Antiviral Chemistry and Chemotherapy

Pegylated interferon alpha-2, alone or in combination with ribavirin, has become the standard therapy for patients with chronic hepatitis C infection. Pegylation of interferon alpha-2 results in a substantially extended half-life that permits once-weekly dosing, because of reduced clearance and more sustained absorption. The size of PEG moiety appears to influence the relative antiviral activities of peginterferon alpha-2. Increasing the size of the polyethylene glycol (PEG) moiety results in a reduction of the specific antiviral activity of the pegylated protein. For example, peginterferon alpha-2b (12 kDa) has an in vitro antiviral specific activity 25–35-fold higher than peginterferon alpha-2a (40 kDa). The antiviral activity of pegylated interferon alpha-2 is also governed by the site of pegylation of the interferon alpha core proteins. Interferon alpha-2a is monopegylated at four major positional lysine (Lys) residues. The major site of interferon alpha-2b monopegylation is histidine (His³⁴), with additional pegylation sites at lysine and cysteine residues. The 12 kDa pegylated His³⁴ positional isomer of peginterferon alpha-2b has the highest antiviral and antiproliferative in vitro specific activity compared with both the 12 kDa Lys positional isomers and the 40 kDa Lys positional isomers. The correlative effects of size and site of pegylation on the JAK/STAT signalling pathway, as evidenced by differences in the formation of the Stat1 homodimer complex, are suggestive of a receptor-mediated mechanism that governs the antiviral activity of pegylated interferons. The elucidation of the in vitro effects of pegylation is important and this will ultimately have a positive impact on the in vivo efficacy of treatment for patients with hepatitis C.

Antiviral therapy of chronic HBV infection remains a clinical challenge. Once this infection has been-established, the viral genome persists for life, either as an integrated genome or as episomal covalently closed circular DNA (cccDNA). The latter is the source of renewed viral replication in case of immune depression or after antiviral drug withdrawal. The mechanisms of clearance of infected cells involve CD8+ cell-mediated cytolytic and non-cytolytic pathways. Antiviral therapy, using nucleoside analogues that inhibit the viral polymerase, induces a slow depletion of intrahepatic cccDNA. The persistence of low-grade viral replication under antiviral therapy may then lead to the selection of drug-resistant mutants. New assays have been developed to study the functional consequences of these polymerase mutations in terms of replication capacity and drug susceptibility. Together with the development of new HBV polymerase inhibitors and novel immunostimulatory approaches, this should lead to the design and evaluation of rational treatment combinations for a better control of viral replication and prevention of drug resistance.

Adefovir dipivoxil (Hepsera™), a first-line therapy for chronic hepatitis B, is an esterase-activated prodrug of PMEA. Dose-limiting nephrotoxicity necessitates suboptimal dosing at 10 mg/day. Remofovir mesylate (MB06866Q) (Hepavir B) is a CYP3A4-activated prodrug of PMEA based on the HepDirect™ technology that targets PMEA to the liver. In a whole body autoradiography study in rats after oral dosing (30 mg/kg) of [¹⁴C]adefovir dipivoxil or [¹⁴C]remofovir mesylate, remofovir yielded 15 times higher concentrations of radioactivity in the liver than adefovir dipivoxil, but only one-third of the concentrations in the kidney. After oral dosing (4 mg/kg) of the same radiolabelled agents in cynomolgus monkeys, remofovir mesylate yielded 60 times higher levels of total radioactivity in the liver, but only two-thirds of total radioactivity levels in the kidney. Thus, remofovir mesylate may provide better efficacy and reduced nephrotoxicity. In portal vein-cannulated rats (30 mg/kg) after a single oral dose of [¹⁴C]adefovir dipivoxil or [¹⁴C]remofovir mesylate, no PMEA was detectable in rat portal plasma early after dosing, indicating that intestinal CYP3A4 does not play a role in conversion of remofovir mesylate to PMEA. The portal/systemic extraction ratio was quite high in both models, suggesting good liver-targeting properties. Portal and systemic remofovir/PMEA ratio indicates that the liver is the site of conversion of remofovir to PMEA. 28-Day toxicity studies demonstrated renal toxicity in rats at doses of 100 mg/kg or higher with no safety concerns at 30 mg/kg and acceptable safety in monkeys at doses up to 60 mg/kg. Thus, in rats and non-human primates, remofovir mesylate has liver-targeting properties and is safer than adefovir dipivoxil.

A series of novel acyclic nucleoside analogues containing bis-(hydroxymethyl)phosphinic acid (BHPA) or tris(hydroxymethyl)phosphine oxide (THPO) coupled with DNA nucleobases or with 5-fluorouracil were prepared and their antiviral activity was studied against cytomegalovirus (CMV), varicella-zoster virus (VZV), parainfluenza-virus type 3, reovirus-type 1, sindbis, coxsackie B4, punta toro, vesicular stomatitis and respiratory syncytial virus, herpes simplex virus-type 1 (KOS) and type 2 (G), vaccinia virus and herpes simplex virus-1 (TK-KOS ACVr). No specific antiviral effects were noted for any of test compounds against viruses evaluated, except thymine, cytosine and adenine derivatives of BHPA exerting borderline activity against respiratory syncytial virus at the 80 mg/ml concentration.

We have recently reported that 2′,3'dideoxy analogues of our exquisitely potent anti-VZV furanopyrimidine deoxynucleosides are shifted to selective anti-HCMV agents. We now find that the fully deoxygenated 2′,3′,5′-trideoxy analogue is fully antivirally active. This is taken as proof that these agents act by a novel non-nucleoside mechanism of action.

Bicyclic furanopyrimidine nucleoside analogues (BCNAs) have been previously reported as potent and selective anti-varicella-zoster virus (VZV) agents. Few modifications on the sugar moiety have been considered so far but some of them have shown interesting activity against human cytomegalovirus (HCMV) while losing activity against VZV. In addition, recent work has led to an entirely new series of anti-HCMV bicyclic furopyrimidine agents, acting through a non-nucleoside mechanism. In order to further investigate structure-activity relationship studies on the sugar moiety, some 3′- and 5′-chloro derivatives and 5′-deoxygenated derivatives have been synthesized. The lack of anti-VZV activity of the 5′-modified derivatives is further proof of a mechanism of action involving VZV thymidine kinase (TK)-mediated phosphorylation. Similarly, the replacement of the 3′-OH with chlorine showed a decrease of antiviral activity, which can be correlated to the lack of interaction with VZV TK as demonstrated by enzyme assays. These results confirm free 5′-OH and 3′-OH as necessary requirements for efficient recognition by VZV TK and for potent anti-VZV activity in cell culture.