Population pharmacokinetic/pharmacodynamic modelling of eplontersen, an antisense oligonucleotide in development for transthyretin amyloidosis.

AIMS: Transthyretin-mediated amyloidosis is a progressive and fatal disease caused by the build-up of misfolded transthyretin (TTR) protein. Eplontersen is a triantennary N-acetyl galactosamine (GalNAc3)-conjugated antisense oligonucleotide targeting TTR messenger ribonucleic acid (mRNA) to inhibit production of both variant and wild-type TTR. We aimed to develop a population pharmacokinetic/pharmacodynamic (PK/PD) model for eplontersen and to evaluate the impact of covariates on exposure and response. METHODS: Plasma eplontersen and serum TTR concentration data were obtained from two phase 1 studies in healthy volunteers (ClinicalTrials.gov: NCT03728634, NCT04302064). Model development was conducted using a nonlinear mixed-effects approach. RESULTS: Eplontersen PK was well described by a two-compartment model. Evaluation of demographics identified significant covariates of lean body mass on clearance and body weight on intercompartmental clearance and volumes of distribution. Population PK modelling showed the absorption rate was 29.6% greater with injection into the abdomen versus the arm. The typical population terminal elimination half-life was 25.5 days. Serum TTR was well described by an indirect response model with inhibition of TTR production by eplontersen. Maximum fractional inhibition (Imax ) was 0.970 (0.549%RSE) and the half maximal inhibitory concentration (IC50 ) was 0.0283 ng/ml (13.3%RSE). Simulations showed subjects with lower weight had higher exposure (AUC, Cmax ), while higher Cmax was observed when comparing site of administration (ratio abdomen/arm = 1.18), but differences in exposure did not significantly impact response at evaluated doses. CONCLUSION: The exposure-response relationship of eplontersen was well characterised by the PKPD model. Weight and injection site were found to affect systemic exposure, but this effect does not seem to result in clinically relevant variation in response.

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.

No effect on QT intervals of mipomersen, a 2'-O-methoxyethyl modified antisense oligonucleotide targeting ApoB-100 mRNA, in a phase I dose escalation placebo-controlled study, and confirmed by a thorough QT (tQT) study, in healthy subjects.

PURPOSE: The aim of this study to evaluate the effect of mipomersen on QT intervals in a phase I dose escalation, placebo-controlled study, and a thorough QT (tQT) study in healthy subjects. METHODS: In the initial phase I study, 29 healthy subjects received either single or multiple (for 4 weeks) ascending doses of mipomersen (50-400 mg) administered subcutaneously (SC) or via a 2-h intravenous (IV) infusion, and 7 subjects received placebo. In the confirmative tQT study, 58 healthy subjects received placebo, 400 mg IV moxifloxacin, 200 mg SC, or 200 mg IV of mipomersen in a double-blind, 4-way crossover design with a minimum 5-day washout between treatments. ECG measurements were performed at baseline and selected time points (including Tmax). The correlation between QTcF intervals corrected for baseline and time-matched placebo when available with PK plasma exposure was evaluated by linear regression analysis. RESULTS: In the phase I study, no positive correlation between the PK exposure and ∆QTcF or ∆∆QTcF was observed within the wide dose or exposure range tested. Similar results were observed in the tQT study, where the predicted ΔΔQTcF and its upper bound of the 90% CI at Cmax of therapeutic and supratherapeutic dose were approximately -1.7 and 2.9 ms, respectively. CONCLUSIONS: Mipomersen showed no effect on QT intervals in both the phase I dose escalation study and the tQT study. These results support the proposal that QT assessment can be made in a phase I dose escalation study, and no tQT study may be necessary if the phase I dose escalation study showed a negative QT effect.

Local and systemic tolerability of a 2'O-methoxyethyl antisense oligonucleotide targeting interleukin-4 receptor-α delivery by inhalation in mouse and monkey.

