Partial Purification and Properties of Human Brain Aldehyde Dehydrogenases

Acetaldehyde and biogenic aldehydes were used as substrates to investigate the subcellular distribution of aldehyde dehydrogenase activity in autopsied human brain. With 10 microM acetaldehyde as substrate, over 50% of the total activity was found in the mitochondrial fraction and 38% was associated with the cytosol. However, with 4 microM 3,4-dihydroxyphenylacetaldehyde and 10 microM indoleacetaldehyde as substrates, 40-50% of the total activity was found in the soluble fraction, the mitochondrial fraction accounting for only 15-30% of the total activity. These data suggested the presence of distinct aldehyde dehydrogenase isozymes in the different compartments. The mitochondrial and cytosolic fractions were, therefore, subjected to salt fractionation and ion-exchange chromatography to purify further the isozymes present in both fractions. The kinetic data on the partially purified isozymes revealed the presence of a low Km isozyme in both the mitochondria and the cytosol, with Km values for acetaldehyde of 1.7 microM and 10.2 microM, respectively. However, the cytosolic isozyme exhibited lower Km values for the biogenic aldehydes. Both isozymes were activated by Mg2+ and Ca2+ in phosphate buffers (pH 7.4). Also, high Km isozymes were found in the mitochondria and in the microsomes.     

In vitro selection and characterization of influenza A (A/N9) virus variants resistant to a novel neuraminidase inhibitor, A-315675

With the recent introduction of neuraminidase (NA) inhibitors into clinical practice for the treatment of influenza virus infections, considerable attention has been focused on the potential for resistance development and cross-resistance between different agents from this class. A-315675 is a novel influenza virus NA inhibitor that has potent enzyme activity and is highly active in cell culture against a variety of strains of influenza A and B viruses. To further assess the therapeutic potential of this compound, in vitro resistance studies have been conducted and a comparative assessment has been made relative to oseltamivir carboxylate. The development of viral resistance to A-315675 was studied by in vitro serial passage of influenza A/N9 virus strains grown in MDCK cells in the presence of increasing concentrations of A-315675. Parallel passaging experiments were conducted with oseltamivir carboxylate, the active form of a currently marketed oral agent for the treatment of influenza virus infections. Passage experiments with A-315675 identified a variant at passage 8 that was 60-fold less susceptible to the compound. Sequencing of the viral population identified an E119D mutation in the NA gene, but no mutations were observed in the hemagglutinin (HA) gene. However, by passage 10 (2.56 microM A-315675), two mutations (R233K, S339P) in the HA gene appeared in addition to the E119D mutation in the NA gene, resulting in a 310-fold-lower susceptibility to A-315675. Further passaging at higher drug concentrations had no effect on the generation of further NA or HA mutations (20.5 microM A-315675). This P15 virus displayed 355-fold-lower susceptibility to A-315675 and >175-fold-lower susceptibility to zanamivir than did wild-type virus, but it retained a high degree of susceptibility to oseltamivir carboxylate. By comparison, virus variants recovered from passaging against oseltamivir carboxylate (passage 14) harbored an E119V mutation and displayed a 6,000-fold-lower susceptibility to oseltamivir carboxylate and a 175-fold-lower susceptibility to zanamivir than did wild-type virus. Interestingly, this mutant still retained susceptibility to A-315675 (42-fold loss). This suggests that cross-resistance between A-315675- and oseltamivir carboxylate-selected variants in vitro is minimal.     

Inhibitors of HCV NS5B polymerase: synthesis and structure-activity relationships of unsymmetrical 1-hydroxy-4,4-dialkyl-3-oxo-3,4-dihydronaphthalene benzothiadiazine derivatives

Substituted 1-hydroxy-4,4-dialkyl-3-oxo-3,4-dihydronaphthalene benzothiadiazine derivatives were investigated as inhibitors of genotype 1 HCV polymerase. Structure-activity relationship patterns for this class of compounds are discussed. It was found that the saturated alkane dialkyl units provided the most active analogs.     

Design, synthesis, and structural analysis of inhibitors of influenza neuraminidase containing a 2,3-disubstituted tetrahydrofuran-5-carboxylic acid core

(+/-)-(2R,3R,5R)-[2-(1'-S-acetamido-3'-methyl)butyl-3-methoxycarbonyl]tetrahydrofuran-5-carboxylic acid (9) and (+/-)-(2R,3R,5R)-[2-(1'-S-acetamido-3'-methyl)butyl-3-(4'-imidazolyl)]tetrahydrofuran 5-carboxylic acid (14) were synthesized as inhibitors of influenza neuraminidase (NA). Both compounds 9 and 14 inhibit influenza NA A with an IC(50) of about 0.5 microM and NA B with an IC(50) of 1.0 microM.     

