MDCK cells transfected with the human ␤-galactoside ␣-2,6-sialyltransferase 1 gene (AX-4 cells) were used to determine the drug susceptibility and pharmacodynamically linked variable of oseltamivir for influenza virus. For dose-ranging studies, five hollow-fiber units were charged with 10 2 A/Sydney/5/97 (H3N2) influenza virus-infected AX-4 cells and 10 8 uninfected AX-4 cells. Each unit was treated continuously with different oseltamivir carboxylate concentrations in virus growth medium for 6 days. For dose fractionation studies, one hollow-fiber unit received no drug, one unit received a 1؋ 50% effective concentration (EC 50 ) exposure to oseltamivir by continuous infusion, one unit received the same AUC 0-24 (area under the concentration-time curve from 0 to 24 h) by 1-h infusion every 24 h, one unit received the same total exposure in two equal fractions every 12 h, and one unit received the same total exposure in three equal fractions every 8 h. Each infusion dose was followed by a no-drug washout, producing the appropriate half-life for this drug. The effect of the drug on virus replication was determined by sampling the units daily, measuring the amount of released virus by plaque assay, and performing a hemagglutination assay. The drug concentration in the hollow-fiber infection model systems was determined at various times by liquid chromatography-tandem mass spectrometry. The dose-ranging study showed that the EC 50 s for oseltamivir carboxylate for the A/Sydney/5/97 strain of influenza virus was about 1.0 ng/ml. The dose fractionation study showed that all treatment arms suppressed virus replication to the same extent, indicating that the pharmacodynamically linked variable was the AUC 0-24 /EC 50 ratio. This implies that it may be possible to treat influenza virus infection once daily with a dose of 150 mg/day.

One of the biggest challenges in the effort to treat and contain influenza A virus infections is the emergence of resistance during treatment. It is well documented that resistance to amantadine arises rapidly during the course of treatment due to mutations in the gene coding for the M2 protein. To address this problem, it is critical to develop experimental systems that can accurately model the selection of resistance under drug pressure as seen in humans. We used the hollow-fiber infection model (HFIM) system to examine the effect of amantadine on the replication of influenza virus, A/Albany/1/98 (H3N2), grown in MDCK cells. At 24 and 48 h postinfection, virus replication was inhibited in a dose-dependent fashion. At 72 and 96 h postinfection, virus replication was no longer inhibited, suggesting the emergence of amantadine-resistant virus. Sequencing of the M2 gene revealed that mutations appeared at between 48 and 72 h of drug treatment and that the mutations were identical to those identified in the clinic for amantadine-resistant viruses (e.g., V27A, A30T, and S31N). Interestingly, we found that the type of mutation was strongly affected by the dose of the drug. The data suggest that the HFIM is a good model for influenza virus infection and resistance generation in humans. The HFIM has the advantage of being a highly controlled system where multiplicity parameters can be directly and accurately controlled and measured.

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). An unanswered question is how to translate this information into therapy for poxvirus infections in people. In a proof-of-principle study, we used a novel in vitro hollow-fiber infection model system to determine the pharmacodynamics of vaccinia virus infection of HeLa-S3 cells treated with cidofovir. Our results demonstrate that the currently licensed dose of cidofovir of 5 mg/kg of body weight weekly with probenecid (which ameliorates nephrotoxicity) is unlikely to provide protection for patients intentionally exposed to Variola major virus. We further demonstrate that the antiviral effect is independent of the schedule of drug administration. Exposures (area under the concentration-time curve) to cidofovir that will have a robust protective effect will require doses that are 5 to 10 times that currently administered to humans. Such doses may cause nephrotoxicity, and therefore, approaches that include probenecid administration as well as schedules of administration that will help ameliorate the uptake of cidofovir into renal tubular epithelial cells need to be considered when addressing such treatment for people.With the threat of terrorist attacks in the United States and abroad, the accidental or intentional exposure to category A pathogens is a real possibility. Of the many bacterial and viral agents of bioterrorism, Variola major virus, the cause of smallpox, is of greatest concern because it can be produced in large quantities, is very stable, easily aerosolized, and can kill up to 30% of unvaccinated people who are infected with the virus (27). A live attenuated vaccine, Dryvax, for the prevention of smallpox is available and was used successfully by the World Health Organization to wipe out the natural disease (5, 42). However, in rare cases, the vaccine had serious side effects, particularly in immunocompromised recipients, leading to the cessation of routine smallpox vaccination programs. Since universal vaccination for the prevention of smallpox was discontinued in the 1970s, large segments of the world's population are susceptible to infection with poxviruses. Despite a large effort to produce effective and safer vaccines to protect the world's population against infection with poxviruses, at present, no safer smallpox vaccine has been licensed for use in humans.In the absence of safe vaccines for smallpox, there is an ongoing search for safe and effective chemotherapeutic agents for the prevention and therapy of smallpox. This effort has identified several antiviral compounds that show in vitro and in vivo efficacy for poxvirus infections (1, 3, 4, 6, 7, 14-16, 21-23, 25-26, 28, 30-34, 36-41, 45). One of these compounds, cidofovir, is approved for treatment of cytomegalovirus retinitis in immunocompromised individuals (11). A recent publication suggests that cidofovir and an experimental nucleoside phosphonate, 6-[2-(phosphonomethoxy)alkoxy]-2,4-diaminopyrimidine (HPMPO-DAPy), were more effective than an acute postexposure vaccination regimen with the Elstree strain of vaccinia virus (VV) for ...

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