TECHNICAL FIELD

[0001] The present disclosure relates to arachidonic acid for use in the prevention or decrease of significant post-operative bleeding, preferably excessive post-operative bleeding, including when an extracorporeal circulation procedure as cardiopulmonary bypass is used. Furthermore, the present disclosure relates to compositions comprising arachidonic acid, namely ingestible and pharmaceutical compositions.

BACKGROUND

[0002] Excessive post-operative bleeding, also known as significant postoperative haemorrhage/ significant post-operative bleeding refers to an abnormal and potentially dangerous amount of bleeding that occurs after a surgical procedure. It occurs when blood vessels that were cut or manipulated during surgery fail to properly clot and seal, leading to prolonged or heavy bleeding at the surgical site. This can result in various complications and may necessitate further medical intervention to control the bleeding and prevent complications such as anemia, infection, or delayed wound healing. Excessive post-operative bleeding can vary in severity, ranging from minor oozing to more significant and lifethreatening hemorrhage. The risk of excessive bleeding can be influenced by factors such as the type of surgery performed, the patient's overall health, the use of certain medications (like blood thinners), and the surgical techniques employed. Surgeons take precautions to minimize the risk of excessive bleeding during surgery, but it remains an important consideration in post-operative care and management.

[0003] Excessive bleeding is a frequent complication after cardiac surgery with cardiopulmonary bypass (CPB), frequently requiring the use of allogenic blood products. Cardiac surgery accounts for 10-15% of transfusions in surgical patients in the United States yearly [1,2], and more than half of patients receive transfusion of blood products during hospital stay [3], Use of transfusions is not harmless, significantly impacting patients' outcome, increasing both morbidity and mortality, and healthcare costs [4], Moreover, postoperative bleeding is also associated with a higher rate of reoperation, which is independently associated with poorer outcomes [5], [0004] CPB has a distinctive and significant contribution for the disruption of hemostasis in cardiac surgery [6,7], Platelet-function deficit is considered as the main CPB-induced hemostatic deficiency [8], CPB decreases the number of platelets by dilution, adhesion, destruction, and consumption [9] and this reduction is associated with excessive postoperative bleeding [9], Moreover, remaining platelets are functionally impaired, but mechanisms remain unclear [10], Even patients with a normal platelet count show higher prevalence of platelet dysfunction of unknown-cause [11], Although circulating platelets appear structurally normal, recovering within minutes after CPB, bleeding times increase and remain prolonged for several hours [12], Flow cytometry in whole-blood after CPB revealed no significant changes in platelet surface receptors [8] and few advances have been made in the field recently.

[0005] While lipid metabolism is essential for platelet activity and function [13], little is known regarding platelet metabolic changes associated with CPB-dysfunction.

[0006] Polyunsaturated fatty acids (PUFA), such as omega-6 fatty-acids, have been associated with lower risk of cardiovascular events, especially due to anti-inflammatory proprieties [2,3], PUFA may regulate platelet activation enhancing endothelial repair, but current evidence is limited. Additionally, it is well established that Arachidonic acid (AA) and its metabolites modulate platelet aggregation. AA-triggered aggregation is significantly decreased after CPB [1], but there is no information on the relation of peri-operative levels of AA and its metabolism on postoperative haemorrhage.

[0007] AA concentration influences both normal cellular functions and the development of platelet dysfunction. AA metabolites act as local hormones and/ or signaling molecules in response to basal metabolism or upon regulation by immune response stimuli, such as the production of cytokines [24], CPB is associated with a significant systemic inflammatory response, with a complex and incompletely understood cross-talk between inflammation and coagulation [24,25], It is known that endothelial dysfunction during CPB induces the production of cytokines and platelet-induction factors [24,25], Some AA metabolites, such as 12-HETE, have an important role in immune mediated platelet activation [23], Although not yet completely understood, 12-HETE potentiates dense granule secretion via NADPH-oxidase activation [23,24] and the activation of surface immunoreceptors [25], Moreover, platelets after CPB have decreased levels of 12-HETE and a depressed activation of LOX activity, contributing to postoperative bleeding risk through an innate immunity-dependent perturbation of coagulation factors function [30], [0008] Although platelet transfusion is widely used to restore perioperative hemostasis, the decision is mainly empirical since there is no consensus regarding dose, trigger and efficacy [18], Moreover, platelet-transfusions are associated with several complications, including increased use of vasoactive drugs, extended mechanical ventilation, prolonged stay in the ICU and hospital, increased risk of postoperative infection and transfusion-related acute kidney injury [12,13], In fact, in a previous study, patients with postoperative significant bleeding had higher rates of hemodynamic support, mechanical ventilation for more than 6h and neurologic complications. Additionally, platelet transfusion is associated with considerable healthcare costs [21], The effort is now centered on reducing the use of blood products to decrease transfusion-related complications and costs. Therefore, it is essential to find new strategies able to reduce postoperative bleeding, leading to a positively impact on the patient's health and reducing the need of blood transfusions.

