Supplementary DATA s9

18

Supplementary DATA

Bococizumab, a Monoclonal Antibody Against Proprotein Convertase Subtilisin/Kexin Type 9: Results from a Randomized, Placebo-Controlled, Dose-Ranging Study in Statin-Treated Subjects with Hypercholesterolemia

Christie M. Ballantyne, MDa,*, Joel Neutel, MDb, Anne Cropp, PharmDc, William Duggan, PhDc, Ellen Q. Wang, PhDd, David Plowchalk, PhDc, Kevin Sweeney, PhDc, Nitin Kaila, PhDc, John Vincent, MD, PhDc, Harold Bays, MDe

aDepartment of Medicine, Section of Cardiovascular Research, Division of Atherosclerosis, Baylor College of Medicine and Houston Methodist DeBakey Heart and Vascular Center, Houston, Texas, USA. bOrange County Research Center, Tustin, California, USA. cPfizer Inc., Groton, Connecticut, USA. dPfizer Inc., New York, New York, USA. eLouisville Metabolic and Atherosclerosis Research Center, Louisville, Kentucky, USA.

*Corresponding author: Christie M. Ballantyne, MD, Department of Medicine, Section of Cardiovascular Research, Division of Atherosclerosis, Baylor College of Medicine, 6565 Fannin Street, Mail Stop: A-601, Houston, Texas 77030, USA. Tel.: 713-798-5034; Fax: 713-798-3057; E-mail: .

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Supplementary Methods

Key inclusion criteria

Men and women had to meet all of the following inclusion criteria to enter the study. Subjects were aged ≥18 years, on a stable dose of statin for ≥6 weeks before screening, with no change in statin dose permitted during the study. Subjects were permitted to take non-statin lipid-lowering drugs if these were already initiated, and if the dose had remained unchanged for ≥6 weeks before screening; again no change in dose was permitted during the study. Subjects also had to have a fasting low-density lipoprotein cholesterol (LDL-C) level of ≥80 mg/dL and a fasting triglyceride level of ≤400 mg/dL at both screening visits. The fasting LDL-C value at the second screening visit (within 7 days of randomization) could not be lower than 20% of the initial value. Provision of a signed and dated informed consent document and being willing and able to comply with all study procedures were also required. All male and female subjects of child-bearing potential had to use a highly effective method of contraception during the active treatment period and for at least 63 days after the last dose of study medication.

Key exclusion criteria

Subjects with any of the following were excluded from entering the study: history of a cardiovascular or cerebrovascular event or procedure during the previous 6 months; congestive heart failure, New York Heart Association functional classes III or IV; poorly controlled type 1 or type 2 diabetes mellitus (defined as glycosylated hemoglobin [HbA1c] >9%); poorly controlled hypertension (defined as the average of 2 blood pressure measurements >160 mmHg systolic or >100 mm Hg diastolic). Subjects who had any other severe acute or chronic medical or psychiatric condition or laboratory or ECG abnormality that might have increased the risk associated with participating in this study or may might have interfered with the interpretation of study results were excluded. Pregnant or breastfeeding women were excluded. Subjects who had previously received bococizumab (RN316/PF-04950615) or who had anti-drug antibodies for bococizumab at baseline, or who had initiated or changed dose of non-lipid-lowering prescription drugs, herbal medicines or supplements (except multivitamins) within 6 weeks of screening were excluded as were subjects who initiated lipid-lowering herbs or supplements within 6 weeks of screening. Subjects who were not on stable doses of lipid-lowering herbs or supplements and those receiving medications contraindicated with statins were also excluded. Subjects who had been treated with an antibody within 6 months (or 5 half-lives of this compound), or those who had a history of allergic or anaphylactic reaction to any therapeutic or diagnostic monoclonal antibody, or molecules made of components of monoclonal antibodies, or those receiving systemic corticosteroids, or topical estrogen or androgens or mineralocorticoid replacement were excluded. Subjects who were receiving regular intramuscular (IM) injections, or those who were anticipated to be receiving IM injections, were excluded due to the possible elevations on creatine kinase (CK).

Selection of doses of study drug

The doses selected were based on a preliminary population pharmacokinetic/pharmacodynamic (PK/PD) analysis of the lipid-lowering effect of bococizumab. PK/PD modeling was performed on data from phase 1 studies, together with interim data from a phase 2a study. The simulations assumed that the distribution of baseline LDL-C and other demographic characteristics in this study were identical to those in the phase 2a study. Doses of 50 mg, 100 mg, and 150 mg subcutaneous (SC) administered Q14d, and 200 mg and 300 mg SC administered Q28d, were selected based on the simulation results.

Administration of study drug

Placebo or bococizumab was administered as a single subcutaneous (SC) injection of 2 mL for the Q14D dose group and as 2 SC injections of 2 mL each for the Q28D dose groups, into a quadrant of the abdomen, over approximately 20 seconds. The subject was blinded to all dosing.

