Composition and method for treating patients for mitochondrial diseases
SUBSTANCE: method involves administering pyrimidine nucleotide precursors at a daily dose of 0.05-0.3 g/kg of body weight. The method is applicable under condition that cytotoxic chemotherapy agent does not belong to pyrimidine nucleoside analogs.
EFFECT: enhanced effectiveness in eliminating mitochondrial respiration chain dysfunction.
The scope of the invention
This invention relates in General to compounds and methods for the treatment and prevention of disease, disability and symptoms associated with mitochondrial dysfunction. Predecessors of pyrimidinediamine administered to a mammal, including man, to compensate for mitochondrial dysfunction and improve mitochondrial function.
Background of the invention
Mitochondria are cellular organelles found in most eukaryotic cells. One of their primary functions is oxidative phosphorylation, the process by which the energy generated by metabolism of fuel, such as glucose or fatty acids, is converted into ATP, which is then used to run various challenging biosynthetic reactions and other metabolic processes. Mitochondria have their own genome, separate from the nuclear DNA, containing the rings of DNA with approximately 16,000 BP in human cells. Each mitochondrion may have multiple copies of its genome, and individual cells can have hundreds of mitochondria.
Mitochondrial dysfunction contributes to many pathological conditions. Some mitochondrial diseases caused by mutations or deletions in mitochondria the genome. Mitochondria divide and proliferate with a higher rate of turnover than their cell-owners, and their replication is under the control of the nuclear genome. If the threshold percentage of mitochondria in a cell is defective, and if the threshold the proportion of such cells in tissue is defective mitochondria may experience the symptoms of dysfunction of a tissue or organ. Almost any fabric can be damaged, and may have a wide variety of symptoms, depending on the extent to which involved various tissues.
The fertilized egg can contain both normal and genetically defective mitochondria. Segregation of defective mitochondria in various tissues during division of this egg is a stochastic process, as the ratio of defective mitochondria to normal mitochondria in a particular tissue or cell (although it may occur either positive or negative selection in relation to defective mitochondrial genomes during the turnover of mitochondria in the cells). Thus, many different pathological phenotypes can arise from the specific point heteroplasmy of a mutation in mitochondrial DNA. On the contrary, the same phenotypes can arise from mutations or deletions affecting different genes in mitochondrial DNA. Clinical symptoms in the case of congenital mitochondrial diseases is any often occur in postmitotic tissues with high energy requirements, such as brain tissue, muscles, optic nerve and cardiac muscle, but also involved other tissues, including the endocrine glands, liver, gastrointestinal tract, kidney and hematopoietic tissue, again depending in part on the segregation of mitochondria during development and the dynamics of mitochondrial turnover in time.
In addition to congenital disorders are hereditary, defective mitochondria acquired mitochondrial dysfunction contributes to diseases, in particular neurodegenerative disorders associated with aging, such as Parkinson's, Alzheimer's, Huntington. The frequency of somatic mutations in mitochondrial DNA increases exponentially with age; reduced activity of the respiratory chain is found everywhere in aging people. Mitochondrial dysfunction is also involved in excitotoxicity neural damage, such as damage associated with epileptic seizures or ischemia.
The treatment of diseases involving mitochondrial dysfunction, still provided for the introduction of vitamins and cofactors used specific elements of the mitochondrial respiratory chain. Coenzyme Q (ubiquinone), nicotinamide, Riboflavin, carnitine, Biotin and lipoic acid used for patients with mitochondrial the major disease sometimes reaching success, in particular, disorders, directly originating from primary failure of one of these factors. However, although these factors were useful in some cases, none of these metabolic cofactors or vitamins has not been shown that they are of General applicability in clinical practice in the treatment of mitochondrial diseases. Similarly, dichloracetic acid (DCU, DCA) was used to treat mitochondrial cytopathy, such as MELAS; DCU inhibits the formation of lactate and mainly applicable in cases of mitochondrial disease, which itself is an excessive accumulation of lactate is one of the symptoms. However, DCU has no effect on the symptoms associated with the mitochondrial insufficiency, and may be toxic for some patients, depending on the underlying disease molecular defects.
Mitochondrial diseases include disorders caused by a huge variety of molecular damage or defects, and the phenotypic manifestation of the disease is further complicated by the stochastic distributions of mitochondria in various tissues.
In patent United States 5583117 described acylated derivative cytidine and uridine. In the application PCT/US 96/10067 described the use of acylated Piri is dinucleoside to reduce the toxicity of chemotherapy and antiviral analogues pyrimidinylidene.
The purpose of the invention
The purpose of this invention is the provision of compositions and methods of treating disorders or pathophysiological consequences associated with mitochondrial dysfunction or dysfunction of the mitochondrial respiratory chain in a mammal, including humans.
The purpose of this invention is the provision of compounds and compositions that improve tissue resistance to mitochondrial dysfunction in vivo.
The purpose of this invention is the provision of compositions and methods of treatment of mitochondrial diseases.
The purpose of this invention is the provision of agents, which are widely compensate for mitochondrial disorders, which includes a large variety of molecular pathology, because, in many cases, accurate diagnosis of molecular damage in mitochondrial disorders is difficult.
The purpose of this invention is the provision of a practical method for the treatment of mitochondrial diseases, which is advantageous in the case of violations of the mitochondrial chain electron transfer regardless of the specific molecular defects.
The purpose of this invention is the provision of treatment is not only relatively rare congenital diseases associated with defects in mitochondrial DNA, but also somethinghere-muscular disorders and disorders of the Central nervous system, associated with the development that appear in childhood, and normal age-related degenerative diseases such as Alzheimer's or Parkinson's disease.
The purpose of this invention is the provision of compositions and methods of treatment and prevention of neurodegenerative and neuromuscular disorders.
The purpose of this invention is the provision of compositions and methods of treatment and prevention excitotoxicity damage to the nervous tissue.
The purpose of this invention is the provision of compositions and methods of treatment and prevention of epilepsy.
The purpose of this invention is the provision of compositions and methods of treatment and prevention of migraine.
The purpose of this invention is to provide compositions and methods for prevention of necrosis or dysfunction postmitotic cells in a mammal, including humans.
The purpose of this invention is the provision of compositions and methods of treating disorders associated with delayed development of the nervous system.
The next objective of this invention is the provision of a composition for the treatment or prevention of tissue damage caused by hypoxia or ischemia.
The next objective of this invention is the provision of compositions and methods of treating or preventing ovarian dysfunction, menop the bonds or secondary effects of menopause.
The next objective of this invention is the provision of compositions and methods to reduce the side effects of cancer chemotherapy, due to induced by chemotherapy damage to the mitochondria.
The next objective of this invention is the provision of a method for the diagnosis of mitochondrial disease and mitochondrial dysfunction.
This invention provides a method of treating pathophysiological consequences of the defect of mitochondrial respiratory chain in a mammal, providing for the introduction to such mammal in need of such treatment, the number of precursor pyrimidinediamine, effective in reducing these pathophysiological consequences. In addition, this invention provides a method for prevention of pathophysiological consequences of defects of the mitochondrial respiratory chain, involving the administration to a mammal of a number of precursor pyrimidinediamine, effective in preventing these pathophysiological consequences.
When mitochondrial disease compounds and compositions of this invention are useful for attenuative clinical complications occurring from defects in the respiratory chain. The defects of the respiratory chain underlying mitochondrial disease, izymautsia various factors, including congenital or hereditary mutations and deletions in mitochondrial DNA defects in nuclear-encoded proteins acting on the activity of the respiratory chain, as well as somatic mutations, a high content of intracellular calcium, excitotoxicity, nitric oxide, hypoxia and defects aksonova transfer.
This invention relates to the treatment of disorders and conditions, which are described here as States, which contribute to mitochondrial defects and are therefore targets for treatment with the compounds and compositions of the present invention. They include the side effects of cancer chemotherapy, such as peripheral neuropathy, nephropathy, fatigue, and early menopause, as well as violations of ovulation and the normal menopause.
The invention and its other aims, characteristics and advantages will be better understood and fully from the following detailed description when read with reference to the accompanying experimental results discussed in the examples below.
Detailed description of the invention
This invention relates to compounds, compositions and methods of treating or preventing a variety of clinical disorders that are secondary to mitochondrial dysfunction, in particular, d is ficiton in the activity of the components of the mitochondrial respiratory chain. Such abnormalities include congenital mitochondrial cytopathy, delays in the development of the nervous system, age-related neurodegenerative diseases, as well as specific diseases affecting the heart, peripheral and related to the autonomic system, nerves, skeletal muscle, pancreas and other tissues and organs.
The term "mitochondrial disorder" refers to disorders in which deficits in activity of the mitochondrial respiratory chain contribute to the pathophysiology of such disorders in a mammal. This category includes (1) congenital genetic deficits activity of one or several components of the mitochondrial respiratory chain; 2) acquired deficits activity of one or several components of the mitochondrial respiratory chain, and these deficits are caused, inter alia, (a) oxidative damage during aging; (b) a high content of intracellular calcium; and C) exposure of infected cells by nitric oxide; (d) hypoxia or ischemia; (e) associates with microtubules deficits aksonova transfer of mitochondria or f) expression of mitochondrial uncoupling proteins.
Mitochondrial respiratory chain (also known as the chain of electron transfer) contains 5 major complexes:
It is the complex I NADH:originaldatabase
Complex II succinate:originaldatabase
Complex III original:cytochrome C reductase
Complex IV cytochrome C oxidase
Complex V ATP synthase
Complexes I and II perform the transfer of electrons from metabolic molecules of fuel, such as products of glycolysis and fatty acids, ubiquinone (Coenzyme Q), turning it into original. Original turns back to ubiquinone by electron transfer to cytochrome-C in Complex III. The cytochrome C re-oxidized in the Complex IV of the electron transfer to molecular oxygen to form water. The complex uses the potential energy of the proton gradient produced across the mitochondrial membrane these transfers electrons, converting ADP into ATP, which then provides the energy for metabolic reactions in the cell.
Dihydroorotatdehydrogenase (DHODH, DHODH) is an enzyme involved in de novo synthesis of originalaction. DHODH activity associated with the respiratory chain through the transfer of electrons from dihydroorotate to ubiquinone; these electrons are then transferred to cytochrome C and oxygen through Complexes III and IV, respectively. Only Complexes III and IV are directly involved in the biosynthesis of pyrimidines. The orotate produced by the action DHODH into originalfotot phosphoriboisomerase and decarboxylation.
"The pre is shestvennik of pyrimidinediamine" in the context of this invention are intermediates (intermediates) on the way, or de novo, or "utilizandolo" synthesis of pyridinylamino, which either come in the synthesis of pyrimidines distal relative to DHODH (for example, orotate), or do not require DHODH activity to result in pyrimidinediamine (for example, citizen, uridine or acylphosphate cytidine or uridine). In the scope of the present invention also includes pyrimidinylpiperazine (e.g., nucleotides, cytidinediphosphocholine, uridinediphosphate); these compounds are decomposed to the level of uridine or cytidine before entering into cells and anabolism. Acylphosphate cytidine and uridine have better oral availability than the original nucleosides or nucleotides. Orotic acid and its esters are transformed into originality and is also applicable to perform the tasks of the present invention.
C. the compounds of this invention
The first sign of this invention is the unexpected discovery that the introduction of the precursors of pyrimidinediamine is effective in treating a wide variety of symptoms and pathological conditions associated with mitochondrial dysfunction.
Tissue levels of pyrimidinediamine increase the introduction of any of several predecessors. The uridine and citizen included in the cellular nucleotide pools by phosphorylation at position 5'; citizen and originality I have are vzaimoprevrascheny through enzymatic reaction, amination and diaminononane. Orotic acid is a key intermediate in the de novo biosynthesis of pyrimidinediamine. The inclusion orotovoy acid in a pool of nucleotides requires cell phosphoribosylpyrophosphate (FRPF, PRPP). An alternative (or in addition to providing exogenous precursors of nucleotides), the availability of uridine to the tissues is increased by the introduction of compounds that inhibit urediniospores, the first enzyme in the pathway of degradation of uridine. The compounds of this invention are applicable in the treatment of mitochondrial diseases and related disorders include, uridine, citizen, orotate, orally bioavailable acylphosphate or esters of data precursors pyrimidinediamine and inhibitors of the enzyme urediniospores.
