Meldonium Decreases the Dietincreased Plasma Levels Of
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Brief Report The Journal of Clinical Pharmacology Meldonium Decreases the Diet‐Increased Plasma XX(XX) 1–4 © The Author(s) 2013 Levels of Trimethylamine N‐Oxide, a Metabolite DOI: 10.1002/jcph.135 Associated With Atherosclerosis Maija Dambrova, PhD1,2, Elina Skapare‐Makarova, PhD1, Ilze Konrade, PhD2, Osvalds Pugovics, PhD1, Solveiga Grinberga, PhD1, Dace Tirzite, PhD1, Ramona Petrovska, MSc3, Ivars Kalvins, PhD1, and Edgars Liepins, PhD1 Keywords trimethylamine, trimethylamine N‐oxide, meldonium, urinary excretion, UPLC/MS/MS Meldonium (mildronate; 3‐(2,2,2‐trimethylhydrazinium) in plasma and the urine samples of healthy volunteers. In propionate dehydrate) is an anti‐ischemic and anti‐athero- addition, the effects of meldonium and TMAO on in vitro sclerotic drug that exerts its cardioprotective action through L‐carnitine uptake were studied in human embryonic 1,2 an L‐carnitine‐lowering effect. The long‐term administra- kidney 293 (HEK293) cells. tion of mildronate also significantly lowers the L‐carnitine plasma concentrations in non‐vegetarian, healthy volun- Subjects and Methods 3 teers. Meldonium regulates the bioavailability of L‐carnitine Subjects and Study Design by inhibiting its biosynthesis via the gamma‐butyrobetaine This study was performed with the approval of the Central hydroxylase enzyme4 and its reabsorption in the kidneys via Medical Ethics Committee of Latvia, and informed consent carnitine/organic cation transporter (OCTN) 2 proteins.5 In was obtained from all subjects. This open label, interven- experimental models, meldonium increases the levels of tional study included eight healthy volunteers: four female gamma‐butyrobetaine, a precursor of L‐carnitine biosynthe- and four male. The duration of the study was 3 weeks. All sis.6 However, the effects of meldonium treatment on other study participants were European descent. The criteria for metabolites of L‐carnitine have not been studied. the volunteers to participate in the study were as follows: The homeostasis of L‐carnitine is maintained through good health, as assessed by clinical examination, non‐ biosynthesis, efficient kidney reabsorption, and food smoking, not pregnant, and no history of drug or alcohol intake, particularly from meat and dairy products.7 In the abuse. The data collected at the study inception included intestine, L‐carnitine is degraded to trimethylamine age, medical history, and anthropometric indices. (TMA),8,9 a simple tertiary amine that is subjected to Before beginning the study, the participants had not metabolism by flavin‐containing monooxygenase (FMO) included any fish or marine products in their diet for and oxidized to odorless trimethylamine N‐oxide 7 days. The subjects arrived at the trial unit at 8:30 am after (TMAO). Both TMA and TMAO are common metabolites overnight fasting, and basal blood samples were collected. in animals.10 TMAO decomposes to TMA, which is the For the next 7 days, the participants consumed a TMA‐rich main odorant characteristic of degrading seafood and is an lunch that included approximately 150 g of fish and sea osmolyte found at high concentrations in fish.11 A recent study has provided strong associations between systemic TMAO levels and both angiographic measures of coronary 1 12 Latvian Institute of Organic Synthesis, Riga, Latvia artery atherosclerosis and cardiac risk. Furthermore, 2Riga Stradins University, Riga, Latvia plasma L‐carnitine levels in patients with concurrently high 3Latvian Biomedical Research and Study Centre, Riga, Latvia TMAO levels predict increased risk for both prevalent cardiovascular disease and incident major adverse cardiac Grant sponsor: BIOMEDICINE. 13 events. Thus far, no pharmacological agent has been Submitted for publication 26 April 2013; accepted 16 June 2013. shown to lower TMAO concentrations in vivo. Corresponding Author: L‐ ‐ Taking into account the carnitine lowering activity of Maija Dambrova, PhD, Latvian Institute of Organic Synthesis, Aizkrau- meldonium, the aim of the present study was to investigate kles Str. 21, LV1006, Riga, Latvia. the effects of meldonium on diet‐increased TMAO levels Email: [email protected] 2 The Journal of Clinical Pharmacology / Vol XX No XX (2013) products (salmon, cod, herring, or shrimps). Plasma grade, Sigma–Aldrich)/methanol (gradient grade, Merck, samples were collected at the end of the period, and a 7‐ Darmstadt, Germany) mixture (3/1). The urine sample was day meldonium (Mildronate®, Grindeks, Latvia) treat- diluted with deionized water (1/50), and 100 mLwasmixed ment then began in addition to the TMA‐rich diet (the with 700 mL internal standard (3‐(2,2‐dimethyl‐2‐prop‐1‐yl‐ choice of fish products was similar to the previous week). hydrazinium)propionate, 200 ng/mL) solution in an acetoni- Meldonium was administered perorally at