Distraneurin®) on CYP2E1 Activity, Transaminases, Liver Fat

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CLINICAL STUDY PROTOCOL K419

Effect of Clomethiazole (Distraneurin®) on CYP2E1 Activity, transaminases, Liver fat

content, and liver stiffness during Alcohol Detoxification – a pilot Study (CALvADoS)

Phase: I

EudraCT No: 2012-005730-11

Protocol-Code: K419

Version: 1.1 FINAL

SPONSOR KRANKENHAUS SALEM DER EVANG. STADTMISSION HEIDELBERG GGMBH Jürgen Unrath Executive Director Zeppelinstr. 11-33 69121 Heidelberg Telefon: +49 (0) 6221 483359 Fax: +49 (0) 6221 483108

Hohmann N, et al. Gut 2021;0:1–2. doi: 10.1136/gutjnl-2021-324727 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Gut

SITE OF INVESTIGATION SALEM MEDICAL CENTER Department of Internal Medicine Zeppelinstraße 11-33 69121 Heidelberg Germany

MEDICAL DIRECTOR Prof. Dr. med. Dr h.c. Helmut-Karl Seitz Distinguished Professor of Medicine, Gastroenterology and Alcohol Research Heidelberg University Department of Medicine Salem Medical Center/University of Heidelberg Zeppelinstraße 11-33 69121 Heidelberg Germany Tel.: +49 (0) 6221 483200 Fax: +49 (0) 6221 483494 Email: [email protected]

INVESTIGATOR Prof. Dr. med. Dr h.c. Helmut-Karl Seitz

INVESTIGATOR’S DEPUTY Prof. Dr. med. Sebastian Mueller Vice Head and Research Director Department of Medicine (Gastroenterology, Hepatology) Salem Medical Center and Center for Alcohol Research, University of Heidelberg Zeppelinstraße 11 – 33 69121 Heidelberg, Germany Tel.: +49 (0) 6221 483210 Fax: +49 (0) 6221 483494 Email: [email protected]

INVOLVED LABORATORIES ANALYTICAL CHEMISTRY LAB Department of Clinical Pharmacology and Pharmacoepidemiology Dr. rer.nat. Jürgen Burhenne Email: [email protected] University Hospital of Heidelberg Im Neuenheimer Feld 410, 69120 Heidelberg, Germany

EXPERT ADVICE (CLINICAL PHARMACOLOGY) DEPARTMENT OF CLINICAL PHARMACOLOGY AND PHARMACOEPIDEMIOLOGY

Prof. Dr. med. Walter E. Haefeli Medical director Email: [email protected] heidelberg.de Tel.: +49 (0) 6221 568740 Fax: +49 (0) 6221 564642 University Hospital of Heidelberg Im Neuenheimer Feld 410, 69120 Heidelberg, Germany

Dr. med. Nicolas Hohmann Clinical Research Unit (Project Management, Pharmacovigilance) Email: [email protected] Tel.: +49 (0) 6221 56 39572 Fax: +49 (0) 6221 56 8527

BIOMETRY Dr. sc. hum. Thomas Bruckner Department of Medical Biometry INF 305 69120 Heidelberg Tel.: +49 (0) 6221 564371 Email: [email protected]

DECLARATION OF INVESTIGATOR ...... 6_ 1_ Abbreviations ...... 7_ 2_ Synopsis of K419 ...... 8_ 3_ Rationale ...... 11_ 3.1_ Idea of the study ...... 11_ 3.2_ Project plan ...... 11_ 4_ Study Objectives ...... 12_ 4.1_ Objectives ...... 12_ 4.2_ Criteria to be studied ...... 12_ Primary endpoint ...... 12_ Secondary endpoints ...... 12_ 5_ Study Design ...... 13_ 5.1_ Clinical Phase ...... 13_ 5.2_ Study Population ...... 13_ 5.3_ Randomization ...... 13_ 5.4_ Visits ...... 13_ 5.5_ Study Overview ...... 13_ 5.6_ Study visits and specific actions ...... 14_ 5.7_ Planned dates of the clinical trial ...... 16_ 6_ Study Medication ...... 17_ 6.1_ Clomethiazole (Distraneurin®) ...... 17_ 6.1.2_ Introduction ...... 17_ 6.1.3_ Pharmacological properties ...... 17_ 6.1.4_ Dosage ...... 18_ 6.1.5_ Adverse effects ...... 18_ 6.1.6_ Contraindications ...... 18_ 6.2_ Clorazepate (Tranxilium®) ...... 18_ 6.2.1_ Introduction ...... 18_ 6.2.2_ Pharmacological properties ...... 19_ 6.2.3_ Dosage ...... 19_ 6.2.4_ Adverse effects ...... 19_ 6.2.5_ Contraindications ...... 20_ 6.3_ Chlorzoxazone (Paraflex®) ...... 20_ 6.3.1_ Introduction ...... 20_ 6.3.2_ Pharmacological properties ...... 20_ 6.3.3_ Pharmaceutical properties ...... 21_ 6.3.4_ Dosage ...... 21_ 6.3.5_ Adverse effects ...... 21_ 6.3.6_ Contraindications ...... 22_ 6.4_ Concomitant medication ...... 22_ 6.4.1_ Permitted concomitant medication ...... 22_ 6.4.2_ Prohibited concomitant medication ...... 22_ 7_ Study Population ...... 23_ 7.1_ Type of population ...... 23_ 7.2_ Planned number of individuals ...... 23_ 7.3_ Inclusion criteria ...... 23_ 7.4_ Exclusion criteria ...... 23_ 7.5_ Admission to the trial ...... 24_ 8_ Study Procedures ...... 25_ 8.1_ Pre-study examination ...... 25_ Study design (see 5) ...... 26_ 8.2_ Blood sampling ...... 26_ 8.3_ Post-Study examination ...... 26_

8.4_ Assessment and recording of adverse events (AE) ...... 26_ 8.5_ Priority and sequence of trial activities ...... 26_ 9_ Monitoring ...... 28_ 10_ End of Study ...... 29_ 11_ Withdrawal ...... 30_ 11.1_ Withdrawal of patients ...... 30_ 11.2_ Drop-outs ...... 30_ 11.3_ Follow up – Treatment ...... 30_ 12_ Early termination of the study ...... 31_ 13_ Study analyses ...... 32_ 13.1_ Determination of standard laboratory parameters ...... 32_ 13.2_ Determination of liver stiffness ...... 32_ 13.3_ Determination of liver fat content ...... 32_ 13.4_ Determination of drug concentrations in biological matrices ...... 32_ 13.5_ Pharmacokinetic analysis ...... 32_ 13.6_ Statistical evaluation ...... 32_ 13.7_ Procedure for handling missing, unused, and inconsistent data ...... 32_ 14_ Ethical and Legal Aspects ...... 33_ 14.1_ General ...... 33_ 14.2_ Benefit-to-risk ratio ...... 33_ 14.3_ Submission to the Ethics Committee and Competent Authority (BfArM) ...... 33_ 14.4_ Notification of authorities ...... 34_ 14.5_ Participant information and informed consent ...... 34_ 14.6_ Responsibilities ...... 34_ 14.7_ Insurance ...... 34_ 14.8_ Confidentiality ...... 34_ 14.9_ Archiving ...... 34_ 15_ Organization of the study ...... 35_ 15.1_ Protocol modifications and amendments ...... 35_ 15.2_ Access to source data, quality control, and data handling ...... 35_ 15.2.1_ Declaration of informed consent ...... 35_ 15.2.2_ Case report form (CRF) ...... 35_ 15.2.3_ List of patients (patients’ log) ...... 35_ 15.2.4_ Patient medical records / source data ...... 35_ 15.2.5_ Data Handling ...... 36_ 15.2.6_ Drug accountability sheet ...... 36_ 15.3_ Financing ...... 36_ 15.4_ Publication of the study results ...... 36_ 16_ References ...... 37_ Appendices ...... 38_ APPENDIX 1: _ Responsibilities of the Principal Investigator ...... 38_ APPENDIX 2: _ Definition of adverse drug events ...... 39_ APPENDIX 3:_ List of raw data relating to the study ...... 42_

1 Abbreviations

ALD Alcoholic Liver Disease ALAT Alanine aminotransferase ASAT Aspartate aminotransferase AUC Area under the time-concentration curve BP Blood pressure CAP Controlled attenuation parameter CMZ Clomethiazole CNS Central nervous system CPT Clorazepate CYP Cytochrome P450 CZX Chlorzoxazone DBS Dried blood spot ECG Electrocardiogram EU European Union GABA Gamma amino butyric acid HR Heart rate ICD-10 International Statistical Classification of Diseases and Related Health Problems, 10 th revision IMP Investigational medicinal product INR International Normalized Ratio LLN Lower limit of the normal MR(2h) Metabolic ratio 2 hours after administration of the study drug MS/MS Tandem mass spectrometry NYHA New York Heart Association PK Pharmacokinetics PXR Pregnane X receptor RFLP Restriction fragment length polymorphism ROS Reactive oxydative species t1/2 Half-life ULN Upper limit of normal UPLC Ultra performantce liquid chromatography

2 Synopsis of K419

Title Effect of Clomethiazole (Distraneurin®) on CYP2E1 Activity, transaminases, Liver fat content, and liver stiffness during Alcohol Detoxification – a pilot Study (CALvADoS)

Study Phase I

Objectives Primary endpoint 1. Changes in ASAT

Secondary endpoints 2. Changes in ALAT 3. Changes in liver fat content 4. Changes in liver stiffness 5. Changes in other function parameters (INR, albumin, bilirubin) 6. Changes in chlorzoxazone pharmacokinetics (AUC0-8, t1/2, clearance, hydroxylation index, metabolic ratio 2 and 4 h post dose) 7. Duration of hospitalisation

Design

Study drugs Distraneurin® (clomethiazole), starting dose 500 mg p.o. 6-10x daily, dose adaptation according to individual needs and local standard of treatment Tranxilium® (clorazepate), 25–50 mg mg p.o. 4 - 6x daily, dose adaptation according to individual needs and local standard of treatment

Diagnostic Substances Paraflex® (chlorzoxazone), 500 mg p.o. 1 x daily at V1, V2, V3 and V4.

