Use of oleuropein and hydroxytyrosol to prevent neonatal hypoxic-ischemic encephalopathy

Abstract

The invention relates to the use of oleuropein (Ole) and the active metabolite thereof, hydroxytyrosol (Htyr) to prevent neonatal hypoxic-ischemic encephalopathy (HIE). Pretreatment with Ole and / or Htyr provides dose-dependent neuroprotection in an in vivo model of neonatal hypoxic-ischemic encephalopathy. Described are compositions comprising oleuropein and / or hydroxytyrosol for use in preventing this disease, both for direct administration to the foetus and / or newborn, and for administration via the mother during pregnancy and / or breastfeeding.

Description

[0001] USE OF OLEUROPEIN AND HYDROXYTYROSOL FOR THE PREVENTION OF NEONATAL HYPOXIC-ISCHEMIC ENCEPHALOPATHY

[0002] DESCRIPTION

[0003] TECHNICAL FIELD

[0004] The present invention falls within the pharmaceutical sector and is intended for the area of ​​prevention of neonatal hypoxic-ischemic encephalopathy.

[0005] STATE OF THE ART

[0006] Neonatal hypoxic-ischemic encephalopathy (HIE) is an encephalopathy caused by inadequate blood flow and oxygen supply to the brain following an episode of perinatal asphyxia. Hypoxic-ischemic (HI) refers to the fact that it is caused by a lack of oxygen (hypoxia - H) and a lack of blood flow (ischemia - I) to the brain. This condition affects 1-8 per 1,000 live births and remains a significant cause of death and disability in the neonatal population. Common neurological deficits associated with neonatal HIE include cerebral palsy, seizures, visual and hearing impairment, learning and behavioral problems, and motor deficits.

[0007] The pathophysiology of neonatal hypoxic-ischemic encephalopathy (HI) involves two phases: primary energy failure and secondary energy failure. Primary energy failure results from reduced cerebral blood flow, leading to oxygen and glucose deprivation and causing mitochondrial dysfunction, ATP depletion, and overproduction of reactive oxygen species (ROS). Glucose and oxygen depletion disrupts normal ion gradients, inducing neuronal depolarization and calcium influx. This leads to excessive glutamate release, causing excitotoxicity and initiating the ischemic cascade. The elevated calcium influx causes cerebral edema and triggers apoptosis and necrosis.Minutes after HI injury, a strong inflammatory response occurs, including activation of microglia and astrocytes, release of proinflammatory cytokines (e.g., TNF-α, IL-1, and IL-6), and disruption of the blood-brain barrier. Following primary energy failure, there is a latent phase during which cerebral circulation is restored, representing the optimal window for therapeutic interventions such as hypothermia. The secondary energy failure phase begins 6–48 hours after HI injury and can last for several days. During this phase, oxidative stress, excitotoxicity, and inflammation are exacerbated, leading to seizures, cytotoxic edema, impaired cerebral energy metabolism, and neuronal cell death.Since the last decade, therapeutic hypothermia has become the standard treatment for term or near-term newborns (>36 weeks of gestation) with moderate to severe hypoxic-ischemic encephalopathy (HIE). Therapeutic hypothermia is associated with a significant reduction in mortality and neurodevelopmental disability, but 40–50% of neonates with HIE still die or develop chronic neurological sequelae.

[0008] Since hypothermia does not confer complete neuroprotection, it is imperative to develop novel neuroprotective strategies for the prevention / treatment of neonatal HIE. To this end, in recent years there has been growing interest in investigating natural compounds whose bioactive properties can target the ischemic cascade and thus reduce brain injury from HIE [1]. Natural compounds appear as attractive alternatives to conventional drugs due to their low toxicity, affordability, and wide accessibility. In vivo studies have shown that phenolic compounds with antioxidant, anti-apoptotic, and anti-inflammatory activity can exert neuroprotection against neonatal HIE when administered as pretreatment. For example, prophylaxis with resveratrol, pterostilbene, quercetin, or iodide has been shown to improve brain damage, cognitive impairment, and sensorimotor deficits secondary to neonatal HIE.

[0009] Oleuropein (Ole, also called oleuropeoside), a naturally occurring phenolic compound, is a glycosylated secoibidoid found in the olive tree (Olea europaea L.) and is the main bioactive compound in the leaves and unripe, unprocessed olives. Ole has demonstrated neuroprotective effects against various neurological disorders, including Alzheimer's disease, traumatic brain injury, spinal cord injury, and stroke [2].

[0010] Regarding cerebral ischemia / reperfusion injury, Ole has demonstrated anti-apoptotic, antioxidant, anti-inflammatory, and antithrombotic activities in vivo, reducing brain injury and neurological deficits when administered as pre-treatment [3], as post-treatment [4;5], and as a dietary supplement of olive leaf extract enriched with Ole [6]. Pre-treatment with the active metabolite of Ole, hydroxytyrosol (Htyr), has shown similar biological protective activities in vivo against cerebral ischemia / reperfusion injury [7].

[0011] However, to date little is known about the potential neuroprotective benefits of Ole or its metabolites in the context of neonatal HI, with the exception of a recent study that showed that adjuvant post-treatment with Ole in combination with hypothermia protected against white matter loss and preserved myelination in a porcine model of neonatal HI [8].

[0012] DESCRIPTION OF THE INVENTION

[0013] Based on the properties of Ole and Htyr in different neurological disorders resulting from ischemia / reperfusion, the present invention proposes pretreatment with Ole or Htyr to protect the neonatal brain and reduce histological and cellular damage induced by HI.

[0014] The effects of Ole administration prior to HI induction were studied in the widely used Rice-Vannucci model of neonatal HI in postnatal day seven (P7) mice, which is a postnatal stage in which the maturity of the central nervous system in rodents is similar to that of full-term human neonates (34-36 weeks of gestation) [9, 10]. Specifically, the effect on infarct size, white matter integrity, astrocytes, and microglia was studied.

[0015] The results obtained show for the first time that pretreatment with Ole confers dose-dependent neuroprotection in an in vivo model of neonatal HI encephalopathy.

[0016] The neuroprotective effect of pretreatment with Ole occurs without the use of therapeutic hypothermia, the standard treatment for hypoxic-ischemic encephalopathy (HI). This represents a substantial advance over the prior art, where the use of Ole independently of therapeutic hypothermia had not been studied in the context of perinatal HI. Eliminating the need for therapeutic hypothermia results in a significantly less invasive treatment for the neonate. Furthermore, it increases accessibility for a wider population, as access to therapeutic hypothermia is primarily restricted to highly specialized hospitals.

[0017] The adjuvant treatment with Ole described in the literature was limited to the treatment of HIE and was not considered as a possible pretreatment prior to the occurrence of an HI episode, as described in the present invention. Therefore, the present invention provides a preventive neuroprotective mechanism against neonatal HI encephalopathy by preventing or reducing HI damage before it occurs, rather than treating or minimizing it once HI damage has already been caused.

[0018] When designing a preventive treatment for the effects of neonatal HI, one must consider the extreme difficulty, almost impossibility, of directly administering any compound to the embryo / fetus during pregnancy, in addition to the severity of the consequences for embryonic, fetal or neonatal development that could result from administering compounds that are not entirely safe or that could have some type of side effect.

[0019] In the present invention, pretreatment with Ole has been carried out by two routes of administration: directly on the neonate, in this case by intraperitoneal (IP) route; and indirectly, by maternal route, by supplementing the mother with Ole both during pregnancy and during lactation.

