A Phase 1, Randomized, Single-blind, Placebo-Controlled ... · Clinical Study Protocol Amendment 2...

Clinical Study Protocol Amendment 2

A Phase 1, Randomized, Single-blind, Placebo-Controlled, Single Ascending Dose, Safety, Tolerability and Pharmacokinetics Study of

ALN-PCS02 in Subjects with Elevated LDL-Cholesterol (LDL-C)

Protocol Number:

ALN-PCS02-001

EudraCT Number:

2011-000581-36

Product:

ALN-PCS02

Indication:

Hypercholesterolemia

Clinical Phase:

Phase 1

Chief Investigator:

Professor James Ritter BM BCh, MRCP, FRCP (Quintiles)

Sponsor:

Alnylam Pharmaceuticals, Inc

Protocol Amendment 2:

Summary

Amendment 2 Date:

12 January 2012

Amendment 1 Date:

18 October 2011

Date of Final Protocol: 09 August 2011

This protocol is the confidential property of Alnylam Pharmaceuticals, Inc and is intended solely for the guidance of this clinical investigation. This protocol may not be disclosed to parties not associated with the clinical investigation or used for any other purpose without the prior written consent of Alnylam Pharmaceuticals, Inc.

Confidential Clinical Study Protocol Amendment 2 Version: Summary Date 20 August 2013

Protocol Number:ALN-PCS02-001 EudraCT Number:2011-000581-36

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1 STUDY CONTACTS

CHIEF INVESTIGATOR: Professor James Ritter, BM BCh, MRCP, FRCP

Quintiles Drug Research Unit at Guy's Hospital, Quintiles Ltd,

6 Newcomen Street, London SE1 1YR, United Kingdom

PRINCIPAL INVESTIGATOR: Dr Joseph Chiesa MD FFPM FICR CSci

Covance Clinical Research Unit Ltd,

Springfield House, Leeds, LS2 9LH, United Kingdom

INVESTIGATIONAL SITES: Quintiles Drug Research Unit at Guy's Hospital, Quintiles Ltd,

6 Newcomen Street, London SE1 1YR, United Kingdom

Covance,

Springfield House, Hyde Street, Leeds LS2 9LH, United Kingdom

ALNYLAM MEDICAL MONITOR: Amy Simon, MD

Director, Clinical Research


300 Third Street, Cambridge, MA 02142, United States of America



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SPONSOR REPRESENTATIVE: Verena Karsten, PhD

Manager, Clinical Operations



SPONSOR: Alnylam Pharmaceuticals, Inc


SPONSOR SIGNATORY: Akshay Vaishnaw, MD, PhD, Senior Vice President, Clinical Research



BIOANALYTICAL ANALYSIS (PK lipids): Tandem Laboratories

1121 East 3900 South, Suite C-105, Salt Lake City, UT 84124, United States of America

BIOANALYTICAL ANALYSIS (PK siRNA): Quest Pharmaceutical Services,

Delaware Technology Park, 3 Innovation Way, Suite 240, Newark DE 19711,

United States of America

PHARMACOKINETIC ANALYSIS: Alnylam Pharmaceuticals, Inc




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LABORATORY ANALYSIS: Quintiles Drug Research Unit at Guy's Hospital

Pathology Laboratory, Quintiles Ltd, 6 Newcomen Street, London SE1 1YR,

United Kingdom

Covance Clinical Pathology Services,

Otley Road, Harrogate HD3 1PY, United Kingdom

ADDITIONAL LABORATORY SAFETY ANALYSIS (cytokines, complement [Bb and C3a], antibodies) Charles River Laboratories International, Inc

22022 Transcanadienne, Senneville, Quebec, Canada H9X 3R3

ADDITIONAL LABORATORY SAFETY ANALYSIS (beta-quantification of total cholesterol and HDL-C) Medpace Reference Laboratories BVBA

Technologielaan 19, B-3001 Leuven, Belgium

ADDITIONAL LABORATORY ANALYSIS (hepatitis B antibodies)[a] The Doctors Laboratory Ltd

60 Whitfield Street, London, W1T 4EU, United Kingdom

ADDITIONAL LABORATORY ANALYSIS (tryptase) ViraCor Laboratories,

1001 NW Technology Drive, Lee's Summit, MO 64086, United States of America

PHARMACODYNAMIC ANALYSIS (beta-quantification of LDL-C) Medpace Reference Laboratories BVBA

Technologielaan 19, B-3001 Leuven, Belgium



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PHARMACODYNAMIC ANALYSIS (PCSK9 protein) Charles River Laboratories International, Inc


PHARMACODYNAMIC ANALYSIS (PCSK9 mRNA) Alnylam Pharmaceuticals, Inc


EXPLORATORY BIOMARKERS (hepatocyte derived proteins) Charles River Laboratories International, Inc




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2 TABLE OF CONTENTS

1 STUDY CONTACTS .................................................................................. 2

2 TABLE OF CONTENTS ............................................................................ 6

3 TABULATED PROTOCOL SUMMARY ................................................ 10

4 LIST OF ABBREVIATIONS .................................................................... 14

5 INTRODUCTION ..................................................................................... 17 5.1 Background Information ............................................................................ 17 5.1.1 Disease Overview ...................................................................................... 17 5.1.2 ALN-PCS02, an RNAi Therapeutic for Hypercholesterolemia ................ 18

5.1.3 Non-Clinical Development ........................................................................ 19 5.1.4 Clinical Development ................................................................................ 20

5.2 Rationale .................................................................................................... 21 5.2.1 Dose Selection ........................................................................................... 21

5.2.2 Dosing of Subjects ..................................................................................... 21 5.3 Risk Assessment ........................................................................................ 22 5.3.1 Infusion-Related Reactions ........................................................................ 22

5.3.2 Other AEs .................................................................................................. 22

5.4 Urgent Safety Measures ............................................................................. 22

6 STUDY OBJECTIVES ............................................................................. 23 6.1 Primary Objective ...................................................................................... 23

6.2 Secondary Objectives ................................................................................ 23

7 INVESTIGATIONAL PLAN .................................................................... 24

7.1 Overall Study Design ................................................................................. 24 7.1.1 Dosing Strategy ......................................................................................... 25 7.1.2 Safety Review Committee ......................................................................... 25

7.1.3 Dose-Escalation and Optional Cohorts ...................................................... 26

7.1.3.1 Dose-Escalation ......................................................................................... 26 7.1.3.2 Optional Cohorts ........................................................................................ 26 7.1.4 Study Stopping Rules ................................................................................ 27

7.1.4.1 Infusion-Related Reactions ........................................................................ 27 7.1.4.2 Other DLTs ................................................................................................ 27 7.1.4.3 Stopping Rules ........................................................................................... 28 7.1.5 Discussion of Study Design ....................................................................... 28 7.2 Study Population ........................................................................................ 29

7.2.1 Subject Inclusion Criteria .......................................................................... 29 7.2.2 Subject Exclusion Criteria ......................................................................... 30 7.2.3 Restrictions ................................................................................................ 32

7.2.3.1 Avoidance of Pregnancy ............................................................................ 33 7.2.3.2 Acceptable Forms of Contraception .......................................................... 34 7.2.3.3 Time Period for the Collection of Pregnancy Information ........................ 34



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7.2.3.4 Follow-up in the Event of a Pregnancy ..................................................... 35 7.2.4 Subject Withdrawals .................................................................................. 35

8 STUDY TREATMENT ............................................................................. 36 8.1 Investigational Product(s) .......................................................................... 36 8.1.1 Supply, Packaging and Labelling .............................................................. 36 8.1.2 Storage and Handling Procedures .............................................................. 36 8.1.3 Accountability ............................................................................................ 36

8.2 Dosage and Administration ....................................................................... 37

8.2.1 ALN-PCS02 or Placebo ............................................................................. 37 8.2.2 Premedication Regimen ............................................................................. 37

8.3 Treatment Strategy ..................................................................................... 38 8.4 Warnings and Precautions ......................................................................... 38 8.5 Prior and Concomitant Medication ............................................................ 38 8.6 Method of Assigning Subjects to Treatment Groups ................................ 38 8.7 Randomisation Procedures ........................................................................ 39

8.8 Maintenance of Randomisation Codes ...................................................... 39

8.9 Blinding ..................................................................................................... 39

9 STUDY PROCEDURES ........................................................................... 40

9.1 Pharmacokinetic Assessments ................................................................... 40

9.1.1 Plasma Samples ......................................................................................... 40

9.1.2 Urine Samples ............................................................................................ 41 9.1.3 Bioanalysis ................................................................................................. 41

9.1.4 PCSK9 Protein Concentration ................................................................... 42 9.1.5 Beta Quantification of LDL-C, HDL-C and Cholesterol .......................... 42 9.1.6 PCSK9 mRNA ........................................................................................... 43

9.1.7 Adverse Events .......................................................................................... 43 9.1.7.1 Adverse Event (AE) ................................................................................... 43

9.1.7.2 Adverse Drug Reaction (ADR) ................................................................. 43 9.1.7.3 Serious Adverse Event (SAE) ................................................................... 43 9.1.7.4 Unexpected Adverse Reactions ................................................................. 44 9.1.8 Reporting of Adverse Events ..................................................................... 44

9.1.9 Categorisation of Adverse Events ............................................................. 45 9.1.10 Causal Relationship Assessment ............................................................... 45

9.1.11 Action Taken for Adverse Event ............................................................... 46 9.1.12 Outcome of Adverse Event ........................................................................ 46 9.1.13 Coding of Adverse Events ......................................................................... 46 9.2 Clinical Laboratory Safety Tests ............................................................... 46 9.2.1 Local Laboratory Safety Tests ................................................................... 46

9.2.2 Central Laboratory Safety Tests ................................................................ 48 9.3 Clinical Safety Assessments ...................................................................... 48 9.3.1 Vital Signs ................................................................................................. 48 9.3.2 12 Lead ECG ............................................................................................. 49 9.3.3 Continuous Lead II ECG Monitoring ........................................................ 49

9.3.4 Medical History ......................................................................................... 49 9.3.5 Physical Examination ................................................................................ 49



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9.3.6 Pulse Oximetry .......................................................................................... 49 9.3.7 Infusion-Related Reactions ........................................................................ 49

9.4 Exploratory Biomarkers ............................................................................. 49

10 DATA COLLECTION .............................................................................. 51

11 EVALUATION OF STUDY DATA ......................................................... 52 11.1 Evaluation of Pharmacokinetic Parameters ............................................... 52 11.2 Evaluation of Pharmacodynamic Measures ............................................... 53

11.3 Evaluation of Safety .................................................................................. 53

12 STATISTICAL METHODS ...................................................................... 54 12.1 Primary and Secondary Target Variables .................................................. 54

12.2 Statistical Analysis Plan ............................................................................ 54 12.3 Sample Size Determination ....................................................................... 54 12.4 Subject Population for Analyses ................................................................ 54

12.5 General Methods ........................................................................................ 54 12.6 Safety Data Analysis .................................................................................. 55 12.7 Pharmacokinetic Analysis ......................................................................... 55

12.8 Pharmacokinetic and Pharmacodynamic Analysis .................................... 56

13 STUDY REPORT ...................................................................................... 57

14 REGULATORY AND ETHICAL ISSUES .............................................. 58 14.1 Regulatory and Ethics Review and Approval ............................................ 58

14.2 Informed Consent ...................................................................................... 58 14.3 Indemnity and Compensation .................................................................... 59

15 STUDY MANAGEMENT ........................................................................ 60 15.1 Communication Plan ................................................................................. 60 15.2 Quality Assurance and Quality Control ..................................................... 60 15.3 Protocol Adherence ................................................................................... 60

15.4 Documents Necessary for Initiation of Study ............................................ 60 15.5 Study Monitoring ....................................................................................... 61

15.6 Study Closure ............................................................................................. 61 15.7 Study Record Retention ............................................................................. 61 15.8 Publication Policy ...................................................................................... 62

16 REFERENCES .......................................................................................... 63

17 STUDY SCHEDULE ................................................................................ 66

18 PROTOCOL SIGNATURES .................................................................... 72



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LIST OF TABLES Table 7.1 Dosing Strategy ............................................................................................ 25

Table 7.2 Categorisation of Infusion-Related ReactionsError! Bookmark not

defined. Table 8.1 Assignment of Subjects to Treatment .......................................................... 39 Table 9.1 Approximate Blood Volumes ...................................................................... 40 Table 17.1 Study Schedule: Screening through to Day 180 .......................................... 67

Table 17.2 Study Schedule: Day 0 to Day 4 .................................................................. 70



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3 TABULATED PROTOCOL SUMMARY

Name of

Sponsor/Company:


Name of Finished

Product(s):

ALN-PCS02 Solution for Injection

Name of Active

Ingredient(s):

ALN-PCS02 (siRNA targeting the PCSK9 mRNA

formulated for IV injection in lipid nanoparticles)

Title of Study:

A Phase 1, Randomized, Single-blind, Placebo-Controlled, Single Ascending Dose,

Safety, Tolerability and Pharmacokinetics Study of ALN-PCS02 in Subjects with

Elevated LDL-Cholesterol (LDL-C)

Principal Investigators:

James Ritter BM BCh, MRCP, FRCP (Chief Investigator)

Joseph Chiesa FFPM FICR CSci

Investigational Sites:

The study will be conducted at up to 2 sites in the United Kingdom:

Quintiles Drug Research Unit at Guy's Hospital, Quintiles Ltd, 6 Newcomen Street,

London SE1 1YR, United Kingdom

Covance, Springfield House, Hyde Street, Leeds LS2 9LH, United Kingdom

Clinical Phase: Phase 1

Objectives:

Primary Objective

To evaluate the safety and tolerability of ALN-PCS02 when administered to

subjects with elevated low density lipoprotein cholesterol (LDL-C).

Secondary Objectives

To characterize the pharmacokinetics (PK) of ALN-PCS02 in subjects with

elevated LDL-C;

To assess the pharmacodynamic (PD) effect of ALN-PCS02 on plasma levels

of proprotein convertase subtilisin kexin 9 (PCSK9) and serum levels of

LDL-C.



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Methodology:

This is a randomized, single-blind, placebo-controlled, single ascending dose study in

up to 8 sequential cohorts (6 dose-escalation cohorts and up to 2 optional cohorts to

further explore safety, pharmacodynamic activity [PD], and or pharmacokinetics [PK])

of healthy subjects with elevated LDL-C. Only the subjects will be blinded to

treatment.

Consenting subjects meeting the eligibility criteria will be enrolled into up to

8 sequential cohorts. Each cohort will comprise 4 subjects randomized

(3:1 ALN-PCS02:placebo) to receive a single intravenous infusion of ALN-PCS02 or

placebo. No subject will be a member of more than one treatment group.

Subjects will be screened from -45 to -7 days prior to dose administration. The

Investigator may include a second screening visit for additional measurement of

LDL-C to confirm eligibility. Eligible subjects will be admitted to the clinical unit on

Day -3 for eligibility checks and pre-treatment assessments. All subjects will receive

oral premedication with dexamethasone, paracetamol, and H2 and H1 blockers the

night before and in the morning prior to the dose of ALN-PCS02 or placebo. On Day 0

of the admission period subjects will receive a single dose of ALN-PCS02 or placebo

administered as a 1-hour infusion. Subjects will be discharged from the unit 4 days

after dose administration and will return to the unit for out-patient visits for safety, PK

and PD monitoring up to 180 days post-dose.

