The design and detailed results from the first 4 years of this randomized, double-blind, placebo-controlled, parallel-group, multicenter, Phase 3 trial conducted at 48 sites in Austria, Canada, Czech Republic, Denmark, France, Germany, Italy, Poland, Russia and Slovakia, have been reported previously [12,13]. The trial originally included 3 years of active treatment and 1 year of treatment-free follow-up to investigate the long-term efficacy of the 300IR 5-grass pollen tablet. At the end of year 3, the Data Safety Monitoring Board recommended that the study be extended for an additional year of treatment-free follow-up (year 5).
In brief, during the first 3 years of the study (2007–09), patients were randomized 1:1:1 using a computer-generated list to either placebo or 300IR 5-grass pollen tablet, administered according to a discontinuous pre- and coseasonal protocol beginning at 4 or 2 months (300IR (4 M) and 300IR (2 M), respectively) prior to the expected start of the grass pollen season and maintained until season’s end. Patients did not receive investigational product during years 4 and 5 of the study (2010–11). Patients and investigators remained blinded until the end of the fifth year of the study.
The study protocol (European Union Drug Regulating Authorities Clinical Trials (EudraCT) number 2006-003258-98) was reviewed and approved by local regulatory authorities and independent ethics committees in each country, and the study and year 5 extension were conducted according to the Declaration of Helsinki and Good Clinical Practice-International Conference on Harmonisation guidelines. Patients provided their written informed consent before the start of any study-associated procedure, and reconsented prior to participation in the year 5 extension.
Patients were aged 18–50 years with documented grass pollen-related ARC for at least the two previous grass pollen seasons, a positive skin-prick test (wheal diameter >3 mm) to 5-grass-pollen-mix extract, Phleum pratense (timothy grass)-specific serum IgE ≥0.7 kU/L, and a retrospective rhinoconjunctivitis total symptom score (RRTSS) ≥12 (on a 0–18 scale), based on the most severe days in the grass pollen season preceding enrolment.
Exclusion criteria were ARC symptoms during the grass pollen season due to allergens other than grass pollen, asthma requiring treatment with more than a beta-2 agonist, or presence of any other disease that might affect the participation or outcome of the study.
During the first 3 years of the study, patients received a daily sublingual tablet containing either 300IR 5-grass pollen extract or placebo, according to the discontinuous dosing schedule described above. Years 4 and 5 of the study (i.e., the fourth and fifth pollen season) were 300IR 5-grass pollen tablet treatment-free.
To maintain blinding, active and placebo tablets were identical in appearance and taste, and patients randomized to the 2 M dosing regimen received placebo for the 2 months prior to starting active treatment.
The use of oral antihistamines, antihistamine eye drops, nasal corticosteroids and oral corticosteroids as rescue medications was permitted throughout the study, following a stepwise regimen defined in the study protocol, in which oral corticosteroids could only be considered once the other types of medications had proven inadequate at managing severe or intolerable ARC symptoms.
Grass pollen season
During each pollen season, pollen counts were monitored and recorded daily in the regions where study sites were located. The pollen period for statistical analysis was defined as starting on the first of 3 consecutive days with a grass pollen count of ≥30 grains/m3 and ending on the last of 3 consecutive days with a grass pollen count of ≥30 grains/m3.
The main outcome measures have been reported previously [12,13]. From approximately 3 weeks before each pollen season until the end of that season, patients recorded in a daily diary six individual rhinoconjunctivitis symptom scores (RSSs: sneezing, rhinorrhea, nasal pruritus, nasal congestion, ocular pruritus and watery eyes) and use of rescue medication during the previous 24 h. Severity of symptoms was rated using a 4-point descriptor scale from 0 (absent) to 3 (severe), and the sum of all six individual RSSs comprised the rhinoconjunctivitis total symptom score (RTSS, range 0–18) . The rescue medication score (RMS, range 0–3) was calculated using the following scale: 0, no rescue medication; 1, use of antihistamine (oral and/or eye drops); 2, use of nasal corticosteroid; and 3, use of oral corticosteroid. If a patient took two or more rescue medications on the same day, the highest score was used for the RMS . Efficacy analyses were performed on the full analysis set (FAS). In contrast to other symptoms scores, no last observation carried forward data adjustment is carried out with the Average adjusted symptom score (AAdSS), thereby reducing the potential for skewing of the data.
