Clinical trial NCT02948777
Conditions: Type 2 Diabetes Mellitus
Keywords: apolipoprotein, CVD, chylomicrons, evolocumab, kinetics
Sponsor: Marja-Riitta Taskinen
Start date: 2016-10-01; Completion date: 2018-06-01
Enrollment: 14 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
Postprandial lipemia is highly prevalent in type 2 diabetes subjects even with normal fasting triglyceride values. Humans are mostly in a postprandial rather than in a fasting state and therefore non-fasting triglyceride values reflect more accurately the continuous exposure of arterial wall to the substantial cholesterol load from remnant particles. Evolocumab lowers blood LDL-cholesterol. This study evaluates the effect of evolocumab on postprandial lipid metabolism in type 2 diabetes. All participants in this study receive evolocumab treatment.

Detailed description:

Arms: arm 1: Evolocumab 140 mg subcutaneous injection once every 2 weeks for 12 weeks
Interventions: intervention 1: Evolocumab
Intervention descriptions: intervention 1: None

Protocol documents:
Prot_SAP_000.pdf: 
EVOLKIN / 27.5.16 
 
1 
FCAB6 
PROTOCOL: Effects of PCSK9 inhibition by Evolocumab on postprandial lipid 
metabolism in type 2 diabetes  
SHORT TITLE: EVOLKIN 
 
EUDRACT NUMBER: 2016-001176-30 
PROTOCOL CODE NUMBER: 20167169  
 
INVESTIGATORS AND STUDY STAFF: 
 
HELSINKI, Clinical Research institute Huch, Ltd. and Research Programs Unit (RPU), University of 
Helsinki:  
- Marja-Riitta Taskinen, MD, PhD, Professor, emerita, Principal Investigator 
- Juhani Kahri, MD, PhD, docent, Principal clinical investigator 
- Sanni Söderlund, MD, PhD, clinical investigator 
- Niina Matikainen, MD, PhD, docent, clinical investigator 
- Helinä Perttunen-Nio, Medical Laboratory Technologist, Laboratory coordinator 
- Elisa Koivisto, Medical Laboratory Technologist, Study Nurse 
- Virve Naatti, Medical Laboratory Technologist, Study Nurse 
- Hannele Hilden, Medical Laboratory Technologist, Laboratory technician 
- Laura Impivaara, MSSc, Research administrator 
 
HELSINKI, Helsinki Medical Imaging Center, University of Helsinki, Helsinki, Finland 
- Nina Lundbom, MD, PhD, docent 
- Antti Hakkarainen, B.Sc 
 
GOTHENBURG, Department of Molecular and Clinical Medicine, Institute of Medicine, University 
of Gothenburg and Sahlgrenska University Hospital, Gothenburg; and Royal Institute of Technology, 
Stockholm, Sweden: 
- Jan Borén, MD, PhD, Professor, chairman, Co-principal Investigator 
- Stefano Romeo, MD, PhD, Senior Lecturer, clinical investigator 
- Elias Björnson, BS, systems biology/statistics, multicompartmental modelling 
- Martin Adiels, PhD, Senior Lecturer, statistics, multicompartmental modelling 
- Adil Mardinoglu, PhD, Assoc Professor, bioinformatics, systems biology/statistics 
- Linda Andersson, PhD, Staff Scientist 
- Marcus Ståhlman, PhD, Staff Scientist 
- Angelika Svensson, Laboratory technician 
- Elin Stenfeldt, Molecular biologist, Senior laboratory technician 
- Eva Hedman Sabler, Medical Laboratory Technologist, Senior laboratory technician 
 
 
 
 
 


EVOLKIN / 27.5.16 
 
2 
TABLE OF CONTENTS 
1. 
OBJECTIVES 
1.1 
Primary efficacy assessments 
1.2 
Secondary efficacy assessments  
2. 
BACKGROUND AND RATIONALE 
2.1 
Disease 
2.2 
Rationale 
2.3 
Hypotheses 
3. 
EXPERIMENTAL PLAN 
3.1 
Study Design 
3.2 
Number of Centers 
3.3 
Number of Subjects 
3.4 
Estimated Study Duration 
4. 
SUBJECT ENROLLMENT AND ELIGIBILITY 
4.1 
Inclusion Criteria 
4.2 
Exclusion Criteria 
5. 
TREATMENT PROCEDURES 
5.1 
The run-in period 
5.2 
Treatment period 
5.2.1 
Evolocumab 
5.2.2 
Dosage, Administration, and Schedule 
5.3 
Metformin and atorvastatin 
5.3.1 
Dosage, administration and schedule 
6. 
STUDY PROCEDURES 
6.1 
General Study Procedures 
6.2 
Sample Storage and Destruction 
7. 
REMOVAL AND REPLACEMENT OF SUBJECTS 
8. 
SAFETY DATA COLLECTION, RECORDING, AND REPORTING 
8.1 
Adverse Events 
8.1.1 
Definition of Adverse Events 
8.1.2 
Reporting Procedures for Adverse Events 
8.2 
Serious Adverse Events 
8.2.1 
Definition of Serious Adverse Events 
 
