Comparison of Corticotropin-Releasing Factor, Dexamethasone, … · grade gliomas, both on MRI and...

Cancer Therapy: Preclinical

Comparison of Corticotropin-Releasing Factor, Dexamethasone,and Temozolomide: Treatment Efficacy and Toxicity in U87 andC6 Intracranial Gliomas

Maxim A. Moroz1, Ruimin Huang1, Tatiana Kochetkov1, Weiji Shi4, Howard Thaler4,Elisa de Stanchina3, Idoia Gamez5, Robert P. Ryan5, and Ronald G. Blasberg1–3

AbstractPurpose/Experimental Design: Treatment of cerebral tumors and peritumoral brain edema remains a

clinical challenge and is associated with highmorbidity andmortality. Dexamethasone is an effective drug for

treating brain edema, but it is associated with well-documented side effects. Corticorelin acetate (Xerecept)

or human corticotrophin-releasing factor (hCRF) is a comparatively new drug and has been evaluated in two

orthotopic glioma models (U87 and C6), by a direct comparison with dexamethasone and temozolomide.

Results: In vitro combination therapy and monotherapy showed a variable response in 6 different

glioma cell lines. In vivo studies showed a dose-dependent effect of hCRF (0.03 and 0.1 mg/kg q12h)

on survival of U87 intracranial xenograft–bearing animals [median survival: control – 41 days (95%

CI 25–61); "low-hCRF" 74.5 days (95% CI 41–88); "high-hCRF" >130 days (95% CI not reached)].Dexamethasone treatment had no effect on survival, but significant toxicity was observed. A survival

benefit was observed with temozolomide and temozolomide þ hCRF-treated animals but with significanttemozolomide toxicity. C6-bearing animals showed no survival benefit, but there were similar treatment

toxicities. The difference in hCRF treatment response between U87 and C6 intracranial gliomas can be

explained by a difference in receptor expression. RT-PCR identified CRF2r mRNA in U87 xenografts; no

CRF receptors were identified in C6 xenografts.

Conclusions: hCRF was more effective than either dexamethasone or temozolomide in the treatment

of U87 xenografts, and results included improved prognosis with long-term survivors and only mild

toxicity. The therapeutic efficacy of hCRF seems to be dependent on tumor hCRF receptor (CRFr)

expression. These results support further clinical assessment of the therapeutic efficacy of hCRF and levels

of CRFr expression in different human gliomas. Clin Cancer Res; 17(10); 3282–92. �2011 AACR.

Introduction

Peritumoral brain edema is a significant cause of mor-bidity and mortality, and there have been relatively fewadvances in brain edema treatment since the introductionof dexamethasone in the 1970s. Relatively new treatmentsthat have shown a significant impact on tumor-associatedbrain edema include human corticotropin-releasing fac-tor (hCRF; ref. 1–4) and antiangiogenic therapy (5, 6),although the latter (bevacizumab) has recently been

associated with potentiation of tumor-cell invasion andrapid progression following the cessation of therapy (7–10). This study focuses on hCRF monotherapy in 2xenograft mouse models and provides a direct compar-ison between hCRF, dexamethasone, and temozolomidemonotherapy.

hCRF is a 41-residue neuropeptide, which was initiallyisolated from sheep hypothalamic extracts in 1981 by Valeand colleagues (11). It is produced in the hypothalamusand is an important component in regulating the hypotha-lamic–pituitary–adrenal (HPA) axis. CRF is the predomi-nant regulator of adrenocorticotropic hormone (ACTH)formation and release by the pituitary (12). In addition toits primary location in the hypothalamic paraventricularnucleus, it has been identified in cerebral cortical inter-neurons, the limbic system, brainstem, and spinalcord (13).

Furthermore, it has been shown that hCRF possessesantiedema properties. As an antiedematous agent, hCRFprevents vascular leakage induced by inflammatory med-iators that selectively act on postcapillary venules andveins in the skin (14), from alveolar capillaries (15–18),

Authors' Affiliations: Departments of 1Neurology and 2Radiology, 3SloanKettering Institute Molecular Pharmacology and Chemistry Program;4Department of Epidemiology and Biostatistics, Memorial Sloan-KetteringCancer Center; and 5Celtic Pharmaceutical Development Services Amer-ica, Inc., New York

Corresponding Author: Ronald G. Blasberg, Departments of Neurologyand Radiology, MH (Box 52) Molecular Pharmacology & Chemistry Pro-gram, SKI Memorial Sloan-Kettering Cancer Center, Zuckerman ResearchCenter, Z-2060 1275 York Avenue, New York, NY 10065. Phone: 646-888-2211; Fax: 646-422-0408. E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-10-3203

�2011 American Association for Cancer Research.