Sentinel dosing will be used as follows: the first 2 subjects (1 on ALN-PCS02 and 1 on

placebo) in each cohort may be dosed at the same time. If the doses are safe and

tolerated as determined by a Safety Review Committee (SRC), dosing of the third

subject (ALN-PCS02) will occur no sooner than 72 ± 2 hours after dosing of the first

subject receiving ALN-PCS02 . Subject 4 (ALN-PCS02) will be dosed no sooner than

24 ± 2 hours after the third subject.

Prior to dosing the next cohort, safety data on 4 subjects from the prior cohort through

at least Day 2 post-dose will be reviewed by the SRC. If stopping criteria have not

been met, the SRC may decide to escalate to the next planned dose level, escalate to an

intermediate dose level, repeat the dose level, or decrease to a lower intermediate dose

level (see Section 7.1 for details).

Up to 2 optional cohorts may be enrolled, each with 4 subjects (maintaining the 3:1

randomization of ALN-PCS02:placebo). These optional cohort(s) will use the same

dosing strategy as cohorts 1 to 5.

If at any point in the study stopping criteria are met, the study will be stopped and no

further subjects will be dosed.



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Number of Subjects:

Up to 32 subjects are planned (including 2 optional cohorts of 4 subjects each).

Main Criteria for Inclusion:

Adult males and females (of non-childbearing potential), with a fasting LDL-C of

>3.0 mmol/L and <5.7 mmol/L (based on the mean of the measurements at screening

and admission) will be eligible for enrolment. The subjects should otherwise be

healthy, aged 18 to 65 years inclusive at the screening visit. Subjects must have body

weight >60.0 kg and body mass index must be at least 19.00 kg/m2 and <35.00 kg/m

2

at the screening visit. Subjects must have a fasting triglyceride concentration of

≤2.8 mmol/L. Subjects should not have received any lipid lowering drug/agent within

30 days prior to the screening visit. All subjects must be able and willing to give

written informed consent.

Test Product, Dose and Route of Administration:

ALN-PCS02 (small interfering RNA [siRNA] targeting the PCSK9 mRNA) is

formulated for intravenous (IV) infusion in lipid nanoparticles (LNP) at doses of 15,

45, 90, 150, 250 and 400 μg/kg, administered over 1 hour.

Reference Therapy, Dose and Route of Administration:

Placebo solution (sterile saline [0.9% NaCl]) administered as a matched intravenous

infusion over 1 hour.

Duration of Treatment:

Single dose administration as a 1-hour IV infusion.

Criteria for Evaluation

Pharmacokinetics:

The PK evaluation will include plasma-concentration time profiles for PCS siRNA and

the novel lipid components DLin-MC3-DMA and PEG2000-C-DMG. PCS siRNA

concentration will be determined at specified time points up to 42 days post-dose.

DLin-MC3-DMA and PEG2000-C-DMG concentrations will be determined at specified

time points up to 180 days post-dose. An assessment of plasma concentrations of free

and encapsulated siRNA will be determined at specified time points.

Urine will be collected to determine PCS siRNA excreted in urine and renal clearance

(CLR) of PCS siRNAafter dosing with ALN-PCS02.

Pharmacodynamics:

The pharmacodynamic evaluation will include assessment of plasma concentrations of

PCSK9 and serum samples of LDL-C.

Safety:

The safety evaluation will include assessment of adverse events, 12-lead



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electrocardiograms (ECGs), lead II ECG monitoring, arterial oxygen saturation using

pulse oximetry, vital signs (blood pressure, pulse, oral body temperature and

respiration rate), clinical laboratory safety tests (hematology, serum chemistry,

coagulation parameters, urinalysis, HDL-C, total cholesterol, cytokines, activation

fragment of complement factor [Bb]) and physical examinations.

Statistical Methods

Pharmacokinetic Parameters:

PK analyses will be conducted using non-compartmental and/or compartmental

evaluation of PCS siRNA, DLin-MC3-DMA (lipid) and PEG2000-C-DMG (lipid)

plasma concentration-time data to determine PK parameter using a validated software

program, WinNonlin®. PK parameter estimates such as Cmax, AUC, CL, Vss and t1/2

will be assessed descriptively whenever possible. Statistical analysis on PK parameter

estimates will be assessed descriptively whenever possible and by exploratory

statistical comparisons.

Pharmacodynamic Parameters:

PD data will be assessed descriptively whenever possible and by exploratory statistical

comparisons.

Safety Parameters:

Individual and summary vital signs, 12-lead ECG parameters, arterial oxygen

saturation and clinical laboratory data will be presented in tabular form with mean,

median, standard deviation and range (minimum and maximum) as appropriate.

For the laboratory safety data, 12-lead ECG parameters and vital signs values outside

the reference range will be flagged in the data listings and a list of clinically

significantly abnormal values will be presented.

Treatment Emergent Adverse Events (TEAE) will be tabulated and summarised

according to the current version of Medical Dictionary for Regulatory Activities

(MedDRA).



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4 LIST OF ABBREVIATIONS

ABPI Association of the British Pharmaceutical Industry

ADR Adverse drug reaction

AE Adverse event

ALP Alkaline phosphatase

ALT Alanine aminotransferase

Apo Apolipoprotein

aPTT Activated partial thromboplastin time

AST Aspartate aminotransferase

ATTR Transthyretin amyloidosis

AUC Area under the plasma concentration-time curve

AUCp Partial area under the plasma concentration-time curve

AUC0-t Area under the plasma concentration-time curve to the last

measurable concentration

AUC0-last Area under the plasma concentration-time curve from zero to the

last measurable time point

AUC0-∞ Area under the plasma concentration-time curve extrapolated to

infinity

Bb Activation fragment of complement factor B

BMI Body mass index

C3a Complement component 3a

CHD Coronary heart disease

CPK Creatine phosphokinase

CPK-MB Creatine phosphokinase - myocardial band

CL Systemic clearance

CLR Renal clearance

Cmax Observed maximum concentration

CPMP Committee for Proprietary Medicinal Products

CRF Case report form

CRP C reactive protein

DLT Dose limiting toxicity

EC50 50% effective concentration

ECG Electrocardiogram

ED50 50% effective dose

ELISA Enzyme linked immunosorbent assay

EMA European Medicines Agency

EOI End of infusion

ET Early termination

EU European Union

FSH Follicle stimulating hormone

GCP Good clinical practice

GGT Gamma glutamyl transpeptidase

GMP Good manufacturing practice

H1 blocker Histamine H1 receptor antagonist



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H2 blocker Histamine H2 receptor antagonist

HBsAb Hepatitis B surface antibodies

HBsAg Hepatitis B surface antigen

HBV Hepatitis B virus

HCV Hepatitis C virus

HDL High density lipoprotein

HDL-C High density lipoprotein cholesterol

HED Human equivalent dose

HIV Human immunodeficiency virus

IB Investigator’s brochure

IC50 Half maximal inhibitory concentration

ICF Informed consent form

ICH International Conference on Harmonization

IEC Independent Ethics Committee

IgE Immunoglobulin E

IMP Investigational medicinal product

IMPD Investigational medicinal product dossier

INR International normalized ratio

IRR Infusion related reaction

IUD Intrauterine device

IUS Intrauterine system

IV Intravenous

KSP Kinesin spindle protein

λz Elimination rate constant

LC/MS/MS Liquid chromatography/mass spectrometry (tandem)

LDH Lactate dehydrogenase

LDL Low density lipoprotein

LDL-C Low density lipoprotein cholesterol

LDLR Low density lipoprotein receptor

LFT Liver function test

LNP Liposomal nanoparticles

MedDRA Medical Dictionary for Regulatory Activities

MHRA Medicines and Healthcare products Regulatory Agency (UK)

mRNA Messenger RNA

MTD Maximum tolerated dose

NHP Non-human primate

NOAEL No observed adverse effects level

NSAID Non-steroidal anti-inflammatory

PCSK9 Proprotein convertase subtilisin kexin 9

PD Pharmacodynamic

PK Pharmacokinetic

PT Prothrombin time

QC Quality control

QP Qualified Person

QT Measure of the time between the start of the Q wave and the end of

the T wave in the heart

QTcB QT interval corrected for heart rate using Bazett's formula



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RNAi RNA interference

RSV Respiratory syncytial virus

SAE Serious adverse event

SaO2 Arterial oxygen saturation

SAP Statistical analysis plan

SAS® Statistical Analysis System

SI Statutory Instrument

siRNA Small interfering RNA

SNALP Stable nucleic acid lipid nanoparticles

SRC Safety Review Committee

SUSAR Suspected unexpected serious adverse reaction

TEAE Treatment emergent adverse event

t1/2β and t1/2α Terminal elimination half-life

Tmax Time of observed maximum concentration

TTR Transthyretin

ULN Upper limit of the normal reference range

VEGF Vascular endothelial growth factor

VLDL Very low density lipoprotein

Vss Volume of distribution at steady state

Vz Volume of distribution based on the terminal phase

WHO World Health Organization



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5 INTRODUCTION

5.1 Background Information

5.1.1 Disease Overview

According to the World Health Organization (WHO), cardiovascular disease, comprised

mainly of coronary heart disease (CHD) and stroke, is the leading cause of death

worldwide, resulting in 17 million deaths annually (WHO Cardiovascular Statistics,

2011). Data from the INTERHEART case-control study estimates that 45% of

myocardial infarctions in Western Europe and 35% of myocardial infarctions in Central

and Eastern Europe are due to elevations in blood lipids (Yusef et al, 2004). The

prevalence of elevated total cholesterol >6.5 mmol/L (normal total cholesterol level

<5 mmol/L) in the European population has been shown to vary considerably by region,

with a range from 8% (Novosibirsk, Russia) to 53% (Ticino, Switzerland) (Truelsen et al,

2003). In particular, elevated low density lipoprotein cholesterol (LDL-C), has been

shown in multiple studies to be one of the major risk factors for CHD with a continuous

and graded relationship between plasma LDL-C concentration and CHD risk: for every

30 mg/dL (0.78 mmol/L) change in LDL-C, the relative risk for CHD changes by

approximately 30% (Grundy et al, 2004). Despite the extensive use of statins, current

therapies for the management of elevated LDL-C remain inadequate throughout the

world. This is particularly true in patients with pre-existing CHD and/or diabetes, who

are at highest risk, and require the most intensive and aggressive management of

hypercholesterolemia (Nag et al, 2007, Davidson et al, 2005). Among these high risk

patients, as many as 45% did not achieve their target LDL-C goal 6 months post statin

treatment despite close monitoring and drug regimen optimization (Foley et al 2003).

Thus, there remains a clear unmet medical need for lowering LDL-C especially in certain

patient populations.

Proprotein convertase subtilisin kexin 9 (PCSK9) is a member of the subtilisin serine

protease family. PCSK9 is predominantly expressed by the liver (Mousavi et al, 2009),

and is critical for the down regulation of hepatocyte LDL receptor (LDLR) expression.

LDL-C levels in plasma are highly elevated in humans with gain of function mutations in

PCSK9, classifying them as having severe hypercholesterolemia (Abifadel et al, 2003).

Data from genetic association studies have identified loss of function alleles in human

PCSK9 that result in lower PCSK9 protein levels and lower LDL-C levels. In one

published study, heterozygous individuals (carrying a single copy of a loss of function

PCSK9 mutation) had significantly lower LDL-C with median levels of approximately

70 mg/dL (1.81 mmol/L). Over a 15-year period of retrospective data analysis, this

sustained lowering in LDL-C levels translated to an 88% lowering of CHD (Cohen et al,

2006). Follow-up publications describe two adult individuals that are compound



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heterozygous for loss of function alleles of PCSK9. These individuals lack detectable

plasma PCSK9 protein, have LDL-C levels ≤ 20 mg/dL, and yet are otherwise healthy

(Zhao et al, 2006; Hooper et al, 2007). Additionally, recent pre-clinical and clinical data

suggest that antibodies that are capable of blocking the ability of PCSK9 to bind to the

LDLR significantly lower LDL-C levels in both non-human primates (NHP) and in

humans with no reported safety issues (Ni et al, 2011, Chan et al, 2009, Regeneron

presentation, 2011). Finally, additional validation of PCSK9 as a therapeutic target

comes from both human and mouse studies suggesting that an elevation of PCSK9 levels

occurs with statin treatment, and that this upregulation limits the magnitude of the statin

response (Davignon et al, 2009). Specifically, PCSK9 loss of function in mice was found

to be synergistic with statin effects in increasing LDLR levels and lowering LDL-C levels

(Rashid et al, 2005). The overall scientific data suggests that PCSK9 is a well-validated

drug target whose inhibition results in significant LDL-C lowering without otherwise

negatively impacting overall health. (Zhao et al, 2006; Hooper et al, 2007). Alnylam

Pharmaceuticals is developing ALN-PCS02, a synthetic investigational ribonucleic acid

interference (RNAi) therapeutic comprising a small interfering RNA (siRNA) targeting

the PCSK9 messenger RNA (mRNA).

5.1.2 ALN-PCS02, an RNAi Therapeutic for Hypercholesterolemia

RNA interference (RNAi) is a naturally occurring cellular mechanism for regulating gene

expression that is mediated by “small interfering RNAs” (siRNAs) (Vaishnaw et al,

2010). Typically, synthetic siRNAs are 19 to 23 base pair double-stranded

oligonucleotides in a staggered duplex with a 2-nucleotide overhang at one or both of the

3’ ends. Such siRNAs can be designed to target an endogenous or virally-expressed gene.

When introduced into cells, the net effect of an RNAi-based pharmacological approach is

the binding of the siRNA to its complementary mRNA sequence, cleavage of this target

mRNA, and suppression of the target protein (Elbashir et al, 2001). The ability to

selectively and potently degrade the mRNA encoding the PCSK9 protein using an siRNA

offers a potent and specific approach for the treatment of hypercholesterolemia.

Unformulated siRNAs, and those without significant modification, are rapidly degraded

and eliminated upon systemic administration, and thus do not achieve significant tissue

distribution (Soutschek et al, 2004). As a result, various formulations are used to target

siRNAs distribution to tissues, and to facilitate their uptake into the relevant cell type.

One approach that has been used successfully in vivo, including in rodents and NHPs,

employs intravenous (IV) delivery of siRNAs in liposomal nanoparticles (LNPs)

(Geisbert et al, 2006; Judge et al, 2006; Morrissey et al, 2005; Soutschek et al, 2004;

Zimmermann et al, 2006). These LNPs, with their small size (<100 nm) and low surface

charge can pass through the fenestrated vascular endothelium of the liver. Endocytosis of

the intact LNPs , followed by fusion with the endosomal membrane (Maurer et al, 1999,



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Akinc et al, 2010) and release of the siRNA into the cytoplasm, results in the siRNA

engaging the endogenous RNAi machinery described above to cause targeted degradation

of the mRNA, and a consequent reduction in target protein levels.

