Antiplatelet Effects of Evolocumab in Patients With Peripheral Arterial Disease
Conditions: Peripheral Arterial Disease
Sponsor: Inova Health Care Services

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Antiplatelet Effects of Evolocumab in Patients with Peripheral Arterial Disease 
 
 
   
 
Principal Investigator: Paul A. Gurbel, M.D. 
 
Co-Investigators: Richard F Neville, M.D. 
 
                            
 
 
Drug Name: Evolocumab 
 
 
 
Sponsor: Inova Heart and Vascular Institute at Inova Fairfax Hospital,  
Falls Church, VA 
 
 
 
 
Site of Investigation: Inova Heart and Vascular Institute at Inova Fairfax Hospital,  
Falls Church, VA 
 
 
 
Protocol Version Date: 19-December-2018 
 
 
 
 
Contact Information:  
Paul A. Gurbel, M.D. 
Inova Center for Thrombosis Research and Drug Development,  
Inova Heart and Vascular Institute 
3300 Gallows Road 
Falls Church, VA 22042 
Phone: 410-367-2590, 
Fax: 410-367-2596 
Email: paul.gurbel@inova.org 
 
 
PROTOCOL SYNOPSIS 

2 
 
 
Title: Antiplatelet Effects of Evolocumab in Patients with Peripheral Arterial Disease 
 
Short Title:  Antiplatelet effects of Evolocumab 
 
Rationale: Peripheral arterial disease (PAD) affects about 8.5 million Americans aged ≥40 
years. PAD is the third leading cause of atherosclerotic vascular morbidity after coronary heart 
disease and stroke.1,2  Statin therapy has been shown to improve walking performance, resting 
and post-exercise ABI and claudication.3 In addition, higher statin doses and lower LDL- 
cholesterol levels have been shown to be independently associated with improved outcomes in 
PAD patients.4 The latter finding indicates that significantly lower LDL-cholesterol may be 
associated with better clinical outcomes and supports proprotein convertase subtilisin/kexin type 
9 (PCSK9) antibody therapy in patients with PAD. A relation between dyslipidemia, oxLDL 
levels, high plasma fibrinogen levels and platelet activation has been demonstrated in patients 
with CVD.5-7 In addition to LDL cholesterol reduction and plaque stabilization, other 
mechanisms of PCSK9 antibodies may further mitigate cardiovascular risk in patients with PAD.   
 
Objectives 
 
Primary: To study the effect of evolocumab + high dose statins on platelet activation and  
                 platelet aggregation. 
 
Secondary: To study the effect of evolocumab + high dose statins on lipid profile, oxLDL,  
                    soluble LOX-1 receptor, fibrinogen, hsCRP, p-selectin, atherox (Soluble markers),   
                    Urinary 11-dh-TxB2 and 8-iso-prostaglandin F2 levels. 
 
Exploratory: To study the effect of evolocumab + high dose statins on platelet-fibrin clot  
                       characteristics and shear-induced platelet aggregation.  
 
Study Type: Single arm-open label study design.  
 
Study Design: This is a phase IV, prospective pharmacodynamic study. 
 
Study Methodology: This investigation will be conducted in subjects >18 years of age with  
PAD. Platelet activation and aggregation, and biomarkers associated with platelet activation, 
oxidative stress, and inflammation will be assessed at the screening (baseline), after 8 weeks of 
high-dose statins and 24 hours and 8 weeks after high dose statin + evolocumab therapy (study 
flow diagram).  
 
Patients: Sixty patients with a history of Peripheral Artery Disease (PAD)including carotid 
artery disease.  
 
Statistical Methodology: Categorical variables will be compared using 2 test or the Fisher 
exact test whereas continuous variables will be assessed using either paired test or Wilcoxon 
signed rank test after checking for normal distribution. Analyses will be performed with SPSS 
software (SPSS, Inc., Chicago, IL) and p<0.05 will be considered significant 

3 
 
 
 
INTRODUCTION 
1.1 
Specific Aims 
To measure platelet activation and aggregation, soluble biomarkers, clot characteristics and 
shear-induced platelet aggregation at the baseline, after 8 weeks of high-dose statins and 24 hr 
and 8 weeks after high-dose statins + evolocumab therapy (study flow diagram).  
 Platelet Aggregation:  
Adenosine diphosphate (ADP)-, Arachidomic acid (AA)-, and collagen-induced  
platelet aggregation by light transmittance aggregometry. 
 Platelet activation markers: 
P-selectin, CD36, LOX-1, basigin (CD147, receptor for glycoprotein VI), oxLDL on   
platelets by flow cytometry. 
 Lipid profile and soluble markers:  
Lipid profile including LDL-C measurement by conventional laboratory methods 
and soluble markers such as oxLDL, LOX-1 receptor, fibrinogen, hsCRP, p-selectin, 
Atherox in plasma by enzyme linked immunoassay. 
 Platelet-fibrin clot characteristics: 
Thrombin-induced platelet-fibrin-clot strength, time to initial thrombin generation,  
 and functional fibrinogen by thrombelastography (TEG6S). 
 Shear-induced platelet aggregation:  
Shear-induced platelet aggregation by T-TAS perfusion flow chamber analysis. 
 Urine 11-dh-TxB2 and 8-iso-prostaglandin F2 
Urinary 11- dehydrothromboxane B2 level using The AspirinWorks™ ELISA 
(Corgenix) and urinary 8-iso-prostaglandin F2 analysis using  Cayman’s 8-Isoprotane 
ELISA kit. 
 