ClinicalCancer

Research

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and from muscle capillaries (19). The antiedematouseffects of hCRF on systemic vessels seem to be mediated,in part, through activation of CRF2 receptors, whereasin the brain and in brain tumors, this effect is mediatedthrough both CRF1 and CRF2 receptors (20). Theseobservations suggest that hCRF acts throughout themicrocirculation to preserve endothelial cell integrity.Several possible mechanisms of hCRF action have beenreviewed in the literature (21), but the exact mechanismsare not fully established as yet.hCRF has been proposed as a new treatment option for

peritumoral brain edema. Strong preclinical data sup-ported hCRF as an effective anti-edematous agent forthe brain that has substantially less toxicity than dexa-methasone (22). The first study of exogenous CRF admin-istration and toxicity assessment in patients was reportedby Chrousos and colleagues (23). More recently, Xerecept(corticorelin acetate or hCRF) has been shown to be awell-tolerated drug, based on data from ongoing clinical

trials involving nearly 200 patients. The side effects ofclinical treatment with hCRF in i.v. single doses (1–5 mg/kg) and continuous i.v. infusions of up to 2,000 mg/24 hper patient were not associated with any significant sideeffects. Only at much higher i.v. single doses (up to 30 mg/kg) did significant symptoms, including hypoten-sion, tachycardia, arrhythmias, and mental "absences,"develop.

Results from ongoing clinical trials involving nearly 200patients who have received hCRF indicate that s.c. admin-istration of the drug, often for extended periods, is welltolerated (24–26). The evolving clinical efficacy and safetydata support the use of hCRF as a dexamethasone-sparingtreatment (if not an alternative to dexamethasone) for themanagement of symptomatic peritumoral brain edema (4).In contrast to the well-known systemic side effects asso-ciated with chronic Dexamethasone administration, whichcan be more debilitating than the primary disease process,hCRF has shown much less toxicity (27, 28), with theprimary effect being transient local reactions at or nearthe injection site.

The evolving clinical efficacy and safety data indicatethat Xerecept might provide a dexamethasone-sparingtreatment (if not an alternative to dexamethasone) forthe management of symptomatic peritumoral brainedema (24, 25). In addition, our previous preclinicalstudies indicated that hCRF may have a direct antitumoreffect (1, 29) and a well-documented antiedematouseffect (1–4). In the present study, we assessed the efficacyand toxicity of hCRF (Xerecept; Celtic Pharma; ref. 30)treatment, both in vitro and in 2 orthotopic glioma animalmodels. First, we assessed hCRF and dexamethasone foradditive effects in cell-culture viability assays involving1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and temo-zolomide treatment of 6 different glioma cell lines. Then,we focused on a comparison of hCRF and dexamethasonemonotherapy and a comparison between temozolomidemonotherapy and combination therapy with temozolo-mide þ hCRF.

Materials and Methods

Cell lines, transduction, and fluorescence-activatedcell sorting

RG2 and C6 rat glioma and U87 and LN229 humanglioma cells were obtained from the American TypeCulture Collection. The cell lines were maintained in75-cm2 flasks with minimum essential medium (MEM;RG2 and U87) or Dulbecco’s modified Eagle’s medium(DMEM; C6 and LN229) containing FBS (10%), penicil-lin (100 U/mL), and streptomycin (100 mg/mL). All cellswere maintained in a humidified atmosphere (5% CO2/95% air) at 37�C.

To monitor intracranial glioma growth by biolumines-cence imaging (BLI), we transduced U87 and C6 cells witha retroviral reporter vector containing a firefly luciferase–IRES–green fluorescent protein (GFP) cassette previouslydeveloped in our laboratory (31). Both the C6 and U87 cell

Translational Relevance

Tumor-associated edema is a major negative fac-tor in patients with brain tumors and is responsiblefor high morbidity and mortality in this patientgroup. Dexamethasone has been the most effectivedrug, which is commonly used for treating brainedema for decades, but it is associated with well-known side effects. Recently, bevacizumab has shownremarkable transient responses in patients with high-grade gliomas, both on MRI and in clinical per-formance that is at least partially related to its anti-edematous effect. In this study, we evaluated theefficacy and toxicity of human corticotropin-releasingfactor (hCRF) in the treatment of 2 orthotopic intra-cranial brain tumors (C6, rat; and U87, human) in anude mouse model. In addition, comparisons weremade with dexamethasone and temozolomide treat-ment regimens.Three important observations were made: (i) hCRF

was more effective than either dexamethasone ortemozolomide in the treatment of U87 intracranialxenografts; (ii) hCRF treatment was associated withsignificantly less toxicity than that associated witheither dexamethasone or temozolomide treatment;(iii) hCRF efficacy was dependent on hCRF receptorexpression in the tumor (e.g., C6 gliomas showed noresponse to hCRF and had nomeasurable levels of CRFmRNA on RT-PCR). These results support the devel-opment of hCRF-releasing formulations to optimizethe therapeutic antitumoral effect in human subjectswith brain tumors because several clinical trials haveshown that hCRF is safe and enables reductions ofsteroid dosing.

