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

Recent reports from the World Anti-Doping Agency (WADA) indicate an alarming prevalence in the use of meldonium among elite athletes. Therefore, in January 2016, meldonium was added to WADA's prohibited list after being monitored since 2015. Meldonium has been shown to have beneficial effects in cardiovascular, neurological and metabolic diseases due to its anti-ischaemic and cardioprotective properties, which are ascribed mainly to its inhibition of ß-oxidation and its activation of glycolysis. Despite its widespread use, there are only a few clinical studies or clinical trials available. Meldonium is registered in most Baltic countries and is easily accessible through the internet with no serious adverse effects reported by the manufacturer so far. Among athletes, meldonium is used with the purpose of increasing recovery rate or exercise performance. The benefit of taking meldonium in view of performance enhancement in athletes is quite speculative and is discussed without sound scientific evidence. This narrative review provides a detailed overview of the drug meldonium, focusing on the main topics pharmacology and biochemical actions, clinical applications, pharmacokinetics, methods of detection and potential for performance enhancement in athletes.

Microencapsulation of mildronate in biodegradable and non-biodegradable polymers.

The extremely high hygroscopicity (solubility in water ≥2 g/ml) of the pharmaceutical preparation mildronate defines specific requirements to both packaging material and storage conditions. To overcome the above mentioned inconveniences, microencapsulated form of mildronate was developed using polystyrene (PS) and poly (lactic acid) (PLA) as watertight coating materials. Drug/polymer interaction as well as influence of the microencapsulation process variables on microparticle properties was studied in detail. Water-in-oil-in-water double emulsion technique was adapted and applied for the preparation of PS/mildronate microparticles with total drug load up to 77 %wt and PLA/mildronate microparticles with total drug load up to 80 %wt. The repeatability of the microencapsulation process was ±4% and the encapsulation efficiency of the active ingredient reached 60 %wt. The drug release kinetics from the obtained microparticles was evaluated and it was found that drug release in vivo could be successfully sustained if polystyrene matrix has been used.

UPLC-MS/MS method for bioequivalence study of oral drugs of meldonium.

A rapid and simple method based on ultra-performance liquid chromatography on a hydrophilic interaction chromatography column with tandem mass-selective detection (UPLC-MS/MS) to determine meldonium in human plasma was developed. The calibration curve acquired in the range of 10-6000 ng/mL had quadratic form. Method validation proved the conformity of its properties (selectivity, matrix effect, lower limit of quantification, accuracy, precision and recovery) with the established requirements. The stability tests necessary for bioanalytical studies were performed. For the first time, the method was successfully applied to the bioequivalence studies of generic and brand name oral drugs of meldonium in capsules. Based on data from 24 volunteers, it was determined that the mean pharmacokinetic curves of the drugs are characterized by a double peak profile.

Mildronate, a regulator of energy metabolism, reduces atherosclerosis in apoE/LDLR-/- mice.

BACKGROUND/AIMS: Mildronate, an inhibitor of L-carnitine biosynthesis and transport, is used in clinics as a modulator of cellular energy metabolism and is a cardioprotective drug. L-Carnitine is a pivotal molecule in fatty acid oxidation pathways and its regulation in vasculature might be a promising approach for antiatherosclerotic treatment. This study was performed to evaluate the effects of mildronate treatment on the progression of atherosclerosis and the content of L-carnitine in the vascular wall. METHODS: ApoE/LDLR(-/-) mice received mildronate at doses of 30 and 100 mg/kg for 4 months. Lipid profile was measured in plasma and atherosclerotic lesions were analyzed in whole aorta and aortic sinus. L-Carnitine concentration was assessed in rat aortic tissues after 2 weeks of treatment with mildronate at a dose of 100 mg/kg. RESULTS: The chronic treatment with mildronate at a dose of 100 mg/kg significantly reduced the size of atherosclerotic plaques in the aortic roots and in the whole aorta, and slightly decreased the free cholesterol level. In addition, mildronate treatment decreased L-carnitine concentration in rat aortic tissues. CONCLUSIONS: Long-term mildronate treatment decreases L-carnitine content in aortic tissues and attenuates the development of atherosclerosis in apoE/LDLR(-/-) mice.