[0009] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.

G EN ERAL DESCRIPTION

[0010] The present disclosure relates to arachidonic acid for use in the prevention or decrease of significant post-operative bleeding, preferably excessive post-operative bleeding, including when an extracorporeal circulation procedure as cardiopulmonary bypass is used. Furthermore, the present disclosure relates to compositions comprising arachidonic acid, namely ingestible and pharmaceutical compositions.

[0011] In an embodiment, the daily form consists of a tablet, suppository, ampoule or other device, comprising a definitive amount of arachidonic acid, the whole of which is intended to be administered as a single dose or in multiple parts.

[0012] An aspect of the present disclosure relates to arachidonic acid or a pharmaceutically acceptable salt thereof for use in the prevention or decrease of significant post-operative bleeding; preferably excessive post-operative bleeding.

[0013] In an embodiment, the arachidonic acid or a pharmaceutically acceptable salt thereof may be use in the prevention or decrease of excessive post-operative cardiac surgery bleeding.

[0014] In an embodiment, the arachidonic acid or a pharmaceutically acceptable salt thereof may be use in surgery comprising extracorporeal circulation.

[0015] In an embodiment the extracorporeal circulation is cardiopulmonary bypass. [0016] Another aspect of the present disclosure relates to a pharmaceutical composition comprising arachidonic acid or a pharmaceutically acceptable salts thereof in a therapeutically effective amount and a pharmaceutical acceptable carrier, adjuvant, excipient, or mixtures thereof for use in the prevention or decrease of significant post-operative bleeding; preferably excessive post-operative bleeding.

[0017] In a preferred embodiment, the pharmaceutical composition is an oral pharmaceutical composition, to be administered before surgery, including urgent, emergent and elective surgery.

[0018] In an embodiment, the dosage amount ranges from 50 to 3600 mg/day; preferably 50 to 1800 mg/day; more preferably 50 to 800 mg/day; even more preferably 75 to 600 mg/day.

[0019] In a preferred embodiment, the dosage amount ranges from 1000 to 3000 mg/day.

[0020] In a preferred embodiment, the dosage amount is 1000 mg/day.

[0021] In another preferred embodiment, the dosage amount is 3000 mg/day.

[0022] In an embodiment the compound/composition of the present disclosure may be administrated before a surgical procedure. In an embodiment the compound/composition of the present disclosure may be administered every 24 hours at least in the 30 days preceding the surgical procedure; preferably at least in the 21 days preceding the surgical procedure; more preferably at least in the 7 days preceding the surgical procedure.

[0023] In an embodiment, the compound/composition of the present disclosure may be administered orally, in a dosage ranging from 50-3600 mg/day, at least 1 to 4 weeks before a surgical procedure.

[0024] In a preferred embodiment, the dosage amount may be 1000 mg/day and the composition is administered every 24 hours in the 21 days preceding the surgical procedure.

[0025] In another preferred embodiment, the dosage amount is 3000 mg/day and the composition is administered every 24 hours in the 21 days preceding the surgical procedure.

[0026] In an embodiment, the compound/composition of the present disclosure may be administered as a preoperative bolus (oral bolus), to be administered less than 24 hours before the surgical procedure; preferably less than 12h before the surgical procedure; or right before the surgery.

[0027] In an embodiment, the amount of arachidonic acid in the preoperative bolus ranges from 50-3600 mg/single dose; preferably ranges from 900-3600 mg/single dose. [0028] In an embodiment, the present disclosure relates to the use of an ingestible composition, capsule or tablet comprising the arachidonic acid or a suitable salt thereof, in pre-operative administration to prevent or decrease significant post-operative bleeding, preferably excessive post-operative bleeding.

[0029] In an embodiment, the administration is performed every 24 hours at least in the 7 days preceding the surgical procedure; preferably at least in the 21 days preceding the surgical procedure; more preferably at least in the 30 days preceding the surgical procedure.

