Eliciting the low-activity aldehyde dehydrogenase Asian phenotype by an antisense mechanism results in an aversion to ethanol.

A mutation in the gene encoding for the liver mitochondrial aldehyde dehydrogenase (ALDH2-2), present in some Asian populations, lowers or abolishes the activity of this enzyme and results in elevations in blood acetaldehyde upon ethanol consumption, a phenotype that greatly protects against alcohol abuse and alcoholism. We have determined whether the administration of antisense phosphorothioate oligonucleotides (ASOs) can mimic the low-activity ALDH2-2 Asian phenotype. Rat hepatoma cells incubated for 24 h with an antisense oligonucleotide (ASO-9) showed reductions in ALDH2 mRNA levels of 85% and ALDH2 (half-life of 22 h) activity of 55% equivalent to a >90% inhibition in ALDH2 synthesis. Glutamate dehydrogenase mRNA and activity remained unchanged. Base mismatches in the oligonucleotide rendered ASO-9 virtually inactive, confirming an antisense effect. Administration of ASO-9 (20 mg/kg/day for 4 d) to rats resulted in a 50% reduction in liver ALDH2 mRNA, a 40% inhibition in ALDH2 activity, and a fourfold (P < 0.001) increase in circulating plasma acetaldehyde levels after ethanol (1 g/kg) administration. Administration of ASO-9 to rats by osmotic pumps led to an aversion (-61%, P < 0.02) to ethanol. These studies provide a proof of principle that specific inhibition of gene expression can be used to mimic the protective effects afforded by the ALDH2-2 phenotype.

Bicyclic N-hydroxyurea inhibitors of 5-lipoxygenase: pharmacodynamic, pharmacokinetic, and in vitro metabolic studies characterizing N-hydroxy-N-(2,3-dihydro-6-(phenylmethoxy)-3-benzofuranyl)urea.

A series of N-hydroxyurea derivatives have been prepared and examined as inhibitors of 5-lipoxygenase. Oral activity was established by examining the inhibition of LTB4 biosynthesis in an ex vivo assay in the mouse. The pharmacodynamic performance in the mouse of selected compounds was assessed using an ex vivo LTB4 assay and an adoptive peritoneal anaphylaxis assay at extended pretreat times. Compounds with an extended duration of action were re-examined as the individual enantiomers in the ex vivo assay, and the (S) enantiomer of N-hydroxy-N-[2,3-dihydro-6-(phenylmethoxy)-3-benzofuranyl]urea, (+)-1a (SB 202235), was selected as the compound with the best overall profile. Higher plasma concentrations and longer plasma half-lives were found for (+)-1a relative to its enantiomer in the mouse, monkey, and dog. In vitro metabolic studies in mouse liver microsomes established enantiospecific glucuronidation as a likely mechanism for the observed differences between the enantiomers of 1a. Enantioselective glucuronidation favoring (-)-1a was also found in human liver microsomes.

Effects of heating on the emanation rates of radon-222 from a suite of natural minerals.

The emanating power of radon provides information on the internal structure of a mineral and the radiation damage caused by the decay of 238U, 235U and 232Th (and their daughters) that are present in the mineral. The concentration of 222Rn in groundwater is primarily controlled by the concentration of U and Th in the underlying rocks, as well as the emanation coefficient for that particular rock. The variations in the emanation coefficient for 222Rn caused when subsurface rocks are subjected to tectonic forces results in changes in 222Rn in groundwater. Increased emanation rates of radon from a mineral grain can potentially alter the 238U-206Pb, 235U-207Pb and 232Th-208Pb chronological clocks. We have measured radon emanation coefficients on a suite of minerals comprised of one oxide (uraninite), three silicates (thorite, zircon, and cerite) and one phosphate (monazite) at room temperature and after heating at 200 degrees C and 600 degrees C. Annealing of some of the nuclear tracks within a mineral significantly reduces the emanation rates of radon in these minerals, suggesting that the tracks created by decay events serve as conduit pathways for the release of 222Rn. Higher emanation rates of 222Rn from mineral grains that are surrounded by liquid as compared to air indicate that a major portion of the escaping 222Rn in air gets embedded into adjacent mineral grains and/or opposite walls of a pore.

Disposition of metabolically radiolabeled CE9.1--a macaque-human chimeric anti-human CD4 monoclonal antibody--in transgenic mice bearing human CD4.