The following definitions apply to allpresan cytidine and uridine.
The term "acylphosphate" as applied here refers to the derived pyrimidinylidene, which is essentially non-toxic organic acyl sub-Deputy derived from carboxylic acids attached to one or more free hydroxyl groups ribose part of oxidoreductase hard-essential communications, and/or in which such Deputy is attached to the amine substituents on the purine ring cytidine amide bond. Such acyl substituents derived from CT is nowych acids, which include, but are not limited to, compounds selected from the group consisting of fatty acids, amino acids, nicotinic acid, dicarboxylic acids, lactic acid, p-aminobenzoic acid and orotovoy acid. Preferred acyl substituents are compounds that are normally present in the body, either as a food component or as an intermediate metabolites.
The term "pharmaceutically acceptable salt" as applied here refers to the additive salt data derivatives and pharmaceutically acceptable acids or bases, which include, but are not limited to, salts of sulfuric, hydrochloric or phosphoric acids, or, in the case orotata, salt with sodium hydroxide or calcium, and salt with a cationic amino acids, particularly lysine.
The term "amino acid" as applied here includes, but is not limited to, glycine, L-forms of alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, Proline, hydroxyproline, serine, threonine, cysteine, cystine, methionine, tryptophan, aspartic acid, arginine, lysine, histidine, ornithine, hydroxylysine, carnitine and other naturally occurring amino acids.
The term "fatty acid" as applied here refers to aliphatic carboxylic acids having 2-22 carbon atoms. Such fatty acids which may be saturated, partially saturated or polyunsaturated.
The term "dicarboxylic acids" as applied here refers to fatty acids with a second by Deputy carboxylic acid.
The compounds of this invention have the following structure:
In all cases, except for these letters, and letters with subscript characters, representing different substituents in the chemical structures of the compounds of this invention are applicable only to the structure immediately prior to the description of this character.
(1) Acylphosphate uridine having the formula:
where R1, R2, R3and R4are the same or different and each denotes hydrogen or the acyl radical of a metabolite, provided that at least one of the substituents R is not hydrogen, or
its pharmaceutically acceptable salt.
(2) Acylphosphate cytidine having the formula:
where R1, R2, R3and R4are the same or different and each denotes hydrogen or the acyl radical of a metabolite, provided that at least one of the substituents R is not hydrogen, or
its pharmaceutically acceptable salt.
The compounds of this invention are applicable in the treatment of mitochondrial diseases is s, include:
(3) Acylphosphate uridine having the formula:
where R1, R2and R3are the same or different and each denotes hydrogen or acyl radical
A. unbranched fatty acid with 2-22 carbon atoms,
b. amino acids selected from the group consisting of glycine, the L forms of alanine, valine, leucine, isoleucine, tyrosine, Proline, hydroxyproline, serine, threonine, cystine, cysteine, aspartic acid, glutamic acid, arginine, lysine, histidine, carnitine, and ornithine,
C. dicarboxylic acid having 3-22 carbon atoms,
d. carboxylic acid selected from the group consisting of glycolic acid, pyruvic acid, lactic acid, enolpyruvate acid, lipoic acid, Pantothenic acid, acetoacetic acid, p-aminobenzoic acid, beta-hydroxybutiric acid, orotovoy acid and creatine.
(4) Acylphosphate cytidine having the formula:
where R1, R2, R3and R4are the same or different and each denotes hydrogen or an acyl radical;
A. unbranched fatty acid with 2-22 carbon atoms,
b. amino acids selected from the group consisting of glycine, the L forms of phenylalanine, alanine, valine, leucine, and is alatina, tyrosine, Proline, hydroxyproline, serine, threonine, cystine, cysteine, aspartic acid, glutamic acid, arginine, lysine, histidine, carnitine, and ornithine,
C. dicarboxylic acid having 3-22 carbon atoms,
d. carboxylic acid selected from the group consisting of glycolic acid, pyruvic acid, lactic acid, enolpyruvate acid, lipoic acid, Pantothenic acid, acetoacetic acid, p-aminobenzoic acid, beta-hydroxybutiric acid, orotovoy acid and creatine.
(5) Acylphosphate uridine having the formula:
where at least one of R1, R2or R3means hydrocarbonengineering part containing 2-26 carbon atoms and the remaining substituents R represent independently hydrocarbonengineering or hydrocarbonbearing part or H or phosphate.
(6) Acylphosphate cytidine having the formula:
where at least one of R1, R2, R3or R4means hydrocarbonengineering part containing 2-26 carbon atoms and the remaining substituents R represent independently hydrocarbonengineering or hydrocarbonbearing part or H or phosphate.
(7) orotic acid or a salt thereof:
Pharmaceutically acceptable salts orotovoy acid include salts in which the cation component of the salt is sodium, calcium, essential amino acid such as arginine or lysine, methylglucamine, choline, or any other essentially non-toxic water-soluble cation, with molecular weight less than about 1000 daltons.
8) Sportsarena derivatives orotata:
where R1denotes the radical of an alcohol containing 1-20 carbon atoms, attached to the orotate through hard-essential communication.
The invention also includes pharmaceutically acceptable salts of the above compounds.
The preferred compounds of this invention are esters of short-chain (2-6 carbon atoms) fatty acids and uridine or cytidine. Especially preferred compounds are triacetyluridine or triacetyluridine. Such compounds have better oral bioavailability than the original nucleosides, and quickly dezazetiliruetsa after absorption after oral administration.
Pyruvic acid is applicable to treatment of cells with defective mitochondrial function. Cells with reduced ability mitochondriale oxidative phosphorylation must rely on glycolysis for generowania ATP. Glycolysis reg is niruetsya redox state of cells. Specifically, for the optimal supply of glucose requires NAD+producing NADH in the process. To maximize the energy production from glycolysis NADH must be re-oxidized to NAD+. Exogenous pyruvate may re-oxidize NADH, partly by means of the enzyme of the plasma membrane NADH-oxidase.
Unidentified(2',3',5'-three-O-providedin) provides benefits both pyrimidines and pyruvate, delivering both components in a single chemical molecular particle and avoiding load of sodium, calcium or other cations in the corresponding salts of pyruvic acid.
An alternative or complementary strategy for the treatment of mitochondrial disease is the inhibition of uridine catabolism enzyme inhibitor urediniospores.
Examples of inhibitors urediniospores that are applicable for the treatment of mitochondrial diseases include, but are not limited to, derivatives of 5-benzylbenzoate or 5-benzylidenemalonate, including 5-benzylbromide, 5-benzyloxybenzoate, 5-benzyloxybenzyl-1-[(1-hydroxy-2-ethoxy)methyl]barbiturate, 5-benzyloxybenzyl-1-[(1-hydroxy-2-ethoxy)methyl] barbiturate and 5-methoxybenzenediazonium, 2,2'-anhydrous-5-amiloride, 5-ethyl-2-deoxyuridine and connections and clonidine, in particular, 5-benzylamine connection group ticlopidine, including, but not limited by them, benzylacrylamide, benzyloxybenzaldehyde, aminomethylenemalonate, aminomethyl-benzyloxybenzaldehyde, hydroxyethylmethylcellulose and hydroxyethylmethylcellulose. Cm. also WO 89/09603 and WO 91/16315, incorporated herein by reference.
C. Composition of the present invention
In one embodiment of the present invention, new pharmaceutical compositions contain as the active agent one or more precursors of pyrimidinediamine selected from the group consisting of uridine, cytidine, orotovoy acid or its salts or esters, and allpresan these predecessors of pyrimidinediamine together with a pharmaceutically acceptable carrier.
Composition, depending on the intended use and the route of administration, is prepared in the form of liquids, suspensions, sprays, microcapsules, tablets, capsules, pills, injectable solution or suppository (see the discussion of the composition below).
In another embodiment of the present invention, the composition comprises at least one precursor of pyrimidinediamine and an agent that inhibits the degradation of uridine, such as enzyme inhibitor urediniospores. Examples of inhibitors urediniospores include himself, but not limited to, derivatives of 5-benzylbenzoate or 5-benzylidenemalonate, including 5-benzylbromide, 5-benzyloxybenzoate, 5-benzyloxybenzyl-1-[(1-hydroxy-2-ethoxy)methyl]barbiturate, 5-benzyloxybenzyl-1-[(1-hydroxy-2-ethoxy)methyl]barbiturate and 5-methoxybenzenediazonium, 2,2'-anhydrous-5-amiloride and connections ticlopidine, in particular, 5-benzylamine connection group ticlopidine, including, but not limited by them, benzylacrylamide, benzyloxybenzyl-allowedin, aminomethylenemalonate, aminomethyl-benzyloxybenzaldehyde, hydroxyethylmethylcellulose and hydroxyethylmethylcellulose. In addition, the scope of the present invention includes the use as an inhibitor only urediniospores, without joint introduction predecessor pyrimidinediamine, for the treatment of mitochondrial diseases or pathophysiological conditions associated with dysfunction of the mitochondrial respiratory chain.
The following variants of the invention include the predecessor of pyrimidinediamine combined with one or more other agents with protective or supporting activity against mitochondrial structure and function. Such agents are presented with recommended daily doses for Mitko trialnyh diseases, include, but are not limited to, pyruvate (1-10 g/day), Coenzyme Q (1-4 mg/kg/day), alanine (1-10 g/day), lipoic acid (1-10 mg/kg/day), carnitine (10-100 mg/kg/day), Riboflavin (20-100 mg/day), Biotin (1-10 mg/day), nicotinamide (20-100 mg/day), Niacin (20-100 mg/day), Vitamin C (100-1000 mg/kg / day), Vitamin E (200-400 mg/day) and dichloracetic acid or its salts. In the case of pyruvate, this active agent may be introduced in the form of pyruvic acid, its pharmaceutically acceptable salt or ester of pyruvic acid with an alcohol residue containing 2-10 carbon atoms.
D. Therapeutic use of compounds and compositions of this invention
Diseases associated with dysfunction of the mitochondrial respiratory chain, can be divided into several categories based on the origin of mitochondrial defects.
Congenital mitochondrial diseases are diseases associated with genetic mutations, deletions, or other defects in mitochondrial DNA or in nuclear genes that regulate the integrity of the mitochondrial DNA or in nuclear genes encoding proteins that are critical for the function of the mitochondrial respiratory chain.
Acquired mitochondrial defects primarily involve 1) damage to mitochondrial DNA, due to OK dlitelnyj processes or aging; 2) mitochondrial dysfunction due to excessive accumulation of intracellular and intramitochondrial calcium; 3) inhibition of complexes of the respiratory chain endogenous or exogenous inhibitors of the respiratory chain; 4) acute or chronic oxygen deficiency and 5) impaired nuclear-mitochondrial interactions, for example, violated the Shuttle movement of mitochondria in the long akanah due to defects of microtubules and 6) the expression of mitochondrial uncoupling proteins in response to lipids, oxidative damage or inflammation.
The most fundamental mechanisms involved in acquired mitochondrial defects and the underlying pathogenesis of various forms of dysfunction of organs and tissues include:
The accumulation of calcium: the Main mechanism of cell damage, especially in the excited tissues, includes a excessive penetration of calcium into the cells as a result of seepage through the plasma membrane, or defects in the mechanisms of manipulation of intracellular calcium. Mitochondria are the main sites of sequestration of calcium and preferably they use energy from the respiratory chain to absorb calcium, but not for ATP synthesis, which leads to the increasing failure of the mitochondria, as the absorption of calcium in the mitochondria p is igodit to a reduced ability transduction (transfer) of energy.
