bmjopen.bmj.com · For peer review only Supplementary material - COCKTAIL-study 2 Jan 2, 2018 Supplementary table 1 (cont.) Rationale and evidence gap to fill . B. Metabolism, cardiometabolic

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For peer review only

Impact of body weight, low energy diet and gastric bypass on drug bioavailability, cardiovascular risk factors and

metabolic biomarkers. The COCKTAIL study – an open, non-randomised, three-

armed single centre study

Journal: BMJ Open

Manuscript ID bmjopen-2018-021878

Article Type: Protocol

Date Submitted by the Author: 24-Jan-2018

Complete List of Authors: Hjelmesæth, Jøran; Vestfold Hospital Trust, The Morbid Obesity Center; University of Oslo, Department of Endocrinology, Morbid Obesity and Preventive Medicine, Institute of Clinical Medicine Åsberg, Anders ; University of Oslo , Department of Pharmaceutical Biosciences, School of Pharmacy; Oslo University Hospital, Rikshospitalet , Department of Transplantation Medicine Andersson, Shalini; AstraZeneca Gothenburg , Drug Metabolism and Pharmacokinetics, Cardiovascular and Metabolic Diseases, IMED Biotech Unit Sandbu, Rune; Vestfold Hospital Trust, The Morbid Obesity Center Robertsen, Ida; University of Oslo , Department of Pharmaceutical Biosciences, School of Pharmacy Johnson, Line Kristin; Vestfold Hospital Trust, The Morbid Obesity Center Angeles, Philip Carlo; Vestfold Hospital Trust, The Morbid Obesity Center Hertel, Jens Kristoffer; Vestfold Hospital Trust, The Morbid Obesity Center Skovlund, E; Norwegian University of Science and Technology, NTNU, Department of Public Health and Nursing Heijer, Maria; AstraZeneca Gothenburg , Study Operations, Early Clinical Development, IMED Biotech Unit Ek, Anna-Lena; AstraZeneca Gothenburg , Study Operations, Early Clinical Development, IMED Biotech Unit Krogstad, Veronica; University of Oslo , Department of Pharmaceutical Biosciences, School of Pharmacy Karlsen, Tor-Ivar; Vestfold Hospital Trust, The Morbid Obesity Center; Universitetet i Agder, Faculty of Health and Sports Science Christensen, Hege; University of Oslo , Department of Pharmaceutical Biosciences, School of Pharmacy Andersson, Tommy B; AstraZeneca Gothenburg , Drug Metabolism and Pharmacokinetics, Cardiovascular and Metabolic Diseases, IMED Biotech Unit; Karolinska institutet , Department of Physiology and Pharmacology, Section of Pharmacogenetics Karlsson, Cecilia; AstraZeneca Gothenburg , CVMD Translational Medicine Unit, Early Clinical Development, IMED Biotech Unit ; AstraZeneca Gothenburg , CVMD GMed, Global Medicines Development

Keywords: BASIC SCIENCES, CLINICAL PHARMACOLOGY, DIABETES & ENDOCRINOLOGY, Cardiology < INTERNAL MEDICINE, NUTRITION & DIETETICS

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COCKTAIL protocol manuscript 1 Jan 24, 2018

Impact of body weight, low energy diet and gastric bypass on drug

bioavailability, cardiovascular risk factors and metabolic biomarkers.

The COCKTAIL study – an open, non-randomised, three-armed single centre study Authors

Hjelmesæth J*1, 2, Åsberg A*3,4, Andersson S5, Sandbu R1, Robertsen I3, Johnson LK1, Angeles PC1, Hertel JK1, Skovlund E6, Heijer M7, Ek A-L7, Krogstad V3, Karlsen TI1,8, Christensen H3, Andersson TB5,9, Karlsson C10, 11 *Shared 1st authorship

Author affiliations

1. Morbid Obesity Centre, Vestfold Hospital Trust, Tønsberg, Norway 2. Department of Endocrinology, Morbid Obesity and Preventive Medicine, Institute of Clinical

Medicine, University of Oslo, Norway 3. Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Norway 4. Department of Transplantation Medicine, Oslo University Hospital - Rikshospitalet, Oslo,

Norway. 5. Drug Metabolism and Pharmacokinetics, Cardiovascular and Metabolic Diseases, IMED

Biotech Unit, AstraZeneca Gothenburg, Sweden 6. Department of Public Health and Nursing, Norwegian University of Science and Technology,

NTNU, Trondheim, Norway 7. Study Operations, Early Clinical Development, IMED Biotech Unit, AstraZeneca Gothenburg,

Sweden. 8. Faculty of Health and Sports Science, University of Agder, Norway 9. Department of Physiology and Pharmacology, Section of Pharmacogenetics, Karolinska

Institutet, Stockholm, Sweden 10. CVMD Translational Medicine Unit, Early Clinical Development, IMED Biotech Unit,

AstraZeneca Gothenburg, Sweden 11. Current position: CVMD GMed, Global Medicines Development, AstraZeneca Gothenburg,

Sweden

Word count Manuscript: 4803 Abstract: 247 January 24, 2018

Corresponding author: Jøran Hjelmesæth (MD, PhD) Professor, Head Morbid Obesity Centre and Section of Endocrinology, Department of Medicine Vestfold Hospital Trust Boks 2168, 3103 Tønsberg, Norway Telephone +47 33 34 32 48 / Mobile +47 40 21 73 49 / Fax +47 33 34 39 91 Email: [email protected]

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Abstract

Introduction

Roux-en-Y gastric bypass (GBP) is associated with changes in cardiometabolic risk factors and

bioavailability of drugs, but whether these changes are induced by calorie restriction, the weight loss

or surgery per se, remains uncertain. The COCKTAIL study was designed to disentangle the short-

term (6-week) metabolic and pharmacokinetic effects of GBP and a very low energy diet (VLED) by

inducing a similar weight loss in the two groups.

Methods and analysis

This open, non-randomized, three-armed single centre study is performed at a tertiary care centre in

Norway. It aims to compare the short-term (6-week) and long-term (2-year) effects of GBP and VLED

on, first, bioavailability and pharmacokinetics (24-hour) of probe drugs and biomarkers, and, second,

their effects on metabolism, cardiometabolic risk factors and biomarkers. The primary outcomes will

be measured as changes in, (1), absolute bioavailability (AUCoral/AUCiv) of midazolam (CYP3A4 probe)

and endogenous CYP3A biomarkers , or drug: metabolite ratio, as appropriate, for the other probe

drugs, and genotypic variation, changes in the expression, and activity data of the drug-metabolizing,

-transport and -regulatory proteins in biopsies from various organs, and, (2), body composition,

cardiometabolic risk factors and metabolic biomarkers.

Ethics and dissemination

The COCKTAIL-protocol was reviewed and approved by the Regional Committee for Medical and

Health Research Ethics (Ref: 2013/2379/REK sørøst A). The results will be disseminated to academic

and health professional audiences and the public via presentations at conferences, publications in

peer-reviewed journals and press releases, and provided to all participants.

Trial registration number (www.ClinicalTrials.gov) NCT02386917.

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Strengths and limitations of this study

1. The main strength of the present study is its potential to disentangle the short-term (6-week)

metabolic and pharmacokinetic effects of bariatric surgery per se and calorie restriction (very

low energy diet) by inducing a similar weight loss in the two groups.

2. Paired tissue biopsies from the GI-tract and the liver for “omics” investigations in

combination with in vivo drug disposition activity measures from a cocktail of probes will be

provided.

3. This study establishes a high-quality tissue biobank for global “omics” or targeted

biochemical analyses.

4. The explorative study design limits the clinical generalisability of the results.

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INTRODUCTION

Obesity represents a global epidemic [1] associated with premature mortality and increased risk for

type 2 diabetes, cardiovascular disease and cancer.[2 3] Weight loss is the primary treatment of

obesity and its comorbidities, and even a small weight reduction has beneficial effects on several

cardiometabolic risk factors.[4] Weight loss can be achieved by calorie restriction, exercise,

pharmacotherapy or bariatric surgery.[5] Interestingly, the literature indicates that Roux-en-Y gastric

bypass (GBP) may have separate, weight loss independent, beneficial effects on glucose metabolism

and type 2 diabetes, e.g. improvements in insulin sensitivity.[6-8] The mechanisms behind the acute

metabolic improvements seen after bariatric surgery remain, however, less clear.[9] It is also difficult

to distinguish the relative contributions of calorie restriction and weight loss from the surgical

procedure. The limited human data available has focused on changes in glucose homeostasis, with

few studies assessing involved pathways, of most detailed mechanistic studies conducted are done in

rodents.[8 10-13]

It is important to individualize drug doses in order to both obtain an adequate effect and

minimize side effects.[14] For most drugs, dose individualization is performed by dosing per kg total

body weight, although this may not necessarily be the best approach.[15] In patients with morbid

obesity, systemic clearance of a cytochrome P450 3A (CYP3A) substrate was found to be similar,

while oral bioavailability and volume of distribution were higher compared to patients with normal

weight.[16] However, other studies indicated that drug clearance might be deviant in patients with

severe obesity.[17-19] A recent study demonstrated a significant inverse correlation between body

mass index (BMI) and protein expression of CYP3A and oral clearance of another CYP3A4

substrate.[20] Hence, subjects with severe obesity might be at risk of drug over-exposure.[21] The

mechanisms behind the altered expression and activity of this CYP enzyme are unknown, but it has

been hypothesised that changed inflammatory state [22-24] as well as hepatic dysfunction may be

involved.[25]

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Several, but not all, bariatric surgery techniques reduce the absorptive surface area by bypassing

parts of the intestine.[26] Accordingly, bariatric surgery may affect the bioavailability of a range of

drugs.[27-29] Previous studies have shown that the bioavailability of atorvastatin is increased early

(months) after surgical reduction of the intestinal surface area.[30 31] It might be speculated that the

net effect of bypassing the metabolic most active parts of the intestine results in this, at a first

glance, contradictory effect. Interestingly, an adaptive process in the intestine seems to normalize

the bioavailability over a longer time span.[32] Even though the literature is sparse and based on

small studies, reduced uptake early after intestinal bypass has been shown for drugs substrates of

other intestinal enzymes.[33 34]

Oral bioavailability of drugs is restricted by a variety of transporters and metabolizing

enzymes in both the enterocytes and hepatocytes. Genetic, environmental and disease-related

factors induce variations in expression and activity of these proteins. Genotypic variations can easily

be assessed from a single blood sample, but in order to investigate the phenotypic variation, specific

probe drugs have to be used in vivo. Several approaches using a cocktail of probe drugs (targeting

different CYP enzymes and drug transporters) have been described.[35]

The present study was designed to disentangle the short-term (6-week) metabolic and

pharmacokinetic effects of GBP and a very low energy diet (VLED) by inducing a similar weight loss in

the two groups. It will include repeated cocktail investigations of a set of key drug metabolizing

enzymes and transporters restricting bioavailability for many drugs. The design allows revealing

pharmacokinetic (PK) changes as well as mechanisms of metabolic changes specifically induced by

GBP and calorie restriction per se. Combining the determination of in vivo CYP enzyme and

transporter activities with protein expression and ex vivo CYP activity of the same proteins is a

powerful tool for further elucidation of the mechanisms of body weight change, GBP, and calorie

restriction per se on the disposition of drugs, and it allows for improved in vitro-in vivo extrapolations

in the future.[20 35 36] A more detailed description of the study rationale and evidence gap to fill is

shown in the online supplementary table 1.[9 10 20 31 32 37-49]

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Study objectives

The primary objectives of this study are related to I) drug bioavailability and disposition, and II)

metabolism, cardiometabolic risk factors and biomarkers.

I. Drug bioavailability and disposition

a. The study aims to investigate the relationship between body composition and the

liver/intestine expression and activity of proteins (drug metabolizing enzymes,

transporters and regulatory factors) important for drug bioavailability and

disposition in the range from normal body weight to morbid obesity cross sectional

in 3 study groups: patients undergoing cholecystectomy, GBP, and VLED.

b. The study aims to compare the short-term (6-week) and long-term (2-year) effects of

GBP and a VLED, with a similar 6-week weight loss, on bioavailability and

pharmacokinetics of probe drugs and biomarkers (and adjoining protein expressions)

for CYP1A2, CYP2C9, CYP2C19, CYP3A, P-glycoprotein (P-gp) and organic anion

transporting polypeptide 1B1 (OATP1B1).

II. Metabolism, cardiometabolic risk factors and biomarkers

a. The study aims to compare the 3 study groups (GBP, VLED and cholecystectomy) at

baseline with respect to body composition, cardiometabolic risk factors and

metabolic biomarkers.

b. The study aims to compare the short-term (6-week) changes in glucose metabolism,

blood pressure, blood lipids and body composition of similar weight loss (GBP vs.

VLED), and long-term effects (2 year) of GBP and VLED on body composition,


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The secondary objectives are:

a) to compare the short- and long-term (6-week and 2-year) effects of GBP and VLED on

physical activity, energy expenditure, health related quality of life, anxiety/depression, eating

behaviour, and obstructive sleep apnoea.

b) to assess the relation between proteins and nucleotides at all investigated sites (biopsies,

blood).

c) to perform an in-depth analysis of the correlation between CYP protein expression in vivo

and ex vivo CYP activity in jejunum and liver biopsies sampled from the same site in each of

the GBP patients, as well as liver biopsies from the cholecystectomy patients.

d) to investigate the impact of inflammation, gut microbiota/antimicrobial peptides,

proteins/peptides, nucleotides and internal body time on cardiometabolic diseases, signalling

pathways and pharmacokinetics parameters.

e) to assess differences in signalling pathways in patients with type 2-diabetes, impaired fasting

glucose and normal glucose levels on the protein/peptide, nucleotide, metabolite, lipid and

bile acid level with the aim to reveal mechanisms of importance for cardiometabolic

diseases.

f) to investigate differences in signalling pathways in persons with a wide range of BMIs and in

patients undergoing similar weight loss by VLED as opposed to GBP with the aim to further

elucidate mechanisms important to health and disease.

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METHODS AND ANALYSIS

Design and setting

This open, non-randomized, three-armed single centre study is performed at a tertiary care centre

(Morbid Obesity Centre, Vestfold Hospital Trust) in Norway. The study was designed to follow the

current routine treatment procedures at the centre with the exception of the 6-week VLED. The

investigations are performed in patients scheduled either for cholecystectomy, or weight loss with

GBP or VLED based on clinical indications, and the treatment procedures are not influenced by the

present protocol (Figure 1).

Patient selection and recruitment

Consecutive patients scheduled for surgical (GBP) or medical (VLED) weight loss treatment as well as

patients scheduled for cholecystectomy, were provided with an invitation letter with information on

what taking part in the study would entail, and those who made contact were assessed according to

the eligibility criteria. Informed consent was obtained before any protocol determined activities,

according to the Declaration of Helsinki and good clinical practice (GCP). Patients and the informing

physician signed the patient information; original stored at the centre and the patient received a

copy. The patients were also informed that they are covered by liability insurance and that they are

allowed to withdraw from the study at any time without giving any reason for doing so.

Inclusion criteria

• willing and able to give informed consent for participation in the study

• scheduled for GBP, VLED or cholecystectomy

• BMI ≥ 18.5 kg/m2

• aged 18 years or above

• able and willing to donate biopsies, perform 24-hour PK-investigations and other

assessments as required by the clinical study protocol

• stable body weight (< 5 kg self-reported weight change) during the last 3 months before

inclusion

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Exclusion criteria

• concomitant treatment with medications and/or other substances that may influence the

cocktail drug pharmacokinetics such as grapefruit juice, Seville oranges, Pomelo juice, St.

John’s wort, nicotine and coffee/tea in close approximation to the investigations

• bradyarrhythmia, Wolff-Parkinson-White (WPW)-syndrome, atrioventricular block 2-3

• electrolyte disturbances (particularly hypokalaemia or hypomagnesaemia)

• estimated GFR < 30 mL/min/1.73 m2

• blood donations the last 4 months before inclusion

• previous bariatric or upper gastrointestinal surgery

• taking glitazones, insulin or sulfonylureas

• pregnancy (checked with HCG in urine at screening) and breast-feeding mothers

• known hypersensitivity (including allergy) to drugs included in the cocktail and/or local

anaesthesia

• taking anticoagulants with associated risk in combination with biopsies

• suspected non-compliance with regards to visits and/or diet

Participant flow and follow-up (Figure 1 and Appendix 1)

A total of 196 patients were screened, out of whom 88 patients were excluded, leaving 108 patients

to be included in the study (Figure 1). After inclusion, both weight-loss groups were subjected to a

24-hour PK cocktail investigation (baseline 1) and both groups started a 3-week low energy diet (LED,

1200 kcal/day) directly after. At the 3-week follow up (baseline 2; day before surgery for GBP group),

the 24-hour PK cocktail investigation was repeated. A third group, patients scheduled for

cholecystectomy, was subjected to a 24-hour PK cocktail investigation the day before surgery.

The day after the second PK investigation (week 0, baseline 2), VLED-patients started the 800

kcal /day diet while GBP-patients were subjected to surgery and biopsies were obtained from

different tissues. The cholecystectomy patients were also subjected to surgery and biopsies the day

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after their 24-hour PK cocktail investigation. This group did not undergo any further investigations or

follow-up. The VLED- and GBP-groups were followed with 24-hour PK cocktail investigations at the 6-

week follow-up, which also will be repeated at the 2-year follow-up. Intestinal biopsies are obtained

at the same location in the intestine as during the GBP surgery in these patients at both the 6-week

and 2-year follow-up. Biobanking of samples was, in addition to the visits mentioned above, also

performed in both groups at the safety visits (2-week, 4-month and 1-year follow-up, see Appendix

1).