8.2.2 
Reporting Procedures for Serious Adverse Events 
9. 
STATISTICAL CONSIDERATIONS 
10. 
PROTOCOL-SPECIFIED PRODUCT(S) 
 


EVOLKIN / 27.5.16 
 
3 
11. 
REGULATORY OBLIGATIONS 
11.1 
Informed Consent 
11.2 
Independent Ethics Committee/Institutional Review Board 
11.3 
Subject Confidentiality 
12. 
ADMINISTRATIVE AND LEGAL OBLIGATIONS 
12.1 
General considerations 
12.2 
Protocol Amendments and Study Termination 
12.3 
Study Documentation and Archive 
12.4 
Study Monitoring and Data Collection 
12.5 
Publication Policy 
12.6 
Compensation 
13. 
REFERENCES 
14. 
APPENDICES 
 
LIST OF APPENDICES 
Appendix 1.  Clinical data files  
Appendix 2.  Adverse Event reporting procedure 
 
 
 
 
 
 
 
 
 


EVOLKIN / 27.5.16 
 
4 
1. 
OBJECTIVES 
The overall aim is to clarify the postprandial dynamics of PCSK9 on the pathophysiology of 
postprandial hypertriglyceridemia in people with type 2 diabetes. The effect of 12 weeks 
treatment with Evolocumab 140 mg s.c. Q2W on postprandial lipid and lipoprotein metabolism 
will be assessed in patients with type 2 diabetes (n=12) in an one-arm unblinded clinical trial.  
1.1 
Primary efficacy assessment 
a. ApoB48 production rate 
1.2 
Secondary efficacy assessments:  
 
a. Chylomicron (Sf 400) TG IAUC and apoB48 IAUC during the standardized fat-rich mixed 
meal test 
b. VLDL1, VLDL2 and LDL production and fractional catabolic rates 
c. Visceral fat volume and liver fat content (%) determined by MRI 
2. 
BACKGROUND AND RATIONALE 
2.1 
Disease 
Insulin resistance and type 2 diabetes (T2D) incidence is constantly increasing in the Western 
countries with a forecast of up to 550 million people affected by 2030.1 People with T2D remain 
at high risk of cardiovascular events despite being on current standard of care pharmacological 
therapy and the majority of individuals with T2D die from accelerated atherosclerotic 
cardiovascular disease (CVD). Part of this residual risk is due to a characteristic dyslipidemia 
consisting of high levels of both fasting and postprandial triglyceride rich lipoproteins (TRLs), 
accumulation of small dense LDL particles and low levels of high density lipoproteins (HDL).2  
TRLs in the plasma consist of chylomicrons (CM) carrying triglycerides (TG) from the diet, liver-
derived VLDL particles and their respective remnant particles. Since remnant particles not only 
contain TGs but also approximately 40 times higher levels of cholesteryl esters per particle 
compared with LDL,3 elevated levels of remnants may lead to accelerated atherosclerosis and 
CVD. Recent epidemiological studies have identified non-fasting (postprandial) triglycerides and 
the remnant particles as clinically significant risk factors for CVD.4-8 Although the genetic basis 
of hypertriglyceridemia is complex; very recent genetic data have established that TRLs and their 
remnants are causally related to coronary artery disease (CAD).9, 10 Thus, there is strong evidence 
that postprandial dyslipidemia is atherogenic, making TRLs plasma levels lowering a crucial 
therapeutic goal in the management of diabetic dyslipidemia and in patients with high residual 
risk. Statins fail to correct the characteristic dyslipoproteinemia observed in diabetic and insulin 
resistant patients,11 and clinical trials with fibrates, niacin and omega-3 fatty acids, showed 
efficient reduction of plasma triglycerides but failed to show significant effects on the hard end 
points considered when added to statins. 
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protease that plays an 
important role in lipoproteins metabolism by mediating the degradation of the LDL receptor. 
PSCK9 loss-of-function (LOF) mutations in humans lead to lifelong low LDL-cholesterol and 
reduced risk of CVD, whereas gain-of-function mutations lead to autosomal dominant 
hypercholesterolemia (ADH-3).12, 13 These findings paved the way for PCSK9 inhibitors as new 
tools to effectively lower LDL cholesterol and to treat hypercholesterolemia. Alirocumab 
(formerly SAR236553/REGN727) and Evolocumab (formerly AMG145) are humanized 
monoclonal antibodies against PCSK9 that have been shown to significantly reduce LDL-
cholesterol as greater than 50% as monotherapies and when combined with other lipid-lowering 
drugs in phase 2 and 3 studies.14, 15 Robust lipid lowering effects of Evolocumab have been 
reported also in patients on statins and ezetimibe.16, 17 Long-term studies have reported sustainable 
robust reduction of LDL-cholesterol (approximately 55 to 75%) and reduced incidence of 


