Effect on Liver Fat, Adipose Tissue and Metabolic Parameters When Switching a Protease Inhibitor or Efavirenz to Once Daily Raltegravir in HIV+ Patients With Body Mass Index Over 25 kg/m2 and With at Least One Metabolic Syndrome Component
Phase: PHASE4
Conditions: HIV Seropositivity, Metabolic Syndrome, Fatty Liver
Sponsor: Helsinki University Central Hospital

 
CLINICAL STUDY PROTOCOL 
 
 
 
A randomized, open, parallel design study to evaluate the effect on 
liver fat, adipose tissue and metabolic parameters when switching a 
protease inhibitor or efavirenz to once daily raltegravir in HIV-infected 
patients with body mass index over 25 kg/m2 and with at least one 
metabolic syndrome component. 
 
 
Study code:  
 
 
OBERAL 
 
ClinicalTrials:  
 
NCT03374358 
 
EudraCT number:  
2017-003430-85 
 
Version:   
 
 
1.2 
 
Principal Investigator: 
Docent Jussi Sutinen 
 
Contact Information: 
Infectious Diseases 
 
Inflammation Center  
Helsinki University Hospital 
 
PL 340, 00029 HUS 
 
Phone:   
 
 
+358 9 47175989 
Fax:  
 
 
 
+358 9 4717 5901 
e-mail:  
 
 
 
jussi.sutinen@hus.fi 
 
 
Date:  
 
 
 
28.5.2019 
 
 
 
 

 
 
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Co-investigators and proposed roles: 
 
 
Kirsi Pietiläinen, Gyllenberg Professor in Clinical Metabolism (Head of Obesity Research Unit, 
clinical procedures, data analysis, writing), Obesity Research Unit, University of Helsinki 
 
Anna Hanttu, MD, Specialist in Internal Medicine (Study coordination, recruiting, clinical 
procedures, data analysis, writing), Infectious Diseases, Inflammation Center, Helsinki University 
Hospital 
  
Matti Ristola, Docent, Specialist in Internal Medicine and Infectious Diseases (Study recruiting, 
clinical follow-up, data analysis, writing), Infectious Diseases, Inflammation Center, Helsinki 
University Hospital 
 
 
 
 
 
 
Pia Kivelä, MD PhD, Specialist in Infectious Diseases (Study recruiting, clinical follow-up, data 
analysis, writing), Infectious Diseases, Inflammation Center, Helsinki University Hospital 
 
Nina Lundbom, Docent, Specialist in Radiology (MRS and MRI analysis), Helsinki Medical 
Imaging Centre, Radiology, Helsinki University Hospital and University of Helsinki 
 
Antti Hakkarainen, PhD, (MRS and MRI analysis), Helsinki Medical Imaging Centre, Radiology, 
Helsinki University Hospital and University of Helsinki 
 
Jesper Lundbom, PhD, (MRS and MRI analysis), Helsinki Medical Imaging Centre, Radiology, 
Helsinki University Hospital and University of Helsinki 
 
Perttu Arkkila, Docent, Specialist in Internal Medicine and Gastroenterology (transient 
elastography analysis), Gastroenterology, Abdominal Center, Helsinki University Hospital 
 
Organization of the research: 
 
This study is an investigator initiated study in collaboration with Infectious Diseases Department, 
HUH and with Obesity Research Unit, University of Helsinki. 
The research project is coordinated by PI Jussi Sutinen and co-investigator Anna Hanttu. 
The recruiting and clinical follow-up of study subjects will take place in Infectious Diseases Clinic, 
Helsinki. There will be two study visits in the Obesity Research Unit, where the metabolic 
procedures will be done. Additionally, liver fat and abdominal fat distribution will be measured by 
MRI/MRS and liver fibrosis by transient elastography, both at the Helsinki University Hospital.    
 
Funding of this project comes from Merck Sharp & Dohme Corp. With this funding, we anticipate 
to organize the study visits of the study subjects in Obesity Research Unit and all the metabolic 
and imaging studies planned for this study. In the future, we will apply funding for stool sample 
analyzes (microbiome studies) and for the gene expression studies of the fat biopsies. 

 
 
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1. Background and rationale of the study 
 
The prevalences of overweight (body-mass-index, BMI 25-30 kg/m2) and obesity (BMI>30 kg/m2) 
are steadily increasing among HIV-infected patients globally. In our HIV outpatient clinic in 
Helsinki, approximately half of the patients are overweight or obese (unpublished data). Obesity 
is often associated with metabolic syndrome, which manifests with dyslipidemia (with high 
triglycerides and low high-density lipoprotein cholesterol (HDL) levels), impaired fasting glucose 
(≥ 5.6 mmol/l), high blood pressure (BP≥ 130/≥ 85 mmHg) and increased visceral adipose tissue 
mass. Inflammation markers, such as hsCRP and IL6 are often increased in serum and there are 
various alterations in bowel microbiota supporting ongoing low-grade inflammation. Similar 
changes in inflammation markers and in metabolism are seen in HIV-infected patients even with 
normal body weight.  
 