To measure ankle-brachial index (ABI) at baseline, after 8 weeks of high dose statin therapy 
(Visit 2),  and 8 weeks after high-dose statins + evolocumab therapy (Visit 5)  to determine the 
severity of peripheral arterial disease (PAD) and response to  evolocumab treatment on 
endothelial function.   
 
1.2 
Hypothesis 
Treatment with evolocumab plus high-dose statin is associated with a reduction in platelet 
activation and aggregation and biomarkers associated with platelet activation, oxidative 
stress, and inflammation. 
 
1.3 
Background and Significance  

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Peripheral arterial disease affects about 8.5 million Americans aged ≥40 years. PAD is the 
third leading cause of atherosclerotic vascular morbidity after coronary heart disease and 
stroke.1,2  PAD is a manifestation of diffuse systemic atherosclerosis that coexists with 
coronary artery and cerebroarterial disease. Patients with PAD often have a very large 
atherosclerotic burden and large atherosclerotic burden has been associated with heightened 
platelet reactivity measured in systemic circulation.8 About 10–20% of people with PAD 
have intermittent claudication, another 50% have atypical leg symptoms, and those without 
exertional leg pain have poor mobility compared with individuals without peripheral 
symptoms. Patients with PAD have roughly a three-fold increase in risk of mortality 
compared with those without peripheral artery disease.2 Ankle-brachial index (ABI) 
measurement is a well-established tool, to identify PAD.  
 
With the expanding size of the elderly population, PAD is similarly a major growing clinical 
problem with limited current therapeutic options. Patients with PAD primarily die from 
thrombotic events - heart attack and stroke. The current antithrombotic agents administered 
for prophylaxis are aspirin and clopidogrel.9 High LDL is a major risk factor for PAD and 
therefore lipid-lowering therapy constitutes another important therapeutic intervention for 
patients with PAD. Statin therapy has been shown to improve walking performance, resting 
and post-exercise ABI and claudication.3 In addition, higher statin doses and lower LDL- 
cholesterol levels have been shown to be independently associated with improved outcomes 
in PAD patients.4 The latter finding indicates that significantly lower LDL-cholesterol may 
be associated with better clinical outcomes and supports proprotein convertase 
subtilisin/kexin type 9 (PCSK9) antibody therapy in patients with PAD.  
 
Monoclonal antibodies against PCSK9 are innovative agents that provide very potent LDL 
reduction when administered on top of statins. PCSK9 antibodies prevent LDL receptor 
degradation and enhance circulatory LDL cholesterol clearance. The lower LDL cholesterol 
levels potentially enhance the stabilization of vulnerable plaque in both culprit and non-
culprit vessels following the index event.10 Increased PCSK9 levels have been correlated 
with cardiovascular event occurrence.11,12  Elevated LDL levels result in the generation of 
proinflammatory oxidized (ox) LDL and augmentation of plaque vulnerability. Levels of 
circulating lectin-like oxLDL receptor-1 (LOX-1), a major ox-LDL receptor, are elevated in 
patients with ACS, with a diagnostic sensitivity and specificity superior to high-sensitivity C-
reactive protein to identify patients with ACS.  LOX-1 receptor expression is inducible by 
proinflammatory stimuli in a dynamic fashion.13,14 LOX-1 receptor activation induces 
oxLDL uptake, increased production of cell-surface adhesion molecules and a state of 
oxidative stress and inflammation- key steps involved in the progression of atherosclerosis 
and destabilization of plaque. Recent studies indicate a cross-reactive mechanism of PCSK9 
and LOX-1 that leads to a mutually amplified proinflammatory response.15  
 
In addition, a relation between dyslipidemia, oxLDL levels, high plasma fibrinogen levels 
and platelet activation has been demonstrated in patients with CVD.5-7 OxLDL activates 
platelets and promotes adhesion to dysfunctional endothelium.16,17 A proposed mechanism of 
oxLDL-induced platelet activation includes binding of oxLDL to the ApoB/E receptor 
(APOB/E-R) and platelet endothelial adhesion molecule (PECAM-1) leading to the 
generation of TxA2 mediated by p38MAPK and cytosolic phospholipase A2 (cPLA2). The 
latter platelet activation is synergistic with platelet responses to other agonists such as 

5 
 
adenosine diphosphate, collagen, and thrombin leading to platelet aggregation. The 
interaction between oxLDL and scavenger receptor A (SRA) and CD36 are also known to 
activate platelets via cPLA2 and p38MAPK-mediated mechanisms.  A recent animal study 
suggested that platelet activation associated with oxLDL and hyperlipidemia is due to 
generation of reactive oxygen species (ROS) through a CD36-protein kinase C (PKC) 
pathway and modulation of cGMP signaling.18 Finally, plasma PCSK9 levels were also 
positively and independently correlated with increased platelet count and plateletcrit.19  
  
The mainstay therapy of patients with PAD is aspirin and statin therapy. Chaudhary et al. 
demonstrated that patients with atherosclerosis on aspirin and lipid lowering therapy was 
associated with significantly lower levels of inflammation and thrombogenicity as measured 
by urinary 11-dTxB2.20 Secondly, another urinary measure which has shown to be a marker 
of  inflammation /oxidative stress is Urinary 8-iso-PGF2α . There is no information on the 
relationship of PCSK9 therapy on  urinary 11-dTxB2 and 8-iso-PGF2α levels. 21 
 
 
2 
STUDY DESIGN AND SUBJECT SELECTION 
 
This is an single-arm open label study that will be conducted in subjects with PAD including 
carotid artery disease. Sixty subjects will be treated with high dose statins for 8 weeks 
followed by 8 weeks of high dose statin + evolocumab (420mg Q 4weeks x 2 doses) therapy. 
The laboratory assessments (see section 2.2) will be performed at the screening (baseline), 
after 8 weeks of high-dose statin therapy, and 24 hr and 8 weeks after high dose statin + 
evolocumab therapy (see Figure 1). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Figure 1.  
 
