Studies on the Chemotherapy of Experimental Brain Tumors: Eval Uation of L,3-Bis(2-Chloroethyl)-L-Nitrosourea, Cyclophosphamide, Mithramycin, and Methotrexate

[CANCER RESEARCH 30, 2401-2413, September 1970]

Studies on the Chemotherapy of Experimental Brain Tumors: Eval uation of l,3-Bis(2-chloroethyl)-l-nitrosourea, Cyclophosphamide, Mithramycin, and Methotrexate

William R. Shapiro,1 James I. Ausman,2 and David P. Rail

Office of the Associate Scientific Director for Experimental Therapeutics, National Cancer Institute, NIH, Bethesda, Maryland 20014

SUMMARY BCNU is one of a series of nitrosoureas developed at Southern Research Institute following the observation that the A brain tumor model has been utilized to test 4 related nitroguanidines produced a weak but significant chemotherapeutic agents. The model consists of C57BL male prolongation in life-span of L1210-inoculated mice (13, 34). mice implanted intracerebrally with carcinogen-induced Among the nitrosoureas, BCNU is most efficacious in gliomas. Four tumors were used, all histologically ependymo- eliminating LI210 leukemia in such mice, even when blastomas, but each differing in its biological behavior. Four meningeal leukemia is induced or occurs spontaneously (34). agents were tested, each administered i.p. A comparison was BCNU is lipid soluble and readily enters the brain and made between treated animals and nontreated control animals cerebrospinal fluid (26). For these reasons, it was chosen as with respect to median survival time and long-term survival the first drug to be tested in the model. It is thought to act as and was statistically evaluated by a modification of the an alkylating agent, probably through an intermediary Wilcoxon rank-sum analysis. diazoalkane (27). l,3-Bis(2-chloroethyl)-l-nitrosourea (NSC 409962) signifi Cyclophosphamide (7V,./V-bis(j3-chloroethyl)-yv',O-propylene- cantly prolonged the life-span of mice bearing the intracerebral phosphoric acid ester diarnide) was first synthesized in 1958 gliomas. Increased life-span varied from 10 to 137%. by Arnold and Bourseaux (1). It is effective against both rat Cyclophosphamide (NSC 26271) less consistently increased tumors and murine leukemia (20) as well as in human survival of glioma-bearing mice. Neither mithramycin (NSC leukemia (43). The drug is given in an "inactive" form and 24559) nor methotrexate (NSC 740) on multiple dose must be metabolized in vivo to an "active" form by the liver. schedules was capable of increasing the life-spans of the The active form is water soluble and does not appear to enter animals. the brain (7). Cyclophosphamide has almost no activity against The implications of these data with respect to blood-brain i.c.-inoculated L1210 leukemia, but it is effective against a barrier, brain tumor permeability, and the value of the model s.c.-implanted experimental brain tumor (41). as a screen for human brain tumor chemotherapy are discussed. Mithramycin, an antibiotic derived from Streptomyces tanashiensis, has marked antibacterial activity in vitro and is INTRODUCTION cytotoxic in tissue culture (31). It shows only moderate antineoplastic activity against experimental tumors, but it has In a previous report (3), a method was described for the produced striking clinical remissions in embryonal cell establishment of an experimental brain tumor model with carcinoma of the testes (5). Mithramycin has been tested i.e.3-implanted fragments of carcinogen-induced brain tumors against a s.c.-implanted mouse glioma (19) and has been used into C57BL male mice. Four tumors were used with respective clinically in human brain tumors (18, 30). It was tested in our survival times of: ependymoblastoma (Zimmerman), 29 Â±3 model because it is currently under investigation by the Brain days (S.D.); Glioma 261 (Shear), 24 Â±2 days; Glioma 26 Tumor Therapy Study Group (23). It appears to act by (Sugiura), 24 Â± 3 days; and the mutant subline of inhibiting RNA synthesis (50). ependymoblastoma, Ependymoblastoma A, 19 Â±1day. Methotrexate was included in the current series as an example of a weakly ionized, lipid-insoluble chemotherapeutic The present study describes chemotherapeutic trials with agent which is cell-cycle active. It has been shown to occupy this model. Four drugs were tested: BCNU (NSC 409962), the extracellular space of the i.c.-implanted murine glioma Cyclophosphamide (NSC 26271), mithramycin (NSC 24559), (22). and methotrexate (NSC 740). 'Present address: Neuropsychiatrie Service, Memorial Sloan- Kettering Cancer Center, New York, N. Y. 10021. MATERIALS AND METHODS 2Present address: Department of Neurosurgery, University of Minnesota Hospitals, Minneapolis, Minn. 55455. TUÂ» i j F â€¢ â€¢ â€¢ i r r 'The abbreviations used are: i.e., intracerebral; BCNU, l,3-bis(2- . â„¢ me,thÂ°d fÂ°r,thf 1X' ""Plantation of tumor fragments chloroethylH-nitrosourea; MLS, median life-span; ILS, increase in nas been described (3). Three groups of animals were used for life-span; MNU, 1-methyl-l-nitrosourea. each experimental drug trial (Table 1). Group I consisted of 25 ReceivedJanuary 23,1970; accepted May 29,1970. tumor-bearing control mice that did not receive drug. Group II

