This was linked dose-dependently to MetAP-2 inhibition [15]. B cells into plasma cells. and B cell assay by blocking interleukin-21-mediated (IL-21-mediated) differentiation of B cells to plasma cells. Furthermore, in a marmoset immunization model, the treatment of animals with PPI-2458 resulted in inhibition of T cell-dependent antibody production and depletion of B cells in the germinal centre. Materials and methods Isolation of human B cells Human tonsil tissue was obtained with informed patient consent and ethical approval from Peterborough Hospital, Peterborough, UK. The B cells were isolated from the tonsil tissue by a standard Ficoll-Hypaque gradient method followed by unfavorable depletion of the mononuclear cell populace using anti-CD3 and anti-CD14 magnetic beads (Dynabeads; Dynal, Oslo, Norway). Flow cytometry was carried out after pre-blocking Fc receptors with extra human immunoglobulin (Ig)G (Cambridge Bioscience, Cambridge, UK). CD19-allophycocyanin (APC), Colec10 CD3-fluorescein isothiocyanate and CD14-APC antibodies were all from Becton Dickinson ML133 hydrochloride (Oxford, UK) and were titrated for optimal staining prior to use. Samples were read using a Becton Dickinson fluorescence activated cell sorter (FACS) Canto using FACS Diva software. Preparations were typically 97% CD19+ by flow cytometry [CD3+ contamination 2%, negligible ( 1%) monocyte (CD14+) contamination]. The MetAP-2 enzyme assay optimization and Ki generation The MetAP-2 assay was carried out using 50 M MGWMDF, a suitable MetAP-2 peptide substrate, and 15 nM recombinant human MetAP-2 (baculovirus expression in-house) in 10 l reactions (low-volume 384-well plate). Assay buffer consisted of 50 mM Hepes, 100 M MnCl2, 100 mM NaCl, 0005% (w/v) bovine serum albumin (BSA), 0006% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]propanesulphonic acid, pH 75. Inhibitor studies were carried out using dilutions of PPI-2458 in the presence of dimethylsulphoxide (DMSO) at a final concentration of 1% (v/v). The release of N-terminal methionine from the peptide substrate was assessed using a coupled enzyme assay comprised of l-amino acid oxidase and horseradish peroxidase. The oxidation of Amplex Red was followed using a SpectraMAX Gemini plate reader (Molecular Devices, Workingham, UK) and the data analysed using grafit version 5.0.12 software (East Grinstead, UK). Generation of PK data for PPI-2458 Marmosets (= 4) were given a single oral dose of PPI-2458 in a methycellulose vehicle and the blood samples were taken into sodium heparin anti-coagulant over a 4-h period to evaluate plasma concentrations of the compound by liquid chromatography/mass spectrometryCmass spectrometry. Primary immunization model All the immunization experiments ML133 hydrochloride were carried out in marmosets (= 3), PPI-2458 at 5 mg kg?1 (= 2) with the compound dosed from day ?1, twice daily, until day 16, when the study was terminated and tissues harvested for histological examination. Peripheral blood was taken on days ?1, 3, 10, 13 and 19. Anti-TNP-specific antibodies were detected ML133 hydrochloride in serum by enzyme-linked immunosorbent assay (ELISA). Secondary immunization model The marmosets were sensitized subcutaneously on day 0 and boosted on day 22 with 05 ml of KLH-TNP (100 g) (Biosearch Technologies, Novato, CA, USA) made up of 1 mg of alum. The treatment groups consisted of vehicle (05% methylcellulose; Sigma, Poole, UK) (= 3) and PPI-2458 at 1 mg kg?1 (= 3) once daily. Dosing was started 1 day prior to sensitization and continued until the study was terminated on day 41 and tissues harvested for histological examination. Peripheral blood was taken at days ?1, 7, 14, 20, 28 and 35 and serum harvested and stored at ?80C until the assay was carried out. PPI-2458 (Fig. 1a) was synthesized at GlaxoSmithKline. The methodology for synthesis of PPI-2458 is usually covered by the Praeceis Pharmaceuticals (Waltham, MA, USA) patent WO 02/422952002, priority US704251 2000. Open in a separate windows Fig. 1 Phenotypic B cell differentiation and immunoglobulin (Ig)G secretion in response to exogenous interleukin-21 (IL-21). Tonsil B cells, isolated by magnetic depletion of contaminating cells, were co-stimulated with F(ab)2 fragments to human IgM, and recombinant human (rh)CD40L, in the presence or absence of IL-21, as described in the Materials and methods section. Figure 1a shows the structure of [(1R)-1-carbamoyl-2-methyl-propyl)]-carbamic acid-(3R,4S, 5S, 6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)oxiranyl]-1-oxaspiro(25)oct-6-yl ester] (PPI-2458). The results of flow cytometric analysis in (b) and (c) show the expression levels of CD38, a B blast cell marker,.