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Antimicrobial Agents and Chemotherapy, January 2009, p. 129-135, Vol. 53, No. 1
0066-4804/09/$08.00+0     doi:10.1128/AAC.00708-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Pharmacodynamics of Cidofovir for Vaccinia Virus Infection in an In Vitro Hollow-Fiber Infection Model System

James J. McSharry, Mark R. Deziel, Kris Zager, Qingmei Weng, and George L. Drusano^*

Antiviral Pharmacodynamics Laboratory, Emerging Infections and Host Defense Group, Ordway Research Institute, 150 New Scotland Avenue, Albany, New York 12208

Received 29 May 2008/ Returned for modification 18 August 2008/ Accepted 3 October 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Variola major virus remains a potent weapon of bioterror. There is currently an investigational-new-drug application for cidofovir for the therapy of variola major virus infections. Stittelaar and colleagues compared the levels of effectiveness of postexposure smallpox vaccination (Elstree-RIVM) and antiviral treatment with cidofovir or an acyclic nucleoside phosphonate analogue {6-[2-(phosphonomethoxy)alkoxy]-2,4-diaminopyrimidine (HPMPO-DAPy)} after lethal intratracheal infection of cynomolgus monkeys with monkeypox virus, a variola virus surrogate. Their results demonstrated that either compound was more effective than vaccination with the Ellstree vaccine (K. J. Stittelaar et al., Nature 439:745-748, 2006). 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.

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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 the control of monkeypox virus (MPXV) infection in cynomolgus monkeys (43). However, at present, none of these antiviral compounds are approved by the Food and Drug Administration for the treatment of people infected with poxviruses. There is an investigational-new-drug proposal for the use of cidofovir for the treatment of poxvirus infections in humans. The pharmacokinetics and pharmacodynamics of cidofovir for human cytomegalovirus infections in immunocompromised patients has been determined (10, 11). Cidofovir is not orally bioavailable. When given by intravenous infusion, less than 5% of the compound is converted into the active moiety, with the majority being excreted through the kidney, leading to nephrotoxicity. There is little information regarding how to choose the correct dose and schedule of administration of cidofovir to treat people with poxviruses. Due to the morbidity and mortality associated with poxvirus infections, the usual phase II/III clinical trials cannot be performed to determine the pharmacodynamics of cidofovir for poxviruses in humans. Therefore, we employed an in vitro hollow-fiber infection model (HFIM) pharmacodynamic system (Fig. 1) to determine the relationship between exposure and virological response as well as the role of schedule of administration for the use of cidofovir for the treatment of poxvirus infections in humans. This system has been employed by our laboratory for a number of years and has successfully predicted the exposure response and schedule response documented in clinical trials of different classes of antiretroviral agents (2, 17, 18, 29). In this proof-of-principle study, we used the HFIM system to determine the appropriate dose and schedule of administration of cidofovir for a VV infection of HeLa-S3 cells. Our data show that a single dose that yields an area under the concentration-time curve from 0 to 72 h (AUC_0-72) of circa 2,800 µM·h of cidofovir produces about 50% inhibition of VV infection in HeLa S3 cells. The effect produced is independent of schedule of administration with a single dose, with the correct human half-life producing the same effect as a continuous infusion of the drug when evaluated at 72 h when the AUC_0-72s of the two modes of administration matched.

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FIG. 1. HFIM system. Each hollow-fiber cartridge contains semipermeable hollow fibers which allow gases and small-molecular-weight nutrients to pass through the membranes while keeping cells and viruses outside the membranes. Uninfected and virus-infected cells are added to the cartridge through one of the sampling ports on the top of the cartridge. Medium from the reservoir is pumped through the hollow fibers to nourish the cells that grow outside of the hollow fibers. The contents of the ECS of the hollow-fiber units are sampled for cells, cell-free virus, and drug from the ports on the top of each unit, and the concentration of drug entering the hollow-fiber unit can be determined by sampling the medium as it enters the hollow-fiber unit.

