Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C59/00—Compounds having carboxyl groups bound to acyclic carbon atoms and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
  • C07C59/40—Unsaturated compounds
  • C07C59/42—Unsaturated compounds containing hydroxy or O-metal groups
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
  • A61K31/201—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having one or two double bonds, e.g. oleic, linoleic acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/04—Anorexiants; Antiobesity agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/06—Antihyperlipidemics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/12—Antihypertensives
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
  • C07C51/42—Separation; Purification; Stabilisation; Use of additives
  • C07C51/487—Separation; Purification; Stabilisation; Use of additives by treatment giving rise to chemical modification
  • C07C51/493—Separation; Purification; Stabilisation; Use of additives by treatment giving rise to chemical modification whereby carboxylic acid esters are formed
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
  • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
  • C07B2200/07—Optical isomers

Definitions

• the present invention refers to a method of synthesis of racemic products and the separation of their optical isomers [-] (which corresponds to the S enantiomer) and [+] (which corresponds to the R enantiomer) of 2-hydroxy derivative compounds from fatty acids, to the isolated enantiomers themselves, to pharmaceutical compositions that comprise them, and to their use as medicines in the treatment of diseases whose common etiology is based on alterations (of any origin) of cell membrane lipids such as, for example : alterations in the level, in the composition or in the structure of said lipids, as well as in the treatment of diseases in which the regulation of the composition and membrane lipid structure induces the reversal of the pathological state.
• the therapeutic effect is preferably achieved through the regulation (activation or inhibition) of the activity of the enzyme ceramide: phosphocholine choline phosphotransferase (also known as sphingomyelin synthase or EC 2.7.8.27, IUBMB Enzyme Nomenclature), or the level of its product, sphingomyelin (SM).
• ceramide phosphocholine choline phosphotransferase
• SM sphingomyelin
• Both PFAG and DFIFR can therefore be used for the detection of the therapeutic efficacy of drugs for the treatment of diseases mediated by the activity of sphingomyelin synthase or SM itself.
• the present invention also refers to the development of a kit that is based on the detection of said diseases.
• both the enzyme EC 2.7.8.27, and sphingomyelin (SM) can be used as molecular markers of the effect of the compounds of the present invention on the diseases described above. Therefore, the present invention also encompasses an in vitro method for the diagnosis / prognosis of said diseases based on the evaluation of the regulation of the activity or level of said EC enzyme 2.7.8.27, and / or the level of MS, PFAG or DFIFR; as well as kits that include means specially designed to carry out said diagnoses / forecasts. These methods and kits would be based on the determination of the changes induced by treatments with the molecules mentioned in the present invention or in the possibility of changing the molecular entities indicated above with said treatments.
• the enzyme EC 2.7.8.27 and / or SM can be used as therapeutic targets to which to direct molecules capable of reversing their altered state and, consequently, treating those pathological processes that had developed, or were to be developed in a future, as a result of the abnormal activity of the enzyme EC 2.7.8.27 or the inappropriate level of SM.
• both the enzyme EC 2.7.8.27, as well as the SM itself, can be the basis for the design of screening procedures for candidate compounds in order to achieve molecules that, such as enantiomers [-] (also called S) and [+] (corresponding to the R form) of 2-hydroxy derivative fatty acid compounds of the invention, have the ability to regulate the activity of the enzyme EC 2.7.8.27 and / or the level of SM, exerting a therapeutic effect.
• the present invention due to its application spectrum, is likely to be encompassed in the field of medicine and pharmacy in general. It should be noted that since regulatory agencies in pharmaceutical matters require the existence of methods or kits to monitor the efficacy of a compound with a certain therapeutic activity, both the description of the compounds, their synthesis, their therapeutic scope and their detection should be considered parts of this invention.
• Cell membranes are structures that define the entity of the cells and the organelles contained therein. In the membranes or in their vicinity most of the biological processes occur. Lipids not only have a structural role, but regulate the activity of important processes. Moreover, the regulation of the lipid composition of the membrane also influences the location or function of important proteins involved in the control of cellular physiology, such as G proteins or PKC (Escribá et al, 1995; Escribá et al, 1997; Yang et al, 2005; Mart ⁇ nez et al, 2005). These and other studies demonstrate the importance of lipids in the control of important cellular functions.
• the lipid composition of cell membranes is influenced by the type and abundance of ingested lipids (Perona et al, 2007). From this it follows that lipid intake can regulate the lipid composition of membranes, which in turn can control the interaction (and therefore the activity) of important cellular signaling proteins (Yang et al, 2005).
• membrane lipids can control cell signaling means that they can also regulate the physiological state of the cells.
• both negative and positive effects of lipids on health have been described (Escribá et al, 2006; Escribá et al, 2008).
• Preliminary studies have shown that 2-hydroxyoleic acid, which is a monounsaturated fatty acid, is capable of reversing certain pathological processes, such as overweight, hypertension or cancer (Alemany et al, 2004; Mart ⁇ nez et al, 2005; Vógler et al, 2008).
• Cardiovascular diseases are frequently associated with hyperproliferation of the cells that constitute the cardiac and vascular tissues. This hyperproliferation of cardiovascular cells results in deposits in the internal lumen of the vessels and cavities of the cardiovascular system that result in a wide range of diseases, such as hypertension, atherosclerosis, ischemia, heart attacks, etc. (Schwartz et al, 1985). In fact, the development of drugs that prevent cell proliferation for the prevention and treatment of cardiovascular diseases has been suggested (Jackson et al, 1992).
• Obesity or overweight is caused by an alteration between the balance of intake and energy expenditure that is due, in part, to alterations in the mechanisms that regulate these processes.
• this pathology is characterized by hyperplasia (increase in the number of cells) or hypertrophy (increase in size) of adipose tissue cells, adipocytes.
• fatty acids either free or as part of other molecules, can influence a series of parameters related to energy homeostasis, such as body fat mass, lipid metabolism, thermogenesis or intake, among others ( Vógler et al, 2008). In this sense, the modification of fatty acids could be a strategy to regulate energy homeostasis and, therefore, body weight.
