In this study, we found that anabasum, a selective CB2 receptor agonist with minimal cannabimetic CNS activity, exerts potent anti‐inflammatory, anti‐infective, as well as a pro‐resolution effect in a model of self‐resolving acute inflammation in healthy human volunteers. The anti‐inflammatory properties of anabasum arose from the inhibition of the neutrophil chemoattractant LTB₄, thereby reducing neutrophil infiltration and hence the potential for further neutrophil‐mediated tissue damage and propagation of inflammation. Moreover, in line with the classic definition of resolution, anabasum hastened antigen clearance, which alongside increased biosynthesis of SPMs at the 20 mg b.i.d. dose collectively resulted in effects that were superior to that of prednisolone. We propose, therefore, that anabasum is the first of a new generation of anti‐inflammatory/pro‐resolving drug that acts on diverse aspects of the immune response bringing back into homeostatic check multiple proinflammatory pathways that are activated by infection or injury. These unique pharmacological properties of anabasum appear to exert a more favorable effect on human pathophysiology compared to the properties of steroids, which as a consequence of their broad actions carry with them a burden of immunosuppressive side effects not seen with anabasum.

Treating neutrophils with anabasum for 4 h followed by LPS for 15 min did not alter their ability to engulf bacteria compared to untreated controls. If we extended the LPS‐stimulation time to 4 h then anabasum significantly enhanced the ability to neutrophils to phagocytose labeled E. coli bioparticles. These experimental conditions, which are akin to the 4 h blister setting and where anabasum significantly inhibits endotoxin levels, suggest that anabasum either triggered the release of prophagocytic factor/s or inhibited the synthesis of factor/s that impair bacterial uptake. While SPMs have been shown to enhance bacterial killing in various settings,25 anabasum did not alter levels of these lipid mediators at 4 h. However, it did dose‐dependently and significantly inhibit synthesis of both PGE₂ and TxB₂. Moreover, adding these prostanoids back to the ex vivo phagocytosis assays reversed anabasum's uptake of labeled bacteria. PGE₂ has been shown to inhibit NADPH oxidase‐mediated bacterial killing via upregulation of cAMP26 and also inhibit FcγR‐mediated phagocytosis22, 27; indeed, we have shown that its elevation in cirrhosis patients underpins their susceptibility to infection that is typical of these individuals.28 Moreover, a post‐hoc analysis revealed a significant correlation between levels of blister fluid endotoxin at 4 h and PGE₂ as well as TXB₂ such that when levels of these prostanoids were inhibited, so also were levels of endotoxin. Thus, inhibiting prostanoid biosynthesis could explain how anabasum reduced endotoxin levels.

There was also a strong correlation between the inhibition of TxB₂ by anabasum (5 mg b.i.d.) and endotoxin levels at 4 h in elicited blister fluid. While there is little evidence that thromboxane can directly affect neutrophil and/or macrophage bacterial phagocytosis, we did find that TxB₂ blocked anabasum‐mediated uptake of E. coli bioparticles by neutrophils. However, there is also compelling evidence that TxA₂‐mediated vasoconstriction may decrease the numbers of bacteria leaving the site of infection,29 supporting the hypothesis that this may be an important mechanism by which TxA₂ may regulate immune responses to infection in vivo. Indeed, we found that levels of TxB₂ after anabasum at 4 h (5 mg b.i.d.) correlated inversely (P = 0.0167 and r = 1) with local vascular blood flow as determined by laser Doppler. Thus, the mechanism by which anabasum lowered endotoxin levels could be due to increased phagocytosis and/or reverse migration to systemic circulation; however, the latter seems unlikely, as there was no spike in systemic CRP levels.

While there are many effective anti‐inflammatory treatment regimens on the market (nonsteroidal anti‐inflammatory drugs (NSAIDs), anticytokine therapies, steroids, etc.), switching off the underlying disease process is much more challenging. In terms of diseases driven by ongoing inflammation, the rationale is therefore to activate pro‐resolution pathways that might be pathologically silenced and/or activate functional pro‐resolution pathways to ameliorate persistent inflammation.4 Indeed, it may transpire that combining pro‐resolution with anti‐inflammatory technology may represent a superior strategy to existing approaches. Along these lines, it was originally feared that hastening resolution might lead to incomplete clearance of the original trigger, especially in the context of infections. Specialized pro‐resolution mediators, for instance, have to date been shown not just to augment resolution processes such as nonphlogistic monocyte recruitment and efferocytosis of neutrophils, but also antimicrobial properties.30 Indeed, while NSAIDs are clearly anti‐inflammatory, they also augment host defense. Therefore, it must be emphasized that dampening inflammation is not necessarily associated with compromised host defense.

