Rosie Z. Yu

Reduction of liver Fas expression by an antisense oligonucleotide protects mice from fulminant hepatitis.

Aberrant apoptosis-mediated cell death is believed to result in a number of different human diseases. For example, excessive apoptosis in the liver can result in fulminant and autoimmune forms of hepatitis. We have explored the possibility that inhibition of Fas expression in mice would reduce the severity of fulminant hepatitis. To do this, we have developed a chemically modified 2′-O-(2-methoxy)ethyl antisense oligonucleotide (ISIS 22023) inhibitor of mouse Fas expression. In tissue culture, this oligonucleotide induced a reduction in Fas mRNA expression that was both concentration- and sequence-specific. In Balb/c mice, dosing with ISIS 22023 reduced Fas mRNA and protein expressions in liver by 90%. The ID50 for this response was 8-10 mg kg−1 daily dosing, and the reduction was highly dependent on oligonucleotide sequence, oligonucleotide concentration in liver, and treatment time. Pretreatment with ISIS 22023 completely protected mice from fulminant hepatitis induced by agonistic Fas antibody, by a mechanism entirely consistent with an oligonucleotide antisense mechanism of action. In addition, oligonucleotide-mediated suppression of Fas expression reduced the severity of acetaminophen-mediated fulminant hepatitis, but was without effect on concanavalin A-mediated hepatitis. Our results demonstrate that 2′-O-(2-methoxy)ethyl containing antisense oligonucleotides targeting Fas can exert in vivo pharmacological activity in liver, and suggest that oligonucleotide inhibitors of Fas may be useful in the treatment of human liver disease.

Cross-species pharmacokinetic comparison from mouse to man of a second-generation antisense oligonucleotide, ISIS 301012, targeting human apolipoprotein B-100

The pharmacokinetics of a 2′-O-(2-methoxyethyl)-modified oligonucleotide, ISIS 301012 [targeting human apolipoprotein B-100 (apoB-100)], was characterized in mouse, rat, monkey, and human. Plasma pharmacokinetics following parental administration was similar across species, exhibiting a rapid distribution phase with t1/2α of several hours and a prolonged elimination phase with t1/2β of days. The prolonged elimination phase represents equilibrium between tissues and circulating drug due to slow elimination from tissues. Absorption was nearly complete following s.c. injection, with bioavailability ranging from 80 to 100% in monkeys. Plasma clearance scaled well across species as a function of body weight alone, and this correlation was improved when corrected for plasma protein binding. In all of the animal models studied, the highest tissue concentrations of ISIS 301012 were observed in kidney and liver. Urinary excretion was less than 3% in monkeys and human in the first 24 h. ISIS 301012 is highly bound to plasma proteins, probably preventing rapid removal by renal filtration. However, following 25 mg/kg s.c. administration in mouse and 5-mg/kg i.v. bolus administration in rat, plasma concentrations of ISIS 301012 exceeded their respective protein binding capacity. Thus, urinary excretion increased to 16% or greater within the first 24 h. Albeit slow, urinary excretion of ISIS 301012 and its shortened metabolites is the ultimate elimination pathway of this compound, as demonstrated by 32% of dose recovered in total excreta by 14 days in a rat mass balance study. The pharmacokinetics of ISIS 301012 in human is predictable from the pharmacokinetics measured in animals. The pharmacokinetic properties of ISIS 301012 provide guidance for clinical development and support infrequent dose administration.

Phase I Clinical/Pharmacokinetic and Pharmacodynamic Trial of the c-raf-1 Antisense Oligonucleotide ISIS 5132 (CGP 69846A)

PURPOSE Raf-1 is a protein kinase that plays a broad role in oncogenic signaling and acts as a downstream effector of Ras in the mitogen-activated protein kinase pathway. The present study was designed to determine the maximum-tolerated dose (MTD), toxicity profile, pharmacokinetics, and antitumor activity of the c-raf-1 antisense oligodeoxynucleotide ISIS 5132 (CGP 69846A; ISIS Pharmaceuticals Inc, Carlsbad, CA). The effect of ISIS 5132 on c-raf-1 gene expression in peripheral-blood mononuclear cells (PBMCs) of treated patients was studied using a reverse transcriptase polymerase chain reaction assay. PATIENTS AND METHODS Patients with refractory malignancies received ISIS 5132 as a 2-hour intravenous infusion three times weekly for 3 consecutive weeks. Pharmacokinetic sampling was performed during the first cycle in all patients; PBMCs for c-raf-1 mRNA analysis were collected at baseline and on days 3, 5, 8, and 15 of cycle 1 and on day 1 of each cycle thereafter. RESULTS Thirty-one patients received ISIS 5132 at one of nine dose levels ranging from 0.5 mg/kg to 6.0 mg/kg. Clinical toxicities included fever and fatigue, but these were not dose limiting. A clinically defined MTD was not reached. The harmonic mean half-life of ISIS 5132 was 59.8 minutes (range, 35.5 to 107.3 minutes). The area under the concentration-time curve increased linearly with dose, and mean plasma clearance was 1.86 mL/kg/min (range, 1.21 to 2.41 mL/kg/min). Two patients experienced prolonged stable disease lasting more than 7 months, which was associated with persistent reduction in c-raf-1 expression in PBMCs. Significant decreases in c-raf-1 expression were identified at time points after the baseline value (P <.05) at doses >/= 2.5 mg/kg. CONCLUSION ISIS 5132 is well tolerated at doses up to 6.0 mg/kg when administered as a thrice weekly 2-hour infusion for 3 consecutive weeks. The pharmacokinetic behavior of the drug is reproducible, and suppression of target gene expression is observed in circulating PBMCs.

