Study on the influence of 7-β-hydroxy-γ-aryloxypropylxanthinyl- 8-thioalkanic acid derivatives on the lipid metabolism in experiment

Statin side effects are not a rare occurrence, in particular dyspeptic disorders, insomnia, headache, skin erythema, rash are often noted. All of this determines scientists to find new effective and low-toxic hypolipidemic agents. Various natural and synthetic xanthine derivatives have been recognized as therapeutically potential compounds and reported to control various diseases. Therefore, the study of new xanthine derivatives and their hypolipidemic effects, which would have a significant therapeutic effect with minimal side effects, is relevant.

According to the World Health Organization Expert Committee, atherosclerosis is a variable combination of changes in the intima of arterial vessels that includes the accumulation of lipids, lipoproteins, complex carbohydrates, fibrous tissue, blood components, calcifications and concomitant changes in the vascular tunica media. The vessels of elastic and muscular-elastic type are damaged mostly (aorta, brain vessels, coronary arteries) and less often -vessels of the lower extremities. The term "atherosclerosis» was firstly formulated in 1904 by Felix Jacob Marshand [1].
The most effective and popular hypolipidemic medicines from the 90s to the present time are statins. During this time, the great number of clinical studies were conducted to investigate statins. The main ones are: ALLHAT, PROSPER, WOSCOPS, PROVE-IT, CARE -to study pravastatin, HPS, IDEAL, A to Z, 4S -simvastatin, ASCOT LLA, CARDS -atorvastatin, AF-CAPS -lovastatin, LIPS -fluvastatin, CORONA, JUPITER -rosuvastatin. IMPROVE-IT, FOURIER and ODYSSEY studies have formed the database to enhance low-density lipoprotein (LDL) cholesterol lowering therapy in addition to statins [1,4].
Statins are usually used for a long time and are relatively well tolerated. However, the side effects of statins are not rare, in particular, dyspeptic disorders, insomnia, headache, erythema of the skin, rash. Dose-dependent side effectshepatotoxicity (with or without elevated transaminases)can be caused by the all types of statins. The most severe side effect of all statins is myopathy, which is manifested by the development of skeletal muscle myodystrophies and others. All of that has led scientists to discover new effective and low-toxic lipid-lowering medicines [5][6][7].
A promising direction in this regard is the search for effective and low-toxic hypolipidemic medicines, in particular, on the basis of xanthine derivatives. Among the alkaloids, xanthine and its derivatives occupy one of the leading positions in the field of medical care. These compounds are natural heterocyclic alkaloids based on purine, which were firstly discovered in 1817 by German chemist Emil Fischer, and later in 1899, the designation «xanthine» was introduced. The structural similarity to two important purine derivatives, adenine and guanine, makes xanthine an interesting therapeutic molecule. Xanthines are known for their diverse biological role, including inhibition of various cellular signaling enzymes and exhibition of polysystemic pharmacological activity, such as antidepressant, antibacterial, metabolic drugs, etc. Natural xanthine derivatives such as caffeine, theophylline and theobromine, are nitrogenous compounds based on purine, which have broad medicinal properties [8].
Various natural and synthetic xanthine derivatives have been recognized as therapeutically potent compounds and have been described to fight various diseases. As for the potential health benefits, interest in these compounds is constantly growing from the sides of scientists, medical professionals and consumers. Therefore, the study on new xanthine derivatives and their hypolipidemic effects, which would have a significant therapeutic effect with minimal side effects, is relevant [9].

Aim
The aim of the study was to investigate the effects of 7-β-hydroxy-γ-aryloxypropylxanthinyl-8-thioalkane acid derivatives on the lipid profile in laboratory rats experimentally.

