Current Status of Anti-Picornavirus Therapies

Current Pharmaceutical Design, 2006, 12, 000-000

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Current Status of Anti-Picornavirus Therapies Dale L. Barnard* Institute for Antiviral Research, Utah State University, 5600 Old Main Hill, Logan, Utah, 84322-5600 USA Abstract: Picornaviruses are important human pathogens causing severe morbidity and some mortality with the potential to cause worldwide crippling disease. Currently, there are few treatments for many of the viruses in the Picornaviridae, For rhinoviruses, there are no approved treatments, although ruprintrivir looks promising in clinical trials and pyridazinyl oxime ethers may prove useful. Poliovirus treatments are needed to supplement the World Health Organization’s polio eradication plan in order to treat infections caused by reversion of the attenuated vaccine virus and to supplement vaccine coverage control in polio endemic areas. However, no promising compounds for treatment of poliovirus have been developed due to the efficacy of the vaccines in use. Broad-spectrum inhibitors developed for other picornavirus may be useful for poliovirus infections. Coxsackievirus infections in children and in infants are being treated with pleconaril with some efficacy in reducing mortality and improving recovery, albeit the treatment is often on a compassionate use basis. There are no therapies for echovirus infections. Very little drug discovery research is being done to develop inhibitors for echovirus infections, probably due to the broad-spectrum inhibition exhibited by capsid binding agents and protease inhibitors discovered for treatment of other picornaviruses. For example, pyridazinyl oxime ethers are inhibitory to most echoviruses. Treatments for enterovirus infections are also limited, although in a small clinical trial, milrinone seemed to reduce mortality and improve recovery from EV71-induced pulmonary edema. Thus, these results strongly emphasize the need for the development of potent and nontoxic compounds for the treatment of picornavirus infections.

Key Words: Picornavirus, rhinovirus, echovirus, coxsackievirus, poliovirus, enterovirus, antiviral, therapy. 1.1. INTRODUCTION

2.1. VIROLOGY

The Picornaviridae includes many human pathogens, some of which are capable of causing severe morbidity and mortality. In this family are viruses causing the common cold and other upper respiratory tract diseases, a hepatitis virus that causes food-borne hepatitis and the enteroviruses that cause a plethora of diseases, including meningitis, encephalitis, otitis media, upper respiratory tract infections, pleurodynia (Bornholm disease), mycocarditis, hand, foot and mouth disease, herpangina, hemorrhagic conjunctivitis, and neonatal coxsackie B infections [1].

The picornaviruses contain RNA that is single stranded and of positive polarity (acts as mRNA or template for minus strand RNA synthesis). The RNA is replicated by virus RNA-dependent RNA polymerase. The single stranded RNA is often associated with a Vpg protein covalently bound to its 5' end, probably acting as primer for RNA synthesis. The RNA is surrounded by an icosahedral capsid made up of four main proteins, usually designated as VP1, VP2, VP3 and VP4. The capsid consists of sixty repeating protomeric units [2]. Variations of amino acids within the four capsid proteins results in the diverse serotypes found within the Picornaviridae. VP4 anchors the capsid to the RNA genome. Uncoating occurs when this protein is destabilized. A compound that would prevent this destabilization presumably would be an effective anti-picornavirus agent. Several virus-specific proteases are used to postranslationally process virus proteins that will become part of the capsid. VP1-VP3 forms the outer structure of the capsid and these proteins are responsible for binding and attachment to cells. These viral protein structures are relatively stable, which makes the viruses in this group somewhat resistant to hostile environmental conditions, including various virucidal agents. For many of the viruses in this family, ICAM-1 generally, but not always, serves as the cellular receptor to which the viruses bind [7]. Other host receptors include integrins, nucleolin, and decay accelerating factor [2].

The family consists of several genera that are important for human disease; the Enterovirus genus which includes polioviruses, coxsackie viruses A1-A22, A24 and B1-B6, echoviruses 1-21, 24-33, the enteroviruses 68-71; the Paraechovirus genus with two serotypes, the Rhinovirus genus which has over 100 serotypes, and the Hepatovirus genus with Hepatitis A as the human pathogen [2]. In the following paragraphs, I will briefly describe the nature of the “typical” picornavirus and explain the targets of current therapeutic approaches based on this knowledge picornavirus structure and replication followed by a review of current picornavirus chemotherapy. Since many reviews are available that cover the field of picornavirus chemotherapy from its beginning [4-11], this paper will review only the current status of chemotherapeutic agents for each of the human pathogen groups in the Picornaviridae from the past several years. *Address correspondence to this author at the Institute for Antiviral Research, Utah State University, 5600 Old Main Hill, Logan, Utah, 843225600, USA; Tel: 1-435-797-2696; Fax: 1-435-797-2696; E-mail: [email protected] 1381-6128/06 $50.00+.00

3.1. TARGETS THERAPY

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PICORNAVIRUS

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Viral attachment to the host cell is one target for chemotherapeutic intervention. In fact, a number of compounds © 2006 Bentham Science Publishers Ltd.

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Current Pharmaceutical Design, 2006, Vol. 12, No. 00

have been synthesized to prevent the virus from attaching to its corresponding host receptor [reviewed in 3, 4, 7, 8]. The strategy to block this interaction is two fold. One way is to make mimetics of the receptor so they act as decoys to ‘steer" the virus away from binding to the host. ICAM-1 mimetics have been synthesized with this purpose in mind [7]. The other is to synthesize molecules that interact with the capsid. Associated with each protomeric unit of the virus capsid is a deep cleft or canyon (pocket of free space) into which the amino acids with the host receptor slide. By designing peptides that fit into the canyons to “plug” them, attachment to the host cell can be blocked [7]. Uncoating is another target. VP4 anchors the capsid to the RNA genome and uncoating occurs when VP-4 is destabilized. A compound that would prevent this destabilization presumably would be an effective anti-picornavirus agent [5]. Many of the current inhibitors evaluated have been designed to block uncoating, including pirodavir and pleconaril [11]. The blocking of the synthesis of plus strand RNA during replication represents another site for chemotherapeutic interdiction. For example, enviroxime has been shown to block this synthesis by interacting with the 3A-coding region of the genome [12]. Directly inhibiting the RNA-dependent RNA polymerase is also an option [3], although no drugs have yet been developed that can do this selectively. The viral mRNA is translated as one large polyprotein, which is then processed by at least two virus-specific proteases to produce virus-structural and non-structural proteins [13]. These proteases, designated as 2A and 3C, are also viable targets for inhibiting viral maturation [reviewed in 6]. The 3C protease of rhinovirus has successfully been targeted, and one inhibitor of 3C protease, ruprintrivir, has been in clinical trials [14]. Other targets for therapeutic intervention include the host endosome whose function is necessary for the virus to be transported to cytoplasm for uncoating, and viroporins, viral proteins that permeabilize the membrane of the host cell to allow entry of the virus [4]. In addition, compounds that could block translation of viral proteins using hydrophilic translation inhibitors represent another method of inhibition [4]. 4.1. RHINOVIRUSES 4.1.2. Diseases The human rhinoviruses cause most of the upper respiratory tract infections (URTI) in humans, accounting from 40-60% of the common colds in humans [2]. These infections are often mild, self-limiting and seasonal. Human rhinoviruses (HRV) infect people of all ages, during all seasons, although seasonal disease prevalence occurs in September and October and in March and April in the northern hemisphere [15, 16]. HRVs also cause serious lower respiratory tract infections [reviewed in 17]. HRV is reported to be the second most frequently detected microbe in infants and young children diagnosed with pneumonia and bronchiolitis [17]. Primary URTIs caused by HRV are one of the predisposing factors for otitis media in children [18]. HRV infections are also important in the induction of acute asthma in adults [19] and in children [20]. HRV can exacerbate disease in asthmatics [21], in people with chronic obstructive pulmonary

Dale L. Barnard

disease and the immunocompromised [reviewed in 22-24], be they patients who are on immunosuppressive therapy or immunocompromised because of pre-existing disease [17]. HRV is also associated with lower respiratory tract infections in other patient populations such as those with cystic fibrosis [25], patients with chronic bronchitis [18], the elderly [26], and infants less than 12 months old [27]. P are also susceptible to complications from HRV infections of the lower respiratory tract [18]. Economically, rhinovirus infections have a significant impact on human productivity (lost days of work or absence from school) in the industrialized nations and on medical costs in terms of number of visits to the physicians’ offices and treatment costs [5, 25]. Thus, effective treatments for human rhinoviruses would significantly reduce morbidity due to the common cold as well as enhance human productivity. 4.1.2. Experimental Therapies Recently, a number of excellent reviews have been published on rhinovirus inhibitors [4-6, 8-11, 18, 28-32]. Therefore, this review will concentrate on advance in rhinovirus chemotherapy for the two to three years. 4.1.2.1. Synthetic Compounds 4.1.2.1.1. Protease Inhibitors A series of tripeptidyl alpha-ketoamides were synthesized as human (HRV) 3C protease inhibitors. The most potent inhibitor was 4I, which inhibited the HR-14 3C protease (IC50 < 0.5 µM) and inhibited viral HRV-14 replication in vitro as well [33]. In a previous study, various 2-pyridone-containing HRV 3C protease inhibitors were synthesized and one was found to be orally bioavailable in the beagle, but when tested in CM-monkeys, it was not [34]. Thus, another synthesis study was done to modify the compound to have better oral bioavailability [35]. As a result, a series of compounds were synthesized composed of a 2-pyridone-containing peptidomimetic binding determinant and an alpha, betaunsaturated ester Michael acceptor moiety to form an irreversible covalent adduct with the active site cysteine residue of the 3C enzyme. Two compounds containing an alpha, beta-unsaturated ethyl ester fragment and either an ethyl or propargyl P(2) moiety displayed the most promising combination of 3C enzyme inhibition (k(obs)/[I] = 170 000-223 000 M(-1) s(-1)), mean antiviral activity (EC50) = 0.0470.058 µM, against seven HRV serotypes), and pharmacokinetics following oral administration (7 h dog plasma levels = 0.248-0.682 µM; 7 h CM-monkey plasma levels = 0.0570.896 µM). These results suggest that these two compounds may be used as lead compounds to develop even more suitable protease inhibitors for clinical use. Several peptide-based fluoromethyl ketones (FMK, caspase inhibitors) were shown to inhibit picornavirus 2A proteinase (RV2APRO) of rhinovirus 14 (HRV14) [36], although the caspases for which the FMKs were designed to inhibit are not related to RV2APRO. The caspase inhibitors, benzoyloxycarbonyl-Val-Ala-Asp(OMe)-FMK and benzoyloxycarbonyl-Ile-Glu-(OMe)-Thr-Asp(OMe)-FMK, inhibited