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.

Efficacy and Safety of Mipomersen, an Antisense Inhibitor of Apolipoprotein B, in Hypercholesterolemic Subjects Receiving Stable Statin Therapy

OBJECTIVES The aim of this study was to evaluate the efficacy and safety of mipomersen in hypercholesterolemic subjects taking stable statin therapy. BACKGROUND Mipomersen is an apolipoprotein (apo) B synthesis inhibitor that has demonstrated significant reductions in apo B and low-density lipoprotein (LDL) cholesterol in Phase 1 clinical trials in healthy volunteers. METHODS A randomized, placebo-controlled, dose-escalation Phase 2 study was designed to evaluate the effects of mipomersen in hypercholesterolemic subjects taking stable statin therapy. Seventy-four subjects were enrolled sequentially into 1 of 6 dose cohorts at a 4:1 (active/placebo) ratio. Subjects received 7 doses of 30 to 400 mg over 5 weeks in the first 5 cohorts and 15 doses of 200 mg over 13 weeks in the sixth cohort. Pre-specified end points included percentage change from baseline in apo B and LDL cholesterol. Safety was assessed with laboratory test results and by the incidence and severity of adverse events. RESULTS The apo B and LDL cholesterol were reduced by 19% to 54% and 21% to 52%, respectively, at doses of 100 mg/week mipomersen and higher in the 5-week treatment cohorts. Efficacy seemed to increase upon treatment for 13 weeks at a dose of 200 mg/week. Injection site reactions (mild to moderate erythema [90%]) and hepatic transaminase increases (17%) were the most common adverse events, leading to discontinuation in 2 subjects and 1 subject, respectively. In the 13-week treatment cohort, 5 of 10 subjects (50%) had elevations >or=3x the upper limit of normal, 4 of which persisted on 2 consecutive occasions. CONCLUSIONS Mipomersen might hold promise for treatment of patients not reaching target LDL cholesterol levels on stable statin therapy. Further studies are needed to address the mechanisms and clinical relevance of transaminase changes after mipomersen administration. (Dose-Escalating Safety Study in Subjects on Stable Statin Therapy; NCT00231569).

Phase I Trial of ISIS 104838, a 2′-Methoxyethyl Modified Antisense Oligonucleotide Targeting Tumor Necrosis Factor-α

ISIS 104838 is a 20-mer phosphorothioate antisense oligonucleotide (ASO) that binds tumor necrosis factor-α (TNF-α) mRNA. It carries a 2′-methoxyethyl modification on the five 3′ and 5′ nucleotide sugars, with 10 central unmodified deoxynucleotides. ISIS 104838 was identified from a 264 ASO screen in phorbol myristate acetate-activated keratinocytes, and the dose response was assessed in lipopolysaccharide (LPS)-activated monocytes. Healthy males received multiple intravenous (i.v.) ISIS 104838 infusions in a placebo-controlled dose escalation trial (0.1–6 mg/kg). Additional volunteers received single or multiple subcutaneous (s.c.) injections. ISIS 104838 suppressed TNF-α protein by 85% in stimulated keratinocytes. The IC50 for TNF-α mRNA inhibition in stimulated monocytes was <1 μM. For i.v.,C max occurred at the end of infusion. The effective plasma half-life was 15 to 45 min at 0.1 to 0.5 mg/kg and 1 to 1.8 h for higher doses. The apparent terminal plasma elimination half-life approximated 25 days. Obese subjects had higher plasma levels following equivalent mg/kg doses. For s.c. injections,C max occurred at 2 to 4 h and was lower than with equivalent i.v. dosing. Plasma bioavailability compared with i.v. was 82% following a 200 mg/ml s.c. injection. Transient activated partial thromboplastin time prolongation occurred after i.v. infusions and minimally after s.c. injections. Two subjects experienced rash, one a reversible platelet decrease, and mild injection site tenderness was noted. TNF-α production by peripheral blood leukocytes, induced ex vivo by LPS, was decreased by ISIS 104838 (p < 0.01). ISIS 104838, a second-generation antisense oligonucleotide, was generally well tolerated intravenously and subcutaneously. The pharmacokinetics support an infrequent dosing interval. Inhibition of TNF-α production ex vivo was demonstrated.

