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Human cytomegalovirus (HCMV) and herpes simplex virus (HSV) can cause a wide variety of clinical manifestations in man. Ganciclovir (GCV) is effective against HCMV infection when administered by the intravenous route and may be used orally in large doses for prophylaxis of HCMV infections in organ transplantation patients and in AIDS patients. In previous studies with acyclovir (ACV), we found that covalent attachment of an alkyl glycerol phosphate moiety greatly increased oral bioavailability and increased antiviral activity against hepatitis B virus. Adducts of ACV with alkyl propanediol phosphate were more active than the alkyl glycerol phosphate analogue in vitro in 2.2.15 cells, which constitutively produce hepatitis B virus. To see if this strategy would work for two other poorly absorbed nucleoside analogues, we synthesized 1-O-hexadecylpropanediol-3-phospho-GCV (HDP-P-GCV) and 1-O-hexadecyl-propanediol-3-phospho-penciclovir (HDP-P-PCV), and evaluated the in vitro antiviral activity, selectivity and oral antiviral activity of both compounds versus GCV or PCV in mice infected with HSV-1 or HDP-P-GCV versus murine cytomegalovirus (MCMV). HDP-P-GCV is orally active in both MCMV and HSV-1 infection in mice with antiviral activity equivalent to (HSV-1) or greater than oral GCV (MCMV). Oral HDP-P-PCV was more active than PCV orally versus intranasal HSV-1 infection in mice.

Dendrimers are large highly branched macromolecules synthesized from a polyfunctional core. They have shown a variety of biological properties, including, in some instances, antiviral activity. In this study, five dendrimers were evaluated for in vitro activity against herpes simplex virus (HSV) types 1 and 2 by cytopathic effect (CPE) inhibition and plaque reduction (PR) assay in human foreskin fibroblast cells. All of the compounds were active against both virus types in the CPE inhibition assay, in which drug was added to the cells prior to the addition of virus. Antiviral activity was reduced or lost in the PR assays, in which the cells were incubated with the virus before the drug was added. The prophylactic efficacy suggested that the dendrimers might have potential as topical microbicides, products intended to be applied to the vaginal or rectal mucosa to protect against sexually transmitted infections. Three dendrimers were evaluated for this application against genital HSV infection in mice. Two of the compounds, BRI-2999 and BRI-6741, significantly reduced infection rates when 15 microl of a 100-mg/ml solution was administered immediately prior to intravaginal challenge, and the most effective compound, BRI-2999, provided significant protection even when applied 30 min before challenge. This is the first report of microbicidal activity by dendrimers in vivo.

In recent studies we found that the topical effectiveness of acyclovir (ACV) formulations was a single-valued function of C-the target site free drug concentration. The topical efficacy was the same when the therapy was initiated 0, 1, or 2 days after intracutaneous herpes simplex virus type-1 (HSV-1) inoculation in hairless mice. The purpose of the present study was to examine the hypothesis that the topical effectiveness of cidofovir (HPMPC) would not be a single valued function of C and that it would be dependent upon when the therapy was initiated relative to the time of viral infection. Formulations of HPMPC and ACV in 95% DMSO as a vehicle were used. Hairless mice intracutaneously infected with HSV-1 were used, and 20 microL of the test formulation was topically applied twice a day. In protocol A, the treatment was continued until the fourth day after virus inoculation, whereas in protocol B the treatment was terminated on the day of virus inoculation. Treatment was initiated on various days ranging from day -6 to day 4, and the lesions were scored on day 5. Treatment of ACV according to protocol A proved efficacious whether started as early as 6 days before virus inoculation or later, whereas the efficacy of ACV was annihilated if applied following protocol B. For HPMPC, on the other hand, the in vivo efficacies were found to be strongly dependent on how early the therapy was initiated, and significant efficacy was observed even when the treatment was terminated on the day of virus inoculation. This difference was attributed to the virus-independent intracellular phosphorylation of HPMPC and slow clearance of its metabolites from the cell. It was also noted that, similar to ACV, for HPMPC the topical efficacy is likely to be a function of C for a fixed protocol. However, unlike for ACV, for HPMPC the efficacy was not a single-valued function of C.

