Safety and Efficacy of the 10-Day Melarsoprol Schedule for the Treatment of Second Stage Rhodesiense Sleeping Sickness

Objective Assessment of the safety and efficacy of a 10-day melarsoprol schedule in second stage T.b. rhodesiense patients and the effect of suramin-pretreatment on the incidence of encephalopathic syndrome (ES) during melarsoprol therapy. Design Sequential conduct of a proof-of-concept trial (n = 60) and a utilization study (n = 78) using historic controls as comparator. Setting Two trial centres in the T.b. rhodesiense endemic regions of Tanzania and Uganda. Participants: Consenting patients with confirmed second stage disease and a minimum age of 6 years were eligible for participation. Unconscious and pregnant patients were excluded. Main Outcome Measures The primary outcome measures were safety and efficacy at end of treatment. The secondary outcome measure was efficacy during follow-up after 3, 6 and 12 months. Results The incidence of ES in the trial population was 11.2% (CI 5–17%) and 13% (CI 9–17%) in the historic data. The respective case fatality rates were 8.4% (CI 3–13.8%) and 9.3% (CI 6–12.6%). All patients discharged alive were free of parasites at end of treatment. Twelve months after discharge, 96% of patients were clinically cured. The mean hospitalization time was reduced from 29 to 13 days (p<0.0001) per patient. Conclusions The 10-day melarsoprol schedule does not expose patients to a higher risk of ES or death than does treatment according to national schedules in current use. The efficacy of the 10-day melarsoprol schedule was highly satisfactory. No benefit could be attributed to the suramin pre-treatment. Trial Registration Current Controlled Trials ISRCTN40537886


Overview of Human African Trypanosomiasis
Human African Trypanosomiasis (HAT), better known as sleeping sickness is caused by the protozoan . However, the importance of HAT with regard to public health lies not in the annual incidence, but in its potential for development of explosive epidemics 3 . If only the incidence is considered, HAT appears to be a minor health problem compared to other parasitic diseases like malaria and helminthic infections. However, even single cases or endemic outbreaks can severely affect whole families, the local economic situation and large scale epidemics were fatal for many thousand people in the past. If the DALY figures (e.g. loss of healthy life years by premature mortality and disability) are considered, the social and economic impact of trypanosomiasis even ranks third of all parasitic diseases behind malaria and schistosomiasis in sub-Saharan Africa 4 .
The clinical profile of HAT is too diffuse to allow for a direct differential diagnosis. Only the proof of the parasites presence in blood, lymph nodes or cerebrospinal fluid is sufficient. T.b. gambiense can be sero-diagnosed with the CATT test (Card Agglutination Test) followed by microscopic confirmation of the parasite in blood and/or lymph. The diagnosis of T.b. rhodesiense has to entirely rely on microscopy. The sensitivity of this method is dependent on the level of parasitaemia and the technicians' experience. Unfortunately, no other diagnostic methods are available. Once the presence of trypanosomes in blood and/or lymph is confirmed, a lumbar puncture has to follow for stage determination. The two parasites are morphologically identical and differentiation is only possible by analysis of isoenzymes and /or DNA-analysis.
The clinical presentation of Rhodesian and Gambiense sleeping sickness is remarkably different. The highly virulent T.b. rhodesiense causes a precipitated evolution of the disease (down to 8 days between infective bite and the presence of trypanosomes in the cerebrospinal fluid 5 , while the less virulent T.b. gambiense about low parasitaemias, elusive and mild symptoms for months, and insidious evolution towards the nervous stage 6 .
T.b. rhodesiense infections presents as an acute febrile illness that is fatal within weeks or months if left untreated 7 . At the site of the infective bite with T.b. rhodesiense, parasites proliferate and, occasionally, lead to nodule or ulcer called a chancre, involving widespread lymph node enlargement.
It is very difficult to distinguish such symptoms in the beginning from other tropical fevers, such as malaria, bacterial meningitis or enteric fever. The parasite becomes established in the blood and it multiplies, with intermittent fever, splenomegaly and often signs of myocardial involvement. The study of the duration of symptoms and the case fatality of T.b. rhodesiense showed that the disease progressed to the stage of central nervous system involvement between three weeks to two months of infection. Compared to the Gambiense form, less demarcation between first and second stage illness is observed. The CNS involvement in T.b. rhodesiense infections can be clinically limited to drowsiness and tremor 8 . Most (> 80%) deaths occurred within six months of illness 9 , often due to cardiac failure or secondary infections.
Clinical symptoms of Gambiense trypanosomiasis commence by an uncharacteristic general malaise.
Typical but not characteristic signs and symptoms are fever, headache, joint pains, transient oedema, In the second stage of the disease neuropsychiatric signs and symptoms occur. Severe endocrinological and mental disturbances, such as impotence, infertility, amenorrhea, delirium, mania, paranoia, schizoid attacks, aggressive behaviour and severe motor problems are the main signs.
Until today, the underlying mechanisms of CNS invasion are not known in detail. Frequently observed is meningoencephalitis. The meninges are infiltrated with lymphocytes, plasma cells and occasional morular (Mott) cells. The inflammatory cell infiltrate extends along the Virchow-Robin spaces into the substance of the brain producing the characteristic picture of perivascular cuffing 10

Melarsoprol Treatment
Treatment regimens of melarsoprol used to be empirically determined and vary considerably between countries and treatment centres. The treatment regimens for melarsoprol embody a real omniumgatherum throughout Africa.
Melarsoprol treatment in T.b. rhodesiense patients is in general preceded by one or two injections of suramin. The pre-treatment is intended to clear parasitaemia in blood and lymph before the treatment Company Confidential -Swiss Tropical Institute Protocol P-001-05-01-03 with melarsoprol starts. This is thought to prevent from an initial high antigen release which might trigger major adverse reactions in second stage treatment and the introduction of trypanosomes in the CNS while performing the diagnostic LP. However, there is no solid scientific evidence for this approach.
The current treatment schedules for melarsoprol in East Africa (see Table 1 combination treatments are ongoing. Likely such a scenario will sooner or later also be required for treatment of T.b. rhodesiense in the lack of alternatives. Since eflornithine is not sufficiently effective in T.b. rhodesiense combinations with melarsoprol will be used. To achieve this, the abridged treatment schedule will be most helpful. Recent reports by field workers about suramin resistance (unpublished) are a further concern. The parasites are exposed in first as well as in second stage treatment to suramin. Especially in second stage treatment very low doses are applied. The frequent uses of the drug at low doses may influence the development and dynamics of resistance.

Development of a new, abridged melarsoprol schedule against late stage infections
Even though melarsoprol was introduced in 1949 only recently its pharmacokinetic and pharmacological properties have been investigated [17][18][19][20] .