[0020] The intraperitoneal route, or peritoneal infusion, is a method used to administer fluids and medications directly into the abdominal cavity through a thin tube. It is the preferred route of administration for newborns, where the oral or intravenous routes present significant technical difficulties for efficient administration, and where the intracranial route poses a high risk to the newborn.

[0021] The maternal route of administration was carried out by supplementing the mother with Ole either during late pregnancy and early lactation (E12.5 - P7), or only during lactation (P0-P7). This simulated the effects of administering Ole to humans via this indirect route during late pregnancy in mice, since the degree of brain maturation in mice at P7 is similar to that of human neonates in full-term pregnancies.

[0022] In all cases, and through all experimentally tested routes of administration, it was possible to detect how Ole and its bioactive metabolite, Htyr, accumulate in the brain of the offspring, confirming that both compounds are able to cross the blood-brain barrier and reach the brain effectively, where their neuroprotective action will take place.

[0023] On the other hand, the low toxicity of these two compounds, Ole and Htyr, allows for the design of preventive treatments in which these substances are administered to newborns before an episode of hypoxic-ischemic encephalopathy (HIE) can occur, and in the case of maternal administration during pregnancy, well before delivery. The preventive use of these compounds, Ole and Htyr, for the prevention of HIE can be extended to the entire pregnant population by incorporating them as part of routine supplementation during pregnancy.

[0024] Given the affordability and wide availability of Ole, it would be the preferred compound for any of the described administration routes. Htyr, on the other hand, presents certain limitations in terms of its availability and handling, as it is a less abundant compound naturally present in olive extracts and tends to oxidize very quickly. Therefore, for its effective handling, it is recommended to combine it with an antioxidant compound to prevent its oxidation.

[0025] Therefore, a first aspect of the invention describes oleuropein (Ole) and its bioactive metabolite, hydroxytyrosol (Htyr), hereinafter referred to as the “compounds of the invention”, for use in the prevention of neonatal hypoxic-ischemic encephalopathy (HIE).

[0026] Oleuropein (Ole) is the compound of formula (I) shown below:

[0027] Oleuropein - Formula (I)

[0028] IIIPAC Name: (4S,5E,6S)-4-[2-[2-(3,4-dihydroxyphenyl)ethoxy]-2-oxoethyl]-5-ethylidene-6-[[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)-2-tetrahydropyranyl]oxy]-4H-pyran-3-carboxylic acid. CAS Number: 32619-42-4.

[0029] The secoiridoid Ole is found in the Oleaceae family, where its content is high in the leaves and green olives. During olive ripening, the Ole content decreases as it is hydrolyzed by endogenous α-glucosidases, producing glucose, oleuropein aglycone, hydroxytyrosol (Htyr), and elenolic acid (Figure 1). A similar process occurs during the production of extra virgin olive oil (EVOO).

[0030] Hydroxytyrosol (Htyr) is the compound with formula (II) shown below:

[0031] Hydroxytyrosol - Formula (II)

[0032] 3,4-Dihydroxyphenylethanol; DOPET; CAS Number: 10597-60-1. Htyr, the bioactive metabolite of Ole, shares neuroprotective properties with Ole, as widely described in the literature [11-13], where it has been shown to similarly enhance cellular antioxidant defense in neuronal systems through Nrf2 activation. Thus, the mechanism of action by which the neuroprotective effect of both compounds operates is presumed to be identical, giving both compounds not only structural but also functional similarity.

[0033] The trials carried out show that Ole accumulates in neonates indiscriminately in the form of Ole or Htyr, depending largely on the route of administration used.

[0034] Intraperitoneal (IP) administration of Ole results in the accumulation of both Ole and Htyr in plasma following administration, with a peak in the levels of both compounds occurring shortly after administration (20 minutes), which then gradually declines to baseline levels. The peak Ole concentration is significantly higher than the levels reached by Htyr, which is expected since the compound is administered as Ole. Ole can be detected not only in plasma but also in the brain, demonstrating its ability to cross the blood-brain barrier. Htyr is not detected in the brain because there is insufficient time for Ole to be metabolized to Htyr and accumulate in that form in the brain.

[0035] On the other hand, when Ole is administered maternally, the compound tends to accumulate as Htyr. In the trials conducted, Htyr was detected in the brains of offspring exposed to maternal Ole supplementation during pregnancy and lactation (E12.5 to P7) or during lactation only (P0 to P7), while Ole was detected at much lower levels. This route of administration involves a much longer and more sustained use of Ole, so this compound is hydrolyzed to Htyr to a much greater extent than in the case of intraperitoneal (IP) administration, resulting in a majority accumulation as Htyr. By P7, Ole has been almost completely hydrolyzed to Htyr, and this is the form in which the compound accumulates in the neonate's brain.

[0036] The terms "prevent" and "prevention" refer to avoiding, either partially or completely, or minimizing the adverse effects or their severity, of a disease or condition—in this case, neonatal hypoxic-ischemic encephalopathy—before it occurs. The reduction in disease severity caused by the hypoxic-ischemic (HI) event, compared to the disease progression that occurs without treatment, will vary depending on several factors, including the dose of the compound administered and the route of administration.

[0037] In a preferred embodiment of this aspect of the invention, Ole and Htyr are characterized in that they are suitable for administration to neonatal mammals.

[0038] In another preferred embodiment of this aspect of the invention, Ole and Htyr are characterized in that they are suitable for administration to pregnant or lactating mammals.

[0039] A second aspect of the invention relates to a composition comprising oleuropein and / or hydroxytyrosol, hereinafter the “composition of the invention” for use in the prevention of neonatal hypoxic-ischemic encephalopathy.

[0040] In a preferred embodiment of this aspect of the invention, the composition of the invention is a pharmaceutical composition.

[0041] In a more preferred embodiment of this aspect of the invention, the pharmaceutical composition of the invention is suitable for administration to neonatal mammals, preferably for intraperitoneal administration.

[0042] In another preferred embodiment of this aspect of the invention, the pharmaceutical composition of the invention is suitable for administration to pregnant or lactating mammals, and preferably further comprises at least one drug.

[0043] In another preferred embodiment of this aspect of the invention, the composition is a nutraceutical composition for administration to pregnant or lactating mammals. Preferably, the nutraceutical composition further comprises at least one compound for supplementing the pregnant or lactating mammal.

[0044] In a more preferred embodiment of these aspects of the invention, where the composition is designed for administration to a pregnant or lactating mammal, it is preferably also suitable for oral administration.

[0045] As used herein, the term "pharmaceutical composition" refers to any substance used for the diagnosis, prevention, relief, treatment, or cure of a disease in humans or animals. The pharmaceutical composition of the invention may be used alone or in combination with other pharmaceutical compositions. In one particular embodiment, the pharmaceutical composition of the invention further comprises a pharmaceutically acceptable carrier or excipient. The term "pharmaceutically acceptable excipient" refers to a substance that aids in the absorption of the pharmaceutical composition comprising the composition of the invention, stabilizes said pharmaceutical composition, or assists in its manufacture by imparting consistency, shape, flavor, or any other specific functional characteristic.Thus, excipients could have the function of holding the ingredients together, such as starches, sugars or celluloses, a sweetening function, a coloring function, a protective function, such as isolating it from air and / or moisture, a filling function for a tablet, capsule or any other form of formulation, such as dibasic calcium phosphate, a disintegrating function to facilitate the dissolution of the components and their absorption, without excluding other types of excipients not mentioned in this paragraph.

