INTRODUCTION

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Herpes simplex virus (HSV) type 1 (HSV-1) and HSV type 2 (HSV-2) are known to establish latent infections in the sensory ganglia that innervate the site of primary infection. The latent virus periodically reactivates to produce infectious virus, which can cause recurrent disease (31, 39). Following a productive infection in permissive epithelial cells of skin or mucosal surfaces, HSV gains access to sensory nerve endings which innervate the peripheral inoculation site and migrates within axons in the retrograde axonal flow to neuronal cell bodies in sensory ganglia (38). Once it is in neurons, the virus can enter a productive cycle, resulting in the release of progeny virions, or can establish true latency (1, 39). During latency, the viral DNA is circularized or exists as a concatemer (4, 29, 30). It is not integrated into the host cell genome but is organized in a structure similar to that of host nuclear chromatin (3, 21). Recurrent disease caused by HSV results from reactivation of latent virus in ganglia, centripetal spread of the virus in axons, and viral replication at the initial portal of entry. This leads to virus shedding with or without clinical symptoms and allows the virus to disseminate in susceptible hosts (39). The ability of HSV to periodically reactivate from latency in sensory ganglia is a key event in the pathogenesis of recurrent infection and thus represents an important target for intervention to prevent not only recurrent diseases but also its spread through the population.

Topical treatments with antiviral agents that specifically inhibit herpesvirus DNA synthesis and viral replication, such as acyclovir, phosphonoacetic acid, and foscarnet, and that were administered shortly after virus inoculation were shown to prevent in most cases the colonization of trigeminal ganglia in a murine model of HSV-1 orofacial infection (9, 12-14). The latent genome copy number correlates with the probability of in vivo reactivation of herpesviruses (18, 32, 33). Therefore, it is proposed that these drugs, if given sufficiently early in the course of infection, may reduce the quantity of virus that accesses the sensory neurons, resulting in a smaller reservoir of latent virus from which subsequent reactivation may arise (6).

Previous studies from our laboratory have demonstrated that the efficacy of 5% acyclovir incorporated into a polymer composed of polyoxyethylene and polyoxypropylene was better than that of the commercial acyclovir ointment (Zovirax) in reducing the development of herpetic skin lesions in mice after a single application 24 h postinfection (27). The improved efficacy of the gel formulation of acyclovir was attributed to the semiviscous character of the polymer, which allows a more efficient drug penetration into the skin. However, foscarnet incorporated into this polymer had no marked effect under the same treatment regimen.

Sodium lauryl sulfate (SLS), an anionic surfactant, possesses properties that enhance drug penetration into the skin because it increases the fluidity of epidermal lipids (7, 19, 24, 25). Using a zosteriform model of HSV-1 infection in hairless mice, we have previously demonstrated that topical treatment with a gel formulation containing 3% foscarnet in combination with 5% SLS was superior to a formulation containing 3% foscarnet alone in reducing the development of herpetic skin lesions when it was given only once 24 h after infection (26). Treatment of mice with the gel containing 5% SLS alone did not reduce the severity of herpetic skin lesions but significantly decreased the mortality rate associated with infection compared to that for untreated infected mice. In addition, our previous studies showed that SLS is a potent inhibitor of the infectivities of herpesviruses in vitro and in vivo (28). These observations suggest that SLS could block or reduce the spread of the virus to the central nervous system. In the present study, we have used a murine model of HSV-1 orofacial infection to evaluate the efficacies of gel formulations containing foscarnet, alone or combined with SLS, on the development of herpetic skin lesions as well as on the establishment of latency and reactivation of latent virus.

	MATERIALS AND METHODS

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Materials. Foscarnet (trisodium phosphonoformate) and SLS were obtained from Sigma Chemical Co. (St. Louis, Mo.).

Preparation of topical formulations. For all experiments, we have used as a vehicle a polymer composed of polyoxypropylene and polyoxyethylene. The polymer was suspended in phosphate buffer (200 mM; pH 6.0) at 4°C to obtain a final concentration of 18% (wt/wt). We selected a pH of 6.0 to correspond to the pH of the skin. For formulations containing foscarnet and/or SLS, the drug and/or SLS was added to the polymer powder, and these were then dissolved in phosphate buffer (200 mM; pH 6.0) at 4°C in amounts needed to achieve final concentrations of 3 and 5% (wt/wt), respectively.