Antisense oligonucleotides (ASOs) bind and facilitate degradation of RNA and inhibit protein expression in pathways not easily targeted with small molecules or antibodies. Interleukin (IL)-4 and IL-13 potentiate signaling through the shared IL-4 receptor-α (IL-4Rα) subunit of their receptors. ASO targeting of IL-4Rα mRNA in a mouse model of asthma led to attenuation of airway hyperactivity, demonstrating potential benefit in asthma patients. This study focused on tolerability of inhaled IL-4Rα-targeting ASOs. Toxicity studies were performed with mouse- (ISIS 23189) and human-specific (ISIS 369645) sequences administered by inhalation. Four week (monkey) or 13 week (mouse) repeat doses at levels of up to 15 mg/kg/exposure (exp) and 50 mg/kg/exp, respectively, demonstrated dose-dependent effects limited to increases in macrophage size and number in lung and tracheobronchial lymph nodes. The changes were largely non-specific, reflecting adaptive responses that occur during active exposure and deposition of ASO and other material in the lung. Reversibility was observed at a rate consistent with the kinetics of tissue clearance of ASO. Systemic bioavailability was minimal, and no systemic toxicity was observed at exposure levels appreciably above pharmacological doses and doses proposed for clinical trials.

Switching from inotersen to eplontersen in patients with hereditary transthyretin-mediated amyloidosis with polyneuropathy: analysis from NEURO-TTRansform.

BACKGROUND: The phase 3 NEURO-TTRansform trial showed eplontersen treatment for 65 weeks reduced transthyretin (TTR), halted progression of neuropathy impairment, and improved quality of life (QoL) in adult patients with hereditary TTR-mediated amyloidosis with polyneuropathy (ATTRv-PN), vs. historical placebo. METHODS: NEURO-TTRansform enrolled patients with ATTRv-PN. A subset of patients were randomized to receive subcutaneous inotersen 300 mg weekly (Weeks 1-34) and subsequently switched to subcutaneous eplontersen 45 mg every 4 weeks (Weeks 37-81). Change in serum TTR and treatment-emergent adverse events (TEAEs) were evaluated through Week 85. Effects on neuropathy impairment, QoL, and nutritional status were also evaluated. RESULTS: Of 24 patients randomized to inotersen, 20 (83%) switched to eplontersen at Week 37 and four discontinued due to AEs/investigator decision. Absolute change in serum TTR was greater after switching from inotersen (-74.3%; Week 35) to eplontersen (-80.6%; Week 85). From the end of inotersen treatment, neuropathy impairment and QoL were stable (i.e., did not progress) while on eplontersen, and there was no deterioration in nutritional status. TEAEs were fewer with eplontersen (Weeks 37-85; 19/20 [95%] patients) compared with inotersen (up to Week 35; 24/24 [100%] patients). Mean platelet counts decreased during inotersen treatment (mean nadir reduction ‒40.7%) and returned to baseline during eplontersen treatment (mean nadir reduction, ‒3.2%). CONCLUSIONS: Switching from inotersen to eplontersen further reduced serum TTR, halted disease progression, stabilized QoL, restored platelet count, and improved tolerability, without deterioration in nutritional status. This supports a positive benefit-risk profile for patients with ATTRv-PN who switch from inotersen to eplontersen.

Predicting the pharmacokinetics and pharmacodynamics of antisense oligonucleotides: an overview of various approaches and opportunities for PBPK/PD modelling.

INTRODUCTION: Advances in research and development (R&D) have enabled many approvals of antisense oligonucleotides (ASOs). Its administration expanded from systemic to local for treating various diseases, where predicting target tissue exposures and pharmacokinetics (PK) and pharmacodynamics (PD) in human can be critical. AREAS COVERED: A literature search for PBPK/PD models of ASOs was conducted using PubMed and Embase (to 1 April 2023). ASO PK and PD in animals and humans and modeling approaches including physiologically based (PB) are summarized; and relevance and impacts of PBPK/PD modeling are assessed. EXPERT OPINION: Allometric scaling and compartmental PK/PD modeling have been successful to predict human ASO PK/PD, addressing most R&D needs. Understanding tissue distribution of ASOs can be crucial for their efficacy and safety especially for intrathecal (IT), pulmonary, or other local routes. PBPK/PD modeling is expected to improve such understanding, for which, efforts have been sporadic. However, developing a PBPK/PD model requires careful review of known biology/pharmacology and thoughtful experimental designs. Resulting models have the potential to predict target/specified tissue exposures and responses in human adults and pediatrics. Ultimately, a PBPK/PD modeling approach can lead to more efficient and rational clinical development, resulting in well-informed decision making and a shortened timeline.