A genital HSV-2 infection of guinea pigs and a cutaneous HSV-1 infection in mice were used to examine the ability of antioxidant components CRT1 to reduce lesion development, duration, and severity. CRT is a patented antioxidant formulation developed by Warner-Lambert Worldwide Consumer Healthcare R. and D. CRT contains three components that work synergistically: vitamin E, sodium pyruvate and membrane stabilizing fatty acids. The MS strain of HSV-2 was utilized for intravaginal inoculation of guinea pigs with approximately 1.2 x 10(5) plaque forming units. The guinea pigs were treated on the external genital skin four times daily for 10 days beginning 48 h post viral inoculation. This study was designed to optimize the CRT formulations that would be used to quantify the synergistic effect of the CRT components. SKH-1 male hairless mice were inoculated with 1 x 10(7) HSV-1 (McIntyre strain) on the dorsal surface of the mouse and treated with CRT formulations starting on the afternoon of the day of infection, and treated for the following 14 days. In the guinea pig model, the CRT formula that contained all three CRT components, worked synergistically to reduce lesion development, duration and severity scores significantly compared to vehicle control or acyclovir. Acyclovir was the only compound that reduced viral titers, but in contrast to CRT, acyclovir did not reduce lesion development, duration or severity. The quantitative effect of the three CRT components was demonstrated in the mouse model.

The purpose of this study was to carry out an extensive examination of the C* concept for prediction of the topical antiviral efficacies of acyclovir (ACV) formulations in a hairless mouse model for the treatment of cutaneous herpes simplex virus type-1 (HSV-1) infections. This method is based on estimation of the free drug concentration at the target site (C*), which is presumed to be the basal cell layer of the epidermis. Five different formulations (containing 5% ACV) were examined in a finite dose multiple dosing regimen (twice a day application) to simulate the clinical situation. For determination of C*, in vitro ACV fluxes across the hairless mouse skin were measured in an in vivo-in vitro experimental design that approximated the in vivo antiviral treatment protocol. Then, the in vivo antiviral efficacies were measured using a 1-day delayed (after HSV-1 virus inoculation) 4-day treatment protocol. 10 microL/cm2 dose of ACV formulation was applied every 12 h for 4 days after which the lesions were scored and efficacies were calculated. Our results indicate that, over a wide range of efficacies, the predictions based on C* (estimated from the experimental fluxes) are in good agreement with the in vivo antiviral efficacies. These studies, therefore, support the validity of the C* concept for various ACV formulations and suggest that the C* approach has potential for future practical situations.

The use of controlled transdermal delivery of acyclovir (ACV) in the treatment of cutaneous herpes simplex virus type 1 infections in hairless mice was investigated. Using an in vivo animal model (A. Gonsho, et al. Int. J. Pharm. 65:183-194 (1990)) made it possible to quantify both, the topical and the systemic antiviral efficacy of ACV transdermal patches as a function of the drug delivery rate of the patches. Drug delivery rates required to attain systemic efficacy were found to be higher than the rates required to attain the same magnitude of topical efficacy. The ACV concentrations in the basal cell layer of the epidermis for 50% topical efficacy and 50% systemic efficacy were estimated. The basal epidermis layer was considered to be the site of antiviral drug activity (skin target site). Systemic plasma levels were obtained from pharmacokinetic studies and were used to estimate the ACV concentration achieved systemically in the basal epidermis layer. A computational model for drug permeation across skin was employed to estimate the ACV concentration achieved topically in the basal epidermis layer. Equal topical and systemic efficacies were found to correspond to equal drug concentrations at the site of antiviral activity. The length of the effective diffusion pathway of drug molecules in the dermis prior to entering the blood circulation was assumed to be approximately equal to 1/20 of the anatomical dermis thickness because of dermis vascularization.

