[CANCER RESEARCH 45,1692-1701, April! 985]

Regional Measurements of [14C]Misonidazole Distribution and Blood Flow in

Subcutaneous RT-9 Experimental Tumors

Ronald Blasberg,1 Marc Horowitz,2 John Strong, Peter Molnar,3 Clifford Patlak, Ernest Owens,4 andJoseph Fenstermacher5

Laboratory of Medicinal Chemistry and Pharmacology, Division of Cancer Treatment, National Cancer Institute [R. B., M. H., J. S., P. M., E. O., J. F.], and TheoreticalStatistics and Mathematics Branch, National Institutes of Mental Health [C. P.], Bethesda, Maryland 20205

ABSTRACT

Regional [14C]misonidazole-derived radioactivity (MISO*) was

measured by quantitative autoradiography in s.c. RT-9 experi

mental tumors 0.5, 2, and 4 h after an i.v. bolus (25 mg) andconstant infusion (10 mg/h) in rats. Misonidazole (MISO) concentration in plasma, tumor, and other tissues was also measuredby high-pressure liquid chromatography. The distribution ofMISO* in the tumors always resulted in a characteristic pattern

with high peripheral and low central values. The high-activityregions in the tumor rim achieved tissue: plasma MISO* activity

ratios of 0.97 and 2.2 by 0.5 and 4 h, respectively; for centraltumor regions, this ratio was 0.20 and 0.32 for the same periods,respectively. The limited distribution of MISO* to central tumor

regions could be correlated to low values of blood flow (measuredwith [131l]iodoantipyrine) and to diffusion from peripheral tumor

regions. Low blood flow in the central regions of these tumorswill significantly limit the distribution of MISO and other drugs toviable-appearing cells in these areas and could account in partfor the failures of chemotherapy in certain solid tumors. Phar-

macokinetic modeling indicates that 1 to 9 h may be necessaryfor MISO concentrations in some tumor regions to reach 50% ofthat in plasma.

INTRODUCTION

Two central issues being addressed by many investigators inthe field of radiation biology are the location and identification ofhypoxic cell populations within the tumor and the application ofvarious modalities to increase the sensitivity of hypoxic cellswithin solid tumors to radiotherapy. The administration of aradiosensitizing compound, such as MISO,6 prior to radiation is

one therapeutic modality currently under intensive investigation.This has resulted in numerous pharmacological studies in animalsand humans that were designed to evaluate the delivery anddistribution of these compounds in a variety of tumors and to

1Present address: Nuctear Mediane Department, Clinical Center, NIH, Be

thesda, MD 20205. To whom requests for reprints should be addressed, at Building10, Room 1C401. NIH, Bethesda, MD 20205.

2 Present address: Department of Hematotogy and Oncology, St. Jude Children's

Research Hospital, 332 North lauderdale. Memphis, TN 38101.'Present address: Department of Pathology, University Medical School of

Debrecen, H-4012 Debrecen, POB 23, Hungary.'Present address: Nuclear Medicine Department, Clinical Center, NIH, Be

thesda. MD 20205.8Present address: Department of Neurological Surgery, Health Sciences Center,

T12-082, State University of New York at Stony Brook, Stony Brook, NY 11794.'The abbreviations used are: MISO, misonidazole; MISO', [14C]misonidazole

plus misonidazole-denved "C-labeled metabolites: DESMISO, desmethylmisoni-dazoie: MlSOf, MISO equivalents (misonidazole calculated from "C radioactivityand specific activity data); [131I)IAP,"'(-labeled todoantipyrine; HPLC, high-pressure

liquid chromatography; A-V, arteriovenous; QAR, quantitative autoradiography; i.e.,intracerebral.

Received 2/13/84; revised 7/16/84.12/11/84; accepted 1/2/85.

determine the optimum time for radiation after drug administration. For example, Rich ef at. (23) have made serial measurements of MISO concentration in tumor tissue, compared themto plasma values, and reviewed similar measurements obtainedduring the many clinical trials which have been undertaken withthis drug. Average values of MISO in human tumors ranged from12 to >100% of the plasma concentrations 4 h after p.o. administration of the drug. Strong ef a/. (26) measured serial tumorMISO levels in 2 patients following a 5-min i.v. infusion and

showed that the tumor concentration reached a maximum, 80%of the plasma drug level, as early as 10 to 20 min after MISOadministration. All of these measurements, however, determinedmean MISO levels in the whole tumor or representative samplesof the tumor. Ash ef a/. (1) found only modest variation of MISOlevels in various regions of tumors that they sampled usingdissection techniques, whereas Rich ef a/. (23) demonstratedthat drug levels in tumor samples correlated inversely with thepercentage of necrosis observed in histological sections prepared from adjacent tissue. The importance of necrosis, a common feature of malignant cell populations, and its relationship toregional variations in drug concentrations is generally recognized(12), but the quantitative aspects of drug delivery to the tumorand its relationship to regional blood flow in the tumors is lessappreciated (20). Nevertheless, the adequate delivery of MISOto the tumor and knowledge (or some estimate) of the timecourse of MISO concentration within the hypoxic tumor regionsis essential vis-à-vis timing of radiotherapy for optimal tumoricidal

effect.The purpose of this study was to assess the regional distri

bution of [14C]MISO-derived radioactivity in s.c. implanted RT-9

tumors in rats after 30 min, 2 h, and 4 h of constant plasmalevels using quantitative autoradiographic techniques. MISO (determined by HPLC) and MISO* were measured in blood, tumor,

and various organ tissues to assess drug delivery and the extentof drug metabolism during continuous i.v. administration. Relationships between the regional distribution of MISO*, blood flow,

and specific histological features of the experimental RT-9 flanktumor were investigated using double-label quantitative autora

diographic and computerized imaging techniques.

MATERIALS AND METHODS

Animal Model and Preparation. RT-9 tumor cells, originally inducedby i.v. injections of A/-nitrosomethylurea in CD Fisher rats (3) were

provided by Paul T. Komblith from continuous cell culture. Tissue culture,preservation, and transplantation methods have been described previously (25). In vitro cultures of RT-9 tumor cells were harvested after 48 h

by trypsinization (0.25% for 10 to 15 min) and adjusted to a concentrationof 4.5 to 5.5 x 10* cells/5 n\ in Hanks solutions. Adult male CD Fischer

rats, 160 to 200 g body weight, were anesthetized with ether, and 5 M!

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INTRATUMOR MISO DISTRIBUTION AND BLOOD FLOW

of the cell suspension were inoculated into the right flank. MISO distribution and blood flow experiments were carried out 18 to 21 days afterinoculation when the s.c. tumors were approximately 1 cm in diameter.