EVOLKIN / 27.5.16 
 
5 
cardiovascular events with both users of Evolocumab18 and Alirocumab19 as compared to placebo. 
Three large clinical trials (Fourier, Odyssey Outcomes and SPIRE I/II) aimed at addressing the 
effects of PCSK9 antibodies on CVD outcomes are currently ongoing.20 Available data indicate 
that these agents are well tolerated and the rate of adverse events is comparable to control groups.21, 
22  
PCSK9 is highly expressed in intestinal cells23 and genetically modified mice lacking PCSK9 are 
protected against postprandial triglyceridemia.24 Mice studies and in vitro studies using Caco-2 
cells have confirmed a role of PCSK9 in the regulation of intestinal lipid metabolism.23-26 Rashid 
et al. recently proposed that PCSK9 has a central role as a driver of chylomicron synthesis in 
intestinal cells in vitro by altering transcriptional and posttranscriptional processes.23, 26 PCSK9 
downregulates LDL-receptors in enterocytes. Furthermore, it associates with increased 
stabilization of apoB that would favor chylomicron assembly. PCSK9 also seems to affect 
cholesterol transport via action on CD36 and NPC1L1, SRE gene activation and activity of MTP.27 
Notably NPC1L1 is the target for ezetimibe that suppresses the activity resulting in the reduction 
of cholesterol absorption.28 Thus, PSCK9 inhibition may reduce cholesterol absorption in parallel 
to the action of ezetimibe.29 Overall the data suggested that PCSK9 deficiency reduce TRLs and 
their remnants and may thus protect against CVD. This has raised the interest to clarify and explore 
further the potential effect of PCSK9 inhibition on chylomicron assembly and secretion and on 
metabolism of TRLs and their remnant.30, 31  
So far the data on the potential effects of PCSK9 inhibitors on plasma triglycerides and TRL 
metabolism is limited and mostly observational. The data from clinical trials is variable but 
Evolocumab has been reported to reduce triglyceride levels in a pooled analysis of 1359 patients 
by about 1223 %.30, 31 Recently, Cariou et al. reported that a short term high fructose diet 
increased serum PCSK9 levels associated with increase of postprandial triglycerides.32 This is in 
agreement with recent results by Chan et al. who showed that both circulating PCSK9 and apoCIII 
associated positively with postprandial triglyceride and apoB48 responses but inversely to the 
fractional catabolic rate (FCR) of apoB48 to oral fat load in obese subjects. In stepwise regression 
analyses plasma PCSK9 was the best predictor of both plasma apoB48 responses as well as the 
FCR of apoB48.33 However, PCSK9 plasma levels were not associated with VLDL-TG 
metabolism in another recent kinetic assessment study.34 Notably the measurement of PCSK9 
concentrations by ELISAs is challenging due to the presence of different forms of circulating 
PCSK9.35  
Postprandial lipemia is a consistent component of the atherogenic dyslipidemia in T2D patients. 
Sterol regulatory element-binding protein 2 (SREBP-2) is the most important nuclear receptor 
transactivator of PCSK936 but also SREBP-1c regulates PSCK9.37 In this context insulin has been 
shown to positively modulate PCSK9 transcription via stimulation of SREBP-1c.37, 38 Recently, 
insulin was reported to induce directly PCSK9 expression in vitro and in vivo in mice.39 
Furthermore, insulin seems to enhance the degradation of LDL-receptors in a PCSK9 dependent 
manner. Blocking insulin signaling in different models consistently reduced PCSK9 mRNA and 
plasma levels by about 5575 %. The data also suggest that insulin resistance may modify the 
functional relationship between insulin and PCSK9 by unmasking the potential effects of other 
factors like glucagon.  
Notably, stimulation of SREBP-1c by insulin and carbohydrate-responsive element-binding 
protein (ChREPB) by glucose activates the pathway of de novo lipogenesis (DNL) that contributes 
to the overproduction of VLDL by the liver in T2D.40 In addition, in insulin resistance state the 
metabolic milieu of hyperglycaemia and hyperinsulinemia may theoretically lead to enhanced 
circulating PCSK9 levels. Recently, PCSK9 was reported to induce the degradation of CD36 
whereas the absence of PCSK9 seems to upregulate CD36 protein the major protein regulating 
fluxes of fatty acids and consequently triglyceride storage in both adipose tissue as well as in the 