Study period Planned start of recruitment: 07. JAN. 2015 Planned data base lock: 01. DEC. 2015 Planned final trial report: 01. MAR. 2016

Sample size N = 30 in each study arm

Inclusion Criteria 1. Age: 18 – 70 years 2. Ability to understand and willingness to comply with study interventions and restrictions. 3. Capacity to consent and ability to provide a written informed consent 4. Voluntarily signed informed consent after full explanation of the study to the participant. 5. Diagnosis of alcohol addiction as defined by ICD-10 6. Patient showing or telling alcohol withdrawal symptoms OR patient who drinks alcohol to avoid the occurrence of alcohol withdrawal symptoms OR patient who in average drinks more than 120 g of alcohol (males) or 80 g of alcohol (females) per day. 7. Hospital admission for alcohol detoxification 8. ASAT >ULN 9. Females of child bearing potential are only included if using reliable contraception with a Pearl Index <1% (i.e. two independent effective contraceptive methods) during the study and two weeks after the last administration of study medication.

Exclusion Criteria 1. Advanced liver disease (liver cirrhosis) or Fibroscan >12kPa 2. Hemoglobin <10 g/dl 3. Uncontrolled diabetes mellitus (defined as fasting blood glucose _200 mg/dl) 4. Significant uncontrolled cardiovascular disease or cardiac insufficiency (New York Heart Association [NYHA] classes III-IV) 5. Renal insufficiency (creatinine clearance calculated with Cockcroft & Gault formula _30 ml/min) 6. Therapy with drugs that act as inhibitors or inducers of CYP2E1 (except ethanol) in the last 14 days. 7. Any antibiotic therapy due to infections 2 weeks prior to first study drug administration 8. Major surgery _4 weeks prior to first study drug administration or ongoing side effects of such surgery 9. Any active systemic infection 10. Systemic diseases (cardiovascular, renal, hepatic, etc.) that would prevent study treatment 11. Allergies (except for mild forms of hay fever) or history of hypersensitivity reactions 12. Any other acute illness or clinically relevant findings in the pre-study examination that could interfere with the patient’s safety or study objectives according to the investigator’s judgement. 13. Treatment with a systemic investigational agent within 28 days prior to the screening visit 14. Inability to communicate well with the investigator due to language problems or poor mental development 15. Contraindication against clomethiazole, chlorazepate, or chlorzoxazone or known intolerance of one of the substances or their additives 16. For female participants: pregnancy or lactation

Methods Measurement of liver stiffness with Fibroscan® Measurement of transaminsases Measurement of liver fat content with Controlled attenuation parameter Sonography UPLC/MS-MS for measurement of chlorzoxazone plasma and urinary concentrations UPLC/MS-MS for measurement of clomethiazole plasma concentrations

Table 1: Study procedures

3 Rationale

3.1 Idea of the study Only limited data is available on the pathophysiology of alcoholic liver disease (ALD) in humans. Induction of cytochrome P450 2E1 (CYP2E1) by chronic alcohol intake appears to play a critical role in ALD physiopathology (Seitz HK et al. Alcohol Clin Exp Res 2005; 29: 1341). CYP2E1 is induced in the majority of patients and induction resolves after alcohol withdrawal (Oneta CM et al. J Hepatol 2002; 36: 47-52). It is time- and dose-dependent and subject to interindividual variability. CYP2E1 produces reactive oxygen species (ROS) that bind to proteins and may cause a change in protein function (Seitz HK et al. Alcohol Clin Exp Res 2005; 29: 1341). Inhibition of CYP2E1 with clomethiazole leads to an important reduction of alcoholic liver damage in animal models (Cederbaum AI. Dig Dis 2010; 28: 802). CYP2E1 knock-out mice do not develop ALD after chronic alcohol ingestion (Lu Y et al. Free Radic Biol Med 2010;49:1406-16). Clomethiazole is the only known selective and potent CYP2E1 inhibitor routinely used in the detoxification therapy of alcoholics. Patients are treated with clomethiazole (Distraneurin®) for eight to ten days until they have overcome the detoxification. This therapy has been practiced for decades and has negligible side effects (Tubridy P. Br J Addict 1988;83:581-5.). The following study will investigate whether inhibition of CYP2E1 by clomethiazole improves liver function recovery and has a hepatoprotective effect assessed as changes in transaminases, liver fat content, and liver stiffness.

3.2 Project plan To investigate the effect of CYP2E1 inhibition on the recovery of liver function in ALD after withdrawal from alcohol, we will compare two drugs routinely used during detoxification. Eligible patients admitted to the hospital for alcohol detoxification will be randomized to receive either clomethiazole (Distraneurin®) or clorazepate (Tranxilium®). Clorazepate is also routinely used in the detoxification of alcoholics but has no effect on CYP2E1 activity. We will evaluate the difference of both treatments in changes of transaminases, liver fat content, and liver stiffness to baseline after 1, 10 (5-12), and 30 (25 -30) days. An additional blood sample will be taken at each study visit for the optional evaluation of experimental biomarkers of ALD and liver damage (e.g. hyaluronate). We will assess CYP2E1 activity with a CYP phenotyping test using the FDA-recommended probe substrate chlorzoxazone at baseline, after 1, 10 (5-12), and 30 days. Therefore, we will quantify chlorzoxazone and its metabolites in plasma, urine, and venous capillary blood after collection on filter paper cards (dried blood spots, DBS). Based on the patient’s general condition of health, willingness for participation, and need for a fast initiation of therapy a study physician will assign the patient either to the limited or extensive PK sampling cohort. We will draw only two PK blood samples (pre-dose and 2 h post-dose) from patients in the limited sampling cohort. In the extensive PK sampling cohort a full PK profile of chlorzoxazone will be collected. Because interindividual pharmacokinetic variability is large – especially for clomethiazole – additional daily blood samples will be drawn to measure clomethiazole exposure.

4 Study Objectives

4.1 Objectives The study objective is to assess whether clomethiazole has a favourable effect on the recovery from alcoholic liver damage. Because long-term clomethiazole intake may be addictive (Fachinformation Distraneurin®), it can only be given for a short time for alcohol detoxification. Thus the effect of clomethiazole can only be assessed for the period of hospitalisation covering the usual treatment period (~ 5-12 days) for the detoxification of alcoholics (Majumdar SK. Drug Alcohol Depend 1991;27:201-7).

4.2 Criteria to be studied The following parameters will be studied at hospital admission, after 1, after 10 (5-12), and after 30 days: 1. Transaminases (ASAT, ALAT) 2. Liver fat content measured via controlled attenuation parameter (CAP) 3. Liver stiffness measured with Fibroscan® 4. Changes in CYP2E1 activity 5. Changes in biomarkers of liver damage such as hyaluronate, M30 and M65.

Primary endpoint 1. Changes in ASAT

5 Study Design

5.1 Clinical Phase Phase I study in patients with chronic alcoholism undergoing alcohol detoxification in a hospital setting.

5.2 Study Population N=60 patients with alcohol dependency, 30 in each study arm.

5.3 Randomization The patients will be randomly assigned to one of the treatment arms. The 1:1 assignment will occur through block randomization. Randomization lists will be provided by the Department of Clinical Pharmacology and Pharmacoepidemiology.

5.4 Visits Patients undergoing hospital admission for alcohol detoxification are eligible for this study. After signing the informed consent form, screening will take place upon hospital admission. There will be 3 study visits for each participant. Two of the visits will take place during hospitalisation. The last visit will be an ambulatory visit to the study centre. The end-of-study visit will take place immediately after visit 3 or within 3 weeks thereof.

5.5 Study Overview Patients will be orally treated according to local standard of treatment either with 6 to 10x500 mg/d of Distraneurin® (clomethiazole) or 4x25 mg to 6x50 mg/d of Tranxilium® (clorazepate) according to treatment assignment. This dose will be adapted based on clinical necessity and gradually reduced with the aim to discontinue the drugs after 5 to 12 days. The patients are hospitalized and closely observed during this treatment phase. Upon hospital admission, blood analyses (including liver enzymes, renal function, hematology, CRP, electrolytes, iron metabolism), abdominal sonography to measure the liver fat content and a Fibroscan® measurement to assess the fibrosis stage will be performed. These examinations are part of clinical routine and thus not part of this study. These examinations are repeated on the day of planned discharge (usually day 10 [5-12] after hospitalization. 30±5 days after hospital admission the patients are called in for an ambulatory visit (V4).

5.6 Study visits and specific actions

Screening (Day 0) ฀Signed informed consent ฀Relevant medical history ฀Current medical condition ฀Concomitant medication ฀Body height, weight, vital signs, physical examination ฀Blood sampling: hematology (2.7 ml), coagulation (3 ml), clinical chemistry ( 7.5 ml) ฀Urinalysis ฀Assessment of liver stiffness with Fibroscan® ฀Eligibility ฀Randomization ฀Assignment to limited or extensive PK sampling cohort

V1 (day 0) Before treatment with CMZ/CPT: ฀Abdominal sonography ฀Assessment of liver fat content with CAP ฀Blood drawings for biomarkers (9 ml), and chlorzoxazone PK predose (4.9 ml) ฀Administration of 500 mg of chlorzoxazone p.o.

Limited PK sampling cohort: ฀Blood sampling 2 h post-dose (4.9 ml). ฀Collection of DBS 2 h post-dose.

Extensive PK sampling cohort: ฀Urine for chlorzoxazone pre-dose ฀Blood sampling 0.5, 1, 2, 3, 4, 6, and 8 h post-dose (4.9 ml each). ฀Collection of DBS 0.5, 1, 2, 3, 4, 6, and 8 h post-dose. ฀8-hour urine collection starting with administration of chlorzoxazone Initiation of treatment with either clomethiazole or clorazepate according to treatment arm.

