Inhibition of Cytomegalovirus Replication with Extended-Half-Life Synthetic Ozonides

Artesunate (AS), a semisynthetic artemisinin approved for malaria therapy, inhibits human cytomegalovirus (HCMV) replication in vitro, but therapeutic success in humans has been variable. We hypothesized that the short in vivo half-life of AS may contribute to the different treatment outcomes.

ABSTRACT

Artesunate (AS), a semisynthetic artemisinin approved for malaria therapy, inhibits human cytomegalovirus (HCMV) replication in vitro, but therapeutic success in humans has been variable. We hypothesized that the short in vivo half-life of AS may contribute to the different treatment outcomes. We tested novel synthetic ozonides with longer half-lives against HCMV in vitro and mouse cytomegalovirus (MCMV) in vivo. Screening of the activities of four ozonides against a pp28-luciferase-expressing HCMV Towne recombinant identified OZ418 to have the best selectivity; its effective concentration inhibiting viral growth by 50% (EC₅₀) was 9.8 ± 0.2 µM, and cytotoxicity in noninfected human fibroblasts (the concentration inhibiting cell growth by 50% [CC₅₀]) was 128.1 ± 8.0 µM. In plaque reduction assays, OZ418 inhibited HCMV TB40 in a concentration-dependent manner as well as a ganciclovir (GCV)-resistant HCMV isolate. The combination of OZ418 and GCV was synergistic in HCMV inhibition in vitro. Virus inhibition by OZ418 occurred at an early stage and was dependent on the cell density at the time of infection. OZ418 treatment reversed HCMV-mediated cell cycle progression and correlated with the reduction of HCMV-induced expression of pRb, E2F1, and cyclin-dependent kinases 1, 2, 4, and 6. In an MCMV model, once-daily oral administration of OZ418 had significantly improved efficacy against MCMV compared to that of twice-daily oral AS. A parallel pharmacokinetic study with a single oral dose of OZ418 or AS showed a prolonged plasma half-life and higher unbound concentrations of OZ418 than unbound concentrations of AS. In summary, ozonides are proposed to be potential therapeutics, alone or in combination with GCV, for HCMV infection in humans.

INTRODUCTION

Infection with human cytomegalovirus (HCMV), a member of the Herpesvirus family, is common in humans. Seroprevalence rates increase with age, reaching 90% in individuals older than 80 years of age (1). While infection is usually asymptomatic, HCMV continues to be a serious threat for transplant recipients and patients with AIDS. It is also the most common congenital infection worldwide, causing hearing loss, mental retardation, and central nervous system damage in children (2–6).

The systemic anti-HCMV drugs target the viral DNA polymerase and efficiently suppress virus replication. However, their use is associated with considerable toxicities to the bone marrow (ganciclovir [GCV]) and kidneys (foscarnet and cidofovir) and the emergence of resistant viruses (7–10). Until recently, intravenous GCV was the only drug approved for the treatment of congenital HCMV infection with central nervous system involvement, based on a phase III clinical trial that documented the prevention of hearing loss in treated children (11). A phase III clinical trial of oral valganciclovir (the valyl ester prodrug of GCV) in congenitally infected infants suggests that 6 months of therapy may produce a better neurological outcome than 6 weeks of therapy, but GCV-resistant mutants emerge (12). The widespread use of a limited number of drugs often leads to the development of drug-resistant strains (13, 14). Thus, new drugs are needed for HCMV therapy. The terminase inhibitor letermovir, used as prophylaxis, was recently reported to decrease the incidence of HCMV infection through 24 weeks following hematopoietic stem cell transplantation (HSCT) and received FDA approval for this indication (15).

Semisynthetic artemisinins are highly effective drugs for the treatment of malaria, and interest in these agents has increased because of their anti-HCMV activities (16–21). Artesunate (AS) and artemether are orally available and well tolerated. Millions of children have been treated with artemisinin derivatives for malaria, with no significant adverse effects for the standard 3-day treatment regimen or a repeated course of therapy. AS has previously been administered to several immunocompromised patients with HCMV disease, but the results were variable (22–26). The short half-life (t_1/2) of AS and its active metabolite, dihydroartemisinin (DHA), could be the basis for this variable efficacy, since HCMV suppression likely depends on the area under the curve (AUC) and the time that drug levels remain above the MIC, with an extended duration likely being required (27, 28). To consider peroxide-based drugs for HCMV therapy, compounds possessing a longer t_1/2 and good safety profiles over prolonged courses of therapy are desired. Towards this goal, we tested the anti-HCMV activity of several ozonides (OZ; 1,2,4-trioxolanes), compounds that represent a new category of bioactive peroxides and that have shown extended t_1/2 in animal models (29, 30). These include the antimalarial ozonide OZ439, which has just completed phase IIa clinical trials (29, 31, 32); the antischistosomal ozonide OZ418 (29); and two closely related analogs, OZ513 (32) and OZ721 (33). From these, OZ418 was identified to be a promising ozonide, and it was tested in vivo in a mouse cytomegalovirus (MCMV) model. Once-daily dosing of OZ418 in mice was efficacious in MCMV inhibition.

RESULTS

Ozonide inhibition of HCMV replication.

