Soluble epoxide hydrolase inhibitors for use in the treatment of chronic inflammatory diseases

Abstract

A first aspect of the invention relates to a method of treating or preventing tissue damage, or treating or preventing tissue destruction, in a subject, said method comprising administering to the subject a therapeutically or prophylactically effective amount of a soluble epoxide hydrolase (sEH) inhibitor. A further aspect of the invention relates to a method of treating or preventing leishmaniasis in a subject, said method comprising administering to the subject a therapeutically or prophylactically effective amount of a soluble epoxide hydrolase inhibitor.

Description

[0001] METHODS

[0002] The present invention relates to therapeutic methods for treating or preventing tissue damage or tissue destruction in a subject.

[0003] BACKGROUND TO THE INVENTION

[0004] Chronic inflammation is slow, long-term inflammation that lasts for prolonged periods of several months to years. Chronic inflammatory diseases are a significant cause of death in the world, with the World Health Organisation (WHO) ranking chronic inflammatory disease as one of the greatest threats to human health.

[0005] There is much to learn about the regulatory mechanisms that control the severity of inflammatory responses to infection as well as those that bring about its resolution. While ongoing research has identified many soluble mediators and cellular processes necessary for effective resolution, the lack of curative therapeutics calls for more research into understanding the complexity of inflammatory responses.

[0006] Classical approaches to treating chronic inflammation have focused on alleviating the clinical symptoms (heat, swelling, redness and pain), rather than the underlying cause. None of the treatments available to date cure the underlying pathology or reverse disease progress.

[0007] The present invention seeks to provide an alternative therapeutic approach which targets the underlying mechanistic pathways. In particular, this offers a different approach to treating chronic inflammatory diseases in a manner that targets disease pathology progression, and furthermore opens up new therapeutic opportunities for treating or preventing tissue damage that may be independent of the classical clinical symptoms of inflammation.

[0008] STATEMENT OF INVENTION

[0009] The present invention relates to various therapeutic applications of soluble epoxide hydrolase (sEH) inhibitors, and the role they play in modulating the inflammatory cascade. In certain embodiments, the invention relates to the therapeutic potential of sEH inhibitors to block the expansion of a particular population of immune cells (namely intermediate monocytes) that are implicated in the pathway and progression of several disparate inflammatory diseases.

[0010] As such, the methods of the invention described herein seek to treat the underlying pathways that drive inflammatory disease, which are mechanistically independent of the classical clinical symptoms of inflammation, namely, heat, redness, swelling and pain. One aspect of the invention therefore relates to a method of treating or preventing tissue damage, or treating or preventing tissue destruction, in a subject, said method comprising administering to the subject a therapeutically or prophylactically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.

[0011] A further aspect of the invention relates to a method of inhibiting the expansion of the intermediate monocyte cell population in a subject, said method comprising administering to the subject a therapeutically or prophylactically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.

[0012] A further aspect of the invention relates to a method of inhibiting disease progression in a subject having a chronic inflammatory disease, an autoimmune disease, cancer, cardiovascular disease, dermatitis, dementia, age-related inflammation, cirrhosis or atherosclerosis, said method comprising administering to the subject a therapeutically effective amount of a soluble epoxide hydrolase (sEH) inhibitor, whereby the sEH inhibitor inhibits the expansion of the intermediate monocyte cell population in the subject.

[0013] A further aspect of the invention relates to a method of treating or preventing tissue damage, or treating or preventing tissue destruction, in a subject, said method comprising administering to the subject a therapeutically or prophylactically effective amount of a soluble epoxide hydrolase (sEH) inhibitor, whereby the sEH inhibitor inhibits the expansion of the intermediate monocyte cell population in the subject.

[0014] A further aspect of the invention relates to a method of treating the pathogenesis of chronic inflammatory disease or chronic inflammation in a subject, said method comprising administering to the subject a therapeutically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.

[0015] A further aspect of the invention relates to a method of treating the pathogenesis of chronic inflammatory disease or chronic inflammation in a subject, said method comprising administering to the subject a soluble epoxide hydrolase (sEH) inhibitor in an amount sufficient to inhibit the expansion of the intermediate monocyte cell population in the subject.

[0016] A further aspect of the invention relates to a method of inhibiting the monocyte differentiation pathway in a subject having chronic inflammatory disease or chronic inflammation, said method comprising administering to the subject a therapeutically effective amount of a soluble epoxide hydrolase (sEH) inhibitor. A further aspect of the invention relates to a method of invoking an innate immune response in a subject having chronic inflammation, said method comprising administering to the subject a soluble epoxide hydrolase (sEH) inhibitor in an amount sufficient to inhibit the expansion of the intermediate monocyte cell population in the subject.

[0017] A further aspect of the invention relates to a method of reducing the number of circulating intermediate monocytes in a subject having chronic inflammation, said method comprising administering to the subject a therapeutically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.

[0018] A further aspect of the invention relates to a method of inhibiting the development of chronic inflammation at a tissue site distal to a primary site of chronic inflammation in a subject, said method comprising administering to the subject a therapeutically effective amount of an sEH inhibitor.

[0019] A further aspect of the invention relates to a method of decelerating the development of chronic inflammation at a tissue site distal to a primary site of chronic inflammation in a subject, said method comprising administering to the subject a therapeutically effective amount of an sEH inhibitor.

[0020] A further aspect of the invention relates to a method of treating or preventing internal organ damage in a subject, said method comprising administering to the subject a therapeutically or prophylactically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.

[0021] A further aspect of the invention relates to a method of treating or preventing leishmaniasis in a subject, said method comprising administering to the subject a therapeutically or prophylactically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.

[0022] A further aspect of the invention relates to a method of treating or preventing tissue damage, or treating or preventing tissue destruction, or treating or preventing tissue lesions in a subject having leishmaniasis, or a subject infected with Leishmania, said method comprising administering to the subject a therapeutically or prophylactically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.

[0023] A further aspect of the invention relates to a method of inhibiting the expansion of the intermediate monocyte cell population in a subject having leishmaniasis or a subject infected with Leishmania, said method comprising administering to the subject a therapeutically or prophylactically effective amount of a soluble epoxide hydrolase (sEH) inhibitor. A further aspect of the invention relates to a method of inhibiting the monocyte differentiation pathway in a subject having leishmaniasis, or a subject infected with Leishmania, said method comprising administering to the subject a therapeutically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.

[0024] A further aspect of the invention relates to a method of invoking an innate immune response in a subject having leishmaniasis, or a subject infected with Leishmania, said method comprising administering to the subject a soluble epoxide hydrolase (sEH) inhibitor in an amount sufficient to inhibit the expansion of the intermediate monocyte cell population in the subject.

[0025] A further aspect of the invention relates to a method of reducing the number of circulating intermediate monocytes in a subject having leishmaniasis, or a subject infected with Leishmania, said method comprising administering to the subject a therapeutically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.

[0026] DETAILED DESCRIPTION

[0027] The methods of the present invention are suitable for treating a range of different therapeutic disorders. In particular, the methods are suitable for treating chronic disorders that involve a dysregulated immune system which drives some or all aspects of disease pathogenesis.

[0028] When considering the immune system’s role in such diseases, there are two very distinct processes at play. The first is “inflammation” as illustrated by Celsus (25 BC - circa 50 AD) who described the four cardinal signs of heat, redness, swelling and pain (see left side of the schematic in Figure 9, “Inflammation”).

[0029] These represent the outward manifestations of our immune system’s waywardness and are mediated by soluble hormones such as cytokines, which have been the subject of much investigation over the years. Indeed, treatment of inflammatory diseases is based largely on inhibiting hormones that drive the clinical features of heat, redness, swelling, and pain. These include NSAIDs and biologies. However, none are curative, many have side effects, and a significant proportion of patients do not respond, or become refractory to them. This presents a challenge to think differently about how diseases occur and progress over time to meet the massive unmet clinical need of treating chronic diseases.

[0030] On this note, there is another conspicuous but nonetheless insidious process mediated by cells of the immune system deep in the tissues and peripheral blood circulatory system which slowly, but progressively, digests tissues leading to organ failure and premature death. These cells are called intermediate monocytes. These processes are distinct to heat, redness swelling. Importantly, there are no treatments that slow down or reverse this distinct process, making this the ultimate goal in the treatment of chronic diseases. See right side of the schematic in Figure 9, “Tissue destruction”.

[0031] Through studies using human experimental models of immunity, the Applicant has discovered the enzyme that controls the expansion of tissue damaging intermediate monocytes, called soluble epoxide hydrolase (sEH). In essence, these cells expand in number preceding the clinical symptoms presented by patients such that inhibiting of the expansion of these cells halts tissue damage and allow tissues to regenerate.

[0032] Without wishing to be bound by theory, it is believed that blocking sEH and the expansion of tissue damaging intermediate monocytes in diseases like rheumatoid arthritis, cancer, systemic lupus erythematosus, dermatitis, dementia, cirrhosis and atherosclerosis, will halt disease progression and be tissue restorative.

[0033] A first aspect of the invention relates to a method of treating or preventing tissue damage, or treating or preventing tissue destruction, in a subject, said method comprising administering to the subject a therapeutically or prophylactically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.

[0034] Soluble epoxide hydrolase (EPHX2, also known as sEH) is one of the primary enzymes involved in the metabolic breakdown of epoxy-oxylipins.

[0035] The epoxy-oxylipins are short lived epoxide lipids that have immuno-modulatory properties which include inhibiting NF-KB activation and blocking p38 MAPK phosphorylation resulting in reduced expression of pro-inflammatory cytokines and cell adhesion molecules (Jiang et al, 2015; Node et al, 1999). Epoxy-oxylipins were first postulated to have anti-inflammatory activity back in 1999 (Node et al., 1999). Since then, a multitude of murine and in vitro models have set out to interrogate the anti-inflammatory properties of soluble epoxide hydrolase inhibition and subsequent elevation of the epoxy-oxylipins. While the murine sEH and human sEH are near identical in their active site, tertiary, and quaternary structure, there are fundamental differences between the Cytochrome P450 enzymes in mouse and human (Gomez et al., 2004). Little is known about how epoxy-oxylipins impact inflammation and resolution in humans.

[0036] The CYP450 epoxygenase enzymes convert polyunsaturated fatty acids (PLIFAs) into epoxyoxylipins. Linoleic acid (LA) is metabolised into epoxy-octadecenoic acids (EpOMEs), arachidonic acid (AA) into epoxy-eicosatetraenoic acids (EETs), docosahexaenoic acid (DHA) into epoxy-docosapentanoic acids and eicosapentaenoic acid (EPA) into epoxyeicosatetraenoic acids (EpETEs). After production, the epoxy-oxylipins are rapidly metabolised into biologically more stable and water soluble counterparts by a group of enzymes known as the epoxide hydrolases (EH), including microsomal EH (mEH) and soluble epoxide hydrolase (sEH). Hence, by inhibiting EH, epoxy-oxylipins are elevated prolonging their biological half-life and as such represents a useful tool to understand their role in health and disease. sEH metabolises epoxy-oxylipins via hydrolysis of the epoxides to the corresponding vicinal diols (Arand et al., 2005). sEH is expressed in numerous tissues in humans. The tissues with the highest sEH activity are the liver, kidney, intestine and vascular tissue, however, expression has also been detected in lung, brain, spleen and white blood cells. Routine use of RNA-seq has allowed for full expression profiling in all human tissues (EPHX2 Gene - GeneCards | HYES Protein | HYES Antibody, n.d.; Sura et al., 2008; VanRollins et al., 1993; Yannick Senouvo et al., 2011 ; Yu et al., 2004; D. Zhang et al., 2012).

[0037] Studies by the Applicant have demonstrated an sEH inhibitor-induced reduction in numbers of intermediate monocytes during experimental inflammation in humans which indicates that epoxy-oxylipins are one of the mechanistic drivers that control the monocyte differentiation pathway.

[0038] In an experimental human model of skin inflammation triggered by bacterial injection, it was found that inhibiting sEH significantly elevated the levels of epoxy-oxylipins in plasma. In control experiments, numbers of Intermediate (CD14+CD16+) and Non-Classical (CD14-CD16+) monocytes in peripheral blood increased significantly between 4 and 24 hours following bacterial skin challenge. However, administering an sEH inhibitor (for example, GSK2256294) was shown to prevent this increase. Further details of these experiments are presented in the accompanying examples.

[0039] In one preferred embodiment, the method is for treating tissue damage or tissue destruction in a subject.

[0040] In one preferred embodiment, the method is for preventing tissue damage or tissue destruction in a subject.

[0041] In one preferred embodiment, the method is for treating or preventing tissue damage or tissue destruction in the absence of classical clinical symptoms of heat, swelling redness or pain.

[0042] In one preferred embodiment, the method is for treating or preventing tissue damage or tissue destruction in chronic inflammation or chronic inflammatory disease. In one preferred embodiment, the subject has a disease selected from a chronic inflammatory disease, an autoimmune disease, cardiovascular disease, cancer, dermatitis, dementia, age- related inflammation, cirrhosis and atherosclerosis.

[0043] In one preferred embodiment, the subject has a chronic inflammatory disease or chronic inflammation. Chronic inflammatory disease or chronic inflammation typically refers to slow, long-term inflammation lasting several months to years. Generally, the extent and effects of chronic inflammation varies with the cause of the injury and the ability of the body to repair and overcome the damage. Chronic inflammatory diseases include, but are not limited to, Crohn’s disease, ulcerative colitis, rheumatoid arthritis, psoriasis, systemic lupus erythematosus, asthma, chronic obstructive pulmonary disease (COPD).

[0044] In one preferred embodiment, the chronic inflammatory disease is inflammatory bowel disease, more preferably Crohn’s disease or ulcerative colitis.

[0045] In one preferred embodiment, the chronic inflammatory disease is Crohn’s disease.

[0046] In one preferred embodiment, the chronic inflammatory disease is ulcerative colitis.

[0047] In one preferred embodiment, the subject has an autoimmune disease. Autoimmine diease is a is a condition that results from an anomalous response of the adaptive immune system, wherein it mistakenly targets and attacks healthy, functioning parts of the body as if they were foreign organisms. Autoimmine dieases include, but are not limited to, celiac disease, type 1 diabetes, Graves' disease, inflammatory bowel diseases (such as Crohn's disease and ulcerative colitis), multiple sclerosis, alopecia areata, Addison's disease, pernicious anemia, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.

[0048] In one preferred embodiment, the autoimmune disease is rheumatoid arthritis, psoriatic arthritis, reactive arthritis, juvenile idiopathic arthritis or systemic lupus erythematosus.

[0049] In one preferred embodiment, the autoimmune disease is rheumatoid arthritis. Rheumatoid arthritis is a chronic autoimmune disease and is characterised by inflammation of the synovium and subsequent damage to joints. Patients living with this disease have expanded numbers of intermediate monocytes both in peripheral blood and within the synovium (Highton et al., 1995; Kawanaka et al., 2002; Rossol et al., 2012; Ruiz-Limon et al., 2019). Monocytes have been shown to contribute to the pathogenesis of this disease by infiltrating into the synovium and establishing as inflammatory macrophages that are capable of driving T cell activation, in particular IL-17 producing T cells and releasing pro-inflammatory cytokines A (Evans et al., 2009; Kinne et al., 2000; Roberts et al., 2015; Walter et al., 2013). In one preferred embodiment, the autoimmune disease is systemic lupus erythematosus. Expanded numbers of intermediate monocytes have been observed in patients with Systemic Lupus Erythematosus (SLE), stroke, Crohn’s disease, sarcoidosis and atherosclerosis (Grip et al., 2007; Hijdra et al., 2012; Hirose et al., 2019; Urra et al., 2009; H. Zhu et al., 2016).

[0050] In one preferred embodiment, the method is for treating or preventing tissue damage in the kidneys of subjects having systemic lupus erythematosus. This is a specific example of a therapeutic application in which the classical hallmarks of inflammation, namely heat, redness, swelling and pain, are absent.

[0051] In one preferred embodiment, the subject has age-related inflammation. Aging is characterised by systemic chronic inflammation which is accompanied by cellular senescence, immunosenescence, organ dysfunction, and age-related diseases. Most older individuals develop inflammageing, a condition characterized by elevated levels of blood inflammatory markers that carries high susceptibility to chronic morbidity, disability, frailty, and premature death. Inflammageing is also a risk factor for cardiovascular diseases.

