Long-term mipomersen treatment is associated with a reduction in cardiovascular events in patients with familial hypercholesterolemia

Accepted Manuscript Long-Term Mipomersen Treatment Is Associated With a Reduction In Cardiovascular Events In Patients With Familial Hypercholesterolemia P.Barton Duell, Raul D. Santos, Bridget-Anne Kirwan, Joseph L. Witztum, Sotirios Tsimikas, John J.P. Kastelein PII:

S1933-2874(16)30209-4

DOI:

10.1016/j.jacl.2016.04.013

Reference:

JACL 934

To appear in:

Journal of Clinical Lipidology

Received Date: 18 February 2016 Revised Date:

24 April 2016

Accepted Date: 29 April 2016

Please cite this article as: Duell PB, Santos RD, Kirwan B-A, Witztum JL, Tsimikas S, Kastelein JJP, Long-Term Mipomersen Treatment Is Associated With a Reduction In Cardiovascular Events In Patients With Familial Hypercholesterolemia, Journal of Clinical Lipidology (2016), doi: 10.1016/ j.jacl.2016.04.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Long-Term Mipomersen Treatment Is Associated With a Reduction In

RI PT

Cardiovascular Events In Patients With Familial Hypercholesterolemia

SC

P. Barton Duell1, Raul D. Santos2, Bridget-Anne Kirwan3, Joseph L Witztum4,

M AN U

Sotirios Tsimikas5,6, John J.P. Kastelein7

TE D

From the 1Knight Cardiovascular Institute, Oregon Health and Sciences University, Portland OR, USA; 2Lipid Clinic Heart Institute (InCor), University of São Paulo Medical School Hospital, São Paulo, Brazil; 3SOCAR RESEARCH SA and London School of Hygiene and Tropical Medicine, Switzerland; 4Division of Endocrinology and Metabolism, University of California San Diego, La Jolla, CA, USA; 5Division of Cardiology, Sulpizio Cardiovascular Center, University of California San Diego, La Jolla, CA, USA; 6Ionis Pharmaceuticals; and the 7Department of Vascular Medicine, Academic Medical Center/University of Amsterdam, Amsterdam, The Netherlands Running title: mipomersen reduces MACE

Key words: mipomersen, antisense, LDL-cholesterol, major adverse cardiovascular events,

EP

familial hypercholesterolemia

AC C

Running title: Mipomersen reduces CVD events

Address for correspondence: John J.P. Kastelein, Academic Medical Center, University of Amsterdam, Rm. F4.159-2, Meibergdreef 9, P.O. Box 22660, 1100 DD Amsterdam, the Netherlands, email: [email protected], phone: +31 20 566 6612, fax: +31 20 566 9343

MIPO_publication_JCL_V20_draft23ST.docx

ACCEPTED MANUSCRIPT

Abstract Background Familial Hypercholesterolemia (FH) is characterized by severely elevated LDLcholesterol and up to a 20-fold increase in premature cardiovascular disease (CVD).

RI PT

Objective Mipomersen has been shown to lower levels of these atherogenic lipoproteins, but whether it lowers major adverse cardiac events (MACE) has not been addressed.

Methods This post-hoc analysis of prospectively collected data of three randomized trials and an open label-extension phase included patients that were exposed to ≥12 months of

SC

mipomersen. MACE rates that occurred during 24 months prior to randomization in the

mipomersen group were compared to MACE rates after initiation of mipomersen. Data from the trials included in this report are registered in Clinicaltrials.gov (NCT00607373,

M AN U

NCT00706849, NCT00794664, NCT00694109). The occurrence of MACE events, defined as cardiovascular death, non-fatal acute myocardial infarction, hospitalization for unstable angina, coronary revascularization and nonfatal ischemic stroke, was obtained from medical history data pre-treatment and adjudicated by an independent adjudication committee for events occurring post-treatment with mipomersen.

TE D

Results MACE were identified in 61.5% of patients (64 patients with 146 events [39 myocardial infarctions, 99 coronary revascularizations, five unstable angina episodes, three ischemic strokes]) during 24 months prior to mipomersen treatment, and in 9.6% of patients (10 patients with 13 events [one cardiovascular death, two myocardial infarctions, six coronary

EP

interventions, four unstable angina episodes]) during a mean of 24.4 months after initiation of mipomersen (MACE rate 25.7/1000 patient-months vs. 3.9/1000 patient-months, OR = 0.053

AC C

[95% CI 0.016 – 0.168], p<0.0001 by the exact McNemar’s test). The reduction in MACE coincided with a mean absolute reduction in LDL-C of 70 mg/dL (-28%) and of non-HDL cholesterol of 74 mg/dL (-26%) as well as reduction in Lp(a) of 11 mg/dL (-17%). Conclusion Long-term mipomersen treatment not only lowers levels of atherogenic lipoproteins but may also lead to a reduction in cardiovascular events in FH patients.

MIPO_publication_JCL_V20_draft23ST.docx

2

ACCEPTED MANUSCRIPT

Abbreviations Homozygous Familial Hypercholesterolemia

HoFH

Heterozygous Familial Hypercholesterolemia

MACE

Major adverse cardiac events

LDL-C

Low density lipoprotein cholesterol

LDLR

LDL receptor

CVD

Cardiovascular disease

Lp(a)

Lipoprotein(a)

AMI

acute myocardial infarction

RCT

Randomized controlled clinical trial

AC C

EP

TE D

M AN U

SC

RI PT

HoFH

MIPO_publication_JCL_V20_draft23ST.docx

3

ACCEPTED MANUSCRIPT

Introduction Patients with the autosomal co-dominant genetic disorder, Familial Hypercholesterolemia (FH), have extreme elevations of LDL cholesterol (LDL-C) that are

RI PT

associated with a 10 to 20-fold increase in risk of premature CVD events.1 If untreated, patients with heterozygous FH (HeFH) have an approximately 50% chance of developing ASCVD by the age of 50 years in men and 60-65 years in women.2, 3 Hence, FH is a highly atherogenic disorder that warrants aggressive lipid-lowering therapy.

SC

Unfortunately, many patients with FH are unable to achieve sufficient LDL-C lowering in response to treatment with standard therapies that now include statins, ezetimibe, and bile acid sequestrants. Such medications act through upregulation of hepatic LDL receptor (LDLR)

M AN U

expression, and thus, the efficacy of these drugs is diminished in patients with severe forms of FH, in which hepatic LDLR expression is diminished or absent.4 For this reason, FH patients with refractory hypercholesterolemia require the addition of an adjunctive therapy that does not rely on upregulation of LDLR expression, but in contrast, inhibits the synthesis of apolipoprotein B-100 (apoB-100), the major apolipoprotein of atherogenic lipoproteins.

