Therapeutic compositions (ii)
The invention relates to the field of medicine and for the application of cyclic ether (R)-3-hydroxybutyrate of formula (1) for the treatment of disease conditions mediated by free radicals, toxic agents, such as peptides and proteins, and genetic defects, harmful to the cell metabolism, insulin resistance or other defects in glucose metabolism or conditions that cause the defect, ischemia, head injury, and/or improve the efficiency of the cells. The compound of formula (1) has a high effectiveness in improving (R)-3-hydroxybutiric acid, maintaining a high level for several hours, the possibility of introducing without counterion and easy to obtain. 9 N. and 7 C.p. f-crystals, 1 tab., 1 Il.
The present invention relates to compositions which are suitable for administration to humans and animals and which are characterized by the ability to increase the level of (R)-3-hydroxybutyrate ((R)-3-hydroxipropionic acid or D--hydroxybutyrate) in their introduction, especially when introduced orally, topically, subcutaneously or parenterally, but the most successful oral.
Introduction: there is now. Among other things this includes increasing the activity of the cardiac muscle, for example, in heart failure, providing an alternative to glucose, an energy source, for example, diabetes and conditions resistance to insulin, and treatment of disorders caused by damage to nerve cells, for example cells of the CNS, in particular by slowing or preventing brain damage, such as observed in Alzheimer's disease and Parkinson's disease and similar diseases and conditions.
It is established that hydroxybutyrate sodium increases cerebral blood flow and local vasomotor reflexes to 40% inclusive (Biull. Eksp. Biol. Med Vol 88 11, pp.555-557).
In the patent EP 0780123 A1 described later using acetoacetate,-hydroxybutyrate, esters with monatomic, diatomic and triatomic alcohols or linear oligomers containing from 2 to 10 units-hydroxybutyrate to suppress swelling of the brain, protecting the brain activity, the correction of the energetic metabolism of the brain and reduce the extent of cerebral infarction.
Intravenous infusion of sodium salts of (R)-3-hydroxybutyrate was performed in healthy individuals and patients with several diseases, for example, the cat is about to reduce the concentration of glucose, free fatty acids and glycerol, but not effective in reducing the oxidation of leucine.
The present invention further established that the compounds and compositions that increase the level of (R)-3-hydroxybutiric acid and/or acetoacetate in blood, are useful for reducing free radicals in vivo and, thus, have implications for the treatment of diseases associated with free radical processes.
(R)-3-Hydroxybutyrate and acetoacetate, commonly called ketone bodies, create a normal physiological alternative to conventional substances, producing energy, glucose and fatty acids. During prolonged fasting in humans, fatty acids are converted in the liver to the (R)-3-hydroxybutiric acid and acetoacetate that can be used by most of the major body tissues except the liver. In such circumstances, the total amount of ketone bodies in the blood increases to approximately 7 mm. When they increase in blood moderately extrahepatic tissues such as the brain, heart and skeletal muscle using these ketone bodies in the mitochondria to provide regenerative energy in the form of NADH, which is the primary substrate of the system t is N mitochondria reduces the ratio of free mitochondrial [NAD+]/[NADH] and the ratio of cytosolic [NADP+]/[NADPH], which relates the ratio of mitochondrial [NAD+]/[NADH]. At the same time, the catabolism of ketone shifts the ratio of mitochondrial [NAD+]/[NADH] in the direction of reduced forms and he shifts the ratio of mitochondrial [ubiquinone]/[originala], [Q]/[QH2] towards the oxidized form. Semiquinonic form original represents the main source of formation of peroxide, O2the mitochondria. By reducing the restored form QH2and its semifinala you can reduce the formation of free radicals by mitochondria, while increasing at the same time traps for free radicals associated with NADP, such as glutathione.
Thus, the authors of the present invention have found that the free radical lesion arising from excess restored Q or inhibition of NADH-dehydrogenase, such as may occur when the toxicity induced RAM, can be reduced by introducing agents that increase the level of ketone bodies in vivo.
Free radical destruction involved in a large number of diseases, including neurological diseases: Parkinson's disease, aminiya lesions implicated as playing a role in coronary reperfusion, diabetic angiopathy, inflammatory bowel disease and pancreatitis.
In a parallel patent application WO 98/41201 described the introduction of a linear esters of (R)-3-hydroxybutiric acid and/or acetoacetate for the production of elevated levels of free compounds in vivo. It was shown that oral administration of 4 mm solution of oligomeric Tetra-(R)-3-hydroxybutyrate or acetokinase ether increases the level of ketone bodies in the blood, resulting in this measured level (R)-3-hydroxybutyrate were increased from 1 to 2 mm during the period exceeding 2 hours.
Currently, the authors of this application have found that the introduction of component (R)-3-hydroxybutiric acid such compositions in the form of cyclic oligomer provides unexpected advantages. These advantages may include, among other things, (a) increased efficiency in raising the level of (R)-3-hydroxybutiric acid, resulting in this level may increase by more than 2 mm, including achievement level, almost corresponding to the hungry and above, (b) maintaining an elevated level for several hours, (C) the possibility of introducing without counterion, such as sodium or methylglucamine, when it is undesirable to increase SOLEV the TBA pure compounds from polymeric starting materials, obtained using bioculture.
This application, in particular, addresses the problem of neurodegenerative diseases, in particular diseases, when neurons are exposed to neurotoxic action of pathogenic agents, such as protein plaques and oxidative damage, and additionally relates to compositions for use in the treatment of these and the above diseases.
In the preferred implementation of the present invention relates to increase of ketones in the blood necessary for the correction of the defects described above and which can be achieved with parenteral or enteral administration. In particular it does not require the introduction of potentially toxic pharmacological agents. Improved efficiency of the present invention in increasing, in particular level in the blood, ketone bodies, provides therapeutic effects of classical ketogenic diet, which is detected by itself is not toxic in children and in adults it causes no side effects compared with those who did not undergo treatment. Moreover, the authors of the present invention found that, in parallel with the correction of the above metabolic and toxic Def the Hanks to improve energy status of neuronal cells and higher trophic stimulation results in increased synthesis of the neurotransmitter, for example acetylcholine.
