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AM6538 is a cannabinoid antagonist that binds CB1 receptors expressed in HEK-293 cells in a wash-resistant manner. The effects of AM6538 in live animals has not previously been established. We characterized the antagonist effects of AM6538 in male mice, using a warm-water tail-withdrawal assay, and in male squirrel monkeys trained to discriminate the CB1 agonist AM4054 from vehicle. The cannabinoid agonists WIN 55,212, Δ(9)-tetrahydrocannabinol (THC), and AM4054 all produced 100% maximum possible antinociceptive effects in mice following vehicle pretreatment. One-hour pretreatment with increasing doses of AM6538 (0.1–10 mg/kg) produced first rightward, then downward shifts of the agonist dose-effect functions. Rimonabant, 1–10 mg/kg, produced parallel rightward shifts of the AM4054 dose-effect functions, and baseline effects of AM4054 were nearly recovered within 24 hours following 10 mg/kg of rimonabant. In contrast, in mice treated with 10 mg/kg of AM6538, antagonism of THC or AM4054 lasted up to 7 days. AM6538 also antagonized the discriminative stimulus effects of AM4054 in squirrel monkeys in a dose-related manner, and the effects of 3.2 mg/kg of AM6538 endured for more than 7 days. The effective reduction in CB1 receptor reserve was used to calculate the relative efficacy (tau values) of WIN 55,212, THC, and AM4054 in mice and of AM4054 monkeys, with results indicating that THC has a lower efficacy than WIN 55,212 or AM4054 in mice. These results demonstrate that AM6538 is a long-acting CB antagonist in vivo, and further suggest that differences in CB efficacy can be revealed in behavioral assays following AM6538 treatment.

The Wnt/β-catenin pathway is among the most frequently altered signaling networks in human cancers. Despite decades of preclinical and clinical research, efficient therapeutic targeting of Wnt/β-catenin has been elusive. RNA interference (RNAi) technology silences genes at the mRNA level and therefore can be applied to previously undruggable targets. Lipid nanoparticles (LNP) represent an elegant solution for the delivery of RNAi-triggering oligonucleotides to disease-relevant tissues, but have been mostly restricted to applications in the liver. In this study, we systematically tuned the composition of a prototype LNP to enable tumor-selective delivery of a Dicer-substrate siRNA (DsiRNA) targeting CTNNB1, the gene encoding β-catenin. This formulation, termed EnCore-R, demonstrated pharmacodynamic activity in subcutaneous human tumor xenografts, orthotopic patient-derived xenograft (PDX) tumors, disseminated hematopoietic tumors, genetically induced primary liver tumors, metastatic colorectal tumors, and murine metastatic melanoma. DsiRNA delivery was homogeneous in tumor sections, selective over normal liver and independent of apolipoprotein-E binding. Significant tumor growth inhibition was achieved in Wnt-dependent colorectal and hepatocellular carcinoma models, but not in Wnt-independent tumors. Finally, no evidence of accelerated blood clearance or sustained liver transaminase elevation was observed after repeated dosing in nonhuman primates. These data support further investigation to gain mechanistic insight, optimize dose regimens, and identify efficacious combinations with standard-of-care therapeutics. Mol Cancer Ther; 15(9); 2143–54. ©2016 AACR.

Daily treatment with cannabinoids results in tolerance to many, but not all, of their behavioral and physiologic effects. The present studies investigated the effects of 7-day exposure to 10 mg/kg daily of Δ(9)-tetrahydrocannabinol (THC) on the diuretic and antinociceptive effects of THC and the synthetic cannabinoid AM4054. Comparison studies determined diuretic responses to the κ-opioid agonist U50,488 and furosemide. After determination of control dose-response functions, mice received 10 mg/kg daily of THC for 7 days, and dose-response functions were re-determined 24 hours, 7 days, or 14 days later. THC and AM4054 had biphasic diuretic effects under control conditions with maximum effects of 30 and 35 ml/kg of urine, respectively. In contrast, antinociceptive effects of both drugs increased monotonically with dose to >90% of maximal possible effect. Treatment with THC produced 9- and 7-fold rightward shifts of the diuresis and antinociception dose-response curves for THC and, respectively, 7- and 3-fold rightward shifts in the AM4054 dose-response functions. U50,488 and furosemide increased urine output to >35 ml/kg under control conditions. The effects of U50,488 were attenuated after 7-day treatment with THC, whereas the effects of furosemide were unaltered. Diuretic effects of THC and AM4054 recovered to near-baseline levels within 14 days after stopping daily THC injections, whereas tolerance to the antinociceptive effects persisted longer than 14 days. The tolerance induced by 7-day treatment with THC was accompanied by a 55% decrease in the Bmax value for cannabinoid receptors (CB1). These data indicate that repeated exposure to THC produces similar rightward shifts in the ascending and descending limbs of cannabinoid diuresis dose-effect curves and to antinociceptive effects while resulting in a flattening of the U50,488 diuresis dose-effect function.

