ANTICANCER RESEARCH 30: 5037-5042 (2010)

Corticorelin Acetate, a Synthetic Corticotropin-releasing Factor with Preclinical Antitumor Activity, alone and with Bevacizumab, against Human Brain Tumor Models

IDOIA GAMEZ1, ROBERT P. RYAN1 and STEPHEN T. KEIR2

1Celtic Pharmaceutical Development Services America, Inc., New York, NY 10022, U.S.A.; 2The Preston Robert Tisch Brain Tumor Center, Duke University, Durham, NC 27710, U.S.A.

Abstract. Background: Corticorelin acetate (CrA) is a brain tumors. The cause of PBE is believed to result in part synthetic form of corticotropin-releasing factor that is from the leakage of edematous brain tumor fluid from currently undergoing clinical trials in the treatment of abnormal tumor vasculature into the surrounding tissue (1, peritumoral brain edema (PBE). This study preclinically 2). The mechanism(s) of action by which CrA exerts its investigated its potential as an antitumor agent against beneficial effect in the therapy of PBE has not been human brain tumor xenografts. Materials and Methods: The elucidated, but two factors appear to be relevant: decreased in vivo efficacy of CrA as a single agent and in combination vascular leakage and preserved integrity of the endothelial with the antiangiogenic agent, bevacizumab, was examined cells, which ultimately helps to maintain the blood-brain in three different patient-derived human brain tumor barrier (3, 4). xenografts implanted orthotopically (intracranially) or CrA appears to mediate its activity through two subtypes subcutaneously in athymic mice. Results: CrA significantly of G-protein coupled receptors: CRF receptor-1 (CRFR1) increased the lifespan of mice implanted orthotopically with and CRF receptor-2 (CRFR2) (5). These CRFRs are widely two different pediatric brain tumor xenograft models. In one distributed in the central nervous system, peripheral tissues, of these tumor models, the combination of CrA with and some types of human tumors (6, 7). It is known that bevacizumab produced a therapeutic outcome superior to CRFR2 plays a critical role in the tonic inhibition of adult that found using either of the two agents alone. Conclusion: neovascularization (8). Moreover, it has been found that The application of CrA for the treatment of PBE likely CRFR2 agonism inhibited the growth of hepatocellular involves its activity as an anti-angiogenic agent, which may carcinoma and reduced tumor microvessel density in nude be one possible mechanism to explain its observed mice (9). Hepatoma cells do not express CRFRs, whereas preclinical antitumor activity against orthotopic human brain associated blood vessels do, mainly CRFR2 (9). tumor models. Additional studies to investigate other Additionally, it was recently described that CrA has activity possible mechanisms of action are underway. as an antiangiogenic in the corneal micropocket assay (15). CrA is a well-tolerated drug based on data from ongoing Corticoreline acetate (Xerecept®, CrA) is a synthetic clinical trials involving nearly 200 patients who have formulation of the endogenous neurohormone, corticotropin- received it subcutaneously (s.c.), often for extended periods releasing factor (CRF). The 41-amino acid sequence of CrA (10-12). The evolving clinical efficacy and safety data is identical to that of the human hormone and it is being indicate that CrA might provide a dexamethasone-sparing developed as an alternative to dexamethasone in the treatment (if not an alternative to dexamethasone) for the treatment of peritumoral brain edema (PBE). Patients are management of symptomatic PBE (13). typically debilitated by the effects of PBE associated with A preclinical study involving the use of CrA in the treatment of mice bearing an orthotopically implanted human brain tumor yielded encouraging positive data (14). Recently, the activity of CrA in the treatment of human solid tumor Correspondence to: Idoia Gamez, Celtic Pharma Development xenografts, particularly when combined with bevacizumab Services, 663 Fifth Ave, 7th Floor, NY, NY 10022, U.S.A. Tel: +1 (Avastin®, BEV; Genentech, South San Francisco, CA, 2126164084, Fax: +1 2126164099, e-mail: idoia.gamez@ dev.celticpharma.com USA), was described (15). The objective of the present study was to investigate further, preclinically, the potential of CrA Key Words: Xerecept®, bevacizumab, Avastin®, corticorelin acetate, as an antitumor agent in the treatment of brain tumors. brain tumor model. Additionally, this study evaluated the ability of CrA to

