Cardiovascular Evaluation of Etrasimod, a Selective Sphingosine 1-phosphate Receptor Modulator, in Healthy Adults: Results of a Randomized, Thorough QT/QTc Study.

Etrasimod is an investigational, once-daily, oral, selective sphingosine 1-phosphate receptor 1,4,5 modulator used as an oral treatment option for immune-mediated inflammatory disorders. This randomized, double-blind, placebo- and positive-controlled, parallel-group, healthy adult study investigated etrasimod's effect on the QT interval and other electrocardiogram parameters. All participants received etrasimod-matched placebo on day 1. Group A received once-daily, multiple ascending doses of etrasimod (2-4 mg) on days 1-14 and moxifloxacin-matched placebo on days 1 and 15. Group B received etrasimod-matched placebo on days 1-14 and either moxifloxacin 400 mg or moxifloxacin-matched placebo on days 1 and 15. The primary analysis was a concentration-QTc analysis using a corrected QT interval by Fridericia (QTcF). The etrasimod concentration-QTc analysis predicted placebo-corrected change from baseline QTcF (ΔΔQTcF) values and associated 90% confidence intervals remained <10 milliseconds over the observed etrasimod plasma concentration range (≤279 ng/mL). Etrasimod was associated with mild, transient, asymptomatic heart rate slowing that was most pronounced on day 1 (2 mg, first dose). The largest-by-time point mean placebo-corrected changes in heart rate from time-matched day -1 baseline (∆∆HR) on days 1, 7 (2 mg, last dose), and 14 (4 mg, last dose) were -15.1, -8.5, and -6.0 bpm, respectively. Etrasimod's effects on PR interval were small, with the largest least squares mean placebo-corrected change from baseline in PR interval (∆∆PR) being 6.6 milliseconds. No episodes of atrioventricular block were observed. Thus, multiple ascending doses of etrasimod were not associated with clinically relevant QT/QTc effects in healthy adults and only had a mild, transient, and asymptomatic impact on heart rate.

Safety, Pharmacokinetics, and Pharmacodynamics of Etrasimod: Single and Multiple Ascending Dose Studies in Healthy Adults.

Etrasimod is an investigational, once-daily, oral, selective sphingosine 1-phosphate receptor 1,4,5 modulator in development for immune-mediated inflammatory diseases (IMIDs). Here, we report the human safety, pharmacokinetics, and pharmacodynamics of etrasimod obtained from both a single ascending dose (SAD; 0.1-5 mg) study and a multiple ascending dose (MAD; 0.35-3 mg once daily) study. Overall, 99 healthy volunteers (SAD n = 40, MAD n = 59) completed the 2 studies. Evaluated single and multiple doses were well tolerated up to 3 mg without severe adverse events (AEs). Gastrointestinal disorders were the most common etrasimod-related AEs. Over the evaluated single- and multiple-dose ranges, dose-proportional and marginally greater-than-dose-proportional etrasimod plasma exposure were observed, respectively. At steady state, etrasimod oral clearance and half-life mean values ranged from 1.0 to 1.2 L/h and 29.7 to 36.4 hours, respectively. Dose-dependent total peripheral lymphocyte reductions occurred following etrasimod single and multiple dosing. Etrasimod multiple dosing resulted in reductions from baseline in total lymphocyte counts ranging from 41.1% to 68.8% after 21 days. Lymphocyte counts returned to normal range within 7 days following treatment discontinuation. Heart rate lowering from pretreatment baseline on etrasimod dosing was typically mild, with mean reductions seen after the first dose of up to 19.5 bpm (5 mg dose). The favorable safety, pharmacokinetic, and pharmacodynamic properties of etrasimod in humans supported its further development and warranted its investigation for treatment of IMIDs.

Disposition and Mass Balance of Etrasimod in Healthy Subjects and In Vitro Determination of the Enzymes Responsible for Its Oxidative Metabolism.