During the first 9 weeks of the study (including the 3-week run-in LED diet) patients were closely

followed and motivated by the clinical nutrition team to ensure a similar weight loss between GBP

and VLED groups. Between the 6-week and the 2-year follow-up the patients were offered

individually tailored follow-up by the dietitian or other members of the multidisciplinary team at the

outpatient clinic.

Sample size

This study has a number of primary study objectives, and the literature on bioavailability after

bariatric surgery generally includes very small samples.[50-56] Based on previous studies,[20 32] we

decided to base the sample size calculation on midazolam oral bioavailability in the intervention

groups. In order to evaluate the change in midazolam bioavailability from before to 6 weeks after

GBP/VLED with an 80% power and a 5% significance level, assuming a bioavailability ratio (6 weeks:

baseline) of 1.4 in the GBP group and no change in the VLED group and a SD of 0.5 in both groups,[20

32] at least 25 patients should be included in each group. Due to the explorative nature of the

present protocol an additional number of patients were included in order to ensure relevant

assessments of other outcome variables. Hence, a total of 80 patients were planned to be included in

the GBP (n=40) and in the VLED (n=40) groups, and we aimed to substitute premature withdrawals as

far as practically possible. The cholecystectomy control group was planned to consist of 20 patients,

based on best guess since no previous data was available.

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Interventions and biopsy procedures

Gastric bypass (GBP)

A routine laparoscopic GBP was performed by implementing an antegastric antecolic Roux-en-Y

configuration with an omega loop.[57] Standard port placement was applied with four bladeless

trocars and a Nathanson liver retractor (Cook Medical). All stapling was performed using a linear

stapler. The pouch was created by stapling the stomach horizontally from the minor curvature and

vertically to create a gastric pouch of about 25 mL. The gastrojejunostomy was created using a 45

mm stapler and completed with a running suture. The biliopancreatic limb was 60 cm. The omentum

was not transected routinely. The entero-enteric anastomosis was created using a side-to-side

technique, using a 45 mm stapler and completed with a running suture. Liver, fat and muscle biopsies

were taken at the beginning of the procedure to maximise time to monitor haemostasis.

Cholecystectomy

A standard four-port laparoscopic cholecystectomy was performed with the optical port inserted

supra-umbilically.

Biopsies

Subcutaneous fat biopsies were obtained in the morning of every visit, on all patients. This was done

by aspiration through a 14Gx3-1/4" G needle, with manual suction on a 20 mL syringe. Infiltration

anaesthesia, Lidocaine 10 mg/mL with adrenaline 5 microg/L, was used.

True-cut biopsies of liver, visceral fat and abdominal muscle were collected from all patients

undergoing cholecystectomy or gastric bypass at the beginning of each procedure to ensure safe

monitoring for bleeding. Bipolar diathermia was used for haemostasis on the cut surface, after the

biopsy was taken. A section of the jejunum, where the anastomosis was placed, was also sampled

from the GBP patients.

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Pinch biopsies of intestinal mucosa in the gastric ventricle, jejunum and ileum were obtained from all

GBP patients. This was performed at the moment the intestines were opened for making the

anastomoses. Biopsies of the gastric ventricle and jejunum will be repeated with the same pinch

technique, at the same site in the intestine, by endoscopy at 6 weeks and 2 years after surgery.

Calorie restriction (interventions)

Low energy diet (LED)

The LED diet aimed for an energy intake <1200 kcal per day during the 3-week run-in period in both

intervention groups (week -3 to 0). It was based on whole-grain crisp bread (Wasa, Barilla Norge AS,

Hamar, Norway) combined with low-fat, high-protein products for three of four daily meals. The

fourth meal (dinner) consisted of a specified amount of fish, poultry or lean meat combined with

vegetables, potatoes, rice or pasta. Participants were instructed to drink at least 1.5 L of water or

energy-free liquid per day. Supplement of one daily multivitamin and mineral tablet was

recommended (Nycoplus Multi, Takeda A/S, Asker, Norway). Additionally, participants could eat an

unlimited amount of vegetables and one fruit per day.

Very low energy diet (VLED)

The VLED diet aimed for an energy intake < 800 kcal per day during additional 6 weeks after the

completion of the LED (week 0 to 6) implementing the same dietary approach as the LED, but with

more limited amounts of all food items including vegetables.

GBP calorie restriction

During the first postoperative week, only liquid meals consisting of protein enriched soups and dairy

products every 2nd or 3rd hour through the day were prescribed, and in the 2nd and 3rd postoperative

week, a VLED with high-protein, low-fat mashed foods were included. During week 4-6, the surgical

patients were advised to use the VLED.

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Supplementary vitamins and minerals

Standard vitamin- and mineral-supplementation after GBP-surgery were prescribed [two

multivitamin/mineral tablets daily (Nycoplus Multi, Takeda A/S, Asker, Norway), chewable vitamin

D/calcium tablets taken morning and evening (each containing 10 µg D3/500 mg calcium carbonate,

Calcigran Forte, Takeda AS, Asker, Norway), iron, 100 mg ferrous sulphate for fertile women or if

needed (Duroferon, ACO HUD AB, Väsby, Sweden), vitamin B12 given intramuscularly 1 month after

surgery and thereafter every 3rd month (1 mg Vitamin B12 Depot, Takeda AS, Asker, Norway)].

Schedule of 24-hour pharmacokinetic investigations (PKs) and measurements

For detailed schedule see online supplementary table 2. In short, patients are to withhold caffeine-

containing beverages from two days before the investigation and to start food and drug fasting from

22:00 the day before the investigation. At 07:30 patients meet at the laboratory for baseline blood

sampling followed by urine and faeces sampling, subcutaneous adipose tissue biopsies and

administration of the cocktail of probe drugs. Blood and urine samples are frequently obtained over

the next 12 hours and the patients also return to the laboratory for 23- and 24-hour samples the

following day.

The drug cocktail consists of; caffeine (100 mg, oral), CYP1A2; losartan (25 mg, oral), CYP2C9;

omeprazole (20 mg, oral), CYP2C19; midazolam (total dose 2.5 mg; 1.5 mg oral at baseline and 1.0

mg iv after four hours), CYP3A; digoxin (0.5 mg, oral), P-gp; rosuvastatin (20 mg, oral), OATP1B1.

Sampling and storage procedures of biological material (blood, urine, faeces and biopsies)

are shown in online supplementary table 3. The samples of biological material will be collected in

accordance with the schedule of assessments and procedures during the PK-investigations (Appendix

1).

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Laboratory methods

Cocktail probe drug (and metabolite) concentrations and other biomarkers (including cytokines) will

be analysed in plasma and urine with validated methods based on at that time current guidelines

from the US Food and Drug Administration and the European Medicines Agency.

Clinical chemistry analyses will be analysed in serum/plasma, primarily at the Department for

Medical Biochemistry at the Vestfold Hospital Trust. They will always include: electrolytes (Na+, K+,

Ca2+), creatinine, ALP, ASAT, ALAT, total-cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides,

free fatty acids, fasting glucose, fasting insulin (average of two samples obtained 15 min apart),

HbA1c, haematocrit and haemoglobin.

Genetic and nucleotide analyses: DNA will be extracted from EDTA whole-blood samples throughout

the study in VLED and GBP groups (online supplementary table 4). Approximately 20 µg of DNA will

be extracted from each sample. Next-generation sequencing will be applied to capture different

types of genetic variation (e.g. single nucleotide polymorphisms (SNPs), copy number variations

(CNVs), whole exons and intergenetic regions, and epigenetic modifications) in genetic regions

relevant for the probe drug PK, obesity, obesity-related diseases and cardiometabolic status.

Analyses of relevant nucleotide expressions and epigenetic analyses in biopsies and potentially urine

will be determined with semi quantitative RT-PCR methods, or other appropriate methods.

Protein, metabolite and biomarker analyses: Analyses of biopsy samples will be performed by state

of the art methodology (e.g. quantitative global and targeted LC-MS/MS analyses or similar).

Analyses of lipids, bile acids and metabolites may be conducted with state of the art methodology.

High sensitive C-reactive protein (hsCRP), interleukin-6 (IL-6), and insulin will be analysed with

commercial ELISA-kits.

Microbiota analyses: An external collaborator will perform analyses of microbiota in faeces samples.

Also analysis of antimicrobial peptides in the jejunum is planned.

CYP activity ex vivo: Analyses of CYP activities will be performed in jejunum and liver biopsies from

the GBP patients and in liver biopsies from the cholecystectomy patients obtained at the time of

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surgery. Hepatic and intestinal microsomes will be prepared and activities of seven CYP enzymes

(CYP3A, CYP2C9, CYP2C8, CYP2D6, CYP2B6, CYP1A2 and CYP2C19) will be assessed by incubation with

selective probe drugs followed by LC-MS/MS quantification of metabolite formation.

Internal body time: Samples for assessments of internal body time are obtained and will be analysed

by validated LC-MS/MS methods.

Registration of questionnaire based data on patient reported outcome measures (PROMs)

All collection of questionnaire-based data including health related quality of life (SF-36,[58 59]

IWQOL-lite,[60] OWQOL and WRSM,[61 62]) anxiety/depression (HADS [63 64]), and eating

behaviour (TFEQ [65 66]), will be web based by the use of SurveyXact™, an Internet based survey

system. Patients will be guided into a quiet office at the study centre and asked to fill in the

questionnaires (online supplementary table 5). The completion time is estimated to about 45

minutes.

Registration of physical activity

Physical activity will be monitored by use of an accelerometer (version GT3X+ from ActiGraph, LLC,

Pensacola, FL, USA). The accelerometer provides an objective measure of overall physical activity and

will be used the first week in the LED-period as well as week 1 and week 6 after GBP or start of the

VLED-period, respectively, and again at the 1- and 2-year follow-up investigation.

Food records

Participants will be asked to record all foods and beverages consumed during a 4-day period (three

week-days and either Saturday or Sunday) at the following time points; the 1st, 3rd and 6th week after

GBP surgery, respectively start of the VLED-period. At the 2-year follow-up, habitual dietary intake in

both groups will be assessed by a structured interview performed by registered dieticians, using an

optically readable food frequency questionnaire (Department of Nutrition, University of Oslo, Oslo,

Norway).

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Measurements

The Appendix 1 shows a summary of the measurements collected.

Sociodemographic characteristics

Age and sex were registered.

Medical history

Medical history, menopausal status, alcohol drinking habits, nicotine use, concomitant drugs

(including dietary supplements and OTC drugs like omega-3, St. John’s wort and similar substances),

possible food/drink interactions, changes in physical activity and/or diet the last 3 months, co-

morbidities that might affect drug bioavailability was recorded.

In addition, obstructive sleep apnoea was assessed with apnoea ApneaLinkTM.

Anthropometric measurements

Body weight was recorded with patients wearing light clothing to the nearest 0.1 kg using an

electronic scale (Inbody 720, Body Composition Analyzer, Biospace Co. Ltd., Korea). Height was

measured to the nearest 1 cm with a wall mounted measuring tape (Soehnle Professional 5002.01,

Backnang, Germany) with patients wearing no shoes. BMI is calculated as weight in kilograms divided

by the square of the height in meters. Waist-circumference (WC) and hip-circumference (HC) was

measured with a stretch-resistant tape parallel to the floor and midway between the 12th rib and the

iliac crest, and around the widest portion of the buttocks, respectively. Bioelectrical impedance

measures were collected using the Inbody 720, Body Composition Analyzer (Biospace Co. Ltd.,

Korea).

Withdrawals, retention and premature study termination

Patients are free to withdraw at any time in accordance with GCP. Premature withdrawals will lower

the power of the study and therefore each withdrawn patient was substituted when possible. In

case of unnatural high AE frequencies as compared to the standard clinical practice the Steering

Committee will re-evaluate if it is ethically possible to continue the study.

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Analysis plan

Primary outcomes


a. Short- (6-week) and long-term (2-year) changes in absolute bioavailability

(AUCoral/AUCiv) of midazolam (CYP3A4) and AUC0-last , or drug: metabolite ratio, as

appropriate, for the other probe drugs and endogenous CYP3A4 biomarkers, after

GBP and VLED respectively.

b. Baseline expression, genotypic variation and activity data of the drug-metabolizing, -

transport and -regulatory proteins in biopsies from ileum, liver, visceral fat,

subcutaneous fat and skeletal muscle in the GBP and cholecystectomy groups.

II. Metabolism

a. Short-term (6-week) changes in cardiovascular risk factors such as fasting glucose,

HbA1c, insulin sensitivity, blood pressure, blood lipid levels, total body fat, BMI,

waist- and hip-circumference and measured cardiometabolic biomarkers, and long-

term (2-year) changes in fasting glucose, HbA1c, insulin sensitivity, blood pressure,

blood lipid levels, total body fat, BMI, waist- and hip-circumference, sleep apnoea

and cardiometabolic biomarkers in the GBP and VLED groups.

b. Cardiovascular risk factors such as fasting plasma glucose, HbA1c, insulin sensitivity,

blood pressure, blood lipid levels, total body fat, BMI, sleep apnoea and

cardiometabolic biomarkers (cross sectional comparison of participants undergoing

GBP, VLED and cholecystectomy at baseline).

Secondary outcomes

1. Changes in health related quality of life (SF-36, IWQOL-lite, OWLQOL, WRSM),

anxiety/depression (HADS), eating behaviour (TFEQ) and obstructive sleep apnoea

(ApneaLink) within the GBP and VLED groups from baseline to the 2-year follow-up. Changes

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in physical activity (accelerometer) and total energy expenditure within the GBP and VLED

groups from baseline to the 6-week, 1-year and 2-year follow-up.

2. Baseline characteristics and changes after intervention in proteins/peptides, nucleotides,

metabolites, lipids, bile acids and other metabolic/inflammatory/signalling pathways/internal

body time parameters in plasma and tissue samples from all patients.

3. Short- (6-week) and long-term (2-year) changes in the expression, nucleotide sequence and

activity data of the drug-metabolizing, -transport and -regulatory proteins in biopsies from

i. the gastric ventricle and jejunum in the GBP-group

ii. subcutaneous adipose tissue in the GBP- and VLED-groups

4. CYP protein expression and microsomal CYP activity measured by specific activity ex vivo in

microsomes from intestinal and hepatic biopsy material from each of the GBP patients.

5. Genetic variants and epigenetic alterations in genes encoding relevant proteins with regards

to obesity and diabetes status.

6. Gut microbiota in the 3 groups at baseline and in the GBP and VLED groups over time and the

association with cardiometabolic disease signalling pathways and pharmacokinetic variables.

7. Internal body time in the 3 groups at baseline and in the GBP and VLED groups over time and

the association with cardiometabolic disease signalling pathways and PK variables.

8. Changes in urine composition predicting development of urine stones in the GBP and VLED

groups.

9. Changes in urine metabolomics in GBP and VLED groups over time.

Statistical analyses

Repeated measurements over time (2 years, 4 time points) will be compared between the GBP and

VLCD group by linear mixed models, with the primary endpoints after 6 weeks and 2 years.

Additional sensitivity analyses will include ANOVA for repeated measurements both applying an

intention to treat principle imputing missing observations by multiple imputation techniques and by

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a per protocol analysis. Linear regression will be applied to assess associations between clinical

variables as well as different protein expressions and other continuous variables.

A descriptive analysis of the body composition, glucose metabolism, cardiovascular disease

and metabolic biomarkers will be performed between the three groups at baseline, including

association analyses. Simple and multiple linear regression models will explore the association with

clinical variables in the study.

In the first secondary endpoint, the group effect on health related quality of life,

anxiety/depression, eating behaviour and obstructive sleep apnoea will be assessed by ANOVA for

repeated measurements. Other secondary endpoints will be tabulated and analysed with

appropriate methods.

Time Schedule

Inclusion first patient: 15 April 2015.

Inclusion last patient: 31 May 2017

Recruitment time: approximately 2 years.

Follow-up: up to 2 years for each patient.

End of study: LPLV, approximately 4 years after study start (2 years after last patient included), May-

June 2019

Organization

The COCKTAIL study is a collaboration between the Morbid Obesity Centre, Vestfold Hospital Trust,

Norway (sponsor), the School of Pharmacy, University of Oslo, Norway and AstraZeneca Gothenburg,

Mölndal, Sweden.

Trial Steering Committee

The Trial Steering Committee (TSC) includes 7 persons, 2 members from each of the two Norwegian

collaborating groups (Vestfold Hospital Trust and University of Oslo, Norway) and 3 members from

AstraZeneca Gothenburg, Sweden, and is led by Professor Anders Åsberg (Department of

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Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Norway). The principal

investigator is Professor Jøran Hjelmesæth (Vestfold Hospital Trust). The TSC will provide oversight of

all matters relating to participant safety. Due to the low risk nature of the COCKTAIL study and that it

is a pragmatic open-label trial, the TSC also has the role of the Data Monitoring Committee. However,

there are no early stopping rules, and all AEs are evaluated unblinded by the trial management group

as well as the TSC according to standard definitions as outlined in online supplementary table 6. In

case of unnatural high AE frequencies as compared to the standard clinical practice, the TSC will

evaluate if it is ethically possible to continue the study.

The TSC has reviewed the study protocol, statistical analysis plan and the suitability of the

proposed safety data to be collected. No interim analysis is planned for this trial.

ETHICS AND DISSEMINATION

The study is performed according to GCP, ICH guidelines and the Declaration of Helsinki. The protocol

(Version 2, 5 September 2014) was reviewed and approved by the Regional Committee for Medical

and Health Research Ethics November 5 2014 (Ref: 2013/2379/REK sørøst A) prior to study start 18

March 2015. The last version of the study protocol with minor amendment (Version 4; 12 August

2015) was approved by the Regional Committee for Medical and Health Research Ethics 5 November

2014 (Ref: 2013/2379/REK sørøst A).