Materials and methods
The objects of the study were 11 derivatives of 7-β-hydroxy-γ-aryloxypropylxanthinyl-8-thioalkane acids ( Table 1), synthesized at the Department of Biological Chemistry of Zaporizhzhia State Medical University headed by Professor M. I. Romanenko, which are promising in search for hypolipidemic medicines. A number of modern physicochemical methods of analysis confirmed the structure of the compounds: IR-, PMR-spectroscopy, mass-spectrometry. The experiments were performed in 112 white laboratory Wistar rats aged between 6-8 months, weighing 180-220 g. The rats were divided into groups: group 1 (normal) -intact animals; group 2 (control) -animals with experimental hiperlipidemia (without treatment); groups 3-13 -animals with hiperlipidemia and tested compounds; group 14comparison drug -atorvastatin. Each group consisted of 8 animals. The rats were obtained from the animal farm of the State Institution "Institute of Pharmacology and Toxicology of the Academy of Medical Sciences of Ukraine". The animals were kept on the standard diet in natural light regime "day and night".
The tween model as an experimental hyperlipidemia was used: intraperitoneal administration of the tween-80 in a dose of 200 mg/100 g body weight. Tested compounds were administered orally, simultaneously with tween, for 6 days. 12 hours after the last administration of tween-80 or tested compounds, the animals underwent laparotomy under ether narcosis, blood was taken from glomerular artery, centrifuged to obtain a serum. This model is advantageous since leads to a rapid (8-10 hours) increase in blood lipids (especially triglycerides) and a decrease in HDL cholesterol. The tested compounds were administered at the dose of 1/10 of the LD 50 (previously calculated by the express method of Prozorovsky) [10]. The comparison option was the medicine from the group of statins -atorvastatin ("Atorvastatin" (Ananta Medicare (India)) at the dose of 20 mg/kg body weight.
The level of total cholesterol (TC) was determined by colorimetric, enzymatic methods, diagnostic kits Cormay Laboratory tests were carried out on the semi-automatic biochemical analyzer "Screen point" (Italy).
For the integral assessment of the derivative hypolipidemic action, the conditional efficacy index Ʃ (EI) was used representing the generalized value of lipid metabolism (sum of reductions in percentages of TC, LDL cholesterol, TG: EI = TC (%) + LDL cholesterol (%) + TG (%) [11].
During the experiment, the rules and regulations were followed according to the Protocol no. 5 (April 17, 2019) of the Commission on Bioethics Session of ZSMU.
The results of the study were processed on a personal computer using the statistical package of the licensed program Statistica, version 13 (Copyright 1984-2018 TIBCO Software Inc. All rights reserved. License No. JPZ8041382130ARCN10-J). The normality of the quantitative variables distribution was analyzed using the Shapiro-Wilk test. Descriptive statistics was provided in the form of a median with interquartile range -Me [Q 25 ; Q 75 ], because the parameters had the distribution different from the normal. The Mann-Whitney test was used to find differences between the groups. A level of P < 0.05 was considered statistically significant.
The highest hypolipidemic activity (TC) relative to the control group was shown by compounds 6043  Analyzing the dependence of lipid profile on the structure of the tested derivatives, we were able to draw some preliminary conclusions. O-methyl-substituted derivatives of 3-methylxanthine (compounds 2439, 2486) and theophylline (compound 6047) showed the highest activity. Moreover, the amide (compound 2439) was more active than acids (derivatives 2486 and 6047). Elongation of the carbon chain in the thioalkanoic acid residue at position 8 of the xanthine molecule practically led to loss of hypocholesterolemic and hypolipidemic effect (compound 2487). Similar effects occurred when the methyl was replaced by the methoxy group (compounds 5705 and 6042). The significant decrease in hypolipidemic activity was observed in a large aromatic substitution (compounds 8402, 8403). For the more detailed analysis of the dependence in the series "structure-action", it is necessary to expand significantly the chemical library of the tested compounds.

Discussion
In recent years, heterocyclic compounds, including xanthine derivatives, have received considerable attention due to their importance in pharmacological research. In particular, the data obtained by us are confirmed by the research results of D. H. Ivanchenko and N. Singh: the use of 3,7-dimethylxanthine (theobromine) is associated with a reduced risk of cardiovascular disease. The main rationale for this is that theobromine has a beneficial effect on fasting serum lipids. It can be associated with inhibition of  Original research phosphodies terases, which, by destroying cellular cyclic adenosine monophosphate (cAMP), increase the activity of ABCA1 (a gene encoding the protein that regulates cellular cholesterol and phospholipid homeostasis), which plays a role in a removal of cholesterol from macrophage foam cells to apolipoprotein A-I (the main apolipoprotein of HDL-cholesterol). Therefore, theobromine can increase HDL-cholesterol levels and have a cardioprotective effect [12][13][14][15]. Another generalized study that significantly confirms our results was that the compound KMUP-1 (7-[2-[4-(2-chlorophenyl)piperazinyl]ethyl]-1,3-dimethylxanthine), by inhibiting phosphodiesterase, modulates G-protein-coupled receptors (GPCR) to reduce hyperlipidemia and lose weight. KMUP-1 reduced the accumulation of triglycerides, indicating inhibition of adipogenesis in cells, and the expression of mitogen-activated protein kinase (MAPK) and immunoreactivity at the stage of adipogenesis. Appropriate biochemical reactions contributed to the inhibition of adipocyte differentiation. These data suggest that KMUP-1 may inhibit hyperadiposis in adipocytes [16].
In addition, literature data confirms that KMUP-1 can cause the decrease in liver fat. However, the mechanisms of KMUP-1 action in obesity-induced steatohepatitis remain unclear. Prolonged administration of KMUP-1 to mice on a high-fat diet reduced body weight, triglyceride and glucose levels, which is completely correlated with our data. Furthermore, KMUP-1 reduced the amount of MMP-9 (matrix metalloproteinase 9 -extracellular zinc-dependent endopeptidase capable of destroying extracellular matrix protein) and reactive oxygen species (ROS), and increased the content of anti-inflammatory cytokine IL-10 in the liver of mice on the high-fat diet. Thus, it was shown that KMUP-1 reduces the accumulation of lipids in liver tissue, which is the promising aspect in the fatty liver disease treatment [17][18][19].
What is more, according to X. Zhu, it was shown that incubation of preadipocytes with a solution containing xanthines significantly reduced the incorporation of triglycerides during adipogenesis without affecting cell viability. Finally, the active study of xanthine derivatives with hypolipidemic activity continues [16]. 4. The results of experimental studies clearly indicate the feasibility and prospects for further search on original lipid-lowering drugs among xanthine derivatives.
The prospect for further research is the study of 7-β-hydroxy-γ-aryloxypropylxanthinyl-8-thioalkane acid derivatives on the ability to enhance a hypolipidemic effect and to influence the values of oxidative stress in various models of hyperlipidemia.