Arzanol, a prenylated heterodimeric phloroglucinyl pyrone, inhibits eicosanoid biosynthesis and exhibits anti-inflammatory efficacy in vivo

Abstract

Based on its capacity to inhibit in vitro HIV-1 replication in T cells and the release of pro-inflammatory cytokines in monocytes, the prenylated heterodimeric phloroglucinyl α-pyrone arzanol was identified as the major anti-inflammatory and anti-viral constituent from Helichrysum italicum. We have now investigated the activity of arzanol on the biosynthesis of pro-inflammatory eicosanoids, evaluating its anti-inflammatory efficacy in vitro and in vivo. Arzanol inhibited 5-lipoxygenase (EC 7.13.11.34) activity and related leukotriene formation in neutrophils, as well as the activity of cyclooxygenase (COX)-1 (EC 1.14.99.1) and the formation of COX-2-derived prostaglandin (PG)E₂ in vitro (IC₅₀ = 2.3–9 μM). Detailed studies revealed that arzanol primarily inhibits microsomal PGE₂ synthase (mPGES)-1 (EC 5.3.99.3, IC₅₀ = 0.4 μM) rather than COX-2. In fact, arzanol could block COX-2/mPGES-1-mediated PGE₂ biosynthesis in lipopolysaccharide-stimulated human monocytes and human whole blood, but not the concomitant COX-2-derived biosynthesis of thromboxane B₂ or of 6-keto PGF_1α, and the expression of COX-2 or mPGES-1 protein was not affected. Arzanol potently suppressed the inflammatory response of the carrageenan-induced pleurisy in rats (3.6 mg/kg, i.p.), with significantly reduced levels of PGE₂ in the pleural exudates. Taken together, our data show that arzanol potently inhibits the biosynthesis of pro-inflammatory lipid mediators like PGE₂ in vitro and in vivo, providing a mechanistic rationale for the anti-inflammatory activity of H. italicum, and a rationale for further pre-clinical evaluation of this novel anti-inflammatory lead.

Introduction

Inflammation is a complex biological response to harmful stimuli required to remove an offensive agent and to initiate the healing process. However, chronic inflammation can lead to diseases (e.g., rheumatoid arthritis, atherosclerosis, and autoimmune disorders) that still represent unmet clinical needs. The prostanoids and leukotrienes (LTs) formed from arachidonic acid (AA) via the cyclooxygenase (COX)-1/2 (EC 1.14.99.1) and 5-lipoxygenase (5-LO) (EC 7.13.11.34) pathway, respectively, mediate inflammatory responses, chronic tissue remodelling, cancer, asthma, and autoimmune disorders, but also possess homeostatic functions in the gastrointestinal tract, uterus, brain, kidney, vasculature, and host defence [1]. In fact, the clinical use of non-steroidal anti-inflammatory drugs (NSAIDs), a class of drugs that block formation of all prostanoids, is hampered by severe side effects including gastrointestinal injury, renal irritations, and cardiovascular risks [2]. Therefore, anti-inflammatory agents interfering with eicosanoid biosynthesis require a well-balanced pharmacological profile to minimize these on-target side effects [2]. Current anti-inflammatory research aims at identifying compounds that can suppress the massive formation of pro-inflammatory prostaglandin (PG)E₂ without affecting homeostatic PGE₂ and PGI₂ synthesis, and also inhibit LT formation [3], [4]. While there is no shortage of anti-inflammatory natural products that suppress eicosanoid generation by inhibiting 5-LO and/or COX enzymes [5], [6], the activity of most of them has only been evaluated in terms of expression and/or activity of COX enzymes and 5-LO in vitro, and high concentrations, unrealistic to translate in a clinical setting, are often required for activity [5]. Moreover, in many cases the anti-inflammatory activity was not evaluated in vivo, or, if so, it was not clearly related to an interference with the biosynthesis of eicosanoids.

Arzanol, a prenylated heterodimeric phloroglucinyl α-pyrone (Fig. 1A), was identified as the major anti-inflammatory constituent of Helichrysum italicum (H. italicum), a Mediterranean plant used in folk medicine to treat inflammatory diseases and infections [7], [8]. In fact, extracts of H. italicum have been reported to exhibit anti-oxidant [7], anti-bacterial [9], anti-fungal [10] and anti-viral [11] effects. Extensive clinical research on the activity of extracts from H. italicum to manage inflammatory conditions was carried out by the Italian physician Leonardo Santini in the 40s and 50s. However, his findings were published in little known Journals, and, after his death, were largely overlooked [8]. Arzanol showed anti-oxidant activity in various systems of lipid peroxidation in vitro, and protected VERO cells against tert-butyl hydroperoxide (TBH)-induced oxidative stress [12]. Moreover, arzanol inhibited nuclear factor kappa beta (NFκB) activation and HIV-1 replication in T cells (IC₅₀ ∼ 5 μg/ml and 5 μM, respectively) as well as the release of pro-inflammatory mediators such as interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)α, and PGE₂ in lipopolysaccharide (LPS)-stimulated monocytes [8]. Nevertheless, additional molecular target(s) of arzanol may exist and its anti-inflammatory efficacy in vivo remains to be investigated. To this aim, we have investigated the activity of arzanol on the biosynthesis of prostanoids and LTs, and we have evaluated its anti-inflammatory efficacy in vitro and in vivo. We show that arzanol potently inhibits the inducible microsomal PGE₂ synthase (mPGES)-1 (EC 5.3.99.3), COX-1, and 5-LO in various test systems with IC₅₀ values between 0.4 and 9 μM, and significantly reduces the inflammatory response and the PGE₂ levels in the carrageenan-induced pleurisy in rats. These observations validate arzanol as a novel anti-inflammatory chemotype worth further development.