0250-7005/2010 $2.00+.40 5037 ANTICANCER RESEARCH 30: 5037-5042 (2010) provide an enhanced therapeutic benefit when given with In all experiments, control mice were treated with drug vehicle. BEV and so the tumor models selected were those known to Mice were weighed twice weekly, individually, and drugs were be sensitive to BEV therapy. administered on a mg/kg basis according to the last weight recorded for each mouse. Injection volumes were delivered in 0.01 ml/g of Materials and Methods body weight. Toxicity assessment Animals. Female athymic mice (nu/nu genotype, Balb/c background, Body weight. The average body weight of mice from each six weeks or older) were used for all antitumor studies. Animals treatment group and appropriate relative untreated control set was were maintained in filter top cages in Thoren units. The guidelines determined just prior to treatment initiation. The average body of the Institutional Animal Care and Use Committee of Duke weights of the same sets of mice were determined after (typically University (where all in vivo studies were performed) were utilized one day) completion of treatment. The difference in average body along with the National Institutes of Health guidelines. weights was used to estimate treatment related toxicity. A loss of average body weight greater than 20% was considered excessively Compounds. CrA was supplied in its clinical vials (1.2 mg/vial) by toxic. None of the treatments in any of the experiments was judged Celtic Pharma Development Services America (New York, NY, to have been excessively toxic by this criterion. In addition, no USA). Clinical vials of BEV (25 mg/ml) were purchased treatment-related deaths occurred in any of the experiments commercially (Genentech). performed. Tumor xenografts and implantation. The following patient-derived Efficacy assessment human tumor xenografts maintained by Duke University were used for Survival. Therapeutic results are presented in terms of: (i) increases experimentation: intracranially (i.c.) -implanted D-341 MED pediatric in lifespan reflected by the relative median survival time (MST) of medulloblastoma and D-456 MG pediatric multiforme glioblastoma, treated (T) mice versus control (C) mice (i.e., % T/C); and (ii) long- and s.c.-implanted D-245 MG adult multiforme glioblastoma. term survivors (LTS). A T/C of 125% was used as the threshold of For intracranial tumor transplantation, tumors were excised, activity when accompanied by a statistical test indicating minced, and cells separated with a cytosieve into Zinc Option significance (p<0.05) (17). Mice surviving to the end of an solution (Biowhitter Inc, Walkersville, MD, USA). After experiment are referred to as LTS. Statistical evaluation of survival centrifugation, supernatant was removed, and cells were mixed 1:1 time was performed using a log-rank test (18). with methylcellulose. Total homogenate (10 μl) was then injected Tumor growth delay. Therapeutic results are presented in terms with a Hamilton syringe (Hamilton, Co., Reno, NV, USA) as of: (i) tumor growth delay reflected by the relative median time for previously described (16). Subcutaneous tumor transplantation into T (treated) mice versus C (control) mice to achieve tumor target size the right flank of the animals with an inoculation volume of 50 μl (i.e., T-C in days); or (ii) percent tumor growth inhibition (%TGI). was carried out using a brei prepared from xenografts as previously Estimates of the median time for a group to achieve five times the described (16). original tumor size were calculated using a proprietary and customized Duke SAS statistical software program. The relative Tumor measurements. All tumors were measured twice weekly with times for treated and control mice to achieve tumor target size (five hand-held vernier calipers (Scientific Products, McGraw, IL, USA). times the initial) was subjected to a Wilcoxon statistical test for Tumor volume was calculated in cubic millimeters, according to the significance (19). following formula: V=[(width)2×(length)]/2. Efficacy was expressed in terms of percent tumor growth inhibition (% TGI), calculated using the formula: Antitumor drug therapy. For the i.c. tumor studies, groups of ten mice were randomized three days after i.c. tumor implantation, at which time all treatments were begun. CrA was administered at [1–(Tt/To × Co/Ct)] 0.1 and/or 0.2 mg/kg/injection, s.c., twice daily (bid). BEV was %TGI = ×100 administered at a dose of 1, 2.5 or 5 mg/kg/inj, intraperitoneally 1 – Co/Ct (i.p.), twice weekly, depending upon the experiment. Treatments were continued until a median day of death was reached per where Ct is the median control tumor size at the end of a group. selected observation period of no less a duration than one tumor For the s.c. D-245 MG tumor study, groups of ten mice were volume doubling time (which was 7.9 days in the D-245 MG distributed per control and treatment sets based on tumor experiment), Co is the median control tumor size at the volume, such that each set’s median tumor size was within 10% initiation of the selected observation period, Tt is median tumor of the overall median tumor size of all mice included in the size of a treated group at the end of the selected observation experiment. This distribution occurred when all the tumored mice period and To is median tumor size of a treated group at the had tumors within the range of 62 to 107 mm3 and treatments initiation of the selected observation period. The selected were initiated on that day, which was day ten post-tumor implant. observation period was chosen to optimize the likelihood of Treatment continued until an approximate median was reached detecting a difference between treatment and control groups. for each s.c. tumor-bearing group of mice using an endpoint of Activity was defined as achievement of %TGI in excess of 50% five times the initial tumor size of each mouse in a treatment (20). Statistical significance using a Mann-Whitney non- group. All mice bearing s.c. tumors were observed (and their parametric test (21) had to be achieved for each day of tumor tumors measured) until a tumor size of 1 gram (1,000 mm3) was measurement included in the selected observation period (other attained. than the initial day).