Four ozonides were tested for virus inhibition and toxicity in noninfected human foreskin fibroblasts (HFFs) (Table 1; Fig. 1). Luciferase activity was used as a measure of HCMV pp28 expression, and a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to test for cytotoxicity. Of the four ozonides (OZ) tested, OZ418 and OZ439 showed similar activity against HCMV and MCMV, based on the concentration inhibiting cell growth by 50% (CC₅₀) and the effective concentration inhibiting viral growth by 50% (EC₅₀) at 3 days postinfection (Table 1 and Fig. 2A; see also Table S1 in the supplemental material). However, in plaque reduction assays using HCMV TB40, OZ418 had better selectivity than OZ439; its EC₅₀ was 8.5 ± 0.1 µM, and in a parallel cell viability assay, its CC₅₀ was 165 ± 9.2 µM, while the CC₅₀ of OZ439 was 58 ± 0.8 µM (Fig. 2C). The expression of cytomegalovirus (CMV) proteins IE2, UL44, and pp65 was inhibited by OZ418 (Fig. 2B). The nonperoxidic OZ418 isostere carbaOZ418 did not inhibit HCMV, as evidenced by pp28-luciferase activity and the results of a plaque reduction assay (Fig. 2D and E).

TABLE 1.

EC₅₀, CC₅₀, and selectivity index of ozonide analogs^a

Compound	EC50 (μM)	CC50 (μM)	Selectivity index
OZ418	9.8 ± 0.2	128.1 ± 8.0	13.1 ± 0.5
OZ439	13.2 ± 1.37	113.6 ± 13.2	8.6 ± 0.8
OZ721	5.7 ± 0.7	45.5 ± 6.8	7.9 ± 0.3
OZ513b	15.7 ± 0.9	49.4 ± 3.7	3.1 ± 0.2

^aThe data represent the means ± SDs of triplicate determinations from three independent experiments.

FIG 1.

Chemical structures of the ozonides and AS.

FIG 2.

In vitro anti-HCMV activity of OZ418. (A) Concentration-dependent activity of OZ418 against HCMV. HFFs were infected with pp28-luciferase-expressing HCMV Towne and treated with OZ418 at the indicated concentrations. Luciferase activity was measured at 72 hpi. (B) Inhibition of HCMV protein expression by OZ418. HFFs were infected with HCMV Towne and treated with OZ418 (30 μM) or GCV (5 μM). Expression of viral proteins IE1/2, UL44, and pp65 was detected by Western blotting at 72 hpi. β-Actin was used as a loading control. The numbers to the left of the gel are molecular masses (in kilodaltons). (C) HFFs were infected with HCMV TB40 (100 PFU/well) and treated with OZ418 and GCV at the indicated concentrations. Plaques were stained and counted after 10 days. (D and E) Comparison of the activities of OZ418 and carbaOZ418 against HCMV by luciferase assays (D) and plaque assays (E). (D) HFFs were infected with HCMV Towne and treated with OZ418 (30 μM), carbaOZ418 (30 μM), or GCV (5 μM). Luciferase activity was measured at 72 hpi. (E) HFFs were infected with HCMV Towne (200 PFU/well) and treated with OZ418 (30 μM), carbaOZ418 (30 μM), or GCV (5 μM). Plaques were stained and counted after 10 days. The data represent the mean ± SD of triplicate determinations from a single experiment. The asterisks indicate a statistically significant difference in normalized luciferase units or plaque titers between groups that were infected only and groups that were infected and compound treated. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Inhibition of GCV-resistant HCMV with OZ418.

The activity of OZ418 was tested against GCV-resistant pp28 Towne. The EC₅₀ of OZ418 was 14.2 ± 0.8 µM (Fig. 3A), and at 30 µM it efficiently inhibited luciferase activity, while 5 µM GCV had no effect on this HCMV strain (Fig. 3B). Viral protein expression and plaque formation were inhibited by OZ418 but not by GCV (Fig. 3C and D).

FIG 3.

Activity of OZ418 against a GCV-resistant HCMV strain. (A) HFFs were infected with the GCV-resistant HCMV pp28 Towne strain and treated with OZ418 at the indicated concentrations. Luciferase activity was measured at 72 hpi. (B) HFFs were infected with wild-type HCMV Towne (Towne) or GCV-resistant HCMV Towne (Towne-GCV^R) and treated with OZ418 (30 μM) or GCV (5 μM). Luciferase activity was measured at 72 hpi. (C) HFFs were infected with wild-type Towne or GCV-resistant Towne and treated with OZ418 (30 μM) or GCV (5 μM). Expression of viral proteins IE1/2, UL44, and pp65 was detected by Western blotting at 72 hpi. β-Actin was used as a loading control. The numbers to the left of the gel are molecular masses (in kilodaltons). (D) HFFs were infected with GCV-resistant strain Towne (100 PFU/well) and treated with OZ418 (30 μM) or GCV (5 μM). Plaques were stained and counted after 10 days. The data represent the mean ± SD of triplicate determinations from a single experiment. The asterisks indicate a statistically significant difference in normalized luciferase units or plaque titers between groups that were infected only and groups that were infected and compound treated. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Timing of HCMV inhibition by OZ418.

