Antimicrobial Agents and Chemotherapy, May 2004, p . 1749-1755, Vol . 48, No . 5

Activities of Benzimidazole D- and L-Ribonucleosides in Animal Models of Cytomegalovirus Infections

Earl R . Kern,^1* Caroll B . Hartline,¹ Rachel J . Rybak,¹ John C . Drach,² Leroy B . Townsend,² Karen K . Biron,³ and Deborah J . Bidanset¹

University of Alabama School of Medicine, Birmingham, Alabama,¹ University of Michigan, Ann Arbor, Michigan,² GlaxoSmithKline, Inc., Research Triangle Park, North Carolina³

Received 4 November 2003/ Returned for modification 8 January 2004/ Accepted 3 February 2004

There are a number of previous studies documenting the in vitro activity of the benzimidazole ribonucleosides against HCMV (4, 12, 24, 31, 36, 39) . Although the original compounds 2,5,6-trichloro-(1-ß-D-ribofuranosyl) benzimidazole and its 2-bromo homolog (BDCRB) were potent and selective inhibitors of HCMV replication in vitro and were bioavailable when delivered orally, they had a short plasma half-life (11) . In order to obtain more stable compounds, a number of new analogs were synthesized (32), including the ribopyranosyl analog of BDCRB, termed GW 275175X (175X) (9, 32, 34),and 2-isopropylamino-5,6-dichloro-(1-ß-L-ribofuranosyl)benzimidazole (1263W94 or maribavir [MBV]) . These compounds had antiviral activity against HCMV that was comparable or better than that of GCV and were active against GCV- or PFA-resistant isolates (4, 36) . Additionally, both 175X and MBV were also active against Epstein-Barr virus (36, 38) . None of the three nucleoside analogs was active against herpes simplex virus type 1 or 2, varicella-zoster virus, human herpesvirus type 6, or human herpesvirus type 8 (36) . The compounds were not active against murine, rat, or guinea pig cytomegalovirus (CMV) strains, which has precluded the evaluation of these compounds in animal models for HCMV infections (36) .

The pharmacokinetics and toxicity of MBV have been evaluated both in animals and in humans, and good oral bioavailability and low toxicity were reported (18, 35) . One clinical trial has been conducted in which a small number of patients with HCMV were given MBV, and a reduction in titers of HCMV in semen was reported (21) .

Currently, there are few animal models that can be used to study the biology of HCMV and determine the efficacy of various antiviral therapies . This is largely due to the fact that HCMV infection and replication are limited to human cells . For this reason, the use of immunosuppressed or immunocompromised animals as hosts for human xenografts and later infection of the grafts with HCMV was developed to provide a model for in vivo determination of antiviral drug efficacy (2, 3, 16) . In previous studies, we have used severe combined immunodeficient (SCID) mice as hosts for human fetal retinal implants and have been able to successfully show that HCMV replicates in the implanted tissue and that GCV and CDV, which are efficacious in the treatment of HCMV infections, are also effective in this model (2, 16) .

In addition to retinal tissue, fetal thymus and liver (thy/liv) tissues have been implanted under the kidney capsule of SCID mice and used to examine the replication of HCMV (5, 13) . Currently we are also utilizing this model for hosts for human fetal thy/liv tissue implants to determine the efficacy of various antiviral therapies against HCMV replication (16) . In this model, thy/liv tissues implanted under the kidney capsule have been shown to fuse and become vascularized . The hematopoietic progenitor cells of the thy/liv implant can then differentiate and proliferate within the environment of the kidney capsule . Like the retinal implants, this tissue can then be infected with HCMV and used to evaluate the efficacy of new potential antiviral therapies for HCMV infection .

The purpose of the studies reported here was to evaluate the effectiveness of therapy with BDCRB, 175X, and MBV with these two animal models for HCMV . Efficacy was compared directly with that obtained with GCV .

Tissue, cells, and viruses. Human fetal tissue was obtained from Advanced Biosciences Resources (Alameda, Calif.) and prepared as described below . Human foreskin fibroblast cells were prepared as primary cultures and used in assays with HCMV . These cells were propagated in MEM containing 10% FBS, L-glutamine, and antibiotics as indicated above . All viruses were propagated by using standard virological methods . The Toledo strain of HCMV was obtained from Edward Mocarski (Palo Alto, Calif.) .

