SNS-595, A NOVEL CELL CYCLE INHIBITOR IN PHA
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SNS-595, A NOVEL CELL CYCLE INHIBITOR IN PHASE I CLINICAL TRIALS, CAUSES TUMOR REGRESSIONS, CELL-CYCLE ARREST, AND APOPTOSIS IN MURINE MODELS OF CANCER Ute Hoch, Jennifer Hyde, Michelle A. Nannini, Marc J. Evanchik, Chris E. Lawrence, Michelle R. Arkin, Duncan H. Walker, Jeffrey A. Silverman, Sunesis Pharmaceuticals Inc, South San Francisco, CA ABSTRACT #2277 BIOLOGIC ACTIVITY OF SNS-595 IN HCT116 TUMORS CURES IN COLO-26 SYNGENEIC MODEL WITH SPARSE DOSING SNS-595 is a novel cell cycle modulator currently in clinical trials for the treatment of advanced solid malignancies. ACTIVATION OF CELL CYCLE AND APOPTOSIS MARKERS IN HCT116 TUMORS SNS-595 shows tumor regression and cures in the highly metastatic syngeneic Colo-26 tumor model known to be SNS-595 shows excellent antitumor activity in a broad spectrum of murine syngeneic and human xenograft tumor refractory to many of the major classes of cytotoxic drugs. SNS-595 is highly active on all schedules with complete After administration of 20 mg/kg to tumor-bearing mice, SNS-595 results in activation of checkpoint markers measured by the appearance of cdc2 models, including drug-resistant models. Detailed cell cycle and pathway analyses in cell culture reveal that SNS-595 phosphorylation and p21 expression, as well as activation of apoptotic markers measured by the appearance of p53 and p73 phosphorylations. The regressions and cures when dosed at the MTD of 20 mg/kg. When dosed at ½ the MTD, the weekly and biweekly acts during S-phase and causes a significant S-phase lag associated with rapid appearance of checkpoint markers and regimens still result in cures, whereas a reduction of the curative effect was apparent with the least frequent regimen. responses in tumors are rapid and consistent with those observed in cell culture. sustained and irreversible arrest with 4N DNA content. This cell cycle arrest is rapidly followed by the onset of apoptosis, which is mediated through p53 independent and dependent mechanisms. To confirm the biological activities observed in cell culture in vivo, we analyzed tumor homogenates after single administration of SNS-595 to TIME COURSE OF SNS-595 PATHWAY ACTIVATION (20 mg/kg DOSE, n=4) SNS-595 10 mg/kg (1/2 MTD) CELLULAR STRESS 10000 mice bearing advanced HCT-116 tumors. Within 24 hr of SNS-595 administration, modulations of pharmacodynamic Median Tumor Volume, Predose markers consistent with G2 cell cycle arrest (cyclin B and cdc 2) and apoptosis (including p53, p21, and caspase-3) 1/10 CR were detected. The pharmacodynamic effects in tumors correlated with tumor exposure to SNS-595 and contributed to p No Treatment 16 hr p p 1000 4 hr 8 hr 2 hr significant growth delay of subcutaneously implanted HCT116 tumors when dosed on a weekly schedule. In addition to CHK1/2 p53 p73 c-Abl Fraction Maximal Marker Cytoplasmic 1.2 2/10 CR a weekly schedule (qwk x 3), less frequent schedules (q2wk x 3 and q3wk x 3) were studied in mice bearing Gemcitabine Sequestration p 1 3 subcutaneous Colo-26 tumors, a highly metastatic syngeneic colorectal tumor model known to be refractory to many p mm cdc25 0.8 p21 p73 Phos-p53(Y15) Activation of the major classes of cytotoxic drugs. SNS-595 proved to be highly active on all schedules with complete Cdk2 0.6 100 SNS-595 qwkx3 regressions and cures when dosed at the MTD. When dosed at ½ the MTD, the weekly and biweekly regimens still Cyclin E Phos-p73(Y99) 0.4 resulted in cures, whereas a reduction of the curative effect was apparent with the least frequent regimen. These p p 0.2 SNS-595 q2wkx3 p studies with SNS-595 provide evidence that the strong tumor responses in animal models are mediated by biological cdc25 Rb Rb Phos-Cdc2(Y15) 8/10 CR p p Y15 p E2F 0 10 SNS-595 q3wkx3 activities consistent with those observed in cell culture. cdc2 cdc2 -0.2 Cyclin B Cyclin B E2F BAX p21 -0.4 -5 0 5 10 15 20 9/10 CR 1 BACKGROUND Time (Hrs) Active Arrest Arrest Apoptosis 0 10 20 30 40 50 60 70 80 Mitosis SNS-595, a naphthyridine derivative, is a novel cytotoxic agent intended for the treatment of several tumor types. The cytotoxic activity of SNS-595 has been demonstrated in more than 20 different tumor cell lines, and antitumor activity Days Post Initial Treatment has been observed in 11 human xenograft tumor models and 3 syngeneic models in mice. SNS-595 has a unique ACTIVATION OF CASPASE-3 IN HCT-116 TUMORS mechanism of action: It causes an S-phase lag, a rapid onset of apoptosis and an irreversible G2 arrest. SNS-595 Activation of Caspase-3 in a tumor sections. Tumors were removed 4 hr after Activation of Caspase-3 in tumor lysates. SNS-595 20 mg/kg (MTD) distinguishes itself from other therapeutics with rapid checkpoint signaling leading to immediate cell death or administration of 20 mg/kg SNS-595 and stained with a Caspase-3 antibody. Tumors (n=3) were removed 6 hr after 10000 senescence (abstracts #2285 and #2293). Median Tumor Volume, administration of 40 mg/kg SNS-595. Vehicle SNS-595 RAPID APOPTOSIS IN CELLS MARKER ACTIVATION IN CELLS 1.2 1/10 CR 1000 No Treatment Caspase Activation (HCT116 Cells) (HCT116 Cells) 1 1 1 SNS-595 O Gemcitabine 3 Maximal Caspase-3 mm 0.8 Doxorubicin 0.8 Maximal Marker CO2H 0.8 Phos-p53(Y15) 100 Activation Etoposide Activation 0.6 0.6 H3C Phos-p73(Y99) N N N SNS-595 qwkx3 Cisplatin HN 0.6 0.4 0.4 Phos-Cdc2(Y15) Docetaxel S N 8/10 CR 0.2 0.2 p21 H3CO 0.4 10 SNS-595 q2wkx3 Gemcitabine 0 Irinotecan 0 8/10 CR 0.2 SNS-595 q3wkx3 0 5 10 15 20 25 0 5 10 15 SNS-595 8/10 CR Time (Hrs) Time (Hrs) 0 1 Vehicle 40 mg/kg 0 10 20 30 40 50 60 70 80 METHODS Days Post Initial Treatment TUMOR AND PLASMA LEVELS AFTER 20 mg/kg IV DOSE HCT116 XENOGRAFT RESPONSE All in vivo studies were performed in accordance with IACUC guidelines and in harmony with the Guide for SNS-595 shows good tumor/plasma ratios and tumor concentrations SNS-595 shows tumor growth inhibition in HCT-116 xenograft model when Laboratory Animal Care and Use. above 10x the cellular GI50 (2560 nM or 1000 ng/mL) for 16 hr. dosed IV at or below the MTD of 20 mg/kg on a weekly dosing regimen. 1500.0 Pharmacodynamic Study: 10000 CONCLUSIONS SNS-595 (ng/mL or ng/g) SNS-595 was administered intravenously to animals with established HCT116 tumors (300 mm3 in size). Tumor and blood (n=4 per time point) were Tumor Weight (mg) 10x GI50 T-C 18.9 days collected at the indicated times post dose. A 50 mg tumor piece was homogenized with 5X buffer. SNS-595 levels in plasma and tumor homogenate were T-C 22.5 days determined after protein precipitation by LC-ESI-MS/MS. The remainder of the tumor was ground into powder for Western or ELISA analysis. Tumor 1000 SNS-595 shows excellent distribution to tumor tissue resulting in tumor to plasma ratios of lysates were prepared and 10-20 µg total protein was run on 10% NuPage Bis-Tri Gel and then transferred to a Nitrocellulose membrane and probed using 1000.0 T-C 25.8 days 1° and 2° mAb (p21 Cellsignaling 2946, phos-p53(Y15) Cellsignaling 9284, phos-p73(Y99) Cellsignaling 4665). 50 µg total protein was used in a capture >10 in the tumor models tested ELISA. Maxisorp Plate→anti-caspase-3 (BD 610322) → lysate → anti-cleaved caspase-3 (Cellsignaling 9661) → 2nd mAb-HRP (Chemicon) 100 HCT-116 Tumor Model: SNS-595 shows biological activities consistent with those observed in cell culture HCT-116 colon carcinoma cells were implanted subcutaneously in the flank. Tumors were allowed to grow to approximately 100 mm3 in size. Mice were 500.0 • Activation of cell cycle markers in HCT-116 tumors, with maximum activation 2 to 16 hr pair-matched into no treatment, Irinotecan (100 mg/kg, IP, qwx5), and SNS-595 (20 and 15 mg/kg, IV, qwx5) treatment groups. Mice were weighed, first 10 daily than twice a weekly and examined frequently for clinical signs of adverse effects. Acceptable toxicity was defined as a mean group weight loss of 20% post dose or less and not more than one toxic death among 8 treated animals. Animals were euthanized when the tumor size reached 1500 mm3. Mean tumor volume was plotted until >25% of animals were lost per group. • Activation of apoptosis markers in HCT-116 tumors, with maximum activation 4 hr post 1 0.0 dose Colo-26 Tumor Model: 0 5 10 15 20 25 • Stress signals observed in vitro lead to in vivo activity Colon carcinoma 26 cells were implanted subcutaneously in the flank. Tumors were allowed to grow to approximately 90 mm3 in size. Mice were pair- Time (hr) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 matched into no treatment, Gemzar (160 mg/kg, IP, q3dx4), and SNS-595 (20 and 15 mg/kg, IV, qwx3, q2wx3, q3wx3) treatment groups. Mice were weighed, first daily than twice a weekly and examined frequently for clinical signs of adverse effects. Acceptable toxicity was defined as a mean group Days Post Initial Treatment Preclinically, SNS-595 retains curative effects in the highly metastatic syngeneic Colo-26 weight loss of 20% or less and not more than one toxic death among 10 treated animals. Animals were euthanized when the tumor size reached 2000 Tumor mm3. Mean tumor volume was plotted until >20% of animals were lost per group. No Treatment Irinotecan 100 mpk qwx5 tumor model, even when dosed every three weeks Plasma SNS-595 20 mpk qwx5 SNS-595 15 mpk qwx3 We would like to acknowledge Piedmont Research Center for their contributions to the HCT-116 and Colo-26 tumor models.