ZEST II for Osteoporotic Fracture Prevention
Phase: PHASE4
Conditions: Osteoporotic Fractures, Osteoporosis, Postmenopausal
Sponsor: Susan L. Greenspan

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Study Protocol  
 
 
 
 
 
 
Official Title: Zoledronic Acid for Osteoporotic Fracture Prevention 
(ZEST II) 
  
 
 
ClinicalTrials.gov ID (NCT number): NCT02589600 
 
 
 
Protocol Date: 3/30/2023 
 
 
 
 

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Scientific Background 
 
Impact of Osteoporosis and Fractures in Long-Term Care Residents: 
Osteoporosis is devastating, particularly for the elderly. Each year 250,000 Americans suffer a 
hip fracture, of whom 20% die in the subsequent year, 25% are institutionalized, and ≤50% fully 
recover. Because the fastest-growing segment of the U.S. population is women >85 years, and 
1/3 of those who fracture are over age 90, the number of patients with hip fractures will double 
in the next 15 years (1,2) and the current annual cost of $17 billion will double by 2040 (3). 
Osteoporosis’ impact for America’s 2 million long-term care (LTC) residents is even more 
dramatic, with a prevalence of 85% (4) and an 11% annual incidence of nonvertebral fractures. 
Such residents were hospitalized ≥15 times as often as those without fracture (5) and those with 
hip or vertebral fractures are at 2-3x greater risk for future fracture (6,7). A vertebral fracture is 
associated with back pain (8-11), decreased mobility, increased bed rest (12), and higher rates 
of hospitalization and mortality (13,14). A recent review of 60,000 nursing home patients 
reported that following a hip fracture, 36% died and 53% died or developed new total 
dependence in 6 months (15). 
 
Osteoporosis Therapy in the Elderly: 
Osteoporosis Therapy is Safe and Efficacious in Healthy Elderly: The PI reported on the first 
prospective, randomized, placebo-controlled, multi-center trial of daily bisphosphonate therapy 
in healthy LTC residents (16). At 2 years, spine and femoral neck bone density had increased 
by 4.4% and 3.2%, respectively, with the bisphosphonate alendronate. This study demonstrated 
that daily alendronate was safe, well-tolerated, and improved bone mass in independent, 
ambulatory, cognitively intact women, able to take a daily oral bisphosphonate by themselves. 
The study excluded women with multiple medical problems, limited mobility, cancers, cognitive 
impairment, or diseases that impact bone and did not address the question if such therapy was 
safe or efficacious in institutionalized elderly. 
Post-hoc pivotal trial subgroup analyses comparing those >75 to those <75 years suggest 
zoledronic acid (17), teriparatide (18) and denosumab (19) are efficacious in older very healthy 
women who qualify for a clinical trial. These trials only included women who were healthy, 
cognitively intact, fully ambulatory, and able to sign their own consent. Fracture reduction data 
in frail elderly are lacking. 
 
Osteoporosis Therapy in Frail Elderly may not be Clinically Effective: 
There are little data on frail elderly or non-healthy LTC residents (20,21). In addition, the limited 
available data questions the efficacy of bisphosphonate in frail elderly. For example, in the HIP 
study, fracture reduction was not demonstrated with an oral bisphosphonate in women over age 
80 enrolled based on clinical risk factors only (22). Furthermore, the PI’s ZEST study in frail LTC 
residents, demonstrated improvement of BMD at the hip and spine with zoledronic acid, 
however no fracture reduction trend was observed. The majority of these patients were 
cognitively impaired, sedentary and had multiple comorbid conditions, excluding them from an 
industry trial. Fracture reduction data are lacking in these vulnerable patients. The March 2011 
NIH/ASBMR Forum on Aging and Skeletal Health echoed the critical need for osteoporosis 
research, a fracture reduction study and treatment for frail LTC residents (23). 
 
Why would therapy fail in frail elderly? Both skeletal and nonskeletal factors in frail elderly may 
mask a fracture reduction benefit from a potent bisphosphonate. Despite the improvement in 
bone density and reduction in bone turnover (both independent contributors to fracture 
reduction), frail individuals may not benefit due to severely compromised skeletal integrity from 
poor trabecular connectivity (disconnected rods and plates), increased trabecular fragments, 

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perforations in trabecular and cortical bone, impaired microstructure, or unhealed stress 
fractures (24-27). This concept was hypothesized by Parfitt (28,29). Bisphosphonates can’t 
reconnect broken fragments. Alternatively, nonskeletal factors may contribute to lack of benefit 
including minimal mobility (less weight bearing) or short life expectancy (too short to see a 
benefit), in addition to other factors such as increased falls (27). Our previous ZEST 1 study was 
not powered to examine fracture reduction. However, neither the limited statistical power nor the 
point estimates observed can be used to infer that fracture reduction would or would not be 
seen in a larger study (30); strong computational evidence suggests that only a fracture 
reduction study will suffice. Based on our data demonstrating a positive impact on both bone 
mass and bone turnover, we believe a fracture reduction study is justified and essential. 
 
