Method and apparatus for determining the efficacy of statins for treating inflammatory diseases in individual patients

Abstract

A method, device, computer program and related immunoassay are disclosed for assessing the efficacy of a statin selected from, for example, selected from RvT1 (7,13,20-trihydroxy-8,10,14,16Z,18-docosapentaenoic acid), RvT2 (7,12,13-trihydroxy-8,10,14,16Z,19Z-docosapentaenoic acid), RvT3 (7,8,13-trihydroxy-9,11,14,16Z,19Z-docosapentaenoic acid) and RvT4 (7,13-dihydroxy-8,10,14,16Z,19Z-docosapentaenoic acid), for use in the treatment of an inflammatory condition in an individual patient, which comprises measuring the levels of at least one 13-series resolvin in biological samples obtained from the patient before and after administration of the statin, wherein an increase in the level of the resolvin after administration of the statin is indicative of efficacy of the statin. Also disclosed is a method of storing a biological sample to preserve lipid mediators in the sample comprising placing the sample in an organic solvent and storing the sample at a temperature of ≤−75° C.

Claims

1. A method of treating an inflammatory condition in a patient with a statin, the method comprising assessing the efficacy of the statin for use in the treatment of the inflammatory condition in the patient wherein the inflammatory condition is cardiovascular disease (CVD) or rheumatoid arthritis, wherein said assessing comprises measuring the levels of at least one 13-series resolvin in biological samples obtained from the patient before and after administration of the statin, wherein an increase in the level of the resolvin after administration of the statin is indicative of efficacy of the statin, and administering the statin to the patient.

2. A method as claimed in claim 1, wherein the at least one 13-series resolvin is selected from RvT1 (7,13,20-trihydroxy-8,10,14,16Z,18-docosapentaenoic acid), RvT2 (7,12,13-trihydroxy-8,10,14,16Z,19Z-docosapentaenoic acid), RvT3 (7,8,13-trihydroxy-9,11,14,16Z,19Z-docosapentaenoic acid) and RvT4 (7,13-dihydroxy-8,10,14,16Z,19Z-docosapentaenoic acid).

3. A method as claimed in claim 2, wherein the levels of two or more of the 13-series resolvins in the biological samples are measured.

4. A method as claimed in claim 2, wherein the levels of three or all four of the 13-series resolvins in the biological samples are measured.

5. A method as claimed in claim 1, wherein the samples are blood, serum or plasma samples.

6. A method as claimed in claim 1, wherein the levels of the at least one 13-series resolvin in the samples are measured using liquid chromatography tandem mass spectrometry (LC-MS/MS).

7. A method as claimed in claim 6, wherein one or more internal labelled standards are added to the samples and quantitation is carried out using linear regression curves constructed using said one or more labelled standards.

8. A method as claimed in claim 1, wherein the levels of the at least one 13-series resolvin in the samples are measured using an immunoassay.

9. A method as claimed in claim 8, wherein the immunoassay is an enzyme immunoassay (EIA).

10. A method as claimed in claim 8, wherein the immunoassay is competitive or non-competitive.

11. A method as claimed in claim 1, wherein the statin is selected from atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin.

12. A method as claimed in claim 1, wherein the sample obtained from the patient after administration of the statin is taken at least 30 minutes after administration of the statin.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In the drawings:

(2) FIGS. 1A-1F: Increased RvT in paws from mice given atorvastatin and pravastatin during inflammatory arthritis. Arthritogenic K/B×N serum (100 μL, i.p.) was given to mice to initiate disease and (A) disease progression was monitored daily using clinical scores. Arrows denote days when mice were given statins or vehicle. (B-D) 0.2 mg/Kg atorvastatin, pravastatin, simvastatin or vehicle (DPBS containing 0.05% ethanol) were given i.v. on days 3, 5 and 7 after disease onset. Paws were collected on day 8 and lipid mediators were identified and quantified using lipid mediator profiling. (B) Representative MRM (multiple reaction monitoring) chromatograms of identified lipid mediators derived from docosahexaenoic acid, n-3 docosapentaenoic acid, eicosapentaenoic acid and arachidonic acid. (C) MS/MS spectra employed in the identification of RvT1, RvT2, RvT3 and RvT4. (D) Percent regulation of RvT1, RvT2, RvT3 and RvT4 compared to vehicle. (E) Representative MRM chromatograms of identified RvT. (F) Quantification of total RvT, RvT1, RvT2, RvT3 and RvT4 compared to vehicle. Results for A, B and E are representative of n=12 mice, for C and F are mean±s.e.m.; n=4 mice per group. * p<0.05 and ** p<0.01 vs. vehicle using one-way ANOVA with post hoc Dunnett's multiple comparisons test. Results are mean±s.e.m.; n=9 for vehicle, 11 for atorvastatin, 11 for pravastatin and 9 for simvastatin treated mice from 4 independent experiments. * p<0.05 vs. vehicle.