Novel hepatitis C virus replicon inhibitors: synthesis and structure-activity relationships of fused pyrimidine derivatives

The synthesis of several pyrido[2,3-d]pyrimidine and pyrimido[4,5-d]pyrimidine analogs is described with one such analog possessing subnanomolar potency in both genotype 1a and 1b cell culture HCV replicon assays.     

Inhibitors of HCV NS5B polymerase: synthesis and structure-activity relationships of N-alkyl-4-hydroxyquinolon-3-yl-benzothiadiazine sulfamides

Substituted N-alkyl-4-hydroxyquinolon-3-yl-benzothiadiazine sulfamides were investigated as inhibitors of genotype 1 HCV polymerase. Structure-activity relationship patterns for this class of compounds are discussed.     

Determination of 5-fluorouracil in plasma with HPLC-tandem mass spectrometry

In this article, we describe a fast and specific method to measure 5FU with HPLC tandem-mass spectrometry. Reversed-phase HPLC was combined with electrospray ionization tandem mass spectrometry and detection was performed by multiple-reaction monitoring. Stable-isotope-labeled 5FU (1,3-15N2-5FU) was used as an internal standard. 5FU was measured within a single analytical run of 16 min with a lower limit of detection of 0.05 microM. The intra-assay variation and inter-assay variation of plasma with added 5FU (1 microM, 10 microM, 100 microM) was less then 6%. Recoveries of the added 5FU in plasma were > 97%. Analysis of the 5FU levels in plasma samples from patients with the HPLC tandem mass spectrometry method and a HPLC-UV method yielded comparable results (r2 = 0.98). Thus, HPLC with electrospray ionization tandem mass spectrometry allows the rapid analysis of 5FU levels in plasma and could, therefore, be used for therapeutic drug monitoring.     

Pharmacokinetics of 5-Fluorouracil in Patients Heterozygous for the IVS14+1G > A Mutation in the Dihydropyrimidine Dehydrogenase Gene

5-Fluorouracil (5FU) and capecitabine are two of the most frequently prescribed chemotherapeutic drugs for the treatment of patients with cancer. Administration of test doses of 5FU to eight patients heterozygous for the IVS14+1G > A mutation and five control patients showed that the AUC and clearance were weak parameters with respect to the identification of patients with a DPD deficiency. However, highly significant differences were observed for the terminal half life of 5FU between DPD patients and controls. Thus, a DPD deficiency could be predicted from 5FU blood concentrations measured after the administration of a test dose of 5FU.     

Des-A-ring benzothiadiazines: inhibitors of HCV genotype 1 NS5B RNA-dependent RNA polymerase

In our program to discover non-nucleoside, small molecule inhibitors of genotype 1 HCV polymerase, we investigated a series of promising analogs based on a benzothiadiazine screening hit that contains an ABCD ring system. After demonstrating that a methylsulfonylamino D-ring substituent increased the enzyme potency into the low nanomolar range, we explored a minimum core required for activity by truncating to a three-ring system. Described herein are the syntheses and structure-activity relationship of a set of inhibitors lacking the A-ring of an ABCD ring system. We observed that small aromatic rings and alkenyl groups appended to the 5-position of the B-ring were optimal, resulting in inhibitors with low nanomolar potencies.     

Partitioning of TSE infectivity during ethanol fractionation of human plasma.

The practice of validating processes for their capacity to inactivate a range of non-enveloped and enveloped viruses also provides confidence that plasma products will be safe from emerging viral pathogens with known aetiology. Of greater concern are diseases of unknown or poorly defined aetiology such as the group of neurological diseases collectively called the transmissible spongiform encephalopathies (TSEs), or prion diseases, for which the best known human disease is Creutzfeldt-Jakob Disease (CJD) and its variant form (vCJD). The goal of the current study was to investigate the potential for manufacturing steps used in the production of albumin and immunoglobulin products by Kistler-Nitschmann fractionation, and the utility of nanofiltration of immunoglobulin to remove TSE agents. Two different scrapie model systems were used. In the first system infectious material used for spiking was scrapie sheep brain homogenate with infectivity titres being measured in hamsters. In the second system purified scrapie agent was used (PrP fibrils) with Western blot analysis measuring reduction in the proteinase K resistant form being used as a measure of removal. The data demonstrated substantial removal of the infectious agent by the manufacturing process in both model systems although some differences were observed in partitioning of the two different infectious materials. The hamster infectivity studies were shown to be approximately 1000 fold more sensitive than the Western Blot assay. The data from both studies provide added confidence that these plasma products are safe with respect to their potential to transmit TSE.