Inhibition of Coagulation by a Phosphorothioate Oligonucleotide

Abstract

In the development of antisense therapeutics, there have been a number of hybridization-independent effects characterized for phosphorothioate oligodeoxynucleotides. One such effect is the transient prolongation of clotting times following intravenous infusion of high doses. In this study, inhibition of clotting times was characterized by determining the time course of both APTT and plasma oligonucleotide following intravenous infusion of ISIS 2302 in cynomolgus monkeys. Prolongation of APTT was also achieved by addition of ISIS 2302 to citrated blood from untreated monkeys, allowing the investigation of the mechanism of inhibition in vitro. Results from this study clearly indicate that the intrinsic pathway (APTT) was more sensitive to inhibition than the extrinsic pathway (PT). The prolongation of APTT was also shown to be transient and closely correlated with plasma oligonucleotide concentrations. The extent of APTT prolongation can be controlled by minimizing peak plasma oligonucleotide concentrations through lowering the dose or prolonging infusion duration. Direct addition of ISIS 2302 to blood produced quantitatively similar inhibition of clotting times. This effect was similar for a number of different phosphorothioate oligodeoxynucleotides, but oligonucleotides containing phosphodiester linkages and 2'-propoxy linkages were much less inhibitory. Additional in vitro studies indicated that the mechanism of inhibition was independent of that of heparin and possibly involved selective inhibition of the intrinsic pathway as well as the common clotting pathway. Investigation of selective clotting factors indicated that there was no direct inhibition of the enzymatic activity of factor Xa, XIa, or thrombin using chromogenic substrates. However, ISIS 2302 did produce a concentration-dependent increase in clotting time when fibrinogen was used as the substrate for thrombin.

siRNA-based approaches in cancer therapy

“…17 However, the presence of an internucleoside charge in oligonucleotides generally increases the possibility of oligonucleotide binding to intracellular proteins and provides a reactive center for potential drug interactions. [18][19][20][21][22][23][24] Some of these problems have been solved in 'second-generation' nucleotides with alkyl modifications at the 2 0 position of the ribose and the development of novel chemically modified nucleotides with improved properties such as enhanced serum stability, higher target affinity and low toxicity. 25 One such modification in oligomer chemistry has led to the development of the phosphorodiamidate morpholino oligomers (PMO) by AVI BioPharma Inc. (Portland, OR), which are non-ionic antisense agents that inhibit gene expression by binding to RNA and sterically blocking processing or translation in an RNaseH-independent manner.…”

Antisense oligonucleotide-based therapeutics for cancer

“…Both of these toxicities are thought to be related to the polyanionic nature of the molecules and the binding of these compounds to specific protein factors in plasma and, as such, relate to peak plasma concentration. Prolongation of clotting times following administration of different phosphorothioate oligodeoxynucleotides is characterized by a transient concentration-dependent prolongation of activated partial thromboplastin times (aPTT) and has been observed in multiple species (Henry et al, 1997b). Recent data suggest that there is a specific allosteric inhibition of the tenase complex as well as binding to thrombin (Sheehan and Phan, 2001).…”

“…Recent data suggest that there is a specific allosteric inhibition of the tenase complex as well as binding to thrombin (Sheehan and Phan, 2001). Complement activation has been described primarily in non-human primates (Galbraith et al, 1994;Henry et al, 1997a). To avoid the potential for complement activation, most studies have increased infusion times, or administered the drug by subcutaneous injection, with the goal of keeping peak plasma concentrations below a threshold concentration (Henry et al, 1997a).…”

“…Complement activation has been described primarily in non-human primates (Galbraith et al, 1994;Henry et al, 1997a). To avoid the potential for complement activation, most studies have increased infusion times, or administered the drug by subcutaneous injection, with the goal of keeping peak plasma concentrations below a threshold concentration (Henry et al, 1997a). It should be noted that phosphorothioate oligodeoxynucleotides have been administered by intravenous infusion to more than 3000 patients and volunteers without any significant indication of activation of the alternative complement cascade.…”

Potential roles of antisense technology in cancer chemotherapy

“…On other schedules, this e ect is transient and does not appear to be associated with a signi®cant risk of bleeding or prolonged bleeding times. The relationship between plasma concentration of drug and complement activation appears to be more complex than previously described (Henry et al, 1997;submitted). However, it may well be multifactorial in origin such that the plasma concentration relationship with complement activation is in fact correct though on certain schedules cytokine release may be a secondary cause.…”