Conditions: Nephrotic Syndrome Edema Sodium Retention
Keywords: Nephrotic Syndrome ENaC Diuretic therapy Amiloride Proteasuria
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 20
Healthy volunteers: no

Brief summary:
The AMILOR study compares treatment of edema in nephrotic syndrome with Amiloride vs. Furosemide.

Detailed description:
The monocenter randomized-controlled AMILOR trial investigates the efficacy of the ENaC blocker amiloride in reducing edema in nephrotic syndrome compared with standard therapy with the loop diuretic furosemide.

Patients with acute nephrotic syndrome are randomized to receive amiloride (starting dose 5 mg) or furosemide (starting dose 40 mg) for 16 days. The target number of patients is n = 18 per arm. Exclusion criteria include GFR \<30ml/min/1.73m², AKIN 1 and 2, hypotension, hyper-/ hypokalemia, and hyponatremia. Overhydration is quantified by bioimpedance spectroscopy. Depending on the course of overhydration, dose adjustments (day 2, 5, 8, 12) or addition of HCT (day 8) are performed during the course of the study.

Primary endpoint is decrease in overhydration at day 8, secondary endpoints include decrease in overhydration at day 16, as well as body weight, edema volume, blood pressure, urine volume, natriuresis at day 8 and 16, and need for dose adjustments and co-medication with HCT. Plasma potassium, sodium, and creatinine concentrations are measured as safety parameters.

Arms:
arm 1: Treatment wirh Amiloride, start dose 5 mg arm 2: Treatment with Furosemide, start dose 40 mg

Interventions: intervention 1: Amiloride intervention 2: Furosemide
Intervention descriptions: intervention 1: Treatment with amiloride, start dose 5 mg intervention 2: Treatment with furosemide, start dose 40 mg

Protocol document:
Prot_000.pdf: 
 
Page 1 of 70 
 
 
Randomized, controlled interventional trial to investigate the efficacy of 
amiloride for the treatment of edema in human nephrotic syndrome  
 
Short Title of Clinical Study 
Amiloride in nephrotic syndrome (AMILOR) 
 
 
Version / Protocol Code: 
2.1 / AmiloridNS-01 
 
 
EudraCT-number 
2019-002607-18 
 
 
Date of Protocol 
01.03.2021 
 
 
Register-number 
 
 
 
Sponsor of the Study  
University Hospital Tuebingen,   
Medical Director, Prof. Dr. med. M. Bamberg  
Director of Administration, G. Sonntag  
Geissweg 3 
72076 Tuebingen 
Germany 
 
 
Representative 
Prof. Dr. med. Ferruh Artunc 
University Hospital Tuebingen 
Department of Internal Medicine IV 
Otfried-Mueller-Strasse 10 
72076 Tuebingen 
Germany 
phone: +49 (0)7071-29 82712 
fax: +49 (0)7071-29 25215 
e-mail: ferruh.artunc@med.uni-tuebingen.de 
 
 
Investigational drug 
Amiloride 
 
 
Comparator 
Furosemide 
 
 
Summary of the revision history 
(amendments) 
Version 1.1 from 10.01.2020 
Amendment 1 from 01.03.2021 
 
 
CONFIDENTIAL This protocol contains confidential information and is intended solely for the guidance of clinical investigation. 
This protocol may not be disclosed to parties not associated with the clinical investigation or used for any purpose without the 
prior written consent of the coordinating Investigator 
 


Protocol code: AmiloridNS-01 
Amiloride in nephrotic syndrome 
(AMILOR) 
Study Protocol Version 2.1 
 
 
Page 2 of 70 
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I. Table of Contents 
I. 
Table of Contents 
2 
I.a) 
List of Tables 
6 
I.b) 
List of Figures 
6 
II. 
Signature Page 
7 
III. 
Contacts 
9 
IV. 
Abbreviations 
11 
V. 
Synopsis 
13 
1. 
Introduction 
20 
1.1. 
Trial Rationale / Justification 
22 
1.2. 
Benefit / Risk Assessment 
23 
1.3. 
Advisory Committes 
24 
2. 
Study Objectives 
25 
2.1. 
Primary Objective and Endpoint 
25 
2.2. 
Secondary Objectives and Endpoints 
25 
3. 
Study Design 
26 
3.1. 
Study Duration and Schedule 
27 
3.2. 
End of Study 
27 
4. 
Study Population 
28 
4.1. 
General Criteria for Subject Selection 
28 
4.1.1. 
Inclusion Criteria 
28 
4.1.2. 
Exclusion Criteria 
29 
5. 
Requirements for Trial Site and Investigator 
31 
6. 
General Information on the Study Medication 
32 
6.1. 
Manufacturing of the Study Medication 
33 
6.2. 
Labeling of the Study Medication 
34 
6.3. 
Storage of the Medical Product 
35 


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6.4. 
Drug Accountability, Therapy Compliance and Disposal 
35 
6.5. 
Method of Treatment Assignment 
35 
6.6. 
Dose Schedule 
35 
6.6.1. 
Dose Modification 
36 
7. 
Study Procedures and Examination Method 
39 
7.1. 
Study Entry 
40 
7.1.1. 
Patient’s Informed Consent (see also 11.2.) 
40 
7.1.2. 
Enrollment 
40 
7.1.3. 
Screening 
40 
7.1.4. 
Randomisation 
41 
7.1.5. 
Concomitant Medication and Treatments 
41 
7.1.6. 
Permitted Prior and Concomitant Medications and Treatments 
41 
7.1.7. 
Prohibited Prior and Concomitant Medications and Treatments 
42 
7.1.8. 
Required Prior and Concomitant Medications and Treatments 
43 
7.2. 
Treatment Phase 
43 
7.2.1. 
Description of Patients’ Visits 
43 
7.3. 
Assessment of Efficacy 
44 
7.3.1. 
Efficacy Parameters 
44 
7.3.2. 
Methods and Timing for Assessing, Recording, and Analysing of Efficacy 
Parameters 
45 
7.4. 
Assessment of Safety 
47 
7.4.1. 
Safety Parameters 
47 
7.4.2. 
Methods and Timing for Assessing, Recording, and Analysing Safety 
Parameters 
47 
7.5. 
Premature termination of clinical trial for a trial subject 
48 
7.6. 
Premature closure of a trial site 
48 
7.7. 
Premature termination of the trial 
48 
7.8. 
End of Study of Subjects 
49 


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7.8.1. 
Plan for Treatment or Care after End of Study 
49 
8. 
Quality Control and Quality insurance 
50 
8.1. 
Monitoring 
50 
8.2. 
Audits/ Inspections 
50 
8.3. 
Documentation: Collection, Handling, Storage and Archiving of Data 
51 
8.3.1. 
Case Report Form 
51 
8.3.2. 
Source Data 
51 
8.3.3. 
Data Handling 
51 
8.3.4. 
Storage and Archiving of Data 
51 
9. 
Statistical Analyses 
53 
9.1. 
Study Population Definition 
53 
9.1.1. 
Sample Size and Power Consideration 
53 
9.1.2. 
Intention-to-Treat Population 
53 
9.2. 
Analysis of Primary Variable 
53 
9.3. 
Analysis of Secondary Variables 
54 
9.4. 
Safety Analysis 
54 
9.5. 
Descriptive Analysis 
54 
9.6. 
Handling of Missing Data 
54 
9.7. 
Subgroup Analysis 
54 
9.8. 
Biometric Report 
55 
10. 
Safety 
56 
10.1. 
Definition of Adverse Events and Side Effects 
56 
10.1.1. 
Adverse Events 
56 
10.1.2. 
Adverse Drug Reaction 
56 
10.1.3. 
Unexpected Adverse Drug Reaction 
56 
10.1.4. 
Serious Adverse Event and Serous Adverse Reaction 
57 
10.2. 
Period of Observation 
57 


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Study Protocol Version 2.1 
 
 
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10.3. 
Documentation and Reporting of Adverse Events 
57 
10.3.1. 
Documentation and Reporting of Adverse Events by the Investigator 
57 
10.3.2. 
Assessment of Severity and Causality 
58 
10.3.3. 
Sponsors Assessment of the SAEs 
59 
10.3.4. 
Follow-up of Initial Report 
59 
10.3.5. 
Suspected Unexpected Serious Adverse Reaction (SUSAR) 
59 
10.3.6. 
Expedited Reporting to the Regulatory Authorities 
60 
10.3.7. 
Examination and Report of Changes in the Risk to Benefit Ratio 
60 
10.3.8. 
Report to the Investigator 
60 
10.4. 
Annual Safety Report 
61 
10.5. 
Deviations from the protocol 
61 
10.6. 
Reporting of pregnancy 
61 
11. 
Regulatory Consideration 
63 
11.1. 
Ethical Conduct of Clinical Study 
63 
11.1.1. 
Good Clinical Practice, Declaration of Helsinki and legal Provision 
63 
11.2. 
Subject Information and Informed Consent 
63 
11.3. 
Insurance 
63 
11.4. 
Confidentiality 
64 
11.5. 
Responsibility of the the Investigator 
64 
11.6. 
Registration of the Trial 
64 
11.7. 
Continuous Information to Independent Ethic Committee 
64 
11.8. 
Approval of Protocol and Subsequent Amendments 
65 
12. 
Publications 
66 
12.1. 
Reports 
66 
12.2. 
Publication 
66 
13. 
Financing 
67 
14. 
Literature 
68 


Protocol code: AmiloridNS-01 
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Study Protocol Version 2.1 
 
 
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15. 
Appendices 
70 
15.1. 
Patient Information and Informed Consent 
70 
15.2. 
Instruction sheet for participants 
70 
15.3. 
Label of the Investigational Medical Product Amiloride 
70 
15.4. 
CE certificate of the Body Composition Monitor 
70 
 
I.a) 
List of Tables 
Table 1: Study Timelines 
27 
Table 2 Table of Events 
39 
Table 3: Budget draft 
67 
 
I.b) 
List of Figures 
Figure 1: Scheme illustrating the pathophysiology of EnaC-mediated sodium retention (a) 
and model of sodium retention in nephrotic syndrome (b) [3] 
21 
Figure 2: Overall study design 
26 
 






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Study Protocol Version 2.1 
 
 
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III. Contacts 
Sponsor  
 
 
 
 
Universitätsklinikum Tuebingen 
Geissweg 3 
72076 Tuebingen 
 
Investigator   
 
 
 
Prof. Dr. med. Ferruh Artunc 
University Hospital Tuebingen 
Department of Internal Medicine IV 
Otfried-Mueller-Strasse 10 
72076 Tuebingen 
Germany 
phone: +49 (0)7071-29 82712 
fax: +49 (0)7071-29 25215 
e-mail: ferruh.artunc@med.uni-tuebingen.de 
 
Biometrician  
 
 
 
Marion Schütt 
Institute for clinical epidemiology and applied 
biometry 
Silcherstrasse 5 
72076 Tuebingen 
Germany 
phone: +49 (0)7071-29 87326 
e-mail: marion.schuett@med.uni-tuebingen.de 
 
Project management 
 
 
Dr. med. Anja Schork 
University Hospital Tuebingen 
Department of Internal Medicine IV 
Otfried-Mueller-Strasse 10 
72076 Tuebingen 
Germany 
phone: +49 (0)7071-29 82712 
fax: +49 (0)7071-29 5705 
e-mail: anja.schork@med.uni-tuebingen.de 
 
Monitoring 
 
 
 
 
Zentrum für Klinische Studien (ZKS) Tübingen 
Frondsbergstraße 23 
72076 Tuebingen 
Germany 
phone: +49 (0)7071-29 85635 
fax: +49 (0)7071-29 25080 
e-mail: zks-mo@med.uni-tuebingen.de 
 
Data management  
 
 
Ruth Bösel 
Institute for clinical epidemiology and applied 
biometry 
Silcherstrasse 5 
72076 Tuebingen 
Germany 
phone: +49 (0)7071-29 85901 
e-mail: ruth.boesel@med.uni-tuebingen.de 
 
 


Protocol code: AmiloridNS-01 
Amiloride in nephrotic syndrome 
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Study Protocol Version 2.1 
 
 
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SAE-Management 
 
 
 
Zentrum für Klinische Studien (ZKS) Tübingen 
Frondsbergstraße 23 
72076 Tuebingen 
Germany 
phone: +49 (0)7071-29 85635 
e-mail: zks-pv@med.uni-tuebingen.de 
 
For SAE reporting: 
fax: +49 (0)7071-29 25205 
 


Protocol code: AmiloridNS-01 
Amiloride in nephrotic syndrome 
(AMILOR) 
Study Protocol Version 2.1 
 
 
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IV. Abbreviations 
ACE 
 
 
Angiotensin converting enzyme 
ADR 
 
 
Adverse Drug Reaction 
AE 
 
 
Adverse Event 
AMG  
 
German Drug Law (Deutsches Arzneimittelgesetz)  
BCM  
 
Body Composition Monitor, Fresenius Medical Care AG & Co 
BDSG  
 
Bundesdatenschutzgesetz 
BfArM  
 
Bundesinstitut für Arzneimittel und Medizinprodukte 
BGA 
 
 
Blood gas analysis 
CRF 
 
 
Case Report Form 
CTCAE 
 
Common Toxicity Criteria for Adverse Events 
CI 
 
 
Coordinating Investigator  
DBL 
 
 
Data Base Lock 
EC 
 
 
Ethics Committee 
ECW  
 
Extracellular water 
ENaC  
 
Epithelial sodium channel 
FCBP  
 
Females of childbearing potential 
FSI 
 
 
First Subject In 
GCP  
 
Good Clinical Practice 
GCP-V 
 
Good Clinical Practice Ordinance (GCP-Verordnung) 
HCT 
 
 
Hydrochlorothiazide 
IC 
 
 
Informed Consent  
ICH 
International Conference on Harmonization of Technical Requirements 
for Registration of Pharmaceuticals for Human Use 
IIT 
Investigator Initiated Trial 
IKEAB 
Institut für Klinische Epidemiologie und angewandte Biometrie 
Tübingen 
IMP 
Investigational Medicinal Product 
ISF 
Investigator Site File 
K 
Potassium (chemical element) 
LSI 
 
 
Last Subject In 


Protocol code: AmiloridNS-01 
Amiloride in nephrotic syndrome 
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Study Protocol Version 2.1 
 
 
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LSO 
 
 
Last Subject Out 
n.a. 
 
 
Not applicable 
Na 
 
 
Sodium (chemical element) 
NKCC2 
 
Sodium-potassium-chloride cotransporter 
OH 
 
 
Overhydration 
PEI 
 
 
Paul-Ehrlich-Institut 
RAAS  
 
Renin-angiotensine-aldosterone system 
SAP 
 
 
Statistical Analysis Plan 
SAE 
 
 
Serious Adverse Event 
SmPC  
 
Summary of Product Characteristics 
SDV 
 
 
Source Data Verification 
SUSAR 
 
Suspected Unexpected Serious Adverse Reaction 
TMF 
 
 
Trial Master File 
 
 
 


Protocol code: AmiloridNS-01 
Amiloride in nephrotic syndrome 
(AMILOR) 
Study Protocol Version 2.1 
 
 
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V. Synopsis 
Sponsor 
University Hospital of Tuebingen represented by  
Medical Director: Prof. Dr. med. M. Bamberg  
Director of Administration: G. Sonntag 
Title 
Randomized controlled interventional trial to investigate the 
efficacy of amiloride for the treatment of edema in human 
nephrotic syndrome 
Short Title 
Amiloride in nephrotic syndrome 
EudraCT-Number 
2019-002607-18 
Internal Study Code 
AmiloridNS / AMILOR 
Investigator and Sponsor’s 
authorized person 
Prof. Dr. med. Ferruh Artunc 
University Hospital Tuebingen 
Department of Internal Medicine IV 
Otfried-Mueller-Strasse 10, 72076 Tuebingen 
phone: 07071-29 82712; fax: 07071-29 25215 
e-mail: ferruh.artunc@med.uni-tuebingen.de 
Study Design: 
Phase IIIb, monocenter, interventional, two-arm, randomized, 
controlled clinical trial 
Number of Patients  
n = 44 (n = 18 per group, parallel design) 
Patient Population 
Patients with nephrotic syndrome 


Protocol code: AmiloridNS-01 
Amiloride in nephrotic syndrome 
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Study Protocol Version 2.1 
 
 
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Length of study/ Time 
Lines 
Duration of total trial (estimated):  
36 months 
Duration of clinical part (FSI – LSO)  
24 months 
Duration for individual patient:  
 
23 days  
 
(Study treatment: 16 days + Follow-up: 7 days) 
FSI (First Subject In): 
06/2020 
LSI (Last Subject In): 
05/2022 
LSO (Last Subject Out): 
06/2022 
DBL (Data Base Lock): 
09/2022 
Statistical Analyses Completed: 
12/2022 
Trial Report Completed: 
06/2023 
Aim of the Study 
 
To prove the efficacy of amiloride for reduction of edema and 
overhydration in human nephrotic syndrome in comparison to 
standard medication with furosemide 
Objectives/Endpoints 
Primary endpoint: 
Decrease of overhydration (OH) after 8 days, compared to 
baseline. OH is measured using bioimpedance spectroscopy 
with the device Body Composition Monitor (Fresenius 
Medical Care AG & Co; BCM), and expressed as percent of 
extracellular water (% ECW). 
Secondary endpoints: 
1. Decrease of OH after 16 days 
2. Decrease of body weight after 8 and 16 days 
3. Decrease of edema circumference after 8 and 16 days 
4. Decrease of systolic and diastolic blood pressure after 8 
and 16 days 
5. Increase of urine volume and natriuresis after 8 and 16 
days 
6. Course of plasma renin activity and serum aldosterone 
concentration after 8 and 16 days 
7. Number of required changes of dose of study medication 
8. Need for co-medication with HCT after 8 days 
9. Occurrence of adverse events 