(3) FIGS. 2A and 2B: Pravastatin dose-dependently increased RvT in human neutrophil-endothelial cell co-incubations. HUVEC (8.5×10.sup.5 cells/cm.sup.2) were incubated with IL-1β (10 ng/mL) and TNF-α (10 ng/mL) for 16 h. These were then incubated with the indicated concentrations of atorvastatin, pravastatin or vehicle (DPBS containing 0.05% ethanol) for 30 minutes, then neutrophils (4×10.sup.6 cells/well) were added. Incubations were quenched after 1 h with 2 volumes of ice cold methanol and RvT were identified and quantified using lipid mediator profiling. (A) Representative MRM chromatograms of identified RvT. (B) RvT1, RvT2, RvT3 and RvT4 regulation compared with vehicle-treated incubations. Results are mean of 4 healthy donors from 4 independent experiments. *p<0.05 and **p<0.01 vs. vehicle using two-way ANOVA with post hoc Tukey's multiple comparisons test.

(4) FIGS. 3A-3E: Atorvastatin and pravastatin reduced disease severity and protected joint architecture. Arthritogenic K/B×N serum was given to mice on days 0 and 2. Disease progression was monitored using a 26-point clinical score in mice given (A) atorvastatin (0.2 mg/Kg), (B) pravastatin (0.2 mg/Kg), (C) simvastatin (0.2 mg/Kg) or vehicle (DPBS containing 0.05% ethanol) on days 3, 5 and 7. Results are mean±s.e.m.; n=8 for vehicle, 10 for atorvastatin, 10 for pravastatin and 6 for simvastatin treated mice from 3 independent experiments. ** p<0.01 and *** p<0.005 vs. vehicle using ordinary two-way ANOVA (analysis of variance). (D) Maximum percentage increase in midfoot pad thickness. Results are mean±s.e.m.; n=8 for vehicle, 10 for atorvastatin, 10 for pravastatin and 6 for simvastatin treated mice from 3 independent experiments. * p<0.05 vs. vehicle using one-way ANOVA with post hoc Dunnett's multiple comparisons test. (E) Representative H&E-stained knee sections of mice (×4 magnification) collected on day 8 using EVOS FL imaging system. Results are representative of n=8 for vehicle, 10 for atorvastatin, 10 for pravastatin and 6 for simvastatin treated mice from 3 independent experiments. F, femur; T, tibia; m, meniscus; IFP, infrapatellar fat pad; PF, pannus formation. Arrows denote leukocyte infiltration.

(5) FIGS. 4A-4D: Differential regulation of circulating leukocyte and platelet activation by each of the statins in inflammatory arthritis. Serum-induced arthritis was initiated in mice and atorvastatin, pravastatin, simvastatin (0.2 mg/Kg each) or vehicle (DPBS containing 0.05% ethanol) were given on days 3, 5 and 7. On day 8 blood was collected. Leukocyte subsets and activation were identified using fluorescently labelled antibodies and flow cytometry. Activation markers on (A) non-classical monocytes, (B) classical monocytes, (C) neutrophils and (D) platelets were assessed as percentage decrease from vehicle. Results are mean±s.e.m.; n=9 for vehicle, 11 for atorvastatin, 11 for pravastatin and 9 for simvastatin treated mice from 4 independent experiments. * p<0.05 vs. vehicle using one-way ANOVA with post hoc Dunnett's multiple comparisons test.

(6) FIGS. 5A-5C: Reduction of monocyte, neutrophil and macrophage activation as well as trafficking to the joint by atorvastatin and pravastatin in inflammatory arthritis. Serum-induced arthritis was initiated in mice and atorvastatin, pravastatin, simvastatin (0.2 mg/Kg each) or vehicle (DPBS containing 0.05% ethanol) were given on days 3, 5 and 7. Front paws were collected on day 8 and digested to liberate infiltrating leukocytes. Leukocyte subsets were defined using antibodies against specific markers and flow cytometry. Trafficking and activation of (A) non-classical monocytes, (B) neutrophils, (C) monocyte-derived macrophages were assessed. Results are mean±s.e.m.; n=9 for vehicle, 11 for atorvastatin, 11 for pravastatin and 9 for simvastatin treated mice from 4 independent experiments. * p<0.05 vs. vehicle using one-way ANOVA with post hoc Dunnett's multiple comparisons test.

(7) FIGS. 6A-6D: Inhibition of RvT production by celecoxib reverses the joint protective actions of atorvastatin and pravastatin. Inflammatory arthritis was initiated using arthritogenic serum (see methods for details). On days 3, 5 and 7 and mice were administered celecoxib (10 mg/Kg) or vehicle (DPBS containing 0.05% ethanol) and after 1 hour given (A) atorvastatin (0.2 mg/Kg), (B) pravastatin (0.2 mg/Kg) or vehicle (PBS containing 0.05% ethanol). Disease activity was assessed daily * p<0.05 vs. vehicle using ordinary two-way ANOVA. (C) On day 8 paws were collected and RvT were identified and quantified using LC-MS-MS based lipid mediator profiling. * p<0.05 and ** p<0.01 vs. atorvastatin or pravastatin alone using one-way ANOVA with post hoc Sidak's multiple comparisons test. (D) Representative H&E-stained knee sections of mice (×4 magnification) collected on day 8 using EVOS FL imaging system. Results are mean±s.e.m.; n=9 for vehicle, 11 for atorvastatin, 11 for pravastatin, 7 for celecoxib plus atorvastatin and 6 for celecoxib plus pravastatin treated mice per group from 2-3 independent experiments.

