Meldonium (purity > 99.9%) was purchased from Yuanye Pharmaceutical Co. (Shanghai, China). L-carnitine (purity > 99.9%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). A stable isotope labeled internal standard compound, acetonitrile and methanol, were obtained from Merck Chemical Co., (Darmstadt, Germany). Formic acid and ammonium acetate were purchased from Thermo Scientific (Waltham, United States). All the other chemicals were of analytical grade or better.

The Liquid chromatography-tandem mass spectrometry (LC-MS/MS) system consisted of a liquid chromatograph (Waters Technologies, Massachusetts, United States) and coupled with an AB SCIEX 6500 triple quadrupole mass spectrometer (Sciex Corp., Framingham, United States). A FLYDWC50-1A animal simulated hypobaric hypoxia chamber was purchased from Guizhou Fenglei Oxygen Chamber Co., Ltd. (Anshun, China). An FLPI-2 laser doppler blood flow monitor (Moor Instruments, Axminster, UK), a high-content imaging analysis microscope (Molecular Devices, California, United States), an ABL90 Flex blood gas analyzer (Radiometer Medical, Copenhagen, Denmark), and an IX51 light microscope (Olympus, Japan) were also applied in this study.

Specific pathogen-free (SPF) Sprague–Dawley (SD) rats weighing 200 ± 20 g were purchased from SIBEIFU Biotechnology Co., Ltd. (Beijing, China). SD rats were housed with an environmental temperature of 22°C ± 2°C and 40%–70% relative humidity. All the experimental animals were acclimated under the above conditions for 3 days and fasted overnight before the experiments. Operations for animal experiments were carried out the Guide for the Care and Use of Laboratory Animals (NBCDSER-IACUC-2018-096, Institutional Animal Care and Use Committee of Laboratory Animal Center, Beijing, China).

To evaluate the effect of pre-administrated with meldonium under acute high-altitude hypoxia, SD rats (6 in each group) were administered by gavage and randomly divided into normoxia group, hypoxia group (model group), hypoxia groups with differential dose meldonium (25, 50, 100, 200, and 400 mg/kg), and hypoxia with acetazolamide group (positive control). In addition, the pharmacokinetics of meldonium (25, 50, 100 mg/kg) was investigated under acute high-altitude hypoxia in SD rats administered by gavage and/or intravenously. Normoxia group rats given saline lived at an average altitude of 43.5 m, and the acute hypoxia groups were placed in a simulated hypobaric hypoxia chamber with an altitude of 7,000 m for 24 h after 3 days of continuous administration with meldonium.

Rats were anesthetized upon exit from the hypobaric hypoxia chamber. Blood samples were drawn from the abdominal aorta into polypropylene syringes containing heparin and immediately transferred to a biochip to keep blood gases near physiological values. Acid-base balance, metabolites, and electrolytes changes, including blood bicarbonate (pHCO₃ ⁻), pH, partial pressure of carbon dioxide (pCO₂), partial pressure of oxygen (pO₂), glucose (Glu), lactic acid (Lac), sodium (Na⁺), potassium (K⁺), and chloride (Cl⁻) were assessed using a blood gas analyzer (ABL90, Radiometer, Copenhagen, Denmark).

The screening included an evaluation of vital functions (systolic and diastolic blood pressure, and mean arterial pressure), biochemical blood analysis, and routine blood examination, which were used for the initial assessment of hypoxia injury and efficacy of meldonium.

Initial assessment of liver injury included measuring levels of serum alanine and aspartate aminotransferases (ALT and AST). Serum creatinine acid (CREA) and blood urea nitrogen (BUN) were used as indicators for kidney damage. Creatine kinase (CK) and serum lactate dehydrogenase (LDH) were used as biochemical markers of cardiac injury. Complement C3 and C4, involved in the immune response, were also detected. In addition, red blood cells (RBC), white blood cells (WBC), hemoglobin, and lymphocytes (LYMPH) were evaluated.

