ALPHA-HYDROXYLATED FATTY-ACID METABOLITES, MEDICAL USES AND USE AS BIOMARKERS

Abstract

Described are fatty acids with one or more unsaturations, having an odd hydrocarbon chain, the fatty acids having the chemical structure of the therapeutically active metabolites of even-chain mono- or polyunsaturated alpha-hydroxylated fatty acids. Also described are compositions comprising said fatty acids, medical uses thereof, and the use thereof as indicators of the efficacy of and/or response to the treatment of a patient with the even-chain mono- or polyunsaturated alpha-hydroxylated fatty acids from which they are derived.

Claims

1.-31. (canceled)

32. A pharmaceutically or nutraceutically acceptable salt or ester of a compound selected from the group consisting of: a compound of formula (II):
COOH—(CH.sub.2).sub.a—(CH═CH—CH.sub.2).sub.b—(CH.sub.2).sub.c—CH.sub.3   (II) and a compound of formula (III):
COOH—(CH.sub.2).sub.a—(CH═CH—CH.sub.2).sub.m—(CH.sub.2).sub.3—(CH═CH—CH.sub.2).sub.(b−1−m)—(CH.sub.2).sub.c—CH.sub.3   (III); wherein a is an integer between 1 and 14; b is an integer between 1 and 7; c is an integer between 0 and 14; m is an integer between 0 and b−11); and wherein a+3b+c+3 is an even integer.

33. A salt or ester according to claim 32, wherein c is 0, 3 or 6 and m=0.

34. A salt or ester according to claim 32, comprising a compound of formula (II), wherein: 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.

35. A salt or ester according to claim 32, comprising a compound of formula (III), wherein a=1, b=6, c=0 and m=0.

36. A salt or ester according to claim 32, wherein said pharmaceutically or nutraceutically acceptable salt is a sodium salt.

37. A method of inducing neuroregeneration or a method of preventing and/or treating a disease or pathology selected from the group consisting of: a neurological or neurodegenerative disease; a cancer; a neoplasm; an inflammatory disease; a cardiovascular disease; a skin and subcutaneous tissue pathology; a metabolic pathology; neuropathic pain; paralysis; sleep disorders; a digestive pathology; a musculoskeletal and connective tissue disease; a genitourinary pathology; and a metabolic disease in a patient, wherein said methods comprise administering an effective amount of a salt or an ester according to claim 32 to said patient.

38. The method according to claim 37, wherein the prevention and/or treatment or induction of neuroregeneration are characterized by the administration of a compound of formula (I), or a pharmaceutically acceptable salt or ester thereof:
COOH—CHOH—(CH.sub.2).sub.a—(CH═CH—CH.sub.2).sub.b—(CH.sub.2).sub.c—CH.sub.3   (I) 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 of the compound of formula (III); and wherein said compound of formula (I) is metabolized to produce a therapeutically effective amount of a compound of formula (II) or a compound of formula (III).

39. The method according to claim 37, wherein said compound is administered prior to, after, or in conjunction with a compound of formula (I) or a pharmaceutically acceptable salt or ester thereof:
COOH—CHOH—(CH.sub.2).sub.a—(CH═CH—CH.sub.2).sub.b—(CH.sub.2).sub.c—CH.sub.3   (I) wherein a is an integer between 1 and 14; b is an integer between 1 and 7; c is an integer between 0 and 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).

40. A pharmaceutical or nutraceutical composition comprising at least a first compound that is a salt or an ester according to claim 32 and at least one pharmaceutically or nutraceutically acceptable excipient.

41. The pharmaceutical or nutraceutical composition according to claim 40, further comprising a second compound of formula (I), or a pharmaceutically or nutraceutically acceptable salt or ester thereof:
COOH—CHOH—(CH.sub.2).sub.a—(CH═CH—CH.sub.2).sub.b—(CH.sub.2).sub.c—CH.sub.3   (I) wherein a is an integer between 1 and 14; b is an integer between 1 and 7; c is an integer between 0 and 14 and a+3b+c+3 is an even integer; and wherein said values of a, b and c of the second compound are equal to or different from the values of a, b and c of the at least first compound.

42. The pharmaceutical or nutraceutical composition according to claim 40, wherein c is 0, 3 or 6 and m=0.

43. The pharmaceutical or nutraceutical composition according to claim 40, wherein at least the first compound is a pharmaceutically or nutraceutically acceptable salt or ester of a compound of formula (II), wherein: 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.

44. The pharmaceutical or nutraceutical composition according to claim 40, wherein the at least first compound is a pharmaceutically or nutraceutically acceptable salt or ester of a compound of formula (III), wherein a=1, b=6, c=0 and m=0.

45. The pharmaceutical or nutraceutical composition according to claim 40, wherein the pharmaceutically acceptable salt is a sodium salt.

46. A method of inducing neuroregeneration or a method of preventing and/or treating a disease or pathology selected from the group consisting of: a neurological or neurodegenerative disease; a cancer; a neoplasm; an inflammatory disease; a cardiovascular disease; a skin and subcutaneous tissue pathology; a metabolic pathology; neuropathic pain; paralysis; sleep disorders; a digestive pathology; a musculoskeletal and connective tissue disease; a genitourinary pathology; and a metabolic disease in a patient, wherein said methods comprise administering an effective amount of the pharmaceutical composition according to any one of claims 40-45 to said patient.

47. A method of preventing a disease or pathology selected from the group consisting of: a neurological or neurodegenerative disease; a cancer; a neoplasm; an inflammatory disease; a cardiovascular disease; a skin and subcutaneous tissue pathology; a metabolic pathology; neuropathic pain; paralysis; sleep disorders; a digestive pathology; a musculoskeletal and connective tissue disease; a genitourinary pathology; and a metabolic disease in a patient, wherein said method comprises administering an effective amount of the nutraceutical composition according to any one of claims 40-45 to said patient.

48. 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 with a pharmaceutically acceptable salt or ester thereof:
COOH—CHOH—(CH.sub.2).sub.a—(CH═CH—CH.sub.2).sub.b—(CH.sub.2).sub.c—CH.sub.3   (I) in a subject, wherein said method comprises determining in vitro in a biological sample of said subject, the amount of a compound: of formula (II):
COOH—(CH.sub.2).sub.a—(CH═CH—CH.sub.2).sub.b—(CH.sub.2).sub.c—CH.sub.3   (II) or of formula (Ill):
COOH—(CH.sub.2).sub.a—(CH═CH—CH.sub.2).sub.m—(CH.sub.2).sub.3—(CH═CH—CH.sub.2).sub.(b−1−m)—(CH.sub.2).sub.c—CH.sub.3   (III) or of its carboxylate anion, or of a derivative formed therefrom in vivo or in vitro, wherein said amount is related to the efficacy of treating said disease or pathology; and wherein a is an integer between 1 and 14; b is an integer between 1 and 7; c is an integer between 0 and 14; m is an integer between 0 and (b−1); and wherein a+3b+c+3 is an even integer.

49. The method according to claim 48, wherein c is 0, 3 or 6 and m=0.

50. The method according to claim 48, wherein said method comprises determining in vitro in a biological sample of said subject, the amount of a compound of formula (II):
COOH—(CH.sub.2).sub.a—(CH═CH—CH.sub.2).sub.b—(CH.sub.2).sub.c—CH.sub.3   (II) or of its carboxylate anion, or a derivative formed therefrom in vivo or in vitro, and wherein: 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.

51. The method according to claim 48, wherein said method comprises determining in vitro in a biological sample of said subject, the amount of a compound of formula (III):
COOH—(CH.sub.2).sub.a—(CH═CH—CH.sub.2).sub.m—(CH.sub.2).sub.3—(CH═CH—CH.sub.2).sub.(b−1−m)—(CH.sub.2).sub.c—CH.sub.3   (III) or its carboxylate anion, or a derivative formed therefrom in vivo or in vitro, and in which a=1, b=6, c=0 and m=0.

52. The method according to claim 48, wherein the pharmaceutically acceptable salt is a sodium salt.

53. A compound of formula (II):
COOH—(CH.sub.2).sub.a—(CH═CH—CH.sub.2).sub.b—(CH.sub.2).sub.c—CH.sub.3   (II) wherein: 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.

54. A method of inducing inducing neuroregeneration or a method of preventing and/or treating a disease or pathology selected from the group consisting of: a neurological or neurodegenerative disease; a cancer; a neoplasm; an inflammatory disease; a cardiovascular disease; a skin and subcutaneous tissue pathology; a metabolic pathology; neuropathic pain; paralysis; sleep disorders; a digestive pathology; a musculoskeletal and connective tissue disease; a genitourinary pathology; and a metabolic disease in a patient, wherein said methods comprise administering an effective amount of the compound according to claim 53 to said patient.

55. The method according to claim 54, wherein the prevention and/or treatment or induction of neuroregeneration is characterized by administering a compound of formula (I), or a pharmaceutically acceptable salt or ester thereof:
COOH—CHOH—(CH.sub.2).sub.a—(CH═CH—CH.sub.2).sub.b—(CH.sub.2).sub.c—CH.sub.3   (I) 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 of the compound of formula (III); and wherein said compound of formula (I) is metabolized to produce a therapeutically effective amount of a compound of formula (II) or a compound of formula (III).

56. The method according to claim 54, wherein said compound is administered before, after or in conjunction with a compound of formula (I) or a pharmaceutically acceptable salt or ester thereof:
COOH—CHOH—(CH.sub.2).sub.a—(CH═CH—CH.sub.2).sub.b—(CH.sub.2).sub.c—CH.sub.3   (I) wherein a is an integer between 1 and 14; b is an integer between 1 and 7; c is an integer between 0 and 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).

57. A pharmaceutical or nutraceutical composition comprising at least a first compound of formula (II) according to claim 53 and at least one pharmaceutically or nutraceutically acceptable excipient.

58. The pharmaceutical or nutraceutical composition according to claim 57, further comprising a second compound of formula (I), or a pharmaceutically or nutraceutically acceptable salt or ester thereof:
COOH—CHOH—(CH.sub.2).sub.a—(CH═CH—CH.sub.2).sub.b—(CH.sub.2).sub.c—CH.sub.3   (I) wherein a is an integer between 1 and 14; b is an integer between 1 and 7; c is an integer between 0 and 14 and a+3b+c+3 is an even integer; and wherein said values of a, b and c of the second compound are equal to or different from the values of a, b and c of the at least first compound.

59. A method of inducing neuroregeneration or a method of preventing and/or treating a disease or pathology selected from the group consisting of: a neurological or neurodegenerative disease; a cancer; a neoplasm; an inflammatory disease; a cardiovascular disease; a skin and subcutaneous tissue pathology; a metabolic pathology; neuropathic pain; paralysis; sleep disorders; a digestive pathology; a musculoskeletal and connective tissue disease; a genitourinary pathology; and a metabolic disease in a patient, wherein said methods comprise administering an effective amount of the pharmaceutical composition according to claim 57 to said patient.

