WO2006110118A1

WO2006110118A1 - Cytoprotection preparation

Info

Publication number: WO2006110118A1
Application number: PCT/UA2006/000019
Authority: WO
Inventors: Victor Pavlovich Kutnyak; Vadym Alekseyevich Kozlovskiy; Yurii Iosifovich Kudryavets
Original assignee: Victor Pavlovich Kutnyak; Vadym Alekseyevich Kozlovskiy; Yurii Iosifovich Kudryavets
Priority date: 2005-04-15
Filing date: 2006-04-12
Publication date: 2006-10-19

Abstract

The inventive cytoprotection preparation is based on an organic acid and used for preventing and treating a hypoxy of different aetiology. Said preparation contains at least gluconic acid anions, in particular, in the content of a pure gluconic acid or a pure magnesium gluconate. In the preferred embodiment, the preparation is presented in the form of a diGAMK-MG-gluconate compound or a binary salt selected from a group consisting of a magnesium succinate-gluconate, magnesium ascorbate-gluconate, magnesium glycinate-gluconate and the mixtures thereof.

Description

CYTROTECTIVE DRUG

Technical field

The invention relates to the composition of cytoprotective preparations based on r-gluconic acid, and preferably based on its coordination compounds and double salts.

State of the art

It is well known that disturbances in the blood supply to organs of the human body due to thrombosis, embolism, atherosclerosis, angiospasm, increased blood pressure and other causes are accompanied by changes in the metabolic activity of cells. Accordingly, the synthesis of ATP, as the main source of energy, and creatine phosphate, as a transport form of ATP, is inhibited. This weakens the vital activity of cells and can cause various diseases (depending on which organ lacks blood supply).

The most dangerous disorders of the blood supply to the myocardium and brain, which are the most important causes of sudden death (Lutai MI, Dear AL. Sickness and mortality due to spine infection in the Ukrainian blood circulation Il Nova medicine. - 2002, NeZ; in Russian: Lutay MI, Dear A.P. Morbidity and mortality from diseases of the circulatory system in Ukraine // New Medicine. - 2002, NsЗ).

At the biochemical level, a decrease in blood flow (ischemia) is always accompanied by hypoxia and impaired activity of enzyme systems, which ensure the synthesis of the required amount of ATP from nutrients and oxygen coming from the blood.

So, against the background of hypoxia, the concentration of ATP in the cells progressively decreases within 30-60 s (Nakapo A., Cohen M., Dowpeu J. lshemic preconditioning from bacis mechapisms to lypisal arllictiops // Pharmacol. Ther. 2000). Further, almost all biochemical processes are distorted and, due to an increase in the concentration of Na ⁺ and Ca ^{2+ ions} and a decrease in the concentration of K ^ and Mg ^{2+ ions} , the ion balance is disturbed and the electrophysiological parameters of the cells deteriorate.

However, even while maintaining normal blood circulation, cellular hypoxia can occur under the influence of other factors such as mechanical or asthmatic asphyxiation, poisoning (in particular, cyanides or fluorides, which block the enzymes responsible for the absorption of oxygen inside the cell) or the action of ionizing radiation, which inhibits the activity of any enzymes , etc.

Thus, the term “hypoxia” in the broad sense means a violation of the normal utilization of oxygen by the cell at the biochemical level. Therefore, in the treatment of this kind of condition, you can use tools that:

(1) exhibit anti-ischemic (vasodilating) action and thereby improve blood circulation to organs and tissues,

(2) reduce the functional activity of cells and (3) improve oxygen uptake by cells.

Indeed, vasodilator drugs can have an antihypoxic effect (Chekman I.S., Gorchakova H.A., Frantsuzova SB., Mintser V.O. Cardioprotectors: aspects of pharmacodynamics / International Medical Journal. - 2002, Ns 1-2). These drugs have long been nitro compounds (nitroglycerin, isosorbite dinitrate, etc.), papaverine and its synthetic analogue drotaverine, calcium channel blockers and, less commonly, class E prostaglandins (Mashkovsky MD. Medicines. In 2 volumes 14th edition, revised, amended and supplemented. - M .: New Publishing House LLC, 2002). Unfortunately, the classic anti-ischemic drugs based on nitro compounds (Mosbu's, 2003) are quite toxic and effective only for ischemia of blood vessels that feed the myocardium. Indeed, the use of nitro compounds against the background of cerebrovascular accident reduces the rate of blood outflow from the cranial cavity, which can cause swelling, edema and, as a result, compression of the brain with impaired central nervous system functions.

