATMENT Cytoxon Cytoxan 30 Cytoxan/ Ame/hopier/n

o 25 v Cytoxon LIJ

l-- ._J 20 > ,I 1> 15- L Z 1 /, l 0 w IE I0' CONTROLS

: DAY OF TREATMENT INITIATION llljl,li],li], l,l,]i 1,1,1, I,i,I 0.28 0.78 22 6.0 17 47 130 28 78 216 47 130 360 1303601000 LEVEL OF INDIVIDUAL TREATMENT(M(j./kg) CItAaT 1.--Influence of the schedule of treatment on the Ten mice per treated group; twenty untreated controls. anti-leukemic effectiveness of Cytoxan. BDF1, female mice. (Exp. 9).

I0 I0 I0 8 9 I0 I0 I0 8 9 I0 5 I

-~ 35 tilt t\ - ttlll tl--- 4 _ 03 ~ I I )- Ameth ~ Cytoxon I Ameth I Cytoxon It No \ No l No 11 No .... 30 - _ .. ~ Tumor ~>~.q Tumor- - I,, Tumor I Tumor- LIJ /4mern ~/ '/ L-,2IO ~/ I

m L~210 Cytoxon I ~ l-- Cytoxon I.- 25 J <~ > " " -1l r \ i ' r

15 Z

a Tumor.... Controls ._J L_ Tumor Controls 4 ~ I0 , Day of Treatment Initiation ., Day of Treatment Initiatio'tl I I I I I I I I I I I I I I I ~ I I I I I I I I I I I I L 5 0.?_.8 0.78 2.2 6.0 17 47 130 0.?..8 0.78 2.?.. 6.0 17 47 130 DAILY DOSE (Mg./kg.) CHART ~.--Comparison of Cytoxan and amethopterin with pared. The numbers above the vertical arrows indicate the respect tothelimitationonanti-leukemiceffectivenessimposed number of survivors out of ten mice in each normal (no by the cumulative toxicity on daily treatment. Drug effects tumor) group treated daily from day 7 through day 55. in normal and leukemic BDFI male and female mice are corn- (Exp. 10).

I 1 1 1 I I I I "1 I i I I' 35 _1 I i"1 I I I" 1 I ! I 1 I DALLY TREATMENT BEGINNING DAY 3 ,DAILY TREATMENT BEGINNING DAY 8 CYf• ~(on "-" 3O (f) TUMOR, L- 1210-M46R TUMOR, L- 1210-M46R

C y/oxon (3 v 25 bd ~E I- _I 20

m .> \ _ Amethoplenh 15 Amethopterin / -L.- oo / Z J t CONTROLS i - ,- CONTROLS I0 k_ Ld i ~E I I 1 I 1 I I 1 1 1 I 1 ] I ] I 1 I I I I 1 I I I 1 0.39 I.I 3.0 8.4 23 65 180 059 I.I 30 8.4 25 65 180 DALLY DOSE (Mg./k9.) CrIaHT 3.--Relative effectiveness of Cytoxan, amethopterin, with early or advanced amethopterin-resistant LI~ 10 (M46R). and 6-mercaptopurine in increasing the surviwll time of mice Ten CDBA male mice per group. (Exp. l 1).