The animals were anesthestized with halothane:nitrous oxide:oxygen(1.5:70:30, v/v/v). Bilateral femoral artery and vein catheters (PE-50,

polyethylene tubing) were inserted, the animal was heparinized with 0.2mg (approximately 28 units) heparin in 1 ml 0.9% NaCI solution (saline)and a single extracorporal A-V shunt was prepared as described previ

ously (5). The animals were immobilized below the midthorax by wrappingthem with a 2-inch plaster bandage that did not impinge on the s.c.

tumor, taped to a lead brick, and allowed to recover from anesthesia forperiods 1 to 4 h. Body temperature was continuously monitored andmaintained at 35-37 °Cusing heat lamps. Arterial blood pressure, pO2,

pCO2, and pH were monitored during the recovery period and just priorto each experiment; arterial blood pressure was monitored throughoutthe experimental period.

Experimental Procedures. Regional and whole-tissue measurementsof MISO [1-(2-nitro-1-imidazolyl)-3-methoxy-2-propanol] (NSC 261037;Ro-07-0582) were made after i.V. administration. [14C]MISO, [2-14C]-(2-

nitroimidazolyl)-3-methoxy-2-propanol, 76 ¿iCi/mg,was supplied by Mor

ris Leaffer (Stanford Research Institute, Menlo Park, CA) under a NationalCancer Institute contract. Radiochemical purity was determined by thin-

layer chromatography using a Silica Gel 60F glass plate (E. Merck, St.Louis, MO) and an ethyl acetate or acetone solvent system; radiochem-

ical purity was found to be 98%.The concentration of MISO and its metabolite, DESMISO, in blood

and tissue were determined by a modification of the HPLC method ofWorkman ef al. (28). Aliquots of blood, 50 ¡A,were analyzed for theparent drug and metabolite by adding 180 n\ of water and 0.8 ml ofmethanol containing 8 ^g of the internal standard, 1-(2-nitro-1 -imidazolyl)-3-fluoro-2-propanol (NSC 292930; Ro-07-0741 ). The sample was thor

oughly mixed by vortex and centrifugea at 800 x g for 5 min to removethe precipitated protein. A 50-^1 aliquot of the supernatant was analyzed

by HPLC (Waters Associates, Inc., Milford, MA). Separation of thecompounds was achieved with a Partisil PxS 5/25 ODS column (Whatman, Inc., Clifton, NJ). The mobile phase was 19% methanol and water;the flow rate was 2 ml/min. Elution times were 2.4, 3.5, and 5.2 min forDESMISO, the internal standard, and MISO, respectively. The averageconcentration of MISO and DESMISO in tissue samples was determinedby homogenizing a representative sample (200 mg, wet weight) with 0.8ml of the internal standard solution described above using a 4-ml Teflonpestle homogenizer (Arther H. Thomas Co., Philadelphia, PA). The ho-mogenate was centrifuged at 800 x g for 5 min, and a 50-¿ilaliquot of

the supernatant analyzed by HPLC as described for the blood samples.All drug concentrations are reported as ^g of MISO per g, wet weight,of tissue.

The distribution of MISO, DESMISO, and MISO* in blood and tissue

was determined from experiments in which unlabeled MISO was addedto the radioactive compound to achieve a saline concentration of 25 mg/ml; this concentration of MISO resulted in a specific activity of 0.87 pC\/mg. Approximately 22 pd of [14C]MISO in 1 ml of neutral saline were

administered to each experimental animal by an i.v. injection over 10s.At 10 min, a constant i.v. infusion of the same [14C]MISO solution, 0.4

ml/h, was initiated and continued for the duration of the experiment.Timed blood samples, approximately 0.1 ml, were rapidly drawn fromthe 3-way stopcock in the A-V loop, cooled, and processed for HPLC

analysis and radioactivity determination by ßliquid scintillation countingusing a Beckman LS 350 spectrometer and external standard quenchcorrection. Three experimental time periods were studied: 0.5, 2, and4 h. The animal was decapitated at the end of the experimental period;the tumor was rapidly extracted, bisected, and frozen in liquid Freonwhich was cooled to -40°C with liquid nitrogen and dry ice. The time

from decapitation to tumor freezing was approximately 1 min. One halfof the tumor was assayed for total 14Cradioactivity by liquid scintillation

spectrometry and for MISO and DESMISO by HPLC; the other half wasprocessed for quantitative autoradiography.

In an additional group of animals, sequential measurements of regionalMISO* and blood flow were made in the same animal using double-label

quantitative autoradiographic techniques. In these studies, [131I]IAP, 4-[131I], 3 mCi/mmol, was used to measure blood flow. [13'I]IAP was

synthesized by an iodine-exchange reaction using a method developedby Dr. Ira Katz.7 Radiochemical purity was determined by chromatogra

phy and was found to be greater than 98%. The experimental procedurewas similar to that described above. Animals were given a 1-ml injectionof [14C]MISO into a femoral vein which was followed by a constant i.v.

infusion 10 min later to achieve constant arterial plasma levels of MISO.Timed arterial blood samples were obtained. At 28.5 min after [14C]MISOinjection, an increasing infusion schedule of [131I]IAP (1 mCi total) was

initiated, and serial 10-s integrated arterial samples were obtained fromthe extracorporal A-V loop. Ninety s after beginning the infusion of [131I]-IAP and 30 min after the administration of [14C]MISO, the animal was

decapitated, and the tissue was rapidly frozen.For samples containing both [131I]IAPand [14C]MISO, 131Iactivity was

determined first in a Packard 5385 gamma spectrometer using standardnarrow-channel peak analysis and the appropriate correction for isotopedecay during the period of sample counting. The efficiency of 13'l countingwas 33%. The samples were then stored at 4°C, and the 13'l was

allowed to decay away for at least 15 half-lives. Samples were checkedfor the absence of -y-émissionsand then processed for ßcounting; 14C

radioactivity was measured as described above.Quantitative Autoradiography and Histology. Tissue sections were

prepared for histology and QAR as described previously (5). Tumorswere mounted on planchéis,and 8 serial sections were cut 20-Mm thickat -20°C in a cryomicrotome at intervals of 400 to 600 Mm. Sections 3

through 6 were placed on glass coverslips, dried rapidly on a slidewarmer at 65°C, and glued to pressed cardboard (Bainbridge Board

172) for autoradiography. Sections 1,2 and 7,8 were placed on microscopic glass slides, fixed in a formalin:ammonium bromide solution (2 gNH4Br/100 ml 10% formaldehyde), and stained with hematoxylin andeosin or cresyl violet. When precise correlation of the histological andautoradiographic images was required, the sections used for autoradiography were fixed and stained following X-ray film exposure. The driedtissue sections were placed in a casette along with 15 [14C]methylme-

thacrylate standards (Amersham Corp., Arlington Heights, IL) and single-coated X-ray film (MR-1 ; Kodak, Rochester, NY) for a 6-week period ofexposure. The [14C]methylmethacrylate standards had been previously

calibrated to reference 20-Mm-thick brain sections of known radioactivityand ranged between 4.5 and 2354 nCi/g. To convert the X-ray film

images to tissue radioactivity (nCi/g), the mean absorbance of each¡mageproduced by the 14C standards was measured and a standard

curve which related absorbance to tissue radioactivity was generatedfor each film. Sequential measurements of absorbance within 50- x 50-

nm elements of the tissue autoradiographic image were made using acomputerized high-speed scanning microdensitometer. The absorbance

data were scored by the computer and could be converted to radioactivityusing the standard curve described above and displayed on a videomonitor; this was accomplished by a system similar to that described byGoocheeefa/. (17).