RATIONALE: Lower lip retraction (LLR) in rats has been described as a distinctive effect of 5-HT(1A) agonists. In the course of evaluating behavioral effects of cannabinoid agonists in rats, LLR effects were evident following injection of several cannabinoid agonists. OBJECTIVES: To pharmacologically characterize cannabinoid-induced LLR in rats. METHODS: Lower lip retraction was scored using a 3-point scale for up to 6 hours after injection of the cannabinoid agonists Δ(9)-tetrahydrocannabinol (Δ(9)-THC; 1–10 mg/kg), AM7499 (0.01–1.0 mg/kg), or AM2389 (0.003–0.1 mg/kg), or, for comparison, the 5-HT(1A) agonist 8-OH-DPAT (0.01–0.3 mg/kg). Next, antagonist effects of rimonabant (1–10 mg/kg) and WAY100635 (0.3 mg/kg) on LLR produced by cannabinoid or 5-HT(1A) agonists were evaluated. Lastly, effects of 8-OH-DPAT were determined following pretreatment with AM2389 (0.003–0.01 mg/kg) or Δ(9)-THC (1 mg/kg). RESULTS: All three cannabinoid agonists produced LLR. Effects of AM2389 were attenuated by both rimonabant and WAY100635 whereas effects of 8-OH-DPAT were antagonized by WAY 100635 but not by rimonabant. Pretreatment with 1 mg/kg Δ(9)-THC or 0.01 mg/kg AM2389 shifted the 8-OH-DPAT dose-effect function for LLR to the left and isobolographic analysis of the data indicates CB1 and 5-HT(1A) interactions can be supraadditive. CONCLUSIONS: Cannabinoid agonists produce LLR in rats, an effect heretofore ascribed only to activity at 5-HT(1A) receptors, via CB1 receptor-mediated actions. Co-administration of a cannabinoid agonist and the 5-HT(1A) agonist 8-OH-DPAT results in a synergistic effect on LLR.

Colorectal carcinomas harbor well-defined genetic abnormalities, including aberrant activation of Wnt/β-catenin and MAPK pathways, often simultaneously. Although the MAPK pathway can be targeted using potent small-molecule drugs, including BRAF and MEK inhibitors, β-catenin inhibition has been historically challenging. RNAi approaches have advanced to the stage of clinical viability and are especially well suited for transcriptional modulators, such as β-catenin. In this study, we report therapeutic effects of combined targeting of these pathways with pharmacologic agents. Using a recently described tumor-selective nanoparticle containing a β-catenin–targeting RNAi trigger, in combination with the FDA-approved MEK inhibitor (MEKi) trametinib, we demonstrate synergistic tumor growth inhibition in in vivo models of colorectal cancer, melanoma, and hepatocellular carcinoma. At dose levels that were insufficient to significantly impact tumor growth as monotherapies, combination regimens resulted in synergistic efficacy and complete tumor growth inhibition. Importantly, dual MEKi/RNAi therapy dramatically improved survival of mice bearing colorectal cancer liver metastases. In addition, pharmacologic silencing of β-catenin mRNA was effective against tumors that are inherently resistant or that acquire drug-induced resistance to trametinib. These results provide a strong rationale for clinical evaluation of this dual-targeting approach for cancers harboring Wnt/β-catenin and MAPK pathway mutations. Mol Cancer Ther; 17(2); 544–53. ©2017 AACR.

Despite recent advances in early detection and therapeutic intervention, colorectal cancer (CRC) remains one of the most deadly cancers in the United States. The Wnt/β-catenin and KRAS/BRAF-driven MAPK pathways were reported to cooperate in inducing CRC initiation and progression. Several studies have also identified Wnt signaling as a resistance mechanism in MAPK pathway-activated CRCs. Thus far, independent targeting of these single oncogenic events in CRC has yielded limited therapeutic success. RNA interference (RNAi) triggers are capable of silencing any expressed mRNA with high potency and specificity, including previously un-druggable targets such as β-catenin, an oncogene frequently dysregulated in CRC. DCR-BCAT is a chemically-optimized DsiRNA targeting CTNNB1, the gene which encodes β-catenin, formulated in a second-generation EnCore lipid nanoparticle. We have previously reported extensive preclinical pharmacology for DCR-BCAT in mouse tumor models of diverse origin. (Ganesh et al, Mol Cancer Ther 2016). Here we demonstrate that the dual inhibition of β-catenin and MEK demonstrates synergistic efficacy in not only primary CRC, but also in aggressive liver metastases of CRC in preclinical models. This combination of DCR-BCAT and the MEK inhibitor trametinib was also efficacious in tumors which are known to be resistant to trametinib, as well as tumors which acquire resistance due to drug exposure. These data support clinical development of these combination approaches for this first-in-class RNAi therapeutic. Citation Format: Shanthi Ganesh, Serena Shui, Girish Chopda, Martin Koser, Kevin Craig, Chaitali Dutta, Cheng Lai, Hank Dudek, Weimin Wang, Bob D. Brown, Marc Abrams. RNAi-based β-catenin inhibition in combination with MEK inhibition promotes synergistic anti-tumor efficacy and overcomes resistance with MEK inhibition promotes synergistic anti-tumor efficacy and overcomes resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1229. doi:10.1158/1538-7445.AM2017-1229

