RU2841595C1 - Metabolites of alpha-hydroxylated fatty acids, medical applications and use thereof as biomarkers - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: present invention relates to a sodium salt selected from the sodium salt of a compound of formula COOH-(CH₂)_a-(CH=CH-CH₂)_b-(CH₂)_c-CH₃ (II), where: a=6, b=1 and c=6; a=6, b=2 and c=3; a=6, b=3 and c=0; a=3, b=3 and c=3; a=2, b=4 and c=3; and a=2, b=5 and c=0; and a sodium salt of a compound of formula COOH-(CH₂)_a-(CH=CH-CH₂)_m-(CH₂)₃-(CH=CH-CH₂)_(b-1-m)-(CH₂)_c-CH₃, where a=1, b=6, c=0, and m=0. Disclosed compounds can be used to treat a disease or pathology associated with a change in the lipid composition of cell membranes, selected from the group consisting of neurological or neurodegenerative disease, cancer, neoplasm and neuropathic pain.

EFFECT: providing therapeutically active compounds (metabolites) which are obtained from other compounds or prodrugs, so that administration of such metabolites, separately, by means of their prodrugs or in combination with their corresponding prodrugs, modulation of the therapeutic effect and possible adverse side effects, depending on the patient's condition and the pathological condition to be treated.

14 cl, 24 dwg, 6 tbl, 7 ex

Description

AREA OF TECHNOLOGY

The present invention relates to fatty acids with one or more sites of unsaturation having an odd number of carbon atoms in the hydrocarbon chain, wherein the chemical structure of said fatty acids corresponds to the chemical structure of metabolites of mono- or polyunsaturated alpha-hydroxylated fatty acids. The present invention also relates to compositions containing said fatty acids with an odd number of carbon atoms in the chain, to their medical applications, as well as to their use as indicators of efficacy for treating a patient with mono- or polyunsaturated alpha-hydroxylated fatty acids, the metabolites of which they are.

LEVEL OF TECHNOLOGY

It is known that changes in the lipid composition of membranes affect cellular signaling, which can lead to the development of diseases, or to their regression, as well as prevent them. Similarly, therapeutic interventions aimed at regulating membrane lipid levels can prevent and cause regression of pathological processes (cure them).

In general, fatty acids with an odd-carbon chain structure are not considered to be therapeutically significant because in humans and mammals in general, the vast majority of fatty acids present have an even-carbon chain, typically 14 to 24 carbon atoms, and the presence of odd-carbon chain fatty acids is very rare and limited to trace amounts.

Currently, the data available in the scientific literature indicate that small structural differences in fatty acids have an important impact on biological activity and therefore on therapeutic activity. It is well known that many drugs have known adverse effects or are toxic to various cells or tissues. Prodrugs are compounds that, when ingested, undergo metabolic reactions and are converted into a drug or medicament, its metabolite, which have an effect on the health of the patient or subject.

Thus, on the one hand, the administration of a therapeutically active compound as a prodrug allows the distribution and absorption of said compound to be modulated over time, since its metabolism ensures the formation of the drug, i.e. the metabolite, only in those cells or tissues in which metabolic reactions occur that ensure the conversion of said prodrug into its active metabolite. In this regard, these prodrugs have other advantages, such as the possibility of delayed or controlled administration of the active metabolite, avoiding its accumulation, which can have an adverse effect on the body. On the other hand, the identification and synthesis of said therapeutically active metabolites allow a more intense action, which makes it possible to administer higher therapeutically active doses and in controlled time frames than those that would occur with spontaneous metabolism of the corresponding prodrug. Thus, the aim of the present invention is to provide therapeutically active compounds (metabolites) which are derived from other compounds or prodrugs, so that the administration of such metabolites, alone, via their prodrugs or in combination with their corresponding prodrugs, allows modulation of the therapeutic effect and possible adverse side effects, depending on the condition of the patient and the pathological condition to be treated.

BRIEF DESCRIPTION OF THE INVENTION

A compound selected from the group consisting of: a compound of formula (II) or a pharmaceutically or nutraceutically acceptable salt or ester thereof:

and a compound of formula (III) or a pharmaceutically or nutraceutically acceptable salt or ester thereof:

where a is an integer from 1 to 14; b is an integer from 1 to 7; c is 0, 3, or 6 and m=0; and where a+3b+c+3 is an even integer.

In particular, the present invention relates to a compound selected from the group consisting of:

compounds of formula (II) or a pharmaceutically or nutraceutically acceptable salt or ester thereof:

where: a=6, b=1 and c=6; or a=6, b=2 and c=3; or a=6, b=3 and c=0; or a=3, b=3 and c=3; or a=2, b=4 and c=3; or a=2, b=5 and c=0;

and a compound of formula (III) or a pharmaceutically or nutraceutically acceptable salt or ester thereof:

where a=1, b=6, c=0 and m=0.

The present invention also relates to a compound selected from the group consisting of a compound of formula (II) and a compound of formula (III) as described herein, for use as a medicine and in particular for use in inducing neuroregeneration and in preventing and/or treating (including supportive treatment) a disease or pathology selected from the group consisting of: a neurological or neurodegenerative disease; cancer; neoplasm; inflammatory disease; cardiovascular disease; pathology of the skin and subcutaneous tissue; metabolic pathology; neuropathic pain; paralysis; sleep disorders; digestive pathology; musculoskeletal and connective tissue disease; genitourinary pathology; and metabolic disease.

The present invention also relates to a pharmaceutical or nutraceutical composition comprising at least a first compound selected from the group consisting of a compound of formula (II) and a compound of formula (III), and optionally a compound of formula (I), as described herein.

Finally, the present invention relates to an in vitro method for determining the efficacy of a therapeutic or preventive treatment of a disease or pathology with a compound of formula (I) or a pharmaceutically acceptable salt or ester thereof in a subject, wherein said method comprises determining in vitro in a biological sample of said subject an amount of a compound of formula (II) or formula (III) as described herein, or a carboxylate anion thereof, or a derivative formed therefrom in vivo or in vitro, wherein said amount is associated with the efficacy of treating said disease or pathology.

DESCRIPTION OF THE INVENTION

The present invention relates to a compound selected from the group consisting of: a compound of formula (II) or a pharmaceutically or nutraceutically acceptable salt or ester thereof:

and a compound of formula (III) or a pharmaceutically or nutraceutically acceptable salt or ester thereof:

where a is an integer from 1 to 14; b is an integer from 1 to 7; c is an integer from 0 to 14; m is an integer from 0 to (b-1); and where a+3b+c+3 is an even integer.

All values of a, b, c and m according to the present invention are integers greater than or equal to zero. Once the value of b is defined in a formula, the value of m is defined as an integer from 0 to (b-1), where the specified value of b is a value that has already been defined as being from 1 to 7. In the context of the present invention, when a specific formula or composition is not specified, the values of a, b, c and m apply to all formulas and compositions described herein.

In one embodiment of the invention, a is an integer from 1 to 7; b is an integer from 2 to 7; c is 0, 3, or 6, m is 0, and a+3b+c+3 is an even integer. In another embodiment of the invention, a is an integer from 1 to 14; b is 1; c is 0, 3, or 6, m is 0; and where a+3b+c+3 is an even integer.

Most preferably, for all embodiments of the present invention, m=0 and thus the present invention relates to a compound selected from the group consisting of: a compound of formula (II) or a pharmaceutically or nutraceutically acceptable salt or ester thereof:

and a compound of formula (III) or a pharmaceutically or nutraceutically acceptable salt or ester thereof:

where a is an integer from 1 to 14; b is an integer from 1 to 7; c is 0, 3, or 6; and where a+3b+c+3 is an even integer.

A preferred embodiment of the invention relates to a pharmaceutically or nutraceutically acceptable salt or ester of a compound of formula (II) or formula (III). More preferably, said salt is a sodium salt and said ester is a methyl or ethyl ester.

The compounds of formula (II) or formula (III) according to the present invention correspond to the formulas of the metabolites of the compound of formula (I) or a pharmaceutically or nutraceutically acceptable salt or ester thereof:

where a is an integer from 1 to 14; b is an integer from 1 to 7; c is an integer from 0 to 14; m is an integer from 0 to (b-1); and where a+3b+c+3 is an even integer.

Thus, the compounds of formula (I) are mono- or polyunsaturated alpha-hydroxylated fatty acids with an even number of carbon atoms (a+3b+c+3 represents an even integer).

On the one hand, 2-hydroxy monounsaturated fatty acids of formula (I) having a chain with an even number of carbon atoms are prodrugs of other monounsaturated fatty acids of formula (II) having a chain with an odd number of carbon atoms, since these prodrugs undergo a decarboxylation process.

On the other hand, 2-hydroxypolyunsaturated fatty acids of formula (I) having a chain with an even number of carbon atoms are prodrugs of other mono- or polyunsaturated fatty acids having a chain with an odd number of carbon atoms, where in the case where decarboxylation occurs but no hydrogenation of one of the double bonds of the compound of formula (I) occurs, the compound obtained from the prodrug of formula (I) will be a compound of formula (II), while in the case where hydrogenation of one of the double bonds and decarboxylation of the compound of formula (I) occurs, the compound obtained from the prodrug of formula (I) will be a compound of formula (III), in which the hydrogenated double bond may be in a different position depending on the value of m.

By way of illustration, Figure 1A and Figure 8F show a metabolic pathway for the α-oxidation of 2-hydroxydocosahexaenoic acid (DHA-H), a compound of formula (I), at the cellular level to produce (6Z,9Z,12Z,15Z,18Z)-heneicosa-6,9,12,15,18-pentaenoic acid (HPA), its metabolite of formula (III). According to this pathway, the prodrug DHA-H is a 2-hydroxylated polyunsaturated fatty acid that is converted to HPA via a sequence of metabolic steps: (1) activation of DHA-H by conjugation with coenzyme A; (2) cleavage mediated by 2-hydroxyacyl-CoA lyase to form an odd-carbon aldehyde; (3) the action of aldehyde dehydrogenase on the aldehyde, hydrogenating one of the double bonds, transforming it into an acid (HPA).

Thus, the HPA of the invention is a compound of formula (III), where m=0, if the HPA is a metabolite of a prodrug of formula (I), where a=1, b=6, c=0, which is an omega-3 polyunsaturated alpha-hydroxylated fatty acid with 22 carbon atoms and 6 conjugated double bonds (DHA-H). However, the HPA can be a compound of formula (II), for cases where the HPA is a metabolite of a prodrug of formula (I), where a=4, b=5 and c=0, which is an omega-3 polyunsaturated alpha-hydroxylated fatty acid with 22 carbon atoms and 5 double bonds (2-hydroxydocosapentaenoic acid). One embodiment of the present invention relates to a compound of formula (III), where a=1, b=6, c=0 and m=0.

In addition, Figure 8 shows metabolic schemes of other fatty acids of formula (I). In particular, Figure 8A shows a metabolic scheme of 2-hydroxylinoleic acid (LA-H), a compound of formula (I) in which a=6, b=2 and c=3, to yield, after decarboxylation, (8Z,11Z)-heptadeca-8,11-dienoic acid (HDA, C17:2 ω-6), which is a compound of formula (II) in which a=6, b=2 and c=3. Figure 8B shows a metabolic pathway for 2-hydroxy-alpha (α)-linolenic acid (ALA-H), which is a compound of formula (I) in which a=6, b=3 and c=0, to yield, after decarboxylation, (8Z,11Z,14Z)-heptadeca-8,11,14-trienoic acid (HTA ω-3, C17:3 ω-3), which is a compound of formula (II) in which a=6, b=3 and c=0. In turn, Figure 8C shows a metabolic scheme of 2-hydroxy-gamma-(γ)-linolenic acid (GLA-H), which is a compound of formula (I), in which a=3, b=3 and c=3, to yield, after decarboxylation, (5Z,8Z,11Z)-heptadeca-5,8,11-trienoic acid (HTA ω-6, C17:3 ω-6), which is a compound of formula (II), in which a=3, b=3 and c=3. Figure 8D shows a metabolic pathway for 2-hydroxyarachidonic acid (ARA-H), which is a compound of formula (I) in which a=2, b=4 and c=3, to yield, after decarboxylation, (4Z,7Z,10Z,13Z)-nanodeca-4,7,10,13-tetraenoic acid (NTA, C: 19:4 ω-6), which is a compound of formula (II) in which a=2, b=4 and c=3. Finally, Figure 8E shows a metabolic pathway for 2-hydroxyeicosapentaenoic acid (EPA-H), which is a compound of formula (I) in which a=2, b=5 and c=0, to yield, after decarboxylation, (4Z,7Z,10Z,13Z,16Z)-nonadeca-4,7,10,13,16-pentaenoic acid (NPA, C19:5 ω-3), which is a compound of formula (II) in which a=2, b=5 and c=0.

In turn, according to the metabolic scheme shown in Figure 11, the compounds of formula (I) can also be monounsaturated alpha-hydroxylated fatty acids with an even number of carbon atoms. According to this pathway, the prodrug, sodium salt of 2-hydroxyoleic acid (2OHOA), is a 2-hydroxylated monounsaturated fatty acid, which is converted to 8Z-heptadecenoic acid (8Z-heptadecenoic or C17:1n-9) through a sequence of metabolic steps: (1) activation by acyl-CoA ligase in a process dependent on ATP (adenosine triphosphate) and magnesium (Mg ²⁺ ); (2) 2OHPA-CoA will undergo 2-hydroxyphytanoyl-CoA lyase (2-hydroxyacyl-CoA lyase 1, HACL1) activity to form a monounsaturated aldehyde intermediate; (3) the aldehyde dehydrogenase enzyme will be responsible for converting said aldehyde intermediate to 8Z-heptadecenoic acid in a NAD ⁺ (nicotinamide adenine dinucleotide) dependent process. Thus, 8Z-heptadecenoic acid according to the invention is a compound of formula (II), where a=6, b=1 and c=6, which is an omega-9-monounsaturated alpha-hydroxylated fatty acid of 17 carbon atoms obtained by metabolizing 2-hydroxyoleic acid or a pharmaceutically acceptable salt or ester thereof, which is a prodrug of formula (I).

Although the medical uses of the compounds of formula (I) are known, the present invention describes the formula of their metabolites, compounds of formula (II) or formula (III), which provide a specific and differentiated therapeutic effect, in the body, after the metabolism of said compounds of formula (I), which therefore act as their prodrugs. Thus, the present invention provides a method for adapting therapeutic treatment depending on the nature of the disease and the prognosis of the patient to be treated.

In particular, the present invention discloses specific formulas of metabolites of alpha-hydroxylated, mono- or polyunsaturated fatty acids, which are therapeutically effective. Thus, the present invention further describes the uses as a medicine of said metabolites of formula (II) or formula (III), as such, allowing to control the administered amount; or their use as a medicine in combination with their prodrug of formula (I); or their use as a medicine by administering said prodrug of formula (I), allowing to adjust the intensity and dose administered over time during treatment. In addition, the administration of compounds of formula (II) or formula (III) by administering their prodrug of formula (I) thus allows to modulate the distribution and absorption of the drug, since its metabolism allows to obtain the corresponding drug only in those cells or tissues in which metabolic reactions occur, converting said prodrug, with the production of the active compound in said cells. In any case, this description applies to both compounds of formula (II) and compounds of formula (III), regardless of whether they are obtained by chemical synthesis (see example 1) or in the process of metabolism of compounds of formula (I).

In this regard, in the context of the present invention, the term "metabolites" is used to designate such compounds of formula (II) or formula (III), regardless of whether they are obtained as a result of metabolism of a compound of formula (I) in the body of a subject or whether such compounds of formula (II) or formula (III) are products obtained by a synthetic route. Thus, in the context of the present invention, the term "compound of formula (I)" is used interchangeably with the term "prodrug of formula (I)" and, similarly, the terms "compound of formula (II)" and "compound of formula (III)" are used interchangeably with, respectively, the terms "metabolite of formula (II)" and "metabolite of formula (III)", since said compounds of formula (II) and formula (III), regardless of whether they are obtained by chemical synthesis or are a result of natural metabolism of a compound of formula (I), have the chemical structure or the chemical formula of a metabolite of a compound of formula (I), which therefore acts as a prodrug thereof.

In order to illustrate the invention, the examples of the present invention demonstrate how the therapeutic effect exerted by the prodrugs of formula (I) is directly related to the therapeutic effect exerted by the metabolite of formula (II) or formula (III) on the body, and also demonstrate the therapeutic effect exerted by the compounds of formula (II) and formula (III) as such. Thus, as shown in Example 7.3, the administration of the sodium salt of 2-hydroxyoleic acid (OHOA), a compound of formula (I), led to a greater reduction in the size of xenograft tumors in mice, the greater the cellular accumulation of the metabolite C17:1n-9 of formula (II). Thus, the therapeutic effect of the sodium salt of 2OHOA is partly due to its conversion to the metabolite C17:1n-9. In turn, Example 6.2 of the present invention demonstrates that the formation of the metabolite C17:1n-9 (8Z-heptadecenoic acid) as a result of 2OHAO incorporation differs in tumor and non-tumor cells. Glioma cells showed a significant increase in their levels of C17:1n-9 compared to 2OHAO (Figure 13B and D), while in non-tumor cells the detected levels of 2OHAO were significantly higher than those of its metabolite C17:1n-9. Furthermore, the Examples and Figures demonstrate that the antiproliferative effect mediated by the compounds of formula (I), exemplified by DHA-H, is mediated at least in part by its metabolite HPA (the compound of formula (III)), since inhibition of the formation of this compound from DHA-H results in a lower antiproliferative effect of DHA-H (Figure 4B). These results show that the therapeutic effect of the compounds of formula (I) is partly due to the biological activity of their metabolite of formula (II) or formula (III), and that said compound of formula (I) acts as a true prodrug of the compound of formula (II).

In turn, administration of a compound of formula (II) or formula (III), such as HPA, under the same experimental conditions as administration of a compound of formula (I), such as DHA-H, provides HPA levels that are an order of magnitude higher than those mediated by DHA-H, suggesting that the therapeutic activity of HPA may be greater than that of DHA-H. This effect is due to structural differences between the prodrug of formula (I) (DHA-H) and its metabolite of formula (III) (HPA). In fact, the present invention shows that the uptake of the alpha-hydroxylated form of DHA (DHA-H) is prevented compared to the uptake of the non-hydroxylated analog (Figure 5C). If the uptake of DHA-H is prevented compared to non-hydroxylated fatty acids, the different intracellular HPA levels observed following administration of DHA-H and HPA must be due to the different uptake of these compounds by cells from the extracellular environment. In addition, as shown in Example 6.3, the IC50 values of the metabolite C17:1n-9 were lower than those of its prodrug 2ОНОА, confirming its greater antiproliferative activity. Furthermore, as shown in Figure 3B, tumor HPA levels were inversely correlated with tumor size in xenograft models. Thus, the therapeutic effect of the compound of formula (I), a prodrug, is enhanced by elevated metabolite levels.