Determination of mildronate in human plasma and urine by liquid chromatography-tandem mass spectrometry.

A sensitive and selective analytical method based on liquid chromatography-triple-quadrupole mass spectrometer has been developed to determine mildronate in human plasma and urine. The aim of this work was to find a valid method to study the pharmacokinetic profiles of mildronate in humans. Mildronate is a heart protection medicine, a carnitine's structural analogue, so levocarnitine was used as an internal standard for quantification. Under the electrospray ionization source positive ion mode, calibration curves with good linearities (r=0.9998 for plasma sample and r=0.9999 for urine sample) were obtained in the range of 1.0-20,000 ng ml(-1) for mildronate. The detection limit was 1 ng ml(-1). Recoveries were around 90% for the extraction from human plasma, and good precision and accuracy were achieved. This method is feasible for the evaluation of pharmacokinetic profiles of mildronate in humans, and to the best of our knowledge, this is the first report on LC-MS-MS analysis of mildronate in plasma and urine.

Pharmacokinetics and pharmacodynamics of meldonium in exercised thoroughbred horses.

Although developed as a therapeutic medication, meldonium has found widespread use in human sports and was recently added to the World Anti-Doping Agency's list of prohibited substances. Its reported abuse potential in human sports has led to concern by regulatory authorities about the possible misuse of meldonium in equine athletics. The potential abuse in equine athletes along with the limited data available regarding the pharmacokinetics and pharmacodynamics of meldonium in horses necessitates further study. Eight exercised adult thoroughbred horses received a single oral dose of 3.5, 7.1, 14.3 or 21.4 mg/kg of meldonium. Blood and urine samples were collected and analyzed using liquid chromatography tandem mass spectrometry. Pharmacokinetic parameters were determined using non-compartmental analysis. Maximum serum concentrations ranged from 440.2 to 1147 ng/mL and the elimination half-life from 422 to 647.8 h. Serum concentrations were below the limit of quantitation by days 4, 7, 12 and 12 for doses of 3.5, 7.1, 14.3 and 21.4 mg/kg, respectively. Urine concentrations were below the limit of detection by day 44 following administration of 3.5 mg/kg and day 51 for all other dose groups. No adverse effects were observed following meldonium administration. While the group numbers were small, changes in heart rate were observed in the 3.5 mg/kg dose group (n = 1). Glucose concentrations changed significantly in all dose groups studied (n = 2 per dose group). Similar to that reported for humans, the detection time of meldonium in biological samples collected from horses is prolonged, which should allow for satisfactory regulation in performance horses. Copyright © 2017 John Wiley & Sons, Ltd.

Carnitine and γ-Butyrobetaine Stimulate Elimination of Meldonium due to Competition for OCTN2-mediated Transport.

Meldonium (3-(2,2,2-trimethylhydrazinium)propionate) is the most potent clinically used inhibitor of organic cation transporter 2 (OCTN2). Inhibition of OCTN2 leads to a decrease in carnitine and acylcarnitine contents in tissues and energy metabolism optimization-related cardioprotective effects. The recent inclusion of meldonium in the World Anti-Doping Agency List of Prohibited Substances and Methods has raised questions about the pharmacokinetics of meldonium and its unusually long elimination time. Therefore, in this study, the rate of meldonium washout after the end of the treatment was tested with and without administration of carnitine, γ-butyrobetaine (GBB) and furosemide to evaluate the importance of competition for OCTN2 transport in mice. Here, we show that carnitine and GBB administration during the washout period effectively stimulated the elimination of meldonium. GBB induced a more pronounced effect on meldonium elimination than carnitine due to the higher affinity of GBB for OCTN2. The diuretic effect of furosemide did not significantly affect the elimination of meldonium, carnitine and GBB. In conclusion, the competition of meldonium, carnitine and GBB for OCTN2-mediated transport determines the pharmacokinetic properties of meldonium. Thus, due to their affinity for OCTN2, GBB and carnitine but not furosemide stimulated meldonium elimination. During long-term treatment, OCTN2-mediated transport ensures a high muscle content of meldonium, while tissue clearance depends on relatively slow diffusion, thus resulting in the unusually long complete elimination period of meldonium.