[0052] In one preferred embodiment, the subject has cardiovascular disease. Systemic and local inflammation have a central role in the development and progression of cardiovascular disease (CVD), from endothelial dysfunction to clinical syndromes. Inflammatory biomarkers have been shown to predict CVD, independently of traditional risk factors. Several acute and chronic conditions, including the traditional risk factors, psychological stress, autoimmune disease, microbial and viral infections, and ageing, can activate endothelial damage and dysfunction. In turn, this promotes a vascular low-grade inflammatory response, leading to the progression of atherosclerosis. Hence, inflammation is a common mechanism linking traditional and emerging CV risk factors to the development of atherosclerosis, leading to CAD, large artery thrombotic stroke, and cerebral aneurysms (Henein et al 2022).

[0053] In one preferred embodiment, the subject has cirrhosis. Cirrhosis is a gradual scarring process that is triggered by chronic inflammation in the liver. One of the key elements involved in cirrhosis physiopathology is systemic inflammation, described as one of the components in the cirrhosis- associated immune dysfunction syndrome. This syndrome refers to the combination of immune deficiency and exacerbated inflammation that coexist during the course of cirrhosis and relates to the appearance of clinical complications (Dirchwolf et al 2015).

[0054] In one preferred embodiment, the subject has cancer. Cancer development and its response to therapy are regulated by inflammation, which either promotes or suppresses tumor progression, potentially displaying opposing effects on therapeutic outcomes. Chronic inflammation facilitates tumor progression and treatment resistance, whereas induction of acute inflammatory reactions often stimulates the maturation of dendritic cells (DCs) and antigen presentation, leading to antitumor immune responses (Zhao et al, 2021).

[0055] In one preferred embodiment, the subject has dermatitis. Dermatitis is inflammation of the skin, typically characterized by itchiness, redness and a rash. There are several types of dermatitis including atopic dermatitis, contact dermatitis, stasis dermatitis and seborrhoeic dermatitis.

[0056] In one preferred embodiment, the method is for treating tissue damage in wound healing.

[0057] In one preferred embodiment, the method is for treating or preventing internal organ damage. In this aspect, the classical hallmarks of inflammation, namely heat, redness, swelling and pain, are absent.

[0058] In one preferred embodiment, the sEH inhibitor inhibits the expansion of the intermediate monocyte cell population in the subject.

[0059] A further aspect of the invention relates to a method of inhibiting the expansion of the intermediate monocyte cell population in a subject, said method comprising administering to the subject a therapeutically or prophylactically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.

[0060] Preferred diseases are as described for the above-mentioned first aspect.

[0061] A further aspect of the invention relates to a method of inhibiting disease progression in a subject having a chronic inflammatory disease, an autoimmune disease, cancer, cardiovascular disease, dermatitis, dementia, age-related inflammation, cirrhosis or atherosclerosis, said method comprising administering to the subject a therapeutically effective amount of a soluble epoxide hydrolase (sEH) inhibitor, whereby the sEH inhibitor inhibits the expansion of the intermediate monocyte cell population in the subject.

[0062] In one preferred embodiment, the subject has an autoimmune disease, preferably rheumatoid arthritis, psoriatic arthritis, reactive arthritis, juvenile idiopathic arthritis or systemic lupus erythematosus

[0063] In one preferred embodiment, the subject has a chronic inflammatory disease, preferably an inflammatory bowel disease, more preferably Crohn’s disease or ulcerative colitis.

[0064] In one preferred embodiment, the subject has multimorbidities, or multiple long term conditions. For example, in one preferred embodiment, the subject has two or more chronic illnesses. In such cases, there may be an accelerated development of a secondary disease as a consequence of the primary disease. For instance, it is known that patients suffering from arthritis can have a 3 or 4-fold increased incidence of atherosclerosis. The common connection between a subject’s joints and large blood vessels is the circulatory system and the immune cells therein. Accordingly, an expanded population of intermediate monocytes can lead to an increased incidence of a secondary disease.

[0065] A further aspect of the invention relates to a method of treating or preventing tissue damage, or treating or preventing tissue destruction, in a subject, said method comprising administering to the subject a therapeutically or prophylactically effective amount of a soluble epoxide hydrolase (sEH) inhibitor, whereby the sEH inhibitor inhibits the expansion of the intermediate monocyte cell population in the subject.

[0066] Preferred diseases are as described for the above-mentioned first aspect.

[0067] A further aspect of the invention relates to a method of treating the pathogenesis of chronic inflammatory disease or chronic inflammation in a subject, said method comprising administering to the subject a therapeutically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.

[0068] In one preferred embodiment, the sEH inhibitor inhibits the expansion of the intermediate monocyte cell population in the subject.

[0069] A further aspect of the invention relates to a method of treating the pathogenesis of chronic inflammatory disease or chronic inflammation in a subject, said method comprising administering to the subject a soluble epoxide hydrolase (sEH) inhibitor in an amount sufficient to inhibit the expansion of the intermediate monocyte cell population in the subject.

[0070] A further aspect of the invention relates to a method of inhibiting the monocyte differentiation pathway in a subject having chronic inflammatory disease or chronic inflammation, said method comprising administering to the subject a therapeutically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.

[0071] In one preferred embodiment, the sEH inhibitor inhibits the expansion of the intermediate monocyte cell population in the subject.

[0072] In one preferred embodiment, the sEH inhibitor reduces the population of intermediate monocytes in peripheral blood. In one preferred embodiment, the sEH inhibitor reduces the population of intermediate monocytes at a primary site of inflammation.

[0073] In one preferred embodiment, the sEH inhibitor reduces the population of intermediate monocytes at a secondary tissue site distal to a primary site of inflammation or disease.

[0074] An expanded monocyte population can accelerate the development of chronic inflammation at tissue sites distal to the primary or index disease. For instance, subjects suffering from a primary chronic inflammatory disease (e.g. rheumatoid arthritis) can often have an increased incidence and / or accelerated development of a secondary inflammatory disease (e.g. atheroscleroisis) as a consequence of the expanded intermediate monocyte population driving the primary disease. Thus, in one preferred embodiment, the sEH inhibitor reduces the population of intermediate monocytes such that chronic inflammation is decreased at one or more secondary sites which are distal to the primary site of chronic inflammation.

[0075] In one preferred embodiment, the sEH inhibitor reduces the population of intermediate monocytes at a primary site of inflammation and at a secondary tissue site distal to the primary site of inflammation or disease, where there is accelerated development of chronic inflammatory disease in said secondary tissue site as a consequence of the primary disease being driven by expanded intermediate monocytes.

[0076] Another aspect of the invention relates to a method of inhibiting the development of chronic inflammation at a tissue site distal to a primary site of chronic inflammation in a subject, said method comprising administering to the subject a therapeutically effective amount of an sEH inhibitor.

[0077] Another aspect of the invention relates to a method of decelerating the development of chronic inflammation at a tissue site distal to a primary site of chronic inflammation in a subject, said method comprising administering to the subject a therapeutically effective amount of an sEH inhibitor.

[0078] An further aspect of the invention relates to a method of invoking an innate immune response in a subject having chronic inflammation, said method comprising administering to the subject a soluble epoxide hydrolase (sEH) inhibitor in an amount sufficient to inhibit the expansion of the intermediate monocyte cell population in the subject.

[0079] A further aspect of the invention relates to a method of reducing the number of circulating intermediate monocytes in a subject having chronic inflammation, said method comprising administering to the subject a therapeutically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.

[0080] A further aspect of the invention relates to a method of treating or preventing internal organ damage in a subject, said method comprising administering to the subject a therapeutically or prophylactically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.

[0081] INTERMEDIATE MONOCYTES

[0082] Studies by the Applicant have shown that sEH inhibition affects the population of intermediate monotyes, a particular subset of immune cells that are involved in the inflammatory cascade.

[0083] Monocytes are circulating leukocytes that are made in the bone marrow and travel through the blood to tissues in the body where they become macrophages or dendritic cells. They constitute a critical component in both innate and adaptive immunity, primarily functioning in immune defence, inflammation and tissue remodelling. In the mouse there are 2 subsets of monocytes whilst there are 3 subsets in humans, that are mobilised from the bone marrow and recruited to sites of inflammation.

[0084] Monocytes in humans can be classified into three subsets: classical (CM), intermediate (IM) and non-classical (NCM). Monocytes make up around 10% of peripheral circulating leukocytes and, during steady state, classical monocytes make up around -85% of monocytes, with intermediates and non-classicals making up the remainder (Cormican & Griffin, 2020). Traditionally, the different monocyte subsets have been defined based on the expression of the LPS co-receptor, CD14, and Fc-gamma receptor IIIA (FCyRIIIA) (also known as CD16), with classical monocytes defined as CD14+CD16-, intermediates as CD14+CD16+ and non- classicals as CD14-CD16+ (Griffin et al., 1981 ; Passlick et al., 1989; H. W. L. Ziegler-Heitbrock, 2000; L. Ziegler-Heitbrock et al., 2010).

[0085] The function of non-classical monocytes is patrolling, clearance of debris, clearance of apoptotic cells and anti-viral responses. The function of classical monocytes is anti-microbial immunity through direct responses including phagocytosis and cytokine production in addition to regulating other cells of the innate and adaptive immune systems.

[0086] The intermediate monocyte subset (CD14+CD16+) is a phenotypically distinct subset with different functionality to classical and non-classical monocytes. Intermediate monocytes exhibit both phagocytic function and anti-inflammatory effects, as well as higher levels of intracellular I L-1 p and tumour necrosis factor (TNF)-a at steady state. Their function is antigen presentation, ROS production, regulation of apoptosis, angiogenesis pro-inflammatory cytokines and T-cell stimulation. In many diseases it has been noted that CD14+CD16+ monocytes are expanded and likely contribute to the pathogenesis of disease. This expansion of intermediates was noted in sepsis patients back in 1993 and has since been noted in many other disease states (Fingerle et al., 1993). Indeed numerous studies have noted increases in circulating CD14+CD16+ in cardiovascular disease, trauma, sepsis, and autoimmunity, suggesting that this maturation / polarization response may be an important mechanism in regulating immune responses and in some cases contributing to disease pathogenesis.

[0087] During normal infection, the conversion of classical to intermediate monocytes is accelerated and the number of intermediate monocytes expand rapidly (Kratofil et al. , 2017). The expansion of the CD16+ monocyte population is a hallmark of numerous chronic inflammatory diseases, including cardiovascular disease, HIV, tuberculosis and autoimmune disease (Balboa et al., 2015; Castano et al., 2011 ; Grip et al., 2007; Han et al., 2009; Highton et al., 1995; Hijdra et al., 2012; Hirose et al., 2019; Kawanaka et al., 2002; Lugo-Villarino & Neyrolles, 2013; Rossol et al., 2012; Ruiz-Limon et al., 2019; llrra et al., 2009; H. Zhu et al., 2016).

[0088] Studies by the Applicant have demonstrated that inhibiting the soluble epoxide hydrolase enzyme and thus elevating the levels of the epoxy-oxylipin, 12,13-EpOME, reduces the number of circulating intermediate monocytes during inflammation in human volunteers.

[0089] TREATMENT OF LEISHMANIASIS

[0090] Studies by the Applicant have demonstrated that intermediate monocytes play an important role in the pathogenic response associated with the disease leishmaniasis. In particular, studies were undertaken to investigate the profile of circulating and lesional monocytes / macrophages in patients with cutaneous leishmaniasis (CL). The results indicated that CL patients show a significant increase in circulating CD16+(non-classical) and CD14+CD16+(intermediate) monocytes, while CD14+CD16“ (classical) monocytes remain unchanged compared to controls. Notably, CD14+CD16+(intermediate) monocytes, are more inflammatory, and may play a key role in driving chronic inflammation and tissue damage in CL lesions, suggesting their involvement in the exacerbation of disease pathology. Based on the underlying mechanistic rationale described herein, soluble epoxide hydrolase (sEH) inhibitors are capable of inhibiting the expansion of the intermediate monocyte cell population. Further details of these experiments are set out in the accompanying Examples section. A further aspect of the invention therefore relates to a method of treating or preventing leishmaniasis in a subject, said method comprising administering to the subject a therapeutically or prophylactically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.

[0091] Leishmaniasis is a parasitic disease caused by infection with Leishmania parasites. These parasites are transmitted to humans by the bite of an infected female phlebotomine sandfly, a tiny 2-3 mm long insect vector. There are more than 20 different Leishmania species.

[0092] The clinical presentation of Leishmania infection is highly variable, ranging from single, selfresolving skin lesions to severe chronic ulcers, including disseminated and mucosal forms, all of which can be disfiguring (Scott et al, 2016). This variability is largely driven by the host immune response, which plays a crucial role in both protection and pathology. Possible treatments depend on clinical presentations, parasite species and strain, and the country in which infection was acquired.

[0093] Leishmaniasis can present in three main ways: cutaneous, mucocutaneous, or visceral.

[0094] In one preferred embodiment, the leishmaniasis is cutaneous leishmaniasis. In one preferred embodiment, the leishmaniasis is diffuse cutaneous leishmaniasis. In one preferred embodiment, the leishmaniasis is disseminated cutaneous leishmaniasis.

[0095] CL is a severely neglected parasitic disease that leads to immune-mediated skin pathology, characterized by the development of destructive cutaneous lesions. This disease affects individuals in 98 countries, including Commonwealth nations (WHO 2022). Brazil is endemic for this infectious disease, with Leishmania braziliensis being the primary causative agent (WHO 2022). In the context of CL, the immune response plays a central role in host protection (Scott et al, 2016). However, it also contributes to the pathology of skin lesions and the development of classical ulcers. Notably, some of the most severe forms of the disease are characterized by chronic inflammation despite parasite control (Scott et al, 2016). High levels of gamma interferon (IFN-y), the pro-inflammatory cytokine interleukin-1 p (IL-1 P), and tumor necrosis factor-alpha (TNF-a) have been positively correlated with lesion size and disease severity (Novais et al, 2017; Melby et al, 1994). Moreover, cytotoxic cells contribute to skin pathology during human Leishmania infections (da Silva Santos et al, 2014), and there is a strong correlation between disease severity and the abundance of CD8+T cells, independent of parasite burden within the lesions (Santos et al, 2013). Parasite-targeted drug treatments, such as pentavalent antimony, are associated with high failure rates in some endemic areas, and no vaccine is currently available for the disease. Thus, identifying mechanisms that drive destructive immunopathological responses could reveal novel therapeutic targets to mitigate the most severe forms of cutaneous leishmaniasis.

[0096] Another aspect of the invention relates to a method of treating or preventing tissue damage, or treating or preventing tissue destruction, or treating or preventing tissue lesions, in a subject having leishmaniasis, or a subject infected with Leishmania, said method comprising administering to the subject a therapeutically or prophylactically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.

[0097] Another aspect of the invention relates to a method of inhibiting the expansion of the intermediate monocyte cell population in a subject having leishmaniasis or a subject infected with Leishmania, said method comprising administering to the subject a therapeutically or prophylactically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.

[0098] Another aspect of the invention relates to a method of inhibiting the monocyte differentiation pathway in a subject having leishmaniasis, or a subject infected with Leishmania, said method comprising administering to the subject a therapeutically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.

[0099] In one preferred embodiment, the sEH inhibitor inhibits the expansion of the intermediate monocyte cell population in the subject.

[0100] In one preferred embodiment, the sEH inhibitor reduces the population of intermediate monocytes at a site of inflammation.

[0101] Another aspect of the invention relates to a method of invoking an innate immune response in a subject having leishmaniasis, or a subject infected with Leishmania, said method comprising administering to the subject a soluble epoxide hydrolase (sEH) inhibitor in an amount sufficient to inhibit the expansion of the intermediate monocyte cell population in the subject.

[0102] Another aspect of the invention relates to a method of reducing the number of circulating intermediate monocytes in a subject having leishmaniasis, or a subject infected with Leishmania, said method comprising administering to the subject a therapeutically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.