V2 (day 1) After initiation of treatment ฀Blood sampling for biomarkers (9 ml), chlorzoxazone and clomethiazole PK pre-dose (9 ml) ฀Spot urine for chlorzoxazone pre-dose (extensive PK sampling cohort only) ฀Administration of 500 mg of chlorzoxazone p.o. Pre-dose blood samples should be taken before the morning dose of clomethiazole. The chlorzoxazone dose and the morning dose of clomethiazole should be taken simultaneously.

Limited PK sampling cohort: ฀Blood sampling 2 h post-dose (4.9 ml). ฀Collection of DBS 2 h post-dose.

Extensive PK sampling cohort: ฀Blood sampling 0.5, 1, 2, 3, 4, 6, and 8 h post-dose (4.9 ml each). ฀Collection of DBS 0.5, 1, 2, 3, 4, 6, and 8 h post-dose. ฀8-hour urine collection starting with administration of chlorzoxazone During hospitalisation ฀Daily blood sampling for clomethiazole concentrations (before first clomethiazole dose in the morning) (4.9 ml each) ฀Blood sampling for transaminases (2.7 ml each) day 1, 2, 3, 5, 7, 9, 11 after initiation of treatment

V3 (Day 5-12) Upon planned discharge from hospital: ฀Blood drawings for biomarkers (9 ml) and chlorzoxazone PK predose (4.9 ml) ฀Spot urine for chlorzoxazone pre-dose (extensive PK sampling cohort only) ฀Administration of 500 mg of chlorzoxazone p.o.

Limited PK sampling cohort:

฀Blood sampling 2 h post-dose (4.9 ml). ฀Collection of DBS 2 h post-dose.

Extensive PK sampling cohort: ฀Blood sampling 0.5, 1, 2, 3, 4, 6, and 8 h post-dose (4.9 ml each). ฀Collection of DBS 0.5, 1, 2, 3, 4, 6, and 8 h post-dose. ฀8-hour urine collection starting with administration of chlorzoxazone ฀Current medical condition ฀Body height, weight, vital signs, physical examination ฀Blood drawing: hematology (2.7 ml), coagulation (3 ml), clinical chemistry ( ml) ฀Urinalysis ฀Abdominal sonography ฀Assessment of liver stiffness with Fibroscan® ฀Assessment of liver fat content with CAP

Discharge V4 (Day 25-35) Arrival at hospital ฀Blood sampling for biomarkers (9 ml) and chlorzoxazone PK predose (4.9 ml each) ฀Spot urine for chlorzoxazone pre-dose (extensive PK sampling cohort only) ฀Administration of 500 mg of chlorzoxazone p.o.

Limited PK sampling cohort: ฀Blood sampling 2 h post-dose (4.9 ml). ฀Collection of DBS 2 h post-dose.

Extensive PK sampling cohort: ฀Blood drawings 0.5, 1, 2, 3, 4, 6, and 8 h post-dose (4.9 ml each). ฀Collection of DBS 0.5, 1, 2, 3, 4, 6, and 8 h post-dose. ฀8 hours urine collection starting with administration of chlorzoxazone ฀Assessment of liver stiffness with Fibroscan® ฀Assessment of liver fat content with CAP ฀Blood drawing: hematology (2.7 ml), coagulation (3 ml), clinical chemistry ( ml) ฀Urinalysis End of Study Visit (up to 21 days after V4) ฀Current medical condition ฀Body height, weight, vital signs, physical examination ฀Blood drawing: hematology (2.7 ml), coagulation (3 ml), clinical chemistry (ml) (not applicable if the same day as V4) ฀Urinalysis (not applicable if the same day as V4) Discharge from hospital

5.7 Planned dates of the clinical trial Planned start of recruitment: 01. FEB 2015 Planned data base lock: 31. DEC. 2015 Planned final trial report: 30. MAR. 2016 EudraCT No: 2012-005730-11 Protocol Code: K419 Version: 1.1 FINAL 19/01/2015 Page 23 of 55

6 Study Medication The study drugs are approved in the European Union (EU). Distraneurin® (clomethiazole) and Tranxilium® (clorazepate) are approved in Germany. Paraflex® (chlorzoxazone) is an approved drug in Sweden. Common marketed drugs will be used and supplied by the pharmacy of the University Hospital. There will be no additional labelling.

6.1 Clomethiazole (Distraneurin®) We will use the following marketed drug: Distraneurin® Mixtur, 50 mg/ml Solution (PZN 0215192; 1Approval Nr. 6083948.00.00) available from Cephlapharm Arzneimittel GmbH, Bahnhofstr. 1a, 17498 Mesekenhagen, Germany.

6.1.2 Introduction Clomethiazole is a hypnotic and sedative drug that increases the electrophysiological reaction of the inhibitory neurotransmitters GABA and glycine. It is used for the treatment of alcohol withdrawal syndrome during acute detoxification under hospitalization, for the treatment of confusion, agitation and restlessness in the elderly, and for the treatment of severe insomnia in the elderly (Fachinformation Distraneurin®).

6.1.3 Pharmacological properties Pharmacodynamic properties The structure of clomethiazole is related to the vitamin B1-molecule. It has sedative, hypnotic, and anticonvulsive properties. Clomethiazole increases the electrophysiological reaction of the inhibitory neurotransmitters GABA and glycine without interfering with the inhibitory reactions to acetylcholine and adenosine. In contrast to barbiturates, clomethiazole does not increase the electrophysiological reactions to excitatory amino acids. As a consequence, an increase of GABA may occur at sites associated to the chloride ion channel of the GABAAreceptor. These findings indicate that the mode of actions differs from barbiturates and benzodiazepines (Fachinformation Distraneurin®). Pharmacokinetic properties After oral administration, clomethiazole is readily resorbed and underlies an extensive firstpass metabolism in the liver before entering the circulation. Bioavailability is low and extremely variable. Depending on the liver function, bioavailability is 5–60% after administration of 2 capsules (384mg) of clomethiazole. There is a marked increase of bioavailability in patients with severe alcoholic liver cirrhosis. Maximal plasma concentrations are measured 90 minutes after application of a capsule or 60 min after intake of the solution. Plasma concentrations 15 minutes after intake of 2 capsules are 426 (±238) ng/ml. The apparent volume of distribution is 9 l/kg in young adults and 13 l/kg in older adults. Plasma protein binding is 60-70%. Clomethiazole is completely metabolized and less than 1% of the dose is excreted unchanged via the urine. The main metabolism occurs through dehalogenation and oxidative degradation of the C5 side chain. The circulating metabolite 5- acetyl-4-methylthiazole is pharmacologically active. The half-life in the distribution phase is 0.42 to 0.54 h, the _-elimination half-life is 2.3 to 5 h in healthy volunteers and up to 9 h in alcoholic liver cirrhosis. The clearance of clomethiazole is reduced in the elderly: terminal elimination half-life is 5–15 h, peak plasma concentrations and the AUC are several fold increased (Fachinformation Distraneurin). Clomethiazole is a CYP2E1 inhibitor: While CYP2E1 activity measured as chlorzoxazone metabolic ratio is increased in patients with alcoholic liver disease (Dilger et al. J Hepatol 1997; 27: 1009) the chlorzoxazone AUC of patients with alcoholism taking clomethiazole is increased 3.4-fold compared to moderately drinking healthy volunteers without clomethiazole (Eap CB et al. Clin Pharmacol Ther 1998; 64: 52).

6.1.4 Dosage Because the pharmacokinetics and responses to clomethiazole are subject to a high intersubject variability, dosing for the individual patient is best figured out by administering an initial dose and then titrating the dose according to symptoms and clinical situation (Fachinformation Distraneurin). Following the local standard of treatment, patients assigned to the clomethiazole arm will receive an initial dose of 6 to 10 x 500mg clomethiazole. This dose will be adapted based on clinical necessity and gradually reduced with the aim to discontinue it after 5 to 12 days.

6.1.5 Adverse effects Immune system disorders Facial angioedema#, allergic reactions#, anaphylactic shock#, Psychiatric disorders Addiction# Nervous system disorders Lightheadedness#, hypesthesia#, dysaesthesia#. Cardiac disorders Palpitations, cardiac arrest#, hypotension#. Respiratory disorders Increased bronchial secretion*, cough***, burning sensation in the throat***, severe respiratory depression†, upper respiratory tract infection†, pneumonia† Gastrointestinal system disorders Increased salivation*, gastric pain***, nausea***, vomiting***, diarrhea***, heartburn***, increased transaminases#, jaundice#, cholestasis#. Skin or subcutaneous disorders Pruritus#, exanthema#, urticarial#, bullous skin disorders#. Eye disorders Conjunctivitis#. General and application site disorders General discomfort***, severe tiredness#. Frequency: *very common ≥1/10; **frequent ≥1/100 to <1/10; ***infrequent ≥1/1,000 to <1/100; † rare ≥ 1/10,000 to < 1/1,000; π very rare <10,000; # unknown.

6.1.6 Contraindications ฀Known hypersensitivity to clomethiazole or one of the excipients. ฀Sleep apnea syndrome ฀Central respiratory depression ฀Acute alcohol intoxication ฀Concomitant use of other CNS depressant substance ฀Addiction to alcohol or other psychotropic substances outside of the treatment of acute detoxification, predelirium or delirium tremens. ฀Bronchial asthma or bronchial hyperreagibility

6.2 Clorazepate (Tranxilium®) We will use the following marketed drug: Tranxilium® 10 mg (10 mg Hartkapseln) (PZN 6064432; Approval Nr.: 26137.01.01) and Tranxilium® 20 mg (20 mg Hartkapseln) (PZN 6064449; Approval Nr.: 26137.02.01) available from Sanofi-Aventis Deutschland GmbH, 65908 Frankfurt/Main, Germany.

6.2.1 Introduction Clorazepate is a long-acting benzodiazepine and has as such anxiolytic, sedative, hypnotic, and muscle relaxing properties. Its primary mode of action is the allosteric enhancement of inhibitory GABAergic neurotransmission. It is used in the treatment of acute or chronic agitation or anxiety, in the treatment of acute detoxification of alcoholics, and as

premedication before diagnostic or operative interventions.

