key: cord-0010763-nwp2if8d
authors: Hayden, Frederick G.; Herrington, Darrell T.; Coats, Teresa L.; Kim, Kenneth; Cooper, Ellen C.; Villano, Stephen A.; Liu, Siyu; Hudson, Spencer; Pevear, Daniel C.; Collett, Marc; McKinlay, Mark
title: Efficacy and Safety of Oral Pleconaril for Treatment of Colds Due to Picornaviruses in Adults: Results of 2 Double-Blind, Randomized, Placebo-Controlled Trials
date: 2003-06-15
journal: Clin Infect Dis
DOI: 10.1086/375069
sha: 5b45ceac6f9e015319c237a01577f46ad9f72a80
doc_id: 10763
cord_uid: nwp2if8d

The novel capsid-binding antiviral pleconaril inhibits in vitro replication of most rhinoviruses and enteroviruses. Oral pleconaril treatment was studied in 2 parallel randomized, double-blind, placebo-controlled trials. Among 1363 picornavirus-infected participants (65%) in the studies combined, the median time to alleviation of illness was 1 day shorter for pleconaril recipients than for placebo recipients (P > .001). Cold symptom scores and frequency of picornavirus cultured from nasal mucus specimens were lower among pleconaril recipients by day 2 of treatment. No treatment effects were seen in those without picornavirus infection. Pleconaril was associated with a higher incidence of nausea (6% vs. 4%) and diarrhea (9% vs. 7%) and with small increases in mean serum cholesterol levels and platelet counts, compared with baseline measurements. A subsequent 6-week prophylaxis study found that pleconaril induces cytochrome P-450 3A enzymes, which metabolize a variety of drugs, including ethinyl estradiol. Early pleconaril treatment was well tolerated and significantly reduced the duration and severity of colds due to picornaviruses in adults.

gether cause ∼50% of colds annually [1] . The incidence of colds due to picornaviruses increases to 60%-80% during peak months in the fall and spring in the Northern Hemisphere [1, 2] . No antiviral therapy of proven value for colds due to picornaviruses is currently available, and prior studies of investigational antivirals did not show therapeutic benefit for established colds [3, 4] . The pathogenesis of cold symptoms is not fully understood [5] , and the importance of ongoing viral replication to symptom causation remains uncertain.

Pleconaril is a novel, orally absorbed viral capsidfunction inhibitor that specifically inhibits the replication of ∼90% of rhinoviruses and 199% of enteroviruses [6] [7] [8] . In experimentally induced human coxsackievirus A21 infections, oral pleconaril significantly reduced viral shedding and illness measures [9] . More recently, retrospective analysis of 2 phase II randomized, double-blind, placebo-controlled studies found that pleconaril treatment provided clinical benefit for colds due to picornaviruses in previously healthy adults [10] . Consequently, 2 large, randomized, double-blind, placebo-controlled, multicenter trials were conducted to evaluate the efficacy and safety of oral pleconaril for treatment of naturally occurring colds presumptively due to picornaviruses in adults.

Participants in both studies were otherwise healthy adults (age, р18 years) with self-diagnosed colds who were enrolled within 24 h after symptom onset. Participants had moderate to severe rhinorrhea and у1 other respiratory symptom (nasal congestion, cough, or sore throat) that was rated moderate or greater in severity.

Subjects were excluded if they had fever (oral temperature, 137.8ЊC), if they had allergic rhinitis that had been treated within the previous 2 weeks, if they had received asthma treatment within the previous 2 months, or if they had chronic cough, any known immunodeficiency, or an underlying medical condition that would confound the study results. Pregnant or nursing women were excluded, and urine pregnancy tests were done at entry. Smokers were allowed. The institutional review board of each participating site approved the protocol, and written informed consent was obtained from each participant at the time of enrollment into the study. Participants were compensated for participation.

