NUCLEIC ACID THERAPEUTICS Volume 24, Number 2, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/nat.2013.0456

Study of CRTC2 Pharmacology Using Antisense Oligonuceotides Robert Dullea,1 Christopher Salatto,1 Simone Sciabola,2 Tracy Chen,2 Debra DiMattia,1 Harmeet Gandhok,1 John Kreeger,3 Yan Weng,4 Tracey Clark,4 Chandra Vage,4 and Robert Stanton 2

The cAMP response element binding protein (CREB)-regulated transcriptional coactivator 2 (CRTC2) is a key component of the transcription complex regulating glucagon driven hepatic glucose production and previous evidence suggests that ‘‘inhibition’’ of CRTC2 improves glucose homeostasis in multiple rodent models of type 2 diabetes. Here we describe a process of identifying potential therapeutic antisense oligonucleotides (ASOs) directed against CRTC2. These ASOs were designed as locked nucleic acid (LNA) gapmers and a panel of approximately 400 sequences were first screened in vitro within both human and mouse liver cell lines. A group of active and selective compounds were then profiled in acute studies in mice to determine the level of CRTC2 mRNA reduction in liver as well as to obtain a preliminary indication of safety and tolerability. The compounds with the best activity and safety profiles were then evaluated in subchronic efficacy studies using the diet induced obese (DIO) mouse model of type 2 diabetes and primary human hepatocytes. Efficacy findings broadly confirmed the beneficial effect of reducing CRTC2 mRNA levels towards improving glucose control and other markers of metabolic function. Additionally, for the first time, translation to human cells has been established with demonstration of a reduction in glucagon-mediated glucose production in primary human hepatocytes and a potential clinical biomarker source identified to assess modulation of CRTC2 mRNA following ASO treatment. While the compounds identified herein did not demonstrate a therapeutic index sufficient for further development, this study should facilitate more efficient prosecution of compounds within an in vivo setting.

Introduction

H

yperglycemia driven by increased hepatic glucose output is a prevalent pathophysiology associated with insulin resistance ultimately contributing to the development of type 2 diabetes mellitus (T2DM) (Consoli et al., 1989). The pancreas-derived glucagon hormone is a key mediator initiating the gluconeogenic program in the liver where upon binding to its receptor activates a transcription complex containing the cAMP response element binding protein (CREB)regulated transcription coactivator 2 (CRTC2). Activation of CRTC2 is then responsible for the increased expression of genes involved in the conversion of three carbon precursors to glucose, principally phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) (Hoeffler et al., 1988; Gonzalez and Montminy, 1989; Habener, 1990). CRTC2 activity is tightly regulated downstream of nutrient sensing signals by multiple post-translation modifications including phosphorylation and acetylation. In the fed state, it is seques-

tered in a phosphorylation-dependent inactive form within the cytoplasm while after fasting and subsequent dephosphorylation translocates to the nucleus where it promotes target gene transcription (Screaton et al., 2004; Canettieri et al., 2005; Koo et al., 2005). Interestingly, phosphorylation deficient (i.e., constitutively active) CRTC2 protein due to O-linked glycosylation at the inhibitory serine-171 phosphorylation site in the liver has been described in insulin-resistance states (Dentin et al., 2008).Through physical interaction, active CRTC2 appears to increase occupancy of the CREB for its target promoters. This association is likely the critical event for promoting glucagon-mediated gluconeogenesis as deletion of the CREB binding domain in CRTC2 lowers blood glucose and improves insulin sensitivity in diet induced obese (DIO) mice (Wang et al., 2009). In addition to the ability of CRTC2 to enhance the expression of genes associated with hepatic glucose output, it has been demonstrated that CREB/CRTC2 activity modulates transcriptional response in adipose tissue to enhance whole

1

Cardiovascular and Metabolic Disease Research Unit and 2Oligonucleotide Therapeutic Unit, Pfizer, Cambridge, Massachusetts. Safety Science, Groton, Connecticut. 4 Pharmacokinetics, Dynamics and Metabolism, Groton, Connecticut. 3

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128 body insulin resistance. This phenomenon appears to function through the ability of CREB/CRTC2 to stimulate the expression of activating transcription factor 3 (ATF3) that in turn decreases the expression of glucose transporter 4 (GLUT4) and adiponectin (Qi et al., 2009). Applying multiple gene function approaches to explore the effect of disrupting CRTC2 activity on whole body physiology in preclinical systems has largely confirmed its role as a key mediator of glucose homeostasis. Specifically, a whole body mouse knockout demonstrated reduced G6Pase and PEPCK mRNA expression within the liver that translated to improved insulin sensitivity measured by oral glucose tolerance test and insulin tolerance test on both normal chow and high fat diet independent of body weight and food intake (Wang et al., 2010b). Furthermore, utilizing a liposomally formulated small interfering RNA (siRNA) that mediated, liver-restricted knockdown of CRTC2, Pepck, and G6Pase target genes improved hepatic insulin sensitivity and glucose homeostasis in high fat diet fed mice and Zucker diabetic fatty rats (Saberi et al., 2009). These studies also revealed that liver-specific ‘‘inhibition’’ of CRTC2 enhanced peripheral insulin sensitivity measured by increased insulin-stimulated glucose disposal rate and decreased triglycerides in muscle as well as a reduction in plasma free fatty acids and an increase in fed insulin levels (Saberi et al., 2009). Taken together, these findings demonstrate the potential therapeutic benefit of improving whole body glucose homeostasis and lipotoxicity through decreasing the liver mRNA expression of CRTC2. While multiple drug modalities exist to translate early target validation experiments into clinical settings, antisense oligonucleotide (ASO) approaches offer a number of advantages. Second generation ASOs are designed as gapmers, in which nucleosides constrained to the RNA conformation flank a central DNA core region (Monia et al., 1993). The central DNA allows for the recruitment of RNaseH, while the flanking or ‘‘wing’’ residues improve the binding energy of the ASO and help slow nuclease degradation. Several modified nucleosides have been developed with the properties necessary for the wing residues, these include locked nucleic acids (LNAs) (Koshkin et al., 1998; Obika et al., 1998; Wahlestedt et al., 2000; Jepsen et al., 2004; Frieden and Ørum, 2008) and 2¢methoxyethyl (MOEs) (Sazani et al., 2003). Antisense compounds have the additional benefit for targets such as CRTC2 of accumulating in the liver and kidney, without the need for additional targeting or formulation agents. In the present study, we use ASOs to broadly confirm phenotypic findings associated with the in vitro and in vivo reduction of CRTC2 and extend these observations to inhibition of glucagon-mediated glucose production in primary human hepatocytes. Additionally, this pharmacodynamic (PD) response is put into context with the pharmacokinetic (PK) profile of potent CRTC2 LNA ASOs as a means to understand these relationships and begin to explore potential translation to the clinic of future ASO-based therapeutics. Materials and Methods Compound design An in-house design workflow was applied for the selection of a set of antisense oligonucleotides sequences able to trigger effective and selective CRTC2 mRNA degradation. The largest human splice variant (ENST00000368633) was enumer-