Exitotoxicity: Excessive stimulation of neurons excite amino acids is a common mechanism in necrosis, or damage to cells in the Central nervous system. Activation of glutamate receptors, in particular, subtype, called NMDA receptors leads to mitochondrial dysfunction, partly by increasing the level of intracellular calcium during excitotoxicity stimulation. In contrast, deficiency in mitochondrial respiration and oxidative phosphorylation sencibilisiruet cells makes them more sensitive to excitotoxicity incentives, leading to necrosis or damage to the cells during exposure to levels exitotoxicity neurotransmitters or toxins that would be harmless to normal cells.
Exposure by nitric oxide: nitric Oxide (˜1 micromolar) inhibits cytochrome oxidase (Complex IV) and thereby inhibits mitochondrial respiration (Brown GC, Mol. Cell. Biochem. 174:189-192, 1997); in addition, prolonged exposure NO irreversibly reduces the activity of Complex I. Physiological or pathophysiological concentrations of NO inhibit thereby biosynthesis of pyrimidines. Nitric oxide is involved in many neurodegenerative disorders, including inflammatory and autoimmune diseases of the Central nervous system and is involved in copper is the formation of excitotoxicity and post-hypoxic neuronal damage.
Hypoxia: Oxygen is the terminal electron acceptor in the respiratory chain. The lack of oxygen disrupts the activity of the chain transfer of electrons, leading to reduced synthesis of pyrimidines, as well as reduced ATP synthesis through oxidative phosphorylation. Human cells proliferate and remain viable when in fact anaerobic conditions if they are provided by uridine and pyruvate (or similar effective agent for oxidation of NADH to optimize the glycolytic production of ATP).
Nuclear-mitochondrial interactions: Transcription of mitochondrial DNA, encoding components of the respiratory chain, requires nuclear factors. In axona nerve cells mitochondria should move back and forth to the core to maintain the activity of the respiratory chain. If axony transfer violated by hypoxia or drugs, such as Taxol, which affect the stability of microtubules, mitochondria, distant from the nucleus, are loss cytochromoxidase activity.
Mitochondrial uncoupling proteins: Mitochondria are the primary source of free radicals and reactive (active) molecules of oxygen due to spillover of the mitochondrial respiratory chain, in particular, when the defects in one or more components of the respiratory chain which violate the right to transfer electrons from metabolic intermediates to molecular oxygen. To reduce oxidative damage, cells can be compensated by the expression of mitochondrial uncoupling proteins (UCP), some of which were identified. UCP-2 is transcribed in response to oxidative damage, inflammatory cytokines or excessive lipid load, for example, fatty infiltration of the liver and steatohepatitis. UCP reduces spillover reactive oxygen molecules from the mitochondria by unloading the proton gradient across the inner membrane of mitochondria, effectively wasting the energy produced by metabolism, and making the cells sensitive to energy stress, as a tradeoff for reduced oxidative damage.
In the nervous system, in particular, defects in the mitochondrial respiratory chain have two generalized consequences: 1) delayed or aberrant (abnormal) the development of reflex arcs (routes of nervous excitement when implementing a reflex act) in the nervous system and 2) accelerated degeneration of neurons and reflex arcs or in the form of acute symptoms, or over a period of several years, depending on the severity of mitochondrial defects and other influencing factors. A similar pattern of impaired development and accelerated degeneration also apply to non-neural tissue is Yam and systems.
Mitochondrial dysfunction and biosynthesis of pyrimidines
Cells with severely damaged mitochondria (including total deletion of mitochondrial DNA, and the subsequent shut down of the activity of the respiratory chain) can survive in culture while providing them with two agents that compensate for critical mitochondrial functions: uridine and pyruvate. The uridine is required in vitro, as the rate-limiting enzyme for de novo synthesis of originalaction, dihydroorotatdehydrogenase (DHODH), is associated with mitochondrial respiratory chain via ubiquinone as proximal electron acceptor, cytochrome C as an intermediate product and oxygen as a terminal electron acceptor (Loffler et al., Mol. Cell. Biochem. 174:125-129, 1997). DHODH required for the synthesis orotata, which is then phosphoriboisomerase and decarboxylated to form urediniospore (MFIs). All other pyrimidines in the cells are formed from MFIs. Cells from patients with mitochondrial disease caused by defects in mitochondrial DNA, require exogenous uridine for survival outside the environment of the body, and the pyrimidines derived from other cells or food and transported through the bloodstream, are, at first glance, sufficient to maintain their viability Bourgeron, et al., Neuromusc. Disord. 3:605-608, 1993). It is important that deliberate Engibarov is their DHODH drugs such as Brequinar or Leflunomide, leads to limiting the dose of cytotoxic damage to the hematopoietic system and the mucous membrane of the gastrointestinal tract, in contrast to the prevalent involvement in postmitotic tissues such as the nervous system and muscles, in clinical mitochondrial disease.
Pathophysiological consequences of dysfunction of the respiratory chain
Mitochondria are critical for the survival and proper functioning of almost all types of eukaryotic cells. Mitochondria of virtually any cell type can be congenital or acquired defects that affect their function. Thus, clinically significant signs and symptoms of mitochondrial defects that affect the function of the respiratory chain, are heterogeneous and variable depending on the distribution of defective mitochondria among cells and the severity of their defects and physiological needs of the affected cells. Nonproliferating tissues with high energy demands, for example, nervous tissue, skeletal muscle and cardiac muscle, are particularly sensitive to dysfunction of the mitochondrial respiratory chain, but can be affected any system of bodies.
Diseases and symptoms, listed below, include the known pathophysiological consequences dysfunc the AI mitochondrial respiratory chain and, as such, are impaired, in which case the compounds and compositions of this invention have therapeutic applicability.
Symptoms secondary to mitochondrial dysfunction is usually associated with 1) spillovers free radicals from the respiratory chain; 2) defects in the synthesis of ATP, leading to failure of cellular energy, or 3) apoptosis triggered by release of mitochondrial signals, such as cytochrome C, which initiate or mediashout cascades of apoptosis. An unexpected characteristic of this invention is the observation that the precursor pyrimidinediamine of the present invention have therapeutic activity against a wide variety of symptoms in patientos with mitochondrial disease, as shown in the examples. This is an important clarification of existing dogmas in understanding the pathogenesis of diseases involving mitochondrial dysfunction, and understanding of how to treat such disorders.
Treatment of congenital mitochondrial cytopathy
Defects in mitochondrial DNA
A number of clinical syndromes associated with mutations or deletions in mitochondrial DNA. Mitochondrial DNA is inherited through the maternal line, with virtually all of the mitochondria in the body, originating from the mitochondria provide the oocyte. If the oocyte there is a mixture of defective and normal mitochondrial distribution and segregation of mitochondria is a stochastic process. Thus, mitochondrial diseases are often Multisystem disorders, and specific point mutation in mitochondrial DNA, for example, may lead to a different set of signs and symptoms in different patients. On the contrary, mutations in two different genes in mitochondrial DNA can lead to the same sets of symptoms.
However, some common symptom picture appeared in connection with the identified defects in mitochondrial DNA, and they include the classic "mitochondrial disease", some of which are listed directly below. However, an important aspect of this invention is the recognition that the concept of mitochondrial disease and its treatment by the compounds and compositions of the present invention applies to many other pathological conditions, which are also described here.
Some of the classic main phenotypes of mitochondrial diseases associated with mutations or deletions of mitochondrial DNA, include:
MELAS: Mitochondrial Encephalomyopathies the Lactic acid Acidemia and shock attacks
MERRF: Mikl the technical Epilepsy with Ragged Red" (muscle) Fibers
MNGIE: Mitochondrial Neuregelung-intestinal Encephalomyopathy
NARP: Neurogenic muscle weakness, Ataxia and Retinitis pigmentosa
LHON: Hereditary Optic neuropathy Leber's
Syndrome Lee (Leigh) (Subacute Necrotizing Encephalomyopathy)
REO: Progressive Outer Ophthalmoplegia
Syndrome Kearns-Sara (REO, pigmentary retinopathy, ataxia and cardiomyopathy)
Other common symptoms of mitochondrial diseases that may be present alone or in combination with these syndromes include cardiomyopathy, muscle weakness and atrophy, delayed development (including motor (motor), speech, cognitive or Executive function), ataxia, epilepsy, renal tubular acidosis, peripheral neuropathy, optic neuropathy, autonomic neuropathy, neurogenic dysfunction of the digestive tract, sensorineural deafness, neurogenic dysfunction of the bladder, gelatinous cardiomyopathy, migraine, liver failure, lactic acid azidemiu and diabetes.
In addition to gene products and tRNA encoded by mitochondrial DNA, many proteins involved in mitochondrial respiration and oxidative phosphorylation or influencing them, are encoded by nuclear DNA. In fact, approximately 3000 proteins, or 20%,of all proteins encoded by the nuclear genome, physically included in the mitochondria or associated with mitochondria and mitochondrial function or biogenesis, although only about 100 of them are directly involved as structural components of the respiratory chain. Thus, mitochondrial disease involves not only the gene products of mitochondrial DNA, but also nuclear-encoded proteins that affect the function of the respiratory chain and the structure of the mitochondria.
Metabolic stress factors (stress factors), such as infections, may unmask mitochondrial defects, which do not necessarily give the symptoms under normal conditions. Neuromuscular or neurological lesions during infection are the distinguishing feature of mitochondrial disease. On the contrary, dysfunction of the mitochondrial respiratory chain may make cells sensitive to stress factors, which otherwise would have been harmless.
Diagnosis of congenital mitochondrial diseases is questionable due to the heterogeneity of symptoms, even among patients with the same molecular defect. Defects in the function of cells and tissues due to mitochondrial dysfunction can mimic tissue dysfunction caused by problems that do not include directly Meath is hondaline defects. Several clinically applicable and practical schemes for the diagnosis of mitochondrial diseases known in this area; they usually include some basic criteria (for example, the classic clinical phenotypes, such as MELAS, NARP or Leigh Syndrome, extreme (>80%) inhibit the activity of complexes of the respiratory chain in samples of fresh tissue) with a good degree of certainty in establishing the role of dysfunction of the respiratory chain in the pathogenesis of the disease, and a large number of minor criteria (for example, mild biochemical abnormalities characteristic defects of the respiratory chain, the symptoms of mitochondrial diseases without full representation one of the above classical phenotypes), which separately are less convincing than some basic criteria, but which together provide strong evidence of the contribution of the defects of the respiratory chain in the clinical presentation of the patient, as described in Walker et al. (Eur. Neurol., 36:260-7, 1996), incorporated herein by reference.
As shown in the examples, the compounds and compositions of the present invention is applicable for treatment of a wide spectrum of signs and symptoms of mitochondrial diseases with different underlying molecular pathologies. Improvements have given the s and additional patients, include, but are not limited to, reducing the frequency and severity of seizures, migraines and shock attacks, improve the added weight in children with "lack of development", the weakening of renal tubular acidosis with a simultaneous decrease in the need for additional bicarbonate, improved muscle strength, improved language development, improvement of ataxia, decrease the frequency and severity of sinus and ear infections, improving memory and relieving symptoms of autonomic and peripheral neuropathy. Improvements observed in a wide variety of symptoms, which were essentially insensitive to other forms of metabolic support, for example, vitamins and cofactors, which were known that they are necessary for normal mitochondrial function (which is the argument against ascribing these improvements to the placebo effect, as the restoration of symptoms when stopping pyrimidine support)demonstrate basic unexpected understanding of the present invention that the functional or caused by environmental factors, lack of pyrimidines underlies a wide variety of dominant symptoms in patients with mitochondrial diseases and that the filling of the pyrimidines is sufficient to improve or ameliorate a wide variety of symptom the in these patients. Still the symptoms of mitochondrial disease were attributed to lack of ATP molecules active oxygen generated by a defective respiratory chain or cell necrosis triggered mitochondrial components of the cascade of apoptosis. DLT (dose limiting toxicity of inhibitors of de novo synthesis of pyrimidines is usually caused by inhibition of proliferation of rapidly dividing cell types such as stem cells in the bone marrow and intestinal mucosa. Unexpectedly, therapeutic advantages of the compounds and methods of the present invention on patients and experimental animals have been demonstrated in tissues containing non-fissile postmitotic cells, for example, neurons of the Central nervous system and peripheral neurons and skeletal and cardiac muscle.