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1959 American Association for Cancer Research. 992 Cancer Research Vol. 19, October, 1959 ing to tumor growth, and those showing recovery periments: (a) Following the achievement of a from the leukemic implant (13). Animals succumb- maximum median survival time with increasing ing to drug toxicity, in general, lost considerable dose on daily treatment (Chart 1) the median body weight, displayed no local tumors at the time survival time diminished precipitously with fur- of death, and died prior to untreated controls. All ther increase in dosage. (b) At the optimal dose on the mice receiving daily treatment succumbed, and daily treatment for mice with advanced leukemia, no clear separation of toxic deaths from tumor there was considerable evidence of weight loss. (c) deaths could be made. Single treatment appeared to be more effective A single treatment with Cytoxan administered than multiple treatment against early leukemia, early in the course of the disease was particularly suggesting that the effectiveness of multiple treat- effective (Table 3). Extensive median survival ment was limited by the cumulative toxicity of times (beyond 60 days) were obtained, accom- the drug (Table 3). panied by a relatively high percentage of total The results of an experiment designed to test recovery from the leukemic implant. Out of 60 the limitation of drug toxicity for the host are mice treated with Cytoxan, over a series of dosc shown in Chart 2. Comparison was made of the levels, there were eighteen 60-day survivors show- survival time of normal mice and mice with ad- ing no evidence of tumor. vanced leukemia L1210, employing daily treat- On single treatment Cytoxan was considerably ment with Cytoxan and with amethopterin. This more effective than amethopterin. The maximum was done in both male and female mice. The data median survival time achieved with a single treat- show that, in both the male and female mice, the ment with amethopterin was 21.0 days, and the toxicity of Cytoxan provided a greater limitation last mouse succumbed on the 28th day. on this schedule of treatment to extended therapy On daily treatment of early leukemia L1210 the than did the toxicity of amethopterin. At the daily maximum median survival time achieved with dose yielding the maximum increase in survival Cytoxan was 29 days, which was considerably less time in leukemic mice, Cytoxan toxicity for normal than that observed with amethopterin on the same mice was fairly extensive, although not as severe schedule (median survival time 89.0 days) (Table as had been observed with 5-fluorouracil (84). As 8). With daily treatment beginning early, neither had been observed in earlier studies (84), ameth- Cytoxan nor amethopterin produced any in- opterin, at its optimal daily dose in leukemic mice, stances of total recovery from the leukemic im- produced little indication of toxicity in normal plant. However, at doses effective in increasing mice. Thus, the data suggest that, although at survival time, both Cytoxan and amethopterin re- optimal dose levels on daily treatment the two tarded the growth of the local tumor at the site of compounds displayed nearly equal effectiveness in leukemic inoculation. prolonging the life of mice with advanced leu- Because of a previous report (19) that Cytoxan kemia, drug toxicity played a more prominent role behaves as a nicotinamide antagonist, an attempt in the death of animals receiving Cytoxan than in was made to block the antileukemic action of animals receiving amethopterin. This emphasizes Cytoxan with that vitamin, both on the single and the importance of investigations of means of re- multiple treatment schedules (Table 8). Doses of ducing the toxicity for the host of drugs such as ~50 mg/kg and 500 mg/kg of nicotinamide were Cytoxan. administered 15 minutes prior to Cytoxan. No evi- In view of the marked activity observed in the dence was obtained that nicotinamide could block treatment of early or advanced leukemia L1210 the antileukemic action of Cytoxan. Nicotinamide with Cytoxan, the studies were extended to an did not reverse the increase in survival time elicited amethopterin-resistant subline and an antipurine- by single or daily administration of Cytoxan, nor resistant subline of leukemia L1210. did it overcome the inhibition of local tumor growth A comparison was made of the relative effective- at the site of leukemic inoculation. No reduction ness of Cytoxan, amethopterin, and 6-mercapto- was observed in the number of "no takes" ob- purine in increasing the survival time of mice with tained on single-dose administration of Cytoxan. the M46R resistant variant of leukemia L1210 Nicotinamide also failed to diminish the toxicity when treatment was started at 3 days and also at of the alkylating agent for the host. 8 days following tumor inoculation (Chart 8). The It was considered desirable to obtain further M46R variant of L1210, in previous studies, had information on the extent to which the cumulative displayed marked resistance to amethopterin, and toxicity of Cytoxan may serve to limit the thera- considerable resistance to 8'5'-dichloroameth- peutic efficacy of the drug. The importance of the opterin (20). In the current experiment the median limitation of toxicity was indicated in several ex- survival time of the control mice was 11 days.

