Clinical trial NCT03307252
Conditions: Healthy
Sponsor: Boehringer Ingelheim
Start date: 2017-10-25; Completion date: 2018-05-02
Enrollment: 45 participants; Allocation: RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
The primary objective of this trial is to investigate the relative bioavailabilities of digoxin, furosemide, metformin and rosuvastatin given as a cocktail alone and as a cocktail together with the following drug transporter inhibitors:

Part 1: verapamil (P-gp) and rifampin (OATP1B1/1B3) Part 2: cimetidine (OCT2/MATE) Part 3: probenecid (OAT1/3) The secondary objective is to investigate other potential changes in pharmacokinetics (e.g. in clearance, volume of distribution, etc.) of digoxin, furosemide, metformin and rosuvastatin given as a cocktail alone and as a cocktail together with the inhibitors describe above.

Detailed description:

Arms: arm 1: None arm 2: None arm 3: None arm 4: None arm 5: None arm 6: None arm 7: None arm 8: None arm 9: None
Interventions: intervention 1: Digoxin intervention 2: Furosemide intervention 3: Metformin intervention 4: Rosuvastatin intervention 5: Verapamil intervention 6: Rifampin intervention 7: Cimetidine intervention 8: Probenecid
Intervention descriptions: intervention 1: Tablet intervention 2: Oral solution intervention 3: Oral solution intervention 4: Film-coated tablet intervention 5: Film-coated tablet intervention 6: Film-coated tablet intervention 7: Tablet intervention 8: Tablet

Protocol documents:
Prot_001.pdf: 
001-MCS-40-106-RD-13 (5.0) / Saved on: 25 Jun 2014
Clinical Trial Protocol
TITLE PAGE
Document Number:
c16810802-04
EudraCT No.:
BI Trial No.:
2017-001549-29
0352-2100
Investigational
Products:
Digoxin, furosemide, metformin, rosuvastatin, verapamil, probenecid, 
cimetidine, rifampin
Title:
The effect of potent inhibitors of drug transporters (verapamil, 
rifampin, cimetidine, probenecid) on pharmacokinetics of a transporter 
probe drug cocktail consisting of digoxin, furosemide, metformin and 
rosuvastatin (an open-label, randomised, crossover trial in three parts)
Lay Title
This study tests the effect of certain medicines on the transport of 
other medicines in the body of healthy men.
Clinical Phase:
I
Trial Clinical Monitor:
Phone:
Fax: 
Principal Investigator:
Phone: 
Fax:
Status:
Final Protocol (Revised Protocol based on global amendment 3)
Version and Date:
Version: 4.0
Date: 01 DEC 2017
Page 1 of 93
Proprietary confidential information
© 2017 Boehringer Ingelheim International GmbH or one or more of its affiliated companies. All rights reserved.
This document may not - in full or in part - be passed on, reproduced, published or otherwise used without prior written permission


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BI Trial No.: 0352.2100
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Trial Protocol
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CLINICAL TRIAL PROTOCOL SYNOPSIS
Name of company:
Tabulated
Trial Protocol
Boehringer Ingelheim
Name of finished product:
Not applicable
Name of active ingredient: digoxin, metformin, 
furosemide, rosuvastatin, verapamil, cimetidine, 
probenecid, rifampin
Protocol date:
Trial number:
Revision date:
01 DEC 2017
18 AUG 2017
0352-2100
Title of trial:
The effect of potent inhibitors of drug transporters (verapamil, rifampin, cimetidine, 
probenecid) on pharmacokinetics of a transporter probe drug cocktail consisting of 
digoxin, furosemide, metformin, and rosuvastatin (an open-label, randomised, 
crossover trial in three parts)
Principal Investigator: 
Trial site:
Clinical phase:
I
Objectives:
1. To investigate pharmacokinetic changes of sensitive drug transporter substrates 
digoxin (P-gp), furosemide (OAT1/3), metformin (OCT2/MATE) and rosuvastatin 
(OATP1B1/1B3) that are given as a cocktail alone and as a cocktail together with the 
following drug transporter inhibitors in healthy male subjects: 
Part 1: verapamil (P-gp) and rifampin (OATP1B1/1B3)
Part 2: cimetidine (OCT2/MATE)
Part 3: probenecid (OAT1/3)
2. To compare the inhibitory effect of cimetidine and probenecid on subtherapeutic 
doses of metformin and furosemide (used in the cocktail) with therapeutic doses of 
both compounds.
Methodology:
Part 1: Open-label, randomised, 3-way crossover, with at least 13 days wash-out
Part 2: Open-label, randomised, 4-way crossover, with at least 7 days wash-out
Part 3: Open-label, randomised, 4-way crossover, with at least 7 days wash-out
No. of subjects: 
47 (but not more than 56, if subjects have to be replaced)
total entered:
Part 1: 15 (+ 3 replacement subjects)
Part 2: 16 (+ 3 replacement subjects)
Part 3: 16 (+ 3 replacement subjects)
each treatment:
Part 1: 15
Part 2: 16
Part 3: 16
Diagnosis:
Not applicable
Main criteria
for inclusion:
Healthy male subjects, age of 18 to 55 years, body mass index (BMI) of 18.5 to 
29.9 kg/m2


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Trial Protocol
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Name of company:
Tabulated
Trial Protocol
Boehringer Ingelheim
Name of finished product:
Not applicable
Name of active ingredient: digoxin, metformin, 
furosemide, rosuvastatin, verapamil, cimetidine, 
probenecid, rifampin
Protocol date:
Trial number:
Revision date:
01 DEC 2017
18 AUG 2017
0352-2100
Trial product 1:
Lanicor® 0.25 mg Digoxin / Tablette (digoxin)
dose:
1 x 1 tablet (0.25 mg digoxin), as part of the Cocktail (Parts 1, 2, and 3)
mode of admin.:
Oral, with 280 mL of water after an overnight fast of at least 10 h
Trial product 2:
Lasix® liquidum 10 mg/mL Lösung zum Einnehmen (furosemide)
dose:
1 x 0.1 mL oral solution (1 mg furosemide) as part of the Cocktail (Parts 1, 2, 3)
1 x 4 mL oral solution (40 mg furosemide) as therapeutic dose (Part 3 only)
mode of admin.:
Oral, with 280 mL of water after an overnight fast of at least 10 h
Trial product 3:
MetfoLiquid GeriaSan®, 1000 mg / 5 mL Lösung zum Einnehmen (metformin)
dose:
1 x 0.05 mL oral solution (10 mg metformin) as part of the Cocktail (Parts 1, 2, and 3)
1 x 2.5 mL oral solution (500 mg metformin) as therapeutic dose (Part 2 only)
mode of admin.:
Oral, with 280 mL of water after an overnight fast of at least 10 h
Trial product 4:
CRESTOR® 10 mg Filmtabletten (rosuvastatin)
dose:
1 x 1 tablet (10 mg rosuvastatin), as part of the Cocktail (Parts 1, 2, and 3)
mode of admin.:
Oral, with 280 mL of water after an overnight fast of at least 10 h
Trial product 5:
Isoptin® 120 mg Filmtabletten (verapamil)
dose:
1 x 1 tablet (=120 mg verapamil), in Part 1 only
mode of admin.:
Oral, with 240 mL of water after an overnight fast of at least 10 h
Trial product 6:
Eremfat® 600 mg (rifampin)
dose:
1 x 1 tablet (=600 mg rifampin), in Part 1 only
mode of admin.:
Oral, with 280 mL of water after an overnight fast of at least 10 h
Trial product 7:
Cimetidin acis® 400 mg, Tabletten (cimetidine)
dose:
4 x 1 tablet on Day 1 (daily dose = 1600 mg cimetidine), Part 2 only
2 x 1 tablet on Day 2 (daily dose = 800 mg cimetidine), Part 2 only
mode of admin.:
Oral, with 240 mL of water after an overnight fast of at least 10 h
Trial product 8:
Probenecid Weimer®, Tabletten (probenecid)
dose:
1 x 2 tablets on Day -1 (daily dose = 1000 mg probenecid), Part 3 only
1 x 2 tablets on Day 1 (daily dose = 1000 mg probenecid), Part 3 only
mode of admin.:
Oral, with 240 mL of water after an overnight fast of at least 10 h


Boehringer Ingelheim
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Name of company:
Tabulated
Trial Protocol
Boehringer Ingelheim
Name of finished product:
Not applicable
Name of active ingredient: digoxin, metformin, 
furosemide, rosuvastatin, verapamil, cimetidine, 
probenecid, rifampin
Protocol date:
Trial number:
Revision date:
01 DEC 2017
18 AUG 2017
0352-2100
Duration of treatment: Part 1
Treatment R1 (Reference 1 = Cocktail): 
0.25 mg digoxin, 1 mg furosemide, 10 mg metformin, and 10 mg rosuvastatin, all four 
drugs given together as single dose
Treatment T1 (Test 1): 
120 mg verapamil, single dose, 1 h prior to Cocktail
Treatment T2 (Test 2): 
600 mg rifampin, single dose, together with Cocktail
The randomised treatments are separated by a wash-out period of at least 13 days.
Part 2
Treatment R1 (Reference 1 = Cocktail): 
0.25 mg digoxin, 1 mg furosemide, 10 mg metformin, and 10 mg rosuvastatin, all four 
drugs given together as single dose
Treatment T3 (Test 3): 
4 x 400 mg cimetidine on Day 1 (first dose 1 h prior to Cocktail);
2 x 400 mg cimetidine on Day 2
Treatment R2 (Reference 2 = therapeutic dose of metformin):
500 mg metformin, single dose 
Treatment T5 (Test 5): 
4 x 400 mg cimetidine on Day 1(first dose 1 h prior to 500 mg metformin); 
2 x 400 mg cimetidine on Day 2 
The randomised treatments are separated by a wash-out period of at least 7 days 
between metformin and/or cocktail administrations. 
Part 3
Treatment R1 (Reference 1 = Cocktail): 
0.25 mg digoxin, 1 mg furosemide, 10 mg metformin, and 10 mg rosuvastatin, all four 
drugs given together as single dose
Treatment T4 (Test 4):
1000 mg probenecid on Day -1 and on Day 1 (1 h prior to Cocktail) 
Treatment R3 (Reference 3 = therapeutic dose of furosemide): 
40 mg furosemide, single dose 
Treatment T6 (Test 6):
1000 mg probenecid on Day -1 and on Day 1 (1 h prior to 40 mg furosemide) 
The randomised treatments are separated by a wash-out period of at least 7 days 
between furosemide and/or cocktail administrations.


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Name of company:
Tabulated
Trial Protocol
Boehringer Ingelheim
Name of finished product:
Not applicable
Name of active ingredient: digoxin, metformin, 
furosemide, rosuvastatin, verapamil, cimetidine, 
probenecid, rifampin
Protocol date:
Trial number:
Revision date:
01 DEC 2017
18 AUG 2017
0352-2100
Criteria for 
pharmacokinetics:
Primary endpoints: 
AUC0-tz and Cmax of digoxin, furosemide, metformin, rosuvastatin (at cocktail doses)
Secondary endpoints: 
AUC0-of digoxin, furosemide, metformin, rosuvastatin (at cocktail doses)
Other parameters of interest:
For digoxin, furosemide, metformin and rosuvastatin given as a cocktail: tmax, z, t1/2, 
%AUCtz-, MRTpo, CL/F, Vz/F, Aet1-t2, fet1-t2, CLR,t1-t2
For metformin and furosemide given at therapeutic doses: AUC0-tz , AUC0-, Cmax, tmax, 
z, t1/2, %AUCtz-, MRTpo, CL/F, Vz/F, Aet1-t2, fet1-t2, CLR,t1-t2
Criteria for safety:
Adverse events (AEs) including clinically relevant findings from the physical 
examination, safety laboratory tests, 12-lead electrocardiogram (ECG), vital signs 
(blood pressure [BP], pulse rate [PR]) 
Statistical methods:
The statistical analysis will be conducted for each trial part (1, 2, 3) separately. This 
applies to all assessments and endpoints.
The pharmacokinetic changes of the investigated sensitive drug transporter substrates 
digoxin, furosemide, metformin, rosuvastatin will be estimated based on the pairwise 
ratios (treatment Ti to treatment R1, i=1,…, 4) of the geometric means (gMeans) of 
the primary and secondary endpoints. Additionally, their two-sided 90% confidence 
intervals (CIs) will be provided. This method corresponds to the two one-sided t-tests 
procedure, each at the 5% significance level. Since the main focus is on estimation 
and not testing, an acceptance range was not specified. The statistical model of the 
primary analysis will be an analysis of variance (ANOVA) on the logarithmic scale 
including effects for ‘sequence’, ‘subjects nested within sequences’, ‘period’ and 
‘treatment’. CIs will be calculated based on the residual error from the ANOVA.
As a sensitivity analysis, all evaluable data of treatment R1 across the 3 trial parts will 
be pooled to analyse the effects within each trial part. The statistical model of the 
sensitivity analysis will be an ANOVA on the logarithmic scale including effects for
‘trial part’, ‘sequence’, ‘subjects nested within sequences’, ‘period’ and ‘treatment’. In 
part 2, the effect of cimetidine on exposure following the therapeutic dose of metfor-
min and in part 3 the effect of probenecid on exposure following the therapeutic dose 
of furosemide are investigated. Therefore, the statistical model of the primary analysis 
is applied to estimate T5 vs. R2 in part 2 and T6 vs. R3 in part 3. These effects will 
then be compared descriptively with the effects of T3 vs. R1 (gMean ratios of 
metformin) and of T4 vs. R1 (gMean ratios of furosemide), respectively. 
As a secondary analysis, the same methods of the primary analysis will be applied to 
CLR,t1-t2 and fet1-t2 of both doses of metformin and furosemide.
Descriptive statistics will be calculated for all endpoints.


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FLOW CHART – PART 1
Period
Visit
Day
Planned time (relative to 
drug administration)
[h:min]
Approximate clock time  
of actual day [h:min]
Event and comment
Safety laboratory
PK blood
PK urine
7
12-lead ECG
Continuous monitoring9
Vital signs (BP, PR)
Questioning for AEs and 
concomitant therapy 6
SCR
1
-21 to -1
Screening (SCR)1
x
x
x
1 / 2 / 3   (separated by a wash-out of least 13 days)
2 / 3 / 4 
-1
-14:30
18:30
Admission to trial site2, allocation to 
treatment (visit 2 only)2
x5
x
-14:00
19:00
Dinner
1
-2:00
07:00
x8
x2
x2
x2
x2
-1:00
08.00
Verapamil administration9
0:00
09:00
Trial drug administration10
▲
▲
0:20
09:20
x
│
│
0:40
09:40
x
│
│
1:00
10:00
x
│
x9
│
x9
1:30
10:30
x
│
│
2:00
11:00
240 mL fluid intake
x
│
x9
│
x9
2:30
11:30
x
│
│
3:00
12:00
x
│
│
4:00
13:00
240 mL fluid intake, thereafter lunch3
x
┼
x
▼
x
x
5:00
14:00
x
│
6:00
15:00
x
│
8:00
17:00
Snack (voluntary)3
x
┼
10:00
19:00
x
│
11:00
20:00
Dinner
│
12:00
21:00
x
┼
x 
2
24:00
09:00
Discharge, breakfast (voluntary) 3
x
x
┼
x
x
x
36:00
21:00
Ambulatory visit
x
▼
x
3
47:00
08:00
Ambulatory visit
x
x
4
71:00
08:00
Ambulatory visit
x
x
5
95:00
08:00
Ambulatory visit
x
x
EOT
6
8 to 20
End of trial (EOT) examination4
x
x
x
x
1.
Subject must be informed and written informed consent obtained prior to starting any screening procedures. Screening 
procedures include physical examination, check of vital signs, ECG (including rhythm stripe over 15 min), safety 
laboratory (including drug screening), demographics (including determination of body height and weight, smoking 
status and alcohol history), relevant medical history, concomitant therapy and review of inclusion/exclusion criteria. 
2.
The time is approximate; the procedure is to be performed and completed within 3 h prior to drug administration.
3.
If several actions are indicated at the same time point, the intake of meals will be the last action.
4.
End of trial examination includes physical examination, vital signs, ECG, safety laboratory, recording of AEs and 
concomitant therapies. 
5.
Only urine drug screening and alcohol breath test.
6.
AEs and concomitant therapies will be recorded throughout the trial, but will be specifically asked for at the time points 
indicated in the Flow Chart above.
7.
A blank urine sample (x) is to be obtained prior to administration of trial medication. Other urine samples are to be 
collected over the stated post-dose intervals (◄––│––│––►) 0-4, 4-8, 8-12, 12-24 and 24-36 h.
8.
To be taken within 20 min prior to first study drug administration
9.
Only in treatment T1
10
In treatments R1 and T1: Cocktail; in treatment T2: Cocktail + rifampin


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FLOW CHART – PART 2
Period
Visit
Day
Planned time (relative to 
drug administration)
[h:min]
Approximate clock time  
of actual day [h:min]
Event and comment
Safety laboratory
PK blood
PK urine
7
12-lead ECG
Vital signs (BP, PR)
Questioning for AEs and 
concomitant therapy 6
SCR
1
-21 to -1
Screening (SCR)1
x
x
x
1 / 2 / 3 / 4  (separated by a wash-out of least 7 days)
2 / 3 / 4 / 5
-1
-14:30
18:30
Admission to trial site2, allocation to 
treatment (visit 2 only)2
x5
x
-14:00
19:00
Dinner
1
-2:00
07:00
x8
x2
x2,9
x2,9
x2
-1:00
08.00
Cimetidine intake10  
0:00
09:00
Trial drug administration11
▲
0:20
09:20
x
│
0:40
09:40
x
│
1:00
10:00
x
│
1:30
10:30
x
│
2:00
11:00
240 mL fluid intake
x
│
2:30
11:30
x
│
3:00
12:00
x
│
4:00
13:00
Cimetidine intake10,12, thereafter lunch3
x
┼
x9
x9
x
5:00
14:00
x
│
6:00
15:00
x
│
8:00
17:00
Cimetidine intake10,12,snack (voluntary)3
x
┼
10:00
19:00
x
│
11:00
20:00
Dinner
│
12:00
21:00
Cimetidine intake10,12
x
┼
x 
2
24:00
09:00
Cimetidine intake10,12, discharge, 
breakfast (voluntary)3
x
x
┼
│
x9
x9
x
36:00
21:00
Ambulatory visit, cimetidine intake10,12
x
▼
x
3
47:00
08:00
Ambulatory visit
x
x
4
71:00
08:00
Ambulatory visit
x9
x9
5
95:00
08:00
Ambulatory visit
x9
x9
EOT
6
8 to 20
End of trial (EOT) examination4
x
x
x
x
1.
Subject must be informed and written informed consent obtained prior to starting any screening procedures. Screening 
procedures include physical examination, check of vital signs, ECG, safety laboratory (including drug screening), 
demographics (including determination of body height and weight, smoking status and alcohol history), relevant 
medical history, concomitant therapy and review of inclusion/exclusion criteria. 
2.
The time is approximate; the procedure is to be performed and completed within 3 h prior to drug administration.
3.
If several actions are indicated at the same time point, the intake of meals will be the last action.
4.
End of trial examination includes physical examination, vital signs, ECG, safety laboratory, recording of AEs and 
concomitant therapies.
5.
Only urine drug screening and alcohol breath test.
6.
AEs and concomitant therapies will be recorded throughout the trial, but will be specifically asked for at the time points 
indicated in the Flow Chart above.
7.
A blank urine sample (x) is to be obtained prior to administration of trial medication. Other urine samples are to be 
collected over the stated post-dose intervals (◄––│––│––►) 0-4, 4-8, 8-12, 12-24 and 24-36 h.
8.
To be taken within 20 min prior to first study drug administration
9.
Only in treatment periods with digoxin (R1 and T3) 
10. Only in treatment T3 and T5
11. In treatments R1 and T3: Cocktail; in treatments R2 and T5: 500 mg metformin
12. In T3 and T5 only: cimetidine intake to be done within 5 min after PK-sampling 


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FLOW CHART – PART 3
Period
Visit
Day
Planned time (relative to 
drug administration)
[h:min]
Approximate clock time  
of actual day [h:min]
Event and comment
Safety laboratory
PK blood
PK urine
7
12-lead ECG
Vital signs (BP, PR)
Questioning for AEs and 
concomitant therapy 6
SCR
1
-21 to -1
Screening (SCR)1
x
x
x
1 / 2 / 3 / 4 (separated by a wash-out of least 7 days)
2 / 3 / 4 / 5
-1
-14.30
18.30
Admission to trial site2, allocation to 
treatment (visit 2 only)2
x5
x
-14.00
19.00
Dinner
-13.00
20.00
probenecid intake10
1
-2:00
07:00
x8
x2
x2,9
x2,9
x2
-1.00
08.00
probenecid intake10
0:00
09:00
Trial drug administration11
▲
0:20
09:20
x
│
0:40
09:40
x
│
1:00
10:00
x
│
1:30
10:30
x
│
2:00
11:00
240 mL fluid intake
x
│
2:30
11:30
x
│
3:00
12:00
x
│
4:00
13:00
240 mL fluid intake, thereafter lunch3
x
┼
x9
x9
x
5:00
14:00
x
│
6:00
15:00
x
│
8:00
17:00
Snack (voluntary)3
x
┼
10:00
19:00
x
│
11:00
20:00
Dinner
│
12:00
21:00
x
┼
x 
2
24:00
09:00
Discharge, breakfast (voluntary) 3
x
x
┼
x9
x9
x
36:00
21:00
Ambulatory visit
x9
▼
x9
3
47:00
08:00
Ambulatory visit
x9
x9
4
71:00
08:00
Ambulatory visit
x9
x9
5
95:00
08:00
Ambulatory visit
x9
x9
EOT
6
8 to 20
End of trial (EOT) examination4
x
x
x
x
1.
Subject must be informed and written informed consent obtained prior to starting any screening procedures. Screening 
procedures include physical examination, check of vital signs, ECG, safety laboratory (including drug screening), 
demographics (including determination of body height and weight, smoking status and alcohol history), relevant 
medical history, concomitant therapy and review of inclusion/exclusion criteria.
2.
The time is approximate; the procedure is to be performed and completed within 3 h prior to drug administration.
3.
If several actions are indicated at the same time point, the intake of meals will be the last action.
4.
End of trial examination includes physical examination, vital signs, ECG, safety laboratory, recording of AEs and 
concomitant therapies. 
5.
Only urine drug screening and alcohol breath test.
6.
AEs and concomitant therapies will be recorded throughout the trial, but will be specifically asked for at the time points 
indicated in the Flow Chart above.
7.
A blank urine sample (x) is to be obtained prior to administration of trial medication. Other urine samples are to be 
collected over the stated post-dose intervals (◄––│––│––►) 0-4, 4-8, 8-12, 12-24 and 24-36 h.
8.
To be taken within 20 min prior to first study drug administration
9.
Only in treatment periods with digoxin (R1 and T4)
10. Only in treatment periods T4 and T6
11. In treatment periods R1 and T4: Cocktail; in treatment periods R3 and T6: 40 mg furosemide 


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TABLE OF CONTENTS
TITLE PAGE ......................................................................................................1
CLINICAL TRIAL PROTOCOL SYNOPSIS ................................................2
FLOW CHART – PART 1.................................................................................6
FLOW CHART – PART 2.................................................................................7
FLOW CHART – PART 3.................................................................................8
TABLE OF CONTENTS ...................................................................................9
ABBREVIATIONS.......................................................................................... 13
1.
INTRODUCTION.................................................................................... 17
1.1
MEDICAL BACKGROUND............................................................................18
1.1.1
Drug transporters .................................................................................18
1.1.2
Drug transporter substrates.................................................................20
1.1.3
Mutual interactions between digoxin, furosemide, metformin, 
rosuvastatin ...........................................................................................20
1.1.4
Drug transporter inhibitors .................................................................22
1.1.5
Endogenous markers for transporter activity....................................26
1.2
DRUG PROFILE...............................................................................................26
1.2.1
Digoxin ...................................................................................................26
1.2.2
Furosemide ............................................................................................27
1.2.3
Metformin..............................................................................................27
1.2.4
Rosuvastatin ..........................................................................................28
1.2.5
Verapamil ..............................................................................................28
1.2.6
Rifampin ................................................................................................29
1.2.7
Cimetidine..............................................................................................30
1.2.8
Probenecid .............................................................................................30
2.
RATIONALE, OBJECTIVES, BENEFIT - RISK 
ASSESSMENT ......................................................................................... 32
2.1
RATIONALE FOR PERFORMING THE TRIAL........................................32
2.2
TRIAL OBJECTIVES.......................................................................................32
2.3
BENEFIT - RISK ASSESSMENT ...................................................................33
2.3.1
Procedure-related risks ........................................................................33
2.3.2
Drug-related risks.................................................................................33
2.3.3
Risks related to the interaction of transporter substrates with
inhibitors................................................................................................34
2.3.4
Safety measures.....................................................................................36
2.3.5
Overall assessment................................................................................36
3.
DESCRIPTION OF DESIGN AND TRIAL POPULATION.............. 37
3.1
OVERALL TRIAL DESIGN AND PLAN ......................................................37
3.1.1
Administrative structure of the trial...................................................38


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3.2
DISCUSSION OF TRIAL DESIGN, INCLUDING THE CHOICE OF 
CONTROL GROUPS........................................................................................39
3.3
SELECTION OF TRIAL POPULATION ......................................................39
3.3.1
Main diagnosis for study entry............................................................39
3.3.2
Inclusion criteria...................................................................................39
3.3.3
Exclusion criteria ..................................................................................40
3.3.4
Removal of subjects from therapy or assessments.............................41
3.3.4.1
Removal of individual subjects................................................41
3.3.4.2
Discontinuation of the trial by the sponsor..............................42
3.3.5
Replacement of subjects .......................................................................42
4.
TREATMENTS........................................................................................ 43
4.1
TREATMENTS TO BE ADMINISTERED....................................................43
4.1.1
Identity of investigational products.....................................................43
4.1.2
Method of assigning subjects to treatment groups ............................45
4.1.3
Selection of doses in the trial................................................................45
4.1.4
Drug assignment and administration of doses for each subject .......46
4.1.5
Blinding and procedures for unblinding ............................................49
4.1.6
Packaging, labelling, and re-supply ....................................................49
4.1.7
Storage conditions.................................................................................50
4.1.8
Drug accountability ..............................................................................50
4.2
OTHER TREATMENTS, EMERGENCY PROCEDURES, 
RESTRICTIONS ...............................................................................................50
4.2.1
Other treatments and emergency procedures....................................50
4.2.2
Restrictions............................................................................................50
4.2.2.1
Restrictions regarding concomitant treatment .........................50
4.2.2.2
Restrictions on diet and life style.............................................51
4.3
TREATMENT COMPLIANCE.......................................................................51
5.
VARIABLES AND THEIR ASSESSMENT ......................................... 52
5.1
EFFICACY - CLINICAL PHARMACOLOGY.............................................52
5.1.1
Endpoints of efficacy.............................................................................52
5.1.2
Assessment of efficacy...........................................................................52
5.2
SAFETY..............................................................................................................52
5.2.1
Endpoints of safety................................................................................52
5.2.2
Assessment of adverse events...............................................................52
5.2.2.1
Definitions of adverse events...................................................52
5.2.2.2
Adverse event collection and reporting ...................................54
5.2.3
Assessment of safety laboratory parameters......................................56
5.2.4
Electrocardiogram................................................................................58
5.2.4.1
12-lead resting ECG.................................................................58
5.2.4.2
Continuous ECG monitoring ...................................................59
5.2.5
Assessment of other safety parameters...............................................59
5.2.5.1
Vital signs ................................................................................59


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5.2.5.2
Medical examinations..............................................................59
5.3
OTHER...............................................................................................................59
5.4
APPROPRIATENESS OF MEASUREMENTS.............................................59
5.5
DRUG CONCENTRATION MEASUREMENTS AND 
PHARMACOKINETICS..................................................................................60
5.5.1
Pharmacokinetic endpoints..................................................................60
5.5.1.1
Primary endpoints....................................................................60
5.5.1.2
Secondary endpoints................................................................60
5.5.1.3
Further parameters of interest..................................................60
5.5.2
Methods of sample collection...............................................................62
5.5.2.1
Plasma sampling for pharmacokinetic analysis.......................62
5.5.2.2
Urine sampling for pharmacokinetic analysis..........................64
5.5.2.3
Further investigations...............................................................65
5.5.3
Analytical determinations ....................................................................65
5.5.3.1
Analytical determination of analyte plasma concentration......65
5.5.3.2
Analytical determination of analyte urine concentration.........66
6.
INVESTIGATIONAL PLAN.................................................................. 67
6.1
VISIT SCHEDULE............................................................................................67
6.2
DETAILS OF TRIAL PROCEDURES AT SELECTED VISITS ................67
6.2.1
Screening period....................................................................................67
6.2.2
Treatment periods.................................................................................67
6.2.3
End of trial period.................................................................................68
7.
STATISTICAL METHODS AND DETERMINATION OF
SAMPLE SIZE......................................................................................... 69
7.1
STATISTICAL DESIGN – MODEL ...............................................................69
7.1.1
Objectives...............................................................................................69
7.1.2
Endpoints...............................................................................................69
7.1.3
Model......................................................................................................69
7.2
NULL AND ALTERNATIVE HYPOTHESES ..............................................70
7.3
PLANNED ANALYSES....................................................................................71
7.3.1
Primary analyses...................................................................................71
7.3.2
Secondary analyses ...............................................................................74
7.3.3
Safety analyses.......................................................................................74
7.3.4
Interim analyses ....................................................................................74
7.3.5
Pharmacokinetic analyses ....................................................................75
7.4
HANDLING OF MISSING DATA ..................................................................75
7.4.1
Safety......................................................................................................75
7.4.2
Plasma/urine drug concentration - time profiles ...............................75
7.4.3
Pharmacokinetic parameters...............................................................75
7.5
RANDOMISATION ..........................................................................................76
7.6
DETERMINATION OF SAMPLE SIZE ........................................................76


