Studies on the Chemotherapy of Experimental Brain Tumors...

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

Studies on the Chemotherapy of Experimental Brain Tumors: Evaluation 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

A brain tumor model has been utilized to test 4chemotherapeutic agents. The model consists of C57BL malemice implanted intracerebrally with carcinogen-inducedgliomas. Four tumors were used, all histologically ependymo-blastomas, but each differing in its biological behavior. Fouragents were tested, each administered i.p. A comparison wasmade between treated animals and nontreated control animalswith respect to median survival time and long-term survivaland was statistically evaluated by a modification of theWilcoxon rank-sum analysis.

l,3-Bis(2-chloroethyl)-l-nitrosourea (NSC 409962) significantly prolonged the life-span of mice bearing the intracerebralgliomas. Increased life-span varied from 10 to 137%.Cyclophosphamide (NSC 26271) less consistently increasedsurvival of glioma-bearing mice. Neither mithramycin (NSC24559) nor methotrexate (NSC 740) on multiple doseschedules was capable of increasing the life-spans of theanimals.

The implications of these data with respect to blood-brainbarrier, brain tumor permeability, and the value of the modelas a screen for human brain tumor chemotherapy are discussed.

INTRODUCTION

In a previous report (3), a method was described for theestablishment of an experimental brain tumor model withi.e.3-implanted fragments of carcinogen-induced brain tumors

into C57BL male mice. Four tumors were used with respectivesurvival times of: ependymoblastoma (Zimmerman), 29 Â±3days (S.D.); Glioma 261 (Shear), 24 Â±2 days; Glioma 26(Sugiura), 24 Â± 3 days; and the mutant subline ofependymoblastoma, Ependymoblastoma A, 19 Â±1 day.

The present study describes chemotherapeutic trials withthis model. Four drugs were tested: BCNU (NSC 409962),Cyclophosphamide (NSC 26271), mithramycin (NSC 24559),and methotrexate (NSC 740).

BCNU is one of a series of nitrosoureas developed atSouthern Research Institute following the observation that therelated nitroguanidines produced a weak but significantprolongation in life-span of L1210-inoculated mice (13, 34).Among the nitrosoureas, BCNU is most efficacious ineliminating LI210 leukemia in such mice, even whenmeningeal leukemia is induced or occurs spontaneously (34).BCNU is lipid soluble and readily enters the brain andcerebrospinal fluid (26). For these reasons, it was chosen asthe first drug to be tested in the model. It is thought to act asan alkylating agent, probably through an intermediarydiazoalkane (27).

Cyclophosphamide (7V,./V-bis(j3-chloroethyl)-yv',O-propylene-

phosphoric acid ester diarnide) was first synthesized in 1958by Arnold and Bourseaux (1). It is effective against both rattumors and murine leukemia (20) as well as in humanleukemia (43). The drug is given in an "inactive" form andmust be metabolized in vivo to an "active" form by the liver.

The active form is water soluble and does not appear to enterthe brain (7). Cyclophosphamide has almost no activity againsti.c.-inoculated L1210 leukemia, but it is effective against as.c.-implanted experimental brain tumor (41).

Mithramycin, an antibiotic derived from Streptomycestanashiensis, has marked antibacterial activity in vitro and iscytotoxic in tissue culture (31). It shows only moderateantineoplastic activity against experimental tumors, but it hasproduced striking clinical remissions in embryonal cellcarcinoma of the testes (5). Mithramycin has been testedagainst a s.c.-implanted mouse glioma (19) and has been usedclinically in human brain tumors (18, 30). It was tested in ourmodel because it is currently under investigation by the BrainTumor Therapy Study Group (23). It appears to act byinhibiting RNA synthesis (50).

Methotrexate was included in the current series as anexample of a weakly ionized, lipid-insoluble chemotherapeuticagent which is cell-cycle active. It has been shown to occupythe extracellular space of the i.c.-implanted murine glioma(22).

MATERIALS AND METHODS'Present address: Neuropsychiatrie Service, Memorial Sloan-

Kettering Cancer Center, New York, N. Y. 10021.2Present 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 forlife-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

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W. R. Shapiro, J. I. Amman, and D. P. Rail

consisted of 5 groups of 10 tumor-bearing animals per group.The 5 groups received 5 different doses of drug. Group IIIhad 5 groups of 10 animals apiece, but without tumors; theseanimals received the same dose of drug as Group II and actedas nontumor-bearing drug controls.

All animals were weighed on the day of tumor implantationand on the days of drug injection. In selected experiments,daily to thrice weekly weights were followed throughout thecourse of the experiment.

The animals were observed following drug injection, and theday of death was recorded for each mouse. Animals survivinglonger than 60 days were followed for 6 to 18 months toobserve for tumor recurrence. However, a few surviving micewere killed by cervical dislocation and the brain was submittedfor histological examination. As noted in a previous report(36), the incidence of "no-takes" among nontreated control

animals was: ependymoblastoma, 0.8%; Glioma 261, 0.9%;Glioma 26, none; and Ependymoblastoma A, 2%.

