potentiation of interleukin lor mediated antitumor effects by ketoconazole1

[CANCER RESEARCH 50. 4709-4717. August 1, 1990]

Potentiation of Interleukin lor Mediated Antitumor Effects by Ketoconazole1

Paul G. Braunschweiger,2 Nirmal Kumar, loannis Constantinidis,3 Janna P. Wehrle, Jerry D. Glickson,Candace S. Johnson,4 and Philip Furmanski5

Laboratories of Experimental Therapeutics Â¡P.G. B., N. K.J. Experimental Hematology [C. S. J./ and Cell Biolog)' [P. F.], AMC Cancer Research Center, Denver,

Colorado 80214 and Division ofNMR Research. Department of Radiolog); Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Â¡I.C., J. P. W.,J. D. G.I

ABSTRACT

In the present studies, the regulatory role of adrenal hormones on theantitumor activity of recombinant human interleukin la (IL-la) wasinvestigated. Ketoconazole, a potent but transient inhibitor of adrenalsteroid hormone biosynthesis, inhibited IL-la induced increases inplasma corticosterone. In s.c. RIF-1 tumors (C3H/HeJ mice) ketocona-zole potentiated IL-la induced hemorrhagic necrosis ("Fe labeled RBCuptake) and prolonged intervals of low tumor perfusion (""Kli1 uptake)

and attendant depletion of tumor high energy phosphate reserves asdetermined by in vivo 31P nuclear magnetic resonance spectroscopy. Innormal muscle and skin the ketoconazole-IL-la combination had noeffect on RBC content and little or no effect on tissue perfusion. Ketoconazole potentiation of IL-la induced tumor pathophysiologies was accompanied by time and ketoconazole dose dependent potentiation of RIF-1tumor clonogenic cell killing. Although ketoconazole at 40 mg/kg andIL-la at 25 Mg/kg alone each produced approximately 50% clonogeniccell kill, a combined treatment (IL-la l h after ketoconazole) resulted insurviving fractions of approximately 1.5%. In vitro, ketoconazole and IL-la induced only additive clonogenic cell kill in primary RIF-1 expiantcultures. The effect of elevated plasma corticosterone levels, induced byketamine-acepromazine anesthesia, on IL-la responsiveness was alsostudied in the RIF-1 tumor model. In C3H/HeJ mice, anesthesia increased plasma corticosterone levels within 30 min, abrogated the IL-laeffect on tumor perfusion, and prevented depletion of tumor high energyphosphate metabolite reserves. Our results are consistent with the hypothesis that IL-la mediated adrenal hormone responses exert a profound negative feedback on IL-la antitumor activities. Our data alsoindicate that adrenal steroid hormone biosynthetic pathways could provide a focus for modulation strategies to increase the efficacy of cytokinebased therapeutic interventions.

INTRODUCTION

Interleukin \a is a multifunctional cytokine with markedantitumor activity in murine tumor models (I, 2). The patho-physiology of the IL-1Â«6response in RIF-1 tumors is charac

terized by vasodilation, vascular congestion, reduced tumorperfusion, extravascular hemorrhage, increased extracellularwater content, a transient reduction in bioenergetic status, andclonogenic cell kill (1, 3).

Corticosteroid hormones are known to inhibit immune functions and ACTH induced increases in plasma corticosteroneafter IL-1Â«treatment in vivo (4, 5) are thought to impart a

Received 10/3/89; revised 4/16/90.The costs of publication of this article were defrayed in part by the payment

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

1This work supported in part by Grants CA 48077. CA 33188. CA 49143,

and CA 44703 from the Department of Health. Education, and Welfare and agift to AMC Cancer Research Center from Gerald M. Quait.

2To whom requests for reprints should be addressed.3 Present address: Department of Radiology. Emory University School of

Medicine, Atlanta, GA.4 Present address: Department of Otolaryngology. University of Pittsburgh

School of Medicine. Pittsburgh. PA 15213.*Present address: Department of Biology. New York University, New York,

NY 10003.'The abbreviations used are: IL-1Â«, recombinant human interleukin la;

ACTH, adrenocorticotrophic hormone; ID, injected dose; NMR, nuclear magnetic resonance; NTP. nucleoside triphosphate: PCr, phosphocreatinine: PDE,phosphodiesters; PME, phosphomonoesters; TNF, tumor necrosis factor.

negative feedback on IL-1Â«stimulated immune responses (6).Recently, we reported that Corticosteroid hormones inhibit IL-la antitumor activities in RIF-1 tumors (7, 8). In these studies,IL-1Â«mediated RIF-1 tumor clonogenic cell kill was substantially greater in adrenalectomized mice than in intact mice.

The antifungal properties of ketoconazole are mediatedthrough the inhibition of cytochrome P-450 dependent enzymesrequired for the conversion of lanosterol to ergosterol, a majorsterol and important membrane component in fungi (9). Inanimals and humans, ketoconazole is known to inhibit thebiosynthesis of adrenal steroids and to "blunt" responses to

ACTH through the inhibition of cytochrome P-450 dependent110-hydroxylase (10), 17Â«-hydroxylase (II), and 17,20-lyase(12). Ketoconazole has palliative activity against hormone dependent prostate cancers (13) and in long term culture it isdirectly cytotoxic to several tumor cell lines (14).

On the other hand, some anesthetic agents are known toinduce marked increases in circulating Corticosteroid levelsthrough the stimulation of ACTH release (15). Since our previous findings suggested that modulation of adrenal responsesto IL-1Â«might be an efficacious approach to enhance IL-1Â«antitumor activities (7, 8), the present experiments were conducted to study the effect of transient adrenal suppression byketoconazole and the effect of adrenal stimulation by ketamine-acepromazine anesthesia on IL-1Â«mediated antitumor activityin the RIF-1 tumor model system.

