Antiviral Therapy for Enteroviruses and Rhinoviruses

Antiviral Chemistry & Chemotherapy 11:261–271

Review Antiviral therapy for enteroviruses and rhinoviruses

Harley A Rotbart

University of Colorado School of Medicine, Departments of Pediatrics and Microbiology, Denver, Col., USA

For correspondence: Tel: +1 303 315 8501; Fax: +1 303 315 7909; E-mail: [email protected]

The picornaviruses are a diverse group of viral This article will focus on the rhinoviruses and pathogens that together comprise the most enteroviruses, agents for which substantial effort common causes of infection of humans in the has been expended, and recent successes reported, developed world. Within the picornavirus family toward the development of safe and effective are three well-known groups of human pathogens antiviral therapy. – the rhinoviruses, the enteroviruses (including polioviruses, coxsackieviruses and echoviruses) Keywords: enteroviruses, rhinoviruses, antiviral and the hepatoviruses (including hepatitis A virus). therapy, interferon, pleconaril, protease inhibitors

Virology

The enteroviruses (EVs) comprise more than 60, and the intermediate form of RNA ( Johnson & Sarnow, 1995). A rhinoviruses (RVs) more than 100, distinct serotypes single reading frame begins several hundred nucleotides (Miller, 1997) within the family Picornaviridae (‘pico’ from the 5′ end and terminates several dozen nucleotides meaning small, ‘rna’ for ribonucleic acid). The EVs are also from the 3′ end, just upstream from a poly-A tail. The frequently classified by their sub-genus names: poliovirus- untranslated sequences at both the 5′ and 3′ ends are es, coxsackieviruses A and B, echoviruses and the newer involved in viral regulatory activities such as replication and numbered EVs. Picornaviruses are small (27–30 nm diam- translation. A single polyprotein is translated from the eter; 1.34 g/ml buoyant density), consisting of a simple viral open reading frame. Post-translational modification is capsid and a single strand of positive (message) sense RNA. accomplished by virus-encoded proteases and results in EVs are acid- and ether-stable and grow optimally at core generation of the four capsid proteins as well as the body temperature (36–37°C); RVs are acid-labile and grow enzymes necessary for replication and translation (Hellen best at the temperature in the nasal passages (33°C). The & Wimmer, 1995). Within hours of infecting a host cell, picornaviral capsid contains four proteins, VP1–VP4, all host cell function is aborted and the cell becomes a fac- arranged in 60 repeating protomeric units of an icosahe- tory for viral replication. Cell death is by lysis, with release dron (Rueckert, 1990). Variations within capsid proteins of progeny virus into the immediate environment of the cell VP1–VP3 are responsible for the antigenic diversity among and the resumption of the viral infection cycle with attach- the viruses. VP4 is not present on the viral surface; rather, ment of new virus particles to neighbouring cells. it is in close association with the RNA core, functioning as an anchor to the viral capsid. Destabilization of VP4 results Clinical importance in viral uncoating. The atomic structure of numerous EVs and RVs have been resolved by X-ray crystallographic stud- Rhinoviruses ies, and reveal a deep cleft or canyon in the centre of each The RVs are responsible for more than half of all cases of protomeric unit into which the specific cellular receptor for the ‘common cold’ (Makela et al., 1998), which in turn is the viruses fits when virus encounters a susceptible host cell responsible for 25 million days of missed work, 23 million (Hogle et al., 1985; Racaniello, 1995). This canyon is days of missed school and 27 million physician visits each important in several of the antiviral strategies discussed year in the USA alone (Turner, 1998). The common cold is below. also the most frequent cause of inappropriate antibiotic use The encapsulated RNA of the EVs and RVs is approxi- in the USA (Gonzales et al., 1998; Nyquist et al., 1998). mately 7.4 kb in length and serves as a template for The median duration of RV colds is 11 days, but up to 25% both viral protein translation and RNA replication, the lat- last ≥2 weeks (Arruda et al., 1997). In a recent study of ter accomplished via a double-stranded replicative more severe viral respiratory tract infections, the median