In turn, as shown in Figure 5, treatment with the sodium salt of HPA (a metabolite obtained by chemical synthesis as described in Example 1) causes a much more pronounced degree of mortality in the culture than treatment with the sodium salt of DHA-H (a prodrug) or the sodium salt of DHA (a natural analogue), under the same conditions. Thus, the introduction of a metabolite allows for a more pronounced therapeutic effect than that obtained with the introduction of a prodrug.

However, as shown in Example 6.3, C17:1n-9 exerted an antiproliferative effect when administered directly in place of its prodrug in both tumor cells and non-tumor cells, whereby administration of said metabolite of formula (II), C17:1n-9, via its prodrug of formula (I), 2OHOA, provides a selective way of achieving a therapeutic effect, allowing for a longer period of time for said therapy without causing undesirable adverse effects, and is equally applicable in maintenance therapy. In particular, odd-chain fatty acids are metabolized by β-oxidation to produce propionyl-CoA, in contrast to even-chain fatty acids, whose metabolism results in the production of acetyl-CoA, which in turn is metabolized via the Krebs cycle. Propionyl-CoA can be converted to propionic acid, which, when it accumulates, causes, as an adverse effect, metabolic acidosis. Propionyl-CoA can be metabolically converted to succinyl-CoA (which is metabolized via the Krebs cycle) in a process dependent on biotin and vitamin B12. This process is not a normal fatty acid metabolic pathway, since the vast majority of fatty acids in mammals have an even-number chain. Accordingly, this metabolic pathway acts selectively on polyunsaturated fatty acids with an odd number of carbon atoms in the chain, such as the metabolites of formula (II) and formula (III), and can be saturated in the case of the presence of too high intracellular concentrations of such metabolites with an odd number of carbon atoms in the chain, or in the case of certain pathological situations leading to a deficiency of biotin or vitamin B12, which leads to the above-mentioned adverse effect expressed in propionic acidosis.

Thus, the metabolite administered directly to the cells has a toxin, which in some cases is undesirable. This toxicity can be modulated by administering a prodrug or a compound of formula (I), which allows the action and toxicity of the metabolite of formula (II) or (III) to be regulated. In this regard, a slower absorption of the prodrug compared to non-hydroxylated fatty acids can be useful in preventing excessively high intracellular concentrations of the metabolite and, therefore, a possible accumulation of propionic acid. Thus, depending on the metabolic state, the administration regimen and the pathology to be treated, and in particular in cases where a more intense therapeutic effect is required, or in a treatment with a reduced duration, it may be desirable to use a metabolite of formula (II) or formula (III) or a pharmaceutically acceptable salt or ester thereof; while in other cases, such as in long-term treatment or maintenance treatment, it may be desirable to employ time-controlled administration using a prodrug of formula (I) or a pharmaceutically acceptable salt or ester thereof, such as sodium DHA-H. Thus, administration of metabolites of formula (II) or formula (III) using even-chain compounds of formula (I) as prodrugs allows for time-controlled administration of their odd-chain metabolites of formula (II) or formula (III).

For all these reasons, the present invention shows how the administration of compounds of formula (II) or formula (III) via the administration of their prodrugs of formula (I) allows for the time-controlled administration of their odd-carbon metabolites.

Thus, the present invention allows to adapt the therapy, depending on the nature of the disease and the prognosis of the patient to be treated, for the use of a metabolite or a compound of formula (I), i.e. a prodrug, as a medicinal product. On the one hand, in cases where short-term therapeutic activity of acute conditions is required, the use of a metabolite would be more appropriate or a priority in order to obtain a rapid and significant effect. In turn, when long-term treatment is required, for example in chronic diseases, or if maintenance treatment is required, it is possible to recommend or give priority to the use of a prodrug or compositions containing a combination of a prodrug and a metabolite in different ratios, depending on the time and situation/severity of the disease.

Thus, in general, the use of compounds of formula (II) and (III) and their pharmaceutically or nutraceutically acceptable salts is beneficial for the body, as shown in the examples of the present application. The action of these compounds of formula (II) and (III), by administering the corresponding prodrug of formula (I), allows to avoid unfavorable effects caused by their metabolism and accumulation, when long-term or high-dose administration is required, when administering said compounds of formula (II) and (III) in a controlled manner, as a result of their metabolism. Thus, prodrugs having formula (I) provide a method of administering metabolites of formula (II) or formula (III) with a lower risk of occurrence of unfavorable side effects and provide a therapeutically effective amount of said metabolites in a stable manner over time, since the prodrug having formula (I) is metabolized after administration. Thus, depending on the metabolic state, the administration regimen and the pathology to be treated, it may be desirable to directly administer the compound of formula (II) or formula (III) or a pharmaceutically acceptable salt or ester thereof (metabolites), or it may be desirable to employ time-controlled administration using a prodrug of formula (I) or a pharmaceutically acceptable salt or ester thereof (prodrug), or a combination thereof (prodrug + metabolites).

Thus, one aspect of the invention relates to a compound selected from the group consisting of a compound of formula (II) or a pharmaceutically acceptable salt or ester thereof:

and a compound of formula (III) or a pharmaceutically acceptable salt or ester thereof:

where a is an integer from 1 to 14; b is an integer from 1 to 7; c is an integer from 0 to 14; m is an integer from 0 to (b-1); and where a+3b+c+3 is an even integer, for use as a medicinal product, and in particular for use in inducing neuroregeneration and in preventing and/or treating (including supportive treatment) a disease or pathology selected from the group consisting of: a neurological or neurodegenerative disease; cancer; neoplasm; inflammatory disease; cardiovascular disease; skin and subcutaneous tissue pathology; metabolic pathology; neuropathic pain; paralysis; sleep disorders; digestive pathology; musculoskeletal and connective tissue disease; genitourinary pathology; and metabolic disease.

In the context of the present invention, the term "maintenance treatment" or "maintenance therapy" is defined as a therapeutic treatment administered as an adjunct to primary treatment or therapy, with the aim of preventing or delaying the recurrence of a disease that has been completely or partially stopped after the primary treatment or therapy, or to slow down the progression of the disease after the end of primary therapy.

Preferably, the present invention relates to a compound of formula (II) or a compound of formula (III), or a pharmaceutically acceptable salt or ester thereof, for use in the treatment or supportive care of a disease or pathology, and more preferably for the treatment or supportive care of cancer.

Thus, one embodiment of the invention relates to the use of a compound selected from the group consisting of a compound of formula (II) or a pharmaceutically acceptable salt or ester thereof and a compound of formula (III) or a pharmaceutically acceptable salt or ester thereof, as described herein, for the manufacture of a medicament for inducing neuroregeneration or for the prevention and/or treatment (including supportive treatment) of a disease or pathology selected from the group consisting of: a neurological or neurodegenerative disease; cancer; neoplasm; inflammatory disease; cardiovascular disease; pathology of the skin and subcutaneous tissue; metabolic pathology; neuropathic pain; paralysis; sleep disorders; digestive pathology; musculoskeletal and connective tissue disease; genitourinary pathology; and metabolic disease.

Another embodiment of the present invention relates to a method for preventing and/or treating a disease or pathology, or to a method for inducing neuroregeneration in a patient, wherein said method comprises administering to said patient an effective amount of a compound selected from the group consisting of a compound of formula (II) or a pharmaceutically acceptable salt or ester thereof:

and a compound of formula (III) or a pharmaceutically acceptable salt or ester thereof:

where a is an integer from 1 to 14; b is an integer from 1 to 7; c is an integer from 0 to 14; m is an integer from 0 to (b-1); and where a+3b+c+3 is an even integer.

In the context of the present invention, an effective amount or a therapeutically effective amount is understood to be an amount that provides a therapeutic effect without causing unacceptable toxic effects to the patient. An effective amount or dose of a drug depends on the compound and the condition or disease being treated, and, for example, on the age, body weight and clinical condition of the patient being treated, the form of administration, the clinical history of the patient, the severity of the disease and the effectiveness of the compound being administered.

Furthermore, one embodiment of the invention relates to a compound selected from the group consisting of a compound of formula (II):

and compounds of formula (III):

for use in the prevention and/or treatment of a disease or pathology, or for the induction of neuroregeneration, wherein the prevention and/or treatment, or the induction of neuroregeneration, is characterized by the administration of a compound or prodrug of formula (I), or a pharmaceutically acceptable salt or ester thereof:

and wherein said compound of formula (I) is metabolized to produce a therapeutically effective amount of:

compounds of formula (II):

or compounds of formula (III):

and where a is an integer from 1 to 14; b is an integer from 1 to 7; c is an integer from 0 to 14; m is an integer from 0 to (b-1); and where a+3b+c+3 is an even integer.

Preferably, said disease is selected from the group consisting of: a neurological or neurodegenerative disease; cancer; neoplasm; inflammatory disease; cardiovascular disease; skin and subcutaneous tissue pathology; metabolic pathology; neuropathic pain; paralysis; sleep disorders; digestive pathology; musculoskeletal and connective tissue disease; genitourinary pathology; and metabolic disease.

Thus, another aspect of the present invention relates to a method of administering an effective amount of a compound of formula (II) or a compound of formula (III), as described herein, for the prevention and/or treatment of a disease or pathology, or for the induction of neuroregeneration and/or the prevention of neurodegeneration, wherein said compound of formula (II) or said compound of formula (III) is administered as a prodrug of formula (I) or as a pharmaceutically acceptable salt or ester thereof, as described herein.

The invention also relates to a method for preventing and/or treating a disease or pathology, wherein said method comprises administering an effective amount of a prodrug of a compound of formula (II) or a pharmaceutically acceptable salt or ester thereof; or a prodrug of a compound of formula (III) or a pharmaceutically acceptable salt or ester thereof, as described herein; wherein said prodrug of compounds of formula (II) or formula (III) has formula (I) or is a pharmaceutically acceptable salt or ester thereof, as described herein.

Furthermore, the present invention relates to a method for preventing and/or treating a disease or pathology, or inducing neuroregeneration and/or preventing neurodegeneration, wherein said method comprises administering to a patient in need thereof an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or ester thereof:

wherein the compound of formula (I) is metabolized in the body of said patient to produce a therapeutically effective amount of the metabolite:

- having the formula (II):

or

- having formula (III):

where a is an integer from 1 to 14; b is an integer from 1 to 7; c is an integer from 0 to 14; m is an integer from 0 to (b-1); and a+3b+c+3 is an even integer, wherein said metabolite provides prevention and/or treatment of said disease or pathology and induction of neuroregeneration and/or prevention of neurodegeneration in a patient. Preferably, when an effective amount of a compound of formula (I) is administered, a metabolite having formula (II) or having formula (III) is present in the body of said patient.

In a preferred embodiment, said compound of formula (I) is metabolized by more than 1%, 10%, more than 40%, more than 50% and up to 99% into a metabolite of formula (II) or formula (III) upon administration.

Another embodiment relates to a method for preventing and/or treating a disease or pathology selected from the group consisting of: a neurological or neurodegenerative disease; cancer; neoplasm; inflammatory disease; cardiovascular disease; skin and subcutaneous tissue pathology; metabolic pathology; neuropathic pain; paralysis; sleep disorders; digestive pathology; musculoskeletal and connective tissue disease; genitourinary pathology; and a metabolic disease, and for inducing neuroregeneration and/or preventing neurodegeneration; wherein said method comprises administering to a patient an effective amount of a prodrug having a structure of formula (I), or a pharmaceutically acceptable salt or ester thereof:

wherein said prodrug is converted in vivo to release the active compound into the cells of said patient; wherein said active compound has the structure:

- formulas (II):

or

- formulas (III):

where a is an integer from 1 to 14; b is an integer from 1 to 7; c is an integer from 0 to 14; m is an integer from 0 to (b-1); and a+3b+c+3 is an even integer.

Preferably, said transformation is a chemical or physiological process. In the context of the present invention, the term "chemical process" refers to the transformation of a prodrug in vivo with the release of an active compound by means of a chemical reaction, where the prodrug is a reactant or substrate of the chemical reaction, and the active compound is the reaction product. Furthermore, in the context of the present invention, the term "physiological process" refers to the transformation due to an event or process occurring naturally in the body, for example, due to enzyme activity.

Furthermore, although the compounds of formula (I) exert their therapeutic effect through their metabolites of formula (II) or formula (III), said compounds of formula (I) also exhibit biological activity independent of said metabolic pathway, as shown in Example 6.4 of the present invention.

Thus, another embodiment of the invention relates to a compound selected from the group consisting of a compound of formula (II) and a compound of formula (III), or a pharmaceutically acceptable salt or ester thereof, as described herein, for use as a medicine and, in particular, for use in the induction of neuroregeneration and/or the prevention of neurodegeneration, and/or for use in the prevention and/or treatment of a disease or pathology, according to the present invention, characterized in that said compound is administered before, after or together with a compound of formula (I), or a pharmaceutically acceptable salt or ester thereof:

where a is an integer from 1 to 14; b is an integer from 1 to 7; c is an integer from 0 to 14 and a+3b+c+3 is an even integer; and where said values of a, b and c are equal to or different from the values of a, b and c of the compound of formula (II) or the compound of formula (III).

Furthermore, the present invention relates to a method for inducing neurodegeneration and/or preventing neurodegeneration, or to a method for preventing and/or treating a disease or pathology, comprising administering to a patient an effective amount of a compound of formula (II) or a compound of formula (III), or a pharmaceutically acceptable salt or ester thereof, and where said method is characterized in that it comprises additional administration of a compound of formula (I) or a pharmaceutically acceptable salt or ester thereof, as described herein; and where said compound of formula (I) is administered before, after or together with said compound of formula (II) or formula (III).

In the context of the present invention, the term "prodrug" refers to a compound that, when administered to a subject, is converted by a metabolic process into a second therapeutically active compound.

In turn, the term “subject” refers, in the context of the present invention, to a human or an animal.

The term "pharmaceutically acceptable" refers, in the context of the present invention, to a compound or substance that is approved or authorized by a regulatory agency of the federal or state government or listed in the European, American or other recognized pharmacopeia for use in animals or humans. In the present specification, this term refers primarily to salts and esters of the compounds of formulas (I), (II) and (III) as defined in accordance with the present invention.

Thus, the term "pharmaceutically acceptable salt" refers to a salt of a compound that also possesses the desired pharmacological activity of the parent compound from which it is derived. Preferably, the pharmaceutically acceptable salt is the sodium salt.

In the context of the present invention, the term "ester" refers to any compound in which the hydroxyl group belonging to the carboxylic acid moiety has been replaced by an alkoxide group. In a preferred embodiment of the invention, the ester is a methyl or ethyl ester. More preferably, the ester is an ethyl ester.

The term "nutraceutically acceptable" refers, in the context of the present invention, to anything used in nutraceutical products. Thus, in the context of the present invention, the term "nutraceutical" or "nutraceutical composition" refers to a food supplement that is to be taken alone or in combination with other food products and that has a beneficial effect on the health of the subject who takes it, especially in the prevention of diseases. In the present description, this term refers especially to salts and esters of the compounds of formulas (I), (II) and (III), which are defined according to the present invention.

In the context of the present invention, the term "stereoisomer" refers to those compounds that have the same chemical formula and the same atomic sequence, but have different three-dimensional orientation in space, and includes R- and S-stereoisomers (for which the nomenclature (+) and (-) is also used), resulting from the presence of a chiral carbon atom, as well as E- and Z-stereoisomers (for which the nomenclature cis-/trans- is also used), resulting from the arrangement of substituents of carbon atoms that form a double bond. Thus, since the prodrugs of formula (I) contain a chiral carbon atom (the alpha carbon for the carboxyl group), the invention also includes two stereoisomers, R- and S-, as well as any mixture thereof, with respect to the configuration of said chiral carbon atom. In turn, since both the prodrugs of formula (I) and their metabolites of formula (II) or (III) contain C=C double bonds, the invention also includes all E- and Z-stereoisomers for each of their double bonds. In a preferred embodiment, all double bonds of the prodrug of formula (I), the compound of formula (II) and the compound of formula (III) have a cis-configuration. Thus, if the prodrug of formula (I) has a cis/trans (or E/Z) stereochemical configuration determined by its double bonds, then the metabolite of formula (II) or formula (III) will also have such a configuration for the double bonds contained therein.

In the context of the present invention, the term "comprises" indicates that it includes a group of certain features (for example, a group of features A, B and C) and is interpreted as meaning that it includes these features (A, B and C), but does not exclude the presence of other features (for example, features D or E), provided that they do not make the claim unfeasible. In addition, the terms "includes", "has" or "comprises", and their plural forms in the context of the present invention should be understood as synonyms for the term "comprises". In turn, if the term "consists of" is used, then the device/method/product does not have additional features other than those that follow the said term. In this regard, in the context of the present invention, the term "comprises" may be replaced by any of the terms "consists of" or "essentially consists of". Accordingly, the term "comprises" may refer to a group of features A, B and C, which may further include other features such as E and D, provided that such features do not render the claim unenforceable, but such term "comprises" also includes the situation in which the group of features "consists of" or "essentially consists of" A, B and C.

Furthermore, the present invention allows the administration of a compound of formula (I), in particular 2OHOA, or a pharmaceutically acceptable salt or ester thereof, more preferably the sodium salt of 2OHOA, in maintenance treatment (maintenance therapy), wherein said compound of formula (I) or a pharmaceutically acceptable salt or ester thereof is administered at different intervals over a certain period of time, wherein the cumulative concentration of its metabolite of formula (II) or formula (III) is a measure of the effectiveness of the treatment. Thus, said in vitro method for determining the effectiveness of treatment with a compound of formula (I) or a pharmaceutically acceptable salt or ester thereof comprises determining the amount of a compound of formula (II) or formula (III), or its carboxylate anion, or a derivative formed from it.

In this regard, in the context of the present invention, the term "biomarker" refers to a first compound or substance, or a derivative of said first compound or substance, which can be used to determine the response and/or efficacy of treatment with a second compound or substance. Thus, in the context of the present invention, metabolites of formula (II) or formula (III) can be used as biomarkers to determine the response and/or efficacy of treatment with a compound of formula (I).

Thus, another aspect of the invention relates to an in vitro method for determining the effectiveness of a therapeutic or preventive treatment of a disease or pathology or a treatment with the induction of neuroregeneration, with a compound of formula (I) or a pharmaceutically acceptable salt or ester thereof:

in a subject, wherein said method comprises determining in vitro in a biological sample of said subject an amount of a compound:

- formulas (II):

or

- formulas (III):

or its carboxylate anion, or a derivative formed therefrom in vivo or in vitro, wherein said amount is related to the effectiveness of the treatment; and wherein a is an integer from 1 to 14; b is an integer from 1 to 7; c is an integer from 0 to 14; m is an integer from 0 to (b-1); and where a+3b+c+3 is an even integer.