Plaque reduction assay for HCMV with liquid overlay. Human foreskin fibroblast cells were seeded into six-well plates and incubated at 37°C . Two days later, drug was serially diluted 1:5 in MEM with 2% FBS using six concentrations of drug with a starting concentration of 100 µg/ml . GCV was used as a positive control . The virus to be used was diluted in MEM containing 10% FBS to a desired concentration which gave 20 to 30 plaques per well . Medium was aspirated from the wells, and 0.2 ml of virus suspension was added to each well in triplicate, with 0.2 ml of medium being added to drug toxicity wells . Plates were incubated for 1 h with shaking every 15 min, and drug was added to each well . After incubation for 8 days, cells were stained with a 5% neutral red solution in phosphate-buffered saline . The stain was aspirated, wells were washed with phosphate-buffered saline, and plaques were counted by using a stereomicroscope . Under these assay conditions, secondary plaque formation did not alter susceptibility levels . By comparing drug-treated wells with untreated wells, 50% effective concentrations were calculated by using MacSynergy II software (27) .

SCID-hu mouse model for HCMV replication in ocular tissue. Implantation of human fetal retinal tissue and later infection of these implants were performed as described previously (2) . In brief, 4- to 8- week-old male SCID mice were anesthetized with an intraperitoneal (i.p.) injection of ketamine (100 mg/kg of body weight) and xylazine (15 mg/kg), and the topical anesthetic, proparacaine-HCl (0.5%), was instilled in the eyes . A 27- by 1/2-in-gauge winged infusion needle containing mechanically dissociated human fetal retinal tissue was then inserted into the nasal sclera and into the anterior chamber . At the temporal side of the anterior chamber, approximately 5 µl of tissue was injected, and the needle was removed . Using similar procedures, the mice were again anesthetized 6 to 9 weeks after implantation, and 10 µl of 2,000 to 7,500 PFU of HCMV, depending on the experiment, was injected into the anterior chamber containing the implant . Within a given experiment all animals received the same size of inoculum . Beginning 24 h after viral inoculation, the mice were treated with 0.1 ml of drug i.p . or 0.2 ml of drug orally once or twice daily for 28 days, according to the treatment schedules below . On days 7, 14, 21, and 28, four to six animals per group were sacrificed, and their eyes were harvested .

SCID-hu mouse model for HCMV infection in thy/liv tissue. For the second experimental infection, 4- to 6-week-old male SCID mice were anesthetized, and fragments of human fetal thy/liv were implanted under the capsule of one kidney with an 18-gauge trocar, using techniques described previously (5, 13, 25) . Following an implant growth period of 12 to 14 weeks, the grafts were inoculated with 2,000 to 9,000 PFU of HCMV, depending on the particular experiment . Beginning 24 h after viral inoculation, the mice were treated daily for 28 days, according to the treatment schedule described below . On days 14, 21, 28, and 35, 5 to 11 implants were biopsied (approximately 50% of the graft size), homogenized, and frozen at –70°C until assayed for HCMV . Biopsies were also obtained 1 week after treatment ended (day 35) to determine if viral replication rebounded after cessation of treatment .

Viral replication in implant tissue. To monitor HCMV replication in retinal implant tissue, animals were sacrificed at various times after infection . The eyes were removed, temporarily stored in sterile irrigating balanced salt solution, and homogenized in MEM containing 10% FBS, 2 mM L-glutamine, 200 U of penicillin/ml, 50 µg of gentamicin/ml, and 3 µg of Fungizone/ml . Eyes were homogenized in 1.0 ml of medium, using a Kontes tissue grinder (Vineland, N.J.) regardless of weight, so titers were calculated as numbers of PFU per milliliter of homogenate . The homogenate was centrifuged at 1,500 rpm for 15 min at 4°C in an Eppendorf 5810R centrifuge, and the supernatant was removed and frozen at –70°C until assayed for HCMV using standard plaque assay techniques . Biopsy samples of thy/liv implants were weighed and homogenized as 10% (wt/vol) suspensions, centrifuged, and assayed as described above . Titers were expressed as log₁₀ PFU/gram of tissue .

Evaluation of efficacy: statistics. In order to determine therapeutic efficacy with these models, animals treated with the benzimidazoles, GCV, or CDV were compared to vehicle-treated animals . Percentages of implants positive for HCMV infection were calculated and compared using a general linear regression model, and titers of virus (no . of PFU/ml ± standard deviation or log₁₀ PFU/g) were compared using a stratified Wilcoxon rank sum test . The results obtained throughout the entire 28-day treatment period were used to calculate significance . In general, a P value of 0.05 or less was considered significant .