duration of illness as defined by complete resolution of Table 1. Clinical illnesses caused by picornavirus both respiratory and systemic symptoms was 14 days infections (Hayden et al., 1999). Rhinoviruses Rhinovirus infections are associated with a number of Upper respiratory tract illness upper and lower respiratory tract complications in both Otitis media children and adults (Table 1). Viral respiratory tract infec- Sinusitis tions, particularly due to the RVs, are the most important Exacerbations of asthma, cystic fibrosis, predisposing factor to acute otitis media (Pitkaranta et al., chronic bronchitis 1998). Most cases of acute sinusitis are thought to result Lower respiratory tract illness – infants, from bacterial disease secondary to a preceding viral upper elderly, immunocompromised respiratory tract infection. Sinus abnormalities are also frequently detectable during uncomplicated colds (Gwaltney Enteroviruses et al., 1994). RV infection is a major factor in the induction Non-specific febrile illnesses of acute exacerbation of asthma in adults (Nicholson et al., Upper respiratory tract illness 1993) and in children ( Johnston et al., 1995). RV infections Otitis media are also associated with lower respiratory tract syndromes in Haemorrhagic conjunctivitis, herpangina, other specific patient populations. In children with cystic hand–foot–mouth syndrome fibrosis, picornaviruses (RVs or EVs) were detected in Pleurodynia about 25% of exacerbations and colds were associated with Myocarditis deterioration in pulmonary function testing (Collinson et Aseptic meningitis al., 1996). Among adults aged 60–90 years residing in the Encephalitis community, RV infection was associated with lower respi- Neonatal sepsis-like syndrome ratory tract symptoms in 65%; 40% consulted their doctor and 76% of these received antibiotics (Nicholson et al., 1996). The overall burden of RVs in elderly people may infants in whom differentiation of viral illness from more approach that of influenza (Nicholson et al., 1996). Up to alarming causes of fever and rash is extremely difficult. One 40% of exacerbations in patients with chronic bronchitis prospective study found that 13% of babies born in the may be associated with RV infections (Gwaltney, 1989). In summer months were infected with EVs during the first infants aged <12 months, RV infections have been associ- month of life; 21% of the infected babies were hospitalized ated with hospitalization for lower respiratory tract illness, for suspected bacterial sepsis and received antibiotics or particularly bronchiolitis (Schmidt & Fink, 1991), deterio- antiherpes therapy ( Jenista et al., 1984). Most EV-associat- ration in those with bronchopulmonary dysplasia ed respiratory illnesses are indistinguishable from those due (Chidekel et al., 1994) and fatal pneumonia in myelosup- to other respiratory viruses, including the RVs. Several EV pressed patients (Ghosh et al., 1999). respiratory syndromes have distinctive characteristics, including haemorrhagic conjunctivitis, herpangina, hand– Enteroviruses foot–mouth syndrome and pleurodynia (Cherry, 1998). The paralytic potential of the polioviruses, the prototypic Recent pandemics of EV haemorrhagic conjunctivitis EVs, was recognized as early as the 14th century BC in (Wadia et al., 1983) and hand–foot–mouth syndrome (Ho Egyptian art. Summer epidemics, of paralytic poliomyelitis et al., 1999) have been associated with concurrent severe ravaged the USA through the 1950s. Since the introduction neurological and/or pulmonary complications. of vaccines in the late 1950s and early 1960s, much of the The EVs are among the most commonly identified aeti- developed world is now virtually free of poliovirus disease. In ologies of myocarditis, causing between 25–35% of cases many developing countries, eradication programmes have for which a cause is found (Martino et al., 1995). Neonates made dramatic progress in the fight against poliomyelitis and young infants (≤6 months of age) are particularly sus- (Centers for Disease Control and Prevention, 1996, 1997). ceptible to EV myocarditis, but most cases occur in young Now that poliovirus infections are under control in adults between the ages of 20–39 years. much of the world, more attention has been direccted to The EVs are also the most common cause of meningitis the non-polio EVs. It is estimated that between 10 and 15 in the USA. The severity of EV meningitis varies with host million people in the USA develop symptomatic non-polio age and immune status (Rotbart, 1997). The duration of EV infections each year (Table 1). Most of these patients illness due to EV meningitis is typically longer than 1 have non-specific febrile syndromes, often with constitu- week, but many patients, particularly adults, may have tional and/or respiratory symptoms, with or without rashes symptoms that persist for 2 or more weeks (Rotbart et al., (Strikas et al., 1986). Most affected patients are young 1998); younger children have a shorter disease duration.

