Alendronate

The primary endpoint was the proportion of subjects in each treatment group who were adherent to treatment at the end of the first year. Efficacy analyses used the intent-to-treat principle and included all randomized subjects for the first year, and all crossover subjects for the second year. Data from both years are reported in this analysis because data that were missing at the time of the prior report [21 ] could be collected during the second year. Exploratory analyses of BMD and BMQ included all observed data at the time point of interest.
Safety endpoints included subject incidences of adverse events and serious adverse events. The safety population within each year of study included all subjects who received at least one dose of study medication in that year. If a subject accidentally received both study treatments in a single period, they were considered to have received denosumab for safety analyses in that period.
Statistical hypothesis tests were conducted at the 0.05 significance level. Point estimates and 95% confidence intervals (CI) were determined for the absolute rate reduction and for the rate ratio between treatment groups for non-adherence, non-compliance, and non-persistence. These endpoints were compared between treatment groups using a Cochran–Mantel–Haenszel test stratified by center and prior osteoporotic fracture. Ordinal, categorical, patient-reported endpoints were compared between treatment groups in each treatment period using a van Elteren non-parametric test, stratified by investigational site and prior osteoporotic fracture. Treatment-by-period interactions were assessed for significance (p value < 0.1) by statistical methods with data from both treatment periods. Time to non-adherence was defined as the time to treatment non-compliance or non-persistence, whichever occurred earliest. Non-adherence to alendronate could begin at any time. The time to denosumab non-adherence (for non-adherent subjects) was defined as 6 months and 4 weeks after the most recent injection. Time to treatment non-adherence was described with Kaplan–Meier methods without statistical comparisons.
Logistic regression analyses of non-adherence, non-compliance, and non-persistence were stratified by prior osteoporotic fracture. Potential explanatory variables explored individually in the model were baseline values (i.e., prior to study entry) for age, age group (<65 or ≥65 years), race (Caucasian or non-Caucasian), prior bone-loss therapy, parental hip fracture (yes or no), smoking history, alcohol intake, and time since menopause, as well as values from the start of each treatment period for total hip BMD and BMQ scores. The sample size was determined as described previously [21 ].

Typically, PEG-CTA (4.00 mg), METAC (8.3 mg, 0.04 mmol), PAA (2.9 mg, 0.04 mmol), BAC (2.1 mg, 8.0 × 10⁻³ mmol), HMP (2-hydroxy-2-methyl-1-propanone, 0.1 mg, 8.00 × 10⁻⁴ mmol), and NaCl (3.5 mg, 0.06 mmol) were dissolved in 2-ml water. The pH of the solution was adjusted to 6.5. The tube was sealed and de-oxygenated by three freeze-vacuum-thaw cycles in nitrogen atmosphere. Then, the tube was exposed to UV light for 3 hours. The NPs were separated from the template by centrifugation using 1.5 M NaCl as eluent for three times with an Amicon Ultra centrifugal filter (Millipore; molecular weight cutoff = 100 kDa) followed by dialyzing against water to remove the salt. Alendronate-decorated core-shell polyelectrolyte NPs were synthesized as abovementioned method using alendronate-modified AD-PEG-CTA. The “bare” PMETAC NPs (no PEG shell) were synthesized according to a previous report (27 ).
For conjugation with siRNA, NPs were mixed with siRNA in culture medium/DEPC (diethyl pyrocarbonate) water/sterile saline and incubated for about 3 min. Then, the medium was added into cultured cells.