1496 YOKOTE K et al. Circ J 2017; 81: 1496 – 1505 ORIGINAL ARTICLE doi: 10.1253/circj.CJ-16-1310 Ischemic Heart Disease

Efficacy and Safety of Bococizumab (RN316/PF-04950615), a Against Proprotein Convertase Subtilisin/Kexin Type 9, in Hypercholesterolemic Japanese Subjects Receiving a Stable Dose of or Treatment-Naive ― Results From a Randomized, Placebo-Controlled, Dose-Ranging Study ―

Koutaro Yokote, MD, PhD; Shigeto Kanada, MD; Osamu Matsuoka, MD, PhD; Hisakuni Sekino, MD, PhD; Keiji Imai; Junichi Tabira; Nobushige Matsuoka, PhD; Sandip Chaudhuri, MD; Tamio Teramoto, MD, PhD

Background: A Phase 2, dose-ranging study of bococizumab, a monoclonal anti-proprotein convertase subtilisin/kexin type 9 antibody, was conducted in Japanese subjects to assess its efficacy, safety, and tolerability in this population.

Methods and Results: Two different hypercholesterolemic study populations were enrolled concurrently: Japanese subjects with uncontrolled low-density lipoprotein (LDL-C) despite atorvastatin treatment (LDL-C ≥100 mg/dL; n=121), and Japanese subjects naive to lipid-lowering agents and with LDL-C ≥130 mg/dL (n=97). Subjects within each study population were randomized to bococizumab 50, 100, or 150 mg, or placebo, q14D for 16 weeks; an open-label 10 mg daily arm was also included for the atorvastatin-treated population. Significant, dose-dependent reductions in fasting LDL-C levels were observed in all bococizumab arms of both study populations at Weeks 12 and 16 (adjusted mean percent changes from baseline: 54.1–76.7% for atorvastatin- treated subjects and 47.7–66.8% for treatment-naive subjects; P<0.001 vs. placebo for all). Bococizumab also caused dose-dependent changes in other lipid parameters in both study populations at Weeks 12 and 16. No serious adverse events (AEs) related to bococizumab treatment occurred and all treatment-emergent AEs were mild or moderate in severity. No dose-dependent relationship between bococizumab treatment and development of anti-drug antibodies was observed.

Conclusions: Bococizumab was well tolerated and significantly reduced fasting LDL-C in atorvastatin-treated and treatment-naive hypercholesterolemic Japanese subjects. (Clinicaltrials.gov identifier: NCT02055976.)

Key Words: Bococizumab; ; Japan; PCSK9 inhibitor; Phase 2 study

ypercholesterolemia caused by high levels of low- density lipoprotein cholesterol (LDL-C) has been Editorial p 1386 H demonstrated by numerous epidemiologic studies to be a risk factor for atherosclerotic cardiovascular (CV) involved in regulation of serum LDL-C levels via its bind- disease, including myocardial infarction and ischemic ing to, and downregulation of, the LDL receptor (LDLR) stroke.1,2 Furthermore, numerous prospective clinical out- expressed on hepatocytes. This reduction in cell-surface come trials and meta-analyses have established that lower- LDLR results in reduced hepatocellular uptake of LDL-C ing LDL-C decreases CV morbidity and mortality.3 and, consequently, higher LDL-C levels within the circula- Proprotein convertase subtilisin/kexin type 9 (PCSK9) is tion. Bococizumab (RN316/PF-04950615) is a humanized

Received January 24, 2017; revised manuscript received April 10, 2017; accepted April 13, 2017; released online May 23, 2017 Time for primary review: 24 days Department of Clinical Cell Biology and Medicine, Chiba University Graduate School of Medicine, Chiba (K.Y.); Heishinkai Medical Group Incorporated, Osaka (S.K.); ToCROM Clinic, Heishinkai Medical Group Incorporated, Tokyo (O.M.); Sekino Hospital, Tokyo (H.S.); Development Japan, Japan Inc., Tokyo (K.I., J.T., N.M.), Japan; Worldwide Research and Development, Pfizer Ltd, Sandwich (S.C.), UK; and Teikyo Academic Research Center, Teikyo University, Tokyo (T.T.), Japan Mailing address: Professor Koutaro Yokote, MD, PhD, Department of Clinical Cell Biology and Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail: [email protected] ISSN-1346-9843 All rights are reserved to the Japanese Circulation Society. For permissions, please e-mail: [email protected]