[0046] A "pharmaceutically acceptable carrier" (or "pharmacologically acceptable") refers to any substance, or combination of substances, known in the pharmaceutical industry, used in the manufacture of dosage forms and includes, among others, solids, liquids, solvents, or surfactants. The carrier may be an inert substance or have an action similar to any of the compounds of the present invention, serving to facilitate the incorporation of the drug, as well as other compounds, allowing for improved dosage and administration, or providing consistency and form to the pharmaceutical composition. When the dosage form is liquid, the carrier is the diluent. The term "pharmacologically acceptable" refers to the fact that the compound in question is permitted and evaluated to be harmless to the organisms to which it is administered.

[0047] The pharmaceutical composition of the invention can be administered via any route of administration, and as such, said composition will be formulated in the pharmaceutical form appropriate to the chosen route of administration. Thus, the pharmaceutical composition of the invention can be administered orally, nasally, ocularly, topically, intradermally, intracranially, intravenously, or intraperitoneally. Preferably, it is administered intraperitoneally in the case of direct administration to neonates and orally in the case of indirect administration via the mother.

[0048] The pharmaceutical composition of the invention may further comprise another compound useful in the prevention of neonatal hypoxic-ischemic encephalopathy, in which case the pharmaceutical composition may include a single composition or separate compositions.

[0049] The composition of the invention may include an effective amount of Ole and / or Htyr. The term "effective amount" used herein refers to an amount sufficient to prevent neonatal hypoxic-ischemic encephalopathy. The effective amount may be appropriately selected by a person skilled in the art, depending on the individual being administered and whether the formulation is administered directly to the neonate or indirectly to the mother during pregnancy or lactation. Thus, the composition may be suitable for administration to neonatal mammals or for administration to pregnant or lactating mammals.

[0050] In the first case, when we refer to a composition suitable for administration to newborn mammals, the "effective amount" will depend mainly on the body weight of the newborn and its degree of maturity based on the weeks of gestation carried to term and its age.

[0051] In the second case, when we refer to a composition suitable for administration to pregnant or lactating mammals, other factors may also come into play, such as age, health conditions, duration of administration, route of administration, excretion rate and other factors of the pregnant animal, including the use of a drug in combination with the pharmaceutical composition or at the same time as it, and other factors known in the field of medicine.

[0052] Therapy is considered "personalized" when the compound administered to the individual to treat a disease is specially adapted to the genotypic and phenotypic characteristics of the individual to be treated, thus avoiding wasting time with ineffective therapies.

[0053] The nutraceutical composition, “medical food” or food composition of the invention comprises Ole and / or Htyr in an amount effective for the prevention of neonatal hypoxic-ischemic encephalopathy in mammals, including a human being, for administration via the maternal route to the pregnant and / or lactating woman.

[0054] The preferred nutraceutical compositions are selected from the list consisting of: a beverage, milk, yogurt, cheese, fermented milk, flavored milk beverage, soy milk, precooked cereals, bread, cakes, butter, margarine, sauces, frying oils, vegetable oils, corn oil, olive oil, soybean oil, palm oil, sunflower oil, cottonseed oil, condiments, salad dressings, fruit juices, syrups, desserts, icings and fillings, soft frozen products, sweets, chewing gum, and intermediate foods. The nutraceutical composition of the invention may be a nutritional or dietary supplement. In a preferred embodiment, the nutritional or dietary supplement comprises a sterile composition containing Ole and / or Htyr, preferably provided with a gastric acid-resistant coating, and is a delayed-release composition.In another preferred embodiment, the food composition, including Ole and / or Htyr and / or the nutritional or dietary supplement, comprises suitable "carriers" such as diluents, adjuvants, excipients, or vehicles with which the compound of the invention is administered. Suitable excipients include, but are not limited to, starch, glucose, fructose, lactose, sucrose, gelatin, malt, rice, flour, calcium sulfate, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene glycol, water, ethanol, and the like.

[0055] The nutraceutical composition may also include another compound useful in the supplementation of the pregnant or lactating mammal, in which case the nutraceutical composition may include a single composition or separate compositions.

[0056] The term "patient," "individual," or "subject," as used herein, refers to a mammal and includes, but is not limited to, domestic and farm animals, primates, and humans, e.g., humans, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents such as rats and mice. In a preferred embodiment, the subject is a human being.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning commonly understood by a person skilled in the art to which this invention pertains. Similar or equivalent methods and materials to those described herein may be used in the practice of the present invention. Throughout the description and claims, the word "comprises" and its variations are not intended to exclude other technical features, additives, components, or steps. Other objects, advantages, and features of the invention will be evident to those skilled in the art upon examination of the description or may be discovered through the practice of the invention. The following examples and drawings are provided for illustrative purposes and are not intended to limit the scope of the present invention.

[0058] BRIEF DESCRIPTION OF THE FIGURES

[0059] Figure 1: Enzymatic hydrolysis of Ole.

[0060] Figure 2: Semi-quantitative histopathological scoring system for assessing brain damage from HI based on histological images. Coronal sections of P14 brains at the level of the striatum and hippocampus were stained with methylene green and photographed using a stereomicroscope. Several brain structures of the ipsilateral hemisphere were assessed by comparing their macroscopic characteristics with their counterparts in the contralateral (control) hemisphere. At the striatum level, the lateral ventricle was scored 0–2 points: 0, no dilation; 1, slightly dilated; and 2, markedly dilated. The striatum was scored 0–3 points: 1 for hypotrophy of the caudate putamen, 2 if this hypotrophy was accompanied by disruption of the external capsule, and 3 for extensive infarction of the entire structure.The cortex was assessed in two sections, skeletal (anterior) and hippocampal (posterior), with a 0–3 point score: 0 for no observable damage, 1 for focal damage accompanied by cell loss, 2 for infarction and tissue loss of up to half the cortex, and 3 for extensive infarction and tissue loss of most of the cortex. The CA1, CA2 / 3, and dentate gyrus regions of the hippocampus were assessed with a 0–5 point score, both in terms of hypotrophy (0–2 points) and cell density (0–2 points). Five points were awarded when the structure was completely absent. In addition, a final macroscopic assessment considered whether the ipsilateral hemisphere was moderately or severely asymmetric (1 or 2 points). The total histopathological score ranged from 0 to 28 points and was the sum of all the above assessments. Scale bar: 1 mm.

[0061] Figure 3: (AE) Representative micrographs of coronal sections with methylene green from the different experimental groups at P14, one week after HI injury. S-sal (A) and S-ole100 (B), with normal brain morphology; n=10 each; (C) Hl-sal, showing cerebral infarction accompanied by loss of hippocampal and cortical tissue, dilation of the lateral ventricle and asymmetry of the ipsilateral hemisphere; n=18', (D) Hl-ole20, showing hippocampal hypotrophy and dilation of the lateral ventricle but without cortical damage; n=19', (E) Hl-ole100, with a macroscopic morphology similar to that of the control group; n=18. The median of the histological damage score of each group was used to select a representative image. Enlargement of the CA2 / 3 region of the hippocampus (A'-E') and of the parietal cortex (A"-E"), as shown in A. Hl-sal shows cell loss and neuronal damage in both brain regions, which are reduced in the Hl-ole20 and Hl-ole100 groups.Scale bars: 1000 pm (AE), 100 pm (A'-E. 1 ).

