Efficacy of pritelivir and acyclovir in the treatment of herpes simplex virus infections in a mouse model of herpes simplex encephalitis.

Pritelivir, a helicase-primase inhibitor, has excellent in vitro and in vivo activity against human herpes simplex virus (HSV). Mice lethally infected with HSV type 1 or 2, including acyclovir-resistant strains, were treated 72 h after infection for 7 days with pritelivir or acyclovir. Both drugs were administered orally twice daily either alone or in combination. Dosages of pritelivir from 0.3 to 30 mg/kg reduced mortality (P < 0.001) against HSV-1, E-377. With an acyclovir resistant HSV-1, 11360, pritelivir at 1 and 3 mg/kg increased survival (P < 0.005). With HSV-2, MS infected mice, all dosages higher than the 0.3 mg/kg dose of pritelivir were effective (P < 0.005). For acyclovir resistant HSV-2, strain 12247, pritelivir dosages of 1-3 mg/kg significantly improved survival (P < 0.0001). Combination therapies of pritelivir at 0.1 or 0.3 mg/kg/dose with acyclovir (10 mg/kg/dose) were protective (P < 0.0001) when compared to the vehicle treated group against HSV-2, strain MS (in line with previous data using HSV-1). An increased mean days to death (P < 0.05) was also observed and was indicative of a potential synergy. Pharmacokinetic studies were performed to determine pritelivir concentrations and a dose dependent relationship was found in both plasma and brain samples regardless of infection status or time of initiation of dosing. In summary, pritelivir was shown to be active when treatment was delayed to 72 h post viral inoculation and appeared to synergistically inhibit mortality in this model in combination with acyclovir. We conclude pritelivir has potent and resistance-breaking antiviral efficacy with potential for the treatment of potentially life-threatening HSV type 1 and 2 infections, including herpes simplex encephalitis.

Efficacy of orally administered low dose N-methanocarbathymidine against lethal herpes simplex virus type-2 infections of mice.

BACKGROUND: N-methanocarbathymidine (N-MCT) has previously been shown to be effective against lethal orthopoxvirus and herpes simplex virus type-1 infections in mice. In this investigation, the antiviral activity of N-MCT was assessed against herpes simplex virus type-2 (HSV-2) in BALB/c mice. METHODS: BALB/c mice were infected intranasally with a lethal challenge dose of HSV-2. N-MCT was administered orally twice daily to mice using doses of 0.01 to 100 mg/kg to determine effects on survival and on viral replication in organ and central nervous system (CNS) samples. RESULTS: N-MCT provided significant protection from mortality even when treatments were delayed until 3 days after viral infection. Viral replication in organ and CNS samples from N-MCT-treated mice was reduced below the limit of detection after 4 days of treatment. CONCLUSIONS: These results indicated that low dose N-MCT treatment was more effective than acyclovir therapy. N-MCT may be effective against HSV disease in humans and is currently undergoing preclinical evaluation. In particular, its potential use as a combination therapy for HSV, with its differing metabolism from acyclovir, make it a promising compound to develop for human use.

Efficacy of CMX001 against herpes simplex virus infections in mice and correlations with drug distribution studies.

CMX001, an orally active lipid conjugate of cidofovir, is 50 times more active in vitro against herpes simplex virus (HSV) replication than acyclovir or cidofovir. These studies compared the efficacy of CMX001 to acyclovir in BALB/c mice inoculated intranasally with HSV types 1 or 2. CMX001 was effective in reducing mortality using doses of 5 to 1.25 mg/kg administered orally once daily, even when treatments were delayed 48-72 h post viral inoculation. Organ samples obtained from mice treated with CMX001 had titers 3-5 log(10) plaque-forming units per gram of tissue lower than samples obtained from mice treated with acyclovir, including 5 different regions of the brain. Detectable concentrations of drug-related radioactivity were documented in the central nervous system of mice after oral administration of (14)C-CMX001. These studies indicate that CMX001 penetrates the blood-brain barrier, is a potent inhibitor of HSV replication in disseminated infections and central nervous system infections, and is superior to acyclovir.