Encephalitis due to the EVs is well-documented but and RVs at atomic resolution identified the ‘canyon’ on uncommon (Whitley et al., 1989; Modlin et al., 1991). each protomeric face of the virus into which the host cell Unlike aseptic meningitis, which may have prolonged mor- receptor fits, and beneath which a ‘pore’ opens into a bidity but from which recovery usually occurs, encephalitis hydrophobic pocket; capsid-inhibiting compounds act in due to the EV may have more profound acute disease and that pocket (Hogle et al., 1985; Racaniello, 1995). Specific long-term consequences. host cell receptors have now been identified for numerous The infected neonate appears to be at greatest risk of EV serotypes (Racaniello, 1995; Bergelson et al., 1994, severe disease when illness develops in the early days of life; 1997), providing yet another potential target in antiviral this pattern suggests possible transplacental acquisition strategies. Finally, rapid and sensitive molecular diagnostic (Abzug et al., 1993). Disseminated intravascular coagula- methods for the EVs have been developed (Rotbart et al., tion and other characteristics of ‘sepsis’ result in an illness 1994), providing an important prerequisite for clinical tri- that may be indistinguishable from that due to overwhelm- als. ing bacterial infection. Mortality is typically due to hepatic failure or myocarditis. In vitro and in vivo models EVs have been implicated in several chronic illnesses (Dalakas, 1995; Rewers & Atkinson, 1995), including juve- The picornaviruses are readily propagated in tissue culture nile onset diabetes mellitus, chronic fatigue syndrome, der- using cells of human or monkey derivation. Buffalo green matomyositis and polymyositis, congenital hydrocephalus monkey kidney (BGM), human rhabdomyosarcoma (RD), and amyotropic lateral sclerosis. Evidence for these associ- human embryonic lung and primary cynomolgous monkey ations has been largely from serological or from nucleic acid kidney cells are the preferred lines used to isolate the EVs hybridization studies; definitive proof is lacking and confir- (Dagan & Menegus, 1986). The group B coxsackieviruses matory studies remain to be carried out (Dalakas, 1995; grow best in BGM cells, whereas echoviruses grow best in Muir et al., 1996). Persistent EV infections occur in agam- RD cells. The RVs grow optimally in human embryonic maglobulinaemic patients and manifestations almost lung fibroblast (HELF) lines such as WI-38 and MRC-5. always include meningoencephalitis (McKinney et al., Human embryonic kidney cells and several HeLa cell 1987; Webster et al., 1993). Half of all patients with per- clones also support the growth of RVs (Landry, 1999). sistent EV meningoencephalitis have concomitant der- Ideal growth conditions for the EVs are at core body tem- matomyositis or polymyositis. These observations confirm perature (37°C), whereas RVs grow optimally at tempera- the important role of antibody in EV clearance, an unusu- tures closer to that of the nasopharynx (33°C). Antiviral al phenomenon because many other viruses are contained assays demonstrating the inhibitory effects of an anti- largely by cell-mediated immunity. A syndrome of late- picornaviral compound generally involve the demonstra- onset muscular atrophy and pain has been reported in indi- tion of protection of virus-induced cytopathic effect in sus- viduals who have suffered paralytic poliomyelitis 20–40 ceptible cell lines (Buontempo et al., 1997; Woods et al., years previously (Dalakas et al., 1984); evidence for persist- 1989). Plaque reduction, virus yield and cytopathic effect ent or latent infection in these individuals has been con- reduction assays in 96-well format are traditionally used to flicting. assess antiviral activity. Novel approaches to detecting capsid inhibitor compounds involving ICAM-1 (Last-Barney Milestones in the development of antiviral et al., 1993) and thermal stabilization (Rombaut et al., therapy for the EVs and RVs 1991) have also been employed. The mouse is the most commonly used species as a Numerous scientific accomplishments over the last 5 model of human EV infections. Suckling mice can be decades have paved the way for development and testing of readily infected with the Barty strain of echovirus 9 antiviral therapy for the EVs and RVs. The first successful (McKinlay et al., 1986) and coxsackievirus A9 (Woods et propagation of virus in continuous cell culture lines was al., 1989; Melnick & Godman, 1951). Infected animals achieved with poliovirus by Enders et al. (1949). This develop flaccid limb paralysis as a result of infection and Nobel Prize-winning advance facilitated the development intragastric administration of a capsid-inhibitor agent is of poliovirus vaccines, allowed for the identification of the capable of preventing the development of paralysis other picornavirus serotypes and established the gold stan- (Woods et al., 1989; McKinlay et al., 1986). Adult mice dard diagnostic test for many viruses. The determination of can be infected intracranially with poliovirus and protect- the complete genomic sequence for many of the RVs and ed from development of paralysis by oral administration EVs provided us with genetic structure–function informa- of capsid-inhibitor agents (Buontempo et al., 1997; tion with which antiviral strategies can be devised (Hellen McKinlay & Steinburg, 1986; Jubeit et al., 1989). & Wimmer, 1995). The 3D capsid structure for the EVs Similarly, coxsackievirus B3 can infect adult mice result-

