Summary of Nonclinical Pharmacology Studies in Monkeys

Toxicokinetics (TK) is generation of kinetic data for systemic exposure and toxicity assessment of the drug. These studies help us to estimate the observed toxicity to that dose. TK evaluation is very important in drug development phase in both regulatory and scientific perspective. There are several guidelines to conduct TK study in animals recommended by regulatory bodies (OECD). TK evaluation is useful in selection of dose, dosing form, alternative dosing route, evaluation of toxicological mechanism, and also used for the setting safe dose level in clinical phases. This TK studies also used to reduces the animal number (replacement, reduction and refinement). On the other hand, TK data are practically used for the purpose of drug discovery such as lead-optimization and candidate-selection. Email:marketing@medicilon.com.cn Web:www.medicilon.com

The studies presented demonstrate that RB006 is a potent anticoagulant in monkeys, capable of achieving essentially complete inhibition of FDC activity for 24 hours or longer following a single bolus IV injection of the drug at supra-clinical doses. Comparison of in vitro studies of the anticoagulant activity of RB006 in monkeys with the APTT and toxicokinetic data from this safety pharmacology study demonstrates a good correspondence between the expected and observed prolongation of the APTT versus the plasma RB006 concentration. Therefore, the APTT assay will serve as a useful tool to monitor anticoagulation induced by RB006 administration. The similarity between the in vitro human and monkey RB006- APTT dose-response curves suggests that the data derived from this monkey study (REGl -TOXOOl), as well as the large general toxicity study conducted in monkeys (REGl -TOX003) will serve as a useful guide in predicting the human response to administration of RB006. Finally, the APTT and toxicokinetic data from REGl -TOXOOl demonstrate that RB007 is a very effective antidote for RB006. Within 15 minutes following bolus IV administration of RB007 in RB006-treated animals, mean APTT times returned to pre-RB006 treatment levels and remained at this baseline level for the entire monitoring period (up to 120 hours). The observed neutralization of the RB006 anticoagulant activity by RB007 was fully supported by toxicokinetic data, and is consistent with the measured thermodynamic stability of the RB006-RB007 complex. Toxicokinetic studies demonstrated that free RB006 levels decreased to below the LLOQ of the assay within 15 minutes post RB007 administration, concomitant with a significant rise in the concentration of complexed RB006, and without an appreciable increase in free RB006 levels for the duration of the toxicokinetic analysis (24 hours post RB006 administration). Therefore, the data obtained in monkey studies demonstrated that the REGl anticoagulation system behaves as intended with respect to achieving stable, durable and monitorable anticoagulation from a single IV injection of the aptamer, followed by rapid, complete, and durable neutralization of aptamer activity upon IV bolus injection of the antidote. This performance of the REGl anticoagulation system was achieved at low to high multiples of the intended clinical dose range (i.e., appropriate doses for toxicity studies), but without adverse effects on the animals.

REGl Toxicokinetics

Bioanalytical methods were developed and validated to enable quantification of the concentrations of free aptamer (RB006), free antidote and aptamer/antidote (RB006/RB007) complex in plasma from monkeys and mice. These methods were applied to analysis of samples collected from the safety pharmacology study in monkeys (Study No. REG1-TOX001), the 14-day study in mice, and the single/repeat-dose study in monkeys. For all three studies, separate groups of animals were included that received either the aptamer alone, or the antidote alone, or the aptamer followed 3 hours later by the antidote. Multiple dose levels of each treatment condition were tested in all of the studies, and two of these studies (the 14-day study in mice and the single/repeat-dose study in monkeys) also employed repeated administration of the test articles. The dose levels of the aptamer tested in these studies ranged from 0.25 to 45 mg/kg in monkeys and 2.5 to 22.5 mg/kg in mice. The doses of the antidote tested were twice those of the aptamer (i.e., up to 90 mg/kg in monkeys and 45 mg/kg in mice). This ratio is analogous to that intended for use in clinical trials. For all three studies, the toxicokinetic results were similar with respect to documenting the following properties of the REGl anticoagulation system: • The plasma concentrations of the aptamer following intravenous injection were dose- proportional over a broad dose range, with a modest degree of inter-animal variation. No gender differences were apparent in either monkeys or mice.

• The clearance of the aptamer from plasma was relatively slow (i.e., the estimated half- life was at least 12 hours in monkeys and ~8 hours in mice). This slow clearance was expected based on the PEGylated structure of the aptamer and is consistent with literature reports on the pharmacokinetics of other PEGylated oligonucleotides. The minimal clearance of the aptamer, in combination with its high factor DC inhibitory potency, provided for a relatively stable degree of anticoagulation over a 6-hour period, based on measurement of pharmacodynamic markers, i.e., activated partial thromboplastin time and activated clotting time. This profile is a desirable property of the aptamer component of the REGl anticoagulation system.

• Intravenous injection of the antidote alone (without prior treatment with aptamer) yielded very low levels in plasma, even at the first sampling time following injection (10-15 minutes). The antidote levels measured at these early times were orders of magnitude lower than those of the aptamer (i.e., as compared to the aptamer levels in those groups that had received aptamer alone) despite the fact that the antidote dose levels were twice as high. Collectively, the data for the antidote indicate that it has a very short half-life in plasma when given alone. No accumulation of the antidote in plasma occurred when it was administered at a relatively high dose level (30 mg/kg) to monkeys every other day for 7 doses (14 days).

• For the groups that received aptamer followed 3 hours later by the antidote (i.e., the complete REGl anticoagulation system), the concentration of free aptamer was sharply reduced within minutes following antidote administration to below or slightly above the limits of quantification (using a highly sensitive hybridization-type assay), indicating complete binding of the circulating aptamer by the antidote. As was seen with the antidote-alone treatment, there were very low levels of free antidote under these conditions. The binding of the aptamer by the antidote was associated with virtually complete neutralization of aptamer activity (i.e., normalization of coagulation parameters), consistent with the intended performance of the REGl anticoagulation system.

• Concurrent with elimination of free aptamer, the aptamer/antidote complex was detected in plasma at levels consistent with the complete binding of aptamer by the antidote. The complex was eliminated from plasma at a rate slightly faster than that of the free aptamer (i.e., by comparison to the rate of aptamer clearance in groups treated with aptamer only) but at a much lower rate than free antidote, as would be expected from the presence of the polyethylene glycol moiety within the complex (derived from the aptamer). Extensive elimination of the aptamer/antidote complex from plasma was evident within 21 hours following antidote dosing. With repeated administration of the aptamer and antidote (the REGl coagulation system) to monkeys every day for two weeks, there was no accumulation of the complex in the blood or the free aptamer, no change in aptamer pharmacokinetics (i.e., during the period prior to antidote dosing), and no evidence of cumulative anticoagulation exerted by the aptamer.

• The only difference between the pharmacokinetics in mice and monkeys was the moderately longer half-life of the aptamer in monkeys (at least 12 hours, compared to ~8 hours in mice).