In vitro susceptibilities of wild-type or drug-resistant hepatitis B virus to (-)-beta-D-2,6-diaminopurine dioxolane and 2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil.

Prolonged treatment of chronic hepatitis B virus (HBV) infection with lamivudine ([-]-beta-L-2',3'-dideoxy-3' thiacytidine) or famciclovir may select for viral mutants that are drug resistant due to point mutations in the polymerase gene. Determining whether such HBV mutants are sensitive to new antiviral agents is therefore important. We used a transient transfection system to compare the sensitivities of wild-type HBV and four lamivudine- and/or famciclovir-resistant HBV mutants to adefovir [9-(2-phosphonyl-methoxyethyl)-adenine; PMEA] and the nucleoside analogues (-)-beta-D-2, 6-diaminopurine dioxolane (DAPD) and 2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil (L-FMAU). The drug-resistant mutants contained amino acid substitutions in the polymerase protein. We found that the M550I and M550V plus L526M substitutions, which confer lamivudine resistance, did not confer cross-resistance to adefovir or DAPD, but conferred cross-resistance to L-FMAU. The M550V substitution in isolation conferred a similar phenotype to M550I, except that it did not confer significant resistance to L-FMAU. The L526M substitution, which is associated with famciclovir resistance, conferred cross-resistance to L-FMAU but not to adefovir or DAPD. Inhibition of HBV secretion by DAPD, L-FMAU, and adefovir did not always correlate with inhibition of the generation of intracellular HBV replicative intermediates, suggesting that these analogs may preferentially inhibit specific stages of the viral replication cycle.

Kinetics of hepadnavirus loss from the liver during inhibition of viral DNA synthesis.

Hepadnaviruses replicate by reverse transcription, which takes place in the cytoplasm of the infected hepatocyte. Viral RNAs, including the pregenome, are transcribed from a covalently closed circular (ccc) viral DNA that is found in the nucleus. Inhibitors of the viral reverse transcriptase can block new DNA synthesis but have no direct effect on the up to 50 or more copies of cccDNA that maintain the infected state. Thus, during antiviral therapy, the rates of loss of cccDNA, infected hepatocytes (1 or more molecules of cccDNA), and replicating DNAs may be quite different. In the present study, we asked how these losses compared when woodchucks chronically infected with woodchuck hepatitis virus were treated with L-FMAU [1-(2-fluoro-5-methyl-beta-L-arabinofuranosyl) uracil], an inhibitor of viral DNA synthesis. Viremia was suppressed for at least 8 months, after which drug-resistant virus began replicating to high titers. In addition, replicating viral DNAs were virtually absent from the liver after 6 weeks of treatment. In contrast, cccDNA declined more slowly, consistent with a half-life of approximately 33 to 50 days. The loss of cccDNA was comparable to that expected from the estimated death rate of hepatocytes in these woodchucks, suggesting that death of infected cells was one of the major routes for elimination of cccDNA. However, the decline in the actual number of infected hepatocytes lagged behind the decline in cccDNA, so that the average cccDNA copy number in infected cells dropped during the early phase of therapy. This observation was consistent with the possibility that some fraction of cccDNA was distributed to daughter cells in those infected hepatocytes that passed through mitosis.

Mechanism of action of 1-beta-D-2,6-diaminopurine dioxolane, a prodrug of the human immunodeficiency virus type 1 inhibitor 1-beta-D-dioxolane guanosine.