Antiviral Chemistry & Chemotherapy 11:4 3 HA Rotbart

ing in myocarditis with involvement of multiple organ Table 2. Therapeutic strategies and candidate systems (Klingel et al., 1996); again, a capsid function compounds for treatment of picornaviral infections inhibitor has been shown to be orally effective in marked- Target Compound class ly reducing viral titres in all organs tested and preventing death of the mice in this latter model system (Pevear et al., Cell susceptibility lnterferons 1999). Human RVs have only been reported to success- Viral attachment and binding Antibodies, soluble ICAM fully infect primates (Dick, 1968; Pinto & Haff, 1969) to host cells and no practical animal model has been developed for the Viral uncoating/capsid function Capsid-function inhibitors RVs. Viral replication Enviroxime-like compounds Therapeutic strategies and clinical trials Viral protein synthesis 3C protease inhibitors

As with other viral pathogens, there are several steps in the The clinical efficacy of intranasal interferon as prophy- replication cycle of the picornaviruses that are potential laxis for RV colds has been demonstrated in several studies targets in antiviral therapy. Cell susceptibility, viral attach- (Hayden et al., 1986; Hayden & Gwaltney, 1983; Merigan ment, viral uncoating, viral RNA replication and viral et al., 1973; Samo et al., 1983; Greenburg et al., 1982). protein synthesis have all been studied as targets of anti- Additional studies demonstrated significant efficacy picornaviral compounds (Table 2). The following sections against naturally acquired RV infections and against con- briefly review these targets, the mechanisms of action of tact spread of RVs within family groups after experimental anti-picornavirus compounds directed at these targets and induction of a natural cold (Hayden et al., 1986; Douglas et clinical trials performed to date. al., 1986). Side-effects of interferon included nasal irrita- tion and stuffiness, and mucosal ulceration (Hayden et al., Interferon 1986; Samo et al., 1983). Administered therapeutically 1 lnterferons are potent, selective mediators of cellular day after experimental RV infection, intranasal interferon changes which induce a number of antiviral, anti-prolifera- had no effect on development of infection or symptoms, tive and immunological effects, all of which collectively but did result in moderate reductions of virus shedding and affect host cell susceptibility to picornavirus infection cold symptoms (Hayden & Gwaltney, 1984). Additional (Capobianchi et al., 1991; Geniteau-Legrendre et al., 1987; studies with low-dose intranasal interferon also demon- Kandolf et al., 1985; Kishimoto et al., 1988; Langford et al., strated a lack of efficacy in post-exposure prophylaxis of 1985, 1988; Lopez-Guerrero et al., 1990; Okada et al., RV infections in families (Monto et al., 1989). Despite in 1992; Sasaki et al., 1986). The cellular antiviral effects of vitro efficacy noted above, interferons have not been clini- interferons are mediated through specific receptor-signal cally evaluated in EV infections. transduction pathways. In conjunction with double-stranded RNA, interferons induce the expression of proteins, Immunoglobulins some of which mediate an antiviral activity. The best The primary mechanism of clearance of EVs by the host is described pathways are: (i) 2′,5′-adenylate synthetase; (ii) via humoral immunity. As noted above, patients who lack double-stranded RNA-dependent protein kinase; and (iii) antibody because of congenital or acquired immunodefi- the Mx proteins. Through transfection/expression systems, ciencies are uniquely susceptible to infections with the EVs an isoform of the 2′,5′−adenylate synthetase system has (McKinney et al., 1987). Similarly, normal neonates are at been linked to the inhibition of replication of picornavirus- high risk for severe EV disease because of a relative defi- es (Chebath & Benech, 1987). Clinically, children with ciency of EV antibodies (Abzug et al., 1993; Modlin et al., acute EV meningitis have significant elevations in endoge- 1981). Antibodies act by binding to EVs and preventing nous interferon levels in the cerebrospinal fluid (CSF) attachment and binding to host cells, which correlates with (Chonmaitree & Baron, 1991; Ichimura et al., 1985), which ‘neutralization’ of EVs observed in cell cultures treated with may be important in recovery from the infection. Although antibody. alpha interferon itself is a very potent inhibitor of picorna- Immune serum globulin has been used prophylactically virus infection, additive or synergistic protective effects are and therapeutically against the EVs in two clinical settings: seen when used in conjunction with capsid-inhibiting com- the neonate and the immunocompromised host. As noted pounds (Langford et al., 1985), nucleoside analogues above, neonates may develop an overwhelming sepsis syn- (Okada et al., 1992) or gamma interferon (Pitkaranta et al., drome from transplacental/peripartum acquisition of EV 1991). Interferons may also work in conjunction with infection. The high mortality rate of this disease, coupled humoral antibodies and macrophages to eliminate picor- with the known association of severe EV disease with navirus infections (Geniteau-Legrendre et al., 1987). absolute or relative antibody-deficiency states, has prompted

