Study Exclusion Criteria

Data Supplement

Active menstruation, acute coronary syndrome within the last 30 days, personal history of hypercoagulable state or coagulopathy, history of thrombocytopenia associated with abnormal bleeding or bleeding risk or baseline platelet count <100,000 x 109/L, history of thrombocytosis or baseline platelet count >600,000 x 109/L, family history of coagulopathy, peptic ulcer disease

<3 years or gastrointestinal or genitourinary bleeding <3 months, severe trauma or surgery <3 months, history of stroke, intracranial bleed or aneurysm, severe persistent hypertension (systolic pressure >180 mm Hg or diastolic >110 mm Hg), baseline hemoglobin <12.0 g/dL for males or

<11.0 g/dL for females, prothrombin time (PT) > upper limit of normal (ULN), international normalized ratio >1.2, aPTT >ULN, clinically significant liver dysfunction, clinically significant renal dysfunction (creatinine clearance <60 mL/min, according to the Cockroft-Gault formula) or serum creatinine >1.5 mg/dL, treatment with other anticoagulant or antiplatelet agents < 7 days, planned cardiac catheterization or revascularization within 7 days of enrollment, use of any investigational drug within the past 30 days, or any other factor engendering an unacceptable risk of adverse effects.

1 Bleeding Questionnaire

The bleeding questionnaire was developed by Harrington, R.A., and Alexander, J.H., to assess minor bleeding in the Anti-Platelet Useful Dose (APLAUD) phase II study of lotrafiban in patients with stable coronary or cerebral atherosclerotic disease.1 The clinical utility of the questionnaire in APLAUD was supported by the stepwise increase in minor bleeding with increasing doses of lotrafiban (Table 1), which correlated closely with its known pharmacodynamic effect (Figure 1). Limitations of the questionnaire include the different antithrombotic platform, and the single bolus vs. maintenance regimen, in APLAUD vs. REG 1b.

2 3 4 5 Table 1. Bleeding Rates in APLAUD

Lotrafiban, mg Placebo (n=95) 5 (n=112) 20 (n=98) 50 (n=104) 100 (n=34) Major bleeding, % 0.9 3.1 2.9 12.1 2.1 Minor bleeding, % 35.8 53.6 61.8 69.7 34.7

Reproduced with permission from Circulation.1

6 Figure 1. Percentage ADP-induced Platelet Aggregation by Dose Level in APLAUD

Reproduced with permission from Circulation.1

7 Pharmacodynamic Monitoring of the REG1 Coagulation Response

We selected the plasma aPTT as our key pharmacodynamic efficacy outcome measure for the following reasons:

1. The plasma aPTT closely reflects the level of factor IXa inhibition of human whole

blood calibrated across a wide range of RB006 concentrations in vitro (Figure 2).

2. Earlier proof-of-concept studies using in vitro human blood treated with earlier

generations of drug and antidote have shown a strong correlation of aPTT with clot time

and F1.2 levels, while small and large animal models have demonstrated a close

relationship between the level of aPTT achieved and inhibition of thrombus formation

by RB006 in vivo.2–4

3. It is a widely available laboratory assay.

8 Figure 2. Factor IXa vs. Plasma aPTT Calibration Curve

9 Complement Activation

Anaphylactoid-like reactions—occasionally leading to hemodynamic shock and cardiac arrest— are a dose-dependent effect observed in primates receiving rapid high-dose intravenous infusions of first generation oligonucleotides, which contained specific sulfur modifications within the oligonucleotide backbone to reduce metabolism of these compounds by tissue nucleases.5,6 While use of different stabilization strategies has reduced the risk of complement activation, it is considered a potential class-specific toxicity of oligonucleotide products, and is thus typically evaluated in clinical studies of such products. This response is attributed to activation of the complement system, as evidenced by a decrease in CH50 and increase in C3a and C5a, complement split products common to the classical and alternative pathway, within minutes of starting a high-dose infusion. These effects are abolished in primates pre-treated with a recombinant inhibitor of complement activation (complement activation blocker-2) before oligonucleotide treatment.7

The alternative pathway of complement activation plays a dominant role in this response, as evidenced by the selective increase of split products specific for the alternative system (Bb fragment), but not the classical pathway (C4a fragment), in response to an oligonucleotide infusion (Figure 3).