SEPTEMBER 1970 2401

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1970 American Association for Cancer Research. W. R. Shapiro, J. I. Amman, and D. P. Rail consisted of 5 groups of 10 tumor-bearing animals per group. Drug toxicity was determined in advance on nontumor- The 5 groups received 5 different doses of drug. Group III bearing animals and the LD50, LD10, and respective 95% had 5 groups of 10 animals apiece, but without tumors; these confidence limits for each drug were calculated by the method animals received the same dose of drug as Group II and acted of Litchfield and Wilcoxon (24). as nontumor-bearing drug controls. All animals were weighed on the day of tumor implantation and on the days of drug injection. In selected experiments, Drug Trials daily to thrice weekly weights were followed throughout the course of the experiment. BCNU is relatively insoluble in water. The weighed powder The animals were observed following drug injection, and the was first dissolved in 95% ethanol, and 0.9% NaCl solution was day of death was recorded for each mouse. Animals surviving then added to yield the proper concentrations. The drug was longer than 60 days were followed for 6 to 18 months to made up to volume such that doses could be administered 0.01 observe for tumor recurrence. However, a few surviving mice ml/g of body weight. In 9 experiments, BCNU was given as a were killed by cervical dislocation and the brain was submitted single i.p. injection on Day 2 following tumor implantation. for histological examination. As noted in a previous report Two experiments were done in which BCNU was given as a (36), the incidence of "no-takes" among nontreated control single i.p. injection on Day 14 in ependymoblastoma animals. animals was: ependymoblastoma, 0.8%; Glioma 261, 0.9%; Ependymoblastoma A, the mutant subline of ependymo Glioma 26, none; and Ependymoblastoma A, 2%. blastoma, was also tested with BCNU in 1 experiment on Day Drugs were obtained from the Cancer Chemotherapy 2 only. National Service Center and were administered i.p. For BCNU, the LD5 0 of single i.p. injection was 44 mg/kg with 95% confidence limits of 40.8 to 74.4 mg/kg. The LD10 was 34 mg/kg; 95% confidence limits were 30.9 to 37.4 mg/kg. Table 1 These data were determined at 30 days because of the Design of each experiment propensity for BCNU to produce delayed toxicity (8, 21). About 90% of animals that died as a result of BCNU did so Group I: II: III: before Day 21, however. 25tumorNo 50tumorTreatment 50nontumorTreatment Cyclophosphamide was weighed and dissolved in 0.9% NaCl treatmentGroup at5 at5 solution. Warm tap water was allowed to bathe the bottle in dose levelsGroup dose levels order to facilitate dissolution. The drug concentration was

Table 2 Effect of BCNU against i.e. ependymoblastoma, i.p., Day 2 only, 1 dose/day

Dose day of Wilcoxon (mg/kg)Experiment20 death(T/CÂ°)40.5/3135/2637/3338.5/2641/3337.5/3150.5/2640/2845/3311.5/3117/2615/2746/338/3110/2612/288/338/318/27Significance,testp (% control)131135112148124121194143136376556139263843242630Meanlife-span/dose126136149743328Survivors>60days1/100/101/100/101/101/103/100/101/101/101/102/103/100/100/102/100/100/100/10

12525 <0.001p <0.0001p <0.001p

2530 <0.0001p <0.00001p

123S40 <0.05p <0.0001p <0.0001p <0.00001LethalN.

124550 S.Lethalp

<0.0001LethalLethalLethalLethalLethalLethalMLS

123S60

14Median

a The abbreviations used in the tables are: T/C (T =median day of death of treated animals, C = median day of death of control animals); N. S. not significant by Wilcoxon analysis. See text for details.

2402 CANCER RESEARCH VOL. 30

Table 3 made up to yield doses at 0.01 ml/g of body weight. The drug Effect of BCNUagainst i.e. ependymoblastoma, i.p., displayed greater toxicity in tumor-bearing animals than in Day 14 only, 1 dose/day normals. In normal animals, the LD50 was 370 mg/kg (95% confidence limits, 330 to 414 mg/kg) and the LD10 was 270 Median day of Significance, mg/kg (95% confidence limits, 241 to 302 mg/kg) at 21 days. Dose Experi- death Wilcoxon MLS Survivors In the tumor-bearing mice, the LD50 was 264 mg/kg (95% (mg/kg) ment (T/O test (% control; >60 days confidence limits, 247 to 282 mg/kg) and the LD10 was 210 mg/kg (95% confidence limits, 191 to 231 mg/kg) at 21 days. 201241/31 < 0.001 Differences in drug toxicity between tumor- and nontumor- 41/28p p < 0.001132 1461/101/10 bearing animals are not uncommon, although their quantita 25 48/28 p < 0.01 171 1/10 tive determination is often difficult because of early tumor-induced deaths (40). Skipper and Schmidt (40) 3040SO60121 < 0.05 considered such differences of little importance in evaluating 33.5/2858/31Bimodal0p 120187129824/104/10 drug efficacy, and such would seem to be the case in systems < 0.001 of rapidly growing tumors like LI210. In our model, tumor 21 36/2825.5/31BimodalLethal 1/102/10 deaths rarely occurred before Day 15, making possible evaluation of drug toxicity in both tumor- and nontumor-

