[CANCER RESEARCH54, 3785—3792,July 15, 19941

Clinical application of tumor-selective acidification by hyperglycemia has met with several reservations, however. Differences in angiogenesis can cause variable responses among related tumors (16)and even within a single tumor because of heterogeneity in partialoxygen pressure (17). Moreover, due to systemic hemoconcentrationand increased RBC rigidity, hyperglycemia bears the risk of stronglyreducing tumor blood flow with inhibitions ranging from 57% toalmost 100% (11, 18—20).Reduced tumor blood flow by hyperglycemia and the osmotic shifts induced by i.p. glucose administration insome experimental settings (1 1) may seriously and selectively impairthe access of pH-sensitive drugs to the target tissue. It is also important to establish whether hyperglycemic conditions preferentiallyacidify the pH13 or rather the pH@of the interstitial fluids. AlthoughpH@and pH1 are not unrelated, low pH@ is most important in theactivation of pH-labile prodrugs and some bioreductive agents or theuptake of weakly acidic drugs (1, 5). Conversely, intracellular activation of platinum-containing drugs and the reactivity of alkylatingagents with target DNA are mainly dependent on increased intracellular H@ ion activity (3, 4). To date, lowest basal pH values andadditional decreases after glucose administration have been recordedusing H@ ion-sensitive electrodes, which probably measure pH@or“aggregated―values of pH@and pH1 (7, 10, 12—14).With 31P MRSanalyses (1 1, 12, 21, 22) or pH-sensitive fluorescent dyes (23) variable effects of extra glucose on pH1 have been reported, ranging frommoderate increases (22) to reductions with up to 0.74 pH unit (1 1).Finally, in view of unpredictable pH responses and because of theinaccessibility of most human tumors to pH measurements withelectrodes, noninvasive monitoring of the response of clinical tumorsto glucose administrations has been advocated (16).

We have addressed these questions from a joint study of fourinstitutions, each contributing by its specific expertise. Based onprevious observations (24), the possibility of effectively decreasingintratumoral pH was explored by combining lower amounts of glucose with a single dose of the mitochondriotropic agent MIBG. MIBGinhibits mitochondrial respiration at site I of the respiratory chain.Mammalian cells in culture respond to MIBG with enhanced lacticacid production from stimulated glycolytic fluxes to compensate forreduced AlP output from the respiratory chain (25). Synergism between MIBG and extra glucose in vivo may lower overall glucoserequirements and prevent severe reduction of tumor blood flow.

Moreover, the mitochondrial inhibitor may also compensate for theeffects of intra- and intertumor variation in partial oxygen pressure(11, 17). By converting oxic cells into functionally hypoxic cells, amore consistent and homogeneous tumor acidification than withglucose alone may be obtained. In addition, the pH data obtainedwith semimicroelectrodes were compared with MRS recordings ofshifts in peaks within the 31P, spectra that reflect mainly pH, (26).Finally, we have noninvasively monitored the manipulation of

3 The abbreviations used are: pH, intracellular pH; pHi, extracellular pH; MIBG,

meta-iodobenzylguanidine; MRS, magnetic resonance spectroscopy; PET, positron emission tomography; [‘8FIFDG, 2-['8F@fluoro-2-deoxy-o-glucose; [3HJDG, 2-deoxy-o-l-(3H@glucose.

3785

Reduction of Intratumoral pH by the Mitochondrial Inhibitorm-Iodobenzylguanidine and Moderate Hyperglycemia'

A. Kuin, L Smets,2T. Volk, A. Paans,G. Adams, A. Atema, E. Jähde,A. Maas, M. F. Rajewsky, G. Visser,and P. Wood

The Netherlands Cancer institute@ Division of Experimental Therapy, 120 Plesmanlaan, NL-llXió CX, Amsterdam, the Netherlands fA. K, L. S., A. A., A. M./; Institute of CellBiology (Cancer Research), University ofEssen, Essen, Germany [T. V., E. J., M. F. R./; University Hospita4 PET Center, Groningen, the Netherlands fA. P., G. V.J; and MRCRadiobiology Unia Chilton@ United Kingdom fG. A., P. W.J

ABSTRACT

The interstitial pH of RIF-1 tumors was selectively lowered by Lp.administration of the mitochondrial inhibitor meta-iodobenzylguamdlne

. (MIBG; 40—100 mg(knj, supported by sustained moderate hyperglycemia

@ (plasma glucose concentration, 14 mM) in rats or by a single i.p. bolusinjection of glucose (1.5 glkgj in mice. Responses were evaluated in a

. multicenter study by pH measurements with semimicroelectrodes and

31P magnetic resonance spectroscopy, by biochemical analysis of tissueand plasma levels ofglucose and lactate, and by positron emission tomography analysis of 2-['8Fjfluoro-2-deoxy-n-glucose uptake. In both achedules, treatment with MIBG and glucose reduced the mean intratumoralpH as recorded with semimicroelectrodes to 6.2. In the mouse model,treatment with MIBG plus glucose was accompanied by a 2-3-fold slim