Drugs were obtained from the Cancer ChemotherapyNational Service Center and were administered i.p.

Table 1Design of each experiment

Group I :25tumorNo

treatmentGroup

II:50tumorTreatment

at5dose levelsGroup

III:50nontumorTreatment

at5dose levels

Drug toxicity was determined in advance on nontumor-bearing animals and the LD50, LD10, and respective 95%confidence limits for each drug were calculated by the methodof Litchfield and Wilcoxon (24).

Drug Trials

BCNU is relatively insoluble in water. The weighed powderwas first dissolved in 95% ethanol, and 0.9% NaCl solution wasthen added to yield the proper concentrations. The drug wasmade up to volume such that doses could be administered 0.01ml/g of body weight. In 9 experiments, BCNU was given as asingle i.p. injection on Day 2 following tumor implantation.Two experiments were done in which BCNU was given as asingle i.p. injection on Day 14 in ependymoblastoma animals.Ependymoblastoma A, the mutant subline of ependymoblastoma, was also tested with BCNU in 1 experiment on Day2 only.

For BCNU, the LD5 0 of single i.p. injection was 44 mg/kgwith 95% confidence limits of 40.8 to 74.4 mg/kg. The LD10was 34 mg/kg; 95% confidence limits were 30.9 to 37.4 mg/kg.These data were determined at 30 days because of thepropensity for BCNU to produce delayed toxicity (8, 21).About 90% of animals that died as a result of BCNU did sobefore Day 21, however.

Cyclophosphamide was weighed and dissolved in 0.9% NaClsolution. Warm tap water was allowed to bathe the bottle inorder to facilitate dissolution. The drug concentration was

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

Dose(mg/kg)Experiment20

12525

2530

123S40

124550

123S60

14Median

day ofdeath(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,

Wilcoxontestp

<0.001p<0.0001p<0.001p

<0.0001p<0.00001p

<0.05p<0.0001p<0.0001p<0.00001LethalN.

S.Lethalp

<0.0001LethalLethalLethalLethalLethalLethalMLS

(% control)131135112148124121194143136376556139263843242630Meanlife-span/dose126136149743328Survivors>60days1/100/101/100/101/101/103/100/101/101/101/102/103/100/100/102/100/100/100/10

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



Brain Tumor Chemotherapy: Drug Studies

Table 3Effect of BCNUagainst i.e. ependymoblastoma, i.p.,

Day 14 only, 1 dose/day

Medianday of Significance,

Dose Experi- death Wilcoxon MLS Survivors(mg/kg) ment (T/O test (% control; >60 days

201241/3141/28p

< 0.001p < 0.001132 1461/101/10

25 48/28 p < 0.01 171 1/10

3040SO60121

21

210

Distribution52/31

33.5/2858/31

36/2825.5/31

20.5/2821/31includes

earlyp

< 0.05Bimodal0p

< 0.001BimodalLethal

LethalLethaland

late deaths.168

12018712982

7368Wilcoxon1/10

4/104/10

1/102/10

0/100/10analysis

notpossible. See text for details.

Table 4Effect of BCNU against i.e. Ependymoblastoma A, i.p.,

Day 2 only, 1 dose/day

Dose(mg/kg)2025304050Experiment11111Medianday

ofdeath(T/O19/1733/1738/1736.5/178/17Significance,WilcoxontestN.S.p

<0.00001p<0.00001p<0.05LethalMLS(%

control)11219422421547Survivors>60days0/101/101/102/100/10

made up to yield doses at 0.01 ml/g of body weight. The drugdisplayed greater toxicity in tumor-bearing animals than innormals. In normal animals, the LD50 was 370 mg/kg (95%confidence limits, 330 to 414 mg/kg) and the LD10 was 270mg/kg (95% confidence limits, 241 to 302 mg/kg) at 21 days.In the tumor-bearing mice, the LD50 was 264 mg/kg (95%confidence limits, 247 to 282 mg/kg) and the LD10 was 210mg/kg (95% confidence limits, 191 to 231 mg/kg) at 21 days.Differences in drug toxicity between tumor- and nontumor-bearing animals are not uncommon, although their quantitative determination is often difficult because of earlytumor-induced deaths (40). Skipper and Schmidt (40)considered such differences of little importance in evaluatingdrug efficacy, and such would seem to be the case in systemsof rapidly growing tumors like LI210. In our model, tumordeaths rarely occurred before Day 15, making possibleevaluation of drug toxicity in both tumor- and nontumor-bearing animals.

Mithramycin was supplied in stoppered vials containing 2.5mg/vial. Sterile water was added to yield dose levels at 0.01ml/g of body weight. The drug was administered as a singleinjection i.p. on Day 2 following tumor inoculation in 4experiments, and as daily injections i.p. on Days 2 to 8 in 6experiments. The single dose LD50 was 2.9 mg/kg, and theLD10 1.35 mg/kg. In the multiple dose experiments, the LDSOwas 1.4 mg/kg/day and the LDi 0 was 0.9 mg/kg/day for 5daily doses. Confidence limits of 95% were not obtained andno difference in lethalities between tumor and nontumoranimals was seen.

