Supplementary Materials and Methods In vitro pharmacology: In vitro screens to identify a human CRP-specific ASO were performed using human hepatocyte-derived Hep3B cells. As CRP mRNA and protein levels are very low in unstimulated cells, to enhance the expression of CRP mRNA and protein secretion (to provide a sufficient signal for quantifying potential drug activity) the Hep3B cells were stimulated with dexamethasone plus IL-6 and IL-1β (Zhang et al., 1995; Lozanski et al., 1996). Approximately 640 ASOs complementary to the human CRP transcript were evaluated to identify the human CRP drug candidates. From these screens, several ASOs were selected that were highly potent in blunting the expression of human CRP mRNA in response to the cytokine stimulus. Subsequently, detailed dose response studies were conducted. ISIS 329993 was found to be one of the most potent inhibitors of human CRP mRNA with an IC50 value of ~8nM in cytokineinduced Hep3B cells after 48 hours treatment (Fig. S1A). Additional in vitro activity assessments were performed in human primary hepatocytes following similar cytokine induction. In this case ISIS 329993 blunted CRP mRNA levels by >70% with a derived IC50 of ~20nM (Fig. S1B). Agreement between arthritis clinical scoring system and arthritic joint damage in collageninduced arthritis: The clinical scoring system we used to monitor collagen-induced arthritis (CIA) was previously described (Brand et al., 2007). In this system a score of 0 = no evidence of erythema and swelling, 1 = erythema and mild swelling confined to the tarsals or ankle joint, 2 = erythema and mild swelling extending from the ankle to the tarsals, 3 = erythema and moderate swelling extending from the ankle to the metatarsal joints, 4 = erythema and severe swelling encompassing the ankle, foot, and digits, or ankylosis of the limb. We used multiple individuals to score mice (NRL, MAP, AJS) and we verified the accuracy of our scoring by micro- computerized tomography (micro-CT) as described by Barck et al. (2004) coupled with histological analysis of representative arthritic limbs (Fig. S2). Human Studies: A randomized, placebo-controlled, double blind dose-escalation phase 1 study was designed to evaluate the safety, tolerability, and PK profile of ISIS 329993 in healthy human volunteers. Eligible subjects were healthy, from 18 to 65 years old, and without clinically significant abnormalities in their medical history, physical examination or laboratory evaluations. Eighty-two of 425 screened subjects (Fig. S3) were randomized 3:1 to ISIS 329993 versus placebo. The study included regimens to assess the effects of ISIS 329993 delivered by SC injection versus 2-hr IV infusion, either as a single or multiple doses (Fig. S3). Doses ranged from 50 to 400 mg SC (n=4 per dose cohort) and 50 to 600 mg IV (n=4 per dose cohort). Multiple dosed individuals received drug once every other day for a total of 3 doses (Fig. S4). Subjects were followed for safety assessments until Day 15 in the single dose cohorts and Day 75 in the multiple dose cohorts. After satisfactory completion of dose escalation to 600 mg IV, the study protocol was amended to include a fifth dose regimen to explore the pharmacodynamics (PD) of ISIS 329993; this fifth regimen represents the 8 multiple dose PD subjects in Figure S3 and is referred to as Cohort-III in Figure S4. Importantly, to be enrolled in Cohort-III subjects had to have CRP levels between 2 and 10 mg/L on two independent measurements during screening. In Cohort-III 600 mg study drug was administered by 2-hr IV infusion on Days 1, 3, 5, 8 and 15 for a total of 5 doses (see Figure 6 of the main article). This dose regimen was designed to achieve an estimated drug level in hepatic tissue of 65-75% of projected steady-state by day 8 and approximately 80-85% of projected steady-state by day 17. Subjects were followed for safety assessments until Day 57 in this cohort. ISIS 329993 was dissolved in 0.9% sterile saline and was supplied by Isis Pharmaceuticals, Inc. (Carlsbad, CA) in 2-mL stoppered glass vials as a 200 mg/mL solution for single use only. Placebo was 0.9% sterile saline. The human study protocol was approved by a central institutional review board (Institutional Review Board Services, Canada), subjects were enrolled at a single site (PharmaNet Canada, Montreal), all participants gave written informed consent, and