Circulation Journal Vol.81, October 2017 Bococizumab: Japanese Phase 2 Dose-Ranging Study 1497 monoclonal anti-PCSK9 antibody with high affinity for Study Treatment the evolutionarily conserved LDLR-binding domain of The doses of bococizumab selected for this study were PCSK9.4 Bococizumab has been investigated worldwide based on a global Phase 2b dose-ranging study and PK/PD as an add-on therapy to the standard of care in patients modeling.7,8 During the treatment period (Days 1–113), with hypercholesterolemia or heterozygous familial subjects randomized to bococizumab received their assigned hypercholesterolemia. Phase 1 and Phase 2a studies have dose by subcutaneous administration q14D, whereas those shown that bococizumab is well tolerated and provides randomized to open-label ezetimibe took a daily 10-mg substantial reductions in fasting LDL-C levels when tablet after food. Subjects received the assigned dose of administered either as single or multiple doses, both bococizumab for the first 12 weeks, with dose adjustment alone and in combination with other lipid-lowering on or after Week 12 (Day 85) for any subject who met the agents.5,6 Based on the results of a global Phase 2b study7 dose-adjustment criteria. A subject’s scheduled dose of and a modeling simulation of the available pharmacokinetic bococizumab was adjusted if they displayed 2 consecutive (PK) and pharmacodynamic (PD) data,8 a twice-monthly LDL-C measurements ≤25 mg/dL at the end of the dosing (q14D) regimen is considered the preferred dosing interval at Weeks 8, 10, and 12, with the dose being adjusted frequency for bococizumab.7 at Week 12 (Day 85) or Week 14 (Day 99) as follows: The current Phase 2 study conducted in Japan bococizumab 150 mg→75 mg; bococizumab 100 mg→50 mg; (NCT02055976) evaluated the efficacy, safety, and bococizumab 50 mg→placebo. All subjects in the atorvastatin- tolerability of bococizumab when administered subcuta- treated population continued to self-administer their daily neously q14D. dose of atorvastatin as background therapy throughout the study period. Only subjects without changes in their Methods daily dose of atorvastatin for at least 6 weeks prior to screening were included, and each subject’s daily atorvas- Subjects tatin dose could not be adjusted during the trial unless an Men and women aged ≥20 years and able to comply with associated serious adverse event (SAE) occurred. The all study procedures were enrolled and included if they, atorvastatin-treated subjects were also allowed to remain based on the Japan Atherosclerosis Society guidelines for on stable doses of lipid-lowering drugs other than atorvas- prevention of atherosclerotic CV diseases,2 displayed tatin, ezetimibe, and (e.g., resins, , and fasting LDL-C ≥100 mg/dL and fasting triglycerides (TG) omega-3 fatty acids) if these had already been initiated and ≤400 mg/dL despite taking a stable dose of atorvastatin if the dose had remained unchanged for at least 6 weeks (‘atorvastatin-treated subjects’), or if they had not received prior to screening. No dose adjustments for these any lipid-lowering agents and displayed fasting LDL-C concomitant lipid-lowering were allowed and TG levels of ≥130 mg/dL and ≤400 mg/dL, respectively during the trial. (‘treatment-naive subjects’). Greater detail on inclusion and exclusion criteria can be found in Supplementary File 1. Endpoints The co-primary efficacy endpoints were the percent change Study Design from baseline in fasting LDL-C at Week 12 (Day 85) and This was a double-blind, parallel-group, placebo-controlled, Week 16 (Day 113). Secondary efficacy endpoints included randomized, dose-ranging trial conducted in Japan and the absolute value and absolute change from baseline in involving 2 different study populations with a treatment LDL-C at Weeks 12 and 16, as well as the absolute value, duration of 16 weeks (4 months). A total of 9 parallel treat- absolute change, and percent change from baseline in other ment arms with 24 subjects per arm was planned (Figure 1). serum lipid parameters (total cholesterol, high-density The atorvastatin plus ezetimibe 10 mg daily arm was open- lipoprotein cholesterol [HDL-C], TG, non-HDL-C, total label, whereas the 8 other arms were blinded. Institutional cholesterol/HDL-C ratio, apolipoprotein B [apoB], apoA-I, review board(s) and/or independent ethics committee(s) at apoA-II, lipoprotein(a) [Lp(a)], very low-density lipoprotein each of the 9 participating centers approved the protocol, cholesterol [VLDL-C], and apoB/apoA-I ratio) and the and all subjects provided written informed consent before proportion of subjects achieving specific LDL-C values initiation of study-specific procedures. The study was during the treatment period (<100, <70, <40, <25, and conducted in accordance with the principles set forth in the <10 mg/dL). Safety endpoints included the incidence of International Ethical Guidelines for Biomedical Research adverse events (AEs), clinical laboratory abnormalities, Involving Human Subjects, the Guidelines for Good and anti-drug antibodies (ADAs). Clinical Practice, and the Declaration of Helsinki. A computer-generated randomization schedule was Laboratory Methods used to assign a study drug in each of the 2 study popula- Serum LDL-C measurements used for dose adjustments tions, with subjects in each population stratified into and analyzing lipid-lowering effects were calculated by the ‘low LDL-C’ (≤130 mg/dL for atorvastatin-treated subjects Friedewald formula, with values obtained by direct and ≤150 mg/dL for treatment-naive subjects) or ‘high measurement used if the calculated value was <25 mg/dL LDL-C’ (>130 mg/dL for atorvastatin-treated subjects or if TG were ≥400 mg/dL. and >150 mg/dL for treatment-naive subjects) groups prior to randomization. Statistical Analysis The participating subjects, investigators, site staff, and The full analysis set was the primary analysis set for the the sponsor (excluding prespecified, unblinded site staff analyses of efficacy data and included all randomized such as unblinded pharmacists involved in study drug subjects administered ≥1 dose of study treatment. For all preparation) remained blinded to the assigned study drug efficacy analyses, subjects were analyzed according to and lipid results (except TG) for the duration of the study. their randomized dose regardless of any subsequent dose reductions. The safety analysis set included all subjects

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Figure 1. (A,B) Subject disposition in the 2 study populations. AE, adverse event; LDL-C, low-density lipoprotein cholesterol.