6.2.2 Pharmacological properties Pharmacodynamic properties Clorazepate is a psychotropic substance and a member of the class of 1,4-benzodiazepines. Clorazepate itself has a low affinity to the benzodiazepine-receptor in the CNS while its EudraCT No: 2012-005730-11 Protocol Code: K419 Version: 1.1 FINAL 19/01/2015 Page 26 of 55 active main metabolite N-desmethyldiazepam (Nordazepam) has a high affinity. Thus it enhances the inhibitory GABAergic neurotransmission in different neuronal groups. The anxiolytic, hypnotic, and sedative properties of clorazepate are due to the binding to benzodiazepine receptors. Furthermore clorazepate has muscle relaxing and anticonvulsive properties (Fachinformation Tranxilium). Clorazepate is used to control and ease withdrawal symptoms in patients with alcoholism undergoing detoxification (Mielke DH et al. Curr Ther Res Clin Exp 1976;19:506). Pharmacokinetic properties The bioavailability of clorazepate is 10-16%. After oral intake, clorazepate is transformed into the main active metabolite nordazepam. The peak plasma concentrations of clorazepate are reached 30 to 60 minutes after intake, the peak plasma concentration of nordazepam after 60 minutes. The terminal plasma half-life of clorazepate is 2-2.5 h, whereas the half-life of the active metabolite nordazepam is 25-82 hours and, hence, steady-state concentrations of nordazepam are reached after 6 to 11 days. The plasma protein binding of nordazepam is 95%. Clorazepate and its metabolites are renally eliminated (Fachinformation Tranxilium). Clorazepate has no known effect on CYP2E1 activity (Stockley’s Drug Interactions).

6.2.3 Dosage The dose and duration of therapy have to be adapted to the patient’s individual response to the indication and the severity of discontinuation symptoms. Generally, dose and duration have to be kept as short as possible. If used for the therapy of acute or chronic anxiety, agitation, or tension the daily dose in an ambulatory setting is 10-20 mg of dipotassium clorazepate 2-3 times per day or as a single dose in the evening. If necessary the dose can be adjusted to a maximum of 150 mg/d. During hospitalization the daily dose of dipotassium clorazepate can be increased to a maximum of 300 mg daily for a short time. Patients randomized to the clorazepate study arm will receive an initial dose of 4x25 mg to 6x50 mg Tranxilium® (clorazepate) according to local standard of treatment using clorazepate to alleviate withdrawal symptoms. Dosage will be adapted based on the clinical situation and symptoms of the patient.

6.2.4 Adverse effects Psychiatric Reduced libido#, lack of concentration#, fatigue#, unmasking of pre-existing depression#, paradox reactions# such as unrest, agitation, aggressive behaviour, anger, acute excitation, anxiety, dream-like confusional state, hallucinations, psychosis, suicidality, muscle spasms, insomnia (especially in children and the elderly), addiction#, rebound-phenomena and withdrawal symptoms after end of therapy#. Neurological Drowsiness#, dizziness#, vertigo#, headache#, anterograde amnesia#, muscular hypotension#, fatigue#, languidness#, reversible disturbance of articulation#, reversible gait insecurity#. Ophthalmological Reversible disturbance of vision# (blurred

vision, diplopia, nystagmus beats). Vascular Mild arterial hypotension# Pulmonary, thoracic and mediastinal Respiratory depression# Gastrointestinal Disturbances of the gastro-intestinal tract#. Hepatobiliary Temporary increase of transaminases#. Dermatological Hypersensitivity reactions of the skin# (maculopapular rash or pruritic rash). Sex organs Dysmenorrhoea#. General Increased risk of falls#.

Frequency: *very common ≥1/10; **frequent ≥1/100 to <1/10; ***infrequent ≥1/1,000 to <1/100; † rare ≥ 1/10,000 to < 1/1,000; π very rare <10,000; # unknown.

6.2.5 Contraindications ฀Known hypersensitivity against clorazepate, other benzodiazepines, or another excipient of Tranxilium®. ฀Decompensated respiratory insufficiency ฀History of drug abuse (medicaments, illegal drugs, alcohol) ฀Acute intoxication with drugs active in the central nervous system like alcohol, analgesics, sleeping agents, neuroleptics, antidepressants, and lithium. ฀Sleep apnoea syndrome ฀Myasthenia gravis ฀Severe liver damage, because hepatic encephalopathy may be triggered by benzodiazepines ฀Spinal and cerebellar ataxia Clorazepate may only be used with caution in patients with ฀Chronic respiratory insufficiency

6.3 Chlorzoxazone (Paraflex®) We will use the following marketed drug: Paraflex® 250 mg (Approval Nr. 6157) available from BioPhausia AB, Blasieholmsgatan 2, 11148 Stockholm, Sveden.

6.3.1 Introduction Chlorzoxazone is a centrally acting benzoxazole derivative with a weak muscle relaxing effect. The dose used in this study protocol is a single 500 mg dose to assess the CYP2E1 enzyme activity (phenotyping) in patients undergoing alcohol withdrawal. The usual indication for chlorzoxazone in combination with paracetamol is the treatment of painful muscle spasms. Drowsiness, weakness, dizziness, and gastrointestinal complaints are the most frequent unwanted effects (Myler’s side effects of drugs). Chlorzoxazone tablets are indicated as an adjunct to rest, physical therapy, and other measures for the relief of discomfort associated with acute, painful musculoskeletal conditions. The mode of action for this drug is not clearly established, but may be related to its sedative properties. Chlorzoxazone does not directly relax tense muscles in man (Fachinformation Parafon® Kellmann).

6.3.2 Pharmacological properties Pharmacodynamic properties Chlorzoxazone is a centrally-acting agent for painful musculoskeletal conditions. Data available from animal experiments as well as human studies indicate that chlorzoxazone acts primarily at the level of the spinal cord and subcortical areas of the brain where it inhibits

multisynaptic reflex arcs involved in producing and maintaining skeletal muscle spasm of variable etiology. The clinical result is a reduction of the skeletal muscle spasm with relief of pain and increased mobility of the involved muscles (Parafon DSC. Chlorzoxazone tablet) Keltman pharmaceuticals inc.). Pharmacokinetic properties Blood levels of chlorzoxazone can be detected during the first 30 minutes and in most patients peak levels are reached 1 to 2 hours after oral administration of chlorzoxazone. Chlorzoxazone is rapidly metabolized by CYP2E1 to 6-OH-chlorzoxazone and is excreted in the urine, primarily in a conjugated form as a glucuronide. Less than one percent of a dose of chlorzoxazone is excreted unchanged in the urine in 24 h (Parafon DSC. Chlorzoxazone tablet Keltman pharmaceuticals inc.). Chlorzoxazone has extensively been investigated as a probe drug for CYP2E1 activity in human. The metabolic ratio 2 or 4 h post-dose or the hydroxylation-index are suitable parameters for this purpose (Frye RF in J Clin Pharmacol 1998; 38:82.). Ethanol induces CYP2E1 activity by inhibiting CYP2E1 mRNA decay. Thus chlorzoxazone AUC decreases and the MR increases after chronic administration of alcohol (Onmeta CM et al). Disulfiram is an inhibitor of CYP2E1 activity leading to an increase of chlorzoxazone AUC and a decrease in MR (Karash ED et al in Clin Pharmacol Ther 1993; 53: 643).

6.3.3 Pharmaceutical properties

6.3.4 Dosage For CYP2E1-phenotyping, single oral doses of 500 mg of chlorzoxazone will be administered at four different occasions (V1-4).

6.3.5 Adverse effects

Nervous system A spasmodic torticollis-like syndrome, repeatedly evoked after the ingestion of chlorzoxazone, is an unusual adverse effect of the drug (Rosin MA. Chlorzoxazone-induced spasmodic torticollis. JAMA 1981; 246: 2575.)

Liver The most serious adverse event of chlorzoxazone, which fortunately occurs only rarely, is hepatotoxicity. In one case, jaundice occurred and rechallenge with a single tablet of Parafon Forte (Chlorzoxazone plus paracetamol) resulted in a dramatic reaction after 5 hours, with fever, chills, nausea, vomiting, and recurrence of icterus. Paracetamol on its own had no adverse effect in this case. The authors reviewed some 23 other cases (SEDA-12, 119) here were two deaths involving hepatic failure. Reports of adverse reactions among six commonly used analgesic—muscle relaxants in Sweden have indicated a low, but comparatively greater, incidence of hepatotoxic reactions associated with a chlorzoxazone-containing compound. (Powers BJ, Cattau EL jr, Zimmermann HJ. Chlorzoxazone hepatotoxic reactions. An analysis of 21 identified or presumed cases. Arch Intern Med 1986;146:1183- 6.).

Nervous system Fatigue**, dizziness**, drowsiness** Gastrointestinal system Reflux**, nausea**, diarrhoea**, gastrointestinal bleeding† Dermis Rash†, pruritus†, urticaria† Immune system Allergic reactions†, angioedema#, anaphylactic reaction# Hepatobiliary Jaundice†, liver damage#, liver impairment#

Frequency: *very common ≥1/10; **frequent ≥1/100 to <1/10; ***infrequent ≥1/1,000 to <1/100; † rare ≥ 1/10,000 to < 1/1,000; π very rare <10,000; # unknown.

6.3.6 Contraindications Chlorzoxazone tablets are contraindicated in patients with known intolerance to the drug.

6.4 Concomitant medication

6.4.1 Permitted concomitant medication All previous medication may be continued.

6.4.2 Prohibited concomitant medication Known inducers (isoniazid) or inhibitors of CYP2E1 (disulfiram). A study physician will screen the concomitant medication taken. Based the study physician’s judgement, individual patients may be excluded from the study if taking medication that puts the patient at risk for clinically relevant drug interactions with the study medication or might jeopardize the study’s scientific value.