210 20.5/2821/31includesLethalLethaland 7368Wilcoxon1/100/100/10analysisbearing animals. Mithramycin was supplied in stoppered vials containing 2.5 mg/vial. Sterile water was added to yield dose levels at 0.01 ml/g of body weight. The drug was administered as a single Distribution52/31 earlyp late deaths.168 not injection i.p. on Day 2 following tumor inoculation in 4 possible. See text for details. experiments, and as daily injections i.p. on Days 2 to 8 in 6 experiments. The single dose LD50 was 2.9 mg/kg, and the LD10 1.35 mg/kg. In the multiple dose experiments, the LDSO was 1.4 mg/kg/day and the LDi 0 was 0.9 mg/kg/day for 5 Table 4 daily doses. Confidence limits of 95% were not obtained and Effect of BCNU against i.e. Ependymoblastoma A, i.p., Day 2 only, 1 dose/day no difference in lethalities between tumor and nontumor animals was seen. Methotrexate was weighed and diluted with 2% sodium ofdeath(T/O19/1733/1738/1736.5/178/17Significance,WilcoxontestN.S.pbicarbonate in sterile water to yield dose levels at 0.01 ml/g of Dose(mg/kg)2025304050Experi body weight. The LD50 was 86 mg/kg/dose (95% confidence ment11111Medianday control)11219422421547Survivors>60days0/101/101/102/100/10 limits, 73 to 101 mg/kg/dose), and the LD10 was 29 mg/kg/dose (95% confidence limits, 25 to 34 mg/kg/dose) for <0.00001p 4 injections, given once each day every 4 days. Since the last <0.00001p dose was given on Day 14 after tumor implantation, toxicity <0.05LethalMLS(% in tumor-bearing animals could not be determined. In order to span the toxicity range from LD0 to LD, 00, the experiments

Table 5 Effect of BCNU against i.e. Glioma 261, i.p., Day 2 only, 1 doseÂ¡day

Dose(mg/kg)2025304050Experiment1 day of Wilcoxon death(T/Q34.5/26 testp (% control)133 life-span/dose14318115910434Survivors>60days3/10

< 0.01 21 41/2732.5/26 p <0.0001p 152125 3/101/10

< 0.01 21 64/2721.5/26 p <0.001Bimodal" 2378323538 5/102/10

21 63.5/2710/26 p <0.0001N.S. 5/101/10

21 45.5/2710/26 BimodalLethal 16938 4/100/10

2Median 8/27Significance, LethalMLS 30Mean 0/10 1Distribution includes early and late deaths. Wilcoxon analysis not possible. See text for details.

SEPTEMBER 1970 2403

Table 6 Effect of BCNUagainst i.e. Glioma 26, i.p. Day 2 only, 1 dose/day

Dose(mg/kg)2025304050Experiment1 day of Wilcoxon death(T/Q22/2029/2525/20testp (% control)110 life-span/dose11312713211940Survivors>60days1/10

< 0.05 21 p <0.01p 116125 0/100/10

< 0.001 21 32/2527/20 p <0.0001p 128135 0/100/10

< 0.001 21 32/2530/20 p <0.01p 128150 1/100/10

< 0.01 21 22/257/20 Bimodal0Lethal 8835 0/100/10

2Median 11/25Significance, LethalMLS 44Mean 0/10 0 Distribution includes early and late deaths. Wilcoxon analysis not possible. See text for details.

Table 7 Effect of cyclophosphamide against i.e. ependymoblastoma, Lp., Day 2 only, 1 dose/day

Dose day of Wilcoxon (mg/kg)150200250350600Experiment1 death(T/O29.5/29 testN.S. (% control)102 life-span/dose116111713310Survivors>60days0/10

2 33.5/31 N.S. 108137124 0/10 31 42.5/3136/29 p <0.0001p 0/100/10

< 0.01 2 30/31 N.S. 97 0/10 31 35/3118.5/29 Bimodal"Bimodal 11364115 0/100/10

2 35.5/31 p < 0.05 0/10 31 10.5/317/29 LethalLethal 3424 0/100/10

2 11.5/31 Lethal 373910 0/10 31 12/313/29 LethalLethal 0/100/10

2 3/31 Lethal 10 0/10 3Median 3/31Significance, LethalMLS 10Mean 0/10 "Distribution includes early and late deaths. Wilcoxon analysis not possible. See text for details. were divided into a "low range" of doses including 4, 8, 16, to the median day of survival of the tumor-bearing, nontreated 32, and 64 mg/kg/day every 4 days for 4 doses, and a "high control groups (C) and the % MLS of the treated animals was range" including 16, 32, 64, 128, and 256 mg/kg/day every 4 determined according to the formulation: days for 4 doses. As noted in the previous report, the mutation of ependymoblastoma prevented testing of methotrexate MLS = (T/C) X Ino (.A) against this tumor; Ependymoblastoma A, the mutant subline, was used instead MLS values for each dose in the various experiments were averaged to yield the mean life span/dose. An MLS exceeding 100% implied that the drug induced an increase in life-span; Analysis of Data MLS values less than 100% were usually related to drug lethality. The results were analyzed by 3 methods. In the first, the A 2nd method of analysis had the purpose of determining median day of survival of the treated groups (T) was compared whether the treated animals survived significantly longer than

2404 CANCER RESEARCH VOL. 30

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1970 American Association for Cancer Research. Brain Tumor Chemotherapy: Drug Studies the untreated controls (see "Discussion"). This involved a increase. The ranks of the treated animals were then summed modification of the rank-sum test of Wilcoxon (10, 48) in and set equal to T in the following formula which the days of death of each of the treated animals was , .. ranked along with those of the nontreated animals. The null T â€”M -â€” ^â€”=â€” identicalhypothesis populations. was then tested that the 2 groups were from 2 ' /NM (M +_ N + 0 ,r>\'"

