Synthesis and antiviral evaluation of alkoxyalkyl derivatives of 9-(S)-(3-hydroxy-2-phosphonomethoxypropyl)adenine against cytomegalovirus and orthopoxviruses

9-(S)-(3-Hydroxy-2-phosphonomethoxypropyl)adenine [(S)-HPMPA] was one of the first acyclic nucleoside phosphonates described and has been reported to have good antiviral activity against most double-stranded DNA viruses, including the herpes group viruses and the orthopoxviruses. However, (S)-HPMPA is not orally bioavailable and has not been developed for clinical use. We have prepared orally bioavailable lipid esters of (S)-HPMPA and report their synthesis and antiviral evaluation against cytomegalovirus and orthopoxviruses. These esters were evaluated in vitro in cells infected with human cytomegalovirus (HCMV), murine cytomegalovirus (MCMV), vaccinia (VV), and cowpox viruses (CV). The most active compound, oleyloxyethyl-(S)-HPMPA, was found to have EC50 value of 0.003 microM against HCMV vs 1.4 microM for unmodified HPMPA. In cells infected with VV and CV, octadecyloxyethyl-(S)-HPMPA had EC50 values of 0.01-0.02 microM versus 2.7-4.0 microM for unmodified HPMPA. When compared with the alkoxyalkyl esters of cidofovir, the corresponding alkoxyalkyl esters of (S)-HPMPA were equally active against HCMV and MCMV but were 15-20-fold more active against VV and CV in vitro. The alkoxyalkyl esters of (S)-HPMPA are promising new compounds worthy of further investigation for treatment of infections caused by herpes viruses and orthopoxviruses.     

Synthesis and antiviral evaluation of alkoxyalkyl-phosphate conjugates of cidofovir and adefovir

Esterification of cidofovir (CDV), an antiviral nucleoside phosphonate, with alkyl or alkoxyalkyl groups increases antiviral activity by enhancing cell uptake and conversion to CDV diphosphate. Hexadecyloxypropyl-CDV (HDP-CDV) has been shown to be 40-100 times more active than CDV in vitro in cells infected with herpes group viruses, variola, cowpox, vaccinia or ectromelia viruses. Since the first phosphorylation of CDV may be rate limiting, we synthesized the hexadecyloxypropyl-phosphate (HDP-P-) and octadecyloxyethyl-phosphate (ODE-P-) conjugates of CDV and phosphonomethoxy-ethyl-adenine (PMEA, adefovir). We tested the CDV analogs in cells infected with human cytomegalovirus, herpes simplex virus, cowpox virus and vaccinia virus; the analogs of PMEA were tested in cells infected with the human immunodeficiency virus, type 1. In general, the alkoxyalkyl-phosphate conjugates of CDV were substantially more active than CDV. HDP-P-CDV and ODE-P-CDV were 4.6-40 times more active against HCMV and 7-30 times more active against cowpox and vaccinia in vitro. Although the compounds of this type were more cytotoxic than the unmodified bases, their selectivity for virally infected cells was generally greater than the parent nucleotides except that HDP-P-PMEA showed little or no selectivity in HIV-1 infected MT-2 cells. Although the new compounds with an interposed phosphate were generally less active than the corresponding alkoxyalkyl esters of CDV and PMEA, the present approach provides a possible alternative method for enhancing the antiviral activity of drugs of this class.     

Increased antiviral activity of 1-O-hexadecyloxypropyl-[2-(14)C]cidofovir in MRC-5 human lung fibroblasts is explained by unique cellular uptake and metabolism

Recently, there has been renewed interest in finding orally active drugs against smallpox. Cidofovir (CDV) given by parenteral injection has been shown to protect against lethal poxvirus infection. We have been interested in the synthesis and evaluation of orally active derivatives of CDV. Previous studies showed that the CDV and cyclic cidofovir (cCDV) analogs 1-O-hexa-decyloxypropyl-CDV (HDP-CDV) and 1-O-hexadecyloxypropyl-cCDV (HDP-cCDV), show >100-fold increases in antiviral activity versus the unmodified nucleosides against cells infected with orthopoxviruses, cowpox, and vaccinia virus. In contrast to CDV, HDP-CDV is orally bioavailable and has been reported to be orally active in lethal cowpox virus infection in mice. To assess the metabolic basis for the increased antiviral activity of HDP-CDV in vitro, we studied the cellular uptake and anabolic metabolism of (14)C-labeled CDV, cCDV, and their alkoxyalkanol esters HDP-CDV and HDP-cCDV. HDP-CDV and HDP-cCDV were taken up rapidly by MRC-5 human lung fibroblasts in vitro, but uptake of CDV and cCDV was much slower. Analysis of cellular metabolites showed that levels of cidofovir diphosphate (CDV-DP), the active antiviral compound, were >100 times greater with HDP-CDV than levels observed with CDV. When cells were exposed to HDP-CDV, the intracellular half-life of CDV-DP was 10 days versus 2.7 days reported when cells are exposed to CDV. HDP-CDV seems to circumvent poor cellular uptake by rapid association with cellular membrane phospholipids, whereas CDV uptake proceeds via the slow process of fluid endocytosis.     

