A novel corneal explant model system to evaluate antiviral drugs against feline herpesvirus type 1 (FHV-1).

Feline herpesvirus type-1 (FHV-1) is the most common viral cause of ocular surface disease in cats. Many antiviral drugs are used to treat FHV-1, but require frequent topical application and most lack well-controlled in vivo studies to justify their clinical use. Therefore, better validation of current and novel treatment options are urgently needed. Here, we report on the development of a feline whole corneal explant model that supports FHV-1 replication and thus can be used as a novel model system to evaluate the efficacy of antiviral drugs. The anti-herpes nucleoside analogues cidofovir and acyclovir, which are used clinically to treat ocular herpesvirus infection in cats and have previously been evaluated in traditional two-dimensional feline cell cultures in vitro, were evaluated in this explant model. Both drugs suppressed FHV-1 replication when given every 12 h, with cidofovir showing greater efficacy. In addition, the potential efficacy of the retroviral integrase inhibitor raltegravir against FHV-1 was evaluated in cell culture as well as in the explant model. Raltegravir was not toxic to feline cells or corneas, and most significantly, inhibited FHV-1 replication at 500 µM in both systems. Importantly, this drug was effective when given only once every 24 h. Taken together, our data indicate that the feline whole corneal explant model is a useful tool for the evaluation of antiviral drugs and, furthermore, that raltegravir appears a promising novel antiviral drug to treat ocular herpesvirus infection in cats.

The lipid moiety of brincidofovir is required for in vitro antiviral activity against Ebola virus.

Brincidofovir (BCV) is the 3-hexadecyloxy-1-propanol (HDP) lipid conjugate of the acyclic nucleoside phosphonate cidofovir (CDV). BCV has established broad-spectrum activity against double-stranded DNA (dsDNA) viruses; however, its activity against RNA viruses has been less thoroughly evaluated. Here, we report that BCV inhibited infection of Ebola virus in multiple human cell lines. Unlike the mechanism of action for BCV against cytomegalovirus and other dsDNA viruses, phosphorylation of CDV to the diphosphate form appeared unnecessary. Instead, antiviral activity required the lipid moiety and in vitro activity against EBOV was observed for several HDP-nucleotide conjugates.

Herpes simplex virus and varicella zoster virus: recent advances in therapy.

PURPOSE OF REVIEW: The mainstay of antiviral therapy for the alpha-herpesviruses [herpes simplex virus (HSV)-1, HSV-2, and varicella zoster virus (VZV)] over the past 40 years has been the nucleoside analogues such as aciclovir. Although conventional antiviral therapy has reduced mortality in severe disease, novel agents are needed to address the emergence of resistance and toxicity associated with current second-line therapy. Treatment and prophylaxis of VZV and HSV reactivations remains a challenge. RECENT FINDINGS: A number of compounds have recently been evaluated in human clinical trials, amongst them brincidofovir, an intracellularly acting derivative of cidofovir currently undergoing phase III trials. The helicase-primase inhibitors are a new class of antiviral agent and may circumvent resistance to existing agents. Amenamevir and pritelivir are two examples of these agents that have been evaluated clinically along with novel nucleoside analogues such as valomaciclovir and FV-100. Tenofovir, an agent used in HIV and hepatitis B therapy, may also have a role in the prevention of HSV-2 acquisition and reduce viral shedding. SUMMARY: Although several novel antiviral agents have undergone clinical trials in recent years, all are yet to gain licensure. Brincidofovir appears to be the candidate with most promise for adoption into routine practice in the near future.

Hydroxymethylcytosine and demethylation of the γ-globin gene promoter during erythroid differentiation.

