Elsevier

Biochemical Pharmacology

Volume 77, Issue 5, 1 March 2009, Pages 910-919
Biochemical Pharmacology

Cross-species comparison of in vivo PK/PD relationships for second-generation antisense oligonucleotides targeting apolipoprotein B-100

https://doi.org/10.1016/j.bcp.2008.11.005Get rights and content

Abstract

The in vivo pharmacokinetics/pharmacodynamics of 2′-O-(2-methoxyethyl) (2′-MOE) modified antisense oligonucleotides (ASOs), targeting apolipoprotein B-100 (apoB-100), were characterized in multiple species. The species-specific apoB antisense inhibitors demonstrated target apoB mRNA reduction in a drug concentration and time-dependent fashion in mice, monkeys, and humans. Consistent with the concentration-dependent decreases in liver apoB mRNA, reductions in serum apoB, and LDL-C, and total cholesterol were concurrently observed in animal models and humans. Additionally, the long duration of effect after cessation of dosing correlated well with the elimination half-life of 2′-MOE modified apoB ASOs studied in mice (t1/2  20 days) and humans (t1/2  30 days) following parental administrations. The plasma concentrations of ISIS 301012, observed in the terminal elimination phase of both mice and monkeys were in equilibrium with liver. The partition ratios between liver and plasma were similar, approximately 6000:1, across species, and thus provide a surrogate for tissue exposure in humans. Using an inhibitory Emax model, the ASO liver EC50s were 101 ± 32, 119 ± 15, and 300 ± 191 μg/g of ASO in high-fat-fed (HF) mice, transgenic mice containing the human apoB transgene, and monkeys, respectively. The estimated liver EC50 in man, extrapolated from trough plasma exposure, was 81 ± 122 μg/g. Therefore, extraordinary consistency of the exposure–response relationship for the apoB antisense inhibitor was observed across species, including human. The cross-species PK/PD relationships provide confidence in the use of pharmacology animal models to predict human dosing for second-generation ASOs targeting the liver.

Graphical abstract

Similar exposure-response relationships of ISIS 301012 in human ApoB transgenic mice and in humans.

Introduction

Applying pharmacokinetic and pharmacodynamic analyses to guide and expedite drug development is well recognized and has received increasing interest in recent years [1]. Although these principles are well accepted and widely used for low molecular weight drugs and proteins, there have been a growing number of reports describing pharmacokinetic and pharmacodynamic relationships with antisense therapeutic agents [2], [3], [4], [5].

The pharmacokinetics of 2′-methoxyethyl (2′-MOE) chimeric phosphorothioate antisense oligonucleotides (ASOs), or second-generation ASOs, have been described in a number of species including man [6], [7], [8], [9], [10]. The structure of the second-generation ASOs is characterized by five 2′-MOE modifications on the ribose sugar at both 5′- and 3′-termini, flanking a 10-nucleotide oligodeoxynucleotide gap (5-10-5 2′MOE chimeras) with phosphorothioate backbone. Although there are slight sequence dependent differences, the pharmacokinetics of the second-generation ASOs are remarkably similar and are characterized by predictable distribution and prolonged tissue elimination (14–30 days) as compared to first-generation oligonucleotides.

Although, ASOs have been shown to be able to work by multiple mechanisms once bound to the target RNA [11], all of the 2′MOE chimeric ASOs in development have been shown to work by forming RNA–ASO duplexes that serve as a substrate for RNase H1[12], [13]. RNase H1 cleaves the RNA to which the ASO is bound, resulting in loss of the target RNA and eventually the protein, both of which have been measured as a direct means of evaluating pharmacodynamic effects.

Consequently, the ASO prevents the translation of the encoded protein product in a highly sequence-specific manner. Because of the unique mechanism of action of antisense therapeutics, investigations of the pharmacological effects of antisense oligonucleotide in vivo have focused primarily on target mRNA reduction and the subsequent reduction in protein translation and the downstream effects resulting from target protein reduction, which are dependent on the target studied. Moreover, establishment of the correlation between plasma equilibrium concentrations with the concentrations at the target sites is pertinent, enabling plasma concentrations to be used as a surrogate in clinical studies to establish the pharmacodynamics and pharmacokinetics relationships. Translation of preclinical to clinical PK/PD relationships require predictive pharmacokinetics and reliable PD biomarkers that can be assessed in real time. In this report, we present data from multiple species for potent apolipoprotein B-100 (apoB-100) 2′-MOE chimeric antisense compounds that provides both of these prerequisites; predictable cross-species pharmacokinetics [9] and reliable biomarkers, apoB itself and its related LDL-cholesterol particles measured in serum in real time.