[0062] Figure 4: (A) Percentage of tissue loss in ipsilateral hemispheres, measured from methylene green-stained coronal brain sections at P14, one week post-HI injury. (B) Total histologic damage score, from 0 to 28 points, assessed from histologic images. (C) Histologic damage score in different brain regions; pie charts represent the proportion of samples with a given score. The total histologic damage score was the sum of all assessments in C. Histologic images were assessed blindly and independently by two investigators; all S-sal and S-ole100 samples received a score of 0 points and are therefore not shown in BC.Statistical significance was calculated using Welch's one-way ANOVA followed by Dunnett's T3 multiple comparisons test (A,B) and the Chi square test (C); * P<0.5, ** P<0.01, *** P<0.001, **** P<0.0001; n=10-19 per group.

[0063] Figure 5: Histological evaluation of brain damage and tissue loss at the striatal level. Representative whole-brain micrographs of coronal methylene green sections at the striatal level from different experimental groups at P14, one week after HI injury. (A) S-sal and (B) S-ole100, with normal brain morphology; n=10 each. (C) Hl-sal, showing hypotrophy in the striatum (st) and cortex (ct) accompanied by lateral ventricle dilation (Iv) and ipsilateral hemisphere asymmetry; n=18. (D) Hl-ole20, with striatal hypotrophy and lateral ventricle dilation but no cortical damage; n=19. (E) Hl-ole100, with mild lateral ventricle dilation and macroscopic morphology similar to the control; n=18. The median histological score for each group was used to select a representative image. (A'-E') Enlargement of the cortex, as shown in A.The cortical layers appeared disorganized in Hl-sal, but not in the pretreated Hl-ole groups. Scale bars: 1000 pm (AE), 100 pm (A). 1 - E'). (F) Percentage of tissue loss in the ipsilateral hemisphere at the hippocampal level, measured from histological images. Statistical significance was calculated using the Kruskal-Wallis test followed by Dunn's multiple comparisons test; only significant changes are indicated. * P<0.5, *** P<0.001; n=10-19 per group.

[0064] Figure 6: (A) Representative whole-brain micrographs immunolabeled for myelin basic protein (MBP) at P14, one week post-HI injury. Sham-sal, n=10', (B) Sham-ole100, n=10', Hl-sal, n=18, showing MBP loss in the external capsule, cortex, and striatum; Hlole20, n=19, showing MBP loss in the striatum; and Hl-ole100, n=18, similar to the control groups. The median MBP area coverage for each group was used to select a representative image; scale bar: 1 mm. (B) Analysis of MBP area coverage by hemisphere in the different experimental groups. Two-way repeated-measures ANOVA followed by Sídák's (between-hemisphere comparisons, #) and Tukey's (between-group comparisons, *) multiple comparisons tests; n=10-19 per group. (C) Correlation between MBP area coverage and tissue loss in ipsilateral hemispheres; linear regression fits are shown as dashed lines.Even in brains with severe tissue loss (>60%, marked with a gray box in C), Ole-pretreated samples show greater MBP coverage than untreated samples, as illustrated by the micrographs (D) Hl-ole20, Hl-ole100, and Hl-sal. Figure 7: (A) Microglia were visualized using Iba1 immunohistochemistry at P14, one week after HI injury. Iba1 cell density was analyzed in the regions highlighted in boxes: the supplementary motor cortex M2 (cortex), the midline of the corpus callosum, and the striatum. (B) Representative coronal sections at the striatal level immunolabeled for Iba1. Hl-sal showed a higher number of microglia in the cortex, corpus callosum, and especially in the striatum, while Hl-ole20 and Hl-ole100 were similar to the simulated controls. The median number of Iba1+ cells per mm² was used. 2to select a representative image from each group. (C) Quantification of microglial cell density, expressed as the number of Iba1+ cells per mm 2 in the aforementioned brain regions. Two-way repeated-measures ANOVA followed by Sídák's multiple comparisons test (comparisons between hemispheres, #) and Tukey's test (comparisons between groups, *); n=10-19 per group. Scale bars: 1000 pm (A), 100 pm (B).

[0065] Figure 8: (A) Astroglia were visualized by GFAP immunohistochemistry at P14, one week after HI injury. GFAP expression was analyzed in the regions highlighted in boxes: the frontal neocortex, the parietal neocortex, and the thalamus. (B) Representative coronal sections at the hippocampal level immunolabeled for GFAP. Astroglia were overexpressed in all three brain regions in the Hl-sal group and less intensely in the Hl-ole20 and Hl-ole100 groups. The median GFAP area coverage was used to select a representative image from each group. (C) Analysis of the astroglial reactive response, expressed as the percentage of area covered by GFAP staining in the aforementioned brain regions. Two-way repeated-measures ANOVA followed by Sídák's multiple comparisons test (comparisons between hemispheres, #) and Tukey's test (comparisons between groups, *). n=10- 19 per group. Scale bars: 1000 pm (A), 100 pm (B).

[0066] Figure 9: Quantification of Ole and Htyr by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS / MS) in brain and plasma samples. (A) Plasma Ole levels, (B) plasma Htyr levels, and (C) brain Ole levels after intraperitoneal injection of Ole (100 mg / kg; green) or saline vehicle (10 ml / kg; blue) in P7 pups. UPLC-MS / MS quantification was performed at different time points after injection: before ischemia (20 min, T1), before hypoxia (2:20 h, T2), and after hypoxia (3:50 h, T3). No Htyr levels were detected in the brain samples. (D) Quantification of Htyr levels in offspring brain samples following exposure to maternal Ole supplementation during lactation (P0-P7) or during pregnancy and lactation (E12.5-P7). A pregnant mother received 100 mg / kg / day of Ole from E12.5 to P7 orally.In P0, five identically aged pups from an unsupplemented mother were transferred to the ole-supplemented mother. Brain and plasma samples were collected at P7 for UPLC-MS / MS; no ole or Htyr levels were detected in the plasma samples, and only low levels of ole (0.2–0.4 ng / g brain) were detected in 2 / 5 samples from the P0–P7 group and in 3 / 5 samples from the E12.5–P7 group (not shown). In AC, symbols represent the mean ± SD of n=5 samples per group, at least two replicates per sample were quantified, nd, not detected; no suppl., not supplemented.

[0067] DETAILED DESCRIPTION OF THE INVENTION

[0068] Example: Neuroprotective effect of administering Ole to neonates prior to HI induction

[0069] Neonatal mouse model of hypoxia-ischemia

[0070] Hypoxic-ischemic brain injury was induced in seven-day-old CD1 mouse pups following the Rice-Vannucci method

[0013] , with some modifications. Briefly, the pups were anesthetized with inhaled isoflurane (4% for induction for 3 min, 2% for maintenance, and 1% for suturing), a small anterior incision was made in the neck, and the left common carotid artery was isolated and ligated twice with 6-0 surgical silk suture. The skin incision was then sutured; the entire surgical procedure took about 15 minutes per animal.

[0071] Following surgery, the pups were allowed to recover from anesthesia and were returned to their biological mothers (2:19 ± 0:47 h, n=87). One hour after the surgery of the last ischemic mouse, the HI pups were exposed to 90 min of hypoxia with 9% O2 in a hypoxic chamber (O2Control InVivo Glove Box, Coy Laboratory Products Inc.). The animals were placed on a warming mat during surgery, recovery, and hypoxia to maintain nesting temperature. The sham group pups underwent anesthesia and isolation of the left common carotid artery, as in the HI group, but there was no ligation or exposure to hypoxia. All offspring were returned to their mothers until they were euthanized 24h or 7 days after HI, on postnatal day 8 (P8) for biochemical studies with brain homogenates or on postnatal day 14 (P14), after motor tests, for histological and immunohistochemical studies.