@ ulation of 2-['8Fjfluoro-2-deoxy-n-glucose uptake and a corresponding. increase in tumor glucose content. Responses were maximal In male mice

with tumors of 0.2-0.8 g. 31P magnetic resonance spectroscopy analysisrevealed no changes in intracellular pH or metabolic status, indicatingthat only extracellular pH was affected. MIBG was synergistic withbolus or continuous glucose administrations by a dual mechanism.The drug reduced by up to 5-fold the amount of glucose required foreffective reduction ofintratumoral pH and promoted the availability of

. (extra) glucose to tumor tissue in a stress-related, sympathomimetic

response. Moreover, by converting oxic tumor cells into functionally@ hypoxic cells, combined treatment resulted In a more homogeneous

decrease In intratumoral pH which included better perfused peripheralI tumor areas. The effects of combined treatment on tumor glucose

: metabolism could be monitored noninvasively by 2.['8Fjfluoro

@ 2-deoxy-D-glucose positron emission tomography analysis.

INTRODUCTION

The hypoxic and acidic microenvironment in tumors is of recognized importance for several nonsurgical anticancer treatment modalities that include heat, ionizing radiation, and the activation of biore

@ ductive agents (1, 2). Low pH also potentiates the cytotoxic activity ofalkylating and platinum-containing anticancer drugs (3, 4) and promotes the local activation of acid-labile prodrugs and immunoconjugates (5). Manipulation of intratumoral pH has therefore significantpotential to improve the therapeutic index of several conventional andexperimental, pH-sensitive (pro)drugs (for a review, see Ref. 6).

Stimulation of the intrinsically higher glycolytic capacity of tumors@ by extra glucose supply is an effective strategy to selectively increase

. intratumoral H@ ion activity in clinical (7—9) and transplanted human

and animal tumors (10—15).Whereas bolus i.v. or i.p. glucose admin@ istration generally results in rapid but transient pH reductions (10—

12), sustained low intratumoral levels can be achieved by continuousand controlled glucose infusions, maintaining plasma values at preset

@ values (13, 14).

Received2/7/94;accepted5/16/94.Thecostsof publicationof thisarticleweredefrayedin partby thepaymentof page

charges. This article must therefore be hereby marked advertisement in accordance with. 18 U.S.C. Section 1734 solely to indicate this fact.

I Supported by the Dutch Cancer Foundation, Grant NIU 91—14; Dr. Mildred Scheel

@ StiftungfürKrebsforschung,W 15/88Ra3; MRSstudiessupportedby MRCandICRF@ (United Kingdom).

2 To whom requests for reprints should be addressed.

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MODULATIONOF TUMOR pH

glucose metabolism in vivo by measuring [‘8F]FDGuptake withPET and metabolic status with MRS. These studies were allperformed in a common model, namely the mouse RIF-1 tumorgrowing s.c. in syngeneic mice or as xenografts in nude rats. Theanalyses were complemented with biochemical studies of lactateand glucose levels in plasma, tumors, and several normal controltissues of the host animals.

MATERIALS AND METhODS

Animal Tumors and Treatment Protocols. RIF-1 cells from liquid nitrogen stocks were propagated in tissue culture and transplanted at restrictedpassage numbers according to previously described protocols (27). Cells (10@)in 0.1 ml were injected s.c. in the back (biochemical, MRS. and pH studies) orthe posterior leg (PET studies) of syngeneic C3H/Km mice or female mu/murats (24). Tumors of >0.1 g developed within 2 weeks. MIBG was synthesizedfrom m-iodobenzylamine as described previously (28). Tumor-bearing andtumor-free C3HIKm mice were given a bolus i.p. injection of 40 mg/kg MIBG(0.01 ml/g body weight of a 4-mg/ml solution in phosphate-buffered saline)and of 1.5 g/kg D-glucose (0.01 ml/g body weight of a 0.15-g/ml solution),alone or in combination. At this dose MIBG is not toxic during repeated dailyinjections (28). Control mice received corresponding volumes of saline.

Female nude rats bearing the RIF-1 tumor were continuously infused withglucose (20% w/v) via central venous catheters, and plasma glucose concentrations were repeatedly monitored as described in previous reports (14, 24).Within 30—45 mm, plasma glucose concentration increased from 6 ± 1 (SD)

to 14 ±3 mMand was maintained at this level by appropriate adjustment ofthe rate of glucose infusion. MIBO, in a total dose of 100 mg/kg body weight,was coinfused during 60 mm from the start of glucose infusions in a totalvolume of 8 ml.

