R ABSTRACT BACKGROUND ECO-4601 is a structurally novel farnesylated dibenzodiazepinone discovered using Ecopia's genomic platform through analysis of actinomycete gene loci encoding the pathways leading to bioactive compounds (Nature Biotechnology 2003, 21:187). This small molecule was shown to inhibit cellular proliferation of different types of tumor cell lines and to have potent antitumor activity against rat glioma, human breast and prostate xenografts (AACR-NCI-EORTC, November 2005 Abstract No. A28). Moreover, ECO-4601 is well tolerated, rapidly absorbed and distributed to different organs including the brain. Preliminary studies performed on Sprague-Dawley rat and Cynomolgus monkey liver, kidneys and urine treated with ECO-4601 indicated that the compound was mainly metabolized into and secreted as a glucuronide. In the present study, we further characterized the biodistribution of ECO-4601, by evaluating the binding of the compound to plasma proteins, its pharmacokinetic profile as well as its metabolism in cryopreserved hepatocytes. These experiments were conducted in parallel in the rodent (Sprague-Dawley rat) and non-rodent (Cynomolgus monkey and human) species to determine if the rat and monkey are appropriate species for pre-clinical toxicology studies. • No significant differences in ECO-4601 plasma protein binding between rat, monkey and human species were observed. Protein binding was estimated to be ~ 70%. • The PK profiles of ECO-4601 found in Sprague-Dawley rats and Cynomolgus monkeys were similar. A single IV bolus injection of the compound resulted in a high C max , followed by relatively rapid distribution and elimination. • ECO-4601 glucuronide (resulting from a Phase II metabolism reaction) was detected as the major metabolite in all 3 species analyzed. Our findings indicate that the rat (rodent) and monkey (non-rodent) are appropriate species for pre-clinical toxicology studies. Furthermore, no major modification in the pharmacological behaviour is expected from clinical studies of ECO-4601 in humans. CONCLUSIONS ECO-4601 (MW 463) is a structurally novel compound discovered using Ecopia's genomic DECIPHER® technology platform. The in vitro cytotoxicity and in vivo antitumor efficacy studies suggest that ECO-4601 is a potent candidate for clinical studies against brain and other solid cancers. In the work presented here, we first evaluated plasma protein binding of [ 3 H] ECO- 4601 (specific activity, 1 Ci/mmol) using the size exclusion method. Percent bound fractions of the compound were found in the same range across rat (70.6% ± 4.6), monkey (70.8% ± 1.4) and human (66.9% ± 1.5) species. Plasma pharmacokinetic parameters of ECO-4601 were also evaluated in rodent (Sprague-Dawley rat) and non- rodent (Cynomolgus monkey) models. In rats (n=3 per gender), pharmacokinetic of a single 30 mg/kg IV bolus injection of ECO-4601 followed 2 phases, best described by a 2-compartment model. The half-life (t 1/2 ) values for the a and b phases were 4.8 min and 2.5 h, respectively. In monkeys (n=3 per gender), the equivalent surface area dose of ECO-4601 (15 mg/kg) was administrated by a single IV bolus injection. Similar biphasic pharmacokinetic profiles were observed, with a t 1/2 a of 20 min and t 1/2 b of ~8 h. Metabolism studies of ECO-4601 were conducted in cryopreserved hepatocytes from the same species. Cells were incubated in the presence of 10 mM and 25 mM ECO-4601 and biotransformation was followed for 4h using LC-MS-MS analysis. A single glucuronide was the major metabolite identified in hepatocytes obtained from rats, monkeys and humans. Further biodistribution analysis of ECO-4601 in the Sprague- Dawley rats and Cynomolgus monkeys has detected the glucuronide in liver and kidneys, with larger amounts of this metabolite in urine. In monkey feces, relatively high concentrations of ECO-4601 were detected suggesting that the compound is excreted unchanged through the bile. Altogether, these data suggest that ECO-4601 has a short half-life in the vascular compartment where it binds to plasma proteins. The compound does not accumulate in tissues. It is partly eliminated in the bile and partly metabolized into a glucuronide that is subsequently eliminated in the urine. To summarize, the pharmacokinetic parameters of ECO-4601, the free compound fraction available to diffuse from the plasma into organs, as well as the metabolic products were found to be similar across species (rat, monkey