ANIMAL STUDIES: ACUTE INFECTIONS

Acyclovir Treatment of Cutaneous Herpes in Guinea Pigs and

Herpes Encephalitis in Mice

PETER COLLINS, Ph.D. NICHOLAS M. OLIVER

Beckenham, United Kingdom

Animal models of cutaneous herpes simplex and herpetic enceph- alitis were used to demonstrate the antiviral potential of acyclovir. Of the various topical formulations of acyclovir tested against herpes simplex in guinea pigs, 5 percent acyclovir in modified aqueous cream was the most effective. Propylene glycol was included in this preparation to increase the aqueous solubility of acyclovir. Acyclovir and vidarabine were found to be equally effective against herpetic encephalitis in mice when administered either orally or subcuta- neously at 100 mg/kg.

Acyclovir l (9-[ 2-hydroxyethoxymethyl] guanine) has been shown to be highly effective against herpes simplex virus in both cell culture [l-3] and animal models [4-81. However, the route of administration, timing of treatment, and the vehicle may be crucial to maximum antiviral effect. Two animal models were chosen to investigate this: topical formulations for cutaneous herpes in guinea pigs [9], and routes of systemic administration for herpes encephalitis in mice.

The cutaneous herpes simplex infection induced in guinea pigs resembles that in humans. The infection is self-limiting and lasts 12 to 14 days, with active viral replication detected during the first four to six days. The lesions then crust over and heal.

lntracerebral infection of mice with herpes simplex virus induces a rapid, fatal encephalitis with death usually occurring three to six days after infection. Although this model does not reflect the course of encephalitis in humans, successful treatment of such infections may indicate therapeutic potential for an antiviral compound [lo]. This model was used to compare the systemic activity of acyclovir with vidarabine, a compound with antiviral potential against this condition in humans [ 1 l].

MATERIALS AND METHODS

Virus Strains. In the guinea pig skin model the H31 strain of herpes simplex virus type 1 was used as a crude cell extract. Virus stocks were prepared in Vero cells and 1 ml aliquots of cell-extracted supernatant material were stored at -7OOC.

From the Antiviral Chemotherapy Section, Well- come Research Laboratories, Beckenham, Kent, United Kingdom. Requests for reprints should be addressed to Dr. Peter Collins, Antiviral Chemo- therapy Section, Wellcome Research Laborato- ries, Langley Court, Beckenham, Kent, BR3 3B.S. United Kingdom.

To induce encephalitis in mice the Beckenham type 1 and the 9F type 2 strains of herpes simplex virus were used. Stock virus was prepared by passing cell culture extracted virus once in mouse brain. Infected brains were then ground to a paste in a pestle and mortar, resuspended in phosphate buffered saline, clarified, and mixed 2:l with glycerol. Aliquots of the sus- pension were stored at -2OOC. Animals. For the skin model, 12 to 14 week old female albino guinea pigs weighing 600 to 700 g were used (Porcellus Animal Breeding Ltd., Heathfield, United Kingdom). The animals were housed singly or in pairs and given feed

l Zovirax,@ Burroughs Wellcome.

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ANIMAL STUDIES: ACUTE INFECTIONS-COLLINS, OLIVER

Figure 1. A comparison of acyclovir treatments of cutaneous herpes simplex in guinea pigs. AC V = acyclovir; MAC = modified aqueous cream; PEG = poly- ethylene glycol ointment. Treatment was twice daily for three days except for 5 percent ACVIPEG (W), which was four times daily for three days.

50 -

40 -

fi 30. In

20 -

10 _

o- 1 2 3 4 5 6 10

Time after treatment begun (days)

(F.P.G. diet, Oxoid Labs., H.C. Styles (Bewdley Ltd.), Bewdley, United Kingdom) and water ad lib.