5038 Gamez et al: Antitumor Activity of Corticorelin Acetate Against Human Brain Tumor Models

Results Table I. Human brain tumor xenograft experimental designs.

The designs of the in vivo antitumor studies are presented in Tumor model, Expt. n CrA BEV implant site no. (mg/kg/inj, (mg/kg/inj, i.p., Table I, and the results from these same experiments are s.c., bid to end) 2×/wk to end) summarized in Table II. D-341 MED, i.c.1 10 - - Intracerebrally-implanted tumor models. In the first of two 1 10 0.1 - experiments involving the i.c. D-341 MED pediatric 1 10 0.2 - 110- 5 medulloblastoma, tumor-bearing mice treated with 0.1 mg/kg/inj of CrA had an MST of 39 days compared to the 2 10 - - control group’s MST of 25 days, or a 156% T/C value 2 10 0.2 - (p<0.001). At the higher dose of 0.2 mg/kg/inj of CrA, the 210- 1 MST realized was 30 days, or a 120% T/C value, which was 2 10 - 2.5 210- 5 statistically different from that of the control group (p<0.05). 2 10 0.2 1 There were no cures or LTS in either CrA-treated group. A 2 10 0.2 2.5 calculation was made as a result of combining both CrA D-456 MG, i.c 110- - treatment groups and comparing their combined outcome to 1 10 0.2 - that of the control group. The combined CrA MST was 34.5 110- 5 1 10 0.2 5 days, or a 138% T/C (p<0.001). Figure 1 depicts the combined D-245 MG, i.c 110- - CrA treatment groups and control. 1 10 0.2 - In the second D-341 MED i.c.-implanted tumor experiment, 110- 5 CrA (0.2 mg/kg/inj, bid, s.c.) when used as a single agent did 1 10 0.2 5 not reach the criteria threshold designated as active in this model (T/C of 117%; p<0.001) despite achieving a marked statistical difference from the control. BEV however, was active at all three dose levels evaluated. When administered s.c.) was inactive. In contrast, BEV produced a delay in i.p. twice weekly at 1, 2.5 or 5 mg/kg/inj, BEV produced tumor growth of 24.3 days distinctly different from the significant (p<0.001) increases in survival corresponding to control (p<0.001). BEV was clearly active by %TGI. When T/C values of 162%, 217%, and 219%, respectively. Doubling assessed during the period from 21 days to 35 days post- the dose of BEV from 2.5 to 5 mg/kg/inj produced essentially implant, BEV produced a %TGI of 94% (p<0.001). The no additional therapeutic benefit. combination of these two treatments however, did not result When CrA (0.2 mg/kg/inj, bid) was administered in in any statistically significant improvement compared to the combination with BEV (at 1 and 2.5 mg/kg/inj, twice tumor growth inhibition caused by BEV alone (71% versus weekly) to mice bearing i.c. D-341 tumors, there was no 94% TGI, respectively, under the doses and schedules used statistically significant potentiation of the activity of BEV in this study). under the doses and schedules used in this study. CrA plus 1 mg/kg/inj, twice weekly, of BEV yielded exactly the same Discussion increase in lifespan (T/C of 162%) as that same regimen of BEV alone. Similarly, CrA plus 2.5 mg/kg/inj, twice weekly, CrA was evaluated in three patient-derived human brain of BEV yielded a 207% T/C value, approximating the T/C tumor models selected for their sensitivity to the anti- of 217% associated with the same regimen of BEV when angiogenic drug, BEV. The strong activity of BEV was used alone. observed in each of those models in the experiments In the D-456 MG i.c.-implanted tumor experiment, CrA described in this report, thus validating the basis for the (0.2 mg/kg/inj, bid, s.c.) was active, achieving a T/C of tumor models used to evaluate CrA. 142% (p<0.001). Likewise, BEV (5 mg/kg/inj, twice weekly, CrA showed activity in the first of two i.c. implanted D- i.p.) produced an active result, a T/C of 164% (p<0.001). 341 MED human pediatric medulloblastoma experiments. In When the two drugs on these same regimens were applied in the confirmatory study, CrA produced a similar statistically combination, the resulting active T/C of 192% (p<0.001) significant increase in lifespan compared to the control represented a therapeutic effect that was statistically distinct group. The combination of CrA and BEV was assessed in (p<0.05) from either single-drug treatment (Figure 2). the second D-341 MED experiment, but the combination of CrA with BEV did not result in any statistically significant Subcutaneously implanted tumor model. In the D-245 MG difference in activity compared to BEV alone under the s.c.-implanted tumor experiment, CrA (0.2 mg/kg/inj, bid, doses and schedules used in this study.

5039 ANTICANCER RESEARCH 30: 5037-5042 (2010)

Figure 1. Mice implanted i.c. with D-341 MED medulloblastoma were Figure 2. Effect of CrA and BEV, individually and in combination versus treated with CrA (combined treatment groups receiving 0.1 and 0.2 i.c. -implanted D-456 MG human pediatric glioblastoma. Control; mg/kg/inj, bid, s.c.) I or vehicle . Treatments were continued until a I CrA 0.2 mg/kg/inj, bid, s.c.; L BEV 5 mg/kg/inj, 2×/wk, i.p; M CrA median day of death was reached. 0.2 mg/kg/inj, bid, s.c. and BEV 5 mg/kg/inj, 2×/wk, i.p.

Table II. Effect of CrA and BEV on human brain tumor models.

Tumor, site CrA BEV Median % Median T-C Activityb Associated (Expt. no.) (mg/kg/inj, (mg/kg/inj, survival T/C time to (days) p-value s.c., bid) i.p., 2×/wk) time (days)a target (days)

