Antiviral treatment: from concept to reality

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1 Antiviral Chemistry & Chemotherapy 1997 Volume 8, Supplement 1: 5-10 Antiviral treatment: from concept to reality RBoon Clinical Investigations, AntHnfectives, SmithKline Beecham, Harlow, UK. For correspondence: Tel: ; Fax: major advances in antiviral therapy. Many challenges, however, still lie ahead. Keywords: antiviral therapy; anti herpes agents; ocrc1ovir; valaciclovir; famaciclovir. ~~ -:::.-'::'::-'~~_ '...-, Introduction Summary The initial development of antiviral compounds was slow, with the first clinical antiviral agent not available until the 1960s. Early development was hindered by the lack of understanding of virus life-cycles and the absence of an assay system for antiviral activity. The appearance of the first assay system, the fertilized egg yolk sac, led to the identification of methisazone. This was the first antiviral agent to be used clinically. The improvement in antiviral assay systems, and the start of directed research programmes led to the development of the first anti herpes agents idoxuridine, trifluorothymidine (TFT) and vidarabine. However, all of these agents were associated with significant adverse effects. Antiherpes therapy took a major step forward with the development of the acyclic nucleoside analogue, aciclovir. Aciclovir is much more potent than previous anti herpesvirus agents. Its mode of action results in selectivity for herpesvirus-infected cells, thus significantly reducing the side-effects seen with earlier agents. Because of this, it became the standard therapy for herpes simplex virus type 1 (HSY-1), HSY-2 and varicella zoster virus infections. However, the bioavailability of oral aciclovir is poor, requiring high and frequent doses. This led to the search for better absorbed agents and to the development of two prodrugs, famciclovir and valaciclovir. Both famciclovir and valaciclovir offer much improved bioavailability of the nucleosides penciclovir and aciclovir, compared with aciclovir. Once in the body the conversion of valaciclovir to aciclovir means that the two agents have similar pharmacokinetic properties. Similarly, famciclovir is converted to penciclovir in the body. Penciclovir, when phosphorylated in virus-infected cells, persists within the cell for a much longer period than aciclovir triphosphate. Over the last 10 years we have seen an acceleration in the development of antiviral agents and some Within the last 40 years, the field of antiviral therapy has grown from the development ofthe first clinically available compound to the current, ever expanding list of antiviral agents. This growth is the result of directed research programmes, which have flourished with the development of viral assay systems, and our increased understanding of viral replication. The products of these research programmes now mean that safe and effective treatments are available for a range of diseases from the mild to the chronically debilitating. With continued research, the list of treatable viral infections should continue to expand. Historical perspective The development of antiviral agents has been a much slower process than that of antibacterial compounds. Antibacterial agents were in clinical use in the 1930s, and many diverse agents have since been developed. In contrast, the first antivirals were discovered in the 1950s, and did not come into clinical use until the 1960s. Since the 1980s there has been a marked acceleration in the development of antiviral compounds. However, there are still relatively few antiviral agents in clinical use and these are effective in only a limited number of indications (Table 1). There were two main reasons for the slow development ofantiviral compounds. Firstly, the lack ofknowledge concerning the virus life-cycle, coupled with the fact that viruses are obligate intracellular parasites, led to concerns that agents toxic to the virus would also prove toxic to the host cell. This expectation was borne out by some of the early antiviral compounds which were associated with significant toxicity. However, subsequent molecular research 1997 International Medical Press Ltd 5