Uganda
Uganda is the only country affected by both forms of the disease, T.b. rhodesiense in the South-East and T.b. gambiense in North-West of the country. Recently, the two closest foci in the centre of the country increased in size and approached each other significantly. A potential overlap of the two diseases has become possible and must be monitored carefully 25

Tanzania
Rhodesiense sleeping sickness, which was first reported in the 1920s and 1930s, was endemic in

Situation in other East African countries
Kenya, Mozambique, Rwanda, Zambia and Zimbabwe report sporadic less than 50 cases annually.
Botswana, Burundi, Ethiopia, Namibia and Swaziland have not reported any cases since 1990 24 .
But large civil-and livestock movements can easily import T.b. rhodesiense to new areas, or historical foci can re-emerge.

Conclusion and Justification
The new, abridged protocol for melarsoprol 30  In the planning of any clinical program in T.b. rhodesiense sleeping sickness, the difficulties in patient's access and diagnosis and the overall limited total number of patients have to be kept in mind.
All investigations have therefore to be limited to proof-of-concept studies. This clinical trial protocol covers two sequential proof-of-concept trials studies with total enrolment number of 60 patients.
One objective of the IMPAMEL III project is to investigate the importance of the suramin pre-treatment.
Currently, the pre-treatment with suramin is administered for two reasons: − To avoid the introduction of parasites into the CSF during the diagnostic LP.
− To clear parasites from blood and lymph in order to reduce the antigen release prior to melarsoprol administration.
The introduction of trypanosomes during LP is theoretically possible. However, the usage of proper materials and technical skills make a LP without blood vessel damage possible. In the rare case of trypanosome introduction to the CSF, the parasites are hampered in growth and survival as the CSF is a suboptimal medium 31 . Some authors believe that a high initial antigen release can trigger immunological overreactions 32 , e.g. increase the risk of ES and consequently increase mortality.
However, the dynamics of parasite elimination in the first hours after drug contact are very similar for melarsoprol and suramin 33 ; the effect of the low-dosed suramin pre-treatment on the parasites is doubtful and will be assessed in these trials. Suramin pre-treatment policies are heterogeneous: in Uganda, only one test dose of 5mg/kg is administered; in Tanzania the test dose (5mg/kg) is followed by one full dose (20mg/kg). In Malawi the suramin pre-treatment is centre specific: some hospitals follow the same schedule as in Tanzania; others do not administer a suramin pre-treatment at all. Also Kenya's doesn't apply the pre-treatment with suramin anymore.
The parasite's exposure to the same drug in first as well as in second stage therapy is suboptimal.
The insufficient doses used in the pre-treatment of late stage disease are rather prone to trigger drug resistances. Field reports of suramin treatment failures/resistances exist, but unfortunately no scientific data is currently available.
Company Confidential -Swiss Tropical Institute Protocol P-001-05-01-03 The additional use of suramin may also increase the risk of the patient for adverse drug reactions like renal impairment. During the IMPAMEL III trials, proteinuria will be monitored by urine analysis.
Suramin (Germanin™) is delivered in vials of 1g suramin sodium powder, which upon usage has to be dissolved in water and should be used within 15 minutes. Quantities remaining in the vial are not suitable for further use. The suramin pre-treatment tends to be uneconomic and the risk-benefit ratio should be re-considered.
The main objective of the IMPAMEL III trials is to assess the safety, tolerability and efficacy of the 10day melarsoprol schedule against late stage T. Africa: not only to be able to offer a substantial better treatment on the basis of an existing drug, but also to form the basis of a harmonization process of all East African treatment protocols in use. Most importantly, it can not be excluded, that melarsoprol will loose some of its efficacy against T.b.
rhodesiense, similar to the phenomenon recently observed in some T.b. gambiense areas 35 . To test and potentially introduce the 10-day schedule in East Africa is a preparatory step for upcoming combination treatments as no alternative drug will be available in the next 5 to 8 years. For Company Confidential -Swiss Tropical Institute Protocol P-001-05-01-03 pharmacological and practical reasons, all attempts for combination treatment with melarsoprol (with nifurtimox and eflornithine) are based on the abridged 10-day schedule.
The incidence rate of the encephalopathic syndromes (ES) under melarsoprol treatment is reported to be 8% in Rhodesiense and 5% in Gambiense sleeping sickness 14 . For about 50% of those cases the ES is a fatal event. According to current guidelines in drug safety, melarsoprol would never pass today's standards. Still today, little knowledge and data on the ES is available. In Angola and DRC, an association study was recently carried out where the ES was correlated with the HLA-genotype 14 .
Unfortunately, the study could not provide enough samples in order to present significant results.
Within the IMPAMEL III trials each patient is asked to provide a blood sample in order to investigate this correlation. Until today, no such data for T.b. rhodesiense is available.
HAT treatment requires a long-term follow-up of each patient in order to monitor drug safety and cure.
Usually, patients are asked to present for follow-up visits after 3  is only active against T.b. gambiense. In case of an overlap of the two disease zones, which treatment will a late stage patient receive? How to treat mixed infections? To have at least the IMPAMEL schedule tested and implemented in both forms of the disease would greatly facilitate such a scenario.
As a conclusion, only the successful translation of knowledge and results gained on the level of trial sites to the national level will provide a significantly positive contribution to the patient's quality of life.

Primary Objective
The primary objective is to further assess the safety, tolerability and efficacy of the 10-day melarsoprol schedule without suramin pre-treatment against late stage T.b. rhodesiense infections.

Secondary Objective
In a sub-study blood samples are collected in order to: Distinction of relapses and re-infections in cases of positive follow-up results.
Investigation of a possible correlation between the HLA-genotype and the occurrence of the ES.

Investigational Plan
The trial protocol was developed in accordance with the Declaration of Helsinki and the ICH Guideline on Good Clinical Practice.

Overall Study Design
This is a two-centred, multinational utilization study. A minimum of 70 subjects with confirmed late stage T.b. rhodesiense sleeping sickness will receive 10 consecutive melarsoprol injections at a dosage of 2.2mg/kg (but a maximum of 180mg).
A minimum of 70 subjects who satisfy the inclusion and exclusion criteria will be enrolled in order to evaluate the primary efficacy endpoint (end of treatment). Enrolment is planned to begin in the second quarter 2007 and to be completed by January 2008. The last follow-up evaluation is expected to be completed approximately 17 months after the initiation of enrolment. Each patient is expected to participate for 12 months including the follow-up period.
Company Confidential -Swiss Tropical Institute Protocol P-001-05-01-03 The two trial sites are in Uganda and Tanzania:

Lwala Hospital, Kaberamaido District, Uganda
Lwala is close to Lake Kyoga and accessible by road throughout the year in a maximum 2 hours drive from Soroti. The hospital has 100 beds, the laboratory is sufficiently equipped and electricity is available from 7 to 10pm. The hospital us financed through a Diocese and also receives some government funds. . Only in the beginning of 2004 it has been established as HAT treatment centre, as a reaction to a new T.b. rhodesiense focus.