Methotrexate was weighed and diluted with 2% sodiumbicarbonate in sterile water to yield dose levels at 0.01 ml/g ofbody weight. The LD50 was 86 mg/kg/dose (95% confidencelimits, 73 to 101 mg/kg/dose), and the LD10 was 29mg/kg/dose (95% confidence limits, 25 to 34 mg/kg/dose) for4 injections, given once each day every 4 days. Since the lastdose was given on Day 14 after tumor implantation, toxicityin tumor-bearing animals could not be determined. In order to

span the toxicity range from LD0 to LD, 00, the experiments

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

Dose(mg/kg)2025304050Experiment1

21

21

21

21

2Median

day ofdeath(T/Q34.5/26

41/2732.5/26

64/2721.5/26

63.5/2710/26

45.5/2710/26

8/27Significance,

Wilcoxontestp

< 0.01p <0.0001p

< 0.01p <0.001Bimodal"

p <0.0001N.S.

BimodalLethal

LethalMLS

(% control)133

152125

2378323538

16938

30Mean

life-span/dose14318115910434Survivors>60days3/10

3/101/10

5/102/10

5/101/10

4/100/10

0/10

1Distribution includes early and late deaths. Wilcoxon analysis not possible. See text for details.

SEPTEMBER 1970 2403



R. Shapiro, J. I. Amman, and D. P. Rail

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


21

21

21

21

2Median

day ofdeath(T/Q22/2029/2525/20

32/2527/20

32/2530/20

22/257/20

11/25Significance,

Wilcoxontestp

< 0.05p <0.01p

< 0.001p <0.0001p

< 0.001p <0.01p

< 0.01Bimodal0Lethal

LethalMLS

(% control)110

116125

128135

128150

8835

44Mean


0/100/10

0/100/10

1/100/10

0/100/10

0/10

0 Distribution includes early and late deaths. Wilcoxon analysis not possible. See text for details.

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


231

231

231

231

23Median

day ofdeath(T/O29.5/29

33.5/3142.5/3136/29

30/3135/3118.5/29

35.5/3110.5/317/29

11.5/3112/313/29

3/313/31Significance,

WilcoxontestN.S.

N.S.p <0.0001p

< 0.01N.S.Bimodal"Bimodal

p < 0.05LethalLethal

LethalLethalLethal

LethalLethalMLS

(% control)102

108137124

9711364115

3424

373910

1010Mean


0/100/100/10

0/100/100/10

0/100/100/10

0/100/100/10

0/100/10

"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, nontreated32, and 64 mg/kg/day every 4 days for 4 doses, and a "high control groups (C) and the % MLS of the treated animals wasrange" 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 mutationof 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 exceeding100% implied that the drug induced an increase in life-span;

Analysis of Data MLS values less than 100% were usually related to druglethality.

The results were analyzed by 3 methods. In the first, the A 2nd method of analysis had the purpose of determiningmedian day of survival of the treated groups (T) was compared whether the treated animals survived significantly longer than





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€”hypothesis was then tested that the 2 groups were from 2 _ ,r>\identical populations. ' /NM (M + N + 0 ' "

The test was performed in the following manner. The V 12individual day of death for each nontreated, tumor-bearingcontrol 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 wascompiled 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 8Effect of cyclophosphamide against i.e. Glioma 261, i.p., Day 2 only, 1 dose/day


21

21

21

212Median

day ofdeath(T/Q29.5/24

33/2336/24

34/239.5/24

20/234/24

7.5/234/24

3/23Significance,

Wilcoxontestp

< 0.05p <0.001p

< 0.0001p <0.0001LethalN.S.Lethal

LethalLethal

LethalMLS

(%control)123143.5150

14840

87173317

13Mean


2/101/103/100/10

0/100/10

0/100/10

0/10

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


231231

231

23123Median

day ofdeath(T/Q31/25

33.5/2634/2934.5/25

35/2635/2932/25

24/2627.5/299/25

7.5/267.5/293/25

3/263/29Significance,

Wilcoxontestp

< 0.001p < 0.001p <0.01p

< 0.01p < 0.01p <0.01Bimodal0

BimodalBimodalLethal

LethalLethalLethal

LethalLethalMLS

(%control)124

129117138

135121128

929536

292612

1210Mean


0/101/100/10

1/100/100/10

0/100/100/10

0/100/100/10

0/100/10

0 Distribution includes early and late deaths. Wilcoxon analysis not possible. See text for details.

SEPTEMBER 1970 2405



W. R. Shapiro, J. I. Austrian, and D. P. Rail

Table 10Effect 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

21

21

21

21

2Median

day ofdeath(T/O26.5/26.5

29.5/2723/26.5

28/2726.5/26.5

31.5/2725/26.5

26.5/276/26.5

7/27Significance,

WilcoxontestN.S.N.S.N.S.