To determine the time during HCMV replication that OZ418 was active, an entry assay was performed. Using an immunofluorescence assay for pp65, neither OZ418 nor GCV inhibited viral entry, but heparin, used as a positive control, did (Fig. 4A). In add-on and removal assays, OZ418, AS, or GCV was added or removed at 0, 6, 12, 24, 36, and 48 h postinfection (hpi), and luciferase activity was measured at 72 hpi (Fig. 4B and C). While the removal of all three drugs displayed the same timing pattern (Fig. 4C), in the add-on assay OZ418 showed a timing of maximal activity that overlapped that of GCV, while addition of AS after the 24-h time point decreased the inhibitory activity against HCMV. These data illustrate that OZ418 is inhibitory at the early stage of HCMV replication.

FIG 4.

OZ418 is an early HCMV inhibitor. (A) Entry assay. HFFs were treated with OZ418 (30 μM) or GCV (5 μM) at 24 h prior to infection. Cells were infected with HCMV Towne and washed with citric acid buffer (pH = 3) to remove viral particles that adhered to the cell surface. The cells were treated with the compounds for 2 h, and pp65 was detected by Western blotting. (B and C) Add-on and removal assays of OZ418, AS, and GCV. HFFs were infected with HCMV Towne, and the compounds were added (B) or removed (C) at 0, 6, 12, 24, 36, and 48 hpi. Luciferase activity was measured at 72 hpi. (D and E) HFFs were infected with HCMV Towne and treated with a combination of OZ418 and GCV (D) or AS and GCV (E) at the indicated concentrations. Luciferase activity was measured at 72 hpi. The drug combination was analyzed using the Bliss model. The data represent the mean ± SD of triplicate determinations from a representative of three independent experiments.

Synergistic HCMV inhibition with the OZ418 and GCV combination.

The effect of OZ418 alone or in combination with GCV was tested in HCMV-infected HFFs. The combinations of OZ418 plus GCV and AS plus GCV were synergistic (Fig. 4D and E). No cellular toxicity was measured for the combinations used.

Cell confluence at the time of infection determines the anti-HCMV activity of OZ418.

HCMV drives the cell cycle in adherent cells from G₁ to S followed by G₁/S arrest (34, 35). We previously reported that AS reverts HCMV-mediated cell cycle progression to G₁/S and that its anti-HCMV activity is improved at early G₁ and is markedly reduced in a heterogeneous/cycling cell population (36). The add-on and removal assay of OZ418 showed a longer time of activity; therefore, the contribution of cell density/contact inhibition to the anti-HCMV activity of OZ418 was tested. Virus inhibition by OZ418 was also dependent on cell confluence (Fig. 5). A high cell confluence provided an environment more conducive for OZ418 activity (but not GCV activity) than a lower cell density, based on the results of the plaque reduction assay (Fig. 5A) and pp28-luciferase activity (Fig. 5B). At 72 hpi, HCMV infection induced the expression of cyclin-dependent kinases (CDKs) 1, 2, 4, and 6 and E2F1 and the phosphorylation of pRb (Ser 807/811) at both a low and a high cell density (Fig. 5C). Virus suppression by OZ418 was marked by the reversal of CDK induction, and a lack of HCMV inhibition by OZ418 correlated with higher levels of phosphorylated pRb and CDKs. In infected confluent (high-density) cells, the expression level of pRb, E2F1, and CDKs 1, 2, 4, and 6 was reduced with OZ418 treatment, but in low-density cells, the expression of these proteins was unchanged (Fig. 5C). Irrespective of the cell density, GCV had no effect on the expression of these proteins.

FIG 5.

HCMV inhibition by OZ418 is cell density dependent. (A) HFFs were seeded on 12-well plates at 0.5 × 10⁶ or 2 × 10⁶ cells/well, infected with 100 PFU/well of Towne HCMV, and treated with OZ418 (15 or 30 μM) or GCV (5 μM). At day 8, plaques were stained with crystal violet and counted. The data shown are averages for four wells (±SD) from a representative experiment from two independent experiments. (B) Cells were plated at densities of 0.5 × 10⁶ and 2 × 10⁶, infected at an MOI of 1 PFU/cell, and treated with the indicated concentrations of OZ418 or GCV. At 72 hpi, cell lysates were collected for luciferase assay. Data are presented as the mean ± SD of triplicates from a single experiment from two independent experiments. (C) Cells were plated and infected as described in the legend to panel B, followed by treatment with OZ418 (30 µM) or GCV (5 µM). At 72 hpi, lysates were used for Western blotting of the indicated proteins. β-Actin was used as a loading control. The numbers to the left of the gel are molecular masses (in kilodaltons). The numbers below the gels represent relative intensities of individual bands, determined by densitometry. Data are representative of those from two independent experiments. Densitometry was performed by the use of ImageJ software (NIH).

Changes in cell cycle progression by OZ418 and AS.

Similar to AS, OZ418 could not inhibit HCMV in low-density cells; therefore, its effect on cell cycle progression was determined in noninfected cells (Fig. 6) and HCMV-infected cells (Fig. 7). Serum-starved cells were either released from starvation (with 10% fetal bovine serum [FBS]) or released and treated with OZ418, AS, or GCV. At 24 h after release from serum starvation, only AS arrested HFFs at early G₁ (Fig. 6A), and at 72 h after release, all cells were contact inhibited, irrespective of the drug used. In agreement with the changes in cell cycle progression, OZ418 treatment did not reduce the expression of CDKs 1, 2, 4, and 6 or pRb phosphorylation at 24 or 72 h (Fig. 6C and D). Similarly, AS reportedly did not reduce the expression of CDKs or pRb in high-density noninfected HFFs (36). Changes in the expression of these proteins were induced only by HCMV infection. Cell cycle analysis in infected cells showed G₁ arrest at 24 h (Fig. 7A) and progression to G₂/M at 72 hpi. Both AS and OZ418 reverted the cell cycle back to G₁, although the effect of AS was stronger (Fig. 7B). At 24 hpi, OZ418 decreased CDK1 expression, and its effects on all tested CDKs and pRb phosphorylation were augmented at 72 hpi (Fig. 7C and D). As previously reported, in HCMV-infected cells, virus inhibition by AS correlated with reduced pRb and CDK2 levels and a modest reduction in CDK4 levels, while the levels of CDK6 were unchanged. GCV did not exhibit any effects on pRb or CDK expression (Fig. 7C and D), confirming the specificity of the cell cycle protein profile of OZ418 and AS.