An important feature of this invention is the unexpected result that the treatment of patients with mitochondrial disease caused by many underlying molecular defects, leads to clinical improvement in a diverse set of symptoms in vivo in patients (examples 1-4). Important and also unexpected is the fact that the clinical benefit was observed even in patients with normal activity of two complexes of the respiratory chain (III and IV), which are directly involved in the transfers of electrons specification is logically required for the biosynthesis of pyrimidines.
In addition, unexpected and important aspect of the present invention is that the optimal effects of the treatment of patients with mitochondrial cytopathies usually require higher doses of the precursor pyrimidinediamine of the present invention than the dose required for adequate treatment of patients with almost complete blocking de novo synthesis of pyrimidines, for example, patients homozygous in respect of aciduria with orotovoy acid in the urine of type I. the Optimal dose of the compounds of the present invention, for example, triacetyluridine (which is effectively absorbed after oral administration)for the treatment of congenital mitochondrial diseases in children are in the range of 1-6 g / m2the surface area of the body (50-300 mg/kg, preferably 100-300 mg/kg), whereas total daily de novo synthesis of pyrimidines is approximately one gram per day in adults (approximately 0.5 g/m2).
Wide applicability of the methods of the present invention is unexpected and separates the compounds and compositions of the invention from other therapies for mitochondrial diseases, which tried to apply, for example, Coenzyme Q, vitamin, carnitine and lipoic acid, which is usually aimed at very specific reactions and cofactors involved in mitochondrial function, and that SL is therefore applicable only in specific cases. However, these metabolic intervention with antioxidants and cofactors complexes of the respiratory chain is compatible with concurrent treatment with the compounds and compositions of the present invention, and they are used to best advantage in combination with the compounds and compositions of this invention.
Treatment of neuromuscular degenerative disorders
The genetic defect that underlies ataxia, Fridrich (FA), the most common hereditary ataxia, was recently identified and named "pradakshina". In FA, after a period of normal development, develop defects in coordination, which progress to paralysis and death, usually between the ages of 30 and 40 years. The most severely affected tissues are the spinal cord, peripheral nerves, heart and pancreas. Patients usually lose a motor (motor) control and invalid chairs and usually suffer from heart failure and diabetes.
The genetic basis of FA includes three nucleotide GAA repeats in intron of the gene encoding frataxin. The presence of these repeats leads to reduced transcription and expression of this gene. Frataxin involved in the regulation of the content of iron in the mitochondria. When reduced compared to the norm maintained and frataxin in mitochondria accumulates excess iron, stimulating oxidative damage and subsequent degeneration and mitochondrial dysfunction.
In the presence of intermediate quantities of GAA repeats in the intron of the gene frataxin, severe clinical phenotype fxii may not develop. However, these three nucleotide elongation intermediate length found in 25-30% of patients with non-insulin-dependent diabetes mellitus, compared with approximately 5% no diabetes population.
Compounds and compositions of the present invention is applicable for the treatment of patients with disorders related to deficiency or defects in frataxin, including ataxia, Fridrich, myocardial dysfunction, diabetes and diabetic complications such as peripheral neuropathy. On the contrary, the diagnostic tests on the estimated deficiency of frataxin, including PCR tests GAA repeats intron applicable to identify patients who will benefit from treatment with the compounds and compositions of this invention.
Muscular dystrophy is called a family of diseases that includes damage to the neuromuscular structure and function, often leading to atrophy of skeletal muscle and myocardial dysfunction. In the case of Duchenne muscular dystrophy, mutations or defects in specific protein, distrofia, are involved in its etiology. The mouse and the activated genes dystrophin find some characteristics of muscular dystrophy and have approximately 50% deficiency in activity of the mitochondrial respiratory chain. Regular end by for neuromuscular degeneration in most cases, is a calcium-mediated disturbance of mitochondrial function. Compounds and compositions of the present invention is applicable to reduce speed reduce muscle functional capacity and to improve muscle functional status in patients with muscular dystrophy.
Multiple sclerosis (MS) is a neuromuscular disease characterized by focal inflammatory and autoimmune degeneration of the cerebral white matter. Periodic exacerbations or attacks significantly correlated with infections of the upper respiratory tract and other infections, both bacterial and viral, indicating that mitochondrial dysfunction plays a role in MS. It is assumed that the inhibition of neural activity of the mitochondrial respiratory chain, caused by nitric oxide (produced by astrocytes and other cells involved in inflammation), is a molecular mechanism contributing MS.
Compounds and compositions of the present invention is applicable for the treatment of patients with multiple sclerosis, both preventive and during episodes of exacerbation.
Treatment of disorders of the neural instability
Treatment of disorders with epilepticus and seizures
Epilepsy often occurs in patients with mitochondrial cytopathies involving a range of severity and frequency of seizures, such as absence seizures, tonic, atonic, myoclonic and continuous epileptic seizures occurring in separate attacks or many times during the day.
In the case of patients with seizures secondary to mitochondrial dysfunction, the compounds and compositions of the present invention is applicable to reduce the frequency and severity of activity of seizures.
The treatment and prevention of migraine
Metabolic studies in patients with recurrent migraine headaches show that defects in mitochondrial activity usually associated with this disorder, manifesting itself in the form of impaired oxidative phosphorylation and excessive production of lactate. Such defects are not necessarily caused by genetic defects in mitochondrial DNA. Migraines are sensitive to nitric oxide, the endogenous inhibitor of the cytochrome C oxidase. In addition, patients with mitochondrial cytopathies, for example, MELAS, often have recurrent migraines.
In the case of patients with recurrent migraines, compounds, compositions and methods of the present invention is applicable to prevention and treatment, in particular, in the case of headaches, resisted the data to compounds of ergot or antagonists of serotonin receptors.
As shown in example 1, the compounds and compositions of the present invention is applicable for the treatment of migraines associated with mitochondrial dysfunction.
Treatment delays development
Delays in neurological or neuropsychological development are frequently found in children with mitochondrial diseases. The development and correction of neural connections requires intensive biosynthetic activity, in particular, participation of the synthesis of the membranes of nerve cells and myelin, both of which require pyrimidinediamine as cofactors. Originality involved in the inactivation and transfer of sugars to glycolipids and glycoproteins. Citizenoriented formed from originaltitel and are crucial for the synthesis of the major phospholipid components of membranes, such as phosphatidylcholine, which receives its Kalinovo part of cytidinediphosphocholine. In the case of mitochondrial dysfunction (caused either by defects in the mitochondrial DNA, or any of the acquired or caused by environmental factors defects, such as excitotoxicity or mediated by nitric oxide and mitochondrial dysfunction, as described above) or other conditions leading to impaired synthesis of pyrimidines, cell proliferation and distribution of axons are violated at key stages in the development of relationships and the reflector is Doug's neurons, leading to delayed or stopped the development of neuropsychological functions, such as speech, motor (motor) function, communication function, Executive function and cognitive skills. For example, in the case of autism, measurements using spectroscopy nuclear magnetic resonance shows that there is a global decrease of the synthesis of membranes and precursors of membranes, detected at low levels depositsfrom and derivatives citizenoriented involved in the synthesis of membranes (Minshew et al., Biological Psychiatry 33:762-773, 1993).
Disorders characterized by developmental delay, include rett syndrome, pervasive delay development PDD (or PDD-NOS: "pervasive developmental delay - not otherwise described, in order to distinguish it from specific sub-categories such as autism)autism, Asperger syndrome and disturbance, manifested in attention deficit/hyperactivity disorder (ADHD), which is recognized as a delay or lag in the development of reflex arcs that underlie Executive functions.
Compounds and compositions of the present invention is applicable for the treatment of patients with developmental delays nervous system, covering motor (motor), language, Executive function and cognitive skills. Methods of treatment of such conditions, for example, ADHD, currently include has the Jn-like stimulants, that reinforce neirotransmissiu in some affected underdeveloped reflex arcs, but such agents, which can improve the control disorders behavioral reactions do not improve cognitive function (the function of cognition), as they are not aimed at the underlying disease defects in the structure and interconnectedness involved in the process of reflex arcs.
Compounds and compositions of the present invention is applicable also in the case of other delays or stops neurological and neuropsychological development in the nervous system and somatic development in non-neural tissues, such as muscle and endocrine glands.
Treatment of neurodegenerative disorders
Two of the most important heavy neurodegenerative diseases associated with aging, Alzheimer's disease (ad) and Parkinson's (BP)have mitochondrial dysfunction in their pathogenesis. In particular, the defects of Complex I often found not only in the neurons of the efferent connection of the black substance with a striped body, which degenerate in Parkinson's disease, but also in peripheral tissues and cells, such as muscle and platelets of patients with Parkinson's disease.
In the case of Alzheimer's disease, the activity of the mitochondrial respiratory chain is often suppressed, in particular, the activity of Complex IV (cytochrome C oxidase). In addition to t the th, mitochondrial respiratory function in General is suppressed as a consequence of aging, further exacerbating the harmful effects of additional molecular povrejdeniy affecting the function of the respiratory chain.
Other factors, in addition to primary mitochondrial dysfunction underlie neurodegeneration in BA, BP, and related disorders. Exitotoxicity stimulation and nitric oxide participate in both diseases, the factors that both exacerbate the defects of the mitochondrial respiratory chain, and the devastating effects are particularly strong on the background of dysfunction of the respiratory chain.
Huntington's disease also involves mitochondrial dysfunction in the affected areas of the brain, with the joint interactions exitotoxicity stimulation and mitochondrial dysfunction contribute to neuronal degeneration. In example 8, the compound of the present invention, triacetyluridine, prevents necrosis of nerve cells in a mouse model of Huntington's disease.
Compounds and compositions of the present invention is applicable for the treatment and attenuation of progression associated with age neurodegenerative diseases, including ad and PD.
Amyotrophic lateral sclerosis
One of the major genetic defects in patients with amyotrophic lateral sclerosis (ALS; b the disease Lou Gehrig; progressive degeneration of motor neurons, atrophy of skeletal muscles, inevitably leading to paralysis and death) is a mutation or defect in the copper-zinc-superoxide dismutase (SOD-1), an antioxidant enzyme. Mitochondria produce active oxygen molecules and are also the primary targets of molecules of active oxygen. Inefficient transfer of electrons to oxygen in the mitochondria is the most important physiological source of free radicals in the systems of mammals. Lack of antioxidants or antioxidant enzymes may lead to degeneration of mitochondria or exacerbate the degeneration of mitochondria. Transgenic mice with mutated SOD-1 develop symptoms and pathology, with the same symptoms and pathology of non-human ALS. It was shown that the development of this disease in these animals includes oxidizing dextrally mitochondria with subsequent functional decline in motor neurons and the appearance of clinical symptoms (Kong and Xu, J. Neurosci. 18:3241-3250, 1998). Skeletal muscle from patients with ALS has a low activity of mitochondrial Complex I (Wiedemann et al., J. Neurol. Sci. 156:65-72, 1998).
Compounds, compositions and methods of the present invention is applicable for the treatment of ALS, to reverse the development or slow the progression of clinical symptoms.
Protection is otiv ischemia and hypoxia
Oxygen deficiency leads to direct inhibition of the activity of the mitochondrial respiratory chain by depriving cells of the terminal electron acceptor for pereokislenie cytochrome C to Complex IV, and indirect inhibition, particularly in the nervous system, through secondary postanoxic exitotoxicity and the formation of nitric oxide.
In conditions such as cerebral anoxia, the crises in angina or sickle cell anemia, tissue are relatively hypoxic. In such cases, the compounds of this invention provide protection of affected tissues from the harmful effects of hypoxia, weaken secondary delayed necrosis of cells and accelerate the recovery from hypoxic tissue stress and damage.
Compounds and compositions of the present invention is applicable to prevent delayed cell necrosis (apoptosis at sites such as the hippocampus or the cortex of the brain occurring after approximately 2-5 days after onset of cerebral ischemia) after ischemic or hypoxic stroke brain.