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8.
INFORMED CONSENT, DATA PROTECTION, TRIAL
RECORDS ................................................................................................ 78
8.1
STUDY APPROVAL, SUBJECT INFORMATION, AND INFORMED 
CONSENT ..........................................................................................................78
8.2
DATA QUALITY ASSURANCE .....................................................................78
8.3
RECORDS..........................................................................................................79
8.3.1
Source documents .................................................................................79
8.3.2
Direct access to source data and documents.......................................79
8.3.3
Storage period of records.....................................................................79
8.4
EXPEDITED REPORTING OF ADVERSE EVENTS .................................79
8.5
STATEMENT OF CONFIDENTIALITY.......................................................79
8.6
COMPLETION OF TRIAL..............................................................................80
9.
REFERENCES......................................................................................... 81
9.1
PUBLISHED REFERENCES...........................................................................81
9.2
UNPUBLISHED REFERENCES.....................................................................88
10. APPENDICES .......................................................................................... 89
11. DESCRIPTION OF GLOBAL AMENDMENT(S).............................. 90


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ABBREVIATIONS
ABC
ATP-binding cassette
ABCB1
Gene encoding P-gp
ABCG2
Gene encoding BCRP
AE
Adverse event
AESI
Adverse events of special interest
Aet1-t2
Amount of analyte eliminated in urine over the time interval t1 to t2
ANOVA
Analysis of variance
AUC
Area under the concentration-time curve of the analyte in plasma
AUC0-∞
Area under the concentration-time curve of the analyte in plasma over the 
time interval from 0 extrapolated to infinity
AUC0-tz
Area under the concentration-time curve of the analyte in plasma over the 
time interval from 0 to the last quantifiable data point
AUCt1-t2
Area under the concentration-time curve of the analyte in plasma over the 
time interval t1 to t2
%AUCtz-∞
The percentage of AUC0-∞obtained by extrapolation
BA
Bioavailability
BCRP
Breast cancer resistance protein
BI
Boehringer Ingelheim
BLQ
Below limit of quantification
BMI
Body mass index (weight divided by height squared)
BP
Blood pressure
CA
Competent authority
CI
Confidence interval
CLR
Renal clearance of the analyte in plasma
CLR,t1-t2
Renal clearance of the analyte in plasma over the time interval from time 
interval t1 to t2
CL/F
Apparent clearance of the analyte in plasma after extravascular 
administration
Cmax
Maximum measured concentration of the analyte in plasma
CML
Clinical Monitor Local
CRA
Clinical Research Associate
CRF
Case Report Form
CRO
Clinical Research Organization
CT
Concomitant treatment
CTP
Clinical trial protocol
CTR
Clinical trial report
CYP
Cytochrome P450


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DDI
Drug-drug interaction
DNA
Desoxyribonucleic acid
ECG
Electrocardiogram
EDTA
Ethylenediaminetetraacetic acid
EMA
European Medicines Agency
EOT
End of trial
F
Absolute bioavailability factor
FDA
U.S. Food and Drug Administration
fet1-t2
Fraction of administered drug excreted unchanged in urine over the time 
interval from t1 to t2
GCP
Good Clinical Practice
gCV
Geometric coefficient of variation
gMean
Geometric mean
HMG-CoA
3-Hydroxy-3-methylglutaryl-coenzyme A
HR
Heart rate
IC50
Half-maximal inhibitory concentration
IEC
Independent Ethics Committee
IRB
Institutional Review Board
ISF
Investigator site file
ITC
International Transporter Consortium
Ki
Inhibition constant
K3-EDTA
Tripotassium ethylenediaminetetraacetic acid
z
Terminal rate constant of the analyte in plasma
LC-MS/MS
Liquid chromatography tandem mass spectrometry
MATE
Multidrug and toxin extrusion protein
MATE1
Multidrug and toxin extrusion protein 1
MATE2-K
Multidrug and toxin extrusion protein 2-K
MedDRA
Medical Dictionary for Regulatory Activities
MRTpo
Mean residence time of the analyte in the body after oral administration
NOA
Not analysed
NOR
No valid result
NOS
No sample available
OAT1
Organic anion transporter 1
OAT3
Organic anion transporter 2
OATP
Organic anion transporting polypeptide
OATP1B1
Organic anion transporting polypeptide 1B1
OATP1B3
Organic anion transporting polypeptide 1B3
OCT
Organic cation transporter


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OCT2
Organic cation transporter 2
PD
Pharmacodynamic(s)
PE
Polyethylene
P-gp
P-glycoprotein
PK
Pharmacokinetic(s)
PKS
Pharmacokinetic set
PP
Polypropylene
PR
Pulse rate
q.d.
Quaque die, once daily
QT
Time between start of the Q-wave and the end of the T-wave in an 
electrocardiogram
QTc
Heart frequency-corrected QT interval
R
Reference treatment
RDC
Remote Data Capture
REP
Residual effect period
RS
Randomised set
SAE
Serious adverse event
SCR
Screening
SLC
Solute Carrier
SLC22A2
Gene encoding OCT2
SLC22A6
Gene encoding OAT1
SLC22A8
Gene encoding OAT3
SLC47A1
Gene encoding MATE1
SLC47A2
Gene encoding MATE2-K
SLCO1B1
Gene encoding OATP1B1
SLCO1B3
Gene encoding OATP1B3
SmPC
Summary of Product Characteristics
SOP
Standard Operating Procedure
ss
(at) steady state
SUSAR
Suspected Unexpected Serious Adverse Reaction
T
Test treatment
t1/2
Terminal half-life of the analyte in plasma
T2DM
Type 2 diabetes mellitus
TDMAP
Trial Data Management and Analysis Plan
tmax
Time from (last) dosing to the maximum measured concentration of the 
analyte in plasma
TMF
Trial master file
TS
Treated set


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TSAP
Trial statistical analysis plan
tz
Time of last measurable concentration of the analyte in plasma
ULN
Upper limit of normal
Vz/F
Apparent volume of distribution during the terminal phase after 
extravascular administration


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1.
INTRODUCTION
Drug transporters play an important role in drug absorption, distribution, and excretion. 
Inhibition of drug transporters by concomitantly administered drugs may cause clinically 
relevant drug-drug interactions (DDI) [P14-07656], [R10-1157]. With increasing recognition 
and understanding of the involvement of drug transporters in clinically relevant DDIs, 
thorough investigation of transporter-mediated DDI has become indispensable during drug 
development. Accordingly, regulatory guidances of both the European Medicines Agency 
(EMA) [P15-06991] and U.S. Food and Drug Administration (FDA, draft guidance) 
[P12-05791] recommend in vitro studies on the effect of new investigational products on 
drug transporters known to be involved in clinically relevant in vivo drug interactions.
If in vivo inhibition of a drug transporter known to be relevant for drug disposition cannot be 
excluded based on in vitro data, an in vivo study is recommended by regulatory agencies. A 
valuable approach to investigate several separate mechanisms underlying DDIs with the 
investigational product as the perpetrator in one single study is the “cocktail study”. This 
method, in which a mixture of well-characterized probe drugs is administered together with 
the new investigational product, is well established for investigation of cytochrome P450 
(CYP)-mediated DDIs. Both the EMA and FDA recommend the use of cocktail studies for 
investigation of transporter-mediated DDIs [P15-06991], [P12-05791]. 
The implementation of a cocktail study approach for transporter-mediated DDIs would be 
valuable because it allows for investigation of several key drug transporters in one single 
standardized clinical trial format, thereby reducing the total number of DDI studies (and 
volunteers involved) during a development program. 
Therefore, Boehringer Ingelheim aims at establishing a “transporter cocktail” consisting of 
well-characterized probe drugs. Based on available literature and our own in vitro data 
[R15-3479], four suitable market-approved substances (digoxin, metformin, furosemide, and 
rosuvastatin) were identified as probe drugs for key drug transporters involved in clinically 
relevant DDIs. 
In a first clinical trial in healthy volunteers, mutual interactions between these four substances 
were investigated in an in vivo study (BI trial number 352.2082, EudraCT number 2014-
001940-40, [c03246006-01]). In this trial, when all four probe drugs were given together, 
both the Cmax (maximum measured concentration of the analyte in plasma) and AUC0-tz (area 
under the concentration-time curve of the analyte in plasma over the time interval from 0 to 
the last quantifiable data point) of rosuvastatin were ~40% higher compared with when dosed 
alone (for details see Section 1.2.5). The relevance of this effect for the sensitivity of the 
cocktail to qualitatively and quantitatively detect and measure transporter-mediated DDIs is 
unclear; however, it is clear that a cocktail without interactions between the probe substrates 
is preferable [P15-06991], [P12-05791].
Based on available in vitro and in vivo data, as well as expert advice, it was hypothesized that 
either metformin or furosemide was the perpetrator of the Cmax and AUC increase of 
rosuvastatin. A second clinical trial in healthy volunteers was performed, in which the effect 
of different doses of furosemide or metformin on systemic rosuvastatin exposure was 


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investigated, in order to identify DDI-free doses (BI trial number 352.2094, EudraCT number 
2015-003052-46, [c08983809-01]). In this trial, rosuvastatin Cmax and AUC0-tz were increased 
by approximately 18% and 16% when given together with 5 mg furosemide. When 
administered concomitantly with 500 mg metformin hydrochloride, rosuvastatin Cmax and 
AUC0-tz were increased by approximately 54% and 52%, respectively. However, 10 mg and 
50 mg metformin hydrochloride or 1 mg furosemide had only minor effects on systemic 
rosuvastatin exposure.
Therefore, the transporter cocktail doses were adjusted accordingly in order to minimize 
mutual interactions. The adjusted cocktail consists of 0.25 mg digoxin and 10 mg 
rosuvastatin (as in the first cocktail trial, 352.2082 [c03246006-01]) and of reduced doses of 
10 mg metformin and 1 mg furosemide (as determined from the second trial, 352.2094
[c08983809-01]). In the third clinical trial (BI trial number 352.2096 [c13060859-01], 
EudraCT number 2016-001893-14, report not yet available), it could be demonstrated that the 
cocktail components used in the adjusted dose strengths lack any mutual interaction, as the 
90% confidence intervals of digoxin, furosemide, metformin and rosuvastatin AUC and Cmax
(gMean) were all within the standard bioequivalence acceptance range.
The aim of the current trial is to investigate the effect of known drug transporter inhibitors on 
the transporter probe substrates of the described cocktail. Special attention is also given to the 
question of whether different inhibitory effects are observed between subtherapeutic and 
therapeutic doses of metformin (500 mg metformin to be tested in Part 2) and furosemide 
(40 mg furosemide to be tested in Part 3). 
1.1
MEDICAL BACKGROUND
1.1.1
Drug transporters
As stated in the International Transporter Consortium’s (ITC) white paper [R10-1157], a 
limited number of solute carrier (SLC) and ATP-binding cassette (ABC) transporters have
been identified as being relevantly involved in drug disposition. New investigational drugs 
may cause DDIs by inhibition or induction of one or more of these transporters. 
Based on the ITC’s recommendations, regulatory agencies explicitly state drug transporters 
that should be investigated in vitro for inhibition by new investigational drugs during drug 
development. The specific transporters explicitly referred to by both the EMA guideline 
[P15-06991] and the FDA draft guidance [P12-05791] are the ABC transporters, P-
glycoprotein (P-gp, gene symbol ABCB1) and breast cancer resistance protein (BCRP, 
ABCG2), and the SLC transporters, organic anion transporting polypeptide 1B1 (OATP1B1, 
SLCO1B1), OATP1B3 (SLCO1B3), organic cation transporter 2 (OCT2, SLC22A2), organic 
anion transporter 1 (OAT1, SLC22A6), and OAT3 (SLC22A8).
P-gp is localized at the luminal membrane of the small intestine and the blood brain barrier, 
where it is involved in limitation of oral bioavailability or limitation of entry into the central 
nervous system, respectively. Moreover, expressed in the apical membrane of hepatocytes 
and renal proximal tubule cells, P-gp is involved in excretion of drugs such as the cardiac 
glycoside digoxin. Administration of strong P-gp inhibitors such as quinidine, clarithromycin, 
or ritonavir may result in increased plasma concentrations and decreased clearance of drugs 
that are P-gp substrates [P14-07656], [R10-1157].


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Both OATP1B1 and OATP1B3 are expressed highly, if not exclusively, in the basolateral 
membrane of hepatocytes, where these transporters mediate the uptake of organic anions such 
as inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) [P13-11134]. 
Inhibition of hepatocellular OATPs by, for example, the calcineurin inhibitor cyclosporine,
increases plasma concentrations of OATP substrates such as the HMG-CoA inhibitor 
rosuvastatin without relevantly affecting terminal elimination half-life (t1/2) [P14-07833].
OCT2 is localized at the basolateral membrane of renal proximal tubule cells where it 
mediates uptake of endogenous compounds such as creatinine or of drugs such as the 
antidiabetic metformin [R10-1157]. The H2-receptor antagonist cimetidine decreases 
metformin renal clearance (CLR) and increases metformin plasma concentrations, and this 
effect has for some time been attributed to OCT2 inhibition [R10-1157], [P12-05791]. In 
vitro obtained half-maximal inhibitory concentrations (IC50) and inhibition constants (Ki) of 
cimetidine for inhibition of OCT2-mediated metformin transport, however, largely exceed 
clinically reached unbound cimetidine plasma concentrations, and this fact has put the role of 
OCT2 in this interaction into question [R14-2106], [R13-3303], [P13-11134]. However, due 
to its exclusive role in proximal tubular cell uptake of organic cations, inhibition of OCT2 is 
expected to cause clinically relevant DDIs, and the ITC recommends OCT2 inhibition to be 
investigated in vitro and in vivo for new molecular entities [R13-3303].
OAT1 and OAT3 mediate renal proximal tubule cell uptake of organic anions such as the 
loop diuretic furosemide [R10-1157]. OAT inhibition by e.g., probenecid, causes a decrease 
in furosemide CLR and an increase of furosemide plasma concentrations [P14-07656]. 
BCRP expressed in the luminal membrane of the gastrointestinal tract and blood brain barrier 
is involved in limitation of oral bioavailability or limitation of entry into the central nervous 
system, respectively [R10-1157]. Moreover, BCRP is found in liver, kidney, brain 
endothelium, mammary tissue, testis, and placenta [R10-1157]. Rosuvastatin is a substrate of 
BCRP, and genetic variants of BCRP may relevantly affect rosuvastatin pharmacokinetics 
(PK) and action [P13-15579]. 
In addition to these transporters, others may be relevantly involved in drug disposition and 
DDIs. The EMA guideline states that investigation of inhibition of MATE1 (SLC47A1) and 
MATE2-K (SLC47A2) may be considered during drug development. 
MATE1 and MATE2-K are localized to the apical membrane of renal tubule cells and 
mediate the export step in renal tubular secretion of organic cations [R13-3303]. Inhibition of 
MATEs is now believed to be the major mechanism underlying the cimetidine-metformin 
interaction [R13-3303]. The ITC recommends investigating MATE inhibition by new 
compounds in development in vitro and, if necessary, in vivo [R13-3303].
Based on available literature data, expert opinion and our own in vitro data, four drugs were 
proposed as probes for clinically relevant drug transporters: The cardiac glycoside digoxin 
(P-gp), the loop diuretic furosemide (OAT1 and OAT3), the antidiabetic metformin (OCT2, 
MATE1, and MATE2-K), and the HMG-CoA reductase inhibitor rosuvastatin (OATP1B1, 
OATP1B3, and BCRP) [R15-3479]. Metabolism of these drugs is minor or negligible, which 
is expected to allow for investigating transporter-mediated DDIs without enzyme inhibition 
relevantly confounding results. All of these drugs are characterized as substrates of the 
respective transporters in vitro, and in vivo interactions by inhibitors of these transporters are 
well documented [P14-07656], [P13-11134], [P14-07833], [R13-3303], [R10-1157].


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1.1.2
Drug transporter substrates
Digoxin is a substrate of P-gp. It is recommended by the EMA as an in vivo probe drug for P-
gp-mediated drug-drug interactions when inhibition of renal tubular secretion by the putative 
perpetrator is of interest [P15-06991]. DDIs mediated by P-gp inhibition involving digoxin 
are well documented [P14-07656]. 
Furosemide is a substrate of both OAT1 and OAT3, and OAT inhibition by e.g., probenecid, 
decreases furosemide CLR and increases furosemide plasma concentrations [P14-07656], 
[P12-05791].
Metformin is a prototypical substrate of OCTs and MATEs for both in vitro and in vivo
investigations [P14-07656], [P13-11134]. Accordingly, the ITC recommends metformin as in 
vivo probe for investigation of DDIs mediated by inhibition of OCT2 and/or MATE1 or 
MATE2-K [R13-3303].
Rosuvastatin is a substrate of OATP1B1, OATP1B3 and BCRP, and inhibition of these 
proteins by, e.g., gemfibrozil (OATP1B1 and OATP1B3) or fostamatinib (BCRP), has been 
reported to result in increased rosuvastatin plasma concentrations [P14-07656], [R16-2202].
1.1.3
Mutual interactions between digoxin, furosemide, metformin, rosuvastatin
Mutual interactions between digoxin, furosemide, metformin and rosuvastatin have been 
investigated in 3 clinical trials (BI trial numbers 352.2082 [c03246006-01], 352.2094 
[c08983809-01], and 352.2096 [c13060859-01]). 
In the first study, 352.2082 [c03246006-01], the combined administration of 0.25 mg 
digoxin, 5 mg furosemide, 500 mg metformin and 10 mg rosuvastatin resulted in a 40% 
increase of rosuvastatin Cmax and AUC0-tz and in a 20% decrease of furosemide Cmax
compared to the single administration of rosuvastatin and furosemide. Both effects were
augmented by a higher dose of 1000 mg metformin (also tested in this study), indicating a 
causal role of metformin in these interactions, which is in line with literature [P14-10279], 
[R02-2556].
Focusing on potential offender drugs, the trial 352.2094 [c08983809-01] investigated the 
effect of different doses of metformin (10 mg, 50 mg, 500 mg) and furosemide (1 mg, 5 mg) 
on rosuvastatin kinetics. The highest doses of both perpetrators caused a significant increase 
of rosuvastatin exposure, while the tested lower doses had only minor effects (see 
Table 1.1.3.1 and 1.1.3.2).
Table 1.1.3: 1
Pharmacokinetic parameters [gMean] of rosuvastatin after single oral intake 
of 10 mg rosuvastatin alone (R), 10 mg rosuvastatin + 10 mg metformin (T1),
10 mg rosuvastatin + 50 mg metformin (T2) and 10 mg rosuvastatin + 500 
mg metformin (T3) [c08983809-01]
R (N=16)
T1 (N=15)
T2 (N=16)
T3 (N=15)
AUC0-tz [nmol*h/L]
81.5
87.8
88.7
126
% change to R
-
+7.7%
+8.8%
+54.6%
Cmax [nmol/L]
9.04
9.59
9.94
14.0
-
+6.1%
+10%
+54.9%


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Table 1.1.3: 2
Pharmacokinetic parameters [gMean] of rosuvastatin after single oral intake
of 10 mg rosuvastatin alone (R), 10 mg rosuvastatin + 1 mg furosemide (T4) 
and 10 mg rosuvastatin + 5 mg furosemide (T5) [c08983809-01]
R (N=16)
T4 (N=15)
T5 (N=16)
AUC0-tz [nmol*h/L]
81.5
91.5
97.2
% change to R
-
+12.3%
+19.2%
Cmax [nmol/L]
9.04
9.9
10.8
-
+9.5%
+19.5%
Based on these results, the doses of metformin and furosemide were reduced to 10 mg and 1 
mg (digoxin and rosuvastatin doses remained unchanged). The mutual interactions of 0.25 
mg digoxin, 1 mg furosemide, 10 mg metformin and 10 mg rosuvastatin were investigated in 
trial 352.2096 [c13060859-01]. After dose adjustment, no interactions between the cocktail 
components could be observed - the 90% confidence intervals of digoxin, furosemide, 
metformin and rosuvastatin AUC and Cmax values were within the bioequivalence 
boundaries:
Table 1.1.3: 3
Adjusted gMean values and relative bioavailability of digoxin, furosemide, 
metformin and rosuvastatin given as monotherapy (Reference) or cocktail 
(Test) in trial 352.2096 [c13060859-01]
Cocktail (T)
Monotherapy (R)
Ratio T/R [%]
90% CI
Digoxin AUC0-tz [nmol*h/L]
Digoxin Cmax [nmol/L]
11.549 (N=28)
1.262 (N=28)
11.981 (N=28)
1.355 (N=28)
96.39
93.17
88.22; 105.33
83.49; 103.97
Furosemide AUC0-tz [nmol*h/L]
Furosemide Cmax [nmol/L]
163.614 (N=28)
86.275 (N=28)
159.434 (N=30)
82.990 (N=30)
102.62
103.96
93.82; 112.25
93.60;115.46
Metformin AUC0-tz [nmol*h/L]
Metformin Cmax [nmol/L]
1283.797 (N=28)
225.156 (N=28)
1316.790 (N=29)
229.171(N=29)
97.49
98.25
93.54; 101.61
91.85;105.09
Rosuvastatin AUC0-tz [nmol*h/L]
Rosuvastatin Cmax [nmol/L]
81.925 (N=28)
8.135 (N=28)
78.016 (N=29)
7.801(N=29)
105.01
104.28
96.39; 114.40
94.95; 114.53
Given the lack of mutual interactions, 0.25 mg digoxin, 1 mg furosemide, 10 mg metformin 
and 10 mg rosuvastatin can be administered together as a cocktail.


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1.1.4
Drug transporter inhibitors
Verapamil
According to the FDA guideline on drug-drug-interactions, several compounds may be used 
as in vivo inhibitors of P-gp [P12-05791]. Among them, verapamil is one of the best 
characterised P-gp inhibitors. Several interactions of verapamil with sensitive P-gp substrates 
like digoxin [R07-2637], fexofenadine [R07-4432] and dabigatran etexilate [U09-1052-01] 
have been reported.
Verapamil-digoxin: The combined administration of digoxin (0.125 mg t.i.d.) and verapamil 
(80 mg t.i.d./120 mg t.i.d.) to 36 healthy men over a treatment period of 2 weeks increased 
the digoxin plasma concentration by 77% /61% [R10-5948]. Rodin et al report an increase of 
digoxin (0.25 mg bid for 14 days) AUC and Cmax of 50% and 44% after co-administration of 
verapamil (80 mg b.i.d. for 4 days followed by 80 mg t.i.d. for 10 days) in healthy men 
[R07-2637]. 
Pedersen et al report an increase of digoxin concentration from 0.21 ng/mL to 0.34 ng/mL 
(about +60%) after one week co-administration of verapamil 80 mg t.i.d. in healthy subjects. 
Following 5 further weeks of digoxin (62.5 µg b.i.d.) and verapamil co-administration, the 
digoxin concentrations reduced to 0.27 ng/mL (about +30% compared to baseline) 
[R10 5949]. These data indicate that the short-term effect of verapamil on digoxin exposure 
may be attenuated after long-term verapamil administration. 
This observation has been confirmed by Lemma et al, who investigated the effect of a 38 day 
treatment period with verapamil (240 mg daily) on fexofenadine exposure. As with digoxin,
fexofenadine is a model substrate of P-gp [P12-05791]. While the first dosing of verapamil 
increased the fexofenadine Cmax from 114 to 165 ng/mL, continuous verapamil administration 
caused a reduction of fexofenadine Cmax to 148 ng/mL on Day 10 and to 126 ng/mL on Day 
38 [R07-4432].
According to Lemma et al, the underlying mechanism is a P-gp induction that counteracts the 
initial inhibition. Considering this assumption, a single dose of verapamil is expected to cause 
even greater effects on digoxin exposure compared to those reported by Belz and Rodin. In 
both trials, verapamil had been administered over 2 weeks, indicating on overlap of inhibitory 
and inductive effects. After a single dose of verapamil, this overlap should not be present: the 
inhibitory effect should not be attenuated by supposed induction.
In the verapamil-dabigatran study, multiple dosing of verapamil (120 mg q.d., 120 mg b.i.d.) 
increased the bioavailability of dabigatran by about 40-60%, while the single dose 
administration of 120 mg verapamil increased dabigatran bioavailability by more than 100%. 
The effect size also depended on the timing of perpetrator drug administration: while 120 mg 
verapamil (single dose) given concomitantly with dabigatran increased its Cmax and AUC by 
+143% and +108%, the maximum effect was achieved when verapamil was given 1 h prior to 
dabigatran (179% and +143%) [U09-1052-01].
Considering the reported long-term effects of verapamil administration and taking into 
account the experience gained in the verapamil-dabigatran study, a single dose of 120 mg 
verapamil given 1 h prior to the cocktail will be used in this trial to achieve a maximum effect 
on digoxin exposure (worst-case scenario).


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Verapamil-rosuvastatin: The interaction of verapamil with rosuvastatin or other probe drugs 
of OATP1B1/1B3 has not been investigated so far in vivo or in vitro.
Verapamil-metformin: The combined administration of verapamil (given for a total of 4 days) 
and metformin (single dose of 750 mg) increased AUC and Cmax of metformin by 7.5% and 
9%, respectively [R17-1791].
Verapamil-furosemide: The interaction of verapamil with furosemide or other probe drugs of 
OAT1 and OAT3 has not been investigated so far in vivo or in vitro.
Rifampin
According to the FDA guideline on drug-drug-interactions, several compounds may be used 
as in vivo inhibitors of OATP1B1/1B3 [P12-05791]. Among them, rifampin is one of the best 
characterised inhibitors. Several interactions of rifampin with sensitive OATP1B1 and/or 1B3 
substrates, like bosentan [R17-1793], ezetimibe [R17-1792], atorvastatin [R16-5125], 
rosuvastatin and pitavastatin [R15-4771], have been described. 
In these studies, a single dose of 600 mg rifampin has been used for OATP inhibition. This is 
to avoid counteracting induction effects, as rifampin is a known inducer of the pregnane X-
receptor (PXR), which mediates the transcriptional activation of metabolic enzymes and drug 
transporters [R17-1789], [S10-2044], [R07-2252]. Thus, a single dose of 600 mg rifampin 
caused a 5-fold increase of bosentan trough levels, while continuous administration of 600 
mg rifampin over 6 days reduced bosentan Cmax to 47%. Bosentan is a model substrate of 
OATP1B1 and is metabolised by CYP3A4. The effect of rifampin single dose is attributed to 
OATP inhibition, while multiple doses of rifampin caused an induction of CYP3A4 which 
resulted in a substantial decline of bosentan exposure [R17-1793].
Rifampin-rosuvastatin: Part 1 of this trial aims to demonstrate the effect of the OATP1B1/ 
1B3 inhibitor rifampin on the exposure of the OATP1B1/1B3 model substrate rosuvastatin. 
In the literature, a 10-fold increase of rosuvastatin Cmax has been reported after co-
administration of a single dose of 600 mg rifampin [R15-4771], [R17-1790]. In both cases,
the effect of rifampin on rosuvastatin AUC was less pronounced (about 5-fold increase). A 
single dose of 600 mg rifampin will also be used in the current trial.
Rifampin-digoxin: The overlap of acute inhibition and chronic induction (of intestinal P-
glycoprotein) determines the net effect of rifampin on digoxin exposure. Therefore, a timely 
separation of the victim drug (here: digoxin) administration is crucial for detection of 
rifampin inductive effects. After administration of 600 mg rifampin for 28 days, the exposure 
of 0.5 mg digoxin was determined on Day 28 (digoxin was given 1 h after the last rifampin 
dose), Day 35 (i.e. 7 days after the last rifampin dose), on Day 42 and 56. Referring to Day 
56 values (=100%), AUC and Cmax of digoxin were 146%/149% (Day 28), 68%/69% (Day 
35), and 98%/88% (Day 42) [R13-2865]. 
The Day 28 results confirm a stronger influence of acute inhibitory effects, although a full 
induction of intestinal P-gp has to be assumed after 4 weeks rifampin dosing [R05-1427]. For 
Part 1 of the current trial, a much greater rifampin effect on digoxin exposure is to be 
expected, because a counteracting induction is not given when a single dose of rifampin is 
given together with the P-gp substrate digoxin as a cocktail component. 


