A novel electrochemical immunosensor for the sensitive detection of tiamulin based on staphylococcal protein A and silver nanoparticle-graphene oxide nanocomposites.

Tiamulin (TML) is a pleuromutilin antibiotic and mainly used to treat pulmonary and gastrointestinal infections. However, excessive use of TML can bring health threats to consumers. In this work, a label-free electrochemical immunosensor was proposed for sensitive detection of TML in pork and pork liver. Silver nanoparticles (AgNPs) were synthesized in situ on graphene oxide (GO), in which GO acted as a carrier for loading more AgNPs and AgNPs exhibited both strong conductivity and good redox property. In addition, staphylococcal protein A (SPA) was applied to oriented immobilization of fragment crystallizable (Fc) region of the TML monoclonal antibody. Under the optimal condition, the developed electrochemical immunosensor exhibited a good linear response with a concentration of TML ranging from 0.05 ng mL-1 to 100 ng mL-1 and the limit of detection (LOD) was 0.04 ng mL-1. Furthermore, the designed immunosensor was applied to detect TML in real samples with a good accuracy. Therefore, the label-free electrochemical immunosensor could be used as a potential method to detect TML and other antibiotic residues in animal derived foods.

Chemo-enzymatic synthesis of the ALG1-CDG biomarker and evaluation of its immunogenicity.

Congenital disorders of glycosylation (CDG) are a growing group diseases that result from defects in genes involved in glycan biosynthesis pathways. One tetrasaccharide, i.e., Neu5Ac-α2, 6-Gal-ß1, 4-GlcNAc-ß1, 4-GlcNAc, was recently reported as the biomarker of ALG1-CDG, the disease caused by ALG1 deficiency. To develop a novel diagnostic method for ALG1-CDG, chemo-enzymatic synthesis of the tetrasaccharide biomarker linked to phytanyl phosphate and the biomarker's immune stimulation were investigated in this study. The immunization study using liposomes bearing phytanyl-linked tetrasaccharide revealed that they stimulated a moderate immune response. The induced antibody showed strong binding specificity for the ALG1-CDG biomarker, indicating its potential in medical applications.

Immunochemical Detection of Protein Modification Derived from Metabolic Activation of 8-Epidiosbulbin E Acetate.

Furanoid 8-epidiosbulbin E acetate (EEA) is one of the most abundant diterpenoid lactones in herbal medicine Dioscorea bulbifera L. (DB). Our early work proved that EEA could be metabolized to EEA-derived cis-enedial (EDE), a reactive intermediate, which is required for the hepatotoxicity observed in experimental animals exposed to EEA. Also, we found that EDE could modify hepatic protein by reaction with thiol groups and/or primary amines of protein. The present study was inclined to develop polyclonal antibodies to detect protein modified by EDE. An immunogen was prepared by reaction of EDE with keyhole limpet hemocyanin (KLH), and polyclonal antibodies were raised in rabbits immunized with the immunogen. Antisera collected from the immunized rabbits demonstrated high titers evaluated by enzyme-linked immunosorbent assays (ELISAs). Immunoblot analysis showed that the polyclonal antibodies recognized EDE-modified bovine serum albumin (BSA) in a hapten load-dependent manner but did not cross-react with native BSA. Competitive inhibition experiments elicited high selectivity of the antibodies toward EDE-modified BSA. The antibodies allowed us to detect and enrich EDE-modified protein in liver homogenates obtained from EEA-treated mice. The developed immunoprecipitation technique, along with mass spectrometry, enabled us to succeed in identifying multiple hepatic proteins of animals given EEA. We have successfully developed polyclonal antibodies with the ability to recognize EDE-derived protein adducts, which is a unique tool for us to define the mechanisms of toxic action of EEA.

Screening and Identification for Immunological Active Components from Andrographis Herba Using Macrophage Biospecific Extraction Coupled with UPLC/Q-TOF-MS.