Renal tubular acidosis
Acidosis is caused by renal dysfunction is often observed in patients with mitochondrial disease, regardless of whether the underlying disease dysfunction dy is athelney chain congenital or induced by ischemia or cytotoxic agents, such as cisplatin. Renal tubular acidosis often requires the introduction of exogenous sodium bicarbonate to maintain pH of blood and tissues.
In example 3, the introduction of the compounds of this invention caused a immediate reverse the development of renal tubular acidosis in a patient with a severe defect of Complex I. the Compounds and compositions of the present invention is applicable for the treatment of renal tubular acidosis and other forms of renal dysfunction caused by defects in the mitochondrial respiratory chain.
Associated with age, neurodegeneration and reduced cognitive abilities
During normal aging is the progressive decline in the function of the mitochondrial respiratory chain. Starting with an age of approximately 40 years, is the exponential increase of defect accumulation of mitochondrial DNA in humans and parallel reduction of nuclear-adjustable elements mitochondrial respiratory activity.
In the work of de Grey (Bioassays, 19:161-167, 1998) mechanisms underlying the observation that many mitochondrial DNA have a selective advantage during mitochondrial turnover, particularly in postmitotic cells. The proposed mechanism is that mitochondria are defective respiratory chain produces less oxidize is inoe damage in respect of itself, than mitochondria with intact functional respiratory chain (mitochondrial respiration is the primary source of free radicals in the body). Thus, a normally functioning mitochondria accumulate oxidative damage of lipid membranes more quickly than do defective mitochondria, and are, therefore, marked for degradation by lysosomes. Because the mitochondria in the cells have a half life of approximately 10 days, the selective advantage can lead to a rapid replacement of functional mitochondria mitochondria with reduced respiratory activity, especially in slowly dividing cells. The overall result is that, as soon as there is a mutation in the gene for mitochondrial protein that reduces oxidative destruction of mitochondria, such defective mitochondria will quickly populate the cell, reducing or eliminating the ability to breath. The accumulation of these cells leads to aging or degenerative disease at the level of the organism. This is consistent with a progressive mosaic cells with defective activity of the electron transport in muscle cells, almost devoid of cytochrome-C oxidase (SOH) activity, randomly scattered among cells with normal activity, and a higher incidence of MOR-negative cleto is in biopsies from older subjects. Thus, the organism during aging or in various mitochondrial diseases, is in a situation in which irreplaceable postmitotic cells (e.g. neurons, the cells of skeletal and cardiac muscles) should be preserved and their function must be supported to a considerable extent the face of relentless progressive decrease the function of the mitochondrial respiratory chain. Neurons with dysfunctional mitochondria become a progressive way more sensitive to such damage, as excitotoxicity damage. Mitochondrial insufficiency contributes to the majority of degenerative diseases (in particular, neurodegeneration), which accompany aging.
Congenital mitochondrial diseases often include early onset neurodegeneration with similar basic mechanism violations that take place during aging people born with normal mitochondria. The demonstration described in the examples that the compounds and compositions of the present invention is applicable in the treatment of congenital mitochondrial diseases or early appearance of mitochondrial diseases, provides direct support to the applicability of the compounds and compositions of the present invention for the treatment of age-related degeneration of tissues.
Joint and compositions of the present invention is applicable for treating or attenuating the decline of cognitive ability and other degenerative effects of aging.
Mitochondria and cancer chemotherapy
Mitochondrial DNA is generally more sensitive to damage than nuclear DNA due to several reasons:
1. Mitochondrial DNA has a more complicated system repair than nuclear DNA.
2. Virtually all chain mitochondrial DNA encode important proteins, so that any defect will potentially damage mitochondrial function. Nuclear DNA contains long regions that do not encode proteins in which mutations or damage remain essentially no consequences.
3. Defective mitochondria often have a selective advantage over normal, active mitochondria during cell proliferation and turnover of mitochondria.
4. Mitochondrially DNA is not protected by histones.
Empirically, damage to mitochondrial DNA is more extensive and persists longer than nuclear damage DNA in cells subjected to oxidative stress or action agents, cancer chemotherapy, such as cisplatin, due to both higher sensitivity and less efficient repair of mitochondrial DNA. Although mitochondrial DNA may be more sensitive to damage than nuclear DNA, in some circumstances, it is relatively resistant to mutagenesis under the action of chemical carcinogens. This is due to those who, that mitochondria are responsible for certain types of mitochondrial DNA destruction of their defective genomes, rather than attempting their reparation. This leads to a global dysfunction of mitochondria within a certain period after cytotoxic chemotherapy. Clinical application of chemotherapeutic agents such as cisplatin, mitomycin and cytoxan, often accompanied by debilitating "chemotherapy fatigue, prolonged periods of weakness and intolerance to exercise, which can persist even after recovery from hematologic and gastrointestinal toxicities of these agents.
Compounds, compositions and methods of the present invention is applicable for the treatment and prevention of side effects of cancer chemotherapy-related mitochondrial dysfunction. This application predecessors pyrimidinediamine to alleviate side effects of cancer chemotherapy is mechanically and biochemically different from reducing the toxicity of cytotoxic anticancer pyrimidine analogues using precursors pyrimidinediamine, which is mediated biochemical level competition nucleotide antimetabolites.
Example 5 illustrates the protective effect of oral input triacetyluridine protection against induzirovanny the th Taxol neuropathy.
In addition, the redox state of the mitochondria of the liver is one of the means of regulation of appetite. Cancer patients often find "early satiety", Pro-anorexia, weight loss and cachexia. Energy metabolism is often severely disrupted in cancer patient with useless rastraivaysya energy cycles hyperactive tumor glycolysis, producing rolling in blood lactate, which in the liver is converted again into glucose. Induced by chemotherapy mitochondrial damage contributes to metabolic disorders.
As shown in example 2, treatment of the compound of the present invention leads to improved appetite of a patient with mitochondrial disease.
Mitochondria and ovarian function
The crucial function of the ovary is maintaining the integrity of the mitochondrial genome to the oocyte, as all of mitochondria moving in the fruit comes from the mitochondria present in the oocyte during fertilization (conception). Deletions in mitochondrial DNA become detectivesyme around the age of menopause and are also associated with abnormal menstrual cycles. As the cells cannot directly detect the defects of mitochondrial DNA and answer them, but can deterioate tol the secondary effects, acting in the cytoplasm, such as impaired breathing, redox state, or defects in the synthesis of pyrimidines, such products of mitochondrial function involved in signal quality for the selection of oocytes and follicular atresia, eventually triggering menopause, when there can be further guaranteed mitochondrial genome accuracy and functional activity. This is similar to apoptosis in cells with DNA damage, which are the active process of cell suicide, when genomic accuracy can no longer be achieved through repair processes. Women with mitochondrial cytopathy acting on the sex glands, often prone to premature menopause or primary manifestation of abnormal menstrual cycles. Cytotoxic cancer chemotherapy often induces premature menopause with a consequent increased risk of osteoporosis. Induced by chemotherapy amenorrhea is usually due to primary ovarian failure. The frequency of occurrence induced by chemotherapy amenorrhea increases as a function of age in premenopausal women receiving chemotherapy, indicating the involvement of mitochondria. Inhibitors of mitochondrial respiration or inhibit protein synthesis induced by hormones ovulation and, in addition, ing is berout the production of steroid hormones in the ovary in response to the pituitary gonadotropins. Women with down syndrome usually prematurely exposed to the menopause and also are subject to an early development of Alzheimer's-like dementia. The low activity of cytochrome C oxidase is constantly found in the tissues of patients with down syndrome and late Alzheimer's disease.
Thus, proper maintenance of mitochondrial function or compensate for mitochondrial dysfunction is useful for protecting against age-related or chemotherapy-induced menopause or menstrual cycles or ovulation. Compounds and compositions of the present invention, including antioxidants and mitochondrial cofactors, applicable for the treatment and prevention of amenorrhoea, abnormal ovulation, menopause or secondary effects of menopause.
In example 1, treatment of the compound of the present invention resulted in shortening of the menstrual cycle. Since the patient was in a persistent luteal phase, the response indicates that the predecessor of pyrimidinediamine paid a reduced sensitivity to gonadotropins by the pituitary gland, which, apparently, was increased to compensate for the decreased sensitivity of the ovaries of mitochondrial origin.
Diagnosis of mitochondrial disease
Amazing response re the Ktsia patients with mitochondrial disease by the introduction of the compounds of the present invention suggests, what clinical response to the predecessor of pyrimidinediamine, introduced in accordance with the methods of the present invention has a diagnostic utility for the detection of possible mitochondrial disease. Molecular diagnostics molecular lesions underlying mitochondrial dysfunction, is difficult and expensive, especially when the defect is not one of the more common mutations or deletions of mitochondrial DNA. Accurate diagnosis of mitochondrial disease often requires a muscle biopsy, but even this invasive measure is applied only if the mitochondrial defects present in the muscle. Because the compounds and compositions of this invention are safe when taken in accordance with the methods of the present invention, therapeutic introduction of precursor pyrimidinediamine is an important diagnostic probe for alleged mitochondrial disease, especially when applied together with tests on various other aspects of mitochondrial dysfunction.
For the diagnosis of congenital mitochondrial cytopathy daily doses of 50-300 mg/kg predecessor pyrimidinediamine of the present invention is administered to the patient during the period from one to twelve weeks and monitor changes in clinical signs and the symptoms of. Improvements observed in patients described in the examples, and additional patients include, but are not limited to, reducing the frequency and severity of seizures, migraines and shock attacks, improving weight gain in children with "lack of development", the weakening of renal tubular acidosis with concurrent reduction in the need for additional bicarbonate, improved muscle strength, improved language development, weakening ataxia, improving hypotension, decrease the frequency and severity of sinus and ear infections, improving memory and relieving symptoms of autonomic and peripheral neuropathy. In one embodiment, the present invention also use other tests of mitochondrial dysfunction to provide proof of diagnosis of mitochondrial disease. Diagnosis usually requires comprehensive consideration of a number of confirmatory tests with varying degrees of reliability, as described in Walker et al. (Eur. Neurol., 36:260-7, 1996). Therapeutic sensitivity to precursor pyrimidinediamine of the present invention is applicable primarily as an additional minor criteria in this diagnostic scheme, since it is possible that after the introduction of the predecessors of pyrimidinediamine can receive therapeutic benefits, mediated only compensatie the defects of the respiratory chain. Since the nature and severity of symptoms of mitochondrial diseases are a heterogeneous and variable between patients, the effectiveness of exogenous precursors pyrimidinediamine usually appreciate the selection of the dominant symptoms of the patient and monitoring their severity as quantified to the extent possible, during therapy. If you anticipate a possible placebo effect, "blind" transfer of a patient with a drug in an appropriate placebo use sometimes in relation to the individual patient. To evaluate the clinical benefit may require considerable skill and experience, but such qualification is in the competence of practitioners in the treatment of patients with Multisystem metabolic diseases and, as such, does not require undue experimentation in view of the gravity of this class of diseases. The following examples are clinical treatment of patients with mitochondrial diseases triacetyluridine, the compound of the present invention demonstrate the ability to determine the clinical benefit of individual patients.
That is, the Introduction and preparation of the compounds and compositions of this invention
In case of any specific therapeutic targets for the treatment of mitochondrial diseases predecessors is pyrimidinediamine, the compounds of this invention are typically administered from one to three times a day. Acylphosphate uridine and cytidine administered orally in doses of 10-500 mg/kg of body weight per day, with variations within this range depending on the quantity required for optimal clinical benefit. Usually the optimal dose is between 50 and 300 mg/kg/day (mostly 100-300 mg/kg/day), divided into two or three separate doses, taken with an interval of 6-12 hours. The uridine and citizen absorbed less efficiently than acylphosphate these two nukes, so require higher doses for therapeutic benefits comparable to the benefits achieved with acylphosphatase. Osmotic diarrhea limits the number of uridine or cytidine (or other derivatives, such as cytidinediphosphocholine), which can be administered to the patient, so in most cases acylphosphate cytidine and uridine are more effective than the parent compound, with fewer side effects. Dose cytidine and uridine used for the purposes of this invention are in the range from 50 to 1000 mg/kg/day, preferably 100-1000 mg/kg/day, depending on the balance between therapeutic efficacy and tolerability. The orotate or esters orotata with alcohols administered orally in doses of from 20 to 200 mg/kg/day, again in zavisimost and the number, required to achieve optimal therapeutic effect in a specific pathological condition, including dysfunction of the mitochondrial respiratory chain. Dose predecessor pyrimidinediamine of the present invention required for a particular disease or a particular patient will depend in part on the severity of the disease.
