Identification of 5-lipoxygenase and microsomal prostaglandin E2 synthase-1 as functional targets of the anti-inflammatory and anti-carcinogenic garcinol

Garcinol (camboginol) from the fruit rind of Guttiferae species shows anti-carcinogenic and anti-inflammatory properties, but the underlying molecular mechanisms are unclear. Here we show that garcinol potently interferes with 5-lipoxygenase (EC 7.13.11.34) and microsomal prostaglandin (PG)E₂ synthase (mPGES)-1 (EC 5.3.99.3), enzymes that play pivotal roles in inflammation and tumorigenesis. In cell-free assays, garcinol inhibited the activity of purified 5-lipoxygenase and blocked the mPGES-1-mediated conversion of PGH₂ to PGE₂ with IC₅₀ values of 0.1 and 0.3 μM, respectively. Garcinol suppressed 5-lipoxygenase product formation also in intact human neutrophils and reduced PGE₂ formation in interleukin-1β-stimulated A549 human lung carcinoma cells as well as in human whole blood stimulated by lipopolysaccharide. Moreover, garcinol interfered with isolated cyclooxygenase (COX)-1 (EC 1.14.99.1, IC₅₀ = 12 μM) and with the formation of COX-1-derived 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid and thromboxane B₂ in human platelets. In contrast, neither Ca²⁺-ionophore (A23187)-induced arachidonic acid release in neutrophils nor COX-2 activity in A549 cells or whole blood, measured as formation of 6-keto PGF_1α, or isolated human recombinant COX-2 were significantly affected by garcinol (≤30 μM). Together, the high potency of garcinol to selectively suppress PGE₂ synthesis and 5-lipoxygenase product formation provides a molecular basis for the anti-inflammatory and anti-carcinogenic effects of garcinol and rationalizes its therapeutic use.     

Analysis of CNS sympatho-inhibition produced by guanabenz

The effects of guanabenz on several autonomic systems were observed in vagotomized, anesthetized cats with the aim of determining, in a quantitative sense, the degree to which guanabenz produces a clonidine-like central nervous system action. Guanabenz given as a single dose (50 μg/kg i.v.) produced a transient hypertension associated with a more sustained bradycardia and depression of centrally (hypothalamic) evoked electrodermal responses (EDR). Increasing cumulative doses of guanabenz (3–1000 μg/kg i.v.) also resulted in a dose-related depression of EDR amplitude, transient hypertension followed by hypotension, sustained bradycardia, and mydriasis. All responses were antagonized by pretreatment with yohimbine (0.5 mg/kg i.v.). The ED₅₀ for depression of the centrally evoked EDR was in the range of 50–100 μg/kg i.v. in the non-pretreated preparations. Guanabenz (100 μg/kg i.v.) was shown to be devoid of significant ganglionic blocking properties. These experiments suggest that guanabenz acts like clonidine in the CNS and that an α₂-adrenergic inhibitory mechanism is involved in its myriad of central autonomic effects.     

Effects of AF3442 [N-(9-ethyl-9H-carbazol-3-yl)-2-(trifluoromethyl)benzamide], a novel inhibitor of human microsomal prostaglandin E synthase-1, on prostanoid biosynthesis in human monocytes in vitro