administered ≥1 dose of study treatment. All efficacy and least 1 dose of study treatment (Figure 1B). Of the subjects safety analyses were conducted in each study population receiving bococizumab, dose titrations according to the separately. The co-primary endpoints were compared with prespecified algorithm occurred in 3 subjects (13%) in the the corresponding placebo arm using a mixed-effects 150-mg arm. model for repeated measures (MMRM). The secondary efficacy endpoints were analyzed using the same MMRM Baseline Demographics analysis. For the statistical test of the co-primary endpoints, All subjects were ethnically Japanese, the age range was multiplicity was adjusted to control family-wise type I 26–80 years, and 54% were men. The baseline characteristics error at α=0.025 (one-sided). The model-adjusted mean within each study population were similar across the percent changes from baseline are reported. Data from the treatment arms (Table 1). Baseline mean LDL-C concentra- open-label ezetimibe arm were summarized descriptively. tions ranged from 123.85 to 135.90 mg/dL in the atorvastatin- AEs and SAEs were summarized using the ICH Medical treated subjects, and from 155.22 to 164.22 mg/dL in the Dictionary for Regulatory Activities (MedDRA) version treatment-naive subjects. 17.1. Safety laboratory parameters and incidence of positive ADA results were summarized. Efficacy Atorvastatin-Treated Subjects Significant dose-dependent Results reductions in fasting LDL-C levels were observed in all bococizumab treatment arms compared with placebo at Subject Disposition both Week 12 and Week 16 (adjusted mean percent reduc- Atorvastatin-Treated Subjects A total of 121 subjects tions of 54.1–76.7%; P<0.001 for all 3 bococizumab arms were randomized across 5 treatment arms and all received at both time points; Figure 2A). The adjusted mean absolute at least 1 dose of study treatment (Figure 1A). Of the reductions in fasting LDL-C levels observed in the bococi- subjects receiving bococizumab, dose titrations according zumab arms were also dose-dependent and significant to the prespecified algorithm occurred in 1 subject (4%) in compared with placebo at Week 12 and Week 16 (P<0.001 the 50-mg arm, 8 subjects (33%) in the 100-mg arm, and 8 for all 3 arms at both time points; Table S1). The down- subjects (33%) in the 150-mg arm. titration of dose on or after Week 12 was reflected in the Treatment-Naive Subjects A total of 97 subjects were LDL-C-lowering effects observed in the bococizumab 100- randomized across the 4 treatment arms and all received at mg and 150-mg arms at Week 16 being slightly reduced

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Table 1. Baseline Demographics of Atorvastatin-Treated and Treatment-Naive Japanese Study Subjects Atorvastatin-treated subjects* Bococizumab Variable† Placebo Ezetimibe (n=26) 50 mg 100 mg 150 mg 10 mg (n=22) (n=25) (n=24) (n=24) Age (years) 58.2±11.2 55.9±8.9 59.6±12.2 59.6±8.6 58.2±10.7 Male sex 13 (50.0) 17 (68.0) 14 (58.3) 12 (50.0) 12 (54.5) Weight (kg) 67.6±13.4 67.7±13.0 66.4±15.3 63.5±11.6 66.6±10.9 BMI (kg/m2) 25.6±3.3 24.4±3.4 24.9±4.1 24.2±3.0 24.7±2.6 CVD risk factors present‡ Hypertension 13 (50.0) 8 (32.0) 12 (50.0) 10 (41.7) 8 (36.4) DM 4 (15.4) 3 (12.0) 9 (37.5) 7 (29.2) 4 (18.2) CAD 1 (3.8) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Stroke 0 (0.0) 1 (4.0) 0 (0.0) 0 (0.0) 0 (0.0) PAD 0 (0.0) 0 (0.0) 1 (4.2) 0 (0.0) 1 (4.5) CKD 2 (7.7) 3 (12.0) 3 (12.5) 2 (8.3) 4 (18.2) LDL-C (mg/dL) 135.90±24.70 135.36±23.65 123.85±20.59 129.19±17.77 135.36±24.75 Total cholesterol (mg/dL) 222.44±31.32 214.16±30.66 210.29±21.59 211.71±23.07 222.84±25.40 HDL-C (mg/dL) 58.54±14.68 54.58±9.05 54.75±11.79 57.00±12.86 56.98±9.61 Triglycerides (mg/dL) 128.50 107.50 141.00 114.25 156.25 (106.00, 185.50) (86.00, 138.00) (112.75, 180.75) (85.25, 152.25) (110.00, 177.00) Non-HDL-C (mg/dL) 163.90±25.27 159.58±29.03 155.54±20.71 154.71±22.51 165.86±26.66 PCSK9 (ng/mL) 269.19±66.84 266.64±61.55 261.23±72.36 285.96±75.79 –

Treatment-naive subjects Bococizumab Variable† Placebo (n=23) 50 mg 100 mg 150 mg (n=25) (n=25) (n=24) Age (years) 58.2±11.7 60.1±8.4 57.7±10.6 53.3±8.1 Male sex 13 (56.5) 12 (48.0) 10 (40.0) 15 (62.5) Weight (kg) 63.4±14.0 65.3±12.5 65.9±11.9 68.0±17.0 BMI (kg/m2) 23.7±3.9 24.9±3.1 25.6±3.8 24.7±5.1 CVD risk factors present‡ Hypertension 8 (34.8) 9 (36.0) 8 (32.0) 8 (33.3) DM 3 (13.0) 4 (16.0) 3 (12.0) 2 (8.3) CAD 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Stroke 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) PAD 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) CKD 1 (4.3) 0 (0.0) 1 (4.0) 1 (4.2) LDL-C (mg/dL) 155.22±23.10 164.22±25.84 158.00±20.00 159.90±19.80 Total cholesterol (mg/dL) 242.04±22.96 245.94±30.43 243.26±21.97 244.19±29.99 HDL-C (mg/dL) 60.87±10.61 57.22±14.15 60.40±13.23 62.02±21.46 Triglycerides (mg/dL) 125.00 111.50 109.50 96.75 (99.00, 151.00) (83.00, 159.00) (95.50, 137.00) (87.75, 139.00) Non-HDL-C (mg/dL) 181.17±23.58 188.72±30.22 182.86±22.84 182.17±21.22 PCSK9 (ng/mL) 210.22±48.55 228.80±50.00 215.50±43.01 234.42±77.54 *Hypercholesterolemic subjects with fasting LDL-C ≥100 mg/dL and not controlled by stable dose of atorvastatin. †Values are mean±standard deviation or n (%), except values for triglycerides, which are presented as median (Q1, Q3). ‡According to the coding definitions of the ICH Medical Dictionary for Regulatory Activities (MedDRA) version 17.1. BMI, body mass index; CAD, coronary artery disease; CKD, chronic kidney disease; CVD, ; DM, diabetes mellitus; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipopro- tein cholesterol; PAD, peripheral arterial disease; PCSK9, proprotein convertase subtilisin/kexin type 9.