Alnylam Pharmaceuticals is developing ALN-PCS02 Solution for Injection (hereafter

referred to as ALN-PCS02), a synthetic investigational RNAi therapeutic comprising an

PCS siRNA targeting the PCSK9 mRNA formulated in a LNP formulation termed AF-

011. The LNP enables delivery of the siRNA primarily to the liver upon systemic

administration, resulting in the down-regulation of hepatic PCSK9 expression, and in

turn, reducing plasma LDL-C levels. The proposed indication for ALN-PCS02 is for the

treatment of hypercholesterolemia. ALN-PCS02 is intended for administration as an IV

infusion over 1 hour.

5.1.3 Non-Clinical Development

As part of the non-clinical development of ALN-PCS02, pharmacology, pharmacokinetic

(PK), metabolism, and safety/toxicity studies have been conducted. Cynomolgus

monkeys are pharmacologically responsive to ALN-PCS02 in terms of silencing of

endogenous mRNA for PCSK9, lowering of systemic PCSK9 protein, and lowering of

LDL-C. While ALN-PCS02 is not pharmacologically active in rats, rats were included in

the nonclinical toxicity assessment as a highly sensitive species for evaluation of general

toxicities related to the lipid nanoparticle formulated siRNA.

The nonclinical safety/toxicity of ALN-PCS02 was evaluated in a series of in vitro and in

vivo safety/toxicity studies (see ALN-PCS02 Investigator’s Brochure [IB]).

In the safety pharmacology study conducted in cynomolgus monkeys, there were no

ALN-PCS02-related electrocardiogram (ECG) abnormalities or effects on blood pressure,

blood gases, and respiratory rate or neurologic signs at the highest dosage evaluated

(6.0 mg/kg). There was a dose-dependent increase in body temperature (1.5-2ºC) and

heart rate at dosages >3.0 mg/kg.The nonclinical toxicity of ALN-PCS02 was evaluated

in single-dose toxicity studies in Sprague-Dawley rats and cynomolgus monkeys at

dosages up to 3.0 mg/kg (1 hour IV infusion). The target organs of toxicity in the rat

were liver, spleen, heart, and/or kidney. In monkeys the target organs of toxicity were

liver and spleen. The toxicities observed in both rats and cynomolgus monkeys were

largely reversible within 15 days of dosing and completely reversible by the end of a 60-

day recovery period.

Genotoxicity studies (Ames and chromosome aberration assays) demonstrated that

ALN-PCS02 was not mutagenic or clastogenic.

Further information can be found in the IB.



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5.1.4 Clinical Development

Though no clinical studies have been conducted with ALN-PCS02, clinical trials have

been conducted with several RNAi medicinal products, with local or systemic

administration, in indications including age-related macular degeneration, respiratory

syncytial virus (RSV) infection, oncology and renal failure (reviewed Vaishnaw et al,

2010). Alnylam has two ongoing systemic delivery programs in the clinic which use a

similar but not identical LNP formulation termed SNALP (stable nucleic acid lipid

particle). ALN-VSP02, comprising two siRNAs targeting vascular endothelial growth

factor (VEGF) and kinesin spindle protein (KSP) formulated in SNALP, has completed

enrolling patients in a multi-dose Phase 1 trial in the USA (IND # 102,876) and Spain

(EudraCT #s 2009-015801-38 and 2009-015802-20) for the treatment of advanced solid

tumors in the liver, and currently has an ongoing open-label extension study for patients

with stable disease. ALN-TTR01, employing an siRNA targeting mutant and wild type

transthyretin (TTR) in SNALP, is being evaluated in a single-dose Phase 1 study in

Portugal, Sweden, the United Kingdom and France (EudraCT # 2009-017383-16) for the

treatment of TTR amyloidosis (ATTR).

To date, a total of 164 doses of ALN-VSP02 (mean of 4 doses per patient; range

1-21 doses) have been administered to 41 patients across 7 dose levels (0.1-1.5 mg/kg).

The highest dose administered was 1.5 mg/kg ALN-VSP02. The maximum tolerated

dose (MTD) was defined as 1.0 mg/kg ALN-VSP02. During the dose-escalation phase of

the study (N=31), ALN-VSP02 was generally well-tolerated, with no dose-dependent

trends in clinical or laboratory adverse events (AEs), including no dose-dependent

changes in LFTs. Preliminary evidence of pharmacodynamic activity (reduction of blood

flow) was demonstrated in the ALN-VSP02 study. Additionally, the specific mRNA

fragment, which results from VEGF siRNA mediated cleavage, was detected in liver

biopsy samples from some of the ALN-VSP02-treated patients, demonstrating that RNA

interference is occurring in man in the targeted tissue (Cervantes et al, 2011 (in press)).

To date, 15 subjects have received ALN-TTR01 at single doses up to 0.4 mg/kg with

encouraging safety and tolerability, and dose-escalation up to 1.0 mg/kg is ongoing. The

emerging results from the Phase 1 ALN-VSP02 and ALN-TTR01 clinical trials indicate

that the SNALP formulation is generally safe and well tolerated when administered as a

15-minute IV infusion; additionally, preliminary evidence of pharmacological activity as

well as RNAi-mediated mechanism of action was demonstrated in the ALN-VSP02 study.

Thus, the nonclinical data with ALN-PCS02 in rats and cynomolgus monkeys, as well as

the clinical experience with SNALP-formulated siRNAs, support the proposed Phase 1,

randomized, single-blind, single ascending dose ALN-PCS02-001 study.

Further information can be found in the IB.



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The Sponsor will immediately notify the Principal Investigators if any relevant new safety

or toxicology information becomes available during the study.

5.2 Rationale

The primary objective of this study is to determine the safety and tolerability of a single

1-hour IV infusion of ALN-PCS02 in subjects with elevated LDL-C. Secondary

objectives include the characterization of plasma and urine PK for ALN-PCS02 as well as

PD assessment of the impact of ALN-PCS02 on plasma levels of PCSK9 and LDL-C.

5.2.1 Dose Selection

A single dose of ALN-PCS02 or placebo will be administered to subjects in a single-

blinded fashion by a 1-hour IV infusion. The dose levels proposed are 15, 45, 90, 150,

250 μg/kg and 400 μg/kg.

We have proposed to have a starting dose of 15 μg/kg (or 0.015 mg/kg) for this study

based on the adequate safety margins in rat and monkey. The highest dose level

proposed, 400 μg/kg, is based on adequate safety margin in the monkey (see the

Investigator’s Brochure for more detail).

5.2.2 Dosing of Subjects

Taking the European Medicines Agency (EMA) “Guideline on Strategies to Identify and

Mitigate Risks for First-in-Human clinical Trials with Investigational Medicinal Products

CHMP/SWP/28367/07)” into consideration, a conservative approach is proposed for the

dosing of subjects within each cohort and between cohorts. The first 2 subjects in each

cohort may be dosed at the same time (see Section 7.1). The study will be randomized to

ensure that one of these 2 subjects receives placebo. If the dose is safe and tolerated, as

determined by the Safety Review Committee (SRC; see Section 7.1 for details), dosing of

the third subject at that same dose level will be allowed to occur no less than 72 ± 2 hours

after the dosing of the first subject receiving ALN-PCS02. The fourth subject will be

dosed no sooner than 24 ± 2 hours after the third subject. Collective cohort safety and

tolerability data on the 4 subjects from the prior cohort through at least Day 2 post-dose

will be reviewed by the SRC prior to approving dosing for the next protocol-specified

dose level.

Up to 2 optional cohorts may be enrolled, each comprised of 4 subjects (3:1

randomization of ALN-PCS02:placebo). An optional cohort may only occur if stopping

rules have not been met and would be included to further confirm safety, PD and/or PK

effects. The optional cohorts will follow the same treatment schema as cohorts 1 to 5.



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5.3 Risk Assessment

The subjects taking part in this clinical trial will not gain any health benefit from their

participation other than a general health check.

5.3.1 Infusion-Related Reactions

Infusion related reactions (IRRs) can occur with systemic administration of certain

biological agents such as liposomes or monoclonal antibodies. These may include acute

reactions or delayed febrile reactions. Premedicating subjects with corticosteroids and/or

antihistamines, or slowing of the infusion rate, are approaches that have been taken to

reduce the incidence and/or severity of IRRs (Gomes et al, 2005, Solensky, 2006).

Consistent with this, a premedication regimen (dexamethasone, paracetamol, H1/H2

blocker) similar to that proposed for this study was used in the prior ALN-VSP02 and

ALN-TTR01 clinical studies that employed siRNAs in LNP formulations (Section 5.1.4).

In order to reduce the potential for an IRR with ALN-PCS02, all subjects in this study

will be premedicated prior to dosing with ALN-PCS02 or placebo. The premedication

regimen will include orally administered dexamethasone, paracetamol, and histamine H1

receptor and H2 receptor blockers (as described in Section 8.2). The rate of infusion

(over the course of 1 hour) will also help to reduce the potential for acute IRRs.

5.3.2 Other AEs

Adverse events are monitored carefully by a cautious dose-escalation schema in each

dosing cohort with safety monitoring after the first 2 subjects are dosed and again after

completing a cohort (n=4 subjects). For definitions and guidance regarding stopping

rules and dose limiting toxicities (DLTs), see Section 7.1.4.

5.4 Urgent Safety Measures

In accordance with UK Law [Medicines for Human Use (Clinical Trials) as amended:

Statuatory Instrument (SI) 1031 Part 4 Section 30] the Sponsor and Investigator may take

appropriate urgent safety measures in order to protect the subjects of a clinical trial

against any immediate hazard to their health or safety. If such measures are taken the

Sponsor shall immediately (no later than 3 days from the date the measures are taken)

give written notice to the licensing authority and the relevant independent ethics

committee (IEC) of the measures taken and the circumstances giving rise to those

measures.



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6 STUDY OBJECTIVES

6.1 Primary Objective

To evaluate the safety and tolerability of ALN-PCS02 when administered to subjects

with elevated LDL-C.

6.2 Secondary Objectives

To characterize the PK of ALN-PCS02 in subjects with elevated LDL-C;

To assess the PD effect of ALN-PCS02 on plasma levels of PCSK9 and serum levels

of LDL-C.



Page 24 of 74

7 INVESTIGATIONAL PLAN

7.1 Overall Study Design

This is a randomized, single-blind, placebo-controlled, single ascending dose study in up

to 8 sequential cohorts of subjects with elevated LDL-C. Only the subjects will be

blinded to treatment. Up to 32 subjects are anticipated to be enrolled at up to 2 clinical

sites.

Single ascending doses of ALN-PCS02 (15, 45, 90, 150, 250 and 400 μg/kg) will be

investigated in 6 sequential cohorts. Each cohort will comprise 4 subjects randomized

(3:1 ALN-PCS02:placebo) to receive a single IV infusion of ALN-PCS02 or placebo. No

subject will be a member of more than one treatment group. Two optional cohorts may

also be included to further explore the safety, PD and/or PK of ALN-PCS02 (see

Section 7.1.3).

Subjects will be screened from -45 to -7 days prior to dose administration. The

Investigator may include additional screening visit(s) for a repeat measurement of fasted

LDL-C to confirm eligibility. Eligible subjects will be admitted to the clinical unit on

Day -3 for eligibility checks and pre-treatment assessments (performed over Day -3

through Day 0). All subjects will receive oral premedication with dexamethasone,

paracetamol, and H1 and H2 blockers the night before and in the morning prior to the

dose of ALN-PCS02 or placebo to reduce the potential for an IRR (see Section 8.2). On

Day 0 of the admission period subjects will receive a single dose of ALN-PCS02 or

placebo administered as a 1-hour IV infusion. Subjects will be discharged from the unit

4 days after ALN-PCS02 or placebo dose administration and will return to the unit for

out-patient visits for safety, PK and PD monitoring up to 180 days post-dose (see

Section 17 for details).

Safety monitoring will include assessment of AEs, 12-lead ECGs, lead II ECG

monitoring, arterial oxygen saturation (SaO2) using pulse oximetry, vital signs (blood

pressure, pulse rate, oral body temperature and respiratory rate), clinical laboratory safety

tests (hematology, serum chemistry, coagulation, urinalysis, HDL-C, total cholesterol,

cytokines and the activation fragment of complement factor B [Bb]) and physical

examinations. Subjects will be closely monitored for both acute and delayed IRRs. All

IRRs will be recorded as AEs.

The PK evaluation will include plasma-concentration time profiles for PCS siRNA and

the novel lipid components DLin-MC3-DMA and PEG2000-C-DMG. PCS siRNA

concentrations will be determined at specified time points up to 42 days post-dose.

PEG2000-C-DMG and DLin-MC3-DMA will be determined at specified time points up to

180 days post-dose. An assessment of plasma concentrations of free and encapsulated



Page 25 of 74

siRNA will be determined at specified time points from cohort 4 onwards and any

optional cohort(s). Urine will be collected to determine PCS siRNA excreted in urine

and renal clearance (CLR) of PCS siRNA after dosing with ALN-PCS02.

The PD evaluation will include serial assessment of plasma concentrations of PCSK9 and

serum concentrations of LDL-C.

7.1.1 Dosing Strategy

A sentinel dosing strategy will be used for this study (see Section 0). The SRC will

evaluate safety and tolerability data in the study and determine if it remains acceptable to

continue dosing (within cohorts) or to dose escalate (between cohorts).

The dosing strategy is summarised in Table 7.1. Sentinel dosing will be as follows: the

first 2 subjects (1 on ALN-PCS02 and 1 on placebo) in each cohort may be dosed at the

same time. If the doses are safe and tolerated, as determined by the SRC, dosing of the

third subject (ALN-PCS02) will occur no sooner than Day 3, 72 ± 2 hours after dosing of

the first subject receiving ALN-PCS02 (see Table 7.1). Subject 4 (ALN-PCS02) will be

dosed no sooner than 24 ± 2 hours after the third subject.

Table 7.1 Dosing Strategy

Subject Dosing Observation Periods & SRC Reviews

Sentinel subjects 1 & 2 dosed

(1 ALN-PCS02 & 1 placebo)

72 ± 2 h observation period after dosing the first subject

with ALN-PCS02

SRC Review of available safety data (Subjects 1+ 2)

Subject 3 dosed

(ALN-PCS02)

24 ± 2 h observation period

Subject 4 dosed

(ALN-PCS02)

SRC Review of available safety data of 4 subjects

through at least Day 2 post dose prior to dose-escalation

7.1.2 Safety Review Committee

The SRC will evaluate safety and tolerability data and determine if it remains acceptable

to continue dosing (within cohorts) or to dose escalate (between cohorts). The SRC will



Page 26 of 74

also be convened in the event of a DLT to determine whether stopping rules for accrual to

a particular dose level have been met (see Section 7.1.4 for study stopping rules).

To ensure timely safety information exchange across the participating study centres, the

SRC will be comprised of the Chief Investigator (Quintiles Drug Research Unit at Guy's

Hospital) and Principal Investigator (Covance Clinical Research Unit) or their designee,

the Alnylam Medical Monitor, and the Medpace Medical Monitor. The SRC will

evaluate the available safety data post infusion including but not limited to: AEs, vital

signs, pulse oximetry, 12-lead ECGs and available laboratory parameters.

The options based on SRC discussion (if stopping criteria have not been met) are to:

Escalate the dose as planned;

Escalate the dose to an intermediate level;

Repeat the dose only in the event of a safety signal (not considered severe) that

indicates that any further dose escalation may affect the safety of the study subjects

(or to re-evaluate the PK data or to further characterize PD markers);

Decrease to a lower intermediate dose;

Terminate the study.