HCMV is species specific and does not infect experimental animals . Consequently, surrogate animal strains of CMV, such as murine, rat, guinea pig, and to a lesser extent rhesus monkey strains, have been utilized as animal model infections for the study of the biology and development of antiviral drugs directed against CMV infection in humans (1, 28) . The activities of BDCRB, MBV, and 175X were compared with those of GCV against human, murine, rat, and guinea pig CMV strains, and these results have been reported previously (36) . Although there was appreciable variability in the toxicity of the compounds in primary mouse and guinea pig cells, calculation of a selectivity index indicated that there was little activity of the three compounds against the murine and rat viruses compared with that of GCV, and only BDCRB showed activity against guinea pig CMV .

Effect of treatment with BDCRB on HCMV replication in a SCID-hu mouse ocular model. Since the benzimidazoles are not active against the usual surrogate murine, rat, and guinea pig CMV strains used for evaluating efficacy, we have utilized two models of HCMV in SCID mice . In the first experiment, we determined the efficacy in the SCID-hu mouse ocular model . SCID mice with retinal implants infected with the Toledo strain of HCMV were treated i.p . with BDCRB over the course of 28 days as follows . Starting 1 day after infection, mice were treated with PEG-400 vehicle, either 50 or 25 mg of BDCRB/kg in PEG-400 or 45 mg of GCV/kg in H₂O . The PEG-400 control and the 50-mg/kg BDCRB dose were administered once daily for 28 days . The 25-mg/kg BDCRB dose was administered twice daily for 1 week followed by once daily for the remaining 3 weeks, and the 45-mg/kg GCV dose was administered twice daily for 2 weeks followed by once daily for the final 2 weeks . On 7, 14, 21, and 28 days after infection, eyes were removed from vehicle-, BDCRB-, and GCV-treated animals and homogenized, and HCMV replication was quantified by plaque assay . The results are shown in Table 1 . In comparison with PEG-400-treated animals, treatment with GCV significantly reduced mean titers of virus in implants on day 28 from 1,399 ± 1,777 PFU/ml in control implants to 15 ± 33 PFU/ml . Implants from mice treated with 50 mg of BDCRB/kg showed a slight but not statistically significant reduction in mean titers of virus, whereas results from animals treated with BDCRB at 25 mg/kg showed no reduction in mean titers of virus . These data suggested that BDCRB was only slightly effective against HCMV at 50 mg/kg and indicated the need to determine the efficacy of BDCRB at higher concentrations .

 
In a second experiment, SCID-hu retinal implants infected with HCMV were treated i.p . with CDV or BDCRB over the course of 28 days . Starting 1 day after infection, mice were treated once daily with vehicle (corn oil), 25 mg of CDV/kg, or 75 mg of BDCRB/kg . On days 7, 14, 21, and 28 after infection, mice were sacrificed, eyes were removed and homogenized, and HCMV replication was quantified by plaque assay . These results are also shown in Table 1 . In comparison with implants from animals treated with vehicle, treatment with CDV significantly reduced HCMV replication at all time points examined . On day 28, HCMV replication was highest in the vehicle control group, with titers of 774 ± 865 PFU/ml . Replication was reduced significantly (P < 0.0001) in the CDV treatment group but not in implants of animals treated with 75 mg of BDCRB/kg . These data indicated that both GCV and CDV were effective in reducing HCMV replication in retinal implant tissue, but BDCRB did not reduce virus titers significantly .

Effect of MBV and 175X on HCMV replication in a SCID-hu mouse ocular model. The short plasma half-life of BDCRB in rodents (7) led to the synthesis of a number of additional analogs, including MBV and 175X . To determine the activities of these compounds, we next utilized SCID mice containing retinal implants infected with the Toledo strain of HCMV . Mice were treated orally with MBV or 175X over the course of 28 days . Starting 1 day after infection, animals were treated either once daily i.p . with 33 mg of GCV/kg in 0.5% methylcellulose or twice daily orally with vehicle (0.5% methylcellulose), 75 or 25 mg of MBV/kg, or 75 or 25 mg of 175X/kg . On 7, 14, 21, and 28 days after infection, mice were sacrificed, eyes were removed and homogenized, and HCMV replication was quantified by plaque assay . The results are shown in Table 2 . In comparison to implants from vehicle-treated animals, HCMV replication in implants of animals treated with GCV was significantly reduced at all time points . At 21 days after infection, peak titers of virus in implants from animals treated with vehicle were 656 ± 1,010 PFU/ml . In contrast, titers of virus in implants of animals treated with 33 mg of GCV/kg were approximately threefold lower (228 ± 482 PFU/ml) . The overall effect of GCV was significant at P values of <0.01 . In addition, HCMV replication in implants from animals treated with either dose of MBV was significantly reduced by approximately threefold . At 75 mg of MBV/kg, titers of virus were reduced to 183 ± 271 PFU/ml, and at 25 mg/kg, titers of virus were 209 ± 432 PFU/ml . Although treatment with 175X was effective at 14 days after infection, it appeared that the drug was ineffective at both doses 21 and 28 days after infection . These data indicate that MBV appears to be an excellent candidate for further study, whereas 175X was less effective than MBV or GCV against HCMV replication in the SCID-hu retinal implant model .