The test was performed in the following manner. The V 12 individual day of death for each nontreated, tumor-bearing control animal was listed along with the day of death of each where Z = statistic, M = no. of animals in the treated group; of the treated animals for 1 dose. A new list was then TV= no. of animals in the control group. The statistic Z was compiled assigning the value 1 for the first day listed and then compared in a significance table and the p value was deter- ranking the subsequent days of death serially by integer mined. By appropriate computation, surviving animals were

Table 8 Effect of cyclophosphamide against i.e. Glioma 261, i.p., Day 2 only, 1 dose/day

Dose day of Wilcoxon (mg/kg)150200250350600Experiment1 death(T/Q29.5/24 testp (%control)123143.5150life-span/dose133149642515Survivors>60days0/10

< 0.05 21 33/2336/24 p <0.001p 2/101/103/100/10

< 0.0001 21 34/239.5/24 p <0.0001LethalN.S.Lethal 14840

21 20/234/24 87173317 0/100/10

212Median 7.5/234/24 LethalLethal 0/100/10

3/23Significance, LethalMLS 13Mean 0/10

Table 9 Effect of cyclophosphamide against i.e. Glioma 26, i.p., Day 2 only, 1 dose/day

Dose(mg/kg)150200250350600Experiment1 day of Wilcoxon death(T/Q31/25 testp (%control)124 life-span/dose1231311053011Survivors>60days0/10

< 0.001 2 33.5/26 p < 0.001 129 0/10 31231 34/2934.5/25 p <0.01p 117138 1/100/10 < 0.01 35/26 p < 0.01 135121128 1/10 35/2932/25 p <0.01Bimodal0 0/100/10

2 24/26 Bimodal 92 0/10 31 27.5/299/25 BimodalLethal 9536 0/100/10

2 7.5/26 Lethal 29 0/10 3123Median 7.5/293/25 LethalLethal 2612 0/100/10

3/26 Lethal 12 0/10 3/29Significance, LethalMLS 10Mean 0/10 0 Distribution includes early and late deaths. Wilcoxon analysis not possible. See text for details.

SEPTEMBER 1970 2405

Table 10 Effect of mithramycin against i.e. ependymoblastoma, i.p., Day 3 to 8 only, 1 dose/day

Dose(mg/kg)0.250.50.751.02.0Experiment1 day of Wilcoxon death(T/O26.5/26.5 testN.S.N.S.N.S. (% control)100 life-span/dose104.595.5108.59624.3Survivors>60days0/10

21 29.5/2723/26.5 10987 0/100/10

21 28/2726.5/26.5 N.S.N.S. 10410011794 0/100/10

21 31.5/2725/26.5 p <0.001N.S. 0/100/10

21 26.5/276/26.5 N.S.Lethal 9822.6 0/100/10

2Median 7/27Significance, LethalMLS 26Mean 0/10

Table 11 Effect of mithramycin against Glioma 261, i.p., Day 3 to 8 only, 1 dose/day

Dose day of Wilcoxon (mg/kg)0.250.50.751.02.0Experiment1 death(T/O24.5/2427.5/2524/24testN.S. (% control)102 life-span/dose1061011009327.5Survivors>60days0/10

21 N.S.N.S. 110100 3/101/10

21 25.5/2522.5/24 N.S.N.S. 10294 0/100/10

21 26.5/2521.5/24 N.S.N.S. 10690 1/100/10

21 24/258/24 N.S.Lethal 963322Mean 0/100/10

2Median 5.5/25Significance, LethalMLS 0/10 included as censored data and the Z value was calculated. ependymoblastoma. The drug produced significant prolonged Where a drug dose exceeded the LD5 0, statistically significant survival at 3 doses below the LD] 0 (20, 25, and 30 mg/kg). At reduction in survival time occurred. Such doses were desig 40 mg/kg, most of the experiments were bimodal with early nated as "lethal." The test was reduced to a computer program drug deaths and prolonged life-span of the drug survivors. for ease of analysis. There was considerable variation among the MLS values, The 3rd method of analysis consisted of tabulating the although in general increased dosage led to increased survival long-term survivors, i.e., animals that survived longer than 60 time. Long-term survival rates ranged from 10 to 30%. days. The value of 60 days was chosen arbitrarily on the basis An attempt was made to determine whether BCNU acted by that over 99% of nontreated tumor animals died of their preventing tumor take only or if the drug actually impeded neoplasms by that time and those that did not were considered tumor growth. In 2 experiments, administration of BCNU was no-takes. Thus, the chance that a long-term-surviving treated delayed until 14 days after tumor inoculation. The results are animal did not have a tumor was less than 1 in 100. shown in Table 3. Only 2 doses below the LD10 were used in experiment 1, but in this experiment 40 mg/kg produced only RESULTS 1 toxic death. The results are comparable to those obtained at Day 2 treatment, indicating that BCNU had an effect beyond BCNU that of preventing tumor take. Table 4 shows the results of BCNU against Ependymo Ependymoblastoma. Table 2 shows the results of BCNU blastoma A, the mutant subline of ependymoblastoma. Within administered as a single injection on Day 2 against the degree of variation, a similar efficacy was demonstrated.