Twenty-four hours post simulated high-altitude hypoxia exposure, rat brain tissue was perfused with 0.9% normal saline and 4% paraformaldehyde solution. The brain tissue was removed, fixed, dehydrated by graded ethanol, embedded in paraffin, and cut into 5 μm thick sections. Sliced sections were stained with hematoxylin and eosin (H&E) and examined using an optical microscope (XDS-113, Olympus Corporation, Japan).

Relative cortical cerebral blood flow was measured with a laser Doppler probe secured to the dura matter. Rats were euthanized, and the skull was exposed by cutting the skin of the head and washed with 150 µL saline. Blood flow in cerebral vessels was captured continuously by low-frequency pulsed ultrasound using laser doppler flowmeters (Moor, England). Results were recorded and analyzed by Moor FLPI-2 Review V50 software.

High-performance liquid chromatography (HPLC) and mass spectrometer analyses of meldonium were performed on a Thermo Hypercarb C18 column (3 mm × 100 mm, 5 μm, Thermo Fisher Scientific, Germany). Autosampler temperature was maintained at 15°C and the injection volume was 5 μL. The flow rate was set at 500 μL min^-1. The mobile phase consisted of methanol and 5 mM ammonium acetate buffer solution containing 0.1% formic acid. The mass spectrometer was operated in the positive ESI mode with a drying gas temperature of 350°C, nebulizer pressure of 40 psi, capillary voltage of 4000 V, and multiple reaction monitoring (MRM) for meldonium with scan time100 m/s (m/z, 147.1 → 58.2).

To explore the pharmacokinetics of meldonium under acute high-altitude hypoxia, 0.2 mL of venous blood was collected in an anticoagulation tube (EDTA-K2) before (time 0) and at 0.08, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h post meldonium administration, and then centrifuged at 2,500 g for 15 min at 4°C. The blood collection time for i.v., administration was increased by 2 min.

Rat liver homogenate, as the incubation system, was taken and added 100 μL of meldonium working solution concentrations (400 μg/mL, 200 μg/mL, 100 μg/mL, 40 μg/mL, 20 μg/mL, 10 μg/mL, and 4 μg/mL) to vortex and mix, and placed in a 37°C water bath for incubation. The samples supernatant was taken at 0 min, 15 min, 30 min, 1 h, 2 h and 4 h, respectively, for determination in LC-MS/MS.

Pharmacokinetic parameters of meldonium including peak time (T_max) and peak concentration (C_max), area under curve (AUC_all) of plasma concentration-time, the apparent volume of distribution (V_z/F), clearance rate (CL/F), and last mean residence time (MRT_last) were calculated using Phoenix WinNonlin software 8.0 (Certara, Princeton, United States). Data are expressed as mean ± SEM. Statistical analyses were performed using GraphPad Prism 6 (GraphPad Software, La Jolla, US) and assessed by the one-way ANOVA followed by Tukey’s test. p < 0.05 were considered statistically significant.

SD rats were exposed to simulated high-altitude environment for the hypoxia test, and the normoxia group was used as parallel control (Figure 1A). Within the first 15 ∼ 20 min of increasing altitude by 30 m/s, compared with the normoxia group, increased activity frequency, faster abdominal breathing, and head-up posture and neck extension were observed in the hypoxia group. With prolonged periods of hypoxia, activity frequency of rats gradually decreased. When reaching the specified altitude of 7,000 m, rat huddled together, and trembling continued until the end of hypoxia treatment. This hypoxia model can induce abnormal activities or affect rat behavior.

By comparison, we found that heart rate tended to increase under acute hypoxia compared to the normoxia group, while heart rate in the meldonium (50 mg/kg and 200 mg/kg) groups were significantly lower after prophylactic administration (p < 0.05). We then compared changes in systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP) in normoxia and hypoxia group as well as after meldonium treatment and found a significant increase in SBP, DBP, and MAP in the hypoxia group (p < 0.05). Further, SBP, DBP, and MAP values in the meldonium pretreatment group by i.g., administeration (50 mg/kg) significantly improved (p < 0.05) and were superior to the positive control drug acetazolamide (Figure 1B).