 
Effect of treatment with BDCRB, MBV, or 175X on HCMV replication in SCID-hu thy/liv tissue implants. Since infection of the eye with HCMV presents a strong blood-eye barrier to the systemic delivery of an antiviral compound, we next used SCID mice implanted with human fetal thy/liv tissue under the kidney capsule to determine the efficacy of the benzimidazole ribonucleosides against HCMV replication in a visceral organ . In this model, the implanted thy/liv tissues have been shown to fuse and become vascularized . The hematopoietic progenitor cells of the thy/liv implant can then differentiate and proliferate within the environment of the kidney capsule (25) . It might be expected that antiviral drugs that do not penetrate well into the eye may be more efficacious in this model .

The effect of BDCRB was first examined in the thy/liv tissue implant model . In the first experiment, 17-week-old thy/liv implants were infected with 6,700 PFU of the Toledo strain of HCMV . Starting 24 h after infection, mice were treated with vehicle (corn oil), 25 mg of GCV/kg, or 100 or 33 mg of BDCRB/kg for 28 days . At 14, 21, 28, and 35 days after infection, implants were biopsied and HCMV titers were quantified by plaque assay . The results (Table 3) indicated that both GCV and BDCRB were efficacious in this model . At 28 days after infection, during the peak of HCMV replication, GCV inhibited HCMV replication by greater than 3 log₁₀ PFU/g and both doses of BDCRB inhibited viral replication by approximately 2 to 3 log₁₀ PFU/g .

 
The effects of the benzimidazole nucleosides, 175X and MBV, on the replication of HCMV were further investigated with the thy/liv tissue implant model . In this experiment, thy/liv implants were infected with 7,500 PFU of the Toledo strain of HCMV . Beginning 24 h after infection, mice were treated with either vehicle (0.5% methycellulose in saline) orally twice daily, 25 mg of GCV/kg in H₂O i.p . once daily, 75 mg of 175X/kg in vehicle orally twice daily, or 75 mg of MBV/kg in vehicle orally twice daily . This regimen was continued for 28 days, with implant biopsies performed 14, 21, 28, and 35 days after infection . The biopsies were homogenized, and HCMV titers were determined by plaque assay . The results in Table 4 indicated that both 175X and MBV were as effective as GCV in inhibiting the replication of HCMV in thy/liv tissue implants . During benzimidazole nucleoside drug administration, HCMV replication was significantly reduced by 1.5 to 3 log₁₀ PFU/g when compared to the vehicle control .

 
To complete these studies, an experiment was performed to determine the dose response of treatment with 100 and 33 mg of these two compounds/kg . In this experiment, thy/liv implant tissue was infected with 4,000 PFU of the Toledo strain of HCMV . Beginning 24 h after infection, animals were treated once daily i.p . with 33 mg of GCV/kg or twice daily orally with vehicle (0.5% methylcellulose in saline), 100 or 33 mg of MBV/kg, or 100 or 33 mg of 175X/kg . Treatment was continued for 28 days, and implants were biopsied 14, 21, 28, and 35 days postinfection . Implant tissue was homogenized, and HCMV titers were quantified by plaque assays . The results, shown in Table 5, indicate that only treatments with 33 mg of GCV/kg or 100 mg of MBV/kg were effective in inhibiting the replication of HCMV . Based on stratified Wilcoxon rank sum analysis of the data, this inhibition was found to be statistically significant, with P values of 0.0003 for treatment with 33 mg of GCV/kg and 0.0002 for treatment with 100 mg of MBV/kg . At 28 days postinfection, titers of HCMV were 4.97 ± 5.02, 1.22 ± 1.69, 0 ± 0, and 3.72 ± 3.89 log₁₀ PFU/g in implants from animals treated with vehicle, 33 mg of GCV/kg, 100 mg of MBV/kg, and 100 mg of 175X/kg, respectively . Although we observed a 1-log decrease in titer of virus after treatment with 100 mg of 175X/kg, this decrease was not statistically significant . These results suggest that of the three benzimidazole ribonucleoside compounds examined, MBV appears to be the best candidate for further evaluation .

 
Overall, these experiments with the benzimidazole nucleosides indicated that these compounds had significant activity against HCMV replication in vivo both in the retinal tissue implant and in the thy/liv implant model . These compounds appear to be excellent candidates for further development as inhibitors of HCMV replication .