All data reviewed at the SRC meetings will be subjected to a quality control (QC) review

and will be distributed to both study site Investigators prior to each SRC meeting.

7.1.3 Dose-Escalation and Optional Cohorts

7.1.3.1 Dose-Escalation

Prior to escalating to the next dose the SRC will review data from 4 evaluable subjects

from the prior cohort through at least Day 2 post-dose. Progression to the next higher

dose will only occur if the SRC deem the previous dose level to be safe and tolerated and

no stopping criteria have been met.

7.1.3.2 Optional Cohorts

If no stopping criteria have been met, the SRC may decide to enroll up to 2 optional

cohorts, each with 4 subjects (maintaining the 3:1 randomization of ALN-PCS02:

placebo). The dosing schema will be the same as for cohorts 1 to 6. Optional cohorts

may be used to:

Escalate or de-escalate the dose to an intermediate dose level;

Repeat a dose level if a safety signal (not considered severe nor a stopping criteria)

indicates that any further dose escalation may affect the safety of the study

participants;

Repeat a dose level to further characterise PD markers;



Page 27 of 74

Repeat a dose level to re-evaluate PK data.

The IEC will be informed of any optional cohorts.

7.1.4 Study Stopping Rules

The actions to be taken in the event of DLTs are described below. If stopping criteria as

defined below are met, no further dosing of subjects will occur.

7.1.4.1 Infusion-Related Reactions

All infusion reactions will be recorded as AEs.

Acute (commencing during the infusion) IRRs will be categorised as follows:

Mild (no interruption of infusion): no DLT.

Moderate (infusion interrupted):

- failure to tolerate re-challenge (resumption of infusion at slower rate) in

2 subjects is considered a DLT;

Severe (prolonged or recurrent signs and symptoms after stopping infusion):

- occurrence in 1 subject is considered a DLT;

Life-threatening:

- occurrence in 1 subject requires dosing to be stopped; this is considered a

DLT.

Delayed (commencing after the completion of the infusion) IRRs will be categorised as

follows:

Mild (no intervention beyond paracetamol as needed): no DLT.

Moderate (requires additional medical intervention, e.g., narcotics, oxygen, IV fluids):

- occurrence in 2 subjects is considered a DLT;

Severe (prolonged or recurrent signs and symptoms after stopping infusion):

- occurrence in 1 subject is considered a DLT;

Life-threatening:

- Occurrence in 1 subject requires dosing to be stopped; this is a DLT.

7.1.4.2 Other DLTs

An elevation in any one of the following LFTs:

- ALT >3 x upper limit of the normal reference range (ULN) or >3 x baseline,

whichever is higher;



Page 28 of 74

- Total bilirubin >2.5 x the subject’s baseline level at admission (Day -1) and

is >2 x ULN;

- ALP >1.5 x ULN;

An increase in the QTcB of 60 ms over baseline on two consecutive ECG

measurements or any QTcB >500 ms;

Any other toxicity which in the opinion of the SRC would have precluded

administration of a second dose if this had been a multi-dose study.

7.1.4.3 Stopping Rules

If 2 acute or delayed moderate IRRs or 1 acute or delayed severe or life-threatening IRR

occur within a dose level, the stopping criteria are met and no further dosing can occur.

Additionally, if there are 2 or more DLTs from the other categories (i.e. non-IRR) within

a dose level, or if there is 1 severe or life-threatening non-IRR toxicity or any SAE

considered to be at least possibly related to ALN-PCS02, the stopping criteria are met and

no further dosing can occur. If the stopping rules are met, the SRC must communicate to

the IEC and Medicines and Healthcare products Regulatory Agency (MHRA). Dose

limiting toxicities that result from IRRs and non-IRR etiologies are not to be considered

additive, but are to be considered as separate categories for stopping criteria.

7.1.5 Discussion of Study Design

This randomized, single-blind, placebo-controlled, single ascending dose study is a

scientifically acceptable design for a first-in-human study in healthy subjects with

elevated LDL-C. Inclusion of subjects with elevated LDL-C may provide additional

information on the PD effect of ALN-PCS02. Inclusion of placebo reduces bias in

interpreting data, especially evaluation of the possible relationship of safety observations

to ALN-PCS02. Up to 32 subjects are anticipated to be enrolled at up to 2 clinical sites.

The small cohort size limits exposure at each dose level and the precautions taken

between dosing of subjects mitigates the risk of the design in case there are unexpected

dose related side effects. The dosing strategy enables an appropriate period of

observation between the administration of ALN-PCS02 to subsequent subjects in a cohort

to observe and interpret reactions and AEs. By the inclusion of one subject receiving

placebo and 1 subject receiving ALN-PCS02 comparison of AEs is possible. The use of a

single placebo subject means a double-blind design would be inappropriate since the

treatment received by the third and fourth subjects would be known to the Investigator.

This cautious approach to dosing makes a single-blind design appropriate for this study.

An SRC will evaluate safety and tolerability data and determine if it remains acceptable

to continue dosing (within cohorts) or to dose escalate (between cohorts). The SRC will



Page 29 of 74

be comprised of both sites' study investigators to enable communication of important

safety information between the study sites.

7.2 Study Population

Up to 32 subjects with elevated LDL-C are expected to be enrolled on this study. Male

and female volunteers will be entered into this study provided that they satisfy the

following inclusion/exclusion criteria.

7.2.1 Subject Inclusion Criteria

1. Adult males and females aged 18 to 65 years inclusive at the screening visit with a

fasting LDL-C of >3.0 mmol/L and <5.7 mmol/L based on the mean of at least

two measurements from screening to admission;

2. Females subjects must be of non-childbearing potential e.g., post-menopausal or

pre-menopausal with surgical sterilization;

3. Subjects who have not received any lipid lowering drug/agent within the 30 days

prior to the screening visit;

4. Subjects who are otherwise healthy as determined by pre study medical history,

physical examination and 12-lead ECG;

5. Subjects with body weight >60.0 kg; body mass index (BMI) must be at least

19.00 kg/m2 and <35.00 kg/m

2 at the screening visit;

6. Subjects who, after 10 minutes supine rest, have a systolic blood pressure

<150 mmHg and a diastolic blood pressure of <90 mmHg;

7. Subjects with a fasting triglyceride concentration ≤2.8 mmol/L at screening and on

admission (Day -3);

8. Subjects with ALT, AST, and gamma glutamyl transpeptidase (GGT)

<1.5 x ULN, total bilirubin and ALP within the reference range, albumin

≥3.9 g/dL at the screening visit and on admission;

9. Subjects with complete blood count test results that are not clinically relevant and

are acceptable to the Investigator;

10. Subjects with an international normalised ratio (INR) ≤1.2 measured at screening;

11. Subjects who are non-smokers for at least 3 months preceding screening;

12. Male subjects who agree to use appropriate 2 effective means of contraception

(see Section 7.2.3.2) throughout study participation until 6 months after dose

administration;

13. Medical history must be verified by either a personal physician or medical

practitioner as appropriate;



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14. Subjects who are able and willing to give written informed consent and are willing

to comply with the study requirements.

7.2.2 Subject Exclusion Criteria

1. Subjects who do not conform to the above inclusion criteria;

2. Female subjects who are pregnant, trying to become pregnant or lactating;

3. Subjects with a history of serious mental illness, that includes, but is not limited to

schizophrenia, bipolar disorder, severe depression requiring hospitalization or

pharmacological intervention;

4. Subjects who have a clinically relevant history or presence of respiratory,

gastrointestinal, renal, cardiovascular, hepatic, hematological, lymphatic,

neurological, psychiatric, musculoskeletal, genitourinary, immunological,

dermatological, connective tissue diseases or disorders;

5. Subjects with a history of multiple drug allergies or history of allergic reaction to a

liposomal product or to an oligonucleotide;

6. Subjects who have a clinically relevant surgical history;

7. Subjects who have a clinically relevant family history;

8. Subjects who have a history of drug abuse;

9. Subjects who have a history of alcoholism;

10. Subjects who consume more than 14 (female subjects) or 21 (male subjects) units

of alcohol a week (unit = 1 glass of wine (125 mL) = 1 measure of spirits = ½ pint

of beer);

11. Subjects with a positive screen for alcohol or drugs of abuse at screening or

admission;

12. Subjects with safety laboratory test results deemed clinical significant by the

Investigator;

13. Subjects with known hepatitis B surface antigen (HBsAg), hepatitis B virus

(HBV), hepatitis C virus (HCV) or human immunodeficiency virus (HIV)

infection;

14. Subjects with a clinically significant illness within the 7 days prior to the

administration of ALN-PCS02 or placebo;

15. Subjects with a history of an adverse reaction to steroids (i.e., history of psychosis,

glaucoma, hypertension or diabetes), paracetamol, antihistamines or H2 blockers;



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16. Subjects who have donated more than 500 mL of blood within the 3 months prior

to ALN-PCS02 or placebo administration;

17. Male subjects who do not agree to use medically acceptable methods of

contraception (as defined in Section 7.2.3.2);

18. Subjects who have received an investigational agent within the 3 months prior to

study entry (admission);

19. Subjects who have received the last dose of an investigational agent greater than

3 months ago but who are on extended follow-up;

20. Subjects who have received treatment with lipid-lowering therapy within the

30 days prior to screening;

21. Subjects who have used prescription drugs within 4 weeks of first dosing;

22. Subjects who have used over the counter medication excluding routine vitamins

within 7 days of first dosing, unless agreed as not clinically relevant by the

Principal Investigator and Sponsor;

23. Subjects who have received megadose (intake of 20 to 600 times the

recommended daily dose) vitamin therapy or dietary supplements (e.g., omega 3

fatty acids, plant sterols, niacin, red yeast extracts) within 4 weeks prior to

screening;

24. Subjects who are vegans, vegetarians, or have medical dietary restrictions;

25. Subjects who have initiated or changed their exercise program within 4 weeks of

screening;

26. Subjects who cannot communicate reliably with the Investigator;

27. Subjects who are unlikely to co-operate with the requirements of the study;

28. Subjects with any conditions which, in the opinion of the Investigator, would

make the subject unsuitable for enrolment or could interfere with the subject’s

participation in, or completion of, the study;

29. Subject is an employee or family member of the sponsor or the clinical study site

personnel.



Page 32 of 74

7.2.3 Restrictions

If subjects exercise, they should be on a stable exercise regimen from at least 4 weeks

prior to screening through Day 180 of the study. Subjects should refrain from strenuous

exercise for 24 hours before the screening medical examination, before admission and

during confinement to the clinical unit, and for 24 hours prior to each out-patient visit.

Subjects should refrain from alcohol and/or xanthine containing products (e.g., caffeine)

for 48 hours before screening and admission, during the period of confinement to the

clinical unit (in-patient) and for 48 hours prior to each out-patient visit.

Subjects should refrain from smoking for the duration of the study (through Day 180).

Subjects should refrain from taking medications during the study as described in

Section 8.5.

Subjects should not donate blood for 3 months after last study visit (Day 180).

Subjects will be admitted to the clinical unit on Day -3 of the study and will remain in the

clinic under supervision until discharge on Day 4. Subjects will be required to attend

out-patient visits as described in Table 17.1.

Subject will be required to fast for at least 8 hours, but no greater than 14 hours, prior to

screening, admission and each blood sample collection during out-patient visit.

Subjects will receive a standard diet whilst resident in the clinical unit. The study menu is

formulated to provide subjects with a varied, nutritious diet that abides by the protocol

guidelines for xanthine, cruciferous vegetables, does not contain charbroiled food,

grapefruit, and citrus containing products and the nutritional requirements to promote

health and prevent diet-related diseases set by the British Nutrition Foundation, 2004:

www.nutrition.org.uk.

Recommended daily requirements for adults are:

Kcal 1940-2550

Protein % 15% ± 2%

Carbohydrate % ~50%

Fat % <35%

Saturated fat % <11%

Fiber: 12-24 g/day

Salt: <6 g/day

On Day 0 the approximate daily intake (± 5%) will be as follows:

Kcal 2165.7



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Protein (g) 89.2

Carbohydrate (g) 363.97

Fat (g) 49.7

Saturated fat (g) 13.99

Fiber (g) 19

Sodium (mg) 1652.8

Whilst resident in the unit meals will be provided at the following times:

Breakfast: after collection of the fasted blood samples and on Day 0 this will be prior

to the start of the infusion and after administration of premedication

(dexamethasone, paracetamol, and H1 and H2 blockers) and collection of the post

dexamethasone fasted samples;

Lunch: will be provided an appropriately scheduled time, except on Day 0 when it will

be provided approximately 4 h following completion of the infusion;

Dinner: will be provided at an appropriately scheduled time;

Evening snack: 8-14 h prior to the following morning’s fasted blood sample collection.

The time when meals are consumed and the duration of fasting for each subject will be

documented.

7.2.3.1 Avoidance of Pregnancy

Women of Non Child-Bearing Potential

Female subjects of non-childbearing potential will be considered for this study (see

Section 7.2). Female subjects of non child-bearing potential are defined as: bilateral

oopherectomy; hysterectomy or post-menopausal females being amenorrhoeic for greater

than 2 years with an appropriate clinical profile, e.g., age appropriate, history of

vasomotor symptoms.

Instructions for Male Subjects

There is no information about effects that ALN-PCS02 could have on the development of

the fetus in humans. Therefore, it is important that the partners of male subjects do not

become pregnant during the study and for a total period of 180 days after the male subject

has taken ALN-PCS02.

As a precaution, all male subjects should avoid fathering a child by either true abstinence

or the use of 2 effective means of contraception (see Section 7.2.3.2).



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As there is no information for ALN-PCS02 being secreted in the ejaculate, male subjects

(including men who have had vasectomies) whose partners are currently pregnant should

use barrier methods for the duration of the study (i.e. 180 days after dose administration).

7.2.3.2 Acceptable Forms of Contraception

Highly effective methods of birth control are defined as those which result in a low

failure rate (i.e., less than 1% per year) when used consistently and correctly.

These include female sterilization (i.e., documented bilateral tubal ligation), hormonal

methods of contraception (oral, implanted or transdermal) or an intrauterine device (IUD)

in combination with a barrier method (condom, diaphragm).

Individually, hormonal, barrier or IUD methods alone are not acceptable.

Acceptable forms of effective contraception are:

Established use of oral, transdermal, injected or implanted hormonal methods of

contraception;

Placement of an IUD or intrauterine system (IUS);

Barrier methods of contraception: condom or occlusive cap (diaphragm or

cervical/vault caps) with spermicidal foam/gel/film/cream/suppository. The use of

barrier contraceptives should always be supplemented with the use of a spermicide.

The following should be noted:

- Failure rates indicate that, when used alone, the diaphragm and condom are

not highly effective forms of contraception. Therefore the use of additional

spermicides does confer additional theoretical contraceptive protection.

- However, spermicides alone are inefficient at preventing pregnancy when the

whole ejaculate is spilled. Therefore, spermicides are not a barrier method of

contraception and should not be used alone.

Male sterilization (with the appropriate post-vasectomy documentation of the absence

of sperm in the ejaculate);

True abstinence: when this is in line with the preferred and usual lifestyle of the

subject. Periodic abstinence (e.g., calendar, ovulation, symptothermal, post-

ovulation methods) and withdrawal are not acceptable methods of contraception.