The methodological validation of meldonium in rat plasma is shown in Table 1. High levels of meldonium linearity were achieved within a range of 0.5–50 μg/mL (r ² = 0.9960) with the lower limit of quantification at 0.5 μg/mL. Inter-batch precision and accuracy were determined by measuring six replicates of quality control (QC) samples at three concentration levels in rat plasma. Results show that the intra-batch precision of meldonium was less than 5.57%, inter-batch precision was less than 8.06%, and accuracy was less than 15%. The recoveries of internal standard calibration in rat plasma at three concentration levels of meldonium ranged from 99.07% to 105.28%. For ionization, the total relative standard deviations of the internal standard normalized matrix effect factors corrected for internal standard at low and high concentrations of meldonium were 4.84% and 4.64%, respectively, suggesting that there was no measurable matrix effect that interfered with meldonium determination in rat plasma. The meldonium solution was stable at room temperature.

Based on a preliminary pharmacodynamic examination, three dose groups of meldonium (low dose, 25 mg/10 mL/kg; medium dose, 50 mg/10 mL/kg; high dose, 100 mg/10 mL/kg) were selected for pharmacokinetic studies. Figure 5A show mean plasma concentration time profiles for meldonium. The normoxia dose groups showed that the AUC of meldonium administered by gavage exhibited non-linear clearance in the range of 25–50 mg/kg. However, concentration-time curves after exposure at 7,000 m high-altitude for 24 h showed an AUC of meldonium with a dose-dependent increase within the range of 25–100 mg/kg by intragastrical administration. Plasma C_max, AUC_all, and MRT_last in meldonium low dose group (25 mg/kg) by intragastric administration under hypoxia showed significantly higher than those of the same dose normoxia group (Table 2).

In order to more visually examine the pharmacokinetic changes of meldonium under normoxia and acute plateau hypoxia, we selected a medium dose of meldonium by intragastric administration for further investigation. As shown in Figure 5B and Table 2, compared to the normoxia group, reduced C_max (24,904.97 ± 22,618.59 Vs. 11,118.71 ± 3,515.13 mL/h/kg, p < 0.05) and increased Vz/F (2,773.03 ± 756.42 Vs. 5,032.12 ± 1,679.6 mL/kg, p < 0.05) of meldonium in plasma under acute high-altitude conditions were observed. In addition, the pharmacokinetics of low-dose intravenous and i.g., administration under normoxia and acute high-altitude hypoxic conditions were evaluated (Figure 5C; Table 3). Similar to clearance of the medium dose of meldonium, plasma clearance for low-dose meldonium (25 mg/kg) given by i.g., administration was significantly lower (1,400 ± 362 Vs. 230 ± 150 mL/h/kg, p < 0.05) and MRT_last was prolonged (4.34 ± 0.58 Vs. 8.40 ± 1.36 h, p < 0.05) under acute high-altitude hypoxia, compared to the normoxia group. However, no significant changes in these PK parameters were observed after low-dose intravenous administration, with comparable MRT_last (3.14 ± 0.68 Vs. 3.43 ± 1.44 h) and clearance (487 ± 34 Vs. 743 ± 252 mL/h/kg, p < 0.05) for normoxia and to high-altitude groups. Therefore, the pharmacokinetics of the different meldonium administration methods differ significantly under acute high-altitude hypoxia.

To investigate the in vitro metabolite of meldonium, succinate and 3-hydroxypropionic acid, rat liver homogenates with serial concentrations of meldonium were incubated. The study results showed that the metabolite 3-hydroxypropionic acid showed an increasing trend with concentration and time in the high concentration group (100–400 μg/mL), while no increasing trend was found with increasing content in the low concentration group (4–40 μg/mL), but an increasing trend was found after 4 h of incubation. The metabolite succinic acid showed an insignificant trend with concentration, but showed an increasing trend with increasing incubation time (Figure 5D).