Kaliua Health Centre, Urambo District, Tanzania
Kaliua is accessible by road (in dry season only) in a maximum 3 hours drive from Tabora. The Kaliua Health Centre is a missionary hospital and has 50 beds. In the area of Tabora and Kigoma it has a very good reputation as HAT treatment centre. If needed, electricity is available. The infrastructure of the laboratory has been improved before the study initiation of the IMPAMEL III proof-of-concept trial.

Patient recruitment
Most of the patients are passively detected. In case of very low patient numbers active case search is planned high prevalence villages (for a time period of 3 to 6 days).
Also, referrals from other hospitals and health centres in the area are possible.
All subjects will undergo baseline microscopic blood examination to demonstrate the presence of trypanosomes. In case of a positive result, the diagnostic LP for stage determination (trypanosomes in CSF and/or WBC > 5 cells/mm 3 ) follows. Subjects will undergo safety evaluations including physical examination, adverse event monitoring, and collection of concomitant medications throughout the treatment period and at the end of treatment evaluation.
All subjects will undergo blood sampling and LP at baseline evaluation; end of treatment and at followup visits.
Children ≥ 6 years can be enrolled in all sites.

Efficacy
Primary efficacy will be evaluated in blood and CSF at the end of treatment (absence of trypanosomes). Secondary efficacy will be evaluated in blood and CSF (absence of trypanosomes) at each follow-up visit, 3, 6 and 12 months post-treatment.
Lumbar puncture will be performed at end of treatment and at all follow-up visits. In the case that trypanosomes and/or WBC > 5 cells/mm 3 are demonstrated, the patients will be re-treated according the centre policy.
In case of a positive blood test after the end of treatment or at follow-up visits, an additional blood sample will be taken (300µl, applied on Gentra Cards) to perform genotyping of the parasite (reference sample taken at baseline evaluation) and to define the case as a relapse or re-infection (PCR). Reinfections will not be considered as relapses or treatment failure.
Company Confidential -Swiss Tropical Institute Protocol P-001-05-01-03 Additional assessments of clinical efficacy will be performed at 12 month post treatment (last follow-up visit). The 12 month post-treatment evaluation is defined as the test of cure evaluation, based on the Draft Informal Consultation from WHO (9Sept2004).

Safety
The incidence of the encephalopathic syndrome (ES) with a fatal outcome and fatalities with other causes of death (e.g. disease related, opportunistic infections) have been set as combined endpoint of ≥10% (see 5.1.4).

Blood sample collection for PCR-analysis
Patients are asked to provide 3, in case of a positive follow-up result, 4 blood samples. Blood sample analysis will provide knowledge on: The possible strain-overlap in Tanzania and Uganda. The presence of the so-called SRA gene should be given in each sample.
At baseline evaluation, a reference blood sample from each patient will be taken. In case of trypanosome positive results during any follow-up visit, the genotyping of the parasites allows to make the distinction between relapses and re-infections.
Genotyping of the HLA-system will be put in correlation with the occurrence of ES 14 . At baseline evaluation a blood sample of each patient will be collected. Analysis will follow within 6 to 9 months after sample collection.
At any time during the study, the patient, or investigator, may elect to discontinue a patient's participation in the study.

Discussion of Study Design
The clinical data collected during the proof-of-concept trial gives evidence that (i) the suramin pre- consider is that children can not give informed consent. Within the IMPAMEL III trials, parents will be asked to give consent for their children.
In order to reduce fever and pain, 1g of paracetamol will be administered twice a day. Paracetamol administration will start on the day patients have to undergo LP.
All trial patients will receive 10mg of prednisone half an hour prior to melarsoprol injection. Any other concomitant treatment, e.g. anticonvulsive drugs will be administered on a centre-specific basis and Two trial sites will enrol subjects. Both trial sites are equipped with all necessary laboratory materials.
Anti-trypanocidal drugs are available through WHO. For the supply of all other drugs, the STI will purchase the required amount needed for the IMPAMEL III trials.
The PCR-analysis for T.b. rhodesiense identification (SRA-gene) will be done in Uganda or Switzerland.
The PCR analysis for distinction of relapses and re-infections will be done at Yale University, USA.

Study Population
Both, male and female subjects ≥ 6 years who meet all the inclusion criteria listed in Section 5.2.1 and exhibit none of the exclusion criteria listed in Section 5.2.2 of this protocol, will be eligible for enrolment.

Criteria for Inclusion
The patient has second stage T.b. rhodesiense infection; i.e. parasitologically confirmed infection in the blood or trypanosomes in CSF and/or WBC > 5 cells/mm 3 in CSF detected by microscopic examination.
Patient is male or female and ≥ 6 years of age.

Criteria for Exclusion
The patient has first stage T.b. rhodesiense infection; i.e. presence of trypanosomes in blood upon microscopic examination and no trypanosomes in CSF and/or ≤ 5cells/mm 3 .
Moribund or unconscious patients are excluded from the trial. The eligibility is evaluated following the Glasgow coma scale (see Appendix 1). Patients with less than 8 points are excluded from the trial.
Pregnant women are excluded from the trial.
Active clinical relevant medical conditions that in the Investigators opinion may jeopardize subject safety or interfere with participation in the study, including but not limited: significant liver disease, chronic pulmonary disease, significant cardiovascular disease, diabetes and open tuberculosis.
Critically ill patients with any condition which necessitates immediate and concomitant treatment not listed above.
The subject has been previously enrolled in the study.

HIV
On request, patients have the possibility to test their HIV status (in both trial sites HIV testing and counselling is normal practise).
HIV positive patients will be referred to the countries respective National Control Program in order to receive treatment.
HIV positive cases will be included in the trial when they meet all inclusion and exclusion criteria.

Discontinuation of Individual Subjects
A subject can be discontinued from the study for the following reasons: 1.
Withdraws voluntarily from the study.

2.
At the discretion of the Principal Investigator if the patient is not compliant to the requirements of the protocol.
If for any reason a subject is discontinued form the study before the end of treatment evaluation, the Investigator is required, as feasible, to perform the safety procedures planned for the end of treatment.
If a subject develops an ES, he/she will remain under observation until the adverse event is resolved and stabilized.
Subjects who discontinue the trial for any other reasons than drug or disease related reasons will be replaced (e.g. abandon of hospital, withdrawal of informed consent).

