Methods For Assessing Risk Of Cardiovascular Disease And Methods And Compounds For Use In Treating Or Preventing Cardiovascular Disease
20200215014 ยท 2020-07-09
Inventors
Cpc classification
G01N33/5308
PHYSICS
G16H20/10
PHYSICS
A61P9/10
HUMAN NECESSITIES
G01N33/92
PHYSICS
G16H50/30
PHYSICS
G01N2800/52
PHYSICS
A61K31/202
HUMAN NECESSITIES
A61K9/0053
HUMAN NECESSITIES
G16H10/40
PHYSICS
G16H50/70
PHYSICS
International classification
A61K31/202
HUMAN NECESSITIES
A61K9/00
HUMAN NECESSITIES
G01N33/92
PHYSICS
G16H10/40
PHYSICS
G16H20/10
PHYSICS
Abstract
A method of treating or preventing cardiovascular disease which comprises administering a therapeutically effective amount of at least one n-3 DPA-derived resolvin and/or upregulating or increasing the biosynthesis or activity of at least one n-3 DPA-derived resolvin. n-3 DPA-derived resolvins are normally regulated diurnally in the body and are linked to activation of platelets and leucocytes and formation of platelet-leukocyte aggregates. Dysfunctional regulation of n-3 DPA-derived resolvins may lead to systemic inflammation because of excessive inflammation-inducing eicosanoids, especially in the early hours of the morning. Further, decreased 5-LOX/15-LOX expression and increased systemic adenosine concentrations are found to be associated with reduced resolvin levels and increased risk of cardiovascular disease. n-3 DPA-derived resolvins are administered to achieve maximum absorption in the early hours. Also disclosed are n-3 DPA-derived resolvins for use in the treatment or prevention of cardiovascular disease, and methods for measuring the levels of n-3 DPA-derived resolvins and/or the expression or activity of adenosine or 5-LOX/15-LOX in biological samples obtained from a subject for assessing the subject's risk of cardiovascular disease.
Claims
1. An n-3 DPA-derived resolvin for use in a method of treating or preventing cardiovascular disease.
2. An n-3 DPA-derived resolvin for use as claimed in claim 1 which reduces activation of platelets and/or leukocytes, particularly monocytes.
3. An n-3 DPA-derived resolvin for use as claimed in claim 1 or claim 2 which reduces the formation of platelet-leukocyte aggregates.
4. An n-3 DPA-derived resolvin for use as claimed in any preceding claim, wherein the method comprises administering the n-3 DPA-derived resolvin to achieve C.sub.max in the early hours of the morning, preferably between about 7 AM and about 9 AM.
5. An n-3 DPA-derived resolvin for use as claimed in any preceding claim which is formulated to give a peak plasma concentration of n-3 DPA-derived resolvin of at least 10 pg/mL.
6. An n-3 DPA-derived resolvin for use as claimed in any preceding claim, wherein the cardiovascular disease is coronary artery disease, myocardial infarction, strokes, transient ischaemic attack, peripheral arterial disease, aortic disease, angina, heart failure or aortic aneurysm.
7. An n-3 DPA-derived resolvin for use as claimed in claim 1, wherein said n-3 DPA-derived lipid mediator is RvD1.sub.n-3 DPA, RvD2.sub.n-3 DPA and/or RvD5.sub.n-3 DPA.
8. A pharmaceutical composition comprising one or more n-3 DPA-derived resolvins as defined in any of claims 1-7 and one or more pharmaceutically acceptable excipients.
9. A pharmaceutical composition as claimed in claim 9 which is formulated for controlled and/or delayed release of the one or more n-3 DPA-derived resolvins to provide maximal absorption in the morning, preferably a delay between administration and T.sub.max of 9-12 hours.
10. A pharmaceutical composition as claimed in claim 8 or claim 9 which is for oral administration.
11. A method of treating or preventing cardiovascular disease which comprises administering a therapeutically effective amount of at least one n-3 DPA-derived resolvin to a subject in need thereof and/or increasing the biosynthesis, activity or expression levels of at least one n-3 DPA-derived resolvin in a subject in need thereof.
12. A method of treating or preventing vascular inflammation and/or myocardial infarction, which comprises administering a therapeutically effective amount of at least one n-3 DPA-derived resolvin to a subject in need thereof and/or increasing the biosynthesis, activity or expression levels of at least one n-3 DPA-derived resolvin in a subject in need thereof.
13. A method of treating dysfunctional diurnal regulation of one or more n-3 DPA-derived resolvins which comprises administering a therapeutically effective amount of at least one n-3 DPA-derived resolvin to a subject in need thereof and/or increasing the biosynthesis, activity or expression levels of at least one n-3 DPA-derived resolvin in a subject in need thereof.
14. A method of attenuating activation of platelets and/or leukocytes in a human subject in need thereof, which comprises administering a therapeutically effective amount of at least one n-3 DPA-derived resolvin and/or increasing the biosynthesis, activity or expression levels of at least one n-3 DPA-derived resolvin in a subject in need thereof.
15. A method of reducing formation of platelet-leukocytes aggregates in a human subject in need thereof, which comprises administering a therapeutically effective amount of at least one n-3 DPA-derived resolvin and/or increasing the biosynthesis, activity or expression levels of at least one n-3 DPA-derived resolvin in a subject in need thereof.
16. A method as claimed in any one of claims 11-15, wherein the at least one n-3 DPA-derived resolvin is formulated for immediate or delayed and/or controlled release, and the n-3 DPA-derived resolvin is administered such that t.sub.max occurs in the early hours of the morning.
17. A method as claimed in any one of claims 11-16, wherein the at least one n-3 DPA-derived resolvin is administered in a dosage that is calculated to achieve a peak plasma concentration of n-3 DPA-derived resolvin of at least 10 pg/mL, preferably 15-25 pg/mL.
18. A method as claimed in any one of claims 11-17, wherein the at least one n-3 DPA-derived resolvin is RvD1.sub.n-3 DPA, RvD2.sub.n-3 DPA and/or RvD5.sub.n-3 DPA.
19. A method as claimed in any one of claims 11-18, which comprises the simultaneous, sequential or separate administration of a combination of two or more n-3 DPA-derived resolvins to the subject.
20. A method as claimed in any one of claims 11-19, wherein the at least one n-3 DPA-derived resolvin is administered orally.
21. A method as claimed in any one of claims 11-20, which comprises reducing the activity or expression levels of adenosine and/or increasing the activity or expression levels of 5-LOX and/or 15-LOX in the subject.
22. A method of assessing the risk of cardiovascular disease in a subject which comprises comparing the levels of one or more n-3 DPA-derived resolvins and/or the activity or expression levels of adenosine or 5-LOX/15-LOX in a biological sample obtained from the subject's blood in the early morning with reference levels for the one or more n-3 DPA-derived resolvins, adenosine or 5/LOX/15-LOX in healthy subjects.
23. A method of assessing the risk of cardiovascular disease in a subject which comprises comparing the levels of one or more n-3 DPA-derived resolvins and/or the expression or activity levels of adenosine and/or 5-LOX/15-LOX in a first biological sample obtained from the subject's blood in the early morning with corresponding levels of the one or more n-3 DPA-derived resolvins, adenosine or 5-LOX/15-LOX in a second biological sample obtained from the subject's blood at another time of day.
24. A method as claimed in claim 23, which further comprises assessing changes in activation of white blood cells or platelets in the subject's blood.
25. A method as claimed in claim 24, wherein activation of the white blood cells or platelets is measured using activation markers, e.g. CD62P, CD11b and/or CD41.
26. A method as claimed in any of claims 22-25, wherein the one or more n-3 DPA-derived resolvins are selected from RvD1.sub.n-3 DPA, RvD2.sub.n-3 DPA and/or RvD5.sub.n-3 DPA.
27. A method as claimed in any of claims 22-26, wherein the levels of two or more n-3 DPA-derived resolvins are measured.
28. A method as claimed in any of claims 22-27, wherein the biological sample is a whole blood, serum or plasma sample.
29. A method as claimed in any of claims 22-28, wherein the levels of the one or more n-3 DPA-derived resolvins are measured using liquid chromatography tandem mass spectrometry (LC-MS/MS).
30. A method as claimed in any of claims 22-28, wherein the levels of the one or more n-3 DPA-derived resolvins are measured using an immunoassay.
31. A method as claimed in any of claims 22-30, which comprises assessing the ratio of n-3 DPA-derived resolvins to inflammation-initiating eicosanoids in the patient's blood, e.g. prostaglandins, leukotriene B4 and/or TxB2.
32. A method of assessing the efficacy of a therapeutic or preventative treatment for cardiovascular disease in one or more subjects, which comprises assessing the levels of one or more n-3 DPA-derived resolvins and/or the expression or activity of adenosine or 5-LOX/15-LOX in samples obtained from the subjects' blood after commencing the treatment, wherein the blood samples are obtained early in the morning and an increase in the levels of the n-3 DPA-derived resolvins or a decrease in the expression or activity of adenosine or an increase in the expression or activity of 5-LOX/15-LOX in the samples is indicative of efficacy of the medicament.
33. A method as claimed in claim 32, wherein the levels of the one or more n-3 DPA-derived resolvins or the levels of expression or activity of adenosine or 5-LOX/15-LOX are compared with corresponding levels of the one or more n-3 DPA-derived resolvins or the levels of expression or activity of adenosine or 5-LOX/15-LOX in samples obtained from the blood of the one or more subjects before treatment.
34. A method as claimed in claim 32, wherein the levels of the one or more n-3 DPA-derived resolvins or the levels of expression or activity of adenosine or 5-LOX/15-LOX are monitored in a series of two or more samples obtained from the or each of the subjects after initiating treatment with the medicament.
35. A method as claimed in claim 32, wherein the levels of the one or more n-3 DPA-derived resolvins in samples obtained from the blood of one or more subjects early in the morning are compared with corresponding levels of the one or more n-3 DPA-derived resolvins in samples obtained from the subjects at a different time of day; wherein an increase in the difference between the levels of the one or more n-3 DPA-derived resolvins in the blood samples obtained in the early morning and the different time of day, after initiating treatment, is indicative of efficacy of the medicament.
36. An immunoassay for measuring the level of an n-3 DPA-derived resolvin in a biological sample, the immunoassay comprising antibodies to the n-3 DPA-derived resolvin that are coated on a surface for capturing the n-3 DPA-derived resolvin in the sample and/or tagged with a label that is altered in a detectable manner by binding to the n-3 DPA-derived resolvin in the sample, or an amount of the n-3 DPA-derived resolvin, which is the same as the one to be quantitated in the sample, that is immobilised on a surface for capturing antibodies to the n-3 DPA-derived resolvin after mixing with the sample.
37. An immunoassay as claimed in claim 36, which is a competitive assay, further comprising a known amount of the n-3 DPA-derived resolvin, which is the same as the one to be quantitated in the sample, that is tagged with a detectable label, the labelled n-3 DPA-derived resolvin being affinity-bound to a surface by an antibody to the n-3 DPA-derived resolvin.
38. An immunoassay as claimed in claim 36, wherein the immunoassay comprises surface-bound n-3 DPA-derived resolvin, which is the same as the n-3 DPA-derived resolvin that is to be quantitated in the sample, and a known amount of antibodies to the n-3 DPA-derived resolvin in solution in excess.
39. An immunoassay as claimed in claim 36, wherein the immunoassay comprises a labelled secondary antibody to the n-3 DPA-derived resolvin or to a primary antibody to the n-3 DPA-derived resolvin for quantifying the amount of the n-3 DPA-derived resolvin bound to surface-bound antibodies to the n-3 DPA-derived resolvin or the amount of the primary antibody bound to the n-3 DPA-derived resolvin immobilised on a surface.
40. Equipment for measuring the level of a specific n-3 DPA-derived resolvin in a blood sample comprising a sample collection device and an immunoassay as claimed in any of claims 36-39.
41. Equipment as claimed in claim 40, further comprising a detector for detecting labelled n-3 DPA-derived resolvin or labelled antibodies to the n-3 DPA-derived resolvin in the immunoassay.
42. A device for measuring the level of at least one n-3 DPA-derived resolvin in a biological sample obtained from a subject, the device comprising one or more parts defining an internal channel having an inlet port and a reaction zone, in which a n-3 DPA-derived resolvin in a sample may be reacted with an immobilised primary antibody for the n-3 DPA-derived resolvin for capturing the n-3 DPA-derived resolvin, or a primary antibody for the n-3 DPA-derived resolvin in excess in solution after mixing with the sample upstream of the reaction zone may be reacted with n-3 DPA-derived resolvin, which is the same as the one to be measured in the sample, that is immobilised on a surface within the reaction zone, for quantifying directly or indirectly the amount of the n-3 DPA-derived resolvin in the sample.
43. A device as claimed in claim 42, wherein the device comprises a plurality of channels, each with its own inlet port, for measuring the levels of a plurality of different n-3 DPA-derived resolvin in the sample in parallel.
44. A computer-implemented method of assessing the efficacy of a therapeutic or preventative treatment for cardiovascular disease in a subject, which comprises receiving in a computer sample data representing the levels of at least one n-3 DPA-derived resolvin in biological samples obtained from the blood of the subject early in the morning, before and after commencing the treatment, and executing software on the computer to compare the levels of the at least one n-3 DPA-derived resolvin in the samples, an increase in the level of the at least one n-3 DPA-derived resolvin after treatment being indicative of efficacy of the medicament, and to output efficacy data representing the efficacy of the treatment on the basis of the comparison.