Figure 1. Chemical structures of representative immunoglobulin preparations that were subsequently shown capsid function-inhibitor compounds with anti- to contain high antibody titres to the infecting serotype picorna-viral activity (Abzug et al., 1993). Individuals with congenital or acquired

Cl antibody deficiencies are also at risk of severe EV infections. Prior to the availability of intravenous immunoglobulin

O preparations, mixed results were reported with intramuscular and/or intrathecal administration of immunoglobulin prepa- Cl rations. As with neonatal sepsis, some antibody-deficient OCH 3 patients appeared to benefit from supplemental

OH2CH 3C OCH3 immunoglobulin, others progressed and died despite therapy (McKinney et al., 1987). Since known antibody-deficient C patients have begun receiving maintenance supplementation OH O with intravenous immunoglobulin, the incidence of chronic,

CH3 progressive EV meningoencephalitis has fallen (demonstrat-

N ing the prophylactic benefit of these preparations) and the O N clinical profile of patients developing such infections has O O been modified (Webster et al., 1993). Therapeutic efficacy in Cl established EV meningoencephalitis in antibody-deficient N patients has only been anecdotally studied. CH O 3

O O N Capsid-inhibiting compounds Cl CH 3 Capsid-inhibiting compounds block viral uncoating and/or N N viral attachment to host cell receptors. As noted above, the OH3CNN resolved 3D structure of the EVs reveals a ‘canyon’ formed by the junctions of VP1 and VP3. Beneath the canyon lies

a ‘pore’ which leads to a hydrophobic pocket into which a O variety of diverse hydrophobic compounds can integrate

H3CN OC OCH2CH3 (Table 2, Figure 1). Although the compounds integrate into a virus capsid via a number of non-covalent, N hydrophobic-type interactions, the affinity is high, with O × –8 × –7 N constants ranging from 2.0 10 to 2.9 10 M (Fox et al., 1991). Several factors appear to correlate with the abilities OCH3 Cl Cl of a compound to function within the hydrophobic pocket

and to manifest antiviral activity, including: the number of O Cl molecules of compound active in each virion; the location of the compound activity within the hydrophobic pocket; Cl O O the length and space-filling properties of the molecule; and, CH3

H3C H3C most importantly, the extent of hydrophobic interactions CF3 N with amino acids in the pocket. With regard to those fac-