Thus, the said method includes determining the amount of a compound of formula (II) or formula (III), their corresponding carboxylate anions or a derivative formed from them.

Said derivative of the compound of formula (II) or (III) may be formed in vitro by reacting said compound of formula (II) or (III) contained in a sample in vitro with a substance to obtain a derivative thereof. In this case, the method according to the invention comprises determining the amount of said derivative of formula (II) or formula (III) formed in vitro. For example, some methods for detecting fatty acids require their prior chemical modification, and thus, detection by gas chromatography usually requires that a fatty acid sample (in this case, a compound of formula (II) or formula (III)) be converted to its corresponding methyl ester for detection and quantification.

In turn, the said derivative of the compound of formula (II) or formula (III) may be a metabolic derivative or a derivative formed in vivo (as a result of a reaction occurring in vivo), formed as a result of the reaction of the said compound of formula (II) or formula (III) with another lipid, protein, enzyme, nucleotide, carbohydrate, etc. Thus, said derivative may be an ester of said compound of formula (II) or formula (III), such as, for example, inter alia, a glycerophospholipid (such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid or any of its S-forms, such as lysophosphatidylcholine, lysophosphatidylethanolamine, etc.), a plasmalogen (alkyl or alkenyl), a cholesterol ester, a glycerolipid such as a triacylglyceride (triglyceride) or diglycerol, a cardiolipin, a sphingolipid, a thioester with a coenzyme (acyl-CoA) or an acylcarnitine. In this case, the method according to the invention comprises determining in vitro the amount of said metabolic derivative (or a derivative formed in vivo) in a biological sample.

Thus, the amount of said compound of formula (II), or formula (III), or their corresponding carboxylate anions, or a derivative formed from them in vivo or in vitro, is associated with the effectiveness of the treatment and/or prevention of a disease or pathology, or with a neuroregeneration-inducing treatment, in a subject with a compound of formula (I), wherein the levels of said compound of formula (II), or formula (III), or its carboxylate anion, or a derivative thereof, compared to a control group, are associated with the effectiveness of the therapeutic or preventive treatment of a disease or pathology with said compound of formula (I) or a pharmaceutically acceptable salt or ester thereof.

Another aspect of the present invention relates to the use of a compound:

- formulas (II):

or

- formulas (III):

or the corresponding carboxylate anion, or a derivative obtained therefrom in vivo or in vitro, for determining in vitro the efficacy of a therapeutic or preventive treatment of a disease or pathology, or a neuroregeneration-inducing treatment with a compound of formula (I) or a pharmaceutically acceptable salt or ester thereof:

in a subject, where a is an integer from 1 to 14; b is an integer from 1 to 7; c is an integer from 0 to 14; m is an integer from 0 to (b-1); and where a+3b+c+3 is an even integer.

More preferably, the disease or pathology is selected from the group consisting of a neurological or neurodegenerative disease; cancer; neoplasm; inflammatory disease; cardiovascular disease; skin and subcutaneous tissue pathology; metabolic pathology; neuropathic pain; paralysis; sleep disorders; digestive pathology; musculoskeletal and connective tissue disease; genitourinary pathology; and metabolic disease. Even more preferably, the disease or pathology is selected from a neurological or neurodegenerative disease; cancer; inflammatory disease; and metabolic disease.

In a more preferred embodiment, the method determines the efficacy of treatment with a pharmaceutically acceptable salt of a compound of formula (I), and even more preferably a sodium salt of a compound of formula (I).

In one embodiment of the invention, the biological sample is a blood sample (including plasma or serum), a urine sample, a saliva sample, a tissue biopsy, cerebrospinal fluid, or a sweat sample.

The present invention also relates to a pharmaceutical composition comprising at least a first compound selected from the group consisting of:

- compounds of formula (II) or a pharmaceutically acceptable salt or ester thereof:

And

- compounds of formula (III) or a pharmaceutically acceptable salt or ester thereof:

wherein said composition optionally comprises a second compound of formula (I) or a pharmaceutically acceptable salt or ester thereof:

and where a is an integer from 1 to 14; b is an integer from 1 to 7; c is an integer from 0 to 14; m is an integer from 0 to (b-1); and a+3b+c+3 is an even integer; and at least one pharmaceutically acceptable excipient.

The present invention also relates to a pharmaceutical composition comprising at least a first compound selected from the group consisting of:

- pharmaceutically acceptable salt or ester of the compound of formula (II):

And

- pharmaceutically acceptable salt or ester of the compound of formula (III):

wherein said composition optionally comprises a pharmaceutically acceptable salt or ester of a compound of formula (I):

and where a is an integer from 1 to 14; b is an integer from 1 to 7; c is an integer from 0 to 14; m is an integer from 0 to (b-1); and a+3b+c+3 is an even integer; and at least one pharmaceutically acceptable excipient.

Another embodiment of the invention relates to a pharmaceutical composition comprising at least a first compound selected from the group consisting of a compound of formula (II) and a compound of formula (III), wherein said composition optionally comprises a pharmaceutically acceptable salt or ester of a compound of formula (I), as described above; and at least one pharmaceutically acceptable excipient; for use as a medicine; and in particular for use in inducing neuroregeneration and/or preventing neurodegeneration, and for use in preventing and/or treating a disease or pathology selected from the group consisting of: a neurological or neurodegenerative disease; cancer; neoplasm; inflammatory disease; cardiovascular disease; skin and subcutaneous tissue pathology; metabolic pathology; neuropathic pain; paralysis; sleep disorders; digestive pathology; musculoskeletal and connective tissue disease; genitourinary pathology; and metabolic disease.

In a preferred embodiment of the invention, said at least first compound is a pharmaceutically acceptable salt or ester of a compound of formula (II) or a compound of formula (III), and/or said second compound is a pharmaceutically acceptable salt or ester of a compound of formula (I).

One embodiment of the invention relates to the use of a pharmaceutical composition comprising at least a first compound selected from the group consisting of a compound of formula (II) and a compound of formula (III), or a pharmaceutically acceptable salt or ester thereof, wherein said composition optionally comprises a compound of formula (I) or a pharmaceutically acceptable salt or ester thereof, as described above; and at least one pharmaceutically acceptable excipient; for the manufacture of a medicament for inducing neuroregeneration and/or preventing neurodegeneration, and/or for preventing and/or treating a disease or pathology.

Another embodiment of the invention relates to a method for preventing and/or treating a disease or pathology or for inducing neuroregeneration and/or preventing neurodegeneration; wherein said method comprises administering to a patient in need thereof an effective amount of a pharmaceutical composition comprising at least a first compound selected from the group consisting of a compound of formula (II) and a compound of formula (III), or a pharmaceutically acceptable salt or ester thereof, wherein said composition optionally comprises a compound of formula (I) or a pharmaceutically acceptable salt or ester thereof, as described above; and at least one pharmaceutically acceptable excipient.

Preferably, said disease or pathology is selected from the group consisting of: a neurological or neurodegenerative disease; cancer; neoplasm; inflammatory disease; cardiovascular disease; skin and subcutaneous tissue pathology; metabolic pathology; neuropathic pain; paralysis; sleep disorders; digestive pathology; musculoskeletal and connective tissue disease; genitourinary pathology; and metabolic disease.

One skilled in the art can select one or more pharmaceutically acceptable carriers or excipients known in the art so that the pharmaceutical compositions are suitable for administration to both a human and an animal subject.

In a preferred embodiment of the invention, said excipient is an albumin, for example: ovalbumin, lactalbumin, native or recombinant albumin of human, bovine, mouse or rabbit origin, more preferably human serum albumin or bovine serum albumin.

The pharmaceutical compositions disclosed in the present invention can also be administered together with, before or after additional therapy. Preferably, such additional therapy is radiation therapy, electric fields for treating tumors (fields for treating tumors), immunotherapy or chemotherapy. More preferably, the pharmaceutical compositions disclosed in the present invention can also be administered together with, before or after therapy comprising the administration of temozolomide.

Such administration may be part of the treatment of an adult or pediatric patient. In a preferred embodiment, said pharmaceutical composition is administered together with, before or after radiotherapeutic treatment, chemotherapeutic treatment, treatment with electrical fields for the treatment of tumors (fields for the treatment of tumors) or immunotherapeutic treatment.

In one embodiment of the invention, the pharmaceutical compositions disclosed herein comprise at least one additional therapeutic component or active compound. Said additional therapeutic component or active compound provides additive or synergistic biological activities. In the context of the present invention, the terms "active compound" or "therapeutic component" shall be understood as a chemical or biological compound that has a therapeutic effect when administered to humans or animals. Such an active compound or additional therapeutic component may be, inter alia, cell therapy, small molecule therapy, immunotherapy, radiation therapy.

Additional therapeutic components or active compounds include compounds for the treatment of neurodegenerative diseases, anti-cancer agents, compounds that regulate metabolism, cardiovascular agents, and agents that regulate obesity and overweight.

Also included among the therapeutic components or additional active compounds are compounds for the treatment of neurodegenerative diseases, chemotherapeutic agents, compounds regulating metabolism, cardiovascular agents and agents regulating obesity and overweight. Preferably, said active compound or said therapy is a chemotherapeutic agent, a cell therapy agent or an immunotherapeutic agent.

In a preferred embodiment, said pharmaceutical composition further comprises a chemotherapeutic agent selected from the group consisting of: platinum-based antitumor agents; antimitotic chemotherapeutic agents; polyadenosine diphosphate ribose polymerase (PARP) inhibitor; type I topoisomerase inhibitors; type II topoisomerase inhibitors; epothilones; agents acting on the cycloskeleton; alkylating agents; histone deacetylase inhibitors; kinase inhibitors; antifolates; peptide antibiotics; retinoids; vinca alkaloids and thymidylate synthase inhibitors. More preferably, the chemotherapeutic agent is selected from the group consisting of: bevacizumab, carmustine, cyclophosphamide, melphalan, ifosfamide, busulfan, temozolomide, mechlorethamine, chlorambucil, melphalan, dacarbazine, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valubicin, paclitaxel, docetaxel, abraxane, taxotere, epothilone, vorinostat, romidepsin, irinotecan, topotecan, camptothecin, exatecan, lurtotecan, etoposide, teniposide, tafluposide, bortezomib, erlotinib, gefitinib, imatinib, vemurafenib, vismodegib, azacitidine, azathioprine, capecitabine, cytarabine, cladribine, fludarabine, doxifluridine, fluorouracil, gemcitabine, hydroxyurea, mercaptopurine, methotrexate, pemetrexed, azathioprine, thioguanine, retinoic acid, bleomycin, actinomycin, carboplatin, cisplatin, oxaliplatin, tretinoin, alitretinoin, bexarotene, topotecan, vinblastine, vincristine, vindesine, and vinorelbine. More preferably, the additional chemotherapeutic agent is temozolomide.

In this regard, the fact that lipids, when integrated into the cell membrane, can control cell signaling suggests that they can also regulate the physiological state of cells and, therefore, the general state of health. Thus, the compounds disclosed herein are suitable for inducing neuroregeneration and for preventing and/or treating various diseases and pathologies, in particular selected from the group consisting of a neurological or neurodegenerative disease; cancer; neoplasm; inflammatory disease; cardiovascular disease; pathology of the skin and subcutaneous tissue; metabolic pathology; neuropathic pain; paralysis; sleep disorders; digestive pathology; diseases of the musculoskeletal system and connective tissue; pathology of the genitourinary system; and metabolic disease.

In the context of the present invention, diseases of the nervous system are all those diseases that affect the nervous system (both central and peripheral). This group includes neurodegenerative diseases, which in the context of the present invention are a heterogeneous group of disorders characterized by progressive degeneration of the structure and function of the central nervous system or the peripheral nervous system.

Preferably, the neurodegenerative disease is selected from the group consisting of spinal cord injury and pain of neurological origin. In the context of the present invention, the term "induction of neuroregeneration" refers to the regeneration of neurological functions. In turn, for the purposes of the present invention, the term "prevention of neurodegeneration" means that the treatment leads to a stop in an already ongoing neurodegenerative process, or that the treatment prevents the onset or progression of neurodegeneration.

Some of these neurodegenerative processes are associated with significant cognitive decline or motor impairment in patients. Neurodegenerative processes, neurological disorders and neuropsychiatric disorders have a common basis of degeneration of neurons or changes in their components, such as lipids (e.g. myelin) or membrane proteins (e.g. adrenergic receptors, serotonergic receptors, etc.).

In particular, the neurodegenerative diseases are selected from the group consisting of: (i) inflammatory diseases of the central nervous system such as bacterial meningitis, non-bacterial meningitis, acute hemorrhagic necrotizing encephalopathy, other encephalitis, myelitis and encephalomyelitis, unspecified (NOS) cerebral ventriculitis, intracranial and intravertebral abscess and granuloma, extradural and subdural abscess, phlebitis, intracranial thrombophlebitis in the spinal canal cavity and sequelae of inflammatory diseases of the central nervous system; (ii) systemic atrophies affecting mainly the central nervous system such as Guillain-Barré syndrome, diabetic neuropathy, Wallerian degeneration, dementia with Lewy bodies, frontotemporal dementia, Huntington's chorea, Huntington's dementia, hereditary ataxia; spinal muscular atrophy and related syndromes such as Werdnig-Hoffmann syndrome; systemic atrophies affecting mainly the central nervous system, post-polio syndrome; motor neuron diseases such as amyotrophic lateral sclerosis and progressive bulbar palsy; (iii) extrapyramidal and movement disorders such as Parkinson's disease, secondary parkinsonism, neuroleptic malignant syndrome, drug-induced secondary parkinsonism, postencephalitic parkinsonism, vascular parkinsonism, degenerative diseases of the basal ganglia, Hallervorden-Spatz syndrome, progressive supranuclear ophthalmoplegia, progressive supranuclear palsy, striatonigral degeneration, essential dystonic tremor, drug-induced tremor, myoclonus, drug-induced chorea, drug-induced tics, organic tics, drug-induced movement disorders, akathisia, restless legs syndrome, stiff person syndrome and benign tremor attacks; (iv) other degenerative diseases of the nervous system such as Alzheimer's disease, early-onset or late-onset Alzheimer's disease, frontotemporal dementia such as Pick's disease, alcohol-induced nervous system degeneration, Alpers' disease, Leigh disease, dementia with Lewy bodies, mild cognitive impairment, corticobasal degeneration, primary degenerative dementia including Alzheimer's dementia, senile and presenile forms, stroke; (v) demyelinating diseases of the central nervous system such as multiple sclerosis of the medulla oblongata, brain stem, disseminated, generalized or unspecified (NOS); acute disseminated demyelination, diffuse sclerosis of the central nervous system; (vi) episodic and paroxysmal disorders such as recurrent epilepsy and seizures, idiopathic epilepsy and seizures, grand mal seizure, non-specific atonic or clonic epilepsy, Lennox-Gastaut syndrome, epileptic spasms, epilepsy of unspecified type, migraine, headache, transient cerebral ischemic attacks and related syndromes, sleep disorders and vertigo; (vii) disorders of the nerves, nerve roots and plexuses such as trigeminal nerve disorders, facial nerve disorders, cranial nerve disorders, nerve root and plexus disorders, mononeuropathies of the upper or lower limbs and Wallerian degeneration; (viii) polyneuropathies and other disorders of the peripheral nervous system including hereditary and idiopathic neuropathies such as Roussy-Levy syndrome, Refsum disease; inflammatory polyneuropathy; sequelae of polyneuropathies such as sequelae of Guillain-Barré syndrome, serum neuropathy; other polyneuropathies such as drug-induced, alcoholic, toxic agent-induced, radiation neuropathy; complications of inflammatory and toxic polyneuropathies; (ix) diseases of the muscle and neuromuscular junction such as myasthenia gravis and other myoneural disorders, disorders of the muscle and neuromuscular junction; (x) cerebral palsy and other paralytic syndromes including hemiplegia, paraplegia, tetraplegia; (xi) other disorders of the nervous system such as complex regional pain syndrome, neuropathic pain, atonal disorders of the nervous system, peripheral autonomic neuropathy, hydrocephalus, brain cysts, Riley-Day syndrome, multisystem degeneration of the autonomic nervous system, hippocampal sclerosis, mesial sclerosis, diabetic neuropathy or Wolfham syndrome, adrenoleukodystrophy, leukodystrophy and spinal cord injury; and (xii) mental and behavioral disorders due to physiological conditions such as vascular dementia, unspecified dementia, depression; behavioral disorders associated with substance abuse; schizophrenia, schizotypal disorder, delusional disorder and other non-mood psychotic disorders; mood (affective) disorders such as manic episode, bipolar disorder, major depressive disorder, cyclothymic disorder, dysthymic disorder; anxiety disorder, dissociative, stress-related and other non-psychotic somatoform mental disorders; behavioral syndromes associated with physiological disorders and physical factors such as eating disorders, sleep disorders; personality disorders, impulsive disorder, pathological craving for gambling; mental retardation; speech disorders, writing disorders, learning disorders, disorders caused by psychoactive substance use and addictive behavior.

In the context of the present invention, "neuropathic pain" is defined as pain caused by damage or disease of the somatosensory nervous system, as defined by the International Association for the Study of Pain (IASP). The somatosensory nervous system contains sensory neurons and neural pathways that respond to changes on the surface or in the body. The term "paralysis" in the context of the present invention refers to a partial or complete loss of mobility in a certain part of the body caused by damage or disease of the central or peripheral nervous system. In turn, the term "sleep disorders" refers to those disorders that include problems in initiating and maintaining sleep caused by a disorder or pathology of the central or peripheral nervous system. Non-limiting examples of such sleep disorders include, among others, insomnia, hypersomnia such as narcolepsy, sleep apnea, restless legs syndrome, circadian rhythm disorders and parasomnia.

Some neurodegenerative diseases can lead to processes that lead to blindness, hearing problems, disorientation, mood disorders, etc. An example of a well-characterized neurodegenerative disorder is Alzheimer's disease, which is characterized by the formation of plaques, mainly formed by the β-amyloid peptide, which is formed by altered protein processing, with subsequent accumulation outside the cells. In addition, neurofibrillary tangles of hyperphosphorylated tau protein appear inside the cell. This process is associated with changes in cholesterol metabolism and subsequent changes in the levels of certain membrane lipids, such as docosahexaenoic acid. In turn, several neurodegenerative pathologies such as Parkinson's disease, Alzheimer's disease, senile dementia (or dementia with Lewy bodies) have been associated with the pathological accumulation of fibrillar aggregates of the protein α-synuclein, which lead to a significant change in the metabolism of cellular triglycerides. Indeed, the development of these and other neurodegenerative diseases is associated with changes in the serum or cellular levels of lipids such as cholesterol, triglycerides, sphingomyelin, phosphatidylethanolamine, etc. This, again, suggests that lipids play a crucial role in the proper functioning of neurons, glial cells, nerves, brain, cerebellum and spinal cord, which is logical given the large amount of lipids in the central nervous system.