• lipid intake also determines the appearance of other pathological processes, such as hypercholesterolemia, hypertriglyceridemia, diabetes or metabolic syndrome (Sloan et al, 2008)
• Neurodegenerative processes give rise to a series of diseases with different manifestations, but with the common characteristic of being caused by degeneration of the cells of the central and / or peripheral nervous system. Some of these neurodegenerative processes involve a significant reduction in the cognitive capacity of patients, such as Alzheimer's disease or senile dementia. Others lead to motor-type alterations, such as Parkinson's disease or different types of sclerosis. Finally, certain neurodegenerative diseases can lead to processes in which blindness, hearing problems, disorientation, mood alterations, etc. develop.
• Alzheimer's disease in which the formation of senile plaques has been observed, composed of remains of membrane proteins (such as the ⁇ -amyloid peptide) erroneously processed, which accumulate outside of the cells, and clews of neurofilaments of Tau protein, which appear inside the cell.
• membrane proteins such as the ⁇ -amyloid peptide
• clews of neurofilaments of Tau protein which appear inside the cell.
• This process has been associated with alterations in cholesterol metabolism and the consequent alteration of cholesterol levels in membranes (Sagin et al, 2008).
• the development of this disease is related to other diseases in which alterations of lipid metabolism have been described, and more specifically of cholesterol, such as cardiovascular diseases.
• sclerosis and other neurodegenerative processes are related to "demyelination", whose net result is the loss of lipids in the neuronal axon sheath, with the consequent alterations in the process of propagation of electrical signals that this implies.
• Myelin is a lipid layer that surrounds the axons of many neurons and is formed by a succession of spiral folds of the glia cell plasma membrane (Schwann cells). Therefore, it is clear that lipids play an important role in the development of neurodegenerative diseases.
• unmodified natural polyunsaturated fatty acids have a moderate preventive effect on the development of neurodegenerative processes (La ⁇ e et al, 2005).
• Metabolic diseases form a set of pathologies characterized by the accumulation or deficit of certain molecules.
• a typical example is the accumulation of cholesterol and / or triglycerides above normal levels.
• the increase in cholesterol and / or triglyceride levels, both at the systemic level (for example the increase in plasma levels) and at the cellular level (for example in cell membranes) is associated with alterations in cell signaling that lead to dysfunctions at various levels, and that are normally due to errors in the activity of certain enzymes or the control of said proteins.
• hypercholesterolemia high cholesterol levels
• hypertriglyceridemia high triglyceride levels.
• the molecules described in the present invention have the structural characteristics that determine a positive effect on the health of certain natural fatty acids, together with molecular modifications that enhance the effect of the original molecules and also prevent their rapid metabolization, both essential characteristics to determine its pharmacological activity.
• sphingolipids Deepening the importance of cell membrane lipids, sphingolipids or sphingophospholipids are an important class of cell membrane lipids and are the most abundant in the tissues of more complex organisms. Sphingolipid molecules have unfriendly properties, that is, both hydrophobic and hydrophilic, which allows them to play an important role in the formation of biological membranes. Some of the glycosphingolipids are found on the surface of red blood cells and the rest of cells acting as antigens and constituting blood groups.
• SM sphingolipids are of great biological importance because of the role of cell signaling they play.
• SM is a very abundant type of sphingolipid in the cell membranes of all organisms (Huitema et al, 2004). It is mainly located in the outer monolayer of the plasma membrane where it has an essential function in the formation of microdomains called lipid rafts, which are specialized areas of the cell membrane with important functions in cell signaling, since these domains concentrate proteins that interact with each other thanks to the approximation that derives from their binding to lipids (Simons and Toomre, 2000) .
• the enzyme EC 2.7.8.27 is responsible for the synthesis of MS by transferring a phosphocholine (from phosphatidylethanolamine or phosphatidylcholine) to the primary hydroxyl group of ceramide to form MS and 1,2-diacylglycerol (DAG). This enzyme occupies a central position in the metabolism of sphingolipids and glycerophospholipids.
• EC 2.7.8.27 is located in the plasma membrane, in the Golgi apparatus and its activity in the nuclear membrane and in chromatin has also been detected (Albi et al, 1999).
• EC 2.7.8.27 also acts as a regulator of ceramide and diacylglycerol (DAG) levels, both of which are molecules that regulate cell death programmed by apoptosis and autophagy (Jiang et al, 2011; Van Helvoort et al, 1994 ; Tafesse et al, 2006).
• the polar head of the SM is very bulky and prevents the anchoring of proteins, such as Ras, which have branched lipids (such as isoprenyl, farnesyl or geranylgeranyl moieties), while favoring the anchoring of other proteins that have saturated fatty acid residues ( as myristic or palmitic acids).
• the present invention refers to the synthesis of a series of 2-hydroxy derivative compounds of fatty acids, the separation of their racemic forms and their therapeutic applications.
• the present invention also includes the description of the cell targets of their activity and, in addition, of biomarkers that allow determining the efficacy of said compounds, as well as the processes used for this.
• Ceramide produced by the activity of EC 2.3.1.50 is a lipid molecule of great biological interest.
• An important role related to ceramide is its participation in the induction of apoptosis, also called programmed cell death (Lladó et al, 2010).
• Apoptosis is a very regulated biological process that serves to eliminate cells that are not useful or that compromise the health of the body. In this sense, it is common for tumor cells to develop molecular mechanisms to escape apoptosis (Lladó et al, 2010).
• the present invention focuses on resolving alterations in the cellular levels of SM, resulting in compounds capable of reversing the altered expression level of the enzyme EC 2.7.8.27 by activation (in case the enzyme is under-expressed or that has a reduced activity) or through its inhibition (in case the enzyme is overexpressed or has an increased activity), managing to control the levels of SM synthesized by said enzyme and, consequently, reverse the processes pathological due to enzyme deregulation or abnormal levels of MS.
• a method of synthesis of molecules in their racemic form and subsequent isolation of the [-] and [+] enantiomers of 2-hydroxy derivative fatty acid compounds was carried out.
• the present invention refers to pharmaceutical compositions comprising said enantiomers, and their use as medicaments in the treatment of diseases whose common etiology is based on alterations (of any origin) of cell membrane lipids such as: alterations in the level, in the composition or in the structure of said lipids, as well as in the treatment of diseases in which the regulation of the composition and membrane lipid structure induces the reversal of the pathological state.
• the therapeutic effect is preferably achieved through the regulation (activation or inhibition) of the activity of the enzyme EC 2.7.8.27, and / or the level of its product, the SM, or even the level of PFAG and / or of DHFR.