Antiviral evaluation of octadecyloxyethyl esters of (S)-3-hydroxy-2-(phosphonomethoxy)propyl nucleosides against herpesviruses and orthopoxviruses

Our previous studies showed that esterification of 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine (HPMPA) or 1-(S)-[3-hydroxy-2-(phosphonomethoxy)-propyl]cytosine (HPMPC) with alkoxyalkyl groups such as hexadecyloxypropyl (HDP) or octadecyloxyethyl (ODE) resulted in large increases in antiviral activity and oral bioavailability. The HDP and ODE esters of HPMPA were shown to be active in cells infected with human immunodeficiency virus, type 1 (HIV-1), while HPMPA itself was virtually inactive. To explore this approach in greater detail, we synthesized four new compounds in this series, the ODE esters of 9-(S)-[3-hydroxy-2-(phosphonomethoxy)-propyl]guanine (HPMPG), 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]thymine (HPMPT), 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-2,6-diaminopurine (HPMPDAP) and 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-2-amino-6-cyclopropylaminopurine (HPMP-cPrDAP) and evaluated their antiviral activity against herpes simplex virus, type 1 (HSV-1), human cytomegalovirus (HCMV), and vaccinia, cowpox and ectromelia. Against HSV-1, subnanomolar EC₅₀ values were observed with ODE–HPMPA and ODE–HPMPC while ODE–HPMPG had intermediate antiviral activity with an EC₅₀ of 40 nM. In HFF cells infected with HCMV, the lowest EC₅₀ values were observed with ODE–HPMPC, 0.9 nM. ODE–HPMPA was highly active with an EC₅₀ of 3 nM, while ODE–HPMPG and ODE–HPMPDAP were also highly active with EC₅₀s of 22 and 77 nM, respectively. Against vaccinia and cowpox viruses, ODE–HPMPG and ODE–HPMPDAP were the most active and selective compounds with EC₅₀ values of 20–60 nM and selectivity index values of 600–3500. ODE–HPMPG was also active against ectromelia virus with an EC₅₀ value of 410 nM and a selectivity index value of 166. ODE–HPMPG and ODE–HPMPDAP are proposed for further preclinical evaluation as possible candidates for treatment of HSV, HCMV or orthopoxvirus diseases.     

新型非环核苷膦酸L-氨基酸酯类化合物的设计、合成与抗乙型肝炎病毒活性

设计合成具有抗乙型肝炎病毒活性的非环核苷膦酸双L-氨基酸酯系列衍生物。以adefovir dipivoxil为先导化合物， 根据核苷L-氨基酸酯前药提高生物利用度及抗病毒活性的研究结果对先导化合物进行结构优化， 设计并合成一系列新型非环核苷膦酸双L-氨基酸类化合物， 确定其结构， 并通过测定其对HepG2 2.2.15细胞分泌的HBV-DNA的抑制作用评价其体外抗病毒活性。结果发现8个adefovir双L-氨基酸类化合物可显示不同程度活性， 其中化合物11的抗病毒活性最强、 选择性指数最高(EC₅₀ 0.095 2 μmol·L^-1, SI 69523)。以上研究提示L-氨基酸酯策略可适用于非环核苷膦酸的前药修饰， 以期发现有效抗HBV药物。     

In vitro activity of cycloSal-nucleoside monophosphates and polyhydroxycarboxylates against orthopoxviruses