N O O tors, enhanced potency appears to correlate with increased O N number of molecules of compound per pocket (Fox et al., H3C 1991; Zhang et al., 1991), proximity of compound to the opening of the pocket (Zhang et al., 1991), increased numerous investigators to administer antibody preparations hydrophobic energy resulting from filling a greater propor- to neonates with EV sepsis. Anecdotal reports of clinical suc- tion of the pocket by the compound (Fox et al., 1991; cess with maternal serum or plasma ( Jantausch et al., 1995) Zhang et al., 1991, 1992), and absence of bulky amino acid or commercial immunoglobulin preparations (Black, 1983; substitutions (mutations) within the pocket structure Johnston & Overall, 1989) against a variety of EV serotypes (Heinz et al., 1989; Heinz, 1990; Pevear et al., 1989). causing neonatal sepsis have been reported; other reports Several hypotheses have been proposed for the mecha- describe progressive disease and death despite such therapy nism of picornavirus inhibition by compounds that affect the (Wong et al., 1989). A blinded, randomized, controlled study function of the virus capsid. Filling the hydrophobic pocket was too small to demonstrate clinical benefit but did show a results in increased stability of the virus, making the virus reduction in viral titre in babies receiving intravenous more resistant to uncoating. The increased stability of the

Antiviral Chemistry & Chemotherapy 11:4 5 HA Rotbart

virus–compound complex is evidenced by the resistance to concentrations above the in vitro minimal inhibitory con- thermal inactivation (Rombaut et al., 1985). This property centrations, both RV trials failed to show efficacy of WIN can be used as a rapid screen in order to identify molecules 54954 (Turner et al., 1993); very low concentrations of the with binding avidity; the majority of, but not all, compounds drug were found in nasal wash samples, the site of the exper- with potent antiviral activity also result in thermal stability. It imental infection. In contrast, WIN 54954 significantly is also possible that a degree of capsid flexibility may be reduced the number and severity of colds induced by cox- required for uncoating, and activity of these compounds sackievirus A21, and also significantly reduced nasal mucous within the hydrophobic pocket may reduce this necessary discharge, respiratory and systemic symptoms, and viral titres flexibility, inducing a more rigid structure. Alternatively, (Schiff et al., 1992). The overall symptomatic attack rate was changes in the conformation of the canyon floor as a result reduced from 15/23 patients in the placebo group to 3/27 in of drug activity within the underlying pocket may affect the the WIN 54954-treated groups (P=0.0001). This study rep- attachment of the virus to the host cell receptor (Langford et resents the first demonstration of oral efficacy of an anti-EV al., 1988). It has been shown, however, that such perturba- agent; the differences in results compared with those in the tions in the canyon floor do not absolutely correlate with RVs studies using the same compound are enigmatic, since antiviral potency (Zhang et al., 1991, 1992). The capsid- the MIC for one of the RV serotypes was identical to that of inhibiting compounds vary in their spectrum of activity, per- the coxsackievirus A21 strain used. The fact that EV infec- haps as a result of factors such as pocket fit, discussed above. tions are systemic, usually with a viraemic phase, may explain Certain compounds demonstrate both anti-EV and anti-RV the enhanced EV efficacy of an orally-active compound that activity (Otto et al., 1985; Pevear, 1999), others are more achieves good blood levels over the effect seen in RV infec- selective to one picornavirus genus or the other (Buontempo