D-341 MED, - - 25 - - - i.c. (1) 0.1 39 156 - - Active 0.0002 0.2 30 120 - - NAc 0.0106 0.1 & 0.2 - 34.5 138 Active 0.0020 D-341 MED, - - 21.0 - - i.c. (2) 0.2 - 24.5 117 - - NA 0.005 - 1 34 162 - - Active <0.001 - 2.5 45.5 217 - - Active <0.001 - 5 46.0 219 - - Active <0.001 0.2 1 34.0 162 - - Active <0.001 0.2 2.5 43.5 207 - Active <0.001 D-456 MG, i.c. - - 33.0 - - - 0.2 - 47.0 142 - - Active 0.0006 - 5 54.0 164 - - Active <0.001 0.2 5 63.5 192 - - Active <0.001 Combination>either (0.011 vs. CrA; drug alone 0.032 vs. BEV) D-245 MG, s.c. - - - - 25.8 - 0.2 - - - 29.4 3.6 NA >0.05 - 5 - - 50.1 24.3 Active (94% TGI) <0.001

200 5 - - 43.1 17.3 Active (71% TGI) <0.001 aExpressed in days post-tumor implant. bActivity noted versus control group; Other comparisons are explained. NA, Not active; TGI, tumor growth inhibition.

CrA was active as a single agent in the i.c. implanted D- produced an enhanced therapeutic outcome superior to that 456 MG pediatric multiforme glioblastoma. This activity obtained with either agent alone. represents the third human brain tumor model implanted Although non-cytotoxic in vitro (unpublished data), previous orthotopically that has responded to CrA with greater than a activity for CrA has been reported in an orthotopically implanted 25% increase in survival among the tumor-bearing mice. human brain tumor, the U87 glioma (14). CrA has also been When combined with BEV in this tumor model, CrA reported to have activity in the human breast carcinoma model,