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Different net effects between staggered (to measure only induction) and simultaneous (to 
measure inductive plus inhibitory effects) administration of rifampin and digoxin have also 
been confirmed by Kirby et al. The co-administration of a single dose of 0.5 mg digoxin and 
600 mg rifampin (last dose of a 14-days treatment period) increased digoxin AUC0-4h and 
Cmax by 37% and 55%, respectively. In contrast, staggered dosing of digoxin (12 h after 
rifampin) reduced digoxin AUC0-4h and Cmax by 24% [P17-05612].
Rifampin-metformin: Administration of 600 mg rifampin over 10 days caused only a slight 
increase (+10%) of metformin exposure in healthy subjects [R11-2145]. This might indicate 
that rifampin does not affect metformin disposition. On the other hand, an overlap of 
potential inhibitory and inductive effects following a 10 day rifampin treatment cannot be 
excluded. Therefore, the result described above might not be predictive for the interaction to 
be expected in the current trial, whereby a single dose of rifampin is used (no induction).
Rifampin-furosemide: The interaction of rifampin and furosemide has not been investigated 
in vivo. Based on in vitro data, rifampin is an inhibitor of OAT1 and OAT3 with IC50-values 
of 48.1 µM and 30.2 µM [R17-1794]. Peak concentrations after a single dose of 600 mg 
rifampin are in the range of 7-10 µg/mL (12 µM) [R16-5125], which is 2-4 fold below the 
IC50-values. Based on cut-off values given in the respective DDI-guidelines [P12-05791], 
[P15-06991] an interaction between rifampin and furosemide mediated by OAT-inhibition 
has to be regarded as possible.
Cimetidine
According to the International Transporter Consortium, cimetidine is a recommended 
compound for testing inhibition of OCT2, MATE1 and MATE2-K, which share overlapping 
substrates. The hydrophilic organic cation metformin is a probe substrate of OCT2 and 
MATEs [R13-3303]. 
Cimetidine-metformin: The effect of cimetidine on metformin was investigated first by 
Somogyi. The combined administration of cimetidine (400 mg b.i.d.) and metformin (250 mg 
q.d.) for 5 days increased metformin Cmax and AUC by 73% and 46%, respectively 
[R99-0743]. This effect size was confirmed by Wang et al, who investigated the cimetidine 
metformin interaction depending on the OCT genotype (referring to the 808G>T SNP) in 
healthy male Chinese subjects. In wild-type carriers (GG), cimetidine increased AUC and 
Cmax of a single dose of 500 mg metformin by 50% (no effect in GT and TT group). In this 
trial, 400 mg cimetidine b.i.d. was given for 6 days and the last cimetidine dose was 
administered in the morning of Day 7, two hours prior to metformin [R09-6148].
Cimetidine-digoxin: In 6 healthy subjects, cimetidine caused a significant increase of digoxin 
exposure (Cmax: +50%; AUC: +23%). Both drugs were given single dose [R17-1845]. In 
patients, the disposition of i.v. digoxin has not been affected by cimetidine; in particular, 
renal clearance and urinary excretion of digoxin did not change following cimetidine 
administration [R17-1844]. According to in vitro data, cimetidine did not inhibit P-
glycoprotein [R11-4702].
Cimetidine-rosuvastatin: The interaction of cimetidine and rosuvastatin has not been 
investigated so far in vivo. Uptake of estradiol 17ß-D-glucuronide by OATP1B1 transfected 
HEK cells was not inhibited by cimetidine [R11-4702].


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Cimetidine-furosemide: The interaction of cimetidine and furosemide has not been 
investigated so far in vivo. In vitro, the uptake of 17α-ethinylestradiol-3-O-sulfate (substrate 
of OAT3 and OAT4, but not of OCT2 and MATE) by OAT3 transfected HEK-293 cells was 
inhibited by cimetidine in a concentration dependent manner [R17-1842]. This is in line with 
the FDA Draft Guideline, which lists cimetidine as an inhibitor of OAT3 [P12-05791]. Thus,
cimetidine may potentially influence kinetics of the OAT3 substrate furosemide.
In drug-drug interaction studies using cimetidine as a perpetrator compound, different dosing 
regimens have been applied. Most of these trials used a pre-dosing of 400 mg cimetidine 
b.i.d. for 1-6 days [R09-6148], [R99-0743], [R99-0753], [P86-21478]. However, in the 
cimetidine – procainamide interaction study, the first dose of cimetidine (400 mg) was given 
1 h prior to procainamide. Further doses of 200 mg cimetidine were administered after 4 h, 8 
h and 12 h [R96-0654]. 
Considering the cimetidine plasma half-life of 2 h, pre-dosing days are not planned in the 
current trial. Following the dosing schedule of the cimetidine-procainamide study, cimetidine 
will be administered 1 h prior to and 4h, 8h, 12h after metformin dosing on Day 1. Two 
additional cimetidine doses will be given on Day 2 in order to cover the elimination phase of 
metformin completely. Each dose of cimetidine will be 400 mg, resulting in a total daily dose 
of 1600 mg on Day 1, which is in line with the highest therapeutic dose that is used in the 
clinic [R17-1838].
Probenecid
According to the FDA guideline, probenecid is the recommended compound to investigate 
the inhibition of OAT1 and OAT3 in vivo [P12-05791]. Several interactions of probenecid 
with sensitive substrates of OAT1 and OAT3, like acyclovir [R17-1858], zidovudine 
[R17-1860] and furosemide [R17-1861], have been reported.
Probenecid-furosemide: The influence of probenecid on orally administered furosemide was 
first described by Smith et al. Probenecid increased the AUC of a single dose of 40 mg 
furosemide by a factor of 3.1, while the renal clearance of the loop diuretic was reduced to 
20% [R17-1861]. Comparable effects have been observed by Vree et al, who describe an 
increase of furosemide (single dose of 80 mg) AUC by a factor of 2.7 and a reduction of its 
renal clearance to 34% [R17-1859]. 
Probenecid-digoxin: The interaction of probenecid with the P-gp substrate digoxin has not 
been investigated so far in vivo or in vitro.
Probenecid-metformin: The interaction of probenecid with metformin has not been 
investigated so far in vivo or in vitro. Probenecid significantly reduced the renal clearance of 
cimetidine (given intravenously) by decreasing the GFR and the secretory clearance. Plasma 
concentrations of cimetidine were not affected [R96-0527]. As with metformin, cimetidine is 
a model substrate of OCT2 [P12-05791]. Thus, the described effect could be at least partly 
mediated by OCT2 inhibition by probenecid and, thus, a potential probenecid-metformin 
interaction cannot be ruled out.
Probenecid-rosuvastatin: The interaction of probenecid with rosuvastatin or other probe 
drugs of OATP1B1/1B3 has not been investigated so far in vivo or in vitro.


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In drug-drug interaction studies using probenecid
as a perpetrator compound, different 
dosing regimens have been applied, including a pre-dosing of probenecid over 3 days. The 
current trial follows the dosing regimen of the Smith study that reported the greatest effect on 
furosemide exposure [R17-1861]: 1000 mg probenecid will be administered the evening 
before and again 1 h prior to furosemide administration. Considering the short elimination 
half-life of furosemide (of about 1 h), further administrations of the inhibitor are not required. 
1.1.5
Endogenous markers for transporter activity
Endogenous compounds including compounds taken with food such as thiamine, carnitine, 
N1-methylnicotinamide, creatinine, 6β-hydroxycortisol, fatty acid dicarboxylates, 
coproporphyrins, bile acids, bile acid conjugates and others are substrates of membranous
transport proteins and have been proposed as endogenous markers for the activity of drug 
transporters [R16-2177], R16-2178]. The use of endogenous compounds as markers for the 
activity of drug transporters (or enzymes) may contribute to the assessment of the DDI risk of 
new compounds (see e.g. [R16-2176]); however, further research is necessary in this area.
Such research is not part of this trial, although following the planned analyses of this trial,
additional, left-over and/or back-up samples may be used for such examinations (as “further 
use” or “further investigations”, see Section 5.5.2.3).
1.2
DRUG PROFILE
1.2.1
Digoxin
The cardiac glycoside digoxin is a potent and specific inhibitor of the membrane-bound 
Na+/K+ ATPase. In cardiomyocytes, Na+/K+ ATPase inhibition increases intracellular Ca++
concentrations during electromechanic coupling. Digoxin has positive inotropic and 
bathmotropic and negative chronotropic and dromotropic cardial effects. Use of digoxin is 
indicated in patients with tachyarrhythmia absoluta due to atrial fibrillation or flutter, with 
congestive heart failure due to systolic dysfunction, or with paroxysmal atrial fibrillation or 
flutter [R16-2173].
Digoxin oral bioavailability is ~60-80%. Its volume of distribution is large at 510 L in 
healthy volunteers; plasma protein binding is ~20%. Digoxin is eliminated principally 
(~80%) by the kidney, with a t1/2 of approximately 40 h [R16-2173].
In therapy, digoxin is given first as a loading regime of up to 0.75 mg digoxin/day, followed 
by a maintenance regime controlled by therapeutic drug monitoring.
Adverse reactions to digoxin include cardiac arrhythmia, gastrointestinal complaints and 
diverse symptoms of the central nervous system such as headache or psychiatric disorders. 
Moreover, disturbances of colour vision may occur. In rare cases, gynecomasty, myasthenia, 
thrombocytopenia, hypersensitivity reactions or lupus erythematodes have been observed 
[R16-2173]. A complete listing of adverse reactions, including frequency of occurrence, may 
be found in the current SmPC.
Symptoms of digoxin toxicity include cardial, gastrointestinal, and central side effects. 
Moreover, hyperkalemia may occur in acute overdosing. Life-threatening intoxications were 
observed after doses of ≥ 10 mg digoxin [R16-2173].
For a more detailed description of digoxin`s profile please refer to the SmPC [R16-2173].


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1.2.2
Furosemide
Furosemide is a loop diuretic indicated for treatment of oedema (cardiac, hepatic, renal, or 
due to burns), arterial hypertension, oliguria or pulmonary oedema. By inhibition of the 
Na+/2Cl-/K+ carrier in the distal ascending limp of Henle’s loop, furosemide increases 
diuresis and excretion of sodium, potassium, calcium, and magnesium [R16-2538].
After administration of furosemide as an oral solution, oral bioavailability is ~80%. Maximal 
plasma concentrations (tmax) are observed at ~1 h. Plasma protein binding is 95%, and VZ/F is 
0.2 L/kg. Furosemide is eliminated principally as unchanged substance, to two thirds by the 
kidney (glomerular filtration and secretion) and to one third by excretion into bile 
[R16-2538].
In therapy, initial doses of oral furosemide are normally 40 mg, but higher doses of over 
200 mg are possible in individual cases when diuresis with lower doses is not sufficient. 
Maintenance dose is normally 40-80 mg/day [R16-2538].
Adverse reactions include mainly electrolyte disorders including dehydration and 
hypovolemia, hearing disorders and allergic reactions including skin reactions. For a 
complete listing of adverse reactions, including frequency of occurrence, please refer to the 
current version of the SmPC [R16-2538].
Symptoms of overdosing are characterized by excessive loss of electrolytes which may lead 
to hypotension, syncope, or delirium [R16-2538].
For a more detailed description of furosemide`s profile please refer to the SmPC [R16-2538].
1.2.3
Metformin
Metformin is an oral antidiabetic that reduces plasma glucose concentrations by decreasing 
intestinal glucose absorption and hepatic glucose production and by enhancing glucose 
utilization in peripheral tissues. Thus, metformin reduces basal (fasting) and postprandial 
plasma glucose in patients with type 2 diabetes mellitus (T2DM). However, metformin does 
not stimulate insulin secretion and is not causally related to hypoglycaemia in patients with 
T2DM or in healthy volunteers. Metformin is used in patients with T2DM if sufficient 
reduction of plasma glucose is not reached by diet and exercise alone [R17-3544].
After oral administration, tmax is reached at ~2.5 h, and oral bioavailability (of 500-850 mg) is 
~50-60%. Plasma protein binding is negligible, however, metformin enters erythrocytes 
which probably compose a deep distribution compartment. CLR of metformin is high 
(estimated population mean of 507 ± 129 mL/min) and the principal mode of elimination,
with a t1/2 of approximately 6.5 h [R17-3544], [P11-01873]. 
The initial metformin dose is normally 500-850 mg up to three times daily. The dose may be 
increased to up to 3000 mg per day [R17-3544].
Most frequent adverse reactions to metformin are gastrointestinal complaints such as nausea, 
vomiting, diarrhoea, abdominal pain, or appetite loss. Moreover, metformin may cause 
changed taste (e.g., metallic taste), and, very rarely or with unknown frequency, respectively, 
skin reactions or abnormal liver function tests. For a complete listing of adverse reactions,
including frequency of occurrence, please refer to the current version of the SmPC 
[R17-3544].


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In addition, metformin may, very rarely, cause lactic acidosis. This is a life-threatening 
disorder caused by metformin accumulation, principally in diabetic patients with severe renal 
insufficiency. In case of unspecific symptoms such as muscle cramps in combination with 
gastrointestinal disorder or severe asthenia, lactic acidosis needs to be taken into account. 
Other possible symptoms are dyspnoea, abdominal disorders, hypothermia, and coma 
[R17-3544]. To our knowledge, lactic acidosis has not been observed in healthy volunteers, 
so far. High doses of up to 85 g metformin did not cause hypoglycaemia. However, lactic 
acidosis has been observed with this severe overdosing [R17-3544].
For a more detailed description of metformin`s profile please refer to the SmPC [R17-3544].
1.2.4
Rosuvastatin
Rosuvastatin is an HMG-CoA reductase inhibitor indicated for treatment of 
hypercholesterolemia and for prophylaxis of cardiovascular events. Inhibition of HMG-CoA 
decreases hepatic cholesterol production which, in turn, stimulates hepatocellular uptake of 
low-density lipoproteins [R17-2886]. 
After oral administration, maximal rosuvastatin plasma concentrations are reached at ~5 h. 
Oral bioavailability is ~20%, plasma protein binding is ~90%, and VZ/F is ~134 L. The liver 
is a principal compartment of distribution, with hepatocellular uptake being mediated mainly 
by OATP1B1 and, to a lesser degree, by OATP1B3. Elimination is mainly via the feces and 
to a lesser degree, via urine (principally via renal tubular secretion), with a t1/2 of 19 h
[R17-2886], [P14-07833].
In therapy, the initial rosuvastatin dose is 5-10 mg once daily (q.d.), and during therapy, the 
daily dose may be increased to up to 40 mg [R17-2886].
Adverse reactions to rosuvastatin are normally mild and transient. Myalgia and myopathy 
with concomitant increase of creatine kinase, and, in rare cases, rhabdomyolysis have been 
observed during rosuvastatin therapy. Moreover, as for other HMG-CoA reductase inhibitors, 
a dose-dependent increase of liver transaminases may be observed [R17-2886].
For a complete listing of adverse reactions, including frequency of occurrence, please refer to 
the SmPC [R17-2886].
1.2.5
Verapamil
Verapamil, a calcium antagonist, inhibits the calcium stream across the muscle cell
membranes. It is approved for the treatment of angina pectoris, arterial hypertension and
paroxysmal supraventricular tachycardias.
It acts as a vasodilator with selectivity for the arterial portion of the peripheral vasculature.
As a result the systemic vascular resistance is reduced, usually without orthostatic hypoten-
sion or reflex tachycardia. Verapamil also dilates the main coronary arteries and coronary 
arterioles, both in normal and ischemic regions, and is a potent inhibitor of coronary artery 
spasm, whether spontaneous or ergonovine-induced. Verapamil regularly reduces the total 
systemic resistance (afterload) against which the heart works both at rest and at a given level 
of exercise by dilating peripheral arterioles. It prolongs the atrio-ventricular conduction
and has negative inotropic effects on the heart.


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After oral administration, 90 to 100% of verapamil is absorbed, but due to high first-pass
metabolism, its bioavailability is only ~20%. Verapamil is extensively metabolized via 
hepatic CYP3A4, CYP1A2, CYP2C8, CYP2C9 and CYP2C18, O-demethylation, N-
dealkylation and first-pass metabolism. Approximately 70% of an administered dose is
excreted renally as metabolites and 3 to 4% unchanged; 16% are excreted via faeces. The 
elimination half-life of the immediate release formulation is between 3 h and 7 h [R10-5872].
For a more detailed description of verapamil`s profile please refer to the SmPC [R17-2887].
1.2.6
Rifampin
Rifampin is a broad-spectrum antibiotic that can be used to treat many infectious agents of 
clinical importance. Rifampin inhibits bacterial RNA polymerase by blocking the path of the 
elongating RNA. Rifampin is used mainly in the treatment of tuberculosis, but it is also a 
useful orally active alternative in the treatment of other infections, such as those caused by 
methicillin-sensitive or methicillin-resistant staphylococcus [P03-08008]. The dosage of 
rifampin in adults in the treatment of tuberculosis is usually between 450 and 600 mg/day. 
Rifampin is readily absorbed from the gastrointestinal tract. Two hours after a single 450 mg 
oral dose of rifampin in healthy adults, the mean peak serum concentration is 5-13 mg/L. 
Absorption of rifampin was reduced by about 30% when the drug was ingested with food. 
Rifampin has a large volume of distribution. The lipophilic compound easily distributes into 
other tissues – concentrations in the liver are up to 20-fold higher compared to serum level, 
concentrations in the kidney up to 5-fold higher. Approximately 70-90% is plasma-protein 
bound. It is metabolized in the liver (main metabolite: 25-desacetyl-rifampin) and rapidly 
eliminated in the bile followed by entero-hepatic circulation. Up to 10-15% of a dose is 
excreted in the urine, with about half of this being unchanged drug. In healthy adults, the 
initial mean biological half-life of rifampin is 3-16 hours. Autoinduction of hepatic enzymes 
results in a reduction of half-life in the first weeks of treatment [R16-5180]. 
Rifampin is transported by organic anion transport proteins OATP1B1 and OATP1B3 
[R10-1157], [R12-0828]. Competitive inhibition of OATP1B1/1B3 by rifampin may cause 
reduced hepatic uptake. On the other hand, rifampin induces a number of drug-metabolising 
enzymes, including CYP 3A in the liver and in the small intestine [R01-0515], [R07-0089]. A 
5-day treatment with rifampin (600 mg daily) reduced the AUC of oral midazolam by about 
96–98%. In addition, rifampin induces expression of drug transporters including intestinal P-
glycoprotein and MRP2 [R09-5227] and hepatic P-gp [P03-08008]. Full induction is reached 
in about 1 week after starting rifampin treatment, and persists until roughly 2 weeks after 
discontinuing rifampin [P03-08008]. Therefore, under chronic rifampin administration, the 
inductive effect on metabolising enzymes and efflux transporters may abolish the rifampin
inhibition effect on uptake transporters [R10-1157].
For a more detailed description of rifampin’s profile (including clinical safety in patients),
please refer to the SmPC [R16-5180].


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1.2.7
Cimetidine
Cimetidine is a histamine H2-receptor antagonist which inhibits gastric acid secretion. The 
therapeutic use of cimetidine is indicated in the treatment of duodenal and gastric ulcer, 
Zollinger-Ellison syndrome and oesophageal reflux.
The usual daily dose is 800 mg. In the treatment of oesophageal reflux, an increase of daily 
dose up to 1600 mg might be required. In patients with Zollinger-Ellison, daily doses of 
1000-2000 mg cimetidine are indicated.
Cimetidine is rapidly absorbed from the gastro-intestinal tract. The bioavailability of 
cimetidine after oral intake is 50-70%. Peak plasma concentrations are reached in about 1-2 
hours. The elimination half-life is about 2 hours in healthy persons. Cimetidine is primarily 
excreted unchanged (60%) via the kidneys and only a small fraction is metabolised in the
liver [R17-1838].
Cimetidine is a highly interactive compound. Inhibition of CYP1A2, 2C9, 2D6, 3A4 and 
2C18 may result in increased plasma concentrations of warfarin, tricyclic antidepressants 
(e.g. amitriptyline), antiarrhythmics (e.g. lidocaine), calcium channel blockers (e.g. nifedipi-
ne), sulfonylureas, phenytoin, theophylline and metoprolol. Alteration of gastric pH may 
enhance (e.g. atazanavir) or reduce (e.g. ketoconazole and itraconazole) absorption of drugs 
[R17-1901].
Inhibition of tubular secretion may result in increased plasma levels of certain drugs 
including metformin, procainamide and pindolol [R09-6148], [R96-0654], [R99-0753].
For a more detailed description of cimetidine’s profile (including clinical safety in patients),
please refer to the SmPC [R17-1838].
1.2.8
Probenecid
Probenecid is a uricosuric and renal tubular blocking agent. It inhibits the tubular 
reabsorption of urate, thus increasing the urinary excretion of uric acid and decreasing serum 
urate levels. Effective uricosuria reduces the miscible urate pool, retards urate deposition, and 
promotes resorption of urate deposits. The recommended dosage of probenecid in adults in 
the treatment of hyperuricaemia is 2x250 mg/day for the first week of treatment and 
afterwards 2x500 mg/day [R12-0767]. The dose of probenecid may need to be increased if 
patients are also given drugs, such as diuretics or pyrazinamide, that increase the blood 
concentration of uric acid. Salicylates, including aspirin, and probenecid are mutually 
antagonistic and should not be given together.
Probenecid is completely absorbed from the gastrointestinal tract and peak plasma 
concentrations occur 2 to 4 hours after a dose. It is extensively bound to plasma proteins (83 
to 94%). The plasma half-life is dose-dependent and ranges from 2-6 hours for therapeutic 
doses (500 to 1000 mg/day) to 4-12 hours at daily doses of 2000 mg and above [R12-0767]. 
Probenecid is metabolised by the liver, and excreted in the urine mainly as metabolites. 
Excretion of unchanged probenecid is increased in alkaline urine.
Probenecid interacts with both organic anion transporters (OATs) and organic cation
transporters (OCTs) that are involved in the active renal secretion of drug molecules 
[R12-0773]. By inhibiting renal tubular secretion, it has the potential to increase the toxicity 
and/or to enhance the therapeutic efficacy of drugs excreted by that route. In some instances,


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a reduction in dose is essential to counteract an increase in toxicity, as is the case with 
methotrexate. Some combinations, such as that with ketorolac, should be avoided.
Conversely, probenecid may be given with some antibacterials, such as the penicillins and 
cephalosporins, to increase their effects [P12-02347].
Altered excretion may also increase serum concentrations of other antibacterials 
(aminosalicylic acid, conjugated sulfonamides, dapsone, meropenem, some quinolones, 
rifampicin), some antivirals (aciclovir, ganciclovir, zalcitabine, zidovudine, and possibly 
famciclovir), some benzodiazepines (adinazolam, lorazepam, and nitrazepam), some 
angiotensin-converting enzyme inhibitors (captopril and enalapril), some non-steroidal anti-
inflammatory drugs (diflunisal, indometacin, ketoprofen, meclofenamate, naproxen), 
paracetamol, and sulfonylurea hypoglycaemic drugs. The clinical significance of such 
interactions is not entirely clear [P12-02347].
For a more detailed description of probenecid`s profile please refer to the SmPC [R12-0767].


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2.
RATIONALE, OBJECTIVES, BENEFIT - RISK ASSESSMENT
2.1
RATIONALE FOR PERFORMING THE TRIAL
Current regulatory guidelines on the investigation of DDIs by the FDA (draft version, 
[P12-05791) and EMA [P15-06991] recommend the use of cocktail studies. Cocktails 
consisting of carefully selected probe drugs for DDIs on the level of drug-metabolising 
enzymes have been established and are a recognized tool in drug development.
Besides drug-metabolising enzymes, drug transporters are also involved in clinically relevant 
drug-drug interactions. To develop a validated cocktail of probe drugs of key drug 
transporters, the following steps have to be taken:
(1) selection of sensitive probe drugs for targeted drug transporters
(2) test for mutual interactions
(3) test the effect of recommended inhibitors of key drug transporters on cocktail probe drugs
Based on in vitro research and available in vivo data, four drugs have been identified as 
suitable probe drugs for DDIs on the level of drug transporters: digoxin for P-gp, furosemide
for OAT1 and OAT3, metformin for OCT2, MATE1, and MATE2-K, and rosuvastatin for
OATP1B1, OATP1B3, and BCRP (step 1). In the trial 352.2096, it could be demonstrated 
that there is no mutual interaction between these drug candidates (step 2).
This trial targets step 3 of cocktail validation – testing of drug transporter inhibitors. The 
effect of verapamil (P-gp) and rifampin (OATP) is tested in Part 1. The effect of cimetidine 
(OCT2, MATE) is tested in Part 2, the effect of probenecid (OAT1/3) is tested in Part 3.
In the cocktail, subtherapeutic doses of furosemide (1 mg) and metformin (10 mg) are used. 
To examine if there are differences in the inhibitory effect between subtherapeutic and thera-
peutic doses of both drugs, additional arms are included in Part 2 (investigating a therapeutic 
dose of 500 mg metformin) and Part 3 (testing a therapeutic dose of 40 mg furosemide).
2.2
TRIAL OBJECTIVES
The primary objective of this trial is to investigate the relative bioavailabilities of digoxin, 
furosemide, metformin and rosuvastatin given as a cocktail alone and as a cocktail together 
with the following drug transporter inhibitors: 
Part 1: verapamil (P-gp) and rifampin (OATP1B1/1B3)
Part 2: cimetidine (OCT2/MATE)
Part 3: probenecid (OAT1/3)
The secondary objective is to investigate other potential changes in pharmacokinetics (e.g. in 
clearance, volume of distribution, etc.) of digoxin, furosemide, metformin and rosuvastatin 
given as a cocktail alone and as a cocktail together with the inhibitors describe above. 
Further objectives of this study are:
-
To compare the inhibitory effect of cimetidine and probenecid on subtherapeutic and 
therapeutic doses of metformin (Part 2) and furosemide (Part 3)
-
The assessment of safety and tolerability
A description of the endpoints to be determined is provided in Section 5.


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2.3
BENEFIT - RISK ASSESSMENT
Participation in this study is without any (therapeutic) benefit for healthy subjects. Their 
participation in the study, however, is of major importance to the development of a cocktail 
for investigation of transporter-mediated DDIs. Such a cocktail will be a valuable tool to 
assess DDIs of several important drug transporters in a standardised clinical format. It is 
expected to allow reduction of the total number of DDI studies during a development 
program by focusing on targeted follow-up studies and by avoiding unnecessary DDI studies.
Thus, the development of new drugs in nearly all therapeutic areas can be supported while the 
exposure of healthy subjects to new compounds in DDI trials may be reduced.
The subjects are exposed to the risks of the study procedures, the risks related to the exposure 
to the trial medication and to the potential risks resulting from drug-drug interactions.
2.3.1
Procedure-related risks
The use of an indwelling venous catheter for the purpose of blood sampling may be 
accompanied by mild bruising and also, in rare cases, by transient severe inflammation of the 
wall of the vein. In addition, in rare cases a nerve might be injured while inserting the venous 
catheter, potentially resulting in paresthesia, reduced sensibility, and/or pain for an indefinite 
period. Venous thrombosis and fainting of the subject may also occur. The same risks apply 
to venipuncture for blood sampling.
The total volume of blood withdrawn during the entire study per subject will not exceed the 
volume of a normal blood donation (500 mL). No health-related risk to healthy subjects is
expected from this blood withdrawal.
2.3.2
Drug-related risks 
All eight drugs used in this study as investigational products are market-approved drugs. The 
used doses are within or below the therapeutic range (see Table 2.3.2:1).
Table 2.3.2: 1
Doses of investigational products used in this trial compared to their thera-
peutic doses and to reported doses that have been given to healthy subjects:
Drug
Highest daily 
dose in this trial
therapeutic daily dose 
Dose tolerated by healthy 
subjects
digoxin
0.25 mg SD
0.2 – 0.4 mg [R16-2173]
0.75 mg SD [R17-1846]
furosemide
40 mg SD
40 – 80 mg [R16-2538]
80 mg SD [R17-1848]
metformin
500 mg SD
up to 3000 mg [R17-1837]
1000 mg SD [U13-2366-01]
rosuvastatin
10 mg SD
10 mg – 40 mg [R17-2886]
80 mg SD [R13-4572]
verapamil
120 mg SD
240 – 480 mg [R17-2887]
240 mg qd [R07-4432]
rifampin
600 mg SD
450 – 600 mg [R16-5180]
600 mg qd [U10-1328-01]
cimetidine
4 x 400 mg
up to 2000 mg [R17-1838]
1600 mg qd [R17-1843]
probenecid
1000 mg
1000 mg [R12-0767]
1000 mg bid [R07-4435] 


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Digoxin (0.75 mg), metformin (1000 mg), rosuvastatin (80 mg), rifampin (600 mg), and 
cimetidine (1600 mg q.d.) have been well tolerated by healthy subjects in the clinical trials 
given as references in the table above.
The administration of a single dose of 80 mg furosemide to 12 healthy subjects caused dry 
mouth, dizziness, dyspepsia, nausea, vomiting, asthenia and headache in 1 subject 
[R17-1848]. These adverse events are manageable in the setting of a phase I trial. While the 
cocktail (to be tested in all trial parts) contains 1 mg furosemide, in part 3 of this trial a single 
dose of 40 mg is administered. No undue risk to healthy subjects is expected from this dose.
The administration of 240 mg verapamil once daily over 38 days to 12 healthy subjects 
caused bradycardia in 3 subjects, 3 cases of mild to moderate headache, 2 cases of bilateral 
ankle oedema and 1 case of constipation. All adverse events resolved without intervention 
and did not result in premature discontinuation from the trial [R07-4432]. These known side 
effects of verapamil are manageable in the setting of a phase I trial. In contrast to the cited 
report, in this study only a single dose of 120 mg verapamil will be administered to healthy 
subjects in part 1. This does not represent an undue risk to healthy subjects.
The administration of 1000 mg probenecid twice daily over 6 days to 12 healthy male 
subjects caused mild stomach disturbances in 3 cases [R07-4435]. This is manageable in the 
setting of a phase I trial and does not represent an undue risk to healthy subjects. In part 3 of 
this trial, two single doses of 1000 mg probenecid will be given on Days -1 and 1.
In summary, the administration of reference treatments (0.25 mg digoxin, 1 mg and 40 mg 
furosemide, 10 mg and 500 mg metformin and 10 mg rosuvastatin), as well as the 
administration of drug transporter inhibitors according to the planned dosing regimen (120 
mg verapamil SD, 600 mg rifampin SD, cimetidine 4 x 400 mg on Day 1 followed by 2 x 400 
mg on Day 2 and 1000 mg probenecid on Day -1 and Day 1), does not represent undue risks 
to healthy subjects participating in this phase I trial.
Complete lists of possible adverse reactions listed for the therapeutic use of digoxin, 
furosemide, metformin, rosuvastatin, verapamil, rifampin, cimetidine and probenecid in 
patients can be found in the current SmPCs of these compounds [R16-2173], [R16-2538], 
[R17-3544], [R17-2886], [R17-2887], [R16-5180], [R17-1838], [R12-0767].
2.3.3
Risks related to the interaction of transporter substrates with inhibitors
Digoxin
In Part 1 of this trial, the interaction between digoxin and verapamil will be tested. This has 
been done by several working groups so far. The greatest effect on digoxin plasma 
concentration was an increase of +77% observed by Belz et al after multiple dosing with 
verapamil [R10-5948]. In this trial, even greater effects might be expected, because a single 
dose of 120 mg verapamil will be used. Thus, P-gp inhibition is not attenuated by induction 
which is supposed to occur after multiple dosing with verapamil (see Section 1.1.4). 
In this trial, 0.25 mg digoxin will be given. According to the literature, single doses of 0.75 
mg digoxin have been well tolerated by healthy subjects [R17-1846], [R17-1847]. Assuming 
dose-proportional kinetics, this would provide a safety margin of factor 3, which is assessed 
to be sufficient to cover the potential increase of digoxin exposure after verapamil mediated 
inhibition of P-gp.