Assessment of the Effect of Organ Impairment on the Pharmacokinetics of 2'-MOE and Phosphorothioate Modified Antisense Oligonucleotides.

The pharmacokinetics (PK) of 2'-O-methoxyethyl and phosphorothioate antisense oligonucleotides (ASOs), with or without N-acetyl galactosamine conjugation, have been well characterized following subcutaneous or intravenous drug administration. However, the effect of organ impairment on ASO PK, primarily hepatic or renal impairment, has not yet been reported. ASOs distribute extensively to the liver and kidneys, where they are metabolized slowly by endo- and exonucleases, with minimal renal excretion as parent drug (<1%-3%). This short review evaluated the effect of organ impairment on ASO PK using 3 case studies: (1) a phase 1 renal impairment study evaluating a N-acetyl galactosamine-conjugated ASO in healthy study participants and study participants with moderate renal impairment, (2) a phase 2 study evaluating an unconjugated ASO in patients with end-stage renal disease; and (3) a phase 3 study evaluating an unconjugated ASO, which included patients with mild hepatic or renal impairment. Results showed that patients with end-stage renal disease had a mild increase (≈34%) in total plasma exposure, whereas mild or moderate renal impairment showed no effect on plasma PK. The effect of hepatic impairment on ASO PK could not be fully evaluated due to lack of data in moderate and severe hepatic impairment study participants. Nonetheless, available data suggest that mild hepatic impairment had no effect on ASO exposure.

Pharmacodynamics and subchronic toxicity in mice and monkeys of ISIS 388626, a second-generation antisense oligonucleotide that targets human sodium glucose cotransporter 2.

ISIS 388626, a 2'-methoxyethyl (MOE)-modified antisense oligonucleotide (ASO) that targets human sodium glucose cotransporter 2 (SGLT2) mRNA, is in clinical trials for the management of diabetes. SGLT2 plays a pivotal role in renal glucose reabsorption, and inhibition of SGLT2 is anticipated to reduce hyperglycemia in diabetic subjects by increasing urinary glucose elimination. To selectively inhibit SGLT2 in the kidney, ISIS 388626 was designed as a "shortmer" ASO, consisting of only 12 nucleotides with two 2'-MOE-modified nucleotides at the termini. Mice and monkeys received up to 30 mg/kg/week ISIS 388626 via subcutaneous injection for 6 or 13 weeks. Dose-dependent decreases in renal SGLT2 mRNA expression were observed, which correlated with dose-related increases in glucosuria without concomitant hypoglycemia. There were no histologic changes in the kidney attributed to SGLT2 inhibition after 6 or 13 weeks of treatment. The remaining changes observed in these studies were typical of those produced in these species by the administration of oligonucleotides, correlated with high doses of ISIS 388626, and were unrelated to the inhibition of SGLT2 expression. The kidney contained the highest concentration of ISIS 388626, and dose-dependent basophilic granule accumulation in tubular epithelial cells of the kidney, which is evidence of oligonucleotide accumulation in these cells, was the only histologic change identified. No changes in kidney function were observed. These results revealed only readily reversible changes after the administration of ISIS 388626 and support the continued investigation of the safety and efficacy of ISIS 388626 in human trials.

Phase 2 Study of the Factor XI Antisense Inhibitor IONIS-FXIRx in Patients With ESRD.