Current Status of Anti-Picornavirus Therapies

HRV14 replication in HeLa cells and the RV2APRO at high concentrations (200 µM) of each compound. The compounds may have inhibited rhinovirus-induced apoptosis [37], cellular eIF4GI cleavage, the RV2APRO, or combination of two or more of the mechanisms to achieve the antiviral effect. Further studies need to be done to demonstrate a selective antiviral effect with these compounds or to demonstrate that inhibition of virus-induced apoptosis represents a viable option for treating HRV infections. 4.1.2.1.2. Capsid Binder Inhibitors Pirodavir (ethyl 4-[2-[1-(6-methyl-3-pyridazinyl)-4-piperidinyl] ethoxy]benzoate) is an inhibitor of picornaviruses, especially numerous HRV serotypes. However, in clinical trials for treatment of HRV infections it was not efficacious, probably because of poor pharmacokinetic properties [38] and its sensitivity to facile hydrolysis, yielding an inactive product [39]. Using pirodavir as a template, a series of pyridazinylpiperidinyl capsid-binding compounds with novel bicyclic substituents were synthesized with purpose of making a more orally bioavailable compound with similar potency to pirodavir [40]. Several 2-alkoxy- and 2-alkylthiobenzoxazole and benzothiazole derivatives were very inhibitory to the HRV strains tested. When evaluated for inhibitory activity against a panel of 16 representative HRV types, a 2-ethoxybenzoxazole derivative was the most potent inhibitor of HRV replication (median EC50 = 3.88 ng/ml). The substitution with the stable 2-ethoxybenzoxazole moiety presumably will enhance the half-life of the compound in vivo and thus improve oral bioavailability. 4.1.2.1.3. Viral RNA Synthesis Inhibitors Enviroxime [2-amino-1-(isopropylsulfonyl)-6-benzimidazole phenyl ketone oxime] has long been known to be an inhibitor of picornaviruses, including HRV [3], but do to lack of efficacy, and poor bioavailability, and due to deleterious side effects such as nausea and vomiting, it will not be used as a single therapy in a clinical situation [3, 4]. Thus, a series of imidazo[1,2-b]pyridazines based on the structure of enviroxime were synthesized, designed to have similarly potent broad-spectrum anti-picornavirus activity and better oral bioavailability [41]. The oxime derivative, 2-amino-3(4-fluorophenyl)-6-benzoylimidazo[1,2] pyridazine oxime, was the most potent inhibitor of HRV14 (IC50 = 0.05 µg/ml). The compound was not toxic up to and including 10 µg/ml. As predicted, the compound was also broad spectrum, inhibiting both coxsackie and polioviruses (IC50 = 0,02-0.04 µg/ml). Fulfillment of the critical criterion, enhanced oral bioavailability, has not yet been reported. Previously, flavonoids had been shown to inhibit a variety of viruses, including rhinoviruses [reviewed in 42]. The exact mechanism of inhibition of this broad class of compounds seems to be different depending on the compound. In general the flavonoids probably inhibit uncoating or some step during viral genome replication [42, 43, 44]. Recently, a new class of flavonoids, 2-styrylchromones, was made that inhibited by rhinovirus groups A and B [45]. However, the most promising inhibitor was not a styrylchromone (3hydroxy-2- styrylchromone was a potent viral inhibitor, but cytotoxic), but a 1-(2-hydroxyphenyl)-5-phenyl-2,4-pentadien-1-one substituted with a chloro and nitrate group on

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each phenyl ring. It inhibited both RV1 (EC50 = 0.89 µM) and RV14 (EC50 = 0.092 µM). The compound was not cytotoxic at the concentrations tested. However, it was only soluble in organic solvents, which will impact its toxicity and oral bioavailability. It has been shown that 5,7,3',4'-tetrahydroxy-3-O-methylflavone exhibits antipicornaviral activity [46]. Since the compound would need to be used intranasally, the potential of 5,7,3',4'-tetrahydroxy-3-O-methylflavone as an antirhinoviral compound for nasal application was studied in combination with several delivery vehicles. The effect of 5,7,3',4'tetrahydroxy-3-O-methylflavone on the ciliary beat frequency (CBF) of human nasal epithelial cells was studied in vitro in the absence or presence of solubility/absorption enhancers (hydroxypropyl-beta-cyclodextrin (HP-beta-CD) or polysorbate 80). HP-beta-CD administered alone did not affect the function of these cells at 1-3% concentration. At 2 and 10 µg/ml, 5,7,3',4'-tetrahydroxy-3-O-methylflavone alone showed some reversible cilio-stimulatory effects. Combined with 3% HP-beta-CD, no ciliotoxic effect could be observed for 5,7,3',4'-tetrahydroxy-3-O-methylflavone up to 20 µg/ml. Thus, this formulation for nasal application of 5,7,3',4'tetrahydroxy-3-O-methylflavone should be further explored. 4.1.2.1.4. Other Innovative Inhibitors RNA silencing or interference (RNAi) is a sequencespecific, post-transcriptional process of mRNA degradation. The degradation of target gene mRNA can be induced by short dsRNA molecules (21-25-nt) corresponding to the sequence of the target gene to be silenced. Short dsRNA molecules have been shown to be very effective in inducing RNA silencing in cell culture [47]. In a recent study, short dsRNA molecules corresponding to the HRV-16 genome inhibited viral replication in cell culture [48]. The siRNAs targeting the gene for VP4, 2C and 3D inhibited virus replication at the lowest concentrations (IC50 = 0.73-0.93 nM). The dosedependent inhibition was sequence-specific, since siRNAs targeting unrelated genes were not inhibitory. However , the molecules had to be transfected into cells to inhibit virus. This necessity of transfection in vitro, is problematic for in vivo studies. In addition, siRNAs are subject to degradation and that obstacle must be overcome before any in vivo testing can occur. Nevertheless, further studies are warranted to determine if such reagents are of clinical relevance. A new method of identifying targets and their inhibitors in virus-infected cells has been developed [49]. It identifies transdominant factors expressed in virus susceptible cells that are small peptides or RNAs (“peturbagens”), which perturb a cellular process to induce a desired phenotype, i.e., resistance to a certain viral infection. By this method, a small protein from a HeLa cell clone (I421dp3) was identified that partially inhibited HRV replication at or before virus RNA replication. The peptide consists of a highly charged peptide segment followed by a very hydrophobic segment. It is possible that small molecule mimetics of this peptide that penetrate cells better could be useful inhibitors of HRV infection. Blockage of virus receptors on cells by monoclonal antibodies is a common strategy to inhibit viral infection [50]. To compete effectively against viruses for sustained receptor binding, viral receptor blockers need to exhibit superior

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binding avidities; otherwise, viruses would have the competitive advantage. The avidity of a single molecule can be enhanced exponentially by a complex that contains two, three or more identical molecular subunits. Applying this mechanism to the design of receptor blockers against HRV major receptor intercellular adhesion molecule-1 (ICAM-1), a multivalent recombinant antibody Fab fusion protein was made that has shown significantly higher avidities than conventional monoclonals [51]. The new drug candidate, designated as CFY196, is a tetrameric chimera, and was more than 200-fold more effective than the corresponding Fab fragment at binding to ICAM-1and preventing HRV infection. Replication of HRV-15 was inhibited at 0.97 nM in a HeLa cell culture assay. This reagent also exhibited a much improved avidity over conventional monoclonal antibodies. This multivalent recombinant Fab fusion protein may represent a new generation of potent antiviral receptor blockers and other therapeutic molecules. Finally, a very theoretical technology must be mentioned, the role of human beta-defensins as therapeutic agents. Defensins are cationic, antimicrobial peptides and are major components of the human neutrophil azurophilic granules [52] and have been reported to impair viral infections in vitro [53]. It has recently been shown that HRVs can induce the production of human beta-defensin 2 [54]. Infection of primary cultures of human epithelial cells with several HRV serotypes induced expression of human beta-defensin 2 mRNA and protein, indicating that human beta-defensin 2 production was independent of viral receptor usage or mechanisms of viral RNA internalization. Induction of human beta-defensin 2 was dependent upon viral replication [54]. Human beta-defensin 2 showed no direct antiviral activity against HRV, although HRV replication was reduced when human beta-defensin 2 was induced. In vivo infection of normal human subjects with HRV-16 induced expression of mRNA for human beta-defensin 2 in nasal epithelial scrapings. Increases in mRNA correlated with viral titer and with increased levels of human beta-defensin 2 protein in nasal lavages. This suggests that peptide mimetics of defensins might be evaluated as potential antiviral therapies for viruses, including HRV [55]. 4.1.2.1. Clinical Studies A number clinical studies have recently been done to evaluate the efficacy of a variety of compounds, including 3C protease inhibitor, ruprintrivir [14] and natural products such as echinacea [56-59] and a rutoside, troxuretin [60]. In addition, the efficacy of zinc has retrospectively been revisited with some interesting conclusions [61]. Several clinical trials to assess prevention or treatment efficacy of echinacea have been or are being done. The results from a limited number of clinical trials have thus far been inconclusive [11]. This inconsistency may be the result of investigators utilizing poorly standardized echinacea products, likely devoid of sufficient quantities of active constituents necessary to exert a definitive clinical effect [11]. Therefore, some Phase II [58] and Phase III clinical trials [59] are being done using more standardized products. Results form the latter two are not yet available, although the phase II study is now closed for enrollment.

