Antiviral Chemistry and Chemotherapy

Cidofovir is a potent, broad spectrum antiviral agent with activity in vitro and in vivo against cytomegalovirus and other members of the herpesvirus family, as well as certain other DNA viruses. After uptake into cells it is converted enzymatically to cidofovir diphosphate, a structural analogue of deoxycytidine triphosphate, which selectively inhibits viral DNA polymerases relative to host cell polymerases. Cross-resistance to cidofovir is not usually seen with human cytomegalovirus isolates that are foscarnet-resistant, or isolates that are ganciclovir-resistant due to a deficiency in ganciclovir phosphorylation. Cross-resistance is seen, however, with isolates that are ganciclovir resistant due to polymerase mutations. A prolonged elimination phase seen in vivo, correlates with a long intracellular half-life seen in vitro and allows for efficacy in animal models of virus infection with infrequent dosing or prophylaxis. Clinical studies of intravenous cidofovir in cytomegalovirus retinitis in patients with AIDS are claimed to show delay of retinitis progression with maintenance doses given once every 2 weeks.

The replication of herpes simplex virus type 1 (HSV-1) or HSV-2 in MRC-5 cells infected at 0.01 pfu cell⁻¹ treated continuously for 72 h, was inhibited more efficiently by penciclovir than aciclovir (p = 0.0001). However, multiple cycles of replication were required in order to distinguish the compounds. Virus from cultures treated for 72 h with either compound, at 3 or 10 μg ml⁻¹ was resistant to penciclovir and aciclovir (50% effective concentrations > 10 μg ml⁻¹), but infectivity titres of supernatants from these aciclovirtreated cultures were higher than for penciclovir. Increased production of resistant virus in aciclovirtreated cultures may be the consequence of the less potent inhibition of virus replication by aciclovir. Penciclovir caused prolonged inhibition of HSV-1 and HSV-2 replication in three human cell lines infected at 1 pfu cell⁻¹ following treatment for 18 h, whereas virus replication resumed rapidly after withdrawal of aciclovir. Neither compound showed prolonged activity after 18 h treatment, when the multiplicity of infection was reduced to 0.01 pfu cell⁻¹. This surprising observation prompted experiments testing the effect of repeated pulse treatment in cultures infected at low multiplicity. Penciclovir inhibited HSV-1 replication significantly more effectively than aciclovir in MRC-5 cells infected at 10⁻⁴ pfu cell⁻¹ treated daily for 6 h (p < 0.001, n = 5) but only a trend was observed for HSV-2 (p = 0.06, n = 6).

Following an earlier demonstration of an antiviral effect against varicella-zoster virus (VZV, human herpesvirus 3) using 2-hydroxymyristic acid (2-hydroxytetradecanoic acid; 2-HM), an inhibitor of protein myristoylation, both 2-HM and 2-hydroxypalmitic acid (2-hydroxyhexadecanoic acid; 2-HP) have been tested against a range of viruses. Although both compounds inhibit the replication of varicella-zoster virus (VZV; human herpesvirus 3) they do not inhibit the replication of closely related herpesviruses. They do, however, inhibit the replication of both poliovirus (a member of the Picornaviridae) and the human immunodeficiency virus type 1 (HIV-1; a member of the Retroviridae). Neither compound is toxic to adherent cells by dye uptake assay, although limited toxicity is apparent to non-adherent cell lines at high concentrations. The mechanisms underlying these effects are discussed. A diminished effect of 2-hydroxymyristic acid when the compound is dissolved in dimethyl sulphoxide (DMSO) rather than ethanol is reported, and the implications for the use of DMSO as a ‘universal solvent’ for compound screening noted. Finally, it is suggested that targeting of ‘virus-essential’ cellular functions may provide an alternative route for inhibiting viral replication.

Various new aurintricarboxylic acid (ATA) polymer analogues have been evaluated for their antiviral activity against a wide array of DNA and RNA viruses, and their mechanism of action against human cytomegalovirus (HCMV) and human immunodeficiency virus type 1 (HIV-1). Most of the polymers exhibited marked antiviral activity against a variety of enveloped viruses, but not against non-enveloped viruses. The ATA polymers displayed the most pronounced activity against HIV-1, HCMV and human herpesvirus type 6 (HHV-6). Their action against HCMV and HIV could be ascribed to inhibition of the initial attachment of virus particles to the cells. Using radiolabelled virus, we proved that the polymers inhibit the binding of HCMV to HEL fibroblasts. By flow cytometric analysis, we demonstrated that these new polymers interfere with (i) the binding of OKT4A monoclonal antibody (mAb) to the cellular CD4 receptor, (ii) the binding of anti-gp120 mAb to HIV-1 glycoprotein (gp) 120, and (iii) the adsorption of HIV-1 virions and recombinant HIV-1gp120 (rgp120) to MT-4 cells. The presence of a salicylic acid substituent on the central bridging carbon in the parent compound ATA seems to play an important role in the anti-HIV activity of these ATA related polymer analogues.

