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[CANCER RESEARCH 33, 1735 1746, July 1973]

Kinetics of Entry and Distribution of 5-Fluorouracil in CerebrospinalFluid and Brain following Intravenous Injection in a Primate1

Robert S. Bourke,2 Charles R. West, Girish Chheda, and Donald B. Tower

Departments of Neurosurgery [R. S. B., C. R. W.]and General Clinical Research Cenler [G. C.], Roswell Park Memorial Institute, Buffalo, New York,14203. and Laboratory of Neurochemistry [D. B. T.], National Institute of Neurological Diseases and Stroke, N IH. Bethesda. Maryland 20014

SUMMARY

The distribution of systemically or locally administered5-fluorouracil-2-14C (5-FU) in brain and cerebrospinal fluid(CSF) of the monkey (Macaca mulatta) has been investigated. Following controlled i.v. injection, there was rapidpenetration of the 5-FU into brain and CSF, concomitantwith a rapid loss of 5-FU from the systemic plasmacompartment. CSF repeatedly sampled from various sites(lumbar, cisternal, intraventricular, and cortical surface)displayed characteristic and different kinetic profiles (drugconcentrations versus time) following initial i.v. injection of5-FU. Despite the fact that 91% of the injected dose of 5-FUwas cleared from the vascular compartment within 5 minafter the start of the i.v. injection (5 ml at 1.086 ml/min)and 98% of the injected dose was cleared by 1 hr,approximately 0.126% of the total administered dose represented the average integrated content of the drug in theentire CSF compartment over the experimental hr. Similarly, the average integrated content of the drug in the brainover the experimental hr represented 0.172% of the totaladministered dose of 5-FU, as determined by samplingcerebral and cerebellar cortex, subcortical cerebral andcerebellar white matter, striatum, pons, and medulla ob-longata.

Labeled 5-FU gained access to the CSF from the plasmaby simple diffusion. The average integrated concentration of5-FU in CSF over the experimental hr was increased by 48%when the rate of i.v. injection was increased fourfold. Asdemonstrated by bilateral perfusion over the exposed cerebral cortex or by bilateral ventriculocisternal perfusion,5-FU in the CSF was an adequate source for delivery of thedrug to contiguous brain tissue. Studies of the regional braincontents of 5-FU following combined i.v. and ventriculocisternal administration of the drug are presented, togetherwith investigations of the physicochemical and metaboliccharacteristics of the drug. Our studies provide in theprimate a model for the determination of the kinetics ofdistribution of antineoplastic agents in brain and CSFfollowing i.v. administration or following perfusion into theCSF pathway.

INTRODUCTION

The rational use of chemotherapeutic agents in thetreatment of primary or metastatic neoplastic diseaseinvolving the CNS3 requires a knowledge of the accessibility

of brain tissue and CSF to antineoplastic agents as afunction of the route and rate of administration. It isgenerally appreciated that accessibility of intracranial tissues and fluids by blood-borne solutes cannot be inferredfrom studies of visceral or somatic tissues since blood-bornesolutes may gain access to the CNS slowly or not at allwhile entering other organ systems with ease (15, 39).Systematic study of the accessibility of brain and CSF tomost antineoplastic agents is lacking. Despite the passage of15 years since the introduction of pyrimidine analogs intothe armamentarium of antineoplastic drugs by Heidelbergeret al. (19, 20), there is conflicting evidence regarding theaccessibility (or lack of it) of the brain CSF to 5-FU (6, 22).Since the 5-FU is useful in the treatment of cancers thatmetastasize to brain (24), we considered it as a suitableagent for investigation. We undertook this study to developa systematic approach to the determination of the kineticsof distribution of any antineoplastic drug in the CSFpathway and CNS following initial instillation in thevascular or CSF compartments in a primate animal model.Insofar as possible, we have reported complete data for thesimultaneous distribution of 5-FU in blood plasma, CSF,and brain tissue following administration of the drug.

MATERIALS AND METHODS

Animals and Materials. Adult monkeys (Macaca mulatta,3.5 to 4.5 kg), as supplied commercially to Roswell ParkMemorial Institute, were used in all studies. Experimentswere carried out under phenylcyclidine hydrochloride anesthesia (0.25 mg/kg body weight i.m.; Sernylan, Park, Davisand Co., Detroit, Mich.) and atropine sulfate (0. l mg i.m.).

The following isotopically labeled compounds were used:36C1(390 MCi/ml as chloride in 2.7 N HC1); 3eCl standardsource (2.29 x 10sdpm/ml); /V-antipyrine-methyl-MC (15.6mCi/mmole); toluene-14C standard source (4.45 x 10s

'Supported by Contract NIH, NINDS-72-2328.2To whom requests for reprints should be sent.

Received January 5, 1973; accepted March 28, 1973.3The abbreviations used are: CNS, central nervous system: CSF,

cerebrospinal fluid; 5-FU, 5-fluorouracil.

JULY 1973 1735

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R. S. Bourke, C. R. West, G. Chheda, and D. B. Tower

dpm/ml); all obtained from New England Nuclear (Boston,Mass.). Human radioiodinated serum albumin-131! (0.05mCi/mg) was obtained from E. R. Squibb and Sons, Inc.(New Brunswick, N. J.). 5-FU-2-I4C (15 mCi/mmole;specific radioactivity as used was 17.1 x IO6cpm/^mole)

was obtained from Schwarz/Mann Division of Becton,Dickinson Co. (Orangeburg, N. Y.). Radiochemical purityof the 5-FU-2-14C as determined prior to use by paper

chromatography was >99%. All radiochemicals were madeup in the perfusate solutions at the concentration specifiedfor each experiment. All other chemicals were of the highestgrade commercially available. Unlabeled 5-FU was obtained from Hoffman-La Roche, Inc. (Nutley, N.J.).

Procedures for Injection and Sampling. After achievementof a satisfactory depth of anesthesia, as manifested byquiescence of the experimental animal without compromising spontaneous ventilation or patency of the airway, thefemoral artery and vein on 1 side were cannulated to alength of 7.5 cm with 30-cm segments of polyethylene tubing (inner diameter, 0.045 inch; outer diameter, 0.062 inch;PE 160; Clay-Adams, Boston, Mass.) to provide access fori.v. infusion and arterial sampling. The femoral artery onthe other side was similarly cannulated for the recording ofarterial blood pressure, by connection to a calibratedStatham (Hato Rey, P. R.) transducer. Model P23AA anda Sanborn (Waltham, Mass.) recording system. Thereafter,the animal was placed in the prone, head-up position withthe head fixed in a stereotaxic apparatus so that theorbitomeatal line of the skull was parallel to the operatingtable. An inlying rectal temperature probe was connected tothe recording telethermometer (Model 42SC, YellowSprings Instrument Co., Inc., Yellow Springs, Ohio).

