Pig-to-human xenotransplantation of islet cells or of vascularized organs would offer a welcome treatment alternative for the ever-increasing number of patients with end-stage organ failure who are waiting for a suitable allograph. The main hurdle are preexisting antibodies, most of which are specific for 'Linear-B', carbohydrate epitopes terminated by the unbranched Gal-alpha(1,3)Gal disaccharide. These antibodies are responsible for the 'hyper-acute rejection' of the xenograft by complement mediated hemorrhage. For depletion of such antibodies we have developed an artificial injectable antigen, a glycopolymer (GAS914) with a charge neutral poly-lysine backbone (degree of polymerization n = 1000) and 25% of its side chains coupled to Linear-B-trisaccharide. With an average molecular weight of 400 to 500 kD, presenting 250 trisaccharide epitopes per molecule, this multivalent array binds anti-alpha Gal antibodies with at least three orders of magnitude higher avidity on a per-saccharide basis than the monomeric epitope. In vivo experiments with non-human primates documented that rather low doses - 1 to 5 mg/kg of GAS914 injected i.v. - efficiently reduce the load of anti-Linear-B antibodies (quickly by at least 80%. This treatment can be repeated without any sensitization to GAS914. Interestingly, although the antibody levels start raising 12 h after injection, they do not reach pretreatment levels. The polymer is degraded and excreted within hours, with a minute fraction remaining in lymphoid tissue of anti-alpha Gal producing animals only, probably binding to and inhibiting antibody-producing B-cells. The results of pig-to-non-human primate xenotransplantations established GAS914 as a relevant therapeutic option for pig-to-human transplantations as well. The synthesis of GAS914 was successfully scaled up to kg amounts needed for first clinical Studies. Key was the use of galactosyl transferases and UDP-galactose for the synthesis of the trisaccharide.

As Alzheimer's disease pathogenesis is associated with the formation of insoluble aggregates of amyloid beta-peptide, approaches allowing the direct, noninvasive visualization of plaque growth in vivo would be beneficial for biomedical research. Here we describe the synthesis and characterization of the near-infrared fluorescence oxazine dye AOI987, which readily penetrates the intact blood-brain barrier and binds to amyloid plaques. Using near-infrared fluorescence imaging, we demonstrated specific interaction of AOI987 with amyloid plaques in APP23 transgenic mice in vivo, as confirmed by postmortem analysis of brain slices. Quantitative analysis revealed increasing fluorescence signal intensity with increasing plaque load of the animals, and significant binding of AOI987 was observed for APP23 transgenic mice aged 9 months and older. Thus, AOI987 is an attractive probe to noninvasively monitor disease progression in animal models of Alzheimer disease and to evaluate effects of potential Alzheimer disease drugs on the plaque load.

Keywords 2,5-Dimethyl-1,3,4-thiadiazole (1a) reacts which aromatic carboxylic acid esters 8a - u in the presence of excessive sodium hydride under condensation to give sodium enolates which afford on hydrolysis the phenacyl-1,3,4-thiadiazoles 9a - u. The action of aromatic carboxylic acid chlorides on 1a in the presence of triethylamine gives rise to the formation of mixtures of diacylated thiadiazole derivatives 16 and 18. In some cases the pure 3-acyl-phenacylidene-2,3-dihydro-1,3,4-thiadiazoles 16 can be isolated. Generally the compounds 16 are rearranged on heating in higher boiling solvents to give the enolbenzoates 18. Hydrolysis of the diacylated thiadiazoles 16 and 18 yields the phenacylthiadiazoles 9a, c, d, g - j, v, w.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Noninvasive near-infrared fluorescence reflectance imaging (FRI) is an in vivo technique to assess physiological and molecular processes in the intact organism. Here we describe a method to assess gastric emptying in mice. TentaGel™ beads with covalently bound cyanine dye (Cy5.5) conjugates as fluorescent probe were administered by oral gavage. The amount of intragastric beads/label was derived from the fluorescence signal intensity measured in a region of interest corresponding to the mouse stomach. The FRI signal intensity decreased as a function of time reflecting gastric emptying. In control mice, the gastric half-emptying time was in agreement with literature data. Pharmacological modulation of gastric motility allowed the evaluation of the sensitivity of the FRI-based method. Gastric emptying was either stimulated or inhibited by treatment with the 5-HT4 receptor agonists tegaserod (Zelnorm®) and cisapride or the α2-receptor agonist clonidine, respectively. Tegaserod and cisapride dose-dependently accelerated gastric emptying. In contrast, clonidine dose-dependently delayed gastric emptying. In conclusion, FRI using fluorescently labeled beads allows the reliable determination of gastric emptying as well as the assessment of pharmacological interventions. The technique thus offers the potential to characterize molecular targets and pathways involved in physiological regulation and pharmacological modulation of gastric emptying.

2,5-Dimethyl-1,3,4-thiadiazole (1a) reacts which aromatic carboxylic acid esters 8a-u in the presence of excessive sodium hydride under condensation to give sodium enolates which afford on hydrolysis the phenacyl-1,3,4-thiadiazoles 9a-u. The action of aromatic carboxylic acid chlorides on la in the presence of triethylamine gives rise to the formation of mixtures of diacylated thiadiazole derivatives 16 and 18. In some cases the pure 3-acyl-phenacylidene-2,3-dihydro-1,3,4-thiadiazoles 16 can be isolated. Generally the compounds 16 are rearranged on heating in higher boiling solvents to give the enolbenzoates 18. Hydrolysis of the diacylated thiadiazoles 16 and 18 yields the phenacyl-thiadiazoles 9a, c, d, g-j, v, w.