2 R Boon Table 1. Summary of antiviral nucleoside developed to dote" class of antiviral agent Pyrimidine nucleoside Acyclic nucleoside Acyclic nucleoside phosphonates Pyrimidine dideoxynucleoside Purine dideoxynucleoside Generic name Brivudin Sorivudine Aciclovir Penciclovir Ganeiclovir Cidolovir Adefovir Zidovudine, Stavudine Zalcitabine Lamivudine Didanosine Carbovir Target virus HSV-l, VZV HSV l, HSV-2, VZV, CMV HSV-l, HSV-2, VZV HSV-l, HSV-2, VZV, CMV HSV- 1, HSV-2, VZV, EBY, CMY, HHV-6, adeno-, pox-, popillomoand hepadnaviruses HSV-l, HSV-2, VZV, EBY, CMV, HHV-6, HIV-l, HIV-2, HBV HIV- 1, HIV-2 HIV-l, HIV-2, HBV HIV- 1, HIV-2 a Adapted from De Clercq (1995). HSV,herpes simplex virus; VN, varicella zoster virus; CMV,cytomegalovirus; EBV, Epstein-Barr virus; HIV, human immunodeficiency virus. into the mechanisms of viral replication has revealed numerous processes that are specific to the virus. Many of these processes provide potential target sites for antiviral agents, for example, absorption of the virus into the host cell, replication of the viral genome, synthesis and translation of the viral RNA, and assembly and release ofvirus particles. This information has facilitated the development ofantiviral agents. The second problem was the absence of systems for testing compounds for antiviral activity. Without a reliable and reproducible system for assaying viral growth, the possible inhibitory effects of compounds could not be examined. This was overcome by the development of assay systems, first in eggs, then in mice, and finally in tissue culture systems. Most screening for antiviral agents is now carried out using cell culture systems. However, for some viruses simple culture systems are not yet available, while others such as human immunodefrciency virus (HIV) require elaborate containment facilities, which restricts screening procedures. Compounds showing antiviral activityin these screening systems can then be further developed for testing in humans. The first virus assay system was developed by Brownlee & Hamre (1951). This assay involved injecting virus into fertilized egg yolk sacs. The survival time of the embryos was found to be inversely proportional to the dose of vaccinia virus injected into eggs on the sixth day of incubation. Compounds were tested for antiviral activity by injecting them into eggs which had been infected with the vaccinia virus. Their effects on embryo survival were then measured. Increases in embryo survival time indicated antiviral activity. The development ofthis assay system marked an important stage in the history of antiviral agents, since a system was now available in which closely related compounds could be tested and compared. Thus, the effects ofmodifications in the structure ofthe compounds could be measured, and a series of related compounds could be produced and tested in this system to determine the most potent agent; structureactivity relationships could therefore be studied. The development of tissue culture systems further promoted the search for new antiviral agents. The early tissue culture systems were only qualitative and were still very labour intensive. These were gradually refined, with the introduction of the plaque inhibition assay in 1960, which provided an easier quantitative screening system for large numbers ofcompounds. The development ofmodern tissue culture techniques reduced the workload and allowed large numbers ofcompounds to be tested in different cell types. The frrst set of compounds tested in the egg yolk assay system were the sulphonamides, which were originally developed as antibacterial agents. These tests revealed that methisazone (para-benzaldehyde thiosemicarbazone) was effective against vaccinia virus infection, producing the equivalent of a 70% reduction in the infecting dose of the virus (Hamre et al., 1950). In 1962, methisazone was successfully used to treat a 7-month-old boy with eczema vaccinatum. The infant had contracted vaccinia from his vaccinated mother and had extensive lesions on the scalp, face and limbs with pyrexia, International Medical Press ltd

3 ...? Antiviral therapy prostration and toxaemia. Previous treatment with antivaccinia virus gamma-globulin had been ineffective. This was probably the first clinical use of an antiviral agent (Turner et al., 1962). Methisazone was subsequently used to treat other cases of eczema vaccinatum and later the more serious infective complication ofvaccinia, vaccinia gangrenosa. Methisazone was found to be equally effective against both conditions. Methisazone was later tested as a prophylatic agent against smallpox (Bauer et al., 1969). In a trial involving nearly 5500 patients, less than 1% ofmethisazone-treated patients developed smallpox within 16 days of contact with the virus, compared