TE D

Mipomersen, a second-generation antisense oligonucleotide, inhibits translation of apolipoprotein B-100 mRNA, thereby reducing hepatic synthesis of apolipoprotein B-100 and lowering the concentration of apoB-100 containing atherogenic lipoproteins.5-10 In fact, levels of plasma total cholesterol, LDL-C, non-HDL cholesterol, apoB and lipoprotein(a) [Lp(a)] have been

EP

consistently and significantly reduced in response to treatment with mipomersen. Since mipomersen produces large absolute reductions in plasma LDL-C in patients with

AC C

FH, we hypothesized that treatment with this novel therapy would significantly reduce CVD events. This hypothesis was bolstered by our observation that major adverse cardiac events (MACE) were infrequent among FH patients during experimental treatment with mipomersen, despite the fact that all the patients had a diagnosis of CVD prior to study entry. We therefore tested the hypothesis that mipomersen therapy would decrease CV events by assessing the prevalence of MACE during 2 years prior to initiation of mipomersen compared to the incidence of MACE after at least 12 months and up to 4.5 years of treatment with mipomersen.

MIPO_publication_JCL_V20_draft23ST.docx

4

ACCEPTED MANUSCRIPT

Methods

Study design and selection of patients Individual patient data are derived from three phase 3 randomized clinical trials (RCTs)

RI PT

conducted in patients with FH from the mipomersen program.5, 7, 8 Briefly, patients who had participated in one of the three phase 3 blinded, randomized, placebo-controlled trials of sixmonths duration (‘’index studies’’), in addition to the open-label extension study

(NCT00694109) and who had a minimum of 12 months of exposure from the combined RCT and

SC

open label extension phases to mipomersen were eligible for inclusion.9 Subjects with less than 12 months of exposure to mipomersen were excluded because lipoprotein lowering for at least

M AN U

12 months is typically required to demonstrate discernible reductions in MACE.11 The design of the index studies is depicted in Error! Reference source not found.. One third of patients (n=34) were randomly allocated to placebo in the index RCT study followed by an open label study with treatment with mipomersen for at least 12 months. Two thirds (n=70) were randomly allocated to blinded mipomersen for 6 months in the index RCT studies followed by at least 6 months

TE D

treatment with mipomersen in the open label study. In total, 104 subjects fulfilled the selection criteria for inclusion in this post-hoc analysis. The prevalence and incidence of MACE events was then determined in these 104 subjects before and after initiation of mipomersen according to the protocol defined below.

EP

All RCTs included in this post-hoc analysis were performed in accordance with the Declaration of Helsinki and Good Clinical Practice. The appropriate national and institutional

AC C

regulatory authorities and ethics committees approved all study protocols. All subjects provided written informed consent. MACE included in this report were cardiovascular death, non-fatal acute myocardial infarction (AMI), hospitalization for unstable angina (UA), coronary revascularization (percutaneous coronary intervention (PCI and/or coronary artery bypass graft (CABG) and ischemic stroke. The protocol for the identification of MACE events prior to and after initiation of treatment with mipomersen was done as follows: Prior to initiation of mipomersen: For patients randomized to (blinded) mipomersen in the index study, information was obtained from the

MIPO_publication_JCL_V20_draft23ST.docx

5

ACCEPTED MANUSCRIPT

medical history section of the case report forms, using the investigator-reported terms for events consistent with MACE that had an onset date no earlier than 24 months prior to inclusion in the index study. For patients randomized to placebo, MACE events were similarly

RI PT

identified from the case report forms that had an onset date no earlier than 18 months prior to inclusion in the index study, as well as MACE events that were reported during their 6-month participation in the placebo phase of the index study. After initiation of mipomersen: All events that occurred after the initiation of mipomersen in the identified 104 subjects were adjudicated

SC

for the possibility of MACE even if an event occurred during the first six month of therapy in the index study. An independent cardiovascular endpoints committee, blinded to treatment group and study phase, adjudicated all prospective deaths and all serious cardiovascular events

M AN U

suggestive of MACE that occurred after enrolment in an index study.

The three index studies included patients as follows: Study NCT00607373 investigated patients with HoFH which included genetic confirmation of HoFH or a clinical diagnosis based on an untreated LDL-C >500 mg/dL together with either xanthoma before 10 years of age or evidence of HeFH in both the parents5; StudyNCT00794664 investigated patients with severe

TE D

hypercholesterolemia, which included a diagnosis of severe hypercholesterolemia having an LDL-C ≥200 mg/dL7; and Study NCT00706849 investigated patients with HeFH with CAD which included a diagnosis of HeFH plus LDL-C ≥100 mg/dL and triglycerides <200 mg/dL and a diagnosis of CAD.8 All the 3 trials were conducted with the same study design at 26 clinical

EP

centers in 6 countries and each trial randomized patients 2:1 to weekly, subcutaneous injections of mipomersen 200 mg or placebo for 26 weeks on a background of maximally

AC C

tolerated lipid-lowering therapy. In addition, all patients from these 3 trials who are included in this analysis also participated in an open label extension study (NCT00694109) during which they used mipomersen for a sufficient time so that they had at least 12 months of exposure to mipomersen. 9

Outcomes Using data completed in the medical history case report forms, we investigated the ‘prevalent’ MACE rate during the two-year interval prior to randomization in the index study

MIPO_publication_JCL_V20_draft23ST.docx

6

ACCEPTED MANUSCRIPT

compared to the MACE rate in the same patients after initiation of mipomersen in subjects who had at least 12 months of treatment with mipomersen. Patients acted as their own control. A fatal event was considered to be a cardiovascular (CV) death if it was due to an acute

RI PT

MI or heart failure or stroke or any other (cardio) vascular cause. Deaths of ‘unknown cause’ were classified as a CV death in the absence of evidence from the provided information that there may have been a non-CV cause. The criteria used to adjudicate non-fatal MI were consistent with ESC/AHA internationally accepted definitions for AMI.12 An event was

SC

adjudicated as being a non-fatal MI if there was evidence of myocardial necrosis by changes in cardiac biomarkers or and supporting information derived from the clinical presentation, electrocardiographic changes, or the results of myocardial or coronary artery imaging. The

M AN U

criteria used to adjudicate hospitalization for UA required the presence of symptoms suggestive of myocardial ischemia at rest, ischemic discomfort including angina, or symptoms thought to be equivalent, which lasted more than 10 minutes and occurred either at rest, or in an accelerating pattern with frequent episodes associated with progressively decreased exercise capacity. In addition, patients had to be hospitalized within 24 hours of the most recent

TE D

symptoms with hospitalization defined as an admission to an inpatient unit or a visit to an emergency department that resulted in a hospital stay of at least 24 hours or, at a minimum, a change in calendar date if the hospital admission or discharge times were not available. In addition, patients had to present with new or worsening ST or T-wave changes on a resting ECG

EP

(in the absence of LBBB or LVH) or with evidence of inducible myocardial ischemia or angiographic evidence of new or worsening CAD with ≥ 70% lesion and/or thrombus in an

AC C

epicardial coronary artery or required coronary revascularization (PCI or CABG) for the presumed culprit lesion(s) undertaken during the unscheduled hospitalization, or subsequent to transfer to another institution without interceding home discharge. The patient should not have presented with evidence of an acute MI. Ischemic stroke was defined as the rapid onset of a new persistent neurological deficit attributed to an obstruction in cerebral blood flow with no apparent non-vascular cause (e.g. tumor, trauma, infection, aneurysm or other nonatherosclerotic vascular abnormality). Available neuroimaging studies were used to verify the clinical diagnosis and to determine if there was a demonstrable lesion compatible with an acute

MIPO_publication_JCL_V20_draft23ST.docx

7

ACCEPTED MANUSCRIPT

stroke. A coronary revascularization was defined as any coronary revascularization procedure (PCI or CABG) that was not performed in an emergency setting for either unstable angina and or an acute coronary syndrome

For the identification of the pre-mipomersen coronary interventions, it was not possible

RI PT

from the review of records to distinguish between planned and unplanned or emergency

revascularization procedures, so any coronary intervention was counted as an event. For the post-mipomersen coronary interventions, planned and unplanned emergency coronary

interventions were adjudicated separately, but for the purpose of this analysis, all interventions

SC

(planned/unplanned) were counted together to maintain consistency with how the pre-

mipomersen coronary interventions were counted. Finally, the analysis was not a time-to-first

M AN U

event analysis; therefore all events were counted individually and a patient could contribute more than one event to the overall event rates.