As the prevalences of obesity and metabolic disorders among HIV patients gradually increase, 
the risk of non-alcoholic fatty liver disease (NAFLD) increases parallel, as shown by the ECHAM 
(European Cohort on HIV, Ageing and Metabolic liver disease) Study (1). NAFLD is characterized 
by excessive hepatic fat accumulation and defined by the presence of steatosis in >5% of 
hepatocytes according to histological analysis or by a weight fraction >5.6% assessed by proton 
magnetic resonance spectroscopy (1HMRS) or proton density fat fraction – a quantitative 
chemical shift-based water and fat separation -magnetic resonance imaging (MRI), which are the 
golden standards in diagnostics (2). The diagnosis of NAFLD requires the exclusion of secondary 
causes, such as excessive daily alcohol consumption (3). NAFLD, however, is a spectrum of 
pathologically distinct liver diseases, where simple steatosis is considered to be a non-
progressive form of NAFLD, and non-alcoholic steatohepatitis (NASH) is considered to be the 
progressive form of NAFLD. NASH manifests with inflammation and fibrosis and can lead to 
cirrhosis and hepatocellular carcinoma (HCC) (2). Clinically alarming are the data which suggest 
that HIV infected individuals have higher rates of progressive form of NAFLD than non-HIV 
infected age, gender and BMI matched controls (4). 
 
Antiretroviral therapy (ART) for HIV-infected patients may promote unfavourable changes in 
metabolism and in trunk fat redistribution. Antiretroviral drugs may increase biosynthesis and 
reduce hepatic clearance of serum cholesterol. In a systematic review regarding ART-associated 
changes in metabolism, association between ART exposure and mean serum total cholesterol 
level and triglycerides was significantly stronger with the use of protease inhibitors (PI), compared 
with the use of other ART regimens (5). Yet the clinical effect of individual agents within the PI 
class varies. Other antiretroviral medications may contribute to dyslipidemia as well. Of the 
NNRTI-group (non-nucleoside reverse transcriptase inhibitor), efavirenz has been associated with 
the worst lipid profile, causing elevated triglycerides and total cholesterol and unfavourable body 
fat redistribution (5).  
 
According to several studies, raltegravir (RAL) has been shown to have neutral metabolic effects 
in the management of HIV. In (ACTG) A5257 study, when treatment-naive adult subjects were 
randomized either to ritonavir-boosted protease inhibitor atazanavir (ATV/r) or darunavir (DRV/r), 
or RAL-based ART, RAL produced the most favorable lipid profile (6).  In SPIRAL-trial, ART-
treated, virologically suppressed HIV-infected patients switching from a PI/r containing ART to 
RAL-based ART showed a favourable impact on lipids (lowering of triglycerides, total and LDL 
cholesterol) as compared to those continuing with the PI/r based ART. (7). In the same study, 
insulin and some inflammatory biomarkers decreased in the RAL-arm (hsCRP, IL-6, TNF-α). In a 
study conducted by Nguyen et al (SWITCH-ER study), favourable changes in total cholesterol, 
triglycerides and LDL cholesterol were seen in the RAL-arm compared to the control arm 
continuing their current efavirenz-containing ART (8).  
 
Although raltegravir has been demonstrated to have beneficial impact on some metabolic 
parameters compared to the PI class or to efavirenz, very little is known whether raltegravir could 
improve hepatic steatosis in HIV-associated metabolic syndrome. Recent studies presented in 
CROI (Conference on Retroviruses and Opportunistic Infections) 2017 showed that serum 

 
 
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biomarker Chi3L1 (hepatic steatosis/fibrosis marker) decreased in HIV-infected women with 
lipohypertrophy when switching from NNRTI- or PI-based regimens to raltegravir. In the same 
study adiponectin concentration decreased, which is surprising knowing the inverse correlation 
between serum/plasma adiponectin concentration and liver fat content (9, 10). STERAL study 
presented also in CROI 2017, showed that switching from efavirenz to raltegravir decreased 
hepatic steatosis, as measured by controlled attenuation parameter (CAP) at 48 weeks, 
compared with those continuing with efavirenz in HIV/HCV co-infected patients (11).  
 
There are confounding data on the effect of raltegravir on body weight and fat tissue. In a study 
with thirty-nine obese women, a switch to raltegravir from a PI or a NNRTI did not reduce visceral 
fat as measured by computed tomography (12). In SPIRAL-LIP substudy there were no 
significant changes in total body fat between maintaining a PI-based regimen or switching to a 
raltegravir–based regimen as measured by DEXA and CT. However, maintaining a PI-based 
regimen was associated with a significant increase in visceral adipose tissue and total adipose 
tissue (13). In a retrospective analysis of the ACTG A5257 study, where ART-naïve HIV-infected 
individuals were randomized to one of 3 regimens: atazanavir/ritonavir (ATV/r), darunavir/ritonavir 
(DRV/r), or raltegravir (RAL) each in combination with tenofovir/emtricitabine, the odds of severe 
weight gain were significantly higher for RAL than for ATV/r and for DRV/r (14). 
 