[0103] In one preferred embodiment, the leishmaniasis is mucosal leishmaniasis. Mucosal leishmaniasis (also known as mucocutaneous leishmaniasis) causes both skin and mucosal ulcers with damage primarily of the nose and mouth. In one preferred embodiment, the leishmaniasis is viseral leishmaniasis. Visceral leishmaniasis or kala-azar ('black fever') is the most serious form and is generally fatal if untreated. Other consequences, which can occur a few months to years after infection, include fever, damage to the spleen and liver, and anemia.

[0104] In one preferred embodiment, the subject is infected with a Leishmania species selected from L donovani, L infantum (synonym: L chagasi), Leishmania martiniquensis, L braziliensis, L guyanensis , L panamensis , L amazonensis , L aethiopica and L tropica.

[0105] A further apect of the invention relates to a soluble epoxide hydrolase (sEH) inhibitor for use in treating or preventing leishmaniasis, or for use in treating or preventing an infection with Leishmania.

[0106] In one preferred embodiment, the sEH inhibitor inhibits the expansion of the intermediate monocyte cell population in the subject. sEH inhibitors for aspects of the invention directed to the treatment or prevention of leishmaniasis are as described below.

[0107] A further apect of the invention relates to the use of a soluble epoxide hydrolase (sEH) inhibitor in the preparation of a medicament for treating or preventing leishmaniasis, or for treating or preventing an infection with Leishmania. sEH Inhibitors

[0108] The methods of the present invention involve the administration of an sEH inhibitor.

[0109] Suitable sEH inhibitors are known in the art. The skilled person would be able to identify suitable sEH inhibitors by assays known in the art. For example, one suitable assay is to assess activity of soluble epoxide hydrolase as per the protocol outlined in Lazaar et al (2016). Briefly, whole blood samples are assessed for sEH activity via the addition of a known sEH inhibitor and 14,15- EET-deuterated (d11), which is metabolised to 14,15-DHET-d11 by sEH. LC / MS / MS is used to quantify the amount of 14,15-EET-d11 and 14,15-DHET-d11. sEH activity is evaluated by the formation of 14,15-DHET-d11. Preferably, the sEH inhibitor has an enzyme inhibition of at least 60 %, or at least 70, or at least 80 % or at least 90 % or at least 95 %, or at least 98 % or at least 99 % in the above mentioned assay.

[0110] In one highly preferred embodiment, the sEH inhibitor has an enzyme inhibition of about 99% in the above mentioned assay. In one preferred embodiment, the sEH inhibitor is selected from GSK2256294, sorafenib, EC5026, Bl 1935, glimepiride, TPPU, dCP2PU, t-AUCB, AUDA and t-TUCB.

[0111] In one preferred embodiment, the sEH inhibitor is GSK2256294, or a pharmaceutically acceptable salt thereof.

[0112] GSK2256294 (CAS No.: 1142090-23-0) is the compound known as (1 R,3S)-N-(4-cyano-2- (trifluoromethyl)benzyl)-3-((4-methyl-6-(methylamino)-1 ,3,5-triazin-2-yl)amino)cyclohexane-1- carboxamide (see also W02009049157), having the chemical structure shown below:

[0113] GSK2256294

[0114] GSK-2256294 is a potent, reversible, tight binding inhibitor of isolated recombinant human sEH (soluble epoxide hydrolase) (IC50 = 27 pM; t1 / 2 = 121 min) and displays potent inhibition against the rat (IC50 = 61 pM) and murine (ICso = 189 pM) orthologs of sEH. GSK-2256294 also displays potent cellular inhibition (IC50 = 0.66 nM) of sEH in an assay developed using a cell line transfected with the human sEH enzyme. GSK-2256294 is well-tolerated and demonstrates sustained inhibition of sEH enzyme activity.

[0115] Characterisation of the pharmacokinetics of the drug has shown that GSK2256294 exhibits a good soluble aqueous profile and moderate lipophilicity. GSK2256294 demonstrates moderate plasma protein binding at 12.8% free fraction at 1 ug / ml in humans (Podolin et al., 2013).

[0116] Studies conducted by Lazaar et al., have shown dose dependent inhibition of the soluble epoxide hydrolase and is safe and well tolerated in human volunteers (Lazaar et al., 2016). Maximum systemic concentrations of GSK2256294 were achieved 1-2 hours after dosing and the observed half-life was 20-30 hours (Lazaar et al., 2016). Lazaar et al., showed that following single doses of GSK2256294 at different concentrations, the average inhibition of sEH was 41.9% for a 2mg dose and 99.8% following a 20mg dose and that the duration of this inhibition was sustained for up to 24 hours (Lazaar et al., 2016).

[0117] Human studies using GSK2256294 have so far investigated the effects of sEH inhibition in COPD, diabetes and aneurysmal subarachnoid haemorrhage. Endothelial function was improved in patients with COPD and pro-inflammatory cytokine expression was reduced in aneurysmal subarachnoid haemorrhage patients with soluble epoxide hydrolase inhibition (Martini et al., 2022; L. Yang et al., 2017). In adipose tissue collected from obese pre-diabetic patients, GSK2256294 reduced the percentage of pro-inflammatory T cells producing IFN-y. Treatment reduces the amount of TNF-a secreted but did not affect the levels of IL-17A (Mashayekhi et al., 2022).

[0118] In one preferred embodiment, the sEH inhibitor is Sorafenib.

[0119] Sorafenib, sold under the brand name Nexavar, is a kinase inhibitor drug approved for the treatment of primary kidney cancer (advanced renal cell carcinoma), advanced primary liver cancer (hepatocellular carcinom a), FLT3-ITD positive AML and radioactive iodine resistant advanced thyroid carcinoma. Sorafenib is the compound known as 4-[4-[[4-chloro-3- (trifluoromethyl)phenyl]carbamoylamino]phenoxy]-A / -methylpyridine-2-carboxamide having the chemical structure below:

[0120] Sorafenib

[0121] It is typically used in tosylate salt form (Nexavar®)

[0122] In one preferred embodiment, the sEH inhibitor is EC5026.

[0123] EC5026 (BPN-19186; CAS No.: 1809885-32-2) is a non-opioid and orally active soluble sEH inhibitor. EC5026 is the compound 1-[3-fluoro-4-(trifluoromethoxy)phenyl]-3-[1-[(2S)-2- methylbutanoyl]piperidin-4-yl]urea, having the chemical structure shown below:

[0124] EC5026

[0125] In one preferred embodiment, the sEH inhibitor is Bl 1935. BI1935 (CAS No.: 940954-41-6) is the compound known as 1-(2-ethoxyethyl)-6-oxo-N-[5-[3- pyridin-3-yl-5-(trifluoromethyl)pyrazol-1-yl]pyridin-2-yl]pyridine-3-carboxamide, having the chemical structure shown below:

[0126] Bl 1935

[0127] BI-1935 is a potent and selective small molecule inhibitor of sEH. It is Example 3 in W02007067836.

[0128] In one preferred embodiment, the sEH inhibitor is Glimepiride.

[0129] Glimepiride (CAS No.: 93479-97-1) is the compound 4-ethyl-3-methyl-A / -[2-[4-[(4- methylcyclohexyl)carbamoyl-lsulfamoyl]phenyl]ethyl]-5-oxo-2 / - / -pyrrole-1 -carboxamide having the chemical structure below:

[0130] Glimepiride

[0131] In one preferred embodiment, the sEH inhibitor is TPPLI. TPPLI (CAS No.: 1222780-33-7) is the compound known as N-[1-(1-oxopropyl)-4-piperidinyl]-

[0132] N’-[4-(trifluoromethoxy)phenyl)-urea, having the chemical structure shown below:

[0133] TPPU TPPLI is a soluble sEH inhibitor with IC50 values of 37 and 3.7 nM for monkey and human sEH, respectively.

[0134] In one preferred embodiment, the sEH inhibitor is dCP2PU. dCP2PU is the compound known as 1-(3,4-dichlorophenyl)-3-(4-phenoxyphenyl)urea, having the chemical structure shown below: dCP2PU

[0135] In one preferred embodiment, the sEH inhibitor is t-AUCB. t-AUCB is the compound known as frans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid, having the chemical structure shown below: t-AUCB

[0136] In one preferred embodiment, the sEH inhibitor is ALIDA.

[0137] ALIDA (CAS No.: 479413-70-2) is the compound known as 12-(1-adamantylcarbamoyl- amino)dodecanoic acid, having the chemical structure shown below:

[0138] AU DA

[0139] AUDA is a potent soluble sEH inhibitor with IC50s of 18 and 69 nM for the mouse and human sEH, respectively.

[0140] In one preferred embodiment, the sEH inhibitor is t-TUCB. t-TUCB (UC-1728; CAS No.: 948304-40-3) is the compound known as 4-[(1 R,4R)-4-[[4- (trifluoromethoxy)phenyl]carbamoylamino]cyclohexyl]oxybenzoic acid, having the chemical structure shown below: t-TUCB t-TUCB is a potent soluble sEH inhibitor, with an IC50 of 0.9 nM.

[0141] PHARMACEUTICAL COMPOSITIONS

[0142] The sEH inhibitors or physiologically acceptable salt, ester or other physiologically functional derivative thereof, described herein, may be presented as a pharmaceutical formulation, comprising the compounds or physiologically acceptable salt, ester or other physiologically functional derivative thereof, together with one or more pharmaceutically acceptable carriers, excipients or diluents therefor and optionally other therapeutic and / or prophylactic ingredients. The carrier(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The pharmaceutical compositions may be for human or animal usage in human and veterinary medicine.

[0143] Examples of such suitable excipients for the various different forms of pharmaceutical compositions described herein may be found in the “Handbook of Pharmaceutical Excipients, 2ndEdition, (1994), Edited by A Wade and PJ Weller. The carrier, or, if more than one be present, each of the carriers, must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient.

[0144] Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).

[0145] Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like. Examples of suitable diluents include ethanol, glycerol and water.

[0146] The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s), buffer(s), flavouring agent(s), surface active agent(s), thickener(s), preservative(s) (including antioxidants) and the like, and substances included for the purpose of rendering the formulation isotonic with the blood of the intended recipient.

[0147] Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol.

[0148] Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.

[0149] Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.

[0150] Pharmaceutical formulations include those suitable for oral, topical (including dermal, buccal and sublingual), rectal or parenteral (including subcutaneous, intradermal, intramuscular and intravenous), nasal and pulmonary administration e.g., by inhalation. The formulation may, where appropriate, be conveniently presented in discrete dosage units and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association an active compound with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.

[0151] Pharmaceutical formulations suitable for oral administration wherein the carrier is a solid are most preferably presented as unit dose formulations such as boluses, capsules or tablets each containing a predetermined amount of active compound. A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine an active compound in a free-flowing form such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, lubricating agent, surface-active agent or dispersing agent. Moulded tablets may be made by moulding an active compound with an inert liquid diluent. Tablets may be optionally coated and, if uncoated, may optionally be scored. Capsules may be prepared by filling an active compound, either alone or in admixture with one or more accessory ingredients, into the capsule shells and then sealing them in the usual manner. Cachets are analogous to capsules wherein an active compound together with any accessory ingredient(s) is sealed in a rice paper envelope. An active compound may also be formulated as dispersible granules, which may for example be suspended in water before administration, or sprinkled on food. The granules may be packaged, e.g., in a sachet. Formulations suitable for oral administration wherein the carrier is a liquid may be presented as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil- in-water liquid emulsion.

[0152] Formulations for oral administration include controlled release dosage forms, e.g., tablets wherein an active compound is formulated in an appropriate release - controlling matrix, or is coated with a suitable release - controlling film. Such formulations may be particularly convenient for prophylactic use.

[0153] Pharmaceutical formulations suitable for rectal administration wherein the carrier is a solid are most preferably presented as unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories may be conveniently formed by admixture of an active compound with the softened or melted carrier(s) followed by chilling and shaping in moulds. Pharmaceutical formulations suitable for parenteral administration include sterile solutions or suspensions of an active compound in aqueous or oleaginous vehicles.

[0154] Injectable preparations may be adapted for bolus injection or continuous infusion. Such preparations are conveniently presented in unit dose or multi-dose containers which are sealed after introduction of the formulation until required for use. Alternatively, an active compound may be in powder form which is constituted with a suitable vehicle, such as sterile, pyrogen-free water, before use.

[0155] An active compound may also be formulated as long-acting depot preparations, which may be administered by intramuscular injection or by implantation, e.g., subcutaneously or intramuscularly. Depot preparations may include, for example, suitable polymeric or hydrophobic materials, or ion-exchange resins. Such long-acting formulations are particularly convenient for prophylactic use.

[0156] Formulations suitable for pulmonary administration via the buccal cavity are presented such that particles containing an active compound and desirably having a diameter in the range of 0.5 to 7 microns are delivered in the bronchial tree of the recipient.

[0157] As one possibility such formulations are in the form of finely comminuted powders which may conveniently be presented either in a pierceable capsule, suitably of, for example, gelatin, for use in an inhalation device, or alternatively as a self-propelling formulation comprising an active compound, a suitable liquid or gaseous propellant and optionally other ingredients such as a surfactant and / or a solid diluent. Suitable liquid propellants include propane and the chlorofluorocarbons, and suitable gaseous propellants include carbon dioxide. Self-propelling formulations may also be employed wherein an active compound is dispensed in the form of droplets of solution or suspension.

[0158] Such self-propelling formulations are analogous to those known in the art and may be prepared by established procedures. Suitably they are presented in a container provided with either a manually-operable or automatically functioning valve having the desired spray characteristics; advantageously the valve is of a metered type delivering a fixed volume, for example, 25 to 100 microlitres, upon each operation thereof.

[0159] As a further possibility an active compound may be in the form of a solution or suspension for use in an atomizer or nebuliser whereby an accelerated airstream or ultrasonic agitation is employed to produce a fine droplet mist for inhalation.

[0160] Formulations suitable for nasal administration include preparations generally similar to those described above for pulmonary administration. When dispensed such formulations should desirably have a particle diameter in the range 10 to 200 microns to enable retention in the nasal cavity; this may be achieved by, as appropriate, use of a powder of a suitable particle size or choice of an appropriate valve. Other suitable formulations include coarse powders having a particle diameter in the range 20 to 500 microns, for administration by rapid inhalation through the nasal passage from a container held close up to the nose, and nasal drops comprising 0.2 to 5% w / v of an active compound in aqueous or oily solution or suspension.

[0161] Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.1 M and preferably 0.05 M phosphate buffer or 0.8% saline. Additionally, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like.

[0162] Formulations suitable for topical formulation may be provided for example as gels, creams or ointments. Such preparations may be applied e.g. to a wound or ulcer either directly spread upon the surface of the wound or ulcer or carried on a suitable support such as a bandage, gauze, mesh or the like which may be applied to and over the area to be treated.

[0163] Liquid or powder formulations may also be provided which can be sprayed or sprinkled directly onto the site to be treated, e.g. a wound or ulcer. Alternatively, a carrier such as a bandage, gauze, mesh or the like can be sprayed or sprinkle with the formulation and then applied to the site to be treated.

[0164] Pharmaceutical or veterinary compositions as described above can be prepared by bringing the active compound(s) into association with the carrier, for example by admixture.

[0165] In general, the formulations are prepared by uniformly and intimately bringing into association the active agent with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

[0166] SALTS / ESTERS

[0167] The SEH inhibitors for use in the methods of the present invention can be present as salts or esters, in particular, pharmaceutically and veterinarily acceptable salts or esters.

[0168] Pharmaceutically acceptable salts of the compounds of the invention include suitable acid addition or base salts thereof. A review of suitable pharmaceutical salts may be found in Berge et al, J Pharm Sci, 66, 1-19 (1977). Salts are formed, for example with strong inorganic acids such as mineral acids, e.g. hydrohalic acids such as hydrochloride, hydrobromide and hydroiodide, sulphuric acid, phosphoric acid sulphate, bisulphate, hemisulphate, thiocyanate, persulphate and sulphonic acids; with strong organic carboxylic acids, such as alkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acids, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (Ci-C4)-alkyl- or arylsulfonic acids which are unsubstituted or substituted (for example, by a halogen) such as methane- or p-toluene sulfonic acid. Salts which are not pharmaceutically or veterinarily acceptable may still be valuable as intermediates.

