Renal uptake and tolerability of a 2′-O-methoxyethyl modified antisense oligonucleotide (ISIS 113715) in monkey
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.
References (27)
- et al.
Short communication: renal tubular vacuolation in animals treated with polyethylene-glycol-conjugated proteins
Toxicol. Sci.
(1998) - et al.
Effect of phosphorothioate modification of oligodeoxynucleotides on specific protein binding
J. Biol. Chem.
(1994) - et al.
A nonradioisotope biomedical assay for intact oligonucleotide and its chain-shortened metabolites used for determination of exposure and elimination half-life of antisense drugs in tissue
Anal. Biochem.
(1999) - et al.
Evaluation of the toxicity of ISIS 2302, a phosphorothioate oligonucleotide, in a 4-week study in cynomolgus monkeys
Toxicology
(1997) - et al.
Synthesis of an antisense oligonucleotide targeted against C-raf kinase: efficient oligonucleotide synthesis without chlorinated solvents
Bioorg. Med. Chem.
(1999) A review of issues in the pharmacokinetics and toxicology of phosphorothioate antisense oligonucleotides
Biochim. Biophys. Acta
(1999)- et al.
Light- and electron-microscopic evaluation of renal tubular cell vacuolation induced by administration of nitrilotriacetate or sucrose
Food Chem. Toxicol.
(1985) - et al.
Preclinical evaluation of the effects of a novel antisense compound targeting C-raf kinase in mice and monkeys
Toxicol. Sci.
(1998) - et al.
Renal uptake of an 18-mer phosphorothioate oligonucleotide
Kidney Int.
(1995) - et al.
Transport of phosphorothioate oligonucleotides in kidney: implications for molecular therapy
Kidney Int.
(1995)
Tissue disposition of a 2′-O-(2-methoxy) ethyl modified antisense oligonucleotides in monkeys
J. Pharm. Sci.
Cellular distribution of phosphorothioate oligodeoxynucleotides in normal rodent tissues
Lab. Invest.
Histological localization of phosphorothioate oligodeoxynucleotides in normal rodent tissue
Nucleosides Nucleotides
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