The mechanism responsible for developmental stage-specific regulation of γ-globin gene expression involves DNA methylation. Previous results have shown that the γ-globin promoter is nearly fully demethylated during fetal liver erythroid differentiation and partially demethylated during adult bone marrow erythroid differentiation. The hypothesis that 5-hydroxymethylcytosine (5 hmC), a known intermediate in DNA demethylation pathways, is involved in demethylation of the γ-globin gene promoter during erythroid differentiation was investigated by analyzing levels of 5-methylcytosine (5 mC) and 5 hmC at a CCGG site within the 5' γ-globin gene promoter region in FACS-purified cells from baboon bone marrow and fetal liver enriched for different stages of erythroid differentiation. Our results show that 5 mC and 5 hmC levels at the γ-globin promoter are dynamically modulated during erythroid differentiation with peak levels of 5 hmC preceding and/or coinciding with demethylation. The Tet2 and Tet3 dioxygenases that catalyze formation of 5 hmC are expressed during early stages of erythroid differentiation and Tet3 expression increases as differentiation proceeds. In baboon CD34+ bone marrow-derived erythroid progenitor cell cultures, γ-globin expression was positively correlated with 5 hmC and negatively correlated with 5 mC at the γ-globin promoter. Supplementation of culture media with Vitamin C, a cofactor of the Tet dioxygenases, reduced γ-globin promoter DNA methylation and increased γ-globin expression when added alone and in an additive manner in combination with either DNA methyltransferase or LSD1 inhibitors. These results strongly support the hypothesis that the Tet-mediated 5 hmC pathway is involved in developmental stage-specific regulation of γ-globin expression by mediating demethylation of the γ-globin promoter.

Development of CMX001 (Brincidofovir) for the treatment of serious diseases or conditions caused by dsDNA viruses.

CMX001 (hexadecyloxypropyl-cidofovir, Brincidofovir) is a broad spectrum, lipid conjugate of cidofovir that is converted intracellularly into the active antiviral, cidofovir diphosphate. The lipid conjugation results in oral bioavailability, higher intracellular concentrations of active drug, lower plasma concentrations of cidofovir and increased antiviral potency against dsDNA viruses.

Effect of vocal fold injection of cidofovir and bevacizumab in a porcine model.

IMPORTANCE: Recurrent respiratory papillomatosis (RRP) is a common and often chronic disorder. Intralaryngeal bevacizumab has gained recent interest as an adjuvant therapy for RRP. However, no histologic model has been published describing the effects of bevacizumab on the vocal fold. OBJECTIVE: To investigate the histologic effects of bevacizumab injections into the vocal fold and compare these findings with those for cidofovir and saline control injections. DESIGN AND SETTING: In vivo animal study involving eighteen 1-year-old Yorkshire crossbreed pigs, with a blinded review of pathologic findings conducted in a veterinary research laboratory. INTERVENTIONS: The pigs were randomly divided into six study groups receiving 2.5 or 5.0 mg of cidofovir or bevacizumab alone or in combination. Each pig received an injection of 0.5 mL of the test drug in the right vocal fold and 0.5 mL of saline in the left vocal fold. These injections were performed 4 times during the course of 8 weeks. One pig from each group was killed humanely and the larynges harvested 2 weeks after the last injection. The remaining pigs were killed 4 months after the last injection on the remaining pigs. The vocal folds were fixed and stained with hematoxylin-eosin and trichrome and reviewed for histologic changes by 3 blinded pathologists. MAIN OUTCOMES AND MEASURES: Histologic changes to the vocal folds. RESULTS: Minimal inflammation, edema, and atypia were found in all treatment groups. No appreciable histologic differences were found among the 3 treatment groups and their controls. No difference was seen in the vocal folds that were harvested late (4 months) vs early (2 weeks) after last injection. No fibrosis was found in any of the specimens. CONCLUSIONS AND RELEVANCE: No histologic evidence suggests that intralaryngeal cidofovir or bevacizumab alone or in combination resulted in significant changes to the porcine vocal fold. Future studies may build on this model to test higher dosages and/or may combine injections with potassium titanyl phosphate laser therapy.

