Pharmacology Chemotherapy 2003;49:126–131 DOI: 10.1159/000070618 Received: September 11, 2002 Accepted after revision: February 14, 2003 Comparative Effects of Cidofovir and Cyclic HPMPC on Lethal Cowpox and Vaccinia Virus Respiratory Infections in Mice Donald F. Smee Kevin W. Bailey Robert W. Sidwell Department of Animal, Dairy and Veterinary Sciences, Institute for Antiviral Research, Utah State University, Logan, Utah, USA Abstract Background: Cidofovir is approved for the treatment of cytomegalovirus retinitis in humans. Although highly effective, the drug can cause renal toxicity in patients. There is much interest in cidofovir as a potential treatment for smallpox, monkeypox and other orthopoxvirus infections. A cyclic phosphonate form of cidofovir, 1-[((S)-2-hydroxy-2-oxo-1,4,2-dioxaphosphorinan-5-yl)methyl]cytosine (cyclic HPMPC), was reported to be less nephrotoxic than cidofovir in animals. Thus, it was deemed important to directly compare the activities of cidofovir and cyclic HPMPC against poxvirus infections in mouse models. Methods: The compounds were evaluated by intraperitoneal and intranasal infection routes using multiple doses of each agent, with single doses of compound given 24 h after virus challenge. Results: By intraperitoneal route, cidofovir protected mice from mortality at 40, 80 and 160 mg/kg, whereas cyclic HPMPC was similarly protective only at 160 mg/kg. By intranasal route, cidofovir was active down to 5 mg/kg, compared to cyclic HPMPC efficacy at 20 and 40 mg/kg. Intraperito- ABC © 2003 S. Karger AG, Basel 0009–3157/03/0493–0126$19.50/0 Fax + 41 61 306 12 34 E-Mail firstname.lastname@example.org www.karger.com Accessible online at: www.karger.com/che neal doses of 40, 80 and 160 mg/kg cidofovir significantly reduced mortality from vaccinia virus infections, compared to doses of 80 and 160 mg/kg cyclic HPMPC. Intranasal treatment with cidofovir at 5–40 mg/kg was comparably effective to cyclic HPMPC doses of 20 and 40 mg/kg in vaccinia virus infections. Active doses significantly reduced lung virus titers and lung consolidation. Overall, the potency of cyclic HPMPC was about 4 times less than that of cidofovir. Conclusions: Although cyclic HPMPC is reported to exhibit reduced nephrotoxicity in vivo, it is also less potent than cidofovir against orthopoxvirus infections. For this reason, cyclic HPMPC may not offer any advantage over cidofovir in treating these infections in humans. Copyright © 2003 S. Karger AG, Basel Introduction Cidofovir has been shown to be active in the treatment of lethal respiratory infections caused by cowpox and vaccinia viruses [1–3]. This drug, approved for the treatment of cytomegalovirus retinitis in humans, is also effective against many other DNA viruses. De Clercq  recently reviewed the activity of this compound for the treatment of various poxvirus infections. Orthopoxviruses such as Donald F. Smee Institute for Antiviral Research, Department of Animal, Dairy and Veterinary Sciences 5600 Old Main Hill, Utah State University Logan, UT 84322-5600 (USA) Tel. +1 435 797 2897, Fax +1 435 797 3959, E-Mail email@example.com Downloaded by: Vanderbilt University Library 188.8.131.52 - 10/26/2017 4:59:49 PM Key Words Cowpox virus W Vaccinia virus W Cidofovir W Cyclic HPMPC W Antiviral treatment Materials and Methods Viruses and Cells Cowpox virus (Brighton strain) was obtained from John Huggins, U.S. Army Medical Research Institute of Infectious Diseases (Ft. Detrick, Frederick, Md., USA). The virus originated from the Centers for Disease Control and Prevention (Atlanta, Ga., USA). Vaccinia virus (WR strain) was purchased from the American Type Culture Collection (ATCC; Manassas, Va., USA). The viruses were propagated in African green monkey kidney (MA-104) cells (BioWhittaker, Walkersville, Md., USA). Plaque assays of the viruses were done in African green monkey kidney (Vero) cells (from ATCC). The MA104 cells were cultured in Eagle’s medium (MEM) containing 9% fetal bovine serum (FBS), whereas the Vero cells were grown in Medium 199 with 5% FBS. MEM with 2% FBS and gentamicin (50 Ìg/ml) was used for viral propagation and plaque assays. Mouse Infection Studies Female BALB/c mice (13–15 g) were purchased from B & K Universal (Fremont, Calif., USA) for the studies. The animals were quarantined 48 h before use. An infectious vaccinia virus challenge of 5 ! 