Comparison of hCRF and Dexamethasone Efficacy in Glioma Models

www.aacrjournals.org Clin Cancer Res; 17(10) May 15, 2011 3283




lines were stably transduced as previously described (32).After transduction, the cells were expanded in culture forseveral days and prepared for subsequent fluorescence-activated cell-sorting (FACS) analysis and cell sorting aspreviously described (33).

Therapeutic drugshCRF (corticorelin acetate, Xerecept) was generously

provided by Celtic Pharmaceutical Development ServicesAmerica, Inc. Dexamethasone and BCNU were obtainedfrom the Memorial Sloan-Kettering Cancer Center(MSKCC) pharmacy. Temozolomide was generously pro-vided by the Developmental Therapeutics Program,National Cancer Institute (Rockville, MD).

In vitro cytotoxicity assaysCytotoxicity assays were conducted using wild-type (WT)

and firefly luciferase (FLuc)-transduced cell lines, as pre-viously described (34). Briefly, cells (5,000 per well) wereseeded onto 96-well microplates and incubated for 4, 24,and 72 hours under control (nontreatment) condition andwith different doses of BCNU and temozolomide, bothalone and in combination with low and high doses ofdexamethasone (0.01 and 1 mmol/L) and hCRF (0.01 and1 nmol/L), treated simultaneously. The in vitro exposuretimes for hCRF were selected on the basis of unpublisheddata obtained from Celtic Pharma. The exposure time forBCNU was selected on the basis of previously publishedwork (35, 36). The total number of experiments was at least6 for each cell line. The WST-1 reagent (Roche) was used tomeasure cell viability after drug incubation. Measurementof the absorbance of the samples against a backgroundcontrol used as the blank was done using the Packard ELISAreader (Packard Bioscience Company). EC50 was assessedusing Excel and Sigma Plot programs.

Semiquantitative reverse transcriptase PCRExpression levels of CRF receptors (CRF1 and CRF2)

were determined by semiquantitative reverse transcriptasePCR (RT-PCR). Briefly, total RNAs from cultured cells and

tumor xenografts were isolated using TRIzol reagent (Invi-trogen) and then treated with RNase-free DNase I(AmBion) according to the manufacturer’s instructions.The first strand of cDNA was synthesized by GoScriptReverse Transcriptase (Promega). PCR was done usingthe following primer sets, which are located in the con-served domains of CRF1, CRF2, and b-actin gene from thehuman, mouse, and rat species: CRF1: (F) 50-cctggtggcctttgtcctc-30; (R) 50-tggggccctggtagatgta-30; CRF2:(F) 50-cctactgcaacacgacctt-30; (R) 50-tagcagccttccacaaaca-30;and b-actin: (F) 50-ggctggccgggacctgac-30; (R) 50-tactcctg-cttgctgat-30.

In vivo studiesMale nu/nu mice were obtained from the National

Cancer Institute [(NCI), Bethesda, MD], and all studieswere conducted in accordance with a Memorial Sloan-Kettering Cancer Center Institutional Animal Care andUse Committee–approved protocol. Xenografts were estab-lished in 3-month-old mice by intracranial injection of 5�104 cells of either U87-FLuc or C6-FLuc reporter cells, usinga stereotactic device as previously described (29).

Eight treatment groups of 10 athymic rnu/rnu mice pergroup, each bearing a U87-FLuc or a C6-FLuc xenograft,were developed as outlined in Table 1. Treatment withhCRF, dexamethasone, temozolomide, or a combinationof temozolomide þ hCRF was initiated 3 weeks aftertumor-cell implantation and lasted for 7 weeks.

For animal groups 1 and 2, the stock clinical gradesolution of hCRF (Xerecept; 1 mg/mL) was diluted with0.9% sodium chloride solution to the required "low" and"high" concentrations prior to each injection (Table 1). Foranimal groups 3 and 4, clinical grade i.v.-injectable dex-amethasone (1 mg/mL) was diluted with 0.9% sodiumchloride solution to the required concentrations. Treat-ment with either hCRF or dexamethasone was done bys.c. injections every 12 hours for 7 weeks, starting in thesecond week after implantation. For animal group 5, ani-mals were treated with a single daily dose of temozolo-mide. The temozolomide solution was freshly prepared

Table 1.

