In monkeys, the typical observation of this change occurs at doses of 10 mg/kg administered by 1-h IV infusion where the peak plasma concentration of 90–100 g/ml is associated with a 20–30% increase in APTT (Figure 12.2). The return to normal values over the next couple of hours is attributed to clearance of oligonucleotide from plasma. Specific mechanistic studies have not been performed for 2 -MOE ASOs, but it is presumed that the increase in APTT is due to the reversible interaction between oligonucleotide and components of the intrinsic tenase complex as described for PS ODNs [50,51].

Consistent with the stable pharmacokinetic properties, the APTT values are highest at the plasma Cmax and return to baseline within hours after each dose administration. There is no progression of the effect over repeated administration, even in chronic studies. In subjects, similar dose-dependent increases in APTT that peak at the end of infusion and return to baseline have been reported for 2 -MOE ASO [48].

Despite these effects on APTT, there are typically no signs of altered coagulation status, with no evidence of internal organ hemorrhage. Fibrinogen and d-dimer levels are also routinely measured in monkey studies, but are unaffected. Superficial bruising associated with the site of venipuncture or restraint is the one observation associated with prolongation of clotting times at doses 50 mg/kg [52], but the observation has been less frequent with 2 -MOE ASOs even with treatment up to 80 mg/kg by SC injection.

One unique application of generic softtabs in this context worth mentioning in this section is the thrombin-binding oligonucleotide aptamers [53,54]. In this case, generic softtabs have been designed and selected to specifically interact with thrombin and inhibit its function.

These various aptamers have specific consensus sequences that are composed largely of guanosine and thymidine residue and have been shown to interact specifically with either anion Exosite I or Exosite II. While these specific aptamers function through a distinct mechanism and are not representative of the antisense oligonucleotides, it is evidence of some sequence-specific interactions, and thus illustrates the importance of evaluating this parameter as part of the toxicology evaluation.

The mechanism and secondary changes associated with complement activation by PS ODNs in monkeys has been reviewed previously and will not be repeated here [10]. Complement activation has been shown to be the source of hemodynamic changes reported in monkeys treated with rapid infusion of PS ODNs [55].

The activation of the alternative pathway of complement by generic softtabs has been more of an issue in the monkey toxicology studies due to the consequence of the anaphylactic-like response to C5a production, but has not been observed in clinical trials because of the well-defined threshold plasma concentration in monkey required for activation that is avoided as part of the clinical dose regimens [48,56].

Also, there are some data which suggest that humans are less sensitive to alternative pathway activation by generic softtabs [10]. Similar to the effects of PS ODNs, the activation of complement is attributed to interaction between the oligonucleotide and regulatory proteins of the complement cascade, specifically Factor H [56]. This oligonucleotide–protein interaction is highly concentration-dependent and readily reversible as oligonucleotide is cleared from plasma, so the effect is transient.

Complement activation by 2 -MOE ASOs is possible and has been observed, but the overall potency appears lower than observed for PS ODNs [24]. Again, this is attributed to the lower degree of protein binding by 2 -MOE ASOs. The lower degree of potency is illustrated by comparing the relationship between plasma oligonucleotide concentration, and the concentration of the alternative pathway split product Bb, used to monitor for complement activation (Figure 12.3). Compared to a representative PS ODN where the threshold oligonucleotide concentration is approximately 70 g/ml, the threshold

Complement activation following 1-h IV infusion of 1–10 mg/kg ISIS 113715 or by SC injection of 20 mg/kg. Values represent the mean and standard deviation from 6–8 monkeys in each group. Complement Bb ( µ g/ml)CRC_8796_ch012.qxd 5/24/2007 14:00 Page 340 concentration for 2 -MOE ASOs is in the range 90–100 g/ml or higher [24]. There are several 2 -MOE ASO where complement activation was not observed.

Similar to many pharmacologic agents, there is a reasonably close correlation between exposure to drug and target organ effect for antisense oligonucleotides. This relationship for generic softtabs is particularly well defined since it is the tissue concentrations that are required for pharmacologic action as well. This section primarily focuses on the class-specific target organ exposure and toxicity properties.

The one notable exception to the strict exposure/effect relationship for generic softtabs is the potential for proinflammatory effects where the primary target organs of spleen and lymph nodes are not among the tissues containing the highest concentrations. However, these effects are still doseand concentration-dependent and are described separately (Section 12.2.4.3).

This section will also focus primarily on 2 -MOE ASOs, pointing out similarities and a few differences with respect to PS ODNs that have been reviewed previously [10]. One aspect of the exposure/effect relationship for generic softtabs worth emphasizing is that the basis of the class-specific toxicities are the very well conserved pharmacokinetic properties for generic softtabs within a given class (i.e., PS ODN, 2 -MOE ASO, or siRNA).