Protocol code: AmiloridNS-01 
Amiloride in nephrotic syndrome 
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Study Protocol Version 2.1 
 
 
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Inclusion Criteria  
1. Acute nephrotic syndrome with proteinuria > 3 g/day 
and formation of edema. 
2. Age ≥ 18 years at the time of signing the informed 
consent. 
3. Understand and voluntarily sign an informed consent 
document prior to any study related 
assessments/procedures. 
4. Ability to adhere to the study visit schedule and other 
protocol requirements.  
5. Use of adequate thrombosis prophylaxis due to the 
increased risk of thrombosis in nephrotic syndrome 
and the expected fluctuations in volume balance 
during study participation 
6. Subject  (male or female) is willing to use highly 
effective methods of contraception according to the 
“Clinical trial fertility group” recommendations 
(http://www.hma.eu/fileadmin/dateien/Human_Medicin
es/01About_HMA/Working_Groups/CTFG/2014_09_H
MA_CTFG_Contraception.pdf) during treatment and 
for 28 days (male or female) after the end of treatment 
(adequate: intrauterine device, intrauterine hormone-
releasoing system, bilateral tubal occlusion, 
vasectomized partner1, sexual abstinence2; due to the 
increased risk of thrombosis in nephrotic syndrome, no 
hormonal contraceptives are recommended). 
7. Female Patients of childbearing potential (WOCBP)3 
must agree to pregnancy testing before inclusion in the 
study. 
8. Female Patients must agree to abstain from 
breastfeeding during study participation and 28 days 
after study drug discontinuation. 
9. All subjects must agree not to share medication. 
1 Vasectomized partner is a highly effective birth control method provided that 
partner is the sole sexual partner of the WOCBP trial participant and that the 
vasectomized partner has received medical assessment of the surgical success 
2 In the context of this guidance sexual abstinence is considered a highly effective 
method only if defined as refraining from heterosexual intercourse during the entire 
period of risk associated with the study treatments. The reliability of sexual 
abstinence needs to be evaluated in relation to the duration of the clinical trial and 
the preferred and usual lifestyle of the subject  
3 For the purpose of this document, a woman is considered of childbearing potential 
(WOCBP), i.e. fertile, following menarche and until becoming post-menopausal 
unless permanently sterile. Permanent sterilisation methods include hysterectomy, 
bilateral salpingectomy and bilateral oophorectomy. A postmenopausal state is 
defined as no menses for 12 months without an alternative medical cause. A high 
follicle stimulating hormone (FSH) level in the postmenopausal range may be used to 
confirm a post-menopausal state in women not using hormonal contraception or 
hormonal replacement therapy. However in the absence of 12 months of 
amenorrhea, a single FSH measurement is insufficient. 


Protocol code: AmiloridNS-01 
Amiloride in nephrotic syndrome 
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Study Protocol Version 2.1 
 
 
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Exclusion Criteria 
1. Severe reduction of kidney function: Creatinine 
clearance or calculated GFR < 30 mL/min/1.73m² or 
acute kidney injury KDIGO stage 2 or 3 or anuria. 
2. Hypovolemia or dehydration. 
3. Uncontrolled diabetes mellitus. 
4. Hypotension, systolic blood pressure < 90 mmHg 
5. Hyperkalemia, plasma potassium concentration > 4.8 
mmol/l 
6. Hypokalemia, plasma potassium concentration < 3.3 
mmol/l 
7. Hyponatremia, plasma sodium concentration < 128 
mmol/l 
8. Hypercalcemia, ionized calcium > 2.0 mmol/l or total 
albumin corrected calcium > 3.0 mmol/ 
9. Signs of cardiac decompensation (orthopnoe, dyspnoe 
NYHA IV) 
10. Hepatic coma or precoma 
11. Symptoms of gout 
12. Current therapy with potassium-sparing diuretics (e.g. 
spironolactone) or potassium supplements 
13. Women during pregnancy and lactation. 
14. History of hypersensitivity to the investigational 
medicinal product, comparator or co-medication or to 
any drug with similar chemical structure or to any 
excipient present in the pharmaceutical form of the 
investigational medicinal product, comparator or co-
medication. 
15. Any other clinical condition that would jeopardize the 
patient’s safety while participating in this clinical trial. 
16. Active participation in other clinical trials or observation 
period of competing trials. 


Protocol code: AmiloridNS-01 
Amiloride in nephrotic syndrome 
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Study Protocol Version 2.1 
 
 
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Statistics, Safety Variables 
and Stopping Rules 
Statistics: 
Primary endpoint of the study is the decrease of overhydration 
(measured using bioimpedance spectroscopy) after 8 days, 
compared to baseline. Based on data from a baseline sample 
(n = 14) and clinical experience on reduction of overhydration, 
baseline overhydration is expected to be 26.5 % of 
extracellular 
water 
(standard 
deviation 
12.3; 
normal 
distribution is assumed) and it is expected that the 
overhydration will be reduced by 90% in the amiloride group 
and by 50% in the furosemide group. For the final t-test for 
two groups, a one-tailed significance level of 0.05 and a power 
of 80% should be ensured. From these conditions results a 
sample size of n = 18 patients per group (total n = 36; 
calculated for a t-test with the nQuery 7.0 program). In order 
to take into account dropouts, the sample size is defined as n 
= 22 per group (total n = 44). 
 
Safety variables: 
- 
General safety parameters: adverse events, 
concomitant medication and check of study medication  
- 
Safety parameters for specific risks of the study: serum 
potassium and sodium levels, plasma creatinine 
concentration 
 


Protocol code: AmiloridNS-01 
Amiloride in nephrotic syndrome 
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Study Protocol Version 2.1 
 
 
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Study Intervention/ Study 
Medication 
Substance 1 (Investigational medical product):  
Amiloride (Modamide®), initial dose 5 mg  
Manufacturer: Laboratoires GERDA, 24 rue Erlanger, 75016 
Paris / France 
Unit strength: 5 mg 
Route of administration: oral, once daily 
Substance 2 (Comparator):  
Furosemide (Furosemid-ratiopharm®), initial dose 40 mg 
Manufacturer: ratiopharm GmbH, Graf-Arco-Straße 3, 89079 
Ulm, Germany 
Unit strength: 40 mg 
Route of administration: oral, once daily 
Substance 3 (Co-medication):  
Hydrochlorothiazide (HCT-Hexal®) 12,5 mg 
Manufacturer: Hexal AG, Industriestraße 25, 83607 
Holzkirchen / Germany 
Unit strength: 25 mg 
Route of administration: oral, once daily 
 
Participants are randomized to start medication with Amiloride 
5 mg (trial arm 1) or Furosemide 40 mg (trial arm 2) at day 0. 
Dose adjustments depending on course of overhydration, 
sodium and potassium level in blood gas analysis and plasma 
creatinine are performed on day 2, 5, 8 and 12. In case of 
therapy-refractory overhydration co-medication with HCT is 
started at day 8. 
Participating Centers 
University Hospital Tuebingen, Department of Internal 
Medicine IV 


Protocol code: AmiloridNS-01 
Amiloride in nephrotic syndrome 
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Study Protocol Version 2.1 
 
 
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GCP-compliance 
The present trial will be conducted in accordance with the 
valid versions of the trial protocol and the internationally 
recognised Good Clinical Practice Guidelines (ICH-GCP), 
including archiving of essential documents and the applicable 
regulatory requirements. 
Financing 
Intramural funding by the AKF (“Angewandte Klinische 
Forschung”) program has been approved. 
 
 


Protocol code: AmiloridNS-01 
Amiloride in nephrotic syndrome 
(AMILOR) 
Study Protocol Version 2.1 
 
 
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1. Introduction 
The nephrotic syndrome is characterized by a high ("nephrotic") proteinuria> 3.5 g / day with 
resulting hypoalbuminemia, as well as by edema and hyperlipidemia. Affected patients suffer 
from massive generalized edema involving the face and eyelids, effusions into the body 
cavities pleura, peritoneum and rarely pericardium as well as increased body weight. [1] 
Mechanisms of edema formation are still not fully understood, but two models have been 
established [2]: The underfill hypothesis describes the development of edema as a 
consequence of decreased oncotic pressure due to hypoproteinemia that is caused by renal 
protein loss. As a result, intravascular volume deficiency developes, that activates the renin-
angiotensin-aldosterone-system (RAAS) leading to a secondary renal sodium retention. In 
contrast, the overfill theory postulates a primary stimulation of renal sodium and water 
retention by the diseased kidney, resulting in volume overload with increased hydrostatic 
pressure and volume shift into the interstitium. [3] In this situation, RAAS components are 
typically suppressed [4]. 
Recent animal studies have demonstrated that primary renal sodium and water retention 
could be caused by activation of the epithelial sodium channel (ENaC) in the distal tubule 
system of the kidney by aberrantly filtered serine proteases (Figure 1) [5-7]. ENaC activation 
in the nephrotic mouse model can be prevented by the serine protease inhibitor aprotinin [8]. 
The pathophysiologically relevant serine proteases originate from the plasma and their 
identification is the subject of current investigations of our group [9]. So far, urokinase, 
plasminogen or plasma kallikrein have been identified as serin proteases that can activate 
the ENaC in vitro and are also aprotinin-sensitive [3, 7, 8, 10-12]. Data from urine samples 
from nephrotic humans and mice are identical with regard to the content of various serine 
proteases [9]. We have reported an aprotinin-sensitive proteasuria also in human proteinuric 
urine [8] and plasminuria was associated with overhydration in chronic kidney disease [13]. 
Therefore, it can be assumed that the pathophysiological processes described in the mouse 
model also apply to humans. 
 


Protocol code: AmiloridNS-01 
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Study Protocol Version 2.1 
 
 
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a 
 
b 
Figure 1: Scheme illustrating the pathophysiology of ENaC-mediated sodium retention (a) and 
model of sodium retention in nephrotic syndrome (b) [3] 
Aprotinin-sensitive serine proteases enter the urine in glomerular disease and can activate ENaC 
endoluminally by cleavage at the γ subunit (a). Proteasuria, as part of nephrotic proteinuria, results in 
sodium retention according to the overflow theory through this direct ENaC activation; if the proteinuria 
is sufficient to induce hypoalbuminemia and an "underfill", the ENaC might be activated additionally by 
RAAS (b). 
 
In addition to the disease specific treatment commonly involving immunosupression, 
supportive therapy of patients with acute nephrotic syndrome includes reduction of 
proteinuria by RAAS blockade, blood pressure control, lipid lowering, thromboembolic 
prophylaxis and the correction of volume overload [1], which is achieved by a dietary salt 
restriction and diuretic therapy. Currently, loop diuretics are preferred as they have the 
highest diuretic potency [14]. However, the diuretic effect of loop diuretics is lower in 
nephrotic syndrome than in other forms of edema or in healthy volunteers [1, 15]. A possible 
cause of this resistance to loop diuretics could be activation of ENaC [16] that is located 
distally to the sodium-potassium-chloride cotransporter (NKCC2), the target of loop diuretics. 
Sodium retention via ENaC may therefore counteract the inhibition of NKCC2.  To overcome 
refractory edema, either higher doses of loop diuretics or combination with thiazide diuretics 
(e.g., hydrochlorothiazide, HCT) for sequential nephron blockade or with distally acting 
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Considering the recent evidence of increased EnaC activity after activation by aberrantly 
filtered serine proteases, EnaC inhibition appears to be a promising approach to treat 
nephrotic edema syndrome [3, 16]. In nephrotic mice with overactivated ENaC, there is a 
stronger natriuresis after administration of the same dose of the EnaC inhibitor amiloride 
compared to the healthy state, and a daily treatment with amiloride prevents edema 
formation completely [8, 11]. There are a few smaller systematic studies on the use of 
amiloride, in particular with regard to the treatment of arterial hypertension in patients with 
proteinuria without nephrotic syndrome [17-19]. In nephrotic syndrome, the use of the ENaC 
inhibitors amiloride or triamterene has been reported in case reports, but there have been no 
controlled clinical trials yet. In an impressive case report by Hinrichs et al., a 38-year-old 
patient with type 1 diabetes and severe nephrotic syndrome, who underwent intensive 
combined antihypertensive and diuretic therapy without achieving blood pressure control and 
amelioration of edema, was treated with additional amiloride 5 mg daily, which lead to a 
marked decrease of systolic blood pressure, weight and edema, and increase of sodium 
excretion [20]. ENaC inhibitor triamterene has been tested with positive results for its 
application in nephrotic syndrome longer time ago already [21, 22] and was also used in a 
recently reported case [16]. Our own clinical experience of treating nephrotic patients with 
amiloride (in combination with hydrochlorothiazide) also confirms the very good efficacy of 
amiloride-based diuretic therapy.  
 
To give a résumé, activation of ENaC in nephrotic syndrome has been characterized in 
different mouse models by our group and others [3, 7, 8, 12, 23]; our investigations in 
patients with proteinuric kidney disease and nephrotic syndrome show that activation of 
ENaC by serine proteases also appears to be of central importance as a mechanism of 
edema formation in humans [12, 13]. These findings are now to be translated into an 
improved treatment of sodium retention and overhydration in patients with nephrotic 
syndrome. This randomized interventional trial investigates the superiority of amiloride to 
reduce edema in the nephrotic syndrome in comparison to the loop diuretic furosemide.  
 
1.1. 
Trial Rationale / Justification 
Treatment of resistant edema and overhydration in nephrotic syndrome can be challenging 
and we successfully use ENaC inhibitors (in combination with hydrochlorothiazide) in these 
patients. However, most clinicians treat nephrotic edema with higher doses of loop diuretics, 
or combination therapy with thiazide diuretics. Worldwide, ENaC inhibitors are rarely used in 


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nephrotic syndrome. This is due to lack of evidence of the role of activated ENaC and the 
effectiveness of ENaC inhibitors in human nephrotic syndrome. In this clinical trial, we aim to 
investigate the therapeutic effect of amiloride on the reduction of edema and overhydration in 
the nephrotic syndrome. A recent review from January 2019 on this topic highlights the 
urgent need for such a study and concludes with the phrase "it is time to undertake new 
clinical trials to direct clinical practice [in nephrotic syndrome]" [24]. 
In our study, patients with nephrotic syndrome will be randomized to medication with the 
investigational drug amiloride (study arm 1) or the comparator furosemide (study arm 2) for a 
study period of 16 days. In case of treatment resistant edema, hydrochlorothiazid (HCT) is 
added at day 8 in both study arms. To prevent bias, patients included in the study are 
randomized to start treatment with amiloride or furosemide. We have opted against blinding, 
as laboratory results required for safety reasons and for definition of dose adjustments will 
inevitably reveal to which study group each patient was randomized (hypokalaemia – 
furosemide, hyperkaliemia – amiloride). Duration of follow up period was determined based 
on clinical experience on the duration of the reversion of edema and overhydration. During 
follow up, the parameters body weight, extracellular water and overhydration (measured by 
bioimpedance spectroscopy), 24-hour natriuresis, blood pressure, plasma creatinine 
concentration, plasma renin activity, serum aldosterone concentration and serum sodium and 
potassium concentration will be measured. Overhydration as primary endpoint can be 
measured with bioimpedance spectroscopy as a validated, investigator-independent, 
reproducible method [25] and directly reflects changes of volume status that are to be 
evaluated. The number of n = 36 patients (n = 18 per group) was chosen on the basis of 
statistical considerations in order to be able to statistically significantly evaluate the primary 
endpoint of the study. The potential for recruiting the required number of patients has been 
assessed by counting patients with nephrotic syndrome presenting in the outpatient clinic of 
the department of nephrology of the University Hospital of Tuebingen during the last years. 
Additionally, agreement from Nephrologists running offices nearby to send in suitable 
patients was obtained. 
 
1.2. 
Benefit / Risk Assessment 
Overhydration and edema are serious symptoms that affect patients with nephrotic 
syndrome. Establishment of a more effective treatment of edema with adding ENaC 
inhibitors to standard treatment in human nephrotic syndrome has the potential to improve 
the clinical outcome of patients suffering from sodium retention. Known side effects of 


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amiloride, furosemide and HCT are listed in the respective Summary of Product 
Characteristics (SmPC). All drugs have been used for a long time, and their effects and side 
effects are well known and can be well estimated. Study specific relevant side effects are 
increase of plasma potassium level by amiloride, decrease of plasma potassium level by 
furosemide, and decrease of sodium level, increase of urine volume and decrease of body 
fluid by both diuretic regimens. To minimize risk of these potential side effects, the following 
precautions have been taken: to avoid occurrence of potential side effects, additional risk 
factors, such as decreased glomerular filtration rate or pre-existing electrolyte disorders are 
considered in the exclusion criteria; to recognize potential side effects early, volume status 
and electrolytes are monitored closely during study intervention phase; to rule out negative 
consequences of potential side effects, dose of study medication is adjusted according to 
changes of fluid status and electrolyte levels as well as renal function as specified in this 
protocol. All patients participating in the study receive adequate diuretic therapy of their 
overhydration due to nephrotic syndrome. Study participants benefit from gaining access to 
an intense treatment at our University hospital and its resources. 
All measurements performed in the study are without health risks and all devices used in the 
study are used according to their intended purpose. Venipuncture can be uncomfortable and 
slightly painful for the patient. The total volume of blood drawings during the entire study 
period does not exceed 100 mL within 8 days and does not pose participants at health risk. 
 
1.3. 
Advisory Committes 
A Data and Safety Monitoring Board and a Scientific Advisory Board are not forseen for this 
small scale exploratory study. 
 