7 Study Population

7.1 Type of population Patients with alcohol addiction (as defined by ICD-10) admitted to Salem Hospital for alcohol detoxification.

7.2 Planned number of individuals 60 individuals are planned to be enrolled in the trial, 30 in each study arm (1:1 randomization).

7.3 Inclusion criteria 1. Age: 18–70 years 2. Ability to understand and willingness to comply with study interventions and restrictions. 3. Capacity to consent and ability to provide a written informed consent 4. Voluntarily signed informed consent after full explanation of the study to the participant. 5. Diagnosis of alcohol addiction as defined by ICD-10 6. Patient showing or telling alcohol withdrawal symptoms OR patient who drinks alcohol to avoid the occurrence of alcohol withdrawal symptoms OR patient who in average drinks more than 120 g of alcohol (males) or 80 g of alcohol (females) per day. 7. Hospital admission for alcohol detoxification 8. ASAT >ULN 9. Females of child bearing potential are only included if using reliable contraception with a Pearl Index <1% (i.e. two independent effective contraceptive methods) during the study and two weeks after the last administration of study medication.

7.4 Exclusion criteria 1. Advanced liver disease (liver cirrhosis) or Fibroscan >12kPa 2. Hemoglobin <10 g/dl 3. Uncontrolled diabetes mellitus (defined as fasting blood glucose _200 mg/dl) 4. Significant uncontrolled cardiovascular disease or cardiac insufficiency (New York Heart Association [NYHA] classes III-IV) 5. Renal insufficiency (creatinine clearance calculated with Cockcroft & Gault formula _30 ml/min) 6. Therapy with drugs that act as inhibitors or inducers of CYP2E1 (except ethanol) in the last 14 days. 7. Any antibiotic therapy due to infections 2 weeks prior to first study drug administration 8. Major surgery _4 weeks prior to first study drug administration or ongoing side effects of such surgery 9. Any active systemic infection 10. Systemic diseases (cardiovascular, renal, hepatic, etc.) that would prevent study treatment 11. Allergies (except for mild forms of hay fever) or history of hypersensitivity reactions 12. Any other acute illness or clinically relevant findings in the pre-study examination that could interfere with the patient’s safety or study objectives according to the investigator’s judgement. 13. Treatment with a systemic investigational compound within 28 days prior to the screening visit 14. Inability to communicate well with the investigator due to language problems or poor mental development 15. Contraindication against clomethiazole, chlorazepate, chlorzoxazone or known intolerance of one of the substances or their additives 16. For female participants: pregnancy or lactation

7.5 Admission to the trial A patient will only be admitted to the trial if he or she meets all inclusion criteria and none of the exclusion criteria and if all pre-study clinical, medical, and laboratory examinations have EudraCT No: 2012-005730-11 Protocol Code: K419 Version: 1.1 FINAL 19/01/2015 Page 32 of 55 been evaluated in a positive manner. A pregnancy test will be performed for women of childbearing potential at the screening visit, and at the follow-up visit (V3) and end-of-study visit. A study physician will certify that according to his/her knowledge the individual is eligible by signing the statement of eligibility in the case report form of the individual. EudraCT No: 2012-005730-11 Protocol Code: K419 Version: 1.1 FINAL 19/01/2015 Page 33 of 55

8 Study Procedures

8.1 Pre-study examination In-patients of Salem Hospital admitted for alcohol detoxification treatment that have given their informed consent and signed the informed consent form (ICF) will be screened on the day of admission. Procedures to ensure the patient’s ability to give informed consent Our considerations are based on the decision of the OLG Hamm (OLG Hamm, 20.02.2011 - III-3 RVs 104/10) that ability to consent may also be given with blood alcohol concentrations above 2.0 ‰, but further explanations of relevant circumstances are necessary, e.g. the presence of neurological deficits, previous drinking habits, and further circumstances that allow the assessment of the impairment of the accused’s cognitive capacity through the degree of alcoholisation. The patient’s ability to consent will be assessed immediately before oral explanation and information about the study. A recent blood alcohol concentration value (e.g. from the routine lab upon hospital admittance) must be available for the assessment, alternatively breath alcohol concentration may be used. After assessing the patient’s actual cognitive capacity, adequate behavior, patient’s orientation to person, time, place, and situation, as well as the medical examination (especially the neurological examination), the investigator documents his assessment of the patient’s ability to consent • If the investigator assesses the patient as not able to give consent, the patient cannot be included into the study. • If the investigator has doubts about the patient’s ability to consent OR the blood alcohol concentration (or breath alcohol concentration) is > 2.0 ‰, an independent specialist for internal medicine or psychiatry may be consulted to assess the patient’s capacitiy to give informed consent. The patient may only be considered for the study if the independent specialist assesses the patient to be able to give informed consent. • If the investigator assesses the patient as able to give informed consent AND the blood alcohol concentration (or breath alcohol concentration) is < 2.0 ‰, the patient may be considered for the study and included, if all other inclusion criteria and exclusion criteria are met.

The screening procedures consist of the following: ฀Medical history and current medical conditions ฀Physical examination including measurement of height and weight, blood pressure, pulse rate and temperature, a review of organ systems including lungs, heart, abdomen, liver, kidneys, and peripheral pulses, eyes, nose, throat, skin, and neurological status. ฀Safety laboratory screening (red blood cell count, haematocrit, hemoglobin, white blood cell count, ASAT, ALAT, AP, GGT, CK, LDH, lipase, iron, transferrin, transferrin saturation, total bilirubin, direct bilirubin, cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, C reactive protein (CRP), creatinine, total protein, albumin, electrophoresis of serum proteins, INR, aPTT, electrolytes (sodium, potassium, calcium, and chloride), blood glucose, glycosylated haemoglobin (HbA1c), blood alcohol concentration, Antinuclear antibodies (ANA), screening for hepatitis A, B, and C. ฀Urinalysis: dipstick testing, microscopic examination if dipstick result is pathological. ฀Urine screening for illicit drugs (opioids). ฀Qualitative pregnancy test in female participants. ฀Assessment of liver stiffness with Fibroscan® Results of examinations routinely performed upon admission may be used for screening if they were done within 24 h of the screening day. Blood and urine samples collected at the study site for determination of safety laboratory will be analysed by the Dept. for laboratory medicine of the Salem Hospital and by MVZ Dr. Limbach und Kollegen, Im Breitspiel 15, 69126 Heidelberg, Germany.

Study design (see 5)

8.2 Blood sampling

Screening Safety lab (2.7 ml + 7.5 ml + 3 ml) 13.2 ml

Hospitalisation phase DAY 0: Pre-treatment CZX PK (8x4.9 ml) ≤ 39.2 ml DAY 0: Pre-treatment biomarkers (9 ml) 9 ml DAY 1: CZX/CMZ PK (8x9 ml) ≤ 72 ml DAY 1: Biomarkers (9 ml) 9 ml Daily: CMZTrough levels (_8x4.9 ml) ≤ 39.2 ml Transaminases (_8x2.9 ml) ≤ 21.6 ml DAY 10: CZX PK (8x4.9 ml) ≤ 39.2 ml DAY 10: Biomarkers (9 ml) 9 ml Ambulatory visit DAY 30: CZX PK (8x4.9 ml) ≤ 39.2 ml DAY 30: Biomarkers (9 ml) 9 ml End of study visit Safety lab (2.7 ml + 7.5 ml + 3 ml) 13.2 ml

TOTAL ≤ 312.8 ml

8.3 Other procedures The following routine procedures will also be done in this study according to local standard of care: ฀Assessment of liver stiffness with Fibroscan® ฀Assessment of liver fat content through CAP ฀Abdominal sonography

8.4 Post-Study examination The post-study examination will take place at visit 4 after all study related procedures have been performed before discharging the patient from the study centre or within 21 days of visit 4. The examination consists of the following procedures: ฀Medical history and current medical conditions ฀Physical examination including measurement of height and weight, blood pressure, pulse rate and temperature, a review of organ systems including lungs, heart, abdomen, liver, kidneys, and peripheral pulses, eyes, nose, throat, skin, and neurological status. ฀Safety laboratory screening (red blood cell count, haematocrit, hemoglobin, white blood cell count, ASAT, ALAT, AP, GGT, CK, LDH, lipase, iron, transferrin, transferrin saturation, total bilirubin, direct bilirubin, cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, C reactive protein (CRP), creatinine, total protein, albumin, electrophoresis of serum proteins, INR, aPTT, electrolytes (sodium, potassium, calcium, and chloride), blood glucose, glycosylated haemoglobin (HbA1c), blood alcohol concentration. ฀Urinalysis: dipstick testing, microscopic examination if dipstick result is pathological. ฀Qualitative pregnancy test in female participants If deemed necessary by a study physician based on the results of the end of study visit, the patient may be called in for an additional safety follow-up visit.

8.5 Assessment and recording of adverse events (AE) Any AE must be documented and suspected unexpected serious adverse reactions (SUSAR) will be reported to the responsible authorities (for definitions see appendix 2) like

the Ethics Committee, the Federal Institute for Drugs and Medical Devices (BfArM), and the Eudra Vigilance Database. In all cases the EU-report form must show the identity of the investigator and be dated and signed. The description of the AE will include the onset, duration, seriousness, intensity, outcome, relationship to the study drug, and any treatment required. The investigator will assess the intensity and causality of any AE (for classification see Appendix 2). The observation period starts with visit 1 and lasts until the post-study examination. The investigator follows the outcome of AE or abnormal laboratory findings that are related to study activities until recovery or stabilisation of the participant’s state. Further therapeutic consequences will be documented, for example any medical treatment or drop-out of the study. In the case of a SAE, the Safety Officer (Dr. Nicolas Hohmann, Dept. of Clinical Pharmacology and Pharmacoepidemiology, University Hospital Heidelberg) will be informed within 24 h using a the SAE form (Fax number: +49 (0) 6221-568523). The Safety Officer will inform the investigator or his deputy. The investigator or his deputy will communicate the SAE themselves to the other members of the study team.

