**DEPARTMENT OF HEALTH & HUMAN SERVICES**
![](media/image1.png){width="1.2729166666666667in"
height="1.0555555555555556in"}

> Public Health Service
>
> Food and Drug Administration

Rockville, MD 20857

**Date:** February 20, 2002

**To:** Antiviral Drug Products Advisory Committee Members and Guests

**From:** Pleconaril Review Team

**Through:** Debra Birnkrant, MD

Director, DAVDP

**Re:** Background Package for NDA 21-245: Picovir (pleconaril)

# I. Summary of Regulatory Issues and Purpose of Meeting

This memorandum serves as an introduction to the FDA presentation at the
upcoming March 19, 2002, meeting of the Antiviral Drug Products'
Advisory Committee (AVAC). At this meeting we will ask you to discuss
the NDA for ViroPharma's Picovir (pleconaril) for the treatment of
acute picornaviral viral respiratory infection (VRI, "common cold") in
adults.

> The background materials from FDA represent the findings and opinions
> of the primary reviewers of each discipline, based on their reviews of
> the respective submissions from ViroPharma. It must be emphasized that
> this document represents the review team's preliminary findings, and
> that no regulatory decision has been made on the status of the
> application. Indeed, the advice the AVAC provides will be critical in
> our regulatory decision making.

Pleconaril represents the first antiviral drug to be considered for
approval for treatment of the common cold. Currently, only treatments
that target individual symptoms of VRI are approved, e.g., analgesics,
antihistamines, and decongestants. Under the Food, Drug and Cosmetics
Act (FD&C Act), the FDA approves drugs based on "substantial evidence"
of safety and effectiveness. A recommendation for approval by the
committee needs to take into account the FD&C Act's evidentiary standard
and the overall risk/benefit ratio for the drug in question. In the case
of pleconaril, the risk/benefit consideration relates to the
identification of infected patients, the variability of efficacy
results, the efficacy results in smokers versus non-smokers and males
versus females, and the overall safety profile of pleconaril.

Beyond the overall examination of the safety and efficacy data for
pleconaril, some critical issues for you to consider as you read the
Agency information and Applicant's background materials include the
study results, indication, resistance, and whether additional data would
be necessary to support approval. We look forward to a very interesting
meeting and thank you in advance for your time and efforts in this
important meeting.

We will ask you to consider the following issues during your
deliberations on this application:

-   **Variable Study Results**

In two large phase 2 studies (843-020 and 843-032), pleconaril produced
a median 0.0 to 0.5 day treatment effect in both the all randomized
(ITT) patient population and those subjects the applicant determined to
be picornavirus infected (PCR+, ITT-I). Low rates of PCR positivity,
high rates of concomitant cold medication use, the inclusion of smokers,
and a stringent endpoint which required all symptoms to have completely
resolved may have contributed to the lack of a clearly demonstrated
treatment effect in the phase 2 program.

Studies 843-043 and 843-044 were identically designed, multi-center,
double-blind, randomized, placebo-controlled phase 3 studies. For the
primary endpoint, the treatment effect for pleconaril was a median of
0.5 (study 843-043) and 1.5 days (study 843-044) compared to placebo. In
both studies, the difference between pleconaril and placebo was
statistically significant in the ITT-I population. In the ITT
population, the difference reached statistical significance only in
study 843-044.

Symptomatic rhinoviral infection is generally a mild, self-limited
illness that typically subsides in adults in 7 to 11 days, depending on
the reference cited. The median time to resolution of VRI in patients
treated with pleconaril in the two pivotal trials ranged from 6.0 and
8.0 days, depending on how the analyses handled patients who did not
have resolution of their VRI or who prematurely discontinued; however,
the overall treatment effect was maintained.

# [Secondary Endpoints]{.underline}

Multiple secondary clinical endpoints were analyzed and included: time
to patient assessment of no illness, time to reduction in symptom
severity and resolution of individual symptoms, tissue use[^1], time to
return to baseline activity level, and the impact of concomitant cold
symptom relief medication use.

FDA generally places the greatest emphasis on the primary endpoint.
Secondary endpoints may be supportive but results should be interpreted
with caution because of large numbers of potential comparisons, required
adjustments for p values, and, in some cases, the small number of
patients included in some analyses. In general, most secondary endpoints
showed similar trends in favor of pleconaril as observed for the primary
endpoint.

##### Patient Subgroups

##### Smokers versus Non-Smokers

Randomization was stratified on smoking history; 30% of study patients
were classified as smokers. Compared to non-smokers, smokers experienced
an overall longer duration of VRI. For smokers, time to resolution of
symptoms was numerically greater in the pleconaril group compared to the
placebo group.

##### Analysis by Gender 

Gender analysis is required under FDA regulations. Analysis by gender
demonstrated that the magnitude of the treatment effect among men was
0.4 days and in women it was approximately 1.0 day.

##### 

##### Special Populations

The studies were conducted in young, healthy individuals. Phase 1
studies suggest that no dose adjustment would likely be necessary when
pleconaril is administered to elderly ([\>]{.underline}65 years)
patients. In the pivotal studies, only 4% of patients were
[\>]{.underline}65 years of age or older which precluded an assessment
of safety or efficacy in this population. There are no data on treatment
of patients with underlying cardiovascular, pulmonary disease, patients
with allergic rhinitis, or patients who are immunocompromised or
otherwise seriously ill. Pediatric VRI treatment trials are ongoing.

# Identification of Picornavirus-Infected Patients and Resistance

The applicant proposes the picornavirus-infected patients (PCR+),
designated ITT-I, as the primary population for assessing efficacy.
Arguably, the all randomized population (ITT) is also a valid population
for assessing efficacy since pleconaril will be prescribed based on
presenting symptoms without a readily available PCR assay.

Review of the NDA raised a number of questions about the methods
employed to identify picornavirus-infected patients. The applicant
states that 65% of patients in the two pivotal trials were infected with
picornaviruses (PCR+). Infected patients were identified by an
experimental TaqMan assay. TaqMan negative samples were subsequently
tested for picornavirus using an experimental ELOSA system. All PCR+
samples were subsequently cultured in a rhinovirus-specific culture
system; 63% of PCR+ samples were culture positive. Methodological
questions include:

**Primer and probe selection.** The probe sequence submitted by
ViroPharma contains a region of eight consecutive Gs and Cs, while the
primer sequences each contain three Gs and/or Cs and on one primer these
are consecutive. Tighter binding by these primers may inhibit primer
dissociation and could lead to variable PCR products.

**Number of cycles**. The TaqMan assay is run for a total of 60 cycles.
The 60-cycle reaction could allow for an increase in non-specific
amplification in samples that contain weakly homologous regions. Any
non-specific amplification could also have an adverse effect on the
sensitivity and specificity of the assay.

**Determination of threshold fluorescence level for detection of
picornarvirus PCR**. The threshold set by the applicant was 0.1. Based
on information from the manufacturer of the assay (ViroMed), this
threshold level may be too low and a more appropriate threshold might be
in the range of 0.4 -- 0.6. Thus, incorrect threshold placement could
result in an incorrect assay definition of positivity. Also, a low
threshold value could subject individual samples to assay background
that could impact the relative quantitative results of the assay.

# Clinical sensitivity. According to the applicant, the TaqMan assay had a sensitivity of 93% (88-97%) and specificity of 75% (72-78%). An "expanded gold standard" was used to re-evaluate sensitivity and specificity. "True positives" were considered to be either (i) virus culture positives regardless of PCR results, or (ii) TaqMan and ELOSA positives regardless of culture results. Statistically, in this analysis, it is likely that the results would favor the PCR result since one PCR assay is being "confirmed" by a second PCR assay. None of the applicant's results were independently confirmed.

# Virus Culture. Two virus culture systems were utilized in the clinical studies. Only samples positive in the TaqMan assays were analyzed by cell culture, no negative specimens were tested. Positive cultures were not assessed for picornavirus positivity. Although cultures were deemed positive for plaque forming units, this was not confirmed to be due to picornavirus by pH lability, identification of the serotype, immunofluorescent assay, or DNA sequence analysis.

Additional studies to assess the performance of the TaqMan assay to
identify picornavirus-infected patients are ongoing and results are
expected in early March; these data will be available for discussion at
the AVAC meeting. (For additional discussion about these issues, please
see Appendix A).

Approximately 24% of isolates examined exhibited either a baseline lack
of susceptibility (13%) or a loss of susceptibility (10.7%) to
pleconaril against picornaviruses.