MATERIALS AND METHODS

Tumor Models. Maintenance of the RIF-1 tumor model has beendescribed in detail previously (16-19). RIF-1 tumor cells were propagated in vitro in RPMI 1640 medium (Mediatech, Washington, DC)supplemented with 7.5% newborn bovine serum and 7.5% colostrumfree bovine serum (Irvine Scientific, Santa Ana, CA), 2 HIMglutamine,and l Mg/rnl gentamicin (GIBCO, Grand Island, NY). RIF-1 tumorcells grow as well in this culture system as in RPMI media supplementedwith 15% fetal calf serum, as used previously (1).

Female C3H/HeJ mice, 6-10 weeks of age, were inoculated s.c. with5 x IO5log phase RIF-1 tissue culture cells, on the right flank. Studies

were initiated 14 days later when tumors were approximately 0.5 g. Allmice were obtained from The Jackson Laboratory (Bar Harbor, ME)and quarantined for a period of 2 weeks prior to entering studies.Randomly selected mice were tested and found to be free of adventitiousmurine viruses. All mice were housed 4-5/cage, in a temperature andhumidity controlled, American Association for the Accreditation ofLaboratory Animal Care approved facility with a 12-h light-dark cycle(lights on at 6 a.m. local time). Mice were provided standard mousechow and water ad libitum. All treatments were routinely initiatedbetween 7 and 9 a.m. local time.

Interleukin la and Ketoconazole Treatments. Recombinant humanIL-la was generously provided by Dr. Peter Lomedico (Hoffmann-LaRoche Inc., Nutley, NJ). The IL-1Â«used in these studies was highlypurified (2.5 x IO9D,0 units/mg protein) and essentially free of endo-toxin contamination (<0.125 endotoxin unit/mg protein). The IL-1Â«was diluted in nonpyrogenic 0.9% NaCl, containing 0.05% bovineserum albumin and administered at 25 Mg/kg (6.25 x IO7DIOunits/kg)body weight (â€”0.5Mg/mouse) in 0.2 ml, total volume, by i.p. injection.This dose is similar to that used in our previous studies (1,7) and 2-3

4709

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IL-la ANTITUMOR ACTIVITY

times the dose we previously showed to minimally stimulate murinehematopoiesis (20).

Ketoconazole was purchased from Sigma Chemical Company (St.Louis, MO), dissolved in l N HC1, diluted in Hanks' balanced salt

solution to 0.1 v and administered in 0.1 ml, total volume, by i.p.injection.

Quantitation of Antitumor Activity. The hemorrhagic response inRIF-1 tumors treated with ketoconazole and IL-la was quantitated bydetermining the packed RBC volume using an 59Felabeled RBC methoddescribed previously (1, 7, 19). One Â¿iCiof 59Fecitrate (NEN, Boston,

MA) was administered i.p. 7 days after tumor inoculation. Studies wereinitiated 7 days later to provide adequate time for labeled erythrocytesto enter circulation and for unincorporated 59Fe to be cleared (21). At24 h after treatment with ketoconazole and/or IL-la, peripheral venousblood was sampled with heparinized hematocrit tubes from the postorbital venous plexus and centrifuged. The animal was then killed bycervical luxation, and the tumor was quickly removed, weighed, andcounted for radioactivity in a gamma scintillation spectrometer (Packard Instruments, Downers Grove, IL). The packed RBC in the hematocrit tubes were also counted for radioactivity and both the tumor andRBC radioactivity expressed as a fraction of the injected dose. Thepacked RBC volume/g of tissue (iil/g) was calculated as the activityratio of the tumor (ID/g) and the venous RBC (ID/Ml packed RBC).

Blood Flow. Perfusion in tumor, femoralis muscle, and skin (pina)was estimated by the 86RbCl distribution assay first described by Sapir-stein (22) and used by us in previous studies with the RIF-1 tumormodel (1,7). Briefly, approximately 200,000 cpm/20 g mouse of 86RbCl

(NEN) was injected via the lateral tail vein. Forty-five s later the micewere killed by cervical luxation, and tissue samples were quickly collected and weighed. Radioactivity was measured using a gamma wellscintillation spectrometer (Packard) and expressed as a fraction of theID. The ID/g tissue is taken to be the distribution of the cardiac outputto the tissue (22).

"P NMR Spectroscopy. 3'P NMR spectra of s.c. implanted tumorswere obtained with a Bruker AM-360 wide bore NMR spectrometerequipped with an 8.5-T/8.9-cm superconducting magnet and interfacedto an Aspect 3000 computer as described before (3). A home built probecontaining a 3-turn solenoidal radiofrequency coil, 1.5 cm in diameter,doubly tuned to 'H and "P (23), was used for these measurements. The

magnetic field was optimized for each tumor to obtain an H2O resonance with a maximum line width of 100 Hz. Each spectrum wasobtained by accumulating 200 scans using a 60-degree flip angle (8 ps)and IK data points for 8 kHz spectral width. A repetition time of 3 swas used. Given the magnitude of the phosphorus changes observed,any saturation effects would be negligible. Resolution was enhanced bythe convolution difference method (24) using 1000 Hz and 20 Hzexponential multiplication. Chemical shifts are reported with respectto the a-NTP resonance, which was assigned to -10 ppm as an internal

reference. Integrated resonance areas were measured using a Lorentzianline fitting program (GLINFIT; Bruker Users Society). In the cases ofPME and PÂ¡,where two Lorentzians were sometimes required forproper fitting, and PDE, where three Lorentzians were required, thereported values are the sum of Lorentzians fitted. Similarly, pH valueswere calculated from the average PÂ¡chemical shifts, weighted by thearea of each PÂ¡resonance.

In order to study the effects of ketoconazole and IL-la on tumorphosphorus metabolites by in vivo 3'P NMR spectroscopy, 48 RIF-1

tumor bearing mice were divided into 8 groups of 6 animals each. Thefirst 3 groups were used to obtain control spectra (0, 10, and 48 h)while the mice in groups 4-8 were treated with ketoconazole (40 mg/kg; Sigma) 1 h prior to the administration of a single dose of IL-1Â«(25Â¿"g/kg)-3'P NMR spectra were obtained from cohorts at 2, 6, 12, 24,and 48 h after IL-la treatment. In these studies, anesthesia was inducedby an i.p. injection of a ketamine hydrochloride (50 mg/kg; Ketaset;Bristol Laboratories, Syracuse, NY), acepromazine malÃ©ate(5 mg/kg;ProMace; AVECO, Inc., Fort Dodge, IA) cocktail, 10 to 15 min priorto examination. Another group of 18 tumor bearing mice was used tostudy the time dependent effects of ketoconazole (40 mg/kg) alone onRIF-1 tumor 31PNMR spectra. As in the combination study, treatments

were initiated between 7 and 9 a.m. local time.