(8) FIGS. 7A-7F: COX-2 inhibition reverses the protective actions of atorvastatin on both systemic and joint leukocytes. Serum-induced arthritis was initiated, on days 3, 5 and 7 and mice were administered celecoxib (10 mg/Kg) or vehicle (DPBS containing 0.05% ethanol) and after 1 hour given atorvastatin (0.2 mg/Kg) or vehicle (DPBS containing 0.05% ethanol). Blood was collected on day 8 and leukocyte subsets and activation were identified using fluorescently labelled antibodies and flow cytometry. (A-C) Activation markers on circulating (A) non-classical monocytes, (B) neutrophils and (C) platelets. Results are presented as percentage decrease from vehicle. (D-F) Leukocytes recovered from the inflamed paws (see methods for details) on day 8. Trafficking and activation profile for (D) non-classical monocytes (E) neutrophils, and (F) monocyte-derived macrophages were assessed using flow cytometry. Results are mean±s.e.m.; n=9 for vehicle, 11 for atorvastatin, 11 for pravastatin, 7 for celecoxib plus atorvastatin treated mice from 2 independent experiments. * p<0.05 vs. vehicle; #p<0.05 vs atorvastatin using one-way ANOVA with post hoc Dunnett's multiple comparisons test.

(9) FIG. 8 is a flow diagram illustrating an example of a method of assessing the efficacy of a statin (atorvastatin) for treating inflammation in an individual patient in accordance with the present invention.

(10) FIG. 9 is a schematic diagram of apparatus for carrying out methods of the present invention using a microfluidic device to carry out an immunoassay.

(11) FIG. 10 is a schematic diagram of a microfluidic device according to the present invention which incorporates an immunoassay of the invention.

(12) FIG. 11 shows the results of experiments comparing the stability of lipid mediators over extended periods of time when stored at −80° C. under nitrogen and methanol respectively.

EXAMPLES

Example 1

(13) Animals

(14) Male C57BL/6 mice (11 weeks old) were procured from Charles River (Kent, UK). All animals were provided with standard laboratory diet and water ad libitum and kept on a 12 h light/dark cycle.

(15) Inflammatory Arthritis

(16) The mice were administered K/BxN serum (100 μL, i.p.) on days 0 and 2 to initiate inflammatory arthritis. The mice were then given atorvastatin, pravastatin, simvastatin (0.2 mg/Kg each) or vehicle (DPBS−/− containing 0.05% ethanol) via i.v. injection on days 3, 5 and 7. Clinical scores were monitored daily using a 26-point arthritic scoring system. Swelling and redness of ankles/wrists, pads and digits of mice were inspected daily as described in Norling L V, Headland S E, Dalli J, et al. “Proresolving and cartilage-protective actions of resolvin D1 in inflammatory arthritis”. JCI Insight. 2016; 1(5):e85922. Blood and paws were collected at the indicated time intervals.

(17) In select experiments, mice were given 10 mg/Kg celecoxib 1 hour prior to statin injections. Blood and paws were collected either on day 8 after arthritis onset or 2 h after statin injection on day 7.

(18) Lipid Mediator Profiling

(19) Ice-cold methanol containing 500 pg of each deuterated (d) internal standard: d.sub.8-SSHydroxyeicosatetraenoic, d.sub.4-Leukotriene (LT) B.sub.4, d.sub.5-Lipoxin (LX) A.sub.4, d-Prostaglandin (PG) E.sub.2 and d.sub.5-Resolvin D.sub.2, was added to samples. Lipid mediators were extracted and profiling conducted as described in Dalli J et al. 2015 (ibid), Colas R A et al. 2014 (ibid) and Rathod K S, Kapil V, Velmurugan S, et al. “Accelerated resolution of inflammation underlies sex differences in inflammatory responses in humans”. J Clin Invest. 2017; 127(1):169-182.

(20) Flow Cytometry

(21) Whole blood was collected using heparin-lined syringes via cardiac puncture. Cells were incubated with Fc-blocking IgG and fluorescent-labelled antibodies for 45 minutes on ice. Cells were washed and incubated with 0.1% Live/Dead stain for 30 minutes on ice. Red blood cells were lysed and fixed using Whole Blood Lysing Reagent Kit. Staining was then evaluated using a flow cytometry analyser and analysed using suitable software.