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Furosemide
In Part 3 of this trial, the interaction of probenecid and furosemide will be tested. Smith and 
Vree observed an approximately 3-fold increase of furosemide AUC after combined 
administration of probenecid and 40 mg [R17-1861] and 80 mg [R17-1859] furosemide. This 
effect size is also expected for the current trial.
The administration of 20 mg furosemide (as oral solution) in the trial 352.2082 resulted in a 
mean AUC0-24 of 4.12 µmol/L*h (=1.36 µg/mL*h) and Cmax of 1.83 µmol/L (0.604 µg/mL) 
[c03246006-01]. Considering dose proportional kinetics, the administration of 40 mg 
furosemide (as oral solution) planned in Part 3 of this trial is expected to cause an AUC0-24 of 
about 8.24 µmol/L*h (=2.72 µg/mL*h) and a Cmax of 3.66 µmol/L (1.21 µg/mL). Combined 
administration with probenecid is expected to increase the exposure by a factor of 3 (AUC0-24
of about 8.2 µg/mL*h; Cmax of about 3.6 µg/mL), which only slightly exceeds the exposure 
reported by Shoaf et al after administration of 80 mg furosemide (AUC0-24 of 7.06 µg/mL*h; 
Cmax of 2.9 µg/mL) to healthy subjects with an acceptable tolerability [R17-1848].
Considering this and taking into account that the spectrum of adverse events reported by 
Shoaf et al in the furosemide-tolvaptan study (see Section 2.3.2) is manageable in the setting 
of a phase I study, the combined administration of 40 mg furosemide and 1000 mg 
probenecid planned for Part 3 of this trial puts the healthy subjects at an acceptable risk.
Andreasen et al administered up to 120 mg furosemide i.v. to healthy subjects to investigate 
dose-dependency of furosemide induced sodium excretion [R14-2147]. Considering the 60-
70% bioavailability of solid furosemide formulations [R17-1836], the achieved exposure 
after 120 mg furosemide i.v. might be twice as high compared to the values obtained in the 
furosemide-tolvaptan study reported by Shoaf, in which 2 x 40 mg tablets furosemide have 
been dosed. Unfortunately, the Andreasen paper does not contain any information about the 
tolerability of these high furosemide doses in healthy subjects.
Metformin
In Part 2 of this trial, the interaction of cimetidine and metformin will be tested. The greatest 
effect of cimetidine on metformin exposure has been reported by Somogyi et al: the 
combined administration of cimetidine (400 mg b.i.d.) and metformin (250 mg q.d.) for 5 
days increased metformin Cmax and AUC by 73% and 46%, respectively [R99-0743]. In the 
current trial, a higher dose of the offender drug cimetidine will be used (4 x 400 mg on Day 
1, followed by 2 x 400 mg on Day 2), which might result in a slightly higher effect size.
The dose of the victim drug metformin is 500 mg in this trial. Considering that doses of 1000 
mg metformin have been well tolerated by healthy subjects, no undue risks are expected from 
the cimetidine-metformin interaction investigated in Part 2 of this trial.
Rosuvastatin
In Part 1 of this trial, the interaction of the OATP-inhibitor rifampin and rosuvastatin will be 
tested. This interaction has been investigated by Lai et al and Pruesaritanont et al, who 
describe an about 10-13 fold increase of rosuvastatin Cmax and a 5-fold increase of 
rosuvastatin AUC after co-medication with 600 mg rifampin [R15-4771], [R17-1790]. This 
effect size is also expected for the current trial.


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The administration of 10 mg rosuvastatin in the trial 352.2082 resulted in a mean AUC0-inf of 
93.4 nM*h (=44.9 ng/mL*h) and Cmax of 8.61 nM (4.14 ng/mL) [c03246006-01]. A rifampin 
mediated 5-fold increase of AUC and 13-fold increase of Cmax would result in a rosuvastatin 
AUC of 225 ng/mL*h and Cmax of 54 ng/mL, which is still below the exposure reported by 
Cooper et al (rosuvastatin AUC of 571 ng/mL*h and Cmax of 61 ng/mL), that was well 
tolerated by healthy subjects [R13-4572]. Therefore, no undue risk is expected from the 
rifampin-rosuvastatin interaction tested in this study.
2.3.4
Safety measures
For safety measures and assessments such as screening examination, AE/CT questioning, 
laboratory examinations, in-house periods or ECG/vital signs measurements, refer to the 
Flow Chart and Section 5.2. The safety measures are adequate to address the potential risks 
of the trial drugs to the volunteers.
2.3.5
Overall assessment
This trial aims to perform step 3 of cocktail validation (see Section 2.1). It should be 
investigated if the cocktail of drug transporter substrates is sensitive towards recommended 
drug transporter inhibitors. As subtherapeutic doses of metformin and furosemide are used, a 
further target of this study is to investigate if the inhibitor mediated effect size observed with 
the lower cocktail doses is comparable to the effect size observed with therapeutic doses of 
metformin (Part 2) and furosemide (Part 3). 
A successful validation of this drug transporter cocktail would provide a valuable tool in the 
drug development process, that allows evaluation of several potential DDI risks of a 
developmental compound within one clinical study instead of several trials. Thus, the 
exposure of healthy subjects to investigational new drugs might be substantially reduced in 
the future.
Victim and offender drugs are administered in doses that have shown at least an acceptable 
tolerability in healthy subjects (see Section 2.3.2). Digoxin, metformin and rosuvastatin have
also been well tolerated after increase of their exposure mediated by those inhibitors that are 
used in this trial. The expected drug exposure of rosuvastatin, metformin and digoxin after 
administration of the respective inhibitor are covered by the literature (Section 2.3.3).
For furosemide, the expected exposure after combined administration with probenecid in this 
trial might slightly exceed the highest exposure for which data on furosemide tolerability in 
healthy subjects could be found in the literature (see Section 2.3.3). However, the spectrum of 
adverse events described here is manageable within the setting of a phase I study, so that no 
undue risk is expected from this interaction.
Taken together, the sponsor feels that the benefit of a successful development of a drug 
transporter cocktail outweighs the potential risks to healthy participants.


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3.
DESCRIPTION OF DESIGN AND TRIAL POPULATION
3.1
OVERALL TRIAL DESIGN AND PLAN
This trial consists of 3 parts testing different drug transporter inhibitors.
Part 1
Part 1 of the study will be performed in healthy male subjects following a randomised, open-
label, three-way crossover design in order to compare the reference treatment R1 to the test 
treatments T1 and T2.
The treatments will be the following (for details refer to Section 4.1):

Treatment R1 (= Cocktail): 0.25 mg digoxin, 1 mg furosemide, 10 mg metformin, and 
10 mg rosuvastatin given together as a single dose

Treatment T1: 120 mg verapamil, single dose, 1 h prior to Cocktail

Treatment T2: 600 mg rifampin, single dose, together with Cocktail
The subjects will be randomly allocated to one of the three treatment sequences (R1-T1-T2; 
T1-T2-R1; T2-R1-T1). There will be a washout period of at least 13 days between the trial 
cocktail administrations of consecutive treatment periods.
Part 2
Part 2 of the study will be performed in healthy male subjects following a randomised, open-
label, four-way crossover design in order to compare the reference treatment R1 to the test 
treatment T3 and to compare the reference treatment R2 to the test treatment T5.
The treatments will be the following (for details refer to Section 4.1):

Treatment R1 (= Cocktail): 0.25 mg digoxin, 1 mg furosemide, 10 mg metformin, and 
10 mg rosuvastatin, all four drugs given together as single dose

Treatment T3: 4 x 400 mg cimetidine on Day 1 (first dose 1 h prior to Cocktail); 2 x 
400 mg on Day 2

Treatment R2: 500 mg metformin, single dose 

Treatment T5: 4 x 400 mg cimetidine on Day 1 (first dose 1 h prior to 500 mg 
metformin); 2 x 400 mg on Day 2 
The subjects will be randomly allocated to one of the four treatment sequences (R1-R2-T3-
T5; R2- T5- R1-T3; T3- R1- T5-R2; T5-T3-R2-R1). There will be a washout period of at 
least 7 days between metformin administrations of consecutive treatment periods.
Part 3
Part 3 of the study will be performed in healthy male subjects following a randomised, open-
label, four-way crossover design in order to compare the reference treatment R1 to the test 
treatment T4 and to compare the reference treatment R3 to the test treatment T6.
The treatments will be the following (for details refer to Section 4.1):


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
Treatment R1 (= Cocktail): 0.25 mg digoxin, 1 mg furosemide, 10 mg metformin, and 
10 mg rosuvastatin given together as a single dose

Treatment T4: 1000 mg probenecid on Day -1 and on Day 1 (1 h prior to Cocktail) 

Treatment R3: 40 mg furosemide, single dose 

Treatment T6: 1000 mg probenecid on Day -1 and on Day 1 (1 h prior to 40 mg 
furosemide) 
The subjects will be randomly allocated to one of the four treatment sequences (R1-R3-T4-
T6; R3 -T6-R1-T4; T4 -R1-T6-R3; T6-T4-R3-R1). There will be a washout period of at least 
7 days between furosemide administrations of consecutive treatment periods.
An overview of all relevant trial activities is provided in the Flow Chart. For visit schedule 
and details of trial procedures at selected visits, refer to Sections 6.1 and Section 6.2, 
respectively.
3.1.1
Administrative structure of the trial
The trial is sponsored by Boehringer Ingelheim (BI) Pharma GmbH & Co. KG, Germany.
BI has appointed a Trial Clinical Monitor, responsible for coordinating all required activities, 
in order to 
-
manage the trial in accordance with applicable regulations and internal SOPs,
-
direct the clinical trial team in the preparation, conduct, and reporting of the trial,
-
ensure appropriate training and information of local clinical monitors (CML), Clinical 
Research Associates (CRAs), and participating trial sites. 
The trial medication will be purchased by the clinical site at a public pharmacy. 
The trial will be conducted at
under the supervision of the Principal 
Investigator.
Safety laboratory tests will be performed by the local laboratory of the trial site 
(Universitätsklinikum Hamburg-Eppendorf, Institut für Klinische Chemie und 
Laboratoriumsmedizin, Hamburg, Germany).
The analyses of digoxin, furosemide, metformin, rosuvastatin and cimetidine concentrations 
in plasma and urine will be performed under the responsibility of the Department of Drug 
Metabolism and Pharmacokinetics, BI Pharma GmbH & Co. KG, Biberach, Germany at 
contract research organisations (CROs)
On-site monitoring will be performed by BI or a contract research organisation appointed by 
BI.
Data management and statistical evaluation will be done by BI according to BI SOPs.
Tasks and functions assigned in order to organise, manage, and evaluate the trial are defined 
according to BI SOPs. A list of responsible persons and relevant local information can be 
found in the ISF.


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3.2
DISCUSSION OF TRIAL DESIGN, INCLUDING THE CHOICE OF 
CONTROL GROUPS
For DDI trials, the crossover design is preferred due to its efficiency: since each subject 
serves as his own control, the comparison between treatments is based on a comparison 
within subjects rather than between subjects. This trial design therefore removes inter-subject 
variability from the comparison between treatments [cf. R94-1529].
In part 1, a wash-out interval of at least 13 days has been chosen to avoid overlapping effects 
of rifampin mediated P-gp induction. Rifampin is known to induce expression of intestinal 
and hepatic P-gp. Although induction is expected to start after first rifampin administration, 
full induction is reached in about 1 week rifampin treatment and persists until roughly 2 
weeks after treatment discontinuation [P03-08008]. 
In parts 2 and 3, a wash-out period of 7 days has been chosen. In previous trials in this project 
(352.2082 [c03246006-01], 352.2096 [c13060859-01]), this time period was sufficient to 
wash out digoxin before drug administration in the subsequent treatment period. The half-life 
of other compounds is shorter compared to digoxin and sufficiently covered by a wash-out 
period of at least 7 days.
In parts 2 and 3, therapeutic doses of metformin and furosemide are tested with the respective 
inhibitor. This is to investigate if there are differences in the inhibitor mediated effect size 
between subtherapeutic (used in the cocktail) and therapeutic doses of both substrates. 
Blinding is not necessary: The open-label treatment in all trial parts is not expected to bias 
results, since the primary and secondary study endpoints are derived from measurement of 
plasma concentrations of the analytes.
3.3
SELECTION OF TRIAL POPULATION
It is planned that a total of 47 healthy male subjects (Part 1: 15; Part 2: 16; Part 3: 16) will 
enter the study. The actual number of entered subjects may exceed 47, if drop-out subjects 
have to be replaced (maximum 3 per trial part), but will not exceed 56 subjects. They will be 
recruited from the volunteers' pool of the trial site.
Only male subjects will be included into the study to reduce inter-individual variability of PK 
data and to avoid any potential confounding effect of hormonal cycle or hormonal 
contraception on the study results. 
A log of all subjects enrolled into the trial (i.e. having given informed consent) will be 
maintained in the ISF at the investigational site irrespective of whether they have been treated 
with investigational drug or not.
3.3.1
Main diagnosis for study entry
The study will be performed in healthy male subjects.
3.3.2
Inclusion criteria
Subjects will only be included into the trial if they meet the following criteria:


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1. Healthy male subjects according to the investigator’s assessment, based on a complete 
medical history including a physical examination, vital signs (BP, PR), 12-lead ECG, and 
clinical laboratory tests 
2. Age of 18 to 55 years (incl.) 
3. BMI of 18.5 to 29.9 kg/m2 (incl.) 
4. Signed and dated written informed consent prior to admission to the study in accordance 
with GCP and local legislation
3.3.3
Exclusion criteria
Subjects will not be allowed to participate if any of the following general criteria apply:
1. Any finding in the medical examination (including BP, PR or ECG) is deviating from 
normal and judged as clinically relevant by the investigator
2. Repeated measurement of systolic blood pressure outside the range of 90 to 140 mmHg, 
diastolic blood pressure outside the range of 50 to 90 mmHg, or pulse rate outside the 
range of 50 to 90 bpm
3. Any laboratory value outside the reference range that the investigator considers to be of 
clinical relevance
4. Any evidence of a concomitant disease judged as clinically relevant by the investigator
5. Gastrointestinal, hepatic, renal, respiratory, cardiovascular, metabolic, immunological or 
hormonal disorders
6. Cholecystectomy and/or surgery of the gastrointestinal tract that could interfere with the 
pharmacokinetics of the trial medication (except appendectomy and simple hernia repair)
7. Diseases of the central nervous system (including but not limited to any kind of seizures 
or stroke), and other relevant neurological or psychiatric disorders 
8. History of relevant orthostatic hypotension, fainting spells, or blackouts
9. Chronic or relevant acute infections
10. History of relevant allergy or hypersensitivity (including allergy to the trial medication or 
its excipients, sulphonamides, or cardiac glycosides)
11. Use of drugs within 30 days prior to administration of trial medication if that might 
reasonably influence the results of the trial (incl. QT/QTc interval prolongation)
12. Participation in another trial where an investigational drug has been administered within 
60 days prior to planned administration of trial medication, or current participation in 
another trial involving administration of investigational drug
13. Smoker (more than 10 cigarettes or 3 cigars or 3 pipes per day)
14. Inability to refrain from smoking on specified trial days
15. Alcohol abuse (consumption of more than 24 g per day for males)
16. Drug abuse or positive drug screening
17. Blood donation of more than 100 mL within 30 days prior to administration of trial 
medication or intended donation during the trial
18. Intention to perform excessive physical activities within one week prior to administration 
of trial medication or during the trial


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19. Inability to comply with dietary regimen of trial site
20. Subject is assessed as unsuitable for inclusion by the investigator, for instance, because 
considered not able to understand and comply with study requirements, or has a condition 
that would not allow safe participation in the study
21. Male subjects with WOCBP partner who are unwilling to use male contraception 
(condom or sexual abstinence) from the first administration of trial medication until 30 
days after last administration of trial medication
In addition, the following trial-specific exclusion criteria apply:
22. Hypokalemia, hypomagnesemia, or hypercalcemia
23. PQ interval greater than 220 ms in the ECG at screening
24. Marked conductivity disorders (e.g. sinu-atrial blocks of II° or III°)
25. Myopathy
26. Known pre-excitation syndrome (WPW- or LGL syndrome)
27. known sick sinus syndrome
28. Hereditary galactose or fructose intolerance, lactase deficiency, or glucose-galactose 
malabsorption
29. History of nephrolithiasis
30. Gout or clinically relevant elevation of uric acid 
31. Creatinine clearance (according to CKD EPI formula) is lower than 80 ml/min
For study restrictions, refer to Section 4.2.2.
3.3.4
Removal of subjects from therapy or assessments
3.3.4.1
Removal of individual subjects
An individual subject is to be removed from the trial if:
1. The subject withdraws consent for trial treatment or trial participation, without the need to 
justify the decision
2. The subject needs to take concomitant drugs that interfere with the investigational product 
or other trial medication
3. The subject is no longer able to participate for other medical reasons (such as surgery, 
adverse events, or diseases)
4. The subject shows an elevation of AST and/or ALT ≥3-fold ULN combined with an 
elevation of total bilirubin ≥2-fold ULN (measured in the same blood sample).
A subject can also be removed from the trial if eligibility criteria are being violated or if the 
subject fails to comply with the protocol (for instance, by non-adherence to dietary rules, or 
non-attendance at study assessments).
If a subject is removed from or withdraws from the trial prior to first administration of trial 
medication, the data of this subject will not be entered in the case report form (CRF) or trial 
database and will not be reported in the clinical trial report (CTR). If a subject is removed 
from or withdraws from the trial after first administration of trial medication, this will be 
documented and the reason for discontinuation must be recorded in the CRF. In this case, the 


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data will be included in the CRF/trial database and will be reported in the CTR. At the time 
of discontinuation, a complete end of trial examination will be performed, if possible, and the 
information will be recorded in the CRFs. 
If the discontinuation occurs before the end of the REP (see Section 5.2.2.2), the discontinued 
subject should, if possible, be questioned for AEs and concomitant therapies at or after the 
end of the REP in order to ascertain collection of AEs and concomitant therapies throughout 
the REP, if not contrary to any consent withdrawal of the subject. These discontinuations will 
be discussed in the CTR.
3.3.4.2
Discontinuation of the trial by the sponsor
Boehringer Ingelheim reserves the right to discontinue the trial overall or at a particular trial 
site at any time for any of the following reasons:
1. The trial will be terminated if more than 50% of the subjects show drug-related and 
clinically relevant adverse events of moderate or severe intensity, or if at least one drug-
related serious adverse event is reported.
2. The expected enrolment goals overall or at a particular trial site are not met
3. Violation of GCP, or the CTP, or the contract with BI by a trial site or investigator, 
disturbing the appropriate conduct of the trial
4. The sponsor decides to discontinue the further development of the drug transporter 
cocktail.
The investigator / the trial site will be reimbursed for reasonable expenses incurred in case of 
trial termination (except in case of the third reason).
3.3.5
Replacement of subjects
In case some subjects do not complete the trial, the trial clinical monitor together with the 
trial pharmacokineticist and the trial statistician are to decide if and how many subjects will 
be replaced. A maximum of 3 subjects can be replaced per trial part. A replacement subject 
will be assigned a unique study subject number and will be assigned to the same treatment 
sequence as the subject he replaces.


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4.
TREATMENTS
4.1
TREATMENTS TO BE ADMINISTERED
The investigational products are commercially available. The clinical site will obtain the 
investigational products from a public pharmacy.
4.1.1
Identity of investigational products
The characteristics of the trial products are given below:
Trial product 1 
Name:
Lanicor® 0,25 mg Digoxin / Tablette
Substance:
Digoxin
Pharmaceutical formulation:
Tablet
Holder or marketing authorization: Teofarma S.r.l., Italy
Unit strength:
0.25 mg
Posology:
1 – 0 – 0 (cocktail component, Part 1-3)
Route of administration:
Oral
Trial product 2
Name:
Lasix liquidum 10 mg/ml Lösung zum Einnehmen
Substance:
Furosemide (as furosemide sodium)
Pharmaceutical formulation:
Oral solution
Holder of marketing authorization: Sanofi-Aventis Deutschland GmbH, Germany
Unit strength:
10 mg/mL
Posology:
0.1 mL (1 mg) – 0 – 0 (as cocktail component, Part 1-3)
4 mL – 0 – 0 (as therapeutic dose, Part 3 only)
Route of administration:
Oral
Trial product 3
Name:
MetfoLiquid GeriaSan® 1000 mg/5 ml Lösung zum 
Einnehmen
Substance:
Metformin hydrochloride
Pharmaceutical formulation:
Oral solution
Holder of marketing authorization: Rosemont Pharmaceuticals Ltd, United Kingdom
Co-marketing: INFECTOPHARM Arzneimittel und 
Consilium GmbH, Germany
Unit strength:
1000 mg/5 mL
Posology:
0.05 mL(10 mg) – 0 – 0 (as cocktail component, Part 1-
3)
2.5 mL (500 mg) – 0 – 0 (as therapeutic dose, Part 2 
only)
Route of administration:
Oral


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Trial product 4
Name:
CRESTOR® 10 mg Filmtabletten
Substance:
Rosuvastatin (as rosuvastatin calcium)
Pharmaceutical formulation:
Film-coated tablet
Holder of marketing authorization: AstraZeneca GmbH, Germany
Unit strength:
10 mg
Posology:
1 – 0 – 0 (Cocktail component, Part 1-3)
Route of administration:
Oral
Trial product 5
Name:
Isoptin® 120 mg Filmtabletten
Substance:
Verapamil
Pharmaceutical formulation:
Film-coated tablet
Holder or marketing authorization: Mylan Healthcare GmbH, Germany
Unit strength:
120 mg
Posology:
1 – 0 – 0 (Part 1 only)
Route of administration:
Oral
Trial product 6
Name:
Eremfat® 600 mg
Substance:
Rifampin
Pharmaceutical formulation:
Film-coated tablet
Holder or marketing authorization: RIEMSER Pharma GmbH, Germany
Unit strength:
600 mg
Posology:
1 – 0 – 0 (Part 1 only)
Route of administration:
Oral
Trial product 7
Name:
Cimetidin acis® 400 mg, Tabletten
Substance:
Cimetidine
Pharmaceutical formulation:
Tablet
Holder or marketing authorization: acis Arzneimittel GmbH, Germany
Unit strength:
400 mg
Posology:
1-1-1-1 (Day 1); 1-0-0-1 (Day 2); Part 2 only
Route of administration:
Oral
Trial product 8
Name:
Probenecid Weimer®, Tabletten
Substance:
Probenecid
Pharmaceutical formulation:
Tablet
Holder or marketing authorization: Biokanol Pharma GmbH, Germany
Unit strength:
500 mg
Posology:
0-0-2 on Day -1; 2-0-0 on Day 1; Part 3 only
Route of administration:
Oral


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4.1.2
Method of assigning subjects to treatment groups
The randomisation list of study subject numbers and assigned treatment sequences will be 
provided to the trial site in advance.
Prior to the screening visit, subjects will be contacted and informed about the planned visit 
dates. The subjects willing to participate will be recruited according to their temporal 
availability.
In the morning of Day 1/Visit 2, subjects will be allocated to treatment sequences prior to 
first study drug administration. For this purpose, subjects will be allocated to a study subject 
number by drawing lots. By the use of a randomisation list, subjects are assigned to a 
treatment sequence. As the study includes healthy subjects from a homogenous population, 
relevant imbalances between the treatment sequences are not expected.
Once a subject number has been assigned, it cannot be reassigned to any other subject.
The randomisation procedure is described in Section 7.5.
4.1.3
Selection of doses in the trial
Based on results of trials 352.2082 [c03246006-01], 352.2094 [c08983809-01], and 352.2096 
[c13060859-01], the proposed cocktail to be tested in this trial consists of 0.25 mg digoxin, 1 
mg furosemide, 10 mg metformin hydrochloride and 10 mg rosuvastatin (see Section 1, 
Introduction).
The inhibitors verapamil, rifampicin, cimetidine and probenecid are used in therapeutic doses 
that have been shown to provide a sufficient inhibition of the target transporters (see 
Section 1.1.4).


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4.1.4
Drug assignment and administration of doses for each subject
Part 1
This trial part is a three-way crossover study. All subjects will receive the three treatments in 
a randomised order. The treatments to be evaluated are outlined in Table 4.1.4: 1 below:
Table 4.1.4: 1
Dosage and treatment schedule
Treatment
Substance
Formulation
Unit strength
Dosage
Total dose
R1 (cocktail)
Digoxin
Furosemide
Metformin
Rosuvastatin
Tablet
Oral solution
Oral solution
Coated tablet
0.25 mg
10 mg/mL
1000 mg/5mL
10 mg
1 tablet as single dose
0.1 mL (1 mg) as single dose
0.05 mL (10 mg) as single dose
1 tablet as single dose
0.25 mg
1 mg
10 mg
10 mg
T1
Verapamil
Digoxin
Furosemide
Metformin
Rosuvastatin
Tablet
Tablet
Oral solution
Oral solution
Coated tablet
120 mg
0.25 mg
10 mg/mL
1000 mg/5mL
10 mg
1 tablet as single dose             
given 1 h prior to the cocktail
1 tablet as single dose
0.1 mL (1 mg) as single dose
0.05 mL (10 mg) as single dose
1 tablet as single dose
120 mg
0.25 mg
1 mg
10 mg
10 mg
T2
Rifampicin
Digoxin
Furosemide
Metformin
Rosuvastatin
Coated tablet
Tablet
Oral solution
Oral solution
Coated tablet
600 mg
0.25 mg
10 mg/mL
1000 mg/5mL
10 mg
1 tablet as single dose             
given together with the cocktail
1 tablet as single dose
0.1 mL (1 mg) as single dose
0.05 mL (10 mg) as single dose
1 tablet as single dose
600 mg
0.25 mg
1 mg
10 mg
10 mg
The medication will be administered with the following amounts of non-sparkling drinking 
water to a subject in the sitting or standing position under supervision of the investigating 
physician or an authorised designee:
R1: cocktail with 280 mL of water
T1: verapamil with 240 mL of water, 1 h prior to the cocktail with 280 mL of water
T2: cocktail + rifampicin with 280 mL of water
The so-called four-eye principle (two-person rule) will be applied for administration of trial 
medication and – if applicable – its preparation. Administration will be performed following 
an overnight fast starting no later than 10 h before scheduled dosing. 


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Part 2
This trial part is a four-way crossover study. All subjects will receive the four treatments in a 
randomised order. The treatments to be evaluated are outlined in Table 4.1.4: 2 below:
Table 4.1.4: 2
Dosage and treatment schedule
Treatment
Substance
Formulation
Unit strength
Dosage
Total dose
R1 (cocktail)
Digoxin
Furosemide
Metformin
Rosuvastatin
Tablet
Oral solution
Oral solution
Coated tablet
0.25 mg
10 mg/mL
1000 mg/5mL
10 mg
1 tablet as single dose
0.1 mL (1 mg) as single dose
0.05 mL (10 mg) as single dose
1 tablet as single dose
0.25 mg
1 mg
10 mg
10 mg
T3
Cimetidine
Digoxin
Furosemide
Metformin
Rosuvastatin
Tablet
Tablet
Oral solution
Oral solution
Coated tablet
400 mg
0.25 mg
10 mg/mL
1000 mg/5mL
10 mg
1 tablet to be given                       
1 h prior to the cocktail
4 h after the cocktail
8 h after the cocktail
12 h after the cocktail
24 h after the cocktail (Day 2)
36 h after the cocktail (day 2)
1 tablet as single dose
0.1 mL (1 mg) as single dose
0.05 mL (10 mg) as single dose
1 tablet as single dose
2400 mg
0.25 mg
1 mg
10 mg
10 mg
R2
Metformin
Oral solution
1000 mg/5mL
2.5 mL (500 mg) as single dose
500 mg
T5
Cimetidine
Metformin
Tablet
Oral solution
400 mg
1000 mg/5mL
1 tablet to be given                       
1 h prior to 500 mg metformin
4 h after 500 mg metformin
8 h after 500 mg metformin
12 h after 500 mg metformin
24 h after 500 mg metformin 
36 h after 500 mg metformin
2.5 mL (500 mg) as single dose
2400 mg
500 mg
The medication will be administered with the following amounts of non-sparkling drinking 
water to a subject in the sitting or standing position under supervision of the investigating 
physician or an authorised designee:
R1, T3: cocktail to be taken with 280 mL of water
R2, T5: therapeutic doses of metformin to be taken with 280 mL of water
T3, T5: all cimetidine doses to be taken with 240 mL water
The so-called four-eye principle (two-person rule) will be applied for administration of trial 
medication and – if applicable – its preparation. Administration will be performed following 
an overnight fast starting no later than 10 h before scheduled dosing.