Efficacy of apolipoprotein B synthesis inhibition in subjects with mild-to-moderate hyperlipidaemia

AIMS Mipomersen, an apolipoprotein (apo) B synthesis inhibitor, has been shown to produce potent reductions in apoB and LDL-cholesterol levels in animal models as well as healthy human volunteers. A randomized, double-blind, placebo-controlled, dose-escalation study was designed to evaluate the efficacy and safety of mipomersen monotherapy with or without dose loading in subjects with mild-to-moderate hyperlipidaemia. METHODS AND RESULTS Fifty subjects with LDL-cholesterol levels between 119 and 266 mg/dL were enrolled into five cohorts at a 4:1 randomization ratio of active to placebo. Two 13-week dose regimens were evaluated at doses ranging from 50 to 400 mg/week. Mipomersen produced dose-dependent reductions in all apoB containing lipoproteins. In the 200 and 300 mg/week dose cohorts, mean reductions from baseline in LDL cholesterol were -45 ± 10% (P= 0.000) and -61 ± 8% (P= 0.000), corresponding to a -46 ± 11% (P= 0.000) and -61 ± 7% (P= 0.000) decrease in apoB levels. Triglyceride levels were also lowered with median reductions up to 53% (P= 0.021). The most common adverse events were injection site reactions. Seven of 40 subjects (18%) showed consecutive transaminase elevations >3× upper limit of normal. Five of these subjects received 400 mg/week, four of whom had apoB levels below the limit of detection. As a consequence, the 400 mg/week cohort was discontinued. CONCLUSIONS Mipomersen administered as monotherapy in subjects with mild-to-moderate hyperlipidaemia produced potent reductions in all apoB-containing lipoproteins. Higher doses were associated with hepatic transaminase increases.

Lack of Pharmacokinetic Interaction of Mipomersen Sodium (ISIS 301012), a 2′-— O -Methoxyethyl Modified Antisense Oligonucleotide Targeting Apolipoprotein B-100 Messenger RNA, with Simvastatin and Ezetimibe

Background and objectivesMipomersen sodium (ISIS 301012) is a 20-mer phosphorothioate antisense oligonucleotide that is complementary to human apolipoprotein B-100 (apoB-100) messenger RNA and subsequently reduces translation of ApoB-100 protein, the major apolipoprotein of very low-density lipoprotein, intermediate-density lipoprotein and low-density lipoprotein (LDL). Mipomersen sodium is currently being studied in phase II/III clinical studies to determine its clinical utility as add-on therapy to HMG-CoA reductase inhibitors or other lipid-lowering agents in subjects with hypercholesterolaemia. The aim of this study was to characterize the pharmacokinetic interactions of mipomersen sodium with simvastatin and ezetimibe. Another aim was to evaluate the ability of mipomersen sodium to inhibit major cytochrome P450 (CYP) isoenzymes in vitro.MethodsIn a phase I clinical study, ten healthy subjects per cohort received a single oral dose of simvastatin 40 mg or ezetimibe 10 mg followed by four 2-hour intravenous doses of mipomersen sodium 200 mg over an 8-day period, with simvastatin 40 mg or ezetimibe 10 mg being administered again with the last dose of mipomersen sodium. Mipomersen sodium pharmacokinetic profiles were assessed following the first dose (mipomersen sodium alone) and the last dose (mipomersen sodium in combination with simvastatin or ezetimibe). Plasma samples for measurement of simvastatin, simvastatin acid, and free and total ezetimibe concentrations were collected at various timepoints following their first and last oral dosing. A comparative pharmacokinetic analysis was performed to determine if there were any effects resulting from coadministration of mipomersen sodium with these lipid-lowering drugs. In addition to the clinical pharmacokinetic analysis, the ability of mipomersen sodium to inhibit the major CYP isoform enzymes (namely CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) was evaluated in cryo-preserved human hepatocytes in vitro.ResultsThe area under the plasma concentration-time curve (AUC) from 0 to 24 hours (AUC24), maximum plasma concentration and apparent elimination half-life values of mipomersen sodium were similar when administered alone and in combination with oral simvastatin or oral ezetimibe. The 90% confidence intervals of the geometric least squares means ratios (%Reference) of the mipomersen sodium AUC24 values were 93.6, 107 when administered together with simvastatin, and 92.4, 111 when administered with ezetimibe. Therefore, there were no large deviations outside the default no-effect boundaries (80–125%) for total exposure (the AUC) of mipomersen sodium in combination with either simvastatin or ezetimibe. Similarly, large deviations outside the default no-effect boundaries were not observed for simvastatin, simvastatin acid, or free and total ezetimibe exposure in combination with mipomersen sodium. In cryo-preserved human hepatocytes, mipomersen sodium exhibited no cytotoxicity. Significant cell uptake was demonstrated by analysing cell-associated concentrations of mipomersen sodium. All evaluated enzyme activities had <10% inhibition at tested concentrations up to 800 μg/mL (∼100 μmol/L) of mipomersen sodium, and dose-dependent inhibition was not observed. Therefore, mipomersen sodium is not considered an inhibitor of CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 enzyme activities.ConclusionsThese data provide evidence that mipomersen sodium exhibits no clinically relevant pharmacokinetic interactions with the disposition and clearance of simvastatin or ezetimibe, and vice versa. Moreover, mipomersen sodium does not inhibit any of the major CYP enzymes that were evaluated. Taken together, the results from this study support the use of mipomersen sodium in combination with oral lipid-lowering agents.