The method of cell biospecific extraction coupled with UPLC/Q-TOF-MS has been developed as a tool for the screening and identification of potential immunological active components from Andrographis Herba (AH). In our study, a macrophage cell line (RAW264.7) was used to extract cell-combining compounds from the ethanol extract of AH. The cell binding system was then analyzed and identified by UPLC/Q-TOF-MS analysis. Finally, nine compounds, which could combine with macrophages, in an ethanol extract of AH were detected by comparing basic peak intensity (BPI) profiles of macrophages before and after treatment with AH. Then they were identified as Andrographidine E (1), Andrographidine D (2), Neoandrographolide (3), Dehydroandrographolide (4), 5, 7, 2', 3'-tetramethoxyflavone (5), ß-sitosterol (7), 5-hydroxy-7, 2', 3'-trimethoxyflavone (8) and 5-hydroxy-7, 8, 2', 3'-tetramethoxyflavone (9), which could classified into five flavonoids, three diterpene lactones, and one sterol. Their structures were recognized by their characteristic fragment ions and fragmentations pattern of diterpene lactones and flavonoids. Additionally, the activity of compounds 3, 4, and 7 was tested in vitro. Results showed that these three compounds could decrease the release of NO (p < 0.01) in macrophages remarkably. Moreover, 3, 4, and 7 showed satisfactory dose-effect relationships and their IC50 values were 9.03, 18.18, and 13.76 µg/mL, respectively. This study is the first reported work on the screening of immunological active components from AH. The potential immunological activity of flavonoids from AH has not been reported previously.

Ovatodiolide suppresses colon tumorigenesis and prevents polarization of M2 tumor-associated macrophages through YAP oncogenic pathways.

BACKGROUND: An increased expression of Yes-associated protein (YAP1) has been shown to promote tumorigenesis in many cancer types including colon. However, the role of YAP1 in promoting colon tumorigenesis remains unclear. Here, we demonstrate that YAP1 expression is associated with M2 tumor-associated macrophage polarization and the generation of colon cancer stem-like cells. YAP1 downregulation by gene silencing or a phytochemical, ovatodiolide, not only suppresses colon cancer tumorigenesis but also prevents M2 TAM polarization. METHODS: Human monocytic cells, THP-1, and colon cancer cell lines, HCT116 and DLD-1, were co-cultured to mimic the interactions between tumor and its microenvironment. M2 polarization of the THP-1 cells were examined using both flow cytometry and q-PCR technique. The inhibition of YAP1 signaling was achieved by gene-silencing technique or ovatodiolide. The molecular consequences of YAP1 inhibition was demonstrated via colony formation, migration, and colon-sphere formation assays. 5-FU and ovatodiolide were used in drug combination studies. Xenograft and syngeneic mouse models were used to investigate the role of YAP1 in colon tumorigenesis and TAM generation. RESULTS: An increased YAP1 expression was found to be associated with a poor prognosis in patients with colon cancer using bioinformatics approach. We showed an increased YAP1 expression in the colon spheres, and colon cancer cells co-cultured with M2 TAMs. YAP1-silencing led to the concomitant decreased expression of major oncogenic pathways including Kras, mTOR, ß-catenin, and M2-promoting IL-4 and tumor-promoting IL-6 cytokines. TAM co-cultured colon spheres showed a significantly higher tumor-initiating ability in vivo. Ovatodiolide treatment alone and in combination with 5-FU significantly suppressed in vivo tumorigenesis and less TAM infiltration in CT26 syngeneic mouse model. CONCLUSIONS: We have identified the dual function of YAP1 where its suppression not only inhibited tumorigenesis but also prevented the generation of cancer stem-like cells and M2 TAM polarization. Ovatodiolide treatment suppressed YAP1 oncogenic pathways to inhibit colon tumorigenesis and M2 TAM generation both in vitro and in vivo. Ovatodiolide should be considered for its potential for adjuvant therapeutic development.

Fluorescence polarization immunoassay using IgY antibodies for detection of valnemulin in swine tissue.

Immunoglobulin Y (IgY) is derived from egg yolk and has been identified as a cheap and high-yield immunoreagent. The application of IgY in immunoassays for the detection of chemical contaminants in food samples has rarely been reported. In this work, we describe a rapid and sensitive fluorescence polarization immunoassay (FPIA) for valnemulin (VAL) using IgY which was produced using a previously prepared immunogen. Three fluorophore-labeled VAL tracers were synthesized and the sensitivity of the best tracer (VAL-DTAF) in the optimized FPIA with antibody IgY100 demonstrated an IC50 value of 12 ng mL(-1) in buffer. After evaluation of several extraction procedures, acidified acetonitrile was selected to extract VAL from swine tissue. The recoveries of VAL in spiked swine tissue at three levels (50, 100, and 200 µg kg(-1)) were higher than 79% with coefficients of variation (CVs) lower than 12%. The limit of detection (LOD) of the FPIA in swine tissue was 26 µg kg(-1) and was lower than the maximum residue limit (MRL) of VAL set by the European Union. The study showed that IgY could be a good substitute for IgG when developing a high-throughput assay for chemical residues.