The study was registered at https://clinicaltrials.gov/ct2/show/NCT02386917 24 February

2015 and last updated 5 May 2017. Any new protocol modifications will be sent for review by the

research ethics committee and will be amended at the clinical trial registry.

Details on safety aspects with the different drugs were thoroughly considered and judged to be

acceptable before study start (online supplementary table 7). During the PK investigations patients

will be closely monitored, assessing pulse and general wellbeing throughout the day. In addition, the

investigation room is equipped with acute medication and necessary equipment to take care of any

emergency situation before the hospital on-call team will be in place.

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The results will be disseminated to academic and health professional audiences via

presentations at conferences and publications in peer-reviewed journals. Participants will be sent a

summary of the trial findings at the time when the main article is published, and if appropriate, the

results will be communicated to policymakers and commissioners of weight management services

through briefing papers summarising the main findings.

Acknowledgments

The crisp bread used for the LED and VLED is provided by Wasa, Barilla Norge AS, Hamar, Norway.

Figure 1 legend

Participant flow from screening to last follow up.

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62 Patrick DL, Bushnell DM, Rothman M. Performance of two self-report measures for evaluating obesity and weight loss. Obesity research 2004;12:48-57 doi:10.1038/oby.2004.8.

63 Herrmann C. International experiences with the Hospital Anxiety and Depression Scale--a review of validation data and clinical results. Journal of psychosomatic research 1997;42:17-41

64 Brunes A, Augestad LB, Gudmundsdottir SL. Personality, physical activity, and symptoms of anxiety and depression: the HUNT study. Social psychiatry and psychiatric epidemiology 2013;48:745-56 doi:10.1007/s00127-012-0594-6.

65 Karlsson J, Persson LO, Sjostrom L, et al. Psychometric properties and factor structure of the Three-Factor Eating Questionnaire (TFEQ) in obese men and women. Results from the Swedish Obese Subjects (SOS) study. Int J Obes Relat Metab Disord 2000;24:1715-25

66 Cappelleri JC, Bushmakin AG, Gerber RA, et al. Psychometric analysis of the Three-Factor Eating Questionnaire-R21: results from a large diverse sample of obese and non-obese participants. International journal of obesity 2009;33:611-20 doi:10.1038/ijo.2009.74.

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Contributors

• JH, AÅ, TBA and CK designed the study, and SA, RS, IR, LKJ, PCA, JKH, ES, MH, ALE, VK, TIK and HC contributed substantially to develop the protocol and the current work.

• JH and AÅ drafted the first version of the manuscript, and all authors revised the work critically for important intellectual content.

• All authors approved the submitted version of the manuscript • All authors agree to be accountable for all aspects of the work in ensuring that questions

related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

• JH is the principal investigator • ES is the trial statistician.

Funding

• This research is funded by the 3 collaborators: 1.Vestfold Hospital Trust, Tønsberg, Norway, 2. School of Pharmacy, University of Oslo, Oslo, Norway and 3. AstraZeneca Gothenburg, Sweden. The sponsor of the trial is Vestfold Hospital Trust. The results of the study will be published upon completion following appropriate review by the steering committee, and the funding organisations will have no influence on decisions regarding publication.

Competing interests

• JH, AÅ, RS, LKJ, JKH, ES, VK, TIK, and HC receive no personal financial benefits from the trial. PCA has received a PhD-grant and IR has received a post-doc grant from the study budget. CK, TBA, ALE, MH, and SA are employed by AstraZeneca, and CK, ALE and MH own shares in AstraZeneca.

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279x215mm (300 x 300 DPI)

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Appendix 1.

Sreening Baseline 1 Baseline 2 "Surgery" Safety Follow-up PK Follow-up biomarkers Safety Follow-up PK

Time -3 weeks -1 day 0 Week 2 Week 6 Month 4 Year 1 Year 2

Time window ± 1 week Day -1 to -4 0 ±5 days ±1 week ±2 weeks ±2 months ±1 months

Check inclusion/exclusion criteria B, D, C B, D B, D, C B, D, C B, D B, D B, D B, D B, D

Patient informed consent, ECG B, D, C

Demographics, medical and surgical history B, D C

Menopausal status B, D, C

Concomittant drugs and doses B, D, C B, D B, D, C B, C B, D B, D

Height B, D, C

Vital signs, body compositiona, heart monitoringb, alcohol use B, D, C B, D B, D, C B, D B, D B, D B, D B, D

AE reporting B, D B, D, C B, C B, D B, D B, D B, D B, D

FRQoL, HADS and TFEQ B, D B, D B, D B, D

ApneaLink™ B, D B, D B, D

Controlled diet (LED/VLED) B, D B, D B, D B, D B, D D D D

Prophylactic antibioticscB

Cocktail investigation (blood and urine) B, D B, D, C B, D B, D

Blood for 4ß-OH-cholesterol B, D B, D, C B, D B, D B, D B, D B, D

Blood and urine for biobanking B, D B, D, C B, D B, D B, D B, D B, D

Blood for cytokines and FFA B, D B, D, C B, D B, D B, D B, D B, D

Blood for internal body time B, D B, D, C B, D B, D

Blood for DNA/genotyping B, D C B, D B, D

Standard clinical chemistryd B, D, C B, D B, D, C B, D B, D B, D B, D B, D

Biopsies

-- gastric ventricle B B B

-- ileum B

-- jejunum (for ex vivo investigation) B

-- jejunum B B B

-- liver B, C

-- skeletal muscle B, C

-- visceral fat B, C

-- subcutaneous adipose tissuec

B, D B, D, C B, C B, D B, D B, D B, D B, D

Faeces B, D B, D, C B, D B, D B, D B, D B, D

Patient groups:

Bariatric surgery patients

Diet-controls

Cholecystectomy patientsaWeight, bioimpedance, waist-hip ratiobBlood pressure, heart rate, ECGcsc-adipose tissue both before antibiotics and simultaneously with other biopsyingdincluding: Na, K, Ca, creat, ALP, ASAT, ALAT, tot-Chol, LDL, HDL, TG, fasting glucose, fasting insulin, HbA1c, hct, Hgb

SCHEDULE OF ASSESSMENTS AND PROCEDURES

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Supplementary material - COCKTAIL-study 1 Jan 2, 2018

Supplementary table 1. Rationale and evidence gap to fill

A. Drug bioavailability and disposition The current literature shows that body weight, bariatric surgery and weight loss alter oral bioavailability and disposition of drugs, which accordingly may affect both drug efficacy and safety. This study aims to provide novel information and fill a number of gaps in the current knowledge of this topic:

1. Body weight. The CYP3A4 metabolizing capacity in individuals with obesity is reduced,[20] but it is unclear what body size measure (e.g. body mass index, ideal body weight, fat free mass) that best correlate with drug metabolizing activities and what the mechanisms behind the regulation are.[42] It is also unclear to what degree other relevant drug metabolizing enzymes and transporters are affected.

2. Gastric bypass (GBP) reduces the total surface area available for drug absorption and bypasses the proximal intestine that is rich in metabolizing enzymes (i.e. mainly CYP3A), absorptive (e.g. OATP1A1) and antiabsorptive transporters (e.g. P-gp). These effects will have opposing effects on oral bioavailability of drugs and affect drugs differently depending on their physiochemical characteristics. Detailed information of the expression of metabolizing enzymes and drug transporter at different sites of the GI-tract is limited.[43] Substrates for CYP3A and/or P-gp will show increased bioavailability [31] but limited data on substrates for other CYP:s and transporters is available. The reduced absorptive surface area will mainly affect drugs with low capacity for passive diffusion over cell membranes, but there is little data on the relative contribution of these opposing effects on bioavailability for drugs.[44]

3. Very low energy diet (VLED). The specific effects of calorie restriction and dietary induced weight loss on local expression of metabolizing enzymes and drug transporters in the GI-tract and liver and the effects on oral bioavailability of different drugs are unknown.

4. Intestinal adaption. Previous studies in GBP patients indicate that bypassing the proximal intestine increases the oral bioavailability acutely (weeks). This effect seems to be reduced over time (years).[32] We hypothesise that there may be an adaptive process in the intestine over time; that drug bioavailability relevant protein expression in the distal part of the intestine is upregulated when it becomes “proximal” after surgery. The current project will provide intestinal expression data to further elucidate this by assessing biopsies obtained via gastroscopy 2 years after surgery.

5. Environment/disease regulation. The regulation of drug metabolizing enzymes and transporters is affected by a wide range of factors, such as disease state, other drugs, food, and pollutants in the environment.[37-41] This study will provide a clearer mechanistic understanding of the impact of inflammation, metabolic status, gut microbiota peptides/proteins and nucleotides on drug metabolizing enzymes and transporters.

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Supplementary table 1 (cont.) Rationale and evidence gap to fill

B. Metabolism, cardiometabolic risk factors and biomarkers The interplay between metabolically active tissues such as skeletal muscle, visceral fat, subcutaneous fat, liver and the gastrointestinal tract is complex and fine-tuned and yet far from understood. This study is designed to disentangle the short-term (6-weeks) metabolic and pharmacokinetic effects of GBP and a VLED by inducing a similar weight loss in the two groups combined with collection of a high-quality biobank and detailed measurements covering different aspects of cardiometabolic diseases. This study thus aims to provide novel information and fill a number of gaps in the current knowledge of this topic:

1. The mechanisms behind development of obesity and obesity-related co-morbidities and improvements of these conditions after weight loss are still not fully understood. This study will generate data to further elucidate these mechanisms.

2. It is still unclear what are the mechanisms behind the acute and dramatic metabolic improvements seen with bariatric surgery.[9] Especially the relative contributions from weight loss and from surgical procedure, respectively, have been inherently challenging to tease out and most mechanistic studies conducted so far are done in pair-fed rodents.[10] However, the mechanisms after bariatric surgery in rodents differ from humans warranting properly designed human studies. This human study has a design that would allow revealing what mechanisms and metabolic changes that are specifically induced by the surgery procedure and by the weight loss per se.

3. The majority of human data on regulation of metabolic pathways in different tissues have been obtained from cross sectional studies, while few prospective studies have addressed short- and long-term changes in tissue compartments after weight loss. This lack of evidence may partly be explained by practical difficulties to obtain serial biopsies from different tissues (e.g. the GI-tract). This study combines in depth analyses of several tissues after serial sampling (subcutaneous adipose tissue, jejunum, gastric ventricle), after single sampling (liver, visceral adipose tissue, skeletal muscle, ileum) and also analyses of serial samples of plasma/urine/faces to provide novel knowledge on how the regulation differs between tissues over time after weight loss induced by different means.

4. Humans possess a circadian clock regulating cell activity in different biological processes with an endogenous, self-sustained period of about 24h.[45] There are growing evidence that internal body time plays a role in the development of diseases.[46] as well as the effect of treatments. For example, the timing of meals in a weight-loss study influenced how much weight people lost [47] and an increased efficacy and reduced toxicity has been shown with chronotherapy in cancer treatment.[48] Interestingly, bromocriptine, a sympatholytic D2-dopamine agonist approved for the treatment of type 2 diabetes in some countries, seems to work by impacting the internal body time.[49] In this study we will repeatedly assess the internal body time as well as inflammation, gut microbiota/antimicrobial peptides, proteins/peptides, nucleotides, lipids, bile acids and investigate how these measures impact/are impacted by/correlate with e.g. body size measures, weight loss induced by diet or gastric bypass surgery, obesity and obesity-related co-morbidities, measures of cardiometabolic diseases, signaling pathways and PK parameters.

5. The current information about genetic variants associated to the topics of the present protocol is somewhat limited when it comes to the metabolic and body composition aspects. This study is small but may provide some important pilot data in this regard.

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Supplementary table 2. Schedule of 24-h pharmacokinetic investigations (PKs) and

measurements

Prior to the investigation

The patients are contacted a few days prior to the investigation day and are asked which drugs they have used since the last visit to confirm that no potential interacting drugs are administered prior to the investigation.

20:00 two days before the PK investigation. From this time patients are not allowed to drink caffeine-containing beverages. 22:00 the day before the PK-investigation patients start to fast. Only water is allowed after this time.

On the investigation day

07:30 patients meet at the laboratory with a mid-stream morning spot urine and feces sample collected maximum 24-hr before the visit. Physical examination including vital signs, blood pressure, heart rate, EKG, weight, waist- and hip-circumference and bioimpedance and AE-reporting are performed. Medical history, concomitant drugs and doses are noted in the CRF. Pregnancy test for fertile women is performed. A peripheral venous catheter is inserted and the following blood samples are drawn: standard clinical chemistry and hematology, baseline samples for study specific analyses as well as baseline cocktail sample, cytokines, 4-β-OH cholesterol and blood samples for epi- and genotyping (selected visits). In addition, a subcutaneous adipose tissue samples is obtained during the first hour.

08:00 first blood sample for internal body time and insulin assessment are drawn followed by administration of caffeine (100 mg).

08:15 the second fasting insulin sample is drawn.

08:30 urine sampling where all urine voiding is collected into a bottle is started.

09:00 the rest of the drug cocktail is administered with water. Losartan tablets (2x12.5 mg), omeprazole enterotablets (1x20 mg), digoxin tablet (0.5 mg), midazolam oral syrup (1.5 mg) and rosuvastatin tablet (20 mg).

Blood samples for analysis of cocktail drugs are drawn at the following time-points: before (0 hours) and 0.25, 0.5, 1, 1.5, 2, 3, 4, 4.25, 4.5, 5, 5.5, 6, 8, 10, 12, 23 and 24 hours following administration. Actual times (24-hour clock) of cocktail administration as well as blood samplings are registered.

11:00 caffeine free breakfast is served.

12:00 blood sample for internal body time assessment is drawn.

13:00 intravenous midazolam (1.0 mg) is administered by a slow infusion over at least 2 minutes.


17:00 the last urine is collected (for losartan drug: metabolite measurement).

The cocktail drugs are analysed in the following samples: caffeine and omeprazole (12:00 sample),

digoxin, midazolam and rosuvastatin (in a selection from all blood samples obtained during the 24-

hr) and losartan (8-hr urine).

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Supplementary table 3. Sampling and storage procedures of biological material

The blood samples during the PK investigations for drug analysis are drawn in pre-chilled K2-EDTA vacutainer tubes (no gel or plastic beads) on ice, centrifuged for 10 minutes at 4°C (1800 g), plasma decanted into Cryovials (in 3 parallels, 5 parallels for the 12:00 sample) and frozen within 1 hour at -70°C.

The blood sample (K2-EDTA) drawn for DNA purification and subsequent genotyping are stored at -

70°C.

The blood samples for 4ß-hydroxycholesterol (and comparable endogenous biomarkers) and internal

body time determinations are drawn in pre-chilled K2-EDTA vacutainer tubes (no gel or plastic beads),

centrifuged for 10 minutes at 20°C (2200 g) and plasma decanted into Cryovials (in 3 parallels) and

frozen immediately at -70°C. Assessment of internal body time will be performed by assessment of at

least one steroid and at least one amino acid, e.g. cortisol and tryptophan.

The blood samples (K2-EDTA) for biobanking and determination of cytokines will be drawn in pre-

chilled vacutainers on ice centrifuged for 10 minutes at 4°C (1800 g) and plasma decanted into

Cryovials (at least 3 parallels, approximately 1 mL each, for each sample) and frozen immediately at -

70°C.

Biopsies are collected in accordance with the SAP (Appendix 1) at the time of surgery and at

additional time points for some of the tissue biopsies. Biopsies are obtained from gastric ventricle,

jejunum, ileum, liver, skeletal muscle, visceral and subcutaneous adipose tissue. All biopsies are snap

frozen in liquid nitrogen and stored at -70°C within 2 hours.

Faeces are sampled at all scheduled visits (Appendix 1) in specific containers. Five spoons (supplied

with the kit) of faeces will be placed in the container and the sealed container will be stored at - 70°C

within 48 hours after faeces were collected.

Urine will be sampled at all scheduled visits (Appendix 1). The patients are asked to collect morning urine prior to coming to the visit. The urine sample is decanted into two 10 mL tubes and frozen immediately at -70 °C. At the PK investigations urine is also collected during the whole day (8:30-17:00) in a container. The total volume of urine collected is noted and three vials are frozen immediately at -70 °C. One urine sample will be analysed for losartan and two urine samples are stored in the biobank.