[0169] Preferred salts include, for example, acetate, trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate, malate, pantothenate, adipate, alginate, aspartate, benzoate, butyrate, digluconate, cyclopentanate, glucoheptanate, glycerophosphate, oxalate, heptanoate, hexanoate, fumarate, nicotinate, palmoate, pectinate, 3-phenylpropionate, picrate, pivalate, proprionate, tartrate, lactobionate, pivolate, camphorate, undecanoate and succinate, organic sulphonic acids such as methanesulphonate, ethanesulphonate, 2-hydroxyethane sulphonate, camphorsulphonate, 2-naphthalenesulphonate, benzenesulphonate, p-chlorobenzenesulphonate and p- toluenesulphonate; and inorganic acids such as hydrochloride, hydrobromide, hydroiodide, sulphate, bisulphate, hemisulphate, thiocyanate, persulphate, phosphoric and sulphonic acids.

[0170] Esters are formed either using organic acids or alcohols / hydroxides, depending on the functional group being esterified. Organic acids include carboxylic acids, such as alkanecarboxylic acids of 1 to 12 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acid, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (Ci-C4)-alkyl- or aryl-sulfonic acids which are unsubstituted or substituted (for example, by a halogen) such as methane- or p-toluene sulfonic acid. Suitable hydroxides include inorganic hydroxides, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminium hydroxide. Alcohols include alkanealcohols of 1-12 carbon atoms which may be unsubstituted or substituted, e.g. by a halogen).

[0171] ENANTIOMERS / TAUTOMERS

[0172] In all aspects of the present invention previously discussed, the invention includes, where appropriate all enantiomers, diastereoisomers and tautomers of the sEH inhibitors of the invention. The person skilled in the art will recognise compounds that possess optical properties (one or more chiral carbon atoms) or tautomeric characteristics. The corresponding enantiomers and / or tautomers may be isolated / prepared by methods known in the art.

[0173] Enantiomers are characterised by the absolute configuration of their chiral centres and described by the R- and S-sequencing rules of Cahn, Ingold and Prelog. Such conventions are well known in the art (e.g. see ‘Advanced Organic Chemistry’, 3rdedition, ed. March, J., John Wiley and Sons, New York, 1985).

[0174] Compounds containing a chiral centre may be used as a racemic mixture, an enantiomerically enriched mixture, or the racemic mixture may be separated using well-known techniques and an individual enantiomer may be used alone.

[0175] STEREO AND GEOMETRIC ISOMERS

[0176] Some of the sEH inhibitors for use according to the methods of the invention may exist as stereoisomers and / or geometric isomers - e.g. they may possess one or more asymmetric and / or geometric centres and so may exist in two or more stereoisomeric and / or geometric forms. The present invention contemplates the use of all the individual stereoisomers and geometric isomers of those compounds, and mixtures thereof. The terms used in the claims encompass these forms, provided said forms retain the appropriate functional activity (though not necessarily to the same degree).

[0177] The present invention also includes all suitable isotopic variations of the sEH inhibitors or a pharmaceutically acceptable salt thereof. An isotopic variation is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature. Examples of isotopes that can be incorporated into the agent and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine such as2H,3H,11C,13C,14C,15N,170,180,31P,32P,35S,18F and36CI, respectively. Certain isotopic variations of the agent and pharmaceutically acceptable salts thereof, for example, those in which a radioactive isotope such as3H or14C is incorporated, are useful in drug and / or substrate tissue distribution studies. Tritiated, i.e. ,3H, and carbon-14, i.e. ,14C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with isotopes such as deuterium, i.e.,2H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be preferred in some circumstances. For example, the invention includes compounds where any hydrogen atom has been replaced by a deuterium atom. Isotopic variations of the agent of the present invention and pharmaceutically acceptable salts thereof of this invention can generally be prepared by conventional procedures using appropriate isotopic variations of suitable reagents.

[0178] ATROPISOMERS

[0179] Some of the sEH inhibitors for use in the methods of the invention may exist as atropisomers. Atropisomers are stereoisomers arising because of hindered rotation about a single bond, where energy differences due to steric strain or other contributors create a barrier to rotation that is high enough to allow for isolation of individual conformers. The invention encompasses all such atropisomers.

[0180] PRODRUGS

[0181] The invention further includes the sEH inhibitors for use in the methods of the present invention in prodrug form, i.e. covalently bonded compounds which release the active parent drug in vivo. Such prodrugs are generally compounds of the invention wherein one or more appropriate groups have been modified such that the modification may be reversed upon administration to a human or mammalian subject. Reversion is usually performed by an enzyme naturally present in such subject, though it is possible for a second agent to be administered together with such a prodrug in order to perform the reversion in vivo. Examples of such modifications include ester (for example, any of those described above), wherein the reversion may be carried out by an esterase etc. Other such systems will be well known to those skilled in the art.

[0182] SOLVATES

[0183] The present invention also includes solvate forms of the sEH inhibitors for use in the methods of the present invention. The terms used in the claims encompass these forms. Preferably, the solvate is a hydrate.

[0184] POLYMORPHS

[0185] The invention further relates to the sEH inhibitors for use in the methods of the present invention in their various crystalline forms, polymorphic forms and (an)hydrous forms. It is well established within the pharmaceutical industry that chemical compounds may be isolated in any of such forms by slightly varying the method of purification and or isolation form the solvents used in the synthetic preparation of such compounds.

[0186] ADMINISTRATION

[0187] The sEH inhibitors / pharmaceutical compositions for use in the methods of the present invention may be adapted for rectal, nasal, intrabronchial, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intraarterial and intradermal), intraperitoneal or intrathecal administration. Preferably the formulation is an orally administered formulation. The formulations may conveniently be presented in unit dosage form, i.e. , in the form of discrete portions containing a unit dose, or a multiple or sub-unit of a unit dose. By way of example, the formulations may be in the form of tablets and sustained release capsules, and may be prepared by any method well known in the art of pharmacy.

[0188] In one preferred embodiment, the sEH inhibitor is administered orally.

[0189] Formulations for oral administration in the present invention may be presented as: discrete units such as capsules, gellules, drops, cachets, pills or tablets each containing a predetermined amount of the active agent; as a powder or granules; as a solution, emulsion or a suspension of the active agent in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; or as a bolus etc. Preferably, these compositions contain from 1 to 250 mg and more preferably from 10-100 mg, of active ingredient per dose.

[0190] For compositions for oral administration (e.g. tablets and capsules), the term “acceptable carrier” includes vehicles such as common excipients e.g. binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropyl-methylcellulose, sucrose and starch; fillers and carriers, for example corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid; and lubricants such as magnesium stearate, sodium stearate and other metallic stearates, glycerol stearate stearic acid, silicone fluid, talc waxes, oils and colloidal silica. Flavouring agents such as peppermint, oil of Wintergreen, cherry flavouring and the like can also be used. It may be desirable to add a colouring agent to make the dosage form readily identifiable. Tablets may also be coated by methods well known in the art.

[0191] A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active agent in a free flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may be optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active agent.

[0192] Other formulations suitable for oral administration include lozenges comprising the active agent in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active agent in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active agent in a suitable liquid carrier.

[0193] Other forms of administration comprise solutions or emulsions which may be injected intravenously, intraarterially, intrathecally, subcutaneously, intradermally, intraperitoneally or intramuscularly, and which are prepared from sterile or sterilisable solutions. Injectable forms typically contain between 10 - 1000 mg, preferably between 10 - 250 mg, of active ingredient per dose.

[0194] The pharmaceutical compositions of the present invention may also be in form of suppositories, pessaries, suspensions, emulsions, lotions, ointments, creams, gels, sprays, solutions or dusting powders. An alternative means of transdermal administration is by use of a skin patch. For example, the active ingredient can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin. The active ingredient can also be incorporated, at a concentration of between 1 and 10% by weight, into an ointment consisting of a white wax or white soft paraffin base together with such stabilisers and preservatives as may be required.

[0195] DOSAGE

[0196] A person of ordinary skill in the art can easily determine an appropriate dose of one of the instant compositions to administer to a subject without undue experimentation. Typically, a physician will determine the actual dosage which will be most suitable for an individual patient, and it will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. The dosages disclosed herein are exemplary of the average case. There can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

[0197] The dosage amount will further be modified according to the mode of administration of the compound. For example, to achieve an “effective amount” for acute therapy, parenteral administration of a compound is typically preferred. An intravenous infusion of the compound in 5% dextrose in water or normal saline, or a similar formulation with suitable excipients, is most effective, although an intramuscular bolus injection is also useful. Typically, the parenteral dose will be about 0.01 to about 100 mg; preferably between 0.1 and 20 mg, in a manner to maintain the concentration of drug in the plasma at a therapeutically effective concentration. The compounds may be administered one to four times daily at a level to achieve a total daily dose of about 0.4 to about 400 mg. The precise amount of an inventive compound which is therapeutically effective, and the route by which such compound is best administered, is readily determined by one of ordinary skill in the art by comparing the blood level of the agent to the concentration required to have a therapeutic effect.

[0198] The compounds of this invention may also be administered orally to the patient, in a manner such that the concentration of drug is sufficient to achieve one or more of the therapeutic indications disclosed herein. Typically, a pharmaceutical composition containing the compound is administered at an oral dose of between about 0.1 to about 500 mg or about 0.1 to about 50 mg in a manner consistent with the condition of the patient. Preferably the oral dose would be about 0.5 to about 50 mg or about 0.5 to about 20 mg. No unacceptable toxicological effects are expected when compounds of the present invention are administered in accordance with the present invention. The compounds of this invention, which may have good bioavailability, may be tested in one of several biological assays to determine the concentration of a compound which is required to have a given pharmacological effect.

[0199] In one preferred embodiment, the sEH inhibitor is administered once a day, preferably orally.

[0200] In one preferred embodiment, the sEH inhibitor is GSK2256294, and is administered at a dose of about 10 to about 50 mg / kg, preferably about 20 to about 40 mg / kg, more preferably, about 25 to about 35 mg / kg, even more preferably about 30 mg / kg based on the body weight of the subject.

[0201] The invention is further described by way of the following non-limiting examples, and figures, wherein:

[0202] Figure 1 shows a schematic of the Study Design: (A) Schematic of prophylactic time course. Participants were dosed with GSK2256294 2 hrs prior to the onset of inflammation. Control participants did not receive any placebo. Subsequently, the forearms of participants were injected intradermally with UVKEc resulting in a local and peripheral inflammatory response. Peripheral blood was collected prior to dose of GSK2256294, 2 hrs after dosing, and then 4, 24 and 48 hrs after the onset of inflammation. Negative pressure blisters were raised at 4 and 24 hrs after the onset of inflammation. (B) Schematic of therapeutic time course. Participants were dosed with GSK2256294 4 hrs after the onset of inflammation. Control participants did not receive any placebo. The forearms of participants were injected intradermally with UVKEc resulting in a local and peripheral inflammatory response. Peripheral blood was collected at baseline, and then 4, 24 and 48 hrs after the onset of inflammation. Negative pressure blisters were raised at 24 and 48 hrs after the onset of inflammation.

[0203] Figure 2 shows a plot representative of a baseline blood sample. An initial gate around all leukocytes is assigned. These cells are taken forward to exclude doublets and dead cells. NK cells, T cells, B cells and neutrophils are removed from the sample by gating for the FITC positive cells, which includes markers for CD3, CD56, CD19, CD20 and CD66b. T cells and B cells are gated in orange and NK cells in yellow. Neutrophils are assigned based on high SSC and high CD 16 expression. The FITC negative cells are taken forward and gated for AF700 H LA-DR positive cells. Classical (CD14+CD16-), Intermediate (CD14+CD16+) and Non-Classical (CD14- CD16+) subsets are assigned according to CD14 and CD16 expression. pDCs are taken from the CD14-CD16- and assigned as CD123+. eDCs are taken from the CD123- cells and assigned according to expression of CD141 (cDC1) and CD1c (cDC2).

[0204] Figure 3 shows how Cytochrome P450-derived lipids are elevated with prophylactic and therapeutic sEH inhibition in plasma (concentration in pg / ml versus time); (A) Quantification of (i) 14,15-EET, (ii) 14,15-DHET, (iii) the ratio of 14,15-EET:14,15-DHET, (iv) 12,13-EpOME, (v)

[0205] 12.13-DiHOME and (vi) 12,13-EpOME:12,13-DiHOME prior to inflammation in participants dosed with GSK2256294 for 2 hrs. (B) Quantification of (i) 14,15-EET, (ii) 14,15-DHET, (iii) the ratio of 14,15-EET:14,15-DHET, (iv) 12,13-EpOME, (v) 12,13-DiHOME and (vi) 12,13- EpOME:12,13-DiHOME during inflammation in the prophylactic arm of the study. (C) Quantification of (i) 14,15-EET, (ii) 14,15-DHET, (iii) the ratio of 14,15-EET:14,15-DHET, (iv)

[0206] 12.13-EpOME, (v) 12,13-DiHOME and (vi) 12,13-EpOME:12,13-DiHOME in the therapeutic arm of the study.

[0207] Figure 4 shows sEH inhibition both prophylactically and therapeutically inhibits expansion of intermediate monocytes; (A) LIMAP of monocyte populations identified in peripheral blood in the therapeutic arm of the study. Total monocytes were extracted from FCS files in FlowJo and then subsequently clustered based on the expression of HLA-DR, CD14, CD16, CD163, CD11c, CCR2, CD86, CD206, CD205, TIM-4 and CD64 using the package CATALYST. Six monocyte populations were identified and labelled. (B) Heatmap of marker expression in each cluster. (C) Monocyte subsets are traditionally split into three groups based on the expression of CD14 and CD16. Classical monocytes are CD14+CD16-, intermediate monocytes are CD14+CD16+ and non-classical monocytes are CD14-CD16+. (i) Classical, (ii) intermediate and (iii) non-classical monocyte numbers during inflammation with prophylactic sEH inhibition, (iv) Classical, (v) intermediate and (vi) non-classical monocytes during inflammation with therapeutic sEH inhibition. (D) (i) LIMAP of monocyte populations faceted by time and treatment in the therapeutic arm of the study, (ii) % abundance of intermediate monocytes quantified by unbiased clustering in the therapeutic arm of the study.

[0208] Figure 5 shows sEH inhibition significantly increases the ratio of 12,13-EpOME:12,13-DiHOME and reduces numbers of intermediate monocytes at the inflammatory site. (A) Quantification of (i) 12,13-EpOME, (ii) 12,13-DiHOME and (iii) 12,13-EpOME:12,13-DiHOME in blister fluid at 4 and 24 hrs in the prophylactic arm of the study. Quantification of (iv) 12,13-EpOME, (v) 12,13- DiHOME and (vi) 12,13-EpOME:12,13-DiHOME, (vii) 14,15-EET, (viii) 14,15-DHET and (ix) the ratio of 14,15-EET:14,15-DHET in blister fluid at 24 and 48 hrs in the therapeutic arm of the study. (B) LIMAP of monocyte populations identified in local inflammatory exudate in the therapeutic arm of the study. Total monocytes were extracted from FCS files in FlowJo and then subsequently clustered based on the expression of HLA-DR, CD14, CD16, CD163, CD11c, CCR2, CD86, CD206, CD205, TIM-4 and CD64 using the package CATALYST. Eight monocyte populations were identified and labelled. (C) LIMAP of monocyte populations faceted by time and treatment in the therapeutic arm of the study. (D) Heatmap of marker expression in each cluster. (E) Monocytes in the blister were identified as Lineage-HLA-DR+ cells and further identified into classical, intermediate and non-classical subsets based on the expression of CD14 and CD16. (i) Classical, (ii) Intermediate and (iii) Non-classical monocytes per blister in the prophylactic arm of the study, (iv) Classical, (v) Intermediate and (vi) Non-classical monocytes per blister in the therapeutic arm of the study.

