Sodium houttuyfonate: A review of its antimicrobial, anti-inflammatory and cardiovascular protective effects.

There is an almost unlimited interest in searching and developing new drugs, especially when we are in an era that are witnessing more and more emerging pathogens. Natural products from traditional medicines represent a large library for searching lead compounds with novel bioactivities. Sodium houttuyfonate is such one bioactive compound derived from Houttuynia cordata Thunb which has been employed in traditional medicine for treating infectious and inflammatory diseases. Sodium houttuyfonate has demonstrated multiple kinds of pharmacological effects, including antifungal, antibacterial, anti-inflammatory, and cardiovascular protective activities, which are discussed here to provide insights into our understanding of the pharmacological effects of SH and the underlying mechanisms.

Novel 2,7-Diazaspiro[4,4]nonane Derivatives to Inhibit Mouse and Human Osteoclast Activities and Prevent Bone Loss in Ovariectomized Mice without Affecting Bone Formation.

Osteoporosis is currently treated with drugs targeting the differentiation or viability osteoclasts, the cells responsible for physiological and pathological bone resorption. Nevertheless, osteoporosis drugs that target only osteoclast activity are expected to preserve bone formation by osteoblasts in contrast to current treatments. We report here the design, synthesis, and biological characterization of a series of novel N-arylsufonamides featuring a diazaspiro[4,4]nonane nucleus to target the guanine nucleotide exchange activity of DOCK5, which is essential for bone resorption by osteoclasts. These compounds can inhibit both mouse and human osteoclast activity. In particular, 4-chlorobenzyl-4-hydroxy-2-phenyl-1-thia-2,7-diazaspiro[4,4]nonane 1,1-dioxide (compound E197) prevented pathological bone loss in mice. Most interestingly, treatment with E197 did not affect osteoclast and osteoblast numbers and hence did not impair bone formation. E197 could represent a lead molecule to develop new antiosteoporotic drugs targeting the mechanism of osteoclast adhesion onto the bone.

Isokotomolide A from Cinnamomum kotoense Induce Melanoma Autophagy and Apoptosis In Vivo and In Vitro.

Melanoma is an aggressive cancer with high lethality. In order to find new anticancer agents, isokotomolide A (Iso A) and secokotomolide A (Sec A) isolated from Cinnamomum kotoense were identified to be potential bioactive agents against human melanoma but without strong antioxidative properties. Cell proliferation assay displayed Iso A and Sec A treated in the normal human skin cells showed high viabilities. It also verified that two of them possess strong antimelanoma effect in concentration-dependent manners, especially on B16F10, A2058, MeWo, and A375 cells. Wound healing assay presented their excellent antimigratory effects. Through 3-N,3-N,6-N,6-N-Tetramethylacridine-3,6-diamine (acridine orange, AO) staining and Western blot, the autophagy induced by treatment was confirmed, including autophagy-related proteins (Atgs). By using annexin V-FITC/PI double-stain, the apoptosis was confirmed, and both components also triggered the cell cycle arrest and DNA damage. We demonstrated the correlations between the mitogen-activated protein kinase (MAPK) pathway and antimelanoma, such as caspase cascade activations. To further evaluate in vivo experiments, the inhibition of tumor cell growth was verified through the histopathological staining in a xenograft model. In this study, it was confirmed that Iso A and Sec A can encourage melanoma cell death via early autophagy and late apoptosis processes.

Drug targeting CYP2E1 for the treatment of early-stage alcoholic steatohepatitis.

BACKGROUND AND AIMS: Alcoholic steatohepatitis (ASH)-the inflammation of fatty liver-is caused by chronic alcohol consumption and represents one of the leading chronic liver diseases in Western Countries. ASH can lead to organ dysfunction or progress to hepatocellular carcinoma (HCC). Long-term alcohol abstinence reduces this probability and is the prerequisite for liver transplantation-the only effective therapy option at present. Elevated enzymatic activity of cytochrome P450 2E1 (CYP2E1) is known to be critically responsible for the development of ASH due to excessively high levels of reactive oxygen species (ROS) during metabolization of ethanol. Up to now, no rational drug discovery process was successfully initiated to target CYP2E1 for the treatment of ASH. METHODS: In this study, we applied a rational drug design concept to develop drug candidates (NCE) including preclinical studies. RESULTS: A new class of drug candidates was generated successfully. Two of the most promising small compounds named 12-Imidazolyl-1-dodecanol (abbr.: I-ol) and 1-Imidazolyldodecane (abbr.: I-an) were selected at the end of this process of drug discovery and developability. These new ω-imidazolyl-alkyl derivatives act as strong chimeric CYP2E1 inhibitors at a nanomolar range. They restore redox balance, reduce inflammation process as well as the fat content in the liver and rescue the physiological liver architecture of rats consuming continuously a high amount of alcohol. CONCLUSIONS: Due to its oral application and therapeutic superiority over an off-label use of the hepatoprotector ursodeoxycholic acid (UDCA), this new class of inhibitors marks the first rational, pharmaceutical concept in long-term treatment of ASH.