The double-level quantitative autoradiographic techniques have also

been described (4, 5). Briefly, tissue sections were prepared and processed as described above. Standards of [13'I]IAP over a wide activity

range (45 to 30,500 nCi/g) were prepared from homogenized calf brain,sectioned, and processed in a manner identical to that for the tissuesections. A single sheet of plastic Mylar film, 3 mils thick, was placed inthe casette between the tissue sections and standards and the X-rayfilm (SB-5; Kodak). The Mylar film absorbed the /i-emissions from 14C

decay but permitted exposure of the X-ray film to the more energeticemissions from 131Idecay (4). Following this exposure, the tissue and131l-labeled standard sections were removed and carefully stored under

dry atmospheric conditions for at least 4 months (15 half-lives) to permit

71.Katz, personalcommunication.

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INTRATUMOR MISO DISTRIBUTION AND BLOOD FLOW

1311decay. The absence of -y-émissionsfrom residual 131Ior contaminants

after the 4-month storage period was determined by spectroscopy before

proceeding with the second autoradiographic exposure. The secondexposure (from 1 to 3 weeks) of SB-5 X-ray film to the tissue sectionswas without interposition of the Mylar film; it also included the [14C]-methylmethacrylate standards and the original 131Istandards to confirmthe absence of an 131Ior contaminant-produced image.

The autoradiographic images obtained from the first exposure werealmost entirely due to 131Idecay. Most of the 14C /i-emissions were

absorbed by the Mylar film; this was determined by the lack of an imageproduced by the [14C]methylmethacrylate standards for the appropriaterange of 14Cradioactivity in the tissue (4). The autoradiographic imagesobtained from the second exposure were entirely produced by 14C-/3-

emissions; this was determined by the absence of an image on thatportion of the film exposed to the 131Istandards. Thus, 2 autoradiographic

X-ray films were obtained in which the images on the first film representthe distribution of 13'l in the tissue and the images on the second filmrepresent the distribution of 14Cin the same tissue sections.

Conversion of the 14C-generated images from the second exposure

to tissue radioactivity (nCi/g) has been described above. A similar procedure was used for the conversion of the 131l-generated images from

the first exposure to tissue radioactivity nCi/g). The mean absorbanceof all 50- x 50-jjm areas of measurement within each of the autoradiographic images produced by the 15 131l-standards (45 to 30,500 nCi/g)

on the X-ray films from the first exposures were determined. A standard

curve which related the mean absorbance of the autoradiographic standards to their respective levels of radioactivity was made using thecomputerized scanning microdensitometer system described above.

Calculations and Measurements. MISO equivalents (MISOt) can becalculated from the measured radioactivity and specific activity of the|'4C]MISO administered assuming no metabolism (MISOt = sampleradioactivity/[14C]MISO specific activity). The MISO:MISOt ratio reflectsthe nonmetabolized fraction of [14C]MISO in both blood and tissue.

The measurement of blood flow (f) in these studies was performedby a slight modification of the method described by Sakurada ef al. (24)using [13'I]IAP and a double-label quantitative autoradiographic technique

described previously (18).Regional measurements of MISO* were obtained using a computer-

controlled cursor-outlining routine on the video monitor which could

demarcate selected tissue areas for measurement. The selected tissueareas were constructed on the basis of the image from an adjacenthistological section which was portrayed on a second monitor. Thehistological-QAR image correlation provided the possibility to perform

regional QAR measurements which were primarily defined by the histological and morphological features of the tumor. Tumor measurementsare given by the mean ±SD of all measurements plus the cross-sectionalarea of measurement (400 individual 50- x 50-^m measurements comprise a 1-sq mm area of interest).

A regional tumor analysis was performed in the following manner sinceall tumor autoradiographs had peripheral (high) to central (low) radioactivity gradients and the histology did not clearly define a tumor rim asdistinct from more central tumor regions. An absorbance slicing routinethat could demarcate the range of absorbances to be analyzed in theautoradiographic image (Tables 1 to 3) was used to match as closely aspossible a peripheral tumor area (high-absorbance range) and a centraltumor area (low-absorbance range) that corresponded to tumor regionsof more densely packed and viable-appearing cells and tumor regions

with more loosely organized cells, necrosis, and cysts, respectively. Thelower absorbance range measurements within small centrally locatedregions of several tumors approached that of the X-ray film background.

The lower limit of acceptable measurements was defined as backgroundplus 1 SD (0.012 absorbance unit), and the cross-sectional area of thetumor below this level was determined and designated as the "nonmea-sureable region." The upper limit of acceptable measurements was never

exceeded.The amount of MISO* delivered to the tissue by the vascular route

(Md") can be calculated from knowledge of tissue blood flow (F) and thearterial plasma concentration-time integral of MISO*:

Ma' = FV, So Cp(f)df (A)

where Cp is the plasma concentration at time f, T is the length of theexperiment, and V, is a dimensionless term (v/v) which represents thefraction of whole blood involved in the blood-tissue transfer process.

Since plasma and blood concentrations of MISO are essentially equivalent and MISO rapidly equilibrates across RBC membranes,8 V, wastaken to be 1.0 for MISO. An image of regional Md' in the tumors wasgenerated by the computer from the blood flow ([13'I]IAP) autoradiograms

and Equation A. Corresponding histological sections were digitized by avideo camera system (Sierra Scientific Corp., Mountain View, CA) andcould also be portrayed on the monitors. In order to perform preciseregional analysis of the autoradiographic data, the images of histology,Md', and MISO* were aligned (e.g., superimposed) and registered in 3

different channels of the image array processor (Gould-DeAnza, SanJose, CA). In this way, the relationship between Md' and MISO* could

be analyzed precisely and directly correlated to specific histologicalfeatures of the tumors.

RESULTS

Tumor Description. The s.c. flank tumors consisted of anirregularly round mass surrounded by a thin pseudocapsulecomposed of connective and fat tissue. The pseudocapsule wasincomplete with several foci of tumorous invasion. The tumorsconsisted of a relatively narrow rim of anaplastic and pleomorphiccells and a relatively large central region that was composed ofirregular regions of necrosis and cysts. The central degenerativeregions were separated by irregular cords and less compactbundles of tumor cells which appeared viable by light microscopy(Fig. 1). Pseudopalisading was occasionally seen along the border zones between necrotic and viable tissue. The bulk of theviable tumor fraction was composed of irregular compact bundlesof polyhedral-shaped cells that were separated by sheets ofspindle-shaped, sarcomatous-appearing cells. The tumor cell

nuclei were large, hyperchromatic, and without a nucleolus. Thenucleancytoplasmic ratio was high; the cytoplasm was pale and,in some cells, hardly discernible.