Biochemical Measurements. Glucose and lactate levels in plasma andtissues were determined 3 or 5 h after treatment. Immediately after excision,metabolic activity of tissues was blocked by freezing at —70°C.Sodiumfluoride and potassium oxalate were added to the blood samples (2 mg/ml

each). Glucose and lactate were estimated spectrophotometrically at 340 nm bycommercial assays (from Boehringer Mannheim and Du Pont, Wilmington,

respectively) based on the reduction of NAD@ and NADP@ to NADH andNADPH, respectively. The protein content of tissue samples was determinedaccording to the protocol of Bradford et a!. (29).

pH Recordings with Semimicroelectrodes. pH values in tumors andnormal tissues were recorded with the use of semimicro glass electrodes asdescribed in detail elsewhere (14, 24). Before measurements, the animals wereanesthetized with 30 or 90 mg/kg body weight of sodium pentobarbital for ratsand mice, respectively. The electrodes were automatically inserted at a speedof 0.5 mm/min with mechanically driven remote control micromanipulatorsand the electrode signals were continuously recorded. Unless stated otherwise,all frequency distributions and mean values ±SEM refer to multiple, i.e.,140—270,single-point measurements in groups of 8—10tumors.

Deoxyglucose Uptake. 2-Deoxy-D-[1-3H]glucose(specific activity, 17.4Ci/mmol; Amersham) was diluted with NaCI (0.9%) to 0.05 @Ci/g.&l.One h

after i.v. injection of 10 MCi,blood and tissue samples were obtained. Tissuesamples (“50mg) were blotted with mannitol (0.3 M,4°C)to remove adheringfluid and extracted in 2 ml perchloric acid (0.5 M)during 48 h. Radioactivityof tissue extracts or of 100-pi plasma samples was assessed by liquid scintillation counting and expressed in percentage of injected dose/g tissue or ml

plasma, respectively.PET Scan Studies. Due to the 110-mmhalf-life, ‘8F-labeledfluorodeoxy

gIucose is a convenient radiopharmaceutical for the in vivo measurement ofglucose consumption (30). ‘8Fwas produced via the ‘80(p,n)'8F reaction

using the 17-MeV proton beam of a Scanditronix MC-17F cyclotron and‘8O-enriched water as target material. No carrier-added [‘8FJFDG was syn

thesized according to the method of Hamacher et aL (31) with a chemicalpurity of >98%. Due to the limited amount of blood available in the mouse,measurement of the input function, i.e., the arterial plasma concentration as afunction of time, was not possible. The uptake in tumors and control tissueswas therefore expressed by the differential absorption ratio defmed as theamount of radioactivity/g of tissue relative to the total activity in mouse/g. Adual-head, uncollimated scintillation camera system, operated in a coincidence

mode, was used as imaging device (32). This system, with an in-planeresolution of 5.5 mm full width at half-maximum and a sensitivity of 2.7

cps/kBq (100 cps/p@Ci),images one, selectableplane and allows for dynamicstudies. Images were reconstructed by back-projection in a 64 X 64 matrix and

corrected for the nonuniform sensitivity of the camera system. Due to the goodenergy resolution (10%) and the fact that only photo-peak events were selected, the scatter fraction was rather limited using small animals (<5%). In thecalibrated system the data could be translated into kBq/cm2. After injection,the animals were placed in a jig in the selected plane of the camera and weremonitored for 45 mm, using a dynamic study protocol. After correction fornonuniform sensitivity and decay the images were analyzed via a region of

interest technique. The regions of interest selected were the total animal, heart,tumor in right hind leg, and corresponding muscle area in the left leg. Allrecordings contained a reference intensity bar in color. To avoid errors resulting from the conversion of into black and white prints, these references wereomitted from the PET scans.

31P MRS Studies. RIF-1 tumor-bearingmice were unanesthetizedforMRS experimentsbut gently restrainedin jigs (33). 31PMRS was carriedoutusing a 4.7-Tesla, 30-cm horizontal bore magnet (Oxford Instruments) interfaced with a SISCO 200 spectrometer. According to tumor size, a 7-mm or1-cm-diameter surface coil was used for R@transmission and signal collection.

Acquisition parameters were set to minimize signal from underlying tissues.Each spectrum consisted of 256 scans with a 2-s delay, giving a total acquisition time of 8 mm. The 31P MRSs were analyzed using a baseline and astandard Lorentzian curve-fitting program developed in house.4 Changes intumor energy status were expressed as the ratio of the P, peak area to the sumof all peak areas, or Pg/total.pH measured by 31PMRS which is predominantlypH3was calculated from the chemical shift of the P. peak relative to that of a-or ‘y-ATP(26). Since phosphocreatine is not present in all tumors, this was notused as a reference.