Laboratory evaluations

The LDL-C values used in the analyses were based on calculated LDL-C reported in the Lipid Research Panel, which utilized the Friedewald calculation. Reflex ultracentrifugation was used for all LDL-C values ≤25 mg/dL. Furthermore, if ultracentrifugation results were available for any other time point they were used in the analysis instead of those derived using the Friedewald calculation. The decision was made to use calculated LDL-C rather than reflex ultracentrifugation values for most readings due to the quicker timelines for the receipt of calculated versus ultracentrifugation values. This ensured that data were available at the time of the next subject visit. The ‘reflex’ approach selected for this study permitted more certainty at the lower end of the range of the observed LDL-C values as a precautionary measure. Moreover, the intention was for the data from this study to be meaningful to the physicians involved in day-to-day practice.

Statistical analyses

The primary efficacy endpoint of the study was the absolute change from baseline to week 12 in LDL-C. The sample size required for each treatment group was 50 subjects (a total of 350 subjects). This sample size determination was based on the need for the provision of safety data and assumed a study discontinuation rate of 20% and thus 40 subjects per treatment group completing the study. Forty subjects per treatment group would provide a 95% confidence interval for obtaining a placebo-adjusted treatment difference of ± 11.1 mg/dL. This calculation was based on a standard deviation of 25 mg/dL and on using the normal approximation of the test statistic for the comparison between 2 means. This sample size of 50 subjects per treatment group also provided more than sufficient power to detect meaningful treatment differences. With α = 0.05, the power to detect a treatment difference of 25 mg/dL, 21 mg/dL, and 19 mg/dL was 99%, 95%, and 90%, respectively. Due to the exploratory nature of this study, no adjustments for the multiple comparisons with placebo were made.

Supplementary Results

Additional safety data

In general, the presence of ADAs did not have an impact on subject LDL-C, with the exception of 1 female subject (0.4%) randomized to the bococizumab 150-mg Q14d dose group. After missing her doses on days 15, 29, 43 and 57, the subject received her next dose of 150 mg on day 71 after which she continued the dosing schedule as per protocol. At baseline the serum LDL-C was 95 mg/dL, on day 15, this subject’s serum LDL-C was 17 mg/dL; by day 43, this had risen to 112 mg/dL. Following the resumption of bococizumab dosing on day 71, her LDL-C fell to 29 mg/dL on day 99. The bococizumab dose was decreased to 100 mg on day 127. She received her last dose of 100 mg on day 155. ADA titers were positive on days 113, 141, 169, 197 and 225, coincident with a decrease in plasma bococizumab concentrations to below the limit of quantification after the day 127 dose. LDL-C responses in this subject on days 113 and beyond were lower than earlier time points when the subject was ADA negative, with serum LDL-C levels ranging from 38 to 115 mg/dL. The subject had adverse events (AEs) of moderate lower abdominal pain on days 122 to 125 and mild abnormal uterine bleeding on days 123 to 158, which were deemed not related to study treatment. There is some evidence to suggest that intermittent dosing increases the incidence of ADAs and it is possible this may have been a factor with this subject given that she received a dose on day 1 and then missed the next 4 doses.

Two subjects who were receiving bococizumab (0.8%) experienced non-serious AEs of memory loss: 1 in the bococizumab 100-mg Q14d group (considered treatment-related) and 1 in the bococizumab 300-mg Q28d group (not considered treatment-related). Neither subject had their bococizumab doses reduced due to persistent LDL-C values ≤25 mg/dL nor discontinued treatment. The subject receiving bococizumab 100-mg Q14d had a minimum LDL-C value of 43 mg/dL recorded. In addition to being on concomitant statin therapy with labelling indicating that it can cause amnesia, this subject was also receiving zolpidem tartrate, which has been associated with amnesia (US Package Insert; http://products.sanofi.us/ambien/ambien.html). This subject also reported a serious AE of retinal detachment. The subject receiving bococizumab 300-mg Q28d had LDL-C levels of 18 mg/dL on day 15, 17 mg/dL on day 43, and 7 mg/dL on day 71. This subject was also receiving concomitant statin therapy with labelling indicating that it can cause amnesia.

The incidences of AEs of special interest are listed in Supplementary Table 3.

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Supplementary Tables

Supplementary Table 1

Lipoprotein efficacy outcomes from baseline at week 12 in subjects randomized to placebo or bococizumab dosed every 14 days or every 28 days*