45. A computer-implemented method of assessing the efficacy of a therapeutic or preventative treatment for cardiovascular disease in a subject, which comprises receiving in a computer sample data representing the levels of at least one n-3 DPA-derived resolvin in a series of at least two groups of biological samples obtained from the blood of the subject, one sample in each group being obtained from the subject early in the morning, and the other sample in each group being obtained from the subject a different time of day, and executing software in the computer to calculate the difference in the levels of the at least one n-3 DPA-derived resolvin between the early morning and different time of day samples in each group, and to compare the differences in levels for the groups of samples of the series; wherein an increase in the difference between the levels of the at least one n-3 DPA-derived resolvin in the early morning and different time of day samples following initiation of treatment is indicative of the efficacy of the treatment.
46. Computer-executable software for carrying out the method of claim 44 or claim 45.
47. Computer apparatus for assessing the efficacy of a therapeutic or preventative treatment for cardiovascular disease in a subject, which comprises a first device incorporating a computer, a second computer and a communication channel between the first device and second computer for the transmission of data therebetween; wherein the first device is arranged to receive sample data representing the levels of at least one n-3 DPA-derived resolvin in biological samples obtained from the subject early in the morning before and following commencing the treatment and to transmit the sample data to the second computer via the communication channel, and the second computer is arranged to execute software to compare the levels of the at least one n-3 DPA-derived resolvin in the samples to determine the efficacy of the treatment for the subject, an increase in the level of the at least one n-3 DPA-derived resolvin following treatment being indicative of efficacy, and output efficacy data representing the efficacy of the treatment.
48. Computer apparatus for assessing the efficacy of a therapeutic or preventative treatment for cardiovascular disease in a subject, which comprises a first device incorporating a computer, a second computer and a communication channel between the first device and second computer for the transmission of data therebetween; wherein the first device is arranged to receive sample data representing the levels of at least one n-3 DPA-derived resolvin in a series of pairs of biological samples obtained from the subject undergoing treatment with the medicament, one sample in each pair being obtained from the subject early in the morning, and the other sample in each pair being obtained from the subject at a different time of day, and to transmit the sample data to the second computer via the communication channel; and the second computer is arranged to execute software to calculate the difference in the levels of the at least one n-3 DPA-derived resolvin between the early morning and different time of day samples in each pair of samples and to compare the differences in the levels between the pairs of samples in the series, an increase in the difference between the early morning and different time of day n-3 DPA-derived resolvin after treatment being indicative of efficacy of the treatment.
49. Computer apparatus as claimed in claim 47 or claim 48, wherein the second computer is arranged to transmit the efficacy data to the first device via the communication channel, or to a third computer.
50. Computer apparatus as claimed in any of claims 47-49, wherein the first device incorporates an immunoassay according to any of claims 36-39, equipment according to claim 40 or 41 or a device according to claim 42 or 43 for measuring the level of at least one n-3 DPA-derived resolvin in a blood sample.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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EXAMPLES
[0246] As disclosed in Examples 1 and 9 below, lipid mediator (LM) profiling of plasma from healthy volunteers demonstrated a significant increase in n-3 DPA-derived resolvins (RvD.sub.n-3 DPA) between 7 AM and 9 AM. At these time intervals, increases in the expression of monocyte, platelet and neutrophil activation markers were found in healthy volunteer peripheral blood. As disclosed in Example 3 below, patients with cardiovascular disease demonstrated reduced plasma RvD.sub.n-3 DPA, a loss in diurnal regulation of these molecules and increases in the activation of circulating platelets, neutrophils and monocytes. Incubation of peripheral blood from these patients with RvD2.sub.n-3 DPA and RvD5.sub.n-3 DPA reduced the expression of specific platelet, monocyte and neutrophil activation markers, as disclosed in Examples 5 and 13 below. Furthermore, as disclosed in Examples 6 and 14 below, administration of RvD5.sub.n-3 DPA to Apolipoprotein E deficient (Apo E).sup./ mice reduced systemic leukocyte and platelet activation and protected from vascular disease.
[0247] Materials and Methods
[0248] Healthy Volunteers Blood Collection
[0249] Venous peripheral blood was collected at indicated intervals in sodium citrate (3.2%) from fasting volunteers that declared not taking NSAIDS for at least 14 days, caffeine and alcohol for at least 24 h and fatty fish for 48 h. Blood was collected via sequel bleeds from the same volunteers on the same day. Food was provided after the 12:00 h blood-draw to all volunteers. Volunteers gave written consent in accordance with a Queen Mary Research Ethics Committee (QMREC 2014:61) and the Helsinki declaration. Blood was then taken for flow cytometry and lipid mediator profiling analysis.
[0250] CVD Patients' Blood Collection
[0251] Fasting patients were screened and those that met the inclusion/exclusion criteria were consented for blood to be obtained between 8:00 to 9:00 h, 12:00 h and between 16:00 to 18:00 h in accordance with East of England-Cambridge Central Research Ethics Committee and the Joint Research Management Office (JRMO), Queen Mary University of London.
[0252] The inclusion criteria were i) severe coronary artery disease requiring treatment; ii) hospital admission for percutaneous coronary intervention (PCI); iii) >24 hour post PCI; iv) able to provide informed consent; v) >18 years and vi) at least 2 of the following risk factors: hypertension, high cholesterol, smoker, diabetes, known ischemic heart disease
[0253] The exclusion criteria were: i) sustained ventricular tachycardia and/or ventricular fibrillation or appropriate ICD valve disease requiring intervention; ii) contra-indications to PCI; iii) women who are pregnant; iv) <18 years and v) enrolled in other studies.
[0254] These blood samples were processed within 60 minutes of collection for lipid mediator profiling and whole blood stimulations as detailed below.
[0255] Targeted Lipid Mediator Profiling
[0256] Plasma was obtained from peripheral blood of healthy volunteers and patients following centrifugation at 1500g for 10 min at room temperature. Descending aortas were weighed, placed in ice-cold methanol and homogenized using a glass dounce.
[0257] All samples for LC-MS-MS-based profiling were extracted using solid-phase extraction columns as described in Dalli et al. 2013 and Rathod et al. 2017, the contents of which are incorporated herein by reference.
[0258] Prior to sample extraction, deuterated internal standards, representing each region in the chromatographic analysis (500 pg each) were added to facilitate quantification in 4V of cold methanol.
[0259] Samples were kept at 20 C. for a minimum of 45 min to allow protein precipitation.
[0260] Supernatants were subjected to solid phase extraction, methyl formate fraction collected, brought to dryness and suspended in phase (methanol/water, 1:1, vol/vol) for injection on a Shimadzu LC-20AD HPLC and a Shimadzu SIL-20AC autoinjector, paired with a triple quadrupole mass spectrometer with or without a linear ion trap.
[0261] An Agilent Poroshell 120 EC-C18 column (100 mm4.6 mm2.7 m) was kept at 50 C. and mediators eluted using a mobile phase consisting of methanol-water-acetic acid of 20:80:0.01 (vol/vol/vol) that was ramped to 50:50:0.01 (vol/vol/vol) over 0.5 min and then to 80:20:0.01 (vol/vol/vol) from 2 min to 11 min, maintained till 14.5 min and then rapidly ramped to 98:2:0.01 (vol/vol/vol) for the next 0.1 min. This was subsequently maintained at 98:2:0.01 (vol/vol/vol) for 5.4 min, and the flow rate was maintained at 0.5 ml/min. Mass spectrometer was operated using a multiple reaction monitoring method as in Walker M E et al. 13-Series resolvins mediate the leukocyte-platelet actions of atorvastatin and pravastatin in inflammatory arthritis. FASEB J. 2017 August; 31(8):3636-3648, the contents of which are incorporated herein by reference.
[0262] Each LM was identified using established criteria including matching retention time to synthetic and authentic materials and at least six diagnostic ions (Walker M E et al. 2017. ibid.)
[0263] Calibration curves were obtained for each using synthetic compound mixtures at 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, 100, and 200 pg that gave linear calibration curves with an r.sup.2 values of 0.98-0.99.
[0264] Profiling of Acetylcholine, Norepinephrine and Adenosine.
[0265] Plasma was placed in ice cold MeOH containing deuterated (d.sub.9)-choline and kept at 20 C. for 45 min to allow for protein precipitation. Samples were then centrifuged for 10 minutes at 4000g. Supernatant were collected and evaporated under a gentle stream of nitrogen gas using a TurboVap LV (Biotage) at 37 C. until dryness. Products were then suspended in MeOH profiled using an LC/MS-MS system. A Qtrap 5500 (AB Sciex) equipped with a Shimadzu SIL-20AC autoinjector and LC-20AD binary pump (Shimadzu Corp.) was used with an Agilent Eclipse Plus C18 column (1004.6 mm1.8 m). The mobile phase consisted of methanol/water/acetic acid, 80:20:0.01 (vol:vol:vol) for 2.5 min that was ramped to 98:2:0.01 (vol:vol:vol) over 0.2 min and maintained for 1.3 min. The flow rate was maintained at 0.5 ml/min. To monitor and quantify the levels of acetylcholine and norepinephrine, the Qtrap 5500 was operated in positive mode and a multiple reaction monitoring (MRM) method was developed with signature ion fragments (m/z) for each molecule monitoring the parent ion (Q1) and a daughter ion (Q3). The MRM transition employed for Acetylcholine was 146>87, for norepinephrine was 170>152 and for adenosine 268>136.
[0266] Preparation of RvD1.sub.n-3 DPA and RvD2.sub.n-3 DPA. RvD1.sub.n-3 DPA and RvD2.sub.n-3 DPA were prepared and isolated as described in Dalli J, Colas R A, Serhan C N. Novel n-3 immunoresolvents: structures and actions. Sci Rep. 2013; 3:1940, the contents of which are incorporated by reference. n-3 DPA (10 M) was incubated with 100 U/ml isolated soybean-LOX (Borate buffer, 4 C., pH 9.2). 17S-HpDPA was isolated using UV-RP-HPLC (Infinity 1260; Agilent Technologies). 17S-HpDPA (10 g) was then incubated with human neutrophils (80106 cells/ml; PBS.sup.+/+) and calcium ionophore (5 M, 37 C.). After 45 min the reaction was quenched using 2 volumes ice-cold methanol, reduced using sodium borohydrate, and products extracted using C18 SPE. RvD1.sub.n-3 DPA and RvD2.sub.n-3 DPA were isolated using RP-HPLC (Infinity 1260; Agilent Technologies). Here, an Agilent Poroshell 120 EC-C18 column (100 mm4.6 mm2.7 m) was kept at 50 C. and LM isolated with a mobile phase consisting of methanol-water-acetic acid of 60:40:0.01 (vol/vol/vol) maintained for 2 minutes, then ramped to 80:20:0.01 (vol/vol/vol) from 2 min to 16 min and to 98:2:0.01 (vol/vol/vol) over 3 minutes. This was maintained for 2 min. Flow rate was kept at 0.5 mL/min.
[0267] Human Whole Blood Incubations
[0268] In Examples 1 and 9 below, venous blood from healthy volunteers was collected and incubated with acetylcholine (ACh) at 0.1 M for 45 min (37 C.) Plasma was then separated by centrifugation at 1,500g for 10 min for LM profiling.
[0269] In Examples 3, 5, 11 and 13 below, whole blood was incubated with RvD1.sub.n-3 DPA, RvD2.sub.n-3 DPA, RvD5.sub.n-3 DPA (0.1, 1, 10 nM) or vehicle (PBS) for 15 min (37 C.) Blood was then incubated with PAF (100 nM) for 30 min (37 C.). After stimulation, samples were washed twice with PBS for 12 min at 800g. Samples were stained for flow cytometry as described below.
[0270] Flow Chamber:
[0271] Using an automated syringe pump (Harvard Apparatus) connected to small-diameter tubing (1.6 mm inner diameter) and chamber slides (15-Slide VI.sup.0.4, Ibidi), whole blood was perfused at a sheer rate of 0.1 dyne/cm.sup.2 (low sheer rate) and at 0.3 dyne/cm.sup.2 (high sheer rate) for 15 min. In selected experiment, blood was incubated with 10 nM Ach or 1 u of ADA for 20 min prior perfusing.
[0272] PRP Incubations:
[0273] Peripheral blood from healthy volunteers was collected in acidified-citrate-dextrose. Blood was centrifuged at 500g for 20 min. PRP was collected and cells incubated with RvD5.sub.n-3 DPA or vehicle (0.01% EtOH+PBS) for 15 min at 37 C. Cells were then incubated with PAF (100 nM) or Vehicle (0.01% EtOH) for 30 min at 37 C. Cells were then washed with PBS and cellular activation was assessed using flow cytometry as detailed below.
[0274] Apo E.sup./ Mice
[0275] Experiments described in Examples 6 and 14 below strictly adhered to UK Home Office regulations (Guidance on the Operation of Animals, Scientific Procedures Act, 1986) and Laboratory Animal Science Association (LASA) Guidelines (Guiding Principles on Good Practice for Animal Welfare and Ethical Review Bodies, 3rd Edition, 2015). Apo E.sup./ mice were a kind gift from Prof Fulvio D'Acquisto (Queen Mary University of London).
[0276] Mice (male and female) were fed a western diet for 6 weeks from 4 weeks of age and kept of a 12 h light dark cycle. At 8 weeks of age, mice were given RvD5.sub.n-3 DPA (100 ng/mouse; i.v.) or vehicle on alternate days for a 2-week period. Mice were culled, aortic arches were collected and stained using oil-red O as in Khambata R S et al. Anti-inflammatory actions of inorganic nitrate stabilize the atherosclerotic plaque. Proc Natt Acad Sci USA. 2017; 114(4):E550-E559, the contents of which are incorporated herein by reference. Staining intensity was determined using image processing software and expressed as relative units per mm.sup.2. The descending aorta was collected, placed in ice-cold methanol and lipid mediators identified and quantified as described above.
