Study Participants

Participants were 18 to 45 years of age and had a median serum neutralizing antibody titer for the Memphis-37b challenge virus of 973 Mu (range, 187 to 2023 Mu), which represented the lowest quartile of the screened population.14,15 The selection criteria are available in the Supplementary Appendix and in the protocol, which is available at NEJM.org.

Study Design

We conducted this randomized, double-blind, placebo-controlled study over three separate study periods. Up to 22 eligible participants per period were confined to a specialized quarantine unit for 14 days (starting 2 days before inoculation and ending 12 days after inoculation [study days −2 to 12]), and outpatient assessments were conducted on study days 16 and 28 (Fig. S1 in the Supplementary Appendix). On study day 0, participants were inoculated intranasally with 4 log 10 plaque-forming-units (PFUs) of the RSV-A Memphis 37b challenge virus.16 From study day 2 until randomization, we monitored participants twice daily for RSV infection, using a Simplexa (Focus Diagnostics) qualitative reverse-transcriptase–polymerase-chain-reaction (RT-PCR) assay. The samples tested were fresh, nonfrozen, nasal washings. Participants underwent randomization and received the first dose of ALS-008176 or placebo approximately 12 hours after the qualitative detection of RSV or on the morning of day 6, whichever occurred first.

The number of participants receiving each dose and the dosing regimens for the first study period were predefined. The protocol allowed for the modification of these specifications in subsequent study periods, by a monitoring group whose members were aware of the study-group assignments, on the basis of emerging data on the numbers of participants who became infected after inoculation, the timing of that infection relative to randomization, safety, and pharmacokinetic and viral kinetic data (see Section 4.0 and Fig. S1 in the Supplementary Appendix, and the protocol).

In each of the three study periods, ALS-008176 or matching placebo was administered orally every 12 hours over 5 days (10 total doses). Three ALS-008176 dosing regimens were evaluated over the three study periods: a single loading dose of 750 mg followed by 9 maintenance doses of 500 mg (group 1), a single loading dose of 750 mg followed by 9 maintenance doses of 150 mg (group 2), and 10 doses of 375 mg (group 3). The study medication was prepared by a pharmacist who was aware of the treatment assignments. The placebo consisted of methylcellulose and water, which was also used to administer ALS-008176. The investigators and all other members of the study team were unaware of the treatment assignments.

Study Oversight

The study was conducted in accordance with the Declaration of Helsinki (1996 version), the International Conference on Harmonisation Good Clinical Practice guidelines, applicable regional and local regulations, and the study protocol. The protocol was approved by the Medicines and Healthcare Products Regulatory Agency and the Cambridge East Regional Ethics Committee of the National Research Ethics Service, all in the United Kingdom. All participants provided written informed consent. The study was sponsored by Alios BioPharma and was designed and managed by the sponsor and the first author; the sponsor and the first author were also responsible for the data analysis. All authors were involved in the collection, analysis, or interpretation of the data. The authors vouch for the accuracy and completeness of the data and analyses and for the fidelity of the study to the protocol.

End Points and Assessments

The prespecified primary end point was the area under the curve (AUC) for viral load in nasal washes, as determined by the measurement of RSV RNA with the use of a quantitative RT-PCR assay.14,15,17,18 Measurements were obtained immediately before administration of the first dose of the study drug or placebo through day 12. Secondary end points included time to nondetectability of the virus, the slope of the curve for viral load during the first 24 to 48 hours after the start of dosing, peak viral load, scores for symptoms related to RSV, and the AUC for mucus weight. All efficacy data were collected and analyzed by study team members and technicians who were unaware of the treatment assignments.

After the inoculation of participants on study day 0, the RSV load in nasal-wash samples was measured twice daily from study days 2 through 12 and once on study days 16 and 28.18 Specimens were stored at a temperature of −80°C from the time of sample collection until the time of analysis. Results were reported as log 10 PFU equivalents (PFUe) per milliliter. In aliquots of nasal washes, we also amplified and sequenced the RSV L gene region that encodes the polymerase domain (amino acids 550 to 1100) to evaluate for mutations developing during treatment that conferred resistance to the study drug. This region includes the sites of all amino acid substitutions previously shown to confer in vitro resistance to ALS-008112 (M628L, A789V, L795I, I796V). There are no other known sites of ALS-008112 resistance. Amino acid sequences were compared with a participant’s first available RSV-positive sample and with the reference RSV Memphis 37b sequence.16 The criteria for selecting participants for the evaluation of resistance are defined in Section 5.0 in the Supplementary Appendix. Although none of the participants met the prespecified criteria for the evaluation of resistance, 29 participants (17 receiving ALS-008176 and 12 receiving placebo) were selected for evaluation on the basis of less stringent, post hoc criteria (defined in Section 5.0 of the Supplementary Appendix).

Symptoms of RSV infection were assessed three times daily during confinement and once on study day 16 with the use of an established participant-reported symptom score.15 Mucus weight was determined every 24 hours by measuring the net weight of secretions within facial tissues.

Safety data were collected through study day 28 (±3 days). The principal investigator, who was unaware of the treatment assignments, regularly assessed adverse events for the possibility of a relationship between an event and the study medication. Symptoms recorded on diary cards by participants were documented as adverse events only if the investigator considered them to be substantial. Plasma samples were collected and analyzed with the use of high-performance liquid chromatography and tandem mass spectrometry to characterize the pharmacokinetic assessments of ALS-008176, ALS-008112, and other metabolites after each dose of ALS-008176.

Statistical Analysis

All participants who were inoculated with the challenge virus and received at least one dose of study drug were included in the safety (intention-to-treat [ITT]) population (Fig. S2 in the Supplementary Appendix). Participants who were RSV-positive according to the results of quantitative RT-PCR assay immediately before dosing (baseline) or who subsequently became positive on at least two occasions after baseline were defined as infected and were included in the primary analysis population for efficacy evaluation (ITT-infected population). Pharmacokinetic analysis was performed on all participants in the ITT population from whom at least one blood specimen had been obtained for the purpose of pharmacokinetic measurement.

On the basis of an enrollment of 20 participants per study period and three study periods, the study had a power of approximately 80% to detect a 50% reduction in the AUC for viral load for the comparison of active, drug-containing regimens with placebo, assuming a 45% coefficient of variation in the AUC for viral load. Up to 22 participants per study period were enrolled to allow for dropouts. Randomization was performed with the use of a permuted-block algorithm.

The primary end point was assessed for each of the three regimens for ALS-008176 as compared with placebo with the use of a mixed-effects model that included a repeated-measures approach to allow for unequal variances, with baseline viral load as a covariate. The errors were assumed to be normally distributed, and a standard covariance structure was assumed. The null hypothesis was that there was no difference between each of the three active treatment regimens and placebo. This same model was used to assess the following secondary end points: the slope of the viral load during the 24-hour and 48-hour periods after first dose, peak viral load, and total RSV symptom score for the AUC (from the time just before administration of the first dose to study day 16).

Between-group differences were evaluated with the use of an analysis-of-variance model, with treatment as the dependent variable for the following secondary end points: time to the point at which RSV was not detectable, time to symptom resolution, and the AUC for mucus weight (from the time just before administration of the first dose to study day 12). ALS-008176 dosing regimens were individually compared with the pooled placebo group, and all comparisons were two-sided, with the level of significance set at 0.05. The results for participants receiving the same dosing regimen in different study periods were pooled and analyzed as a single treatment regimen.