N.S.N.S.

p <0.001N.S.

N.S.Lethal

LethalMLS

(% control)100

10987

10410011794

9822.6

26Mean

life-span/dose104.595.5108.59624.3Survivors>60days0/10

0/100/10

0/100/10

0/100/10

0/100/10

0/10

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


21

21

21

21

2Median

day ofdeath(T/O24.5/2427.5/2524/24

25.5/2522.5/24

26.5/2521.5/24

24/258/24

5.5/25Significance,

WilcoxontestN.S.

N.S.N.S.

N.S.N.S.

N.S.N.S.

N.S.Lethal

LethalMLS

(% control)102

110100

10294

10690

963322Mean

life-span/dose1061011009327.5Survivors>60days0/10

3/101/10

0/100/10

1/100/10

0/100/10

0/10

included as censored data and the Z value was calculated.Where a drug dose exceeded the LD5 0, statistically significantreduction in survival time occurred. Such doses were designated as "lethal." The test was reduced to a computer program

for ease of analysis.The 3rd method of analysis consisted of tabulating the

long-term survivors, i.e., animals that survived longer than 60days. The value of 60 days was chosen arbitrarily on the basisthat over 99% of nontreated tumor animals died of theirneoplasms by that time and those that did not were consideredno-takes. Thus, the chance that a long-term-surviving treatedanimal did not have a tumor was less than 1 in 100.

RESULTS

BCNU

Ependymoblastoma. Table 2 shows the results of BCNUadministered as a single injection on Day 2 against

ependymoblastoma. The drug produced significant prolongedsurvival at 3 doses below the LD] 0 (20, 25, and 30 mg/kg). At40 mg/kg, most of the experiments were bimodal with earlydrug deaths and prolonged life-span of the drug survivors.There was considerable variation among the MLS values,although in general increased dosage led to increased survivaltime. Long-term survival rates ranged from 10 to 30%.

An attempt was made to determine whether BCNU acted bypreventing tumor take only or if the drug actually impededtumor growth. In 2 experiments, administration of BCNU wasdelayed until 14 days after tumor inoculation. The results areshown in Table 3. Only 2 doses below the LD10 were used inexperiment 1, but in this experiment 40 mg/kg produced only1 toxic death. The results are comparable to those obtained atDay 2 treatment, indicating that BCNU had an effect beyondthat of preventing tumor take.

Table 4 shows the results of BCNU against Ependymoblastoma A, the mutant subline of ependymoblastoma. Withinthe degree of variation, a similar efficacy was demonstrated.




Glioma 261. Table 5 depicts the results of BCNU againstGlioma 261. At 3 dose levels below the LD10, the drugconsistently produced an increase in life-span. MLS values inExperiment 2 were quite high and, at the lower 3 dosages, thenumber of long-term survivors exceeded 20% in all but 1 case.


Glioma 261 appeared to be more sensitive to BCNU thaneither ependymoblastoma or Glioma 26.

Glioma 26. Significant increased survival time was inducedby BCNU in mice bearing i.e. Glioma 26 (Table 6), althoughMLS values were consistently small. The number of long-term

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


21

21

212Median

day ofdeath(T/Q27.5/2725.5/2527/27

24.5/2526/27

25.5/2526.5/27

20.5/256/27

6.5/25Significance,

WilcoxontestN.S.

N.S.N.S.

N.S.N.S.

N.S.N.S.

N.S.Lethal

LethalMLS

(% control)102

102100

989610298

822226Mean


1/100/10

1/100/10

1/100/10

1/100/10

0/10

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


12348

123416

1234532

1234564

12345128

5256

5Median

day ofdeath(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,

WilcoxontestN.S.N.S.N.S.N.S.N.S.N.S.N.S.N.S.p

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

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

<0.01LethalLethalLethalLethalLethalLethalMLS

(%control)9510090.59710810390.595110105959510311610090.587106126745753756139MeanSurvivors

life-span/dose >60days0/1095.6

2/100/100/102/1099

2/100/100/103/101/10102

0/101/100/101/104/10100

0/103/100/103/100/1077

0/100/100/100/100/10

SEPTEMBER 1970 2407




survivors was also low. Glioma 26 appeared to be less sensitiveto BCNU than both ependymoblastoma and Glioma 261.

Cyclophosphamide

Ependymoblastoma was inconsistently affected by Cyclophosphamide administered as a single injection on Day 2following tumor inoculation (Table 7). However, cyclo-phosphamide significantly prolonged the lives of mice bearingGlioma 261 (Table 8) and Glioma 26 (Table 9) at 2 dose levelsbelow the tumor-bearing LD10 (210 mg/kg). The maximumMLS values were seen in Glioma 261, as were the largestnumber of long-term survivors. One experiment in whichcyclophosphamide was used against Glioma 261 producedexcessive lethality in the drug control animals and wasdiscarded.