Inhibitors of microsomal prostaglandin (PG) E synthase-1 (mPGES-1) are being developed for the relief of pain. Redirection of the PGH₂ substrate to other PG synthases, found both in vitro and in vivo, in mPGES-1 knockout mice, may influence their efficacy and safety. We characterized the contribution of mPGES-1 to PGH₂ metabolism in lipopolysaccharide (LPS)-stimulated isolated human monocytes and whole blood by studying the synthesis of prostanoids [PGE₂, thromboxane (TX)B₂, PGF_2α and 6-keto-PGF_1α] and expression of cyclooxygenase (COX)-isozymes and down-stream synthases in the presence of pharmacological inhibition by the novel mPGES-1 inhibitor AF3442 [N-(9-ethyl-9H-carbazol-3-yl)-2-(trifluoromethyl)benzamide]. AF3442 caused a concentration-dependent inhibition of PGE₂ in human recombinant mPGES-1 with an IC₅₀ of 0.06 μM. In LPS-stimulated monocytes, AF3442 caused a concentration-dependent reduction of PGE₂ biosynthesis with an IC₅₀ of 0.41 μM. At 1 μM, AF3442 caused maximal selective inhibitory effect of PGE₂ biosynthesis by 61 ± 3.3% (mean ± SEM, P < 0.01 versus DMSO vehicle) without significantly affecting other prostanoids (i.e. TXB₂, PGF_2α and 6-keto-PGF_1α). In LPS-stimulated whole blood, AF3442 inhibited in a concentration-dependent fashion inducible PGE₂ biosynthesis with an IC₅₀ of 29 μM. A statistically significant inhibition of mPGES-1 activity was detected at 10 and 100 μM (38 ± 14%, P < 0.05, and 69 ± 5%, P < 0.01, respectively). Up to 100 μM, the other prostanoids were not significantly affected. In conclusion, AF3442 is a selective mPGES-1 inhibitor which reduced monocyte PGE₂ generation also in the presence of plasma proteins. Pharmacological inhibition of mPGES-1 did not translate into redirection of PGH₂ metabolism towards other terminal PG synthases in monocytes. The functional relevance of this observation deserves to be investigated in vivo.     

Paracetamol does not potentiate the acetylsalicylate inhibition of prostaglandin release from macrophages

Prostaglandin (PGE₂ and PGI₂) synthesis can be stimulated in macrophages by the tumour promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) and inhibited by acetylsalicyclic acid (IC₅₀ = 8.2 × 10^?6 M) and paracetamol (IC₅₀ = 1.9 × 10^?4 M). When mixtures of both drugs were tested only an additive effect was observed. This result is in contrast to recent reports describing both potentiation of acetylsalicylate-induced PG synthesis inhibtion and enhancement of PG production by paracetamol.     

Anti-allergic activity of emodin on IgE-mediated activation in RBL-2H3 cells

BackgroundEmodin (1,3,8-trihydroxy-6-methylanthraquinone) is a Chinese herbal anthraquinone derivative from the rhizome of rhubarb (Rheum palmatum L.) that exhibits numerous biological activities, such as antitumor, antibacterial, antiinflammatory, and immunosuppressive. In the present studies, the anti-allergic activities of emodin were investigated to elucidate the underlying active mechanisms.MethodsThe inhibitory effects of emodin on the IgE-mediated allergic response in rat basophilic leukemia (RBL-2H3) cells were evaluated by measuring the release of granules and cytokines. The Ca²⁺ mobilization in RBL-2H3 cells loaded with the Ca²⁺-reactive fluorescent probe Fluo-4 AM was also measured by laser scanning confocal microscope.ResultsEmodin inhibited the release of β-hexosaminidase (β-HEX; IC₅₀ = 5.5 μM) and tumor necrosis factor (TNF)-α (IC₅₀ = 11.5 μM) from RBL-2H3 cells induced by 2,4-dinitrophenylated bovine serum albumin (DNP-BSA) and displayed stronger inhibition of β-HEX release than ketotifen fumarate salt (IC₅₀ = 63.8 μM). Emodin at a concentration of 12.5 μM also inhibited the DNP-BSA-induced influx of extracellular Ca²⁺ in RBL-2H3 cells.ConclusionsThese results suggested that emodin likely exhibits anti-allergic activities via increasing the stability of the cell membrane and inhibiting extracellular Ca²⁺ influx.     

1,5-Disubstituted Indazol-3-ols with Anti-Inflammatory Activity

A series of new indazol-3-ol derivatives was synthesized. Some of these compounds exhibit interesting anti-inflammatory activities in various models of inflammation. 5-Methoxy-1-[quinoline-2-yl-methoxy)-benzyl]-1H-indazol-3-ol ( 27 ) strongly inhibits the oxidation of arachidonic acid to 5-hydroperoxyeicosatetraenoic acid catalyzed by 5-lipoxygenase (IC₅₀ = 44 nM). 27 also inhibits the contraction of sensitized guinea pig tracheal segments (IC₅₀ = 2.9 μM). In guinea pigs treated with 27 (1 mg/kg i.p.) 2 h before antigen provocation, there was a marked inhibition (47%) of the antigen-induced airway eosinophilia. After topical application of 1 μg/ear 27 inhibits the arachidonic acid induced mouse ear edema (41%). Indazole derivatives     