Single- and Multiple-dose Pharmacokinetic, Safety and Tolerability Study of Mildronate Injection in Healthy Chinese Subjects Pharmacokinetic of Mildronate Injection.

Mildronate is an agent for cardioprotection and neuroprotection. This study aimed to evaluate the pharmacokinetic (PK) profiles, safety and tolerability of mildronate injection after single escalating doses and multiple doses in healthy Chinese subjects. We performed a randomized, open-label, single- and multiple-dose phase I trial including 3 doses of mildronate: 250, 500 and 750 mg. Plasma and urine samples were collected and concentrations of mildronate were analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). PK parameters were calculated using noncompartmental analysis. Safety and tolerability was assessed throughout noting subjects' vital signs and monitoring adverse events (AEs) and conduct a comprehensive physical examination and laboratory analyses before and after the study. There was no significant difference in C 0, AUC0-t, AUC0-∞ among 3 single-dose groups, whereas T 1/2 had significant statistical difference which may be caused by the inhibition of metabolic enzymes. Single- and multiple-dose intravenous injection of mildronate exhibited linear PK profiles in the range of 250-750 mg. An unconspicuous accumulation phenomenon was found after multiple-dose mildronate administration. No significant gender difference was found and mildronate is primarily excreted by the kidney. No serious AEs were observed. The formulation was safe and well tolerated from 250 to 750 mg.

Free radical activation of monomethyl and dimethyl hydrazines in isolated hepatocytes and liver microsomes.

Isolated hepatocytes and liver microsomes incubated with monomethyl-1,1 dimethyl- and 1,2 dimethyl-hydrazines produced free radical intermediates which were detected by ESR spectroscopy by using 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) as spin trapping agent. The spectral features of the spin adducts derived from all three hydrazine derivatives corresponded to the values reported for the methyl free radical adduct of 4-POBN. In the microsomal preparations inhibitors of the mixed function oxidase system and the destruction of cytochrome P450 by pretreating the rats with CoCl2 all decreased the free radical formation. Methimazole, an inhibitor of FAD-containing monoxygenase system, similarly decreased the activation of 1,1 dimethyl-hydrazine, but not that of monomethyl- and 1,2 dimethyl-hydrazines. The addition to liver microsomes of physiological concentrations of glutathione (GSH) lowered by approx. 80% the intensities of the ESR signals. Consistently, incubation of isolated hepatocytes with methyl-hydrazines decreased the intracellular GSH content, suggesting that GSH can effectively scavenge the methyl free radicals. The results obtained suggest that methyl free radicals could be the alkylating species responsible for the toxic and/or carcinogenic effect of methyl-hydrazines.

Nonlinear pharmacokinetic properties of mildronate capsules: a randomized, open-label, single- and multiple-dose study in healthy volunteers.

Mildronate has been used as antianginal drug in parts of Europe for many years, but its pharmacokinetic (PK) properties in humans remain unclear. This study was designed to assess and compare the PK properties of mildronate capsules after single escalating oral dose and multiple doses in healthy Chinese volunteers. Volunteers were randomly assigned to receive a single dose of 250, 500, 1000, 1250 or 1500 mg of mildronate capsules. Those who received the 500-mg dose continued on the multiple-dose phase and received 500 mg three times a day for 13 days. Plasma drug concentrations were analysed by ultraperformance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). Tolerability was assessed throughout the study. A total of 40 Chinese volunteers were enrolled in the study. No period or sequence effect was observed. Area under the concentration and C(max) were increased proportionally with the dose levels, whereas t(1/2) and V(d)/f were dependent on the dose. Nonlinear PK properties were found at doses of 250-1500 mg. There was an accumulation after multiple-dose administration. No serious adverse events (AEs) were reported in the PK study. The formulation was well tolerated.

