Rosanne M. Crooke

Trimethylamine-N-oxide, a metabolite associated with atherosclerosis, exhibits complex genetic and dietary regulation.

Circulating trimethylamine-N-oxide (TMAO) levels are strongly associated with atherosclerosis. We now examine genetic, dietary, and hormonal factors regulating TMAO levels. We demonstrate that two flavin mono-oxygenase family members, FMO1 and FMO3, oxidize trimethylamine (TMA), derived from gut flora metabolism of choline, to TMAO. Further, we show that FMO3 exhibits 10-fold higher specific activity than FMO1. FMO3 overexpression in mice significantly increases plasma TMAO levels while silencing FMO3 decreases TMAO levels. In both humans and mice, hepatic FMO3 expression is reduced in males compared to females. In mice, this reduction in FMO3 expression is due primarily to downregulation by androgens. FMO3 expression is induced by dietary bile acids by a mechanism that involves the farnesoid X receptor (FXR), a bile acid-activated nuclear receptor. Analysis of natural genetic variation among inbred strains of mice indicates that FMO3 and TMAO are significantly correlated, and TMAO levels explain 11% of the variation in atherosclerosis.

Antisense inhibition of proprotein convertase subtilisin/kexin type 9 reduces serum LDL in hyperlipidemic mice

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of a family of proteases that is thought to promote the degradation of the low density lipoprotein receptor (LDLR) through an as yet undefined mechanism. We developed second generation antisense oligonucleotide (ASO) inhibitors targeting murine PCSK9 to determine their potential as lipid-lowering agents. Administration of a PCSK9 ASO to high fat-fed mice for 6 weeks reduced total cholesterol and LDL by 53% and 38%, respectively. Moreover, inhibition of PCSK9 expression resulted in a 2-fold increase in hepatic LDLR protein levels. This phenotype closely resembles that reported previously in Pcsk9-deficient mice. The absence of cholesterol lowering in Ldlr-deficient mice effectively demonstrated a critical role for this receptor in mediating the lipid-lowering effects of PCSK9 inhibition. Antisense inhibition of PCSK9 is an attractive and novel therapeutic approach for treating hypercholesterolemia in human.

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

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.

Antisense Inhibition of Apolipoprotein C-III in Patients with Hypertriglyceridemia

BACKGROUND Apolipoprotein C-III (APOC3) is a key regulator of plasma triglyceride levels. Elevated triglyceride levels are associated with a risk of adverse cardiovascular events and pancreatitis. ISIS 304801 is a second-generation antisense inhibitor of APOC3 synthesis. METHODS We conducted a randomized, double-blind, placebo-controlled, dose-ranging, phase 2 study to evaluate ISIS 304801 in untreated patients with fasting triglyceride levels between 350 mg per deciliter (4.0 mmol per liter) and 2000 mg per deciliter (22.6 mmol per liter) (ISIS 304801 monotherapy cohort), as well as in patients receiving stable fibrate therapy who had fasting triglyceride levels between 225 mg per deciliter (2.5 mmol per liter) and 2000 mg per deciliter (ISIS 304801-fibrate cohort). Eligible patients were randomly assigned to receive either ISIS 304801, at doses ranging from 100 to 300 mg, or placebo, once weekly for 13 weeks. The primary outcome was the percentage change in APOC3 level from baseline. RESULTS A total of 57 patients were treated in the ISIS 304801 monotherapy cohort (41 received active agent, and 16 received placebo), and 28 patients were treated in the ISIS 304801-fibrate cohort (20 received active agent, and 8 received placebo). The mean (±SD) baseline triglyceride levels in the two cohorts were 581±291 mg per deciliter (6.6±3.3 mmol per liter) and 376±188 mg per deciliter (4.2±2.1 mmol per liter), respectively. Treatment with ISIS 304801 resulted in dose-dependent and prolonged decreases in plasma APOC3 levels when the drug was administered as a single agent (decreases of 40.0±32.0% in the 100-mg group, 63.8±22.3% in the 200-mg group, and 79.6±9.3% in the 300-mg group, vs. an increase of 4.2±41.7% in the placebo group) and when it was administered as an add-on to fibrates (decreases of 60.2±12.5% in the 200-mg group and 70.9±13.0% in the 300-mg group, vs. a decrease of 2.2±25.2% in the placebo group). Concordant reductions of 31.3 to 70.9% were observed in triglyceride levels. No safety concerns were identified in this short-term study. CONCLUSIONS We found that treatment with ISIS 304801 was associated with significant lowering of triglyceride levels, among patients with a broad range of baseline levels, through selective antisense inhibition of APOC3 synthesis. (Funded by Isis Pharmaceuticals; ClinicalTrials.gov number, NCT01529424.).

