METADOXINE REGULATION OF ELIMINATION OF ETHANOL AND ITS METABOLITES FROM THE RAT’S BODY

Development of pathogenetic therapy of acute ethanol poisoning is of undoubted interest since such intoxication is often accompanied by a severe course and high lethality. Taking into account the peculiarities of the ethanol pathological action the treatment of such patients involves the use of drugs with the complex neuroand hepatoprotective effects. Thereby, the studies for searching new pharmaceutical agents with a wide range of the pharmacological activity and economic availability continue. For a long time the object of researchers’ attention is the chemical compound – pyridoxineL-2-pyrrolidone-5-carboxylate (metadoxine). Till this moment a large number of the experimental data showing a diverse pharmacological profile of the drug action (antioxidant, antisteatotic, anti-inflammatory, antifibrotic, neurotropic) has been accumulated. Paying attention to the facts mentioned above our attention was focused on the detoxification action of metadoxine. Therefore, the aim of this work was to study the effect of metadoxine on the rate of excretion of ethanol and its metabolites in white rats in a preventive single introduction of the drug. The parameters of excretion of acetaldehyde, acetate and the unchanged ethanol with urine and feces have been chosen as a criterion for assessing the effectiveness of the process. Excretion of ethanol by lungs and sweat glands has not been studied because of a negligible contribution of these pathways in the process of ethanol elimination. It has been determined that intraperitoneal introduction of metadoхine in the dose of 200 mg/kg 30 min prior ethanol introduction (orally in the dose of 1 g/kg) accelerates the rate of elimination of ethanol and its metabolites (acetaldehyde and acetate) in rats.

D evelopment of pathogenetic therapy of acute ethanol poisoning is of undoubted interest since such intoxication is often severe and highly lethal. Treatment of acute poisoning with ethyl alcohol traditionally includes a set of measures maintaining the vital functions and constant internal environment, as well as measures aimed to accelerate ethanol elimination [5,14]. Currently, there is large number of drugs used in medical practice for the treatment of the effects of the ethanol action on the human body. Taking into account the peculiarities of ethanol pathological action the treatment of such patients involves the use of drugs with the complex neuro-and hepatoprotective effects.
Thereby, the studies for searching new pharmaceutical agents with a wide range of the pharmacological activity and economic availability continue. For a long time the object of researchers' attention is the chemical compoundpyridoxine-L-2-pyrrolidone-5-carb-oxylate (metadoxine), which is an equimolar combination of pyrrolidone carboxylate (A) and pyridoxine (B) as an ionic salt ( Fig.).
L-2-pyrrolidone-5-carboxylate (pyroglutamate, 5-oxoproline) is included to the substance due to the possibility of this cyclic lactam of glutamic acid (an intermediate of the gamma -glutamine cycle) to be involved in the glutathione metabolism [15].
The cationic part of metadoxine is presented as pyridoxine possessing the vitamin activity (one of vitamin B 6 three forms). The compound plays an important role in metabolism of the substances involved in the synthesis of neurotransmitters. The phosphorylated form participates in the processes of decarboxylation, transamination and deamination of amino acids, improves the use of unsaturated fatty acids, reduces the level of cholesterol and lipids in the blood [7,10].
As the active pharmaceutical ingredient metadoxine has been developed by "Laboratory Balda-chi S.P.A." (Italy). The generic of this drug is manufactured in Ukraine by ALC "INTERCHEM" in the form of tablets ("Alkodez ® ІС" and "Liveriya ® ІС").
Till this moment a large number of the experimental data showing a diverse pharmacological profile of the drug action (antioxidant, antisteatotic, anti-inflammatory, antifibrotic, neurotropic) has been accumulated.
Paying attention to the facts mentioned above our attention was focused on the detoxification action of metadoxine.
Therefore, the aim of this work was to study the effect of metadoxine on the rate of excretion of ethanol and its metabolites in white rats in a preventive single introduction of the drug. The parameters of excretion of acetaldehyde, acetate and unchanged ethanol with urine and feces were chosen as a criterion for assessing the effectiveness of the process. Excretion of ethanol by lungs and sweat glands has been not studied because of a negligible contribution of these pathways in the process of ethanol elimination [11].