Proposed in the present invention the treatment is beyond the scope of ketone bodies in the circulation, since the proposed treatment of the cells are unable to function due to neurodegeneration and/or metabolic defects, in particular in the metabolism of glucose, for example, caused by neurotoxic agents such as peptides, proteins, free radical damage or the result of genetic abnormalities. Treatment includes action themselves ketone bodies in the cells, and not on the flow of blood.
Thus, in the first embodiment, the present invention relates to a cyclic ether (R)-3-hydroxybutyrate of formula (I)
where n is an integer of 1 or more,
or its complex with one or more cations or salts thereof for use in therapy or nutrition.
For oral delivery may be preferred free of cyclic oligomer. When the complex cations are present, preferred cations are sodium, potassium, magnesium and calcium, and they are balanced in the proposed salt complex with a physiologically acceptable counterion.
Examples of physiologically acceptable salts of sledaka as methylglucamine salt.
Preferably, n is an integer from 1 to 200, more preferably from 1 to 20, most preferably from 1 to 10 and especially useful is 1, i.e., (R,R,R)-4,8,12-trimethyl-1,5,9-dioxadodecane-2,6,10-Trion, 2, 3, 4 or 5.
Cyclic ethers according to the invention is preferably used for the treatment of painful conditions, mediated by free radicals, toxic agents, such as peptides and proteins, genetic defects, harmful for the metabolism of nerve cells, insulin resistance or other defects in glucose metabolism, or conditions that induce defect, ischemia, head trauma and/or to improve the efficiency of the cells, for example the efficiency of the heart cells, for example, heart failure.
In the second embodiment, the invention relates to methods for treating cells with functional failure due to free radicals, toxic agents, such as peptides and proteins, genetic defects, harmful to the cell metabolism, resistance to insulin, or other defects in glucose metabolism, or conditions that induce defect, ischemia, head trauma, and/or to improve the efficiency of the cells, characterswhen conditions in humans and/or animals.
Select this option to include such use as use as a neural stimulator, for example, can stimulate the growth of axons and/or dendrites and/or nerve cells, for example, in cells of the hippocampus or substance nigra, in vivo or in vitro, in particular, under conditions when the neurodegeneration has serious clinical consequences, by increasing its effect on the level of (R)-3-hydroxybutyrate and acetoacetate in blood and plasma.
In the third embodiment, the invention relates to a method of enteral or parenteral nutrition, preferably, oral feeding method involving the introduction of a cyclic oligomer of formula (I).
In the fourth embodiment, the invention relates to the use of a cyclic ether of the formula I to obtain drugs for the treatment of painful conditions, mediated by free radicals, toxic agents, such as peptides and proteins, genetic defects, harmful to the cell metabolism, insulin resistance or other defects in glucose metabolism, or conditions that induce defect, ischemia, head trauma and/or to improve the efficiency of the cell.
In the fifth embodiment, the invention relates to compositions, characterized Cesky acceptable carrier.
In particular, the composition is suitable for parenteral or enteral administration, in particular for oral administration. When the composition is intended for parenteral use, it is sterile and free from pyrogens. The composition for oral administration may include food based and can be in the form of an emulsion or a simple mixture of solid food.
In particular, cyclic(s) oligomer(s) include(s) an effective amount of the total composition, for example, at least 2% or more, for example at least 5% of the composition by weight, more preferably 20% or more, and most preferably from 50% to 100%. The composition may be intended for oral, parenteral or any other convenient forms of introduction.
In preferred forms of all the variants of the invention the compound of formula (I) is administered in conjunction with acetoacetate or metabolic precursor of acetoacetate in the physiological ratio. The term of its metabolic precursor, in particular, refers to compounds that include acetoacetyl parts, such as acetoacetyl-1,3-butanediol, preferably acetoacetyl-(R)-1,3-butanediol, acetoacetyl-(R)-3-hydroxybutyrate and acetoacetic higher, for example, glycosylase, alcohols.
In patients with diabetes the use of cyclic oligomers allows you to maintain low blood glucose levels without the risk of complications of hypoglycemia. In healthy, non-diabetic subjects, blood sugar with values on an empty stomach from 80 to 90 mg % (4,4-5 mm) increases to 130 mg % (7.2 mm) after eating. In patients with diabetes "close control" diabetes were offered for a long time as a way to delay vascular complications, but in practice, the doctors discovered that it is difficult to maintain blood sugar tightly controlled below 150 mg % (8.3 mm) after eating due to hypoglycemic episodes. In healthy subjects regularly observed hypoglycemic coma in the fall of sugar to 2 mm. As discussed previously (62, 63), in the presence of 5 mm of ketone bodies is not observed neurological symptoms in the fall blood sugar below 1 mm.
In the present invention discovered that getting sick mellitus type II cyclic oligomers of the invention allows to provide better control of blood sugar, thereby preventing vascular changes in the eyes and kidneys, which are currently observed after 20 years of diabetes, and which are the main cause of severe t is kami, such as untreatable epilepsy, because they treat the ketogenic diet therapy is improved by application of cyclic oligomers, by reducing or eliminating high content of lipids, and carbohydrates. Such patients include those who have genetic disorders in the transport system of glucose in the brain, in glycolysis or in the PDG (pyruvaldehyde), such as suffering from the syndrome Lei, endotoxic shock or conditions of severe stress.
Specific disorders that are treated with these medicines, apply to all States, including the blockade of SOEs, including the condition that occurs after a head injury or involving the reduction or exclusion provision of mitochondrial acetyl-COA, such as insulin coma and hypoglycemia, defects in the transport of glucose to the brain or somewhere else (80) or in the stages of the enzymes of glycolysis.
When a drug or food additive includes acetoacetate, it is preferable that they are not stored for a long time or were not exposed to temperatures exceeding 40°C. Acetoacetate unstable when heated and rapidly decomposes at 100°C in acetone and CO2. In such conditions, it is preferable that consequently, that the composition included the ether precursor acetoacetate.
In the sixth embodiment, the invention relates to a method of treating neuronal cells of the human or animal body, such as brain cells, subjected to a disease associated with cell damage, in particular, which leads to cell death that is specified for the second and fourth variants, in particular a neurodegenerative disease, for example, such that belongs to neurotoxic conditions such as the presence of amyloid protein, such as a disease associated with memory or movement, such as Alzheimer's or Parkinson's disease, epileptic seizures, and this method includes the introduction of the subject, at least one of the materials for use in embodiments of the invention from the first to the fifth.