DULLEA ET AL. ated using a 14-mer antisense design length, which resulted in 2631 potential sequences. High similarity with alternative CRTC2 human splice variants and other species (mouse, rat, and macaque) represented a key filter in the selection and only 14-mer sequences having fewer than 2 mismatches to any other CRTC2 transcript of interest were retained (data shown in Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/nat). Additionally, sequences were filtered out if they aligned with: (1) known human SNPs, (2) sequence motifs associated with nonspecific binding, (3) low complexity regions in the mRNA, and (4) other transcripts in the genome. The surviving sequences were scored based on potency predicted with an in-house statistical model and the final selection accounted for the location of the oligonucleotide sequence, such that the chosen compounds were not clustered in a particular mRNA region but, instead, were spread out along the target complementary DNA (cDNA). A small number of compounds were included to explore regions of the sequence that were poorly covered or not mentioned in existing patents. At the end of this process, *400 14-mer oligonucleotides were selected for synthesis. To improve nuclease stability as well as target affinity, the chosen sequences were chemically modified to have a phosphorothioated backbone and the 3-8-3 LNA gapmer design. Additionally, any CpG motifs were methylated to reduce the potential for any immune stimulatory response. A nontargeting control sequence 2 was also used in the in vivo studies. It was designed to have no perfect matches to mouse or human cDNA sequences. Oligonucleotide synthesis and QC All oligonucleotides were synthesized using fast-deprotecting phosphoramidite protocols on a MerMade 12 or MerMade 192 oligonucleotide synthesizer (BioAutomation) at 200- to 1000-nmole scales employing standard CPG supports (BioSearch) or Glen UnySupport (Glen Research). After synthesis, the oligonucleotides were cleaved from the support and deprotected using ammonium hydroxide/methylamine. The crude 4, 4’-dimethoxytrityl (DMTr) on oligonucleotides were purified via 4, 4¢-dimethoxytrityl (DMTr)-selective cartridge purification techniques and, if necessary, further purified via reversed phase high-performance liquid chromatography (RP HPLC) and desalted via cartridge-based methods. A Waters Acquity ultra performance liquid chromatography system connected in-line to a Waters LCT Premier time of flight mass spectrometer was used to confirm purity. Oligonucleotide concentration was determined using a Molecular Devices SpectraMax M5 absorbance reader at 260 nm and a C Technologies Solo VP Slope reader equipped with ‘‘Quick Slope’’ software and by utilizing Beer’s Law. Extinction coefficients were calculated using the nearest neighbor model. In vitro assays Human hepatocellular carcinoma cell line Hep3B and mouse hepatoma cell line Hepa1-6 (ATCC) were used in this study. Cells were maintained in Eagle’s minimum essential medium (Hep3B) or Dulbecco’s modified Eagle’s medium (Hepa1-6) with 10% fetal bovine serum (FBS) (ATCC). Subculture was done according to the provider’s recommendations. The day before lipid transfection, cells were plated at a density of 8,000 cells in 80 mL complete growth media per well in

CRTC2 ASOs 96-well plates. On the day of transfection, LNAs were prepared at 10 · of the final concentration (final 3 nM) in Opti-Mem (Invitrogen). Lipofectamine 2000 (Invitrogen) was diluted at 1:25 in Opti-Mem. An equal volume of the 10 · LNA and Lipofectamine 2000 (40 mL each) was mixed and incubated at room temperature for 30 minutes. Then, 20 mL of the mixture was added into each well of the cells, bringing the final volume to 100 mL per well. Cells were returned to the incubator and incubated at 37C and 5% CO2 for 24 hours before harvesting. For the unassisted delivery studies, a compound plate was prepared at 20 · of final concentration on the day of transfection. Hepa1-6 cells were trypsinized and resuspended in Gibco Media 199 (Invitrogen) with 10% FBS. A volume of 95 mL cell suspension was added to a 96-well plate at the density of 4,000 cells per well. Then, 5 mL of compounds were added to each well. The plate was incubated at 37C for 7 days. QuantiGene 2.0 assays (Affymetrix) for the mouse and human CRTC2 transcripts along with the housekeeper, cylophilin B (PPIB), were utilized to determine mRNA expression levels following the manufacturer’s procedures. On the day of harvesting, 200 mL per well of lysis buffer (with 1:100 protease K) was added to the cells. A total of 20 mL of lysate was used for mouse CRTC2 and PPIB probes, while 80 mL and 40 mL lysate was used for human CRTC2 and PPIB probes, respectively. Assay plates were read on the GloRunner Microplate Luminometer (Promega) and data reported was normalized against PPIB. In vivo activity and tolerability Eight-week-old, male, C57BL/6 mice were obtained from Charles River Laboratories. All mice were housed under standard conditions with a 12-hour light/dark cycle and free access to chow diet (Rodent Diet 5001) and water. All procedures involving animals were conducted under animal use protocols approved by Pfizer’s Institutional Animal Care and Use Committee in compliance with the Guide for Care and Use of Laboratory Animals, and all applicable federal regulations. To measure acute activity, mice were administered single dose of compound in saline at either 3 mg/kg or 10 mg/ kg by subcutaneous injection. Forty-eight hours after compound administration, the mice were euthanized. Biopsies of the kidney and left lateral liver lobe were collected and flash frozen for mRNA analysis and tissue exposure. To assess the preliminary safety profile of early lead sequences, mice were administered compound in saline at 25 mg/kg s.c. twice a week. On day 14, mice were euthanized and serum was collected from the vena cava for analysis of serum markers of liver and kidney toxicity. Tissues (liver, kidney, heart, spleen, bone marrow, and mid-jejunum) were fixed in 10% neutral buffered formalin, and processed for histopathology. Serum alanine aminotransferase (ALT) and aspartate transaminase (AST) levels were then determined on an ADVIA 2400 Chemistry System (Siemens Healthcare Diagnostics). In vivo efficacy To characterize the efficacy of compound 1, one-week-old, male C57Bl6/J mice ( Jackson Labs) maintained on a 60% kcal fat diet (Research Diets) were acclimated to the local vivarium for 3 weeks. Age-matched C57Bl6 mice on normal diet (LabDiet) served as a normal control group. Prior to study initiation, mice were assigned to treatment groups based upon