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2. Study Objectives 
2.1. 
Primary Objective and Endpoint 
In the light of our current results on ENaC activation by serine proteases aberrantly filtered 
into urine in nephrotic syndrome, we hypothesize that targeting ENaC with amiloride will lead 
to a more effective reduction of overhydration in nephrotic syndrome than standard treatment 
with the loop diuretic furosemide.  
Primary objective of the study is therefore to prove the efficacy and superiority of amiloride 
for reduction of edema and overhydration (OH) in human nephrotic syndrome in comparison 
to standard medication with furosemide. Primary endpoint of the study is the decrease of 
overhydration after 8 days, compared to baseline. OH is measured using bioimpedance 
spectroscopy with the device Body Composition Monitor, Fresenius Medical Care AG & Co; 
BCM) and expressed as percent of ectracellular water (% ECW). 
 
2.2. 
Secondary Objectives and Endpoints 
Secondary endpoints represent further parameters to evaluate course of overhydration and 
regulation of body volume status after initiation of study medication with amiloride or 
furosemide. Secondary endpoints include  
1. decrease of OH after 16 days, measured using bioimpedance spectroscopy and 
expressed as % ECW 
2. decrease of body weight after 8 and 16 days 
3. decrease of edema circumference after 8 and 16 days, measured as the largest 
extent on the lower leg 
4. decrease of systolic and diastolic blood pressure after 8 and 16 days 
5. increase of urine volume and natriuresis after 8 and 16 days, determined from urine 
collection over 24 hours  
6. course of plasma renin activity and serum aldosterone concentration after 8 and 16 
days 
7. number of required changes of dose of study medication 
8. need for co-medication with HCT after 8 days 
9. occurrence of adverse events 


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3. Study Design 
The study design is a phase IIIb, monocenter, interventional, two-arm, randomized, open-
label controlled clinical trial. We decided for a study type with randomization as method 
against bias and have omitted blinding, as laboratory results required for safety reasons and 
dose adjustments will inevitably reveal to which study group patients have been randomized. 
Overall study design is shown in figure 2. Participants are randomized to start medication 
with amiloride 5 mg (trial arm 1) or furosemide 40 mg (trial arm 2) at day 0. Dose 
adjustments depending on course of overhydration, sodium and potassium level in blood gas 
analysis and plasma creatinine are performed on day 2, 5, 8 and 12 as defined in section 
6.6.1. In case of therapy-refractory overhydration, co-medication with HCT 12,5 mg is started 
at day 8. Last clinical study visit is at day 16. Follow up ends at day 
23.
 
 
 
Figure 2: Overall study design 


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3.1. 
Study Duration and Schedule 
The duration of the trial for each subject is 16 days of study treatment and 7 days of follow 
up. 
The overall duration of the trial is expected to be approximately 3.0 years including 
preparatory and analysis / report phase. Recruitment of subjects is planned to start in 
06/2020. The actual overall duration or recruitment may vary. The Study timelines are 
described in Table 1. 
 
Table 1: Study Timelines 
Total trial duration 
36 months 
Duration of clinical part (FSI – LSO) 
24 months 
Duration for in individual patient 
Treatment: 16 days 
Follow up: 7 days 
FSI (First Subject In) 
06/2020 
LSI (Last Subject In) 
05/2022 
LSO (Last Subject Out) 
06/2022 
DBL (Data Base Lock) 
09/2022 
Statistical Analyses Completed 
12/2022 
Trial Report Completed 
06/2023 
 
3.2. 
End of Study 
The end of the study is defined as the date of the last study visit of the last patient in the trial. 
Last patient last visit (LPLV) is either the date of the last visit of the last patient to complete 
the study, or the date at which the last data point from the last patient, which was required for 
statistical analysis (i.e. key safety and efficacy results for decision making), was received, 
whichever is the later date. 
 


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4. Study Population 
This study will include patients with acute nephrotic syndrome (definition see below) due to 
different underlying diseases (e.g. diabetic nephropathy, membranous nephropathy, minimal 
change disease, focal segmental glomerulosclerosis). The patients will be recruited from the 
nephrological outpatient department of the University Hospital Tuebingen or after referral 
from local nephrological practices. 
 
4.1. 
General Criteria for Subject Selection 
Adult male and female patients with acute nephrotic syndrome and fulfilling the below 
outlined inclusion criteria will be enrolled into the study.  
Trial population will consist of both genders. Gender distribution in the trial is supposed to 
reflect the distribution in the real patient’s population, i.e. there will be no prior defined 
quantitative ratio between females and males. No statistical analysis based on genders or 
differences between genders will be performed. 
 
4.1.1. 
Inclusion Criteria 
Subjects meeting all of the following criteria will be considered for admission to the trial: 
1. Acute nephrotic syndrome with proteinuria > 3 g/day and formation of edema. 
2. Age ≥ 18 years at the time of signing the informed consent. 
3. Understand and voluntarily sign an informed consent document prior to any study 
related assessments/procedures. 
4. Ability to adhere to the study visit schedule and other protocol requirements.  
5. Use of adequate thrombosis prophylaxis due to the increased risk of thrombosis in 
nephrotic syndrome and the expected fluctuations in volume balance during study 
participation 
6. Subject (male or female) is willing to use highly effective methods of contraception 
according 
to 
the 
“Clinical 
trial 
fertility 
group” 
recommendations 
(http://www.hma.eu/fileadmin/dateien/Human_Medicines/01About_HMA/Working_Gr
oups/CTFG/2014_09_HMA_CTFG_Contraception.pdf) during treatment and for 28 
days (male or female) after the end of treatment (adequate: intrauterine device, 
intrauterine hormone-releasoing system, bilateral tubal occlusion, vasectomized 


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partner1, sexual abstinence2; due to the increased risk of thrombosis in nephrotic 
syndrome, no hormonal contraceptives are recommended). 
7. Female Patients of childbearing potential (WOCBP)3 must agree to pregnancy testing 
before inclusion in the study. 
8. Female Patients must agree to abstain from breastfeeding during study participation 
and 28 days after study drug discontinuation. 
9. All subjects must agree not to share medication. 
 
1 Vasectomized partner is a highly effective birth control method provided that partner is the sole 
sexual partner of the WOCBP trial participant and that the vasectomized partner has received medical 
assessment of the surgical success 
2 In the context of this guidance sexual abstinence is considered a highly effective method only if 
defined as refraining from heterosexual intercourse during the entire period of risk associated with the 
study treatments. The reliability of sexual abstinence needs to be evaluated in relation to the duration 
of the clinical trial and the preferred and usual lifestyle of the subject  
3 For the purpose of this document, a woman is considered of childbearing potential (WOCBP), i.e. 
fertile, following menarche and until becoming post-menopausal unless permanently sterile. 
Permanent sterilisation methods include hysterectomy, bilateral salpingectomy and bilateral 
oophorectomy. A postmenopausal state is defined as no menses for 12 months without an alternative 
medical cause. A high follicle stimulating hormone (FSH) level in the postmenopausal range may be 
used to confirm a post-menopausal state in women not using hormonal contraception or hormonal 
replacement therapy. However in the absence of 12 months of amenorrhea, a single FSH 
measurement is insufficient. 
 
4.1.2. 
Exclusion Criteria 
Subjects presenting with any of the following criteria will not be included in the trial: 
1. Severe reduction of kidney function: Creatinine clearance or calculated GFR < 30 
mL/min/1.73m² or acute kidney injury KDIGO stage 2 or 3 or anuria. 
2. Hypovolemia or dehydration. 
3. Uncontrolled diabetes mellitus. 
4. Hypotension, systolic blood pressure < 90 mmHg. 
5. Hyperkalemia, plasma potassium concentration > 4.8 mmol/l. 
6. Hypokalemia, plasma potassium concentration < 3.3 mmol/l. 
7. Hyponatremia, plasma sodium concentration < 128 mmol/l. 
8. Hypercalcemia, ionized calcium > 2.0 mmol/l or total albumin corrected calcium > 3.0 
mmol/. 


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9. Signs of cardiac decompensation (orthopnoe, dyspnoe NYHA IV). 
10. Hepatic coma or precoma. 
11. Symptoms of gout. 
12. Current therapy with potassium-sparing diuretics (e.g. spironolactone) or potassium 
supplements. 
13. Women during pregnancy and lactation. 
14. History of hypersensitivity to the investigational medicinal product, comparator or co-
medication or to any drug with similar chemical structure or to any excipient present 
in the pharmaceutical form of the investigational medicinal product, comparator or co-
medication. 
15. Any other clinical condition that would jeopardize the patient’s safety while 
participating in this clinical trial. 
16. Active participation in other clinical trials or observation period of competing trials. 
 


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5. Requirements for Trial Site and Investigator 
The necessary equipment, including Body Composition Monitor, is permanent available at 
the trial site.  
There are no special training measures required to use the investigational medical product or 
comparator. 
 


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6. General Information on the Study Medication 
Investigational medicinal product: Amiloride 5 mg 
Registered trade name: Modamide® 
International Non-proprietary Name (INN): Amiloride 
ATC code, if officially registered: C03DB01 
Pharmaceutical formulation: tablet 
Route of administration: oral 
Storage conditions: No specific storage conditions 
Stabilitiy: 3 years 
Manufacturer: Laboratoires GERDA, 24 rue Erlanger, 75016 Paris / France 
Source: Pharmacy of the University Hospital Tuebingen 
Components: Quinoline Yellow, Red Iron Oxide, Corn Starch, Calcium Hydrogen Phosphate, 
Magnesium Stearate, Lactose 
Indications: Cardiac edema; edema and ascites in cirrhosis; arterial hypertension 
Contraindications: Hyperkalemia (above 5.5 mmol/l); therapy with other potassium-sparing 
diruetics; therapy with potassium supplements; impaired renal function (anuria, acute renal 
failure, severe progressive kidney disease and diabetic nephropathy); hypersensitivity to any 
component of the drug 
 
Comparator: Furosemide 40 mg 
Administration method: oral 
Registered trade names: Furosemid-ratiopharm® 
International Non-proprietary Name (INN): Furosemide 
ATC code, if officially registered: C03CA01 
Pharmaceutical formulation: tablet 
Route of administration: oral 
Storage conditions: No specific storage conditions 
Stabilitiy: 5 years 
Manufacturer: ratiopharm GmbH, Graf-Arco-Straße 3, 89079 Ulm, Germany 
Source: Pharmacy of the University Hospital Tuebingen 


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Components: Lactose monohydrate, microcrystalline cellulose, Carboxymethyl starch sodium 
(type A), povidone K25, fumed silica, magnesium stearate 
Indications: Cardiac or hepatic edema; renal edema; edema due to burns; arterial 
hypertension 
Contraindications: Kidney failure with anuria; hepatic coma and praecoma; severe 
hypokalemia; severe hyponatremia, hypovolemia or dehydration; lactating women; 
hypersensitivity to any component of the drug 
 
Co-medication: Hydrochlorothiazide (HCT) 12.5 mg 
Administration method: oral 
Registered trade names: HCT-Hexal® 
International Non-proprietary Name (INN): Hydrochlorothiazide (HCT) 
ATC code, if officially registered: C03AA03 
Pharmaceutical formulation: tablet 
Route of administration: oral 
Storage conditions: No specific storage conditions 
Stabilitiy: 3 years 
Manufacturer: Hexal AG, Industriestraße 25, 83607 Holzkirchen / Germany 
Source: Pharmacy of the University Hospital Tuebingen 
Components: Corn starch, lactose monohydrate, microcrystalline cellulose, fumed silica, talc, 
poly (O-carboxymethyl) starch sodium salt, magnesium stearate 
Indications: Arterial hypertension; cardiac, hepatic or renal edema; adjuvant symptomatic 
therapy of chronic heart failure in addition to ACE inhibitos  
Contraindications: severe renal impairment (renal insufficiency with oliguria or anuria, 
creatinine clearance <30 ml/min and / or serum creatinine >1.8 mg/100 ml); acute 
glomerulonephritis; hepatic coma and praecoma; hypokalemia; hyponatremia; hypovolemia 
or dehydration; hypercalcaemia; gout; hypersensitivity to any component of the drug 
 
6.1. 
Manufacturing of the Study Medication 
Study medication (investigational medical product amiloride, comparator furosemide and co-
medication hydrochlorothiazide) is commercially available and will be obtained via the 
Pharmacy of the University Hosptial Tuebingen directly from the respective manufacturer and 


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used for the study without further manufacturing processes or repackaging. The 
investigational medical product amiloride is not available as individual substance in Germany 
and will be imported from Laboratoires GERDA, 24 rue Erlanger, 75016 Paris / France. The 
comparator furosemide and co-medication hydrochlorothiazide are available in Germany 
from German manufactorers. 
 
6.2. 
Labeling of the Study Medication 
The trial medication furosemide and HCT will be provided by the pharmacy of the University 
Hospital Tuebingen as part of standard therapy medication from German manufacturers. As 
the trial is not blinded, no additional labelling has to be performed for furosemide and HCT. 
 
The trial medication amiloride will be imported and labelled by the Pharmacy of the University 
Hospital Tuebingen according to § 5 (1) of GCP-V. The label will contain information in 
German language to ensure identification of the medical product and the clinical trial and to 
ensure a proper use of the medical product. 
The label of the Investigational Medical Product Amilorid will contain the following details: 
- 
Name, address and phone number of the representative of the trial sponsor 
- 
Name and address of the Pharmacy 
- 
Name and potency of investigational medicinal product 
- 
Batch number or code number of the trial 
- 
Administration form 
- 
Content specified by weight, volume or quantity 
- 
Application form 
- 
Dosing instructions or a reference to instructions by the clinical investigators 
- 
Expiry date using note “usable until” 
- 
Discharge date from the pharmacy  
- 
Code of Trial Protocol 
- 
EudraCT-Number 
- 
Note: “For use in clinical trial” 
- 
Note: “Keep inaccessible for children” 
- 
Note: “Bring this package including blister to every study visit” 
- 
Note: “Return unused tablets at the end of the trial” 
- 
Note: “Store below 25 °C” 
 


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The template of the label is shown in appendix 15.3. As accompanying document an 
instruction sheet, which can be seen in appendix 15.2, will be issued to every subject with 
study enrollment. 
 
6.3. 
Storage of the Medical Product 
All trial medication must be kept in a locked area with access restricted to designated trial 
staff. Amiloride and Furosemide must be stored below 25 °C. Otherwise, no trial medication 
requires any specific storage conditions according to the manufacturers’ instructions. 
 
6.4. 
Drug Accountability, Therapy Compliance and Disposal 
The site investigator will keep an account of the trial medication and acknowledge the receipt 
of all shipments of the trial medication.  
Trial medication will be dispensed to the subjects by the investigator at day 0 of study 
participation. The investigator will document the date of dispensary, subject identification, 
batch/ serial numbers or other identification of trial medication. The investigator will also keep 
accurate records of the quantities of trial medication dispensed, used, and returned by each 
subject. Subjects will be instructed to intake the IMP / comparator daily and to bring all 
remaining trial medication and empty blisters to the trial site at every study visit. Compliance 
will be assessed by count of tablets. Details will be recorded in the Case Report Form (CRF). 
The University Hospital Tübingen is responsible to discard the unused trial medication at the 
end of the trial.  
 
6.5. 
Method of Treatment Assignment 
See Section 7.1.4 (Randomisation). 
 
6.6. 
Dose Schedule 
Participating patients will take the study medication orally once daily in the morning in fasting 
state, unchewed with adequate amount of fluid. Initial dose will be amiloride 5 mg or 
furosemide 40 mg. Initial dose of amilorde was chosen by virtue of clinical expertise and 
former study results from studies using amiloride in related groups of patients [17-19], with 
reflection of clinical effectiveness and prevention of adverse events. Initial dose of 
furosemide was chosen as a typically used initial dose. The duration of treatment is 16 days, 


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since the major loss of overhydration is expected to be reached in this time period. 
Depending on course of overhydration, co-medication with HCT 12,5 mg is started at ay 8. 
6.6.1. 
Dose Modification 
The following considerations were taken into account when determining the dose 
adjustments:  
- 
The initial dosages determined on the basis of clinical experience and common clinical 
practice are amiloride 5 mg and furosemide 40 mg. In the treatment of overhydration in 
nephrotic syndrome with furosemide, treatment resistance must be expected [26, 27]. 
The dose should therefore be increased depending on the clinical effect. To ensure the 
comparability of the results of the two study arms, the dose should be increased by steps 
of one whole tablet of each, amiloride or furosemide. Based on available clinical data [17-
20], maximum dose of amiloride is 15 mg and of furosemide should be 120 mg.  
- 
Based on data from trial measurements of baseline overhydration in nephrotic patients 
prior to treatment (n = 14), an overhydration of 1 L corresponds to 4.25 %ECW. The daily 
weight loss of water should not exceed 1 L (corresponding to 4.25 %ECW) according to 
usual recommendations (safety criterion).  
- 
With a weight loss of less than 0.4 L (corresponding to 1.7 %ECW) per day, the response 
to the therapy is too low and the diuretic therapy should be increased (efficacy criterion). 
- 
If weight loss under medication with amilorid or furosemide is treatment refractory, co-
medication with HCT should be initiated after 8 days, with maximum dose 25 mg. 
- 
Electrolyte disorders should lead to dose adjustments in case of plasma potassium < 3.3 
mmol/l or > 5.3 mmol/l and plasma sodium < 125 mmol/l (safety criteria). 
- 
Indications of acute kidney injury (increase of serum creatinine by ≥ 0.4 mg/dl) should 
lead to a dose reduction. 
- 
Low blood pressure (BP) should lead to a dose reduction. 
- 
When overhydration is no longer present, the dose should not be increased further, but a 
maintenance dose based on previous needed dose should be continued. Overhydration 
is no longer present, if OH is within the normal range of -1 to +1 L, corresponding to -4.25 
to +4.25 %ECW. 
 
The dose after dose adjustment is valid until the next study visit. If a safety criterion 
and an efficacy criterion are both met, the dose adjustment shall be performed on the 
basis of the safety criterion. 
 