[0209] Figure 6 shows that CD4 T cells are reduced at the inflammatory site with therapeutic sEH inhibition. The forearms of participants were intradermally injected with UV-killed E. coli (UV- KEc) resulting in a local and peripheral inflammatory response. Untreated participants did not receive any drug or placebo (pink circles). 2 hrs prior to (prophylactic) or 4 hrs after (therapeutic) UV-KEc injection, participants were dosed with 15 mg of GSK2256294, a soluble epoxide hydrolase inhibitor (solid blue squares). In the therapeutic arm, at baseline and 4 hrs these participants were not given any drug or placebo (unfilled blue squares). Local inflammatory exudate was collected, blister fluid was subject to lipidomic analysis using mass spectrometry and leukocytes were analysed by multiparameter flow cytometry at 4 and 24 hrs in participants with prophylactic dosing and 24 and 48 hrs in participants with therapeutic dosing post UV-KEc injection. (A) Time course of monocyte subset numbers at 4, 24 and 48 hrs following UVKEc injection, with quantification of CD4 and CD8 T cells at 24 hrs in untreated participants. (B) (i) CD4, (ii) CD4 T regulatory and (iii) CD8 T cell numbers per blister at 48 hours in treated and untreated participants in the therapeutic arm of the study. (C) The percentage of dead T cells at the inflammatory site at 24 and 48 hrs in the therapeutic arm of the study. (D) Concentration of IL-1 a in pg / ml in blister fluid at 24 and 48 hrs with therapeutic dosing. (E) Intermediate monocytes were sorted using FACS and co-cultured with CD4 T cells at a 5:1 (T celkmonocyte) ratio for 48 hours and subsequently analysed by multi-parameter flow cytometry for activation markers, (i) Representative flow plots showing the expression of CD69 on CD4 T cells cultured alone and with intermediate monocytes for 48 hrs. (ii) Quantification of CD69+ CD4 T cells after 48 hrs when cultured alone and or with intermediate monocytes, n = 3 experimental repeats. (F) Concentration of IL-i p, IL-6, IL-8 and IL-15 in blister fluid at 4, 24 and 48 hrs in control participants. (G) Classical, intermediate and non-classical monocytes and CD8 T cells were sorted using FACS and co-cultured for 4 days at a 5:1 (T celkmonocyte) ratio with a cocktail of pro-inflammatory cytokines (5 ng / ml IL-6, 20 ng / ml IL-8, 10 ng / ml I L-1 p, 20 ng / ml IL-18 and 10 ng / ml IL-15). After 4 days a cytotoxicity assay against the K562 (MHC-deplete) target cell line was performed and specific lysis of K562 cells calculated, n = 2 experimental repeats.

[0210] Figure 7(A) shows neutrophil numbers per blister in the therapeutic arm of the study. Figure 7(B) show dendritic cell populations in blister fluid were categorized into cDC1 (CD141+), cDC2 (CD1c+) and pDC (CD123+). Number of (i) cDC1 , (ii) cDC2 and (iii) pDCs in blister fluid at 24 and 48 hrs in the therapeutic arm of the study. Figure 7(C) Classical, intermediate and non- classical monocytes and CD8 T cells were sorted using FACS and either cultured alone or cocultured for 4 days at a 5:1 (T celkmonocyte) ratio with or without a cocktail of pro-inflammatory cytokines (5 ng / ml IL-6, 20 ng / ml IL-8, 10 ng / ml IL-1 p, 20 ng / ml IL-18 and 10 ng / ml IL-15). After 4 days a cytotoxicity assay against the K562 (MHC-deplete) target cell line was performed and specific lysis of K562 cells calculated, n = 2 experimental repeats.

[0211] Figure 8 shows the profile of circulating and lesional monocytes / macrophages in patients with cutaneous leishmaniasis (CL). (A-B) CD14 and CD16 expression in circulating monocytes from healthy volunteers (HC) and patients with CL. Monocytes were identified as CD45+HLADR+CD3-CD56-CD19-CD20- by flowcytometry. (C) Immune cell deconvolution using ImmQuant software. Values represent relative score based on mean of all samples in each cell type. (D) Dotplots and boxplots of selected cell populations from deconvolution analysis where groups are compared by Wilcoxon rank-sum test. HC, Healthy controls; LCL, Localized Cutaneous Leishmaniasis patients. (E) Representative image of cellular infiltration in CL lesions. Staining was performed according to the indicated markers by immunofluorescence and analyzed by Halo software.

[0212] Figure 9 shows the two faces of chronic immune disorders, namely inflammation and tissue destruction.

[0213] EXAMPLES

[0214] Studies by the Applicant explored how soluble epoxide hydrolase inhibition, and the subsequent elevation of epoxy-oxylipins, affected the resolution of the inflammatory response both systemically and locally in humans. The study used an established UV-killed E. coli (UV-KEc) model of human inflammation, a resolving model that provokes immune effects both in the peripheral blood and at the inflammatory site.

[0215] Materials and Methods

[0216] GSK2256294 was obtained from GSK. GSK2256294 was used in the UV-killed E. coli (UV- KEc) model to induce dermal inflammation in human volunteers to determine the effects of sEH inhibition on immune profiles and soluble mediators of the inflammatory cascade both systemically and locally, alongside clinical signs such as heat, pain and microvascular reactivity.

[0217] The Study Design

[0218] A schematic of the Study Design is shown in Figure 1.

[0219] Prophylactic: The initial visit (-24h) for individuals allocated to the sEH inhibited group included pre-dose recording of baseline inflammatory symptoms and blood sample collection. Prior (-2h) to intradermal injection of UV-killed E. coli, participants were orally dosed with 15 mg of the sEH inhibitor GSK2256294. Untreated participants did not receive a placebo control. Oh time point, dermal inflammation was induced. The protocol for the subsequent visits for the sEH inhibited volunteers was identical to the control group; at the beginning of every visit (0, 4, 24 and 48 h post induction of inflammation) peripheral blood was collected at each timepoint for plasma, cellular analysis by flow cytometry, and storing PBMCs. At the 4 and 24 h time points, the inflammatory site was sampled by creation of suction blisters.

[0220] Therapeutic: Participants attended sessions in the lab four times over the course of the experiment: at 0 hrs to induce inflammation, 4, 24, and 48 hrs after the onset of inflammation. Participants in the drug arm of the study received a single 15 mg dose of GSK2256294 at the 4 hr timepoint after the UV-KEc injection. Untreated participants did not receive a placebo control. Peripheral blood was collected at each timepoint for plasma, cellular analysis by flow cytometry, and storing PBMCs. At the 24 and 48 h time points, the inflammatory site was sampled by creation of suction blisters.

[0221] The UVKEc model

[0222] There is a need to be able to study inflammation and its resolution in vivo and in humans. The human intradermal UV-killed Escherichia coli (E. coli) (UVKEc) model of inflammation was developed by Motwani and colleagues in the Gilroy laboratory to study resolving inflammation in response to a localised bacterial insult (Motwani et al., 2016). Briefly, UV-killed E. coli is administered intradermally into the forearms of a participant. This results in localised inflammation at the site of injection, characterised by mild pain, redness, and swelling. Using negative pressure blister suction cups, two blisters (one on each forearm) can be raised at the inflammatory site to investigate local cellular infiltrate and cytokine, chemokine, and lipid mediator profiles. The UVKEc model also results in systemic changes in peripheral blood, which is collected at each time point for plasma analysis and cellular characterisation. Bacterial growth and preparation

[0223] An antibiotic sensitive strain of E. co / / was purchased from Public Health England (NCTC 10418) and prepared as previously described (Motwani et al., 2016) .

[0224] Under aseptic conditions, the ampoule was opened, and the E. coli was reconstituted in 500 l of sterilised Lysogeny broth (LB). To isolate a single colony, bacteria were spread onto agar plates and incubated overnight at 37°C. LB was used as a negative control. A single colony forming unit (CFU) was picked and transferred to a conical flask containing 10 ml of LB autoclaved broth and cultured for 6 hrs at 37°C at 220 revolutions per minute (RPM) in a bacterial shaking incubator. 750 pl of starter culture was added to 750 ml of LB broth and cultured overnight under the same conditions. Bacteria were pelleted by centrifugation at 4000 g at 4°C for 20 minutes and subsequently washed twice in Phosphate Buffered Saline (PBS) (Corning, 21-040-CV) and resuspended in 40 ml of sterile saline. Bacteria were then subsequently exposed to UV light (302 nm, ChemiDoc, trans-UV mode) for 60 minutes in petri dishes. To assess viability, bacteria were spread onto LB agar plates and incubated overnight at 37°C. The density of non-viable bacteria was counted using a spectrophotometer, with an optical densityeoo of 0.365 taken as 1.5 x 108E. co / z / ml (formulated by Dr Andrew Smith, Division of Medicine, UCL). 300 pl aliquots of 1.5 x 108E. co / z / ml stored at -80°C. To confirm non-viability, samples were sent to the microbiology department at UCL Hospital prior to injection.

[0225] Injection of UVKEc

[0226] UVKEc was injected 7 cm from the crook of the elbow. Prior to injection, the injection site was disinfected. 1.5 x 107UV-Killed E. coli in 100pl saline were injected intradermally into a predefined site on the volar aspect of each forearm.

[0227] Blister technique

[0228] To raise blisters, suction cups were placed over the inflammatory site. Negative pressure machines (Electronic diversities Ltd) were used to create negative pressure in the cup starting from 2-inch Mercury (Hg). Negative pressure was raised over time according to the protocol in Table 1. If the negative pressure was below 9 upon the formation of a bleb, the pressure was not increased, and a blister was left to form. Once the blister was fully formed, the pressure was decreased at 1 Hg / minute. Table 1 Time course for raising a blister using negative pressure blister suction cups

[0229] Blister exudate was collected by piercing the base of the blister with a 26.5-guage needle (BD Biosciences) and aspirating the fluid using a p200 pipette with a filter tip. To ensure the collection of all fluid, a 1 ml syringe was rolled over the blister site and the remaining fluid aspirated.

[0230] Following the collection of the blister fluid, the skin of the blister was further punctured using a needle to prevent the formation of a second blister. The area was cleaned with antiseptic and covered with a plaster.

[0231] Processing of blister sample

[0232] Blister fluid was aspirated and transferred into 50 l of Sodium Citrate (3% in PBS) in a 96-well plate on ice. Cells were centrifuged at 1000g for 5 minutes at 4°C. Blister fluid was aspirated, the volume of fluid recorded and stored in 30 pl aliquots in ProteinLo bind eppendorfs (022431081) at -80°C for further cytokine, chemokine, and lipidomic analysis. Cells were resuspended in 150 pl Ammonium-Chloride-Potassium (ACK) Lysis buffer (Gibco, A10492-01) and incubated at room temperature (RT) for 3 minutes. The sample was centrifuged at 1000g for 5 minutes at 4°C prior to staining for flow cytometry.

[0233] Peripheral blood processing

[0234] Peripheral blood was collected by venepuncture using aseptic technique.

[0235] Peripheral blood for analysis by flow cytometry was collected into an EDTA vacutainer tube (4 ml, Griener Bio-One). For the removal of red blood cells from peripheral blood, 9 ml of ACK lysis buffer was added to 1 ml of whole blood and incubated at RT for 7 minutes. Cells were spun at 400g for 5 minutes at 4°C and the supernatant was aspirated. The pellet was resuspended, washed in PBS, and spun for a second time at 400 g for 5 minutes at 4°C. Cell number was enumerated. The remaining cells were spun at 400g for 5 minutes at 4°C. Cells were transferred to a 96-well plate prior to staining for flow cytometry. Multiparametric Flow Cytometry

[0236] Samples were processed appropriately in preparation for staining. All samples were stained in a volume of 100 pl. Antibody cocktails were prepared with 50 pl of Brilliant Stain Buffer (BSB) (BD Biosciences, 566349), with the remainder of the volume made up with FACS buffer (PBS, 5% Foetal Calf Serum, 2 mM EDTA). All panels were compensated appropriately, either using cells as vehicles for single stains, UltraComp eBeads™ compensation beads (Thermo Fisher, 01-2222-41), or ArC™ Amine Reactive compensation bead kit (Thermo Fisher, A10346). Unstained sample and Fluorescence minus-one (FMO) and, where appropriate, isotype control antibodies were used as a control. Data were acquired on a BD Fortessa UV X20 flow cytometer using BD FACS DIVA software. Data analysis was performed using FlowJo software.

[0237] Cell Surface staining

[0238] All samples with solely extra-cellular staining were stained for 30 minutes at 4°C and subsequently washed with 200 pl of FACS buffer and spun at 400 g for 5 minutes at 4°C. The supernatant was aspirated, and cells were resuspended in 300 pl of 4% Paraformaldehyde (PFA) (SC-281692) prior to acquisition on the flow cytometer.

[0239] The gating strategy for is outlined below. The list of antibodies used is in Table 2. Briefly, monocyte populations were gated as HLA-DR+ and according to the expression of CD14 and CD16. Classical monocytes were gated as CD14+CD16-, intermediate monocytes as CD14+CD16+, and non-classical monocytes as CD14-CD16+.

[0240] Table 2: Antibody list for panel 1

[0241] Gating strategy for monocytes

[0242] Plot representative of a baseline blood sample is shown in Figure 2. An initial gate around all leukocytes assigned. These cells are taken forward to exclude doublets and dead cells. NK cells, T cells, B cells and neutrophils are removed from the sample by gating for the FITC positive cells, which includes markers for CD3, CD56, CD19, CD20 and CD66b. T cells and B cells are gated in orange and NK cells in yellow. Neutrophils are assigned based on high SSC and high CD 16 expression. The FITC negative cells are taken forward and gated for AF700 H LA-DR positive cells. Classical (CD14+CD16-), Intermediate (CD14+CD16+) and Non-Classical (CD14- CD16+) subsets are assigned according to CD14 and CD16 expression. pDCs are taken from the CD14-CD16- and assigned as CD123+. eDCs are taken from the CD123- cells and assigned according to expression of CD141 (cDC1) and CD1c (cDC2). Lipidomics

[0243] Peripheral blood was collected by venepuncture using an aseptic technique. Plasma was isolated by centrifugation of sodium heparin vacutainer tubes (10 ml; Griener Bio-One) at 20,000 xg for 10 min (room temperature). Both plasma and blister fluid tubes were blinded and sent for lipid analysis at the Zeldin laboratory (National Institute for Environmental Health Sciences). The samples were prepared and analysed as previously described (PMID 25114171 ; J Lipid Res. 2014 Oct; 55(10): 2124-2136). Briefly, samples were acidified and washed with wash solution (5 % methanol and 0.1 % acetic acid). To prevent epoxy-oxylipin catabolism t-AUCB (sEH-l; 10 pM final concentration) was added to each sample. Post addition of internal standards, lipid extraction was performed by using 1.5 ml ethyl acetate liquid: liquid extraction. The samples were dried and reconstituted in 50 pl of 30 % glycerol. Liquid-chromatography mass spectrometry was used for the analysis of extracted lipids in 10 ul of sample as previously described (PMID 38786655; FASEB J; 2024 May 31 ; 38(10):e2369).

[0244] Fluorescent activated cell sorting (FACS)

[0245] Cell sorting was performed on the BD FACSDiscover S8 cell sorter. PBMCs were isolated, enumerated and stained in 0.2 pl antibody (CD14 BUV805, CD16 BV711 , HLA-DR PE, CD56 BUV395, TCRap BUV615, CD8 APC-Cy7, CD19 / CD20 FITC, Live / Dead Zombie UV) per 1 million cells for 30 minutes at 4°C. Samples were washed and resuspended in PBS prior to sort.

[0246] CD8 Cytotoxicity Assays

[0247] NK, CD8 T cells and monocyte subsets were isolated using FACS, plated and incubated with and without cytokines (5 ng / ml IL-6 (Thermofisher, A42541), 20 ng / ml IL-8 (Thermofisher, PHC0084), 10 ng / ml IL-1 (3 (Thermofisher, A42509), 20 ng / ml IL-18 (R&D, 9124-IL-050 / CF) and 10 ng / ml IL-15 (Thermofisher, 200-15) for four days. CD8 T cells and monocytes were cultured at a 5:1 ratio.