129 fasted glucose, fasted insulin, and body weight values. Pretreatment fasted plasma levels were determined for triglyceride, total cholesterol, fructosamine, lactate, and betahyroxybutyrate (Roche Diagnostics). Additionally, body composition was measured (EchoMRI) prior to beginning the study. Mice were administered the indicated dose of LNA ASO compound formulated in saline or saline alone as vehicle at a frequency of twice per week by subcutaneous injection into the right scapular region. At the initiation of the daily light cycle on day 14 of the study, a pyruvate tolerance test (PTT) was performed where food was removed from all mice within the vehicle and 10 mg/kg LNA ASO treated groups and, following a 6-hour fast, blood glucose was measured using the AlphaTrak blood glucose meter (Abbott). Mice were then challenged with 1 g/kg, i.p. sodium pyruvate (Sigma) and blood glucose was monitored at 15, 30, 60, and 120 minutes post injection. An oral glucose tolerance test was then performed on all treatment groups following a 6-hour fast on day 27 of the study. Baseline blood glucose was measured using the AlphaTrak blood glucose meter (Abbott). Mice were then challenged with 1 g/kg, p.o. dextrose and blood glucose was monitored at 15, 30, 60, and 120 minutes post injection. On study day 28, mice were again fasted for 6 hours prior to sacrifice where at termination plasma, liver tissue, white adipose tissue, and subcutaneous fat (left scapular region) were collected. Similar protocols were followed for the evaluation of the second active CRTC2 compound, compound 3, except that dosing occurred at once weekly intervals and the PTT was performed on study day 13 (6 days following the second dose). The experiment was then terminated immediately following the PTT. RNA isolation and single-stranded cDNA preparation Mouse liver biopsy punches (3mm, 30 mg) were harvested into sterile 2-mL screw-capped Sarstedt tubes and flash frozen in liquid nitrogen. RLT buffer (Qiagen) was added to the samples followed by tissue homogenization using the TissueLyser Mixer Mill. Total RNA was isolated using the RNeasy 96 kit (Qiagen) following the manufacturer’s instructions. RNA quality and concentration was assessed by the SpectraMax M2 instrument (Molecular Devices) where optical density readings were taken at wavelengths of 260nm, 280nm, and 320nm. A maximum of 2.0 mg RNA was converted to single-stranded (ss) cDNA using high capacity RNA-to-cDNA Master Mix (Applied Biosystems, ABI) in a final volume of 20 mL. Final reactions were either used immediately in real time quantitative polymerase chain reactions or stored at - 20C. Real-time quantitative polymerase chain reaction Amplification of CRTC2 cDNA was carried out in triplicate in 384-well format, using with the 7900HT Fast Real-Time PCR System (ABI) and TaqMan Gene Expression Assays (Mm00433832_m1, ABI). Employing the 5¢ nuclease activity of Taq DNA polymerase to generate a real-time quantitative analysis (Heid et al., 1996), 20-mL amplification reactions contained: 5 mL of 1-to-10 dilution of sscDNA (1 part ss cDNA, 9 parts molecular biology grade water), 1 · TaqMan Gene Expression Master Mix (ABI), and 1 · Target Assay Mix (ABI). Similar 20 mL reference reactions were prepared in triplicate for amplification of the housekeeping gene HPRT1. Thermal

130 cycling conditions for all reactions were as follows: 2 minutes at 50C, 10 minutes at 95C, then 40 cycles of 15 seconds at 95C and 1 minute at 60C. The relative mRNA expression level of the CRTC2 mRNA was determined by normalizing its reaction CT to the HPRT1 CT, where CT is the cycle number at which emitted fluorescence exceeds 10 · the standard deviation of baseline emission (measured from cycle 3 to 15). The DCT value, calculated by subtracting the average HPRT1 CT from the average CRTC2 CT, was then substituted into the formula 2.0 - [DCt] to provide the relative mRNA expression (Meijerink et al., 2001). PK/PD studies The DIO mice were administered the CRTC2 LNA ASO, compound 1, by weekly subcutaneous injection at 10 mg/kg for 3 weeks. Animals were sacrificed at 6, 24, 48, and 168 hours after dose 1; and at 24, 168, 336, 504, and 672 hours post dose 3. Liver ASO concentration and CRTC2 mRNA were measured at each time point. ASO concentrations in the liver were measured using a liquid chromatography-tandem mass spectrometry (LC/MS/MS–based method. A portion of liver tissue was sectioned, weighed and homogenized (1 part tissue in 4 parts water; w/v) using a BioSpec Beadbeater for up to 5 minutes with 2-mm Zirconia beads in a cooled block. Calibration and quality control standards were prepared in control tissue homogenate. Samples were further diluted with control homogenate in order for concentrations to fall within the dynamic range of the assay. One hundred mL of homogenate was added to 96-well plate wells followed by the addition of 15 mL of internal standard (IS, GCATTGGTATT CATTTTTT, phosphothioate-linked DNA) at 5 mg/mL in K2EDTA plasma. Twenty microleters of chloroform:phenol (1:2; v:w) was added to each well, the plate was gently vortexed for 1 minute and then centrifuged at 1500 g for 1 hour. Sixty microliters of supernatant was removed and added to 60 mL water in a 96-well plate containing silanized q-glass vials. Injections of 10 mL were made with a CTC Analytics LEAP Technologies HTs PAL Autosampler System leap autoinjector onto a Hypersil Gold 50 mm · 2.1 mm; 1.9 mm column at 60C with a flow rate of 0.180 mL per minute delivered by a Shimadzu HPLC system. Mobile phase A (MP A) = 1.7 mM triethylamine (TEA), 100 mM hexafluoroisopropanol (HFIP) in water and mobile phase B (MP B) = methanol. LC conditions: start 5% MP B, gradient to 30% MP B by 10 min, re-equilibrate to 5% MP B by 14 minutes. LC flow was directed to the MS from 5.0 to 8.0 minutes. Multiple reaction monitoring in negative ion mode was done using a Sciex API 4000 Q-Trap. Sample concentrations were determined by interpolation from a standard curve with a linear 1/(x2) fit using Analyst 1.4.2. PK/PD analysis Time to maximal concentration (Tmax) and terminal half-life (t1/2) were determined from mean liver concentration time data using noncompartmental analysis in WinNonlin (version 5.2, Pharsight). Tmax was determined as time at which the maximal tissue concentration was observed. Terminal half-life was calculated based on linear regression of logtransformed time versus concentration curve using the last three time points. CRTC2 mRNA levels following ASO treatment were reported as percent of saline-treated mRNA levels (equation 1) and used as the direct PD biomarker for