Antisense therapy targeting apolipoprotein(a): a randomised, double-blind, placebo-controlled phase 1 study

BACKGROUND Lipoprotein(a) (Lp[a]) is a risk factor for cardiovascular disease and calcific aortic valve stenosis. No effective therapies to lower plasma Lp(a) concentrations exist. We have assessed the safety, pharmacokinetics, and pharmacodynamics of ISIS-APO(a)Rx, a second-generation antisense drug designed to reduce the synthesis of apolipoprotein(a) (apo[a]) in the liver. METHODS In this randomised, double-blind, placebo-controlled, phase 1 study at the PAREXEL Clinical Pharmacology Research Unit (Harrow, Middlesex, UK), we screened for healthy adults aged 18-65 years, with a body-mass index less than 32·0 kg/m(2), and Lp(a) concentration of 25 nmol/L (100 mg/L) or more. Via a randomisation technique, we randomly assigned participants to receive a single subcutaneous injection of ISIS-APO(a)Rx (50 mg, 100 mg, 200 mg, or 400 mg) or placebo (3:1) in the single-dose part of the study or to receive six subcutaneous injections of ISIS-APO(a)Rx (100 mg, 200 mg, or 300 mg, for a total dose exposure of 600 mg, 1200 mg, or 1800 mg) or placebo (4:1) during a 4 week period in the multi-dose part of the study. Participants, investigators, and study staff were masked to the treatment assignment, except for the pharmacist who prepared the ISIS-APO(a)Rx or placebo. The primary efficacy endpoint was the percentage change from baseline in Lp(a) concentration at 30 days in the single-dose cohorts and at 36 days for the multi-dose cohorts. Safety and tolerability was assessed 1 week after last dose and included determination of the incidence, severity, and dose relation of adverse events and changes in laboratory variables, including lipid panel, routine haematology, blood chemistry, urinalysis, coagulation, and complement variables. Other assessments included vital signs, a physical examination, and 12-lead electrocardiograph. This trial is registered with European Clinical Trials Database, number 2012-004909-27. FINDINGS Between Feb 27, 2013, and July 15, 2013, 47 (23%) of 206 screened volunteers were randomly assigned to receive ISIS-APO(a)Rx as a single-dose or multi-dose of ascending concentrations or placebo. In the single-dose study, we assigned three participants to receive 50 mg ISIS-APO(a)Rx, three participants to receive 100 mg ISIS-APO(a)Rx, three participants to receive 200 mg ISIS-APO(a)Rx, three participants to receive 400 mg ISIS-APO(a)Rx, and four participants to receive placebo. All 16 participants completed treatment and follow-up and were included in the pharmacodynamics, pharmacokinetics, and safety analyses. For the multi-dose study, we assigned eight participants to receive six doses of 100 mg ISIS-APO(a)Rx, nine participants to receive six doses of 200 mg ISIS-APO(a)Rx, eight participants to receive six doses of 300 mg ISIS-APO(a)Rx, and six participants to receive six doses of placebo. Whereas single doses of ISIS-APO(a)Rx (50-400 mg) did not decrease Lp(a) concentrations at day 30, six doses of ISIS-APO(a)Rx (100-300 mg) resulted in dose-dependent, mean percentage decreases in plasma Lp(a) concentration of 39·6% from baseline in the 100 mg group (p=0·005), 59·0% in the 200 mg group (p=0·001), and 77·8% in the 300 mg group (p=0·001). Similar reductions were observed in the amount of oxidized phospholipids associated with apolipoprotein B-100 and apolipoprotein(a). Mild injection site reactions were the most common adverse events. INTERPRETATION ISIS-APO(a)Rx results in potent, dose-dependent, selective reductions of plasma Lp(a). The safety and tolerability support continued clinical development of ISIS-APO(a)Rx as a potential therapeutic drug to reduce the risk of cardiovascular disease and calcific aortic valve stenosis in patients with elevated Lp(a) concentration. FUNDING Isis Pharmaceuticals.