Treatment

Groups Dose(mg/kg)

Route Volume (mL) Frequency

hCRF (high dose) 0.1 s.c. 0.05 Twice dailyhCRF (low dose) 0.03 s.c. 0.05 Twice dailyDEX (high dose) 1 s.c. 0.1 Twice dailyDEX (low dose) 0.3 s.c. 0.1 Twice dailyTMZ 40 i.p. 0.2 Daily, 5 d/wkTMZ þ hCRF combination (high dose) 40 þ 0.1 i.p. þ s.c. Daily, 5 d/wk þ twice dailyTMZ þ hCRF combination (low dose) 40 þ 0.03 i.p. þ s.c. Daily, 5 d/wk þ twice dailyControl (vehicle) – – Daily

Abbreviations: DEX, dexamethasone; TMZ, temozolomide.

Moroz et al.

Clin Cancer Res; 17(10) May 15, 2011 Clinical Cancer Research3284




daily and administered by i.p. injection on 5 of 7 days eachweek, for a total of 7 weeks. Temozolomide powder wasdissolved in pure dimethyl sulfoxide (DMSO) and furtherdiluted at a ratio of 1:5 with 0.9% sodium chloride solutionto the desired concentration. For animal groups 6 and 7,the effects of combined temozolomide and "low" or "high"hCRF were studied (Table 1). The preparation and admin-istration of the drugs were the same as described above,respectively. For animal group 8 (control), animals weretreated with a single daily dose of drug vehicle. All survivinganimals were euthanized on day 150.

In vivo bioluminescence imagingIn vivo BLI was done 7 days after implantation of the

xenografts and then repeated weekly for the duration of theexperiment, using an IVIS-200 Imaging System. Imagingwas carried out 10 minutes after i.p. injection of D-luciferin(2 mg per animal; Xenogen), with mice lying in the proneposition. Five mice were imaged at the same time with afield of view of 25 cm. An imaging time of 3 minutes withmedium binning and an f-stop of 1 were used initially; thiswas sequentially reduced as the xenografts grew and satura-tion levels of BLI signal intensity were approached. Mea-surements of signal intensity were obtained from region-of-interest analysis using Living Image software (Xenogen).The images displayed in each data set were normalized tothe appropriate color intensity scale. BLI intensity wasexpressed as total photons per region of interest per secondof imaging.

Statistical analysesResults are reported as mean � SD. We applied ANOVA

to the EC50 results for each drug (BCNU and temozolo-mide) and each cell line (C6, LN229, RG2, and U87)separately and included the date of experiment as an effectin the model for quality control. We compared each of 4treatments—low- and high-dose dexamethasone and low-and high-dose hCRF against BCNU or temozolomidealone—and adjusted for multiple comparisons within eachexperiment (combination of drug and cell line) usingDunnett’s method. We considered P < 0.05, after adjust-ment for multiple comparisons, to be statistically signifi-cant.To account for possible differences in global levels or

calibration of BLI measurement between animals, we usedthe change from the average of week 1 and week 2 to theaverage of week 5 and week 6 as a summary value. Survivalwas measured from time of administration until death,sacrifice, or end of study. Animals that were moribund atthe time of sacrifice were considered equivalent as animalsthat died, and other animals were considered as censoredobservations in survival analysis. Using parallel factorialdesigns, we applied ANOVA to change in BLI measure-ments and the Cox proportional hazard model to survivaldata. Furthermore, we included each animal’s change inBLI as a potential predictor of survival in the Cox model.Analysis showed that there was no significantly differenteffect on survival between high versus low doses of hCRF,

dexamethasone, or temozolomide þ hCRF. We combinedhigh and low doses of each treatment in further analyses.

Results

Effects of hCRF and dexamethasone on BCNU andtemozolomide cytotoxicity in cell culture

Cytotoxicity studies were conducted in 6 glioma cell lines(U87, U87-FLuc, C6, C6-FLuc, Ln229, and RG2) withincreasing doses of hCRF (0.0001–1,000 nmol/L) anddexamethasone (0.001–10 mmol/L). Minimal loss of cellviability (

hCRF treatment of intracranial gliomasA dose-dependent effect of hCRF on the survival of

animals bearing U87-FLuc intracranial xenografts wasobserved (Fig. 1A). Both "low" (0.03 mg/kg q12h) and"high" doses (0.1 mg/kg q12h) of hCRF increased survivalcompared with controls, with median survival rangingfrom 41 days (95% CI: 25–61) to 74.5 days (95% CI:41–88; P < 0.05) as well as to more than 130 days (95% CInot reached; P < 0.05), respectively. In contrast, hCRF hadno effect on the survival of animals bearing intracranial C6-FLuc xenografts (Fig. 2A).