Famciclovir (FCV) is the oral form of the potent and selective antiherpesvirus agent penciclovir (PCV). In order to provide more information on the spectrum of activity of PCV, the activities of FCV and PCV against duck hepatitis B virus (DHBV) in chronically infected ducks were examined. As part of this investigation, the oral pharmacokinetics of FCV and PCV were determined in ducks. Oral treatment of DHBV-infected ducks (twice daily for 21 days) with either PCV (20 mg kg⁻¹ or 100 mg kg⁻¹) or FCV (5 mg kg⁻¹ or 25 mg kg⁻¹) suppressed virus replication, as measured by both plasma viral DNA and DNA polymerase levels. Both markers were reduced to undetectable levels within 4 days of the start of treatment, and after the cessation of treatment there was a delay of 2 to 8 days before plasma DHBV DNA and DNA polymerase levels began to increase, indicating that virus replication had resumed. The demonstration of efficacy of PCV and its oral form FCV against DHBV suggested that the two compounds may have clinical benefits. against human hepatitis B virus. Clinical trials are currently ongoing.

Foscarnet and (-)9-[4-hydroxy-2-(hydroxymethyl)butyl] guanine (H2G) have already been shown to inhibit herpesviruses in vitro and also to inhibit viral antigen production in primary SIV infection in monkeys. Attempts have been made to determine if these invivo effects on SIV were due to a direct effect on SIV or were mediated through inhibition of endogenous transactivating herpesviruses. The possible involvement of herpesviruses in primary SIV_sm infection in monkeys was studied by the use of various inhibitors of herpesvirus replication. Subcutaneous injections of 3 × 5 mg kg⁻¹ day⁻¹ of aciclovir, 3 × 5 mg kg⁻¹ day⁻¹ of ganciclovir and 3 × 28 mg kg⁻¹ day⁻¹ of phosphonoacetic acid had no effect on primary SIV_sm infection in cynomolgus monkeys. These doses of aciclovir, ganciclovir and phosphonoacetic acid are inhibitory to several herpesviruses. The results suggest that the effects of foscarnet and H2G on primary SIV_sm infection in monkeys are direct and not mediated through inhibition of a replicating herpesvirus.

Lipophilic prodrugs of 2′,3′-dideoxycytidine (ddC), 4,5′-diacetyI-ddC (DAC), 4,5′-ditrimethylacetyl-ddC (DTMAC), 4,5′-dicyclopentylpropionyl-ddC (DCYPP) and 5′-cholesteryl-ddC (CHOL), were evaluated for their utility in improving brain delivery of the parent nucleoside. The lipophilicity of the prodrugs was greater, compared to ddC., with partition coefficient values increasing from 0.03 for ddC to 0.37,28, 63 and 483 for DAC., DTMAC., DCYPP and CHOL., respectively. Aqueous solubility was decreased proportionally to the increase in lipophilicity. Bioconversion studies were performed in phosphate buffer (pH 7.4), human serum, mouse serum, and mouse brain and liver homogenates. Whereas CHOL was stable in vitro in all media, DAC., DTMAC and DCYPP exhibited stability only in buffer, indicating that the hydrolytic reaction for these compounds was, predominately, enzymatically triggered. DCYPP was rapidly hydrolysed in mouse serum and liver and brain homogenates with degradation half-life values of 0.04, 0.35 and 0.34 h respectively. DAC had a longer half-life in mouse serum than did DTMAC (0.82 h vs. 0.38 h), however, in mouse brain homogenate DTMAC (t_1/2=3.9 h) was more stable than DAC (t_1/2= 1.6 h). Both of these pro-drugs were rapidly metabolized in the mouse liver homogenate with half-life values of 0.36 h for DAC and 0.23 h for DTMAC. In-vivo studies performed for ddC., DAC and DTMAC in mice showed that the relative brain exposure (r_e) of ddC was not improved by administering the prodrugs. DTMAC yielded a r_e value of 0.023 which was similar to that for ddC (r_e = 0.028), while no ddC was detected in brain after DAC administration. Thus, although all of the prodrugs were more lipophilic than ddC., delivery of ddC to the brain was not enhanced in vivo.

1-(3′-Fluoro-2′,3′,6′-trideoxy-β-D-allofuranosyl)thymine [7] and 1-(3′-fluoro-2′,3′,6′-trideoxy-α-L-talofuranosyl) thymine [8] were synthesized starting from the corresponding 2,3′-anhydro nucleoside derivatives. The fluorine was introduced stereoselectively by opening of the anhydro bridge in the presence of HF/AIF₃. The 5′-C-methyl derivatives, [7] and [8], of 3′-fluoro-3′-deoxythymidine (FLT) were evaluated for their inhibitory effect against human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2). The compounds [7] and [8] had antiviral activity which was three orders of magnitude lower than the reference compound 3′-fluoro-3′-deoxythymidine. None of the compounds showed appreciable activity against other RNA or DNA viruses at subtoxic concentrations.