Discontinuation of the Entire Study
The entire study will be terminated if the combined endpoint (fatal ES and fatalities with other cause of death) is ≥10%, which corresponds to ≥ 7 patients in a group of 30 patients.
The STI may terminate this study prematurely; either for safety reasons, political unrests or census of false data. In the case of discontinuation of the entire study, the STI immediately notify the Investigator and subsequently provide written instructions for study termination.

Scientific Advisory Board
The study will be supervised by a SAB which supports and guides the IMPAMEL III program.

Treatment schedule
The IMPAMEL III program aims at the further assessment of the safety, tolerability and efficacy of the 10-day melarsoprol schedule without suramin pre-treatment in late stage T.b. rhodesiense patients. In total, both trial sites will enrol a minimum of 70 patients. After ethical clearance is received, the study initiation will follow immediately. Best would be to start in both countries within close time periods in order to assure the availability of the results at similar time points. The defined study population will be treated with melarsoprol at a dosage of 2.2 mg/kg (but a maximum of 180mg) for 10 days continuously. No suramin will be administered. Other drugs centre-specific (best supportive care) Facilities participating in the study will exclusively use the schedule described for all patients who gave consent. In facilities not participating, the standard treatment schedule and supplementary medication as foreseen by the National Authorities will be used without modifications.

Additional Medication
If National rules exist they are applicable for additional medication and therapy. Any additional medication applied that deviates from the standard procedures in a respective centre must be listed in detail in the CRF (i.e. date of prescription, drug, duration, justification).

Drugs to avoid during the study
The use of thiabendazole should be strictly omitted because it is under suspicion of precipitating reactive encephalopathies 39 .

4.3.4. Supplementary medication before melarsoprol treatment (standard if no rules exist)
Malaria: centre specific diagnosis and treatment.
Helminthic infections: centre specific diagnosis and treatment.
Mebendazole: Day one to three: 100 mg total dose twice daily.
Paracetamol: before and after LP.

Encephalopathy
If preliminary signs of reactive encephalopathy are observed in a hospitalized patient (onset of fever and severe headaches) he has to be surveyed strictly. In case symptoms of reactive encephalopathy occur (convulsions, and/or confusion, coma) the melarsoprol treatment is interrupted. If no medical doctor is on site all efforts have to be made to get hold of him. All efforts have to be made that he is examined by a medical doctor immediately.
If reactive encephalopathy is diagnosed melarsoprol treatment is suspended and the patient is treated following the rules of IMC or any participating partner. The interventions below may serve as guidelines.
³ Immediate treatment in case of reactive encephalopathy: -Adrenaline (epinephrine) may be given at 0.5 mg s.c. -The patient is to be kept under permanent surveillance and good general nursing until the condition improves, then the melarsoprol treatment can be resumed after 1 to 3 days. Treatment is resumed at the position where the encephalopathy has occurred. If two or less doses are missing the treatment will be considered complete and will not be resumed.

Neuropathies
Neuropathies (sensitivity problems, severe pain when walking) are a phenomenon which occurs at largely different frequencies in different locations. If paraesthesias or severe pain occur which make an intervention necessary, the melarsoprol treatment is interrupted until improvement of the symptoms. Then treatment is resumed.

Skin Eruptions
In case skin eruptions (especially severe maculopapular eruptions or the begin of bullous lesions) ("exfoliative dermatitis") occur the medical doctor has to be informed. A differential diagnosis is made in order to discriminate possible treatment related problems and concomitant infections.
The melarsoprol treatment is suspended if: After recovery the melarsoprol treatment is resumed with a third of the last used dose, at the following injection two thirds are applied, and then the full dose can be resumed. The patient has to remain hospitalized and kept under strict control. If two or less doses are missing the treatment will be considered complete and will not be resumed.

Hypotension or collapse
Collapse with slight hypotension:

Compliance
The local investigator of the executing organization or Authority is responsible for complete compliance with the investigators manual of all the medical doctors, nurses and staff involved.
-The investigator must agree and sign a document confirming that he or she understands the investigation and will work according to the protocol and Good Clinical Practice.
-The investigator is responsible for ensuring that the protocol is strictly followed. The investigator should not make any changes without the agreement of the STI, except when necessary to eliminate an apparent immediate hazard or danger to a subject.
-The investigator may take any steps judged necessary to protect the safety of the patients, whether specified in the protocol or not. Any such steps must be documented.
-During the treatment the records are maintained by the responsible nurse or the medical doctor. All entries have to be made clearly readable with a pen. The records are controlled by the medical doctor before discharge of the patient, where ever possible.
-The investigator must be thoroughly familiar with the properties, effects and safety of the investigational pharmaceutical product.
-Drugs are only applied by a medical doctor or a trained nurse assigned and adequately instructed.
Exceptions in case of human resource constraints are allowed after discussion with the program director.

Pre-treatment Period
The subjects will be recruited (I) among the patients reporting to one of the trial sites and (II) during active surveillance in high prevalence villages.
The patients, who qualify for the study, will be asked to provide written informed consent prior to undergoing any study specific procedures or treatment.
For baseline evaluation following tests and procedures are to be performed: Demographic information with height, weight, body mass index (calculated).
Complete medical history. For all women the fertility status will be noted (childbirth within 9 months, no birth within last 2 years, no birth within last 5 years or menopause). Concomitant diseases and pre-treatment medications for the 7-day period prior to doing.
Vital signs including blood pressure, heart rate and temperature, taken in sitting position.
Physical examination and Glasgow Coma Scale (Appendix 1).
Pregnancy test.
Confirmatory tests for presence of trypanosomes in blood and/or lymph, and lumbar puncture for stage determination.
The following tests will be performed in the order indicated to confirm the diagnosis of the screening examination with a maximum of 3 days prior to treatment initiation. Tests must be performed in order as indicated below; if a test is found to be positive for trypanosomes, subsequent tests need not to be performed: Microscopic examination of blood (thin and/or thick smear).

Haematocrit centrifugation (WOO).
A sample of CSF (5ml) will be obtained by LP and the following tests will be performed: WBC count for stage determination (average of 3 countings).
Modified single centrifugation technique 41 : microscopic examination of CSF for trypanosomes.
The subjects enrolled at a site will undergo following exams within 48 hours prior to dosing: Blood sampling: for the performance of the tests listed, max. 5ml whole blood will be taken of each patient.
Quantifying the level of parasitaemia (thick blood film).