(22) Paws were harvested and leukocytes isolated as described in Dalli J et al. 2015 (ibid). Briefly, paws were incubated in RPMI-1640 (containing 0.5 μg/mL collagenase D and 40 g/mL DNAse I) at 37° C. for 30 minutes with vigorous agitation. Isolated cells were passed through a 70 μM strainer and suspended in RPMI-1640 containing 2 U/mL penicillin, 100 mg/mL streptomycin and 10% FBS, then centrifugated at 400×g, 10 minutes. Isolated cells were suspended in DPBS−/− containing 0.02% BSA and 1% Fc-blocking IgG (v/v), and incubated with 0.1% Live/Dead stain for 20 minutes on ice. Cells were washed using DPBS−/− and incubated with fluorescent-labelled antibodies for 45 minutes on ice. These were then washed and fixed using 1% paraformaldehyde. Absolute counting beads were used for leukocyte enumeration. Staining was then evaluated using a flow cytometry analyser and analysed using suitable software.

(23) Human Neutrophil—Endothelial Cell Isolation

(24) Umbilical cords were collected by the midwifery staff of the Maternity Unit, Royal London Hospital (protocol approved by East London and The City Health Authority Research Ethics Committee Number: 06/Q0605/40) and human umbilical vein endothelial cells were isolated as described in Gittens B R, Wright R D, Cooper D. Methods for assessing the effects of galectins on leukocyte trafficking. Methods Mol Biol. 2015; 1207:133-151. Cells were then incubated with Interleukin (IL)-1β and Tumour Necrosis Factor (TNF)-α (10 ng/mL each, 16 h, 37° C., 5% CO.sub.2).

(25) Neutrophils were isolated from blood of healthy consenting donors in accordance with the Declaration of Helsinki and Queen Mary Research Ethics Committee approved protocol (QMREC 2014:61). Incubations were conducted as described in Dalli J et al. 2015 (ibid).

(26) Statistics

(27) Results are presented as mean±s.e.m. Differences between groups were tested using GraphPad Prism 7 (GraphPad Software) and using one-way ANOVA with post hoc Dunnett's, Sidak's or Tukey's multiple comparisons test. Where appropriate one-sample t-test compared to normalized vehicle or two-way ANOVA were used. The criterion for statistical significance was p<0.05.

(28) Results

(29) Differential Regulation of Local and Systemic RvT by Atorvastatin, Pravastatin and Simvastatin During Inflammatory Arthritis

(30) It was first investigated whether atorvastatin regulated RvT formation during inflammatory arthritis and whether this action was unique to this statin or was shared with other clinically relevant statins, namely pravastatin and simvastatin. To test this, arthritogenic serum from K/BxN mice was administered on days 0 and 2. This serum leads to a Fcγ mediated immune response with a rapid onset and severe inflammatory arthritis.

(31) Mice were then given atorvastatin (0.2 mg/Kg), pravastatin (0.2 mg/Kg) simvastatin (0.2 mg/Kg) or vehicle in a therapeutic paradigm on days 3, 5 and 7-post serum administration, at a time where clinical signs of disease were observed (FIG. 1A).

(32) Plasma and paws were collected 24 h after the last statin dose, and lipid mediators were identified and quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based lipid mediator profiling. In paws from arthritic mice mediators from all four major bioactive metabolomes were identified, including D-series resolvins and RvT (FIG. 1B, C). These mediators were identified in accordance with published criteria including matching retention times to authentic or synthetic standards (FIG. 1B) and at least 6 ions in the MS-MS as described in Colas R A et al. 2014 (ibid) (FIG. 1C).

(33) Using multiple reaction monitoring the concentrations of mediators identified in these paws were quantified. Here it was found that in joints from mice receiving atorvastatin there was a 43% increase in overall RvT amounts that was due to increased RvT1, RvT2, RvT3 and RvT4 when compared with paws from vehicle treated mice (FIG. 1D). Of note, the concentrations of these mediators were within their described bioactive ranges as reported in Dalli J et al. 2015.

(34) Pravastatin also increased paw RvT by ˜20% with increases in joint RvT1 and RvT2 concentrations, whereas simvastatin did not significantly increase joint RvT concentrations (FIG. 1D).

(35) Assessment of plasma mediator levels demonstrated decreases in LTB.sub.4, PGD.sub.2, PGE.sub.2, PGF.sub.2α and TxB.sub.2 in mice given either pravastatin or atorvastatin. In these mice, an increase in plasma levels of RvT concentrations were also found, with RvT1 being increased by both atorvastatin and pravastatin whereas RvT4 was only increased by atorvastatin.

(36) Given that statins are rapidly cleared from the circulation, with a half-life for atorvastatin of ˜14 h and pravastatin of ˜3 h, it was next investigated whether systemic regulation of RvT biosynthesis by these statins was more pronounced immediately after dosing. For this purpose, arthritis was initiated using K/B×N serum and mice were given atorvastatin and pravastatin as described above. Blood was then collected 2 h after the last statin dose on day 7 and lipid mediators were identified and quantified using LM profiling.