Targeting APOC3 in the Familial Chylomicronemia Syndrome

The familial chylomicronemia syndrome is a genetic disorder characterized by severe hypertriglyceridemia and recurrent pancreatitis due to a deficiency in lipoprotein lipase (LPL). Currently, there are no effective therapies except for extreme restriction in the consumption of dietary fat. Apolipoprotein C-III (APOC3) is known to inhibit LPL, although there is also evidence that APOC3 increases the level of plasma triglycerides through an LPL-independent mechanism. We administered an inhibitor of APOC3 messenger RNA (mRNA), called ISIS 304801, to treat three patients with the familial chylomicronemia syndrome and triglyceride levels ranging from 1406 to 2083 mg per deciliter (15.9 to 23.5 mmol per liter). After 13 weeks of study-drug administration, plasma APOC3 levels were reduced by 71 to 90% and triglyceride levels by 56 to 86%. During the study, all patients had a triglyceride level of less than 500 mg per deciliter (5.7 mmol per liter) with treatment. These data support the role of APOC3 as a key regulator of LPL-independent pathways of triglyceride metabolism.

Antisense oligonucleotides targeting apolipoprotein(a) in people with raised lipoprotein(a): two randomised, double-blind, placebo-controlled, dose-ranging trials

BACKGROUND Elevated lipoprotein(a) (Lp[a]) is a highly prevalent (around 20% of people) genetic risk factor for cardiovascular disease and calcific aortic valve stenosis, but no approved specific therapy exists to substantially lower Lp(a) concentrations. We aimed to assess the efficacy, safety, and tolerability of two unique antisense oligonucleotides designed to lower Lp(a) concentrations. METHODS We did two randomised, double-blind, placebo-controlled trials. In a phase 2 trial (done in 13 study centres in Canada, the Netherlands, Germany, Denmark, and the UK), we assessed the effect of IONIS-APO(a)Rx, an oligonucleotide targeting apolipoprotein(a). Participants with elevated Lp(a) concentrations (125-437 nmol/L in cohort A; ≥438 nmol/L in cohort B) were randomly assigned (in a 1:1 ratio in cohort A and in a 4:1 ratio in cohort B) with an interactive response system to escalating-dose subcutaneous IONIS-APO(a)Rx (100 mg, 200 mg, and then 300 mg, once a week for 4 weeks each) or injections of saline placebo, once a week, for 12 weeks. Primary endpoints were mean percentage change in fasting plasma Lp(a) concentration at day 85 or 99 in the per-protocol population (participants who received more than six doses of study drug) and safety and tolerability in the safety population. In a phase 1/2a first-in-man trial, we assessed the effect of IONIS-APO(a)-LRx, a ligand-conjugated antisense oligonucleotide designed to be highly and selectively taken up by hepatocytes, at the BioPharma Services phase 1 unit (Toronto, ON, Canada). Healthy volunteers (Lp[a] ≥75 nmol/L) were randomly assigned to receive a single dose of 10-120 mg IONIS-APO(a)LRx subcutaneously in an ascending-dose design or placebo (in a 3:1 ratio; single-ascending-dose phase), or multiple doses of 10 mg, 20 mg, or 40 mg IONIS-APO(a)LRx subcutaneously in an ascending-dose design or placebo (in an 8:2 ratio) at day 1, 3, 5, 8, 15, and 22 (multiple-ascending-dose phase). Primary endpoints were mean percentage change in fasting plasma Lp(a) concentration, safety, and tolerability at day 30 in the single-ascending-dose phase and day 36 in the multiple-ascending-dose phase in participants who were randomised and received at least one dose of study drug. In both trials, the randomised allocation sequence was generated by Ionis Biometrics or external vendor with a permuted-block randomisation method. Participants, investigators, sponsor personnel, and clinical research organisation staff who analysed the data were all masked to the treatment assignments. Both trials are registered with ClinicalTrials.gov, numbers NCT02160899 and NCT02414594. FINDINGS From June 25, 2014, to Nov 18, 2015, we enrolled 64 participants to the phase 2 trial (51 in cohort A and 13 in cohort B). 