Antiviral activity of a selective ribonucleotide reductase inhibitor against acyclovir-resistant herpes simplex virus type 1 in vivo

Abstract

The present study reports the activity of BILD 1633 SE against acyclovir (ACV)-resistant herpes simplex virus (HSV) infections in athymic nude (nu/nu) mice. BILD 1633 SE is a novel peptidomimetic inhibitor of HSV ribonucleotide reductase (RR). In vitro, it is more potent than ACV against several strains of wild-type as well as ACV-resistant HSV mutants. Its in vivo activity was tested against cutaneous viral infections in athymic nude mice infected with the ACV-resistant isolates HSV type 1 (HSV-1) dlsptk and PAAr5, which contain mutations in the viral thymidine kinase gene and the polymerase gene, respectively. Following cutaneous infection of athymic nude mice, both HSV-1 dlsptk and PAAr5 induced significant, reproducible, and persistent cutaneous lesions that lasted for more than 2 weeks. A 10-day treatment regimen with ACV given topically four times a day as a 5% cream or orally at up to 5 mg/ml in drinking water was partially effective against HSV-1 PAAr5 infection with a reduction of the area under the concentration-time curve (AUC) of 34 to 48%. The effects of ACV against HSV-1 dlsptk infection were not significant when it was administered topically and were only marginal when it was given in drinking water. Treatment under identical conditions with 5% topical BILD 1633 SE significantly reduced the cutaneous lesions caused by both HSV-1 dlsptk and PAAr5 infections. The effect of BILD 1633 SE against HSV-1 PAAr5 infections was more prominent and was inoculum and dose dependent, with AUC reductions of 96 and 67% against infections with 10(6) and 10(7) PFU per inoculation site, respectively. BILD 1633 SE also significantly decreased the lesions caused by HSV-1 dlsptk infection (28 to 51% AUC reduction). Combination therapy with topical BILD 1633 SE (5%) and ACV in drinking water (5 mg/ml) produced an antiviral effect against HSV-1 dlsptk and PAAr5 infections that was more than the sum of the effects of both drugs. This is the first report that a selective HSV RR subunit association inhibitor can be effective against ACV-resistant HSV infections in vivo.

FIG. 1

Molecular structure of BILD 1633 SE. Me, methyl.

FIG. 2

Effect of ACV in combination with BILD 1633 SE against wild-type and ACV-resistant HSV in vitro. Selected concentrations of BILD 1633 SE were tested in combination with various doses of ACV, and the EC₅₀s for HSV KOS, F, PAA^r5, and dlsptk were determined by the ELISA-based antiviral assay as described in Materials and Methods. These values were used to calculate FEC₅₀ (ACV), and FEC₅₀ (ACV) was plotted as a function of the ratio of the fixed concentration of BILD 1633 SE to the EC₅₀ of BILD 1633 SE for the respective viruses to generate the isobologram. (A) HSV-1 F; (B) HSV-1 KOS; (C) HSV-1 dlsptk; (D) HSV-1 PAA^r5.

FIG. 3

Comparative effects of ACV and BILD 1633 SE against HSV-1 PAA^r5 infection. Animals were cutaneously inoculated with 10⁶ PFU/site, as described in Materials and Methods. ACV (5%) and BILD 1633 SE (5%) were applied topically four times per day. (A) Mean lesion scores. The mean lesion score was significantly reduced by ACV treatment on days 12-14 (n = 10; P < 0.05) and was further reduced by BILD 1633 SE on days 10 to 24 (n = 24; the result was significantly different [P < 0.05] from those for all other groups). (B) AUCs of the lesion scores. The AUCs of lesion scores are presented as means ± SEMs. ∗, P < 0.05 compared with the results for all other groups.

FIG. 4

Effects of BILD 1633 SE and ACV against HSV-1 PAA^r5 infection. Animals were cutaneously inoculated with 10⁷ PFU/site, as described in Materials and Methods. (A and B) BILD 1633 SE and ACV were applied in 5% topical formulation. (C and D) BILD 1633 SE was applied four times a day at concentrations of 0, 0.8, 2, and 5%. The AUCs of the lesion scores are presented as means ± SEMs (n = 12). ∗, P < 0.05 compared with the results for the vehicle group; †, P < 0.05 compared with the results for the vehicle and 0.8% BILD 1633 SE groups.

FIG. 5

Antiviral effects of ACV in drinking water against HSV-1 PAA^r5 infection. Animals were cutaneously inoculated with 10⁷ PFU/site, as described in Materials and Methods. ACV was given in the drinking water at 0, 1, 3, and 5 mg/ml. (A) Mean lesion scores. (B) AUC of the lesion scores. The AUCs of the lesion scores are presented as means ± SEMs (n = 12). ∗, P < 0.05 compared with the results for the vehicle group.

FIG. 6

Antiviral effects of oral ACV and topical BILD 1633 SE against HSV-1 PAA^r5 infection. Animals were cutaneously inoculated with 10⁷ PFU/site, as described in Materials and Methods. ACV was given in the drinking water at a dose of 1.5 mg/ml. BILD 1633 SE (0.8%) was applied topically four times per day. (A) Mean lesion scores. (B) AUC of the lesion scores. The AUCs of the lesion scores are presented as means ± SEMs (n = 24). ∗, P < 0.05 compared with the results for the vehicle groups.

FIG. 7

Comparative effects of oral ACV and topical BILD 1633 SE against HSV-1 dlsptk infection. Animals were cutaneously inoculated with 10⁷ PFU/site, as described in Materials and Methods. ACV (5%) and BILD 1633 SE (5%) were applied topically four times per day. (A) Mean lesion scores. (B) AUCs of the lesion scores. ACV only transiently (n = 12; P < 0.05) reduced the mean lesion score on days 13 to 15 compared with the scores for the no-treatment group (n = 12) and the vehicle group (n = 24). BILD 1633 SE more effectively reduced the mean lesion score (n = 24; P < 0.05 on days 8 to 24). The AUCs of the lesion scores are presented as means ± SEMs. ∗, P < 0.05 compared with the results for the vehicle groups.

FIG. 8

Antiviral effects of oral ACV and topical BILD 1633 SE against HSV-1 dlsptk infection. Animals were cutaneously inoculated with 10⁷ PFU/site, as described in Materials and Methods. ACV was given in drinking water at a dose of 5 mg/ml. BILD 1633 SE (5%) was applied topically four times per day. (A) Mean lesion scores. (B) AUCs of the lesion scores. ACV transiently (P < 0.05) reduced the mean lesion score on days 12 to 15 compared with the scores for the vehicle control group. BILD 1633 SE more effectively reduced the mean lesion score (P < 0.05 on days 4 to 24 versus both the vehicle and the ACV groups). Combination therapy further reduced the mean lesion scores significantly from those for all other groups on days 3 to 24. The AUCs of lesion scores are presented as means ± SEMs (n = 24). ∗, P < 0.05 compared with the results for all other groups.