Etrasimod (APD334) is an investigational, once-daily, oral, selective sphingosine 1-phosphate receptor 1,4,5 modulator (S1P1,4,5 ) in development for treatment of various immune-mediated inflammatory disorders. The disposition and mass balance of a single 2-mg [14 C]etrasimod dose were evaluated in 8 healthy males. An in vitro study was also conducted to identify etrasimod's oxidative metabolizing enzymes. Peak concentrations of etrasimod and total radioactivity in plasma and whole blood were typically reached 4-7 hours postdose. Etrasimod constituted 49.3% of total radioactivity plasma exposure, with multiple minor/trace metabolites making up the remainder. Etrasimod was slowly cleared mainly via biotransformation, predominantly by oxidative metabolism, with unchanged etrasimod recovered in feces accounting for only 11.2% of the dose and none in urine. The mean apparent terminal half-lives of etrasimod and total radioactivity in plasma were 37.8 and 89.0 hours, respectively. Mean cumulative recovery of radioactivity in excreta over 336 hours was 86.9% of the dose, mostly in feces. The prevalent metabolites eliminated in feces were M3 (hydroxy-etrasimod) and M36 (oxy-etrasimod sulfate), accounting for 22.1% and 18.9% of the dose, respectively. From in vitro reaction phenotyping, the predominant enzymes involved in the oxidation of etrasimod were CYP2C8, CYP2C9, and CYP3A4, with minor contributions from CYP2C19 and CYP2J2.

Safety, tolerability, and pharmacokinetics of the selective prostacyclin receptor agonist ralinepag in single and multiple dosing studies of an immediate-release oral formulation in healthy volunteers.

Ralinepag (APD811), an oral, potent, and selective prostacyclin receptor (IP) agonist is being developed for treatment of pulmonary arterial hypertension. Two, single-center, randomized, double-blind, placebo-controlled, Phase 1 studies (single ascending dose and multiple ascending dose) evaluated an oral immediate-release capsule formulation of ralinepag in healthy subjects. Blood samples assessed plasma pharmacokinetics and safety and tolerability data monitored adverse events, vital signs, laboratory findings, physical examination, and electrocardiograms. Eighty-two healthy subjects (single ascending dose (n = 32) and multiple ascending dose (n = 50)) completed the studies. No clinically significant safety issues were observed, except one serious adverse event of atrial fibrillation considered moderate in intensity. In the single ascending dose study, ralinepag was tolerated up to 100 µg (single dose), but not 200 µg due to nausea and vomiting. Dose proportional mean ralinepag plasma exposure measures were observed. Maximum plasma concentrations were reached within 1.0-1.5 h post-dose and mean terminal elimination half-life values from 20.5-26.4 h. In the multiple ascending dose study, ralinepag tolerability decreased with increasing QD or BID dose. Dose proportional steady-state plasma exposure measures were observed where evaluable, with mean steady-state peak-to-trough ratios ranging from 3.34-4.49 (QD dosing) and 1.95-2.36 (BID dosing). Mean effective half-life values ranged from 17.5-18.4 h, reflecting ∼1.7-fold (QD dosing) and ∼2.6-fold (BID dosing) accumulation in plasma exposure. Safety and tolerability of oral immediate-release ralinepag was generally consistent with expectations for this drug class, but more individualized dose escalation appears warranted. Ralinepag exhibited favorable pharmacokinetic properties, with BID dosing producing desired minimal steady-state peak-to-trough fluctuation. Overall, results supported further clinical investigation of ralinepag and guided development of an extended-release formulation to facilitate QD dosing.

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.

Predictive dose-based estimation of systemic exposure multiples in mouse and monkey relative to human for antisense oligonucleotides with 2'-o-(2-methoxyethyl) modifications.

Evaluation of species differences and systemic exposure multiples (or ratios) in toxicological animal species versus human is an ongoing exercise during the course of drug development. The systemic exposure ratios are best estimated by directly comparing area under the plasma concentration-time curves (AUCs), and sometimes by comparing the dose administered, with the dose being adjusted either by body surface area (BSA) or body weight (BW). In this study, the association between AUC ratio and the administered dose ratio from animals to human were studied using a retrospective data-driven approach. The dataset included nine antisense oligonucleotides (ASOs) with 2'-O-(2-methoxyethyl) modifications, evaluated in two animal species (mouse and monkey) following single and repeated parenteral administrations. We found that plasma AUCs were similar between ASOs within the same species, and are predictable to human exposure using a single animal species, either mouse or monkey. Between monkey and human, the plasma exposure ratio can be predicted directly based on BW-adjusted dose ratios, whereas between mouse and human, the exposure ratio would be nearly fivefold lower in mouse compared to human based on BW-adjusted dose values. Thus, multiplying a factor of 5 for the mouse BW-adjusted dose would likely provide a reasonable AUC exposure estimate in human at steady-state.

Pharmacology of a central nervous system delivered 2'-O-methoxyethyl-modified survival of motor neuron splicing oligonucleotide in mice and nonhuman primates.