DULLEA ET AL. exposure-effect relationship analysis (Yu et al., 2007; Yu et al., 2009). The pharmacological effect of liver ASO exposure on CRTC2 mRNA was characterized using an inhibitory Emax model described by equation 2 (Yu et al., 2009) in NONMEM VI. The liver CRTC2 mRNA and the ASO concentration data were represented as E and C, respectively, in the PK/PD model. The baseline mRNA levels, maximal pharmacological effect, liver ASO concentration for half-maximal effect, and hill coefficient were represented as E0, Emax, EC50, and n, respectively, in the PK/PD model. E¼

mRNAtreatment · 100 mRNAcontrol

E ¼ E0 

Emax · Cn ECn50 þ Cn

equation 1

equation 2

Glucose output assay in primary cryopreserved human hepatocytes Cryopreserved primary human hepatocytes (Celsis) were thawed and pelleted by centrifugation at 50 g for 5 minutes. The supernatant was discarded, cells were resuspended in hepatocyte culture medium, and viability was assessed using trypan blue exclusion (typically 70%–90%). Liver-derived nonparenchymal cells, as judged by their size ( < 10 mm diameter) and morphology (nonpolygonal), were consistently found to be less than 1% in these preparations. To create micropatterned cocultures, primary cryopreserved hepatocytes were first seeded on 96-well collagen-patterned substrates that mediate selective cell adhesion. The cells were washed with medium 2–3 hours later to remove unattached cells and incubated in hepatocyte medium overnight. Stromal support cells were seeded the following day to create cocultures. Cultures were treated with 10 mM of the indicated LNA ASO compound or vehicle (in the absence of transfection reagent) in 0.1% serum medium on experimental days 0 and 2. On day 4, cultures were washed three times with phosphate-buffered saline (PBS) and received compound in glucose free, 0.1% serum media containing 100 nM glucagon to deplete glycogen stores. On day 6, glucose free, glucose production media was prepared containing 0.1% serum supplemented with 20 mM lactate and 2 mM pyruvate. Each of the compounds was diluted to 10 mM in this media and each volume of compound was split in half to allow for + / - glucagon conditions. One aliquot of each compound was spiked with glucagon for a final concentration of 100nM. Cultures were washed three times with PBS. Half of the wells were treated with compound in the presence of glucagon and the other half received doses without glucagon. At 1, 2, 4, 7, and 20 hours after treatment, glucose production media was collected for determination of glucose levels by Amplex Red detection of glucose oxidase activity (Invitrogen) and RNA isolated in 100mL RLT buffer (Qiagen) to assess CRTC2 gene expression by real-time quantitative polymerase chain reaction (qRT-PCR). Statistical analysis Unless otherwise noted in the figure legends, results were analyzed using a two-tailed Student’s t-test. A p-value less than 0.05 was considered statistically significant.

CRTC2 ASOs

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Results In vitro assessment of ASO-mediated CRTC2 mRNA reduction A series of *400 ASO compounds were designed to target CRTC2 specifically over the rest of the genome. A fully phosphorothioated 3-8-3 gapmer design was used, in which the 3 nucleotide wings were LNA surrounding a central 8nucleotide DNA gap. The sequences for all compounds are given in Supplementary Table S1. The compounds were evaluated for the ability to reduce CRTC2 mRNA expression in vitro using both lipid transfection and unassisted delivery protocols. For liposomal formulated delivery transfection, efficiency was optimized in both cell lines using a positive control LNA ASO of the same length and design to achieve > 80% message reduction in the absence of cytotoxicity as measured by changes in housekeeping gene expression as well as two independent cell proliferation methods (data not shown). The results of these assays are shown in Table 1 for compounds that were then moved on into in vivo evaluation; results for the remaining compounds are shown in Supplementary Table S2. The compounds were screened in both human (Hep3B) and mouse (Hepa1-6) cell lines, as while the drug was intended for use in humans, many of the preclinical studies would be run in mouse. Although selected using the same criteria, the compounds were found to differ significantly in their ability to regulate CRTC2 transcript levels. Using transfection, dose-dependent CRTC2 message reduction was observed for the majority of compounds in both human and mouse cell lines. The mouse cell line generally showed less activity as compared to the human cells although this may be a function of differences in the assay hybridization probe rather than the ASO. While the rank ordering of compounds changes slightly, the overall classification of compounds into those with high and low gene knock down remains the same across both species. A subset of *130 of the most active compounds was also evaluated using unassisted delivery (sometimes referred to as gymnotic delivery) which require much higher doses of compound (10mM) and extended cell culture viability (7 or 10 days). The mechanism of in vitro uptake under these

conditions is not well understood although a role has been proposed for scavenger receptors (Crooke et al., 1995; Bijsterbosch et al., 1997). These conditions require careful controls for toxicity and cell number when analyzing the data. In this case, housekeeper (PPIB) gene expression was monitored and those sequences that reduced PPIB mRNA levels by > 20% or decreased cell number relative to vehicle were removed from subsequent consideration. The effect of target mRNA reduction under unassisted conditions has been shown to be better predictive of in vivo activity in some studies (Stein et al., 2010), and here it was used as an additional criteria for selecting which compounds to move forward into in vivo studies. The data in Table 1 (and Supplementary Table S2) show that for the majority of compounds, the activity translates well between transfection and unassisted assay conditions; however, there are a number of compounds (e.g., 3 or 7) that show a reduced activity in the unassisted assay. Compounds were advanced into an assessment of in vivo activity and preliminary hepatotoxicity evaluation based on the in vitro data with some compounds selected based on a high degree of activity under only one of the two assay conditions. This selection process was used due to uncertainty in the translation of in vitro activity for these two transfection techniques versus in vivo potency. In vivo assessment of ASO-mediated CRTC2 mRNA reduction The acute in vivo activity of CRTC2 compounds was assessed in the livers of wild-type mice fed a standard chow diet 48 hours following treatment using 3 and 10 or 5 mg/kg doses as summarized in Table 2 and Supplementary Table S3, respectively. In total, 41 compounds were evaluated in vivo including a scrambled control 3-8-3 LNA gapmer ASO, compound 2, which was designed to have 2 or greater mismatches against any sequence in the mouse and human genome. This scrambled control compound was also shown by multiple cell proliferation methods not to effect cell viability in vitro (data not shown), in vivo by monitoring plasma liver enzymes (Table 2) as well as histopathology (data not shown) and additionally did not reduce CRTC2 levels in vitro (Fig. 4A

Table 1. Percentage of CRTC2 mRNA Knockdown in Human (Hep3B) and Mouse (Hepa1-6) Cell Lines Hep3B ID

T-1nM

1 3 4 5 6 7 8 9 10 11 12 13 14

87.4 72

T-3nM

Hepa1-6 T-10nM

U-10mM

T-1nM

T-10nM

U-10mM

89.6 92

80.8 59.4

52.6 32.6

85.8 79.3 94.6 92 106 94.9 89.6 100.8 87.9 65.5

84.6 72.9 51.7 66 62.2 69.3 80.9 74.8 60.1 69

63.8 64.4 48.4 50.6 43 39.8 40.3 45.6 36 61.9

87.6 74.6 79.5 90.5 88.3 74.4 75.3 76.1 70.2 85.2 84.1 74.6 76.3

90.4 29.7 80.7 87.2 72.1 32.2 - 27 55.1 69.4 84.8 72.6 59 - 16.6

68.9 86.1 90.9 79.8 84.9 73.9 80.4 74 87 65.3 86.2

T, lipid transfection at 1, 3, and 10 nm; U, unassisted delivery at 10m4m.