8.6 Priority and sequence of trial activities In case of coinciding activities (activities that are scheduled at the same time of the clinical trial), for instance blood sampling and drug administration, the activities will be performed in the following sequence: 1) blood sampling, 2) drug administration, and 3) clinical observations/examinations.

9 Monitoring Qualified personnel from the Department of Clinical Pharmacology and Pharmacoepidemiology will carry out the clinical monitoring. The clinical monitor is responsible for checking the quality of data and adherence to the trial protocol, the legal and ethical requirements according to local laws, and all applicable principles of GCP (Good Clinical Practice). Source data verification is an essential part of the monitoring process and the investigator must grant direct access to all data. The clinical monitor will verify (according to the current version of SOP 6-02009 of the Department of Clinical Pharmacology and Pharmacoepidemiology) ฀Informed consent ฀Inclusion/exclusion criteria ฀Demographic data (identity number, age, and gender) ฀Drug accountability ฀AEs and especially SAEs ฀Premature trial termination (drop-outs and withdrawals) ฀Adherence to the protocol and to Good Clinical Practices ฀Ensure that study medication is being properly stored, dispensed, and accounted for according to protocol specifications In order to comply with the legal requirements of SAE reporting, the procedures and time limits for documentation and submission of reports will be strictly followed. It is important for the investigator to ensure that CRFs (Case Report Forms) are filled in as soon as any procedure is carried out.

10 End of Study End of study is defined as the last safety follow-up visit of the last participant.

11 Withdrawal

11.1 Withdrawal of patients A participant may withdraw his/her consent for further participation at any time. The participant is not required to state the reason for withdrawal. The investigator may decide to withdraw a participant, if the participant suffers from a serious or severe AE regardless of whether it is drug-related or not, if the participant’s safety is compromised for any other reason, or if trial procedures cannot be completed, if the participant violates the protocol, or any other event occurs that is known to interfere with investigation of the target criteria. Allergic reactions related to the study drugs during the study will lead to exclusion of the patient for safety reasons. Severe complications of alcohol withdrawal, e.g. convulsions, will lead to exclusion of the subject due to safety aspects.

11.2 Drop-outs All drop-outs must be recorded by the investigator and will be replaced. After drop-out, individuals can be re-included in the study based on the principal investigator’s judgement. Their inclusion and treatment numbers must not be re-used.

11.3 Follow up – Treatment Depending on the patient’s clinical outcome the investigator or a physician member of the study team may decide that hospitalisation in a rehab facility after acute detoxification is necessary. For these patients the end of study-visit will be after V3 shortly before discharge from Salem Hospital. Participation at V4 is not possible for these patients. Hospitalisation in a rehab centre for follow-up treatment is not considered a SAE (see appendix 2).

12 Early termination of the study The Principal Investigator may decide at any time to terminate the study e.g. in case of SAE or an unexpected high frequency of AE.

13 Study analyses

13.1 Determination of standard laboratory parameters Standard laboratory parameters will be measured by MVZ Dr. Limbach und Kollegen, Im Breitspiel 15, 69126 Heidelberg, Germany. EudraCT No: 2012-005730-11 Protocol Code: K419 Version: 1.1 FINAL 19/01/2015 Page 39 of 55

13.2 Determination of liver stiffness Liver stiffness will be determined with Fibroscan® by a physician or otherwise qualified person in the Salem Hospital.

13.3 Determination of liver fat content Liver fat content will be measured using CAP by a physician or otherwise qualified person in the Salem Hospital.

13.4 Determination of drug concentrations in biological matrices Concentrations of the study drugs clomethiazole and chlorzoxazone and their metabolites if necessary will be determined in biological matrices (e.g. plasma, urine) by an LC/MS/MS method validated according to FDA standards. Analyses are planned to be conducted in the Analytical Chemistry Laboratory of the Department of Clinical Pharmacology and Pharmacoepidemiology.

13.5 Pharmacokinetic analysis Calculation of standard pharmacokinetic parameters of chlorzoxazone and clomethiazole will be performed using Kinetica 5.0 software (Thermo Fisher Scientific, Waltham, MA, USA).

13.6 Statistical evaluation This is a pilot study. For this reason no sample size estimation has been performed. For practical reasons a random convenience sample of n = 30 per treatment arm was defined. It is possible to calculate the effect size based on distances between means (Cohen’s d) of approximately 0.75 with a power of 80% and a significance level of _ = 5%. Because this study is explorative (pilot study), the analysis will be purely descriptive. This is also true for the result of statistical tests. The empirical distribution function will be described through arithmetic mean, standard deviation, median, and range in the case of continuous characters and scores, and through absolute and relative frequency in the case of categorical characteristics. Potential differences in both treatment arms in the course of the study will be analyzed with the help of analysis of variance (ANOVA) with repeated measures for continuous parameters. Baseline values will be integrated into the model as continuous covariates.

13.7 Procedure for handling missing, unused, and inconsistent data Individual missing or inconsistent data will be subject to a simple edit query process. Eventually missing data will not be imputed.

14 Ethical and Legal Aspects

14.1 General The study was planned and will be carried out in accordance with: ฀The legal requirements as laid down in the German “Arzneimittelgesetz” (AMG), the ”Bundesdatenschutzgesetz” (BDSG), and the medical professional code of conduct (Berufsordnung der Ärzte in Baden-Württemberg). ฀The actual version of the Declaration of Helsinki adopted by the World Medical Association in June 1964. ฀The ICH recommendations on Good Clinical Practice.

14.2 Benefit-to-risk ratio Concerning treatment: The medication administered during the study is routinely used during alcohol detoxification (Hillemacher T et al. Pharmacopsychiatry 2008;41:134-7.; Haddox VG et al. West J Med 1987;146:695-6.) and the drugs used are equal alternatives in alcoholic liver disease (Caspari D et al. Psychiatr Prax 1992;19:23-7.). The study drugs are approved treatments and standard of care for patients with alcohol withdrawal symptoms. Concerning CYP2E1 phenotyping: We will administer a single 500 mg oral dose of chlorzoxazone to the patients at 4 different occasions. Chlorzoxazone, an approved drug in the European Union, is the recommended probe drug for CYP2E1 phenotyping (Frye RF et al. J Clin Pharmacol 1998; 38: 82). The safety of this procedure has been established in healthy volunteers (Minshin VM et al. Am J Gastroenterol 1998; 93:215) and in patients suffering from alcoholism (Onmeta CM et al. J Hepatol 2001; 32: 47). The administered doses are the standard doses but dosing frequency is well below the therapeutic schedule of 3-4x500 mg daily. There is no risk of drug accumulation and the risk for adverse events or toxicity is small. Concerning blood sampling: On PK days, blood will be drawn from an indwelling catheter, on non-PK days blood will be drawn after phlebotomy. The maximum amount of blood drawn is 321.8 ml over ~30 d. This amount is well below the volume drawn when donating blood. Therefore the risk of anemia or hypovolemia is negligible. The venous puncture for blood withdrawal and the amount of blood withdrawn is associated with minimal risks for the patient. Concerning other diagnostic procedures: All examinations (laboratory values, sonography, or fibroscan) are routine procedures at hospital admission and before discharge. Sonography and fibroscan® are non-invasive procedures negligible risk of adverse effects. Most procedures of this study are standard of care. The only study-related additional examinations for the patients are the chlorzoxazone phenotyping test and an additional visit 30 days after the initiation of alcohol therapy with laboratory analyses, sonography, and fibroscan®. Overall, the risks of this study appear very small and justified considering the potential clinical benefits of identifying a superior treatment with regard to liver damage and liver regeneration and the scientific benefit of understanding more about the involvement of CYP2E1 and consequences of CYP2E1 inhibitions in humans.

14.3 Submission to the Ethics Committee and Competent Authority (BfArM) The investigator submits the required documents to the Ethics Committee of the Medical Faculty of the University of Heidelberg and obtains the opinion of the Committee in writing. The investigator also submits the required documents to the BfArM (Bundesinstitut für Arzneimittel und Medizinprodukte) and obtains their approval in writing. Participants will not be included until receiving unconditional approval of the BfArM and the Ethics Committee. Both, Ethics Committee and BfArM will be informed of all suspected unexpected serious AE

(SUSAR) according to GCP guidelines.

14.4 Notification of authorities According to § 67 of the German ”Arzneimittelgesetz” the ”Regierungspräsidium Karlsruhe” will be notified about the study after approval of the competent authority (BfArM).

14.5 Participant information and informed consent Before an individual is included in the study, the objectives, risks, and methods of the study are explained to him or her orally and in writing. The individual gives his/her consent in signing an informed consent form. With their consent, participants agree that their data are given, if necessary, in pseudonymised form to the competent authorities and that the personnel of the Department of Internal Medicine (Salem Hospital) and of the Department of Clinical Pharmacology and Pharmacoepidemiology (University Hospital of Heidelberg) can take insight into their data as far as it is necessary for reviewing the procedure of the study.

14.6 Responsibilities The investigators whose names and addresses are on the cover page agree to assume personal responsibility as described in the Appendix 1 (see below). Approval of the clinical study protocol by the investigator implies that he/she agrees to comply with Good Clinical Practice recommendations as far as relevant for drug trials not performed for drug approval.

14.7 Insurance According to the legal requirements (§40 AMG) an insurance policy has been issued before the beginning of the study.

14.8 Confidentiality The data obtained in the course of the trial will be treated pursuant to the Federal Data Protection Law (Bundesdatenschutzgesetz, BDSG). During the clinical trial, participants will be identified solely by means of year of birth and an individual identification code (participant number). Trial findings stored on a computer will be stored in accordance with the local data protection law and will be handled in the strictest confidence. For protection of these data, organisational procedures are implemented to prevent distribution of data to unauthorised persons. The appropriate regulations of local data handling policies will be fulfilled in its entirety. The participant consents in writing to release the investigator from his/her professional discretion in as far as to allow inspection of original data for monitoring purposes by health authorities and authorised persons. Authorised persons (clinical monitors, inspectors, and auditors) may inspect the participant-related data collected during the trial ensuring the data protection law. The investigator will maintain a participant identification list (participant numbers with the corresponding participant names) to enable records to be identified. Volunteers who do not consent to circulate their pseudonymised data will not be included into the trial.