When administered orally, papaverine and drotaverin are too slowly absorbed from the gastrointestinal tract and exhibit a weak vasodilating effect, while their intravenous administration can drastically lower blood pressure (up to collapse) and disrupt the heart rhythm (sometimes even to the point of stopping the heart). Therefore, these drugs are preferably administered intramuscularly. This reduces the duration of the manifestation of the therapeutic effect to 15-20 minutes and virtually eliminates these complications.

However, nitro compounds, papaverine and drotaverin do not exhibit a pronounced cytoprotective effect. Calcium channel blockers (verapamil, diltiazem, nifedipine, cinnarizine, nimodipine, etc.) are less toxic than nitro compounds, are quite easily absorbed, and can exhibit a cytoprotective effect.

However, their use as such is limited due to side effects. So, verapamil and diltiazem often inhibit the work of the heart. Nifedipine has little effect on the myocardium, but often causes hypotension. Cinnarizine has almost no side effects, but its effectiveness in treating brain hypoxia is negligible. Not relatively effective in this quality is the relatively new drug of this class - nimodipine (which, by the way, gives noticeable side effects). Β-adrenoreceptor blockers, hypnotics and tranquilizers can also affect the functional activity of cells (and the corresponding tissues and organs). However, they are “highly specialized)). In particular, β-blockers primarily act on the heart, and tranquilizers and hypnotics on the brain. Therefore, to suppress pathological processes in several vital organs of their have to be used in combination, which significantly increases the risk of side effects.

Further, all β-blockers used in medicine disrupt the central nervous system, causing distraction, depression and weakness, the respiratory apparatus, causing bronchospasm (up to the development of asthma), and the cardiovascular system, inhibiting the cardiac conduction system and myocardial contractility and causing hypotension , bradycardia and Raynaud's syndrome.

Finally, β-blockers are undesirable in pregnancy and diabetes. Side effects of the use of tranquilizers and sleeping pills include dysfunction of the central nervous system (up to coma) and suppression of the functions of the cardiovascular system, which limits their use in clinical practice. Indeed, the cytoprotective effect of these drugs was demonstrated mainly experimentally, and they are rarely used in clinical practice (Polishchuk H., Treshchinsky A. Intensive care for acute ischemic stroke Il Dostor. - 2003, NsЗ, p. 21-23).

Therefore, special attention is paid to drugs that positively affect the biochemical processes in the cells. Long-known antioxidants are among them (Povarova O. V., Kalenikova E. I., Gorodetskaya E. I., Medvedev O.S. Antioxidants as neuroprotectors for ischemic stroke Il Experimental and clinical pharmacology. - 2003, Ns4 , pp. 69-73) and relatively new nootropic drugs.

Nootropics regulate the functioning of the cardiovascular system as a whole (I. Tyurenkov, VN Perfilova. The role of the GABAergic brain system in the regulation of blood circulation / Experimental and Clinical Pharmacology. - 2001, Nab, p. 68-72), and the practically non-toxic reference drug piracetam is effective for hypoxia of the brain and myocardium and is recommended for widespread use (see: 1. оrсit Mashkovsky MD, t. 1, p. 224; 2. Orgogozo JM РіРісетат ип те treеatttef оf асіте strіке Il PHARMACORSUSHIATRU. - 1999, v. 32, Srl 1, pp. 25-32).

Unfortunately, piracetam is effective as a cytoprotector only in large doses (6-12 g per dose), which is difficult to achieve even under clinical conditions.

Therefore, the search for more effective, safe and easy-to-use cytoprotective agents with a spectrum of action almost independent of etiological factors has long been begun and continues to continue.

Of these, magnesium compounds are now most used, because Mg ^{2+ ions} participate in many enzymatic reactions and therefore play an important role in the regulation of metabolism, including ATP synthesis. In addition, magnesium ions are able to modulate the activity of receptors and, thereby, regulate cellular activity.

