Inflammation is a host response to various noxious stimuli, including thermal, mechanical, chemical injuries, as well as pathogenic infections. The primary goal of the inflammatory response is to remove harmful stimuli and promote the healing process, thereby restoring the organism's homeostasis. If resolution of inflammation fails, the process can become chronic, contributing to a broad spectrum of chronic inflammatory diseases such as asthma, heart disease, cancer, obesity, and many others [1]. The inflammatory response is tightly regulated by lipid mediators (LMs) such as prostanoids, leukotrienes (LTs), epoxyeicosatrienoic acids (EETs), and specialized pro-resolving mediators (SPMs), whose biosynthesis starts with the release of polyunsaturated fatty acids (PUFAs) from membrane phospholipids. In particular, free arachidonic acid (AA) serves as substrate for three enzymatic branches: cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP450) pathways, each generating a broad range of LMs [2]. All these metabolites have been intensely investigated for their crucial role in orchestrating the propagation and the resolution of the inflammatory cascade. Prostanoids are produced by the activity of COXs, and primarily play a pro-inflammatory role. However, some prostanoids, such as PGE2 and PGD2, can exert an anti-inflammatory actions depending on the context and the cellular target [3].

Pro-inflammatory LTs derive from the 5-LOX pathway, involving the 5-LOX-activating protein (FLAP) that is devoid of enzymatic activity and assists 5-LOX in the initial steps of LT biosynthesis, providing AA as substrate [4,5]. 5-LOX also metabolizes other PUFAs such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) into bioactive LMs, which contribute to resolving inflammation [6]. However, the involvement of FLAP in SPM biosynthesis via 5-LOX depends on the PUFA, where EPA and AA but not DHA are utilized by FLAP as 5-LOX substrates for SPM production [7,8].

EETs derive from AA after conversion by CYP450, and their role as endogenous anti-inflammatory LMs has been well characterized. Nevertheless, upon formation, EETs survive only for a very short time in the bloodstream due to their hydrolysis and inactivation by soluble epoxide hydrolase (sEH), which converts them into the corresponding pro-inflammatory dihydroxyeicosatrienoic acid (DiHET) derivatives [9]. EETs possess anti-inflammatory, analgesic, anti-migratory, and fibrinolytic properties, and their pathway modulation represents a fruitful therapeutic option in the anti-inflammatory field [10,11]. In this regard, inhibition of sEH leads to an increased concentration of EETs and a concomitant reduction in the production of pro-inflammatory DiHETs, thereby attenuating inflammation in experimental models of acute and chronic inflammation [12,13].

To date, nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit prostanoid biosynthesis, represent the pillar of the anti-inflammatory treatment; however, they are plagued by several and serious side effects, especially at cardiovascular and gastrointestinal levels [[14], [15], [16]]. Current developments in the design of new anti-inflammatory compounds are oriented toward multitarget approaches, intended to facilitate simultaneous interaction with two or more enzymes involved in the formation and degradation of LMs. This path is particularly pursued because it enables the enhancement of efficacy and safety in the treatment of inflammation-related disorders [[17], [18], [19]].

Recently, we conducted a drug discovery campaign with the aim of identifying novel anti-inflammatory compounds that act via alternative mechanisms than those of NSAIDs. We developed a dual 5-LOX/sEH inhibitor (compound 73, Fig. 1), which has been validated using in vivo murine models of inflammation [20]. Moving forward this research and exploiting the previously acquired information, we obtained a selective sEH inhibitor (compound 28, Fig. 1), with in vivo anti-inflammatory activities [21]. Taking advantage of our previous findings in this field, here we report the identification of a dual FLAP/sEH inhibitor (compound 6, Fig. 1), obtained through in-silico design. Derivative 6 was validated by in vitro testing its inhibitory activity on human isolated sEH, verifying the lack of activity against 5-LOX, and assessing its activity in human neutrophils in the absence or presence of exogenous AA. Thus, we investigated about compound 6 selectivity, observing no activity against COXs and mPGES1 and finally we continued with its in vivo pharmacological characterization, profiling the mice lipidome after derivative 6 administration.

We shifted our interests toward FLAP instead of 5-LOX, considering the aim of inhibiting pro-inflammatory LT production, while preserving SPM formation from DHA. At the same time, sEH inhibition allows the increase of EET levels, exploiting their anti-inflammatory properties [[22], [23], [24], [25], [26]]. This pharmacological strategy has been well validated, as demonstrated by the number of sEH inhibitors subjected to clinical trials [[27], [28], [29]]. In contrast, despite several FLAP inhibitors disclosed so far, none of them have reached the pharmaceutical market, principally because of their poor pharmacokinetic [30,31]. Contextually, FLAP/sEH dual inhibition remains a largely unexplored area, with diflapolin and its derivatives (Fig. 1) currently the only known compounds exhibiting this profile [32,33].