(37) In plasma from mice given atorvastatin a significant increase (>200%) in RvT was found, with increases in RvT1, RvT2 and RvT3 when compared to vehicle treated mice (FIGS. 1E and 1F). In these mice, decreased circulating amounts of inflammation-initiating eicosanoids were found, including PGD.sub.2 and PGE.sub.2. Assessment of plasma LM profiles from mice given pravastatin also demonstrated a marked increase (>100%) in peripheral blood RvT with RvT1 demonstrating the highest increase when compared with vehicle treated mice (FIGS. 1E and 1F). In these mice, decreases in circulating PGD.sub.2 (˜46%) and PGE.sub.2 (˜29%) were also observed.

(38) Together these results demonstrate that atorvastatin and pravastatin increase both joint and plasma RvT and decrease systemic inflammation during inflammatory arthritis.

(39) Given that in the vasculature RvT are produced during neutrophil endothelial interactions, it was next questioned whether the increased RvT observed in murine systems by pravastatin were also translatable to humans. For this human neutrophil-endothelial cell co-cultures with pravastatin were incubated and its ability to regulate RvT was assessed.

(40) Here it was found that pravastatin dose-dependently upregulated the concentrations of all four RvT to a similar extent as that observed by atorvastatin (FIGS. 2A and 2B).

(41) Atorvastatin and Pravastatin Reduce Joint Inflammation and Protect Against Leukocyte Mediated Tissue Damage

(42) It was next investigated whether atorvastatin and pravastatin at doses that increased RvT also reduced joint inflammation. Arthritis was initiated and mice were treated and disease progression monitored as described above. In mice given vehicle, signs of disease were observed as early as day 2, disease severity reached a maximum at day 6 with a score of 11.9±0.9 after which the disease activity plateaued to day 7 (FIG. 3A).

(43) When mice were given atorvastatin disease progression was dampened as early as day 4 (1 day after treatment initiation), with disease scores reaching a maximum score of 9.1±1.2 at day at 5. This reduction in disease activity was sustained through to day 7 (FIG. 3A).

(44) Similarly, when mice were given pravastatin disease activity at day 5 was found to be lower when compared to mice given vehicle alone with a ˜23% reduction in disease activity that was maintained through to day 7 (FIG. 3B).

(45) Administration of simvastatin at equal doses to that of atorvastatin and pravastatin did not significantly reduce disease activity (FIG. 3C). These findings were also reflected in the extent of paw swelling where atorvastatin and pravastatin reduced joint swelling as measured by a decrease in midfoot pad thickness (FIG. 3D).

(46) It was next assessed whether atorvastatin, pravastatin and simvastatin displayed joint protective actions. Haemotoxylin and eosin (H&E) stained sections of knee joints from mice given atorvastatin and pravastatin demonstrated reduced leukocyte infiltration, pannus formation and joint damage when compared with vehicle treated mice, whereas these parameters appeared to be unaltered in mice given simvastatin (FIG. 3E).

(47) Together these findings demonstrate that atorvastatin and pravastatin are more potent than simvastatin at regulating local inflammation and protecting from leukocyte mediated joint damage in inflammatory arthritis.

(48) Decreased Leukocyte Activation in Joints and Blood from Arthritic Mice by Atorvastatin and Pravastatin

(49) To ascertain whether these statins regulated systemic inflammation in inflammatory arthritis, the levels of platelet-leukocyte aggregates in peripheral blood from arthritic mice were assessed, given the relationship of these heterotypic aggregates and cellular activation with CVD.

(50) Using flow cytometry it was found that atorvastatin regulated the expression of CD11b on both non-classical and classical monocytes as well as platelet-monocyte aggregates, measured by a decrease in CD62P (FIGS. 4A and B) and CD41 expression (n=9 mice) on these monocyte subsets.

(51) Atorvastatin administration also regulated neutrophil and platelet responses, significantly reducing neutrophil CD11b expression, platelet-neutrophil aggregates (FIG. 4C) and decreasing platelet CD62P expression by ˜60% (FIG. 4D), when compared with mice given vehicle alone. Similar findings were made with peripheral blood leukocytes and platelets from mice given pravastatin, where there was a reduction in monocyte CD11b expression (˜10% for non-classical and ˜40% for classical monocytes), platelet leukocyte aggregates (˜35% for both monocyte subsets and ˜25% for neutrophils) and platelet activation, with a reduction of ˜70% in CD62P expression (FIG. 4). Simvastatin did not significantly regulate leukocyte CD11b expression, whereas heterotypic aggregates formed by classical monocytes and platelets and platelet CD62P expression were reduced (FIG. 4). These results demonstrate that atorvastatin and pravastatin regulate systemic inflammation dampening circulating monocyte, neutrophil and platelet activation during inflammatory arthritis.

(52) It was next tested whether these actions also translated to a regulation of leukocyte trafficking and activation in the joint.

(53) First, the trafficking of non-classical monocytes to the inflamed joints was assessed, given their role in disease onset and propagation of K/B×N serum initiated inflammatory arthritis.