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Part 3
This trial part is a four-way crossover study. All subjects will receive the four treatments in a 
randomised order. The treatments to be evaluated are outlined in Table 4.1.4: 3 below:
Table 4.1.4: 3
Dosage and treatment schedule
Treatment
Substance
Formulation
Unit strength
Dosage
Total dose
R1 (cocktail)
Digoxin
Furosemide
Metformin
Rosuvastatin
Tablet
Oral solution
Oral solution
Coated tablet
0.25 mg
10 mg/mL
1000 mg/5mL
10 mg
1 tablet as single dose
0.1 mL (1 mg) as single dose
0.05 mL (10 mg) as single dose
1 tablet as single dose
0.25 mg
1 mg
10 mg
10 mg
T4
Probenecid
Digoxin
Furosemide
Metformin
Rosuvastatin
Tablet
Tablet
Oral solution
Oral solution
Coated tablet
500 mg
0.25 mg
10 mg/mL
1000 mg/5mL
10 mg
2 tablets to be given                       
13 h prior to the cocktail and
1 h prior to the cocktail
1 tablet as single dose
0.1 mL (1 mg) as single dose
0.05 mL (10 mg) as single dose
1 tablet as single dose
2000 mg
0.25 mg
1 mg
10 mg
10 mg
R3
Furosemide
Oral solution
10 mg/mL
4 mL (40 mg) as single dose
40 mg
T6
Probenecid
Furosemide
Tablet
Oral solution
500 mg
10 mg/mL
2 tablets to be given                       
13 h prior to 40 mg furosemide
1 h prior to 40 mg furosemide
4 mL (40 mg) as single dose
2000 mg
40 mg
The medication will be administered with the following amounts of non-sparkling drinking 
water to a subject in the sitting or standing position under supervision of the investigating 
physician or an authorised designee:
R1, T4: cocktail to be taken with 280 mL of water
R3, T6: all furosemide doses to be taken with 280 mL of water
T4, T6: all probenecid doses to be taken with 240 mL of water
The so-called four-eye principle (two-person rule) will be applied for administration of trial 
medication and – if applicable – its preparation. Administration will be performed following 
an overnight fast starting no later than 10 h before scheduled dosing. 
Administration of the cocktail (in R1, T1, T2, T3, T4)
When digoxin, furosemide, metformin and rosuvastatin are given together as cocktail, the 
administration will be performed as follows: 
1) About 20 mL of non-sparkling water is given into an appropriate glass container with 
cap.
2) 100 µL of furosemide 10 mg/mL oral solution is added to the glass container by use 
of an appropriate dosing device (e.g., syringe or pipette).


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3) 50 µL of metformin 1000 mg/5 mL oral solution is added to glass container by use of 
an appropriate dosing device (e.g., syringe or pipette).
4) After addition of furosemide and metformin oral solution, the glass container is closed 
using the corresponding cap. The content of the glass container is mixed thoroughly.
5) Immediately thereafter, the subject drinks the 20 mL of water containing the oral drug 
solution from the glass container (diluted furosemide and metformin oral solution).
6) Thereafter, the glass container is rinsed once with about 20 mL of non-sparkling 
water, and the subject drinks the rinsing water. 
7) Thereafter, one digoxin tablet and one rosuvastatin film-coated tablet are administered 
with about 240 mL of non-sparkling water.
Thereafter, complete drug administration is assumed.
After administration (in all trial parts)
Subjects will be kept under close medical surveillance until 24 h following drug 
administration. During the first 4 h after drug administration, they are not allowed to lie down 
(i.e. no declination of the upper body of more than 45 degrees from upright posture), unless 
required for medical procedures. For restrictions with regard to diet, see Section 4.2.2.2.
Wash-out intervals
In Part 1, the cocktail administrations will be separated by a wash-out phase of at least 13 
days.
In Part 2, the treatments will be separated by a wash-out phase of at least 7 days between 
metformin administrations of consecutive treatment periods.
In Part 3, the treatments will be separated by a wash-out phase of at least 7 days between 
furosemide administrations of consecutive treatment periods.
4.1.5
Blinding and procedures for unblinding
This Phase I trial will be handled in an open fashion throughout (that is, during the conduct,
including data cleaning and preparation of the analysis). This is considered acceptable 
because the potential for bias seems to be low and does not outweigh practical considerations.
Emergency envelopes will not be provided, since this study is open-label.
4.1.6
Packaging, labelling, and re-supply
All investigational drugs used in this trial are market-approved drugs, will be purchased by 
at a public pharmacy, and will be dispensed out of original and 
unmodified packages.


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4.1.7
Storage conditions
Drug supplies will be kept in their original packaging and in a secure limited access storage 
area according to the recommended (labelled) storage conditions. Where necessary, a 
temperature log must be maintained to make certain that the drug supplies are stored at the 
correct temperature. If the storage conditions are found to be outside the specified range, the 
local clinical monitor (as provided in the list of contacts) is to be immediately contacted.
4.1.8
Drug accountability
Only authorised personnel, as documented in the form ‘Trial Staff List’, may dispense
medication to trial subjects. The trial medication must be administered in the manner 
specified in the CTP. All unused medication will be disposed locally by the trial site upon 
written authorisation by the clinical monitor. Receipt, usage and disposal must be 
documented on the respective forms. Account must be given for any discrepancies.
The investigator must maintain records of the product’s delivery to the trial site, the inventory 
at the site, the use by each subject, and the disposal of unused products.
These records will include dates, quantities, batch / serial numbers, expiry (‘use-by’) dates, 
and the unique code numbers assigned to the investigational products and trial subjects. The 
investigator will maintain records that document adequately that the subjects were provided 
the doses specified by the CTP, and that reconcile all investigational products received from 
the sponsor. At the time of disposal, the investigator must verify that no remaining supplies 
are in the investigator’s possession.
4.2
OTHER TREATMENTS, EMERGENCY PROCEDURES,
RESTRICTIONS
4.2.1
Other treatments and emergency procedures
There are no special emergency procedures to be followed. No additional treatment is 
planned. However, in case of adverse events in need of treatment, the investigator can 
authorise symptomatic therapy. In those cases, subjects will be treated as necessary and, if 
required, kept under supervision at the trial site or transferred to a hospital until all medical 
evaluation results have returned to an acceptable level.
4.2.2
Restrictions
4.2.2.1
Restrictions regarding concomitant treatment
In principle, no concomitant therapy is allowed. All concomitant or rescue therapies will be 
recorded (including time of intake on study days) on the appropriate pages of the CRF.
Ibuprofen, diclofenac, acetylsalicylic acid and other non-steroidal anti-inflammatory drugs 
should be avoided from 24 h before each trial drug administration until the last PK sample of 
the respective period.


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4.2.2.2
Restrictions on diet and life style
While admitted to the trial site, the subjects are restricted from consuming any other foods or 
drinks than those provided by the staff. Standardised meals will be served at the time points 
described in the Flow Chart. No food is allowed for at least 4 h after drug intake.
From 1 h before drug intake until lunch, fluid intake is restricted to the water administered 
with the drug, and an additional 240 mL of water at 2 h and 4 h post –dose (mandatory for all 
subjects). The term “post-dose” refers to 

the administration of the cocktail in R1, T1, T2, T3, and T4

the administration of 500 mg metformin in R2 and T5

the administration of 40 mg furosemide in R3 and T6
In T3 and T5 (Part 2), the water at 4 h post-dose will be used for cimetidine intake. 
In all periods total fluid intake is restricted to 3000 mL from lunch until 24 h post-dose.
Green tea, grapefruits, Seville oranges (sour or bitter oranges) and their juices, and dietary 
supplements and products including St. John's wort (Hypericum perforatum) are not 
permitted starting 7 days before the first administration of trial medication until after the last 
PK sample of each study period is collected.
Alcoholic beverages are not permitted starting 48 h before each administration of trial 
medication until after the last PK sample of each treatment period is collected.
Methylxanthine-containing drinks or foods (such as coffee, white or black tea, cola, energy 
drinks, and chocolate) are not allowed from 12 h before administration of trial medication 
until subsequent discharge from the study site.
Smoking is not allowed during in-house confinement at the trial site.
Excessive physical activity (such as competitive sport) should be avoided starting 7 days 
before the first administration of trial medication until the end of trial examination.
4.3
TREATMENT COMPLIANCE
Compliance will be assured by administration of all trial medication in the study centre under 
supervision of the investigating physician or a designee. The measured plasma concentrations 
and urinary excretion will provide additional confirmation of compliance.
Subjects who are non-compliant (for instance, who do not appear for scheduled visits or 
violate trial restrictions) may be removed from the trial and the CRF will be completed 
accordingly (for further procedures, please see Section 3.3.4.1).


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5.
VARIABLES AND THEIR ASSESSMENT
5.1
EFFICACY - CLINICAL PHARMACOLOGY
5.1.1
Endpoints of efficacy
No efficacy endpoints will be evaluated in this trial.
5.1.2
Assessment of efficacy
Not applicable.
5.2
SAFETY
5.2.1
Endpoints of safety
There is no specific endpoint regarding safety.
Safety and tolerability of the investigational drugs will be assessed based on:

Adverse events (including clinically relevant findings from the physical examination)

Safety laboratory tests

12-lead ECG

Vital signs (blood pressure, pulse rate)
These parameters will be evaluated in a descriptive way only, see Section 7.3.
5.2.2
Assessment of adverse events
5.2.2.1
Definitions of adverse events
Adverse event
An adverse event (AE) is defined as any untoward medical occurrence in a patient or clinical 
investigation subject administered a medicinal product and which does not necessarily have 
to have a causal relationship with this treatment.
An AE can therefore be any unfavourable and unintended sign (including an abnormal 
laboratory finding), symptom, or disease temporally associated with the use of a medicinal 
product, whether or not considered related to the medicinal product.
Serious adverse event
A serious adverse event (SAE) is defined as any AE which fulfils at least one of the 
following criteria:

results in death, 

is life-threatening, which refers to an event in which the patient was at risk of death 
at the time of the event; it does not refer to an event that hypothetically might have 
caused death if more severe,


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
requires inpatient hospitalisation or 

requires prolongation of existing hospitalisation,

results in persistent or significant disability or incapacity, or

is a congenital anomaly/birth defect, 
or

is deemed serious for any other reason if it is an important medical event when based 
upon appropriate medical judgment which may jeopardise the patient and may 
require medical or surgical intervention to prevent one of the other outcomes listed in 
the above definitions.
Examples of such events are intensive treatment in an emergency room or at home for 
allergic bronchospasm, blood dyscrasias or convulsions that do not result in hospitalisation or 
development of dependency or abuse. 
AEs considered ‘Always Serious’
Cancers of new histology and exacerbations of existing cancer must be classified as a serious 
event regardless of the duration between discontinuation of the drug and must be reported as 
described in Section 5.2.2.2 subsections ‘AE collection’ and ‘AE reporting to sponsor and 
timelines’.
In accordance with the European Medicines Agency initiative on Important Medical Events, 
Boehringer Ingelheim has set up a list of further AEs, which by their nature, can always be 
considered to be ‘serious’ even though they may not have met the criteria of an SAE as 
defined above.
The latest list of ‘Always Serious AEs’ can be found in the RDC system. These events should 
always be reported as SAEs as described above.
Adverse events of special interest (AESIs)
The term AESI relates to any specific AE that has been identified at the project level as being 
of particular concern for prospective safety monitoring and safety assessment within this trial, 
e.g. the potential for AEs based on knowledge from other compounds in the same class. 
AESIs need to be reported to the sponsor’s Pharmacovigilance Department within the same 
timeframe that applies to SAE, please see above.
No AESIs have been defined for this trial.
Intensity (severity) of AEs
The intensity (severity) of the AE should be judged based on the following:
Mild:
Awareness of sign(s) or symptom(s) that is/are easily tolerated
Moderate:
Sufficient discomfort to cause interference with usual activity
Severe:
Incapacitating or causing inability to work or to perform usual activities
Causal relationship of AEs
Medical judgment should be used to determine the relationship, considering all relevant 
factors, including pattern of reaction, temporal relationship, de-challenge or re-challenge, 


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confounding factors such as concomitant medication, concomitant diseases and relevant 
history.
Arguments that may suggest that there is a reasonable possibility of a causal relationship 
could be:

The event is consistent with the known pharmacology of the drug 

The event is known to be caused by or attributed to the drug class.

A plausible time to onset of the event relative to the time of drug exposure.

Evidence that the event is reproducible when the drug is re-introduced

No medically sound alternative aetiologies that could explain the event (e.g. pre-
existing or concomitant diseases, or co-medications).

The event is typically drug-related and infrequent in the general population not 
exposed to drugs (e.g. Stevens-Johnson syndrome).

An indication of dose-response (i.e. greater effect size if the dose is increased, smaller 
effect size if dose is diminished).
Arguments that may suggest that there is no reasonable possibility of a causal relationship 
could be:

No plausible time to onset of the event relative to the time of drug exposure is evident 
(e.g. pre-treatment cases, diagnosis of cancer or chronic disease within days / weeks 
of drug administration; an allergic reaction weeks after discontinuation of the drug 
concerned)

Continuation of the event despite the withdrawal of the medication, taking into 
account the pharmacological properties of the compound (e.g. after 5 half-lives). Of 
note, this criterion may not be applicable to events whose time course is prolonged 
despite removing the original trigger.

Additional arguments amongst those stated before, like alternative explanation (e.g. 
situations where other drugs or underlying diseases appear to provide a more likely 
explanation for the observed event than the drug concerned).

Disappearance of the event even though the trial drug treatment continues or remains 
unchanged.
5.2.2.2
Adverse event collection and reporting
AEs collection
Upon enrolment into a trial, the subject’s baseline condition is assessed (for instance, by 
documentation of medical history/concomitant diagnoses), and relevant changes from 
baseline are noted subsequently.
Subjects will be required to report spontaneously any AEs as well as the time of onset, end, 
and intensity of these events. In addition, each subject will be regularly assessed by the 
medical staff throughout the clinical trial and whenever the investigator deems necessary. As 
a minimum, subjects will be questioned for AEs (and concomitant therapies) at the time 
points indicated in the Flow Chart. Assessment will be made using non-specific questions 


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such as ‘How do you feel?’. Specific questions will be asked wherever necessary in order to 
more precisely describe an AE.
A careful written record of all AEs shall be kept by the investigator in charge of the trial. 
Records of AEs shall include data on the time of onset, end time, and intensity of the event as 
well as any treatment or action required for the event and its outcome.
The following must be collected and documented on the appropriate CRF(s) by the 
investigator:

From signing the informed consent onwards until an individual subject’s end of trial:
o
All AEs (serious and non-serious) and all AESIs.
o
The only exception to this rule are AEs (serious and non-serious) and AESIs 
in Phase I trials in healthy volunteers, when subjects discontinue from the trial 
due to screening failures prior to administration of any trial medication. In 
these cases, the subjects’ data must be collected at trial site but will not be 
entered in the CRF or trial database and will not be reported in the CTR.

After the individual subject’s end of trial:
o
The investigator does not need to actively monitor the subject for AEs but 
should only report related SAEs and related AESIs of which the investigator
may become aware of by any means of communication, e.g. phone call. Those 
AEs should, however, not be reported in the CRF.
The REP for digoxin, when measurable drug levels or PD effects are still likely to be present, 
is defined as 7 days after administration. The REP for the other 7 probe drugs is shorter. In 
order to avoid reporting bias (i.e., shorter lengths of on-treatment periods after single 
component (mono-treatment) administration compared to probe drug cocktail administration 
could lead to a higher number of AEs after administration of the test treatment), all AEs 
which occur from first drug administration until 7 days thereafter in each treatment period 
will be considered as on treatment; please see Section 7.3.3. A REP of 7 days will be used for 
each treatment period.
Thus, in Part 1 the wash-out interval of at least 13 days between drug administrations of 
consecutive treatment periods is split into 2 intervals. AEs occurring in the first interval after 


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first drug administration (extending over 7 days) will be assigned to the preceding treatment
and AEs occurring in the remaining interval of at least 6 days until the next drug 
administration will be assigned to “post-treatment”.
The follow-up period describes the period of time after the last administration of trial 
medication until the end of trial examination (last per protocol visit). AEs occurring after the 
REP but prior to the last per-protocol contact are defined as “follow-up” events.
AE reporting to sponsor and timelines
The Investigator must report SAEs, AESIs and non-serious AEs which are relevant for the 
reported SAE or AESI, on the BI SAE form via fax immediately (within 24 hours) to the 
sponsor’s unique entry point (country specific contact details will be provided in the ISF). 
The same timeline applies if follow-up information becomes available. In specific occasions 
the Investigator could inform the sponsor upfront via telephone. This does not replace the 
requirement to complete and fax the BI SAE form. 
With receipt of any further information to these events, a follow-up SAE form has to be 
provided. For follow-up information the same rules and timeline apply as for initial 
information.
Information required
For each AE, the investigator should provide the information requested on the appropriate 
CRF pages and the BI SAE form if applicable. The investigator should determine the causal 
relationship to the trial medication. The following should also be recorded as an (S)AE in the 
CRF and on the BI SAE form (if applicable):

Worsening of the underlying disease or of other pre-existing conditions 

Changes in vital signs, ECG, physical examination and laboratory test results, if they 
are judged clinically relevant by the investigator.
If such abnormalities already pre-exist prior to trial inclusion they will be considered as 
baseline conditions and should be collected in the CRF only.
All (S)AEs, including those persisting after individual subject’s end of trial must be followed 
up until they have resolved, have been assessed as ‘chronic’ or ‘stable’, or no further 
information can be obtained.
5.2.3
Assessment of safety laboratory parameters
For the assessment of laboratory parameters, blood and urine samples will be collected by the 
trial site at the time points indicated in the Flow Chart.
The parameters that will be determined are listed in Table 5.2.3: 1 and Table 5.2.3: 2. 
Reference ranges will be provided in the ISF, Section 10.
Manual differential white blood cell count or urine sediment examinations will only be 
performed if there is an abnormality in the automatic blood cell count (i.e. atypical or 
pathological cells) or in the urinalysis, respectively.


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Table 5.2.3: 1
Routine laboratory tests
Functional lab group
Test name 
Set A1
Set B2
Set C1
Haematology
Haematocrit 
Haemoglobin 
Red blood cells (RBC)
White blood cells (WBC)
Platelets
X
X
X
X
X
-
-
-
-
-
X
X
X
X
X
Automatic differential 
WBC (relative and 
absolute)
Neutrophils 
Eosinophils 
Basophils 
Monocytes 
Lymphocytes 
X
X
X
X
X
-
-
-
-
-
X
X
X
X
X
Coagulation
Activated partial thromboplastin time (aPTT)
Prothrombin Time (Quick and INR)
X
X
-
-
-
-
Enzymes
Aspartate aminotransferase (AST/GOT)
Alanine aminotransferase (ALT/GPT)
Gamma-glutamyltransferase (GGT)
Creatine kinase (CK)
X
X
X
X
X
X
X
X
X
X
X
X
Substrates
Plasma glucose 
Creatinine
Creatinine-Clearance (CKD EPI formula)
C-reactive protein
X
X
X
X
-
X
-
-
-
X
-
X
Electrolytes
Calcium
Sodium
Potassium
Magnesium
X
X
X
X
X
X
X
X
X
X
X
X
Hormones
Thyroid stimulating hormone (TSH)
X
-
-
Urinalysis (Stix)
Urine nitrite
Urine protein
Urine glucose
Urine ketone
Urobilinogen
Urine bilirubin
Urine erythrocytes
Urine leukocytes
Urine pH
X
X
X
X
X
X
X
X
X
-
-
-
-
-
-
-
-
-
X
X
X
X
X
X
X
X
X
1 Parameters will be determined at screening (A) and end-of-trial (C) examination.
2 Parameters of Set B will be determined during the study.
The tests listed in Table 5.2.3: 2 are exclusionary laboratory tests which may be repeated as 
required. The results will not be entered in the CRF/database and will not be reported in the 
CTR. Except for drug screening it is planned to perform these tests during screening only. 
Drug screening will be performed at screening and prior to each treatment period.


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Table 5.2.3: 2
Exclusionary laboratory tests
Functional lab group
Test name 
Drug screening (urine)
Amphetamine/MDA
Barbiturates
Benzodiazepine
Cannabis
Cocaine 
Methadone
Methamphetamines/MDMA/XTC
Opiates 
Phencyclidine
Tricyclic antidepressants
Infectious serology (blood)
Hepatitis B surface antigen (qualitative)
Hepatitis B core antibody (qualitative)
Hepatitis C antibodies (qualitative)
HIV-1 and HIV-2 antibody (qualitative)
To encourage compliance with alcoholic restrictions, a breath alcohol test (Alcotest® 6510, 
Dräger AG, Lübeck, Germany) will be performed prior to each treatment period, and may be 
repeated at any time during the study at the discretion of an investigator or designee. The 
results will not be included in the CTR.
The laboratory tests listed in Table 5.2.3: 1 and 5.2.3: 2 will be performed at Universitäts-
klinikum Hamburg-Eppendorf, Institut für Klinische Chemie und Laboratoriumsmedizin, 
Hamburg, Germany with the exception of the drug screening tests. These tests will be 
performed at the trial site using Diagnostik Nord 10 test. Laboratory data will be transmitted 
in paper format from the laboratory to the trial site.
5.2.4
Electrocardiogram
5.2.4.1
12-lead resting ECG
Twelve-lead ECGs (I, II, III, aVR, aVL, aVF, V1 - V6) will be recorded using a
computerised electrocardiograph (CardioSoft EKG System, GE Medical Systems, Freiburg,
Germany) at the time points given in the Flow Chart.
All ECGs will be recorded for a 10-sec duration after the subjects have rested for at least 5
min in a supine position . ECG assessment will always precede all other study procedures of 
the same time point (except blood drawing from an intravenous cannula which is already in 
place) to avoid impact of sampling on the ECG quality. 
All ECGs will be stored electronically on the Muse CV Cardiology System (GE Medical 
Systems, Freiburg, Germany). Electrode placement will be performed according to the 
method of Wilson, Goldberger and Einthoven. Precise electrode placement will be marked 
with an indelible mark on the skin to allow reproducible placement throughout the study.
All locally printed ECGs will be evaluated by the investigator or a designee. ECGs may be 
repeated for quality reasons (like alternating current artefacts, muscle movements, electrode 
dislocation) and the repeated ECG will be used for analysis. Additional (unscheduled) ECGs 
may be collected by the investigator for safety reasons. ECG recording will not be transferred 
to the clinical trial database, but stored locally.


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Abnormal findings will be reported as AEs (during the trial) or baseline conditions (at 
screening) if judged clinically relevant by the investigator. Any ECG abnormalities will be 
monitored carefully and, if necessary, the subject will be removed from the trial and will 
receive the appropriate medical treatment. 
5.2.4.2
Continuous ECG monitoring
In Treatment T1 (Part 1) cardiac rhythm (including heart rate) will be monitored by means of 
continuous 5-lead ECG recording for at least 15 min before verapamil intake (for baseline 
assessment) and 4 h following the cocktail intake using the ApexProTM (GE Healthcare, 
Freiburg, Germany). This data is only generated for safety monitoring and will not be 
transferred into the database. Abnormal findings during continuous ECG recording will be 
recorded as AEs if judged clinically relevant by the investigator.
5.2.5
Assessment of other safety parameters
5.2.5.1
Vital signs
Systolic and diastolic blood pressures (BP) as well as pulse rate (PR) or heart rate (heart rate 
is considered to be equal to pulse rate) will be measured by a blood pressure monitor 
(Dinamap Pro 200, GE Medical Systems, Freiburg, Germany) at the times indicated in the 
Flow Chart, after subjects have rested for at least 5 min in a supine position. All recordings 
should be made using the same type of blood pressure recording instrument on the same arm.
5.2.5.2
Medical examinations
At screening, the medical examination will include demographics including height and body 
weight, smoking and alcohol history, relevant medical history and concomitant therapy, 
review of inclusion and exclusion criteria, review of vital signs (BP, PR), 12-lead ECG, 
laboratory tests, and a physical examination. At the end of trial examination, it will include 
review of vital signs, 12-lead ECG, laboratory tests, and a physical examination.
5.3
OTHER
Not applicable.
5.4
APPROPRIATENESS OF MEASUREMENTS
All measurements performed during this trial are standard measurements and will be 
performed in order to monitor subjects’ safety and to determine pharmacokinetic parameters 
in an appropriate way. The safety assessments are standard, are accepted for evaluation of 
safety and tolerability of orally administered drugs, and are widely used in clinical trials. The 
pharmacokinetic parameters and measurements outlined in Section 5.5 are generally used 
assessments of drug exposure. 


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5.5
DRUG CONCENTRATION MEASUREMENTS AND 
PHARMACOKINETICS
Date and exact clock time of drug administration and pharmacokinetic sampling will be 
recorded. The actual sampling times will be used for determination of pharmacokinetic 
parameters.
5.5.1
Pharmacokinetic endpoints
5.5.1.1
Primary endpoints
The following primary endpoints will be determined for digoxin, furosemide, metformin, and 
rosuvastatin (at cocktail doses):

AUC0-tz (area under the concentration-time curve of the analyte in plasma over the 
time interval from 0 to the last quantifiable data point)

Cmax (maximum measured concentration of the analyte in plasma)
5.5.1.2
Secondary endpoints
The following secondary endpoint will be evaluated for digoxin, furosemide, metformin, and 
rosuvastatin (at cocktail doses):

AUC0-∞(area under the concentration-time curve of the analyte in plasma over the 
time interval from 0 extrapolated to infinity)
For digoxin, AUC0-∞will be evaluated only if determination with sufficient precision is 
possible.
5.5.1.3
Further parameters of interest
The following additional endpoints will be evaluated for digoxin, furosemide, metformin, and 
rosuvastatin given at cocktail doses:

%AUCtz-∞(the percentage of AUC0-∞obtained by extrapolation)

tmax (time from dosing to maximum measured concentration of the analyte in plasma)

λz (terminal rate constant in plasma)

t1/2 (terminal half-life of the analyte in plasma)

MRTpo (mean residence time of the analyte in the body after oral administration)

CL/F (apparent clearance of the analyte in the plasma after extravascular 
administration)

Vz/F (apparent volume of distribution during the terminal phase after extravascular 
administration)

Aet1-t2 (amount of analyte that is eliminated in urine from the time interval t1 to t2)

fet1-t2 (fraction of given drug excreted unchanged in urine from time point t1 to t2) 


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
CLR, t1-t2 (renal clearance of the analyte in plasma from the time point t1 to t2)
The following additional endpoints will be evaluated for furosemide and metformin given at 
therapeutic doses:

AUC0-tz (area under the concentration-time curve of the analyte in plasma over the 
time interval from 0 to the last quantifiable data point)

AUC0-∞(area under the concentration-time curve of the analyte in plasma over the 
time interval from 0 extrapolated to infinity)

%AUCtz-∞(the percentage of AUC0-∞obtained by extrapolation)

Cmax (maximum measured concentration of the analyte in plasma)

tmax (time from dosing to maximum measured concentration of the analyte in plasma)

λz (terminal rate constant in plasma)

t1/2 (terminal half-life of the analyte in plasma)

MRTpo (mean residence time of the analyte in the body after oral administration)

CL/F (apparent clearance of the analyte in the plasma after extravascular 
administration)

Vz/F (apparent volume of distribution during the terminal phase after extravascular 
administration)

Aet1-t2 (amount of analyte that is eliminated in urine from the time interval t1 to t2)

fet1-t2 (fraction of given drug excreted unchanged in urine from time point t1 to t2) 

CLR, t1-t2 (renal clearance of the analyte in plasma from the time point t1 to t2)
Further PK parameters may be calculated as appropriate.