Diterpenoids with Immunosuppressive Activities from Cinnamomum cassia.

Three new diterpenoids with unprecedented carbon skeletons, cinncassiols F (1) and G (2) and 16-O-ß-D-glucopyranosyl-19-deoxycinncassiol G (3), a new isoryanodane diterpenoid, 18-hydroxyperseanol (4), six known isoryanodane diterpenoids, 5-10, and a known ryanodane diterpenoid, 11, were isolated from the stem bark of Cinnamomum cassia. Compound 1 possesses an 11,13:12,13-diepoxy-6,11-epoxy:12,13-disecoisoryanodane diterpenoid skeleton bearing ketal and hemiketal functionalities, whereas compounds 2 and 3 feature an 11,12-secoisoryanodane diterpenoid skeleton with an 11,6-lactone moiety. The structures of the four new diterpenoids, 1-4, and their absolute configurations were established using HRESIMS, NMR, ECD, single-crystal X-ray diffraction, and chemical methods. Compounds 2 and 11 significantly inhibited the proliferation of murine T cells induced by ConA.

Formation of the unusual semivolatile diterpene rhizathalene by the Arabidopsis class I terpene synthase TPS08 in the root stele is involved in defense against belowground herbivory.

Secondary metabolites are major constituents of plant defense against herbivore attack. Relatively little is known about the cell type-specific formation and antiherbivore activities of secondary compounds in roots despite the substantial impact of root herbivory on plant performance and fitness. Here, we describe the constitutive formation of semivolatile diterpenes called rhizathalenes by the class I terpene synthase (TPS) 08 in roots of Arabidopsis thaliana. The primary enzymatic product of TPS08, rhizathalene A, which is produced from the substrate all-trans geranylgeranyl diphosphate, represents a so far unidentified class of tricyclic diterpene carbon skeletons with an unusual tricyclic spiro-hydrindane structure. Protein targeting and administration of stable isotope precursors indicate that rhizathalenes are biosynthesized in root leucoplasts. TPS08 expression is largely localized to the root stele, suggesting a centric and gradual release of its diterpene products into the peripheral root cell layers. We demonstrate that roots of Arabidopsis tps08 mutant plants, grown aeroponically and in potting substrate, are more susceptible to herbivory by the opportunistic root herbivore fungus gnat (Bradysia spp) and suffer substantial removal of peripheral tissue at larval feeding sites. Our work provides evidence for the in vivo role of semivolatile diterpene metabolites as local antifeedants in belowground direct defense against root-feeding insects.

Production of antibodies and development of enzyme-linked immunosorbent assay for valnemulin in porcine liver.

Polyclonal and monoclonal antibodies against valnemulin, a new semi-synthetic antibiotic derivative of pleuromutilin administered to treat swine dysentery and pneumonia, were generated. To achieve high enzyme-linked immunosorbent assay sensitivity for valnemulin, several heterologous coating antigens were prepared and evaluated, differing in the length of the spacer arm and the conjugation site between valnemulin and carrier protein. After the optimisation of immuno-reagents dilution, the enzyme-linked immunosorbent assay, based on polyclonal antibody number 2 and one heterologous coating antigen, showed the highest sensitivity with an IC(50) value for valnemulin of 0.96 ng/mL in buffer. For spiked porcine liver, an extraction procedure with a mixture of acetonitrile and 0.01 M hydrochloric acid (40:60 v/v) was proposed and no further sample pre-treatment other than 10 times dilution of the extract was necessary prior to analysis, which gave recovery values ranging from 75.7% to 89.4%. The dynamic assay range and the limit of detection of the assay were 2.4-49.9 and 1.67 µg/kg for porcine liver, respectively. The assay was compared with a confirmation method based on LC-MS/MS by using valnemulin-treated samples, and a satisfactory correlation between both methods was observed.