Dale L. Barnard

One study was a randomized, double-blind, placebocontrolled clinical trial to evaluate the ability of Echinacea purpurea to prevent infection with RV-39 [56]. Forty-eight previously healthy adults received echinacea or placebo, 2.5 ml 3 times per day, for 7 days before and 7 days after intranasal inoculation with RV-39. Symptoms were assessed to evaluate clinical illness. A total of 92% of echinacea recipients and 95% of placebo recipients were infected. Colds developed in 58% of echinacea recipients and 82% of placebo recipients, although the difference was not statistically significant. Thus, administration of echinacea before and after exposure to rhinovirus did not decrease the rate of infection. However, as in past studies, this study suffered from a small sample size, not allowing the investigators to detect differences in the frequency and severity of illness. In a Canadian study, a formulation containing alkamides, cichoric acid, and polysaccharides at concentrations of 0.25, 2.5, and 25 mg/ml, respectively, was prepared from freshly harvested Echinacea purpurea plants to achieve a more standardized product [57]. The objective of this study was to test the therapeutic efficacy of this highly standardized formulation in reducing the severity and duration of symptoms of a naturally acquired common cold. In a randomized, double-blind, placebo-controlled trial, 282 subjects in good health were recruited, aged 18-65 years, with a history of two or more colds in the previous year. The subjects were randomized to receive either echinacea or placebo. They were instructed to start the echinacea or placebo at the onset of the first symptom related to a cold, consuming ten doses the first day and four doses per day on subsequent days for seven days. A total of 128 subjects were diagnosed with a common cold (59 in echinacea group, 69 in placebo group). For those who strictly followed the study protocol, the total daily symptom scores were 23.1% lower in the echinacea group, which was statistically significant. Throughout the treatment period, the response rate to treatments was greater in the echinacea group and few adverse event profiles were observed in both groups. The investigators concluded that early intervention with a standardized formulation of echinacea resulted in reduced symptom severity in subjects with naturally acquired upper respiratory tract infection. However, given the rather frequent dosing required to achieve this modest effect, the benefits seem rather meager. More recently, a retrospective review of the clinical trials reported in the literature concluded that echinacea does not have therapeutic efficacy for treating HRV infections [62]. Ruprintrivir selectively inhibits HRV 3C protease and has been shown to be a potent, broad-spectrum anti-HRV activity in vitro [63]. Ruprintrivir has been evaluated in a double-blind, placebo-controlled treatment and prophylaxis study [14]. For this study, 202 healthy volunteers were recruited to assess the activity of ruprintrivir in an experimental HRV infection. Subjects received intranasal ruprintrivir (8 mg) or placebo sprays prophylactically two or five times daily for five days starting six h before infection or therapeutically 5x/day for 4 days starting 24 h after infection. Ruprintrivir prophylaxis significantly reduced the proportion of subjects with positive viral cultures, but did not decrease the frequency of colds. Ruprintrivir treatment significantly reduced the mean total daily symptom score by 33%. Secondary endpoints, including viral titers, individual symptom


scores, and nasal discharge weights, were also reduced by ruprintrivir treatment. Ruprintrivir was well tolerated, although blood-tinged mucus and nasal passage irritation were the most common adverse effects reported. These studies demonstrate that in an experimental rhinoviral intranasal ruprintrivir shows efficacy and might be useful in treating and preventing natural HRV infection. The rutosides are naturally occurring flavonoids that have documented effects on capillary permeability and edema [64, 65]. Since nasal edema contributes to the pathogenesis of the common cold, a study was done to determine the effect of the rutoside, troxerutin, on ameliorating some of the symptoms of the common cold. Ninety-four volunteers with common cold symptoms were recruited for participation in the study. Volunteers were randomly selected to receive treatment (n=49) with troxerutin (50 mg) plus 25 mg Zn gluconate or control treatment (n=45) with 10 mg Zn gluconate. Symptoms were assessed by subjective symptom scores prior to treatment and then daily for the next four days. The total symptom scores were not significantly different over the four days of study treatment. The total daily symptom score on day one was significantly reduced by 11% compared to baseline in the treatment group and by 1% in the control group. The total rhinorrhea score over the course of the study was significant lower in the active group compared to the control group. Daily rhinorrhea scores were significantly lower in the active group on study days one and three as well. Unfortunately, the effects of troxerutin might have been confounded by the presence of zinc gluconate, which has also been shown to have anti-rhinovirus effect, although the control group also received zinc gluconate, but at lesser dose than troxerutin-treated group. Thus, no firm conclusions can be made about efficacy of this compound in ameliorating symptoms of the common cold. A retrospective study was done on the efficacy of zinc by reviewing all past literature describing the use of zinc compounds for treating HRV infections [61]. Clinical tests of zinc for treatment of common colds have been inconsistent, primarily because of study design, small sample size, lack of blinding, and the use of different formulations of lozenge contents. Early formulations of lozenges were also unpalatable, ruling out an oral formulation as useful. In three trials with similar study designs, methodologies, and efficacy assessments, zinc effectively and significantly shortened the duration of the common cold when it was administered within 24 hours of the onset of symptoms. Zinc gluconate, in the form of a nasal gel, seemed to do the same and has been shown to be effective in reducing the duration and severity of common cold symptoms in patients with established illness. Thus, some clinical trial data support the value of zinc as an agent that can reduce the duration and severity of symptoms of the common cold when administered within 24 hours of the onset of common cold symptoms. Finally, there is some interest in the finding that increased generation of nitric oxide during rhinovirus infections is associated with fewer symptoms and more rapid viral clearance [66]. Recent evidence suggests that nitric oxide is an important contributor to the host response during colds [67. 68]. Consistent with these observations are pilot studies that seem to associate the increased generation of nitric oxide


during HRV infections with fewer symptoms and more effective clearance of virus [66]. Further detailed studies, especially in animal models, are needed to evaluate the role of nitric oxide in colds to determine the value of nitric oxide donor compounds for treating for viral infections that occur in airway diseases. 5.1. ENTEROVIRUSES The enteroviruses include the coxsackieviruses, echoviruses, enteroviruses 68-71, polioviruses, and the parechoviruses. For the latter species, which contains the former echoviruses 22 and 23, there is not much information regarding therapeutic agents for treatments of infections that this virus group causes. One assumes that they may be closely enough related to echoviruses that some of those agents inhibiting echoviruses may also inhibit the parechoviruses, especially the broad-spectrum inhibitors such as the capsid inhibitors. 5.1.2. Polioviruses 5.1.2.1. Diseases Poliovirus is the prototypic virus for the genus Enterovirus. There a four serotypes, all of which are capable of causing a spectrum of diseases from “summer colds” to poliomyelitis. With the advent of two effective vaccines, the need for antiviral agents seemed to have diminished. However, the recent appearance of polio cases in the US due to reversion of the live-attenuated vaccine to wild type virus, the emergence of post-poliomyelitis syndrome, and the failure of certain countries to meet eradication goals set by the World Health Organization (WHO) has increased interest in developing antiviral agents to supplement effective immunization programs. In addition, WHO has now called for the development of anti-polio drugs to control post-eradication outbreaks that may arise from the use of the attenuated vaccine once polio has been certified eradicated [69]. 5.1.2.2. Experimental Therapies Because of effective immunization programs in the US where polio has been declared eradicated, well funded drug discovery programs searching for poliovirus inhibitors are not numerous. Therefore, many studies to identify inhibitors have been done by individual investigators with little followup in animal studies. 5.1.2.2.1. Synthetic Compounds Enviroxime has previously been shown to act synergistically with disoxaril [5-7-4-(4,5dihydro-2-oxazolyl)phenoxy heptyl-3-methyl isoxazole] to inhibit polio type 1 in cell culture [70]. Fortuitously, the combination of the two drugs inhibited resistant mutants to each type of drug. This combination has now been found not to be synergistically toxic at most every set of concentrations tested [71]. This is very enlightening, since the compounds are toxic at high oral doses [4]. These data suggest that this combinational therapy should be pursued as a potential therapy for poliovirus infections. siRNAs have been developed that directly inhibit poliovirus replication [72]. These act by silencing targeted genes by triggering RNase degradation of the siRNA bound sequence.

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However, escape mutants could be generated, so the investigator targeted longer stretches of RNA that contained multiple genes to prevent the establishment of escape mutants [72]. These were also efficacious. Should such agents be formulated for delivery in animals, they promise to be very potent and specific inhibitors meriting further development. A novel, alternative method for treating poliovirus infections is to inhibit nuclear fragmentation induced by the virus in infected cells [73]. Using chlorophyllin, a more soluble derivative of chlorophyll, investigators demonstrated that nuclear fragmentation in poliovirus-infected cells was greatly reduced. The advantage that this compound may have is that it is already in use as a food additive, as a wound healer and as an agent for controlling noxious human odors. Thus, it has a long record of use and a well-documented safety profile. 5.1.2.2.1. Natural Products Recently, investigators have concentrated on designing and synthesizing compounds based on natural products previously shown to inhibit poliovirus replication [reviewed in 3]. For example, extracts from the Australian aboriginal plant, Dianella longifolia, were shown to inhibit several poliovirus serotypes. An active compound isolated from that plant, chrysophanic acid (1,8-dihydroxy-3-methyl anthraquinone) inhibited poliovirus types 2 and 3 [74]. The compound inhibited virus replication at an early stage at a concentrations ranging from 0.02 to 0.21 µg/ml. The compound was also relatively toxic, but the antiviral activity was well below the cytotoxic concentrations [selective index (SI) = 625] and independent of any virucidal activity. Known, related anthraquinones were not as active and were often more cytotoxic. Previously, flavonoids had been shown to inhibit a variety of viruses, including polioviruses [reviewed in 42]. Based on this knowledge, Robin et al. [75] isolated some flavonoids from methanolic extracts of a medicinal plant, Psiada dentata (Cass.). 3-Methylkaempferol (5,7,4'trihydroxy-3-methylflavone) was one of the flavonoids isolated that inhibited poliovirus type 1 replication (EC50 = 0.45 µM, CC50 = 100 µM). This activity was independent of any potential virucidal activity. It inhibits genomic RNA synthesis [75]. Salvati et al. [43] evaluated 3(2H)-isoflavone, a synthetic derivative of 4',6-dichloroflavan (derived from a plant extract) that inhibits rhinovirus uncoating [44]. 3(2H)Isoflavone also appeared to inhibit the uncoating of poliovirus type 2 by stabilizing any conformational changes in VP1 and VP4 that affect viral capsid flexibility [43]. Other less characterized natural products inhibiting poliovirus included methanolic extracts from Piper aduncum, an Indonesian medicinal plant [76] and five methanolic extracts from plants from the island of Le Réunion [72]. However, it was unclear whether the inhibition by these extracts was merely due to physically disrupted the virus or due to inhibition of some part of the virus replication cycle.

