Supplement: The story behind meldonium–From pharmacology to performance enhancement: A narrative review

Pharmacology and biochemical actions of L- and meldonium:

The design of meldonium was aimed at interference with L-carnitine metabolism. L-carnitine, a quaternary , is a water-soluble molecule that is especially important in mammalian metabolism for the mitochondrial oxidation of fatty acids. Meldonium acts as a carrier for the transport of activated long-chain fatty acids from the cytosol into the mitochondria, where - oxidation and ATP synthesis take place. The balance of L-carnitine is maintained through , kidney reabsorption, and nutrition intake, particularly from red meat and milk. [1,2]

About 25% of L-carnitine in the human organism is derived by endogenous biosynthesis from the amino acids and methionine in the liver and kidney. More than 99% of the body’s L- carnitine can be found intracellularly, mainly in the liver, heart, and skeletal muscle. Because L- carnitine cannot be degraded or metabolised, it needs to be eliminated, mainly in the urine.

In the pathway of carnitine biosynthesis, enzymatic processes performed by mitochondrial trimethyllysine aldolase (TMLD), 3-hydroxy-trimethyllysine aldolase (HTMLA) and trimethylaminobutyrate dehydrogenase (TMABA) by the formation of -butyrobetaine (GBB) play a central role. In the final step, GBB is hydroxylated by GBB hydroxylase (BBOX) in order to form L-carnitine.[3]

The transport of L-carnitine through cell membranes is performed via Na+ -dependent organic cation transporters (OCTN), mainly OCTN2 and to a lesser extent OCTN1 (figure to supplement). After the activation of fatty acids with a CoA group, the acyl-CoA units are converted into carnitine esters, catalysed by the carnitine palmytoiltransferase I (CPT I), resulting in long-chain acylcarnitine esters. The CPT I-mediated L-carnitine transport is one of the rate-limiting steps of free fatty-acid oxidation, because low carnitine concentrations can inhibit free fatty-acid transport to mitochondria.[4,5] These esters are transported over the inner mitochondrial membrane by carnitine-acylcarnitine translocase (CACT) and are further transesterified to intramitochondrial CoA on the mitochondrial matrix side by carnitine palmitoyltransferase II (CPT II). Acyl-CoA is formed, and these long-chain CoA complexes enter

-oxidation, resulting in the formation of acetyl-CoA needed for the citric acid cycle. Catalysed by carnitine acetyltransferase (CAT), the released L-carnitine forms complexes with acetyl residues, which can then leave the mitochondria via CACT. In addition, L-carnitine is involved in the recycling of CoA, facilitating the shuttling of short-chain acyl groups from the mitochondria to the cytosol (), which results in an increase of mitochondrial free CoA and thus better availability for further energy production.[6] L-Carnitine is also involved in the regulation of glucose metabolism via the modulation of the free /acetyl-coenzyme A ratio.[7]

The main target of meldonium is the enzyme GBB hydroxylase, which can be inhibited competitively as well non-competitively.[8,9] The following main actions of meldonium have been reported (for a review, see [10-12] and figure to supplement). In humans, meldonium decreased the concentrations of L-carnitine and increased the plasma concentration of GBB.[13] The inhibition of -oxidation induced an increase in fatty-acid levels in the sera of treated rats.

Meldonium blocked the transport of carnitine inside the mitochondria by a weak inhibition of CAT

[14] and inhibited Na+ -dependent carnitine transport and CPT I activity. In addition, weak competitive inhibition of CACT was reported.[15] Meldonium increased the protein content of hexokinase type 1 and sarcoplasmic reticulum Ca2+-ATPase, activated the reuptake of Ca2+ by the sarcoplasmic reticulum, [16-18] and improved the uptake of Ca2+ ions in the sarcoplasmic reticulum.[19] In addition, it induced an increase in renal L-carnitine excretion [13] and a competitive inhibition of OCNT2 proteins by the renal brush-border membrane.[9] In total, the consequences of meldonium applications are: a) a shift of cell metabolism from highly oxygen- consuming fatty-acid oxidation to increased glucose consumption and increased effectiveness of

ATP generation and b) a protection of the mitochondria against free fatty-acid overload by the reduction of long-chain acylcarnitines, the activation of mitochondrial free fatty-acid utilization, and the redirection of fatty-acid metabolism from the mitochondria to the peroxisomes. Legend to Figures:

Figure to Supplement:

Cellular transport of L-carnitine and fatty acids and mitochondrial energy-metabolism pathways including the sites of meldonium actions (adapted from [4, 19]).

Abbreviations: inhibition by meldonium; activation by meldonium; OMM, outer mitochondrial membrane; IMM, inner mitochondrial membrane; OCTN2, organic cation/carnitine transporter 2; CPT, carnitine palmytoil-transferase; CACT, carnitine acyl-carnitine translocase;

LC-Acyl-CoA, long-chain acyl-coenzyme A; CoASH, free coenzyme A; glucose-6-P, glucose-6- phosphate; PDC, pyruvate dehydrogenase complex; LC-acylcarnitine, long-chain acylcarnitine;

LC-acyl-CoA, long-chain acyl-coenzyme A; CAT, carnitine acetyl-transferase Reference List

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