Spinal muscular atrophy (SMA) is a debilitating neuromuscular disease caused by the loss of survival of motor neuron (SMN) protein. Previously, we demonstrated that ISIS 396443, an antisense oligonucleotide (ASO) targeted to the SMN2 pre-mRNA, is a potent inducer of SMN2 exon 7 inclusion and SMN protein expression, and improves function and survival of mild and severe SMA mouse models. Here, we demonstrate that ISIS 396443 is the most potent ASO in central nervous system (CNS) tissues of adult mice, compared with several other chemically modified ASOs. We evaluated methods of ISIS 396443 delivery to the CNS and characterized its pharmacokinetics and pharmacodynamics in rodents and nonhuman primates (NHPs). Intracerebroventricular bolus injection is a more efficient method of delivering ISIS 396443 to the CNS of rodents, compared with i.c.v. infusion. For both methods of delivery, the duration of ISIS 396443-mediated SMN2 splicing correction is long lasting, with maximal effects still observed 6 months after treatment discontinuation. Administration of ISIS 396443 to the CNS of NHPs by a single intrathecal bolus injection results in widespread distribution throughout the spinal cord. Based upon these preclinical studies, we have advanced ISIS 396443 into clinical development.

Lack of clinical pharmacodynamic and pharmacokinetic drug-drug interactions between warfarin and the antisense oligonucleotide mipomersen.

Mipomersen is a second-generation antisense oligonucleotide indicated as an adjunct therapy for homozygous familial hypercholesterolemia (HoFH). Warfarin is commonly prescribed for a variety of cardiac disorders in homozygous familial hypercholesterolemia population, and concurrent use of warfarin and mipomersen is likely. This open-label, single-sequence 2-period phase 1 study in healthy subjects evaluated the potential drug-drug interactions between mipomersen and warfarin. The subjects received a single oral 25 mg dose of warfarin alone on day 1, and after a 7-day washout period, received 200 mg mipomersen alone subcutaneously every other day on days 8-12, and received both concurrently on day 14. Coadministration of mipomersen did not change the pharmacodynamics (international normalized ratio, prothrombin time, and activated partial thromboplastin time) and pharmacokinetics (PK) of warfarin. There were no clinically significant changes in the PK of mipomersen with concurrent administration of warfarin. There were no events indicative of an increase in bleeding tendency when warfarin was coadministered with mipomersen, and the adverse event profile of mipomersen did not appear to be altered in combination with warfarin, as compared with that of the respective reference treatment. The combination of these 2 medications appeared to be safe and well tolerated. These results suggest that the dosage adjustment of warfarin or mipomersen is not expected to be necessary with coadministration.

Changes in mipomersen dosing regimen provide similar exposure with improved tolerability in randomized placebo-controlled study of healthy volunteers.

BACKGROUND: Mipomersen, an apolipoprotein B synthesis inhibitor, demonstrated significant reductions in low-density lipoprotein (LDL) cholesterol, non-high density lipoprotein cholesterol, and apolipoprotein B in 4 phase 3 studies at the FDA-approved subcutaneous dose of 200 mg once weekly. METHODS AND RESULTS: A short-term phase 1 study in healthy volunteers was conducted to evaluate the relative bioavailability, safety, and tolerability of mipomersen in 2 test dose regimens in reference to the 200 mg weekly dose regimen. Eighty-four adults were randomized to 1 of 3 cohorts (30 mg once daily, 70 mg 3 times weekly, or 200 mg once weekly) and then mipomersen or placebo (3:1 ratio) for 3 weeks of treatment. Comparable mipomersen post-distribution phase plasma concentrations were observed across the 3 dose regimens suggesting similar tissue exposure. Injection site reactions were reported, but did not lead to treatment discontinuation. The median incidence of these responses per injection was decreased by lowering the dose. Signals from a diverse panel of systemic inflammation markers were essentially indistinguishable between dose regimens and placebo treatment. The one exception was a modest transient post-dose elevation of C-reactive protein (CRP) in the mipomersen 200 mg weekly group. This elevation was not associated with an increase in other proinflammatory markers. CONCLUSIONS: This study demonstrated a similar drug exposure and overall safety profile between the 3 dosing regimens. Exploratory assessment of a diverse panel of biomarkers found no indication of a systemic inflammatory response to mipomersen treatment. These results support assessment of alternative dose regimens in longer-term studies. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT01061814.

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.