105 plaque-forming units (about 10 50% lethal doses) per mouse was used for the experiments. This was based upon previously conducted lethality titrations with the viruses in mice. Virus was administered i.n. in a 50-Ìl volume following anesthesia with ketamine (100 mg/kg given by i.p. injection). A single i.p. treatment with cidofovir, cyclic HPMPC or placebo was given 24 h after virus exposure. This regimen using cidofovir was shown to be effective against cowpox  and vaccinia (WR strain) [2, 3] virus respiratory infections. Alternatively, single i.n. treatments, proven to be effective using cidofovir against cowpox virus , were also given in separate experiments. Animals were individually weighed every 2–3 days and deaths were recorded for 21 days. There were 10 mice per group held for death determinations and 5 mice per group sacrificed per day for virus titer determinations. Uninfected toxicity control animals were not used in these experiments since single doses have been reported as nontoxic [1–3, 23]. Lungs from sacrificed cowpox virus-infected mice were removed and frozen at –80 ° C on day 6 of the infection for virus titer determinations. Lung infection parameters for the vaccinia virus experiments were determined in a manner similar to those reported for influenza virus  in groups of infected mice. On day 5 of the vaccinia virus infection (mice died more rapidly with this infection than with the cowpox virus infection, thus the difference in day of sacrifice), lungs from sacrificed mice were collected, given a severity score based upon lung discoloration ranging from 0 (normal) to 4 (100% of lung area exhibiting a plum coloration), weighed and frozen for later virus titration. With the cowpox virus infection, lungs did not become enlarged or discolored as they did with the vaccinia virus infection, and thus were not scored. Virus titers from lung samples were determined by plaque assay in Vero cells as described previously . Antiviral Compounds Norbert Bischofberger and Mick Hitchcock of Gilead Sciences (Foster City, Calif., USA) kindly provided cidofovir and cyclic HPMPC, respectively. The compounds were dissolved in sterile saline at a maximum concentration of 22.4 mg/ml for intraperitoneal (i.p.) injection or at a maximum concentration of 11.2 mg/ml for intranasal (i.n.) instillation into 14-gram mice. Treatment volumes were 0.1 or 0.05 mg/mouse for these respective treatments. Sterile saline served as the placebo control. Statistical Methods Statistical comparisons were made between the drug-treated groups and the placebo controls by two-tailed analyses. The Fisher exact test was used to interpret differences in numbers of survivors. Mean day of death, mean lung virus titers, mean lung scores and mean lung weight comparisons were statistically analyzed by the Mann-Whitney U test. Calculations were made using the InStat computer program (GraphPad Software, San Diego, Calif., USA). Effects of Cidofovir and Cyclic HPMPC Chemotherapy 2003;49:126–131 127 Downloaded by: Vanderbilt University Library 184.108.40.206 - 10/26/2017 4:59:49 PM smallpox and monkeypox have become a concern due to their bioterrorism potential [5–8]. Treatments for progressive vaccinia , molluscum contagiosum [10–13] and orf virus infections  are also being sought. Although cidofovir is a very effective drug against cytomegalovirus infections in humans, it is not orally active and may cause renal toxicity [15, 16]. Derivatives of cidofovir are being investigated that may be safer and orally effective. One such compound, 1-[((S)-2-hydroxy-2oxo-1,4,2-dioxaphosphorinan-5-yl)methyl]cytosine (cyclic HPMPC), is a cyclic phosphonate derivative of cidofovir that has been reported to have reduced nephrotoxicity in rodents [17–19] and in humans . Certain salicylate ester prodrugs of cyclic HPMPC have been synthesized that exhibit up to 46% oral bioavailability . Thus, prodrug forms of cyclic HPMPC hold the promise of exhibiting both oral bioavailability and reduced nephrotoxicity relative to the parent drug cidofovir. Such a compound may be useful in treating orthopoxvirus infections. Recently, Bray et al.  reported on the activities of cidofovir and cyclic HPMPC by aerosol route to treat cowpox virus infections in mice. Although it is difficult to accurately quantify drug uptake by this treatment method (the dosages were reported as ranges rather than as discrete mg/kg doses), the authors concluded that cyclic HPMPC was less potent than cidofovir in the treatment of the infections. In the present studies, we evaluated the activities of the two compounds under more stringent treatment regimens in order to determine differences in potency. This would help determine if increases in the safety of cyclic HPMPC (due to decreased renal toxicity) would be negated by decreases in potency against infection (i.e. more drug would be needed to counteract the infection). The results of the present studies indicate that cyclic HPMPC is indeed less potent than cidofovir against infections caused by cowpox and vaccinia viruses in mice. Table 1. Effects of i.p. treatment with cidofovir and cyclic HPMPC on a cowpox virus respiratory infection in mice Compound Dose mg/kg Survivors/ total Mean day of death B SD Mean lung virus titer B SD Cidofovir 160 80 40 20 160 80 40 20 – 10/10*** 10/10*** 10/10*** 0/10 8/10*** 4/10 3/10 0/10 0/10 – – – 14.1B3.6*** 10.0B1.4 16.7B2.7*** 12.7B3.0*** 10.4B1.7* 8.7B1.2 7.7B0.3** 7.8B0.1** 8.1B0.1** 7.8B0.1** 7.9B0.3* 8.0B0.3* 8.2B0.2 8.2B0.3 8.5B0.2 Cyclic HPMPC Placebo Treatment was given as a single injection 24 h after virus exposure. The mean day of death was recorded in those mice that died prior to day 21. Mean lung virus titer was determined as log10 plaque-forming units/g (5 mice/group) on day 6 of the infection. * p ! 0.05; ** p ! 0.01; *** p ! 0.001. Table 2. Effects of i.n. treatment with cidofovir and cyclic HPMPC on a cowpox virus respiratory infection in mice Compound Dose mg/kg Survivors/ total Mean day of death B SD Mean lung virus titer B SD Cidofovir 40 20 10 5 40 20 10 5 – 8/10*** 8/10*** 10/10*** 8/10*** 9/10*** 8/10*** 0/10 2/10 0/10 17.5B3.5* 9.5B0.7 – 9.0B0.0 10.0B0.0 10.0B0.0 12.1B2.7* 11.0B2.2* 8.5B0.5 5.7B0.7*** 7.1B0.6** 6.9B0.4** 7.5B0.2** 6.8B0.6** 7.6B0.4** 7.5B0.1** 8.0B0.5 8.5B0.2 Cyclic HPMPC Placebo Treatment was given as a single dose 24 h after virus exposure. The mean day of death was recorded in those mice that died prior to day 21. Mean lung virus titer was determined as log10 plaque-forming units/g (5 mice/group) on day 6 of the infection. * p ! 0.05; ** p ! 0.01; *** p ! 0.001. Effect of i.p. Treatment on Cowpox Virus Infection Intraperitoneal treatment with cidofovir at 40, 80 and 160 mg/kg prevented mortality by 100% and significantly reduced cowpox virus titers in lungs on day 6 of the infection (table 1). At the 20 mg/kg dose, all of the mice died, but the time to death was delayed over 5 days relative to deaths in the placebo group. Cyclic HPMPC at 160 mg/kg protected 80% of mice from death. At 80 mg/kg, there were 40% survivors, which was not quite significantly different from the placebo group. At 160 and 80 mg/kg, virus 128 Chemotherapy 2003;49:126–131 titers were reduced compared to the placebo group. Mice dying the 20, 40 and 80 mg/kg cyclic HPMPC groups lived significantly longer than placebo controls. Cidofovir was approximately 4 times more potent than cyclic HPMPC against the infection. Effect of i.n. Treatment on Cowpox Virus Infection It has been previously shown that i.n. cidofovir treatment of cowpox virus infections requires much less compound for efficacy than by i.p. administration . Also, the suppression of lung virus titers in mice treated i.n. was greater than in i.p. treated mice. In the present study, i.n. Smee/Bailey/Sidwell Downloaded by: Vanderbilt University Library 220.127.116.11 - 10/26/2017 4:59:49 PM Results Table 3. Effects of i.p. treatment with cidofovir and cyclic HPMPC on a vaccinia virus respiratory infection in mice Compound