60. A method of preventing a disease or pathology selected from the group consisting of: a neurological or neurodegenerative disease; a cancer; a neoplasm; an inflammatory disease; a cardiovascular disease; a skin and subcutaneous tissue pathology; a metabolic pathology; neuropathic pain; paralysis; sleep disorders; a digestive pathology; a musculoskeletal and connective tissue disease; a genitourinary pathology; and a metabolic disease in a patient, wherein said method comprises administering an effective amount of the nutraceutical composition according to claim 57 to said patient.

61. A method of inducing neuroregeneration or preventing and/or treating a disease or pathology selected from the group consisting of: a neurological or neurodegenerative disease, neuropathic pain, and paralysis in a patient, wherein said methods comprise administering an effective amount of the compound of formula (III):
COOH—(CH.sub.2).sub.a+3—(CH═CH—CH.sub.2).sub.(b−1)—(CH.sub.2).sub.c—CH.sub.3   (III); wherein a=1, b=6, c=0, and m=0, to said patient.

62. A method of inducing neuroregeneration or preventing and/or treating a disease or pathology selected from the group consisting of: a neurological or neurodegenerative disease, neuropathic pain, and paralysis in a patient, wherein said methods comprise administering an effective amount of a pharmaceutical composition comprising at least a first compound of formula (III):
COOH—(CH.sub.2).sub.a+3—(CH═CH—CH.sub.2).sub.(b−1)—(CH.sub.2).sub.c—CH.sub.3   (III); wherein a=1, b=6, c=0, and m=0, and at least one pharmaceutically acceptable excipient, to said patient.

63. The method according to claim 62, further comprising administering a second compound of formula (I), or a pharmaceutically acceptable salt or ester thereof:
COOH—CHOH—(CH.sub.2).sub.a—(CH═CH—CH.sub.2).sub.b—(CH.sub.2).sub.c—CH.sub.3   (I) wherein a is an integer between 1 and 14; b is an integer between 1 and 7; c is an integer between 0 and 14 and a+3b+c+3 is an even integer; and wherein said values of a, b and c of the second compound are equal to or different from the values of a, b and c of the at least first compound.

64. A method of preventing a disease or pathology selected from the group consisting of: a neurological or neurodegenerative disease, neuropathic pain, and paralysis in a patient, wherein said methods comprise administering an effective amount of a nutraceutical composition comprising at least a first compound of formula (Ill):
COOH—(CH.sub.2).sub.a+3—(CH═CH—CH.sub.2).sub.(b−1)—(CH.sub.2).sub.c—CH.sub.3   (III); wherein a=1, b=6, c=0, and m=0, and at least one nutraceutically acceptable excipient, to said patient.

65. The method according to claim 64, further comprising administering a second compound of formula (I), or a nutraceutically acceptable salt or ester thereof:
COOH—CHOH—(CH.sub.2).sub.a—(CH═CH—CH.sub.2).sub.b—(CH.sub.2).sub.c—CH.sub.3   (I) wherein a is an integer between 1 and 14; b is an integer between 1 and 7; c is an integer between 0 and 14 and a+3b+c+3 is an even integer; and wherein said values of a, b and c of the second compound are equal to or different from the values of a, b and c of the at least first compound.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0142] FIG. 1. A Scheme illustrating the cellular metabolism of 2-hydroxidocosahexaenoic acid (DHA-H) resulting in (6Z,9Z,12Z,15Z,18Z)-heneicose-6,9,12,15,18-pentaenoicacid (HPA) by α-oxidation. DHA-H requires activation by an Acyl-CoA synthetase, in a process dependent on ATP (adenosine triphosphate) and magnesium (Mg.sup.2+). DHA-H-CoA would be subject to the activity of 2-hydroxyfitanoyl-CoA lyase (2-hydroxyacyl-CoA lyase 1, HACL1), leading to the formation of an intermediate polyunsaturated aldehyde that should contain 5 or 6 double bonds. The activity of HACL1 is dependent on thiamine pyrophosphate (TPP) and Mg.sup.2+, and can be inhibited by a competitive antagonist (e.g. oxythiamine). The aldehyde dehydrogenase enzyme would be responsible for the conversion of the intermediate aldehyde to HPA in a a process dependent on NAD.sup.+ (Nicotinamide Adenine Dinucleotide). B. DHA-H is metabolically transformed into HPA by α-oxidation in HEK293T cells. Intracellular levels of DHA-H (B1 and B3) and HPA (B2 and B4) are represented in the ordinate axis (nmoles/mg protein), versus treatment concentration with DHA-H sodium salt (μM) for 24 hours (B1 and B2) or incubation time (h) with a constant concentration of DHA-H sodium salt of 30 μM (B3 and B4), including untreated controls (C), in the abscissa axis. Black bars represent the result in cells without additional stimulus, white bars represent the result after simultaneous treatment with 1 mM oxythiamine, and striped bars represent the result after treatment with 10 mM oxythiamine. Both DHA-H and HPA increased as a function of concentration and incubation time, with HPA levels significantly higher than those of DHA-H at 30 μM exposure of DHA-H sodium salt from 24 hours. This increase in HPA is inhibited in the presence of 10 mM oxythiamine (which inhibits the HCLA1 enzyme), demonstrating the involvement of α-oxidation in this metabolic conversion. The bars represent the mean±standard error, and the statistical analysis was performed using one-way ANOVA and the Tukey multiple evaluation test: *p<0.05 when comparing HPA levels with those of DHA-H under the same condition; #, p<0.05 when comparing values in the presence and absence of 10 mM oxythiamine. C. Endogenous levels of DHA (non-hydroxy native form of docosahexaenoic acid) in HEK293T are not altered after treatment with DHA-H sodium salt. Intracellular levels of DHA are represented on the ordinate axis (nmoles/mg protein), versus treatment concentration with DHA-H sodium salt (μM) for 24 hours (C1) or incubation time (h) with a constant concentration of DHA-H sodium salt of 30 μM (C2), including untreated controls (C), on the abscissa axis. Treatment with DHA-H sodium salt did not have any significant effect on DHA levels either as a function of concentration or incubation time. The bars represent the mean±standard error, and the statistical analysis was done by unidirectional ANOVA and Tukey multiple evaluation test. From these results it can be concluded that the administration of DHA-H, or its sodium salt in this case, does not alter the endogenous levels of DHA (docosahexaenoic acid), nor of other cellular fatty acids studied, but that treatment with DHA-H results in an increase exclusively in the levels of HPA, implying that the therapeutic effect obtained through the treatment with DHA-H, and in particular with its sodium salt, is mediated by HPA and not by a modulation of the levels of other fatty acids of endogenous origin.

[0143] FIG. 2. A. Mice treated with DHA-H sodium salt exhibit dose-dependent brain accumulation of HPA, with DHA-H being undetectable in the brain. Brain levels of HPA (Al) or DHA (A2) (nmoles/mg of protein) are represented in the ordinate axis, with respect to treatment doses with the sodium salt of DHA-H (A1) and the sodium salt of DHA (A1 and A2) (mg/kg). A1: • animals WT; ∘ 5xFAD. A2: Black bars refer to WT animals and white bars to 5xFAD. The levels of HPA and DHA were determined in the brain of WT and 5xFAD mice after chronic administration of DHA-H sodium salt (4 months; 5 doses/week M-F; between 3 and 7 months of age; sacrifice at 7 months). HPA accumulates in the brain of both mouse strains similarly, depending on the dose of DHA-H sodium salt administered (A1: • r2=0.9292, p=0.0002; ∘ r2=0.9704, p<0.0001). DHA levels did not vary significantly between experimental conditions (A2). Data are shown as mean±standard error, and statistical analysis was done by one-way ANOVA and Tukey multiple evaluation test: *p<0.05 compared to control (mice treated with vehicle). Therefore, when the prodrug DHA-H, in particular its sodium salt, is administered in healthy mice and transgenic models of Alzheimer's disease, a significant accumulation of HPA occurs at the brain level, without the presence of the prodrug (DHA-H) being detected, nor changes in endogenous levels of DHA. B. 5xFAD mice treated with DHA-H sodium salt exhibit cognitive improvement that directly correlates with brain HPA levels. The number of total errors (B1), reference memory errors (RME) (B2) or working memory errors (WME) (B3), committed, are represented in the ordinate axis against the brain levels of HPA (nmol/mg of protein) in the abscissa axis. The cognitive assessment was done by testing the 8-arm radial labyrinth during the last month of treatment of the same animals shown in FIG. 2A. Individual experimental points from the entire study animal population were plotted and the data from the 5xFAD animals were adjusted to an inverse polynomial regression f(x)=y.sub.0+(a/x). The values of r.sup.2 and p corresponding to the regression of each parameter are shown below: total errors, RME and WME versus the brain concentration of HPA: C1: r.sup.2=0.9146 and p=0.0311; C2: r.sup.2=0.9252 and p=0.0243; C3: r.sup.2=0.7785 and p=0.0346. The data obtained suggest that minimal increases in brain levels of HPA are associated with an improvement in spatial cognition. Each point in the graphs represents the average±standard error for each pathology/treatment condition: • WT+vehicle, .Math. WT+DHA-H 20 mg/kg; .diamond-solid. WT+DHA-H 200 mg/kg; ∘ 5xFAD+vehicle; Δ 5xFAD+DHA-H 5mg/kg; ∇ 5xFAD+DHA-H 20mg/kg; □ 5xFAD+DHA-H 50 mg/kg; ⋄ 5xFAD+DHA-H 200mg/kg. These results show that moderate increases in brain HPA levels are significantly related to an improvement in spatial cognition, which is one of the most affected cognitive abilities in Alzheimer's disease.

[0144] FIG. 3. A. Mice treated with the sodium salt of DHA-H exhibit tumor accumulation of HPA, being undetectable DHA-H, in U118 cell xenographic tumors. The levels of DHA (black bars) and HPA (white bars) (pmoles/mg of tissue) in the tumor are represented in the ordinate axis, versus the treatment condition (vehicle and DHA-H 200 mg/kg) in the abscissa axis. NUDE (immunosuppressed) mice at 3 months of age were injected subcutaneously with 7.5.10.sup.6 U118 cells (grade IV human multiform glioblastoma). Tumor growth was allowed at the subcutaneous level for 10 days prior to initiation of oral treatments (vehicle or DHA-H sodium salt 200 mg/kg), which were maintained 42 days until sacrifice. Lipid analysis of the xenographic tumors revealed the absence (undetectable levels) of DHA-H. The bars represent the mean±standard error for each treatment condition. B. Tumor HPA levels correlate inversely with tumor size in xenographic models. The size of the tumor (cm.sup.3) is represented, in the ordinate axis, versus the levels of HPA (pmoles/mg of tissue) in the tumor, in the abscissa axis, for two treatment conditions: ∘ vehicle and sodium • salt of DHA-H 200 mg/kg. The presence of HPA in the tumor of animals under treatment with DHA-H sodium salt has astatistically significant linear relationship with tumor volume (A2), where: r.sup.2=0.4296 and p=0.0029. The results showed that HPA levels in xenographic tumors have a statistically significant inverse linear relationship to tumor size. In the absence of the DHA-H parent molecule in the target organ, these evidences show that the presence of HPA in the target organ has an in vivo therapeutic effect.