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Supplementary table 4. List of possible candidate genes/genetic variants/genetic regions relevant for drug transport/metabolism, obesity and metabolic traits Next-generation sequencing will be applied to capture different types of genetic variation relevant for the probe drug PK, obesity, obesity-related diseases and cardiometabolic status. In this project, we will focus on genes linked to drug metabolism, obesity and obesity-related diseases by adding different filtering strategies. Drug transport and drug metabolism: CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2B6, CYP3A4, CYP3A5, CYP2D6, UGT1A1, OATP1B1, OATP1B3, OATP2B1, BCRP, MDR1, MRP1, MRP2, MRP4, OCT1, OAT2, NTCP, MATE1, PEPT1. Obesity and/or BMI: FTO (rs9939609), INSIG2 (rs7566605), PFKP (rs6602024), CTNNBL1 (rs6013029), FDFT1 (rs7001819), MC4R (rs17782313), TMEM18 (rs6548238), SH2B1/ATP2A1 (rs7498665), KCTD15 (rs11084753), NEGR1 (rs2568958), GNPDA2 (rs10938397), MTCH2 (rs10838738), BDNF/LIN7C/LGR4 (rs925946), SEC16B/RASAL2 (rs10913469), FAIM2/BCDIN3D (rs7138803), ETV5/SFRS10/DGKG (rs7647305), NPC1 (rs1805081), MAF (rs1424233), PTER (rs10508503), PRL (rs4712652), RBJ/ADCY3/POMC (rs713586), GPRC5B/IQCK (rs12444979), MAP2K5/LBXCOR1 (rs2241423), QPCTL/GIPR (rs2287019), TNNI3K (rs1514175), SLC39A8 (rs13107325), FLJ35779/HMGCR (rs2112347), LRRN6C (rs10968576), TMEM160/ZC3H4 (rs3810291), FANCL (rs887912), CADM2 (rs13078807), PRKD1 (11847697), LRP1B (rs2890652), PTBP2 (rs1555543), MTIF3/GTF3A (rs4771122), ZNF608 (rs4836133), RPL27A/TUB (rs4929949), NUDT3 (rs206936), NRXN3 (rs10150332), TFAP2B (rs987237), TNKS/MSRA (rs17150703), SDCCAG8 (rs12145833), KCNMA1 (rs2116830), OLFM4 (rs9568856) and HOX5B (rs9299), MC4R (rs12970134), TFAP2B (rs987237), MSRA (rs545854), NRXN3 (rs10146997), LYPLAL1 (rs2605100), RSPO3 (rs9491696), VEGFA (rs6905288), TBX15/WARS2 (rs984222), NFE2L3 (rs1055144), GRB14 (rs10195252), DNM3/PIGC (rs1011731), ITPR2/SSPN (rs7183149), LY86 (rs1294421), HOXC13 (rs1443512), ADAMTS9 (rs6795735), ZNRF3/KREMEN1 (rs4823006), NISCH/STAB1 (rs6784615), MCHR1 (rs133072) and CPEB4 (rs6861681). Type 2 Diabetes and metabolic traits: PROX1 (rs340874), NOTCH2 (rs10923931), GRB14 (rs3923113), BCL11A (rs243021), RBMS1 (rs7593730), GCKR (rs780094), IRS1 (rs2943641), THADA (rs7578597), ST6GAL1 (rs16861329), ADCY5 (rs11708067), ADAMTS9 (rs4607103), IGF2BP2 (rs4402960), PPARG (rs1801282), WFS1 (rs1801214), ZBED3 (rs4457053), CDKAL1 (rs7754840), KLF14 (rs972283), DGKB (rs972283), GCK (rs4607517), JAZF1 (rs864745), TP53INP1 (rs896854), SLC30A8 (rs13266634), TLE4 (rs13292136), PTPRD (rs17584499), CDKN2A/B (rs10811661), VPS26A (rs1802295), CDC123 (rs12779790), HHEX (rs1111875), TCF7L2 (rs7903146), ARAP1 (rs1552224), HMGA2 (rs1531343), MTNR1B (rs10830963), KCNQ1 (rs2237892, rs231362), KCNJ11 (rs5219), HNF1A (rs7957197), TSPAN8 (rs7961581), SPRY2 (rs1359730), AP3S2 (rs2028299), HMG20A (rs7178572), C2CD4A (rs11071657), ZFAND6 (rs11634397), PRC1 (rs8042680), FTO (rs8050136, rs9939609), SRR (rs391300), HNF1B (rs757210), HNF4A (rs4812829) and DUSP9 (rs5945326).

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Supplementary table 5. PROMs Questionnaires

Generic Health Related Quality of Life (HRQoL) Short Form-36 Health Survey (SF-36) Generic HRQL was assessed using validated Norwegian versions of the Short Form-36 Health Survey (SF-36), 4-week recall, version 2.0 (Vestfold Hospital Trust).[58, 59] The forms were analysed with certified scoring software (QualityMetric Health OutcomesTM Scoring Software 4.0/4.5). Scores for each of the 8 domains and summary scores for physical and mental health were calculated and norm-adjusted using the US 98 population norms to facilitate comparison. Obesity specific HRQOL IWQOL-Lite The IWQOL-Lite is a 31-item measure of weight-related quality of life. There are five domain scores (Physical Function, Self-Esteem, Sexual Life, Public Distress and Work) and a total score. Scores for all domains and total score range from 0-100, with lower scores indicating greater impairment.[60] Obesity and Weight-Loss Quality of Life (OWLQOL) The validated obesity specific OWLQOL measures feelings and emotions resulting from being obese and trying to lose weight. [20] The instrument consists of 17 statements rated from zero (“not at all”) to six (“a very great deal) on a seven-point scale. The 17 items form a sum scale ranging from 0-102, with higher scores indicating better emotional HRQOL. As proposed by the authors the scoring syntax converts the scale to 0-100. The questionnaire will be administered at both baseline, one and two-year follow-up.[61, 62] Weight-related Symptom Measure (WRSM) The validated obesity specific WRSM measures 20 symptoms commonly related to being overweight or obese, including foot problems, joint pain, sensitivity to cold, shortness of breath, etc. using two different sets of items. The first set assesses whether or not a patient is experiencing specific symptoms, and the second set rates the level of the distress of the symptoms with values from zero (“not at all”) to six (“bothers a very great deal”). The first set creates an additive scale summing symptoms from 0-20, while the second forms a symptom distress scale ranging from 0-120. This was administered at both baseline, one and two-year follow-up. [19,20] Hospital Anxiety and Depression Scale (HADS) The validated generic HADS measures symptoms of anxiety and depression using 14 items scored from 0-3.[63] It is decomposed into two domains measuring depression (HADS-D) and anxiety (HADS-A), both consisting of seven items yielding a score from 0-21. Norwegian normative data are available.[64] Three Factor Eating Questionnaire –R 21 (TFEQ-R21) The validated generic TFEQ-R21 measures eating behaviour and has been validated for use in individuals with obesity.[65, 66] It consists of 21 items comprising three domain scores; (1) uncontrolled eating; assessing the tendency to lose control over eating when feeling hungry or when exposed to external stimuli, (2) cognitive restraint; assessing the conscious restriction of food intake to control body weight or body shape, and (3) emotional eating; assessing overeating related to negative mood states. The domain scores were transformed to 0-100 scales to facilitate comparison; a higher score indicates more uncontrolled, restraint, or emotional eating.

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Supplementary table 6. Adverse and serious adverse events defined

Adverse events (AE) include all medical occurrences during a treatment of study subject with the drug cocktail in the present study. Abnormalities in laboratory values are also included. Changes in laboratory values are only considered to be adverse events if they are judged to be clinically significant, e.g. if some action or intervention is required. If abnormal laboratory values are result of pathology for which there is an overall diagnosis (e.g. increased creatinine in renal failure), the diagnostic term should be reported as the adverse event. A serious adverse event (SAE) includes every occurrence that at any time point during the study run. 1. Results in death 2. Is life threatening 3. Requires inpatient hospitalization or prolongation of existing hospitalization 4. Results in persistent or significant disability/incapacity 5. Is a congenital anomaly/birth defect 6. Important medical events that may require intervention to prevent one of 1 to 5 above or

may expose the subject to danger, even though the event is not immediately life threatening or fatal, or does not result in hospitalization.

Each SAE that is at least possibly related to a study drug is to be classified by the principal investigator as expected or unexpected. A SAE that is at least possibly related to study drug, and unexpected, is defined as a suspected unexpected serious adverse reaction (SUSAR). It is “expected” if it is already known from earlier studies or is mentioned in available summary of product characteristics (SmPC).

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Supplementary table 7. Drug cocktail-safety aspects Caffeine: 100 mg caffeine is given, which is approximately equivalent to one cup of coffee and

is not considered to have any safety issues. Caffeine is not expected to give any side effects but people who never drink coffee, cola or tea can in rare cases experience headache, nausea or palpitations when ingesting caffeine.

Losartan: Losartan is an angiotensin II-antagonist with antihypertensive effect and the normal

therapeutic dose is 50 mg once daily. Half this dose, 25 mg, will be administered in the drug cocktail. The most common side effect of losartan is dizziness, which can occur in about 2% of the patients.

Omeprazole: Omeprazole is a proton pump inhibitor used for the treatment of acid related

gastrointestinal diseases. The therapeutic dose is 20-40 mg once daily and in the present drug cocktail 20 mg is included. In therapeutic doses rare cases of headache, nausea, constipation or diarrhoea can be seen.

Midazolam: The total dose of 2.5 mg midazolam used in the present study (1.5 mg oral and an

additional 1.0 mg iv four hours later) is the lowest dose used clinically when administered for its relaxing effect in adjunction to invasive procedures. No special precautions need to be considered in obese patients and it has previously been used in combination with the other cocktail drugs.

Digoxin: A single dose of 0.5 mg is considered safe. A simulation of a single dose of 0.5 mg in

an obese patient, using RightDose (www.lapk.org), resulted in maximal peak concentrations less than 0.65 µg/L and a trough concentration of 0.9 µg/L and is considered safe.

Rosuvastatin: Statins are generally well tolerated. The most common adverse effects are myopathy

and liver toxicity, however, these usually come after multiple dosing and a single dose of 20 mg has not previously been associated with significant adverse effects.

Interactions between the different components of the cocktail: There is no published information about any relevant pharmacokinetic interactions between the drugs in the present drug cocktail.

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Impact of body weight, low energy diet and gastric bypass on drug bioavailability, cardiovascular risk factors and

metabolic biomarkers: protocol for an open, non-randomised, three-armed single

centre study (COCKTAIL)

Journal: BMJ Open

Manuscript ID bmjopen-2018-021878.R1

Article Type: Protocol

Date Submitted by the Author: 20-Apr-2018

Complete List of Authors: Hjelmesæth, Jøran; Vestfold Hospital Trust, The Morbid Obesity Center; University of Oslo, Department of Endocrinology, Morbid Obesity and Preventive Medicine, Institute of Clinical Medicine Åsberg, Anders ; University of Oslo , Department of Pharmaceutical Biosciences, School of Pharmacy; Oslo University Hospital, Rikshospitalet , Department of Transplantation Medicine Andersson, Shalini; AstraZeneca Gothenburg , Drug Metabolism and Pharmacokinetics, Cardiovascular, Renal and Metabolism, IMED Biotech Unit Sandbu, Rune; Vestfold Hospital Trust, The Morbid Obesity Center Robertsen, Ida; University of Oslo , Department of Pharmaceutical Biosciences, School of Pharmacy Johnson, Line Kristin; Vestfold Hospital Trust, The Morbid Obesity Center Angeles, Philip Carlo; Vestfold Hospital Trust, The Morbid Obesity Center Hertel, Jens Kristoffer; Vestfold Hospital Trust, The Morbid Obesity Center Skovlund, E; Norwegian University of Science and Technology, NTNU, Department of Public Health and Nursing Heijer, Maria; AstraZeneca Gothenburg , Study Operations, Early Clinical Development, IMED Biotech Unit Ek, Anna-Lena; AstraZeneca Gothenburg , Study Operations, Early Clinical Development, IMED Biotech Unit Krogstad, Veronica; University of Oslo , Department of Pharmaceutical Biosciences, School of Pharmacy Karlsen, Tor-Ivar; Vestfold Hospital Trust, The Morbid Obesity Center; Universitetet i Agder, Faculty of Health and Sports Science Christensen, Hege; University of Oslo , Department of Pharmaceutical Biosciences, School of Pharmacy Andersson, Tommy B; AstraZeneca Gothenburg , Drug Metabolism and Pharmacokinetics, Cardiovascular, Renal and Metabolism, IMED Biotech Unit; Karolinska institutet , Department of Physiology and Pharmacology, Section of Pharmacogenetics Karlsson, Cecilia; AstraZeneca Gothenburg , CVMD Translational Medicine Unit, Early Clinical Development, IMED Biotech Unit ; Sahlgrenska Academy, University of Gothenburg, Department of Molecular and Clinical Medicine, Institute of Medicine


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<b>Primary Subject Heading</b>:

Pharmacology and therapeutics

Secondary Subject Heading: Nutrition and metabolism, Cardiovascular medicine, Diabetes and endocrinology, Medical management, Surgery

Keywords: BASIC SCIENCES, CLINICAL PHARMACOLOGY, DIABETES & ENDOCRINOLOGY, Cardiology < INTERNAL MEDICINE, NUTRITION & DIETETICS

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COCKTAIL protocol manuscript, R1 1 Apr 20, 2018

Impact of body weight, low energy diet and gastric bypass on drug

bioavailability, cardiovascular risk factors and metabolic biomarkers:

protocol for an open, non-randomised, three-armed single centre study (COCKTAIL)

Authors

Hjelmesæth J*1,2 , Åsberg A*3,4, Andersson S5, Sandbu R1, Robertsen I3, Johnson LK1, Angeles PC1, Hertel JK1, Skovlund E6, Heijer M7, Ek A-L7, Krogstad V3, Karlsen TI1,8, Christensen H3, Andersson TB5,9, Karlsson C10,11 *Shared 1st authorship

Author affiliations

1. Morbid Obesity Centre, Vestfold Hospital Trust, Tønsberg, Norway 2. Department of Endocrinology, Morbid Obesity and Preventive Medicine, Institute of Clinical

Medicine, University of Oslo, Norway 3. Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Norway 4. Department of Transplantation Medicine, Oslo University Hospital - Rikshospitalet, Oslo,

Norway 5. Drug Metabolism and Pharmacokinetics, Cardiovascular, Renal and Metabolism, IMED

Biotech Unit, AstraZeneca Gothenburg, Sweden 6. Department of Public Health and Nursing, Norwegian University of Science and Technology,

NTNU, Trondheim, Norway 7. Study Operations, Early Clinical Development, IMED Biotech Unit, AstraZeneca Gothenburg,

Sweden 8. Faculty of Health and Sports Science, University of Agder, Norway 9. Department of Physiology and Pharmacology, Section of Pharmacogenetics, Karolinska

Institutet, Stockholm, Sweden 10. Cardiovascular, Renal and Metabolism Translational Medicine Unit, Early Clinical

Development, IMED Biotech Unit, AstraZeneca Gothenburg, Sweden 11. Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Academy,

University of Gothenburg, Gothenburg, Sweden

Word count Manuscript: 4923 Abstract: 251 April 20, 2018

Corresponding author: Jøran Hjelmesæth (MD, PhD) Professor, Head Morbid Obesity Centre and Section of Endocrinology, Department of Medicine Vestfold Hospital Trust Boks 2168, 3103 Tønsberg, Norway Telephone +47 33 34 32 48 / Mobile +47 40 21 73 49 / Fax +47 33 34 39 91 Email: [email protected]

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Abstract

Introduction

Roux-en-Y gastric bypass (GBP) is associated with changes in cardiometabolic risk factors and

bioavailability of drugs, but whether these changes are induced by calorie restriction, the weight loss

or surgery per se, remains uncertain. The COCKTAIL study was designed to disentangle the short-

term (6-week) metabolic and pharmacokinetic effects of GBP and a very low energy diet (VLED) by

inducing a similar weight loss in the two groups.

Methods and analysis

This open, non-randomized, three-armed single centre study is performed at a tertiary care centre in

Norway. It aims to compare the short-term (6-week) and long-term (2-year) effects of GBP and VLED

on, first, bioavailability and pharmacokinetics (24-hour) of probe drugs and biomarkers, and, second,

their effects on metabolism, cardiometabolic risk factors and biomarkers. The primary outcomes will

be measured as changes in, (1), all six probe-drugs by absolute bioavailability (AUCoral/AUCiv) of

midazolam (CYP3A4 probe), systemic exposure (AUCoral) of digoxin and rosuvastatin and drug:

metabolite ratios for omeprazole, losartan and caffeine, levels of endogenous CYP3A biomarkers and

genotypic variation, changes in the expression, and activity data of the drug-metabolizing, -transport

and -regulatory proteins in biopsies from various organs, and, (2), body composition, cardiometabolic

risk factors and metabolic biomarkers.


The COCKTAIL-protocol was reviewed and approved by the Regional Committee for Medical and

Health Research Ethics (Ref: 2013/2379/REK sørøst A). The results will be disseminated to academic

and health professional audiences and the public via presentations at conferences, publications in

peer-reviewed journals and press releases, and provided to all participants.

Trial registration number (www.ClinicalTrials.gov) NCT02386917.

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Strengths and limitations of this study

1. The main strength of the present study is its potential to disentangle the short-term (6-week)

metabolic and pharmacokinetic effects of bariatric surgery per se and calorie restriction (very

low energy diet) by inducing a similar weight loss in the two groups.

2. Paired tissue biopsies from the GI-tract and the liver for “omics” investigations in

combination with in vivo drug disposition activity measures from a cocktail of probes will be

provided.

3. This study establishes a high-quality tissue biobank for global “omics” or targeted

biochemical analyses.