Cidofovir Cyclic HPMPC Dose mg/kg Survivors/ total Mean day of death B SD 160 80 40 20 160 80 40 20 0 7/10** 7/10** 7/10** 3/10 6/10* 6/10* 2/10 0/10 0/10 3.3B2.3 6.7B0.6 6.0B0.0 9.4B2.8** 6.5B0.6 8.8B2.1* 7.1B2.0 7.6B1.8 6.4B0.8 Mean lung parameters B SD score weight, mg virus titer, log10 PFU/g 0.3B0.4* 0.3B0.3** 0.6B0.7* 1.3B0.6 0.8B0.8 1.1B0.8 0.9B0.5 2.5B0.4 2.1B0.9 208B90 190B37* 188B79 218B61 214B104 226B68 174B76* 266B34 292B49 7.8B1.3* 8.6B0.2 8.3B1.4 8.5B1.1 8.2B0.3* 8.5B0.2 8.5B0.2 8.8B0.1 8.8B0.2 Treatment was given as a single injection 24 h after virus exposure. The mean day of death was recorded in those mice that died prior to day 21. Mean lung parameters were determined from 5 mice per group on day 5 of the infection. Lung consolidation scores ranged from 0 (normal) to 4 (entire lung discolored). PFU = Plaque-forming units. * p ! 0.05; ** p ! 0.01. Effect of i.p. Treatment on Vaccinia Virus Infection Intraperitoneal treatment with cidofovir administered at 40, 80 and 160 mg/kg prevented mortality by 70% and significantly reduced vaccinia virus titers in lungs at the 160 mg/kg dose on day 6 of the infection (table 3). Lung consolidation scores were significantly reduced at the top three doses. Reductions in lung weights were evident compared to the placebo group, but lung weights were only significantly different at the 80 mg/kg dose. Lung weights were quite variable from animal to animal, which impacted on statistical significance. Cyclic HPMPC prevented mortality by 60% at 80 and 160 mg/kg. In these groups of mice, lung virus titers were not significantly different from those in placebo controls. Lung consolidation scores differed from those in placebos at 40, 80 and 160 mg/kg, but the results were not statistically signifi- Effects of Cidofovir and Cyclic HPMPC cant. At 40 mg/kg, the lung weights differed significantly from placebo animals. Cyclic HPMPC was less protective to the lungs than was cidofovir, based upon lung scores. In this experiment and based upon mortality results, cidofovir was approximately 2 times more potent than cyclic HPMPC against the infection. Effect of i.n. Treatment on Vaccinia Virus Infection In this experiment, i.n. drug doses of 5, 10, 20 and 40 mg/kg prevented mortality in 70–80% of the animals (table 4). At the 20 and 40 mg/kg doses, mice dying from the infection lived several days longer than those dying in the placebo group. Virus titers were reduced in lungs of mice treated with all of these doses compared to virus in lungs of placebo-treated mice. Treatment with cidofovir at 20 and 40 mg/kg significantly reduced lung scores and lung weights. Cyclic HPMPC was effective in preventing death by 50 and 70% at doses of 20 and 40 mg/kg, respectively. Doses of 5 and 10 mg/kg did not prevent mortality, however. Virus titers were reduced by treatments of 5– 40 mg/kg. The highest two doses of cyclic HPMPC were not as effective as the highest doses of cidofovir in reducing lung virus titers, lung consolidation scores and lung weights. Cidofovir was approximately 4 times more potent than cyclic HPMPC against the infection. Chemotherapy 2003;49:126–131 129 Downloaded by: Vanderbilt University Library 18.104.22.168 - 10/26/2017 4:59:49 PM drug doses of 5, 10, 20 and 40 mg/kg prevented mortality in 80–100% of the animals (table 2). Greater virus titer reductions were seen in lungs compared to lungs of mice treated i.p. (table 1). Cyclic HPMPC was effective at 20 and 40 mg/kg in preventing mortality and reducing lung virus titers. Reductions in lung virus titers with cyclic HPMPC at 20 and 40 mg/kg were similar to those achieved by HPMPC at 5 and 10 mg/kg. In this experiment, cidofovir was approximately 4 times more potent than cyclic HPMPC against the infection. Table 4. Effects of i.n. treatment with cidofovir and cyclic HPMPC on a vaccinia virus respiratory infection in mice Compound Cidofovir Cyclic HPMPC Dose mg/kg Survivors/ total Mean day of death B SD 40 20 10 5 40 20 10 5 0 8/10*** 7/10** 8/10*** 7/10** 7/10** 5/10* 0/10 0/10 0/10 12.0B0.0*** 8.7B1.2** 8.5B2.1 8.3B1.5 7.7B1.5 10.8B4.6 8.8B1.5 7.8B0.6 6.9B0.3 Mean lung