a dosage of trile/methanol mixture (3/1). The samples were centrifuged 500 mg, twice daily between meals. Plasma samples were for 10 min at 13,000 rpm, and the clear supernatants were collected at the end of the period. Urine samples were separated and used for the analysis. The chromatographic collected every morning and evening during the experi- separation of analytes was achieved using an Acquity UPLC mental period. Urine aliquots and plasma samples were system (Waters, Milford, MA, USA). The analytes were stored at À20°C prior to analysis. separatedonanHILICBEHcolumn(2.1mm 100 mm, 1.7 mm, Waters) using a gradient elution from 75% to 55% The Inhibition of OCTN2‐Mediated Transport of L‐ acetonitrile in 10 mM aqueous ammonium acetate (pH 4) at a Carnitine flow rate of 0.25 mL/min. The TMAO analyses were The transport of L‐carnitine was measured as L‐[N‐ performed in the multiple reaction mode (MRM). Two methyl‐3H] carnitine hydrochloride (specific activity, 85 precursors to product ion transitions (m/z 75.8 > 58.3 for Ci/mmol, Biotrend, Köln, Germany) uptake by HEK293 TMAO and m/z 175.4 > 86.0 for the internal standard) were (ATCC collection code CRL‐1573, LGC Standards AB, monitored. Data acquisition and processing were performed Boras, Sweden) cells. The cells were grown in 24‐well using MassLynx V4.1 and QuanLynx V4.1 software plates in DMEM/F‐12 medium (Sigma–Aldrich, Stein- (Waters). heim, Germany) supplemented with 10% fetal bovine serum (FBS, Sigma–Aldrich) until approximately 90% Statistical Analyses and Calculations confluence was reached. Prior to the assay, the cells were The results are expressed as the mean Æ standard error washed twice with 0.5 mL DMEM/F‐12 without FBS; the means (SEM). Statistically significant differences in the assay was performed in 300 mL of medium. The cells mean values were tested by a repeated measures ANOVA were pre‐incubated with meldonium or TMAO (Sigma– and Tukey’s post‐test. The differences were considered Aldrich; 10, 30, 100, 300, and 1,000 mM) for 15 min at significant when P < .05. The data were analyzed using 37°C, and uptake was initiated by the simultaneous GraphPad Prism 3.0 statistical software (GraphPad Inc., addition of unlabeled L‐carnitine (10 mM) and L‐[N‐ La Jolla, CA, USA). The amount of TMAO excreted in methyl‐3H] carnitine (4 nM, 12 kBq/mL). After incuba- the urine was calculated as the area under the curve tion for 60 min at 37°C, the medium was removed, and the after taking into account the average creatinine excretion cells were washed three times with 300 mL ice‐cold rate determined by the Jaffe method, as described phosphate‐buffered saline (PBS). The cells were then previously.3 lysed directly in the plate with 100 mL 0.1% sodium dodecyl sulfate (SDS) in 1 M NaOH. A 200‐mL aliquot of scintillation cocktail was added to 50 mL of the cell lysate, Results and Discussion and the radioactivity was measured using Wallac Because fish products are rich in TMA and it has been MicroBeta Trilux scintillation counter (PerkinElmer shown that the consumption of fish products increases the Inc., Waltham, Massachusetts, USA). The data were urinary concentrations of TMAO,14,15 we used fish as part normalized to the protein content, as determined using the of a TMA‐rich diet in the present study. The average age Lowry method. The control measurement of L‐carnitine and body‐mass index of the study participants did not uptake in the absence of additional substances was taken differ significantly between the genders and was 30 Æ 2 as 100%. The measurements were performed in triplicate years and 23 Æ 1 kg/m2, respectively. No adverse events for each compound concentration, and the data were were observed during the study. As shown in Figure 1A, analyzed using GraphPad Prism 3.0 software. the average plasma concentration of TMAO was 4.9 Æ 1.3 mM before the start of the study, and the UPLC/MS/MS Analysis plasma TMAO concentration increased 16‐fold The TMAO concentrations in both the plasma and urine (81.5 Æ 8.6 mM) after 7 days of the TMA‐rich diet. samples were determined using a UPLC/MS/MS method and The addition of meldonium to the TMA‐rich diet increased aQuattroMicrotriple–quadrupole mass spectrometer the plasma concentration of TMAO only ninefold (Micromass, Waters, Milford, MA, USA) using electrospray (43.0 Æ 3.8 mM; Figure 1A). The excretion of TMAO ionization in the positive ion mode. The plasma sample to be in the urine followed the plasma analysis patterns: the tested (25 mL) was mixed with 500 mL internal standard (3‐ concentration of TMAO was 2.8 Æ 0.5 mmol/mg creati- (2,2‐dimethyl‐2‐prop‐1‐yl‐hydrazinium)propionate, 200 ng/ nine  7 days at the beginning of experiment, with a mL, synthesized in‐house) solution in acetonitrile (gradient sixfold increase (18.2 Æ 2.2 mmol/mg creatinine  7 Dambrova et al 3 Figure 2. The inhibition of the OCTN2‐mediated uptake of L‐carnitine 3 by meldonium and TMAO.