T.b. rhodesiense identification by PCR methodology (presence of SRA-gene).
Baseline data for each patient (blood collected on Gentra Cards). In case of a positive follow-up result, an additional blood sample will be taken. By comparison of the parasites genotype (PCR-Methodology) the distinction of relapse vs. re-infection is possible.
Subjects can require an HIV test (in the case of positive result they will be referred to the National Control Program). HIV positive patients can be included in the trial population.
Eligibility will be determined by the inclusion / exclusion criteria. Subjects are admitted as in-house patients to the clinical site for the entire treatment period.
Diagnosis and treatment of concomitant diseases such as malaria will be done according to current guidelines at the site before initiation of treatment with suramin. Treatment of filariasis will be postponed until completion of the application of trypanocidal drugs. Malaria will be tested on thin Company Confidential -Swiss Tropical Institute Protocol P-001-05-01-03 and/or thick smear of blood. Any diagnosis will be recorded as a part of the Medical History and any medications prescribed to treat newly diagnosed diseases will be recorded.

Study Drug administration
Melarsoprol will be given to the patient under direct observation of an authorized staff member.
Patients receive 10 melarsoprol injections at a dosage of 2.2mg/kg, the injections are spaced by 24 hours.
Prednisone (10mg) is given to all trial patients half an hour before melarsoprol injection. Paracetamol will be given to the patients twice a day (every 12 hours). Patients with ≥ 30kg body weight will receive 2x1000mg and patients with ≤ 30kg body weight 2x250mg. In the mornings, paracetamol administration follows just after melarsoprol administration. 12 hours later the patients receive the second dose pf paracetamol.
Other concomitant treatments will be administered according to the centres guidelines (= best supportive care).

Procedures and Observations
Any concurrent medication taken during the treatment period will be recorded.
Any adverse signs and symptoms reported by the patient or noted during contact with the patient arising during the treatment period will be recorded. Refer to Appendix 4 for information regarding adverse event reporting requirements.
Vital signs will be recorded daily in the monitoring after each treatment dose.

End of Treatment
On study day 11 (after treatment) following tests will be performed in the order indicated: Microscopic examination of blood (thin and/or thick smear).

Vital signs
In case of treatment failure: Lumbar Puncture Blood will be collected for the distinction of a possible relapse or re-infection (PCR).
Signs or symptoms of HAT will be queried and graded as outlined in Concurrent medication taken during the study post treatment period (last day of study drug through discharge from the treatment centre) will be noted.
Any adverse signs and symptoms reported by the patient or noted during contact with the patient arising between the initial dose of study drug and end of treatment evaluation will be recorded. For the purpose of this study, disease progression and relapse will be considered as treatment failure, not as an adverse event. Refer to Appendix 4 for information regarding adverse event reporting requirements.
Before final discharge from the treatment centre, a physical examination, including vital signs and Glasgow Coma Scale, will be performed. Any deterioration in physical examination compared to the baseline examination should be reported as an adverse event.
All subjects and parents or guardians of adolescents will be educated to watch for potential adverse events that may develop after the patients is discharged from the treatment centre, including signs and symptoms of ES. Should adverse events present, the subject should immediately return to the treatment centre for evaluation.

Subject Long-Term Follow-up
The treating organization is responsible for the correct follow-up of the subjects. At the day of discharge each patient will receive a sleeping sickness treatment card that clearly indicated the dates of his/her follow-up visits. If support in transport is required, the IMPAMEL III program will cover the cost.
In addition to the regular forms of the National Sleeping Sickness Program, the supplementary CRF forms provided for follow-up by STI must be used for every patient.
All subjects will undergo the following exams at the 3 month post treatment visit: Microscopic examination of blood (thin and/or thick smear).
Subjects will additional undergo a lumbar puncture. The WBC in the CSF will be counted. Microscopic examination of the CSF for trypanosomes will be performed (single modified centrifugation).
In case of positive results an additional blood sample will be taken for PCR-analysis in order to determine if it is a relapse or re-infection.
All subjects will undergo the following exams at the 6 month post treatment visit: Microscopic examination of blood (thin and/or thick smear).
Company Confidential -Swiss Tropical Institute Protocol P-001-05-01-03 Subjects will additional undergo lumbar puncture. The WBC in the CSF will be counted. Microscopic examination of the CSF for trypanosomes will be performed (single modified centrifugation).
In case of positive results an additional blood sample will be taken for PCR-analysis in order to determine if it is a relapse or re-infection.
All subjects will undergo the following exams at the 12 month post treatment visit: Microscopic examination of blood (thin and/or thick smear).
Subjects will additional undergo lumbar puncture. The WBC in the CSF will be counted. Microscopic examination of the CSF for trypanosomes will be performed (single modified centrifugation).
In case of positive results an additional blood sample will be taken for PCR-analysis in order to determine if it is a relapse or re-infection.
At month 12 an oral interview and physical examination will be performed.
For the purpose of determining appropriate clinical evaluation and follow-up, subjects will be classified according to the following descriptions as favourable evolution, uncertain evolution, relapse, reinfection or death.
In case of suspicion of relapse, whole blood will be collected for PCR testing. Any adverse signs and symptoms which are spontaneously reported between the end of treatment evaluation and 30 days post-treatment will be recorded. The Investigator will report all serious adverse events, regardless of the time of the event relative to the completion of treatment. Refer to Appendix 4 for information regarding adverse event reporting requirements. The patient will be asked about his/her current health status (well or unwell). If the patient is not available at any post treatment evaluation, data about the status of the patient (alive and well or unwell; death and cause of death) will be gathered from family, friends or local authorities.

Monitoring for Safety
Treatment emergent adverse signs or symptoms will be recorded in the adverse event section of the Case Report Form, along with date(s) of occurrence, duration, degree of severity, and probable relationship to study drug. The observation time for adverse events starts when the treatment is Company Confidential -Swiss Tropical Institute Protocol P-001-05-01-03 initiated and continues until discharge from the facility at the end of treatment. Any adverse signs and symptoms which are spontaneously reported between the end of treatment evaluation and 30 days post treatment will be recorded. The Investigator will report all serious adverse events, regardless of the time of the event relative to the completion of treatment. For the purpose of this trial, disease progression and relapse will be considered as treatment failure, not as an adverse event. Toxicity will be graded on a scale of 0 (no toxicity) to 4 using criteria in Appendix 3.

Collection and Reporting of Adverse Events
Instructions for definitions, collecting and reporting of adverse events are included in Appendix 4.

Appropriateness of Measurements
Refer to Section 5.1. for discussion of the measurements to be used in the trial.

Primary Efficacy Variables
The primary efficacy variable will be the combined rate of clinical and parasitological cure (Table 5)

Secondary Efficacy Variables
Parasitological cure, clinical cure, relapse, re-infection and death rates at the 3, 6, and 12 month evaluations will be determined. Parasitological cure, relapse, re-infection and death rates will also be assessed at the 12 month test of cure evaluation. Clinical cure will be considered equivalent to the parasitological cure at the 12 month evaluation. Clinical Response Definitions, based on WHO Draft Informal Consultation are outlined in Table 5 (below). For purposes of statistical analyses, each patient will be defined within one of the following categories at each post treatment assessment, based on the appropriate characteristics.