[0030] In an embodiment, the daily form consists of a tablet, capsule, ampoule or other dosage form or device, comprising a definitive amount of 50-3600 mg.

[0031] Another aspect of the present disclosure relates to the use of arachidonic acid or a pharmaceutically acceptable salt thereof or the composition herein described for the manufacture of a medicament for the prevention or decrease of significant post-operative bleeding; preferably excessive post-operative bleeding.

[0032] Another aspect of the present disclosure relates to a method for preventing or decreasing significant post-operative bleeding; preferably excessive post-operative bleeding. In a subject, the method comprising administering arachidonic acid or a pharmaceutically acceptable salt thereof or the composition herein described to the subject.

BRI EF DESCRI PTION OF THE DRAWINGS

[0033] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.

[0034] Figure 1: Platelet count (A), variability in percentage (B), platelet mean volume (C) and platelet distribution width (D) of pre-operative and 6h and 24h postoperative patients. Values are presented in median with interquartile range.

[0035] Figure 2: (A) Correlation of postoperative bleeding (mL) within the first postoperative 12h and preoperative quantification of arachidonic acid, with X presenting patients with higher levels of AA and non-significant postoperative bleeding and triangles presenting patients with low levels of AA and significant postoperative bleeding. Circles present arachidonic acid values and postoperative bleeding of the rest of the patients. (B) Preoperative arachidonic acid levels between patients with (white) or without (black) a significant postoperative bleeding (defined by postoperative chest tube blood loss of more than 600mL within 12 hours); C, Median postoperative chest tube blood within 12h according to arachidonic acid quantification. [0036] Figure 3: (A) Postoperative transfusion rate considering preoperative arachidonic acid quantification and the reduction in percentage at postoperative 6h. (B) Receiver operating characteristic (ROC) curve based on the preoperative arachidonic acid measurements and represented by an area under the curve (AUC) of 0.73 indicating a satisfactory predictive ability. (C) Correlation between human interleukin 6 (hlL6) levels and variability in arachidonic acid levels at 6h postoperative (percent change compared to preoperative levels).

[0037] Figure 4: (A) HPLC-MS/MS quantification of arachidonic acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), conjugated linoleic acids (CLA), glutamic acid, 12- hydroxyeicosatetraenoic acid (12-HETE), platelet activating factor (PAF), 8,11,14-eicosatrienoic acid and thromboxane B2 (TxB2) in blood at 6h and 24h after surgery, comparing to preoperative quantification. (B) Relative reduction in levels of arachidonic acid, EPA, DHA, CLA, glutamic acid, 12-HETE, PAF, 8,11,14-eicosatrienoic acid and TxB2 in blood at 6h and 24h after surgery, compared to preoperative levels (percent change). Values are presented in median with interquartile range. The following symbols were used in figures to indicate statistical significance: ns: non-significant; p<0.05 (*); p<0.01 (**); p<0.001 (***); p<0.0001 (****).

[0038] Figure 5A and 5B: HPLC-MS/MS quantification of arachidonic acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), conjugated linoleic acids (CLA), glutamic acid, 12- hydroxyeicosatetraenoic acid (12-HETE), platelet activating factor (PAF), thromboxane B2 (TxB2) and 8,11,14-eicosatrienoic acid in blood at 6h and 24h after surgery, comparing to preoperative quantification, between patients with or without preoperative antiagreggation with acetylsalicylic acid; p<0.05 (*); p<0. 01 (**); p<0.001 (***); p<0.0001 (****).

[0039] Figure 6A: Relative reduction in levels of arachidonic acid, EPA, CLA, glutamic acid, PAF and TxB2 in blood at 6h and 24h after surgery, compared to preoperative levels (percent change), between patients with or without antiagreggation with acetylsalicylic acid. Values are presented in median with interquartile range. The following symbols were used in figures to indicate statistical significance: ns: non-significant; p<0.05 (*); p<0.01 (**); p<0.001 (***); p<0.0001 (****).