EVOLKIN / 27.5.16 
 
6 
liver.41 Interestingly, the authors demonstrated that CD36 levels were increased about 3-fold in 
livers of PCSK9 deficient mice reflected in increased lipid droplets in the livers of these mice. In 
face of these complex relationships it is not surprising that the limited available data on PCSK9 
levels in subjects with T2D are inconsistent and both elevated and normal levels have been 
reported in small cohort studies.33, 36, 42 
It is clear that our understanding of endogenous regulation of PCSK9 in people with diabetes is 
insufficient to optimize the use of PCSK9 inhibitors in people with diabetes. Current findings 
suggest PCSK9 inhibition as a possible effective approach to reduce the lipid burden in the 
postprandial state and manage the typical T2D dyslipidemia, thus contributing to CVD risk 
reduction in these high risk patients. So far, no studies exist on the effects of PCSK9 on 
postprandial lipid metabolism or on circulating TRL fluxes and the underlying molecular 
mechanisms. Therefore, the evaluation of the impact of PCSK9 inhibition on the postprandial 
responses and hepatic lipid metabolism is of particular interest in T2D subjects.43 
2.2 
Rationale 
The overall aim of this research program is to clarify the postprandial dynamics of PCSK9 on 
the pathophysiology of postprandial hypertriglyceridemia in people with type 2 diabetes. Our 
team has the expertise, state-of-the-art equipment, and logistics to perform these studies. This 
is proven by our longstanding track of successful kinetic assessments  of lipoprotein metabolism 
and of our track in lipid and lipoprotein associated inflammatory pathways. 
2.3 
Hypotheses 
In this study we will answer the following clinically highly important questions: 
a. Is the postprandial reduction of triglyceride levels mediated by Evolocumab associated with 
reduced secretion and/or improved clearance of TRLs? 
b. Is decreased secretion of chylomicrons by Evolocumab associated with inhibition of apoB48 
synthesis in intestinal cells and/or by reduced lipid incorporation into chylomicrons  
c. Is the reduced production of intestinal TRLs (after treatment with Evolocumab) associated 
with improved metabolism of liver-derived VLDL? 
d. Does Evolocumab reduce intestinal cholesterol absorption?  
e. Are the changes of TRL fluxes induced by Evolocumab reflected in altered LDL kinetics? 
3. 
EXPERIMENTAL PLAN 
3.1 
Study Design 
The study is designed to investigate of the impact of Evolocumab on the postprandial kinetics of 
TRLs metabolism and LDL fluxes. Lipoprotein metabolism is a complex system in which 
abnormal concentrations of various pro- and anti-atherogenic particles can result from alterations 
in their rates of production, conversion and/or catabolism. Traditional methods that measure 
plasma lipoprotein concentrations only provide static estimates of lipoprotein metabolism and 
hence limited mechanistic information. By contrast, we use tracers labelled with stable isotopes 
(Figure 1 and Table 1), which provide us with a powerful and cutting-edge tool for probing lipid 
and lipoprotein kinetics in vivo, thus improving the understanding of the pathogenesis of 
dyslipoproteinaemia.  
The study specifically clarifies the postprandial response of TRLs and focuses on the synthesis 
and turnover of chylomicrons and hepatically derived large triglyceride-rich VLDL1 and smaller 
VLDL2, and the interaction between these lipoproteins. This will also allow us also to follow the 
effects of changes in VLDL metabolism on LDL kinetics. The subjects will be extensively 
phenotyped including imaging of liver fat and measurements of hepatic de novo lipogenesis 
(DNL), biomarkers of cholesterol absorption and synthesis. We will also perform PCSK9 


EVOLKIN / 27.5.16 
 
7 
genotyping in the study subjects. 
Figure 1. Outline of a kinetic procedure that includes three major steps: (i) clinical visit protocol, 
(ii) sample preparation/processing and enrichment analysis by GC/MS, and (iii) mathematical 
modeling. 
Table 1. Stable isotopes 
2H5-Glycerol 
Injected tracer that is esterified to triglycerides in the liver but not in the intestine 
as it lacks glycerol kinase activity. It is thus only incorporated into VLDL–
triglycerides. 
2H3-Leucine 
Injected tracer that is incorporated into apoB100 and apoB48. Chylomicrons 
secreted from the enterocytes contain apoB48, while VLDL secreted from the liver 
contain apoB100 
13C4-
Palmitate 
The dietary tracer [1,2,3,4-13C]-palmitate will be administered in the high fat meal 
to track the contribution of dietary lipids on VLDL–triglyceride secretion 
2H2O (heavy 
water) 
Given orally. Incorporated into all fatty acids. Used to quantify the amount of fatty 
acids derived from de novo lipogenesis in the liver in VLDL1 and VLDL2 
These studies will clarify the dynamics of PCSK9 after an oral fat load, and will correlate the 
kinetics of PCSK9 with the postprandial kinetics of chylomicrons and liver derived VLDL and 
LDL in extensively phenotyped obese subjects. 
Clinical protocol: Each subject enrolled will participate in two kinetic procedures to compare 
kinetic data before and after 12 weeks treatment with Evolocumab. On the evening of the first 
visit, 
2H2O (3 
g/kg) 
will 
be 
served between 8 
PM and 10 PM to 
assess 
the 
contribution 
of 
de 
novo 
lipogenesis 
to 
fatty 
acids 
in 
chylomicrons 
and 
VLDL 
(Figure 2).  
 