# Safety

The adult VRI safety database includes information on 2,488 patients who
received at least one dose of pleconaril. Of these, 1,046 patients
received the proposed regimen, 400 mg TID for 5 days (15 doses), using
the proposed commercial tablet formulation. Nearly 2,000 patients
received placebo in VRI studies with the tablet being of the same
composition without the active ingredient.

# [General Adverse Events]{.underline}

Gastrointestinal events (abdominal pain, nausea, vomiting, and
diarrhea), and headache were the most common adverse events reported in
the adult VRI safety database, with headache occurring slightly more
frequently among patients who received pleconaril. There were no
significant laboratory abnormalities identified.

**[Menstrual Disorders]{.underline}**

There was a significantly higher rate of menstrual abnormalities in
women treated with pleconaril while taking estrogen/progestin-based oral
contraceptives (OC) compared to women taking OCs and placebo. The
frequency for pleconaril/OC users was 2.4% in 5-7 day treatment trials
compared to \<1% for placebo/OC users. In a six-week VRI prevention
study, the frequencies were 53%, 69%, and 25% among women taking OCs
with pleconaril 400 mg QD, 400 mg BID, and placebo, respectively. This
will be discussed in greater detail in section V, below.

##### Palpitations and Tachycardia

In a theophylline/pleconaril interaction study, three of 15 subjects
experienced tachycardia and palpitations when the two drugs were
co-administered. In this study, subjects were naïve to theophylline
prior to enrollment.

Patients in the clinical trials were otherwise healthy adults with no
history of underlying cardiovascular or pulmonary disease. Palpitations
and/or tachycardia with and without dizziness or syncope were reported
by seven patients treated with pleconaril (0.3%) compared to two treated
with placebo (0.1%). Four of the pleconaril-treated patients
discontinued from studies due to these events.

# II. Clinical Development Summary

# 

The original pleconaril IND was filed June 23, 1993 by Sterling Winthrop
and transferred to ViroPharma on July 5, 1995.

Pleconaril had been investigated as a treatment for enteroviral
meningitis. Difficulty in identifying a correct dose, identifying
patients with enteroviral meningitis with a PCR assay, determining an
objective endpoint, and the inability to consistently demonstrate a
treatment effect led to termination of development for this indication.
Likewise, the applicant investigated pleconaril as a possible treatment
for pediatric hand, foot, and mouth disease, but was not able to
establish efficacy.

### Pleconaril is currently under investigation for treatment of VRI in pediatric patients, prevention of VRI and treatment of enteroviral sepsis syndrome. It has been provided to patients with presumed or documented severe enteroviral diseases under a compassionate use protocol. 

Development of pleconaril for treatment of acute VRI followed results of
a study in which it appeared that prophylactic administration of
pleconaril reduced the severity and duration of VRI in a coxsackie A-21
prevention study. Subsequently, the applicant conducted multiple phase 2
studies to assess pleconaril's activity in patients with VRI, and to
prevent asthma exacerbations in patients with VRI.

The NDA contains data from six clinical trials, including two pivotal
studies (843-043 and 843-044). Both pivotal studies were identical in
design, and were conducted during the same time period
(September-December 2000), in a similar geographic distribution (US and
Canada). Both studies evaluated the safety and efficacy of pleconaril
versus placebo in patients presenting with symptoms consistent with a
viral upper respiratory tract infection.

Supportive studies 843-010, 843-020, and 843-032 investigated various
doses (200 mg and 400 mg) and dosing schedules (BID or TID for 5 or 7
days) in patients who presented with symptoms of a VRI. Study 843-013
attempted to evaluate pleconaril's utility in preventing acute
exacerbations in patients with a VRI and a history of asthma. A summary
of the six trials included in the NDA is presented in Table 1.

Table 1. Adult VRI Studies

+----------+-------------------+----------+------------+--------------+
| **       | **Design**        | **Re     | **No.      | **1^o^       |
| Protocol |                   | gimens** | Patients** | Endpoint**   |
| No.**    |                   |          |            |              |
+----------+-------------------+----------+------------+--------------+
| 843-010  | Double-blind,     | 200 mg   | P          | Time to      |
|          | randomized,\      | TID\     | leconaril\ | complete     |
|          | pl                | 400 mg   | 200 mg:    | resolution   |
|          | acebo-controlled, | TID\     | 73\        | of all       |
|          | parallel group in | Placebo  | 400 mg:    | symptoms     |
|          | patients with     |          | 74\        | (score of 0) |
|          | Summer Flu        | 7 days   | Placebo:   | for 48 hours |
|          |                   |          | 74         |              |
+----------+-------------------+----------+------------+--------------+
| 843-013  | Double-blind,     | 200 mg   | P          | Number and   |
|          | randomized,\      | TID\     | leconaril\ | proportion   |
|          | pl                | 400 mg   | 200 mg:    | of patients  |
|          | acebo-controlled, | TID\     | 87\        | with an      |
|          | parallel group in | Placebo  | 400 mg:    | asthma       |
|          | patients with     |          | 84\        | exacerbation |
|          | bronchial asthma  | 7 days   | Placebo:   |              |
|          |                   |          | 83         |              |
+----------+-------------------+----------+------------+--------------+
| 843-020  | Double-blind,     | 400 mg   | P          | Time to      |
|          | randomized,\      | BID\     | leconaril: | complete     |
|          | pl                | 400 mg   |            | resolution   |
|          | acebo-controlled, | TID\     | BID: 335   | of the signs |
|          | parallel group in | Placebo  |            | and symptoms |
|          | patients with     |          | TID: 349   | of VRI       |
|          | picornarvirus VRI | 7 days   |            | (symptom     |
|          |                   |          | Placebo:   | score of 0)  |
|          |                   |          | 340        | sustained    |
|          |                   |          |            | for 48 hours |
+----------+-------------------+----------+------------+--------------+
| 843-032  | Double-blind,     | 400 mg   | P          | Time to      |
|          | randomized,\      | TID\     | leconaril: | alleviation  |
|          | pl                | Placebo  | 436\       | of all VRI   |
|          | acebo-controlled, |          | Placebo:   | symptoms     |
|          | parallel group in | 7 days   | 439        | sustained    |
|          | patients with     |          |            | for 24 hours |
|          | picornavirus VRI  |          |            |              |
+----------+-------------------+----------+------------+--------------+
| 843-043  | Double-blind,     | 400 mg   | (843-043)  | Time to      |
|          | randomized,\      | TID\     |            | resolution   |
| 843-044  | pl                | Placebo  | P          | of           |
|          | acebo-controlled, |          | leconaril: | rhinorrhea   |
|          | parallel group in | 5 days   | 526        | and          |
|          | patients with     |          |            | alleviation  |
|          | picornavirus VRI  |          | Placebo:   | (absent or   |
|          |                   |          | 526        | mild) of     |
|          |                   |          |            | other VRI    |
|          |                   |          | (843-044)  | symptoms     |
|          |                   |          |            | without use  |
|          |                   |          | P          | of           |
|          |                   |          | leconaril: | concomitant  |
|          |                   |          | 520        | cold         |
|          |                   |          |            | medications  |
|          |                   |          | Placebo:   | for 48 hours |
|          |                   |          | 524        |              |
+----------+-------------------+----------+------------+--------------+

For a review of the design and results of studies 843-010, 843-013,
843-020, and 843-032, please see Appendix B.

# III. Summary of Efficacy

# A. Dose and Regimen

The applicant selected 400 mg (2 x 200 mg capsules) TID for five days
(15 doses) as adequate for the inhibition of picornaviral replication in
the treatment of VRI. For additional information on pharmacokinetics,
dose, and regimen, please see Appendix C.

**B. Phase 3 Studies 843-043 and 843-044**

-   **Study Design and Baseline Characteristics**

Studies 843-043 and 843-044 were identically designed multi-center,
double blind, randomized, placebo-controlled studies. Study 843-043 was
conducted in 97 centers and 843-044 was conducted in 106 centers. Both
studies were conducted in Canada and the U.S. between August and
December 2000.

Patients with moderate to severe rhinorrhea and at least one other
symptom of VRI (cough, nasal congestion, pharyngeal symptoms, malaise
and myalgia) were randomized to receive either pleconaril 400 mg TID x 5
days or matching placebo (total 15 doses). The first dose of study
medication was to be administered within 36 hours of first symptom onset
(later revised to 24 hours based on post-hoc analysis of study 843-032).