Statistical analysis

Statistical analyses were done independently in collaboration with the funder. Baseline

TE D

characteristics were summarized as frequencies for categorical variables and descriptive statistics for continuous variables. For the lipids and lipoprotein concentrations, percentage change from baseline was calculated as the (change divided by the baseline value)*100. Mean and standard error of the mean were plotted. Due to non-normality of these data, a non-

EP

parametric Wilcoxon signed rank test was used to evaluate if the mean change and mean % change from baseline at 12 months were statistically different from 0. An exact McNemar's test

AC C

(i.e. inequality test for two correlated proportions) was used to determine if there was a statistically significant difference in the proportion of subjects with MACE events pre and posttreatment with mipomersen. Event rates were expressed as the number of patients with at least one event divided by the total follow-up time in months (X 1000). All statistical analyses were done with SAS (version 9.2).

MIPO_publication_JCL_V20_draft23ST.docx

8

ACCEPTED MANUSCRIPT

Role of funding source The funder of the study (Genzyme, A Sanofi Company and Ionis Pharmaceuticals) supervised enrolment of patients and study conduct at the clinical sites and collaborated with

RI PT

the authors in data interpretation and writing of the report, but had no role in the adjudication of the MACE events nor the data analyses. The corresponding author had full access to all data and had final responsibility for the decision to submit for publication.

SC

Results Study Design and patient Flow

Figure 1 depicts the study design and patient disposition from enrolment in the index

M AN U

studies through the open label treatment study. In total, 233 patients were randomized in one of the three index studies and 141 of these patients continued in the open-label study (OLE). At the time this analysis was performed, 104 of the 141 patients had completed at least 12-months of treatment with mipomersen and were thus included in this post-hoc analysis. The mean

TE D

follow-up following initiation of mipomersen was 24.4 months.

Baseline characteristics and changes in lipid parameters at 1 year The baseline characteristics of the 104 subjects are shown in Table 1. In total, 72% of patients were treated with a maximum statin dose and 79% with the combination of a statin

EP

and ezetimibe. Table 2a shows the baseline and 12-month levels (absolute and mean % change) of total cholesterol, LDL-C, HDL-C, non-HDL-C, triglycerides, apoB and Lp(a) for the overall

AC C

population (i.e. 104 subjects) and Tables 2b and 2c show the same data for the Homozygous FH and Heterozygous FH subjects respectively. Figure 2 depicts the mean absolute changes in addition to the percentage changes from baseline after at least 12 months of treatment with mipomersen for key lipid and lipoprotein levels for the 104 patients included in this analysis.

Evaluation of MACE rates Table 3a summarizes and figure 3 depicts the MACE events that occurred up to 24 months prior to and after initiation of mipomersen treatment for the 104 subjects included in

MIPO_publication_JCL_V20_draft23ST.docx

9

ACCEPTED MANUSCRIPT

this analysis. Table 3b summarizes this data Homozygous FH and Heterozygous FH subjects respectively. Prior to treatment with mipomersen, 146 MACE (39 myocardial infarctions, 99 coronary revascularizations, five unstable angina episodes, three ischemic strokes) were

RI PT

identified for 64 subjects (61.5% of subjects). In total, 145 of these events occurred prior to inclusion in the index study and were recorded in the medical history and one event occurred during participation in an index study for a subject randomized to placebo.

In contrast, after the initiation of mipomersen treatment, there were only 13 MACE (one

SC

cardiovascular death, two myocardial infarctions, six coronary interventions and four unstable angina episodes) adjudicated for 10 (9.6%) subjects. Two MACE events occurred during the initial 6-month index study for patients randomized to mipomersen, and 11 occurred during the

M AN U

open label study phase. There was a statistically significant difference in the proportion of patients with a MACE pre and post treatment with mipomersen: – rate: 25.7/1000 patientmonths of follow-up prior to treatment with mipomersen and 3.9/1000 patient-months of

Discussion

TE D

follow-up post-mipomersen treatment, Odds ratio = 0.053 [95%: CI 0.016 – 0.168], P<.0001.

In this retrospective analysis of the impact of mipomersen treatment on MACE in highrisk FH patients, we found a significant 85% decrease in the rate of new MACE during a follow-

EP

up period of up to 4.5 years (average 24.4 months) after initiation of mipomersen therapy compared to the two years preceding this therapy. Despite the fact that the majority of subjects

AC C

were on a maximal dose of statin (72%) and/or adjunctive therapy with ezetimibe (79%) in combination with standard cardioprotective medications such as aspirin and beta-blockers, the majority of subjects (61.5%) had experienced MACE during the two years prior to subject recruitment into the index study. Because these subjects all have FH and had a near lifetime exposure to exceedingly high levels of atherogenic apoB-100 containing lipoproteins (their mean age was 50.5 years at initiation of therapy), their baseline risk for MACE was extremely high.1, 2, 13 Despite being on maximally tolerated hypolipidemic therapies, mean baseline LDL-C was still 230 mg/dL, which is far above the current goals of therapy for such high-risk subjects.

MIPO_publication_JCL_V20_draft23ST.docx

10

ACCEPTED MANUSCRIPT

Recent data suggest that an optimal LDL-C level may be 50 mg/dL or even lower in patients at high risk such as those with FH or pre-existing CVD.14-16, 21 Mipomersen therapy inhibits the hepatic production of apoB-100, a rate-limiting step in

RI PT

the formation of VLDL and LDL, and thus should be effective in lowering elevated LDL-C levels in patients with FH. Indeed, in response to mipomersen, there was a mean decrease of LDL-C of 70 mg/dL (-28%) and of non-HDL-cholesterol of 74 mg/dL (-26%) consistent with a reduction in VLDL, IDL and LDL atherogenic apoB-100-containing lipoproteins. In parallel, mean plasma apoB levels decreased by 54 mg/dL (-29%). Furthermore, reflective of the fact that ~ 50% of FH

SC

subjects have elevated Lp(a) values, an independent risk factor for CVD,2 their mean baseline Lp(a) was 61 mg/dL, a value significantly higher than the median levels of 10-15 mg/dL in

M AN U

populations at large.17, 18 Mipomersen therapy also reduced mean Lp(a) levels by 11 mg/dL (17%). Thus, mipomersen therapy effectively reduced the plasma levels of all atherogenic lipoproteins in these severely hypercholesterolemic FH subjects.

