L. Naesens

Differential antiherpesvirus and antiretrovirus effects of the (S) and (R) enantiomers of acyclic nucleoside phosphonates: potent and selective in vitro and in vivo antiretrovirus activities of (R)-9-(2-phosphonomethoxypropyl)-2,6-diaminopurine.

The (S)- and (R)-enantiomers of acyclic purine nucleoside phosphonate analogs (i.e., 3-hydroxy-2-phosphonomethoxypropyl [HPMP] derivatives, 3-fluoro-2-phosphonomethoxypropyl [FPMP] derivatives, and 2-phosphonomethoxypropyl [PMP] derivatives of adenine [A], 2-aminopurine, 2,6-diaminopurine [DAP], and guanine [G]) have been synthesized and evaluated for antiviral activity. As a rule, the HPMP derivatives proved effective against DNA viruses but not RNA viruses or retroviruses. In particular, (S)-HPMPA, (S)-HPMPDAP, and (R)- and (S)-HPMPG were exquisitely inhibitory to herpes simplex virus type 1 (50% effective concentrations, 0.63, 0.22, 0.10, and 0.66 microM, respectively). The FPMP and PMP derivatives showed marked inhibitory activities against retroviruses but not DNA viruses. The (S)-enantiomer of FPMPA and the (R)-enantiomer of PMPA were approximately 30- to 100-fold more effective against human immunodeficiency virus and Moloney murine sarcoma virus (MSV) than their enantiomeric counterparts. In contrast, both (S)- and (R)-enantiomers of the DAP and G derivatives proved equally effective against retroviruses, except for (R)-PMPDAP, which was 15- to 40-fold more inhibitory than (S)-PMPDAP. (R)-PMPDAP emerged as the most potent and selective inhibitor of MSV-induced transformation of murine C3H/3T3 cells and human immunodeficiency virus-induced cytopathicity in MT-4 and CEM cells (50% effective concentration, approximately 0.1 to 0.6 microM). When administered intraperitoneally at a single dose as low as 2 mg/kg, (R)-PMPDAP efficiently decreased MSV-induced tumor formation in newborn NMRI mice and significantly increased the survival time of MSV-infected mice. In addition, upon oral administration to MSV-infected mice, (R)-PMPDAP showed marked antiretroviral efficacy.

(R)-9-(2-phosphonylmethoxypropyl)-2,6-diaminopurine is a potent inhibitor of feline immunodeficiency virus infection.

The antiviral efficacy of acyclic nucleoside phosphonates, including 9-(2-phosphonylmethoxyethyl)adenine (PMEA) and (R)-9-(2-phosphonylmethoxypropyl)-2,6-diaminopurine [(R)-PMPDAP] against feline immunodeficiency virus (FIV) infection was determined. (R)-PMPDAP showed the highest selectivity index (> 2,000) in vitro. Treatment of experimentally FIV-infected asymptomatic cats with PMEA or (R)-PMPDAP had no effect on the CD4+/CD8+ ratio. However, mean plasma viral RNA concentrations decreased significantly in the (R)-PMPDAP-treated cats. Our data show that, in comparison to PMEA, (R)-PMPDAP is a more potent and less toxic inhibitor of FIV replication both in vitro and in vivo.

Mouse Adenovirus Type 1 Infection in SCID Mice: an Experimental Model for Antiviral Therapy of Systemic Adenovirus Infections

ABSTRACT The importance of human adenovirus infections in immunocompromised patients urges for new and adequate antiadenovirus compounds. Since human adenoviruses are species specific, animal models for systemic adenovirus infections rely on a nonhuman adenovirus. We established mouse adenovirus type 1 (MAV-1) infection of BALB/c SCID mice as a model for the evaluation of antiadenovirus therapy. In vitro studies with mouse embryonic fibroblasts pointed to the acyclic nucleoside phosphonate cidofovir and the N-7-substituted acyclic derivative 2-amino-7-(1,3-dihydroxy-2-propoxymethyl)purine (S-2242) as markedly active compounds against MAV-1. SCID mice, infected intranasally with MAV-1, developed a fatal disseminated infection after approximately 19 days, characterized by hemorrhagic enteritis. Several techniques were optimized to monitor viral, immunological, and pathological aspects of MAV-1 infection. Real-time PCR quantification of viral DNA revealed that after replication in the lungs, virus disseminated to several organs, including the brain, liver, spleen, intestine, heart, and kidneys (resulting in viruria). Immunohistochemical staining showed that MAV-1 was localized in the endothelial cells of the affected organs. Using reverse transcription-PCR, tissue levels of proinflammatory cytokines (i.e., interleukin-1β and tumor necrosis factor alpha) were found to be markedly increased. The MAV-1/SCID model appears to be an appropriate model for in vivo evaluation of antiadenovirus agents. Treatment with cidofovir or S-2242 at a dose of 100 mg per kg of body weight resulted in a significant delay in MAV-1-related death, although these antivirals were unable to completely suppress virus replication despite continued drug treatment. These findings suggest that complete virus clearance during antiviral therapy for disseminated adenovirus infection may require an efficient adaptive immune response from the host.