In vitro metabolic stabilities and metabolism of 2'-O-(methoxyethyl) partially modified phosphorothioate antisense oligonucleotides in preincubated rat or human whole liver homogenates.

In vitro metabolic stability testing of phosphorothioate 2'-O-methoxyethyl (2'-MOE) partially modified antisense oligonucleotides (ASOs) is not routinely performed to help screen discovery compounds (eg, predict in vivo half-lives), as no suitable in vitro test system currently exists. The aims of this work were to develop, optimize, and evaluate an in vitro whole liver homogenate (rat or human) test system. The test system was used to evaluate in vitro metabolic stabilities (intrinsic clearance) of selected ASOs, with results compared to reported in vivo half-lives, and generated metabolites also identified. Test system optimization involved preincubating whole liver homogenates at 37°C for ≥24 hours, which increased in vitro ASO metabolism rate. From calculated in vitro intrinsic clearance (CL(int)) values in preincubated rat or human whole liver homogenates, metabolic stabilities of fully phosphorothioated 2'-MOE ASOs (ISIS 104838 and ISIS 301012) were, as expected, greater (ie, lower CL(int)) than a 2'-MOE ASO containing a single phosphodiester substitution (ISIS 104838PO10). However, comparable-to-lower in vitro metabolic stability for ISIS 301012 was seen compared to ISIS 104838, in contrast to reported ∼2-fold longer in vivo tissue elimination half-lives for ISIS 301012. Identified in vitro metabolic products of ISIS 301012 were consistent with previously reported in vivo observations.

Lack of pharmacokinetic interactions between steady-state zonisamide and valproic acid in patients with epilepsy.

OBJECTIVES: This study evaluated the effect of the addition of zonisamide on valproic acid (valproate sodium) pharmacokinetics under steady-state conditions in patients with epilepsy. A second aim was to characterise zonisamide pharmacokinetics in the presence of valproic acid. METHODS: Twenty-two patients (males and females, 18-55 years of age) with their seizure disorder stabilised on valproic acid monotherapy were included in a two-centre, open-label, one-way drug-interaction trial. The zonisamide dose was gradually increased from 100 mg/day to 400 mg/day. Three pharmacokinetic profiles were obtained: on days -7 and -1, to assess pharmacokinetic parameters of oral valproic acid administered alone, and on day 35, after 14 days of zonisamide treatment at the maximal tolerated dose, to evaluate the effect of zonisamide on valproic acid pharmacokinetics and to characterise zonisamide pharmacokinetics in the presence of valproic acid. RESULTS: Seventeen patients completed the study, with 16 patients contributing to the pharmacokinetic analyses. Coadministration of zonisamide and valproic acid appeared reasonably well tolerated. Steady-state dosing of zonisamide (200mg twice daily) had no statistically significant effect on the mean (+/- SD) maximum observed plasma concentration (C(max)) [70.8 +/- 20.5 vs 69.2 +/- 27.0 microg/mL], area under the plasma concentration-time curve from the time of dosing to 12 hours post-dose (AUC(12)) [689.3 +/- 250.4 vs 661.8 +/- 251.3 microg . h/mL] or other evaluated pharmacokinetic parameters for valproic acid measured before and after zonisamide administration. Furthermore, 90% confidence intervals for the ratio of the geometric means (day 35/day -1) of valproic acid pharmacokinetic exposure measures fell only slightly outside the 'no effect' range of 0.80-1.25. In the presence of valproic acid, mean zonisamide oral clearance (1.23 L/h) and elimination half-life (52.5 hours) are generally consistent with values reported for healthy volunteers receiving zonisamide monotherapy. CONCLUSION: There is no apparent clinically significant effect of steady-state dosing of zonisamide on valproic acid pharmacokinetics, and valproic acid did not appear to affect the pharmacokinetics of zonisamide, indicating that no dosage adjustment of either drug should be required when they are used in combination in patients with epilepsy.

Efficacy and toxicity of the antisense oligonucleotide aprinocarsen directed against protein kinase C-alpha delivered as a 21-day continuous intravenous infusion in patients with recurrent high-grade astrocytomas.