[0277] Flow Cytometry
[0278] Whole blood was incubated with lineage-specific markers for 45 min (4 C., in DPBS containing 0.02% BSA). The following anti-human antibodies were used: VioBlue-anti-CD41, PE-Cy5-anti-CD62P, Brilliant Violet 711-anti-CD11b, APC-Cy7-anti-CD16, Alexa Fluor 647-anti-CD14. After staining, red blood cells were lysed using Whole Blood Lysing Reagent Kit, according to the manufacturer's instructions. Data was collected using a flow cytometer and analysis was conducted using appropriate software.
[0279] In separate experiments blood was collected from Apo E.sup./ mice using heparin-lined syringes via cardiac puncture. Cells were incubated with Fc-blocking IgG and anti-mouse CD11b-PE-Texas Red, CD62P-Brilliant Violet650, CD115-Brilliant Violet 711, and CD41-Brilliant Violet 510 (Biolegend). for 45 minutes on ice. Red blood cells were lysed and fixed using Whole Blood Lysing Reagent Kit. Staining was then evaluated using a flow cytometer and analysis was conducted using appropriate software.
[0280] For the analysis of the biosynthetic enzymes, whole blood was incubated with lineage-specific markers for 30 min (4 C., in DPBS containing 0.02% BSA). The following anti-human antibodies were used: Brilliant Violet 786-anti-CD14, APC-Cy7-anti-CD16, PerCP-Cy5.5-anti-CD4. After staining, red blood cells were lysed using Whole Blood Lysing Reagent Kit, according to the manufacturer's instructions. Samples were washed twice with PBS for 12 min at 800g, and incubated with Fc block for 20 min at RT (dilution 1:2, in Permeabilization buffer). Next followed the intracellular staining for 30 min (RT, in Permeabilization buffer). The following anti-human antibodies were used: Alexa Fluor 647-anti-15-LOX, Dylight 405-anti-5-LOX. Staining was then evaluated using LSRFortessa cell analyser (BD Biosciences) and analysed using FlowJo software (Tree Star Inc., V10).
[0281] In select experiments platelet adhesion molecule expression was assessed. Here platelets were incubated with fluorescently labelled mouse anti-human VioBlue-anti-CD41, PE-Cy5-anti-CD62P, PerCP/Cy5.5-anti-CD63 and FITC-ant-CD42b for 30 min at 4 C. Cells were then washed and fluorescence, staining evaluated using LSRFortessa cell analyser (BD Biosciences) and analysed using FlowJo software (Tree Star Inc., V10).
[0282] ImageStream.
[0283] Whole blood was incubated with lineage-specific markers for 45 min (4 C., in DPBS containing 0.02% BSA). The following anti-human antibodies were used: eFluor450-anti-CD41, PE-Cy5-anti-CD62P, APC-Cy7-anti-CD16, FITC-anti-CD14. After staining, red blood cells were lysed using Whole Blood Lysing Reagent Kit, according to the manufacturer's instructions. Staining was then assessed using ImageStream X MK2 and analysis was performed using IDEAS (Image Data Exploration and Analysis Software, Version 6.0).
[0284] Statistical Analysis
[0285] Results are expressed as means.e.m. Normality and equal distribution of variance between the different groups analysed were assumed. Sample sizes for each experiment were determined on the variability observed in preliminary experiments. Differences between groups were assessed using one-sample t test (normalized data), Student's t test (2 groups), 1-way ANOVA (multiple groups) followed by post hoc Dunnett's test. Investigators were not blinded to group allocation or outcome assessment. The criterion for statistical significance was p0.05. Sample sizes for each experiment were determined on the variability observed in prior experiments (Rathod K S et al. 2017) and preliminary experiments. Partial least squares-discrimination analysis (PLS-DA) and principal component analysis (PCA)19 were performed using SIMCA 14.1 software (Umetrics, Umea, Sweden) following mean centering and unit variance scaling of LM levels. PLS-DA is based on a linear multivariate model that identifies variables that contribute to class separation of observations (Blister exudates) on the basis of their variables (LM levels). During classification, observations were projected onto their respective class model. The score plot illustrates the systematic clusters among the observations (closer plots presenting higher similarity in the data matrix). Loading plot interpretation identified the variables with the best discriminatory power (Variable Importance in Projection greater then 1) that were associated with the distinct intervals and contributed to the tight clusters observed in the Score plot.
[0286] Results
Example 1: Diurnal Changes in Peripheral Blood n-3 DPA-Derived SPM are Regulated by Acetylcholine
[0287] To investigate whether peripheral blood SPM concentrations are diurnally regulated, plasma was obtained from healthy volunteers at distinct intervals during a 24 h period (see Table 4 below for demographics).
TABLE-US-00004 TABLE 4 Healthy volunteers demographics Sex Age (years) Weight (Kg) BMI (Kg/m.sup.2) 3M/4F 34 4.1 65.6 11.3 23.2 3.0
[0288] LM were then extracted using C18 solid phase extraction and identified and quantified using liquid chromatography-tandem mass spectrometry (LC/MS-MS).
[0289] In plasma from healthy volunteers, mediators from all four major essential fatty acid metabolomes were identified, including the EPA derived E-series resolvins, n-3 DPA-derived resolvins and protectins, DHA-derived protectins and maresins and the arachidonic acid (AA)-derived prostaglandins and leukotrienes (see
[0290] These mediators were identified in accordance with published criteria that include matching retention time in liquid chromatography and at least six diagnostic ions in the tandem mass spectrum (Dalli et al. 2013) as illustrated for RvD5.sub.n-3 DPA (
[0291] Retention times for RvD1.sub.n-3 DPA, RvD2.sub.n-3 DPA and RvD5.sub.n-3 DPA are disclosed above in Table 3.
[0292] Diagnostic ions for RvD1.sub.n-3 DPA, RvD2.sub.n-3 DPA and RvD5.sub.n-3 DPA are disclosed in Table 5 below.
TABLE-US-00005 TABLE 5 Diagnostic ions for RvD1.sub.n-3 DPA, RvD2.sub.n-3 DPA and RvD5.sub.n-3 DPA RvD.sub.n-3 DPA Fragmentation pattern ions (m/z)
TABLE-US-00006 TABLE 6 Diurnal lipid mediator profiles in healthy volunteer peripheral blood. Peripheral blood was collected from healthy volunteers at the indicated intervals. Plasma was placed in ice- cold methanol and lipid mediators (LM) were assessed using LM-profiling (see methods for details). Q1, M-H (parent ion) and Q3, diagnostic ion in the MS-MS (daughter ion). Results are mean s.e.m. and expressed as pg/mL. n = 7 volunteers per interval. The detection limit was ~0.1 pg. , Below limits of detection. Healthy volunteer plasma DHA bioactive Lipid mediators' concentration (pg/mL) metabolome Q1 Q3 18:00 7:00 9:00 12:00 15:00 RvD1 375 141 0.8 0.4 0.8 0.3 1.4 0.4 0.8 0.3 0.8 0.2 RvD2 375 141 0.9 0.4 0.9 0.2 0.6 0.3 0.3 0.2 0.3 0.2 RvD3 375 147 0.1 0.0 0.6 0.4 0.4 0.2 0.2 0.1 0.1 0.0 RvD4 375 101 0.3 0.2 0.3 0.2 0.3 0.2 1.3 0.8 0.8 0.6 RvD5 359 199 0.6 0.2 0.3 0.2 0.3 0.1 0.5 0.2 0.5 0.1 RvD6 359 101 0.6 0.2 0.4 0.1 0.7 0.4 0.7 0.2 0.8 0.3 17R-RvD1 375 141 0.2 0.1 0.4 0.2 0.3 0.2 0.4 0.2 0.4 0.1 17R-RvD3 375 147 0.1 0.1 0.2 0.1 0.1 0.1 0.1 0.1 0.2 0.1 PD1 359 153 0.6 0.2 0.7 0.4 0.8 0.3 1.0 0.4 1.0 0.6 17R-PD1 359 153 0.6 0.3 0.4 0.3 0.4 0.2 0.3 0.2 0.3 0.2 10S,17S-diHDHA 359 153 0.6 0.2 1.4 0.4 1.1 0.6 0.7 0.4 0.7 0.2 22-OH-PD1 375 153 0.8 0.4 1.3 0.7 1.1 0.5 1.4 0.7 2.7 2.1 MaR1 359 221 1.0 0.3 0.6 0.2 1.2 0.2 1.1 0.4 1.0 0.4 7S,14S-diHDHA 359 221 1.1 0.4 1.0 0.3 0.6 0.4 1.0 0.3 0.8 0.4 4S,14S-diHDHA 359 101 11.3 8.5 8.5 5.7 7.8 4.8 9.1 5.6 8.1 5.5 n-3 DPA bioactive metabolome RvD1.sub.n-3 DPA 377 143 1.9 0.4 10.9 4.2 7.5 2.3 6.0 1.8 1.6 0.3 RvD2.sub.n-3 DPA 377 261 2.3 1.3 1.8 1.0 2.5 1.1 1.7 1.2 1.1 0.7 RvD5.sub.n-3 DPA 361 263 2.6 1.2 2.6 1.2 4.5 2.3 2.3 1.2 3.2 2.2 PD1.sub.n-3 DPA 361 183 1.1 0.3 2.3 0.5 1.6 0.3 1.6 0.3 1.1 0.3 MaR1.sub.n-3 DPA 361 249 1.7 0.7 3.5 1.9 3.2 1.4 0.9 1.0 1.3 0.6 RvT1 377 193 0.1 0.1 0.6 0.2 0.3 0.1 0.4 0.2 0.0 0.0 RvT2 377 143 0.3 0.2 0.5 0.3 0.5 0.3 0.5 0.3 0.3 0.2 RvT3 377 255 RvT4 359 193 2.0 0.8 2.6 1.4 3.0 1.3 1.4 0.6 1.4 0.6 EPA bioactive metabolome RvE1 349 195 3.9 1.5 3.7 1.5 4.5 1.5 4.9 1.9 4.9 2.0 RvE2 333 199 2.3 0.6 1.9 0.7 2.5 0.8 2.3 0.8 2.7 1.1 RvE3 333 201 1.2 0.3 1.4 0.5 1.6 0.4 1.4 0.6 1.3 0.6 AA bioactive metabolome LXA.sub.4 351 217 0.3 0.1 0.8 0.4 0.7 0.3 0.6 0.2 0.6 0.2 LXB.sub.4 351 221 0.9 0.4 0.8 0.3 0.2 0.2 0.5 0.2 0.6 0.2 5S,15S-diHETE 335 235 11.7 3.6 9.2 3.5 19.0 11.2 8.9 2.4 8.5 3.0 15epi-LXA.sub.4 351 217 7.0 3.1 8.3 5.5 4.2 1.2 4.6 1.2 4.2 1.1 15epi-LXB.sub.4 351 221 1.7 0.9 1.0 0.2 1.5 0.4 0.5 0.2 2.3 1.0 LTB.sub.4 335 195 2.0 0.5 2.5 0.7 2.6 1.2 1.8 0.6 1.9 0.5 5S,12S-diHETE 335 195 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 20-OH-LTB.sub.4 351 195 0.1 0.0 0.3 0.2 0.3 0.1 0.1 0.1 0.2 0.1 PGD.sub.2 351 189 5.8 1.7 7.5 1.5 4.7 0.4 6.6 1.8 7.6 1.6 PGE.sub.2 351 189 5.9 0.8 8.7 3.6 4.4 1.2 5.3 1.2 6.5 1.5 PGF.sub.2 353 193 6.1 1.5 11.8 2.5 7.0 1.3 6.3 0.9 8.9 0.7 TxB.sub.2 369 169 233.6 149.7 364.5 176.9 92.3 34.2 183.6 100.0 415.8 305.8
[0293] Diurnal changes were also found in plasma Thromboxane (TxB.sub.2), the inactive further metabolite of the potent platelet agonist TxA.sub.2 (Samuelsson B. Role of basic science in the development of new medicines: examples from the eicosanoid field. J Biol Chem. 2012; 287(13):10070-10080) (see Table 6 above). Of note, concentrations of n-3 DPA derived SPM were within their reported bioactive ranges (Serhan C N. 2017; Arnardottir H H et al. Resolvin D3 Is Dysregulated in Arthritis and Reduces Arthritic Inflammation. J Immunol. 2016; 197(6):2362-2368) suggesting that they may be involved in regulating vascular responses.