 
8 PM    10 PM                        8 AM                                          6 PM
Plasma
VLDL1, VLDL2, 
CM, LDL
2H20 (3 g/kg)
Bolus inj of 2H5-glycerol 
and 2H3-leucine
Test meal with 
13C4 palmitate


EVOLKIN / 27.5.16 
 
8 
At 8 AM the following morning, the subjects will receive a bolus injection of 2H5-glycerol and 
2H3-leucine to assess VLDL and LDL-apoB100 and chylomicron–apoB48 kinetics; two hours 
later they will consume a defined mixed high fat meal (57 g fat, 63 g carbohydrates and 40 g 
protein, total 934 kcal) also containing 13C4-palmitate tracer. These procedures will be repeated 
identically after the Evolocumab treatment.  
The study design does not contain a control group. Instead, each subject will function as their 
own control. This study design is common in kinetic studies since it minimizes variation, and thus 
the number of study subjects needed. This is critical since kinetic studies are extremely labor 
intensive and time demanding. 
3.2 
Number of Centers 
The study is a single-center one-arm unblinded clinical trial. The clinical part of the study will 
be conducted in the research facilities of Research Programs’ Unit, Diabetes & Obesity, 
University of Helsinki; Clinical Research Institute, Huch ltd. Biomedicum Helsinki, Finland. 
Laboratory work and analysis will be made in the Helsinki and Gothenburg centers. All 
enrichment studies of stable isotopes, proteomics and lipidomics analyses, as well as statistical 
and modelling work will be performed in Gothenburg, Sweden. 
3.3 
Number of Subjects 
Total 12 subjects (i.e., 24 kinetic procedures). 
3.4 
Estimated Study Duration 
The duration of the run-in period is at least 4 weeks. The study procedure comprises about 10 
days followed by treatment over 12 weeks followed by another 10 days period for study 
procedures. 
4. 
SUBJECT ENROLLMENT AND ELIGIBILITY 
 
Subjects will be recruited from previous studies and by using an online recruitment service that 
is provided by Clariness GmbH, Germany.  
4.1 
Inclusion Criteria 
- Male or female (non-fertile or using a medically approved birth control method) 
overweight/obese subjects with T2D treated with lifestyle counselling and a stable 
metformin dose for at least three months 
- age 18–77 yrs. 
- BMI 25–40 kg/m2 
- triglycerides between 1.5–4.5 mmol/L and LDL cholesterol >1.8 but ≤4.0 mmol/L (on 
Atorvastatin 20 mg/day) 
- HbA1c: ≤9%.  
- Each patient will attend a pre-screening visit (at week 5) where eligibility criteria will be 
evaluated. If the patient uses another statin than atorvastatin (20 mg) at screening visit the 
used statin is stopped and atorvastatin 20 mg will be initiated. If the patient is not using any 
statin, atorvastatin 20 mg will be initiated and the lipid values will be checked after 4 weeks 
when all inclusion/exclusion criteria will be assessed. 
4.2 
Exclusion Criteria 
- Type 1 diabetes 
- apoE2/2 phenotype 
- ALT/AST > 3×ULN  
- CK>3×ULN 


EVOLKIN / 27.5.16 
 
9 
- GFR <60 ml/min  
- clinically significant TSH outside the normal range 
- BMI >40 kg/m2  
- HbA1C > 9.0 %  
- fasting TG > 4.5 mmol/l  
- total chol > 7.0 mmol/l  
- positive urine or serum pregnancy test  
- untreated or inadequately treated hypertension defined as blood pressure >160 mmHg 
systolic and/or >105 mmHg diastolic, use of thiazide diuretics at a dose of ≥25 mg/day 
- subject not on a stable dose of atorvastatin (20 mg/ day before randomization)  
- lipid-lowering drugs other than statins within 3 months  
- any other diabetes medication except diet + metformin  
- history/diagnosis of diabetes nephropathy / retinopathy  
- current smoking 
- weekly alcohol use over 24 doses for men and 16 for women  
- history of MI, ACS or coronary revascularization (PCI or CABG) within the last 6 mos. 
- planned revascularization (eg CABG, PCI, carotid or peripheral revascularization 
procedures) within 3 months of screening 
- NYHA class III/IV congestive heart failure persisting despite treatment 
- history of hemorrhagic stroke 
- hypersensitivity to (evolocumab or) any of the excipients found in the drug product  
- use of estrogen therapy  
- current use of antithrombotic or anticoagulant therapy 
- known bleeding tendency that would be an contraindication to heparin test  
- history of cancer within the past 5 years (except for adequately treated basal cell skin 
cancer, squamous cell skin cancer or in situ cervical cancer)  
- women of childbearing potential not protected by effective birth control method and/or 
not willing to be tested for pregnancy 
- 
patient considered by the investigator or any sub-investigator as inappropriate for this 
study for any reason 
Restrictions during the study:  
● No aspirin or non-steroidal anti-inflammatory drugs (NSAIDs) within 1 week before 
heparin tests 
● No alcohol or strenuous exercise within 72 hours of each special test 
5.  
TREATMENT PROCEDURES 
The protocol comprises two periods: 1. Run-in period and 2. Treatment period 
5.1 
The run-in period  
The run-in period starts by the screening visit when the eligibility to enter the treatment period 
will be evaluated. If the subjects is not using statin at all or is using another statin, then 
atorvastatin is initiated at a dose of 20 mg/ day. The qualification labs are obtained a minimum 
of 4 weeks after the change in statin or statin dose. 
5.2 
Treatment period 
Eligible subjects will be assigned to Evolocumab monotherapy 140 mg subcutaneously every 2 
weeks (Q2W) as add on to the statin therapy. Intervention with Evolocumab will start at the last 
day of the first kinetic visit and continue over a period of 12 weeks. The patients are invited to 
visit the clinic monthly for safety blood samples. The 2nd kinetic procedure will be scheduled to 