 
2.1 
Recruitment 
Recruitment will occur at the Inova Heart and Vascular Institute. The expected length of the 
recruitment period is 12 months. If the study conduct (e.g. recruitment rate; drop-out rate; 
data quality; protocol compliance) does not suggest a proper completion of the trial within 
the reasonable time frame as agreed upon, the recruitment period may be extended to reach 
the desired sample size.  
 
 
 
 
 
 
 

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2.1.1 
Eligibility criteria 
 Inclusion criteria 
o History of  Peripheral Artery Disease (PAD).  
Defined as one or more of the following: 
- 
Documented history of PAD  
- 
Previous limb or foot amputation for arterial vascular disease (i.e., 
excludes trauma),  
- 
Carotid artery disease (defined as >50% stenosis or prior   
 revascularization )  
o Subject may be of either sex and of any race, and must be >18 years of age 
o Subject agrees to not participate in any other investigational or invasive 
clinical study for a period of 4 months during the study period 
o Subject must be willing and able to give appropriate informed consent 
o The subject is able to read and has signed and dated the informed consent 
document including authorization permitting release of personal health 
information approved by the investigator's Institutional Review Board 
(IRB) 
o Subjects who are either statin-naïve or already on statin and are willing to 
be started on high dose (HD) statin therapy:   
- 
If not on statinstart HD statin therapy (atorvastatin 40mg or 
rosuvastatin 20mg as according to investigator discretion) 
- 
If on atorvastatin 40 mg once a daycontinue 
- 
If on lovastatin, pitavastatin, simvastatin, or pravastatinchange 
to atorvastatin 40 mg once a day 
- 
If on atorvastatin 80mg once a daycontinue 
- 
If on rosuvastatin 20 to 40mg once a daycontinue 
- 
If on rosuvastatin 10mg once a dayincrease to 20mg once a day 
 
 
 Exclusion criteria: Subjects will be excluded from entry if ANY of the criteria 
listed below are met: 
o Prior use of any PCSK9 inhibition treatment  
o Participation in any investigational study within the last 60 days. 
o Severe renal dysfunction, defined as an eGFR <20 mL/min/1.73 m2, or 
creatinine kinase (CK) level at 10 X ULN at screening. 
o Active liver disease or hepatic dysfunction, defined as AST or ALT >3 x  
ULN as determined by central laboratory analysis at screening 
o Recipient of any major organ transplant (e.g., lung, liver, heart, bone 
marrow, renal) 
o Known major active infection or major hematologic, renal, metabolic, 
gastrointestinal, or endocrine dysfunction in the judgment of the 
investigator 

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o Malignancy (except non-melanoma skin cancers, cervical in situ 
carcinoma, breast ductal carcinoma in situ, or stage 1 prostate carcinoma) 
within the last 5 years 
o Subject has received drugs via a systemic route that have known major 
interactions with background statin therapy within 1 month before 
randomization or is likely to require such treatment during the study 
period (e.g. cyclosporine, clarithromycin, HIV protease inhibitors, 
gemfibrozil) 
o Female subject who is unwilling to use at least 2 effective birth control 
methods for at least 1 month before screening and 15 weeks after the end 
of treatment with investigational products, unless the subject is sterilized 
or postmenopausal 
o Subject is pregnant or breast feeding, or planning to become pregnant or to 
breastfeed during receipt of investigational products and within 15 weeks 
after the end of study treatment 
o Known previous hypersensitivity reaction/s to the investigational 
products’ active components and excipients  
o Subjects treated with any anticoagulant 
o Patients treated with Prasugrel or Ticagrelor 
o Subject likely to not be available to complete all protocol-required study 
visits or procedures, to the best of the subject's and investigator's 
knowledge 
o History or evidence of any other clinically significant disorder, condition, 
or disease other than those outlined above that, in the opinion of the 
investigator, may compromise the ability of the subject to give written 
informed consent, would pose a risk to subject safety or interfere with the 
study evaluation, procedures, or completion 
 
* Acceptable methods of contraception (birth control) while taking part in this study are: 
• Total Abstinence (no sexual intercourse), absence of menstrual periods in women for 
more than one year after menopause (change of life), sterilization surgery, including tubal 
ligation (tubes tied) or hysterectomy (removal of the uterus or womb) in women or a 
vasectomy in men. 
• Oral contraceptives (birth control pills), intrauterine device (IUD), implantable or 
injectable contraceptives (Norplant or Depo-Provera), contraceptive patch, vaginal ring 
or use of condom with spermicide.  These methods must be used exactly as directed. 
 
2.2 
Study methods 
2.2.1 
Blood sampling 
               Phlebotomy sites will be carefully chosen to minimize risk and platelet activation. 
After discarding the first 2-3mL of free flowing blood, the blood collection tubes 
will be filled to capacity and gently inverted 3 to 5 times to ensure complete 
mixing of the anticoagulant. Tubes containing 3.2% trisodium citrate (n=3) will 
be used for flow cytometry, light transmittance aggregometry and 
thrombelastography.  Tube containing hirudin will be used for shear-induced 
platelet aggregation and tubes containing 3.2% trisodium citrate   will be used for 
platelet activation marker analysis. 