4-Aryl-1-oxa-4,9-diazaspiro[5.5]undecane Derivatives as Dual µ-Opioid Receptor Agonists and σ1 Receptor Antagonists for the Treatment of Pain.

The synthesis and pharmacological activity of a new series of 1-oxa-4,9-diazaspiro[5.5]undecane derivatives as potent dual ligands for the sigma-1 receptor (σ1R) and the µ-opioid receptor (MOR) are reported. The different positions of the central scaffold, designed using a merging strategy of both target pharmacophores, were explored using a versatile synthetic approach. Phenethyl derivatives in position 9, substituted pyridyl moieties in position 4 and small alkyl groups in position 2 provided the best profiles. One of the best compounds, 15au, showed a balanced dual profile (i.e., MOR agonism and sigma antagonism) and a potent analgesic activity, comparable to the MOR agonist oxycodone in the paw pressure test in mice. Contrary to oxycodone, as expected from the addition of σ1R antagonism, 15au showed local, peripheral activity in this test, which was reversed by the σ1R agonist PRE-084. At equianalgesic doses, 15au showed less constipation than oxycodone, providing evidence that dual MOR agonism and σ1R antagonism may be a useful strategy for obtaining potent and safer analgesics.

Synthesis, biological evaluation and molecular modeling of 1-oxa-4-thiaspiro- and 1,4-dithiaspiro[4.5]decane derivatives as potent and selective 5-HT1A receptor agonists.

Recently, 1-(1,4-dioxaspiro[4,5]dec-2-ylmethyl)-4-(2-methoxyphenyl)piperazine (1) was reported as a potent 5-HT1AR agonist with a moderate 5-HT1AR selectivity. In an extension of this work a series of derivatives of 1, obtained by combining different heterocyclic rings with a more flexible amine chain, was synthesized and tested for binding affinity and activity at 5-HT1AR and α1 adrenoceptors. The results led to the identification of 14 and 15 as novel 5-HT1AR partial agonists, the first being outstanding for selectivity (5-HT1A/α1d = 80), the latter for potency (pD2 = 9.58) and efficacy (Emax = 74%). Theoretical studies of ADME properties shows a good profile for the entire series and MDCKII-MDR1 cells permeability data predict a good BBB permeability of compound 15, which possess a promising neuroprotective activity. Furthermore, in mouse formalin test, compound 15 shows a potent antinociceptive activity suggesting a new strategy for pain control.

Compositions and anti-tumor activity of Pyropolyporus fomentarius petroleum ether fraction in vitro and in vivo.

The chemical compositions and anti-tumor activities of the petroleum ether fraction (PE), from mushroom Pyropolyporus fomentarius, were studied. Upon gas chromatography-mass spectrometry (GC-MS) analysis, nine major constituents were identified in the fraction. In vitro, the PE showed cytotoxic activity against murine sarcoma S180 (S180) cells in a dose- and time-dependent manner, and the cytotoxic effects were associated with apoptosis. The mitochondrial membrane potential loss and the intracellular ROS generation were greatly increased in the Pyropolyporus fomentarius PE treated group, suggesting cell apoptosis, induced by the PE in S180 cells, might be mitochondria dependent and ROS mediated. Consistent with in vitro findings, the in vivo study showed that the Pyropolyporus fomentarius PE was also effective in inhibiting the tumor growth induced by S180 cells and had lower immune organ toxicity. We found that the Pyropolyporus fomentarius PE has significant anti-tumor activity and great potential in screening anti-tumor drugs.

Discovery of 2,8-diazaspiro[4.5]decane-based trisubstituted urea derivatives as highly potent soluble epoxide hydrolase inhibitors and orally active drug candidates for treating hypertension.

