Cancer Chemotherapy and Pharmacology

, Volume 61, Issue 6, pp 911–921 | Cite as

Identification, characterization and potent antitumor activity of ECO-4601, a novel peripheral benzodiazepine receptor ligand

  • Henriette GourdeauEmail author
  • James B. McAlpine
  • Maxime Ranger
  • Bryan Simard
  • Francois Berger
  • Francis Beaudry
  • Pierre Falardeau
Original Article



ECO-4601 is a structurally novel farnesylated dibenzodiazepinone discovered through DECIPHER® technology, Thallion’s proprietary drug discovery platform. The compound was shown to have a broad cytotoxic activity in the low micromolar range when tested in the NCI 60 cell line panel. In the work presented here, ECO-4601 was further evaluated against brain tumor cell lines. Preliminary mechanistic studies as well as in vivo antitumor evaluation were performed.


Since ECO-4601 has a benzodiazepinone moiety, we first investigated if it binds the central and/or peripheral benzodiazepine receptors. ECO-4601 was tested in radioligand binding assays on benzodiazepine receptors obtained from rat hearts. The ability of ECO-4601 to inhibit the growth of CNS cancers was evaluated on a panel of mouse, rat and human glioma cell lines using a standard MTT assay. Antitumor efficacy studies were performed on gliomas (rat and human), human breast and human prostate mouse tumor xenografts. Antitumor activity and pharmacokinetic analysis of ECO-4601 was evaluated following intravenous (IV), subcutaneous (SC), and intraperitoneal (IP) bolus administrations.


ECO-4601 was shown to bind the peripheral but not the central benzodiazepine receptor and inhibited the growth of CNS tumor cell lines. Bolus SC and IP administration gave rise to low but sustained drug exposure, and resulted in moderate to significant antitumor activity at doses that were well tolerated. In a rat glioma (C6) xenograft model, ECO-4601 produced up to 70% tumor growth inhibition (TGI) while in a human glioma (U-87MG) xenograft, TGI was 34%. Antitumor activity was highly significant in both human hormone-independent breast (MDA-MB-231) and prostate (PC-3) xenografts, resulting in TGI of 72 and 100%, respectively. On the other hand, IV dosing was followed by rapid elimination of the drug and was ineffective.


Antitumor efficacy of ECO-4601 appears to be associated with the exposure parameter AUC and/or sustained drug levels rather than C max. These in vivo data constitute a rationale for clinical studies testing prolonged continuous administration of ECO-4601.


Dibenzodiazepine Peripheral benzodiazepine receptor (PBR) Cancer Glioma Xenografts 



International Association for Assessment and Accreditation of Laboratory Animal Care


Area under the curve


Canadian Council on Animal Care


Central benzodiazepine receptor


5% dextrose

q3d × 7

Every 3 days for 7 cycles






Mitochondrial permeability transition pore

q1d × 5

Once daily for 5 consecutive days


Once every other day


Peripheral benzodiazepine receptor





The authors would like to thank Anna Avrutskaya, Robert Mullin and Jennifer Streicker (From Piedmont Reserch Center) and Simon Taylor and Kresimir Pucaj (Nucro-Technics) for performing animal studies. We also thank Mario Chevrette for helpful discussions and revisions.