[0145] FIG. 4. A. DHA-H is a prodrug that is metabolically transformed into HPA by α-oxidation in U118 cells. The intracellular levels of DHA (black bars), DHA-H (white bars) and HPA (striped bars) are represented on the ordinate axis (nmoles/mg of protein), with respect to the treatment conditions: Control (C) and sodium salt of DHA-H 150 μM (48 h), in the presence or absence of simultaneous treatment with oxythiamine 1 and 10 mM, in the abscissa axis. Both DHA-H and HPA increased in cells treated with the sodium salt of DHA-H. This increase in HPA is inhibited in the presence of 1 and 10 mM oxythiamine, demonstrating the involvement of α-oxidation in this metabolic conversion. The bars represent the mean±standard error, and the statistical analysis was performed using one-way ANOVA and the Tukey multiple evaluation test: *p<0.05 when comparing only HPA levels. B. Metabolic conversion of DHA-H to HPA is necessary for an anti-tumor effect to exist. The cell viability (% of the control -C- without oxythiamine) is represented in the ordinate axis, with respect to the treatment conditions: Control (C- black bars) and the sodium salt of DHA-H 150 μM, 48 h (white bars) in the presence and absence of simultaneous treatment with 1 mM oxythiamine, in the abscissa axis. Treatment with DHA-H on U118 cells significantly reduces the viability of the culture, while treatment with oxythiamine (alone) has no effect on cell viability. However, when treatment with the sodium salt of DHA-H is done simultaneously with oxythiamine, the anti-proliferative effect of this compound decreases significantly compared to the effect without oxythiamine. The bars represent the mean±standard error, and the statistical analysis was performed using one-way ANOVA and the Tukey multiple evaluation test: *p<0.05 when compared to control (C); #p<0.05 when comparing the effect of DHA-H in the presence and absence of oxythiamine.

[0146] FIG. 5. A. Viability of U118 cells in culture after treatment with DHA-H sodium salt, DHA sodium salt, and HPA. The cell viability (% of the control without treatment) is represented in the ordinate axis, versus the different treatment conditions in the abscissa axis: Control (black bar), DHA-H sodium salt (150 μM, 48 h—white bar), DHA (150 μM, 48 h—striped bar) and HPA (150 μM, 48 h—grid bar). Treatment with HPA under the same conditions induces a much more evident degree of mortality on the culture than that induced by DHA-H (prodrug) or DHA (natural analogue). This effect could be due to a mixture of anti-proliferative effect and toxic effects typical of HPA, which would not be attributable to DHA-H or DHA under the same experimental conditions. The bars represent the mean±standard error, and the statistical analysis was performed using one-way ANOVA and the Tukey multiple evaluation test: *p<0.05 when compared to control. B. Intracellular levels of HPA in U118 cells in culture, treated with the sodium salt of DHA-H and HPA. The levels of HPA (nmoles/mg of protein) are represented in the ordinate axis, versus the treatment conditions in the abscissa axis: sodium salt of DHA-H (150 μM, 48 h—black bar) and HPA (5-150 μM, 48 h—white bars). Administration of 150 μM of the DHA-H sodium salt results in HPA levels equivalent to those generated by the HPA treatment itself at 5 μM. Treatment with 150 μM HPA results in significantly higher intracellular levels of HPA than those generated by the same concentration of administration of the prodrug. The bars represent the mean±standard error.

[0147] C. Levels of DHA-H and DHA in HEK293T cells in the presence (C1) or absence (C2) of culture medium. The levels of DHA-H (•) and DHA (∘) in the culture medium (% of the initial levels at time 0) are represented in the ordinate axis, versus the incubation time (h) in the abscissa axis. The concentration of the lipid in the culture medium is 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 decreased significantly at 48 and 72 h, as a consequence of DHA uptake by the cells, while DHA-H levels remained unchanged up to 72 h. In the absence of cell culture (C2), the levels of both DHA and DHA-H remained constant over time. The bars represent the mean±standard error, and the statistical analysis was performed using one-way ANOVA and the Tukey multiple evaluation test: *p<0.05 when compared to control.

[0148] FIG. 6. A, B and C: Chronic treatment with HPA acid or its prodrug, DHA-H, prevents cognitive decline typical of Alzheimer's disease in the murine transgenic model (5xFAD). Cognitive assessment was performed by the 8-arm Radial Labyrinth test. The animals received treatment between 3 and 7 months of age and the test was performed during the last month of treatment. During this test, the total errors made during the test (A), the reference memory errors (RME) (B) and the working memory errors (WME) (C) were taken into account. Each column represents the mean±SEM of errors during the last week of the radial labyrinth test. The black columns represent the errors made by WT mice. The blank columns represent the errors made by the 5xFAD transgenic mice treated with the vehicle (5% ethanol). Striped columns represent errors made by 5xFAD mice treated with DHA-H (20 mg/kg/day). Boxed columns represent errors made by 5xFAD mice treated with HPA (20 mg/kg/day). Results show a cognitive improvement of 5xFAD mice treated with DHA-H and HPA in a similar manner. The bars represent the mean±standard error for each treatment condition and the statistical analysis was performed by unidirectional ANOVA and the Tukey multiple evaluation test: *p<0.05 when compared to healthy control (WT) and #p<0.05 when compared to 5xFAD control condition (treated with the vehicle).

[0149] FIG. 7. A: Tumor growth is inhibited in vivo in the presence of treatment with HPA sodium salt or its prodrug, DHA-H, in xenographic models. The size of the tumor (cm.sup.3) is represented in the ordinate axis, versus the days of treatment elapsed in the abscissa axis. To induce xenographic tumors in NUDE (immunosuppressed) mice 3 months of age, 7.5.10.sup.6 human grade IV (U-118 MG) glioblastoma cells were inoculated subcutaneously on both sides of the animal's dorsal flank (8-12 weeks of age, 30-35 g). After 10 days, the tumors became visible with an approximate volume of 0.1 cm.sup.3. Animals were randomly divided into groups with a similar mean tumor volume and received daily oral treatments for 42 days: o vehicle (untreated control), DHA-H .box-tangle-solidup. (200 mg/kg/day) and .square-solid. HPA (200 mg/kg/day). Tumor volumes (v) were calculated as v=A.sup.2×L/2, where A is the width of the tumor and L is its length. The data obtained for each treatment condition were adjusted to an exponential growth curve. B: HPA and the prodrug, DHA-H, reduce the volume of the xenographic tumor significantly compared to the untreated control. The volume of the tumors induced 42 days after the start of the treatments is represented in the ordinate axis, versus the treatment conditions in the abscissa axis. The individualised data of the animals participating in the study are represented: o carrier (untreated control), DHA-H .box-tangle-solidup. (200 mg/kg/day) and .square-solid. HPA (200 mg/kg/day). The bars represent the mean±standard error for each treatment condition and the statistical analysis was performed by unidirectional ANOVA and the Tukey multiple evaluation test: *p<0.05 when compared to the control condition.

[0150] FIG. 8. Illustrative schemes of the cellular metabolism of 2-hydroxylated polyunsaturated fatty acids (prodrugs, PUFA-H) giving rise via α-oxidation to their corresponding non-hydroxylated metabolites, the latter having one carbon atom less than the initial molecule. Hydroxylated fatty acid requires activation by an Acyl-CoA synthetase, in a process dependent on ATP (adenosine triphosphate) and magnesium (Mg.sup.2+). This PUFA-H-CoA would be subject to the activity of 2-hydroxyacyl-CoA lyase (HACL, isoforms 1 or 2 depending on the cell type), which would lead to the formation of an intermediate polyunsaturated aldehyde. The activity of HACL depends on thiamine pyrophosphate (TPP) and Mg.sup.2+, and can be inhibited by a competitive antagonist, such as oxythiamine. The aldehyde dehydrogenase enzyme would be responsible for the conversion of the intermediate aldehyde into the final fatty acid in a process dependent on a process dependent on NAD.sup.+ (Nicotinamide Adenine Dinucleotide). A. Scheme of cell conversion of 2-hydroxy-linoleic acid (LA-H) resulting in (8Z,11Z)-heptadeca-8,11-dienoicacid (HDA). B. Scheme of cell 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 cell conversion of 2-hydroxy-gamma (γ)-linolenic acid (GLA-H) resulting in (5Z,8Z,11Z)-heptadeca-5,8,11-trienoicacid (HTA ω-6). D. Scheme of cell conversion of 2-hydroxy-arachidonic acid (ARA-H) resulting in (4Z,7Z,10Z,13Z)-nonadeca-4,7,10,13-tetraenoicacid (NTA). E. Scheme of cell conversion of 2-hydroxy-eicosapentaenoic acid (EPA-H) resulting in (4Z,7Z,10Z,13Z,16Z)-nonadeca-4,7,10,13,16-pentaenoicacid (NPA). F. Scheme of cell conversion of 2-hydroxidocosahexaenoic acid (DHA-H) resulting in (6Z,9Z,12Z,15Z,18Z)-heneicosa-6,9,12,15,18-pentaenoicacid (HPA).

[0151] FIG. 9. Amplified regions of the different chromatograms obtained by gas chromatography with flame ionisation detector (GC-FID) by treating HEK293T cells with the corresponding prodrug: A. (1) control (vehicle) and (2) LA-H (100 μM, 24 h). The white arrow indicates the chromatographic peak of the LA-H parent molecule, the black arrow indicates the chromatographic peak corresponding to the HDA metabolite. The formation of HDA is inhibited in the presence of 10 mM oxyamine. B. (1) control (vehicle) and (2) ALA-H (100 μM, 24 h). The white arrow indicates the chromatographic peak of the ALA-H parent molecule, the black arrow indicates the chromatographic peak corresponding to the metabolite HTA ω-3. The formation of ω-3 HTA is inhibited in the presence of 10 mM oxythiamine. C. (1) control (vehicle) and (2) GLA-H (100 μM, 24 h). The white arrow indicates the chromatographic peak of the parent molecule GLA-H, the black arrow indicates the chromatographic peak corresponding to the metabolite HTA ω-6. The formation of ω-6 HTA is inhibited in the presence of 10 mM oxythiamine. D. (1) control (vehicle) and (2) ARA-H (100 μM, 24 h). The white arrow indicates the chromatographic peak of the ARA-H parent molecule, the black arrow indicates 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 indicates the chromatographic peak of the EPA-H parent molecule, the black arrow indicates the chromatographic peak corresponding to the NPA metabolite. NPA formation is inhibited in the presence of 10 mM oxyamine. F. (1) control (vehicle) and (2) DHA-H (100 μM, 24 h). The white arrow indicates the chromatographic peak of the HPA parent molecule, the black arrow indicates the chromatographic peak corresponding to the HPA metabolite. The formation of HPA is inhibited in the presence of 10 mM oxythiamine.