and human). Consequently, the rat and monkey represent appropriate species for pre-clinical toxicology studies and no major modification in the pharmacological behaviour is expected for clinical studies of ECO-4601 in humans. RESULTS 3094 97 th AACR Washington D.C. April 1-5, 2006 7290 Frederick-Banting Saint-Laurent, Quebec www.ecopiabio.com H4S 2A1 Canada info@ecopiabio.com Pharmacokinetic and ADME Studies of the Antitumor Compound ECO-4601 M-A . Wioland 1 , A. Ibrahim 1 , F. Beaudry 2 , F.K. Yeboah 1 , M. Ranger 1 , J. McAlpine 1 and H. Gourdeau 1 1. Ecopia BioSciences Inc., Ville Saint-Laurent, QC H4S 2A1, Canada; 2. University of Montreal, Saint-Hyacinthe, QC J2S 2M2, Canada Plasma protein binding of ECO-4601 in rat, monkey and human plasma Plasma samples were incubated with ECO-4601 at 2mM or 5mM for 2 hours at 37 o C, loaded onto NAP-5 Sephadex G-25 columns and eluted. [ 3 H]Warfarin (specific activity, 14.1 Ci/mmol) and [ 3 H]methotrexate (specific activity, 46.8 Ci/mmol) incubated with 90% human plasma were used as positive controls while [ 3 H]ECO-4601 (specific activity, 1 Ci/mmol) in the absence of plasma was used as negative control.  No significant differences were found in the PPB percentages for ECO-4601 across species at the 2 tested concentrations, with values of 63.2% to 75.2% (average 69.4%).  The PPB values of warfarin (76.2% ± 2.1) and methotrexate (5.1% ±1.3) were ~25-30% lower than published data (98%-100% for warfarin and 34%-57% for methotrexate). Consequently, the PPB value for ECO- 4601 of ~70% may be considered as an underestimate. The in vitro metabolism of ECO-4601 was evaluated in female and male cryopreserved hepatocytes obtained from Sprague-Dawley rats, Cynomolgus monkeys and humans. ECO-4601 was tested at 10 mM and 25 mM and the parent drug and its metabolites were followed at different time point (0, 15, 30, 60, 120, 180 and 240 min) using HPLC/MS/MS analysis. In vitro metabolism of ECO-4601 in rat, monkey and human hepatocytes  When results were expressed as a ratio of ECO-4601 glucuronide/ECO-4601 parent drug, depending on the species and cell viability, the percentage of glucuronide varied between 14.0% to 33.9% after a 4h incubation period.  Glucuronidation was the main metabolite in all three species analyzed. PK profile of ECO-4601 following bolus IV administration The pharmacokinetic profiles of ECO-4601 following a single IV bolus injection was investigated in Sprague-Dawley rats (30 mg/kg) and Cynomolgus monkeys (15 mg/kg, equivalent on a mg/m 2 basis to the dose administrated to rats). Plasma samples were obtained (n = 6 per time point) at 2, 5, 15, 30 min and 1, 2, 4, 8, 12 (rats) and 24h.  The PK profile was similar in both species and for both genders.  The PK bolus IV dosing resulted in high C max (~500 mM in both species).  The plasma concentrations of ECO-4601 declined rapidly due to the short half-life (t1/2 a = 4.8 min and 20 min for rat and monkey, respectively). Dose C0 AUC ¥ CL Vss Vz Species (mg/kg) (mg/mL*h) (L/h/kg) (L/kg) (L/kg) (h) Rat 30 249 537 86 0.35 0.07 1.2 2.4 Monkey 15 255 550 67 0.23 0.22 2.1 6.4 Rat 0 50 100 150 200 250 0 5 10 15 20 25 Time (min) Glucuronide/ECO-4601 at time zero (%) Monkey 0 50 100 150 200 250 0 10 20 30 Time (min) Human 0 50 100 150 200 250 0 10 20 30 40 50 Time (min) Glucuronide/ECO-4601 at time zero (%) Glucuronide/ECO-4601 at time zero (%) 25 mM 10 mM Species PPB (%) Mean (%) SEM 66.9 63.2 67.4 72.2 69.3 66.0 75.2 70.6 70.8 1.5 1.4 4.6 70.7 66.2 Rat Human Monkey 0 5 10 15 20 25 10 100 1,000 10,000 100,000 1,000,000 Hours Plasma Concentration (ng/mL) Cynomolgus Monkeys Males Females Sprague-Dawley Rats 0 2 4 6 8 10 12 10 100 1000 10000 100000 Hours Plasma Concentration (ng/mL) Males Females t 1/2 z (mM) (mg/mL) Methotrexate (5 mM) + Human Plasma (90%) 0 10 20 30 40 1 3 5 7 9 11 13 15 Eluate Radioactivity (%) Warfarin (5 mM) + Human Plasma (90%) 0 20 40 1 3 5 7 9 11 13 15 Eluate Radioactivity (%) 0 10 20 30 40 1 3 5 7 9 11 Eluate Radioactivity (%) ECO-4601 (5 mM) + Human Plasma (90%) Bound fraction Unbound fraction t1/2 (min) Intrinsic Clearance (μL/min/10 6 cells) % Glucuronidation at 4h Species E CO-4601 25 μM E CO-4601 25 μM E CO-4601 25 μM Male rat 43.1 ± 8.6 4.4 ± 0.1 14.0 ± 1.2 Female rat 67.5 ± 22.5 21.3 ± 3.1 28.7 ± 4.4 Male monkey 79.7 ± 15.2 5.7 ± 1.2 14.4 ± 3.8 Female monkey 44.7 ± 12.8 8.0 ± 0.01 16.9 ± 2.8 Male human 77.8 ± 7.4 6.2 ± 0.6 20.2 ± 4.4 Female human 39.1 ± 9.9 5.2 ± 0.2 33.9 ± 4.3