Prior to the first dose, nasal mucus was collected for PCR analysis to
detect picornaviral RNA using an experimental TaqMan assay system.
TaqMan negative samples were re-tested using a modified experimental
Enzyme-Linked Oligo Sorbant Assay (ELOSA). Only samples that were
determined to be PCR+ by one of these techniques were sent for viral
culture. Follow-up nasal mucus specimens for PCR were obtained on study
days 3 and 6.

Patients underwent baseline assessment of VRI symptoms including
rhinorrhea, nasal congestion, pharyngeal symptoms (sore throat), cough,
myalgia and malaise using an ordinal numerical scoring system for
severity. Rhinorrhea was scored as absent (not present, score=0), mild
(1 tissue per hour, score=1), moderate (2-5 tissues per hour, score=2)
or severe (more than 5 tissues per hour, score=3). Nasal congestion,
pharyngeal symptoms, cough, malaise and myalgia were similarly scored as
absent (not present), mild (noticeable), moderate (bothersome), or
severe (interferes with activity) using the same ordinal numeric scoring
system.

As shown in Table 2, both studies predominantly enrolled female
Caucasians with a mean age of 36 years.

Table 2. Baseline demographic characteristics of all-randomized patients

+-------------+-------------+-------------+-------------+-------------+
|             | # St        |             | **Study     |             |
|             | udy 843-043 |             | 843-044**   |             |
+-------------+-------------+-------------+-------------+-------------+
|             | **P         | **Placebo** | **P         | **Placebo** |
|             | leconaril** |             | leconaril** |             |
|             |             | **(n=526)** |             | **(n=524)** |
|             | **(n=526)** |             | **(n=520)** |             |
+-------------+-------------+-------------+-------------+-------------+
| ## Age      | 36          | 36          | 36          | 37          |
|             |             |             |             |             |
| Mean        | 17-77       | 18-77       | 18-82       | 18-86       |
| (years)     |             |             |             |             |
|             |             |             |             |             |
| Range       |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
| ## Gender   | 33%         | 34%         | 30%         | 28%         |
|             |             |             |             |             |
| Male        | 67%         | 66%         | 70%         | 72%         |
|             |             |             |             |             |
| Female      |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
| ## Race     | 75%         | 79%         | 90%         | 88%         |
|             |             |             |             |             |
| White       | 11%         | 10%         | 4%          | 5%          |
|             |             |             |             |             |
| Black       | 10%         | 7%          | 3%          | 4%          |
|             |             |             |             |             |
| Hispanic    | 2%          | 2%          | 1%          | 1%          |
|             |             |             |             |             |
| Asian       | 2%          | 2%          | 1%          | 2%          |
|             |             |             |             |             |
| Other       |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
| ## Smokers  | 29%         | 29%         | 28%         | 27%         |
|             |             |             |             |             |
| ##  M       | 12          | 15          | 12          | 12          |
| ean (years) |             |             |             |             |
|             | \<1-48      | \<1-50      | \<1-60      | \<1-50      |
| Range       |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
| **Pre-study | 28%         | 29%         | 32%         | 30%         |
| cold**      |             |             |             |             |
|             |             |             |             |             |
| *           |             |             |             |             |
| *medication |             |             |             |             |
| use**       |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
| **Time      | 18.4        | 18.4        | 18          | 17.8        |
| between**   |             |             |             |             |
|             | 3.2-26.5    | 2.2-25.3    | 1.8-39.1    | 2.4-43.2    |
| **first     |             |             |             |             |
| symptom and |             |             |             |             |
| first dose  |             |             |             |             |
| of study    |             |             |             |             |
| m           |             |             |             |             |
| edication** |             |             |             |             |
|             |             |             |             |             |
| Mean        |             |             |             |             |
| (hours)     |             |             |             |             |
|             |             |             |             |             |
| Range       |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
| **Baseline  | 9           | 9           | 9           | 9           |
| symptom     |             |             |             |             |
| score^a^**  | 4-16        | 4-16        | 4-18        | 4-18        |
|             |             |             |             |             |
| Range       |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+
| **PCR+^b^** | 337 (64%)   | 326 (62%)   | 344 (66%)   | 356 (68%)   |
+-------------+-------------+-------------+-------------+-------------+
| **Viral     | 201 (38%)   | 196 (37%)   | 206 (40%)   | 224 (43%)   |
| culture +   |             |             |             |             |
| at          |             |             |             |             |
| ba          |             |             |             |             |
| seline^c^** |             |             |             |             |
+-------------+-------------+-------------+-------------+-------------+

a\. Maximum score=18

b\. PCR + on days 1, 3 and/or 6 by research based TaqMan or modified
ELOSA.

c\. Viral cultures were conducted only on samples that were positive by
either the TaqMan or ELOSA.

PCR+ patients had slightly higher severity scores for rhinorrhea,
congestion, and sore throat compared to PCR- patients. Also, PCR+
patients were more likely to have used cold symptom relief medications
prior to study entry.

-   **Review of Efficacy**

**[Primary Efficacy Population]{.underline}**

The applicant's primary efficacy population is all patients whose nasal
mucus tested positive for picornavirus PCR during the study (infected
patients, ITT-I). For completeness, analyses of efficacy in the
all-randomized patient population (ITT) are also presented.

**[Summary of Clinical Virology]{.underline}**

Nasal samples were tested for picornavirus using an experimental TaqMan
PCR assay. Samples that tested negative by TaqMan were re-tested using
an experimental Enzyme-Linked Oligo Sorbent Assay (ELOSA) in an attempt
to identify additional picornavirus-infected patients. On study days 3
and 6 nasal mucus samples were collected and tested using only the
TaqMan assay (instillation of 0.5 ml of saline was used to obtain nasal
mucus in patients who could not expectorate mucus). Based on these
procedures, the applicant concluded that 65% of patients were
picornavirus PCR+ at some time during the studies.

PCR+ samples were cultured. Cultures were considered positive for
rhinovirus based on presence of plaque forming units. Cultures were not
further evaluated to confirm that the cultures were, in fact,
rhinoviruses versus entero or other viruses.

In studies 843-043 and 043-044, 13% of baseline isolates (96/744) were
not inhibited by pleconaril concentrations of 10 μg/ml. Further, during
the 5-day course of treatment, reduced susceptibility to pleconaril
(defined as \>10-fold decrease in susceptibility compared to baseline
pre-treatment isolate) emerged in 10.7% of virus isolates tested (28/263
isolates).

**[Primary Efficacy Endpoint and Analysis]{.underline}**

The primary endpoint for studies 843-043 and 843-044 was the median time
(days) to resolution of rhinorrhea and alleviation (absent or mild) of
other symptoms of VRI without use of concomitant cold medications for 48
hours.[^2]

The manner in which patients who did not reach the primary endpoint were
handled impacts the analysis of efficacy. The applicant censored
patients at the time immediately after their last recorded observation.
The FDA analysis considered any patient who did not have resolution of
their VRI or prematurely discontinued the study as treatment failures
and assigned them the last possible observation as the day of healing
(day 18.5). The applicant and FDA's analysis of the primary endpoint is
presented in Table 3; you will note that although the treatment effect
remains stable, the overall duration of illness is increased by \>1 day.