Because variola virus might be used as a pathogen in biological attacks, there is an urgent need to provide effective antiviral drugs for the treatment of orthopoxvirus infections. Thus, the aim of the present study was to test the antiviral activity of 3 pro-nucleotides of the acyclic nucleoside analogues aciclovir (ACV), 3 of penciclovir (PCV) and 38 of the cyclic nucleoside analogue brivudin (BVDU), on the basis of cycloSaligenyl-nucleoside monophosphate approach against vaccinia virus and cowpox virus in vitro. In further experiments, 13 synthetic humic acid-like polymers, so-called polyhydroxycarboxylates, were examined. Antiviral screening was performed by means of the plaque reduction assay and for quantification of the cytotoxicity of the test compounds the XTT-based tetrazolium reduction assay EZ4U was used. As result, three cycloSal-monophosphate derivatives of ACV proved to be potent inhibitors of both vaccinia virus and cowpox virus replication in vitro. Among the tested monophosphate derivatives of cycloSal-PCV and cycloSal-BVDU, selected substances showed a promising antiviral activity against vaccinia virus and cowpox virus. For the polyanionic compounds, no relevant antiviral activity was detected. In conclusion, by the delivery of nucleoside monophosphates from neutral, membrane-permeable prodrugs on the basis of the cycloSaligenyl-nucleotide concept, different ACV, PCV and BVDU derivatives can act as potent and selective inhibitors of orthopoxvirus replication. However, most of the cycloSal-monophosphate derivatives of BVDU had a higher cytotoxicity than their parent nucleosides.     

Treatment of lethal cowpox virus respiratory infections in mice with 2-amino-7-[(1,3-dihydroxy-2-propoxy)methyl]purine and its orally active diacetate ester prodrug

The acyclic purine nucleoside analog, 2-amino-7-[(1,3-dihydroxy-2-propoxy)methyl]purine (S2242) and its orally active diacetate ester prodrug (HOE961) were reported to be potent inhibitors of vaccinia virus replication in cell culture and in infected mice. These compounds were evaluated further, using infections with the related cowpox virus. Against a wild-type (WT) cowpox virus strain in mouse C127I cell culture, 50% effective concentrations (EC(50), determined by plaque reduction assays) of S2242 and cidofovir (a positive control) were 3.5 and 1.0 microM, respectively. EC(50) values obtained against a cidofovir-resistant strain of the virus were 33 and 230 microM, respectively. Compounds were at least ten-fold less potent against WT virus in Vero cells than C127I cells. S2242 and cidofovir were 50% inhibitory to the proliferation of uninfected C127I cells at 340 and 180 microM, respectively, but neither compound inhibited Vero cell growth at 1000 microM. Mice were lethally infected with cowpox virus by intranasal inoculation, followed 24 h later by antiviral treatment for 5 consecutive days. Once or twice daily intraperitoneal (i.p.) treatments with either S2242 or HOE961 at 100 mg/kg per day resulted in > or = 70 survival compared with no survivors in the placebo group. Lower doses of these compounds (10 and 30 mg/kg per day) were not protective, however. Cidofovir was 100% protective at 30 mg/kg per day. A 10-day course of treatment gave comparable survival results and demonstrated the oral efficacy of HOE961. Treatments with S2242 (100 mg/kg per day) and cidofovir (30 mg/kg per day) each reduced lung and nasal virus titers by approximately ten-fold, whereas, HOE961 (100 mg/kg per day) was less active. Overall, S2242 and HOE961 were found to be effective against cowpox virus infections in mice but were less potent than cidofovir. Since, HOE961 was orally active, it may have advantages over the other parenterally administered compounds for treating orthopoxvirus infections.     

Development in lipid drugs

Lipopeptide lipid moieties induce dendritic cell (DC) internalization and epitopes are recognized by MHC, the major histocompatibility complex. HIV-1 (human immunodeficiency virus type 1) lipopeptide vaccine candidate elicits immune responses, and sustains HIV control after highly active antiretroviral therapy (HAART). Mp- and Dp-MART (anti-melanoma lipopeptides) induce strong CTL (cytolytic T lymphocyte) response. New BGTC, BGDA, TGKC lipoplexes mediate gene delivery, e.g., into mouse pancreatic tumor nodules. Triterpene glycyrrhizic acid (GL) inhibits SARS-CoV (severe acute respiratory syndrome associated coronavirus) replication. Compared to CDV (cidofovir), CDV ether lipid esters have enhanced activity against vaccinia (VV) and cowpox (CV) viruses in vitro. Oral treatment of VV and CV infected mice with CDV ether lipid esters, as effective as i.p. CDV, may be useful against orthopoxvirus infections in humans.     