Preformed and elicited Ab's against the Galalpha1,3Gal terminating carbohydrate chains (alphaGal Ab's) are the primary cause of hyperacute and acute vascular xenograft rejection in pig-to-primate transplantation. alphaGal Ab's are produced by long-lived Ab-producing cells that are not susceptible to pharmacological immunosuppression. We reasoned that antigen-specific elimination of alphaGal Ab's might be achieved in vivo by systemic administration of nonimmunogenic polyvalent alphaGal structures with high avidity for alphaGal Ab's. We devised GAS914, a soluble trisaccharide-polylysine conjugate of approximately 500 kDa that effectively competes for alphaGal binding by alphaGal IgM (IC(50), 43 nM) and IgG (IC(50), 28 nM) in vitro. Injections of GAS914 in cynomolgus monkeys, at the dose of 1 mg/kg, resulted in the immediate decrease of more than 90% of circulating alphaGal Ab's and serum anti-pig cytotoxicity. In baboons, repeated injections of GAS914 effectively reduced both circulating alphaGal Ab's and cytotoxicity over several months. Studies with [(14)C]GAS914 in rhesus monkeys and Gal(-/-) mice indicate that GAS914 binds to circulating alphaGal Ab's and that the complex is quickly metabolized by the liver and excreted by the kidney. Remarkably, posttreatment alphaGal Ab titers never exceeded pretreatment levels and no sensitization to either alphaGal or the polylysine backbone has been observed. Furthermore there was no apparent acute or chronic toxicity associated with GAS914 treatment in primates. We conclude that GAS914 may be used therapeutically for the specific removal of alphaGal Ab's.

Wie funktionieren retentive Glycosyltransferasen? Bei der Beantwortung dieser Frage muss berücksichtigt werden, dass von den Disubstratanaloga 1 überraschenderweise 1β und nicht 1α die α(1-3)-Galactosyltransferase sehr gut hemmt. Das Verständnis dieser Hemmung ist ein wichtiger Schritt für die pharmakologische Vermeidung hyperakuter Abstoßungsreaktionen bei der Xenotransplantation von Schweineorganen auf Primaten. In den Verbindungen 1 sind die Enzymsubstrate, UDP-Gal und Galactose, kovalent verknüpft.

The synthesis of Galα1→3Gal-quinic acid pseudo-trisaccharides and their elaboration into trimeric clusters is described.

How do retaining glycosyltransferases function? To answer this question, UDP-Gal and galactose were covalently linked to form disubstrate analogues 1, of which surprisingly 1β and not 1α inhibited α(1-3)-galactosyltransferases very well. An understanding of this inhibition is a key to the pharmacological prevention of hyperacute rejection in pig to primate xenotransplantation.

The major glycosphingolipid in pig vascular endothelium is the ceramide pentasaccharide Galα(1→3)Galβ(1→4)GlcNAcβ(1→3)Galβ(1→4)Glcβ(1→0)Cer (1), which binds specifically to human anti-Gal antibody and is involved in the hyperacute rejection response in xenotransplantation from pig to man. The synthesis of 1 and its methyl glycoside 2 is described.

An early step of the inflammatory response-the rolling of leukocytes on activated endothelial cells-is mediated by selectin/carbohydrate interactions. The tetrasaccharide sialyl Lewis(x) (sLe(x)) 1 is a ligand for E-, P-, and L-selectin and, therefore, serves as a lead structure to develop analogues which allow the control of acute and chronic inflammation. Here we describe the efficient synthesis (10 linear steps) of the potent sLe(x) mimetic 2. Compared to sLe(x), compound 2 showed a 30-fold improved affinity in a static, cell-free E-selectin-ligand binding assay (IC(50) = 36 microM). These data were confirmed by a marked inhibition in an in vitro cell-cell rolling assay which simulates in vivo conditions (IC(50) approximately 40 microM). The assays are predictive for the in vivo efficacy of test compounds as indicated by a marked inhibitory effect of 2 in a thioglycollate induced peritonitis model of acute inflammation in mice (ED(50) approximately 15 mg/kg).

Abstracttert‐Butyldimethylsilyl 2‐azido‐4,6‐O‐benzylideneglucopyranoside 5 proved to be a versatile starting material for the synthesis of the Lex antigen family. 3‐O‐Fucosylation of 5, ensuing reductive benzylidene ring cleavage, and then 4‐O‐galactosylation afforded Lex trisaccharide building block 12 which was readily converted into trisaccharide donor 14α,β and into trisaccharide 3c‐O‐acceptor 16. Their reaction in acetonitrile as solvent at low temperatures (nitrile effect) afforded exclusively the β‐connected hexasaccharide 17, which was transformed via the same steps into hexasaccharide donor 20α,β and acceptor 22. The use of these building blocks and known lactose derivative 23b, and 3‐O‐benzoylprotected azidosphingosine 32 as acceptors led to ready formation of the target molecules. Thus, from 20α, β and 23b octasaccharide 24b was obtained which was converted via 25b and 26b into O‐unprotected octasaccharide 27; the derived O‐acyl‐protected donor 29bα was linked to 32, thus providing by application of the “azidosphingosine glycosylation procedure” dimeric Lex 1B. From 20α, β and 16 nonasaccharide 35a was obtained; its transformation via 36a and 37a into donor 38aα,β gave by reaction with 23b undecasaccharide 39a; the derived donor 43aα furnished by treatment with 32 trimeric Lex 1C. Similarly, from 20α, β and 22 dodecasaccharide 35b was generated which gave via 36b, 37b, and donor 38bα, β by reaction with 23b as acceptor tetradecasaccharide 39b; 39b, after transformation into the corresponding donor 43bα, β gave by treatment with 32 as acceptor tetrameric Lex 1D. Additionally, octasaccharide donor 29bα, β was attached to spacer 30, thus yielding spacer‐connected dimeric Lex antigen 2. magnified image