ApoB-100 is the protein component of atherogenic lipids and triglycerides, including LDL-cholesterol. ApoB-100 is synthesized and packaged into lipoprotein particles principally in the liver of all species [14], [15], [16]. Circulating LDL-C, which typically constitutes 60–70% of serum cholesterol, is widely recognized as a major risk factor for coronary heart disease (CHD) and has been implicated in the inflammation associated with the pathogenesis of atherosclerosis. Similarly, apoB-100 is now recognized as a risk factor for atherosclerosis. This led to the development of an antisense inhibitor of the molecular target, apolipoprotein B (apoB), for use in lowering apoB-100 and subsequent lowering LDL-C.

Since the sequence of mRNAs for apoB-100 differs depending on the species and 2′-MOE ASOs are highly specific, we have used species-specific apoB-100 ASOs to demonstrate potent dose-dependent reduction of apoB-100 mRNA and protein in the liver of all species tested and concomitant reductions in plasma apoB and apoB-100 containing lipoproteins [17], [18]. Species-specific apoB antisense inhibitors had been evaluated in multiple animals species, including mice, hamsters, rabbits and monkeys (lean and HF-fed) and it had been demonstrated that administration of the apoB-100 antisense inhibitor produced significant pharmacological effects, i.e., significant reductions in mRNA and liver protein with concomitant reductions in serum apoB, LDL-C, and total cholesterol [18]. All the apoB antisense inhibitors evaluated are 20-mer phosphorothioate oligonucleotide with 2′-O-(2-methoxy) ethyl (MOE) modification on the 5 nucleotides on both 3′ and 5′ termini. MOE modifications provide enhanced resistance to nucleases, a longer target organ half-life, and reduced toxicity [19], [20]. In addition, these modifications increase the affinity of an antisense oligonucleotide for complementary target mRNA, resulting in enhanced potency and specificity [20], [21], [22].

ISIS 301012 (Mipomersen), the human specific apoB-100 antisense inhibitor, is currently in Phase 3 clinical development in familial hypercholesteremia. ISIS 301012 has been shown to produce consistent and predictable dose-dependent and exposure-dependent reduction in serum atherogenic lipids and lipoproteins in human subjects [23], [24], [25], [26]. These dose- and exposure–response correlations have been demonstrated in all clinical subject populations studied to date, including healthy volunteers, subjects with mild hypercholesterolemia, polygenic hypercholesterolemia subjects on stable statin therapy, and homozygous familial hypercholesterolemia subjects on stable statin therapy.

In this paper, we present for the first time remarkable cross-species correlates of PK/PD associations at the molecular level for apoB-100 antisense oligonucleotides. Herein we will summarize the pharmacokinetics and pharmacodynamics of apoB antisense inhibitors in fat fed mice, transgenic human apoB-100 mice, monkeys and humans using species-specific apoB antisense inhibitors. The comparisons in pharmacodynamics across species provide guidance in selection of predictive animal models for the development of future antisense oligonucleotides in this chemical class.

Section snippets

Oligonucleotides

ISIS 147764, ISIS 326358 and ISIS 301012 are 20-nucleotide second-generation antisense oligonucleotides targeting apoB mRNA in mice, monkeys and humans, respectively. All the compounds have MOE modifications at positions 1–5 and 15–20 (Table 1). ISIS 13866, a 2′MOE -modified oligonucleotide at positions 15–21 (underlined) with a sequence of 5′-GCG TTT GCT CTT CTTMCTTGMCG TTT TTT-3′, was used as the internal standard for quantitation of ASO in tissues. In addition, all the cytosines of the

Pharmacokinetics

The clearance of apoB ASOs from tissues was slow in all species studied. The elimination half-life for the mouse-specific apoB antisense inhibitor, ISIS 147764 in mouse liver was 20 days, while the elimination half-lives for ISIS 301012 were 24 and 34 days in mice and monkeys, respectively (Table 2). Elimination half-life was not determined for ISIS 326358 because ISIS 326358 is a monkey-specific apoB inhibitor and is not going to be developed for use in humans. The monkey study with ISIS

Discussion

Because the direct pharmacological response of antisense therapeutics is target mRNA reduction, establishment of the correlation of target organ concentration and target mRNA reduction is most widely used in studying the pharmacokinetic/pharmacodynamic relationships of antisense oligonucleotides in animal models. The pharmacological effect of antisense inhibitors occurs in cells when the antisense inhibitor binds to its cognate mRNA and the RNA strand of the heteroduplex is degraded by RNase H

Acknowledgements

The authors wish to thank Drs. Stanley Crooke and Jeff Jonas for scientific discussion and critical review of the manuscript. Finally, this manuscript would not be possible without the administrative support provided by Robert Saunders, for which we are grateful.

Authors are employees of ISIS Pharmaceuticals and own shares in ISIS Pharmaceuticals.

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