Of particular interest for these studies is a comparison of the Toledo strain of CMV used in the animal models with other laboratory and clinical isolates . It has been reported that the Toledo isolate contains DNA sequences not found in the AD169 or Towne strains and more closely resembling those of wild-type CMV (6) . It has also been reported that the Toledo isolate replicated to significantly greater titers in thy/liv implants in SCID mice (5), but we have reported that Toledo, AD169, and Towne replicate to similar levels in thy/liv or retinal tissue implants (3, 16) . The Toledo strain was similar to other laboratory and clinical isolates in its susceptibility to GCV and the benzimidazole ribonucleosides (36), which validates its use for the animal model studies .

Of the three compounds, only MBV has been extensively evaluated for pharmacokinetic properties and toxicity . These preclinical studies indicated that the compound was about 90% orally bioavailable in the rat and about 50% in monkeys but was poorly distributed in the brain, cerebrospinal fluid, or vitreous humor and was highly protein bound . The results of the toxicology studies indicated a favorable safety profile (18) . Based upon the results from the preclinical and toxicology studies and in the absence of an animal model for CMV infection, MBV was evaluated in a phase I safety and pharmacokinetic trial . The results indicated that the drug was rapidly absorbed after oral administration, had few if any severe adverse effects (35), and in a second phase I dose escalation trial with human immunodeficiency virus-infected men reduced titers of HCMV in semen by about 3 log₁₀ PFU/ml (21) .

Animal models for CMV utilizing murine, rat, and guinea pig CMV have played an important role in the development of antiviral drugs for CMV, including GCV, CDV, and PFA (10, 14, 15, 22, 23, 26, 29, 30) . However, many of the newer compounds, including the benzimidazole nucleosides, are not active against those surrogate viruses (36) . To provide animal models for HCMV, we have utilized SCID mice implanted with either human fetal retinal tissue in the eye or thy/liv tissue under the kidney capsule . The implant tissue can be infected with human CMV (8, 25) after a few weeks and used to evaluate the efficacies of new antiviral agents (3, 16) . To validate the two models, the efficacies of GCV and CDV were determined in both ocular and thy/liv implants . The results indicated that both models are useful in determining activity against HCMV in vivo and appear to be predictive for efficacy in humans (16) .

In the present study, we compared the activities of BDCRB, MBV, and 175X with GCV in both ocular and thy/liv implants . CDV was also used in one retinal study . In the retinal model, GCV given i.p . was very active in significantly reducing viral replication in the implant and reducing infection rates, as was CDV . However, BDCRB did not alter titers of virus, which was not surprising given its short plasma half-life (11) . In contrast, oral MBV at either 75 or 25 mg/kg significantly reduced viral replication, although infection rates were not altered . In spite of the information from the preclinical studies, which indicated that MBV was poorly absorbed into the eye and was highly protein bound (18), twice-daily therapy was highly efficacious . In mice treated with 175X, a significant reduction in titers of virus was observed in animals that received 25 mg/kg but was not observed with 75 mg/kg . There is little preclinical information available on this analog to help interpret those results .

In the next series of experiments, the activities of GCV, BDCRB, MBV, and 175X were determined in SCID mice with thy/liv implants under the kidney capsule . As expected, GCV was very effective in this model of infection, reducing HCMV titers by about 3 log₁₀ PFU/g . Similar results have been reported by Mocarski and colleagues (25) . Although BDCRB was not efficacious in the retinal model, it was highly effective in reducing viral replication in the SCID-hu thy/liv implants . In this model of infection, both MBV and 175X significantly reduced HCMV replication at concentrations of 75 mg/kg twice daily . Lower doses of 33 mg/kg were not effective .

These results support the concept that CMV infection of the visceral organs is more easily treated than those of the eye or the central nervous system . Although GCV and MBV worked in both models, suggesting that MBV may be efficacious in human CMV infection when given orally, it is difficult to make a direct comparison between oral GCV and oral MBV, since GCV is poorly orally bioavailable in humans but relatively highly bioavailable in mice . In general, in our studies with these two models, GCV and CDV have been more effective in treating the thy/liv infection than those in the eye (16) . Similar observations were made in these studies for both BDCRB and MBV .

While these two experimental infections do not provide exact models for CMV retinitis and disseminated CMV in transplant patients, they do provide excellent in vivo systems for determining the effect of treatment on the replication of CMV in growing tissue with innervated blood vessels . The efficacy of MBV in these studies supports the clinical observation that treatment of CMV-infected human immunodeficiency virus patients reduced viral replication in semen and further supports the evaluation of this compound in additional clinical studies .

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