7.2.3.3 Time Period for the Collection of Pregnancy Information

All pregnancies in the female partners of male subjects receiving at least one dose of IMP

will be recorded from first dose to 180 days after infusion.



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7.2.3.4 Follow-up in the Event of a Pregnancy

If the female partner of a male subject, who has received IMP becomes pregnant the

pregnancy will be recorded. The IEC and the Sponsor will be informed. The subject will

be asked to provide information on the outcome of the pregnancy, including premature

termination should the case arise.

Spontaneous miscarriage and congenital abnormalities will be reported as serious adverse

events (SAEs).

The follow-up period will be deemed to have ended when the health status of the child

has been determined upon birth.

7.2.4 Subject Withdrawals

A subject may withdraw for any reason. The Investigator will advise the Sponsor of the

withdrawal of any subject.

A subject may be withdrawn in any of the following circumstances:

AEs (including infusion reactions);

Protocol violation;

Withdrawal of consent;

Termination of the study by the Investigator or Sponsor.

Subjects who voluntarily withdraw are termed dropouts. Dropouts may be replaced

following discussion with the Investigator and Sponsor.

Subjects who are withdrawn due to AEs during infusion of the ALN-PCS02 or placebo

will not be replaced.

If a subject is withdrawn/withdraws the discharge procedures should be performed and an

early termination visit scheduled as follows:

If a subject is withdrawn/withdraws prior to Day 28 then the Day 28 assessments

should be completed;

If a subject is withdrawn/withdraws after Day 28 and prior to Day 180 then the Day

180 assessments should be completed.

If a subject is withdrawn due to an AE, appropriate medical care should be provided and

the AE should be followed to resolution. Follow-up procedures should be conducted as

scheduled.



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8 STUDY TREATMENT

8.1 Investigational Product(s)

ALN-PCS02 Solution for Injection is an RNAi therapeutic consisting of (siRNA targeting

PCSK9 mRNA) formulated in LNPs. The ALN-PCS02 drug product is a sterile

formulation of a PCS siRNA with lipid excipients (DLin-MC3-DMA, DSPC, cholesterol

and PEG2000-C-DMG) in isotonic phosphate buffered saline. ALN-PCS02 Solution for

Injection contains 2 mg/mL of PCS siRNA .

The placebo solution is vehicle control (sterile saline [0.9% NaCl]) for injection.

8.1.1 Supply, Packaging and Labelling

The ALN-PCS02 drug product is packaged in 10 mL glass vials with a fill volume of

5.5 mL. The container closure system consists of a USP/EP Type I borosilicate glass vial,

a teflon-faced butyl rubber stopper and an aluminum flip-off cap.

Each investigational site will be responsible for assembly and labelling according to

separate IMP handling instructions and allocating treatments to the subjects according to

the randomization code.

All packaging and labelling as well as the production of study medication will be in

compliance with GMP specifications, as described in the Manufacture of Investigational

Medicinal Products Volume 4 Annex 13 and any other or local applicable regulations.

A release document signed by a legally authorized Qualified Person (QP) will be placed

in the appropriate section of the Trial Master File to document labelling and dispensing of

IMP to the subject. A technical agreement will be in place for each investigational site to

cover all pharmacy related activities, detailing roles and responsibilities prior to receipt of

the Investigational Product at each investigational site.

8.1.2 Storage and Handling Procedures

All study medication will be stored refrigerated at approximately 5 ± 3oC, protected from

light in the storage area of the investigational site pharmacy, in a secure, temperature

controlled, locked environment with restricted access.

No special procedures for the safe handling of ALN-PCS02 are required. The Sponsor

will be permitted upon request to audit the supplies, storage, dispensing procedures and

records provided that the blind of the study is not compromised.

8.1.3 Accountability

In accordance with GCP, the Investigational Site will account for all supplies of

ALN-PCS02 and placebo. Details of receipt, storage, assembly and return will be

recorded.



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All unused supplies of ALN-PCS02 and placebo will either be destroyed by the

investigational sites or returned to the study Sponsor at the end of the study in accordance

with instruction by the Sponsor.

8.2 Dosage and Administration

8.2.1 ALN-PCS02 or Placebo

Following a standard breakfast (given at approximately 30 minutes prior to the start of

infusion) subjects will receive a single dose of ALN-PCS02 or placebo by IV infusion

over 60 minutes by a controlled infusion device. The infusion time may be extended up

to 3 hours in the event of an acute infusion reaction. The subjects will be in a supine

position during the IV infusion.

Four subjects will be dosed per group (3 subjects on ALN-PCS02 and 1 subject on

placebo) using a sentinel dosing strategy as described in Section 7.1. The planned dosing

schedule is as follows:

Group 1: 15 µg/kg ALN-PCS02 or placebo;





Group 6 (optional cohort): 250 µg/kg ALN-PCS02 or placebo;

Group 7: 400 μg/kg ALN-PCS02 or placebo;

Group 8 (optional cohort): 400ug/kg ALN-PCS02 or placebo or intermediate dose

level.

Subjects ≥105 kg in body weight will receive ALN-PCS02 dosing based on a calculated

lean bodyweight (see www.medcalc.com).

8.2.2 Premedication Regimen

All subjects will be premedicated prior to dosing with ALN-PCS02 or placebo to reduce

the potential for an infusion reaction. Premedication will be administered as follows:

Dexamethasone single oral 8 mg dose administered the evening before dosing and 20

mg 30 to 60 minutes prior to start of infusion of ALN-PCS02 or placebo;

Paracetamol, oral 500 mg the evening before dosing and 30 to 60 minutes prior to the

start of infusion of ALN-PCS02 or placebo;



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Oral H2 blocker (i.e., ranitidine 150 mg or famotidine 20 mg or equivalent other H2

blocker dose) the evening before dosing and 30 to 60 minutes prior to start of

infusion of ALN-PCS02 or placebo;

Oral H1 blocker, 10 mg cetirizine (hydroxyzine 25 mg or fexofenadine may be

substituted if subject does not tolerate cetirizine) the evening before dosing and

30 to 60 minutes prior to start of infusion of ALN-PCS02 or placebo;

8.3 Treatment Strategy

The clinical staff at the investigational sites are all responsible for the ongoing safety and

well-being of the volunteers while they are in the clinical unit. In the case of an

emergency, cardiac resuscitation trolleys are found in each clinical unit. These trolleys

contain drugs, equipment for airway insertion, circulation lines, defibrillation etc, together

with oxygen cylinders and portable suction machines. There will be a physician on site

until at least 24 hours post-infusion. In addition, if necessary the clinical staff can contact

further on-call physicians or public emergencies services, including the ambulance and

local resuscitation teams in the event of a serious medical event. Equipment and

emergency drugs are available to treat common medical emergencies that might occur in

a Phase 1 study.

8.4 Warnings and Precautions

As this is the first administration of ALN-PCS02 in humans, all effects cannot be reliably

predicted. The preclinical data suggest an acceptable safety margin. Facilities and staff

for resuscitation and the treatment of other medical emergencies will be provided. Details

of the handling of IRRs are given in Section 5.3.1.

8.5 Prior and Concomitant Medication

The participants are not allowed to take any prescription medication within 4 weeks of

dosing. Subjects are not allowed to take any non-prescription medication within 7 days

prior to first dose administration until 28 days post-infusion.

Subjects are not allowed to take megadose (intake of 20 to 600 times the recommended

daily dose) vitamin therapy or dietary supplements (e.g. omega 3 fatty acids, plant sterols,

niacin, red yeast extracts) within 4 weeks prior to screening

In the interests of subject safety and acceptable standards of medical care the Investigator

will be permitted to prescribe treatment(s) at his/her discretion. All treatments must be

recorded in the subjects’ case report form (CRF; medication, dose, treatment duration and

indication).

8.6 Method of Assigning Subjects to Treatment Groups

At screening potential study subjects will be assigned a screening number. Following

confirmation of eligibility, at admission, subjects will be assigned a subject number in the



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order in which they are enrolled in the study, as shown below. The subject number will

determine the allocation of treatment.

Table 8.1 Assignment of Subjects to Treatment

Cohort Subject Numbers

1 0001-0004 2 0005-0008 3 0009-0012 4 0013-0016 5 0017-0020 6 (optional cohort) 0021-0024 7 0025-0028 8 (optional cohort) 0029-0032

If applicable, replacement subjects will receive the same treatment allocation as those

whom they replace.

Subjects who are replaced (dropouts) will be allocated the same treatment number with

the number 1, e.g., if treatment number 0001 is replaced then the replacement number

will be 1001.

8.7 Randomisation Procedures

Allocation to treatment will be according to a predetermined random order.

Randomization of ALN-PCS02 or placebo will take place for each group separately.

Prometrika, LLC will generate the randomization list using the statistical analysis system

(SAS®) computer package.

Four subjects will be dosed per cohort (3 subjects on ALN-PCS02 and 1 subject on

placebo) using a sentinel dosing strategy. Two subjects will be dosed first; 1 subject will

be randomised to receive ALN-PCS02 and 1 subject to receive placebo. The remaining 2

subjects of each cohort will receive ALN-PCS02.

8.8 Maintenance of Randomisation Codes

Randomization codes will be provided to the investigational site pharmacist and the

person(s) performing the PK analysis at the start of the study. The pharmacist will use

the randomization code list for preparing subject doses throughout the study.

8.9 Blinding

The study is single-blind. Only the subjects will be blinded to treatment.



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9 STUDY PROCEDURES

The schedule of study procedures is presented in Section 17.

The blood volumes to be collected from each subject are presented in Table 9.1.

Laboratory sample handling details will be presented in a separate Laboratory Manual.

Table 9.1 Approximate Blood Volumes

Assessment

Sample Volume [a]

(mL) No. of

Samples

Total Volume [a]

(mL)

Safety

Serum chemistry 3.5 13 45.5 Coagulation 1.8 3 5.4 Hematology 2.0 9 18.0 Viral serology 3.5 1 3.5 Serum pregnancy test [b] 3.5 2 7.0 Follicle stimulating hormone (FSH) [b] 3.5 1 3.5 Plasma tryptase and C3a [c] 2.0 3 6.0 Cytokines 3.0 4 12.0 Complement (Bb) 2.0 4 8.0 Pharmacokinetic DLin-MC3-DMA, PEG2000C-DMG, free total PCS

siRNA

3.0 21 63.0

Free and encapsulated PCS siRNA[d] 3.0 4 12.0

Pharmacodynamic PCSK9 2.0 18 36.0 Serum LDL-C [e] 12.0 19 228 PCSK9 mRNA 12.0 2 24.0 Other

Exploratory biomarkers (hepatocyte derived proteins): Plasma 1.0 15 15.0

Serum 1.0 15 15.0 Total Males: 491.4 Females: 501.9 [a] Sample volumes are based on direct venipuncture; where a canula is used an extra 1 mL will be drawn and discarded. [b] Female subjects only [c] These samples are only to be collected in the event of an infusion reaction [d] These samples are only collected from cohort 4 onwards (including any optional cohorts) [e] HDL-C and total cholesterol beta-quantification safety tests will also be analysed from this sample.

9.1 Pharmacokinetic Assessments

9.1.1 Plasma Samples

The PK parameter estimates and dose proportionality profiles of ALN-PCS02 will be

determined for ALN-PCS02 siRNA and liposome lipid components, DLin-MC3-DMA

and PEG2000-C-DMG. The plasma concentration-time profiles for PCS siRNA, DLin-

MC3-DMA and PEG2000-C-DMG will be determined and used in the PK analysis for all

dose cohorts. In addition free and encapsulated concentrations of siRNA will be



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determined in plasma at specified time points (see Section 17) from cohort 4 onwards

(including any optional cohorts). The drug concentrations in the lower cohorts are

expected to be undetectable for this analysis.

Blood sample collection times are included in the schedule of events (see Section 17).

Plasma PK samples (siRNA and lipids) will be collected pre-dose (within 1 hour of

planned dosing start), at end of infusion (EOI), at 5, 10, and 30 minutes, and at 1, 2, 4, 6,

8, 24, and 48 hours post infusion. Samples will also be collected on Days 3, 4, 7, 14, 21,

28, 42, 90 and 180 post infusion as well as at the early termination (ET) visit (if

applicable).

A separate plasma PK sample will be collected on Day 0 at EOI and then 2, 24 and

48 hours post-infusion from subjects from cohort 4 onwards, (including any optional

cohorts). These samples will be analysed for free and encapsulated siRNA.

For the PK blood draws, the sampling windows are ± 1 minute for the 5 and 10 minute

draws; ± 2 minutes for the 30 minute draws; ± 5 minutes for the 1, 2, 4, 6, and 8 hour

draws; and ± 30 minutes for the 24 and 48 hour draws.

The EOI will usually occur at 60 minutes after the start of infusion, except in patients

where the infusion has been prolonged. If the infusion is stopped, a PK blood sample will

be taken when the infusion was stopped. All remaining PK samples will be collected

according to the schedule of assessments when the dose is completed (i.e., all post-

infusion times are relative to the EOI, regardless of the duration of infusion).

Details of sample collection and processing for PK analyses will be included in a

laboratory manual.

9.1.2 Urine Samples

Urine will be collected to determine the PCS siRNA fraction excreted in urine and CLR of

siRNA after dosing with ALN-PCS02. Urine sample collection times are included in the

schedule of events (see Section 17). Urine will be collected into suitable containers and

kept refrigerated. Pre-dose samples will be collected within 1 hour of the planned dosing

start. For the 0 to 6 hour collection, the subject should void at time 0 hours and discard

the specimen. Details of sample collection and processing for PK analyses will be

included in a laboratory manual.

9.1.3 Bioanalysis

PCS siRNA concentrations will be determined at specified time points up to 42 days post-

dose. PEG2000-C-DMG and DLin-MC3-DMA will be determined at specified time points

up to 180 days post-dose.

Plasma and urine samples will be evaluated by Quest Pharmaceutical Services (Newark,

Delaware, USA) using a validated ATTO-Probe-HPLC assay to determine PCS siRNA



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concentration. The concentrations of DLin-MC3-DMA and PEG2000-C-DMG in plasma

samples will be evaluated by a validated liquid chromatography/mass spectrometry

(tandem) (LC/MS/MS) assay at Tandem Labs (Salt Lake City, Utah, USA).

Details of how samples will be collected, processed, and stored will be in the Study

Laboratory Manual. The laboratory for PCS siRNA and plasma lipids DLin-MC3-DMA

and PEG2000C-DMG bioanalysis for PK samples is:

For PCS siRNA PK Samples Bioanalysis:

Quest Pharmaceutical Services

Delaware Technology Park

3 Innovation Way, Suite 240

Newark, DE 19711

For DLin-MC3-DMA and PEG2000-C-DMG lipids PK Samples Bioanalysis:

Tandem Laboratories:

1121 East 3900 South, Suite C-110

Salt Lake City, UT 84124, USA

Pharmacodynamic/Efficacy Assessments

9.1.4 PCSK9 Protein Concentration

Blood sample collection times are included in the schedule of events (see Section 17).