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5.5.2
Methods of sample collection
5.5.2.1
Plasma sampling for pharmacokinetic analysis
Part 1 (R1, T1, T2), Part 2 (only R1, T3) and Part 3 (only R1, T4) – Cocktail components
For quantification of digoxin, furosemide, metformin and rosuvastatin (cocktail components) 
plasma concentrations, 7.5 mL of blood will be taken from an antecubital or forearm vein 
into K3-EDTA (tripotassium ethylenediaminetetraacetic acid)-anticoagulant blood drawing 
tubes at the times indicated in the Flow Chart. Blood will be withdrawn by means of either an 
indwelling venous catheter or by venipuncture with a metal needle.
The EDTA-anticoagulated blood samples will be centrifuged for about 10 min at about 
2000 g to 4000 g and at 4 to 8 °C. Six plasma aliquots (4 primaries and 2 back-ups) will be 
obtained and stored in polypropylene tubes. The aliquots should contain at least 0.4 mL 
plasma.
The process from blood collection until transfer of plasma aliquots into the freezer should be 
completed within 90 min, with interim storage of blood samples in ice water or on ice until 
centrifugation. For each aliquot, the time when the sample was placed in the freezer will be 
documented. Until transfer on dry ice, the aliquots will be stored upright at about -20°C or 
below at the trial site, except for the second back-up aliquot (“BU-2”), which will be stored at 
about -70 °C or below at the trial site.
For all treatment periods, the primary aliquots will be sent to the bioanalytical laboratory 
first. At the bioanalytical laboratory, the primary aliquots will be stored at about -20°C or 
below until analysis.
The back-up aliquots will be stored at the clinical site until the analysis of the primary 
aliquots has been finished. Thereafter the trial bioanalyst will decide on further transfer. 
At a minimum, the sample tube labels should list the following information: BI trial number, 
subject number, trial part, visit number (e.g.: first treatment period = visit number 2), aliquot* 
and planned sampling time. Further information such as matrix may also be provided. 
* aliquot names are planned as follows:
-
“D” 
= primary for digoxin (R1, T1-T4)
-
“F-C” 
= primary for furosemide given in the cocktail (R1, T1-T4)
-
“M-C”
= primary for metformin given in the cocktail (R1, T1-T4)
-
“R” 
= primary for rosuvastatin (R1, T1-T4)
-
“C-BU” 
= back-up for cocktail (R1, T1-T4)
-
“BU-2”
= second back-up (all treatments)


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Part 2 (only R2, T5) and Part 3 (only R3, T6) – therapeutic doses of furosemide / metformin
For quantification of metformin (Part 2) and furosemide (Part 3) plasma concentrations, 
2.7 mL of blood will be taken from an antecubital or forearm vein into K3-EDTA 
(tripotassium ethylenediamine-tetraacetic acid)-anticoagulant blood drawing tubes at the 
times indicated in the Flow Chart. Blood will be withdrawn by means of either an indwelling 
venous catheter or by venipuncture with a metal needle.
The EDTA-anticoagulated blood samples will be centrifuged for about 10 min at about 
2000 g to 4000 g and at 4 to 8 °C. Three plasma aliquots (1 primary and 2 back-ups) will be 
obtained and stored in polypropylene tubes. The first aliquot should contain at least 0.35 mL 
plasma, the back-up 0.35 mL or less.
The process from blood collection until transfer of plasma aliquots into the freezer should be 
completed within 90 min, with interim storage of blood samples in ice water or on ice until 
centrifugation. For each aliquot, the time when the sample was placed in the freezer will be 
documented. Until transfer on dry ice, the aliquots will be stored upright at about -20°C or 
below at the trial site, except for the second back-up aliquot (“BU-2”), which will be stored at 
about -70 °C or below at the trial site.
For all treatment periods, the primary aliquots will be sent to the bioanalytical laboratory 
first. At the bioanalytical laboratory, the primary aliquots will be stored at about -20°C or 
below until analysis.
The back-up aliquots will be stored at the clinical site until the analysis of the primary 
aliquots has been finished. Thereafter the trial bioanalyst will decide on further transfer.
At a minimum, the sample tube labels should list the following information: BI trial number, 
subject number, trial part, visit number (e.g.: first treatment period = visit number 2), aliquot* 
and planned sampling time. Further information such as matrix may also be provided.
* aliquot names are planned as follows:
-
“MTD” 
= primary for metformin given as therapeutic dose (R2, T5)
-
“FTD” 
= primary for furosemide given as therapeutic dose (R3, T6)
-
“M-BU” 
= back-up for metformin given as therapeutic dose (R2, T5)
-
“F-BU” 
= back-up for furosemide given as therapeutic dose (R3, T6)
-
“BU-2”
= second back-up (all treatments)


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5.5.2.2
Urine sampling for pharmacokinetic analysis
A blank urine sample will be collected before administration of trial medication (within 3 h 
before drug dosing) and aliquots will be retained to check for analytical interference. All 
urine voided during the sampling intervals indicated in the Flow Chart will be collected in 2 
L polyethylene (PE) containers and stored at room temperature. Subjects are told to empty 
their bladders at the end of each sampling interval.
The urine weight/volume for each collection interval will be documented (however, no 
correction for the specific gravity of urine is done, i.e. 1 L is defined to be equal to 1 kg). 
Aliquots will be stored in polypropylene (PP) tubes for bioanalytical measurement. In case 
more than one collection container is used in an interval, the contents of all containers are to 
be mixed before aliquots are prepared. Mixing should be done by transferring the entire 
content of all collection containers into a single PE/PP or glass container, and stirring the 
mixed fractions for about 1 min (manually or using a stir bar or other stirring device made of 
PE, PP, Teflon or glass).
In treatment periods with cocktail administration (R1, T1, T2, T3, T4), six urine aliquots of at 
least 1 mL will be obtained per time point or interval (4 primaries and 2 back-ups). In 
treatment periods with therapeutic doses of metformin (Part 2: R2, T5) and furosemide (Part 
3: R3, T6), three urine aliquots of at least 1 mL will be obtained per time point or interval (2
primaries and 1 back-up). Until transfer on dry ice, the urine samples will be stored at 
about -20C or below at the trial site, except for the second back-up aliquot (“BU-2”), which 
will be stored at about -70 °C or below at the trial site.
For all treatment periods, the primary aliquots will be sent to the bioanalytical laboratory 
first. At the bioanalytical laboratory, the primary aliquots will be stored at about -20°C or 
below until analysis.
The back-up aliquots will be stored at the clinical site until the analysis of the primary 
aliquots has been finished. Thereafter the trial bioanalyst will decide on further transfer.
At minimum, the sample tube labels should list the following information: BI trial number, 
subject number, trial part, visit number (e.g.: first treatment period = visit number 2), 
aliquot*, and planned collection interval or time point, as applicable. Further information,
such as matrix, may also be provided. Neither subject name nor initials or birth date will be 
given on the labels.
* aliquot names are planned as follows:
-
“D” 
= primary for digoxin (R1, T1-T4)
-
“F-C” 
= primary for furosemide given in the cocktail (R1, T1-T4)
-
“M-C”
= primary for metformin given in the cocktail (R1, T1-T4)
-
“R” 
= primary for rosuvastatin (R1, T1-T4)
-
“MTD” 
= primary for metformin given as therapeutic dose (R2, T5)
-
“FTD” 
= primary for furosemide given as therapeutic dose (R3, T6)
-
“C-BU” 
= back-up for cocktail (R1, T1-T4)
-
“M-BU” 
= back-up for metformin given as therapeutic dose (R2, T5)
-
“F-BU” 
= back-up for furosemide given as therapeutic dose (R3, T6)
-
“BU-2”
= second back-up (all treatments)


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5.5.2.3
Further investigations
After completion of the analysis of plasma and urine samples for concentrations of digoxin, 
furosemide, metformin, and rosuvastatin, the plasma and urine samples (including back-ups 
and left-over sample volumes from pre-specified analyses) may be used as follows:
-
For further methodological investigations, e.g. for stability testing, assessment of 
metabolites. However, only data related to the analyte and/or its metabolite(s) 
including anti-drug antibodies (if applicable) will be generated by these additional 
investigations.
-
For analyses of endogenous substances (including substances taken up with e.g. food). 
However, only substances that are substrates of membranous transport proteins or 
substrates or products of enzymes or that could otherwise possibly serve as potential 
indicators for the activity of membranous transport proteins or enzymes may be 
analysed.
-
For analyses of the concentrations of inhibitors (verapamil, probenecid, cimetidine, or 
rifampin) and/or their metabolites
Results of further investigations are not planned to be part of the trial report; however, results 
of further investigations may be part of the trial report, if required.
The study samples will be discarded after completion of the additional investigations, but not 
later than 5 years after the final study report has been signed.
5.5.3
Analytical determinations
5.5.3.1
Analytical determination of analyte plasma concentration
Digoxin, furosemide, metformin, and rosuvastatin concentrations in plasma will be 
determined by validated LC-MS/MS (liquid chromatography tandem mass spectrometry) 
assays. All details of the analytical methods will be available prior to the start of analysis.
Plasma samples dedicated to the analysis of digoxin are transferred to:
Plasma samples dedicated to the analysis of furosemide, metformin and rosuvastatin are 
transferred to:


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5.5.3.2
Analytical determination of analyte urine concentration
Digoxin, furosemide, metformin, and rosuvastatin concentrations in urine will be determined 
by validated LC-MS/MS assays. All details of the analytical methods will be available prior 
to the start of sample analysis.Urine samples dedicated to the analysis of digoxin are 
transferred to: 
Urine samples dedicated to the analysis of furosemide, metformin and rosuvastatin are 
transferred to: 


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6.
INVESTIGATIONAL PLAN
6.1
VISIT SCHEDULE
Exact times of measurements outside the permitted time windows will be documented. The 
acceptable time windows for screening and end of trial examination are given in the 
Flow-Chart.
Study measurements and assessments scheduled to occur ‘before’ trial medication 
administration on Day 1 are to be performed and completed within a 3 h period prior to the 
trial drug administration.
The acceptable deviation from the scheduled time for vital signs and ECG will be - 15 min
after study drug administration on Day 1 and ± 60 min on Day 2.
In all trial parts, subjects may have their dinner together at 08.00 p.m.
If scheduled in the Flow Chart at the same time as a meal, blood sampling, vital signs and 12-
lead ECG recordings have to be done first. Furthermore, if several measurements including 
venipuncture are scheduled for the same time, venipuncture should be the last of the 
measurements due to its inconvenience to the subject and possible influence on physiological
parameters.
For planned individual plasma concentration sampling times and urine collection intervals,
refer to the Flow Chart. While these nominal times should be adhered to as closely as 
possible, the actual sampling times will be recorded and used for determination of 
pharmacokinetic parameters.
If a subject misses an appointment, it will be rescheduled if possible. The relevance of 
measurements outside the permitted time windows will be assessed no later than at the Report 
Planning Meeting.
6.2
DETAILS OF TRIAL PROCEDURES AT SELECTED VISITS
6.2.1
Screening period
After having been informed about the trial, all subjects will give their written informed 
consent in accordance with GCP and local legislation prior to enrolment in the study.
For information regarding laboratory tests (including drug and virus screening), ECG, vital 
signs, and physical examination, refer to Sections 5.2.3 to 5.2.5.
6.2.2
Treatment periods
Each subject is expected to participate in three (Part 1) or four (Part 2 and 3) treatment 
periods. Adjacent treatment periods will be separated by at least 13 days (Part 1) or 7 days
(Part 2 and 3) between the victim drugs (cocktail, metformin, furosemide) administration.
On Day -1 of each treatment period, study participants will be admitted to the trial site and 
kept under close medical surveillance for at least 24 h following drug administration. The 


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subjects will then be allowed to leave the trial site after formal assessment and confirmation 
of their fitness. On all other study days, the study will be performed in an ambulatory fashion.
For details on time points and procedures for collection of plasma and urine samples for PK 
analysis, refer to Flow Chart and Section 5.5.2.
The safety measurements performed during the treatment period are specified in Section 5.2
of this protocol and in the Flow Chart. For details on time points for all other trial procedures, 
refer to the Flow Chart. AEs and concomitant therapy will be assessed continuously from 
screening until the end of trial examination.
6.2.3
End of trial period
For AE assessment, laboratory tests, recording of ECG and vital signs, and physical 
examination during the end of trial period, see Sections 5.2.2 to 5.2.5.
Subjects who discontinue treatment before the end of the planned treatment period should 
undergo the end of trial visit.
All abnormal values (including laboratory parameters) that are judged clinically relevant by 
the investigator will be monitored using the appropriate tests until a return to a medically 
acceptable level is achieved. (S)AEs persisting after subject’s end of trial must be followed 
up until they have resolved, have been sufficiently characterised, or no further information 
can be obtained. 
The end of the trial as a whole is defined by the ‘last regular visit completed by last subject’ 
or ‘end date of the last open AE’ or ‘date of the last follow-up test’ or ‘date of an AE has 
been decided as sufficiently followed-up’, whichever is latest.


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7.
STATISTICAL METHODS AND DETERMINATION OF 
SAMPLE SIZE
7.1
STATISTICAL DESIGN – MODEL
7.1.1
Objectives
The primary and secondary objectives of this trial are stated in Section 2.2.
7.1.2
Endpoints
The assessment of effects of drug transporter inhibitors (verapamil, rifampin, probenecid, 
cimetidine) on kinetics of sensitive drug transporter substrates (digoxin, furosemide, 
metformin, and rosuvastatin) at the dose strength used in the cocktail will be based on their
primary and secondary endpoints, see Sections 5.5.1.1 and 5.5.1.2.
Further pharmacokinetic parameters of interest of digoxin, furosemide, metformin, and 
rosuvastatin are stated in Section 5.5.1.3.
7.1.3
Model
The primary and secondary statistical analysis will be applied to each trial part separately. 
The statistical model used for the analysis of primary and secondary endpoints will be an 
ANOVA (analysis of variance) model on the logarithmic scale. This model will include 
effects accounting for the following sources of variation: ‘sequence’, ‘subjects within 
sequences’, ‘period’ and ‘treatment’. The effect ‘subjects within sequences’ will be 
considered as random, whereas the other effects will be considered as fixed. The model is 
described by the following equation:
yijkm = + i + sim + j + k + eijkm, where
yijkm = logarithm of response (primary or secondary endpoint, see Section 5.5.1) 
                      measured on subject m in sequence i receiving treatment k in period j,
= the overall mean,
i = the ith sequence effect, i = 1, 2, 3, for part 1 and i = 1,…, 4 for part 2 and 3
sim = the effect associated with the mth subject in the ith sequence,
                    m = 1,…, 5 for part 1 and m = 1,…, 4 for part 2 and 3
j = the jth period effect, j = 1, 2, 3, for part 1 and j = 1,…, 4 for part 2 and 3
k = the kth treatment effect,  k ∈{R1, T1, T2} for part 1 and 
                                                            k ∈{R1, T3, R2, T5}  for part 2 and 
                                                            k ∈{R1, T4, R3, T6}  for part 3 


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eijkm = the random error associated with the mth subject in sequence i who received
                       treatment k in period j.
where sim ~ N(0, 
) i.i.d., eijkm ~ N(0, 
 ) i.i.d., and sim, eijkm are independent random 
variables (note that the indices ‘B’ and ‘W’ correspond to ‘between’ and ‘within’ variability 
respectively).
For the primary analysis the following pairwise comparisons are of interest:
       for part 1: (k1, k2) ∈{(R1, T1), (R1, T2)}
        for part 2: (k1, k2) ∈{(R1, T3)}
        for part 3: (k1, k2) ∈{(R1, T4)}
There will be two sensitivity analyses of the primary analysis conducted. The one sensitivity 
analysis will be based on the same data as the primary analysis with the model described 
above, but the effect ‘subjects within sequences’ will be considered as a fixed effect.
The other sensitivity analysis will analyse the effects within each trial part based on all 
evaluable data of treatment R1 across the 3 trial parts. The statistical model of this sensitivity 
analysis will be the model described above for the primary analysis with the additional effect 
‘trial part’.
The secondary analysis will apply the primary analysis model to CLR,t1-t2 and to fet1-t2 of each 
investigated dose of the sensitive drug transporter substrates (digoxin, furosemide, 
metformin, and rosuvastatin).
For the further analysis investigating changes in selected PK parameters of the therapeutic 
doses of metformin and furosemide, the primary analysis model described above will be 
applied with (k1, k2) ∈{(R2, T5)} in part 2 and (k1, k2) ∈{(R3, T6)} in part 3.
7.2
NULL AND ALTERNATIVE HYPOTHESES
The pharmacokinetic changes of the investigated sensitive drug transporter substrates 
digoxin, furosemide, metformin and rosuvastatin will be estimated based on the pairwise 
ratios (test treatment Ti , i=1,…, 4, to reference treatment R1) of the geometric means 
(gMeans) of the primary and secondary endpoints. Additionally, their 2- sided 90% CIs will 
be provided.
No significance level adjustment for multiple comparisons will be applied. Note, the
computed CIs must be interpreted in the context of the exploratory character of the study, i.e.
confidence intervals are considered as interval estimates for effects.
Since the main focus is on estimation and not testing, an acceptance range was not specified, 
that is, no hypothesis will be tested.


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7.3
PLANNED ANALYSES
The statistical analysis will be conducted for each trial part separately, if not specified 
otherwise. This applies to all assessments and endpoints.
7.3.1
Primary analyses
The pharmacokinetic endpoints listed in Section 5.5.1 will be calculated according to the BI 
Standard Operating Procedure (SOP) ‘Standards and processes for analyses performed within 
Clinical Pharmacokinetics/Pharmacodynamics’ (001-MCS-36-472).
Plasma and urine concentration data and parameters of a subject will be included in the
statistical pharmacokinetic (PK) analyses if they are not flagged for exclusion due to a 
protocol violation relevant to the evaluation of PK (to be decided no later than in the Report 
Planning Meeting) or due to PK non-evaluability (as revealed during data analysis, based on 
the criteria specified below). Exclusion of a subject’s data will be documented in the CTR.
Relevant protocol violations may be:

Incorrect trial medication taken, i.e. the subject received at least one dose of trial 
medication the subject was not assigned to.

Incorrect dose of trial medication taken.
Plasma and urine concentrations and/or parameters of a subject will be considered as non-
evaluable, if for example

the subject experienced emesis that occurred at or before two times median tmax of the 
respective treatment (median tmax is to be determined excluding the subjects 
experiencing emesis),

a pre-dose concentration is >5% of the Cmax value of that subject,

missing samples/concentration data at important phases of PK disposition curve.
The following analysis sets will be defined for this trial: 
Treated set (TS): This subject set includes all subjects from the RS who were 
documented to have taken at least one dose of study drug. This is the full analysis set 
population in the sense of ICH-E9.

PK parameter analysis set (PKS): The subject set includes all subjects from the TS 
who provide at least one primary or secondary PK endpoint that was not excluded 
according to the description above. Thus, a subject will be included in the PKS, even 
if he contributes only one PK endpoint value for one treatment period to the statistical 
assessment.
Point estimates of the ratios of the geometric means (test/reference treatment) for the primary 
and secondary endpoints (see Section 5.5.1.1, 5.5.1.2), and their two-sided 90% confidence 
intervals (CIs) will be provided.
To this end, the PK endpoints will be log-transformed (natural logarithm) prior to fitting the 
ANOVA model (cf. Section 7.1.3). For each endpoint, the difference between the expected 
means for log(Ti)-log(R1) will be estimated by the difference in the corresponding adjusted 
means (LeastSquares Means), and a two-sided 90% confidence interval based on the 


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t-distribution will be computed. These quantities will then be back-transformed to the original 
scale to provide the point estimate and 90% CIs for each endpoint.
The following Tables display the planned comparisons for all trial objectives regarding 
pharmacokinetics for each trial part separately.
Table 7.3.1: 1
Planned pairwise comparisons of endpoints for the respective analyte to 
assess the trial objectives – part 1
Objective
Endpoints
Test treatment
Reference treatment
Analyte
Primary
Primary and 
secondary PK 
endpoints
T1
R1
Digoxin
T1
R1
Furosemide
T1
R1
Metformin
T1
R1
Rosuvastatin
T2
R1
Digoxin
T2
R1
Furosemide
T2
R1
Metformin
T2
R1
Rosuvastatin
Secondary
CLR, t1-t2 and fet1-t2
T1
R1
Digoxin
T1
R1
Furosemide
T1
R1
Metformin
T1
R1
Rosuvastatin
T2
R1
Digoxin
T2
R1
Furosemide
T2
R1
Metformin
T2
R1
Rosuvastatin
Note: for the description of treatments R1, T1 and T2 please refer to Table 4.1.4: 1 .


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Table 7.3.1: 2
Planned pairwise comparisons of endpoints for the respective analyte to 
assess the trial objectives – part 2
Objective
Endpoints
Test treatment
Reference treatment
Analyte
Primary
Primary and 
secondary PK 
endpoints
T3
R1
Digoxin
T3
R1
Furosemide
T3
R1
Metformin
T3
R1
Rosuvastatin
Secondary
CLR,t1-t2 and fet1-t2
T3
R1
Digoxin
T3
R1
Furosemide
T3
R1
Metformin
T3
R1
Rosuvastatin
Further
AUC0-tz, Cmax, 
AUC0-∞, CLR,t1-t2, 
fet1-t2
T5
R2
Metformin (500mg)
Note: for the description of treatments R1, R2, T3 and T5 please refer to Table 4.1.4: 1 .
Table 7.3.1: 3
Planned pairwise comparisons of endpoints for the respective analyte to 
assess the trial objectives – part 3
Objective
Endpoints
Test treatment
Reference treatment
Analyte
Primary
Primary and 
secondary PK 
endpoints
T4
R1
Digoxin
T4
R1
Furosemide
T4
R1
Metformin
T4
R1
Rosuvastatin
Secondary
CLR,t1-t2 and fet1-t2
T4
R1
Digoxin
T4
R1
Furosemide
T4
R1
Metformin
T4
R1
Rosuvastatin
Further
AUC0-tz, Cmax, 
AUC0-∞, CLR,t1-t2, 
fet1-t2
T6
R3
Furosemide (40mg)
Note: for the description of treatments R1, R3, T4 and T6 please refer to Table 4.1.4: 1 .
To assess the primary trial objective, the above mentioned pairwise comparisons (see 
Tables 7.3.1: 1, 7.3.1: 2, 7.3.1: 3) will be analysed using the primary statistical model and 
both sensitivity analyses (see Section 7.1.3).


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7.3.2
Secondary analyses
The secondary parameters (refer to Section 5.5.1) will be calculated according to the BI SOP 
‘Standards and processes for analyses performed within Clinical Pharmacokinetics/ 
Pharmacodynamics’ (001-MCS-36-472) and will be assessed statistically using the same 
methods as described for the primary endpoints.
The secondary analysis includes the assessments of all pairwise comparisons as described in 
the Tables 7.3.1: 1, 7.3.1: 2 and 7.3.1: 3, above, regarding the secondary and further trial 
objectives. For the secondary analysis, only the primary analysis model will be applied and 
no sensitivity analyses will be conducted.
7.3.3
Safety analyses
Safety will be assessed for the assessments listed in Section 5.2.1. All treated subjects (that is,
all subjects who received at least one dose of study drug), will be included in the safety
analysis. Safety analyses will be descriptive in nature and will be based on BI standards. No 
hypothesis testing is planned.
The analyses will be done by ‘treatment at onset’.
Treatments will be compared in a descriptive way. Tabulations of frequencies/proportions 
will be used for the evaluation of categorical (qualitative) data, and tabulations of descriptive 
statistics will be used to analyse continuous (quantitative) data.
Measurements (such as ECG, vital signs, or laboratory parameters) or AEs will be assigned to 
treatments (see Section 4.1) based on the actual treatment at the planned time of the 
measurement or on the recorded time of AE onset (concept of treatment emergent AEs).
Therefore, measurements planned or AEs recorded prior to first intake of trial medication will 
be assigned to 'screening', those between first trial medication intake and end of the residual 
effect period (see Section 5.2.2.2) will be assigned to the preceding treatment, those between 
end of REP and next administration of trial medication will be assigned to ‘post-treatment’. 
AEs occurring after the REP but prior to the last per-protocol contact are defined as ‘follow-
up’ events. These assignments including the corresponding time intervals will be defined in 
detail in the TSAP.
Adverse events will be coded using the Medical Dictionary for Regulatory Activities
(MedDRA). Frequency, severity and causal relationship of AEs will be tabulated by 
treatment, system organ class and preferred term. SAEs, and other significant AEs (according 
to ICH E3) will be listed separately.
Laboratory data will be compared to their reference ranges. Values outside the reference 
range as well as values defined as clinically relevant will be highlighted in the listings. 
For vital signs, descriptive statistics over time will be evaluated.
Relevant ECG findings will be reported as AEs.
7.3.4
Interim analyses
No interim analysis is planned.


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7.3.5
Pharmacokinetic analyses
The pharmacokinetic parameters listed in Section 5.5.1 for digoxin, furosemide, metformin 
and rosuvastatin (including the therapeutic doses of metformin and furosemide) will be 
calculated according to the relevant SOP of the Sponsor [001-MCS-36-472].
Subjects who are not included in the PKS (refer to Section 7.3.1) will be reported with their 
individual plasma/urine concentrations and individual pharmacokinetic parameters; however, 
they will not be included in descriptive statistics for plasma/urine concentrations, 
pharmacokinetic parameters or other statistical assessment.
Only concentration values within the validated concentration range and actual sampling times 
will be used for the calculation of pharmacokinetic parameters. Concentrations used in the 
pharmacokinetic calculations will be in the same format as provided in the bioanalytical 
report (that is, to the same number of decimal places as provided in the bioanalytical report).
If a predose concentration value is greater than 5% of Cmax, the subject’s pharmacokinetic 
data will not be included in any statistical evaluations, in accordance with international 
guidances. The individual pharmacokinetic parameters of such a subject will be calculated 
and listed separately. If a predose concentration is above BLQ, but less than or equal to 5% of 
the subject’s Cmax value, the subject’s data without any adjustments will be included in all 
pharmacokinetic measurements and calculations.
7.4
HANDLING OF MISSING DATA
7.4.1
Safety
With respect to safety evaluations, it is not planned to impute missing values.
7.4.2
Plasma/urine drug concentration - time profiles
Handling of missing PK data will be performed according to the relevant Corporate 
Procedure of the Sponsor [001-MCS-36-472].
Drug concentration data identified with NOS (no sample available), NOR (no valid result), 
NOA (not analysed), or BLQ (below the lower limit of quantification) will be displayed as 
such and not replaced by zero at any time point (this rule also applies to the lag phase,
including the predose values). 
7.4.3
Pharmacokinetic parameters
Handling of missing PK data will be performed according to the relevant SOP of the Sponsor 
[001-MCS-36-472).
For the non-compartmental analysis, concentration data identified with NOS, NOR or NOA 
will generally not be considered. Concentration values in the lag phase identified as BLQ will 
be set to zero. All other BLQ values of the profile will be ignored. The lag phase is defined as 
the period between time zero and the first time point with a concentration above the 
quantification limit.


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7.5
RANDOMISATION
Part 1: Subjects will be randomised to one of the three treatment sequences in a 1:1:1 ratio.
The block size will be documented in the CTR.
Part 2 and 3: Subjects will be randomised to one of the four treatment sequences in a 1:1:1:1
ratio. The block size will be documented in the CTR.
The sponsor will arrange for the randomisation. The randomisation list will be generated 
using a validated system, which involves a pseudo-random number generator and a supplied 
seed number so that the resulting allocation is both reproducible and non-predictable.
The randomisation list will contain additional blocks to allow for subject replacement (refer 
to Section 3.3.5).
7.6
DETERMINATION OF SAMPLE SIZE
It is planned to enter 15 subjects in part 1 and 16 subjects each in parts 2 and 3; thus, a total 
of 47 subjects will be entered in the trial, because this sample size is considered sufficient to 
achieve the aims of this exploratory trial.
Based on trial 352.2096, the intra-individual geometric coefficients of variation (gCVs) of the 
primary pharmacokinetic endpoints are assumed to range from 10% to 25% [c13060859-01].
Table 7.6: 1 provides an overview of the precision (defined as ratio of upper confidence limit 
over point estimate) and the 2-sided 90% CI of the gMean ratio (Ti/R1) that are expected (see 
Section 1.1.4) for digoxin and rosuvastatin with 95% probability, based on various gCVs and 
different sample sizes of evaluable subjects for part 1. Since this trial part has a 3-way 
crossover design, Table 7.6: 1 shows the precision in case of N=12 and N=15 evaluable 
subjects.
Table 7.6: 1
Expected precision and 2-sided 90% confidence interval for different gCVs, 
sample sizes and gMean ratios Ti/R1 - trial part 1 (3-way crossover)
gCV
N
Precision
90% CI for respective gMean ratio (Ti/R1)
100%
150%
600%
1100%
10%
12
1.092
( 91.57, 109.20)
(137.36, 163.80)
(549.45, 655.21)
(1007.32, 1201.21)
15
1.079
( 92.68, 107.89)
(139.03, 161.84)
(556.10, 647.36)
(1019.52, 1186.83)
15%
12
1.141
( 87.67, 114.07)
(131.50, 171.10)
(526.00, 684.41)
(964.34, 1254.75)
15
1.120
( 89.26, 112.03)
(133.89, 168.05)
(535.56, 672.19)
(981.86, 1232.35)
20%
12
1.191
( 83.97, 119.10)
(125.95, 178.64)
(503.80, 714.57)
(923.63, 1310.05)
15
1.163
( 86.00, 116.28)
(129.00, 174.42)
(515.99, 697.69)
(945.98, 1279.09)
25%
12
1.243
( 80.47, 124.27)
(120.71, 186.40)
(482.83, 745.60)
(885.19, 1366.94)
15
1.206
( 82.90, 120.63)
(124.35, 180.94)
(497.40, 723.76)
(911.90, 1326.90)
Thus, based on a sample size of 12 PK evaluable subjects and assuming a gCV of 25% for a 
specific PK endpoint, the precision (in terms of upper limit to point estimate) of the 2-sided 
90% CI would be 1.243. If the gMean ratio (Ti/R1) would be 150%, then the 90% CI is 
expected to range approximately from 120 to 186%.
Table 7.6: 2 provides an overview of the precision and the 2-sided 90% CI of the gMean ratio 
(Ti/R1) that are expected (see Section 1.1.4) for metformin and furosemide with 95% 
probability, based on various gCVs and different sample sizes of evaluable subjects for parts


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2 and 3, respectively. Since these trial parts have a 4-way crossover design, Table 7.6: 2
shows the precision in case of N=12 and N=16 evaluable subjects.
Table 7.6: 2
Expected precision and 2-sided 90% confidence interval for different gCVs, 
sample sizes and gMean ratios Ti/R1 - trial part 2, 3 (4-way crossover)
gCV
N
Precision
90% CI for respective gMean ratio (Ti/R1)
100%
150%
250%
300%
10%
12
1.087
( 91.99, 108.71)
(137.99, 163.06)
(229.98, 271.77)
(275.97, 326.12)
16
1.072
( 93.26, 107.23)
(139.89, 160.84)
(233.15, 268.07)
(279.78, 321.68)
15%
12
1.133
( 88.26, 113.30)
(132.39, 169.95)
(220.66, 283.24)
(264.79, 339.89)
16
1.110
( 90.09, 111.00)
(135.14, 166.50)
(225.23, 277.50)
(270.27, 333.00)
20%
12
1.180
( 84.73, 118.03)
(127.09, 177.04)
(211.81, 295.07)
(254.18, 354.09)
16
1.149
( 87.06, 114.86)
(130.59, 172.29)
(217.66, 287.15)
(261.19, 344.58)
25%
12
1.229
( 81.38, 122.89)
(122.06, 184.33)
(203.44, 307.21)
(244.13, 368.66)
16
1.188
( 84.18, 118.80)
(126.27, 178.20)
(210.44, 296.99)
(252.53, 356.39)
Thus, based on a sample size of 12 PK evaluable subjects and assuming a gCV of 25% for a 
specific PK endpoint, the precision of the 2-sided 90% CI would be 1.229. If the gMean ratio 
(Ti/R1) would be 300%, then the 90% CI is expected to range approximately from 244 to 
369%.
The expected 90% confidence interval limits in Tables 7.6: 1 and 7.6: 2 were calculated using 
the following equation:
CI limit upper/lower = exp(ln(θ) ± ω)
with θ being the ratio (Ti/R1) on the original scale and ω the distance from the estimate θ to 
either CI limit on the log-scale.
The calculations were performed as described by Kupper and Hafner [R12-0972] using R 
Version 3.2.2.
The previous trials in this project (352.2082, 352.2094 and 352.2096) had a drop-out rate of 6 
to 17%. Therefore, in this trial we do not expect more than 3 drop-outs within each trial part 
and the planned sample size of 47 subjects in total is considered sufficient to account for 
possible subjects with not evaluable primary endpoint data.