[0294] The mechanism(s) by which peripheral blood n-3 DPA derived SPM may be regulated were investigated. Since, as mentioned above, it has been found that acetylcholine (ACh) regulates SPM production in leukocytes, it was assessed whether peripheral blood levels of this neurotransmitter were diurnally regulated. Here it was found that plasma ACh concentrations mirrored those of the RvD.sub.n-3 DPA reaching a maximum at 7:00 h (see
[0295] To test this, whole blood was incubated with ACh, and the n-3 DPA SPM concentrations were investigated using lipid mediator profiling. In these, whole blood incubations mediators from all four n-3 DPA mediator families were identified in accordance with published criteria (
TABLE-US-00007 TABLE 7 ACh regulation of n-3 DPA metabolome in human whole blood. Peripheral blood from healthy volunteers was collected and incubated with ACh (0.1, 1 or 10 M; 45 min; 37 C.) Incubations were quenched with ice-cold methanol and n-3 DPA-derived LM identified and quantified using LM-profiling (see methods described above for details). Q1, M-H (parent ion) and Q3, diagnostic ion in the MS-MS (daughter ion). Results are expressed as pg/mL, mean s.e.m, n = 9 donors per group. = below limits of detection; detection limit = ~0.1 pg. n-3 DPA bioactive Lipid mediators concentration (pg/mL) metabolome Q1 Q3 Vehicle ACh 0.1 M RvD1.sub.n-3 DPA 377 143 3.4 1.8 4.9 2.8 RvD2.sub.n-3 DPA 377 261 2.8 1.2 7.4 2.6* RvD5.sub.n-3 DPA 361 201 0.8 0.7 1.5 0.6 PD1.sub.n-3 DPA 361 155 0.4 0.2 0.5 0.1 MaR1.sub.n-3 DPA 361 223 0.3 0.2 0.9 0.3* RvT1 377 211 0.3 0.3 2.0 1.3 RvT2 377 197 1.2 0.8 1.6 0.9 RvT3 377 255 1.2 0.9 1.4 0.9 RvT4 359 211 0.5 0.3 1.0 0.4 17-HDPA 345 247 64.4 14.9 58.9 13.2 14-HDPA 345 207 112.6 25.5 137.2 25.8* 13-HDPA 345 195 1.4 0.4 1.9 0.6* 7-HDPA 345 143 56.4 19.3 44.9 10.7 DPA 329 285 3703.3 703.0 3873.1 706.3
[0296] Quantitation of the identified molecules demonstrated increases in RvD.sub.n-3 DPA (
Example 2: Circadian Regulation of Systemic Leukocyte and Platelet Activation
[0297] Having found diurnal changes in peripheral blood LM-SPM levels and given the potent actions that RvD.sub.n-3 DPA exert on leukocyte and platelet function (Dalli et al. 2013; Gobbetti T et al. Protectin D1.sub.n-3 DPA and resolvin D5.sub.n-3 DPA are effectors of intestinal protection. Proc Natl Acad Sci USA. 2017; 114(15):3963-3968) it was investigated whether this reflected changes in leukocyte and platelet activation. Flow cytometric analysis of peripheral blood cells demonstrated significant increases in neutrophil CD11b expression as well as an increase in platelet-neutrophil aggregates, measured as increases in the expression of CD41 (Shinohara M, et al. 2014) on peripheral blood neutrophils (
Example 3: RvD.SUB.n-3 DPA .Reduce Leukocyte and Platelet Activation in Peripheral Blood
[0298] The actions of RvD.sub.n-3 DPA in regulating monocyte, neutrophil and platelet activation as well as platelet-leukocyte aggregates were investigated in light of their pathogenic functions in cardiovascular disease (Furman M I et al. 2001; Pfluecke C et al. 2016; Huo Y et al. 2003). For this purpose, human peripheral blood was incubated with platelet activating factor (PAF) in the presence or absence of RvD.sub.n-3 DPA, given PAF's role in propagating vascular inflammation (Palur Ramakrishnan A V et al. 2017).
[0299] The expression of activation markers on peripheral blood cells was assessed using flow cytometry. Incubation of human peripheral blood with RvD2.sub.n-3 DPA led to dose dependent decreases in neutrophil CD11b expression and in the amounts of neutrophil-platelet aggregates measured as decreases in neutrophil CD62P (
[0300] Similar findings were also made when healthy volunteer whole blood was incubated with RvD5.sub.n-3 DPA that resulted in dose-dependent decreases in neutrophil and monocyte CD11b expression as well as in leukocyte-platelet aggregates (see
[0301] RvD1.sub.n-3 DPA was also found partially to regulate neutrophil, monocyte and platelet responses (n=5 donors).
[0302] These data suggest that each of the RvD.sub.n-3 DPA displays specific biological actions in regulating vascular leukocyte and platelet responses.
[0303] These findings also suggest that the observed increases in peripheral blood n-3 DPA SPM in the morning hours (
Example 4: Reduced RvD.SUB.n-3 DPA .and Increased Systemic Inflammation in Peripheral Blood from Patients with Cardiovascular Disease
[0304] It was investigated whether results obtained with healthy volunteers were translatable to the clinical setting. Given that RvD.sub.n-3 DPA increased during the early morning hours, a time window associated with higher incidence of myocardial infarct (Nakashima H et al. Impact of Morning Onset on the Incidence of Recurrent Acute Coronary Syndrome and Progression of Coronary Atherosclerosis in Acute Myocardial Infarction. Circ J. 2017; 81(3):361-367; Muller J E et al. Circadian variation in the frequency of onset of acute myocardial infarction. N Engl J Med. 1985; 313(21):1315-1322) the peripheral blood levels of RvD.sub.n-3 DPA in patients with cardiovascular diseases (CVD) that were also at an increased risk of myocardial infarct were investigated (see Table 8 below for details and methods for risk criteria).
TABLE-US-00008 TABLE 8 CVD-demographics and clinical data Participants 9 Age (years) 65.2 8.6 Sex 7 Male, 2 Female CRP mg/L 35.4 42.2 IL-6 pg/mL 2.5 1.0 TNF- pg/mL 108.2 74.9 Creatine 119.1 90.5 mol/L LDL mmol/L 3.0 0.2 HDL mmol/L 0.5 0.1 Type II 3 Diabetes Hypertension 9 Current 0 Smoking Obese n 4 Previous AMI 1 Previous PCI 4 LVEF 50% 4 Aspirin (n) 9 Statins (n) Atorvastatin (4), Simvastatin (3) and Rosuvastatin (1) Other Allopurinol (1), Amitriptyline (2), Amlodipine (2), Apixaban (1), Bisoprolol medications (6), Candesartan (1), Citalopram (1), Clopidogrel (2), Codeine (1), (n) Cyanacobalamin (1), Dorzolamide (1), Doxazosin (2), Enoxaparin (1), Fentanyl (1), Finasteride (1), Flucloxacillin (1), Fluoxetine (2), Furosemide (2), Isosorbide mononitrate (1), Lansoprazole (4), Lantus Insulin (1), Lisinopril (1), Metformin (1), Nicorandil (1), NoroRapid Insulin (1), Omeprazole (3), Paracetamol (1), Phyllocontine (1), Priadel (1), Ramipril (5), Salbutamol (1), Salmeterol (1), Sertraline (1), Setagliptin (1), Tamoxifen (1), Tamsulosin (2) Temazepam (1), Thiamine (1), Tildiem (1), Timolol (1), Tioropium bromide (1), Warfarin (1), Xalatan (1).
[0305] Using lipid mediator profiling, three RvD.sub.n-3 DPA, including RvD5.sub.n-3 DPA (
TABLE-US-00009 TABLE 9 Peripheral blood LM profiles in patients with CVD. Peripheral blood from CVD patients was collected at 9:00 h (AM) and between 16:00-18:00 h (PM). Plasma was placed in ice-cold methanol containing internal standards. Lipid mediators (LM) were extracted, identified and quantified using LM-profiling (see methods for details). Q1, M-H (parent ion) and Q3, diagnostic ion in the MS-MS (daughter ion). Results are mean s.e.m. and expressed as pg/mL. n = 9 paired patients. The detection limit was ~0.1 pg. , Below limits of detection. Plasma from CVD patients Lipid mediators DHA bioactive concentration (pg/mL) metabolome Q1 Q3 AM PM RvD1 375 141 0.8 0.8 0.8 0.7 RvD2 375 141 0.2 0.2 RvD3 375 147 0.5 0.3 0.1 0.0 RvD4 375 101 2.8 1.9 1.6 0.7 RvD5 359 199 2.7 1.3 1.8 0.6 RvD6 359 101 0.2 0.1 0.4 0.3 17R-RvD1 375 141 0.1 0.1 17R-RvD3 375 147 PD1 359 153 0.7 0.2 0.5 0.2 17R-PD1 359 153 0.0 0.0 0.5 0.5 10S,17S-diHDHA 359 153 0.1 0.1 0.5 0.2 22-OH-PD1 375 153 0.1 0.1 3.5 3.7 MaR1 359 221 0.6 0.5 0.1 0.1 7S,14S-diHDHA 359 221 1.2 0.7 0.3 0.3 4S,14S-diHDHA 359 159 0.5 0.3 0.1 0.1 n-3 DPA bioactive metabolome RvD1.sub.n-3 DPA 377 215 1.8 0.6 1.0 0.4 RvD2.sub.n-3 DPA 377 261 2.2 0.9 1.2 0.6 RvD5.sub.n-3 DPA 361 263 0.2 0.2 0.6 0.3 PD1.sub.n-3 DPA 361 183 0.3 0.1 0.2 0.1 MaR1.sub.n-3 DPA 361 249 RvT1 377 193 0.4 0.3 0.1 0.0 RvT2 377 233 0.5 0.3 RvT3 377 197 RvT4 359 211 0.4 0.2 0.6 0.2 EPA bioactive metabolome RvE1 349 195 2.4 1.9 3.6 3.4 RvE2 333 199 0.2 0.2 0.7 0.7 RvE3 333 201 1.7 1.1 1.1 0.5 AA bioactive metabolome LXA.sub.4 351 115 0.1 0.0 LXB.sub.4 351 221 0.9 0.5 0.5 0.3 5S,15S-diHETE 335 235 8.2 1.7 16.6 4.2 15epi-LXA.sub.4 351 115 0.6 0.3 0.9 0.4 15epi-LXB.sub.4 351 221 30.0 10.7 20.0 7.5 LTB.sub.4 335 195 2.0 0.8 1.8 0.3 5S,12S-diHETE 335 195 1.0 0.7 0.2 0.1 20-OH-LTB.sub.4 351 195 0.2 0.1 0.6 0.3 PGD.sub.2 351 189 1.7 0.3 3.6 1.2 PGE.sub.2 351 189 6.3 1.8 8.1 1.3 PGF.sub.2 353 193 8.1 2.6 6.2 1.2 TxB.sub.2 369 169 10.4 6.2 20.4 13.7
[0306] Assessment of plasma RvD.sub.n-3 DPA levels demonstrated significant decreases in both morning (9:00 h; am) and evening (16:00-18:00 h; pm) concentrations in CVD patients when compared to the respective intervals in healthy volunteers (
[0307] Furthermore, the ratio of plasma RvD.sub.n-3 DPA to inflammation-initiating eicosanoids (prostaglandins, leukotriene B4 and TxA.sub.2) was significantly lower in these patients at both intervals measured when compared to healthy volunteers indicating an elevated systemic inflammatory status (p<0.05).
[0308] This was further supported by the observation that peripheral blood leukocyte and platelets also displayed an increased activation status. Flow cytometric analysis demonstrated increases in the expression of CD11b on both neutrophils and monocytes from CVD patients when compared with healthy volunteers (
[0309] In peripheral blood from these patients, a significant decrease in morning plasma ACh concentrations was found compared to evening values (
Example 5: Reduced Leukocyte Activation by RvD2.SUB.n-3 DPA .and RvD5.SUB.n-3 DPA .in Patient Peripheral Blood
[0310] In order to test whether there was a relationship between the increased systemic inflammation and reduced n-3 DPA derived SPM, it was tested whether RvD.sub.n-3 DPA regulated patient peripheral blood leukocyte responses. For this purpose, whole blood from these patients was incubated with RvD2.sub.n-3 DPA and cellular responses were assessed using flow cytometry. RvD2.sub.n-3 DPA dose-dependently decreased platelet-neutrophil and platelet-monocyte aggregates without significantly regulating CD11b expression (see
[0311] Incubation of whole blood with RvD5.sub.n-3 DPA also led to a reduction in neutrophil platelet and monocyte-platelet aggregates with higher potency than RvD2.sub.n-3 DPA (
[0312] It was tested whether the actions of these two mediators were also retained in the presence of PAF (Shinohara M et al. 2014; Palur Ramakrishnan A V et al. 2017). Incubation of patient whole blood with either RvD2.sub.n-3 DPA or RvD5.sub.n-3 DPA led to decreases in platelet-neutrophil and platelet-monocyte aggregates measured as decreases in CD62P (see
[0313] It was also found that RvD5.sub.n-3 DPA decreased the expression of CD11b on neutrophils and monocytes, an action that was only in part shared with RvD2.sub.n-3 DPA (
[0314] These results suggest that reductions in circulating RvD.sub.n-3 DPA lead to increased circulating leukocyte and platelet activation in CVD patients.
Example 6: n-3 DPA Reduces RvD5 Systemic Leukocyte and Platelet Activation and Protects Against Vascular Disease in Apo E.SUP./ Mice
[0315] It was next investigated whether the protective actions of RvD5.sub.n-3 DPA observed with peripheral blood cells from both healthy volunteers and CVD patients were also retained in vivo. For this purpose, Apo E mice were fed western diet for 6 weeks and RvD5.sub.n-3 DPA (100 ng/mouse; i.v.) was administered on alternative days for a two-week period. RvD5.sub.n-3 DPA administration reduced circulating platelet-monocyte aggregates, as measured by a decrease in both CD41 and CD62P expression on CD115 positive cells, and monocyte activation with a decrease in CD11b expression (
[0316] Since platelet-leukocyte aggregates are involved in the pathogenesis of atherosclerosis (Huo Y et al. 2003), it was investigated whether RvD5.sub.n-3 DPA also protected against vascular disease. Oil red-O staining demonstrated a significant reduction in aortic lesions in mice given RvD5.sub.n-3 DPA when compared to mice given vehicle (
TABLE-US-00010 TABLE 10 Reduced eicosanoids in aortic tissues from Apo E.sup.-/- mice given RvD5.sub.n-3 DPA. Descending aortas were placed in ice-cold methanol containing internal standards. Lipid mediators (LM) were extracted, identified and quantified using LM-profiling (see methods described above for details). Q1, M-H (parent ion) and Q3, diagnostic ion in the MS-MS (daughter ion). Results are mean s.e.m. and expressed as pg/10 mg tissue. n = 4 mice per group. * p < 0.05 vs Vehicle mice. Lipid mediators concentration (pg/10 mg tissue) Q1 Q3 Apo E.sup.-/- + Vehicle Apo E.sup.-/- + RvD5.sub.n-3 DPA PGD.sub.2 351 189 18.5 2.8 15.0 2.2 PGE.sub.2 351 189 21.5 3.2 16.2 2.7 PGF.sub.2a 353 193 10.8 0.7 9.1 2.2 TxB.sub.2 369 169 46.9 6.5 30.7 2.0*
[0317] The above examples demonstrate a diurnal regulation of RvD.sub.n-3 DPA in the vasculature of healthy volunteers. This upregulation in RvD.sub.n-3 DPA coincides with an increase in platelet, monocyte and neutrophil activation during the morning hours. Circadian regulation of these pro-resolving mediators is controlled by the neurotransmitter ACh that is, in turn, also diurnally regulated in plasma of healthy volunteers. In CVD patients, significantly lower RvD.sub.n-3 DPA was found as compared with healthy volunteers. A failure in the upregulation of these molecules during the early morning hours was also found that was linked with a decrease in plasma ACh concentrations and increased peripheral blood leukocyte activation. Incubation of whole blood from both patients and healthy volunteers with RvD2.sub.n-3 DPA or RvD5.sub.n-3 DPA significantly reversed leukocyte and platelet activation. In addition, administration of RvD5.sub.n-3 DPA to Apo E.sup./ mice using a therapeutic paradigm reduced systemic platelet and leukocyte activation and vascular disease. Together these findings indicate that disruption in the ACh-RvD.sub.n-3 DPA axis may result in CVD.