There is now an extensive literature, based on randomized controlled clinical trials, showing that lowering LDL-C in hypercholesterolemic subjects by means of ileal-bypass, bile-

TE D

sequestrants, statins and ezetimibe, effectively reduces MACE.11, 19-22 Indeed, with commonly used therapies to lower LDL-C, there is a linear relationship between absolute reductions in LDLC and reductions in risk. Furthermore, this relationship appears to hold in both primary and secondary prevention settings, and at least for secondary prevention, appears valid even with

EP

starting LDL values as low as 70 mg/dL.15, 16, 21, 23 This has given rise to the concept that “the lower the LDL-C the better” is the goal of therapy in subjects at highest risk.14, 24However, all of

AC C

the interventions to date have lowered LDL-C primarily by enhancing LDL removal from plasma, primarily by induction of hepatic LDLR expression, and this is also true for the newly introduced monoclonal antibodies to PCSK9.15, 16 Although mipomersen produces sizable absolute reductions in LDL-C and other atherogenic lipoproteins, it achieves this primarily by inhibiting hepatic apoB-100 production. It was therefore important to assess whether the reduction in the burden of atherogenic lipoproteins in plasma resulting from this novel therapeutic modality would also produce the predicted decrement in incidence of cardiovascular events. Since it is not ethical to conduct a long-term, randomized, placebo-controlled, blinded study of

MIPO_publication_JCL_V20_draft23ST.docx

11

ACCEPTED MANUSCRIPT

mipomersen therapy in this very high risk population, we utilized a retrospective study design to evaluate events before and after therapy, in a similar manner as has been done to evaluate the impact of statin therapy and LDL apheresis in this population. 3, 25-28

RI PT

We determined the adjudicated MACE rates in this cohort during the period on mipomersen therapy, and then using each subject as their own control, we determined MACE for that subject for the two-year period prior to their randomization into the index study.

Among the 104 subjects who formed the cohort that met the study criteria, there had been 146

SC

MACE in the two years prior to randomization (25.7/1000 patient months) in comparison to only 13 events after initiation of mipomersen (3.9/1000 patient months), which represents an 85% reduction in events. This might even be a conservative estimate of the benefit, as it should

M AN U

be noted that the 13 MACE events for the on-therapy calculation included two events for two subjects whose MACE occurred during the initial six months of mipomersen therapy. These two MACE were included as both subjects continued on with the mipomersen therapy after their events, and thus met the inclusion criteria of 12 months or more of therapy. In the face of the high MACE rates in our patient cohort, the 85% reduction in MACE

TE D

represents a dramatic improvement over predicted rates. Raal et al3 recently performed a retrospective analysis of the impact of statin therapy in HoFH subjects (baseline mean LDL-C of 615 mg/dL) utilizing data from two large lipid clinics in South Africa. They estimated the hazard ratio for the benefit of statin therapy for MACE among statin-treated patients compared with

EP

statin-naive patients to be 0.49 (95% confidence interval 0.22–1.07; P< 0.07). This was associated with a mean relative reduction of LDL-C of only 26.4%. In a similar set of studies,

AC C

retrospective analyses of cardiovascular events were done before and during LDL apheresis therapy of FH patients to judge the effect of LDL-C lowering, which was estimated to result in a time-averaged LDL lowering of ~ 50-60%. These studies consistently reported marked decreases in cardiovascular event rates, which ranged from 49 to 88%.25-28 While such retrospective studies are compromised to some extent by inherent methodological issues, the consistency of the observations strongly support a beneficial benefit on MACE of LDL-C lowering in FH subjects. Thus, the 85% reduction in MACE observed in our study is consistent with what might be predicted from the statin and apheresis studies noted above. In addition to the ability to

MIPO_publication_JCL_V20_draft23ST.docx

12

ACCEPTED MANUSCRIPT

lower LDL-C and non-HDL-C by both mipomersen and apheresis may explain the potential increased 29

rates of event reductions in such trials compared with the statin trials cited above.

Important limitations of our analysis include the absence of a control cohort, the non-

RI PT

randomized nature of this study, the small size of our cohort and the relatively short observation period. In addition, we were unable to adjudicate the pre-mipomersen MACE and did not include subjects who died, whether patients had planned or unplanned

revascularization pre-mipomersen, or who did not take at least 12 months of mipomersen.

SC

These considerations underline the fact that our analysis is hypothesis generating and cannot be considered conclusive. Other important caveats are that we were unable to define the extent to which previous interventions, such as revascularization and use of stents, as well as

M AN U

use of various cardioprotective drugs known to impact MACE outcomes, might have influenced these results. Nevertheless, our data clearly demonstrate that mipomersen induced lowering of hepatic apoB-100 production is highly effective to lower LDL-C in patients with FH and that this

AC C

EP

TE D

is associated with the expected benefit of a marked reduction in CVD outcomes.

MIPO_publication_JCL_V20_draft23ST.docx

13

ACCEPTED MANUSCRIPT

Disclosures ST and JLW are co-inventors of and receive royalties from patents or patent applications owned by the University of California San Diego on antibodies for biotheranostic applications. ST has a

RI PT

dual appointment at UCSD and Ionis Pharmaceuticals, Inc. JLW has received honoraria for consulting for Ionis, CymaBay, and Intercept Pharmaceuticals. PBD has received grants from Genzyme, Retrophin, Regeneron, Amgen and consulting honoraria from Genzyme/Sanofi,

Retrophin, Regeneron and Kaneka. RDS has received a grant form Genzyme and consulting fees

SC

from Amgen, Aegerion, Astra Zeneca, Biolab, Boehringer-Ingelheim, Cerenis, Eli Lilly, Genzyme, Kowa, Pfizer, Sanofi/Regeneron, Torrent and Unilever. JJK is a consultant to and receives honoraria from AstraZeneca, Eli Lilly and Company, Amgen, Sanofi, Regeneron, Genzyme, Ionis,

M AN U

Aegerion, and KOWA. B-AK has no conflicts to disclose.

Contributors

BAK was responsible for the statistical analysis and wrote the first draft of the

TE D

manuscript. All authors interpreted the data and collaborated in the preparation of the manuscript. The authors made the decision to submit the manuscript for publication and vouch for the accuracy of the data and analyses and for the fidelity of this report to the trial protocol. JJK had full access to all the data in the study and had final responsibility for the decision to

AC C

Acknowledgement

EP

submit for publication.

We would like to thank Emilie Perrin (SOCAR Research SA) who provided editorial

assistance with the preparation of the tables, figures and references

MIPO_publication_JCL_V20_draft23ST.docx

14

ACCEPTED MANUSCRIPT

References

Hopkins PN, Toth PP, Ballantyne CM, Rader DJ. Familial hypercholesterolemias: prevalence, genetics, diagnosis and screening recommendations from the National Lipid Association Expert Panel on Familial Hypercholesterolemia. J Clin Lipidol 2011;5(3 Suppl):S9-17.