Each isotope was made up for injection in a 5-ml volumeand injected at 1 of 3 constant rates by means of a Harvardinfusion pump, Model 900. The infusion rates as specified ineach experiment were found on calibration to be (ml/min):1.086 Â±0.005, total injection time 4.6 min; 2.135 Â±0.035,total injection time 2.34 min; 0.535 Â±0.007, total injectiontime 9.35 min. During the period of i.v. infusion of isotope,arterial blood ( 1 ml) was drawn into heparinized tubes everymin for the 1st 7 or 9 min and thereafter at 5-min intervals,depending on the duration of the subsequent experimentalperiod. Plasma was rapidly separated as previously described (10).

For sampling of lumbar or cisternal CSF concurrent withsampling of blood, a 20-gauge hypodermic needle waspercutaneously placed atraumatically in the lumbar orcisterna magna subarachnoid space prior to i.v. injection ofthe isotope. The needle shaft had been previously bent sothat the hub of the needle provided a well in which tocontain CSF. The CSF (0.050 ml) was aspirated by amouth-controlled, calibrated Lang-Levy-type micropipet byimmersing the tip of the pipet to the hub-shaft junction inorder to ensure that the CSF aspirated was representative ofsubarachnoid CSF. The "dead space" of the needle was

0.008 ml. The CSF was aspirated at 2, 5, 7, 10, 12, and 15min and thereafter at 5-min intervals, depending on theduration of the subsequent experimental period. Thus, inany single animal not more than 0.75 ml (total) of CSF was

aspirated over the 60-min (maximum) experimental period.This volume represents approximately 5.8% of the totalCSF volume in the monkey. Aspirated CSF was transferreddirectly to counting vials.

For sampling of CSF from over the convexity of thecerebral cortex and in the lateral ventricles, bilateralfrontoparietal craniectomies were fashioned (9, 11). Whencomplete hemostasis was assured, the dura was opened and0.050-ml samples of supracortical CSF were aspiratedbilaterally with micropipets placed into the supracorticalsubarachnoid spaces bilaterally, and the samples of CSFwere transferred to counting vials. Immediately followingsacrifice (38), a micropipet was introduced through thecraniectomy site and cerebral cortex, and crystal-clearlateral ventricular CSF (0.050 ml) was aspirated andtransferred by the micropipet to counting vials.

For sampling of brain tissue after sacrifice of eachanimal, the skull was rapidly removed and the brainincluding brain stem and cerebellum was removed andgrossly blotted (8). Slices (pial surface slice and subadjacent2nd slice, 0.5 mm thick) of cerebellar and somatosensorycerebral cortex were prepared at once by using an unmoist-ened Stadie-Riggs fresh tissue microtome. The 2 slices (pialand subpial) were combined and weighed on a torsionbalance (combined weights about 300 mg), and each pairwas separately homogenized in 2.0 ml of deionized water inPotter-Elvehjem glass homogenizer tubes fitted with motor-driven Teflon pestles. Similarly, biopsies (of about 300 mgeach) of subcortical cerebellar and cerebral (centrum ovale)white matter were bluntly dissected out with scissors.Samples of medulla oblongata (at the level of the inferiorolives), pons (at the level of the facial colliculi), and corpusstriatum (putamen and globus pallidus only) were similarlytaken. These samples were individually prepared as alreadydescribed.

Perfusion of Cerebral Cortex. The method of preparationof plastic domes placed bilaterally over exposed frontoparietal cerebral cortex has been described elsewhere (7, 27).The configuration of the domes permitted perfusion ofexposed cerebral cortices with artificial CSF, withoutmixing of perfusate on 1 side with that on the other andwithout any contamination of the endogenous CSF samplesfrom the cisterna magna (7, 10, 41 ). For perfusion, bicarbonate-buffered saline-glucose solutions were used with thefollowing composition (m.\i, final composition): glucose,20; NaHCO3, 25; NaCl, 122; KC1, 3; CaCl2, 1.3; MgSO4,1.2; KH2PO4, 0.4. Prior to use, stock solutions of perfusate were bubbled with 95% O2:5% CO2 to maintain thepH of the fluid at 7.40 Â±0.03. Simultaneous bilateralperfusion of the exposed cerebral cortex under the domeswas carried out as previously described (7, 9, 11). Thedual infusion pump delivered warmed perfusate (38Â°)at0.747 ml/min, and effluent perfusate was collected as 1-min samples in calibrated tubes for the 1st 30 min and as5-min samples from 30 to 60 min of the experimentalperiod. The effluent perfusate (0.75 ml/min) was sampledfor radioactivity by transfer of specified volumes (0.5 ml) ofeach 1-min sample into counting vials; 0.5 ml of effluentperfusate from the later 5-min samples was similarly trans-

1736 CANCER RESEARCH VOL. 33



5-FU Distribution in Monkey CNS Tissues and Fluids

ferred to counting vials. Thus, both samples of arterialplasma and samples of effluent perfusate were comparedfor activity (cpm/ml) as a function of time after i.v. administration of 5-FU-2-l4C or other labeled compounds.

Similar experiments were carried out to determine thedistribution in brain of 5-FU-2-14C perfused over exposed,

intact cerebral cortex for 1 hr. The basic experimentaldesign was similar to that already described (and furtherdetailed in "Results"), except that the 5-FU-2-14C was

perfused over the cerebral cortices for the full 60 min.Following sacrifice of the animal at the completion of theexperimental hr, a block of tissue representing only exposedand perfused cortex and subadjacent brain structures (including lateral ventricular wall) was removed and seriallysectioned by use of the hand microtome. Each tissue slice(0.5 mm thick and about 150 mg, wet weight) was homogenized separately in 2.0 ml of deionized water for counting.

Ventriculocisternal Perfusion. The procedure for achieving bilateral Ventriculocisternal perfusion as described byFenstermacher (17) was followed. Following bilateral Ventriculocisternal perfusion with perfusate containing 5-FU-2-14C (see individual experiment in "Results"), a block of

tissue known to be in direct contact with perfusate wasremoved and serially sectioned and prepared for counting asalready described.

Monitoring of Vital Signs and Vital Function. Duringexperimentation, arterial blood pressure monitored as already described was found to be maintained at 120 to165/70 to 90 mm of Hg. Plasma pH, pCO2, and pO2 weredetermined on periodic samples of arterial blood at the startand near the end of the experimental period by microgaso-metric analysis (9). The mean values Â±S.D. for theseparameters in 20 animals were as follows (in mm Hg or pHunits): pCO2, 37.5 Â±4.2; pO2, 85.4 Â±6.2; pH, 7.441 Â±0.031. The values for microhematocrits determined onarterial blood from 20 animals at the start of the experimental period were 38.5 Â± 5.2%. Body temperature wasmaintained at 39Â°by warming blankets.

