RU2169568C2 - Agent for correction of energy metabolism - Google Patents

Abstract

FIELD: biochemistry, pharmacology. SUBSTANCE: invention proposes an agent that is a solution for parenteral administration in doses 50-200 mg/kg of body mass. Invention provides effect that is similar with that of purine mononucleotides and nucleosides. EFFECT: valuable medicinal effect, decreased adverse effect.

Description

 The invention relates to pharmacology and can be used in experimental and practical medicine and veterinary medicine.

 Currently, ribosylated derivatives of purines are used as means for correcting energy metabolism: adenosine triphosphate, which is a part of such preparations as Adenosine Triphosphoric Acid Disodium Salt (Russia), Vita-iodurool (France), Adenosine Monophosphate (domestic preparations: Phosphaden) , "Adenylic acid"), inosine - preparations "Riboxin" (Russia), "Inosie F" (Japan). Domestic muscle preparations have also been proposed: “Muscle adenyl preparation” (“MAP”) containing adenosine monophosphate, and “Miol” containing adenosine triphosphate, adenosine diphosphate and adenosine monophosphate. The mechanism of action of all these drugs includes both regulatory effects that are realized through purinergic receptors and a direct effect on the metabolism, carried out not so much by the drugs themselves, but by their breakdown products.

 The disadvantages of preparations of purine mononucleides and nucleosides include the fact that their production technology is very complex and expensive. In addition, it is known that for phosphorylated metabolites, in particular adenine mononucleotides, the cell membrane is impermeable [Buhl M.R. // Dan. Med. Bull.-1982. - V.29, N 1, - P.1-26]. Under the action of ectoenzymes located on the outer side of the plasma membranes (ecto-ATPase, ecto-ADPase, ecto-5'-nucleotidase, adenylate cyclase, ectonucleotide transferase, pyrophosphohydrolase, non-specific ectophosphatases, ectoprotein kinases) and B. blood enzymes [deformative B. Oleinikov O.D., Ryabova V.V. // Bull. experimental biology and medicine. - 1990. - T.109, N 5, - С.436-438; Dmitrienko N.P. Purine metabolism and its regulation in lymphocytes. - Kiev: Naukova Dumka, 1991.200 p .; Berne R. M., Rubio R. // Adv. Cardiol.-1974.- V.12. - P.303-317; Cooper D.R., Lewis G.P., Lieberman G.E. et. al.// Thrombosis Research. - 1979. - V.14. - P.901-914 .; Ronca-Testoni S., Borghini F. // J. Mol. Cell. Cardiol. -1982. -V. 14, N.3 - P.177-180], the end product of which is adenosine. Inorganic phosphate is also formed, which is often undesirable, since for many diseases that are indications for the use of purine mononucleotide preparations, endogenous hyperphosphatemia occurs due to the catabolism of its own tissue nucleotides.

 Adenosine (like other nucleosides) is able to penetrate the cell, however, a significant part of it is catabolized extracellularly by the action of adenosine deaminase located on the outer surface of the cytolemma [Dmitrienko N.P. // Ukr. biochem. journal - 1981.-T.53, N 1. - S.114-123 .; Dmitrienko N.P. Purine metabolism and its regulation in lymphocytes. - Kiev: Naukova Dumka, 1991, 200 p.], In the endothelium of blood vessels and blood cells [Trams E.G. , Lauter O.J. // Biochim. Biophys.Acta.- 1974.-V.346, N.2 - P. 180-197; Berne RM, Rubio R. // Adv. Cardiol.-1974.-V.12.-P.303-317], and purinucleoside phosphorylase localized in endothelial cells, vascular smooth muscle, and pericytes [Berne RM, Rubio R. // Adv. Cardiol.-1974.-V.12.-P.303-317; Rubio R., Berne R.M., Winn H.R.// Cerebral vascularsmooth muscle and its control / Ed. by M. J. Perves. - Amsterdam: Elsevier-North Holland, 1978.-P.355-372]. Adenosine deaminase converts adenosine into inosine, purine nucleoside phosphorylase breaks down inosine to form hypoxanthine and ribose-1-phosphate. Further, under the action of phosphoribomutase, ribose-1-phosphate can be converted into ribose-5-phosphate, which serves as the initial substrate for a number of biochemical processes [Shultsev G.P., Panchenko V.M., Antonidze A.V. // Nov. medicines. drugs. Express inform. VNIIMI, 1979.-N 7.-C.8-14]. In particular, under the action of phosphoribosyl disphosphate synthetase, phosphoribosyl diphosphate is formed from ribose-5-phosphate, a key substrate for both de novo purine synthesis and nitrogen base reutilization [Buhl MR // Dan.Med.Bull.- 1982.- V.29, No. 1. - P.1-26].