Alzheimer's disease (AD) is a neurodegenerative disorder for which there is currently no effective therapy or cure and whose pathophysiology is still largely unknown. Over the past decade, numerous drugs and treatments have been created and developed to halt or slow down the neurodegenerative process that characterizes the disease. However, no treatment has yet been successfully evaluated in phase III clinical trials in humans. Most of the therapies were inspired by the amyloid cascade hypothesis, which is now being challenged by the near-total failure of anti-amyloid/tau therapy clinical trials.

In turn, various types of sclerosis and other neurodegenerative processes are associated with "demyelination", the end result of which is the loss of lipids in the sheath of neuronal axons, with subsequent changes in the process of propagation of electrical signals that it entails. Myelin is a lipid layer that surrounds the axons of many neurons and is formed by a series of helical folds of the plasma membrane of glial cells (Schwann cells and oligodendrocytes, at the peripheral and central level, respectively). For all these reasons, lipids have been shown to play an important role in the development of neurodegenerative diseases. In addition, natural polyunsaturated fatty acids have been shown to have a modest preventive effect on the development of neurodegenerative processes. Indeed, the most abundant lipid in the central nervous system is docosahexaenoic acid (DHA), the content of which is altered in many neurodegenerative processes, such as Alzheimer's disease.

Furthermore, the metabolic disease is preferably selected from the group consisting of obesity, overweight, hypercholesterolemia, hypertriglyceridemia, diabetes and insulin resistance. Metabolic diseases form a set of pathologies characterized by the accumulation or deficiency of certain molecules. A typical example is the accumulation of glucose, cholesterol and/or triglycerides above normal levels. Elevated levels of glucose, cholesterol and/or triglycerides, both at the systemic level (e.g. elevated plasma levels) and at the cellular level (e.g. in cell membranes), are associated with changes in cellular signaling leading to dysfunctions at various levels and are usually due to errors in the activity of certain enzymes or the regulation of such proteins. The most important metabolic disorders include hypercholesterolemia (high cholesterol levels) and hypertriglyceridemia (high triglyceride levels). These diseases are characterized by high morbidity, severity and mortality rates, so their treatment is a priority. Other important metabolic disorders are diabetes and insulin resistance, which are characterized by impaired glucose regulation. These metabolic pathologies are involved in the occurrence of other pathological processes, such as cancer, hypertension, obesity, atherosclerosis, etc. Another pathological process associated with the metabolic pathologies described above has been identified, which in itself may represent a new metabolic pathology, which is metabolic syndrome.

In the context of the present invention, a neoplasm is defined as an abnormal mass of tissue that occurs when cells multiply more than they should or are not destroyed at the appropriate time. Neoplasms are either benign (non-cancerous) or malignant (cancerous). The term "neoplasm" is equivalent to the term "tumor". There are many types of cancer, including, for example, cancer of the oral cavity and pharynx, cancer of other digestive organs, cancer of other respiratory organs, cancer of bone and articular cartilage, melanoma and other malignant neoplasms of the skin, cancer of the mesothelial and soft tissues, cancer of the genitourinary organs, cancer of the urinary tract, cancer of the eye, brain and other areas of the nervous system, cancer of the thyroid gland and other endocrine glands, neuroendocrine malignancies, cancer of lymphoid, hematopoietic and related tissues, carcinomas in situ, benign neoplasms, neoplasms of undetermined behavior, polycythemia vera and myelodysplastic syndromes, neoplasms of other sites, and neoplasms of undetermined behavior.

Lipid modification of the cell membrane may be used as a strategy for the prevention or treatment of several types of cancer. In one embodiment of the invention, the cancer is selected from the group consisting of: colon cancer, pancreatic cancer, bile duct cancer, neuroblastoma, colon cancer, gastric cancer, liver cancer, glioblastoma, non-Hodgkin's lymphoma, kidney cancer, esophageal cancer, gastric cancer, cervical cancer or lymphoma tumors, colorectal carcinoma, colorectal adenocarcinoma, prostate cancer, prostate adenocarcinoma, prostate carcinoma, breast cancer, breast carcinoma, breast adenocarcinoma, triple-negative breast cancer, brain cancer, brain adenocarcinoma, brain neuroblastoma, lung cancer, lung adenocarcinoma, lung carcinoma, small cell lung cancer, large cell lung cancer, ovarian cancer, ovarian carcinoma, ovarian adenocarcinoma, uterine cancer, gastroesophageal cancer, renal cell carcinoma, clear cell renal cell carcinoma, endometrial cancer, endometrial carcinoma, endometrial stromal sarcoma, cervical carcinoma, thyroid carcinoma, metastatic papillary thyroid carcinoma, follicular thyroid carcinoma, bladder carcinoma, bladder carcinoma, transitional cell carcinoma of the bladder, liver cancer, metastatic liver cancer, pancreatic cancer, neuroendocrine cancers, squamous cell carcinoma, osteosarcoma, rhabdomyosarcoma, embryonal cancers, glioblastoma, glioma, neuroblastoma, medulloblastoma, retinoblastoma, nephroblastoma, hepatoblastoma, melanoma, hematological malignancies such as leukemia, lymphoma and myeloma.

Preferably, the cancer is selected from the group consisting of lung cancer, brain cancer, glioma, glioblastoma, breast cancer, leukemia, liver cancer, endometrial cancer and pancreatic cancer. More preferably, the cancer is selected from the group consisting of lung cancer, brain cancer, breast cancer, leukemia, liver cancer and pancreatic cancer.

In the context of the present invention, a cardiovascular disease is defined as a group of diseases or disorders of the heart and blood vessels. Such cardiovascular diseases are selected from the group consisting of: cerebral ischemic stroke, acute rheumatic fever, chronic heart disease, hypertension, ischemic heart disease, pericarditis, endocarditis, valve disorders, cardiomyopathy, tachycardia, heart failure, amyloid angiopathy, cerebrovascular diseases and disorders, consequences of intracerebral hemorrhage, consequences of cerebral infarction, consequences of cerebrovascular diseases, diseases of arterial and capillary vessels; diseases of veins, vessels and lymph nodes.

The term "pathology of the skin and subcutaneous tissue" in the context of the present invention refers to pathologies of the skin tissue, including: bullous disorders, dermatitis, eczema, papulosquamous disorders, disorders of the skin appendages, postoperative complications, urticaria and erythema.

Inflammatory processes include a wide range of pathologies characterized by the presence of inflammation. In the context of the present invention, said inflammatory processes are selected from the group consisting of: cardiovascular inflammation; inflammation caused by tumors; inflammation of rheumatoid origin; respiratory inflammation; acute and chronic inflammation; inflammatory hyperalgesia; and edema and inflammation caused by trauma or burns.

The term "digestive pathology" in the context of the present invention refers to diseases of the oral cavity and salivary glands; diseases of the esophagus, stomach and duodenum; diseases of the appendix; non-infectious enteritis and colitis; diseases of the peritoneum and retroperitoneal space; diseases of the liver; diseases of the gallbladder, bile ducts and pancreas.

In the context of the present invention, a musculoskeletal and connective tissue disease refers to pathologies of muscles, joints and bones, which may or may not have an autoimmune origin. The said musculoskeletal and connective tissue diseases are selected from the group consisting of: arthropathies, connective tissue disorders, muscle and soft tissue disorders; synovial and tendon membrane disorders; osteopathies and chondropathies.

The term "urogenital pathology" in the context of the present invention refers to glomerular diseases; tubulointerstitial diseases of the kidneys; acute renal failure; chronic renal failure; lithiasis; and inflammatory and non-inflammatory disorders of the urinary system.

The present invention also relates to a nutraceutical composition comprising at least a first compound selected from the group consisting of:

- a compound of formula (II) or a nutraceutically acceptable salt or ester thereof:

And

- a compound of formula (III) or a nutraceutically acceptable salt or ester thereof:

wherein said composition optionally comprises a second compound of formula (I) or a nutraceutically acceptable salt or ester thereof:

and where a is an integer from 1 to 14; b is an integer from 1 to 7; c is an integer from 0 to 14; m is an integer from 0 to (b-1); and a+3b+c+3 is an even integer; and at least one nutraceutically acceptable excipient.

The present invention also relates to a nutraceutical composition comprising at least a first compound selected from the group consisting of:

- a nutraceutically acceptable salt or ester of a compound of formula (II):

And

- a nutraceutically acceptable salt or ester of a compound of formula (III):

wherein said composition optionally comprises a nutraceutically acceptable salt or ester of a compound of formula (I):

and where a is an integer from 1 to 14; b is an integer from 1 to 7; c is an integer from 0 to 14; m is an integer from 0 to (b-1); and a+3b+c+3 is an even integer; and at least one nutraceutically acceptable excipient.

The present invention also relates to a nutraceutical composition comprising at least a first compound selected from the group consisting of a compound of formula (II) and a compound of formula (III), or a nutraceutically acceptable salt or ester thereof, wherein said composition optionally comprises a compound of formula (I) or a nutraceutically acceptable salt or ester thereof, as described above, for use in preventing a disease or pathology.

Furthermore, the present invention also relates to a method for preventing a disease or pathology, wherein said method comprises administering to a subject an effective amount of a nutraceutical composition comprising at least a first compound selected from the group consisting of a compound of formula (II) and a compound of formula (III), or a nutraceutically acceptable salt or ester thereof, wherein said composition optionally comprises a compound of formula (I) or a nutraceutically acceptable salt or ester thereof, as described above.

Preferably, said disease or pathology is selected from the group consisting of: a neurological or neurodegenerative disease; cancer; neoplasm; inflammatory disease; cardiovascular disease; skin and subcutaneous tissue pathology; metabolic pathology; neuropathic pain; paralysis; sleep disorders; digestive pathology; musculoskeletal and connective tissue disease; genitourinary pathology; and metabolic disease.

Preferably, m=0, and each of the disclosed embodiments of the present invention, including those embodiments that relate to compounds of formula (II) or formula (III), pharmaceutical and nutraceutical compositions containing them, their first and second medical uses, methods for inducing neuroregeneration and/or preventing neurodegeneration, or methods for preventing and/or treating a disease or pathology, as well as the use and in vitro method for determining the effectiveness of the treatment, relates to a compound selected from the group consisting of a compound of formula (II) or a pharmaceutically or nutraceutically acceptable salt or ester thereof:

and a compound of formula (III) or a pharmaceutically or nutraceutically acceptable salt or ester thereof:

where a is an integer from 1 to 14; b is an integer from 1 to 7; c is 0, 3, or 6; and where a+3b+c+3 is an even integer.

Also preferably m=0, and each of the disclosed embodiments of the present invention, including those embodiments that relate to compounds of formula (II) or formula (III), pharmaceutical and nutraceutical compositions containing them, their first and second medical uses, methods for inducing neuroregeneration and/or preventing neurodegeneration, or methods for preventing and/or treating a disease or pathology, as well as the use and in vitro method for determining the effectiveness of treatment, relates to a pharmaceutically or nutraceutically acceptable salt or ester of a compound of formula (II):

or a pharmaceutically acceptable salt or ester of a compound of formula (III):

where a is an integer from 1 to 14; b is an integer from 1 to 7; c is 0, 3, or 6; and where a+3b+c+3 is an even integer.

Even more preferably, m=0, and each of the disclosed embodiments of the present invention, including those embodiments that relate to compounds of formula (II) or formula (III), pharmaceutical and nutraceutical compositions containing them, their first and second medical uses, methods for inducing neuroregeneration and/or preventing neurodegeneration, or methods for preventing and/or treating a disease or pathology, as well as the use and in vitro method for determining the effectiveness of the treatment, relates to a compound selected from the group consisting of:

- compounds of formula (II) or a pharmaceutically or nutraceutically acceptable salt or ester thereof:

where: a=6, b=1 and c=6; or a=6, b=2 and c=3; or a=6, b=3 and c=0; or a=3, b=3 and c=3; or a=2, b=4 and c=3; or a=2, b=5 and c=0;

And

- a compound of formula (III) or a pharmaceutically or nutraceutically acceptable salt or ester thereof:

where a=1, b=6 and c=0.

Also more preferably, m=0, and each of the disclosed embodiments of the present invention, including those embodiments that relate to compounds of formula (II) or formula (III), pharmaceutical and nutraceutical compositions containing them, their first and second medical uses, methods for inducing neuroregeneration and/or preventing neurodegeneration, or methods for preventing and/or treating a disease or pathology, as well as the use and in vitro method for determining the effectiveness of treatment, relates to a compound selected from the group consisting of:

- a pharmaceutically or nutraceutically acceptable salt or ester of a compound of formula (II):

where: a=6, b=1 and c=6; or a=6, b=2 and c=3; or a=6, b=3 and c=0; or a=3, b=3 and c=3; or a=2, b=4 and c=3; or a=2, b=5 and c=0;

And

- a pharmaceutically or nutraceutically acceptable salt or ester of a compound of formula (III):

where a=1, b=6 and c=0.

Even more preferably, said salt is a sodium salt and said ester is an ethyl ester.

In one embodiment of the invention, the pharmaceutical and nutraceutical compositions described herein comprise a compound of formula (I) together with a compound of formula (II) or a compound of formula (III) in a concentration of from 0.01% to 99.99% w/w, preferably the composition comprises from 10% to 80% w/w, or even more preferably in a concentration of from 20% to 80% w/w. In another embodiment of the invention, the compositions described herein comprise a prodrug of formula (I) together with a compound of formula (II) or a compound of formula (III), wherein said combination is in a ratio in the range of from 0.01:100 to 100:0.01, preferably from 1:5 to 5:1, and most preferably from 1:2 to 2:1.

In a further aspect, the pharmaceutical or nutraceutical compositions according to the invention can be presented in vials, ampoules, powders, capsules, tablets, sachets, solutions, syrups, ointments, creams, emulsions, gels, patches, controlled release formulations, suppositories, eggs, etc. The preparations are suitable for administration by, inter alia, oral, sublingual, gastrointestinal, rectal, parenteral (intravenous, intraarterial, intramuscular and subcutaneous), inhalation, topical (ophthalmic, intraaural, transdermal) routes. The route of administration can be easily determined by a person skilled in the art.

The compositions of the present invention may be in the form of a gastric-stable composition to prevent degradation of their components under the influence of the low pH of the intragastric environment. In certain embodiments, the composition of the invention further comprises one or more additional components or excipients, such as diluents, antioxidants, sweeteners, gelling agents, flavoring agents, fillers or other carrier media, such as colloidal anhydrous silicon dioxide and glyceryl monostearate. The said compositions may be in the form of a capsule, shell, paper or other packaging. Conventional methods for preparing pharmaceutical compositions may be used to prepare the said compositions. For example, the compounds described above in this document may be mixed with a carrier, or diluted by a carrier, or enclosed in a carrier, which may be in the form of an ampoule, capsule, shell, paper or other packaging. When the carrier is a diluent, it can be a solid, semi-solid or liquid material which acts as a vehicle, excipient or medium for the active compound. Some examples of suitable diluents are water, saline solutions, alcohols, polyethyleneglycols, polyhydroxyethoxylated castor oil, peanut oil, olive oil, lactose, calcium sulfate, sucrose, cyclodextrins, amylose, magnesium stearate, talc, gelatin, agar, pectin, acacia, stearic acid, alkyl ethers of cellulose, silicic acid, fatty acids, fatty acid amines, monoglycerides and diglycerides of fatty acids, pentaerythritol fatty acid esters, polyethylene, hydroxymethyl cellulose and polyvinylpyrrolidone. Likewise, the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax. The compositions may also contain wetting agents, antioxidants, emulsifiers and suspending agents, preservatives, sweeteners and flavors. The compositions of the invention may be designed to provide rapid, sustained or delayed release of the compounds disclosed herein after administration to a patient using methods well known in the art.

The disclosed compositions may be solid compositions or liquid solutions. In one non-limiting embodiment of the invention, the composition is a solid composition that may contain 20-80% of a compound of formula (I) and/or a compound of formula (II) or formula (III), 20-80% of a diluent, 0.1-20% of an antioxidant, 0.01-10% of a sweetener, 0.1-20% of a gelling agent, and 0.01-10% of a flavoring additive. In another non-limiting embodiment of the invention, said composition is an oral solution containing from 20 to 80% of a compound of formula (I) and/or a compound of formula (II) or formula (III), from 20 to 80% of a diluent, from 0.1 to 20% of an antioxidant, from 0.01 to 10% of a sweetener, from 0.1 to 20% of a gelling agent, and from 0.01 to 10% of a flavoring additive.

The pharmaceutical compositions can be sterilized and mixed, if necessary, with auxiliary agents, emulsifiers, salt for influencing the osmotic pressure, buffers and/or colorants and the like substances that do not enter into an undesirable reaction with the compounds disclosed above.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Figure 1. A Schematic illustrating the cellular metabolism of 2-hydroxydocosahexaenoic acid (DHA-H) to produce (6Z,9Z,12Z,15Z,18Z)-heneicosa-6,9,12,15,18-pentaenoic acid (HPA) by α-oxidation. DHA-H requires activation by acyl-CoA synthetase in a process that is ATP (adenosine triphosphate) and magnesium (Mg ²⁺ ) dependent. DHA-H-KoA will undergo 2-hydroxyphytanoyl-CoA lyase activity (2-hydroxyacyl-CoA lyase 1, HACL1), resulting in the formation of a polyunsaturated aldehyde intermediate that must contain 5 or 6 double bonds. HACL1 activity is dependent on thiamine pyrophosphate (TPP) and Mg ²⁺ and can be inhibited by a competitive antagonist (e.g., oxythiamine). The enzyme aldehyde dehydrogenase is responsible for the conversion of the aldehyde intermediate to HPA in a NAD ⁺ (nicotinamide adenine dinucleotide)-dependent process. B. DHA-H is metabolically transformed into HPA by α-oxidation in HEK293T cells. Intracellular DHA-H (B1 and B3) and HPA (B2 and B4) levels are plotted on the ordinate (nmol/mg protein) versus the concentration of sodium DHA-H treatment (μM) for 24 h (B1 and B2) or the time of incubation (h) with a constant concentration of 30 μM sodium DHA-H (B3 and B4), including untreated controls (C) on the abscissa. Black bars represent the result in cells without additional stimulus, white bars represent the result after co-treatment with 1 mM oxythiamine, and hatched bars represent the result after treatment with 10 mM oxythiamine. Both DHA-H and HPA levels increased in a concentration- and time-dependent manner, with HPA levels being significantly higher than DHA-H upon exposure to 30 μM sodium DHA-H for 24 h. This increase in HPA was inhibited in the presence of 10 mM oxythiamine (which inhibits the HCLA1 enzyme), demonstrating the involvement of α-oxidation in this metabolic conversion. Bars represent mean ± SEM, and statistical analysis was performed using one-way ANOVA and Tukey's multiple comparison test: *p<0.05 when comparing HPA levels with DHA-H levels under the same conditions; #, p<0.05 when comparing values in the presence and absence of 10 mM oxythiamine. C. Endogenous DHA (non-hydroxylated native form of docosahexaenoic acid) levels in HEK293T cells are not altered by sodium DHA-H treatment. Intracellular DHA levels are plotted on the ordinate (nmol/mg protein) versus sodium DHA-H treatment concentration (μM) for 24 h (C1) or incubation time (h) with a constant sodium DHA-H concentration of 30 μM (C2), including untreated controls (C) on the abscissa. Sodium DHA-H treatment did not significantly affect DHA levels either by changing the concentration or by changing the incubation time. Bars represent mean ± standard error and statistical analysis was performed using one-way ANOVA and Tukey's multiple comparison test. From these results, it can be concluded that administration of DHA-H or its sodium salt in this case does not change endogenous levels of DHA (docosahexaenoic acid) or other cellular fatty acids examined, but DHA-H treatment results in an increase in HPA levels exclusively, suggesting that the therapeutic effect obtained by treatment with DHA-H and in particular its sodium salt is mediated by HPA and not by modulation of levels of other endogenous fatty acids.