[0318] Plasma RvD.sub.n-3 DPA concentrations were found to increase during the early morning hours (
[0319] Furthermore, RvD.sub.n-3 DPA regulated reduced leukocyte and platelet responses in peripheral blood from both healthy volunteers and patients, and RvD5.sub.n-3 DPA protected against vascular disease in Apo E.sup./ mice (
[0320] Plasma concentrations of the RvD.sub.n-3 DPA pathway marker, and 5-LOX product (Dalli et al. 2013), 7-HDPA were significantly reduced (
[0321] Results from the above examples demonstrate that the vascular levels of this neurotransmitter in healthy volunteers are diurnally regulated and increase during the early morning hours (
[0322] In summary, the above examples demonstrate a protective pathway that is centered on the diurnal regulation of vascular n-3 DPA-derived pro-resolving mediators. Increases in these molecules during the morning hours counter-regulate physiological platelet and leukocyte activation limiting systemic inflammation and potentially vascular disease. In patients with cardiovascular disease, there is a significant loss in the production of these molecules with an increase in peripheral blood cell activation leading to increased systemic inflammation and CVD, including risk of myocardial infarct. In line with this notion, RvD.sub.n-3 DPA reprogrammed circulating leukocyte and platelet activation, which in mice resulted in a significant reduction in vascular disease. Thereby, strategies to restore peripheral blood RvD.sub.n-3 DPA, including n-3 DPA supplementation that was recently shown to increase plasma RvD5.sub.n-3 DPA in healthy volunteers (Markworth J F et al. Divergent shifts in lipid mediator profile following supplementation with n-3 docosapentaenoic acid and eicosapentaenoic acid. FASEB J. 2016; 30(11):3714-3725) may present possible therapeutic options. In addition, therapeutics based on the RvD.sub.n-3 DPA may provide new opportunities for fine-tuning the increased inflammatory status present in these patients, dampening systemic inflammation and reducing vascular disease.
Example 7: Assessment of the Efficacy of a Medicament for the Treatment or Prevention of Cardiovascular Disease
[0323] A method of assessing the efficacy of a medicament for use in the treatment or prevention of cardiovascular disease in an individual patient in accordance with the present invention is illustrated in
[0324] Step 10 indicates the start of the method. First, before the start of treatment with the medicament, a first biological sample is taken from the patient (step 20). In the present example, the biological sample is a plasma sample, but in other embodiments, the sample may be whole blood or serum taken from the patient. The sample is taken from the patient in the early hours of the morning between 7 AM and 9 AM. In the present example, the sample is taken from the patient at 8 AM.
[0325] The patient is then started on a course of treatment with the medicament (step 30).
[0326] The medicament may be any suitable medicament for the treatment or prevention of cardiovascular disease. In particular, the medicament may be a statin, a fibrate, a calcium channel blocker or combinations of medicaments such, for example, as a combination of a statin and calcium channel blocker. Other suitable cardiovascular treatments will be known to those skilled in the art. A suitable medicament is selected by a medical practitioner based on the patient's medical history and symptoms.
[0327] Suitable statins include simvastatin, fluvastatin, atorvastatin, rosuvastatin, pravastatin, lovastatin.
[0328] Suitable fibrates include gemfibrozil, fenofibrate, clofibrate, bezafibrate, ciprofibrate, clinofibrate, clofibride, ronifibrate and simfibrate.
[0329] Suitable calcium channel blockers include amlodipine, aranidipine, azelnidipine, barnidipine, benidipine, cilnidipine, clevidipine, efonidipine, felodipine, isradipine, lacidipine, lercanidipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipine and pranidipine.
[0330] The medicament is administered (including self-administration) to the patient in accordance with the medical practitioner's prescription. Typically, the medicament may be administered one or more times per day.
[0331] After a prescribed period of time after commencing treatment with the medicament, a second biological sample is taken from the patient (step 40). In the present example, the prescribed period of time is 24 hours, but other time periods may be used in different embodiments. Typically, the prescribed period of time may between 1 and 14 or 30 days. In any event, the period of time should be sufficiently long to allow the pharmacological effects of the medicament to manifest themselves in the patient.
[0332] The second sample is also taken from the patient in the early hours of morning at the same time of day as the first sample, i.e. 8 AM in the present example.
[0333] In step 50, the first and second samples taken from the patient in steps 20 and 40, before and after commencing treatment with the medicament, are analysed to quantify the levels of at least one n-3 DPA-derived resolvin (RvD.sub.n-3 DPA) in the samples.
[0334] In the present example, the levels of three n-3 DPA-derived resolvins (RvD1.sub.n-3 DPA, RvD2.sub.n-3 DPA and RvD5.sub.n-3 DPA) are measured in the first and second samples by reverse phase liquid chromatography electrospray tandem mass spectrometry (LC-MS/MS). In different embodiments of the invention, fewer than three of the n-3 DPA-derived resolvins may be analysed, i.e. one or two of the n-3 DPA-derived resolvins. Details of this method of quantitating the levels of the n-3 DPA-derived resolvins in the first and second samples are disclosed in Colas R A et al. 2014 and Dalli et al. 2015, the contents of which are incorporated herein by reference.
[0335] For each of the first and second plasma samples, venous blood (10 mL) is collected in heparin from the patient. Plasma is obtained by centrifugation of heparinised blood (2000 g, 10 minutes) and placed in 4 volumes of methanol before solid-phase extraction as described below.
[0336] Internal labelled standards 5S-HETE-d.sub.8, LTB.sub.4-d.sub.4, LXA.sub.4-d.sub.5, RvD2-d.sub.5 and PGE.sub.2-d.sub.4 (500 pg each) in 4 mL of ice-cold methanol are added to each sample to facilitate quantification and sample recovery. Next, samples are held at 20 C. For 45 minutes to allow protein precipitation and then centrifuged (2000 g, 4 C., 10 minutes). Supernatants are collected and brought to less than 1 mL of methanol content in a gentle stream of nitrogen onto an automated evaporation system with the water bath set to 37 C. and a nitrogen feed with a flow rate of no more than 15 psi. The samples are then centrifuged (2000 g, 4 C., 10 minutes). Samples are then placed in an automated extraction system with the water bath set to 37 C. and a nitrogen feed with a flow rate of no more than 15 psi and products extracted as follows.
[0337] Solid-phase C18 cartridges are washed with 3 mL of methanol and 6 mL of H.sub.2O. 9 mL H.sub.2O (pH 3.5, HCl) is then added to the samples, and the acidified solutions are rapidly loaded onto the conditioned C18 columns that are washed with 4 mL of H.sub.2O to neutralise the acid. Next, 5 mL of hexane are added and the products are eluted with 9 mL methyl formate. Products are brought to dryness using the automated evaporation system and immediately suspended in methanol-water (50:50 vol/vol) for LC-MS/MS automated injections.
[0338] In the present example, for LC-MS/MS, an HPLC and autoinjector, paired with a triple quadrupole mass spectrometer fitted with a high dynamic range pulse counting system, is employed. Alternative suitable LC-MS/MS equipment is available to those skilled in the art. A C18 column is kept in a column oven maintained at 50 C., and the RvD lipid mediators are eluted with a mobile phase consisting of water containing 0.01% acetic acid as a solvent A and methanol containing 0.01% acetic acid as solvent B. The column is equilibrated with mobile phase at 80:20 (A:B) which is ramped to 50:50 (A:B) over 12 seconds. This gradient is maintained for two minutes and then ramped to 80:20 (A:B) over the next 9 minutes. This gradient is then maintained for the next 3.5 minutes, before ramping to 98:2 (A:B). Finally, this gradient is maintained for 5.4 minutes to wash the column. The flow rate is maintained at 0.5 mL/min throughout the process.
[0339] The mass spectrometer is operated in negative ionisation mode using scheduled multiple reaction monitoring (MRM) coupled with information-dependent acquisition and an enhanced product line scan. The scheduled MRM window is 90 seconds, and each lipid mediator parameter is optimised individually.
[0340] The identity of each RvD.sub.n-3 DPA (n-3 DPA-derived resolvin) is confirmed by matching its retention time (RT) to synthetic and authentic materials (
TABLE-US-00011 TABLE 11 Diagnostic ions for RvD.sub.n-3 DPA RvD Q1 Q3 RvD1.sub.n-3 DPA 377 215 RvD2.sub.n-3 DPA 377 261 RvD5.sub.n-3 DPA 361 263
[0341] Calibration curves are obtained for each using authentic compound mixtures and deuterium labelled lipid mediators at 3.12, 6.25, 12.5, 25, 50, 100 and 200 pg. Linear calibration curves are obtained for each LM, which gives r.sup.2 values of 0.98-0.99. Internal standard recoveries, interference of the matrix, and limit of detection are determined.
[0342] Following quantitation of the levels of the RvDs in each of the first and second samples, the levels are compared (step 60).
[0343] A significant increase in the levels of the RvDs in the second sample as compared to the first sample indicates that the medicament administered to the patient may be effective in treating or preventing cardiovascular disease, particularly cardiovascular disease that is mediated by vascular inflammation as a result of dysfunctional diurnal control over pro-inflammatory mediators. On the other hand, no increase in the levels of the RvDs in the second sample as compared with the first sample may indicate that the administered medicament is ineffective in the individual patient.
[0344] Based on these results, the medicament may be prescribed to the patient if it is indicated as being effective (step 80). Alternatively, the method may be repeated using a different medicament.
[0345] Step 90 indicates the end of the method.
Example 8
[0346] Further examples of different aspects of the present invention are described below with reference to
[0347] As best shown in
[0348] The PDMS layer is moulded with a plurality of micro-channels 120a-c which terminate at one end at a sample collector port 125 and at another end at a waste drain 130. In the present example, the microfluidic device 110 has three micro-channels 120a-c, one for measuring the level of a different respective n-3 DPA-derived resolvin (RvD1.sub.n-3 DPA, RvD2.sub.n-3 DPA, RvD5.sub.n-3 DPA) in the blood sample B. In variants of the present example, the microfluidic device may have fewer than three micro-channels, for instance one or two micro-channels for measuring the levels of a corresponding number of RvD.sub.n-3 DPA.
[0349] Between the sample collector port 125 and the waste drain 130, each of the micro-channels 120a-c comprises a reaction zone 150a-c. Suitably, the micro-channels 120a-c may be serpentine in the reaction zone 150a-c to promote mixing of the sample B and reagents added to the device as described below.
[0350] Intermediate the sample collection port 125 and its reaction zone 150a-c, each microchannel 120 a-c is provided with a respective reagent inlet port 140a-c for admitting a series of different reagents into the channels 120a-c for mixing with the sample B. The micro-channels 120a-c and inlet ports 140a-c are provided with suitable micro-valves or the like for controlling the flow of the sample and reagents.
[0351] In each reaction zone 150a-c, a surface of each microchannel 120a-c is coated with a monoclonal antibody to a different respective one of RvD.sub.n-3 DPA to be quantitated in the sample B.
[0352] Adjacent each reaction zone 150a-c, the device 110 incorporates hydrogenated amorphous silicon (a-Si:H) photodiodes 175a-c on the glass slide 114. The photodiodes 175a-c are connected to a suitable interface 120, which is connected to a first computer 200 as shown in
[0353] The interface 120 is arranged to receive signals from the photodiodes 175a-c and to transmit computer-readable data to the first computer 200 representing those signals. The interface 120 may be physically connected to the first computer 200 by a suitable data cable. Alternatively, the interface 120 may be connected wirelessly to the first computer 200 by any suitable wireless data transfer method such, for example, as Bluetooth. In some embodiments, the first computer may comprise a handheld device.
[0354] The first computer 200 comprises a microprocessor, a memory and a storage device, and is arranged to execute software for storing data representing the signals received from the photodiodes 175a-c in association with patient identity data. Where the first computer 200 is a handheld device, the software may be an App.
[0355] The first computer 200 is connected via a suitable data communication channel 300 to a remote second computer 400. In the present embodiment, the data communication channel 300 comprises the Internet, but in other embodiments, the first and second computers 200, 400 may be interconnected on a local or wide area network (not shown). The first and second computers 200, 400 may be physically to each other connected via data communication cables, or they may be interconnected wirelessly using a suitable wireless data communication technology such, example, as IEEE 802.11 a,b,g,n or Bluetooth. Suitably, each of the first and second computers 200, 400 is connected to the Internet 300 through a suitable modem.
