Walter Singleton

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.

Comparison of the Immediate Effects of Five β-Adrenoreceptor-Blocking Drugs with Different Ancillary Properties in Angina Pectoris

We compared the immediate effects of five beta-adrenoreceptor-blocking agents in 16 patients with stable angina pectoris. Acute dose-response studies showed that all five drugs improved exercise tolerance and reduced ST-segment depression, heart rate and blood pressure by a similar degree in comparison with a placebo (P less than 0.01). Near maximum improvement in exercise tolerance occurred when the acute cumulative oral dose had reached 160 mg for propranolol and oxprenolol, 200 mg for metoprolol and tolamolol and 400 mg for practolol. When these drugs were administered as a single doses, increase in walking time before the development of angina and reduction in ST-segment depression, heart rate and systolic blood pressure all occurred within one hour and persisted for eight hours--effects markedly different from the response to a placebo (P less than 0.01). These data show that non-cardioselective agents (propranolol and oxprenolol), cardioselective agents (practolol, metoprolol and tolamolol), as well as drugs with intrinsic sympathomimetic activity (oxprenolol and practolol), were equally effective in the treatment of angina pectoris.

Comparison of five beta-adrenoreceptor antagonists with different ancillary properties during sustained twice daily therapy in angina pectoris☆

The effects of five beta-adrenoreceptor blocking agents and placebo during twice daily sustained therapy were compared in 23 patients with stable, exertional angina pectoris. The study was double blind in design, and each drug was prescribed for a period of one month in a random fashion. The number of anginal attacks and consumption of glyceryl trinitrate tablets during the one month period were significantly reduced by a similar degree during therapy with all five beta blocking drugs in comparison to the placebo (P less than 0.01). Exercise tolerance, when assessed 12 hours after a previous dose had been given and 1 hour after the morning dose was given, also improved by a similar degree with all five drugs in comparison to the placebo (P less than 0.01). The increase in exercise duration was associated with a significant reduction in the S-T segment depression, heart rate, systolic blood pressure, and the product of heart rate and systolic blood pressure, with each of the five drugs--effects markedly different from those obtained with the placebo (P less than 0.01). These data show that noncardioselective (propranolol and oxprenolol) and cardioselective (practolol, metoprolol and tolamolol) agents, as well as drugs with intrinsic sympathomimetic activity (oxprenolol and practolol), were equally effective antianginal agents during sustained therapy. Furthermore, twice daily therapy with any of these drugs was effective in the management of patients with angina pectoris.

A Selective Inhibitor of Human C-reactive Protein Translation Is Efficacious In Vitro and in C-reactive Protein Transgenic Mice and Humans

Observational studies of patients with established rheumatoid arthritis (RA) document a positive correlation between C-reactive protein (CRP) blood concentration and worsening of RA symptoms, but whether this association is causal or not is not known. Using CRP transgenic mice (CRPTg) with collagen-induced arthritis (CIA; a rodent model of RA), we explored causality by testing if CRP lowering via treatment with antisense oligonucleotides (ASOs) targeting human CRP mRNA was efficacious and of clinical benefit. We found that in CRPtg with established CIA, ASO-mediated lowering of blood human CRP levels improved the clinical signs of arthritis. In addition, in healthy human volunteers the ASO was well tolerated and efficacious i.e., treatment achieved significant CRP lowering. ASOs targeting CRP should provide a specific and effective way to lower human CRP levels, which might be an effective therapy in patients with established RA.

AN ANTISENSE INHIBITOR OF APOLIPOPROTEIN C-III SIGNIFICANTLY DECREASES APOLIPOPROTEIN C-III, TRIGLYCERIDES, VERY-LOW-DENSITY LIPOPROTEIN CHOLESTEROL AND PARTICLE NUMBER, AND INCREASES HIGH-DENSITY LIPOPROTEIN CHOLESTEROL AND PARTICLE NUMBER IN HYPERTRIGLYCERIDEMIC PATIENTS ON A FIBRATE

ApoC-III plays a pivotal role in regulating plasma triglyceride (TG) levels by inhibiting the hydrolysis of TG-rich lipoproteins and the receptor-mediated uptake of lipoprotein remnants by the liver and is recognized as a CV risk factor. ISIS-ApoCIIIRx selectively inhibits apoC-III protein synthesis

THU0539 Isis-Crp Rx: Pharmacokinetics, Pharmacodynamics, Safety and Tolerability of an Antisense Oligonucleotide Specific for Inhibition of CRP in Inflammation

Background CRP is elevated in many diseases with inflammatory components such as rheumatoid arthritis, acute coronary syndrome and atrial fibrillation. Elevated CRP levels are associated with increased disease burden and are potentially involved in the propagation of the inflammatory response. Lowering CRP levels could improve disease outcome. ISIS-CRPRx, an antisense oligonucleotide (ASO) that selectively reduces CRP, distributes rapidly from plasma to tissue, especially liver, has predictable plasma and tissue exposure, half-life of 2-3 weeks, and high tissue bioavailability by s.c. or i.v. administration (Jones, 2012; Yu, 2012). Objectives Characterize PK, PD, safety and tolerability of ISIS-CRPRx in healthy volunteers; assess effect of ISIS-CRPRx on the acute phase response following an immune stimulus (endotoxin challenge in healthy volunteers); and evaluate the ability of ISIS-CRPRx to modulate a chronic inflammatory condition (rheumatoid arthritis) in 3 randomized, double-blind, placebo-controlled trials. Methods 80 healthy volunteers in a Phase 1 clinical study (s.c. and i.v., placebo or up to 600 mg per dose), 35 healthy volunteers in an endotoxin challenge study (placebo, 400 mg and 600 mg i.v. per dose) and 51 RA patients in a 12-week Phase 2, parallel group, multicenter study (placebo, 100 mg, 200 mg and 400 mg s.c. per dose). Results In the Phase 1 study ISIS-CRPRx produced statistically significant reductions in non-stimulated CRP levels by a median of 76% compared to baseline at the 600 mg dose and was well tolerated at all doses. In the endotoxin challenge study, pretreatment with ISIS-CRPRx selectively and significantly inhibited the CRP response in a dose-dependent manner, without altering other inflammatory plasma biomarkers or the physiological response to endotoxin. In the Phase 2 RA study there were dose-dependent reductions in CRP (>70% at the 400mg dose) with no serious infections, and no elevations in LFTs, lipids, creatinine or other lab abnormalities related to ISIS-CRPRx. ACR20 was 20.0% for placebo and 36.4% for ISIS-CRPRx 400mg at Day 36 while at Day 92 ACR20 was 54.5% in placebo and 36.4% in the 400 mg group. The DAS28 change from baseline at Day 92 was -1.32 for placebo and -1.13 at 400 mg. There was no statistically significant difference versus placebo for these parameters. Conclusions In these studies ISIS-CRPRx selectively reduced CRP in a rapid manner, and was well tolerated in healthy volunteers and in RA patients. Use of ISIS-CRPRx in other inflammatory conditions may warrant further exploration. References Jones NR, Pegues MA, McCrory MA, Singleton W, Bethune C, Baker BF, Norris DA, Crooke RM, Graham MJ, Szalai AJ. A selective inhibitor of human C-reactive protein translation is efficacious in vitro and in C-reactive protein transgenic mice and humans. Mol Ther Nucleic Acids. 2012;1:e52. doi: 10.1038/mtna.2012.44. Yu RZ, Grundy JS, Geary. Clinical pharmacokinetics of second generation antisense oligonucleotides. Expert Opin Drug Metab Toxicol. 2013; 9: 169-182. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.2538