Anti-inflammatory mechanism of 15,16-epoxy-3α-hydroxylabda-8,13(16),14-trien-7-one via inhibition of LPS-induced multicellular signaling pathways.

Phytochemical investigation of Leonurus japonicus has led to the isolation of a labdane diterpene derivative, 15,16-epoxy-3α-hydroxylabda-8,13(16),14-trien-7-one (1), which was tested for its in vitro anti-inflammatory effects. The results demonstrated that 1 exhibits an inhibitory effect on LPS-stimulated RAW 264.7 macrophages. The anti-inflammatory action shown by 1 suppressed LPS-induced NF-κB activation, resulting in the down-regulation of iNOS and COX-2 protein expression, attributable to the inhibitory action of LPS-induced NO and PGE(2) production. Compound 1 inhibited LPS-induced phosphorylation and the degradation of inhibitory kappa B (IκBα) and decreased the nuclear translocation of p50 and p65. In addition, 1 exhibited an inhibitory effect on LPS-induced NF-κB-DNA and AP-1-DNA binding activity, using an electrophoretic mobility shift assay with NF-κB- and AP-1-specific (32)P-labeled probes. The LPS-induced mitogen-activated protein kinases (p-JNK, p-p38, and p-ERK) and p-Akt were inhibited after 30 and 10 min of LPS stimulation, respectively. In addition, TNF-α production was suppressed by 1.

Synthetic adjuvants for vaccine formulations: evaluation of new phytol derivatives in induction and persistence of specific immune response.

Terpenoids are ubiquitous natural compounds that have been shown to improve vaccine efficacy as adjuvants. To gain an understanding of the structural features important for adjuvanticity, we studied compounds derived from a diterpene phytol and assessed their efficacy. In a previous report, we showed that phytol and one of its derivatives, PHIS-01 (a phytol-derived immunostimulant, phytanol), are excellent adjuvants. To determine the effects of varying the polar terminus of PHIS-01, we designed amine and mannose-terminated phytol derivatives (PHIS-02 and PHIS-03, respectively). We studied their relative efficacy as emulsions with soluble proteins, ovalbumin, and a hapten-protein conjugate phthalate-KLH. Immunological parameters evaluated consisted of specific antibody responses in terms of titers, specificities and isotype profiles, T cell involvement and cytokine production. Our results indicate that these new isoprenoids were safe adjuvants with the ability to significantly augment immunogen-specific IgG1 and IgG2a antibody responses. Moreover, there was no adverse phthalate cross-reactive anti-DNA response. Interestingly, PHIS-01 and PHIS-03 influenced differentially T-helper polarization. We also observed that these compounds modulated the immune response through apoptotic/necrotic effects on target tumor cells using murine lymphomas. Finally, unlike squalene and several other terpenoids reported to date, these phytol derivatives did not appear arthritogenic in murine models.

Magnesium depletion triggers production of an immune modulating diterpenoid in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) is the causative agent of the human disease Tuberculosis, and remains a worldwide health threat responsible for ∼1.7 million deaths annually. During infection, Mtb prevents acidification of the engulfing phagosome, thus blocking endocytic progression and eventually leading to stable residence. The diterpenoid metabolite isotuberculosinol (isoTb) exhibits biological activity indicative of a role in this early arrest of phagosome maturation. Presumably, isoTb production should be induced by phagosomal entry. However, the relevant enzymatic genes are not transcriptionally upregulated during engulfment. Previous examination of the initial biosynthetic enzyme (Rv3377c/MtHPS) involved in isoTb biosynthesis revealed striking inhibition by its Mg(2+) cofactor, leading to the hypothesis that the depletion of Mg(2+) observed upon phagosomal engulfment may act to trigger isoTb biosynthesis. While Mtb is typically grown in relatively high levels of Mg(2+) (0.43 mM), shifting Mtb to media with phagosomal levels (0.1 mM) led to a significant (∼10-fold) increase in accumulation of the MtHPS product, halimadienyl diphosphate, as well as easily detectable amounts of the derived bioactive isoTb. These results demonstrate isoTb production by Mtb specifically under conditions that mimic phagosomal cation concentrations, and further support a role for isoTb in the Mtb infection process.