Vascularization. Differences in blood supply to the tumor periphery cornpared to the center were estimated from the relative density of perfused bloodvessels. Mice bearing the RIF-1 tumor were anesthetized with nernbutal andinjected i.v. with 0.1 ml acridine orange (saturated solution). Twenty s afterinjection mice were sacrificed and the tumor was excised and frozen at —20°C(34). The number of stained vessels per 0.2 mm2 was counted microscopically

in 25 peripheral (outer 1 mm) and central (>2 mm from the edge) areas in 7histological sections of 2 different tumors.

Statistical Analysis. Significance of differences in mean values was determined by Student's t test and included adjustment for multiple testing insubgroups. P < 0.05 was judged to be of statistical significance.

RESULTS

Modification of Intratumoral pH. The effect of MIBG and glucose on intratumoral pH was measured in two schedules. In a ratmodel, a short-time infusion of MIBG was given alone or duringcontinuous glucose infusion. In mice, bolus i.p. injections of glucoseand MIBG were given to control and tumor-bearing animals.

In the rat model, continuous glucose infusions were given to maintam blood levels at constant and preset values of 14 ±3 mr'.s(modcrate hyperglycemia) or 30 ±3 m@i,with or without coinfusion ofMIBG (100 mg/kg). Multiple single-point recordings with semimicroelectrodes revealed a pH downshift by MIBG alone (pH 6.48 ±0.13;P < 0.01) or moderate hyperglycemia alone (jH 6.54 ± 0.22;

P < 0.05) compared to a basal value of pH 6.78 ±0.11 in tumors of

saline-infused control animals. Coinfusion of MIBG and glucose(plasma glucose concentration, 14 mM) caused a further drop in pH toa mean value of 6.23 ±0.38 (P = 0.02), with single readings rangingfrom pH 5.4 to 6.7 (Fig. 1). To achieve similar low values by glucoseinfusion alone, plasma glucose had to be raised to 30 m@i(data notshown; see Refs. 14 and 24). At 30 mr@iplasma glucose, coinfusion ofMIBG resulted in a further pH reduction to a mean valueof 5.94 ±0.27. These pH responses in RIF-1 tumors lasted for about

4 C. Counsell, unpublished observations.

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MODULATION OF TUMOR pH

()zId

aId

I'

10 h and were not accompanied by significant alterations in pH inliver or kidney with mean values of 7.1 (24).

With MIBG alone (Fig. 1C), the range in individual pH recordingswith SEM = 0.13 was smaller than with moderate hyperglycemiaalone (Fig. 1B; SEM 0.22). To investigate whether this was due tolower intertumor variation or to more homogeneous pH reductionswithin individual tumors by MIBG, pH signals were plotted as afunction of the electrode track length. The results (Fig. 2) revealedthat MIBG alone lowered the pH more homogeneously, notably in thetumor periphery, than did glucose alone. According to the studies ofFenton and Way (35), RIF-1 tumors are better vasculanzed in theperiphery than in the interior. To confirm this, perfusion in central andperipheral areas of tumors from untreated animals was estimated fromthe relative densities of perfused vessels, staining with acridine orange(34). The ratioof perfusedvessels in the peripheryover centralareaswas 1.42 ±0.09 (n = 7; P 0.00001).

Studies in the mouse model, although less detailed, revealed that asingle i.p. bolus administration of MIBO (40 mg/kg) plus glucose (1.5gJkg) caused similar reductions in intratumoral pH in male mice asthose achieved in the rat experiments with continuous glucose infusion (Fig. 3). Basal pH in saline-treated control tumors of male mice(jH 6.73 ±0.08) was as recorded in the same tumors when xcnografted in female rats (Fig. IA). Assuming that basal pH wasindependent of the sex of the host animal, tumors in male mice wereclearly more responsive to combined treatment than were those infemale animals.

31PMRS Studies. RIF-1tumorsin male C3H/He mice at volumesof 100—300mm3 (small tumors) or 1000—2000mm@(large tumors)were used for 31P MRS experiments. Control spectra were collectedand mice given injections of 1.5 g/kg glucose alone or immediatelyfollowed by 40 mg/kg MIBG. 31P MRSs were then collected at 15 mmintervals from 3—5h after injection. The spectra were analyzed andPdtotal ratios and pH were calculated. Values of Pdtotal against timegiven in Fig. 4A for large tumors were comparable for small tumors

20

10

AFig. 1. Intratumoral pH measured with semi

microelectrodes in RIF-1 tumor xenografts in nuderats. Recordings were made at 5 h after the start of(A)saline infusion,(B)glucose infusion to maintainplasma glucose values at 14 m@.t,(C) a 1 h infusionof MIBO(100mg/kg),or (D) combinedtreatmentwith MIBG and glucose infusion (plasma glucoseconcentration, 14 mat). Histograms were calculatedfrom 140—270single-point determinations in 8—10tumors in each experimental group.