[0072] Experimental groups and oleuropein administration. Oleuropein was purchased from TCI Chemicals (00420). Oleuropein was dissolved in ethanol and stored at -20 °C. Aliquots were diluted in saline (10% ethanol) prior to use. Oleuropein was administered intraperitoneally to the pups 20 min before induction of ischemia at a dose of 20 or 100 mg / kg body weight, similar to other studies with phenolic compounds [3, 15]. Equal volumes of saline (10% ethanol; 1 ml / kg body weight) were used as the vehicle.

[0073] Oleuropein concentrations were chosen based on previous reports in which oleuropein showed neuroprotective and cardioprotective properties when administered intraperitoneally prior to injury in adult rodent models of cerebral [3] and cardiac

[0015] ischemia-reperfusion.

[0074] The pups were randomly distributed into five experimental groups: the control group pups, subjected to simulated surgery and without hypoxia, were treated with saline solution (Sham-Sal) or oleuropein at 100 mg / kg (Sham-OlelOO), the HI group pups, whose left common carotid artery was ligated and who were subjected to hypoxia, were treated with saline vehicle (Hl-Sal), oleuropein at 20 mg / kg (Hl-Ole20) or oleuropein at 100 mg / kg (Sham-Ole100). A total of n=75 pups from seven litters were used for histological and immunohistochemical studies (n=10 Sham-Sal, n=10 Sham-Ole100, n=18 Hl-Sal, n=19 Hl-Ole20 and n=18 Hl-Ole100), while a total of n=40 pups from five litters were used to prepare brain homogenates for biochemical assays (n=8 S-sal, n=17 Hl-sal, n=15 Hl-ole100). The mean litter size and the age of the mothers were similar across litters.

[0075] Histological and immunohistochemical studies

[0076] Tissue processing

[0077] Seven days after HI injury, at P14, the pups were deeply anesthetized with inhaled isoflurane and intracardially perfused with ice-cold saline (0.9% NaCl) followed by 4% paraformaldehyde dissolved in phosphate-buffered saline (PBS; pH 7.3). The brains were dissected and fixed for 2 h in 4% PFA at 4 °C. After dehydration with graded ethanol and xylene, the brains were embedded in paraffin, and coronal sections 7 µm thick were cut using a microtome, Bregma +1.10–2.80 mm approximately. All sections were stored on coded slides, and the coding was not broken until all measurements were completed.Coronal sections were stained with methylene green or immunostained with antibodies against myelin basic protein (MBP), ionized calcium-binding adaptor molecule 1 (Iba1), or glial fibrillary acidic protein (GFAP), to visualize myelin sheaths, microglia, and astroglia, respectively.

[0078] Methylene green staining

[0079] Paraffin-embedded coronal sections were deparaffinized with Clear SX85 xylene substitute (Casa Álvarez, AF-17SX100E), rehydrated with graded ethanol, and stained for 6 min with methylene green (7.65 mM methylene green, ChemCruz, SC-295475A; 0.4 M boric acid; pH 6.1). The sections were washed with distilled water, dehydrated with graded ethanol, and mounted on coverslips using DPX (PanReac AppliChem, 255254.1610).

[0080] Brain injury assessment

[0081] To quantify the extent of infarction and assess the severity of tissue injury, whole-brain micrographs were taken from methylene green-stained coronal sections using a stereomicroscope (SteREO Discovery v8, Zeiss). For each brain sample, two sections of the striatum and hippocampus were analyzed, corresponding to Bregma 0.50–0.26 mm and -1.58–1.82 mm, respectively. The infarct area was defined as the area exhibiting loss of the normal methylene green staining pattern and / or tissue loss. The cross-sectional areas of both hemispheres were measured using FIJI software (ImageJ). Measurements were performed by a researcher blinded to the experimental group of the samples. The extent of cerebral infarction was calculated as the percentage of ipsilateral damage, using the formula: (C - / ) / C x 100, where C and / are the total areas of the contralateral and ipsilateral hemispheres, respectively.

[0082] The severity of tissue damage was characterized macroscopically based on different structures—the cortex, striatum, and hippocampus—using a semi-quantitative histopathological scoring system shown in Figure 2. The cortex was assessed in two coronal sections, anterior (striatal level) and posterior (hippocampal level), with scores ranging from 0 to 3: 0 for no observable damage, 1 for focal damage accompanied by cell loss, 2 for infarction and tissue loss comprising up to half of the cortex, and 3 for extensive infarction and tissue loss comprising most of the cortex. The striatum was scored from 0 to 3: 1 for hypotrophy of the caudate putamen, 2 if this hypotrophy was accompanied by rupture of the external capsule, and 3 for extensive infarction of the entire structure. The CA1, CA2 / 3 and dentate gyrus regions of the hippocampus were evaluated in bregma -1,58, with 0 to 5 points, both in terms of hypotrophy (0-2 points) and cell density (0-2 points).Five points were awarded when the structure was completely absent. In addition, two other macroscopic assessments considered whether the lateral ventricle was mildly or extensively dilated (1 or 2 points), and whether the ipsilateral hemisphere was moderately or severely asymmetric (1 or 2 points). The total histopathological score ranged from 0 to 28 points and was the sum of all the above assessments.

[0083] Each brain was evaluated by two independent researchers who were unaware of the sample group; the final histopathological score was the average of the evaluation by both researchers.

[0084] Evaluation of microglia by immunohistochemistry Iba1

[0085] Coronal sections at the level of the striatum (Bregma 0.50 to 0.26) were deparaffinized and hydrated as previously described. The sections were boiled in citrate buffer (10 mM citric acid, 0.05% Tween-20 in PBS, pH 6) for antigen recovery. After rinsing the slides with PBS, endogenous peroxidase was deactivated by incubating the sections for 30 minutes with 3% H₂O₂. The sections were then washed with 0.1% Triton X-100 in PBS (hereafter PBST), permeabilized with 0.3% Triton X-100 in PBS, and blocked for 1 h in blocking buffer (1% BSA in PBST). The sections were incubated overnight at 4 °C with rabbit anti-Iba1 antibody (Wako, 019-19741) diluted 1:1000 in blocking buffer. After incubation with the primary antibody, the sections were thoroughly washed with PBST, and incubated with biotinylated donkey anti-rabbit secondary antibody (Jackson ImmunoResearch, 711-065-152), diluted 1:400 in blocking buffer.After washing with PBST, the signal was amplified by incubating the samples for 1 h with the Vectastain Elite ABC-HRP kit (avidin and biotin-radish peroxidase complex; Vector Laboratories, PK-6100), diluted 1:100 in PBST. The sections were washed with PBST and incubated for 10–15 min with 3,3'-diaminobenzidine (Sigma, D8001). Finally, the sections were washed with ice-cold water for 5 min, dehydrated, and mounted with DPX as before.