Evaluation of calcium entry blockers and alpha2-adrenergic antagonists on B-HT 920-induced presser responses in the autonomically blocked dog

Several calcium entry blockers and alpha2-adrenergic receptor antagonists were evaluated for inhibition of presser responses induced by the selective alpha₂ agonist B-HT 920 in pentobarbital-anesthetized dogs pretreated with prazosin (0.5 mg/kg, i.v.), propranolol (0.5 mg/kg, i.v.), and hexamethonium (10 mg/kg i.v.). In this preparation, autonomic blockade (alpha₁, beta, and ganglionic block) persists for approximately 4 hr. The B-HT 920 administered intravenously causes dose-related increases in mean arterial blood pressure (ED₅₀ = 4.90 μg/kg, i.v., dose causing a 50 mm Hg rise in mean arterial blood pressure). Maximum increases in mean arterial pressure approximate 80 mm Hg at 100 μg/kg, i.v. Repeated bolus administration of B-HT 920 over a 4-hr period shows no significant reduction in the pressor response, suggesting good stability of this experimental model and no rapidly developing tolerance. Calcium entry blockers (nifedipine, D-600, and diltiazem) and alphaz-adrenergic receptor antagonists (yohimbine and idazoxan) inhibit the B-HT 920-induced presser response in a dose-related manner. The ED₅₀ values (dose of antagonist that causes a 50% inhibition of B-HT 920-induced pressor response) were calculated. Idazoxan and yohimbine have ED₅₀ values (mg/kg, i.v.) of 0.086 and 0.063, respectively, whereas D-600, nifedipine, and diltiazem have values of 0.074, 0.111, and 0.542, respectively. The data show that calcium entry blockers and alpha2-adrenergic blockers are potent inhibitors of B-HT 920 pressor responses in the autonomically blocked dog. This experimental model is appropriate for the evaluation of calcium entry blockers and/or alpha₂-adrenergic antagonists in vivo.     

Selective inhibition of the cyclooxygenase pathway of the arachidonic acid cascade by the nonsteroidal antiinflammatory drug isoxicam

The effects of the nonsteroidal antiinflammatory drug isoxicam on prostaglandin formation by HSDM₁C₁ mouse fibrosarcoma cells grown in culture and 5-HETE(5(S)-5-hydroxy-6-trans, 8,11,14-cis eicosatetraenoic acid) formation by human leukocytes were determined. Isoxicam inhibited the formation of the cyclooxygenase product prostaglandin E₂ with an IC₅₀ of 2.0 μM. In comparison, the reference standards piroxicam and indomethacin had IC₅₀ values of 0.55 μM and 0.14 μM, respectively. Nordihydroguaiaretic acid (NDGA) was inactive up to 10 μM. Isoxicam, piroxicam, and indomethacin were relatively devoid of any significant inhibitory property on 5-lipoxygenase activity exhibiting IC₅₀ values > 500 μM. In comparison, the compounds BW755C and NDGA, known lipoxygenase inhibitors, had IC₅₀ values of 11 μM and 0.6 μM, respectively. The present findings indicated that isoxicam exhibited a selective inhibition of the cyclooxygenase pathway of the arachidonic acid cascade and that this effect may at least in part be responsible for its antiinflammatory activity observed in animals and humans.     