Study design and drug administration. Two prospective, multicenter, randomized, double-blind trials of identical design were conducted from August through November 2000; each enrolled participants from geographically diverse areas of the United States (150 sites) and Canada (47 sites). Participants were randomized in a 1:1 ratio to receive either pleconaril at 400 mg (two 200-mg tablets; Picovir; ViroPharma) or matching placebo tablets 3 times per day for 5 days. To enhance oral absorption, participants were instructed to take the study medication within 15 min after a meal or snack. Randomization was stratified by the subject's preenrollment smoking status and preenrollment use of cold symptom-relief medication to ensure that these subjects were balanced between the treatment arms. Acetaminophen and dextromethorphan were provided for disabling symptoms, because these agents were unlikely to affect the prominent nasopharyngeal symptoms of colds. The concomitant use of prescription and other over-the-counter cold symptom-relief medications was not permitted.

Clinical monitoring. Participants were evaluated at enrollment and again on study days 3, 6 (end of treatment), and 18 (end of the study) for clinical assessment and obtainment of samples for laboratory testing. Study personnel contacted participants every other day by telephone until their cold had resolved or through day 18.

Participants recorded the severity of 6 individual cold symptoms (rhinorrhea, nasal congestion, sore throat, cough, malaise, and myalgia) in study diaries twice daily, grading each as "not present," "mild" (noticeable but not bothersome), "moderate" (bothersome), or "severe" (limiting usual activities), which were scored as 0, 1, 2, and 3, respectively, for data analysis. Once per day, subjects also recorded data on the number of facial tissues used, sleep disturbance, impairment of daily activity as a result of cold symptoms, and use of cold symptom-relief medications or other medications for any reason. Safety laboratory studies (hematological study, clinical chemistry, and urinalysis) and physical assessments were done at enrollment and on study day 6.

Virology assessments. Nasal mucus samples were obtained for virological studies at baseline and on study days 3 and 6. Subjects were asked to blow nasal mucus directly onto plastic wrap; mucus was induced, if necessary [11] . The sample was transferred into a tube containing viral transport medium (Starswab Multitrans Collection and Transport System; Starplex Scientific) and shipped for storage at Ϫ80ЊC until assayed.

The presence of picornavirus RNA in nasal mucus samples was identified using a real-time, quantitative RT-PCR assay (TaqMan; Applied Biosystems). The PCR primers and probe used in the TaqMan assay were derived from conserved sequences within the 5 nontranslated region of sequenced human rhinovirus (HRV) genomes. The forward primer sequence was 5 -GTGAAGAGCC(G/C)C(A/G)TGTGCT-3 , corresponding to nucleotides 414-432 of HRV89. The reverse primer sequence was 5 -GCT(G/C)CAGGGTTAAGGTTAGCC-3 , corresponding to the reverse complement of nucleotides 461-481 of HRV89. The double-labeled fluorescent probe sequence was 5 -(FAM)-TGAGTCCTCCGGCCCCTGAATG-(TAMRA)-3 , corresponding to nucleotides 438-459 of HRV89. In this assay, the lower limit of detection for the virus (HRV1B) used to generate the standard curve was 10 pfu/mL, or 10,550 genome equivalents/mL (211 genome equivalents per reaction).

If all 3 samples obtained from a patient had negative or indeterminate results for this assay, the baseline sample was retested by a modification of an enzyme-linked oligosorbent RT-PCR assay, which detects all prototype rhinoviruses and culturable enteroviruses [12] [13] [14] [15] . A patient was considered to be positive for picornavirus infection if nasal mucus specimens tested positive with either RT-PCR assay on any sampling day.

For subjects who had positive RT-PCR results, an aliquot of the baseline mucus sample (200 mL) was submitted for viral culture on monolayers of HeLa-I cells by a previously described technique [16] . If culture of the baseline sample yielded positive results, aliquots of samples obtained on days 3 and 6 were also cultured.