compared with those observed at Week 12. Dose-dependent the treatment period. In the open-label ezetimibe arm, the reductions in fasting LDL-C levels were approximately unadjusted mean percent change (standard deviation) maximal from Week 3 onwards (Figure 3A). Compared from baseline in fasting LDL-C was −18.6% (16.0) at with the bococizumab 50-mg and 100-mg arms, the bococi- Week 12 and −20.6% (18.7) at Week 16 (Figure S1A). zumab 150-mg arm showed stable LDL-C reduction during All subjects receiving bococizumab (100%) achieved

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compared with placebo at Week 12 and Week 16 (P<0.001 for all 3 arms at both time points; Table S3). Dose-dependent reductions in fasting LDL-C levels were approximately maximal from Week 3 onwards (Figure 3B). Compared with the bococizumab 50-mg arm, stable LDL-C reductions were observed in the bococizumab 100-mg and 150-mg arms. With the exception of 2 subjects in the 50-mg arm, all subjects receiving bococizumab achieved LDL-C <100 mg/dL during the treatment period, with 64.9%, 92.0%, and 87.5% in the 50-mg, 100-mg, and 150-mg arms, respectively, also achieving LDL-C <70 mg/dL (Table S2). During the treatment period, an LDL-C level <25 mg/dL was observed in 20.8% of subjects in the bococizumab 150-mg arm, but was not observed in the other treatment arms. Bococizumab treatment also caused significant dose- dependent changes in lipid parameters other than LDL-C at both Week 12 and Week 16 (Tables 3,S3). Unadjusted mean percent changes at both time points showed similar patterns to the adjusted values for these parameters (Figure S2B).

Safety Atorvastatin-Treated Subjects There were no deaths in this subgroup. Treatment-emergent SAEs occurred in 1 subject in the bococizumab 50-mg arm (cellulitis) and 1 subject in the placebo arm (angina pectoris), both of which were assessed by the investigator as not being related to the study drug (Table 4). All treatment-emergent AEs were of mild or moderate severity, with injection-site erythema, injection-site pruritus, nasopharyngitis, and upper respira- tory tract inflammation being the most commonly reported AEs in the bococizumab treatment arms. Injection-site erythema and injection-site pruritus were the most frequent treatment-related AEs, although their severity was mild and no subject discontinued study treatment because of an injection-site AE. There was no difference in the incidence of AEs when comparing subjects who achieved fasting LDL-C levels ≤25 mg/dL with those who did not achieve this level (data not shown). There was no increase in Figure 2. (A,B) Primary analysis: adjusted mean percent abnormal function tests over time and no clinically changes in LDL-C in the 2 study populations at Weeks 12 and important changes in any treatment arm. No cases of Hy’s 16. *P<0.001 vs. placebo. CI, confidence interval; LDL-C, law were observed. low-density lipoprotein cholesterol. A total of 2 of the 121 randomized subjects (2%) discon- tinued treatment because of AEs considered related to the study drug: 1 receiving bococizumab 100 mg experienced a mild gastrointestinal disorder, and 1 receiving bococi- LDL-C <100 mg/dL during the treatment period, with all zumab 150 mg experienced moderate nausea and dizziness; except 1 subject (in the 50-mg arm) also achieving LDL-C these events were confirmed to have resolved within 15 days <70 mg/dL (Table S2). During the treatment period, an and 3 days after onset, respectively. LDL-C level <25 mg/dL was observed in 12.0%, 66.7%, Treatment-Naive Subjects There were no deaths in this and 58.3% of subjects in the bococizumab 50-mg, 100-mg, subgroup. Two SAEs occurred in 2 subjects, both of which and 150-mg arms, respectively. were considered unrelated to study drug: 1 subject in the Bococizumab treatment also caused significant changes bococizumab 100-mg arm experienced a severe SAE of in lipid parameters other than LDL-C at both Week 12 spinal compression fracture, and 1 subject in the bococi- and Week 16 (Tables 2,S1). Unadjusted mean percent zumab 50-mg arm was diagnosed with colon cancer in the changes at both time points showed similar patterns to the follow-up period, with this event being confirmed as adjusted values for these parameters (Figure S2A). serious after withdrawal from the study (Table 4). All Treatment-Naive Subjects Significant dose-dependent other treatment-emergent AEs had a maximum severity of reductions in fasting LDL-C levels were observed in all mild or moderate, with the most commonly reported being bococizumab treatment arms compared with placebo at injection-site erythema, injection-site pruritus, nasophar- both Week 12 and Week 16 (adjusted mean percent reduc- yngitis, and pharyngitis. There was no difference in the tions of 47.7–66.8%; P<0.001 for all 3 bococizumab arms incidence of AEs when comparing subjects who achieved at both time points; Figure 2B). The adjusted mean absolute fasting LDL-C levels ≤25 mg/dL with those who did not reductions in fasting LDL-C levels observed in the bococi- achieve this level (data not shown). Four subjects zumab arms were also dose-dependent and significant displayed elevated aspartate aminotransferase and/or alanine

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Figure 3. (A,B) Time course of unadjusted mean percent changes in LDL-C in the 2 study populations. LDL-C, low-density lipoprotein cholesterol; SD, standard deviation.