parameters B SD score weight, mg virus titer, log10 PFU/g 0.5B0.0*** 0.5B0.0*** 3.3B0.3 3.0B0.4 3.0B0.1* 2.4B0.9* 2.1B1.0* 3.3B0.4 3.5B0.4 154B9*** 180B35*** 288B76 252B85* 230B54*** 220B41*** 238B36*** 306B130 384B38 6.4B0.6*** 7.9B0.3*** 8.6B0.2* 8.4B0.3** 8.1B0.2*** 8.3B0.2*** 8.1B0.3*** 8.4B0.3** 8.9B0.1 Treatment was given as a single dose 24 h after virus exposure. The mean day of death was recorded in those mice that died prior to day 21. Mean lung parameters were determined from 5 mice per group on day 5 of the infection. Lung consolidation scores ranged from 0 (normal) to 4 (entire lung discolored). PFU = Plaque-forming units. * p ! 0.05; ** p ! 0.01; *** p ! 0.001. Direct efficacy comparisons of cidofovir and cyclic HPMPC were made against cowpox and vaccinia viruses using two treatment regimens previously found to be effective against cowpox and vaccinia virus infections in mice [1–3, 23]. Cyclic HPMPC demonstrated decreased potency relative to HPMPC by 4-fold in three of the studies and 2-fold in one study. Bray et al.  presented data indicating that cidofovir was more potent than cyclic HPMPC against cowpox virus infections by aerosol administration. In those studies, it was difficult to precisely quantify the drug dose because of the route of administration. Thus, more precise comparisons of the effects of the two compounds were made herein. The responses of mice to antiviral treatment of the cowpox and vaccinia virus infections were similar, although vaccinia infections were harder to treat due to more rapid occurrence of death, more severe lung consolidation and higher lung virus titers than occurred in the cowpox virus infections. Thus, the overall numbers of survivors of the cowpox virus infections treated with cidofovir were greater than those with the vaccinia virus infections. Intranasal treatment of the cowpox virus infection with cidofovir reduced lung virus titers to a greater extent than similar treatments of the vaccinia virus infection. Doses of either compound that caused a positive effect 130 Chemotherapy 2003;49:126–131 against cowpox virus were also effective against vaccinia virus. Against both infections, i.n. treatments were more effective than i.p. treatments in reducing the severity of the various infection parameters. This is understandable, since i.n. treatment deposited the drug directly at the site of the infection. In cell culture studies, cidofovir was reported to be 3.8 times more potent than cyclic HPMPC against cowpox virus and 2.6 times more potent against vaccinia virus (Copenhagen strain) in mouse 3T3 cells . These differences may explain the potency differences seen in vivo. For this to be true, mouse lung cells must behave similarly to 3T3 cells, and the WR strain of vaccinia virus must act similarly to the Copenhagen strain in terms of responses to cyclic HPMPC treatment. In a pharmacokinetic study of cyclic HPMPC performed in humans, the cidofovir excreted in urine in 24 h was only 9.4% of the total cyclic HPMPC dose, indicating low conversion to the active antiviral form . This could contribute negatively to the compound’s antiviral efficacy in man. The authors of that report indicated that cyclic HPMPC had a lower potential for nephrotoxicity in humans compared to cidofovir. Previous investigations showed that cidofovir accumulated in kidneys of rats at a level 20 times higher than that of cyclic HPMPC, whereas the level of radioactive compound in other tissues was similar for both drugs . Cidofovir was reported to be at least 13 times more toxic Smee/Bailey/Sidwell Downloaded by: Vanderbilt University Library 22.214.171.124 - 10/26/2017 4:59:49 PM Discussion to mice than cyclic HPMPC . Taking into account the reduced efficacy of cyclic HPMPC relative to cidofovir against poxviruses with the greater safety profile in mice, cyclic HPMPC may only have a safety margin about 3 times better than cidofovir. The results of human pharmacokinetic data of the conversion rate of cyclic HPMPC to cidofovir suggest that cyclic HPMPC may prove to be no better than the parent drug. 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