Diterpenos en infl amación: las Labiadas como paradigma / Diterpenes in inflammation: Labiatae as paradigm

Esta revisión trata esencialmente sobre los diterpenos antiinflamatorios procedentes de especies de Labiadas. Se estudian las publicaciones posteriores a 2002 referentes a los cuatro tipos estructurales más importantes en esa familia: labdano, abietano, pimarano y kaurano. Además se incluyen algunas moléculas interesantes obtenidas de especies de diferente origen taxonómico. De entre los mecanismos implicados destaca la inhibición del metabolismo del ácido araquidónico a través de ciclooxigenasas y lipoxigenasas, así como el freno a la producción de NO o citocinas. Adicionalmente se ha descrito para algunos principios el aumento de la expresión de hemo-oxigenasa 1. Algunos de los géneros botánicos mejor representados son Lavandula, Isodon, Rosmarinus y Sideritis (AU)

This review is devoted to the antiinflammatory diterpenoids obtained from species of Labiatae. Basically, we study the post-2002 publications concerning the four major structural types in the family: labdane, abietane, pimarane and kaurane. Certain interesting diterpenes derived from plants of different taxonomic origin are also reported. Among the mechanisms involved in the pharmacological effect, we should highlight the inhibition of arachidonic acid metabolism through cyclo-oxygenases and lipoxygenases, and the decrease in the production of NO or cytokines. Additionally, some principles are described as enhancers of the expression of heme oxygenase 1. Some of the botanical genera best represented are Lavandula, Isodon, Rosmarinus or Sideritis (AU)

Diterpenes drive Th1 polarization depending on IL-12.

Sandaracopimaric acid and Sandaracopimaradiene-3beta-ol are diterpenes isolated from the heatwood of Cryptomeria japonica and are pharmacologically active substances. Dendritic cells (DC) are key antigen presenting cells (APC), which link innate and adaptive immunity, ultimately activating antigen-specific T cells. We demonstrate that Sandaracopimaric acid and Sandaracopimaradiene-3beta-ol activate humans DC as documented by phenotypic and functional maturation and altered cytokine production. The expression of the co-stimulatory molecules such as CD83, CD86 and HLA-DR on Sandaracopimaric acid- and Sandaracopimaradiene-3beta-ol-primed DC was enhanced. Sandaracopimaric acid- and Sandaracopimaradiene-3beta-ol-primed DC also enhanced the T cell stimulatory capacity in an allo MLR. Naive T cells co-cultured with Sandaracopimaric acid- or Sandaracopimaradiene-3beta-ol-primed DC turned into typical Th1 cells, which produced large quantities of IFN-gamma and released small amounts of IL-4 depending on IL-12 secretion. Sandaracopimaric acid- or Sandaracopimaradiene-3beta-ol-primed DC had a high migration to macrophage inflammatory protein (MIP)-3beta. These results suggest that some diterpenes modulate human DC function in a fashion that favors Th1 cell polarization and may be used on DC-based vaccines for cancer immunotherapy.

Neutrophils are a key component of the antitumor efficacy of topical chemotherapy with ingenol-3-angelate.

Harnessing neutrophils for the eradication of cancer cells remains an attractive but still controversial notion. In this study, we provide evidence that neutrophils are required to prevent relapse of skin tumors following topical treatment with a new anticancer agent, ingenol-3-angelate (PEP005). Topical PEP005 treatment induces primary necrosis of tumor cells, potently activates protein kinase C, and was associated with an acute T cell-independent inflammatory response characterized by a pronounced neutrophil infiltrate. In Foxn1(nu) mice depleted of neutrophils and in CD18-deficient mice (in which neutrophil extravasation is severely impaired) PEP005 treatment was associated with a >70% increase in tumor relapse rates. NK cell or monocyte/macrophage deficiency had no effect on relapse rates. Both in vitro and in mice, PEP005 induced MIP-2/IL-8, TNF-alpha, and IL-1beta, all mediators of neutrophil recruitment and activation. In vitro, PEP005 activated human endothelial cells resulting in neutrophil adhesion and also induced human neutrophils to generate tumoricidal-reactive oxygen intermediates. Treatment of tumors with PEP005 significantly elevated the level of anticancer Abs, which were able to promote neutrophil-mediated Ab-dependent cellular cytotoxicity (ADCC) in vitro. PEP005 treatment of tumors grown in SCID mice was also associated with >70% increase in tumor relapse rates. Taken together, these data suggest a central role for neutrophil-mediated ADCC in preventing relapse. PEP005-mediated cure of tumors therefore appears to involve initial chemoablation followed by a neutrophil-dependent ADCC-mediated eradication of residual disease, illustrating that neutrophils can be induced to mediate important anticancer activity with specific chemotherapeutic agents.