We identified 2,8-diazaspiro[4.5]decane-based trisubstituted urea derivatives as highly potent soluble epoxide hydrolase (sEH) inhibitors and orally active agents for treating hypertension. Docking studies using human and murine sEH X-ray crystal structures revealed steric hindrance around the side chain of Phe406 of murine sEH. The trifluoromethyl moiety (11) was replaced with a trifluoromethoxy moiety (12) to prevent steric clash, and improved murine sEH inhibitory activity was observed. The oral administration of 12, 20, and 37 at a dose of 30mg/kg reduced blood pressure in spontaneously hypertensive rat, but had little effect on blood pressure in normotensive rat.

The duration of pacing-induced atrial fibrillation is reduced in vivo by inhibition of small conductance Ca(2+)-activated K(+) channels.

Atrial fibrillation (AF) is associated with increased morbidity and is in addition the most prevalent cardiac arrhythmia. Compounds used in pharmacological treatment has traditionally been divided into Na(+) channel inhibitors, ß-blockers, K(+) channel inhibitors, and Ca(2+) channel inhibitors, whereas newer multichannel blockers such as amiodarone and ranolazine have been introduced later. This study was devoted to the evaluation of an acute pacing-induced in vivo model of AF in rats. Antiarrhythmic effects of well-known compounds such as lidocaine, dofetilide, and ranolazine were confirmed in this model. In addition, antiarrhythmic effects of different inhibitors of Ca(2+)-activated small conductance K(+) (SK) channels were demonstrated. Intravenous application of 5 mg/kg of the negative SK channel modulator NS8593 reduced AF duration by 64.5%, and the lowest significantly effective dose was 1.5 mg/kg. A dose-effect relationship was established based on 6 different dose groups. Furthermore, it was demonstrated that the antiarrhythmic effect of NS8593 and other tested drugs was associated with an increase in atrial effective refractory period. The functional role of SK channels was confirmed by 2 other SK channel inhibitors, UCL1684 and apamin, thereby confirming the hypothesis that these channels might constitute a new promising target for antiarrhythmic treatment.

Pharmacokinetic properties and metabolism of a new potent antimalarial N-alkylamidine compound, M64, and its corresponding bioprecursors.

Antimalarial activities and pharmacokinetics of the bis-alkylamidine, M64, and its amidoxime, M64-AH, and O-methylsulfonate, M64-S-Me, derivatives were investigated. M64 and M64-S-Me had the most potent activity against the Plasmodium falciparum growth (IC(50)<12nM). The three compounds can clear the Plasmodium vinckei infection in mice (ED(50)<10mg/kg). A liquid chromatography-mass spectrometry method was validated to simultaneously quantify M64 and M64-AH in human and rat plasma. M64 is partially metabolized to M64-monoamidoxime and M64-monoacetamide by rat and mouse liver microsomes. The amidoxime M64-AH undergoes extensive metabolism forming M64, M64-monoacetamide, M64-diacetamide and M64-monoamidoxime. Strong interspecies differences were observed. The pharmacokinetic profiles of M64, M64-AH and M64-S-Me were studied in rat after intravenous and oral administrations. M64 is partially metabolized to M64-AH; while M64-S-Me is rapidly and totally converted to M64 and M64-AH. M64-AH is mostly oxidized to the inactive M64-diacetamine while its N-reduction to the efficient M64 is a minor metabolic pathway. Oral dose of M64-AH was well absorbed (38%) and converted to M64 and M64-diacetamide. This study generated substantial information about the properties of this class of antimalarial drugs. Other routes of synthesis will be explored to prevent oxidative transformation of the amidoxime and to favour the N-reduction.

Effects of sodium houttuyfonate on expression of NF-κB and MCP-1 in membranous glomerulonephritis.

AIMS OF THE STUDY: Sodium houttuyfonate (SH) is an addition compound of sodium bisulfite and houttuynin. Houttuynin is one of the main ingredients in the volatile oil of Houttuynia cordata Thunb, which has been widely used in traditional Chinese medicines. In this study, we investigated the effect of SH in membranous glomerulonephritis (MGN) induced by cationic Bovine Serum Albumin (C-BSA) in BALB/c mice. MATERIALS AND METHODS: Mice were divided into four groups, including normal vehicle-treated controls (N group), model (M group), low SH of 60 mg/kg body weight (L group), or high SH of 120 mg/kg body weight (H group). Urine protein quantification was detected by the urine protein strip test. Morphological assessment in kidneys was observed by light microscope and electron microscopy. The level of nuclear factor-kappaB (NF-kappaB) in the nuclear was evaluated by Western blot. Immunohistochemical was used to analyze the expression of MCP-1. RESULTS: SH was shown to reverse C-BSA induced increases in urinary protein, and changes in morphology. Treatment with SH at 60-120 mg/kg (L and H groups, respectively) dose-dependently decreased the level of nuclear NF-kappaB and MCP-1 expression compared to that of the M group. CONCLUSIONS: This study reveals that SH could treat C-BSA induced MGN in BALB/c mice by suppressing NF-kappaB activation and MCP-1 expression. Therefore, the most likely mechanism underlying the biological effects of SH is inhibition of an NF-kappaB mediated-cytokine pathway.