[0086] The immunolabeled sections for Iba1 were visualized using a 20x objective on an Olympus DP72 microscope. To quantify the number of microglia cells in various brain regions, images were taken of three 890 x 666 µm (0.59 mm²) regions per hemisphere, corresponding to the supplementary motor cortex M2, the midline corpus callosum, and the striatum. The number of Iba1+ cells was counted systematically and semi-automatically. The images were digitized and converted to 8-bit grayscale, the background was subtracted twice (rolling ball radius of 50 pixels), the contrast was increased by 0.3%, the images were smoothed twice, an automated threshold was applied to binarize the image, and the number of particles sized 200–2000 pixels was counted as the Iba1+ cell count. The areas of the different regions of the contralateral and ipsilateral hemispheres were also measured, since some of them showed infarction.A “blind” researcher analyzed two brain sections per sample to calculate the average number of Iba1+ per mm. 2 of tissue in each brain region.

[0087] Evaluation of white matter integrity using MBP immunohistochemistry

[0088] Coronal sections at the striatum were immunolabeled as before, using rat anti-MBP (myelin basic protein) primary antibody (BioRad, aa82-87) and biotinylated donkey anti-rat secondary antibody (Jackson ImmunoResearch, 712-065-150) diluted 1:500 and 1:400, respectively, in blocking buffer. Sections were imaged using a Leica Thunder microscope with a 10x objective. Approximately 60–80 bright-field mosaics (1824 x 1216 pixels each) were acquired and automatically fused using Leica LAS X software to obtain whole-brain cross-sectional images. A researcher blinded to the sample group used FUI to acquire and analyze two whole-brain images per sample. The microscopic images were digitized at 8 bits, segmented using an automated threshold, and binized to black and white to measure the total pixel area.The area of ​​the contralateral hemisphere was also measured and MBP density was reported as the pixel area of ​​the MBP staining area normalized to the total pixel area of ​​the contralateral hemisphere.

[0089] Evaluation of astroglia by GFAP immunohistochemistry

[0090] Coronal sections at the hippocampal level (Bregma -1.7 to -2) were immunolabeled as before, using a rat anti-GFAP primary antibody (Dako, Z0334) and a biotinylated donkey anti-rat secondary antibody (Jackson ImmunoResearch, 712-065-150) diluted 1:500 and 1:400, respectively, in blocking buffer. Three 650 x 430 µm (0.28 mm²) regions per hemisphere were imaged using a Leica Thunder microscope with a 40x objective, corresponding to the frontal neocortex, parietal neocortex, and thalamus. The microscopic images were digitized at 8 bits, segmented using an automated threshold, and binized to black and white to measure the total pixel area. GFAP density was reported as the pixel area of ​​the GFAP staining area normalized to the pixel area of ​​each image, as some regions showed infarction.A researcher who was unaware of the sample group analyzed two brain sections per sample to calculate the average GFAP area coverage in each brain region.

[0091] Neonatal HI causes ipsilateral brain damage and cell loss that are reduced with Ole pretreatment.

[0092] Coronal sections of the entire brain were histologically evaluated at P14, one week after HI injury (Fig. 3A-E). Histological imaging revealed that the regions most susceptible to HI injury were the hippocampus, cortex, and striatum, and that the severity of brain damage ranged from mild tissue damage in these regions to extensive ipsilateral hemispheric infarction.

[0093] Subfield images of the CA2 / 3 region of the hippocampus and parietal cortex in these methylene green-stained sections (Fig. 3 A'-E', A"-E") showed that HI induced significant cell loss and neuronal damage, with neurons appearing swollen and deformed compared to the control. In the H1-Ole20 brain sections, only mild cell loss and a lower number of damaged neurons were observed in the hippocampus, but not in the cortex.

[0094] The hippocampus and cortex of more than half of the Hl-Ole100 samples had a structure similar to that of the control brains.

[0095] For its part, brain damage and tissue loss at the stalk level were also evaluated (Fig. 5) where Hl-Sal (Fig. 5C) showed hypotrophy in the striatum (st) and cortex (ct) accompanied by dilation of the lateral ventricle (Iv) and asymmetry of the ipsilateral hemisphere; HI-Ole20 (Fig. 5D) showed stalk hypotrophy and dilation of the lateral ventricle but without cortical damage and a higher dose of Ole, Hl-ole100 (Fig. 5D) showed a slight dilation of the lateral ventricle and macroscopic morphology similar to the control.

[0096] Pretreatment with Ole reduces tissue loss and improves HI-induced brain damage in a dose-dependent manner.

[0097] The percentage of tissue loss in the ipsilateral hemisphere was measured as the cerebral infarct area reading (Fig. 4A). The Hl-sal group had a median tissue loss of 42.8% (IQR 25.1–69.3%, n=18). Pretreatment with Ole reduced tissue loss in a dose-dependent manner: the Hl-Ole20 group had a median tissue loss of 13.6% (6.8–42.6%, n=19, P=0.5, Welch's ANOVA), and the Hl-Ole100 group had a median tissue loss of 8.6% (4.5–36.1%, n=18, P=0.043). Macroscopic brain damage was further assessed using a custom-developed histological damage scoring system, ranging from 0 to 28 points, based on other similar scales (Fig. 4B). Regarding tissue loss, pretreatment with Ole improved overall histological damage in a dose-dependent manner: The Hl-Sal group had a median histological damage score of 20 points (IQR 16-26), the Hl-Ole20 group had 14 points (IQR 4-21; P=0.032), and the Hl-Ole100 group had 1 point (IQR 0-19; P=0.0034).

[0098] Both the measurement of tissue loss and the histological score reflected the variability in the severity of brain damage caused by HI, in the untreated group (Hl-Sal) but also in the pups that received pretreatment with Ole.

[0099] Histological damage scoring was also used to classify damage in the regions most susceptible to HI: the hippocampus, cortex, and striatum. In the Hl-Sal group, the hippocampus and striatum showed hypotrophy or tissue loss in most pups (94%, 17 / 18). Cortical tissue loss (67%, 12 / 18), ventriculomegaly, observed as dilation of the ipsilateral lateral ventricle (83%, 15 / 18), and ipsilateral hemispheric asymmetry (94%, 17 / 18) were also common histological features. In the most severe cases, the infarction encompassed almost the entire ipsilateral hemisphere (28%, 5 / 18). In the Hl-Ole20 group, a smaller proportion of brains showed loss of cortical tissue (32%, 6 / 19; P=0.014, Chi square test) and, although most samples showed some degree of hippocampal hypotrophy or tissue loss (90%, 17 / 19), the histological damage was milder than in the Hl-Sal group (P<0.0001).Ventriculomegaly was also common (68%, 12 / 19; P=0.15), but ipsilateral hemisphere asymmetry was less frequent than in the Hl-Sal group (58%, 11 / 19; P<0.0001). More than half of the HI-Qle100 brain samples were macroscopically similar to the simulated ones (56%, 10 / 18), although some showed hypotrophy or loss of hippocampal (40%, 7 / 18; P<0.0001) and stalk (44%, 8 / 18; P<0.0001) tissue, which in the most severe cases was accompanied by loss of cortical tissue (28%, 5 / 18; P<0.0037). Ventriculomegaly (56%, 10 / 18; P=0.0066) and ipsilateral hemisphere asymmetry (39%, 7 / 18; P<0.0001) were less frequent than in the Hl-Sal group (Fig. 4C).

[0100] These results show how pretreatment with Ole improves HI-induced tissue loss and histological damage in a dose-dependent manner, although there is a proportion of severe HI cases that cannot be prevented with Ole.