Safety Variables
The safety variables which will be evaluated through the end of treatment evaluation in this Phase II trial include: adverse events, vital sign measurements, laboratory results (blood, urine), physical examinations, and the use of any concomitant medications. Adverse events which are spontaneously reported between the end of treatment evaluation and 30 days post treatment will also be collected.
The Investigator will report all serious adverse events, regardless of the time of the event relative to the completion of treatment.

Justification of Sample Size
The sample size of 2x30 subjects is not based on formal statistical analysis, but is regarded based on clinical judgment as sufficient in this Phase II trial to assess the safety and efficacy objectives of the study.

Efficacy Analysis
The primary efficacy endpoint is the parasitological cure 24 hours after completion of treatment. A secondary endpoint is the parasitological cure 3, 6 and 12 months after completion of treatment.

Safety and Tolerability Analysis
The primary outcome measure for safety is (I) death in temporal relationship to treatment. This includes the incidence of encephalopathic syndromes with a fatal outcome and fatalities with other cause of death (e.g. disease related, opportunistic infections). A combined endpoint of ≥ 10% has been set (N=30, CI=9.93 -42.28).

Stopping rule
Assuming a combined endpoint ≥ 10% of fatal outcome of treatment and a binomial distribution, then the probability of 7 or more events in 30 patients is approximately 0.026.
Therefore the trial would be stopped if ≥7 patients in a group of 30 patients would have a fatal outcome of their treatment. Final data analysis will be performed after study closure.

Statistical and Analytical Plan
The analytical plan will be developed during the execution of the study and added as an amendment to the protocol.

Protocol Deviations
When a variation from the protocol is deemed necessary for an individual subject, the investigator or other physician in attendance must contact the study coordinator (see Appendix 4).
Such contact must be made as soon as possible to permit a decision as to whether or not the subject is to continue in the study. The deviation from the protocol will be authorized only for that subject.
All deviations related to study inclusion and exclusion criteria and significant deviations to subject management and protocol procedures must be documented on the appropriate case report form.
Company Confidential -Swiss Tropical Institute Protocol P-001-05-01-03 Changes to the protocol (after Signatures of Agreement are obtained) that affect the decision of the Ethics Committee (e.g., more extensive procedures, increased risk to subjects, changes in the subject population, additional safety information, etc.) must be documented in the form of an amendment. This amendment must be signed by the appropriate STI personnel and the Investigator, and approved by the Ethics Committee before it may be implemented. If the amendment is minor or reduces the risk to the subject, the chairperson of the Ethics Committee alone may approve it. Ethics Committee acceptance is not necessary for protocol clarifications that consist of minor protocol changes such as correcting typographical errors, rewording for clarity, changes in monitoring personnel, or for other changes to the protocol that do not affect the conduct of the study, including changes in the plan for statistical analysis.
The only circumstances in which the amendment may be initiated without Ethics Committee acceptance is where the change is necessary to eliminate apparent immediate hazards to the subjects. In that event, the Investigator must notify the Ethics Committee in writing within five (5) working days after the implementation.

Ethical Clearance
The study protocol was developed in accordance with the Declaration of Helsinki and the ICH Guidelines on Good Clinical Practice.
Before study initiation the protocol must be approved by the Ethics Committee of the Kanton beider Basel (EKBB) and the Ethics Committee of the Ugandan Ministry of Health and the Tanzanian National Institute for Medical Research (NIMR).

Ethical Conduct of the Study
The IMPAMEL III trial is registered in the Current Controlled Trials (CCT, sister company of Biomed Central) database, which allow users to search, register and share information about clinical trials.
Registration Number is ISRCTN40537886.
The study will be conducted in accordance with the protocol, all ICH and GCP regulations governing clinical study conduct; ethical principles that have their origin in the Declaration of Helsinki (Appendix 5), and all applicable local laws and regulations. The investigator must assure that the study is conducted complies with prevailing local laws and customs. Responsibilities of the Investigator are specified in Appendix 5.

Subject Information and Consent
Patients will receive the information on the benefits and risks of the treatment and will be asked for consent to get treatment. Every patient is given the choice to participate in the trial or to receive the standard treatment. For inclusion of children, the parents (or guardian or legal representative) are asked to sign the Informed Consent.

Subject Confidentiality
The investigators must ensure that the subject's anonymity will be maintained. Subjects will be identified on the Case Report Forms by the Subject Number and Subject's Initials in addition to centre and study identification information. The investigators will keep a separate confidential enrolment log that matches identifying codes with the subjects' names and residencies.

Data Quality Assurance
The National Coordinates are responsible for an adequate data quality.
Prior to the initiation of the study, an investigator's meeting will be held with the investigators and their study coordinators and staff from STI. This meeting will include a detailed discussion of the protocol, performance of study procedures, CRF completion, and specimen collection methods. In addition to the investigator's meeting, the study personnel at each site will be trained on the study procedures by STI personnel at a study initiation visit.
STI personnel will monitor each site throughout the study. Source document review will be performed against entries on the CRF and a quality assurance check will be performed to ensure that the investigator is complying with the protocol and regulations. In addition, after CRFs are retrieved by the STI personnel. A review of the data will be conducted by a physician and a clinical review team at STI.
All data hand entered in the database will be verified by a double-key entry procedure. Any discrepancies will be reviewed against the hard copy CRF and corrected on-line. After completion of the entry process, computer logic checks will be run to check for such items as inconsistent study dates and outlying laboratory values. Any necessary corrections will be made to the database and documented via addenda or audit trail. A manual review of selected line listings will also be performed at the end of the study.

Source Documents, Case Report Forms (CRFs)
All CRFs will be controlled for completeness locally. The data will be double-entered into a DMSys database prepared by the study coordinator. The quality of the final database is a subject to auditing by the PMU.
Company Confidential -Swiss Tropical Institute Protocol P-001-05-01-03 Subject source documents are the physician's subject records maintained at the study site. In most cases, the source documents will be the hospital's or the physician's chart. In cases where the source documents are the hospital's or the physician's chart, the information collected on the CRFs must match those charts. In some cases, a portion of the source documents for a given subject may be the CRFs.
The CRFs will be printed on CR ("carbon required") paper to permit multiple copies. The bottom copy is to be retained at the site for the Investigator's study file. All questions should be answered using a black ink ballpoint pen. If certain data are not available, not done, or not applicable: "NAV," "ND," or "NAP," respectively, will be entered in the appropriate space.
Twenty-four hour clock should be used for all time entries. Changes and/or additions to data entered on original CRFs must be made in the following manner: The original entry will be lined out with a single line drawn through the error (not erased or "whited out") so as to leave it still legible. The correction will be entered using a black ink ballpoint pen, initialled, and dated by the person making the correction. The Investigator or delegate (e.g., Sub-Investigator or study coordinator), may enter corrections on original CRFs. The monitoring team may make changes to the copies of CRFs based on information supplied by the Investigator and documented in the study file.
An STI staff member or assigned consultant will bring the CRFs to the STI.
An STI staff member or assigned consultant will visit the study site for the purpose of comparing the data on the CRFs with the source documents. The Investigator agrees to make source documents available for this purpose.
The CRFs should be completed as soon as possible after the data are available.