The therapeutic efficacy of magnesium preparations is almost the same in patients with its low and normal concentration in the blood. Their long-term use This improves the condition of patients with coronary heart disease and contributes to the regression of atherosclerosis, and in case of myocardial infarction it reduces the risk of sudden death (Shilov AM, Svyatov I. S, Kravchenko VV et al. Use of magnesium preparations for the prevention of cardiac arrhythmias in patients with acute myocardial infarction Il Russian Journal of Cardiology. - 2002, Ns1, p.16-19). And, finally, Mg ^{2+ ions are} equally useful for regulating coronary and cerebral blood flow and mitigating the effects of ischemia (Chekman, I. S. Biochemical Pharmacodynamics. - Kiev: Zdorovya, 1991).

Unfortunately, magnesium sulfate commonly used in therapy is practically not absorbed from the gastrointestinal tract and has laxative properties. Therefore, it is administered only parenterally. This leads to rapid dissociation of salt, a reduction in the duration of the therapeutic effect and saturation of the body’s liquid media with sulfate anions.

This undesirable effect can be eliminated by the use of an organic salt, namely magnesium orotate (otherwise referred to as magnerot). However, it is also poorly soluble in water-based media and has a laxative effect, and orotate anions with prolonged use of magnerot contribute to fatty liver degeneration, which requires medical correction (Smirnov B.A. Vitamins. M .: Medicine, 1974).

Magnesium asparaginate, usually used in combination with potassium asparaginate in the form of the popular panangun drug (op. Chekman I.S.), has some cytoprotective effect against the background of ischemic and reperfusion injury to the myocardium. It is practically harmless to humans in typical therapeutic doses, because asparaginate anions can be included in metabolic processes and completely utilized in plastic exchanges. This preparation based on organic (aspartic) acid can be considered the closest in technical essence to the drug proposed further.

Unfortunately, a pronounced cytoprotective effect is usually achieved only with a combination of aspartic acid derivatives with other cytoprotective agents. SUMMARY OF THE INVENTION

The basis of the invention is the task of changing the composition to create such a cytoprotective drug based on an organic acid that, when introduced into the human body, would exhibit a complex cytoprotective effect and thereby reduce the risk of complications in the prevention and treatment of hypoxia of various etiologies.

This object is achieved in that the cytoprotective agent based on organic ^'acids according to the invention comprises at least gluconic acid and anions, respectively, in the simplest case represents the net-hand glyukono- acid (otherwise known as 2,3,4,5,6-pentagidpokcikapponovaya or 2,3,4,5,6- pentaoxyhexanoic acid).

As will be shown in the examples below, the use of even pure gluconic acid provides a cytoprotective effect regardless of the causes of hypoxia and the resulting dysfunctions of the cell functions of such important organs as the brain and heart. Gluconic acid is perfectly absorbed by the body, is practically non-toxic, has no side effects and is easily utilized in metabolic processes as a useful anabolite.

An additional difference is that the cytoprotective drug is pure magnesium gluconate, the intake of which is most effective in hypoxic conditions against the background of a deficiency of magnesium cations in the body.

The following additional differences are that the cytoprotective preparation is either a coordination compound duGAMK ~ Mg-gluconam based on magnesium gluconate and γ-aminobutyric acid (hereinafter - GABA), or a double salt selected from the group consisting of succinate gluconate, ascorbate gluconate , magnesium glycinate gluconate and combinations thereof. These drugs are most effective in severe hypoxic conditions of the body as a whole and are easily utilized in metabolic processes.

BEST MODES FOR CARRYING OUT THE INVENTION The essence of the invention is explained below: a description of the methods for producing cytoprotective drugs according to the invention and experimental dosage forms based on them, a description of experiments to test the effectiveness of cytoprotective drugs in laboratory models of cell damage, and guidelines for the use of cytoprotective drugs. 1) Methods for producing cytoprotective drugs

Gluconic acid (empirical formula C ₆ H ₁₂ O ₇ ) as such and magnesium gluconate (empirical formula dihydrate Ci ₂ H ₂₂ MgOi ₄ * 2H ₂ O) are available on the market as chemical reagents that must have a quality of at least “xh "(Chemically pure). If desired, magnesium gluconate can be obtained by neutralizing gluconic acid (usually taken as an aqueous solution of easily hydrolyzable d-gluconolactone) with an almost equimolar amount of magnesium oxide or hydroxide.