Mithramycin

Mithramycin was used in 2 schedules, 1 injection on Day 2only, and daily injections for 5 days from Day 3 to 8. On thesingle injection schedule, doses used included 0.5, 0.75, 1.0,1.5, and 4.5 mg/kg. Two experiments against ependymoblastoma and 1 each against Glioma 261 and Glioma 26 weredone on the single injection schedule. No difference in survivalbetween control animals and treated animals was seen in anyof the experiments, and there were no long-term survivors.

On the multiple injection schedule, 6 experiments, 2 eachwith ependymoblastoma, Glioma 261, and Glioma 26, weredone and are tabulated in Tables 10, 11, and 12. With oneexception (ependymoblastoma, Table 10, Experiment 2, 0.75mg/kg/day), no significant increase in survival time occurred.However, there were a few long-term survivors in both Glioma261 and Glioma 26 (Tables 11 and 12).

Methotrexate

Experiments with methotrexate were divided into low-rangeand high-range dosage schedules of 1 injection/day every 4days, times 4 injections (see "Materials and Methods"). The

results are shown in Tables 13, 14, and 15.Table 13 shows the results of methotrexate against

Ependymoblastoma A, the mutant subline of ependymoblastoma. Experiment 1 had 3 significant points of increasedsurvival time, only 2 of which were at or below the LDt 0 (29mg/kg/dose). In none of the other experiments was asignificant increase in survival shown. However, a number oflong-term survivors occurred at several dose levels.

With Glioma 261, Experiment 1 showed 2 high MLS values,119 and 123% at, respectively, 16 and 32 mg/kg/dose. Thesewere not significant by Wilcoxon analysis. A number oflong-term survivors was also recorded.

No significant increase in survival occurred with methotrexate against Glioma 26 (Table 15), although there wereseveral long-term survivors.

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


128

1216

123432

123464

1234128

34256

34Median

day ofdeath(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,

WilcoxontestN.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





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


128

1216

123432

123464

1234128

34256

34Median

day ofdeath(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,

WilcoxontestN.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

DISCUSSION

Method of Analysis

Three methods were used to analyze the results.Comparison of the median survival times of treated andcontrol groups yielded ILS values which varied over wideranges among several experiments. A change in 1 or 2 days inthe median day of death could alter the ILS by as much as 10to 30%. Under such circumstances, the mean "life-span perdose" was at best â€¢only an approximation. Construction of

dose-response curves was not possible, nor was statisticalcalculation of significant differences.

With the help of Dr. John Slivka (Mathematical Statisticsand Applied Mathematics Section, Biometry Branch, NationalCancer Institute) a method of analysis was adopted utilizingthe rank-sum test of significance first described by Wilcoxon(48) and later modified by Gehan (10). The details of theanalysis are described under "Materials and Methods," but

basically the test consisted of serially ranking the days ofdeath of the animals and comparing the sums of the ranks ofthe control animals with that of the treated animals under thenull hypothesis that the 2 groups of mice were from differentpopulations. By this method, the variation imposed by the rawdata is nullified by preserving the order of day of death buteliminating the variable time among the days of death.Furthermore, unlike the ILS method, which deals with themedian day of death, the Wilcoxon analysis preserves the

contribution of each animal to the statistic. It was thuspossible to determine statistically whether treatment wasindeed better than no treatment.

The test unfortunately did not allow us to determine "howmuch better" treatment was over no treatment. Furthermore,

when 20 to 30% of a treated group died early, presumablybecause of drug intoxicity, and the remainder outlived thecontrol group, the Wilcoxon test could not be utilized, forunder these conditions the treated group was bimodal andcould not be compared with the control group.

Despite these limitations, the Wilcoxon analysis did allow atest of significance, and the ILS approximations could be usedas a rough dose-responsive curve. Thus, it was the Wilcoxontest which dictated when a drug was declared useful by thismodel and it is emphasized that the value "mean life-span perdose" is included principally as a guide.

Finally, the long-term survivors also represent a measure ofdrug response, although permitting only rough comparisons.Nevertheless, such a parameter can be very useful in assessingthe long-term effectiveness of an agent.

BCNU. Of the 4 agents used in this study, only BCNUshowed a consistent inhibiting effect on growth of the i.e.tumors. For each tumor, BCNU-treated animals survivedsignificantly longer than nontreated animals. In general, as thedose of BCNU was increased from 20 through 25 to 30 mg/kg,the length of survival increased, although no specific dose-response relationship could be obtained because of the highdegree of variability.

SEPTEMBER 1970 2409




The effect of BCNU on each of the 3 tumors was similar,although the best results were obtained against Glioma 261,the worst against Glioma 26; ependymoblastoma wasintermediate. We cannot yet explain the difference in resultsamong these 3 tumors. Historically, no consistentdifferential feature has been found. Although our resultssuggest different biological behavior patterns both in responseto BCNU and in mean survival time of nontreated,tumor-bearing animals (see above), only kinetic data could testwhether such a difference exists.