When treatment was started on the 3d day or on DISCUSSION the 8th day following leukemic inoculation, The current studies show that Cytoxan, a cyclic amethopterin was relatively ineffective in increas- derivative of nitrogen mustard, has pronounced ing the survival time of the mice. The most effec- antileukemic action against the sensitive line of tive daily dose employed 9 (3 mg/kg) increased the leukemia LI~10 in mice, and against an antifolic- median survival time by 3.5 days when treatment resistant and an antipurine-resistant variant of was begun on the 3d day and by ~ days when this leukemia. Against advanced stages of the treatment was begun on the 8th day. sensitive line of leukemia LI~10, Cytoxan was ap- 6-Mercaptopurine elicited a 6-day increase in proximately as effective as amethopterin, though survival time beyond that of the controls, when not as effective as the halogenated derivatives of the treatment was started early or late following amethopterin (11) when daily treatments were leukemic inoculation (Chart 3). This is in agree- given. Unlike the results with amethopterin (15) ment with previous observations with this anti- however, the effectiveness of Cytoxan against ad- folic-resistant subline of LI~10 (20). 6-Mercapto- vanced LI~10 increased as the interval between purine also gave similar survival times with the treatments was increased. Against an early stage sensitive LI~10 (18). of leukemia LI~10, it was not quite as effective as Cytoxan produced rather extensive increases in amethopterin on daily treatment but considerably survival time when treatment was started on more effective than amethopterin when ad- either the 3d or the 8th day after tumor inocula- ministered as a single dose. tion (Chart 3). The median survival time was in- The observation that, for the sensitive line of creased by 15 days when treatment was started leukemia L1~10, over a wide dose range, on early early, and by 18.5 days when treatment was begun treatment, Cytoxan was capable of total inhibition at 8 days following leukemic inoculation. The of growth of the leukemic implant indicates that optimal daily dose starting treatment late (65 the drug is capable of cytotoxic action on the tu- mg/kg) was higher than that observed when treat- mor cells. This is supported by the preliminary ment was initiated early (39 mg/kg). This, and observation that, in some cases, transplant of the lesser increase in survival time on early treat- spleen failed to transmit the disease following ment, may, as with the sensitive leukemia, reflect treatment of systemic leukemia with Cytoxan. 3 the earlier appearance of cumulative toxicity In contrast, single or multiple daily treatment of when treatment was initiated early. These in- early leukemia with amethopterin failed to pro- creases in survival time are similar to those ob- duce tumor-free survivors. However, in other ex- served with the sensitive LI~10. Thus, the periments (16) it was demonstrated that two amethopterin-resistant subline of LI~10 (M46R) treatments with high doses of amethopterin spaced was not resistant to Cytoxan. 4 days apart did yield a high percentage of "no Similarly, the 8-azaguanine-resistant subline of takes" in mice with early leukemia. leukemia LI~10 retained its sensitivity to Cytoxan The observation that Cytoxan retained its anti- (Table 4). The median survival time of the con- leukemic effectiveness against an amethopterin- trols was 12 days. 6-Mercaptopurine increased the resistant variant of leukemia L1210 and against an median survival time by only 1 day, giving an 8 8-azaguanine-resistant variant of LI~10 also re- per cent increase in survival time. Cytoxan, on the sistant to 6-mercaptopurine indicates that Cy- other hand, increased the median survival time by toxan may be useful in the exploration of a means 13.5 days, giving a 118 per cent increase in survival to overcome leukemic cell resistance to antifolics time. Amethopterin, as had been reported previ- and antipurines in experimental and clinical ously (33), was particularly effective against this leukemia. Failure of cross-resistance with these subline of LI~10 and in this experiment gave more variant tumors to Cytoxan also indicates that the than a 817 per cent increase in survival time. Thus, drug exerts its action at sites other than those in- with the 8-azaguanine-resistant variant of leu- hibited by metabolic antagonists in folic acid and kemia LI~10 there was no loss in antileukemic ef- purine metabolism. This would suggest that fectiveness for Cytoxan. Cytoxan be employed in studies of synergism in 2 Although definite maxima were not obtained, there was conjunction with folic acid antagonists and purine evidence of toxicity at the highest daily dose employed (3 antagonists in attempts to improve antitumor in- mg/kg), indicating that higher doses would have been less hibitory action and therapeutic effectiveness. effective in increasing survival time. In other experiments in The current demonstration of the use of studies which a clearly optimal daily dose of amethopterin was obtained for the treatment of early LI$10 (M46R), extensions in of cross-resistance to detect differences in the median survival time greater than 3-4 days over controls were s S. R. Humphreys, J. M. Venditti, and A. Goldin, unpub- not realized. lished data.

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1959 American Association for Cancer Research. 994 Cancer Research Vol. 19, October, 1959 mode of antileukemic action of drugs also re-empha- ment of normal and leukemic mice. Studies with sizes the potential usefulness of the employment early leukemia suggest that the action of this of resistant variants in cancer screening and alkylating agent may result in destruction of im- development programs (~5-31, 36, 37, 39). planted tumor cells. The anti-tumor effectiveness The previous demonstration in other tumor of Cytoxan was not diminished in LI~10 sublines systems that Cytoxan acts as a nicotinamide which were resistant to antifolic or antipurine antagonist could not be duplicated with leukemia treatment. The significance of these results rela- LI~10. However, it should be pointed out that, in tive to problems such as the toxicity of known the previous studies, a series of treatments with anti-tumor agents for the host and the origin of nicotinamide was given before and after a single resistance during therapy is discussed. dose of Cytoxan (19). The limitation of host toxicity in therapy with REFERENCES Cytoxan, as evidenced by the toxicity for non- 1. ARNOLD, H.; BOURSEAUX, F.; and BROCK, N. Neuartige leukemic mice of therapeutically active dosages, Krebs-Chemotherapeutika aus der Gruppe der zyklischen appears to be greater than that observed for N-Lost-Phosphamidester. Die Naturwissensehaften, 45: amethopterin. However, methods may be sought 64--66, 1958. ~. BERGEL, F. Design of Alkylating Agents for Selectivity of to reduce host toxicity with the retention of anti- Action. Ann. New York Acad. Sc., 68:1~38-45, 1958. tumor activity. If nicotinamide or other metabo- 3. BROCK, N. 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John M. Venditti, Stewart R. Humphreys and Abraham Goldin

Cancer Res 1959;19:986-995.

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