Novel Anticoagulants

Abstract

While all anticoagulants have, to a certain extent, novel properties, the development of agents that inhibit specific coagulation proteases through structural affinity and can be inhibited themselves by the concomitant production of antidotes (drug–antidote pair construct) has the potential to revolutionize the field. With the evolution of our thinking toward hemostasis and thrombosis has come new pharmacologic constructs for safe and effective treatment. Aptamers are single-stranded nucleic acids that inhibit a protein’s function by folding into a specific three-dimensional structure that defines high-affinity binding to the target protein (White et al., 2000). The term aptamer (from the Latin aptus, “to fit”) was coined by Ellington and Szostak (1990) following their pioneering work published originally in Nature. Based on iterative selection techniques, aptamers that bind essentially any protein or small molecule can be generated. A high-affinity, specific inhibitor that interacts with functional groups (on both the nucleic acid and the protein) can be constructed if a small amount of pure target is available. The initiation point for aptamer development is a combinatorial library composed of single-stranded nucleic acids (RNA, DNA, or modified RNA), typically containing 20 to 40 randomized positions (1024 different sequences). Isolation of high-affinity nucleic acid ligands involves a process known as SELEX (systemic evolution of ligands by exponential enrichment). The starting library is incubated with the protein of interest. Nucleic acid molecules that adopt conformations that allow target protein binding are subsequently partitioned from other sequences (that do not bind the protein). The bound sequences are removed and amplified by reverse transcription and polymerase chain reaction (PCR) (for RNA-based libraries) or PCR alone (for DNA-based libraries). After repeating the process several times, the selected ligands are secured and evaluated for binding affinity and ability to inhibit activity (of the target protein). Postselection optimization steps typically include (1) reduction in aptamer length (from a starting molecule of 80–100 nucleotides to 40 nucleotides); (2) enhanced stability in biologic systems (achieved by substitution of ribonucleotides with 2-amino, 2´-fluoro, or 2´-0-alkyl nucleotides and protection from exonuclease digestion by 3´ end capping); and (3) reduced renal clearance (achieved by increasing the molecules’ mo lecular weight through site-specific addition of polyethylene glycol moieties or other hydrophobic groups.