INTRODUCTION: Patients with end-stage renal disease (ESRD) requiring hemodialysis (HD) have an increased risk of thrombotic events and bleeding. Antisense reduction of factor XI (FXI) with IONIS-FXIRx is a novel strategy that may safely reduce the risk of thrombotic events. METHODS: This multicenter study enrolled 49 patients receiving HD in 2 parts. First, 6 participants (pharmacokinetics [PK] cohort) received 1 open-label 300 mg dose of IONIS-FXIRx both before and after HD. Subsequently, 43 participants were treated in a double-blind, randomized design with 200 mg or 300 mg IONIS-FXIRx or placebo for 12 weeks. The PK, pharmacodynamics (PD), and adverse events of IONIS-FXIRx were evaluated (ClinicalTrials.gov: NCT02553889). RESULTS: The PK of IONIS-FXIRx was consistent with previous studies and similar whether injected before or after HD. No accumulation of IONIS-FXIRx was observed after repeat administration. By day 85, mean levels of FXI activity fell 56.0% in the 200 mg group, 70.7% in the 300 mg group, and 3.9% in the placebo group compared with baseline. FXI antigen levels paralleled FXI activity. Dose-dependent prolongation of activated partial thromboplastin time (aPTT) was observed, with no changes in international normalized ratio (INR). IONIS-FXIRx was not associated with drug-related serious adverse events. In the randomized phase of the study, major bleeding events occurred in 0 (0.0%; 200 mg), 1 (6.7%; 300 mg), and 1 (7.7%; placebo) patients and were not considered related to treatment. CONCLUSION: IONIS-FXIRx reduced FXI activity in patients with ESRD receiving HD. Further studies are needed to determine the benefit-risk profile of FXI as a therapeutic target for patients who require HD.

PK/PD modeling of FXI antisense oligonucleotides to bridge the dose-FXI activity relation from healthy volunteers to end-stage renal disease patients.

IONIS-FXIRX (BAY2306001) is an antisense oligonucleotide that inhibits the synthesis of coagulation factor XI (FXI) and has been investigated in healthy volunteers and patients with end-stage renal disease (ESRD). FXI-LICA (BAY2976217) shares the same RNA sequence as IONIS-FXIRX but contains a GalNAc-conjugation that facilitates asialoglycoprotein receptor (ASGPR)-mediated uptake into hepatocytes. FXI-LICA has been studied in healthy volunteers and is currently investigated in patients with ESRD on hemodialysis. We present a model-informed bridging approach that facilitates the extrapolation of the dose-exposure-FXI relationship from IONIS-FXIRX to FXI-LICA in patients with ESRD and, thus, supports the selection of FX-LICA doses being investigated in patients with ESRD. A two-compartment pharmacokinetic (PK) model, with mixed first- and zero-order subcutaneous absorption and first-order elimination, was combined with an indirect response model for the inhibitory effect on the FXI synthesis rate via an effect compartment. This PK/pharmacodynamic model adequately described the median trends, as well as the interindividual variabilities for plasma drug concentration and FXI activity in healthy volunteers of IONIS-FXIRX and FXI-LICA, and in patients with ESRD of IONIS-FXIRX . The model was then used to predict dose-dependent steady-state FXI activity following repeat once-monthly doses of FXI-LICA in a virtual ESRD patient population. Under the assumption of similar ASGPR expression in patients with ESRD and healthy volunteers, doses of 40 mg, 80 mg, and 120 mg FXI-LICA are expected to cover the target range of clinical interest for steady-state FXI activity in the phase IIb study of FXI-LICA in patients with ESRD undergoing hemodialysis.

Ligand conjugated antisense oligonucleotide for the treatment of transthyretin amyloidosis: preclinical and phase 1 data.