Table 2. Adjusted Mean Percent Changes in the Lipid Profile of Hypercholesterolemic Atorvastatin-Treated Japanese Study Subjects Following Treatment Week 12 Week 16 Bococizumab Bococizumab Variable* Placebo Placebo (n=26) 50 mg 100 mg 150 mg (n=25) 50 mg 100 mg 150 mg (n=25) (n=23) (n=22) (n=24) (n=23) (n=22) LDL-C Adjusted percent change −5.18 −55.01a −71.93a −76.71a −11.77 −54.06a −68.03a −73.15a (−10.45, (−60.39, (−77.55, (−82.35, (−16.78, (−59.17, (−73.31, (−78.49, −0.10) −49.64) −66.31) −71.07) −6.75) −48.95) −62.75) −67.81) Placebo-adjusted adjusted – −49.84 −66.75 −71.53 – −42.29 −56.26 −61.38 percent change (−57.34, (−74.52, (−79.28, (−49.42, (−63.60, (−68.73, −42.34) −58.99) −63.78) −35.17) −48.92) −54.04) TC Adjusted percent change −4.70 −36.34a −44.58a −50.59a −8.95 −34.32a −42.44a −47.71a (−8.50, (−40.17, (−48.56, (−54.62, (−12.71, (−38.11, (−46.34, (−51.69, −0.91) −32.52) −40.61) −46.56) −5.19) −30.53) −38.54) −43.73) HDL-C Adjusted percent change −6.64 6.75a 9.91a 6.65a −3.99 6.02b 12.26a 5.58b (−10.14, (3.19, (6.20, (2.86, (−8.60, (1.32, (7.44, (0.67, −3.14) 10.32) 13.62) 10.44) 0.62) 10.72) 17.09) 10.49) TG Adjusted percent change 4.03 −21.95a −17.08b −27.01a −3.22 −13.28 −27.57b −18.22c (−6.58, (−32.84, (−28.54, (−38.57, (−15.39, (−25.77, (−40.49, (−31.23, 14.63) −11.05) −5.63) −15.44) 8.95) −0.79) −14.66) −5.20) Non-HDL-C Adjusted percent change −4.09 −50.86a −63.29a −70.96a −10.42 −48.41a −61.27a −66.77a (−8.94, (−55.78, (−68.39, (−76.15, (−15.15, (−53.21, (−66.20, (−71.82, 0.76) −45.95) −58.19) −65.77) −5.68) −43.61) −56.34) −61.73) Lp(a) Adjusted percent change −19.26 −46.70b −59.85a −46.42b −23.44 −41.83a −60.23a −60.29a (−33.49, (−61.23, (−74.96, (−61.76, (−30.64, (−49.18, (−67.80, (−67.98, −5.02) −32.18) −44.74) −31.08) −16.24) −34.49) −52.66) −52.59) *Values are presented as mean (95% confidence interval). aP<0.001 vs. placebo; bP<0.01 vs. placebo; cP<0.05 vs. placebo. Abbreviations as in Table 1. aminotransferase activity (>3×the upper limit of the normal A total of 3 of the 97 randomized subjects (3%) discon- range [ULN]), all of whom had mild hepatic function tinued study treatment because of AEs considered related abnormalities at baseline. No cases of Hy’s law were observed. to the study drug: 1 receiving bococizumab 50 mg