[0040] Figure 6B: Relative reduction in levels of DHA, 12-HETE, and 8,11,14-eicosatrienoic acid in blood at 6h and 24h after surgery, compared to preoperative levels (percent change), between patients with or without antiagreggation with acetylsalicylic acid. Values are presented in median with interquartile range. The following symbols were used in figures to indicate statistical significance: ns: non-significant; p<0.05 (*); p<0.01 (**); p<0.001 (***); p<0.0001 (****). [0041] Figure 7: (A) lnterleukin-6 (IL-6) levels of all patients; (B) IL-6 levels comparing patients with and without significant postoperative bleeding. The following symbols were used in figures to indicate statistical significance: ns: non-significant; p<0.05 (*); p<0.01 (**); p<0.001 (***); p<0.0001 (****).

DETAILED DESCRIPTION

[0042] The present disclosure relates to arachidonic acid for use in the prevention or decrease of significant post-operative bleeding, preferably excessive post-operative bleeding including when an extracorporeal circulation procedure as cardiopulmonary bypass is used. Furthermore, the present disclosure relates to compositions comprising arachidonic acid, namely ingestible and pharmaceutical compositions.

[0043] To explore the possible contribution of metabolic perturbations for platelet dysfunction after cardiac surgery with CPB, it was performed an untargeted metabolomic analysis using high-performance liquid chromatography-tandem mass spectrometry (HPLC- MS/MS) to identify specific metabolic signatures that contribute to platelet dysfunction after CPB.

Materials and Methods

Porcine model of cardiopulmonary-induced platelet dysfunction.

[0044] The confirmation of the biological impact of the administration of arachidonic acid is performed with a porcine model of cardiopulmonary-induced platelet dysfunction. Establishing a swine model of cardiopulmonary bypass, we compare a group with previous administration of arachidonic acid with a placebo group, collecting blood postoperatively to measure platelet dysfunction.

Clinical trial

[0045] A clinical trial is performed to compare the supplementation of arachidonic acid versus placebo to test the potential of AA to reduce the risk of excessive postoperative bleeding in heart surgery patients. After pre-operative supplementation with arachidonic acid or placebo, postoperative bleeding, use of allogenic transfusions and platelet dysfunction is evaluated.

Study Population

[0046] The aortic valve replacement for inflammation (SVA-Study) registry is a study to characterize the inflammatory response after surgical aortic valve replacement (SAVR). In an embodiment, patients included were older than 18-years undergoing SAVR. Previous cardiac surgery, concomitant procedures, neoplasia and use of corticosteroids were exclusion criteria. Blood was collected from 33 patients before surgery, and 6h and 24h after surgery. Plasma was prepared and stored at -80°C. Medical records were assessed to obtain clinical data. All patients provided written informed consent and the study was approved by the Institutional Ethics Committee (Comissao Etica-CHLN, Ref.N. ⁹ 23/18, April 2018), in accordance with Declaration of Helsinki and following STROBE guidelines.

Classification of Hemorrhage

[0047] Postoperative chest tube output was quantified in the ICU hourly. Significant bleeding was considered when postoperative chest tube blood loss was above 600mL within 12h, as defined by the International Initiative on Haemostasis Management in Cardiac Surgery [14], The need for transfusion (platelet concentrate, packed red-blood cells, fresh frozen plasma and fibrinogen) was considered according to bleeding and/ or to correct aggregation and coagulation deficiencies [6,15],

Untargeted metabolomics

[0048] An untargeted metabolomics study was performed for supporting information. After generation of blood plasma, proteins were removed by adding 400pL of a methanol/ ethanol mixture(4:l,v/v) to lOOpI of plasma, followed by vigorous vortex shaking for 5 minutes at room-temperature and centrifugating at 4000xg-10min at 4°C. The supernatant was collected, transferred to an Eppendorf, shock frozen with liquid nitrogen, and stored at -80°C until analysis. Extracted samples were thawed on ice, centrifuged for 2 min at 15,000xg, and diluted according to the different sample weight with 0.1% formic acid (RP, reversed-phase) or 50% acetonitrile (ACN) (HILIC, hydrophilic interaction chromatography). 2.5 mL of each diluted sample were pooled and used as a quality control (QC) sample. Samples were randomly assigned into the autosampler and metabolites were separated on a SeQuant ZIC-pH ILIC HPLC column (Merck, 100 3 2.1 mm;5 mm) or a RP-column (Waters, ACQ.UITY UPLC HSS T3 150 3 2.1;1.8mm) with a flow rate of 100 mL/min delivered through an Ultimate 3000 HPLC system (Thermo-Fisher Scientific). The gradient ramp up time takes 25 min from 10% to 80%B in HILIC (A:ACN; B:25 mM ammonium bicarbonate(ABC) in water) and from 1% to 90% B in RP (A:0.1% FA in water;B:0.1% FA in ACN). Metabolites were ionized via electrospray ionization in polarity switching mode after HILIC separation and in positive polarity mode after RP separation. Sample spectra were acquired by data-dependent high-resolution tandem mass spectrometry on a Q-Exactive Focus(Thermo-Fisher Scientific). Ionization potential was set to +3.5/-3.0kV, the sheet gas flow was set to 20, and an auxiliary gas flow of 5 was used. Samples were analysed in a randomized fashion and QC samples were additionally measured in confirmation mode to obtain additional MS/MS spectra for identification. Obtained datasets were processed by compound discoverer 3.0(Thermo-Fisher Scientific). Compound annotation was conducted by searching the mzCloud database with a mass accuracy of 3 ppm for precursor masses and 10 ppm for fragment ion masses as well as ChemSpider with a mass accuracy of 3 ppm using BioCyc, Human Metabolome Database, KEGG, MassBank and MetaboLights as databases.