[0248] K562 target cells were loaded with 15 pM Calcein-AM (Thermofisher) for 30 minutes at 37°C. Cells were washed and seeded at a 20:1 effector to target ratio in a 96 well U-bottom plate. NK cells were used as a positive control (maximum Lysis). K562 cells alone were used as a negative control (spontaneous release). Co-culture plates were spun at 400 g for 1 minute and subsequently incubated for 6 hours at 37°C. Following incubation, samples were spun at 400 g for 5 minutes and 100 pl of supernatant was transferred to a black flat bottom 96 well plate. Fluorescence was quantified using an infinite 200Pro plate reader (Ex: 488 nm, Em: 517 nm). Specific lysis was calculated as: (Sample - Spontaneous release) / (Maximum Lysis - Spontaneous release) * 100.

[0249] Cytochrome P450-derived lipids are elevated with prophylactic and therapeutic sEH inhibition in plasma.

[0250] See Figure 3.

[0251] The forearms of participants were intradermally injected with UV-killed E. coli (UVKEc) resulting in a local and peripheral inflammatory response. Untreated participants did not receive any drug or placebo (pink circles). 2 hrs prior to (prophylactic) or 4 hrs after (therapeutic) UVKEc injection, participants were dosed with 15 mg of GSK2256294, a soluble epoxide hydrolase inhibitor (solid blue squares). In the therapeutic arm, at baseline and 4 hrs these participants were not given any drug or placebo (blue squares). Plasma was collected and subject to lipidomic analysis before dosing with GSK2256294 in the prophylactic arm (-2 hrs), baseline and 4 hrs, 24 hrs and 48 hrs post UVKEc injection. (A) Quantification of (i) 14,15-EET, (ii) 14,15-DHET, (iii) the ratio of 14,15-EET:14,15-DHET, (iv) 12,13-EpOME, (v) 12,13-DiHOME and (vi) 12,13-EpOME:12,13- DiHOME prior to inflammation in participants dosed with GSK2256294 for 2 hrs. (B) Quantification of (i) 14,15-EET, (ii) 14,15-DHET, (iii) the ratio of 14,15-EET:14,15-DHET, (iv) 12,13-EpOME, (v) 12,13-DiHOME and (vi) 12,13-EpOME:12,13-DiHOME during inflammation in the prophylactic arm of the study. (C) Quantification of (i) 14,15-EET, (ii) 14,15-DHET, (iii) the ratio of 14,15-EET:14,15-DHET, (iv) 12,13-EpOME, (v) 12,13-DiHOME and (vi) 12,13- EpOME:12,13-DiHOME in the therapeutic arm of the study. All graphs were analysed using two-way-ANOVA mixed effect analysis with Sidak’s multiple comparisons test, *p <0.05, **p <0.01 , ***p <0.001 , ****p <0.0001.

[0252] Following the pre-dosing regime, within 2 hrs GSK2256294 caused a significant increase in plasma 14,15-EET and a trend towards an increase in 12,13-EpOME, which resulted in a significant increase in the 12,13-EpOME:12,13-DiHOME ratio (Figure 3A). Following induction of inflammation, prophylactic sEH inhibition resulted in a significant increase in the ratio of 12,13- EpOME:12,13-DiHOME at4 and 24 hrs (Figure 3B). In the therapeutic dosing regime, inhibition of sEH resulted in an increase in the levels of 14,15-EET at 24 and 48 hrs and a corresponding signficant increase in the ratio of 14,15-EET:14,15-DHET (Figure 3C). The ratio of 12,13- EpOME:12,13-DiHOME was also singificantly increased at 24 and 48 hrs following therapeutic dosing compared to untreated participants (Figure 3C). Collectively, these data demonstrate efficient inhibition of sEH in plasma by GSK2256294 in this model. sEH inhibition both prophylactically and therapeutically inhibits expansion of intermediate monocytes

[0253] See Figure 4.

[0254] The forearms of participants were intradermally injected with UV-killed E. coli (UVKEc) resulting in a local and peripheral inflammatory response. Untreated participants did not receive any drug or placebo (pink circles). 2 hrs prior to (prophylactic) or 4 hrs after (therapeutic) UVKEc injection, participants were dosed with 15 mg of GSK2256294, a soluble epoxide hydrolase inhibitor (solid blue squares). In the therapeutic arm, at baseline and 4 hrs these participants were not given any drug or placebo (unfilled blue squares). Peripheral blood was collected, and leukocytes analysed at baseline and 4 hrs, 24 hrs and 48 hrs post UVKEc injection using multiparameter flow cytometry. (A) UMAP of monocyte populations identified in peripheral blood in the therapeutic arm of the study. Total monocytes were extracted from FCS files in FlowJo and then subsequently clustered based on the expression of HLA-DR, CD14, CD16, CD163, CD11c, CCR2, CD86, CD206, CD205, TIM-4 and CD64 using the package CATALYST. Six monocyte populations were identified and labelled. (B) Heatmap of marker expression in each cluster. (C) Monocyte subsets are traditionally split into three groups based on the expression of CD14 and CD16. Classical monocytes are CD14+CD16-, intermediate monocytes are CD14+CD16+ and non-classical monocytes are CD14-CD16+. (i) Classical, (ii) intermediate and (iii) non-classical monocyte numbers during inflammation with prophylactic sEH inhibition, (iv) Classical, (v) intermediate and (vi) non-classical monocytes during inflammation with therapeutic sEH inhibition. Data are presented as median ± 95% Cl. All graphs were analysed using two-way- ANOVA mixed effect analysis with Sidak’s multiple comparisons test, *p <0.05, **p <0.01 , ***p <0.001 , ****p <0.0001. (D) (i) UMAP of monocyte populations faceted by time and treatment in the therapeutic arm of the study, (ii) % abundance of intermediate monocytes quantified by unbiased clustering in the therapeutic arm of the study.

[0255] Peripheral blood immune cells were isolated and incubated with antibodies tailored for the identification of mononuclear phagocytes. Data were processed by unbiased multiparametric analysis using the CATALYST package in R which identified six groups - two sub-categories of classical monocytes, one population that is transitioning between classical-intermediate monocytes, one distinct intermediate population, a population transitioning from intermediate to non-classical monocytes and finally nonclassical monocytes; all presented as Uniform Manifold Approximation and Projection (LIMAP) (Figure 4A). Expression markers charted the differentiation of these populations with CD14, CCR2, CD64 and CD205 typically declining as classical monocytes differentiate with a corresponding increase in CD16, HLA-DR, CD86 and CD11c as cells acquire a non-classical phenotype, (Figure 4B). Looking at the effects of GSK2256294 on each of these populations revealed at 24 hrs reduction in numbers of intermediate monocytes following intradermal UV-KEc-driven inflammation, which is quantified and visualised in Figure 4D. Examining these data using conventional gating and expressing the outcomes quantitatively / ml of blood confirmed the inflammation-induced increase in classical monocytes at 4hrs transitioning to successive peaks in intermediate and then non-classical, with prophylactic GSK2256294 blocking the appearance of intermediate monocytes at 24 hrs in the prophylactic arm (Figure 4C). In the therapeutic arm, again classical monocytes expanded in response to UV-KEc with GSK2256294 reducing numbers of intermediate monocyte population at 24 hrs (Figure 4C). These data demonstrate that sEH inhibition blocks the differentiation of classical to intermediate monocytes in a human model of experimental inflammation. sEH inhibition significantly increases the ratio of 12,13-EpOME:12,13-DiHOME and reduces numbers of intermediate monocytes at the inflammatory site

[0256] See Figure 5.

[0257] The forearms of participants were intradermally injected with UV-killed E. coli (UVKEc) resulting in a local and peripheral inflammatory response. Untreated participants did not receive any drug or placebo (pink circles). 2 hrs prior to (prophylactic) or 4 hrs after (therapeutic) UVKEc injection, participants were dosed with 15 mg of GSK2256294, a soluble epoxide hydrolase inhibitor (solid blue squares). In the therapeutic arm, at baseline and 4 hrs these participants were not given any drug or placebo (unfilled blue squares). Local inflammatory exudate was collected, blister fluid was subject to lipidomic analysis using mass spectrometry and leukocytes were analysed by multiparameter flow cytometry at 4 and 24 hrs in participants with prophylactic dosing and 24 and 48 hrs in participants with therapeutic dosing post UVKEc injection. (A) Quantification of (i) 12,13-EpOME, (ii) 12,13-DiHOME and (iii) 12,13-EpOME:12,13-DiHOME in blister fluid at 4 and 24 hrs in the prophylactic arm of the study. Quantification of (iv) 12,13-EpOME, (v) 12,13- DiHOME and (vi) 12,13-EpOME:12,13-DiHOME, (vii) 14,15-EET, (viii) 14,15-DHET and (ix) the ratio of 14,15-EET:14,15-DHET in blister fluid at 24 and 48 hrs in the therapeutic arm of the study. (B) UMAP of monocyte populations identified in local inflammatory exudate in the therapeutic arm of the study. Total monocytes were extracted from FCS files in FlowJo and then subsequently clustered based on the expression of HLA-DR, CD14, CD16, CD163, CD11c, CCR2, CD86, CD206, CD205, TIM-4 and CD64 using the package CATALYST. Eight monocyte populations were identified and labelled. (C) LIMAP of monocyte populations faceted by time and treatment in the therapeutic arm of the study. (D) Heatmap of marker expression in each cluster. (E) Monocytes in the blister were identified as Lineage-HLA-DR+ cells and further identified into classical, intermediate and non-classical subsets based on the expression of CD14 and CD16. (i) Classical, (ii) Intermediate and (iii) Non-classical monocytes per blister in the prophylactic arm of the study, (iv) Classical, (v) Intermediate and (vi) Non-classical monocytes per blister in the therapeutic arm of the study. All graphs were analysed using two- way-ANOVA mixed effect analysis with Sidak’s multiple comparisons test, *p <0.05, **p <0.01 , ***p <0.001 , ****p <0.0001.

[0258] Locally in blister fluid, prophylactic dosing caused a significant increase in 12,13-EpOME only compared to untreated controls and a concomitant reduction in 12,13-DiHOME at 4 hrs, resulting in a significant increase in the ratio of 12,13-EpOME:12,13-DiHOME. A trend towards an increase in 12,13-EpOME and a trend towards a decrease in 12,13-DiHOME resulted in a significant alteration in the ratio at 24 hrs with prophylactic dosing (Figure 5A). 14,15-EET and

[0259] 14.15-DHET was not detected in the blister fluid of participants in the prophylactic arm of the study. Following therapeutic dosing, GSK2256294 had no effect on the levels of 14,15-EET or

[0260] 14.15-DHET, but significantly increased the ratio of 12,13-EpOME:12,13-DiHOME at both 24 and 48 hrs (Figure 5A).

[0261] We applied a similar unbiased analytical approach to identifying mononuclear phagocytes that accumulated at the site of UV-KEc-triggered inflammation. Here we found nine separate populations CD163hiTIM-4l0, CD163hiTIM-4hi, CD206hi, classical monocytes (CM), classical- intermediate monocytes (CM-IM), intermediate monocytes (IM), intermediate to nonclassical (IM-NCM) monocyte transitioning cells, non-classical monocytes (NCM) and finally CD205hiMonocyte-DC (Mo-DC) (Figure 5B) with expression markers for each in Figure 5C. Faceting the LIMAP of mononuclear phagocyte population by time and treatment indicated a reduction in intermediate monocytes at 24 and 48h (Figure 5D). Quantifying monocyte populations per blister with traditional gating revealed that accumulation of intermediate monocytes trended towards a decrease at 24 hrs in the prophylactic participants and at 48 hrs in the therapeutic participants (Figure 5E).

[0262] Prolonged exposure to intermediate monocytes in a pro-inflammatory environment elicits tissue injury

[0263] See Figures 6 and 7. Studies were undertaken to investigate the effect of intermediate monocytes on tissue function in a pro-inflammatory environment. A cell death assay reflective of chronic inflamed tissue was established comprising CD8 T cells with CM, IM and NCM for 4 days and a cocktail of pro- inflammatory cytokines (IL-i p, IL-6, IL-8, IL-16 and IL-18) that are typically absent from resolving tissues (Figure 6F). A CD8 cytotoxicity assay was subsequently performed against a MHC- deplete target cell line. Intermediate monocytes either alone or with CD8 T cells had negligible effects on cell viability (Figure 7). However, the addition of the pro-inflammatory cytokine cocktail caused intermediate monocytes to illicit the greatest level of cell death, Figure 6G. Collectively, these data show that transient intermediate monocytes during inflammatory resolution maintain T cell viability and activity, but their persistence during chronic inflammation and in a pro-inflammatory environment may drive collateral damage leading to loss of tissue function.

[0264] Profile of circulating and lesional monocytes / macrophages in patients with cutaneous leishmaniasis

[0265] Studies were undertaken to investigate the profile of circulating and lesional monocytes / macrophages in patients with cutaneous leishmaniasis.

[0266] See Figures 8A-8E.

[0267] Experimental design:

[0268] Study Subjects: Peripheral blood from 8 untreated cutaneous leishmaniasis (CL) patients were used in this study. The diagnosis of CL was determined by clinical and laboratory criteria. All patients in thestudy tested positive for the PCR / restriction fragment length polymorphism for L. braziliensis and reported no prior Leishmania infections or treatment. The control group consisted of 8 healthy age matched individuals (HC) from non-endemic areas. All study participants were serologically negative for HIV and HBV. They also had no history of chemotherapy, radiotherapy or treatment with immunosuppressive medications within the last 6 months. All volunteers and patients provided written informed consent, and study procedures were performed in accordance with the principles of the Declaration of Helsinki.

[0269] PBMC and T Cells Isolation: PBMCs from CL and HC were isolated by centrifugation of heparinized whole blood through a Ficoll-Hypaque gradient (GE Healthcare, Uppsala, Sweden). Cryopreserved cells from both controls and patients were thawed in RPMI complete medium supplemented with 10% of fetal calf serum. Viability and recovery were measured using trypan blue dye exclusion in haemocytometer. Flow cytometric analysis: was carried out using the following reagents: Live / dead UV Zombie, Lin cocktail FITC (CD3, CD56, CD19, CD20), HLA-DR Brilliant Violet 510, CD16 Brilliant Violet 786, CD14 Alexa fluor 700, CD45 APC-H7 from BD Biosciences. Cell suspensions were incubated with antibody solutions for 30min at 4°C for extracellular staining. Samples were aquired at Fortessa X-20 cytometer (BD Biosciences)and analyzed using FlowJo software (Treestar). Isotype control staining and fluorescence-minus-one controls were used to set the populations.

[0270] Transcriptomic analyses of skin lesions from publicly available data sets: Raw counts matrix from a previous study on the lesional gene expression in CL was obtained from NCBI’s Gene Expression Omnibus (GEO) through accession code:GSE12 7831

[0029] , All analyses of RNA-Seq data were performed using R version 4.0.3 (https: / / www.r-project.org / ) in RStudio 1.4.110. This resulted in count data from 21 skin samples from patients infected with L. braziliensis before treatment and 7 uninfected endemic controls. Patients from this data set consisted of 17 males and 4 females of age 38 (+ / - 16.8) and lesions size ranging from 13 to 1237 mm2. Genes that had less than 10 total counts in all sampleswere removed prior to subsequent analyses, resulting in a count table of 26,545 genes. DESeq2 (Love et al, 2014) that was used to identify differentially expressed genes, considering those with Benjamini-Hochberg p-adjusted value less than 0.05as statistically significant. For visualization and clustering, count data were normalized, and variance stabilized through the vst function available in DESeq2 package. The Principal Component Analysis (PCA) was calculated using plotPCA function, also in the DESeq2 package. The CellAge database (Avelar et al, 2020) from Human Aging Genomic Resources (Tacutu et al, 2018) was adapted with added senescence-associated genes and was used to investigate the senescence signature in the dataset. The STRING database v11.0

[0035] was used to determine human protein-protein interactions (minimum score of 700) within the senescence-associated DEGs through use of the RITAN package. Immune cell deconvolution was performed through ImmQuant software (Frishberg et al, 2016) using the DMAP database, with scores calculated relative to the mean of all samples. Spearman's correlation was calculated between variance stabilized gene expression values and cell population scores predicted by ImmQuant. A correlation matrix was generated with the cor function in R, and the p-values matrix was created using the cor_pmat function in ggcorrplot package. The results were then plotted as a heatmap. Several tools were used for visualizing the results of RNA-Seq analyses. Heatmaps were constructed with relative expression vst transformed values, centred across gene means, through the ComplexHeatmap package. The package graph was employed to construct the network plots. All other plots were generated with ggplot2. Multiplex Immunofluorescence: Subjects diagnosed with cutaneous leishmaniasis (n = 4) were recruited from the Reference Center for Diagnosis and Treatment of c Leishmaniasis in Corte de Pedra - BA, Brazil. Written informed consent was obtained from all participants. Ethical approval for this study was granted by the Hospital Universitario Professor Edgard Santos’s Ethical Committee. FPAparaffin embedded histological sections were obtained from skin biopsies and prepared on poly-L-lysine coated glass slides. These FPA-paraffin embedded sections were dewaxed and rehydrated using xylene ethanol series for immunofluorescence. Permeabilization was performed using a PBS solution with 0.3% Triton X-100. Antigen retrieval was accomplished using Tris-EDTA (pH9) and Citrate (pH6) buffers underpressure conditions. Sections were stained with conjugatedantibodies anti-CD4 (ab280849, Abeam), anti-CD8 (372906, BioLegend), anti-CD68 (ab277276, Abeam) and anti- GranzymeB (14-8822-82, Thermo Fisher) for 18 h at 4°C. Slides were mounted with Fluoroshield Mounting Medium containing DAPI followed by image acquisition using the Axioscan 7, Zeiss, Germany. Segmentation analysis was developed using the Highplex FL module from HALO Software(lndica Labs) where the CD4 T cell population was defined by CD4+, CD8- and CD68- cells.