the study was performed in compliance with the standards of Good Clinical Practice and the Declaration of Helsinki in its revised edition (Washington 2002). Demographics and baseline characteristics of the overall study population and the cohort evaluated for drug pharmacodymamics (Cohort-III) are summarized in Table S1. Pharmacokinetic Analysis: Drug plasma concentrations were measured at Anapharm (Montreal, Canada) and PPD Development (Richmond, VA). Pharmacokinetic properties were assessed by non-compartmental methods for both single and multiple dosing (Phoenix WinNonlin version 6.0; Pharsight Corp, St Louis, MO). The maximum observed drug concentration (Cmax) (Table S2) and predose (trough) concentrations (Table S3) were obtained directly from the concentration-time data (Figure S5). Area-under-the-curve (AUC) was calculated using the linear trapezoidal rule. A similar plasma pharmacokinetic profile was observed for subjects in single and multiple dose cohorts and maximum plasma concentrations were dose-dependent for both routes of administration (Table S2). There was no increase in the mean maximum plasma concentration (Cmax) with repeated dosing by IV infusion or SC injection. Average Cmax values were approximately 6-fold lower by SC administration (0.42 to 12 g/mL, 50 to 400 mg) relative to the equivalent 2-hr IV infusion dose (4.4 to 41 g/mL, 50 to 400 mg) as a result of slow absorption from the injection site. Median time to maximum drug plasma concentration ranged from 3 to 8 hours by SC injection. After attaining Cmax, plasma concentrations decreased in an apparent multi-exponential fashion with time that was characterized by an initial rapid clearance from plasma followed by a relatively slow clearance phase (Figure S5). Absolute plasma bioavailability following SC administration ranged from 53 to 96%. Plasma trough levels appeared to reach near steady-state levels after 4 doses, by day 15, in Cohort III (Table S3). Safety and Laboratory Assessments: Safety and tolerability was assessed by determining the incidence, severity and dose relationship of adverse events (AEs) and changes in clinical laboratory parameters. All subjects who received at least one dose of ISIS 329993 or placebo were included in the safety analyses. Standard laboratory tests were performed by AnaPharm (Montreal, Canada) and included blood chemistry, hematology, coagulation, complement split products and urinalysis. CRP levels were measured by Gamma-Dynacare Medical Laboratories (Montreal, Canada) by rate immuno-turbidimetry (Beckman Coulter) using a high sensitivity CRP assay (lower detection limit of 0.2 mg/L). Other assessments included vital signs, ECGs, and physical examination. There were no discontinuations of drug due to adverse events. The most frequent adverse event (Table S4) was prolonged activated partial thromboplastin time (aPTT), occurring predominantly in those subjects who received ISIS 329993 by IV infusion at the highest doses of 400 and 600 mg (maximum aPTT was75 seconds at the end of the 2-hr IV infusion). Prolonged aPTTs were associated with the peak drug plasma concentration and consequently were transient in nature, returning to baseline aPTT values with time. The second most frequent adverse event was injection site reactions (ISRs) in subjects who received ISIS 329993 by SC injection at a dose of 100 mg or higher. These events were mild in severity and characterized mostly by erythema and pain. ISRs did not worsen with repeated dosing and typically resolved within several days after the injection (Table S4). There was no clear association between ISIS 329993 and changes in other assessments, including vital signs, ECGs, urinalysis and other safety laboratory evaluations such as hematology, complement split products and hepatic and renal chemistries. Statistical Analysis: Demographic and baseline characteristics were summarized descriptively. Baseline CRP for Cohort-III was defined as the median of six CRP values collected during screening (2 values), the Run-In week (3 values), and prior to dosing on Day 1. Change in CRP from baseline was summarized for subjects dosed with ISIS 329993 or placebo in Cohort-III. Supplemental References Lozanski, G, Jiang, SL, Samols, D, and Kushner, I (1996). C-Reactive Protein and Serum amyloid A mRNA stability following induction by cytokines. 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