[0101] Pretreatment with Ole reduces white matter injury and preserves myelination in a dose-dependent manner. Immunohistochemical studies performed on P14, one week after HI induction, revealed substantial loss of MBP immunostaining in the ipsilateral hemispheres of Hl-Sal pups, affecting the cortex, striatum, and, in the most severe cases, the external capsule. Hl-Ole20 brains also showed MBP loss, particularly in the striatum, while most Hl-Ole100 brains were similar to the simulated ones (Fig. 6A).

[0102] White matter integrity was assessed by measuring the area covered with MBP staining relative to the area of ​​the contralateral hemisphere. This analysis corroborated the microscopic observations: compared to the contralateral hemispheres, there was a significant decrease in MBP area coverage in the ipsilateral hemispheres of the Hl-Sal (P<0.0001) and Hl-Ole20 (P=0.0066) groups, but not in the Hl-Ole100 group (P=0.0620). The median MBP area coverage for the Hl-Sal group was 10.8% (IQR 2.5–15.7; n=18), for Hl-Ole20 14.5% (9.6–15.5; n=19), and for Hl-Ole100 15.5% (9.8–16.2; n=18). This MBP coverage was significantly different between Hl-Sal and Hl-Ole100 (P=0.0048) but not between Hl-Sal and Hl-Ole20 (P=0.1495). These results suggest that Ole may protect against HI-induced demyelination in a dose-dependent manner (Fig. 6B).

[0103] As shown in the whole-brain micrographs, ipsilateral tissue loss was typically accompanied by a reduction in MBP area coverage, although this linear correlation was less strong in pups pretreated with Ole (R-squared 0.90 in Hl-Sal, 0.83 in Hl-Ole20, and 0.79 in Hl-Ole100; Fig. 6C). In pups with severe brain damage (tissue loss >60%), untreated pups showed less MBP coverage than treated pups (shaded area in Fig. 6C). Of this subset of pups with severe tissue loss, the Hl-Sal group had a mean ipsilateral MBP coverage of 0.9% (n=5; Fig. 6D), Hl-Ole20 of 5.2% (n=4; Fig. 6D), and Hl-Ole100 of 7.5% (n=3). Fig. 6D). This suggests that, although it does not confer complete neuroprotection, Ole may preserve myelination in cases of severe tissue loss.

[0104] Pretreatment with Ole suppresses HI-induced microglial activation.

[0105] Given the important role of inflammation in the pathophysiology of HI, we investigated whether pretreatment with Ole could modulate the microglial reactive response. To this end, we assessed the number of Iba1+ cells by P14 immunohistochemistry in three brain areas (the cortex, specifically the supplementary motor cortex M2, the midline corpus callosum, and the striatum; Fig. 7A) that had previously shown microglia overexpression following HI injury. The number of microglia in the contralateral hemispheres was similar in all experimental groups (-170 Iba1+ cells per mm²). 2 in the three regions studied; Fig. 7C). As can be seen from the Iba1+ immunostaining images of the ipsilateral hemispheres (Fig. 7B), Hl-Sal showed a significant increase in the number of microglia in the three regions studied: the cortex, with a median of 224 Iba1+ cells per mm 2(IQR 194-338, n=18, P=0.025 compared to the contralateral hemisphere), the midline corpus callosum (237, IQR 192-567; P=0.009), and with greater intensity in the striatum (373, IQR 281-964; P<0.0001). Ole pretreatment reduced microglial overexpression at both doses in these three areas. In the cortex, HI-Qle20 and HI-Qle100 had a median of 174 (147-188, n=19, P=0.025 compared to HI-Sal) and 180 Iba1+ cells per mm², respectively. 2(164-207, n=18, P=0.043), respectively; in the corpus callosum 177 (131-211; P=0.012) and 173 (150-233; P=0.026); and in the striatum 201 (171-606; P=0.013) and 191 (147-407; P=0.004). In the striatum, there was a subset of samples Hl-Qle20 (5 / 19) and HI-Qle100 (5 / 18) that exhibited a high number of microglia. These samples also showed severe brain damage on histological imaging (Fig. 4C), suggesting that Ole pretreatment may reduce HI damage, but there may be some cases of acute HI that cannot be rescued by Ole pretreatment.

[0106] The results indicate that pretreatment with Ole can reduce microglial activation and this neuroprotective effect was similar with doses of 20 and 100 mg / kg.

[0107] The astroglial reactive response induced by HI can be partially reduced by pretreatment with Ole.

[0108] Similar to the studies on Iba1+, the effect of Ole pretreatment on the astroglial reactive response was investigated using immunohistochemistry against glial fibrillary acidic protein (GFAP) at P14. GFAP expression was analyzed in three brain regions at the hippocampal level: the frontal neocortex, the parietal neocortex, and the thalamus (Fig. 8A). Note that some HI samples showed infringement and, therefore, GFAP expression could not be measured in all three regions; the n values ​​for each measurement are indicated.

[0109] There were no differences in astroglia expression, measured as the percentage of area covered by GFAP staining, in the contralateral hemispheres of the different experimental groups. The contralateral frontal neocortex showed GFAP area coverage of ~2%, the parietal neocortex -0.8%, and the thalamus -0.6% (Fig. 8C). Immunostaining images revealed a significant increase in astroglia expression in all HI groups, although it was higher in the untreated Hl-Sal samples than in the Hl-Ole20 and Hl-Ole100 groups (Fig. 8B). In the frontal neocortex, Hl-Sal had a median GFAP area coverage of 6.4% (IQR 4, 1-8.1, n=18, P<0.0001 compared to the contralateral hemisphere), Hl-Ole20 of 3.4% (IQR 2, 3-4.7, n=19, P<0.0001), and Hl-Qle100 of 4.1% (IQR 2, 1-5.6, n=18, P=0.0033).In the parietal neocortex, Hl-Sal had a mean GFAP area coverage of 9.2% (IQR 7.3-16.4, n=10, P<0.0001), HI-01620 of 5.7% (IQR 4.0-6.5, n=15, P=0.0009), and HI-Qle100 of 7.0% (IQR 2.0-11.1, n=17, P<0.0001). Finally, the thalamus showed the greatest overexpression of astroglia: Hl-Sal had a median GFAP area coverage of 8.8% (IQR 5.8-22.1, n=17 P<0.0001), HI-Qle20 of 6.6% (IQR 2.3-13.8, n=19 P<0.0001), and HI-Qle100 of 2.2% (IQR 0.5-11.7, n=18 P=0.0023).

[0110] The results indicate that pretreatment with Ole can partially reverse the astroglial reactive response induced by HI injury.

[0111] After evaluating the neuroprotective properties of Ole against HI, circulating levels of Ole and its metabolites were quantified using UPLC-MS / MS, both in plasma and in the brains of neonates. One study focused on measuring the levels of Ole and its metabolites achieved after intraperitoneal (IP) administration (Example 2). The other study focused on determining whether Ole or its metabolites reached the offspring after maternal supplementation with Ole during pregnancy and / or lactation in order to verify whether maternal supplementation could be an effective route for Ole administration (Example 3). As previously explained, among the Ole metabolites, hydroxytyrosol (Htyr) is of particular interest since it has been shown to increase cellular antioxidant defense in neuronal systems similarly to Ole through the activation of Nrf2 [11-13].

[0112] 2: Levels of Ole and its metabolites after administration

[0113] Experimental groups

[0114] Five groups were included: control pups, who only suffered exposure of the left common carotid artery but not hypoxia and received saline solution (S-sal) or Ole at 100 mg / kg (S-Ole100), and HI pups who received saline solution (Hl-Sal), Ole at 20 mg / kg (Hl-Ole20) or Ole at 100 mg / kg (Hl-Ole100).