Dicer-substrate siRNAs (DsiRNAs) are a potent class of RNA interference (RNAi) triggers capable of silencing any expressed mRNA with high specificity. DCR-MYC, a first-in-class DsiRNA targeting the MYC oncogene is currently in Phase 1b/II clinical trials [ASCO 2015, abstract 11006]. DCR-BCAT is an advanced preclinical development candidate that targets CTNNB1, the gene which encodes β-catenin. The β-catenin/Wnt pathway is consistently activated in human tumors, including >50% of hepatocellular carcinomas (HCC) and >80% of colorectal cancers (CRC). Robust preclinical and genetic evidence strongly suggests that inhibiting β-catenin function would yield broad therapeutic benefit in oncology, but efforts to target it using conventional drug modalities have been unsuccessful to-date. We have previously reported extensive preclinical pharmacology for DCR-BCAT in mouse tumor models of diverse origin. Here, we explore DCR-BCAT as a monotherapy and in combination with both standard-of-care and experimental therapeutics. Interestingly, when mice bearing HCC tumors were treated with a combination of CTNNB1 and MYC DsiRNAs, the antitumor efficacy was additive or synergistic relative to either single agent alone. Additionally, since up to 50% of colorectal tumors have both activated Wnt signaling and mutant KRAS, we also explored combination therapy of DCR-BCAT and FDA-approved MEK inhibitors. A major advantage of DCR-BCAT is that the improved nanoparticle formulation is more selective for siRNA delivery to tumors over liver and other normal tissues. These data support continued development of DCR-BCAT as a first-in-class RNAi therapeutic, and highlight the potential for use in combination with other promising agents. Citation Format: Shanthi Ganesh, Wendy Cyr, Martin Koser, Girish Chopda, Edmond Chipumuro, Zakir Siddiquee, Kevin Craig, Serena Shui, Dongyu Chen, Cheng Lai, Hank Dudek, Weimin Wang, Bob Brown, Marc Abrams. Preclinical characterization of DCR-BCAT as a component of combination therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3827.

MYC and CTNNB1 are well-characterized drivers of numerous tumor types. Human and preclinical genetic evidence suggest that pharmacological intervention to reduce transactivation of MYC and CTNNB1-regulated genes would yield therapeutic benefit to many cancer patients. Since the proteins encoded by these genes are challenging to target via conventional modalities, progress in new therapeutic agents has been slow despite decades of research. RNA interference technology has enabled the inhibition of previously-undruggable genetic targets at the mRNA level, and has advanced to clinical development for several indications. DCR-MYC is a Phase I-stage lipid nanoparticle (LNP)-formulated Dicer substrate siRNA (DsiRNA), representing a potent class of RNAi triggers being developed by Dicerna Pharmaceuticals. Here we describe new preclinical data that increase our understanding of the parameters that impact tumor delivery and activity of DsiRNA. We demonstrate that the cationic lipid and PEG-lipid components of Dicerna's unique EnCore LNP platform can be modulated to improve delivery of DsiRNA to both orthotopic and spontaneous liver tumors, as well as xenograft tumors of diverse non-hepatic tissue origin. Characterization of LNP formulations with respect to plasma PK, tissue exposure and target mRNA knockdown was employed towards understanding the pharmacology of LNP-mediated tumor delivery. Citation Format: Marc Abrams, Shanthi Ganesh, Bo Ying, Girish Chopda, Utsav Saxena, Anee Shah, Martin Koser, Rokhand Arvan, Dongyu Chen, Serena Shui, Rohan Diwanji, Wei Zhou, Benjamin Holmes, Boyoung Kim, Hailin Yang, Mihir Patel, Wendy Cyr, Wendy Cyr, Natalie Pursell, Nicole Avitahl-Curtis, Hank Dudek, Cheng Lai, Weimin Wang, Bob D. Brown. EnCore-LNP mediated tumor delivery of MYC and CTNNB1 Dicer Substrate RNAs (DsiRNAs). [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B20.