Cytokine measurement

[0049] Cytokine concentrations were determined using human interleukin-6 (IL-6) kit (ELISA- MAXTM Deluxe Sets, BioLegend, San Diego, CA, USA), according to manufacturer's protocol and measured on a Tecan spectrophotometer plate-reader.

Statistical Analysis

[0050] Principal Component Analysis was performed with R [16], using the basic package "stats". Remaining statistical analysis was performed using GraphPad Prim 9.0(GraphPad Software). Continuous variables are presented with median with interquartile range and were analyzed using Wilcoxon matched-pairs signed rank test for paired samples and Wilcoxon rank sum test for non-paired samples, adjusted to FDR. Pairwise comparison was performed between pre and postoperative samples. The Bonferroni correction was performed to reduce the chances of obtaining false-positive results (type I errors). Categorical variables are reported in percentage or frequency and were analyzed using chi-squared test. For correlation we used the Spearman's rank-order correlation. Predictive models were performed using logistic regression. Enrichment and pathway metabolomic analysis were performed using the MetaboAnalyst 5.0 tool [17], The following symbols were used in figures to indicate statistical significance: ns: non-significant; p<0.05(*); p<0.01(**); p<0.001(***); p<0.0001(****).

Results

Porcine model of cardiopulmonary-induced platelet dysfunction.

[0051] The confirmation of the biological impact of the administration of arachidonic acid in a dosage ranging from 50-3600 mg is performed with a porcine model of cardiopulmonary- induced platelet dysfunction. It is observed that the administration of AA induces a positive impact on post-cardiopulmonary bypass platelet dysfunction, improving platelet function and reducing postoperative cardiopulmonary-related bleeding. Clinical trial

Example A

[0052] A clinical trial is performed to compare the supplementation of arachidonic acid versus placebo to test the potential of AA to prevent excessive postoperative bleeding in heart surgery patients. It is observed that the administration of AA at a dose of 50-3600 mg/day in the 14-21 days preceding the surgical procedure induces a positive impact on patient's morbidity, reducing the risk of excessive postoperative bleeding and the use of allogenic transfusions and improving postoperative platelet dysfunction.

Example B

[0053] A clinical trial is performed to compare the supplementation of arachidonic acid versus placebo to test the potential of AA to prevent excessive postoperative bleeding in heart surgery patients. It is observed that the administration of AA at a dose of 3000 mg/day in the 21 days preceding the surgical procedure induces a positive impact on patient's morbidity, reducing postoperative bleeding and the use of allogenic transfusions and improving postoperative platelet dysfunction.

Example C

[0054] A clinical trial is performed to compare the supplementation of arachidonic acid versus placebo to test the potential of AA to prevent excessive postoperative bleeding risk in heart surgery patients. It is observed that the administration of AA at a dose of 1000 mg/day in the 14-21 days preceding the surgical procedure induces a positive impact on patient's morbidity, reducing postoperative bleeding and the use of allogenic transfusions and improving postoperative platelet dysfunction.

[0055] It was surprisingly found that the preoperative administration of AA is safe and effective to reduce platelet dysfunction after cardiac surgery with CPB, reducing postoperative bleeding and the necessity of transfusion. The reduction of allogenic transfusions is associated with lower intensive care unit and hospital lengths of stay, and less renal and pulmonary dysfunction, leading to lower healthcare-related costs.