5040 Gamez et al: Antitumor Activity of Corticorelin Acetate Against Human Brain Tumor Models

MX-1, and to synergize with BEV both in this model and the 6 Hillhouse EW and Grammatopoulos OK: The molecular Colo-205 human colon carcinoma model (15). mechanisms underlying the regulation of the biological Clinically, CrA is currently being evaluated as a safer activity of corticotropin releasing hormone receptors: implications for physiology and pathophysiology. Endocr Rev alternative to steroid therapy for the prevention and reduction 27: 260-286, 2006. of edema associated with brain tumors and cytoreductive 7 Reubi JC, Waser W, Vale W and Rivier J: Expression of CRF1 treatments (10-12). and CRF2 receptors in human cancers. J Clin Endocrinol Metab The role of CRFR2 in tumor formation and angiogenesis is 88: 3312-3320, 2003. of continued interest. For example, activation of CRFR2 was 8 Bale TL, Giordano FJ and Vale WW: A new role for observed to suppress angiogenesis and rearrange vasculature corticotrophin-releasing factor receptor-2: suppression of (8). Additionally, activation of CRFR2 inhibited the vascularization. Trends Cardiovasc Med 13: 68-71, 2003. proliferation of rat smooth muscle cells in vitro, subsequent to 9 Wang J, Xu Y, Xu Y, Zhu H, Zhang R, Zhang G and Li S: ’s inhibition of tumor growth and angiogenesis in VEGF suppression, and reduced capillary tube formation in hepatocellular carcinoma via corticotrophin-releasing factor gels (22). It was also reported that CRFR2 agonism receptor 2. Cancer Invest 26: 359-368, 2008. inhibited hepatocellular carcinoma tumor angiogenesis in vitro 10 Recht LD, Mechtler L, Phuphanich S, Hormigo A, Hines V, and reduced tumor microvessel density in vivo (9). Conducting Milsted R, O’Connor PC, Ryan RP and Wong ET: A placebo- in vitro studies with human umbilical endothelial cells, controlled study investigating the dexamethasone-sparing effects Tjuvajev et al. (23) demonstrated that VEGF-induced of corticorelin acetate in patients with primary or metastatic proliferation was inhibited with CRF. Thus, the observed brain tumors and peritumoral edema. J Clin Oncol 27: abstr vascular and antiproliferative effects, in both endothelial and 2078, 2009. 11 Mechtler L, Wong ET, Hormigo A, Pannullo S, Hines V, Milsted tumor cells, may explain the antineoplastic properties of CRF R, O’Connor PO, Ryan RP and Recht L: A long-term open-label in vivo. In support of such a hypothesis, CrA was recently extension study examining the steroid-sparing effects of described as producing a substantial (>70%) inhibition of corticorelin acetate in patients with cerebral tumors. J Clin Oncol neovascularization in a microcorneal pocket assay (15). 