[Study on sugarcane alkane alcohol to quail hyperlipidemia and atherosclerosis model].

OBJECTIVE: To study on pharmacologic actions on quail hyperlipidemia and atherosclerosis model. METHOD: To duplicate quail hyperlipidemia model by ectogenesis cholesterol and high fat forage, induce to atherosclerosis model, observe influence of sugarcane alkane alcohol to model animals' blood fat level, formation of atherosclerosis plaque, pathological changes of coronary vessels and vascular intimal. RESULT: TC, TG, LDL-C level in blood serum of quail hyperlipidemia markedly decreased after administered sugarcane alkane alcohol by dose of 30, 15, 7.5 mg x kg(-1), proliferation of aorta and brachiocephalic artery tunica intima foam cells was suppressed. CONCLUSION: Sugarcane alkane alcohol has satisfactory pharmacologic actions on hyperlipidemia and atherosclerosis animal model by regulating blood fat.

Investigation of the photostability properties of memoquin, a quinone derivative for the treatment of Alzheimer's disease.

The photostability properties of memoquin, a multifunctional compound in preclinical development for the treatment of Alzheimer's disease (AD) were investigated in solutions exposed to radiations, using a xenon arc lamp to simulate the natural sunlight. Reversed phase liquid chromatography coupled with diode array detection and electrospray ionization mass spectrometry (LC-UV/DAD-ESI-MS/MS) was applied to follow the photodegradation and disappearance of memoquin after irradiation. Under optimized chromatographic conditions, memoquin was separated with high resolution from the photoproducts formed in the photoexposed solutions. The results showed that memoquin is more stable at physiological and acid pHs, while it has a slow degradation pattern at more drastic conditions such as basic pH (t(1/2)=389 min) and in methanolic solutions (t(1/2)=465 min). In the irradiated solutions the appearance of photoproducts with lower retention times and molecular weight than memoquin was observed, thus indicating that some fragments were lost from its structure. The photodegradation products were characterized by LC-ESI-MS/MS and LC-UV/DAD analysis. The photoreactive centers were found on the amino groups of the side chains while the 1,4-benzoquinone functionality was maintained. Conversely, memoquin was found to be stable in the dark. These results suggest that, with appropriate handling and storage, memoquin's activity is not impaired.

MTDL design strategy in the context of Alzheimer's disease: from lipocrine to memoquin and beyond.

The multifunctional nature of Alzheimer's disease (AD) provides the logical foundation for the development of an innovative drug design strategy centered on multi-target-directed-ligands (MTDLs). In recent years, the MTDL concept has been exploited to design different ligands hitting different biological targets. Our first rationally designed MTDL was the polyamine caproctamine (1), which provided a synergistic cholinergic action against AD by antagonizing muscarinic M(2) autoreceptors and inhibiting acetylcholinesterase (AChE). Lipocrine (7) represented the next step in our research. Due to its ability to inhibit AChE catalytic and non-catalytic functions together with oxidative stress, 7 emerged as an interesting pharmacological tool for investigating the neurodegenerative mechanism underlying AD. Memoquin (9) is a quinone-bearing polyamine endowed with a unique multifunctional profile. With its development, we arrived at the proof of concept of the MTDL drug discovery approach. Experiments in vitro and in vivo confirmed its multimodal mechanisms of action and its interaction with different end-points of the neurotoxic cascade leading to AD. More recently, the MTDL approach led to carbacrine (12). In addition to the multiple activities displayed by 7, 12 displayed an interesting modulation of NMDA receptor activity. The pivotal role played by this target in AD pathogenesis suggests that 12 may be a promising new chemical entity in the MTDL gold rush.

Memoquin: a multi-target-directed ligand as an innovative therapeutic opportunity for Alzheimer's disease.