Protein kinase C alpha (PKC-alpha) is a cytoplasmic serine threonine kinase involved in regulating cell differentiation and proliferation. Aprinocarsen is an antisense oligonucleotide against PKC-alpha that reduces PKC-alphain human cell lines and inhibits a human glioblastoma tumor cell line in athymic mice. In this phase 2 study, aprinocarsen was administered to patients with recurrent high-grade gliomas by continuous intravenous infusion (2.0 mg/kg/day for 21 days per month). Twenty-one patients entered this trial. Their median age was 46 years (range, 28-68 years), median Karnofsky performance status was 80 (range, 60-100), median tumor volume was 58 cm3 (range, 16-254 cm3), and histology included glioblastoma multiforme (n = 16), anaplastic oligodendroglioma (n = 4), and anaplastic astrocytoma (n = 1). The number of prior chemotherapy regimens included none (n = 3), one (n = 10), and two (n = 8). No tumor responses were observed. Patients on this therapy rapidly developed symptoms of increased intracranial pressure with increased edema, enhancement, and mass effect on neuroimaging. The median time to progression was 36 days, and median survival was 3.4 months. The observed toxicities were mild, reversible, and uncommon (grade 3 thrombocytopenia [n = 3] and grade 4 AST [n = 1]), and no coagulopathy or CNS bleeding resulted from this therapy. Plasma concentrations of aprinocarsen during the infusion exhibited significant interpatient variability (mean = 1.06 mug/ml; range, 0.34-6.08 mug/ml). This is the first study to use an antisense oligonucleotide or a specific PKC-alpha inhibitor in patients with high-grade gliomas. No clinical benefit was seen. The rapid deterioration seen in these patients could result from tumor growth or an effect of aprinocarsen on bloodbrain barrier integrity.

Carbamazepine pharmacokinetics are not affected by zonisamide: in vitro mechanistic study and in vivo clinical study in epileptic patients.

Carbamazepine is metabolized by CYP3A4 and several other cytochrome P450 enzymes. The potential effects of zonisamide on carbamazepine pharmacokinetics (PK) have not been well characterized, with contradictory literature reports. Hence, an in vitro study was designed to evaluate the cytochrome P450 inhibition spectrum of zonisamide using human liver microsomes. Further, an in vivo steady-state study was performed to measure the effect of zonisamide on carbamazepine PK in epileptic patients, and monitor zonisamide PK. In vitro human liver microsomes were incubated with zonisamide (200, 600 or 1000 microM) in the presence of appropriate probe substrates to assess selected cytochrome P450 activities. In vivo, the effect of zonisamide, up to 400 mg/day, on the steady-state PK of carbamazepine and carbamazepine-epoxide (CBZ-E) was studied in 18 epileptic patients. In vitro, zonisamide did not inhibit CYP1A2 and 2D6, and only weakly inhibited CYP2A6, 2C9, 2C19, and 2E1. The estimated Ki for zonisamide inhibition of CYP3A4 was 1076 microM, 12 times higher than typical unbound therapeutic serum zonisamide concentrations. In vivo, no statistically significant differences were observed for mean Cmax, Tmax, and AUC0-12 of total and free carbamazepine and CBZ-E measured before and after zonisamide administration (300-400 mg/day for 14 days). However, CBZ-E renal clearance was significantly (p < 0.05) reduced by zonisamide. The observed mean zonisamide t1/2 (36.3h), relative to approximately 65 h reported in subjects on zonisamide monotherapy, reflects known CYP3A4 induction by carbamazepine. Based on the lack of clinically relevant in vitro and in vivo effects, adjustment of carbamazepine dosing should not be required with concomitant zonisamide administration.

Prediction of clinical responses in a simulated phase III trial of Crohn's patients administered the antisense phosphorothioate oligonucleotide ISIS 2302: comparison of proposed dosing regimens.

ISIS 2302, an antisense phosphorothioate oligonucleotide (ODN) targeting human intercellular adhesion molecule-1 (ICAM-1) mRNA, is currently being evaluated for treatment of patients with Crohn's disease. From data collected in phase II clinical studies with ISIS 2302, validated population pharmacokinetic and exposure-response models were developed and used to simulate the plasma exposure and clinical response results for a proposed phase III trial design involving 100 patients treated with active drug and 50 patients treated with placebo. Simulated results of 1000 replications of the trial were calculated for various proposed dosing regimens. Overall, the simulated results indicated that a fixed dose regimen (250-400 mg, depending on patient sex and total body weight) given three times weekly provides both desirable ISIS 2302 plasma exposure and a high rate of clinical response in this patient population. However, the simulated results also suggest that inclusion of a larger number of patients than projected may be necessary to provide a desirable probability of study success (i.e., >80%), regarding demonstration of statistically significant differences between the active treatment and placebo groups for the primary clinical response measure (CCR rate).