TaqMan assays were performed at ViroMed Biosafety Laboratories (St. Paul, Minnesota), enzyme-linked oligosorbent (3) 17 (3) 13 (2) 19 (4) Lost to follow-up 11 (2) (7) 47 (9) 31 (6) 34 (7) RT-PCR assays were performed at ViroPharma Incorporated (Exton, Pennsylvania), and viral cultures were performed at the University of Virginia (Charlottesville) or the University of Rochester Medical Center (Rochester, New York). Study personnel at each laboratory were blinded to treatment and sample collection day. Efficacy end points. The primary efficacy population included any randomized participant with у1 nasal mucus sample that tested positive for picornavirus RNA on any sampling day by either quantitative or qualitative RT-PCR methods. The secondary efficacy population included all randomized participants. These participants are referred to as the intent-to-treat infected (ITT-I) and intent-to-treat (ITT) populations, respectively.

The primary end point was the time from initiation of therapy to alleviation of illness, defined as the number of days until complete resolution of rhinorrhea and the other 5 cold symptoms self-assessed as absent or mild for у48 h without use of cold symptom-relief medication. Prospectively defined secondary end points were the time to subject-assessed "no cold," times to complete resolution of individual symptoms, total cold symptom severity scores, tissue counts, proportion of nights with disturbed sleep, duration of cold symptom-relief medication use, and frequency of viral shedding in nasal mucus. Other end points were the time to у50% reduction in symptom score and changes in viral RNA levels over time.

Data analysis. The distribution of time to resolution of symptom scores was estimated by the Kaplan-Meier method [17] , and the Wilcoxon-Gehan statistic [18] was used to test the difference in median resolution times between treatment groups. These analyses included stratification for smoking status and preenrollment use of cold symptom-relief medication. Combined analyses of both studies also included stratification by study. In these time-to-event analyses, subjects who discontinued the study were included up to the point of the last recorded observation. The distribution of time to у50% reduction from baseline in total cold symptom severity score was analyzed in the same manner.

The treatment effect for change from baseline in daily total symptom severity score and total symptom severity score over the 18-day study was analyzed by analysis of covariance, with effects for treatment, study, smoking status, preenrollment use of cold symptom-relief medication, and baseline total symptom severity score. The last observation carried forward was used to impute missing individual symptom severity scores.

Treatment effect for presence of picornavirus by culture and percentage of subjects using cold medications was evaluated using Fisher's exact test. Analysis of variance was used to compare the treatment groups for reduction in virus levels (measured by PCR) from baseline to days 3 and 6, proportion of nights with sleep disturbance, and tissue use.

All study participants who received у1 dose of study medication were included in the safety analysis. Adverse events that began or worsened at any time after receipt of the first dose of study drug through 5 days after the last dose were summarized. All analyses were done using SAS statistical software, version 6.12 (SAS Institute) [19] , and a 2-sided test at the 5% level was used for all comparisons.

Sample size.

Calculations indicated that enrollment of 1000 subjects in each study was required to detect a 25% relative difference between treatment groups in the proportion of picornavirus-infected subjects reaching the primary end point (or an estimated 2-day difference in median time) with 90% power (2-sided test at the 5% level of significance [20] ).

The 2 studies randomized 2096 participants (1046 in the pleconaril group and 1050 in the placebo group); 190% of subjects completed treatment (table 1). The (30) 107 (32) 119 (33) 120 (35) a Current smokers or those who stopped smoking р3 months before the start of study. most common reason that subjects did not complete treatment was an adverse event (3% of subjects each in the pleconaril and placebo groups). Overall, 65% of participants were infected with picornavirus, with a narrow range (62%-68%) across treatment groups in each study. The pleconaril and placebo groups were similar at baseline with regard to relevant demographic and illness characteristics (table 2). The mean age of the ITT-I population was 36 years, 69% were female, and 28% were smokers. The median time from symptom onset to receipt of the first dose of study drug was 20 h. Illness resolution. Among the picornavirus-infected (ITT-I) population, the time to reach the primary end point of illness alleviation was significantly shorter among pleconaril-treated subjects than among placebo recipients in each study (figure 1). Overall, pleconaril-treated patients experienced a 1-day reduction in the median duration of illness, compared with placebo-treated subjects ( ; table 3 ). The treatment effect P ! .001 was similar for subjects who had positive results of viral cultures at baseline (combined median time to illness alleviation, 7.9 days for the placebo group and 6.8 days for the pleconaril group;