The authors present invention further found that ketone bodies are provided through the introduction of cyclic oligomers of (R)-3-hydroxybutiric acid in quantities sufficient to increase the total concentration of ketone bodies in the blood to high levels, lead to more than simple maintenance of cell viability, but also really improve the functioning and growth of cells over normal, so refers to the application of cyclic oligomers as agents, can cause neuronal stimulation, i.e., activity, similar to nerve growth factor, increase metabolic rate and increase the degree of functional characteristics of a neuron, such as axons and dendrites. This variant of the present invention relates to a mechanism for improving neuronal function, as well as full delay degradation.
In recent work Hoshi with co-workers (77, 78) has convincingly shown that some amyloid protein, A1-42, the accumulation of which is a distinctive feature of Alzheimer's disease, acts as a stimulator of mitochondrial histidine-protein kinase, which phosphorylase and inactivates multienzyme complex pyruvate-dehydrogenase. Complex PDG is a mitochondrial enzyme that provides the formation of acetyl-COA and NADH from pyruvate formed by glycolysis in the cytoplasm. Formed in the mitochondrial acetyl-COA is involved with oxaloacetate in the cycle of TAC (tricarboxylic acid) Krebs, completely incinerating pyruvate to CO2and providing the mitochondria of regenerative energy, which becomes a substrate for the transport of electrons, which is the energy of the disposal, what is happening in the basal ganglia at the level in the blood is below 1 mm (76). However, when the level of 7.5 mm, and it does so in a healthy person as a result of prolonged starvation, the rate of ketone bodies in the brain is sufficient to ensure the basic needs of brain energy and to prevent symptoms of hypoglycemia even when blood sugar levels, which in normal conditions causes seizures or coma (63).
In a parallel application WO 98/41201 "Therapeutic composition" describes the hypothesis of the inventor that in Alzheimer's disease, when there is a block PDG, which prevents the normal production of energy from glucose, if you can provide an increased, for example, a normal fasting level of ketones, you can bypass the blockade PDG available in these patients, and thereby prevent cell death due to the exhaustion of energy or absence of cholinergic stimulation and thereby slow the progression of memory loss and dementia. Moreover, applying growth/stimulating effects of ketone bodies in relation to nerves, especially (R)-3-hydroxybutyrate or physiological correlation with acetoacetate, you can call the improvement is still living cells outside the state, to which the animals and humans after eating the contents of acetoacetate in the liver, which essentially corresponds to the content in the blood is very low, for example, 0.09 mm, and the content of (R)-3-hydroxybutyrate is 0,123 mm, but it increases after 48 hours of fasting to, for example, 0.65 mm of acetoacetate and 1.8 mm (R)-3-hydroxybutyrate (84).
Ketone bodies are growing at starvation because of the reduction of insulin reduction reesterification fatty acids to triglycerides in adipose tissue, which induces the release of free fatty acids into the bloodstream. Selected fatty acids can then be captured and used as an energy source by muscle, heart, kidneys and liver in the process-oxidation. The liver, however, has the ability to convert free fatty acids into metabolic energy source ketones for use by organs other than the liver, including the brain, as an alternative to glucose during periods of starvation. The synthesis of ketone bodies in the liver is carried out from mitochondrial acetyl-COA generated in the process-oxidation of fatty acids in the liver.
Ketone bodies come in tissues other than the liver, using the same carrier, and other monocarboxylate can act in the kgce to act as competitive inhibitors of the transport of ketone bodies. Because the transport of ketone bodies through the blood-brain barrier is a limiting factor in the utilization of ketone bodies in the brain (76), you should make every effort to preserve the non-physiological concentrations of these enantiomers in the blood at low levels in the process of ketogenic therapy. When the concentration of ketone bodies in the blood rises to a level that can be detected by starvation, heart, muscles, kidneys and the brain uses ketone bodies as the preferred energy substrate.
In the present invention, therefore, determined that mitochondrial acetyl-COA arising out of ketone bodies, formed from cyclic oligomers, referred to in the present invention can, therefore, compensate for the deficiency of acetyl-COA, which occurs when inhibition mnogovershinnoe complex PDG in tissues that are dependent on glucose metabolism, to ensure their metabolic energy. Formed in mitochondria citrate may also be transported into the cytoplasm Transporter three - or dicarboxylic acids, where it can be turned into cytoplasmic acetyl-COA, essential for the synthesis of acetylcholine. Reactions of the Krebs cycle is shown in scheme 1 for additional the acetyl-COA in the liver. Because acetyl-COA is an important precursor fatty acids, it cannot occur in the liver with increased synthesis or fatty acids, or cholesterol, which usually provides more than half of synthesis in the body these two potentially pathogenic materials. The liver is sensitive to the given value of acetoacetate/(R)-3-hydroxybutyrate and must respond to the change in the ratio of free [NAD+]/[NADH] in mitochondria due to the almost equilibrium set-hydroxysteroiddehydrogenase (EC 18.104.22.168) (31).
Among other things, the above also indicates that it is possible to suggest a method of increasing the efficiency of the energy generation in the mitochondria in humans or animals that do not suffer from acute or chronic metabolic disease, which includes the introduction of a person or an animal such amount of cyclic oligomer of formula (I), which is sufficient to increase the level of (R)-3-hydroxybutyrate blood up to a level of from 0.5 to 20 mm.
The easiest way to increase of ketones in the blood is starvation. During prolonged starvation, the level of ketones in the blood reaches 7.5 mm (62, 63). However, this feature is not the same composed mainly of lipids, was used from 1921 to treat epilepsy in children, especially myoclonic and geneticheskih seizures (109), and was shown its effectiveness in cases of resistance to conventional pharmacological agents (71). Oral or parenteral administration of free fatty acids or triglycerides can increase blood ketones, thereby providing a low level of carbohydrates and insulin to prevent reesterification in adipose tissue. In rats fed diets containing 70% corn oil, 20% of casein hydrolysate, 5% cellulose, 5% salt mixture MacCallum, ketone bodies in the blood reached a level of approximately 2 mm. Replacement of pork fat in corn oil increases blood ketones to approximately 5 mm (Veech, unpublished).