U87-FLuc intracranial xenografts treated with hCRFusually showed a BLI signal intensity pattern that paralleledsurvival (Fig. 3). For example, a rapid decrease in BLI signalintensity that remained low for the duration of the experi-ment was associated with a long survival, whereas, anincreasing signal was usually associated with a shortersurvival (Fig. 3B). hCRF treatment had no effect on thepattern of increasing BLI signal intensity of C6-FLuc glio-mas, consistent with the absence of any survival benefit inthese animals (Fig. 2). Further analysis of treatment effectson overall survival, adjusting for experiment and change inBLI effects, showed that hCRF (low or high doses) signifi-cantly prolonged overall survival (P < 0.0001).

Very little or no toxicity was observed in animals treatedwith hCRF alone. The only minor complication was a ran-dom nonspecific skin infection that probably reflects thesystemic effects of corticosteroids on the immune system. Inaddition, hCRF-treated animals bearing U87-FLuc gliomascontinued to gain weight, similar to control animals(Fig. 1B). No difference in animal weight was noted betweencontrol and "low-" or "high"-hCRF-treated animals.

Dexamethasone treatment of intracranial gliomasDexamethasone at both low (0.3 mg/kg) and high doses

(1 mg/kg) was not effective in prolonging animal survivalcompared with control nontreated animals bearing eitherU87-FLuc (Fig. 1A) or C6-FLuc (Fig. 2A) gliomas. Similarly,the patterns of increasing bioluminescence intensity wereindistinguishable from that of nontreated control animals(Fig. 3C). Further statistical analysis showed that animalstreated with hCRF had significantly longer survival thandexamethasone-treated animals (P ¼ 0.02), whereas nodifferences between "low" and "high" doses of dexametha-sone were noted. Treatment-related toxicity was greater inthe dexamethasone-treated animals and included signifi-cant weight loss (Figs. 1B and Fig. 2B), more sever skininfections, and some necrosis at the injection sites.

Temozolomide treatment of intracranial gliomasA significant effect of temozolomide alone (P ¼ 0.008)

or in combination with different doses of hCRF (P¼ 0.003)on the survival of animals bearing U87-FLuc xenografts wasobserved (Fig. 1C), but there was no effect observed on thesurvival of animals bearing C6-FLuc xenografts (Fig. 2C).The temporal profiles of the bioluminescence images weresimilar to those of the survival data in both U87-FLuc (Figs.1B and 4B, respectively) and C6-FLuc (data not shown)xenograft-bearing animals. Many of the animals treatedwith temozolomide showed both reduction and stabiliza-tion of the BLI signal at a low or moderate level for most ofthe treatment and posttreatment period, followed by arapid increase in the last 2 weeks of the animal’s life.However, temozolomide monotherapy and temozolomidecombined with hCRF was accompanied with high toxicity.

Table 2. In vitro cell viability studies

Cell line BCNU aloneBCNU þlow hCRF(0.01 nmol/L)

BCNU þhigh hCRF(1.0 nmol/L)

BCNU þlow DEX(0.01 mmol/L)

BCNU þhigh DEX(1.0 mmol/L)

A. hCRF and DEX effects on BCNU EC50 (mmol/L)U87 226 � 56 (9) 264 � 91 (9) 193 � 54 (9) 311 � 65 (9)a 260 � 59 (9)C6 414 � 43 (8) 419 � 33 (8) 388 � 59 (8) 346 � 94 (8) 304 � 102 (9)aLn229 234 � 43 (7) 261 � 58 (7) 219 � 36 (7) 239 � 47 (7) 201 � 91 (7)RG2 140 � 22 (6) 259 � 96 (6) 173 � 60 (6) 329 � 165 (6)a 147 � 72 (6)

Cell line TMZ aloneTMZ þlow hCRF(0.01 nmol/L)

TMZ þhigh hCRF(1.0 nmol/L)

TMZ þlow DEX(0.01 mmol/L)

TMZ þhigh DEX(1.0 mmol/L)