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 Platelet Aggregation 
Platelet aggregation will be assessed in platelet-rich plasma using a Chronolog 
Lumi-Aggregometer (model 490-4D) with the AggroLink software package after 
stimulation with 2 and 5µM ADP, 1.2 mM AA and 4ug/ml collagen. 
 Platelet Activation by Flow cytometry 
     Expressions of p-selectin, CD36, LOX-1, basigin (CD147, receptor for    
     glycoprotein VI), and oxLDL on platelets will be assessed by flow cytometry  
     using respective antibodies.  
 Platelet-fibrin clot characteristics by TEG6S 
      The TEG6s instrument is a microfluidic fully automated cartridge-based device.  
 Lipid profile and Soluble Biomarkers 
After collection, plasma samples will be frozen at -70°C until analysis by 
commercially available enzyme linked immunoassay kits and ACL TOP 300 
coagulation analyzer. Lipid profile will be assessed by conventional laboratory 
method and soluble markers such as oxLDL, LOX-1 receptor, fibrinogen, hsCRP, 
p-selectin, Atherox in plasma by enzyme linked immunoassay. 
 Safety Labs including Comprehensive Metabolic Panel (CMP) and Creatine 
Kinase (CK). 
2.2.2 
Urine Sampling 
Ten (10) mL of a spot urine specimen will be collected at baseline, 8 weeks post 
high-dose statin therapy and 8 weeks post first dose of evolocumab.  
 
 Urine 11-dh-TxB2 and 8-iso-prostaglandin F2 
Urine 11-dh-TxB2 concentrations by AspirinWorks test kit, an enzyme linked 
immunoassay (ELISA). 
Urinary 8-iso-PGF2α  analysis by 8-iso-Prostaglandin F2 ELISA Kit (Cayman 
Chemical Co. #516351). 
 
3 
STATISTICAL CONSIDERATIONS/DATA ANALYSIS  
3.1 
Primary and secondary endpoints 
Primary endpoint 
Relative difference in maximal ADP-induced platelet aggregation between high-dose 
statin therapy and high-dose statin plus evolocumab therapy 
Relative difference = 100 * (baseline on high dose statin value – post high dose 
statin plus evolocumab value)/ baseline on high dose statin value 
 
3.1.1 Secondary Endpoints 
 Relative differences in AA- and collagen-induced platelet aggregation between  
     high dose statin therapy and evolocumab plus high-dose stain therapy.  

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 Relative differences in platelet bound p-selectin, oxLDL, soluble LOX-1 receptor,     
     between high dose statin therapy and evolocumab plus high-dose stain therapy. 
 Relative differences in lipid profile, oxLDL, soluble LOX-1 receptor, fibrinogen,  
    hsCRP, p-selectin, atherox (soluble markers) between high dose statin therapy and       
    evolocumab plus high-dose stain therapy. 
 Relative differences in platelet-fibrin clot characteristics and shear-induced platelet  
     aggregation between high dose statin therapy and evolocumab and high-dose stain  
     therapy  
 Relative differences in ABI between high dose statin therapy. and evolocumab and  
     high-dose stain therapy  
 Relative differences in urinary 11-dh-thromboxane B2 levels between high dose 
statin therapy and evolocumab plus high-dose statin therapy. 
 Relative differneces in urinary 8-iso-Prostaglandin F2 levels between high dose 
statin therapy and evolocumab plus high-dose statin therapy. 
 
3.2 Statistical calculations 
Categorical variables will be compared using 2 test or the Fisher exact test whereas 
continuous variables will be assessed using either paired test or Wilcoxon signed rank 
test after checking for normal distribution. Analyses will be performed with SPSS 
software (SPSS, Inc., Chicago, IL) and p<0.05 will be considered a significant 
difference between high-dose statin therapy and evolocumab plus high-dose statin 
therapy. 
        No prior data is available regarding direct antiplatelet effects of evolocumab. Sample  
        size calculation is mainly based on our previous experience in pharmacodynamic    
        studies. In this study, 50 patients are needed to detect the absolute difference of 14%  
        (~20% relative difference) in ADP-induced aggregation after evolocumab plus high  
        dose statins compared to previous high dose statin therapy alone in these same patients  
        with a power of 91%, a two sided alpha = 0.05 and SD of 30%.  Assuming a 16% drop  
        out rate, we need 60 patients in total.  Calculations based on a paired t-test. 
3.3 
Clinical endpoints – None 
 
4.0 EVOLOCUMAB 
Two doses of evolocumab will be administered in this trial, given 4 weeks apart (see visit 
schedule). Each evolocumab dose to be administered is 420mg. The dosage form and 
strength of evolocumab supplied for this trial is 140mg/mL solution in a single-use prefilled 
SureClick autoinjector. To administer 420 mg of evolocumab, three separate subcutaneous 
injections, given consecutively within 30 minutes will be administered to subjects. 
Evolocumab will be administered by a qualified member of the study staff. 
 