4. The explorative study design limits the clinical generalisability of the results.

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INTRODUCTION

Obesity represents a global epidemic [1] associated with premature mortality and increased risk for

type 2 diabetes, cardiovascular disease and cancer.[2] Weight loss is the primary treatment of

obesity and its comorbidities, and even a small weight reduction has beneficial effects on several

cardiometabolic risk factors.[3 4] Weight loss can be achieved by calorie restriction, exercise,

pharmacotherapy or bariatric surgery.[5] Interestingly, the literature indicates that Roux-en-Y gastric

bypass (GBP) may have separate, weight loss independent, beneficial effects on glucose metabolism

and type 2 diabetes, e.g. improvements in insulin sensitivity.[6-8] The mechanisms behind the acute

metabolic improvements seen after bariatric surgery remain, however, less clear.[9] It is also difficult

to distinguish the relative contributions of calorie restriction and weight loss from the surgical

procedure. The limited human data available has focused on changes in glucose homeostasis, with

few studies assessing involved pathways, of most detailed mechanistic studies conducted are done in

rodents.[8 10-13]

It is important to individualize drug doses in order to both obtain an adequate effect and

minimize side effects.[14] For most drugs, dose individualization is performed by dosing per kg total

body weight, although this may not necessarily be the best approach.[15] In patients with morbid

obesity, systemic clearance of a cytochrome P450 3A (CYP3A) substrate was found to be similar,

while oral bioavailability and volume of distribution were higher compared to patients with normal

weight.[16] However, other studies indicated that drug clearance might be deviant in patients with

severe obesity.[17-19] A recent study demonstrated a significant inverse correlation between body

mass index (BMI) and protein expression of CYP3A and oral clearance of another CYP3A4

substrate.[20] Hence, subjects with severe obesity might be at risk of drug over-exposure.[21] The

mechanisms behind the altered expression and activity of this CYP enzyme are unknown, but it has

been hypothesised that changed inflammatory state [22-24] as well as hepatic dysfunction may be

involved.[25]

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Several, but not all, bariatric surgery techniques reduce the absorptive surface area by bypassing

parts of the intestine.[26] Accordingly, bariatric surgery may affect the absorption rate and

bioavailability of a range of drugs.[27-31] Previous studies have shown that the bioavailability of

atorvastatin is increased early (months) after surgical reduction of the intestinal surface area.[32 33]

It might be speculated that the net effect of bypassing the metabolic most active parts of the

intestine results in this, at a first glance, contradictory effect. Interestingly, an adaptive process in the

intestine seems to normalize the bioavailability over a longer time span.[34] Even though the

literature is sparse and based on small studies, reduced uptake early after intestinal bypass has been

shown for drugs substrates of other intestinal enzymes.[35 36]

Oral bioavailability of drugs is restricted by a variety of transporters and metabolizing

enzymes in both the enterocytes and hepatocytes. Genetic, environmental and disease-related

factors induce variations in expression and activity of these proteins. Genotypic variations can easily

be assessed from a single blood sample, but in order to investigate the phenotypic variation, specific

probe drugs have to be used in vivo. Several approaches using a cocktail of probe drugs (targeting

different CYP enzymes and drug transporters) have been described.[37]

The present study was designed to disentangle the short-term (6-week) metabolic and

pharmacokinetic effects of GBP and a very low energy diet (VLED) by inducing a similar weight loss in

the two groups. It will include repeated cocktail investigations of a set of key drug metabolizing

enzymes and transporters restricting bioavailability for many drugs. The design allows revealing

pharmacokinetic (PK) changes as well as mechanisms of metabolic changes specifically induced by

GBP and calorie restriction per se. Combining the determination of in vivo CYP enzyme and

transporter activities with protein expression and ex vivo CYP activity of the same proteins is a

powerful tool for further elucidation of the mechanisms of body weight change, GBP, and calorie

restriction per se on the disposition of drugs, and it allows for improved in vitro-in vivo extrapolations

in the future.[20 37 38] A more detailed description of the study rationale and evidence gap to fill is

shown in the online supplementary table 1.[9 10 20 33 34 39-51]

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Study objectives

The primary objectives of this study are related to I) drug bioavailability and disposition, and II)

metabolism, cardiometabolic risk factors and biomarkers.


a. The study aims to investigate the relationship between body composition and the

liver/intestine expression and activity of proteins (drug metabolizing enzymes,

transporters and regulatory factors) important for drug bioavailability and

disposition in the range from normal body weight to morbid obesity cross sectional

in 3 study groups: patients undergoing cholecystectomy, GBP, and VLED.

b. The study aims to compare the short-term (6-week) and long-term (2-year) effects of

GBP and a VLED, with a similar 6-week weight loss, on bioavailability and

pharmacokinetics of probe drugs and biomarkers (and adjoining protein expressions)

for CYP1A2, CYP2C9, CYP2C19, CYP3A, P-glycoprotein (P-gp) and organic anion

transporting polypeptide 1B1 (OATP1B1).

II. Metabolism, cardiometabolic risk factors and biomarkers

a. The study aims to compare the 3 study groups (GBP, VLED and cholecystectomy) at

baseline with respect to body composition, cardiometabolic risk factors and

metabolic biomarkers.

b. The study aims to compare the short-term (6-week) changes in glucose metabolism,

blood pressure, blood lipids and body composition of similar weight loss (GBP vs.

VLED), and long-term effects (2 year) of GBP and VLED on body composition,


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The secondary objectives are:

a) to compare the short- and long-term (6-week and 2-year) effects of GBP and VLED on

physical activity, energy expenditure, health related quality of life, anxiety/depression, eating

behaviour, and obstructive sleep apnoea.

b) to assess the relation between proteins and nucleotides at all investigated sites (biopsies,

blood).

c) to perform an in-depth analysis of the correlation between CYP protein expression in vivo

and ex vivo CYP activity in jejunum and liver biopsies sampled from the same site in each of

the GBP patients, as well as liver biopsies from the cholecystectomy patients.

d) to investigate the impact of inflammation, gut microbiota/antimicrobial peptides,

proteins/peptides, nucleotides and internal body time on cardiometabolic diseases, signalling

pathways and pharmacokinetics parameters.

e) to assess differences in signalling pathways in patients with type 2-diabetes, impaired fasting

glucose and normal glucose levels on the protein/peptide, nucleotide, metabolite, lipid and

bile acid level with the aim to reveal mechanisms of importance for cardiometabolic

diseases.

f) to investigate differences in signalling pathways in persons with a wide range of BMIs and in

patients undergoing similar weight loss by VLED as opposed to GBP with the aim to further

elucidate mechanisms important to health and disease.

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METHODS AND ANALYSIS

Design and setting

This open, non-randomized, three-armed single centre study is performed at a tertiary care centre

(Morbid Obesity Centre, Vestfold Hospital Trust) in Norway. The study was designed to follow the

current routine treatment procedures at the centre with the exception of the 6-week VLED. The

investigations are performed in patients scheduled either for cholecystectomy, or weight loss with

GBP or VLED based on clinical indications, and the treatment procedures are not influenced by the

present protocol (Figure 1).

Patient and Public Involvement

The development of the research questions and outcome measures were not informed by patients`

priorities, experience, or preferences. Participants will be sent a summary of the trial findings when

the main article is published, and if appropriate, the results will be communicated to policymakers

and commissioners of weight management services through briefing papers summarising the main

findings.

Patient selection and recruitment

Consecutive patients scheduled for surgical (GBP) or medical (VLED) weight loss treatment as well as

patients scheduled for cholecystectomy, were contacted by telephone or face to face at the centre

by a project researcher who informed about the study. Patients interested in participation were

provided with an invitation letter with detailed information on what taking part in the study would

entail, and a time for appointment for screening-examination was suggested. At screening patients

were assessed according to the eligibility criteria. Informed consent was obtained before any

protocol determined activities, according to the Declaration of Helsinki and good clinical practice

(GCP). Patients and the informing physician signed the patient information; original stored at the

centre and the patient received a copy. The patients were also informed that they are covered by

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liability insurance and that they are allowed to withdraw from the study at any time without giving

any reason for doing so.

Inclusion criteria

• willing and able to give informed consent for participation in the study

• scheduled for GBP, VLED or cholecystectomy

• BMI ≥ 18.5 kg/m2

• aged 18 years or above

• able and willing to donate biopsies, perform 24-hour PK-investigations and other

assessments as required by the clinical study protocol

• stable body weight (< 5 kg self-reported weight change) during the last 3 months before

inclusion

Exclusion criteria

• concomitant treatment with medications and/or other substances that may influence the

cocktail drug pharmacokinetics such as grapefruit juice, Seville oranges, Pomelo juice, St.

John’s wort, nicotine and coffee/tea in close approximation to the investigations

• bradyarrhythmia, Wolff-Parkinson-White (WPW)-syndrome, atrioventricular block 2-3

• electrolyte disturbances (particularly hypokalaemia or hypomagnesaemia)

• estimated GFR < 30 mL/min/1.73 m2

• blood donations the last 4 months before inclusion

• previous bariatric or upper gastrointestinal surgery

• taking glitazones, insulin or sulfonylureas

• pregnancy (checked with HCG in urine at screening) and breast-feeding mothers

• known hypersensitivity (including allergy) to drugs included in the cocktail and/or local

anaesthesia

• taking anticoagulants with associated risk in combination with biopsies

• suspected non-compliance with regards to visits and/or diet

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Participant flow and follow-up (Figure 1 and Appendix 1)

A total of 196 patients were screened, out of whom 88 patients were excluded, leaving 108 patients

to be included in the study (Figure 1). After inclusion, both weight-loss groups were subjected to a

24-hour PK cocktail investigation (baseline 1) and both groups started a 3-week low energy diet (LED,

1200 kcal/day) directly after. At the 3-week follow up (baseline 2; day before surgery for GBP group),

the 24-hour PK cocktail investigation was repeated. A third group, patients scheduled for

cholecystectomy, was subjected to a 24-hour PK cocktail investigation the day before surgery.

The day after the second PK investigation (week 0, baseline 2), VLED-patients started the 800

kcal /day diet while GBP-patients were subjected to surgery and biopsies were obtained from

different tissues. The cholecystectomy patients were also subjected to surgery and biopsies the day

after their 24-hour PK cocktail investigation. This group did not undergo any further investigations or

follow-up. The VLED- and GBP-groups were followed with 24-hour PK cocktail investigations at the 6-

week follow-up, which also will be repeated at the 2-year follow-up. Intestinal biopsies are obtained

at the same location in the intestine as during the GBP surgery in these patients at both the 6-week

and 2-year follow-up. Biobanking of samples was, in addition to the visits mentioned above, also

performed in both groups at the safety visits (2-week, 4-month and 1-year follow-up, see Appendix

1).

During the first 9 weeks of the study (including the 3-week run-in LED diet) patients were closely

followed and motivated by the clinical nutrition team to ensure a similar weight loss between GBP

and VLED groups. Between the 6-week and the 2-year follow-up the patients were offered

individually tailored follow-up by the dietitian or other members of the multidisciplinary team at the

outpatient clinic.

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Sample size

This study has a number of primary study objectives, and the literature on bioavailability after

bariatric surgery generally includes very small samples.[52-58] Based on previous studies,[20 34] we

decided to base the sample size calculation on midazolam oral bioavailability in the intervention

groups. In order to evaluate the change in midazolam bioavailability from before to 6 weeks after

GBP/VLED with an 80% power and a 5% significance level, assuming a bioavailability ratio (6 weeks:

baseline) of 1.4 in the GBP group and no change in the VLED group and a SD of 0.5 in both groups,[20

34] at least 25 patients should be included in each group. Due to the explorative nature of the

present protocol an additional number of patients were included in order to ensure relevant

assessments of other outcome variables. Hence, a total of 80 patients were planned to be included in

the GBP (n=40) and in the VLED (n=40) groups, and we aimed to substitute premature withdrawals as

far as practically possible. The cholecystectomy control group was planned to consist of 20 patients,

based on best guess since no previous data was available.

Interventions and biopsy procedures

Gastric bypass (GBP)

A routine laparoscopic GBP was performed by implementing an antegastric antecolic Roux-en-Y

configuration with an omega loop.[59] Standard port placement was applied with four bladeless

trocars and a Nathanson liver retractor (Cook Medical). All stapling was performed using a linear

stapler. The pouch was created by stapling the stomach horizontally from the minor curvature and

vertically to create a gastric pouch of about 25 mL. The gastrojejunostomy was created using a 45

mm stapler and completed with a running suture. The biliopancreatic limb was 60 cm. The omentum

was not transected routinely. The entero-enteric anastomosis was created using a side-to-side

technique, using a 45 mm stapler and completed with a running suture. Liver, fat and muscle biopsies

were taken at the beginning of the procedure to maximise time to monitor haemostasis.

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Cholecystectomy

A standard four-port laparoscopic cholecystectomy was performed with the optical port inserted

supra-umbilically.

Biopsies

Subcutaneous fat biopsies were obtained in the morning of every visit, on all patients. This was done

by aspiration through a 14Gx3-1/4" G needle, with manual suction on a 20 mL syringe. Infiltration

anaesthesia, Lidocaine 10 mg/mL with adrenaline 5 microg/L, was used.

True-cut biopsies of liver, visceral fat and abdominal muscle were collected from all patients

undergoing cholecystectomy or gastric bypass at the beginning of each procedure to ensure safe

monitoring for bleeding. Bipolar diathermia was used for haemostasis on the cut surface, after the

biopsy was taken. A section of the jejunum, where the anastomosis was placed, was also sampled

from the GBP patients.

Pinch biopsies of intestinal mucosa in the gastric ventricle, jejunum and ileum were obtained from all

GBP patients. This was performed at the moment the intestines were opened for making the

anastomoses. Biopsies of the gastric ventricle and jejunum will be repeated with the same pinch

technique, at the same site in the intestine, by endoscopy at 6 weeks and 2 years after surgery.

Calorie restriction (interventions)

Low energy diet (LED)

The LED diet aimed for an energy intake <1200 kcal per day during the 3-week run-in period in both

intervention groups (week -3 to 0). It was based on whole-grain crisp bread (Wasa, Barilla Norge AS,

Hamar, Norway) combined with low-fat, high-protein products for three of four daily meals. The

fourth meal (dinner) consisted of a specified amount of fish, poultry or lean meat combined with

vegetables, potatoes, rice or pasta. Participants were instructed to drink at least 1.5 L of water or

energy-free liquid per day. Supplement of one daily multivitamin and mineral tablet was

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recommended (Nycoplus Multi, Takeda A/S, Asker, Norway). Additionally, participants could eat an

unlimited amount of vegetables and one fruit per day.

Very low energy diet (VLED)

The VLED diet aimed for an energy intake < 800 kcal per day during additional 6 weeks after the

completion of the LED (week 0 to 6) implementing the same dietary approach as the LED, but with

more limited amounts of all food items including vegetables.

GBP calorie restriction

During the first postoperative week, only liquid meals consisting of protein enriched soups and dairy

products every 2nd or 3rd hour through the day were prescribed, and in the 2nd and 3rd postoperative

week, a VLED with high-protein, low-fat mashed foods were included. During week 4-6, the surgical

patients were advised to use the VLED.

Supplementary vitamins and minerals

Standard vitamin- and mineral-supplementation after GBP-surgery were prescribed [two

multivitamin/mineral tablets daily (Nycoplus Multi, Takeda A/S, Asker, Norway), chewable vitamin

D/calcium tablets taken morning and evening (each containing 10 µg D3/500 mg calcium carbonate,

Calcigran Forte, Takeda AS, Asker, Norway), iron, 100 mg ferrous sulphate for fertile women or if

needed (Duroferon, ACO HUD AB, Väsby, Sweden), vitamin B12 given intramuscularly 1 month after

surgery and thereafter every 3rd month (1 mg Vitamin B12 Depot, Takeda AS, Asker, Norway)].

Schedule of 24-hour pharmacokinetic investigations (PKs) and measurements

For detailed schedule see online supplementary table 2. In short, patients are to withhold caffeine-

containing beverages from two days before the investigation and to start food and drug fasting from

22:00 the day before the investigation. At 07:30 patients meet at the laboratory for baseline blood

sampling followed by urine and faeces sampling, subcutaneous adipose tissue biopsies and

administration of the cocktail of probe drugs. Blood and urine samples are frequently obtained over

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the next 12 hours and the patients also return to the laboratory for 23- and 24-hour samples the

following day.

The drug cocktail consists of; caffeine (100 mg, oral), CYP1A2; losartan (25 mg, oral), CYP2C9;

omeprazole (20 mg, oral), CYP2C19; midazolam (total dose 2.5 mg; 1.5 mg oral at baseline and 1.0

mg iv after four hours), CYP3A; digoxin (0.5 mg, oral), P-gp; rosuvastatin (20 mg, oral), OATP1B1.

Sampling and storage procedures of biological material (blood, urine, faeces and biopsies)

are shown in online supplementary table 3. The samples of biological material will be collected in

accordance with the schedule of assessments and procedures during the PK-investigations (Appendix

1).

Laboratory methods

Cocktail probe drug (and metabolite) concentrations and other biomarkers (including cytokines) will

be analysed in plasma and urine with validated methods based on at that time current guidelines

from the US Food and Drug Administration and the European Medicines Agency.

Clinical chemistry analyses will be analysed in serum/plasma, primarily at the Department for

Medical Biochemistry at the Vestfold Hospital Trust. They will always include: electrolytes (Na+, K+,

Ca2+), creatinine, ALP, ASAT, ALAT, total-cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides,

free fatty acids, fasting glucose, fasting insulin (average of two samples obtained 15 min apart),

HbA1c, haematocrit and haemoglobin.

Genetic and nucleotide analyses: DNA will be extracted from EDTA whole-blood samples throughout

the study in VLED and GBP groups (online supplementary table 4). Approximately 20 µg of DNA will

be extracted from each sample. Next-generation sequencing will be applied to capture different

types of genetic variation (e.g. single nucleotide polymorphisms (SNPs), copy number variations

(CNVs), whole exons and intergenetic regions, and epigenetic modifications) in genetic regions

relevant for the probe drug PK, obesity, obesity-related diseases and cardiometabolic status.

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Analyses of relevant nucleotide expressions and epigenetic analyses in biopsies and potentially urine

will be determined with semi quantitative RT-PCR methods, or other appropriate methods.

Protein, metabolite and biomarker analyses: Analyses of biopsy samples will be performed by state

of the art methodology (e.g. quantitative global and targeted LC-MS/MS analyses or similar).

Analyses of lipids, bile acids and metabolites may be conducted with state of the art methodology.

High sensitive C-reactive protein (hsCRP), interleukin-6 (IL-6), and insulin will be analysed with

commercial ELISA-kits.

Microbiota analyses: An external collaborator will perform analyses of microbiota in faeces samples.

Also analysis of antimicrobial peptides in the jejunum is planned.

CYP activity ex vivo: Analyses of CYP activities will be performed in jejunum and liver biopsies from

the GBP patients and in liver biopsies from the cholecystectomy patients obtained at the time of

surgery. Hepatic and intestinal microsomes will be prepared and activities of seven CYP enzymes

(CYP3A, CYP2C9, CYP2C8, CYP2D6, CYP2B6, CYP1A2 and CYP2C19) will be assessed by incubation with

selective probe drugs followed by LC-MS/MS quantification of metabolite formation.