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Cells and viruses. HeLa-S3 (ATCC CCL-2.2) and Vero (ATCC CCL 81) cells were obtained from the American Type Culture Collection. HeLa-S3 cells were maintained as monolayers in 75-cm² plastic tissue culture flasks in Spinner minimal essential medium supplemented with 10% fetal bovine serum, 100 units of penicillin, and 100 µg/ml of streptomycin (SMEM) at 37°C under an atmosphere of 5% CO₂. Vero cells were maintained in 75-cm² plastic tissue culture flasks in SMEM. In either case, the cells were split 1:10 once per week.

The WR strain of VV (vP1289) that expresses the human immunodeficiency virus (HIV) gag gene, leading to the production of the HIV p24 antigen upon infection of susceptible cells (20), was obtained from the NIH AIDS Research and Reference Reagent Program, and the IHD-J and IHD-W strains of VV (ATCC VR-156 and ATCC VR-1441) were obtained from the American Type Culture Collection. Virus stocks were prepared by infecting Vero cell monolayers at a multiplicity of infection of 0.01 PFU per cell. When greater than 50% of the monolayer showed cytopathic effect, the medium and cells were collected and frozen at –80°C. The amount of infectious virus in these stocks was determined by plaque assay as described by Earl et al. (19).

Drug. Cidofovir was provided in powdered form by Mick Hitchcock of Gilead Sciences, Foster City, CA. A stock solution was prepared by suspending the powder in deionized, distilled water and bringing the suspension to pH 7.4 to 8 to completely dissolve the compound. The solution was brought to a final concentration of 75 mg/ml, sterilized by filtration through a 0.2-µm filter, and stored at room temperature.

EC₅₀ determinations. To determine 50% effective concentrations (EC₅₀s) for cidofovir for the WR, IHD-J, and IHD-W strains of VV, HeLa-S3 cell monolayers in 25-cm² plastic tissue culture flasks were infected with each of these viruses at a multiplicity of infection of 0.01 PFU/cell. After a 2-h adsorption period, the inoculum was removed, the monolayers were washed one time with medium without drug, 5 ml of SMEM containing various concentrations of cidofovir was added to the flasks, and the infected cells were incubated at 37°C and 5% CO₂ for 3 to 5 days. Then, the medium was collected from each flask and frozen at –80°C for analysis of released virus by plaque assay (19).

HFIM system. To determine the pharmacodynamically linked variable for antiviral compounds effective against viruses, the HFIM system (Fig. 1), was developed by Bilello and colleagues (2). For our studies with cidofovir and VVs, we used 4300-C2011 cartridges (FiberCell Systems, Inc., Frederick, MD) containing high-molecular-weight-cutoff (the average pore size was for particles with a molecular weight cutoff of 20,000) polysulfone hollow fibers with a surface area of 2,100 cm² and a 15-ml extracapillary space (ECS), giving a surface area-to-volume ratio of 140. Before cells were placed in the hollow-fiber units, each hollow-fiber unit was washed with phosphate-buffered saline for 2 days, followed by 1 day of treatment with SMEM. Then, 10⁸ uninfected HeLa S3 cells and 10⁶ VV-infected HeLa S3 cells were suspended in 25 ml of SMEM and injected into the ECS of each of the hollow-fiber units. To demonstrate the growth of the three strains of VV in the HFIM system, the hollow-fiber units containing this mixture of uninfected and virus-infected cells were connected to central reservoirs containing SMEM and infused with medium for several days. To maintain good growth of cells, the medium was changed daily. For dose-range studies, media containing different concentrations of cidofovir were pumped through the systems as continuous infusions for several days. To maintain the proper concentrations of cidofovir, the medium in each reservoir was changed daily. For dose fractionation studies, cidofovir was infused into the central reservoirs over a 1-h period, followed by a no-drug washout over a 3-day period to simulate the correct human half-life. In each case, virus replication was monitored daily by sampling the medium in the ECS for determination of the amount of cell-free virus by plaque assay. In one case, virus yield from an experiment utilizing the recombinant WR strain of VV was determined by p24 enzyme-linked immunosorbent assay (ELISA). The actual concentration of cidofovir in the medium entering each hollow-fiber unit at each time point was determined by liquid chromatography-mass spectrometry (LC-MS) (Agilent Technologies).