In summary, raltegravir seems to have a favourable impact on distinct metabolic components 
compared to PI class or efavirenz. Raltegravir also seems to decrease hepatic steatosis 
measured by CAP in HIV-infected subjects co-infected with HCV. These intriguing results set up 
a hypothesis whether raltegravir could improve liver fat content in HIV-infected population without 
HCV, who are overweight or obese and have other metabolic syndrome components and who 
currently use a PI or efavirenz in their ART. To test this hypothesis, we plan to run a prospective, 
randomized, open, parallel design study to compare the liver fat content in study subjects either 
continuing their current ART regimens (which include a PI or efavirenz) or switching to raltegravir 
based ART.  For the quantification of liver fat content, we aim to use the proton magnetic 
resonance spectroscopy – the golden standard method -, which has not been used previously in 
PI/ efavirenz/ raltegravir-switch studies. 
 
As there are confounding data on body fat composition changes with raltegravir in studies using 
single slice CT scan and DEXA methods, the plan is to characterize the adipose tissue changes 
in more detail with MRI, using 16 slices described in more detail below in methods. To acquire 
more knowledge on metabolic effects in adipose tissue level, we also intend to take 
subcutaneous adipose tissue biopsies together with blood, saliva and feces samples. With these 
study methods we aim to characterize in more detail the metabolic effects of protease inhibitors, 
efavirenz and raltegravir in HIV-infected overweight or obese patients with the presence of at 
least one metabolic syndrome component. 
 

 
 
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2. List of study objectives  
 
Primary objective is: 
 
1. Baseline to 24-week Change in Liver Fat (measured by proton magnetic resonance 
spectroscopy, Δ %) 
 
Secondary objectives are: 
 
1. Baseline to 24-week Change in Body Composition including subcutaneous and visceral 
adipose tissue volume (measured by magnetic resonance imaging), total body fat and lean 
tissue mass (measured by Bioelectrical Impedance Analysis) 
 
2. Baseline to 24-week Change in Liver Stiffness measured by transient elastography 
(Fibroscan ®) 
 
3. Baseline to 24-week Change in Glucose Metabolism including fasting glucose, insulin, 2-h 
oral glucose tolerance test 
 
4. Baseline to 24-week Change in Lipid Profile (LDL and HDL cholesterol, Triglyceride) 
 
5. Baseline to 24-week Change in Metabolic and Inflammatory Biomarkers (e.g. high-sensitivity 
C-reactive protein (hsCRP), adiponectin, etc)  
 
6. To collect subcutaneous adipose tissue samples at baseline and 24 weeks for future gene 
expression studies 
 
7. To collect stool and saliva samples at baseline and 24 weeks for future microbiome studies 
 
8. To collect safety and tolerability data related to the switch to raltegravir 
 
9. Determination of the fatty liver adipose allele (B-PNPLA3) 
 
10. Determination of basal metabolic rate by indirect calorimetry at screening and at the end of 
study 
 
 
 
3. Study subjects 
 
 
Actual subject recruitment will start after ethics committee (EC), competent authority (CA) and 
clinic approvals have been granted. The study subjects will be recruited and screened in a single 
center (Aurora hospital, Infectious diseases clinic, HUH, Helsinki). All the eligible study subject 
candidates will be contacted during regular visits and informed of the study by study investigators 
verbally and with a written information hand out. After adequate time and opportunity to inquire 
about details of the study and to decide whether or not to participate in the study, the candidates 
will sign and personally date an informed consent. After the study has finished, all the study 
subjects will continue their regular follow up with a chance to continue their current ART or to 
switch to another regimen. 
 
The total number of study subjects included in the study is 45. Study subjects will be randomized 
1:1 to continue current ART regimens or to switch their PI or efavirenz to once daily raltegravir 

 
 
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plus continue current NRTIs. The randomization will be stratified by age, sex and antiretroviral 
regimens (PI or efavirenz). 
 
The inclusion criteria are: 
 
• 
Written informed consent (IC) obtained. 
 
• 
HIV-positive adult (age over 18) subjects currently on stable ART, with no changes in the 
ART regimens during the past 6 months. 
 
• 
Current ART includes either a protease inhibitor or efavirenz. 
 
• 
No documented or suspected resistance to integrase inhibitors or to NRTIs. 
 
• 
No prior history of virologic failure. Failure is defined as a confirmed plasma viral load > 200 
cop/ml measured no less than six months after initiation or modification of therapy.  
 
• 
Virological blips accepted only if a single viral load measurement has been between 50-200 
cop/ml followed by VL < 50 cop/ml without the need to initiate a change in ART and no blip 
within 12 month window period prior to screening. 
 
• 
Documented evidence of at least two HIV VL < 50 cop/ml measurements during the past 12 
months prior to inclusion: one within 6 months prior to screening.  
 
• 
HIV VL < 50 cop/ml at screening. 
 