  1. 1.
    Alley MC, Hollingshead MG, Dykes DJ, Waud WR (2004) Human tumor xenograft models in NCI drug development. Cancer Drug Discov Dev 125–152Google Scholar
  2. 2.
    Bachmann BO, McAlpine, JB, Zazopoulos E, Farnet CM, Piraee M (2004) Farnesyl dibenzodiazepinones, processes for their production and their use as pharmaceuticals. US Patent No. 7,101,872Google Scholar
  3. 3.
    Batra S, Larsson I, Boven E (2000) Mitochondrial and microsomal peripheral benzodiazepine receptors in human ovarian cancer xenografts. Int J Mol Med 5:619–623PubMedGoogle Scholar
  4. 4.
    Beinlich A, Strohmeier R, Kaufmann M, Kuhl H (1999) Specific binding of benzodiazepines to human breast cancer cell lines. Life Sci 65:2099–2108PubMedCrossRefGoogle Scholar
  5. 5.
    Benda P, Lightbody J, Sato G, Levine L, Sweet W (1968) Differentiated rat glial cell strain in tissue culture. Science 161:370–371PubMedCrossRefGoogle Scholar
  6. 6.
    Beurdeley-Thomas A, Miccoli L, Oudard S, Dutrillaux B, Poupon MF (2000) The peripheral benzodiazepine receptors: a review. J Neurooncol 46:45–56PubMedCrossRefGoogle Scholar
  7. 7.
    Braestrup C, Squires RF (1977) Specific benzodiazepine receptors in rat brain characterized by high-affinity (3H)diazepam binding. Proc Natl Acad Sci USA 74:3805–3809PubMedCrossRefGoogle Scholar
  8. 8.
    Bribes E, Carriere D, Goubet C, Galiegue S, Casellas P, Simony-Lafontaine J (2004) Immunohistochemical assessment of the peripheral benzodiazepine receptor in human tissues. J Histochem Cytochem 52:19–28PubMedGoogle Scholar
  9. 9.
    Broaddus WC, Bennett JP Jr (1990) Peripheral-type benzodiazepine receptors in human glioblastomas: pharmacologic characterization and photoaffinity labeling of ligand recognition site. Brain Res 518:199–208PubMedCrossRefGoogle Scholar
  10. 10.
    Carmel I, Fares FA, Leschiner S, Scherubl H, Weisinger G, Gavish M (1999) Peripheral-type benzodiazepine receptors in the regulation of proliferation of MCF-7 human breast carcinoma cell line. Biochem Pharmacol 58:273–278PubMedCrossRefGoogle Scholar
  11. 11.
    Charan RD, Schlingmann G, Janso J, Bernan V, Feng X, Carter GT (2004) Diazepinomicin, a new antimicrobial alkaloid from a marine Micromonospora sp. J Nat Prod 67:1431–1433PubMedCrossRefGoogle Scholar
  12. 12.
    Chelli B, Lena A, Vanacore R, Pozzo ED, Costa B, Rossi L, Salvetti A, Scatena F, Ceruti S, Abbracchio MP, Gremigni V, Martini C (2004) Peripheral benzodiazepine receptor ligands: mitochondrial transmembrane potential depolarization and apoptosis induction in rat C6 glioma cells. Biochem Pharmacol 68:125–134PubMedCrossRefGoogle Scholar
  13. 13.
    Clarke GD, Ryan PJ (1980) Tranquillizers can block mitogenesis in 3T3 cells and induce differentiation in friend cells. Nature 287:160–161PubMedCrossRefGoogle Scholar
  14. 14.
    Corbett T, Polin L, LoRusso P, Valeriote F, Panchapor C, Pugh S, White K, Knight J, Demchick L, Jones J, Jones L, Lisow L (2004) In vivo methods for screening and preclinical testing. Cancer Drug Discov Dev 99–123Google Scholar
  15. 15.
    Cornu P, Benavides J, Scatton B, Hauw JJ, Philippon J (1992) Increase in omega 3 (peripheral-type benzodiazepine) binding site densities in different types of human brain tumours. A quantitative autoradiography study. Acta Neurochir (Wien) 119:146–152CrossRefGoogle Scholar