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Table 3. Adjusted Mean Percent Changes in the Lipid Profile of Hypercholesterolemic Treatment-Naive Japanese Study Subjects Following Treatment Week 12 Week 16 Bococizumab Bococizumab Variable* Placebo Placebo (n=23) 50 mg 100 mg 150 mg (n=23) 50 mg 100 mg 150 mg (n=23) (n=22) (n=24) (n=23) (n=22) (n=23) LDL-C Adjusted percent change −1.62 −49.15a −64.24a −65.88a −0.13 −47.70a −63.47a −66.82a (−7.25, (−54.73, (−69.85, (−71.37, (−6.12, (−53.64, (−69.42, (−72.69, 4.01) −43.57) −58.64) −60.40) 5.87) −41.77) −57.52) −60.94) Placebo-adjusted adjusted – −47.53 −62.62 −64.27 – −47.58 −63.35 −66.69 percent change (−55.51, (−70.56, (−72.13, (−56.07, (−71.78, (−75.09, −39.55) −54.69) −56.41) −39.09) −54.92) −58.30) TC Adjusted percent change −3.92 −34.05a −43.44a −45.29a −1.72 −32.56a −41.74a −45.66a (−8.15, (−38.25, (−47.69, (−49.43, (−5.78, (−36.58, (−45.79, (−49.66, 0.32) −29.86) −39.20) −41.14) 2.35) −28.55) −37.68) −41.67) HDL-C Adjusted percent change −7.04 −2.93 2.52a 2.78a −7.44 1.59b 1.10b 1.46b (−11.13, (−7.02, (−1.62, (−1.23, (−11.45, (−2.43, (−2.97, (−2.52, −2.96) 1.15) 6.66) 6.79) −3.43) 5.61) 5.18) 5.44) TG Adjusted percent change −7.43 0.61 −9.94 −7.06 4.96 −8.87 1.58 −10.07d (−20.96, (−12.78, (−23.57, (−20.30, (−8.22, (−21.98, (−11.80, (−23.17, 6.11) 13.99) 3.69) 6.19) 18.15) −4.24) 14.96) 3.02) Non-HDL-C Adjusted percent change −2.78 −42.84a −58.79a −60.36a 0.58 −42.59a −56.12a −60.72a (−7.82, (−47.86, (−63.84, (−65.29, (−4.54, (−47.68, (−61.22, (−65.76, 2.26) −37.81) −53.74) −55.43) 5.70) −37.50) −51.01) −55.69) Lp(a) Adjusted percent change −17.69 −41.85b −36.91c −44.20b −15.54 −48.48a −46.86a −48.39a (−30.21, (−54.23, (−49.50, (−56.47, (−26.62, (−59.47, (−58.03, (−59.35, −5.16) −29.48) −24.32) −31.93) −4.46) −37.49) −35.68) −37.42) *Values are presented as mean (95% confidence interval). aP<0.001 vs. placebo; bP<0.01 vs. placebo; cP<0.05 vs. placebo; dP=0.056 vs. placebo. Abbreviations as in Tables 1,2.

experienced mild injection-site erythema (the same subject at both Week 12 and Week 16. The extent of the LDL-C who subsequently developed the case of colon cancer reduction observed was approximately 55–75% of the described above); 1 receiving bococizumab 50 mg expe- baseline value in atorvastatin-treated subjects and approx- rienced moderate myalgia; and 1 receiving bococizumab imately 50–65% of the baseline value in treatment-naive 100 mg displayed moderately abnormal hepatic function. subjects. The extent of LDL-C reduction we observed in Immunogenicity Overall, 74 (50.3%) of the 147 subjects the atorvastatin-treated study population was therefore receiving bococizumab were ADA-positive. A dose- approximately 5–15% greater than the reduction observed dependent relationship was not observed and there was no in the treatment-naive subjects. As well as causing a reduc- apparent difference in the incidence of ADAs between the tion in serum LDL-C levels, treatment with has been 2 study populations. In general, there were no apparent previously demonstrated to upregulate PCSK9 levels9,10 differences in LDL-C-lowering efficacy between ADA- and therefore modulation of PCSK9 by background therapy positive and ADA-negative subjects across all dosing may have influenced the extent of LDL-C reduction groups in both study populations (data not shown). However, observed in the atorvastatin-treated subjects. the onset of ADA in 1 subject (treatment-naive and receiving The LDL-C-lowering effect observed in our study is bococizumab 50 mg) occurred at the same time as a reduction greater than the effect observed in a pooled analysis of the in plasma concentration of bococizumab and attenuation 6 lipid-lowering trials within the SPIRE (Studies of PCSK9 of the LDL-C-lowering response. The incidence of injection- Inhibition and the Reduction of vascular Events) Phase 3 site reactions was higher in ADA-positive subjects than in clinical development program for bococizumab, and is also ADA-negative subjects (40.5% vs. 13.7%). Four subjects greater than the effect observed in a combined analysis of who had abnormal hepatic aminotransferase laboratory the 2 SPIRE CV outcomes trials.11–13 The pooled analysis results (>3×ULN) were ADA-positive. of 6 lipid-lowering trials showed a placebo-adjusted reduc- tion in LDL-C from baseline of 55% at Week 12 in patients Discussion receiving bococizumab 150 mg q14D, and the combined analysis of the CV outcomes trials showed a placebo- The results from this Phase 2 dose-ranging study in atorv- adjusted reduction of 59% at Week 14 in patients receiving astatin-treated and treatment-naive hypercholesterolemic bococizumab 150 mg q14D. In contrast, the atorvastatin- Japanese subjects demonstrated that bococizumab signifi- treated and treatment-naive subjects who received bococi- cantly reduced fasting LDL-C in a dose-dependent manner zumab 150 mg q14D in our study displayed reductions of