2.

Alonso R, Andres E, Mata N, Fuentes-Jimenez F, Badimon L, Lopez-Miranda J, Padro T, Muniz O, azDiaz JL, Mauri M, Ordovas JM, Mata P. Lipoprotein(a) levels in familial hypercholesterolemia: an important predictor of cardiovascular disease independent of the type of LDL receptor mutation. J Am Coll Cardiol 2014;63(19):1982-9.

3.

Raal FJ, Pilcher GJ, Panz VR, van Deventer HE, Brice BC, Blom DJ, Marais AD. Reduction in mortality in subjects with homozygous familial hypercholesterolemia associated with advances in lipidlowering therapy. Circulation 2011;124(20):2202-7.

4.

Tietge UJ, Kozarsky KF, Donahee MH, Rader DJ. A tetracycline-regulated adenoviral expression system for in vivo delivery of transgenes to lung and liver. J Gene Med 2003;5(7):567-75.

5.

Raal FJ, Santos RD, Blom DJ, Marais AD, Charng MJ, Cromwell WC, Lachmann RH, Gaudet D, Tan JL, Chasan-Taber S, Tribble DL, Flaim JD, Crooke ST. Mipomersen, an apolipoprotein B synthesis inhibitor, for lowering of LDL cholesterol concentrations in patients with homozygous familial hypercholesterolaemia: a randomised, double-blind, placebo-controlled trial. Lancet 2010;375(9719):998-1006.

6.

McGowan MP, Tardif JC, Ceska R, Burgess LJ, Soran H, Gouni-Berthold I, Wagener G, Chasan-Taber S. Randomized, placebo-controlled trial of mipomersen in patients with severe hypercholesterolemia receiving maximally tolerated lipid-lowering therapy. PLoS One 2012;7(11):e49006.

7.

Stein EA, Dufour R, Gagne C, Gaudet D, East C, Donovan JM, Chin W, Tribble DL, McGowan M. Apolipoprotein B synthesis inhibition with mipomersen in heterozygous familial hypercholesterolemia: results of a randomized, double-blind, placebo-controlled trial to assess efficacy and safety as add-on therapy in patients with coronary artery disease. Circulation 2012;126(19):2283-92.

8.

Thomas GS, Cromwell WC, Ali S, Chin W, Flaim JD, Davidson M. Mipomersen, an apolipoprotein B synthesis inhibitor, reduces atherogenic lipoproteins in patients with severe hypercholesterolemia at high cardiovascular risk: a randomized, double-blind, placebo-controlled trial. J Am Coll Cardiol 2013;62(23):2178-84.

9.

Santos RD, Duell PB, East C, Guyton JR, Moriarty PM, Chin W, Mittleman RS. Long-term efficacy and safety of mipomersen in patients with familial hypercholesterolaemia: 2-year interim results of an open-label extension. Eur Heart J 2015;36(9):566-75.

10.

Santos RD, Raal FJ, Catapano AL, Witztum JL, Steinhagen-Thiessen E, Tsimikas S. Mipomersen, an antisense oligonucleotide to apolipoprotein B-100, reduces lipoprotein(a) in various populations

AC C

EP

TE D

M AN U

SC

RI PT

1.

MIPO_publication_JCL_V20_draft23ST.docx

ACCEPTED MANUSCRIPT

with hypercholesterolemia: results of 4 phase III trials. Arterioscler Thromb Vasc Biol 2015;35(3):689-99. Baigent C, Blackwell L, Emberson J, Holland LE, Reith C, Bhala N, Peto R, Barnes EH, Keech A, Simes J, Collins R. Efficacy and safety of more intensive lowering of LDL cholesterol: a meta-analysis of data from 170,000 participants in 26 randomised trials. Lancet 2010;376(9753):1670-81.

12.

Thygesen K, Alpert JS, Jaffe AS, Simoons ML, Chaitman BR, White HD, Thygesen K, Alpert JS, White HD, Jaffe AS, Katus HA, Apple FS, Lindahl B, Morrow DA, Chaitman BR, Clemmensen PM, Johanson P, Hod H, Underwood R, Bax JJ, Bonow JJ, Pinto F, Gibbons RJ, Fox KA, Atar D, Newby LK, Galvani M, Hamm CW, Uretsky BF, Steg PG, Wijns W, Bassand JP, Menasche P, Ravkilde J, Ohman EM, Antman EM, Wallentin LC, Armstrong PW, Simoons ML, Januzzi JL, Nieminen MS, Gheorghiade M, Filippatos G, Luepker RV, Fortmann SP, Rosamond WD, Levy D, Wood D, Smith SC, Hu D, LopezSendon JL, Robertson RM, Weaver D, Tendera M, Bove AA, Parkhomenko AN, Vasilieva EJ, Mendis S, Bax JJ, Baumgartner H, Ceconi C, Dean V, Deaton C, Fagard R, Funck-Brentano C, Hasdai D, Hoes A, Kirchhof P, Knuuti J, Kolh P, McDonagh T, Moulin C, Popescu BA, Reiner Z, Sechtem U, Sirnes PA, Tendera M, Torbicki A, Vahanian A, Windecker S, Morais J, Aguiar C, Almahmeed W, Arnar DO, Barili F, Bloch KD, Bolger AF, Botker HE, Bozkurt B, Bugiardini R, Cannon C, de LJ, Eberli FR, Escobar E, Hlatky M, James S, Kern KB, Moliterno DJ, Mueller C, Neskovic AN, Pieske BM, Schulman SP, Storey RF, Taubert KA, Vranckx P, Wagner DR. Third universal definition of myocardial infarction. J Am Coll Cardiol 2012;60(16):1581-98.

13.

Hobbs HH, Brown MS, Russell DW, Davignon J, Goldstein JL. Deletion in the gene for the lowdensity-lipoprotein receptor in a majority of French Canadians with familial hypercholesterolemia. N Engl J Med 1987;317(12):734-7.

14.

Steinberg D, Glass CK, Witztum JL. Evidence mandating earlier and more aggressive treatment of hypercholesterolemia. Circulation 2008;118(6):672-7.

15.

Robinson JG, Kastelein JJ. PCSK9 Inhibitors and Cardiovascular Events. N Engl J Med 2015;373(8):774.

16.

Sabatine MS, Giugliano RP, Wiviott SD, Raal FJ, Blom DJ, Robinson J, Ballantyne CM, Somaratne R, Legg J, Wasserman SM, Scott R, Koren MJ, Stein EA. Efficacy and safety of evolocumab in reducing lipids and cardiovascular events. N Engl J Med 2015;372(16):1500-9.

17.

Albers JJ, Slee A, O'Brien KD, Robinson JG, Kashyap ML, Kwiterovich PO, Jr., Xu P, Marcovina SM. Relationship of apolipoproteins A-1 and B, and lipoprotein(a) to cardiovascular outcomes: the AIM-HIGH trial (Atherothrombosis Intervention in Metabolic Syndrome with Low HDL/High Triglyceride and Impact on Global Health Outcomes). J Am Coll Cardiol 2013;62(17):1575-9.