 


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Dose adjustments are performed as follows: 
Dose adjustment at day 2: 
Reasons for incease (efficacy 
criteria) 
Reasons for decrease (safety 
criteria) 
Reasons for pause of 
study medication (safety 
criteria) 
- Decrease of OH since day 0 
is less than 3.4 %ECW 
- Decrease of OH since day 0 
is more than 8.5 %ECW 
- Potassium in BGA >5.3 or 
<3.3 mmol/l 
- Systolic BP < 90 mmHg or 
diastolic BP < 60 mmHg 
- Increase of creatinine by 
>0.4 mg/dl since day 0 
- K in BGA >5.6 or <3.0 
mmol/l 
- Na in BGA <125 mmol/l 
 
Dose adjustment at day 5: 
Reasons for incease (efficacy 
criteria) 
Reasons for decrease (safety 
criteria) 
Reasons for pause of 
study medication (safety 
criteria) 
- Decrease of OH since day 2 
is less than 5.1 %ECW 
- Decrease of OH since day 2 
is more than 12.8 %ECW 
- Potassium in BGA >5.3 or 
<3.3 mmol/l 
- Systolic BP < 90 mmHg or 
diastolic BP < 60 mmHg 
- Increase of creatinine by 
>0.4 mg/dl since day 0 
- K in BGA >5.6 or <3.0 
mmol/l 
- Na in BGA <125 mmol/l 
 
Dose adjustment at day 8: 
Reasons for increase (efficacy 
criteria) 
Reasons for decrease (safety 
criteria) 
Reasons for pause of 
study medication (safety 
criteria) 
- Decrease of OH since day 5 
is less than 5.1 %ECW 
- Decrease of OH since day 5 
is more than 12.8 %ECW 
- Potassium in BGA >5.3 or 
<3.3 mmol/l 
- Systolic BP < 90 mmHg or 
diastolic BP < 60 mmHg 
- Increase of creatinine by 
>0.4 mg/dl since day 0 
- K in BGA >5.6 or <3.0 
mmol/l 
- Na in BGA <125 mmol/l 
 
Dose adjustment at day 12: 
Reasons for increase (efficacy 
criteria) 
Reasons for decrease(safety 
criteria) 
Reasons for pause of 
study medication (safety 
criteria) 
Decrease of OH since day 5 is 
less than 6.8 %ECW 
- Decrease of OH since day 8 
is more than 17.0 %ECW 
- Potassium in BGA >5.3 or 
<3.3 mmol/l 
- Systolic BP < 90 mmHg or 
diastolic BP < 60 mmHg 
- Increase of creatinine by 
>0.4 mg/dl since day 0 
- K in BGA >5.6 or <3.0 
mmol/l 
- Na in BGA <125 mmol/l 


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Increase is defined as 
- 
Increase of study medication by amiloride 5 mg or furosemide 40 mg if current dose is 
amiloride 5 or 10 mg or furosemide 40 or 80 mg 
- 
Addition of HCT 12,5 mg or 25 mg  at day 8 or 12 
Decrease is defined as  
- 
pause of study medication for 1 day if current dose is amiloride 5 mg or furosemide 40 
mg and then continue with amiloride 5 mg or furosemide 40mg 
- 
reduction by amiloride 5 mg or furosemide 40 mg if current dose is amilorid 10 or 15 mg 
or furosemide 80 or 120 mg 
- 
reduction of HCT dose at day 12 
Pause of study medication is defined as pause of study medication until the next visit.  
 


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7. Study Procedures and Examination Method 
This Study will consist of the following consecutive phases: Study entry, Treatment and 
Follow-up. Time-points and trial procedures are listed in Table 2. 
Table 2 Table of Events 
 
Study 
entry 
Treatment 
Follow-
up 
Visit number 
1 
2 
3 
4 
5 
6 
7 
8 
Day 
-3 to -1 
0 
2 
5 
8 
12 
16 
23 
Study Entry 
Informed Consent 
X 
 
 
 
 
 
 
 
Demographics, including sex, body 
weight, high, age 
X 
 
 
 
 
 
 
 
Inclusion/Exclusion criteria: 
anamnesis, clinical examination, 
blood pressure, pre-existing 
medication, proteinuria in 24 hours 
collected urine, general laboratory 
control (including blood count, 
creatinine, urea, liver enzymes, lactat 
dehydrogenase, c-reactive protein, 
electrolytes (potassium, sodium, 
calcium), glucose, HbA1c, pregnancy 
test in females) 
X 
 
 
 
 
 
 
 
Safety Assessments 
Adverse events 
 
X 
X 
X 
X 
X 
X 
X 
Concomitant medication 
X 
X 
X 
X 
X 
X 
X 
X 
Check of the study medication 
 
X 
X 
X 
X 
X 
X 
 
Return of any remaining study 
medication 
 
 
 
 
 
 
X 
 
Plasma creatinine 
X 
X 
X 
X 
X 
X 
X 
X 
Potassium and sodium level in blood 
gas analysis 
X 
X 
X 
X 
X 
X 
X 
X 
Efficacy Assessment 
Body weight 
X 
X 
X 
X 
X 
X 
X 
X 
Bioimpedance spectroscopy with 
BCM 
X 
X 
X 
X 
X 
X 
X 
X 


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Edema circumference 
X 
X 
X 
X 
X 
X 
X 
X 
Blood pressure 
X 
X 
X 
X 
X 
X 
X 
X 
Plasma renin activity, serum 
aldosterone concentration 
X 
X 
X 
X 
X 
X 
X 
X 
24 hours urine volume and natriuresis 
X 
 
X 
X 
X 
X 
X 
 
Diet diary during urine collection 
X 
 
X 
X 
X 
X 
X 
 
Check for dose adjustment 
 
X 
X 
X 
X 
X 
 
 
 
7.1. 
Study Entry 
7.1.1. 
Patient’s Informed Consent (see also 11.2.) 
The patient is to be informed both in writing and verbally by the investigator before any study-
specific procedure is performed. Each patient will be informed about the modalities of the 
clinical study in accordance with the provided patient information. The patient is given 
sufficient time (24h) to consider participation in the clinical trial and ask for additional advise if 
needed. Informed consent from the patient will be obtained using a form approved by the 
ethical committee of the University Hospital Tuebingen. The patient and informing physician/ 
investigator must each personally date and sign the informed consent form and declaration 
on data privacy protection (section 11.4) on the same day. The original signed documents 
will be part of the investigator’s site file and retained with it and a copy included the insurance 
policy of the trial will be handed to the patient. 
7.1.2. 
Enrollment 
After the patient’s informed consent, a unique subject number for identification purposes will 
be assigned to the patient in order to maintain his/her pseudonymity. The subject number will 
be used for the patient throughout the study (= ID number). Screening failures will also be 
assigned to a subject number and all collected data will be recorded in the CRF. The PI or a 
nominated investigator will decide on enrolment after screening. 
7.1.3. 
Screening  
Screening will be performed within 3 days prior to first administration of study medication. 
After having signed the informed consent, patients will undergo all assessments listed below: 
• 
Anamnesis including medical history and pre-existing medication 
• 
Questions about participation in other clinical trials or research projects 
• 
Demographics and clinical examination 
• 
Blood pressure 


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• 
Assessment of edema, measurement of highest edema circumference at the lower 
leg 
• 
Blood sampling for measurement of laboratory parameters and blood gas analysis; 
laboratory parameters include blood count, creatinine, urea, liver enzymes, lactat 
dehydrogenase, C-reactive protein, electrolytes (potassium, sodium, calcium), 
glucose, HbA1c, pregnancy test in females 
• 
Proteinuria in 24 hours collected urine 
• 
Natriuresis in 24 hours collected urine 
• 
Diet diary during urine collection 
• 
Bioimpedance spectroscopy (BCM) 
Patients who fulfill all the inclusion criteria and none of the exclusion criteria will be eligible to 
participate in the trial. Screening failures, i.e. screened patients not in compliance with all 
criteria; are to be excluded and the reason will be recorded in the patient notes.  
Information of patient's trial participation can be provided to the patient’s general practitioner 
upon request. 
7.1.4. 
Randomisation  
The randomization will follow a randomization scheme of a 1:1 (amiloride and furosemide). 
Subjects will be assigned to their respective treatment by randomization. A randomisation 
number will be assigned (= Random. number). Subjects will then receive their study 
medication during their personal visit at day 0 of study participation. 
7.1.5. 
Concomitant Medication and Treatments 
Relevant additional medications and treatments administered to the subjects on entry to the 
trial or at any time during the trial are regarded as concomitant medications and treatments 
and must be documented on the appropriate pages of the CRF. 
7.1.6. 
Permitted Prior and Concomitant Medications and Treatments 
The following concomitant medications and treatments are permitted during the trial and for 3 
days prior to enrolment into the clinical trial: 
- 
Proteinuria lowering medication (ACE inhibitors and Angiotensin II receptor) in a low 
dose (maximally 8 mg Candesartan or 2,5 mg Ramipril per day or equivalent) 
- 
Corticosteroids and other immunosuppressive therapy as therapy of underlying 
disease starting from day 8 
- 
Antihypertensive therapy with calcium antagonists. 


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7.1.7. 
Prohibited Prior and Concomitant Medications and Treatments 
Medication that are contraindicated in association with amilorid, furosemide or HCT as 
defined in the respective SmPCs have been considered in the Exclusion criteria, this 
includes in particular potassium sparing diuretics and potassium salts.   
Further drug interactions are reflected in the following. 
The SmPC of Amiloride lists potassium salts, potassium diuretics, angiotensin-converting 
enzyme inhibitors, angiotensin II antagonists, nonsteroidal anti-inflammatory drugs, heparins 
(low molecular weight or unfractionated), immunosuppressants such as ciclosporin or 
tacrolimus, trimethoprim as medications that can promote the occurrence of hyperkaliemia 
under treatment with amiloride. The SmPC points out that this risk is particularly important 
with potassium-sparing diuretics and potassium salts and these drug classes have therefore 
been considered as prohibited prior and concomitant medication and in the exclusion criteria. 
Other listed potential interactions include increased risk of toxicity of HCT (baclofen, 
antidepressants, neuroleptics) or other drugs (lithium, metformin), increased risk of 
hypotension or acute kidey injury especially in case ot hypovolemia (nonsteroidal anti-
inflammatory drugs, ACE inhibitors, iodinated contrast media, other diuretics, alpha blocker 
antihypertensives, amifostine), decreased effect of HCT (corticosteroids). 
The SmPC of furosemide lists interactions with other pharmaceuticals, like increased risk of 
hypokalemia and therefore increased risk of arrhythmia (glucocorticoids, carbenoxolone or 
laxatives), increased risk of acute kidney injury especially if hypovolemia is caused (non-
steroidal anti-inflammatory drugs, antibiotics like aminoglycosides, cephalosporins or 
polymyxins, cisplatin, iodinated contrast medium), weakening of the effect of furosemide 
(probenecid, methotrexate, phenytoin, sucralfate) or of the other drug (antidiabetics, 
sympathomimetics), increased risk of ototoxicity (aminoglycosides, cisplatin, increased risk of 
hypotonia (ACE inhibitors, angiotensin II receprot antagonists), decrease of renal elimination 
(of lithium, probenecid, methotrexate), increased risk of hyperuricemia (cycosporin A).  
The SmPC of HCT lists interactions like increased risk of hypotonia (guanethidin, 
methyldopa, calcium antagonists, ACE inhibitors, angiotensin II receptor blockers, dopamine 
reuptake inhibitor, beta receptor blocker, nitrates, barbiturates, phenothiazine, thicyclic 
antidepressants, vasodilatatirs, ethanol), acute kidney injury (ACE inibitors, non steroidal 
antiinflammatoric drugs), increased risk of hyperglycemia (beta receptor blockers), reduced 
effect of HCT (non steroidal antiinflammatoric drugs), increased risk of hypokalemia and 
therefore increased risk of arrhythmia (other diuretics, glucocorticoids, ACTH, cabenoxolon, 
penicillin G, salicylates, amphotericin B, antiarrhythmics, glycosides, laxans), increased risk 
of hyponatremia (diuretics, antidepressants, neuroleptics, antiepileptics), increased toxicity of 


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other drugs (salicylate, allopurinol, amantadine), decreased effect of HCT (colestyramin, 
colestipol) or other drugs (insulin, anti-hyperurikemic drugs, noradrenalin, adrenalin), 
increased toxicity of other drugs (caclophosphamid, fluorouracil, methotrexate, lithium, 
muscle relaxans, vitamine D, calcium, ciclosporin, diazoxid, methyldopa), increased effect of 
HCT (atropine, biperiden). 
Most of the listed co-medication with potential interactions is not expected to be prescribed or 
indicated in the study cohort of patients with nephrotic syndrome. However, serum creatinine 
levels and serum electrolyte concentrations are monitored closely during the study as safety 
parameters (section 7.4). If any other co-medication with potential interaction is found during 
check of co-medication at screening visit, any condition that could jeopardize the patient’s 
safety is defined as exclusion criteria. Patients are informed in the patient information sheet 
that they may only undergo other medical treatment, except in emergencies, after prior 
consultation with the study investigator, they have to notify the investigator immediately of 
any emergency treatment received and they have to inform other treating physicians about 
the study participation to prevent pharmaceutical interactions. 
 
7.1.8. 
Required Prior and Concomitant Medications and Treatments 
As co-medication according to study protocol, hydrochlorothiazide 12.5 mg to 25 mg in 
addition to the initial medication with amiloride or furosemide is added at day 8 in case of 
treatment refractory edema as defined in section 6.6.1. Otherwise, there are no concomitant 
medications and treatments required during the trial.  
 
7.2. 
Treatment Phase 
During the treatment phase (day 0 – 15) of this trial, participants will receive either amiloride 
with an initial dose of 5 mg once daily or furosemide with an initial dose of 40 mg once daily. 
Dose adjustments will be performed as defined in 6.6.1. Beginning at day 8, participants will 
receive hydrochlorothiazide (initial dose 12.5 mg) as co-medication in addition to the initial 
medication with amiloride or furosemide in case of treatment refractory edema as defined in 
section 6.6.1. 
 
7.2.1. 
Description of Patients’ Visits 
Visit 1 is the screening visit and described in 7.1.3. The patient will be randomized if all 
inclusion criteria and no exclusion criteria are fulfilled. 


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The patient wil be instructed to take study medication in the morning on all study days during 
treatment phase (at day 0 – 15)  
Visit 2 – 7 take place in the morning and all have the same schedule. Examinations 
performed on visit 2 – 7 include: 
- 
interview for adverse events and concomitant medication 
- 
body weight, bioimpedance spectroscopy (BCM) and edema circumference 
- 
blood pressure 
- 
blood sampling: blood gas analysis (1 mL) is carried out immediately; Li-Hep-plasma 
(3 mL) is taken for measurement of serum creatinine directly after blood collection; 
further blood samples including EDTA-plasma (8 mL), Li-Hep-plasma (5 mL) and 
serum (8 mL) are centrifuged and frozen at -20 °C for later measurements) 
- 
24 hours collected urine volume and natriuresis and diet diary during urine collection 
(the patient will be instructed to collect urine for 24 hours and write a diet diary prior to 
the study visit, see instruction sheet, appendix 15.2).  
- 
handing out / check / receive back (visit 7) the patient’s study medication 
- 
check for dose adjustments as defined in section 6.6.1 and completing the medication 
and dose for the next study days on the patient's instruction sheet (see appendix 
15.2). 
After the last examination at study visit 7, treatment phase ends for the respective patient. 
Treatment after the study will be operated as described in section 7.8.1 
At day 23, a follow up (visit 8) will be performed. The follow-up visit invludes: 
- 
interview for adverse events and concomitant medication 
- 
body weight, bioimpedance spectroscopy (BCM) and edema circumference 
- 
blood pressure 
- 
blood sampling: blood gas analysis (1 mL) is carried out immediately; Li-Hep-plasma 
(3 mL) is taken for measurement of serum creatinine directly after blood collection; 
further blood samples including EDTA-plasma (8 mL), Li-Hep-plasma (5 mL) and 
serum (8 mL) are centrifuged and frozen at -20 °C for later measurements) 
 
 
7.3. 
Assessment of Efficacy  
7.3.1. 
Efficacy Parameters 
Aim of the study is to prove the efficacy of amiloride for reduction of edema and 
overhydration in human nephrotic syndrome in comparison to standard medication with 


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furosemide. Overhydration as the primary endpoint variable is determined directly using 
bioimpedance spectrosycopy with the Body Composition Monitor in an investigator 
independent and reproducible way. As further clinical parameters to assess overhydration, 
body weight, edema circumference and systolic and diastolic blood pressure are measured 
in a clinical examination.  
Laboratory parameters performed for assessment of efficacy include plasma renin activity, 
serum aldosterone concentration and urine volume and natriuresis in 24 hours collected 
urine. Plasma renin activity and serum aldosterone concentration are relevant parameters of 
the body’s fluid regulation and suitable laboratory parameters to assess efficacy of diuretic 
medication. Increase of natriuresis represents the mechanism of acting of diuretic medication 
and leads to higher urine volume output with the consequence of reduction of overhydration. 
Therefore, urine volume and natriuresis are necessary parameters to assess the efficacy of 
diuretic medication. To consider that natriuresis is affected by sodium intake in further 
analysis, patients are instructed to write a diet diary to enable estimation of sodium intake. 
Additional efficacy parameters are the number of required changes of the dose of the study 
medication and the need for co-medication with hydrochlorothiazide as defined in this 
protocol. 
 
