Effectiveness of Cytoxan against Intracerebrally and

In a previous study it was found that thelymphoid leukemia (L1210) of mice grew successfully when inoculated intracerebrally (I.C.). Theleukemic cells grew in the brain, infiltrated diifusely in the arachnoid, and became systemic in2—8days following intracerebral inoculation. Also,when the leukemia was inoculated I.C., death occurred somewhat sooner than when the leukemiccells were inoculated subcutaneously (S.C.). Dcspite the more virulent nature of the disease onintracerebral inoculation of leukemic cells, thefolic acid antagonists amethopterin (MTX) andS',S'-dichloroamethopterin (DCM) produced increases in survival time of the mice (5).

The importance of studying the physiologicaldistribution of anticancer agents, particularly withreference to their capacity to cross the blood-brainbarrier, has been emphasized by other investigators (13, 15, 17). It has been shown that in bothanimals and patients with leukemia there is oftenconsiderable infiltration of the brain by leukemiccells. This observation has led to the suggestion

Receivedfor publication August 2. 1961.

that the ultimate failure of therapy of leukemiawith some anticancer drugs was attributable, atleast in part, to the inability of the drugs to reachfoci of leukemic cells which had become localizedin the brain (8, 9, 12).

The technic of introducing leukemia directlyinto the brain has made it possible to study someof the pharmacological aspects involved in thechemotherapy of central nervous system (C.N.S.)leukemia. This paper describes a series of experiments designed to provide information on therelative effectiveness of N,N,-bis-(@-chloroethyl)N',O-propylene phosphoric acid ester diamide (Cytoxan)' against I.C. and S.C. inoculated leukemiaL1210. Cytoxan has demonstrable activity against

a wide variety of transplantable tumors (1—3,11, 16) and is currently being employed in clinicalstudies (6, 10).

Experiments were conducted under the following headings:

1 The Cytoxan used in these studies was obtained from

Mead Johnson &Company through the courtesyof the CancerChemotherapy National Service Center.

187

Subcutaneously Inoculated Mouse LymphoidLeukemia L1210

MICHAEL A. CHIRIGOS, STEWART R. HUMPHREYS, AND ABRAHAM GOLDIN

(Laboratory of Chemical Pharmacology, NatiOnal Cancer Institute, Bethesda, Maryland)

SUMMARY

The current studies indicate that Cytoxan is considerably less effective in the treatment of intracerebrally (I.C.) than of subcutaneously (S.C.) inoculated leukemiaL1210. Two factors which may contribute to the inability of Cytoxan, or its biologicallyactive form, to produce extensive survival times in I.C. inoculated mice are : reducedefficiency of penetration of the drug into the brain; and the favorable environmentafforded by the intracerebral site for the growth of the leukemic cells.

The diminished therapeutic response of I.C. inoculated mice was observed withdirect intracerebral administration as well as with the subcutaneous administration ofCytoxan.

Tissue bioassay experiments following treatment with Cytoxan indicated that therewas less extensive inhibition in I.C. than in S.C. inoculated mice.

Treatment of mice with Cytoxan prior to inoculation of L1210 provided evidencethat, following injection, Cytoxan or an active metabolic product was present in effective levels for approximately 1 hour in blood and tissues, and for a lesser period in thebrain.

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Cancer Research Vol. 22, February 1962188

1. The relative efficacy of Cytoxan therapy in

mice given inoculations I.C. or S.C. of leukemiaL1210.

2. The effect of intracerebral administration ofCytoxan.

3. The effect of Cytoxan on the transplantability of tissues.

4. The duration of effective drug level.

MATERIALS AND METHODS

Ten- to 12-week-old DBA and CDBA2 malemice weighing 22—28gm. were employed in thisstudy. For each experiment a DBA/2J mouse

CHART 1.—The effect of three dose levels of Cytoxan on thesurvival time of mice given inoculations intracerebrally (I.C.)or subcutaneously (S.C.) of three inoculum levels of leukemiaL1210. A single injection of Cytoxan administered 24 hoursafter leu.kemicinoculation.The numbers in parentheses abovethe arrows indicate the number of micesurvivingformorethan60 days.

bearing a subcutaneous implant of leukemia(L1210) in the right inguinal region was sacrificedon the 9th day after inoculation. The mouse wasanesthetized with ether, a midline incision wasmade, and the diaphragm was exposed and cuttransversely. Blood was collected by cardiac puncture into a heparinized syringe and diluted withsterile saline (0.9 per cent NaCl) to the desiredinoculum concentration.