Usually, BCNU was given on Day 2 following tumorinoculation. In 2 experiments (Table 3), drug was given onDay 14 only. Essentially similar results were obtained as withthe early treatment. This would be expected since, as Skipperhas pointed out (38, 39), a noncycle-active agent kills aconstant percentage of the cells for a given dose and not aconstant number of cells. Thus a given dose of drug shouldyield the same mortality time no matter when the drug wasgiven, as long as it was given early enough to cause its effectbefore the animal died. For this reason, all subsequent singledose experiments utilized Day 2 drug injection.

The inhibitory effect of BCNU on murine glioma has beendemonstrated previously by Sugiura (42). He found that 8.0mg/kg/day for 7 days inhibited the s.c. tumor by 96% (averagediameters, tumor index = 0.04) over nontreated controls at 2weeks after implantation, although the treated animals lost 3.5g in the same period while the controls gained 1.5 g of weight.Lower doses of BCNU were less effective. Similar results wereobtained against other solid tumors.

Rosso et al. (33) found that MNU and BCNU significantlyprolonged the survival of mice inoculated i.e. with varioustiters of Sarcoma 180 cells, although MNU did not inhibit s.c.or i.p. Sarcoma 180. Sugiura (42) found that both BCNU andMNU inhibited sarcoma inoculated s.c. as a solid fragment.Similar results were obtained by Rosso et al (33) when theytested MNU against Ehrlich carcinoma inoculated i.e., i.p., ands.c. The drug prolonged the life-span of animals with the i.e.tumor but did not inhibit the s.c. or i.p. growth. In contrast,Sugiura (42) found that MNU and BCNU had a pronouncedinhibitory effect on the s.c. Ehrlich carcinoma.

Cyclophosphamide. On the single dose schedule used,cyclophosphamide significantly increased the survival time ofmice bearing all 3 tumors although it did so least consistentlywith ependymoblastoma. As in the BCNU trials, Glioma 261was most sensitive to the agent, but Glioma 26 wasintermediate with cyclophosphamide, differing from theresults with the nitrosourea. ILS values overall were lower withcyclophosphamide than they were with BCNU.

Rosso et al. (33) used cyclophosphamide to treat micebearing Sarcoma 180 and Ehrlich carcinoma. The drug failedto increase survival time of animals implanted i.e. with eithertumor, although significantly increased life-span was obtainedin animals with i.p.-implanted tumors and significantinhibition occurred in s.c. tumors. Conclusions regarding thenegative results in the i.e. tumor system must be tempered bythe fact that only a single dose level of cyclophosphamide wasused and higher levels might have been effective.

Prior to the present study, the only reported attempt totreat experimental gliomas with cyclophosphamide was that ofSoloway et al. (41). They utilized an ependymoblastoma

grown i.m. in the hind limb of a mouse and measured thechange in cross-sectional area of the tumor after a specifiedtime interval following treatment. Cyclophosphamide washighly effective; the tumor disappeared on the 4th day aftertreatment but recurred about 1 week later.

Mithramycin. Mithramycin failed to prolong significantlythe lives of mice bearing any of the 3 tumors, although afew long-term survivors occurred with Glioma 261 and Glioma26 (Tables 11 and 12). The 2 schedules used, i.e., single doseon Day 2, and 5 daily doses starting on Day 3 following tumorimplantation, spanned the toxicity range for the drug.Although the 5-day schedule was chosen by reference to theliterature as the most effective for this drug, it is possible thata different schedule could have been more effective in thepresent system.

Kennedy et al. (19) evaluated the effect of mithramycin onGlioma 26 implanted s.c. in mice. Utilizing a variety ofschedules, they demonstrated inhibition of tumor growth; thetumor volumes ranged from 32% of controls in animals treated3 times weekly to 42% of controls in animals treated on 4consecutive days at 3-week intervals. Their schedules differedfrom ours; treatment was continued in some cases beyond thepoint when 50% of the treatment animals were dead. In theirstudies, the LDSO in C57 mice for a single injection ofmithramycin was 6.0 mg/kg while in our studies it was 2.0mg/kg. On 5 equal daily doses in C3H mice, the LDSO was 1.7mg/kg/day. In our studies utilizing C57 mice, the LDS 0 for 5daily doses was 1.4 mg/kg/day. Since we evaluated survivaltime, doses which yielded lethal effects greater than LD10were considered nonevaluable.

Despite the differences in schedules and toxicity betweenour studies and those reported by Kennedy et al., the lattergroup demonstrated significant inhibition of brain tumorgrowth when the tumor was implanted s.c. In our studies, thedrug either failed to inhibit growth or did so to too small adegree to affect survival time. DeWys et al. (9) have shown apoor correlation between tumor growth inhibition and survivaltime in the Walker 256 tumor system. Such investigations raiseimportant questions regarding the screening value of s.c. solidtumor models when tumor size at a fixed point in time is theend point.