 Ribose-1-phosphate is also formed during the catabolism of endogenous nucleosides that turn into the corresponding nitrogen base: adenosine into adenine, guanosine into guanine, inosine into hypoxanthine [Nucleic acids / Under. ed. E. Charguff and J. Davidson. - M., 1957 .: Buhl M.R.// Dan.Med.Bull.-1982.-V. 29, N.1-P.1-26]. Under pathological conditions, the rate of decay of purine nucleotides exceeds the rate of their formation from decay products - ribose-1-phosphate and nitrogenous bases. Recycling of nitrogenous bases depends on the availability of phosphoribosyl diphosphate, the synthesis of which is limited in vivo by the availability of ribose-5-phosphate [Dmitrienko N.P. Purine metabolism and its regulation in lymphocytes. -Kiev. : Naukova Dumka, 1991.200 p.: Hershko A., Mager J. // Israel. J. Chem. - 1968. -V.6 -P.100-109; Pilz R. B., Willis R. C., Boss G. R. // J. Biol. Chem. -1984. -V. 259. N 5.-P.2927-2935], but not the activity of phosphoribosyl diphosphate synthetase [Hershko A., Mager J. // Israel. J. Chem. -1968.-V.6.-P.100-109]. The latter under conditions of a decrease in the level of ADP, its inhibitor [Buhl M.R. //Dan.Med.Bull.-1982.-V.29. N1.-P.1-26] and increasing the content of inorganic phosphate activator of this enzyme [Hershko A., Mager J. // Israel. J. Chem. - 1968. -V.6.-P.100-109], can even increase. Thus, the excess of ribose-1-phosphate to phosphoribosyl diphosphate is most often inhibited at the stage of the phosphoribomutase reaction. This may be due to the inhibition of phosphoribomutase inorganic phosphate [Golovatsky I.D., Tsegelsky A.A.//Ukr. biochem. -1987.-T. 59, N 5, C. 45-49], which naturally accumulate under conditions of enhanced nucleotide decay. The use of ribose-1-phosphate for direct resynthesis of purine nucleosides is not possible, since the purinucleoside phosphorylase reaction in vivo is practically irreversible [Buhl M. R. // Dan.Med.Bull. - 1982. - V.29, N1. - P.1-26].

 Hypoxanthine, resulting from the catabolism of exogenous purine mononucleotides and nucleosides, can be involved in a xanthine oxidase reaction and turn into uric acid, giving exacerbations of gout and a number of other diseases associated with impaired purine metabolism. In addition, the activation of xanthine oxidase reaction is associated with overproduction of reactive oxygen species, increased lipid peroxidation and damage to biomembranes [Kireyev M.M., Conway V.D. // Pathogenesis and experimental therapy of terminal conditions. - Omsk, 1976. -P.44-45; Conway V.D.// Pathologic Physiology and Experimental Therapy. - 1982. N 6.-C.78-81; Zolin P.P., Conway V.D. // Bull. an expert. biol. -1997. -T.124, N 12.-S.629-631]. This is all the more dangerous because many diseases in which purine nucleotide and nucleoside preparations are used are accompanied by the accumulation of endogenous hypoxanthine and other purine bases resulting from nucleotide catabolism.

 The purpose of the present invention is to expand the range of tools for the correction of energy metabolism by offering a drug having a pharmacotherapeutic effect similar to the effects of purine mononucleotides and nucleosides that are now used to correct energy metabolism, but with fewer side effects.

 The problem is solved in that it is proposed to use D-ribose as a means for correcting energy metabolism.