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8.
INFORMED CONSENT, DATA PROTECTION, TRIAL
RECORDS
The trial will be carried out in compliance with the protocol, the principles laid down in the 
Declaration of Helsinki, in accordance with the ICH Harmonised Tripartite Guideline for 
Good Clinical Practice (GCP) and relevant BI SOPs.
The investigator should inform the sponsor immediately of any urgent safety measures taken 
to protect the study subjects against any immediate hazard, and also of any serious breaches 
of the protocol or of ICH GCP.
The rights of the investigator and of the sponsor with regard to publication of the results of 
this trial are described in a separate agreement between the investigator or the trial site and 
the sponsor. As a general rule, no trial results should be published prior to finalisation of the 
CTR.
Insurance Coverage: The terms and conditions of the insurance coverage must be given to 
each subject and are made available to the investigator via documentation in the ISF.
8.1
STUDY APPROVAL, SUBJECT INFORMATION, AND INFORMED 
CONSENT
This trial will be initiated only after all required legal documentation has been reviewed and 
approved by the respective Institutional Review Board (IRB) / Independent Ethics Committee 
(IEC) and competent authority (CA) according to national and international regulations. The 
same applies for the implementation of changes introduced by amendments.
Prior to a subject’s participation in the trial, written informed consent must be obtained from 
each subject (or the subject’s legally accepted representative) according to ICH GCP and to 
the regulatory and legal requirements of the participating country. Each signature must be 
personally dated by each signatory and the informed consent and any additional subject 
information form are to be retained by the investigator as part of the trial records. A copy of 
the signed and dated written informed consent and any additional subject information must be 
given to each subject or the subject’s legally accepted representative.
The subject must be informed that his/her personal trial-related data will be used by 
Boehringer Ingelheim in accordance with the local data protection law. The level of 
disclosure must also be explained to the subject.
The subject must be informed that his or her medical records may be examined by authorised 
monitors (Clinical Monitor Local/Clinical Research Associate) or Clinical Quality Assurance 
auditors appointed by Boehringer Ingelheim, by appropriate IRB/IEC members, and by 
inspectors from regulatory authorities.
8.2
DATA QUALITY ASSURANCE
A quality assurance audit/inspection of this trial may be conducted by the sponsor or 
sponsor’s designees, by IRBs/IECs, or by regulatory authorities. The quality assurance 
auditor will have access to all medical records, the investigator’s trial-related files and 
correspondence, and the informed consent documentation of this clinical trial.


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The data management procedures to ensure the quality of the data are described in detail in 
the trial data management and analysis plan (TDMAP) available in the TMF.
8.3
RECORDS
CRFs for individual subjects will be provided by the sponsor. For drug accountability, refer 
to Section 4.1.8.
8.3.1
Source documents
Source documents provide evidence for the existence of the subject and substantiate the 
integrity of the data collected. Source documents are filed at the investigator’s site.
All data reported in the CRFs must be consistent with the source data or the discrepancies 
must be explained.
The investigator may need to request previous medical records or transfer records, depending 
on the trial.
8.3.2
Direct access to source data and documents
The investigator/institution will permit trial-related monitoring, audits, IRB/IEC review and 
regulatory inspection, providing direct access to all related source data/documents. CRFs and 
all source documents, including progress notes (if applicable) and copies of laboratory and 
medical test results must be available at all times for review by the sponsor’s clinical trial 
monitor, auditor and inspection by health authorities (e.g. FDA). The Clinical Research 
Associate/on site monitor and auditor may review all CRFs, and written informed consents. 
The accuracy of the data will be verified by reviewing the documents described in
Section 8.3.1.
8.3.3
Storage period of records
Trial site:
The trial site must retain the source and essential documents (including ISF) according to the 
national or local requirements (whatever is longer) valid at the time of the end of the trial.
Sponsor:
The sponsor must retain the essential documents according to the sponsor’s SOPs.
8.4
EXPEDITED REPORTING OF ADVERSE EVENTS
BI is responsible to fulfil their legal and regulatory reporting obligation in accordance with 
regulatory requirements.
8.5
STATEMENT OF CONFIDENTIALITY
Individual subject medical information obtained as a result of this trial is considered 
confidential and disclosure to third parties is prohibited with the exceptions noted below. 
Subject confidentiality will be ensured by using subject identification code numbers.


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Treatment data may be provided to the subject’s personal physician or to other appropriate 
medical personnel responsible for the subject’s welfare. Data generated as a result of the trial 
need to be available for inspection on request by the participating physicians, the sponsor’s 
representatives, by the IRB/IEC and the regulatory authorities, i.e. the CA.
8.6
COMPLETION OF TRIAL
The EC/competent authority in each participating EU member state needs to be notified about 
the end of the trial (last subject / subject out, unless specified differently in Section 6.2.3 of 
the CTP) or early termination of the trial.


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9.
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9.1
PUBLISHED REFERENCES
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Graham GG, Punt J, Arora M, Day RO, Doogue MP, Duong JK, Furlong 
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Guidance for industry: drug interaction studies - study design, data analysis, 
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Probenecid. http://www.medicinescomplete.com/ (access date: 28 February 
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important determinants of drug disposition and effects. Pharmacol Rev 
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Neuvonen PJ, Backman JT, Niemi M. Pharmacokinetic comparison of the 
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R09-5227
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120: 120 mg/Filmtablette (AWD.pharma), verschreibungspflichtig 
(Fachinformation (Zusammenfassung der Merkmale des 
Arzneimittels/SPC), Stand der Information: Februar 2008).
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Cho SK, Yoon JS, Lee MG, Lee DH, Lim LA, Park K, Park MS, Chung 
JY. Rifampin enhances the glucose-lowering effect of metformin and 
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Probenecid Weimer Tabletten (Biokanol), verschreibungspflichtig 
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Landersdorfer CB, Kirkpatrick CMJ, Kinzig M, Bulitta JB, Holzgrabe U, 
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Lasix liquidum 10mg/ml Loesung zum Einnehmen (Sanofi-Aventis), 
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W, et al. Disposition and sterol-lowering effect of ezetimibe are influenced 
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potential of 22 antituberculosis drugs on organic anion and cation 
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Lasix long 30 mg Retardkapseln, Lasix 40 mg Tabletten/Lasix 500 mg Tabs 
(Sanofi-Aventis), verschreibungspflichtig (Fachinformation, Stand der 
Information: Juni 2016).
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Glucophage 500 mg/- 850 mg/- 1000 mg Filmtabletten (Merck Serono), 
verschreibungspflichtig (Zusammenfassung der Merkmale des 
Arzneimittels, Stand der Informationen: Januar 2017).
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Cimetidin acis 200 mg, 400 mg, 800 mg Tabletten (acis Arzneimittel), 
verschreibungspflichtig (Fachinformation, Stand der Information: Januar 
2014).
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Han YH, Busler D, Hong Y, Tian Y, Chen C, Rodrigues AD. Transporter 
studies with the 3-O-sulfate conjugate of 17alpha-ethinylestradiol: 
assessment of human kidney drug transporters. Drug Metab Dispos 
2010;38(7):1064-1071.
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Ohnhaus EE. Cimetidine and the immuno-response in healthy volunteers. J 
Invest Dermatol 1989;93(6):757-761.
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Garty M, Perry G, Shmueli H, Ilfeld D, Boner G, Pitlik S, et al. Effect of 
cimetidine on digoxin disposition in peptic ulcer patients. Eur J Clin 
Pharmacol 1986;30(4):489-491.
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cimetidine: investigation of the potential for a drug interaction. Hum 
Toxicol 1985;4(4):391-399.
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alter the pharmacokinetics of digoxin? Isr Med Assoc J 2002;4(10):772-
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pharmacokinetics of digoxin in healthy volunteers. Pharmacotherapy 
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pharmacodynamic interaction between tolvaptan, a non-peptide AVP 
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2007;50(2):213-222.
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al. Effects of probenecid on the pharmacokinetics and elimination of 
acyclovir in humans. Antimicrob Agents Chemother 1982;21(5):804-807.


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Probenecid inhibits the renal clearance of frusemide and its acyl 
glucuronide. Br J Clin Pharmacol 1995;39:692-695.
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Hedaya MA, Elmquist WF, Sawchuk RJ. Probenecid inhibits the metabolic 
and renal clearances of zidovudine (AZT) in human volunteers. Pharm Res 
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of furosemide-probenecid interaction in humans. J Pharm Sci 
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Produkteigenschaften/SPC), Stand der Information: Dezember 2016).
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Isoptin 120 mg, Filmtabletten (Mylan Healthcare), verschreibungspflichtig 
(Fachinformation, Stand der Information: 03.2016).
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MetfoLiquid 1000 mg/5 ml Loesung zum Einnehmen (GeriaSan), 
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probenecid on the renal elimination of cimetidine. Clin Pharmacol Ther 
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9.2
UNPUBLISHED REFERENCES
001-MCG-156
Analysis and Presentation of Adverse Event Data from Clinical Trials.
Current Version
001-MCS-36-472 Standards and processes for analyses performed within Clinical 
Pharmacokinetics/Pharmacodynamics. Current version
U09-1052-01
Relative bioavailability of a 
single oral dose of 150 mg dabigatran etexilate with or without oral 
administration of verapamil in two different dosages (240 and 480 mg 
daily), two different formulations (IR vs. ER) in healthy male and female 
volunteers (open-label, fixedsequence design), and relative bioavailability 
of a single oral dose of 150 mg dabigatran given with or without a single 
oral dose of 120 mg verapamil administered at different time points related 
to dabigatran dosing. (open-label, randomised, four-way crossover design).
Trial 1160.74. March 2009.
U10-1328-01
 An open-label, 2-period, 
fixed-sequence, phase I trial to evaluate the effect of multiple doses of 
rifampicin on the multiple-dose pharmacokinetics of linagliptin. Trial 
1218.67. February 2010.
U13-2366-01
 Bioequivalence of 
empagliflozin/metformin fixed dose combination tablets compared to single 
tablets administered together in healthy male and female volunteers under 
fed and fasted conditions (an open-label, randomised, single-dose, 
crossover study). Trial 1276.8. October 2013.
c03246006-01
 Relative bioavailability of a single 
oral dose of digoxin, furosemide, metformin, and rosuvastatin given alone 
and all together as a cocktail, and investigation of the effect of increased 
doses of metformin or furosemide on relative bioavailability of the other 
cocktail compounds in healthy male subjects. Trial 352.2082. July 2015.
c08983809-01
The effect of different doses of 
metformin or furosemide on rosuvastatin pharmacokinetics following oral 
administration in healthy male subjects (an open-label, randomised, single-
dose,six-way crossover study). Trial 352.2094. August 2016.
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pharmacokinetic interactions of digoxin, furosemide, metformin, and 
rosuvastatin given all together as a probe cocktail for key drug transporters 
(an open-label, randomised, single-dose, five-way crossover study). Trial 
352.2096. July 2017


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10.
APPENDICES
Not applicable. 


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11.
DESCRIPTION OF GLOBAL AMENDMENT(S)
Number of global amendment
01
Date of CTP revision
29 SEP 2017
EudraCT number
2017-001549-29
BI Trial number
0352-2100
Investigational Product(s)
Digoxin, furosemide, metformin, rosuvastatin, 
verapamil, probenecid, cimetidine, rifampin
Title of protocol
The effect of potent inhibitors of drug transporters 
(verapamil, rifampin, cimetidine, probenecid) on 
pharmacokinetics of a transporter probe drug cocktail
consisting of digoxin, furosemide, metformin and 
rosuvastatin (an open label, randomised, crossover 
trial in three parts)
To be implemented only after 
approval of the IRB / IEC /
Competent Authorities
This amendment to CTP has been written to fulfil 
requirements of the Competent authority.
To be implemented 
immediately in order to 
eliminate hazard –
IRB / IEC / Competent 
Authority to be notified of 
change with request for 
approval
Can be implemented without 
IRB / IEC / Competent 
Authority approval as changes 
involve logistical or 
administrative aspects only
Section to be changed
1) Synopsis, Section 3.3 and Section 3.3.5
2) Flow Chart Part 1
3.) Section 3.3.3
4.) Section 3.3.4.2
5.) Section 3.3.4.1
Description of change
1.) maximum 3 replacement subjects per trial part
2.) rhythm stripe added to screening examination
3.) new exclusion criteria added
4.) change of criterion for trial discontinuation
5.) there is no DILI checklist in this trial 
Rationale for change
1.)-4.) To fulfil requirements of Competent Authority
5.) correction to be consistent with Section 5.2.2.1


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Number of global amendment
02
Date of CTP revision
24 OCT 2017
EudraCT number
2017-001549-29
BI Trial number
0352-2100
Investigational Product(s)
Digoxin, furosemide, metformin, rosuvastatin, 
verapamil, probenecid, cimetidine, rifampin
Title of protocol
The effect of potent inhibitors of drug transporters 
(verapamil, rifampin, cimetidine, probenecid) on 
pharmacokinetics of a transporter probe drug cocktail 
consisting of digoxin, furosemide, metformin and 
rosuvastatin (an open label, randomised, crossover 
trial in three parts)
To be implemented only after 
approval of the IRB / IEC / 
Competent Authorities
To be implemented 
immediately in order to 
eliminate hazard –
IRB / IEC / Competent 
Authority to be notified of 
change with request for 
approval
Can be implemented without 
IRB / IEC / Competent 
Authority approval as changes 
involve logistical or 
administrative aspects only
The originally planned medication “MetfoLiquid 
GeriaSan 500mg/5mL” is no longer available on the 
German market. It was replaced by “MetfoLiquid 
GeriaSan 1000 mg/5 mL” which will now be used in 
this trial. The Amendment 02 has been written to 
implement the change of medication. To cover the 
higher concentration of the metformin solution the 
volume of application will be reduced, the given doses 
of metformin remain unchanged (10 mg in R1, 500 mg 
in R2). Thus Amendment 02 does not impact safety, 
or physical and mental integrity of the subjects nor the 
scientific value of the trial and is regarded as non-
substantial from a regulatory view-point. Furthermore,
minor changes have been made to correct mistakes.
Section to be changed
1.) Synopsis, section 1.2.3., 4.1.1 and 4.1.4
2.) Flow Chart (Part 2)
3.) Section 4.1.4
4.) Section 4.2.2.2
5.) Section 5.2.3
6.) Section 5.2.4


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Number of global amendment
02
Description of change
1.) change of metformin medication (see above) and 
reduction of metformin dosing volume to 0.05 mL (in 
treatment R1) and 2.5 mL (in treatment R2)
2.) footnote 12 refers to cimetidine intake only
3.) restriction to body posture does not refer to 
medical procedures
4.) Restriction on fluid intake refers to all periods.
5.) Lab data are transferred in a paper form.
6.) Description of ECG continuous monitoring 
Rationale for change
1.) MetfoLiquid GeriaSan 500 mg/5mL is no longer 
available on the German market
2.) to avoid misunderstanding 
3.) ECG/vital signs have to be done in supine position
4.) to avoid misunderstanding
5.) initial entry was not correct
6.) Description of continuous ECG-monitoring in T1 
was missing by mistake.


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Number of global amendment
03
Date of CTP revision
01 DEC 2017
EudraCT number
2017-001549-29
BI Trial number
0352-2100
Investigational Product(s)
Digoxin, furosemide, metformin, rosuvastatin, 
verapamil, probenecid, cimetidine, rifampin
Title of protocol
The effect of potent inhibitors of drug transporters 
(verapamil, rifampin, cimetidine, probenecid) on 
pharmacokinetics of a transporter probe drug cocktail 
consisting of digoxin, furosemide, metformin and 
rosuvastatin (an open label, randomised, crossover 
trial in three parts)
To be implemented only after 
approval of the IRB / IEC / 
Competent Authorities
To be implemented 
immediately in order to 
eliminate hazard –
IRB / IEC / Competent 
Authority to be notified of 
change with request for 
approval
Can be implemented without 
IRB / IEC / Competent 
Authority approval as changes 
involve logistical or 
administrative aspects only
Hypomagnesemia is a defined exclusion 
criterion (22). However this is not reflected in the 
lab table 5.2.3:1 by mistake. Amendment 03 is 
written to correct this mistake. 
To check exclusion criterion 22 the trial site has 
added magnesium to the lab panel right from the 
beginning. Magnesium values have been 
measured for all subjects prior to dosing. Thus, 
the CTP Amendment does not result in changes in 
the clinical performance of the trial. It adapts the 
incorrect CTP to the correct clinical practice. 
Considering this Amendment 03 does not impact the
safety, or physical and mental integrity of the subjects 
nor the scientific value of the trial and is regarded as 
non-substantial from a regulatory view-point.
Section to be changed
Table 5.2.3:1
Description of change
Magnesium added to electrolytes
Rationale for change
Magnesium was missing by mistake. 


 
 
 
 
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Date Signed 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
c16810802
4.0
clinical-trial-protocol-revision-03
The effect of potent inhibitors of drug transporters (verapamil, rifampin, cimetidine,
probenecid) on pharmacokinetics of a transporter probe drug cocktail consisting of
digoxin, furosemide, metformin and rosuvastatin (an open-label, randomised,
crossover trial in three parts)
Author-Trial Clinical Monitor
01 Dec 2017 13:50 CET
Approval-Dept 
or
or
01 Dec 2017 14:10 CET
Verification-Paper Signature
Completion
01 Dec 2017 14:50 CET
Author-Trial Clinical
Pharmacokineticist
01 Dec 2017 14:52 CET
Author-Trial Statistician
04 Dec 2017 14:28 CET


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SAP_000.pdf: 
TITLE PAGE
TRIAL STATISTICAL ANALYSIS PLAN
c21690485-01
BI Trial No.:
0352-2100
Title:
The effect of potent inhibitors of drug transporters (verapamil,
rifampin, cimetidine, probenecid) on pharmacokinetics of a 
transporter probe drug cocktail consisting of digoxin, furosemide, 
metformin and rosuvastatin (an open-label, randomised, crossover 
trial in three parts)
Investigational
Product:
Digoxin, furosemide, metformin, rosuvastatin, 
verapamil, probenecid, cimetidine, rifampin
Responsible trial 
statisticians:
Phone:
Fax:
Phone: 
Fax: 
Date of statistical 
analysis plan:
07 JUN 2018 SIGNED 
Version:
Final
Page 1 of 28
Proprietary confidential information
2018 Boehringer Ingelheim International GmbH or one or more of its affiliated companies. All rights reserved.
This document may not - in full or in part - be passed on, reproduced, published or otherwise used without prior written permission.


Boehringer Ingelheim
TSAP for BI Trial No: 0352-2100
Page 2 of 28
Proprietary confidential information © 2018 Boehringer Ingelheim International GmbH or one or more of its affiliated companies
1.
TABLE OF CONTENTS
TITLE PAGE............................................................................................................................1
1.
TABLE OF CONTENTS......................................................................................2
LIST OF TABLES ...................................................................................................................3
2.
LIST OF ABBREVIATIONS...............................................................................4
3.
INTRODUCTION.................................................................................................6
4.
CHANGES IN THE PLANNED ANALYSIS OF THE STUDY ......................7
5.
ENDPOINTS..........................................................................................................8
5.1
PRIMARY ENDPOINTS .....................................................................................8
5.2
SECONDARY ENDPOINTS ...............................................................................8
5.2.1
Key secondary endpoints ......................................................................................8
5.2.2
Secondary endpoints .............................................................................................8
5.3
FURTHER ENDPOINTS.....................................................................................8
5.4
OTHER VARIABLES ..........................................................................................9
6.
GENERAL ANALYSIS DEFINITIONS...........................................................10
6.1
TREATMENTS...................................................................................................10
6.2
IMPORTANT PROTOCOL VIOLATIONS....................................................14
6.3
SUBJECT SETS ANALYSED ...........................................................................15
6.4
SUBGROUPS.......................................................................................................17
6.5
POOLING OF CENTRES..................................................................................17
6.6
HANDLING OF MISSING DATA AND OUTLIERS ....................................17
6.7
BASELINE, TIME WINDOWS AND CALCULATED VISITS....................17
7.
PLANNED ANALYSIS ......................................................................................18
7.1
DEMOGRAPHIC AND OTHER BASELINE CHARACTERISTICS .........19
7.2
CONCOMITANT DISEASES AND MEDICATION......................................20
7.3
TREATMENT COMPLIANCE ........................................................................20
7.4
PRIMARY ENDPOINTS ...................................................................................20
7.5
SECONDARY ENDPOINTS .............................................................................22
7.5.1
Key secondary endpoints ....................................................................................22
7.5.2
Secondary endpoints ...........................................................................................22
7.6
FURTHER ENDPOINTS...................................................................................22
7.7
EXTENT OF EXPOSURE .................................................................................23
7.8
SAFETY ANALYSIS..........................................................................................23
7.8.1
Adverse events .....................................................................................................23
7.8.2
Laboratory data...................................................................................................24
7.8.3
Vital signs.............................................................................................................25
7.8.4
ECG ......................................................................................................................25
7.8.5
Others ...................................................................................................................25
8.
REFERENCES ....................................................................................................26
9.
ADDITIONAL SECTIONS................................................................................27
10.
HISTORY TABLE..............................................................................................28


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LIST OF TABLES
Table 6.1: 1
Treatments and labels used in the analysis (Trial Part 1).................................10
Table 6.1: 2
Treatments and labels used in the analysis (Trial Part 2).................................10
Table 6.1: 3
Treatments and labels used in the analysis (Trial Part 3).................................11
Table 6.1: 4
Overview of intra-individual comparisons.......................................................13
Table 6.2: 1
Important protocol violations ...........................................................................15
Table 6.3: 1
Subject sets analysed ........................................................................................16
Table 10: 1
History table .....................................................................................................28


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2.
LIST OF ABBREVIATIONS
Term
Definition / description
ADS
Analysis Dataset
AE
Adverse Event
AESI
Adverse Event of Special Interest
ANOVA
Analysis of Variance
AUC0-tz
Area under the concentration-time curve of the analyte in plasma over the 
time interval from 0 to the last quantifiable data point
AUC0-∞
Area under the concentration-time curve of the analyte in plasma over the 
time interval from 0 extrapolated to infinity
BI
Boehringer Ingelheim
BWC
Bioavailability/Bioequivalence, Within-Subject Design, Time-Controlled
CI
Confidence Interval
CLR,t1-t2
Renal clearance of the analyte in plasma from the time point t1 to t2
Cmax
Maximum measured concentration of the analyte in plasma
CRF
Case Report Form
CTP
Clinical Trial Protocol
CTR
Clinical Trial Report
CV
Arithmetic Coefficient of Variation
DBLM
Database Lock Meeting
ECG
Electrocardiogram
fet1-t2
Fraction of given drug excreted unchanged in urine from time point t1 to t2
gCV
Geometric Coefficient of Variation
gMean
Geometric Mean
ICH
International Conference On Harmonisation
LLT
Lower Level Term
Max
Maximum
MedDRA
Medical Dictionary For Regulatory Activities
Min
Minimum
O*C
Oracle Clinical
PK
Pharmacokinetic(s)
PKS
PK parameter analysis set
PT
Preferred Term


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Term
Definition / description
PV
Protocol Violation
qd
Quaque die (daily)
qid
Quater in die (4 times a day)
R
Reference Treatment
RAGe
Report Appendix Generator system
REP
Residual Effect Period
RPM
Report Planning Meeting
RS
Randomised set
SAS®
Statistical Analysis System
SD
Standard Deviation
SOC
System Organ Class
T
Test treatment
tmax
Time from dosing to maximum measured concentration of the analyte in 
plasma
TS
Treated Set
TSAP
Trial Statistical Analysis Plan
WHO-DD
World Health Organization Drug Dictionary
XPKISTAT
Library of SAS® Macros for PK analysis


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3.
INTRODUCTION
As per ICH E9 (1), the purpose of this document is to provide a more technical and detailed 
elaboration of the principal features of the analysis described in the protocol, and to include 
detailed procedures for executing the statistical analysis of the primary and secondary 
variables and other data.
This trial statistical analysis plan (TSAP) assumes familiarity with the Clinical Trial Protocol 
(CTP), including Protocol Amendments. In particular, the TSAP is based on the planned 
analysis specification as written in CTP Section 7 “Statistical Methods and Determination of 
Sample Size”. Therefore, TSAP readers may consult the CTP for more background 
information on the study, e.g., on study objectives, study design and population, treatments, 
definition of measurements and variables, planning of sample size, randomisation.
Study data will be stored in a trial database within the Oracle ClinicalTM (O*C) system.
Pharmacokinetic (PK) parameters will be calculated using Phoenix WinNonlinTM software 
(version 6.3 or higher, Certara USA Inc., Princeton, NJ, USA).
The statistical analyses will be performed within the validated working environment CARE, 
including SASTM (current Version 9.4, by SAS Institute Inc., Cary, NC, USA), and a number 
of SASTM-based tools (e.g., macros for the analyses of AE data or laboratory data; Report 
Appendix Generator system (RAGe) for compilation/formatting of the CTR appendices).


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4.
CHANGES IN THE PLANNED ANALYSIS OF THE STUDY
All analyses as planned in the CTP will be performed and are described in more detail in this 
TSAP. The following change compared to the protocol will be made:
No ‘Randomised set’ (RS) will be defined in the TSAP as data of subjects randomised but 
discontinued before first administration of trial medication will not be entered in the case 
report form. A correct display of the RS would not be possible.
The follow-up and post treatment phase defined in the CTP will be combined to one post-
treatment phase (“Post-trt”) for Listings and Tables of Adverse events.


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5.
ENDPOINTS
5.1
PRIMARY ENDPOINTS
Section 5.5.1.1 of the CTP:
The following primary endpoints will be determined for digoxin, furosemide, metformin, and 
rosuvastatin (at cocktail doses):

AUC0-tz (area under the concentration-time curve of the analyte in plasma over the 
time interval from 0 to the last quantifiable data point)

Cmax (maximum measured concentration of the analyte in plasma)
5.2
SECONDARY ENDPOINTS
5.2.1
Key secondary endpoints
This section is not applicable as no key secondary endpoints have been defined in the CTP.
5.2.2
Secondary endpoints
Section 5.5.1.2 of the CTP: 
The following secondary endpoint will be evaluated for digoxin, furosemide, metformin, and 
rosuvastatin (at cocktail doses):

AUC0-∞(area under the concentration-time curve of the analyte in plasma over the
time interval from 0 extrapolated to infinity)
For digoxin, AUC0-∞will be evaluated only if determination with sufficient precision is
possible.
5.3
FURTHER ENDPOINTS
Pharmacokinetic (PK):
Pharmacokinetic parameters are further parameters of interest. For more details, see CTP 
Section 5.5.1.3.
For secondary PK analyses the following endpoints for digoxin, furosemide, metformin, and 
rosuvastatin (at cocktail doses) are of particular relevance:

fet1-t2 (fraction of given drug excreted unchanged in urine from time point t1 to t2)

CLR, t1-t2 (renal clearance of the analyte in plasma from the time point t1 to t2)


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For further PK analyses, the following endpoints for furosemide and metformin given at 
therapeutic doses are of particular relevance:

AUC0-tz (area under the concentration-time curve of the analyte in plasma over the 
time interval from 0 to the last quantifiable data point)

Cmax (maximum measured concentration of the analyte in plasma)

AUC0-∞(area under the concentration-time curve of the analyte in plasma over the 
time interval from 0 extrapolated to infinity)
Safety:
Further criteria of interest:

AEs (including clinically relevant findings from the physical examination)

Safety laboratory tests

12-lead ECG

Vital signs (blood pressure, pulse rate) 
For detailed information (formula) please refer to Section 7.8.
5.4
OTHER VARIABLES
Section 5.2.5.2 of the CTP: At screening, the medical examination will include demographics 
including height and body weight, smoking and alcohol history, relevant medical history and 
concomitant therapy […].
Age [years] will be determined as the difference between year of informed consent and year 
of birth.
BMI will be calculated as weight [kg] / (0.01 * height [cm])2.