18.

Khera AV, Everett BM, Caulfield MP, Hantash FM, Wohlgemuth J, Ridker PM, Mora S. Lipoprotein(a) concentrations, rosuvastatin therapy, and residual vascular risk: an analysis from the JUPITER Trial (Justification for the Use of Statins in Prevention: an Intervention Trial Evaluating Rosuvastatin). Circulation 2014;129(6):635-42.

19.

The Lipid Research Clinics Coronary Primary Prevention Trial results. I. Reduction in incidence of coronary heart disease. JAMA 1984;251(3):351-64.

AC C

EP

TE D

M AN U

SC

RI PT

11.

MIPO_publication_JCL_V20_draft23ST.docx

16

ACCEPTED MANUSCRIPT

Buchwald H, Varco RL, Matts JP, Long JM, Fitch LL, Campbell GS, Pearce MB, Yellin AE, Edmiston WA, Smink RD, Jr., . Effect of partial ileal bypass surgery on mortality and morbidity from coronary heart disease in patients with hypercholesterolemia. Report of the Program on the Surgical Control of the Hyperlipidemias (POSCH). N Engl J Med 1990;323(14):946-55.

21.

Cannon CP, Blazing MA, Giugliano RP, McCagg A, White JA, Theroux P, Darius H, Lewis BS, Ophuis TO, Jukema JW, De Ferrari GM, Ruzyllo W, De LP, Im K, Bohula EA, Reist C, Wiviott SD, Tershakovec AM, Musliner TA, Braunwald E, Califf RM. Ezetimibe Added to Statin Therapy after Acute Coronary Syndromes. N Engl J Med 2015;372(25):2387-97.

22.

Canner PL, Berge KG, Wenger NK, Stamler J, Friedman L, Prineas RJ, Friedewald W. Fifteen year mortality in Coronary Drug Project patients: long-term benefit with niacin. J Am Coll Cardiol 1986;8(6):1245-55.

23.

Ridker PM, Danielson E, Fonseca FA, Genest J, Gotto AM, Jr., Kastelein JJ, Koenig W, Libby P, Lorenzatti AJ, MacFadyen JG, Nordestgaard BG, Shepherd J, Willerson JT, Glynn RJ. Rosuvastatin to prevent vascular events in men and women with elevated C-reactive protein. N Engl J Med 2008;359(21):2195-207.

24.

Jarcho JA, Keaney JF, Jr. Proof That Lower Is Better--LDL Cholesterol and IMPROVE-IT. N Engl J Med 2015;372(25):2448-50.

25.

Versmissen J, Oosterveer DM, Yazdanpanah M, Defesche JC, Basart DC, Liem AH, Heeringa J, Witteman JC, Lansberg PJ, Kastelein JJ, Sijbrands EJ. Efficacy of statins in familial hypercholesterolaemia: a long term cohort study. BMJ 2008;337:a2423.

26.

Mabuchi H, Koizumi J, Shimizu M, Kajinami K, Miyamoto S, Ueda K, Takegoshi T. Long-term efficacy of low-density lipoprotein apheresis on coronary heart disease in familial hypercholesterolemia. Hokuriku-FH-LDL-Apheresis Study Group. Am J Cardiol 1998;82(12):1489-95.

27.

Leebmann J, Roeseler E, Julius U, Heigl F, Spitthoever R, Heutling D, Breitenberger P, Maerz W, Lehmacher W, Heibges A, Klingel R. Lipoprotein apheresis in patients with maximally tolerated lipid-lowering therapy, lipoprotein(a)-hyperlipoproteinemia, and progressive cardiovascular disease: prospective observational multicenter study. Circulation 2013;128(24):2567-76.

28.

Rosada A, Kassner U, Vogt A, Willhauck M, Parhofer K, Steinhagen-Thiessen E. Does regular lipid apheresis in patients with isolated elevated lipoprotein(a) levels reduce the incidence of cardiovascular events? Artif Organs 2014;38(2):135-41.

29.

Arai K, Orsoni A, Mallat Z, Tedgui A, Witztum JL, Bruckert E, Tselepis AD, Chapman MJ, Tsimikas S. Acute impact of apheresis on oxidized phospholipids in patients with familial hypercholesterolemia. J Lipid Res 2012;53(8):1670-8.

AC C

EP

TE D

M AN U

SC

RI PT

20.

MIPO_publication_JCL_V20_draft23ST.docx

17

ACCEPTED MANUSCRIPT

Figure 1 - Consort diagram

RI PT

Index Studies N = 233

Homozygous FH$ N=51

Placebo N=17

N=23

N=16

Blinded mipomersen N=39

N=6

N=23

EP

Treated with mipomersen for ≥ 12 months N=104

AC C

N=38

TE D

Withdrew consent N=1

Open Label Extension study N=141

SC

Blinded mipomersen N=34

M AN U

Index Studies

Severe FH† N=58

Heterozygous FH‡ N=124

Placebo N=19

Blinded mipomersen N=83

Placebo N=41

N=3

N=55

N=39

N=9¥

N=94

N=6

N=75

Flow of Patients in the index and Open-Label Extension Studies. All patients on stable maximally tolerated lipid-lowering treatment (statin +/-) $ Raal, et al.5 † McGowan, et al.6 ‡ Stein, et al.7 ¥ Eight of 26 sites participated in the index study participated in the open label extension study.

MIPO_publication_JCL_V20_draft23ST.docx

ACCEPTED MANUSCRIPT

Table 1 – Baseline characteristics of the study group All subjects

Homozygous FH

Heterozygous FH

(N=104)

(N=23)

(N=75)

Age (years)*

50.5 (47.6, 53.4)

31.2 (26.1, 36.2)

Male

64 (61.5)

10 (43.5)

Body-mass index (kg/m2)*

28.6 (27.7, 29.6)

26.1 (23.7, 28.5)

LDL-C (mg/dL)

229.6 (151.1)

455.1 (148.7)

HDL-C (mg/dL)

45.9 (13.5)

Non-HDL-C (mg/dL)

Demographics

55.8 (53.5, 58.1) 50 (66.7)

29.3 (28.3, 30.4)

SC

M AN U

Lipids/Lipoprotein

RI PT

Variable

155.4 (55.8)

48.2 (12.9)

253.8 (156.5)

482.7 (159.3)

177.7 (59.2)

Total Cholesterol (mg/dL)

299.7 (152.9)

520.7 (157.9)

225.9 (59.6)

Apolipoprotein B (mg/dL)

173.5 (83.8)

290.6 (90.0)

134.8 (36.9)

Lipoprotein(a) (mg/dL)†

48.0 (13.5, 89.0)

50.8 (35.1)

63.9 (58.3)

Triglycerides (mg/dL)

121.2 (69.0)

138.3 (101.4)

111.3 (45.8)

TE D

38.0 (13.3)

AC C

EP

Table legend: Data are number of subjects (%) or mean (SD) unless otherwise indicated. *Data are mean (95%CI). †Data are the median (25th, 75th). %=percentage; SD=standard deviation; kg/m2=kilogram per square meter; mg/dL=milligram per deciliter.