) and for those with negative results of viral P p .002 culture at baseline (7.0 days for the placebo group and 6.0 days for the pleconaril group;

). No treatment effect was P p .048 observed among participants without detectable picornavirus infection (table 3 ). In the ITT population, the magnitude of the treatment benefit was smaller than that observed among ITT-I subjects but favored pleconaril in both studies.

Among ITT-I subjects, the self-assessed times to "no cold" and to resolution of each individual cold symptom were reduced in pleconaril recipients (table 4). The total cold symptom 

Alleviation of illness is defined as the absence of rhinorrhea and presence of no or mild other cold symptoms for у48 h without use of cold symptom-relief medication. ITT, intent-to-treat; ITT-I, intent-to-treat infected. severity score was reduced by 19% over the duration of the study for pleconaril recipients (table 4) , who also experienced significant reductions from baseline in symptom severity scores by day 2 of treatment, compared with placebo recipients (figure 2). A reduction in total symptom severity score of у50% occurred earlier among pleconaril recipients than among placebo recipients (combined medians of 2.9 and 3.9 days, respectively; ). Pleconaril treatment was associated with fewer tissues P ! .001 used for nose blowing (24% reduction), fewer nights of sleep disturbance (1 night reduction), and fewer days of cold symptom-relief medication use (table 4) .

Analyses of the combined study results were conducted with Figure 2 . Change from baseline value in total cold symptom severity scores among picornavirus-infected subjects, days 1-6. Pleconaril recipients experienced significant ( ) reductions from baseline by day 2 P ! .05 of treatment in each study. 

"Most common" is defined as incidence of у3% in either treatment group. a Total no. of subjects who experienced у1 adverse event, regardless of its relationship to use of the study drug. a Cox regression model to assess 2-way interaction effects between treatment and the variables age, sex, race, smoking status, and preenrollment cold medication use. The only interaction with significant impact on treatment effect was smoking status ( ). The time to reach the primary end point was P p .013 shorter for pleconaril recipients than for placebo recipients among nonsmokers (6.0 vs. 7.3 days;

), but it was not P ! .001 different among smokers (8.3 vs. 7.4 days; ). However, P p .692 additional analysis regarding effects of pleconaril for smokers is limited by the fact that smokers constituted only 28% of the study population.

Virologic analysis. Among subjects with detectable picornavirus RNA in baseline nasal mucus samples, 827 (65%) of 1263 subjects had positive results of viral cultures. Acid stability testing of 69 randomly selected isolates determined that 68 (99%) were acid-labile and presumed rhinoviruses, whereas 1 was acid-stable and a presumed enterovirus.

Among those who had positive results of culture at baseline, fewer pleconaril recipients had positive culture results on day 3 (range, day 2-4) than did placebo recipients (53% vs. 72%;

; figure 3 ). Additional analysis of the subset of subjects P ! .001 who had samples obtained for culture on day 2 revealed that significantly fewer pleconaril recipients than placebo recipients had positive viral culture results (27 [60%] of 45 subjects vs. 49 [84%] of 58 subjects;

). P p .007 Nasal mucus viral RNA levels decreased rapidly in both pleconaril and placebo treatment groups. Subjects in the pleconaril group showed a larger median percentage reduction from baseline in virus levels on study day 3, compared with subjects in the placebo group (97.7% vs. 90.3%;

). By day 6 (range, P ! .001 day [5] [6] [7] [8] [9] , the median percentage reduction in virus levels was 199% in both treatment groups.