40

pH

(data not shown). Clearly neither treatment altered energy status inthis tumor system. The pH measured by 31P MRS was calculated withreference to either a-ATP (Fig. 4B) or y-ATP (Fig. 4C). The effectsof glucose alone or in combination with MIBG on pH in large tumors(Fig. 4, B and C) were identical to results obtained from small tumors(data not shown). Again, neither treatment altered the pH measured by31P MRS in either size of tumor.

Ia.

7.00

6.80

6.60

6.40

6.20

6.000 2 4 6 8 10

depth (mm)

Fig. 2. Local pH values in RIF-1 tumors in rats, recorded at distances of 0.5 mm alongelectrodetracks,insertedautomaticallyintotumors15—20mmindiameter.Treatmentandother experimental details are as in the legend to Fig. 1. 0, saline-infused controls; 0,moderate hyperglycemia (plasma glucose concentration, 14 mM); •,MIBG alone; U,MIBGplusmoderatehyperglycemia.Bars,SEM.

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MODULATION OF TUMOR pH

The results (Fig. 6) revealed the highest but variable uptake in hearttissue. High uptake was also recorded in the glucose-utilizing tissuesmuscle and brain. Metabolic activity of tumor tissue exceeded that ofmuscle or brain, confirming the results of a similar study by Contiet aL (36). Pilot studies on the effect of MIBG and glucose in micesuggested increased uptake by the heart, tumor, and liver as opposed to reduced uptake in muscle tissue by about 50%.

Based on these results, a more detailed analysis of [‘8F]FDGuptakein situ was performed using positron emission tomography. Similarto the [3H]DG studies, the highest but variable uptake was observed in the thoracic area. As illustrated in Fig. 7, the uptake bytumors was sufficiently high to permit imaging of tumors with amedian weight of 0.75 g. This allowed for a correct definition ofthe region of interest in subsequent calculations. In these areas,

I0.

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6.80

6.60

640

6.20

6.00control female male

Fig. 3. Intratumoral pH recorded with semimicroelectrodes in RIF-1 tumors implanteds.c. in syngeneic male or female mice at 4—6h following i.p. bolus injection of saline(controls), MIBG (41) mg/kg), and glucose (1.5 g/kg). Data [mean ±SD (bars)] are of70—90single-point determinations in 3 tumors in each experimental group. The averagetumor weight was 140 mg. Controls are males given injections of NaCI vehicle.

0.25

0.20

Biochemical Studies. In the course of the rat studies, it wasobserved that with coinfusion of MIBG, up to 5-fold lower amountsof glucose were required for maintaining blood glucose levels atpreset values. Typically, in the presence of MIBG the glucose infusionrate at steady state could be reduced from 4 to 0.78 mI/h for maintaming plasma glucose concentration at 14 m@t.

Glucose and lactate levels were determined in plasma, tumors, andseveral normal tissues of male and female mice during single andcombined administration of i.p. bolus glucose and MIBG. The resultsat 5 h after combined treatment of male animals were essentiallysimilar to, or only slightly different from, those recorded after 3 h.Pooled data are shown in Fig. 5, A and B, respectively. Plasma glucose(Fig. SA) and lactate (Fig. SB) both increased after combined treatment with MIBG and glucose. These increases were not or only justobserved with glucose or MIBG alone in plasma, kidney, muscle, or

tumor tissue. Separate experiments with tumor-free male animalsrevealed comparable increases in plasma glucose and lactate levels as

observed in tumor-bearing hosts, indicating a systemic effect of com

bined treatment. Glucose but not lactate content of muscle tissue wasalso increased by combined treatment. No significant changes werenoted in glucose and lactate content of liver (glucose: control,1.19 ±0.50; treated, 1.28 ±0.53; lactate: control, 0.23 ±0.10;treated, 0.18 ±0.05 mmol/g protein) or kidney (Fig. 5). Compared tothese normal tissues, the basal glucose content of tumors was verylow, e.g., 2—3-foldlower than in muscle or kidney, but increased2-fold after combined treatment (Fig. 5A). As in muscle tissue,changes in tumor lactate content were marginal, however.

In agreement with the limited effects on intratumoral pH (Fig.4), nosignificant overall changes in the biochemical parameters of femalehosts were observed on this protocol. The basal glucose content oftumors was equally low as in male hosts. An increase in glucosecontent (from 0.07 ±0.02 to 0.12 ±0.05 mmol/g protein; P = 0.03)and lactate (from 0.31 ± 0.09 to 0.50 ± 0.13 mmol/g protein;P = 0.006) was observed, however, in smaller tumors of <0.4 g in

female hosts on schedules containing higher amounts of glucose(3 g/kg). Compared to males, these responses were delayed and notobvious at <5 h after the start of treatment.

Deoxyglucose Uptake. In cells in culture, MIBG-induced stimulation of glycolysis is associated with increased glucose consumptionand stimulated uptake of the nonmetaboliz@ble analogue [3H]DG (25).I3H]DGwasthereforeusedto monitorglucosemetabolismin vivo.