  16. 16.
    Damm HW, Muller WE, Schlafer U, Wollert U (1978) H]flunitrazepam: its advantages as a ligand for the identification of benzodiazepine receptors in rat brain membranes. Res Commun Chem Pathol Pharmacol 22:597–600PubMedGoogle Scholar
  17. 17.
    Decaudin D (2004) Peripheral benzodiazepine receptor and its clinical targeting. Anticancer Drugs 15:737–745PubMedCrossRefGoogle Scholar
  18. 18.
    Decaudin D, Castedo M, Nemati F, Beurdeley-Thomas A, De Pinieux G, Caron A, Pouillart P, Wijdenes J, Rouillard D, Kroemer G, Poupon MF (2002) Peripheral benzodiazepine receptor ligands reverse apoptosis resistance of cancer cells in vitro and in vivo. Cancer Res 62:1388–1393PubMedGoogle Scholar
  19. 19.
    Dimitriadou V, Gourdeau H, Simard B, Boccard S, Pelletier L, McAlpine JB, Zazopoulos E, Falardeau P, Berger F, Farnet CM (2004) Abstract # 569: a new antitumor compound, ECO-4601: preclinical evaluation and in vivo efficacy in glioma. Presented at the 16th EORTC-NCI-AACR symposiumGoogle Scholar
  20. 20.
    Galiegue S, Tinel N, Casellas P (2003) The peripheral benzodiazepine receptor: a promising therapeutic drug target. Curr Med Chem 10:1563–1572PubMedCrossRefGoogle Scholar
  21. 21.
    Guo P, Ma J, Li S, Guo Z, Adams AL, Gallo JM (2001) Targeted delivery of a peripheral benzodiazepine receptor ligand-gemcitabine conjugate to brain tumors in a xenograft model. Cancer Chemother Pharmacol 48:169–176PubMedCrossRefGoogle Scholar
  22. 22.
    Han Z, Slack RS, Li W, Papadopoulos V (2003) Expression of peripheral benzodiazepine receptor (PBR) in human tumors: relationship to breast, colorectal, and prostate tumor progression. J Recept Signal Transduct Res 23:225–238PubMedCrossRefGoogle Scholar
  23. 23.
    Hans G, Wislet-Gendebien S, Lallemend F, Robe P, Rogister B, Belachew S, Nguyen L, Malgrange B, Moonen G, Rigo JM (2005) Peripheral benzodiazepine receptor (PBR) ligand cytotoxicity unrelated to PBR expression. Biochem Pharmacol 69:819–830PubMedCrossRefGoogle Scholar
  24. 24.
    Hardwick M, Fertikh D, Culty M, Li H, Vidic B, Papadopoulos V (1999) Peripheral-type benzodiazepine receptor (PBR) in human breast cancer: correlation of breast cancer cell aggressive phenotype with PBR expression, nuclear localization, and PBR-mediated cell proliferation and nuclear transport of cholesterol. Cancer Res 59:831–842PubMedGoogle Scholar
  25. 25.
    Katz Y, Eitan A, Gavish M (1990) Increase in peripheral benzodiazepine binding sites in colonic adenocarcinoma. Oncology 47:139–142PubMedCrossRefGoogle Scholar
  26. 26.
    Krueger KE (1995) Molecular and functional properties of mitochondrial benzodiazepine receptors. Biochim Biophys Acta 1241:453–470PubMedGoogle Scholar
  27. 27.
    Landau M, Weizman A, Zoref-Shani E, Beery E, Wasseman L, Landau O, Gavish M, Brenner S, Nordenberg J (1998) Antiproliferative and differentiating effects of benzodiazepine receptor ligands on B16 melanoma cells. Biochem Pharmacol 56:1029–1034PubMedCrossRefGoogle Scholar
  28. 28.
    Le Fur G, Vaucher N, Perrier M, Flamier A, Benavides J, Renault C, Dubroeucq M, Gueremy C, Uzan A (1983) Differentiation between two ligands for peripheral benzodiazepine binding sites, [3H]RO5–4864 and [3H]PK 11195, by thermodynamic studies. Life Sci 33:449–457PubMedCrossRefGoogle Scholar
  29. 29.