Virus strain. HSV-1 strain F (VR-733; American Type Culture Collection, Manassas, Va.) was propagated in Vero cells (American Type Culture Collection) in Eagle's minimum essential medium (Canadian Life Technologies, Burlington, Ontario, Canada) supplemented with 0.22% sodium bicarbonate, 100 U of penicillin-streptomycin per ml, 2 mM L-glutamine, and 2% heat-inactivated fetal bovine serum (EMEM plus 2% FBS) in a 5% CO₂ atmosphere. At approximately 80 to 90% cell lysis, the cells were scraped off from the dishes with a sterile cell scraper. The cellular suspension was centrifuged (1,450 × g for 10 min at 4°C), and the supernatant was retained. The pellet was submitted to three freeze-thaw cycles in liquid nitrogen and then centrifuged again. Supernatants were pooled, filtered on a 0.45-µm-pore-size low-binding membrane (Durapore; Millipore Corp., Bedford, Mass.), centrifuged (100,000 × g for 2 h and 40 min at 4°C), and stored at 80°C until they were used. The viral titer determined in Vero cells was 3 × 10⁸ PFU/ml.

Orofacial model. Female hairless mice (SKH1 mice; age, 5 to 6 weeks; Charles River Laboratories Inc., St. Constant, Québec, Canada) were used throughout the study. Mice were anesthetized by intraperitoneal injection of a mixture containing 70 mg of ketamine hydrochloride (Rogar/STB Inc., Montréal, Québec, Canada) and 11.5 mg of xylazine (Miles Canada Inc., Etobicoke, Ontario, Canada) per kg of body weight. The virus was inoculated on the triangular area of the snout. The skin was slightly abraded with a 27-gauge needle. A viral suspension (5 × 10⁵ PFU/25 µl) was deposited on the abraded area, and then the area was rubbed for 10 s with a cotton-tipped applicator saturated with EMEM plus 2% FBS. The mice were then returned to their cages and observed daily.

Treatments. All topical treatments were given twice daily for 3 days and were initiated at various times after infection (6, 24, and 48 h). Briefly, 25 µl of each of the gel formulations was deposited on the abraded area and then rubbed gently for 10 s on the infected side of the face. The area treated with the topical formulations was not protected against licking and grooming by the mice. The efficacies of the different formulations were evaluated from the mean lesion score, viral titers in skin samples and ipsilateral trigeminal ganglia, and the mean titers of reactivated virus in explant cultures of ipsilateral and contralateral trigeminal ganglia from latently infected mice.

Scoring of skin lesions. The evolution of cutaneous orofacial lesions was evaluated daily for 15 days, and their intensities were graded by the following criteria: 0, normal skin; 1, one to five discrete lesions; 2, six or more discrete lesions; 3, confluent lesions; 4, necrotic lesions.

Viral titration in tissue samples. The mice were killed and skin samples from the snout and the ipsilateral trigeminal ganglia were excised. Tissue samples were maintained in Hank's balanced salt solution (Canadian Life Technologies) at 4°C, blotted, weighed, and diluted with 1 ml of EMEM plus 2% FBS. Viruses were released from tissue samples with three cycles of sonication for 10 s each with a 5-s interval. The suspension obtained was centrifuged (1,100 × g for 15 min at 4°C). The supernatant was collected and stored at 80°C until it was used. Viruses extracted from skin or ipsilateral trigeminal ganglia were diluted appropriately in EMEM plus 2% FBS. Confluent Vero cells seeded in 24-well plates were then infected with 0.5 ml of diluted samples for 2 h at 37°C. Viral suspensions were removed, and cell sheets were overlaid with 0.5 ml of 0.6% SeaPlaque agarose (Mandel Scientific, St. Laurent, Québec, Canada) in EMEM plus 2% FBS and incubated for 2 days at 37°C. The cells were then fixed with 10% formaldehyde in phosphate-buffered saline (pH 7.2) for 20 min, washed with deionized water, and stained with 0.05% methylene blue. Viral titers were calculated as the mean of the log PFU per gram of tissue, but the calculations did not include data for animals from which virus was not isolated. The limit of detection of the assay was 100 PFU/g of tissue.