(-)-beta-D-2,6-Diaminopurine dioxolane (DAPD), is a nucleoside reverse transcriptase (RT) inhibitor with activity against human immunodeficiency virus type 1 (HIV-1). DAPD, which was designed as a water-soluble prodrug, is deaminated by adenosine deaminase to give (-)-beta-D-dioxolane guanine (DXG). By using calf adenosine deaminase a K(m) value of 15 +/- 0.7 microM was determined for DAPD, which was similar to the K(m) value for adenosine. However, the k(cat) for DAPD was 540-fold slower than the k(cat) for adenosine. In CEM cells and peripheral blood mononuclear cells exposed to DAPD or DXG, only the 5'-triphosphate of DXG (DXG-TP) was detected. DXG-TP is a potent alternative substrate inhibitor of HIV-1 RT. Rapid transient kinetic studies show the efficiency of incorporation for DXG-TP to be lower than that measured for the natural substrate, 2'-deoxyguanosine 5'-triphosphate. DXG-TP is a weak inhibitor of human DNA polymerases alpha and beta. Against the large subunit of human DNA polymerase gamma a K(i) value of 4.3 +/- 0.4 microM was determined for DXG-TP. DXG showed little or no cytotoxicity and no mitochondrial toxicity at the concentrations tested.

An analysis of the catalytic cycle of HIV-1 reverse transcriptase: opportunities for chemotherapeutic intervention based on enzyme inhibition.

This review describes each of the steps in the HIV-1 reverse transcriptase catalytic cycle and evaluates each of these steps as a potential point of inhibition of the enzyme and consequently viral replication. To date, two classes of approved drugs act on the reverse transcriptase. They are: (1) the nucleoside reverse transcriptase inhibitors which either directly inhibit the enzyme or serve as alternative substrates for catalysis (resulting in chain termination) and (2) the non-nucleoside reverse transcriptase inhibitors which bind to an allosteric site and adversely affect the function of the enzyme by slowing the rate of chemical catalysis. In order to provide the best possible analysis of the potential of each of the steps in the catalytic cycle as a site of inhibition, the molecular forces which determine the intrinsic binding affinities and specificity of natural components of the catalytic complex will be described in as much detail as possible.

Inhibition of the replication of the DNA polymerase M550V mutation variant of human hepatitis B virus by adefovir, tenofovir, L-FMAU, DAPD, penciclovir and lobucavir.

Several nucleoside analogues (penciclovir, lobucavir, dioxalane guanine [DXG], 1-beta-2,6-diaminopurine dioxalane [DAPD], L-FMAU, lamivudine) and acyclic nucleoside phosphonate analogues (adefovir, tenofovir) that are in clinical use, in clinical trials or under preclinical development for the treatment of hepatitis B virus (HBV) infections, were evaluated for their inhibitory effect on the replication of a la- mivudine-resistant HBV variant containing the methionine --> valine substitution (M550V) in the polymerase nucleoside-binding domain. The antiviral activity was determined in the tetracycline-responsive HepAD38 and HepAD79 cells, which are stably transfected with either a cDNA copy of the wild-type pregenomic RNA or with cDNA containing the M550V mutation. As expected, lamivudine was much less ( approximately 200-fold) effective at inhibiting replication of the M550V mutant virus than the wild-type virus. In contrast, adefovir, tenofovir, lobucavir, L-FMAU, DXG and DAPD proved almost equally effective against both viruses. A second objective of this study was to directly compare the antiviral potency of the anti-HBV agents in HepG2 2.2.15 cells (which are routinely used for anti-HBV drug-screening purposes) with that in HepAD38 cells. HepAD38 cells produce much larger quantities of HBV than HepG2 2.2.15 cells, and thus allow drug screening in a multiwell plate format. All compounds were found to be almost equally effective at inhibiting HBV replication in HepAD38 cells (as in HepG2 2.2.15 cells), except for penciclovir, which was clearly less effective in HepAD38 cells.

Conformation and local environment of nucleotides bound to HIV type 1 reverse transcriptase (HIV-1 RT) in the ground state.

Nuclear Overhauser effect experiments were used to characterize the protein environment and conformations of dTTP, dATP and AZTTP bound to HIV-RT in the ground state. The results show the initial binding sites for the nucleotides overlap but are not completely coincident. All of the bound nucleotides assume the same anti C4'-exo conformation.

Third-party reprocessing of endoscopic accessories.