AIMS: Amyloidogenic transthyretin (ATTR) amyloidosis is a fatal disease characterized by progressive cardiomyopathy and/or polyneuropathy. AKCEA-TTR-LRx (ION-682884) is a ligand-conjugated antisense drug designed for receptor-mediated uptake by hepatocytes, the primary source of circulating transthyretin (TTR). Enhanced delivery of the antisense pharmacophore is expected to increase drug potency and support lower, less frequent dosing in treatment. METHODS AND RESULTS: AKCEA-TTR-LRx demonstrated an approximate 50-fold and 30-fold increase in potency compared with the unconjugated antisense drug, inotersen, in human hepatocyte cell culture and mice expressing a mutated human genomic TTR sequence, respectively. This increase in potency was supported by a preferential distribution of AKCEA-TTR-LRx to liver hepatocytes in the transgenic hTTR mouse model. A randomized, placebo-controlled, phase 1 study was conducted to evaluate AKCEA-TTR-LRx in healthy volunteers (ClinicalTrials.gov: NCT03728634). Eligible participants were assigned to one of three multiple-dose cohorts (45, 60, and 90 mg) or a single-dose cohort (120 mg), and then randomized 10:2 (active : placebo) to receive a total of 4 SC doses (Day 1, 29, 57, and 85) in the multiple-dose cohorts or 1 SC dose in the single-dose cohort. The primary endpoint was safety and tolerability; pharmacokinetics and pharmacodynamics were secondary endpoints. All randomized participants completed treatment. No serious adverse events were reported. In the multiple-dose cohorts, AKCEA-TTR-LRx reduced TTR levels from baseline to 2 weeks after the last dose of 45, 60, or 90 mg by a mean (SD) of -85.7% (8.0), -90.5% (7.4), and -93.8% (3.4), compared with -5.9% (14.0) for pooled placebo (P < 0.001). A maximum mean (SD) reduction in TTR levels of -86.3% (6.5) from baseline was achieved after a single dose of 120 mg AKCEA-TTR-LRx . CONCLUSIONS: These findings suggest an improved safety and tolerability profile with the increase in potency achieved by productive receptor-mediated uptake of AKCEA-TTR-LRx by hepatocytes and supports further development of AKCEA-TTR-LRx for the treatment of ATTR polyneuropathy and cardiomyopathy.

An oral antisense oligonucleotide for PCSK9 inhibition.

Inhibitors of proprotein convertase subtilisin/kexin type 9 (PCSK9) reduce low-density lipoprotein (LDL) cholesterol and are used for treatment of dyslipidemia. Current PCSK9 inhibitors are administered via subcutaneous injection. We present a highly potent, chemically modified PCSK9 antisense oligonucleotide (ASO) with potential for oral delivery. Past attempts at oral delivery using earlier-generation ASO chemistries and transient permeation enhancers provided encouraging data, suggesting that improving potency of the ASO could make oral delivery a reality. The constrained ethyl chemistry and liver targeting enabled by N-acetylgalactosamine conjugation make this ASO highly potent. A single subcutaneous dose of 90 mg reduced PCSK9 by >90% in humans with elevated LDL cholesterol and a monthly subcutaneous dose of around 25 mg is predicted to reduce PCSK9 by 80% at steady state. To investigate the feasibility of oral administration, the ASO was coformulated in a tablet with sodium caprate as permeation enhancer. Repeated oral daily dosing in dogs resulted in a bioavailability of 7% in the liver (target organ), about fivefold greater than the plasma bioavailability. Target engagement after oral administration was confirmed by intrajejunal administration of a rat-specific surrogate ASO in solution with the enhancer to rats and by plasma PCSK9 and LDL cholesterol lowering in cynomolgus monkey after tablet administration. On the basis of an assumption of 5% liver bioavailability after oral administration in humans, a daily dose of 15 mg is predicted to reduce circulating PCSK9 by 80% at steady state, supporting the development of the compound for oral administration to treat dyslipidemia.

Population Pharmacokinetic-Pharmacodynamic Modeling of Inotersen, an Antisense Oligonucleotide for Treatment of Patients with Hereditary Transthyretin Amyloidosis.