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Fig. 4. Energy status and intracellular pH in RIF-1 tumors measured by 31P MRS. Timecourse for the effect of 1.5 g/kg glucose alone (0, 0) or 1.5 g/kg glucose with 40 mg/kgMIBG (•,r in RIF-1 tumors at volumes of 1000—2000mm@.A, energy status (Pg/total).B, change in tumor pH using @‘1P-a-ATP (kArP) as reference. C, change in tumor pHusing 31P-y-ATP (G-ATP) as reference. Data shown are means ±SEM (bars) from 4—5mice. @,control values for tumors prior to treatment.

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1

MODULA11ON OF TUMOR pH

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heart tissue of all treated male animals. To control for possible effectsof animal handling per Se, control experiments were performed withsaline-treated animals as described in the legends to Table 1. In this“notreatment―group T1IFc (1.39 ±0.43) and M@/M@(1.25 ±0.20)values were close to unity, indicating the relative stability of repeatedmeasurements.

12 -.

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p1 mu ki tu

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Fig. 6. Uptake of 2-[3H]deoxyglucose, calculated as percentage of injected dose per gtissue, in heart (H), s.c. RIF-l tumors (Tu), brain (Br), leg muscle (Mu), lung (Lu), spleen(Sp), intestine (I), kidney (Ki), plasma (P1), and liver (Li). Mean ±SD (bars) of 8animals.

p1 mu ki tuFig. 5. Glucose (A) and lactate (B) values in plasma (pi), muscle (am), kidney (ki),

and RIF-1 tumors (ni) in male hosts, recorded in control animals (R) or in animals3-5 h aftertreatmentwithM!BG(40mg/kg)plusglucose(1.5g/kg)(0). Leftordinate,plasma values in mmol/liter; right ordinate, tissue values in mmol/g protein. Values aremean ±SD(bars) from 12—16plasma or tissue samples of5—7independent experiments.Glucose levels before and after treatment were statistically different for plasma(P = 0.002), tumor (P < 0.001), and muscle (P = 0.001); lactate levels were statisticallydifferent for plasma (P < 0.001) and tumor (P < 0.03) according to Student's t test.

tumor-specific uptake was determined in all tumors from thedifferential absorption ratio as specified in “Materialsand Methods.― Fig. 8 (black columns) illustrates that net uptake/g washighest in the smaller tumors and leveled off at tumor weight>0.3—0.4 g. This observation agrees with the reported higherperfusion rate and metabolic activity of smaller tumors (16). Ad

ministration of MIBG alone and of MIBG plus glucose eachstimulated [18F]FDG uptake in the tumors, albeit with considerablevariation (Fig. 8, hatched and white columns, respectively). Theresponses were most consistent in the weight range of 0.2 to 0.8 gwith an average stimulation of [18F]FDG uptake by 2.8-fold (SD±1.5; n = 8). Uptake data of all tumors and of control muscletissues are summarized in Table 1.

Relative tumor [18@p@yj uptake in MIBG-treated animals wassignificantly higher than the control value (set at 1.00) recorded in thesame animal on the previous day. Due to large interanimal variationtracer uptake during treatment with MIBG and glucose (range oftracer uptake in treated animals versus that in control animals E'F/FC]was 0.77—6.10;cf Fig. 8) was only significantly above control in thetumor weight range 0.2—0.8g (see above) but not different from theeffects of treatment with MIBG alone. [‘8F]FDGuptake in muscle(M) tissue was significantly reduced on both schedules. This effect,already noticed in the [3HIDG uptake studies, probably reflects asystemic response to MIBO which was also evident from the consistent stimulation of [‘8F]FDGor [3H]DG uptake in the thoracic area or

Fig. 7. Typical PET total body scans of[18Fjfluoro-deoxyglucose distribution in micewith R!F-1 tumors, implanted in the right posterior leg (arrows). Shown are four animals,positioned head to tail. From top to bottom, tumors weighed 1.16, 0.45, 0.67, and 0.70 g,respectively.Reproducedfromcolorprintoutwithdeletionof referencebar (see“Materials and Methods―).

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TreatmentTjF@M,/M@MIBG

(40 mg/kg)2.04 ±0.86 (n = 7)0.66 ±0.09 (n =6)P0.0090.0001MIBG

(40 mg/kg)2.23 ±1.83 (n = 6)0.64 ±0.28 (n =5)+glucose (1 .5g/kg)P0.080.02

MODULATION OF TUMOR pH

intratumoral pH. In rats, the effect of MIBG with plasma glucoselevels maintained at 14 m@iequalled that of severe hyperglycemiaalone (30 mM). In the mouse model, the reduction in tumor pH bycombined treatment was similar to that obtained with 4—5-foldhigheramounts of glucose alone (5—7.5g/kg) in other studies (1 1, 12) andrequired only moderate, i.e., 30% increase in plasma glucose levels(Fig. 5A). Whether or not the large glucose-sparing effects of MIBGindeed prevent strong reductions in tumor blood flow as observedwith true hyperglycemia, remains to be established.