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6.
GENERAL ANALYSIS DEFINITIONS
6.1
TREATMENTS
For basic study information on investigational products, assignment of treatment sequences,
and selection of doses, please see CTP, Sections 3 and 4.
The study will be performed as a randomised, open-label, crossover trial in three parts. 
In total, it was planned to assign 47 healthy male subjects to the three trial parts; 15 healthy 
male subjects will enter trial part 1, 16 healthy males will enter trial part 2 and 16 healthy 
males will enter trial part 3.
For details of dosage and formulation, see Tables 6.1: 1, 6.1: 2 and 6.1: 3 below.
Table 6.1: 1
Treatments and labels used in the analysis (Trial Part 1)
Treatment
Treatment long label*
Short label
A
R1
Digoxin 0.25mg + Furosemide 1mg + Metformin 10mg + 
Rosuvastatin 10mg
Cocktail (R1)
B
T1
Verapamil 120mg + 
Digoxin 0.25mg + Furosemide 1mg + Metformin 10mg + 
Rosuvastatin 10mg
R1+Vera (T1)
C
T2
Rifampin 600mg + 
Digoxin 0.25mg + Furosemide 1mg + Metformin 10mg + 
Rosuvastatin 10mg
R1+Rifa (T2)
* Please note:
The above listed treatment long labels do not reflect the actual treatment regimens from Oracle Clinical. These treatment long labels are a 
little bit more comprehensive.
Table 6.1: 2
Treatments and labels used in the analysis (Trial Part 2)
Treatment
Treatment long label*
Short label
A
R1
Digoxin 0.25mg + Furosemide 1mg + Metformin 10mg + 
Rosuvastatin 10mg
Cocktail (R1)
D
R2
Metformin, oral solution, 500mg
Met (R2)
E
T3
Day 1: Cimetidine 400mg tablet, qid + 
Digoxin 0.25mg + Furosemide 1mg + Metformin 10mg + 
Rosuvastatin 10mg
Day 2: Cimetidine 400 mg bid
R1+Cime (T3)
F 
T5
Day 1: Cimetidine, 400 mg tablet, qid + 
Metformin, oral solution, 500mg
Day 2: Cimetidine 400 mg bid
R2+Cime (T5)
* Please note:
The above listed treatment long labels do not reflect the actual treatment regimens from Oracle Clinical. These treatment long labels are a 
little bit more comprehensive.


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Table 6.1: 3
Treatments and labels used in the analysis (Trial Part 3)
Treatment
Treatment long label*
Short label
A
R1
Digoxin 0.25mg + Furosemide 1mg + Metformin 10mg + 
Rosuvastatin 10mg
Cocktail (R1)
K
R3
Furosemide, oral solution, 40mg, qd
Furo (R3)
L
T4
Day -1: Probenecid 2*500mg tablet qd
Day 1: Probenecid 2*500mg tablet, qd + 
Digoxin 0.25mg + Furosemide 1mg + Metformin 10mg + 
Rosuvastatin 10mg
R1+Prob (T4)
M 
T6
Day -1: Probenecid 2*500mg tablet qd
Day 1: Probenecid,2*500 mg tablet, qd + 
Furosemide, oral solution, 40mg, qd
R3+Prob (T6)
* Please note:
The above listed treatment long labels do not reflect the actual treatment regimens from Oracle Clinical. These treatment long labels are a 
little bit more comprehensive.
The following separate study phases will be defined for the analyses of AEs:

Screening (ranging from 0:00 h on day of informed consent until first administration 
time of study drug)

On treatment
(separately for each treatment, including residual effect period (REP); i.e. ranging 
from administration time of study drug until 7 days thereafter )

Post-treatment (ranging from end of on treatment phase until next drug 
administration or 0:00 h on the day after trial termination date (labelled “Post-trt”))
Displays of AEs will be presented separately for the treatments described in Table 6.1: 1,
Table 6.1: 2 and Table 6.1: 3 above.
Two types of AE displays will be provided in the report:
A) Section 15.3 (separately for all trial parts) and Appendix 16.1.13.1.8 (for 
ClinicalTrials.gov (separately for all trial parts) and EudraCT (all trial parts combined)) 
of the CTR displays:
In these displays, the on treatment phase will be analysed (labelled with the name of the study 
treatment (short label)). Screening and Post-treatment will not be included in this analysis. 
The following total will be provided in addition:

a total over all on treatment phases included in this analysis ("Total on treatment") 
(Section 15.3 only)


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B) Section 15.4 and Appendix 16.1.13.1.8 (except for ClinicalTrials.gov and EudraCT) of 
the CTR displays:

Screening 

On treatment (labelled with the name of the study treatment (short label))

Post-treatment
In Section 16.1.13.1.8 AE tables (separately for all trial parts), the following totals will be
provided in addition:

a total over all study phases ("Total")
Tables of vital signs and laboratory values will present results by the above mentioned on 
treatment phase.
For detailed information on the handling of the treatments in the O*C views, refer to 
Technical TSAP ADS (analysis data set) plan. 
The following Table 6.1: 4 gives an overview of the planned analyses per trial part with the 
respective endpoints used to assess the pairwise comparisons for the investigated analyses.


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Table 6.1: 4
Overview of intra-individual comparisons
Analysis
Trial part
Analyte
Endpoints
Test
treatment 
(T)
Reference 
treatment 
(R)
Primary + 
sensitivity 
Trial part 1
Digoxin
AUC0-tz
T1
R1
Furosemide
Cmax
T2
R1
Metformin
AUC0-∞
Rosuvastatin
Trial part 2
Digoxin
AUC0-tz
T3
R1
Furosemide
Cmax
Metformin
AUC0-∞
Rosuvastatin
Trial part 3
Digoxin
AUC0-tz
T4
R1
Furosemide
Cmax
Metformin
AUC0-∞
Rosuvastatin
Secondary
Trial part 1
Digoxin
CLR,t1-t2
T1
R1
Furosemide
fe t1-t2
T2
R1
Metformin
Rosuvastatin
Trial part 2
Digoxin
CLR,t1-t2
T3
R1
Furosemide
fe t1-t2
Metformin
Rosuvastatin
Trial part 3
Digoxin
CLR,t1-t2
T4
R1
Furosemide
fe t1-t2
Metformin
Rosuvastatin


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Table 6.1: 4
Overview of intra-individual comparisons (cont.)
Analysis
Trial part
Analyte
Endpoints
Test
treatment 
(T)
Reference 
treatment 
(R)
Further
Trial part 2
Metformin
(500mg)
AUC0-tz
T5
R2
Cmax
AUC0-∞
CLR,t1-t2
fe t1-t2
Trial part 3
Furosemide (40mg)
AUC0-tz
T6
R3
Cmax
AUC0-∞
CLR,t1-t2
fe t1-t2
Please note, that AUC0-∞will be evaluated for digoxin if determination with sufficient 
precision is possible.
6.2
IMPORTANT PROTOCOL VIOLATIONS
Data discrepancies and deviations from the CTP will be identified for all treated subjects.
Listings of protocol deviations and of unresolved discrepancies will be provided to be 
discussed at the combined report planning and database lock meeting (RPM/DBLM), e.g. 
deviations in drug administration, in blood sampling, etc. At this meeting, it will be decided 
whether the discrepant data can be used as they are or whether the data have to be corrected in 
the clinical database.
Each protocol deviation must be assessed to determine whether it is an important protocol 
violation. A protocol violation (PV) is important if it affects the rights or safety of the study 
subjects or if it can potentially influence the primary outcome measure(s) for the respective 
subjects in a way that is neither negligible nor in accordance with the study objectives. This 
last category of important PV forms the basis for the decision of whether a subject does or 
does not belong to an analysis set. PVs that do not influence the subject's rights and safety or 
the evaluability of the subjects for the main study objectives are called non-important PVs.
These are only considered when checking the trial quality in general.
If any important PVs are identified, they are to be summarized into categories and will be 
captured in the RPM/DBLM minutes via an accompanying Excel spreadsheet [001-MCS-50-
413_RD-02] (2). The following table contains the categories which are considered to be 
important protocol violations in this trial. If the data show other important PVs, this table will 
be supplemented accordingly by the time of the RPM/DBLM.


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Table 6.2: 1
Important protocol violations
Category
/Code
Description
A
Entrance criteria not met
A1
Inclusion criteria violated
A2
Exclusion criteria violated
B
Informed consent
B1
Informed consent not available
B2
Informed consent too late
C
Trial medication and randomisation
C1
Incorrect trial medication taken
C2
Randomisation not followed
C3
Non-compliance
C4
Incorrect intake of trial medication
C5
Improper washout between treatments
D
Concomitant medication
D1
Concomitant medication with the potential to affect the assessment of the trial 
medication
E
Missing data 
E1
Certain violations of procedures used to measure primary or secondary data
F
Incorrect timing1
F1
Certain violations of time schedule used to measure primary or secondary data
G
Other trial specific important violations
G1
Appropriate fasting condition not met prior to study drug administration
G2
PVs affecting safety and rights
1 Time deviations will only be flagged as important PV, when leading to exclusion of the entire subject from an analysis set
Source: ‘Protocol Violation Handling Definitions’ [001-MCS-50-413_RD-01] (3)
6.3
SUBJECT SETS ANALYSED

Treated set (TS):
This subject set includes all subjects who were dispensed study medication and were 
documented to have taken at least one dose of study drug.
This is the full analysis set population in the sense of ICH-E9 (1). It is used for safety 
analyses.


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Section 7.3.1 of the CTP: Plasma concentration data and parameters of a subject will be 
included in the statistical pharmacokinetic (PK) analyses, if they are not flagged for exclusion 
due to a protocol violation relevant to the evaluation of PK (to be decided no later than in the 
Report Planning Meeting) or due to PK non-evaluability (as revealed during data analysis, 
based on the criteria specified below). Exclusion of a subject's data will be documented in the 
CTR.
Relevant protocol violations may be:

Incorrect trial medication taken, i.e. the subject received at least one dose of trial 
medication the subject was not assigned to.

Incorrect dose of trial medication taken.

Subject did not receive all study drugs assigned for the specific treatment period.
Plasma and urine concentrations and/or parameters of a subject will be considered as non-
evaluable, if for example

the subject experienced emesis that occurred at or before two times median tmax of the 
respective treatment (median tmax is to be determined excluding the subjects 
experiencing emesis),

a pre-dose concentration is >5% of the Cmax value of that subject,

missing samples/concentration data at important phases of PK disposition curve.
[…]

PK parameter analysis set (PKS):
The subject set includes all subjects from the TS who provide at least one primary or 
secondary PK endpoint that was not excluded according to the description above. Thus, a 
subject will be included in the PKS, even if he contributes only one PK endpoint value for 
one treatment period to the statistical assessment.
The descriptive analysis of PK concentrations will be based on the ADS ADPC as described 
at the beginning of Section 7.
Table 6.3: 1
Subject sets analysed
Subject set
Class of endpoint
TS
PKS
Safety endpoints 
X
Primary, secondary and further PK endpoints 
X
Demographic/baseline endpoints
X
Important PVs/Disposition
X
Disposition
X


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6.4
SUBGROUPS
No subgroup analysis is planned.
6.5
POOLING OF CENTRES
This section is not applicable, because the study was performed in only one centre.
6.6
HANDLING OF MISSING DATA AND OUTLIERS
Handling of missing data and outliers will be performed as described in the CTP, Section 7.4.
The only exception where imputation might be necessary for safety evaluation is AE dates. 
Missing or incomplete AE dates are imputed according to BI standards (see 001-MCG-
156_RD-01 (4)).
Missing data and outliers of PK data are handled according to BI standards (see 001-MCS-36-
472_RD-01) (5).
6.7
BASELINE, TIME WINDOWS AND CALCULATED VISITS
The baseline value is defined as the last measurement before first trial drug administration in 
each treatment period, separately for each trial part. 
Section 6.1 of the CTP: Exact times of measurements outside the permitted time windows 
will be documented. The acceptable time windows for screening and end of trial examination 
are given in CTP Flow Chart .
Study measurements and assessments scheduled to occur ‘before’ trial medication 
administration on Day 1 are to be performed and completed within a 3 h period prior to the 
trial drug administration.
The acceptable deviation from the scheduled time for vital signs and ECG will be - 15 min 
after study drug administration on Day 1 and ± 60 min on Day 2.
Adherence to time windows will be checked via the consistency check listings at the 
RPM/DBLM.


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7.
PLANNED ANALYSIS
All three trial parts will be evaluated separately.
Safety analysis (refer to Section 7.8) will be performed by
 and 
will be presented in Sections 15.1 to 15.4 of the CTR and in Appendix 16.2 and 16.1.13.1.
Inferential statistical analyses of PK endpoints (refer to Sections 7.4 and 7.5.2) will also be 
performed by
 and will be presented in Section 15.5 of the CTR 
and in Appendix 16.1.13.3.
Descriptive data analysis of PK endpoints will be performed by the department Translational 
Medicine and Clin. Pharmacology at BI and will be presented in Section 15.6 of the CTR.
The format of the listings and tables will follow the standards defined in the BI corporate 
guideline “Reporting of Clinical Trials and Project Summaries” [001-MCG-159] (6) with the 
exception of those generated for PK-calculations.
In each trial part, the individual values of all subjects will be listed, sorted by treatment 
sequence, subject number, visit and actual treatment (if appropriate).
The listings will be included in Appendix 16.2 of the CTR.
For end-of-text tables, the set of summary statistics for non-PK parameters is: 
N 
number non-missing observations 
Mean
arithmetic mean
SD
standard deviation 
Min
minimum 
Median
median 
Max
maximum
For analyte concentrations, as well as for all PK parameters, the following descriptive 
statistics will additionally be calculated:
CV 
arithmetic coefficient of variation
gMean
geometric mean
gCV
geometric coefficient of variation
P10 
10th percentile
Q1 
1st quartile
Q3 
3rd quartile
P90 
90th percentile
The data format for descriptive statistics of concentrations will be identical with the data 
format of the respective concentrations. The descriptive statistics of PK parameters will be 
calculated using the individual values with the number of decimal places as provided by the 
evaluation program. Then, the individual values, as well as the descriptive statistics, will be 
reported with three significant digits in the CTR.


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Tabulations of frequencies for categorical data will include all possible categories and will 
display the number of observations in a category, as well as the percentage (%) for each 
treatment sequence/group. Percentages will be rounded to one decimal place and will be 
based on all subjects in the respective subject set, whether they have non-missing values or 
not. The category 'missing' will be displayed only if there are actually missing values.
Units of variables should be given in the titles or column/row descriptors in square brackets 
(e.g. [mg]).
Exclusion of PK parameters
The analysis data set (ADS) ADPP (PK parameters) contains column variables APEXC and 
APEXCO indicating inclusion/exclusion (APEXC) of a PK parameter and an analysis flag 
comment (APEXCO). All analyses based on the PKS will include parameters if they are not 
flagged for exclusion; that is, APEXC is equal to “Included”.
Exclusion of PK concentrations
The ADS ADPC (PK concentrations per time-point or per time-interval) contains column 
variables ACEXC or ACEXCO indicating inclusion/exclusion (ACEXC) of a concentration 
and an analysis flag comment (ACEXCO). Exclusion of a concentration depends on the 
analysis flag comment ACEXCO. For example, if ACEXCO is set to ‘ALL CALC’, the value 
will be excluded for all types of analyses based on concentrations. If ACEXCO is set to 
‘DESC STATS’, the value will be excluded from descriptive evaluations per planned time 
point/time interval. If ACEXCO contains the addition ‘TIME VIOLATION’ or ‘TIME 
DEVIATION’, the value can be used for further analyses based on actual times. If ACEXCO 
is set to ‘HALF LIFE’, the value will be excluded from half-life calculation (and, as a 
consequence, any calculation that relies on λz) only; the value is included for all other 
analyses.
Further details are given in 001-MCS-36-472_RD-01 “Noncompartmental Pharmacokinetic / 
Pharmacodynamic Analyses of Clinical Studies” (5) and 001-MCS-36-472_RD-03 
“Description of Analytical Transfer Files and PK/PD Data Files” (7).
7.1
DEMOGRAPHIC AND OTHER BASELINE CHARACTERISTICS
Only descriptive statistics are planned for this section of the report, based on the TS.
The data will be summarised by treatment sequence and in total.


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7.2
CONCOMITANT DISEASES AND MEDICATION
Frequency tables are planned for this section of the report, based on the TS.
Concomitant diseases will be coded using the coding system of the Medical Dictionary for 
Drug Regulatory Activities (MedDRA). Medications will be coded using the World Health 
Organization Drug Dictionary (WHO-DD). The coding version number will be displayed as a 
footnote in the respective output.
The diagnoses and medications will be listed. Subjects without any concomitant diagnoses or 
concomitant therapies should be marked with a “No” in the respective column.
The relevance of the concomitant therapies to the evaluation of PK will be decided no later 
than at the RPM/DBLM.
7.3
TREATMENT COMPLIANCE
Section 4.3 of the CTP: Compliance will be assured by administration of all trial medication 
in the study centre under supervision of the investigating physician or a designee. The 
measured plasma concentrations and urinary excretion will provide additional confirmation 
of compliance.
It is not intended to list the compliance separately. Any deviations from complete intake will 
be addressed in the RPM/DBLM (cf. TSAP Section 6.2) and described in the CTR.
7.4
PRIMARY ENDPOINTS
Primary analysis
Relative bioavailability is to be determined on the basis of the primary, secondary and some 
further PK parameters (AUC0-tz, Cmax and AUC 0-∞, CLR,t1-t2  and fet1-t2). Those parameters will 
be ln-transformed (natural logarithm) prior to fitting the ANOVA model (see below).
Section 7.1.3 of the CTP: The statistical model used for the analysis of primary and 
secondary and some further endpoints will be an ANOVA (analysis of variance) model on the 
logarithmic scale. This model will include effects accounting for the following sources of 
variation: ‘sequence’, ‘subjects within sequences’, ‘period’ and ‘treatment’. The effect 
‘subjects within sequences’ will be considered as random, whereas the other effects will be 
considered as fixed. The model is described by the following equation:
yijkm = µ+ ζi + sim + πj + τk + eijkm, where
yijkm = logarithm of response (AUC0-tz, Cmax, AUC 0-∞) measured on subject m in 
sequence i receiving treatment k in period j,
µ = the overall mean,
ζi = the ith sequence effect, i = 1, 2, 3 for part 1 and i = 1,…, 4 for part 2 and 3


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sim = the effect associated with the mth subject in the ith sequence, m =1, ..., 5 for part 
1 and m = 1,…, 4 for part 2 and 3
πj = the jth period effect, j = 1, 2, 3, for part 1 and j = 1,… 4 for part 2 and 3
τk =the kth treatment effect, k ∈{R1, T1, T2} for part 1 and
k ∈{R1, T3, R2, T5} for part 2 and
k ∈{R1, T4, R3, T6} for part 3
eijkm = the random error associated with the mthsubject in sequence i who received 
treatment k in period j.
The difference between the expected means for test treatments and reference treatment
ln(T)-ln(R), estimated by the difference in the corresponding Least Square Means (point 
estimate) and two-sided 90% confidence intervals based on the t-distribution, will be 
computed. These quantities will then be back-transformed to the original scale to give the 
point estimator (geometric mean) and interval estimates for the ratio between response under 
test and response under reference.
The analysis will be accomplished by using the XPKISTAT macro, based on PKS, and option 
BWC (Bioavailability/Bioequivalence, within-subject design, time-controlled).
Sensitivity analyses
In addition, a sensitivity analysis will be performed by fitting the model described above, but 
using all effects as fixed. Furthermore, the input dataset will be restricted in such a way that 
treatments not relevant for the comparison of interest will be deleted. 
This analysis will be done using PROC GLM. The following SAS code can be used to fit the 
model:
PROC GLM DATA=indata; 
CLASS subject treatment sequence period; 
MODEL logkp = treatment sequence period subject(sequence); 
LSMEANS treatment / PDIFF=CONTROL(“Ref_trt”) CL ALPHA=0.1; 
RUN;
Another sensitivity analysis will analyse the effects within each trial part based on all 
evaluable data of treatment R1 across the three trial parts. It will be performed by fitting the
primary analysis model described above, including ‘trial part’ as additional (fixed) effect. 
This analysis will be accomplished by using the XPKISTAT macro, and option BWC, 
including ‘trial part’ as covariate (classfix=part).


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7.5
SECONDARY ENDPOINTS
7.5.1
Key secondary endpoints
This section is not applicable as no key secondary endpoints have been specified in the 
protocol.
7.5.2
Secondary endpoints
The same analyses as for the primary endpoints will be performed for the secondary endpoint 
AUC0-∞of furosemide, metformin and rosuvastatin.
Section 5.5.1.2 of the CTP: For digoxin, AUC0-∞will be evaluated only if determination with 
sufficient precision is possible.
7.6
FURTHER ENDPOINTS
Pharmacokinetic (PK)
Secondary analysis
Section 7.1.3 of the CTP: The secondary analysis will apply the primary analysis model to 
CLR,t1-t2 and to fet1-t2 of each investigated dose of the sensitive drug transporter substrates 
(digoxin, furosemide, metformin, and rosuvastatin).
Further analysis
The further analysis will apply the primary analysis model to AUC0-tz, Cmax, AUC0-∞and 
CLR,t1-t2 and to fet1-t2 of the therapeutic doses of metformin and furosemide.
Descriptive statistics of PK parameters
Descriptive statistics of plasma and urine concentrations and PK endpoints will be done by
the department Translational Medicine and Clin. Pharmacology at BI and will be presented in 
Section 15.6 of the CTR.
The analysis of PK parameters, as well as the tables and graphs for the pharmacokinetic non-
compartmental analyses, will follow specific definitions of this TSAP or, otherwise, the BI 
standard procedure “Standards and processes for analyses performed within Clinical 
Pharmacokinetics/Pharmacodynamics” [001-MCS-36-472] (8).
Safety


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Refer to TSAP Section 7.8 for a description of the analysis of safety and tolerability.
7.7
EXTENT OF EXPOSURE
Descriptive statistics are planned for this section of the report based on the TS. The date and 
time of drug administration will be listed for each subject.
7.8
SAFETY ANALYSIS
All safety analyses will be performed on the TS, separately for each trial part.
If not stated otherwise, the safety results will be sorted by actual treatment.
The safety data for treated subjects who failed to complete the study (dropouts or
withdrawals) will be reported as far as their data are available. All withdrawals will be 
documented and the reason for withdrawal recorded.
7.8.1
Adverse events
AEs will be coded with the most recent version of MedDRA.
The analyses of AEs will be descriptive in nature and will be based on BI standards as 
presented in the corporate guideline: “Analysis and Presentation of Adverse Event Data from 
Clinical Trials” [001-MCG-156] (9).
The standard AE analyses will be based on the number of subjects with AEs (and not on the 
number of AEs). 
For analysis multiple AE occurrence data on the case report form (CRF) will be collapsed into 
an AE provided that all of the following applies:

All AE attributes are identical (lower level term (LLT), intensity, action taken, therapy
required, seriousness, reason for seriousness, relationship, outcome, AE of special 
interest)

The occurrences were time-overlapping or time-adjacent (time-adjacency of 
two occurrences is given if the second occurrence started within one hour after end of 
the first occurrence)
For further details on summarization of AE data, please refer to [001-MCG-156] (9).
No AESIs have been defined for this trial.
The analysis of adverse events will be based on the concept of treatment emergent adverse 
events. 
Section 5.2.2.2 of the CTP: The REP for digoxin, when measurable drug levels or PD effects 
are still likely to be present, is defined as 7 days after administration. The REP for the other 7 


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probe drugs is shorter. In order to avoid reporting bias (i.e., shorter lengths of on-treatment 
periods after single component (mono-treatment) administration compared to probe drug 
cocktail administration could lead to a higher number of AEs after administration of the test 
treatment), all AEs which occur from first drug administration until 7 days thereafter in each 
treatment period will be considered as on treatment […].
According to ICH E3 (10), AEs classified as ‘other significant’ needs to be reported and will 
include those non-serious and non-significant adverse events with 
(i) ‘action taken = discontinuation’ or ‘action taken = reduced’, or
(ii) marked haematological and other lab abnormalities or lead to significant concomitant 
therapy as identified by the Clinical Monitor/Investigator at the Report Planning Meeting.
An overall summary of AEs will be presented.
The frequency of subjects with AEs will be summarized by treatment, primary system organ 
class (SOC) and preferred term (PT). Separate tables will be provided for subjects with other 
significant AEs according to ICH E3 (10), for subjects with serious AEs, for subjects with 
drug-related AEs and for subjects with drug related serious adverse.
The SOC and PTs will be sorted by frequency (within SOC). The MedDRA version number 
will be displayed as a footnote in the respective output.
In addition, frequencies of subjects with non-serious AEs that had an incidence of > 5% for at 
least one treatment will be summarised by treatment, primary SOC and PT.
For disclosure of adverse events on EudraCT additional information not included in a 
standard AE analysis will be performed. The following three entries will be created:

Adverse Events per arm for disclosure on EudraCT

Non-serious Adverse Events for disclosure on EudraCT

Serious Adverse Events for disclosure on EudraCT
7.8.2
Laboratory data
The analyses of laboratory data will be descriptive in nature and will be based on BI standards 
[001-MCG-157] (11).
Laboratory data will be analysed qualitatively via comparison of laboratory data to their 
reference ranges. Values outside the reference range as well as values defined as clinically 
relevant will be highlighted in the data listings.
Possibly clinically significant abnormal laboratory values are only those identified either in 
the Investigator’s comments on the CRF or at the RPM/DBLM at the latest. It is the 
investigator's responsibility to decide whether a lab value is clinically significant abnormal or 
not. Standard or project-specific rules for flagging clinically significant values will not be 
applied in this study.


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7.8.3
Vital signs
Descriptive statistics over time including change from baseline will be performed for vital 
signs (blood pressure and pulse rate).
7.8.4
ECG
12-lead ECG
ECG recordings will be checked by the investigator for pathological results. Clinically 
relevant abnormal findings for ECG will be listed under 'Relevant Medical History / Baseline 
Conditions' (when they occurred during screening) or will be reported as AEs (when they 
occurred during treatment).
7.8.5
Others
Physical examination
Physical examination findings will be reported as relevant medical history/baseline condition 
(i.e., a condition already existent before intake of study drug) or as AE and will be
summarised as such. No separate listing or analysis of physical examination findings will be 
prepared.


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8.
REFERENCES
1.
CPMP/ICH/363/96: "Statistical Principles for Clinical Trials", ICH Guideline Topic E9, 
Note For Guidance on Statistical Principles for Clinical Trials, current version.
2.
001-MCS-50-413_RD-02: “Important Manual Protocol Violations Spreadsheet”, current 
version, IDEA for CON.
3.
001-MCS-50-413_RD-01: “Protocol Violation Handling Definitions”, current version, 
IDEA for CON.
4.
001-MCG-156_RD-01: "Handling of Missing and Incomplete AE Dates", current 
version; IDEA for CON.
5.
001-MCS-36-472_RD-01: “Noncompartmental Pharmacokinetic/Pharmacodynamic
Analyses of Clinical Studies”, current version; IDEA for CON.
6.
001-MCG-159: “Reporting of Clinical Trials and Project Summaries”, current version; 
IDEA for CON.
7.
001-MCS-36-472_RD-03: “Description of Analytical Transfer Files and PK/PD Data 
Files”, current version; IDEA for CON.
8.
001-MCS 36-472: "Standards and processes for analyses performed within Clinical 
Pharmacokinetics/Pharmacodynamics", current version; IDEA for CON.
9.
001-MCG-156: “Analysis and Presentation of Adverse Event Data from Clinical Trials”, 
current version; IDEA for CON.
10. CPMP/ICH/137/95: “Structure and Content of Clinical Study Reports”, ICH Guideline 
Topic E3; Note For Guidance on Structure and Content of Clinical Study Reports, current 
version
11. 001-MCG-157: "Handling, Display and Analysis of Laboratory Data", current version; 
IDEA for CON.


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9.
ADDITIONAL SECTIONS
Not applicable as no additional information is needed.


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10.
HISTORY TABLE
Table 10: 1
History table
Version
Date
(DD-MMM-YY)
Author
Sections 
changed
Brief description of change
Final
07-JUN-18
None
This is the final TSAP without any 
modification