A population pharmacokinetic (PK) and pharmacodynamic (PD) model was developed for inotersen to evaluate exposure-response relationships and to optimize therapeutic dosing regimen in patients with hereditary transthyretin (TTR) amyloidosis polyneuropathy (hATTR-PN). Inotersen PK and TTR level (PD) data were composed of one Phase 1 study in healthy subjects, one Phase 2/3 study in hATTR patients, and its one open-label extension study. Effects of intrinsic and extrinsic factors (covariates) on PK and PK/PD of inotersen were evaluated using a full model approach. Inotersen PK was characterized by a two-compartment model with elimination from the central compartment. The population PK analysis identified disease status and lean body mass (LBM) as significant covariates for inotersen PK. Nonetheless, the contribution of disease status and LBM on PK was small, as the difference in clearance (CL/F) was 11.1% between healthy subjects and patients with hATTR-PN and 38% between the lowest and highest LBM quartiles of the patient population. Age, race, sex, baseline renal function estimated glomerular filtration rate, and hepatic function markers (baseline albumin, bilirubin, and alanine aminotransferase values) were not statistically significant covariates affecting inotersen PK. An inhibitory effect indirect-response model (inhibition of TTR production) was used to describe the drug effect on TTR-time profiles, with baseline TTR included as a covariate. The overall population Imax and IC50, together with 95% confidence interval, was estimated to be 0.913 (0.899-0.925) and 9.07 (8.08-10.1) ng/mL, respectively. V30M mutation showed no effect on the estimated IC50 value for hATTR patients. The final population PK and PK/PD model was used to simulate four different treatment regimens. The population PK/PD model developed well described the PK and PD of inotersen in patients with hATTR-PN and has been used for label recommendation and trial simulations.

Immunogenicity Assessment of Inotersen, a 2'-O-(2-Methoxyethyl) Antisense Oligonucleotide in Animals and Humans: Effect on Pharmacokinetics, Pharmacodynamics, and Safety.

Inotersen (TEGSEDI™) is a 2'-O-(2-methoxyethyl)-modified antisense oligonucleotide, intended for treating hereditary transthyretin (TTR) amyloidosis with polyneuropathy. The potential immunogenicity (IM) response to inotersen was evaluated in chronic nonclinical safety studies and the pivotal phase 2/3 clinical study. The evaluation was designed to assess the characteristics of antidrug antibodies (ADAs) and their effects on the pharmacokinetics, pharmacodynamics, clinical efficacy, and safety in animals and humans. No immunogenic response was observed after long-term treatment with inotersen in mice. In monkeys, the incidence rate of IM to inotersen appeared to be dose dependent, with 28.6%-50.0% of animals developing ADAs after 36 weeks of treatment. This was characterized as late onset (median onset of 185 days) with low titers (median titer of 8, or 400 if minimum required dilution of 50 is included). The overall incidence rate of patients who developed ADAs was 30% after 65 weeks of treatment with median onset of 203 days and median peak titer of 300. IM had minimal effect on plasma peak (Cmax) and total exposure (i.e. area under curve, AUC) of inotersen, but showed elevated plasma trough levels in both IM-positive animals and humans. However, ADAs had no effect on tissue exposure, TTR messenger RNA, or plasma TTR levels in the long-term monkey study. Similarly, IM showed no effect on plasma TTR levels in clinical studies. Thus, ADAs antibodies were binding antibodies, but not neutralizing antibodies. Finally, no association was observed between IM and toxicity findings (eg, platelet, complement activation, and histopathology findings) in the inotersen 9-month monkey study. In humans, no difference was observed in hematology, including platelets, kidney function tests, or incidence of adverse events between IM-positive and -negative patients. Overall, IM showed no effect on toxicity or safety of inotersen evaluated in both monkeys and humans. ClinicalTrials.gov Identifier: NCT01737398.

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 OBJECTIVES: Mipomersen 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. METHODS: In 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. RESULTS: The area under the plasma concentration-time curve (AUC) from 0 to 24 hours (AUC(24)), 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 AUC(24) 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 microg/mL (approximately 100 micromol/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. CONCLUSIONS: These 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.

Pharmacokinetics and Clinical Pharmacology Considerations of GalNAc3-Conjugated Antisense Oligonucleotides.