Apart from glucose-sparing effects, the observation of a morehomogeneous lowering of intratumoral pH by MIBG alone than byglucose alone, notably in the peripheral area of tumors in rats (Fig. 2),is of note. The 40% excess in open vessels in the periphery of mousetumors suggested better oxygenation relative to more central areasand corroborated the morphometric studies of Fenton and Way inthis tumor (35). The small number of track points in mouse tumors

prevented a direct comparison between vascular data in mousetumors and pH values in the large rat tumors. Because the vascularstructure is independent of size (35), both observations are considered to yield complementary information. The pH reduction by

MIBG alone in this area is entirely consistent with the capacity ofthe mitochondrial inhibitor to convert normooxic cells into functionally hypoxic cells. With glucose alone, these cells might escapefrom pH modulation and consequently from selective sensitizationto pH-sensitive cytostatic drugs. The shift from oxidative phosphorylation to less efficient glycolysis was not apparent, however,from the MRS data on energy status (Fig. 4A), probably becauseonly a part of the tumor was involved in the response and becauseof efficient glycolytic compensations.

Our observations are in agreement with related studies, demonstrating that increased glucose availability selectively stimulates the accumulation of acidic metabolites in tumors, resulting in local pHreductions (7—14).We did not observe, however, similar large increases in lactate levels as described by other investigators in tumors(10) and tumor effusates (16) of hyperglycemic animals. According tothe MRS data, there were no significant changes in pH1 or in energy

Table 1 Effect oJMIBG and MIBG plus glucose on the relative uptake off'8F/fluorodeoxyglucose in RJF-1 tumors and muscle tissue'

a [‘8FJFDGuptake was measured by region of interest analysis of PET data asdescribed in “Materialsand Methods―in tumors (1') implanted in the posterior leg of maleC3H/Km mice and in control muscle tissue (M) of the contralateral leg. Control uptakelevels were recorded in untreated animals on day 0 (F, and M@,respectively) and set atthe value 1.00 for each individual animal. The next day, the animals received MIBG orMIBG plus glucose. [‘8FJFDGwas injected at 2.5 or 4.5 h following the indicatedtreatment. Recorded uptake values (‘I',and M,) were compared to individual controlvalues. The resulting ratios are means ±SD of 2 separate experiments with 2—4animalsin each treatment group. P values refer to the difference with control values of 1.00.

8-

6-

C,0IL.

2@@ J1T@Ii@ @T@ TttjjjI!IIilJII!iIiI

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2

tumor weight (g)

Fig. 8. [‘°F]FDGlevels in RIF-1 tumors in malemice recorded by PET analysis 24 h before (U) and3—5h after treatment with MIBG (40 mg/kg) alone(@)or withMIBGplus1.5g/kgglucose(0).E‘8F]FDGlevelsareexpressedasspecificuptakeinpercentage of injected dose/g tissue minus background values recorded in the corresponding area inthe contralateral leg and plotted as a function oftotal tumor weight.

3790

DISCUSSION

The results in this report confirm a previous conclusion (24) thatMIBG is a useful adjunct to glucose administration for tumor-specificacidification. Combined treatment resulted in significant and sustamed lowering of intratumoral pH recorded with semimicroelectrodes in RIF-1 tumors hosted by rats or male mice (Figs. 1 and 3). Inthe rat model, the reduction by almost a full pH unit relative to normaltissue and lasting for prolonged periods, i.e., from 3 to 10 h after thestart of treatment, fulfil the requirements for clinically meaningfulpotentiation of various conventional and experimental, pH-sensitivedrugs. Coadministration of MIBG reduced the total amount of glucoserequired for achieving significant tumor acidification. Thus in the ratmodel, MIBG had a marked, about 5-fold, glucose-sparing effect inmaintaining plasma glucose levels at 14 m@iduring infusions. Likewise, in the mouse model, combined i.p. treatment with MIBG andglucose caused increases in tumor and plasma glucose levels whichwere not detected with either agent alone (Fig. 5), suggesting thatMIBG interfered with glucose mobilization and sequestration (seebelow). MIBG was also clearly synergistic with glucose in lowering

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MODULATION OF TUMOR pH

status. Apparently, the tumor cells remained capable of active exportof protons and other acidic waste products to maintain intracellular pHat near-normal values. In this respect, manipulation of tumor pH inour protocols differed mechanistically from “acidictrapping―inducedby venal occlusion after hyperthermia or photodynamic therapy whichdoes include changes in pH1 and energy status (37).

