Analyses of Meldonium (Mildronate) from Blood, Dried Blood Spots (DBS), and Urine Suggest Drug Incorporation into Erythrocytes

Abstract

Initially developed in the late 1970s for veterinary applications due to proposed growth-promoting effects in animals [5], meldonium has become an approved drug in selected Eastern European countries and is the subject of ongoing clinical trials focusing the compound's anti-ischemic and cardioprotective properties [2,3,12,15] as well as potential applications regarding diabetes, neurodegenerative disorders, and bronchopulmonary diseases. In the context of athletic performance, beneficial effects on the individuals' physical working capacity, increased endurance performance, and accelerated recovery after physical activity were discussed [4,10,11], mentioning oral doses of meldonium of up to 2.0 g per day over 2-3 weeks in the course of pre-competition preparation phases [4]. In 2015, the World Anti-Doping Agency (WADA) initiated a one-year monitoring program [22] regarding the prevalence of meldonium (mildronate) in doping controls. Obtained data demonstrated a considerable extent of meldonium use by athletes [8,16], which was further corroborated by a significant number of declarations of use and analytical findings at the Baku 2015 European Games [18]. Subsequently, the WADA Prohibited List that became effective in January 2016 [24] classified meldonium as banned under S4 (Hormone and Metabolic Modulators). Pharmacokinetic properties of meldonium were reported for single-and multiple-dose administration studies with healthy volunteers [25], where the drug's elimination was monitored in plasma over 24 h post-administration and characterized by nonlinear pharmacokinetics. To date, doping controls are based on urine and blood as test matrices, and a variety of alternative matrices including amongst others dried blood spots (DBS) and dried plasma spots (DPS) have been considered lately [20]. Consequently, the knowledge about factors that potentially influence the elimination of target analytes is of particular importance to sports drug testing and, to the best of our knowledge, the role of erythrocytes and their ability to affect detection windows of meldonium in doping controls (e. g., by incorporation) has not been investigated. Therefore, in the context of a pilot study, DBS, whole blood (Na 2 -EDTA), and urine samples were collected from 2 healthy male volunteers who orally administered meldonium either as single dose (500 mg) or as multi-dose (3 × 500 mg/day over a period of 6 consecutive days). The study protocol was approved by the local ethics committee of the National Institute for Sports Research (Bucharest, Romania, approval number #162/2016), written consent was obtained from the study participants, and the study was conducted in accordance with ethical standards in sports medicine and exercise science [9]. DBS samples were collected prior to and post-administration up to 16 days using standard DBS collection cards (Whatman DMPK-C, GE Heathcare Europe, Freiburg, Germany), dried at room temperature, and stored at + 4 °C in a plastic bag with desiccant until analysis. Na 2 -EDTA-stabilized whole blood sp...

Story behind meldonium–from pharmacology to performance enhancement: a narrative review

“…In their recent ‘Notice-Meldonium’,9 WADA put emphasis on this issue by providing the stakeholders with new information on preliminary results obtained from single and multiple drug applications. First light on the discussion on meldonium's elimination brought a recent pilot study by Tretzel et al 60. Preliminary data could demonstrate that there exists a second, very slow elimination phase of meldonium with urinary concentrations of 80 ng/mL 16 days and between 10 and 200 ng/mL for up to 49 days postadministration after a single dose of 500 mg. Urinary meldonium concentration profiles revealed values between 1 and 9 μg/mL after multiple applications (3×500 mg/day for 6 days) until day 33.…”

Meldonium long‐term excretion period and pharmacokinetics in blood and urine of healthy athlete volunteers

“…A study of the pharmacokinetics of single‐use (500 mg) and multiple‐use (500 mg three times per day for six days) of meldonium showed the presence of the second, longer, elimination phase of the drug, with determined concentrations in urine . A single dose of meldonium for 16 days yielded a mean urinary concentration of 80 μg/mL, and at 49 days produced individual concentrations ranging from 10 to 200 μg/mL.…”

“…18 A study of the pharmacokinetics of single-use (500 mg) and multiple-use (500 mg three times per day for six days) of meldonium showed the presence of the second, longer, elimination phase of the drug, with determined concentrations in urine. 19 The effectiveness of this transfer depends on medium pH and pKa values of the drug's corresponding ionogenic group. However, at high-density substrate, which was simulated during the present study, this process may also reach the saturation limit.…”

Pharmacokinetics and pharmacodynamics of meldonium in exercised thoroughbred horses

“…The tissue accumulation may be a result of meldonium's affinity for the organic cation transporter‐2 which leads to reabsorption in the renal tubules as well as uptake in various tissues, including the heart and skeletal muscle, containing this transporter . A recent study in humans demonstrated that meldonium incorporates into red blood cells and the investigators hypothesized that the slow release of the drug from erythrocytes, which have a lifespan of 70–140 days, could explain the long elimination half‐life and prolonged detection time . The lifespan of erythrocytes in horses is approximately 145 days.…”