Sapacitabine for cancer.

INTRODUCTION: Sapacitabine is an orally bioavailable nucleoside analog prodrug that is in clinical trials for hematologic malignancies and solid tumors. The active metabolite of sapacitabine, CNDAC (2'-C-cyano-2'-deoxy-1-ß-D-arabino-pentofuranosylcytosine), exhibits the unique mechanism of action of causing single-strand breaks (SSBs) after incorporation into DNA, which are converted into double-strand breaks (DSBs) when cells enter a second S-phase. CNDAC-induced DSBs are predominantly repaired through homologous recombination (HR). Cells deficient in HR components are greatly sensitized to CNDAC. Therefore, sapacitabine could be specifically effective against tumors that are deficient in this repair pathway. AREAS COVERED: This review summarizes results from supporting evidence for the mechanisms of action of sapacitabine, its preclinical activities and the current results of clinical trials in a variety of cancers. The novel action mechanism of sapacitabine is discussed, with a view to validate it as a chemotherapeutic drug targeting malignancies with defects in HR. EXPERT OPINION: Knowledge of CNDAC mechanism identifies tumors that may be sensitized to sapacitabine, thus enabling a personalized treatment strategy. It also creates the opportunity to overcome resistance to current front-line therapies and identify synergistic interactions with known anticancer drugs. The results of such investigations may provide rationales for the design of sapacitabine-based clinical trials.

Topical treatment of cutaneous vaccinia virus infections in immunosuppressed hairless mice with selected antiviral substances.

BACKGROUND: Certain nucleoside, nucleotide and pyrophosphate analogues may be useful for treating severe complications arising as a result of virus dissemination following smallpox (live vaccinia virus) vaccinations, especially in immunocompromised individuals. We used an immunosuppressed hairless mouse model to study the effects of 10 antiviral agents on progressive vaccinia infections. METHODS: Hairless mice were immunosuppressed by treatment with cyclophosphamide (100 mg/kg) every 4 days starting 1 day prior to vaccinia virus (WR strain) infection of wounded skin. Topical treatments with antiviral agents were applied twice a day for 7 days starting 5 days after virus exposure. RESULTS: Topical 1% cidofovir cream treatment was effective in significantly reducing primary lesion severity and decreasing the number of satellite lesions. Topical 1% cyclic HPMPC and 1% phosphonoacetic acid were not quite as active as cidofovir. Ribavirin (5%) treatment reduced lesion severity and diminished the numbers of satellite lesions, but the mice died significantly sooner than placebos. 2-Amino-7-[(1,3,-dihydroxy-2-propoxy)methyl]purine (compound S2242; 1%) moderately reduced primary lesion sizes. Ineffective treatments included 5% arabinosyladenine, 1% arabinosylcytosine, 1% 5-chloro-arabinosylcytosine, 5% arabinosylhypoxanthine 5-monophosphate and 5% viramidine. CONCLUSIONS: Of the compounds tested, topically applied cidofovir was the most effective treatment of cutaneous vaccinia virus infections in immunosuppressed mice. Topical treatment with cidofovir could be considered as an adjunct to intravenous drug therapy for serious infections.

Atypical cytostatic mechanism of N-1-sulfonylcytosine derivatives determined by in vitro screening and computational analysis.

We have previously shown that N-1-sulfonylpyrimidine derivatives have strong antiproliferative activity on human tumor cell lines, whereby 1-(p-toluenesulfonyl)cytosine showed good selectivity with regard to normal cells and was easily synthesized on a large scale. In the present work we have used an interdisciplinary approach to elucidate the compounds' mechanistic class. An augmented number of cell lines (11) has allowed a computational search for compounds with similar activity profiles and/or mechanistic class by integrating our data with the comprehensive DTP-NCI database. We applied supervised machine learning methodology (Random Forest classifier), which offers information complementary to unsupervised algorithms commonly used for analysis of cytostatic activity profiles, such as self-organizing maps. The computational results taken together with cell cycle perturbation and apoptosis analysis of the cell lines point to an unusual mechanism of cytostatic action, possibly a combination of nucleic acid antimetabolite activity and a novel molecular mechanism.