7.3.2. 
Methods and Timing for Assessing, Recording, and Analysing of 
Efficacy Parameters  
A) Measurement of body fluid status with the Body Composition Monitor: 
Body fluid status will be determined using bioimpedance spectroscopy with the device Body 
Composition Monitor (BCM, Fresenius Medical Care AG & Co). Bioimpedance spectroscopy 
measures the electrical resistance of the body tissue at 50 different frequencies in the range 
from 5 to 1000 kHz. Due to the principle that high-frequency measuring currents flow 
unhindered through the whole body water, while the low-frequency measuring currents can 
not penetrate the cell membrane and flow only through the extracellular space, intra- and 
extracellular water can be determined separately. Based on two physiological models, the 
volume model and the body composition model, the relevant parameters for assessing the 
hydration status are calculated from the measured resistances at the different measurement 
frequencies. The volume model is used to calculate total body water (TBW), ECW and 
intracellular water (ICW). The Body Composition model calculates the so-called 
overhydration (OH) as excess fluid volume out of total body mass, normal hydrated fatty 
tissue mass and normal hydrated lean tissue mass. [25] 


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Measurement with the BCM is performed as follows: The patient is already in a supine 
position for two minutes before taking the measurement. The patient should not be in skin 
contact with metal objects. Two measuring electrodes with at least 3 cm distance are 
attached to the back of the right hand and foot and connected to the BCM. After entering the 
patient data necessary for calculating the results, namely gender, age, daily updated size 
and daily updated weight, the measurement can be started. During the few seconds of 
measurement, the patient must lie still and not speak. After a short calculation time, the 
device displays the results. 
The BCM device carries a CE certificate (see appendix 15.4) and is used during the study 
exclusively for its intended purpose and by trained personnel. 
For the study, overhydration is expressed as percent of ectracellular water (% ECW). 
Measurement with the Body Composition Monitor is performed at every study visit (study 
entry, treatment phase and end of follow up) and documented in the case report form. 
 
B) Measurement of body weight, edema circumference, systolic and diastolic blood 
pressure: 
The body weight is measured with a calibrated scale. For each measurement of the patient's 
body weight during participation in the study the same scale will be used. Body weight will be 
recorded with the patient wearing street clothes without shoes, outerwear or accessories. 
Edema circumference is measured using a calibrated measuring tape. Maximal 
circumference of the lower leg is measured on both sides. 
For blood pressure measurements, a calibrated electric blood pressure monitor with upper 
arm cuff is used. Blood pressure will be measured as office blood pressure in a sitting 
position after at least 5 minutes of rest at the patient’s dominant side (right side if right-
handed, left side if left-handed) twice. The average of the measured values is documented. 
Measurement of body weight, body fluid status, edema circumference, systolic and diastolic 
blood pressure is performed at every study visit and documented immediately in the case 
report form. 
C) Measurement of laboratory parameters: 
Blood and urine samples for measurement of the laboratory parameters are taken at every 
study visit during treatment phase. This includes EDTA-plasma for measurement of plasma 
renin activity, serum for serum-aldosterone concentration and 24 hours collected urine for 
natriuresis. Urine volume of 24 hours collected urine is documented immediately. Test tubes 


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with blood samples are centrifuged and blood and urine samples are frozen at -20 °C. 
Measurement of plasma renin activity, serum aldosterone concentration and urine sodium 
concentration are performed at the end of the patient’s follow up in the nephrological 
laboratory and in the central laboratory of the Univerity Hospital of Tuebingen. To enable 
consideration of salt content of the food in later analysis and interpretation of study results, 
study participants are instructed to write food diaries on the days of the urine collection. 
 
7.4. 
Assessment of Safety  
7.4.1. 
Safety Parameters  
Safety assessment includes adverse events, concomitant medication and check of study 
medication. As safety parameters for specific risks of the study, serum potasium and sodium 
levels and plasma creatinine concentration are monitored during study participation. 
(Serious) Adverse Events (see section 10) 
 
7.4.2. 
Methods and Timing for Assessing, Recording, and Analysing Safety 
Parameters  
Check for adverse events is performed continuously from beginning of study medication until 
7 days after end of study treatment. Check of concomitant medication is performed at 
screening visit and continuously until end of follow up. Check of study medication (integrity, 
correct number of remaining tablets) is checked at every study visit during treatment phase. 
Any remaining study medication is to be returned at the end of treatment phase. 
Specific risks from the study medication are shifts in the electrolyte balance and volume 
status. Parameters of volume status are assessed as efficacy parameters as described 
above (section 7.3). Sodium and potassium levels are measured in blood gas analysis at 
every study visit. Therefore, blood gas analysis is performed directly after blood collection 
using the blood gas analysis device at the dialysis department. If this blood gas analysis 
device is not available at the respective moment, blood gas analysis devices at the 
emergency department or intensive care unit are used alternatively. All BGA devices used 
are subject to continuous quality assurance and inspection. Possible changes in body fluid 
status and electrolyte levels are counterregulated by dose adjustment requirements specified 
in this protocol (section 6.6.1). 
As changes in body fluid status could lead to acute (prerenal) kidney failure, serum creatinine 
levels are measured at every study visit. The blood sample is sent to the central laboratory of 


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the University Hospital Tuebingen directly after blood collection and plasma creatine levels 
are measured immediately. If plasma ceratinine increases by >0.4 mg/dl, study medication is 
discontinued as specified in section 6.6.1.  
 
7.5. 
Premature termination of clinical trial for a trial subject  
Reasons for premature termination of trial for an individual trial subject are: 
1. Death 
2. Withdrawal of consent 
3. Patient lost to follow-up 
4. Major protocol violation  
5. At their own request or at request of the legal representative 
6. If, in the investigator’s opinion, continuation of the trial would be detrimental to the  
subject’s well-being 
7. Occurrence of serious adverse event caused by the investigational medicinal product 
8. For women, if it becomes known that the subject is pregnant 
 
The PI decides about withdrawal of subjects from trial treatment in case of occurrence of 
criteria mentioned above. In all cases, the reason for withdrawal must be recorded in the 
CRF and in the subject’s medical records. In case of withdrawal of a subject at his/ her own 
request, the reason should be determined and documented. All examinations scheduled for 
the last trial day will be performed and documented as far as possible, subject to the consent 
of the patient. These subjects will enter the regular follow up of the trial, unless the subject 
has withdrawn his/her consent to any further study-related procedure. All ongoing Adverse 
Events (AEs)/ Serious Adverse Events (SAEs) of withdrawn subjects have to be followed up 
until no more signs and symptoms are verifiable or the subject is on stable condition. 
 
7.6. 
Premature closure of a trial site 
Not applicable since monocentric study. 
 
7.7. 
Premature termination of the trial 
The trial may be prematurely terminated, if in the opinion of the sponsor and coordinating 
investigator there is sufficient reasonable cause. Written notification documenting the reason 
for study termination will be provided to the investigators. 


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In case of the following situations, a premature termination of the trial has to be considered: 
• Serious adverse drug reactions / not justifiable toxicity 
• Substantial changes in risk-benefit considerations 
• New insights from other trials 
• Insufficient recruitment rate 
 
7.8. 
End of Study of Subjects  
The End of Study for a patient enrolled in this trial is defined as the date of the last visit of the 
respective patient in the trial. 
7.8.1. 
Plan for Treatment or Care after End of Study 
At the end of study tretmant, further medication for nephrotic syndrome will be determined by 
the nephrologist of the patient’s medical practice or the attending physician in the 
nephrologic outpatient clinic of the University Hospital Tübingen. The study medication may 
be continued or adjusted at the discretion of the responsible nephrologist. The responsible 
nephrologist will therefore be informed of the patient’s medication during study participation. 
There are no special requirements regarding the termination of the medication taken during 
the study. If diuretic therapy with amiloride, furosemide or hydrochlorothiazide is continued, 
further controls of the electrolytes and renal function should be performed as they would be 
without any study participation.  


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8. Quality Control and Quality insurance 
8.1. 
Monitoring 
Monitoring for this study is provided by the Zentrum für Klinische Studien Tübingen (ZKS 
Tübingen). The monitoring will be conducted according to ZKS Tübingen’s internal SOPs and 
a dedicated monitoring manual for the study. The monitoring timelines include, for all centres, 
an initiation visit, regular monitor visits during the course of the trial as well a close out visit. 
Monitoring will end with the last visit after full documentation of the last patient enrolled (close 
out visit). All investigators agree that the monitors regularly visit the trial site, assure that the 
monitors will receive appropriate support in their activities and will have access to all trial-
related documents.  
The aims and scope of activities of the monitoring visits will be specified in a monitoring 
manual. Briefly, visits will include: 
• 
To check the informed consent documents  
• 
To monitor trial subject safety (occurrence and documentation/reporting of Adverse 
Events (AEs) and Serious AEs).  
• 
To check the completeness and accuracy of entries on the CRFs.  
• 
To validate the entries on the CRFs against those in the source documents (source 
data verification (SDV) (section 8.3.2).  
• 
To evaluate the progress of the trial. 
• 
To evaluate compliance with the trial protocol. 
• 
To assess whether the trial is being performed according to GCP at the trial site. 
• 
To discuss with the investigator aspects of trial conduct and any deficiencies found. 
• 
A monitoring visit report is prepared for each visit describing the progress of the 
clinical trial and any problems. 
 
8.2. 
Audits/ Inspections 
In addition to the monitoring activities, a comprehensive quality control will be conducted by 
CI in the form of audits. These may include checking the whole course of the study, the 
documentation, statistical analysis, the trial centre, the investigators, and the monitor. The 
competent regulatory authorities may also conduct audits or inspections.  
With his participation in the study, the investigator agrees to support the activities of the 
auditor/inspector, provide her/him with direct access to the source documents and give 
her/him the opportunity to inspect the laboratory facilities, storage of the investigational 
product, etc. 


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8.3. 
Documentation: Collection, Handling, Storage and Archiving of Data 
8.3.1. 
Case Report Form 
The trial case report form (CRF) is the primary data collection instrument for the trial. All data 
requested on the CRF must be recorded. All missing data must be explained.  
For this project, paper Case Report Forms (CRFs) will be used. The investigator is 
responsible for ensuring that all sections of the CRF are completed correctly and that entries 
can be verified against source data. Any errors should have a single line drawn through them 
so that the original entry remains legible and the correct data should be entered at the side 
with the investigator's signature, date and reason for change. Self-explanatory corrections do 
not need to be justified. 
The correctness of entries in CRFs will be confirmed by dated signature of the responsible 
investigator. 
8.3.2. 
Source Data 
Source data is all information, original records of clinical findings, observations or other 
activities in a clinical trial necessary for the reconstruction and evaluation of the trial. Source 
data are contained in source documents. Examples of these original documents and data 
records include: hospital records, clinical and office charts, laboratory notes, memoranda, 
patients’ diaries or evaluation checklists, pharmacy dispensing records, recorded data from 
automated instruments, copies or transcriptions certified after verification as being accurate 
and complete, microfiches, photographic negatives, microfilm or magnetic media, x-rays, 
CTs, MRIs, ultrasound reports, patient files, and records kept at the pharmacy, at the 
laboratories, and at medico-technical departments involved in the clinical trial. 
8.3.3. 
Data Handling 
After first check for plausibility by eye, all data will be entered in a database as recorded in 
the CRF. To ensure data quality a double data entry will be performed.  
The trial master file, the CRFs, and other material supplied for the conduct of the study will 
be retained by Sponsor/CRO according to applicable regulations and laws. 
8.3.4. 
Storage and Archiving of Data 
According to the §13 of the German GCP-Ordinance all important trial documents (e.g. CRF) 
will be archived for at least 10 years after the trial termination.  


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The investigator(s) will archive all trial data (source data and Investigator Site File (ISF) 
including subject identification list and relevant correspondence) according to the section 4.9 
of the ICH Consolidated Guideline on GCP (E6) and to local law or regulations. 


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9. Statistical Analyses 
9.1. 
Study Population Definition 
9.1.1. 
Sample Size and Power Consideration  
Primary endpoint of the study is decrease of overhydration (measured using bioimpedance 
spectroscopy) after 8 days, compared to baseline. Based on data from a baseline sample (n 
= 14) and clinical experience on reduction of overhydration baseline overhydration is 
expected to be 26.5 % of extracellular water (standard deviation 12.3; normal distribution is 
assumed) and it is expected that the overhydration will be reduced by 90% in the amiloride 
group and by 50% in the furosemide group. For the final t-test for two groups, a one-tailed 
significance level of 0.05 and a power of 80% should be ensured. From these conditions 
results a sample size of n = 18 patients per group (total n = 36; calculated for a t-test with the 
nQuery 7.0 program). In order to take into account dropouts, the sample size is defined as n 
= 22 per group (total n = 44). 
9.1.2. 
Intention-to-Treat Population 
All statistical analyses will be based on the Intention-to-Treat Population (ITT). The ITT 
includes all randomised patients with exception of patients who withdraw their informed 
consent for the analysis of their data during the study.  
 
9.2. 
Analysis of Primary Variable 
The primary endpoint variable is change of overhydration (OH) after 8 days compared to 
baseline, measured by bioimpedance spectroscopy using the Body Composition Monitor and 
expressed in percent of ectracellular water (% ECW). The primary endpoint variable will be 
compared between the two study arms, which are patients with amilorid treatment and 
patients with furosemide treatment.  
The null-hypothesis is that there is equal or greater decrease of OH after 8 days in the group 
of patients with furosemide treatment compared to the group of patients with amiloride 
treatment. The alternative hypothesis is that there is a greater decrease of OH after 8 days in 
the group of patients with amiloride treatment compared to the group of patients with 
furosemide treatment. For analysis of the primary endpoint variable a one-sided t-test for two 
groups will be performed to test the null hypothesis against the alternative hypothesis. Effect 
size and 95%-confidence interval for effect size will be estimated. Only the rejection of the 
null hypothesis will be interpreted as statistical evidence for the efficacy of the amiloride 
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9.3. 
Analysis of Secondary Variables 
Secondary endpoint variables are listed in section 2.2. All secondary endpoints will be 
compared and statistically assessed for descriptive purposes and not in a confirmatory 
sense. The aim of the analysis is explorative data analysis, not hypothesis testing or 
generation of evidence for efficacy and no attempt will be made to adjust the p-values of 
statistical tests of the secondary endpoints for multiple testing. If adequate, secondary 
endpoints will be compared and statistically assessed using t-tests for two groups or 
covariance techniques with baseline values as covariates in case of quantitative variables. 
Depending on distribution, non-parametric tests may be indicated. Dichtomous data will be 
compared and statistically assessed using Mantel-Haenszel chi-squared tests including 
relative risks and 95%-confidene intervals for relative risks. 
 
9.4. 
Safety Analysis 
Safety will be assessed by frequency tabulations and line listings. 
 
9.5. 
Descriptive Analysis 
Descriptive analyses will include absolute and percentage frequencies for categorical 
variables, means, medians, standard deviations, quartiles and ranges for quantitative 
variables and medians, quartiles and ranges for ordinal variables. 
 
9.6. 
Handling of Missing Data 
All variables included in the CRF are mandatory. The monitoring will assure quality of the 
assessments. Thus, missing values are to be expected only due to refusal by patients. In the 
analysis of the primary endpoint missing values will be imputed using multiple imputation 
approaches, complete case and last observation carried forward analyses will be performed 
as sensitivity analyses. All other missing data will be given in the descriptive tables but 
excluded from the statistical test. 
 
9.7. 
Subgroup Analysis 
No subgroup analysis is planned. 


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9.8. 
Biometric Report 
The statistical analysis will be conducted by the IKEAB, once the database is declared 
closed. In this study protocol the directions of the planned analyses are given. Before starting 
the final analysis, a detailed statistical analysis plan (SAP) will be written and signed by the 
responsible statistician and the coordinating investigator. If any major deviations in the SAP 
from the original study protocol are necessary, the reason for that will be given in detail. A 
protocol amendment will be adequate for major deviations (e.g. change of primary endpoint, 
sample size). The final SAP will be documented in the final biometrical report. The 
biometrical report will be written by the responsible statistician and should include the 
complete statistical analysis. 


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10. Safety 
10.1. Definition of Adverse Events and Side Effects 
10.1.1. 
Adverse Events 
Any untoward medical occurrence in a patient or clinical investigation subject administered a 
pharmaceutical product and which does not necessarily have a causal relationship with this 
treatment. An adverse event (AE) can therefore be any unfavorable and unintended sign 
(including an abnormal laboratory finding), symptom, or disease temporally associated with 
the use of a medicinal (investigational) product, whether or not related to the medicinal 
(investigational) product. 
An AE may be: 
• 
New symptoms/ medical conditions. 
• 
New diagnosis. 
• 
Changes of laboratory parameters. 
• 
Diseases and accidents. 
• 
Worsening of medical conditions/ diseases existing before clinical trial 
start. 
• 
Recurrence of disease. 
• 
Increase of frequency or intensity of episodical diseases. 
A pre-existing disease or symptom will not be considered an adverse event unless there will 
be an untoward change in its intensity, frequency or quality. This change will be documented 
by the investigator. 
Surgical procedures themselves are not AEs; they are therapeutic measures for conditions 
that require surgery. The condition for which the surgery is required may be an AE. Planned 
surgical measures permitted by the clinical trial protocol and the condition(s) leading to these 
measures are not AEs, if the condition leading to the measure was present prior to inclusion 
into the trial. 
AEs are classified as "non-serious" or "serious". 
10.1.2. 
Adverse Drug Reaction 
Adverse reaction means all untoward and unintended responses to an investigational 
medicinal product unrelated to the dose administered.  
10.1.3. 
Unexpected Adverse Drug Reaction 
An unexpected Adverse Drug Reaction (ADR) is a reaction which nature or severity is not 
consistent with the applicable product information available for the IMP. Expected ADRs are 


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listed in the appropriate reference documents, e.g. Investigator’s Brochure; Summary of 
Product Characteristics (SmPC [Fachinformation in Germany]). 
10.1.4. 
Serious Adverse Event and Serous Adverse Reaction 
AEs are classified as "non-serious" or "serious". 
A serious adverse event (SAE) is one that at any dose: 
• 
Results in death. 
• 
Is life-threatening (the term life-threatening refers to an event in which the 
subject was at risk of death at the time of event and not to an event which 
hypothetically might have cause death if it was more severe).  
• 
Requires subject hospitalization or prolongation of existing hospitalization. 
• 
Results in persistent or significant disability/ incapacity. 
• 
A congenital anomaly / birth defect.  
 