The effect of the anesthesia and immobilization stress, associatedwith the NMR protocol, on the IL-la response was also determined.Tumor bearing mice were anesthetized as above, and a pre-IL-latreatment spectrum was obtained approximately 20 min after theinduction of the anesthesia. The mice were then given i.p. injections ofIL-1Â«(approximately 30 min after anesthesia) and then reexamined 2and 4 h later when IL-1Â«mediated perturbations of tumor phosphatelevels were most profound (3). A total of 6 mice were used for thisstudy.

Clonogenic Cell Survival. Clonogenic tumor cell kill in RIF-1 solidtumors treated with IL-la, and/or ketoconazole was determined by amodification (1) of the excision clonogenic cell survival assay describedby Twentyman et al. (16). RIF-1 tumors were excised aseptically 24 hafter IL-la treatment, weighed, minced with scissors, and incubated

with an enzyme cocktail (trypsin, type 3, 0.75 mg/ml; collagenase, type2, 0.75 mg/ml; and DNase, 0.05 mg/ml; all from Sigma) in Hanks'

balanced salt solution, at room temperature for 45 min with constantagitation. In a typical experiment, tumors were resected, weighed, andbisected, and weighed tissue from 2 to 3 tumors was pooled prior tomincing. Similarly prepared suspensions from untreated control tumorswere included in each experiment. The dissociated cell suspension wasfiltered through sterile gauze and centrifuged. The cells were resus-pended in fresh media containing 15% serum, washed twice, counted,diluted, and plated out at several dilutions in 6-well cluster plates(Costar, Cambridge, MA). Control tumor cell yields were routinely 7-14 x IO7cells/g tissue. The cultures were incubated for 7 days at which

time the colonies were counted and the number of clonogenic cells/gof tissue was determined. Surviving fractions were taken as the ratio ofclonogenic cells/g for treated and control tumors.

The effect of IL-1Â«and ketoconazole on the survival of RIF-1 tumorclonogenic cells in primary expiant cultures was also determined. Inthese experiments primary expiant cultures were established as described previously (1) and treated with IL-1Â«and/or ketoconazole for24 h. Vehicle controls were also studied. Cells were then harvested bytrypsinization, washed, counted, diluted, and replated in fresh medium.Colonies were counted 7 days later and the number of clonogenic cells/flask was calculated. Surviving fractions were calculated as the ratio ofclonogenic cells per flask in treated and vehicle control cultures.

Blood Sampling and Plasma Corticosterone Levels. Plasma corticosterone was determined using a radioimmunoassay kit (ICN Biomedi-cals, Inc., Irvine, CA) according to the directions provided by themanufacturer. Mice were housed 5/large cage and the bedding waschanged 3 days prior to study. IL-la treatments were given between 7and 8:45 a.m. local time to minimize the effect of diurnal variation.Ketoconazole was given 1 h prior to IL-1Â«.Untreated, ketoconazolealone, and IL-la alone controls were concurrently studied. Since previous studies have shown that maximal plasma corticosterone increasescan be expected within 2-3 h after IL-la treatment (4-6), blood sampleswere obtained 2 h after IL-1Â«from the postorbital venous plexus in

heparinized Pasteur pipettes. Cohorts of untreated controls were sampled at 8 and at 11 a.m. local time.

In some experiments, ketamine (50 mg/kg)-acepromazine (5 mg/kg)anesthesia was used to stimulate corticosterone release. The anesthesiawas administered at 8:15 a.m. local time and the mice were immobilizedin a vertical orientation for 15 min to simulate the conditions of theNMR spectroscopy protocol. At 30 or 60 min after induction ofanesthesia, blood samples were obtained, as described above, andplasma corticosterone levels were determined. Unanesthetized and otherwise untreated, undisturbed controls were bled at 8 a.m. to obtainunstimulated control plasma corticosterone levels. Each mouse wassampled only once and to minimize the effects of sampling stress andhandling, the bleeding was completed within 15 s.

Statistical Analysis. Analysis of variance was used to determine if theobserved treatment effects produced by IL-1Â«and ketoconazole aloneor in combination could be accounted for by chance alone. Wheresignificant treatment effects were detected, the Newman-Keuls multiplerange test was used to test the significance of differences between groupmeans. For both tests, P = 0.05 or less was considered adequate toreject the null hypothesis (25).

4710



IL-1Â«ANTITUMOR ACTIVITY

RESULTS

RIF-1 tumors in C3H/HeJ mice are well vascularized andrarely exhibit areas of necrosis. Fig. 1 shows the appearance ofrepresentative RIF-1 tumors resected 24 h after the mice weretreated with IL-1Â«alone or with ketoconazole (40 mg/kg) andIL-1Â«.Hemorrhagic responses in tumors treated with the ke-toconazole-IL-1Â« combination were more pronounced thanthose in tumors treated with IL-1Â«alone. In controls, neitherthe IL-1Â«vehicle nor ketoconazole alone had any noticeableeffect on these tumors.

The effect of ketoconazole on IL-1Â«mediated hemorrhagicresponses was quantitated by the intratumor accumulation of"Fe-RBC (Fig. 2). Hemorrhagic responses measured 24 h aftertreatment indicated that although IL-1Â«alone produced significant increases in tumor packed RBC volumes, ketoconazole

Fig. 1. Gross appearance of representative RIF-I tumors treated with IL-lnalone (2A) or IL-IÂ« at 1 h after 40 mg/kg ketoconazole (2B). Tumors wereresected 24 h after IL-1Â«treatment, bisected along the longest axis with a scalpel,and folded open to expose the center to the tumor. Also shown are representativevehicle alone (1A) and ketoconazole alone (IB) controls. Bar. 1 cm.