For any individual patient with a disease characterized or caused by mitochondrial dysfunction, an effective dose of the precursor pyrimidinediamine of the present invention is usually determined empirically. In the case of congenital mitochondrial diseases, also known as mitochondrial cytopathy or mitochondrial encephalomyopathy, clinical manifestation of signs and symptoms is usually heterogeneous and variable between patients. Clinical benefit after administration of the compounds of this invention is determined by monitoring a number of symptoms and assessment of their severity over time, for example, at monthly intervals. For this purpose, choose three to five dominant symptoms, and the degree of attenuation being evaluated as a component of clinical benefit, is often subject to clinical evaluation. When treating patients with complex metabolic disorders such valuation is not excessive burden is determined as being experimentation, especially under the condition of gravity (often life-threatening) mitochondrial cytopathy and costly care. As early as possible in the lives of patients compensation mitochondrial or other metabolic defects can give a very big difference compared to the intervention, after the development of the brain and the body reaches homeostasis after puberty. So it makes sense to show a significant effort spent on diagnosis and treatment of complex metabolic diseases, in particular, in the case of the development of children. The following examples of clinical improvement after administration of the compounds of this invention to patients with mitochondrial diseases demonstrate the suitability and value of such a treatment and assessment.
In the case of most diseases with less heterogeneity in the clinical manifestation than in the case of mitochondrial disease, in this area there are suitable scale validated assessments to determine the effectiveness of drug treatment. Before conducting clinical trials to determine doses predecessors pyrimidinediamine of the present invention for the treatment of pathological conditions described herein, suitable day for individual patients, are determined by clinical assessment from the maintain reaction (including images, magnetic resonance imaging (MRI) and other indicators, for example, biochemical measurements, which may not necessarily be clinically obvious by simple observation of the patient's symptoms) in accordance with the quantitative rating scale disease. In all cases, the dominant symptoms of a specific pathological conditions are monitored over time to determine whether the improvement in signs and symptoms or weakening of clinical deterioration, as it is usual in the field of medicine. Before determining the dose in blind clinical trials, the response of the particular patient on the predecessor of pyrimidinediamine of the present invention differentiate from possible placebo effect simple "blind" switching from drugs to placebo over a period of several weeks.
In the case of patients unable to receive oral input medicines, the compounds of this invention, in particular, uridine, citizen and esters orotata, may be introduced, if necessary, by continuous intravenous infusion, delivering daily dose of 10-500 mg/kg/day.
Pharmacologically active compound optionally together with a suitable pharmaceutically acceptable carriers containing excipients and auxiliary substances (additives)that facilitate processing of the active compounds. They are administered in the form of tablets, suspensions, solutions, pills, capsules or suppositories. For example, these compositions are introduced orally, rectally, vaginally, or released through the buccal pocket of the oral cavity and can be applied in the form of a solution using injections, oral or local administration. The composition may contain from about 0.1 to 99%, preferably from about 50 to 90% of the active compounds (compounds) with excipients (excipients).
For parenteral administration by injection or intravenous administration, the active compounds suspended or dissolved in aqueous medium, such as sterile water or saline. Injectable solutions or suspensions optionally contain a surface-active agent, such as esters of polyoxyethylenesorbitan, esters sorbitan, ethers of polyoxyethylene, or solubilizing agents, such as propylene glycol or ethanol. The solution typically contains from 0.01 to 5% of the active compounds.
Suitable excipients include fillers such as sugars, for example lactose, saccharose, mannitol or sorbitol, preparations of cellulose and/or calcium phosphates, for example tricalcium phosphate or calcium phosphate, and also binders such as starch paste prepared using, for example, corn to the making a movie, wheat starch, rice or potato starch, gelatin, tragakant, methylcellulose, hypromellose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone.
Auxiliary substances (additives) include regulating the fluidity agents and lubricants, for example silica, talc, stearic acid or its salts, such as magnesium stearate or calcium stearate and/or polyethylene glycol. The Central part of the pills provide suitable coatings which, if desired, are resistant to gastric juice. For this purpose use concentrated solutions of sugars, that may not necessarily contain the Arabian gum, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, glazing solutions and suitable organic solvents or solvent mixtures. To produce coatings resistant to gastric juice, use solutions of suitable preparations of cellulose, such as acetylcholin cellulose or phthalate of hydroxypropylmethylcellulose. Dyes or pigments do not necessarily add to the coating of tablets or coated tablets, for example, for identification or to indicate different doses of the compounds.
The pharmaceutical preparations of this invention are prepared are known per se manner, for example, by conventional methods of blending,granulation, the manufacture of tablets, dissolution or lyophilization. Thus, pharmaceutical preparations for oral administration obtained by combining the active compounds (compounds) with solid eccipienti, optionally grinding the resulting mixture and processing the mixture of granules, after adding suitable additives, if desirable or necessary, to obtain tablets or Central parts of the bean.
Other pharmaceuticals that are applicable for oral delivery include capsules with a tight fitting two halves made of gelatin, and also soft sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Capsules with a tight fitting two halves contain the active connection (connections) in the form of granules, which are not necessarily mixed with fillers such as lactose, binders, such as starches, and/or lubricating agents such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds are preferably dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin or polyethylene glycols. In addition, optional add stabilizers.
The pharmaceutical compositions used rectally include, for example, supp is sicarii, consisting of a combination of active compounds with a base of the suppository. Suitable bases suppositories are, for example, natural or synthetic triglycerides, hydrocarbons of the paraffin series, polyethylene glycols or higher alkanols. In addition, use gelatin rectal capsules comprising the combination of active compounds with a base. The core materials include, for example, liquid triglycerides, polyethylene glycols or paraffin hydrocarbons of the series. In another embodiment of the present invention, using drugs for enemas, which does not necessarily contain increasing viscosity fillers, such as methylcellulose, hypromellose, carboxymethylcellulose, carbopol, polyacrylates glycerin or other hydrogels.
Suitable compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, in the form of water-soluble salts.
In addition, suspensions of the active compounds, if necessary, give an oily injection suspensions. Suitable lipophilic solvents or carriers include fatty oils, for example sesame oil, or synthetic fatty acid esters, such as ethyl acetate, or triglycerides. Aqueous injection suspensions optionally include substances that increase Ascot suspension, which include, for example, sodium-carboximetilzellulozu, sorbitol and/or dextran. The optional suspension contains stabilizers.
F. Synthesis of compounds of this invention
Acylphosphate cytidine and uridine synthesize usually ways acylation involving the reaction of acid chlorides or anhydrides of carboxylic acids with cytidine or uridine.
Synthesis of 2',3',5'-tri-O-providedin shown in example 6.
The following examples are illustrative, but not limiting the methods and compositions of the present invention. Other suitable modifications and adaptations of a variety of conditions and parameters normally encountered in clinical therapy, which are obvious to persons of ordinary skill in this field are within the intent and scope of this invention.
Example 1: Treatment of Multisystem mitochondrial disorders by triacetyluridine
29-year-old woman with partial defect of Complex I, the son was diagnosed with mitochondrial disease, leading to shock seizures, ataxia, and encephalopathy, manifested Multisystem mitochondrial disorder. Signs and symptoms included hemiplegic/fazicheskie migraine, large seizures, neurogenic dysfunction pediatricain is on tract and bladder, requiring catheterization approximately four times a day, dysphagia, autonomic and peripheral polyneuropathy, producing paresthesia syndrome tachycardia/bradycardia and poor functional ability, inability to climb one flight of stairs without stopping to rest, and the decline in cognitive abilities with bouts of clouded consciousness and bad memory, which lasts for a period of from several hours to several days.
After the start of treatment 0.05 mg/kg/day oral input triacetyluridine and during a period of at least 6 months, this patient had no history of seizures or migraines; it paresthesia associated with peripheral neuropathy, has been eliminated. She was able to spontaneously emptied most of the days, and katerizatsii required only once or twice a week. After 6 weeks of treatment triacetyluridine, this patient was able to walk, passing one mile, which she was unable to do in the last two years due to inadequate functional abilities. Attacks aetiology of this patient during sleep and tachycardia during physical strain had a reduced frequency; before treatment, tachycardia with a heart rate over 140 beats per minute occurred when simply standing up, and after 6 weeks triacetyl Idina tachycardia occurred only on slides and ladders. Her mind was clear and defects of memory has improved significantly.
During treatment periods that the patient was shortened from 4 weeks up to two weeks, and she was persistent luteal phase, as determined from measurements of estradiol, progesterone, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). After a few months, her cycle is normalized to 4 weeks.
This patient demonstrates the important characteristics of this invention, namely, that 1) the compound of the present invention caused improvement in almost all the characteristics of a complex Multisystem disease associated with mitochondrial dysfunction in various tissues, and that 2) the compounds of this invention are unexpectedly applicable for the treatment of pathological conditions associated with partial deficiency of Complex I, which affects a portion of the mitochondrial respiratory chain, which is outside the chain transfer electrons directly involved in the de novo biosynthesis of pyrimidines.
Temporary shortening of the menstrual cycle the patient is interpreted as improving ovarian function caused by triacetyluridine, excessive hormonal stimulation, by means of which the neuroendocrine system tried to compensate for the dysfunction of the ovaries. Reverse tie is between the ovaries and the hypothalamus led to a gradual normalization of the cycle time.
Example 2: Treatment of refractory (resistant) epilepsy
11-year-old boy had refractory epilepsy with an age of 4.5 years, obviously, obuslovlennoe syndrome multiple deletions of mitochondrial DNA. In December 1997, his condition deteriorated, resulting in two cases of recognition of the need to be placed in the intensive care about greatly increasing epilepsy. Even with the aggressive schemes of standard anticonvulsant therapy, this patient had 8-10 Grand Mal seizures per night, making it unable to regularly attend school or participate in sports activities. He also developed the automaticity of the upper lip.
In the first three days after the start of treatment, injected oral triacetyluridine (first dose of 0.05 g/kg/day and gradually increase the dose to 0.1 and then to 0.24 g/kg/day for several weeks) seizures was not involuntary movements of the lips ceased. Subsequently there was some recurrence of seizures, especially during infections, although at a much lower frequency than before treatment triacetyluridine. This patient was able to return to school and resume active participation in sportin events. His appetite, cognitive function and fine motor coordination has improved during therapy, leading to academic success and the outstanding result is m sports activity, such as baseball.
Example 3: Treatment of renal tubular acidosis
2-year-old girl with down syndrome Lee (Leigh) (subacute nekrotizirutaya encephalopathy), associated with severe deficiency of Complex I, with the manifestation of renal canalcaveira acidosis requiring intravenous 25 mEq per day sodium bicarbonate. Within a few hours after intragastric treatment triacetyluridine at the dose of 0.1 g/mg/day renal tubular acidosis were eliminated and did not require the introduction of additional bicarbonate to normalize blood pH. Triacetyluridine has also led to a rapid normalization of elevated concentrations of amino acids in the bloodstream and supported lactic acid at low levels after discontinuation of treatment with dichloroacetate, which was previously required to prevent lactic acidosis.
