Safety, pharmacokinetics, and antiviral activity of A77003, a C2 symmetry-based human immunodeficiency virus protease inhibitor.
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A77003, an inhibitor of the human immunodeficiency virus type 1 (HIV-1) protease, was administered to asymptomatic HIV-1-infected patients in a phase I trial. The drug was given by continuous intravenous infusion at dosages of 0.035, 0.07, 0.14, and 0.28 mg/kg of body weight per h. The drug was given first for 24 h and then for up to an additional 4 weeks in a second infusion period following at least a 6-day washout. Apart from reversible increases in hepatic transaminase levels in some patients, no systemic toxicities occurred during extended infusion of the drug. Dose-related local vein irritation, despite dilution of the infusate, however, caused severe infusion site phlebitis precluding dosage escalation beyond 0.28 mg/kg/h. Pharmacokinetic analysis demonstrated dose linear increases in mean steady-state concentrations. However, clearance of the drug from plasma was unexpectedly high, averaging 62 liters/h across all groups. The concentrations of A77003 in plasma achieved the in vitro 50% inhibitory concentration (0.16 microgram/ml) only in the 0.28-mg/kg/h dosage group, but it did not attain the 90% inhibitory concentration (0.48 micrograms/ml). No statistically significant effect on CD4 cell numbers occurred in any of the groups, and there was no evidence of antiviral activity, as determined by HIV-1 p24 antigen level, quantitative plasma and cell culture, and quantitation of viral RNA in plasma. In conclusion, A77003, as formulated in the present study, causes severe phlebitis, which prevents administration of the infusates necessary to achieve high concentrations of the drug in plasma. In conclusion, A77003, as formulated in the present study, causes severe phlebitis, which prevents administration of the infusates necessary to achieve high concentrations of the drug in plasma. The lack of antiviral activity observed in the study may be a consequence of the low concentrations in plasma in all groups. (+info)
Characterization of human immunodeficiency virus type 1 variants with increased resistance to a C2-symmetric protease inhibitor.
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Inhibitors of the human immunodeficiency virus type 1 protease represent a promising class of antiviral drugs for the treatment of AIDS, and several are now in clinical trials. Here, we report the in vitro selection of viral variants with decreased sensitivity to a C2-symmetric protease inhibitor (A-77003). We show that a single amino acid substitution (Arg to Gln or Lys) at position 8 of the protease results in a substantial decrease in the inhibitory activity of the drug on the enzyme and a comparable increase in viral resistance. These findings, when analyzed by using the three-dimensional structure of the protease-drug complex, provide a strategic guide for the future development of inhibitors of the human immunodeficiency virus type 1 protease. (+info)
Binding of Yersinia enterocolitica to purified, native small intestinal mucins from rabbits and humans involves interactions with the mucin carbohydrate moiety.
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Plasmid-bearing (but not plasmid-cured) Yersinia enterocolitica is known to bind to purified small intestinal mucins from rabbits and humans. This study examined which region(s) of the mucin molecule is important for bacterial adherence. Pronase digestion of mucin and removal of nonglycosylated or poorly glycosylated peptide regions had no effect on bacterial binding, suggesting that plasmid-bearing Y. enterocolitica interacts with mucin carbohydrate. Periodate oxidation also did not alter bacterial adherence, indicating that vicinal hydroxyl groups in the mucin sugars are not important for binding. Boiling of mucin, depolymerization by reduction of disulfide bonds, or removal of noncovalently associated lipid actually enhanced bacterial adherence, suggesting that plasmid-bearing Y. enterocolitica can interact with additional domains in the mucin molecule revealed by these treatments. These domains were destroyed by pronase digestion. In delipidated mucin (but not in reduced or boiled mucin), binding to these domains appeared to be hydrophobic since it could be prevented by treatment of bacteria with tetramethyl urea. Oligosaccharides obtained from both human and rabbit small intestinal mucins were capable of inhibiting attachment of plasmid-bearing (but not plasmid-cured) Y. enterocolitica to mucin. After removal of terminal and backbone sugar residues by treatment of mucin with trifluoromethanesulfonic acid, binding of plasmid-bearing bacteria increased significantly when N-acetylgalactosamine, either alone or with galactose attached, was revealed, indicating that core regions of the sugar side chains are involved in bacterial binding. Adherence of plasmid-cured organisms was unaffected by trifluoromethanesulfonic acid treatment of mucin. We concluded that virulent Y. enterocolitica interacts with the carbohydrate moiety of native small intestinal mucin through a plasmid-mediated process. When mucin becomes denatured, binding of the organism can increase through hydrophobic and nonhydrophobic interactions with (most likely) the mucin protein. (+info)
Comparison of immunoturbidimetric and Lowry methods for measuring concentration of very low density lipoprotein apolipoprotein B-100 in plasma.