Virus yield assay. To determine the amount of infectious virus produced in these experiments, Vero cells were grown in 24-well plates at 37°C with 5% CO₂ for 24 to 48 h to form confluent monolayers. The amount of cell-free virus produced in the ECS of each hollow-fiber unit or in each flask was determined by plaque assay (19).

Statistical analysis. An inhibitory sigmoid maximal-effect (E_max) model using the formula effect = control effect – [E_max x exposure^H/(exposure^H + EC₅₀^H)] was fit to the data. Control effect is the measured viral output in the absence of drug, E_max is the greatest reduction in viral output produced by drug exposure, EC₅₀ is the drug exposure producing half the E_max, and H is Hill's constant. The model was fit to the data by nonlinear regression analysis, as performed within the ADAPT II package of programs of D'Argenio and Schumitzky (12).

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EC₅₀s for cidofovir for VVs. To use the HFIM system to determine the pharmacodynamically linked variable for antiviral compounds, the EC₅₀s of the compounds for the viruses under study must be known. To that end, we determined the EC₅₀s of cidofovir for the recombinant WR strain and the IHD-J and IHD-W strains of VV grown in HeLa-S3 cells in flasks. The EC₅₀s of cidofovir for the WR, IHD-J, and IHD-W strains of VV are 30.85 ± 8.78, 18.74 ± 6.02, and 20.61 ± 4.21 µM, respectively. These values are the averages of results from three independent analyses. The EC₅₀s for these strains of VV are not significantly different, and they are similar to published cidofovir EC₅₀s for other strains of VV (14, 15, 21, 23).

Growth of VVs in HeLa S3 cells in the HFIM system. For this proof-of-principle study, it was important to demonstrate that VV will grow in HeLa-S3 cells in the HFIM system. To that end, 10⁶ HeLa-S3 cells infected with the recombinant WR strain or the IHD-W and the IHD-J strain of VV were mixed with 10⁸ uninfected HeLa-S3 cells and placed into three different hollow-fiber units. Each unit showed growth of virus in the system, as determined by sampling the ECS for cell-free virus for 4 days by plaque assay (19). The data in Fig. 2 show that all three strains of VV grew well in HeLa-S3 cells in the HFIM system. The recombinant WR strain was the slowest-growing strain, whereas the IHD-W and IHD-J strains grew more rapidly and to higher titers. The IHD-W strain caused more cytopathic effect than the IHD-J and WR strains, leading to a decline in the amount of cell-free virus produced in the hollow-fiber unit between days 3 and 4. The IHD-J strain, being less cytopathic, grew well in the HeLa-S3 cells, resulting in the highest virus yield at the end of the 4-day experiment. Although the recombinant WR strain did not grow as well as the IHD-J or the IHD-W strain of VV, we used the recombinant WR strain in these experiments because it expresses the HIV p24 antigen in virus-infected cells and the released virus is easily measured by p24 ELISA and plaque assay of cell-free virus. However, for most of these experiments, plaque assay of released virus was the standard method for determining the effect of cidofovir on virus replication in the HFIM system.

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FIG. 2. Growth of VV in the HFIM system. For growth of VV in HeLa-S3 cells, 106 VV-infected HeLa-S3 cells were mixed with 108 uninfected HeLa-S3 cells and placed in hollow-fiber units. The units were connected to reservoirs containing SMEM. The hollow-fiber units were placed in incubators at 36°C with 5% CO2, and medium was circulated through each hollow fiber for 4 days. At various times, the cells and virus produced in the ECS were sampled through the ports at the top of the cartridge. The cells were removed from the sample by centrifugation, and the amount of virus in the clarified supernatant was determined by plaque assay. The data show that all three strains of VV grow well in the HFIM system. The data are from a single experiment that is representative of several independent experiments (n = 3).

Dose-range study for cidofovir for VV in the HFIM. We first examined the effect of a range of exposures of cidofovir on the replication of VV when the drug was given as a continuous infusion in the HFIM system. A range of cidofovir concentrations from 12.5 µM to 200 µM was evaluated along with a no-treatment control for the recombinant WR strain of VV. The dose-range experiment demonstrated (Fig. 3) that cidofovir was able to suppress VV replication nearly completely at continuous infusion values greater than or equal to 50 µM. The cidofovir concentration causing 50% inhibition was approximately 38 µM.