 Ribose is currently used as an additive to culture media for certain microorganisms, as well as a reaction for chemical, biochemical and physiological studies [Big Medical Encyclopedia. - M .: Soviet Encyclopedia, 1984. - T. 22. - S.279-280; Brief chemical encyclopedia. - M., 1965. - T.4. - S.673; Kochetkov N.K. et al. Chemistry of carbohydrates. -M., 1957; Nucleic Acids / Ed. E. Chargaffa and J. Davidson.-M., 1957; Methods in carbohydrate chemistry / Ed.by R. L. Whistler and M. L. Wolform. - New York, London, 1962; Michelson A.M. The chemistry of nucleosides and nucleotides. - London, New York, 1963].

 We found in a series of experiments that ribose, like existing pharmaceuticals of purine mononucleotides and nucleosides, replenishes the pool of purine mononucleotides, which is reduced due to their catabolism. Moreover, ribose has a beneficial effect on the integral indicators of the body: the survival rate of resuscitated animals, the restoration of their neurological status and an indicator of general condition.

 Experiments to study disorders of energy metabolism and the influence of ribose on them were carried out in 1989-97. at the Central Research Laboratory of the Omsk State Medical Academy. In total, we used 302 outbred white male rats kept under normal vivarium conditions and fed standard laboratory food [Zapadnyuk I.P., Zapadnyuk V.I., Zakharia EA, Zapadnyuk B.V. Laboratory animals. Breeding, maintenance, use in the experiment. - Kiev: Vishcha school, 1983.-383 p.]. To reproduce the state of impaired energy metabolism, we chose a model of mechanical asphyxia according to [Shim N.V. / Pathogenesis and experimental therapy of terminal conditions. - Omsk, 1979. - P.57-62], technically not complicated and not associated with the use of any pharmacological agents, with the exception of ether for anesthesia. From our previous work it was known that with clinical death and postresuscitation disease caused by a 6.5-minute mechanical asphyxia, the catabolism of purine mononucleotides increases sharply, nucleosides and nitrogen bases, including hypoxanthine, xanthine and urate, accumulate, which is accompanied by activation of peroxidation lipids [Conway V.D.// Pathologist. physiology and experiment. therapy. - 1982. - N 6. - C.78-81; Conway V.D. Violation of purine metabolism in the liver in the postresuscitative period and its prevention: Diss. ... Dr. honey. Sciences.-Tomsk, 1988. - 426 l.].

 Rats under light ether anesthesia were fixed in the supine position, intubated with a polyethylene tube with a diameter of 2 mm or 2.5 mm (depending on the weight of the rat) using an illuminating device with a light guide. The tube was fixed to the upper lip with a thread sewn through the lip, and the oral cavity was plugged with a moistened napkin. After the establishment of rhythmic breathing, the tube was closed for 6.5 minutes, after which resuscitation measures were carried out, consisting of direct heart massage and artificial respiration.

Immediately after the restoration of the heartbeat (usually this occurred 4-5 minutes after the start of resuscitation), D - (-) - ribose manufactured by Fluka AG, Buchs SG (Switzerland) or Schuchardt Munchen (Germany) was injected into the femoral vein once at a dose of 50 mg • kg ^-1 body weight, or two doses at a dose of 200 mg • kg ^-1 (the second time, 40 minutes after the start of resuscitation), dissolved in 0.9% NaCl (2.5 ml • kg ^-1 ) . These groups of rats were called Resuscitation + Ribose and Resuscitation + Ribose 200, respectively. The animals of the Resuscitation group immediately after the restoration of the heartbeat were injected with an equivalent volume of 0.9% NaCl instead of ribose. The rats of the Control group did not undergo asphyxiation and were injected with 0.9% NaCl. The animals of the Ribose and Ribose 200 groups did not undergo asphyxiation and were given ribose in doses of 50 and 200 mg • kg ^-1 body weight, respectively. These animals, like the rats of the Control group, were subjected to the same effects (ether anesthesia, fixation, intubation), with the exception of asphyxiation and resuscitation.