 

11 
 
Figure 3 Guide to Parts 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

12 
 
4.1 Study Treatment Administration  
Study treatment will be maintained, stored, and distributed by the research pharmacy. Once 
the evolocumab is available for administration from the pharmacy, follow the following 
steps: 
 
 
Step 1: Prepare 
Step 1A: Prepare 3 prefilled autoinjectors for administration. Wait 30 minutes for the 
autoinjectors to reach room temperature before injecting. Do not heat autoinjectors.  
Step 1B; Inspect all SureClick autoinjectors. Check the expiration dates.  
Step 1C: Gather all materials needed for the injection and wash hands well with soap and 
water. 
Step 1D: Prepare and clean an injection site.Body areas that can be used include: thigh; 
abdomen, except for 2-inch area around the navel; and outer area of upper arm. For the 
second and third injections, use a different spot than the last injection. Do not inject into 
areas where the skin is tender, bruised, red, or hard. 
 
Step 2: Get ready 
Step 2A: Pull the orange cap off only when ready to inject. Do not leave the ornage cap 
off for more than five minutes, This can dry out the study drug. 
Step 2B: Stretch or pinch the injection site to create a firm surface. 
 
Step 3: Inject 
Step 3A: Hold the stretched or pinched skin. With the orange cap off, place the yellow 
end of the autoinjector on the skin at 90 degrees. Do not touch the gray start button yet. 
Step 3B: Firmly push down the autoinjector onto the skin until it stops moving. You must 
push all the way down but do not touch the gray start button until ready to inject. 
Step 3C: When tready to inject, press the gray start button. You will hear a click. 
Step 3D: Keep pushing the autoinjector down on the skin. Then lift thumb while still 
holding the autoinjector on skin. Each injection could take about 15 seconds. The 
medicine window will turn from clear to yellow when the injection is done. You may 
hear a second click. 
 
Step 4: Finish 
Step 4A: When the injection is done, throw away the used autoinjector and orange needle 
cap. 
Step 4B: Check the injection site. If there is blood, press a cotton ball or gauze pad on the 
injection site. Apply adhesive bandage if needed. Do not rub the injection site. 
 
 
Repeat steps 1B to 4B for the second and third injections. Total dose administered should 
be 420 mg. Use a different injection site for each injection. Administer injections 
consecutively within 30 minutes. 
 
4.1 Evolocumab storage 
The evolocumab prefilled SureClick autoinjectors are stored in the original carton and are 
kept in refrigerated storage. Allowed temperature excursions while in storage is 36°F to46°F 
(2°C to 8°C). Once removed from the refrigerator, the autoinjectors should be kept at room 

13 
 
temperature at 68°F to 77°F (20°C to 25°C) in the original carton and must be used within 30 
days. The prefilled autoinjectors cannot be frozen. Do not use a prefilled autoinjector that has 
been frozen. 
 
5 DATA MANAGEMENT 
 
5.1 Data collection 
Designated study site staff will record data required by the protocol into the case report forms 
(CRFs) and enter it into the electronic database. Authorized research staff will review the 
CRFs for completeness and accuracy and make any necessary corrections to the data entered 
into the electronic database.  
5.2 Confidentiality, data storage, and retention 
Information about study subjects will be kept confidential and managed according to the 
requirements of the Health Insurance Portability and Accountability Act of 1996 (HIPAA).  
Each subject screened and enrolled will be assigned a subject identification number (ID) and 
a list of subjects with their corresponding subject ID will be maintained separately from 
collected data. 
Physical CRFs will be stored in the research site in a locked office and electronic subject data 
will be locked in a password protected file on a secure internet server, accessed only by 
authorized research staff. All data records will be stored on site until 2 years after the 
investigation is formally discontinued. Paper records will be shredded and recycled. 
 
 
6 VISIT SCHEDULE AND ASSESSMENTS 
 
Informed consent will be obtained from subjects meeting the inclusion criteria and none of 
the exclusion criteria before the initiation of any study-specific procedures. The study 
comprises of 2 periods: (1) high-dose (HD) statin therapy and (2) HD statin + evolocumab 
therapy. Subjects will be on a HD statin therapy for 8 weeks followed by the initiation of 
evolocumab 420mg Q 4 weeks x 2 doses, in addition to HD statin therapy for another 8 
weeks. Required assessments for each study visit are listed in Table 1. Subjects should be 
seen for all visits on the designated day or according to the allowed window period (see 
Table 1).  
 
The study requires 5 clinic visits. Each clinic visit (except visit 4) includes laboratory 
measurements, safety assessment, and health and concomitant medication review. A 
complete physical exam and safety laboratory assessment are required for visits 1 and 2. 
 
6.1 Laboratory measurements  
Laboratory measurements (see section 2.2) will be obtained at baseline (prior to study-
recorded/initiated 8 week HD statin therapy), end of 8 weeks HD statin therapy, 24 hours 
after first evolocumab (420mg Q 4 weeks) dose, and at end of study (after 8 weeks of HD 
statin + evolocumab therapy). The study will require clinic visits during the required 
laboratory measurements.  

14 
 
6.2 Health and concomitant medication review 
During the follow-up clinic visits, changes in participant’s medical condition (any new 
diagnosis, scheduled surgical procedures, etc) and concomitant medications should be 
updated. Eligibility will be re-confirmed, adverse events reported, side effects and symptoms 
assessed. 
 
6.3 Physical Exams 
A physical exam will be conducted by the investigator or research nurse during the screening 
period after obtaining consent to evaluate the general status of the subject and to further 
elucidate patient symptoms, risk factors, or concerns that may increase the subject’s risk for 
adverse reactions to the study treatment. A physical evaluation may be conducted as a 
nursing assessment if a physical exam was performed as standard of care ≤1 month prior to 
screening or at visit 2 if no AE/SAE’s have occured.  
 