In vivo imaging of cidofovir treatment of cowpox virus infection.

Variola virus and other members of the genus Orthopoxviruses constitute a prominent bioterrorism and public health threat. Treatment with the anti-viral drug cidofovir inhibits replication of orthopoxviruses in vitro and in vivo. In this study, we visualized the effect of cidofovir on viral kinetics in orthopoxvirus infected mice by using whole-body fluorescence imaging (FI). We engineered a cowpox virus (CPV) expressing the enhanced green fluorescent protein (GFP). Single-step growth curves and calculated 50% lethal doses (LD(50)) of wild-type CPX (Wt-CPV) and GFP-expressing CPX (GFP-CPV) were comparable. Whole-body FI first detected GFP fluorescence in the mesenteric tissue of untreated animals on post-infection day (PID) 1. On PID 3 GFP signal was detected throughout the mesentery, in all abdominal organs by PID 5 and in most major organs, except for the heart and brain by PID 6. Infected animals treated with 25mg/kg of cidofovir also began showing signs of viral replication on PID 1, however, the fluorescent signal was limited only to discrete foci throughout the course of the infection. This work describes the first use of an established Orthopox model of infection to evaluate drug efficacy and track virus progression on a macroscopic level.

Clinical and in vitro evaluation of cidofovir for treatment of adenovirus infection in pediatric hematopoietic stem cell transplant recipients.

Post-hematopoietic stem cell transplantation (HSCT) adenovirus infections were identified in 31 of 204 consecutive pediatric HSCT patients, 18 of whom had severe manifestations of infection. Cidofovir treatment led to clinical improvement in 8 of 10 patients with severe infection and to virologic clearance in 9 patients. In vitro susceptibility to cidofovir was demonstrated in 12 clinical adenovirus isolates. Cidofovir is a promising treatment option for this population.

The cotton rat model for adenovirus ocular infection: antiviral activity of cidofovir.

To determine the antiviral effects of compounds against ocular adenovirus (AdV) infection, we established an animal model of AdV infection in cotton rat eyes. Cotton rat eyes were inoculated intrastromally and topically with four AdV serotypes 4, 5, 8, and 37, and treated topically with 1% HPMPC (cidofovir) eye drops twice a day. The infected corneas were extracted and homogenized, and virus titers in the cornea specimens were determined by a plaque assay. The virus titer in AdV type 5-inoculated eyes peaked on days 0 through 3 after inoculation and virus shedding was detected for 18.0+/-2.8 days. AdV 5 antigen in the infected corneas was demonstrated in the corneal epithelial cells by immunofluorescence stain. However, for AdV serotypes 4, 8, and 37, no evidence of continued virus replication in cotton rat eyes was noted. Specimens from cidofovir-treated eyes infected with AdV 5 demonstrated a statistically significant reduction in the mean virus titer (days 3-15) (P=0.028) and virus shedding duration (P=0.0014), as compared with those of the control group.

The role of growth factors, angiogenic enzymes and apoptosis in neovascularization and tumor growth-collected publications.