• the present invention demonstrates that, for example in U118 cells, compound 20HOA regulates the activity of other enzymes involved in lipid metabolism.
• the enzyme EC 2.3.1.50 was investigated.
• 20HOA stimulates the activity of CD 2.3.1.50 (Example 8 and Figure 9), causing programmed cell death in human leukemia cells.
• an important reduction in oleic acid levels was also found in the membranes of U118 cells treated with 20HOA, demonstrating that 20HOA is a potent inhibitor of EC 1.14.19.1, an enzyme responsible for the synthesis of oleic acid from of stearic acid (Example 9 and Figure 10).
• both EC 2.7.8.27 and EC 2.3.1.50 are enzymes corresponding to the metabolism of sphingolipids, which are related to belonging to the metabolic pathway of the same type of molecules.
• the enzyme EC 1.14.19.1 is a fatty acid modifier.
• the relationship with the other two enzymes is that sphingolipids always carry fatty acid chains in their structure. Since each fatty acid has different properties than sphingolipids, the fact that they can be modified affects their biological activity. Thus, it can be affirmed that the three enzymes mentioned in the present invention are closely related and therefore it is normal that the same molecule can regulate the activity of the 3 enzymes.
• the enantiomers [-] (also S isomer) and [+] (also R isomer) of the invention differ in the direction of deflection of polarized light. If the optical isomer deflects the polarized light to the right (in orientation with the hands of the clock) it is represented by the sign [+] (it is the dextrogiro isomer or dextrous form). On the other hand, if the optical isomer deflects the polarized light to the left (in a counter-clockwise direction), it is represented by the sign [-] (it is the levographic isomer or levo form).
• the present invention evidences that the [-] enantiomer of 2-hydroxy derivative compounds of fatty acids acts as activator of the enzyme EC 2.7.8.27 by positively regulating the synthesis of SM, a sphingolipid that, as explained above, is mostly in the membranes of human and animal cells, and indispensable for the correct structuring of the lipid bilayer and functioning of the cell.
• said enantiomer [-] can be used for the preparation of a pharmaceutical composition for the treatment of those pathologies whose common etiology is structural and / or functional alterations of lipids located in the cell membrane, such as: cancer, obesity , hypertension, hypertriglyceridemia, hypercholesterolemia or diabetes, etc., due to an abnormally low activity of said enzyme EC 2.7.8.27.
• the racemic form is activating this enzyme, since the activity of the [-] isomer predominates, which is the active one, over the [+] isomer, which does not induce the activity of the enzyme.
• the positive activity of the racemic form is considered to be due to the fact that for the activity of this enzyme an activation that induces the synthesis of new molecules of SM is more important, than the inhibition that can silence enzyme molecules than previously They were not active.
• the activating power of the racemic form is inferior to that of the enantiomer [-], which explains its lower therapeutic activity.
• the [-] enantiomer of 2-hydroxy derivative fatty acid compounds exhibits an improved therapeutic effect with respect to the racemic (which contains equal amounts of the two enantiomers).
• the [-] enantiomer of 2-hydroxy derivative fatty acid compounds has less toxicity and side effects than the racemic one and that the [+] enantiomer (see Table 3, Example 7).
• the present invention also demonstrates that the [+] enantiomer of 2-hydroxy derivative fatty acid compounds acts as an inhibitor of said EC enzyme 2.7.8.27 by negatively regulating the synthesis of SM, and can be used in basic research for the study of regulation of the EC enzyme 2.7.8.27 itself, or for the treatment of diseases characterized by an abnormally high activity of the enzyme EC 2.7.8.27, and / or an abnormally high level of SM, such as cystic fibrosis (Slomiany et al, 1982). On the other hand, high cholesterol and triglyceride levels have been associated with significant cardiovascular disorders.
• lipid rafts liquid ordered, that is, ordered liquid
• the increase in cholesterol favors the increase of these lipid regions, which implies changes in cell signaling that can lead to different diseases or cardiovascular and metabolic alterations. Therefore, reducing the levels of MS in cases of hypercholesterolemia and hypertriglyceridemia can help lower cholesterol and triglyceride levels in plasma and membranes.
• the enantiomer [+] would have a protective role in certain types of disorders, such as hypercholesterolemia and hypertriglyceridemia, since it induces reductions in serum levels of these lipids and reductions in MS levels that would concur in a reduction of The density of lipid rafts in the cells.
• both the enzyme and the SM itself could be considered as molecular markers, usable to lead to carry out a method of diagnosis and / or prognosis in vitro of diseases based on alterations in the cell membrane. Such changes do not occur naturally, but are caused by the activity of the compounds described in the present invention.
• the compounds of the present invention also regulate the levels of DHFR and PFAG proteins. Consequently, the present invention also refers to a method / kit for Carry out the diagnosis or prognosis of pathologies associated with altered levels with respect to the normal DHFR and PFAG proteins.
• Said kit comprises reagents or means capable of evaluating the activity of the enzyme EC2.7.8.27, and / or the level of SM, DFIFR or PFAG and, consequently, implementing said diagnosis / prognosis as a method to follow the efficacy of the treatment of patients
• said enzyme EC2.7.8.27 and / or its product, SM can be considered as therapeutic targets to which to direct molecules capable of regulating the activity of the enzyme, and / or the level of MS, and , consequently, to reverse those pathological processes that would have developed, or were going to develop in the future, as a result of the alteration in the activity of the enzyme or in the level of SM, DFIFR or PFAG.
• the [-] enantiomer of the invention serves to illustrate the possible use of the enzyme EC 2.7.8.27 as a therapeutic target, by activating its enzymatic function in pathological processes in which said function is deficient, being able to restore the level of SM at normal levels.
• the measurement of the activity of the enzyme EC 2.7.8.27, and / or the levels of SM, and / or the levels of PFAG, and / or the levels of DHFR would also be useful for performing selection procedures ( screening, in English) of candidate compounds with the aim of achieving other molecules that, such as the [-] and [+] enantiomers of the invention, had the ability to regulate the activity of the enzyme EC 2.7.8.27, and / or the level of SM, and / or the level of PFAG, and / or the level of DHFR, being able to reverse pathological processes.
• the present invention demonstrates the particular importance of selecting compounds with exclusive structural characteristics such as: fatty acids with at least one double bond, with a total of carbon atoms (C) equal to or less than 20, and a substituted carbon, particularly with a hydroxyl radical (OH), on carbon 2 (or carbon a).