Internal body time: Samples for assessments of internal body time are obtained and will be analysed

by validated LC-MS/MS methods.

Registration of questionnaire based data on patient reported outcome measures (PROMs)

All collection of questionnaire-based data including health related quality of life (SF-36,[60 61]

IWQOL-lite,[62] OWQOL and WRSM,[63 64]) anxiety/depression (HADS [65 66]), and eating

behaviour (TFEQ [67 68]), will be web based by the use of SurveyXact™, an Internet based survey

system. Patients will be guided into a quiet office at the study centre and asked to fill in the

questionnaires (online supplementary table 5). The completion time is estimated to about 45

minutes.

Registration of physical activity

Physical activity will be monitored by use of an accelerometer (version GT3X+ from ActiGraph, LLC,

Pensacola, FL, USA). The accelerometer provides an objective measure of overall physical activity and

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will be used the first week in the LED-period as well as week 6 after GBP or start of the VLED-period,

respectively, and again at the 1- and 2-year follow-up investigation.

Food records

Participants will be asked to record all foods and beverages consumed during a 4-day period (three

week-days and either Saturday or Sunday) at the following time points; the 1st, 3rd and 6th week after

GBP surgery, respectively start of the VLED-period. At the 2-year follow-up, habitual dietary intake in

both groups will be assessed by a structured interview performed by registered dieticians, using an

optically readable food frequency questionnaire (Department of Nutrition, University of Oslo, Oslo,

Norway).

Measurements

The Appendix 1 shows a summary of the measurements collected.

Sociodemographic characteristics

Age and sex were registered.

Medical history

Medical history, menopausal status, alcohol drinking habits, nicotine use, concomitant drugs

(including dietary supplements and OTC drugs like omega-3, St. John’s wort and similar substances),

possible food/drink interactions, changes in physical activity and/or diet the last 3 months, co-

morbidities that might affect drug bioavailability was recorded.

In addition, obstructive sleep apnoea was assessed with apnoea ApneaLinkTM.

Anthropometric measurements

Body weight was recorded with patients wearing light clothing to the nearest 0.1 kg using an

electronic scale (Inbody 720, Body Composition Analyzer, Biospace Co. Ltd., Korea). Height was

measured to the nearest 1 cm with a wall mounted measuring tape (Soehnle Professional 5002.01,

Backnang, Germany) with patients wearing no shoes. BMI is calculated as weight in kilograms divided

by the square of the height in meters. Waist-circumference (WC) and hip-circumference (HC) was

measured with a stretch-resistant tape parallel to the floor and midway between the 12th rib and the

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iliac crest, and around the widest portion of the buttocks, respectively. Bioelectrical impedance

measures were collected using the Inbody 720, Body Composition Analyzer (Biospace Co. Ltd.,

Korea).

Withdrawals, retention and premature study termination

Patients are free to withdraw at any time in accordance with GCP. Premature withdrawals will lower

the power of the study and therefore each withdrawn patient was substituted when possible. In

cases of unnatural high AE frequencies as compared to the standard clinical practice the Steering

Committee will re-evaluate if it is ethically possible to continue the study.

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Analysis plan

Primary outcomes


a. Short- (6-week) and long-term (2-year) changes in absolute bioavailability

(AUCoral/AUCiv) of midazolam (CYP3A4) and AUC0-last, or drug: metabolite ratio, as

appropriate, for the other probe drugs and endogenous CYP3A4 biomarkers, after

GBP and VLED respectively.

b. Baseline expression, genotypic variation and activity data of the drug-metabolizing, -

transport and -regulatory proteins in biopsies from ileum, liver, visceral fat,

subcutaneous fat and skeletal muscle in the GBP and cholecystectomy groups.

II. Metabolism

a. Short-term (6-week) changes in cardiovascular risk factors such as fasting glucose,

HbA1c, insulin sensitivity, blood pressure, blood lipid levels, total body fat, BMI,

waist- and hip-circumference and measured cardiometabolic biomarkers, and long-

term (2-year) changes in fasting glucose, HbA1c, insulin sensitivity, blood pressure,

blood lipid levels, total body fat, BMI, waist- and hip-circumference, sleep apnoea

and cardiometabolic biomarkers in the GBP and VLED groups.

b. Cardiovascular risk factors such as fasting plasma glucose, HbA1c, insulin sensitivity,

blood pressure, blood lipid levels, total body fat, BMI, sleep apnoea and

cardiometabolic biomarkers (cross sectional comparison of participants undergoing

GBP, VLED and cholecystectomy at baseline).

Secondary outcomes

1. Changes in health related quality of life (SF-36, IWQOL-lite, OWLQOL, WRSM),

anxiety/depression (HADS), eating behaviour (TFEQ) and obstructive sleep apnoea

(ApneaLink) within the GBP and VLED groups from baseline to the 2-year follow-up. Changes

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in physical activity (accelerometer) and total energy expenditure within the GBP and VLED

groups from baseline to the 6-week, 1-year and 2-year follow-up.

2. Baseline characteristics and changes after intervention in proteins/peptides, nucleotides,

metabolites, lipids, bile acids and other metabolic/inflammatory/signalling pathways/internal

body time parameters in plasma and tissue samples from all patients.

3. Short- (6-week) and long-term (2-year) changes in the expression, nucleotide sequence and

activity data of the drug-metabolizing, -transport and -regulatory proteins in biopsies from

i. the gastric ventricle and jejunum in the GBP-group

ii. subcutaneous adipose tissue in the GBP- and VLED-groups

4. CYP protein expression and microsomal CYP activity measured by specific activity ex vivo in

microsomes from intestinal and hepatic biopsy material from each of the GBP patients.

5. Genetic variants and epigenetic alterations in genes encoding relevant proteins with regards

to obesity and diabetes status.

6. Gut microbiota in the 3 groups at baseline and in the GBP and VLED groups over time and the

association with cardiometabolic disease signalling pathways and pharmacokinetic variables.

7. Internal body time in the 3 groups at baseline and in the GBP and VLED groups over time and

the association with cardiometabolic disease signalling pathways and PK variables.

8. Changes in urine composition predicting development of urine stones in the GBP and VLED

groups.

9. Changes in urine metabolomics in GBP and VLED groups over time.

Statistical analyses

Repeated measurements over time (2 years, 4 time points) will be compared between the GBP and

VLCD group by linear mixed models, with the primary endpoints after 6 weeks and 2 years.

Additional sensitivity analyses will include ANOVA for repeated measurements both applying an

intention to treat principle imputing missing observations by multiple imputation techniques and by

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a per protocol analysis. Linear regression will be applied to assess associations between clinical

variables as well as different protein expressions and other continuous variables.

A descriptive analysis of the body composition, glucose metabolism, cardiovascular disease

and metabolic biomarkers will be performed between the three groups at baseline, including

association analyses. Simple and multiple linear regression models will explore the association with

clinical variables in the study.

In the first secondary endpoint, the group effect on health related quality of life,

anxiety/depression, eating behaviour and obstructive sleep apnoea will be assessed by ANOVA for

repeated measurements. Other secondary endpoints will be tabulated and analysed with

appropriate methods.

Time Schedule

Inclusion first patient: 15 April 2015.

Inclusion last patient: 31 May 2017

Recruitment time: approximately 2 years.

Follow-up: up to 2 years for each patient.

End of study: LPLV, approximately 4 years after study start (2 years after last patient included), May-

June 2019

Organization

The COCKTAIL study is a collaboration between the Morbid Obesity Centre, Vestfold Hospital Trust,

Norway (sponsor), the School of Pharmacy, University of Oslo, Norway and AstraZeneca Gothenburg,

Mölndal, Sweden.

Trial Steering Committee

The Trial Steering Committee (TSC) includes 7 persons, 2 members from each of the two Norwegian

collaborating groups (Vestfold Hospital Trust and University of Oslo, Norway) and 3 members from

AstraZeneca Gothenburg, Sweden, and is led by Professor Anders Åsberg (Department of

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Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Norway). The principal

investigator is Professor Jøran Hjelmesæth (Vestfold Hospital Trust). The TSC will provide oversight of

all matters relating to participant safety. Due to the low risk nature of the COCKTAIL study and that it

is a pragmatic open-label trial, the TSC also has the role of the Data Monitoring Committee. However,

there are no early stopping rules, and all AEs are evaluated unblinded by the trial management group

as well as the TSC according to standard definitions as outlined in online supplementary table 6. In

case of unnatural high AE frequencies as compared to the standard clinical practice, the TSC will

evaluate if it is ethically possible to continue the study.

The TSC has reviewed the study protocol, statistical analysis plan and the suitability of the

proposed safety data to be collected. No interim analysis is planned for this trial.

ETHICS AND DISSEMINATION

The study is performed according to GCP, ICH guidelines and the Declaration of Helsinki. The protocol

(Version 2, 5 September 2014) was reviewed and approved by the Regional Committee for Medical

and Health Research Ethics November 5 2014 (Ref: 2013/2379/REK sørøst A) prior to study start 18

March 2015. The last version of the study protocol with minor amendment (Version 4; 12 August

2015) was approved by the Regional Committee for Medical and Health Research Ethics 5 November

2014 (Ref: 2013/2379/REK sørøst A).

The study was registered at https://clinicaltrials.gov/ct2/show/NCT02386917 24 February

2015 and last updated 5 May 2017. Any new protocol modifications will be sent for review by the

research ethics committee and will be amended at the clinical trial registry.

Details on safety aspects with the different drugs were thoroughly considered and judged to be

acceptable before study start (online supplementary table 7). During the PK investigations patients

will be closely monitored, assessing pulse and general wellbeing throughout the day. In addition, the

investigation room is equipped with acute medication and necessary equipment to take care of any

emergency situation before the hospital on-call team will be in place.

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The results will be disseminated to academic and health professional audiences via

presentations at conferences and publications in peer-reviewed journals. Participants will be sent a

summary of the trial findings when the main article is published, and if appropriate, the results will

be communicated to policymakers and commissioners of weight management services through

briefing papers summarising the main findings.

Acknowledgments

The crisp bread used for the LED and VLED is provided by Wasa, Barilla Norge AS, Hamar, Norway.

Figure 1 legend

Participant flow from screening to last follow up.

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62 Kolotkin RL, Crosby RD, Kosloski KD, et al. Development of a brief measure to assess quality of life in obesity. Obesity research 2001;9:102-11 doi:10.1038/oby.2001.13.

63 Niero M, Martin M, Finger T, et al. A new approach to multicultural item generation in the development of two obesity-specific measures: the Obesity and Weight Loss Quality of Life (OWLQOL) questionnaire and the Weight-Related Symptom Measure (WRSM). Clin Ther 2002;24:690-700

64 Patrick DL, Bushnell DM, Rothman M. Performance of two self-report measures for evaluating obesity and weight loss. Obesity research 2004;12:48-57 doi:10.1038/oby.2004.8.

65 Herrmann C. International experiences with the Hospital Anxiety and Depression Scale--a review of validation data and clinical results. Journal of psychosomatic research 1997;42:17-41

66 Brunes A, Augestad LB, Gudmundsdottir SL. Personality, physical activity, and symptoms of anxiety and depression: the HUNT study. Social psychiatry and psychiatric epidemiology 2013;48:745-56 doi:10.1007/s00127-012-0594-6.

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67 Karlsson J, Persson LO, Sjostrom L, et al. Psychometric properties and factor structure of the Three-Factor Eating Questionnaire (TFEQ) in obese men and women. Results from the Swedish Obese Subjects (SOS) study. Int J Obes Relat Metab Disord 2000;24:1715-25

68 Cappelleri JC, Bushmakin AG, Gerber RA, et al. Psychometric analysis of the Three-Factor Eating Questionnaire-R21: results from a large diverse sample of obese and non-obese participants. International journal of obesity 2009;33:611-20 doi:10.1038/ijo.2009.74.

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Contributors

• JH, AÅ, TBA and CK designed the study, and SA, RS, IR, LKJ, PCA, JKH, ES, MH, ALE, VK, TIK and HC contributed substantially to develop the protocol and the current work.

• JH and AÅ drafted the first version of the manuscript, and all authors revised the work critically for important intellectual content.

• All authors approved the submitted version of the manuscript • All authors agree to be accountable for all aspects of the work in ensuring that questions

related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

• JH is the principal investigator • ES is the trial statistician.

Funding

• This research is funded by the 3 collaborators: 1. Vestfold Hospital Trust, Tønsberg, Norway, 2. School of Pharmacy, University of Oslo, Oslo, Norway and 3. AstraZeneca Gothenburg, Sweden. The sponsor of the trial is Vestfold Hospital Trust. The results of the study will be published upon completion following appropriate review by the steering committee, and the funding organisations will have no influence on decisions regarding publication.

Competing interests

• JH, AÅ, RS, LKJ, JKH, ES, VK, TIK, and HC receive no personal financial benefits from the trial. PCA has received a PhD-grant and IR has received a post-doc grant from the study budget. CK, TBA, ALE, MH, and SA are employed by AstraZeneca, and CK, ALE and MH own shares in AstraZeneca.

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279x215mm (300 x 300 DPI)

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Appendix 1.

Sreening Baseline 1 Baseline 2 "Surgery" Safety Follow-up PK Follow-up biomarkers Safety Follow-up PK

Time -3 weeks -1 day 0 Week 2 Week 6 Month 4 Year 1 Year 2

Time window ± 1 week Day -1 to -4 0 ±5 days ±1 week ±2 weeks ±2 months ±1 months

Check inclusion/exclusion criteria B, D, C B, D B, D, C B, D, C B, D B, D B, D B, D B, D

Patient informed consent, ECG B, D, C

Demographics, medical and surgical history B, D C

Menopausal status B, D, C

Concomittant drugs and doses B, D, C B, D B, D, C B, C B, D B, D

Height B, D, C

Vital signs, body compositiona, heart monitoringb, alcohol use B, D, C B, D B, D, C B, D B, D B, D B, D B, D

AE reporting B, D B, D, C B, C B, D B, D B, D B, D B, D

FRQoL, HADS and TFEQ B, D B, D B, D B, D

ApneaLink™ B, D B, D B, D

Controlled diet (LED/VLED) B, D B, D B, D B, D B, D D D D

Prophylactic antibioticscB

Cocktail investigation (blood and urine) B, D B, D, C B, D B, D

Blood for 4ß-OH-cholesterol B, D B, D, C B, D B, D B, D B, D B, D

Blood and urine for biobanking B, D B, D, C B, D B, D B, D B, D B, D

Blood for cytokines and FFA B, D B, D, C B, D B, D B, D B, D B, D

Blood for internal body time B, D B, D, C B, D B, D

Blood for DNA/genotyping B, D C B, D B, D

Standard clinical chemistryd B, D, C B, D B, D, C B, D B, D B, D B, D B, D

Biopsies

-- gastric ventricle B B B

-- ileum B

-- jejunum (for ex vivo investigation) B

-- jejunum B B B

-- liver B, C

-- skeletal muscle B, C

-- visceral fat B, C

-- subcutaneous adipose tissuec

B, D B, D, C B, C B, D B, D B, D B, D B, D

Faeces B, D B, D, C B, D B, D B, D B, D B, D

Patient groups:

Bariatric surgery patients

Diet-controls

Cholecystectomy patientsaWeight, bioimpedance, waist-hip ratiobBlood pressure, heart rate, ECGcsc-adipose tissue both before antibiotics and simultaneously with other biopsyingdincluding: Na, K, Ca, creat, ALP, ASAT, ALAT, tot-Chol, LDL, HDL, TG, fasting glucose, fasting insulin, HbA1c, hct, Hgb

SCHEDULE OF ASSESSMENTS AND PROCEDURES

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Supplementary material - COCKTAIL-study 1 April 19, 2018

Supplementary table 1. Rationale and evidence gap to fill

A. Drug bioavailability and disposition The current literature shows that body weight, bariatric surgery and weight loss alter oral bioavailability and disposition of drugs, which accordingly may affect both drug efficacy and safety. This study aims to provide novel information and fill a number of gaps in the current knowledge of this topic:

1. Body weight. The CYP3A4 metabolizing capacity in individuals with obesity is reduced,[20] but it is unclear what body size measure (e.g. body mass index, ideal body weight, fat free mass) that best correlate with drug metabolizing activities and what the mechanisms behind the regulation are.[44] It is also unclear to what degree other relevant drug metabolizing enzymes and transporters are affected.

2. Gastric bypass (GBP) reduces the total surface area available for drug absorption and bypasses the proximal intestine that is rich in metabolizing enzymes (i.e. mainly CYP3A), absorptive (e.g. OATP1A1) and antiabsorptive transporters (e.g. P-gp). These effects will have opposing effects on oral bioavailability of drugs and affect drugs differently depending on their physiochemical characteristics. Detailed information of the expression of metabolizing enzymes and drug transporter at different sites of the GI-tract is limited.[45] Substrates for CYP3A and/or P-gp will show increased bioavailability [33] but limited data on substrates for other CYP:s and transporters is available. The reduced absorptive surface area will mainly affect drugs with low capacity for passive diffusion over cell membranes, but there is little data on the relative contribution of these opposing effects on bioavailability for drugs.[46]

3. Very low energy diet (VLED). The specific effects of calorie restriction and dietary induced weight loss on local expression of metabolizing enzymes and drug transporters in the GI-tract and liver and the effects on oral bioavailability of different drugs are unknown.

4. Intestinal adaption. Previous studies in GBP patients indicate that bypassing the proximal intestine increases the oral bioavailability acutely (weeks). This effect seems to be reduced over time (years).[34] We hypothesise that there may be an adaptive process in the intestine over time; that drug bioavailability relevant protein expression in the distal part of the intestine is upregulated when it becomes “proximal” after surgery. The current project will provide intestinal expression data to further elucidate this by assessing biopsies obtained via gastroscopy 2 years after surgery.