Variable / Q14 days / Q28 days
Placebo
(n = 47) / Bococizumab (mg) / Placebo
(n = 47) / Bococizumab (mg)
50
(n = 45) / 100
(n = 44) / 150
(n = 46) / 200
(n = 48) / 300
(n = 50)
Apolipoprotein B
Mean (SD) change, mg/dL / –3.2 (16.9) / –20.4 (12.9) / –33.7 (19.2) / –33.4 (19.9) / –0.5 (21.9) / –12.2 (17.4) / –24.7 (26.2)
Mean (95% CI) placebo-adjusted change, mg/dL / --- / –18.6
(–25.4, –11.7) / –27.7
(–34.6, –20.8) / –32.1
(–39.0, –25.2) / --- / –14.6
(–22.9, –6.2) / –28.5
(–36.8, –20.2)
P-value / --- / <0.001 / <0.001 / <0.001 / --- / <0.001 / <0.001
Mean (SD) percent change, % / –2.1 (17.7) / –23.2 (14.2) / –34.9 (18.0) / –37.4 (23.5) / 1.5 (17.1) / –13.6 (20.2) / –26.0 (28.2)
Mean (95% CI) placebo- adjusted percent change, % / --- / –21.6
(–29.2, –13.9) / –30.5
(–38.2, –22.9) / –36.0
(–43.6, –28.3) / --- / –17.1
(–25.9, –8.4) / –30.7
(–39.5, –22.0)
P-value / --- / <0.001 / <0.001 / <0.001 / <0.001 / <0.001
Apolipoprotein A-I
Mean (SD) change, mg/dL / 1.5 (18.5) / 4.7 (19.0) / 8.7 (17.5) / 6.7 (24.9) / 2.1 (15.9) / 7.7 (16.8) / 7.7 (16.4)
Mean (95% CI) placebo-adjusted change, mg/dL / --- / 1.8
(–6.3, 9.9) / 5.4
(–2.7, 13.5) / 3.8
(–4.3, 11.9) / --- / 5.6
(–0.9, 12.1) / 4.4
(–2.1, 10.8)
P-value / --- / 0.667 / 0.187 / 0.352 / --- / 0.090 / 0.184
Mean (SD) percent change, % / 1.8 (12.9) / 2.9 (13.0) / 5.9 (11.5) / 9.9 (45.8) / 2.1 (10.3) / 5.6 (11.1) / 5.7 (11.6)
Mean (95% CI) placebo- adjusted percent change, % / --- / –0.7
(–10.1, 8.7) / 2.3
(–7.0, 11.7) / 6.5
(–2.9, 15.9) / --- / 3.5
(–0.8, 7.8) / 2.7
(–1.6, 6.9)
P-value / --- / 0.890 / 0.625 / 0.172 / --- / 0.109 / 0.218
Lipoprotein(a)†
Median (Q1, Q3) change, mg/dL / 0.0
(–4.4, 3.6) / –1.7
(–4.7, 0.6) / –1.6
(–7.6, 0.0) / –2.2
(–8.8, 0.1) / 0.8
(–1.9, 5.3) / 0.0
(–4.4, 0.5) / –1.9
(–8.5, 0.0)
Median (Q1, Q3) percent change, % / 0.0
(–12.0, 19.8) / –7.9
(–18.3, 5.1) / –6.3
(–24.7, 0.0) / –9.0
(–26.9, 3.3) / 3.5
(–11.1, 24.9) / 0.0
(–9.6, 5.0) / –10.7
(–27.1, 0.0)
Values are mean (SD), mean (95% CI), or median (Q1, Q3); n = number of subjects treated. CI = confidence interval; SD = standard deviation.
*These values include subjects who had their bococizumab dose reduced in the weeks prior to week 12.
†The mixed model repeated measures analysis was not undertaken for lipoprotein(a).

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Supplementary Table 2

Incidence of all-causality adverse events in subjects randomized to placebo or bococizumab dosed every 14 days, or placebo or bococizumab dosed every 28 days, with or without an LDL-C ≤25 mg/dL

Variable / Subjects with LDL-C ≤25 mg/dL
Q14 days / Q28 days
Placebo
(n = 0) / Bococizumab (mg) / Placebo
(n = 0) / Bococizumab (mg)
50
(n = 3) / 100
(n = 21) / 150
(n = 25) / 200
(n = 34) / 300
(n = 38)
AEs / 0 / 1 (33%) / 18 (86%) / 20 (80%) / 0 / 30 (88%) / 31 (82%)
Most frequent AEs (≥~10%)*
Nasopharyngitis / 0 / 0 / 3 (14%) / 2 (8%) / 0 / 5 (15%) / 3 (8%)
Injection site erythema / 0 / 0 / 2 (10%) / 3 (12%) / 0 / 4 (12%) / 3 (8%)
Upper respiratory tract infection / 0 / 0 / 3 (14%) / 0 / 0 / 5 (15%) / 2 (5%)
Diarrhea / 0 / 0 / 2 (10%) / 2 (8%) / 0 / 2 (6%) / 4 (11%)
Bronchitis / 0 / 0 / 0 / 4 (16%) / 0 / 3 (9%) / 2 (5%)
Sinusitis / 0 / 0 / 3 (14%) / 2 (8%) / 0 / 2 (6%) / 0
Vomiting / 0 / 0 / 1 (5%) / 3 (12%) / 0 / 2 (6%) / 1 (3%)
Arthralgia / 0 / 0 / 3 (14%) / 3 (12%) / 0 / 0 / 0