(54) Flow cytometric analysis of leukocytes isolated from joints of mice given atorvastatin demonstrated a significant reduction in the total numbers of non-classical monocytes recruited to the joints (>60%). There also was a reduction in CD11b and a significant reduction in MHCII expression on these cells when compared to mice given vehicle alone (FIG. 5A). It was found that neutrophil trafficking was decreased in mice that received atorvastatin when compared to vehicle treated mice (FIG. 5B). In these mice, it was found that statin administration downregulated the expression of neutrophil CD11b and MHCII expression, although this did not reach statistical significance (FIG. 5B). Assessment of macrophage trafficking to the joints also demonstrated a significant reduction in the number of monocyte-derived macrophages as well as in the expression of activation markers CD11b and MHCII (FIG. 5C).

(55) Similar findings were also made with mice given pravastatin that reduced joint monocyte, neutrophil and macrophage numbers as well as activation profile (FIG. 5).

(56) Of note, although simvastatin regulated the expression of some of the activation markers on these cell subsets it did not significantly reduce leukocyte numbers in the paws when compared with mice given vehicle alone (FIG. 5).

(57) Together these findings demonstrate that pravastatin and atorvastatin also regulate joint leukocyte trafficking and activation in inflammatory arthritis.

(58) COX-2 Inhibition Reverses the Protective Actions of Atorvastatin and Pravastatin

(59) In order to assess the contribution of RvT in the protective actions exerted by atorvastatin and pravastatin, it was next investigated whether inhibition of COX-2, the initiating enzyme in the RvT pathway, reversed the protective actions of pravastatin and atorvastatin.

(60) Clinical scores of mice given celecoxib, a COX-2 selective inhibitor, immediately prior to atorvastatin were similar to those of mice receiving vehicle alone and higher than those of mice receiving atorvastatin (FIG. 6A).

(61) Similarly, celecoxib also blunted the anti-inflammatory actions of pravastatin measured by an increase in disease activity when compared with mice receiving the statin alone (FIG. 6B).

(62) This loss of protective actions of pravastatin and atorvastatin in mice given celecoxib was also associated with an ˜60% reduction in joint RvT and a reduction in RvT1 concentration that was >75% when compared to mice given pravastatin or atorvastatin alone (FIG. 6C).

(63) In these mice, a significant reversal of the joint protective actions of both statins was also found where in mice given celecoxib there was an increase in pannus formation and loss of joint architecture when compared to mice given each of the statin alone (FIG. 6D).

(64) It was next investigated whether COX-2 inhibition also reversed the leukocyte directed actions exerted by atorvastatin and pravastatin.

(65) Celecoxib administration blunted the protective actions of atorvastatin on circulating leukocytes and platelets, increasing blood platelet-monocyte and platelet-neutrophil aggregates as well as the expression of CD11b on both leukocyte subsets (FIGS. 7A and B). Celecoxib administration also increased the expression of CD62P on circulating platelets when compared to mice receiving atorvastatin alone (FIG. 7C). Inhibition of COX-2 reversed the actions of atorvastatin on leukocyte trafficking and activation in the joint, increasing the numbers of non-classical monocytes (FIG. 7D), neutrophils (FIG. 7E) and monocyte-derived macrophages (FIG. 7F) recruited to the inflamed joints. Expression of activation markers on these leukocytes was also increased following celecoxib inhibition when compared with mice given atorvastatin alone (FIGS. 7D-F).

(66) Similar findings were also made when systemic and joint leukocyte responses and trafficking in mice given celecoxib together with pravastatin were assessed. Here it was found that COX-2 inhibition returned the activation profile of circulating leukocytes and platelets to that observed in mice receiving vehicle alone. Similarly, leukocyte trafficking and activation in the joints was increased to levels that were similar to those found in vehicle treated mice.

(67) Together these findings demonstrate that inhibition of COX-2 reduces RvT production and abolished the joint and systemic protective actions of pravastatin and atorvastatin in inflammatory arthritis.

(68) These results demonstrate that both atorvastatin and pravastatin increase RvT production in inflammatory arthritis. Upregulation in tissue and blood concentrations of these SPM was associated with a reduction in joint disease activity as well as joint leukocyte trafficking and activation. In addition, both atorvastatin and pravastatin also decreased systemic inflammation reducing platelet, monocyte and neutrophil activation. The protective actions of these statins were reversed by inhibition of COX-2. Of note, simvastatin did not increase RvT and displayed blunted actions in regulation of joint disease and leukocyte responses.

(69) Together these findings establish the rank order potency of atorvastatin, pravastatin and simvastatin in regulating RvT and the role of these molecules in mediating the protective actions of these statins. Joint and systemic increases in these pro-resolving mediators also correlated with the ability of each of these three statins to dampen various aspects of local and systemic inflammation including oedema, leukocyte and platelet activation. Thus, these results establish a novel mechanism of action for atorvastatin and pravastatin in regulating inflammation in arthritis and provide novel functional biomarkers for measuring the efficacy of statins in controlling local and vascular inflammation in patients.

Example 2

(70) A method of assessing the efficacy of a statin for use in the treatment of an inflammatory condition in an individual patient in accordance with the present invention is illustrated in FIG. 8.

