Anti-L-Selectin Aptamers: Binding Characteristics, Pharmacokinetic Parameters, and Activity Against an Intravascular TargetIn Vivo

Abstract

Therapeutic and diagnostic applications have been envisioned for aptamers, a class of oligonucleotide ligands that bind their target molecules with high affinity and specificity (Gold, J. Biol. Chem. 270, 13581-13584, 1995). To identify parameters that are important for the in vivo activity of aptamers acting on intravascular targets, we have studied binding characteristics in vitro, pharmacokinetic parameters in Sprague-Dawley rats, and inhibitory activity in a SCID mouse/human lymphocyte model of lymphocyte trafficking for both 2'F pyrimidine 2'OH purine RNA and ssDNA anti-human L-selectin aptamers. The data indicate that aptamers with low nanomolar affinity are suitable candidates for use as in vivo reagents and that nonspecific binding to vascular cells is not an issue for efficacy. As is often observed for other reagents, plasma clearance is biphasic. Both the distribution phase and the clearance rate strongly affect in vivo activity. Pharmacokinetic parameters and in vivo activity are significantly improved by conjugating aptamers to a carrier molecule, such as polyethylene glycol (PEG). Most active in vivo is 1d40, a 2'F pyrimidine 2'OH purine aptamer conjugated to 40 kDa PEG. At a dose of 5.4 nmol/kg body weight, its duration of effect (time to 50% inhibition) is 11.2 hours, and at 1 mg or 90 nmol/kg, its plasma clearance rate (CL) is 0.4 ml/min/kg. Its ED50 is estimated to be 80 pmol/kg in preinjection dose-response experiments, compared with 4 pmol/kg for the dimeric anti-L-selectin antibody DREG56. Further improvement of in vivo activity is expected from nucleotide modifications that increase resistance to nuclease digestion for aptamers where mass is not rate limiting for clearance. Because the relationship of clearance to conjugate molecular weight (MW) is not the same for all aptamers, it is advisable to determine the relationship at the outset of in vivo studies. In summary, the data suggest that properly formulated aptamers have the capacity to be effective therapeutic agents against intravascular targets.

In vivo properties of an anti-GnRH Spiegelmer: An example of an oligonucleotide-based therapeutic substance class

“…In conjunction with the data from the cell assays, it can be concluded that this effect is caused by reduced clearance. In addition, the sustained duration of the effect provides strong evidence for the enhanced in vivo stability of Spiegelmers compared with aptamers (15).…”

“…In an effort to maximize the pharmacological response of NOX 1255 by increasing its bioavailability, the Spiegelmer was modified by linking a 40-kDa PEG moiety to the 5Ј end, generating NOX 1257 (15,16).…”

Inhibition of rat corneal angiogenesis by a nuclease-resistant RNA aptamer specific for angiopoietin-2

“…A DNA aptamer against plateletderived growth factor has been used to inhibit intimal hyperplasia in a rat carotid injury model (44) and matrix protein accumulation in a rat model of proliferative glomerulonephritis (45). Both DNA and nuclease-resistant RNA aptamers against human L-selectin have been shown to inhibit human lymphocyte trafficking in severe combined immunodeficient (SCID) mice (46,47). The most progress has been made with a polyethylene glycol-conjugated version of a nuclease-resistant RNA aptamer against VEGF, which has now shown promising results in both preclinical and clinical studies for the exudative form of agerelated macular degeneration (41,48,49).…”

Escort aptamers: a delivery service for diagnosis and therapy

“…This desirable clearance rate is more comparable to small peptides than to scFv's or antibodies. At 8-15 kDa, escort aptamers are small enough to permit rapid renal filtration and rapid access to extravascular tissues (for functionblocking aptamers against intravascular proteins, longer blood residence is desirable and is obtained by conjugation to 40 kDa polyethylene glycol [28]). As nuclease cleavage occurs during the time scale of these measurements, 99m Tc-labeled aptamer metabolites will also contribute to clearance.…”