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Table 4. Summary of Study Safety Data Atorvastatin-treated subjects* Treatment-naive subjects Bococizumab Ezetimibe Bococizumab Variable Placebo Placebo 10 mg (open (n=26) 50 mg 100 mg 150 mg (n=23) 50 mg 100 mg 150 mg (n=25) (n=24) (n=24) label; n=22) (n=25) (n=25) (n=24) AE, n (%) 13 (50) 17 (68) 16 (67) 13 (54) 5 (23) 11 (48) 16 (64) 16 (64) 15 (63) Serious AE, n (%) 1 (4) 1 (4) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)** 1 (4) 0 (0) Treatment discontinuation 0 (0) 0 (0) 1 (4) 1 (4) 0 (0) 0 (0) 2 (8) 2 (8) 0 (0) because of AE, n (%) Most frequent (incidence ≥5%) treatment-emergent AEs, n (%) Injection-site erythema 0 (0) 2 (8) 6 (25) 8 (33) 0 (0) 1 (4) 4 (16) 7 (28) 6 (25) Injection-site hemorrhage 2 (8) 0 (0) 0 (0) 1 (4) 0 (0) 0 (0) 1 (4) 1 (4) 0 (0) Injection-site pain 0 (0) 0 (0) 1 (4) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 3 (13) Injection-site swelling 0 (0) 0 (0) 0 (0) 2 (8) 0 (0) 0 (0) 0 (0) 0 (0) 1 (4) Injection-site pruritus 0 (0) 2 (8) 4 (17) 7 (29) 0 (0) 0 (0) 4 (16) 6 (24) 5 (21) Pharyngitis 0 (0) 1 (4) 0 (0) 1 (4) 0 (0) 0 (0) 0 (0) 0 (0) 5 (21) Nasopharyngitis 5 (19) 5 (20) 1 (4) 1 (4) 1 (5) 4 (17) 3 (12) 4 (16) 2 (8) Upper respiratory tract inflammation 2 (8) 2 (8) 3 (13) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Headache 0 (0) 1 (4) 2 (8) 0 (0) 0 (0) 2 (9) 1 (4) 0 (0) 1 (4) Dizziness 0 (0) 0 (0) 0 (0) 2 (8) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Fall 1 (4) 1 (4) 1 (4) 0 (0) 0 (0) 1 (4) 0 (0) 3 (12) 0 (0) Pruritus 0 (0) 0 (0) 2 (8) 0 (0) 0 (0) 0 (0) 1 (4) 0 (0) 0 (0) Myalgia 1 (4) 1 (4) 1 (4) 0 (0) 0 (0) 1 (4) 3 (12) 0 (0) 0 (0) Eczema 0 (0) 1 (4) 0 (0) 0 (0) 1 (5) 1 (4) 3 (12) 0 (0) 0 (0) Blood ALP increased 0 (0) 1 (4) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 2 (8) Laboratory test abnormalities, n (%) CK >2×ULN 5 (19) 0 (0) 3 (13) 3 (13) 1 (5) 1 (4) 0 (0) 5 (20) 1 (4) AST >3×ULN 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 2 (8) 0 (0) ALT >3×ULN 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 2 (8) 2 (8) *Hypercholesterolemic subjects with LDL-C ≥100 mg/dL and not controlled by a stable dose of atorvastatin. **Colon cancer occurred in 1 subject and confirmed as serious after the subject’s withdrawal from the study. AE, adverse event; ALT, alanine aminotransferase; ALP, alka- line phosphatase; AST, aspartate aminotransferase; CK, creatine kinase; LDL-C, low-density lipoprotein cholesterol; ULN, upper limit of the normal range.

72% and 64%, respectively, at Week 12. The LDL-C- caution because the study populations, their baseline lowering effect observed in our study was also larger than characteristics, and the study designs and dosing regimens that observed in a global Phase 2b study of bococizumab show considerable differences. Nevertheless, our results in -treated subjects in whom mean baseline LDL-C add to the growing body of evidence that anti-PCSK9 levels ranged from 105 to 119 mg/dL.7 The global study monoclonal antibody therapies are able to substantially reported mean percent LDL-C reductions from baseline of reduce fasting LDL-C in Japanese patients who do not approximately 34–52% at Week 12, although it did not meet guideline-advocated LDL-C targets despite ongoing include Japanese participants. The greater LDL-C-lowering statin treatment.19 effect observed in the current study compared with the As well as desirable reductions in fasting LDL-C, the pooled analysis of the 6 lipid-lowering trials, combined serum levels of other lipid parameters were significantly analysis of the SPIRE CV outcomes trials, and the global modified by treatment with bococizumab in a dose-dependent Phase 2b study is likely related to the smaller Japanese manner; most notably, there were significant reductions in body size, with weight and body mass index being significant Lp(a). The significance of these changes in other lipid predictors of LDL response in population PK/PD modeling.8 parameters in terms of additional potential cardioprotective The LDL-C-lowering effects observed in our study were of effects is difficult to assess and may only become clearer a similar magnitude to the results from a recent meta- once data from long-term CV outcomes trials have been analysis of 24 Phase 2 and Phase 3 randomized controlled generated, although disentangling the individual effects trials investigating the anti-PCSK9 monoclonal antibodies contributed by each lipid parameter from the overall and (n=10,159), which reported a changes in such a large number of lipids may be challenging. mean reduction in LDL-C of 47.49% (95% confidence In general, the safety profile of bococizumab in our interval: 25.35–69.64%) vs. treatment with no anti-PCSK9 study was similar to that observed in the 6 SPIRE lipid- antibody.14 The LDL-C-lowering effect observed in our lowering trials, 2 SPIRE CV outcomes trials, and the study was also similar to that observed in Japanese trials global Phase 2b study,7,12,13 with the severity of most of evolocumab and alirocumab.15–18 Overall, however, direct treatment-emergent AEs being mild. The reduction in comparisons between the current study of bococizumab LDL-C was substantial and we observed no increase in the and the studies described above should be made with incidence of AEs in those who displayed a profoundly