B. hCRF and DEX effects on TMZ EC50 (mmol/L)U87 4.8 � 3.5 (5) 3.1 � 1.8 (5) 3.8 � 2.8 (5) 4.5 � 3.3 (5) 3.9 � 3.2 (5)C6 2.9 � 1.1 (5) 3.6 � 1.7 (5) 2.9 � 1.0 (5) 4.5 � 3.0 (5) 3.8 � 3.5 (5)Ln229 3.1 � 0.6 (6) 3.2 � 0.5 (6) 3.3 � 0.6 (6) 3.4 � 1.0 (6) 3.5 � 1.0 (6)RG2 9.8 � 2.8 (5) 6.9 � 1.4 (5)* 6.2 � 0.3 (5)a 7.8 � 2.1 (5) 7.9 � 1.6 (5)

Abbreviations: DEX, dexamethasone; TMZ, temozolomide.aAssayswere conducted after 24-hour exposure to the drugs. Significant difference comparedwith BCNUor temozolomide alone;P <0.05 after adjustment for multiple comparisons using Dunnett's method.

Moroz et al.





There was a substantial loss of weight (up to 40%), whichtended to recover during the posttreatment period if theanimal survived. This weight loss is reflected in the photo-graphic images of the animals at week 9, just after com-pleting the course of temozolomide therapy (Fig. 4B andC). A very high incidence (27 of 30) of skin neoplasms wasobserved in animals treated with temozolomide and therewere rare occurrences (2 of 30) of internal abdominaltumors. Spontaneous tumorigenesis was not observed inany of the other treatment groups.

CRF1 and CRF2 receptor expressionU87-FLuc and C6-FLuc cell lines and xenografts were

examined for CRF1 and CRF2 mRNA levels by RT-PCR(Fig. 5). U87 cultured cells and xenografts express measur-able levels of CRF2 but not CRF1 mRNA transcript. Incontrast, neither CRF1 nor CRF2 transcripts could bedetected in the C6 cultured cells and xenografts.

Discussion

We previously reported a dose-dependent decrease invasogenic peritumoral brain edema following treatment ofimmunocompetent Fischer 344 rats bearing RG2 intracra-nial gliomas, with hCRF (Xerecept; 29). Furthermore, wereported a similar result in a single-dose hCRF studyinvolving Sprague Dawley rats bearing W256 intracranialgliomas (1, 17). Both studies showed a significant anti-edematous effect in tumor and peritumoral brain tissue byproton density-weighted and T1-weighted contrast-enhanced MRI and by ex vivo measures of tissue watercontent. In addition, the initial study reported a significantsurvival advantage for hCRF-treated animals comparedwith dexamethasone-treated and control animals (29).

In this study, an immunocompromised animal modelharboring either an intracranial human (U87) or a rat (C6)glioma xenograft was studied and therapeutic efficacy was

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Figure 1. Time profiles of survival (A and C) and body weight (B and D) of animals bearing orthotopic U87-FLuc intracranial xenografts. The treatment periodand end of the experiment (euthanasia of all surviving animals) is indicated by the bracket and arrow, respectively. Control animals received vehicleinjections (black dashed line in A and C). Treated animals in A received either "low dose" hCRF (0.03 mg/kg twice daily; red dotted line), "high dose" hCRF(0.1 mg/kg twice daily; red solid line), "low dose" dexamethasone (0.3 mg/kg twice daily; blue dotted line), or "high dose" dexamethasone (1 mg/kg twicedaily; blue solid line). The corresponding mean weight profiles of surviving animals in B utilize a similar color code where solid symbols refer to "highdose" and "open symbols refer to "low dose." Temozolomide-treated animals are shown in C. Animals received monotherapy (40 mg/kg daily, 5 of7 days; green solid line), or combination therapy with "low" and "high" doses of hCRF (40 mg/kg of temozolomide þ 0.03 mg/kg of hCRF, orangedashed line; or 40 mg/kg of temozolomide þ 0.1 mg/kg of hCRF, orange solid line, respectively). The corresponding mean weight profiles of survivinganimals in B utilize a similar color code where solid symbols refer to "high dose" and "open symbols refer to "low dose."






evaluated in terms of animal survival. We focused on acomparison of hCRF and dexamethasone monotherapy,although a comparison between temozolomide monother-apy and temozolomide þ hCRF combination therapy wasalso done. Interestingly, a highly significant, dose-depen-dent effect of hCRF monotherapy on survival was observedin animals bearing U87 gliomas but not C6 gliomas. Theseresults were highly consistent with tumor growth, regres-sion, and regrowth patterns visualized by sequential weeklybioluminescence reporter gene imaging of the xenograftsbefore, during, and following hCRF treatment. Interest-ingly, dexamethasone treatment had little or no effect oneither the survival or BLI profiles of animals bearing eitherU87 or C6 gliomas, and this may reflect, in part, the toxicside effects of twice-daily s.c. injections of dexamethasone.Temozolomide monotherapy and temozolomide þ hCRFcombination therapy for U87 gliomas yielded similar sur-vival profiles that were significantly longer than those forcontrol animals. Again, no survival advantage was observedin C6 gliomas.