Plasma samples will be evaluated by Charles River Laboratories (Montreal, Canada)

using a validated enzyme linked immunosorbent assay (ELISA) to determine PCSK9

protein concentration. Details of how plasma samples will be collected for PCSK9

protein analysis, processed, and stored will be in the Study Laboratory Manual. The

laboratory for PCSK9 protein analysis is:

Charles River Laboratories

22022 Transcanadienne

Senneville, Quebec

Canada H9X 3R3

9.1.5 Beta Quantification of LDL-C, HDL-C and Cholesterol

Serum samples will be evaluated by Medpace Reference Laboratories (Leuven, Belgium)

using preparative ultracentrifugation (beta-quantification) to isolate very low density

lipoprotein (VLDL) from HDL and LDL at density d=1.006 g/L and subsequent assay of

cholesterol content to determine LDL-C. HDL-C will be measured after isolation of HDL

with dextran-sulphate precitipitation of apo B containing lipoproteins. Total cholesterol



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concentration will be measured on whole serum. All lipid measurements will be done in

the CDC Lipid Standardization Program Part III laboratory. Details of how serum

samples will be collected for LDL-C, HDL-C and cholesterol analysis, processed, and

stored by the beta-quantification analysis will be in the Study Laboratory Manual. The

laboratory for LDL-C, HDL-C and cholesterol by beta-quantification analysis is:

Medpace Reference Laboratories BVBA

Technologielaan 19, 3011 Leuven Belgium

9.1.6 PCSK9 mRNA

Blood for serum PCSK9 mRNA is to be collected (12 mL samples) at the times shown in

Section 17. The goal of assessing PCSK9 mRNA is to further enable the exploratory

evaluation of potential RNAi effects of ALN-PCS02. Alnyalm is developing a

quantitative reverse transcriptase-polymerase chain reaction based assay to recover and

analyze PCSK9 mRNA from serum. Details of how samples will be collected, processed

and stored will be in the Study Laboratory Manual. The laboratory for analysis of PCSK9

mRNA is:

Alnylam Pharmaceuticals

300 Third Street

Cambridge, MA 02142

United States of America

Safety Assessments

9.1.7 Adverse Events

Definitions:

9.1.7.1 Adverse Event (AE)

Any untoward medical occurrence in a patient or clinical investigation subject

administered a pharmaceutical product and which does not necessarily have to have a

causal relationship with this treatment.

An AE can therefore be any clinically significant sign (including an abnormal laboratory

finding, for example), symptom, or disease temporally associated with the use of a

medicinal product, whether or not considered related to the medicinal product.

9.1.7.2 Adverse Drug Reaction (ADR)

Any untoward and unintended response in a subject to an IMP which is related to any

dose administered to that subject.

9.1.7.3 Serious Adverse Event (SAE)

An adverse reaction is ‘serious’ if it:

Results in death;



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Is life-threatening;

Requires hospitalisation or prolongation of existing hospitalisation;

Results in persistent or significant disability or incapacity;

Consists of a congenital anomaly or birth defect;

Is a medically important event.

9.1.7.4 Unexpected Adverse Reactions

An adverse reaction is ‘unexpected’ if its nature or severity are not consistent with the

information about the medicinal product in the IB.

9.1.8 Reporting of Adverse Events

All AEs must be fully recorded for 28 days from the end of infusion (dose) in the AE

book and will be transcribed into the subjects’ CRF, whether or not they are considered to

be drug-related. Each AE should be described in detail: onset time and date, offset time

and date, description of event, severity, relationship to investigational product, action

taken and outcome.

AE should be followed until recovery to the normal state has been achieved. In the event

of a subject not returning to the clinical unit, the outcome of this event will be recorded as

lost at follow up.

Reporting of SAEs and SUSARs

SAEs occurring on this study will be reported to Alnylam or its representative within

24 hours from the time that site personnel first learn about the event. The Investigator

will be requested to complete a separate SAE reporting form in addition to the

information on the CRF. The SAE form and any relevant Source Documentation will be

faxed to Medpace Clinical Safety:

Medpace Clinical Safety; Europe

Nussbaumstr. 10

80331 Munich

Germany

The Medicines for Human Use (Clinical Trials) Regulations 2004 (SI 1031) and

subsequent amendments define the following terms:

A suspected unexpected serious adverse reaction (SUSAR) which is fatal or

life-threatening will be reported to the competent authority and IEC immediately

(within 7 days) after the Sponsor became aware of the event. Any additional

information will be reported within eight days of sending the first report.



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A SUSAR which is not fatal or life-threatening will be reported to the competent

authority and IEC as soon as possible (within 15 days) after the Sponsor becomes

aware of the event.

9.1.9 Categorisation of Adverse Events

The intensity of an AE will be categorised as follows:

Mild: Mild events are those which are easily tolerated with no disruption

of normal daily activity.

Moderate: Moderate events are those which cause sufficient discomfort to

interfere with daily activity.

Severe: Severe events are those which incapacitate and prevent usual

activity.

9.1.10 Causal Relationship Assessment

Causal relationship assessment to drug treatments is required for purposes of reporting

AEs. To promote consistency, the following guidelines should be taken into

considerations along with good clinical and scientific judgment when determining the

relationship of drug treatments to an AE:

Definitely Related: A clinical event, including laboratory test abnormality, occurring in

a plausible time relationship to the medication administration, and

which cannot be explained by concurrent disease or other drugs or

chemicals. The response to withdrawal of the drug should be

clinically plausible.

Possibly Related: A clinical event, including laboratory test abnormality, with a

reasonable time sequence to the medication administration, but

which could also be explained by concurrent disease or other drugs

or chemicals. Information on the drug withdrawal may be lacking

or unclear.

Unlikely Related: A clinical event, including laboratory test abnormality, with little or

no temporal relationship to medication administration, and which

other drugs, chemicals or underlying disease provide plausible

explanations.



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Not Related: A clinical event, including laboratory test abnormality that has no

temporal relationship to the medication or has more likely

alternative etiology.

9.1.11 Action Taken for Adverse Event

Action taken in regards to study medication will be defined as:

None;

Infusion interrupted;

Infusion stopped.

9.1.12 Outcome of Adverse Event

Outcome will be defined as:

Resolved (with or without sequelae);

Ongoing;

Lost to follow up.

9.1.13 Coding of Adverse Events

All AEs will be coded using the current version of Medical Dictionary for Regulatory

Activities (MedDRA).

9.2 Clinical Laboratory Safety Tests

Sample collection times are included in the study schedule (see Section 17). Details of

sample handling will be documented in a separate Laboratory Manual. Details of all

methodology and reference ranges will be provided in the Trial Master File.

9.2.1 Local Laboratory Safety Tests

The following tests will be performed locally at each investigational site (either Quintiles

Pathology Laboratory for Quintiles, London, or at Covance Clinical Pathology Services,

Harrogate, as applicable):

Hematology Parameters

Hematology

White blood cell count Neutrophils absolute and % Red blood cell count Lymphocyctes absolute and % Hemaglobin Monocytes absolute and % Hematocrit Eosinophils absolute and % Mean corpuscular volume Basophils absolute and % Mean corpuscular hemaglobin Platelets Mean corpuscular hemaglobin concentration



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Serum Chemistry Parameters

Serum Biochemistry

Sodium ALP Potassium ALT Urea AST Creatinine GGT Albumin Creatine phosphokinase (CPK) Calcium CPK-myocardial band (CPK-MB) Phosphate Lactate dehydrogenase (LDH) Glucose Total bilirubin Total cholesterol [a] Troponin I LDL-C [a] CRP [b] HDL-C [a] Triglycerides [a] Cholesterol, HDL-C and LDL-C will be assessed locally at each investigational site as part of the serum biochemistry panel to allow review of eligibility at screening and admission and allow for review of PD markers . Separate PD samples for beta-quantification of LDL-C, HDL-C and total cholesterol will be taken at the time points shown in Section 17 and analyzed by Medpace Reference Lab, Leuven, Belgium. [b] CRP will only be reported at screening, Day -3, Day 0 pre-dose and 24 h post end of infusion. If a subject's 24 h CRP is above the upper limit of the reference range then CRP will also be reported at 96 h post end of infusion.

Coagulation Parameters

Coagulation

INR Prothrombin time (PT) Activated partial thromboplastin time (aPTT)

Urinalysis Parameters

Urinalysis

Leucocytes Red blood cells Protein pH Bilirubin Nitrite Urobilinogen Specific gravity Ketones Glucose

Urine microscopy will be performed if clinically indicated.

Viral Serology Parameters

Viral Serology

HIV I and II Hepatitis C Virus HBsAg and hepatitis B surface antibodies [HBAb]) [a]

[a] HBAb will be performed by TDL, London, UK on behalf of Quintiles Pathology Laboratory.



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Drugs of Abuse and Alcohol Parameters

Drugs of Abuse and Alcohol (urine)

Amphetamine / ecstasy Benzodiazepines Ethanol Methadone metabolites Cannabinoids Barbiturates Cocaine Urine creatinine Opiates Cotinine

Female subjects will have serum pregnancy tests at screening and admission and serum

follicle-stimulating hormone (FSH) tests at screening to assess eligibility.

Additional and repeat testing may be performed at the discretion of the Investigator.

9.2.2 Central Laboratory Safety Tests

The following laboratory safety tests will be performed at the laboratories indicated:

Cytokine panel: IL-1RA,IL-6, G-CSF, INF-α, INF-γ, TNF-α, IL-1β, IP-10, IL-12 to be

performed by Charles River Laboratories;

Complement (Bb): to be performed by Charles River Laboratories;

Beta-quantification of HDL-C and cholesterol: to be performed by Medpace Reference

Lab (from the LDL-C PD blood sample; see Section 17 for LDL-C sample

collection times);

C3a (only to be performed in the event of an infusion reaction): to be performed by

Charles River Laboratories;

Antibody detection: to be performed by Charles River Laboratories;

Tryptase (only to be performed in the event of an infusion reaction): to be performed

by ViraCor Laboratories.

9.3 Clinical Safety Assessments

Assessment times are included in the study schedule (see Section 17).

9.3.1 Vital Signs

Blood pressure, pulse rate, oral body temperature and respiratory rate will be measured in

the supine position using an automated instrument after the subject has rested comfortably

for 10 minutes. Additional vital signs may be added for safety of the subjects.

Measurements will be reported in the subject’s CRF.



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9.3.2 12 Lead ECG

Computerised 12-lead ECG recordings will be obtained. Each lead shall be recorded for

at least 3 beats at a speed of 25 mm/s. Triplicate recordings will be made at the time

points indicated in Table 17.2.

The following parameters will be recorded: rhythm, ventricular rate, PR interval, QRS

duration, QT and QTcB.

9.3.3 Continuous Lead II ECG Monitoring

Continuous Lead II ECG monitoring will be performed for a minimum of pre-dose to

4 hours after the end of the infusion.

9.3.4 Medical History

A complete medical history will include evaluation (past or present) of the following:

general, head and neck, eyes, ears, nose throat, chest/respiratory, heart/cardiovascular,

gastrointestinal/liver, gynecological/urogenital, musculoskeletal/extremities, skin,

neurological/psychiatric, endocrine/metabolic, heamotologic/lymphatic, allergies/drug

sensitivities, past surgeries, substance abuse or any other diseases or disorders.

9.3.5 Physical Examination

Physical examinations will be performed by a physician and will include the examination

of the following: general appearance, eyes, ears, nose, throat, chest/respiratory,

heart/cardiovascular, gastrointestinal/liver, musculoskeletal/extremities,

dermatological/skin, thyroid/neck, lymph nodes, neurological/psychiatric.

Body weight will be measured at screening and admission (Day -3) for assessment of

eligibility. The weight at admission (Day -3) will be used for dose calculation.

9.3.6 Pulse Oximetry

SaO2 will be assessed by pulse oximetry.

9.3.7 Infusion-Related Reactions

Infusion related reactions will be recorded as AEs (see Section 7.1.4.1).

9.4 Exploratory Biomarkers

To explore the effect of ALN-PCS02 on the expression of hepatocyte derived proteins

serum and plasma samples will be collected at selected time points (see Section 17).

Analytes may include but are not limited to apolipoprotein B (ApoB), apolipoprotein E

(ApoE) and TTR. Serum samples will also be used to detect antibodies, for example anti-

PEG antibodies. Details for sample collection and processing are included in the lab

manual. The laboratory for this analysis is:



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Charles River Laboratories

22022 Transcanadienne

Senneville, Quebec

Canada H9X 3R3

Biological samples for biomarker research can be retained on behalf of Alnylam for a

maximum of 15 years following the last subject's last visit in the study.



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10 DATA COLLECTION

The Investigator and designees agree to maintain accurate CRFs and source

documentation as part of these case histories. Source documents are the originals of any

documents used by the Investigator or hospital/institution that allow verification of the

existence of the subject and substantiate the integrity of the data collected during the trial.

Alnylam will supply either paper or electronic CRFs for each subject. CRFs must be

completed only by persons designated by the Investigator. Corrections must be made so

as not to obliterate original data and must be identified and dated by the person who made

the correction. All data entered into the CRF must also be available in the source

documents. The Investigator will allow designated Alnylam representatives and

regulatory bodies to have direct access to the source documents to verify the data reported

in the CRFs.

Each completed CRF must be reviewed and signed by the Investigator or designee in a

timely manner. The completed CRF will be collected by the Alnylam monitor as soon as

practical after completion. A copy of the CRF will remain in the Investigator’s files.



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11 EVALUATION OF STUDY DATA

11.1 Evaluation of Pharmacokinetic Parameters

PK analyses will consist of a non-compartmental and/or compartmental evaluation of

ALN-PCS02 siRNA, and of ALN-PCS02 lipids DLin-MC3-DMA and PEG2000-C-DMG

plasma concentration-time profiles to determine PK parameter estimates using a validated

software program, WinNonlin®. PK parameter estimates to be calculated for all subjects

receiving ALN-PCS02 will include, but may not be limited to: Cmax; tmax; t½β; t1/2,

AUCp, AUC0-t, AUC0-last and AUC0-∞; z; CL, Vss and Vz. The dose proportionality of

ALN-PCS02 following a single, 60-minute infusion will also be determined. PK

parameter estimates determination using the concentration-time data will be performed by

Alnylam (Cambridge, MA, USA). The PK profile of each patient will be characterized

by non-compartmental analysis of each siRNA plasma concentration using validated

computer software (WinNonlin, Pharsight Corp., Mountain View, California, USA).

Parameters will include, but may not be limited to: Cmax, Tmax, AUC, CL, Vss and Vz. The

area under the plasma concentration-time curve (AUC) will be calculated using the linear

trapezoidal method (linear interpolation). Other parameters may be calculated, if deemed

necessary. If possible, the terminal elimination phase of the PK profile will be identified

and its slope calculated using log-linear regression. The coefficient of determination of

the line fitted to the terminal elimination phase will be calculated. PK parameters

describing the systemic exposure of the test article in the test system will be estimated

from observed plasma concentration values for each patient.

