Antiviral chemotherapy

Historical perspective

Viruses are resistant to the action of antibiotics that target bacteria and other microbes. As obligate intracellular parasites with very restricted genetic coding capacity, viruses rely heavily on utilizing the metabolic machinery of the cell for their replication. However, replication involves (and is reliant on) one or more specific proteins encoded by viruses and this has led to the development of a number of successful antiviral agents that can, at some level, selectively inhibit viral protein functions and therefore curtail virus infection. Chemotherapeutic agents fall into three broad groups. Virucides include detergents and solvents that directly inactivate viruses. Antiviral inhibit virus replication and aim to achieve this with little or no effect on host cell metabolism. Finally, immunomodulating agents (e.g. therapeutic interleukins and interferons) attempt to enhance the immune response against viruses to promote virus clearance.

Modes of action

Virus replication involves multiple stages, and each stage (attachment and penetration, uncoating of the nucleic acid, transcription and translation, genome replication, and release of mature progeny) can be a target for antiviral drug intervention. To date, the most common target is viral nucleic acid metabolism by compounds known as nucleoside analogs or nonnucleoside inhibitors. Many of the first antivirals targeted against HIV acted against the viral polymerase (reverse transcriptase) such as azidothymidine (AZT) and lamivudine (also effective against HBV). A range of antivirals are effective towards different stages of the influenza virus replication cycle such as amantidine, which blocks uncoating by blocking the ion channel that forms by the viral M2 protein; ribavarin, which inhibits the viral RNA polymerase; and zanamivir, which prevents the final egress of the virus by inhibiting neuraminidase activity.

Many viruses produce a virus-specific protease to process their proteins and this has been a valuable target for drug inhibition against HIV (e.g. ritonavir, saquinavir). A series of inhibitors of Picornaviridae act by directly binding to the virion capsid, blocking the interaction between the virion and the receptor on the cell surface that facilitates penetration and uncoating. Perhaps the most successful antiviral compound is the nucleoside analog acyclovir (ACV– now named aciclovir and sold under the trade name Zovirax), with more than 40 million patients treated. The drug inhibits the replication of HSV and has been administered prophylactically with no ill effects for over 20 years to individuals to suppress recurrences of genital herpes. ACV is an analog of the natural nucleoside guanosine and in this form is inactive and harmless to cells. Activation requires three enzymatic phosphorylation steps, to the active drug molecule ACV triphosphate. The HSV thymidine kinase enzyme can convert ACV to ACV monophosphate (ACV-MP), but ACV is a poor substrate for cellular enzymes, which do not perform this step.

Hence ACV remains inactive in uninfected cells. By contrast, the conversion of ACV to ACV-MP in HSV-infected cells is rapidly followed by conversion to the triphosphate (ACV-TP) form through the action of cellular enzymes and it then enters the nucleotide pool within these cells. ACV-TP competes with guanosine triphosphate as a substrate for HSV DNA polymerase (cellular DNA polymerases are much less sensitive) and as the ACV-TP is linked into the growing chain of replicating viral DNA it forms a chain terminator. There is no 3’-OH group on the ACV sugar moiety to link with the next nucleotide residue and hence the growing chain of virus DNA can extend no further, terminating virus replication. Table  highlights the modes of action of a range of antiviral compounds.

Interferon is a natural human product (a cytokine) that acts on the surface of normal cells to render them immune to virus replication. The compound is used to treat hepatitis B and C infections (usually accompanied by ribavirin) but is toxic, the side effects mimicking the symptoms of influenza. A newer version, polyethylene glycol interferon (Peginterferon; Roche), is less toxic and more stable, allowing a reduced dosing regime of one subcutaneous injection per week, rather than three.

Effective dose, therapeutic index, and drug toxicity

Because of the intimate replication of viruses within cells, and their absolute reliance on cellular protein and energy metabolisms, the development of antiviral compounds faces the difficult challenge of selective toxicity – interference with viral replication needs to be achieved without unacceptable damage to the host (uninfected cells). The activity of antiviral compounds is assessed at an early stage in their development, quantifying their ability to interfere with viral growth in tissue culture. The replication of the target virus is assayed by, for example, TCID50 or plaque assay, comparing levels of infection within cells in the presence of various concentrations of the drug candidate (with drug-free control infections alongside).

