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Antisense Oligonucleotide Inhibition of Apolipoprotein C-III Reduces Plasma Triglycerides in Rodents, Nonhuman Primates, and Humans

Originally publishedhttps://doi.org/10.1161/CIRCRESAHA.111.300367Circulation Research. 2013;112:1479–1490

    Abstract

    Rationale:

    Elevated plasma triglyceride levels have been recognized as a risk factor for the development of coronary heart disease. Apolipoprotein C-III (apoC-III) represents both an independent risk factor and a key regulatory factor of plasma triglyceride concentrations. Furthermore, elevated apoC-III levels have been associated with metabolic syndrome and type 2 diabetes mellitus. To date, no selective apoC-III therapeutic agent has been evaluated in the clinic.

    Objective:

    To test the hypothesis that selective inhibition of apoC-III with antisense drugs in preclinical models and in healthy volunteers would reduce plasma apoC-III and triglyceride levels.

    Methods and Results:

    Rodent- and human-specific second-generation antisense oligonucleotides were identified and evaluated in preclinical models, including rats, mice, human apoC-III transgenic mice, and nonhuman primates. We demonstrated the selective reduction of both apoC-III and triglyceride in all preclinical pharmacological evaluations. We also showed that inhibition of apoC-III was well tolerated and not associated with increased liver triglyceride deposition or hepatotoxicity. A double-blind, placebo-controlled, phase I clinical study was performed in healthy subjects. Administration of the human apoC-III antisense drug resulted in dose-dependent reductions in plasma apoC-III, concomitant lowering of triglyceride levels, and produced no clinically meaningful signals in the safety evaluations.

    Conclusions:

    Antisense inhibition of apoC-III in preclinical models and in a phase I clinical trial with healthy subjects produced potent, selective reductions in plasma apoC-III and triglyceride, 2 known risk factors for cardiovascular disease. This compelling pharmacological profile supports further clinical investigations in hypertriglyceridemic subjects.

    Introduction

    Hypertriglyceridemia has been recognized by the National Cholesterol Education Program Adult Treatment Panel III as an independent risk factor for the development of coronary heart disease (CHD).1 Recent epidemiological studies and meta-analyses have reaffirmed that elevated triglyceride levels are associated with CHD and a greater risk of disease recurrence in patients with stable CHD.2–4 Furthermore, CHD risk is ameliorated when triglyceride levels are reduced.4–6

    In This Issue, see p 1401

    Editorial, see p 1405

    Very high plasma triglyceride levels (>5.7 mmol/L [500 mg/dL]) are also associated with pancreatitis and may account for as much as 10% of all cases of routine acute pancreatitis and 50% of gestational pancreatitis.7,8 Although the risk of pancreatitis is considered significant in any patient with plasma triglyceride levels >11.3 mmol/L (1000 mg/dL), there is also an increased probability of developing the disease associated with even more modest levels.9 The pathogenesis of pancreatitis is still incompletely understood, but thought to be related to the proinflammatory state produced when triglyceride are metabolized by pancreatic lipases.8 Interestingly, elevated triglyceride levels seem to increase the incidence of pancreatitis, but not the severity of attacks.10

    Plasma triglyceride are complex lipids primarily transported on very low–density lipoprotein (VLDL) particles and chylomicrons that are synthesized in the liver and intestine, respectively.11 The plasma triglyceride concentration is a complex polygenic trait, but a variety of genetic determinants have been identified, including apolipoprotein C-III (apoC-III), lipoprotein lipase, and a number of other genes.12–14 ApoC-III genetic variants that enhance apoC-III plasma concentrations were associated with higher plasma triglyceride and an increase in the incidence of nonalcoholic fatty liver disease.15 Conversely, variants that suppress apoC-III levels, as observed in a group of Old Order Amish subjects, were associated with lower triglyceride levels.16 Similar observations have been made with regard to lipoprotein lipase genetic mutations.17–20

    ApoC-III, a key regulator of plasma triglyceride levels, is a 79-aa glycoprotein, synthesized principally in the liver and, to a lesser extent, by the intestine.21,22 Multiple apoC-III protein molecules reside on the surface of apoB-containing lipoproteins and high-density lipoproteins (HDLs), a percentage of which exchange rapidly between these particles. The apoC3 gene is located within the apolipoprotein A-1 and apolipoprotein A-IV gene cluster on chromosome 11q23.23 ApoC-III expression is suppressed by insulin and is induced by glucose via transcriptional regulatory elements in the gene promoter region.24,25 Activation of peroxisome proliferator–activated receptor-α also reduces apoC-III expression, accounting in part for the hypotriglyceridemic action of fibrates.26,27

    Elevated plasma apoC-III protein possesses several proatherogenic properties. ApoC-III is a potent inhibitor of the lipolysis of triglyceride-rich lipoproteins by antagonizing apolipoprotein C-II activation of lipoprotein lipase11,21 and hepatic lipase, which play an important role in both the conversion of VLDL to intermediate-density lipoproteins to low-density lipoprotein (LDL) and in the remodeling of HDL.28,29 It has also been suggested that apoC-III regulates apoB lipoprotein in apoB lipoprotein metabolism by promoting intrahepatic VLDL assembly and secretion,11 reducing triglyceride-rich lipoprotein clearance,28,30 and increasing the formation of atherogenic small dense LDL.31 Additionally, the enrichment of HDL with apoC-III may render a normally protective molecule atherogenic.32 There is also evidence that apoC-III promotes inflammation and endothelial cell dysfunction by enhancing monocytic cell adhesion via increased vascular cell adhesion molecule-1 expression.32–34 Finally, apoC-III levels are increased in type 1 diabetic patients and are also thought to be a cofactor in pancreatic β cell death.4,35 Taken together, these results support the concept that apoC-III is a multifunctional protein that not only regulates the metabolism of triglyceride-rich lipoproteins, but may also contribute to CHD and pathophysiological metabolic states.