A-73209 is a novel oxetanocin derivative with potent in vitro and in vivo activity against VZV, HSV-1, and HSV-2. A-73209 was two logs more potent than acyclovir against five thymidine kinase positive (TK+) strains of VZV in vitro (mean EC50 0.01 vs. 1.22 micrograms/ml). The activity of A-73209 was one log more potent than acyclovir against TK+ HSV-1 strains in vitro (EC50 = 0.03 vs. 0.32 micrograms/ml). A-73209 yielded a mean EC50 of 2.2 micrograms/ml compared to a mean EC50 of 0.37 micrograms/ml for acyclovir against a panel of TK+ HSV-2 strains in vitro. The in vitro activity of A-73209 against thymidine kinase negative or deficient strains of VZV, HSV-1 and HSV-2 was much lower than for the corresponding TK+ strains. A-73209 produced efficacy superior to acyclovir against lethal systemic or intracerebral HSV-1 infections in mice. The greater efficacy of A-73209 relative to acyclovir was especially apparent with oral dosing. Against HSV-2 infections in mice, the efficacy of A-73209 ranged from equal to 1.7 times less active relative to acyclovir with oral dosing. A-73209 was orally bioavailable in mice, with maximal serum concentrations well in excess of in vitro inhibitory concentrations. A-73209 appears to be a potent and selective agent against varicella-zoster virus and herpes simplex virus infections.

This report describes the study of a novel animal model for the topical treatment of cutaneous herpes virus infections, with a focus upon the relationship between the dermal flux of the antiviral agent and the effectiveness of the topical therapy. A recently developed (trans)dermal delivery system (TDS) for controlling acyclovir (ACV) fluxes was employed in the treatment of cutaneous herpes simplex virus type 1 (HSV-1) infections in hairless mice. The TDS's were fabricated with rate-controlling membranes to provide nearly constant fluxes of ACV for up to 3 to 4 days. At the end of each experiment an extraction procedure was used to determine the residual ACV, validating the drug delivery performance of the TDS. Virus was inoculated into the skin of the mice at a site distant from the TDS area, and the induced lesion development was evaluated to distinguish between topical and systemic effectiveness of the therapy. In the main protocol, ACV therapy was initiated 0, 1, 2, and 3 days after virus inoculation and the lesion development "scored" on Day 5. The topical efficacies of 1- and 2-day-delayed treatments were essentially the same as that of a 0-day-delayed treatment, while the topical efficacy of a 3-day-delayed treatment was much poorer. Also, in the cases of 0-, 1-, and 2-day-delayed treatments, topical efficacy increased with increasing flux in the range of 10 to 100 micrograms/cm2-day. When the ACV flux was 100 micrograms/cm2-day or greater, a maximum 100% topical efficacy was obtained. The results for systemic efficacy were shifted to higher fluxes: approximately 10-fold greater ACV fluxes were necessary to provide efficacy equal to the topical efficacy results. The animals treated with a high ACV flux (350-500 micrograms/cm2-day) lived significantly longer than those treated with a low ACV flux (10-125 micrograms/cm2-day) and those of untreated (placebo) animals. Further, their mean survival time decreased with an increase in the time delay for ACV treatment. In contrast, the mean survival time for the animals which received a low ACV flux was similar to that of the control animals and remained unaltered with an increase in the time delay for ACV treatment. The approach developed in this study should be valuable in (a) the screening of new antiviral agents for the topical treatment of cutaneous herpes virus infections and (b) in the optimization of drug delivery systems (i.e., topical formulations).

Murine acquired immunodeficiency syndrome (MAIDS) was induced in C57BL/6 mice following infection with the LP-BM5 retrovirus complex. Infected mice developed splenomegaly, lymphadenopathy and loss of B- and T-cell functions 100 days after virus inoculation. Mice with AIDS were highly susceptible to opportunistic murine cytomegalovirus (MCMV) and herpes simplex virus (HSV-1) infections. The therapeutic activities of two phosphonylmethoxyalkyl derivatives, 9-(2-phosphonylmethoxyethyl)adenine (PMEA) and (S)-1-(3-hydroxy-2-phosphonylmethoxy-propyl)cytosine (HPMPC), were evaluated in MAIDS immunosuppressed mice infected with MCMV or HSV-1. MCMV infection resulted in extensive viral replication in lung, liver and spleen and death occurred five to twelve days post-infection. Treatment with either HPMPC or ganciclovir (DHPG) reduced mortality and viral replication in target organs; however, HPMPC was as effective as DHPG at one-fifth the DHPG dose. Moreover, when a single dose (100 mg/kg) of HPMPC was administered 24 h prior to MCMV infection, it suppressed virus replication at seven and 14 days post-infection, thus resulting in a significant prolongation of life. PMEA was effective against opportunistic HSV-1 infections, but appeared to be less effective than HPMPC against MCMV infections. These results indicate that MAIDS can be used as a model for evaluating antivirals in an immunocompromised host, and suggest that both PMEA and HPMPC may be useful in the treatment of opportunistic CMV and HSV-1 infections.