Effect of an immune enhancer, GPI-0100, on vaccination with live attenuated herpes simplex virus (HSV) type 2 or glycoprotein D on genital HSV-2 infections of guinea pigs.

These studies were performed to determine the effect of AD-472, an attenuated human herpes simplex virus (HSV) type 2 or HSV-2 glycoprotein D (gD) when combined with an adjuvant, GPI-0100, a semi-synthetic Quillaja Saponin analog in a genital HSV-2 infection in guinea pigs. While animals immunized with either vaccine had reduced clinical disease, GPI-0100 only improved the efficacy of gD and did not affect the efficacy of the live vaccine. Neither vaccine had any therapeutic effect if administered 24 h after viral infection.

Effect of oral treatment with (S)-HPMPA, HDP-(S)-HPMPA or ODE-(S)-HPMPA on replication of murine cytomegalovirus (MCMV) or human cytomegalovirus (HCMV) in animal models.

We utilized BALB/c mice infected with murine CMV (MCMV) or severe combined immunodeficient (SCID) mice implanted with human fetal tissue and infected with HCMV to determine the efficacy of (S)-9-[3-hydroxy-2-(phophonomethoxy)propyl]adenine ((S)-HPMPA), hexadecyloxypropyl-(S)-HPMPA (HDP-(S)-HPMPA) or octadecyloxyethyl-(S)-HPMPA (ODE-(S)-HPMPA). In MCMV-infected BALB/c mice, oral HDP-(S)-HPMPA at 30 mg/kg significantly reduced mortality when started 24-48 h post inoculation. In the experimental HCMV infection, oral administration of vehicle or 10mg/kg of (S)-HPMPA, HDP-(S)-HPMPA or ODE-(S)-HPMPA was initiated 24h after infection and continued for 28 consecutive days. Cidofovir (CDV), at 20mg/kg given i.p., was used as a positive control. HDP-(S)-HPMPA or ODE-(S)-HPMPA significantly reduced viral replication compared to vehicle-treated mice, while oral (S)-HPMPA was ineffective.

Effect of oral treatment with hexadecyloxypropyl-[(S)-9-(3-hydroxy-2- phosphonylmethoxypropyl)adenine] [(S)-HPMPA] or octadecyloxyethyl-(S)-HPMPA on cowpox or vaccinia virus infections in mice.

We have previously reported that (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine, or (S)-HPMPA, is active in vitro against cowpox virus (CV) and vaccinia virus (VV) but is not active orally in animals. However, the ether lipid esters of (S)-HPMPA, hexadecyloxypropyl-[(S)-HPMPA] [HDP-(S)-HPMPA] and octadecyloxyethyl-[(S)-HPMPA] [ODE-(S)-HPMPA], had significantly enhanced activity in vitro and are orally bioavailable in mice. In the current study, HDP-(S)-HPMPA and ODE-(S)-HPMPA were prepared in water and administered once daily by oral gavage to mice at doses of 30, 10, and 3 mg/kg of body weight for 5 days beginning 24, 48, or 72 h after inoculation with CV or VV. Oral HDP-(S)-HPMPA and ODE-(S)-HPMPA were both highly effective (P < 0.001) at preventing mortality due to CV at 30 mg/kg, even when treatments were delayed until up to 72 h postinfection. ODE-(S)-HPMPA or HDP-(S)-HPMPA were also highly effective (P < 0.001) at preventing mortality in mice infected with VV at 30 mg/kg when treatments were delayed until to 48 or 72 h postinfection, respectively. Protection against both viruses was associated with a significant reduction of virus replication in the liver, spleen, and kidney but not in the lung. These data indicate that HDP-(S)-HPMPA and ODE-(S)-HPMPA are active when given orally against lethal CV and VV infections in mice, and further evaluation is warranted to provide additional information on the potential of these orally active compounds for treatment of human orthopoxvirus infection.

Effect of immunization with herpes simplex virus type-1 (HSV-1) glycoprotein D (gD) plus the immune enhancer GPI-0100 on infection with HSV-1 or HSV-2.