    In most, but not all, studies plasma apoC-III levels have been positively associated with CHD risk. In mouse models, genetic ablation of apoC-III had no effect on atherosclerosis36; however, overexpression of human apoC-III in the low density lipoprotein receptor (Ldlr)−/− background significantly increased atherosclerosis.37 In humans, it was recently reported in a prospective study analysis that the risk for fatal or nonfatal myocardial infarction was significantly increased in subjects with apoC-III containing VLDL and LDL.30 Furthermore, the genetic variants that suppress plasma apoC-III levels in the Old Order Amish subjects and Ashkenazi Jew populations also exhibited reduced risk of CHD.16,38

    Although a variety of therapeutic agents reduce triglyceride levels (statins, niacin, fibrates, and ω-3 fatty acids),5,39–43 some patients still cannot meet their recommended triglyceride goals, suggesting a need for more effective drugs.2 Therefore, we postulated that a direct inhibitor of apoC-III, itself an independent CHD risk factor, might provide therapeutic benefit because of its broad regulatory effects on triglyceride, triglyceride-rich lipoproteins, and HDL particle homeostasis. In this article, we describe the identification and characterization of a second-generation antisense drug that selectively reduces apoC-III in transgenic mice, nonhuman primates, and healthy human volunteers. We demonstrate the selective dose-dependent reduction of apoC-III with concomitant triglyceride lowering in multiple preclinical models and species, and in humans. Importantly, we also show that apoC-III reduction is well tolerated and is not associated with increased liver triglyceride accumulation or hepatotoxicity. These results support the advancement of the human apoC-III drug to phase 2 investigations in patients with hypertriglyceridemia.

    Methods

    An expanded methods section can be found in the online Data Supplement Material.

    Oligonucleotides

    A series of chimeric 20-mer phosphorothioate antisense oligonucleotides (ASOs) containing 2′-O-methoxyethyl groups at positions 1 to 5 and 16 to 20 targeted to murine, rat, and human apoC-III mRNA, as well as a control ASO, were synthesized and purified on an automated DNA synthesizer using phosphoramidite chemistry as previously described.44

    Preclinical Pharmacology Models

    An institutional animal care and use committee approved all procedures and protocols for the preclinical pharmacology studies. See the expanded online Data Supplement Material section for detailed descriptions of the ASO sequences, animal strains/models, and of all experiments.

    Phase I Clinical Trial in Healthy Human Volunteers

    A randomized, placebo-controlled, double-blind, ascending dose, phase 1 study was conducted in healthy volunteers to evaluate the safety, pharmacokinetics, and pharmacological effects of the human apoC-III ASO, ISIS 308401, in humans. The study protocol was approved by a central institutional review board (Institutional Review Board Services, Canada) and performed in compliance with the standards of good clinical practice and the Declaration of Helsinki in its revised edition.45 See Online Figure I for diagrams of the single and multiple dose cohort schedules, Online Figure II for the flow of participants through the study, and Online Table I for baseline characteristics of subjects assigned to the multiple dose cohorts.

    Results

    Identification of ISIS 304801 (Human ApoC-III ASO) and Rodent-Specific ApoC-III ASOs

    The apoC3 gene, which is conserved in eukaryotes, is ≈500 base pairs in length, containing 3 introns and 4 exons. The human, rhesus monkey, and cynomolgus monkey genes are highly conserved with ≈93% homology. To identify potential human candidates, ≈350 second-generation 2′-O-methoxyethyl chimeric ASOs were screened against ≈200 sites (Online Figure III). All the ASOs were 18 to 20 nucleotides long, and the majority were of the 5-10-5 design, that is, five 2′-O-methoxyethyl nucleotides at the 5′ end, 10 deoxynucleotides in the center, five 2′-O-methoxyethyl nucleotides at the 3' end, and phosphorothioate substitution throughout.46 Active ASOs were further evaluated in dose–response studies. Microwalks around sites where active ASOs were identified were performed to further delineate the selection of lead candidates. Throughout the process, the use of appropriate control ASOs assured that all of the compounds were highly selective.

    The 26 most potent human ASOs were evaluated in human apoC3 transgenic mice and in a 4-week tolerability study in mice at a high dose (100 mg/kg per week). Based on its significant, dose-dependent reductions in human apoC-III mRNA, protein, and triglyceride in the transgenic model (Figure 1) and an attractive tolerability profile in mice (data not shown), ISIS 308401 was selected for further evaluation in nonhuman primates and the clinic. A similar process involving in vitro and in vivo evaluations47 was used to identify the mouse-(ISIS 440726) and rat-specific (ISIS 353982) apoC-III ASOs.