2406 CANCER RESEARCH VOL. 30

Glioma 261. Table 5 depicts the results of BCNU against Glioma 261 appeared to be more sensitive to BCNU than Glioma 261. At 3 dose levels below the LD10, the drug either ependymoblastoma or Glioma 26. consistently produced an increase in life-span. MLS values in Glioma 26. Significant increased survival time was induced Experiment 2 were quite high and, at the lower 3 dosages, the by BCNU in mice bearing i.e. Glioma 26 (Table 6), although number of long-term survivors exceeded 20% in all but 1 case. MLS values were consistently small. The number of long-term

Table 12 Effect of mithramycin against Glioma 26, i.p., Day 3 to 8 only, 1 dose/day

Dose(mg/kg)0.250.50.751.02.0Experiment121 day of Wilcoxon death(T/Q27.5/2725.5/2527/27testN.S. (% control)102 life-span/dose10299999024Survivors>60days1/10

N.S.N.S. 102100 1/100/10

21 24.5/2526/27 N.S.N.S. 989610298 1/100/10

21 25.5/2526.5/27 N.S.N.S. 1/100/10

212Median 20.5/256/27 N.S.Lethal 822226Mean 1/100/10

6.5/25Significance, LethalMLS 0/10

Table 13 Effect of MTX against Ependymoblastoma A, i.p., every 4 days for 4 doses

Dose day of Wilcoxon Survivors (mg/kg)Experiment4 death(T/Q18/1919/1919/2118.5/1920.5/1919.5/1919/2118/1921/1920/1920/2118/1918.5/1822/1919/1919/2116.5/1919/1824/1914/1912/2110/1913.5/1811/187/18Significance,testN.S.N.S.N.S.N.S.N.S.N.S.N.S.N.S.p(%control)9510090.59710810390.595110105959510311610090.587106126745753756139Meanlife-span/dosedays0/1095.6 >60

12348 2/100/100/102/1099

123416 2/100/100/103/101/10102

1234532 <0.05N.S.N.S.N.S.N.S.p

0/101/100/101/104/10100

1234564 <0.05N.S.N.S.N.S.N.S.p

0/103/100/103/100/1077

12345128 <0.01LethalLethalLethalLethalLethalLethalMLS

0/100/100/100/100/10

5256

5Median

SEPTEMBER 1970 2407

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1970 American Association for Cancer Research. W. R. Shapiro, J. I. Amman, and D. P. Rail survivors was also low. Glioma 26 appeared to be less sensitive On the multiple injection schedule, 6 experiments, 2 each to BCNU than both ependymoblastoma and Glioma 261. with ependymoblastoma, Glioma 261, and Glioma 26, were done and are tabulated in Tables 10, 11, and 12. With one exception (ependymoblastoma, Table 10, Experiment 2, 0.75 Cyclophosphamide mg/kg/day), no significant increase in survival time occurred. However, there were a few long-term survivors in both Glioma Ependymoblastoma was inconsistently affected by Cyclo 261 and Glioma 26 (Tables 11 and 12). phosphamide administered as a single injection on Day 2 following tumor inoculation (Table 7). However, cyclophosphamide significantly prolonged the lives of mice bearing Methotrexate Glioma 261 (Table 8) and Glioma 26 (Table 9) at 2 dose levels below the tumor-bearing LD10 (210 mg/kg). The maximum Experiments with methotrexate were divided into low-range MLS values were seen in Glioma 261, as were the largest and high-range dosage schedules of 1 injection/day every 4 number of long-term survivors. One experiment in which days, times 4 injections (see "Materials and Methods"). The cyclophosphamide was used against Glioma 261 produced results are shown in Tables 13, 14, and 15. excessive lethality in the drug control animals and was Table 13 shows the results of methotrexate against discarded. Ependymoblastoma A, the mutant subline of ependymo blastoma. Experiment 1 had 3 significant points of increased survival time, only 2 of which were at or below the LDt 0 (29 Mithramycin mg/kg/dose). In none of the other experiments was a significant increase in survival shown. However, a number of Mithramycin was used in 2 schedules, 1 injection on Day 2 long-term survivors occurred at several dose levels. only, and daily injections for 5 days from Day 3 to 8. On the With Glioma 261, Experiment 1 showed 2 high MLS values, single injection schedule, doses used included 0.5, 0.75, 1.0, 119 and 123% at, respectively, 16 and 32 mg/kg/dose. These 1.5, and 4.5 mg/kg. Two experiments against ependymo were not significant by Wilcoxon analysis. A number of blastoma and 1 each against Glioma 261 and Glioma 26 were long-term survivors was also recorded. done on the single injection schedule. No difference in survival No significant increase in survival occurred with metho between control animals and treated animals was seen in any trexate against Glioma 26 (Table 15), although there were of the experiments, and there were no long-term survivors. several long-term survivors.

Table 14 Effect ofMTX against Glioma 261, i.p., every 4 days for 4 doses

Dose day of Wilcoxon (mg/kg)Experiment4 death(T/Q26.5/2621/2529.0/2625.5/2531/2623.5/2525.5/2724/2432/2624/2527/2728/2926/2621.5/2533/2711.5/299/2710/297/279/29Significance,testN.S.N.S.N.S.N.S.N.S.N.S.N.S.N.S.N.S.N.S.N.S.N.S.N.S.N.S.N.S.LethalLethalLethalLethalLethalMLS(% control)102841121021199494831239610097100861224033352631Meanlife-span/dose9310797.5104873428.5Survivors>60days1/101/101/102/104/100/100/102/102/102/100/102/100/100/101/100/100/100/100/100/10