 
• 
BMI>25 kg/m2 and one metabolic syndrome condition, which are 
o 
 BP ≥ 130/≥ 85 mmHg or hypertension medication currently in use or 
o 
fasting glucose ≥ 5.6 mmol/l or B-HbA1C > 42 mmol/mol or diabetes medication 
currently in use or 
o 
HDL < 1.0 mmol/l in men and < 1.3 mmol/l in women or triglycerides ≥ 1.7 mmol/l 
or a cholesterol-lowering regimen currently in use or 
o 
waist circumference > 94 cm in men and >80 cm in women (or respective cut off 
values for non-European ethnic groups as defined by International Diabetes 
Federation). 
 
OR 
 
• 
ultrasound or biopsy proven hepatosteatosis. 
 
 
Exclusion criteria are: 
 
• 
Within 12 month window period prior to screening, HIV VL measurement of >50 cop/ml. 
 
• 
More than one consecutive HIV VL measurements of > 50 cop/ml in the treatment history 
after initial viral suppression with ART. 
 
• 
Chronic hepatitis B or C. 
 
• 
Daily alcohol consumption ≥ 30 g for men and ≥ 20 g for women. 
 
 
• 
Pregnancy or planned pregnancy during the study period.  
 

 
 
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• 
Lipid or glucose lowering regimen or hormonal supplement started within 3 months 
before the planned study start. 
 
• 
Psychiatric disorder, which prevents a study subject to understand the study protocol.  
 
• 
Other serious disease, which prevents a study subject to participate in the study.  
 
• 
For MRI/spectroscopy imaging: metal objects in the body or claustrophobia. 
 
 
 
4. Study procedures and assessments 
 
This study is an investigator initiated, single-center, prospective, randomized, open-label, parallel 
design study.  
 
Study flow chart consists of 
 
1. Screening visit, signing of the informed consent and randomization 
2. Baseline study visit in the Obesity Research Unit with study measurements detailed 
below in section “Study procedures in Obesity Research Unit”. 
3. Baseline visit in Meilahti hospital, where MR imaging and spectroscopy, and transient 
elastography will be done.  
4. Visit at the Infectious Diseases Clinic, where the current ART will be continued or 
modified according to the randomization.  Patient follow up will take place according to 
the standard of care with two safety laboratory tests at weeks 2 and 8. 
5. 24 week study visit in the Obesity Research Unit with the same study measurements as 
detailed at the baseline (see section below). 
6. 24 week study visit in Meilahti hospital, where MR imaging and spectroscopy, and 
transient elastography will be repeated. 
7. End of study visit (24 weeks after study start) at the Infectious Diseases Clinic, where 
current ART can be continued or modified according to the standard of care. 
 
4.1. Screening visit 
 
A screening visit will be performed prior to first study visit in Obesity Research Unit.  The 
screening visit consists of the following procedures: 
• 
A physical examination will be performed and demographic data will be recorded 
including subject’s gender, date of birth, ethnicity, substance use, recent and current 
alcohol use (AUDIT questionnaire), smoking status, weight, height, BMI, and waist and 
hip circumferences.  
• 
Medical history and current medical conditions and concomitant treatments will be 
inquired and recorded. 
• 
Blood pressure and heart rate will be measured.  
The subject may enter into the study if all the inclusion criteria and none of the exclusion criteria 
are met and will be randomized to one of the treatment arms after signing the informed consent 
(IC). Recording of adverse events (AEs) starts from the time that a study subject signs the IC 
form.  The study subjects will be given a study diary, where they can fill in AEs and possible 
concomitant treatments during the study. They will be asked to fill a dietary and exercise diary for 
three days at the study start and at the end of study. 

 
 
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4.2. Clinical follow up visits in Infectious Diseases Clinic (Aurora hospital) 
 
Table 1 lists all study procedures in Aurora hospital, Helsinki.  
 
Clinical follow up 
Lab measurements 
Other 
first visit, day 1 
HIV viral load, complete blood 
count, Creatinine, ALT, AST, 
CD4 count 
According to the 
randomization, switching of a 
PI or efavirenz to raltegravir, 
or continuing with the previous 
ART. 
 
remote contact by phone 1, 
week 2 
compete blood count, CRP, 
ALT, Glucose, Creatinine  
AEs inquired and reported 
remote contact by phone 2, 
week 8 
complete blood count, ALT, 
AST, Creatinine, HIV viral load 
AEs inquired and reported 
last visit, week 24 
HIV viral load, complete blood 
count, Creatinine, ALT, AST, 
CD4 count 
AEs inquired and reported 
 
 
4.3. Study procedures in the Obesity Research Unit and in the Radiology Department 
 
The study protocol includes two visits in the Obesity Research Unit. The first visit will take place 
after randomization and before the switch of the ART. The second visit will take place after 24 
weeks of follow up. 
 
Study subjects are advised to fast 10-12 hours before each visit in the Obesity Research Unit 
(water is allowed during fasting). Alcohol intake is prohibited from 48 h before each study visit. 
Smoking is prohibited during the visits. Apart from these restrictions the subjects are advised to 
keep to their usual exercise and lifestyle habits for the duration of the study.  
 