IDEC-CE9.1 is a macaque/human chimeric IgG1 monoclonal antibody (mAb) directed against the human T-lymphocyte receptor, CD4. CE9.1 is highly specific for the human receptor and is known to cross-react only with chimpanzee CD4. Thus, limited in vivo investigations have been performed that would be expected to reflect the behavior of this mAb in humans. CE9.1 was metabolically radiolabeled using [3H]leucine, and studies of the distribution and pharmacokinetics of [3H]CE9.1 were performed in transgenic mice bearing either the hCD4 receptor in place of the mouse receptor (CD4+), or no CD4 receptor (CD4-). Single-dose studies were performed after intravenous administration of approximately 0.4 and 100 mg/kg. The disposition of CE9.1 was highly dependent on the presence and distribution of the hCD4 receptor. After a low intravenous dose to CD4+ mice, rapid loss of [3H]CE9.1 from plasma (mean residence time < 1 hr) was accompanied by accumulation of radioactivity in the spleen (a maximum of 18% of the administered dose at 2 hr). By contrast, no significant uptake of radiolabel was observed in the spleen of CD4- mice after a low intravenous dose (< 1%), and plasma radioactivity exceeded 40% of the administered dose at 24 hr. Significant accumulation of radiolabel was observed in the liver of both CD4+ and CD4- mice (maximum of 9-13%), suggesting this process was not CD4-receptor-mediated. After a high intravenous dose to CD4+ mice, the mean residence time of CE9.1 was approximately 24 hr, and dose-normalized plasma area under the concentration vs. time curve was within a factor of 2 of that observed in CD4- mice. Spleen radioactivity was < 1% after a high intravenous dose to CD4+ mice, whereas in the liver, the profile of radioactivity was similar in CD4+ mice at 0.4 and 100 mg/kg.

Paradigm to test a drug-induced aversion to ethanol.

The screening of new agents for aversive therapy of alcoholism requires a simple animal model. Animals trained to ingest ethanol solutions and subsequently administered a drug known to produce an aversion to ethanol in humans, do not readily make the association between the malaise induced by the aversive drug-ethanol reaction and the consumption of the same ethanol-containing solution that has been consumed previously without ill effects. An experimental paradigm is reported in which the malaise of the drug-ethanol reaction is quickly recognized by rats as derived from ethanol. Disulfiram was used as the model drug. Lewis rats were deprived of water for 18 h after which 6% (v/v) ethanol was offered as the only fluid. During the first hour of ethanol access, both controls (vehicle) and disulfiram (100 mg/kg)-treated animals consumed intoxicating amounts of ethanol (0.7-0.9 g ethanol/kg). Plasma acetaldehyde levels developed were 3-5 microM and 40-50 microM in the two groups respectively. After this time, disulfiram-treated animals virtually ceased consuming alcohol (90% inhibition), indicating that the disulfiram-ethanol reaction is associated with alcohol ingestion. Control animals continued consuming the alcohol solution for the additional 4-5 h tested. This model should be of value in the testing of new agents that reduce aldehyde dehydrogenase levels for prolonged periods for their potential as an aversive treatment in alcoholism.

Characterization of the metabolites of the peptidomimetic human immunodeficiency virus type 1 protease inhibitor SK&F 107461 in rats using liquid chromatography/mass spectrometry.

The metabolic fate of SK&F 107461 [Cbz-Ala-Ala-Phe psi [CHOHCH2] Gly-Val-Val-OMe], a potent and specific inhibitor of the protease encoded by human immunodeficiency virus type 1, in male Sprague-Dawley rats is described. SK&F 107461 is a hexapeptide analog containing a hydroxyethylene linkage in place of one of the peptide bonds, and in which the amino terminus is blocked with a carbobenzyloxy group and the carboxy terminus is modified to a methyl ester. The major metabolites of SK&F 107461 found in bile and urine after intravenous administration of 3H-labeled compound were characterized by LC/MS using either thermospray or continuous flow/FAB models of ionization. Approximately 80% of the administered radioactivity was recovered in the bile of bile duct-exteriorized rats following an intravenous dose. Radiochromatographic profiling indicated that SK&F 107461 was subject to extensive biotransformation. Structures were determined for three major biliary and five major urinary metabolites. Two of the major circulating plasma metabolites observed after intravenous bolus administration had similar retention times to metabolites that were observed in both bile and urine. A pathway for the biotransformation of SK&F 107461 in the rat is proposed. The parent molecule underwent two primary modes of metabolism. Hydrolysis of the carboxy-terminal ester or hydrolysis of the Ala-Ala peptide bond near the amino terminus were the primary metabolic events. All of the other metabolites characterized can be accounted for by exopeptidase activity subsequent to one or both of these primary events. There were no major metabolites observed resulting from anything other than hydrolysis of the ester or peptide bonds in the parent molecule.

Gene and antisense delivery in alcoholism research.

This article represents the proceedings of a symposium at the 2001 annual meeting of the Research Society on Alcoholism in Montreal, Canada. Drs. Yedy Israel and Fulton Crews were organizers and co-chairpersons. The presentations were (1) Introduction to the symposium, by Yedy Israel; (2) Gene delivery to the brain, by Fulton T. Crews; (3) Gene therapy in alcoholic liver injury, by Ronald Thurman; and (4) Antisense oligonucleotides and antisense-gene delivery, by Yedy Israel.