In a series of radioisotope studies, together with the ribose solution or saline, the rats immediately after restoration of the heartbeat were injected with ¹⁴ C-hypoxanthine at a dose of 2 MBq • kg ^-1 body weight (for studying the interorgan and interstitial redistribution of hypoxanthine) or 740 kBq • kg ^-1 (for studying hypoxanthine metabolism). Slaughter of animals and tissue sampling for research were performed under ether anesthesia.

 The obtained digital data was subjected to static processing using parametric and non-parametric methods.

It was found that after the pool of purine mononucleotides in the brain, liver and heart of rats was depleted under the influence of 6.5 minutes asphyxiation, intravenous administration of D-ribose at a dose of 50 mg • kg ^-1 led to its completion 30 minutes after the start resuscitation. The favorable effect of ribose, most likely, is realized through its conversion to ribose-5-phosphate under the action of ribokinase, followed by the synthesis of phosphoribosyl disphosphate and enhanced reuse of nitrogenous bases. The following observations indicate this mechanism of ribose action. The incorporation of ¹⁴ C-hypoxanthine into purine liver mononucleotides was statistically significantly decreased 30 min after resuscitation compared to control rats. And in the group of rats, which were immediately injected with ribose, this indicator not only did not decrease, but even was slightly higher than the control. The incorporation of labeled hypoxanthine into purine brain mononucleotides in rats treated with ribose was also higher than the control level. In the heart and blood cells of resuscitated animals receiving ribose, the formation of purine mononucleotides from hypoxanthine was higher than in rats resuscitated without ribose.

By activating the incorporation of hypoxanthine into purine nucleotides, ribose thereby prevented its oxidation by xanthine oxidase to xanthine and uric acid. The content of urate in the blood of rats treated with ribose at a dose of 50 mg • kg ^-1 , 30 minutes after the start of resuscitation was lower than in untreated and control animals. At the same time, xanthine oxidase production of superoxide radicals and hydrogen peroxide and their activation of lipid peroxidation were also susceptible to correction by ribose. The content of lipid peroxidation products - diene conjugates, triene conjugates and lipofuscin-like pigment in the liver of rats resuscitated without treatment was higher than that of the control group, but did not differ from the control in the treatment of ribose. A study of the general biochemical parameters of the liver: its protein content, lipids, cholesterol, triglycerides, the activity of lactate dehydrogenase, alkaline phosphatase, alpha-amylase did not reveal statistically significant differences between the groups.

It is known that the rate of penetration of hypoxanthine into the cell may depend on the intensity of its intracellular metabolism [Vincent M.-F., Van der Berghe G., Hers H.-G.// Biochem.J.-1984.-V.222, N 1.-P.145-155]. The study of interorgan and interstitial redistribution of ¹⁴ C-hypoxanthine revealed additional mechanisms of ribose action. It was found that at the 30th minute of the postresuscitation period in untreated rats, the total inclusion of the label in the cortex of the cerebral hemispheres (especially in the sensorimotor cortex), in the thalamus and other subcortical structures of the brain increased. In the group of rats treated with ribose at a dose of 50 mg • kg ^-1 , the inclusion of hypoxanthine in the brain structures was lower than in the group without treatment. The incorporation of ¹⁴ C-hypoxanthine into the whole brain perchloric acid extract was changed in a similar way. Thus, ribose restricts extracerebral hypoxanthine into the brain and thereby reduces the risk of damage to neurons by reactive oxygen species during oxidation of hypoxanthine by xanthine oxidase, whose activity in brain tissue is very high [Wajner M., Harkness RA // Biochim.Biophys.Acta. -1989.-V.991.- P.79-84]. The administration of a dose of 50 mg • kg ^{-1 to} healthy animals led to a significant decrease in the radioactivity of the whole brain perchloric acid extract and the total radioactivity of the thalamus as compared to the control.