Alzheimer's disease is currently thought to be a complex, multifactorial syndrome, unlikely to arise from a single causal factor; instead, a number of related biological alterations are thought to contribute to its pathogenesis. This may explain why the currently available drugs, developed according to the classic drug discovery paradigm of "one-molecule-one-target," have turned out to be palliative. In light of this, drug combinations that can act at different levels of the neurotoxic cascade offer new avenues toward curing Alzheimer's and other neurodegenerative diseases. In parallel, a new strategy is emerging-that of developing a single chemical entity able to modulate multiple targets simultaneously. This has led to a new paradigm in medicinal chemistry, the "multi-target-directed ligand" design strategy, which has already been successfully exploited at both academic and industrial levels. As a case study, we report here on memoquin, a new molecule developed following this strategy. The in vitro and in vivo biological profile of memoquin demonstrates the suitability of the new strategy for obtaining innovative drug candidates for the treatment of neurodegenerative diseases.

[Study on antiinflammatory effect of different chemotype of Cinnamomum camphora on rat arthritis model induced by Freund's adjuvant].

OBJECTIVE: To study the antiinflammatory effects of naphtha from different chemotypes of Cinnamomum camphora and natural borneol on the rat arthritis model induced by Freund's adjuvant. METHOD: The arthritis model was induced by injecting Freund's adjuvant in rat voix pedis dermis and the rats were randomly divided into seven groups: normal control group, model control group, triptergium wilfordii control group, borneol chemotype naphtha group, camphor chemotype naphtha group, isocamphane chemotype naphtha group and natural borneol group. Rats of the triptergium wilfordii control group were given orally 8.1 mg x kg(-1) triptergium wilfordii for 35 days, rats of the normal control group and model control group were given same volume water, and rats of other groups were given 80 mg x kg(-1) corresponding drug. We observed the rat common condition, weighed the rat body weight weekly, measured the degree of swelling of voix pedis every 4 days, weighed the thymus and spleen on the end of life, and measured the contents of cell factor TNF-alpha, IL-2, and IL-6 in rat blood serum. RESULT: As far as the arthrosis degree of swelling and the contents of cell factor TNF-alpha, IL-2, IL-6 were concerned, rats of model control group were higher than normal control group, and rats of other drug groups were lower than the model control group. The order of inhibition ratios of the arthrosis degree of swelling from high to low principle was isocamphane chemotype naphtha group, camphor chemotype naphtha group, borneol chemotype naphtha group and natural borneol group. All medication administration teams evidently reduced the contents of the IL-2 and IL-6, and the inhibition ratios were higher than 38%. In the case of the contents of TNF-alpha and IL-2, all groups were not evidently different. In the case of inhibition of IL-6, camphor chemotype naphtha group was better than borneol chemotype naphtha group and natural borneol group, the latter was better than isocamphane chemotype naphtha group. As far as the weight, thymus index and spleen index were concerned, all medication administration groups were not different. CONCLUSION: The different chemotypes of C. camphora have anti-inflammatory effect on the rat arthritis model induced by Freund's adjuvant, but pharmacological activity and mechanism of action are different. The study points out the clinical curative effects of the chemotypes of the kindred medicinal plant are different, and please consider the difference of chemotype in clinical application.

Prevention of polymorphic light eruption with a sunscreen of very high protection level against UVB and UVA radiation under standardized photodiagnostic conditions.

Polymorphic light eruption (PLE), with an overall prevalence of 10-20%, is mainly provoked by ultraviolet A (UVA) (320-400 nm) and to a lesser degree by UVB (280-320 nm). The most effective prophylaxis of PLE, application of UV protection clothing, is not feasible for all sun-exposed areas of the skin and UV-hardening is time-consuming and may be associated with side-effects. Most sunscreens protect predominantly against UVB and therefore fail to prevent PLE. The protection level of potent UVA-protective filters remains unresolved. This single-centre, open, placebo-controlled, intra-individual, comparative study, analysed the efficacy of a sunscreen of very high protection level against UVB and UVA, containing methylene bis-benzotriazolyl tetramethylbutylphenol (Tinosorb M), bis-ethylhexyloxyphenol methoxyphenyl triazine (Tinosorb S) and butyl methoxydibenzoylmethane as UVA absorbing filters, in the prevention of PLE under standardized photodiagnostic conditions. After determination of the minimal erythema dose at day 0, photoprovocation was performed in 12 patients with a clinical history of PLE, on days 1, 2 and 3 with 100 J/cm2 UVA and variable doses of UVB, starting with the 1.5-fold minimal erythema dose of UVB. Prior to irradiation, placebo was applied to the right and sunscreen to the left dorsal forearm under COLIPA (European Cosmetic, Toiletry and Perfumery Association) conditions. In 10 patients PLE could be provoked at the placebo site, with positive reactions in 90% of the UVA, 40% of the UVB and 90% of the UVA/UVB irradiated fields. At the site with the active treatment none of these patients developed PLE. These data demonstrate that a sunscreen with effective filters against UVA and UVB can successfully prevent the development of PLE. Further studies are needed to examine whether regular application of sunscreen under everyday conditions, especially in doses less than the tested COLIPA-norm, could be an equivalent alternative to UV-hardening therapy.