6.4 Safety assessments 
Safety assessments will consist of monitoring and recording of adverse events and serious 
adverse events (see section 8). The occurrence of adverse events should be sought by non-
directive questioning of the subject at each visit during the study. AEs may also be detected 
when these are volunteered by the subject during or between visits or through physical 
examination, laboratory test, or other assessments. Medical conditions/diseases present 
before starting the study drug are considered AEs only if they worsen after starting the study 
drug. Abnormal laboratory values or test results constitute AEs only if they induce clinical 
signs or symptoms, are considered clinically significant, or require therapy. Abnormal values 
that constitute a SAE or lead to discontinuation of administration of study drug must be 
reported and recorded as an AE. Adverse event collection will commence after screening and 
initation of the HD statin. 
 
Additional assessments required to ensure safety of subjects should be administered as 
deemed necessary by the investigator on a case by case basis.  
 
6.5 Treatment compliance 
Compliance will be assessed by the investigator and/or study personnel at visit 2 and end of 
study visit. Pill counts will be used to assess compliance with HD statin therapy. This 
information should be captured in the source document at each visit.Non-compliance is 
defined as <80%.  
 
6.6 Patient education 
All subjects will be counseled and given instructions in regards to study treatments. Subject 
education will include study treatment action, side effects, benefits and risks, dosage and 
administration, food-drug/drug-drug interactions, pregnancy/lactation warning, and when to 
call investigator/physician. Subjects will be instructed to inform physicians and dentists of 
study treatment intake and to list all prescription medications, over-the-counter medications, 
or dietary supplements they are taking or plan to take so study personnel knows about other 
treatments that may affect adverse event risk.  
 

15 
 
Table 1 
Schedule of Events 
 
V1 
V2 
V3 
V4 
V5/EOS 
 
Screening/ 
Initiate HD 
statin therapy 
1st dose of 
evolocumab        
8 weeks post HD 
statin therapy + 5 
days 
 
24±4 hours 
post 1st dose 
of 
evolocumab 
2st dose of 
evolocumab       
4 weeks (+3days) 
post 1st dose  
 
8 weeks (+5days) 
post 1st dose of 
evolocumab 
Informed Consent 
X 
 
 
 
 
Inclusion/Exclusion 
Criteria 
X 
 
 
 
 
Height and Weight 
X 
 
 
 
 
Vital Signs (HR, BP) 
X 
X 
X 
X 
X 
Medical History 
X 
X 
X 
X 
X 
Review Prior/Concomitant 
Medications 
X 
X 
X 
X 
X 
Safety Labs (CMP and CK) 
X1 
X 
 
 
X 
Urine Or Serum Pregnancy 
Test2 
X 
 
 
 
X 
Ankle/Brachial Index 
X 
X 
 
 
X 
Physical Exam 
X3 
X3 
 
 
 
Laboratory Measurements – 
Blood Sampling 
X 
X 
X 
 
X 
Laboratory Measurements-
Urine Sampling 
X 
X 
 
 
X 
Dispense Study Drug(s) 
X 
X 
 
X 
 
Assign/Continue HD Statin 
Therapy  
X 
X 
X 
X 
 
Administer Evolocumab 
420mg/Q4 wk x 2 doses 
 
X 
 
X 
 
Drug Accountability 
 
X 
 
X 
X 
Adverse Events 
 
X 
X 
X 
X 
X 
 
1 CMP laboratory results within 3 months of Visit 1 are acceptable. If not available, a blood sample for CMP 
will be obtained and results reviewed prior to HD statin therapy initiation. 
2 Urine or blood specimen for pregnancy test will be collected on women of childbearing potential at screening 
visit and end of study. 
3 May be conducted as a nursing assessment if a physical was performed as standard of care ≤1 month prior to 
screening or at visit 2 if no AE/SAE’s have occurred.  
 
 
7 TIMING OF ASSESSMENTS 
7.1 Visit 1: HD statin therapy 

16 
 
7.1.1.1 
Subject demographic and baseline characteristics  
Demographic data to be collected on all subjects include: date of birth, age, sex, 
race, ethnicity. Height, weight, blood pressure, heart rate, relevant medical 
history/current medical condition present before signing informed consent, 
diagnoses and not symptoms will be recorded. Lipid and other CV medications 
will be recorded in eCRFs. 
 
Obtain safety laboratory test results (comprehensive metabolic panel and CK) to 
check for renal and hepatic function prior to starting/continuing HD statin 
therapy. Urine or serum pregnancy test will be performed for women of 
childbearing potential only. 
 
Laboratory measurements (see 2.2, blood and urine sampling) will be obtained 
prior to initiation of study-HD statin therapy 
 
Perform complete physical examination or nursing assessment if applicable 
 
Measure ankle/brachial index 
 
HD statin therapy 
Eligible subjects may start HD statin therapy once it is confirmed that they meet 
all inclusion criteria and none of the exclusion criteria. Enrolled subjects for this 
study can include subjects who are statin-naïve or already on statin. Statin-naïve 
subjects will be started on HD statin therapy. HD statin therapy allowed for the 
duration of the study includes (a) atorvastatin 40mg and 80mg and (b) 
rosuvastatin 20 to 40mg. Please see below for HD statin therapy guidelines: 
 If not on statinstart HD statin therapy (atorvastatin 40mg or rosuvastatin 
20mg as according to investigator discretion) 
 If on atorvastatin 40 mg once a daycontinue 
 If on lovastatin, pitavastatin, simvastatin, or pravastatinchange to 
atorvastatin 40 mg once a day 
 If on atorvastatin 80mg once a daycontinue 
 If on rosuvastatin 20 to 40mg once a daycontinue 
 If on rosuvastatin 10mg once a dayincrease to 20mg once a day 
 