While anabasum was effective at dampening neutrophil trafficking and hastening antigen clearance in a self‐resolving model of inflammation in healthy volunteers, its ability to exert similar actions on chronic inflammatory disease has been documented as well. In a rat adjuvant arthritis model, anabasum reduced inflammation, weight loss, pannus formation in the joints, and joint tissue injury over the entire 4‐week period of the study.31, 32 Likewise, in bleomycin‐induced lung and dermal inflammation and fibrosis models, anabasum, administered therapeutically after the start of bleomycin, inhibited organ fibrosis, collagen deposition, and myofibroblast differentiation.33, 34 In clinical studies anabasum treatment over a 12‐week period has been shown to reduce skin thickening in systemic sclerosis patients, and the rate and number of pulmonary exacerbations and inflammatory biomarkers in cystic fibrosis patients.35, 36

One interesting observation made during this study was that anabasum caused a dose‐dependent increase in local microvascular hyperemia around the injection site. While this suggested increased “inflammatory flare,” upon clinical examination the site was neither edematous nor painful. Moreover, we found no increase in neutrophils or macrophages in the affected tissue. In order to understand the mechanism underlying this observation, we measured levels of nitrite/nitrate in the blister fluid and found no evidence of increased NO biosynthesis (Figure S5). However, anabasum dose‐dependently inhibited TxB₂, but was without effect on prostacyclin, measured as 6‐keto PGF1_α (Figure S6). This inhibition of a potent vasoconstrictor while keeping levels of the vasodilator prostacyclin unaffected could result in a net increase in local microvascular hyperemia. Indeed, these data are very reminiscent of the beneficial effects of low‐dose aspirin on pathophysiology, namely, the inhibition of vasoconstrictive thromboxane while maintaining prostacyclin levels due to its preference for acetylating platelets in the portal circulation.

While both doses of anabasum inhibited inflammation, the mechanisms by which it exerted this potent effect may be dose‐specific. For instance, at 20 mg b.i.d. anabasum increased levels of LXA₄, LXB₄, RvD1, and RvD3, which is of interest because each one of these mediators carries potent pro‐resolving actions. For instance, LXA₄ and RvD1 reduce human neutrophilic chemotaxis and infiltration,37 while RvD3 increases efferocytosis and phagocytosis, while, alongside aspirin‐triggered RvD3, are potent immunoresolvents.38, 39 In contrast, 5 mg b.i.d. anabasum had no effect on these SPMs. PGE₂ and TxB₂ inhibition occurred with both 5 mg b.i.d. and 20 mg b.i.d. doses. These findings suggest lower concentrations of anabasum or lower levels of CB2 receptor occupancy have metabolic effects on the arachidonic acid pathway, whereas higher concentrations are required to trigger SPM synthesis pathways. Thus, 20 mg b.i.d. may activate both effects of anabasum, whereas 5 mg b.i.d. only activates metabolic effects on the arachidonic acid pathway, at least in the setting of exposure limited to 4 days. These data suggest that 20 mg b.i.d. may be a more effective therapeutic dose of anabasum for treating chronic inflammatory diseases because it engages resolution pathways, with the acknowledgment that a longer duration of exposure to lower doses might also lead to activation of resolution pathways.

In summary, we used UV‐killed E. coli‐driven acute dermal inflammation to report the potent anti‐inflammatory properties of the CB2 receptor agonist, anabasum. Specifically, anabasum was as efficacious as prednisolone in inhibiting neutrophil infiltration and also accelerated the clearance of the injected bacteria, a key determinant for the resolution of inflammation. It transpires that its mode of action is, at least in part, through inhibition of chemoattractant LTB₄, as well as through inhibition of antiphagocytic prostanoids, while maintaining levels of the vasodilator prostacyclin. Anabasum also triggered specialized pro‐resolving lipid mediators including lipoxins and resolvins. Therefore, anabasum exerts its effects on multiple aspects of the inflammatory and resolution cascade, resulting in both anti‐inflammatory and pro‐resolving properties in a manner that does not appear to compromise host defense or vascular homeostasis.