with over 4% of the control group. The prophylactic treatment also produced a similar reduction in mortality. The discovery ofan antiviral agent which could effectively prevent smallpox was a major achievement at the time. Fortunately, with the eradication of smallpox, this therapy is no longer needed. Another important milestone in the development of antiviral treatment came with a demonstration that amantidine given prophylactically could prevent influenza. The antiviral activity ofthis agent was initially demonstrated in mice and egg yolk systems (Davies et al., 1964). Activity against rubella was also observed in the egg yolk system but did not translate into effective antiviral activity in animal models. In a clinical trial carried out by Jackson and coworkers (Jackson et al., 1963), 200 volunteers lacking the influenza antibody were infected with the Asian strain of the virus. Only 37% of the amantidine-treated group, compared with 66% of the placebo-treated group (P<O.Ol), became infected, thus demonstrating a prophylactic activity for amantidine. TIllS finding was confirmed in a study by Couch (1990) involving family contacts of patients with influenza. Daily prophylactic administration of amantidine over 2-3 months during the influenza outbreak showed 70% efficacy in preventing infection. Despite its proven efficacy,amantidine is not used widely. A derivative ofamantidine, rimantidine, has been developed which has a more favourable side-effect profile, but this too is not widely used. Another of the early antiviral agents is ribavirin, a nucleoside analogue with activity against a wide range of DNA and RNA viruses. Ribavirin was developed in the 1970s and found to have a wide spectrum ofactivity but its use was limited by the need to administer it by aerosol. This agent is active against several serious viral infections, for example, Lassa fever. Early antiherpes agents The 1960s signalled the start of an acceleration in the number of antiviral compounds being developed. New antiviral agents were derived both from existing com- pounds and from research into new types of molecules, such as synthetic nucleosides. This decade also saw the start of directed research programmes, in which research was targeted at a specific virus type. The first of these research programmes aimed to develop antiherpes compounds and proved to be one of the most successful' areas ofprogress in antiviral therapy. The first antiherpes agent to be identified in this programme was idoxuridine (IDU), a synthetic nucleoside previously shown to have antineoplastic activity. Kaufman demonstrated its efficacy in herpetic keratitis models, first in rabbits, and then in humans (Kaufman et al., 1964). This lead to the introduction of IDU as a topical agent. Intravenous administration ofiduwas investigated for the treatment ofpatients with herpes encephalitis but was stopped due to unacceptable toxicity and lack ofefficacy. Cytarabine, another compound which was effective in tissue culture experiments against both herpes simplex virus (HSV) and varicella zoster virus (VZV) (Rapp, 1964), proved to be highly toxic and ofdubious clinical efficacy as an antiviral agent. Trifluorothymidine (TFT) was among the most successful of the these early antiherpes agents, again showing activity in the rabbit herpetic keratitis model. In a clinical trial in 78 patients with herpetic keratitis, Wellings et ai. (1972) showed that healing occurred in 92.5% of TFTtreated patients, compared with 60.5% of the IDU-treated group, and that mean healing time was reduced by 2 days for TFT-treated patients. Thus, TFT became the standard treatment for herpetic keratitis. TFT is only suitable as a topical agent. This makes it unsuitable for use against other conditions caused by herpes simplex virus. Vidarabine was also developed around the same time and was the first clinical antiviral agent to be used intravenously. Schabel and colleagues showed that it was active against both cytomegalovirus (CMV) and VZV (Schabel, 1968), but it also showed activity against HSV. In one study in patients with HSV encephalitis (Whitley et al., 1977), vidarabine reduced mortality from 70% for placebo to 28%, and those patientswho received vidarabine early in the course of encephalitis had fewer neurological sequelae. Vidarabine thus became the treatment of choice in herpes encephalitis. Acyclic nucleoside It was the development ofacyclic nucleoside that marked the major step forward in the treatment ofherpesvirus infection. Among the first of these, aciclovir, was found to be 10 times more active than IDU and 600 times more active thanvidarabine, makingit the most potentantiherpes agent available when it was introduced. Aciclovir is effective against acute and recurrent genital herpes and the acute symptoms ofherpes zoster (McKendrick et al., 1986). Antiviral Chemistry & ChemotherapyVolume 8, Supplement1 7