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DULLEA ET AL.

Table 2. CRTC2 Liver mRNA Expression* and Plasma Liver Enzyme Levels** mRNA reduction in liver (%) Compound 1 2 3 5 6 7 8 9 10 11 12 13 14

Liver enzymes 4 · 25 mg/kg (U/L)

3 mg/kg

10 mg/kg

ALT

AST

GLDH

57 7 45 nd 26 6 6 15 -5 9 13 17 9

90 10 84 nd 54 13 42 54 38 24 26 36 37

327 34 79 3890 1735 62 349 7583 197 233 7136 44 367

255 48 78 7229 1100 56 248 1676 114 213 3886 62 397

93 nd 40 nd 986 32 73 4747 71 169 2590 17 140

ALT, alanine aminotransferase; AST, aspartate transaminase; CRTC2, cAMP response element binding protein (CREB)-regulated transcriptional coactivator 2; GLDH, glutamate dehydrogenase; nd, no data. *Liver mRNA expression measured 42 hours following single 3 or 10 mg/kg dosing. **Plasma liver enzymes measured following 2 weeks of 25 mg/kg biweekly dosing.

& 4B) and in vivo (Table 2 and Supplementary Fig. S1). Table 2 captures the initial in vivo strategy of evaluating compound activity at 3 and 10 mg/kg and, in parallel, a preliminary assessment of hepatoxicity by measurement of plasma liver enzyme levels utilizing a 25 mg/kg dose delivered twice per week for 2 weeks. Following completion of these experiments, the in vivo screening process was modified to support higher throughput and reduce compound requirement (Supplementary Table S3). In this design, acute activity within the liver was monitored 48 hours following delivery of a 5 mg/kg dose and hepatoxicity was measured by plasma liver enzyme analysis 72 hours after mice received a single 25 mg/kg dose (except those compounds where values are noted with an asterisk in Supplementary Table S3 that were measured after a 17.5 mg/kg single dose). Table 2 describes a wide response range within a dose group for both compound potency and tolerability indicating the importance of triaging in vitro leads prior to longer duration subchronic efficacy studies. Additionally, a dose response trend for CRTC2 mRNA reduction was observed for most CRTC2 compounds and only a minor, nonsignificant, 10% reduction was seen following treatment with the scrambled control, compound 2. Also noteworthy was that CRTC2 activity did not appear to correlate with elevation in liver enzymes. Compound 1 was utilized to bridge the dose response acute activity studies and multiple dose hepatic safety studies with the increased throughput single dose activity and tolerability screening. As shown in Supplementary Table S3, compound 1 demonstrated robust reduction of CRTC2 mRNA expression and no significant change from vehicle in liver enzyme levels (vehicle mean values were 54, 58, and 14 for ALT, AST, and glutamate dehydrogenase (GLDH), respectively, data not shown). Importantly, this modified strategy was suitable to differentiate CRTC2 compounds, both with respect to activity and hepatoxicity,

FIG. 1. (A) Pyruvate tolerance test in diet induced obese (DIO) mice on day 14: DIO mice were administered the indicated dose of compound (or vehicle) two times weekly for 2 weeks, and following 6-hour fast were given an intrapareteneal dose of pyruvate. Blood glucose measurements were then taken at the indicated times. Symbols represent mean – standard error of the mean (SEM) for eight mice. Significant differences from vehicle treated mice are indicated as *, p < 0.05; and significant differences from mice administered the scrambled negative control compound (Compound 2) as #, p < 0.05, as determined by Wilcoxon pairwise comparison tests. (B) Oral glucose tolerance test in DIO mice on day 28: DIO mice were administered the indicated dose of compound (or vehicle) two times weekly for 4 weeks. C57BL/ 6J mice fed a standard chow diet were given vehicle twice a week for 4 weeks. Following the treatment period, animals were fasted for 6 hours and given an oral glucose solution. Blood glucose measurements were then taken at the indicated times. Symbols within DIO cohorts represent mean – SEM for eight mice, while for the C57BL/6J mice fed standard chow diet the symbols represent mean – SEM for four mice. Significant differences from vehicle treated mice are indicated as *, p < 0.05; significant differences from mice administered the scrambled negative control compound (Compound 2) indicated as #, p < 0.05; and significant differences from mice fed standard chow diet indicated as !, p < 0.05, as determined by Wilcoxon pairwise comparison tests. validating this approach as a means to more efficiently prosecute compounds within an in vivo setting. Efficacy study for compound 1 To evaluate the metabolic consequences of modulating CRTC2 mRNA expression in vivo, two compounds that

CRTC2 ASOs

FIG. 2. Relative cAMP response element binding protein– regulated transcription coactivator 2 (CRTC2) mRNA levels in subcutaneous fat in mice on day 28. DIO mice were administered the indicated dose of compound (or vehicle) two times weekly for 4 weeks. C57BL/6J mice fed standard chow diet were also dosed two times weekly for 4 weeks. Significant differences from vehicle treated mice are indicated as *, p < 0.05; and significant differences from mice administered the scrambled negative control compound (Compound 2) indicated as #, p < 0.05. demonstrated > 75% reduced gene expression and < 10-fold increase in liver enzymes were selected for profiling within the DIO mouse animal model. Following dosing schedules defined previously for liver targets utilizing 2¢methoxy-ethyl (2MOE) gapmer ASOs, compound 1 was administered twice weekly for 28 days (Watts et al., 2005). During this treatment period, non-fasted blood glucose was periodically monitored and by day 10, mice receiving the 10 mg/kg dose of the CRTC2 LNA ASO exhibited significantly lower values compared to saline and the negative control sequence, compound 2, a response that persisted through the duration of the study (data not shown). On study day 14, animals administered the