[0356] In use, a sample of blood B is obtained from a patient, for example using a conventional lancet.
[0357] The sample B is placed on the microfluidic device 110 at the sample inlet port 125. The sample is drawn into the micro-channels 120a-c by capillary action. In alternative embodiments, the sample B may be actively drawn into the micro-channels 120a-c using a micro-pump or under reduced pressure, etc.
[0358] In the reaction zones 150a-c, RvD.sub.n-3 DPA in the samples react with the antibodies coated on the surface of the micro-channels 120a-c. A different RvD.sub.n-3 DPA is captured in each reaction zone 150a-c. The sample B is incubated with the antibodies in the reaction zone for a suitable period of time. A wash solution is then introduced into the micro-channels 120a-c to remove unbound sample. Discarded material from the micro-channels 120a-c is removed from the device 110 via the drain 130.
[0359] After removing unbound sample from the reaction zone 150a-c, a second monoclonal antibody is introduced into each of the micro-channels 120a-c with specificity for the respective RvD.sub.n-3 DPA. Each of the second antibodies is tagged with horseradish peroxidase in the manner well known to those skilled in the art. The second monoclonal antibodies are allowed to incubate with the surface-captured RvD.sub.n-3 DPA in the reaction zones 150a-c. The micro-channels 120a-c are then washed again.
[0360] Next, a substrate for horseradish peroxidase is introduced into each of the micro-channels 120a-c via the inlet ports 140a-c. Suitable substrates are known to those skilled in the art, but in the present example luminol is used, which fluoresces when acted on by horseradish peroxidase. The fluorescence is detected by the photodiodes 175 giving rise to signals that are received by the interface 180. The intensity of the fluorescence is indicative of the amount of second antibody that is bound to the immobilised RvD.sub.n-3 DPA in each of the channels 120a-c. The microfluidic device 110 may be calibrated in a manner known to those skilled in the art so that the level of RvD.sub.n-3 DPA in each of the micro-channels 120a-c can be quantitated.
[0361] Data representing the intensity of fluorescence in each microchannel 120a-c is transmitted from the interface 180 to the first computer 200 as described above. The computer 200 executes the aforementioned software to calculate the level of each RvD.sub.n-3 DPA in the sample B from the intensity of fluorescence measured by the photodiodes 175.
[0362] The microfluidic device is then washed through again with a suitable washing agent.
[0363] In the present example, a first blood sample B.sup.1 is taken from the patient in the evening, for example between about 4 PM and 6 PM, and the levels of the B.sup.1 are measured using the microfluidic device 110 as described above.
[0364] A second sample B.sup.2 is taken from the patient in the early hours of the following morning, for example between about 7 AM and 9 AM. The levels of the one or more RvD.sub.n-3 DPA in the sample B.sup.2 are measured using the same or a similar microfluidic device 110 as described above.
[0365] Data representing the levels of the one or more RvD.sub.n-3 DPA in the samples B.sup.1 and B.sup.2 are calculated and stored by the first computer 200.
[0366] Data representing the levels of the one or more RvD.sub.n-3 DPA in the first and second samples are then transmitted by the first computer 200 to the second computer 400 in association with information identifying the patient P.
[0367] The second computer 400 includes a microprocessor, memory and a storage device and is arranged to execute software for calculating the difference between the levels of the one or more RvD.sub.n-3 DPA in the first and second samples B.sup.1, B.sup.2.
[0368] Since, in a healthy individual, one would normally expect the plasma level of RvD.sub.n-3 DPA to be in the range 10-25 pg/mL in the early morning, with a minimum of around 5 pg/mL in the evening, if the difference in the levels of the RvD.sub.n-3 DPA between the first and second samples B.sup.1, B.sup.2 is less than about 5 pg/mL, the patient may be assessed to be at risk of cardiovascular disease, and data indicating this is transmitted from the second computer back to the first computer 200 where it is saved and/or displayed to a person carrying out the test.
[0369] Similarly, if the level of RvD.sub.n-3 DPA in the patient's blood in the sample B.sup.2 taken early in the morning is less than around 10 pg/mL, particularly if it is less than around 5 pg/mL, this may indicate that the patient is at risk of cardiovascular disease or myocardial infarction.
[0370] On the basis of the comparison of the RvD.sub.n-3 DPA levels in the first and second samples B.sup.1, B.sup.2, the patient may be prescribed a suitable medicament for the treatment or prevention of cardiovascular disease.
[0371] In the present example, the microfluidic device 110 is arranged to carry out a non-competitive, heterogeneous ELISA sandwich immunoassay. However, in variants of the invention, a microfluidic device may be arranged to carry out a homogeneous immunoassay and/or a competitive immunoassay.
[0372] For example, in one variant, each microchannel 120a-c may be coated on a surface within its respective reaction zone 150a-c with a respective RvD.sub.n-3 DPA (e.g. RvD1.sub.n-3 DPA, RvD2.sub.n-3 DPA or RvD5.sub.n-3 DPA) which is the same as the one in the sample B that is to be analysed in the respective reaction zone 150a-c. Intermediate the reaction zone 150a-c and the sample collection port 125, in each microchannel 120a-c the sample B may be mixed with a known amount of a primary antibody to the respective RvD.sub.n-3 DPA. The primary antibody is provided in excess, and remaining antibody will then subsequently react with the surface-bound RvD.sub.n-3 DPA in the reaction zone 150a-c, effectively in competition with the corresponding RvD.sub.n-3 DPA in the sample. After washing, a labelled secondary antibody is introduced into each reaction zone 150a-c through the inlet ports 140a-c which is specific for the respective primary antibody. As described above, the secondary antibody is tagged with an enzyme suitable for use in EIA such, for example, as horseradish peroxidase. The amount of secondary antibody remaining after reaction with the sample can then be measured by admitting a suitable substrate for horseradish peroxidase into the reaction zones 150a-c and measuring the intensity of the fluorescence or colour as described above.
[0373] A microfluidic device in accordance with the invention such, for example, as microfluidic device 110 described above provides a convenient device for performing the methods of the present invention in a point of care setting such, for example, as a healthcare clinic where there is no access to more sophisticated equipment such as LC-MS/MS which may only be found in large laboratories.
Example 9: Diurnal Changes in Peripheral Blood n-3 DPA-Derived SPM are Regulated by Acetylcholine
[0374] To investigate whether peripheral blood SPM concentrations are diurnally regulated we obtained plasma from healthy volunteers at distinct intervals during a 24 h period, with demographics as set out in Table 12 below:
TABLE-US-00012 TABLE 12 Healthy volunteers demographics Sex Age (years) Weight (Kg) BMI (Kg/m.sup.2) 3M/4F 34 4.1 65.6 11.3 23.2 3.0
[0375] In plasma from healthy volunteers we identified mediators from all four major essential fatty acid metabolomes, including the EPA derived E-series resolvins, n-3 DPA-derived resolvins and protectins, DHA-derived protectins and maresins and the arachidonic acid (AA)-derived prostaglandins and leukotrienes. These mediators were identified in accordance with published criteria (Dalli J, Colas R A, Serhan C N. Novel n-3 immunoresolvents: structures and actions. Sci Rep. 2013; 3:1940) as illustrated for RvD5.sub.n-3 DPA (
TABLE-US-00013 TABLE 13 Diurnal lipid mediator profiles in healthy volunteer peripheral blood. Peripheral blood was collected from healthy volunteers at the indicated intervals. Plasma was placed in ice- cold methanol and lipid mediators (LM) were assessed using LM-profiling (see methods for details). Q1, M-H (parent ion) and Q3, diagnostic ion in the MS-MS (daughter ion). Results are mean s.e.m. and expressed as pg/mL. n = 7 volunteers per interval. The detection limit was ~0.1 pg. , Below limits of detection * P < 0.05 vs 18:00 h values using paired Mann-Whitney test. DHA bioactive Healthy volunteer plasma Lipid mediators concentration (pg/mL) metabolome Q1 Q3 18:00 7:00 9:00 12:00 15:00 RvD1 375 141 0.8 0.4 0.8 0.3 1.4 0.4 0.8 0.3 0.8 0.2 RvD2 375 141 0.9 0.4 0.9 0.2 0.6 0.3 0.3 0.2 0.3 0.2 RvD3 375 147 0.1 0.0 0.6 0.4 0.4 0.2 0.2 0.1 0.1 0.0 RvD4 375 101 0.3 0.2 0.3 0.2 0.3 0.2 1.3 0.8 0.8 0.6 RvD5 359 199 0.6 0.2 0.3 0.2 0.3 0.1 0.5 0.2 0.5 0.1 RvD6 359 101 0.6 0.2 0.4 0.1 0.7 0.4 0.7 0.2 0.8 0.3 17R-RvD1 375 141 0.2 0.1 0.4 0.2 0.3 0.2 0.4 0.2 0.4 0.1 17R-RvD3 375 147 0.1 0.1 0.2 0.1 0.1 0.1 0.1 0.1 0.2 0.1 PD1 359 153 0.6 0.2 0.7 0.4 0.8 0.3 1.0 0.4 1.0 0.6 17R-PD1 359 153 0.6 0.3 0.4 0.3 0.4 0.2 0.3 0.2 0.3 0.2 10S,17S-diHDHA 359 153 0.6 0.2 1.4 0.4 1.1 0.6 0.7 0.4 0.7 0.2 22-OH-PD1 375 153 0.8 0.4 1.3 0.7 1.1 0.5 1.4 0.7 2.7 2.1 MaR1 359 221 1.0 0.3 0.6 0.2 1.2 0.2 1.1 0.4 1.0 0.4 7S,14S-diHDHA 359 221 1.1 0.4 1.0 0.3 0.6 0.4 1.0 0.3 0.8 0.4 4S,14S-diHDHA 359 101 11.3 8.5 8.5 5.7 7.8 4.8 9.1 5.6 8.1 5.5 n-3 DPA bioactive metabolome RvD1.sub.n-3 DPA 377 143 1.9 0.4 10.9 4.2* 7.5 2.3* 6.0 1.8 1.6 0.3 RvD2.sub.n-3 DPA 377 261 2.3 1.3 1.8 1.0 2.5 1.1 1.7 1.2 1.1 0.7 RvD5.sub.n-3 DPA 361 263 2.6 1.2 2.6 1.2 4.5 0.3* 2.3 1.2 3.2 2.2 PD1.sub.n-3 DPA 361 183 1.1 0.3 2.3 0.5 1.6 0.3 1.6 0.3 1.1 0.3 MaR1.sub.n-3 DPA 361 249 1.7 0.7 3.5 1.9 3.2 1.4 0.9 1.0 1.3 0.6 RvT1 377 193 0.1 0.1 0.6 0.2 0.3 0.1 0.4 0.2 RvT2 377 143 0.3 0.2 0.5 0.3 0.5 0.3 0.5 0.3 0.3 0.2 RvT3 377 255 RvT4 359 193 2.0 0.8 2.6 1.4 3.0 1.3 1.4 0.6 1.4 0.6 EPA bioactive metabolome RvE1 349 195 3.9 1.5 3.7 1.5 4.5 1.5 4.9 1.9 4.9 2.0 RvE2 333 199 2.3 0.6 1.9 0.7 2.5 0.8 2.3 0.8 2.7 1.1 RvE3 333 201 1.2 0.3 1.4 0.5 1.6 0.4 1.4 0.6 1.3 0.6 AA bioactive metabolome LXA.sub.4 351 217 0.3 0.1 0.8 0.4 0.7 0.3 0.6 0.2 0.6 0.2 LXB.sub.4 351 221 0.9 0.4 0.8 0.3 0.2 0.2 0.5 0.2 0.6 0.2 5S,15S-diHETE 335 235 11.7 3.6 9.2 3.5 19.0 11.2 8.9 2.4 8.5 3.0 15epi-LXA4 351 217 7.0 3.1 8.3 5.5 4.2 1.2 4.6 1.2 4.2 1.1 15epi-LXB4 351 221 1.7 0.9 1.0 0.2 1.5 0.4 0.5 0.2 2.3 1.0 LTB.sub.4 335 195 2.0 0.5 2.5 0.7 2.6 1.2 1.8 0.6 1.9 0.5 5S,12S-diHETE 335 195 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 20-OH-LTB.sub.4 351 195 0.1 0.0 0.3 0.2 0.3 0.1 0.1 0.1 0.2 0.1 PGD.sub.2 351 189 5.8 1.7 7.5 1.5 4.7 0.4 6.6 1.8 7.6 1.6 PGE.sub.2 351 189 5.9 0.8 8.7 3.6 4.4 1.2 5.3 1.2 6.5 1.5 PGF.sub.2 353 193 6.1 1.5 11.8 2.5 7.0 1.3 6.3 0.9 8.9 0.7 TxB.sub.2 369 169 233.6 149.7 364.5 176.9 92.3 34.2 183.6 100.0 415.8 305.8
TABLE-US-00014 TABLE 14 ACh regulation of n-3 DPA metabolome in human whole blood. Peripheral blood from healthy volunteers was collected and incubated with ACh (0.1 M; 45 min; 37oC). Incubations were quenched with ice-cold methanol and n-3 DPA-derived LM identified and quantified using LM-profiling (see methods for details). Q1, M-H (parent ion) and Q3, diagnostic ion in the MS-MS (daughter ion). Results are expressed as pg/mL, mean s.e.m, n = 9 donors per group. * p < 0.05 vs Vehicle group using paired Mann-Whitney test. n-3 DPA bioactive Lipid mediators concentration (pg/mL) metabolome Q1 Q3 Vehicle ACh 0.1 M RvD1.sub.n-3 DPA 377 143 3.4 1.8 4.9 2.8 RvD2.sub.n-3 DPA 377 261 2.8 1.2 7.4 2.6* RvD5.sub.n-3 DPA 361 201 0.8 0.7 1.5 0.6 PD1.sub.n-3 DPA 361 155 0.4 0.2 0.5 0.1 MaR1.sub.n-3 DPA 361 223 0.3 0.2 0.9 0.3* RvT1 377 211 0.3 0.3 2.0 1.3 RvT2 377 197 1.2 0.8 1.6 0.9 RvT3 377 255 1.2 0.9 1.4 0.9 RvT4 359 211 0.5 0.3 1.0 0.4
[0376] These diurnal changes in RvD.sub.n-3 DPA were also associated with a circadian regulation of leukocyte and platelet activation that reaches a maximum between 7:00 and 9:00 h coincident with an increase in RvD.sub.n-3 DPA concentrations (
[0377] We next investigated the mechanism(s) by which peripheral blood n-3 DPA derived SPM may be regulated. Here we found that plasma ACh concentrations mirrored those of the RvD.sub.n-3 DPA reaching a maximum at 7:00 h (
[0378] Of note, this increase was not linked with a selective mobilization of n-3 DPA in peripheral blood, see Table 15:
TABLE-US-00015 TABLE 15 Peripheral blood SPM substrate and precursor concentrations. n-3 DPA bioactive Lipid mediators precursors concentration (pg/mL) metabolome Q1 Q3 Vehicle ACh 0.1 M 17HDHA 343 245 101.9 26.7 103.6 25.9 14HDHA 343 205 205.8 47.3 229.2 41.2 7HDHA 343 141 9.1 3.3 8.6 3.7 4HDHA 343 101 20.4 5.47 19.9 5.1 DHA 327 283 23117.7 7852.2 28104.0 10103.4 18HEPE 317 259 33.8 11.3 36.2 12.2 15HEPE 317 219 35.3 13.6 31.3 8.7 12HEPE 317 179 382.6 116.1 402.4 106.0 5HEPE 317 115 45.5 11.7 42.7 11.3 EPA 301 257 5371.5 514.6 5867.2 713.2 15HETE 319 219 287.6 88.1 336.4 95.2 12HETE 319 179 4751.4 1271.4 5148.0 1153.2 5HETE 319 115 61.2 5.5 69.9 13.5 AA 303 259 20584.4 4222.3 24205.0 4783.4* 17-HDPA 345 247 64.4 14.9 58.9 13.2 14-HDPA 345 207 112.6 25.5 137.2 25.8* 13-HDPA 345 193 1.4 0.4 1.9 0.6 7-HDPA 345 143 56.4 19.3 44.9 10.7 DPA 327 283 3703.3 703.0 3873.1 706.3 Peripheral blood from healthy volunteers was collected and incubated with acetylcholine (ACh) at 0.1 M for 45 min. Plasma was isolated, placed in ice cold methanol containing deuterium labelled internal standards and SPM precursors together with their pathway markers were extracted, identified and quantified using lipid mediator (see methods for details). Q1, M-H (parent ion) and Q3, diagnostic ion in the MS-MS (daughter ion). Results are expressed as pg/mL. Mean SEM of n = 9 per condition. *p < 0.05 vs Vehicle group using paired Mann-Whitney test.