Circulation Journal Vol.81, October 2017 1504 YOKOTE K et al. reduced level of fasting LDL-C (≤25 mg/dL). We observed Conclusions a dose-dependent relationship between bococizumab treatment and the incidence of injection-site reactions, Treatment of hypercholesterolemic Japanese subjects, who which were the most common AE observed. However, were either receiving a stable dose of atorvastatin or were these reactions were generally mild in severity and infre- naive to treatment with lipid-lowering agents, with the quently led to discontinuation of study treatment. anti-PCSK9 monoclonal antibody bococizumab led to sig- Approximately 50% of all bococizumab-treated subjects nificant dose-dependent reductions from baseline in fasting were ADA-positive at some point during the study, but LDL-C of approximately 55–75% at Week 12; similar there was no dose-dependent relationship with bococi- results were obtained at Week 16. Many other serum lipid zumab treatment and no difference in AEs between ADA- parameters were also significantly changed in a dose- positive and ADA-negative subjects, except for injection-site dependent manner following bococizumab treatment. reactions. There was also no apparent difference in mean Bococizumab treatment was well tolerated by both study LDL-C reduction between ADA-positive and ADA-negative populations, with injection-site reactions being the most subjects. However, 1 subject who developed ADAs displayed common AE. Unfortunately, the program was discontin- a concurrent reduction in LDL-C-lowering effect from ued based primarily on its long-term clinical profile. bococizumab treatment, suggesting that a potential neutralizing effect of ADAs on the efficacy of the drug Acknowledgments could not be ruled out in this subject. This observation The authors thank the other principal investigators (Drs Kazuyuki within a single subject in our study is especially interesting Mizuyama, Shintaro Yano, Harunori Oda, Hyeteok Kim, Koichi given that the pooled analysis of 6 SPIRE lipid-lowering Nakamura, and Arihiro Kiyosue), the participating subjects, and the Pfizer Operations Group for their assistance in conducting this trials reported ADA titer-dependent reductions in bococi- study, which was funded by Pfizer Japan Inc. Editorial support was zumab’s LDL-C-lowering efficacy after the Week 12 provided by David Wateridge, PhD, of Engage Scientific and was timepoint.12 Overall, bococizumab was well tolerated in funded by Pfizer. both study populations in our study and our data suggested no previously unknown risks from the use of this agent in Conflict of Interest Statement these subjects. However, on November 1, 2016, Pfizer Inc. K.Y. reports research funding from Astellas; departmental sponsor- announced the discontinuation of the global clinical devel- ship by MSD; subsidies/donations from Astellas, Boehringer Ingelheim, opment program for bococizumab.20 This decision was Bristol-Myers Squibb, Daiichi Sankyo, Sumitomo Dainippon Pharma, based on the totality of clinical information for bococi- Eli Lilly, Kyowa Hakko Kirin, Mochida Pharmaceutical, MSD, Ono Pharmaceutical, Pfizer, Shionogi, Taisho Toyama Pharmaceutical, zumab, as well as the evolving treatment and market Takeda, Mitsubishi Tanabe Pharma, Teijin Pharma, and Toyama landscape for lipid-lowering agents. Pfizer observed an Kagaku Kogyo; and honoraria from Astellas, AstraZeneca, Boehringer unanticipated attenuation of the LDL-C-lowering effect Ingelheim, Daiichi Sankyo, Sumitomo Dainippon Pharma, Eisai, over time, as well as a higher level of immunogenicity Kowa, Kowa Pharmaceutical, Kyowa Hakko Kirin, Mochida Pharmaceutical, MSD, Ono Pharmaceutical, Pfizer, Sanofi, Sanwa and higher rate of injection-site reactions than shown with Kagaku Kenkyusho, Shionogi, Taisho Toyama Pharmaceutical, the other agents in this class. Effective and durable Takeda, and Mitsubishi Tanabe Pharma. T.T. reports research grants lowering of LDL-C is a necessary prerequisite to reduce and honoraria from ASKA Pharmaceutical, Astellas, Bayer, Daiichi the occurrence of CV events such as heart attacks and Sankyo, Kissei, MSD, and Takeda; research grants from Kowa, Mochida Pharmaceutical, and Shionogi; and honoraria from Amgen, stroke in the long term. Kobayashi Pharmaceutical, Pfizer, and Sanofi. S.K., O.M., and H.S. Despite the sponsor’s decision to discontinue the clinical all report no conflict of interest. K.I., J.T., N.M., and S.C. are development of bococizumab, it is our hope that the data employees of Pfizer. generated from this Phase 2 study in Japanese subjects will help to inform the wider, ongoing discussion regarding the Funding role of PCSK9 inhibitors within treatment algorithms for This study was funded by Pfizer Japan Inc. the management of hypercholesterolemia. Even if develop- ment of bococizumab will not be taken further at this References point, the importance of continuing to expand and refine 1. Okamura T. Dyslipidemia and cardiovascular disease: A series our understanding and clinical armamentarium with of epidemiologic studies in Japanese populations. J Epidemiol regard to atherosclerotic CV disease is difficult to overstate; 2010; 20: 259 – 265. it remains the most frequent cause of death in the world.21 2. 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Proprotein convertase substilisin/kexin type 9 antago- of this study is ongoing. Pfizer has publically committed to nism reduces low-density lipoprotein cholesterol in statin-treated hypercholesterolemic nonhuman primates. J Pharmacol Exp publishing data from the clinical studies of bococizumab Ther 2012; 340: 228 – 236. despite their decision to discontinue the drug’s development 5. Gumbiner B, Udata C, Joh T, Liang H, Wan H, Shelton DL, et al. because this disclosure will allow the clinical and research The effects of single dose administration of RN316 (PF-04950615), communities to independently assess what these data a humanized IgG2a monoclonal antibody binding proprotein signify with regard to the much broader topics of PCSK9 convertase subtilisin kexin type 9, in hypercholesterolemic sub- jects treated with and without atorvastatin (abstract). Circulation inhibitors, lipid-lowering agents, and monoclonal antibody 2012; 126: A13322. therapies in general. 6. Gumbiner B, Joh T, Udata C, Forgues P, Baum CM, Garzone PD. Effects of 12 weeks of treatment with RN316 (PF-04950615),

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