Third-party reprocessing of medical devices labeled for single use is a safe, FDA regulated practice that helps hospitals reduce costs without compromising patient care. Simply because a device is labeled as single use does not mean it cannot be safely reprocessed. To the contrary, the single use label is chosen by the manufacturer, sometimes for economic gain, as there are no formal FDA regulations or standards to distinguish between reusable and single use devices. The current FDA regulatory framework for third-party reprocessors, which emphasizes compliance with FDA quality assurance requirements, is presently under review, and the agency is in the process of developing a new regulatory scheme for reprocessing.

Safety assessment, in vitro and in vivo, and pharmacokinetics of emivirine, a potent and selective nonnucleoside reverse transcriptase inhibitor of human immunodeficiency virus type 1.

Emivirine (EMV), formerly known as MKC-442, is 6-benzyl-1-(ethoxymethyl)-5-isopropyl-uracil, a novel nonnucleoside reverse transcriptase inhibitor that displays potent and selective anti-human immunodeficiency virus type 1 (HIV-1) activity in vivo. EMV showed little or no toxicity towards human mitochondria or human bone marrow progenitor cells. Pharmacokinetics were linear for both rats and monkeys, and oral absorption was 68% in rats. Whole-body autoradiography showed widespread distribution in tissue 30 min after rats were given an oral dose of [(14)C]EMV at 10 mg/kg of body weight. In rats given an oral dose of 250 mg/kg, there were equal levels of EMV in the plasma and the brain. In vitro experiments using liver microsomes demonstrated that the metabolism of EMV by human microsomes is approximately a third of that encountered with rat and monkey microsomes. In 1-month, 3-month, and chronic toxicology experiments (6 months with rats and 1 year with cynomolgus monkeys), toxicity was limited to readily reversible effects on the kidney consisting of vacuolation of kidney tubular epithelial cells and mild increases in blood urea nitrogen. Liver weights increased at the higher doses in rats and monkeys and were attributed to the induction of drug-metabolizing enzymes. EMV tested negative for genotoxic activity, and except for decreased feed consumption at the high dose (160 mg/kg/day), with resultant decreases in maternal and fetal body weights, EMV produced no adverse effects in a complete range of reproductive toxicology experiments performed on rats and rabbits. These results support the clinical development of EMV as a treatment for HIV-1 infection in adult and pediatric patient populations.

Screening for potential toxicity of antiviral therapies.

Preclinical screening for the toxicity of antiviral drugs has thus far proven quite successful. Twenty-three antivirals spanning a variety of chemical classes and including a combination product, have been safely developed and are listed in the 1999 Physician's Desk Reference. Several of these antivirals have been administered for many years and in various combinations and we are currently unaware of any being withdrawn for safety-related reasons. Progress in protecting this record includes advances in rational drug design, the development of in vitro and in vivo models available to study both efficacy and safety, and improved bioanalytical capabilities. Coupled with approximately 25 years of experience since vidarabine was approved for the treatment of herpes encephalitis, these advances set the stage for even more precise and focused development of pharmaceuticals for the treatment of life-threatening viral infections, including those caused by HIV, HBV and emerging viruses. Experience to date suggests that each antiviral, particularly the nucleoside analogs, may have unique attributes of efficacy and safety. This brief review is an attempt to highlight a combination of established and recent methods that may be helpful in screening antivirals for potential toxicity. The earliest phases of drug development are mentioned in the introduction, followed by contributions provided by in vitro and in vivo testing in models of viral disease. Finally, current and new methods applicable to screening for toxicity are discussed.

Structural and electrochemical examinations of PACVD TiO2 films in Ringer solution.

The conditions for obtaining titanium dioxide from the substrates titanium tetrachloride and oxygen and applying this to a surgical stainless steel of the type 316L by the plasma assisted chemical vapour deposition method have been determined. It was established that, during the process, titanium dioxide anatase is created, Crystallizing in a tetragonal lattice. During exposure of the 316L steel with the titanium dioxide coating, in Ringer's solution, protective properties of this covering improve. After 120 h the coating adopts superior barrier characteristics. Titanium dioxide covering increases the resistivity of steel of the type 316L to pitting corrosion and general corrosion. Any damage or partial removal of the coating does not cause an increased galvanic corrosion of the substrate.