Counting of Samples. Portions of perfusate (0.5 ml), oftissue homogenates (0.5 ml), or of blood plasma (0.2 ml)containing 36C1or 14C-labeled isotopes were placed in glass

counting vials (Wheaton Glass Co., Millville, N. J.) and anequal volume of Hyamine (Nuclear-Chicago Corp., DesPlaines, 111.)was added. Blanks containing water weresimilarly prepared. Samples of CSF (0.050 ml) or thecorresponding blanks were not pretreated with Hyamine.The vials containing Hyamine were incubated for 10 min at70Â°to ensure solubilization of the proteins in the Hyamine.

After the samples were cooled to room temperature, 10 mlof liquid scintillation phosphor (8) were added and thesamples were counted with a Packard Model 3320 liquidscintillation spectrometer. All samples were corrected forquenching by recounting after addition of a standardamount of the appropriate isotope. Samples containingalbumin-131! were counted in glass vials in a Packard Auto

Gamma spectrometer, Serial No. 11265.Partition Coefficient of 5-FU. The lipid: water partition

coefficient was selected as the index for lipid solubility of5-FU (31). A solution (10 ml) of 5-FU-2-14C in 0.16 M

phosphate buffer (pH 7.4) was shaken in a separatory funnelwith an equal volume of various organic solvents for 5 min.Portions of the separated liquid phases were counted forradioactivity (Table 1).

Binding of 5-FU to Plasma Proteins. 5-FU (0.08 to 0.6mM) was dissolved in 0.01 Mphosphate in 0.14 MNaCl (pH7.2) and dialyzed against equal volumes of diluted plasma(plasma was diluted with 4 parts buffer) for 4 hr at roomtemperature by using a Kontron-Diapack (Princeton, N. J.)equilibrium dialysis system. The concentrations of 5-FU inthe 5-FU-buffer half-cell were determined before and afterdialysis from measurements of absorbance at 260 nm on aCary Model 14 spectrophotometer. The binding of 5-FU toplasma proteins was calculated by subtracting twice thefinal concentration in the buffer half-cell from the originalconcentration therein; this difference was divided by theinitial concentration and expressed as a percentage. Themean values for 5 determinations were 0. Thus, for allpractical purposes, 5-FU did not bind to plasma proteins.

lonization of 5-FU. The pKa of 5-FU is reported to be 8.0Â±0.1 (34). Therefore, since pH = pKa + log[FU-]/[FU]

we calculated that 25% of the 5-FU would be expected toexist in the ionized form at pH 7.4.

Determination of the Molecular Integrity of Plasma-borne5-FU. During the course of experiments periodic samples (1ml each) of arterial blood and CSF were taken to providesufficient quantities of fluid for analysis of the molecularintegrity of the 5-FU-2-14C previously injected i.v. Fol

lowing desalting of the samples of CSF (40), samples ofboth CSF and plasma were each spotted, together with amarker of nonradioactive 5-FU on strips of Whatman No. 3Chromatographie paper. Each sample so prepared wasdeveloped (descending) for 16 hr with each of 3 solventsystems: (a) propan-2-ol: water concentrated NH3 (7:2:1,by volume); (b) ethyl acetate :2-ethoxyethanol: 16% (w/v)formic acid (4:1:2, by volume); (c) ethyl acetate:propan-l-ol:water (4:1:2, by volume). The Chromatographie stripswere scanned for radioactivity with a Packard Model 7201radiochromatogram scanner. The location of the unlabeled5-FU was determined by scanning under UV and thecorresponding areas were cut out and eluted and the eluateswere counted for radioactivity. We determined that 70% ofthe radioactivity (cpm/sample) initially streaked on thepapers was recovered, and the radioactivity recoveredrepresented solely intact 5-FU in the samples of CSF andplasma sampled over the experimental hr in vivo. Thecomplete recovery of total counts of radioactivity elutedfrom the chromatograms was considered to represent lossessecondary to handling. Minor breakdown products (25)might have been overlooked; however, we obtained no directevidence for this possibility.

RESULTS

Profiles of Plasma Levels of Various Labeled Compoundsafter a Single Injection. Previous experimental studies haveshown that the kinetics of entry of most drugs from plasmainto CSF is well described in terms of a Ist-order reaction

JULY 1973 1737




(13, 14, 23, 29). Such experimental approaches to the studyof drug entry into CSF from blood require that theconcentration of the drug in plasma be maintained at aconstant level during the period of repeated sampling ofCSF. This requirement of the experimental design denies tothe investigator the profile of the concentration of the drugin the plasma as a function of time. These latter data may beobtained following a single intravascularly administereddose of the drug and may be correlated with the kineticprofile of influx of the drug into CSF from the plasma.Knowledge of the kinetic profile (concentration as a function of time) of a drug in plasma, together with an estimateof the retention of the drug in the plasma relative to the totalinitial dose administered, is of significant theoretical andclinical value. This information may be used to judge theeffectiveness of the vascular compartment as a source of thedrug for organs shown to be accessible to the blood-bornedrug. In addition, the single, limited dose administrationmore nearly correlates with the demands of the clinicalsetting where systemic drug toxicity and patient needs limitthe dose, rate of administration, and scheduling of systemicchemotherapy.

To gain insight into the kinetics of distribution of 5-FUadministered i.V., several labeled compounds with knowndistributions in body tissues and fluids were compared with5-FU following injection into monkeys under identicalexperimental conditions (Chart 1). Albumin-131! [whichover the 30-min duration of the experiment is retainedwithin the intravascular compartment (5)] appeared toincrease in relative concentration in plasma during the 4.6min of i.v. infusion. However, 36C1 and /V-antipyrine-methyl- 14C(both of which are known to gain ready access toCSF and brain and to distribute widely in bodily tissues andfluids (10, 29)], as well as 5-FU-2-14C, appeared to leave the

vascular compartment during the i.v. injection. Doubling orhalving the total amount of radioactive 5-FU or addingvarious amounts of unlabeled carrier (1, 2.5, or 5 mM) 5-FUto the injection did not alter the relative plasma concentrations of 5-FU-2-MC. Even though albumin-131! exhibited a

different rate of dispersion within the vascular tree thanthose obtained for the other compounds, it is clear that 36C1,

Table IDetermination oflipid solubility of 5-FU

Solutions (10 ml) of 5-FU-2-"C in 0.16 M phosphate (pH 7.4) wereshaken for 5 min in a separatory funnel with equal volumes of variousorganic solvents.