Materials and Methods
Experiments were conducted on white male Wistar rats weighing 200-270 g. Care of the animals and the experimental studies were carried out in accordance with the regulatory requirements [1,2]. The animals were divided into 2 groups: group 1 -control, and group 2 -test. The experimental animals received metadoxine (i.p., 200 mg/kg) 30 min before the main introduction. Ethanol was administered orally in the dose of Ѕ LD 50 (3.5 g/kg) by two groups of animals.
Collection of excretes was in 3; 6; 12; 24 and 30 h. When collecting the urine the funnel was washed 4 times with 10 ml of H 2 O, the total volume of the sample obtained (with overflows) was recorded. To determine the total radioactive material 1.0-2.0 ml of the sample were collected in scintillation vials. Quantification of the content of acetic acid was performed in scintillation vials. Of the total volume 10 ml of the sample were taken, and 1 ml of 10% Na 2 CO 3 was added, then it was evaporated to obtain a semi-dry residue. To determine acetaldehyde 10.0 ml were taken into a vial from the residue of the total sample, and hydrogen peroxide was added to oxidize acetaldehyde containing in the sample to acetic acid. Oxidation was carried out for 20 min, then 1 ml of 10% Na 2 CO 3 was added to adjust the alkaline pH (≥8.2), and it was evaporated to obtain a semi-dry residue. Feces were collected, placed in an oven, and then the samples were weighed. After grinding 50 mg of the samples were collected in each vial, 1 cm 3 of formic acid was added, and hydrolyzed on a water bath. In all cases, 1 cm 3 of Triton X-100 and 10 cm 3 of a scintillator of the Canberra Packard firm were added to the samples. The quantity of the radioactive material in the samples was determined by a Canberra PACKARD TRI CARB 2700 liquid scintillation photometer.
Processing of the results obtained was carried out in accordance with the algorithms [4]. In comparative analysis of the results of our studies Student's t-test was used [3]. The confidence interval in all the experiments was calculated at the significance level of P ≤ 0.05, providing the reliability of the results with the probability of 95%.
All mathematical calculations were performed using the Excel XP software package of the personal computers (Pentium 4).

Results and Discussion
The limiting stage of the ethanol action is the absorption process, which is carried out mainly in the proximal part of the gastrointestinal (GI) tract, in the stomach (70%) and in the duodenum (25%), while 5% of ethanol is absorbed in the distal intestine [13].
The first stage of the metabolism of alcohol occurs in the gastric mucosa with participation of the gastric fraction of alcohol dehydrogenase. According to some sources up to 10% of the ethanol administered is metabolized in the stomach [6]. However, it should be noted that at present time the issue of the stomach as an organ where the first phase of the metabolism of ethanol occurs is discussed. It is suggested that the first phase of the alcohol metabolism takes place in the li-ver [9], but it depends on the rate of absorption in the stomach and small intestine.
It has been found that systemic ethanol oxidation occurs in the liver by three parallel metabolic pathways: 1) by alcohol dehydrogenase providing metabolism of the major amount (90%) of alcohol in the liver, 2) by microsomal monooxygenase containing cytochrome P-450 2E1 (8-10% of ethanol oxidation), 3) by peroxisome catalase (oxidation to 2% of ethanol) [12]. Oxidation of ethanol by alcohol dehydrogenase requires participation of NAD + , due to which the proton transfers from the ethanol molecule to a molecule of NAD + with its reduction to NADH and formation of acetaldehyde. Further, in the mitochondria, acetaldehyde in a NAD-dependent reaction is converted into acetic acid via acetaldehyde dehydrogenase; in its turn, with participation of acetyl coenzyme A synthetase, acetic acid is involved in formation of acetyl-CoA involving in the Krebs cycle or the fatty acids cycle. NADH formed during this reaction oxidizes again. Thus, the alcohol degradation rate by alcohol dehydrogenase and aldehyde dehydrogenase depends on the intensity of the reoxidation of NADH to NAD + . The metabolism of one molecule of alcohol requires two molecules of NAD + . This significantly alters the redox potential of hepatocytes towards the reduction reactions. Degradation of alcohol can be inhibited if NADH is not constantly re-oxidized. The increase in the NADH concentration decelerates the reactions of the Krebs cycle. Under these conditions the excess of acetyl-CoA is consumed in the synthesis of ketone bodies entering the blood. This mechanism of ethanol metabolism is used to create medicines affecting the pathological units appearing during use (especially abuse) of alcohol.
At the first stage the kinetics of the total elimination of ethanol and its metabolites with the nor- mal and preliminary introduction of metadoxine in white rats was studied.
It has been found that metadoxine increases the total amount of radioactive products (Tab. 1) by 30 h after introduction exceeding the similar value after introduction of ethanol in 1.4 times (from 32.6 ± 5.6 to 46.1 ± 5.5% of the injected dose of ethanol). However, increase in the amount of the excreted total metabolites (on the background of metadoxine introduction is observed only in 7.5 h after introduction of ethanol, while by the third hour after introduction their quantity is eliminated higher in the group of intact animals (19.6 ± 2.3% of the injected dose). This difference is explained by the fact that the effect of metadoxine is due to modulation of biochemical processes. In this regard, the important question is not only about the elimination rate of the total amount of metabolites of ethanol, but about their quantitative composition as well.
There is an intensive metabolism of ethanol (increase in the acetate content in the urine is observed with time) as seen from the data presented (Tab. 2). Already from ~ 7 hour after introduction the amount of the excreted unchanged ethanol does not exceed 1% of the injected dose, while the major part of radioactive products is its metabolites -acetaldehyde (15-24%) and acetate (59-84%). Acetate formation in this case is the leading pathogenetic factor since it leads to metabolic acidosis and disturbance of the body homeostasis, while acetaldehyde formed because of its volatility is eliminated from the body not only by renal, but also by respiratory routes, reducing its toxicity.
There is a significant redistribution of the profile of the excreted ethanol and its metabolites against the background of the preliminary introduction of metadoxine. Thus, the relative amount of the excreted unchanged ethanol (from 15.9 ± 0.6 to 77.8 ± 0.6% in the first 3 h after introduction) significantly increases. At the same time the amount of the excreted unchanged acetaldehyde also increases (by 12 h after introduction increases from 18.2 ± 2.8 to 42.3 ± 2.7%). Simultaneously, the relative amount of the excreted acetate decreases in ~ 2.3 times (being 28.1 ± 0.8% in 30 h after introduction). This difference in the ratio of the excreted unchanged ethanol and its metabolites is the result of increasing of its metabolism.
For integral characteristic of the excretion processes based on the amount of the common excreted metabolites (Tab. 1) and their relative composition (Tab. 2) the indicators of the maximum amount of the excreted compounds (Tab. 3) at the theoretically infinite exposure time (Q max ), as well as the kinetic characteristics of these processes (excretion constant, k ex , h -1 ) were calculated. Noteworthy is the fact that the values of the unchanged ethanol Q max (increasing from 6.3 ± 0.6% to 38.5 ± 4.8%) and its final metabolite -acetate (decreasing from 19.9 ± 3.1% to 8.5 ± ± 0.7%) change most of all, while amount of the excreted acetaldehyde undergoes no statistically significant changes (Tab. 3).
The mechanism of the metadoxine stimulating effect on the Table 1