Synergistic cytotoxicity and apoptosis induced in human cholangiocarcinoma cell lines by a combined treatment with tumor necrosis factor-alpha (TNF-alpha) and triptolide.

Cholangiocarcinoma is known to be relatively resistant to chemotherapy. One alternative approach is to use a combination of an immunomodulating agent with an anticancer drug. Here we studied the synergistic actions of TNF-alpha and triptolide (a diterpene epoxide prepared from Tripterygium wilfordii), previously shown to have antitumor activity against hamster cholangiocarcinoma (CCA) cells. Three human CCA cell lines (HuCCA-1, HubCCA-1, KKU-100 cell lines) were subjected to a combined treatment of TNF-alpha (0.1-10 ng/ml) and triptolide (5-50 ng/ml) for 24 hours in microculture plates. The combination of TNF-alpha and triptolide had a significantly increased cytotoxic activity over that of triptolide alone (p < 0.05). Under the same conditions, TNF-alpha by itself was not cytotoxic to these cell lines. Similarly, the combined treatment could also accelerate apoptotic cell death in all three human cholangiocarcinoma cell lines. The combined treatment of TNF-alpha at 10 ng/ml and triptolide at 50 ng/ml for 6-10 hours achieved a percentage of apoptotic cells shown by DAPI staining of 18-65%, compared to only 6-20% apoptotic cells for triptolide alone. Analyzing the possible mechanisms of the combined treatment, we found by Western blot that at 6 hours, there was a poly (ADP-ribose) polymerase (PARP) cleavage which was not detectable by the treatment of either TNF-alpha or triptolide alone. The cleavage of PARP was inhibited when the cells were pretreated with the enzyme inhibitor AC-DEVD-CMK, suggesting that apoptosis induced by the combination of TNF-alpha and triptolide involved activation of caspase 3. These results indicate that apoptosis of human cholangiocarcinoma cell lines as induced by a combination of TNF-alpha and triptolide is mediated through caspase 3 activation.

Human microsomal epoxide hydrolase is the target of germander-induced autoantibodies on the surface of human hepatocytes.

Germander, a plant used in folk medicine, caused an epidemic of cytolytic hepatitis in France. In about half of these patients, a rechallenge caused early recurrence, suggesting an immunoallergic type of hepatitis. Teucrin A (TA) was found responsible for the hepatotoxicity via metabolic activation by CYP3A. In this study, we describe the presence of anti-microsomal epoxide hydrolase (EH) autoantibodies in the sera of patients who drank germander teas for a long period of time. By Western blotting and immunocytochemistry, human microsomal EH was shown to be present in purified plasma membranes of both human hepatocytes and transformed spheroplasts and to be exposed on the cell surface where affinity-purified germander autoantibodies recognized it as their autoantigen. Immunoprecipitation of EH activity by germander-induced autoantibodies confirmed this finding. These autoantibodies were not immunoinhibitory. The plasma membrane-located EH was catalytically competent and may act as target for reactive metabolites from TA. To test this hypothesis CYP3A4 and EH were expressed with human cytochrome P450 reductase and cytochrome b(5) in a "humanized" yeast strain. In the absence of EH only one metabolite was formed. In the presence of EH, two additional metabolites were formed, and a time-dependent inactivation of EH was detected, suggesting that a reactive oxide derived from TA could alkylate the enzyme and trigger an immune response. Antibodies were found to recognize TA-alkylated EH. Recognition of EH present at the surface of human hepatocytes could suggest an (auto)antibody participation in an immune cell destruction.

Structural optimization affording 2-(R)-(1-(R)-3, 5-bis(trifluoromethyl)phenylethoxy)-3-(S)-(4-fluoro)phenyl-4- (3-oxo-1,2,4-triazol-5-yl)methylmorpholine, a potent, orally active, long-acting morpholine acetal human NK-1 receptor antagonist.