1-methyl substituent and stereochemical effects of 2-phenylcyclopropylamines on the inhibition of rat brain mitochondrial monoamine oxidase A and B

(E)-2-Phenylcyclopropylamine ((E)-TCP), (Z)-2-phenylcyclopropylamine ((Z)-TCP), (E)-1-methyl-2-phenylcyclopropylamine ((E)-MTCP), and (Z)-1-methyl-2-phenylcyclopropylamine ((Z)-MTCP) were synthesized and used to determine to what extent 1-methyl substitution and stereochemistry of 2-phenylcyclopropylamines affect inhibition of monoamine oxidase (MAO). Inhibition of rat brain mitochondrial MAO-A and B by the compounds were measured using serotonin and benzylamine as the substrate, respectively and IC₅₀ values obtained with 95% confidence limits by the method of computation. For the inhibition of MAO-A, (E)-MTCP (IC₅₀=6.2×10^?8M) was found to be 37 times more potent than (Z)-MTCP (IC₅₀=2.3×10^?6 M), whereas the activity of (E)-TCP (IC₅₀=2.9×10^?7 M) was slightly less than that of (Z)-TCP (IC₅₀=2.3×10^?7 M). Similarly, for the inhibition of MAO-B, (E)-MTCP (IC₅₀=6.3×10^?8 M) was 7 times more potent than (Z)-MTCP (IC₅₀=4.7×10^?7 M) and (E)-TCP (IC₅₀=7.8×10^?8 M), 0.6 times as potent as (Z)-TCP (IC₅₀=4.4×10^?8 M). The results suggested that while without 1-methyl group, potency of a (Z)-isomer was comparable to that of (E)-isomer, the methyl group in its (Z)-position was very unfavorable to the inhibition of MAO and that in its (E)-position, the methyl group contributed positively to the potency as found by the fact that (E)-MTCP was 1–5 times more potent than (E)-TCP. In view of the selective inhibition of MAO-A or B, all compounds elicited 4–10 times higher preference for the inhibition of MAO-B over MAO-A and 1-methyl substitution as well as the stereochemical factors did not significantly influence the selectivity.     

Synthesis of some 1,3,4-thiadiazole derivatives as inhibitors of Entamoeba histolytica

In the quest for potent anti-amoebic agents, some 1,3,4-thiadiazole derivatives were synthesized and characterized by spectral data. The purity of the compounds was confirmed by elemental analysis. All the compounds were screened in vitro against HM1:IMSS strain of Entamoeba histolytica by microdilution method. The results revealed that compounds 1 (IC₅₀ = 0.670 μM), 3 (IC₅₀ = 1.60 μM) and 8 (IC₅₀ = 0.522 μM) had much better anti-amoebic activity than the reference drug metronidazole (IC₅₀ = 1.80 μM). Further, cytotoxicity of the compounds having IC₅₀ value less than metronidazole was assessed by MTT assay on human breast cancer MCF-7 cell line and all the compounds were found low cytotoxic in the concentration range of 2.5–250 μM. Preliminary results indicate that these three compounds (1, 3 and 8) may be subjected to further investigations and it may be hoped that the present study will stimulate efforts towards the development of novel effective anti-amoebic agents.     

Inhibition of [3H]norepinephrine uptake in peripheral organs of some mammalian species by ouabain

In order to demonstrate the possible involvement of (Na⁺ + K⁺)-ATPase in the high affinity uptake of [³H]-norepinephrine in the sympathetic nerve endings, the effect of ouabain on [³H]norepinephrine uptake in spleen and heart slices of five mammalian species was examined. The ouabain sensitivity of [³H]norepinehrine uptake in the heart slices from various species, as determined by the estimation of IC₅₀, was, in increasing order, lamb (2.3 μM) < calf (2.5 μM) < guinea pig (4 μM) < rabbit (10 μM) << rat (>500 μM). The IC₅₀ values in the spleen slices were: lamb (1 μM) < calf (3.2 μM) < rabbit (9.5 μM) < guinea pig (25 μM) << rat (>500μM). The IC₅₀ values for the inhibition of specific [³H]ouabain binding in the microsomal fractions of spleen and heart of the five mammalian species by ouabain were similar to the IC₅₀ values for the inhibition of [³H]norepinephrine uptake by the cardiac glycoside. Since ouabain is known to bind exclusively to (Na⁺ + K⁺)-ATPase of a microsomal fraction, these results suggest that the inhibition of [³H]norepinephrine uptake in the sympathetic nerve endings by ouabain is mediated by the inhibition of (Na⁺ + K⁺)-ATPase.     