Reactivation of latent virus. The mice were killed 4 weeks after virus inoculation. The ipsilateral and contralateral trigeminal ganglia were removed and maintained in EMEM plus 5% FBS at 4°C. Each ganglion was transferred separately in 24-well plates containing 1 ml of EMEM plus 5% FBS and maintained in explant culture at 37°C in a 5% CO₂ atmosphere. After 10 days in culture, the ganglion explants and culture medium were homogenized with three cycles of sonication for 10 s each with a 5-s interval. The suspension obtained was centrifuged (1,100 × g for 15 min at 4°C), and the supernatant was collected and stored at 80°C until it was used. Samples were assayed for the presence of reactivated virus on confluent Vero cells by a method similar to that described above for the determination of viral titers. Titers of reactivated virus in explant cultures of latently infected mouse trigeminal ganglia were expressed as the mean of the log PFU, but the calculations did not include data for animals from which virus was not isolated. The limit of detection was 10 PFU.

Zosteriform model. Female hairless mice were infected with HSV-1 strain F (5 × 10⁵ PFU/50 µl) in the lower flank and were treated with a single application of topical formulations containing 3% foscarnet alone or 3% foscarnet with increasing SLS concentrations (1, 5, and 10%) 24 h after infection, as we have described previously (26).

Statistical analysis. The area under the curve (AUC) of the mean lesion score between days 4 and 10 was calculated for all animals including those that were asymptomatic. The AUC values for the different treatment groups were compared by a one-way analysis of variance test, followed as appropriate by a t test with Fisher's corrections for multiple simultaneous comparisons. The significance of the differences in (i) viral contents in tissues and (ii) titers of reactivated virus in explant cultures of latently infected trigeminal ganglia of mice treated with the gel alone and drug-treated groups was analyzed by an unpaired t test. The significance of the differences in the proportion of mice with (i) skin lesions, (ii) virus-positive tissue samples, and (iii) reactivated virus in trigeminal ganglion explants and (iv) the significance of the differences in the mortality rates between mice treated with the gel alone and drug-treated groups were evaluated by a chi-square test. All statistical analyses were performed with a computer package (Statview+SE Software; Abacus Concepts, Berkeley, Calif.). A P value of less than 0.05 was considered statistically significant.

	RESULTS

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Lesion scores. Figure 1 shows the time evolution of the mean lesion scores for untreated infected mice and infected mice treated twice daily for 3 days with the gel alone or with the gel containing 3% foscarnet and/or 5% SLS. Topical treatments were initiated at 6 h (Fig. 1A), 24 h (Fig. 1B), and 48 h (Fig. 1C) after virus inoculation. In untreated infected mice, vesicles began to appear 4 days after infection and became progressively coalescent and ulcerated. Herpetic skin lesions regressed spontaneously from day 11 to day 20 postinfection, and all animals survived the infection. Treatment of mice with the gel alone or with the gel containing 5% SLS did not reduce the mean lesion score compared to that for untreated infected mice. Topical formulations containing 3% foscarnet, alone or in combination with 5% SLS, given 6 or 24 h postinfection markedly reduced the development of herpetic skin lesions. Treatment of mice with the gel formulation containing 3% foscarnet 48 h after virus inoculation was less effective than earlier treatment. Of prime interest, the gel containing both 3% foscarnet and 5% SLS still significantly reduced the severities of the lesions. In addition, in all treatment regimens, the number of mice presenting with skin lesions was lower among animals treated with gel formulations containing foscarnet alone or foscarnet combined with SLS than among mice treated with gel alone (Table 1).

View larger version (27K): [in this window] [in a new window]   FIG. 1.   Time evolution of the mean lesion scores for hairless mice infected cutaneously in the triangular area of the snout with HSV-1 strain F and treated twice daily for 3 days with the gel alone (), gel containing 5% SLS (), gel containing 3% foscarnet (), or gel containing 3% foscarnet plus 5% SLS (). Untreated infected mice () were used as controls. Topical treatments were initiated 6 h (A), 24 h (B), or 48 h (C) after virus inoculation. Values represent the means for 8 to 12 animals per group.

Viral titers. Table 2 shows the influence of the time of initiation of topical treatments on viral titers measured in skin samples of the snout and the ipsilateral trigeminal ganglia. In untreated infected mice, high titers of virus were detected in skin tissues and the ispilateral trigeminal ganglia. Treatment of the mice with the gel alone or with the gel containing 5% SLS did not influence the titers of virus in these tissues. Gel formulations that contained 3% foscarnet, alone or in combination with 5% SLS, and that were given 6 h after virus inoculation significantly reduced the viral titers in the ipsilateral ganglia and in both the ipsilateral ganglia and skin tissue, respectively. In addition, these formulations reduced the number of mice with virus-positive tissue samples compared with the number of mice treated with the gel alone with virus-positive tissue samples. The gel formulation containing both foscarnet and SLS was still effective in reducing viral titers in skin tissues but not in ipsilateral trigeminal ganglia when treatment was started 24 h postinfection. In this treatment group, the number of mice with virus-positive skin samples was significantly decreased compared with the number of mice treated with the gel alone with virus-positive skin samples. No reduction of viral titers in the skin or ipsilateral ganglia was observed when treatments were initiated 48 h after virus inoculation.