Pharmacokinetics and biological fate of 3-(2,2, 2-trimethylhydrazinium)propionate dihydrate (MET-88), a novel cardioprotective agent, in rats.

In this study, we examined the disposition, metabolism, and excretion of a novel cardioprotective agent, 3-(2,2, 2-trimethylhydrazinium)propionate dihydrate (MET-88), in rats. The disposition of MET-88 after oral and i.v. administration of 2, 20, and 60 mg/kg indicated that the pharmacokinetics of MET-88 were nonlinear. The profiles of radioactive MET-88 and total radioactivity in plasma were consistent at doses of 20 and 60 mg/kg. However, at 2 mg/kg, the plasma MET-88 levels were obviously lower than the total. The excretion of radioactivity after oral administration of MET-88 indicated that increasing doses led to a shift from exhaled CO(2) to urinary excretion as the major excretion route. Major metabolites in plasma after oral administration of MET-88 were glucose, succinic acid, and 3-hydroxypropionic acid, and in vitro studies revealed that MET-88 was converted to 3-hydroxypropionic acid by gamma-butyrobetaine hydroxylase (EC 1.14. 11.1). An isolated liver perfusion system modified to trap CO(2) gas was used to examine the excretion pathway of MET-88. [(14)C]CO(2) gas was decreased by the addition of iodoacetic acid, DL-fluorocitric acid, or gamma-butyrobetaine to this system, and subsequent thin-layer chromatography analyses of perfusates revealed that MET-88 was first converted to 3-hydroxypropionic acid by gamma-butyrobetaine hydroxylase and then was biosynthesized to glucose and metabolized to CO(2) gas via the glycolytic pathway and tricarboxylic acid cycle.

Cytochrome P450 activity and distribution in the human colon mucosa.

Enzymatic activity associated with the mixed-function oxidase system was determined in microsomes prepared from the mucosal cells extracted from normal human colons. A high activity toward nitrogen oxidation reactions was observed. 1,2-Dimethylhydrazine, a colon-specific carcinogen, was metabolized at a higher rate in vitro by human colon microsomes as compared with the rat, and exhibited a km ten-fold lower, 1.03 mmol/l versus 9.68 mmol/l, respectively. This activity was inhibited by classic cytochrome P450 inhibitors; 70% inhibition was achieved using 70 mmol/l metyrapone (2-methyl-1,2-di-3-pyridyl-1-propanone), 20 mmol/l; SKF-525A (diethylaminoethyl-2,-2-diphenylvalerate HCl), or 350 mumol/l n-octylamine. These data suggest the presence of a stable, active mixed-function oxidase system in the human colon mucosa which has a preferential activity toward nitrogenous compounds and provides a mechanism for the activation of carcinogens. Its distribution in the colon appears to parallel the reported incidence of human colonic carcinomas.

Pharmacokinetic analysis of the cardioprotective effect of 3-(2,2, 2-trimethylhydrazinium) propionate in mice: inhibition of carnitine transport in kidney.

The site of action of 3-(2,2,2-trimethylhydrazinium) propionate (THP), a new cardioprotective agent, was investigated in mice and rats. I.p. administration of THP decreased the concentrations of free carnitine and long-chain acylcarnitine in heart tissue. In isolated myocytes, THP inhibited free carnitine transport with a Ki of 1340 microM, which is considerably higher than the observed serum concentration of THP. The major cause of the decreased free carnitine concentration in heart was found to be the decreased serum concentration of free carnitine that resulted from the increased renal clearance of carnitine by THP. The estimated Ki of THP for inhibiting the reabsorption of free carnitine in kidneys was 52.2 microM, which is consistent with the serum THP concentration range. No inhibition of THP on the carnitine palmitoyltransferase activity in isolated mitochondrial fractions was observed. These results indicate that the principal site of action of THP as a cardioprotective agent is the carnitine transport carrier in the kidney, but not the carrier in the heart.