[0152] FIG. 10. Treatment with HPA and other odd-chain polyunsaturated fatty acids prevents excitotoxicity-induced neuronal death. Neuronal culture was obtained by differentiation from human SH-SY5Y neuroblastomas by retinoic acid and BDNF (Brain Derived Neurotrophic Factor). Neuronal death by excitotoxicity was induced by the addition of NMDA (10 mM) and calcium/glycine (530 μM/10 mM) to the culture medium for 1 hour. To test the neuroprotective effect of the different study compounds (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 and HPA or C21:5 ω-3), pre-treatment was performed for 24 hours: vehicle (black bars), 1 μM (white bars), 3 μM (dotted bars) and 10 μM (striped bars). Treatments tested under these experimental conditions demonstrated that these compounds can prevent excitotoxicity-induced death from a 3 μM concentration. The bars represent the mean±standard error for each treatment condition and the statistical analysis was performed by unidirectional ANOVA and the Tukey multiple evaluation test: *p<0.05 when compared to the control condition (vehicle pre-treatment).

[0153] FIG. 11. Illustrative scheme of 2-OHOA (LAM561) cell metabolism resulting in 8Z-heptadecenoic acid (C17:1n9), by α-oxidation. 2-OHOA requires activation by an Acyl-CoA ligase, in a process dependent on ATP (adenosine triphosphate) and magnesium (Mg2+). 2OHOA-CoA would be subject to the activity of 2-hydroxyfitanoyl-CoA lyase (2-hydroxyacyl-CoA lyase 1, HACL1), leading to the formation of an intermediate monounsaturated aldehyde. The activity of HACL1 is dependent on thiamine pyrophosphate (TPP) and Mg.sup.2+, and can be inhibited by a competitive antagonist (e.g. oxythiamine). The aldehyde dehydrogenase enzyme would be responsible for the conversion of the intermediate aldehyde to 8Z-heptadecenoic in a a process dependent on NAD+(Nicotinamide Adenine Dinucleotide).

[0154] FIG. 12. Analysis of the composition of fatty acids in U-118 MG glioma cells. (A) Representative chromatograms showing the composition of fatty acids in U-118 MG cells incubated in the presence of 400 μM 2OHOA sodium salt or absence of treatment (Control) for 24 h, determined by gas chromatography. Retention times (min): C17:1n-9 (10.12), OA (13.01), 2OHOA (16.87), and C17:0 margaric acid as internal control (10.81). (B) Quantification of different fatty acids identified in the chromatograms (OA, 2OHOA and C17:1n-9). 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. The columns show the mean±SEM of three independent experiments expressed in nmoles and normalized per mg of protein. Statistical significance is determined with a Student's t test (***p<0.001 with respect to the control).

[0155] FIG. 13. Analysis of the composition of fatty acids in different glioma and non-tumor cell lines after treatment with 2OHOA sodium salt. Representative chromatograms showing the composition of fatty acids (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-tumor: (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 hours) determined by gas chromatography 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. C17:0 margaric acid is included as an internal control. The columns show the mean±SEM of three independent experiments expressed in nmoles and normalized per mg of protein. Statistical significance is determined with a Student's t-test (**p<0.01, ***p<0.001 with respect to the control).

[0156] FIG. 14. Effect of 2OHOA sodium salt, OA and C17:1n-9 sodium salt on cell viability and proliferation of glioma cells. Viability curves of different glioma cell lines (A1-A3) U-118 MG; (B1-3) U-251 MG; and (C1-C3) SF-295 treated with increasing doses of 2OHOA (0-1000 μM) sodium salt (A1, B1, and C1); OA (0-300 μM) (A2, B2, and C2); and C17:1n-9 (0-300 μM) sodium salt (A3, B3, and C3) for 72 hours. Viability was determined by violet crystal staining. Each value represents the mean±SEM of three independent experiments with at least three biological replicates, expressed as a percentage with respect to the cells treated with vehicle (100%).

[0157] FIG. 15. Effect of 2OHOA sodium salt, C17:1n-9 sodium salt, and OA on cell viability and proliferation of non-tumor cells. Non-tumor cell viability curves (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 violet crystal staining. Each value represents the mean±SEM of three independent experiments with at least three biological replicates, expressed as a percentage with respect to the cells treated with carrier (100%).

[0158] FIG. 16. Analysis of the effect of different fatty acids on markers of proliferation and death in different cell lines. Immunoblots representative of the effect of fatty acids (200 μM OA, 200 μM C17:1n-9 sodium salt and 400 μM 2OHOA sodium salt) on various proteins involved in the 2OHOA-regulated cell death and signaling pathways in glioma cells: (A) U-118 MG; (B) U-251 MG; and (C) SF-295; and non-tumor: (D) MRC-5 (human fibroblasts); and (E) mouse astrocytes, after 72h of treatment.

[0159] FIG. 17. Analysis of the composition of fatty acids in U-118 MG glioma cells after inhibition of α-oxidation and effect of oxythiamine on the cell survival of U-118 MG glioma cells. (A)

[0160] Quantification of 2OHOA and C17:1n-9 fatty acids in U-118 MG cells treated with 400 μM 2OHOA for 24 hours, pre-incubated with increasing doses (1-10 mM) of oxythiamine (α-oxidation inhibitor) for 90 minutes, determined by gas chromatography. Results are shown as the mean±SEM of three independent experiments expressed in nmoles and normalized per mg protein. The statistical significance is determined with a Student's t test (*p<0.05, ***p<0.001 comparing the amount of 2OHOA with that detected after 400 μM of 2OHOA in the absence of oxythiamine; $$p<0.01, $$$p<0.001 comparing the amount of C17:1n-9 with that formed after 400 μM of 2OHOA in the absence of oxythiamine). (B) Viability of U-118 MG cells pre-incubated with oxythiamine (for 90 minutes) and treated in the absence (Control) or in the presence of 2OHOA sodium salt (400 μM, 72 hours), determined by vital exclusion staining with trypan blue. The results are represented as the mean cell count±SEM of three independent experiments. Statistical significance is determined with a Student's t test (***p<0.001 with respect to the absence of 2OHOA and oxythiamine, Control-0; and $$p<0.01, $$$p<0.001 with respect to treatment with 2OHOA without pre-incubation with oxythiamine).

[0161] FIG. 18. Effect of 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-tumor cells: (C) human MRC-5 fibroblasts and (D) mouse astrocytes; all treated in the absence or presence of 2OHOA sodium salt (400 μM, 72 hours) and pre-incubated or not with oxythiamine (for 90 minutes). Cell viability was determined by vital exclusion staining with trypan blue. The results are represented as the mean cell count±SEM of three independent experiments. Statistical significance is determined with a Student's t test (**p<0.01 and ***p<0.001 with respect to the absence of 2OHOA and oxyamine; and $p<0.05 with respect to treatment with 2OHOA alone).

[0162] FIG. 19. Analysis of the effect of the metabolite C17:1n-9 on the action of 2OHOA in markers of proliferation and death in different cell lines by inhibition of its formation by oxythiamine. Immunoblots representative of the effect of 2OHOA (400 μM) combined or not with oxythiamine (2 mM) on various proteins involved in the 2OHOA-regulated cell death and signaling pathways in glioma cells: (A) U-118 MG; (B) U-251 MG; and (C) SF-295; and non-tumor: (D) MRC-5 (human fibroblasts); and (E) mouse astrocytes, after 72h of treatment.

[0163] FIG. 20. Analysis of the composition of fatty acids in rat plasma after 24 hours of treatment with 2OHOA sodium salt. (A) Representative chromatograms showing the composition of fatty acids in rat plasma samples obtained at different times (0, 1, 2, 3, 4, 6, 8 and 24 hours) after acute treatment with 2OHOA (2mg/Kg, 24 hours) determined by gas chromatography. C17:0 margaric acid was quantified as internal control in the chromatogram. (B) Quantification of the 2OHOA and C17:1n-9 fatty acids identified in the chromatograms. Results are shown as the mean±SEM of 4 animals and expressed in nmoles and normalized per ml of plasma. Statistical significance is determined with a Wilcoxon test (*p<0.05 and **p<0.01 with respect to baseline levels at 0 hours; $p<0.05 and $$p<0.01 with respect to 2OHOA fatty acid levels).

[0164] FIG. 21. Analysis of the composition of fatty acids in rat plasma after 15 days of treatment with 2OHOA sodium salt. (A) Representative chromatograms showing the composition of fatty acids in rat plasma samples obtained at different times (0, 1, 2, 3, 4, 6, 8 and 24 hours) after chronic treatment with 2OHOA (2mg/Kg, 15 days) determined by gas chromatography. C17:0 margaric acid was quantified as internal control. (B) Quantification of the 2OHOA and C17:1n-9 fatty acids identified in the chromatograms. Results are shown as the mean±SEM of 4 animals, expressed in nmoles and normalized per ml of plasma. Statistical significance is determined by a Wilcoxon test (*p<0.05 and **p<0.01 with respect to baseline levels at 0 hours; $p<0.05 and $$p<0.01 with respect to 2OHOA fatty acid levels).

[0165] FIG. 22. Analysis of the composition in fatty acids of xenographic tumors of immunosuppressed mice. (A) Representative chromatograms showing the composition of fatty acids in xenographic tumors originating from U-118 MG glioblastoma cells in mice treated orally and daily with 2OHOA sodium salt (200 mg/kg, 42 days) determined by gas chromatography. (B) Quantification of the OA and C17:1n-9 fatty acids identified in the chromatograms. C17:0 margaric acid was quantified as internal control. The white bar corresponds to the concentration of each fatty acid in the control, and the black bar corresponds to the concentration of the fatty acid after treatment with 2OHOA sodium salt. Results are shown as the mean±SEM of at least 7 xenographic tumors and expressed in nmoles and normalized per g of tissue. Statistical significance is determined by a Mann-Whitney test (* **p<0.01 with respect to control).

[0166] FIG. 23. Inverse correlation between tumor volume and amount of C17:1n-9 metabolite.

[0167] Representation of the amount of metabolite quantified by gas chromatography in xenographic tumours of mice, relative to the tumour volume measured on day 42 of treatment with 200 mg/kg sodium salt of 2OHOA (black boxes) or its vehicle (Control, white circles). Significance determined by Pearson's correlation coefficient (p=0.0001; r=−0.825).