Definition of PK parameters that will be calculated using non-compartmental analysis

and/or compartmental and will include as allowed by data analysis, but may not be

limited to:

Observed maximum concentration (Cmax);

Time of observed maximum concentration (Tmax);

Partial area under the plasma concentration-time curve (AUCp);

Area under the plasma concentration-time curve to the last measurable concentration

(AUC0-t);

Area under the plasma concentration-time curve from zero to the last measurable time

point (AUC0-last);

Area under the plasma concentration-time curve extrapolated to infinity (AUC0-∞);

Terminal elimination half-life (t1/2β and t1/2α);

Elimination rate constant (λz);



Page 53 of 74

Systemic clearance (CL);

Volume of distribution at steady state (Vss);

Volume of distribution based on the terminal phase (Vz).

Other parameters may be calculated if deemed necessary.

In addition an assessment of plasma concentrations of free and encapsulated siRNA will

be determined at specified time points (see Section 17) from cohort 4 onwards

(including any optional cohorts).

The amount of siRNA excreted in the urine over the two separate and combined

collection intervals will be determined and CLR calculated for each patient, if possible.

For the following ALN-PCS02 components (total PCS siRNA ) the following urine PK

parameters will include, but may not be limited to:

Amount of free siRNA excreted in the urine over the 2 separate and combined

collection intervals.

CLR of siRNA.

Other PK parameters may be calculated if deemed necessary.

11.2 Evaluation of Pharmacodynamic Measures

The PD evaluation will include assessment of plasma concentrations of PCSK9 and

serum concentrations of LDL-C.

11.3 Evaluation of Safety

The safety evaluation will include assessment of AEs, 12-lead ECGs, arterial SaO2, vital

signs (blood pressure, pulse rate, oral body temperature and respiratory rate), clinical

laboratory safety tests (hematology, serum chemistry, coagulation, urinalysis, cytokines,

complement (Bb), HDL-C and total cholesterol) and physical examination findings.



Page 54 of 74

12 STATISTICAL METHODS

12.1 Primary and Secondary Target Variables

Primary Target Variables

Safety and tolerability of single infusions of ALN-PCS02.

Secondary Target Variables

PK profile of ALN-PCS02.

PD effect of ALN-PCS02 on plasma PCSK9 and serum LDL-C.

12.2 Statistical Analysis Plan

A statistical analysis plan (SAP) will be written and approved prior to locking the

database.

12.3 Sample Size Determination

This is a Phase 1 study to investigate the safety and tolerability of a novel compound,

ALN-PCS02. The sample size was chosen based on previous experience in Phase 1

studies and was not based on power calculations.

12.4 Subject Population for Analyses

Safety Population

All subjects who receive study medication (ALN-PCS02 or placebo) will be included in

the Safety Population.

Pharmacokinetic Population

All subjects who receive ALN-PCS02 and have at least 1 blood draw to determine plasma

concentrations of PCS siRNA, DLin-MC3-DMA and PEG2000-C-DMG will be included

in the PK data analysis.

Pharmacodynamic Population

All subjects who receive ALN-PCS02 and have at least 1 blood draw to determine plasma

PCSK9 and serum LDL-C will be included in the PD analysis.

12.5 General Methods

All study data will be presented in by-subject data listings. Statistical analyses will be

descriptive only, with no hypothesis testing. Analyses will be performed using SAS® for

Windows. Summary tables will present results for each ALN-PCS02 dose and placebo,

where the placebo subjects will be combined across dose cohorts. Descriptive statistics

will be presented for continuous variables, and frequencies and percentages will be

presented for categorical and ordinal variables.



Page 55 of 74

12.6 Safety Data Analysis

All safety analyses will be performed on the Safety Population.

Adverse events will be coded by System Organ Class and Preferred Term using the

MedDRA dictionary. Incidences of treatment-emergent adverse events (TEAEs, those

events that started after exposure to study drug or worsened in severity after dosing) will

be presented by dose group. Incidences of TEAEs will also be presented by maximum

severity and relationship to study medication.

For each continuous laboratory parameter, results will be categorized as low, normal, or

high based on the laboratory normal ranges. Frequencies and percentages will be

presented by dose group for subjects who had a shift to low and for subjects who had a

shift to high from baseline to any post-dosing assessment. All out-of-range and clinically

significant laboratory results will be identified in subject data listings.

The number and percentage of subjects in each dose group with normal and abnormal

physical examination results will be presented for evaluations at baseline and final visit.

For each body system, changes in the subjects’ findings from baseline to final visit (no

change, normal to abnormal, or abnormal to normal) will be tabulated for each dose

group.

Mean scores for serial vital signs, 12-lead ECG, and pulse oximetry measurements (SaO2)

over time will be plotted by dose group. Since ECG measurements will be obtained in

triplicate for each subject, a single summary measure of the three measurements (mean or

median) will be used for analysis.

Individual and summary blood pressures, pulse, oral body temperature and respiration

rate, ECG parameters, SaO2 and clinical laboratory data (haematology, serum

biochemistry, coagulation, HDL-C, total cholesterol, complement and cytokines) will be

presented in tabular form with mean, median, standard deviation and range (minimum

and maximum) as appropriate.

For the laboratory safety data, out of range values will be flagged in the data listings and a

list of clinically significantly abnormal values will be presented.

AE will be tabulated and summarised according to the current version of MedDRA.

12.7 Pharmacokinetic Analysis

Statistical analysis for PK parameter estimates to be calculated for all subjects receiving

ALN-PCS02 will include, but may not be limited to: Cmax; tmax; t½β; t1/2, AUCp, AUC0-t,

AUC0-last and AUC0-∞; z; CL, Vss and Vz. The dose proportionality of ALN-PCS02

following a single, 60-minute infusion will also be determined.



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Tabulations will be presented by dose level. For categorical variables, summary

tabulations of the number and percentage of patients within each category (with a

category for missing data) will be presented. For continuous variables, the number of

patients, mean, median, standard deviation, minimum, and maximum values will be

presented.

All data will be provided in by-subject listings.

12.8 Pharmacokinetic and Pharmacodynamic Analysis

PK and PD data will be assessed descriptively whenever possible and by exploratory

statistical comparisons. PK/PD analyses will include evaluation with respect to PCS

siRNA of ALN-PCS02 plasma concentrations, exposure, or the relationship of any PK

parameter estimate to PCSK9 or LDL-C, concentration after ALN-PCS02 following a

single, 60-minute infusion. Attempts will be made to perform PK/PD analysis to

determine PK/PD parameters (e.g., 50% effective dose [ED50], 50% effective

concentration [EC50]).



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13 STUDY REPORT

Following completion of the study, a clinical study report compliant with the

requirements of ICH E3 will be prepared.



Page 58 of 74

14 REGULATORY AND ETHICAL ISSUES

14.1 Regulatory and Ethics Review and Approval

This study will be performed in compliance with the protocol, International Conference

on Harmonization (ICH) Good Clinical Practice (GCP) and applicable regulatory

requirements. Aspects of the study concerned with the investigational medicinal

product(s) (IMPs) will meet the requirements of European Union (EU) Good

Manufacturing Practice (GMP).

Regulatory authorities will receive the protocol, amendments, reports on SAEs, interim

safety reports, and the clinical study report according to national regulations.

The study will be submitted for to the Medicines and Healthcare products Regulatory

Agency (MHRA-UK) for review and approval and IEC for ethical review and approval.

The documents submitted will include but are not limited to;

MHRA:

The final protocol;

The IB;

The Investigational Medicinal Product Dossier (IMPD); and

Annex 1: Clinical Trial Application Form (Request for authorisation of a clinical trial

on a medicinal product for human use to the competent authorities and for opinion

of the IEC in the community).

Ethics:

The final protocol;

The IB;

The ethics application form; and

The Informed Consent Form (ICF)

The study will not commence unless the following conditions are satisfied:

An IEC has given a favourable opinion in relation to the clinical trial; and

The clinical trial has been authorised by the licensing authority (MHRA).

14.2 Informed Consent

Informed consent will be given freely after the subject has been informed of the nature,

significance, implications and risks of the trial; and consent is evidenced in writing, dated

and signed, or otherwise marked, by that person so as to indicate his/her consent, prior to

the start of the study. The nature of the informed consent will comply with the principles



Page 59 of 74

that have their origin in the Declaration of Helsinki, the current requirements of GCP

(Committee for Proprietary Medicinal Products (CPMP)/ICH/135/95) and local

regulation (The requirements are set out in Schedule 1 of SI 1031 and amendments)

which ever provides the greater subject protection.

14.3 Indemnity and Compensation

In accordance with SI 1031 and amendments section 15 (5i, j) and the EU Clinical Trials

Directive 2000/20/EC Article 3 (2f), provision is to be made for:

the indemnity or compensation in the event of injury or death attributable to the

clinical trial; and

insurance or indemnity to cover the liability of the Investigator or Sponsor.

Therefore the Sponsor, Alnylam Pharmaceuticals, Inc., will indemnify the Investigators

from all and any third-party claims as provided for in the parties agreement governing the

conduct of this study

In the event that it can be demonstrated that a subject suffers any significant deterioration

in health or well-being or any harmful susceptibility or toxicity as a direct result of their

participation in this study then Alnylam Pharmaceuticals, Inc., will agree to abide by the

current Association of the British Pharmaceutical Industry (ABPI) Guidelines with regard

to compensation payable to the subject. The amount of compensation will be calculated

by reference to the level of damages commonly awarded in UK law for similar injuries at

the time when such injury occurred.

The Investigators declare that they have adequate insurance cover for the malpractice

and/or negligence of their employees and agents, as more specifically provided for in the

parties agreement governing this study.



Page 60 of 74

15 STUDY MANAGEMENT

15.1 Communication Plan

Study related communication will take place via electronic mail (email), conference calls,

and/or face to face meetings. Investigators and team members from both clinical sites,

sponsor representatives and CRO representatives will participate in regular project team

teleconferences to review important study information and ensure coordination of

recruitment activities, if needed. Safety and tolerability information will be reviewed and

discussed at SRC meetings that occur within a cohort and after each cohort. The SRC

meetings will be attended by the medical monitors and the investigators or their delegate

from each site. To ensure timely dissemination and review of important study

information by the SRC on an ad hoc basis email will be used and/or ad hoc phone calls

will take place. Every communication will be documented.

15.2 Quality Assurance and Quality Control

In accordance with the guideline for ICH GCP, the Sponsor has responsibility for

implementing and maintaining quality assurance and QC systems, and the ultimate

responsibility for the quality integrity of the trial data resides with the Sponsor.

15.3 Protocol Adherence

The protocol must be read thoroughly and the instructions followed exactly. Major

changes in this research activity, except those to remove an apparent immediate hazard to

the subject, must be reviewed and approved by Alnylam and the IEC which approved the

study. Amendments to the protocol must be submitted in writing to the IEC and the

Competent Authority for approval prior to subjects being enrolled into the amended

protocol.

Any deviations should be agreed by both the Sponsor and the Investigator, with the

appropriate written and approved protocol amendments made to reflect the changes

agreed upon. Where the deviation occurs for the well-being of the subject, the Sponsor

must be informed of the action agreed upon.

15.4 Documents Necessary for Initiation of Study

The following documents will be available prior to the first administration of the drug to

the first subject:

Regulatory authorisation;

Copy of current IB;

Risk assessment report;

Completed and signed investigator agreement/contract;



Page 61 of 74

Signed original of the final protocol;

IEC approval;

Copy of the constitution of the Research Ethics Committee;

A list of members of the IEC;

A copy of the consent form and subject information to be used;

The curriculum vitae of the Chief Investigator and Principal Investigator;

The QP certification for the release of each batch of IMP;

A technical agreement between the Sponsor and the Investigators defining the

responsibilities of the Sponsor, Investigators and any third parties significantly

involved in the supply chain of the IMP, where applicable;

The product specification file, where applicable; and

A list of laboratory reference range values for parameters measured in the study.

15.5 Study Monitoring

In accordance with ICH GCP, the Sponsor will be responsible for monitoring the conduct

of the study. The Sponsor, or delegate, will conduct pre-study and start-up meeting site

visits and in addition will visit the site during the conduct of the study, review the study

data and conduct a post-study visit. Study data recorded on source documents will be

made available to the study monitor for the purpose of source document verification.

The Investigator will allow trial-related monitoring audits, IEC review, and regulatory

inspection allowing direct access to the source data/documents.

15.6 Study Closure

Premature termination of the study by either party will be governed under the terms of the

clinical business agreement between both parties.

15.7 Study Record Retention

In accordance with SI 1928, the Sponsor and the Investigator at each investigational site

shall ensure that the documents contained, or which have been contained, in the trial

master file are retained for at least 5 years after the conclusion of the trial and that during

that period are:

Readily available to the licensing authority on request; and

Complete and legible.

All data derived from the study will remain the property of Alnylam Pharmaceuticals,

Inc.



Page 62 of 74

All correspondence (e.g. with the Sponsor, IEC) relating to this study should be kept in

the appropriate file folders.

Records of subjects source documents, CRF’s, IMP inventory, pertaining to the study

must be kept on file. Records must be retained according to the current ICH Guidelines

on GCP.

The Sponsor shall appoint named individuals within his organisation to be responsible for

archiving the documents which are, or have been, contained in the trial master file.

Access to those documents shall be restricted to those appointed individuals. If there is

transfer of ownership of data or documents connected with the clinical trial:

The Sponsor shall record the transfer; and

The new owner shall be responsible for data retention and archiving in accordance

SI 1031 and amendments.

If the Investigator moves, withdraws from an investigation or retires, the responsibility

for maintaining the records may be transferred to another person who will accept

responsibility. Notice of transfer must be made to and agreed by the Sponsor.

15.8 Publication Policy

After completion of the study, the Investigator may prepare a joint publication with the

Sponsor.



Page 63 of 74

16 REFERENCES

1. [Abifadel et al, 2003] Abifadel M et al, 2003, Mutations in PCSK9 cause

autosomal dominant hypercholesterolemia. Nat Genet, 34(2):154-156.

2. [Akinc et al, 2010] Akinc A et al, 2010, Targeted delivery of RNAi therapeutics

with endogenous and exogenous ligand-based mechanisms. Mol Ther, 18(7):1357-

1364.

3. [Cervantes et al, 2011 (in press)] Cervantes A et al, 2011 (in press), Phase I dose-

escalation study of ALN-VSP02, a novel RNAi therapeutic for solid tumors with

liver involvement. ASCO Proceedings.

4. .[Chan et al, 2009] Chan JCY et al, 2009, A proprotein convertase subtilisin/kexin

type 9 neutralizing antibody reduces serum cholesterol in mice and nonhuman

primates. Proc Natl Acad Sci U S A. Jun 16;106(24):9820-9825.

5. [Cohen et al, 2006] Cohen JC et al, 2006, Sequence variations in PCSK9, low

LDL, and protection against coronary heart disease. N Engl J Med, 354(12):1264-

1272.

6. [Davidson et al, 2005] Davidson MH et al, 2005, Results of the National

Cholesterol Education (NCEP) Program Evaluation ProjecT Utilizing Novel E-

Technology (NEPTUNE) II survey and implications for treatment under the recent

NCEP Writing Group recommendations. Am J Cardiol. 96(4):556-63.

7. [Davignon et al, 2009] Davignon J and Dubuc G, 2009, Statins and ezetimibe

modulate plasma proprotein convertase subtilisin kexin-9 (PCSK9) levels. Trans

Am Clin Climatol Assoc, 120:163-173.

8. [Elbashir et al, 2001] Elbashir SM et al, 2001, RNA interference is mediated by

21- and 22-nucleotide RNAs. Genes Dev 15, 188-200.