Figure 2. A. Mice treated with sodium DHA-H exhibit dose-dependent accumulation of HPA in the brain, with undetectable DHA-H in the brain. Brain HPA (A1) or DHA (A2) levels (nmol/mg protein) are plotted against the doses of sodium DHA-H (A1) and sodium DHA (A1 and A2) treatment (mg/kg). A1: • WT animals; ο 5xFAD. A2: Black bars represent WT animals and white bars represent 5xFAD. HPA and DHA levels were determined in the brains of WT and 5xFAD mice after chronic sodium DHA-H treatment (4 months; 5 doses/week MF; 3 to 7 months of age; sacrificed at 7 months). HPA accumulates in the brain of both mouse strains similarly, depending on the administered dose of DHA-H sodium salt (A1: • r2 = 0.9292, p = 0.0002; ο r2 = 0.9704, p < 0.0001). DHA levels did not differ significantly between experimental conditions (A2). Data are shown as mean ± standard error and statistical analysis was performed using one-way ANOVA and Tukey's multiple comparison test: * p < 0.05 compared to control (vehicle-treated mice). Therefore, when DHA-H prodrug, in particular its sodium salt, is administered to healthy mice and transgenic models of Alzheimer's disease, there is a significant accumulation of HPA at the brain level, without detecting the presence of the prodrug (DHA-H) or changing endogenous DHA levels. B. 5xFAD mice treated with sodium DHA-H exhibit cognitive improvement that directly correlates with brain HPA levels. The number of total errors (B1), long-term memory errors (RME) (B2), or working memory errors (WME) (B3) made is plotted on the ordinate versus brain HPA levels (nmol/mg protein) on the abscissa. Cognitive performance was assessed by testing the 8-arm radial maze during the last month of treatment in the same animals as in Figure 2A. Individual data points from the entire treatment population were plotted, and data from 5xFAD animals were fitted by inverse polynomial regression f(x)=y ₀ +(a/x). The r ² and p values corresponding to the regression of each parameter are given below: summary errors, RME and WME, depending on the concentration of HPA in the brain: C1: r ² = 0.9146 and p = 0.0311; C2: r ² = 0.9252 and p = 0.0243; C3: r ² = 0.7785 and p = 0.0346. These data indicate that minimal increases in brain HPA levels are associated with improved spatial cognition. Each data point on the graphs represents the mean ± standard error for each pathology/treatment: • WT + vehicle, WT + DHA-H 20 mg/kg; ♦ WT + DHA-H 200 mg/kg; ο 5xFAD + vehicle; Δ 5xFAD + DHA-H 5 mg/kg; ∇ 5xFAD + DHA-H 20 mg/kg; □ 5xFAD + DHA-H 50 mg/kg; ◊ 5xFAD + DHA-H 200 mg/kg. These results indicate that modest increases in brain HPA levels are significantly associated with improvements in spatial cognition, which is one of the most affected cognitive abilities in Alzheimer's disease.

Figure 3. A. DHA-H sodium-treated mice demonstrate tumor accumulation of HPA, in the absence of detectable DHA-H, in U118 cell xenograft tumors. Tumor DHA (black bars) and HPA (white bars) levels (pmol/mg tissue) are plotted on the ordinate versus treatment (vehicle and DHA-H 200 mg/kg) on the abscissa. Three-month-old NUDE mice were injected subcutaneously with 7.5 x 10 ⁶ U118 cells (human glioblastoma multiforme grade IV). Tumors were allowed to grow subcutaneously for 10 days before receiving oral treatment (vehicle or DHA-H sodium 200 mg/kg) for 42 days before sacrifice. Lipid analysis of xenograft tumors revealed the absence (undetectable levels) of DHA-H. Bars represent the mean ± SE for each treatment. B. Tumor HPA levels are inversely correlated with tumor size in xenograft models. Tumor size ( ^cm3 ) is plotted on the ordinate versus tumor HPA levels (pmol/mg tissue) on the abscissa for two treatments: vehicle and 200 mg/kg sodium DHA-H. The presence of HPA in tumors of animals treated with sodium DHA-H had a statistically significant linear relationship with tumor volume (A2), where: ^r2 = 0.4296 and p = 0.0029. The results showed that HPA levels in xenograft tumors had a statistically significant inverse linear relationship with tumor size. In the absence of the parent DHA-H molecule in the target organ, this evidence demonstrates that the presence of HPA in the target organ exerts a therapeutic effect in vivo.

Figure 4. A. DHA-H is a prodrug that is metabolically transformed into HPA by α-oxidation in U118 cells. Intracellular levels of DHA (black bars), DHA-H (white bars), and HPA (hatched bars) are plotted on the ordinate (nmol/mg protein) as a function of treatment: Control (C) and 150 μM sodium DHA-H (48 h), in the presence or absence of co-treatment with 1 and 10 mM oxythiamine, on the abscissa. Both DHA-H and HPA levels were increased in cells treated with sodium DHA-H. This increase in HPA levels was inhibited in the presence of 1 and 10 mM oxythiamine, demonstrating the involvement of α-oxidation in this metabolic transformation. Bars represent mean ± SE and statistical analysis was performed using one-way ANOVA and Tukey's multiple comparison test: *p<0.05 when comparing HPA levels only. B. Metabolic conversion of DHA-H to HPA is required for the antitumor effect. Cell viability (% of control -C- without oxythiamine) is plotted on the ordinate versus treatment conditions: control (C- black bars) and sodium DHA-H 150 μM, 48 h (white bars) in the presence and absence of co-treatment with 1 mM oxythiamine, on the abscissa. DHA-H treatment of U118 cells significantly reduced culture viability, while oxythiamine treatment (alone) did not affect cell viability. However, when sodium salt of DHA-H was co-treated with oxythiamine, the antiproliferative effect of this compound was significantly reduced compared to the effect without oxythiamine. Bars represent mean ± standard error, and statistical analysis was performed using one-way ANOVA and Tukey's multiple comparison test: * p<0.05 compared to control (C); # p<0.05 when comparing the effect of DHA-H in the presence and absence of oxythiamine.

Figure 5. A. Viability of U118 cells in culture after treatment with sodium DHA-H, sodium DHA, and HPA. Cell viability (% of untreated control) is plotted on the ordinate versus the different treatment conditions on the abscissa: control (black bar), sodium DHA-H (150 μM, 48 h - white bar), DHA (150 μM, 48 h - hatched bar), and HPA (150 μM, 48 h - checkerboard bar). HPA treatment under the same conditions causes a much more obvious degree of lethality in culture than that induced by DHA-H (prodrug) or DHA (natural analogue). This effect may be due to a mixture of the antiproliferative effect and toxic effects typical of HPA, which cannot be attributed to DHA-H or DHA under the same experimental conditions. Bars represent mean ± SEM and statistical analysis was performed using one-way ANOVA and Tukey's multiple comparison test: *p<0.05 when compared with control. B. Intracellular HPA levels in U118 cells in culture treated with sodium DHA-H and HPA. HPA levels (nmol/mg protein) are presented on the ordinate versus treatment conditions on the abscissa: sodium DHA-H (150 μM, 48 h - black bar) and HPA (5-150 μM, 48 h - white bars). Administration of 150 μM sodium DHA-H resulted in HPA levels equivalent to those generated by treatment with 5 μM HPA alone. Treatment with 150 μM HPA resulted in significantly higher intracellular HPA levels than those generated by treatment with the same concentration of prodrug. Bars represent mean±SE.

C. DHA-H and DHA levels in HEK293T cells in the presence (C1) or absence (C2) of culture medium. DHA-H (•) and DHA (ο) levels in the culture medium (% of initial levels at time 0) are plotted on the ordinate versus incubation time (h) on the abscissa. The lipid concentration in the culture medium was 30 μM and the culture plates were incubated for up to 72 h. In the presence of cell culture (C1), DHA levels in the medium significantly decreased after 48 and 72 h due to DHA uptake by the cells, while DHA-H levels remained unchanged up to 72 h. In the absence of cell culture (C2), both DHA and DHA-H levels remained constant over time. Bars represent mean±SE, and statistical analysis was performed using one-way ANOVA and Tukey's multiple comparison test: *p<0.05 when compared with control.

Figure 6. A, B, and C: Chronic treatment with HPA or its prodrug, DHA-H, prevents cognitive decline characteristic of Alzheimer's disease in a transgenic mouse model (5xFAD). Cognitive performance was assessed using the 8-arm radial maze test. Animals were treated at 3 to 7 months of age and the test was performed during the last month of treatment. The test included total errors during the test (A), long-term memory errors (RME) (B), and working memory errors (WME) (C). Each bar represents the mean ± SEM of errors during the last week of the radial maze test. Black bars represent errors made by WT mice. Open bars represent errors made by vehicle (5% ethanol)-treated 5xFAD transgenic mice. Shaded bars represent errors made by 5xFAD mice treated with DHA-H (20 mg/kg/day). Checkerboard bars represent errors made by 5xFAD mice treated with HPA (20 mg/kg/day). The results demonstrate cognitive improvement in 5xFAD mice treated with DHA-H and HPA in a similar manner. Bars represent mean±SE for each treatment, and statistical analysis was performed using one-way ANOVA and Tukey’s multiple comparison test: * p<0.05 versus healthy control (WT) and # p<0.05 versus 5xFAD control (vehicle-treated).

Figure 7. A: Tumor growth is inhibited in vivo by treatment with the sodium salt of HPA or its prodrug, DHA-H, in xenograft models. Tumor size (cm ³ ) is plotted on the ordinate versus days of treatment on the abscissa. To induce xenograft tumors in 3-month-old NUDE mice, 7.5⋅10 ⁶ human grade IV glioblastoma cells (U-118 MG) were subcutaneously inoculated on both sides of the back of the animal (8-12 weeks old, 30-35 g). After 10 days, tumors became visible, reaching an approximate volume of 0.1 cm ³ . Animals were randomly divided into groups with similar mean tumor volumes and received daily oral therapy for 42 days with: vehicle (untreated control), DHA-H (200 mg/kg/day) and HPA (200 mg/kg/day). Tumor volumes (v) were calculated as v = A ² × L/2, where A is the tumor width and L is its length. The data obtained for each treatment were approximated by an exponential growth curve. B: HPA and the prodrug, DHA-H, significantly reduced xenograft tumor volume compared with untreated controls. Tumor volume induced 42 days after the start of treatment is shown on the ordinate with treatments on the abscissa. Individualized data for the animals participating in the study are shown: ο vehicle (untreated control), DHA-H (200 mg/kg/day) and NRA (200 mg/kg/day). Bars represent mean ± SE for each treatment, and statistical analysis was performed using one-way ANOVA and Tukey's multiple comparison test: *p<0.05 compared with control group.

Figure 8. Illustrative schemes of the cellular metabolism of 2-hydroxylated polyunsaturated fatty acids (prodrugs, PUFA-H) leading via a-oxidation to their corresponding non-hydroxylated metabolites, the latter having one carbon atom less than the parent molecule. The hydroxylated fatty acid requires activation by acyl-CoA synthetase in a process dependent on ATP (adenosine triphosphate) and magnesium (Mg ²⁺ ). This PUFA-H-KoA will undergo 2-hydroxyacyl-CoA lyase (HACL, isoforms 1 or 2 depending on the cell type) activity, resulting in the formation of a polyunsaturated aldehyde intermediate. HACL activity is dependent on thiamine pyrophosphate (TPP) and Mg ²⁺ and can be inhibited by a competitive antagonist such as oxythiamine. The enzyme aldehyde dehydrogenase is responsible for the conversion of the intermediate aldehyde to the final fatty acid in a NAD ⁺ (nicotinamide adenine dinucleotide)-dependent process. A. Schematic diagram of the intracellular conversion of 2-hydroxylinolenic acid (LA-H) to (8Z,11Z)-heptadeca-8,11-dienoic acid (HDA). B. Schematic diagram of the intracellular conversion of 2-hydroxy-alpha (α)-linolenic acid (ALA-H) to (8Z,11Z,14Z)-heptadeca-8,11,14-trienoic acid (HTA ω-3). C. Scheme of intracellular transformation of 2-hydroxy-gamma (γ)-linolenic acid (GLA-H) to produce (5Z,8Z,11Z)-heptadeca-5,8,11-trienoic acid (NTA ω-6). D. Scheme of intracellular transformation of 2-hydroxyarachidonic acid (ARA-H) to produce (4Z,7Z,10Z,13Z)-nonadeca-4,7,10,13-tetraenoic acid (NTA). E. Scheme of intracellular transformation of 2-hydroxyeicosapentaenoic acid (EPA-H) to produce (4Z,7Z,10Z,13Z,16Z)-nonadeca-4,7,10,13,16-pentaenoic acid (NPA). F. Scheme of intracellular conversion of 2-hydroxydocosahexaenoic acid (DHA-H) to (6Z,9Z,12Z,15Z,18Z)-heneicosa-6,9,12,15,18-pentaenoic acid (HPA).

Figure 9. Amplified regions of different GC-FID chromatograms upon treatment of HEK293T cells with the respective prodrug: A. (1) control (vehicle) and (2) LA-H (100 μM, 24 h). The white arrow indicates the chromatographic peak of the parent LA-H molecule, the black arrow indicates the chromatographic peak corresponding to the HDA metabolite. HDA formation is inhibited in the presence of 10 mM oxythiamine. B. (1) control (vehicle) and (2) ALA-H (100 μM, 24 h). The white arrow indicates the chromatographic peak of the parent ALA-H molecule, the black arrow indicates the chromatographic peak corresponding to the ω-3 NTA metabolite. ω-3 NTA formation is inhibited in the presence of 10 mM oxythiamine. C. (1) Control (vehicle) and (2) GLA-H (100 μM, 24 h). The white arrow points to the chromatographic peak of the parent GLA-H molecule, the black arrow points to the chromatographic peak corresponding to the ω-6 NTA metabolite. The formation of ω-6 NTA is inhibited in the presence of 10 mM oxythiamine. D. (1) Control (vehicle) and (2) ARA-H (100 μM, 24 h). The white arrow points to the chromatographic peak of the parent ARA-H molecule, the black arrow points to the chromatographic peak corresponding to the NTA metabolite. The formation of NTA is inhibited in the presence of 10 mM oxythiamine. E. (1) Control (vehicle) and (2) EPA-H (100 μM, 24 h). The white arrow points to the chromatographic peak of the parent EPA-H molecule, the black arrow points to the chromatographic peak corresponding to the NPA metabolite. NPA formation is inhibited in the presence of 10 mM oxythiamine. F. (1) control (vehicle) and (2) DHA-H (100 μM, 24 h). The white arrow indicates the chromatographic peak of the parent molecule of HPA, the black arrow indicates the chromatographic peak corresponding to the metabolite of HPA. HPA formation is inhibited in the presence of 10 mM oxythiamine.

Figure 10. Treatment with HPA and other odd-chain polyunsaturated fatty acids prevents excitotoxicity-induced neuronal death. Neuronal culture was established by differentiation from human SH-SY5Y neuroblastomas using retinoic acid and BDNF (brain-derived neurotrophic factor). Neuronal death due to excitotoxicity was induced by adding NMDA (10 mM) and calcium/glycine (530 μM/10 mM) to the culture medium for 1 hour. To test the neuroprotective effects of the different test compounds (HDA or C17:2 ω-6, NTA ω-3 or C17:3 ω-3, NTA ω-6 or C17:3 ω-6, NTA or C19:4 ω-6, and HPA or C21:5 ω-3), pretreatments were performed for 24 h: vehicle (black bars), 1 μM (white bars), 3 μM (dotted bars), and 10 μM (hatched bars). The treatments tested under these experimental conditions demonstrated that these compounds could prevent excitotoxicity-induced death from a concentration of 3 μM. Bars represent mean±SE for each treatment, and statistical analysis was performed using one-way ANOVA and Tukey's multiple comparison test: *p<0.05 compared with control group (vehicle pretreatment).

Figure 11. Illustrative schematic of the cellular metabolism of 2-OHAA (LAM561) to 8Z-heptadecenoic acid (C17:1n9) via α-oxidation. 2-OHAA requires activation by acyl-CoA ligase in a process that is ATP (adenosine triphosphate) and magnesium (Mg ²⁺ ) dependent. 2OHOA-CoA will undergo 2-hydroxyphytanoyl-CoA lyase activity (2-hydroxyacyl-CoA lyase 1, HACL1), resulting in the formation of a monounsaturated aldehyde intermediate. HACL1 activity is thiamine pyrophosphate (TPP) and Mg ²⁺ dependent and can be inhibited by a competitive antagonist (e.g., oxythiamine). The enzyme aldehyde dehydrogenase is responsible for converting the intermediate aldehyde to 8Z-heptadecenoic acid in a NAD ⁺ (nicotinamide adenine dinucleotide)-dependent process.

Figure 12. Fatty acid composition analysis of U-118 MG glioma cells. (A) Representative chromatograms showing the fatty acid composition of U-118 MG cells incubated in the presence of 400 μM 2OHOA sodium salt or no treatment (control) for 24 h, as determined by gas chromatography. Retention times (min): C17:1n-9 (10.12), OA (13.01), 2OHOA (16.87), and C17:0 margaric acid as an internal control (10.81). (B) Quantification of the different fatty acids identified in the chromatograms (OA, 2OHOA, and C17:1n-9). The black bar represents the concentration of each fatty acid in the control, and the white bar represents the concentration of the fatty acid after 2OHOA sodium salt treatment. Bars represent the mean ± SEM of three independent experiments, expressed in nmol and normalized per mg protein. Statistical significance was determined using Student's t-test (***p<0.001 relative to control).