    Maaser K, Hopfner M, Jansen A, Weisinger G, Gavish M, Kozikowski AP, Weizman A, Carayon P, Riecken EO, Zeitz M, Scherubl H (2001) Specific ligands of the peripheral benzodiazepine receptor induce apoptosis and cell cycle arrest in human colorectal cancer cells. Br J Cancer 85:1771–1780PubMedCrossRefGoogle Scholar
  30. 30.
    Maaser K, Grabowski P, Sutter AP, Hopfner M, Foss HD, Stein H, Berger G, Gavish M, Zeitz M, Scherubl H (2002) Overexpression of the peripheral benzodiazepine receptor is a relevant prognostic factor in stage III colorectal cancer. Clin Cancer Res 8:3205–3209PubMedGoogle Scholar
  31. 31.
    Maeda J, Suhara T, Zhang MR, Okauchi T, Yasuno F, Ikoma Y, Inaji M, Nagai Y, Takano A, Obayashi S, Suzuki K (2004) Novel peripheral benzodiazepine receptor ligand [11C]DAA1106 for PET: an imaging tool for glial cells in the brain. Synapse 52:283–291PubMedCrossRefGoogle Scholar
  32. 32.
    Matthew E, Laskin JD, Zimmerman EA, Weinstein IB, Hsu KC, Engelhardt DL (1981) Benzodiazepines have high-affinity binding sites and induce melanogenesis in B16/C3 melanoma cells. Proc Natl Acad Sci USA 78:3935–393PubMedCrossRefGoogle Scholar
  33. 33.
    McAlpine JB, Bachmann BO, Piraee M, Tremblay S, Alarco AM, Zazopoulos E, Farnet CM (2005) Microbial genomics as a guide to drug discovery and structural elucidation: ECO-02301, a novel antifungal agent, as an example. J Nat Prod 68:493–496PubMedCrossRefGoogle Scholar
  34. 34.
    McEnery MW (1992) The mitochondrial benzodiazepine receptor: evidence for association with the voltage-dependent anion channel (VDAC). J Bioenerg Biomembr 24:63–69PubMedCrossRefGoogle Scholar
  35. 35.
    McEnery MW, Snowman AM, Trifiletti RR, Snyder SH (1992) Isolation of the mitochondrial benzodiazepine receptor: association with the voltage-dependent anion channel and the adenine nucleotide carrier. Proc Natl Acad Sci USA 89:3170–3174PubMedCrossRefGoogle Scholar
  36. 36.
    Olsen RW, Tobin AJ (1990) Molecular biology of GABAA receptors. Faseb J 4:1469–1480PubMedGoogle Scholar
  37. 37.
    Papadopoulos V (2003) Peripheral benzodiazepine receptor: structure and function in health and disease. Ann Pharm Fr 61(1):30–50PubMedGoogle Scholar
  38. 38.
    Papadopoulos V, Amri H, Li H, Yao Z, Brown RC, Vidic B, Culty M (2001) Structure, function and regulation of the mitochondrial peripheral-type benzodiazepine receptor. Therapie 56:549–556PubMedGoogle Scholar
  39. 39.
    Papadopoulos V, Baraldi M, Guilarte TR, Knudsen TB, Lacapere JJ, Lindemann P, Norenberg MD, Nutt D, Weizman A, Zhang MR, Gavish M (2006) Translocator protein (18kDa): new nomenclature for the peripheral-type benzodiazepine receptor based on its structure and molecular function. Trends Pharmacol Sci 27:402–409PubMedCrossRefGoogle Scholar
  40. 40.
    Pawlikowski M, Kunert-Radek J, Radek A, Stepien H (1988) Inhibition of cell proliferation of human gliomas by benzodiazepines in vitro. Acta Neurol Scand 77:231–233PubMedGoogle Scholar
  41. 41.
    Plumb JA, Milroy R, Kaye SB (1989) Effects of the pH dependence of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide-formazan absorption on chemosensitivity determined by a novel tetrazolium-based assay. Cancer Res 49:4435–4440PubMedGoogle Scholar
  42. 42.