    Figure 1.

    Figure 1. Representative experiment showing dose-dependent reduction in human apolipoprotein C-III (apoC-III) mRNA, plasma apoC-III protein, and triglyceride (TG) in mice containing the human apoC3 transgene after administration of ISIS 304801, the human apoC-III antisense oligonucleotide (ASO). ISIS 304801 was administered once weekly at 50, 15, 5, and 1.5 mg/kg per week (n=3 per group) via intraperitoneal injection for 2 weeks. Data in A are presented as mean±SD for each analyte as a percentage of saline control values. Data in B present apoC-III plasma protein (mg/dL) and triglyceride (mmol/L) levels in absolute concentration ±SD. *Significant difference from saline cohort using 1-way ANOVA post hoc Tukey multicomparison test (P<0.05).

    ApoC-III ASOs Reduce ApoC-III mRNA and Triglyceride in Rodent Preclinical Models

    To fully explore the spectrum of apoC-III ASO pharmacological effects as a function of different dyslipidemic states, we evaluated a variety of mouse and rat strains, diets, and disease models. Species-specific apoC-III ASOs, identified through the process described above, were administered once weekly over 6 weeks to C57BL/6, Ldlr−/−, ob/ob, and cholesteryl ester transfer protein (CETP) transgenic / Ldlr−/− mice, as well as the fructose-fed and Zucker diabetic fatty rat models. As shown in Table 1, hepatic apoC-III mRNA was reduced by 66% to 98% in all treated animals, with the greatest reduction being observed in CETP transgenic, Ldlr−/− mice. Consistent with effects on apoC-III mRNA, fasting triglyceride levels were suppressed by 19% to 89%, with absolute reductions varying depending on the model and diet. Interestingly, perhaps because of its expression of CETP, the CETP transgenic Ldlr−/− mice were the only rodent preclinical model that showed increases in HDL-cholesterol (Online Table VII). More detailed pharmacological and mechanistic studies were performed in C57BL/6 and Ldlr−/− mice fed either normal chow or a Western diet (WD), and the data will be described below.

    Table 1. ApoC-III mRNA and Plasma Triglyceride Levels in Multiple Preclinical Models After Administration of Species-Specific ApoC-III Antisense Oligonucleotides

    Model/DietHepatic ApoC-IIIPlasma TG, mmol/L*TG (% Change) vs Control ASO)
    mRNA (% Reduction)SalineControl ASOApoC-III ASO
    C57BL/6 mice (chow)−661.01±0.100.88±0.050.71±0.03−19
    C57BL/6 mice (Western diet)−901.02±0.061.14±0.060.64±0.04−44
    Ldlr−/− mice (chow)−701.44±0.061.82±0.121.33±0.07−27
    Ldlr−/− mice (Western diet)−956.98±0.926.97±0.683.97±0.53−43
    Ob/Ob mice (chow)−901.91±0.041.61±0.120.83±0.04−48
    CETP transgenic Ldlr−/− mice (Western diet)−986.50±0.627.17±0.341.35±0.32−79
    Sprague Dawley rats (fructose)§−844.77±0.353.84±0.340.66±0.11−83
    ZDF rat (chow)§−907.20±1.005.74±0.640.66±0.01−89

    ApoC-III indicates apolipoprotein C-III; ASO, antisense oligonucleotide; CETP, cholesteryl ester transfer protein; Ob/Ob mice, obese mice with a leptin deficiency; TG, triglyceride; and ZDF, Zucker diabetic fatty.

    *Values represent mean±SEM. Plasma TG levels were evaluated after a 4-h fast.

    Mice were administered 12.5 mg/kg per wk of ISIS 440726 for 6 wk.

    Denotes significantly different (P<0.05) from control ASO.

    §Rats were administered 25 mg/kg per wk of ISIS 353982 for 6 wk.

    ISIS 440726, a mouse-specific apoC-III ASO (3.1, 6.3, and 12.5 mg/kg per week) and a control ASO (12.5 mg/kg per week) were administered by intraperitoneal injections to wild-type C57BL/6 mice fed either normal chow or a WD for 6 weeks. At the end of treatment, mice were fasted for 5 hours, euthanized, and liver apoC-III mRNA, plasma protein, and other parameters were evaluated (Online Table II). ISIS 440726 produced dose-dependent reductions in hepatic apoC-III mRNA and plasma protein in mice fed either diet (Figure 2A and 2B). Significant reductions in triglyceride were produced at the 12.5 mg/kg per week dose in mice fed normal chow (19%) and at all doses in the WD-fed animals (37%, 50%, and 44% in the 3.1, 6.3, and 12.5 mg/kg per week groups, respectively, Online Table II). Fast protein liquid chromatography analysis demonstrated that the triglyceride loss was primarily in the VLDL fraction (Online Figure VIA). Postprandial triglyceride were also reduced by ≈30% (Figure 2C and 2D). This change in postprandial triglyceride was not because of differences in intestinal triglyceride absorption, because after a poloxamer 407 block and oral gavage of 3H-triolein, appearance of 3H radioactivity in plasma was not different between control ASO and apoC-III ASO-treated mice (Figure 2E). To demonstrate that the activities of the apoC-III ASO resulted from target-specific inhibition, the pharmacology of the murine-specific ASO was compared in wild-type chow-fed C57BL/6 and apoC-III−/− mice. Six weeks of treatment with the mouse apoC-III ASO (12.5 mg/kg per week) reduced liver apoC-III mRNA to levels similar to those observed in the whole body apoC3−/− mice (data not shown). ISIS 440726 significantly (P<0.05) reduced fasting plasma triglyceride from 0.95±0.06 mmol/L (84±5 mg/dL) in saline controls to 0.79 mmol/L (70.3 mg/dL) in the wild-type mice. Treatment of apoC-III−/− mice with the apoC-III ASO failed to produce reductions in fasting plasma triglyceride (0.64±0.08 mmol/L [56±7 mg/dL] in saline versus 0.70±0.01 mmol/L [62±1 mg/dL]), supporting the notion that the pharmacological effects observed in other mouse models were attributable to reductions in apoC-III protein.