Organic phasePartition coefficient

ofS-FU"

HeptaneChloroform"Olive oilChloroform :methanol (2:1)"

1.66 x 10-'7.78 x 10-Â«1.83 x 10-'3.27 x IO-3

Â°The partition coefficient was calculated in every case by dividing theconcentration (cpm/ml) of 5-FU-2-"C in the organic phase by that

determined in the aqueous phase."The organic phase was reconstituted to the original volume prior to

determination of concentration of labeled 5-FU in the organic phasespecified.

INJECTION PERIOD

TIME (MIN)Chart I. The concentration (cpm/ml) of tracer in samples of arterial

plasma expressed as a percentage of the total dose (cpm/ml times volume)injected (ordinate) as a function of time after the start of i.v. infusion(abscissa). Monkeys (3.5 to 4.5 kg body weight) received injections i.v. ofthe individual isotopically labeled compounds indicated (IO x IO6to 30 xI0"cpm in 5 ml of balanced 0.9% NaCl solution) at a constant rate of 1.086ml/min. Coincident with and following the 4.6-min period of injection,samples of arterial blood were drawn at the times indicated. Each plottedpoint represents the mean Â±S.D. (vertical half-bars) for 4 or moredeterminations from 14 animals.

/V-antipyrine-methyl-14C and 5-FU-2-14C distributed in avolume of fluid greater than that attributable to the plasmacompartment as defined by albumin.

From the data on Chart 1, we estimated that during theinjection (0 to 4.6 min) there was at least 6- to 15-fold moreof the original total dose of labeled albumin retained withinthe plasma than there was in the case of 5-FU-2-14C.Moreover, subsequent to completion of the i.v. injection (forthe period 5 to 30 min) there was 22- to 52-fold more of theoriginal total dose of labeled albumin retained within theplasma in comparison to that for 5-FU-2-14C. From thealbumin-131! data we estimated the volume of the plasma

compartment in the monkey (M. mulatta) to be 40.1 ml/kgbody weight, a value which is consistent with those fromsimilar determinations for this species obtained by othermethods (18). On the basis of this estimate of plasmavolume, the percentage of the original total dose of 5-FU-2-14Cinjected that was retained within the plasma compartment (for the 5- to 60-min period postinjection) is small(Chart 2). By 0.4 min after completion of the i.v. infusion of5-FU-2-14C, approximately 91% of the original dose had

been cleared from the plasma compartment, and at 56.4 minpostinjection approximately 98% of the original dose of5-FU-2-'4C had been cleared from the plasma compart -

1738 CANCER RESEARCH VOL.33




o

11.0

S 10.0.2.C

S 9.0I

o'S 7.0

3Â«

3 6.0

< 5.0o.z 4.0

\ 3.0

2Ã¼J orÂ»et Z.O

Â¿ LO

7-i 1 r

INJECTIONPERIUD 50 6020 30 40

TIME (MIN )Chart 2. The content of 5-FU-2-"C remaining (as percentage of the

original dose injected) in the plasma compartment (ordinate) as a functionof time (min) following the start of infusion (abscissa). Monkeys (4.0 Â±0.5kg body weight) received injections i.v. of 5-FU-2-"C, 58.8 Â±3.6 x 10s

cpm in 5 ml of balanced 0.9% NaCl solution at 1.086 ml/min. Samples (Iml) of arterial blood were drawn at the times specified. The radioactivity oftracer (cpm/ml) in each sample of plasma was multiplied by the previouslyestimated volume of the plasma compartment, as determined by thedilution of injected albumin-"1! (see "Materials and Methods"), to give

the value for the total 5-FU contained in the plasma compartment. Eachplotted point represents the mean Â±S.D. (bars) for 4 or more determinations in 10 monkeys. , (10 to 60 min) fitted by the method of leastsquares, Â¡ifdescribed by the equation: In y = 1.51 - 0.0137 (Â±0.001])x.

ment. Clearly, the total amount of the drug in the vasculartree available for transfer into intracranial fluids and tissueswill be small in comparison to the total dose administered tothe animal, regardless of the accessibility of the brain andCSF to the drug delivered from the vasculature.

Kinetics of Entry of 5-FU-2-14C into Various Sites in theCSF Pathway from Blood. During and following the i.v.injection of 5-FU-2-14C, both the arterial plasma and theCSF (sampled from various subarachnoid and intraven-tricular sites) were monitored for radioactivity (Chart 3).During the injection, the cpm of 5-FU-2-14C per ml of

plasma rose to a peak; however, levels of tracer at thecompletion of the injection represented only 5% of thatexpected if the drug were totally retained within the plasmacompartment. Following injection, a plot of levels of tracer(cpm/ml) as a function of postinjection time described asteady decline. However, corresponding plots of the tracerin CSF indicated that the drug levels in CSF describeddistinctly different profiles depending on the site sampled.At no time did the levels of 5-FU-2-14C in CSF exceed the

corresponding drug levels in plasma. The profile of tracerlevels in CSF at all sites samples indicated rising levels ofdrug in CSF for variable periods despite the fact that thecorresponding blood levels were falling. Several distinctprofiles of radioactivity (cpm/ml) of 5-FU-2-14C plotted as

a function of postinjection time were seen for the variousCSF sites samples (Chart 3). CSF in contact with highlyvascular cortical regions attained the highest levels ofactivity, with that over cerebral cortex exhibiting levels oftracer that were essentially in equilibrium with corresponding plasma levels of the drug from 30 to 60 min postinjection

50,000

40,00030,00020.0OOJ18.OOO'

16,000

Ã‹ 14,000

eE 12,000

uÂ§ 10,000

8,000

6,000

4,000

2,000

10 50 6O20 30 40TIME (MIN)

Chart 3. Radioactivity of 5-FU-2-"C (cpm/ml) in arterial plasma and

in CSF sampled from different sites plotted as a function of time (min)following the start of i.v. infusion of the drug. Each plotted point representsthe mean Â±S.D. (vertical half-bars) for 3 or more determinations from 12animals. Monkeys (4.0 Â±0.4 kg body weight) received injections i.v. of5-FU-2-14C, 58.7 Â±3.4 x 10"cpm in 5 ml of balanced 0.9% NaCl solution

at 1.086 ml/min. The total period of injection was 4.6 min. The experimental details are fully outlined in "Materials and Methods." Note that rela

tive to the mean plasma concentration ( 13,000 cpm/ml) the amount transferred to the CNS compartment is large [about 5,000 cpm/ml in CSF andabout 1,400cpm/g in brain (Table 3)1.