133 10 mg/kg dose (active and control LNA ASO compounds), as well as the vehicle cohort, were fasted for 6 hours followed by a pyruvate tolerance test. PTT Mice administered the active CRTC2 antisense compound had decreased baseline fasting glucose and reduced gluconeogenic capacity during the PTT compared with vehicle and scrambled control compound cohorts (Fig. 1A). This functional improvement was associated with a > 90% reduction of CRTC2 message levels in the liver (Supplementary Fig. S1) and were independent of changes in food intake or body weight (data not shown). On day 27 of the study, DIO mice receiving the 10 mg/kg dose of antisense compounds and vehicle, as well as vehicle treated lean C57Bl6 mice, were fasted for 6 hours and an oral glucose tolerance test was performed. Consistent with the 2 week findings, baseline fasted glucose was significantly reduced with the CRTC2 compound after 4 weeks of treatment and following glucose load reduced excursion was observed compared with vehicle and control antisense-treated DIO mice (Fig. 2B). Furthermore, this experiment revealed a similar profile between mice given the CRTC2 compound and age-matched lean counterparts indicating a normalization of response upon CRTC2 ‘‘inhibition’’ (Fig. 1B). These effects were once again not attributed to changes in food intake (data not shown) or whole body mass driven (Supplementary Fig. S2). At study conclusion, antisense-mediated effects on the expression of CRTC2 mRNA expression was assessed in liver and subcutaneous fat. In liver there was a dose-responsive reduction in CRTC2 message levels reaching a maximum 97% decrease versus vehicle at the 10 mg/kg dose (data not shown). As an initial attempt to identify a suitable tissue source for clinical biomarker work, the mRNA expression of CRTC2 was measured in subcutaneous fat taken distal to the injection site. Although not as robust as the response seen in the liver, likely attributed to decreased compound exposure, a statistically significant dose-responsive decrease in CRTC2 message expression was found within this clinically assessable fat depot (Fig. 2). Markers of metabolic function were also measured in plasma samples collected after a 6-hour fast prior to study termination and compared to measurements taken under the similar conditions prior to study initiation (Table 3). Although there was no change in whole body mass after the 4-week treatment period, a decrease in the percent body fat accumulated over the course of the experiment in mice on high fat

FIG. 3. Pharmacokinetic-pharmacodynamic (PK/PD) relationship for Compound 1 in mice: antisense oligonucleotide (ASO) exposure and CRTC2 mRNA expression levels in the liver of the DIO mice following 3 weeks of weekly subcutaneous dosing at 10 mg/ kg. Symbols represent mean – standard deviation for five mice.

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diet was observed with the CRTC2 antisense compound at the 10 mg/kg dose compared to both vehicle and compound 2. This finding is consistent with the decreased plasma triglyceride and increased betahydroxybutyrate levels seen at the 10 mg/kg compound 1 dose indicative of reduced hepatic lipid output and enhanced beta oxidation of fatty acids. Given these findings, it is possible that a difference in body weight could be achieved by extending the treatment period. In addition to the improvements in lipid metabolism, treatment with the high dose of CRTC2 antisense compound reduced the increase in circulating insulin associated with the progression of the disease phenotype found in control animals, and the decrease in fructosamine further signifies a general improvement in overall blood glucose control over the time course of the study. These treatment benefits occurred in the apparent absence of mechanistic toxicity, as there were no increases found in circulating lactate with CRTC2 compound 1 treatment. In line with the dose-response relationship seen for the reduction in CRTC2 mRNA expression, intermediate effects in several of the metabolic endpoints were observed following administration of the 3 mg/kg dose (Table 3). To confirm the findings that reduction of CRTC2 gene expression promotes both a decrease in fasted plasma glucose and gluconeogenic capacity, a second CRTC2 LNA antisense oligonucleotide, compound 3, was tested in the DIO mouse model. Here, mice were dosed at 5 and 10 mg/kg at onceweekly intervals for 2 weeks. Six days following the second dose, there was a statistically significant decrease in fasting plasma glucose as well as a reduced glucose excursion during PTT at the 10 mg/kg dose independent of changes in body weight (Supplementary Fig. S3A, B). An intermediate response in fasting plasma glucose (nonsignificant) and PTT (significant at the 15-minute sampling time) was found at the 5 mg/kg dose. Consistent with these results, there was a doseresponsive lowering of hepatic CRTC2 mRNA expression (Supplementary Fig. S4) which, when taken together, independently corroborate the findings obtained for compound 1.

between maximal drug exposure and mRNA knockdown. The accumulation of ASO following the three weekly subcutaneous doses led to maximal mRNA knockdown observed at 24 hours after the third dose. The slow clearance of the ASO from the liver during the recovery phase was in parallel to the slow recovery of CRTC2 mRNA expression levels (Fig. 4), consistent with the mechanism of action for an antisense oligonucleotide. The PK/PD relationship between liver ASO and CRTC2 mRNA expression levels was well defined by an inhibitory Emax model with an EC50 of 412 ng ASO per gram of liver and a maximal effect of 88.1% (Table 4). Glucose output in primary human hepatocytes To begin to assess the translation of inhibiting this signaling mechanism from mouse to human and the downstream therapeutic potential of regulating CRTC2 expression as a treatment for type 2 diabetes, glucose production was monitored in primary human cryopreserved hepatocytes following treatment with the CRTC2 LNA ASO. The HepatoPac platform (Hepregen) was employed in these studies as the technology promotes the longer term viability and metabolic activity of hepatocytes in culture required to allow for ASOmediated mRNA reduction and subsequent effects on functional activity (Wang et al., 2010a). Figures 4A and 4B capture the 7-hour time point of the mRNA expression data. These results serve as a representative example of those observed across the complete time course where treatment with compound 1 demonstrated a statistically significant reduction in CRTC2 mRNA levels of at least 88% versus both vehicle and the scrambled control, compound 2, independent of the presence of glucagon in the media. Furthermore, Fig. 5 shows that the LNA ASO–mediated reduction in CRTC2 gene expression achieved by compound 1 treatment in primary human hepatocytes translated to a statistically significant decrease in glucagon stimulated glucose production at all time points monitored in this study.

PK/PD of compound 1

Discussion

As shown in Fig. 3, the clearance of the ASO from the liver is relatively slow. The elimination half-life was about 13 days, as determined from the recovery phase of the animals following 3 weeks of weekly subcutaneous injection. In contrast to the time of Emax of mRNA knockdown, which was observed at 48 hours after the initial dose, the tissue exposure Tmax was observed at 6 hours, indicating a temporal delay

Insulin resistance within the liver and the associated increase in hepatic glucose output contributes to the chronic hyperglycemia observed in type 2 diabetes (Consoli et al., 1989). Glucagon’s ability to stimulate gluconeogenesis during short-term fasting is thought to be driven through a multiprotein complex consisting of CREB, CBP, TAF4, and CRTC2, which upon nuclear accumulation and subsequent

Table 3. Markers of Metabolic Function Observed in Diet-Induced Obese C57Bl/6J Mice Following 28 Days of Administration with Either Vehicle, Compound 1 or Compound 2 Percent change from study baseline Treatment Vehicle 1 (1 mg/kg) 1 (3 mg/kg) 1 (10 mg/kg) 2 (10 mg/kg)

Body fat

Insulin

Triglycerides

Total cholesterol

Fructosamine

Lactate

BHBA

4.7 – 0.6 4.9 – 1.1 4.7 – 1.0 1.0 – 0.8*# 4.0 – 0.8

85.2 – 52.9 68.9 – 23.3 - 8.7 – 10.6 - 80.2 – 4.1*# 64.8 – 26.7

- 20.5 – 7.3 - 25.3 – 2.6 - 36.9 – 2.7* - 44.5 – 4.0*# - 29.4 – 5.1

16.4 – 6.1 19.7 – 3.7 28.7 – 9.7 36.3 – 8.0 9.9 – 6.3

- 13.8 – 3.4 - 11.9 – 2.3 - 25.2 – 2.8# - 31.3 – 3.2*# - 10.1 – 4.1

- 41.7 – 4.8 - 38.1 – 5.9# - 59.9 – 2.7* - 60.9 – 4.8* - 59.2 – 3.3

- 10.9 – 14.2 7.3 – 17.9 100.9 – 35.9 > 337.0 – 52.0*# 0.0 – 24.8

All values reported are mean – standard error of the mean. Significant differences from vehicle treated mice are indicated as *, p < 0.05, and significant differences from mice administered the scrambled negative control compound, compound 2, as #, p < 0.05. BHBA, beta-hydroxybutyrate.