    Figure 2.

    Figure 2. Administration of the murine apolipoprotein C-III (apoC-III) antisense oligonucleotide (ASO; ISIS 440726) leads to dose-dependent reductions in hepatic apoC-III mRNA and plasma apoC-III protein, while enhancing postprandial triglyceride clearance. C57BL/6 mice (n=5/group), fed either a chow or Western diet, were administered 12.5, 6.3, or 3.1 mg/kg per week of the murine apoC-III ASO for 6 weeks. A, Hepatic murine apoC-III expression was analyzed by quantitative polymerase chain reaction. B, Western blot analysis of apoC-III protein isolated from chow- and Western diet–fed mice. C, Postprandial triglyceride clearance was quantitated in the 12.5 mg/kg per week cohort by administering a fat bolus to chow-fed C57BL/6 fasted mice and measuring plasma triglyceride concentrations every hour for 4 hours. D, The mean area under the curve (AUC) was significantly reduced in apoC-III ASO-treated mice after the fat challenge. E, Postprandial TG absorption was quantitated in the 12.5 mg/kg per week cohort by first administering poloxamer 407 to chow-fed C57BL/6 fasted mice. One hour later, a 3H-triolein bolus was administered via oral gavage, and counts per minute was quantitated in plasma at 90 and 180 minutes after gavage. *Significant difference (P<0.05) from control ASO in chow-fed C56BL/6 mice; †Significant difference (P<0.05) from control ASO in Western diet–fed C57BL/6 mice.

    Because previous publications suggested that apoC-III plays a role in VLDL secretion, we next assessed the effects of apoC-III reduction on VLDL and triglyceride export from the livers of chow- and WD-fed C67BL/6 and apoC3−/− mice administered 12.5 mg/kg/wk for 6 weeks.48 Secretion of triglyceride, as assessed by a poloxamer 407 block, was unaffected by apoC-III ASO treatment (Figure 3A and 3B). As shown in Figure 3C, although apoC-III ASO treatment tended to increase liver triglyceride levels in this mouse model, those changes did not achieve statistical significance.

    Figure 3.

    Figure 3. Administration of the murine apolipoprotein C-III (apoC-III) antisense oligonucleotide (ASO) to chow- and Western diet–fed C57BL/6 mice did not affect the secretion of triglyceride from the liver nor cause hepatic steatosis. C57BL/6 mice (n=4–5/group) fed chow (A), or Western diet (B) were administered 12.5 mg/kg per week of the murine apoC-III ASO for 6 weeks. Hepatic triglyceride secretion in saline, control ASO, and apoC-III ASO groups was compared with that observed in apoC3−/− mice over a 4-hour period by measuring triglyceride accumulation in plasma (mmol/L) after injection of poloxamer 407. C, Liver triglyceride levels in chow- (n=4–5/group) and Western diet–fed C57BL/6 (n=5–13/group) mice after administration of the control and apoC-III ASOs compared with untreated saline and apoC3−/− mice. *Significant difference (P<0.05) from control ASO in Western diet–fed C57BL/6 mice. ΨSignificant difference (P<0.05) from saline in Western diet–fed C57BL/6 mice.

    Because the apoC-III ASO seemed to reduce postprandial triglyceride to a lesser extent than that observed in the apoC3−/− mice (Online Figure IVB), we speculated that this could be attributable to insufficient intestinal apoC-III suppression. Online Figure IVA shows that, as expected, there was less distribution of ASOs to the gut than in liver, thus the apoC-III ASO had a less profound effect on apoC-III mRNA levels in the intestine and on fat clearance. In contrast, in the apoC-III−/− mice, where apoC-III was absent in the intestine (Online Figure IVB), fat clearance was more rapid.

    Effects of ApoC-III Reduction in Ldlr−/− Mice

    To determine the effects of apoC-III reduction in a model of mixed dyslipidemia, we used Ldlr−/− mice fed chow and WDs. Again, we treated these mice for 6 weeks with different doses of the mouse-specific apoC-III ASO. As observed in previously described mouse models, apoC-III mRNA and plasma protein were reduced in a dose-dependent fashion (data not shown). The potency of the drug, effects on plasma lipids, and tolerability measures (Online Table III, Online Figure VIB) approximated those observed in chow- and WD-fed C57BL/6 mice.