The β-catenin/Wnt pathway is consistently activated in human tumors, including >50% of hepatocellular carcinomas (HCC) and >80% of colorectal cancers (CRC). This is often caused directly by tumorigenic mutations in the CTNNB1 gene, which encodes β-catenin protein. Preclinical evidence in genetic cancer models strongly suggests that inhibiting β-catenin function would yield therapeutic benefit to many cancer patients. Since β-catenin is very challenging to target via conventional modalities, no efficacious therapy targeting β-catenin has emerged despite decades of research. RNA interference (RNAi) technology has enabled the inhibition of previously undruggable targets at the mRNA level and has advanced to late-stage clinical development for several indications. However, the clinical application of RNAi has proven to be most successful for targeting mRNAs in the normal liver, and challenges remain with developing formulations that efficiently deliver to extra-hepatic tumors. Here we describe the development and characterization of a platform to treat tumors of diverse primary tissue origin and metastatic location with CTNNB1-targeting DsiRNA. Tumor types tested included xenograft tumors of colorectal origin (CRC), experimental melanoma lung metastases, and difficult-to-transfect leukemia cells which have infiltrated the spleen. In addition to strong silencing of CTNNB1 mRNA, >75% inhibition of tumor growth was observed for a CRC xenograft model. This was achieved by formulating the DisRNA payload in a lipid nanoparticle (LNP) platform termed EnCore (because of its specific Envelope and Core lipid components). EnCore activity against multiple tumors was further improved by optimizing critical cationic lipid and PEG-lipid components. Importantly, studies in knockout mice demonstrate that in vivo tumor activity is independent of Apolipoprotein E, which has been widely described as an essential endogenous ligand for internalization of LNPs into hepatocytes. This strongly suggests that Encore-mediated delivery to tumors in vivo is independent of the ApoE/LDLR system. DCR-MYC, an EnCore-delivered DsiRNA targeting MYC mRNA, is currently in Phase I clinical trials. Further evaluation of LNP-delivered DsiRNAs in PK/PD and efficacy models, including PDX and GEMM will guide a clinical development strategy for CTNNB1 DsiRNA. Citation Format: Shanthi Ganesh, Bo Ying, Martin Koser, Wendy Cyr, Girish Chopda, Hank Dudek, Cheng Lai, Weimin Wang, Bob Brown, Marc T. Abrams. Systemic delivery of CTNNB1 Dicer-substrate siRNAs (DsiRNAs) leads to efficient oncogene silencing in diverse tumor types of extra hepatic origin. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3533. doi:10.1158/1538-7445.AM2015-3533

Hepatoblastoma (HB), the commonest pediatric liver tumor is frequently associated with an N-terminal deletion in the CTNNB1 gene, which encodes stable β-catenin and promotes nuclear localization and transactivation activity. In 80% of patients, nuclear localization of β-catenin and Yes Associated Protein (YAP), a Hippo-related transactivator, is seen together. Further, stable hepatic co-expression of Δ90-CTNNB1 and active-YAP in mice causes rapid and aggressive HB. Dicer-substrate siRNAs (DsiRNAs) are potent RNA interference (RNAi) triggers that that are efficacious in preclinical tumor models of diverse origin, and are currently under clinical evaluation. To determine if CTNNB1-targeting DsiRNAs have potential as a therapy in HB, we encapsulated this oligonucleotide payload into lipid nanoparticles (LNPs), and systemically administered into mice bearing CTNNB1/YAP-induced HB. The LNP platform used, termed EnCore (because of its specific Envelope and Core lipid components), enables high encapsulation efficiency, long-term stability, and consistent analytical criteria. Downstream of LNP-mediated DsiRNA delivery, both qPCR and in situ hybridization were used to visualize changes in CTNNB1 expression in both tumor and normal liver. Robust and specific silencing of CTNNB1 mRNA was achieved in the tumors. The distribution of mRNA knockdown within each tumor nodule suggests efficient extravasation and internalization of the LNP and its payload into the tumor parenchyma, in contrast to previous reports of vascular-channel limited nanoparticle accumulation in tumors. Intriguingly, a “liver-centric” LNP formulation, which delivers cargo efficiently to a normal liver due to rapid hepatic extraction and apolipoprotein-mediated internalization, was inactive in the tumors. Further evaluation demonstrated efficacy of β-catenin targeting in this model. In conclusion, we report a highly relevant modality of RNAi delivery in a mouse model of hepatoblastoma to target a classically-undruggable oncogene. Citation Format: Marc T. Abrams, Junyan Tao, Shanthi Ganesh, Wendy Cyr, Bo Ying, Martin Koser, Rokhand Arvan, Girish Chopda, Hank Dudek, Cheng Lai, Weimin Wang, Bob Brown, Satdarshan Monga. Targeting β-catenin with a Dicer-substrate siRNA (DsiRNA) in a sleeping beauty transposon-driven murine hepatoblastoma model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3536. doi:10.1158/1538-7445.AM2015-3536