EVOLKIN / 27.5.16 
 
10 
be started on day 5 (± 2) after Evolocumab injection at week 12.  
 
Figure 3. Design of the clinical study with study visits (blue arrows) and two kinetic procedures 
to compare kinetic data before and after 12 weeks treatment with Evolocumab (red arrows). An 
additional visit is needed for measurements of liver fat content in the fasting state by magnetic 
resonance imaging (MRI visit). This visit should be performed within 7 days before the start of 
the kinetic procedure. Safety visits at 2, 4 and 8 weeks.  
Thus, the study protocol includes 264 clinical visits (i.e., 22 visits for each study subjects) in 
addition to 50 screening visits. 
5.2.1 Evolocumab 
PCSK9 is a protein secreted by liver. Evolocumab is a fully human monoclonal IgG2 antibody 
against PCSK9. Inactivation of PCSK9 prevents PSCK9/LDL receptor interaction and results 
in the decreased LDL-receptor degradation and lowers LDL-C by enhancing LDL receptor-
mediated clearance. Evolocumab (dose 140 mg) is injected subcutaneously once every 2 weeks. 
This dosing maintains a consistent reduction of LDL cholesterol levels reported to be up to 
5570% in the drug development program. The efficacy, tolerability and safety of PCSK9 
inhibition has been confirmed in intervention trials including ˃8000 subjects. Regulatory 
approval for Evolocumab (Repatha) occurred in Europe and USA in 2015. 
5.2.2 Dosage, Administration, and Schedule 
Intervention with Evolocumab will start at the last day of the first kinetic visit and continue over a 
Table 2. Summary of clinical visits in the study (Appendix 1) 
CLINICAL VISITS 
REASON FOR VISIT(S) 
NUMBER OF VISITS 
Screening visit (1) 
Screening visit (2) 
Screening 
Heparin test 
50 subjects × 1 visit = 50 
12 subjects × 1 = 12 
Kinetic visits 
Injections of stable isotopes 
and blood sampling 
12 subjects × 7 visits × 2 (two kinetic 
procedures) = 168 
MRI visits 
Measurement of hepatic 
and visceral fat 
12 subjects × 1 × 2 (one visit for each 
kinetic procedure) = 24 
Safety visits 
Safety monitoring 
12 subjects × 4 visits = 48 
Final visit 
Heparin test, evaluation 
12 subjects × 1 = 12 
Metformin and 
Atorvastatin 
+ Evolocumab 140 mg Q2W
Figure 3. Study design
(Number of subjects = 12, total 24 kinetic studies)
Screening 
visit
24 weeks
Studies repeated month 0 and 3
Non-fasting kinetics & Liver and abdominal fat
Week 2 Week 4 Week 6
Week 12


