Elsevier

Toxicology

Volume 301, Issues 1–3, 15 November 2012, Pages 13-20
Toxicology

Renal uptake and tolerability of a 2′-O-methoxyethyl modified antisense oligonucleotide (ISIS 113715) in monkey

https://doi.org/10.1016/j.tox.2012.06.005Get rights and content

Abstract

The primary target organ for uptake of systemically administered phosphorothioate oligonucleotides is the kidney cortex and the proximal tubular epithelium in particular. To determine the effect of oligonucleotide uptake on renal function, a detailed renal physiology study was performed in cynomolgus monkeys treated with 10–40 mg/kg/week ISIS 113715 for 4 weeks. The concentrations of oligonucleotide in the kidney cortex ranged from 1400 to 2600 μg/g. These concentrations were associated with histologic changes in proximal tubular epithelial cells that ranged from the appearance of cytoplasmic basophilic granules to atrophic and degenerative changes at higher concentrations. However, there were no renal functional abnormalities as determined by the typical measurements of blood urea nitrogen, serum creatinine, creatinine clearance, or urine specific gravity. Nor were there changes in glomerular filtration rate, or renal blood flow. Specific urinary markers of tubular epithelial cell damage, such as N-acetyl-glucosaminidase, and α-glutathione-s-transferase were not affected. Tubular function was further evaluated by monitoring the urinary excretion of amino acids, β2-microglobulin, or glucose. Renal function was challenged by administering a glucose load and by examining concentrating ability after a 4-h water deprivation. Neither challenge produced any evidence of change in renal function. The only change observed was a low incidence of increased urine protein/creatinine ratio in monkeys treated with ≥40 mg/kg/week which was rapidly reversible. Collectively, these data indicate that ISIS 113715-uptake by the proximal tubular epithelium has little or no effect on renal function at concentrations of 2600 μg/g.

Introduction

It is well documented that the kidney is the primary site for oligonucleotide distribution following parenteral administration, achieving a higher concentration than any other organ, including liver, and accounting for approximately 20% of the administered dose (Geary et al., 2001a, Geary et al., 2001b). This property of therapeutic oligonucleotides is independent of both species and sequence (Levin, 1999). Oligonucleotides circulate in plasma with greater than 90% of molecules bound to proteins such as albumin and α2-macroglobulin, and therefore, are largely restricted from glomerular filtration (Brown et al., 1994, Srinivasan et al., 1995). Nonetheless, some oligonucleotide is filtered, either as free drug or as bound to lower molecular weight proteins, and is distributed to kidney. Localization of oligonucleotide within the kidney has demonstrated that the proximal tubular epithelial cells contain the high levels of oligonucleotide, with far less present in either glomeruli or medullary rays (Butler et al., 1997a, Butler et al., 1997b).

Oligonucleotide may enter the tubular epithelial cells from the apical brush boarder or from the basolateral surfaces, but it appears that uptake is predominantly through reabsorption of filtered material in tubular lumen at the brush boarder (Oberbauer et al., 1995, Rappaport et al., 1995). Oligonucleotides were visualized by immunohistochemistry or fluorescent labeling, as a punctuate pattern within the cytoplasm of the proximal tubular epithelial cells. The uptake of oligonucleotide at the brush boarder appears receptor-mediated, and can be competed with other polyanions such as dextran sulfate, but the precise receptor(s) is not yet known (Sawai et al., 1995, Sawai et al., 1996). The punctuate distribution pattern is thought to reflect the compartmentalization of oligonucleotide within endosomes and lysosomes. An observation supported by ultrastructural analysis (Rappaport et al., 1995).

As a consequence of distribution to proximal tubular epithelium, the kidney is a target tissue for toxicity. Because of the metabolic stability of therapeutic oligonucleotides and their extended tissue half-lives, there is accumulation of oligonucleotide with repeated administration (Geary et al., 2001b, Yu et al., 2004). In toxicology studies, there are typically no overt effects of this accumulation on renal morphology or function at therapeutically active doses, but starting at doses roughly 3–7 times intended therapeutic doses (approximately 10–20 mg/kg/week), there are more subtle changes in histologic and morphologic appearance of these cells including cytoplasmic vacuolation of tubular epithelium, and atrophic/regenerative changes in brush boarder (Henry et al., 1997, Henry et al., 2001, Monteith et al., 1998). Importantly, there is a close correlation between high concentrations of oligonucleotide and morphologic changes in proximal tubular epithelium. Histologic changes are limited to proximal tubular epithelial cells, and there are typically no changes to glomerulus, medulla, or collecting tubules. At high enough doses, degeneration of proximal tubular epithelial cells, and subsequent increase in excretion of low molecular weight proteins can be observed (Monteith et al., 1999). These morphologic changes, including proximal tubular epithelial cell degeneration, occur at doses greater than necessary for pharmacologic activity and have been shown to be reversible upon clearance of oligonucleotide.

The objective of this research was to more thoroughly describe the concentration/effect relationship for a representative 2′-MOE modified oligonucleotide in monkey kidney. In addition to standard histology and clinical pathology, a detailed assessment of renal function was conducted, with an emphasis on evaluation of proximal tubular epithelial cell function and markers for renal function.

Section snippets

Oligonucleotide characteristics and preparation

The oligonucleotide examined is a full phosphorothioate modified oligonucleotide with 2′-O-methoxyethyl (MOE) modification of 5 residues on each the 3′- and 5′-end of the oligonucleotide. The inhibitor is intended to target human and monkey protein tyrosine phosphatase-1b (PTP-1b) for the purpose of increasing inulin sensitivity and the sequence is 5′-GCTCCTTCCACTGATCCTGC-3′. The oligonucleotide was synthesized at Isis Pharmaceuticals, Inc. according to standard solid phase synthesis techniques

Kidney exposure and morphologic changes

The doses and duration of treatment examined in this study were sufficient to produce kidney cortex concentrations of oligonucleotide up to 2600 μg/g. The concentrations of oligonucleotide in kidney cortex increased dose dependently, but were not precisely dose linear (Table 1). Alternative day dosing for 4 doses was used to load the animals on study to approximately 70–80% of steady state concentrations.

Treatment-related microscopic findings in the kidney consisted of dose-dependent basophilic

Discussion

The uptake of oligonucleotide into proximal tubular epithelium is related to the normal functional role of this cell type to scavenge material such as glucose, amino acids, and low molecular weight proteins that are filtered from (Laiken and Fanestil, 1985). This scavenging occurs at the apical surface of the cell at the brush boarder which is rich in receptors and the subcellular organelles necessary for endocytosis (Christensen and Nielsen, 1991). The uptake of oligonucleotide in kidney has

Conflict of interest

Robert Fey and Scott P. Henry are employers of Isis Pharmaceuticals, Inc. Arthur A. Levin formerly employed by Isis Pharmaceuticals, Inc. and shareholder, consulting agreements with various oligonucleotide companies.

Funding

Isis Pharmaceuticals, Inc. funded this research (Robert Fey and Scott P. Henry). Zanardi employed by Isis Pharmaceuticals, Inc. and shareholder.

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