The clinical potential of the acyclic (and cyclic) nucleoside phosphonates: the magic of the phosphonate bond

The use of the acyclic nucleoside phosphonates, starting with (S)-HPMPA as the prototype, yielded three clinically approved antiviral drugs, cidofovir for the treatment of CMV retinitis in AIDS patients, adefovir dipivoxil for the treatment of chronic hepatitis B and tenofovir disoproxil fumarate for the treatment of HIV infections (AIDS) and HBV infections. This era has now grown to many more acyclic (and cyclic) nucleoside phosphonates (such as the “open ring” DAPy and Fd4A phosphonates) and alkoxyalkyl and phosphonoamidate prodrugs thereof, as well as new clinical applications, including new drug combination regimens for the treatment of AIDS, the chemoprophylaxis of HIV infections, and the anticancer potential against some malignant disorders.     

Acyclic nucleoside phosphonates: a key class of antiviral drugs

Almost 20 years after the broad antiviral activity spectrum of the first acyclic nucleoside phosphonates was described, several of these compounds have become important therapies for DNA virus and retrovirus infections. Here, we review the discovery and development of acyclic nucleoside phosphonates, focusing on cidofovir and its potential in the treatment of various herpes-, papilloma-, polyoma-, adeno- and pox-virus infections, adefovir for the treatment of hepatitis B and tenofovir for the treatment of AIDS and the prevention of HIV infections.     

Cidofovir in the therapy and short-term prophylaxis of poxvirus infections

Although it is often stated that only vaccination would be able to contain or protect the population against a catastrophic smallpox outbreak, the acyclic nucleoside phosphonate analog cidofovir offers a valuable alternative for the therapy and short-term pre- and post-exposure prophylaxis, not only of smallpox but also of other poxvirus infections and DNA viruses. Cidofovir has proven effective against vaccinia, cowpox and monkeypox in various animal model infections. In cell culture, cidofovir has demonstrated activity against variola virus, the etiological agent of smallpox, and in patients it has shown marked efficacy against molluscum contagiosum and orf, two poxvirus infections. Cidofovir is available as an aqueous solution for intravenous administration and could be reformulated for topical (cream or gel), intranasal (aerosol) or peroral (as a lipid prodrug) use, should the need arise.     

Novel antiviral C5-substituted pyrimidine acyclic nucleoside phosphonates selected as human thymidylate kinase substrates

Acyclic nucleoside phosphonates (ANPs) are at the cornerstone of DNA virus and retrovirus therapies. They reach their target, the viral DNA polymerase, after two phosphorylation steps catalyzed by cellular kinases. New pyrimidine ANPs have been synthesized with unsaturated acyclic side chains (prop-2-enyl-, but-2-enyl-, pent-2-enyl-) and different substituents at the C5 position of the uracil nucleobase. Several derivatives in the but-2-enyl- series 9d and 9e, with (E) but not with (Z) configuration, were efficient substrates for human thymidine monophosphate (TMP) kinase, but not for uridine monophosphate-cytosine monophosphate (UMP-CMP) kinase, which is in contrast to cidofovir. Human TMP kinase was successfully crystallized in a complex with phosphorylated (E)-thymidine-but-2-enyl phosphonate 9e and ADP. The bis-pivaloyloxymethyl (POM) esters of (E)-9d and (E)-9e were synthesized and shown to exert activity against herpes virus in vitro (IC(50) = 3 μM) and against varicella zoster virus in vitro (IC(50) = 0.19 μM), in contrast to the corresponding inactive (Z) derivatives. Thus, their antiviral activity correlates with their ability to act as thymidylate kinase substrates.     

Esterification of cidofovir with alkoxyalkanols increases oral bioavailability and diminishes drug accumulation in kidney

Smallpox was eradicated by vaccination in the 1970s. However, concerns have arisen about the potential use of variola virus as a biological weapon. Most of the world's population has little residual immunity because systematic vaccination against smallpox ceased in the early 1970s. Vaccination of key elements of the population against smallpox is again being considered. However, there are now large numbers of persons who cannot be safely vaccinated with the current vaccine because of AIDS, immunosuppressive drugs, and certain common skin disorders. It would be useful to have a potent orally active drug as an alternative for these persons in case of an outbreak of smallpox. Alkoxyalkyl esters of cidofovir (CDV) have been shown to be highly active and selective against poxviruses in vitro with activities several logs greater than the activity of unmodified CDV. This is due in large part to increased cellular penetration and conversion to CDV-diphosphate, the active antiviral. In this paper, the oral pharmacokinetics of 14C-labeled hexadecyloxypropyl-cidofir (HDP-CDV), octadecyloxyethyl-cidofir (ODP-CDV), and oleyloxypropyl-cidofir (OLP-CDV) are examined and oral bioavailability and tissue distribution assessed and compared with parenteral CDV. The alkoxyalkyl CDVs are highly orally bioavailable and do not concentrate in kidney, the site of the dose-limiting toxicity of CDV. Plasma and tissue drug levels are many times greater than the in vitro EC(50s) for variola, cowpox, and vaccinia viruses. Thus, the compounds are good candidates for further development for prevention and treatment of smallpox infection and the complications of vaccination.     