Methotrexate. On the schedules used, methotrexate failedto increase overall survival time of mice bearing Glioma 26,Glioma 261, or Ependymoblastoma A, although a smallpercentage of long-term survivors was scattered throughout theexperiments. The choice of an every-4-day regimen was basedon the results with LI210 reported elsewhere (12), and otherschedules possibly might have been effective.

Methotrexate is efficacious against L1210 leukemia exceptthat, when the tumor invades the brain, methotrexate appearsto be completely ineffective (37, 47) (see below). Its apparentinability to reach high enough concentrations in the centralnervous system accounts for its ineffectiveness in this location.In human meningeal leukemia, intrathecal methotrexate and itscongener aminopterin yield prompt reduction in leukemic cellsin the cerebrospinal fluid (15, 32), although frequently noteliminating them entirely from the subarachnoid space (45).

Rosso et al. (33) failed to increase survival of mice bearingi.e. Sarcoma 180 with methotrexate therapy.

Wilson et al. (49) stereotactically placed cultured human




oligodendroglioma cells and carcinogen-induced mammarycarcinoma cells in the thalamus of rat brains and comparedintracisternally with i.p.-administered methotrexate. Animalswith oligodendroglioma treated by each route had survivalssignificantly greater than controls; the intracisternally treatedrats survived a little longer than those treated i.p. The animalswith i.e. mammary carcinoma showed no difference insurvival. The results did not include early animal deathsresulting from the intracisternal treatment procedure, andinclusion of such data would have reduced the mean survivaltime of this treated group.

Brain Tumor Permeability and Chemotherapy

A problem unique to the therapist who would treat braintumors with anticancer agents is the location of the tumor.Many molecular species do not easily enter the normal brainfrom the blood because of restricted permeabilityâ€”thephenomenon called the blood-brain barrier. Does a similar"barrier" also prevent the entry of chemotherapeutic agents

into brain tumors?Leukemic cells in both experimental (47) and human (14)

leukemic meningitis are isolated from many systemicallyadministered chemotherapeutic agents. Thomas et al. (47)demonstrated the methotrexate reduced the number of LI 210cells in the blood and spleen of the animal but had no effecton the number of cells in brain tissue. Similarly, Skipper et al.(37) found that methotrexate therapy did not increase thelife-span of animals with LI 210 cells in the central nervous

system. These investigators and Chirigos et al. (7) reportedthat cyclophosphamide also failed to increase the life-span ofanimals with central nervous system L1210, although the drugreadily eliminated leukemic cells from blood and spleen.Thomas et al. (46) studied the pathology of LI210 leukemiain animals following systemic cyclophosphamide therapy.There was complete destruction of leukemic cells in the liver,spleen, bone marrow, and dura but a persistence of the LI 210cells in the subarachnoid space. A combination ofmethotrexate and cyclophosphamide was reported to eliminate LI210 cells in the blood and reduce their number in thespleen, but failed to decrease the number of leukemic cells inthe brain (6).

Such data appeared to confirm the observation of Rail andZubrod (29) that drug solubility and ionization constant areimportant properties with respect to the permeability of thebrain to pharmacological agents. These investigators demonstrated that water-soluble, ionized molecules could not easilyenter the brain, whereas lipid-soluble, non-ionized speciesreadily crossed the blood-brain barrier. That this principleapplied also to chemotherapeutic agents was shown by Skipperet al. (37) and Schabel et al. (34), who found that, incontradistinction to the water-soluble, ionized agent methotrexate, the lipid-soluble, non-ionized nitrosoureas were highlyeffective against experimental leukemia when administeredsystemically. Skipper et al. (37) demonstrated that MNU giveni.p. increased the life-span of mice with i.e. L1210 cellinoculations. Schabel et al. (34) studied several analogs ofmethylnitrosourea and found that BCNU increased thelife-span of mice with systemic and/or i.e. leukemia. Moreover,


the analogs of MNU which were non-ionized in aqueous mediaat pH 7 and which had a high butyl acetate :NaCl solution(lipid:water) solubility had a significant effect on i.e. L1210leukemia. Finally, systemically administered BCNU inpreliminary trial has shown effectiveness against humanmeningeal leukemia (16).

Although such studies appear to affirm the existence of ablood-brain barrier in meningeal leukemia, there is evidence thatsuch a barrier may not be present within a solid malignantbrain tumor. Electron microscopic studies have shownnumerous differences between normal brain and brain tumors(reviewed in Ref. 35). The endothelium in normal brain issingle layered with no pores and only occasional pinocytoticvesicles, while in glioblastoma tissue, endothelial cells are oftenproliferated, pores may be present, and pinocytotic vesiclesoccur in abundance. Pores are also seen in the basementmembrane of glioblastomas. The neuropile of normal brain istightly packed with a moderately sized interstitial space. Theparenchyma of a glioblastoma consists of loosely organizedtumor cells in a large interstitial space. Although muchvariation may occur depending on from where specimens aretaken within the tumor, such alterations may be themorphological substrate for less restricted permeability thanoccurs in normal brain.