A new antiinflammatory compound, timegadine (N-cyclohexyl-N"-4-[2-methylquinolyl]-N'-2-thiazolylguanidine), which inhibits both prostaglandin and 12-hydroxyeicosatetraenoic acid (12-HETE) formation

Timegadine (N-cyclohexyl-N′′-4-[2-methylquinolyl]-N′-2-thiazolylguanidine), a new antiinflammatory agent, was found to be a potent, competitive inhibitor of prostaglandin synthetase derived from a variety of tissues, including bovine seminal vesicles, rabbit renal papillae, rabbit lung, rabbit platelets and rat brain. The concentration of timegadine required to obtain 50% inhibition (IC₅₀) varied from 5 × 10^?9M (washed rabbit platelets) to 2 × 10^?5M (rat brain). Timegadine was also found to be an inhibitor of lipoxygenase activity in the cytosol fraction of horse platelet homogenates, and in washed rabbit platelets. IC₅₀ was 1 × 10^?4 M in both cases. These effects are discussed in the view of recent evidence, suggesting that dual inhibitors of arachidonate cyclo-oxygenase and lipoxygenase may have an improved profile of antiinflammatory activity.     

Effects of the natural flavone trimethylapigenin on cardiac potassium currents

The natural flavones and polymethylflavone have been reported to have cardiovascular protective effects. In the present study, we determined whether quecertin, apigenin and their methylated compounds (3,7,3′,4′-tetramethylquecertin, 3,5,7,3′,4′-pentamethylquecertin, 7,4′-dimethylapigenin, and 5,7,4′-trimethylapigenin) would block the atrial specific potassium channel hKv1.5 using a whole-cell patch voltage-clamp technique. We found that only trimethylapigenin showed a strong inhibitory effect on hKv1.5 channel current. This compound suppressed hKv1.5 current in HEK 293 cell line (IC₅₀ = 6.4 μM), and the ultra-rapid delayed rectify K⁺ current I_Kur in human atrial myocytes (IC₅₀ = 8.0 μM) by binding to the open channels and showed a use- and frequency-dependent manner. In addition, trimethylapigenin decreased transient outward potassium current (I_to) in human atrial myocytes, inhibited acetylcholine-activated K⁺ current (IC₅₀ = 6.8 μM) in rat atrial myocytes. Interestingly, trimethylapigenin had a weak inhibition of hERG channel current. Our results indicate that trimethyapigenin significantly inhibits the atrial potassium currents hKv1.5/I_Kur and I_KACh, which suggests that trimethylapigenin may be a potential candidate for anti-atrial fibrillation.     

Inhibition of CYP3A by Antimalarial Piperaquine and Its Metabolites in Human Liver Microsomes With IVIV Extrapolation

The potential of the antimalarial piperaquine and its metabolites to inhibit CYP3A was investigated in pooled human liver microsomes. CYP3A activity was measured by liquid chromatography-tandem mass spectrometry as the rate of 1′-hydroxymidazolam formation. Piperaquine was found to be a reversible, potent inhibitor of CYP3A with the following parameter estimates (%CV): IC₅₀ = 0.76 μM (29), K_i = 0.68 μM (29). In addition, piperaquine acted as a time-dependent inhibitor with IC₅₀ declining to 0.32 μM (28) during 30-min pre-incubation. Time-dependent inhibitor estimates were k_inact = 0.024 min^?1 (30) and K_I = 1.63 μM (17). Metabolite M2 was a highly potent reversible inhibitor with estimated IC₅₀ and K_i values of 0.057 μM (17) and 0.043 μM (3), respectively. M1 and M5 metabolites did not show any inhibitory properties within the limits of assay used. Average (95th percentile) simulated in vivo areas under the curve of midazolam increased 2.2-fold (3.7-fold) on the third which is the last day of piperaquine dosing, whereas for its metabolite M2, areas under the curve of midazolam increased 7.7-fold (13-fold).     