FIG 6.

OZ418 does not regulate cell cycle progression in noninfected HFFs. (A and B) HFFs (1 × 10⁶) were seeded and serum starved for 72 h. Cells were then released in DMEM with 0% FBS, 10% FBS, or 10% FBS plus OZ418 (30 μM), AS (30 μM), or GCV (30 μM) for 24 h (A) and 72 h (B). Following staining of DNA with propidium iodide, cells were analyzed by flow cytometry. Numbers above each graph indicate the percentages of cells in G₁, S, and G₂/M in each condition. (C and D) HFFs were treated with OZ418 (30 μM) or GCV (5 μM), and the expression of cellular proteins pRb (Ser 807/811) and CDKs 1, 2, 4, and 6 was detected by Western blotting at 24 h (C) and 72 h (D) posttreatment. β-Actin was used as a loading control. The numbers to the left of the gels are molecular masses (in kilodaltons). The data shown are representative of the results from three independent experiments.

FIG 7.

OZ418 reverts HCMV-induced cell cycle progression. (A and B) Cells (1 × 10⁶) were seeded and serum starved for 72 h and then infected with HCMV Towne, followed by treatment with OZ418 (30 μM), AS (30 μM), or GCV (5 μM) for 24 h (A) and 72 h (B). Noninfected cells were released in DMEM with 10% FBS as a control. Following staining of DNA with propidium iodide, cells were analyzed by flow cytometry. (C and D) HFFs were infected with HCMV Towne and treated with OZ418 (30 μM) or GCV (5 μM). The expression of HCMV pp65 and cellular proteins E2F1, pRb (Ser 807/811), and CDKs 1, 2, 4, and 6 was detected by Western blotting at 24 hpi (C) and 72 hpi (D). β-Actin was used as a loading control. The numbers to the left of the gels are molecular masses (in kilodaltons).

Plasma protein binding of OZ418 and DHA.

The plasma protein binding of OZ418 and DHA was measured to determine the concentrations of unbound compound present in mouse plasma after dosing. The unbound fraction was 0.0063 for OZ418, whereas the value was 0.22 for DHA.

PK and efficacy of OZ418 in a mouse CMV infection model.

The plasma concentration-versus-time profile of a single oral dose of OZ418 (10 mg/kg of body weight) was compared to that of AS (10 mg/kg) in noninfected mice. Following oral administration of AS, only very low and transient plasma concentrations of the parent compound were detected up to 1 h postdose (Fig. 8A). The maximum plasma concentrations of the metabolite, DHA, were observed at 0.25 h postdose (the first sampling time), indicating the rapid conversion of AS to DHA, although the DHA plasma concentrations were also low and could be detected only up to 4 h postdose, at which time the total concentration was about 0.02 µM (or 0.004 µM for the unbound fraction) (Fig. 8A). In contrast, a high level of exposure to OZ418 was observed following oral administration, with the maximum plasma concentration being seen at 1 h postdose, and the concentrations were maintained above 3.0 µM (or 0.029 µM for the unbound fraction) for 24 h (Fig. 8B and Table 2). Given the high concentration of OZ418 observed at 24 h, there would likely be significant accumulation following a once-daily dosing regimen.

FIG 8.

Plasma concentration-versus-time profiles of OZ418 and AS. Profiles were obtained following administration of a 10-mg/kg oral dose of AS (A) or OZ418 (B) to noninfected BALB/c mice. In panel A, the concentrations of DHA formed from AS are also shown. The data represent the mean ± SD for three mice at each time point.

TABLE 2.

Plasma pharmacokinetic parameters after oral dosing in mice^a

Compound	Dose (mg/kg)	Cmax (µM)	Tmax (h)	t1/2 (h)	AUC (µM·h)	Concn at 24 h (µM)	Fraction unbound
AS	10	0.05	0.25	Could not determine	Could not determine	<0.013	Not measured
DHAb		0.35	0.25	Could not determine	0.32	<0.018	0.22
OZ418	10	27	1	3.4	268	3.03	0.0063

^aC_max, maximum concentration in plasma; T_max, time to maximum concentration in plasma; t_1/2, half-life; AUC, area under the concentration-time curve.

^bDHA is the active metabolite of AS.

Once-daily oral administration of OZ418 to infected mice showed a dose-response in MCMV inhibition in salivary glands, liver, and spleen (Fig. 9A to C), with good efficacy against MCMV being seen at a dose of 10 mg/kg/day. To compare MCMV inhibition by OZ418 and AS, mice were treated orally with OZ418 (10 mg/kg) once daily, AS (10 mg/kg) twice daily, or GCV (10 mg/kg) twice daily for a total of 5 days. At day 14 postinfection, mice were euthanized and their organs were harvested for plaque assays. For all tested tissues, once-daily administration of OZ418 showed better activity against MCMV than twice-daily administration of AS (Fig. 9D to F), supporting a role for extended exposure to OZ418 in virus suppression.