[0271] Results: Cytotoxicity and inflammation are key features in the pathogenesis of cutaneous leishmaniasis. In this regard, many immune cells, such as CD4+T cells, CD8+T cells, and macrophages / monocytes, are drivers of these features. Cutaneous leishmaniasis patients show a significant increase in circulating CD16+(non-classical) and CD14+CD16+(intermediate) monocytes, while CD14+CD16“ (classical) monocytes remain unchanged compared to controls (Figure 8A and B). Notably, CD14+CD16+(intermediate) monocytes, are more inflammatory, and may play a key role in driving chronic inflammation and tissue damage in CL lesions, suggesting their involvement in the exacerbation of disease pathology. To investigate the immune cell types involved in the immune response in situ, within CL lesions, we performed a deconvolution analysis of bulk RNA-Seq data using ImmQuant. This analysis revealed distinct immune cell type compositions between patients and healthy controls (Figure 8C). Notably, CD8+effector memory T cells (CD8 TEM), CD8+effector memory T cells that re-express CD45RA (CD8 TEMRA), NK cells, and monocytes were significantly enriched in CL lesions (Figure 8D). These immune cell populations are closely associated with the heightened cytotoxic and inflammatory responses observed in the lesional tissue. Histological analysis of CL lesional skin demonstrated intense cellular infiltration, with prominent areas containing CD4+and CD8+GzB+T cells, as well as CD68+macrophages (Figure 8E). Interestingly, spatial analysis reveals that macrophages interact with these lymphocytes, suggesting a complex interplay that could contribute to the immunopathogenesis of the disease. Conclusion: The results of the study provide important insights into the immune landscape of cutaneous leishmaniasis (CL) and highlight the significant role of monocytes / macrophages in the pathogenic responses associated with the disease. Given the lack of effective vaccines and the challenges associated with current drug therapies, identifying these immune mechanisms offers potential therapeutic targets to modulate the destructive immune responses in CL

[0272] Summary

[0273] Findings from the above studies shed light on the role of intermediate monocyte-derived macrophages during the resolution of acute inflammation. In particular, studies have demonstrated the effect of intermediate monocytes on tissue function in a pro-inflammatory environment, and the significant role of monocytes / macrophages in the pathogenic responses associated with cutaneous leishmaniasis. Looking at the other key cells types present at 24h, the phase when intermediate monocyte-derived macrophages predominate, there was no difference in numbers of neutrophils indicating that as they disappeared uniformly over time in line with established resolution indices, neutrophil clearance was most likely facilitated by classical monocyte-derived macrophages. However, numbers of T cells were significantly reduced at 48h following GSK2256294 coincident with an increase in CD4 T cells acquiring a live dead stain and remnants of immune debris. These findings are consistent with intermediate monocyte-derived macrophages being able to maintain T cell viability, creating a tentative link with helping to imprint long-term tissue immunity following infection. However, when intermediate monocytes expand and turn pathogenic, there is a potential role for sEH inhibition in treating chronic diseases of inflammation as the data show a reduction in the differentiation of classical monocyte population into intermediate and subsequently non-classical monocytes concomitant with a statistically significant reduction in pain and blood flow to the site of infection after 24 hours.

[0274] Various modifications and variations of the described aspects of the invention will be apparant to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims. REFERENCES

[0275] Podolin, P. L., Bolognese, B. J., Foley, J. F., Long, E., Peck, B., Umbrecht, S., Zhang, X., Zhu, P., Schwartz, B., Xie, W., Quinn, C., Qi, H., Sweitzer, S., Chen, S., Galop, M., Ding, Y., Belyanskaya, S. L., Israel, D. I., Morgan, B. A., Callahan, J. F. (2013). In vitro and in vivo characterization of a novel soluble epoxide hydrolase inhibitor. Prostaglandins and Other Lipid Mediators. https: / / doi.Org / 10.1016 / j.prostaglandins.2013.02.001

[0276] Lazaar, A. L., Yang, L., Boardley, R. L., Goyal, N. S., Robertson, J., Baldwin, S. J., Newby, D. E., Wilkinson, I. B., Tai-Singer, R., Mayer, R. J., & Cheriyan, J. (2016). Pharmacokinetics, pharmacodynamics and adverse event profile of GSK2256294, a novel soluble epoxide hydrolase inhibitor. British Journal of Clinical Pharmacology; 81 :5 / 971-979. https: / / doi.org / 10.1111 / bcp.12855

[0277] Luther, J. M., Ray, J., Wei, D., Koethe, J. R., Hannah, L., Dematteo, A., Manning, R., Terker, A. S., Peng, D., Nian, H., Yu, C., Mashayekhi, M., Gamboa, J., & Brown, N. J. (2021). GSK2256294 Decreases sEH (Soluble Epoxide Hydrolase) Activity in Plasma, Muscle, and Adipose and Reduces F2-lsoprostanes but Does Not Alter Insulin Sensitivity in Humans. Hypertension, 78, 1092-1102. https: / / doi.org / 10.1161 / HYPERTENSIONAHA.121.17659

[0278] Martini, R. P., Siler, D., Cetas, J., Alkayed, N. J., Allen, E., & Treggiari, M. M. (2022). A Double-Blind, Randomized, Placebo-Controlled Trial of Soluble Epoxide Hydrolase Inhibition in Patients with Aneurysmal Subarachnoid Hemorrhage. Neurocritical Care, 36(3), 905-915. https: / / doi.Org / 10.1007 / S12028-021-01398-8 / TABLES / 5

[0279] Mashayekhi, M., Wanjalla, C. N., Warren, C. M., Simmons, J. D., Ghoshal, K., Pilkinton, M., Bailin, S. S., Gabriel, C. L., Pozzi, A., Koethe, J. R., Brown, N. J., Kalams, S. A., & Luther, J. M. (2022). The soluble epoxide hydrolase inhibitor GSK2256294 decreases the proportion of adipose pro-inflammatory T cells. Prostaglandins & Other Lipid Mediators, 158, 106604. https: / / doi.Org / 10.1016 / J. PROSTAGLAN DI NS.2021 .106604

[0280] Yang, L., Cheriyan, J., Gutterman, D. D., Mayer, R. J., Ament, Z., Griffin, J. L., Lazaar, A. L., Newby, D. E., Tai-Singer, R., & Wilkinson, I. B. (2017). Mechanisms of Vascular Dysfunction in COPD and Effects of a Novel Soluble Epoxide Hydrolase Inhibitor in Smokers. Chest. https: / / doi.Org / 10.1016 / j.chest.2016.10.058

[0281] Cormican, S., & Griffin, M. D. (2020). Human Monocyte Subset Distinctions and Function: Insights From Gene Expression Analysis. In Frontiers in Immunology. https: / / d0i.0rg / l 0.3389 / fimmu.2020.01070

[0282] Griffin, J. D., Ritz, J., Nadler, L. M., & Schlossman, S. F. (1981). Expression of myeloid differentiation antigens on normal and malignant myeloid cells. The Journal of Clinical Investigation, 68(4), 932-941. https: / / doi.org / 10.1172 / JCI110348 Passlick, B., Flieger, D., & Ziegler-Heitbrock, H. (1989). Identification and Characterization of a Novel Monocyte Subpopulation in Human Peripheral Blood. Blood, 74(7), 2527-2534. https: / / doi.Org / 10.1182 / BLOOD.V74.7.2527.2527

[0283] Ziegler-Heitbrock, H. W. L. (2000). Definition of human blood monocytes. Journal of Leukocyte Biology, 67(5), 603-606. https: / / doi.Org / 10.1002 / JLB.67.5.603

[0284] Ziegler-Heitbrock, L., Ancuta, P., Crowe, S., Dalod, M., Grau, V., Hart, D. N., Leenen, P. J. M., Liu, Y. J., MacPherson, G., Randolph, G. J., Scherberich, J., Schmitz, J., Shortman, K., Sozzani, S., Strobl, H., Zembala, M., Austyn, J. M., & Lutz, M. B. (2010). Nomenclature of monocytes and dendritic cells in blood. In Blood, https: / / doi.org / 10.1182 / blood-2010-02- 258558

[0285] J. X. Jiang etal., EETs Attenuate Ox-LDL-lnduced LTB4 Production and Activity by Inhibiting p38 MAPK Phosphorylation and 5-LO / BLT 1 Receptor Expression in Rat Pulmonary Arterial Endothelial Cells. PLoS One 10, e0128278 (2015).

[0286] K. Node et al., Anti-inflammatory properties of cytochrome P450 epoxygenase-derived eicosanoids. Science (New York, N.Y285, 1276-1279 (1999).

[0287] M. Y. Henein et a / ; Int. J. Mol. Sci. 2022, 23 (21), 12906; https: / / doi.org / 10.3390 / ijms232112906

[0288] Gomez, G. A., Morisseau, C., Hammock, B. D., & Christianson, D. W. (2004). Structure of human epoxide hydrolase reveals mechanistic inferences on bifunctional catalysis in epoxide and phosphate ester hydrolysis. Biochemistry, 43(10), 4716-4723. https: / / doi.org / 10.1021 / BI036189J

[0289] M. Dirchwolf et al, World J Hepatol. 2015 Aug 8; 7(16): 1974-1981. Published online 2015 Aug 8. doi: 10.4254 / wjh.v7.i16.1974

[0290] H. Zhao et al, Signal Transduction and Targeted Therapy volume 6, Article number: 263 (2021)

[0291] Sura, P., Sura, R., Enayetallah, A. E., & Grant, D. F. (2008). Distribution and Expression of Soluble Epoxide Hydrolase in Human Brain. Journal of Histochemistry and Cytochemistry, 56(8), 551. https: / / doi.org / 10.1369 / JHC.2008.950659

[0292] VanRollins, M., Kaduce, T. L., Knapp, H. R., & Spector, A. A. (1993). 14,15- Epoxyeicosatrienoic acid metabolism in endothelial cells. Journal of Lipid Research, 34(11), 1931-1942. https: / / doi.Org / 10.1016 / S0022-2275(20)35111 -7

[0293] Yannick Senouvo, F., Tabet, Y., Morin, C., Albadine, R., Sirois, C., & Rousseau, E. (2011). Improved bioavailability of epoxyeicosatrienoic acids reduces TP-receptor agonist-induced tension in human bronchi. Am J Physiol Lung Cell Mol Physiol, 301, 675-682. https: / / doi.Org / 10.1152 / ajplung.00427.2010. -Epoxyeicosatrienoic

[0294] Yu, Z., Davis, B. B., Morisseau, C., Hammock, B. D., Olson, J. L., Kroetz, D. L., & Weiss,

[0295] R. H. (2004). Vascular localization of soluble epoxide hydrolase in the human kidney. American Journal of Physiology - Renal Physiology, 286(4 55-4), 720-726. https: / / doi.Org / 10.1152 / AJPRENAL.00165.2003

[0296] Zhang, D., Xie, X., Chen, Y., Hammock, B. D., Kong, W., & Zhu, Y. (2012). Homocysteine upregulates soluble epoxide hydrolase in vascular endothelium in vitro and in vivo.

[0297] Circulation Research, 110(6), 808-817. https: / / doi.org / 10.1161 / CIRCRESAHA.111.259325

[0298] Highton, J., Carlisle, B., & Palmer, D. G. (1995). Changes in the phenotype of monocytes / macrophages and expression of cytokine mRNA in peripheral blood and synovial fluid of patients with rheumatoid arthritis. Clinical and Experimental Immunology, 102(3), 541-546. https: / / doi.Org / 10.1111 / J.1365-2249.1995.TB03850.X

[0299] Kawanaka, N., Yamamura, M., Aita, T., Morita, Y., Okamoto, A., Kawashima, M., Iwahashi, M., Ueno, A., Ohmoto, Y., & Makino, H. (2002). CD14+.CD16+ blood monocytes and joint inflammation in rheumatoid arthritis. Arthritis and Rheumatism, 46(10), 2578-2586. https: / / doi.Org / 10.1002 / ART.10545

[0300] Rossol, M., Kraus, S., Pierer, M., Baerwald, C., & Wagner, U. (2012). The CD14(bright) CD16+ monocyte subset is expanded in rheumatoid arthritis and promotes expansion of the Th17 cell population. Arthritis and Rheumatism, 64(3), 671-677. https: / / doi.org / 10.1002 / ART.33418

[0301] Ruiz-Limon, P., Ortega-Castro, R., Barbarroja, N., Perez-Sanchez, C., Jamin, C., Patino- T rives, A. M., Luque-Tevar, M., Ibanez-Costa, A., Perez-Sanchez, L., de La Rosa, I. A., Abalos-Aguilera, M. C., Jimenez-Gomez, Y., Calvo-Gutierrez, J., Font, P., Escudero- Contreras, A., Alarcon-Riquelme, M. E., Collantes-Estevez, E., & Lopez-Pedrera, C. (2019). Molecular characterization of monocyte subsets reveals specific and distinctive molecular signatures associated with cardiovascular disease in rheumatoid arthritis. Frontiers in Immunology, W(MAY), 1111. https: / / doi.org / 10.3389 / FIMMU.2019.01111 / BIBTEX

[0302] Evans, H. G., Gullick, N. J., Kelly, S., Pitzalis, C., Lord, G. M., Kirkham, B. W., & Taams, L.