CRTC2 ASOs

135 Table 4. The Model Parameters Derived from the Pharmacokinetic/Pharmacodynamic Relationship Between Liver Antisense Oligonucleotides Exposure and CRTC2 mRNA Levels Model parameter E0 (%) Emax (%) EC50 (ng/g) n

Estimate – SE 100 fixed 88.1 – 3.26 412 – 47.2 2.09 – 0.4

E0, baseline mRNA levels; Emax, maximal pharmacological effect; EC50, liver antisense oligonucleotides concentration for half-maximal effect; n, hill coefficient.

phorylation (through inactivation of SIK2 by PKA) and acetylation (via CBP/p300) promote a nuclear distribution while phosphorylation (by SIK family kinases and AMPK), as well as deactylation (through SIRT1), inactivate CRTC2 coordinating its nuclear exclusion and subsequent ubiquitin-mediated degradation (Dentin et al., 2007; Liu et al., 2008). Interestingly, in hyperglycemic mouse models where flux through the hexosamine pathway is increased, CRTC2 is Oglycoslytated at the same serine residues required for its degradation leading to nuclear sequestration and enhanced hepatic glucose production (Dentin et al., 2008). These findings provide the framework to investigate the therapeutic potential of ‘‘inhibiting’’ CRTC2 as a strategy to reduce hepatic glucose output associated with type 2 diabetes. The screening process for selecting ASOs for use as in vivo target validation tools is described along with the data generated. Noteworthy within this early phase of testing is that the in vitro response of the top 13 sequences (excluding the negative compound, 2) did not translate to in vivo activity, as

FIG. 4. CRTC2 mRNA levels in primary human hepatocytes cultured in the presence (A) and absence (B) of glucagon. Cells were transfected with 10 mM of the indicated locked nucleic acid (LNA) ASO compound (or vehicle) for 4 days followed by a 2-day incubation in glycogen depletion media. Plates were then incubated in glucose production media in the presence or absence of glucagon and samples collected at 1-, 2-, 4-, 7-, and 20-hour intervals to assess CRTC2 mRNA levels and glucose output. Shown here is the 7-hour time point, which is representative of the results obtained at across the experiment. Bars represent mean – SEM for four treatment wells that were subsequently pooled and run as triplicate real-time quantitative polymerase chain reaction reactions. Significant differences from vehicle are indicated as *, p < 0.05; and significant differences from the scrambled negative control compound (Compound 2) indicated as #, p < 0.05. association at cAMP response elements (CREs) induce the transcription of enzymes essential for the production of glucose including PEPCK and G6Pase (Ravnskjaer et al., 2007). The intracellular location of multiple proteins within this complex including CRTC2 mediates its ability to induce a transcriptional response. Post-translation modifications are largely responsible for functional activity where dephos-

FIG. 5. Glucose output measured in primary human hepatocytes : conditioned media from cells were collected and glucose levels were determined by measurement of glucose oxidase activity at 1, 2, 4, 7, and 20 hours following treatment with the indicated LNA ASO or vehicle. Bars represent mean – SEM for at least three treatment wells. Significant differences from vehicle - glucagon are indicated as *, p < 0.05, significant differences from vehicle + glucagon are indicated as + , p < 0.05, and significant differences from the scrambled negative control compound, Compound 2, indicated as #, p < 0.05.

136 only 4 of 13 yielded greater than a 50% reduction in CRTC2 message levels following a single 10 mg/kg dose. The apparent absence of an in vitro to in vivo correlation might be explained by the discrepancy in uptake and subcellular distribution mechanisms involved in various in vitro systems compared to ASO disposition processes within hepatocytes in vivo. (Stein et al., 2010; Koller et al., 2011). As discussed above, these preliminary in vivo studies also included an assessment of liver toxicity as a second means to triage compounds for further pharmacodynamic evaluation. This characterization was performed at a reasonable throughput relying entirely on measurement of liver enzymes as we determined that elevation of these enzymes predicted downstream histological changes according to preliminary screening data (not shown). Importantly, we observed no correlation with a compound’s activity and effects on liver enzymes indicating that reduction in CRTC2 should not have a deleterious effect on liver function. Applying an election threshold of < 10 · increase in liver enzymes relative to the negative control compound further eliminated two ‘‘active’’ sequences and, as such, compounds 1 and 3 were chosen for further in vivo functional characterization. Treatment with these compounds in high fat diet fed mice reduced hepatic gluconeogenic capacity by pyruvate tolerance testing and normalized oral glucose tolerance to a profile similar to that of standard chow fed animals independent of food intake or body weight. Also apparent was a decrease in plasma insulin levels perhaps indicating improved hepatic insulin selectivity. These findings are consistent with earlier reports where both a liposomally formulated liver-targeted CRTC2 siRNA in diabetic rodent models and CRTC2 knockout mouse exhibited decreased hyperglycemia and enhanced both hepatic and peripheral insulin sensitivity (Saberi et al., 2009; Wang et al., 2010b). Additional agreement was observed for the DIO LNA ASO treated mice and CRTC2 knockout mice fed normal chow with respect to a reduction in circulating triglycerides. Interestingly, the high fat diet fed ASO treated mice also demonstrated increased circulating serum beta-hydroxybutyrate and absolute terminal total plasma cholesterol levels while the normal chow knockout animals did not differ in these endpoints relative to their wild-type cohorts. This disparity might simply reflect a difference in diet; however, our findings also suggest a possible diversion of carbon flux from very low density lipoprotein (VLDL) production to both beta oxidation and cholesterol biosynthesis. The former is a desirable consequence, while the latter is a response that needs to be explored further to determine whether this is a rodent-specific phenomenon driven by differences in lipoprotein profiles and lipid remodeling enzymes compared with human. Importantly, and consistent with these earlier studies, no evidence of mechanism-related toxicity such as hypoglycemia or lactic acidosis was observed. Upon confirming the beneficial role of CRTC2 ‘‘inhibition’’ utilizing an ASO strategy in rodent, efforts were focused on developing a framework for future monitoring of the PK/PD relationship in clinical trials as well as understanding the conservation of the pharmacology in a human system. ASOs including those of a LNA gapmer design have a fairly predictable plasma distribution profile following subcutaneous administration where there is rapid distribution out of the plasma compartment into tissues followed by a prolonged terminal elimination phase where a concentration equilibrium