[0168] FIG. 24. Analysis of composition in fatty acids in human patients with advanced glioma. (A) Representative chromatogram of the fatty acid composition of a patient with glioma responsive to treatment with 2OHOA sodium salt (12 g/day, 21 days) and determined in plasma samples obtained at different treatment times (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 times 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 the first day of the second treatment cycle (574 hours). (C) Quantification of the 2OHOA and C17:1n-9 fatty acids identified in the chromatograms of the same responding and non-responding patients jointly. The C17:0 margaric acid in the chromatograms was quantified as internal control. Results are shown as the mean±SEM of 8 patients (4 responders and 4 non-responders) and expressed in nmoles and normalized per ml of plasma. Statistical significance is determined by a Mann-Whitney test (*p<0.05 and **p<0.01 relative to the amounts of 2OHOA fatty acid).

EXAMPLES

[0169] The examples described below are for purposes of illustration only and are not meant to limit the scope of the present invention.

Example 1

Fatty Acids, Reagents and Organic Solvents

[0170] 1.1. DHA, DHA-H and HPA

[0171] DHA (sodium salt of docosahexaenoic acid; C22:6 n-3), DHA-H (sodium salt of 2-hydroxy-drocosahexaenoic acid; 20H-C22:6 n-3), EPA-H (sodium salt of 2-hydroxy-eicosapentaenoic acid), ARA-H (sodium salt of 2-hydroxy-arachidonic 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-hydroxy-linoleic 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), HTA ω-6 (sodium salt of (5Z,8Z,11Z)-heptadeca-5,8,11-trienoic acid), HTA 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). The margaric acid (C17:0) was purchased from Sigma-Aldrich and the heneicosapentanoic acids (HPA free acid; C21:5 n-3) and (4Z,7Z,10Z,13Z,16Z)-nonadeca-4,7,10,13,16-pentaenoic acid (NPA free acid; C19:5 ω-3) were purchased from Cayman Chemicals (Michigan, United States). The 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 (ethylene diamine tetraacetic acid) and tris-base were acquired from Sigma-Aldrich. In contrast, 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 Laboratorios Calier S.A. (Spain), and oxythiamine hydrochloride from Santa Cruz Biotechnology (Germany).

[0172] For the production of HPA, chemical synthesis is performed from the (5Z,8Z,11Z,14Z,17Z)-eicose-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.

##STR00001##

[0173] Reagents and conditions: a)(COCI).sub.2/PhH 1.5h rt., b)CH.sub.2N.sub.2/ether 20 min. 0° C., c)AgOBz(cat.), Et.sub.3N/THF/H.sub.2O

[0174] The synthesis of the sodium salt of the HPA of the present invention has been made from the compound designated with the number 5 when R is CH.sub.3-CH.sub.2—(CH═CH—CH.sub.2).sub.5—CH.sub.2CH.sub.2—, which corresponds to HPA (C.sub.21). The salt is obtained under an acid base reaction, a liquid-liquid extraction is performed with MTBE/HCI and the pH is adjusted with NaOMe to obtain the sodium salt of HPA with good yields. A similar procedure can be performed for the synthesis of HDA, HTA ω-3, HTA ω-6, NTA, and NPA, by adjusting the starting substrate.

[0175] 1.2. OHOA, OA and C17:1n-9

[0176] The lipid compounds sodium salt of 2OHOA, sodium salt of OA and sodium salt of C17:1n-9 were purchased from Medalchemy, SL (Spain).

[0177] The chemical synthesis of C17:1n-9 is disclosed in WO1997049667. A solution of 8Z-heptadecene (66mg, 0.26mmo1, 1 equivalent) and 2-methyl-2-butane (1.6mL, 15.1 mmol, 58 equivalents) in tBuOH (6.5mL) at 25° C., under an N.sub.2 atmosphere, is treated by adding dropwise (2.5mL) a solution of NaClO.sub.2 (80%, 208 mg, 2.3 mmol, 9 equivalents) and NaH.sub.2PO.sub.4.H.sub.2O (250mg, 1.8 mmol, 7 equivalents) in deionized water. The reaction mixture was allowed to stir for an additional 15 minutes, before being concentrated in vacuo. The residue is treated with water (30 mL) and the aqueous layer is extracted with EtOAc (3x30 mL). The organic layers are dried with (Na.sub.2SO.sub.4), filtered and concentrated in vacuo. Chromatography (SiO.sub.2, 2×13 cm, 10-20% EtOAc-hexane gradient elution) afforded 27 mL (66 mg, 95%) as a clear oil. The synthesis of the sodium salt of C17:1n-9 of the present invention has been made from compound C17:1n-9. The salt is obtained under an acid base reaction, a liquid-liquid extraction is performed with MTBE/HCI and the pH is adjusted with NaOMe to obtain the sodium salt of C17:1n-9 with good yields.

Example 2

Compositions with DHA-H and HPA

[0178] Some examples of compositions that do not limit the scope of the invention are described in general terms below.

TABLE-US-00001 TABLE 1 Example Topical Use Formulation Composition Composition Composition Component % w/W % w/W % w/W DHA-H 3.6 0 1.3 HPA 0 3.6 1.3 DMSO 80.0 80.0 80.0 Water 16.4 16.4 16.4 Total 100 100 100

TABLE-US-00002 TABLE 2 Example oral formulation Composition Composition Composition Component % w/W % w/W % w/W DHA-H 5 0 2.5 HPA 0 5 2.5 Ethanol (96% v/v) 5 5 5 Water 90 90 90 Total 100 100 100

TABLE-US-00003 TABLE 3 Example oral formulation soft capsule Component Composition % w/W HPA 63.3 Triglycerides 25.5 Glyceryl monostearate 6.66 Aroma 2.22 Dismutase superoxide 1.11 Colloidal silica 1.11 Total 100

Example 3

Cellular Assays with DHA-H and HPA

[0179] 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 (Human Embryonic Kidney Cells 293T) cell cultures were employed, which is an embryonic, non-tumoral cell line, widely used in human metabolism studies under physiological conditions.

[0180] HEK293T cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Dubelcco'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. Mouse 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% CO.sub.2 at 37° C.

[0181] HEK293T cells were incubated with DHA-H and DHA at 10, 30 and 100 μM for 24 hours, and at 30 μM for 6, 48 and 72 hours. These cells were also incubated with LA-H, ALA-H, GLA-H, ARA-H and EPA-H at 100 μM for 24 hours. 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. The HEK293T cells were separated from the plates by pipettes with cold phosphate buffer saline solution (PBS). Cells were recovered by centrifugation (1000 xg, 10 min at 4° C.) and washed twice with cold PBS before being frozen at −80° C. To analyze DHA-H and DHA levels in the 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 attached HEK293T cells (5.10.sup.5 cells/plate). The plates were incubated as described above and 1 ml aliquots of the plates were collected at 0, 6, 24, 48 and 72 hours. Aliquots of the cell culture medium were immediately centrifuged at 1000×g for 10 min at 4° C. to remove any cell suspension and the cell-free aliquots were stored at -20° C.

[0182] U-118 MG, MIA-PaCa 2 and A549 cell lines were obtained from the European Collection of Cell Cultures (ECACC) via Sigma-Aldrich Co (St Louis, Mo.) and maintained in RPMI (Roswell Park Memorial Institute) culture medium (U-118 MG and A549) or DMEM (MIA-PaCa 2) supplemented with 10% FBS (Gibco, Thermo-Fisher), in an atmosphere of 5% CO.sub.2 at 37° C. U-118 MG, MIA-PaCa 2, and A549 cells were treated under the conditions described in the description of the assay performed to obtain the results of table 4, eventually in the presence or absence of oxytymine (1 or 10 mM). Cell survival was analyzed in a Barker chamber using trypan blue vital exclusion staining (Scharlab) or by cell proliferation kit II (Roche). Briefly, the cells were seeded in 96-well plates at a density of 3 x 10.sup.3 cells per well 24 h prior to treatment, and then cultured in the presence or absence of compounds of interest at the concentrations and for the times indicated in the figures. After different times, plaque viable cells were determined by the addition of XTT according to the manufacturers instructions. Cells were incubated at 37° C. in 5% CO.sub.2 until a constant color was developed and absorbance was recorded at 495 nm using a microplate reader with a reference wavelength of 650 nm (FLUOstar Omega, BMG LABTECH, Germany).

[0183] SH-SY5Y human neuroblastoma cells were maintained in DMEM-F12 (Invitrogen) supplemented with 10% FBS (Sigma), penicillin/streptomycin (PAA), non-essential amino acids (Sigma), and 2 mM L-Gln (Sigma). Differentiation of these cells to a neuronal phenotype was carried out following a standard procedure. Briefly, the cells were seeded on plates pre-treated with poly-L-lysine and 24 h later, the medium was replaced by a fresh medium supplemented with 10 μM retinoic acid (Sigma). The cells were then incubated in the dark for 5 days and the medium was replaced with a serum-free medium and supplemented with 50 ng/ml of 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 the compounds HDA, HTA ω-3, HTA ω-6, NTA, NPA and HPA, at 1.3 and 10 pm, for 24 h, prior to induction of excitotoxicity with NMDA (n-Methyl-D-Aspartate, 10 mM, Sigma) in a medium containing glycine (530 μM, Sigma) and calcium (10 mM, Sigma).

[0184] Treatment with DHA-H results in high cellular levels of HPA, compared to prodrug levels in cell cultures (FIG. 1B). In FIG. 1B, intracellular levels of DHA-H and HPA are shown in HEK293T cells under treatment with DHA-H. The accumulation of both compounds is evident as a function of treatment concentration or incubation time, but HPA levels are significantly higher than those of the prodrug from 24 hours of incubation and 30 μM of treatment. The increase in HPA levels is inhibited in the presence of concomitant treatment of oxythiamine (partial inhibition with 1 mM and almost total with 10 mM), a competitive antagonist of 2-hydroxyacyl-CoA lyase (see FIG. 1A). In this regard, it has also been found that endogenous levels of DHA (the non-hydroxylated native form) are not altered by this treatment with DHA-H (FIG. 1C).