27: abstr 2079, 2009. In summary, CrA, a synthetic formulation of human CRF, 12 Shapiro WR, Mechtler L, Cher L, Wheeler H, Hines V, has demonstrated its potential as an antitumor agent. It has Milsted R, O’Connor PC, Ryan RP and Recht L: A been shown to significantly increase the lifespan of mice randomized, double-blind study comparing corticorelin acetate with dexamethasone in patients with primary malignant implanted orthotopically with two different pediatric brain glioma who require increased dexamethasone doses to control tumor xenograft models. Furthermore, in one of those tumor symptoms of peritumoral brain edema. J Clin Oncol 27: abstr models, the combination of CrA with the anti-angiogenic drug, 2080, 2009. BEV, produced a therapeutic outcome superior to that found 13 Moliterno JA, Henry E and Pannullo SC: Corticorelin acetate using either of the two agents alone. Additional preclinical injections for the treatment of peritumoral brain edema. Expert antitumor and mechanism of action studies are underway. Opin Investig Drugs 18: 1413-1419, 2009. 14 Ryan R, Moroz M, Berestein T, Evans-Freke S, Gamez I, Hines Acknowledgements V and Blasberg R: Corticorelin acetate exhibits preclinical antitumor activity against the human glioma U87 xenograft. Proc Amer Assoc Canc Res abstr 2326, 2009. The contributions of the research staff from The Preston Robert 15 Gamez I, Ryan RP, Reid LD, Routt SM and Hollister BA: Tisch Brain Tumor Center, Duke University, Durham, NC, USA, are Corticorelin acetate, a synthetic corticotropin-releasing factor duly recognized. with preclinical antitumor activity, alone and with bevacizumab, against human solid tumor models. Cancer Chemother References Pharmacol, Online First™, 2 September 2010. 16 Friedman HS, Colvin OM, Skapek SX, Ludeman SM, Elion GB, 1 Stummer W: Mechanisms of tumor-related brain edema. Schold SC Jr., Jacobsen, PF, Muhlbaier LH and Bigner DD: Neurosurg Focus 22: E8, 2007. 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20 Velaperthi U, Wittman M, Liu P, Carboni JM, Lee FY, Attar R, 22 Hao Z, Huang Y, Cleman J, Jovin IS, Vale WW, Bale TL and Balimane P, Clarke W, Sinz MW, Hulburt W, Patel K, Discenza Giordano FJ: Urocortin 2 inhibits tumor growth via effects on L, Kim S, Gottardis M, Greer A, Li A, Saulnier M, Yang Z, vascularization and cell proliferation. Proc Natl Acad Sci 105: Zimmerman K, Trainor G and Vyas D: Discovery and 3939-3944, 2008. Evaluation of 4-(2-(4-chlor-1H-pyrazol-1-yl) ethylamino)-3-(6- 23 Tjuvajev J, Kolesnikov Y, Joshi R, Sherinski J, Koutcher L, Zhou (1-(3-fluoropropyl) piperidin-4-yl)-4-methyl-1H-benzo (d) Y, Matei C, Koutcher J, Kreek MJ and Blasberg R: Anti- imidazol-2-yl pyridin-2 (1H)-one (BMS-695735), an orally neoplastic properties of human corticotropin releasing factor: efficacious inhibitor of -like growth Factor-I receptor involvement of the nitric oxide pathway. In Vivo 12: 1-10, 1998. kinase with broad-spectrum in vivo antitumor activity. J. Med. Chem 51: 5897–5900, 2008. Note: the referenced formula of interest is provided within supplemental material available on line via the Internet at: http://pubs.acs.org Received October 22, 2010 21 Altman DG: Practical Statistics for Medical Research. London: Revised November 12, 2010 Chapman and Hall, 1991. Accepted November 16, 2010

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