CD.1 mice (Charles River U.K. Ltd., Manston, Kent) weighing 12 to 16 g were used in the experimental enceph- alitis model. The animals were housed in groups of 5 to 10 and given feed (P.C.D. diet, B.P. Nutrition, Whitham, Essex) and water ad lib. lnoculatlon Procedures. Cutaneous herpes in guinea pigs: Both flanks of the guinea pigs were closely shaved and the remaining fur removed with a proprietary depilatory cream. The exposed areas of the skin were then washed and dried. After four hours, eight sites on each flank were lightly scar- ified with a dulled scalpel blade through a drop of virus sus- pension (H31 strain of herpes simplex virus type 1 containing lo6 PFU/ml). Herpetic sncephelltis in mice: Mice were anesthetized with ether and inoculated intracerebrally with 0.025 ml herpes virus suspension containing 300 iDso. Anti&al Compounds. Acyclovir was supplied by Dr. H. J. Schaeffer, boughs Weilcome Co., Research Triangle Park, NC. Topical formulations of acyclovir were prepared by Mr. A. White, Pharmaceutical Development Laboratories, Well- come Foundation Ltd., Dartford, Kent. Vidarabine (9- /%arabinofuranosyladenine) was purchased from the Sigma Chemical Co., Poole, Dorset. For systemic administration to mice, vkiarabine and acyclovir were prepared as suspensions containing 20 mg/ml. Test Procedures. Cutaneous herpes in guinea pigs: The right flanks of infected guinea pigs were treated twice daily with 1 ml of the test preparations, beginning 18 hours after infection and continuing for three days. Extent of infection was assessed daily and the animals were photographed on

completion of treatment and three days later. Variables were assessed as follows: 0, 1,2, or 3 for specific er@ema around the site of infection, and pustule formation, size, number, and whether confluent; or 0, 1, or 2 for induration at the site of infection; scab formation; and area of infection over and above that infected.

The left flanks were either untreated or placebo-treated to serve as controls. Herpetic encephalitis in mice: Mice were treated either orally or subcutaneously with suspensions of acyclovir or vidarabine, 190 mg/kg twice daily for five days. The recip- rocal survival times of the animals were recortled and com- pared.

RESULTS

Cutaneous Herpes in Guinea Pigs. Acyclovir formu- lated as an ointment in polyethylene glycol 300 and polyethylene glycol 1540 (60:40) has been shown to be effective against cutaneous herpes simpkx virus in- fections in animals. Although this was effective in our laboratory the rate of cure was slow. Extensive for- mulation studies were therefore begun. These studies indicated that the donor phase of the formulation was aqueous rather than organic, and therefore any increase in the aqueous content of acyclovir shouldi result in its increased availability. This was achieved by adding 40 percent propylene glycol to aqueous cream which in- creased the aqueous solubility of acyclovir to ap- proximately 0.3 percent. Preliminary studies with a 5 percent preparation of acyclovir in aqueous cream

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ANIMAL STUDIES: ACUTE INFECTIONS-COLLINS, OLIVER

Treatment

- PEG PLACEBO Treatment 4

104 - MAC PLACEBO

rl 5% ACV in PEG

- 5% ACV in MAC

0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7

TIME AFTER TREATMENT BEGUN (DAYS) TIME AFTER TREATMENT BEGUN (DAYS)

Figure 2. Comparative virus isolations from guinea pigs treated with 5 percent acyclovir in MAC or PEG. ACV = acyclovir; MAC = modified aqueous cream; PEG = polyethylene glycol ointment; pfuiml = plaque-forming units/ml. Treatment was twice daily for three days.

containing 40 percent propylene glycol resulted in a rapid and effective cure. This formulation was then compared with the 5 percent acyclovir polyethylene glycol ointment.

These results show clearly that 5 percent acyclovir in MAC produces a more rapid cure than 5 percent acyclovir in PEG ointment. The response after therapy

A total of 40 albino guinea pigs were infected with the H31 strain of herpes simplex virus and were divided

with 5 percent acyclovir in MAC was immediate, and

into groups of 10. The animals’ right flanks were treated twice daily for three days beginning 18 hours after in-

after only four applications few signs of infection were

fection, as follows: group 1, 5 percent acyclovir in polyethylene glycol ointment (PEG); group 2, polyeth- ylene glycol ointment placebo control; group 3, 5 per- cent acyclovir in modified aqueous cream (MAC) con- taining 40 percent propylene glycol; and group 4, modified aqueous cream (MAC) placebo control. The mean daily scores are presented in Figure 1.

evident. Placebo preparations had no effect on the course of infection.