The visits consist of the following study measurements: Oral glucose tolerance test (OGTT), 
measurements of liver fat by 1H-spectroscopy, body composition by MRI, hepatic stiffness by 
transient elastography and calorimetry for measurement of basal metabolic rate. Samples of 
blood, urine, stool and saliva are collected, ECG and subcutaneous adipose tissue biopsies 
taken, and health questionnaire filled in. 
 
Body composition studies 
 
Body composition (total lean and fat mass) will be analyzed by a non-invasive BioElectrical 
Impedance Analyzer System.  
 
Abdominal subcutaneous and visceral fat (MRI) 
 
A series of T1-weighted trans-axial images for the determination of visceral and subcutaneous 
fat will be acquired from a region extending from 8 cm above to 8 cm below the 4th and 5th 
lumbar intervertebral discs (16 slices, field of view 375 x 500 mm2, slice thickness 10 mm, 
breath-hold TR/TE 91  ms/5.24  ms). Water signal is suppressed using a frequency selective 
fat excitation. Visceral and subcutaneous fat volumes will be measured using an image 
analysis program (SliceOmatic, http://www.tomovision.com/products/sliceomatic.html).  
 
Intrahepatic fat content (1H Magnetic resonance spectroscopy, MRS) 
 
Single voxel (25x25x25 mm3) proton spectra from the liver will be acquired using 16  
exitations, a  body matrix surface coil and a 1.5-tesla magnetic resonance device (Avantofit; 

 
 
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Siemens, Erlangen, Germany). A Point resolved spectroscopy (PRESS) single voxel 
technique will be applied, with an echo time of 30 ms. Respiratory motion will be triggered 
using a navigator belt so that repetition time will be kept >  3000 ms. Intensities of water signal 
(Swater) ppm  and methylene signal (Sfat) representing intracellular triglyceride in the liver, 
resonating at 4.8 and 1.4 ppm, respectively,  will be determined. Effects of T2 relaxation will 
be corrected for water and fat intensities and hepatic fat percentage will be calculated by 
dividing Sfat by the sum of Sfat and Swater. Signal ratios will be further converted to mass 
fractions as previously described (15). This measurement of percent hepatic fat by proton 
spectroscopy has been validated against the lipid content of liver biopsies in humans (16).  
MRI and MRS methods involve no ionizing radiation exposure. 
 
Transient elastography (Fibroscan®) 
 
Transient elastography examinations will be carried out by a single gastroenterologist (PA) 
with Fibroscan® (Echosens, France). The examinations will be performed through one 
intercostal space that provides an optimal unobstructed view to the right lobe of the liver. The 
results will be based on a median of ten validated measurements. Transient elastography 
results with an interquartile range (IQR) below 30% of the median value and a success rate of 
at least 60% will be accepted, according to manufacturer’s recommendations.  
 
Indirect calorimetry 
 
Indirect calorimetry (Datex Metabolic Monitor, Datex, Helsinki, Finland) will be used to 
estimate basal metabolic rate from measurements of oxygen (O2) consumption and carbon 
dioxide (CO2) production. Patient is lying supine in bed, breathing calmly and regularly in the 
canopy with a constant air flow (to be adjusted to give O2 and CO2 concentrations within 
workable range). O2 (VO2) consumption, CO2 (VCO2) production, a urinary nitrogen 
excretion during the study will be measured and on the assumption that all the oxygen is used 
to oxidize degradable fuels and all the CO2 thereby evolved is recovered, it is possible to 
estimate the amounts of glucose and lipids oxidized by the body and calculate the basal 
energy expenditure.  
 
Fat biopsy 
 
A subcutaneous fat biopsy (appr. 2-3 g) will be taken under local anesthesia from abdominal 
area. Biopsy will be taken using mini-liposuction technique. Part of the sample will be 
immediately frozen and stored in liquid nitrogen or -80C until used for transcriptional, protein 
or other biochemical analyses. Another part will be used for measurement of adipocyte size 
under a light microscope.  
Fasting blood samples 
 
Routine laboratory tests, including HbA1c, total, LDL and HDL cholesterol, and triglycerides, 
thyroid function (TSH, T4v) and liver enzymes (AST, ALT, GGT, AFOS) will be collected and 
analyzed in HUSLAB. B-PNPLA3 sample will be collected and send to Yhtyneet Medix 
laboratory where the sample will be analyzed for the fatty liver adipose allele. In addition, extra 
samples of plasma and serum will be stored for further analysis, e.g. for metabolomics, 
lipidomics, proteomics, hormones, adipokines. 
 
Oral Glucose Tolerance Test (OGTT)  
 
OGTT (oral glucose tolerance test) will be performed for all patients after an overnight (10-12 
h) fast. A fasting blood sample will be collected after which patients will take a 75 g oral 
glucose dose in about 5 minutes. Zero time is the beginning of the drink and blood samples 
are collected at time points 0, 30, 60 and 120 minutes for measurement of plasma glucose, 
insulin and c-peptide. Extra samples will be collected at each time point for the above 
mentioned purposes. 
 