Figure 13. Fatty acid composition analysis of different glioma cell lines and non-neoplastic cells after treatment with 2OHOA sodium salt. Representative chromatograms showing the fatty acid composition (left) and quantification of different fatty acids identified in the chromatograms (C17:0, OA, 2OHOA, and C17:1n-9) (right) in glioma cells: (A) and (B) U-251 MG; (C) and (D) SF-295; and non-neoplastic cells: (E) and (F) MRC-5 (human fibroblasts); (G) and (H) mouse astrocytes, after treatment in the absence (control) or presence of 2OHOA sodium salt (400 μM, 24 h), determined by gas chromatographic analysis. The black bar corresponds to the concentration of each fatty acid in the control, and the white bar corresponds to the concentration of the fatty acid after treatment with 2OHOA sodium salt. Margaric acid C17:0 is included as an internal control. Bars represent the mean ± SEM of three independent experiments, expressed in nmol and normalized per mg protein. Statistical significance was determined using Student's t-test (**p<0.01, ***p<0.001 relative to control).

Figure 14. Effect of 2OHOA sodium salt, OA, and C17:1n-9 sodium salt on the viability and proliferation of glioma cells. Viability curves of different glioma cell lines (A1-A3) U-118 MG; (B1-B3) U-251 MG; and (C1-C3) SF-295 treated with increasing doses of 2OHOA sodium salt (0-1000 μM) (A1, B1, and C1); OA (0-300 μM) (A2, B2, and C2); and C17:1n-9 sodium salt (0-300 μM) (A3, B3, and C3) for 72 hours. Viability was determined by crystal violet staining. Each value represents the mean ± SEM of three independent experiments with at least three biological replicates, expressed as a percentage relative to vehicle-treated cells (100%).

Figure 15. Effect of 2OHOA sodium salt, C17:1n-9 sodium salt, and OA on the viability and proliferation of non-neoplastic cells. Viability curves of non-neoplastic (A1-A3) MRC-5 (human fibroblasts); and (B1-B3) mouse astrocytes treated with increasing doses of 2OHOA sodium salt (0-1000 μM) (A1 and B1); OA (0-300 μM) (A2 and B2); and C17:1n-9 sodium salt (0-300 μM) (A3 and B3) for 72 hours. Viability was determined by crystal violet staining. Each value represents the mean ± SEM of three independent experiments with at least three biological replicates, expressed as a percentage relative to vehicle-treated cells (100%).

Figure 16. Analysis of the effect of different fatty acids on proliferation and death markers in different cell lines. Immunoblots illustrating the effect of fatty acids (200 μM OA, 200 μM sodium C17:1n-9, and 400 μM sodium 2OHOA) on various proteins involved in 2OHOA-regulated cell death and signaling pathways in glioma cells: (A) U-118 MG; (B) U-251 MG; and (C) SF-295; and non-neoplastic: (D) MRC-5 (human fibroblasts); and (E) mouse astrocytes, after 72 hours of treatment.

Figure 17. Fatty acid composition analysis in U-118 MG glioma cells after inhibition of α-oxidation and the effect of oxythiamine on the survival of U-118 MG glioma cells. (A) Quantification of 2OHOA and C17:1n-9 fatty acids in U-118 MG cells treated with 400 μM 2OHOA for 24 h pre-incubated with increasing doses (1-10 mM) of oxythiamine (an α-oxidation inhibitor) for 90 min, determined by gas chromatography. Results are shown as the mean ± SEM of three independent experiments, expressed in nmol and normalized per mg protein. Statistical significance was determined using Student's t-test (*p<0.05, ***p<0.001 when comparing the amount of 2OHPA with that detected after 400 μM 2OHPA in the absence of oxythiamine; $$p<0.01, $$$p<0.001 when comparing the amount of C17:1n-9 with that formed after treatment with 400 μM 2OHPA in the absence of oxythiamine). (B) Viability of U-118 MG cells preincubated with oxythiamine (for 90 min) and treated in the absence (control) or presence of 2OHPA sodium salt (400 μM, 72 h) was determined by trypan blue staining excluding viable cells. Results are presented as mean cell numbers ± SEM of three independent experiments. Statistical significance was determined using Student's t-test (***p<0.001 relative to the absence of 2OHNOA and oxythiamine, control-0; and $$p<0.01, $$$p<0.001 relative to treatment with 2OHNOA without prior incubation with oxythiamine).

Figure 18. Effect of the metabolite C17:1n-9 on the action of 2OHOA. Viability of different human glioma cell lines: (A) U-251 MG and (B) SF-295; and non-neoplastic cell lines: (C) human fibroblasts MRC-5 and (D) mouse astrocytes; all were treated in the absence or presence of 2OHOA sodium salt (400 μM, 72 h) and pre-incubated with or without oxythiamine (for 90 min). Cell viability was determined by trypan blue staining excluding viable cells. Results are presented as mean cell numbers ± SEM of three independent experiments. Statistical significance was determined using Student's t-test (**p<0.01 and ***p<0.001 relative to the absence of 2OHOA and oxyamine; and $p<0.05 relative to treatment with 2OHOA alone).

Figure 19. Analysis of the effect of the metabolite C17:1n-9 on the effect of 2OHOA on markers of proliferation and death in various cell lines by inhibition of its formation by oxythiamine. Immunoblots illustrating the effect of 2OHOA (400 μM) in combination or not with oxythiamine (2 mM) on various proteins involved in 2OHOA-regulated cell death and signaling pathways in glioma cells: (A) U-118 MG; (B) U-251 MG; and (C) SF-295; and non-neoplastic: (D) MRC-5 (human fibroblasts); and (E) mouse astrocytes, after 72 hours of treatment.

Figure 20. Fatty acid composition analysis of rat plasma after 24 h treatment with 2OHOA sodium salt. (A) Representative chromatograms showing the fatty acid composition of rat plasma samples obtained at different times (0, 1, 2, 3, 4, 6, 8 and 24 h) after short-term treatment with 2OHOA (2 mg/kg, 24 h) determined by gas chromatography. C17:0 margaric acid was quantified as an internal control in the chromatogram. (B) Quantification of 2OHOA and C17:1n-9 fatty acids identified in the chromatograms. Results are shown as mean ± SEM for 4 animals and are expressed in nmol and normalized per ml plasma. Statistical significance was determined using the Wilcoxon test (*p<0.05 and **p<0.01 relative to baseline levels at time 0 h; $p<0.05 and $$p<0.01 relative to 2OHOA fatty acid levels).

Figure 21. Fatty acid composition analysis of rat plasma after 15 days of treatment with 2OHOA sodium salt. (A) Representative chromatograms showing the fatty acid composition of rat plasma samples obtained at different times (0, 1, 2, 3, 4, 6, 8 and 24 hours) after chronic treatment with 2OHOA (2 mg/kg, 15 days) determined by gas chromatography. C17:0 margaric acid was quantified as an internal control. (B) Quantification of 2OHOA and C17:1n-9 fatty acids identified in the chromatograms. Results are shown as the mean ± SEM of 4 animals, expressed in nmol and normalized per ml plasma. Statistical significance was determined using the Wilcoxon test (*p<0.05 and **p<0.01 relative to baseline levels at time 0 h; $p<0.05 and $$p<0.01 relative to 2OHOA fatty acid levels).

Figure 22. Fatty acid composition analysis of xenograft tumors from immunosuppressed mice. (A) Representative chromatograms showing the fatty acid composition of U-118 MG glioblastoma cell-derived xenograft tumors from mice treated orally and daily with 2OHOA sodium salt (200 mg/kg, 42 days) determined by gas chromatography. (B) Quantification of OA and C17:1n-9 fatty acids identified in the chromatograms. C17:0 margaric acid was quantified as an internal control. The white bar represents the concentration of each fatty acid in the control, and the black bar represents the concentration of fatty acid after 2OHOA sodium salt treatment. Results are shown as mean ± SEM for at least 7 xenograft tumors and are expressed in nmol and normalized per g tissue. Statistical significance was determined by the Mann-Whitney test (***p<0.01 vs. control).

Figure 23. Inverse correlation between tumor volume and C17:1n-9 metabolite amount. Representation of the amount of metabolite quantified by gas chromatography in mouse xenograft tumors as a function of tumor volume measured on day 42 of treatment with 200 mg/kg 2OHNOA sodium salt (black squares) or its vehicle (control, white circles). Significance was determined using Pearson's correlation coefficient (p=0.0001; r=-0.825).

Figure 24. Fatty acid composition analysis in representative human patients with advanced glioma. (A) Representative fatty acid composition chromatogram of a patient with glioma responding to 2OHOA sodium salt treatment (12 g/day, 21 days) determined in plasma samples obtained at different time points of treatment (0, 4 and 360 hours, 15 days) by gas chromatography. (B) Quantification of 2OHOA and C17:1n-9 fatty acids identified in the chromatograms of 2OHOA responders and non-responders in plasma samples obtained at different time points on the first day of treatment (0, 1, 2, 4, 6, 8 hours) and on days 8 (192 hours), 15 (360 hours), 21 (504 hours) and on the first day of the second treatment cycle (574 hours). (C) Quantification of 2OHPA and C17:1n-9 fatty acids identified in chromatograms of the same responders and non-responders together. C17:0 margaric acid was quantified in the chromatograms as an internal control. Results are shown as mean ± SEM for 8 patients (4 responders and 4 non-responders) and are expressed in nmol and normalized per ml plasma. Statistical significance was determined by the Mann-Whitney test (*p<0.05 and **p<0.01 relative to 2OHPA fatty acid amounts).

EXAMPLES

The examples described below are intended for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1. Fatty acids, reagents and organic solvents

1.1. DHA, DHA-H and HPA

DHA (sodium salt of docosahexaenoic acid; C22:6 n-3), DHA-H (sodium salt of 2-hydroxydocosahexaenoic acid; 2OH-C22:6 n-3), EPA-H (sodium salt of 2-hydroxyeicosapentaenoic acid), ARA-H (sodium salt of 2-hydroxyarachidonic acid), GLA-H (sodium salt of 2-hydroxy-gamma (γ)-linolenic acid), ALA-H (sodium salt of 2-hydroxy-alpha (α)-linolenic acid), LA-H (2-hydroxylinoleic acid), HPA (sodium salt of (6Z,9Z,12Z,15Z,18Z)-heneicosa-6,9,12,15,18-pentaenoic acid), NTA (sodium salt of (4Z,7Z,10Z,13Z)-nonadeca-4,7,10,13-tetraenoic acid), NTA ω-6 (sodium salt of (5Z,8Z,11Z)-heptadeca-5,8,11-trienoic acid), NTA ω-3 (sodium salt of (8Z,11Z,14Z)-heptadeca-8,11,14-trienoic acid) and HDA ((8Z,11Z)-heptadeca-8,11-dienoic acid) were obtained from Lipopharma Therapeutics (Spain). Margaric acid (C17:0) was purchased from Sigma-Aldrich, and heneicosapentanoic acids (free acid HPA; C21:5 n-3) and (4Z,7Z,10Z,13Z,16Z)-nonadeca-4,7,10,13,16-pentaenoic acid (free acid NPA; C19:5 ω-3) were purchased from Cayman Chemicals (Michigan, USA). D(+)-glucose (cell culture tested), sodium pyruvate, L-Gln (cell culture tested), acetyl chloride and N,O-bis(trimethylsilyl)acetamide, sodium chloride, sodium phosphate, EDTA (ethylenediaminetetraacetic acid), and Tris base were obtained from Sigma-Aldrich. In turn, chloroform, ethanol, methanol, hydrochloric acid and hexane were obtained from Scharlab (Spain). Heparin (5000 units/ml) was purchased from Hospira Invicta S.A. (Spain), ketamine (Anesketin 100 mg/ml) - from Eurovet Animal Health BV (Netherlands), xylazine (Xilagesic 20 mg/ml) - from Laboratories Calier S.A. (Spain) and oxythiamine hydrochloride - from Santa Cruz Biotechnology (Germany).

To obtain HPA, chemical synthesis is carried out from (5Z,8Z,11Z,14Z,17Z)-eicosa-5,8,11,14,17-pentaenoic acid (EPA (C20:5, ω-3)), according to reaction scheme 1. The chemical synthesis of HPA is disclosed in the prior art (Larsen et al., 1997, Lipids 32(7), 707-714. doi: 10.1007/s11745-997-0090-4). The reactions were carried out in the absence of light and in a nitrogen atmosphere.

Reagents and conditions: a)(COCl) ₂ /PhH 1.5 h rt, b) _CH2N2 / _ether 20 min, 0°C, c) AgOBz(cat.), _Et3N /THF/ _H2O

The synthesis of sodium salt of HPA according to the present invention was carried out from the compound designated by number 5 when R is _CH3 - _CH2- (CH=CH- _CH2 ) _{5 - CH2CH2-} , which corresponds to HPA ( _C21 ). This salt was prepared in an acid-base reaction, liquid-liquid extraction was carried out with MTBE (methyl tert-butyl ether)/HCl and the pH was adjusted with NaOMe to obtain sodium salt of HPA in good yields. A similar procedure can be carried out for the synthesis of HDA, NTA ω-3, NTA ω-6, NTA and NPA by selecting the starting substrate.

1.2. OHOA, OA and C17:1n-9

Lipid compounds sodium salt of 2OHOA, sodium salt of OA and sodium salt of C17:1n-9 were purchased from Medalchemy, SL (Spain).

The chemical synthesis of C17:1n-9 is disclosed in WO 1997049667. A solution of 8Z-heptadecene (66 mg, 0.26 mmol, 1 equivalent) and 2-methyl-2-butane (1.6 mL, 15.1 mmol, 58 equivalents) in tBuOH (6.5 mL) at 25 °C under N ₂ was treated by dropwise addition of 2.5 mL of NaClO ₂ solution (80%, 208 mg, 2.3 mmol, 9 equivalents) and NaH ₂ PO ₄ ⋅H ₂ O (250 mg, 1.8 mmol, 7 equivalents) in deionized water. The reaction mixture was allowed to stir for another 15 min, then concentrated in vacuo. The residue was treated with water (30 mL) and the aqueous layer was extracted with EtOAc (3 x 30 mL). The organic layers were dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. Chromatography (SiO ₂ , 2 x 13 cm, gradient elution 10-20% EtOAc-hexane) gave 27 mL (66 mg, 95%) as a clear oil. The synthesis of sodium salt C17:1n-9 according to the present invention was carried out from compound C17:1n-9. Said salt was prepared by acid-base reaction, liquid-liquid extraction was carried out with MTBE (methyl tert-butyl ether)/HCl and pH was adjusted with NaOMe to give sodium salt C17:1n-9 in good yields.

Example 2. Compositions with DHA-H and HPA

Some examples of compositions, which do not limit the scope of the present invention, are described in general terms below.

Example 3. Cellular assays with DHA-H and HPA

To describe the metabolic conversion of DHA-H to HPA (C21:5 ω-3), as well as the conversion of LA-H to HDA (C17:2 ω-6), ALA-H to HTA ω-3 (C17:3 ω-3), GLA-H to HTA ω-6 (C17:3 ω-6), ARA-H to NTA (C19:4 ω-6), EPA-H to NPA (C19:5 ω-3), HEK293T cell cultures (human embryonic kidney 293T cells), which are an embryonic non-tumor cell line widely used in studies of human metabolism under physiological conditions, were used.

HEK293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Dulbecco's modified Eagle's medium, Biowest, France) supplemented with 10% FBS (fetal bovine serum; Gibco, Thermo-Fisher), 2 mM L-Gln, 25 mM D(+)-glucose, 1 mM sodium pyruvate and penicillin/streptomycin. Murine neuroblastoma N2a cells were maintained in a 1:1 (v:v) mixture of DMEM and Opti-Mem (Gibco, Thermo-Fisher) supplemented with 5% FBS and penicillin/streptomycin. Both cell lines were incubated in an atmosphere of 5% _CO2 at 37°C.

HEK293T cells were incubated with DHA-H and DHA at 10, 30, and 100 μM for 24 h and at 30 μM for 6, 48, and 72 h. The cells were also incubated with LA-H, ALA-H, GLA-H, ARA-H, and EPA-H at 100 μM for 24 h. HEK293T cells were also incubated with oxythiamine in the presence of DHA-H under the same conditions at final oxythiamine concentrations of 1 and 10 mM. HEK293T cells were detached from the plates using cold phosphate-buffered saline (PBS) pipettes. Cells were isolated by centrifugation (1000 x g, 10 min at 4°C) and washed twice with cold PBS before freezing at -80°C. To analyze DHA-H and DHA levels in cell culture medium, 90 mm diameter plates were filled with 11 ml of complete cell culture medium containing 30 μM DHA-H or DHA in the presence or absence of adherent HEK293T cells (5 × 10 ⁵ cells/plate). Plates were incubated as described above and 1 ml aliquots of the plates were collected at 0, 6, 24, 48, and 72 h. Aliquots of cell culture medium were immediately centrifuged at 1000 × g for 10 min at 4°C to remove any cell suspension, and cell-free aliquots were stored at -20°C.

U-118 MG, MIA-PaCa 2, and A549 cell lines were obtained from the European Collection of Cell Cultures (ECACC) via Sigma-Aid rich Co (St. Louis, MO) and maintained in RPMI (Roswell Park Memorial Institute) (U-118 MG and A549) or DMEM (MIA-PaCa 2) culture medium supplemented with 10% FBS (Gibco, Thermo-Fisher) in an atmosphere of 5% CO ₂ at 37°C. U-118 MG, MIA-PaCa 2, and A549 cells were treated under the conditions described in the assay description to obtain the results in Table 4, finally in the presence or absence of oxythymine (1 or 10 mM). Cell viability was analyzed in a Bürker chamber using trypan blue staining excluding viable cells (Scharlab) or the Cell Proliferation Detection Kit II (Roche). Briefly, cells were seeded in 96-well plates at a density of 3 x 10 ³ cells/well 24 h before treatment and then cultured in the presence or absence of the compounds of interest at the concentrations and for the times indicated in the figures. At different time points, viable plaque cells were detected by adding XTT according to the manufacturer's instructions. Cells were incubated at 37°C in 5% CO ₂ until color was constant and the optical density was recorded at 495 nm using a microplate reader with a reference wavelength of 650 nm (FLUOstar Omega, BMG LABTECH, Germany).

SH-SY5Y human neuroblastoma cells were maintained in DMEM-F12 (Invitrogen) supplemented with 10% FBS (Sigma), penicillin/streptomycin (PAA), nonessential amino acids (Sigma), and 2 mM L-Gln (Sigma). Differentiation of these cells into the neuronal phenotype was performed using standard procedures. Briefly, cells were seeded on poly-L-lysine-pretreated plates, and after 24 h, the medium was replaced with fresh medium supplemented with 10 μM retinoic acid (Sigma). Cells were then incubated in the dark for 5 days, and the medium was replaced with serum-free medium and supplemented with 50 ng/ml human brain-derived neurotrophic factor (hBDNF; Alomone Labs; Tel Aviv, Israel). Finally, cells were incubated for 6 days to complete differentiation. Neurons were treated for 24 h with HDA, HTA ω-3, HTA ω-6, NTA, NPA, and HPA at 1.3 and 10 μM for 24 h before induction of excitotoxicity with NMDA (n-methyl-D-aspartate, 10 mM, Sigma) in medium containing glycine (530 μM, Sigma) and calcium (10 mM, Sigma).