[0185] Similarly, FIG. 8 shows that this same metabolic pathway is valid for other 2-hydroxylated polyunsaturated fatty acids, employed as prodrugs, such as LA-H, ALA-H, GLA-H, ARA-H, and EPA-H, resulting in HDA, HTA ω-3, HTA ω-6, NTA, NPA, respectively (chromatograms shown in FIG. 9). All of these metabolites have demonstrated therapeutic activity, as shown in FIG. 10 and table 4 below:

TABLE-US-00004 TABLE 4 IC50 values in human glioblastoma (U118 MG), pancreatic cancer (MIA-PaCa 2) and human lung adenocarcinoma (A549) cell lines IC50 (μM) U118 MG MIA-PaCa 2 A549 HDA (C17:2 n-6) 144 ± 32 234 ± 18  159 ± 10 HTA n-3 (C17:3 n-3) 129 ± 03 216 ± 24  166 ± 30 HTA n-6 (C17:3 n-6) 235 ± 26 95 ± 31 212 ± 48 NTA(C19:4 n-6) 113 ± 13 62 ± 02 216 ± 03 NPA (C19:5 n-3)  90 ± 09 91 ± 18 143 ± 39 HPA (C21:5 n-3) 124 ± 16 58 ± 17 228 ± 03

[0186] The anti-tumor activity of the different metabolites described in FIGS. 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, on which the IC50 value for each of these compounds was determined (Inhibitory Concentration 50: concentration of study compound that induces death of 50% of the tumor cell population). The cell cultures used correspond to different types of cancer: U118-MG (human grade IV glioblastoma), MIA-PaCa 2 (pancreatic carcinoma) and A549 (small cell lung adenocarcinoma). The different compounds showed variable IC50 values on the different tumor lines, demonstrating the selectivity of some of them to induce the selective death of certain types of tumor cells.

Example 4

In Vivo Trials with DHA-H and HPA

[0187] The 5xFAD model of Alzheimer's disease is a dual transgenic PS1/APP mouse that harbors five human mutations associated with familial AD (Tg6799 line): Swedish (K670N/M671L), Florida 151(1716V) and London (V717I) in APP; and clinical mutations M146L and L286V in human PS1. Both transgenes are expressed under the control of the Thy-1 promoter and mice show cognitive decline from 4 months of age (Oackley et al., 2006, Neurosci 26(40), 10129-10140. doi: 10.1523/jneurosci.1202-06.2006). 5xFAD transgenic animals and wild type (WT) 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. The animals were housed at a controlled temperature of 22° C. (±2° C.) and a humidity of 70%, in a 12h-12h light-dark cycle, with free access to a standard laboratory diet (Panlab A03, Barcelona, Spain). Transgenic male WT and 5xFAD mice received DHA-H (or DHA) orally, dissolved in 5% ethanol, at a daily dose of 5, 20, 50 and 200 mg/kg, or vehicle alone. On the other hand, in an independent assay, these animals have also been treated with HPA (20 mg/kg) and DHA-H (20 g/kg) to compare the effect of both compounds in this model. These treatments were initiated when the mice reached 3 months of age (dosed 5 days/week) and continued until 7 months of age. During the last month of treatment, all animals were kept on a hypocaloric diet to perform selected behavioral spatial learning and memory testing (radial arm maze). A total of 46 animals were used for the study shown in FIG. 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 of FIG. 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). After the behavioral test, mice were maintained on a normal diet (and treatment) for an additional week, after which they were anesthetized with an intraperitoneal injection of ketamine/xylazine (100/10 mg/kg) and infused intracardiacally with 50 mL of heparinized saline. Animal brains were immediately removed and dissected by the midline on a cold surface. Once the cerebellum was removed, each cerebellum-free half was frozen in liquid nitrogen and stored at −80° C. NUDE (Swiss) Crl:NU (Ico)-Foxn1.sup.nu mice (8-12 weeks old, 30-35 g, Charles River Laboratories, Paris, France) were maintained in a thermostatic cabinet (28° C., EHRET, Labor-U-Pharmatechnik) with a sterile air flow at a relative humidity of 40-60% and with 12-hour dark/light cycles. Their diet consisted of a standard diet with feed (Labdiet 22% rat-mouse breeding, Sodispan) ad libitum. To cause xenographic tumors, 7.5×10.sup.6 U-118 MG cells were inoculated subcutaneously on both sides of the animal's dorsal flank and after one week the tumors became visible with an approximate volume of 100 mm.sup.3. Animals were randomly divided into groups with a similar mean tumor volume and received daily oral treatments for 42 days: vehicle, DHA-H (200 mg/kg) and HPA (200 mg/kg). The study of FIG. 3 comprises animals treated with vehicle (untreated controls;

[0188] n=6) and treated with DHA-H (200 mg/kg; n=9). FIG. 7 shows animals treated with vehicle (untreated controls; n=6), treated with DHA-H (200 mg/kg; n=8) and treated with HPA (200 mg/kg; n=8). Tumor volumes (v) were calculated as v=A.sup.2×L2, where A is the width of the tumor and L is its length. Upon completion of treatment, mice were sacrificed by cervical dislocation and xenographic tumors were dissected and frozen in liquid nitrogen and at −80° C. All the protocols used were approved by the Bioethics Committee of the University of the Balearic Islands, and comply with national and international guidelines on animal welfare. In the use of healthy mice and transgenic models of Alzheimer's disease (5xFAD), it was found that there was a significant accumulation of HPA at the brain level, while the parent molecule (DHA-H) could not be detected, nor changes in the endogenous levels of DHA (non-hydroxylated native form) (FIG. 2A).

[0189] Radial Arm Maze Test

[0190] The spatial behavior test was performed as described above, with some modifications 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 were kept in these conditions for one week before starting the test and until the end of the test. After the dietary restriction and 3 days before the start of the trials, the animals were trained twice a day in the eight-arm radial labyrinth test (LE766/8, Panlab SL, Spain) for 3 days. Each mouse was placed in the center of the maze and allowed to seek the 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 the eight primed arms or failed to complete all the arms in 10 minutes. The movement of each animal was recorded with a digital video tracking system (LE 8300 with Sedacomv1.3 software, Panlab, SL, Spain) and after training, the experimental paradigm began. In all experimental sessions (1 session per day), only four arms were primed compared to the training protocol, and each session ended when the animals managed to find all four primed arms or failed after 10 minutes. The performance was evaluated taking into account: (1) the time to perform the test; (2) the number of Working Memory Errors (WME, re-entry into a previously visited primed arm); (3) the number of Reference Memory Errors (RME, entry into a non-primed arm); and (4) the total number of errors (WME+RME). The test was repeated 5 days/week for 3 weeks. After the test, the animals were fed ad libitum for an extra week before slaughter.

[0191] In this sense, it can be observed that the levels of HPA at the brain level in the Alzheimer's model mice have a statistically significant inverse relationship with behavioral parameters in an evaluation test of spatial and associative memory (radial labyrinth test) (FIG. 2B). These results suggest that moderate increases in brain HPA levels are significantly related to an improvement in spatial cognition. Likewise, direct administration of HPA has similar effects to administration of DHA-H on the parameters of the same behavioral parameters analyzed (FIG. 6).

Example 5

Lipid Extraction and Fatty Acid Transmethylation Relating to Examples 3 and 4

[0192] The HEK293T or U-118 MG cells used in the above examples were lysed with a cold hypotonic buffer (1 mM EDTA, 20 mM Tris-HCl [pH 7.4]) by pipetting up and down. The cell lysates were subjected to ultrasound pulses (4 cycles, 10 s/cycle, 10 W) before lipid extraction. For brain analysis, the tissue of each animal was homogenized in cold PBS at 1:10 (p:v) in the presence of protease inhibitors (Roche), using a blade homogenizer (Polytron PT3100). Homogenates were ultrasounded, aliquots were made and stored at −80 ° C. Only one aliquot of each sample, containing about 8 mg protein/aliquot, was subjected to lipid extraction. Protein content before lipid extraction was determined by a modified Lowry method (Bio-rad DC Protein Assay).

[0193] Margaric acid (C17:0) was added to the samples subjected to lipid extraction as an internal standard and the lipids were extracted with chloroform:methanol (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. This mixture was vortexed for 1 minute and centrifuged at 1000×g for 10 minutes. The lower organic phase was collected and washed with 1 ml of PBS:methanol (1:1,v:v), and the resulting organic phase was dried under argon flow. The film containing the extracted lipids was transmethylated by incubation of the lipid mixture for 90 minutes at 100° C. in 3 ml of methanol:acetyl chloride (10:1, v:v), under an argon atmosphere (Christie, 1993). The resulting fatty acid methyl esters (FAMEs) were extracted with hexane, adding 3 ml of H.sub.2O 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 the FAMEs was collected and the remaining volume was washed twice with 1 ml of hexane. The hexane phases were combined, evaporated under argon flow and resuspended in 60 μl of hexane (for the analysis of cell samples, cell culture medium and blood plasma) or in 200 μl (for the analysis of brain samples). To check if a fatty acid compound is hydroxylated, isolated FAME were subjected to a second derivatization with trimethylsilyl (Alderson et al., 2004, J Biol Chem 279(47), 48562-48568. doi: 10.1074/jbc.M406649200). Briefly, the FAMEs were dried under argon flow and the lipid film was dissolved in N,O-bis (trimethylsilyl) acetamide (0.1-5.0 mg lipid for 200-400 μl trimethylsilylation reagent), which in turn was heated in a capped 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 under study is hydroxylated, the retention time of the FAME changes as a result of this second derivatization. However, if the fatty acid under study is not hydroxylated, the resulting FAME shows the same retention time regardless of the second derivatization.

[0194] The levels of HPA generated from the treatment with the prodrug DHA-H in these cells were evaluated, in the presence or absence of oxythiamine (competitive inhibitor of α-oxidation) (FIG. 4A). The results showed that the addition of DHA-H to a U-118 MG cell culture results in a significant increase in HPA levels. This increase is inhibited in the presence of simultaneous treatment with 1 or 10 mM oxythiamine, demonstrating that the transformation of DHA-H into HPA is mediated by α-oxidation. Treatment with DHA-H on U-118 MG cells in culture had no effect on endogenous levels of DHA (non-hydroxylated native form). On the same cells in culture, viability assays were carried out with DHA-H in the presence or absence of 1 mM oxythiamine (FIG. 4B), proving that 1 mM oxythiamine has no effect on cell viability.

[0195] On the other hand, the addition of DHA-H (150 μM, 48 h) presents a significant anti-proliferative effect on U118-MG cells. However, this effect is partially reversed (in a statistically significant manner) in the presence of 1 mM oxythiamine. At this 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 show that the anti-proliferative effect mediated by DHA-H on U-118 MG cells is mediated, at least in part, by HPA, since the inhibition of the formation of this compound from DHA-H translates into a lower anti-proliferative effect of DHA-H (FIG. 4B). The anti-proliferative effect that HPA has on a culture of U-118 MG cells was also studied, compared to the administration of the DHA-H prodrug and the native form of DHA. The anti-proliferative effect on U-118 MG is much higher for HPA relative to DHA-H and DHA (see FIG. 5A). When compared to DHA-H, this effect can be explained by differences in intracellular levels of HPA, induced by DHA-H and HPA (see FIG. 5B). Indeed, FIG. 5C shows that uptake of the hydroxylated form of DHA is prevented compared to that of the non-hydroxylated analogue.

