Vol. 36, No. 5

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1992, p. 934-937

0066-4804/92/050934-04$02.00/0 Copyright © 1992, American Society for Microbiology

Inactivators of Herpes Simplex Virus Ribonucleotide Reductase: Hematological Profiles and In Vivo Potentiation of the Antiviral Activity of Acyclovir THOMAS

SPECTOR,`*

DAVID C. LOBE,2 M. NIXON ELLIS,2 TODD A. BLUMENKOPF,3 AND GEORGE M. SZCZECH4 Therapy, 1 Virology,2 Organic Chemistry,3 and Toxicology and Pathology,4

Divisions of Experimental Wellcome Research

Laboratories, Research Tniangle Park North Carolina 27709

Received 12 November 1991/Accepted 21 January 1992

A111OU (BW 1110U81) is an inactivator of herpesvirus ribonucleotide reductases and a potentiator of the antiviral activity of acyclovir (ACV) (T. Spector, J. A. Harrington, R. W. Morrison, Jr., C. U. Lambe, D. J. Nelson, D. R. Averett, K. Biron, and P. A. Furman, Proc. Natl. Acad. Sci. USA 86:1051-1055, 1989) that was subsequently found to cause hematological toxicity at high oral doses in rats. Eleven structurally related inactivators of herpes simplex virus (HSV) ribonucleotide reductase were therefore tested in vivo for hematological toxicity and for potentiation of ACV. None of the novel ribonucleotide reductase inactivators was hematologically toxic to rats following oral dosing at 60 mg/kg/day for 30 days. Four of these inactivators statistically improved the antiviral topical potency of ACV on HSV type 1-infected nude mice. A promising compound, 2-acetylpyridine 5-[(2-chloroanilino)thiocarbonyl]thiocarbonohydrazone (BW 348U87), was studied more extensively in two in vivo models: dorsum-infected athymic nude mice and snout-infected hairless mice. BW 348U87 significantly potentiated the antiviral activity of ACV against all virus strains tested, i.e., wild-type (ACV-sensitive) HSV type 1 and HSV type 2 strains and three mutant (ACV-resistant) HSV type 1 strains. The latter included a virus expressing a DNA polymerase resistant to inhibition by ACV triphosphate, a virus deficient in thymidine kinase (the enzyme responsible for phosphorylating ACV), and a virus expressing an altered thymidine kinase, which catalyzes the normal phosphorylation of thymidine but not of ACV. BW 348U87 and ACV are currently being developed as a combination topical therapy for cutaneous herpes infections.

1110U81 at 60 mg/kg/day for 30 days developed hematological toxicity. Although a topically applied compound would not be expected to expose humans to comparable levels of BW 1110U81, this toxicity was nevertheless undesirable. We therefore sought safer inactivators with similar efficacies. In the present study, a series of compounds that had demonstrated apparently selective inactivation of HSV ribonucleotide reductase (1) were tested for hematological toxicity in rats and for antiviral efficacy in combination with ACV on mice. One hematologically nontoxic and highly efficacious compound, 2-acetylpyridine5-[(2-chloroanilino)thiocarbonyl]thiocarbonohydrazone (BW 348U87), was studied further. The mechanism of inactivation of HSV type 1 (HSV-1) ribonucleotide reductase by BW 348U87 has been reported elsewhere (19).

Acyclovir (ACV) is used clinically to treat herpes simplex virus (HSV) and varicella-zoster virus (VZV) infections. It is selectively phosphorylated in herpesvirus-infected cells. The fully activated drug, ACV triphosphate (ACV-TP), competes with dGTP for binding to the target enzyme, herpesvirus DNA polymerase (recently reviewed in references 11 and 17). Because HSV and VZV code for unusual ribonucleotide reductases that are not subject to the customary allosteric product inhibition of the mammalian counterpart enzymes (5, 6, 20), they induce the unrestricted synthesis of deoxynucleotides (reviewed in references 13 and 14). Consequently, HSV (and presumably VZV)-infected cells treated with ACV develop increased pools of dGTP, which could impede the binding of ACV-TP to herpesvirus DNA polymerases (3, 16, 18). We therefore studied inactivators of herpesvirus ribonucleotide reductases as a means of preventing the buildup of dGTP. Surprisingly, these inactivators not only decreased the dGTP pools but also markedly increased the pools of ACV-TP, thereby enhancing the ACV-TP/dGTP ratio by 90-fold (16, 18). Thus, 2-acetylpyri-