Dale L. Barnard

species, A and B. Serotypes A7, A9, B1-B6 are implicated as the etiological agents for “aseptic meningitis [2]. Group B viruses cause serious generalized neonatal disease, often associated with the summer and autumn months, myelopericarditis, and are also thought to be the cause of pleurodynia (Bornholm disease). Group B viruses also cause persistent CNS infections. Group A viruses tend to cause less severe diseases-herpangina (A1-6, 8, 10, 22), conjunctivitis (A24), and hand, foot and mouth disease (A16) [78]. A feature unique to the this group of viruses is their ability to cause pathogenesis in suckling mice, making it easy to evaluate compounds for efficacy in a small animal model. 5.1.3.3. Experimental Therapies 5.1.3.3.1. Synthetic Compounds A number of novel compounds have been synthesized using existing anti-picornavirus agents such as pleconaril as design templates [79-82]. These compounds were synthesized retaining the basic structure of the template compound (i.e., the isoxazole ring of pleconaril that is necessary for activity) and then replacing the remaining structure with other moieties. Adding biphenyl groups to the isoxazole ring of pleconaril resulted in biphenyloxypropyl isoxazoles substituted with various other substituents at the terminal benzene ring [82]. Of these derivatives, a biphenyl substituted with a tetraphenyl group was a very potent inhibitor of a coxsackie B3 (CVB3) isolate (EC50 = 0.013 µg/ml, SI = 962). It also inhibited human rhinovirus at a nearly equal concentration. Other were compounds inhibiting CVB nucleoside analogs modified by the attachment of 3-methylisoxazole rings to the nucleoside [79]. Two of these compounds inhibited CVB at concentrations equivalent to ribavirin, but both were very cytotoxic. Further modification is needed to eliminate the cytotoxicity. Synthesis of a number of pyridyl imidazolidinone analogs resulted in significant anti-coxsackie virus A activity [80]. 1-[6-bromophenoxy)hexyl]-3-pyridin-4-yl-imidazolidin-2-one and 1-(4-pyridyl)-3-{6-[4(trifluoromethyl)phenoxy]hexyl}-imidazolidin-2-one were potent inhibitors of CVA9 (IC50 = 0.47-0.55 µM) and CVA24 (IC50 = 0.470.55 µM). 1-[5-(4-bromophenoxy)pentyl-3-pyridin-4-yl-imidazolidin-2-one, designated as BPR0Z-194, was shown to target the hydrophobic pocket of the VP1 capsid protein [9]. Another class of compounds, pyrazolo[3,4-d]pyrimidines, was made to inhibit enteroviruses, including CVB3 (IC50 = 0.063-0.089 µM) [81]. The pyrazolo[3,4-d]pyrimidines with a thiophene group were the most potent inhibitors of CVB3. The compounds were not toxic at concentrations up to and including 25 µM. Thus, these compounds are relatively non-toxic yet potent and should be pursued.

5.1.3.2. Diseases

Cis-substituted cyclohexanyl nucleosides were synthesized that also inhibited CVB3 [83]. The cyclohexene ring was added to provide protection against hydrolysis. Cis-(±)1-[3'-hydroxymethyl)cyclohexanyl]thymine was the only compound of this series that inhibited CVB3 (~ EC50 =168 µM) without exhibiting some cytotoxicity. This represented just modest inhibition and thus these types of compounds are not likely to be developed further.

Human coxsackieviruses of the Enterovirus genus of the Picornaviridae comprise about 23 serotypes, grouped as two

Another synthetic approach was the synthesis of 3methylthio-5-aryl-4-isothioazolecarbonitriles [84]. A broad-

5.1.3. Coxsackieviruses


spectrum picornavirus inhibitor, isothiazolecarbonitrile (IS50) served as the model compound from which the new compounds were derived. The two most active compounds that inhibited CVB1 (EC50 = 0.5-2.5 µM, CC50 >20 µM) had a butyl (3-methylthio-5-[4-(3-phenoxy-1-butoxy)phenyl]4-isothiazolecarbonitrile) or propyl (3-methylthio-5-[4-(3phenoxy-1-propoxy)phenyl]-4-isothiazolecarbonitrile) group between the two phenoxy rings. These two compounds were also inhibitors of polio and echoviruses. Preliminary data suggest that virus adsorption may be the target of inhibition for these compounds. Peroxynitrite, formed by the reaction of NO and superoxide, is a powerful oxidant produced during inflammation in animals and humans [85]. Peroxynitrite in low concentrations may be beneficial to the host during pathogen infection [86]. Therefore, it was evaluated for inhibition of CVB3 replication. The major finding of this study was that peroxynitrite inhibited viral replication; at 1 µM peroxynitrite decreased viral replication by 1,000-fold. Peroxynitrite appeared to inhibit the entry of viral RNA into the host cell. These data suggest that nitric oxide or reactive nitrogen intermediates of nitric oxide such as peroxynitrite should be studied further as potential antiviral agents. Several groups have synthesized antisense oligonucleotides targeting CVB3 sequences in the coding regions of the capsid genes, the P2A cleavage enzyme gene and the 5' and 3' untranslated regions of CVB3 RNA [87-89]. In one study oligonucleotides (5 µM) targeting the pyrmidine rich tract (SCB1) of VP1 and the beginning of the untranslated region reduced viral replication by 75-90% [88]. A 21-mer antisense phosphorothioate oligonucleotide (10 µM) targeting nucleotides 581-601 of the virus genome in the 5' noncoding region of CVB3 inhibited viral replication in cell culture even at 48 h post virus exposure [87]. In the most recent study it has been for inhibition demonstrated that the most effective target is the proximal terminus of the 3' untranslated region [89]. The antisense oligonucleotide, designated as AS-7, strongly inhibited viral RNA and viral protein synthesis as compared to scrambled antisense oligonucleotides. These studies suggest that antisense molecules may be a feasible approach to treating CVB infections if the compounds can be delivered effectively. An interesting study was done to determine the efficacy of various combinations of anti-picornavirus compounds, many of which having been discontinued as feasible antiviral compounds because of lack of efficacy or toxicity [90]. The investigators hypothesized that the drugs might be efficacious given together and would be effective in combination against viruses resistant to either drug. In this study, it was found that all the combinations tested against CVB1 were strongly synergistic and inhibitory to wild type and resistant viruses alike. The combinations evaluated included enviroxime (viral RNA synthesis inhibitor) + S-7 (ethyl-2methylthio-4-methyl-5pyrmidine carboxylate, uncoating inhibitor), enviroxime + PTU-23 ( N'-phenyl-N'-3-hydroxyphenyl urea, blocks synthesis of viral 37S RNA), disoxaril (uncoating inhibitor) + HBB (2-alpha-hydroxylbenzyl-benzimidazole, RNA polymerase inhibitor), disoxaril + PTU-23, arildone (uncoating inhibitor) + HBB, arildone + PTU-23, and S-7 + HBB. The most effective combinations were HBB


with arildone or disoxaril. Unfortunately, no data was provided on the combinational cytotoxic effects. Finally, an interesting study was done using selenite as a treatment for apparent CVB5 infection in cell culture [91]. This study was inspired by observation that in China in the Keshan province, where soils are deficient in selenium, Keshan disease (childhood cardiomyopathy) is endemic. Previous studies had shown that diet supplementation with selenium effectively reduced the incidence of Keshan disease [92, 93]. The hypothesis was that one of the etiological agents for that disease may be a coxsackievirus [93] and that it could be treated with selenium supplementation [reviewed in 94]. The first part of the hypothesis was supported by several studies in which CVB was isolated from the tissues of patients with Keshan disease [95, 96]. In support of the second part of the hypothesis were laboratory studies demonstrating the occurrence of mutations and increased virulence of coxsackieviruses infecting selenium-deficient mice or in mice carrying a non-functional gene for the seleniumdependent glutathione-peroxidase [97, 98]. Cermelli et al. [91] discovered that 5 µM selenite, but not 10 µM selenate or selenomethionine, modestly reduced viral titers in cell culture by about 2 log10. The efficacy of the selenite could be reversed with zinc, which is an inhibitor of selenite toxicity or with DTT, a sulfhydryl-protecting agent also known to reverse selenite toxicity. Solid epidemiological driven clinical studies need to be done to verify these findings. 5.1.3.3.2. Natural Products Natural products have long been used to treat various diseases. Various investigators have recently evaluated extracts that purported have activity against respiratory disease purported have caused by CVB. Extracts that were tested included hot water extracts of black soybean [99], alcohol extracts of Loranthus yadoriki [100], garlic and Shanghuanglian [101], Xinjierkang granules [100], acetate extract of Tian-hua-fen 102], Chuan-Kang-Ping granules [103] and various extracts of Ganlu Xiaodu Dan [104]. The black soybean extract was found to be a broad-spectrum virus inhibitor and inhibited CVB1 with an IC50 ~ 2.6 µg/ml which was below the CC50 value. The Xinjierkang granules extract were apparently evaluated for efficacy in a mouse myocarditis model for CVB. The compound seemed to reduce death due to virus [99]. These types of products need to be further purified to ascertain the compound(s) within the extract responsible for the antiviral activity detected in the studies mentioned above. 5.1.3.3. Animal Studies Mycophenolate mofetil (MMF), an immunosuppressive agent, inhibited development of CVB3-induced myocarditis in mice [105]. Four-week-old mice infected with CVB3 received MMF twice daily for seven days by oral gavage. Using 300 mg/kg/day, there was a 78% reduction in myocarditis as assessed by morphometric analysis. The compound was well tolerated. Viral titers and total viral RNA in heart tissues were actually increased, so protection must have been due to immunosuppression or some immunomodulatory mechanism. These data suggest that MMF should not be used alone for treating CVB infections, but in combination with an inhibitor of viral replication.

