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Acetyl-boswellic acids (acetyl-BA) are pentacyclic triterpenes derived from the gum resin of frankincense. We have previously shown that these compounds are effective cytotoxic agents, acting through a mechanism that appears to involve the inhibition of topoisomerase activity. We have now investigated the mechanism of action of acetyl-BA and show that these compounds are more potent inhibitors of human topoisomerases I and IIalpha than camptothecin, and amsacrine or etoposide, respectively. Our data demonstrate that acetyl-BA and, to a lesser extent, some other pentacyclic triterpenes, such as betulinic acid, ursolic acid, and oleanolic acid, inhibit topoisomerases I and IIalpha through a mechanism that does not involve stabilization of the cleavable complex or the intercalation of DNA. Surface plasmon resonance analysis revealed that topoisomerases I and IIalpha bind directly to an immobilized derivative of acetyl-BA. This acetyl-BA derivative interacts with human topoisomerases through high-affinity binding sites yielding K(D) values of 70.6 nM for topoisomerase I and 7.6 nM for topoisomerase IIalpha. Based on our data, we propose that acetyl-BA inhibit topoisomerases I and IIalpha through competition with DNA for binding to the enzyme. Thus, acetyl-BA are a unique class of dual catalytic inhibitors of human topoisomerases I and IIalpha.

Expression of proinflammatory cytokines by monocytes is tightly regulated by transcription factors such as NF-κB. In this study, we show that, in LPS-stimulated human peripheral monocytes, the pentacyclic triterpenes acetyl-α-boswellic acid (AαBA) and acetyl-11-keto-β-boswellic acid (AKβBA) down-regulate the TNF-α expression. AαBA and AKβBA inhibited NF-κB signaling both in LPS-stimulated monocytes as detected by EMSA, as well as in a NF-κB-dependent luciferase gene reporter assay. By contrast, the luciferase expression driven by the IFN-stimulated response element was unaffected, implying specificity of the inhibitory effect observed. Both AαBA and AKβBA did not affect binding of recombinant p50/p65 and p50/c-Rel dimers to DNA binding sites as analyzed by surface plasmon resonance. Instead, both pentacyclic triterpenes inhibited the LPS-induced degradation of IκBα, as well as phosphorylation of p65 at Ser536 and its nuclear translocation. AαBA and AKβBA inhibited specifically the phosphorylation of recombinant IκBα and p65 by IκBα kinases (IKKs) immunoprecipitated from LPS-stimulated monocytes. In line with this, AαBA and AKβBA also bound to and inhibited the activities of active human recombinant GST-IKKα and His-IKKβ. The LPS-triggered induction of TNF-α in monocytes is dependent on IKK activity, as confirmed by IKK-specific antisense oligodeoxynucleotides. Thus, via their direct inhibitory effects on IKK, AαBA and AKβBA convey inhibition of NF-κB and subsequent down-regulation of TNF-α expression in activated human monocytes. These findings provide a molecular basis for the anti-inflammatory properties ascribed to AαBA- and AKβBA-containing drugs and suggest acetyl-boswellic acids as tools for the development of novel therapeutic interventions.

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In this study we investigated the effects of concomitant food intake on the bioavailability of distinct boswellic acids (BAs) from the test preparation BSE-018, a dry extract from Boswellia serrata gum resin. In a randomised, open, single-dose, two-way crossover study, healthy male subjects received three capsules of BSE-018 equivalent to 786 mg dry extract of Boswellia serrata gum resin either in the fasted state or together with a standardised high-fat meal. BA plasma concentrations were analysed for up to 60 h after oral dosing by reversed phase HPLC. As compared to the fasted state (treatment A), the administration of BSE-018 concomitantly with a high-fat meal (treatment B) led to several-fold increased areas under the plasma concentration-time curves as well as peak concentrations of beta-boswellic acid (betaBA), 11-keto-beta-boswellic acid (KbetaBA) and acetyl-11-keto-beta-boswellic acid (AKbetaBA). Plasma levels of both acetyl-alpha-boswellic acid (AalphaBA) and alphaBA became only detectable when administered with treatment B, i.e., the high-fat meal. Accordingly, pharmacokinetic data could be calculated for betaBA, KbetaBA and AKbetaBA (treatment A) and for betaBA, KbetaBA, AKbetaBA, alphaBA, and AalphaBA (treatment B). For the first time these data reveal detailed kinetics of BAs after oral dosing of an extract and demonstrate a profound effect of food intake on the pharmacokinetic profile of the BAs. This finding should be very important whenever BAs would be considered for therapeutic use.

Results:Gremlin-1 binds with high affinity to macrophage migration inhibitory factor and attenuates the progression of atherosclerosis. Conclusion:We describe a novel mechanism that regulates foam cell formation and plaque growth. Significance: The findings disclose a new mechanism for the regulation of plaque growth and may open novel therapeutic strategies to control the progression of atherosclerosis.Monocyte infiltration and macrophage formation are pivotal steps in atherosclerosis and plaque vulnerability. Gremlin-1/ Drm is crucial in embryo-/organogenesis and has been shown to be expressed in the adult organism at sites of arterial injury and to inhibit monocyte migration. The purpose of the present study was to evaluate and characterize the role of Gremlin-1 in atherosclerosis. Here we report that Gremlin-1 is highly expressed primarily by monocytes/macrophages in aortic atherosclerotic lesions of ApoE Atherosclerosis is a chronic disease of the arteries characterized by inflammation of the vessel wall (1-3). Endothelial activation, monocyte recruitment, and the formation of macrophages are critical events in the development of atherosclerosis (4). Macrophages secrete cytokines/chemokines and growth factors that promote atheroprogression and contribute substantially to plaque vulnerability and acute complications of the disease such as acute coronary syndromes (5). The cytokine macrophage migration inhibitory factor (MIF) 2 is a noncognate ligand of CXC chemokine receptors and regulates monocyte recruitment toward atherosclerotic lesions (6). Blocking or genetic deletion of MIF reduces macrophage and T-cell content of atherosclerotic plaques and attenuates the progression of atherosclerosis in ApoE Ϫ/Ϫ mice (7). Gremlin-1 and its rat homolog Drm (down-regulated by v-mos) are highly conserved 20.7-kDa glycoproteins (8, 9). Gremlin-1 belongs to the DAN/Cerberus protein family, which is a member of the cysteine knot superfamily that includes TGF-␤ and VEGF (10). Gremlin-1 is a bone morphogenetic protein (BMP) antagonist and binds to BMP-2, -4, and -7 (9). It exists in secreted and cell-associated forms (9). The Gremlin-1 gene encodes a 23-and 28-kDa protein that is glycosylated before secretion (9). Gremlin-1-dependent inhibition of BMPs is important for embryogenesis and development of organs such as limbs, kidney, and lungs (11, 12). Gremlin-1 knock-out mice are neonatally lethal with significant renal and lung defects (13). Transgenic mice overexpressing Gremlin-1 under the control of the osteocalcin promoter show reduced bone formation (14). Gremlin-1 is expressed in endothelial cells exposed to disturbed flow in mouse aorta and in human coro-