Example 6

In Vitro Assays with 2OHOA and C17:1n-9

[0196] The concentrations of 2OHOA sodium salt used in the experiments described below and the duration of the treatments varied according to the type of assay, being either 200 μM or 400 μM and 24 or 72 hours. In some experimental series, C17:1n-9 sodium salt solutions were used at a concentration of 200 μM for 24 or 72h.

[0197] To prepare these solutions, we started from a stock aliquot at 100 mM. To prepare this starting aliquot, the corresponding milligrams of the lipid compound (powder) were dissolved in absolute ethanol and autoclaved distilled water (vol.1:1, normally an aliquot of 1m1 is prepared so that 500 μl of ethanol and 500 μl of water are added) inside the culture hood, the solution was introduced 10 min into the culture oven at 37° C. so that the lipid compound was dissolved and subsequently subjected to stirring.

[0198] 6.1. Incorporation and Metabolization of 2OHOA U-118 MG Glioma and Non-Tumor Cells

[0199] To confirm the incorporation of 2OHOA into glioma cell membranes and to determine if changes in fatty acid profile occur following treatment with 2OHOA, 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 h. Analysis of fatty acid levels in glioma cells revealed an absence of changes in OA levels following treatment with 2OHOA sodium salt relative to control (FIG. 12). In addition, cell incorporation of 2OHOA was observed after 24 hours of treatment due to the identification of a peak in the chromatogram, only in treated cells, which corresponded to its standard. On the other hand, it is worth noting the appearance of a new peak, practically exclusively, in the chromatogram of the treated cells. Elevated levels of this new peak were detected in the treated cells, accumulating almost double (19.71±0.39 nmol/mg protein) than 2OHOA (10.81±0.34 nmol/mg protein). After several studies to determine its identity, it was confirmed that this new peak corresponded to the cis-8-heptadecenoic fatty acid (C17:1n-9). The formation of C17:1n-9 is a consequence of α oxidation of 2OHOA (FIG. 11).

[0200] 6.2. Analysis of the Composition of Fatty Acids in Different Glioma and Tumor Cells after Treatment with 2OHOA Sodium Salt

[0201] The fatty acid composition of the lipid membranes in other glioma cell lines (U-251 MG and SF-295) was analyzed in comparison to non-tumor cells, human fibroblasts (MRC-5), and primary cultures of mouse astrocytes, after incubation in the absence or presence of sodium salt of 2OHOA sodium salt (400 μM, 24 hours) by gas chromatography. No significant change in the amount of OA was observed after treatment with 2OHOA sodium salt in any of the cell lines analyzed (FIG. 13). However, the incorporation of 2OHOA was observed, as well as the formation of the metabolite C17:1n-9, both in other glioma cell lines and in non-tumor cells, after 24 hours of treatment with 2OHOA (FIG. 13). The formation of the metabolite C17:1n-9, from the incorporation of 2OHOA, differs 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 2OHOA (19.16±0.53 vs. 9.21±0.41 and 18.38±1.97 vs. 9.31±1.44 nmol/mg nmol/mg, respectively) (FIGS. 13B and 13d, table 5). In contrast, in non-tumor cells, detected levels of 2OHOA were significantly higher than those of their metabolite C17:1n-9. 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) (FIGS. 13f and 13H, table 5).

TABLE-US-00005 TABLE 5 Levels of the fatty acids 2OHOA and C17:1n-9 in different glioma and non-tumor cell lines after treatment with 2OHOA. Quantification values of 2OHOA and C17:1n-9 fatty acids in different glioma lines, U-118 MG, U-251 MG and SF-295 (above) and non-tumor, MRC-5 and astrocytes, (below) after treatment with 2OHOA (400 μM for 24 hours) determined by gas chromatography. The results correspond to the mean ± SEM of three independent experiments expressed in nmoles and normalized per mg of protein. U-118MG U-251MG SF-295 Control 2OHOA Control 2OHOA Control 2OHOA 2OHOA 0.00 ± 10.81 ± 0.00 ± 9.31 ± 0.00 ± 9.21 ± 0.00 0.34 0.00 1.44 0.00 0.41 C17:1n-9 0.00 ± 19.71 ± 1.65 ± 18.38 ± 1.08 ± 19.16 ± 0.57 0.39 0.66 1.97 0.59 0.53 MRC-5 ASTROCYTES Control 2OHOA Control 2OHOA 2OHOA 0.00 ± 26.31 ± 0.00 ± 12.28 ± 0.00 4.32 0.00 0.90 C17:1n-9 1.08 ± 14.00 ± 2.30 ± 7.58 ± 0.46 1.92 0.34 0.70

[0202] 6.3. Effect of 2OHOA, C17:1n-9 on Cell Viability and Proliferation of Glioma Cells

[0203] In order to evaluate the antiproliferative effect of C17:1n-9, its IC.sub.50, which corresponds to the amount of a compound needed to reduce cell viability in vitro by 50%, as well as its effect on the regulation of proteins involved in the mechanism of action of 2OHOA, were determined. To do this, 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, OA and 2OHOA sodium salt for 72 hours. Upon completion of treatment, IC.sub.50 was determined by violet crystal staining technique. Results of the cell viability assays showed that the three compounds, 2OHOA, OA and C17:1n-9, had an antiproliferative effect on all glioma cells tested, in a concentration-dependent manner, after 72 hours of treatment. Moreover, in the non-tumor cells studied, MRC-5 and astrocytes, no effect of 2OHOA on their cell viability was observed, but the OA and C17:1n-9 fatty acids did produce an antiproliferative effect on the same non-tumor cells (FIGS. 14 and 15). IC.sub.50 values of 2OHOA 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.sub.50 of 2OHOA was 1000 μM for non-tumor cells, MRC-5 and astrocytes. For compound C17:1n-9, the IC.sub.50 values were 222.04±9.09, 220.35±7.93, and 248.85±6.02 μM in glioma cells U-118 MG, U-251 MG, and SF-295, respectively.

[0204] Thus, C17:1n-9 induced a highly similar antiproliferative effect on both glioma and non-tumor cells. Meanwhile, treatment with 2OHOA only affected the viability of the different glioma cell lines, without affecting the viability of the non-tumor cells. The IC.sub.50 values of 2OHOA were 1.90, 1.95, and 1.60 times greater than those of the metabolite C17:1n-9 in the U-118 MG, U-251 MG, and SF-295 glioma cells, respectively (table 6). In addition, the IC.sub.50 values of 2OHOA were 1.92, 1.80 and 1.56 times higher than those of its non-hydroxy analogue OA. The fact that C17:1n-9 has shown a higher antiproliferative potency may be due to the fact that it has a higher accumulation capacity in the cells than 2OHOA.

TABLE-US-00006 TABLE 6 IC.sub.50 values for different cell lines after treatment with 2OHOA, OA and C17:1n-9. Summary of IC.sub.50 of glioma cell lines (U-118 MG, U-251 MG, and SF-295) and non-tumor cells (MRC-5 and astrocytes), calculated from results obtained in FIGS. 16 and 17. The IC.sub.50 values obtained correspond to the average of three independent experiments and calculated using a dose-response equation using the statistical program GraphPad prism 6.0 (sigmoid model). U-118MG U-251 MG SF-295 MRC-5 Astrocytes 2OHOA  432.75 ± 10.77 429.96 ± 9.67  399.14 ± 11.47 1000 1000.00 OA 225.68 ± 7.30 236.96 ± 6.52 256.06 ± 5.79 236.31 ± 6.73 256.355.14 C17:1n-9 222.04 ± 9.09 220.35 ± 7.93 248.85 ± 6.02 248.03 ± 7.28 231.816.41

[0205] 6.4. Analysis of the Effect of Different Fatty Acids on Proliferation and Death Markers in Different Cell Lines

[0206] The effect of the metabolite C17:1n-9 on different signaling pathways that are altered by the effect of 2OHOA was analyzed. For this purpose, the different glioma cell lines (U-118 MG, U-251 MG and SF-295) and non-tumoral (MRC-5 and mouse astrocytes) were treated with doses close to IC.sub.50 of each of the compounds (200 μM C17:1n.9, 200 μM OA or 400 μM 2OHOA) for 72 hours and their effect on different signaling proteins was analyzed by Western Blot.

[0207] The results showed that treatment with 2OHOA increased levels of BIP, CHOP and cJun phosphorylation and decreased phosphorylation of Akt and cyclin D3 levels. Instead, treatment with C17:1n-9 did not produce changes in any of these proteins (FIG. 16). This suggests that death induced by the metabolite C17:1n-9 is triggered via routes other than those of 2OHOA.

[0208] 6.5. Analysis of Fatty Acid Composition in U-118 MG Glioma Cells after Inhibition of α-Oxidation and Determination of the Effect of Oxythiamine on Cell Survival of U-118 MG Glioma Cells

[0209] To confirm the formation of C17:1n-9 acid from 2OHOA by α-oxidation, oxythiamine chloride was used, which inhibits the enzyme 2-hydroxyfitanoyl-CoA lyase (HACL1, key enzyme in α-oxidation), among other functions. To do this, the U-118 MG glioma cells were first pre-incubated with 1 or 10 mM oxythiamine for 90 minutes, then treated with 400 μM of the 2OHOA sodium salt for 24 hours and the fatty acids were analyzed by gas chromatography. In the analysis of certain fatty acids detected by gas chromatography, a significant reduction in C17:1n-9 was observed in U-118 MG glioma cells pre-incubated with oxythiamine and treated with 2OHOA sodium salt relative to cells treated only with 2OHOA sodium salt without oxythiamine (FIG. 17A). This reduction was 51.35% after pre-incubation with 1 mM oxythiamine (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 reached in 2OHOA-treated cells without the presence of oxythiamine. In contrast, no significant differences were detected in the amounts of 2OHOA between cells treated with 2OHOA sodium salt and up to 3 mM oxythiamine compared to those treated with 2OHOA sodium salt only (FIG. 17A). However, a significant increase in the amount of 2OHOA of 12.33% was observed in cells treated with 2OHOA sodium salt and 4mM oxythiamine (12.41±0.75 to 15.74±0.24 nmol/protein); and 52.94% with 10 mM oxythiamine (18.98±0.42 nmol/protein). Thus, oxythiamine inhibited the formation of C17:1n-9 and increased the amounts of 2OHOA from 4 mM, due to inhibition of α-oxidation, confirming the metabolism pathway of 2OHOA to C17:1n-9.