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azole on the packed RBC volume in tumor, muscle, and skin 24 h after treatment.Each group is the mean Â±SE (bars) for 7 samples. C, control.

alone had no significant effect. Treatment with 40 mg/kgketoconazole l h before IL-1 a, however, produced a greater (P< 0.05) response than that seen after IL-1Â« alone. Ketoconazole, IL-1Â«,or the combination had no significant effect on thepacked RBC volume in muscle and skin at this time.

Fig. 3 shows the effect of ketoconazole alone on IL-1Â«induced increases in plasma corticosterone levels in C3H/HeJmice. In untreated, undisturbed control mice, plasma corticosterone levels ranged from 14.5 to 127 ng/ml. Samples obtainedat 8 and at 11 a.m. from controls exhibited similar plasmacorticosterone levels. Analysis of variance indicated highly significant (F = 56.6; PÂ« l x 10~5) treatment effects. Plasma

corticosterone levels 2 h after IL-1Â« alone were markedlyincreased. Although ketoconazole alone at 20 mg/kg resultedin a significant (P < 0.01) increase in plasma corticosterone,the magnitude of the response was significantly (P< 0.05) lessthan that seen after IL-1Â«alone. Plasma corticosterone levels

after 40 or 60 mg/kg ketoconazole alone were not significantlydifferent from those in the untreated controls. When 20 mg/kgketoconazole were given l h prior to IL-1Â«the plasma corticosterone levels were significantly (P< 0.001) increased relativeto those in the untreated control, but not to any greater extentthan that seen after 20 mg/kg ketoconazole alone. When ketoconazole at 40 or 60 mg/kg was given l h prior to IL-1Â«,theIL-1Â« induced rise in plasma corticosterone was completelyabolished.

Several types of anesthesia are known to stimulate the releaseof ACTH and reuslt in increased plasma corticosterone levels(15). The effects of ketamine-acepromazine anesthesia onplasma corticosterone levels determined at 30 or 60 min afteranesthesia induction are shown in Fig. 4. Significant increasesin circulating levels of plasma corticosterone were observedwithin 30 min after anesthesia. The magnitude of this responsewas approximately 60% ofthat seen after IL-1Â«alone.

Fig. 5 shows the effect of ketoconazole on IL-1Â«mediatedchanges in tumor perfusion as measured by the uptake of'"'Rb*.IL-1Â«alone resulted in a reduction in 86Rb+ uptake within 30min after treatment. 86Rb+ uptake at l h after ketoconazole

alone was not significantly different from that in untreatedmice. Treatment with IL-1Â«l h after ketoconazole resulted ina longer interval of reduced 86Rb+ uptake than after IL-1Â«

alone. In tumors treated with the ketoconazole-IL-1Â« combination, recovery was not detected until 48 h. Although markedand sustained reduction in 86Rb+ uptake was observed in tu

mors, the treatment had little or no effect on the uptake of86Rb+in normal muscle and skin (Fig. 6).

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Fig. 3. Effect of IL-1Â«alone(25 ng/kg). ketoconazole alone, and ketoconazolegiven l h prior to IL-In on plasma corticosterone levels. Blood samples wereobtained at 2 h after IL-1Â«and 3 h after ketoconazole. Control blood sampleswere obtained at 8 a.m. and 11 a.m. local time. K20, 20 mg/kg ketoconazole;K40, 40 mg/kg ketoconazole: K60, 60 mg/kg ketoconazole; bars, SE.

4711



IL-1n ANTITUMOR ACTIVITY

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Fig. 4. Effect of kctamine (50 mg/kg)-acepromazine (5 mg/kg) anesthesia(K+A) and handling on the plasma corticosterone levels in mice at 30 and 60 minafter anesthesia induction. The anesthetized mice were physically restrained in avertical position for 15 min, beginning 5 min after anesthesia induction, tosimulate the NMR spectroscopy protocol. Control (C) mice were not immobilizednor were they disturbed in any other way until the blood sample was obtained.Also shown are plasma corticosterone levels for mice 2 h after a single IL-lntreatment. All injections were administered between 8 and 8:15 a.m. local time.Control blood samples were obtained at 8 a.m. Each column is the mean Â±SEM(bars) for 5 mice. Analysis of variance indicated highly significant (P < 0.001)treatment effects.

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Fig. 5. Effect of IL-ln (25 iig/kg) alone (â€¢)and IL-la given l h after 40 mg/kg ketoconazole (O) on the uptake of "Rb* in the RIF-1 tumor model. Each

symbol is the mean Â±SEM (bars) for at least 7 tumors. Analysis of varianceindicated highly significant (P < 0.0001) overall treatment effects. Note thechange in abscissa scaling at 12 h. *, significantly (P < 0.05) different (i test)from the respective age matched IL-la alone cohort. The 86Rb* uptake 1 h after

ketoconazole alone was not significantly different from that in the untreatedcontrols.

Since ketamine-acepromazine anesthesia resulted in promptincreases in plasma corticosterone levels and corticosteroidshad been shown previously to inhibit IL-la antitumor responses, the effect of anesthesia on IL-1Â«mediated changes intumor perfusion was determined (Table 1). Analysis of varianceindicated significant treatment effects for tumor (F'= 22.7, P<IO"5) and muscle (F = 11.7, P < 10~4), but not for skin (F =

2.2). Anesthesia and immobilization (group 2) alone significantly (P < 0.005) reduced 86Rb+ uptake in tumor, but not inmuscle. When IL-la was given 1.75 h prior to anesthesia andimmobilization (group 3), the 86Rb+ uptake in both tumor andmuscle at 2 h after IL-la was significantly less than that seenin untreated (group 1) or anesthesia (group 2) controls. Incontrast to the enhancing effect of ketoconazole on IL-lamediated changes in tumor perfusion, ketamine-acepromazineanesthesia (group 4) abrogated IL-la mediated changes in theperfusion of tumor and muscle. In these mice the 86Rb+uptake

in tumor and muscle was similar to the respective uptakes afteranesthesia alone (group 2) and significantly (P < 0.05) greater

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Fig. 6. Effect of ketoconazole (40 mg/kg)-IL-lÂ« (25 fig/kg, l h after ketoconazole) on ""Kli uptake in tumor (â€¢),femoralis muscle (A), and skin (â€¢).Each

point is the mean Â±SEM (bars) for 7 samples. Tumor data are replotted fromFig. 2. Analysis of variance indicated significant treatment effects for tumor andfemoralis muscle. The combination had no significant effect on the "*Rb* uptakein skin. Note the change in abscissa scaling at 24 h. *, significantly (P < 0.05)different from the pre-IL-ln value (Newman-Keuls multiple range test).