Usually the level of ketone bodies produced in such diets is approximately 2 mm (R)-3-hydroxybutyrate and 1 mm acetoacetate, while the level of free fatty acids is approximately 1 mm. Have exhausted other options composition, including triglycerides with an average length of the chains. In General, the reaction to such a limited diet was poor because of their neopytnosti (56). Diets with a high content of lipids to reduce the availability of glucose for tumors (88), as well as diets that reduce weight in patients suffering or not suffering from diabetes (74, 112), and to improve resistance training (83).
Numerous are restrictions diets, which are lipids, to improve blood ketones to neurological effective level. First, the level of ketone bodies when using based on lipid diets tend to be lower than 3 mm, which is considerably below the level of 7.5 mm, achieved in obese men during prolonged starvation. Secondly, unnecessary feeding of carbohydrates increases insulin secretion and causes a rapid decrease in conversion in the liver, free fatty acids into ketones and subsequent fall of the level of ketones in the blood and the direction of the lipids in the esterification to triglycerides in adipose tissue. Many anecdotal messages are related to the restoration of convulsions in children, "diet which was destroyed birthday cake". Thirdly, low taste and the need to avoid carbohydrates to maintain a high level of ketone bodies makes such a diet with a high content of lipids difficult for use in adults and in patients outside the hospital setting, especially in a society where traditionally consume a lot of refined the sick, in addition to children beyond the age when all the food is prepared at home under strict supervision. Fourthly, feeding such large quantities of lipids of the adult population would lead to a significant hypertriglyceridemia and hypercholesterolemia with pathological complications such as increased destruction of blood vessels and sporadic diseases of the liver and pancreas and, therefore, could not be prescribed from a medical point of view. Feeding diets with a high content of lipids and low carb diet was popular in the 1970-ies for weight loss in spite of the consumption of energy-dense foods offered to carbohydrate intake was low. However, because of the growing understanding of the relationship of elevated blood lipids and atherosclerosis popularity of this diet has fallen sharply.
Substitution of glucose into lipid diet, when glucose was provided 47% of the content of calories, racemic 1,3-butanediol caused approximately 10-fold increase in the concentration of ketones in the blood to 0.98 mm (R)-3-hydroxybutyrate and 0.33 mm acetoacetate (107). These values were slightly lower usually after 48-hour fast and were significantly lower than the 7.5 mm, obtained from a starving man. Rat/img>- natural and D--hydroxybutyrate (respectively, (S)-3-hydroxybutanoic and (R)-3-hydroxybutyrate). Although racemic 1,3-butanediol intensively investigated as a cheap source of calories for animal feed and, even though it was experimentally applied in diets for humans (81, 101), the formation of non-natural L-isomer, seems to be leading, eventually, to the induction of significant toxicity, as it was shown for human consumption unnatural D-lactate (64). One of the weaknesses of the introduction of non-natural L-isomer is that it competes with the natural (R)-3-hydroxybutyrate for transport. Thus, the feeding of (R)-1,3-butanediol as a precursor of ketone bodies is one possibility, in which there is no need of introducing or get unnatural isomer.
Mono - and diacetoacetate esters of racemic 1,3-butanediol are, as expected, a source of calories and tested on pigs (67). Oral administration briketirovannogo diet containing 30% of calories in the form of esters, caused a transient increase in blood ketones to 5 mm, however, the use of racemic 1,3-butanediol with the formation of his unnatural (S)-3-hydroxyacetate not recommended ethers (R) 1,3-butanediol can be used either as such or in the form of ether with acetoacetate. Studies in rats have shown that the feeding of racemic 1,3-butanediol causes a decrease in the ratio of [ABOVE']/[NADH] in the cytosol of the liver from 1,500 to approximately 1000 (87). For comparison: the introduction of ethanol reduces [NAD]/[NADH] in the liver to approximately 200 (106).
Fresh acetoacetate can be applied in solutions for infusion, in which it can be entered in a physiologically normal relations with (R)-3-hydroxybutyrate for optimal activity (95). Due to production requirements, which currently requires a longer shelf life and temperature-sterilized liquid, acetoacetate often given in the form of ether. This was done to increase the shelf life and stability to heat in the sterilization process. It was found that esterna activity in the blood line is approximately 0.1 mmol/min/ml, and in the liver approximately 15 mmol/min/g (68). In addition to esters, combining 1,3-butanediol and acetoacetate, was also conducted an intensive study glycerolipid esters of acetoacetate parenteral (59) and enteral feeding (82). It was reported that such prepare used for treatment of burns (85).
For the preferred implementation of the present invention under optimum conditions, there must be a physiological ratio of ketones with the introduction of cyclic oligomers and acetoacetate. If this does not happen, the whole liver of the animal must set the value of ketones in accordance with its ratio free [NAD+]/[NADH] in mitochondria. If given divergent from the norm the ratio of ketones, the liver must change this value while changing the ratio [ON+]/[NADH] in the liver. Perfusion of the working heart acetoacetate as the sole substrate quickly causes heart failure (99) in contrast to the hearts of rats, perfuziruemah a mixture of glucose, acetoacetate and (R)-3-hydroxybutyrate, when cardiac efficiency was increased physiological ratio of ketone bodies (95).
Cyclic oligomers for use in the present invention it is convenient to synthesize using microorganisms, producing the polyesters. Natural polyesters (R)-3-hydroxybutyrate are solid as paragraphs trade agreements, such as polymers with M. C. 530000 from Alcaligenes eutrophus (Sigma Chemical Co. St. Louis) or as polymers with M. C. 250000 sugar is the size of bacteria was developed in the 1970-ies ICI in the UK and Solvay et Cie in Belgium in the form of potentially biodegradable plastic to cover tampons and other purposes. System for synthesis of poly (R)-3-hydroxybutyrate, to date, cloned, and to provide bacteria substrates obtained variations in the composition of the polymer. The genes responsible for the synthesis of polyalkanoates, were cloned and expressed in number of microorganisms (93, 102, 113), which gives the opportunity to receive this material in many organisms with highly variable conditions.