To further explore the dichotomy in hCRF treatmentresponse between U87 and C6 gliomas, we evaluated thelevels of CRF1 and CRF2 receptor mRNAs in the 2 cell lines

and xenografts. This investigation was based on our pre-vious study, which showed that significant levels of CRF1mRNA (0.25 � 0.01 pg/mg total RNA) were detectable inW256 cells and presumably reflects CRF1-receptor expres-sion on tumor and/or endothelial cell membranes (22).Blockade of CRF receptors with alpha-helical CRF (9–41)analogue abolished the growth inhibitory and differentia-tion-inducing effects of hCRF. Together, these findingssuggested that W256 cells express functional rat CRF recep-tors in vitro and that these receptors are likely tomediate theeffects observed following exposure to hCRF. Our currentresults suggest that C6 cells and xenografts express neitherCRF1 nor CRF2 receptors whereas U87 cells and xeno-grafts express CRF2 but not CRF1 receptors. This differencebetween the 2 cell lines and xenografts could contributeto the difference in hCRF treatment response that wasobserved in this study. In addition, these results suggestthat CRF1 and/or CRF2 receptor expression is required forthe antitumor and antiedematous effects of hCRF.

It was previously shown that CRFR1 is expressed intumor cells and that urocortin (UCN) and CRF, bothmembers of the CRF family, reduce tumor-cell growthvia CRF1 receptor (37, 38). Multiple malignancies have

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Figure 2. Time profiles of survival(A and C) and body weight (B andD) of animals bearing orthotopicC6-FLuc intracranial xenografts.Control animals received vehicleinjections (black dashed line in Aand C); treated animals in Areceived either "low dose" hCRF(0.03mg/kg twice daily; red dottedline), "high dose" hCRF (0.1 mg/kgtwice daily; red solid line), "lowdose" dexamethasone (0.3 mg/kgtwice daily; blue dotted line), or"high dose" dexamethasone (1mg/kg twice daily; blue solid line).The corresponding mean weightprofiles of surviving animals in Butilize a similar color code wheresolid symbols refer to "high dose"and "open symbols refer to "lowdose." Temozolomide-treatedanimals are shown in C. Animalsreceived monotherapy (40 mg/kgdaily, 5 of 7 days; green solid line),or combination therapy with "low"and "high" doses of hCRF (40 mg/kg of temozolomide þ 0.03 mg/kgof hCRF, orange dashed line; or 40mg/kg of temozolomide þ 0.1 mg/kg of hCRF, orange solid line,respectively). The correspondingmean weight profiles of survivinganimals in B utilize a similar colorcode where solid symbols refer to"high dose" and "open symbolsrefer to "low dose."

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been reported to have high levels of CRFR1 and CRFR2expression and to be sensitive to the suppressive effects ofCRF and its agonists. Graziani and colleagues reported thatUCN/CRF inhibited the growth of adenocarcinoma Ishi-kawa cells in a concentration-dependent manner and thatthis effect was mediated by CRF1 receptors (38). It wasreported that UCN inhibited the proliferation of mela-noma cells in vitro and in vivo, also through CRF1 receptor(39) and, in human mammary cancer cells, CRF acted onCRF1 receptor to inhibit the proliferative effects of estro-gens on MCF-7 cells in both paracrine and autocrinemanners (40). Activation of CRF2 receptor was observedto suppress angiogenesis and rearrange the vasculature.

Furthermore, it was reported that CRF2 receptor agonistsinhibited hepatocellular carcinoma tumor angiogenesisin vitro and reduced tumor microvessel density in vivo.Our results are consistent with these observations.

Reports from other animal and human studies confirmthat the antiedematous effects of hCRF- and dexametha-sone-treated animals are similar. Patients treated withhCRF and decreasing doses of dexamethasone show clin-ical improvement in neurologic symptoms as well as haveless steroid-associated myopathy and insulin dependence(41). The substitution of a less toxic, but equally effective,drug for dexamethasone to treat tumor-associated cerebraledema has been a long-explored challenge for clinicians.

Figure 3. BLI time course ofrepresentative control and treatedanimals bearing U87-FLucorthotopic gliomas. The profilesof: control (vehicle-treated) (A),hCRF-treated (B), anddexamethasone (DEX)-treated (C)animals are shown.