Table 1 Effect ofketamine (50 mg/kg). aceproma:ine (5 mg/kg) anesthesiawith and without IL-ln on S6AA*uptake (%ID/g) in tissues from RIF-1 tumor

bearing mice

Experimentalgroups1.

Control2.A43.

IL-IO+A*4.A + IHÂ«/Tumor1.985

Â±0.473 (13)Â°1.301 Â±0.415 (7)f0.066 Â±0.273 (5)'1.212 Â±0.211(13)rÂ°

MRb* %ID/g; mean Â±SD (n).* Anesthesia (A) was administered 2.5Muscle2.743

Â±0.6192.731 Â±0.9561.103 Â±0.373'

2.053 Â±0.422h

prior to "RbClSkin2.604

Â±0.8131.885 Â±1.4112.042 Â±0.8332.004 Â±0.930assay;

mice wereimmobilized in a vertical position for 15 min after anesthesia induction.

' Significantly (P ^ 0.05) different from control (group I).d Anesthesia was administered I h and 45 min after IL-la; 86RbCl assay was

conducted at 2 h after IL-ln.' Significantly (P s 0.05) different from groups 1. 2, and 4.^IL-1Â« was given 30 min after anesthesia. **RbCI assay conducted 2 h after

II I- treatment.

than that seen Â¡ngroup 3 tumors where IL-1Â«was given priorto anesthesia.

The effect of ketoconazole and ketamine-acepromazine anesthesia on IL-la mediated changes in tumor metabolism wasstudied by in vivo 31P NMR spectroscopy. Fig. 7 shows "PNMR spectra from representative size matched RIF-1 tumorstreated with ketoconazoIe-IL-1Â«. The spectra obtained fromcontrol tumors indicated relatively well energized tumor tissuewith detectable PCr resonances and PÂ¡//i-NTP resonance arearatios of approximately 1.0. Marked spectral changes wereobserved within 2 h. PÂ¡resonance intensities were increasedrelative to those for NTP, PCr, and PDE. These spectralchanges persisted for at least 24 h after treatment. At 48 h thespectra were similar to those obtained from the pretreatmentcontrols. During the 48-h study interval, analysis of varianceindicated that the treatment produced no significant change incalculated tumor volumes.

Table 2 shows the calculated metabolite ratios and NMR pHvalues for control tumors and for tumors treated with ketocon-azole-IL-la. In untreated control tumors, significant decreasesin PCr/PE and PDE/PME ratios were seen in the 48-h group.The NMR pH in these 48-h age matched controls (6.80 Â±0.12)was significantly lower than that seen in either the pretreatmentor the 10-h untreated controls. A significant increase in thePME//3-NTP ratio was also noted in these 48-h controls. Thesespectral changes are characteristic of those observed in tumorsduring progressive and unrestricted growth (26). Analysis ofvariance indicated that ketoconazole-IL-la produced marked

4712



IL-I<r ANTITUMOR ACTIVITY

Fig. 7. Representative in vivo 31PNMR spectra obtained from control tumors(A) and from size matched RIF-1 tumors at 2 h (B), 6 h (C), 12 h (O). 24 h (E).and 48 h(F (after the ketoconazole-IL-1 a combination treatment. Tumor bearingmice were treated with IL-la (25 Â¿tg/kg),l h after ketoconazole (40 mg/kg) andanesthetized (ketamine-acepromazine) for 3'P NMR study 5-10 min prior toexamination. Tumor size for controls was 0.53 Â±0.21 cm3. Tumor sizes at 48 hafter treatment (0.60 Â±0.12 cm3) were not significantly greater than that forcontrols and analysis of variance indicated no significant overall effect of treatment on tumor size. Each spectrum was obtained from a total of 200 scans. Totalacquisition time was 10 min.

effects on PÂ¡/0-NTP (F = 35.9), PCr/PÂ¡(F = 14.5), PME//8-NTP (F = 24.2), and PDE/PME (F = 26.2). Significant increases in Pj/0-NTP and PME/ÃŸ-NTP ratios were seen duringthe first 24 h after treatment with the combination. At the sametime, significant decreases were observed for PCr/PÂ¡and PDE/PME ratios. These changes were detected as early as 2 h afterIL-la treatment.

Poor signal/noise for the a-NTP and PCr resonances wasobserved in most spectra during the first 24 h after treatment.Since this could introduce significant errors in the determination of the chemical shifts for these metabolites and the PÂ¡resonance, the NMR pH can be reported only for tumors in the48-h treatment group. At this time, however, a significantalkaline shift was detected. These tumors not only exhibitedhigher NMR pH values than did age matched controls but alsohigher NMR pH values than those seen for tumors in thepretreatment or 10-h control groups.

In mice scanned at 4 or 24 h after ketoconazole alone, nosignificant effect on any of the metabolite ratios or on NMRpH was seen (Table 3).

In contrast to the potentiation of IL-la mediated changes intumor, high phosphate metabolite ratios by ketoconazole-ket-amine-acepromazine anesthesia and NMR scanning prior toIL-la treatment significantly inhibited IL-la mediated changesin tumor bioenergetics (Table 4). Quantitative studies to assessthe effect of anesthesia on IL-la induced hemorrhagic necrosiswere not conducted. Gross examination of tumors at 24 h afterthe anesthesia and IL-la treatment, however, indicated little orno hemorrhagic necrosis.