[0379] We next investigated whether changes in sheer rate, associated with an increase in blood pressure may also regulate RvD.sub.n-3 DPA production. Blood perfusion at 0.3 pascals (Pa), which is associated with an increase in platelet leukocyte aggregates (n=4 donors), led to a significant increase in plasma RvD.sub.n-3 DPA when compared with blood perfused at a sheer rate of 0.1 Pa (
TABLE-US-00016 TABLE 16 SPM concentrations in peripheral blood incubations with cortisol. n-3 DPA bioactive Lipid mediators concentration (pg/mL) metabolome Q1 Q3 Vehicle Cortisol 1 M Cortisol 10 M RvD1.sub.n-3 DPA 377 143 1.4 1.1 1.9 1.2 1.7 1.5 RvD2.sub.n-3 DPA 377 261 1.8 1.3 1.0 0.9 1.2 0.8 RvD5.sub.n-3 DPA 361 201 1.9 0.3 1.1 0.6 1.7 0.3 PD1.sub.n-3 DPA 361 155 0.9 0.2 1.5 1.1 2.5 1.5 MaR1.sub.n-3 DPA 361 223 0.9 0.5 0.3 0.2 0.2 0.2 RvT1 377 211 0.7 0.3 0.5 0.2 0.6 0.2 RvT2 377 197 0.1 0.1 0.1 0.1 0.1 0.0 RvT3 377 255 3.9 2.5 1.6 0.5 2.2 1.5 RvT4 359 211 2.1 1.3 2.7 1.5 3.0 1.8 17-HDPA 345 247 73.3 20.8 74.9 18.2 71.5 17.5 14-HDPA 345 207 282.0 39.0 286.2 91.0 241.0 40.9 13-HDPA 345 193 3.3 0.7 3.2 0.7 3.0 0.6 7-HDPA 345 143 239.7 95.6 268.9 83.5 257.8 109.6 DPA 327 283 21213.7 7408.2 24568.2 6300.4 28823.5 8019.0 Peripheral blood from healthy volunteers was collected and incubated with cortisol (1-10 M) or vehicle for 45 min. Plasma was obtained, placed in ice cold methanol containing deuterium labelled internal standards and lipid mediators were extracted, identified and quantified using lipid mediator profiling (see methods for details). Q1, M-H (parent ion) and Q3, diagnostic ion in the MS-MS (daughter ion). Results are expressed as pg/mL. Mean SEM of n = 5 per condition.
Example 10: RvD.SUB.n-3 DPA .Reduce Leukocyte and Platelet Activation in Peripheral Blood
[0380] We next investigated the actions of RvD.sub.n-3 DPA in regulating monocyte, neutrophil and platelet activation as well as platelet-leukocyte aggregates in light of the pathogenic roles played by cellular activation in cardiovascular disease (Furman M I, Barnard M R, Krueger L A, et al. Circulating monocyte-platelet aggregates are an early marker of acute myocardial infarction. J Am Coll Cardiol. 2001; 38(4):1002-1006; Pfluecke C, Tarnowski D, Plichta L, et al. Monocyte-platelet aggregates and CD11b expression as markers for thrombogenicity in atrial fibrillation. Clin Res Cardiol. 2016; 105(4):314-322; Huo Y, Schober A, Forlow S B, et al. Circulating activated platelets exacerbate atherosclerosis in mice deficient in apolipoprotein E. Nat Med. 2003; 9(1):61-67). Incubation of human peripheral blood with RvD2.sub.n-3 DPA led to dose dependent decreases in neutrophil CD11b expression and in neutrophil-platelet aggregates measured as decreases in neutrophil CD62P (
Example 11: Reduced RvD.SUB.n-3 DPA .Correlate with Peripheral Blood Cell Activation in Patients with Cardiovascular Disease
[0381] We next investigated whether results obtained with healthy volunteers were translatable to the clinical setting. Given that RvD.sub.n-3 DPA increased during the early morning hours, a time window associated with higher incidence of myocardial infarct (Nakashima H, Mashimo Y, Kurobe M, Muto S, Furudono S, Maemura K. Impact of Morning Onset on the Incidence of Recurrent Acute Coronary Syndrome and Progression of Coronary Atherosclerosis in Acute Myocardial Infarction. Circ J. 2017; 81(3):361-367; Muller J E, Stone P H, Turi Z G, et al. Circadian variation in the frequency of onset of acute myocardial infarction. N Engl J Med. 1985; 313(21):1315-1322), we next investigated the peripheral blood levels of RvD.sub.n-3 DPA in patients with cardiovascular diseases (CVD) that were also at an increased risk of myocardial infarct. Details and methods for risk criteria are set out in Table 17 below:
TABLE-US-00017 TABLE 17 CVD-demographics and clinical data Participants 9 Age (years) 65.2 8.6 Sex 7 Male, 2 Female CRP mg/L 35.4 42.2 IL-6 pg/ml 2.5 1.0 TNF- pg/ml 108.2 74.9 Creatine mol/L 119.1 90.5 LDL mmol/L 3.0 0.2 HDL mmol/L 0.5 0.1 Type II Diabetes 3 Hypertension 9 Current Smoking 0 Obese n 4 Previous AMI 1 Previous PCI 4 LVEF 50% 4 Aspirin (n) 9 Statins (n) Atorvastatin (4), Simvastatin (3) and Rosuvastatin (1) Other medications (n) Allopurinol (1), Amitriptyline (2), Amlodipine (2), Apixaban (1), Bisoprolol (6), Candesartan (1), Citalopram (1), Clopidogrel (2), Codeine (1), Cyanacobalamin (1), Dorzolamide (1), Doxazosin (2), Enoxaparin (1), Fentanyl (1), Finasteride (1), Flucloxacillin (1), Fluoxetine (2), Furosemide (2), Isosorbide mononitrate (1), Lansoprazole (4), Lantus Insulin (1), Lisinopril (1), Metformin (1), Nicorandil (1), NoroRapid Insulin (1), Omeprazole (3), Paracetamol (1), Phyllocontine (1), Priadel (1), Ramipril (5), Salbutamol (1), Salmeterol (1), Sertraline (1), Setagliptin (1), Tamoxifen (1), Tamsulosin (2) Temazepam (1), Thiamine (1), Tildiem (1), Timolol (1), Tioropium bromide (1), Warfarin (1), Xalatan (1).
[0382] Using lipid mediator profiling we found significant decreases in morning (9:00 h), midday (12:00 h) and evening (16:00-18:00 h) plasma RvD.sub.n-3 DPA concentrations in CVD patients when compared to the respective values in healthy volunteers (
TABLE-US-00018 TABLE 18 Peripheral blood LM profiles in patients with CVD. Plasma from CVD patients Lipid mediators concentration (pg/mL) Q1 Q3 PM AM Midday DHA bioactive metabolome RvD1 375 141 1.0 0.5 0.6 0.5 0.5 0.2 RvD2 375 141 0.3 0.1 0.2 0.2 0.2 0.1* RvD3 375 147 0.2 0.1 0.4 0.2 0.1 0.1 RvD4 375 101 1.8 0.5 2.5 1.2 2.3 1.6 RvD5 359 199 1.8 0.4 2.1 0.9 0.7 1.3 RvD6 359 101 0.3 0.2 0.3 0.1 0.2 0.2 17R-RvD1 375 141 0.5 0.3 0.5 0.2 1.2 0.9 17R-RvD3 375 147 0.3 0.3 0.3 0.2 1.2 0.8 PD1 359 153 0.3 0.1 0.8 0.2* 0.5 0.5 17R-PD1 359 153 0.4 0.3 0.2 0.1 10S,17S-diHDHA 359 153 0.3 0.2 0.1 0.1 22-OH-PD1 375 153 2.4 2.3 0.1 0.1 0.4 0.3 MaRl 359 221 0.8 0.4 0.9 0.5 1.0 0.7 7S,14S-diHDHA 359 221 0.2 0.2 0.8 0.5 4S,14S-diHDHA 359 101 0.6 0.3 0.4 0.2 0.1 0.1* n-3 DPA bioactive metabolome RvD1.sub.n-3 DPA 377 143 1.1 0.2 1.4 0.4 0.5 0.3* RvD2.sub.n-3 DPA 377 261 1.0 0.4 1.7 0.6 0.5 0.2 RvD5.sub.n-3 DPA 361 263 0.9 0.3 0.9 0.4 1.0 0.6 PD1.sub.n-3 DPA 361 183 0.3 0.1 0.2 0.1 0.7 0.2 MaR1.sub.n-3 DPA 361 249 0.3 0.2 0.6 0.7 RvT1 377 193 0.1 0.1 0.3 0.2 RvT2 377 143 0.3 0.2 RvT3 377 255 0.2 0.1 0.9 0.3 0.2 0.2 RvT4 359 193 0.4 0.2 0.3 0.1 EPA bioactive metabolome RvE1 349 195 2.5 2.2 1.8 1.2 0.4 0.2 RvE2 333 199 0.5 0.4 0.2 0.1 0.1 0.0 RvE3 333 201 1.5 0.9 2.1 0.8 1.7 1.9 AA bioactive metabolome LXA.sub.4 351 217 0.1 0.1 0.4 0.3 LXB.sub.4 351 221 1.1 0.4 1.1 0.6 0.5 0.4* 5S,15S-diHETE 335 235 18.4 5.6 15.8 7.9 8.1 3.0 15epi-LXA.sub.4 351 217 4.6 2.6 1.6 0.6 2.3 1.6 15epi-LXB.sub.4 351 221 13.5 5.3 20.5 7.6 5.5 2.6 LTB.sub.4 335 195 2.2 0.4 2.1 0.6 1.1 0.6 5S,12S-diHETE 335 195 0.3 0.1 0.8 0.5 0.2 0.2 20-OH-LTB.sub.4 351 195 0.4 0.2 0.2 0.1 0.1 0.1 PGD.sub.2 351 189 5.7 2.5 2.6 0.6 5.5 2.4 PGE.sub.2 351 189 13.3 3.6 9.6 2.4* 20.3 9.2 PGF.sub.2 353 193 9.1 4.0 7.4 1.9 14.1 13.7 TxB.sub.2 369 169 36.2 23.9 26.6 20.0 86.1 93.5 Peripheral blood from CVD patients was collected at 9:00 h (AM) 12:00 h (Midday) and between 16:00-18:00 h (PM). Plasma was placed in ice-cold methanol containing internal standards. Lipid mediators (LM) were extracted, identified and quantified using LM-profiling (see methods for details). Q1, M-H (parent ion) and Q3, diagnostic ion in the MS-MS (daughter ion). Results are mean s.e.m. and expressed as pg/mL. n = 9 paired patients. The detection limit was ~0.1 pg. , Below limits of detection *p < 0.05 vs PM values using paired Mann-Whitney test.