Human immunodeficiency virus type-1 reverse transcriptase. Contribution of Met-184 to binding of nucleoside 5'-triphosphate.

Mutations were made in recombinant human immunodeficiency virus type-1 reverse transcriptase (RT) by substituting methionine 184 with alanine (M184A) or valine (M184V), and steady-state and pre-steady-state kinetic constants were determined. The Km values of M184A RT for dNTPs were larger than those of wt RT for RNA-directed synthesis; the kcat values of M184A RT for processive or distributive synthesis were similar. In contrast to M184A RT, the Km and kcat values of M184V RT for dNTP substrates were similar to those of wt RT. The Ki values of M184V RT for 1-beta-L-nucleoside analogs were increased 30-500-fold relative to wt RT for both RNA- and DNA-directed synthesis. The Kd and kp values of wt RT and M184V RT for dCTP and cis-5-fluoro-1-[2-(hydroxymethyl)-1, 3-oxathiolan-5-yl]cytosine 5'-triphosphate (1-beta-L-FTCTP) were estimated from pre-steady-state kinetics for single nucleotide incorporation. The Kd value of M184V RT for 1-beta-L-FTCTP was 19-fold greater than that of wt RT; the kpvalues of the two enzymes were similar. These results support the hypothesis that methionine 184 in the highly conserved YMDD region of wt RT participates in the binding of the nucleoside (analog) 5'-triphosphate.

Recombinant human immunodeficiency virus type 1 reverse transcriptase is heterogeneous.

Recombinant wild type (wt) and T215Y HIV-1 reverse transcriptase (RT) were isolated using three methods designated A, B, and C. The three samples of wt RT were kinetically indistinguishable with respect to dTTP turnover on poly(rA).p(dT)10. However, whereas the kinetic constants for dTTP and AZTTP for both T215Y B and T215Y C were similar to those of wt protein, T215Y A exhibited a twofold increase in Km value for dTTP and a 13-fold increase in Ki value for AZTTP with respect to wt protein purified in the same manner. We further investigated this observation by studying the denaturation of wt RT by urea. The urea denaturation curves monitored by fluorescence and circular dichroism spectroscopy were not coincident with the denaturation curve monitored by enzyme activity and yielded Cm values (the concentration of urea at which 50% of the protein is denatured) of 4.1 and 2.0 M urea, respectively. The noncoincidence of the transition curves reflects two separable, sequential, noncooperative conformational changes in the molecule: (a) from a catalytically active to an inactive conformation, and (b) from a catalytically inactive to a denatured, unfolded conformation. We therefore used denaturation as detected by changes in enzyme activity to compare the conformational stability of the three samples of wt and T215Y RT A, B, and C. The Cm values for T215Y RT did not differ from those of the respective wt; however, differences in Cm values were noted depending on how the protein was isolated. This suggested that the heterogeneity of the recombinant RT was due to small differences in conformation at or near the active site.

Prenatal diagnosis of Freeman-Sheldon syndrome (whistling face).

The diagnosis of Freeman-Sheldon syndrome was made by ultrasonographic evaluation of a 20-week fetus with a positive family history. The ultrasonographic features were abnormalities of the extremities and mouth.

Evaluation of the potent anti-hepatitis B virus agent (-) cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine in a novel in vivo model.

A murine model was developed to investigate the in vivo activity of anti-hepatitis B virus (HBV) agents. Mice with subcutaneous tumors of HBV-producing 2.2.15 cells showed reductions in levels of HBV in serum and in intracellular levels of HBV when the mice were orally dosed with (-) cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine (FTC). No effects on tumor size or alpha-fetoprotein levels were observed. FTC can selectively inhibit HBV replication at nontoxic doses.

Pharmacokinetics, oral bioavailability, and metabolic disposition in rats of (-)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl] cytosine, a nucleoside analog active against human immunodeficiency virus and hepatitis B virus.