10.2. Period of Observation 
For the purpose of this trial, the period of observation for collection of adverse events 
extends from the time the patient starts study medication until 7 days after the last dose 
administrated. 
All adverse events that occur in the course of a clinical trial regardless of the causal 
relationship must be monitored and followed up until the outcome is known.  
 
10.3. Documentation and Reporting of Adverse Events 
10.3.1. 
Documentation and Reporting of Adverse Events by the Investigator 
The investigator must document all adverse events that occur during the observation period 
set in this protocol (see Section 10.2) on the pages provided in the case report form. 
Additional instructions may be provided in the investigator file and in the case report form 
itself. The following approach will be taken for documentation: 
All adverse events (whether serious or non-serious) must be documented on the “adverse 
event” page of the case report form. 
If the adverse event is serious (see Section 10.1.4), the investigator must complete, in 
addition to the “adverse event” page in the case report form, a “serious adverse event report 
form” at the time when the serious adverse event is detected. The investigator will document 
the date when he/she or any employee was first aware of the report and fax all SAE reports 
(initial and follow-up reports), even if they are incomplete, immediately (without culpable 


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delay), 
at 
the 
latest 
24 
hours 
after 
obtaining 
knowledge 
to 
the 
safety 
department/representative of the Sponsor: 
Zentrum für Klinische Studien Tuebingen (ZKS Tuebingen) 
Otfried-Mueller-Str. 45 
72076 Tuebingen, Germany 
Phone.: +49 7071 29-85635 
SAE-Fax: +49 7071 29-25205 
e-mail: zks-pv@med.uni-tuebingen.de  
 
The investigator should also assess the severity and the causal relationship between the 
event and the trial medication (section 10.3.2). 
10.3.2. 
Assessment of Severity and Causality 
The investigator will also provide an assessment of the severity of the event according to 
CTCAE criteria (Version 5.0) and causal relationship between the event and each of the 
investigational products or trial procedures. 
AEs and SAEs should be evaluated for severity according to the following scale: 
• 
Grade 1 - mild event: Causing no limitations of usual activities; the patient may 
experience slight discomfort. 
• 
Grade 2 - moderate event: Causing some limitation of usual activities; the patient 
may experience annoying discomfort. 
• 
Grade 3 - severe event: Causing inability to carry out usual activities; the patient 
may experience intolerable discomfort or pain. 
• 
Grade 4 - life threatening or disabling event 
• 
Grade 5 - death related to event 
 
The investigator must determine also the relationship between the administration of IMP and 
the occurrence of an AE/SAE as defined below: 
 
Related: There is a reasonable possibility that the SAE may be related to the IMP (e.g. 
favourable temporal relationship, positive dechallenge: symptoms are receding when IMP is 
withdrawn or the dose reduced, positive rechallenge: symptoms are reappearing when the 
IMP is reintroduced or the full dose is re-administered) 
 
Not Related: There is no reasonable possibility that the SAE is related to the IMP (e.g. there 
is a plausible alternative cause for the SAE that better explains the occurrence of the SAE) 


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10.3.3. 
Sponsors Assessment of the SAEs  
All SAE will be subject to a second assessment by the trial Sponsor or authorized second 
assessors.  
The second assessor will fill out a ‘Second Assessment Form’ for each SAE containing.  
• 
Event serious yes/no 
• 
Relationship between SAE and IMP 
• 
Expectedness of SAE according to the reference document: SmPCs 
• 
Benefit / risk assessment for the trial regarding change as a result of SAE. 
10.3.4. 
Follow-up of Initial Report 
Information not available at the time of the initial report (e.g. an end date for the adverse 
event or laboratory values received after the report) must be documented on a “Serious 
Adverse Event” form with the box “Follow-up” checked under “Report type”. 
All patients who have adverse events, whether considered associated with the use of the 
investigational products or not, must be monitored to determine the outcome. The clinical 
course of the adverse event will be followed up according to accepted standards of medical 
practice even after the end of the period of observation, until a satisfactory explanation is 
found or the investigator considers it medically justifiable to terminate follow-up. Should the 
adverse event result in death, a full pathologist’s report should be supplied, if possible. 
The sponsor will identify missing information for each SAE report and will require follow up 
information in regular intervals from the investigators until all queries are resolved or no 
further information can be reasonably expected. All responses to queries and supply of 
additional information by the investigator should follow the same reporting route and 
timelines as the initial report. 
10.3.5. 
Suspected Unexpected Serious Adverse Reaction (SUSAR) 
SAEs have to be assessed by the second assessor wether they are both suspected, i.e. 
possibly related to IMP and ‘unexpected’, i.e. the nature and / or severity of which is not 
consistent with the applicable product information. They are then to be classified as 
Suspected Unexpected Serious Adverse Reactions (SUSARs). 
In case, either the investigator or the second assessor classify the SAE as ‘suspected’ i.e. 
either as related or probable, or possible, or unlikely-related to IMP (section 9.3.2) and the 
SAE is unexpected as assessed by the Sponsor / CI it will be categorized as a SUSAR.  


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All SUSARs are subject to an expedited reporting (section 9.3.6) to the responsible ethics 
committee(s), the competent higher federal authority (i.e. either BfArM or PEI) and to all 
participating investigators. 
10.3.6. 
Expedited Reporting to the Regulatory Authorities  
Fatal and life-threatening SUSARs 
The competent authority and the ethics committee responsible must be informed by the 
Sponsor/CI of all fatal or life-threatening SUSARs. This must be done immediately, at the 
latest seven calendar days after becoming aware of the minimum criteria for reporting. In all 
cases, attempts must be made to obtain further relevant information, which must be supplied 
to the competent authority and the ethics committee within a further eight days. Furthermore, 
if a trial subject dies, this information must be passed on to the ethics committee responsible 
for the region in which the death occurred. 
SUSARs that are not fatal or life threatening 
The authority and the ethics committee responsible will be informed without delay by the 
sponsor or CI of all SUSARs, at the latest within 15 calendar days of becoming aware of the 
minimum criteria for reporting. Further relevant details will be passed on as soon as possible. 
If the information at the time of reporting is incomplete, further information to enable 
adequate assessment of the case will be requested from the reporter or other available 
sources. 
10.3.7. 
Examination and Report of Changes in the Risk to Benefit Ratio 
Without delay, and at the latest within 15 days of the decision for the need to do so, the 
Sponsor / CI will inform the competent authority, the ethics committee responsible of any 
events or factors that could result in a review of the risk-benefit ratio of the IMP. These 
consist of especially: 
• 
Individual reports of expected serious ADRs with an unexpected outcome. 
• 
A clinically relevant increase in the rate of occurrence of expected ADRs. 
• 
SUSARs in trial subjects who have already completed the follow-up period of the 
clinical trial (”end-of-trial visit”). 
• 
Factors emerging in connection with trial conduct or the development of the IMP 
that may affect the safety of persons concerned. 
10.3.8. 
Report to the Investigator 
The Sponsor / CI will inform investigators of all SUSARs including all relevant further 
information within the periods set by the authority. 


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If new information becomes known that is different from the scientific information given to the 
investigator, all investigators will be informed of this by the sponsor. 
10.4. Annual Safety Report 
Once a year, the Sponsor / CI will supply a report on the safety of trial subjects with all 
available relevant information concerning patient safety during the reference period to the 
competent authorities. This report will also be supplied to the responsible ethics committee. 
The annual safety report will be compiled according to the corresponding ICH guideline E2F 
„Development Safety Update Report – DSUR“ 
10.5. Deviations from the protocol 
Any deviation from the protocol will be noted in the CRF. The PI or a nominated person will 
evaluate this deviation from the protocol and will decide on the further course of the trial for 
the respective subject. 
10.6. Reporting of pregnancy 
Pregnancies and suspected pregnancies (including a positive pregnancy test regardless of 
age or disease state) of a female patient or the female partner of a male patient occurring 
while the patient is on study drug or within 28 days of the patient’s last dose of study drug are 
considered events to be reported immediately to the Sponsor. All study drugs have to be 
discontinued immediately and the patient instructed to return any unused portion of the study 
drug to the investigator(s). The pregnancy, suspected pregnancy, or positive pregnancy test 
must be immediately reported to Sponsor after investigator’s knowledge using the 
“Pregnancy Reporting Form”.  
The female should be referred to an obstetrician. 
The investigator(s) will follow the female patient until completion of the pregnancy, and must 
notify the Sponsor of the outcome of the pregnancy (including notification of false-positive 
tests) within 24 hours of having knowledge of the event as a follow-up to the initial report. 
If the outcome of the pregnancy meets the SAE-criteria , the investigator(s) should follow the 
procedures for reporting SAEs (i.e., report the event within 24 hours of the investigator’s 
knowledge of the event). 
All neonatal deaths that occur within 28 days of birth should be reported, without regard to 
causality, as SAE.  
If the female is found not to be pregnant, any determination regarding the patient’s continued 
participation in the study will be determined by the investigator(s).  


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In case of study discontinuation due to pregnancy, protocol-required procedures for study 
discontinuation and follow-up must be performed on the patient unless contraindicated by 
pregnancy (e.g., x-ray studies). Other appropriate pregnancy follow-up procedures should be 
considered if indicated. 
 
Information on pregnancy must be collected on the “Pregnancy Reporting Form”. In order for 
Sponsor or designee to collect any pregnancy surveillance information from the female 
partner, the female partner must sign an informed consent form for disclosure of this 
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11. Regulatory Consideration 
11.1. Ethical Conduct of Clinical Study 
11.1.1. 
Good Clinical Practice, Declaration of Helsinki and legal Provision 
The procedures set out in this trial protocol, pertaining to the conduct, evaluation, and 
documentation of this trial, are designed to ensure that all persons involved in the trial act 
according to Good Clinical Practice (GCP) and the ethical principles described in the 
applicable version of the Declaration of Helsinki.  
This is a scientific clinical study; the German Medicines Act (AMG) §40 is applicable without 
restrictions according to section §42.  
 
11.2. Subject Information and Informed Consent 
Each patient will be informed about the modalities of the clinical study in accordance with the 
provided patient informed consent (IC). The patient is to be informed both in writing and 
verbally by the investigator before any study-specific procedure is performed. The patient 
must be given sufficient time (i.e. >24 h) to decide whether to participate in this comparative 
study and to ask questions concerning this trial. It must also be made clear to the patient that 
he / she can withdraw from the study at any time without giving reasons and that he / she will 
not be in any way disadvantaged for this. The subject must give consent in writing. The 
patient and informing physician must each personally date and sign the informed consent 
form with an integrated declaration on data privacy protection, whereby the physician must 
not sign before the patient. Original signed documents will be part of the investigator’s file 
and retained with it. A copy of the signed informed consent document and study insurance 
policy must be given to the subject. The documents must be in a language understandable to 
the subject and must specify who informed the subject. The subjects will be informed as 
soon as possible if new information may influence his/her decision to participate in the trial. 
The communication of this information should be documented in the patient chart. 
 
11.3. Insurance 
Each patient is insured against any health impairment occurring as a result of participation in 
the study in accordance with the laws and regulations of the Deutsches Arzneimittelgesetz. 
The insurance is covered by HDI-Gerling Industrie Versicherung AG, Am Schönenkamp 45, 
40599 Düsseldorf, phone +49 211 177 69 0, fax +49 211 352 345; the Policy number is 
5701031101013 and the insurance is valid throughout the conduct of the study including 16 


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days follow-up for each individual patient. A copy of the insurance policy and conditions are 
distributed to the patient upon enrolment into the study and the patient is advised to adhere 
to the conditions of the insurance policy to safeguard a valid patient insurance. 
 
11.4. Confidentiality 
The data obtained in the course of the trial will be treated according to the European General 
Data Protection Regulation (Datenschutz-Grundverordnung; DSGVO) and the applicable 
local data protection regulations as well as the AMG. The details on confidentiality according 
to EU-DSGVO and AMG are provided in the patient information using the text templates of 
the Medical Ethics Commissions in the Federal Republic of Germany (see appendix 15.1). 
 
11.5. Responsibility of the the Investigator 
The investigator should ensure that all persons assisting with the trial are adequately 
informed about the protocol, any amendments to the protocol, the trial treatments, and their 
trial-related duties and functions. 
The investigator should maintain a list of subinvestigators and other appropriately qualified 
persons to whom he or she has delegated significant trial-related duties. 
 
11.6. Registration of the Trial 
Prior to the beginning of the clinical phase (First Patient In) the Sponsor / CI will register the 
trial in http://www.clinicaltrials.gov. The trial has be given a unique EudraCT number. 
 
11.7. Continuous Information to Independent Ethic Committee 
According to the German Drug Law (AMG) and the GCP Ordinance, the EC and the 
competent authority will be informed of all suspected serious unexpected adverse reactions 
(SUSARs). Both institutions will be informed in case the risk/ benefit assessment did change 
or any others new and significant hazards for subjects’ safety or welfare did occur. 
Furthermore, a report on all observed SAEs will be submitted once a year – Annual Safety 
Report. 
The EC and the regulatory authorities must be informed of the end of the trial. They will be 
provided with a summary of trial results within one year after the end of clinical phase. 
 


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11.8. Approval of Protocol and Subsequent Amendments 
Before the start of the trial, the trial protocol, informed consent document, and any other 
appropriate documents will be submitted to the independent Ethics Committee (EC) as well 
as to the competent authority (BfArM or PEI). A written favourable vote of the EC and an 
(implicit) approval by the competent higher federal authority are a prerequisite for initiation of 
this clinical trial. Before the first subject is enrolled in the trial, all ethical and legal 
requirements must be met. All planned substantial changes (see §10, (1) of German GCP-
Regulation) will be submitted for approval to EC and the competent authority in writing as 
protocol amendments.  
 


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12. Publications 
12.1. Reports 
Within one year of the completion of the trial, the competent authority and the ethics 
committee will be supplied with a summary of the final report on the clinical trial containing 
the principle results. 
All reports to the sponsor will be written in English language. All clinical, analytical and 
statistical results will be presented in a final clinical trial report (CTR). The outline of this 
report will accord to the ICH Topic E3. 
Section 10.4 describes the requirements for Annual Safety Reports on the safety of trial 
subjects. 
 
12.2. Publication 
The final results of this study will be presented at scientific meetings and published in a peer 
reviewed leading medical journal of general medicine or a leading nephrological journal. All 
publications in result of this study are the responsibility of the principal coordinating 
investigator and the authorship will reflect the contributions of each collaborating centre. Any 
publication, abstract or presentation based on patients included in this study must be 
approved by the coordinating investigator.  
 
 


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13. Financing 
The study is financed with intramural funding by the AKF (“Angewandte Klinische 
Forschung”) program of the University Tuebingen.  
Table 3 lists the financial resources requested for funding by the intramural program. There 
are no further financial resources required to complete the study. 
Table 3: Budget draft 
 
Details of requested funds 
Amount of requested 
funds 
Staff resources 
Clinical Investigator: 10% / TV-Ä 
(88.200€/Year) 
Assistant scientist (16,79 €/h x 85h 
/Monat) 
17.640,00 € 
 
17.126,00 € 
Material resources 
Submission Ethics Committee /  Federal 
authority (ZKS/PI) 
Monitoring (ZKS) 
Pharmakovigilance (ZKS) 
Fees first submission 
Fees Amendment 
Fees DSUR 
Biometrics, data management (IKEAB) 
Subjects insurance 
Travel reimbursement 
Material costs (Elektrodes for BCM 
measurement, laboratory material) 
Study medication + Labelling 
Presentation of study results by 
participation in congresses 
Publication costs 
maximum. 9.330,00 € 
 
13.150,00 € 
22.740,00 € 
8.500,00 € 
1.250,00 € 
2.000,00 € 
13.900,00 € 
3.000,00 € 
5.400,00 € 
8.400,00 € 
 