Reactivation of latent virus. Table 4 shows the influence of the time of initiation of topical treatments on the mean titers of reactivated virus in explant cultures of ipsilateral and contralateral trigeminal ganglia from latently infected mice. In untreated infected mice, large amounts of latent virus reactivated from both types of trigeminal ganglia. Treatment of mice with the gel alone or containing 5% SLS had no effect on reactivation of latent virus in both types of ganglia. Topical formulations of foscarnet alone or foscarnet combined with SLS did not significantly influence the mean titers of reactivated virus in explant cultures of latently infected mouse ganglia.

Mortality rate. The mortality rate for hairless mice infected cutaneously with HSV-1 strain F in the lower flank (zosteriform model) and treated topically only once 24 h postinfection with gel formulations containing 3% foscarnet and/or increasing amounts of SLS was also examined. Among the untreated infected mice, 59% of animals died from encephalitis between days 7 and 12. Treatment of mice with the gel alone had no marked effect on mortality rates. The presence of 1 or 5% SLS in the gel formulation decreased the mortality rate to 35% (P < 0.05), whereas the addition of 10% SLS reduced it to 24% (P < 0.01). All formulations containing 3% foscarnet, with or without SLS, decreased the mortality rate to 18% (P < 0.01).

	DISCUSSION

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In the present study we have evaluated the influence of the time of initiation of topical treatment on the efficacies of gel formulations containing foscarnet, alone or in combination with SLS, against HSV-1 cutaneous lesions and on the establishment and reactivation of latent virus in mice. We selected HSV-1 strain F for our studies since its inoculation into the skin of the nose of 5- to 6-week-old mice caused latent infection in trigeminal ganglia in a large and reproducible percentage of the mice. Once established, the latent infection persisted in a reactivatable state for at least 6 months (15). Our data confirm that inoculation of HSV-1 strain F in the snout in hairless mice (orofacial model) allowed the appearance of skin lesions and the establishment of latent infections in the trigeminal ganglia of almost all mice without causing death of the animals. Conversely, the inoculation of HSV-1 strain F in the lumbosacral area of hairless mice (zosteriform model) was associated with an approximately 50 to 60% mortality rate among untreated infected mice (26, 27).

The efficacies of gel formulations containing foscarnet, alone or in combination with SLS, given 6 or 24 h after virus inoculation reduced to a similar extent the development of herpetic orofacial lesions. A greater efficacy was observed for the formulation containing the combination of foscarnet and SLS when treatment was initiated 48 h postinfection. The better efficacy of this gel formulation cannot be attributed to the licking or grooming of the topical formulations by the mice since previous studies from our laboratory have shown that with the zosteriform model of infection, in which the treated lesions were protected with corn cushions, the gel containing both 3% foscarnet and 5% SLS given only once 24 h postinfection was superior to the gel containing 3% foscarnet at reducing the mean lesion score (26). The incorporation of SLS into the gel formulation of foscarnet increases the level of penetration of the drug into the epidermis. This was attributed to the properties of SLS that enhance the fluidity of epidermal lipids (7, 19, 24, 25). The increased lipid fluidity below the applied site allows SLS to diffuse in the radial path without necessarily increasing transdermal drug delivery (24). SLS also inhibited the HSV-1 strain F-induced cytopathic effect probably by affecting newly synthesized viruses. SLS acts by decreasing the infectivities of herpesviruses, as demonstrated in vitro (8, 28).