    Simard B, Gourdeau H, Bichat F, Mirjolet JF, McAlpine J, Farnet C, Berger F, Falardeau Pu (2005) Abstract # 5896: ECO-4601. A Novel anticancer compound, is a peripheral benzodiazepine receptor ligand and induces apoptosis in gliomasGoogle Scholar
  43. 43.
    Stephenson FA (1995) The GABAA receptors. Biochem J 310(Part 1):1–9PubMedGoogle Scholar
  44. 44.
    Sutter AP, Maaser K, Barthel B, Scherubl H (2003) Ligands of the peripheral benzodiazepine receptor induce apoptosis and cell cycle arrest in oesophageal cancer cells: involvement of the p38MAPK signalling pathway. Br J Cancer 89:564–572PubMedCrossRefGoogle Scholar
  45. 45.
    Trapani G, Laquintana V, Latrofa A, Ma J, Reed K, Serra M, Biggio G, Liso G, Gallo JM (2003) Peripheral benzodiazepine receptor ligand-melphalan conjugates for potential selective drug delivery to brain tumors. Bioconjug Chem 14:830–839PubMedCrossRefGoogle Scholar
  46. 46.
    Veenman L, Levin E, Weisinger G, Leschiner S, Spanier I, Snyder SH, Weizman A, Gavish M (2004) Peripheral-type benzodiazepine receptor density and in vitro tumorigenicity of glioma cell lines. Biochem Pharmacol 68:689–698PubMedCrossRefGoogle Scholar
  47. 47.
    Venturini I, Zeneroli ML, Corsi L, Avallone R, Farina F, Alho H, Baraldi C, Ferrarese C, Pecora N, Frigo M, Ardizzone G, Arrigo A, Pellicci R, Baraldi M (1998) Up-regulation of peripheral benzodiazepine receptor system in hepatocellular carcinoma. Life Sci 63:1269–1280PubMedCrossRefGoogle Scholar
  48. 48.
    Venturini I, Alho H, Podkletnova I, Corsi L, Rybnikova E, Pellicci R, Baraldi M, Pelto-Huikko M, Helen P, Zeneroli ML (1999) Increased expression of peripheral benzodiazepine receptors and diazepam binding inhibitor in human tumors sited in the liver. Life Sci 65:2223–2231PubMedCrossRefGoogle Scholar
  49. 49.
    Walter RB, Raden BW, Cronk MR, Bernstein ID, Appelbaum FR, Banker DE (2004) The peripheral benzodiazepine receptor ligand PK11195 overcomes different resistance mechanisms to sensitize AML cells to gemtuzumab ozogamicin. Blood 103:4276–4284PubMedCrossRefGoogle Scholar
  50. 50.
    Wang JK, Morgan JI, Spector S (1984) Differentiation of friend erythroleukemia cells induced by benzodiazepines. Proc Natl Acad Sci USA 81:3770–3772PubMedCrossRefGoogle Scholar
  51. 51.
    Zazopoulos E, Huang K, Staffa A, Liu W, Bachmann BO, Nonaka K, Ahlert J, Thorson JS, Shen B, Farnet CM (2003) A genomics-guided approach for discovering and expressing cryptic metabolic pathways. Nat Biotechnol 21:187–190PubMedCrossRefGoogle Scholar
  52. 52.
    Zisterer DM, Williams DC (1997) Peripheral-type benzodiazepine receptors. Gen Pharmacol 29:305–314PubMedGoogle Scholar
  53. 53.
    Zisterer DM, Campiani G, Nacci V, Williams DC (2000) Pyrrolo-1,5-benzoxazepines induce apoptosis in HL-60, Jurkat, and Hut-78 cells: a new class of apoptotic agents. J Pharmacol Exp Ther 293:48–59PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Henriette Gourdeau
    • 1
    Email author
  • James B. McAlpine
    • 1
  • Maxime Ranger
    • 1
  • Bryan Simard
    • 2
  • Francois Berger
    • 2
  • Francis Beaudry
    • 3
  • Pierre Falardeau
    • 1
  1. 1.Thallion Pharmaceuticals Inc.St LaurentCanada
  2. 2.INSERM U318GrenobleFrance
  3. 3.Department of Veterinary Biomedicine, Faculty of Veterinary MedicineUniversity of MontrealSt HyacintheCanada

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