MATERIALS AND METHODS Ribonucleotide reductase inactivators. BW 1110U81 was synthesized as previously described (18). Other inactivators were synthesized in our laboratories (1). Hematological toxicity. Groups of five male and five female CD rats (Crl:CDBR VAF/Plus), purchased from Charles River Breeding Laboratories, Inc., Raleigh, N.C., were dosed orally for 30 consecutive days with the compounds listed in Table 1 at 60 mg/kg/day. Test substances were suspended in 0.5% methylcellulose and given by gavage. Complete blood counts without differential cell counts were measured once before dosing, after 2 weeks of dosing, on the last day of dosing, and 30 days after dosing. The evaluated parameters are listed in Table 1, footnote a. The data were

dine-4-(2-morpholinoethyl)thiosemicarbazone (A723U), an inactivator of herpesvirus ribonucleotide reductases, potentiates the antiviral activity of ACV in vitro (7, 16) and 2acetylpyridine - 5 - [(dimethylamino)thiocarbonyl]thiocarbonohydrazone (AlllOU or BW 1110U81), a more potent inactivator (10), potentiates the antiviral activity of ACV in vivo (2, 8) as well as in vitro (18). Unfortunately, in this study, rats dosed orally with BW *

Corresponding author. 934

POTENTIATORS OF ACYCLOVIR

VOL. 36, 1992

TABLE 1. In vivo hematological toxicity of an potentiation of the antiviral activity of ACV by inactivators of HSV-1 ribonucleotide reductase CH3

S

S

)KNHN )R HEMATOLOGICAL R=

COMPOUND

BW 111OU81 BW 348U87

N(CH3)2 Cl

POTENTIATIONC

ToxcrrY

OF ANTI-HSV-1 ACTIVITY OF ACV % (S-E.)

positivea

73 (1O)d

negativeb

67 (20)d

NHO BW 349U87

NH-c-I

negative

45 (20)

BW 464U87

CH3O

negative

49 (9)

NHQ NO2

BW 606U87

BW 952UU88

I

negative

53 (10)d

negative

23 (5)

negative

41 (10)

negative

61 (9)d

negative

30 (8)

negative

54 (10)d

negative

28 (7)

negative

32 (7)

NH-) BW 955U88 BW 956U88

cNH C( NHM

BW 1055U88

NH§ BW 1057U88

CH3 NH--CI

BW 1 058U88

BW 1110U88

NH H

Br

CH3O

NHA Cl

Rats orally dosed with BW 110U81 had the following changes, compared with untreated animals: hematocrit, 10.5% decrease; hemoglobin, 10% decrease; erythrocyte count, 24% decrease; erythrocyte distribution width, 53% increase. Less than a 10% change occurred in the platelet count and the leucocyte count (relative and absolute). b Negative, no significant hematological changes (P > 0.05). c Nude mice infected with wild-type HSV-1 on the dorsal flank were either untreated or treated topically with 5% ACV or with 5% ACV plus 3% ribonucleotide reductase inhibitor (see Materials and Methods). AUC values from six experiments ranged from 61 to 72 for untreated animals and from 27 to 33 for ACV-treated animals. The percent potentiation of ACV was equal to 100 {[(AUC of ACV + inhibitor)/(AUC of ACV alone)1100}. d Combination therapies that were significantly more effective (P < 0.05) than therapy with ACV alone. a

-

935

analyzed by a one-way analysis of variance comparing treated animals on dose day 30 to control rats concurrently dosed with 0.5% methylcellulose. Antiviral efficacy. The analogs listed in Table 1 were tested on nude mice (about 10 per group) infected with wild-type HSV-1 on the dorsal flank as previously described (8). Mice were treated with topical preparations of 5% ACV or 3% analog combined with 5% ACV (suspended in modified aqueous cream [2, 8]) three times per day on days 1 to 5 postinfection. Lesion scores were measured (8) for 17 to 20 days, and the mean lesion score versus day was graphed. Areas under the curve (AUC) of the lesion score-day graph were calculated by the trapezoidal rule. AUC values thus summarized the severity of the infection. Combination therapies were compared with treatment with ACV alone by a one-way analysis of variance of the mean AUC values. BW 348U87 was tested for greater-than-additive antiviral therapy with ACV as described elsewhere (2, 8). BW 348U87 was considered to potentiate or to synergize with ACV when the observed AUC of the combination therapy was significantly smaller than the AUC calculated for the sum of the independent antiviral activities of ACV and BW 348U87. The latter was calculated from the following equation:

(AUGC JAUCcontro1)(AUCBW 348U87IAUCcontroI)(AUCcontroI)

Details of the various experiments with BW 348U87 and ACV are described in Table 2, footnote a. Animals and viruses. Athymic nude mice and hairless mice were purchased and caged as described previously (2, 8). The strains of wild-type and ACV-resistant HSV and their in vitro susceptibilities to ACV have been described previously (8). Mice were infected on either the dorsum (8) or the snout