Numerous, irregular, thin-walled vascular channels were ob

served in the compact tumor regions, primarily in the rim, whichfrequently had easily identifiable endothelial cells lining the channels. Few vascular elements could be identified in the centraltumor regions; those which were observed were associated withviable-appearing cords of tumor cells.

MISO Levels and [14C]MISO-derived Radioactivity in Blood

and Representative Tissue Samples. A mean peak plasmalevel of 1100 ±85 (SD) ¿¿g/mlwas measured following a 10-si.v. injection of 25 mg MISO/animal. Ten min after MISO administration, a constant i.v. infusion, 170 ^g MlSO/min, was startedand continued throughout the experimental period. Plasma MISOconcentrations remained relatively constant during the infusion(Chart 1). The average plasma concentration of MISO was 170±39, 145 ±47, and 145 ±19 ng/m\ for the 0.5-h (Table 1), 2-h(Table 2), and 4-h (Table 3) experiments, respectively.

The tissue:plasma ratios for MISO equivalents, MISOt, wereroughly 1 or slightly greater for most tissues (Table 4); kidneyand liver were exceptionally high, whereas the tumor values

* J. Strong, unpublishedobservations.

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Table 1[*4C]MISO-derived radioactivity in RT-9 tumor tissue and plasma 0.5 h after administration

Whole tumorTumor rimTumor centerMeasurable

regionExperimentMS9

MS15MS19MD1MD2MD4MD5Area3

(sqmm)178

7311113310912092Activity

(nCi/g)49±49'

87 ±6178 ±5835 ±3851 ±4747 ±2961 ±44Area

(sqmm)52

325920427552Activity

(nCi/g)97

±29159 + 31116 ±4099 ±26

101 ±3365 ±2485 ±22High

activity6

(nCi/g)155

±18216 ±13186 ±17167 ±24167 ±22114±13130 ±14Range0

(nCi/g)>71

>111>50>65>63>36>59Area

(sqmm)104

414488644538Activity

(nCi/g)30

±2055 + 2723 ±1331 ±1630 ±2025 ±737 ±16Range

(nCi/g)5-71

9-1116-50

11-654-63

16-364-59Non

measurableregion0Area

(sqmm)22

08

25402Range

(nCi/g)<5

<9<6

<11<4

<16<4Plasma

Cp"

(nCi/ml)121

±11201 ±29201 ±36188 ±16211 ±21142 + 16124 ±13

a Maximum cross-sectional area of measurement (there are 400 individual50 x 50-iim measurementsper sq mm tumor cross-sectionalarea).0 Mean value tor 10% of the rim area with the highest activity.c Range of measurement (see "Materials and Methods")." Region of the autoradiographic image where the absorbance measurementswere <0.012 absorbance unit above the film background.eCp,mean plasma level (10 min to the end of the experiment).' Mean ±SD.

Table 2[i4C]MISO-derived radioactivity in RT-9 tumor tissue and plasma 2 h after administration

All valuesare as in Table 1.

Whole tumorTumor rimTumor centerMeasurable

regionExperimentMS10

MS12MS18Area

(sqmm)260

143176Activity

(nCi/g)60±41"

53 ±4277 ±73Area

(sqmm)99

4162Activity

(nCi/g)98

±2996 ±32

157 + 57High

activity(nCi/g)157

±27159 + 33257 ±33Range

(nCi/g)>62

>60>75Area(sq

mm)111

81106Activity

(nCi/g)37

±1329 ±1530 + 19Range

(nCi/g)20-62

11-604-75Nonmeasurable

regionArea(sq

mm)50

218Range

(nCi/g)<20

<11<4Plasma

Cp(nCi/ml)97

± 9147 ±35191 ±18

3 Mean ±SD.

All values are as in Table 1.

Table 3["CJMISO-derived radioactivity in RT-9 tumor tissueand plasma 4 h after administration

Whole tumorTumor rimTumor centerMeasurable

regionExperimentMS8

MS3MS16Area

(sqmm)160

99176Activity

(nCi/g)79±61a

91 +78143 ±109Area

(sqmm)66

3897Activity

(nCi/g)138

±50170 ±60213 ±103High

activity(nCi/g)247

±48286 ±28428 ±61Range

(nCi/g)>78

>74>95Area

(sqmm)90

6179Activity

(nCi/g)39

±2038 ±1660 ±20Range

(nCi/g)5-78

4-748-95Nonmeasurable

regionArea

(sq mm)4

00Range

(nCi/g)<5

<4<8Plasma

Cp(nCi/ml)124

±61159+13152 + 36

8 Mean ±SD.

were exceptionally low. The MISO:MISOt ratio or nonmetabo-lized fraction of the measured 14C radioactivity representing

parent drug was markedly different for different tissues (Table5). Values ranged from 1% or less for kidney and liver, indicatingthat almost all the radioactivity in these organs was due to 14C-

labeled metabolites, to more than 90% for plasma, where almostall of the measured radioactivity was parent compound, [14C]-MISO. [14C]MISO accounted for a substantial amount of the

radioactivity in tumor tissue at 30 min; at 2 and 4 h, however,less than one-third of the radioactivity was due to parent drug.

DESMISO levels were also determined. The plasmaDESMISO:MISO concentration ratios were <0.05 throughoutthe 4-h experiments. The tissue ratios (including the tumor) werealways less than 0.1, usually less than 0.05. These results areconsistent with other reports (26) which observed that less than10% of the MISO administered to rats was recovered as DES

MISO in the urine. When MISO was administered to the isolated-

perfused rat liver, the relative clearance of MISO as DESMISOwas<5%(21).

Distribution of [14C]MISO-derived Radioactivity in Flank Tumors. The regional distribution of MISO* within the flank tumor

during periods of relatively constant plasma levels was studiedby quantitative autoradiography. The most striking feature of theautoradiographic images was their consistent pattern; in all theflank tumors studied, a peripheral to central decreasing radioactivity gradient was observed, and it was similar for all theexperimental time periods, i.e., 0.5, 2, and 4h. The highestactivity was always located in the tumor rim, and the lowestactivity was always within the central region (Tables 1 to 3). Theregional variation or range of the individual measurements isexpressed by the standard deviation of the mean values. Thestandard deviation was very large, whereas the covariance was

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INTRATUMOR MISO DISTRIBUTION AND BLOOD FLOW

900 r

800

_ 700E

l¡j 600

600

O 4002OC/J^ 300

200

100125

mgY**

« » 8 a- 8 8a170

fjg/min100

200BOLUS

TIME (minutes)

Chart 1. Plasma concentrations of MISO (•)and MISO equivalents (O) versustime after i.v. bolus and constant i.v. infusion for experiment.