Table 2
The relative composition (% of the total radioactivity eliminated) of ethanol metabolites following the single introduction of ethanol (control) and on the background of the preliminary introduction of metadoхine (test) process of ethanol elimination has not been completely elucidated.
The elimination rate of ethanol may increase as a result of activation of the primary metabolism of ethanol in the gastrointestinal tract, intensification of the hepatic blood flow, induction of the synthesis of alcohol oxidation enzymes in the liver or re-oxidation of NADH. The latest mechanism deserves the greatest attention. The intensity of the reoxidation NAD(P)H process is a limited element determining the rate of any dehydrogenase reaction, including metabolism of ethanol to acetaldehyde followed by formation of acetic acid. The functioning of these enzymes is limited by the presence and availability of the oxidized form of NAD + . An excess of protons in alcohol intoxication does not allow the NAD/NADH system to transport all of them in the chain of biological oxidation enzymes. In this situation, the catalytic activity of alcohol dehydrogenase and aldehyde dehydrogenase is high, but their actions are blocked. To accelerate the oxidation of ethanol and acetaldehyde the efficiency of NAD/NADH turnover must be increased. There is information [8] that metadoxine under conditions of alcohol intoxication can maintain the normal ratio of NAD + /NADH in cells, and hence the necessary dehydrogenase activity, thus providing a high rate of ethanol biotransformation.
On the other side, such difference is explained by acceleration of acetate disposal (its inclusion in the energy and plastic metabolism), whereby its quantity excreted with the urine significantly decreases. By feedback acetate oxidation leads to the increased amounts of NADH and NADPH (and increase in the ratio of NADPH/NADP + ), which in turn, reduces the activity of alcohol dehydrogenase, and increases the amount of unmetabolized ethanol. It is observed in the experiment.
These mechanisms prevent development of ketosis and metabolic acidosis in acute alcohol intoxication. The increased rate of ethanol elimination is accompanied with the accelerated reduction of intoxication and the toxic effect.
CONCLUSIONS Based on the data obtained it can be concluded that the prior introduction of metadoxine accelerates the metabolic degradation of ethanol and its metabolites, which appearance and accumulation are responsible for development of the toxic effect of alcohol. These results allow considering the further study of the possible use of the drug to be promising for prevention of acute alcohol intoxication.