128

1216

123432

123464

1234128

34256

34Median

2408 CANCER RESEARCH VOL. 30

Table 15 Effect ofMTX against Glioma 26, i.p., every 4 days for 4 doses

Dose(mg/kg) day of Wilcoxon Experiment4 death(7/O23.5/2724/2626.5/2722/2622.5/2721/2624.5/2726/2528/2726.5/2626/2726.5/2519.5/2721.5/2623.5/2721.5/2519/2710.5/2514/2710/25Significance,testN.S.N.S.N.S.N.S.N.S.N.S.N.S.N.S.N.S.N.S.N.S.N.S.LethalN.S.N.S.N.S.LethalLethalLethalLethalMLS(% control)8793988583819110410410296.51067283878670425240Meanlife-span/dose9091.590102825646Survivors>60days1/101/101/100/100/102/102/100/102/102/103/100/101/100/100/100/100/100/100/100/10

128

1216

123432

123464

1234128

34256

34Median

DISCUSSION contribution of each animal to the statistic. It was thus possible to determine statistically whether treatment was indeed better than no treatment. Method of Analysis The test unfortunately did not allow us to determine "how Three methods were used to analyze the results. much better" treatment was over no treatment. Furthermore, Comparison of the median survival times of treated and when 20 to 30% of a treated group died early, presumably control groups yielded ILS values which varied over wide because of drug intoxicity, and the remainder outlived the ranges among several experiments. A change in 1 or 2 days in control group, the Wilcoxon test could not be utilized, for the median day of death could alter the ILS by as much as 10 under these conditions the treated group was bimodal and to 30%. Under such circumstances, the mean "life-span per could not be compared with the control group. dose" was at best â€¢onlyan approximation. Construction of Despite these limitations, the Wilcoxon analysis did allow a dose-response curves was not possible, nor was statistical test of significance, and the ILS approximations could be used calculation of significant differences. as a rough dose-responsive curve. Thus, it was the Wilcoxon With the help of Dr. John Slivka (Mathematical Statistics test which dictated when a drug was declared useful by this and Applied Mathematics Section, Biometry Branch, National model and it is emphasized that the value "mean life-span per Cancer Institute) a method of analysis was adopted utilizing dose" is included principally as a guide. the rank-sum test of significance first described by Wilcoxon Finally, the long-term survivors also represent a measure of (48) and later modified by Gehan (10). The details of the drug response, although permitting only rough comparisons. analysis are described under "Materials and Methods," but Nevertheless, such a parameter can be very useful in assessing basically the test consisted of serially ranking the days of the long-term effectiveness of an agent. death of the animals and comparing the sums of the ranks of BCNU. Of the 4 agents used in this study, only BCNU the control animals with that of the treated animals under the showed a consistent inhibiting effect on growth of the i.e. null hypothesis that the 2 groups of mice were from different tumors. For each tumor, BCNU-treated animals survived populations. By this method, the variation imposed by the raw significantly longer than nontreated animals. In general, as the data is nullified by preserving the order of day of death but dose of BCNU was increased from 20 through 25 to 30 mg/kg, eliminating the variable time among the days of death. the length of survival increased, although no specific dose- Furthermore, unlike the ILS method, which deals with the response relationship could be obtained because of the high median day of death, the Wilcoxon analysis preserves the degree of variability.