Pentacyclic triterpenoids from Mitracarpus frigidus (Willd. ex Roem. & Schult.) K. Shum: in vitro cytotoxic and leishmanicidal and in vivo anti-inflammatory and antioxidative activities

Previous studies had shown that different extracts obtained from Mitracarpus frigidus (Willd. ex Roem. & Schult.) K. Shum, an annual shrub commonly found in South America, present antimicrobial, leishmanicidal, cytotoxic, laxative, and anti-inflammatory activities. Thus, this study aimed to isolate the bioactive compounds from the hexane extract of the aerial parts of this plant and to evaluate some of their biological activities. The bioactivity guided fractionation of the hexane extract of M. frigidus afforded two pentacyclic triterpenoids, ursolic acid and methyl ursolate. Their identities were confirmed unambiguously by way of ¹H, ¹³C, ¹H COSY, HMBC, HMQC, Dept 135, IR and UV–Vis spectroscopy, and mass spectrometry. These compounds, hitherto unknown in the species, were evaluated for their in vitro biological activities against four tumor cell lines (HL60, Jurkat, MCF-7, and HCT) and four Leishmania species (L. amazonensis, L. major, L. braziliensis, and L. chagasi). Also, their in vivo anti-inflammatory effect was evaluated using carrageenan-induced paw edema and carrageenan-induced peritonitis. Ursolic acid was active for all tumor cell lines with ED₅₀ values varying from 4.2 to 35.7 μg/mL, and methyl ursolate was active only for HL60 cells with an ED₅₀ value of 22.7 μg/mL. A pronounced antileishmanial potential was verified for amastigote form of L. major, with IC₅₀ values of 1.3 and 2.1 μg/mL for ursolic acid and methyl ursolate, respectively. They also presented in vivo anti-inflammatory effects in the carrageenan-induced paw edema test. Oral administration of these triterpenoids at 1 mg/kg significantly inhibited edema formation induced by carrageenan by 80 % and peritoneal leukocyte migration by 85 %. The results obtained reinforced that pentacyclic triterpenoids are an important class of natural products for investigation in the search of new bioactive compounds.     

Synthesis and evaluation of thiopyrano[3,4-c]quinoline-9-carboxamide derivatives as inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1)

A series of poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors, 5-oxo-2,4,5,6-tetrahydro-1H-thiopyrano[3,4-c]quinoline-9-carboxamide derivatives, were successfully synthesized and their PARP-1 inhibitory activity was evaluated. These compounds were prepared from carboxylic acid 7 and the appropriate amines, and a number of the synthesized compounds were found to have significant PARP-1 activity. Among them, 9m showed potent activity in a PARP-1 enzymatic assay and cell-based assay (IC₅₀?=?0.045?μM, ED₅₀?=?0.54?μM). Molecular modeling studies confirmed the obtained biological results.     

Steroid constituents from the soft coral Sinularia microspiculata

A methanol extract of the soft coral Sinularia microspiculata revealed five sterols, including two new compounds. Using combined chromatographic and spectroscopic experiments, the new compounds were found to be 7-oxogorgosterol (1) and 16α-hydroxysarcosterol (2). Their structures were determined on the basis of spectroscopic data (¹H and ¹³C NMR, HSQC, HMBC, ¹H-¹H COSY, NOESY, and FT-ICR-MS) and by comparing obtained results to the values indicated in previous studies. Among the isolated compounds, 3 showed weak cytotoxic effects against HL-60 (IC_50? = 89.02 ± 9.93?μM) cell line, whereas 5 was weakly active against HL-60 (IC_50? = 82.80 ± 13.65?μM) and SK-Mel2 (IC_50? = 72.32 ± 1.30?μM) cell lines.     

Diorganotin (IV) derivative of 2-thiophene acetic acid: characterizations and biological activities of {[n-Bu2SnO2C–CH2–C4H3S]2O}2