Although IL-la (25 Mg/kg) and ketoconazole (40 mg/kg)alone produced significant clonogenic tumor cell kill in vivo,ketoconazole potentiated IL-la mediated clonogenic tumor cellkilling in a time dependent fashion (Fig. 8). More than additive(P < 0.01) clonogenic cell kill was seen when ketoconazole wasgiven at 30 or 60 min, but not at 120 min before IL-la.

In vivo clonogenic tumor cell kill was also ketoconazole dosedependent (Fig. 9). When IL-la was given l h after ketoconazole, greater than additive responses were seen for ketoconazoledoses between 40 and 100 mg/kg. Ketoconazole alone, up to100 mg/kg, produced no more than a 50% reduction in RIF-1tumor clonogenic cellularity.

In explant cultures from primary RIF-1 tumors, IL-la (1000units/ml, 24 h) treatment decreased the number of clonogeniccells per flask by approximately 50% (Fig. 10). Ketoconazole(20 Mg/ml, for 24 h) also produced a slight but significantdecrease in the number of clonogenic cells in the explantcultures. Exposure to IL-la and ketoconazole for 24 h resultedin an additive decrease in the number of clonogenic cells perflask. Although a 24-h treatment with 60 /ug/ml ketoconazoleproduced a greater decrease in RIF-1 clonogenic cells/flask (25Â±3%, surviving fraction) in expiant cultures than did 20 /tig/ml, the cytotoxic effect of a 24-h exposure to IL-la (1000 units/ml) and ketoconazole (60 pg/m\) together (surviving fraction,10.7 Â±2.2%) was not different from that expected for anadditive effect.

DISCUSSION

Adrenalectomy profoundly enhanced IL-la mediated hemorrhagic necrosis and clonogenic tumor cell kill in RIF-1 tumors (7). Although hemodynamic toxicity was also increased,dexamethasone, given before or up to 3 h after IL-la, ameliorated toxicity (7, 27). Corticosteroids are known to inhibit thesynthesis of IL-la (28), prostaglandins, and leukotrienes (29,30), to reduce capillary permeability in solid tumors (19, 31);

Table 2 Effect of ketoconazole and IL-la on the phosphate metabolite ratios in RIF-1 tumors

ControlOhÂ«lOh48

hKetoconazole

+IL-1Â«'2h6h12h24

h48hPCr/Pi0.32

Â±0.12*0.41

Â±0.140.17+0.16''0.11

Â±0.05"0.05Â±0.03"0.08Â±0.06"0.01Â±O.Oi"0.59Â±0.33'Pi/0-NTP1.17

Â±0.280.91Â±0.232.22Â±0.944.87

Â±1.00"10.32Â±2.65''7.24Â±2.60''7.84Â±0.90"1.06Â±0.53'PME/fi-NTP1.15

Â±0.410.96Â±0.22'1.94+0.63''2.98

Â±0.32'-"3.65Â±0.72'-"3.19+ 0.79''"1.92

Â±0.41"1.25+ 0.31PDE/PME0.46

Â±0.24'0.59Â±0.17'0.19

+0.05''0.12

Â±0.03"0.09Â±0.06"0.20Â±0.12"0.24Â±0.06"1.01Â±0.19e-''NMR

pH7.05

+0.107.11Â±0.086.80

Â±0.12"7.26

Â±0.06'- "

" Controls were examined at 0, 10, and 48 h after IL-la.Metabolite ratios determined from the integrated areas of the resonances fitted by Lorentzian curves (mean Â±SD, n = 6). NMR pH values at 2, 6, 12, and 24 h

after treatment were not calculated due to poor signal/noise ratios for the PCr and a-NTP resonances.c Significantly different (P < 0.05) from 48-h controls." Significantly different (P < 0.05) from 0- and 10-h controls.' Mice were treated with IL-1Â«(25 Mg/kg) l h after ketoconazole (40 mg/kg) treatment and the tumors were examined at 2, 6, 12, 24, or 48 h after IL-la.

4713



IL-ltt ANTITUMOR ACTIVITY

Table 3 Effect of ketoconazole alone on the high energy phosphate metabolite ratios and pii in RÃŒF-1tumors

Control4hÂ»

24 hANOVA'PCr/Pi0.32

Â±0.12"

0.28 Â±0.240.50 Â±0.19

NSEPi/0-NTP1.

17 Â±0.281.47 + 0.670.69 Â±0.20

NSEPME//3-NTP1.15

Â±0.411.29 + 0.451.01 Â±0.14NSEPDE/PME0.46

Â±0.240.50 Â±0.440.65 Â±0.15NSENMRpH7.05

Â±0.106.92 Â±0.247.15 Â±0.11NSE

" Metabolite ratios determined from the integrated areas of the resonances fitted by Lorentzian curves (mean Â±SD, n = 6).* Time after ketoconazole injection.' ANOVA. analysis of variance; NSE, no significant effect.

Table 4 Effect ofketamine (50 mg/kg)-aceproma:ine (5 mg/kg) anesthesia on IL-la mediated changes in phosphate metabolite ratios

Control2hÂ»IL-la

+AcA+IL-la4

hIL-la+AA

+IL-la12hIL-la

+AA+ IL-laPCr/P,0.32

+0.10Â°0.07

Â±0.070.22Â±0.19P

<0.01''0.08

Â±0.050.21Â±0.12^<

0.010.28

+0.190.19+0.10NSDP,//3-NTP1.17

+0.285.61

+2.731.07Â±0.30P

<0.013.66

Â±2.171.27Â±0.37P

<0.011.92

Â±0.872.38+1.13NSDPME/0-NTP1.15Â±0.413.35

Â±1.410.87Â±0.13P

<0.012.08

Â±0.391.15Â±0.27P

<0.051.74

Â±0.521.71Â±0.38NSDPDE/PME0.47

+0.190.15

Â±0.100.45+0.15P

<0.010.14

Â±0.060.28+0.11NSD0.37

+0.270.24Â±0.18NSDNMRpH7.05

Â±0.106.97

Â±0.076.86+0.27NSD6.94

Â±0.056.97+0.08NSD7.03

Â±0.157.07Â±0.06NSD

Â°Mean metabolite ratios determined from the integrated areas of the resonances fitted to Lorentzian curves (mean + SD, n = 6).* Time after IL-la treatment. Anesthesia given 30 min prior to IL-la (A + IL-la) or 10 min prior to NMR examination (IL-la + A).f Metabolite ratios and NMR pH calculations for IL-ln + A groups have been published previously (3).* Level of significance for the differences between the two treatment groups determined by Student's t test; NSD. no significant difference.