Example 4: Treatment of delays in development
The girl at the age of 4.5 years with epilepsy, ataxia, developmental delay speech and intolerance of fats and aciduria emitting dicarboxylic acids in the urine was treated with triacetyluridine with a daily dose of 0.1-0.3 g/kg/day. This treatment resulted in a 50% reduction in seizure frequency, improvement of ataxia and motor coordination, restoration of tolerance to food fats and rapidly accelerating the development of skills of verbal expression (speech).>
Example 5: Prevention of Taxol-induced neuropathy
Peripheral neuropathy is a frequent DLT (dose limiting side effect of important anticancer agents such as cisplatin and Taxol. In the case of Taxol, sensory neuropathy occurred a few days after injection. The mechanism of action of Taxol includes the stabilization of microtubules, which are useful for the treatment of cancer, but harmful to peripheral neurons. Stabilization of microtubules violates axony transfer of cellular components. Mitochondria move the Shuttle, between the cell body and terminals of neurons, so that the expression of components of the mitochondrial respiratory chain can be regulated nuclear transcription factors. During inhibition of Shuttle movement of mitochondria, mitochondria, distant from the core, subjected to reduction in the expression of subunits of the respiratory chain are encoded by the mitochondrial genome, due to inadequate exposure factors transcription of mtDNA, resulting in a lack of neuronal energy in that particular area and to other consequences of mitochondrial dysfunction.
Two groups of 10 mice each were treated with Taxol, to 21.6 mg/kg/day for 6 consecutive days by intraperitoneal injection. An additional group of 10 mice received injectiable media. One ie groups treated with Taxol mice were treated orally administered triacetyluridine, 4000 mg/kg twice daily (b.i.d.). Nine days after the start of treatment with Taxol nociceptive sensory defects experienced by determining the latent period of sharp movements of the tail after exposure of the tail when focusing thermal radiation of the infrared heating lamps. In this system, the delay in response to a sharp movement of the tail radiated heat correlated with sensory defects of nerve cells.
|Group:||The latent period of sharp movements of the tail|
|Control (without Taxol)||10,8±0.5 seconds|
|Taxol + triacetyluridine||11,9±0.7 seconds|
Taxol treatment violated the response to painful stimuli as an indicator of toxic sensory neuropathy. Oral administration triacetyluridine significantly weakened Taxol-induced changes in the latent period of sharp movements of the tail.
Example 6: Synthesis of writingerotica
A. Obtaining pyrovillia performed by using the simple reaction of alpha, alpha-dichlorodimethyl ether and pyruvic acid using the procedure Ottenheum and Man (Synthesis, 1975, R).
Century of Uridine (3.0 g, 12 mmol) was dried by azeotropic distillation with toluene under vacuum (3) and then was dissolved in DMF (20 ml) and pyridine (20 ml). The resulting solution was cooled to -10°and added dropwise to 6.0 ml of pyrovillia (obtained in stage A). The reaction mixture was stirred at room temperature in an argon atmosphere for 24 hours. The TLC analysis (5% MeOH/CH2Cl2showed expenditure of uridine. The reaction mixture was evaporated to dryness and distributed between CH2Cl2and aqueous sodium bicarbonate. The organic layer was washed with water, aqueous HCl (pH 3.0) and water; dried over sodium sulfate; and concentrated and purified flash chromatography (silica gel, 5% MeOH/CH2Cl2) to obtain 1.4 g of writingerotica or 2',3',5'-tri-O-providedin.
Example 7: therapeutic effects of oral input triacetyluridine in the model using the MPTP Parkinson's disease (BP) and mitochondrial dysfunction
The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is an inhibitor of complex I (NADH-dehydrogenase) of the mitochondrial respiratory chain, which is used for the induction of loss of dopaminergic cells (Varastet et al., Neuroscience, 63:47-56, 1994). This toxin is widely used at present in the model animal for disease Parkinsona (BP) (Bezard et al., Exp. Neurol., 148:288-92, 1997).
Male mice C57/BL6 in which osrate 6-9 months weighing 30-40 g from Taconic Farms used in studies with MPTP (n=7 per group). MPTP (30 mg/kg VB.) was administered twice a day for 1.5 days. TAU (triacetyluridine) was administered P.O. twice a day for 4 g/kg 0.75% hydroxypropylmethylcellulose media with 200 mg TAU (triacetyluridine) per ml of solution, for 2 hours prior to administration of the toxin to the day before the killing.
After 8 days after cessation of MPTP injection, mice were killed with the help of CO2and striatum from both sides were dissected on a cold surface. Striped body was frozen on dry ice. The survival of dopaminergic neurons was assessed by the content of dopamine (DA) striatum. The content of dopamine was assessed radioterminal way in terms of GLP, but YES can be measured using liquid chromatography high pressure with electrochemical detection as described previously (Friedemann & Gerhardt, Neurobiol. Aging, 13: 325-32, 1992). Was obtained reduced mortality in MPTP treated mice as a result of processing triacetyluridine (TAU). Mortality in mice group control + MPTP was 71,4% compared with 28.6% in group processing TAU + MPTP. Received also a neuroprotective effect processing PN401 on the reduction of DA due to MPTP.
The effect of TAU on MPTP-induced decrease of DA in the striatum
|Processing||YES striped t the La *|
|Control + control||147±13,0|
|TAU + control||93,8±10,7|
|Control + MPTP||9,2±2,2|
|TAU + MPTP||37,9±7,4|
* Data are presented as ng YES/mg protein (mean±standard error of the mean)
The second study was performed using MPTP (25 mg/kg VB. twice a day for 2 days). Male mice C57/BL6 at the age of 6-9 months with a weight of 30-40 g from Taconic Farms used in studies with MPTP (n=6 per group). MPTP (30 mg/kg VB.) was administered twice a day for 2 days. TAU (triacetyluridine) was administered P.O. twice a day for 4 g/kg 0.75% hydroxypropylmethylcellulose media with 200 mg TAU (triacetyluridine) per ml of solution, for 2 hours prior to administration of the toxin to the day before the killing. TAU or carrier is administered orally (dose TAU=4 g/kg body weight twice a day)from the day before MPTP administration and ending at day 8. Mice were killed at day 9. This study also showed that TAU showed a protective effect on dopaminergic neurons, as shown weakened by loss-YES striatum as a result of MPTP.
The effect of TAU on MPTP-induced decrease of DA in the striatum
|Processing the||YES striatum *|
|Control + control||71,0±10,6|
|TAU + control||52,0±3,0|
|Control + MPTP||15,9±2,2|
|TAU + MPTP||26,7±0,9|
* Data are presented as ng YES/mg protein (mean±standard error of the mean)
Example 8: therapeutic effect of TAU in the model using 3-nitropropionic acid (NP) Huntington's disease (HD)
Huntington's disease (HD) is characterized by progressive loss of neurons, particularly in the striatum. Patients with HD have a reduced activity of succinate dehydrogenase (complex II)-ubichinonoxidoreductase (complex III). Browne, Mitochondria &Free Radicals in Neurodegenerative Diseases, 361-380 (1997). A widely used model BH uses the inhibitor of succinate dehydrogenase, 3-nitropropionic acid (NP). (Ferrante et al., Mitochondria &Free Radicals in Neurodegenerative Diseases, 229-244, 1997). In particular, NP induces damage to the striatum (Brouillet et al., J. Neurochem., 60: 356-9, 1993).
Males 6-8-month-old Swiss mice (National Cancer Institute; NCI, Frederick, MD) were treated NP (65 mg/kg VB.) once a day for 4 days to induce mortality, loss of nerve cells and behavior disorders based n=8 per group. TAU was administered P.O. twice daily at a dose of 4 g/kg body weight of 0.75% hydroxypropyl intellisnap media with 200 mg TAU (triacetyluridine) per ml of solution and was given to mice one day before the introduction NP and every day until day 8. Day 9 mice were fixed by perfusion with 10% buffered formalin and were processed for silver staining for detection of neuronal damage. Was obtained reduced mortality caused NP, in mice treated with TAU, in comparison with controls, as shown below. In the group NP + TAU was no mortality, but 3 out of 8 mice died in the group media + NP.
Scoring behavior processed NP mice had to determine whether a violation of motor abilities at any time during this study. 88% of mice in the control group + NP with behavioral disorders were detected by macroscopic observation. Was found reduced incidence of violations (only 50%) in mice treated with TAU + NP.
The silver staining was analyzed "blind" pathologist, i.e. the pathologist, which were not known variants of the tissue samples. In mice treated with TAU + NP, found no clear signs of neural damage. However, in mice treated with control + NP, was clearly expressed by the silver staining of synaptic terminala in the area of the striatum (the caudate area of the core/shell) and a black substance. Impregnation with silver axons and/or synaptic terminala in the thalamus, a deep average brain and/or reticular formation (the medulla) was also detected in 80% of mice, about Botanik control + NP. The black substance is in the striatum, and these areas are particularly sensitive to damage NP. The damage of the dark matter and striatum was prevented by triacetyluridine (TAU).
Example 9: therapeutic effect of TAU in the model using 3-nitropropionic acid (NP) epilepsy
3-nitropropionic acid (NP) is a mitochondrial toxin that acts by inhibition of Complex II of the respiratory chain; it is used to induce brain damage similar to the damage typical of Huntington's disease. Seizures can be induced by using NP as a model of epilepsy and mitochondrial dysfunction. Urbanka et al., Eur J. Pharmacol., 359: 55-8 (1998). In all these studies used male mice CD-1 (National Cancer Institute; NCI, Frederick, MD) weighing between 26-40, Mice were divided into groups of 5 mice and animals for each group were selected randomly from different cells in order to avoid the possible influence of age. Mice maintained on a 12-hour cycle of light / dark with free access to feed and water. All experiments were performed during the light period between 9:00 and 16:00 o'clock Mice (n=17-20) gave 160 mg/kg NP and watched seizures. NP was prepared at a concentration of 16 mg or 18 mg/ml in sterile water (pH 7.4). NP introduced V.B. in a volume of 0.1 ml/10 g body weight. TAU was administered at a dose g/kg p/o 0.75% hydroxypropylmethylcellulose media for 2 hours before the introduction of NP. Seizures were evaluated according to previously described methods (Roberts & Keith, J. Pharmacol. Exp. Ther., 270: 505-11, 1994; Urbanka et al., Eur. J. Pharmacol., 359: 55-8 (1998).
Behavioral (behavioral) observations were performed within 120 minutes after application NP. Take into account, and registered three main categories of response of seizures:
1. Clonic movements: movements of the forelimbs, accompanied front convulsions.
2. Explosive clonic movements: the movement of all four extremities, including running, jumping and jumping.
3. Tonic response: including tonic flexion (bending) and tonic extension of all four limbs.
The mortality rate was estimated at 120 minutes after injection NP.
NP induced primarily clonic seizures, some mice developed the behavior of running and jumping, which resulted in mortality. TAU reduced the percentage of occurrence of clonic seizure, seizure in the form of running and mortality caused NP. The first endpoint was the latent period before clonic seizure. TAU increased the latent period before clonic seizure from 25.0 to 40.8 minutes. The results are presented as mean±standard error of the mean.
|Endpoint||Control + NP|
|% clonic seizures||90,0||70,6|
|% of seizures with Jogging||42,9||5,9|
|The latent period before clonic seizure||23, 8±0,7||40,8± and 4.9|
Example 10: therapeutic effect of TAU in the model of excitotoxicity with quinolinic acid (XK)
Quinolinic acid is an agonist of the NMDA receptor, used in models of Huntington's disease and excitotoxicity damage (Beal et al., J. Neurosci., 11: 1649-59, 1991; Beal et al., J. Neurosci., 11: 147-58, 1991; Ferrante et al., Exp. Neurol., 119:46-71, 1993). It can induce severe damage to the Central nervous system when introduced directly into the striatum. Damage and/or mortality caused by the introduction of HC in the striatum due to Central nervous system etiology.
Males 6-8-month-old Swiss mice (National Cancer Institute; NCI, Frederick, MD) was treated with HC (50 or 100 nmol injected bilaterally in both striatum, n=8 per group). TAU have introduced p/o twice daily at a dose of 4 g/kg body weight of 0.75% hydroxypropylmethylcellulose media with 200 mg TAU/ml and was given to mice one day before the introduction of HC and every day until day 6. At day 7, the mice were killed. HC was injected in a volume of 2 μl, as described previously (Tatter et al., Neuroreport, 6: 1125-9, 1995).