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The effect of BCNU on each of the 3 tumors was similar, grown i.m. in the hind limb of a mouse and measured the although the best results were obtained against Glioma 261, change in cross-sectional area of the tumor after a specified the worst against Glioma 26; ependymoblastoma was time interval following treatment. Cyclophosphamide was intermediate. We cannot yet explain the difference in results highly effective; the tumor disappeared on the 4th day after among these 3 tumors. Historically, no consistent treatment but recurred about 1 week later. differential feature has been found. Although our results Mithramycin. Mithramycin failed to prolong significantly suggest different biological behavior patterns both in response the lives of mice bearing any of the 3 tumors, although a to BCNU and in mean survival time of nontreated, few long-term survivors occurred with Glioma 261 and Glioma tumor-bearing animals (see above), only kinetic data could test 26 (Tables 11 and 12). The 2 schedules used, i.e., single dose whether such a difference exists. on Day 2, and 5 daily doses starting on Day 3 following tumor Usually, BCNU was given on Day 2 following tumor implantation, spanned the toxicity range for the drug. inoculation. In 2 experiments (Table 3), drug was given on Although the 5-day schedule was chosen by reference to the Day 14 only. Essentially similar results were obtained as with literature as the most effective for this drug, it is possible that the early treatment. This would be expected since, as Skipper a different schedule could have been more effective in the has pointed out (38, 39), a noncycle-active agent kills a present system. constant percentage of the cells for a given dose and not a Kennedy et al. (19) evaluated the effect of mithramycin on constant number of cells. Thus a given dose of drug should Glioma 26 implanted s.c. in mice. Utilizing a variety of yield the same mortality time no matter when the drug was schedules, they demonstrated inhibition of tumor growth; the given, as long as it was given early enough to cause its effect tumor volumes ranged from 32% of controls in animals treated before the animal died. For this reason, all subsequent single 3 times weekly to 42% of controls in animals treated on 4 dose experiments utilized Day 2 drug injection. consecutive days at 3-week intervals. Their schedules differed The inhibitory effect of BCNU on murine glioma has been from ours; treatment was continued in some cases beyond the demonstrated previously by Sugiura (42). He found that 8.0 point when 50% of the treatment animals were dead. In their mg/kg/day for 7 days inhibited the s.c. tumor by 96% (average studies, the LDSO in C57 mice for a single injection of diameters, tumor index = 0.04) over nontreated controls at 2 mithramycin was 6.0 mg/kg while in our studies it was 2.0 weeks after implantation, although the treated animals lost 3.5 mg/kg. On 5 equal daily doses in C3H mice, the LDSO was 1.7 g in the same period while the controls gained 1.5 g of weight. mg/kg/day. In our studies utilizing C57 mice, the LDS 0 for 5 Lower doses of BCNU were less effective. Similar results were daily doses was 1.4 mg/kg/day. Since we evaluated survival obtained against other solid tumors. time, doses which yielded lethal effects greater than LD10 Rosso et al. (33) found that MNU and BCNU significantly were considered nonevaluable. prolonged the survival of mice inoculated i.e. with various Despite the differences in schedules and toxicity between titers of Sarcoma 180 cells, although MNU did not inhibit s.c. our studies and those reported by Kennedy et al., the latter or i.p. Sarcoma 180. Sugiura (42) found that both BCNU and group demonstrated significant inhibition of brain tumor MNU inhibited sarcoma inoculated s.c. as a solid fragment. growth when the tumor was implanted s.c. In our studies, the Similar results were obtained by Rosso et al (33) when they drug either failed to inhibit growth or did so to too small a tested MNU against Ehrlich carcinoma inoculated i.e., i.p., and degree to affect survival time. DeWys et al. (9) have shown a s.c. The drug prolonged the life-span of animals with the i.e. poor correlation between tumor growth inhibition and survival tumor but did not inhibit the s.c. or i.p. growth. In contrast, time in the Walker 256 tumor system. Such investigations raise Sugiura (42) found that MNU and BCNU had a pronounced important questions regarding the screening value of s.c. solid inhibitory effect on the s.c. Ehrlich carcinoma. tumor models when tumor size at a fixed point in time is the Cyclophosphamide. On the single dose schedule used, end point. cyclophosphamide significantly increased the survival time of Methotrexate. On the schedules used, methotrexate failed mice bearing all 3 tumors although it did so least consistently to increase overall survival time of mice bearing Glioma 26, with ependymoblastoma. As in the BCNU trials, Glioma 261 Glioma 261, or Ependymoblastoma A, although a small was most sensitive to the agent, but Glioma 26 was percentage of long-term survivors was scattered throughout the intermediate with cyclophosphamide, differing from the experiments. The choice of an every-4-day regimen was based results with the nitrosourea. ILS values overall were lower with on the results with LI210 reported elsewhere (12), and other cyclophosphamide than they were with BCNU. schedules possibly might have been effective. Rosso et al. (33) used cyclophosphamide to treat mice Methotrexate is efficacious against L1210 leukemia except bearing Sarcoma 180 and Ehrlich carcinoma. The drug failed that, when the tumor invades the brain, methotrexate appears to increase survival time of animals implanted i.e. with either to be completely ineffective (37, 47) (see below). Its apparent tumor, although significantly increased life-span was obtained inability to reach high enough concentrations in the central in animals with i.p.-implanted tumors and significant nervous system accounts for its ineffectiveness in this location. inhibition occurred in s.c. tumors. Conclusions regarding the In human meningeal leukemia, intrathecal methotrexate and its negative results in the i.e. tumor system must be tempered by congener aminopterin yield prompt reduction in leukemic cells the fact that only a single dose level of cyclophosphamide was in the cerebrospinal fluid (15, 32), although frequently not used and higher levels might have been effective. eliminating them entirely from the subarachnoid space (45). Prior to the present study, the only reported attempt to Rosso et al. (33) failed to increase survival of mice bearing treat experimental gliomas with cyclophosphamide was that of i.e. Sarcoma 180 with methotrexate therapy. Soloway et al. (41). They utilized an ependymoblastoma Wilson et al. (49) stereotactically placed cultured human