Untargeted metabolomics

[0056] Patient's demographic and clinical characteristics are detailed in Table 1. No differences were observed between the two groups (significant bleeding and no significant bleeding) regarding age, gender, comorbidities, left ventricle function and median Euroscorell. Table 1. Surgery and postoperative Data

AKI: acute kidney injury; BMI: Body mass index; LV: left ventricular; IQR: interquartile range; CPB: cardiopulmonary bypass; ICU: intensive care unit

Haemodynamic support: use of vasopressors to maintain adequate perfusion, without the necessity of mechanical support

Neurologic complication: occurrence of stroke, delirium or postoperative cognitive dysfunction.

[0057] Untargeted metabolomics data for the complete population recorded 8668 metabolic features for each plasma sample. Multivariate analysis was employed, mainly pattern recognition tools, such as principal component analysis (PCA), between patients with or without significant post-operative haemorrhage. The PCA plot did not reveal a clear separation in the metabolic profile between both groups. I Then, it was performed a pairwise hypothesis test comparing pre and postoperative metabolite measurements at 24h. The pairwise hypothesis test identified statistically significant differences for 547 metabolites (p- value<0.001). Using the 547-significant metabolites, an enrichment and pathway analysis were performed. From the top 30 pathways identified in our analysis (Table 2), it is identified a platelet-related signature, characterized by an overrepresentation of changes in one known fatty acid metabolism pathway (arachidonic acid [AA] pathway) involved in platelet function. The list of the studied metabolites, associated with the identified pathway, is presented in Table 3. Table 2. Pathway analysis from the comparison between pre-operative and postoperative metabolites. Table 3. Arachidonic acid related metabolites identified by untargeted metabolomics

[0058] It was evaluated the concentrations of the 9 features associated with the AA pathway: AA, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), conjugated linoleic acids (CLA), platelet activating factor (PAF) (as part of the upstream activation of AA metabolism), 8,11,14- eicosatrienoic acid (eicosanoids pathway), 12-HETE and glutamic acid (lipoxygenase pathway) and thromboxane B2(TxB2) [cyclooxygenase pathway],

[0059] Considering all patients with measurements for the three time-points, levels of AA in the preoperative were 5.93xl0 ⁸ [ion counts - all the values are ion counts (area) linearly proportional to the actual concentration of the metabolite] (IQR 4.4xl0 ⁸ -7.4xl0 ⁸ ). Although absolute measurements remained stable at 6h-5.6xlO ⁸ (IQR 4.3xl0 ⁸ -7.1xl0 ⁸ ,p=0.55), they were significantly decreased at 24h 4.17xlO ⁸ (IQR 3.08xl0 ⁸ -5.5xl0 ⁸ ,p<0.0001) (Figure 4 A).

[0060] Revealing a similar pattern, levels of EPA (7.98xl0 ⁷ IQR 5.15-12.67xl0 ⁷ ) and DHA(5.23xlO ⁸ IQR 4.5-8.06xl0 ⁸ ) remained stable at 6h (8.19xl0 ⁷ IQR 4.92-10.8xl0 ⁷ , p=0.247 and 5.43xl0 ⁸ IQR 3.57-7.34xl0 ⁸ ,p=0.505, respectively) but significantly decreased at 24h (4.27xl0 ⁷ IQR 2.95-6.86xl0 ⁷ ,p<0.0001 and 3.57xl0 ⁸ IQR 2.54-5.41xl0 ⁸ ,p<0.0001, respectively) [Supplementary Figure 2A], CLA increased at 6h (3.1xl0 ⁸ IQR 1.8-4.91xl0 ⁸ , p=0.059), and returned to near basal concentrations at 24h (2.3xl0 ⁸ IQRl.36-3.9xlO ⁸ , p=0.972) [Figure 4A], [0061] AA metabolites derived from the lipoxygenase pathway, such as glutamic acid (20.59xl0 ⁸ IQR 15.97-25.02) and 12-HETE (1.45xl0 ⁷ IQR 0.92-1.98), had a significant reduction in the absolute measurements at 6h (13.37x10 ^s IQR 9.97-17.03xl0 ⁸ , p<0.0001 and 1.17xl0 ⁷ IQR 0.77-1.54xl0 ⁷ , p<0.0001, respectively), and 24h (13.03x10 ^s IQR 8.58-16.56xl0 ^s , p<0.001, and 1.13xl0 ⁷ IQR 0.88-1.63xl0 ⁷ , p<0.0001, respectively) [Figure 4A],

[0062] PAF concentrations (16.99x10 ^s IQR 14.05-18.8x10 ^s ) were significantly reduced at 6h (9.65x10 ^s IQR 7.56-11.37x10 ^s , p<0.0001) and 24h (6.89x10 ^s IQR 5.86-8.16xlO ^s , p<0.0001), compatible with the previously reported reduction of platelet activity after CPB. No significant changes were observed in thromboxane B2 concentrations at 6 and 24h.