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FIG. 3. Dose-range experiment for VV and cidofovir. To determine the dose of cidofovir that will inhibit the growth of VV in the HFIM system, six hollow-fiber units were set up as described in the legend of Fig. 2 and the stated concentrations of cidofovir were continuously infused through the different units. The ECS was sampled at the indicated times, and the effect of cidofovir on virus replication was determined by plaque assay. The results show that concentrations of cidofovir equal to or greater than 50 µM inhibited VV replication in HeLa-S3 cells. The data are the result of a single experiment.

Dose fractionation experiment. In the next experiment, we examined continuous-infusion profiles of 3 µM and 250 µM cidofovir. The 3 µM continuous infusion was chosen because it produces the same AUC₇₂ as that produced by an intravenous infusion of cidofovir by the largest dose administered to humans (10 mg/kg of body weight). The 250 µM exposure was chosen to ensure nearly complete suppression of virus replication. In addition, two hollow-fiber systems administered a 1-h infusion of cidofovir, which attained a peak concentration that declined with the human half-life so as to provide an AUC₇₂ that matched that of the systems getting the continuous-infusion profile of 3 µM or 250 µM. In addition, we had a third arm where the exposure was given once, but the AUC_0-72 matched that of a continuous infusion at 100 µM. These results are displayed in Fig. 4.

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FIG. 4. Dose fractionation experiment for VV and cidofovir. To determine the pharmacodynamically linked variable for cidofovir for VV, 108 uninfected HeLa-S3 cells and 106 HeLa-S3 cells infected with the recombinant WR strain of VV were placed into five hollow-fiber units. One unit received no cidofovir. One unit received 250 µM cidofovir as a continuous infusion. One unit received 3 µM cidofovir as a continuous infusion. One unit received 250 µM cidofovir delivered as an intermittent infusion over a 1-h period, followed by a no-drug washout. One unit received 3 µM cidofovir delivered as an intermittent infusion over 1 h, followed by a no-drug washout. One unit received 100 µM cidofovir delivered as an intermittent infusion over a 1-h period and then a no-drug washout. Each hollow-fiber unit was sampled at the indicated times, and the amount of virus produced was measured by p24 ELISA. The data show that 3 µM cidofovir delivered as an intermittent infusion or as a continuous infusion had no effect on the production of VV but that 250 µM cidofovir delivered as an intermittent infusion or as a continuous infusion inhibited virus replication, as did a bolus of 100 µM cidofovir. The data presented are the results of a single experiment.

At hour 72, when the exposures of the two profiles (bolus versus continuous infusion) match, it is clear that both endpoints demonstrate that there is no substantive difference in antiviral effect between the bolus and the continuous-infusion modes of administration. Further, neither the bolus administration nor the continuous infusion of the 10-mg/kg dose (3 µM continuous infusion) produced substantial antiviral effect. Treatment with cidofovir given as a bolus or as a continuous infusion gave essentially the same amount of virus suppression. Therefore, it can be concluded that the pharmacodynamically linked variable for cidofovir for VV is the AUC/EC₅₀ ratio.

To document that the exposures were correct, we sampled the central reservoir of the hollow-fiber systems on seven occasions for each exposure over the duration of the experiment. Samples were assayed for cidofovir by an LC-MS technique. The actual achieved concentrations over time are displayed in Fig. 5 and show that the boluses given at 3 and 250 µM cidofovir peaked at the appropriate times and were followed by a drug-free washout. Furthermore, the continuous infusions were at the correct concentrations and remained constant throughout the experiment.

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FIG. 5. Pharmacokinetic analysis of the dose fractionation experiment. The medium entering each hollow-fiber unit was sampled at the indicated times, and the amount of cidofovir was determined by LC-MS. The data show that the two continuous doses (3 µM and 250 µM) were maintained over the 72-h time course and that three bolus doses (3 µM, 100 µM, and 250 µM) peaked at the end of the 1-h infusion and then declined with the no-drug washout. These results demonstrate that each hollow-fiber unit actually received the intended dose of cidofovir. Contin Inf, continuous infusion.