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1970 American Association for Cancer Research. Brain Tumor Chemotherapy: Drug Studies oligodendroglioma cells and carcinogen-induced mammary the analogs of MNU which were non-ionized in aqueous media carcinoma cells in the thalamus of rat brains and compared at pH 7 and which had a high butyl acetate :NaCl solution intracisternally with i.p.-administered methotrexate. Animals (lipid:water) solubility had a significant effect on i.e. L1210 with oligodendroglioma treated by each route had survivals leukemia. Finally, systemically administered BCNU in significantly greater than controls; the intracisternally treated preliminary trial has shown effectiveness against human rats survived a little longer than those treated i.p. The animals meningeal leukemia (16). with i.e. mammary carcinoma showed no difference in Although such studies appear to affirm the existence of a survival. The results did not include early animal deaths blood-brain barrier in meningeal leukemia, there is evidence that resulting from the intracisternal treatment procedure, and such a barrier may not be present within a solid malignant inclusion of such data would have reduced the mean survival brain tumor. Electron microscopic studies have shown time of this treated group. numerous differences between normal brain and brain tumors (reviewed in Ref. 35). The endothelium in normal brain is single layered with no pores and only occasional pinocytotic Brain Tumor Permeability and Chemotherapy vesicles, while in glioblastoma tissue, endothelial cells are often proliferated, pores may be present, and pinocytotic vesicles A problem unique to the therapist who would treat brain occur in abundance. Pores are also seen in the basement tumors with anticancer agents is the location of the tumor. membrane of glioblastomas. The neuropile of normal brain is Many molecular species do not easily enter the normal brain tightly packed with a moderately sized interstitial space. The from the blood because of restricted permeabilityâ€”the parenchyma of a glioblastoma consists of loosely organized phenomenon called the blood-brain barrier. Does a similar tumor cells in a large interstitial space. Although much "barrier" also prevent the entry of chemotherapeutic agents variation may occur depending on from where specimens are into brain tumors? taken within the tumor, such alterations may be the Leukemic cells in both experimental (47) and human (14) morphological substrate for less restricted permeability than leukemic meningitis are isolated from many systemically occurs in normal brain. administered chemotherapeutic agents. Thomas et al. (47) Physiological data also tend to suggest free permeability demonstrated the methotrexate reduced the number of LI 210 into malignant brain tumors. Numerous studies have been cells in the blood and spleen of the animal but had no effect done since the work of Broman (4) showing trypan blue on the number of cells in brain tissue. Similarly, Skipper et al. staining of tumors, including the studies of Moore (28) with (37) found that methotrexate therapy did not increase the fluorescein and radioactive fluorescein. Radioiodinated life-span of animals with LI 210 cells in the central nervous albumin, chlormerodrin-203Hg and many nuclides following system. These investigators and Chirigos et al. (7) reported systemic administration exhibit tumor to brain ratios that cyclophosphamide also failed to increase the life-span of exceeding 1, indicating ready access of the agents into the animals with central nervous system L1210, although the drug tumor (35). Such studies, of course, failed to localize the readily eliminated leukemic cells from blood and spleen. marker within the tumor, but the data of Jackson et al. (17), Thomas et al. (46) studied the pathology of LI210 leukemia Long et al. (25), Tator et al. (44), and others utilizing in animals following systemic cyclophosphamide therapy. autoradiography showed markers in the extravascular space of There was complete destruction of leukemic cells in the liver, brain tumors implying that there was no vascular barrier to spleen, bone marrow, and dura but a persistence of the LI 210 such markers. cells in the subarachnoid space. A combination of Quantitative characterization of molecular entry into methotrexate and cyclophosphamide was reported to elimi experimental brain tumors was achieved by Ausman and Levin nate LI210 cells in the blood and reduce their number in the (2) and Levin et al. (22) in our laboratory. These investigators spleen, but failed to decrease the number of leukemic cells in evaluated the steady-state distribution spaces of inulin and the brain (6). methotrexate in nephrectomized mice bearing the i.e. Such data appeared to confirm the observation of Rail and ependymoblastoma. The inulin space in the tumor was about Zubrod (29) that drug solubility and ionization constant are 27%, while in distant brain the value was 2.5%. Methotrexate important properties with respect to the permeability of the had a space of 28% in the tumor and 3% in distant brain. brain to pharmacological agents. These investigators demon Simultaneous vascular spaces were less than 1% in both areas. strated that water-soluble, ionized molecules could not easily Important controls in such experiments included the purity of enter the brain, whereas lipid-soluble, non-ionized species the markers, the absence of their metabolism, and the absence readily crossed the blood-brain barrier. That this principle of dissociation of the radioactive isotope from the marker in applied also to chemotherapeutic agents was shown by Skipper vivo. It is especially important that continuous administration et al. (37) and Schabel et al. (34), who found that, in was carried out, thus reducing the problem of reentry of contradistinction to the water-soluble, ionized agent metho administered markers into the vascular system. Other studies, trexate, the lipid-soluble, non-ionized nitrosoureas were highly although showing similar qualitative results, have often failed effective against experimental leukemia when administered to take such controls into consideration. systemically. Skipper et al. (37) demonstrated that MNU given Thus, in meningeal leukemia, the parenchyma of the brain is i.p. increased the life-span of mice with i.e. L1210 cell probably little disturbed and the blood-brain barrier is inoculations. Schabel et al. (34) studied several analogs of preserved. When a solid tumor invades the brain, however, methylnitrosourea and found that BCNU increased the marked changes in the vasculature and parenchyma take place, life-span of mice with systemic and/or i.e. leukemia. Moreover, resulting in far less restriction of permeability within the tumor

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than in normal brain. This would appear to explain the REFERENCES effectiveness of cyclophosphamide in the studies reported here compared to its ineffectiveness against i.e. leukemia. Other 1. Arnold, H., and Bourseaux, F. Synthese und Abbau cytostatisch factors, especially the sensitivity of the tumor itself and the Wirksoner cyclischer A'-Phosphamidester des Bis-((3-cho- relative local concentrations, must be evaluated to account for rathyl)-amins. Angew. Chem., 70: 539-544, 1958. 2. Ausman, J. I., and Levin, V. A. Intra- and Extravascular the poor results with methotrexate and mithramycin. Distribution of Standard Drug Molecules in Brain Tumor and That lipid solubility may still be an important property of Brain. In: C. G. Drake and R. Duvoisin (eds.), Fourth International chemotherapeutic agents useful in brain tumors is shown by Congress of Neurological Surgery, p. 41. New York: Excerpta the highly efficacious results with BCNU. 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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1970 American Association for Cancer Research. Studies on the Chemotherapy of Experimental Brain Tumors: Evaluation of 1,3-Bis(2-chloroethyl)-1-nitrosourea, Cyclophosphamide, Mithramycin, and Methotrexate

William R. Shapiro, James I. Ausman and David P. Rall

Cancer Res 1970;30:2401-2413.

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