Intracerebral inoculation was accomplishedemploying a microliter syringe (Hamilton Co.)with a i-inch 26-gauge needle. The skull waspierced, and 0.05 ml. of leukemic inoculum wasintroduced in the area of the thalamus (5). Theanimals were sedated lightly with ether beforeinoculation.

2 (BALB/cAn X DBA/2.J)F1 hybrid male. The hybrid andDBA/2J mice were obtained from the NIH breeding colony.

For retranspiant experiments, brain or spleentissue, removed from either I.C. or S.C. inoculated mice, was prepared as a mash. The tissuewas inserted into a 1- or 2-mi. syringe and forcedthrough a 20-gauge needle into an appropriatevolume of sterile saline. The mash was passedback and forth through the needle into the syringebarrel until a uniform tissue suspension was obtamed. Whole blood was collected by cardiacpuncture as previously described. Sterile salinewas used for all dilutions.

Cytoxan was dissolved in sterile saline. It wasinjected subcutaneously in the axillary region at0.01 ml/gm body weight of the mouse.

RESULTSThe rela!ive efficacy of Cytoxan therapy in mice

given inoculaiions I.C. or S.C. ofleukemia L1210.—In the first experiment (Chart 1), a single injectionof Cytoxan was administered 24 hours after inoculation of the leukemia I.C. or S.C. The resultsindicate that I.C. inoculated L1210 is more difficult to treat than S.C. inoculated disease. All themice given inoculations I.C. of the 1/10 and 1/100dilution of leukemic blood succumbed to thedisease despite drug therapy. In contrast, with thesame inoculum levels and drug concentrations, allbut two of the S.C. inoculated mice survived andshowed no evidence of tumor at 60 days. At the1/1000 inoculum level, no tumor growth occurredin the control S.C. inoculated mice, but all the control I.C. inoculated mice succumbed to the disease.At the 1/1000 inoculum level, in I.C. inoculatedmice, seven of ten mice survived for greater than60 days with the highest drug concentration. Inthis experiment, the 1/1000 inoculum level I.C.and the 1/10 inoculum level S.C. showed approximately the same median survival time. Howevertreatment of the latter group resulted in somewhat greater therapeutic response.

The results of a second experiment (Chart 2)show the effect of Cytoxan therapy in mice withadvanced L1210. Treatment was initiated 7 daysafter intracerebral or subcutaneous inoculationsand continued every 2d day for a total of seveninjections. Treatment was more effective in theS.C. than in the I.C. inoculated groups. The S.C.inoculated mice showed longer median survivaltimes and a number of 60-day survivors. All themice given intracerebral inoculations subsequentlysuccumbed to the disease despite treatment.

The limitation of toxicity of Cytoxan for thehost is indicated by the survival times observed intreated nonleukemic mice. The median survivaltimes for the S.C. inoculated leukemic mice and

CYTCIAN(MCRC)

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I .. I I I

CHIRIGOS et al.—Cytoxan against Leukemia L1@1O 189

the treated nonleukemic controls were similar forthe two highest doses (78 mg/kg; 130 mg/kg).

In this experiment the inoculum levels wereadjusted in an attempt to have I.C. and S.C. inoculated control groups succumb at the sametime. The mice given inoculations I.C. of the1/1000 dilution and the mice given inoculationsS.C. of the 1/5 dilution of leukemic whole bloodhad the same median survival times (11.0 days)(Chart 2). However, treatment of the latter miceresulted in somewhat greater therapeutic effect.

In a third experiment, the effect of continuous

60

50

40

30 -

Cytoxan employed (36 mg/kg) did diminish ineffectiveness when the initiation of therapy waswithheld. However, at the two higher doses thetime of initiation of therapy did not alter thetherapeutic response. With the intracerebraltherapy, death may be attributable primarily tofailure of therapy, rather than to drug toxicity,since the survival time was considerably less thanfor the treated nonleukemic controls.

The effect of intracerebral administra&m ofCytoxan.—The therapeutic efficacy of direct administration of Cytoxan into the brain of mice with

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continued every other day for a total of seven treatments. Thenumbers in parentheses above the arrows indicate the numberof mice surviving for more than 60 days.