(2). RESULTS Hematological toxicity. BW 111OU81 given to rats by gavage at 60 mg/kg/day for 30 days caused significant hematological toxicity (Table 1). Eleven new compounds recently found to potently inactivate HSV ribonucleotide reductase (1) did not produce hematological toxicity (Table 1). Therefore, all eleven inactivators were tested in combination with ACV for antiviral efficacy in vivo. Antiviral efficacy. Mice infected on the dorsum with wildtype HSV-1 were treated with topical preparations of either 5% ACV or 5% ACV plus 3% inactivator or were untreated. Untreated control mice fully developed "zosteriformlike" rashes in about 7 days. Neither BW 111OU81 (8) nor BW 348U87 (see below) produced detectable individual antiviral activity in this particular model. ACV treatment significantly delayed rash development and showed significant (P < 0.05) antiviral activity. The AUC values for ACV-treated animals were approximately one-half those for untreated animals. BW 111OU81 and the other inactivators, in combination with ACV, markedly suppressed lesion formation, and the AUC values were smaller than those with ACV alone. The percent improvement of the combination therapy over ACV therapy is presented in Table 1. However, only combinations of ACV with BW 111OU81, BW 348U87, BW 606U87, BW 956U87, and BW 1057U88 produced significantly (P < 0.05) smaller AUC values than ACV. The promising activity of BW 348U87 encouraged additional efficacy studies with several wild-type and ACV-resistant HSV strains. Enhanced antiviral activity of BW 348U87 and ACV against ACV-susceptible and ACV-resistant viruses. BW 348U87 and ACV were tested alone and in combination against wild-type (ACV-sensitive) HSV-1 and HSV-2 and three mutant (ACV-

936

SPECTOR ET AL.

ANTIMICROB. AGENTS CHEMOTHER.

TABLE 2. Enhanced topical therapy of BW 348U87 and ACVa Virus and

Expt

1

2

3

4

5

treatment

AUC, % (SE) of control AUC

Wild-type HSV-1 BW 348U87 ACV BW 348U87 + ACV

100 (2) 47 (7) 23 (7), P = 0.01

Wild-type HSV-2 BW 348U87 ACV BW 348U87 + ACV

106 (2) 72 (3) 34 (10), P = 0.004

ACV-resistant HSV-1 (polymerase mutant) BW 348U87 ACV BW 348U87 + ACV

348U87

3

0

-j

2 U

65 (20) 62 (15) 4 (2), P = 0.03

348U87

r plus ACV

0

0 5 treat on days 1-5

ACV-resistant HSV-1 (thymidine kinasedeficient mutant) BW 348U87 ACV BW 348U87 + ACV

96 (10) 72 (4) 30(2), P = 0.002

ACV-resistant HSV-1 (altered thymidine kinase mutant) BW 348U87 ACV BW 348U87 + ACV

74 (4) 71 (5) 6 (2), P = 0.000004

10

15

20

Days Post Infection

a In experiments 1 to 4, 10 to 12 athymic nude mice per group were dorsally infected, treated on days 1 to 5 (1 to 4 for experiment 4) postinfection, and scored for 16 days (15 days in experiment 4). In experiment 5, nine hairless mice per group were infected via the snount, treated on days 0.5 to 4 postinfection, and scored for 10 days. The AUC values (standard errors) for the control (untreated) groups for experiments 1 to 5 were 63 (2), 57 (7), 39 (9), 32 (4), and 24 (2), respectively. The combination therapy was considered enhanced (either synergy or potentiation) when the observed AUC was significantly smaller than the AUC calculated for the sum of the independent antiviral activities of ACV and BW 348U87 (see Materials and Methods). The calculated percent AUC values (standard errors), relative to the controls, for the combination therapy in expreiments 1 to 5 were 47 (7), 76 (10), 40 (20), 70 (10), and 52 (7), respectively. P values, determined with a two-sample Student's t test, represent the level of significance of the difference between the observed and calculated values.

resistant) HSV-1 strains. The latter included a virus (46-fold resistant) expressing a DNA polymerase resistant to inhibition by ACV-TP, a virus (11-fold resistant) deficient in thymidine kinase (the enzyme responsible for phosphorylating ACV), and a virus (14-fold resistant) expressing an altered thymidine kinase, which catalyzes the normal phosphorylation of thymidine but not of ACV. Athymic mice infected on the dorsum (experiments 1 to 4) and hairless mice infected on the snout (experiment 5) were used as models. BW 348U87 significantly potentiated the antiviral efficacy of ACV against all virus strains (Table 2). BW 348U87 demonstrated statistically significant inherent antiviral activity only against HSV-1 expressing a substrate specificity-altered thymidine kinase. The data from experiments with the DNA polymerase and altered thymidine kinase mutants are shown in Fig. 1 and 2. No obvious dermal toxicity was observed in any study. Similar results were noted for athymic nude mice infected with the thymidine kinase-altered (ACV-resistant) HSV-1 mutant (data not

shown).

aCO)

FIG. 1. Synergistic therapy with BW 348U87 and ACV for athymic mice infected with an ACV-resistant HSV-1 mutant expressing an altered DNA polymerase. Shown is a graphic presentation of the data from experiment 3 of Table 2. The topical therapies are indicated.

DISCUSSION BW 1110U81 potentiates or synergizes with ACV as a topical treatment for mice infected with several wild-type and ACV-resistant HSVs (2, 8). It also produces synergistic inhibition of VZV replication in vitro (18). Although the present finding that rats developed hematological toxicity when dosed orally with BW 1110U81 at 60 mg/kg/day for 30 days did not prohibit the development of this topical therapy, it did detract from the overall safety profile and 4

none

TREATMENT/

O C 0

, 2