Bis-quinolinium cyclophanes: highly potent and selective non-peptidic blockers of the apamin-sensitive Ca2+-activated K+ channel.

Small conductance Ca(2+)-activated K(+) (SK(Ca)) channels comprise an important subclass of K(+) channels. Selective blockade of SK(Ca) channels may find application in the therapy of myotonic muscular dystrophy, gastrointestinal dysmotilities, memory disorders, narcolepsy, and alcohol abuse. In the cyclophanes described herein the two 4-aminoquinolinium groups are joined at the ring N atoms (linker L) and at the exocyclic N atoms (linker A). When both the spacer A and L have only one benzene ring, the blocking potency changes dramatically with simple structural variations in the linkers. One of these smaller cyclophanes having A = benzene-1,4-diylbis(methylene) and L = benzene-1,3-diylbis(methylene) shows activity in the low nanomolar range. Furthermore, the results with the present series add significantly to the structure-activity knowledge in the field, since they incorporate the first example of molecules in which the activity depends critically on the nature of the linkers joining the two quinolinium (Q) groups. Later on, a novel series of bis-quinolinium bis-alkylene cyclophanes was described. The biological results of the present series add support to the suggestion that the linkers of the two Q groups do not form direct interactions with the channel protein but comprise a molecular support for the two Q groups. Two important structural features of the pharmacophore for SK(Ca) channel blockade have been identified. These are (1) an optimum distance of ca. 5.8 A between the centroids of the pyridinium rings of the two quinolinium groups, and (2) a preference for conformations having the Q groups in a synperiplanar orientation.

Safety, efficacy and anti-inflammatory activity of rho iso-alpha-acids from hops.

A defined mixture of rho iso-alpha-acids (RIAA), a modified hop extract, was evaluated for anti-inflammatory efficacy and safety. RIAA inhibited LPS-stimulated PGE(2) formation with >200-fold selectivity of COX-2 (IC(50)=1.3 microg/ml) over COX-1 (IC(50)>289 microg/ml). This occurred only when RIAA was added prior to, but not post, LPS stimulation. Consistent with this observation, RIAA produced no physiologically relevant, direct inhibition of COX-1 or COX-2 peroxidase activity. This suggests that RIAA inhibits inducible but not constitutive COX-2. In support, we found RIAA showed minimal PGE(2) inhibition (IC(50)=21mug/ml) relative to celecoxib (IC(50)=0.024 microg/ml), aspirin (IC(50)=0.52 microg/ml) or ibuprofen (IC(50)=0.57 microg/ml) in the AGS gastric mucosal model, where COX-1 and -2 are expressed constitutively. Taken together these results predict RIAA may have lower potential for gastrointestinal and cardiovascular toxicity observed with COX enzyme inhibitors. Following confirmation of bioavailable RIAA administered orally, gastrointestinal safety was assessed using the fecal calprotectin biomarker in a 14-day human clinical study; RIAA (900 mg/day) produced no change compared to naproxen (1000 mg/day), which increased fecal calprotectin 200%. Cardiovascular safety was addressed by PGI-M measurements where RIAA (1000 mg) did not reduce PGI-M or affect the urinary PGI-M/TXB(2) ratio. Drug interaction potential was evaluated against six major CYPs; of relevance, RIAA inhibited CYP2C9. Toxicity was assessed in a 21-day oral, mouse subchronic toxicity study where no dose dependent histopathological effects were noted. Clinically, RIAA (1000 mg/day) produced a 54% reduction in WOMAC Global scores in a 6-week, open-label trial of human subjects exhibiting knee osteoarthritis.