Table 3. Time to resolution (days) of rhinorrhea and alleviation (absent
or mild) of other symptoms of VRI without use of concomitant cold
medications for 48 hours

+-----------------+-------+--------+-------+-------+-------+-------+
|                 | ###   |        |       | # Stu |       |       |
|                 | # Stu |        |       | dy 84 |       |       |
|                 | dy 84 |        |       | 3-044 |       |       |
|                 | 3-043 |        |       |       |       |       |
|                 |       |        |       | **Di  |       |       |
|                 | **Di  |        |       | ff.** |       |       |
|                 | ff.** |        |       |       |       |       |
|                 |       |        |       | **Pl  |       |       |
|                 | **Pl  |        |       | acebo |       |       |
|                 | acebo |        |       | Pleco |       |       |
|                 | Pleco |        |       | naril |       |       |
|                 | naril |        |       | p-va  |       |       |
|                 | p-va  |        |       | lue** |       |       |
|                 | lue** |        |       |       |       |       |
+-----------------+-------+--------+-------+-------+-------+-------+
| ### App         |       |        |       |       |       |       |
| licant Analysis |       |        |       |       |       |       |
+-----------------+-------+--------+-------+-------+-------+-------+
| ## ITT (n)      | 526   | 526    | 0.5   | 524   | 520   | 0.9   |
|                 |       |        |       |       |       |       |
| N w/resolution  | 417   | 430    | 0.201 | 411   | 423   | 0.015 |
|                 | (79%) | (82%)  |       | (78%) | (81%) |       |
| Median          |       |        |       |       |       |       |
|                 | 6.9   | 6.4    |       | 7.1   | 6.2   |       |
+-----------------+-------+--------+-------+-------+-------+-------+
| ## ITT-I (n)    | 326   | 337    | 0.6   | 356   | 344   | 1.5   |
|                 |       |        |       |       |       |       |
| N w/resolution  | 258   | 282    | 0.037 | 286   | 290   | 0.001 |
|                 | (79%) | (84%)  |       | (80%) | (84%) |       |
| Median          |       |        |       |       |       |       |
|                 | 7.2   | 6.6    |       | 7.7   | 6.2   |       |
+-----------------+-------+--------+-------+-------+-------+-------+
| #               |       |        |       |       |       |       |
| ## FDA Analysis |       |        |       |       |       |       |
+-----------------+-------+--------+-------+-------+-------+-------+
| ## ITT (n)      | 526   | 526    | 0.5   | 524   | 520   | 1.0   |
|                 |       |        |       |       |       |       |
| Median          | 7.5   | 7.0    | 0.13  | 80    | 7.0   | 0.014 |
+-----------------+-------+--------+-------+-------+-------+-------+
| ## ITT-I (n)    | 324   | 334    | 0.5   | 350   | 343   | 1.5   |
|                 |       |        |       |       |       |       |
| Median          | 7.5   | 7.0    | 0.03  | 8.5   | 7.0   | 0.004 |
+-----------------+-------+--------+-------+-------+-------+-------+

**[Efficacy Results According to Randomization Strata]{.underline}**

Randomization was stratified based on smoking status and pre-treatment
use of symptomatic cold relief medications.

**Smokers Compared to Non-Smokers**

Approximately 30% of study participants had a smoking history. At
baseline, smokers reported slightly more severe cough, otherwise there
were no significant demographic differences between smokers and
non-smokers. A history of smoking clearly impacted the duration of VRI
overall, and resulted in numerically negative treatment effect for
pleconaril. The FDA analysis of the primary endpoint based on smoking
status is presented in Table 4.

**Table 4. Time (days) to the primary endpoint in smokers and
non-smokers**

+-------------------+----------+----------+-----------+---------------+
|                   | ##       |          | # Stud    |               |
|                   | ## Study |          | y 843-044 |               |
|                   |  843-043 |          |           |               |
|                   |          |          | **Placebo |               |
|                   | *        |          | Ple       |               |
|                   | *Placebo |          | conaril** |               |
|                   | Plec     |          |           |               |
|                   | onaril** |          |           |               |
+-------------------+----------+----------+-----------+---------------+
| **ITT Smokers     | 152      | 153      | 138       | 136           |
| (n)**             |          |          |           |               |
|                   | 8.0      | 7.5      | 7.5       | 8.5           |
| Median            |          |          |           |               |
+-------------------+----------+----------+-----------+---------------+
| **ITT Non-smokers | 374      | 374      | 386       | 384           |
| (n)**             |          |          |           |               |
|                   | 7.5      | 6.5      | 8.0       | 6.0           |
| Median            |          |          |           |               |
+-------------------+----------+----------+-----------+---------------+
| **ITT-I Smokers   | 76       | 102      | 90        | 87            |
| (n)**             |          |          |           |               |
|                   | 8.0      | 9.0      | 8.5       | 9.0           |
| Median            |          |          |           |               |
+-------------------+----------+----------+-----------+---------------+
| **ITT-I           | 224      | 219      | 249       | 240           |
| Non-smokers (n)** |          |          |           |               |
|                   | 7.5      | 6.5      | 8.5       | 6.5           |
| Median            |          |          |           |               |
+-------------------+----------+----------+-----------+---------------+

###### Also, smokers treated with pleconaril experienced more acute complications of VRI (otitis media, bronchitis, sinusitis, and pneumonitis) compared to non-smokers.

**Pre-Treatment Cold Medication Users Compared to Non-Users**

At study entry, patients were stratified on pre-treatment use of cold
symptom relief medications. Approximately 30% of participants in both
studies used at least one dose of symptom relief medication (e.g.,
acetaminophen, ibuprofen, dextromethorphan, pseudoephedrine, Dristan,
Dimetapp, echinacea, zinc, or NyQuill,) prior to initiation of study
medication. Patients with moderate/severe pharyngeal symptoms, myalgia,
and malaise appeared to be more likely to have used pre-treatment cold
relief medications. The analysis of the primary endpoint by use and
non-use of pretreatment cold relief medications is presented in Table 5.

Table 5. Time (days) to primary endpoint in users and non-users of
pre-treatment cold medications

+-----------------------+----------+-----------+----------+----------+
|                       | ##       |           | # Study  |          |
|                       | ## Study |           |  843-044 |          |
|                       |  843-043 |           |          |          |
|                       |          |           | *        |          |
|                       | *        |           | *Placebo |          |
|                       | *Placebo |           | Plec     |          |
|                       | Plec     |           | onaril** |          |
|                       | onaril** |           |          |          |
+-----------------------+----------+-----------+----------+----------+
| **ITT users (n)**     | 138      | 138       | 163      | 162      |
|                       |          |           |          |          |
| Median                | 8.0      | 8.5       | 8.0      | 8.5      |
+-----------------------+----------+-----------+----------+----------+
| **ITT non-users (n)** | 388      | 388       | 361      | 358      |
|                       |          |           |          |          |
| Median                | 7.0      | 6.5       | 8.0      | 6.5      |
+-----------------------+----------+-----------+----------+----------+
| **ITT-I users (n)**   | 85       | 101       | 119      | 120      |
|                       |          |           |          |          |
| Median                | 9.5      | 8.5       | 9.0      | 8.5      |
+-----------------------+----------+-----------+----------+----------+
| #                     | 215      | 220       | 237      | 224      |
| # ITT-I non-users (n) |          |           |          |          |
|                       | 7.5      | 6.5       | 7.4      | 6.0      |
| Median                |          |           |          |          |
+-----------------------+----------+-----------+----------+----------+

**[Efficacy Analysis by Gender]{.underline}**

Analysis of the primary endpoint by gender demonstrated:

-   In men, there was a median 0.4 day treatment effect in favor of
    pleconaril over placebo, 5.7 versus 6.1 days (p=0.210).

-   Women treated with pleconaril experienced a median 1.0-day faster
    time to resolution compared to women treated with placebo, 7.0
    versus 8.0 days.

**[Analysis of Secondary Endpoints]{.underline}**

Multiple secondary clinical endpoints were analyzed and included: time
to patient assessment of no illness, time to reduction in symptom
severity and resolution of individual symptoms, tissue use, time to
return to baseline activity level, and the impact of concomitant cold
symptom relief medication use.

FDA generally places the greatest emphasis on the primary endpoint.
Secondary endpoints may be supportive but results must be interpreted
with caution because of the large number of potential comparisons,
required adjustments for p values, and, in some cases, the small number
of patients included in the analysis. The results of a number of these
analyses are presented for completeness.

Analyses of time to patient assessment of no cold are presented in Table
6.

**Table 6. Time (days) to patient assessment of "no cold"**

+-----------------------+-----------+-----------+-----------+-----------+
|                       | #         |           | # Study   |           |
|                       | ### Study |           |  843-044  |           |
|                       |  843-043  |           |           |           |
|                       |           |           | **Placebo |           |
|                       | **Placebo |           | Ple       |           |
|                       | Ple       |           | conaril** |           |
|                       | conaril** |           |           |           |
+-----------------------+-----------+-----------+-----------+-----------+
| **ITT (n)**           | 526       | 526       | 524       | 520       |
|                       |           |           |           |           |
| Median                | 6.7       | 6.0       | 6.9       | 5.9       |
+-----------------------+-----------+-----------+-----------+-----------+
| **ITT-I (n)**         | 326       | 337       | 356       | 344       |
|                       |           |           |           |           |
| Median                | 6.9       | 6.5       | 7.4       | 6.3       |
+-----------------------+-----------+-----------+-----------+-----------+

Analysis of time to resolution of the individual symptoms of VRI was
assessed and the results are presented in Tables 7a and 7b.