DHA-H treatment results in high cellular levels of HPA compared to prodrug levels in cell cultures (Figure 1B). Figure 1B shows intracellular levels of DHA-H and HPA in HEK293T cells treated with DHA-H. Accumulation of both compounds is evident depending on the concentration used for treatment or the incubation time, but HPA levels are significantly higher than prodrug levels after 24 h of incubation and treatment with a concentration of 30 μM. The increase in HPA levels is inhibited in the presence of co-treatment with oxythiamine (partial inhibition with 1 mM and almost complete with 10 mM), a competitive antagonist of 2-hydroxyacyl-CoA lyase (see Figure 1A). In this regard, it was also found that endogenous DHA levels (non-hydroxylated native form) are not affected by this DHA-H treatment (Figure 1C).

Similarly, Figure 8 shows that the same metabolic pathway is valid for other 2-hydroxylated polyunsaturated fatty acids used as prodrugs such as LA-H, ALA-H, GLA-H, ARA-H and EPA-H, which form HDA, NTA ω-3, NTA ω-6, NTA, NPA, respectively (chromatograms are shown in Figure 9). All these metabolites demonstrated therapeutic activity as shown in Figure 10 and Table 4 below:

The antitumor activity of the various metabolites described in Figures 8 and 9 was determined by direct treatment with these molecules (HDA or C17:2 ω-6, HTA ω-3 or C17:3 ω-3, HTA ω-6 or C17:3 ω-6, NTA or C19:4 ω-6, NPA or C19:5 ω-3, and HPA or C21:5 ω-3) in tumor cell cultures, in which the IC50 value for each of these compounds was determined (inhibitory concentration 50: the concentration of the test compound that induces the death of 50% of the tumor cell population). The cell cultures used correspond to different types of cancer: U118-MG (human glioblastoma grade IV), MIA-PaCa 2 (pancreatic carcinoma) and A549 (small cell adenocarcinoma of the lung). Different compounds showed varying IC50 values on different tumor lines, demonstrating the selectivity of some of them for inducing selective death of some types of tumor cells.

Example 4. In vivo studies with DHA-H and HPA

The 5xFAD model of Alzheimer's disease is a PS1/APP double transgenic mouse carrying five human mutations associated with familial AD (strain Tg6799): Swedish (K670N/M671L), Florida 151 (I716V), and London (V717I) in APP; and the clinical mutations M146L and L286V in human PS1. Both transgenes are expressed under the control of the Thy-1 promoter, and mice exhibit cognitive decline compared with 4 months of age (Oackley et al., 2006, Neurosci 26(40), 10129-10140. doi: 10.1523/jneurosci.1202-06.2006). 5xFAD and wild type (WT) transgenic animals were obtained from Jackson Laboratories (USA) and maintained in a B6/SJL genetic background by crossing heterozygous transgenic mice with B6/SJL WT (F1) reproducers. Animals were kept at a controlled temperature of 22°C (±2°C) and humidity of 70% under a 12 h–12 h light/dark cycle with free access to a standard laboratory diet (Panlab A03, Barcelona, Spain).

Transgenic male WT and 5xFAD mice were given DHA-H (or DHA) orally dissolved in 5% ethanol at daily doses of 5, 20, 50 and 200 mg/kg, or vehicle only. In turn, in an independent analysis, these animals also received HPA (20 mg/kg) and DHA-H (20 g/kg) to compare the effect of both compounds in this model. These procedures were started when mice reached 3 months of age (dosing 5 days per week) and continued until 7 months of age. During the last month of treatment, all animals were kept on a hypocaloric diet to perform the selected behavioral spatial learning and memory testing (radial arm maze). A total of 46 animals were used for the study shown in Figure 2: WT treated with vehicle (n=3), WT treated with DHA-H (20 mg/kg, n=3; and 200 mg/kg, n=3), WT treated with DHA (20 mg/kg, n=3); 5xFAD (n=5) treated with DHA-H (5 mg/kg, n=6; 20 mg/kg, n=5; 50 mg/kg, n=6; and 200 mg/kg, n=7), and 5xFAD (20 mg/kg, n=5) treated with DHA. A total of 20 animals were used in the study shown in Figure 6: WT treated with vehicle (n=5), 5xFAD treated with vehicle (n=5), 5xFAD treated with DHA-H (20 mg/kg, n=5) and HPA (20 mg/kg, n=5). Following the behavioral test, mice were maintained on their standard diet (and treatment) for another week, after which they were anesthetized with an intraperitoneal injection of ketamine/xylazine (100/10 mg/kg) and given an intracardiac infusion of 50 ml of heparinized saline. The brains of the animals were immediately removed and dissected along the midline on a cold surface. After removal of the cerebellum, each cerebellar-free half was frozen in liquid nitrogen and stored at -80°C. NUDE (Swiss) Crl:NU (Ico) -Foxnl ^nu mice (8-12 weeks old, 30-35 g, Charles River Laboratories, Paris, France) were maintained in a thermostatic box (28°C, EHRET, Labor-U-Pharmatechnik) with sterile air flow at a relative humidity of 40-60% and 12-hour light-dark cycles. Their diet consisted of standard chow (Labdiet 22% rat-mouse breeding, Sodispan) provided ad libitum. To induce xenograft tumors, 7.5×10 ⁶ U-118 MG cells were inoculated intramuscularly and subcutaneously on both sides of the animal's back, and after one week the tumors became visible, reaching a volume of approximately 100 mm ³ . Animals were randomly assigned to groups with similar mean tumor volumes and received daily oral therapy for 42 days with vehicle, DHA-H (200 mg/kg), and HPA (200 mg/kg). The study in Figure 3 includes vehicle-treated (untreated controls; n=6) and DHA-H-treated (200 mg/kg; n=9) animals. Figure 7 illustrates vehicle-treated (untreated controls; n=6), DHA-H-treated (200 mg/kg; n=8), and HPA-treated (200 mg/kg; n=8) animals. Tumor volumes (v) were calculated as v = A ² × L/2, where A is the tumor width and L is the tumor length. At the completion of treatment, mice were sacrificed by cervical dislocation and xenograft tumors were dissected and frozen in liquid nitrogen and at -80°C. All protocols used were approved by the Bioethics Committee of the University of the Balearic Islands and comply with national and international guidelines for the humane treatment of animals. Using healthy mice and transgenic models of Alzheimer's disease (5xFAD), a significant accumulation of HPA was detected at the brain level, while the parent molecule (DHA-H) could not be detected, as well as changes in endogenous DHA levels (non-hydroxylated native form) (Figure 2A).

Radial Arm Maze Test

The spatial behavior test was performed as described above with some modifications by Fiol-Deroque (et al., 2013, Biogerontology 14(6), 763-775. doi: 10.1007/s10522-013-9461-4). All animals were isolated and subjected to caloric restriction to 80-85% of normal body weight and maintained under these conditions for one week before the start of the test and until the end of the test. After dietary restriction and 3 days before the start of the test, the animals were trained twice a day in the eight-arm radial maze test (LE766/8, Panlab SL, Spain) for 3 days. Each mouse was placed in the center of the maze and allowed to search for a reward - a 45 mg food pellet (Dustless Precision Pellets, Bio-Serv, USA) located at the end of each arm. Each session ended when the animal managed to find eight charged arms or failed to find all arms within 10 min. The movement of each animal was recorded using a digital video tracking system (LE 8300 with Sedacomv1.3 software, Panlab, SL, Spain) and after training the experimental design started. In all experimental sessions (1 session per day), only four arms were charged compared to the training protocol and each session ended when the animals managed to find all four charged arms or failed to find them after 10 min. Performance was assessed taking into account: (1) the time to complete the trial; (2) the number of working memory errors (WME, re-entering a previously visited charged arm); (3) the number of long-term memory errors (RME, entering an uncharged arm); and (4) the total number of errors (WME+RME). The test was repeated 5 days a week for 3 weeks. Following testing, animals were fed ad libitum for another week before being sacrificed.

In this regard, it can be noted that brain HPA levels in Alzheimer's disease mouse models have a statistically significant inverse relationship with behavioral parameters in a spatial and associative memory assessment test (radial maze test) (Figure 2B). These results suggest that a moderate increase in brain HPA levels is significantly associated with improved spatial cognition. Likewise, direct administration of HPA has a similar effect to DHA-H administration on the same behavioral parameters analyzed (Figure 6).

Example 5. Extraction of lipids and transmethylation of fatty acids related to Examples 3 and 4

HEK293T or U-118 MG cells used in the above examples were lysed with cold hypotonic buffer (1 mM EDTA, 20 mM Tris-HCl [pH 7.4]) by pipetting up and down. Cell lysates were sonicated (4 cycles, 10 sec/cycle, 10 W) prior to lipid extraction. For brain analysis, tissue from each animal was homogenized in cold PBS at 1:10 (p:v) in the presence of protease inhibitors (Roche) using a paddle homogenizer (Polytron PT3100). Homogenates were sonicated, aliquoted, and stored at -80°C. Only one aliquot of each sample, containing approximately 8 mg protein/aliquot, was subjected to lipid extraction. Protein content before lipid extraction was determined using a modified Lowry method (Bio-rad DC Protein Assay).

Margaric acid (C17:0) was added to the samples undergoing lipid extraction as an internal standard, and lipids were extracted with a chloroform:methanol mixture (Eggers and Schwudke, 2016). Briefly, 0.75 volumes of the aqueous phase (which already contained the biological sample) were mixed with 2 volumes of chloroform and 1 volume of methanol. The mixture was vortexed for 1 min and centrifuged at 1000 x g for 10 min. The lower organic phase was collected and washed with 1 ml of EtOH:methanol (1:1, v:v), and the resulting organic phase was dried under argon flow. The film containing the extracted lipids was transmethylated by incubating the lipid mixture for 90 min at 100°C in 3 ml of methanol:acetyl chloride (10:1, v:v) under argon (Christie, 1993). The resulting fatty acid methyl esters (FAME) were extracted with hexane by adding 3 ml of _H2O and 1 ml of hexane to the transmethylation reaction and vortexing the mixture thoroughly. After centrifugation at room temperature (1000×g for 10 min), the upper phase containing FAME was collected and the remaining volume was washed twice with 1 ml of hexane. The hexane phases were combined, evaporated under argon, and resuspended in 60 μl hexane (for analysis of cell samples, cell culture medium, and blood plasma) or 200 μl (for analysis of brain samples). To test whether the fatty acid compound was hydroxylated, the isolated FAME was subjected to a second derivatization with trimethylsilyl (Alderson et al., 2004, J Biol Chem 279(47), 48562-48568. doi: 10.1074/jbc.M406649200). Briefly, FAME was dried under argon flow and the lipid film was dissolved in N,O-bis(trimethylsilyl)acetamide (0.1-5.0 mg lipid per 200-400 μl trimethylsilylation reagent), which in turn was heated in a sealed vial at 70°C for 30 min. The solvent was evaporated and the lipid film was resuspended in hexane for analysis. When the fatty acid of interest is hydroxylated, the retention time of FAME is altered by this second derivatization. However, if the fatty acid of interest is not hydroxylated, the resulting FAME exhibits the same retention time regardless of the second derivatization.

HPA levels resulting from DHA-H prodrug treatment in these cells were assessed in the presence or absence of oxythiamine (a competitive inhibitor of α-oxidation) (Figure 4A). The results showed that addition of DHA-H to U-118 MG cell cultures resulted in a significant increase in HPA levels. This increase was inhibited in the presence of co-treatment with 1 or 10 mM oxythiamine, demonstrating that the conversion of DHA-H to HPA is mediated by α-oxidation. DHA-H treatment of U-118 MG cells in culture had no effect on endogenous DHA (non-hydroxylated native form) levels. Viability assays were also performed on the same cells in culture with DHA-H in the presence or absence of 1 mM oxythiamine (Figure 4B), showing that 1 mM oxythiamine did not affect cell viability.

On the other hand, addition of DHA-H (150 μM, 48 h) exerts a significant antiproliferative effect on U118-MG cells. However, this effect is partially reversible (in a statistically significant manner) in the presence of 1 mM oxythiamine. At the same time, it should be remembered that this concentration of oxythiamine is sufficient to completely inhibit the increase in HPA levels from DHA-H. These results then demonstrate that the DHA-H-mediated antiproliferative effect on U-118 MG cells is mediated, at least in part, by HPA, since inhibition of the formation of this compound from DHA-H results in a lower antiproliferative effect of DHA-H (Figure 4B). The antiproliferative effect that HPA exerts on U-118 MG cell culture was also studied in comparison with the administration of DHA-H prodrug and the native form of DHA. The antiproliferative effect of U-118 MG is significantly higher for HPA compared to DHA-H and DHA (see Figure 5A). Compared to DHA-H, this effect may be explained by the differences in intracellular HPA levels induced by DHA-H and HPA (see Figure 5B). Indeed, Figure 5C shows that the uptake of the hydroxylated form of DHA is prevented compared to the uptake of the non-hydroxylated analogue.

Example 6. In vitro assays with 2ОНОА and С17:1n-9

The concentrations of 2OHOA sodium salt used in the experiments described below and the duration of treatment varied depending on the assay type, being 200 μM or 400 μM and 24 or 72 hours. In some sets of experiments, solutions of C17:1n-9 sodium salt were used at a concentration of 200 μM for 24 or 72 hours.

To prepare these solutions, we started with a starting aliquot at a concentration of 100 mM. To obtain this starting aliquot, the appropriate number of milligrams of the lipid compound (powder) was dissolved in absolute ethanol and autoclaved distilled water (1:1 v/v, typically a 1 ml aliquot was prepared by adding 500 μl ethanol and 500 μl water) inside a culture hood, the solution was injected into a culture oven at 37°C for 10 min to dissolve the lipid compound, and then stirred.

6.1. Incorporation and Metabolism of 2OHAO in U-118 MG Glioma Cells and Non-Tumor Cells

To confirm the incorporation of 2OHOA into the cell membranes of glioma cells and to determine whether changes in the fatty acid profile occur after 2OHOA treatment, total lipids were analyzed by gas chromatography on U-118 MG human glioma cells incubated in the absence (control) or presence of 400 μM 2OHOA sodium salt for 24 hours. Analysis of fatty acid levels in glioma cells showed no change in OA levels after 2OHOA sodium salt treatment compared to the control (Figure 12). Furthermore, the incorporation of 2OHOA into the cells was observed after 24 hours of treatment due to the identification of a peak in the chromatogram, only in the treated cells, which corresponded to its standard. On the other hand, it should be noted that a new peak appeared almost exclusively in the chromatogram of the treated cells. Increased levels of this new peak were detected in treated cells, with the summed level being almost twice as high (19.71±0.39 nmol/mg protein) as that of 2OHPA (10.81±0.34 nmol/mg protein). After several assignment studies, it was confirmed that this new peak corresponded to cis-8-heptadecenoic fatty acid (C17:1n-9). The formation of C17:1n-9 is a consequence of the α-oxidation of 2OHPA (Figure 11).

6.2. Analysis of fatty acid composition in various glioma and tumor cells after treatment with sodium salt 2OHOA

The fatty acid composition of lipid membranes was analyzed in other glioma cell lines (U-251 MG and SF-295) compared to non-neoplastic cells, human fibroblasts (MRC-5), and primary cultures of mouse astrocytes after incubation in the absence or presence of 2OHOA sodium salt (400 μM, 24 h) using gas chromatography. No significant change in OA amounts was observed in any of the cell lines analyzed after 2OHOA sodium salt treatment (Figure 13). However, after 24 h of 2OHOA treatment, 2OHOA incorporation as well as the formation of the metabolite C17:1n-9 was observed in both the other glioma cell lines and non-neoplastic cells (Figure 13). The formation of metabolite C17:1n-9 as a result of 2OHPOA incorporation differed between tumor and non-tumor cells. Glioma cells (U-251 MG and SF-295) showed a significant increase in their C17:1n-9 levels, accumulating 97.42% and 108.03% more than 2OHPOA (19.16±0.53 vs. 9.21±0.41 and 18.38±1.97 vs. 9.31±1.44 nmol/mg nmol/mg, respectively) (Figures 13B and 13d, Table 5). In contrast, in non-tumor cells, the detected 2OHPOA levels were significantly higher than their metabolite C17:1n-9 levels. 46% more 2OHOA was accumulated compared to its metabolite C17:1n-9 in human MRC-5 fibroblasts (26.31±4.32 vs. 14.00±1.92 nmol/mg) and 38.27% in mouse astrocytes (12.28±0.90 vs. 7.58±0.70 nmol/mg) (Figures 13f and 13H, Table 5).

6.3. Effect of 2OHOA, C17:1n-9 on the viability and proliferation of glioma cells

To evaluate the antiproliferative effect of C17:1n-9, its IC ₅₀ , which corresponds to the amount of compound required to reduce cell viability in vitro by 50%, and its effect on the regulation of proteins involved in the mechanism of action of 2OHOA were determined. For this purpose, glioma cell lines (U-118 MG, U-251 MG, and SF-295) and non-tumor cell lines (MRC-5 and astrocytes) were treated with increasing concentrations of C17:1n-9 sodium salt, OA, and 2OHOA for 72 h. Upon completion of the treatment, the IC ₅₀ was determined using crystal violet staining. The results of cell viability assays showed that the three compounds, 2OHOA, OA, and C17:1n-9, exerted antiproliferative effects on all glioma cells tested in a concentration-dependent manner after 72 h of treatment. Furthermore, in the studied non-tumor cells, MRC-5 and astrocytes, no effect of 2OHAO on cell viability was observed, but OA and C17:1n-9 fatty acids exerted antiproliferative effects on the same non-tumor cells (Figures 14 and 15). The IC ₅₀ values of 2OHAO sodium salt were 432.75±10.77, 429.96±9.67, and 399.14±11.47 μM in U-118 MG, U-251 MG, and SF-295 glioma cells, respectively (Table 6). The IC 50 of 2OHAO was 1000 μM for non-tumor cells, MRC-5, and astrocytes. For compound C17:1n-9, the IC ₅₀ values were 222.04±9.09, 220.35±7.93, and 248.85±6.02 μM in U-118 MG, U-251 MG, and SF-295 glioma cells, respectively.