5. Environment/disease regulation. The regulation of drug metabolizing enzymes and transporters is affected by a wide range of factors, such as disease state, other drugs, food, and pollutants in the environment.[39-43] This study will provide a clearer mechanistic understanding of the impact of inflammation, metabolic status, gut microbiota peptides/proteins and nucleotides on drug metabolizing enzymes and transporters.

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Supplementary table 1 (cont.) Rationale and evidence gap to fill

B. Metabolism, cardiometabolic risk factors and biomarkers The interplay between metabolically active tissues such as skeletal muscle, visceral fat, subcutaneous fat, liver and the gastrointestinal tract is complex and fine-tuned and yet far from understood. This study is designed to disentangle the short-term (6-weeks) metabolic and pharmacokinetic effects of GBP and a VLED by inducing a similar weight loss in the two groups combined with collection of a high-quality biobank and detailed measurements covering different aspects of cardiometabolic diseases. This study thus aims to provide novel information and fill a number of gaps in the current knowledge of this topic:

1. The mechanisms behind development of obesity and obesity-related co-morbidities and improvements of these conditions after weight loss are still not fully understood. This study will generate data to further elucidate these mechanisms.

2. It is still unclear what are the mechanisms behind the acute and dramatic metabolic improvements seen with bariatric surgery.[9] Especially the relative contributions from weight loss and from surgical procedure, respectively, have been inherently challenging to tease out and most mechanistic studies conducted so far are done in pair-fed rodents.[10] However, the mechanisms after bariatric surgery in rodents differ from humans warranting properly designed human studies. This human study has a design that would allow revealing what mechanisms and metabolic changes that are specifically induced by the surgery procedure and by the weight loss per se.

3. The majority of human data on regulation of metabolic pathways in different tissues have been obtained from cross sectional studies, while few prospective studies have addressed short- and long-term changes in tissue compartments after weight loss. This lack of evidence may partly be explained by practical difficulties to obtain serial biopsies from different tissues (e.g. the GI-tract). This study combines in depth analyses of several tissues after serial sampling (subcutaneous adipose tissue, jejunum, gastric ventricle), after single sampling (liver, visceral adipose tissue, skeletal muscle, ileum) and also analyses of serial samples of plasma/urine/faces to provide novel knowledge on how the regulation differs between tissues over time after weight loss induced by different means.

4. Humans possess a circadian clock regulating cell activity in different biological processes with an endogenous, self-sustained period of about 24h.[47] There are growing evidence that internal body time plays a role in the development of diseases.[48] as well as the effect of treatments. For example, the timing of meals in a weight-loss study influenced how much weight people lost [49] and an increased efficacy and reduced toxicity has been shown with chronotherapy in cancer treatment.[50] Interestingly, bromocriptine, a sympatholytic D2-dopamine agonist approved for the treatment of type 2 diabetes in some countries, seems to work by impacting the internal body time.[51] In this study we will repeatedly assess the internal body time as well as inflammation, gut microbiota/antimicrobial peptides, proteins/peptides, nucleotides, lipids, bile acids and investigate how these measures impact/are impacted by/correlate with e.g. body size measures, weight loss induced by diet or gastric bypass surgery, obesity and obesity-related co-morbidities, measures of cardiometabolic diseases, signaling pathways and PK parameters.

5. The current information about genetic variants associated to the topics of the present protocol is somewhat limited when it comes to the metabolic and body composition aspects. This study is small but may provide some important pilot data in this regard.

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Supplementary table 2. Schedule of 24-h pharmacokinetic investigations (PKs) and

measurements

Prior to the investigation

The patients are contacted a few days prior to the investigation day and are asked which drugs they have used since the last visit to confirm that no potential interacting drugs are administered prior to the investigation.

20:00 two days before the PK investigation. From this time patients are not allowed to drink caffeine-containing beverages. 22:00 the day before the PK-investigation patients start to fast. Only water is allowed after this time.

On the investigation day

07:30 patients meet at the laboratory with a mid-stream morning spot urine and feces sample collected maximum 24-hr before the visit. Physical examination including vital signs, blood pressure, heart rate, EKG, weight, waist- and hip-circumference and bioimpedance and AE-reporting are performed. Medical history, concomitant drugs and doses are noted in the CRF. Pregnancy test for fertile women is performed. A peripheral venous catheter is inserted and the following blood samples are drawn: standard clinical chemistry and hematology, baseline samples for study specific analyses as well as baseline cocktail sample, cytokines, 4-β-OH cholesterol and blood samples for epi- and genotyping (selected visits). In addition, a subcutaneous adipose tissue samples is obtained during the first hour.

08:00 first blood sample for internal body time and insulin assessment are drawn followed by administration of caffeine (100 mg).

08:15 the second fasting insulin sample is drawn.

08:30 urine sampling where all urine voiding is collected into a bottle is started.

09:00 the rest of the drug cocktail is administered with water. Losartan tablets (2x12.5 mg), omeprazole enterotablets (1x20 mg), digoxin tablet (0.5 mg), midazolam oral syrup (1.5 mg) and rosuvastatin tablet (20 mg).

Blood samples for analysis of cocktail drugs are drawn at the following time-points: before (0 hours) and 0.25, 0.5, 1, 1.5, 2, 3, 4, 4.25, 4.5, 5, 5.5, 6, 8, 10, 12, 23 and 24 hours following administration. Actual times (24-hour clock) of cocktail administration as well as blood samplings are registered.

11:00 caffeine free breakfast is served.


13:00 intravenous midazolam (1.0 mg) is administered by a slow infusion over at least 2 minutes.


17:00 the last urine is collected (for losartan drug: metabolite measurement).

The cocktail drugs are analysed in the following samples: caffeine and omeprazole (12:00 sample),

digoxin, midazolam and rosuvastatin (in a selection from all blood samples obtained during the 24-

hr) and losartan (8-hr urine).

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Supplementary table 3. Sampling and storage procedures of biological material

The blood samples during the PK investigations for drug analysis are drawn in pre-chilled K2-EDTA vacutainer tubes (no gel or plastic beads) on ice, centrifuged for 10 minutes at 4°C (1800 g), plasma decanted into Cryovials (in 3 parallels, 5 parallels for the 12:00 sample) and frozen within 1 hour at -70°C.

The blood sample (K2-EDTA) drawn for DNA purification and subsequent genotyping are stored at -

70°C.

The blood samples for 4ß-hydroxycholesterol (and comparable endogenous biomarkers) and internal

body time determinations are drawn in pre-chilled K2-EDTA vacutainer tubes (no gel or plastic beads),

centrifuged for 10 minutes at 20°C (2200 g) and plasma decanted into Cryovials (in 3 parallels) and

frozen immediately at -70°C. Assessment of internal body time will be performed by assessment of at

least one steroid and at least one amino acid, e.g. cortisol and tryptophan.

The blood samples (K2-EDTA) for biobanking and determination of cytokines will be drawn in pre-

chilled vacutainers on ice centrifuged for 10 minutes at 4°C (1800 g) and plasma decanted into

Cryovials (at least 3 parallels, approximately 1 mL each, for each sample) and frozen immediately at -

70°C.

Biopsies are collected in accordance with the SAP (Appendix 1) at the time of surgery and at

additional time points for some of the tissue biopsies. Biopsies are obtained from gastric ventricle,

jejunum, ileum, liver, skeletal muscle, visceral and subcutaneous adipose tissue. All biopsies are snap

frozen in liquid nitrogen and stored at -70°C within 2 hours.

Faeces are sampled at all scheduled visits (Appendix 1) in specific containers. Five spoons (supplied

with the kit) of faeces will be placed in the container and the sealed container will be stored at - 70°C

within 48 hours after faeces were collected.

Urine will be sampled at all scheduled visits (Appendix 1). The patients are asked to collect morning urine prior to coming to the visit. The urine sample is decanted into two 10 mL tubes and frozen immediately at -70 °C. At the PK investigations urine is also collected during the whole day (8:30-17:00) in a container. The total volume of urine collected is noted and three vials are frozen immediately at -70 °C. One urine sample will be analysed for losartan and two urine samples are stored in the biobank.

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Supplementary table 4. List of possible candidate genes/genetic variants/genetic regions relevant for drug transport/metabolism, obesity and metabolic traits Next-generation sequencing will be applied to capture different types of genetic variation relevant for the probe drug PK, obesity, obesity-related diseases and cardiometabolic status. In this project, we will focus on genes linked to drug metabolism, obesity and obesity-related diseases by adding different filtering strategies. Drug transport and drug metabolism: CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2B6, CYP3A4, CYP3A5, CYP2D6, UGT1A1, OATP1B1, OATP1B3, OATP2B1, BCRP, MDR1, MRP1, MRP2, MRP4, OCT1, OAT2, NTCP, MATE1, PEPT1. Obesity and/or BMI: FTO (rs9939609), INSIG2 (rs7566605), PFKP (rs6602024), CTNNBL1 (rs6013029), FDFT1 (rs7001819), MC4R (rs17782313), TMEM18 (rs6548238), SH2B1/ATP2A1 (rs7498665), KCTD15 (rs11084753), NEGR1 (rs2568958), GNPDA2 (rs10938397), MTCH2 (rs10838738), BDNF/LIN7C/LGR4 (rs925946), SEC16B/RASAL2 (rs10913469), FAIM2/BCDIN3D (rs7138803), ETV5/SFRS10/DGKG (rs7647305), NPC1 (rs1805081), MAF (rs1424233), PTER (rs10508503), PRL (rs4712652), RBJ/ADCY3/POMC (rs713586), GPRC5B/IQCK (rs12444979), MAP2K5/LBXCOR1 (rs2241423), QPCTL/GIPR (rs2287019), TNNI3K (rs1514175), SLC39A8 (rs13107325), FLJ35779/HMGCR (rs2112347), LRRN6C (rs10968576), TMEM160/ZC3H4 (rs3810291), FANCL (rs887912), CADM2 (rs13078807), PRKD1 (11847697), LRP1B (rs2890652), PTBP2 (rs1555543), MTIF3/GTF3A (rs4771122), ZNF608 (rs4836133), RPL27A/TUB (rs4929949), NUDT3 (rs206936), NRXN3 (rs10150332), TFAP2B (rs987237), TNKS/MSRA (rs17150703), SDCCAG8 (rs12145833), KCNMA1 (rs2116830), OLFM4 (rs9568856) and HOX5B (rs9299), MC4R (rs12970134), TFAP2B (rs987237), MSRA (rs545854), NRXN3 (rs10146997), LYPLAL1 (rs2605100), RSPO3 (rs9491696), VEGFA (rs6905288), TBX15/WARS2 (rs984222), NFE2L3 (rs1055144), GRB14 (rs10195252), DNM3/PIGC (rs1011731), ITPR2/SSPN (rs7183149), LY86 (rs1294421), HOXC13 (rs1443512), ADAMTS9 (rs6795735), ZNRF3/KREMEN1 (rs4823006), NISCH/STAB1 (rs6784615), MCHR1 (rs133072) and CPEB4 (rs6861681). Type 2 Diabetes and metabolic traits: PROX1 (rs340874), NOTCH2 (rs10923931), GRB14 (rs3923113), BCL11A (rs243021), RBMS1 (rs7593730), GCKR (rs780094), IRS1 (rs2943641), THADA (rs7578597), ST6GAL1 (rs16861329), ADCY5 (rs11708067), ADAMTS9 (rs4607103), IGF2BP2 (rs4402960), PPARG (rs1801282), WFS1 (rs1801214), ZBED3 (rs4457053), CDKAL1 (rs7754840), KLF14 (rs972283), DGKB (rs972283), GCK (rs4607517), JAZF1 (rs864745), TP53INP1 (rs896854), SLC30A8 (rs13266634), TLE4 (rs13292136), PTPRD (rs17584499), CDKN2A/B (rs10811661), VPS26A (rs1802295), CDC123 (rs12779790), HHEX (rs1111875), TCF7L2 (rs7903146), ARAP1 (rs1552224), HMGA2 (rs1531343), MTNR1B (rs10830963), KCNQ1 (rs2237892, rs231362), KCNJ11 (rs5219), HNF1A (rs7957197), TSPAN8 (rs7961581), SPRY2 (rs1359730), AP3S2 (rs2028299), HMG20A (rs7178572), C2CD4A (rs11071657), ZFAND6 (rs11634397), PRC1 (rs8042680), FTO (rs8050136, rs9939609), SRR (rs391300), HNF1B (rs757210), HNF4A (rs4812829) and DUSP9 (rs5945326).

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Supplementary table 5. PROMs Questionnaires

Generic Health Related Quality of Life (HRQoL) Short Form-36 Health Survey (SF-36) Generic HRQL was assessed using validated Norwegian versions of the Short Form-36 Health Survey (SF-36), 4-week recall, version 2.0 (Vestfold Hospital Trust).[60, 61] The forms were analysed with certified scoring software (QualityMetric Health OutcomesTM Scoring Software 4.0/4.5). Scores for each of the 8 domains and summary scores for physical and mental health were calculated and norm-adjusted using the US 98 population norms to facilitate comparison. Obesity specific HRQOL IWQOL-Lite The IWQOL-Lite is a 31-item measure of weight-related quality of life. There are five domain scores (Physical Function, Self-Esteem, Sexual Life, Public Distress and Work) and a total score. Scores for all domains and total score range from 0-100, with lower scores indicating greater impairment.[62] Obesity and Weight-Loss Quality of Life (OWLQOL) The validated obesity specific OWLQOL measures feelings and emotions resulting from being obese and trying to lose weight. [20] The instrument consists of 17 statements rated from zero (“not at all”) to six (“a very great deal) on a seven-point scale. The 17 items form a sum scale ranging from 0-102, with higher scores indicating better emotional HRQOL. As proposed by the authors the scoring syntax converts the scale to 0-100. The questionnaire will be administered at both baseline, one and two-year follow-up.[63, 64] Weight-related Symptom Measure (WRSM) The validated obesity specific WRSM measures 20 symptoms commonly related to being overweight or obese, including foot problems, joint pain, sensitivity to cold, shortness of breath, etc. using two different sets of items. The first set assesses whether or not a patient is experiencing specific symptoms, and the second set rates the level of the distress of the symptoms with values from zero (“not at all”) to six (“bothers a very great deal”). The first set creates an additive scale summing symptoms from 0-20, while the second forms a symptom distress scale ranging from 0-120. This was administered at both baseline, one and two-year follow-up. [63, 64] Hospital Anxiety and Depression Scale (HADS) The validated generic HADS measures symptoms of anxiety and depression using 14 items scored from 0-3.[65] It is decomposed into two domains measuring depression (HADS-D) and anxiety (HADS-A), both consisting of seven items yielding a score from 0-21. Norwegian normative data are available.[66] Three Factor Eating Questionnaire –R 21 (TFEQ-R21) The validated generic TFEQ-R21 measures eating behaviour and has been validated for use in individuals with obesity.[67, 68] It consists of 21 items comprising three domain scores; (1) uncontrolled eating; assessing the tendency to lose control over eating when feeling hungry or when exposed to external stimuli, (2) cognitive restraint; assessing the conscious restriction of food intake to control body weight or body shape, and (3) emotional eating; assessing overeating related to negative mood states. The domain scores were transformed to 0-100 scales to facilitate comparison; a higher score indicates more uncontrolled, restraint, or emotional eating.

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Supplementary table 6. Adverse and serious adverse events defined

Adverse events (AE) include all medical occurrences during a treatment of study subject with the drug cocktail in the present study. Abnormalities in laboratory values are also included. Changes in laboratory values are only considered to be adverse events if they are judged to be clinically significant, e.g. if some action or intervention is required. If abnormal laboratory values are result of pathology for which there is an overall diagnosis (e.g. increased creatinine in renal failure), the diagnostic term should be reported as the adverse event. A serious adverse event (SAE) includes every occurrence that at any time point during the study run. 1. Results in death 2. Is life threatening 3. Requires inpatient hospitalization or prolongation of existing hospitalization 4. Results in persistent or significant disability/incapacity 5. Is a congenital anomaly/birth defect 6. Important medical events that may require intervention to prevent one of 1 to 5 above or

may expose the subject to danger, even though the event is not immediately life threatening or fatal, or does not result in hospitalization.

Each SAE that is at least possibly related to a study drug is to be classified by the principal investigator as expected or unexpected. A SAE that is at least possibly related to study drug, and unexpected, is defined as a suspected unexpected serious adverse reaction (SUSAR). It is “expected” if it is already known from earlier studies or is mentioned in available summary of product characteristics (SmPC).

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Supplementary table 7. Drug cocktail-safety aspects Caffeine: 100 mg caffeine is given, which is approximately equivalent to one cup of coffee and

is not considered to have any safety issues. Caffeine is not expected to give any side effects but people who never drink coffee, cola or tea can in rare cases experience headache, nausea or palpitations when ingesting caffeine.

Losartan: Losartan is an angiotensin II-antagonist with antihypertensive effect and the normal

therapeutic dose is 50 mg once daily. Half this dose, 25 mg, will be administered in the drug cocktail. The most common side effect of losartan is dizziness, which can occur in about 2% of the patients.

Omeprazole: Omeprazole is a proton pump inhibitor used for the treatment of acid related

gastrointestinal diseases. The therapeutic dose is 20-40 mg once daily and in the present drug cocktail 20 mg is included. In therapeutic doses rare cases of headache, nausea, constipation or diarrhoea can be seen.

Midazolam: The total dose of 2.5 mg midazolam used in the present study (1.5 mg oral and an

additional 1.0 mg iv four hours later) is the lowest dose used clinically when administered for its relaxing effect in adjunction to invasive procedures. No special precautions need to be considered in obese patients and it has previously been used in combination with the other cocktail drugs.