Table 4Tissue:plasma ratio of [ "C//W/SO equivalents* during constant i.v. infusion

Tissue plasma ratio

TissueKidneyLiverLungSpleenHeartMuscleBrainTumor0.5hours2.0±0.461.9

±0.11.2±0.11.0±0.11.1

±0.10.95±0.060.77±0.070.44+ 0.132

hours7.1

±0.15.0±0.81.7

±0.11.2±0.11.0

+0.11.1±0.20.80±0.100.50

±0.044

hours3.3

±0.64.1±0.91.3

±0.20.95±0.091.0

±0.20.87±0.140.71

±0.070.59±0.22

" rC]MISO equivalents (MISOt) = tissue radioactivity/[14C)MISO specific activ

ity (assuming no metabolism).0 Mean ±SE of 4 measurements.

Table 5Fraction oítotal radioactivity representing parent drug (¡Ì4C]MISO)during

constant i.v. infusion'

Fraction of totalradioactivityTissueBloodKidney

LiverMuscleBrainTumor0.5

h0.97±0.02"

0.04 ±0.010.01 ±0.010.76 ±0.100.93 ±0.030.67 ±0.172h0.90

±0.020.01 ±0.01

<0.010.59 ±0.060.69 ±0.170.32 ±0.044h0.89

±0.070.01 ±0.01

<0.010.66 ±0.020.67 ±0.100.30 ±0.02

8 Fraction of nonmetabolized parent drug = MISO/MISOt.6 Mean ±SE of 4 measurements.

approximately 80% for all the tumors and was independent ofthe length of the experimental period.

The ratio of the average measurable MISO* activity within the

central region of the tumor to the mean plasma activity increasedslightly (0.20 to 0.32) between 0.5 and 4 h, but this differencewas not significant. In contrast, the average tissue:mean plasmaactivity ratio for the tumor rim did increase significantly over the

same time period, from about 0.62 at 0.5 h to 1.2 at 4 h (P <0.001, Students i test). Similarly, the highest ratio of tissueactivity in tumor periphery (representing the mean value of 10%of the tumor cross-sectional area) to plasma increased significantly from about 0.97 at 0.5 h to 2.2 at 4 h (P < 0.001, Student's

i test).Blood Flow and [14C]MISO-derived Radioactivity. Concur

rent measurements of regional blood flow and MISO* activity

(using double-label quantitative autoradiography) and specific

correlations between tumor histology and these measurementswere made using image registration and image analysis techniques (Figs. 2 and 3). Blood flow was consistently low in centraltumor regions. Blood flow generally did not correlate well withspecific histological features of these tumors such as the extentof necrosis. Viable-appearing tumor cells in bundles, cords, and

sheets were frequently seen in regions where blood flow wasconsiderably less than 1 ml/hg/min (Figs. 1 and 3). A variablepattern of F was observed in peripheral tumor regions, and thisdid not always correlate with the degree of vascularity observedin the adjacent histological section (Fig. 2A).

The amount of MISO* "delivered" regionally to the tumor, Ma",

can be calculated (Equation A) and compared to the amountactually "measured" after a 30-min experiment (Fig. 2B, Chart

2). It was apparent from this analysis, assuming that blood flowand thus delivery was constant during the experiment, that 2distinctly different patterns of MISO* delivery to peripheral and

central tumor regions exist. Not surprisingly, most peripheraltumor regions had a greater amount of MISO* delivered to thetissue than was measured after 30 min. In contrast, MISO*

activity measured in central tumor regions was slightly greaterthan the activity which could be accounted for by the delivery

5 5DISTANCE FROM THE TUMOR MARGIN: mm

10

Chart 2. A, schematic drawing of a tumor (Experiment MD-1 ; Table 1 and Fig.3). Two levels of sectioning through the tumor are shown; left column correspondsto the images shown in Fig. 3; righi column corresponds to sectioning through theapproximate center and largest cross-sectional area of the tumor (Table 1). Sequential measurements within 0.5- x 0.5-mm tumor regions were obtained acrossthe horizontal plane of both sections (see "Materials and Methods"). The measure

ments in the smaller section (left) include the small area with relatively high bloodflow in the left margin of the tumor (Fig. 3, left). B, graphs of MISO* "delivered" tothe tissue over 30 min (O) and MISO' "measured" in the tissue at 30 min (•)versus

distance from the left margin of both tumor sections described above are shown.The difference between the "measured" and "delivered" values of MISO' is indicated

by the stippled regions between the curves. , mean plasma concentrationof MISO* during the 30-min experiments.

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INTRATUMOR MISO DISTRIBUTION AND BLOOD FLOW

and distribution of MISO* from the blood alone (Chart 2). This

pattern was observed in all tumors (Table 1, MD series).

DISCUSSION

It is generally believed that hypoxic tumor cells exist withinmany solid neoplasms and frequently form a border zone aroundnecrotic tissue areas. Thomlinson and Gray (27) originally proposed the existence of a gradient of oxygen tension betweenthe periphery and center of tumors that have a peripheral sourceof vascularization. As the tumor size increases, an irregularpatchy central necrosis commonly occurs; frequently, cords oftumor cells persist which appear viable by light microscopy. Thishistological pattern plus low oxygen tension measurements (8)and low blood flow values in many solid tumors (22) has supported the considerable effort to develop and use hypoxic cellradiation sensitizers in order to enhance radiotherapy of humanneoplastic disease.

A basic assumption of the radiation sensitizer effort is that thedrug will reach the hypoxic tumor cells in an adequate concentration and make them more sensitive to radiation damage. Theassumption of adequate drug delivery to tumor cells is commonlymade in most chemotherapeutic studies, and therapeutic failureis frequently described in terms of inadequate dosage or tumorinsensitivity. However, this reasoning ignores the well-recog

nized relationship between drug delivery and blood flow (20). Weshow in this paper that low blood flow limits the delivery of MISOto tumor regions that are likely to be hypoxic.

Intratumor Distribution of MISO. Several investigators haveattempted to correlate MISO tumor levels with the morphologicaland histological pattern of tumor tissue in patients (1, 23). Byobtaining multiple tumor samples and performing separate determinations of drug concentration and histological analysis ofrepresentative tumor sections, a wide intratumor variation inMISO concentration could be shown (23). The lowest levels ofdrug were always associated with necrotic regions, and in manycases MISO was not detected in tumor samples containingmostly necrotic tissue.