Introduction: Triantennary N-acetyl galactosamine (GalNAc3) - conjugated antisense oligonucleotides (ASOs) have demonstrated improved hepatocyte uptake and pharmacologic activity over their parent unconjugated ASOs in animals and humans. Areas covered: In this review, the ADME (absorption, distribution, metabolism, and excretion) characteristics of GalNAc3-conjugated ASOs in animals and in humans are summarized, and their clinical relevance is evaluated from the clinical pharmacology perspectives. Expert opinion: ASOs distribute to tissues via receptor-mediated processes, and conjugation to a ligand specific to certain cell types can improve target tissue delivery. GalNAc3-conjugation represents a good example on this regard and has demonstrated ideal characteristics of a prodrug to target delivery of ASOs to hepatocytes via the asialoglycoprotein receptor (ASGPR). The improved potency and safety margin permit more flexible dosing (e.g. monthly or less frequently if needed) taking full advantage of the long half-life of the parent ASO in humans. However, while still speculative, it should be noted that ASGPR-mediated uptake could become nonlinear with dose and factors that impact ASGPR expression or compete with ASGPR-mediated uptake could potentially affect the uptake of GalNAc3-conjugated ASOs, further studies are warranted.

Integrated Assessment of the Clinical Performance of GalNAc3-Conjugated 2'-O-Methoxyethyl Chimeric Antisense Oligonucleotides: I. Human Volunteer Experience.

Advances in medicinal chemistry have produced new chemical classes of antisense oligonucleotides (ASOs) with enhanced therapeutic properties. Conjugation of the triantennary N-acetylgalactosamine (GalNAc3) moiety to the extensively characterized phosphorothioate (PS)-modified 2'-O-methoxyethyl (2'MOE) ASO exemplifies such an advance. This structure-activity optimized moiety effects receptor-mediated uptake of the ASO prodrug through the asialoglycoprotein receptor 1 to support selective targeting of RNAs expressed by hepatocytes. In this study we report the integrated assessment of data available from randomized placebo-controlled dose-ranging studies of this chemical class of ASOs administered systemically to healthy human volunteers. First, we compare the pharmacokinetic and pharmacodynamic profiles of a subset of the GalNAc3-conjugated PS-modified 2'MOE ASOs to the parent PS-modified 2'MOE ASOs for which plasma analytes are available. We then evaluate the safety profile of the full set of GalNAc3-conjugated PS-modified 2'MOE ASO conjugates by the incidence of signals in standardized laboratory tests and by the mean laboratory test results as a function of dose level over time. With hepatocyte targeted delivery, the ED50 for the GalNAc3-conjugated PS-modified 2'MOE ASO subset ranges from 4 to 10 mg/week, up to 30-fold more potent than the parent PS-modified 2'MOE ASO. No GalNAc3-conjugated PS-modified 2'MOE ASO class effects were identified from the assessment of the integrated laboratory test data across all doses tested with either single or multidose regimens. The increase in potency supports an increase in the safety margin for this new chemical class of ASOs now under broad investigation in the clinic. Although the total exposure is limited in the initial phase 1 trials, ongoing and future investigations in patient populations will support evaluation of the effects of long-term exposure.

Potent reduction of apolipoprotein B and low-density lipoprotein cholesterol by short-term administration of an antisense inhibitor of apolipoprotein B.

BACKGROUND: Apolipoprotein B (apoB) is an important structural component of low-density lipoprotein cholesterol (LDL-C) and plays a key role in LDL-C transport and removal. Reduction in apoB synthesis is expected to reduce circulating LDL-C, a proven risk factor of cardiovascular disease. In the present study, we describe the outcome of the first-in-humans study on the safety and efficacy of an antisense oligonucleotide inhibitor of apoB. METHODS AND RESULTS: This study was a double-blind, randomized, placebo-controlled, dose-escalation investigation conducted at a single site in 36 volunteers with mild dyslipidemia. The study utilized an initial single dose of 50 to 400 mg of ISIS 301012, a 20-mer oligonucleotide, followed by a 4-week multiple-dosing regimen with the same assigned dose. Safety was assessed by the incidence, severity, and relationship of adverse events to dose. Efficacy was determined by changes in serum apoB and LDL-C relative to baseline and placebo. The most common adverse event was erythema at the injection site (21 of 29 subjects). ApoB was reduced by a maximum of 50% (P=0.002) from baseline in the 200-mg cohort. This decrease in apoB coincided with a maximum 35% reduction of LDL-C (P=0.001). LDL-C and apoB remained significantly below baseline (P<0.05) up to 3 months after the last dose. CONCLUSIONS: Administration of an antisense oligonucleotide to human apoB resulted in a significant, prolonged, and dose-dependent reduction in apoB and LDL-C. Although injection-site reactions were common, adherence to protocol was unaffected.