Hemangiomas represent the most frequent tumors of infancy. However, the pathogenesis of these tumors is still largely unknown, and current treatment of juvenile hemangiomas remains unsatisfactory. We have presented a novel animal model for hemangiomas. Induction of hemangioma development was achieved by intraperitoneal (i.p.) infection of 4-day-old rats with the mouse polyoma virus (PyV). This led to the development of multiple hemangiomas, which caused death of the untreated animals within 3 weeks p.i. The hemangiomas had the histological and immunohistochemical features reminiscent of human hemangiomas. Moreover, the angiogenesis inhibitor TNP-470 afforded a protective effect in this model. Tumor growth is determined by the balance between cell proliferation and apoptosis and is modulated by angiogenesis. Angiogenesis is a complex process, involving extensive interplay between cells, extracellular matrix components and soluble factors. Each of these factors represents a possible target for pharmacological intervention to inhibit blood vessel formation and subsequently tumor growth. We have focused on specific inhibitors of the angiogenesis inducers basic fibroblast growth factor and Thymidine Phosphorylase and studied their mechanism of action and anti-angiogenic activity. In addition, we have shown that the apoptosis-inducer cidofovir inhibits PyV-induced hemangioma development in rats and the growth of virus-independent, vascular tumors in mice. So far, cidofovir has only been evaluated clinically for the treatment of human papillomavirus (HPV)-associated tumors. Our findings open new perspectives for the use of cidofovir as an anti-tumor agent in the therapy of hemangiomas and other tumors that are not associated with an oncogenic virus.

Synthesis and antiviral activity of oxaselenolane nucleosides.

As dioxolane and oxathiolane nucleosides have exhibited promising antiviral and anticancer activities, it was of interest to synthesize isoelectronically substituted oxaselenolane nucleosides, in which the 3'-CH(2) is replaced by a selenium atom. To study structure-activity relationships, various pyrimidine and purine oxaselenolane nucleosides were synthesized from the key intermediate, (+/-)-2-benzoyloxymethyl-1,2-oxaselenolane 5-acetate (6). Among the synthesized racemic nucleosides, cytosine and 5-fluorocytosine analogues exhibited potent anti-HIV and anti-HBV activities. It was of interest to obtain the enantiomerically pure isomers to determine if they have differential antiviral activities. However, due to the difficult and time-consuming nature of enantiomeric synthesis, a chiral HPLC separation was performed to obtain optical isomers from the corresponding racemic mixtures. Each pair of enantiomers of Se-ddC and Se-FddC was separated by an amylose chiral column using a mobile phase of 100% 2-propanol. The results indicate that most of the anti-HIV activity of both cytosine and fluorocytosine nucleosides resides with the (-)-isomers.

The antisense oligonucleotide ISIS 2922 prevents cytomegalovirus-induced upregulation of IL-8 and ICAM-1 in cultured human fibroblasts.

Human cytomegalovirus (HCMV) infection is associated with excessive proinflammatory immune responses such as cytokine/chemokine production or upregulation of adhesion molecules on the host cells. It is assumed that these features of HCMV-related immunopathology can not be treated effectively with currently available anti HCMV drugs. In the present study the efficacy of ganciclovir (GCV), foscarnet (PFA), cidofovir (HPMPC), and ISIS 2922, an antisense oligonucleotide complementary to HCMV immediate-early (IE) mRNA, was investigated on HCMV-induced secretion and functional activity of the C-X-C chemokine IL-8 and the expression of the intercellular adhesion molecule-1 (ICAM-1). As compared with mock-infected cells IL-8 production was increased up to 9-fold and ICAM-1 expression up to 4-fold in HCMV-infected fibroblasts. Treatment of infected cells with GCV (40 microM), PFA (200 microM) or HPMPC (2 microM) suppressed completely virus replication as demonstrated by quantification of late (L) antigen expression and infectious virus production. These drugs, however, failed to inhibit IE antigen expression and did not prevent HCMV-induced upregulation of IL-8 and ICAM-1. In contrast, ISIS 2922 (1 microM) suppressed both IE and L antigen expression by 99% and inhibited infectious virus production by 10(4)-fold. Moreover, ISIS 2922 significantly suppressed HCMV-induced upregulation of both IL-8 and ICAM-1 expression on the transcriptional and on the protein level. Our results indicate that ISIS 2922 but not inhibitors of HCMV DNA prevents HCMV-induced upregulation of IL-8 and ICAM-1, both hallmarks of inflammatory processes. Thus, inhibition of HCMV IE expression with ISIS 2922 may be an important strategy for the treatment of HCMV-related immunopathogenesis.