The preferred forms of cyclic oligomeric (R)-3-hydroxybutyrate are such that at least partly is easily digested and/or metabolized in humans or animals. Preferred oligomers containing from 2 to 200 repetitions, usually from 2 to 20, and most suitable oligomers ranging in length from 3 to 10 repetitions, in particular from 3 repetitions, i.e. trialed. It should be understood that it is possible with advantage to use mixtures of such oligomers, because there can be obtained the ranking of the characteristics of their capture. In a similar way can be proposed mixed with the monomer or linear oligomers or polymers for modification of the emerging profile level in the blood.
Cyclic oligomers for use in the invention can be obtained, inter alia, by using the methods described by Seebach et al. what the Windows, consisting of 5 to 7 or more units (R)-3-hydroxybutyrate may be preferred because they may break easier in vivo. Described here are methods of synthesis of compounds incorporated herein by reference.
Because the liver is unable to metabolize ketone bodies, and can only change the ratio of (R)-3-hydroxybutyrate/acetoacetate trapped in the blood stream monomers ketone bodies are transported to extrahepatic tissues, where they can be recycled. The achievable level of ketones is not changed by unauthorized taking of carbohydrates, as is the case with existing ketogenic diet. On the contrary, ketones should just be a Supplement to the normal diet, given in sufficient quantity to long maintain their level in the blood, usually from 0.3 to 20 mm, more preferably from 2 to 7.5 mm, over a 24 hour period, depending on the condition being treated. Now suppose that in the case of resistant epilepsy in children is sufficient blood levels of 2 mm. In the case of Alzheimer's disease could even be an attempt to maintain the level of 7.5 mm or higher, achieved in studies to hungry people, for the ware is of arocena activity PDG due to an excess of A1-42amyloid peptide (77, 78).
Discovered by the authors of this invention is the fact that (R)-3-hydroxybutyrate and its mixture with acetoacetate acts as a stimulant of the nervous system, such as a growth stimulant to the nerves and/or stimulator of the growth of axons and dendrites, opens the possibility to increase the level of ketone bodies to a lesser extent than is required when the flow of food, for the treatment of neurodegenerative diseases.
The composition according to the invention are mostly sterile and free from pyrogens, especially from endotoxins.
Secondly, they are preferably in such a way that they could be pleasant to the taste, when applied as a Supplement to your normal diet to increase the compliance of patients in relation to the admission of such additives. Cyclic oligomers usually are odorless. The compositions of cyclic oligomers of (R)-3-hydroxybutyrate and its mixtures with acetoacetate can be covered with masking agents or can be sent to the intestines by using coating them sucking in the gut or encapsulate them in a different way, as is well known in the pharmaceutical and food industry.
As ketone bodies contain approximately the avoidance of obesity.
Special advantages of the use of cyclic oligomers, referred to in the present invention are as follows:
1) they can have with a normal load carbohydrates without reducing the level of ketone bodies in the blood, reduction of which should weaken the effect of treatment,
2) they will not increase VLDL and cholesterol in the blood, which is observed in modern diets containing margarine and cream, thus reducing the risk of accelerated occurrence of vascular diseases, fatty degeneration of the liver and pancreatitis
3) they must have wide scope with a large number of different diseases, including, but not limited to: diabetes type II to prevent episodes of hypoglycemia and coma, in Alzheimer's disease and other neurodegenerative diseases to prevent the death of nerve cells, for example cells of the hippocampus, and untreatable epilepsy, due either to a reduction of glucose transporters in the brain, defects of glycolysis, or the so-called syndromes Leia with congenital defects PDG.
Cyclic oligomers of the invention can be used for oral and parenteral use in emulsions because acetoacetate in naturebureau is she half-life at room temperature of approximately 30 days. In the case where the composition of the invention should include acetoacetate, it can be in the form of a precursor. Convenient to acetoacetate could be offered in the form of esters with (R)-3-hydroxybutyrate, as proposed in the parallel application "therapeutic composition".
Treatment may include giving patients a substantial portion of incoming calories in the form of a cyclic oligomer of (R)-3-hydroxybutyrate or oligomers prepared to obtain a delayed release so as to maintain ketones blood at a higher level, oscillating, for example, in the range from 0.5 to 20 mm, preferably in the range of 2-7,5 mm, within a 24-hour period. Secretion of ketone bodies in the blood can be limited by applying various techniques such as microencapsulation, absorption and the like, which are used in modern practice oral introduction of a large number of pharmaceutical agents. Forms covered for resorption in the intestine, for directed delivery lower stomach can in particular be used when the material does not require hydrolysis or not subject to it in an acidic environment. When this hydrolysis is desired, can be applied form without coating. Some forms of the crobial Polyesters.
Preferred cyclic oligomers, such as trialid, can simply be added as such to food and/or may be supplemented therapeutic diet other sources of formation of ketone bodies with a different profile selection, such as Monomeric (R)-3-hydroxybutyrate. The latter can be represented in the form of an aqueous solution, for example in the form of salts, for example salts of sodium, potassium, magnesium or calcium.
To provide a diet of 1500 calories adult sick person could consume 198 g of cyclic esters (the present invention) in a day. For a diet to 2000 calories in the same proportions he could consume 264 g of ketones in the day. When the consumption of the ketogenic diet ketones in the blood increase to approximately 2 mm, which has been proven somewhat effective for at least 60% of treated children. When consumed ketone diet the level of ketones should be higher because ketones replace fat by caloric equivalent, that is, 1.5 g of ketone/g fat. Accordingly, blood ketones should be approximately 3 mm, which is an effective level of children, but still below the level of 7.5 mm, achieved from a starving man.
There are several advantages is leegomery, which themselves increase the level of ketone bodies in the blood. First, the provision of themselves ketone bodies does not require restrictions on carbohydrates, thereby improving the taste quality of the dietary compositions, especially in communities where diets high in carbohydrates are normal. Secondly, ketone bodies can be metabolized by muscle, heart and brain tissue, but not the liver. Therefore, avoids fatty degeneration of the liver, which may be adverse side effects of the ketogenic diet. Thirdly, the inclusion of carbohydrates in diet formulations increases the chances of acceptance and reveals practical therapeutic approaches to patients with type II diabetes, in which insulin is high, making known the ketogenic diet is not applicable.