4 RBoon Thus, it became standard therapy for these herpes infections. However, the poor oral bioavailability (10-20%) of aciclovir necessitates frequent dosing and may compromise efficacy under some conditions. Bioavailability problems have been overcome to a large degree by the recent introduction of an aciclovir prodrug, valaciclovir, and the prodrug famciclovir, which is converted in the body to penciclovir. These prodrugs have ester groups linked to the parent molecule which facilitate absorption, thus giving higher bioavailabilityofthe respective nucleosides. The conversion of both famciclovir and valaciclovir into their respective nucleosides occurs in the liver. Both prodrugs are absorbed into the blood from the gastrointestinal tract and pass into the portal system. In the liver they are metabolized to their active forms, which then pass into the general circulation (Perry & Wagstaff, 1995; Perry & Faulds, 1996). Studies show that valaciclovir increases the systemic bioavailabilityofaciclovir to 54%, and famciclovir provides even higher levels of the active agent, penciclovir, with bioavailability of 77% (Perry & Wagstaff, 1995; Perry & Faulds, 1996). Prodrugs have the advantage that their pharmacokinetics are largely predictable and consistent. This allows physicians to predict the plasma levels of the active molecule produced by different doses ofthe prodrug. This dose proportionality can be seen in the plasma levels of penciclovir for different doses of famciclovir (Pue & Benet, 1993). Intracellular action of penciclovir and aciclovir Penciclovir and aciclovir inhibit viral replication by broadly similar mechanisms (Fig. 1). Aciclovir and penciclovir diffuse freely across the plasma membrane into cells. Ifthe cells are uninfected, the drugs move in and out of the cell unchanged. In cells infected with a herpesvirus, however, penciclovir and aciclovir are phosphorylated by the viral thymidine kinase enzyme to the monophosphate form which is subsequently converted to the triphosphate form by cellular enzymes. These triphosphates are the active antiviral molecules which block viral DNA synthesis by competing with the natural nucleotides for the viral DNA polymerase. A difference between penciclovir and aciclovir lies in the behaviour of the triphosphate form of the molecule. Intracellular aciclovir triphosphate is much less stable than penciclovir triphosphate, so it is readily converted back into aciclovir, which can easily diffuse out of the cell. Thus the half-life of aciclovir triphosphate in infected cells is only about 1 h. In contrast, penciclovir triphosphate is much more stable. In cells infected with HSV it has a half-life of h and in VZV-infected cells, its half-life is about 9 h (Fig. 2) (Bacon etal., 1996). This means that famciclovir can potentiallybe given less frequently than aciclovir. Clinical efficacy Aciclovir, valaciclovir and famciclovir have all been studied in various clinical settings involving herpesvirus infection (Perry & Wagstaff, 1995; Perry & Faulds, 1996). Articles in this supplement discuss the clinical uses of these antiherpes agents in patients with herpes zoster, herpes labialis and genital herpes. The clinical advantages of these prodrugs, particularly famciclovir, relative to aciclovir are also discussed. Figure 1. Mechanism of the antiviral action of penciclovir, showing its activation pathway within herpesvirus-infected cells. Pencidovir Aciclovir shows the same mode of action InternationalMedical Press Ltd