The gene designated gamma 134.5 maps in the inverted repeats flanking the long unique sequence of herpes simplex virus-1 (HSV-1) DNA, and therefore it is present in two copies per genome. This gene is not essential for viral growth in cell culture. Four recombinant viruses were genetically engineered to test the function of this gene. These were (i) a virus from which both copies of the gene were deleted, (ii) a virus containing a stop codon in both copies of the gene, (iii) a virus containing after the first codon an insert encoding a 16-amino acid epitope known to react with a specific monoclonal antibody, and (iv) a virus in which the deleted sequences were restored. The viruses from which the gene was deleted or which carried stop codons were avirulent on intracerebral inoculation of mice. The virus with the gene tagged by the sequence encoding the epitope was moderately virulent, whereas the restored virus reacquired the phenotype of the parent virus. Significant amounts of virus were recovered only from brains of animals inoculated with virulent viruses. Inasmuch as the product of the gamma 134.5 gene extended the host range of the virus by enabling it to replicate and destroy brain cells, it is a viral neurovirulence factor.

Intranasal inoculation of newborn mice with Herpesvirus hominis (HVH) type 2 provides a model for disseminated herpesvirus infections of human newborn infants. Treatment of this experimental infection with polyinosinic-polycytidylic acid [poly(I:C)] significantly increased the mean survival time and markedly altered the pathogenesis of the infection. No significant protection against final mortality was observed. Poly(I:C) therapy completely inhibited detectable viral replication in all target organs tested except the brain. In the brain there was a 2-day delay in the onset of viral replication in treated animals, which correlated with the 1- to 2-day increase in mean survival time. In general, the control of HVH replication occurred in those target organs in which poly(I:C)-induced interferon was detectable. The failure of poly(I:C) to alter the final mortality of newborn mice infected with HVH appears to be primarily due to the lack of sufficient levels of interferon induced in brain tissue and the failure to prevent viral replication in this critical target organ.

The effect of oral or intraperitoneal administration of acyclovir was evaluated in four experimental models of herpes simplex virus (HSV) encephalitis in mice. Mice were inoculated with HSV-1 or HSV-2 intracerebrally or with HSV-2 intranasally, intraperitoneally, or intravaginally. With all four routes of inoculation, oral acyclovir therapy significantly reduced mortality when started as late as 72 to 96 hours after viral challenge. Intraperitoneal acyclovir was not effective in protecting mice inoculated intravaginally, but was effective if given 24 to 48 hours after intracerebral or intranasal challenge and as late as 96 hours after intraperitoneal infection. Oral acyclovir was more active than intraperitoneal treatment in all four model infections. Levels of acyclovir inhibitory for HSV in cell culture were maintained in plasma and brain tissue throughout oral treatment but lasted only three to six hours after each intraperitoneal treatment. These results suggest that acyclovir may be useful in treating serious HSV infections in humans.

The studies reviewed here are encouraging in that HSV is moderately sensitive to the action of interferon and that infections in experimental animals clearly may be altered by exogenous interferon and interferon inducers. On the other hand it is apparent that the potential of this approach to treatment of herpesvirus infections is limited by the levels of interferon that can be passively administered or induced, the development of hyporeactivity which prevents maintaining high levels of interferon in infected animals after inoculation of inducers, and the failure to alter the course of HSV infection once the virus is established in target organs.

The clinical and virologic course of herpes simplex genitalis in women and men was examined in order to identify measurements useful in antiviral trials. Factors influencing the clinical course included initial disease versus recurrent disease, wet-skin versus dry-skin lesions, female versus male sex. Women with initial genital herpes had higher mean peak lesion virus titers than those with recurrent disease (10(4.5) pfu compared with 10(2.5) pfu) and excreted virus longer (13 to 15 days compared with 6 to 8 days). Men with recurrent lesions had higher mean peak virus titers than women (10(4.0) pfu compared with 10(2.5) pfu), but the duration of virus excretion was shorter (three to four days compared with six to eight days). There was pronounced variation in the clinical and virologic course of recurrent lesions among different patients and even within the same patient. These observations indicate several difficulties that must be considered in conducting careful antiviral trials in patients with herpes simplex genitalis.