In a subsequent experiment, three guinea pigs were

Virus specimens were taken before therapy, during the first four days after infection and again three days

infected with the H31 strain of herpes simplex virus type

later. The virus content of the specimens was measured by plaque assay in Vero cells and the mean virus titers

1 as described, and were treated with 5 percent acy-

are shown in Figure 2. Virus titers were found to peak two to three days after infection when left untreated or

clovir in PEG ointment four times daily for three days.

treated with placebo preparations and virus was cul- tured up to seven days after infection. High titers of virus

Although the extent of virus infection and resultant viral

were detected after one day of therapy with 5 percent acyclovir in PEG ointment but none was detected sub- sequently. Virus was barely detectable after therapy for one day with 5 percent acyclovir in MAC, a further in- dication of this formulation’s rapid effect.

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ANIMAL STUDIES: ACUTE INFECTIONS-COLLINS, OLIVER

damage appeared less than expected, as indicated in the virus control scores, increased frequency of dosing

increased the antiviral effect (Fig. 1).

Herpetic Encephalitis in Mice. A total of 160 CD. 1 mice were inoculated intracerebrally with herpes sim- plex virus: 80 with the Beckenham type 1 strain and 80 with the 9F type 2 strain. Groups of 10 mice infected with each virus type were treated with subcutaneous acyclovir 100 mg/kg, subcutaneous vidarabine 100 mg/kg, oral acyclovir 100 mg/kg, or oral vidarabine 100 mg/kg. The remaining animals were untreated virus controls. Survival times were recorded in days and converted to reciprocals, from which a mean reciprocal survival time was calculated for each treatment group. Reduction in mean reciprocal survival times compared with virus controls indicates an antiviral effect.

The results shown in Table I suggest that acyclovir is no less active than vidarabine in this model of herpes simplex virus encephalitis.

CONCLUSIONS

The reformulation of acyclovir in a modified aqueous cream, in which the acyclovir content in the aqueous phase was increased, was more effective against cu- taneous herpes in guinea pigs than the 5 percent acy- clovir in PEG ointment. This was evident from data on virus shedding and assessments of the extent of in- fection. Although the activity of 5 percent acyclovir in PEG ointment increased with frequency of application, the rapid effect of 5 percent acyclovir in MAC during the

period of active viral replication suggests that this preparation offers advantages in the treatment of cu- taneous herpes simplex infections. Therapy in such self-limiting infections should be started as early as possible to exert a material effect, preferably during the prodromal period if it can be recognized.

TABLE I Comparison of the Mean Reciprocal Survival Times after Acyclovir and Vidarabine Treatment of Herpes Simplex Virus-Induced Encephalitis in Mice

Virus Type Treatment Group Mean Reciprocal

Survival Time

Beckenham Type 1 virus control subcutaneous acyclovir subcutaneous

vidarabine oral acyctovir oral vidarabine

9F Type 2 virus control subcutaneous acyclovir subcutaneous

vidarabine oral acyclovir oral vidarabine

0.301 0.185 0.114’

0.145 0.149 0.285 0.106’ 0.155

0.156 0.185

l 2/ 10 survivors. + 1 / 10 survivor.

These results would indicate that 5 percent acyclovir in MAC administered early in the infection process is likely to be of great use in the treatment of cutaneous herpes simplex virus infections in humans.

The comparative study of vidarabine and acyclovir against herpetic encephalitis in mice highlighted the intractability of this condition. This animal model is a stringent test of antiviral activity since the virus is in- oculated directly into the brain, and therefore any pro- longation of survival time represents a significant antiviral effect. Similar increases in survival times were observed after oral and subcutaneous therapy with vidarabine and acyclovir. These results lead us to conclude that if this activity can be demonstrated in humans, acyclovir may become a useful therapy for this devastating condition.

REFERENCES

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4. Bauer DJ, Collins P, Tucker WE, Macklin AW: Treatment of 10. Bauer DJ: The chemotherapeutic activity of compounds of experimental herpes simplex keratitis with acyclogua- copper, rhodium and certain other metals in mice infected nosine. Br J Ophthalmol 1979; 63: 429-435. with neurovaccinia and ectromelia viruses. Br J Exp Pathol

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