The present study was designed to assess the regional intratumor distribution of MISO in experimental RT-9 s.c. flank tumors. The mean maximum cross-sectional area of the tumors

studied was 141 ±49 (SD) sq mm, similar in size to those whichmight be encountered in humans. In order to simulate tumor andtissue exposure to MISO in this animal model to that considerednecessary to produce significant sensitization in patients (13), aconstant i.v. infusion of the drug was administered to maintain ablood MISO level of approximately 150 ¿¿g/ml-The intratumordistribution of [14C]MISO-derived radioactivity, MISO*, was de

termined in the tumors by quantitative autoradiography anddemonstrated a consistent pattern. These experiments showthat the tumor rim usually attained MISO* concentrations equal

to or greater than those measured in blood and that these levelsrapidly decreased from the periphery to more central tumorregions in all experiments. In addition, direct histological correlation demonstrated that significant portions of the central regions of these tumors contained viable-appearing cells on micro

scopic examination and that these regions corresponded toextremely low levels of MISO* even after 4 h.

Blood Flow Correlations. In roughly 50% of the tumor mass,there was a good correlation between regional tumor blood flow

and MISO* measured after 30 min of constant plasma levels(Figs. 2 and 3). Blood flow and MISO* delivery were extremely

low in central tumor regions where values of F were usuallybelow 1 ml/hg/min. In contrast, the lowest values of F in necroticregions of the same RT-9 tumor when implanted in brain were

between 5 and 20 ml/hg/min (7, 18). This difference in bloodflow could reflect the large difference in tumor size between thei.e. and s.c. RT-9 tumors [8.2 (18) versus 117 sq mm (Table 1)maximum cross-sectional area, respectively] as well as a differ

ence in the potential of the host tissue to support the vascularbed of the tumor (2). Blood flow in the i.e. tumors was 10- to 50-

fold higher than that measured within central regions of the s.c.tumors and did not appreciably limit the delivery of MISO* to

either viable or necrotic i.e. tumor (18).In RT-9 flank tumors, a different pattern was observed. Blood

flow in central tumor regions containing viable-appearing cellswas at or below the limit of measurement, about 0.2 ml/hg/min.The blood flows measured in large portions of these s.c. tumorswere considerably lower than the range of values (1 to 60 ml/hg/min) reported for other systemic tumors (22). The limits ofthe experimental method to accurately measure such low flowsand the ability to perform precise regional measurements areimportant methodological considerations. Thus, any direct comparison of results obtained by different techniques is very difficult.

We believe the autoradiographic method to measure localtumor blood flow and directly correlate these values with morphological features of the tumor offers particular advantages (5,24). For example, from the double-label quantitative autoradiographic studies of blood flow and MISO* distribution, we couldcalculate the total amount of MISO* "delivered" locally to the

tumor over the 30 min in experimental time and compare it tothe amount of MISO* "measured" in the same tumor region at

30 min (Figs. 2 and 3). Delivery to the periphery of most tumorswas usually sufficient to achieve relatively high tissue levels, andconsiderable backflux of MISO* into the blood during the experiment is likely (Fig. 3). In a few cases, the delivery of MISO* to

peripheral tumor regions was quite variable (Fig. 2). However, inall cases the vascular delivery of MISO* to central tumor regionswas insufficient for equilibration with plasma MISO* during the

30 min (Figs. 2 and 3; Table 1), 2 h (Table 2), and 4 h (Table 3)experiments. Thus, one cannot conclude that MISO* does notbind or localize within the central "hypoxic" regions of these s.c.tumors since the primary reason for low MISO* activity in theseregions was the low rate of MISO* delivery to the tissue (i.e.,

low blood flow).The effect of low blood flow on drug delivery and drug con

centration in the tissue can be readily illustrated. In neoplastictissue with a blood flow of 1 ml/hg/min and experimental conditions where first-order transport kinetics, complete exchange

across the capillaries, a uniform equilibrium tissue distributionvolume of 0.8 ml/g, constant plasma levels, and no metabolismor diffusion of the drug prevail, the time for tissue levels of thedrug to reach one-half of the equilibrium value (tv,) is 1 h. For

tissue with a blood flow of 0.1 ml/hg/min and the same distribution conditions described above, tv, is 9 h. This brief modelingexercise emphasizes the importance of the "input function" or

administration schedule on the delivery of drug to viable tumortissue with low blood flow; a more detailed modeling analysis ofdrug delivery and the effects of drug metabolism has beenpresented elsewhere (6).

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The amount of MISO* "measured" in the central regions ofthese tumors was always greater than the total amount "delivered" (Figs. 2 and 3; Chart 2). This difference was alwaysassociated with a peripheral to central MISO* concentrationgradient. In each case, the difference between "measured" and"delivered" was consistent with the physical diffusion of MISO*through the tissue from peripheral to central tumor regions.8 In

the case of one tumor (Fig. 3), certain regions of the tumor rimhad higher MISO* levels measured than the amount of MISO*delivered and suggests that MISO* diffused from surrounding

s.c. tissue into the tumor. These experimental observationssupport the concept of a peripheral-to-central diffusion of drugs

in solid tumors with very low central tumor blood flows (19) andindicate that low regional blood flow can significantly limit thedelivery of drugs to viable tumor cells (20).

Tissue Distribution of MISO and [14C]MISO-derived Radio

activity. Recent studies in mice with EMT 6 (16) and Lewis lungtumors (10) and in tumor spheroids (14, 15) have shown thatMISO* distributes to higher levels in viable appearing tumor cells

that would be expected to be hypoxic than in surrounding cellswhich are not hypoxic. These studies by Chapman ef al. (9)indicate that metabolism-induced binding of MISO* to hypoxic

tumor cells occurs and supports their suggestion that this observation has the potential for specific applications in tumorbiology and clinical medicine. For example, they suggest thatMISO* localization may be used to identify hypoxic cell popula

tions, provides a method for determining whether or not suchhypoxic cells enter a proliferative phase following radiation andchemotherapy, and can be applied in appropriate nuclear medicine-based clinical studies that could impact on diagnosis and

influence treatment (10).The studies reported here and previously (18) in a different

tumor model provides additional evidence to support Chapman'ssuggestions. Although MISO* binding was not specifically mea

sured, it is clear from a comparison of the 30-min (Table 1; Figs.2 and 3), 2-h (Table 2), and 4-h (Table 3) studies that exposure

periods longer than 2 h are required for appreciable concentrations of MISO* (above that in plasma) to develop locally withinthe tumors. By 4 h, the concentration of MISO* (namely, the sum

of the bound and unbound fractions) in some locations of thetumor compared to that in plasma reached a maximum ratio ofabout 2.2. The markedly lower tissue:plasma concentration ratios in tumor center at 4 h were due to low blood flow (lowdelivery) and low exposure of this region to MISO*. We havealso found that a substantial fraction of MISO* radioactivity in a

narrow rim of tissue around necrotic regions of tumor in brainand in some areas of viable-appearing tissue in s.c. tumorsbecomes bound to macromolecules over a 4-h period in contrastto surrounding tumor and host tissue regions (in which MISO*radioactivity is almost totally extractable).9 Although these stud

ies do not identify hypoxic tumor locations in terms of localoxygen tensions, tumor regions which bind MISO* can be cor

related with specific morphological characteristics of the tumorcells using autoradiographic and imaging techniques. They indicate that metabolism-induced binding of pharmacological levelsof MISO* occurs at a relatively slow rate. Tracer MISO* may

result in higher tissue:plasma concentration ratios and morefavorable images. MISO binding may involve a second-order

* Unpublishedanalysis of these data.

process and be saturated at pharmacological drug levels. Thus,we suggest that tracer levels of MISO*, an infusion time ofseveral h and one or more days from MISO* administration toMISO* detection may be required to identify hypoxic cell popu

lations in patients who have large tumors with very low bloodflows.