(71) Step 10 indicates the start of the method. First, a first suitable 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, or a suitable tissue sample.

(72) An amount of the statin to be tested is then administered to the patient (step 30). In accordance with the invention, any statin that is approved for use, either for marketing as a medicinal product or for use as investigational medicinal product (IMP) in clinical studies, may be administered. The statin may be administered according to its recommended initial or maintenance dose. Suitably, the statin may be administered according to its recommended initial dose. In the present example, atorvastatin is used at a dose of 10 mg or 20 mg. However, in other embodiments, a different statin may be used. The amount administered to the patient may be adjusted in accordance with clinical practice.

(73) After a prescribed period of time, a second biological sample is taken from the patient (step 40). In the present example, the prescribed period of time is 2-3 hours, but again, other time periods may be used in different embodiments. The period of time should be sufficiently long to allow the pharmacological effects of the statin to manifest themselves.

(74) In step 50, the first and second samples taken from the patient in steps 20 and 40, before and after administration of the statin, are analysed to quantify the levels of at least one 13-series resolvin (RvT) in the samples. In the present example, the levels of four 13-series resolvins (RvT1, RvT2, RvT3 and RvT4) 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 four of the 13-series resolvins may be analysed, i.e. one, two or three of the 13-series resolvins. Details of this method of quantitating the levels of the 13-series resolvins in the first and second samples are disclosed in Colas R A et al. 2014 (ibid) and Dalli et al. 2015 (ibid), the contents of which are incorporated herein by reference.

(75) 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.

(76) 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.

(77) 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.

(78) 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 RvT 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.

(79) 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.

(80) The identity of each RvT (13-series resolvin) is confirmed by matching its retention time (R.sub.T) to synthetic and authentic materials (FIG. 1B) and at least six diagnostic ions for each RvT (FIG. 1C) and quantified using multiple reaction monitoring of the parent ion (Q1) and characteristic daughter ion (Q3) as described in Table 2 below.

(81) TABLE-US-00002 TABLE 2 RvT Q1 Q3 RvT1 377 193 RvT2 377 215 RvT3 377 143 RvT4 361 193

(82) 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.

(83) Following quantitation of the levels of the RvTs in each of the first and second samples, the levels are compared (step 60).

(84) A significant increase in the levels of the RvTs in the second sample as compared to the first sample indicates that the statin administered to the patient may be effective in controlling inflammation. On the other hand, no increase in the levels of the RvTs in the second sample as compared with the first sample may indicate that the administered statin is ineffective in the individual patient (step 70).

(85) Based on these results, the statin may be prescribed to the patient if it is indicated as being effective (step 80). Alternatively, the method may be repeated, e.g., the following day, using a different statin.

(86) Step 90 indicates the end of the method.

Example 3

(87) Further examples of different aspects of the present invention are described below with reference to FIGS. 9 and 10 in which an immunoassay incorporated into a microfluidic device 110 is used for measuring the level of at least one 13-series resolvin (RvT) in a sample of blood B obtained from a patient P.

(88) As best shown in FIG. 10, the microfluidic device 110 has a general construction of the kind known to those skilled in the art. Specifically, the microfluidic device 110 comprises at least one layer of polydimethylsiloxane (PDMS) 112, a transparent, biocompatible elastomer that allows for channel inspection and optical signal acquisition, bonded to a glass slide 114.

(89) The PDMS layer is moulded with a plurality of micro-channels 120a-d 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 four micro-channels 120a-d, one for measuring the level of a different respective RvT (RvT1, RvT2, RvT3, RvT4) in the blood sample B. As in Example 2 above, in variants of the present example, the microfluidic device may have fewer than four micro-channels, for instance one, two or three micro-channels for measuring the levels of a corresponding number of RvTs.

(90) Between the sample collector port 125 and the waste drain 130, each of the micro-channels 120a-d comprises a reaction zone 150a-d. Suitably, the micro-channels 120a-d may be serpentine in the reaction zone 150a-d to promote mixing of the sample B and reagents added to the device as described below.

(91) Intermediate the sample collection port 125 and its reaction zone 150a-d, each microchannel 120 a-d is provided with a respective reagent inlet port 140a-d for admitting a series of different reagents into the channels 120a-d for mixing with the sample B. The micro-channels 120a-d and inlet ports 140a-d are provided with suitable micro-valves or the like for controlling the flow of the sample and reagents.

(92) In each reaction zone 150a-d, a surface of each microchannel 120a-d is coated with a monoclonal antibody to a different respective one of RvTs to be quantitated in the sample B.

(93) Adjacent each reaction zone 150a-d, the device 110 incorporates hydrogenated amorphous silicon (a-Si:H) photodiodes 175a-d on the glass slide 114. The photodiodes 175a-d are connected to a suitable interface 120, which is connected to a first computer 200. The interface 120 is arranged to receive signals from the photodiodes 175a-d 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.

(94) 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-d in association with patient identity data. Where the first computer 200 is a handheld device, the software may be an app.

(95) 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.