FIG 9.

OZ418 efficiently inhibits MCMV replication. Male and female BALB/c mice (age, 4 to 6 weeks) were infected intraperitoneally with MCMV at 10⁶ PFU/mouse. (A to C) Dose-dependent inhibitory effect of OZ418 on MCMV replication. OZ418 was administered orally once daily at the indicated dosage, and GCV was delivered intraperitoneally twice daily at the indicated dosage. (D to F) Comparison of MCMV inhibition by OZ418 and AS. OZ418 (once daily) and AS (twice daily) were administered orally, and GCV (twice daily) was delivered intraperitoneally. All drugs were dosed at 10 mg/kg. A total of 5 doses of OZ418 and 10 doses of AS or GCV were administered. After 14 days, salivary glands (A and D), liver (B and E), and spleen (C and F) were harvested and plaque assays were performed in mouse embryonic fibroblasts. The data are presented as the mean ± SD of the number of PFU/100 mg of tissue homogenate. The asterisks indicate a statistically significant difference in plaque titers between the groups that were infected only and the groups that were infected and compound treated. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

DISCUSSION

Artemisinins have shown anti-HCMV activities both in vitro and in vivo (16–21), and treatment with AS for HCMV disease in immunocompromised patients has shown modest, although variable, efficacy (22–26). While multiple factors may underlie the observed effects of AS in these patients, we speculated that the pharmacokinetic (PK) characteristics of artemisinins may be less than optimal for HCMV suppression in humans. The very short t_1/2 of AS has been addressed in malaria therapy through its use in combination with another longer-half-life drug. However, for effective HCMV suppression, it is likely that the AUC and the time above the MIC are important considerations (27, 28), leading us to hypothesize that compounds possessing improved PK and extended exposure would result in improved efficacy against HCMV. One such class of compounds is the synthetic ozonides (1,2,4-trioxolanes), which, like the artemisinins, contain a peroxide pharmacophore (29, 30). In comparison to DHA, the active metabolite of AS, OZ439 (and its structural analogs), exhibited a substantial increase in t_1/2 and the duration of exposure in both animal models (29) and humans (31). We therefore assessed the in vitro and in vivo activity of selected ozonides in models of HCMV and MCMV infection.

In our in vitro assays in HFFs and mouse embryonic fibroblasts (MEFs), OZ439 had efficacy similar to that of OZ418 in the 72-h assays (luciferase activity, plaque assays in MEFs and MTT; Table 1; see also Table S1 in the supplemental material), but in plaque reduction assays in HFFs, OZ418 showed better selectivity and was therefore selected for use in all subsequent studies, including in vivo experiments. CarbaOZ418, the nonperoxide analog of OZ418, had no anti-HCMV activity based on a pp28-luciferase assay (Fig. 2D) and a plaque reduction assay (Fig. 2E), confirming a requirement of the peroxide bond for HCMV inhibition.

Similar to AS, OZ418 was active against a GCV-resistant HCMV strain (Fig. 3), it did not inhibit HCMV entry, and it was synergistic with GCV in HCMV inhibition (37) (Fig. 4). Based on the add-on assay, HCMV inhibition by OZ418 was observed even when it was added at 36 hpi, whereas AS was inactive under the same conditions (Fig. 4B). Similar to AS, the anti-HCMV activity of OZ418 correlated with the cell density at the time of infection (Fig. 5A and B). In high-density cells, HCMV was suppressed by OZ418 and the expression level of CDKs, E2F1, and pRb was reduced. In noninfected HFFs, only AS could arrest cell cycle progression at 24 h after release from serum starvation (Fig. 6A), in accordance with the findings of our previous studies (36). OZ418 and GCV did not modulate cell cycle progression or expression of CDKs in noninfected HFFs. However, in HCMV-infected cells, a gradual effect on cell cycle progression was observed for both AS and OZ418 (Fig. 7), with AS showing even stronger effects at 72 hpi. The reversal of HCMV-mediated cell cycle progression by OZ418 correlated with the reduced expression of pRb, E2F1, and CDKs, mostly CDK1. Thus, despite some difference in the timing of activity during infection, AS and OZ418 shared similar in vitro activities involving the cell cycle environment, which is counterproductive for HCMV. Based on the luciferase assay characteristics, the differences in the time of activity may or may not be significant. In addition, apart from their essential peroxide bonds, AS and OZ418 are structurally dissimilar, and differences in lipophilicity have been reported (38). Despite the structural changes, trioxolanes and semisynthetic artemisinins reportedly share an overlapping parasite protein alkylation signature suggestive of a common mechanism of action for the endoperoxides (39). The antiviral activities of 1,2,4-trioxolane and artemisinins may also use similar mechanisms.

The most notable differences between OZ418 and AS were observed in the in vivo MCMV model and likely resulted from their distinctly different PK profiles (Table 2; Fig. 8). AS was rapidly biotransformed to the active metabolite, DHA, which also exhibited a relatively limited exposure profile, with concentrations being below 0.02 µM by 4 h. In contrast, OZ418 concentrations were maintained above 3.0 µM for more than 24 h. Importantly, although the protein binding of OZ418 is substantially greater (i.e., the fraction unbound is lower) than that of DHA, the unbound concentrations of OZ418 were still much greater than those of DHA throughout the postdosing period.