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Our preclinical studies add to the body of data that indi-cates that hCRF has the potential to meet this challenge. Weobserved higher therapeutic efficacy with hCRF than witheither dexamethasone or temozolomide monotherapy inU87 glioma xenografts. Furthermore, no toxic effects orsigns of discomfort were observed inmice that had receivedhCRF treatment in low or high doses for 2 to 6 months. Incomparison, animals that have received dexamethasone ortemozolomide therapy exhibited significant toxicity, man-ifested by a decrease in weight, increased irritability, localskin necrosis, and even spontaneous tumorigenesis.

The current clinical development program for hCRF hasfocused on the treatment of peritumoral brain edema in

patients with metastatic and primary brain tumors requir-ing dexamethasone to control symptoms. Three majorclinical trials (0303, 0501, and 0302) have established 2important points: (i) hCRF is safe in man, and (ii) hCRFenables significant reductions or elimination of steroiddosing in patients with cerebral tumors, with no apparentimpairment of neurocognitive status (26). Blinded, inde-pendent review of MRI scans from patients receiving hCRFshowed that most of these patients experienced prolongedperiods of stable disease and a minority have achieved ameasurable level of tumor regression (24). In a phase1 clinical trial, 10 of the 15 patients who received hCRFhad improvement in neurologic symptoms or physical

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hCRF(0.1 mg/kg)

+TMZ

(40 mg/kg)

hCRF(0.03 mg/kg)

+TMZ

(40 mg/kg)

Figure 4. BLI time course ofrepresentative control andtemozolomide (TMZ)-treatedanimals bearing U87-FLucorthotopic gliomas. The profiles ofcontrol (vehicle-treated) (A),temozolomide monotherapy (B),and temozolomide þ hCRFcombination therapy (C) areshown.

Moroz et al.





findings, with little or no toxicity (4). It is notable that therewas a measurable decrease in steroid-related side effects,especially myopathy and the appearance of Cushingoidfeatures. One patient, who had lymphoma-related pruritusand a long-standing rash resistant to steroids, notedimprovement while receiving hCRF (25). Furthermore,in this preclinical study, we noted differences in toxicityand efficacy of hCRF, dexamethasone, and temozolomidetreatment of the glioma-bearing animals.Overall, we have shown therapeutic efficacy and low

toxicity of hCRF in the treatment of a human-derivedglioma in an orthotopic nude mouse model. Our resultsare consistent with the requirement of CRF receptor

expression in the tumor cells for hCRF therapeutic effi-cacy. Notably, hCRF treatment was less toxic than thatwith dexamethasone or with temozolomide. These resultssupport the development of novel hCRF-releasing for-mulations or platforms for extended constant adminis-tration and studies to optimize the therapeuticantitumoral effect. Further clinical studies will need toevaluate whether long-term hCRF treatment (or the rapidcessation of treatment) is associated with increasedglioma cell invasion and rapid tumor progression, ashas been described for bevacizumab.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Dr. Pat Zanzonico and Ms. Valerie Longo for imaging supportand Dr. Ekaterina Moroz for technical support.

Grant Support

This work was supported by Celtic Pharma award SK#12715 and by NIHgrant P50 CA8643 ICMIC. The MSKCC Small Animal Imaging Core Facilitywas supported by NIH Small-Animal Imaging Research Program grant R24CA83084 and NIH Center grant P30 CA08748.

Celtic Pharmaceutical Development Services America, Inc., New York.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 3, 2010; revised February 17, 2011; acceptedFebruary 24, 2011; published OnlineFirst March 8, 2011.

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1 2 3

U87

CRF1

CRF2

ACTB

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Figure 5. Expression of CRF1 and CRF2 gene transcripts in cultured U87and C6 parental cells (columns 1 and 5) and reporter-transduced cells(columns 2 and 6) and in s.c. xenografts from U87 and C6 parentalcells (columns 3 and 7) and reporter-transduced cells (columns 4 and 8).Normal mouse brain tissue, which expressed both CRF1 and CRF2mRNAs, was used as a positive PCR control. Expression levels of CRF1and CRF2 were normalized by the b-actin gene (ACTB).






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2011;17:3282-3292. Published OnlineFirst March 8, 2011.Clin Cancer Res Maxim A. Moroz, Ruimin Huang, Tatiana Kochetkov, et al. Toxicity in U87 and C6 Intracranial GliomasDexamethasone, and Temozolomide: Treatment Efficacy and Comparison of Corticotropin-Releasing Factor,

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