Identifying Biological Markers for Altitude Exposure and Use of Recombinant Human Erythropoietin (rHuEPO)

Study Overview
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Brief Summary
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The project represents a well powered study of the human response to altitude exposure combined with rHuEPO treatment. A total of 20 male and 20 female non-competing athletes will participate. The participants will be exposed to a period of hypobaric hypoxia at 2.320 m for four weeks and a sea-level intervention period of four weeks. The participants will be blinded and randomly allocated to intra venous injection with 20 IU per kg body weight of recombinant human erythropoietin or placebo every second day for the initial three weeks of each intervention. Included subjects will be runners due to the good possibility of recruitment and conduction of training at altitude. Samples are collected weekly for four weeks prior to each intervention period, during the four week intervention and four weeks after. This approach yields a highly valuable biobank for identification of markers sensitive to initiation of rHuEPO injections as well as termination of injections.

Detailed Description
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The main hypothesis for this study is that metabolomics and proteomics methods can identify biomarkers that are sensitive to altitude exposure and recombinant human erythropoietin treatment, respectively. Secondarily, the hypothesis is that the identified biomarkers can distinguish between altitude exposure and abuse of rHuEPO. We will include 20 Danish men and 20 Danish women aged 18-35 years. The inclusion criteria are a maximum oxygen uptake rate (VO2max) > 48 ml / kg / min for men and > 43 ml / kg / min for women. All trial participants must have > 2 years of history in regular exercise including running (> 2 times a week; > 30 min per session) and capable of performing a 5 km run in 22 min for men and 25 min for women at sea level on a level surface in dry conditions and a temperature between 10 and 20 degrees C. Furthermore, the participants must not have experienced running related injuries within the past two years. The experimental protocol consists of two experimental periods, both of which must be performed by all participants. Both periods contain a baseline period of four weeks followed by four weeks training camp and four weeks follow up. The training camp in one period of experiments is performed at sea level while the training camp in the other period is performed at 2320 m above sea level. Each period is separated by a minimum of two months to ensure that the participants have returned to baseline values. During the two intervention periods, the participants will be transported by air and car to the destination of the respective training camp. It is expected that the participants train for 1-2 hours a day on average and that the time spent for the measurements described is   5-7 hours a week. To investigate whether the described methods can identify sensitive biomarkers for the physiological response due to rHuEPO treatment, the participants will be treated with rHuEPO. The distribution of participants allows the following: 28 participants receive rHuEPO at sea level, thereby identifying biomarkers sensitive for use of rHuEPO 28 participants receive placebo under altitude exposure, whereby biomarkers sensitive to altitude exposure can be identified 12 participants receive placebo at sea level, whereby random fluctuations of the identified biomarkers can be determined. 12 participants receive rHuEPO under altitude exposure, whereby the identified rhuEPO biomarkers and altitude exposure can be tested for whether they can be used to separate individuals receiving rHuEPO under altitude exposure from individuals receiving placebo under altitude exposure. 8 participants receive placebo at sea level and altitude exposure, whereby natural and random individual fluctuations can be determined over a longer period (> 6 months). Since random individual fluctuations can be determined at n = 1, n = 8 will be sufficient to determine this.

Official Title
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Identification of Biological Markers for Hypoxic Exposure and Use of Recombinant Human Erythropoietin

Conditions
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Healthy

Intervention / Treatment
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* Other: Sea level
* Other: Altitude


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Male: VO2max > 48 ml/min/kg bodyweight and 5km run < 22 min, Female: VO2max > 43ml/min/kg bodyweight and 5km run < 25 min A minimum of 2 years injury running training Exclusion Criteria: Age, insufficient fitness or injury

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 35 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Other
Allocation: Randomized
Intervention Model: Parallel Assignment
Interventional Model Description: Explorative design
Masking: Quadruple


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: Sea level<br>Sea level training camp | Other: Sea level<br>* Sea level exposure / sea level exposure combined with microdoses of rHuEPO<br>|
| Experimental: Altitude<br>Altitude training camp | Other: Altitude<br>* Altitude exposure / altitude exposure Iron metabolism<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Identification of metabolites in urine for rHuEPO and altitude exposure | Urine is analyzed by metabolomics and the metabolites for rHuEPO and altitude are verified using an exploratory design. More specifically, the aim is to identify the resulting metabolites from altitude exposure and treatment with rHuEPO as these are not yet known In this WADA-supported research project, the aim is to identify high sensitivity biomarkers for altitude exposure through a metabolomics methods, as 'omics' methods have previously shown promising results in other exposure contexts. However, being the first of its kind in antidoping the resulting metabolites from rHuEPO misuse and altitude have not yet been identified. Thus, an exact description of these is not possible | 5 years | 
| Identification of human metabolites in serum for rHuEPO and altitude exposure | Human serum is analyzed by metabolomics and the metabolites for rHuEPO and altitude are verified using an exploratory design. | 5 years | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Reticulocyte percentage | Reticulocyte percentage, will be measured to determine variation as a result of either 12 weeks exposure to altitude or sea-level (24 weeks in total). | Up to 24 weeks | 
| Hemoglobin concentration | [hemoglobin] will be measured to determine variation as a result of either 12 weeks exposure to altitude or sea-level. | Up to 24 weeks | 
| OFF-score | OFF-score (computed from reticulocyte perscentage and hemoglobin concentration), will be measured to determine variation as a result of either 12 weeks exposure to altitude or sea-level. | Up to 24 weeks | 
| Maximal oxygen uptake (VO2max) | VO2max (ml/min) measured by incremental work load on a treadmill | Up to 24 weeks | 
| Iron metabolism | Markers of iron metabolism such as hepcidin and erythroferrone are collected analyzed for evaluation of iron metabolism during and after a training camp at 2300m above sea level | Up to 24 weeks | 
| Work economy | Running efficiency (%) measured by energy expenditure on a treadmill | Up to 24 weeks | 
| Sustainable workload | Lactate threshold (mmol/L) measured on a treadmill | Up to 24 weeks | 
| Running performance | 5000m time trial performance (time) measured on a 400m running track | Up to 24 weeks | 
| Muscle oxygenation | Muscle oxygenation measured by near-infrared spectroscopy during running on a treadmill | Up to 24 weeks | 
| Total hemoglobin mass | Total hemoglobin mass will be measured by two-minute carbon-monoxide re-breathing to determine variation as a result of either 12 weeks exposure to altitude or sea-level. | Up to 24 weeks |