• the compounds referred to in the present invention are the [-] and [+] enantiomers of Formula I:
• the preferred therapeutic form of the present invention is the [-] enantiomer (corresponding to the steric configuration S) of Formula I, which is presented as the most effective way in the activation of the enzyme EC 2.7. 8.27, ahead of the racemic form, and the [+] enantiomer (corresponding to the steric configuration R) of Formula I which is presented as an inhibitor of the enzyme EC 2.7.8.27.
• the present invention makes special reference to the [+] and [-] enantiomers of Formula I with the following values of a, b and c:
• diseases characterized by a deficit in the activity of the enzyme EC 2.7.8.27, and, consequently, by an abnormally low level of SM in cell membranes, and that could be treated or prevented with the enantiomer [- ] of the invention are:
• Cancer prostate cancer, breast cancer, pancreatic cancer, leukemia, uterine cancer, colon cancer, brain cancer, lung cancer, malignant melanoma and liver cancer (see Table 2).
• vascular pathologies hypertension, arteriosclerosis, cardiomyopathies, angiogenesis, cardiac hyperplasia, etc.
• Metabolic pathologies diabetes, metabolic syndrome or obesity.
• the first aspect of the present invention refers to an [-] or [+] enantiomer of a compound of Formula I and / or at least one of its pharmaceutically acceptable salts
• a, b and c can take independent values between O and or, provided that the total number of carbons is ⁇ 20.
• diseases characterized by an excess in the activity of the enzyme EC 2.7.8.27, and, consequently, by an abnormally high level of SM in cell membranes, and which could be treated or prevented with the enantiomer [+] of the invention are fibrosis cystic, hypercholesterolemia and hypertriglyceridemia.
• a second aspect of the present invention refers to the use of at least one compound of the aforementioned for the preparation of a pharmaceutical composition for the treatment and / or prevention of pathologies whose common etiology is structural and / or functional alterations of the cell membrane , due to deregulation of the activity of the enzyme EC 2.7.8.27, of the level or concentration of SM, of the level of PFAG or of the level of DHFR, in cells in general and in membranes, in particular.
• the present invention covers at least one compound of the aforementioned for use in the treatment and / or prevention of pathologies whose common etiology is structural and / or functional alterations of the cell membrane, due to the deregulation of the activity of the EC enzyme 2.7.8.27, of the level or concentration of SM, of the level of PFAG or of the level of DHFR, in cells in general and in membranes, in particular.
• the present invention refers to the pharmaceutical composition itself comprising at least one of said compounds and, optionally, pharmaceutically acceptable carriers.
• the compounds of the present invention can be administered independently or formulated in pharmaceutical compositions where they are combined with excipients such as: binders, fillers, disintegrators, lubricants, coaters, sweeteners, flavorings, colorants, transporters, etc., and combinations thereof.
• the compounds of the invention can be part of pharmaceutical compositions in combination with other active ingredients.
• the compounds of the invention can be carried out by any route such as, for example, enterally (through the digestive system), orally (by pills, tablets or syrups), rectally (by suppositories or enemas), topically (through creams or patches), inhalation, parenterally injected, intravenous injection, intramuscular injection or subcutaneous injection, as indicated above or in any type of pharmaceutically acceptable form, such as : methyl, ethyl, phosphates, other radicals of the ester, ether, alkyl type, etc.
• any route such as, for example, enterally (through the digestive system), orally (by pills, tablets or syrups), rectally (by suppositories or enemas), topically (through creams or patches), inhalation, parenterally injected, intravenous injection, intramuscular injection or subcutaneous injection, as indicated above or in any type of pharmaceutically acceptable form, such as : methyl, ethyl, phosphates, other radicals of the ester,
• a third aspect of the present invention refers to a method of treatment and / or prevention of pathologies whose common etiology is structural and / or functional alterations of the cell membrane, due to deregulation of the activity of the enzyme EC 2.7.8.27, the SM level, the PFAG level or the DHFR level; which comprises the administration to the patient of a therapeutically effective amount of at least one compound of the aforementioned or compositions comprising them.
• the pathologies are preferably selected from: preferably prostate cancer, breast cancer, pancreas cancer, leukemia, uterine cancer, colon cancer, brain cancer, lung cancer, malignant melanoma and liver cancer; vascular pathologies preferably hypertension, arteriosclerosis, cardiomyopathies, angiogenesis, cardiac hyperplasia or metabolic pathologies; Metabolic pathologies preferably: diabetes, metabolic syndrome or obesity.
• the pathology is, for example, cystic fibrosis, hypercholesterolemia and hypertriglyceridemia.
• therapeutically effective amount is understood as that which reverts the disease or prevents it without showing adverse side effects.
• pharmaceutically effective amount would also be understood as that which, producing a significant therapeutic effect, had an acceptable level of toxicity when the treated disease was very serious or fatal.
• a fourth aspect of the present invention refers to an in vitro method for the selection of candidate compounds useful in the treatment and / or prevention of pathologies whose common etiology is structural and / or functional alterations of the cell membrane, which includes the evaluation of changes produced on the activity of the enzyme EC 2.7.8.27, the level of SM, the level of PFAG or the level of DHFR in the presence of said candidate compound. That is, the present invention comprises the use of the enzyme EC 2.7.8.27, of SM, PFAG or DHFR as therapeutic targets, with the purpose of designing therapeutic tools capable of altering their activity (eg, Figure 5) or levels and to which to direct compounds with the aim of preventing and / or treating the pathologies described above.
• the fifth aspect of the present invention refers to in vitro methods for the prognostic / diagnosis of pathologies whose common etiology is structural and / or functional alterations of the lipids located in the cell membrane comprising the determination of the deregulation of the activity of the enzyme EC 2.7.8.27 and / or the presence of an abnormal level of SM, PFAG or DFIFR in the cell membrane or other cell compartments. That is, the present invention comprises the use of the enzyme EC 2.7.8.27, or of SM, PFAG or DFIFR, as molecular markers through which to make the diagnosis and / or prognosis of the previously described pathologies.
• both SM and the enzyme EC 2.7.8.27, DHFR and PFAG constitute biomarkers for the detection of human diseases, and, as mentioned above, are therapeutic targets for the design of new therapies for humans.
