Six cobalamin-biotin conjugates have been prepared. The cobalamin-biotin conjugates were prepared to evaluate the effect that the location of attachment had on the binding with transcobalamin II (TCII), the cobalamin binding protein in plasma, and to evaluate their potential use for in vitro and in vivo applications. This study focused only on the effect of binding with TCII. To decrease the possibility of steric problems in binding of the cobalamin conjugates with TCII, and biotin's binding with streptavidin or avidin, moieties of 11-18 atoms in length were used as linkers. Four biotin conjugates were prepared which were attached to the corrin ring of the cobalamin molecule (on b-, c-, d-, and e-side chains). One conjugate was attached to the 5'-OH of the ribose moiety, and another conjugate was attached at the cobalt metal (in place of the cyanide moiety of cyanocobalamin). Competitive binding studies were conducted where various amounts of the cobalamin-biotin conjugates and their precursor cobalamin derivatives competed with [57Co]cyanocobalamin for binding of recombinant human TCII (rhTCII). Evaluation of cobalamin derivatives which were conjugated at the 5'-OH of ribose or the cobalt metal center indicated that conjugation at either of these positions had little effect on binding with rhTCII. However, conjugates where the attachment was made on the corrin ring substituents had a large variation in binding with rhTCII. Conjugates on the e-propionamide side chain had little effect (relative affinity was equal to or decreased less than a factor of 3) on binding with rhTCII, conjugates of the b-isomer had decreased binding (relative affinity decreased less than a factor of 10), conjugates of the d-propionamide had further decreased binding (relative affinity decreased between 44 and 69 times), and conjugates on the c-acetamide group had poor binding to rhTCII (relative affinity decreased between 295 and 1160 times). The significance of the side chains on the corrin ring in providing specificity and high-affinity binding with rhTCII is discussed.

pharmacology of robenacoxib: a novel selective inhibitor of cyclooxygenase-2. J. vet. Pharmacol. Therap. 32,[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17] This manuscript reports the results of preclinical studies in the rat with robenacoxib, a novel selective cyclooxygenase (COX)-2 inhibitor. Robenacoxib selectively inhibited COX-2 in vitro as evidenced from COX-1:COX-2 IC 50 ratios of 27:1 in purified enzyme preparations and >967:1 in isolated cell assays. Binding to COX-1 was rapid and readily reversible (dissociation t 1 ⁄ 2 << 1 min), whilst COX-2 binding was slowly reversible (t 1 ⁄ 2 = 25 min). In vivo, robenacoxib inhibited PGE 2 production (an index of COX-2 inhibition) in lipopolysaccharide (LPS)-stimulated air pouches (ID 50 0.3 mg ⁄ kg) and for at least 24 h in zymosan-induced inflammatory exudate (at 2 mg ⁄ kg). Robenacoxib was COX-1 sparing, as it inhibited serum TxB 2 synthesis ex vivo (an index of COX-1 inhibition) only at very high doses (100 mg ⁄ kg but not at 2-30 mg ⁄ kg). Robenacoxib inhibited carrageenan-induced paw oedema (ID 50 0.40-0.48 mg ⁄ kg), LPS-induced fever (ID 50 1.1 mg ⁄ kg) and Randall-Selitto pain (10 mg ⁄ kg). Robenacoxib was highly bound to plasma protein (99.9% at 50 ng ⁄ mL in vitro). After intravenous dosing, clearance was 2.4 mL ⁄ min ⁄ kg and volume of distribution at steady-state was 306 mL ⁄ kg. Robenacoxib was preferentially distributed into inflammatory exudate; the AUC for exudate was 2.9 times higher than for blood and the MRT in exudate (15.9 h) was three times longer than in blood (5.3 h). Robenacoxib produced significantly less gastric ulceration and intestinal permeability as compared with the reference nonsteroidal anti-inflammatory drug (NSAID), diclofenac, and did not inhibit PGE 2 or 6-keto PGF 1a concentrations in the stomach and ileum at 30 mg ⁄ kg. Robenacoxib also had no relevant effects on kidney function at 30 mg ⁄ kg. In summary, results of preclinical studies in rats studies suggest that robenacoxib has an attractive pharmacological profile for potential use in the intended target species, cats and dogs.(Paper