A randomized, double-masked, placebo-controlled study of alicaforsen, an antisense inhibitor of intercellular adhesion molecule 1, for the treatment of subjects with active Crohn's disease.

BACKGROUND & AIMS: The aim of this study was to compare the safety and efficacy of alicaforsen, a first-generation antisense inhibitor of intercellular adhesion molecule 1, with placebo in subjects with active Crohn's disease, a disorder in which intercellular adhesion molecule 1 is overexpressed. METHODS: In 2 identical double-masked, placebo-controlled studies, 331 subjects with active Crohn's disease were treated with either alicaforsen (221 subjects) or placebo (110 subjects) administered via 2-hour intravenous infusion 3 times a week for 4 weeks. Patients then returned for follow-up every 2 weeks. The primary end point was clinical remission by week 12. Secondary end points included clinical response and remission in relation to previous use of other biologics including tumor necrosis factor-alpha antagonists and presence of fistulous disease. RESULTS: The results, whether combined or analyzed individually, failed to demonstrate statistical significance as a measure of its primary outcome (alicaforsen 33.9% vs placebo 34.5%; P = .89). In addition, no statistical differences in response were observed between alicaforsen and placebo in subjects who were previously treated with anti-tumor necrosis factor-alpha therapy or had baseline fistulizing disease. There were no significant differences in adverse events from placebo apart from a higher infusion reaction rate. CONCLUSIONS: In the subject population studied, alicaforsen failed to demonstrate efficacy in any of its primary outcome measures. Alicaforsen was well-tolerated.

Lack of QT Prolongation for 2'-O-Methoxyethyl-Modified Antisense Oligonucleotides Based on Retrospective Exposure/Response Analysis of Ten Phase 1 Dose-Escalation Placebo-Controlled Studies in Healthy Subjects.

The potential of QT prolongation of ten 2'-O-methoxyethyl-modified (2'-MOE) antisense oligonucleotides (ASOs) was evaluated retrospectively via exposure/response (ER) analysis using data from Phase 1 clinical studies in healthy subjects. All Phase 1 studies were double-blind, placebo-controlled, single and multiple ascending dose studies designed to assess the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics of the ASOs in healthy subjects. The active doses in these studies ranged from 50 to 450 mg administered by subcutaneous (SC) injection in single and multiple ascending dose cohorts. Two of the ten studies also included 2-h intravenous (IV) infusions up to 600 mg. Electrocardiogram (ECG) measurements were performed at baseline and selected time points (including Tmax). The correlation between QTcF intervals corrected for baseline (ΔQTcF) and the mean time-matched placebo (ΔΔQTcF) with PK plasma exposure when available was evaluated using a linear mixed-effects approach. There was no evidence for QTc prolongation associated with increasing plasma concentrations in healthy subjects, including exposures with treatment up to 450 mg administered SC or 600 mg by IV infusions, and concentrations that are 4-20 times the Cmax of the therapeutic dose, as assessed by both ΔQTcF and ΔΔQTcF. The ER analysis of the relationship between drug plasma concentration and ΔΔQTcF showed that the slope of the regression line was close to zero, and the upper bound of the 90% confidence interval at twice the mean observed (or predicted) Cmax (2 × Cmax) of the clinical therapeutic dose (ie, the highest clinically relevant plasma concentration) was well below 10 ms for all 10 compounds evaluated. Therefore, no concentration-dependent effect on QT prolongation was observed for any one of the ten 2'-MOE ASOs evaluated in Phase 1 studies. These results confirmed that 2'-MOE ASOs, as a chemical class, do not cause QT prolongation at clinically relevant dose levels.