JULY 1973 1739




(Chart 3). CSF samples from the cisterna magna where itwas in contact with cerebellar cortex attained the nexthighest levels of drug. The CSF in contact with choroidplexus as sampled from the lateral ventricles exhibited lesserlevels of drug but had a similar kinetic profile of tracerradioactivity (cpm/ml) plotted as a function of postinjectiontime, as described by a parabola with both positive andnegative vectors. This general kinetic profile was similar tothat described by blood levels of 5-FU-2-14C. In contrast,

the plot obtained for the levels of tracer in lumbar CSF atdifferent postinjection times described a parabola with onlya positive vector that approached an asymptote and nonegative vector that paralleled the falling blood levels ofdrug. This striking difference may reflect interchange of5-FU-2-14C between different sites in the CSF pathway asdiscussed below. Addition of unlabeled carrier 5-FU (1,2.5, or 5 mM) to the injected dose did not alter the absoluteradioactivity (cpm/ml) or the kinetic profile of radioactivity of the drug for specific CSF sites. Moreover, doublingor halving the total injected dose of 5-FU-2-14C (with allother conditions maintained constant) changed only theabsolute level of the drug in the blood and CSF (at varioussites) without altering the kinetic profiles. These observations may suggest that 5-FU gains access to CSF fromblood by simple diffusion.

Permeability of the CSF to 5-FU-2-14C Deliveredfrom theBlood. From the kinetic profiles (Chart 3) it was possible toestimate at each CSF site the representative concentrationof 5-FU-2-14C that obtained throughout the entire experi

mental period by integration of the radioactivity (cpm/ml)under the plot describing each profile (36). The integratedaverage level of 5-FU-2-I4C determined for different CSFsites sampled during the 60-min experimental period variedby as much as 103%, as exemplified by the integratedaverage level of tracer in lumbar CSF in comparison to thatin cerebrocortical subarachnoid CSF (Table 2). Similarly,the index of permeability of 5-FU-2-14C from blood intoCSF over the entire experimental period varied withdifferent CSF sites samples (Table 2, Groups B to E).

Table 2Permeability of CSF lo 5-FU-2-"C from blood

GroupA

BCD

HFFluid

sourceArterial

plasmaCSF: overcerebralcortexCSF:

cisterna magnaCSF: lumbarCSF: lateralventricleAv.

of all CSFsources(GroupsB F)Integrated

av." cpm/ml(0 60min)13,1967,9855,695

3,9345,1295,686Ratio

ofindexofpermeability

[CSF:plasma(av.cpm/ml)]0.610.43

0.300.390.43

" Values tabulated represent the arithmetical average of the tabulatedintegrated values for radioactivity (cpm/ml) of 5-FU-2-"C counted in thefluids specified. The mean values plotted in Chart 3 provided the data forthe integrations.

Although the volume of each CSF site undoubtedly differedlike the concentrations of drug at each site, we chose as anapproximation to equate each site in order to assess itsimportance for the entire CSF load of drug. On this basis,the concentration in the cisterna magna (Table 2, Group C)appeared to be the most representative of all the CSF sitessamples (Table 2, Group F) under our experimental conditions. Since the volume of CSF in the monkey is 13 ml (32)and since the dose of drug injected was 58.7 Â±3.4 x IO6

cpm/5 ml in 12 monkeys, the integrated average concentration (Table 2, Group F) of tracer in the total CSF during the60-min experimental period was 5686 cpm/ml. Consequently, the averaged content of 5-FU-2-14C in the entire

CSF for the total experimental period represented 0.126%of the original i.v. dose.

The course which described the changes in the level oftracer in cisternal CSF as a function of time (Chart 3) wassimilar to that which described the efflux of 5-Fu-2-14C

from blood into artificial CSF perfused over the exposedcerebral cortex (Chart 4) (7, 9). Thus we were able to obtaina measure of cerebral capillary permeability to 5-FU-2-'4C

together with the corresponding kinetic profile (cpm/ml ofperfusate per sq cm as a function of time following the startof i.v. injection of the drug). A comparison of the kineticprofile for cisternal levels of 5-FU-2-14C (Chart 3) with

that for the supracortical perfusate (Chart 4) demonstratedthat both curves described a parabola with both positive andnegative vectors and both curves appeared to recapitulatethe plasma profile for (he drug (Charts 3 and 4) whenallowances were made for diffusion delays, inflow dilutions,and volume dilutions.

Kinetics of Entry of 5-FU-2-14C into Brain following i.v.Injection. There was a progressive increase in the content(cpm/g) of 5-FU-2-14C in all regions of brain sampled overthe 60-min experimental period. Generally, tissues composed primarily of gray matter (cerebral and cerebellarcortex and corpus striatum) and mixed gray and whitematter (medulla and pons) contained some 60% more tracerthan tissues primarily composed of white matter (subcorti-cal white matter of cerebrum and cerebellum) (Chart 5).The apparent increase in tissue content of tracer during theexperimental period occurred despite the constantly decreasing levels in the arterial plasma. However, throughoutthe experimental period the plasma content of the drugexceeded that in brain tissue by a factor of 6 to 12. Hencethe plasma may well have acted as the source of the drug forbrain tissue, especially since 5-FU is not bound to plasmaconstituents. With a brain weight of 88.5 g for the monkey(M. mulatta) (12) and a calculated average content of5-FU-2-14C in whole brain determined as 1146 Â±260 cpm/gfrom the data in Chart 5 for the entire 60-min experimentalperiod, we could estimate that the average content of5-FU-2-14C in brain represented 0.172% of the i.v. dose of

the drug. In addition to blood, the CSF undoubtedly was asource for the entry of drug into brain tissue (Table 3; Chart6). When 5-FU-2-'4C was presented to the brain via

artificial CSF perfused over exposed and intact cerebralcortex or by ventriculocisternal perfusion, the drug readilypenetrated brain tissue at a distance from the site of initial





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TIME (MIN )Chart 4. Efflux of 5-FU-2-"C (cpm/ml/sq cm) from the cerebral

vasculature into the perfusate overlying the exposed and perfused cerebralcortices (left ordinale) and the corresponding concentrations of 5-FU-2-"C

(cpm/ml) in arterial plasma (right ordinate) plotted as functions of time(min) following the start of i.v. infusion of the drug. Each plotted pointrepresents the mean Â±S.D. (vertical half-bars) for 4 or more determinations from 4 animals. Monkeys (3.9 Â±0.2 kg body weight) receivedinjections i.v. of 5-FU-2-uC, 59.3 Â±0.3 x 10'cpm in 5 ml of 0.9% NaCl

solution at 1.086 ml/min. The total injection period was 4.6 min. Theexperimental details are fully outlined in "Materials and Methods."

perfusion (Chart 6). This observation may suggest thatCSF-borne 5-FU-2-14C gained access to brain tissue waterby simple diffusion. This conclusion would be consistentwith data determined for other compounds (30).