Based on the dose-response of OZ418 in MCMV inhibition (Fig. 9A to C) and its favorable PK characteristics, MCMV inhibition was tested after oral administration of once-daily doses of OZ418 and compared to that achieved with twice-daily doses of AS. Using this regimen, OZ418 showed improved MCMV inhibition in the salivary gland, liver, and spleen (Fig. 9D to F).

Our data provide preliminary evidence that an extended exposure profile for peroxide-based drugs is important for inhibiting CMV replication using a mouse model of disease and support further investigations to explore the utility of OZ418, its derivatives, and additional ozonides for HCMV therapy either alone or, more favorably, in combination with GCV. Since HCMV is likely to require a prolonged dosing period for virus suppression, additional work will be required to better understand the pharmacokinetic/pharmacodynamic (PK/PD) relationship, predict the likely human dose, and establish the safety profile of OZ418 (or its derivatives) under conditions of prolonged administration.

MATERIALS AND METHODS

Compounds.

The synthesis of the ozonides (1,2,4-trioxolanes) has been reported previously (29, 30, 32, 33), and that of carbaOZ418 is described in the supplemental material. OZ513 was tested as its tosylate salt, OZ439 was tested as its mesylate salt, and OZ721 was tested as its sodium salt. AS and ganciclovir (GCV) were obtained from Sigma-Chemical (St. Louis, MO). The compounds were dissolved in dimethyl sulfoxide (DMSO), and 10 mM stock solutions were stored at −80°C. The concentration of each compound was calculated and adjusted by volume such that it was constant throughout the experiment.

Viruses.

The pp28-luciferase-expressing Towne HCMV strain was constructed as previously described (40). The recombinant virus expresses luciferase under the control of the UL99 (pp28) late promoter at 48 to 72 h postinfection (hpi). Luciferase expression from this promoter is almost completely inhibited in the presence of viral DNA polymerase inhibitors, such as GCV and foscarnet. Luciferase activity highly correlates with the results of the plaque reduction assay. The Towne HCMV strain (ATCC VR-977) was used for plaque reduction, DNA replication, and cell cycle assays. Plaque assays were also performed with the HCMV TB40 strain (ATCC VR-1578). Murid herpesvirus (MCMV; ATCC VR-1399) was used for infection of mouse embryonic fibroblasts (MEFs) and mice.

Cell culture, virus infection, and antiviral assays.

Human foreskin fibroblasts (HFFs) at passages 12 to 16 (ATCC CRL-2088) and MEFs at passages 9 to 14 (ATCC CRL-1658) were grown in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA) in a 5% CO₂ incubator at 37°C and used for infection with pp28-luciferase-expressing Towne HCMV and MCMV, respectively.

Plaque assays were performed with the HCMV Towne strain (ATCC VR-977) and MCMV. HFFs and MEFs were seeded at 3 × 10⁶ or 2 × 10⁶ cells/plate in a 12-well plate and infected at 100 PFU/well after 24 h. Following virus adsorption for 90 min, virus was aspirated and DMEM containing 4% FBS with 0.5% carboxymethyl cellulose was added with the compounds at the concentrations indicated above into triplicate wells. After a 10-day incubation at 37°C for HCMV and a 3-day incubation for MCMV, the overlay was removed and the plaques were counted after crystal violet staining at ×40 magnification.

To determine the effect of cell density/cycling on the anti-HCMV activity of the ozonides, HFFs were seeded on 12-well plates at 0.5 × 10⁶ or 2 × 10⁶ cells/plate, infected with 100 PFU/well of Towne HCMV, and treated with OZ418 (15 or 30 μM) or GCV (5 μM). At day 8, plaques were stained with crystal violet and counted. pp28-luciferase activity and protein expression were measured at 72 hpi in cells plated at densities of 0.5 × 10⁶ and 2 × 10⁶ cells/plate, infected at a multiplicity of infection (MOI) of 1 PFU/cell, and treated with OZ418 or GCV.

Entry assay.

The compounds were diluted in serum-free medium and added to HFFs seeded on chamber slides 24 h prior to infection. After infection and treatment, cells were fixed with chilled 100% methanol and blocked for 1 h with 1× phosphate-buffered saline (PBS), 5% serum, 0.3% Triton X-100. The blocking buffer was used as the antibody dilution buffer. Cells were incubated with mouse monoclonal anti-pp65 antibody (Vector Laboratories, Burlingame, CA) at 37°C in humidified chambers for 1 h, washed three times with 0.1% Tween 20 in PBS (PBST), incubated with rhodamine-conjugated anti-mouse IgG (Sigma) at 37°C in humidified chambers for 1 h, washed with PBST (0.1% Tween 20), and air dried. A drop of mounting oil containing DAPI (4′,6-diamidino-2-phenylindole; Santa Cruz) was added to the slides before visualization with a Zeiss Z1 fluorescence microscope. Images were captured at ×40 magnification.

Add-on and removal experiments.

HFFs were infected with pp28-luciferase-expressing HCMV as described above. AS, OZ418, and GCV were used at concentrations resulting in >90% virus inhibition.