[Evaluation of antiviral agents for adenovirus using the MTT method in vitro].

PURPOSE: Currently, there is no effective treatment for adenoviral conjunctivitis. We evaluated the antiviral inhibitory effect of four antiviral agents against adenovirus (ADV) in vitro. METHODS: Viruses used for the experiment were ADV type 4 (ADV 4), type 8 (ADV 8) and type 37 (ADV 37). We examined four antiviral agents, i.e., cidofovir (HPMPC), zalcitabine (ddC), foscarnet (PFA), and acyclovir (ACV). 50% effective concentration (EC50), 50% cytotoxic concentration (CC50) and selectivity index (SI) of compounds were determined for ADV infection in HEp-2 cells using the 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. RESULTS: HPMPC and ddC showed an inhibitory effect against all three ADVs. In particular, ddC showed more potent and selective inhibition against ADV than HPMPC. PFA and ACV were ineffective against ADV. CONCLUSIONS: HPMPC and ddC were inhibitory against ADV 4, ADV 8 and ADV 37 replication in vitro. The MTT method is rapid and simple for the screening of antiviral agents. We think this method is also very useful for the screening of anti-ADV agents.

The antiviral nucleotide analogs cidofovir and adefovir are novel substrates for human and rat renal organic anion transporter 1.

Nephrotoxicity is the dose-limiting clinical adverse effect of cidofovir and adefovir, two potent antiviral therapeutics. Because renal uptake likely plays a role in the etiology of cidofovir- and adefovir-associated nephrotoxicity, we attempted to identify a renal transporter capable of interacting with these therapeutics. A cDNA clone was isolated from a human renal library and designated human organic anion transporter 1 (hOAT1). Northern analysis detected a specific 2.5-kilobase pair hOAT1 transcript only in human kidney. However, reverse transcription-polymerase chain reaction revealed hOAT1 expression in human brain and skeletal muscle, as well. Immunoblot analysis of human kidney cortex demonstrated that hOAT1 is an 80- to 90-kilodalton heterogeneous protein modified by abundant N-glycosylation. Xenopus laevis oocytes expressing hOAT1 supported probenecid-sensitive uptake of [(3)H]p-aminohippurate (K(m) = 4 microM), which was trans-stimulated in oocytes preloaded with glutarate. Importantly, both hOAT1 and rat renal organic anion transporter 1 (rROAT1) mediated saturable, probenecid-sensitive uptake of cidofovir, adefovir, and other nucleoside phosphonate antivirals. The affinity of hOAT1 toward cidofovir and adefovir (K(m) = 46 and 30 microM, respectively) was 5- to 9-fold higher compared with rROAT1 (K(m) = 238 and 270 microM, respectively). These data indicate that hOAT1 may significantly contribute to the accumulation of cidofovir and adefovir in renal proximal tubules and, thus, play an active role in the mechanism of nephrotoxicity associated with these antiviral therapeutics.

Development and survival of Drosophila melanogaster fed a diet containing pyrimidine analogs.

A single generation of Drosophila melanogaster was raised on different media. One of the media was unsupplemented (the control) and the others were supplemented with pyrimidine analog at 10.3 mmol/kg culture medium. The relative numbers of larvae, pupae, and F1 adults reproduced from parent flies on each medium served as an indication of the relative toxicity of the supplements. The relative decreasing order of toxicity of the pyrimidines was as follows: 5-bromouracil < thymine < uracil = orotic acid = control = cytosine, control < UMP. The toxic effects of 5-bromouracil and thymine seem to be associated with the addition of a bromine or methyl group to carbon 5 of the pyrimidine ring. The UMP supplementation increased the number of adult F1 flies above the control group indicating that UMP was not only non toxic but also that it was beneficial.