CHART2.—The effect of three dose levels of Cytoxan on thesurvival time of mice given inoculations I.C. or S.C. of threeinoculum levels of leukemia L1210. Treatment initiated 7 daysafter intracerebral or subcutaneous inoculation of L1210 and

therapy with Cytoxan initiated at three differenttimes was evaluated in I.C. and S.C. inoculatedmice (Chart 3). In this experiment a twentyfoldmore concentrated inoculum level of leukemicwhole blood was employed S.C. With this adjustment in inoculum level, the median survival timewith the intracerebral and subcutaneous routeswas the same (10.0 days). However, despite theadjustment in inoculum level, Cytoxan was moreeffective in prolonging survival time in the S.C.than in the I.C. inoculated mice. With the S.C.inoculated mice the day of initiation of therapydid not alter the effectiveness of therapy appreciably. Similar increases in survival times wereobtained at each of the doses employed. Theanimals apparently succumbed in large measureto drug toxicity, as indicated by extensive body

weight loss accompanying marked inhibition oftumor growth at the site of leukemic inoculation.The survival time range, with therapy, was similarto that observed in treated nonleukemic mice.

In the I.C. inoculated mice the lowest dose of

CHART 8.—The effect of three dose levels of Cytoxan on thesurvival time of mice given inoculations I.C. or S.C. of leukemm L1210. Mice given inoculations I.C. of 0.05 mL of a 1/100dilution, or S.C. of 0.05 mL of a 1/5 dilution of leukemic blood.Therapy initiated 1, 4, or 7 days postinoculation and continueddaily. The number in parentheses above the arrow indicates thenumber of mice surviving for more than 40 days.

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190 Cancer Research Vol. 22, February 1962

9th day. However, the earlier time of death ofrecipient mice implanted with tissues from theI.C. inoculated mice (Group 1) indicated that

these donor tissues contained a greater number ofleukemic cells than the tissues taken from the S.C.inoculated mice (Group 3).

When tissues were removed from S.C. inoculated mice 2 hours after a single injection of Cytoxan S.C., blood, brain, and six out of sevenspleen transplants failed to produce tumor growthin recipient mice (Group 4). Cytoxan also had aninhibitory effect in the mice given inoculationsI.C. of L1210. Treatment with Cytoxan extended

the survival time and diminished the number ofdeaths from blood and spleen transplants. Tumorgrowth from brain transplants was somewhat retarded, and all but one of the recipient micesuccumbed to leukemic growth (Group 2).

In another experiment (Table 3) the mice weregiven injections of a single dose of Cytoxan 8 daysafter subcutaneous inoculation of L1210. Bloodand brain tissue were bioassayed at subsequentintervals.

It is of interest that, on transplantation, braintissue gave a longer survival time than did blood,

TABLE 1

intracerebral L1210 was tested (Table 1). Onlyminimal increases in survival time were observedwhether Cytoxan was administered I.C. or S.C.1 day following leukemic inoculation I.C. Al

though higher concentrations of Cytoxan couldbe employed and more extensive survival timeswere obtained with administration of the drugS.C. (Chart 1), toxicity for the host3 precluded administration I.C. of doses of drug greater than

100 mg/kg.Effe4 of Cytoxan on transplantability of tissues

A series of experiments was conducted to determine the effect of Cytoxan therapy on the transplantability of tissues taken from I.C. and S.C.inoculated mice with leukemia L1210 (Tables 2,3,and 4).

In two experiments (Table 2), a single injectionof Cytoxan was given S.C. on the 7th or 9th dayafter intracerebral or subcutaneous inoculation,and the effect on transplantability of blood, spleen,and brain tissues was determined. Tumor growthand death occurred in all but one of the recipientmice when the tissues of untreated S.C. and I.C.inoculated mice were transferred on the 7th or

3Unpublishedobservations.

EFFEcT OF INTRACEREBRAL ADMINISTRATION OF CYTOXAN IN MICE GIVENiNOCULATIONS INTRACEREBRALLY OF LEUKEMIA L1210

a Leukemic whole blood diluted with 0.9 per cent sterile saline, and inoculated intracerebrally in the mid.brain, 0.05 mi/mouse:

1/10= 1 ml. leukemic whole blood +9 ml. 0.9 per cent sterile saline.1/100=1 ml. of 1/10 leukemic whole blood +9 ml. 0.9 per cent sterile saline.1/1000= 1 ml. of 1/100 leukemic whole blood +9 ml. 0.9 per cent sterile saline.f A singleinjectionof drugadministeredS.C.in the axiilaryregion,or I.C. in the midbrain,24 hours

afterL1210inoculation.@ Mice given injections I.C. of 0.05 ml. of 0.9 per cent sterile saline.