tions, which are limited to the upper airway where drug dis- et al., 1997; Cox et al., 1996). tribution may have been insufficient. WIN 54954 was not Trials of the ‘R’ series of capsid-binding compounds further developed for clinical use because of adverse reactions have been limited to intranasal administration to patients of flushing and rash, possibly related to concomitant alcohol with RV colds (Al-Nakib et al., 1989; Barrow et al., 1990; ingestion by study volunteers. Hayden et al., 1992). Pirodavir (R77975) and R61837 were Pleconaril {3-[3,5-dimethly-4-[(3-methyl-5-isoxa- efficacious in experimentally-induced RV colds when these zolyl)propyl]phenyl]-5-(trifluoromethyl)-1,2,4-oxadiazole} drugs were administered intranasally before or after infec- (Figure 1) is the first of a new generation of metabolically tion, but prior to onset of symptoms (Barrow et al., 1990; stable capsid function-inhibitors. This compound has Hayden et al., 1992); pirodavir required six times daily dos- demonstrated broad spectrum and potent anti-EV and anti- ing, with efficacy loss at three daily doses (Hayden et al., RV activity and is highly orally-bioavailable (Pevear et al., 1992). Another series of capsid-binding compounds, the 1999; Kearns et al., 1998, 1999; Abdel-Rahman & Kearns, phenoxyl imidazoles, are broad spectrum inhibitors of the 1999). In a mouse model of multi-organ system infection EVs and demonstrate therapeutic oral efficacy in animal following intracranial inoculation of EVs, pleconaril has models (Buontempo et al., 1997; Cox et al., 1996). This been shown to reduce viral titres in all affected organs and series has limited potency against the RVs and further to prevent death of the animals (Pevear et al., 1999). High development of candidate drugs has been discontinued. levels of pleconaril are achieved in the central nervous sys- The ‘WIN’ series of compounds has been clinically eval- tem and in the nasal epithelial tract (M McKinlay, personal uated in both RV and EV infections. The first compound of communication). Pharmacokinetic studies of pleconaril this group to advance to clinical trials was disoxaril (WIN have been undertaken in adults, children and neonates 51711; Figure 1). Disoxaril was moderately active against (Kearns et al., 1998, 1999; Abdel-Rahman & Kearns, 1999). RVs in vitro, and very active against EVs both in vitro and in In adults, the pharmacokinetics of pleconaril is best charac- vivo (McKinlay et al., 1996; McKinlay & Steinberg, 1986; terized as a one-compartment open model with first order Otto et al., 1985). The appearance of asymptomatic crystal- absorption (Abdel-Rahman & Kearns, 1999). luria in healthy volunteers prevented further clinical study. Concentrations of pleconaril 12 h after a single oral dose Shortening of the aliphatic chain from n=7 to n=5 and remain 2.5-fold greater than required to inhibit 95% of EVs adding chloro- groups to the phenyl ring (Figure 1) resulted in vitro.Neonates and older children have similar PK pro- in WIN 54954 which had broad, potent anti-RV and anti- files (Kearns et al., 1998, 1999). Oral bioavailability in EV activity in vitro and in vivo (Woods et al., 1989), includ- humans and other animals approaches 70%. ing oral therapeutic efficacy in mice. Clinical efficacy was In pre-clinical trials, pleconaril was devoid of cardiovas- assessed in two RV (rhinovirus 23 and rhinovirus 39) chal- cular and central nervous system side-effects and no differ- lenge trials (Turner et al., 1993) and one EV challenge trial ences from placebo have been noted in adverse events in (coxsackievirus A21)(Schiff et al., 1992). Despite adminis- any of the clinical trials to date (see below). tering the compound prior to infection and achieving serum In a challenge study of coxsackievirus A21 respiratory