At 0, 6, 12, 24, 36, and 48 hpi, the media were replaced with OZ418- or GCV-containing media. For time-of-removal studies, media containing the compounds were removed at 0, 6, 12, 24, 36, and 48 hpi. The cells were then washed three times with PBS, and drug-free medium was added. The cells were incubated at 37°C, and luciferase activity was measured at 72 hpi.

Cell viability

HFFs or MEFs were seeded in 96-well plates, treated with various concentrations of ozonides, and incubated at 37°C for 3 or 7 days. Cell viability was determined by an MTT-based colorimetric assay (Sigma-Aldrich), which was performed at the same time points as the antiviral assay.

Inhibition of mouse CMV replication.

For the infection experiments, 4- to 6-week-old BALB/c mice were purchased from Harlan Laboratories (Indianapolis, IN). The Animal Care and Use Committee of Johns Hopkins University approved the experimental procedures. After 2 to 3 days of adaptation to the housing environment, the mice were randomly divided into 6 groups, as follows: a control group (1 mouse), an infected group (5 mice), groups infected and treated with OZ418 at 1 mg/kg (7 mice), 3 mg/kg (7 mice), or 10 mg/kg (3 mice) orally once daily, and a group infected and treated with GCV at 10 mg/kg (2 mice) twice daily intraperitoneally. For another experiment, mice were randomly divided into 5 groups, as follows: a control group (1 mouse), an infected group (3 mice), an infected group treated with OZ418 at 10 mg/kg orally once daily (7 mice), an infected group treated with AS at 10 mg/kg orally twice daily (7 mice), and an infected group treated with GCV at 10 mg/kg intraperitoneally twice daily (3 mice). OZ418 and AS were formulated by dissolving each in a 70/30 mixture of Tween 80 and ethanol, which was then diluted with water to give the desired dose with a volume of 0.1 ml/mouse. Mice were infected intraperitoneally with 10⁶ PFU tissue culture-derived MCMV (Smith strain; ATCC VR-1399) in 0.1 ml in 0.8% saline, and all treatments were started at 24 hpi. Control mice received 0.1 ml of saline intraperitoneally, and control and infected mice received equivalent volumes of saline. Mice were sacrificed at day 14 after infection. The salivary glands, liver, and spleen were harvested and stored at −80°C. The organs were homogenized in DMEM with 4% FBS at a final concentration of 100 mg/ml. Two million MEFs were seeded into 24-well plates. From each sample, 5% of the salivary gland tissue homogenate or 10% of the liver tissue homogenate was used for infection of MEFs in triplicate. Plaques were counted after 3 days.

Flow cytometry.

To test whether the cell cycle was affected by OZ418, noninfected HFFs were serum starved for 72 h and then released in DMEM with 0% FBS (the control for G₀ arrest), 10% FBS (the control for cell cycle progression), or 10% FBS plus OZ418, AS, or GCV for 24 h and 72 h. To determine the effects of OZ418 on the cell cycle in infected HFFs, 1 × 10⁶ cells were serum starved for 72 h and then infected with the HCMV Towne strain (MOI = 1 PFU/cell), followed by treatment with OZ418 (30 μM), AS (30 μM), or GCV (10 μM) for 24 h and 72 h. Cells were collected by trypsin treatment and washed with PBS. They were then fixed in 5 ml of cold 70% ethanol overnight at 4°C and resuspended in 500 µl of staining buffer (50 μg/ml propidium iodide [Sigma-Aldrich] and 0.2 mg/ml RNase A in PBS) for 45 min at 37°C in 5% CO₂. The samples were analyzed on an LSR II flow cytometer (BD Biosciences, San Jose, CA), and at least 10,000 cells were counted for each sample. The flow data were recorded and obtained using BD FACSDiva software. The percentage of cells in G₀/G₁, S, and G₂/M was calculated under each condition.

SDS-PAGE and immunoblot analysis.

Cell lysates were quantified for total protein content using a Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA). Equivalent amounts of protein were mixed with an equal volume of sample buffer (125 mM Tris-HCl [pH 6.8], 4% SDS, 20% glycerol, 5% β-mercaptoethanol) and boiled at 100°C for 10 min. Denatured proteins were resolved in Tris-glycine polyacrylamide gels (10% to 12%) and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA) by electroblotting. Membranes were incubated in blocking solution (5% nonfat dry milk–bovine serum albumin [BSA] and 0.1% Tween 20 in PBS [PBST]) for 1 h, washed three times with PBST, and incubated with primary antibodies diluted in 5% milk or BSA (for pRb) at 4°C overnight. The membranes were washed with PBST, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies in 5% milk–BSA for 1 h at room temperature. After three washing steps with PBST, the protein bands were visualized by chemiluminescence using the SuperSignal West Pico reagent (Pierce Chemical, Rockford, IL). The following antibodies were used: mouse monoclonal anti-HCMV UL83 (pp65, VP-C422; Vector Laboratories Inc., Burlingame, CA), mouse anti-IE1 and anti-IE2 monoclonal (MAb810; EMD Millipore Corporation, Temecula, CA), mouse anti-UL44 monoclonal (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-phospho Rb antibody (Ser 807/811) (Cell Signaling Technology, Beverly, MA), anti-E2F1 (Cell Signaling Technology, Beverly, MA), rabbit anti-CDK1, -2, -4, and -6 polyclonal (Santa Cruz), mouse anti-β-actin monoclonal (Santa Cruz), HRP-conjugated goat anti-rabbit IgG (Cell Signaling Technology, Beverly, MA), and HRP-conjugated sheep anti-mouse IgG (GE Healthcare, Waukesha, WI).