    Because both VLDL and LDL particle number49 and composition28,50 have been suggested to be influenced by the presence of apoC-III, we determined the effects of ASO treatment on those parameters. ApoC-III protein and triglyceride were reduced in VLDL particles, and an increase in cholesteryl esters was observed at the highest ASO dose (Figure 4A). Except for the absence of apoC-III protein, LDL particle composition was unaffected (data not shown). We also evaluated the effects of the mouse-specific apoC-III ASO on apoB-containing lipoprotein particle size and demonstrated that there was a reduction in large VLDL and chylomicron particles and a modest reduction in small VLDL particles (Figure 4B).

    Figure 4.

    Figure 4. Administration of the murine apolipoprotein C-III (apoC-III) antisense oligonucleotide (ASO) to Western diet–fed Ldlr−/−mice modifies very low–density lipoprotein (VLDL) lipid composition and particle number. A, VLDL particles were isolated by ultracentrifugation from Western diet–fed Ldlr−/− mice that were administered 12.5 mg/kg per week of a control ASO or 12.5 or 3.1 mg/kg per week of the murine apoC-III ASO for 6 weeks. Lipid was quantitated by colorimetric assay, whereas VLDL-associated apoC-III protein was analyzed by Western blot. B, VLDL particle concentration and size from Western diet–fed Ldlr−/− mice that were administered 12.5 mg/kg per week of a control ASO or 12.5, 6.25, and 3.1 mg/kg per week of the murine apoC-III ASO for 6 weeks were quantitated by nuclear magnetic resonance as described in Materials and Methods. *Significantly different (P<0.05) from control ASO.

    ISIS 304801 (Human ApoC-III ASO) Reduces ApoC-III mRNA, Protein, and Triglyceride in Chow-Fed Cynomolgus and Hypertriglyceridemic Rhesus Monkeys

    Because the binding site for the human apoC-III ASO, ISIS 304801, is 100% conserved between cynomolgus and rhesus monkeys, and lipid metabolism in nonhuman primates resembles that observed in humans, we evaluated the effects of that drug on hepatic apoC-III mRNA, plasma apoC-III protein, and triglyceride in monkeys. When a 13-week pilot study (data not shown) was performed in chow-fed cynomolgus monkeys, ISIS 304801 (administered at doses of 4, 8, 12, and 40 mg/kg per week) reduced apoC-III mRNA in a dose-responsive fashion by 47%, 51%, 80%, and 89%, respectively, with a calculated ED50 for apoC-III protein of 6.3 mg/kg per week. Because these animals had very low triglyceride levels, another study was performed in a rhesus monkey model of hypertriglyceridemia.

    Rhesus monkeys fed a normal chow diet were made hypertriglyceridemic via administration of a high-fructose supplement for 16 weeks and then treated with ISIS 304801 (10, 20, and 40 mg/kg per week) for 12 weeks as the high-fructose diet was maintained. With fructose supplementation, plasma triglyceride levels were increased at least 3-fold over initial baseline in all treatment groups (0.49 mmol/L [43 mg/dL] at day 1 versus 1.6 mmol/L [142 mg/dL] at day 112, just before initiation of dosing). As was observed in the normo-lipidemic monkeys described above in the pilot study, ISIS 304801 significantly reduced hepatic apoC-III mRNA (Online Figure VA) and plasma apoC-III protein (Online Figure VB). Because the apoC-III reductions observed at 10 mg/kg per week were similar to results at the higher dose levels, more detailed analyses focused only on the 10 mg/kg per week dosing cohort. Analysis of day 86 samples from the 10 mg/kg per week group revealed that plasma total triglyceride (Figure 5A) and VLDL/chylomicron triglyceride (Figure 5B) were suppressed by 0.30 and 0.29 mmol/L (26.5 and 25.7 mg/dL), respectively, and that HDL levels were increased by 0.21 mmol/L (8.1 mg/dL; Figure 5C). Monkeys administered ISIS 304801 (10 mg/kg per week) for 10 weeks had significantly lower fasting and postprandial plasma triglyceride levels than the PBS control group (Figure 5D, Online Figure VC and Online Table VI). The 38% area under the curve (AUC) reduction in the rhesus monkey postprandial triglyceride excursion was similar to that observed in the C57BL/6 fat clearance study (Figure 2C). Finally, ISIS 304801 did not significantly increase liver triglyceride levels in these animals (Online Figure VD).

    Figure 5.

    Figure 5. The human apolipoprotein C-III (apoC-III) antisense oligonucleotide, ISIS 304801, reduced plasma triglycerides, very low–density lipoprotein (VLDL)+chylomicron triglyceride, and decreased postprandial triglyceride excursion, while increasing high-density lipoprotein-cholesterol (HDL-C) in hypertriglyceridemic rhesus monkeys. Monkeys (n=5 per group) that were made hypertriglyceridemic with fructose supplementation were treated for a total weekly dose of 10 mg/kg per week ISIS 304801 or PBS over 12 weeks and plasma, A, total triglyceride, B, VLDL and chylomicron triglyceride, and C, HDL-C levels were quantified using nuclear magnetic resonance analysis. D, A postprandial triglyceride excursion test was performed in a separate cohort of rhesus monkeys (n=5 per group) that were treated with a total weekly dose of 10 mg/kg per week ISIS 304801 or PBS for 10 weeks. Postprandial mean plasma triglyceride concentrations (mmol/L)±SEM were measured after 0, 1, 2, 3, and 4 hours. Below each of the 4 histograms, a corresponding table provides the mean value for each data point plotted. Data are plotted as mean (mmol/L) change from baseline±SEM. *Significant difference from PBS cohort using 1-way ANOVA post hoc Tukey multicomparison test (P<0.05).