Maintenance of Records
The Investigator will maintain all study documents: a. for a minimum of two years after study closure. OR b. for any longer period that is specified by the regulatory requirements of the country in which the study site is located.
If the Investigator relocates, retires, or for any reason withdraws from the study, the study records may be transferred to an acceptable designee, such as another Investigator, another institution, or to STI.
The Investigator must obtain STI's permission before disposing of any records.
It is the responsibility of the STI to inform the investigator/institution as to when these documents no longer need to be retained.

Completion of the Study
The investigator will conduct this study in compliance with the protocol, and will complete the study in satisfactory compliance with the protocol within 8 weeks after the last evaluation of the last subject or Company Confidential -Swiss Tropical Institute Protocol P-001-05-01-03 within 8 weeks of the designated completion date. The investigator will provide a summary of the study's outcome to the IEC and the STI.

Final Report
Upon request by the Investigator, at the completion of the study and following analysis of the data, STI will supply a listing of the Investigator's subjects' treatment assignments, tabulated data, and statistical analyses, as appropriate. A copy of the final study report and corrected CRFs and/or change requisitions including a receipt to be signed and returned to STI will be provided to each Investigator following its release.

Use of Information
All information concerning the IMPAMEL III project belongs to the STI and is confidential (e.g. basic scientific data, or and not previously published is considered confidential information).
The information developed during the conduct of this clinical study is also considered confidential and will be used by the STI. To allow for the use of the information derived from this clinical study and to ensure complete and thorough analysis, the investigator is obligated to provide STI with complete test results and all data developed in this study and to provide direct access to source data/documents for trial-related monitoring.

Publication
The final results of the investigations will be published in scientific journals and at seminars or conferences.
All results from investigations are considered confidential and shall not be made available to any third part by any member of the investigating team before publication.
A restrictive definition of authorship of any publications will be applied: Any author must have made significant contributions to (a) the conception and design, or analysis and interpretation of the data; and to (b) drafting the article or revising it critically for important intellectual content; and on (c) final approval of the version to be published. Each author must have participated sufficiently in the work to take public responsibility for the total content of the publication.
The guidelines follow recent publication regarding this issue 42 .
The study coordinator will be the first author of the main scientific papers to be published. Country coordinators will be considered as co-authors for publications containing data from their countries, provided a continuous active participation throughout the conduct of the project, e.g. from drafting the study protocol to the writing of the manuscript is warranted.

Appendix 4 Administrative Procedures for the Reporting of Adverse Events Adverse Event
The Investigator will be required to provide appropriate information concerning any findings that suggest significant hazards, contraindications, side effects, or precautions pertinent to the safety of the treatment schedule under investigation.

Types of adverse events
The term adverse event could include any of the following events which develop or increase in the severity during the course of the study: Any signs or symptoms whether thought to be related to the condition under study; Any abnormality detected during physical examination.
These data will be recorded on the appropriate CRFs, regardless of whether they are thought to be associated with the study or the drug under investigation. Associated with the use of the drug means that there is a reasonable possibility that the event may have been caused by the drug, respectively by the treatment schedule.
Adverse signs or symptoms will be graded by the Investigator as mild, moderate, severe, or intolerable according to the following definitions: Grade Definition (1) Mild: (2) Moderate: (3) Severe: (4) Intolerable: Causing no limitations of usual activities Causing some limitations of usual activities Causing inability to carry out usual activities Intolerable or life-threatening The observation time for adverse events starts when the treatment is initiated and continues until 5 days post-treatment. Any adverse signs and symptoms which are spontaneously reported between the end of treatment evaluation and 30 days post treatment will be recorded. For the purpose of the trial, disease progression and relapse will be considered as treatment failure, not as an Adverse Event. Cases of re-infection will not be considered as treatment failure.

Relationship to the Study Drug
The investigator will use the following definitions to assess the relationship of the adverse event to the use of the study drug:

Probably related
An adverse event has a strong temporal relationship to study drug, or recurs on re-challenge and etiology is unlikely or significantly less likely.

Probably not related
An adverse event has a strong temporal relationship to the study drug and an alternative etiology is equally or less likely compared to the potential relationship to the study drug.

Possibly related
An adverse event has little or no temporal relationship to the study drug and/or more likely alternative etiology exists.

Not related
An adverse event is due to an underlying or concurrent illness or effect of another drug and is not related to the study drug (e.g. has no temporal relationship to study drug or has much more likely alternative etiology.
If an investigator's opinion of possibly, probably not, or not related to study drug is given, an alternative etiology must be provided by the investigator of the adverse event.

Serious Adverse Event
A "serious" adverse event is defined as any event that suggests a significant hazard, contraindication, side effect, or precaution. A serious adverse event includes any event that: is fatal is life threatening, meaning, the subject was, in the view of the Investigator, at immediate risk of death from the reaction as it occurred, i.e., does not include a reaction that, had it occurred in a more serious form, might have cause death; is a persistent or significant disability or incapacity, i.e., the event causes a substantial disruption of a person's ability to conduct normal life function; requires, or prolongs in-patient hospitalization; is a congenital anomaly or birth defect; Is an important medical event, based upon appropriate medical judgment that may jeopardize the patient or subject or may require medical or surgical intervention to prevent one of the other outcomes defining serious.

Reporting Obligations
All "serious" events, whether or not unexpected or considered to be associated with the use of drug, and regardless of the timing of the occurrence after the first dose of study drug, must be communicated immediately upon discovery of the event, and within 24 hours of discovery, either by telephone or email.

Follow-up of adverse events
All Serious Adverse Events must be followed with appropriate medical management until resolved or until considered chronic and stable or otherwise explained. If the treatment was interrupted due to an adverse event, it may be resumed if considered both safe and ethical. The minimum coherent duration of treatment must be maintained if the patient is re-treated.

Appendix 5 Ethical Consideration and Human Subject Protection
Investigators and Study Sites The investigators who are responsible for the conduct of this study, in compliance with this protocol, are identified on the Signatures of Agreement page.