• the present invention refers to an in vitro method for the prone diagnosis / diagnosis of pathologies whose common etiology is structural and / or functional alterations of the lipids located in the cell membrane comprising the determination of a deficit in the EC enzyme activity 2.7.8.27, the presence of an abnormally low level of SM in the cell membrane, an abnormally low cellular level of PFAG or an abnormally high cellular level of DFIFR, where, preferably, the pathologies They are selected from: preferably prostate cancer, breast cancer, pancreas cancer, leukemia, uterine cancer, colon cancer, brain cancer, lung cancer, malignant melanoma and liver cancer; vascular pathologies preferably hypertension, arteriosclerosis, cardiomyopathies, stroke, angiogenesis, cardiac hyperplasia or metabolic pathologies; Metabolic pathologies preferably: diabetes, metabolic syndrome or obesity.
• the present invention refers to an in vitro method for the prone diagnosis / diagnosis of pathologies whose common etiology is structural and / or functional alterations of the lipids located in the cell membrane comprising the determination of an excess in the EC enzyme activity 2.7.8.27, the presence of an abnormally high level of SM in the cell membrane, an abnormally high level of PFAG or an abnormally low level of DHFR, where, preferably, the pathology is fibrosis cystic hypercholesterolemia and hypertriglyceridemia.
• kits to be used in the prognostic / diagnostic method defined above which comprises useful means for determining the activity of the enzyme EC 2.7.8.27, of the level of SM in the membrane cellular, DHFR levels / activity and / or GAPF levels.
• useful means comprise the TLC and HPTLC technique, gas chromatography, image analysis, absorption or fluorescence spectroscopy, optical microscopy, fluorescence, confocal microscopy, immunoblotting, immunocytochemistry, ELISA or similar techniques (RIA, dot / slot blot, EIA, etc.).
• the kit is characterized in that the prognosis / diagnosis is carried out through direct quantification of sphingomyelin levels, and / or indirect quantification thereof through its precursors (for example phosphatidylethanolamine, phosphatidylcholine , ceramide, etc.) or its derivatives (for example sphingolipids).
• the precursor for example phosphatidylethanolamine, phosphatidylcholine , ceramide, etc.
• the derivatives for example sphingolipids.
• the precursor is ceramide
• the derivative is BD-Cer or BD-SM and indirect measurement is made through lisenin.
• Another aspect of the present invention refers to a method of synthesis of racemic compounds and the isolation and purification of the enantiomers of the molecules of the invention comprising the following steps:
• step 1) in the case of acid and step 2) in the case of the ester to start reprocessing each of the isolated fractions (acid and ester) until the desired enantiomeric purity is obtained (95% enantiomeric excess, which is equivalent to 97.5% of the desired enantiomer and 2.5% of the unwanted one).
• the separation and isolation of the two enantiomers of 20HOA is not known to date. Especially the isolation of the enantiomer [-] had not been possible to date due to its technical difficulty.
• the present invention also refers to an in vitro method of monitoring the cellular alterations produced on the diseased cells by the effect of the compounds of the present invention, or other compounds that act on the same process or on related cellular processes, producing the healing or improvement of the affected cells in the patient. That is, the present invention comprises the use of the enzyme EC 2.7.8.27, or of SM, DHFR or GAPF as molecular markers whose changes induced by the treatment with the molecules of the present invention allow to know if the patient responds to the treatment and, therefore, determine its effectiveness and the time that must be maintained.
• a preferred aspect of the present invention refers to a method that allows to follow the changes induced by the molecules of the present invention or other molecules that perform a similar effect on pathological cells. Therefore, the use of MS, the enzyme EC 2.7.8.27, DHFR and / or PFAG offers the possibility that the applied therapy can change its levels or activity.
• the purpose of this determination is (1) the prediction of the efficacy of the treatment to avoid potentially non-responding patients following an ineffective therapy and (2) knowing the evolution of the patient to confirm that he responds to the therapy and know the doses to be delivered depending on the phase of the treatment, as well as the duration of these phases and the treatment itself as a whole.
• these aspects are critical to avoid unnecessary pharmaceutical expenditure and to be able to apply the therapy in the most rational way possible, the large regulatory agencies in pharmaceutical matters request the existence of biomarkers and diagnostic systems as described here.
• the present invention refers to a method for the treatment of patients suffering from the pathologies described above, which comprises the determination of the presence of said deregulation in the enzyme EC 2.7.8.27, of the level of MS, of the level of PFAG or DHFR level, and the treatment of the patient presenting said deregulation with the compounds of the invention.
• the present invention refers to a method for selecting therapy for a patient with a pathology described in the present invention which comprises determining the presence of said deregulation in the enzyme EC 2.7.8.27, of the level of MS, of the level of PFAG or DFIFR level and the selection, based on said determination, of a therapy based on the compounds of the present invention.
• High / low level of PFAG A high level of PFAG is considered to be double or more (> 200%) of the normal levels present in glia cells, in reference to mg of total protein.
• a low level of PFAG is considered to imply the presence of levels of half or less ( ⁇ 50%) with respect to the normal levels present in glia cells, in reference to mg of total protein.
• ⁇ High / low level of DHFR A high level of DHFR is considered to be double or more (> 200%) of the normal levels present in quiescent cells of any type, in reference to mg of total protein.
• a low level of DHFR is considered to imply the presence of levels of half or less ( ⁇ 50%) with respect to the normal levels present in quiescent cells of any type, in reference to mg of total protein.
• the compound 20HOA induces a significant increase in the synthesis of MS in different human brain cancer cell lines (U118, SF767 and 1321N1), human lung cancer cells (A549) and human leukemia cells (Jurkat), but not in non-tumor cells (human lung fibroblast cells MRC5). This increase is, therefore, induced and specific for tumor cells. Likewise, the antitumor effect on these and other tumor lines has also been studied (see Table 2). The cells were treated vehicle (water with 5% ethanol, Control) or with the compound 20HOA, for 24 hours at a concentration of 200 ⁇ .
• the levels of MS measured in tumor cells are significantly lower than those of normal cells and the 20HOA compound only induced significant changes in cancer cells.
• the enzyme EC 2.7.8.27 is a therapeutic target for the treatment of cancer and a biomarker for the monitoring of the pathology and its evolution during the applied therapies, and that, on the other hand, the compound 20HOA is an activator of this enzyme and reverses the low levels of SM presented by tumor cells.
• other techniques have been tested that have yielded very similar results, such as gas chromatography, confocal microscopy, fluorescence spectroscopy, etc.