DiGAMK-Mg-gluconate (empirical dihydrate formula

C ₂ oH ₄ oMg N ₂ Oi _{8 "} 2H ₂ O) was prepared from magnesium gluconate and γ-aminobutyric acid (empirical formula C ₄ H ₉ NO ₂ ) as follows. First, slowly dissolved in distilled water with stirring and heating in a water bath a certain amount (for example, 0.1 mole) of magnesium gluconate The solution was cooled to a temperature of 35-4O ⁰ C and gradually the calculated amount of GABA was introduced with stirring in a molar ratio of magnesium gluconate of 2: 1. b the compound was isolated from the solution by spray drying.

Magnesium succinate gluconate (empirical formula Ci ₀ Hi ₆ MgOi i) and magnesium ascorbate-gluconate (empirical formula C ₁₂ H _2b MgO ₁₃ -2H ₂ O) were obtained by dissolving in a 50% aqueous gluconic acid solution of equimolar amounts of succinic or ascorbic acid gradual introduction into the resulting solution with vigorous stirring of an equimolar (calculated as a cation) amount of magnesium oxide (or hydroxide). After complete assimilation of the magnesium source, the solution was cooled in an ice bath, the remaining water was separated on a Shot filter, and the precipitate was dried to constant weight. Magnesium glycinate gluconate (empirical formula C ₈ H ₁₅ Mg NO ₉ ) was prepared by dissolving equimolar amounts of magnesium glycinate and magnesium gluconate, which are available on the pharmaceutical market in water heated to 40-50 ^° C with constant stirring. Then the water was evaporated, and the residue was dried to constant weight. Experimental dosage forms of these compounds were prepared by dissolving them in an isotonic aqueous solution of sodium chloride or glucose.

2) Experiments in laboratory models of hypoxic cell damage

To assess the cytoprotective activity and action characteristics of the above drugs, we used the following experimental models of hypoxic cell damage: brain hypoxia due to occlusion of both carotid arteries in rats (Spasov AA., Kosolapoe BA, Ostrovsky O.V. et al. (Anti-ischemic properties of the new antioxidant Enoxifol drugs Il Experimental and clinical pharmacology. - 2003, N ° 4, p.17); heart hypoxia due to perfusion of isolated rat hearts according to Lanrendorf; general hypoxia of the body due to hypercap ii) general hypoxia due to ionizing radiation of the whole organism. Manipulations (except ionizing radiation) were performed under sodium sodium anesthesia at a dose of 40 mg / kg. Cytoprotective activity was evaluated by the number of surviving animals, biochemical indicators of the activity of tissue respiration enzymes and the amount of ATP, as the main energy substance, the preparations were administered in isotoxic doses (5% of LD ₅₀ ), and piracetam was used as the reference drug. Numerical data were processed statistically. Table 1

RAT SURVIVAL WITH BRAIN HYPOXIA on the background of preliminary administration of cytoprotective drugs

* p <0.05 compared with the control.

As can be seen from table 1, drugs based on gluconic acid contribute to the survival of animals with hypoxia of the brain. Biochemical analyzes (see tab. 2) confirm this conclusion.

table 2

The activity of redox enzymes in brain hypoxia

* p <0.05 compared with control

From table 2 it is seen that gluconic acid-based cytoprotectors (especially diHAMK-Mg-gluconate) practically normalize tissue respiration in the brain compared to the control, whereas piracetam has almost no effect on the activity of tissue enzymes. Similar data (see tab. 3) were obtained in the study of experimental myocardial hypoxia in rats (7 animals in each group).

Table 3, Part 1

The activity of redox enzymes in myocardial hypoxia

* p <0.05 compared with control

Table 3, part 2

The activity of redox enzymes in myocardial hypoxia

* p <0.05 compared with control

As can be seen from the tab. 3, derivatives of gluconic acid exhibit pronounced cytoprotective activity during myocardial hypoxia, significantly exceeding piracetam. However, most metabolic indicators are not different from the control.

To ensure hypercapnic hypoxia, the rats were placed in an airtight chamber, in the volume of which the oxygen concentration progressively decreased as it breathed and the concentration of carbon dioxide increased. The criterion of cytoprotective activity was the average life span of rats under hypercapnia. The results are given in table 4.