The Effect of Altered Rate and Site of Administrationof aStandard Dose of 5-FU-2-14C on Levels of the Drug in CSF,Brain, and Blood. Two monkeys matched for weightreceived injections i.v. of identical doses of 5-FU-2-14C in5-ml volumes. The speed of injection into the 1 monkeyexceeded that into the other by a factor of 4. From 15 to 60min after the start of the injection the plasma concentrations of the tracer were quite similar despite the markeddifference in the rates of injection (Chart 7). However, theintegrated average level of radioactivity of drug (cpm/ml)during the 0- to 15-min period after the start of injection(which encompassed the injection "spike") was 46% greater

following the rapid injection than following the slowerinjection. Hence the integrated average concentration of5-FU-2-MC in plasma over the entire 0- to 60-min experi-

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TIME (MIN)Chart 5. Content of 5-FU-2-"C in brain tissue (cpm/g) sampled from

the sites indicated (left ordinate) and the corresponding concentrations of5-FU-2-14C in arterial plasma (IO"3 cpm/ml) (right ordinale) plotted as

functions of time (min) following the start of i.v. infusion of the drug. Eachplotted point represents I or more determinations from 4 animals: data for2 or more determinations are plotted as means Â±S.D. (bars). Monkeys(4.0 Â±0.3 kg body weight) received injections i.v. of 5-FU-2-14C, 58.8 Â±2.6 x 10' cpm in 5 ml of 0.9% NaCl solution at 1.086 ml/min. The total

injection period was 4.6 min. Arterial blood samples were drawn at timesindicated. Animals were sacrificed at 5, 10, 30, and 60 min and brain tissuewas sampled (as described in "Materials and Methods"). The integratedaverage values for the content of 5-FU-2-"C in various brain regions overthe entire 60-min experimental period are as follows (in cpm/g): cerebralcortex, 1504; subcortical cerebral white matter, 725; striatum, 1350;cerebellar cortex, 1127: subcortical cerebellar white matter, 918: pons.1207; medulla, 1197. The arithmetical average content of 5-FU-2-"C in

brain as a whole was calculated from the above data as 1146 Â±260 cpm/g.

mental period was 22.3% greater following rapid injectionthan following slower injection. Similarly, the averageconcentration of 5-FU-2-MC determined in samples ofcisterna! CSF over the entire experimental period followingrapid i.v. injection exceeded by 48% the average concentration of the drug in CSF following slow i.v. injection (Chart7). These observations received further support from plotsof the ratios of the levels of the drug in samples of cisterna!CSF to corresponding levels of 5-FU-2-I4C in plasma as a

function of time following i.v. injection (Chart 8). Theincreasing ratios with time after the rapid injection represented an absolute increase in the CSF concentration of thetracer in comparison to that for the CSF after slowerinjection.

Without alteration of the total dose administered, thebrain content of the drug could be readily increased bychanging the site of drug administration (Table 3). Animals

JULY 1973 1741



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5-FU Distribution in Monkev CMS Tissues and Fluids

Distance (mm) from Lateral Ventricle Surface

9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0

20 3O 4.0 5.0 6.0 7.0 aO 9.0 10.0Distance (mm) from Pial Surface

Chart 6. Content of 5-FU-2-14C in brain tissue (cpm/g) followingperfusion over bilaterally exposed intact cerebral cortex with isotope-richfluid (left ordinale) or following bilateral ventriculocisternal perfusion withisotope-rich fluid (right ordinale) plotted as functions of the distance (mm)from the site of initial perfusion of the drug in the respective experiments.In each experiment a monkey (3.9 kg body weight) underwent eithercerebrocortical or lateral ventriculocisternal perfusion bilaterally with theperfusion solutions containing 5-FU-2-"C, 2.5 x I05cpm/ml for 60 min.

Thereafter the animals were sacrificed and a block of tissue from the areain direct contact with perfusate and extending from the pial cerebrocorticalsurface to the lateral ventricular wall was removed. Tissue sections (0.5 mmthick) were serially cut and counted for radioactivity. At each 10-mm pointthe sections were approximately at the piai or ventricular surface,respectively. The plotted points represent single values. During theperfusions arterial blood samples were drawn at 10-min intervals; theradioactivity in the plasma (cpm/ml) gradually increased over the hr.However, the maximum radioactivity in plasma did not exceed 400cpm/ml. Thus, even a the greatest distance from the sources of the drug,the plasma content could not have significantly affected the tissue levels.Further experimental details are given in "Materials and Methods."

of similar body weight were given approximately the sametotal dose of 5-FU-2-14C. When 3.9% of the total dose was

administered by ventriculocisternal perfusion and the remainder of the drug was administered by simultaneous i.v.infusion, the average content of drug in brain was increasedabout 2-fold above that which obtained following administration of the drug by i.v. infusion alone (Table 3, Groups Aand B). When 20% of the total dose of 5-FU-2-14C was

administered by ventriculocisternal perfusion and the remaining 80% of the dose was administered by simultaneousi.v. infusion, the average content of drug in brain at the endof the experimental period was increased approximately10-fold over that obtained by i.v. injection alone (Table 3,Groups A and C). The individual tissue sites in the brainexhibited increases in drug content consistent with thatalready described for whole brain (all tissue loci averaged).These findings suggested that metastatic neoplasia to brainmay be treated by a program designed to treat the primaryneoplastic disease while simultaneously gaining optimaltreatment of brain lesions using a chemotherapeutic agentthat is freely diffusible from blood to brain.

DISCUSSION

Effective chemotherapy of metastatic and disseminatedneoplasia involving the brain anticipates attainment ofeffective levels of the antineoplastic agents in all sites ofmalignant cellularity. The large, solid tumors metastatic tothe brain are thought to contain microvasculature that is

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Chart 7. Radioactivity of 5-FU-2-"C (IO'J cpm/ml) in arterial plasma

and CSF sampled from cisterna magna (ordinale) plotted as a function oftime (min) after the start of i.v. infusion of the drug (abscissa). Each plottedpoint represents a single determination. In the individual experiments amonkey (3.8 kg body weight) received 5-FU-2-"C (61.4 x 10*cpm in a

volume of 5 ml of 0.9% NaCI solution) by i.v. infusion. The rate of i.v.injection into I animal was 0.535 ml/min (â€¢),and the duration of injectionwas 9.35 min. The rate of i.v. injection into the other animal was 2.135mi/min (O), and the duration of i.v. injection was 2.34 min. During the60-min experimental period, samples of arterial blood (I ml) and cisternalCSF (0.050 ml) were taken for counting radioactivity, at the timesindicated. Following the rapid i.v. injection (2.135 ml/min) the integratedaverage concentration of 5-FU-2-"C in plasma and CSF was (cpm/ml):

plasma (0 to 60 min), 14,024; plasma (0 to 15 min), 24.935; CSF (0 to 60min), 6,788. Following the slow i.v. injection (0.535 ml/min) the integratedaverage concentration of 5-FU-2-"C in plasma and CSF was as follows

(cpm/ml): plasma (Oto 60 min), 11,471;plasma (Oto 15min), 17,138;CSF(O lo 60 min), 4,576.