Structural modifications requiring novel synthetic chemistry were made to the morpholine acetal human neurokinin-1 (hNK-1) receptor antagonist 4, and this resulted in the discovery of 2-(R)-(1-(R)-3, 5-bis(trifluoromethyl)phenylethoxy)-3-(S)-(4-fluoro)phenyl-4-(3-ox o-1 ,2,4-triazol-5-yl)methyl morpholine (17). This modified compound is a potent, long-acting hNK-1 receptor antagonist as evidenced by its ability to displace [125I]Substance P from hNK-1 receptors stably expressed in CHO cells (IC50 = 0.09 +/- 0.06 nM) and by the measurement of the rates of association (k1 = 2.8 +/- 1.1 x 10(8) M-1 min-1) and dissociation (k-1 = 0.0054 +/- 0.003 min-1) of 17 from hNK-1 expressed in Sf9 membranes which yields Kd = 19 +/- 12 pM and a t1/2 for receptor occupancy equal to 154 +/- 75 min. Inflammation in the guinea pig induced by a resiniferatoxin challenge (with NK-1 receptor activation mediating the subsequent increase in vascular permeability) is inhibited in a dose-dependent manner by the oral preadmininstration of 17 (IC50 (1 h) = 0.008 mg/kg; IC90 (24 h) = 1.8 mg/kg), indicating that this compound has good oral bioavailbility and peripheral duration of action. Central hNK-1 receptor stimulation is also inhibited by the systemic preadministration of 17 as shown by its ability to block an NK-1 agonist-induced foot tapping response in gerbils (IC50 (4 h) = 0.04 +/- 0.006 mg/kg; IC50 (24 h) = 0.33 +/- 0.017 mg/kg) and by its antiemetic actions in the ferret against cisplatin challenge. The activity of 17 at extended time points in these preclinical animal models sets it apart from earlier morpholine antagonists (such as 4), and the piperidine antagonists 2 and 3 and could prove to be an advantage in the treatment of chronic disorders related to the actions of Substance P. In part on the basis of these data, 17 has been identified as a potential clinical candidate for the treatment of peripheral pain, migraine, chemotherapy-induced emesis, and various psychiatric disorders.

Identification of contact allergens in unmodified rosin using a combination of patch testing and analytical chemistry techniques.

In order to investigate the contact allergens in the unmodified colophony (rosin) used in routine patch testing, preparative chromatographic techniques were used to separate its components which were then patch tested on colophony-sensitive individuals. The chemical structure of the dermatologically active components was elucidated using mass spectrometry, nuclear magnetic resonance and infra-red techniques. The study shows that oxidized resin acids are stronger sensitizers than the resin acids themselves, the most potent contact allergen being 7-oxydehydroabietic acid (purity 92%). At naturally occurring concentrations (relative to abietic acid) all unoxidized resin acids were found to play an insignificant part, individually, in colophony dermatitis, except abietic acid (purity 99%), which was found to be a weak but important allergen. The commercial Trolab abietic acid preparation (unknown purity) was found to be dermatologically more active than purified abietic acid.

Contact allergy to resin acid hydroperoxides. Hapten binding via free radicals and epoxides.

For a better understanding of the mechanisms of contact allergic reactions, the patterns of cross-reactivity between different resin acid oxidation products were studied. The 13,14(alpha)-epoxide and the 13,14(beta)-epoxide of abietic acid and 15-hydroperoxydehydroabietic acid (15-HPDA) were shown in experimental sensitization studies to be contact allergens. Cross-reactivity was observed between the alpha- and beta-epoxides and also between the epoxides and the previously identified rosin allergen 15-hydroperoxyabietic acid (15-HPA). This indicates that 15-HPA may form an epoxide which then reacts with skin protein to generate the complete antigen. 15-HPA and 15-HPDA cross-reacted as well. This can be explained by the formation of similar alkoxy radicals from both hydroperoxides which further react with skin protein. Cross-reactivity patterns of the resin acid oxidation products indicate that 15-HPA may react with skin proteins either as a radical or as an epoxide, thus generating different antigens. The presence in rosin of the epoxides of abietic acid was also studied. The beta-epoxide was detected in gum rosin. Moreover, the epoxides elicited reactions in rosin-allergic individuals. Thus, the 13,14(beta)-epoxide of abietic acid was identified as a new, important rosin allergen.