Metabolic scheme of sense strand (A) and antisense strand (B). The sense strand contains three GalNAc moieties attached to a linker, which can undergo cleavage of the amide bonds at positions 1 (linker 1) through 3 (linker 3). The antisense strand (B) can undergo metabolism by exonucleases (red) that work on the end of the strand, resulting in release of mononucleotides, whereas endonucleases (blue) cleave internally and result in strands of varying lengths. The GalNAc-siRNAs evaluated in these series of studies contain consistent chemical modifications of the 2′ ribose including substitution of a fluoro (green) or O-methyl (black). The GalNAc in the ESC and above classes also contain phosphorothioate (PS) in place of phosphorodiester bonds in the six labeled positions (orange line). The earlier class of GalNAc-siRNAs that revusiran and siRNA20 are from, called STC, contain only two PS bonds at the overhang nucleotides in the antisense strand at the 3′ end.

Mean ± S.D. siRNA5 (A) or siRNA1 (B) duplex plasma concentration-time profiles based on AS and sense strand (SS) after a single subcutaneous administra tion of varying but similar dose levels in rat, monkey, and human. The data are plotted as the mean and S.D. (error bars) of n = 4, 6, or 10 data points per time point. The lower limit of quantitation (LLOQ) for assay was 10 ng/ml (dashed line). The PK parameters were described using noncompartmental methods. Statistical signifi cance was determined using a two-tailed, unpaired Student’s t test with unequal variance. There was no significant difference between AS and SS concentrations at any time points or in the derived PK parameters. (C) The Pearson correlation coefficient for the AUC (h*µg/ml) values derived from multiple GalNAc-siRNAs tested at increasing dose levels in rat (x-axis) and monkey (y-axis). (D) The Pearson correlation coefficient for the AUC (h*µg/ml) values derived from multiple GalNAc-siR NAs tested at increasing dose levels in monkey (x-axis) and human (y-axis). (E) The AUC (h*µg/ml) values derived for increasing subcutaneous dose levels of seven GalNAc-siRNA in human, and (F) the same data derived in monkey. In these panels, siRNA15 is depicted by the blue circle, siRNA2 by the red square, siRNA21 by the green upward triangle, siRNA5 by the purple downward triangle, siRNA6 by the orange diamond, siRNA7 by the black circle, and siRNA13 by the brown square. (G) The concentration-time profile for siRNA1 (blue circle; closed is after intravenous administration of 10 mg/kg, and open is after subcutaneous administration of 10 mg/kg), siRNA2 (red square; closed is after intravenous administration of 10 mg/kg, and open is after subcutaneous administration of 10 mg/kg), and siRNA5 (green tri angle; closed is after intravenous administration of 5 mg/kg, and open is after subcutaneous administration of 5 mg/kg). The table shows the derived PK parameters and the estimated % bioavailability (%F) from these studies.

Dose-normalized C_max (A–D) and AUC (E–H) values in male and female livers after subcutaneous administration to rat (A and E), subcutaneous administration to monkey (B and F), intravenous administration to rat (C and G), and intravenous administration to monkey (D and H). The horizontal line represents the median data point, and the vertical lines represent the 90% confidence intervals in (A), (B), (E), and (F), where multiple dose levels were evaluated after subcutaneous administration. For intravenous dosing, only a single dose level was administered, and the data points represent the composite mean value for n = 3 rats or monkeys. In these panels, siRNA1 is represented by a blue circle, siRNA2 by a red triangle, siRNA3 by a green upward triangle, siRNA4 by a purple downward triangle, siRNA5 by an orange diamond, siRNA6 by a black square, and siRNA7 by a brown square. Statistical significance between PK parameters across GalNAc-siRNA between males and females was evaluated using a two-tailed, unpaired Student’s t test with unequal variance, and there were no statistically significant findings. (I and J) The AUC (h*µg/g) determined in liver following increasing subcutaneous dose levels of siRNA1 through siRNA22 (depicted with varying color circles) in rat (I) and monkey (J). The black line represents the mean linear regression for the data set, whereas the blue lines represent the slopes between 2- and 0.5-fold of the mean.

Tissue distribution profile in rats after subcutaneous administration of radiolabeled siRNA2 (3 mg/kg), siRNA5 (3 mg/kg), and siRNA20 (10 mg/kg) to rats based on the estimated dose-normalized AUC (h*µg/g) (A) from the QWBA analysis. The QWBA data represents the composite value of n = 3 animals per time point. (B and C) The microautoradiography results after 3 mg/kg administration of siRNA5 to rats. In these pictures, the annotations of 10, 11, 13, and 14 represent the central vein, the centrilobular region, the periportal region, the portal vein, and the bile duct, respectively.

Metabolite profiles for siRNA2, siRNA5, siRNA6, siRNA9, and siRNA13 in homogenate prepared from monkey livers after subcutaneous dosing. The percentage of full-length (blue) compared with 3′N-1 (orange) and other active metabolites (green) is derived from the AUC profile either as a pool or discrete time point samples. In all cases, authentic standards were used to determine the quantitative levels of full-length or metabolites, and the sum of the individual components was used to derive the percentage of total.

After subcutaneous administration of [³H]siRNA2 in rats, a secondary peak of radioactivity was observed starting around 120 hours postdosing. (A) The primary and secondary radioactivity peaks, which already account for the release of tritiated water via deduction. Evaluation of the radiochromatogram from plasma samples taken at the maximum of this secondary peak (B) demonstrates that all of the radioactivity elutes early, where shortmer (>3 but <7 oligonucleotides), dimer (2 oligonucleotides), or monomers (single nucleotides and/or nucleosides) elute. There were no peaks observed at the elution times for siRNA2 (13.9 minutes) or the active metabolite AS(N-1)3′ (13.5 minutes).

Pharmacodynamics were measured after subcutaneous administration of 1 mg/kg siRNA11 to intact female rats (blue) or bile duct–cannulated female rats (red) using a commercially available ELISA kit toward the target protein in serum. The y-axis is the relative amount of target remaining, and the x-axis is the time point in hours. Each point represents the mean of three animals per time point, with the bars representing the S.D. A two-tailed, unpaired Student’s t test with unequal variance was used to evaluate whether there were any statistical differences between the intact and BDC rats at each time point. There were no statistically significant differences between the groups observed.

The time profile (x-axis) for concentrations of siRNA17 and two active metabolites was evaluated over 99 days after a single s.c. administration of 3 mg/kg in monkey (n = 3) in plasma (A) (y-axis), liver (B) (y-axis), and RISC (C) (y-axis). In addition, the measurement of target in serum samples was determined using a commercially available ELISA kit to measure pharmacodynamics as a ratio of baseline (predose) levels (D) (y-axis). Each point represents the composite mean per time point.

siRNA7 was administered by a single subcutaneous injection to mouse (red), cynomologus monkey (cyno) (black), and human (blue) at 0.3 (squares) or 3 mg/kg (circle). The extent and duration of target reduction was evaluated through serum measurements over time. The relative target (y-axis) over time (x-axis) is displayed for n = 3 (mouse and monkey) and n = 6 (human).