Study Objectives 
 
Primary Outcome: 
Effectiveness of fracture reduction will be demonstrated by total non-traumatic incidental 
fractures (vertebral and nonvertebral [identified by x-ray, CT, MRI, VFA imaging) except those 
viewed as severe trauma (fall from a height higher than a stool or chair or severe trauma other 
than a fall), cancer-related or fractures of the toes, finger or facial bones. Incident morphometric 
vertebral fractures will be defined using the Genant criteria (134) as a reduction in vertebral 
height of at least 20% by quantiative morphometry or by an increase of one severity grade or 
more on semiquantitative analysis (134, 135). We will be able to assess primary outcome in all 
randomized women, optimally obtaining partial data in terms of person months of exposure and 
corresponding counts of fractures for analysis from those expiring/dropping out/moving before 3 
years. 
 
Secondary/Exploratory Outcomes: 
(1) New nonvertebral fractures 
(2) New vertebral fractures 
 
Other Secondary/Exploratory Skeletal Outcomes: 
(1) Hip and Spine BMD 
(2) Trabecular bone score (TBS) 
(3) Markers of bone turnover (CTX and P1NP) 
 
Laboratory Safety Outcomes: 
(1) H&H 
(2) Serum Calcium 
(3) Albumin 
(4) Alkaline phosphatase 
(5) Serum 25 OH Vitamin D 
(6) eGFR 
 
Adverse Events Outcomes: 
(1) All and serious adverse events, both by MedRA category and any-event basis 
(2) All and serious acute phase adverse events ≤3 days from an infusion 
(3) Special attention to falls, atrial fibrillation, stroke and symptomatic hypocalcemia 
 
 

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Study Design & Methods 
 
This is a 3-year, randomized, double-blind, calcium and vitamin D controlled trial with the 
antiresorptive agent zoledronic acid (Reclast). 
 
The study consists of 5 main visits (Screening, Randomization, Month 12, Month 24, and Month 
36), with pre and post infusion safety check visits. Pre-infusion safety check visits will be done 
up to 2 weeks prior to receiving the annual IV study drug or placebo. Post-infusion safety check 
visits will be done 1-3 days after infusion. 
 
We have chosen yearly intravenous zoledronic acid because it has been shown to significantly 
reduce vertebral fractures by 70%, all clinical fractures by 35%, hip fractures by 41% in the 
pivotal trial that included older healthy women. Additionally, in a sub-analysis including women ≥ 
75 years old, yearly zoledronic acid decreased vertebral fractures by 66% over 3 years. 
Moreover, fracture reduction after 2 and 3 infusions were 47% and 39%, respectively. Because 
nursing home patients are difficult to treat and resources are limited, the intravenous injection 
has several advantages. It does not require fasting, administration at a specific time of day, or 
sitting up, which are all significant practical advantages over a weekly oral bisphosphonate. All 
patients without liver abnormalities will be offered acetaminophen prior to the infusion and for 2 
days post-infusion to decrease acute phase reactions and maintain the blind. 
 
Rationale for yearly dose versus single dose: Despite the improvement in density over 2 years 
after a single dose in our initial ZEST study, we will follow conventional yearly dosing that 
demonstrated fracture reduction in the pivotal trials. In the event we do not find a fracture 
reduction benefit, we wanted to be certain that it would not be due to under-dosing. 
 
At the request of the DSMB, we will be offering a Follow-up Extension phase to this study to 
continue data collection on the skeletal health of participants in the ZEST II study. Therapy and 
blood work will not be provided throughout the follow-up extension. There will be a maximum of 
three visits in the follow-up extension. The number of visits that a participant will have can be 
determined by when they have completed their Month 36 visit. Participants who have completed 
the Month 36 visit on or before July 31, 2019 will have three follow-up visits. Participants who 
have completed the Month 36 visit on or before July 31, 2020 will have two follow-up visits. 
Participants who have completed the Month 36 visit on or before July 31, 2021 will have one 
follow-up visit. Participants who do not complete the Month 36 visit before August 1, 2021 will 
not be eligible to participate in the follow-up extension. 
 