8


An interferon inducer, ampligen (Poly I : C12U), has been shown to protect mice from CVB3-induced myocarditis by 98% when administered at 20 mg/kg/day [106]. Another important finding from this study was that ampligen also reduced virus RNA and infectious titers in the heart, and the electrocardiogram in treated mice returned to normal. Ampligen at the concentrations used was well tolerated. In this study, human interferon also seemed to partially protect mice from myocarditis indicating the significance having a good interferon response to CVB3 infections in vivo. In another study it was demonstrated that 2-(3,4dichloro-phenoxy)-5-nitrobenzonitrile (DNB), a compound that was earlier shown to exhibit broad-spectrum antipicornavirus activity was also markedly active against CBV replication in primary human myocard fibroblasts [107]. DNB was also evaluated to determine if the compound was able to prevent the development of CBV-induced myocarditis in a murine model [107]. Subcutaneous administration of DNB at 250 mg/kg/day for seven days, starting at 1 day before infection and administered at multiple injection sites to 4-week old C3H-mice, resulted in a 62% reduction in the number of myocarditis foci as compared to the untreated control animals, with a concomitant reduction in viral titers in the heart. These findings suggest that selective inhibition of the replication of CBV by DNB may have a beneficial effect on the development of viral myocarditis. In another study, the efficacy of nitric oxide donors (in vitro inhibitors of 2A and 3C picornavirus proteases) in protecting mice against CVB3-induced myocarditis was also demonstrated [108]. The nitric oxide donors and vasodilators, glyceryl trinitrate (GTN) and isosorbide dinitrate (ISDN), were given by “feeding” (GTN) or orally administering (ISDN) starting 1 h prior to virus exposure and daily thereafter for 14 days (GTN) or 28 days ISDN). GTN enhanced the survival of mice (86% survival rate) and significantly reduced the signs of myocarditis. ISDN was only efficacious by measured physiological parameters after 28 days of treatment. However, 100% of the treated mice survived 28 days. In this study, it was apparent that endogenous NO production was not sufficient to provide protection against myocarditis, but had to be supplemented with NO-releasing drugs. Treatment of CVB3-infected mice with WIN 54954 (isoxazole derivative), an inhibitor of viral uncoating, intragastrically with 100 mg of compound at one hr pre-virus exposure and then twice daily for five days, seemed to reduce tissue necrosis, viral RNA levels in the heart tissue, and myocardiocyte apoptosis [109]. The compound was also well tolerated at this dosage. The data suggest a new mechanism whereby CVB3 infection may be inhibited or ameliorated by preventing or reducing myocardiocyte apoptosis. However, it still needs to be determined if apoptosis is directly induced in healthy cells by a viral protein or if results from the host response to severely damaged cells from the virus infection. 5.1.3.3. Clinical Studies Four premature newborn infants (two sets of twins) with systemic CVB infection were treated with an oral suspension of pleconaril (5 mg/kg/day for 7-10 days). The patients had myocarditis, fulminant hepatitis, meningoencephalitis, and

Dale L. Barnard

disseminated intravascular coagulopathy (DIC) [110]. Prior to pleconaril treatment, two patients had received intravenous immunoglobulin (IVIG), but continued to decline. Pleconaril was obviously administered on compassionate use basis, and remarkably, the patients recovered with no adverse events associated with drug therapy. In the three infants evaluated for presence of virus, virus was not detected 10 days after beginning pleconaril treatment in throat, stool or cerebral spinal fluid. In another study, ten neonates presented with CVB infection, characterized by meningoencephalitis, thrombocytopenia, cardiomyopathy, DIC, and hepatitis with an average duration of three months [1]. Seven of theses infants were treated with pleconaril (5 mg/kg/three times daily). Three other patients were diagnosed with mild disease and not treated. Four of the seven patients receiving pleconaril were also treated with IVIG. Of the patients receiving pleconaril, the two with the most severe cardiomyopathy and DIC did not survive as opposed to the two other infants with cardiomyopathy who did survive. Thus, pleconaril may decrease viral load and enhance survival, but it cannot reverse the affects of extensive tissue damage. These two case studies suggest that early diagnosis along with early intervention is probably the answer to successful use of pleconaril in this patient population. In addition, clinical trials should be done to evaluate the use of pleconaril in this type of population. 5.1.4. Echoviruses 5.1.4.1. Diseases Echoviruses (enteric cytopathogenic human orphan viruses) are a group of enteroviruses that include 31 serotypes (1-9, 11-27, 29-33) [2]. They are significant cause of aseptic meningitis (ECHO 3, 4, 6, 7, 9, 11, 14, 16, 30, 31), especially chronic meningoencephalitis [78]. They are also associated with maculopapular exanthema (“rubelliform”, ECHO 9; “Boston exanthem”, ECHO 16), upper respiratory tract disease (ECHO 11, 20) and can cause neonatal carditis (ECHO, 9,22), encephalitis (ECHO 11), and hepatitis (ECHO 11). 5.1.4.2. Experimental Therapies In comparison to rhinoviruses and coxsackieviruses, very few antiviral agents are being developed for echovirus infections. However, a few compounds have been evaluated in vitro for efficacy against echoviruses. 5.1.4.2.1. Synthetic Compounds The antiviral properties (non-virucidal) of betulin, a lupane triterpene, and some betulin derivatives were tested for efficacy against echovirus 6 [111]. Betulinic acid potently inhibited virus replication (EC50 = 0.007 µM), which compared favorably with pleconaril. The compound was also not very cytotoxic with selective index value of >4100 µM. Some new 3-methylthio-5-aryl-4-isothiazolecarbonitriles were also synthesized that had broad-spectrum antiviral activity [74]. Isothiazoles with longer alkyl chains were found to inhibit ECHO 9 virus. Thus, four compounds in that series were found to inhibit echovirus with IC50 values from 0.20.5 µM. All four were generally not cytotoxic at the concentrations tested (CC50>20 µM). 3-Methylthio-5-[4-3-phen-


oxy-1-propoxy)phenyl]- 4-isothiazolecarbonitrile was the most specific inhibitor of ECHO 9, while the other three compounds also potently inhibited polio, coxsackie and/or rhinoviruses. Pirodavir was one of the most promising capsid-binding compounds to show efficacy in human clinical trials for chemoprophylaxis of the common cold. Because pirodavir was susceptible to hydrolysis when used as an oral agent, new agents have needed to be synthesized that are orally bioavailable and not as susceptible to hydrolysis. Compounds such as pyridazinyl oxime ethers have been shown to be as potent as pirodavir [112]. Benzaldehyde 4-[2-[1-(6chloro - 3 - pyridazinyl) - 4 - piperidinyl]ethoxy] - O - ethyloxime (BTA39), and the ethyl oxime ether pyridazinamine, and benzaldehyde 4 - [2 - [1 - (6 - methyl - 3 - pyridazinyl) - 4 - piperidi nyl]ethoxy]-O-ethyloxime (BTA188) inhibited many viruses in the Picornaviridae, including echoviruses (IC50 = 193 to 5,155 nM). Both compounds were relatively nontoxic in actively growing cells (50% cytotoxic doses, ≥ 4,588 nM). These data suggest that these oxime ethers warrant further investigation as potential agents for treating selected PV infections. 5.1.4.2.2. Natural Products Shuanghuanglian and garlic have been evaluated for inhibition of ECHO 11 virus [101. At 2.5 µg/ml, the Shuanghuanglian reduced plaques by 81%, although the compound was also very toxic. Garlic also inhibited echovirus replication, but was very toxic as well. Lactoferrin, an iron-binding glycoprotein in milk with natural antiviral properties, was also inhibitory to echovirus replication [113]. ECHO 6 virusinduced apoptosis was inhibited by bovine lactoferrin. 5.1.4.2. Clinical Studies Pleconaril has been used on a compassionate use basis to treat echovirus infections [114]. An echovirus 19 infection in a transplant patient with flaccid paralysis was treated with pleconaril and intravenous immunoglobulin. Recovery and improvement of laboratory evidence correlated with onset of treatment. 5.1.4. Enteroviruses 5.1.5.1. Diseases Enteroviruses (EV) 68-71 of the Enterovirus genus are distinct viruses from the other enteroviruses [78]. These serotypes seem to be associated with a polio-like illness (EV71); aseptic meningitis (EV70, 71); hand, foot, and mouth disease (EV71) and epidemic conjunctivitis (EV70). EV71 consists of three genotypes designated as A, B and C. In 1998 in Taiwan, there was an outbreak of EV71 infections manifested as by hand, foot and mouth disease, aseptic meningitis/encephalitis, and flaccid paralysis in which 80 children died [115]. This emphasizes the severity of the disease sequelae from an EV71 infection. 5.1.5.2. Experimental Therapies A limited number few antiviral agents are being developed for treating enterovirus 68-71 infections and most of these have been evaluated against EV71 because of its ability causes flaccid paralysis.