 
 
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Saliva 
 
Saliva will be collected after stimulation by chewing parafilm. Saliva samples will be stored for 
later purposes and can be used e.g. for microbiota, cortisol, and metabolomics analyses. 
 
Feces 
 
Feces will be collected either at home or at the research center. Collection specimen and 
instructions will be given during the first visit. Feces samples can be used later to analyze the 
fecal metabolites, including fatty acid composition and the microbiota. 
 
Urine 
 
Urine samples will be collected on a study morning to perform a pregnancy test for females at 
childbearing age. Additionally urine samples can be stored for later analyzes, e.g. 
metabolomics. 
 
 
 
5. Safety assessments and ethical considerations 
 
5.1. Study sites and study procedures 
 
The investigator is responsible for ensuring adequate medical expertise and facilities to 
conduct the study and to handle possible emergency situations during the study. All 
procedures and possible hazards, risks and discomforts are explained to the subjects both 
verbally and in writing (see informed consent). 
 
For the blood sampling, a forearm vein will be cannulated.  In this study, the maximum volume 
of blood drawn per subject during the visits in Obesity Research Unit will be approximately 
300 ml. 
 
There is no ionizing radiation in MRI/MRS/ transient elastography studies.  
 
The subcutaneous abdominal fat biopsies will be taken under local anesthesia with sterile 
conditions. The needle used for local anesthesia can cause slight discomfort but the biopsies 
themselves are painless. A fat biopsy will be taken using a mini-liposuction technique. The 
incision is closed with skin tape and covered by pressure bandages. The subjects will be 
adviced to avoid strenuous exercise for two days after the procedure. The biopsy site might 
have a dull ache for 24-48 h for which the subjects are provided pain-killers. However, 
medications such as aspirin, or ibuprofen are not recommended since they may contribute to 
bleeding and/or bruising during the post-biopsy period.  
 
Study subjects may experience moderate discomfort from the blood samples and biopsies. 
 
The subjects will have to reserve two days for the study visits in Obesity Research Unit 
besides the normal follow up in Aurora hospital. Travel costs incurred as a result of a study 
visit will be reimbursed on the basis of supporting documents according to actual costs.  
 
Study personnel will conduct all procedures according to GCP (good clinical practice) 
guidelines. In the case of a needle stick injury, personnel is advised to contact immediately the 
study investigator or the infectious diseases specialist on duty in the Infectious Diseases Clinic 
for the evaluation of the need for HIV post exposure prophylaxis.   

 
 
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5.2 Raltegravir 
 
Raltegravir (trade name Isentress®) is an HIV medicine that is used in combination with other 
HIV medicines to treat adults and children who are infected with human immunodeficiency 
virus (HIV-1). It is developed and manufactured by Merck Sharp & Dohme Corp (MSD). The 
European Commission granted a conditional marketing authorisation valid throughout the 
European Union for Isentress on 20th December 2007. This was switched to a full marketing 
authorisation on 14th July 2009. A new formulation of Isentress 600mg coated tablets was 
granted a marketing authorisation by European Comission on 18th July 2017. The 
recommended daily dose for the new formulation is 1200 mg (two 600 mg tablets) once daily. 
In this study, we plan to use the once daily formulation of raltegravir. This study drug is 
provided by Merck Sharp & Dohme Corp. 
 
Raltegravir is one of the best tolerated HIV-medicines. The most common reported adverse 
effects with Isentress (seen in between 1 and 10 patients in 100) are abnormal dreams, 
nightmares, insomnia, depression, abnormal behavior, dizziness, headache, psychomotor 
hyperactivity, vertigo, decreased appetite, abdominal distension, abdominal pain, diarrhea, 
flatulence, nausea, vomiting, dyspepsia, rash, asthenia, fatigue, pyrexia, atypical lymphocytes, 
and increased blood levels of some enzymes (alanine aminotransferase, aspartate 
aminotransferase, lipase and pancreatic amylase) and triglycerides. For more details of the 
study drug, see SmPC of Isentress attached. 
 
 
5.3. Adverse events assessment 
 
An AE is any untoward medical occurrence in a study subject administered a pharmaceutical 
product and which does not necessarily have a causal relationship with this treatment. An AE 
can therefore be any unfavourable and unintended sign (including an abnormal laboratory 
finding), symptom, or disease temporally associated with the use of an investigational 
medicinal product (IMP), whether or not related to the IMP. The definition also covers 
medication errors and uses outside what is foreseen in the protocol, including misuse and 
abuse of the product. 
 
Thus, an AE may be an appearance or worsening of any undesirable sign or symptom, any 
worsening of the current medical conditions or onset of a new disease, compared with the 
previous observations or a clinically significant adverse change in a laboratory variable or 
other diagnostic finding. 
 