14.9 Archiving The investigator is responsible for archiving the participants’ files and also all documents concerning the study for at least 10 years after the end of the study.

15 Organization of the study

15.1 Protocol modifications and amendments If the study protocol has to be changed after approval, a written amendment is required that must be signed by the same persons as the study protocol. Any protocol amendment will only be implemented after approval has been granted by Ethics Committee and Competent Authorities. Substantial amendments affecting the benefit-to-risk ratio must be approved by Ethics Committee and BfArM. These procedures must be completed before any modifications can come into operation, except when they are necessary to eliminate immediate hazards for the study participants

15.2 Access to source data, quality control, and data handling For the purpose of ensuring compliance with the Clinical Trial Protocol, Good Clinical Practice, and applicable regulatory requirements, the investigator should permit auditing/inspection by responsible regulatory authorities. The investigator will grant the auditors/inspectors direct access to the study records for review, being understood that this personnel is bound by professional secrecy and as such will not disclose any personal identity or personal medical information. The investigator will make every effort to help with the performance of the audits and inspections, giving access to all necessary facilities, data, and documents. The confidentiality of the data verified and the protection of the participants should be respected during these inspections.

15.2.1 Declaration of informed consent For each participant in the clinical trial, a written declaration of informed consent has to be kept by the Investigator in the participant's medical record (especially after completion of the study) or - during the study - in the Investigator's study file.

15.2.2 Case report form (CRF) All data to be recorded according to this study protocol must be documented in a CRF. For each patient a separate CRF must be completed. Each page of the CRF must carry the study number and an allocation / random number. The Investigator must ensure that the patient’s pseudonymity will be maintained. Entries in the CRF must only be made by the Investigator or persons authorized by him. If corrections in the CRF are necessary, the initial entry must be crossed out but remain legible. The new entry must be dated and signed. If appropriate, the reason for the change must be provided.

15.2.3 List of patients (patients’ log) The Investigator keeps a confidential list with full names, date of birth, and addresses of all patients participating in the study, giving reference to the patients’ records. For enrolled patients, the date of enrolment and the allocation / random number must be recorded. With the help of this list it must be possible to identify the patients and their medical records, e.g. ifquestions arise after completion of the study. Care must be taken that this list is kept confidential by the Investigator. The identity of the patients must not be revealed to unauthorized persons.

15.2.4 Patient medical records / source data Patient medical records must be kept for all patients taking part in the study. Participation in the study, administration of study medication, and key data recorded during the study must be documented in the patient medical record as source data unless specified otherwise in this study protocol. Data in the patient medical record (including original laboratory reports etc.) are the ‘source data’ and entries in the CRF have to be checked against these data

(source data verification).

15.2.5 Data Handling All data will be entered in a database with double data entry. After checking for plausibility, consistency, and completeness, queries will be produced. If no data are missing, database will be closed and used for statistical analysis.

15.2.6 Drug accountability sheet On the drug accountability sheet, the Investigator must document receipt of study medication, supply of study medication to patients, and return of study medication.

15.3 Financing This study is a non-commercial investigator initiated trial. The study is financed by the dept. of internal medicine, Salem Hospital. Analytical chemistry is financed by the dept. of clinical pharmacology and pharmacoepidemiology, University Hospital Heidelberg.

15.4 Publication of the study results The results of the study will be published in an international scientific peer-reviewed journal.

16 References ฀Seitz HK, Salaspuro M, Savolainen M, Haber P, Ishii H, Teschke R, Moshage H, Lieber CS. From alcohol toxicity to treatment. Alcohol Clin Exp Res 2005;29:1341-50. ฀Oneta CM, Lieber CS, Li J, Rüttimann S, Schmid B, Lattmann J, Rosman AS, Seitz HK. Dynamics of cytochrome P4502E1 activity in man: induction by ethanol and disappearance during withdrawal phase. J Hepatol 2002;36:47-52. ฀Cederbaum AI. Role of CYP2E1 in ethanol-induced oxidant stress, fatty liver and hepatotoxicity. Dig Dis 2010;28:802-11. ฀Lu Y, Wu D, Wang X, Ward SC, Cederbaum AI. Chronic alcohol-induced liver injury and oxidant stress are decreased in cytochrome P4502E1 knockout mice and restored in humanized cytochrome P4502E1 knock-in mice. Free Radic Biol Med 2010;49:1406-16. ฀Tubridy P. Alprazolam versus chlormethiazole in acute alcohol withdrawal. Br J Addict 1988;83:581-5. ฀Fachinformation Distraneurin®, Stand Februar 2011. ฀Dilger K, Metzler J, Bode JC, Klotz U. CYP2E1 activity in patients with alcoholic liver disease. J Hepatol 1997;27:1009-14. ฀Eap CB, Schnyder C, Besson J, Savary L, Buclin T. Inhibition of CYP2E1 by chlormethiazole as measured by chlorzoxazone pharmacokinetics in patients with alcoholism and in healthy volunteers. Clin Pharmacol Ther 1998;64:52-7. ฀Fachinformation Tranxilium®, Stand September 2013. ฀Mielke DH, Gallant DM, McFarlain RA. Clorazepate dipotassium (Tranxene): a controlled evaluation in alcoholic patients after withdrawal. Curr Ther Res Clin Exp 1976;19:506-11. ฀Meyler’s Side Effects of Drugs, Elsevier Ltd, Oxford; Auflage: 15th Revised edition (REV). (25. September 2006) ฀Stockley's Drug Interactions, 9th Edition. Baxter K., EditorPharmaceutical Press, London. ฀Drug information Parafon DSC chlorzoxazone tablet, Keltman pharmaceuticals. Available under: http://dailymed.nlm.nih.gov/dailymed/lookup.cfm?setid=5af155d7-3780- 435b-9a87-0ce01f1ed6ec. Last accessed: 11th July 2014. ฀Summary of Products characteristics Paraflex®, Biophausia Schweden, Stand 2010. ฀Frye RF, Adedoyin A, Mauro K, Matzke GR, Branch RA. Use of chlorzoxazone as an in vivo probe of cytochrome P450 2E1: choice of dose and phenotypic trait measure. J Clin Pharmacol 1998;38:82-9. EudraCT No: 2012-005730-11 Protocol Code: K419 Version: 1.1 FINAL 19/01/2015 Page 47 of 55 ฀Kharasch ED, Thummel KE, Mhyre J, Lillibridge JH. Single-dose disulfiram inhibition of chlorzoxazone metabolism: a clinical probe for P450 2E1. Clin Pharmacol Ther 1993;53:643-50. ฀Choi MG, Camilleri M, Burton DD, Zinsmeister AR, Forstrom LA, Nair KS. Reproducibility and simplification of 13C-octanoic acid breath test for gastric emptying of solids. Am J Gastroenterol 1998;93:92-8. ฀Majumdar SK. Chlormethiazole: current status in the treatment of the acute ethanol withdrawal syndrome. Drug Alcohol Depend 1991;27:201-7. ฀Caspari D, Wappler M, Bellaire W. [Treatment of delirium tremens--a comparison between clomethiazole and clorazepate with reference to effectiveness and rate of side effects]. Psychiatr Prax 1992;19:23-7. ฀Hillemacher T, Weinland C, Heberlein A, Wilhelm J, Bayerlein K, Kornhuber J, Frieling H, Bleich S. Treatment with clomethiazole is associated with lower rates of premature discharge during alcohol withdrawal. Pharmacopsychiatry 2008;41:134-7. ฀Haddox VG, Bidder TG, Waldron LE, Derby P, Achen SM. Clorazepate use may prevent alcohol withdrawal convulsions. West J Med 1987;146:695-6. EudraCT No: 2012-005730-11 Protocol Code: K419 Version: 1.1 FINAL 19/01/2015 Page 48 of 55

Appendices

APPENDIX 1: Responsibilities of the Principal Investigator During the conduct of the study the Principal Investigator is responsible for the following points. In accordance with Good Clinical Practice, he should:

1. Before the beginning of the study ฀Have all study documents concerning the study drugs. ฀Ensure that he and his team will be available to conduct the study under good conditions. ฀Organise the technical facilities at his disposition so that the study is carried out correctly. ฀Submit the protocol, the compound information sheet and the information and informed consent form (as applicable) to the Ethics Committee. ฀Ensure that the batches of study drugs are stored under good storage conditions in a specific place inaccessible to third parties. ฀Sign to confirm his agreement to carry out the clinical study protocol in compliance with ICH-GCP, GCP-V, and AMG. ฀Supply each participant with the required information concerning the study drug and the study procedure before the inclusion in the study

2. During the study ฀Ensure that the protocol and its appendices are faithfully followed. Notably, if treatment is discontinued for any participant, every effort will be made to obtain information about this participant as promptly as possible after discontinuation of treatment. ฀Give the participant any new information concerning the study drug which may modify his consent to participate in the study. ฀If there is a modification to the risk/benefit ratio, submit these to the Ethics Committee. ฀Guarantee that the drugs will be administered exclusively within the context of the protocol. ฀Mark the participants’ files to indicate their participation in a clinical study, showing dates and data involved in the study. ฀Promptly establish a case report form in case of a SAE. ฀Make a list of personnel involved in the study and update in case of any changes.

3. After the study ฀Retain the list of names of participants included in the study (to be attached to the study documentation). ฀Ensure that the participants’ files and all documents relating to the study are archived for at least 10 years after the end of the study (the patient identification list has to be archived for at least 15 years). ฀Date and sign the final report. ฀All corrections or additions to a final report must be made in the form of a justified amendment, signed, and dated by the investigator.

Responsibilities of the members of the study team ฀Have all study documents concerning the study drugs. ฀Ensure that he will be available to conduct the study under good conditions. ฀Organise the technical facilities so that the study is carried out correctly. ฀Ensure that the batches of study drugs are stored under good storage conditions in a specific place inaccessible to third parties.