5 - Antiviral therapy Figure 2. Comparisons of the intracellular half-life of penciclovir triphosphate (PCV-TP) and aciclovir triphosphate (ACV-TP) in cells infectedwith HSV-l, HSV-2 or VlV. "I...!!! Qj u c ~ 1000 'E (5 E 100.9:- c.j? 10 2 C Q) u cou o.s: 0- C r--""::>"'T""---r--"":>'..,..--..,..::::"'_..., ~ t-= Time (h) Data from VereHodge & Cheng (1993) and Bacon et al. (1996). PCV-TP / HSV-l PCV-TP / HSV-2 PCV"TP / VlII ACV-TP / HSV-1 ACV-TP / HSV-2 ACV-TP / VlII Half-life (h) Future challenges for antiviral research These antiherpes agents are only a few of the antiviral agents that have been developed in the last years. Other antiviral agents currently in development or in clinical use include acyclic nucleoside phosphonates (such as adefovirand cidofovir) (De Clercq, 1995) pyrimidine nucleoside (for example brivudine and netivudine) (De Clercq, 1995) and dideoxynucleoside, which have formed the basis ofmost HIV treatment. An entirely new class of antiviral agents, the protease inhibitors, has also appeared recently. This range of compounds, together with the acyclic nucleoside discussed earlier, allows the physician to treat a wide range ofvirus infections, including HSV-1, HSV-2, CMV, VZv, Epstein-Barr virus, and human herpesvirus 6, 7 and 8. However, despite the recent advances in antiviral treatments, numerous challenges lie ahead. Effective treatments for certain viral infections, such as HIV and CMV, still need to be found. We still need to address the problems oflatency, andwhether the cycle oflatency and reactivation can be interrupted. This issue is addressed in an article included in this supplement by Field & Thackray, (pp ). The development oftherapeutic vaccines for herpes simplex, and possibly herpes zoster, is another area of active research. The needfor earlier, more accurate diagnoses ofviral diseases and the possibilities of co-therapy for viral infections such as hepatitis B are other challenges to be addressed. Finally, there are the possibilities for treatment offered by gene therapy and by expression vectors carrying antiviral agents. References Bacon TH, GilbartJ, Howard BA & Standing-Cox R (1996) Inhibition of varicella-zoster virus by penciclovir in cell culture and mechanism of action. Antiviml Chemistry and Chemothempy 7: Bauer DJ, St Vincent L, Kempe CH, Young PA & Downie AVV (1969) Prophylaxis of smallpox with methisazone. American journalifepidemiology 90: Brownlee KA & Hamre DA (1951) Studies on chemotherapy of vaccinia virus. 1. An experimental design for testing antiviral agents. journal ifbacteriology 61: Couch RB (1990) Respiratory diseases. In AntivimlAgents and Viral DiseasesifMan. Edited by GJ Galasso, RJ Whitley & TC Merigan. New York: Raven Press, pp De Clercq E (1995) Trends in the development of new antiviral agents for tile chemotherapy of infections caused by herpesviruses and retroviruses. Reviews in Medical Virology 5: Davies VVL, Grunert RR, HaffRF et al. (1964) Antiviral activity of L-adamantanamine (Amantadine). Science144: Hamre DA, Bernstein J & Donovick R (1950) Activity of p-aminobenzaldehyde, 3-thiosemicarbazone on vaccinia virus in the chick embryo and in the mouse. Proceedingsifthe Societyfor Experimental Biology andmedicine 73: Jackson GG, Muldoon RL & Akers LW (1963) Serological evidence for prevention of influenzal infection in volunteers by an anti-influenzal drug adamantanamine hydrochloride. AntimicrobialAgents and Chemothempy 3: Kaufman HE & Heidelberger C (1964) Therapeutic antiviral action of 5-trifluoromethyl-2'deoxyuridine in herpes simplex keratitis. Science145: McKenderick MW, McGill]1, White JE& Wood MJ (1986) Oral acyclovir in acute herpeszoster. British Medicaljournal 93: Antiviral Chemistry& ChemotherapyValume 8, Supplement1 9

6 R Boon Perry CM & WagstaffAJ (1995) Famciclovir: a review ofits pharmacological properties and therapeutic efficacy in herpesvirus infections. Drugs 50: Perry CM & Faulds D (1996) Valaciclovir: a review of its antiviral activity, pharmacokinetic properties and therapeutic efficacy in herpesvirus infections. Drugs 52: Pue MA & Benet LZ (1993) Pharmacokinetics of famciclovirin man. Antiviral Chemis17y and Chemotherapy4 (Supplement 1): Rapp F (1964) Inhibition by metabolic of plaque fomation by herpes zoster and herpes simplex viruses. journalifimmunology 93: Schabel FMJr (1968) The antiviral activity of9-~-d-arabinofuranosyladenine (ARA-A). Chemotherapy 13: Turner W, Bauer DJ & Nimmo-Smith RH (1962) Eczema vaccinatum treated with N-methylisatin ~-thiosemicarbazone. British Medical journal1: Vere Hodge RA & Cheng Y-C (1993) The mode of action of penciclovir.antiviral Chemistry and Chemotherapy 4 (Supplement 1): Wellings PC, Awdry PN, Bors FH, Jones BR, Brown DC & Kaufman HE (1972) Clinical evaluation of trifluorothymidine in the treatment of herpes simplex corneal ulcers. American journalif Ophthalmology 73: Whitley RJ, Soong S-J, Dolin R, Galasso GJ, Chi'en LT & Alford CA (1977) Adenine arabinoside therapy ofbiopsy-proved herpes simplex encephalitis. New EnglandjournalifMedicine 297: International Medical PressLtd