(96) In use, a sample of blood B is obtained from a patient, for example using a conventional lancet. 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-d by capillary action. In alternative embodiments, the sample B may be actively drawn into the micro-channels 120a-d using a micro-pump or under reduced pressure, etc.

(97) In the reaction zones 150a-d, RvTs in the samples react with the antibodies coated on the surface of the micro-channels 120a-d. A different RvT is captured in each reaction zone 150a-d. 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-d to remove unbound sample. Discarded material from the micro-channels 120a-d is removed from the device 110 via the drain 130.

(98) After removing unbound sample from the reaction zone 150a-d, a second monoclonal antibody is introduced into each of the micro-channels 120a-d with specificity for the respective RvT. 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 RvTs in the reaction zones 150a-d. The micro-channels 120a-d are then washed again.

(99) Next, a substrate for horseradish peroxidase is introduced into each of the micro-channels 120a-d via the inlet ports 140a-d. 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 RvT in each of the channels 120a-d. The microfluidic device 110 may be calibrated in a manner known to those skilled in the art so that the level of RvT in each of the micro-channels 120a-d can be quantitated.

(100) Data representing the intensity of fluorescence in each microchannel 120a-d 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 RvT in the sample B from the intensity of fluorescence measured by the photodiodes 175.

(101) The microfluidic device is then washed through again with a suitable washing agent.

(102) As described above in Example 2, a statin is then administered to the patient P. Suitably, the statin is administered according to its recommended initial or maintenance dose. Since details of administration of the statin have been described above, they are not repeated here.

(103) After a suitable period of time—for example 2-3 hours—a second sample B is obtained from the patient P and tested using another microfluidic device 110 that is similar to the one described above. The levels of the RvTs in the second sample are measured in the same way, and data representing the levels are calculated and stored by the first computer 200.

(104) Data representing the levels of RvTs 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.

(105) The second computer 400 includes a microprocessor, memory and a storage device and is arranged to execute software for comparing the levels of the RvTs in the first and second samples to determine whether or not the levels of RvTs in the second sample are increased (by a biologically relevant amount) as compared with the levels in the first sample. If the levels of the RvTs are increased in the second sample relative to the first sample, the statin is assessed to be effective for treating an inflammatory disorder in the patient P, 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. On the basis of the result of the comparison of the RvT levels in the first and second samples, the patient may be prescribed the statin. On the other hand, if the levels of RvTs in the second sample are not significantly increased relative to the levels in the first sample, the statin is assessed to be ineffective in the patient P for treating inflammatory condition. The test may then be repeated, e.g., the next day, with a different statin.

(106) 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.

(107) For example, in one variant, each microchannel 120a-d may be coated on a surface within its respective reaction zone 150a-d with a respective RvT (i.e. RvT1, RvT2, RvT3 or RvT4) which is the same as the one in the sample B that is to be analysed in the respective reaction zone 150a-d. Intermediate the reaction zone 150a-d and the sample collection port 125, in each microchannel 120a-d the sample B may be mixed with a known amount of a primary antibody to the respective RvT. The primary antibody is provided in excess, and remaining antibody will then subsequently react with the surface-bound RvT in the reaction zone 150a-d, effectively in competition with the corresponding RvT in the sample. After washing, a labelled secondary antibody is introduced into each reaction zone 150a-d through the inlet ports 140a-d 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-d and measuring the intensity of the fluorescence or colour as described above.

(108) 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 4: Methods for Enhancing the Stability of Lipid Mediators

(109) Human serum was either snap frozen and stored under nitrogen (Method 1 below) or placed in methanol containing deuterium labelled internal standards (Method 2). At the intervals indicated in FIG. 11, lipid mediators were extracted, and the concentrations of the indicated products were determined. These were then compared to published values (reference) were samples were only snap frozen without additional treatment. Results are mean of n=1 experiment and 3 determinations and are illustrated in FIG. 11.

(110) Method 1

(111) a) Prepare serum following appropriate methods b) Collect serum and transfer to appropriate container. c) Purge tube with nitrogen for an appropriate amount of time to replace air in the headspace above the serum with nitrogen. Note: This step needs to be performed immediately after sample collection and without exposing samples to temperatures above room temperature. d) Immediately snap-freeze the sample by placing in liquid nitrogen until frozen. e) Transfer tubes to appropriate container and store at −80° C. or lower. Note: Samples should not be thawed and refrozen at any point.
Method 2 a) To prepare methanol for each 1 mL of serum add 500 pg of each of the deuterium labelled internal standards to 4 ml of mass spectrometry grade methanol. b) Store at −20° C. for at least 1 h prior to use. c) Prepare serum following appropriate methods. d) Collect serum and transfer to appropriate container. e) Add 4 mL of methanol containing deuterium labelled internal standards per 1 mL of serum f) If samples are to be immediately processed these should be placed at −20° C. for at least 45 min prior to lipid mediator extraction g) If samples are to be stored these should be stored at −80° C. or lower.

REFERENCE METHOD

(112) Colas R A et al. 2014 (ibid).