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1 This manuscript presents the preclinical profile of lumiracoxib, a novel cyclooxygenase-2 (COX-2) selective inhibitor. 2 Lumiracoxib inhibited purified COX-1 and COX-2 with K i values of 3 and 0.06 mM, respectively. In cellular assays, lumiracoxib had an IC 50 of 0.14 mM in COX-2-expressing dermal fibroblasts, but caused no inhibition of COX-1 at concentrations up to 30 mM (HEK 293 cells transfected with human COX-1). 3 In a human whole blood assay, IC 50 values for lumiracoxib were 0.13 mM for COX-2 and 67 mM for COX-1 (COX-1/COX-2 selectivity ratio 515). 4 Lumiracoxib was rapidly absorbed following oral administration in rats with peak plasma levels being reached between 0.5 and 1 h. 5 Ex vivo, lumiracoxib inhibited COX-1-derived thromboxane B 2 (TxB 2 ) generation with an ID 50 of 33 mg kg À1, whereas COX-2-derived production of prostaglandin E 2 (PGE 2 ) in the lipopolysaccharidestimulated rat air pouch was inhibited with an ID 50 value of 0.24 mg kg À1 . 6 Efficacy of lumiracoxib in rat models of hyperalgesia, oedema, pyresis and arthritis was dosedependent and similar to diclofenac. However, consistent with its low COX-1 inhibitory activity, lumiracoxib at a dose of 100 mg kg À1 orally caused no ulcers and was significantly less ulcerogenic than diclofenac (Po0.05). 7 Lumiracoxib is a highly selective COX-2 inhibitor with anti-inflammatory, analgesic and antipyretic activities comparable with diclofenac, the reference NSAID, but with much improved gastrointestinal safety. British Journal of Pharmacology (2005) 144, 538-550. doi:10.1038/sj.bjp.0706078 Published online 17 January 2005 Keywords: Lumiracoxib; COX-2; cyclooxygenase-2 selective inhibitor; preclinical Abbreviations: AUC, area-under-curve of the concentration vs time curve; C max , maximum drug plasma concentration; CFA, complete Freund's adjuvant; 51 Cr-EDTA, chromium-51 labelled EDTA; COX, cyclooxygenase; D 30 , dose at which 30% inhibition was achieved; DMSO, dimethyl sulphoxide; F 0 , fraction of uninhibited enzyme at equilibrium; GI, gastrointestinal; HEK, human embryonic kidney; IL-1, interleukin-1; K i , inhibitor constant; k on , second-order rate constant representing speed at which an inhibitor binds to an enzyme; I, inhibitor concentration; LC/MS/MS, liquid chromatography/mass spectrometry/mass spectrometry; LPS, lipopolysaccharide; NSAID, nonsteroidal anti-inflammatory drug; O 2 , oxygen; PGE 2 , prostaglandin E 2 ; s, arachidonic acid concentration; t 1/2 , half-life; t opt , time to optimal velocity; TxB 2 , thromboxane B 2 ; V 0 , velocity in the absence of inhibitor; V obs , observed velocity in the presence of inhibitor; V opt , highest observed O 2 consumption velocity; V max , Michaelis-Menten constant for the maximal calculated velocity

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Objective. All ␥-chain cytokines signal through JAK-3 and JAK-1 acting in tandem. We undertook this study to determine whether the JAK-3 selective inhibitor WYE-151650 would be sufficient to disrupt cytokine signaling and to ameliorate autoimmune disease pathology without inhibiting other pathways mediated by JAK-1, JAK-2, and Tyk-2.Methods. JAK-3 kinase selective compounds were characterized by kinase assay and JAK-3-dependent (interleukin-2 [IL-2]) and -independent (IL-6, granulocyte-macrophage colony-stimulating factor [GM-CSF]) cell-based assays measuring proliferation or STAT phosphorylation. In vivo, off-target signaling was measured by IL-22-and erythropoietin (EPO)-mediated models, while on-target signaling was measured by IL-2-mediated signaling. Efficacy of JAK-3 inhibitors was determined using delayed-type hypersensitivity (DTH) and collagen-induced arthritis (CIA) models in mice.Results. In vitro, WYE-151650 potently suppressed IL-2-induced STAT-5 phosphorylation and cell proliferation, while exhibiting 10-29-fold less activity against JAK-3-independent IL-6-or GM-CSF-induced STAT phosphorylation. Ex vivo, WYE-151650 suppressed IL-2-induced STAT phosphorylation, but not IL-6-induced STAT phosphorylation, as measured in whole blood. In vivo, WYE-151650 inhibited JAK-3-mediated IL-2-induced interferon-␥ production and decreased the natural killer cell population in mice, while not affecting IL-22-induced serum amyloid A production or EPO-induced reticulocytosis. WYE-151650 was efficacious in mouse DTH and CIA models.Conclusion. In vitro, ex vivo, and in vivo assays demonstrate that WYE-151650 is efficacious in mouse CIA despite JAK-3 selectivity. These data question the need to broadly inhibit JAK-1-, JAK-2-, or Tyk-2-dependent cytokine pathways for efficacy.

This study was designed to identify the cellular component of the intestinal villus where transcobalamin II (TCII) is synthesized, because this protein provides an essential function in the intestinal absorption of vitamin B12 (cobalamin, Cbl). When a segment of proximal or distal small intestine of the guinea pig is cultured in medium containing [57Co]Cbl, TCII-[57Co]Cbl appears within 15 min. Northern blot analysis of RNA from both proximal and distal small intestine identified the TCII transcript. In situ hybridization of the distal ileum with35S-labeled TCII antisense transcript localized grains predominantly in crypts and in the lower third and central core of the villi. Grains were also evident at the base of the enterocytes in close apposition with the vascular network, whereas few grains appeared in the apical region of the columnar cells. This study provides evidence that TCII is constitutively expressed in the intestinal villi where vascular endothelium is abundant. In the distal ileum, where the intrinsic factor (IF) receptor is expressed, after uptake of IF-Cbl and the subsequent binding of free Cbl to TCII synthesized in the villi, the TCII-Cbl complex enters the microcirculation and passes into the portal blood.

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