Table 4

The activity of drugs with hypercapnic hypoxia (chamber volume 600 cm ³ )

* <0.05 compared with control

As follows from Table 4, all the studied compounds exhibit a cytoprotective effect, increasing the life span of animals in hypercapnia by 14–40%. In this case, the maximum efficiency was observed in diGAMA-Mg-gluconate.

The ionizing radiation of the rat organism was generally provided using an x-ray apparatus with an operating voltage of 190 kV, an operating current of 10 mA, and a field area of 40x40 cm ² . Rats weighing 150-220 g were kept in the indicated field for a time sufficient to obtain an absorbed dose of 6 Gy.

The studied drugs were administered within 1 hour after irradiation. The conclusion about the radioprotective effect was made on the basis of analysis of bone marrow punctate on the 9th day after irradiation. The data obtained are shown in table 5. Table 5

KEY INDICATORS OF MYELOGRAMS FOR THE 9TH DAY AFTER IRRADIATION

* <0.05 compared with control (radiation)

Table 5 shows that, in contrast to piracetam, the preparations according to the invention showed a cytoprotective (in particular, radioprotective) effect on the bone marrow.

Procedures for working with laboratory animals are illustrated by examples. Example 1. In a rat weighing 150 g under ethanol-sodium anesthesia, bilateral occlusion of the carotid arteries was performed. After 8.2 hours, the animal died. Example 2. A rat weighing 160 g was injected intraperitoneally with magnesium gluconate in a dose

250 mg / kg. After 30 minutes, bilateral occlusion of the carotid arteries was performed under ethinal sodium anesthesia. The rat remained alive for 48 hours, after which it was euthanized.

Example 3. A rat weighing 160 g was injected intraperitoneally with diHAMK-Mg-muconate at a dose of 250 mg / kg. After 30 minutes, bilateral occlusion of the carotid arteries was performed under ethinal sodium anesthesia. The rat remained alive for 48 hours, after which it was euthanized.

Example 4. A rat weighing 135 g was placed in a sealed chamber with a volume of 600 cm ³ . After 12 minutes, the animal died from asphyxiation. Example 5. A rat weighing 130 g was injected intraperitoneally with diHAMK-Mg-gluconate at a dose of 250 mg / kg. After 30 minutes, it was placed in a sealed chamber of 600 cm ³ . After 17 minutes, the rat developed cramps and she died.

These examples confirm the cytoprotective efficacy of the preparations according to the invention for arbitrary hypoxia. 3) Guidelines for the use of cytoprotective drugs

The cytoprotective preparations according to the invention can be used as therapeutic and prophylactic in hypoxic conditions of arbitrary etiology. In particular, intravenous administration is indicated for the treatment of tissue hypoxia in acute cerebrovascular accident, traumatic brain injuries, carbon monoxide poisoning or other tissue poisons, and for violations of pulmonary circulation.

Parenteral administration can be used to prevent hypoxia in firefighters, mountain rescuers and climbers.

In addition, the manniferous preparations according to the invention are expediently used in the complex treatment of electrolyte metabolic disturbances as a means of correcting the concentration of magnesium in body fluids.

Industrial applicability

The cytoprotective preparations according to the invention can be easily manufactured industrially and used for the prevention and outpatient or clinical treatment of hypoxic conditions of arbitrary etiology. They have a wide spectrum of cytoprotective activity, are easily absorbed and in therapeutic doses are practically harmless to warm-blooded organisms.

Claims

CLAIM

1. A cytoprotective preparation based on an organic acid, characterized in that it contains at least anions of gluconic acid.

2. The cytoprotective preparation according to claim 1, characterized in that it is pure gluconic acid.

3. The cytoprotective drug according to n. 1, characterized in that it is a pure magnesium rkzhonat.

4. The cytoprotective drug according to claim 1, characterized in that it is a coordination compound of diHAMK-Mg-gluconate based on γ-aminobutyric acid and magnesium gluconate.

5. The cytoprotective preparation according to claim 1, characterized in that it is a double salt selected from the group consisting of succinate gluconate, ascorbate gluconate, magnesium glycinate gluconate, and combinations thereof.