BODIPY(®) FL histamine as a new modality for quantitative detection of histamine receptor upregulation upon IgE sensitization in murine bone marrow-derived mast cells.

Flow cytometry is one of the most widely used methods for the qualitative and quantitative analysis of cell surface expressed proteins by making use of fluorescent specific antibodies. Lacking an antibody validated for flow cytometry, an alternative approach for labeling cell surface receptors is the use of fluorescently tagged ligands. In this study, histamine H4 receptor transfected Chinese hamster ovary cells and murine bone marrow-derived mast cells (mBMMCs) were selected for studying the possibility of staining individual histamine receptors using BODIPY(®) FL histamine and selective antagonists. Flow cytometric measurements and supporting calculations showed that BODIPY FL histamine is suitable tool for quantitating cell surface histamine receptors. The binding, and competitive inhibition of this fluorescent ligand were characterized, which were in good agreement with a semi-empirical model constructed from fundamental protein-binding relationships. Using this method it was shown for the first time that even though mature mBMMCs express H2R and H4R to the same extent, immunoglobulin E sensitization results in H4R upregulation only, while the surface expression of H2R remains unchanged.

Histamine receptor H4 regulates mast cell degranulation and IgE induced FcεRI upregulation in murine bone marrow-derived mast cells.

There is increasing evidence that histamine regulates the immune system via histamine H4 receptors, therefore we sought to investigate the functions of the H4 receptor on mast cells. Mast cells were differentiated from murine bone marrow stem cells, and the expression of mast cell surface markers FcεRI and CD117 were measured using flow cytometry. Real-time qRT-PCR was used to determine the expression of mH4R; as a measure of antigen-dependent degranulation, ß-hexosaminidase release assay was carried out using IgE sensitized mast cells. We determined that the expression kinetics of FcεRI and mH4R can be described with a function that has one maximum value in the time range of the culture's differentiation. Antigen-dependent degranulation of murine bone marrow-derived mast cells could be inhibited by a selective H4 antagonist/inverse agonist only when it was present during the IgE sensitization phase of degranulation. In addition, flow cytometric analysis revealed that the H4 antagonist/inverse agonist also inhibited IgE induced FcεRI upregulation. The inhibition percentage of H4 antagonist on IgE induced FcεRI upregulation was determined to be dependent upon the maturity of the mast cell cultures, and this time-dependency was consistent with the expression kinetics of both mH4R and FcεRI. These results imply that H4R has regulatory roles in FcεRI expression and FcεRI mediated functions in mast cells. In conclusion the present study shows that H4 receptors potentially play a role in IgE induced FcεRI upregulation and in the sensitization phase but not the effector phase of mast cell degranulation.

Insulin in flavin-containing monooxygenase regulation. Flavin-containing monooxygenase and cytochrome P450 activities in experimental diabetes.

Microsomal monooxygenases - cytochrome P450 (CYP, EC 1.14.14.1) and flavin-containing monooxygenase (FMO, EC 1.14.13.8) - have profound roles in drug metabolism. While the induction of some metabolic enzymes such as hepatic FMO and intestinal CYP1A, CYP2B is recognized in experimental diabetes, the effect of insulin treatment on FMO and intestinal CYP3A in diabetic animals has not been reported before. Changes in abundance and activity of hepatic and intestinal microsomal CYPs and FMO were studied in streptozotocin-induced diabetic rats either treated or not with insulin. Hepatic FMO1 activity increased in diabetic rats, but it was restored to control value on insulin treatment. Insulin itself had no effect on FMO1 activity in non-diabetic animals. A remarkable increase of total CYP content was accompanied by a reduced CYP3A specific enzyme activity in the small intestine of diabetic animals. The extent of these changes decreased on insulin treatment. Both, hepatic FMO1 and intestinal CYP3A activity correlated with average blood glucose concentration in untreated diabetic rats. These results indicate that insulin is involved in the regulation of hepatic FMO1 and intestinal CYP3A in rats. Blood glucose level is a good marker for FMO induction. The marked reduction of intestinal CYP3A capacity suggests that diabetes exerts a substantial effect on the activity of most determining intestinal CYP enzyme.

Identification of nitric oxide donors by biomimetic HTS application.

Metalloporphyrin catalyzed biomimetic oxidation was used for the identification of nitric oxide (NO) donors with diverse chemical structure. Methodology was validated by testing known NO donors. Efficient automation of the test allowed us to investigate a subset of our corporate library. Several hits identified in this campaign were validated in both the chemical and also microsomal model that revealed all hits to be active in the biological system, as well. One of the hits showed comparable activity to V-PYRRO/NO, the prototypic liver selective NO donor.

Assessment of the N-oxidation of deprenyl, methamphetamine, and amphetamine enantiomers by chiral capillary electrophoresis: an in vitro metabolism study.