Digoxin: A single dose of 0.5 mg is considered safe. A simulation of a single dose of 0.5 mg in

an obese patient, using RightDose (www.lapk.org), resulted in maximal peak concentrations less than 0.65 µg/L and a trough concentration of 0.9 µg/L and is considered safe.

Rosuvastatin: Statins are generally well tolerated. The most common adverse effects are myopathy

and liver toxicity, however, these usually come after multiple dosing and a single dose of 20 mg has not previously been associated with significant adverse effects.

Interactions between the different components of the cocktail: There is no published information about any relevant pharmacokinetic interactions between the drugs in the present drug cocktail.

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SPIRIT Checklist COCKTAIL April 19, 2018 1

SPIRIT 2013 Checklist: Recommended items to address in a clinical trial protocol and

related documents*

Section/item ItemNo

Description

Administrative information

Title 1 Descriptive title identifying the study design, population, interventions,

and, if applicable, trial acronym

Checked, see manuscript page 1 (Title page)

Trial registration 2a Trial identifier and registry name. If not yet registered, name of

intended registry

Checked, see page 2 (abstract) and page 20 (Ethics and

dissemination)

2b All items from the World Health Organization Trial Registration Data

Set

n/a

Protocol version 3 Date and version identifier

Checked, see page 21 (Ethics and dissemination)

Funding 4 Sources and types of financial, material, and other support

Checked, see page 28 (Funding and Competing interests)

Roles and

responsibilities

5a Names, affiliations, and roles of protocol contributors

Checked, see page 1 Authors and Author affiliations, and page 28

Contributors

5b Name and contact information for the trial sponsor

Checked, page 1 Corresponding author

5c Role of study sponsor and funders, if any, in study design; collection,

management, analysis, and interpretation of data; writing of the report;

and the decision to submit the report for publication, including whether

they will have ultimate authority over any of these activities

Checked, page 28 Funding.

5d Composition, roles, and responsibilities of the coordinating centre,

steering committee, endpoint adjudication committee, data

management team, and other individuals or groups overseeing the

trial, if applicable (see Item 21a for data monitoring committee)

Checked, pages 20-21. Trial Steering Committee

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Introduction

Background and

rationale

6a Description of research question and justification for undertaking the

trial, including summary of relevant studies (published and

unpublished) examining benefits and harms for each intervention

Checked, pages 4-5 Introduction, pages 6-7 Study objectives and

Supplementary table 1. Rationale and evidence gap to fill.

6b Explanation for choice of comparators

Checked, see comments to item 6a and page 5 last paragraph

Objectives 7 Specific objectives or hypotheses

Checked, see pages 6-7 and supplementary table 1.

Trial design 8 Description of trial design including type of trial (eg, parallel group,

crossover, factorial, single group), allocation ratio, and framework (eg,

superiority, equivalence, noninferiority, exploratory)

Checked, see page 2 abstract and page 8 Design and setting.

Methods: Participants, interventions, and outcomes

Study setting 9 Description of study settings (eg, community clinic, academic hospital)

and list of countries where data will be collected. Reference to where

list of study sites can be obtained

Checked, page 8 Design and setting, single-centre study

Eligibility criteria 10 Inclusion and exclusion criteria for participants. If applicable, eligibility

criteria for study centres and individuals who will perform the

interventions (eg, surgeons, psychotherapists)

Checked, pages 8 and 9

Interventions 11a Interventions for each group with sufficient detail to allow replication,

including how and when they will be administered

Checked, pages 11-12

11b Criteria for discontinuing or modifying allocated interventions for a

given trial participant (eg, drug dose change in response to harms,

participant request, or improving/worsening disease)

Checked, page 8 second paragraph:” allowed to withdraw from the

study at any time without giving any reason for doing so”, and page16

last paragraph “Patients are free to withdraw at any time in

accordance with GCP. “, and page 21 first paragraph “there are no

early stopping rules” and “AEs are evaluated unblinded by the trial

management group as well as the TSC according to standard

definitions as outlined in online supplementary table 6”, and “In case

of unnatural high AE frequencies as compared to the standard clinical

practice, the TSC will evaluate if it is ethically possible to continue the

study.”

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11c Strategies to improve adherence to intervention protocols, and any

procedures for monitoring adherence (eg, drug tablet return,

laboratory tests)

Checked, see page 8 Design and setting. The study was designed to

follow the current routine treatment procedures at the centre with the

exception of the 6-week VLED. To improve compliance in the VLED-

period, the study nutritionist contacted participants by telephone once

a week to discuss weight development, encourage adherence to the

diet and answer questions. Participants could also contact the study

nutritionist and/or the study physician themselves at any time by

mobile phone or text messages. In addition, close and frequent

communication was established between study personnel and

participants as usual in clinical practise. See more details at page 10

“During the first 9 weeks of the study (including the 3-week run-in LED

diet) patients were closely followed and motivated by the clinical

nutrition team to ensure a similar weight loss between GBP and VLED

groups. Between the 6-week and the 2-year follow-up the patients

were offered individually tailored follow-up by the dietitian or other

members of the multidisciplinary team at the outpatient clinic.”

11d Relevant concomitant care and interventions that are permitted or

prohibited during the trial

n/a

Outcomes 12 Primary, secondary, and other outcomes, including the specific

measurement variable (eg, systolic blood pressure), analysis metric

(eg, change from baseline, final value, time to event), method of

aggregation (eg, median, proportion), and time point for each

outcome. Explanation of the clinical relevance of chosen efficacy and

harm outcomes is strongly recommended

Checked, page 1 (abstract) and pages 17-18

Participant

timeline

13 Time schedule of enrolment, interventions (including any run-ins and

washouts), assessments, and visits for participants. A schematic

diagram is highly recommended (see Figure)

Checked, see page 9 Participant flow and follow-up and Figure 1 and

Appendix 1 (SAP)

Sample size 14 Estimated number of participants needed to achieve study objectives

and how it was determined, including clinical and statistical

assumptions supporting any sample size calculations

Checked, see page 10 Sample size

Recruitment 15 Strategies for achieving adequate participant enrolment to reach

target sample size

Checked, page 10 Sample size and all participants are enrolled

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Methods: Assignment of interventions (for controlled trials)

Allocation:

Sequence

generation

16a Method of generating the allocation sequence (eg, computer-

generated random numbers), and list of any factors for stratification.

To reduce predictability of a random sequence, details of any planned

restriction (eg, blocking) should be provided in a separate document

that is unavailable to those who enrol participants or assign

interventions

n/a

Allocation

concealment

mechanism

16b Mechanism of implementing the allocation sequence (eg, central

telephone; sequentially numbered, opaque, sealed envelopes),

describing any steps to conceal the sequence until interventions are

assigned

n/a

Implementation 16c Who will generate the allocation sequence, who will enrol participants,

and who will assign participants to interventions

n/a

Blinding

(masking)

17a Who will be blinded after assignment to interventions (eg, trial

participants, care providers, outcome assessors, data analysts), and

how

n/a

17b If blinded, circumstances under which unblinding is permissible, and

procedure for revealing a participant’s allocated intervention during

the trial

n/a

Methods: Data collection, management, and analysis

Data collection

methods

18a Plans for assessment and collection of outcome, baseline, and other

trial data, including any related processes to promote data quality (eg,

duplicate measurements, training of assessors) and a description of

study instruments (eg, questionnaires, laboratory tests) along with

their reliability and validity, if known. Reference to where data

collection forms can be found, if not in the protocol

Checked, see pages 9-10 “Participant flow and follow-up” , Figure 1,

Appendix 1, online supplementary table 2

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18b Plans to promote participant retention and complete follow-up,

including list of any outcome data to be collected for participants who

discontinue or deviate from intervention protocols

Checked, see page 8 Design and setting. “The study was designed to

follow the current routine treatment procedures at the centre with the

exception of the 6-week VLED. To improve compliance in the VLED-

period, the study nutritionist contacted participants by telephone once

a week to discuss weight development, encourage adherence to the

diet and answer questions. All participants could also contact the

study nutritionist and/or the study physician themselves at any time by

mobile phone or text messages. In addition, close and frequent

communication was established between study personnel and

participants as usual in clinical practice”

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Data

management

19 Plans for data entry, coding, security, and storage, including any

related processes to promote data quality (eg, double data entry;

range checks for data values). Reference to where details of data

management procedures can be found, if not in the protocol

Checked, page 15: Registration of questionnaire based data on

patient reported outcome measures (PROMs). All PROMs

questionnaires will be web based by the use of SurveyXact™, an

internet based survey system. Patients will be guided into a quiet

office and asked to complete the questionnaires. They may ask a

study nurse for help, if necessary. The completion time is estimated to

about 45 minutes. The completed web-based questionnaires will only

contain the respondents project number as identification. After

completion, the data on the SurveyXact™ server will be downloaded

to a server at the University of Agder, and the data will be stored

encrypted and password protected behind the University’s firewalls as

a part of the University’s ordinary information safety protocols. Only

study personnel at the University of Agder (n=1) has access to the

patient non-identifiable database of PROMs data.

All patient data is recorded in paper versions of Case Report Files

(CRF) before entered into electronic CRF (eCRFs, single data entry).

A paper version of CRFs is stored in locked archive storage shelves,

separated from other data and patient identification keys. All electronic

study data is stored on a password-protected server for storage of

personal and health related research (TSD2.0) at the University of

Oslo. Only study personnel responsible for data management (n=2)

have access to this server. The eCRF is stored at the TSD2.0-server

and study personnel transfer encrypted electronic files from the

different laboratories for secure storage on the TSD2.0-server. Patient

data recorded in eCRFs are continuously controlled. Quality checks of

all manually entered data are controlled with visual plots of ranges

and correlations. Study data out of natural range data are queried. All

discrepancies/queries and the following changes in the database are

logged specifically. A final validation of the database will be performed

during a database lock and clean data is extracted. After this locking

the database cannot be changed.

Statistical

methods

20a Statistical methods for analysing primary and secondary outcomes.

Reference to where other details of the statistical analysis plan can be

found, if not in the protocol

Checked, see pages 19-20 Statistical analyses

20b Methods for any additional analyses (eg, subgroup and adjusted

analyses)

n/a

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20c Definition of analysis population relating to protocol non-adherence

(eg, as randomised analysis), and any statistical methods to handle

missing data (eg, multiple imputation)

Checked, see pages 19-20 Statistical analyses

Methods: Monitoring

Data monitoring 21a Composition of data monitoring committee (DMC); summary of its role

and reporting structure; statement of whether it is independent from

the sponsor and competing interests; and reference to where further

details about its charter can be found, if not in the protocol.

Alternatively, an explanation of why a DMC is not needed

Checked, see page 21 first paragraph. “Due to the low risk nature of

the COCKTAIL study and that it is a pragmatic open-label trial, the

TSC (Trial Steering Committee) also has the role of the Data

Monitoring Committee.

21b Description of any interim analyses and stopping guidelines, including

who will have access to these interim results and make the final

decision to terminate the trial

Checked, see page 21 first paragraph: “ no early stopping rules” and

second paragraph “No interim analysis is planned for this trial”.

Adverse events are evaluated unblinded by the trial management

group as well as the TSC according to standard definitions as outlined

in online supplementary table 6. Adverse and serious adverse events

defined

Harms 22 Plans for collecting, assessing, reporting, and managing solicited and

spontaneously reported adverse events and other unintended effects

of trial interventions or trial conduct

Checked, see page 16(-17) last sentence: “In cases of unnatural high

AE frequencies as compared to the standard clinical practice the

Steering Committee will re-evaluate if it is ethically possible to

continue the study. See also page 21:” TSC also has the role of the

Data Monitoring Committee. However, there are no early stopping

rules, and all AEs are evaluated unblinded by the trial management

group as well as the TSC according to standard definitions as outlined

in online supplementary table 6. In case of unnatural high AE

frequencies as compared to the standard clinical practice, the TSC will

evaluate if it is ethically possible to continue the study.” See last

sentence page 21: ”During the PK investigations patients will be

closely monitored, assessing pulse and general wellbeing throughout

the day. In addition, the investigation room is equipped with acute

medication and necessary equipment to take care of any emergency

situation before the hospital on-call team will be in place.”

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Auditing 23 Frequency and procedures for auditing trial conduct, if any, and

whether the process will be independent from investigators and the

sponsor

n/a (not mandatory in this type of studies)


Research ethics

approval

24 Plans for seeking research ethics committee/institutional review board

(REC/IRB) approval

Checked, approved, see page 21 “ETHICS AND DISSEMINATION”

Protocol

amendments

25 Plans for communicating important protocol modifications (eg,

changes to eligibility criteria, outcomes, analyses) to relevant parties

(eg, investigators, REC/IRBs, trial participants, trial registries, journals,

regulators)

Checked, see page 21 last paragraph: “Any new protocol

modifications will be sent for review by the research ethics committee

and will be amended at the clinical trial registry.”

Consent or assent 26a Who will obtain informed consent or assent from potential trial

participants or authorised surrogates, and how (see Item 32)

Checked, see page 8 second paragraph: “Patients and the informing

physician signed the patient information; original stored at the centre

and the patient received a copy.” Potential participants were invited to

a screening where the trial was thoroughly explained by the nutritionist

and the study physician. The informed consent form was then

reviewed by the physician and patient together in order to ensure full

understanding, before both patient and physician signed the consent

form. The participant received a copy. The original was stored in

locked archive storage shelves, separated from other data and patient

identification keys.

26b Additional consent provisions for collection and use of participant data

and biological specimens in ancillary studies, if applicable

n/a

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Confidentiality 27 How personal information about potential and enrolled participants will

be collected, shared, and maintained in order to protect confidentiality

before, during, and after the trial

Checked: All patient data is recorded in paper versions of Case

Report Files (CRF) before being entered into electronic CRF (eCRFs,

single data entry). Paper copies of CRFs are stored in locked archive

storage shelves, separated from other data and patient identification

keys. All electronic study data is stored on a password-protected

server for storage of personal and health related research (TSD2.0) at

the University of Oslo. Only study personnel responsible for data

management (n=2) have access to this server. The eCRF is stored at

the TSD2.0-server and study personnel transfer encrypted electronic

files from the different laboratories for secure storage on the TSD2.0-

server.

Declaration of

interests

28 Financial and other competing interests for principal investigators for

the overall trial and each study site

Checked: see page 28 “Competing interests”

Access to data 29 Statement of who will have access to the final trial dataset, and

disclosure of contractual agreements that limit such access for

investigators

Checked: The TSC (which includes the investigators) and head

statistician of the trial will have access to the final trial dataset.

Ancillary and

post-trial care

30 Provisions, if any, for ancillary and post-trial care, and for

compensation to those who suffer harm from trial participation

n/a

Dissemination

policy

31a Plans for investigators and sponsor to communicate trial results to

participants, healthcare professionals, the public, and other relevant

groups (eg, via publication, reporting in results databases, or other

data sharing arrangements), including any publication restrictions

Checked: see page 22. “The results will be disseminated to academic

and health professional audiences via presentations at conferences

and publications in peer-reviewed journals. Participants will be sent a

summary of the trial findings when the main article is published, and if

appropriate, the results will be communicated to policymakers and

commissioners of weight management services through briefing

papers summarising the main findings.”

31b Authorship eligibility guidelines and any intended use of professional

writers

Checked: The results of the study will be published upon completion

following appropriate review by the steering committee, and author

eligibility will be assessed according to the Recommendations for the

Conduct, Reporting, Editing, and Publication of Scholarly Work in

Medical Journals http://www.icmje.org/icmje-recommendations.pdf

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31c Plans, if any, for granting public access to the full protocol, participant-

level dataset, and statistical code

Checked: The full protocol and the informed consent form will be

made available via the Norwegian publication database

(www.christin.no) once this protocol paper has been published.

According to individual data protection laws in Norway we are not

allowed to publish participant-level datasets. The datasets may be

available for audit/inspection in special cases on a need-basis only,

after a specific application to the Norwegian data inspectorate. We do

not plan to publish general statistical codes.

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Appendices

Informed consent

materials

32 Model consent form and other related documentation given to

participants and authorised surrogates

Checked: see page 8 second paragraph: “Informed consent was

obtained before any protocol determined activities, according to the

Declaration of Helsinki and good clinical practice (GCP). Patients and

the informing physician signed the patient information; the original was

stored at the centre and the patient received a copy. Patients were

also informed that they were covered by liability insurance and that

they were allowed to withdraw from the study at any time without

giving any reason for doing so.” The consent form was approved by

the Regional Committee for Medical and Health Research Ethics and

may be provided if requested. The form will be made available via the

Norwegian publication database (www.christin.no) once this protocol

paper has been published.

Biological

specimens

33 Plans for collection, laboratory evaluation, and storage of biological

specimens for genetic or molecular analysis in the current trial and for

future use in ancillary studies, if applicable

Checked: see pages 9-10 Participant flow and follow-up including

Figure 1 and Appendix 1. See also Supplementary table 3. Sampling

and storage procedures of biological material and Supplementary

table 4. List of possible candidate genes/genetic variants/genetic

regions relevant for drug transport/metabolism, obesity and metabolic

traits. The collection of the samples and generation of data in the

present study are anticipated to lead to future ad hoc analyses based

upon the initial results.

*It is strongly recommended that this checklist be read in conjunction with the SPIRIT 2013

Explanation & Elaboration for important clarification on the items. Amendments to the

protocol should be tracked and dated. The SPIRIT checklist is copyrighted by the SPIRIT

Group under the Creative Commons “Attribution-NonCommercial-NoDerivs 3.0 Unported”

license.

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Documents

bmjopen.bmj.com · For peer review only Supplementary material - COCKTAIL-study 2 Jan 2, 2018 Supplementary table 1 (cont.) Rationale and evidence gap to fill . B. Metabolism, cardiometabolic