Thus, C17:1n-9 induced very similar antiproliferative effects on both glioma and non-neoplastic cells. However, 2OHOA treatment affected only the viability of various glioma cell lines without affecting the viability of non-neoplastic cells. The IC ₅₀ values of 2OHOA were 1.90-, 1.95-, and 1.60-fold higher than those of the C17:1n-9 metabolite in U-118 MG, U-251 MG, and SF-295 glioma cells, respectively (Table 6). In addition, the IC ₅₀ values of 2OHOA were 1.92-, 1.80-, and 1.56-fold higher than those of its non-hydroxylated analog OA. The fact that C17:1n-9 showed higher antiproliferative activity may be due to its higher cellular accumulation capacity than 2OHOA.

6.4 Analysis of the effect of different fatty acids on markers of proliferation and death in different cell lines

The effect of the metabolite C17:1n-9 on various signaling pathways that are altered by 2OHOA was analyzed. For this purpose, various glioma cell lines (U-118 MG, U-251 MG, and SF-295) and non-tumor cells (MRC-5 and murine astrocytes) were treated with doses close to the IC ₅₀ of each compound (200 μM C17:1n.9, 200 μM OA, or 400 μM 2OHOA) for 72 h and their effects on various signaling proteins were analyzed by Western blotting.

The results showed that 2OHOA treatment increased the phosphorylation levels of BIP, CHOP, and cJun and decreased the phosphorylation of Akt and cyclin D3 levels. In contrast, C17:1n-9 treatment did not cause changes in any of these proteins (Figure 16). This suggests that C17:1n-9-induced death is mediated by pathways other than 2OHOA.

6.5. Analysis of fatty acid composition in U-118 MG glioma cells after inhibition of a-oxidation and determination of the effect of oxythiamine on the survival of U-118 MG glioma cells

To confirm the formation of C17:1n-9 acid from 2OHPA via α-oxidation, oxythiamine chloride was used, which, among other functions, inhibits the enzyme 2-hydroxyphytanoyl-CoA lyase (HACL1, a key enzyme in α-oxidation). For this, U-118 MG glioma cells were first pre-incubated with 1 or 10 mM oxythiamine for 90 minutes, then treated with 400 μM sodium salt of 2OHPA for 24 hours, and fatty acids were analyzed by gas chromatography. When analyzing specific fatty acids detected by gas chromatography, a significant reduction in C17:1n-9 was observed in U-118 MG glioma cells preincubated with oxythiamine and treated with 2OHOA sodium salt compared to cells treated with 2OHOA sodium salt alone without oxythiamine (Figure 17A). This reduction was 51.35% after preincubation with 1 mM oxythiamine (from 17.17±1.07 to 8.35±0.36 nmol/mg protein); and 58.45% with 10 mM oxythiamine (7.13±0.39 nmol/protein) relative to the amount of metabolite that was achieved in cells treated with 2OHOA without the presence of oxythiamine. In contrast, no significant differences were found in the amounts of 2OHPA between cells treated with 2OHPA sodium salt and up to 3 mM oxythiamine compared to cells treated with 2OHPA sodium salt alone (Figure 17A). However, a significant increase in the amount of 2OHPA by 12.33% was observed in cells treated with 2OHPA sodium salt and 4 mM oxythiamine (from 12.41±0.75 to 15.74±0.24 nmol/protein); and 52.94% with 10 mM oxythiamine treatment (18.98±0.42 nmol/protein). Thus, oxythiamine inhibited the formation of C17:1n-9 and increased the amount of 2OHPOA from 4 mM by inhibiting α-oxidation, which confirms the metabolic pathway of 2OHPOA to C17:1n-9.

Next, to determine whether inhibition of 2OHPA metabolism via 2OHPA α-oxidation affects cell viability, the survival of U-118 MG glioma cells after incubation in the absence or presence of 2OHPA (400 mM, 72 h) and pre-incubated with oxythiamine at the doses described above was examined using trypan blue staining to exclude viable cells. Oxythiamine induced a significant decrease in the survival of U-118 MG glioma cells. In more detail, 1 mM oxythiamine induced 12.16±0.5% cell death, 21.17±1.76% cell death at 2 mM, until reaching a maximum inhibition of cell survival of 27.13±0.41% at 10 mM (Figure 7B). These results confirm the in vitro antitumor activity of oxythiamine, which was already known.

On the other hand, incubation of cells with 2OHPOA sodium salt induced cell death by 24.71±1.88%; and after combination with 1 mM oxyamine, cell viability was restored by 5% (20.94±1.97% death); and by 17.26% (11.71±1.14% death) in the case of 2 mM oxyamine (Figure 17B).

In view of Figures 17A and B it is noted that the levels of C17:1n-9 do not vary much between the lowest and highest concentrations of oxythiamine. This can be interpreted to mean that at low concentrations (1.2 mM) oxythiamine itself is less cytotoxic, but its effect in reducing C17:1n-9 production is very evident. At these concentrations we see a reducing effect on C17:1n-9 levels, but at higher concentrations of oxythiamine we begin to see greater cytotoxicity caused by oxythiamine itself, an effect that appears to be enhanced by high concentrations of the sodium salt of 2OHOA. At high concentrations a greater accumulation of 2OHOA is found, indicating that administration of the sodium salt of 2OHOA also causes cytotoxicity by itself and that this effect is additive to the cytotoxic effect of oxythiamine at these concentrations.

6.6. Effect of the metabolite C17:1n-9 on the action of 2OHOA

To investigate whether the metabolite C17:1n-9 could be involved in the action of 2OHOA, the effect of pre-incubation with oxythiamine on cell survival and proteins regulated by 2OHOA was studied. For this purpose, cell survival of various glioma and non-tumor cell lines treated with 2OHOA (400 μM, 72 h) and pre-incubated or not with 2 mM oxythiamine (90 min) was analyzed by cell counting using trypan blue staining excluding viable cells. In addition, Western blots of 2OHOA-modulated proteins were examined. In glioma cells, a significant decrease in cell survival was observed after incubation with 2 mM oxythiamine for 72 h. Oxythiamine induced 18.51±0.58% and 17.35±0.63% cell death in U-251 MG and SF-295 cells, respectively (Figure 18A and 18B). These results confirm the in vitro antitumor effect of oxythiamine on glioma cells. Treatment of cells with 2ОНОА induced 23.22±1.32% and 23.97±1.25% cell death in U-251 MG and SF-295, respectively. After combination with 2 mM oxythiamine, significant recovery of cell viability was observed: 12% (14.07±1.62% death in U-251 MG cells) and 17.25% (10.85±0.58% death) in SF-295 cells. In contrast, in non-tumor cells, none of the treatments studied had any effect on cell survival (Figure 18C and 18D).

Regarding the study of proteins involved in various signaling pathways and cell death in glioma cells, oxythiamine affected the levels of BiP, CHOP, c-Jun phosphorylation, Akt and cyclin D3 phosphorylation in glioma cells in a similar manner as 2OHOA, although weaker (Figure 19), and when combined, the modulation induced by 2OHOA was inhibited. This finding confirms that the metabolism of 2OHOA to C17:1n-9 is necessary for the potentiation of its antitumor effect. In contrast, no changes in such signaling proteins were observed in non-tumor cell lines after any of the treatments (Figure 19). Although 2OHOA has antiproliferative activity by inhibiting its metabolism to C17:1n-9, the formation of the C17:1n-9 metabolite has a major impact on the mechanism of action of 2OHOA, enhancing its antiproliferative effect, and confirms that 2OHOA is also a prodrug that leads to the formation of the active metabolite 17:1n-9.

Example 7. In vivo studies with 2ОНОА and С17:1n-9

7.1 Analysis of fatty acid composition in rat plasma after 24 hours of 2OHOA treatment

The pharmacokinetic profile of 2ОНОА and its metabolite С17:1n-9 in animal plasma was studied. In this case, rats were used as an animal model for the experiments. Rats have a larger volume than mice and are the most suitable model for studying the effect of prolonged administration of the maximum tolerated dose of 2ОНОА (2 g/kg), determined in preclinical studies.

In the present study, rats aged 12-14 weeks were orally administered 2 g 2OHOA/kg as sodium salt for 15 days. Then, plasma samples were collected at different times (0, 1, 2, 3, 4, 6, 8 and 24 hours) from day 1 (short-term treatment) and day 15 (long-term treatment). Finally, the fatty acid profile of the plasma samples was analyzed by gas chromatography. After analyzing the chromatograms, 2OHOA and C17:1n-9 fatty acids were detected in the plasma samples collected after the short-term treatment (administration on day 1) (Figure 20A).

The two compounds, 20HOA and C17:1n-9, showed very similar pharmacokinetic profile in rat plasma after short-term treatment (Figure 20B). There was a significant increase in 2OHOA and C17:1n-9 levels, which reached their maximum plasma concentrations 2 hours after 2OHOA administration (26.23±5.79 nmol 2OHOA/mL plasma and 60.47±6.53 nmol C17:1n-9/mL plasma). Thereafter, there was a decrease in 2OHOA and C17:1n-9 plasma levels, reaching their minimum values after 24 hours (2.80±0.69 and 14.03±2.20 nmol/mL plasma, respectively). However, baseline levels were not achieved, particularly for C17:1n-9 (1.22±0.33 and 8.45±2.52 nmol/mL plasma, respectively). C17:1n-9 metabolite levels after long-term treatment were higher than 2OHHOA levels (Figure 21B). The differences between the two compounds were significant before 2OHOA administration (0 h; 1.22±0.33 nmol 2OHOA/ml plasma, versus 8.45±2.52 nmol C17:1n-9/plasma), after 8 h (7.12±1.56 nmol 2OHOA/ml plasma, versus 23.31±5.18 nmol C17:1n-9/plasma), and after 24 h (2.80±0.69 nmol 2OHOA/ml plasma, versus 14.03±2.21 nmol C17:1n-9/plasma).

7.2. Analysis of fatty acid composition in xenograft tumors of immunosuppressed mice

To study the effect of C17:1n-9 formation as a product of 2OHOA metabolization via α-oxidation in animal models, levels of the metabolite C17:1n-9, compared to 2OHOA levels, were determined and analyzed in a xenograft tumor model in immunosuppressed mice. For this purpose, U-118 MG glioblastoma cells were administered to immunosuppressed mice and after one week, the mice were treated with vehicle or 2OHOA sodium salt (200 mg/kg) orally and daily for 42 days. After completion of treatment, the mice were sacrificed and the tumors were removed, lipids were analyzed for 2OHOA and C17:1n-9 fatty acids by gas chromatography. The fatty acid 2OHOA was not detected in xenograft tumors from mice treated with this compound, as no peak was observed at the retention time corresponding to 2OHOA (Figure 22A). In contrast, the metabolite of 2OHOA, the fatty acid C17:1n-9 (0.25±0.04 nmol C17:1n-9/g tissue), was detected in tumors from mice treated with 2OHOA (Figure 22B).

7.3 Correlation between tumor volume and the amount of C17:1n-9 metabolite

A possible correlation between the levels of C17:1n-9 metabolite present in tumors and tumor volume was studied as an evidence of the relationship between 2OHOA incorporation and metabolization and the efficacy of this compound in tumors. The obtained graphs showed a negative correlation between the amount of C17:1n-9 present in tumors and tumor volume (Figure 23). The determination coefficient r-0.8248 and p-value 0.0001 were obtained for tumors of mice treated with 2OHOA between the amount of C17:1n-9 and tumor volume. That is, the smaller the tumor volume, the more C17:1n-9 metabolite was detected in it. These results indicate that C17:1n-9 metabolite has pronounced antitumor activity and 2OHOA is an effective prodrug of this compound.

7.4 Fatty acid composition analysis in human patients with late-stage glioma after 2OHOA treatment

2OHOA and C17:1n-9 fatty acids were detected and quantified in plasma samples from 8 patients who responded or did not respond to treatment with 12 g/day sodium 2OHOA for at least one 3-week cycle in the 2OHOA Phase I/IIA clinical trial (MIN-001-1203). Plasma samples were obtained at different times (0, 2, 4, 6, 8 hours and 8, 15, 21 and 28 days after 2OHOA treatment) and then collected for fatty acid analysis using a gas chromatographic method.

In the chromatograms, 2OHOA and its metabolite C17:1n-9 were detected in all plasma samples from the analyzed patients (Figure 24A). A very similar pharmacokinetic profile was observed in all patients, both those who showed a clinical response (responders) and those who did not (non-responders) (Figure 24B). Both compounds reached peak levels 4 hours after 2OHOA administration. When analyzing the results of all responders and non-responders, the values of 53.08 ± 6.52 nmol 2OHOA/mL plasma and 122.80 ± 10.61 nmol C17:1 n-9/mL plasma were obtained 4 hours after the first drug administration (Figure 24C). Then, the levels of 2OHPA and C17:1n-9 gradually decreased until 8 hours after administration (25.39±3.99 and 92.89±9.39 nmol/mL plasma, respectively). After 8 days of treatment (192 hours), a significant increase in the amounts of the two compounds in plasma was observed at 192 hours (25.39±3.99 nmol/mL plasma 2OHPA and 141.10±16.35 nmol/mL plasma C17:1n-9). The compounds 2OHPA and C17:1n-9 accumulated in the plasma of patients over time, which was observed after 15 days (360 hours) of 2OHPA treatment (184.70±25.60 and 366.9±72.47 nmol/mL plasma 2OHPA and C17:1n.9, respectively) (Figure 24C).

Of note, similar to what occurred in cells and animals, plasma levels of the C17:1n-9 metabolite in all patients were higher than 2OHOA levels (Figure 14B), being significantly higher after 8 hours of 2OHOA administration (Figure 24C). C17:1n-9 levels were 3.66- and 2.20-fold higher than 2OHOA in patients who received 2OHOA for 8 and 15 days, respectively (92.89±9.39 and 311.10±37.38 nmol C17:1n-9/mL plasma compared with 25.39±3.99 and 141.10±16.35 nmol 2OHOA/mL plasma, respectively). Finally, after 21 days of 2OHOA treatment, C17:1n-9 levels were 1.90-fold higher than 2OHOA levels (366.9±72.47 nmol C17:1n-9/ml plasma versus 184.70±25.60 nmol 2OHOA/ml plasma, respectively).

Claims (34)

1. A sodium salt selected from the group consisting of: sodium salt of the compound of formula (II): where: a=6, b=1 and c=6; a=6, b=2 and c=3; a=6, b=3 and c=0; a=3, b=3 and c=3; a=2, b=4 and c=3; and a=2, b=5 and c=0; And sodium salt of the compound of formula (III): where a=1, b=6, c=0, and m=0. 2. Use of the salt according to claim 1 for the treatment of a disease or pathology associated with a change in the lipid composition of cell membranes and selected from the group consisting of: a neurological or neurodegenerative disease; cancer; neoplasm; and neuropathic pain. 3. The use according to claim 2, wherein said salt is a sodium salt of a compound of formula (II), where a=6, b=2 and c=3, and said neurological or neurodegenerative disease is Alzheimer's disease. 4. Use according to claim 2 or 3, wherein the treatment of a disease or pathology is characterized by the administration of a compound of formula (I): or a pharmaceutically acceptable salt thereof, wherein the values of a, b and c are equal to the values of a, b and c of the compound of formula (II) or the compound of formula (III), as defined in claim 1; and wherein said compound of formula (I) is metabolized to yield a therapeutically effective amount of the compound of formula (II) or the compound of formula (III). 5. Use according to claim 2 or 3, wherein said salt is administered before, after or together with the compound of formula (I): or a pharmaceutically acceptable salt or ester thereof, wherein a is an integer from 1 to 14; b is an integer from 1 to 7; c is an integer from 0 to 14, and a+3b+c+3 is an even integer; and wherein said values of a, b and c are equal to or different from the values of a, b and c of the compound of formula (II) or the compound of formula (III) as defined in claim 1. 6. A pharmaceutical composition for treating a disease or pathology associated with a change in the lipid composition of cell membranes and selected from the group consisting of: a neurological or neurodegenerative disease; cancer; neoplasm; and neuropathic pain, wherein said composition contains at least a first compound and at least one pharmaceutically acceptable excipient, wherein said first compound is a salt according to claim 1. 7. The composition according to claim 6, further comprising a second compound of formula (I): or a pharmaceutically acceptable salt or ester thereof, wherein a is an integer from 1 to 14; b is an integer from 1 to 7; c is an integer from 0 to 14, and a+3b+c+3 is an even integer; and wherein said values of a, b and c of said second compound are equal to or different from the values of a, b and c of said at least first compound, as defined in paragraph 1. 8. Use of a pharmaceutical composition according to claim 6 or 7 for the treatment of a disease or pathology associated with a change in the lipid composition of cell membranes and selected from the group consisting of: a neurological or neurodegenerative disease; cancer; neoplasm; and neuropathic pain. 9. The use according to claim 8, wherein said salt is a sodium salt of a compound of formula (II), wherein a=6, b=2 and c=3, and said neurological or neurodegenerative disease is Alzheimer's disease. 10. An in vitro method for determining the effectiveness of therapeutic treatment of a disease or pathology associated with a change in the lipid composition of cell membranes, using a compound of formula (I): or a pharmaceutically acceptable salt or ester thereof in a subject, wherein a is an integer from 1 to 14; b is an integer from 1 to 7; c is an integer from 0 to 14; m is an integer from 0 to (b-1); and wherein a+3b+c+3 is an even integer, wherein said disease or pathology is selected from the group consisting of: a neurological or neurodegenerative disease; cancer; neoplasm; and neuropathic pain; and wherein said method comprises determining in vitro in a biological sample of said subject an amount of a compound of formula (II): where a is an integer from 1 to 14; b is an integer from 1 to 7; c is an integer from 0 to 14; m is an integer from 0 to (b-1); and where a+3b+c+3 is an even integer, or compounds of formula (III): where a is an integer from 1 to 14; b is an integer from 1 to 7; c is an integer from 0 to 14; m is an integer from 0 to (b-1); and where a+3b+c+3 is an even integer, or a carboxylate anion of said compound of formula (II) or (III), or a derivative formed in vivo or in vitro from said compound of formula (II) or (III), wherein said amount is related to the effectiveness of treating said disease or pathology. 11. The method according to claim 10, wherein in the compound of formula (I) or (II) c is 0, 3 or 6, or wherein in the compound of formula (III) c is 0, 3 or 6, and m=0. 12. The method of claim 10, wherein said method comprises determining in vitro in a biological sample of said subject an amount of a compound of formula (II) or a carboxylate anion of said compound of formula (II), or a derivative formed in vivo or in vitro from said compound of formula (II), and where: a=6, b=1 and c=6; or a=6, b=2 and c=3; or a=6, b=3 and c=0; or a=3, b=3 and c=3; or a=2, b=4 and c=3; or a=2, b=5 and c=0. 13. The method according to claim 10, wherein said method comprises determining in vitro in a biological sample of said subject the amount of a compound of formula (III) or a carboxylate anion of said compound of formula (III), or a derivative formed in vivo or in vitro from said compound of formula (III), and where a=1, b=6, c=0, and m=0. 14. The method according to any one of claims 10-13, wherein the pharmaceutically acceptable salt is a sodium salt.