JULY 1973 1743




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TIME (MIN)Chart 8. Ratios of the concentrations of 5-FU-2-"C in CSF sampled

from cisterna magna to the concentrations of 5-FU-2-"C in arterial

plasma (ordinate) plotted as a function of time (min) following the start ofi.v. infusion of the drug (abscissa). Monkeys (3.4 to 4.1 kg body weight)received injections of isotopically labeled drug (41.1 x IO6cpm in 5 ml of0.9% NaCl solution). The rate of i.v. injection of labeled 5-FU in 1group ofmonkeys was 2.135 ml/min (O), and the duration of i.v. injection was 2.34min. The rate of i.v. injection of labeled 5-FU in the other group of animalswas 0.535 ml/min (â€¢),and the duration of injection was 9.35 min. Duringthe 60-min experimental period samples of arterial blood (1 ml) andcisternal CSF (0.050 ml) were taken for counting radioactivity at the timesindicated. Each plotted point represents the mean Â±S.D. (represented byvertical half-bars) for 3 determinations of ratios for 6 animals.

structurally abnormal and apparently quite permeable toblood-borne solutes, including compounds not usuallythought to cross the blood-brain barrier (1, 28, 37, 42, 43).However, small nests of malignant cells, such as leukemicinfiltrates or malignant cell implants, undoubtedly areeffectively bathed only by those solutes that normally crossthe blood-brain barrier. Thus, a cytotoxic agent that usuallygains only limited access to the brain and CSF followingintravascular administration would be of little value in thetreatment of small, malignant cellular nests or disseminatedcancers that do not have a well-developed intrinsic mi-crovasculature. Moreover, effective levels of antineoplasticagents in the CNS must not be achieved at the expense ofunacceptable systemic drug toxicity. The physician may notuse inordinate dosage of drugs to compensate for relativelyrestricted accessibility of these agents to the brain and CSF.

Consequently, an understanding of the kinetics of distribution of antineoplastic agents in the CNS as a function of theroute and rate of administration will provide the insightrequired for safe and effective use of drugs in the mostpractical manner. Indeed, the clinical correlate for thepractical application of systemic and CNS chemotherapy toprevent recrudescence of a malignant disease is demonstrable in the current trends in the management of childhoodlymphoblastic leukemia in which adequate treatment of thesystemic disease requires adequate treatment of the subclin-ical disease in the central nervous system (2).

The blood-borne solutes that do gain ready access tobrain and CSF following parenteral administration usuallydo not achieve homogeneous distribution in either CSF orbrain tissue sampled from various anatomical sites (Refs. 3and 35 and the data presented herein). The rate of influx of5-FU-2-14C into CSF varied as a function of the locus

within the CSF pathway that was sampled, and at any single time the concentration of 5-FU-2-I4C in CSF sampledfrom various sites in the CSF pathway varied by as much asa factor of 8 (Chart 3). Clearly, there was no single locus inthe CSF pathway that was entirely representative of theCSF as a whole, although CSF sampled from the cisternamagna (Table 2; Chart 3) appeared to be the mostrepresentative under our experimental conditions. In contrast, levels of 5-FU-2-14C in CSF sampled from the lumbar

subarachnoid space demonstrated quite a different kineticprofile (Chart 3). While levels of 5-FU-2-I4C in CSFsampled from other sites were falling during the 30- to60-min postinjection, the levels of the drug in lumbar fluidcontinued to rise. This profile may reflect entry of the druginto lumbar CSF from other CSF sites "upstream."

Progressive relative lumbar sequestration of solutes initiallyintroduced directly into CSF by intracisternal or intraven-tricular injection has been demonstrated for a number ofcompounds under various experimental conditions (8, 21).Similar findings have been reported following initial instillation of an indicator solute into lumbar CSF (32). Both thekinetic profile of 5-FU-2-14C and the integrated average

concentration of the drug in lumbar CSF during theexperimental period are at best poorly representative of theCSF as a whole and bear little relationship to the kinetics ofdistribution of the drug in the intracranial fluids bathing thebrain.

It may be of considerable potential clinical importancethat the rapid i.v. injection of 5-FU-2-14C significantly

increases the concentration of the drug in the CSF incomparison to that attained with a slower rate of i.v.injection (Chart 7). This finding is important because theCSF can be an adequate and significant source of the drugfor brain tissue, as demonstrated in this study (Chart 6; seealso Ref. 30) and can well complement the blood as a sourceof 5-FU-2-I4C. Since the radioactivity (cpm/ml) in blood

over the experimental hr (Table 1) represented intact5-FU-2-14C and not a metabolite, we can be quite confident

that the isotope sampled in CSF following the i.v. infusionrepresented labeled 5-FU that readily diffused into the CSFfrom the blood. Clearly, in order to attain an overallincrease of 5-FU-2-14C in brain, the administration of the





drug by rapid i.v. infusion together with simultaneousventriculocisternal perfusion of the drug would be theadministration scheme of choice (Table 3). The combinedrapid i.v. infusion of drug plus simultaneous ventriculocisternal perfusion of the drug achieved increased levels ofdrug in all regions of brain without increasing the total doseof drug given to the animal. The results with ventriculocisternal perfusion essentially paralleled those with ven-triculosubarachnoid perfusion (Chart 6), and the latterprocedure has been shown to be clinically applicable for theperfusion of antineoplastic agents via a s.c. ventricularreservoir (26, 33). Ventriculosubarachnoid perfusion alone,however, may lead to highly variable levels of drug in tissuesin direct contact with drug-ladened perfusate or in tissuesaccessible to the drug by diffusion from the perfusate (30).Moreover, variably effective perfusion or variable distribution profiles of drug in brain tissue as reflections of thechemical characteristics of a particular compound per semight deprive some regions of the brain of chemo-therapeutically effective levels of drug (16). And i.v. administration alone of a solute like 5-FU that readily diffusesinto brain and CSF may lead to variable levels of the drug inbrain tissue (Chart 5) that may be chemotherapeuticallyinadequate. One reason for this last possibility is the rapidclearance of the drug from the systemic vasculature (Charts1 and 2). At least in the case of 5-FU-2-14C, there is acorrespondence between the ready permeability of cerebralcapillaries to the blood-borne drug (Chart 4) and the generalsystemic capillary permeability suggested in Chart 2. Thus,the plasma source of 5-FU is effectively reduced by its rapidloss from the systemic vascular tree following i.v. injection.