[0210] Next, to determine whether inhibition of 2OHOA metabolism through α-oxidation of 2OHOA has effects on cell viability, cell survival of U-118 MG glioma cells following incubation in the absence or presence of 2OHOA (400 mM, 72 hours), and pre-incubated with oxythiamine at the doses described above, was studied by vital exclusion staining with trypan blue. Oxythiamine induced a significant decrease in the survival of U-118 MG glioma cells. In detail, at 1 mM induced 12.16±0.5% death, 21.17±1.76% death at 2 mM, until reaching a maximum cell survival inhibition of 27.13±0.41% at 10 mM oxythiamine (FIG. 7B). These results confirm the in vitro antitumor effect of oxythiamine that was already known.

[0211] On the other hand, incubation of the cells with 2OHOA sodium salt induced 24.71±1.88% cell death; and after the combination with 1 mM oxyamine there was a recovery in cell viability of 5% (20.94±1.97% death); and of 17.26% (11.71±1.14% death) in the case of 2 mM oxyamine (FIG. 17B).

[0212] In view of FIGS. 17A and B, it is observed that the levels of C17:1n-9 do not vary much between the lowest and highest concentration of oxythiamine. This can be interpreted on the basis that, at low concentrations (1.2 mM), oxythiamine is less cytotoxic on its own, but its effect in decreasing the production of C17:1n-9 are very evident. At these concentrations we see the effect of lowering levels of C17:1n-9, but at higher concentrations of oxythiamine, we begin to see more cytotoxicity produced by oxythiamine itself, an effect that seems to be enhanced with high concentrations of 2OHOA sodium salt. At high concentrations more accumulated 2OHOA is detected, which would indicate that the administration of the sodium salt of 2OHOA also produces cytotoxicity by itself and that it adds to the cytotoxic effect of oxythiamine at such concentrations. 6.6. Effect of Metabolite C17:1n-9 on the Action of 2OHOA

[0213] To study whether the metabolite C17:1n-9 can participate in the action of 2OHOA, the effect of pre-incubation with oxythiamine on cell survival and 2OHOA-regulated proteins was studied. To do this, the cell survival of different glioma and non-tumor cell lines treated with 2OHOA (400 μM, 72 hours) and pre-incubated or not with 2 mM oxyamine (90 minutes) was analyzed by counting the cells with the vital exclusion stain with trypan blue. In addition, Western-blot 2OHOA-modulated proteins were studied. In glioma cells, a significant decrease in cell survival was observed after incubation with 2 mM oxythiamine for 72 hours. Oxythiamine induced 18.51±0.58% and 17.35±0.63% cell death in U-251 MG and SF-295 cells, respectively (FIGS. 18A and 18b). These results support the in vitro anti-tumor effect of oxythiamine on glioma cells.

[0214] Treatment of cells with 2OHOA induced 23.22±1.32% and 23.97±1.25% cell death in U-251 MG and SF-295, respectively. Following combination with 2 mM oxythiamine, there was a significant recovery in cell viability of 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 tested produced an effect on cell survival (FIGS. 18c and 18d).

[0215] As for the study of proteins involved in different signaling pathways and cell death in glioma cells, oxythiamine had an effect on the levels of BiP, CHOP, c-Jun phosphorylation, Akt phosphorylation, and D3 cyclin in glioma cells in the same sense as 2OHOA, although milder (FIG. 19), when combined, modulation induced by 2OHOA was inhibited. This fact confirms that the metabolism of 2OHOA in C17:1n-9 is necessary to enhance its anti-tumor action. On the contrary, no change in such signaling proteins in non-tumor cell lines was observed after any of the treatments (FIG. 19). Although 2OHOA has an antiproliferative activity when its metabolism in C17:1n-9 is inhibited, the formation of the metabolite C17:1n-9 has a great impact on the mechanism of action of 2OHOA, enhancing its antiproliferative effect, and confirms that 2OHOA is also a prodrug that gives rise to an active metabolite 17:1n-9.

Example 7:

In vivo Trials with 2OHOA and C17:1n-9

[0216] 7.1 Analysis of the Composition of Fatty Acids in Rat Plasma after 24 Hours of Treatment with 2OHOA

[0217] The pharmacokinetic profile of 2OHOA and its metabolite C17:1n-9 in animal plasma was studied. In this case, rats were used as an animal model of experimentation. Rats have a higher volume than mice, and are the most suitable model for studying the effect of continued administration of the maximum tolerated dose of 2OHOA (2 g/Kg) defined in preclinical studies.

[0218] For the present study, 2 g of 2OHOA/Kg sodium salt was administered to rats 12-14 weeks of age orally for 15 days. Subsequently, plasma samples were extracted at different times (0, 1, 2, 3, 4, 6, 8 and 24 h) from day 1 (acute treatment) and 15 (chronic treatment). Finally, the fatty acid profile in plasma samples was analyzed by gas chromatography. After analyzing the chromatograms, the detection of 2OHOA and C17:1n-9 fatty acids in plasma samples collected after acute treatment (first day administration) was notable (FIG. 20A).

[0219] The two compounds, 2OHOA and C17:1n-9, showed a very similar pharmacokinetic profile in rat plasma following acute treatment (FIG. 20B). A significant increase in 2OHOA and C17:1n-9 levels was observed, reaching a maximum plasma concentration at 2 hours of administration with 2OHOA (26.23±5.79 nmol 2OHOA/ml plasma and 60.47±6.53 nmol C17:1n-9/m1 plasma). There was a subsequent decrease in plasma 2OHOA and C17:1n-9 levels reaching trough values at 24 hours (2.80±0.69 and 14.03±2.20 nmol/ml plasma, respectively). However, the initial levels were not reached especially in the case of C17:1n-9 (1.22±0.33 and 8.45±2.52 nmol/ml plasma, respectively). The levels of the C17:1n-9 metabolite after chronic treatment were higher than those of 2OHOA (FIG. 21B). The differences between the two compounds were significant prior to administration of 2OHOA (0 hours; 1.22±0.33 nmol 2OHOA/mL plasma, relative to 8.45±2.52 nmol C17:1n-9/plasma), after 8 hours (7.12±1.56 nmol 2OHOA/mL plasma, relative to 23.31±5.18 nmol C17:1n-9/plasma) and at 24 hours (2.80±0.69 nmol 2OHOA/mL plasma, relative to 14.03±2.21 nmol C17:1n-9/plasma).

[0220] 7.2. Analysis of the Composition in Fatty Acids of Xenographic Tumors of Immunosuppressed Mice

[0221] In order to study the effects in animal models of the formation of C17:1n-9 as a product of the metabolization of 2OHOA by α-oxidation, the levels of the metabolite C17:1n-9, compared to those of 2OHOA, were detected and analyzed in a model of xenographic tumors in immunodepressed mice. To do this, U-118 MG glioblastoma cells were injected into immudepressed mice and, one week later, treatment of mice with vehicle or 2OHOA sodium salt (200 mg/kg) was initiated orally and daily for 42 days. Once treatment was complete, mice were euthanized and tumors were removed, lipids were processed for 2OHOA and C17:1n-9 fatty acids by gas chromatography. Fatty acid 2OHOA was not detected in the xenographic tumors of mice treated with this compound, as no peak in the retention time corresponding to 2OHOA was observed (FIG. 22A). In contrast, the metabolite of 2OHOA, C17:1n-9 fatty acid (0.25±0.04 nmol C17:1n-9/g tissue), was detected in the tumors of mice treated with 2OHOA (FIG. 22B).

[0222] 7.3. Correlation between the Volume of Tumours and the Amount of the Metabolite C17:1n-9

[0223] The possible correlation was studied between the levels of the C17:1n-9 metabolite present in tumors with respect to the volume of tumors, as an indication of the relationship between the incorporation and metabolization of 2OHOA and the efficacy of the compound in tumors. In the graphs obtained, a negative correlation was observed between the amount of C17:1n-9present in the tumors and the volume of the tumors (FIG. 23). A coefficient of determination r of −0.8248 and a p-value of 0.0001 were obtained for the tumors of mice treated with 2OHOA between the amount of C17:1n-9 and the volume of the tumors. That is, the smaller the volume of the tumor, the more of the C17:1n-9 metabolite was detected in it. These results show that the metabolite C17:1n-9 has marked antitumor activity and 2OHOA is an effective prodrug of this compound.

[0224] 7.4. Fatty Acid Composition Analysis in Human Patients with Advanced Glioma after Treatment with 2OHOA

[0225] 2OHOA and C17:1n-9 fatty acids were detected and quantified in plasma samples from 8 patients who responded, or not, to treatment with 12 g/day of 2OHOA sodium salt for at least one 3-week cycle in clinical phase I/IIA of 2OHOA (MIN-001-1203). Plasma samples were obtained at different times (0, 2, 4, 6, 8 hours and after 8, 15, 21 and 28 days after treatment with 2OHOA) and were subsequently given for fatty acid analysis using the gas chromatography technique.

[0226] In the chromatograms, 2OHOA and its C17:1n-9 metabolite were detected in all plasma samples from patients analyzed (FIG. 24A). A very similar pharmacokinetic profile was observed in all patients, both those who showed clinical response (responders) and those who did not (non-responders) (FIG. 24B). Both compounds reached peak levels at 4 hours of administration with 2OHOA. Analyzing the results of all patients, responders and non-responders, values of 53.08±6.52 nmol of 2OHOA/ml of plasma and 122.80±10.61 nmol of C17:1n-91m1 of plasma were obtained 4 hours after the first intake of the drug (FIG. 24C). Subsequently, the levels of 2OHOA and C17:1n-9 gradually decreased until 8 hours after treatment (25.39±3.99 and 92.89±9.39 nmol/ml of plasma, respectively). At 8 days of treatment (192 hours) a significant increase in the amounts of the two plasma compounds, 192 hours (25.39±3.99 nmol/ml 2OHOA plasma and 141.10±16.35 nmol/ml C17:1n-9 plasma) was observed. Compounds 2OHOA and C17:1n-9 accumulated in patients' plasma over time, as observed after 15 days (360 hours) of treatment with 2OHOA (184.70±25.60 and 366.9±72.47 nmol/ml 2OHOA and C17:1n.9 plasma, respectively) (FIG. 24C).

[0227] It should be noted that, similarly to what happened in cells and animals, the levels of the metabolite C17:1n-9 in the plasma of all patients were higher than those of 2OHOA (FIG. 14B), being significantly higher after 8 hours of administration of 2OHOA (FIG. 24C). C17:1n-9 levels were observed to be 3.66 and 2.20 times higher than 2OHOA in patients who had been treated with 2OHOA for 8 and 15 days, respectively (92.89±9.39 and 311.10±37.38 and nmol C17:1n-9/m1 plasma compared to 25.39±3.99 and 141.10±16.35 nmol 2OHOA/ml plasma, respectively). Finally, at 21 days of treatment with 2OHOA C17:1n-9 levels were 1.90 times higher than those of 2OHOA (366.9±72.47 nmol C17:1n-9/m1 plasma compared to 184.70±25.60 nmol 2OHOA/ml plasma, respectively).