1.00

0.10

0.01

1 Additivity Â±_95% CL

1 24

Mrs. Between Ketoconazole and IL-lot Treatment

Fig. 8. Sequence dependent clonogenic cell kill in RIF-1 tumors treated withketoconazole (40 mg/kg) and IL-la (â€¢);survival after IL-la alone (O) andketoconazole alone (A) are also shown. Each point is the mean + SE (bars) for atleast 2 experiments using pooled tissue from 2 tumors/experiment. CL, confidence limits.

1.000

0.100

0.010

0.0010 20 40 60 80 100 120

KETOCONAZOLE (mg/kg)Fig. 9. Ketoconazole dose dependent potentiation of IL-la mediated clono

genic tumor cell kill (â€¢).Ketoconazole was given l h prior to IL-la (25 jjg/kg).Also shown is the surviving fraction after ketoconazole alone (â€¢).Each symbol isthe mean Â±SE (bars) for at least two experiments using pooled tissue from twotumors in each experiment. CL, confidence limits.

O<o:

OL3

and to inhibit the induction of IL-1Â«mediated hemorrhagicnecrosis in solid tumors. When adrenalectomized tumor bearing mice were treated with IL-1Â«and 2 h later with dexameth-asone, the surviving fraction for clonogenic tumor cells (lessthan 2%) was more than 1 log lower than that seen in intactmice receiving the same IL-lÂ«-dexamethasone treatment (7).These results indicated that it might be possible through modulation of adrenal negative feedback responses to substantiallyincrease antitumor activity without attendant increases in tox-icity.

Pretreatment with ketoconazole, an agent known to inhibitACTH stimulated corticosterone biosynthesis (10, 11, 32, 33),inhibited IL-1Â«mediated corticosterone responses in C3H/HeJmice and potentiated IL-1Â« mediated antitumor responses.Potentiation of IL-1Â«mediated clonogenic tumor cell kill wasseen when ketoconazole, at 40 mg/kg and above, was given l hprior to IL-la. Although ketoconazole at the 20-mg/kg doselevel inhibited IL-1Â«induced increases in circulating corticos-

Control1.0 r Ketoconazole

Og

<n

1Ã•1IL-1

a

T1KetoconazoleIL-laI1

0.1Fig. 10. Survival of RIF-1 clonogenic tumor cells in primary expiant cultures

exposed to IL-la alone (1000 units/ml), ketoconazole alone (20 ng/ml). and IL-la + ketoconazole for 24 h. Each column is the mean Â±SE (bars) for 3 cultures.

4714




terone levels, treatment with 20 mg/kg ketoconazole producedsignificantly increased plasma corticosterone levels. The reasonfor this increase is unclear; however, it may be due to handlingstress or the stress imposed by the administration of ketoconazole by i.p. injection. The increases in plasma corticosteronelevels after 20 mg/kg ketoconazole were similar to those seenafter the ketamine-acepromazine anesthesia and may providean explanation for only additive cell kill with the ketoconazole(20 mg/kg)-IL-la combination. At higher ketoconazole doses(40 and 60 mg/kg) the increases in plasma corticosterone levelsattendant with the handling stress and IL-la administrationwere ablated.

These results are consistent with our finding that potentiationof IL-1 Â«mediated clonogenic tumor cell killing by ketoconazoleoccurred at 40, 60, and 100 mg/kg but not at the 20-mg/kgdose level. Although time course studies were not conducted,the short plasma half-life (~35 min) for ketoconazole in rodents(34), the lack of severe hemodynamic toxicity seen with thecombination, and the reversibility of ketoconazole inhibition ofadrenal function (10, 32, 33) would suggest that adrenalsuppression was temporary and that adrenal responses to thecombination treatment were delayed rather than abolished.Although additional studies will be necessary to confirm thevalidity of this hypothesis, our findings that potentiation of IL-la mediated cell killing by 40 mg/kg ketoconazole was achievedwith a 30- or 60-min, but not a 120-min, sequence interval areconsistent with this mechanism of action.

Ketoconazole has been reported to enhance the activity ofmacrophages against fungi in vitro (35) and to be cytotoxic tocell lines continuously exposed to the agent in vitro (14). Exposure of RIF-1 tumor primary expiant cultures to ketoconazole for 24 h resulted in only a modest reduction in clonogen-icity and cell kill after in vitro exposure of explant cultures toIL-la and ketoconazole suggested only additive cytotoxicity.These findings strengthen the hypothesis that ketoconazolepotentiation of IL-la mediated tumor cell kill in vivo is not dueto a direct interaction between the 2 agents but is mediatedindirectly through the inhibition of adrenal hormone negativefeedback pathways.

The marked increase in hemorrhagic necrosis in tumorstreated with ketoconazole-IL-la was reflected in treatmentmediated changes in tumor perfusion and tumor high energyphosphate metabolite ratios. Although the magnitude of thedecreases in tumor perfusion seen after treatment with ketocon-azoIe-IL-la was similar to those seen after IL-la alone, theduration of restricted tumor perfusion after the combinationwas longer than that seen after IL-la alone. While the combination produced marked effects on tumor perfusion, little orno effect on 86Rb* uptake in skin and muscle was observed.