After the study had commenced, guidance from health authorities and professional societies regarding the primary efficacy endpoint of AIT clinical trials subsequently recommended the use of a score that considers both symptoms and rescue medication use, to avoid an underestimation of symptoms severity. In response to this guidance, the average adjusted symptom score (AAdSS)  was introduced as primary efficacy measure [12,13]. Results for the AAdSS have been reported previously [12,13].
The AAdSS was calculated per patient as the average of the non-missing daily adjusted symptom scores (AdSSs, range 0–18), which were derived from the daily RTSS (DRTSS) adjusted for rescue medication use over 2 consecutive days .
Secondary efficacy measures included individual RSSs; Average Combined Score (ACS, range 0–3); DRTSS; daily RMS (DRMS); ARTSS (range 0–18); and average RMS (range 0–3). All average scores were patient-specific and calculated as the average of the non-missing daily scores during the pollen season while the patient was on treatment . In response to regulatory authorities recommending use of a combined symptom and rescue medication score , a post-hoc analysis was performed using an alternative measure for the evaluation of efficacy: DCS (range 0–3), which assigns equivalent weighting to both symptom and rescue medication scores, and is calculated as [(DRTSS/6) + DRMS)]/2 [8,14]. DCS is a genuine combined score, unlike AdSS, which is not a combined score per se, but a symptom score that is adjusted by taking into account the use of allergy rescue medication .
In this paper, results using the alternative efficacy measure, DCS, are presented.
Safety was assessed through monitoring of adverse events (AEs), laboratory parameters, physical examination and vital signs. AEs were recorded throughout the study and classified according to the Medical Dictionary for Regulatory Activities (MedDRA; version 9.1).
The results of a previous pivotal, dose-finding study in Europe of the 5-grass pollen tablet for the treatment of ARC  suggested that a sample size of 144 patients per group would have 80% power to detect a difference of 20% between the active-treatment and placebo groups in AAdSS during the third on-treatment pollen season (primary endpoint), given a type I error of 5% and a common standard deviation (SD) of 3.6.
For post-treatment, long-term efficacy assessment, assuming a screening failure rate of 12% each treatment year and a comparable 12% drop-out rate for the fourth year, 127 patients per group would have 75% power to detect a 20% difference in AAdSS for post-treatment long-term efficacy. No formal pre-defined power calculation was performed for the analysis of year 4 and year 5.
Statistical analyses were performed using SAS® software (version 9.1.3, SAS Institute Inc., Cary, NC, USA). The safety sets in year 4 and year 5 included all patients who received ≥1 dose of 300IR 5-grass pollen tablet during years 1–3, and who completed the first visit in year 4 (visit 19) or year 5 (visit 23), respectively. The FAS for years 1–3 included all patients who started treatment and had ≥1 AdSS recorded during the pollen season of that year. The FAS for years 4 and 5 included all patients who started the respective post-treatment year and had ≥1 AdSS recorded during the pollen season of that year. The per-protocol sets included all FAS patients with ≥14 valid AdSS days during the pollen season or valid AdSS days for ≥50% of the pollen period, and who completed the study without any major protocol deviations.
Average scores were analyzed using a linear model and an analysis of covariance (ANCOVA) with pooled site, age, gender, asthma status and sensitization status as covariates. The post-hoc analysis of the DCS was performed using a linear mixed model with repeated measures and an ANCOVA. Variables which could potentially impact the clinical score (pooled site, age, gender, asthma status and sensitization status) were included in the model as covariates. Treatment was the main effect and the day was the indicator of time. All effects were fixed, except the patient who was considered as a random effect. A point estimate and 95% confidence interval (CI95%) for the difference in adjusted means between active-treatment and placebo groups were calculated. The threshold for statistical significance was set at p ≤ 0.05 and all inferential tests were two-sided.
DRTSS and DRMS, the components of the DCS, were analyzed similarly to the DCS.