Safety. Pleconaril was generally well tolerated. The most commonly reported adverse events were headache, diarrhea, and nausea (table 5); у95% of adverse events were mild or moderate in severity. Four subjects receiving pleconaril and 2 receiving placebo reported a serious adverse event, none of which was considered by the investigators to be related to study drug except for 1 case of inadvertent overdose of pleconaril (with no adverse sequelae).

No differences were noted in vital signs or physical examination findings in either treatment group, and there were no clinically significant laboratory abnormalities (data not shown). The only laboratory findings associated with pleconaril use were small median increases from baseline values in the pleconaril group for nonfasting serum cholesterol levels (an increase of 5 mg/dL [or 3%], compared with a decrease of 4 mg/dL [or 2%] in placebo recipients;

) and for platelet counts (an in-P ! .001 crease of platelets/mm 3 [or 6%], compared with an 3 15 ϫ 10 increase of platelets/mm 3 [or 3%] in placebo recipients; 3 7 ϫ 10 ). P ! .001 

These prospective, double-blind, placebo-controlled studies found that early pleconaril treatment significantly reduces the duration and severity of colds due to picornaviruses in adults; these findings establish pleconaril as the first antiviral to have proven therapeutic value for such illnesses. These results confirm and extend those of an earlier retrospective analysis of adults with colds due to picornaviruses, which found a 1.5-day reduction in the time to alleviation of illness in pleconaril recipients, compared with placebo recipients [10] . Pleconaril use was associated with significant reductions in symptom severity scores and the frequency of recovery of picornaviruses within 1 day after initiation of therapy. In addition, pleconaril therapy resulted in a significant reduction in the duration of each individual cold symptom monitored in the study, a finding consistent with the hypothesis that ongoing viral replication is an important contributor to the pathogenesis of cold symptoms.

The rapid decrease in viral RNA levels in both pleconaril and placebo groups illustrates the importance of early initiation of antiviral therapy. We also observed that substantial proportions of both placebo recipients and, less often, pleconaril recipients continued to have positive culture results on study day 6 or later, although at very low levels of viral RNA. This prolonged recovery of virus is consistent with earlier data from natural and experimentally induced rhinovirus infections [21, 22] , but it raises the issue of emergence of drug-resistant variants. In the current studies, viruses with reduced susceptibility to pleconaril (у10-fold change from baseline value) were recovered during or after therapy from ∼10% of patients who received pleconaril. However, subgroup analyses indicate that clinical benefit for these participants was as good as or better than that for pleconaril recipients with no reduction in virus susceptibility to the drug (unpublished data). Further phenotypic and genetic characterization of viruses from these and other pleconaril trials is ongoing, to determine relationships between in vitro susceptibility and clinical outcomes.

Pleconaril was shown to be safe and generally well tolerated. Compared with placebo, there were only small (2%) excess frequencies of headaches, nausea, and diarrhea in patients receiving pleconaril. The small increases from baseline in cholesterol levels and platelet counts are not clinically significant, an observation that was confirmed in a subsequent 6-week prophylaxis study (unpublished observations). In that study, an excess of mild or moderate menstrual disorders (most commonly breakthrough bleeding or spotting) was reported from women taking oral contraceptives and pleconaril. Subsequent investigations revealed that pleconaril induces hepatic cytochrome P-450 3A enzymes. Pleconaril reduced the area under the curve of plasma levels of ethinyl estradiol by 34% following single-dose administration (G. Rhodes, personal communication). Retrospective review of all randomized, placebo-controlled trials in which pleconaril was administered for 5-7 days revealed that menstrual irregularities were reported by 3.5% of 310 pleconaril-treated women who were using oral contraceptives and by none of 291 placebo-treated women. None of the menstrual irregularities led to discontinuation of treatment. Additional studies are ongoing to better characterize the magnitude and duration of cytochrome P-450 3A induction and to determine the clinical significance for coadministration of pleconaril with other drugs metabolized by cytochrome P-450 3A.