• the ordinate axis represents the quantification of the fluorescence intensity (arbitrary units) by confocal microscopy in sections of the tumors shown in the upper panel (mean ⁇ standard error of the mean), from vehicle treated animals (Control ), or 20HOA at 600 mg / kg -day (T600) or 900 mg / kg ⁇ day (T900) for 50 days (po). *** P ⁇ 0.001.
• A Ul 18 human glioma cancer cells treated at a concentration of 200 ⁇ of 20HOA at different times (2, 6, 12, 24, 48 and 72 hours). The white bars correspond to cells treated with vehicle (control).
• B U118 cells treated for 24 hours at different concentrations of 20HOA (25-400 ⁇ ).
• FIG. 3 The 20HOA compound induces an increase in nuclear SM.
• U118 cancer cells were treated for 24 hours with vehicle (control) or with 20HOA at a concentration of 200 ⁇ .
• the compound 20HOA acts directly on the enzyme EC 2.7.8.27.
• the cells or cell extracts or incubation medium were incubated with the fluorescent substrate of this BD-Cer enzyme (nitrobenzoxadiazol-yl) ceramide], in the presence or absence (control, white bars) of 20HOA (black bars).
• Figure 5 Structure-function relationship in the activation of the enzyme EC 2.7.8.27. Increase of SM in U118 cells induced by different fatty acids (200 ⁇ , 24 h).
• Control vehicle; 18: ln-9 (octadecenoic acid); 2Me-18: ln-9 (2-methyloctadecenoic acid); 20H16: 1 (2-hydroxyhexadecenoic acid); 2OH-18: 0 (2-hydroxyoctadecanoic acid); 20HOA (20H-18: ln-9, 2-hydroxyoctadecenoic acid); 20H- 18: 2n-6 (2-hydroxylinoleic acid); 20H-18: 3n-6 (2-hydroxy-y-linolenic acid); 20H- 18: 3n-3 (2-hydroxy-a-linolenic acid); 2OH-20: 4n-6 (2-hydroxyarachidonic acid); 2OH-20: 5n-3 (2-hydroxyeicosapentaenoic acid); 20H-22: 6n-3
• A Changes in cell membrane composition are shown to induce the translocation of Ras protein from the membrane to the cytoplasm. Elevated membrane levels of compound 20HOA and SM prevent anchoring of the Ras protein in the membrane.
• the figure shows phase contrast (Ph.C) optical microscopy images of human glioma cells (SF767), and confocal microscopy, using a specific antibody against fluorescently labeled Ras, after 10 minutes (confocal 1) and 24 hours (confocal 2) treatment with vehicle (control) or compound 20HOA.
• Ras protein prevents the inactivation of the Raf protein, as well as the next activation in the signaling cascade of the MEK protein, as observed by the reduction in state of the phosphorylated (active) form of both proteins, determined by immunoblot in U118 cells grown in absence (Control) no presence of 150, 200 or 250 ⁇ of 20HOA.
• C The previous molecular / cellular events lead to a dramatic reduction in the activity status of the MAP kinase protein (ERK1 and ERK2), also determined by immunoblotting with specific antibodies, at the concentrations of 20HOA indicated in B.
• D Activity states (phosphorylated form levels) of Akt and EGFR also decrease after incubation with 250 ⁇ of 20HOA.
• the ordinate axis indicates the percentage of phosphoprotein with respect to the untreated (control) SF767 cells.
• the abscissa axis indicates the concentration (micromolar) of compound 20HOA.
• Lower panel Expression of DHFR in human lung cancer cells (A549).
• DHFR levels vary in response to treatment with the molecules of the invention, so that the activity or levels of this protein can be used as biomarkers to track the therapeutic efficacy of the compounds described herein or compounds acting as similar form. It also follows that tumors in which DHFR levels are high may respond well to treatment with the compounds of the present invention, so this protein is a good biomarker to predict the possible efficacy of enantiomers of fatty acid derivatives. and subsequently demonstrate the efficacy of treatment with these compounds.
• F Left panel: PAFG levels in serum of animals with human tumors (glioma) determined by immunoblotting.
• mice were infected with SF767 cells and treated with vehicle (Glioma) or with 20HOA (T, 600 mg / kg ⁇ day, po, 28 days). Immunoreactivity against the fragmented peptide of the PFAG (see photo) in the serum of these animals, expressed as a percentage, was compared with that found in tumor-free and untreated animals (Control). Right panel: Idem, but determined by serum ELISA of animals treated for 7 days (7d600) or 28 days (28d600) with 600 mg / kg of 20HOA (po). A specific antibody against PFAG was used for all experiments.
• PFAG levels vary in response to treatment with the molecules of the invention, whereby the levels of this protein can be used as biomarkers to follow the therapeutic efficacy of the compounds described herein or compounds that act similarly.
• tumors in which PFAG levels are low may respond well to treatment with the compounds of the present invention, so this protein is a good biomarker to predict the possible efficacy of enantiomers of fatty acid derivatives. and subsequently demonstrate the efficacy of treatment with these compounds.
• Various techniques have been used to measure DHFR and PFAG, including electrophoresis, immunoblot, ELISA and the like, RT-PCR, etc. All these and similar techniques can be used to determine the levels or activity of DHFR and PFAG.
• Figure 7 A) Specificity of the action of the [-] and [+] enantiomers, and of the +/- racemic mixture of 20HOA, in the activity of the enzyme EC 2.7.8.27.
• the figure shows the levels of SM (percentage of SM over total lipids) in U118 glioma cells after treatment with the racemic mixture of 2-hydroxyoleic acid (+/-), with compound [-] 20HOA and with compound [+] 20HOA.
• the cells were treated 24 hours at a concentration of 50 ⁇ (50) and 100 ⁇ (100).
• the enantiomer [-] produces increases in SM, which indicates that it is an activator of the enzyme EC 2.7.8.27, and the enantiomer produces a reduction in the levels of SM (especially obvious at a concentration of 100 ⁇ [+ J20HOA), which indicates that it is an inhibitor of This enzyme
• Figure 8 This figure shows the effect of racemic 20HOA (+/-) and optical isomers [-] 20HOA and [+ J20HOA on different pathological processes in animal models.