EVOLKIN / 27.5.16 
 
11 
period of 12 weeks. The patients are invited to visit the clinic at weeks 2, 4, and 8 for safety blood 
samples. The 2nd kinetic procedure will be scheduled to start on day 5 (± 2) after Evolocumab 
injection at week 12. The dose of Evolocumab will be 140 mg s.c. Q2W without any dose 
adjustments or escalation.  
5.3 
Metformin and Atorvastatin 
5.3.1 Dosage, Administration, and Schedule  
The subjects will use metformin at a stable dose (5003000 mg per day) throughout the study. 
If the patient uses another statin than atorvastatin (20 mg) at screening visit the used statin is 
stopped and atorvastatin 20 mg is initiated. If the patient is not using any statin, atorvastatin 20 
mg will be initiated and the lipid values will be checked after 4 weeks. If eligible (LDL>1.8 
mmol/L), the patient can be recruited. The subjects will continue using atorvastatin 20 mg 
throughout the study. The subjects will use the metformin as a prescription medication. 
Atorvastatin will be administered by the study centre to the subjects. 
6. 
STUDY PROCEDURES 
6.1 
General Study Procedures 
Blood samples will be taken at the time points indicated by the red dots in Figure 2 and at days 
1, 2, 3, 4, 5 and 7 and lipoprotein fractions (chylomicrons, VLDL1 and VLDL2) will be separated 
by density gradient ultracentrifugation. LDL will be isolated separately by ultracentrifugation 
using a fixed angle rotor. Concentrations of lipids and apolipoproteins (apoB100, apoB48) will 
be performed in whole plasma and in all fractions at each time point and enrichments of isotopes 
in lipids (TG, FAs) and proteins (apoB100, apoB48, PCSK9, apoCIII) by mass spectrometry 
analysis at each time point. Hepatic DNL will be calculated from enrichment of deuterated water 
ingested during the kinetic procedure at specified time points (0, 4 and 8 hrs). The following 
biomarkers will be measured: 
 PCSK9, ApoCIII, apoA5, FGF21 and resistin in samples at fasting, 4 and 8 hrs. 
 Biomarkers of cholesterol absorption (cholestanol, campesterol, sitosterol, avenasterol) and 
synthesis (cholestenol, desmosterol, lathosterol) in samples at fasting, 4 and 8 hrs. 
 Markers of energy balance (FFA, β-OHB), glucose and insulin during the mixed meal 
 Fasting samples: apoB100, apoAI, apoAII, Lp(a), adiponectin, and ghrelin, leptin, and 
LDL- and HDL size 
 Lipolytic enzymes: we will measure lipoprotein lipase and hepatic lipase activities as well 
as lipoprotein lipase mass after injection of 75 IE/kg heparin. Blood samples will be taken 
before and after 10 min heparin injection. See Appendix 1.  
Determination of liver, subcutaneous and intra-abdominal fat: Proton magnetic resonance 
spectroscopy will be performed to determine liver fat content, and magnetic resonance imaging 
to determine subcutaneous abdominal and intra-abdominal fat (a separate visit).  
Modeling of the data: We will use multicompartmental models to describe the kinetics and 
dynamics of material. Enrichment of deuterated water to measure DNL is measured in fasting 
and at 4-h and 8-h samples. 
6.2. Sample Storage and Destruction 
Samples will be stored at Sahlgrenska University Hospital, Gothenburg, Sweden in -80C 
freezers in locked freezers that have alarm, in locked rooms.  
7. 
REMOVAL AND REPLACEMENT OF SUBJECTS 


EVOLKIN / 27.5.16 
 
12 
We estimate that 10 successfully finalized subjects are needed. Therefore, we enroll 12 subjects 
in the study.  
Withdrawal from the study: 
● 
Withdrawal of consent 
● 
Clinical information/condition/adverse event that potentially endangers study subject/ study 
results judged by the PI of the study 
● 
any information or clinical event described in Evolocumab SPC that is a contraindication 
for the use of Evolocumab 
All study withdrawals will be recorded in the CRFs and in the patient’s medical records when 
considered as confirmed.  
8. 
SAFETY DATA COLLECTION, RECORDING, AND REPORTING 
Safety visits will occur at weeks 2, 4 and 8 during the intervention and 3 months after the 
termination of the study drug. Safety visits include inspection of the injection sites, evaluation 
and recording of possible side effects, clinical examination (blood pressure, pulse, weight, waist 
circumference), and blood sampling (blood cell count, ALT, AST, cholesterol values, blood 
glucose, and creatinine kinase (CK)). 
8.1  Adverse Events 
 
In addition to adverse events defined below, safety data collection includes also collection 
and recording of adverse device effects and other safety events such as overdose, 
misuse/abuse, lack of effect.  
8.1.1 Definition of Adverse Events 
An adverse event (AE) is any untoward medical occurrence in a patient or clinical investigation.  
8.1.2 Reporting Procedures for Adverse Events 
All AEs, regardless of seriousness, or relationship to IMP, are to be recorded to CRF and 
reported in compliance with all applicable regulations. All adverse events will be collected from 
the first study-related activity (from the signing of the informed consent) and in all following 
contacts with the study-subject through-out the project. This includes events from the first trial 
related activity after the subject has signed the informed consent, and until the post treatment 
follow-up period. Laboratory or vital signs are to be recorded as AEs only if they are 
symptomatic, require corrective treatment or consultation, lead to IMP discontinuation, or are 
considered as clinically relevant. 
8.1.3 Reporting Procedures for Product Complaints 
Any concerns or irregularities about the packaging, appearance or usage of the prefilled 
AI/Pen are to be reported to Amgen within 24 hours of discovery or notification of the 
concern or irregularity. Should any such concerns or irregularities occur the IP will not be 
used until Amgen confirms that it is permissible to use the product. 
 