    Tolerability of Species-Specific ApoC-III ASOs in Preclinical Studies

    In addition to specific pharmacological end points, we routinely monitor various factors, including plasma liver transaminases, and other metabolic parameters (ketones as well as liver, spleen, and body weights). In all preclinical rodent and nonhuman primate studies, there were no significant changes in plasma liver transaminases (Online Tables II and III), or other metabolic parameters when compared with controls, indicating that the apoC-III ASO was well tolerated (data not shown). Additionally, our data indicate that there were no statistically significant increases in hepatic triglyceride in mice or monkeys when compared with control animals (Figure 3C, Online Figure VD).

    Pharmacodynamic Evaluations

    Based on the preclinical efficacy data, a phase I double-blind, placebo-controlled, dose escalation clinical study (ISIS 304801-CS1) was performed to assess the safety, tolerability, pharmacokinetics, and pharmacodynamics of ISIS 304801 in healthy human volunteers (see online Data Supplement material for study details). Consistent with preclinical data, ISIS 304801 administered to healthy human volunteers produced dose-dependent and prolonged reductions in apoC-III with concomitant lowering of triglyceride levels (Figures 6A andB, Table 2). For example, median percentage reductions from baseline values in apoC-III of 19.7%, 17.3%, 70.5%, and >77.5% were observed in the 50-, 100-, 200-, and 400-mg multiple dose groups, respectively, 1 week after the last dose (day 29). Consistent with the changes in apoC-III, triglyceride levels were also reduced by 19.5%, 25.0%, 43.1%, and 43.8% in the same dosing cohorts. Despite the small sample size (n=3), the median percentage change from baseline in apoC-III at day 29 was the lowest P value possible at the 200- and 400-mg doses (P=0.0571, versus pooled placebo). At the 400-mg dose, 2 of 3 treated subjects had apoC-III levels that were reduced below the level of assay detection (day 22, 2 subjects; days 23 and 29, 1 subject). Although greater variability in triglyceride levels was observed, the AUC of the percentage change in triglyceride from baseline to day 29 achieved near significance in the 200- and 400-mg dose groups compared with placebo, and a correlation was observed between changes in triglyceride and apoC-III levels (r=0.899). Although the size of the study precludes a definitive conclusion about the effects of the 50 and 100 mg doses, the reductions on apoC-III and triglyceride were substantial. Reductions in apoC-III and triglyceride levels were sustained for at least 4 weeks after the last dose in the higher dose cohorts consistent with the long terminal elimination half-life of the drug.

    Table 2. Dose-Dependent Reduction in ApoC-III and Triglyceride Levels in Healthy Human Volunteers After Short-Term Multiple Dose Treatment With ISIS 304801

    Median Age (%) Change From Baseline 1 wk After Last Dose
    Placebo (n=4)50 mg (n=3)100 mg (n=3)200 mg (n=3)400 mg (n=3)
    ApoC-III−11.0−19.7−17.3−70.5*>−77.5*
    TG28.5−19.5−25.0−43.1−43.8
    HDL-C2.119.00.013.98.0
    LDL-C5.518.4−3.6−3.2−3.9

    ApoC-III indicates apolipoprotein C-III; HDL, high-density lipoprotein; LDL, low-density lipoprotein; and TG, triglyceride.

    Values are the median percentage change from baseline, 1 wk after the last dose of study drug (day 29). The percentage change from baseline for each dose group was compared with pooled placebo using the exact Wilcoxon Rank Sum test.

    *Statistic test result (P=0.0571) was the lowest P value possible for the sample size.

    Figure 6.

    Figure 6. Administration of ISIS 304801 to healthy human volunteers produced dose- and time-dependent reductions in apolipoprotein C-III (apoC-III) and triglycerides levels. Data represent the median percentage change from baseline (BSLN) of A, apoC-III and B, triglyceride levels obtained from healthy volunteers in the phase I trial. Time period ranges from day 1 to day 50. Arrows indicate dosing days.

    Other lipid parameters were also evaluated (Table 2). As expected, LDL values did not change in this small study in normal subjects, whereas HDL levels tended to increase, although these changes were not dose dependent.

    Pharmacokinetic Profile After Single and Multiple Dosing

    Clinical pharmacokinetics were similar to those observed for other drugs of this chemical class.51 ISIS 304801 demonstrated dose-dependent increases in plasma exposure as measured by peak concentrations (Cmax) and AUC0–24. After a single subcutaneous injection (day 1), mean time to maximum plasma concentrations (Tmax) was between 2 and 4 hours, depending on the dose (Online Table IV). Exposure as measured by AUC0–24 also increased as a function of dose. The mean residence time in plasma was relatively short, consistent with the rapid distribution from the plasma into tissues. In the multiple dose cohorts (Online Table V), there was no plasma accumulation after multiple doses, with Cmax and AUC values remaining similar after the first and last doses in each dose group. Plasma elimination half-life values ranged from 11.7 to 31.2 days, with clearance primarily a result of urinary excretion of metabolites.