10 Figure 3. Temporal Trend of Circulating Complement Split Products with High-dose

Infusion of the Oligonucleotide ISIS 2302

Reproduced with permission from The Journal of Pharmacology and Experimental

Therapeutics.5

Our study showed no significant increase in complement Bb levels over time at all dose levels of RB006 and RB007:

11 Table 2. Complement Bb Profile

12 13 14 P=0.48 across all dose groups from 10 minutes to 24 hours. Pairwise comparisons: p=0.04 for 15 mg vs. placebo, p=0.90 for 30 mg vs. placebo, p=0.96 for 50 mg vs. placebo and p=0.90 for 75 mg vs. placebo.

15 Platelet Response to RB006 in Phase 1a Healthy Volunteer Study

In non-human primate toxicologic studies, extreme doses of RB006 (45 mg/kg) followed by

RB007 (90 mg/kg) did not impair the aggregation response measured by light transmittance aggregometry (data on file, Regado Biosciences). In the phase 1a healthy volunteer study, subjects in the 2 highest dose levels did not experience prolongation of closure times measured using the PFA-100® analyzer (Table 3) These data support a neutral effect of RB006 and RB007 on platelet–platelet aggregation and shear-dependent platelet hemostatic capacity on non-human primates and healthy volunteers respectively.

16 Table 3. PFA-100 Closure Times in REG 1a Healthy Volunteer Study

17 Rationale for Statistical Quality Control of Coagulation Samples

The rationale for using PT as a quality control measure was 2-fold. First, RB006 and RB007 have high molecular specificity for factor IXa and are therefore not expected to prolong the PT.

Second, RB006, RB007, and their combination did not exert any effect on PT measured onsite in prior clinical studies8 or in non-clinical studies conducted in multiple species dosed at high clinical multiples (Regado Biosciences, unpublished data).

The PT (prothrombin time) and aPTT (activated partial thromboplastin time) are sensitive to several pre-analytical variables.9

1. Tube filled to <90% of fill volume

2. Hematocrit elevated >55%

3. Sample not drawn into the appropriate citrate concentration

4. Blood clot present

5. Heparin flush used

6. Improper storage with sample for aPTT testing left >4 hours at room temperature

before analysis

7. Sample stored in a "frost-free" freezer prior to testing

8. Sample grossly hemolyzed, lipemic, or icteric resulting in optical interference

Because samples were aliquoted prior to freezing and shipping, variables 1, 4, 6, and 7 would be undetected by the quality control (QC) step at the core laboratory in a multicenter trial.

In order to obtain precise aPTT estimates, we therefore applied an additional PT-based statistical

QC step prior to unblinding and pharmacodynamic analysis.

18 Robust Outlier Identification

This trimming algorithm developed by Tukey uses a statistical algorithm to detect biologically implausible outliers, and is described as follows: treat any value greater than the 75th percentile plus 1.5 times the interquartile range or less than the 25th percentile minus 1.5 times the interquartile distance as an outlier.10 This technique is considered more robust because it uses the quartile values instead of variance to describe the spread of the data, and quartiles are less influenced by extreme values. Because suboptimal handling conditions have a unidirectional effect on coagulation measures (prolongation of prothrombin time or activated partial thromboplastin time),9 we modified the algorithm to produce a 1-sided trimming effect on quartile 4 only.

SAS Macro for Robust Outlier Identification

We assumed that the majority of samples would be handled appropriately, and we verified this assumption by comparing the similarity between this PT cut-off with the core laboratory upper limit of normal. APTT measurements obtained at the same time point as a spuriously prolonged PT measurement were thus excluded from the pharmacodynamic analysis.

19 The algorithm yielded a PT cut-off value of 16.75 seconds, which approximated the reference upper limit of 15.4 seconds (Figure 4). A total of 57/931 (6.1%) core laboratory aPTT observations from the pharmacodynamic population had corresponding PT values above 16.75 seconds and were thus excluded from the pharmacodynamic analysis.

Figure 4. Histogram Showing Distribution of PT Values, Core Laboratory ULN (15.4 sec) and Trimming Algorithm Cutpoint

The proportion of core coagulation observations identified by the trimming algorithm did not differ between dose groups, implying that RB006 did not cause a dose-dependent increase in prothrombin time (Table 4).

20 Table 4. Samples from Subjects in RB006-only Group Excluded by Statistical Quality

Control

Dose Placebo 15 mg 30 mg 50 mg 75 mg group (n=8) RB006 RB006 RB006 RB006

(n=3) (n=3) (n=4) (n=4) Total no. 416 153 156 205 214 of samples Samples 6 1 0 0 0 with PT

>16.75 seconds

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