Under conditions of accumulation of hypoxanthine in the body, its increased incorporation into the liver is the main organ of hypoxanthine metabolism [Vincent M.-F., Van der Berghe G., Hers H.-G.//Biochem.J.-1984.-V.222, N. 1 -P. 145-155] would have a compensatory value. However, the incorporation of ¹⁴ C-hypoxanthine into the liver 30 minutes after resuscitation without treatment remains at the control level and is statistically significantly increased only in the group of rats that were injected with ribose. Blood radioactivity increased half an hour after resuscitation, and in the untreated group, due to uniform elements, and in the group with ribose treatment, due to plasma. The inclusion of ¹⁴ C-hypoxanthine in the intestines, heart and adrenal glands of rats of the above groups did not differ significantly from the control, but the inclusion in other organs and tissues (cortical and brain layers of the kidneys, spleen, thymus, lungs, pancreas, etc.) changed in each case is unique, in accordance with the fabric specificity. Administration of a dose of 50 mg • kg ^{-1 to} healthy animals did not lead to a significant change in label inclusion in extracerebral tissues.

 30 minutes after resuscitation in rat groups without treatment and with treatment with ribose, as well as in the group with the introduction of a healthy animal ribose, the plasma content of total protein, urea, creatinine, glucose, cholesterol and triglycerides, as well as total lipids, did not differ significantly from the control. The plasma and erythrocyte content of lipid peroxidation products also did not change significantly.

In a separate series of experiments, we studied the comparative effect of ribose and its closest analogue, inosine, on the indicator of the general condition of resuscitated rats, measured by [Lysenkov SP, Korpachev VG, Tel L.Z. Clinic, pathogenesis and treatment of emergency conditions . - Novosibirsk, 1982. - S. 8-13], the severity of their neurological deficit, measured according to [Hendrickx HHL, Rao GR, Safar P., Gisvold SE // Resuscitation.-1984.-N 12. - P.97- 116], and animal survival in the postresuscitative period. It was found that in the group of rats, which in the resuscitation process immediately after restoration of blood circulation was injected with a dose of 50 mg • kg ^-1 , the severity of neurological deficit was significantly lower than in rats resuscitated without treatment (that is, with saline). In animals treated in the same way with inosine, administered in a dose equimolar to ribose (16 mg • kg ^-1 ), the neurological deficit also decreased, but it was not significant. The effect of a single administration of ribose on the index of the general condition of resuscitated rats was also more pronounced than the effect of inosine, and lasted longer. Moreover, the administration of resuscitated rats with ribose at a dose of 50 mg • kg ^-1 , as well as inositol in an equimolar dosage, did not lead to a statistically significant decrease in postresuscitation mortality.

The administration of 200 mg • kg ^-1 to rats in rats, in addition to the beneficial effect on the index of the general condition of resuscitated rats and restoration of their neurological status, led to a decrease in mortality 3 days after resuscitation from 39% in the untreated group to 10% in the group with the introduction of a ribose solution. The significance of the differences was P = 0.02 according to the non-parametric Fisher criterion, which can be considered statistically significant [Ivanov Yu.I., Pogorelyuk ON. Statistical processing of the results of biomedical research on microcalculators according to the programs. - M .: Medicine, 1990. - 224 p.].

Thus, the administration of ribose to resuscitated rats has a beneficial effect on the integral indicators of the body: animal survival, restoration of their neurological status and general condition indicator. The effect of ribose, administered at a dose of 50 mg • kg ^-1 , was more pronounced than the effect of its closest pharmacopoeial analogue of inosine (inosine pharmaceuticals - Riboxin, Inosin F), administered in an equimolar dosage. Our results of biochemical and radioisotope studies indicate that the pharmacotherapeutic effect of exogenous ribose is associated with its conversion into ribose-5-phosphate, from which phosphoribosyl diphosphate is then synthesized, which is a key substrate for de novo purine synthesis and nitrogen base reutilization. It has been shown that exogenous ribose enhances the reutilization of hypoxanthine in the liver, brain and heart, as a result of which it prevents its involvement in the xanthine oxidase reaction and activation of lipid peroxidation. Due to the increased recycling of nitrogenous bases, as well as, possibly, to other mechanisms, ribose replenishes the pool of purine mononucleotides, which is reduced due to asphyxiation, thereby contributing to the normalization of energy metabolism.

Claims (1)

 Means for the correction of energy metabolism based on D-ribose, characterized in that it is a solution for parenteral administration in doses of 50-200 mg / kg body weight.