These studies were performed to determine the effects of GPI-0100, a semi synthetic Quillaja Saponin analog, formulated with herpes simplex virus type-1 (HSV-1) glycoprotein D (gD) on immunity to HSV. SKH-1 hairless mice, used as a model of herpes labialis, inoculated with HSV-1 results in facial lesions, virus replication and mortality. Mortality rates, lesion scores and viral titers were significantly reduced in SKH-1 mice immunized with gD/GPI-0100 prior to cutaneous inoculation with HSV-1 and the protective effects were greater than those using the standard alum adjuvant. Genital HSV-2 infections in guinea pigs were also utilized to determine if gD combined with GPI-0100 was protective against infection, disease severity and viral shedding. Guinea pigs immunized with HSV-1 gD with or without GPI-0100 had significantly reduced area under the curve lesion scores, but infection rates and virus shedding was not altered. When Tween 40 was added to gD and GPI-0100, mean peak lesion scores were also significantly reduced. The results obtained in a genital HSV-2 infection of guinea pigs did not indicate enhanced protection or reduced virus shedding following immunization with GPI-0100 and gD. There was, however, a significant improvement in clinical herpetic genital disease with the combination of gD plus the immune enhancer GPI-0100.

Evaluation of AD472, a live attenuated recombinant herpes simplex virus type 2 vaccine in guinea pigs.

An attenuated recombinant herpes simplex virus type 2 (HSV-2), designated as AD472, was constructed by deleting both copies of the gamma(1)34.5 gene, UL55-56, UL43.5, and the US10-12 region from HSV-2 strain G. This virus was engineered to be a safe and effective live attenuated HSV-2 vaccine and was tested in the guinea pig model of genital herpes to evaluate its ability to protect from disease upon challenge with the wild type (wt) virus, HSV-2 (G). AD472 administered intramuscularly did not prevent infection or virus replication in the vaginal tract, but did reduce both lesion development and severity in a dose-dependent manner in guinea pigs challenged with the wt virus. Frequency of reactivation from latency was low compared with that of the parent virus, HSV-2 (G). Immunization with AD472 at doses of 1x10(5)PFU generally precluded colonization of the ganglia or establishment of latency by the challenge virus. Results presented here support the concept of a rationally engineered live attenuated vaccine for the prevention of the genital disease associated with infection by HSV-2.

Oral treatment of murine cytomegalovirus infections with ether lipid esters of cidofovir.

To improve the oral bioavailability of cidofovir (CDV), a series of ether lipid ester prodrugs were synthesized and evaluated for activity against murine cytomegalovirus (MCMV) infection. Four of these analogs, hexadecyloxypropyl (HDP)-CDV, octadecyloxyethyl (ODE)-CDV, oleyloxyethyl (OLE)-CDV, and oleyloxypropyl (OLP)-CDV, were found to have greater activity than CDV against human CMV and MCMV in vitro. The efficacy of oral treatment with these compounds against MCMV infections in BALB/c mice was then determined. Treatment with HDP-CDV, ODE-CDV, OLE-CDV, or OLP-CDV at 2.0 to 6.7 mg/kg of body weight provided significant protection when daily treatments were initiated 24 to 48 h after viral inoculation. Additionally, HDP-CDV or ODE-CDV administered twice weekly or as a single dose of 1.25 to 10 mg/kg was effective in reducing mortality when treatment was initiated at 24 h, 48 h, or, in some cases, 72 h after viral inoculation. In animals treated daily with HDP-CDV or ODE-CDV, virus titers in lung, liver, spleen, kidney, pancreas, salivary gland, and blood were reduced 3 to 5 log(10)-fold, which was comparable to CDV given intraperitoneally. These results indicated that HDP-CDV or ODE-CDV given orally was as effective as parenteral CDV for the treatment of experimental MCMV infection and suggest that further evaluation for use in CMV infections in humans is warranted.

Cutaneous infections of mice with vaccinia or cowpox viruses and efficacy of cidofovir.