35 were randomly assigned to IONIS-APO(a)Rx and 29 to placebo. At day 85/99, participants assigned to IONIS-APO(a)Rx had mean Lp(a) reductions of 66·8% (SD 20·6) in cohort A and 71·6% (13·0) in cohort B (both p<0·0001 vs pooled placebo). From April 15, 2015, to Jan 11, 2016, we enrolled 58 healthy volunteers to the phase 1/2a trial of IONIS-APO(a)-LRx. Of 28 participants in the single-ascending-dose phase, three were randomly assigned to 10 mg, three to 20 mg, three to 40 mg, six to 80 mg, six to 120 mg, and seven to placebo. Of 30 participants in the multiple-ascending-dose phase, eight were randomly assigned to 10 mg, eight to 20 mg, eight to 40 mg, and six to placebo. Significant dose-dependent reductions in mean Lp(a) concentrations were noted in all single-dose IONIS-APO(a)-LRx groups at day 30. In the multidose groups, IONIS-APO(a)-LRx resulted in mean reductions in Lp(a) of 66% (SD 21·8) in the 10 mg group, 80% (SD 13·7%) in the 20 mg group, and 92% (6·5) in the 40 mg group (p=0·0007 for all vs placebo) at day 36. Both antisense oligonucleotides were safe. There were two serious adverse events (myocardial infarctions) in the IONIS-APO(a)Rx phase 2 trial, one in the IONIS-APO(a)Rx and one in the placebo group, but neither were thought to be treatment related. 12% of injections with IONIS-APO(a)Rx were associated with injection-site reactions. IONIS-APO(a)-LRx was associated with no injection-site reactions. INTERPRETATION IONIS-APO(a)-LRx is a novel, tolerable, potent therapy to reduce Lp(a) concentrations. IONIS-APO(a)-LRx might mitigate Lp(a)-mediated cardiovascular risk and is being developed for patients with elevated Lp(a) concentrations with existing cardiovascular disease or calcific aortic valve stenosis. FUNDING Ionis Pharmaceuticals.

Antisense Oligonucleotide Directed to Human Apolipoprotein B-100 Reduces Lipoprotein(a) Levels and Oxidized Phospholipids on Human Apolipoprotein B-100 Particles in Lipoprotein(a) Transgenic Mice

Background— Lipoprotein (a) [Lp(a)] is a genetic cardiovascular risk factor that preferentially binds oxidized phospholipids (OxPL) in plasma. There is a lack of therapeutic agents that reduce plasma Lp(a) levels. Methods and Results— Transgenic mice overexpressing human apolipoprotein B-100 (h-apoB-100 [h-apoB mice]) or h-apoB-100 plus human apo(a) to generate genuine Lp(a) particles [Lp(a) mice] were treated with the antisense oligonucleotide mipomersen directed to h-apoB-100 mRNA or control antisense oligonucleotide for 11 weeks by intraperitoneal injection. Mice were then followed up for an additional 10 weeks off therapy. Lp(a) levels [apo(a) bound to apoB-100] and apo(a) levels [“free” apo(a) plus apo(a) bound to apoB-100] were measured by chemiluminescent enzyme-linked immunoassay and commercial assays, respectively. The content of OxPL on h-apoB-100 particles (OxPL/h-apoB) was measured by capturing h-apoB-100 in microtiter wells and detecting OxPL by antibody E06. As expected, mipomersen significantly reduced plasma h-apoB-100 levels in both groups of mice. In Lp(a) mice, mipomersen significantly reduced Lp(a) levels by ≈75% compared with baseline (P<0.0001) but had no effect on apo(a) levels or hepatic apo(a) mRNA expression. OxPL/h-apoB levels were much higher at baseline in Lp(a) mice compared with h-ApoB mice (P<0.0001) but decreased in a time-dependent fashion with mipomersen. There was no effect of the control antisense oligonucleotide on lipoprotein levels or oxidative parameters. Conclusions— Mipomersen significantly reduced Lp(a) and OxPL/apoB levels in Lp(a) mice. The present study demonstrates that h-apoB-100 is a limiting factor in Lp(a) particle synthesis in this Lp(a) transgenic model. If applicable to humans, mipomersen may represent a novel therapeutic approach to reducing Lp(a) levels and their associated OxPL.