infection, 33 volunteers were randomized to receive either gitis) each resolved 1–2 days sooner in the pleconaril-treat- 400 mg of pleconaril or matching placebo, orally, 14 h before ed patients. inoculation with virus (Schiff et al., 1996). Beginning after In all clinical studies to date (Schiff et al., 1996; Sawyer inoculation, subjects received 200 mg capsules twice daily for et al., 1999; Shafran et al., 1999; Rotbart & Webster, 1999; 6 days. Pleconaril had a significant beneficial effect on symp- Hayden et al., 1999), a very favourable safety profile has tom scores, global assessment, fever and nasal mucous pro- been observed with pleconaril. There have been no differ- duction, with 41% of placebo treated subjects experiencing ences in adverse events between treatment and placebo moderate colds versus none in the pleconaril-treated group. groups. This observation is probably the result of the Peak viral titres that occurred on the peak day of symptoms unique site of action of the compound on the viral capsid were reduced by more than 99% in the pleconaril group and the paucity of metabolic by-products. compared to the placebo group (Schiff et al., 1996). In a placebo-controlled trial of pleconaril in 221 paedi- Enviroxime-related compounds atric patients with EV meningitis, significant reductions in Enviroxime [2-amino-l-(isopropyisulphonyl)-6-benzimi- the total morbidity (composite measurement of all disease dazole phenyl ketone oxime] is a prototype compound for symptoms) and global assessment (caregiver’s assessment of a series of molecules with broad anti-EV and anti-RV patient’s illness) scores were documented for the overall activity developed by Lilly Pharmaceuticals (DeLong et al., study population treated with pleconaril (Sawyer et al., 1978a,b; Wikel et al., 1980). The mechanism of action of 1999). Headache duration was significantly reduced by ple- these compounds has been suggested to be the inhibition of conaril treatment in children older than 8 years. Responses RNA replication via the targeting of the 3A protein coding were noted as early as 24 h after initiation of treatment. Viral region of the viruses (Heinz & Vance, 1995). The drugs shedding from the throat was also reduced in the pleconaril- apparently prevent formation of the RNA replicative inter- treated group compared with placebo (Sawyer et al., 1999). mediate, a complex dependent on both viral proteins 3A Pleconaril has also been studied in adult patients with EV and 3AB (Heinz & Vance, 1995), and hence the formation meningitis (Shafran et al., 1999). In a double-blind, placebo- of new plus-strand RNA molecules. Vinyl acetylene benz- controlled trial, 198 patients aged 14–65 years received either imidazoles derivatives of enviroxime provide improved 200 mg of pleconaril three times per day for 7 days, or place- bioavailability of the compounds; flouridation of these lat- bo. Those receiving pleconaril had a 2-day reduction in dura- ter structures further enhances blood levels in animal mod- tion of headache and a 2-day faster resolution of all symp- els (Tebbe et al., 1997a,b). This class of compounds can be toms of meningitis. Pleconaril-treated patients also returned added to tissue culture systems several hours after viral to work or school 2 days earlier (Shafran et al., 1999). inoculation without loss of antiviral activity, again reflect- Pleconaril has been used in a compassionate release pro- ing their action at a later stage of the viral life cycle (RNA tocol for more than 90 patients with potentially life-threat- replication). ening EV infections, 38 of whom have been followed long Enviroxime resulted in modest clinical and virological enough to assess therapeutic responses (Rotbart & benefit in some studies (Phillpotts et al., 1981, 1983) and Webster, 1999). Among 16 antibody-deficient patients no benefit in others (Hayden & Gwaltney, 1982; Miller et with chronic EV meningoencephalitis, 12 showed some al., 1985). Problems with poor pharmacokinetics and unde- clinical improvement and three others stabilized concurrent sirable toxicology and side-effects resulted in discontinua- with therapy. Six of eight of these patients cleared the virus tion of that programme. Newer derivatives of enviroxime and eight of nine showed an improvement in other labora- promise better bioavailability and tolerance, but have not tory parameters. Clinical responses were also seen in three been clinically evaluated. of four patients with severe neonatal EV disease, three of four cases of myocarditis, three of three patients with 3C protease inhibitors chronic EV infection related to bone marrow transplant, A series of compounds are under development that target two of three patients with vaccine-associated or wild-type the 3C protease of picornaviruses resulting in inhibition of poliomyelitis, and one (of one) patient with post-polio viral protein synthesis, via blocking viral-specific protein muscular atrophy syndrome (Rotbart & Webster, 1999). processing (Patick et al., 1997). Published results are limit- In a double-blind, placebo-controlled study of 1024 ed to those with tripeptide aldehydes derived from the adults with viral respiratory infection during the fall rhino- sequence of a natural 3C cleavage site, Leu-Phe-Gln. Anti- virus season, patients receiving pleconaril recovered from all enzyme activity is potent (Ki=6 nM) with high therapeutic cold symptoms and returned to overall wellness (measured indices in vitro. Like the RNA inhibitors discussed previ- via a global assessment score) 3.5 days sooner than patients ously, time of addition with the protease inhibitors is sever- receiving placebo (Hayden et al., 1999). Individual symp- al hours without loss of antiviral activity. These compounds toms (including nasal congestion, rhinorrhoea and pharyn- appear to have both anti-RV and anti-EV activity. Clinical

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Pharmacology 39:613–618. trials in RV upper respiratory infections have been under- Abzug MJ, Levin MJ & Rotbart HA (1993) Profile of enterovirus taken, but results are not yet available. disease in the first two weeks of life. Pediatric Infectious Disease Journal 12:820–824. Drug resistance Al-Nakib W, Higgins PG, Barrow GI, Tyrrell DAJ, Andries K, Bussche, GV, Tayior N & Janssen PAJ (1989) Suppression of colds Enterovirus mutants that are resistant to the antiviral effects in human volunteers challenged with rhinovirus by a new synthetic drug (R61837). Antimicrobial Agents and Chemotherapy of the capsid inhibitors have been extensively studied; resist- 33:522–525. ance to other anti-picornaviral compounds has not been Arruda E, Pitkaranta A, Witek TJ, Doyle CA & Hayden FG (1997) thoroughly studied due to the lack of progress of these other Frequency and natural history of rhinovirus infections in adults compounds in the clinical arena. Mutants have only been during autumn. 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