# Table 7a. Median time (days) to resolution (score=0) of symptoms of VRI, ITT

+----------------------+-----------+----------+-----------+----------+
|                      | #         |          | # Stud    |          |
|                      | ### Study |          | y 843-044 |          |
|                      |  843-043. |          |           |          |
|                      |           |          | ##        |          |
|                      | ##        |          | ######  P |          |
|                      | ######  P |          | lacebo Pl |          |
|                      | lacebo Pl |          | econaril  |          |
|                      | econaril  |          |           |          |
+----------------------+-----------+----------+-----------+----------+
| **Rhinorrhea (n)**   | 490       | 486      | 495       | 489      |
|                      |           |          |           |          |
|                      | 6.0       | 6.5      | 6.5       | 5.5      |
+----------------------+-----------+----------+-----------+----------+
| **Congestion (n)**   | 467       | 472      | 482       | 471      |
|                      |           |          |           |          |
|                      | 60        | 6.0      | 6.0       | 6.0      |
+----------------------+-----------+----------+-----------+----------+
| **Cough (n)**        | 411       | 387      | 402       | 399      |
|                      |           |          |           |          |
|                      | 6.0       | 6.0      | 7.0       | 5.5      |
+----------------------+-----------+----------+-----------+----------+
| **Pharyngitis (n)**  | 396       | 352      | 375       | 380      |
|                      |           |          |           |          |
|                      | 4.0       | 3.5      | 3.5       | 3.0      |
+----------------------+-----------+----------+-----------+----------+
| **Malaise (n)**      | 448       | 448      | 454       | 445      |
|                      |           |          |           |          |
|                      | 4.5       | 4.0      | 4.0       | 4.0      |
+----------------------+-----------+----------+-----------+----------+
| **Myalgia (n)**      | 366       | 328      | 337       | 344      |
|                      |           |          |           |          |
|                      | 4.0       | 3.0      | 4.0       | 3.5      |
+----------------------+-----------+----------+-----------+----------+

Table 7b. Median time (days) to resolution (score=0) of symptoms of VRI,
ITT-I

+----------------------+-----------+----------+-----------+----------+
|                      | #         |          | # Stud    |          |
|                      | ### Study |          | y 843-044 |          |
|                      |  843-043. |          |           |          |
|                      |           |          | ##        |          |
|                      | ##        |          | ######  P |          |
|                      | ######  P |          | lacebo Pl |          |
|                      | lacebo Pl |          | econaril  |          |
|                      | econaril  |          |           |          |
+----------------------+-----------+----------+-----------+----------+
| **Rhinorrhea (n)**   | 284       | 302      | 323       | 311      |
|                      |           |          |           |          |
|                      | 7.0       | 6.0      | 7.5       | 6.0      |
+----------------------+-----------+----------+-----------+----------+
| **Congestion (n)**   | 274       | 294      | 318       | 305      |
|                      |           |          |           |          |
|                      | 6.5       | 5.5      | 6.5       | 6.0      |
+----------------------+-----------+----------+-----------+----------+
| **Cough (n)**        | 242       | 238      | 262       | 258      |
|                      |           |          |           |          |
|                      | 6.5       | 6.5      | 7.0       | 6.0      |
+----------------------+-----------+----------+-----------+----------+
| **Pharyngitis (n)**  | 228       | 212      | 243       | 234      |
|                      |           |          |           |          |
|                      | 3.5       | 3.0      | 3.5       | 3.0      |
+----------------------+-----------+----------+-----------+----------+
| **Malaise (n)**      | 261       | 276      | 307       | 287      |
|                      |           |          |           |          |
|                      | 4.5       | 4.0      | 4.0       | 3.5      |
+----------------------+-----------+----------+-----------+----------+
| **Myalgia (n)**      | 206       | 192      | 223       | 222      |
|                      |           |          |           |          |
|                      | 3.5       | 3.0      | 3.5       | 3.0      |
+----------------------+-----------+----------+-----------+----------+

Analysis of nights disturbed by VRI symptoms is presented in Table 8.

# Table 8. Median nights with sleep disturbance due to VRI symptoms

+-----------------------+-----------+-----------+-----------+-----------+
|                       | #         |           | # Study   |           |
|                       | ### Study |           |  843-044  |           |
|                       |  843-043  |           |           |           |
|                       |           |           | **Placebo |           |
|                       | **Placebo |           | Ple       |           |
|                       | Ple       |           | conaril** |           |
|                       | conaril** |           |           |           |
+-----------------------+-----------+-----------+-----------+-----------+
| **ITT w/sleep         | 440       | 414       | 427       | 419       |
| disturbance (n)**     |           |           |           |           |
|                       | 3.0       | 2.0       | 3.0       | 2.0       |
| Median                |           |           |           |           |
+-----------------------+-----------+-----------+-----------+-----------+
| **ITT-I w/sleep       | 282       | 276       | 300       | 289       |
| disturbance (n)**     |           |           |           |           |
|                       | 3.0       | 2.0       | 3.0       | 2.0       |
| Median                |           |           |           |           |
+-----------------------+-----------+-----------+-----------+-----------+

There was no difference in time to return to baseline level of activity
between pleconaril and placebo for the ITT or ITT-I populations, median
3.0 days.

At baseline each patient was dispensed acetaminophen and
dextromethorphan and instructed to use these relief medications only if
necessary. All use was to be documented in the patient diaries.
Approximately 50% of study participants used symptomatic relief
medications during the study, and there were no differences in the
frequency or duration of use between pleconaril and placebo-treated
patients.

**V. Safety Summary**

The adult VRI safety database includes information on 2,488 patients who
received at least one dose of pleconaril; 1,046 of whom received the
proposed dose of 400 mg TID for 5 days (15 doses), using the proposed
commercial tablet formulation.

Gastrointestinal events (abdominal pain, nausea, vomiting, diarrhea),
and headache were the most common adverse events reported in the adult
VRI safety database. Of significant concern is the apparent higher rate
of menstrual disorders among females treated with pleconaril and
concomitant progestin and/or estrogen-based oral contraceptives (OCs).
Another potential concern was the occurrence of tachycardia and
palpitations that was temporally related to administration of pleconaril
alone and in combination with theophylline. There were no significant
changes in clinical laboratory or hematologic parameters in the phase 2
and 3 VRI trials reviewed where pleconaril was dosed for 5-7 days.

Treatment emergent adverse events (all cause and all severity) observed
in the two pivotal VRI studies are presented in Table 9.

Table 9. Treatment-emergent adverse events in pivotal adult VRI studies,
(%)

+-----------------+------------------------+--------------------------+
| **Event**       | **Pleconaril**         | **Placebo**              |
|                 |                        |                          |
|                 | **(n=1046)**           | **(n=1040)**             |
+-----------------+------------------------+--------------------------+
| Headache        | 23                     | 21                       |
+-----------------+------------------------+--------------------------+
| Diarrhea        | 7                      | 7                        |
+-----------------+------------------------+--------------------------+
| Nausea          | 6                      | 4                        |
+-----------------+------------------------+--------------------------+
| Vomiting        | 2                      | 2                        |
+-----------------+------------------------+--------------------------+
| Abdominal pain  | 2                      | 4                        |
+-----------------+------------------------+--------------------------+
| Sinusitis       | 3                      | 2                        |
+-----------------+------------------------+--------------------------+
| Bronchitis      | 3                      | 3                        |
+-----------------+------------------------+--------------------------+
| Increased cough | 3                      | 3                        |
+-----------------+------------------------+--------------------------+
| Pain            | 3                      | 2                        |
+-----------------+------------------------+--------------------------+
| Fever           | 1                      | 2                        |
+-----------------+------------------------+--------------------------+
| Dizziness       | 2                      | 1                        |
+-----------------+------------------------+--------------------------+
| Hematuria       | 1                      | \<1                      |
+-----------------+------------------------+--------------------------+
| Otitis media    | 1                      | \<1                      |
+-----------------+------------------------+--------------------------+
| Rhinitis        | 2                      | 3                        |
+-----------------+------------------------+--------------------------+
| Allergic        | \<1                    | \<1                      |
| reactions       |                        |                          |
+-----------------+------------------------+--------------------------+
| Rash            | \<1                    | \<1                      |
+-----------------+------------------------+--------------------------+
| Menstrual       | 2                      | 1                        |
| disorders       |                        |                          |
+-----------------+------------------------+--------------------------+
| Abnormal LFTs   | \<1                    | 1                        |
+-----------------+------------------------+--------------------------+
| Palpitati       | \<1                    | \<1                      |
| ons/tachycardia |                        |                          |
+-----------------+------------------------+--------------------------+

There were no significant differences in the frequency of adverse events
between PCR+ and PCR- patients in studies 843-043 and 843-044.