9. [Manufacture of Investigational Medicinal Products Volume 4 Annex 13]

EudraLex: The Rules Governing Medicinal Products in the European Union,

Volume 4, EU Guidelines to Good Manufacturing Practice, Medicinal Products

for Human and Veterinary Use, Annex 13, Investigational Medicinal Products.

10. [Foley et al, 2003] Foley KA et al, 2003, Effectiveness of statin titration on low-

density lipoprotein cholesterol goal attainment in patients at high risk of

atherogenic events. Am J Cardiol, 92(1):79-81.

11. [Geisbert et al, 2006] Geisbert TW et al, 2006, Postexposure protection of guinea

pigs against a lethal ebola virus challenge is conferred by RNA interference. J

Infect Dis 193, 1650-1657.



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12. [Gomes et al, 2005] Gomes ER and Demoly P, 2005, Epidemiology of

hypersensitivity drug reactions. Current Opinion in Allergy & Clin. Immunol,

5(4):309-316.

13. [Grundy et al, 2004] Grundy SM et al; National Heart, Lung, and Blood Institute;

American College of Cardiology Foundation; American Heart Association.; 2004,

Implications of recent clinical trials for the National Cholesterol Education

Program Adult Treatment Panel III guidelines, Circulation. 110(2):227-39.

14. [Hooper et al, 2007] Hooper AJ et al, 2007, The C679X mutation in PCSK9 is

present and lowers blood cholesterol in a Southern African population.

Atherosclerosis, 193(2):445-448.

15. [IB] Investigator's Brochure for ALN-PCS02, Edition 1, 2011.

16. [Judge et al, 2006] Judge AD et al, 2006, Design of noninflammatory synthetic

siRNA mediating potent gene silencing in vivo. Mol Ther 13, 494-505.

17. [Maurer et al, 1999] Maurer N Et al, 1999, Lipid-based systems for the

intracellular delivery of genetic drugs. Mol Membr Biol 16, 129-140.

18. [www.medcalc.com], .medcalc.com/body.html.

19. [Morrissey et al, 2005] Morrissey DV et al 2005, Potent and persistent in vivo

anti-HBV activity of chemically modified siRNAs. Nat Biotechnol 23, 1002-1007.

20. [Mousavi et al, 2009] Mousavi SA et al, 2009, The unique role of proprotein

convertase subtilisin/kexin 9 in cholesterol homeostasis. J Intern Med.

266(6):507-19.

21. [Nag et al, 2007] Nag SS et al, 2007, LDL-C goal attainment among patients

newly diagnosed with coronary heart disease or diabetes in a commercial HMO. J

Manag Care Pharm, 13(8):652-663.

22. [Ni et al, 2011] Ni YG et al, 2011, A PCSK9-binding antibody that structurally

mimics the EGF(A) domain of LDL-receptor reduces LDL cholesterol in vivo. J

Lipid Res, 52(1):78-86.

23. [Rashid et al, 2005] Rashid S et al, 2005, Decreased plasma cholesterol and

hypersensitivity to statins in mice lacking Pcsk9. Proc Natl Acad Sci USA,

102(15):5374-5379.

24. [Regeneron presentation, 2011] http://investor.regeneron.com/presentations.cfm

April 26, 2011.

25. [Solensky, 2006] Solensky R, 2006, Drug hypersensitivity. Med Clin North Am

90: 233-260.

http://investor.regeneron.com/presentations.cfm



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26. [Soutschek et al, 2004] Soutschek J et al, 2004, Therapeutic silencing of an

endogenous gene by systemic administration of modified siRNAs. Nature 432,

173-178.

27. [Truelsen et al, 2003] Truelsen T et al, 2003, Trends in Stroke and Coronary Heart

Disease in the WHO MONICA Project. Stroke, 34(6):1346-1352.

28. [Vaishnaw et al, 2010] Vaishnaw AK et al, 2010, A status report on RNAi

therapeutics. Silence, 1(1):14.

29. [WHO Cardiovascular Statistics, 2011], World Health Organization

Cardiovascular Statistics, 2011,

http://www.who.int/mediacentre/factsheets/fs317/en/index.html.

30. [Yusef et al, 2004], Yusef et al, 2004, Effect of potentially modifiable risk factors

associated with myocardial infarction in 52 countries (the INTERHEART study):

case-control study. Lancet 364: 937-952.

31. [Zhao et al, 2006] Zhao Z et al, 2006, Molecular characterization of loss-of-

function mutations in PCSK9 and identification of a compound heterozygote. Am

J Hum Genet. 79(3):514-523.

32. [Zimmermann et al, 2006] Zimmermann TS et al, 2006, RNAi-mediated gene

silencing in non-human primates. Nature.;441(7089):111-4. Epub 2006 Mar 26.

http://www.who.int/mediacentre/factsheets/fs317/en/index.html



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17 STUDY SCHEDULE

The following 'priority order' will be followed when more than one assessment is required

at a particular time point:

1. ECG

2. Vital signs

3. PK blood sample (at the protocol scheduled time)

4. PD blood sample (as close to the protocol scheduled time as possible)

5. Laboratory safety blood samples

Pre-dose assessments may be performed up to 60 minutes prior to the intended time of

each dose unless specified otherwise.

For the PK blood draws, sampling windows allowed follow:

±1 minute for the 5 and 10 minute draws;

±2 minutes for the 30 minute draws;

±5 minutes for the 1, 2, 4, 6, and 8 hour draws; and

±30 minutes for the 24 and 48 hour draws.

The same time windows should be applied for all other timed assessments with the

following exceptions: PK urine collections, vital signs, 12-lead ECGs and pulse oximetry

should be taken ±15 minutes of the protocol scheduled time.

If an assessment or collection is scheduled by day, and not by a specific time, then no

time window will apply although generally the time of day (AM or PM) should be the

same throughout the study.

Meals will be provided at the times indicated in Section 7.2.3.

Confidential Clinical Study Protocol Amendment 2 Version: Final Date 20 August 2013


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Table 17.1 Study Schedule: Screening through to Day 180

Residential Period Out Patient/ Follow Up

Scr Adm

[t]

Pre-dose

Procedures -45 to -7 -3 -2 -1 0 0 1 2 3 4

[a] 7±1 10±1 14±2 17±2 21±2

28[b]

+3

42[b

±3 90

[b]

±7 180

[b]

±14 Day

Informed consent X

Medical history[s]

X X

X

Physical examination X X X

X X X X

X

X

X

Body weight X X

Vital signs [c]

X X X[d]

X[d]

X[d]

X X X

X

X

X

12-lead ECG X

X[e]

X[e]

X[e]

X

Lead II ECG monitoring

[f]

X X

Pulse oximetry

X[g] X[g] X[g]

Fasting PCSK9 [h]

X X X[i] X

[i]

X X X X X X X X X X X X X

PCSK9 mRNA

X

X

Fasting LDL-C, HDL-C and cholesterol by beta-quantification

[h]

X X X X[i] X

[i]


Fasting serum chemistry

X[j] X

[j] X X

[i] X

[j]

X

[j] X

X

[j] X X

X

X

X

Haematology X X X

X X

X X

X

X

Urinalysis X X X

X X

X X

X

X Coagulation: (PT/aPTT/INR)

X

X

X

Cytokine panel

X[k]

X[k]

X[k]

Complement ( Bb)

X[l] X

[l] X

[l]

If infusion reaction: tryptase and C3a

X[m]

X[m]

Viral serology tests X

Urine drugs of abuse and alcohol test

X X

Pregnancy test (females)

X X



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FSH (females) X

Inclusion/exclusion criteria

X X X X X

Pre-med administration

X[n] X[n]

ALN-PCS02 or placebo administration

X

Plasma PK [o]


Plasma PK (free/encapsulated siRNA)

[p]

X X X

Urine PK [q]

X X X

Exploratory biomarkers

X X X X X X X X X X X X X X X

Concomitant medications X---------------------------------------------------------------------------------------------------------------------X

Review/record AEs[r]

X-------------------------------------------------------------------------------------------------------X

Scr = Screening Adm = Admission. Notes: [a] Discharge (following completion of Day 4 assessments). [b] Or early termination as follows: if a subject is withdrawn/withdraws prior to Day 28 then Day 28 assessments should be performed. If a subject is withdrawn/withdraws after Day 28 and prior to Day 180 then Day 180 assessments should be performed. [c] Vital Signs are blood pressure, pulse rate, oral body temperature and respiratory rate. [d] Serial vital signs: to be measured at the times shown in Table 17.2. [e] Serial ECGs: collected in triplicate at the times shown in Table 17.2. [f] Lead II ECG monitoring will be performed as shown in Table 17.2. [g] Serial pulse oximetry: to be measured at the times shown in Table 17.2. [h] Fasting no less than 8 hours and no greater than 14 hours prior to draws; draw at approximately the same time each day and prior to 9 AM in the morning. [i] On Day -1 collect blood sample prior to evening premedication administration. On Day 0 collect blood samples pre and post morning dexamethasone administration. [j] CRP will only be reported at screening, Day -3, Day 0 pre-dose and 24 h post end of infusion. If a subject's 24 h CRP is above the upper limit of the reference range then CRP will also be reported at 96 h post end of infusion. [k] Cytokines: to be measured at the times shown in Table 17.2. [l] Complement: to be measured at the times shown in Table 17.2. [m] Tryptase and C3a are to be measured only in the event of an infusion reaction (acute or delayed): at time of event or as soon as possible after onset, 1 hour and 24 hours after the event. [n] Premedication will be administered the evening before dosing (Day -1) and 30-60 minutes prior to dosing (Day 0) as described in Section 8.2.2.



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[o] Plasma PK samples (siRNA and lipids) will be collected on Days 0 to 4 at the times shown in Table 17.2. Samples will be additionally collected on Days 7, 14, 21, 28, 42, 90 and 180 post infusion as well as at the early termination visit (if applicable). PCS siRNA concentrations will be determined at specified time points up to 42days post-dose. PEG2000-C-DMG and DLin-MC3-DMA will be determined at specified time points up to 180 days post-dose. [p] Plasma PK on Day 0 at end of infusion, 2, 24 and 48 hours post-infusion will be collected for subjects from cohort 4, onwards (including any optional cohorts) for analysis of free and encapsulated siRNA. [q] Urine PK sample collection periods to be at the times shown in Table 17.2. [r] Any infusion reactions will be recorded as adverse events as described in Section 7.1.4.1. [s] Changes in medical history during the follow-up period will be captured as adverse event(s) or concomitant medication(s) as appropriate. [t] Subjects who are admitted on Day -3 as a reserve who are not dosed or discharged from the clinic only require Day -1 assessments to be repeated. Assessments already performed for Day -2 and Day -3 do not need to be repeated.



Page 70 of 74

Table 17.2 Study Schedule: Day 0 to Day 4

Day 0

1 2 3 4

Procedures Pre-dose

(mins) SOI EOI

5 mins post EOI

10 mins post EOI

30 mins post EOI

1 h post EOI

2 h post EOI

4 h

post EOI

6 h post EOI

8 h post EOI

12 h post EOI

24 h post EOI

48 h post EOI

72 h post EOI

96 h post EOI

Physical exam X

X[a]

X[a]

X[a]

X[a]

Fasting PCSK9, X[b]

X X X X

PCSK9 mRNA X[l]

X

Fasting LDL-C, HDL-C and cholesterol by beta-quantification

X[b]

X X X X

Fasting serum chemistry X[b], [c]

X[c]

X X[c]

Haematology X

X X X

Urinalysis X

X X X

Coagulation X

X

Cytokine panel X (-10)

X X X

Complement ( Bb) X (-10)

X X X

If infusion reaction: tryptase and C3a

X-----------------------------------------------------------------------------------------------------------------------X[d]

Dexamethasone X (-60 to -30)

H1 and H2 blockers X (-60 to -30)

Paracetamol X (-60 to -30)

Vital signs X (-60) X X X X X X X X X X X

12-lead ECGs[e]

X (-60) X X X X X X

Lead II ECG monitoring X--------------------------------------------------------------------------------------X

Meals X (-30)

X X X[f]

X[g]

X[g]

X[g]

X[g]

ALN-PCS02 infusion

Start Stop

Pulse oximetry X (-60) X X X X X X X X

Plasma PK X ( ≤-60) X[h]

X X X X X X X X X X X X

Plasma PK (free/encapsulated siRNA) [i]

X X X X

Urine PK X[j] X-----------------------------------------------------------------------------------------------------------------------X

[k]

Exploratory biomarkers X (-60)

X X X X

Adverse events X-------------------------------------------------------------------------------------------------------------------------------------------------------------------X

SOI = Start of Infusion; EOI = End of Infusion.



Page 71 of 74

Notes: [a] Physical examination update: only changes to the physical examination will be recorded in the study database. [b] Samples to be collected pre and post morning dexamethasone administration. Post dexamethasone samples are not required to be fasted and should be collected after breakfast, as close as possible to the start of study drug infusion, taking into account other pre-dose assessments. [c] CRP will only be reported at screening, Day -3, Day 0 pre-dose and 24 h post end of infusion. If a subject's 24 h CRP is above the upper limit of the reference range then CRP will also be reported at 96 h post end of infusion. [d] If an infusion reaction (acute or delayed) occurs then blood samples for analysis of tryptase and C3a should be taken at the time of the event or as soon as possible after onset and 1 hour and 24 hours after the time of the event. [e] 12-lead ECGs are collected in triplicate. [f] Evening snack [g] On non-dosing days breakfast will be served after fasted blood samples have been collected; other meals will be served at standard times as described in Section 7.2.3. [h] If the infusion is stopped, a PK blood sample will be taken when the infusion was stopped. [i] Samples will be collected from subjects from cohort 4 onwards (including any optional cohorts) for analysis of both free and encapsulated siRNA. [j] The pre-dose sample should be taken within 1 hour of the planned dosing start. [k] Collection times: 0-6 hours, 6-12 hours, 12-24 hours and 24-48 hours post EOI. [l] Pre-dose sample to be collected after premedication administration.

Confidential Clinical Study Protocol Amendment 2 Version: Final Date 12 20 August 2013


Page 72 of 74

18 PROTOCOL SIGNATURES

INVESTIGATOR PROTOCOL APPROVAL:

I agree with the content of this protocol and the confidential nature of the documentation

made as part of this study. I also acknowledge that the Sponsor of the study has the right

to discontinue the study at any time. I have read the protocol and understand it and will

work according to it and according to the principles of Good Clinical Practices, applicable

laws and regulations, and the principles that have their origin in the Declaration of

Helsinki.

Chief/Principal Investigator (signature) Date

________________________________

Chief/Principal Investigator (print name)



Page 73 of 74

SPONSOR PROTOCOL APPROVAL:

I agree with the content of this protocol and the confidential nature of the documentation

made as part of this study. I also acknowledge that the Investigator has the right to

discontinue the study at any time. I have read the protocol and understand it and will

ensure that the clinical trial is conducted according to it and according to the principles of

Good Clinical Practices, applicable laws and regulations, and the principles that have

their origin in the Declaration of Helsinki.

Senior Vice President, Clinical Research Date

Akshay Vaishnaw, MD, PhD

Alnylam Pharmaceuticals, Inc.



Page 74 of 74

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