A serious adverse event (SAE) is any untoward medical occurence that  
• 
results in death 
• 
is life-threatening 
• 
requires inpatient hospitalization or prolongation of existing hospitalization 
• 
results in persistent or significant disability/incapacity 
• 
is a congenital anomaly/ birth defect, or 
• 
is an important medical event jeopardizing the patient or requiring intervention to 
prevent serious outcome (examples of such are intensive treatment in an emergency 
room, convulsions that do not result in hospitalization, etc.) 
 
Any case of pregnancy during a clinical study will be reported by the investigator in the same way 
as an SAE.  
 
Other significant AEs are AEs (other than those meeting the definition of serious) that are of 
clinical importance and lead to: 
• a diagnostic or therapeutic intervention 
• discontinuation of the IMP 

 
 
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• significant additional concomitant treatment, or 
• marked hematological and other laboratory abnormalities 
 
Suspected Unexpected Serious Adverse Reaction or “SUSAR” shall mean any Serious 
Adverse Event, the nature, severity or frequency of which is not consistent with information in the 
most current Summary of Product Characteristics (SmPC) or Package Insert. 
 
All AEs must be elicited, documented and reported by the investigator. The investigator will 
assess and record the causality and severity of the AEs due to IMP (see criteria for causality and 
severity below). SAEs and other significant AEs should be followed up until resolved or until the 
event is considered a chronic or stable outcome, or both.   
 
The investigator is responsible for expediting all suspected unexpected serious adverse reactions 
(SUSARS) as well as other safety issues requiring expedited reporting to the relevant authorities 
within applicable timelines. 
 
Principal Investigator shall forward to MSD’s Global Safety (“MSD GS”) group, any SAE and 
SUSAR or Incident information, including, but not limited to, all initial and follow-up information 
involving any study subject in the study.  Notification shall be in the form of a completed CIOMS 
I/MedWatch (or other mutually agreed upon format) within two (2) business days of learning of 
the information.   
 
Causality criteria: 
 
Related to the IMP: The temporal relationship of the AE/SAE onset to the administration of the 
IMP makes a causal relationship possible, and other drugs, therapeutic intervention or underlying 
conditions do not provide a sufficient explanation for the AE/SAE. 
 
Not related to the IMP: The temporal relationship of the AE/SAE onset to the administration of the 
IMP makes a causal relationship unlikely, or other drugs, therapeutic interventions or underlying 
conditions provide a sufficient explanation for the AE/SAE. 
 
Severity criteria: 
 
Mild: Discomfort noticed, but it does not affect normal activity. 
 
Moderate: Discomfort sufficient to reduce or affect normal daily activity. 
 
Severe: Incapacitating with inability to perform normal daily activity. 
 
Clinical safety assessments are detailed in section “4.2. Clinical follow up visits in Aurora 
hospital.”  Additionally pregnancy test for females of child-bearing potential is done during the first 
visit in Obesity Research Unit. 
 
Possible unexpected adverse events due to study regimens are covered by the Pharmaceutical 
Injury Insurance (Lääkevahinkovakuutus). 
 
All subjects are insured by the patient insurance of Helsinki University Hospital. 

 
 
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6. Data analysis 
 
The data will be analyzed by the investigators and the study group. 
 
All variables of the data will be analyzed after the last subject’s last visit, when the data have 
been declared complete. 
The data will be analyzed using IBM SPSS Statistics v. 22. The differences between the groups 
will be analyzed by using chi-square, Fisher’s exact, Mann-Whitney U test and others, as 
appropriate. 
As there are no earlier ART-switch studies using 1H-Magnetic resonance spectroscopy for the 
quantification of liver fat, the calculation of the study power is an estimate. In the STERAL study 
evaluating the effect of switching from efavirenz (EFV) to RAL vs. continuing EFV-based ART, a 
statistically significant difference in the liver fat content using CAP (controlled attenuated 
parameter) was shown with a study sample of 19 +18 subjects after 48 weeks (7,3% decrease in 
the RAL vs. 11,0% increase in the EFV arm, p = 0,019) (11). 
The power of this study is calculated with the assumption of liver fat being 4.4-5.0% in this study 
group at baseline. This estimation is based on our previous results published in 2002, when the 
median liver fat was 8% with lipodystrophic patients and 3% with non-lipodystrophic patients (17). 
Hadigan et al reported liver fat content being >5% in 42% of HIV positive subjects (18). As the  
liver fat content correlates with visceral fat mass, studying overweight or obese patients with at 
least one metabolic syndrome component includes patients at high risk for elevated liver fat 
content.  
In the power calculation, we have used a coefficient of variation (CV) of 1H-Magnetic resonance 
spectroscopy being 11% (17). To detect a 9% difference between study arms with 80% power, a 
minimum sample size of 25 + 25 subjects with a 10% drop-out rate is needed. 
 
Publication plan 
 
We anticipate to submit one main manuscript with the results of the primary objective (liver fat 
change) and secondary objectives (1-3: body composition change as well as metabolic markers). 
Additional manuscripts will be submitted after further analysis of subcutaneous fat samples (for 
transcriptional, protein and other biochemical analyses), fecal samples (microbiota) and blood 
samples (inflammatory markers and metabolomics studies).   
 