Drug combination and analysis.

Drug combination studies were performed as previously reported (37). Briefly, 2 × 10⁶ HFFs/plate were seeded in 96-well plates and infected with the pp28-luciferase-expressing Towne HCMV strain (MOI = 1 PFU/cell). First, a dose-response curve was generated for each drug individually to determine its EC₅₀. Then, the drugs were combined at twice their EC₅₀, diluted in DMEM with 4% FBS followed by serial dilution, and added together after infection. The luciferase activity of the combination and each drug individually was quantified at 72 hpi. The Bliss model was used to calculate the effect of each drug combination on pp28-luciferase activity. In this model, the activity of the drug combination represents the product of two probabilistically independent events, as described in the following equation (41):

$$F_{U1+2}=F_{U1}\hspace{0.17em}\cdot \hspace{0.17em}{F}_{U2}=\frac{1}{1\hspace{0.17em}+\hspace{0.17em}{\left(\frac{D_1}{{\text{EC}}_{\text{50(1)}}}\right)}^{m_1}}\hspace{0.17em}\cdot \hspace{0.17em}\frac{1}{1\hspace{0.17em}+\hspace{0.17em}{\left(\frac{D_2}{{\text{EC}}_{\text{50(2)}}}\right)}^{m_2}}$$

where D₁ and D₂ are the concentrations of drugs 1 and 2, respectively; m₁ and m₂ are the slopes of drugs 1 and 2, respectively; and EC₅₀₍₁₎ and EC₅₀₍₂₎ are the effective concentration resulting in 50% virus inhibition for drugs 1 and 2, respectively. The combined effect of two inhibitors (fraction unaffected [F_U1+2]) is computed as the product of the individual effects of the two inhibitors, F_U1 and F_U2. If the ratio of the observed fold inhibition divided by the expected fold inhibition is greater than 1, the compounds are synergistic. If the ratio is less than 1, the combination is considered antagonistic, and if it equals 1, the combination is additive.

Plasma protein binding.

The protein binding of DHA and OZ418 in mouse plasma was determined by ultracentrifugation as described previously (42). The stability of the compounds at 37°C was assessed over the 4-h assay period by measuring the initial and final concentrations in plasma control samples by liquid chromatography-mass spectrometry (LC-MS).

PK of AS, DHA, and OZ418.

Pharmacokinetic (PK) studies were conducted using established procedures in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, and the study protocols were reviewed and approved by the Monash Institute of Pharmaceutical Sciences Animal Ethics Committee. The systemic exposure to AS (and the DHA formed from AS) and OZ418 was studied in noninfected, nonfasted BALB/c mice weighing 22 to 31 g. The mice had access to food and water ad libitum. AS and OZ418 were prepared by dissolving the solid in a mixture of Tween 80 and ethanol (70:30), which was then diluted with water prior to dosing orally by gavage (0.1 ml per mouse) at a dose of 10 mg/kg (expressed relative to the amount of free acid). Blood samples were collected into tubes containing heparin and complete protease inhibitor cocktail for up to 6 h postdose for AS and 48 h postdose for OZ418 (n = 3 mice per time point). The sampling procedure was the same for both compounds. There were two samples for each mouse; the first sample from each mouse was taken via submandibular bleed, and the final sample was taken via cardiac puncture while the mouse was under anesthesia.

Blood samples were centrifuged immediately upon collection, and plasma was separated for analysis by LC-MS. PK parameters were determined using standard noncompartmental methods.

Plasma concentrations were determined following precipitation of proteins using a 2-fold volume ratio of acetonitrile. The supernatant was injected onto a Supelco Ascentis Express RP amide column (50 by 2.1 mm; particle size, 2.7 µm) and eluted under gradient conditions using a mobile phase of water and acetonitrile, each containing 0.05% formic acid. The flow rate was 0.4 ml/min, and the injection volume was 2 to 3 µl. Detection was via positive (AS or DHA) or negative (OZ418) electrospray ionization with multiple reaction monitoring on either a Waters Xevo TQ (AS, DHA) or TQD (OZ418) triple quadrupole mass spectrometer coupled to a Waters Acquity ultraperformance liquid chromatography system. Mass spectrum transitions (m/z) were as follows: for OZ418, 413.12 to 355.06; for AS, 267.21 to 163.11; and for DHA, 267.15 to 163.24. The cone voltage and collision-induced dissociation energies were as follows: for OZ418, 35 V and 35 V, respectively; for AS, 30 V and 7 V, respectively; and for DHA, 25 V and 7 V, respectively. Quantitation was conducted by comparison of the peak area (as a ratio to the internal standard, diazepam) to that for calibration standards prepared by spiking blank mouse plasma.

Statistical analysis.

A Student’s t test was performed using SigmaPlot (Systat Software, San Jose, CA) and GraphPad Prism (GraphPad Software, La Jolla, CA) software. P values of <0.05 were considered significant. The curve-fitting toolbox MATLAB software (v7.10; MathWorks, Natick, MA) was used to determine EC₅₀ and CC₅₀ values using a four-parameter logistic regression. Densitometric analysis was done by the use of ImageJ software (NIH).