5.1.5.1.1. Synthetic Compounds Using the basic structure of the WIN compounds, computer-assisted design led to the synthesis of a number of pyridyl imidazolidinone analogs with significant anti-EV71 activity [80]. 1-[5-biphenyl-4-yloxy)pentyl]-3-pyridin-4-ylimidazolidin-2-one and its hydrochloride salt were the potent and least toxic of these compounds. The IC50 value for this compound was 0.06 µM and the CC50 value was >25 µM. 1[5 - (4-Bromophenoxy)pentyl- 3 -pyridin- 4 -yl-imidazolidin - 2 one, designated as BPR0Z-194, also inhibited all genotypes of EV71 and was shown to target the hydrophobic pocket of the VP1 capsid protein [9]. Another class of compounds, pyrazolo[3,4-d]pyrimidines, was made to inhibit enteroviruses [81]. The first compound, from the initial synthesis was found to inhibit EV68, all genotypes of EV71 and CVB3. Subsequent compounds were synthesized that were somewhat more potent inhibitors of EV71 (genotype B), especially those with a thiophene group. EC50 values for the EV71 active compounds ranged from 0.32-0.6 µM. The compounds were not toxic at concentrations up to and including 25 µM. Pyridazinyl oxime ethers were also potent inhibitors of enterovirus 71. In a neutral red uptake assay, BTA39 significantly inhibited enterovirus 71 replication with an IC50 value of 1 nM and an IC90 value of 2 nM. BTA188 inhibited the virus with an IC50 and IC90 of 82 and 109 nM, respectively [112]. Pirodavir was a much less effective inhibitor of the virus, with an IC50 of 5,420 nM and an IC90 of >13,350 nM. RNA interference (RNAi) is a sequence-specific, posttranscriptional process of mRNA degradation induced by small interfering RNA molecules. In one study, RNAi strategy was exploited to treat the EV71 infection [116]. Short hairpin RNA (shRNA) expression plasmids were constructed that significantly inhibited viral protein expression in a sequence-specific and dose-dependent fashion after transient transfection in cell cultures. Stable expression of shRNAs in cultured cells exhibited marked viral resistance in every step assessed in the viral replication. Using cytotoxicity of shRNA-expressing cells as a surrogate marker, it was shown that replication of EV71 was specifically attenuated by these plasmid-derived shRNAs, while replication of other related enteroviruses examined was not. These studies demonstrated the feasibility of this approach for the therapy of EV71induced disease. 5.1.5.1.1. Natural Products Allophycocyanin isolated from a blue-green alga Spirulina platensis inhibited apoptosis induced by EV71 [9]. In a plaque reduction assay, the IC50 value was equal to 0.1 µM. Most of this inhibitory activity that was due to a virucidal effect and the fraction of the inhibition that was due to binding to the capsid to prevent adsorption or uncoating was not determined in this study. However, the partially purified allophycocyanin was also very cytotoxic (CC50 = 1.5-1.6 µM). The toxicity of the compound does not bode well for future development of the compound. Lactoferrin was also inhibitory to echovirus replication [117]. EV71 virus-replication was inhibited by both human

10


and bovine lactoferrin, although the bovine version was a 510 fold more potent (IC50 = 10.5-24.5 µg/ml). The extent of this inhibitory activity attributable to a virucidal effect or due to binding to the capsid to prevent adsorption or uncoating was not determined in this study. 5.1.5.1 Clinical Studies Brain-stem encephalitis and pulmonary edema are severe complications of EV71 that can lead to death. A study was designed to evaluate the potential therapeutic effect of milrinone, a phosphodiesterase (PDE) inhibitor, in the treatment of patients with EV71-induced pulmonary edema [118]. A historically controlled trial of 24 children (≤ 5 yrs old) with severe EV71-induced pulmonary edema from April 1998June 2003 in southern Taiwan was done. Patients were divided into groups treated before and after the introduction of milrinone therapy. All 24 patients who were positive by viral culture and confirmed by immunofluorescence and neutralization tests were below 5 years of age. The mortality was lower in the milrinone-treated vs. nontreated group (36.4% vs. 92.3%). Sympathetic tachycardia was decreased. There was a significant decrease in IL-13 in milrinone-treated patients compared to controls and in white blood cell and platelet counts compared to controls. These results were associated with a general improvement in sympathetic regulation and recovery from disease. Thus, milrinone therapy may provide a useful therapeutic approach for treating lifethreatening EV71 infections. ABBREVIATION HRV = Human rhinovirus EV

= Enterovirus

Dale L. Barnard

[10] [11] [12] [13] [14]

[15] [16] [17] [18]

[19] [20] [21] [22] [23] [24]

CVA = Coxsackievirus A CVB = Coxsackievirus B IC50 = 50% inhibitory concentration EC50 = 50% effective concentration

[25] [26] [27]

CC50 = 50% cell cytotoxic concentration SI

= Selective index

REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9]

Bryant PA, Tingay D, Dargaville PA, Starr M, Curtis N. Neonatal coxsackie B virus infection-a treatable disease? Eur J Pediatr 2004; 163: 223-8. Ruekert RR. In: Knipe DM, Howley PM Eds, Fields Virology. Philadelphia, Lippinscott Williams & Wilkins; 685-722. Carrasco L. Picornavirus inhibitors. Pharmacol Ther 1994; 64: 21590. Diana GD, Pevear DC. Antipicornavirus drugs: current status. Antivir Chem Chemother 1997; 8: 401-8. McKinlay MA. Recent advances in the treatment of rhinovirus infections. Curr Opin Pharmacol 2001; 1: 477-81. Wang QM. Protease inhibitors as potential antiviral agents for the treatment of picornaviral infections. Prog Drug Res 2001; Spec No: 229-53. Turner RB. The treatment of rhinovirus infections: progress and potential. Antiviral Res 2001; 49: 1-14. Rotbart HA. Treatment of picornavirus infections. Antiviral Res 2002; 53: 83-98. Shih SR, Chen SJ, Hakimelahi GH, Liu HJ, Tseng CT, Shia KS. Selective human enterovirus and rhinovirus inhibitors: An over-

[28] [29] [30] [31] [32] [33]

[34]

[35]

view of capsid-binding and protease-inhibiting molecules. Med Res Rev 2004; 24: 449-74. Charles CH, Yelmene M, Luo GX. Recent advances in rhinovirus therapeutics. Curr Drug Targets Infect Disord 2004; 4: 331-7. Turner RB. New considerations in the treatment and prevention of rhinovirus infections. Pediatr Ann 2005; 34: 53-7. Heinz BA, Vance LM. The antiviral compound enviroxime targets the 3A coding region of rhinovirus and poliovirus. J Virol 1995; 69: 4189-97. Hellen CUT, Wimmer E. In: Rotbart HA Ed, Human enterovirus infections. Washington DC, ASM Press 1995; 25-72. Hayden FG, Turner RB, Gwaltney JM, Chi-Burris K, Gersten M, Hsyu P, et al. Phase II, randomized, double-blind, placebocontrolled studies of ruprintrivir nasal spray 2-percent suspension for prevention and treatment of experimentally induced rhinovirus colds in healthy volunteers. Antimicrob Agents Chemother 2003; 47: 3907-16. Arruda E, Pitkaranta A, Witek TJ Jr, Doyle CA, Hayden FG. Frequency and natural history of rhinovirus infections in adults during autumn. J Clin Microbiol 1997; 35: 2864-8. Makela MJ, Puhakka T, Ruuskanen O, Leinonen M, Saikku P, Kimpimaki M, et al. Viruses and bacteria in the etiology of the common cold. J Clin Microbiol 1998; 36: 539-42. Hayden FG. Rhinovirus and the lower respiratory tract. Rev Med Virol 2004; 14: 17-31. Nokso-Koivisto J, Raty R, Blomqvist S, Kleemola M, Syrjanen R, Pitkaranta A, et al . Presence of specific viruses in the middle ear fluids and respiratory secretions of young children with acute otitis media. J Med Virol 2004; 72: 241-8. Tan WC. Viruses in asthma exacerbations. Curr Opin Pulm Med 2005; 11: 21-6. Gern JE. Viral respiratory infection and the link to asthma. Pediatr Infect Dis J 2004; 23(1 Suppl): S78-86. Gern JE. Mechanisms of virus-induced asthma. J Pediatr 2003; 142(2 Suppl): S9-13. Bianco A, Mazzarella G, Bresciani M, Paciocco G, Spiteri MA. Virus-induced asthma. Monaldi Arch Chest Dis 2002; 57: 188-90. Osur SL. Viral respiratory infections in association with asthma and sinusitis: a review. Ann Allergy Asthma Immunol 2002; 89: 553-60. Seemungal TA, Wedzicha JA. Viral infections in obstructive airway diseases. Curr Opin Pulm Med 2003; 9: 111-6. Greenberg SB. Respiratory viral infections in adults. Curr Opin Pulm Med 2002; 8: 201-8. Widdicombe J, Kamath S. Acute cough in the elderly: aetiology, diagnosis and therapy. Drugs Aging 2004; 21: 243-58. Korppi M, Kotaniemi-Syrjanen A, Waris M, Vainionpaa R, Reijonen TM. Rhinovirus-associated wheezing in infancy: comparison with respiratory syncytial virus bronchiolitis. Pediatr Infect Dis J 2004; 23: 995-9. Magden J, Kaariainen L, Ahola T. Inhibitors of virus replication: recent developments and prospects. Appl Microbiol Biotechnol 2005; 66: 612-21. Weinberger M. Respiratory infections and asthma: current treatment strategies. Drug Discov Today 2004; 9: 831-7. Anzueto A, Niederman MS. Diagnosis and treatment of rhinovirus respiratory infections. Chest 2003; 123: 1664-72. Fendrick AM. Viral respiratory infections due to rhinoviruses: current knowledge, new developments. Am J Ther 2003; 10: 193202. Turner RB. The treatment of rhinovirus infections: progress and potential. Antiviral Res 2001; 49: 1-14. Chen SH, Lamar J, Victor F, Snyder N, Johnson R, Heinz BA, Wakulchik M, et al. Synthesis and evaluation of tripeptidyl alphaketoamides as human rhinovirus 3C protease inhibitors. Bioorg Med Chem Lett 2003; 13: 3531-6. Dragovich PS, Prins TJ, Zhou R, Brown EL, Maldonado FC, Fuhrman SA, et al. Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 6. Structure-activity studies of orally bioavailable, 2-pyridonecontaining peptidomimetics. J Med Chem 2002; 45: 1607-23. Dragovich PS, Prins TJ, Zhou R, Johnson TO, Hua Y, Luu HT, et al. Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 8. Pharmacological optimization of orally bioavailable 2-pyridone-containing peptidomimetics. J Med Chem 2003; 46: 4572-8