In the present invention found that, although it may be desirable any increase in the level of ketone bodies, the preferred amount of cyclic ether with any acetoacetyl component should be sufficient to increase the level of ketone bodies in the blood to a value of from 0.5 to 20 mm, preferably to a value of from 2 mm to 7.5 mm and above, especially when trying to suspend the death of brain cells, prny, should be expected to suspend further damage and at least some recovery of function.
The total number of ketone bodies used for the treatment of neurodegenerative diseases such as Alzheimer's and parkinsonism should preferably increase the level of ketone bodies in the blood on the value of from 0.5 mm to 20 mm. In the present invention found that to achieve this could take anywhere from 200 to 300 grams (0.5 pounds) equivalent of ketone bodies on patient per day. When the treatment is intended to protect cells from the effects of the neurotoxin, this can be achieved at a level sufficient to act as a significant source of calories, for example from 2 to 7.5 mm in the blood. When it comes to action so produced (R)-3-hydroxybutyrate as a factor stimulating the nerve cells, the amount of preparation can be lower, for example, to ensure an increase in the amount of from 0.2 to 4 mm, but may of course be great if one disease or another.
It should be understood that the treatment of neurodegenerative diseases such as Alzheimer's disease or parkinsonism, should be most effective when assigning soon after identifying pedroluchini of a positive test result on one or more States, selected from group (i) mutation of the gene of the protein precursor of amyloid in chromosome 21, (ii) mutations of the gene presenilin in chromosome 14, (iii) the presence of isoforms of apolipoprotein E. of Course, can be applied and other tests, which shows that they are indicators of Alzheimer's disease.
After such a positive test result would be rational to prevent the development of memory loss and/or other neurological dysfunction by increasing the total concentration of ketone bodies, (R)-3-hydroxybutyrate and/or acetoacetate, in the blood or plasma of the patient, for example, between 1.5 and 10 mm, more preferably from 2 to 8 mm, using one of the following methods. Preferably, the patient is given a diet containing a sufficient amount of the compounds of formula (I), optional parenterally, but preferably and conveniently, enterline.
It should be understood that the hypoglycemic dysfunction of the brain should also be treated using treatment options, compositions and compounds of the present invention. The next distinctive feature associated with this treatment, should be the improvement of the overall health of the muscles.
The supply of food and medicines on the basis of cyclic oligomer is materials, of which can be obtained cyclic (R)-3-hydroxipropionic acid (see Microbial Polyesters Yoshiharu Doi. ISBN 0-89573-746-9, Chapter 1.1, 3.2 and 8). The presence of genes suitable for insertion in the organisms that produce food products, provides the basis for creating products such as yogurt and cheese, which enriched cyclic oligomeric (R)-3-hydroxybutiric acid, or by Monomeric substance after the destruction of enzymes able to degrade these polymers (see Doi. Chapter 8).
Methods of obtaining poly-(R)-3-hydroxybutyrate is not specifically stated, as they are known in science. For example, Shang et al, (1994) Appli. Environ. Environ. 60: 1198-1205. This polymer is commercially available from the company Fluka Chemical Co. P1082, cat# 81329, 1993-94, 980. Second St. Ronkonkoma NY 11779-7238, 8003585287.
The present invention is hereinafter described only to illustrate by reference to the following figure and experimental examples. In their world for professionals will be clear later implementation in the scope of the invention.
The drawing is a graph showing the level of (R)-3-hydroxybutiric acid in the blood depending on the time after feeding rats trialed (R)-3-hydroxybutiric acid, cyclic oligomer obtained in example 1, the composition of the yoghurt and the control of the LM is,6,10 triona: trialid (R)-3-hydroxybutiric acid
The synthesis was described in Angew, Chem. Int. Ed. Engl. (1992), 31, 434. A mixture of poly[(R)-3-hydroxybutiric acid] (50 g) and monohydrate toluene-4-sulfonic acids (21,5 g, 0,113 mol) in toluene (840 ml) and 1,2-dichloroethane (210 ml) was stirred and heated under reflux for 20 hours. The water was removed with traps Dean-stark for 15 hours, after which the brown solution was cooled to room temperature, washed first Polynesians solution of sodium carbonate, and then with a saturated solution of sodium chloride, dried over magnesium sulfate and the solvent was removed in vacuum. Brown semi-solid residue was distilled using apparatus Kugeler (Kugelrohr) at 120-130°C / 0.15 mm RT.article (20 n/m2) to obtain white solids (18,1 g). At temperatures above 130°With waxy solid begins to athanase - at this point the distillation was stopped. So pl. distilled material was 100-102°C (literature data so pl. 110-110,5°C). Recrystallization from hexane gave colorless crystals with a yield of 15.3, So pl.=107-108°C; D-35,1 (=1,005, l3), (published data = -33,9).1H NMR (300 MHz, CDCl3):=20,86 (CH3), 42,21 (CH2), 68,92 (CH), 170, 12 (CO). Elemental analysis: calculated for C12H18O6: 55,81; N 7,02; found: 55,67; N 7,15.
Comparative example 1: Obtain oligomers of (R)-3-hydroxybutiric acid (R)-3-hydroxybutyrate
(R)-3-hydroxybutiric acid (Fluka - 5,0 g: 0,048 mol), p-toluensulfonate (0.025 g) and benzene (100 ml) was stirred at reflux device with trap Dean-stark within 24 hours. The reaction mixture was cooled and the benzene was evaporated in vacuo (0.5 mm RT.article (66,66 n/m2) ). There was obtained 4.4 g of colorless oil, 20 mg of a sample which was converted into methyl esters for analysis of the number of Monomeric repeats using NMR. This study shows that the product is a mixture of oligomers of D--hydroxybutyrate with a moderate number of repetitions of 3.75, predominantly a mixture of make-up artists, tetramers and pentameron, and only presents the most material is a tetramer. The mixture of products was soluble in 1 equivalent of sodium hydroxide.