Because of the large difference in effect between the 3 and 250 µM exposures, we repeated the dose fractionation experiment with continuous-infusion exposures and bolus exposures of 30 and 100 µM and employed a change in the number of PFU/ml as the endpoint. As before, there were no substantive differences in antiviral effect between the bolus and continuous-infusion modes of administration (Fig. 6), again documenting AUC as the driver for the antiviral effect.

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FIG. 6. Repeat of the dose fractionation study. Since the original dose fractionation study (Fig. 4) indicated that low doses (3 µM) given as a continuous infusion or as an intermittent administration followed by a no-drug washout had no effect on VV replication in HeLa-S3 cells, the dose fractionation study was repeated at higher doses. The results show that 30 µM drug given as a continuous infusion or as an intermittent administration followed by a no-drug washout had the same antiviral effect. Similar results occurred when 100 µM drug was given as a continuous infusion or as an intermittent administration followed by a no-drug washout. These results show that the pharmacodynamically linked variable for cidofovir is the AUC0-72/EC50 ratio. These results are from a single representative experiment.

We then employed an inhibitory sigmoid E_max model to link exposure to response. The results are displayed in Fig. 7. Analysis of all the data estimates that the 10-mg/kg-equivalent exposure (bolus administration or continuous infusion at 3 µM) generated about 8% of the E_max. Both large exposures produced a near E_max. The bolus administration equivalent to a 100 µM continuous infusion produced 81% of the E_max.

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FIG. 7. Relationship between the antiviral effect and the measured AUC0-72. The antiviral activity as measured by p24 output from the WR strain of VV at 72 h was the dependent variable, and the AUC0-72 of cidofovir was the independent variable. The AUC0-72 required to achieve 50% of the Emax was 2,804 µM·h.

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We have demonstrated that doses of 5 mg/kg of cidofovir (10 mg/kg gives a 3 µM continuous infusion; AUC₂₄, 72 µM·h) will not significantly inhibit VV replication in our HFIM system but that doses that exceed 50 µM as a continuous infusion (AUC₂₄, >1,200 µM·h) will nearly completely suppress virus replication. Since delivery of cidofovir as a continuous infusion and as an infusion followed by a no-drug washout with the appropriate half-life yield the same amount of suppression of virus replication, we can conclude that the pharmacodynamically linked variable for cidofovir for VVs is the AUC/EC₅₀ ratio.

Vaccinia virus replicates in many cell lines in vitro and in many organs in vivo. We chose the HeLa-S3 cell line to perform our in vitro hollow-fiber experiments because VV grows well in this cell line (19), the cell line grows well in the hollow-fiber units, and the cells do not attach firmly to the fibers, enabling easy sampling of virus-infected cells from the ECS. Other adherent cells bind too tightly to the fibers, making analysis of cell growth problematic. Thus, for this proof-of-principle experiment, we used HeLa-S3 cells.

It is important to place the effects of cidofovir on the suppression of VV replication into perspective. Stittelaar and colleagues (43) examined cidofovir and other anti-pox virus agents in a lethal nonhuman primate model of MPXV infection. They employed either five or six cidofovir doses of 5 mg/kg over 13 days. Lacy and colleagues demonstrated that a 2.5-mg/kg cidofovir dose in cynomolgus monkeys with probenecid produced an AUC of about 14.4 mg·h/liter of cidofovir (24). Scaled to the 5-mg/kg dose and transformed to µM·h, the AUC for a single dose is 102.8 µM·h. For the five- and six-dose regimens, the total AUCs are then 514 µM·h and 616.8 µM·h, respectively. These doses allowed one (five doses) and two (six doses) deaths. When one examines viral titers, the five-dose regimen causes little reduction in plasma viremia. We estimate from interpolation from the graph that the five-dose regimen decreased viral load by <10%. There was greater antiviral effect with the six-dose regimen (circa 30% of the E_max).