฀Sign to confirm his agreement to carry out the clinical study protocol in compliance with GCP. ฀Supply each participant with the required information concerning the study drug and the study procedure before the inclusion in the study. ฀Ensure that the protocol and its appendices are faithfully followed. Notably, if treatment is discontinued for any participant, every effort will be made to obtain information about this participant as promptly as possible after discontinuation of treatment. ฀Give the participant any new information concerning the study drug which may modify his consent to participate in the study. ฀Guarantee that the drugs will be administered exclusively within the context of the protocol. ฀Correctly fill in the CRFs or check that they have been correctly filled in by the person delegated to do this. In case of erroneous data, all modifications should be initialled and dated. The original data must remain legible. Date and sign the CRF. ฀Promptly establish a case report form in case of a SAE.

APPENDIX 2: Definition of adverse events Definition of an adverse event (AE) An AE is any unfavourable and unintended sign (including an abnormal laboratory finding), symptom, or disease temporally associated with the use of a medical treatment or procedure that may or may not be considered related to the medical treatment or procedure. Every change in laboratory values of vital function of the patient that leads to discontinuation of the study medication or discontinuation of the study should be regarded as AE. Definition of a serious adverse event (SAE) A SAE is an AE that at any time during the period of observation: ฀results in death ฀is immediately life-threatening ฀requires or prolongs hospitalisation ฀results in persistent or significant disability or incapacity ฀involves congenital anomaly ฀is medically important ฀requires medical intervention to prevent permanent impairment or damage Exceptions from SAE In the following cases AEs will not be regarded as SAEs: ฀Relapse to alcoholism ฀Re-admittance for alcohol detoxification to Salem Hospital after relapse ฀Admittance to a rehab centre for follow-up treatment ฀Planned hospitalisation exclusively for diagnostic procedures Definition of unexpected adverse event An unexpected AE is an AE of which the nature and severity is not consistent with the applicable product informaion. Definition of suspected unexpected serious adverse reaction (SUSAR) A SUSAR is a suspected unexpected serious adverse reaction. All AE that are suspected to be related to an investigational medicinal product and that are both unexpected and serious are considered to be SUSARs. Intensity

The assessment of intensity will be based on the investigator’s clinical judgment using the following definitions:

Mild: an event that is easily tolerated by the patient, causing minimal discomfort and not interfering with everyday activities.

Moderate: an event that is sufficiently discomforting to interfere with normal everyday

activities.

Severe: An event that prevents normal everyday activities. Causality The causal relationship of an AE to treatment (treatment indicates all investigational medicinal products including placebo) has to be assessed in accordance with WHO criteria in a modified version: Edwards IR, Biriell C. Harmonisation in Pharmaco-vigilance. Drug Safety 2004;10:93-102.

The investigator will use clinical judgment to determine the relationship. Alternative causes, such as natural history of the underlying diseases, concomitant therapy, other risk factors etc. will be considered. The investigator will also consult the Investigator Brochure or other product information. The relationship between the drug and the occurrence of each AE will be evaluated and classified as:

Certain: A clinical event, including laboratory test abnormality, occurs in a plausible time relationship to drug administration, and which cannot be explained by concurrent disease or other drugs or chemicals. The response to withdrawal of the drug (dechallenge) should be clinically plausible. The event must be definitive pharmacologically or phenomenologically, using a satisfactory rechallenge procedure if necessary.

Probable: A clinical event, including laboratory test abnormality, with a reasonable time sequence to administration of the drug, unlikely to be attributed to concurrent disease or other drugs or chemicals, and which follows a clinically reasonable response on withdrawal (dechallenge). Rechallenge information is not required to fulfil this definition.

Possible: A clinical event, including laboratory test abnormality, with a reasonable time sequence to administration of the drug, but which could also be explained by concurrent disease or other drugs or chemicals. Information on drug withdrawal may be lacking or unclear.

Unlikely: A clinical event, including laboratory test abnormality, with a temporal relationship to drug administration which makes a causal relationship improbable, and in which other drugs, chemicals or underlying disease provide plausible explanations.

Unclassified: A clinical event, including laboratory test abnormality, reported as an adverse reaction, about which more data are essential for a proper assessment or the additional data are under examination.

Unclassifiable: A report suggesting an adverse reaction which cannot be judged because information is insufficient or contradictory, and which cannot be supplemented or verified.

Not related: A clinical event, including laboratory test abnormality, with no relationship to drug administration, and in which other drugs, chemicals or circumstances provide plausible explanations.

Reporting of Serious Adverse Events by Investigator All SAEs must be reported by the investigator to the responsible Safety Officer (Principle Investigator) within 24h after the SAE becomes using the “Serious Adverse Event” form. The initial report must be as complete as possible including details of the current illness and (serious) AE and an assessment of the causal relationship between the event and the trial medication. (The investigator must also inform the site monitor in all cases). SUSAR - Responsibilities of the Principal Investigator The Principal Investigator is responsible for the notification of the SUSAR to the Ethics Committee and competent authority of each concerned Member State of findings that could adversely affect the health of participants, impact on the conduct of the study, or alter the

competent authorisation to continue the study in accordance with Directive 2001/20/EC. In the case of SUSARs that resulted in death or were life-threatening the competent authority, the Ethics Committee, and all investigators should be notified as soon as possible but no later than 7 calendar days after the Principal Investigator has first knowledge of the minimum criteria for expedited reporting. In each case relevant follow-up information should be sought and a report completed as soon as possible. It should be communicated to the competent authority and the ethics committee in the concerned Member States within an additional eight calendar days. All other SUSARs and safety issues must be reported to the competent authority and the Ethics Committee in the concerned Member States as soon as possible but no later than 15 calendar days after the Principal Investigator has first knowledge of the minimum criteria for expedited reporting. Further relevant follow-up information should be given as soon as possible. Person-related data have to be pseudonymised by using a participant code number. Each SUSAR has to be reported as a single case to the competent authority in the concerned Member State. Expedited reporting is not usually required: for reactions which are serious but expected.

For regulatory purposes, initial expedited reports should be submitted within the time limits as soon as the minimum following criteria are met: ฀A suspected investigational medicinal product ฀An identifiable participant (pseudonymised) ฀An AE assessed as serious and unexpected, and for which there is a reasonable suspected causal relationship ฀An identifiable reporting source ฀A unique study identification number (EudraCT number) ฀An assessment about causality, intensity, data about the started therapies and the outcome. Outcome of an adverse event The outcome of an AE at the time of the last observation will be classified as:

‘Recovered/resolved’: All signs and symptoms of an AE disappeared without any sequels at the time of the last interrogation.

‘Recovering/resolving’: The intensity of signs and symptoms has been diminishing and/or their clinical pattern has been changing up to the time of the last interrogation in a way typical for its resolution.

‘Not recovered/not resolved’: Signs and symptoms of an AE are mostly unchanged at the time of the last interrogation.

‘Recovered/resolved with sequel’: Actual signs / symptoms of an AE disappeared but there are sequels related to AE.

‘Fatal’: Resulting in death. If there are more than one AE only the AE leading to death (possibly related) will be characterised as “fatal”.

‘Unknown’: The outcome is unknown or implausible and the information cannot be supplemented or verified.

Action taken with IMP The action taken with IMP will be assigned to one of the following categories: ‘Dose not changed’: no change in the dose of IMP.

‘Dose reduced’: reduction in the dose of IMP. ‘Dose increased’: increase in the dose of IMP. ‘Drug withdrawn’: discontinuation of IMP. ‘Unknown’: the information in unknown or implausible and it cannot be supplemented or verified. ‘Not applicable’: the question is implausible (e.g. the participant is dead.) ‘Countermeasures‘ refers to the specific actions taken to treat or alleviate AE or to avoid their sequels. Following categories will be used to categorise the countermeasures to AE. None: no action taken.

Drug treatment: newly prescribed medication or change in dose of a medication

Others: other countermeasures, e.g. a surgical procedure

Procedures for reporting of SUSAR In accordance with the internal University regulations the Principle Investigator of an Investigator Initiated Trial also acts as sponsor. All serious AEs (including SUSARS) are documented on the international CIOMS form (SOP 6-02005). According to §42 and §63b AMG (Arzneimittelgesetz/German Drug Law) serious adverse reactions fulfilling specified criteria have to be reported by the Principal Investigator within 15 days to the Ethics Committee, the competent authorities of the member states in whose territory the clinical study is conducted, and to the Investigators participating in the study. SUSARS that are fatal or life-threatening have to be reported even within 7 days (GCP-V §13).

References ฀Detailed guidance on the European database of Suspected Unexpected Serious Adverse Reactions. ฀Eudravigilance – Clinical Trial Module, April 2004 ฀Verordnung über die Anwendung der Guten Klinischen Praxis bei der Durchführung von klinischen Prüfungen mit Arzneimitteln zur Anwendung am Menschen ฀(GCP Verordnung-GCP-V), Bundesgesetzblatt Jahrgang 2004 Teil I Nr. 42. ฀9. August 2004 ฀Clinical Safety Data Management: Data elements for transmission of individual case safety reports, CPMP/ICH/287/95 ฀The European Agency for the Evaluation of Medicinal products, London, ฀16. November 2000 ฀Note for Guidance on Definitions and Standards for expedited reporting (CPMP/ICH/3945/03) ฀The European Agency for the Evaluation of Medicinal products, London, ฀November 2003

APPENDIX 3: List of raw data relating to the study Study data which should be included in the source dossier: ฀Participant’s identity ฀Medical history, physical examination, ECG, and safety laboratory values ฀Dates of administration of the study drug ฀Intermittent (concomitant) treatments ฀Dates of participation in the study ฀Additional examinations or assessments carried out during the study ฀AE forms (+ follow-up) ฀Date of drop-out and reason

Hohmann N, et al. Gut 2021;0:1–2. doi: 10.1136/gutjnl-2021-324727