Headache, primarily mild in severity, has been reported consistently by
18-20% of patients in the VRI safety database. In the two pivotal
studies, the frequency of headache was similar as was the number of
patients who discontinued due to headache, four pleconaril and six
placebo. Although some cases of headache were quite likely due to the
underlying VRI, many were reported during treatment and therefore may
have been treatment related. Other etiologies for headaches have not
been suggested.

Gastrointestinal-related adverse events were reported at a similar
frequency across the studies in the VRI safety database as shown in
Table 9. In the two pivotal studies, 17 (1.6%) and six (.05%) pleconaril
and placebo-treated patients, respectively, discontinued due to GI
adverse events. Review of discontinuations due to GI events demonstrated
a pattern: onset of symptoms was within 1-2 days of initiation of study
medication with resolution within 1-2 days of discontinuation. In
general, gastrointestinal symptoms are not part of the typical
constellation of symptoms reported by patients with VRI. One possibility
is that the GI events are formulation related. The placebo and
pleconaril formulations used in the pivotal studies contain sodium
laurel sulfate (SLS).[^3] Other etiologies for these gastrointestinal
events have not been suggested.

**Menstrual Disorders**

There was a significant increase in menstrual disorders (such as
epimenorrhea, intermenstrual bleeding, menstrual disorder, amenorrhea,
menorrhagia, early menses, prolonged menstrual cycle, and delayed
menses) among women treated with pleconaril and using
progestin/estrogen-containing OCs.

Menstrual disorders were identified in 7/229 (3.1%) of women who were
taking OCs during 5-7 days of treatment with pleconaril compared to
0/223 who were treated with placebo. Among women who received pleconaril
but were not using OCs, the rate of menstrual disorders was 0.1%.

Additional information on menstrual disorders from a recently completed
six-week prophylaxis study were submitted and reviewed. The observed
results are suggestive of a dose response relationship. At least one of
the menstrual disorders listed above was observed in 20 (25%), 52 (53%),
and 40 (69%) women taking placebo, pleconaril 400 mg QD, or pleconaril
400 mg BID and an OC. The most common events were epimenorrhea and
intermenstrual bleeding.

During week one of the prophylaxis study, the prevalence of menstrual
disorders was 3%, which was the same as the prevalence in the 5-7 day
treatment trials. The prevalence peaked by week three to 28%, and began
to decrease by week seven.

Also, there appeared to be a potential dose response for the occurrence
of epimenorrhea and intermenstrual bleeding among women receiving
pleconaril and not taking OCs; however the numbers of patients in this
analysis are very small.

In animal studies, pleconaril was not found to be mutagenic,
teratogenic, or genotoxic, and did not negatively impact viability or
normal development, including reproductive performance of the offspring.
In a 6-month toxicology study conducted in rats, mild to moderate
macroscopic uterine horn distention was observed at necropsy in low (10
mg/kg BID) and high dose animals (50 mg/kg BID). Corresponding
microscopic uterine horn dilatation was observed in two of two low dose
animals and five of 15 high dose animals. These findings were also
present in one high dose animal at the end of the two-month recovery
period. At the time the toxicology study was reviewed, these findings
were not considered sufficient to warrant a hold for a dosing duration
of five days, since no similar toxicities were noted in one-month dog
and rat studies or in six month dog studies.

A total of 13 (eight pleconaril and five placebo) pregnancies have been
reported in women receiving pleconaril in 5-7 day treatment and six-week
prevention studies. Of the eight pregnancies that occurred in pleconaril
recipients, two occurred in women taking an OC at the time they became
pregnant. One pregnancy terminated in an abortion and one pregnancy is
ongoing. All the other women were using barrier methods at the time they
became pregnant.

The etiology of the menstrual disorders has not yet been determined.
Possible mechanisms include CYP 3A4 induction, changes in intestinal
flora, interference in enterohepatic circulation, displacement of
albumin binding of estrogen, changes in sex hormone binding globulin,
changes in steroid receptor binding, and partial antagonism of estrogen
receptors.

Two studies investigating changes in serum progesterone, luteinizing
hormone and follicle-stimulating hormone are ongoing. A third study
investigating the possibility of induction of the CYP 3A4 enzyme system
using midazolam is also ongoing. Very preliminary data suggests that
pleconaril causes induction of CYP 3A4 and decreases ethinyl estradiol
exposures. Additional data from these studies will be presented at the
AVAC meeting.

**Tachycardia/Palpitations**

A potential pharmacodynamic interaction between pleconaril and
theophylline was identified. During the conduct of an interaction study,
four of 14 subjects experienced palpitations, tachycardia, dizziness,
and/or syncope, during the combination dosing period. In addition,
general adverse events of palpitations, nausea, vomiting, abdominal
pain, and dizziness were reported more frequently during the
theophylline/pleconaril periods. At the time of these events, no
significant changes in either theophylline or pleconaril
pharmacokinetics noted were noted.

In the adult VRI database 18 patients (eight placebo and 10 pleconaril)
reported cardiac-related adverse events for which a cardiac etiology
could not be ruled out. Nine patients, seven pleconaril and two placebo,
complained of tachycardia and/or palpitations. Of these, six of the
pleconaril patients reported tachycardia and/or palpitations within one
hour of ingestion of pleconaril, four discontinued from treatment due to
tachycardia and/or palpitations, one patient's event was considered
serious. No patients underwent re-challenge following resolution of the
events.

There were no apparent differences in heart rate or blood pressure noted
in the VRI safety database.

# Proposed Phase 4 Studies

The applicant has proposed the following post-approval activities:

-   Conduct additional pediatric VRI studies using an endpoint of time
    to 50% reduction in symptoms

```{=html}
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```
-   Assess pleconaril's ability to prevent VRI in patients with
    underlying pulmonary disease (e.g., chronic asthma and COPD)

```{=html}
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```
-   Assess transmission of VRI and development of resistance

VII\. Issues for Discussion

Listed below are a number of issues for you to consider during the
discussion period. Please note that these issues may change prior to the
meeting.

1.  Has the applicant established efficacy of pleconaril for treatment
    of acute picornavirus VRI in adults?

Please consider the following points as you discuss this question:

-   Methods used to identify infected patients

-   Variability of treatment effect across the phase 2 studies, and
    studies 843-043 and 843-044

-   Determination of the primary efficacy population

-   The need to institute treatment within 24 hours of onset of the
    first symptoms

-   Results in males versus females

-   Results in smokers versus non-smokers

1b. If the answer to question 1 is yes, please provide your
recommendations for labeling. Please include a discussion of the
populations and sub-populations for which pleconaril should or should
not be labeled, and any recommendations for statements to clarify how
pleconaril should be used.

1c. If the answer to question 1 is no, please discuss what additional
data would be helpful to establish pleconaril's efficacy.

2.Has the applicant demonstrated safety in adult patients with symptoms
of acute VRI?

Please consider the following points as you discuss this question:

-   The types and frequencies of adverse events are generally similar
    between pleconaril and placebo, with headache and gastrointestinal
    symptoms occurring most frequently.

-   The frequency of menstrual disorders occurring in females using OCs
    while being treated with pleconaril.

-   Tachycardia and palpitations were reported in \<1% of otherwise
    healthy patients treated with pleconaril, and were pronounced in a
    theophylline/pleconaril interaction study. Patients with significant
    pulmonary and cardiovascular disease were not included in any of the
    studies.

2a. If the answer to question 2 is yes, please provide any
recommendations for labeling. Please include a discussion of the
populations and sub-populations for which pleconaril should or should
not be labeled, and recommendations for specific safety related
statements that should be included in the labeling.

2b. If the answer to question 2 is no, please discuss what additional
data would be helpful to establish pleconaril's safety.

3.Please provide comments on the applicant's proposed phase 4 studies.
Please also provide suggestions for any other study designs or patient
populations that should be studied as phase 4 commitments.

**APPENDIX A**

**SUMMARY OF VIROLOGIC METHODS**

# 

Additional studies to assess the performance of the TaqMan assay to
identify picornavirus-infected patients are ongoing and results are
expected in early March; these data will be available for discussion at
the AVAC meeting and may supercede the concerns outlined below.