One limitation of the current studies is that most participants were generally healthy young adults. Other studies have established that rhinoviruses can cause both upper and lower respiratory tract complications, including asthma exacerbations in both adults and children [23] [24] [25] , acute exacerbations of chronic obstructive pulmonary disease [26] , acute otitis media in children [27] , and sinusitis in adults [13] . Others at risk for lower respiratory complications due to rhinovirus infection include patients with cystic fibrosis [28] , elderly individuals [29] , and immunocompromised persons [30] . The positive findings in the current trials indicate that studies of pleconaril should be extended to children, smokers, and those with underlying airway disease.

In summary, early pleconaril treatment of colds due to picornaviruses reduces the duration and severity of illness in adults. Pleconaril at this dosage was well tolerated, although additional data are needed to better characterize its potential for drug interactions.

Tucson: A. Adamczyk; Brandywine Clinical Research, Downingtown, PA: L. Alwine; Credit Valley Hospital

Armstrong

TN: I. Biaggioni; Primary Physicians Research

Brankston; Alpine Clinical Research Center

Central Austin Internists

Herridge Community Health Clinic

W. Gaman; Allergy and Asthma Practice

Miami Valley Clinical Trial Resources

Children's & Adult's Clinical Research Foundation, Chapel Hill, NC: F. Henderson; New Hanover Medical Research, Wilmington, NC: C. Herring; West Texas Medical Associates, San Angelo: D. Herrington; NSDEA

Welstar Health System, Marietta

Medical Group at Marple Commons

D. Orchard; NFLD Drive Family Practice

Pierone; Clinical Research

AR: K. Roberts; University of Missouri Columbia, Columbia

Yeoman; Northern California Research, Fair Oaks: D. Young; The Male Health Centres

Viruses and bacteria in the etiology of the common cold

Frequency and natural history of rhinovirus infections in adults during autumn

Intranasal recombinant alfa-2b interferon treatment of naturally occurring common colds

Intranasal pirodavir (R77,975) treatment of rhinovirus colds

Clinical virology

Analysis of three structurally related antiviral compounds in complex with human rhinovirus 16

Treatment of the picornavirus common cold by inhibitors of viral uncoating and attachment

Activity of pleconaril against enteroviruses

Clinical activity of pleconaril in an experimentally induced coxsackievirus A21 respiratory infection

Oral pleconaril treatment of picornavirus-associated viral respiratory illness in adults: efficacy and tolerability in phase II clinical trials

Improved method for collection of nasal mucus

Detection of human rhinovirus RNA in nasal washings by PCR

Detection of rhinovirus in sinus brushings of subjects with acute community-acquired sinusitis by reverse transcription-PCR

Rapid detection of human rhinoviruses in nasopharyngeal aspirates by a microwell reverse transcription-PCR-hybridization assay

Detection of herpes simplex virus DNA in cerebrospinal fluid samples using the polymerase chain reaction and microplate hybridization

Comparative susceptibility of human embryonic fibroblasts and HeLa cells for isolation of human rhinoviruses

Nonparametric estimation from incomplete observations

Cancer therapy: prognostic factors and criteria

SAS/STAT user's guide. Version 6

Tables of the number of patients required in clinical trials using the log rank test

The Seattle virus watch. V. Epidemiologic observations of rhinovirus infections, 1965-1969, in families with young children

Sites of rhinovirus recovery after point inoculation of the upper airway

Respiratory viruses and exacerbations of asthma in adults

The relationship between upper respiratory infections and hospital admissions for asthma: a time trend analysis

Association of rhinovirus infections with asthma

Respiratory viral infections in adults with and without chronic obstructive pulmonary disease

Detection of rhinovirus, respiratory syncytial virus, and coronavirus infections in acute otitis media by reverse transcriptase polymerase chain reaction

Severe viral respiratory infections in infants with cystic fibrosis

Risk factors for lower respiratory complications of rhinovirus infections in elderly people living in the community: prospective cohort study

Community respiratory virus infections in immunocompromised patients with cancer

We thank Marissa Seligman and Sandra Norris for editorial assistance in manuscript preparation.