• A. Effect on the volume of tumors derived from human lung cancer cells (A549): immunosuppressed mice were infected with human lung cancer cells and 7 days later (when the tumors were observable) vehicle treatments were started (control ), Racemic 20HOA (20HOA), and the optical isomers [-] 20HOA and [+] 20HOA (600 mg / kg ⁇ day, 15 days, oral). The bars correspond to mean values ⁇ SEM of the increase in volume (percentage with respect to the volume in control animals at the beginning of the treatment, considered as 100%) of the tumors (n 6).
• racemic 20HOA Treatment with racemic 20HOA induced significant reductions (P ⁇ 0.01) with respect to control.
• the optical isomer [-] 20HOA induced significant reductions compared to all other treatments (P ⁇ 0.01).
• Figure 9 Regulation of enzyme activity 2.3.1.50.
• the ordinate axis (y) shows the levels of incorporation of [ 3 H] palmitate (dpm / mg protein) in different lipid fractions of U118 cells incubated in the absence (Control, white bars) or presence of 20HOA (black bars). Only a significant increase in radioactivity was found in the sphingomyelin (SM) and ceramide (Cer) fractions, which clearly indicates the selective activation of EC 2.7.8.27 and the enzyme 2.3.1.50.
• SM sphingomyelin
• Cer ceramide
• Figure 10 Regulation of enzyme activity 1.14.19.1.
• the ordinate (Y) axis shows the levels of incorporation of [ 3 H] oleate (dpm / mg protein) into membranes of Ul 18 cells incubated in the absence (Control, white bar) or presence of 20HOA (black bar).
• the reduction in incorporation of tritiated oleic into the lipids of cell membranes incubated in the presence of 20HOA demonstrates the inhibition of the enzyme 1.14.19.1.
• Example 1 Methodology for obtaining racemic compounds and separating the enantiomers [+] and [-] of the 2-hydroxy derivatives of fatty acids.
• the methodology described below details the synthesis of sodium salts of 2-hydroxy fatty acids with a purity of 99%, by generating the oleic acid dianion by reaction with LDA and subsequent hydroxylation with molecular oxygen; The crude obtained is treated with NaOH to form the sodium salt and purified by two recrystallizations in MeOH / water (avoiding the use of chromatographic columns).
• the crude product is dissolved in 7.2 L of methanol at 40 ° C in a 10 L reactor and treated with an aqueous solution of sodium hydroxide (134 g in 1.4 L of water), increasing the T to 50 ° C for obtain a homogeneous solution. Cool to 5 ° C for at least 6 h to precipitate the sodium salt, which is filtered and washed with acetone. The solid is recrystallized twice in (10%) H 2 0 / MeOH (0.5 LH 2 O / 5.0 L MeOH) at 50 ° C until complete dissolution and cooling for a minimum of 6 h at 0-5 ° C. The final product is filtered, washed with acetone and dried in vacuo.
• sodium hydroxide 134 g in 1.4 L of water
• the method has been carried out at scales of 1-150 g, obtaining similar and reproducible results.
• the enantiomers can be separated by crystallization, which facilitates the kilos scale process.
• the loss of performance is due to the manipulation and workup processes, however, new impurities are not generated and the washing and crystallization waters could be combined and reprocessed again.
• Example 2 Tumor cells have lower levels of SM in their membranes, which increase after treatment with the molecules of the invention.
• a diagnostic method has been generated for the detection of the described pathological processes and pathologies in which the levels of MS are altered which allows to predict whether the treatment with the molecules of the present invention can be effective, thus as for monitoring the efficacy of the therapy applied using the molecules of the present invention and others of similar activity.
• the present invention also It includes diagnostic kits to assess a priori the potential utility of this therapy and to track the efficacy of treatment with the molecules of the present invention of these diseases a posteriori.
• Antiproliferative effect + cell growth inhibition, - absence of effect.
• Technique used for the analysis 1) TLC or HTPLC, 2) gas chromatography, 3) image analysis, 4) fluorescence spectroscopy, 5) confocal or fluorescence microscope.
• Example 3 raises the levels of MS by activating the enzyme EC 2.7.8.27.
• SM is a membrane lipid that prevents the binding of certain molecules involved in cell proliferation, such as the Ras protein.
• Ras levels were measured by confocal microscopy, using a fluorescently labeled antibody, and it could be detected that the tide passed from the membrane ( Figure 6: 95-99% of the total fluorescence detected in membranes before treatment ) to the cytoplasm (94-97% of the fluorescence detected inside the cell after treatment with 20HOA) in human glioma, lung cancer and leukemia cells.
• the translocation of Ras from the membrane to the cytoplasm implies the absence of productive interactions between Ras and the Receptor-Tyrosine-Kinase (RTK) or between Ras and Raf, so that the proteins of the MAP kinase pathway do not receive activation and Tumor cells stop proliferating and cell death programs begin.
• the changes induced by the molecules of the present invention in cell physiology have as intermediate effects (prior to the death of cancer cells or the regulation of activity in other cells) the induction of dramatic reductions in DHFR levels and dramatic increase of PFAG levels (Figure 6).
• Another effect associated with the reduction of cell proliferation is the increase in nuclear MS levels.
• the 20HOA treatments resulted in increases in nuclear MS levels ( Figure 3), demonstrating that cell proliferation inhibition is induced.
• Example 4. Regulation of the enzyme EC 2.7.8.27 depends on the molecular structure of the fatty acid.
• the present invention refers to the use of the aforementioned compounds as activators (enantiomer [-]), or inhibitors (enantiomer [+]) specific to the enzyme EC 2.7.8.27.
• the activation of the enzyme EC 2.7.8.27 depends on the number of carbon atoms that the fatty acid has, so that fatty acids with more than 20 C atoms do not produce significant changes in the activity of this enzyme ( Figure 5).
• other requirements for activation of the enzyme EC 2.7.8.27 are the presence of an OH group on carbon 2 and one or more double bonds, since the presence of other radicals (such as H or CH 3 ) and the lack of Double bonds in the fatty acid structure gave rise to inactive molecules (Figure 5).
• MS has a molecular structure that determines the effects produced on cell physiology and justifies the reversal of different pathological processes.
• Figure 7A shows the activation of said enzyme, measured through the levels of SM in U118 human glioma cell membranes, after 24 hours of incubation in the presence of 50 and 100 mM of each of the isomers, as well as the racemic mixture (which contains approximately the same amount of each of them).
