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. 2017 Dec;27(6):345-353.
doi: 10.1089/nat.2017.0683. Epub 2017 Sep 29.

Pharmacokinetic Properties of DNA Aptamers With Base Modifications

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Free PMC article

Pharmacokinetic Properties of DNA Aptamers With Base Modifications

Shashi Gupta et al. Nucleic Acid Ther. .
Free PMC article

Abstract

The addition of novel side chains at the 5-position of uracil is an effective means to increase chemical diversity of aptamers and hence the success rate for discovery of high-affinity ligands to protein targets. Such modifications also increase nuclease resistance, which is useful in a range of applications, especially for therapeutics. In this study, we assess the impact of these side chains on plasma pharmacokinetics of modified aptamers conjugated to a 40 kDa polyethylene glycol. We show that clearance from plasma depends on relative hydrophobicity: side chains with a negative cLogP (more hydrophilic) result in slower plasma clearance compared with side chains with a positive cLogP (more hydrophobic). We show that clearance increases with the number of side chains in sequences of ≥28 synthons, but this effect is dramatically diminished in shorter sequences. These results serve as a guide for the design of new therapeutic aptamers with diversity-enhancing side chains.

Keywords: aptamer pharmacokinetics; modified nucleotides; plasma clearance; side chains.

Conflict of interest statement

S.G., D.W.D., S.K.W., S.M.W., J.C.R., J.D.C., W.S.M., M.R.O., C.R.F., D.J.S., and N.J. are employees and stakeholders of SomaLogic, Inc. T.S., M.H., and Y.I. are employees and stakeholders of Otsuka Pharmaceutical Co., Ltd.

Figures

<b>FIG. 1.</b>
FIG. 1.
Effect of side chains. (A) Sequences (5′→3′) of SL1026, SL1033, and variants thereof, utilized for determination of the effect of side chains on plasma PK. Standard single-letter designations (A, C, G, and U) are used for the unmodified dehydroxy nucleobases, whereas positions designated Z, E, and P show the locations of 5-(N-benzylcarboxamide), 5-[N-(2-phenylethyl)carboxamide], and 5-[N-(1-naphthylmethyl)carboxamide]-modified uridine (highlighted in red text). The positions containing 2′-OMe ribose are shaded gray. All sequences contain a 3′-idT cap and have a 40 kDa PEG attached to the 5′-terminus (not shown). SL1026 contains 2′-OMe ribose at positions 3, 6, 16, 19, 20, and 28, whereas SL1033 contains C3 spacer (L) at positions 1, 14, and 15. The variants (V1 and V2) are fully modified with 2′-OMe ribose. Plasma half-life (t1/2) and plasma clearance values are shown alongside each sequence. (B) Plasma concentration versus time curves. 2′-OMe, 2′-methoxy; PEG, polyethylene glycol; PK, pharmacokinetic.
<b>FIG. 2.</b>
FIG. 2.
Effect of hydrophobicity of side chains. (A) Sequences (5′→3′) in Series A and B utilized for determination of the effect of different side chains (see below) on plasma PK. Standard single-letter designations (A, C, G, and U) are used for the unmodified dehydroxy nucleobases, whereas positions designated X highlighted in red text show the location of side chains. The positions containing 2′-OMe ribose are shaded gray. All sequences contain a 3′-idT cap, are fully modified with 2′-OMe ribose, except for positions containing a C3 spacer (L) in place of a nucleotide (positions 13 and 14 of Series B) and have a 40 kDa PEG attached to the 5′-terminus (not shown). (B) Structures of 13 different side chains are shown along with their cLogP and plasma clearance values obtained for sequences from Series A and B with each side chain. The abbreviations for the side chains (X group) are Im [(2-imidazolylethyl)carboxamide], MOE [(morpholino-2-ethyl)carboxamide], Thr [(R-2-hydroxypropyl)carboxamide], RTHF (R-tetrahydrofuranylmethylcarboxamide), Pyr [(4-pyridylmethyl)carboxamide], Trp [(3-indole-2-ethyl)carboxamide], MBn [(3,4-methylenedioxybenzyl)carboxamide], Bn (benzylcarboxamide), FBn [(4-fluorobenzyl)carboxamide], cHM [(1-cyclohexylmethyl)carboxamide], Pe [(2-phenylethyl)carboxamide], Nap [(1-naphthylmethyl)carboxamide], and 2Nap [(2-naphthylmethyl)carboxamide]. (C) Correlation between cLogP and CHI for Series A and B. (D) Rat plasma clearance (CL) values are plotted versus the cLogP values of the side chains. Data points are grouped by negative or positive cLogP values using gray boxes. (E) Volumes of distribution at steady state (Vss) are plotted for sequences with a negative cLogP or positive cLogP for Series A and B. Median values for each group are indicated with a horizontal bar. CHI, chromatographic hydrophobicity index.
<b>FIG. 3.</b>
FIG. 3.
Effect of number of side chains. (A) Individual sequences (5′→3′) that make up Series C, D, E, and F designed for the determination of the effect of the number of side chains on rat plasma PK. Standard single-letter designations (A, C, G, and U) are used for the unmodified dehydroxy nucleobases, whereas positions designated Z and P (highlighted in red text) are as described in Fig. 1. The positions containing 2′-OMe ribose are shaded gray. All sequences contain a 3′-idT cap, are fully modified with 2′-OMe ribose except for positions containing a C3 spacer (L) in place of a nucleotide (positions 13 and 14 of Series D and F) and have a 40 kDa PEG attached to the 5′-terminus (not shown). The number of side chains and plasma clearance is shown next to each sequence. (B) Linear regression analyses of plasma clearance (CL) versus the number of side chains in Series C and D. (C) Linear regression analyses of plasma clearance (CL) versus the number of side chains in Series E and F.
<b>FIG. 4.</b>
FIG. 4.
Effect of sequence length. (A) Individual sequences (5′→3′) that make up modified aptamer Series G and H designed for the determination of the effect of aptamer length on plasma PK. Standard single-letter designations (A, C, G, and U) are used for the unmodified dehydroxy nucleobases, whereas positions designated Z or P (highlighted in red text) are as described in Fig. 1. The positions containing 2′-OMe ribose are shaded gray. All sequences contain a 3′-idT cap, are fully modified with 2′-OMe ribose, except for positions containing a C3 spacer (L) in place of a nucleotide (positions 13 and 14 of Series H) and have a 40 kDa PEG attached to the 5′-terminus (not shown). Aptamer length and plasma clearance (CL) are shown alongside each sequence. (B) Linear regression analyses of plasma clearance versus aptamer length in Series G and H.

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