The gel formulation of foscarnet in combination with SLS but not the gel containing 3% foscarnet alone significantly decreased the viral content in skin tissues when both treatments were given within 24 h postinfection. This effect is probably due to the increased skin penetration of foscarnet observed in the presence of SLS (26). However, this difference was seen only when viral titers were measured on day 4 postinfection, which corresponds to approximately 16 h after the cessation of therapy. Indeed, a rebound of infectious virus occurred on the following days, reaching levels similar to those in untreated infected mice. Thackray and Field (36, 37) have also reported a rebound of infectious virus in tissues following the cessation of therapy with valacyclovir. The high viral titers detected in the skin, despite the local application of a potent inhibitor of HSV replication, may be caused by a rapid decrease in the foscarnet concentration in this tissue following the cessation of therapy. Therefore, the remaining virus in skin tissues or a supply of virus coming back from the ganglia may still replicate to cause a rebound of infectious virus.

Gel formulations of foscarnet, alone or in combination with SLS, applied topically 6 h after virus inoculation also reduced viral titers in ipsilateral trigeminal ganglia when the titers were measured on day 4 postinfection. It was already shown that topical formulations containing acyclovir, phosphonoacetic acid, or foscarnet administered early in the course of infection could prevent in most cases the colonization of the trigeminal ganglia in a murine model of HSV-1 orofacial infection (9, 12-14). However, as in skin samples, we have also observed a rebound of infectious virus in the ipsilateral trigeminal ganglia on the following days. We have previously shown that foscarnet could not be detected in plasma after topical application of the gel with foscarnet even when SLS was added to the formulation (26). This suggests that foscarnet may decrease the amount of virus that migrates from the skin to the trigeminal ganglia, but once virus has reached the ganglia, the antiviral agent may have no effect on viral multiplication therein.

Latent infections can reproducibly be detected by maintenance of latently infected ganglia in explant culture for a few days followed by homogenization of ganglion explants in culture media and assay for the presence of infectious virus (6, 35). The mean titer of reactivated HSV in explant cultures of latently infected mouse trigeminal ganglia was maximal at day 9 or 10 (11). Titers of reactivated virus were highly reproducible, and the model was used to test the effects of drugs on the in vitro reactivation of HSV on latently infected ganglia (10, 11). A gel containing 3% foscarnet alone had no effect on the mean titers of reactivated virus in explant cultures of ipsilateral and contralateral trigeminal ganglia from latently infected mice. Klein et al. (9) have also reported that foscarnet did not prevent the reactivation of latent virus in trigeminal ganglia when topical treatment was initiated as early as 3 h after infection.

A gel formulation containing 5% SLS alone had no effect on the development of herpetic skin lesions, the amount of virus in skin tissues, the colonization of ipsilateral trigeminal ganglia, or the reactivation of latent virus in trigeminal ganglia. However, treatment of mice with this formulation only once at 24 h postinfection reduced significantly the rate of mortality associated with infection in the zosteriform model of infection. The administration of potent polyclonal and monoclonal immunoglobulin G (IgG) antibodies with high virus-neutralizing activities also protected mice from death even when they were given 1 or 2 days after infection (2, 20, 23, 34). Although the mechanism of action is not clearly understood, IgG antibodies may participate in antibody-dependent cellular cytotoxicity and antibody-dependent complement-mediated cytolysis (22, 34). Similar to SLS, antibodies could neutralize the infectivity of virus released from dying cells, thereby preventing local and distant virus dissemination (17). Those investigators postulated that IgG protects mice from death because it can block the spread of HSV after the onset of infection. However, they showed that mice receiving IgG tolerated a lethal dose of HSV-1 but shed more virus and had high virus titers in the eyes and trigeminal ganglia. In addition, in vitro reactivation of latent virus occurred in all trigeminal ganglion explants.

Treatment of mice with the gel formulation containing both foscarnet and SLS 6 h after infection had no effect on the mean titers of reactivated virus in explant cultures of latently infected ganglia. Similarly, these two active ingredients had no additive effect on the prevention of mortality of mice. This suggests that foscarnet may reduce or antagonize the effect exerted by SLS on HSV infection. In fact, we have recently demonstrated that combinations of foscarnet and SLS exert subsynergistic to subantagonistic effects on the HSV-1-induced cytopathic effect on Vero cells, depending on the concentrations used (26).

In conclusion, our results show that incorporation of SLS into a polymer matrix composed of polyoxypropylene and polyoxyethylene containing foscarnet increased the efficacy of the polymer matrix with foscarnet against herpetic cutaneous lesions. This formulation may represent an attractive approach for the treatment of mucocutaneous infections, especially those caused by acyclovir-resistant strains, which are less able to establish latent infections in ganglia (5, 16). In addition, SLS could be a convenient tool in studies of virus dissemination in the central nervous system.