Conjugation of adenine arabinoside 5'-monophosphate to arabinogalactan: synthesis, characterization, and antiviral activity.

A conjugate consisting of the antiviral nucleotide analogue adenine arabinoside 5'-monophosphate (araAMP, vidarabine monophosphate) and the naturally occurring polysaccharide arabinogalactan was synthesized. The conjugate consisted of 7.9 araAMP residues per molecule of arabinogalactan. The proposed structure of the conjugate was consistent with 13C NMR spectroscopic studies. Daily injections of the conjugate, at a dose of 3 mg of araAMP/kg, into woodchuck carriers of woodchuck hepatitis virus (WHV) decreased serum levels of WHV DNA. A dose of 3 mg/kg of unconjugated araAMP was ineffective, while a higher dose of araAMP (15 mg/kg, 14 days) produced a drop in WHV DNA. After cessation of dosing with the conjugate, serum viral DNA levels remained depressed for 42 days. In contrast, after cessation of dosing with araAMP, WHV DNA rapidly returned to original levels.

Arabinogalactan for hepatic drug delivery.

Arabinogalactan, a polysaccharide from the tree Larix occidentalis, has been purified and its biological and physical properties described. Intravenous injection of radiolabeled arabinogalactan (4 mg/kg) in rats resulted in 52.5% of the dose being present in the liver, while prior injection of asialofetuin (100 mg/kg) reduced hepatic radioactivity to 3.54%. Gel chromatography indicates arabinogalactan is a single species of 19 kDa, while light scattering gave a molecular weight of 40 kDa. Glycosyl linkage analysis of arabinogalactan is consistent with a highly branched structure comprising a backbone of 1,3-linked galactopyranose connected by 1,3-glycosidic linkages, comprised of 3,4,6-,3,6-, and 3,4- as well as 3-linked residues. In the carbon-13 NMR spectra, the major resonances of arabinogalactan are assigned to beta-galactopyranose, beta-arabinofuranose, and beta-arabinopyranose. Arabinogalactan produced no adverse reactions in single intravenous dose (mouse, 5000 mg/kg) and repeat dose toxicity studies (rats, 500 mg/kg/day, 90 days). When tritiated arabinogalactan was injected, radioactivity cleared from the liver with a half-life of 3.42 days. Arabinogalactan has properties that make it suitable as a carrier for delivering diagnostic or therapeutic agents to hepatocytes via the asialoglycoprotein receptor.

Regulation of neutrophil superoxide by antichymotrypsin-chymotrypsin complexes.

The ability of neutrophils to generate free radicals is a crucial component of host defense (Babior, B. M. (1978) N. Engl. J. Med. 298, 659-668, 721-725. Neutrophil oxidants, however, can cause significant host tissue destruction (Weiss, S. J. (1989) N. Engl. J. Med. 320, 365-376), and the regulation of free radical production is not well understood. We have previously shown that recombinant antichymotrypsin (rACT), a serine protease inhibitor, inhibits superoxide production in intact neutrophils (Kilpatrick, L., Johnson, J. L., Nickbarg, E. B., Wang, Z., Clifford, T. F., Banach, M., Cooperman, B. S., Douglas, S. D., and Rubin, H. (1991) J. Immunol. 146, 2388-2393). Using a cell-free NADPH oxidase preparation, we now demonstrate that rACT alone has no effect on superoxide production and that antichymotrypsin-chymotrypsin (rACT.CT) complexes are required to inhibit superoxide, suggesting that neutrophil chymotrypsin-like proteases produce conformational changes in ACT, allowing it to become active in regulating superoxide production. Additionally, we have identified NADPH oxidase itself as the target for rACT.CT and have demonstrated that rACT.CT interferes specifically with activation of the NADPH oxidase without changing the Km for NADPH or the rate constant describing the rate-limiting step in activation. These observations suggest an important role for antichymotrypsin in the regulation of NADPH-oxidase activation, which is a prerequisite for neutrophil superoxide production, and predict possible therapeutic uses for rACT in conditions where unregulated neutrophil-free radical production has been implicated in the mechanism of tissue destruction.

Characterization of the urinary metabolites of 5-amino-1-naphthol in the rat.

The metabolic fate of [14C]5-amino-1-naphthol (5A1N) was investigated in Sprague-Dawley rats. [14C]5A1N was administered by gastric intubation to male rats at doses 1, 37 and 135 mg/kg body weight. In a separate experiment the rats were also dosed with 150 mg/kg of unlabeled 5A1N daily for 4 consecutive days. Between 74% and 85% of the administered dose was excreted in the urine. Over 98% of the urinary radioactivity was characterized as unchanged 5A1N, 5-acetamido-1-naphthol (5AA1N) and glucuronic and sulfuric acid conjugates of both 5A1N and 5AA1N. Unchanged 5A1N and 5AA1N accounted for less than 3% of the dose. The amount of 5A1N converted to 5AA1N and its conjugates varied inversely with the dose. Two minor metabolites were not identified. Rats dosed repeatedly with 150 mg/kg of 5A1N showed no significant change in metabolite excretion patterns compared to rats dosed singly. These findings indicate that in the rodent model the metabolism of 5A1N was dose dependent, and occurred predominantly by phase II reactions involving N-acetylation and conjugation with glucuronic and sulfuric acids. N-Acetylation predominated at lower doses and O-sulfate conjugation at higher doses. There was no evidence for the formation of N-hydroxylated metabolites over the dose range studied.