# General Overview of the Assay

The basis for Taqman PCR is to continuously measure PCR product
accumulation using a dual-labeled flourogenic oligonucleotide probe
called a TaqMan^®^ probe. This probe is composed of a short (ca. 20-25
bases) oligodeoxynucleotide labeled with two different fluorescent dyes.
On the 5\' terminus is a reporter dye and on the 3\' terminus is a
quenching dye. This oligonucleotide probe sequence is homologous to an
internal target sequence present in the PCR amplicon. When the probe is
intact, energy transfer occurs between the two flourophors and the
quencher quenches emissions from the reporter. During the extension
phase of PCR, the probe is cleaved by 5\' nuclease activity of Taq
polymerase thereby releasing the reporter from the
oligonucleotide-quencher and producing an increase in reporter emission
intensity

The fluorescence intensity of reporter and quencher dyes is measured and
the increase in normalized reporter emission intensity over the course
of the amplification is calculated. The results are then plotted versus
time, represented by cycle number, to produce a continuous measure of
PCR amplification. To provide precise quantification of initial target
in each PCR reaction, the amplification plot is examined at a point
during the exponential phase of amplification. This is accomplished by
assigning a fluorescence threshold above background and determining the
time point at which each sample\'s amplification plot reaches the
threshold (defined as the threshold cycle number or CT). Differences in
threshold cycle number can be used to quantify the relative amount of
PCR target contained within each tube.

![](media/image2.png){width="5.208333333333333in" height="5.1in"}

# 

# Critical Assay Parameters

# [TaqMan® Probe and Primers]{.underline} 

1\) As noted by Applied Biosystems, the manufacturer of the TaqMan
system, the use of Primer Express® software in designing primers and
probes is critical for the successful development of an assay. The assay
under review did use this software for primer and probe design, however
there are at least two areas of concern. As noted by the Primer Express
software:

**TaqMan® Probe**:

-   Avoid runs of an identical nucleotide. This is especially true for
    guanine (G), where runs of four or more Gs should be avoided

**TaqMan® Primers:**

-   The five nucleotides at the 3\' end should have no more than two G
    and/or C bases

2)  Because of heterogeneity between rhino and enteroviruses, and that
    the assay used only two primers, it is not clear how effective the
    assay can be at detecting all various serotypes. In addition, as
    noted below there was significant variation in the level of
    detection of the serotypes. Therefore, these primer-binding
    characteristics have the ability to significantly affect both the
    sensitivity and specificity of the assay.

**[Threshold Value]{.underline}**

The threshold is the numerical value assigned for each run that reflects
the average DRn (change in fluorescence) during the initial cycles of
PCR (baseline). The threshold should be placed in the region of
exponential amplification across all of the amplification plots. This
region is depicted in the log view of the amplification plots as the
portion of the plot that is linear. The threshold line should neither be
placed in the plateau phase or in the initial linear phase of
amplification. In the case of the ABI 5700 the threshold is manually
adjusted so there may be variation in where individual investigators
believe placement should be set.

*It is possible that the threshold currently set for this assay, 0.1,
may be too low and a more appropriate threshold would be in the range of
0.4 -- 0.6.1. While there is a range of threshold values that could be
appropriate, incorrect threshold placement could result in an incorrect
assay definition of positivity.*

*A low threshold value would subject individual samples to assay
background and could impact the relative quantitative results of the
assay.*

*In the clinical studies, 67% (6/9) of the assay failures were due to
the negative control crossing the threshold value.*

**[Number of Cycles]{.underline}**

The current assay is run for a total of 60 cycles. In general, most
assays are run for a maximum of 45 cycles.

*The 60-cycle reaction could allow for an increase in non-specific
amplification in samples that contain weakly homologous regions.*

*Any non-specific amplification would also have an adverse effect on the
sensitivity and specificity of the assay.*

*Possible resolutions to this issue would include analyzing the reaction
products by ethidium bromide-stained agarose (acrylamide) gel
electrophoresis, Southern blotting/probe hybridization, or fluorescence
assay. These were requested of ViroPharma but have not been received to
date.*

**Pre-Clinical Studies**

**[Validation Reports]{.underline}**

1)  The validation report in the original NDA submission identified the
    primers and probe selected for the assay using the appropriate
    Primer Express software and these would generate a 67-68 nucleotide
    (nt) fragment.

*A validation report from ViroMed, who performed the TaqMan assays for
the applicant, states that the primers and probe selected using the
Primer Express software generated a 150 nt fragment; ViroPharma will
clarify the exact length of the nt fragment.*

**[Detection Limits (Analytical Sensitivity)]{.underline}**

1)  Detection limits vary widely between serotypes. Detection limits
    were expressed in genome equivalents (GE)/plaque forming units
    (pfu). Five human rhinovirus (HRV) serotypes representing both major
    (serotypes 14,16, and 89) and minor (serotypes 1A and 1B) receptor
    binding groups were used to establish the limit of detection.

  ----------------------- ----------------------- -----------------------
                          Analytical Sensitivity  

  HRV                     GE (RNA) per reaction   GE/pfu

  1A                      30                      132

  1B                      50                      1055

  14                      1289                    12675

  16                      47                      408

  89                      2036                    33,068
  ----------------------- ----------------------- -----------------------

*Given that particle/pfu ratios for picornaviruses are generally between
200 --1000^2^ and since there should be no more than one genome per
virus particle (or less), these GE/pfu ratios appear to be overstated.*

*The analytical sensitivity varied over 60-fold, from 30 to 2,036 GE per
reaction depending on virus serotype.*

*There is a discrepancy between the claimed limit of detection (LOD) of
the TaqMan assay. In the ViroMed validation report the LOD is stated as
1.0 pfu/ml while in the studies submitted by ViroPharma, the listed LOD
is 10 pfu/ml.*

*The exact quantitative nature of the TaqMan assay cannot be evaluated
as the calibrators also serve as the positive control for the assay and
therefore will only control for substantial reagent failure. When
control and calibrators are the same material, the calibrators will
appear to function within the parameters of each assay but there may be
substantial inter-assay variation that will not be detected.*

*When used with spiked clinical material (nasal mucus) the detection
limits of the TaqMan assay varied among the serotypes by 2000 fold.*

*.*

# Clinical Data

# [Virus Culture]{.underline}

1)  Two virus culture systems were utilized in the clinical studies.

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a)  In one, samples of nasal mucus specimens were inoculated into WI-38
    cells (human embryonic lung cells), inculbated at 33° C, and
    examined for cytopathic effect (cpe) over the course of 14 days.

b)  The second culture system employed Hela I-CAM cells with other
    conditions being similar.

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2)  Only samples positive in the TaqMan assay were analyzed by cell
    culture, no negative specimens were tested.

3)  Positive cultures were not assessed for picornavirus positivity.
    Although cultures were deemed positive for cpe, this was not
    confirmed to be due to picornavirus by pH lability, identification
    of the serotypes, immunofluorescent assay, or DNA sequence analysis.

*Thus, while a positive culture was suggestive of picornavirus, the
applicant provided no evidence for either the presence or absence of
actual picornavirus infection.*

# [Clinical Sensitivity]{.underline}

1)  In Study 843-032 when compared to culture, the TaqMan assay had a
    sensitivity of 93% (88-97%) and specificity of 75% (72-78%).

2)  An "expanded gold standard" was used to re-evaluate sensitivity and
    specificity. "True positives" were considered to be either (i) virus
    culture positives regardless of PCR results, or (ii) TaqMan and
    ELOSA positives regardless of culture results.

*There is no valid measure for specificity for either assay, and neither
result has been independently confirmed.*

*Statistically this analysis will always favor the PCR result, since one
PCR assay is being "confirmed" by a second PCR assay.*

**APPENDIX B**

REVIEW OF SUPPORTIVE STUDIES

The design and primary endpoint analysis for each of four phase 2
studies are presented below. Presented below are analyses of the primary
endpoint for each study.

Study 843-010 was the first study to investigate the safety and efficacy
of pleconaril in the treatment of VRI. The primary endpoint was time to
complete resolution of all systemic symptoms (score of 0) for 48 hours
among the all randomized (ITT) patient population. Nasal mucus was PCR+
for picornavirus in 29% percent of patients, too few upon which to
conduct an analysis.