Physiological data also tend to suggest free permeabilityinto malignant brain tumors. Numerous studies have beendone since the work of Broman (4) showing trypan bluestaining of tumors, including the studies of Moore (28) withfluorescein and radioactive fluorescein. Radioiodinatedalbumin, chlormerodrin-203Hg and many nuclides following

systemic administration exhibit tumor to brain ratiosexceeding 1, indicating ready access of the agents into thetumor (35). Such studies, of course, failed to localize themarker within the tumor, but the data of Jackson et al. (17),Long et al. (25), Tator et al. (44), and others utilizingautoradiography showed markers in the extravascular space ofbrain tumors implying that there was no vascular barrier tosuch markers.

Quantitative characterization of molecular entry intoexperimental brain tumors was achieved by Ausman and Levin(2) and Levin et al. (22) in our laboratory. These investigatorsevaluated the steady-state distribution spaces of inulin andmethotrexate in nephrectomized mice bearing the i.e.ependymoblastoma. The inulin space in the tumor was about27%, while in distant brain the value was 2.5%. Methotrexatehad a space of 28% in the tumor and 3% in distant brain.Simultaneous vascular spaces were less than 1% in both areas.Important controls in such experiments included the purity ofthe markers, the absence of their metabolism, and the absenceof dissociation of the radioactive isotope from the marker invivo. It is especially important that continuous administrationwas carried out, thus reducing the problem of reentry ofadministered markers into the vascular system. Other studies,although showing similar qualitative results, have often failedto take such controls into consideration.

Thus, in meningeal leukemia, the parenchyma of the brain isprobably little disturbed and the blood-brain barrier ispreserved. When a solid tumor invades the brain, however,marked changes in the vasculature and parenchyma take place,resulting in far less restriction of permeability within the tumor

SEPTEMBER 1970 241!



W R. Shapiro, J. I. Amman, and D. P. Rali

than in normal brain. This would appear to explain theeffectiveness of cyclophosphamide in the studies reported herecompared to its ineffectiveness against i.e. leukemia. Otherfactors, especially the sensitivity of the tumor itself and therelative local concentrations, must be evaluated to account forthe poor results with methotrexate and mithramycin.

That lipid solubility may still be an important property ofchemotherapeutic agents useful in brain tumors is shown bythe highly efficacious results with BCNU. One possibleexplanation for this finding may relate to the poorpermeability of normal brain to water-soluble molecules. Suchdrugs, for example, methotrexate and mithramycin, althoughcapable of entering the tumor per se, may rapidly lower theirconcentration within the tumor by (a) diffusion intosurrounding brain where the drug concentration would beessentially zero and (b) return to the vascular system.Depending on the critical level for effectiveness, the resultantdrop in concentration within the tumor could remove theagent before it had a chance to act. Lipid-soluble drugs enterboth the tumor and the surrounding normal brain, preventingthe formation of a concentration gradient and permitting timefor the agent to take effect. Only further work with these 2classes of agents can affirm or deny this hypothesis.

Screening Value of Experimental Brain Tumors

In 1966, Goldin (11) examined the ability of animal tumorchemotherapy screens to predict clinical usefulness. Criteriafor clinical activity were well established for the leukemiasand, to a lesser extent, for the lymphomas; but such was notalways true for other solid tumors. Nevertheless, the authorsfound that all of 20 clinically active drugs were predicted byvarious combinations of 12 screening systems. Indeed, 4 of thesystems were sufficient in themselves.

It is too early to predict the usefulness of our model withrespect to human malignant brain tumors. Nor is it possible todetermine if this model will more likely be predictive thanother systems. There are several chemotherapy models whichuse animal brain tumors and, in spite of differences in thetumor systems, all the tumors respond to various drugs in asimilar fashion. Differences between the results obtained inour model and those utilizing s.c. or i.p. implantation may beonly quantitative. We believe, however, that a model utilizingi.e. tumors is more appropriate to brain tumor chemotherapystudies than those using tumors implanted in extracerebral sites.Furthermore, studies which make use of nonglial tumorspresume that only minor differences exist among neoplasmsderived from various sites, a view not supported when thetumors have been closely examined. Human gliomas appear tohave greater similarity, at least histologically, to the murineependymoblastoma than they do to nonglial animal tumors. Inthe final analysis, however, only closely correlated clinicaltrials compared to results in animal systems can adequatelyprove the screening value of a given model.

ACKNOWLEDGMENTS

The technical assistance of Mr. Charles E. Ester is gratefullyacknowledged.

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SEPTEMBER 1970 2413



1970;30:2401-2413. Cancer Res William R. Shapiro, James I. Ausman and David P. Rall Cyclophosphamide, Mithramycin, and MethotrexateEvaluation of 1,3-Bis(2-chloroethyl)-1-nitrosourea, Studies on the Chemotherapy of Experimental Brain Tumors:

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