The biochemical and metabolic studies were much complicated bylarge interanimal variation. Experimental error was always higher incases of glucose administrations than in controls (Fig. 5) or treatmentwith MIBG alone (Table 1). Variable responses to glucose administration have been observed in relation with tumor implantation siteand also among s.c. transplants of histologically similar tumors (16).This variation and the inaccessibility of most human tumors in situ tomeasurements with pH electrodes stresses the need for noninvasivemonitoring of glucose metabolism in clinical tumors considered fortherapies involving pH manipulation. It would appear from our datathat PET analysis of the uptake of [‘8F]FDG,a convenient radiopharmaceutical of glucose consumption (30), could serve this purpose.Nevertheless, [‘8FIFDGuptake is only a qualitative measure ofchanges in glucose metabolism. As shown in Table 1, MIBG with orwithout glucose had similar effects on [‘8F]FDGuptake while havingdifferential effects on intratumoral pH. During combined treatmentthere was an increase in glucose levels in plasma, tumors, and muscletissue which reduces the effective specific radioactivity of the tracer.Since tumor glucose uptake is well below saturation (38) and increases proportional by glucose availability (39), [‘8F]FDGuptakeprobably underestimates total glucose uptake in tumors during cornbined treatment. Conversely, lower [18F]FDG uptake by muscle tissue(Table 1) under conditions of glucose mobilization does not necessarily reflect a proportional decrease in net glucose utilization.

MIBG has been developed from the neuron-blocking agents bretylhum and guanethidine for the in vivo scintigraphy by [‘31I]MIBGofthe myocardial and neuroadrenergic tissues. MIBG localizes to catecholamine-storing granules of various normal and malignant neuroadrenergic cells, displacing endogenous biogenic amines (40). At highdoses, as applied in the present experiments, systemic (indirect)sympathomimetic effects by release of noradrenaline can be anticipated, therefore. These effects can include increased blood pressure;stimulation of glycogenolysis in kidney, liver, and muscle; as well asrises in plasma glucose and lactate (41). Pharmacological effects ofMIBG, typical of the above-described sympathomimetic responses,were indeed indicated in mice. Stimulated heart activity was obviousfrom increased uptake of [3H]DG in this organ and of [‘8FJFDGuptake in the thoracic area in MIBG-treated animals. The glucosesparing effect of MIBG in both animal models are also consistent withstimulated gluconeogenesis. Finally, in mice the sex-related differences in response probably reflect a generally higher level of sympathetic innervation and catecholamine levels in males. Similar differences may exist in rats but were not apparent in the present study withfemale hosts only.

All this raises the question of whether MIBG contributes to tumoracidification by biochemical mechanisms at the cellular level ormerely by promoting glucose availability to tumors by systemic,pharmacological mechanisms. Increased cardiac output from a MIBGinduced stress response can conceivably promote glucose uptake fromnormoglycemic blood and was actually suggested by PET analysis of[‘8F]FDGuptake (Fig. 8; Table 1, MIBG alone). Likewise, in rats(Fig. 1) MIBG could facilitate the access of moderately hyperglycemic blood to the tumor tissue, diminishing the need for a steepgradient between plasma and intratumoral glucose concentrations asobserved under conditions of severe hyperglycemia. On the otherhand, the pH effects of MIBG in the presumed normooxic tumorperiphery (Fig. 2) are consistent with reported cellular effects of the

drug (25). Moreover, a true synergism between MIBG and elevatedglucose levels in the acidification of tumor cells grown in tissueequivalent densities in vitro has been repeatedly confirmed in thislaboratory. On balance, it would appear that potentiation of glucoseinduced acidification by MIBG in vivo is based on both cellular andsystemic actions of the drug. The relative contribution of each mechanism to pH modulation remains to be established, however.

In conclusion, treatment with a mitochondrial inhibitor supportedby extra glucose supply appears to be an effective, nontoxic intervention for prolonged and selective stimulation of H@ ion activity in theinterstitial fluid of tumors, the effect of which can be noninvasivelymonitored by PET analysis. It may be superior to severe hyperglycemia because of much lower glucose requirements and of more consistent and homogeneous tumor acidification by including well-perfused areas in the response. In view of predominant reduction of pH@,acid-labile prodrugs and bioreductive agents are the first candidatecompounds to be tested for an improved therapeutic index in cornbined treatments.

ACKNOWLEDGMENTS

The assistance of Marianne Hulsbergen, Department of Clinical Chemistry,in glucose and lactate determinations; the assistance of Karin Buurman inthe animal experiments; the technical assistance of Janet Sansom with the

MRS experiments; and critical comment of Dr. J. Bernards are gratefullyacknowledged.

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1994;54:3785-3792. Cancer Res   A. Kuin, L. Smets, T. Volk, et al.   -Iodobenzylguanidine and Moderate Hyperglycemia

mReduction of Intratumoral pH by the Mitochondrial Inhibitor

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