The OBR is tannich TAU mice was reduced mortality, called HC. The percentage of mice surviving for 7 days, treated with 50 nmol HC, was 64% in the control group + HC and 73% in the group TAU + HC, and in the case of mice treated with 100 nmol HC, only 4% survived in the control group + HC, while 19% survived in the group TAU + HC. TAU showed a neuroprotective effect on exitotoxicity called HC.
Although this invention has been described in the form of preferred options, it should be clear that variations and modifications will be apparent to persons skilled in this field. Thus, it is understood that the appended claims cover all such equivalent variations and modifications are included in the claimed scope of this invention.
1. The way to reduce the side effects of cytotoxic agents cancer chemotherapy by administration of a precursor pyrimidinediamine effective amount of from 0.05 to 0.3 g of the specified predecessor per 1 kg of body weight per day, where the specified agent cytotoxic chemotherapy is not an analogue of pyrimidinedione.
2. The method according to claim 1, where the mentioned side effects of cytotoxic cancer chemotherapy is selected from the group consisting of peripheral neuropathy, chemotherapy-induced menopause, is associated with chemotherapy fatigue and reduced appetite.
FIELD: organic chemistry, medicine, biochemistry.
SUBSTANCE: invention proposes using acizol representing bis-(1-vinylimidazol-N) zinc diacetate as an adaptogen. Acizol is known early as antidote. Acizol exceeds plant Eleutherecoccus senticosus by activity with respect to indices of functional state of the body adaptation systems due to providing the higher intensity of tissue respiration, improvement of energetic metabolism and regenerative processes.
EFFECT: enhanced effectiveness and valuable medicinal and biochemical properties of adaptogen.
1 dwg, 8 tbl
FIELD: medicine, anesthesiology, resuscitation.
SUBSTANCE: one should correct hemostasis disorders intra-operationally due to introducing fraxiparine about 40-80 min against the onset of operation along with intravenous injection of mexidol, by drops at the dosage of about 5-6 mg/kg patient's body weight dissolved in 400 ml 0.9%-NaCl solution at the rate of 60 drops/min. The present innovation enables to create the peak of fraxiparine and mexidol concentrations at traumatic stage of operation that enables to correct hemostasiological disorders during operative interference and at early post-operational period due to combined action of preparations onto different links of hemostasis.
EFFECT: higher efficiency and accuracy of correction.
FIELD: organic chemistry, medicine, neurology, pharmacy.
SUBSTANCE: invention relates to derivatives of pyridazinone or triazinone represented by the following formula, their salts or their hydrates: wherein each among A1, A2 and A3 represents independently of one another phenyl group that can be optionally substituted with one or some groups chosen from the group including (1) hydroxy-group, (2) halogen atom, (3) nitrile group, (4) nitro-group, (5) (C1-C6)-alkyl group that can be substituted with at least one hydroxy-group, (6) (C1-C6)-alkoxy-group that can be substituted with at least one group chosen from the group including di-(C1-C6-alkyl)-alkylamino-group, hydroxy-group and pyridyl group, (7) (C1-C6)-alkylthio-group, (8) amino-group, (9) (C1-C6)-alkylsulfonyl group, (10) formyl group, (11) phenyl group, (12) trifluoromethylsulfonyloxy-group; pyridyl group that can be substituted with nitrile group or halogen atom or it can be N-oxidized; pyrimidyl group; pyrazinyl group; thienyl group; thiazolyl group; naphthyl group; benzodioxolyl group; Q represents oxygen atom (O); Z represents carbon atom (C) or nitrogen atom (N); each among X1, X2 and X3 represents independently of one another a simple bond or (C1-C6)-alkylene group optionally substituted with hydroxyl group; R1 represents hydrogen atom or (C1-C6)-alkyl group; R2 represents hydrogen atom; or R1 and R2 can be bound so that the group CR2-ZR1 forms a double carbon-carbon bond represented as C=C (under condition that when Z represents nitrogen atom (N) then R1 represents the unshared electron pair); R3 represents hydrogen atom or can be bound with any atom in A1 or A3 to form 5-6-membered heterocyclic ring comprising oxygen atom that is optionally substituted with hydroxyl group (under condition that (1) when Z represents nitrogen atom (N) then each among X1, X2 and X3 represents a simple bond; and each among A1, A2 and A3 represents phenyl group, (2) when Z represents nitrogen atom (N) then each among X1, X2 and X3 represents a simple bond; A1 represents o,p-dimethylphenyl group; A2 represents o-methylphenyl group, and A3 represents phenyl group, or (3) when Z represents nitrogen atom (N) then each among X1, X2 and X3 represents a simple bond; A1 represents o-methylphenyl group; A2 represents p-methoxyphenyl group, and A3 represents phenyl group, and at least one among R2 and R means the group distinct from hydrogen atom) with exception of some compounds determined in definite cases (1), (3)-(8), (10)-(16) and (19) given in claim 1 of the invention. Compounds of the formula (I) elicit inhibitory activity with respect to AMPA receptors and/or kainate receptors. Also, invention relates to a pharmaceutical composition used in treatment or prophylaxis of disease, such as epilepsy or demyelinization disease, such as cerebrospinal sclerosis wherein AMPA receptors take part, a method for treatment or prophylaxis of abovementioned diseases and using compound of the formula (I) for preparing a medicinal agent used in treatment or prophylaxis of abovementioned diseases.
EFFECT: valuable medicinal properties of compounds and pharmaceutical composition.
32 cl, 10 tbl, 129 ex
FIELD: organic chemistry, medicine, pharmacy.
SUBSTANCE: invention relates to new compounds of the following formulae:
and , and to a pharmaceutical composition possessing the PPAR-ligand binding activity and comprising the indicated compound, and a pharmaceutically acceptable vehicle. Also, invention relates to a method for treatment of patient suffering with physiological disorder that can be modulated with the compound possessing the PPAR-ligand binding activity. Method involves administration to the patient the pharmaceutically effective dose of indicated compound or its pharmaceutically acceptable salt.
EFFECT: valuable medicinal properties of compounds and pharmaceutical composition.
10 cl, 1 tbl, 104 ex
FIELD: topical phyto-mineral compositions for prophylaxis and treatment of infective, respiratory, skin, tumor and other diseases.
SUBSTANCE: claimed composition represents aqueous solution containing as drug plants: everlasting, milfoil, goosegrass, celandine, calendula, bay leave, pine buds, birch buds; as cereals: oat, millet (corn), buckwheat (corn) and wheal (corn); and as mineral components: sea salt and laminaria in amount of 7-9 g per 1000 ml of water, correspondently. In autumn-spring and winter seasons it contains additionally phyto-component such as tutsan, in amount of 7-9 g.
EFFECT: topical composition having no side-effects, with combined component action and increased penetration effect through hypoderm.
SUBSTANCE: method involves carrying out irrigoscopy examination under artificial hypotonia of large intestine with single-stage double opacification of large intestine done with fine peptized aqueous barium cocktail heated to 36°C composed of water - 100 ml, sodium citrate - 1.5 g, sugar - 8 g, barium sulfate - 200 g, carboxymethyl cellulose 0.5 g, raw hen eggs - 2 eggs. Irrigoscopy examination is repeatedly carried out in 24 h. Ribbon-like shadows being observed, intestinal heminthiasis is to be diagnosed.
EFFECT: high reliability in determining shape and localization of helminth even in the cases of negative coprological and serological examination results.
SUBSTANCE: method involves taking into consideration dose-exposure plot, age, hourly diuresis, hemolysis, exotoxic shock, larynx edema, hemoglobinuria availability, measuring body temperature and evaluating each symptom in points. The points are summed. Their sum being less than -17, favorable prognosis is formulated and line emergency team gives medical care. The sum of points being from -17 to +23, outcome prognosis is doubtful. The sum being greater than +23, the prognosis becomes unfavorable. The last two cases Poison dose is additionally determined irrespectively of exposure, vomiting and its nature, gastroesophageal hemorrhage, hemolysis, oliguria, larynx edema exotoxic shock, hemolysis, hemoglobinuria availability, measuring body temperature and evaluating each symptom in points. The points are summed. Their sum being less than -20, line emergency team gives medical care. The sum of points being greater than +20, resuscitation aid and calling for resuscitation group of emergency team on himself with state stabilization and transporting patient with the resuscitation group to the nearest resuscitation department following. Premedication, analgesics, spasmolytics, corticosteroids and hemostatics are administered with probe-mediated gastric lavage and infusion therapy being started.
EFFECT: enhanced effectiveness of treatment; high objectivity in evaluating patient health state.
1 dwg, 6 tbl
SUBSTANCE: the present innovation deals with the preparation of antimutagenic activity towards chemical mutagens as betulinic acid previously known as an antimutagen towards ultraviolet and as an antioxidant. The decrease of chromosomal aberrations has been shown in animals towards chemical mutagens of dioxidine and cyclophosphamide due to the action of betulinic acid within wide range of dosages.
EFFECT: higher efficiency.
FIELD: medicine, therapy.
SUBSTANCE: the present innovation deals with preventing post-injection necrosis of soft tissues as a result of false subcutaneous injection of 10%-calcium chloride solution. One should detect the volume of the introduced 10%-calcium chloride solution; then it is necessary to introduce 5-fold volume of 5%-sodium citrate solution against the volume of calcium chloride injected; then one should apply a sac with ice onto the site of injection for the period of 30 min, not less. The innovation suggested enables to inhibit the processes for tissue damage due to binding calcium cations with anions of sodium citrate along transforming soluble calcium salt into insoluble one to stop calcium transport into the cells and, thus, prevents its damaging action.
EFFECT: higher efficiency.
SUBSTANCE: method involves introducing drug of gas transportation activity in distal position with respect to blood circulation arrest place immediately after blood circulation arrest. Perfluoran is selected as the drug of gas transportation activity at a dose not exceeding 30 ml per 1 kg of injured zone mass.
EFFECT: improved oxygen supply to ischemic tissues; reduced risk of reperfusion complications.
FIELD: medicine, virology, chemical-pharmaceutical industry, pharmacy.
SUBSTANCE: invention relates to a synergetic composition comprising azidothymidine and glycyrrhizic acid penta-O-nicotinate - niglizine taken in physiologically blood concentrations: 0.0037-0.0254 mcM for azidothymidine and 0.0052-9.64 mcM for niglizine. Using the proposed composition provides effective inhibition of HIV-1 reproduction and significant reducing consumptions required for treatment. The composition shows high bioavailability and high effectiveness.
EFFECT: improved and valuable medicinal properties of composition.
6 tbl, 1 dwg
FIELD: medicine, virology, pharmacy.
SUBSTANCE: invention relates to treatment of HIV-infection and AIDS and involves a method for decreasing the content of ceramides in a patient suffering these diseases and a set for its realization. Method involves administration in a patient the medicinal preparation comprising L-carnitine or acyl-L-carnitine wherein a linear or branched acyl group comprises 2-6 carbon atoms, or their pharmacologically acceptable salts in combination with an antiretroviral medicinal preparation taken among the group of nucleoside-like inhibitors of reverse transcriptase, non-nucleoside inhibitors of reverse transcriptase, inhibitors of HIV protease. A set for decreasing the content of the content of ceramides involves an antiretroviral medicinal agent taken among the abovementioned group and enhancing agent taken among the group consisting of L-carnitine or acyl-L-carnitine wherein linear or branched acyl group comprises 2-6 carbon atoms, or their pharmacologically acceptable salts or mixtures. Invention provides enhancing activity of antiretroviral agents and to protect immune system due to decreasing the concentration of ceramides that results to inhibition of HIV expression and cellular apoptosis.
EFFECT: improved and valuable medicinal properties of agent.
20 cl, 2 tbl, 2 ex
< / BR>where R represents isopropyl, neopentyl or cyclohexyl,
containing pharmaceutical compositions
SUBSTANCE: method involves administering Capecitabine during 14 days within 15 days long treatment course in everyday mode and Oxyplatin at a dose of 50 mg/m2 at the first, eighth and fifteenth treatment day. Pause between treatment courses is 2-3 weeks long.
EFFECT: enhanced effectiveness of treatment.