Provide patient education and medication instructions form for HD statin use 
 
Drug Accounatbility of statin therapy  
 
7.2 Visit 2: HD statin + evolocumab 420mg Q 4 weeks therapy 
7.2.1.1 
Subjects will return to clinic for lab measurements (see section 2.2, blood 
and urine sampling) after 8 weeks (+ 5 days) of HD statin therapy. Lab 
measurements will be obtained prior to first dose administration of 
evolocumab therapy.  
7.2.1.2 
Assessment of treatment compliance (i.e. HD statin therapy) 
7.2.1.3 
Perform complete physical examination 
7.2.1.4 
First dose administration of evolocumab 420mg 
7.2.1.5 
Provide patient information hand-out for study drug. Second dose of study  
            drug-evolocumab will be 4 weeks after first dose. Subject will continue  
            assigned HD statin therapy. 
7.2.1.6 
Safety laboratory test 
7.2.1.7 
Health and medication review 
7.2.1.8 
Adverse events assessment 

17 
 
7.2.1.9 
Measure ankle/brachial index 
7.2.1.10 
Collect vital signs (HR, BP) 
 
7.3 Visit 3: 24 hours post first dose of evolocumab 
7.3.1.1 
Subjects will return to clinic 24 ±4 hours post first dose of evolocumab for      
            lab measurements (see section 2.2.1) 
7.3.1.2 
Adverse events assessment 
7.3.1.3 
Health and medication review 
7.3.1.4 
Collect vital signs (HR, BP) 
 
7.4 Visit 4: HD statin + evolocumab 420mg continue Q 4 weeks therapy 
7.4.1.1        Subjects will return to clinic after 12 weeks (+ 3 days) of HD statin     
         therapy.  
7.4.1.2 
Assessment of treatment compliance (i.e. HD statin therapy) 
7.4.1.3 
Second dose administration of evolocumab 420mg 
7.4.1.4        Adverse events assessment 
7.4.1.5 
Health and medication review 
7.4.1.6 
Collect vital signs (HR, BP) 
 
7.5 End of study visit 
7.5.1.1 
Subjects will return to clinic 8 weeks (±5 days) post first dose of  
evolocumab for lab measurements (see section 2.2.1, blood and urine 
sampling).  
7.5.1.2 
Adverse events assessment 
7.5.1.3 
Safety laboratory tests 
7.5.1.4 
Health and medication review 
7.5.1.5 
Assessment of treatment compliance (HD statin therapy) 
7.5.1.6 
Urine or serum pregnancy test for women of childbearing potential 
7.5.1.7 
Subjects to continue original statin therapy 
7.5.1.8 
Measure ankle/brachial index 
7.5.1.9 
Collect vital signs (HR, BP) 
 
 
 
8 ADVERSE EVENTS 
8.1 Adverse Event (AE) Definitions   
An AE is defined as any untoward medical occurrence in a patient or clinical investigation 
subject administered a pharmaceutical product which does not necessarily have to have a 
causal relationship with this treatment. An adverse event can be any unfavorable and 
unintended sign (e.g., including an abnormal laboratory finding), symptom, or disease 
temporally associated with the use of a drug, without any judgment about causality (i.e., 
whether or not it is considered to be drug-related). This includes any newly occurring event 
or previous condition that has increased in severity or frequency since the administration of 
the study treatment/intervention. 
A suspected adverse reaction means any adverse event for which there is a reasonable 
possibility that the drug caused the adverse event. A suspected adverse reaction implies a 

18 
 
lesser degree of certainty about causality than adverse reaction, which means any adverse 
event caused by a drug. 
An adverse reaction means any adverse event caused by a drug. Adverse reactions are a 
subset of all suspected adverse reactions for which there is reason to conclude that the drug 
caused the event. An adverse event or suspected adverse reaction is considered “unexpected” 
if it is not specifically mentioned as occurring with the particular drug under investigation. 
Information about common side effects already known about the study drug/s drug can be 
found in the study drug/s package inserts. This information will be included in the subject’s 
informed consent and should be discussed with the subject during the study as needed. 
 
8.2 Serious Adverse Event (SAE) Definition 
An SAE is defined as an event that: 
 is fatal or life-threatening;  
 results in persistent or significant  disability/incapacity; 
 constitutes a congenital anomaly/birth defect; 
 requires inpatient hospitalization or prolongation of existing hospitalization, unless 
hospitalization is for: 
 routine treatment or monitoring of the studied indication, not associated with 
any deterioration in condition 
 elective or pre-planned treatment for a pre-existing condition that is unrelated 
to the indication under study and has not worsened since the start of study drug 
 treatment on an emergency outpatient basis for an event not fulfilling any of 
the definitions of a SAE given above and not resulting in hospital admission  
 social reasons and respite care in the absence of any deterioration in the 
subject’s general condition 
 is medically significant, i.e., defined as an event that jeopardizes the subject or 
may require medical or surgical intervention to prevent one of the outcomes listed 
above 
 
8.3 AE Grading Scale 
The descriptions and grading scales found in NCI Common Terminology Criteria for 
Adverse Events (CTCAE) version 4.03 will be used for AE reporting. Each AE term is 
associated with a 5-point severity scale.  
 