Cardiovascular and Metabolic Effects of ANGPTL3 Antisense Oligonucleotides

BACKGROUND Epidemiologic and genomewide association studies have linked loss‐of‐function variants in ANGPTL3, encoding angiopoietin‐like 3, with low levels of plasma lipoproteins. METHODS We evaluated antisense oligonucleotides (ASOs) targeting Angptl3 messenger RNA (mRNA) for effects on plasma lipid levels, triglyceride clearance, liver triglyceride content, insulin sensitivity, and atherosclerosis in mice. Subsequently, 44 human participants (with triglyceride levels of either 90 to 150 mg per deciliter [1.0 to 1.7 mmol per liter] or >150 mg per deciliter, depending on the dose group) were randomly assigned to receive subcutaneous injections of placebo or an antisense oligonucleotide targeting ANGPTL3 mRNA in a single dose (20, 40, or 80 mg) or multiple doses (10, 20, 40, or 60 mg per week for 6 weeks). The main end points were safety, side‐effect profile, pharmacokinetic and pharmacodynamic measures, and changes in levels of lipids and lipoproteins. RESULTS The treated mice had dose‐dependent reductions in levels of hepatic Angptl3 mRNA, Angptl3 protein, triglycerides, and low‐density lipoprotein (LDL) cholesterol, as well as reductions in liver triglyceride content and atherosclerosis progression and increases in insulin sensitivity. After 6 weeks of treatment, persons in the multiple‐dose groups had reductions in levels of ANGPTL3 protein (reductions of 46.6 to 84.5% from baseline, P<0.01 for all doses vs. placebo) and in levels of triglycerides (reductions of 33.2 to 63.1%), LDL cholesterol (1.3 to 32.9%), very‐low‐density lipoprotein cholesterol (27.9 to 60.0%), non–high‐density lipoprotein cholesterol (10.0 to 36.6%), apolipoprotein B (3.4 to 25.7%), and apolipoprotein C‐III (18.9 to 58.8%). Three participants who received the antisense oligonucleotide and three who received placebo reported dizziness or headache. There were no serious adverse events. CONCLUSIONS Oligonucleotides targeting mouse Angptl3 retarded the progression of atherosclerosis and reduced levels of atherogenic lipoproteins in mice. Use of the same strategy to target human ANGPTL3 reduced levels of atherogenic lipoproteins in humans. (Funded by Ionis Pharmaceuticals; ClinicalTrials.gov number, NCT02709850.)

Clinical pharmacokinetics of second generation antisense oligonucleotides

Introduction: Multiple “second generation” gapmer antisense oligonucleotides (ASOs) of varying chemistries have been evaluated as potential therapeutic agents in the clinic. Compared to first generation chemistries, second generation ASOs consistently demonstrate greater biological stability, greater in vitro/in vivo potency, and less non-hybridization based toxicities. Areas covered: The authors summarize previously publshed clinical pharmacokinetic (PK) properties of second generation ASOs following intravenous or subcutaneous administration. Expert opinion: Our understanding of potential roles of RNAs in maintaining normal health and contribution to various diseases is increasing; thus directly targeting RNAs (with second generation ASOs) present a compelling therapeutic strategy. Further, the similar clinical PK properties across the class of second generation ASOs helps facilitate their clinical development. The majority of published information available for assessment is restricted to acute/sub-acute early clinical development. A limited but growing database on chronic dosing of second generation ASOs, across various patient and special populations, and also with non-systemic local delivery approaches, will help further characterize the clinical PK properties of these compounds and better quantify the extent and sources of any observed PK variability and potential impact on clinical response.