The TMAO-Generating Enzyme Flavin Monooxygenase 3 Is a Central Regulator of Cholesterol Balance

Circulating levels of the gut microbe-derived metabolite trimethylamine-N-oxide (TMAO) have recently been linked to cardiovascular disease (CVD) risk. Here, we performed transcriptional profiling in mouse models of altered reverse cholesterol transport (RCT) and serendipitously identified the TMAO-generating enzyme flavin monooxygenase 3 (FMO3) as a powerful modifier of cholesterol metabolism and RCT. Knockdown of FMO3 in cholesterol-fed mice alters biliary lipid secretion, blunts intestinal cholesterol absorption, and limits the production of hepatic oxysterols and cholesteryl esters. Furthermore, FMO3 knockdown stimulates basal and liver X receptor (LXR)-stimulated macrophage RCT, thereby improving cholesterol balance. Conversely, FMO3 knockdown exacerbates hepatic endoplasmic reticulum (ER) stress and inflammation in part by decreasing hepatic oxysterol levels and subsequent LXR activation. FMO3 is thus identified as a central integrator of hepatic cholesterol and triacylglycerol metabolism, inflammation, and ER stress. These studies suggest that the gut microbiota-driven TMA/FMO3/TMAO pathway is a key regulator of lipid metabolism and inflammation.

Aberrant Hepatic Expression of PPARγ2 Stimulates Hepatic Lipogenesis in a Mouse Model of Obesity, Insulin Resistance, Dyslipidemia, and Hepatic Steatosis

Insulin-resistant apoB/BATless mice have hypertriglyceridemia because of increased assembly and secretion of very low density apolipoprotein B (apoB) and triglycerides compared with mice expressing only apoB (Siri, P., Candela, N., Ko, C., Zhang, Y., Eusufzai, S., Ginsberg, H. N., and Huang, L. S. (2001) J. Biol. Chem. 276, 46064-46072). Despite increased very low density lipoprotein secretion, apoB/BATless mice have fatty livers. We found that hepatic mRNA levels of key lipogenic enzymes, acetyl-CoA carboxylase, fatty-acid synthase, and stearoyl-CoA desaturase-1 were increased in apoB/BATless mice compared with levels in apoB mice, suggesting increased lipogenesis in apoB/BATless mice. This was confirmed by determining incorporation of tritiated water into fatty acids. Neither the hepatic mRNA of the lipogenic transcription factor, SREBP-1c (sterol-response element-binding protein 1c), nor the nuclear levels of the mature form of SREBP-1 protein were elevated in apoB/BATless mice. By contrast, hepatic levels of peroxisomal proliferator-activated receptor 2 (PPARγ2) mRNA and protein were specifically increased in apoB/BATless mice, as were hepatic mRNA levels of two targets of PPARγ, CD36 and aP2. Treatment of apoB/BATless mice for 4 weeks with intraperitoneal injections of a PPARγ antisense oligonucleotide resulted in dramatic reductions of both PPARγ1 and PPARγ2 mRNA, PPARγ2 protein, and mRNA levels of fatty-acid synthase and acetyl-CoA carboxylase. These changes were associated with decreased hepatic de novo lipogenesis and hepatic triglyceride concentrations. We conclude that hepatic steatosis in apoB/BATless mice is associated with elevated rates of hepatic lipogenesis that are linked directly to increased hepatic expression of PPARγ2. The mechanism whereby hepatic Pparγ2 gene expression is increased and how PPARγ2 stimulates lipogenesis is under investigation.