MISO is known to be extensively metabolized by variousanimal species including humans. Total recoveries of MISO andits metabolite DESMISO in urine and feces have been reportedby numerous investigators, and these data have been summarized by Strong eíal. (26). The activity of MISO* measured in

these autoradiographic studies do not only reflect MISO concentration. The fraction of plasma MISO* that represents MISO (andnot 14C-labeled metabolites) was high, 0.9 or greater (Table 5).

This fraction for RT-9 s.c. tumor tissue fell to approximately 0.7

at 30 min and to 0.3 at 2 and 4 h (Table 5). A smaller decreasein the nonmetabolized fraction of parent drug was measured inbrain and skeletal muscle, whereas extremely rapid metabolismof 14C-MISO was measured in kidney and liver (11).

Conclusions and Implications for Chemotherapy. A heterogeneous regional distribution of MISO* activity was measured in

RT-9 s.c. flank tumors, and a peripheral to central concentration

gradient was consistently observed. Blood flow was low in thesetumors, and a good correlation between regional MISO* concen

tration and blood flow was observed. In approximately one-halfof the tumor mass where MISO* concentrations were low, thetotal amount of MISO* in the tissue could be related to theamount of MISO* delivered to the tissue by the blood; namely,

blood flow and the blood concentration-time integral. Physical

diffusion from peripheral tumor and s.c. tissue regions down aconcentration gradient is also likely to contribute to the distribution and delivery of MISO* to central tumor regions.

It is clear from this study and the calculations made abovethat low blood flow can significantly limit the delivery of drugs toneoplastic tissue. For drugs that are rapidly metabolized toinactive products in tissue and/or have rapid plasma clearance,tissue-drug exposures (C x f) will be very low in tumors with lowblood flow (6). From this perspective, the differences in chemo-therapeutic response between the hematopoietic tumors andmany solid tumors could be due, at least in part, to differencesin drug delivery, namely, to differences in tumor blood flow.

In addition to the pharmacokinetic principles of regional drugdelivery in solid tumors that were demonstrated, this studysupports the potential application of MISO* imaging in patients

provided that hypoxic tumor regions receive an adequate exposure to the drug for a sufficient period of time. For s.c. RT-9tumors, a 4-h period of constant MISO* blood levels was insuf

ficient for significant tissue localization and binding to occur.Insufficient delivery of MISO* to central "hypoxic" tumor regions

during this 4-h period was due to very low blood flow in thesetissue regions. Thus, adequate delivery of MISO* to tumor tissue

is an essential prerequisite to the possibility of identifying hypoxictumor regions as originally suggested by Chapman ef al. (10).

REFERENCES

1. Ash, D. V., Smith, M. R., and Bugden, R. D. Distribution of misonidazole inhuman tumours and normal tissues. Br. J. Cancer, 39: 503-509, 1979.

2. Auerbach. R.. and Auerbach, W. Regionaldifferences in the growth of normaland neoplastic cells. Science(Wash. DC).275: 127-134, 1982.

3. Benda, P. H., Someda, K., Messer, J., and Sweet, W. H. Morphological and

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immunochemical studies of rat glial tumors and clonal strains propagated inculture. J. Neurosurg., 34: 310-323,1971.

4 Blasberg, R. G., Gazendam, J., Patlak, C. S., and Fenstermacher, J. D.Quantitative autoradiographic studies of brain edema and a comparison ofmulti-isotopeautoradiographictechniques.In J. Cervos-NavarrosandS. Ferszt(eds.), Brain Edema, pp. 255-270. New York: Raven Press, 1980.

5 Blasberg, R. G., Groothius, D., and Molnar, P. Application of quantitativeautoradiographic measurements in experimental brain tumor models. Semin.Neurol., 7:203-223, 1981.

6. Blasberg, R. G., Molnar, P., Groothuis, D., Patlak, C. S., Owens, E., andFenstermacher,J. Concurrent measurementsof blood flow and transcapillarytransport in ASV-induced experimental brain tumors: implications for braintumor chemotherapy. J. Pharmacol.Exp. Then, 231: 724-735,1984.

7. Blasberg, R. G., Molnar, P., Horowitz, M.. Kornblith, P.. Pleasants, R., andFentermacher,J. Regionalblood flow in RT-9 brain tumors. J. Neurosurg., 58:863-873. 1983.

8. Cater. D. B., and Silver, I. A.: Quantitative measurements of oxygen tensionin normal tissues and in tumors of patients before and after radiotherapy.ActaRadiol.,53. 233-256,1960.

9. Chapman, J. D., Baer, K., and Lee, J. Characteristics of the metabolism-inducedbindingof misonidazoleto hypoxic mammaliancells. Cancer Res., 43:1523-1528,1983.

10. Chapman, J. D., Franko, A. J., and Sharplin, J. A marker for hypoxic cells intumors with potential clinical applicability. Br. J. Cancer, 43: 546-550, 1981.

11. Chin, J. B., and Rauth, A. M. The metabolism and pharmacokinetics of thehypoxic cell radiosensitizer and cytotoxic agent, misonidazole, in C3H mice.Radiât.Res., 86: 341-357,1981.

12. Donelli. M. G., Broggini, M., Colombo, T., and Garattini, S. Importance of thepresence of necrosis in studying drug distribution within tumor tissue. Eur. J.Drug Metab. Pharmacokinet.,2: 63-67, 1977.

13. Foster,J. L, Flockhart, I. R., Dische,S., Gray,A.. Lenox-Smith,I., and Smithen,C. E. Serum concentration measurements in man of the radiosensitizer Ro-07-0582: some preliminary results. Br. J. Cancer, 37: 679-683, 1975.

14. Franko, A. J., and Chapman,J. D. Bindingof 14C-misonidazoleto hypoxic cellsin V79 spheroids. Br. J. Cancer, 44: 694-699. 1982.

15. Franko. A. J., Chapman, J. D., and Koch, C. J. Binding of misonidazole toEMT6 and V79 spheroids. Int. J. Radiât.Oncol. Biol. Phys., 8: 737-739.1982.