New organotin (IV) derivatives of thiophene acetic acid have been prepared and characterized by IR, ¹H- and ¹³C-NMR spectroscopic techniques. A single crystal of {[n-Bu₂SnO₂C–CH₂–C₄H₃S]₂O}₂ has been synthesized and its cell parameters were measured. It crystallizes in the monoclinic system (P2₁) [a?=?15.1337(7), b?=?12.2587(5), c?=?18.8766(9), and β?=?105.811(5)°]. The compound showed selective inhibitory effect against β-glucuronidase enzyme (IC₅₀ 3.1?±?0.1?μM), which is more potent than our standard, d-saccharic acid 1,4-lactone (IC₅₀ 48.4?±?1.3?μM). Also, it exhibited immunomodulatory activity, and cytotoxicity against PC-3 cell line (IC₅₀?=?16.9?±?1.3?μM, and 1.2?±?0.1?μM, respectively), which are close to the standards ibuprofen (IC₅₀ 11.8?±?1.8?μM), and doxorubicin (IC₅₀ 0.9?±?0.1?μM), respectively. This compound did not show any significant inhibition for other biological test such as α-chymotrypsin, urease, phosphodiesterase enzymes, and antiglycation activity.     

Expression of proliferative and inflammatory markers in a full-thickness human skin equivalent following exposure to the model sulfur mustard vesicant, 2-chloroethyl ethyl sulfide

Sulfur mustard is a potent vesicant that induces inflammation, edema and blistering following dermal exposure. To assess molecular mechanisms mediating these responses, we analyzed the effects of the model sulfur mustard vesicant, 2-chloroethyl ethyl sulfide, on EpiDerm-FT™, a commercially available full-thickness human skin equivalent. CEES (100-1000 μM) caused a concentration-dependent increase in pyknotic nuclei and vacuolization in basal keratinocytes; at high concentrations (300-1000 μM), CEES also disrupted keratin filament architecture in the stratum corneum. This was associated with time-dependent increases in expression of proliferating cell nuclear antigen, a marker of cell proliferation, and poly(ADP-ribose) polymerase (PARP) and phosphorylated histone H2AX, markers of DNA damage. Concentration- and time-dependent increases in mRNA and protein expression of eicosanoid biosynthetic enzymes including COX-2, 5-lipoxygenase, microsomal PGE₂ synthases, leukotriene (LT) A₄ hydrolase and LTC₄ synthase were observed in CEES-treated skin equivalents, as well as in antioxidant enzymes, glutathione S-transferases A1-2 (GSTA1-2), GSTA3 and GSTA4. These data demonstrate that CEES induces rapid cellular damage, cytotoxicity and inflammation in full-thickness skin equivalents. These effects are similar to human responses to vesicants in vivo and suggest that the full thickness skin equivalent is a useful in vitro model to characterize the biological effects of mustards and to develop potential therapeutics.     

On the inhibition of 5-lipoxygenase product formation by tryptanthrin: mechanistic studies and efficacy in vivo

BACKGROUND AND PURPOSE

Leukotrienes (LTs) are pro-inflammatory mediators produced by 5-lipoxygenase (5-LO). Currently available 5-LO inhibitors either lack efficacy or are toxic and novel approaches are required to establish a successful anti-LT therapy. Here we provide a detailed evaluation of the effectiveness of the plant-derived alkaloid tryptanthrin as an inhibitor of LT biosynthesis.

EXPERIMENTAL APPROACH

We analysed LT formation and performed mechanistic studies in human neutrophils stimulated with pathophysiologically relevant stimuli (LPS and formyl peptide), as well as in cell-free assays (neutrophil homogenates or recombinant human 5-LO) and in human whole blood. The in vivo effectiveness of tryptanthrin was evaluated in the rat model of carrageenan-induced pleurisy.

KEY RESULTS

Tryptanthrin potently reduced LT-formation in human neutrophils (IC₅₀ = 0.6 µM). However, tryptanthrin is not a redox-active compound and did not directly interfere with 5-LO activity in cell-free assays. Similarly, tryptanthrin did not inhibit the release of arachidonic acid, the activation of MAPKs, or the increase in [Ca²⁺]_i, but it modified the subcellular localization of 5-LO. Moreover, tryptanthrin potently suppressed LT formation in human whole blood (IC₅₀ = 10 µM) and reduced LTB₄ levels in the rat pleurisy model after a single oral dose of 10 mg·kg⁻¹.

CONCLUSIONS AND IMPLICATIONS

Our data reveal that tryptanthrin is a potent natural inhibitor of cellular LT biosynthesis with proven efficacy in whole blood and is effective in vivo after oral administration. Its unique pharmacological profile supports further analysis to exploit its pharmacological potential.