The EC₅₀ of cidofovir for MPXV and VV in Vero cells was shown by Smee and colleagues to not differ significantly from the value that we found for VV WR in our system (40). Figure 4 demonstrates that exposures to cidofovir like that seen in the Stittelaar et al. study (43) are expected to produce slightly less than 20% of the E_max, which is concordant with the outcome observed in this report. The EC₅₀ of the MPXV employed for challenge was not reported and could influence the observed outcome. Nonetheless, the in vitro findings correlate with the in vivo results. It is also important to recognize that the regimens employed still allowed deaths. Further, viral titers were only minimally affected, leading to the question of whether larger doses of cidofovir would be necessary to optimize clinical outcomes for poxvirus infections. For a 50% reduction in viral output, our results indicate that an AUC of about 2,800 µM·h would be required.

A single 5-mg/kg dose of cidofovir in humans produces an AUC_0- of about 75 to 100 µM·h (10, 11). To recapitulate the exposures seen in the Stittelaar et al. study, a dose of 25 to 30 mg/kg would be required. For better viral suppression, even more cidofovir would be required. Again, the outcome seen would be modulated by the EC₅₀ of the virus, so that while the EC₅₀s for MPXV and VV are similar (40), the EC₅₀s of cidofovir for Variola major virus have been reported to be considerably lower by some investigators (6 to 8 µM) (15), but those for MPXV and VV have be reported to be similar by others (27 µM) (23).

Doses of 25 to 30 mg/kg or greater exceed by a factor of 2 to 3 the largest dose of cidofovir ever given to humans. Consequently, considerable pharmacokinetic and safety evaluations would have to be done before the use of such doses could be countenanced. Cidofovir's major dose-limiting toxicity is nephrotoxicity. The drug must enter the renal tubular epithelial cells to cause toxicity. Human OAT1 is the major transporter of cidofovir into the target cells (8, 9). Probenecid blocks the intracellular uptake of cidofovir and provides some protection against nephrotoxicity. Obviously, probenecid will be needed in any regimen of cidofovir to be employed clinically. However, we can also gain insight into ameliorating toxicity through a prudent schedule of administration. Aminoglycoside antibiotics are tubulotoxins. Here, as with cidofovir, there is saturable uptake into tubular epithelial cells. Administering the whole dose once instead of in a fractionated fashion decreases the uptake of drug and, therefore, decreases the likelihood of nephrotoxicity. This has been demonstrated in animals (44), in humans (13), and in a randomized clinical trial comparing once-daily with twice-daily drug administration (35). We speculate that it may be prudent, therefore, to examine the administration of half the total drug dose once weekly times 2 instead of administering a smaller (5-mg/kg) dose every other day for 6 doses (i.e., 15 mg/kg twice separated by 6 or 7 days compared to 5 mg/kg every other day times 6). Our data indicate that the pharmacodynamic driver is AUC and, therefore, that there is a low likelihood of loss of therapeutic activity by intermittent dosing. Finally, it should be recognized that the current administration of cidofovir is once weekly for its licensed indication, cytomegalovirus disease.

In summary, we have been able to demonstrate that AUC is the measure of drug exposure driving antiviral effect for VV. We also demonstrate that large cidofovir exposures are required to substantially reduce viral titers for this agent. These results are concordant with the in vivo model of Stittelaar et al. (43). Larger doses of cidofovir (30 mg/kg or more) need animal model evaluation for VV infection and MPXV infection, as well as infection by Variola major virus, and an evaluation of toxicity at these higher doses.

 
This work was supported by a grant from the Charitable Leadership Foundation, Clifton Park, NY.

 
* Corresponding author. Mailing address: Ordway Research Institute, 150 New Scotland Avenue, Albany, NY 12208. Phone: (518) 641-6434. Fax: (518) 641-6304. E-mail: GDrusano{at}ordwayresearch.org

Published ahead of print on 13 October 2008.

Deceased.

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Antimicrobial Agents and Chemotherapy, January 2009, p. 129-135, Vol. 53, No. 1
0066-4804/09/$08.00+0     doi:10.1128/AAC.00708-08
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