A chiral capillary electrophoresis method using hydroxypropyl-beta-cyclodextrin as chiral selector was developed and validated for the quantification of the N-oxygenated metabolites of deprenyl, methamphetamine, and amphetamine enantiomers, formed in vitro. The influence of various parameters (selector concentration, buffer pH, temperature, polymer additive, etc.) on the simultaneous separation of the optical isomers of the parent drugs and their metabolites has been evaluated. The buffer pH had the greatest impact on the separation selectivity of the N-oxygenated compounds. Linear calibration curves were obtained over the concentration range of 2.5-50 microM for the enantiomers of amphetamine-hydroxylamine, methamphetamine-hydroxylamine, and deprenyl-N-oxide. The inter- and intra-assay precision and accuracy varied by less than 15% for all analytes at concentrations of 5, 10, and 30 microM, and less than 20% at the lower limit of quantitation (2.5 microM). The sample extraction recovery ranged between 109 and 129% at the three concentration levels. The drug enantiomers were incubated with recombinant human flavin-containing monooxygenase enzymes (FMO3 and FMO1), and human liver microsomes, respectively. The enantioselectivity of the substrate preference, as well as the stereoselective formation of the new chiral center upon the oxidation of the prochiral tertiary nitrogen of deprenyl were assessed. FMO1, the extrahepatic form of the enzyme in man, was shown to be more active in the N-oxygenation of both deprenyl and methamphetamine isomers than FMO3. Deprenyl enantiomers and S-methamphetamine were substrates of human recombinant FMO3. Conversion of amphetamine to its hydroxylamine derivative could not be observed on incubation with either FMO1 or FMO3. Formation of the new chiral center on the nitrogen, during N-oxidation of the tertiary amine deprenyl, was found stereoselective. The two FMO isoforms have shown opposite preference in the formation of this chiral center. Methamphetamine-hydroxylamine formed from methamphetamine was further transformed by FMO, amphetamine-hydroxylamine was identified as the product of a demethylation reaction.

Identification of metabolic pathways involved in the biotransformation of tolperisone by human microsomal enzymes.

The in vitro metabolism of tolperisone, 1-(4-methyl-phenyl)-2-methyl-3-(1-piperidino)-1-propanone-hydrochloride, a centrally acting muscle relaxant, was examined in human liver microsomes (HLM) and recombinant enzymes. Liquid chromatography-mass spectrometry measurements revealed methyl-hydroxylation (metabolite at m/z 261; M1) as the main metabolic route in HLM, however, metabolites of two mass units greater than the parent compound and the hydroxy-metabolite were also detected (m/z 247 and m/z 263, respectively). The latter was identified as carbonyl-reduced M1, the former was assumed to be the carbonyl-reduced parent compound. Isoform-specific cytochrome P450 (P450) inhibitors, inhibitory antibodies, and experiments with recombinant P450s pointed to CYP2D6 as the prominent enzyme in tolperisone metabolism. CYP2C19, CYP2B6, and CYP1A2 are also involved to a smaller extent. Hydroxymethyl-tolperisone formation was mediated by CYP2D6, CYP2C19, CYP1A2, but not by CYP2B6. Tolperisone competitively inhibited dextromethorphan O-demethylation and bufuralol hydroxylation (K(i) = 17 and 30 microM, respectively). Tolperisone inhibited methyl p-tolyl sulfide oxidation (K(i) = 1200 microM) in recombinant flavin-containing monooxygenase 3 (FMO3) and resulted in a 3-fold (p < 0.01) higher turnover number using rFMO3 than that of control microsomes. Experiments using nonspecific P450 inhibitors-SKF-525A, 1-aminobenzotriazole, 1-benzylimidazole, and anti-NADPH-P450-reductase antibodies-resulted in 61, 47, 49, and 43% inhibition of intrinsic clearance in HLM, respectively, whereas hydroxymethyl-metabolite formation was inhibited completely by nonspecific chemical inhibitors and by 80% with antibodies. Therefore, it was concluded that tolperisone undergoes P450-dependent and P450-independent microsomal biotransformations to the same extent. On the basis of metabolites formed and indirect evidences of inhibition studies, a considerable involvement of a microsomal reductase is assumed.

Peptide based vaccine design: synthesis and immunological characterization of branched polypeptide conjugates comprising the 276-284 immunodominant epitope of HSV-1 glycoprotein D.

The importance of the length and conjugation site of a protective epitope peptide (276SALLEDPVG284) from glycoprotein D of herpes simplex virus in branched polypeptide conjugates has been investigated. A new set of peptides, with a single attachment site and truncated sequences, was prepared. The immunogenicity of conjugates and the specificity of antibody responses elicited were investigated in BALB/c, C57/B1/6 and CBA mice. It was found that the covalent coupling of the peptide comprising the 276-284 sequence of gD through its Asp residue at position 281 did not influence the immunogenic properties of the epitope, while involvement of the side chain of Glu at position 280 almost completely abolished immunogenicity. These results clearly indicated that the conjugation site of the epitope peptide influenced the intensity and specificity of antibody responses. Comparison of the immunological properties of conjugates containing truncated gD peptides revealed the presence of two epitopes within the 276-284 region. One of the proposed epitopes is situated at the N-terminal (276-281) region, while the other is located at the C-terminal end of the sequence (279-284). Binding data demonstrated that some of the peptides comprising these epitopes induced gD-specific responses in their conjugated form and also elicited an immune response that conferred protection against lethal HSV-1 infection. The correlation of peptide- and gD-specific antibody responses with the protective effect of the immune response is discussed.