§MicegiveninoculationsIC. of0.05ml.ofa1/10dilutionofnormalCDBAwholeblood.

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CHIRIG0s et al.—Cytoxan against Leukemia L1210 191

despite the fact that a 50 per cent concentration ofbrain tissue had been employed. This suggeststhat, by the 7th day following subcutaneous inoculation of the disease, there was little, if any,localization of leukemic cells in the brain tissueper Se, or that the leukemic cells were still confined to the blood circulating in the brain. In anyevent, under these circumstances, treatment withCytoxan inhibited the transplantability of leukemia from blood and brain tissue for a period of6 days. Leukemic growth was again observed ontransplantation of blood and brain on the 8thday after treatment.

In the final experiment (Table 4) Cytoxan wasadministered as a single dose 7 days after intracerebral inoculation of L1210, and, as in theprevious experiment, blood and brain tissues werebioassayed at subsequent times. The injection ofCytoxan into the I.C. inoculated mice was onlymoderately effective in inhibiting leukemic growthon transplantation. The inhibitory effect was moreevident on transplantation of blood than of brain.The inhibition was less extensive and of shorter

duration as compared with that observed on treatment of S.C. inoculated mice (Table 3).

Duration of effective drug level.—Experimentswere conducted in order to obtain information onthe duration of effective drug level following administration of Cytoxan (Charts 4—6).In theseexperiments, Cytoxan was administered as asingle treatment before or after inoculation of theleukemic cells.

In the first experiment, Cytoxan (250 mg/kg)was administered S.C. at different time intervals,into mice given inoculations I.C. of L1210 (Chart4). At three inoculum levels no appreciable increase in survival time occurred in mice treatedwith Cytoxan at 1 hour prior to L1210 inoculation. However, a small but definite increase in survival time did occur in mice which received Cytoxan simultaneously4 with or at 1 hour afterL1210 inoculation.

In another experiment, Cytoxan (250 mg/kg)was administered, at narrower time intervals, to

4 Cytoxan injected immediately prior to intracerebral in

oculation of leukemia L1210.

TABLE 2

BIOASSAY OF TISSUFS FROM DONOR MICE GIVEN iNOCULATIONSI.C. OR S.C. OF LEUKEMIA L1210

Results of two separate experiments: Median survival time of nontreated 1.C. inoculated mice(9.0 days); S.C. inoculated mice (18.0 days).

a Donor mice sacrificed on day 7 (Exp. 1) or day 9 (Exp. 9). Average tumor diameter of S.C. inoculated mice (7.4 mm.).

t A1/20inoculumdilutionofleukemicwholebloodused;0.05ml.inoculatedi.C.in themidbrainor S.C. in the right inguinal region.

@ 0.1 ml. of undiluted whole blood, 0.1 ml. of a 10 per cent concentration of spleen, or 0.1 ml. of a50 per cent concentration of brain inoculated S.C. in the right inguinal region of recipient mice.

§Rangeofdeathformicethatdied.Experimentterminatedafter30days.#Theincreaseinsurvivaltimewithbraintransplantwassharplydiminished(1dayincreaseover

controls) in the second experiment.

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ADMINISTRATION

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BIOASSATIN RECIPIENTMICEBloodtBraintD/TMST

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a Donor mice received a single injection of Cytoxan (250 mg/kg), (S.C., axilary region)on the 8th day postinoculation ofLl2lO spleen mash (S.C. in the right inguinalregion). Averagetumor diameter of donor mice on day 8, 11 .8 mm.

Median Survival Time (MST) for untreated donor mice (day 10).Median Survival Time (MST) for treated donor mice (day 21).t 0.1ml.ofa10percentconcentrationofdonormousewholebloodand0.1ml.ofa50per

cent concentration of donor mouse brain inoculated S.C. in the right inguinalregion of recipientmice. Range of death for mice that died. Experiment terminated after 35 days.

@ Untreated controls=untreated donor mice sacrificed on the 8th day postinoculationof L1210.