86Rb*uptake reflects the distribution of the cardiac output to

the tissue and is thus sensitive to treatment mediated changesin cardiac output. Although cardiac output could not be measured in the mice from these studies, changes in this parameterwould likely be transient and reflected by the changes in 86Rb+

uptake in muscle and skin. For example, cardiodepressive effects of Nembutal are reflected by increases in 86Rb+ uptake in

tumor, decreased uptake in muscle, and no effect in skin (36).In the present studies a transient decrease in muscle 86Rb+uptake was seen, but only at 2 h after IL-la. No significantchange was seen at any of the other study intervals. In contrast,restricted uptake of 86Rb+was seen in the tumor for at least 24

h after treatment.Perfusion is thought to be a major factor contributing to high

energy phosphate metabolite reserves in RIF-1 tumors (37, 38).

In vivo "P NMR studies of tumors treated with ketoconazole-

IL-la indicated a profound, treatment mediated deteriorationin the high energy phosphate stores in the tumor. Although themagnitude of the response was similar to that seen after IL-laalone (3), high PÂ¡/Â«-NTPand low PCr/P, ratios persisted forup to 24 h after treatment. In tumors treated with IL-la alone,control spectral characteristics were seen by 12 h after treatment. The time course of the changes in RIF-1 tumor highenergy phosphate content was consistent with the time coursefor the restriction and subsequent recovery of tumor perfusionafter ketoconazole-IL-la.

Poor signal/noise ratios for the PCr and aNTP resonances,during most of the posttreatment study interval, precludedreliable estimation of the NMR pH. However, by 48 h the 31P

NMR spectra indicated reenergization of at least part of theresidual tumor mass. At this time a significant alkaline shift inthe NMR pH was observed. Alkaline shifts in the NMR pHhave been observed in tumors after chemotherapy cytoreduction(25, 39, 40) where increases in perfusion may accompany intervals of increased cell proliferation. In previous studies using IL-la as a single agent, reenergization of the tumor was temporallycoincident with increases in perfusion and increases in theproliferation of surviving tumor cells (3). Alternatively, thealkaline shift might reflect an alteration in the Na+/H+ ex

change system (41), the generation of large necrotic areas (42,43), or the presence of as yet unidentified alkaline catabolicproducts.

In contrast to the effect of ketoconazole on plasma corticosterone levels and on IL-la antitumor responses, pretreatmentof the tumor bearing mice with ketamine-acepromazine anesthesia abrogated the effects of IL-la on tumor perfusion andprevented the deterioration of high energy phosphate metabolite reserves in the tumor. Since exogenous corticosteroids givenprior to IL-la can inhibit IL-1 mediated antitumor responses(7), the prompt increases in plasma corticosterone induced bythe combination of anesthesia and handling may have inhibitedthe response to the subsequent IL-la treatment. Althoughneither tumor hemorrhage nor clonogenic tumor cell kill wasquantitated in these studies, hemorrhagic necrosis was notapparent in tumors from similarly treated mice at 24 h afterthe IL-la treatment. These results might suggest that plasmacorticosterone levels at the time of IL-la treatment couldprofoundly affect IL-la responses and suggest that systemicstress, nutritional status, and perhaps diurnal rhythms shouldbe carefully controlled in cytokine based experiments in rodents. Such effects could become extremely important in thepreclinical evaluation of IL-la based combination therapeuticstrategies where manipulations (e.g., anesthesia, restraint, drugtreatments, surgery) applied prior to IL-la might activate systemic negative feedback pathways that could inhibit IL-laactivities.

Although the mechanisms by which IL-la mediated adrenalhormone responses protect the vasculature from IL-la inducedinjury are yet to be clearly defined, structural differences between tumor and normal tissue vasculature and/or differencesin the density of receptors for effectors or inhibitors of IL-laaction could provide a basis for differential injuries in thesetissues. The mechanism by which greater than additive cell killwas obtained in tumors treated with ketoconazole-IL-la likelyinvolves the prolonged restriction of tumor perfusion and theextended period of tumor high energy phosphate depletion.

The acute hemorrhagic necrosis and pathophysiological effects of IL-la in solid tumors are quite similar to the injuriesinduced by TNF (44, 45). TNF has been shown to exhibit dose

4715




dependent inhibition of tumor perfusion (46) and to profoundlydeplete tumor high energy phosphate reserves (47, 48). Thesimilarities between the histolÃ³gica! and metabolic responsesto IL-la and TNF in solid tumors might indicate that eitherboth cytokines are involved in the genesis of the acute hemor-rhagic response seen after injection of one or the other cytokineor the underlying mechanism of action for both cytokines issimilar. In this regard, IL-la and TNF have been shown tohave several overlapping and synergistic activities (49, 50), andeach has been shown to stimulate the production of the other(51).

Since other cytokines such as interferons may also inducemicrovascular injuries (52) and adrenal hormone responses(53), ketoconazole or other similarly acting drugs may offer anovel approach for enhancing the efficiency of cytokine basedtherapeutic strategies. Our studies would also indicate thatpathophysiological responses to IL-la and perhaps other inflammatory cytokines could significantly affect the response oftumors to other more conventional therapies. For example,intervals of reduced tumor blood flow after IL-la might reducethe intratumor distribution and effectiveness of systemicchemotherapy. On the other hand, conditions of reduced tumorblood flow and reduced high energy phosphates after IL-latreatment might substantially increase the sensitivity of tumorsto bioreductive alkylating agents, hypoxic cell toxins, and hy-perthermia. In this regard we have reported preliminary evidence which indicates that IL-la can increase both thermalsensitivity and mitomycin C cytotoxicity in RIF-1 solid tumors(54,55).

ACKNOWLEDGMENTS

The authors acknowledge the technical assistance of Virginia Ord,Joeth Derby, Penny Jenkins, and Gary Cromwell.

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1990;50:4709-4717. Cancer Res Paul G. Braunschweiger, Nirmal Kumar, Ioannis Constantinidis, et al. Ketoconazole

Mediated Antitumor Effects byαPotentiation of Interleukin 1

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potentiation of interleukin lor mediated antitumor effects by ketoconazole1

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