Current Status of Anti-Picornavirus Therapies [36] [37] [38] [39] [40]

[41]

[42] [43]

[44] [45] [46]

[47] [48]

[49] [50] [51]

[52] [53] [54] [55] [56] [57]

[58]

Deszcz L, Seipelt J, Vassilieva E, Roetzer A, Kuechler E. Antiviral activity of caspase inhibitors: effect on picornaviral 2A proteinase. FEBS Lett 2004; 560: 51-5. Deszcz L, Gaudernak E, Kuechler E, Seipelt J. Apoptotic events induced by human rhinovirus infection. J Gen Virol 2005; 86(Pt 5): 1379-89. Hayden FG, Hipskind GJ, Woerner DH, Eisen GF, Janssens M, Janssen PA, Andries K. Intranasal pirodavir (R77,975) treatment of rhinovirus colds. Antimicrob Agents Chemother 1995; 39: 290-4. Andries K. In: Adams J, Merluzzi VJ Eds, The search for antiviral drugs. Boston, Birkhauser 1993; 179-209. Brown RN, Cameron R, Chalmers DK, Hamilton S, Luttick A, Krippner GY, McConnell DB, et al. 2-Ethoxybenzoxazole as a bioisosteric replacement of an ethyl benzoate group in a human rhinovirus (HRV) capsid binder. Bioorg Med Chem Lett 2005; 15: 2051-5. Hamdouchi C, Sanchez-Martinez C, Gruber J, Del Prado M, Lopez J, Rubio A, Heinz BA. Imidazo[1,2-b]pyridazines, novel nucleus with potent and broad spectrum activity against human picornaviruses: design, synthesis, and biological evaluation. J Med Chem 2003; 46: 4333-41. Hudson JB. Antiviral compounds from plants. Boca Raton, CRC Press 1990; 119-32. Salvati AL, De Dominicis A, Tait S, Canitano A, Lahm A, Fiore L. Related Articles, Mechanism of action at the molecular level of the antiviral drug 3(2H)-isoflavene against type 2 poliovirus. Antimicrob Agents Chemother 2004; 48: 2233-43. Tisdale M, Selway JW. Effect of dichloroflavan (BW683C) on the stability and uncoating of rhinovirus type 1B. J Antimicrob Chemother 1984; 14 Suppl A: 97-105. Desideri N, Mastromarino P, Conti C. Synthesis and evaluation of antirhinovirus activity of 3-hydroxy and 3-methoxy 2styrylchromones. Antivir Chem Chemother 2003; 14: 195-203. Dimova S, Mugabowindekwe R, Willems T, Brewster ME, Noppe M, Ludwig A, et al. Safety-assessment of 3-methoxyquercetin as an antirhinoviral compound for nasal application: effect on ciliary beat frequency. Int J Pharm 2003; 263: 95-103. Tan FL, Yin JQ. RNAi, a new therapeutic strategy against viral infection. Cell Res 2004; 14: 460-6. Phipps KM, Martinez A, Lu J, Heinz BA, Zhao G. Small interfering RNA molecules as potential anti-human rhinovirus agents: in vitro potency, specificity, and mechanism. Antiviral Res 2004; 61: 49-55. Poritz MA, Malmstrom S, Schmitt A, Kim MK, Zharkikh L, Kamb A, Teng DH. Isolation of a peptide inhibitor of human rhinovirus. Virology 2003; 313: 170-83. Fang F, Yu M. Viral receptor blockage by multivalent recombinant antibody fusion proteins: inhibiting human rhinovirus (HRV) infection with CFY196. J Antimicrob Chemother 2004; 53: 23-5. Charles CH, Luo GX, Kohlstaedt LA, Morantte IG, Gorfain E, Cao L, et al. Prevention of human rhinovirus infection by multivalent Fab molecules directed against ICAM-1. Antimicrobial Agents and Chemotherapy 2003; 47: 1503-1508. Proud D, Sanders SP, Wiehler S. Human rhinovirus infection induces airway epithelial cell production of human beta-defensin 2 both in vitro and in vivo. J Immunol 2004; 172: 4637-45. Gropp R, Frye M, Wagner TO, Bargon J. Epithelial defensins impair adenoviral infection: implication for adenovirus-mediated gene therapy. Hum Gene Ther 1999; 10: 957-64. Wu Z, Cocchi F, Gentles D, Ericksen B, Lubkowski J, Devico A, et al. Human neutrophil alpha-defensin 4 inhibits HIV-1 infection in vitro. FEBS Lett 2005; 579: 162-6. van Wetering S, Sterk PJ, Rabe KF, Hiemstra PS. Defensins: key players or bystanders in infection, injury, and repair in the lung? J Allergy Clin Immunol 1999; 104: 1131-8. Sperber SJ, Shah LP, Gilbert RD, Ritchey TW, Monto AS. Echinacea purpurea for prevention of experimental rhinovirus colds. Clin Infect Dis 2004; 38: 1367-7 Goel V, Lovlin R, Barton R, Lyon MR, Bauer R, Lee TD, Basu. TK. Efficacy of a standardized echinacea preparation (Echinilin) for the treatment of the common cold: a randomized, double-blind, placebo-controlled trial. J Clin Pharm Ther 2004; 29: 75-83. ClinicalTrials.gov Principle investigator: Turner RB. Evaluation of echinacea versus for the common cold. Phase II. http://clinicaltrials.gov.

Current Pharmaceutical Design, 2006, Vol. 12, No. 00 11 [59] [60] [61] [62] [63]

[64]

[65] [66] [67]

[68] [69] [70] [71] [72] [73]

[74] [75] [76] [77] [78] [79] [80]

[81]

[82]

[83]

ClinicalTrials.gov Principle investigator: Barrett B. Echinacea versus placebo effect in common cold. Phase II. http://clinicaltrials.gov. Turner RB, Fowler SL, Berg K. Treatment of the common cold with troxerutin. APMIS 2004; 112: 605-11. Hulisz D. Efficacy of zinc against common cold viruses: an overview. J Am Pharm Assoc (Wash DC) 2004; 44: 594-603. Caruso TJ, Gwaltney JM Jr. Treatment of the common cold with echinacea: a structured review. Clin Infect Dis 2005; 40: 807-10. Patick AK, Binford SL, Brothers MA, Jackson RL, Ford CE, Diem MD, et al. In vitro antiviral activity of AG7088, a potent inhibitor of human rhinovirus 3C protease. Antimicrob Agents Chemother 1999; 43: 2444-50. Rehn D, Nocker W, Diebschlag W, Golden G. Time course of the anti-oedematous effect of different dose regimens of O-(betahydroxyethyl) rutosides in healthy volunteers. Arzneimittelforschung 1993; 43: 335-8. Renton S, Leon M, Belcaro G, Nicolaides AN. The effect of hydroxyethylrutosides on capillary filtration in moderate venous hypertension: a double blind study. Int Angiol 1994; 13: 259-62. Proud D. Nitric oxide and the common cold. Curr Opin Allergy Clin Immunol 2005; 5: 37-42. Sanders SP, Siekierski ES, Richards SM, Porter JD, Imani F, Proud D. Rhinovirus infection induces expression of type 2 nitric oxide synthase in human respiratory epithelial cells in vitro and in vivo. J Allergy Clin Immunol 2001; 107:235–243. Sanders SP, Proud D, Permutt S, Siekierski ES, Yachechko R, Liu MC. Role of nasal nitric oxide in the resolution of experimental rhinovirus infection. J Allergy Clin Immunol 2004; 113: 697–702. Martin Enserink. Wanted: Drug for a Disappearing Disease. Science 2004; 303: 1971. Nikolaeva L, Galabov AS. Synergistic inhibitory effect of enviroxime and disoxaril on poliovirus type 1 replication. Acta Virol 1995; 39: 235-41. Nikolaeva L, Galabov AS. Cytotoxicity of the synergistic antienteroviral combination of enviroxime and disoxaril. Acta Virol 1999; 43: 263-5. Gitlin L, Stone JK, Andino R. Poliovirus escape from RNA interference: short interfering RNA-target recognition and implications for therapeutic approaches. J Virol 2005; 79: 1027-35. Botelho MV, Orlandi JM, de Melo FL, Mantovani MS, Linhares RE, Nozawa C. Chlorophyllin protects HEp-2 cells from nuclear fragmentation induced by poliovirus. Lett Appl Microbiol 2004; 39: 174-7. Semple SJ, Pyke SM, Reynolds GD, Flower RL. In vitro antiviral activity of the anthraquinone chrysophanic acid against poliovirus. Antiviral Res 2001; 49: 169-78. Robin V, Irurzun A, Amoros M, Boustie J, Carrasco L. Antipoliovirus flavonoids from Psiadia dentata. Antivir Chem Chemother 2001; 12: 283-91. Lohézic-Le Devehat F, Bakhtiar A, Bezivin C, Amoros M, Boustie J. Antiviral and cytotoxic activities of some Indonesian plants. Fitoterapia 2002; 73: 400-5. Fortin H, Vigor C, Lohezic-Le Devehat F, Robin V, Le Bosse B, Boustie J, et al . In vitro antiviral activity of thirty-six plants from La Reunion Island. Fitoterapia 2002; 73: 346-50. Minor, PD, Morgan-Capner P, Muir P. In: Zuckerman AJ, Banatvala JE, Pattison JR Eds, Principles and practice of clinical virology. New York, Wiley &Sons 2000; 427-49. Lee YS, Kim BH. Heterocyclic nucleoside analogues: design and synthesis of antiviral, modified nucleosides containing isoxazole heterocycles. Bioorg Med Chem Lett 2002; 12: 1395-7. Shia KS, Li WT, Chang CM, Hsu MC, Chern JH, Leong MK, et al. Design, synthesis, and structure-activity relationship of pyridyl imidazolidinones: a novel class of potent and selective human enterovirus 71 inhibitors. J Med Chem 2002; 45: 1644-55. Chern JH, Shia KS, Hsu TA, Tai CL, Lee CC, Lee YC, et al . Design, synthesis, and structure-activity relationships of pyrazolo[3,4d]pyrimidines: a novel class of potent enterovirus inhibitors. Bioorg Med Chem Lett 2004; 14: 2519-25. Makarov VA, Riabova OB, Granik VG, Wutzler P, Schmidtke M. Novel [(biphenyloxy)propyl]isoxazole derivatives for inhibition of human rhinovirus 2 and coxsackievirus B3 replication. J Antimicrob Chemother 2005; 55: 483-8. Barral K, Courcambeck J, Pepe G, Balzarini J, Neyts J, De Clercq E, et al. Synthesis and antiviral evaluation of cis-substituted cyclo-