TABLE 4

BIOASSAY OF BLooD AND BRAIN TISSUES FROM I.C.-IN0cuLATED MICE WITH AD

VANCED LEUKEMIA L1210 PRIOR TO AND AFTER CYTOXAN ADMINISTRATION

a Donor mice received a single injection of Cytoxan (250 mg/kg) S.C. in the axillary regionon the 7th day after intracerebral inoculation of 0.05 ml. of a 1/100 dilution of leukemic wholeblood.

Median survival time for untreated donor mice=day 9.Median survival time for treated donor mice—day 13.t 0.1 ml. of a 10per cent concentrationof donormouse wholeblood and 0.1 ml. of a 50 per

cent concentration of donor mouse brain, inoculated S.C. in the right inguinal region of recipientmite.

@ Range of death for mice that died. Experiment terminated after SOdays.§Untreatedcontrols=untreateddonormicesacrificedonthe7thdaypostinoculationof

L1210.

TABLE S

BIOASSAY OF BLooD AND BRAiN TissuEs FROM S.C.-IN0cULATED MICE WITH AD

VANCED LEUKEMIA L1910 PRIOR TO AND AFTER CYTOXAN ADMINISTRATION

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DIOURS)@ -4-2-I-I/2-V40*V2•I•4

CHmrnos et al.—Cytoxan against Leukemia L1@1O 198

mice given inoculations I.C. of L1210 (Chart 5).A small but definite increase in survival time occurred in I.C. inoculated mice receiving Cytoxan4hourpriortoL1210inoculation.Cytoxanactivity was diminished when the drug was administered 1 hour prior to leukemic inoculation.

In a third experiment, Cytoxan (125 mg/kg)was administered to mice given inoculations I.C.or S.C. of L1210. With this dosage a small increasein survival time occurred in the I.C. inoculatedmice receiving Cytoxan at 15 minutes prior toinoculation of L1210. This was reduced when thedrug was administered 30 minutes early. Withsubcutaneous inoculation of the leukemia, treat

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CHART 4.—Duration of Cytoxan effectiveness in mice giveninoculations I.C. of three inoculum levels of leukemia L19i0. Asingle subcutaneous injection of Cytoxan (250 mg/kg) administered preceding (—)or following (+) inoculation of leukemicblood. No-tumor controls represent mice given injections ofdrug. The number in parentheses indicates the number of micesurviving for more than SO days.

CHART 6.—Duration of Cytoxan effectiveness in mice given

inoculations I.C. or S.C. of 0.05 ml. of a 1/100 dilution ofleukemic whole blood. A single subcutaneous injection of Cytoxan (194 mg/kg) administered preceding (—)or following(+) inoculationof leukemicblood.No-tumorcontrolsrepre

ment was effective when the drug was administered 30 minutes early, but showed a sharp dropin effectiveness when administered 1 hour early. Asobserved in previous experiments, treatment of theS.C. inoculated leukemia was considerably moreeffective than treatment of the I.C. inoculateddisease.

DISCUSSIONThe current studies show that S.C. administered

Cytoxan is considerably less effective in the treatment of I.C. than of S.C. inoculated leukemiaL1210.Thus, when Cytoxanwas administered early

Caairr 5.—Duration of Cytoxan effectiveness in mice giveninoculations I.C. of leukemia L12i0. A single subcutaneous injection of Cytoxan (950 mg/kg) administered preceding (—)orfollowing (+) inoculation of leukemic blood. No-tumor controls represent mice given injections of drug. The number inparentheses indicates the number of mice surviving for morethan SO days.

sent mice given injections of drug. The numbers in parenthesesindicate the number of mice surviving for more than SO days.The S.C. inoculated group receiving drug at —2hours contamed only nine mice.

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Vol. @22,February 1962194 Cancer Research

in the course of the disease, when the leukemic cellsare localized essentially at the site of inoculation,extensive survival times and “notakes― were ohserved at relatively high inoculum levels with thesubcutaneous route of inoculation of the disease.This is in agreement with the previous observations of Venditti et a!. (16) and Lane (11). Atcomparable inoculum levels Cytoxan elicited onlyminimal increases in survival time in the I.C. inoculated mice. With this route of leukemic inoculation, it was necessary to employ a considerablyreduced inoculum level to obtain extensive increases in survival time or “notakes.― The currentobservations are in agreement with those ofSkipper et a!. (15), who demonstrated markedlyreduced effectiveness of Cytoxan and other antineoplastic agents in the treatment of leukemia inoculated into the brain.