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To assess whether the Lowry-tetramethylurea method for measuring apolipoprotein B-100 (apo-B) in very low density lipoprotein (VLDL) could be replaced by direct assay of VLDL apo-B using a highly practicable immunological method. Seventy five fasting blood samples were collected from patients attending the lipid clinic at this hospital. Plasma was separated immediately and VLDL isolated by preparative ultracentrifugation at solution density 0.93-1.006 kg/l. Apo-B was precipitated from an aliquot of the VLDL fraction using the tetramethylurea (TMU) technique and protein mass determined by the Lowry method (LM); mean apo-B 83.02 micrograms/ml (SD 74.85). Apo-B was also measured in VLDL using direct immunoturbidimetry on the Cobas-Fara analyser; mean apo-B 82.32 micrograms/ml (SD 72.88). There was a very close correlation between methods (immunoturbidimetry = 0.94.LM + 3.95, r = 0.97, p < 0.001). The mean difference between methods (constant error) was small (0.70 microgram/ml) and not significant (p = 0.742). Random error was 13.01 micrograms/ml by analysis of variance. It is concluded that immunoturbidimetry, a more rapid and convenient test, may replace the LM and TMU techniques for measuring VLDL apo-B concentration and that this method could be applied to research studies requiring analysis of large numbers of samples. (+info)
Nonribosomal nature of novel, stable ribonucleic acid species accumulated by blue-green bacteria.
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The facultatively chemoheterotrophic blue-green bacterium Aphanocapsa 6714 accumulates two novel, stable ribonucleic acid species when deprived of sources of carbon and energy. At least one of these species is nonribosomal. (+info)
Selection of multiple human immunodeficiency virus type 1 variants that encode viral proteases with decreased sensitivity to an inhibitor of the viral protease.
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Inhibitors of the human immunodeficiency virus type 1 (HIV-1) protease represent a promising addition to the available agents used to inhibit virus replication in a therapeutic setting. HIV-1 is capable of generating phenotypic variants in the face of a variety of selective pressures. The potential to generate variants with reduced sensitivity to a protease inhibitor was examined by selecting for virus growth in cell culture in the presence of the protease inhibitor A-77003. Virus variants grew out in the presence of the inhibitor, and these variants encoded proteases with reduced sensitivity to the inhibitor. Variants were identified that encoded changes in each of the three subsites of the protease that interact with the inhibitor. HIV-1 displays significant potential for altering its interaction with this protease inhibitor, suggesting the need for multiple protease inhibitors with varying specificities. (+info)
Preclinical evaluation of antiviral activity and toxicity of Abbott A77003, an inhibitor of the human immunodeficiency virus type 1 protease.
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A synthetic, symmetry-based inhibitor of the human immunodeficiency virus type 1 (HIV-1) protease, A77003, was evaluated for antiviral activity and cytotoxicity in vitro in human peripheral blood lymphocytes or cell lines H9, CEM, and U937. Toxicity and antiviral activity of the HIV-1 protease inhibitor were compared with those of the reverse transcriptase inhibitors zidovudine and 2',3'-dideoxy-2',3'-didehydrothymidine and human recombinant alpha and beta interferons. Production of infectious virus particles, cell-free p24 antigen, and cell-associated viral proteins was reduced 50% by the HIV-1 protease inhibitor at concentrations of 0.12 to 0.26 microM (50% effective concentration [EC50]) in acute infection and 0.2 to 1.7 microM (EC50) in persistent infection. Fluorescence-activated cell sorter analysis of U937 cells persistently infected with HIVIIIB using a monoclonal antibody to HIV also showed a reduction of cell-associated viral protein in A77003-treated cells. Furthermore, toxicity of A77003 assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay was not observed at greater than 100 times the EC50. A77003 was more effective in persistent HIV-1 infection than alpha and beta interferons (1,000 U/ml), while zidovudine and 2',3'-dideoxy-2',3'-didehydrothymidine were not active. (+info)
Partial inhibition of the human immunodeficiency virus type 1 protease results in aberrant virus assembly and the formation of noninfectious particles.
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The production of infectious particles by human immunodeficiency virus type 1 is dependent on the accurate cleavage of its Gag and Gag/Pol precursors by a virally encoded protease. In the absence of protease activity, morphologically abnormal particles which are noninfectious are formed. Recently, inhibitors of the protease of human immunodeficiency virus type 1 have been developed as potential therapeutic agents. We have examined the basis for the loss of infectivity at the limiting inhibitor concentrations that are likely to be achieved in clinical settings. We found that subtle defects in processing are correlated with profound deficits in infectivity. Further, we correlated this partially disrupted processing with an altered virion morphology. These data suggest that accurate and complete processing is essential to the formation of infectious, morphologically normal virions and that the pathway by which these precursors are processed and assembled is sensitive to partial inhibition of the protease by an inhibitor disproportionate to the effect of the inhibitor on the viral protease itself. (+info)