Generally, drugs that would be expected a priori to crossthe blood-brain barrier following intravascular infusion areboth highly lipid soluble and undissociated at physiologicalpH (23, 29, 31). Nevertheless, 5-FU, which readily crossesthe blood-brain barrier, exhibits little or no lipid solubility(Table 1) but does exhibit significant (~25%) ionization atphysiological pH. This important observation suggests thateach antineoplastic drug should be experimentally andsystematically evaluated to determine the accessibility ofbrain tissue and CSF following its i.v. administration.Moreover, optimal routes and rates of administrationshould be determined when anticipating use of the agentagainst neoplastic disease involving the CNS.

REFERENCES

1. Aleu, F. P., Edelman, F. L., Katzman, R., and Scheinberg. L. C.Ultrastructural and Biochemical Analysis in Cerebral Edema Associated with Experimental Mouse Gliomas. J. Neuropathol. Exptl.Neurol., 23: 253 263, 1964.

2. Aur, R. J. A.. Simone, J., Hustu, H. O., Walters, T., Borella, L.,Pratt, C., and Pinkel, D. Central Nervous System Therapy andCombination Chemotherapy of Childhood Lymphocytic Leukemia.Blood. 37: 272 281, 1971.

3. Bakay, L. The Blood-Brain Barrier, with Special Regard to the Use ofRadioactive Isotopes, p. 154. Springfield. 111.:Charles C ThomasPublisher. 1956.

4. Bakay, L. The Extracellular Space in Brain Tumors. II. The SucroseSpace. Brain, 93: 699 708, 1970.

5. Bender, M. A. Blood Volume of the Rhesus Monkey. Science, 122:156, 1955.

6. Bering, E. A., Wilson, C. B., and Norell, H. A. The KentuckyConference on Brain Tumor Chemotherapy. J. Neurosurg., 27: 1 10,1967.

7. Boretos, J. W., Bourke, R. S., Nelson, K. M., Naumann, R. A., andOmmaya, A. K. Technique for Unilateral Isolation of the SubduralSpace in the Intact Primate. J. Neurosurg., 35: 101 107, 1971.

8. Bourke, R. S.. Greenberg, E. S., and Tower, D. B. Variation ofCerebral Cortex Fluid Spaces in Vivo as a Function of Species BrainSize. Am. J. Physiol., 208: 682 692, 1965.

9. Bourke, R. S., and Nelson, K. M. Further Studies on the K*-depend-

ent Swelling of Primate Cerebral Cortex in Vivo:The Enzymatic Basisof the K'-dependent Transport of Chloride. J. Neurochem., 19:663-685, 1972.

10. Bourke, R. S., and Nelson, K. M. Studies on the Site of MediatedTransport of Chloride from Blood into Cerebrospinal Fluid: Effects ofAcetazolamide. J. Neurochem., 19: 1225 1232, 1972.

11. Bourke, R. S., Nelson, K. M., Naumann, R. A., and Young, O. M.Studies of the Production and Subsequent Reduction of Swelling inPrimate Cerebral Cortex under Isosmotic Conditions in Vivo. Exptl.Brain Res., 10: 427 446, 1970.

12. Count, E. W. Brain and Body Weight in Man: Their Antecedents inGrowth and Evolution. Ann. N. Y. Acad. Sci., 46: 993 1122, 1947.

13. Davson, H. A Comparative Study of the Aqueous Humor andCerebrospinal Fluid in the Rabbit. J. Physiol. London, 129: 111 133;454, 1955.

14. Davson, H. Physiology of Ocular and Cerebrospinal Fluids, p. 388.London: J. and A. Churchill, Ltd., 1956.

15. Dobbing, J.The Blood Bain Barrier. Physiol. Rev..41: 130 188, 1961.16. Feldberg, W. A Pharmacological Approach to the Brain from Its

Inner and Outer Surface, pp. 8 30. Baltimore: The Williams &Wilkins Co., 1963.

17. Fenstermacher, J. D. Ventriculocisternal Perfusion as a Technique forStudying Transport and Metabolism within the Brain. In: N. Marksand R. Rodnight (eds.), Research Methods in Neurochemistry, pp.165 178. New York: Plenum Press, 1972.

18. Gregersen, M. I., Sear, H., Rawson. R. A., Chien, S., and Sarger, G.L. Cell Volume, Plasma Volume, Total Blood Volume and F Factor inthe Rhesus Monkey. Am. J. Physiol., 196: 184-187, 1959.

19. Heidelberger, C., and Ansfield. F. J. Experimental and Clinical Use ofFluorinated Pyrimidines in Cancer Chemotherapy. Cancer Res., 23:1226 1243, 1963.

20. Heidelberger, C. Chaudhuri, N. K., Danneberg, P., Mooren. D..Griesback, L., Duschinsky, R., Schnitzer, R. J.. Pleven, E., andScheiner, J. Fluorinated Pyrimidines. a New Class of Tumor-inhibitory Compounds. Nature, 779: 663 666, 1957.

21. Korobkin, R. K., Lorenzo, A. V., and Cutler, R. W. P. Distribution ofC "-Sucrose and I '"-Iodide in the Central Nervous System of the Cat

after Various Routes of Injection into the Cerebrospinal Fluid. J.Pharmacol. Exptl. Therap., 164: 4\2 420, 1968.

22. Kotsilimbas. D. G., Karpf, R., Meredith, S., and Scheinberg, L. C.Evaluation of Parenteral 5-FU on Experimental Brain Tumors.Neurology, 16: 916 918, 1966.

23. Mayer, S., Maickel, R. P., and Brodie, B. B. Kinetics of Penetration ofDrugs and Other Foreign Compounds into Cerebrospinal Fluid andBrain. J. Pharmacol. Exptl. Therap., 727: 205-211, 1959.

24. Moore, G. E. Effects of 5-Fluorouracil (NSC-19893) in 389 Patientswith Cancer. Cancer Chemotherapy Rept.. 52: 641 653, 1968.

25. Mukherjee, K. L., Boohar. J.. Wentland, D., Ansfield. F. J., andHeidelberger, C. Studies on Fluorinated Pyrimidines. XVI. Metabolism of 5-Fluorouracil-2-C" and 5-Fluoro-2-deoxyuridine 2-C" in

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1973;33:1735-1746. Cancer Res Robert S. Bourke, Charles R. West, Girish Chheda, et al. a Primate

inCerebrospinal Fluid and Brain following Intravenous Injection Kinetics of Entry and Distribution of 5-Fluorouracil in

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