The resulting level of virus infectivity is plotted against the drug concentration and the concentration of compound that reduces virus titer by 50% is expressed as the effective dose 50 concentration (ED50), if achievable, the ED90 concentration is also determined. Tissue culture can also be used to assess the toxicity of compounds, although this is often tested in animals and humans. Animals play a vital role in the study of toxicity and a number of statutory tests are carried out to determine the risks and side effects before new compounds enter clinical trials. Drug toxicity is expressed as the selective index, which determines the ratio between the concentration at which the drug inhibits cell proliferation or DNA synthesis (i.e. it is toxic to uninfected cells), and the concentration at which the drug inhibits virus replication. A high value indicates a selective drug with low toxicity, while a ratio approaching equivalence indicates that the compound is toxic.

For compounds with a less than ideal selective index, a consideration of benefit/risk ratio can be appropriate, as side effects may be tolerated more if the risk from the disease is high (e.g. AIDS). Some antiviral agents have a level of toxicity which, while acceptable for some diseases (e.g. AIDS), would not be tolerated for less severe diseases.

Drug targeting, design, and clinical trials

Initially antiviral compounds were discovered in an empirical fashion; chemical synthesis of a wide range of compounds followed by testing of antiviral activity in cell culture. The increasing knowledge of the molecular basis of virus replication processes, the availability of complete nucleotide sequences of virus genomes and three-dimensional protein structure information means that compounds can now be more precisely designed to interact with specific targets and active sites on essential viral proteins. In practice the most useful targets are viral enzymes that have properties or activities different to those of the counterpart enzymes in host cells (e.g. thymidine kinase, DNA polymerase, reverse transcriptase, and protease).

Through gene cloning, it is possible to express, purify, crystallize, and examine the three-dimensional structure of individual virus proteins and use this information to determine molecular structures predicted to interact with particular sites in the viral protein. In addition, features such as low toxicity and production costs are highly important, given the enormous cost of developing a potential candidate to clinical application and market. The majority of costs are split between the scientific research necessary to identify and test the compounds, and clinical trials that establish its effectiveness and safety in vivo. Clinical trials must pass, in order, through several tightly regulated phases.

Phase I involves administering the drug candidate to healthy human volunteers where studies on the pharmacokinetics, pharmacology, and metabolism of the compound are monitored.

In Phase II the compound is administered to disease patients, with a view to assessing data similar to those of Phase I, as the metabolism may be different compared with a healthy individual. Usually in excess of 100 patients are needed for these trials to give reliable data.

In Phase III the drug is usually tested for its clinical efficacy by comparing with placebos or existing drugs. The main aim is to determine the benefit/risk ratio for the therapeutic course, and this phase requires 100–1000 patients. In many, but not all Phase III trials, the drug and placebo are administered randomly, such that neither the test subjects nor the trial administrators know which individual has received which treatment. This is known as a double-blind trial and is important to safeguard the unbiased observation, recording, and interpretation of clinical outcomes. In certain circumstances Phase III trials are conducted without placebos (i.e. all patients receive the drug), as withholding of the drug and its potential therapeutic value would be unethical.

Phase IV studies are usually conducted following marketing approval and increased experience of treating patients, providing more information on safety and efficacy. Sadly most potential antiviral compounds, although good inhibitors of virus protein activity in biochemical tests, or even of viral replication in cell culture, fail to pass successfully through all of the phases of clinical studies and are ultimately rejected.

Drug resistance

Probably the greatest efforts in recent years have been devoted to the development of antiretroviral agents to combat the replication of HIV. Effective treatments for this virus must involve combinations of drugs to attempt to overcome the problem of virus resistance to antivirals. The high rate of virus replication in a host means that there is a high rate at which mutations occur in the virus genome – compounded in RNA viruses by the lack of any proofreading activity in RNA polymerases.

As a result, changes occur in the amino acid sequences of virus proteins, including those that serve as targets of antivirals. Drug binding and activity are diminished or abolished and hence the virus adapts to become resistant to drugs that had previously been effective. The development of resistance to antivirals is a persistent problem with HIV, where the integration of the HIV genome into the chromosome of host cells makes for very long-term replication over months and years. HIV mutations that give rise to resistance to AZT occur with a very high frequency, and treatment of HIV infection requires combining drugs that target different virus enzymes, e.g. two nucleoside analogs combined with an anti-protease inhibitor. Such regimes have been highly effective in reducing virus loads and raising CD4 cell counts in HIV-infected individuals and in reducing the risk of viral resistance. Most important are measures that insure that patients adhere to the regime of drug taking, which in some cases is highly complex and involves a number of different drugs routinely. Due to genomic integration, antiviral agents are unlikely to eliminate HIV from the patient, but they are effective in reducing symptoms, delaying the progression of infection to AIDS by several years.