    Safety and Tolerability

    ISIS 308401 was generally well tolerated. There were no serious adverse events and no early terminations of dosing because of an adverse event. The most common adverse event was mild injection site reaction, a typical response to subcutaneously administered drugs.52 No subject dosed with placebo complained of injection site reactions, although 13 of 25 (52%) subjects dosed with ISIS 304801 experienced at least one. Approximately 1 of 6 injections (median, 17%) led to an injection site reaction, the majority of which resolved within an hour. Single transient increases in C-reactive protein were reported in 7 of 25 (28%) subjects who received ISIS 304801. These increases were dose dependent, ranging from 7 to 29 mg/L, and had no associated symptoms. All other safety evaluations were clinically unremarkable across treatment groups.

    Discussion

    Because of the central role of apoC-III in triglyceride homeostasis and a compelling body of human genetic evidence linking elevated plasma apoC-III levels with hypertriglyceridemia, metabolic syndrome, and proinflammatory conditions,15,16,38 we developed species-specific antisense inhibitors to suppress apoC-III biosynthesis and evaluated their pharmacology in relevant rodent and nonhuman primate dyslipidemia and disease preclinical models and, more recently, in a phase I trial conducted in healthy human subjects. These studies demonstrated that apoC-III inhibition led to a variety of potential cardioprotective effects, including (1) significantly reduced plasma apoC-III and triglyceride, (2) enhanced postprandial triglyceride clearance, (3) favorable effects on VLDL particle composition, and (4) suggestions of enhanced HDL levels.

    Several key observations that were essential for understanding the pharmacology of apoC-III inhibition are derived from rodent and monkey studies, including those performed in the well-characterized murine hypotriglyceridemic apoC3−/− model.53 For example, administration of the species-specific ASO to those mice did not affect their already low plasma triglyceride levels, indicating that the effects of the compound were apoC-III dependent. As described above, apoC-III ASOs also significantly enhanced postprandial triglyceride clearance in mice and nonhuman primates, data consistent with observations in the apoC3−/− mice, and in the Old World Amish possessing the R19X null mutation.16 Interestingly, postprandial clearance was more extensive in apoC3−/− mice, a whole body knockout of the target, compared with apoC-III ASO-treated mice, where suppression is more extensive in liver when compared with the proximal small intestine, where apoC-III plays an important role in chylomicron formation. These data suggest that intestinal apoC-III expression also plays an integral role in plasma triglyceride homeostasis.

    Although the pharmacological outcome of inhibiting apoC-III on triglyceride and VLDL is predictable, the relationship to LDL levels is less clear. Some triglyceride-lowering agents, such as fish oils, have been shown to increase LDL in some instances.54 Although those agents were not evaluated in these studies, it is noteworthy that apoC-III ASO treatment alone did not modulate LDL levels in rodents, hypertriglyceridemic monkeys, and healthy volunteers in the phase I study. There is, however, considerable evidence that apoC-III present on apoB-containing particles is proatherogenic. For example, in the Cholesterol and Recurrent Events trial, it was shown that in patients with coronary artery disease, increased apoC-III concentrations in VLDL+LDL were a significant predictor for a recurrent coronary event.55 Furthermore, increased serum apoC-III concentrations were associated with elevated levels of the small, dense LDL particles.28,55 Additionally, in patients with type 2 diabetes mellitus, apoC-III–enriched LDL particles had increased binding to arterial wall proteoglycans.56 Our data showing the depletion of apoC-III protein from VLDL in Western diet–fed Ldlr−/− mice suggest that antisense inhibition of apoC-III may produce less proinflammatory apoB-containing lipoprotein particles. Further studies in more hypertriglyceridemic states (>5.7 mmol/L [>500 mg/dL]) will be necessary to elucidate the effects of apoC-III inhibition in mixed dyslipidemia.

    The well-established link between high plasma triglyceride and low HDL would suggest that the plasma triglyceride–lowering effects of apoC-III inhibition could lead to increases in HDL. Furthermore, there is evidence that apoC-III bound to HDL particles may produce a dysfunctional state with reduced cardioprotective properties.32 In preclinical models possessing CETP, that is, CETP transgenic Ldlr−/− mice, nonhuman primates, as well as in the phase I clinical trial, apoC-III inhibition led to either significant increases (Online Table VII) or elevation trends (nonhuman primate study and phase I study) in HDL levels. These effects are, once again, consistent with observations in the Old Order Amish.16 It is important to note that both the nonhuman primates and subjects in our clinical trial had normal to modestly elevated triglyceride levels, raising the possibility that the HDL effects of apoC-III inhibition could be more robust in a hypertriglyceridemic state. Furthermore, as HDL increases were not seen in CETP-deficient models, this may also suggest that the HDL raising properties of apoC-III are CETP dependent. Whether these effects are attributable to changes in CETP substrate availability or direct effects on CETP enzymatic activity are currently under investigation.