8.2 
Serious Adverse Events 
8.2.1 Definition of Serious Adverse Events 
A serious adverse event (SAE) is any untoward medical occurrence that at any dose: results in 
death, or is life-threatening, or requires inpatient hospitalization or prolongation of existing 
hospitalization, or results in persistent or significant disability/incapacity, or is a congenital 
anomaly / birth defect, or is a medically important event. 
8.2.2 Reporting Procedures for Serious Adverse Events 


Evolocumab / 27.5.16 
 
13 
All SAEs will be reported in compliance with all applicable regulations. An SAE is to be reported 
within 24 hours. The investigators will copy Amgen Finland Safety department (EU-Nordic 
Baltic Drug Safety nordic.baltic.drugsafety@amgen.com when expediting to FIMEA any 
serious adverse reactions (SAR) or suspected unexpected serious adverse reactions (SUSAR) 
which occurred during the use of Evolocumab in the study (Appendix 2).  
9. 
STATISTICAL CONSIDERATIONS 
The primary and secondary efficacy assessment will be compared before and after Evolocumab 
using paired t-test and ANCOVA. Efficacy assessments 1.1.a and 1.2.b are outputs from the 
mathematical model (the production and clearance rates), 1.2.a is calculated from time series data 
using the trapetzoid method (AUC for chylomicron triglycerides and apoB48 ) and 1.2.c is 
calculated from within the software in the MRI/MRS scanner (Visceral fat volume and liver fat 
content).  
Using 10 subjects we estimate that we can detect a difference of at least 20% for TG kinetics and 
15% for apoB kinetics (both under 1.2.b) at an alpha of 0.05 and a power of 80% (Reproducibility 
of glucose, fatty acid and VLDL kinetics and multi-organ insulin sensitivity in obese subjects 
with non-alcoholic fatty liver disease. Magkos et al Int J Obes 2011).  
Based on our paper Improved Estimation of Human Lipoprotein Kinetics with Mixed Effects 
Models by Berglund M et al. PLoS One. 2015 Sep 30;10(9):e0138538, we hope to further increase 
study power. It is hard to perform this type of estimates in kinetic assessments since the effect of 
the intervention is unclear. Therefore, we enroll 12 subjects in the study.  
Subjects completing the protocol are included in the statistical analyses. Paired t-test and ancova 
will be used to compare estimates of kinetic parameters in all subjects, from kinetic studies before 
and after Evolocumab intervention.  
10. 
PROTOCOL-SPECIFIED PRODUCT(S) 
Evolocumab (AMG 145) is supplied as a sterile, single-use, preservative free solution for 
subcutaneous injection in a disposable, spring-based prefilled autoinjector (AI)/pen. The AI/pen 
contains a 1.0 mL deliverable volume of 140 mg/mL Evolocumab in 220 mM proline, 20 mM 
acetate, 0.01% (w/v) polysorbate 80, pH 5.0. Evolocumab will be administered 140 mg s.c. 
Q2W without any dose adjustments or escalation. 
11. 
REGULATORY OBLIGATIONS 
11.1 Informed Consent 
Only subjects who sign an informed consent are included to the study. No study specific 
interventions will take place before the patient signs informed consent. 
11.2 Independent Ethics Committee/Institutional Review Board 
The study protocol will be subjected to the consideration of the Medical Ethics committee of the 
Helsinki University Central Hospital and permission from the Finnish Medicines Agency 
(Fimea) will be obtained. The study will be conducted according to GCP and GLP standards. 
11.3 Subject Confidentiality 
All information disclosed or produced during this clinical study (the trial protocol, CRFs, the 
results) are confidential prior to the publication of results and treated in compliance with the 
applicable laws and regulations.  
12. 
ADMINISTRATIVE AND LEGAL OBLIGATIONS 


Evolocumab / 27.5.16 
 
14 
12.1 General considerations 
The clinical part of the study will be conducted in the research facilities of Research Programs’ 
Unit, Diabetes & Obesity, University of Helsinki; Clinical Research Institute, Huch ltd. 
Biomedicum Helsinki, Finland. Laboratory work and analysis will be made in Helsinki and 
Gothenburg centers. The study protocol will be subjected to the consideration of the Medical 
Ethics committee of the Helsinki University Central Hospital and permission from the Finnish 
Medicines Agency (Fimea) will be obtained. The study will be conducted according to GCP and 
GLP standards. The subjects taking part to the study are insured by a patient insurance taken out 
by the Helsinki University Central Hospital. 
12.2 Protocol Amendments and Study Termination 
In case of any protocol amendment, the amendment will be submitted to the Ethics committee 
and requires written approval by the Ethics committee. The written amendment should be 
signed by the investigator and filed with the clinical protocol. Amgen will be informed of 
protocol amendments and will provide a review.  
12.3 Data Collection, study Documentation and Archive 
All study documentation and data collected during the study will be documented and archived 
in compliance with all applicable regulations. 
12.4  Publication Policy 
The Principal Investigator agrees to provide Amgen with a copy of any manuscript, at least 
forty-five (45) days prior to submission. In accordance with scientific custom, the contribution 
of Amgen will be expressly noted in all written or public disclosures, by acknowledgment, as 
appropriate.  
12.5 Compensation 
Amgen will provide funding in support of the Study. The funding constitutes an Investigator-
Initiated Research Grant for this Study.  
13. 
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14. 
APPENDICES 
LIST OF APPENDICES 
Appendix 1.  Clinical data files  
Appendix 2.  Adverse Event reporting procedure