[0063] Interestingly, the concentrations of 8,11,14-eicosatrienoic acid (1.83x10 ^s IQR 1.37- 2.32x10 ^s ) were unchanged at 6h (2.06x10 ^s IQR 1.51-2.38x10 ^s , p=0.271) but reduced at 24h (1.24x10 ^s IQR 1-1.53x10 ^s , p<0.001).

[0064] Because two postoperative measurements were tested for the nine metabolites, a Bonferroni-adjusted significance level of 0.00278 was calculated to account for the increased possibility of type-1 error.

[0065] To evaluate if the variation of the metabolites followed a similar trend than the absolute value, it was calculated the change in the metabolites measurements at 6h and 24h (percentage of change comparing to pre-operatory levels) (Figure 4B and Table 4). Although a high percentage of patients had a pronounced reduction at 6h, median reduction of AA, EPA and DHA was not significant at 6h. However, levels were significantly reduced at 24h. On the other hand, CLA had an increase at 6h with a non-significant variation at 24h. Glutamic acid and 12-HETE had a similar pattern of reduction both at 6h and 24h. Although PAF measurement were reduced at 6h, the reduction was even more pronounced at 24h. In contrast, TxB2 values were more reduced at 6h than at 24h. 8,11,14-eicosatrienoic acid showed a particular pattern, with a median increase of 19.4% (-15;44.8), significantly decreasing at 24h. Table 4. Metabolites absolute values and relative change in percentage compared to preoperative levels

IQR: interquartile range; EPA: eicosapentaenoic acid; DHA: docosahexaenoic acid; CLA: conjugated linoleic acids; 12-HETE: 12-hydroxyeicosatetraenoic acid; PAF: platelet activating factor; TxB2: thromboxane B2; Pre-op: pre-operative; Post-op: postoperative

Platelet count and morphology and antiplatelet therapy

[0066] In the population of the present study, platelet count decreased after surgery (Figure 1A), with a median -32.8% (-39.5;-28.8) at 6h and -28.5% (-35;-18.2) at 24h (Figure IB). The reduction was consistent within all patients (Figure IB), and was not correlated to any comorbidity or the use of anticoagulation and/ or anti-aggregation. Consistent with previous reports, mean platelet volume (MPV) was also reduced at 6h and 24h (Figure 1C). However, platelet distribution width (PDW) remained stable at all times (Figure ID). [0067] Importantly, the variation in the postoperative metabolite levels was not induced by the cardiopulmonary bypass reduction of platelet count, since platelet count at pre-operative, 6h and 24h was not correlated with metabolites measurement neither with the percentage of variation in the same timepoint. The absolute value and the variation of the metabolites were also not correlated with the variation in the MPV and PDW.

[0068] Considering that a substantial percentage of patients were medicated with platelet inhibitors (AAT), it was assessed if preoperative AAT influenced preoperative levels and their variation. None of our patients was taking non-steroidal anti-inflammatory drugs and 45.5% (15 patients) were taking acetylsalicylic acid (ASA) [Table 1], Patients taking ASA had similar levels of the studied metabolites in the preoperative (Figure 5). No significant differences were observed in any of the analyzed compounds in absolute levels for each timepoint, and for the percentage of reduction at 6h and 24h (Figure 5-6).

Arachidonic acid signature and postoperative bleeding

[0069] Based on the AA metabolic signature observed, it was tested whether pre-operatory levels of AA were associated with post-operative bleeding. It was surprisingly found that levels of preoperative AA were inversely correlated with postoperative chest tube blood loss at 24h (R=-0.3957;*p=0.03) (Figure 2A). In contrast, the postoperative blood loss was not correlated with platelet count in the preoperative (R=-0.04, p=0.815), at 6h (R=-0.098, p=0.597) or at 24h (R=-0.06,p=0.741). Considering patients with a significant postoperative blood-loss (at least 600mL in the first 12h after surgery), the preoperative levels of AA were considerably lower (4.8xl0 ⁸ vs 6.8xl0 ⁸ , *p=0.032) (Figure 2B). The subjects with low levels of pre-operatory AA (first quartile of no significant bleeding) had increased risk of postoperative bleeding with a relative risk (RR) 2.8 [95% confidence interval (Cl) 1.19-6.68] and odds ratio (OR) 7 (95% Cl 1.35-30.25), *p=0.045. Among individuals with preoperative levels of AA below 5xl0 ⁸ , the postoperative blood loss (600cc IQR 325-600 vs 350cc IQR 300-400, *p=0.029) were substantially higher (Figure 2C).