Recovery of Humoral Immunity Is Critical for Successful Antiviral Therapy in Disseminated Mouse Adenovirus Type 1 Infection

ABSTRACT Severe adenovirus infections in transplant recipients undergoing immunosuppressive therapy are of increasing concern. Controversy exists on the contribution of antiviral therapy and the host immune response to recovery from these infections. Here, we established a systemic mouse adenovirus type 1 (MAV-1) infection in cyclophosphamide (CyP)-treated BALB/c mice. CyP was administered at 100 mg per kg of body weight every other day for 2, 3, or 4 weeks, thereby inducing general but reversible leukopenia, with a major suppression of the B-cell numbers and functionality that was more pronounced than that seen with T cells. The outcome of MAV-1 infection was dependent on the duration of CyP therapy, as the mice with the most severe immunosuppression were the most vulnerable to MAV-1-induced hemorrhagic enteritis and mortality. The protective effect of concomitant antiviral therapy with cidofovir depended on the level of immunosuppression. The combination of cidofovir treatment with the withdrawal of immunosuppression was the most successful regimen for increasing survival rates. Survival was clearly correlated with the clearance of virus and increased titers of MAV-1-specific antibodies in sera. In addition, the passive transfer of MAV-1-specific immunoglobulin G into MAV-1-infected SCID BALB/c mice caused a marked delay in mortality, the extent of the delay being dependent on the titer of MAV-1-specific antibodies. Based on the critical role of the humoral immune response in the early defense against disseminated adenovirus infection, the concomitant use of adenovirus-specific immunoglobulins and antiviral therapy should be considered for transplant patients at risk for severe adenovirus infections.

Therapeutic Approaches to HHV-6 Infection

Publisher Summary In the absence of human herpesvirus-6 (HHV-6)-specific therapies, treatment of HHV-6 infections relies on the relatively broad-spectrum antiherpetic agents (val)ganciclovir and foscarnet. In case of ganciclovir, the anti-HHV-6 activity is diminished by two factors: inefficient phosphorylation of ganciclovir by the HHV-6 pU69 protein kinase and relatively low affinity of ganciclovir triphosphate for the HHV-6 DNA polymerase. Although these drugs offer an indisputable benefit in the therapy or prophylaxis of cytomegalovirus (CMV), their clinical efficacy against HHV-6 can only be estimated from a number of heterogeneous case reports. A definite proof of anti-HHV-6 activity requires an assay to directly measure the inhibitory effect of the compound on HHV-6 replication. In antigen-based assays, HHV-6 proteins are detected with HHV-6-specific monoclonal or polyclonal antibodies. After indirect immunofluorescence staining, the fluorescence intensity in the HHV-6-infected cells is quantified by fluorescence microscopy or fluorescenceactivated cell sorter (FACS) analysis In case of HHV-6, some investigators have used the continuous T-lymphoblasts HSB-2 and Molt-3, which are the preferred host cell lines for propagation of HHV-6A or HHV-6B strains, respectively.

Metabolism and anti-HIV activity of phosphoramidate derivatives of D4T-MP with variations in the amino acid moiety

The metabolism of different phosphoramidate prodrugs of d4T-MP, in which the phosphate group is linked to a phenyl group and the alkyl ester of an amino acid was studied in crude CEM cell extracts. Significant (80-100%) conversion to the amino acyl d4T-MP metabolite was obtained with derivatives containing L-alanine or methyl-L-aspartic acid. A lower degree of conversion was seen with derivatives containing L-phenylalanine, L-methionine, methyl-L-glutamic acid or L-leucine. Derivatives containing D-alanine, beta-alanine, glycine, L-valine or L-lactate showed no conversion to the amino acyl d4T-MP metabolite. Overall, there was a close correlation between the anti-HIV activity of these prodrugs and their conversion rate to the amino acyl d4T-MP metabolite. Our data suggest that the enzymes involved in the formation of the amino acyl d4T-MP metabolite have a rather stringent specificity for L-alanine as the amino acid moiety. In addition, these enzymes were found to be markedly species-dependent, their activities being highest in mouse serum, followed by guinea pig serum, but only minimal in human serum. Mouse serum therefore appears to be the medium of choice to isolate and identify the enzymes that are involved in the metabolism of these phosphoramidate prodrugs.