MIPO_publication_JCL_V20_draft23ST.docx

ACCEPTED MANUSCRIPT

Table 2a – Lipids and Lipoproteins – Descriptive statistics for all subjects All subjects (N=104)

1 Year after start of mipomersen treatment Absolute Change % Change

AC C

EP

TE D

M AN U

SC

RI PT

Baseline LDL-C (mg/dL) Mean (SD) 229.6 (151.1) 159.2 (114.2) -70.3 (78.0) Median 165.5 117.0 -47.5 (Q1,Q3) (124.5, 278.0) (90.0, 168.0) (-91.3, -26.0) HDL-C (mg/dL) Mean (SD) 45.9 (13.5) 48.0 (15.4) 2.1 (9.3) Median 46.0 46.5 2.0 (Q1,Q3) (36.0, 55.0) (37.7, 53.5) (-4.0, 7.3) Non-HDL-C (mg/dL) Mean (SD) 253.8 (156.5) 179.6 (120.1) -74.2 (83.7) Median 193.0 136.3 -48.0 (Q1,Q3) (143.0, 305.0) (108.3, 185.5) (-99.3, -28.0) Apolipoprotein B (mg/dL) Mean (SD) 173.5 (83.8) 119.9 (65.3) -53.6 (52.1) Median 143.0 97.3 -37.3 (Q1,Q3) (119.5, 201.0) (82.0, 133.0) (-68.0, -23.0) Lipoprotein(a) (mg/dL) Mean (SD) 60.9 (54.9) 49.9 (50.2) -10.9 (17.1) Median 48.0 35.5 -6.0 (Q1,Q3) (13.5, 89.0) (11.0, 62.3) (-20.3, 0.0) Total Cholesterol (mg/dL) Mean (SD) 299.7 (152.9) 227.7 (116.4) -72.1 (82.3) Median 237.5 183.0 -46.0 (Q1,Q3) (187.5, 348.0) (157.0, 244.0) (-101.0, -23.8) Triglycerides (mg/dL) Mean (SD) 121.2 (69.0) 100.2 (56.0) -21.0 (54.1) Median 101.0 83.3 -9.5 (Q1,Q3) (75.0, 144.5) (66.0, 122.3) (-37.5, 13.0) mg/dL=milligram per decilitre; SD=standard deviation; Q1=25th percentile; Q3=75th percentile.

MIPO_publication_JCL_V20_draft23ST.docx

-28.0 (22.5) -27.9 (-44.8, -11.9) 6.5 (21.9) 5.3 (-8.1, 19.8) -26.5 (21.9) -25.3 (-43.3, -12.9) -29.2 (20.5) -29.1 (-40.3, -15.2) -16.6 (36.1) -19.5 (-33.0, 0.0) -21.1 (18.9) -20.0 (-34.3, -8.7) -10.4 (30.7) -8.9 (-34.6, 12.4)

ACCEPTED MANUSCRIPT

Table 2b – Lipids and Lipoproteins – Descriptive statistics for Homozygous FH subjects

AC C

EP

TE D

M AN U

SC

RI PT

Homozygous FH subjects 1 Year after start of mipomersen treatment (N=23) Baseline Absolute Change % Change LDL-C (mg/dL) 455.1 (148.7) 330.9 (130.0) -124.2 (106.3) -26.9 (18.7) Mean (SD) 482.0 348.0 -109.0 -21.8 Median (311.0, 569.0) (252.0, 431.0) (-199.0, -40.0) (-42.1, -10.5) (Q1,Q3) HDL-C (mg/dL) 38.0 (13.3) 42.2 (14.1) 4.3 (8.1) 14.8 (27.9) Mean (SD) 37.0 40.0 4.0 10.6 Median (24.0, 46.0) (31.5, 54.0) (-3.0, 10.0) (-6.5, 30.0) (Q1,Q3) Non-HDL-C (mg/dL) 482.7 (159.3) 356.8 (142.0) -126.0 (114.009) -25.7 (18.5) Mean (SD) 503.0 361.5 -93.0 -19.0 Median (368.0, 603.0) (266.0, 461.0) (-192.0, -40.0) (-41.7, -9.2) (Q1,Q3) Apolipoprotein B (mg/dL) 290.6 (90.0) 207. 7(79.9) -82.9 (71.0) -28.2 (19.4) Mean (SD) 300.0 213.5 -57.0 -21.2 Median (245.0, 335.0) (157.0, 279.0) (-128.0, -34.0) (-40.3, -14.9) (Q1,Q3) Lipoprotein(a) (mg/dL) 50.8 (35.1) 41.1 (33.612) -9.8 (11.8) -21.5 (26.1) Mean (SD) 51.0 35.0 -6.0 -23.8 Median (29.0, 60.0) (21.0, 49.5) (-21.0, -3.0) (-39.8, -4.9) (Q1,Q3) Total Cholesterol (mg/dL) 520.7 (157.9) 399.0 (137.4) -121.7 (111.7) -22.4 (16.8) Mean (SD) 537.0 398.5 -87.5 -18.6 Median (403.0, 647.0) (322.0, 493.0) (-200.0, -32.5) (-34.3, -7.1) (Q1,Q3) Triglycerides (mg/dL) 138.3 (101.4) 122.3 (80.9) -16.0 (70.8) 0.9 (33.5) Mean (SD) 98.0 95.0 2.0 2.4 Median (61.0, 175.0) (68.5, 170.5) (-13.0, 21.0) (-15.9, 26.2) (Q1,Q3) th th mg/dL=milligram per decilitre; SD=standard deviation; Q1=25 percentile; Q3=75 percentile.

MIPO_publication_JCL_V20_draft23ST.docx

21

ACCEPTED MANUSCRIPT

Table 2c – Lipids and Lipoproteins – Descriptive statistics for Heterozygous FH subjects