DULLEA ET AL. is established between liver and plasma (Geary et al., 2007; Yu et al., 2007; Yu et al., 2009). As a means to understand the underlying processes governing the exposure/pharmacodynamic effect relationship in the liver, we constructed a PK/PD model in DIO mice as a surrogate for predicting response in type 2 diabetes. The model drew upon information collected through a kinetic study of whole liver tissue exposure and CRTC2 mRNA levels and established an EC50 of 412 – 47 ng/g for compound 1. A key component to understanding whether or not the mechanism has actually been tested in the clinic is to establish a biomarker that is as proximal to the compound target interaction as possible. As monitoring the binding interaction between ASO and mRNA is not technically feasible and obtaining liver biopsy samples in a clinical setting is challenging from a consensual basis, we assessed whether compound activity could be monitored in subcutaneous fat samples obtained from high fat diet fed mice. Although a lower response when compared with liver was observed there was a significant dose-dependent reduction in CRTC2 mRNA levels within this fat depot following a subchronic dosing paradigm. Taken together, these studies provide an outline towards assessing and understanding downstream clinical data. Confidence in preclinical model translation to the human condition and response is a particularly challenging exercise and the inability to understand this link at an early stage likely contributes to a considerable percentage of phase 2 compound attrition. In the absence of any human genetics information demonstrating a causal relationship of CRTC2 with hyperglycemia, we attempted to determine the importance of the glucagon-mediated CRTC2 driven glucose production in primary cryopreserved human hepatocytes. A common hurdle inherent in marrying primary cell culture and oligonucleotides is the time necessary to reduce gene and ultimately protein expression while maintaining the viability and functionality of these cells. The Hepregen platform utilizes a fibroblast coculture system and has demonstrated drug metabolism, canicular transport properties as well as functional response in primary human hepatocytes for multiple weeks in culture (Khetani and Bhatia, 2008). Incorporating this in vitro technology demonstrated that LNA ASO CRTC2–mediated mRNA reduction comparable to that achieved within an in vivo diabetic mouse model is capable of significantly reducing glucagon-mediated glucose production. To our understanding, this is the first example illustrating target-mediated pharmacology for CRTC2 within a human system and provides additional confidence in rationale for pursuing its reduction as a therapeutic approach for treating type 2 diabetes. While there are multiple options for target validation, the studies presented here demonstrate the effectiveness of antisense gapmers for targets in the liver. Unlike siRNA, short ASOs do not require the use of transfection agents or hydrodynamic injection for cell entry, both methods that can confound the interpretation of in vivo studies. While a large number of ASOs were investigated in vivo for this target, this was partly done to develop an understanding of the behavior and toxicity of this modality and hopefully limit the number of in vivo studies necessary for future gene targets. As an example of this learning process, the screening paradigms used to evaluate compounds for early signs of hepatotoxicity were changed following the characterization of an initial set of

CRTC2 ASOs compounds. These data indicated that the majority of compounds with hepatotoxicity liabilities could be identified by liver function test elevations after a single acute dose rather than requiring a 2-week multidose evaluation. While the shorter protocol is an improvement saving time and resources, the true goal remains a mechanistic understanding of the root cause of the ASO-induced hepatotoxicity and the development of in vitro assays or in silico filters to eliminate such compounds from the initial design. In conclusion, CRTC2 ASOs have been generated and shown to have dose-dependent mRNA activity and efficacy in a 4-week model run in DIO mice. For the first time, the translation to human cells has been demonstrated with primary human hepatocyte data and a clinical biomarker identified. The testing funnel and associated data is presented describing the process of moving from a gene sequence with a reported function to having in vivo tool compounds. While the compounds do not have an adequate therapeutic index for further development, they should facilitate additional studies of CRTC2 and may form the basis for future development of antisense-based therapeutics. Acknowledgments The authors would like to thank Hepregen, Vinicus Bonato, Marc Roy, Deb Moshinsky, and Lishan Chen for their contributions. Author Disclosure Statement No competing financial interests exist. References BIJSTERBOSCH, M.K., MANOHARAN, M., RUMP, E.T., DE VRUEH, R.L.A., VAN VEGHEL, R., TIVEL, K.L., BIESSEN, E.A.L., BENNETT, C.F., COOK, P.D., and VAN BERKEL, T.J.C. (1997). In vivo fate of phosphorothioate antisense oligodeoxynucleotides: predominant uptake by scavenger receptors on endothelial liver cells. Nucleic Acids Res. 25, 3290– 3296. CANETTIERI, G., KOO, S., BERDEAUX, R., HEREDIA, J., HEDRICK, S., ZHANG, X., and MONTMINY, M.R. (2005). Dual role of the coactivator TORC2 in modulating hepatic glucose output and insulin signaling. Cell Metab. 2, 331–338. CONSOLI, A., NURJHAN, N., CAPANI, F., and GERICH, J. (1989). Predominant role of gluconeogenesis in increased hepatic glucose production in NIDDM. Diabetes 38, 550–557. CROOKE, R.M., GRAHAM, M.J., COOKE, M.E., and CROOKE, S.T. (1995). In vitro pharmacokinetics of phosphorothioate antisense oligonucleotides. J Pharmacol. Exp. Ther. 275, 462–473. DENTIN, R., HEDRICK, S., XIE, J., YATES, J., and MONTMINY, M.R. (2008). Hepatic glucose sensing via the CREB coactivator CRTC2. Science 319, 1402–1405. DENTIN, R., LIU, Y., KOO, S.-H., HEDRICK, S., VARGAS, T., HEREDIA, J., YATES, J., and MONTMINY, M. (2007). Insulin modulates gluconeogenesis by inhibition of the coactivator TORC2. Nature 449, 366–369. FRIEDEN, M., and ØRUM, H. (2008). Locked nucleic acid holds promise in the treatment of cancer. Curr. Pharm. Des.14, 1138– 1142. GEARY, R.S., YU, R., SIWKOWSKI, A., and LEVIN, A.A. (2007). Pharmacokinetic/pharmacodynamic properties of phosphor-

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Address correspondence to: Robert Stanton, PhD Worldwide Medicinal Chemistry Pfizer 620 Memorial Drive Cambridge, MA 02139 E-mail: [email protected] Submitted for publication September 9, 2013; accepted after revision November 14, 2013.