Comparative example 2: obtain the acetoacetic ester oligomeric (R)-3-hydroxybutiric acid
The other party colorless oil Ether nitrogen. Additionally added diketene (3.8 g) and the reaction mixture was heated for another hour, then cooled, diluted with ether, washed with water and was extracted with saturated sodium bicarbonate (5×100 ml). The combined extract was washed with ether and then acidified with concentrated Hcl (added dropwise). After extraction with ethyl acetate (3×50 ml) were dried over magnesium sulfate and evaporation in vacuo. There was obtained a yellow mixture of solid and oil substances (7.6 g), which was chromatographically on a column of silica gel using dichloromethane/methanol (98:2) to obtain a light amber oil product. Were selected impurities with greater mobility (1.6 g), and after re-chromatography with carbon tetrachloride/methanol (99:1) was obtained 0.8 g of oil, which, as was shown by NMR and massspectrometry is desired mixture acetoacetanilide oligomers of (R)-3-hydroxybutyrate. The mixture of products was Rf0.44 in the system of dichloromethane/methanol (90:1) and was soluble in 1 equivalent of sodium hydroxide.
Both products of comparative examples 1 and 2 are amenable to separation into individual components by means of preparative HPLC.
Oral administration to rats t is s in blood were measured as follows. The day before the start of the experiment 12 Wistar rats weighing 316±10 g were placed in separate cells. They were not given access to food for 15 hours before granting containing trialed songs, but water was given without restrictions.
On the morning of the day of the experiment were mixed 0.64 g trialed with 5 g of yogurt Black Cherry labeled Co-op in a separate feeders for 9 rats. The remaining 3 rats received 5 g of yoghurt without trialed as a control. Containing yogurt feeders were placed in cages and recorded the time of eating his rats. Two of the three control rats ate all the yogurt, and four of the six rats that received yogurt with triolith, ate about half of them suggested. The remaining six rats slept.
Control rats (n=2) were scored after 60 and 180 minutes after ingestion of yogurt, and rats fed trialid, scored over 80, 140, 150, and 155 minutes. He took blood samples for determination of (R)-3-hydroxybutyrate. The brain was frozen using a socket and later were extracted with perchloric acid extracts neutralize and analyzed. Level (R)-3-hydroxybutyrate in the blood was measured using NAD-/EDTA analysis Anal. Biochem (1983) 131, R-482. To each cuvette was added 1.0 ml of a solution prepared from 2-amino-2-methyl-1-propanol (10
Two control rats ate 5,2±0,1 g of yoghurt and the concentration of (R)-3-hydroxybutyrate in plasma was approximately 0.45 mm after 60 and 180 minutes. Four ate trialed rats consumed to 0.39±0.03 g trialed and 2.6 ± 0.2 g of yogurt. The concentration of (R)-3-hydroxybutyrate in plasma was 0.8 mm through 80 minutes and 1.1 mm in the group, crammed in about 150 minutes. All rats were observed harmful effects of consumption trialed. Thus, there has been an increase of (R)-3-hydroxybutyrate in serum 0.65 mm after eating only 0.4 g trialed. It should be noted that, because the rats were hungry, the initial level of (R)-3-hydroxybutyrate were increased after feeding with 0.1 mm to about 0.45 mm.
Thus, we tested rats shows an increase of (R)-3-hydroxybutyrate in plasma for at least 3 hours with no ill effects. It should be noted that the other two rats, potrebica approximately 1.5 g trialed each comprising cookies ‘Hob-Nob’it has no known harmful effects after two weeks.
It should be noted that elevated levels of (R)-3-hydroxybutyrate should be reflected at the level of acetoacetate, which is not measured as in vivo produced shall be between 40 and 100% of (R)-3-hydroxybutyrate.
Comparative example 3: oral rat (R)-3-hydroxybutyrate, oligomers of (R)-3-hydroxybutyrate and acetoacetic ester oligomers of (R)-3-hydroxybutyrate
The ability of oral input (R)-3-hydroxybutyrate and linear oligomers of comparative examples 1 and 2 to increase the level of ketone bodies blood was examined as follows. Rats were starved overnight and then they were fed 100 μl/100 g body weight 4 M (R)-3-hydroxybutyrate with a pH brought to 7.4 with methylglucamine. Measured plasma level (R)-3-hydroxybutyrate after 30 minutes amounted to 0.62 mm, whereas the use of 9 M (R)-3-hydroxybutyrate he was equal to 3 mm.
This procedure was repeated with 2 M solutions of mixtures of oligomers of (R)-3-hydroxybutyrate and acetoacetic esters, described in comparative examples 1 and 2. The pH value of the oligomer (R)-3-hydroxybutyrate (19/1) and acetoacetic ester (20/4) brought to 7.6 using methylglucamine and the level of (R)-3-hydroxybutyrate blood was tracked using the above test procedures. Been shown to increase (R)-3-hydroxybutyrate blood from 0.2 to 0.5 mm at 60 and 120 minutes after feeding.
An example of the ketogenic diet for 1500 calories (6280,2 j) using the cyclic oligomer is cal/g carbohydrate (37,68 kJ/g) and 4 kcal/g of protein (x 16.75 kJ/g). The oligomers were replaced to obtain an equal number of calories (see table).
Example 6: Effect of (R)-3-hydroxybutyrate on the cells of the hippocampus
Cultural environment and chemicals
Used 0 to day 4 serum-free medium containing Neuropathology environment by adding B47, diluted 50-fold (Life Technology, Gaithersburg, MD), to which was added 0.5 mm L-glutamine, 25 μm Na L-glutamate, 100 U/ml penicillin and 100 μg/ml streptomycin. After 4 days using DMEM/F12 (modified by Dulbecco Wednesday needle) containing 5 μm insulin, 30 nm l-thyroxine, 20 nm progesterone, 30 nm Selenite Na, 100 U/ml penicillin and 100 μg/ml streptomycin.