8.4 AE Reporting 
All SAEs and AEs which are not serious but which lead to permanent discontinuation of 
study medication will be captured in the CRF. Non-serious AEs which do not lead to 
discontinuation of study medication will not be collected.  
 
 
 

19 
 
8.5 Procedures for Recording and Reporting of Adverse Events 
All AEswill be reported to the principal investigator. For both serious and non-serious AEs, 
the investigator has the primary responsibility for AE. identification, documentation, grading, 
and assignment of attribution to the study treatment/intervention. The sponsor will consider 
the investigator’s view when assessing the safety of the drug and determining whether to 
report expeditiously to the FDA, local institutional review board (IRB), and other regulatory 
agencies.  
AEs, occurring from the start of study medication administration through the last day of 
study participation must be recorded on the AE CRF with the following information:  
 The intensity grade (grade 1, 2, 3, 4, 5; see CTCAE v4.03 grading) 
 The relationship to the study drug(s)  
 Attribution: An assessment of the relationship between the AE and the medical 
intervention (i.e., study drug administration). After naming and grading the 
event, the clinical investigator must assign an attribution to the AE using the 
following attribution categories: 
 The duration (start and end dates or if continuing at final exam) 
 Occurrence (known risks for study drug/s, underlying illness or population) 
o Expected 
o Unexpected 
 Other contributing causes 
 Action taken with study drug 
 Any other actions in response to event 
 Outcome  
o Death related to AE 
o Recovered/resolved with sequelae 
o Not recovered/resolved 
o Recovered/resolved without sequelae 
o Recovering/resolving 
o Intervention for AE continues 
o Unknown 
 Whether it constitutes a serious adverse event (SAE) 
 
For serious adverse events not previously documented in study drug/s Package Insert (new 
occurrence) and are thought to be related to the study drug/s, the investigator may urgently 
require further information from the investigator for Health Authority reporting. Amgen or 
designee may need to issue an IND Safety Letter (Investigator Notification) to inform all 
investigators involved in any study with the same drug that this SAE has been reported. 
All suspected unexpected serious adverse reactions (SUSARs) related or possibly related to 
evolocumab and their follow-up reports must be reported to Amgen within 24 hours of 
submission to the regulatory agency, IRB or IEC. A copy of any safety report involving an 
Amgen drug (e.g. evolocumab) submitted to the regulatory agency, IRB or IEC, must be 
faxed to Amgen, within 24 hours of such submission. 
 

20 
 
8.6 Reporting of Pregnancies 
The sponsor will report all pregnancies and pregnancies occurring in the partner of a patient 
participating in the study or potential infant exposure through lactation within 10 calendar 
days of sponsor’s awareness to amgen.  
Pregnant, lactating women, or women planning to become pregnant or to breastfeed during 
receipt of investigational products and within 15 weeks after the end of study treatment are 
excluded from participating in this trial. Women of child-bearing potential recruited into the 
study must have a negative urine or serum pregnancy test prior to initiation of HD statin 
therapy (screening visit) and will undergo urine or serum pregnancy testing at the end of 
study visit. Women of child-bearing potential must agree to use at least 2 forms of medically 
accepted method of contraception during the entire study duration. Male participants will be 
advised to use a medically accepted method of contraception during the study duration. If a 
female partner conceives and becomes pregnant while the male subject is participating in this 
study, the sponsor will be notified as per the procedures described above. No further doses of 
IP would be administered in these cases. 
 
8.7 Adverse Event Treatment  
All AEs should be treated appropriately and managed as according to standard of care, at the 
discretion of the investigator. The action taken to treat the AE should be recorded on the AE 
CRF. A detected AE deemed related to study drug/s that led to permanent study drug 
discontinuation should be followed until its resolution or until the subject completes the 
study. Assessment should be made at each visit (or more frequently, if necessary) of any 
changes in severity, relationship to the study drug, the interventions required to treat it, and 
the outcome. 
 
9 SUBJECT COST AND FUNDING 
This is an investigator initiated study funded by Amgen Inc. Amgen will provide  
evolocumab.  
 
The subject or their insurance company will not be billed for this study. All study related 
tests and procedures will be paid for by the research site. Subjects will be compensated $40 
per visit ($200 for entire study).  
 
10 CONFLICTS OF INTEREST   
Dr. Gurbel reports personal fees from AstraZeneca, Boehringer Ingelheim, Daiichi 
Sankyo/Lilly, Merck, Janssen Pharmaceuticals, New Haven Pharmaceuticals, Bayer, and 
Haemonetics; grants from Haemonetics, Merck, Duke Clinical Research Institute, Harvard 
Clinical Research Institute, National Institutes of Health, New Haven Pharmaceuticals, 
Coramed Technologies, MedImmune, and Sinnowa; a patent for platelet function testing; and 
stock options in Merck. 
 

21 
 
11 FACILITIES AND EQUIPMENT  
The research site is equipped with its own laboratory equipment, which includes state of the 
art technologies for platelet assays, centrifuges, refrigerators, and freezers for study specimen 
processing and storage. For outpatient visits, subjects will be seen in the site’s outpatient 
clinic room equipped with supplies and equipment for necessary for subject assessment. 
 
12 OUTSIDE CONSULTANTS/COLLABORATORS  
There are no outside consultants/collaborators participating. 
 
10  CONTRACTURAL AGREEMENTS     
There are no outside consultants/collaborators participating. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

22 
 
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