Two factors could contribute to the diminishedeffectiveness of Cytoxan in the treatment of I.C.inoculated leukemia:

a) Reduced efficiency of penetration of Cytoxan, or an active form of the drug (7), to thebrain site.

b) The intracerebral site would appear to provide a favorable environment for the inoculationof the leukemia. At equal inoculum levels in untreated controls reduced survival times were ohserved with I.C. as compared with S.C. inoculatedmice. In previous studies (Chirigos et al. [5]), ithad been shown that from the intracerebral siteof inoculation the disease may become systemic in2—Sdays. Effectively, with equal initial inoculuin,Cytoxan would have to act against a greaternumber of viable cells or a more virulent form ofthe disease. It is of interest that when the inoculum level was adjusted so that equal survival timeswere obtained with both routes of inoculation inthe untreated control mice, subcutaneous treatment still remained somewhat less effectiveagainst the intracerebral leukemia.

The diminished efficacy of therapy of intracerebral as compared with subcutaneous leukemiawas also observed in the treatment of the advanced disease. Here, too, treatment of intracerebral leukemia was less effective not only when theinitial inocula were equal, but also when the subcutaneous inoculum was increased so that untreated I.C. and S.C. inoculated controls hadequal survival times. Again, diminished effectiveness of therapy may be attributable to pro

portionately greater localization of leukemic cellsin the brain with I.C. inoculated leukemia andfailure of sufficient drug to reach the I.C. site.Disproportionate distribution of leukemic cells inother nonaccessible areas also remains a possibil

The experiments involving transplantability oftissues before and after therapy provide additionalpertinent information. The reduced survival timesfollowing transplantation of blood, spleen, andbrain of untreated controls with advanced leukemia indicate the more extensive infiltration ofthese tissues following intracerebral inoculation ofthe leukemia.

The results of transplantation of these tissuesfollowing therapy also reflected diminished effectiveness of Cytoxan against the I.C. inoculatedleukemia.

The transplantability experiments also providedinformation on the rapidity and duration of inhibitory action of Cytoxan. Against the S.C. inoculated leukemia Cytoxan appeared to exertmaximum effect by approximately 1—2hours, andthe effect persisted for approximately a week. Therapidity of action against the I.C. inoculated leukemia was approximately the same as for thesubcutaneous route, but recovery from the inhibitory effect occurred in approximately 3 days.Thus, the inhibitory effect of Cytoxan against theI.C. inoculated leukemia was not only dimin

ished, but it was of shorter duration. Burchenalet al. (4) similarly demonstrated that a single doseof Cytoxan given mice with advanced leukemiasL1210, P815, P1081, P1534, B82T, and with Sar

coma 180, rendered these tumors nontransplantable when transplantation was attempted 4—8hours after drug injection.

Not only does the therapeutic action of Cytoxan appear to be rapid, but the effective level inthe host, following injection of therapeutic doses,appears to be of short duration. Thus, pre-inoculation of the drug was ineffective when administered 1 hour prior to inoculation of leukemic cellsS.C. or IC. The drug had a marked inhibitoryactivity when administered@ hour prior to subcutaneous inoculation of leukemic cells but noeffect unless it was administered 15 minutes priorto intracerebral inoculation of the disease.

The observations on the rapidity of action ofCytoxan are in agreement with the previous findings of Foley et al. (7). These investigators demonstrated that serum collected as early as 15 or 30minutes following administration of Cytoxan exhibited marked inhibitory activity in mammaliancell cultures.

The possible contribution of brain infiltration tothe origin of a refractory state in therapy has beenstressed (4—5,8, 9, 12—14,17). The current observation that intracerebral leukemia is considerablymore difficult to treat than subcutaneous diseaseemphasizes the importance of the influence oflocalization of leukemic cells on the effectivenessof therapy.ity.

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CHIRIG0s et al.—Cytoxan against Leukemia L1210 195

REFERENCES

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1962;22:187-195. Cancer Res Michael A. Chirigos, Stewart R. Humphreys and Abraham Goldin Subcutaneously Inoculated Mouse Lymphoid Leukemia L1210Effectiveness of Cytoxan against Intracerebrally and

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