1.150,00 € 
5.000,00 € 
 
2.500,00 € 
Total amount 
 
130.436,00 € 
 


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14. Literature 
 
1. 
Radhakrishnan J. Pathophysiology and treatment of edema in patients with the 
nephrotic syndrome UpToDate2017 [updated May 19, 201707.08.2018]. 
2. 
Bockenhauer D. Over- or underfill: not all nephrotic states are created equal. Pediatric 
nephrology (Berlin, Germany). 2013;28(8):1153-6. Epub 2013/03/27. doi: 10.1007/s00467-
013-2435-6. PubMed PMID: 23529637. 
3. 
Artunc F, Worn M, Schork A, Bohnert BN. Proteasuria - the impact of active urinary 
proteases on sodium retention in nephrotic syndrome. Acta physiologica (Oxford, England). 
2019:e13249. Epub 2019/01/01. doi: 10.1111/apha.13249. PubMed PMID: 30597733. 
4. 
Dorhout EJ, Roos JC, Boer P, Yoe OH, Simatupang TA. Observations on edema 
formation in the nephrotic syndrome in adults with minimal lesions. The American journal of 
medicine. 1979;67(3):378-84. Epub 1979/09/01. PubMed PMID: 474584. 
5. 
Passero CJ, Hughey RP, Kleyman TR. New role for plasmin in sodium homeostasis. 
Curr Opin Nephrol Hypertens. 2010;19(1):13-9. Epub 2009/10/30. doi: 
10.1097/MNH.0b013e3283330fb2. PubMed PMID: 19864949; PubMed Central PMCID: 
PMCPMC2894526. 
6. 
Passero CJ, Mueller GM, Rondon-Berrios H, Tofovic SP, Hughey RP, Kleyman TR. 
Plasmin activates epithelial Na+ channels by cleaving the gamma subunit. J Biol Chem. 
2008;283(52):36586-91. Epub 2008/11/05. doi: 10.1074/jbc.M805676200. PubMed PMID: 
18981180; PubMed Central PMCID: PMCPMC2605981. 
7. 
Svenningsen P, Bistrup C, Friis UG, Bertog M, Haerteis S, Krueger B, et al. Plasmin 
in nephrotic urine activates the epithelial sodium channel. Journal of the American Society of 
Nephrology : JASN. 2009;20(2):299-310. Epub 2008/12/17. doi: 10.1681/asn.2008040364. 
PubMed PMID: 19073825; PubMed Central PMCID: PMCPMC2637049. 
8. 
Bohnert BN, Menacher M, Janessa A, Worn M, Schork A, Daiminger S, et al. 
Aprotinin prevents proteolytic epithelial sodium channel (ENaC) activation and volume 
retention in nephrotic syndrome. Kidney Int. 2018;93(1):159-72. Epub 2017/10/19. doi: 
10.1016/j.kint.2017.07.023. PubMed PMID: 29042083. 
9. 
Wörn M, Boldt K, Ueffing M, Heyne N, Artunc F. Identification of serine proteases in 
urine from patients with nephrotic syndrome using a proteomic approach. 10th Annual 
Meeting of the German Society of Nephrology. 2018;Abstract number FV 17. 
10. 
Bohnert BN, Artunc F. Induction of Nephrotic Syndrome in Mice by Retrobulbar 
Injection of Doxorubicin and Prevention of Volume Retention by Sustained Release 
Aprotinin. Journal of visualized experiments : JoVE. 2018;(135). Epub 2018/05/22. doi: 
10.3791/57642. PubMed PMID: 29782000. 
11. 
Bohnert BN, Daiminger S, Worn M, Sure F, Staudner T, Ilyaskin AV, et al. Urokinase-
type plasminogen activator (uPA) is not essential for epithelial sodium channel (ENaC)-
mediated sodium retention in experimental nephrotic syndrome. Acta physiologica (Oxford, 
England). 2019:e13286. Epub 2019/04/22. doi: 10.1111/apha.13286. PubMed PMID: 
31006168. 
12. 
Haerteis S, Schork A, Dorffel T, Bohnert BN, Nacken R, Worn M, et al. Plasma 
kallikrein activates the epithelial sodium channel in vitro but is not essential for volume 
retention in nephrotic mice. Acta physiologica (Oxford, England). 2018:e13060. Epub 
2018/03/01. doi: 10.1111/apha.13060. PubMed PMID: 29489063. 
13. 
Schork A, Woern M, Kalbacher H, Voelter W, Nacken R, Bertog M, et al. Association 
of Plasminuria with Overhydration in Patients with CKD. Clin J Am Soc Nephrol. 
2016;11(5):761-9. Epub 2016/03/05. doi: 10.2215/cjn.12261115. PubMed PMID: 26933188; 
PubMed Central PMCID: PMCPMC4858495. 


Protocol code: AmiloridNS-01 
Amiloride in nephrotic syndrome 
(AMILOR) 
Study Protocol Version 2.1 
 
 
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01.03.2021 
 
14. 
Elwaleed A. Elhassan RWS. Disorders of Extracellular Volume. In: Richard J Johnson 
JF, Jürgen Floege, editor. Comprehensive clinical nephrology. 5: Elsevier Saunders; 2015. p. 
80-94. 
15. 
Danielsen H, Pedersen EB, Madsen M, Jensen T. Abnormal renal sodium excretion 
in the nephrotic syndrome after furosemide: relation to glomerular filtration rate. Acta medica 
Scandinavica. 1985;217(5):513-8. Epub 1985/01/01. PubMed PMID: 4025007. 
16. 
Hoorn EJ, Ellison DH. Diuretic Resistance. American journal of kidney diseases : the 
official journal of the National Kidney Foundation. 2017;69(1):136-42. Epub 2016/11/07. doi: 
10.1053/j.ajkd.2016.08.027. PubMed PMID: 27814935; PubMed Central PMCID: 
PMCPMC5182087. 
17. 
Andersen H, Hansen PB, Bistrup C, Nielsen F, Henriksen JE, Jensen BL. Significant 
natriuretic and antihypertensive action of the epithelial sodium channel blocker amiloride in 
diabetic patients with and without nephropathy. Journal of hypertension. 2016;34(8):1621-9. 
Epub 2016/05/24. doi: 10.1097/hjh.0000000000000967. PubMed PMID: 27214087. 
18. 
Oxlund CS, Buhl KB, Jacobsen IA, Hansen MR, Gram J, Henriksen JE, et al. 
Amiloride lowers blood pressure and attenuates urine plasminogen activation in patients with 
treatment-resistant hypertension. Journal of the American Society of Hypertension : JASH. 
2014;8(12):872-81. Epub 2014/12/11. doi: 10.1016/j.jash.2014.09.019. PubMed PMID: 
25492830. 
19. 
Unruh ML, Pankratz VS, Demko JE, Ray EC, Hughey RP, Kleyman TR. Trial of 
Amiloride in Type 2 Diabetes with Proteinuria. Kidney Int Rep. 2017;2(5):893-904. Epub 
2017/09/12. doi: 10.1016/j.ekir.2017.05.008. PubMed PMID: 28890943; PubMed Central 
PMCID: PMCPMC5584552. 
20. 
Hinrichs GR, Mortensen LA, Jensen BL, Bistrup C. Amiloride resolves resistant 
edema and hypertension in a patient with nephrotic syndrome; a case report. Physiological 
Reports. 2018;6(12):e13743. doi: 10.14814/phy2.13743. PubMed PMID: PMC6016639. 
21. 
Campanacci D, Bonomini V, Zucchelli P. [The use of triamterene in nephrotic 
syndromes]. Minerva medica. 1963;54(81 Suppl):3035-9. Epub 1963/10/10. PubMed PMID: 
5877337. 
22. 
Cavazzuti GB. [Results of treatment with triamterene in nephrosic edemas]. Minerva 
nefrologica. 1965;12(3):183-8. Epub 1965/07/01. PubMed PMID: 5843347. 
23. 
Deschenes G, Wittner M, Stefano A, Jounier S, Doucet A. Collecting duct is a site of 
sodium retention in PAN nephrosis: a rationale for amiloride therapy. Journal of the American 
Society of Nephrology : JASN. 2001;12(3):598-601. Epub 2001/02/22. PubMed PMID: 
11181809. 
24. 
Gupta S, Pepper RJ, Ashman N, Walsh SB. Nephrotic Syndrome: Oedema Formation 
and Its Treatment With Diuretics. Frontiers in physiology. 2019;9:1868. Epub 2019/01/31. 
doi: 10.3389/fphys.2018.01868. PubMed PMID: 30697163; PubMed Central PMCID: 
PMCPMC6341062. 
25. 
Moissl UM, Wabel P, Chamney PW, Bosaeus I, Levin NW, Bosy-Westphal A, et al. 
Body fluid volume determination via body composition spectroscopy in health and disease. 
Physiological measurement. 2006;27(9):921-33. Epub 2006/07/27. doi: 10.1088/0967-
3334/27/9/012. PubMed PMID: 16868355. 
26. 
Keller E, Hoppe-Seyler G, Schollmeyer P. Disposition and diuretic effect of 
furosemide in the nephrotic syndrome. Clin Pharmacol Ther. 1982;32(4):442-9. Epub 
1982/10/01. doi: 10.1038/clpt.1982.187. PubMed PMID: 7116760. 
27. 
Smith DE, Hyneck ML, Berardi RR, Port FK. Urinary protein binding, kinetics, and 
dynamics of furosemide in nephrotic patients. J Pharm Sci. 1985;74(6):603-7. Epub 
1985/06/01. doi: 10.1002/jps.2600740604. PubMed PMID: 4020645. 
 


Protocol code: AmiloridNS-01 
Amiloride in nephrotic syndrome 
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Study Protocol Version 2.1 
 
 
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15. Appendices  
15.1. Patient Information and Informed Consent 
15.2. Instruction sheet for participants 
15.3. Label of the Investigational Medical Product Amiloride 
15.4. CE certificate of the Body Composition Monitor 

SAP_001.pdf: 
17.11.2023


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Confidentiality statement 
The contents of this document are the property of the applicants at the Eberhard-Karls 
University of Tübingen Germany. The contents may not be used or disclosed in any way 
without the express permission of the applicants.  
 
 
Note 
This SAP was performed according to the SOP BI03_V02 (valid since 11.02.2022) of the ZKS 
Tübingen. The analysis tables and listings will be independently validated by a second 
statistician according the SOP BI06 of the ZKS Tübingen. 
 


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Table of Contents 
 
Abbreviations ........................................................................................................................ 4 
1 
Introduction ................................................................................................................ 5 
1.1 
Preface ...................................................................................................................... 5 
1.2 
Purpose of the Study.................................................................................................. 5 
2 
Study Objectives ........................................................................................................ 6 
2.1 
Primary Objectives and Endpoint ............................................................................... 6 
2.2 
Secondary Objectives and Endpoints ......................................................................... 6 
3 
Study Methods ........................................................................................................... 7 
3.1 
General Study Design and Plan ................................................................................. 7 
3.2 
Study Treatment ........................................................................................................ 7 
3.3 
General Study Population and Inclusion/Exclusion Criteria ........................................ 8 
3.3.1 Inclusion Criteria ........................................................................................................ 8 
3.3.2 Exclusion Criteria ....................................................................................................... 9 
3.4 
Randomisation ..........................................................................................................10 
3.5 
Blinding .....................................................................................................................10 
3.6 
Study Procedures .....................................................................................................10 
4 
Sample Size ..............................................................................................................11 
5 
General Considerations ............................................................................................11 
5.1 
Timing of Analyses ....................................................................................................11 
5.2 
Analysis Population ...................................................................................................11 
5.2.1 Intention-to-treat Population (ITT) .............................................................................11 
5.2.2 Safety Population (Safety) ........................................................................................12 
6 
Data and Data Management .....................................................................................12 
6.1 
Case Report Form ....................................................................................................12 
6.2 
Data Handling ...........................................................................................................12 
6.3 
Missing Data .............................................................................................................12 
6.4 
Derived Variables ......................................................................................................12 
7 
Statistical Analyses ...................................................................................................13 
7.1 
Study Information ......................................................................................................13 
7.2 
Protocol Deviations ...................................................................................................13 
7.2.1 Major Protocol Deviations .........................................................................................13 
7.2.2 Minor Protocol Deviations .........................................................................................13 
7.3 
Descriptive Statistics .................................................................................................13 
7.4 
Efficacy Analyses ......................................................................................................14 
7.4.1 Primary Endpoint ......................................................................................................14 
7.4.2 Secondary Endpoints ................................................................................................14 
7.5 
Safety Analysis .........................................................................................................14 
8 
Reporting Conventions ..............................................................................................14 
9 
Technical Details .......................................................................................................15 
11  
Time Table ................................................................................................................15 
12 
Planned Tables, Figures and Listings .......................................................................15 


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Abbreviations 
 
AE 
 
Adverse Event 
BCM  
Body Composition Monitor, Fresenius Medical Care AG & Co 
CRF 
 
Case Report Form 
ECW  
Extracellular water 
ENaC  
Epithelial sodium channel 
FCBP  
Females of childbearing potential 
FSH 
 
Follicle stimulating hormone 
GFR 
 
Glomerular filtration rate 
HbA1c  
Haemoglobin A1c 
HCT 
 
Hydrochlorothiazide 
IKEAB 
Institut für Klinische Epidemiologie und angewandte Biometrie Tübingen 
KDIGO 
Kidney Disease – Improving Global Outcomes 
 
NKCC2 
Sodium-potassium-chloride cotransporter 
NYHA 
New York Heart Association 
OH 
Overhydration 
RAAS 
Renin-angiotensin-aldosterone system 
SAP 
Statistical Analysis Plan 
SAE 
Serious Adverse Event 
SOP 
Standard Operating Procedure 
TMF 
Trial Master File 
WOCBP 
Women of childbearing potential 
ZKS   
Zentrum für Klinische Studien Tübingen  
 
 


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1 
Introduction 
This SAP is based on the study protocol Version 1.1 as of 10. January 2020 with amendment 
1 as of 01. March 2021 (EudraCT-No: 2019-002607-18). Text citation directly form the study 
protocol is given in Italic letters. 
 
1.1 
Preface 
The nephrotic syndrome is characterized by a high ("nephrotic") proteinuria> 3.5 g / day with 
resulting hypoalbuminemia, as well as by oedema and hyperlipidaemia. Affected patients 
suffer from massive generalized oedema involving the face and eyelids, effusions into the 
body cavities pleura, peritoneum and rarely pericardium as well as increased body weight. 
In addition to the disease specific treatment commonly involving immunosuppression, 
supportive therapy of patients with acute nephrotic syndrome includes reduction of proteinuria 
by the renin-angiotensin-aldosterone-system (RAAS) blockade, blood pressure control, lipid 
lowering, thromboembolic prophylaxis and the correction of volume overload, which is 
achieved by a dietary salt restriction and diuretic therapy. 
Currently, loop diuretics are preferred as they have the highest diuretic potency. However, the 
diuretic effect of loop diuretics is lower in nephrotic syndrome than in other forms of oedema 
or in healthy volunteers. A possible cause of this resistance to loop diuretics could be the 
activation of the epithelial sodium channel (ENaC) that is located distally to the sodium-
potassium-chloride cotransporter (NKCC2), the target of loop diuretics. Sodium retention via 
ENaC may therefore counteract the inhibition of NKCC2.  
To overcome refractory oedema, either higher doses of loop diuretics or combination with 
thiazide diuretics (e.g., hydrochlorothiazide, HCT) for sequential nephron blockade or with 
distally acting diuretics such as the ENaC inhibitors amiloride and triamterene blockers are 
conceivable. 
Additionally, our investigations in patients with protein uric kidney disease and nephrotic 
syndrome show that activation of ENaC by serine proteases also appears to be of central 
importance as a mechanism of oedema formation in humans 
 
1.2 
Purpose of the Study  
Treatment of resistant oedema and overhydration in nephrotic syndrome can be challenging 
and we successfully use ENaC inhibitors (in combination with hydrochlorothiazide) in these 
patients. However, most clinicians treat nephrotic oedema with higher doses of loop diuretics, 
or combination therapy with thiazide diuretics. Worldwide, ENaC inhibitors are rarely used in 
nephrotic syndrome. This is due to lack of evidence of the role of activated ENaC and the 
effectiveness of ENaC inhibitors in human nephrotic syndrome. In this clinical trial, we aim to 
investigate the therapeutic effect of amiloride on the reduction of oedema and overhydration 
in the nephrotic syndrome. 
 
 
 


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2 
Study Objectives  
In the study, patients with nephrotic syndrome will be randomized to medication with the 
investigational drug amiloride (study arm 1) or the comparator furosemide (study arm 2) for a 
study period of 16 days. In case of treatment resistant oedema, hydrochlorothiazide (HCT) is 
added at day 8 in both study arms. To prevent bias, patients included in the study are 
randomized to start treatment with amiloride or furosemide. 
 
2.1 
Primary Objectives and Endpoint 
In the light of our current results on ENaC activation by serine proteases aberrantly filtered into 
urine in nephrotic syndrome, we hypothesize that targeting ENaC with amiloride will lead to a 
more effective reduction of overhydration in nephrotic syndrome than standard treatment with 
the loop diuretic furosemide.  
Primary objective of the study is therefore to prove the efficacy and superiority of amiloride for 
reduction of oedema and overhydration (OH) in human nephrotic syndrome in comparison to 
standard medication with furosemide. Primary endpoint of the study is the decrease of 
overhydration after 8 days, compared to baseline. OH is measured using bioimpedance 
spectroscopy with the device Body Composition Monitor, Fresenius Medical Care AG & Co; 
BCM) and expressed as percent of extracellular water (% ECW). 
 
2.2 
Secondary Objectives and Endpoints 
Secondary endpoints represent further parameters to evaluate course of overhydration and 
regulation of body volume status after initiation of study medication with amiloride or 
furosemide. Secondary endpoints include  
1. decrease of OH after 16 days, measured using bioimpedance spectroscopy and 
expressed as % ECW 
2. decrease of body weight after 8 and 16 days 
3. decrease of oedema circumference after 8 and 16 days, measured as the largest extent 
on the lower leg 
4. decrease of systolic and diastolic blood pressure after 8 and 16 days 
5. increase of urine volume and natriuresis after 8 and 16 days, determined from urine 
collection over 24 hours  
6. course of plasma renin activity and serum aldosterone concentration after 8 and 16 
days 
7. number of required changes of dose of study medication 
8. need for co-medication with HCT after 8 days 
9. occurrence of adverse events 
 
 
 
 


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3 
Study Methods 
3.1 
General Study Design and Plan 
The study design is a phase IIIb, monocentre, interventional, two-arm, randomized, open-label 
controlled clinical trial. We decided for a study type with randomization as method against bias 
and have omitted blinding, as laboratory results required for safety reasons and dose 
adjustments will inevitably reveal to which study group patients have been randomized. 
Overall study design is shown in figure 1.  
 
 
 
Figure 1 
Overall study design (according to study protocol) 
 
3.2 
Study Treatment 
Participants are randomized to start medication with amiloride 5 mg (trial arm 1) or furosemide 
40 mg (trial arm 2) at day 0. Dose adjustments depending on course of overhydration, sodium 
and potassium level in blood gas analysis and plasma creatinine are performed on day 2, 5, 8 
and 12. In case of therapy-refractory overhydration, co-medication with HCT 12,5 mg is started 
at day 8.  
 