The pharmacokinetics and metabolism of the potent anti-human immunodeficiency virus and anti-hepatitis B virus compound, (-)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl] cytosine (FTC), were investigated in male CD rats. Plasma clearance of 10 mg of FTC per kg of body weight was biexponential in rats, with a half-life at alpha phase of 4.7 +/- 1.1 min (mean +/- standard deviation) and a half-life at beta phase of 44 +/- 8.8 min (n = 5). The total body clearance of FTC was 1.8 +/- 0.1 liters/h/kg, and the oral bioavailability was 90% +/- 8%. The volume of distribution at steady state (Vss) was 1.5 +/- 0.1 liters/kg. Increasing the dose to 100 mg/kg slowed clearance to 1.5 +/- 0.2 liters/kg/h, lowered the Vss to 1.2 +/- 0.2 liters/kg, and reduced the oral bioavailability to 65% +/- 15%. FTC in the brains of rats was initially less than 2% of the plasma concentration but increased to 6% by 2 h postdose. Probenecid elevated levels of FTC in plasma as well as in brains but did not alter the brain-to-plasma ratio. The urinary and fecal recoveries of unchanged FTC after a 10-mg/kg intravenous dose were 87% +/- 3% and 5% +/- 1.6%, respectively. After a 10-mg/kg oral dose, respective urinary and fecal recoveries were 70% +/- 2.5% and 25% +/- 1.6%. Two sulfoxides of FTC were observed in the urine, accounting for 0.4% +/- 0.03% and 2.7% +/- 0.2% of the intravenous dose and 0.4% +/- 0.06% and 2.5% +/- 0.3% of the oral dose. Also observed were 5-fluorocytosine, representing 0.4% +/- 0.06% of the intravenous dose and 0.4% +/- 0.07% of the oral dose, and FTC glucuronide, representing 0.7% +/- 0.2% of the oral dose and 0.4% +/- 0.2% of the intravenous dose. Neither deaminated FTC nor 5-fluorouracil was observed in the urine (less than 0.2% of dose). The high oral availability and minimal metabolism of FTC encourage its further preclinical development.

Identification of the nucleotide binding site of HIV-1 reverse transcriptase using dTTP as a photoaffinity label.

We have utilized UV-induced cross-linking of [methyl-3H]dTTP to identify the nucleotide binding site on heterodimeric HIV-1 reverse transcriptase (RT). RT was derivatized by irradiating a solution containing [methyl-3H]dTTP and purified recombinant RT for 10 min. The UV-induced cross-linking reaction between dTTP and RT is linear with time of UV exposure up to 10 min, and it has been determined previously that dTTP cross-linking is half-maximal at 90 microM [Cheng, N., Painter, G. R., & Furmann, P.A. (1991) Biochem. Biophys. Res. Commun. 174, 785-789]. Under these reaction conditions, only the 66-kDa subunit of the 66-kDa/51-kDa RT heterodimer was labeled with dTTP. The [methyl-3H]dTTP-labeled RT was fragmented with trypsin and endoproteinase Asp-N, and peptides were purified on reversed phase HPLC. The peptide covalently linked to [methyl-3H]dTTP was subjected to amino acid sequence analysis. The sequencing data localized the nucleotide binding site of RT to Lys-73 in the vicinity of several mutation sites linked to antiviral drug resistance. Since most effective anti-AIDS compounds are inhibitors of RT, information about its dNTP binding site may make it possible to understand the basis for the antiviral activity of nucleoside analogs such as AZT, ddI, and ddC. This information may also be useful for a more rationally based design of anti-HIV agents.

The 5'-triphosphates of the (-) and (+) enantiomers of cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolane-5-yl]cytosine equally inhibit human immunodeficiency virus type 1 reverse transcriptase.

The (-) enantiomer of cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolane-5-yl]cytosine is a potent inhibitor of human immunodeficiency virus type 1 in vitro. The 5'-triphosphates of the (-) and (+) enantiomers equally inhibited the production of full-length minus-strand DNA in an endogenous reverse transcriptase reaction, each competitively inhibited DNA synthesis, and each was used as a chain-terminating substrate.