    In all of the preclinical studies, apoC-III ASOs were well tolerated. In rodent models, there was no change in body weights (data not shown) or plasma transaminases with any apoC-III ASO dose. Although previous studies have indicated that high-fat fed apoC3−/− mice developed hepatic steatosis,53 we did not observe an increase in liver triglyceride in mice or nonhuman primates. The fact that ASO administration does not affect triglyceride secretion is consistent with these observations.

    The human apoC-III antisense drug, ISIS 304801, was well tolerated in healthy human volunteers, with no unexpected safety signals identified across the full dose range tested. The pharmacokinetic and pharmacodynamic profiles were predictable based on the preclinical results and previous preclinical and clinical experience with other ASO drugs in its class.47,51,57,58 A profound dose-dependent reduction of apoC-III up to ≈90% from baseline occurred with an associated dose-dependent reduction in triglyceride up to ≈80% in subjects administered active drug for 4 weeks. This pharmacological response was durable, with median apoC-III levels remaining below baseline for at least 1 month at the higher doses of 200 and 400 mg.

    In conclusion, ASO-mediated suppression of apoC-III has demonstrated robust triglyceride and VLDL lowering across multiple rodent models, nonhuman primates, and man. In addition, in hypertriglyceridemic monkeys and healthy human subjects, apoC-III inhibition seemed to raise HDL levels with no adverse effects on LDL. Based on the favorable preclinical and phase I clinical pharmacodynamics and tolerability profile, the human apoC-III drug has advanced to phase 2 trials. Initial patient populations for an apoC-III antisense drug will focus on individuals with plasma triglyceride levels >5.7 mmol/L (>500 mg/dL) on maximally tolerated first-line agents, such as niacin, fish oil, and fibrates, attributable to their increased risk of acute pancreatitis and cardiovascular event risk. Such subjects might include individuals with lipolysis deficiencies attributable to mutations in lipoprotein lipase,19 apolipoprotein C-II,59 or glycosylphosphatidylinositol-anchored protein of capillary endothelial cells genes.60,61 Future clinical studies will evaluate the potential role of apoC-III in metabolic syndrome, diabetes mellitus, and HDL homeostasis to further broaden the potential therapeutic benefit of this first-in-class apoC-III antisense drug.

    Acknowledgments

    We thank Drs Brett P. Monia and Frank C. Bennett for their thoughtful review of this article. In addition, we also acknowledge Tracy Reigle for her valuable help in formatting all of the figures and tables.

    Nonstandard Abbreviations and Acronyms

    apoC-III

    apolipoprotein C-III

    ASO

    antisense oligonucleotide

    AUC

    area under the curve

    CETP

    cholesteryl ester transfer protein

    CHD

    coronary heart disease

    HDL

    high-density lipoprotein

    LDL

    low-density lipoprotein

    VLDL

    very low–density lipoprotein

    WD

    Western diet

    Footnotes

    In February 2013, the average time from submission to first decision for all original research papers submitted to Circulation Research was 11.98 days.

    *These authors contributed equally.

    The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.111.300367/-/DC1.

    Correspondence to Mark J. Graham, Isis Pharmaceuticals, 2855 Gazelle Court, Carlsbad, CA 92010. E-mail

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    Novelty and Significance

    What Is Known?

    • Elevated triglycerides are an independent risk factor for cardiovascular disease, and very high triglyceride levels (>5.7 mmol/L) are associated with an enhanced risk of pancreatitis.

    • Current therapeutic agents for the treatment of very high triglyceride levels are limited.

    • Apolipoprotein C-III (apoC-III) is synthesized principally in the liver, and it regulates serum triglyceride levels.

    • Loss-of-function variants of apoC-III in a group of Old Order Amish are associated with lower serum triglyceride levels and are cardioprotective.

    What New Information Does This Article Contribute?

    • ApoC-III biosynthesis could be selectively inhibited by antisense oligonucleotides.

    • ApoC-III antisense oligonucleotide treatment produces consistent and significant reductions in serum apoC-III and triglyceride levels in rodents, nonhuman primates, and man.

    • ApoC-III antisense oligonucleotide drugs are well tolerated in preclinical models and in a clinical setting, with no evidence of hepatotoxicity.

    Individuals with very high triglyceride (>5.7 mmol/L) and apoC-III levels are at increased risk for developing cardiovascular disease, metabolic syndrome, diabetes mellitus, and pancreatitis. Although several therapeutic agents affect triglyceride levels, there is an unmet need for more effective therapies. Given the importance of apoC-III in the regulation of triglyceride homeostasis, we developed antisense inhibitors to demonstrate that reduction of apoC-III could produce therapeutic benefit. In preclinical rodent and nonhuman primate models and, most importantly, in man, we demonstrate that selective inhibition of apoC-III is well tolerated, and that it results in significant, prolonged reductions in apoC-III and triglyceride levels, enhanced postprandial triglyceride clearance. The human apoC-III antisense drug may be used as a monotherapy or in combination with other agents, for example, fibrates. Drug–drug interactions are not anticipated because antisense oligonucleotides are metabolized by nucleases rather than by metabolic pathways used by traditional small molecules, such as the cytochrome P450 system. These findings support clinical evaluation of this apoC-III drug in subjects with severely elevated triglyceride levels and type 2 diabetics with moderately elevated triglyceride levels and uncontrolled glucose levels.

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