[0115] Brain and plasma samples were collected at various time points (T0-T3) on postnatal day 7 (P7) for ultra-performance liquid chromatography-mass spectrometry (UPLC-MS / MS). Brain homogenates were prepared on postnatal day 8 (P8) for biochemical assays, while histology, immunohistochemistry, and motor tests were performed on postnatal day 14 (P14). All samples used in the study were coded, and all experiments were conducted by blinded investigators.

[0116] A total of n=35 P7 pups from four litters were used to quantify olein (Ole) and some of its metabolites (hydroxytyrosol, tyrosol, and oleacin) by UPLC-MS / MS after intraperitoneal (IP) administration of Ole. Fifteen pups received saline vehicle, and another fifteen received Ole at 100 mg / kg. Pups in both groups were sacrificed at three time points after IP injection: 20 min (T1), before unilateral ligation of the left common carotid artery (LOCA) began; 2:20 h (T2), pups that underwent LOCA ligation, before hypoxia; and 3:50 min (T3), pups that underwent LOCA ligation and 90 min of hypoxia. Finally, five pups that did not receive any IP injection were used as reference controls (T0).

[0117] Quantification of Ole and related metabolites in brain and plasma by UPLC-MS / MS

[0118] The pups were deeply anesthetized with inhaled isoflurane, and blood was collected from a small incision in the right ear using heparinized microhematochtho capillary tubes (Deltalab, 7301) and centrifuged to obtain plasma (2,500 g, 15 min at 4 °C). The pups were then intracardially perfused with ice-cold saline, and their entire brains were dissected. Brain and plasma samples were frozen in liquid nitrogen and stored at -80 °C before being extracted and analyzed by UPLC-MS / MS by an investigator blinded to the identity of the samples.

[0119] The circulating levels of Ole and related metabolites in plasma and those that can reach the brain after intraperitoneal (IP) administration were quantified.

[0120] The results showed that IP injection of Ole at 100 mg / kg produced an approximately 10,000-fold increase in plasma Ole levels at T1 (20 min after IP), reaching a concentration of 3.8±1.2x10 6 ng / ml of plasma (mean ± SD, n=5), which returned to baseline levels 2:20 h after IP, in T2 (Fig. 9A).

[0121] During the same time period there was also an approximately 10-fold increase in plasma levels of Htyr, which reached a mean concentration of 15±5 ng / ml (n=5), and then returned to baseline levels at T2 (Fig. 9B).

[0122] Therefore, a peak of Ole in plasma was detected 20 min after IP, reaching a concentration in the mg / ml range, while a peak in the ng / ml range was also observed for Htyr, a bioactive metabolite of Ole, during the same time interval. An approximately 7-fold increase in Ole levels was also observed in brain samples, although the concentration reached in the brain (2.1 ± 1.1 ng / g brain, n = 5) was much lower than in plasma. This increase in Ole levels in the brain was maintained at T2 and returned to baseline at T3, 3:50 h after IP (Fig. 9C). There were some baseline plasma Ole levels (32 ± 9 ng / ml plasma, n = 5; not shown) that did not vary between groups or time points, suggesting that they were not related to Ole administration.

[0123] Tyrosol was not detected in any of the samples.

[0124] IP injection resulted in high bioavailability and a short time to reach peak plasma concentration compared to oral administration

[0015] as, for example, a study in adult rats showed that unmodified Ole reached its peak plasma concentration 2 h after a single oral dose of 100 mg / kg, reaching a peak value of 200 ng / ml, whereas Htyr was not detected

[0016] .

[0125] Small levels of Ole were also detected in the brain after IP (in the ng / g range), indicating that Ole can cross the blood-brain barrier.

[0126] Example 3: Levels of Ole and its metabolites after maternal supplementation with Ole during pregnancy and lactation

[0127] Experimental groups

[0128] Ole was administered at 100 mg / kg / day to a pregnant mouse from pregnancy day E12.5 to postnatal day P7 orally, similar to other maternal supplementation studies for HI [17,18]. At P0, five same-aged pups were transferred from an unsupplemented mother to the Ole-supplemented mother.

[0129] Quantification of Ole and related metabolites in brain and plasma by UPLC-MS / MS

[0130] Brain and plasma samples were collected at P7 to quantify Ole and some of its metabolites (hydroxytyrosol, tyrosol, and oleacin) using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS / MS) as explained in Example 2.

[0131] There was some accumulation of Htyr in the brains of P7 pups exposed to maternal Ole supplementation during pregnancy and lactation, from E12.5 to P7 (24±16 ng / g brain, n=5) or during lactation only, from P0 to P7 (27±12 ng / g brain, n=5; Fig. 9D). Therefore, hydroxytyrosol accumulates in the neonatal brain after maternal Ole supplementation.

[0132] Neither Ole nor Htyr were detected in the plasma samples and only low levels of Ole (0.2-0.4 ng / g brain) were detected in some of the brain samples (in 3 / 5 samples from group E12.5-P7 and 2 / 5 from group P0-P7; not shown).

[0133] Therefore, maternal supplementation with Ole during pregnancy and / or lactation leads to some accumulation of its metabolite hydroxytyrosol in the brain of the offspring.

[0134] References

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Claims

CLAIMS 1. Oleuropein or hydroxytyrosol for use in the prevention of neonatal hypoxic-ischemic encephalopathy.

2. Oleuropein or hydroxytyrosol for use according to the preceding claim, characterized in that the compound is suitable for administration to term mammalian fetuses and / or neonates.

3. Oleuropein or hydroxytyrosol for use according to the preceding claim, characterized in that the compound is suitable for administration to human fetuses and / or neonates from week 34 of gestation.

4. Oleuropein or hydroxytyrosol for use according to claim 1 characterized in that the compound is suitable for administration to pregnant or lactating mammals.

5. Oleuropein or hydroxytyrosol for use according to the preceding claim, characterized in that the compound is suitable for administration to pregnant or lactating humans.

6. Composition comprising oleuropein and / or hydroxytyrosol for use in the prevention of neonatal hypoxic-ischemic encephalopathy.

7. Composition for use according to the preceding claim characterized in that it is a pharmaceutical composition.

8. Pharmaceutical composition for use according to the preceding claim characterized in that it is suitable for administration to term mammalian fetuses and / or neonates.

9. Pharmaceutical composition for use according to the preceding claim characterized in that it is suitable for administration to human fetuses and / or neonates from week 34 of gestation.

10. Pharmaceutical composition for use according to the preceding claim characterized in that it is suitable for intraperitoneal administration.

11. Composition for use according to any of claims 6 to 10 further comprising at least one drug for the prevention of neonatal hypoxic-ischemic encephalopathy.

12. Composition for use according to claim 6 characterized in that it is suitable for administration to pregnant or lactating mammals.

13. Composition for use according to the preceding claim characterized in that it is suitable for administration to pregnant or lactating humans.

14. Composition for use according to the preceding claim characterized in that it is a nutraceutical composition.

15. Nutraceutical composition for use according to the preceding claim further comprising at least one compound for supplementation of the pregnant or lactating mammal.

16. Composition for use according to claim 6 further comprising at least one drug for administration to a pregnant or lactating mammal.

17. Composition for use according to any of claims 12 to 16 characterized in that it is suitable for oral administration.

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