Microstructure culture of hippocampus
Primary cultures of hippocampus were obtained from embryos of Wistar rats on day 18 of development and dispersively a slight shaking in the pipette. The suspension was centrifuged at 1500 × g for 10 min and supernatant was discarded. Sediment resuspendable in the new environment at the final cell concentration of 0.4-0.5×106cells/ml Ten ml of this suspension was applied in the center covered with poly-D-lysine culture wells and cups were incubated at 38°C for 4 h, then was added 400 μl of fresh neuropathology environment. Later the medium was changed to DMEM/F12, containing 5 μm insulin, 30 nm 1-thyroxine, 20 nm progesterone, 30 nm Selenite Na, 100 U/ml penicillin and 100 μg/ml streptomycin. The wells were divided into 4 groups: half of the wells was added (R)-3-hydroxybutyrate to a final concentration of 8 mm, and the other half of the wells was added 5 nm amyloid1-42(Sigma). The medium was changed after 2 days (day 8) and the cells were fixed on day 10 and were stained with anti-MIR (Boehringer Manheim, Indianapolis IN) to visualize neurons and vimentin and using GFAP (Boehringer) for visualization of glial cells.
Adding (R)-3-hydroxybutyrate in the incubation system has led to an increase in the number of neuronal cells in microstroke on average from 30 to 70 cells on microstrobos. Adding amyloid1-42to the cultures reduced the number of cells with 70 to 30 cells on microstrobos, which is confirmed by observations Hoshi et al., that the amyloid1-42is toxic to neurons in the hippocampus. Adding (R)-3-hydroxybutyrate to cultures containing amyloid1-42that increased the number of cells on average from 30 to 70 cells on microstrobos. Nanowrimo nutrients which are glucose, pyruvate and L-glutamine, slows the rate of cell death in culture. Is further that (R)-3-hydroxybutyrate can reduce the increased rate of cell death in the hippocampus induced by adding to the culture of amyloid1-42.
In the presence of (R)-3-hydroxybutyrate was observed as an increased number of dendritic processes, and the length of the axons, regardless of the presence1-42. This indicates characteristics similar to those of nerve growth factor.
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1. The use of cyclic ether (R)-3-hydroxybutyrate of formula (I)
or its complex with one or more cations or salts thereof for the treatment of disease conditions mediated by free radicals, toxic agents, such as peptides and proteins, and genetic defects, harmful to the cell metabolism, insulin resistance or other defects in glucose metabolism or conditions that cause the defect, ischemia, head injury, and/or to improve the efficiency of the Sabbath.' potassium, magnesium and calcium, or where the connection is free algorithm, not bound in the complex.
3. Treatment of cells with impaired function due to the action of free radicals, toxic agents, such as peptides and proteins, and genetic defects, harmful to the cell metabolism, insulin resistance or other defects in glucose metabolism, or conditions that cause the defect, ischemia, head injury, and/or to improve the efficiency of the cells, characterized in that it includes the introduction of a cyclic ether (R)-3-hydroxybutyrate of formula (I).
4. The method according to p. 3, wherein the cyclic ether (R)-3-hydroxybutyrate of formula (I) acts as a neural stimulator, for example, can stimulate the growth of axons and/or dendrites of nerve cells, for example cells of the hippocampus or substance nigra, in vivo or in vitro, particularly in the States, when the neurodegeneration has serious clinical consequences.
5. The method of implementation of enteral or parenteral nutrition, preferably oral feeding method involving the introduction of a cyclic ether (R)-3-hydroxybutyrate of formula (I) into a physiologically acceptable form optional physiologists in the now, comprising oral administration of a cyclic ether (R)-3-hydroxybutyrate of formula (I).
7. The method according to p. 6, in which a person or animal eats the diet, the fat content of which is less than 50 wt.% from its caloric content.
8. The method according to p. 6, in which a person or animal eats the diet, the fat content of which ranges from 0 to 25 wt.% from its caloric content.
9. The method according to p. 5 or 6 used in relation to a patient in need of treatment one or more of the following diseases: Alzheimer's disease, parkinsonism, amyotrophic lateral sclerosis, epilepsy, a disease mediated by free radicals, heart failure, diabetes type II, deficiency or blockade of pyruvaldehyde, the inability to implement glycolysis in cells of one or more types of muscular dystrophy of Duchenne.
10. The way to ensure caloric substitution of carbohydrates for lowering the level of glucose in the blood, including the introduction of a composition containing a cyclic ether (R)-3-hydroxybutyrate of formula (I), a person or an animal in need of such substitution.
11. The way to ensure caloric substitution of carbohydrates for reducing the content level of the formula (I), a person or an animal in need of such reduction.
12. The way to increase the efficiency of the energy generation in the mitochondria of a human or an animal not suffering from acute or chronic metabolic disease involving the introduction of a human or animal cyclic ether (R)-3-hydroxybutyrate of formula (I) in a quantity sufficient to increase the level of (R)-3-hydroxybutyrate in the blood to a value of from 0.5 to 20 mm.
13. The method according to p. 12, in which the level is increased to a value of from 1 to 10 mm.
14. The use of cyclic ether (R)-3-hydroxybutyrate of formula (I) in obtaining medications for the treatment of disease conditions mediated by free radicals, toxic agents, such as peptides and proteins, genetic defects, harmful to the cell metabolism, insulin resistance or other defects in glucose metabolism, or conditions that cause the defect, ischemia, head injury, or to improve the efficiency of the cell.
15. Composition for treating medical conditions mediated by free radicals, toxic agents, such as peptides and proteins, and genetic defects, harmful to the cell metabolism, insulin resistance or other defects of the exchange rate is the notes of the cells, characterized in that it contains a cyclic ether (R)-3-hydroxybutyrate of formula (I) into a physiologically acceptable form.
16. The composition according to p. 15, characterized in that it contains a physiologically acceptable medium.
where: A means-OR1-C(O)N(R1R2or-N(R1R21; each X, Y and Z independently represents N or C(R19); each U represents N or C(R5), provided that U is N only when X represents N, and Z and Y denote CR19; each W represents N or CH; V denotes: (1) N(R4); (2) C(R4)H; or (3) the groupdirectly related to the group -(C(R14R20)n-A,denotes a 5-6-membered N-heterocyclyl, optionally containing 6-membered ring additional heteroatom selected from oxygen, sulfur and NR6where R6denotes hydrogen, optionally substituted phenyl, 6-membered heterocyclyl containing 1-2 nitrogen atom, optionally substituted 5-membered heterocyclyl containing 1-2 nitrogen atom, aminosulfonyl, monoalkylammonium, dialkylaminoalkyl,1-6alkoxycarbonyl, acetyl, etc