AC C

EP

TE D

M AN U

SC

RI PT

Heterozygous FH subjects 1 Year after start of mipomersen treatment (N=75) Baseline Absolute Change % Change LDL-C (mg/dL) Mean (SD) 155.4 (55.8) 107.8 (32.3) -47.6 (50.9) -26.8 (22.7) Median 143.0 105.0 -36.0 -28.0 (Q1,Q3) (117.0, 181.0) (85.0, 128.0) (-65.5, -19.0) (-43.5, -11.6) HDL-C (mg/dL) Mean (SD) 48.2 (12.9) 49.4 (15.3) 1.1 (8.8) 3.2 (17.5) Median 48.0 47.7 2.0 3.9 (Q1,Q3) (37.0, 56.0) (38.0, 53.0) (-5.0, 6.0) (-8.7, 13.8) Non-HDL-C (mg/dL) Mean (SD) 177.7 (59.2) 126.6 (35.3) -51.1 (55.5) -25.1 (22.0) Median 161.0 122.0 -38.0 -25.1 (Q1,Q3) (136.0, 203.0) (102.5, 153.0) (-74.0, -20.0) (-41.7, -12.6) Apolipoprotein B (mg/dL) Mean (SD) 134.8 (36.9) 94.4 (28.7) -40.4 (37.4) -27.9 (20.0) Median 129.0 90.0 -34.0 -29.4 (Q1,Q3) (109.0, 152.0) (74.0, 112.0) (-54.0, -21.0) (-38.9, -13.9) Lipoprotein(a) (mg/dL) Mean (SD) 63.9 (58.3) 52.9 (53.2) -11.0 (18.9) -12.7 (38.4) Median 47.0 39.5 -6.0 -17.9 (Q1,Q3) (13.0, 97.0) (11.0, 84.5) (-20.0, 0.0) (-30.9, 0.0) Total Cholesterol (mg/dL) Mean (SD) 225.9 (59.6) 175.9 (34.9) -50.0 (56.1) -19.2 (18.6) Median 222.0 168.5 -42.0 -19.8 (Q1,Q3) (180.0, 255.0) (153.0, 198.0) (-73.7, -18.0) (-31.4, -9.8) Triglycerides (mg/dL) Mean (SD) 111.3 (45.8) 93.6 (46.7) -17.8 (37.3) -12.5 (28.6) Median 103.0 82.0 -10.0 -9.9 (Q1,Q3) (76.0, 138.0) (65.5, 113.0) (-39.0, 9.5) (-37.1, 10.1) mg/dL=milligram per decilitre; SD=standard deviation; Q1=25th percentile; Q3=75th percentile.

MIPO_publication_JCL_V20_draft23ST.docx

ACCEPTED MANUSCRIPT

Figure 2 – Lipids and Lipoproteins – absolute and % change from baseline to one year of treatment with mipomersen A P value

<.0001*

.0226*

<.0001*

LDL-C

HDL-C

Non-HDL-C

104

104

104

<.0001*

.0086*

<.0001*

<.0001*

<.0001*

<.0001*

.0004*

RI PT

10 0

Mean change (mg/dL) (±SEM)

-10 -20 -30

SC

-40 -50

-70 -80 -90

N available

B P value

5 0 -5

104

104

104

104

<.0001*

<.0001*

<.0001*

.0010*

EP

-10 -15 -20

AC C

Mean percentage change (%) (±SEM)

Apolipoprotein B Lipoprotein(a) Total Cholesterol Triglycerides

TE D

10

M AN U

-60

-25 -30 -35

N available

LDL-C

HDL-C

Non-HDL-C

104

104

104

Apolipoprotein B Lipoprotein(a) Total Cholesterol Triglycerides

104

104

104

104

Figure legend: (A) Mean absolute change from baseline to one year of treatment with mipomersen. SEM=Standard error of the mean; mg/dL=milligram per deciliter; N available=Number of subjects for whom value was available. *p-values are based on the

MIPO_publication_JCL_V20_draft23ST.docx

ACCEPTED MANUSCRIPT

Wilcoxon Signed Rank test. (B) Mean percent change from baseline to one year of treatment with mipomersen. SEM=Standard error of the mean; N available=Number of subjects for whom

AC C

EP

TE D

M AN U

SC

RI PT

value was available. *p-values are based on the Wilcoxon Signed Rank test.

MIPO_publication_JCL_V20_draft23ST.docx

24

ACCEPTED MANUSCRIPT

Table 3a – MACE incidence pre mipomersen and post mipomersen

Total # of events (incidence rate†)

# Subjects with event (%)

Total # of events (incidence rate†)

146 (25.7)

64 (61.5%)

13 (3.9)

0

Acute Myocardial Infarction

39

Coronary revascularization*

99

Unstable Angina

5

10 (9.6%)

0.053 (0.016 – 0.168)

<.0001

1

2

6

TE D

Cardiovascular death

P value

M AN U

Individual MACE events

Odds ratio (95%CI)

# Subjects with event (%)

SC

MACE events

≥12 months treatment with mipomersen (N=104)

RI PT

Prior to treatment withmipomersen (N=104)

4

AC C

EP

3 0 Ischemic Stroke Figure legend: Data presented are mean (SD) or number of events or number of subjects (%). † The number of subjects with at least one event divided by the total follow-up time in months (X 1000). MACE=Major Adverse Cardiac Events; MI=Myocardial Infarction; Coronary revascularization includes percutaneous Coronary Intervention and Coronary Artery Bypass Grafting. *Coronary revascularization includes both planned and unplanned revascularization.

MIPO_publication_JCL_V20_draft23ST.docx

ACCEPTED MANUSCRIPT

Table 3b – MACE incidence pre mipomersen and post mipomersen for the Homozygous FH subgroup

# Subjects with event (%)

12 (12.7)

7 (30.4%)

0

Acute Myocardial Infarction

1

Coronary revascularization*

9

Unstable Angina

0

Ischemic Stroke

2

Heterozygous FH subjects (N=75)

Cardiovascular death Acute Myocardial Infarction Coronary revascularization* Unstable Angina

53 (70.7%)

EP

Individual MACE events

128 (29.5)

4 (9.5)

# Subjects with event (%) 4 (17.4%)

1 0 1 2 0

9 (3.0)

0

0

38

2

84

5

5

2

AC C

MACE events

TE D

Cardiovascular death

M AN U

Individual MACE events

Total # of events (incidence rate†)

SC

Total # of events (incidence rate†) Homozygous FH subjects (N=23) MACE events

≥12 months treatment with mipomersen

RI PT

Prior to treatment withmipomersen

6 (8.0%)

1 0 Ischemic Stroke Figure legend: Data presented are mean (SD) or number of events or number of subjects (%). † The number of subjects with at least one event divided by the total follow-up time in months (X 1000). MACE=Major Adverse Cardiac Events; MI=Myocardial Infarction;

MIPO_publication_JCL_V20_draft23ST.docx

26

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Coronary revascularization includes percutaneous Coronary Intervention and Coronary Artery Bypass Grafting. *Coronary revascularization includes both planned and unplanned revascularization.

MIPO_publication_JCL_V20_draft23ST.docx

27

ACCEPTED MANUSCRIPT

Figure 3 – Incidence of MACE pre and post initiation of mipomersen treatment

100

RI PT

OR = 0.053 [95% CI: 0.016, 0.168], P<.0001

64%

61.5%

SC

60

9.6%

M AN U

% subjects with MACE event

80

40

20

0

TE D

10%

Prior to mipomersen treatment

104

104

EP

N of subjects

≥ 12 months mipomersen treatment

AC C

Figure legend: OR=odds ratio; CI=confidence interval; MACE=major adverse cardiac events.

MIPO_publication_JCL_V20_draft23ST.docx

ACCEPTED MANUSCRIPT

1- Patients with FH have a 10-20x increase in risk of premature CVD. 2- Many patients cannot reach LDL-C goals despite statins, ezetimibe and PCSK9 inhibitors. 3- Mipomersen reduces the LDL-C, non-HDL cholesterol, apoB and Lp(a).

RI PT

4- 61.5% of patients had MACE prior to mipomersen but only 9.4% after mipomersen.

AC C

EP

TE D

M AN U

SC

5- Long-term mipomersen treatment may also lead to a reduction in CVD events in FH.