[0070] Moreover, the transfusion rate was also higher in patients with reduced pre-operative AA levels and with a post-operative reduction. In patients with pre-op AA levels below 5xl0 ⁸ ion count 42% required post-operative transfusion, versus 22% in patients with higher levels (p=0.418) (Figure 3A. Additionally, patients with reduced levels of AA at 6h also had a higher rate of transfusion, compared to those with unchanged or increased values (40% vs 28%, p=0.489)(Figure 3A], Regarding other outcomes in patients with postoperative bleeding, the necessity of hemodynamic support was higher, although not statistically significant, as well as mechanical ventilation for more than 6h and neurologic complications (Table 1). Interestingly, postoperative atrial fibrillation was more common in the group without significant bleeding.

[0071] AA was later evaluated for predictive accuracy for identifying patients with an increased risk of significant bleeding. From the ROC curve, AA was identified as a predictor of postoperative significant bleeding with an AUC of 73.2% (Figure 3B).

[0072] As CPB is associated with a systemic inflammatory response, it was also measured IL-6 levels (Figure 7A) and correlated them with postoperative AA variability (Figure 3C). It was observed a strong correlation between IL-6 levels and the AA variability(r=- 0.602,***p=0.0004), suggesting a possible role of the inflammatory setting in AA metabolism perturbation. Curiously, IL6 levels were lower in patients with significant postoperative bleeding Figure 7B).

[0073] It was observed that IL6 concentrations 6h after surgery are strongly correlated with the variability in AA levels in our patients, suggesting that inflammation plays an essential role in postoperative AA dysfunction. Interestingly, not all the pathways of AA-activation may have the same relevance in post-operative bleeding. We have seen that levels of TxB2 remain stable, consistent with previous studies that have also reported that TxB2 plasma levels one hour and 24h after the operation had returned to preoperative values [12], Moreover, although concentrations of metabolites from both eicosanoids and lipoxygenase pathways were reduced at 6h and 24h, the reduction remains approximately the same at 6h and 24h, while the reduction of the metabolite representing lipoxygenase is much pronounced at 24h. Thus, while cyclooxygenase pathway may remain unchanged, eicosanoid and lipoxygenase pathways may be differently disrupted. Curiously, patients with significant postoperative bleeding had lower levels of IL6, especially beginning 12h post-operative. It has been documented that AA can induce the release of IL-6 from inflammatory cells, inducing the production of acute-phase proteins to balance some of the detrimental effects of AA metabolites in shock [31], IL-6 augments platelet count and function and the thrombotic potential of IL6 is greater than its fibrinolytic effect in hemorrhage, especially in the early stages of bleeding [32], The fact that IL6 levels are lower in patients with significant hemorrhage is a possible consequence of the AA metabolism perturbation.

[0074] The present disclosure shows that preoperative AA administration is effective in reducing platelet dysfunction after cardiac surgery with CPB, reducing postoperative bleeding and the necessity of transfusion. AA supplementation at a dose of 50-3600 mg/day in the days preceding the surgical procedure is safe and increases plasmatic levels of AA in a dosedependent manner

[0075] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0076] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. The above described embodiments are combinable.

[0077] Where ranges are provided, the range limits are included. Furthermore, it should be understood that unless otherwise indicated or otherwise evident from the context and/or understanding of a technical expert, the values which are expressed as ranges may assume any specific value within the ranges indicated in different achievements of the invention, at one tenth of the lower limit of the interval, unless the context clearly indicates the contrary. It should also be understood that, unless otherwise indicated or otherwise evident from the context and/or understanding of a technical expert, values expressed as range may assume any sub-range within the given range, where the limits of the sub-range are expressed with the same degree of precision as the tenth of the unit of the lower limit of the range.

[0078] The following dependent claims further set out particular embodiments of the disclosure.

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