 
 


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3.3 
General Study Population and Inclusion/Exclusion Criteria 
This study will include patients with acute nephrotic syndrome (definition see below) due to 
different underlying diseases (e.g. diabetic nephropathy, membranous nephropathy, minimal 
change disease, focal segmental glomerulosclerosis). The patients will be recruited from the 
nephrological outpatient department of the University Hospital Tuebingen or after referral from 
local nephrological practices. 
Adult male and female patients with acute nephrotic syndrome and fulfilling the below outlined 
inclusion criteria will be enrolled into the study.  
Trial population will consist of both genders. Gender distribution in the trial is supposed to 
reflect the distribution in the real patient’s population, i.e. there will be no prior defined 
quantitative ratio between females and males. No statistical analysis based on genders or 
differences between genders will be performed. 
 
3.3.1 Inclusion Criteria 
According to the study protocol subjects meeting all of the following criteria will be considered 
for admission to the trial: 
1. Acute nephrotic syndrome with proteinuria > 3 g/day and formation of oedema. 
2. Age ≥ 18 years at the time of signing the informed consent. 
3. Understand and voluntarily sign an informed consent document prior to any study 
related assessments/procedures. 
4. Ability to adhere to the study visit schedule and other protocol requirements.  
5. Use of adequate thrombosis prophylaxis due to the increased risk of thrombosis in 
nephrotic syndrome and the expected fluctuations in volume balance during study 
participation 
6. Subject (male or female) is willing to use highly effective methods of contraception 
according 
to 
the 
“Clinical 
trial 
fertility 
group” 
recommendations 
(http://www.hma.eu/fileadmin/dateien/Human_Medicines/01About_HMA/Working_Gr
oups/CTFG/2014_09_HMA_CTFG_Contraception.pdf) during treatment and for 28 
days (male or female) after the end of treatment (adequate: intrauterine device, 
intrauterine hormone-releasing system, bilateral tubal occlusion, vasectomized 
partner1, sexual abstinence2; due to the increased risk of thrombosis in nephrotic 
syndrome, no hormonal contraceptives are recommended). 
7. Female Patients of childbearing potential (WOCBP)3 must agree to pregnancy testing 
before inclusion in the study. 
8. Female Patients must agree to abstain from breastfeeding during study participation 
and 28 days after study drug discontinuation. 
9. All subjects must agree not to share medication. 
 
1 Vasectomized partner is a highly effective birth control method provided that partner is the sole sexual 
partner of the WOCBP trial participant and that the vasectomized partner has received medical 
assessment of the surgical success 


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2 In the context of this guidance sexual abstinence is considered a highly effective method only if defined 
as refraining from heterosexual intercourse during the entire period of risk associated with the study 
treatments. The reliability of sexual abstinence needs to be evaluated in relation to the duration of the 
clinical trial and the preferred and usual lifestyle of the subject  
3 For the purpose of this document, a woman is considered of childbearing potential (WOCBP), i.e. 
fertile, following menarche and until becoming post-menopausal unless permanently sterile. Permanent 
sterilisation methods include hysterectomy, bilateral salpingectomy and bilateral oophorectomy. A 
postmenopausal state is defined as no menses for 12 months without an alternative medical cause. A 
high follicle stimulating hormone (FSH) level in the postmenopausal range may be used to confirm a 
post-menopausal state in women not using hormonal contraception or hormonal replacement therapy. 
However, in the absence of 12 months of amenorrhea, a single FSH measurement is insufficient. 
 
3.3.2 Exclusion Criteria 
Subjects presenting with any of the following criteria will not be included in the trial: 
1. Severe reduction of kidney function: Creatinine clearance or calculated GFR < 30 
mL/min/1.73m² or acute kidney injury KDIGO stage 2 or 3 or anuria. 
2. Hypovolemia or dehydration. 
3. Uncontrolled diabetes mellitus. 
4. Hypotension, systolic blood pressure < 90 mm Hg. 
5. Hyperkalaemia, plasma potassium concentration > 4.8 mmol/l. 
6. Hypokalaemia, plasma potassium concentration < 3.3 mmol/l. 
7. Hyponatremia, plasma sodium concentration < 128 mmol/l. 
8. Hypercalcemia, ionized calcium > 2.0 mmol/l or total albumin corrected calcium > 3.0 
mmol/. 
9. Signs of cardiac decompensation (orthopnoea, dyspnoea NYHA IV). 
10. Hepatic coma or precoma. 
11. Symptoms of gout. 
12. Current therapy with potassium-sparing diuretics (e.g. spironolactone) or potassium 
supplements. 
13. Women during pregnancy and lactation. 
14. History of hypersensitivity to the investigational medicinal product, comparator or co-
medication or to any drug with similar chemical structure or to any excipient present in 
the pharmaceutical form of the investigational medicinal product, comparator or co-
medication. 
15. Any other clinical condition that would jeopardize the patient’s safety while participating 
in this clinical trial. 
16. Active participation in other clinical trials or observation period of competing trials. 
 
 


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3.4 
Randomisation 
The randomization will follow a randomization scheme of a 1:1 (amiloride and furosemide). 
Subjects will be assigned to their respective treatment by randomization. A randomisation 
number will be assigned (= Random. number). Subjects will then receive their study medication 
during their personal visit at day 0 of study participation. 
 
3.5 
Blinding 
The principal investigator  and the study team have opted against blinding, as laboratory results 
required for safety reasons and for definition of dose adjustments will inevitably reveal to which 
study group each patient was randomized (hypokalaemia – furosemide, hyperkalaemia – 
amiloride). 
 
3.6 
Study Procedures  
This Study consists of the following consecutive phases:  
 
Study entry 
 
Treatment  
 
Follow-up. 
Time-points and trial procedures are listed in Table 1. 
 
Table 1 
Table of Events 
 
Study 
entry 
Treatment 
Follow-
up 
Visit number 
1 
2 
3 
4 
5 
6 
7 
8 
Day 
-3 to -1 
0 
2 
5 
8 
12 
16 
23 
Study Entry 
Informed Consent 
X 
 
 
 
 
 
 
 
Demographics, including sex, body weight, 
high, age 
X 
 
 
 
 
 
 
 
Inclusion/Exclusion criteria: anamnesis, 
clinical examination, blood pressure, pre-
existing medication, proteinuria in 24 hours 
collected urine, general laboratory control 
(including blood count, creatinine, urea, 
liver enzymes, lactate dehydrogenase, c-
reactive protein, electrolytes (potassium, 
sodium, calcium), glucose, HbA1c, 
pregnancy test in females) 
X 
 
 
 
 
 
 
 
Safety Assessments 
Adverse events 
 
X 
X 
X 
X 
X 
X 
X 
Concomitant medication 
X 
X 
X 
X 
X 
X 
X 
X 
Check of the study medication 
 
X 
X 
X 
X 
X 
X 
 
Return of any remaining study medication 
 
 
 
 
 
 
X 
 
Plasma creatinine 
X 
X 
X 
X 
X 
X 
X 
X 
Potassium and sodium level in blood gas 
analysis 
X 
X 
X 
X 
X 
X 
X 
X 
 


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Continued Table 1 
Table of Events 
 
Study 
entry 
Treatment 
Follow-
up 
Visit number 
1 
2 
3 
4 
5 
6 
7 
8 
Day 
-3 to -1 
0 
2 
5 
8 
12 
16 
23 
Efficacy Assessment 
Body weight 
X 
X 
X 
X 
X 
X 
X 
X 
Bioimpedance spectroscopy with BCM 
X 
X 
X 
X 
X 
X 
X 
X 
Oedema circumference 
X 
X 
X 
X 
X 
X 
X 
X 
Blood pressure 
X 
X 
X 
X 
X 
X 
X 
X 
Plasma renin activity, serum aldosterone 
concentration 
X 
X 
X 
X 
X 
X 
X 
X 
24 hours urine volume and natriuresis 
X 
 
X 
X 
X 
X 
X 
 
Diet diary during urine collection 
X 
 
X 
X 
X 
X 
X 
 
Check for dose adjustment 
 
X 
X 
X 
X 
X 
 
 
 
 
4 
Sample Size 
Primary endpoint of the study is decrease of overhydration (measured using bioimpedance 
spectroscopy) after 8 days, compared to baseline. Based on data from a baseline sample (n = 
14) and clinical experience on reduction of overhydration baseline overhydration is expected 
to be 26.5 % of extracellular water (standard deviation 12.3; normal distribution is assumed) 
and it is expected that the overhydration will be reduced by 90 % in the amiloride group and 
by 50 % in the furosemide group. For the final t-test for two groups, a one-tailed significance 
level of 0.05 and a power of 80 % should be ensured. From these conditions results a sample 
size of n = 18 patients per group (total n = 36; calculated for a t-test with the nQuery® Advisor 
7.0 program). In order to take dropouts into account, the sample size is defined as n = 22 per 
group (total n = 44). 
 
5 
General Considerations 
5.1 
Timing of Analyses  
The analysis will be performed after finalization and approval of this SAP. The analysis will be 
performed on a database-snapshot with clean data regarding the primary endpoint, in- and 
exclusion criteria and a few demographic variables. After the close-out visits took place the 
final analysis will be performed on the data which have been declared clean according to ZKS-
SOP DA05. All changes to the database after the snap-shot will be documented in the TMF. 
 
5.2 
Analysis Population  
5.2.1 Intention-to-treat Population (ITT)  
All statistical analyses will be based on the Intention-to-Treat Population (ITT). The ITT 
includes all randomized patients with exception of patients who withdraw their informed 
consent for the analysis of their data during the study.  
 


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5.2.2 Safety Population (Safety)  
The safety population includes all subjects who received any study treatment. 
 
6 
Data and Data Management 
6.1 
Case Report Form  
The trial case report form (CRF) is the primary data collection instrument for the trial. All data 
requested on the CRF must be recorded. All missing data must be explained.  
For this project, paper Case Report Forms (CRFs) will be used. The investigator is responsible 
for ensuring that all sections of the CRF are completed correctly and that entries can be verified 
against source data. The correctness of entries in CRFs will be confirmed by dated signature 
of the responsible investigator. 
 
6.2 
Data Handling  
After first check for plausibility by eye, all data will be entered in a database as recorded in the 
CRF. To ensure data quality a double data entry will be performed. After completion of data 
entry, checks for plausibility, consistency, and completeness of the data will be performed. 
Based on these checks, queries will be produced combined with the queries generated by 
visual control. 
 
6.3 
Missing Data  
All variables included in the CRF are mandatory. The monitoring will assure quality of the 
assessments. Thus, missing values are to be expected only due to refusal by patients. In the 
analysis of the primary endpoint missing values will be imputed using multiple imputation 
approaches, complete case and last observation carried forward analyses will be performed 
as sensitivity analyses. All other missing data will be given in the descriptive tables but 
excluded from the statistical test. 
 
6.4 
Derived Variables  
Endpoints that are derived variables, i.e. not collected via the CRF: 
 
Change of overhydration from baseline to day 8 (see primary endpoint) 
 
Change of overhydration from baseline to day 16 
 
Change of body weight, oedema circumference, systolic and diastolic blood pressure 
after 8 and 16 days  
 
Change of urine volume, natriuresis, plasma renin activity and serum aldosterone 
concentration after 8 and 16 days 
 
 
 
 


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7 
Statistical Analyses 
7.1 
Study Information  
For all study subjects the disposition during the study and the duration including reasons 
(death, toxicity, treatment failure, withdraw consent) for pre-termination  will be shown. All data 
for the CONSORT 2010 flow diagram will be given, i.e. the allocation to the analysis population 
and the number of subjects dropped out.  
 
7.2 
Protocol Deviations  
The specific protocol deviations during the randomized treatment phase will be reviewed in the 
blind data review regarding the impact for the confirmatory analysis and categorized into major 
and minor. For all major protocol deviations summary statistics will be produced. 
 
7.2.1 Major Protocol Deviations  
The following events were classified as major protocol deviations: 
 
Deviation against inclusion or exclusion criteria  
 
Missing visit 5 (day 8) → no calculation of primary endpoint possible 
 
Late visit until day 8 
 
7.2.2 Minor Protocol Deviations  
The following events were classified as minor protocol deviations: 
 
Replacement of missing laboratory values from clinical patient record 
 
Late follow-up visit (1 – 5 days) 
 
Due to practical reasons blood and urine samples were taken at the routine ambulance 
visit which was declared as inclusion visit, and took place before the formal informed 
consent  no consequences for patient safety or data integrity. 
 
Stop of increasing the dose. 
According to the blind data review (finalized 02.11.2023) no major protocol deviations 
occurred. Note-to-files were documented during the course of the study for all minor protocol 
deviations. 
 
7.3 
Descriptive Statistics  
For all study data descriptive summary statistics will be produced. Descriptive analyses will 
include absolute and percentage frequencies for categorical variables, means, medians, 
standard deviations, quartiles and ranges for quantitative variables and medians, quartiles and 
ranges for ordinal variables. 
 


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7.4 
Efficacy Analyses  
7.4.1 Primary Endpoint  
The primary endpoint variable is the change of overhydration (OH) after 8 days compared to 
baseline, measured by bioimpedance spectroscopy using the Body Composition Monitor and 
expressed in percent of extracellular water (% ECW). The primary endpoint variable will be 
compared between the two study arms, which are patients with amiloride treatment and 
patients with furosemide treatment.  
The null-hypothesis is that there is equal or greater decrease of OH after 8 days in the group 
of patients with furosemide treatment compared to the group of patients with amiloride 
treatment. The alternative hypothesis is that there is a greater decrease of OH after 8 days in 
the group of patients with amiloride treatment compared to the group of patients with 
furosemide treatment. For analysis of the primary endpoint variable a one-sided t-test for two 
groups will be performed to test the null hypothesis against the alternative hypothesis. Effect 
size and 95%-confidence interval for effect size will be estimated. Only the rejection of the null 
hypothesis will be interpreted as statistical evidence for the efficacy of the amiloride treatment.   
 
7.4.2 Secondary Endpoints  
Secondary endpoint variables are listed in section 2.2. All secondary endpoints will be 
compared and statistically assessed for descriptive purposes and not in a confirmatory sense. 
The aim of the analysis is explorative data analysis, not hypothesis testing or generation of 
evidence for efficacy and no attempt will be made to adjust the p-values of statistical tests of 
the secondary endpoints for multiple testing. If adequate, secondary endpoints will be 
compared and statistically assessed using t-tests for two groups or covariance techniques with 
baseline values as covariates in case of quantitative variables. Depending on distribution, non-
parametric tests may be indicated. Dichotomous data will be compared and statistically 
assessed using Mantel-Haenszel chi-squared tests including relative risks and 95%-
confidence intervals for relative risks. 
 
7.5 
Safety Analysis  
Safety will be assessed by frequency tabulations and line listings for AEs and SAEs. 
 
8 
Reporting Conventions  
P-values ≥ 0.001 will be reported to 3 decimal places; p-values less than 0.001 will be reported 
as “< 0.001”. The mean, standard deviation, and any other statistics other than quantiles, will 
be reported to one decimal place greater than the original data. Quantiles, such as median, or 
minimum and maximum will use the same number of decimal places as the original data or 
one decimal place greater than the original data. Estimated parameters, not the same scale 
as raw observations (e.g. regression coefficients) will be reported within tables or figures. 
 


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9 
Technical Details  
At the time of writing this SAP the study specific documents used data base will be stored at 
Koordobas 5.1.0, the software package for statistical analysis will be SAS 9.4, and the 
operating system of the computer will be Windows Server 2019. 
 
11  
Time Table 
Actual for data analysis and statistical report the following time table is planned 
Approval of the SAP (signed) 
 
 
 
 
 
11/2023 
Final clean file for data analysis  
 
 
  
 
11/2023 
Submission of the final statistical report  
 
 
 
12/2023 
 
12 
Planned Tables, Figures and Listings 
Actual the following tables, figures and listings are planned for the statistical report:  
Table 2 
Planned tables, figures and listings 
No 
Title/Item 
1 
Study information 
1.1 
Consort 2010 statement (figure) 
1.2 
Inclusion-/exclusion criteria, informed consent 
1.3 
Study duration 
1.4 
Termination for subjects 
1.5 
Protocol deviations (major) 
2 
Descriptive Analysis (ITT population) 
2.1 
Study entry 
2.1.1 
Demographic data 
2.1.2 
Clinical examination 
2.1.3 
Blood analysis 
2.1.4 
Urine analysis 
2.1.5 
Additional laboratory data 
2.1.6 
Medical history 
2.1.7  
Previous medication 
2.1.8 
Study medication 
2.2 
Treatment phase 
2.2.1 
Clinical examination 
2.2.2 
Blood analysis 
2.2.3 
Urine analysis 
2.2.4 
Additional laboratory data 
2.2.5 
Dose of study medication 
2.2.6  
Concomitant medication 
 
 


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IKEAB, UK Tübingen 
 
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Continued Table 2 
Planned tables, figures and listings 
No 
Title/Item 
3 
Statistical Analysis (ITT population) 
3.1 
Primary endpoint 
3.2 
Secondary endpoints 
4 
Safety (safety population) 
4.1 
Extend of exposure 
4.2 
AEs 
4.3 
SAEs 
5 
Listings (ITT population) 
5.1 
Study information 
5.2 
Inclusion-/exclusion criteria 
5.3 
Demographic and anamnestic parameters 
5.4 
Clinical examination 
5.5 
Pre-existing, concomitant and study medication 
5.6 
Laboratory parameters and pregnancy test 
5.7 
Bioimpedance spectroscopy (BCM) 
5.8 
Items of the diet diary 
5.9 
AEs and SAEs