[0383] Furthermore, there was a marked impairment in the diurnal regulation of these mediators in CVD patients, where morning RvD.sub.n-3 DPA concentrations were only slightly but not significantly elevated compared with evening values (
[0384] Flow cytometric analysis of peripheral blood leukocyte from these patients demonstrated increases in the expression of CD11b on both neutrophils and monocytes from CVD patients when compared with healthy volunteers (
[0385] In order to establish the mechanisms leading to the downregulation of RvD.sub.n-3 DPA biosynthesis we next assessed the expression of the RvD.sub.n-3 DPA biosynthetic enzymes in peripheral blood leukocytes. Flow cytometric assessment of peripheral blood myeloid cells from both healthy volunteers demonstrated that myeloid cell expression of both 15-LOX and 5-LOX was upregulated early in the morning (
Example 12: Elevated Plasma Adenosine Reduces RvD.SUB.n-3 DPA .Biosynthesis in CVD Patients
[0386] Given that we found a significant reduction in 7-HDPA concentrations in peripheral blood from CVD patients (
Example 13: Reduced Leukocyte Activation by RvD2.SUB.n-3 DPA .and RvD5.SUB.n-3 DPA .in Patient Peripheral Blood
[0387] In order to test whether there was a relationship between the increased systemic inflammation and reduced n-3 DPA derived SPM we next investigated whether RvD.sub.n-3 DPA regulated patient peripheral blood leukocyte responses. RvD2.sub.n-3 DPA dose-dependently decreased platelet-neutrophil and platelet-monocyte aggregates without significantly regulating CD11b expression (
[0388] We next tested whether the actions of these two mediators were also retained in the presence of PAF (Shinohara M, Kibi M, Riley I R, et al. Cell-cell interactions and bronchoconstrictor eicosanoid reduction with inhaled carbon monoxide and resolvin D1. Am J Physiol Lung Cell Mol Physiol. 2014; 307(10):L746-757; Palur Ramakrishnan A V, Varghese T P, Vanapalli S, Nair N K, Mingate M D. Platelet activating factor: A potential biomarker in acute coronary syndrome? Cardiovasc Ther. 2017; 35(1):64-70). Incubation of patient whole blood with either RvD2.sub.n-3 DPA or RvD5.sub.n-3 DPA led to decreases in platelet-neutrophil and platelet-monocyte aggregates measured as decreases in CD62P (
Example 14: RvD5.SUB.n-3 DPA .Reduces Systemic Leukocyte and Platelet Activation and Protects Against Vascular Disease in ApoE/ Mice
[0389] We next investigated whether the protective actions of RvD5.sub.n-3 DPA observed with peripheral blood cells from both healthy volunteers and CVD patients were also retained in vivo. For this purpose ApoE.sup./ mice were fed western diet for 6 weeks and RvD5.sub.n-3 DPA (100 ng/mouse; i.v.) was administered on alternative days for a two-week period. RvD5.sub.n-3 DPA administration reduced circulating platelet monocyte-aggregates, as measured by a decrease in both CD41 and CD62P expression on CD115 positive cells, and monocyte activation with a decrease in CD11b expression (
[0390] Since platelet-leukocyte aggregates are involved in the pathogenesis of atherosclerosis (Huo Y, Schober A, Forlow S B, et al. Circulating activated platelets exacerbate atherosclerosis in mice deficient in apolipoprotein E. Nat Med. 2003; 9(1):61-67) we next investigated whether RvD5.sub.n-3 DPA also protected against vascular disease. LC/MS-MS analysis of aortic sections from mice given RvD5.sub.n-3 DPA demonstrated distinct lipid mediator profiles when compared with mice given vehicle. This was characterized by a significant upregulation of DHA and AA derived SPM including MaR1 and 15-epi-LXA4 (
TABLE-US-00019 TABLE 19 RyD5.sub.n-3 DPA administration upregulated SPM and reduced pro-inflammatory eicosanoids in aortic tissues from ApoE.sup./. ApoE.sup./ WD ApoE.sup./ WD + RvD5.sub.n-3 DPA DHA Bioactive Metabolome Q1 Q3 (pg/10 mg aorta) (pg/10 mg aorta) RvD1 375 233 3.7 1.1 1.7 0.9* RvD2 375 215 0.7 0.7 0.4 0.4 RvD3 375 147 5.7 3.9 2.4 0.6 RvD4 375 225 0.2 0.1 RvD5 359 199 0.3 0.1 0.4 0.2 RvD6 359 159 1.0 0.8 1.5 1.1 17R-RvD1 375 215 0.6 0.1 1.6 0.9 17R-RvD3 375 147 1.2 0.7 1.3 0.3 PD1 359 153 2.1 0.7 2.6 0.7 10S,17S-diHDHA 359 153 32.2 6.0 66.0 35.4 17R-PD1 359 137 0.8 0.4 0.3 0.2 22-OH-PD1 375 153 7.2 1.7 6.0 1.0 MaR1 359 177 17.7 2.3 35.4 22.7* 7S,14S-diHDHA 359 177 0.3 0.3 1.8 0.3* 4,14-diHDHA 359 159 3.5 2.1 4.9 2.0 n-3 DPA Bioactive Metabolome RvT1 377 211 RvT2 377 255 RvT3 377 173 RvT4 361 193 RvD1.sub.n-3 DPA 377 215 0.1 0.1 0.1 0.1 RvD2.sub.n-3 DPA 377 261 0.1 0.1 RvD5.sub.n-3 DPA 361 263 0.1 0.1 0.1 0.1 PD1.sub.n-3 DPA 361 183 0.1 0.1 0.0 0.0 MaR1.sub.n-3 DPA 361 223 EPA Bioactive Metabolome RvEl 349 161 1.8 0.6 1.8 0.5 RvE2 333 159 1.2 1.3 0.1 0.1 RvE3 333 201 0.3 0.2 0.7 0.1 AA Bioactive Metabolome LXA.sub.4 351 115 0.4 0.2 0.3 0.0 LXB.sub.4 351 221 1.3 0.8 0.6 0.4 5S,15S-diHETE 335 235 35.6 24.0 51.9 15.3 15-epi-LXA.sub.4 351 115 6.2 2.2 9.6 1.9* 15-epi-LXB.sub.4 351 221 4.6 4.3 1.4 0.5 LTB.sub.4 335 195 1.4 0.3 1.7 0.3 5S,12S-diHETE 335 195 1.3 0.4 0.6 0.2 20-OH-LTB.sub.4 351 195 PGE.sub.2 351 189 24.1 2.2 20.5 4.5* PGD.sub.2 351 189 18.5 2.9 14.9 3.5 PGF.sub.2 353 193 10.8 0.7 10.0 1.7 TxB.sub.2 369 169 46.9 6.5 34.5 4.1* ApoE.sup./ mice were fed a Western diet (WD) for 6 weeks. On week 4 mice were administered vehicle or 100 ng/mouse RvD5n-3 DPA (via i.v. injection) on alternative days. Descending aortas were harvested and placed in ice-cold methanol containing internal standards. Lipid mediators (LM) were extracted, identified and quantified using LM-profiling (see methods for details). Q1, M-H (parent ion) and Q3, diagnostic ion in the MS-MS (daughter ion). Results are mean s.e.m. and expressed as pg/10 mg tissue. n = 4 mice per group. *p < 0.05 vs Vehicle mice using Mann-Whitney test.
[0391] We also found significant reductions in aortic prostanoids with concentrations of TxB2 being reduced by >35% in mice given RvD5.sub.n-3 DPA (
[0392] In the present studies we uncovered a novel host protective mechanism centered on the diurnal regulation of systemic RvD.sub.n-3 DPA in healthy volunteers. Disruption in the production of these mediators correlated with increased peripheral blood leukocyte and platelet activation in patients with CVD. It is now widely appreciated that physiological processes including cardiovascular function and the immune system are under the control of a molecular clock that oscillates following a diurnal pattern (Ingle K A, Kain V, Goel M, Prabhu S D, Young M E, Halade G V. Cardiomyocyte-specific Bmal1 deletion in mice triggers diastolic dysfunction, extracellular matrix response, and impaired resolution of inflammation. Am J Physiol Heart Circ Physiol. 2015; 309(11):H1827-1836; McAlpine C S, Swirski F K. Circadian Influence on Metabolism and Inflammation in Atherosclerosis. Circ Res. 2016; 119(1):131-141). In the vasculature, platelet activation is at a maximum during the early hours of the day with the upregulation of several activation markers including CD62P (Scheer F A, Michelson A D, Frelinger A L, 3rd, et al. The human endogenous circadian system causes greatest platelet activation during the biological morning independent of behaviors. PLoS One. 2011; 6(9):e24549). Of note, this increase in platelet activation is coincident with an increase in plasma plasminogen activator inhibitor-1, a serine protease inhibitor that functions as the principal inhibitor of tissue plasminogen activator and urokinase, thereby increasing the risk of thrombosis (Sakata K, Hoshino T, Yoshida H, et al. Circadian fluctuations of tissue plasminogen activator antigen and plasminogen activator inhibitor-1 antigens in vasospastic angina. Am Heart J. 1992; 124(4):854-860).
[0393] Platelet CD62P mediates platelet-leukocyte interactions, a process that in addition to facilitating leukocyte recruitment to the vascular endothelium is also involved in leukocyte activation and the production of inflammatory mediators including cysteinyl leukotrienes (Shinohara M, Kibi M, Riley I R, et al. Cell-cell interactions and bronchoconstrictor eicosanoid reduction with inhaled carbon monoxide and resolvin D1. Am J Physiol Lung Cell Mol Physiol. 2014; 307(10):L746-757), tumor necrosis factor- and CC motif ligand-2 (Furman M I, Barnard M R, Krueger L A, et al. Circulating monocyte-platelet aggregates are an early marker of acute myocardial infarction. J Am Coll Cardiol. 2001; 38(4):1002-1006; Pfluecke C, Tarnowski D, Plichta L, et al. Monocyte-platelet aggregates and CD11b expression as markers for thrombogenicity in atrial fibrillation. Clin Res Cardiol. 2016; 105(4):314-322). CD62P enhances platelet adhesion to endothelial cells expressing fratelkine, and triggers the release of Weibel-Palade-bodies in endothelial cells, thus perpetuating the pro-inflammatory and pro-thrombotic status during the early hours of the day. In addition, platelet-leukocyte aggregates are implicated in vascular disease pathogenesis, including atherosclerosis (Huo Y, Schober A, Forlow S B, et al. Circulating activated platelets exacerbate atherosclerosis in mice deficient in apolipoprotein E. Nat Med. 2003; 9(1):61-67). Thus, these observations suggest that in healthy individuals endogenous, diurnally regulated, protective mechanisms are engaged that counterregulate this physiological inflammation to prevent vascular inflammation and thrombus formation. In the present study, we found that plasma RvD.sub.n-3 DPA concentrations increase during the early morning hours (
[0394] In patients with CVD we found a significant decrease (3 fold lower) in peripheral blood RvD.sub.n-3 DPA during the early morning hours that was also observed at other intervals during the day. This reduction in RvD.sub.n-3 DPA concentrations was associated with an increased leukocyte and platelet activation suggesting that RvD.sub.n-3 DPA are endogenous protective signals that control physiological platelet and leukocyte activation. This is further supported by the observation that RvD.sub.n-3 DPA reduced leukocyte and platelet activation in peripheral blood from both healthy volunteers and patients. RvD5.sub.n-3 DPA reduced platelet-leukocytes aggregates in vivo and modulated vascular lipid mediator profiles reducing concentrations of the pro-thrombogenic mediator TxA.sub.2 (measured as its metaboliteTxB.sub.2) and upregulating the formation of pro-resolving mediators including MaR1 and 15-epi-LXA.sub.4. Furthermore RvD5.sub.n-3 DPA also decreased early aortic lesions in ApoE.sup./ mice (
[0395] Tissue pro-resolving mediator biosynthesis is also regulated by the vagus nerve via release of the neurotransmitter ACh, a mechanism that is central in maintaining tissue resolution tone (Dalli J, Colas R A, Arnardottir H, Serhan C N. Vagal Regulation of Group 3 Innate Lymphoid Cells and the Immunoresolvent PCTR1 Controls Infection Resolution. Immunity 2017; 46(1):92-105). Results from the present study demonstrate that the vascular levels of this neurotransmitter in healthy volunteers are diurnally regulated and increase during the early morning hours (
[0396] In summation, the present findings uncover a novel protective pathway centered on the diurnal regulation of vascular n-3 DPA-derived resolvins. Increases in these molecules during the morning hours counterregulate physiological platelet and leukocyte activation limiting systemic inflammation and potentially vascular disease thereby ensuring vascular homeostasis. In patients with cardiovascular disease, there is a significant loss in both production and circadian regulation of these molecules that is associated with an increase in peripheral blood cell activation leading to increased systemic inflammation and susceptibility to myocardial infarct. In line with this notion, RvD.sub.n-3 DPA reprogrammed circulating leukocyte and platelet activation, which in mice resulted in a significant reduction in vascular disease. Thereby, strategies to restore peripheral blood RvD.sub.n-3 DPA, including n-3 DPA supplementation that was recently shown to increase plasma RvD5.sub.n-3 DPA in healthy volunteers, may be a useful therapeutic option. In addition, therapeutics based on the RvD.sub.n-3 DPA may also provide new opportunities for fine-tuning the increased inflammatory status present in these patients, dampening systemic inflammation and reducing vascular disease.