Orthopoxviruses, including smallpox, monkeypox and molluscipox, pose risks to human health through bioterrorist acts or natural transmission. There is no approved therapy for orthopoxvirus infections; however, cidofovir (CDV) has been approved as an investigational new drug for emergency treatment of adverse effects following smallpox vaccination. For evaluation of new therapies directed against orthopoxvirus infections, we have utilized immunocompetent, hairless mice (SKH-1) inoculated by a cutaneous route with cowpox virus (CV) or vaccinia virus (VV). Mice subsequently developed skin lesions and virus was recovered from the site of inoculation and quantified. Skin biopsies were evaluated microscopically, revealing brick-like eosinophilic, intracytoplasmic inclusion bodies characteristic of orthopoxvirus infection. SKH-1 mice fully recovered from either CV or VV infection. Immunodeficient Athymic or Rhino mice inoculated with CV or VV had more lesions and severe disease than SKH-1 mice. CV-infected SKH-1 mice were treated either with systemic or topical CDV. Although some protection was achieved with systemic treatment, 5% topical CDV was most effective at reducing virus titers in skin, lung, kidney, and spleen. These models may provide a means for evaluating efficacy of new therapies directed against orthopoxvirus diseases and further confirm the topical activity of CDV against cutaneous infections.

Oral treatment of cowpox and vaccinia virus infections in mice with ether lipid esters of cidofovir.

Four newly synthesized ether lipid esters of cidofovir (CDV), hexadecyloxypropyl-CDV (HDP-CDV), octadecyloxyethyl-CDV (ODE-CDV), oleyloxypropyl-CDV (OLP-CDV), and oleyloxyethyl-CDV (OLE-CDV), were found to have enhanced activities against vaccinia virus (VV) and cowpox virus (CV) in vitro compared to those of CDV. The compounds were administered orally and were evaluated for their efficacies against lethal CV or VV infections in mice. HDP-CDV, ODE-CDV, and OLE-CDV were effective at preventing mortality from CV infection when treatments were initiated 24 h after viral inoculation, but only HDP-CDV and ODE-CDV maintained efficacy when treatments were initiated as late as 72 h postinfection. Oral pretreatment with HDP-CDV and ODE-CDV were also effective when they were given 5, 3, or 1 day prior to inoculation with CV, even when each compound was administered as a single dose. Both HDP-CDV and ODE-CDV were also effective against VV infections when they were administered orally 24 or 48 h after infection. In animals treated with HDP-CDV or ODE-CDV, the titers of both CV and VV in the liver, spleen, and kidney were reduced 3 to 7 log(10). In contrast, virus replication in the lungs was not significantly reduced. These data indicate that HDP-CDV or ODE-CDV given orally is as effective as CDV given parenterally for the treatment of experimental CV and VV infections and suggest that these compounds may be useful for the treatment of orthopoxvirus infections in humans.

Efficacy of multiple- or single-dose cidofovir against vaccinia and cowpox virus infections in mice.

Orthopoxviruses, including variola and monkeypox, pose risks to human health through natural transmission or potential bioterrorist activities. Since vaccination has not recently been utilized for control of these infections, there is renewed effort in the development of antiviral agents not only for postexposure smallpox therapy but also for treatment of adverse reactions following vaccination. The objectives of this study were to expand on the results of others that cidofovir (CDV) is effective in mice inoculated with cowpox virus (CV) or vaccinia virus (VV) and to document the efficacy of single and interval dosing beginning prior to or after infection, particularly including evaluations using suboptimal doses of CDV. We utilized BALB/c or SCID mice inoculated with CV or VV as models for systemic poxvirus infections. BALB/c mice were inoculated intranasally with CV or VV and treated with CDV prior to or after virus inoculation. CDV, at concentrations as low as 0.7 to 6.7 mg/kg of body weight/day for 5 days, conferred significant protection when treatment was initiated as late as 72 to 96 h postinfection. A single-dose pretreatment or posttreatment with CDV at 3 to 100 mg/kg was effective when given as early as 5 days prior to infection or as late as 3 days after infection with either VV or CV. Interval treatments given every third day beginning 72 h postinfection using 6.7 or 2 mg of CDV/kg also proved effective against CV infections. When SCID mice were inoculated intraperitoneally with CV or VV and treated for 7 to 30 days with CDV, all the mice eventually died during or after cessation of treatment; however, significant delays in time to death and reduction of virus replication in organs occurred in most treated groups, and no resistance to CDV was detected.