Cidofovir in the treatment of poxvirus infections

Cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine, HPMPC] has since 1996 been licensed for clinical use in the treatment of cytomegalovirus (CMV) retinitis in AIDS patients. Cidofovir has broad-spectrum activity against virtually all DNA viruses, including herpes-, adeno-, polyoma-, papilloma- and poxviruses. Among the poxviruses, vaccinia, variola (smallpox), cowpox, monkeypox, camelpox, molluscum contagiosum and orf have proven sensitive to the inhibitory effects of cidofovir. In vivo, cidofovir has shown high efficacy, even after administration of a single systemic (intraperitoneal) or intranasal (aerosolized) dose, in protecting mice from a lethal respiratory infection with either vaccinia or cowpox. Cidofovir has also demonstrated high effectiveness in the treatment of vaccinia virus infection in severe combined immune deficiency mice. In humans, cidofovir has been used successfully in the treatment, by both the topical and intravenous route, of recalcitrant molluscum contagiosum and orf in immunocompromised patients. Taken together, these data indicate that cidofovir should be effective in the therapy and short-term prophylaxis of smallpox and related poxvirus infections in humans, as well as the treatment of the complications of vaccinia that may arise in immunocompromised patients inadvertently inoculated with the smallpox vaccine (vaccinia).     

Therapy and short-term prophylaxis of poxvirus infections: historical background and perspectives

The era of antiviral chemotherapy started more than 50 years with the findings by Domagk and his colleagues that thiosemicarbazones showed activity against vaccinia virus. One of the derivatives, methisazone, was even investigated in the prophylaxis of smallpox. With the successful implementation of the smallpox vaccine, the use of methisazone was not further pursued. Should there be a threat of smallpox or other poxvirus infections, that could not be immediately controlled by vaccination, a therapeutic intervention could be envisaged based on several therapeutic strategies targeted at such cellular enzymes as IMP dehydrogenase, SAH hydrolase, OMP decarboxylase and CTP synthetase, as well as viral enzymes such as the DNA polymerase. Most advanced as a therapeutic or early prophylactic modality to tackle poxvirus infection is cidofovir, which was found active (i) in vitro against all poxviruses studied so far; (ii) in vivo, against vaccinia and cowpox virus infections in experimental animal models; as well as (iii) some human poxvirus infections, such as molluscum contagiosum. In case of an inadvertent poxvirus epidemic, antiviral therapy (i.e. with cidofovir) will offer the possibility to provide short-term prophylaxis, or therapy. Cidofovir should also allow to treat severe complications of vaccination as may happen in for example immunosuppressed patients.     

Cell line dependency for antiviral activity and in vivo efficacy of N-methanocarbathymidine against orthopoxvirus infections in mice

A novel carbocyclic thymidine analog, N-methanocarbathymidine [(N)-MCT], was evaluated for inhibition of orthopoxvirus infections. Efficacy in vitro was assessed by plaque reduction assays against wild-type and cidofovir-resistant strains of cowpox and vaccinia viruses in nine different cell lines. Minimal differences were seen in antiviral activity against wild-type and cidofovir-resistant viruses. (N)-MCT's efficacy was affected by the cell line used for assay, with 50% poxvirus-inhibitory concentrations in cells as follows: mouse=0.6-2.2 microM, rabbit=52-90 microM, monkey=87 to >1000 microM, and human=39-220 microM. Limited studies performed with carbocyclic thymidine indicated a similar cell line dependency for antiviral activity. (N)-MCT did not inhibit actively dividing uninfected cells at 1000 microM. The potency of (N)-MCT against an S-variant thymidine kinase-deficient vaccinia virus was similar to that seen against S-variant and wild-type viruses in mouse, monkey, and human cells, implicating a cellular enzyme in the phosphorylation of the compound. Mice were intranasally infected with cowpox and vaccinia viruses followed 24h later by intraperitoneal treatment with (N)-MCT (twice a day for 7 days) or cidofovir (once a day for 2 days). (N)-MCT treatment at 100 and 30 mg/kg/day resulted in 90 and 20% survival from cowpox virus infection, respectively, compared to 0% survival in the placebo group. Statistically significant reductions in lung virus titers on day 5 occurred in 10, 30, and 100mg/kg/day treated mice. These same doses were also active against a lethal vaccinia virus (WR strain) challenge, and protection was seen down to 10mg/kg/day against a lethal vaccinia virus (IHD strain) infection. Cidofovir (100mg/kg/day) protected animals from death in all three infections.     