• Figure 7A shows how racemic 20HOA produces significant increases in levels of SM in U118 cell membranes.
• the optical [-] 20HOA isomer (corresponding to the S enantiomer) is capable of producing even greater increases in the levels of SM.
• the present invention protects the use for therapeutic applications of the optical isomer [-] 20HOA (S) as activator of the enzyme EC 2.7.8.27 and of the optical isomer [+ J20HOA (R) as a specific inhibitor of this enzyme.
• S optical isomer
• R optical isomer
• These results can be extended to all unsaturated fatty acids between 14 carbon atoms or more and 20 carbon atoms or less, which have one or more double bonds and a hydroxyl radical on the C2 carbon (alpha carbon: Figure 7B).
• Example 6 The [-] and [+] isomers of C18 fatty acids, such as [-] 20HOA and [+ J20HOA, used as therapeutic agents for the treatment of human diseases.
• the enantiomer [-] 20HOA and derivatives shown in this invention have therapeutic applications in different areas, such as cancer treatment, obesity, cardiovascular pathologies, diabetes, metabolic syndrome, spinal cord injury, Alzheimer's disease and other processes. Figures 6 and 8; Tables 2-5).
• the enantiomer [+ J20HOA had a positive effect on cholesterol and triglyceride levels, since it induced significant reductions in the plasma levels of these lipids, whose elevated levels are considered negative for human health ( Figure 8 ). Therefore, the therapeutic effects of each of the isomers, as well as the racemic mixture, in different pathological models were studied.
• the effect of the optical isomer [-] 20HOA is superior to that of the racemic.
• after 15-day treatments with the optical isomer [+ J20HOA there were no significant changes in the volume of the tumors. This result demonstrates that the [-] 20HOA isomer is what activates the enzyme EC 2.7.8.27 and has the antitumor therapeutic activity.
• the animals treated with the [- J20HOA isomer showed reductions in blood pressure greater than 60 mm Hg their systolic pressure, while those treated with the [+ J20HOA isomer and with the racemic product had a remarkable therapeutic effect but of less amplitude .
• rats treated with the racemic lost 11 grams of weight (approximately 3% of body weight), while animals in the group treated with the enantiomer [-] 20HOA lost 21 grams and the animals of the group treated with the enantiomer [+ J20HOA only lost 7 grams of weight.
• this invention shows that the optical isomers [-] [+] of unsaturated fatty hydroxy acids of 18 C atoms are effective molecules with therapeutic activity for the treatment of the pathologies indicated above.
• one and only one of the enantiomers showed to have an activity superior to that of the other molecular forms (statistical significance always P ⁇ 0.05).
• the [-] enantiomer was shown to be more effective in curing cancer, obesity, diabetes, hypertension, etc., while the enantiomer [+] showed to be more effective in controlling hypercholesterolemia or hypertriglyceridemia.
• racemic compound was shown as an intermediate situation, capable of emulating the positive effects of any of the optical isomers, but with a lower potency, due to the lower concentration of the active enantiomer in each case.
• use of the unsuitable enantiomer induced unwanted side effects that were avoided at therapeutic doses with the most active enantiomer (Table 3).
• Example 7 Efficacy, toxicity and side effects of the enantiomers of the invention.
• This example reflects the study conducted in immunosuppressed mice infected with human glioma cells (SF767) and treated for 15 days (oral) with the indicated doses (Table 3).
• This table shows the volume of the tumors at the end of the treatment and the symptoms observed during the days that it lasted. Note, once again, that the efficacy of the [-] 20HOA enantiomer is superior to that of the 20HOA racemic compound, and that the [+ J20HOA enantiomer showed no activity after 15 days of treatment at the indicated doses.
• the dose that induced reductions of approximately one third of the tumor volume did not induce any side effects in animals treated with [- J20HOA (50 mg / kg), while animals treated with the racemic compound 20HOA at Doses that induced similar reductions in tumor volume (600 mg / kg) showed significant side effects (Table 3).
• the [-] 20HOA enantiomer induced tumor volume reductions of 89% without observable adverse effects, while at the same dose, the racemic compound only induced or 23% reductions in the volume of the tumors and some of the animals presented adverse effects in response to treatment.
• the [-] 20HOA enantiomer has a greater potency and that at the maximum therapeutic doses it does not produce adverse effects, while the racemic compound has a more modest effect and induces certain unwanted effects at therapeutic doses.
• the differences in efficacy between the [-] 20HOA and the racemic enantiomer can mean differences of months or years in the life expectancy of patients.
• differences in efficacy can result in the cure or not of a certain cancer in a patient.
• differences in toxicity at therapeutic doses can result in a higher quality of life in patients receiving the enantiomeric form [-] 20HOA.
• U118 human glioma cells were incubated in the presence or absence (control) of 20HOA (200 ⁇ , 24 h) and then incubated with [ 3 H] palmitate for 5 minutes. After said incubation periods, cell lipids were extracted and separated by TLC. The bands corresponding to each lipid species were extracted and the amount of radioactive palmitate incorporated was measured by liquid scintillation. As can be seen, there was not only an important incorporation in the SM fraction (indicator of activation of EC 2.7.8.27), but also in the ceramide fraction, which demonstrates the activation of the enzyme EC 2.3.1.50 ( serine palmitoyl transferase).
• Oleic acid is synthesized from stearic acid through the activity of the enzyme stearoyl-CoA desaturase (EC 1.14.19.1). This enzyme is crucial in lipid metabolism, since it is limiting in the synthesis of fatty acids.
• Ul 18 cells were incubated in the presence or absence of 20HOA (200 ⁇ , 24 h) and, subsequently, in the presence of [ 3 H] oleate (5 minutes ). It was found that the incorporation of radioactive oleic acid into U118 cell membranes incubated in the presence of 20HOA was significantly lower than in control cells ( Figure 10). This result indicates that 20HOA is a potent inhibitor of CD 1.14.19.1, whose regulation has been suggested to be important in the treatment of different human pathologies.
• Type PV Sastre M, Garc ⁇ a-Sevilla JA. (1995) Disruption of cellular signaling pathways by daunomycin through destabilization of nonlamellar membrane structures. Proc Nati Acad Sci U S A. 92: 7595-7599.
• Arachidonic and docosahexanoic acids reduce the growth of A549 human lung tumor cells increasing lipid peroxidation and PPARs. Chem Biol Interact. 165: 239-50.

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