12


[84]

[85]

[86]

[87]

[88] [89]

[90] [91]

[92] [93]

[94] [95] [96]

[97]

[98] [99]

[100]

hexenyl and cyclohexanyl nucleosides. J Med Chem 2005; 48: 4506. Cutrí CC, Garozzo A, Siracusa MA, Castro A, Tempera G, Sarva MC, et al. Synthesis of new 3-methylthio-5-aryl-4-isothiazolecarbonitriles with broad antiviral spectrum. Antiviral Res 2002; 55: 357-68. Espeya MG, Miranda KM, Thomas DD, Xavier S, Citrin D, Vitek MP, et al. A chemical perspective on the interplay between NO, reactive oxygen species, and reactive nitrogen oxide species. Annals New York Acad Sci 2002; 962: 195-206. Padalko E, Ohnishi T, Matsushita K, Sun H, Fox-Talbot K, Bao C, et al. Peroxynitrite inhibition of Coxsackievirus infection by prevention of viral RNA entry. Proc Natl Acad Sci U S A 2004; 101: 11731-6. Qi X, Li X, Liu M, et al. Study on the inhibitory effect of antisense phosphorothioate oligodeoxynucleotide on coxsackie virus B replication in vitro. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi 2000; 14: 253-6. Sun H, Liu Z, Zhang T. Antisense oligonucleotides resistance to coxsackievirus B3 infection in HeLa cells. Zhonghua Liu Xing Bing Xue Za Zhi 2000; 21: 295-7. Yuan J, Cheung PK, Zhang H, Chau D, Yanagawa B, Cheung C, et al. A phosphorothioate antisense oligodeoxynucleotide specifically inhibits coxsackievirus B3 replication in cardiomyocytes and mouse hearts. Lab Invest 2004; 84: 703-14. Nikolaeva-Glomb L, Galabov AS. Synergistic drug combinations against the in vitro replication of Coxsackie B1 virus. Antiviral Res 2004; 62: 9-19. Cermelli C, Vinceti M, Scaltriti E, Bazzani E, Beretti F, Vivoli G, et al . Selenite inhibition of Coxsackie virus B5 replication: implications on the etiology of Keshan disease. J Trace Elem Med Biol 2002; 16: 41-6. Yang GQ, Chen JS, Wen ZM, Ge KY, Zhu LZ, Chen XC, et al. The role of selenium in Keshan disease. Adv Nutr Res 1984; 6: 203-31. Xu GL, Wang SC, Gu BQ, Yang YX, Song HB, Xue WL, et al . Further investigation on the role of selenium deficiency in the aetiology and pathogenesis of Keshan disease. Biomed Environ Sci 1997; 10: 316-26. Beck MA, Levander OA, Handy J. Selenium deficiency and viral infection. J Nutr 2003; 133 (Suppl 1): 1463S-7S. Guanqing H. On the etiology of Keshan disease: two hypotheses. Chin Med J (Engl) 1979; 92: 416-22. Levander OA, Beck MA. Interacting nutritional and infectious etiologies of Keshan disease. Insights from coxsackie virus Binduced myocarditis in mice deficient in selenium or vitamin E. Biol Trace Elem Res 1997; 56: 5-21. Beck MA, Shi Q, Morris VC, Levander OA. Rapid genomic evolution of a non-virulent coxsackievirus B3 in selenium-deficient mice results in selection of identical virulent isolates. Nat Med 1995; 1: 433-6. Beck MA, Esworthy RS, Ho YS, Chu FF. Glutathione peroxidase protects mice from viral-induced myocarditis. FASEB J 1998; 12: 1143-9. Yamai M, Tsumura K, Kimura M, Fukuda S, Murakami T, Kimura Y. Antiviral activity of a hot water extract of black soybean against a human respiratory illness virus. Biosci Biotechnol Biochem 2003; 67: 1071-9. Wang ZJ, Yang ZQ, Huang TN, Wen L, Liu YW. Experimental research on inhibitory effect of alcohol extracts from Loranthus yadoriki Sieb. on coxsackie B3 virus. Zhongguo Zhong Yao Za Zhi 2000; 25: 685-7.

Dale L. Barnard [101] [102] [103] [104] [105] [106]

[107] [108]

[109]

[110] [111]

[112]

[113]

[114]

[115] [116] [117] [118]

Luo R, Dong Y, Fang F. The experimental study of the antienterovirus effects of drugs in vitro. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi 2001; 15: 135-8. Li ZH, Nie BM, Chen H, Chen SY, He P, Lu Y, et al. In vitro anticoxsackievirus B(3) effect of ethyl acetate extract of Tian-hua-fen. World J Gastroenterol 2004; 10: 2263-6. Li Z, Dong G, Li Z. Experimental study of the virus inhibitory effect of Chuan-Kang-Ping Granule. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi 2001; 15: 83-5. He Y, Wu C, Zhao G. Experimental study on inhibitory effect of ganlu xiaodu Dan on coxackie virus in vitro. Zhongguo Zhong Xi Yi Jie He Za Zhi 1998; 18: 737-40. Padalko E, Verbeken E, Matthys P, Aerts JL, De Clercq E, Neyts J. Mycophenolate mofetil inhibits the development of Coxsackie B3virus-induced myocarditis in mice. BMC Microbiol 2003; 3: 25. Padalko E, Nuyens D, De Palma A, Verbeken E, Aerts JL, De Clercq E, et al. The interferon inducer ampligen [poly(I)poly(C12U)] markedly protects mice against coxsackie B3 virusinduced myocarditis. Antimicrob Agents Chemother 2004; 48: 26774. Padalko E, Verbeken E, De Clercq E, Neyts J. Inhibition of coxsackie B3 virus induced myocarditis in mice by 2-(3,4dichlorophenoxy)-5-nitrobenzonitrile. J Med Virol 2004; 72: 263-7. Zell R, Markgraf R, Schmidtke M, Gorlach M, Stelzner A, Henke A, et al. Nitric oxide donors inhibit the coxsackievirus B3 proteinases 2A and 3C in vitro, virus production in cells, and signs of myocarditis in virus-infected mice. Med Microbiol Immunol (Berl) 2004; 193: 91-100. Kytö V, Saraste A, Fohlman J, Ilback NG, Harvala H, Vuorinen T, et al. Cardiomyocyte apoptosis after antiviral WIN 54954 treatment in murine coxsackievirus B3 myocarditis. Scand Cardiovasc J 2002; 36: 187-92. Bauer S, Gottesman G, Sirota L, Litmanovitz I, Ashkenazi S, Levi I. Severe Coxsackie virus B infection in preterm newborns treated with pleconaril. Eur J Pediatr 2002; 161: 491-3. Pavlova NI, Savinova OV, Nikolaeva SN, Boreko EI, Flekhter OB. Antiviral activity of betulin, betulinic and betulonic acids against some enveloped and non-enveloped viruses. Fitoterapia 2003; 74: 489-92. Barnard DL, Hubbard VD, Smee DF, Sidwell RW, Watson KG, Tucker SP, et al. In vitro activity of expanded-spectrum pyridazinyl oxime ethers related to pirodavir: novel capsid-binding inhibitors with potent antipicornavirus activity. Antimicrob Agents Chemother 2004; 48: 1766-72. Tinari A, Pietrantoni A, Ammendolia MG, Valenti P, Superti F. Inhibitory activity of bovine lactoferrin against echovirus induced programmed cell death in vitro. Int J Antimicrob Agents 2005; 25: 433-8. Starlin R, Reed N, Leeman B, Black J, Trulock E, Mundy LM. Acute flaccid paralysis syndrome associated with echovirus 19, managed with pleconaril and intravenous immunoglobulin. Clin Infect Dis 2001; 33: 730-2. Ho M, Chen ER, Hsu KH, Twu SJ, Chen KT, Tsai SF, et al. An epidemic of enterovirus 71 infection in Taiwan. Taiwan Enterovirus Epidemic Working Group. N Engl J Med 1999; 341: 929-35. Lu WW, Hsu YY, Yang JY, Kung SH. Selective inhibition of enterovirus 71 replication by short hairpin RNAs. Biochem Biophys Res Commun 2004; 325: 494-9. Lin TY, Chu C, Chiu CH. Lactoferrin inhibits enterovirus 71 infection of human embryonal rhabdomyosarcoma cells in vitro. J Infect Dis 2002; 186: 1161-4. Wang SM, Lei HY, Huang MC, Wu JM, Chen CT, Wang JN, et al. Therapeutic efficacy of milrinone in the management of enterovirus 71-induced pulmonary edema. Pediatr Pulmonol 2005; 39: 219-23.