Ethics Committee Acceptance
It is required that a valid Ethics Committee approves in writing the conduct of this clinical study, together with the Investigator's informed consent document, prior to study initiation.
The trial protocol was developed in accordance with the Declaration of Helsinki and the ICH Guidance on Good Clinical Practice.
In performing this study, both the Investigator and Sponsor endorse, as a minimum, the standards for conduct of clinical research activities as set forth in the Declaration of Helsinki, ICH guidelines and local country laws and regulations.
The Study Director will submit the protocol and informed consent for Ethics Committee acceptance.
This will be appropriately documented. The Ethics Committee should be asked to give its acceptance in writing. The names and qualifications of the members of the review committee will be recorded and submitted to the Swiss Tropical Institute together with the written acceptance for the conduct of the study.
The members of the Ethics Committee accepting must be independent of the sponsor and the Investigator.
The written acceptance should consist of a completed Ethics Committee Acceptance form or written documentation from the Ethics Committee containing the same information.
Until written acceptance by the Ethics Committee has been received by the Sponsor, no subject may undergo any procedures solely for the purpose of determining eligibility for this study.
Protocol amendments must also be reviewed and accepted by the Ethics Committee and written acceptance from the committee or at least the chairperson (or a designated committee member) must be received by the Swiss Tropical Institute before implementation. This written approval will consist of a completed Ethics Committee Acceptance form or written documentation from the Ethics Committee containing the same information.
Additional, the IMPAMEL III trial is registered in the Current Controlled Trials (CCT, sister company of The purpose of biomedical research involving human subjects must be to improve diagnostic, therapeutic and prophylactic procedures and the understanding of the etiology and pathogenesis of disease.
In current medical practice most diagnostic, therapeutic or prophylactic procedures involve hazards.
This applies especially to biomedical research.
Medical progress is based on research which ultimately must rest in part on experimentation involving human subjects. In the field of biomedical research a fundamental distinction must be recognised between medical research in which the aim is essentially diagnostic or therapeutic for a patient, and medical research the essential object of which is purely scientific and without implying direct diagnostic or therapeutic value to the person subjected to the research.
Special caution must be exercised in the conduct of research which may affect the environment, and the welfare of animals used for research must be respected.
Because it is essential that the results of laboratory experiments be applied to human beings to further scientific knowledge and to help suffering humanity, the World Medical Association has prepared the following recommendations as a guide to every physician in biomedical research involving human subjects. They should be kept under review in the future. It must be stressed that the standards as drafted are only a guide to physicians all over the world. Physicians are not relieved from criminal, civil and ethical responsibilities under the law of their own countries.
I. Basic Principles

1.
Biomedical research involving human subjects must conform to generally accept scientific principles and should be based on adequately performed laboratory and animal experimentation and on a thorough knowledge of the scientific literature.

2.
The design and performance of each experimental procedure involving human subjects should be clearly formulated in an experimental protocol which should be transmitted to a specially appointed independent committee for consideration, comment and guidance.

3.
Biomedical research involving human subjects should be conducted only by scientifically qualified persons and under the supervision of a clinically competent medical person. The responsibility for the human subject must always rest with a medically qualified person and never rest on the subject of the research, even though the subject has given his or her consent.

4.
Biomedical research involving human subjects cannot legitimately be carried out unless the importance of the objective is in proportion to the inherent risk to the subject.

5.
Every biomedical research project involving human subjects should be preceded by careful assessment of predictable risks in comparison with foreseeable benefits to the subject or to others.
Concern for the interests of the subject must always prevail over the interests of science and society.

6.
The right of the research subject to safeguard his or her integrity must always be respected.
Every precaution should be taken to respect the privacy of the subject and to minimize the impact of the study on the subject's physical and mental integrity and on the personality of the subject.

7.
Physicians should abstain from engaging in research projects involving human subjects unless they are satisfied that the hazards involved are believed to be predictable. Physicians should cease any investigation if the hazards are found to outweigh the potential benefits.

8.
In publication of the results of his or her research, the physician is obliged to preserve the accuracy of the results. Reports of experimentation not in accordance with the principles laid down in this Declaration should not be accepted for publication.

9.
In any research on human beings, each potential subject must be adequately informed of the aims, methods, anticipated benefits and potential hazards of the study and the discomfort it may entail. He or she should be informed that he or she is at liberty to abstain from participation in the study and that he or she is free to withdraw visor her consent to participation at any time. The physician should then obtain the subject's freely given informed consent, preferably inheriting.

10.
When obtaining informed consent for the research project the physician should be particularly cautious if the subject is in dependent relationship to him or her or may consent under duress. In that case the informed consent should be obtained by a physician who isn't engaged in the investigation and who is completely independent of this official relationship.

11.
In case of legal incompetence, informed consent should be obtained from the legal guardian in accordance with national legislation. Where physical or mental incapacity makes it impossible to obtain informed consent, or when the subject is a minor, permission from the responsible relative replaces that of the subject in accordance with national legislation. Whenever the minor child is in fact able to give consent, the minor's consent must be obtained in addition to the consent of the minor's legal guardian. 1.
In the treatment of the sick person, the physician must be free to use a new diagnostic and therapeutic measure, if in his or her judgement it offers hope of saving life, re-establishing health or alleviating suffering.

2.
The potential benefits, hazards and discomfort of a new method should be weighed against the advantages of the best current diagnostic and therapeutic methods.

3.
In any medical study, every patient-including those of a control group, if any-should be assured of the best proven diagnostic and therapeutic method.

4.
The refusal of the patient to participate in a study must never interfere with the physicianpatient relationship.

5.
If the physician considers it essential not to obtain informed consent, the specific reasons for this proposal should be stated in the experimental protocol for transmission to the independent committee (1, 2).

6.
The physician can combine medical research with professional care, the objective being the acquisition of new medical knowledge, only to the extent that medical research is justified by its potential diagnostic or therapeutic value for the patient.

III. Non-Therapeutic Biomedical Research Involving Human Subjects (Non-Clinical Biomedical
Research) 1.
In the purely scientific application of medical research carried out on a human being, it is the duty of the physician to remain the protector of the life and health of that person on whom biomedical research is being carried out.

2.
The subjects should be volunteers-either healthy persons or patients for whom the experimental design is not related to the patient's illness.

3.
The investigator or the investigating team should discontinue the research if in his/her or their judgment it may, if continued, be harmful to the individual.

4.
In research on man, the interest of science and society should never take precedence over considerations related to the well-being of the subject.

Informed Consent
The Investigator will obtain informed consent from each subject enrolled in the study, in accordance with the Declaration of Helsinki, the current versions of the ICH guidelines and the laws and regulations of the country in which the investigation is being conducted.
The Ethics Committee must accept the informed consent document to be used by the Investigator. It is the responsibility of the Investigators to assure that the patient (or guardian or legal representative) has signed the Informed Consent before any activity or treatment is undertaken which is not part of routine care. This includes, but is not limited to, the performance of diagnostic ort therapeutic procedures and the administration of the first dose of the study medication.