Potential antiviral therapeutics for smallpox,monkeypox and other orthopoxvirus infections

We assessed the activities of 24 different antiviral compounds against smallpox (two strains of variola major and one of variola minor), monkeypox, vaccinia and cowpox viruses by a neutral red uptake assay. To establish assay parameters, we examined viral replication and its inhibition at various times postinfection and at several multiplicities of infection. Drugs were selected to target a range of functions involved in viral replication. Eight compounds (cidofovir, cyclic HPMPC (cHPMPC), HPMPA, ribavirin, tiazofurin, carbocyclic 3-deazaadenosine, 3-deazaneplanocin A and DFBA (1-(2,4-difluorobenzyloxy)adenosine perchlorate)-a derivative of adenosine N1-oxide) inhibited the replication of all three variola strains and the other orthopoxviruses at drug concentrations within a pharmacologically achievable range. Two others (methisazone and bis-POM-PMEA) showed a lesser degree of antiviral effect, while the remainder were inactive. To examine possible naturally occurring drug resistance among a large number of variola isolates obtained from different geographical regions and at different times, we examined the sensitivity of 35 different strains of variola as well as other orthopoxviruses to a subset of three of the most active compounds: cidofovir, cHPMPC, and ribavirin. Preliminary data indicate that nearly all isolates appear to have similar drug sensitivities. These findings are currently being verified and expanded.     

Inhibition of cowpox virus and monkeypox virus infection by mitoxantrone

Mitoxantrone, an FDA-approved therapeutic for the treatment of cancer and multiple sclerosis, was previously reported to exhibit antiviral activity against vaccinia virus. To determine whether this activity extends to other orthopoxviruses, mitoxantrone was tested against cowpox and monkeypox. Mitoxantrone demonstrated an EC₅₀ of 0.25 μM against cowpox and 0.8 μM against monkeypox. Intraperitoneal treatment of cowpox virus-challenged C57Bl/6 mice with 0.5 mg/kg mitoxantrone resulted in 25% survival and a significant increase in survival time. In an effort to improve its efficacy, mitoxantrone was tested for synergistic activity with cidofovir. In vitro tests demonstrated significant synergy between the two drugs against cowpox; however, no synergistic effect on animal survival or median time-to-death was seen in intranasally-infected BALB/c mice. Significantly fewer animals survived when treated with a combination of 0.5 mg/kg mitoxantrone and 100 mg/kg cidofovir than with 100 mg/kg cidofovir alone. This is, to our knowledge, the first report of limited anti-orthopoxvirus activity by mitoxantrone in an animal model.     

In vitro evaluation of the anti-orf virus activity of alkoxyalkyl esters of CDV, cCDV and (S)-HPMPA

Acyclic nucleoside phosphonates (ANPs) and in particular (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (HPMPC, cidofovir, CDV, Vistide) and its adenine counterpart (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine [(S)-HPMPA] are highly active against orf virus infections. This parapoxvirus commonly causes infection in sheep, goats, but also humans. Alkoxyalkyl esters of CDV have an increased oral bioavailability and are more active against orthopoxviruses than the parent compounds. In the present study, the potency of several alkoxyalkyl esters of CDV, cyclic cidofovir (cCDV) and (S)-HPMPA was evaluated against different orf virus isolates in two cell types, human embryonic lung (HEL) fibroblast and primary lamb keratinocytes. Each prodrug was at least 10-fold more active than its parent compound in both cell types. Of all the compounds tested, the (S)-HPMPA alkoxyalkyl esters showed the highest activity and selectivity against orf virus. Our results support the development of alkoxyalkyl esters of ANPs as antivirals not only for the treatment of complicated human orf lesions, but also in the therapy and prophylaxis of contagious ecthyma in sheep and goats.