Intranasal inoculation of newborn mice with Herpesvirus hominis type 2 provides an experimental infection that closely resembles disseminated herpesvirus infection of human newborn infants. After inoculation of mice, the virus multiplies in the respiratory tract and is disseminated through the blood to the liver and spleen and to the brain by both a viremia and nerve route transmission. Although therapy with 1-beta-d-arabinofuranosylcytosine (ara-C) did not reduce final mortality, it did increase the mean survival time by 1 day. This effect on the mean survival time was associated with a 1-day delay in the appearance of herpesvirus in the blood, liver, and spleen and a reduction of virus replication in lung and brain for 1 day as compared with untreated control animals. Treatment with 9-beta-d-arabinofuranosyladenine (ara-A) likewise had no effect on final mortality, but increased the mean survival time by 2 days. Therapy with ara-A delayed or suppressed virus replication in blood, lung, liver, spleen, and brain for 2 days. Although treatment with either ara-C or ara-A in this experimental H. hominis type 2 infection resulted in a temporary delay and/or suppression of viral replication in several target organs, neither compound was completely effective in inhibiting viral replication or in protecting animals from eventual death due to the infection.

Intranasal inoculation of weanling mice with herpes simplex virus type 2 (HSV-2) provides an experimental infection that closely resembles disseminated and central nervous system HSV infections of human neonates. Intraperitoneal treatment with acyclovir (ACV) successfully reduced mortality even when therapy was begun as late as 2 days and oral therapy as late as 4 days after viral challenge. Treatment with ACV beginning on day 1 completely inhibited HSV-2 replication in lung, spleen, kidney, olfactory lobe, and cerebrum and decreased viral titers in the pons by 2-3 logs. Comparison of these data with our previous experiments using adenine arabinoside and adenine arabinoside 5' monophosphate indicates that ACV is more effective in the murine model of neonatal disease and suggests that ACV may also be more effective in treating the disease in humans.

Genital infection of mice with Herpesvirus hominis type 2 provides an experimental model for screening potential antiviral chemotherapeutic agents before clinical trials in humans. Intravaginal treatment with phosphonoacetic acid (at a dose of 500 mg/kg in saline or as a 5% cream) initiated 3 hr after inoculation with H. hominis type 2 completely inhibited viral replication in the genital tract and prevented subsequent mortality. Although therapy initiated 24-72 hr after infection significantly reduced titers of virus in vaginal secretions from three- to 100-fold, most mice eventually died of encephalitis. Topical treatment with either adenine arabinoside or adenine arabinoside 5'-monophosphate at a dose of 500 mg/kg in saline or as a 10% cream failed to alter viral replication in the genital tract or to protect the mice from death due to encephalitis. Treatment by the intraperitoneal route with any of these three agents had no effect on local viral replication or final mortality.

The effectiveness of 2-deoxy-D-glucose (2-DG) was evaluated in the treatment of cutaneous infections with herpes simplex virus type 1 (HSV-1) in mice and genital infections with HSV type 2 (HSV-2) in mice and guinea pigs. Groups of mice were inoculated in the lumbosacral or orofacial area with HSV-1 and treated topically three times a day with 0.2% or 0.5% 2-DG solution beginning 3 hr after inoculation. No effect on skin lesions, mortality, or latency was observed. Mice were inoculated intravaginally with HSV-2 and treated intravaginally three times a day with 0.2%-5% 2-DG in solution or suspended in miconazole nitrate cream beginning 6 hr, 24 hr, or 48 hr after inoculation. Replication of HSV-2 in the vagina and final mortality were not affected. Guinea pigs were infected intravaginally with HSV-2 and treated both intravaginally and topically on the external genitalia four times a day with 1% or 5% 2-DG in miconazole nitrate cream. Treatment initiated just prior to development of lesions (on day 3 after inoculation) did not alter vaginal virus replication, lesion development and severity, or virus titers in lesions.