Table 2. Time (days) to complete resolution of all systemic symptoms for
48 hours

+-----------------+-----------------+-----------------+-----------------+
|                 | **Placebo**     | **Pleconaril    | **Pleconaril    |
|                 |                 | 200 mg TID**    | 400 mg TID**    |
+-----------------+-----------------+-----------------+-----------------+
| **ITT (n)**     | 74              | 73              | 74              |
|                 |                 |                 |                 |
| Median          | 10              | 9               | 10              |
|                 |                 |                 |                 |
| p-value versus  |                 | 0.501           | 0.504           |
| placebo         |                 |                 |                 |
+-----------------+-----------------+-----------------+-----------------+

Study 843-013 evaluated two doses of pleconaril (200 mg or 400 mg TID
for seven days) in the treatment of rhinovirus VRI in patients with
bronchial asthma. The purpose was to determine if treatment of VRI in
patients with asthma might reduce the morbidity and mortality associated
with such exacerbations. The low rates of asthma exacerbations and
picornavirus PCR+ patients precluded any efficacy assessments.

**Study 843-020** evaluated the safety and efficacy of two doses of
pleconaril (400 mg BID or TID for seven days) for treatment of
picornavirus VRI. The primary endpoint was time to reduction of a
composite symptom score to 0 (all signs and symptoms absent) for two
consecutive days (48-hours) in adult patients with acute picornavirus
VRI. Nasal mucus was PCR+ for picornavirus in 41% of patients.

Table 3. Time (days) to resolution of all VRI symptoms sustained for 48
hours

+----------------+-----------------+----------------+-----------------+
|                | **Combined      | **Pleconaril   | **Pleconaril    |
|                | Placebo**       | 400mg BID**    | 400mg TID**     |
+----------------+-----------------+----------------+-----------------+
| #              | 340             | 335            | 347             |
| ###### ITT (n) |                 |                |                 |
|                | 8               | 8              | 8               |
| Median         |                 |                |                 |
|                |                 | 0.31           | 0.011           |
| p-value versus |                 |                |                 |
| placebo        |                 |                |                 |
+----------------+-----------------+----------------+-----------------+
| ###            | 128             | 136            | 147             |
| #### ITT-I (n) |                 |                |                 |
|                | 8               | 8              | 9               |
| Median         |                 |                |                 |
|                |                 | 0.69           | 0.70            |
| p-value versus |                 |                |                 |
| placebo        |                 |                |                 |
+----------------+-----------------+----------------+-----------------+

**Study 843-032** evaluated the safety and efficacy of pleconaril 400 mg
TID or matching placebo TID for seven days (total 21 doses) in patients
\>14 years of age presenting with acute VRI. The first dose of study
medication was to be administered within 36 hours of the onset of
symptoms. The primary endpoint was time to alleviation of all VRI
symptoms sustained for 24 hours. Nasal mucus was PCR+ for picornavirus
in 43% of patients.

Table 4. Time (days) to resolution of all VRI symptoms sustained for 24
hours

+-----------------+-----------------+-----------------+-----------------+
|                 | **Placebo**     | **Pleconaril**  | **Difference**  |
|                 |                 |                 |                 |
|                 |                 | **400mg TID**   | **p-value**     |
+-----------------+-----------------+-----------------+-----------------+
| ####### ITT (n) | 439             | 436             | 0.0             |
|                 |                 |                 |                 |
| Median          | 9.0             | 9.0             | 0.7             |
+-----------------+-----------------+-----------------+-----------------+
| **ITT-I (n)**   | 280             | 240             | 0.5             |
|                 |                 |                 |                 |
| Median          | 9.5             | 9.0             | 0.11            |
+-----------------+-----------------+-----------------+-----------------+

**APPENDIX C**

# SUMMARY OF PHARMACOKINETICS, DOSE AND REGIMEN

The applicant conducted an extensive PK development program. Unless
otherwise noted, the PK studies involved healthy volunteers between 18
and 75 years of age. Relevant findings and issues raised by
pharmacokinetic studies that are applicable to the adult VRI indication
include:

-   Pleconaril reaches maximal plasma concentration (C~max~) about 3.0
    hours (range 2.0-5.0 hours) after oral administration.

-   The bioavailability of pleconaril is increased by 4-6.5 fold after
    administration of pleconaril with a meal high in calories, fat, and
    protein, as compared to administration of pleconaril in a fasted
    state.

-   Pleconaril is highly bound (99.8%) to plasma proteins.

-   Following oral absorption, pleconaril displays a bi-exponential
    disposition profile with a short alpha half-life (2-3 hours) and a
    long terminal half-life (180 hours).

-   The pharmacokinetics following a single dose may not predict
    multiple dose pharmacokinetics, due to the prolonged half-life after
    multiple doses (1010 hours after 400 mg TID for 5 days) compared to
    that after single dose (180 hours).

-   Plasma accumulation of pleconaril is modest (2-fold) after
    pleconaril 400 mg TID for 5 days. Steady state is not reached after
    5 days of treatment with pleconaril.

-   Mass balance was not achieved following 288 hours of collection of
    urine and feces after a single radiolabeled oral dose of 200 mg
    pleconaril under fed conditions (37.6% of administered radioactivity
    recovered in urine and feces).

-   Pleconaril is excreted in urine and feces as multiple inactive
    metabolites. The cytochrome P450 drug metabolizing enzymes are not
    significantly involved in the major biotransformation pathways of
    pleconaril. Therefore, co-administered drugs that are CYP450
    substrates, inhibitors or inducers are not expected to alter the
    pharmacokinetic profile of pleconaril.

-   *In vitro* studies showed that pleconaril may be a weak CYP1A2 (Ki:
    8.8 μM), CYP2C9 (Ki: 6.4 μM), and CYP2C19 (Ki: 10 μM) inhibitor. The
    *in vivo* pleconaril-theophylline drug-drug interaction study showed
    that pleconaril increased theophylline AUC by 15%, but not Cmax.
    Palpitations, syncope and tachycardia were observed during the
    theophylline and pleconaril combination treatment but not during
    theophylline alone treatment. These symptoms appeared to be
    unrelated to theophylline AUC or Cmax. Pleconaril had no effect on
    pharmacokinetics of S- or R-warfarin.

-   An increased incidence of menstrual disorders, including
    break-through bleeding, was observed in pleconaril treated women who
    took oral contraceptives, both in prophylaxis and treatment studies.
    The potential for pleconaril induction of CYP450 enzymes is
    currently under investigation.

-   The pharmacokinetic profile of pleconaril is not clinically
    significantly changed due to age or renal impairment.

-   Hepatic impaired subjects have 40% lower pleconaril exposures
    compared to healthy subjects after a single 400 mg pleconaril oral
    liquid dose. The difference may be due to lower fat and meal
    consumption in hepatic impaired subjects compared to healthy
    subjects during the study or reduced albumin level in hepatic
    impaired subjects. Thus, the results of this study are inconclusive.

-   The population PK study was inconclusive due to insufficient blood
    samples, inappropriately fixed parameters (using ka, Q/F, V~2~/F and
    t~lag~ values from Phase I studies with different formulations of
    pleconaril), and poor prediction of observed concentrations.

-   The dose selected (400 mg TID for 5 days) may not be optimal based
    on the free drug concentrations achieved in the plasma and estimated
    nasal tissue concentration from a rat model, compared to non-protein
    adjusted MIC~90~ of pleconaril for piconavirus. Also, no higher dose
    was studied and no exposure-response was evaluated.

[^1]: Tissue use was measured during the study as a surrogate for the
    severity of rhinorrhea.

[^2]: The endpoint evolved through numerous derivations and symptom
    scoring systems, and represents conclusions reached primarily from
    post-hoc analyses of previously completed studies. The endpoint
    represents assessments of four components hypothesized to be
    important in assessing treatment response in patients with VRI.
    Absence of rhinorrhea was chosen because it represents the
    predominant symptom of VRI. Alleviation of other symptoms to absent
    or mild provides outcome data on the more acute phase of VRI. The
    48-hour observation period was chosen to ensure that sufficient time
    elapsed to document that the VRI had ended. Finally, absence of
    concomitant cold medication would likely ensure that patients would
    not report a reduced duration of VRI that could have been attributed
    to use of symptom relief cold medications.

[^3]: SLS is a generally recognized as safe (GRAS) anionic emulsifying,
    detergent used as a tablet lubricant and wetting agent in cosmetics
    and topical pharmaceutical products. It is a moderately toxic agent
    with irritating effects to the eyes, skin, mucous membranes, upper
    respiratory tract, and stomach. Handbook of Pharmaceutical
    Excipients, 3^rd^ edition, Kibbe AH, ed; 2000, 487-89.