 
7. Study budget 
 
Study budget is detailed in a separate sheet “Study Budget” attached.  

 
 
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8. References 
 
1. Lemoine M, Assoumou L, Ingiliz P, et al. Liver Steatosis and Fibrosis in at-risk European HIV-
monoinfected Patients. Conference on Retroviruses and Oppotunistic Infections, February 13-16, 
2017, Seattle, USA 2017; Abstract 703. 
2. European Association for the Study of the Liver (EASL), European Association for the Study of 
Diabetes (EASD) and European Association for the Study of Obesity (EASO): EASL–EASD–
EASO Clinical Practice Guidelines for the management of non-alcoholic fatty liver disease. J 
Hepatol 2016; 64:1388–1402. 
3. Ratziu V, Bellentani S, Cortez-Pinto H, Day C, Marchesini G. A position statement on 
NAFLD/NASH based on the EASL 2009 special conference. J Hepatol 2010;53:372–384. 
4. Vodkin I, Valasek MA, Bettencourt R, et al. Clinical, biochemical and histological differences 
between HIV associated NAFLD and primary NAFLD: a case–control study. Aliment Pharmacol 
Ther 2015; 41: 368–378.  
5. Non LR, Escota GV, Powderly WG. HIV and its relationship to insulin resistance and lipid 
abnormalities. Translational Research 2017; 183: 41-56. 
6. Ofotokun I,  Na LH, Landovitz RJ, et al. Comparison of the metabolic effects of ritonavir-
boosted darunavir or atazanavir versus raltegravir, and the impact of ritonavir plasma exposure: 
ACTG 5257. Clin Infect Dis 2015;60:1842-51. 
7. Martínez E, D'Albuquerque PM, Llibre JM, et al. Changes in cardiovascular biomarkers in HIV-
infected patients switching from ritonavir-boosted protease inhibitors to raltegravir. AIDS 
2012 ;26:2315-26. 
8. Nguyen A, Calmy A, Delhumeau C, et al. A randomized cross-over study to compare 
raltegravir and efavirenz (SWITCH-ER study). AIDS 2011;25:1481-7. 
9. Renaldi O, Pramono B, Sinorita H, et al. Hypoadiponectinemia: a risk factor for metabolic 
syndrome. Acta Med Indones 2009; 41: 20–4. 
10. Hara K, Yamauchi T, Kadowaki T. Adiponectin: an adipokine linking adipocytes and type 2 
diabetes in humans. Curr Diab Rep 2005; 5 : 136–40 
11. Macías J, Merino D,Téllez F, et al. Changes in liver steatosis after switching efavirenz to 
raltegravir: the STERAL study. Conference on Retroviruses and Oppotunistic Infections,  
February 13-16, 2017, Seattle, USA 2017; Abstract 697. 
12. Lake JE, McComsey GA, Hulgan T, et al. Switch to Raltegravir From Protease Inhibitor or 
Nonnucleoside Reverse-Transcriptase Inhibitor Does not Reduce Visceral Fat In Human 
Immunodeficiency Virus-Infected Women With Central Adiposity. Open Forum Infect Dis; 2015 
Apr; 2(2)  
13. Curran A, Martinez E, Saumoy M, et al. Body composition changes after switching from 
protease inhibitors to raltegravir: SPIRAL-LIP substudy.AIDS 2012;26:475-81 
14. Bhagwat P, Ofotokun I, McComsey GA, et al. Predictors of severe weight/body mass index 
gain following antiretroviral initiation. Conference on Retroviruses and Oppotunistic Infections,  
February 13-16, 2017, Seattle, USA 2017; Abstract 695. 
15. Kotronen A, Peltonen M, Hakkarainen A, et al. Prediction of non-alcoholic fatty liver disease 
and liver fat using metabolic and genetic factors. Gastroenterology 2009, 137(3):865-72. 
16. Ryysy L, Häkkinen AM, Goto T, et al. Hepatic fat content and insulin action on free fatty acids 
and glucose metabolism rather than insulin absorption are associated with insulin requirements 
during insulin therapy in type 2 diabetic patients. Diabetes 2000; 49: 749-758. 
17. Sutinen J, Häkkinen AM, Westerbacka J, et al. Increased fat accumulation in the liver in HIV-
infected patients with antiretroviral therapy-associated lipodystrophy. AIDS 2002;16:2183-93. 
18. Hadigan C, Liebau J, Andersen R, et al. Magnetic resonance spectroscopy of hepatic lipid 
content and associated risk factors in HIV infection. J Acquir Immune Defic Syndr 2007;46:312-7. 
  

 
 
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9. List of attachments: 
 
1. Synopsis 
2. CVs of Principal Investigator and Head of the Obesity Research Unit 
3. SmPC of Isentress   
4. Study budget 
5. Patient registry report 
6. Summary of the current state of the report to the national competent authority (Fimea)