16. Garrecht, B. M., and Chapman, J. D. The labelingof EMT-6 tumors in BALB/c mice with "C-misonidazole. Br. J. Radiol., 56: 745-753,1983.

17. Goochee, C., Rasband,W., and Sokoloff, L. Computerized densitometry andcolor coding of "C-deoxyglucose autoradiography.Ann. Neurol.. 7: 359-370,1980.

18. Horowitz, M., Blasberg. R., Molnar. P., Strong, J.. Kornblith, P., Pleasants,R.,and Fenstermacher,J. Regional['4C]misonidazoledistribution in experimentalRT-9 brain tumors. Cancer Res., 43: 3800-3807.1983.

19. Levin, V. A., Patlak, C. S., and Landahl, H. D. Heuristic modeling of drugdelivery to malignantbrain tumors. J. Pharmacokinet.Biopharm..8: 257-296,1980.

20. Levin, V. A., Wright, D. C., Landahl,H. D., Patlak, C. S., and Csejtey, J. In situdrug delivery. Br. J. Cancer,41 (Suppl. 4): 74-78, 1980.

21. McManus, M. E., Monks, A., Collins, J. M., White, R., and Strong, J. M.Nonlinearpharmacokineticsof misonidazoleand desmethylmisonidazolein theisolated perfused rat liver. J. Pharmacol.Exp. Ther., 279: 669-674, 1981.

22. Peterson, H. I. Tumor blood flow compared with normal tissue blood flow. In:H. I. Peterson (ed.), Tumor Blood Circulation, pp. 103-114 Boca Raton, FL.CRC Press. 1979.

23. Rich, T. A., Dische, S., Saunders, M. I., Stratford. M. R. L., and Minchinton,A. A serial study of the concentrations of misonidazole in human tumorscorrelated with histologicalstructure. Int. J. Radiât.Oncol. Biol. Phys.. 7: 197-203,1981.

24. Sakurada,O., Kennedy,C., Jehle,J., Brown, J. D., Carbin,G. L., and Sokoloff,L. Measurementof local cerebral blood flow with iodo('4C)-antipyrine.Am. J.Physiol.,234: H59-H66,1978.

25. Schmidek, H. H., Nielsen, S. L., Shiller, A. L., and Messer, J. Morphologicalstudies of the rat brain induced by A/-nitrosomethylurea.J. Neurosurg., 34:335-340,1971.

26. Strong, J. M., Schwade, J. G.. Gangji, D., Shoemaker, D. D., and Upton, D. K.Misonidazoledose and tumor level relationships:effects of individualvariationin rate of misonidazole metabolism and absorption from the gastrointestinaltract. Cancer Clin. Trials, 4: 41-46, 1981.

27. Thomlinson, R. G., and Gray, L. N. The histological structure of some humanlung cancers and the possible implicationsfor radiotherapy. Br. J. Cancer, 9:539-549, 1955.

28. Workman, P., Little, C. J., Marten, T. R., Dale, A. D., Ruane, R. F.. Flockhart,I. R., and Bleehan,N.M. Estimationof the hypoxic cell-sensitizermisonidazoleand its O-demethylated metabolite in biological materials by reversed-phasehigh-performance liquid chromatography. J. Chromatogr., 745: 507-512,1978.

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INTRATUMOR MISO DISTRIBUTION AND BLOOD FLOW

^w^&^m^m>¿*&*%?^S.*f¿!&$^s*^~fe^:^' . >*^>%i^. *-V*-¥-*¿í^-- v «*•-^af,.^%5^T * ^ *? '

<T-9 tumor growing in the s.c. tissue of the flank showing areas with cellular fragmentation, Tarificationviable-appearing cells with irregularly stained and variable size nuclei. (Experiment MD5; Table 1 and Fi

SllsR2%fl**Fig. 1. Histological section of the central portion of an RT-9 tumor grc

necrosis (lower right) adjacent to cords of densely packed, viable-appearuH &E, x 180.

Fig. 2. Left, macroscopic histology and corresponding pseudocolor image of regional blood flow, color coded to a range of specific values (see "Materials andMethods') for a section cut through a s.c. tumor growing in the flank (Experiment MD1; Table 1). Note the irregular patterns of tumor necrosis (pale areas) and the

marked variability of blood flow in the tumor rim. Two small regions of the tumor rim have relatively high blood flow compared to other portions of the tumor and areassociated with vascular structures; however, similar vascular structures were identified in other regions of the tumor rim that corresponded to low flow values. Mostregions in this tumor had blood flow less than 0.5 ml/hg/min. Despite these very low flows, "viable" appearing cords and islands of tumor cells were abundant and formedover 50% of the tumor mass in this section. Right, corresponding pseudocolor images of the total amount of MISO" "delivered" locally to the tumor over 30 min and theamount of MISO' "measured" locally at 30 min in the same tissue section described above (see "Materials and Methods"). Except for the 2 areas of the tumor rim withrelatively high blood flow (left), the amount of MISO' per g of tissue "measured" at 30 min generally exceeded the total amount "delivered," suggesting diffusion of MISO'into the tumor from surrounding s.c. tissue. The local concentration of MISO" "measured" in the tumor was always equal to or less than the mean plasma concentration

(188nCi/ml).

Fig. 3. Left, macroscopic histology and corresponding pseudocolor autoradiographic reconstruction of regional blood flow, color coded to a range of specific values(see "Materials and Methods") for a section cut through the center of a s.c. tumor growing in the flank (Experiment MD5; Table 1 and Fig. 1). Note the irregular patterns

of tumor necrosis (pale areas) and blood flow. In the tumor center, blood flow was less than 1 ml/hg/min; the lowest values were less than 0.4 ml/hg/min. Right.corresponding pseudocolor images of the total amount of MISO' "delivered" locally to the tumor over 30 min and the amount of MISO' "measured" locally ai 30 min inthe same tissue section described above (see "Materials and Methods"). Note that the total amount per g tissue "delivered" to the peripheral regions or rim of the tumoris considerably greater than the amount "measured." This indicates considerable backflux of MISO' from the tumor rim into blood during the 30-min experimental period.The tissue concentration "measured" in peripheral tumor regions at 30 min is slightly less than the mean concentration of MISO' in plasma (124 nCi/ml). In most centraltumor regions, the opposite relationship is seen; the amount of MISO' per g tissue "measured" was greater than the amount "delivered." Note that the amount of MISO'"measured" locally in the tumor does not represent MISO* binding since most of the radioactivity in the tissue is readily extracted by methanol and xylene following 30min experiments.8

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1985;45:1692-1701. Cancer Res   Ronald Blasberg, Marc Horowitz, John Strong, et al.   Blood Flow in Subcutaneous RT-9 Experimental Tumors

C]Misonidazole Distribution and14Regional Measurements of [

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