[0303] S. (2009). In vivo activated monocytes from the site of inflammation in humans specifically promote Th 17 responses. Proceedings of the National Academy of Sciences of the United States of America, 106( 5), 6232-6237. https: / / doi.org / 10.1073 / PNAS.0808144106 / SUPPL_FILE / 0808144106SI.PDF

[0304] Kinne, R. W., Brauer, R., Stuhlmuller, B., Palombo-Kinne, E., & Burmester, G. R. (2000). Macrophages in rheumatoid arthritis. Arthritis Research, 2(3), 189-202. https: / / d0i.0rg / l 0.1186 / AR86 Roberts, C. A., Dickinson, A. K., & Taams, L. S. (2015). The interplay between monocytes / macrophages and CD4+ T cell subsets in rheumatoid arthritis. Frontiers in Immunology, 6(NOV), 571. https: / / doi.org / 10.3389 / FIMMU.2015.00571 / XML / NLM

[0305] Walter, G. J., Evans, H. G., Menon, B., Gullick, N. J., Kirkham, B. W., Cope, A. P., Geissmann, F., & Taams, L. S. (2013). Interaction with activated monocytes enhances cytokine expression and suppressive activity of human CD4+CD45ro+CD25+CD127low regulatory T cells. Arthritis & Rheumatism, 65(3), 627-638. https: / / doi.org / 10.1002 / ART.37832

[0306] Grip, O., Bredberg, A., Lindgren, S., & Henriksson, G. (2007). Increased subpopulations of CD16+ and CD56+ blood monocytes in patients with active Crohn’s disease. Inflammatory Bowel Diseases, 13(5), 566-572. https: / / doi.org / 10.1002 / IBD.20025

[0307] Hirose, S., Lin, Q., Ohtsuji, M., Nishimura, H., & Verbeek, J. S. (2019). Monocyte subsets involved in the development of systemic lupus erythematosus and rheumatoid arthritis. International Immunology. https: / / doi.Org / 10.1093 / intimm / dxz036

[0308] Urra, X., Villamor, N., Amaro, S., Gomez-Choco, M., Obach, V., Oleaga, L., Planas, A. M., & Chamorro, A. (2009). Monocyte subtypes predict clinical course and prognosis in human stroke. Journal of Cerebral Blood Flow and Metabolism, 29(5), 994-1002. https: / / doi.org / 10.1038 / jcbfm.2009.25

[0309] Zhu, H., Hu, F., Sun, X., Zhang, X., Zhu, L., Liu, X., Li, X., Xu, L., Shi, L., Gan, Y., & Su, Y. (2016). CD16+ monocyte subset was enriched and functionally exacerbated in driving T- cell activation and B-cell response in systemic lupus erythematosus. Frontiers in Immunology, https: / / doi.org / 10.3389 / fimmu.2016.00512

[0310] Fingerle, G., Pforte, A., Passlick, B., Blumenstein, M., Strobel, M., & Ziegler- Heitbrock, H. (1993). The Novel Subset of CD14+ / CD16+ Blood Monocytes Is Expanded in Sepsis Patients. Blood, 82(10), 3170-3176. https: / / doi.org / 10.1182 / BLGOD.V82.10.3170.3170

[0311] Balboa, L., Barrios-Payan, J., Gonzalez-Dominguez, E., Lastrucci, C., Lugo-Villarino, G., Mata-Espinoza, D., Schierloh, P., Kviatcovsky, D., Neyrolles, O., Maridonneau-Parini, I., Sanchez-Torres, C., Sasiain, M. del C., & Hernandez-Pando, R. (2015). Diverging biological roles among human monocyte subsets in the context of tuberculosis infection. Clinical Science (London, England : 1979), 129(4), 319-330. https: / / doi.Org / 10.1042 / CS20150021

[0312] Castano, D., Garcia, L. F., & Rojas, M. (2011). Increased frequency and cell death of CD16+ monocytes with Mycobacterium tuberculosis infection. Tuberculosis (Edinburgh, Scotland), 91(5), 348-360. https: / / doi.Org / 10.1016 / J.TUBE.2011.04.002 Kratofil, R. M., Kubes, P., & Deniset, J. F. (2017). Monocyte conversion during inflammation and injury. In Arteriosclerosis, Thrombosis, and Vascular Biology. https: / / doi.Org / 10.1161 / ATVBAHA.116.308198

[0313] Han, J., Wang, B., Han, N., Zhao, Y., Song, C., Feng, X., Mao, Y., Zhang, F., Zhao, H., & Zeng, H. (2009). CD14(high)CD16(+) rather than CD14(low)CD16(+) monocytes correlate with disease progression in chronic HIV-infected patients. Journal of Acquired Immune Deficiency Syndromes (1999), 52(5), 553-559. https: / / doi.Org / 10.1097 / QAI .0B013E3181 C1 D4FE

[0314] Hijdra, D., Vorselaars, A. D., Grutters, J. C., Claessen, A. M., & Rijkers, G. T. (2012).

[0315] Differential expression of TNFR1 (CD120a) and TNFR2 (CD120b) on subpopulations of human monocytes. Journal of Inflammation (London, England), 9, 38. https: / / doi.org / 10.1186 / 1476-9255-9-38

[0316] Scott, P. & Novais, F. O. Cutaneous leishmaniasis: Immune responses in protection and pathogenesis. Nat Rev Immunol 16, 581-592 (2016).

[0317] WHO. World Health Organoization. Leihmaniasis Geneva: WHO. 2022. (2022).

[0318] Novais, F. O. et al. CD8+ T cell cytotoxicity mediates pathology in the skin by inflammasome activation and IL-1 p production. PLoS Pathog 13, e1006196 (2017).

[0319] Melby, P. C. et al. Increased expression of proinflammatory cytokines in chronic lesions of human cutaneous leishmaniasis. Infect Immun 62, 837-842 (1994). da Silva Santos, C. & Brodskyn, C. I. The Role of CD4 and CD8 T Cells in Human Cutaneous Leishmaniasis. Front Public Health 2, Article 165, 1-6 (2014).

[0320] Santos, C. D. S. et al. CD8+ granzyme B+ -mediated tissue injury vs. CD4 + IFNy+ - mediated parasite killing in human cutaneous leishmaniasis. Journal of Investigative Dermatology 133, 1533-1540 (2013). https: / / www.r-proje ct.org / . R Core Team. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing. (2020).

[0321] Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15, 550 (2014).

[0322] Avelar, R. A. et al. A multidimensional systems biology analysis of cellular senescence in aging and disease. Genome Biol 21 , 91 (2020).

[0323] Tacutu, R. et al. Human Ageing Genomic Resources: new and updated databases.

[0324] Nucleic Acids Res 46, D1083-D1090 (2018). Frishberg, A., Brodt, A., Steuerman, Y. & Gat-Viks, I. ImmQuant: a user-friendly tool for inferring immune cell-type composition from gene-expression data. Bioinformatics 32, 3842-3843 (2016).

[0325] Motwani MP, Flint JD, De Maeyer RP, Fullerton JN, Smith AM, Marks DJ, Gilroy DW; Novel translational model of resolving inflammation triggered by UV-killed E. coli; J Pathol Clin Res. 2016 May 4;2(3): 154-65. doi: 10.1002 / cjp2.43. eCollection 2016

[0326] Jul.PMID: 27499924

[0327] Lugo-Villarino G, Neyrolles O.; Dressed not to kill: CD16+ monocytes impair immune defence against tuberculosis; Eur J Immunol. 2013 Feb;43(2):327-30. doi:

[0328] 10.1002 / eji.201243256. PM ID: 23322255

[0329] Han J, Wang B, Han N, Zhao Y, Song C, Feng X, Mao Y, Zhang F, Zhao H, Zeng H.; CD14(high)CD16(+) rather than CD14(low)CD16(+) monocytes correlate with disease progression in chronic HIV-infected patients; J Acquir Immune Defic Syndr. 2009 Dec;52(5):553-9. doi: 10.1097 / qai.0b013e3181c1d4fe.PMID: 19950429

Claims

CLAIMS1 . A method of treating or preventing tissue damage, or treating or preventing tissue destruction, in a subject, said method comprising administering to the subject a therapeutically or prophylactically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.

2. A method according to claim 1 for treating tissue damage or tissue destruction in a subject, said method comprising administering to the subject a therapeutically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.

3. A method according to claim 1 for preventing tissue damage or tissue destruction in a subject, said method comprising administering to the subject a prophylactically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.

4. A method according to any preceding claim, wherein the subject has a disease selected from a chronic inflammatory disease, an autoimmune disease, cardiovascular disease, cancer, dermatitis, dementia, age-related inflammation, cirrhosis and atherosclerosis.

5. A method according to claim 4, wherein the subject has a chronic inflammatory disease, preferably an inflammatory bowel disease, more preferably Crohn’s disease or ulcerative colitis.

6. A method according to claim 4, wherein the subject has an autoimmune disease, preferably rheumatoid arthritis, psoriatic arthritis, reactive arthritis, juvenile idiopathic arthritis or systemic lupus erythematosus.

7. A method according to any one of claims 1 to 3 for treating or preventing internal organ damage.

8. A method according to any preceding claim, wherein the sEH inhibitor inhibits the expansion of the intermediate monocyte cell population in the subject.

9. A method of inhibiting the expansion of the intermediate monocyte cell population in a subject, said method comprising administering to the subject a therapeutically or prophylactically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.

10. A method according to claim 9 wherein the subject has a disease selected from a chronic inflammatory disease, an autoimmune disease, cancer, cardiovascular disease, dermatitis, dementia, age-related inflammation, cirrhosis and atherosclerosis.

11. A method according to claim 10, wherein the subject has a chronic inflammatory disease, preferably an inflammatory bowel disease, more preferably Crohn’s disease or ulcerative colitis.

12. A method according to claim 10, wherein the subject has an autoimmune disease, preferably rheumatoid arthritis, psoriatic arthritis, reactive arthritis, juvenile idiopathic arthritis or systemic lupus erythematosus.

13. A method of inhibiting disease progression in a subject having a chronic inflammatory disease, an autoimmune disease, cancer, cardiovascular disease, dermatitis, dementia, age-related inflammation, cirrhosis or atherosclerosis, said method comprising administering to the subject a therapeutically effective amount of a soluble epoxide hydrolase (sEH) inhibitor, whereby the sEH inhibitor inhibits the expansion of the intermediate monocyte cell population in the subject.

14. A method according to claim 13 wherein the subject has an autoimmune disease, preferably rheumatoid arthritis, psoriatic arthritis, reactive arthritis, juvenile idiopathic arthritis or systemic lupus erythematosus15. A method according to claim 13, wherein the subject has a chronic inflammatory disease, preferably an inflammatory bowel disease, more preferably Crohn’s disease or ulcerative colitis.

16. A method of treating or preventing tissue damage, or treating or preventing tissue destruction, in a subject, said method comprising administering to the subject a therapeutically or prophylactically effective amount of a soluble epoxide hydrolase (sEH) inhibitor, whereby the sEH inhibitor inhibits the expansion of the intermediate monocyte cell population in the subject.

17. A method according to claim 16 wherein the subject has a disease selected from a chronic inflammatory disease, an autoimmune disease, cancer, cardiovascular disease, dermatitis, dementia, age-related inflammation, cirrhosis and atherosclerosis.

18. A method according to claim 16 wherein the subject has an autoimmune disease, preferably rheumatoid arthritis, psoriatic arthritis, reactive arthritis, juvenile idiopathic arthritis or systemic lupus erythematosus.

19. A method according to claim 16, wherein the subject has a chronic inflammatory disease, preferably an inflammatory bowel disease, more preferably Crohn’s disease or ulcerative colitis.

20. A method of treating the pathogenesis of chronic inflammatory disease or chronic inflammation in a subject, said method comprising administering to the subject a therapeutically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.21 . A method according to claim 20, wherein the sEH inhibitor inhibits the expansion of the intermediate monocyte cell population in the subject.

22. A method of treating the pathogenesis of chronic inflammatory disease or chronic inflammation in a subject, said method comprising administering to the subject a soluble epoxide hydrolase (sEH) inhibitor in an amount sufficient to inhibit the expansion of the intermediate monocyte cell population in the subject.

23. A method of inhibiting the monocyte differentiation pathway in a subject having chronic inflammatory disease or chronic inflammation, said method comprising administering to the subject a therapeutically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.

24. A method according to claim 23, wherein the sEH inhibitor inhibits the expansion of the intermediate monocyte cell population in the subject.

25. A method according to claim 23, wherein the sEH inhibitor reduces the population of intermediate monocytes in peripheral blood.

26. A method according to claim 23, wherein the sEH inhibitor reduces the population of intermediate monocytes at a site of inflammation.

27. A method according to claim 23, wherein the sEH inhibitor reduces the population of intermediate monocytes at a tissue site distal to a primary site of inflammation or disease.

28. A method of invoking an innate immune response in a subject having chronic inflammation, said method comprising administering to the subject a soluble epoxide hydrolase (sEH) inhibitor in an amount sufficient to inhibit the expansion of the intermediate monocyte cell population in the subject.

29. A method of reducing the number of circulating intermediate monocytes in a subject having chronic inflammation, said method comprising administering to the subject a therapeutically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.

30. A method of inhibiting the development of chronic inflammation at a tissue site distal to a primary site of chronic inflammation in a subject, said method comprising administering to the subject a therapeutically effective amount of an sEH inhibitor.

31. A method of decelerating the development of chronic inflammation at a tissue site distal to a primary site of chronic inflammation in a subject, said method comprising administering to the subject a therapeutically effective amount of an sEH inhibitor.

32. A method of treating or preventing internal organ damage in a subject, said method comprising administering to the subject a therapeutically or prophylactically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.

33. A method of treating or preventing leishmaniasis in a subject, said method comprising administering to the subject a therapeutically or prophylactically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.

34. A method of treating or preventing tissue damage, or treating or preventing tissue destruction, or treating or preventing tissue lesions, in a subject having leishmaniasis, or a subject infected with Leishmania, said method comprising administering to the subject a therapeutically or prophylactically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.

35. A method according to claim 33 or claim 34, wherein the sEH inhibitor inhibits the expansion of the intermediate monocyte cell population in the subject.

36. A method of inhibiting the expansion of the intermediate monocyte cell population in a subject having leishmaniasis or a subject infected with Leishmania, said methodcomprising administering to the subject a therapeutically or prophylactically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.

37. A method of inhibiting the monocyte differentiation pathway in a subject having leishmaniasis, or a subject infected with Leishmania, said method comprising administering to the subject a therapeutically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.

38. A method according to claim 37, wherein the sEH inhibitor inhibits the expansion of the intermediate monocyte cell population in the subject.

39. A method according to any one of claims 33 to 38, wherein the sEH inhibitor reduces the population of intermediate monocytes at a site of inflammation.

40. A method of invoking an innate immune response in a subject having leishmaniasis, or a subject infected with Leishmania, said method comprising administering to the subject a soluble epoxide hydrolase (sEH) inhibitor in an amount sufficient to inhibit the expansion of the intermediate monocyte cell population in the subject.41 . A method of reducing the number of circulating intermediate monocytes in a subject having leishmaniasis, or a subject infected with Leishmania, said method comprising administering to the subject a therapeutically effective amount of a soluble epoxide hydrolase (sEH) inhibitor.

42. A method according to any one of claims 33 to 41 wherein the leishmaniasis is cutaneous leishmaniasis.

43. A method according to any one of claims 33 to 41 wherein the leishmaniasis is mucosal leishmaniasis.

44. A method according to any one of claims 33 to 41 wherein the leishmaniasis is visceral leishmaniasis.

45. A method according to any one of claims 33 to 44 wherein the subject is infected with a Leishmania species selected from L donovani, L infantum (synonym: L chagasi), Leishmania martiniquensis, L braziliensis, L guyanensis , L panamensis , L amazonensis , L aethiopica and L tropica.

46. A method according to any preceding claim wherein the sEH inhibitor is selected from GSK2256294, sorafenib, EC5026, BI1935, glimepiride, TPPU, dCP2PU, t-AUCB, AUDA and t-TUCB.

47. A method according to any preceding claim wherein the sEH inhibitor is GSK2256294, or a pharmaceutically acceptable salt thereof.

48. A method according to any preceding claim wherein the sEH inhibitor is administered orally.

49. A method according to any preceding claim wherein the sEH inhibitor is administered once a day.

50. A method according to any one of claims 47 to 49 wherein the GSK2256294 is administered at a dose of about 10 to about 50 mg / kg, preferably about 20 to about 40 mg / kg, more preferably, about 25 to about 35 mg / kg, even more preferably about 30 mg / kg based on the body weight of the subject.

51. A method according to any preceding claim wherein the sEH inhibitor is administered in the form of a pharmaceutical composition comprising a pharmaceutically acceptable diluent, excipient or carrier.

52. A soluble epoxide hydrolase (sEH) inhibitor for use in treating or preventing leishmaniasis, or for use in treating or preventing an infection with Leishmania.

53. A soluble epoxide hydrolase (sEH) inhibitor for use according to claim 52, wherein the sEH inhibitor inhibits the expansion of the intermediate monocyte cell population in the subject.

54. A soluble epoxide hydrolase (sEH) inhibitor for use according to 52 or claim 53 wherein the sEH inhibitor is selected from GSK2256294, sorafenib, EC5026, Bl 1935, glimepiride, TPPU, dCP2PU, t-AUCB, AUDA and t-TUCB, and is more preferably, GSK2256294 or a pharmaceutically acceptable salt thereof.