SKELETAL ASSESSMENTS 
 
Fragility Fractures: From baseline to the end of follow-up, nonvertebral and clinical vertebral 
fractures will be identified by the electronic medical record alert and by Theradoc (a real-time, 
stand-alone clinical alert and decision support application used at UPMC LTC facilities, with 
custom rules to be written for this study to provide detection and identification of participants 
presenting to any UPMC facility so that generated alerts will be sent to the study team via e-
mail). In addition, participants/charts will be queried at approx. 6 month intervals for clinical 
fragility fractures defined as a fracture following a fall from a standing or sitting height. All 
reported fractures will be verified by obtaining the radiology report confirming the fractures. We 
exclude fingers, toes, face, and skull fractures. All femur fractures will be examined for atypical 
fractures using ASBMR criteria (128). 

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Vertebral Fractures: We will use the presence of thoracic or lumbar vertebral fractures as 
determined by Vertebral Fracture Assessment (VFA is DXA-derived) as the primary 
classification of vertebral fracture performed annually (53, 73, 129). The fractures will be scored 
according to the method of Genant (130, 131) and considered a grade 1 mild vertebral 
deformity, a grade 2 moderate vertebral deformity, or a grade 3 severe vertebral deformity. We 
will assess vertebral fractures at baseline, 12, 24, and 36 months, as well as at follow-up visits 
1, 2, and 3. Conventional x-rays or a MRI that demonstrates a fracture will also be used. 
Indices of Bone Mineral Metabolism: Serum will be assayed for 25-hydroxyvitamin D2/D3 for the 
secondary aim. We will collect serum for bone turnover markers at baseline and M36 for 
responder analysis for the exploratory aim (60, 65-67, 132, 133). To assess bone resorption, we 
will measure serum CTX. We will assess bone formation with P1NP. Serum will be archived for 
future analyses. 
 
Trabecular Bone Score (TBS): TBS, a new gray-level texture measurement is evaluated in the 
same region of interest as the spine BMD using the iNsight Software version 1.9 (54-58). TBS 
utilizes the 2-D DXA spine images and provides 3-D microarchitectural images and parameters 
(57, 59). The precision for TBS ranged from 1.1 to 1.9% (55). In previous studies, TBS was 
independent of BMD for fracture prediction and significantly improved risk protection (80, 81). 
TBS is only minimally affected by sclerosis/osteoarthritis (57). For the exploratory aim, we will 
examine TBS from DXA images. 
 
Eligibility Criteria  
 
Inclusion Criteria:  
Our goal is to include older women with osteoporosis or low bone mass (with fracture 
risk) unless there is a known contraindication to therapy. We will enroll ambulatory 
women age ≥65 years including those using assistive devices to maximize 
generalizability, if they: 
1) Reside in long-term care (LTC) including Managed Senior Communities which 
provide the following: (a) An environment engineered to prevent falls (handrails, ramps) 
and are ADA accessible; (b) Facilitate access to medical attention (pull cords, life-alerts, 
or other electronic means); (c) ADA accessible transportation services available; (d) 
Provide or coordinate meals; (e) Secure building (automatically locked door or buzzer 
system, security cameras). 
2) Have a) osteoporosis by axial bone density (spine, hip or forearm BMD T-score ≤ -
2.5 SD)(99,100); or b) a previous adult fragility fracture of the spine or hip; or, c) would 
be treated based on FRAX (101,102) NOF treatment thresholds of a 10 year risk of ≥ 
20% for a major osteoporotic fracture or ≥ 3% for hip fracture using femoral neck BMD 
(103). 
 
Follow-up Extension Inclusion Critera: 
1) Complete Month 36 Visit on or before July 31, 2021. 
Exclusion Criteria:  

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We will exclude: 
1) Men because osteoporosis is less common in men and our initial ZEST 1 study only 
included women. 
2) Institutionalized women with subacute illnesses surviving or discharged in < 3 years. 
3) Women currently on bisphosphonate, denosumab, or teriparatide therapy or who 
have been on a bisphosphonate for greater than 1 year during the previous 2 years 
because bisphosphonates are long acting (104). 
4) Patients with a calculated creatinine clearance < 35 ml/min (105,106) or who have a 
contraindication for bisphosphonates (allergy, hypocalcemia). 
5) Women scheduled for a tooth extraction to avoid jaw osteonecrosis (107-109). 
 
We will screen with a detailed history, chart review, and baseline laboratory analyses. 
Women with vitamin D levels <25 ng/mL (110,111) will be treated with vitamin D 50,000 
IU/wk for 8 weeks (112,113). The patient will be enrolled if the follow-up vitamin D level 
is ≥ 25 ng/mL. Vitamin D levels <25 ng/mL are associated with falls (114,115) and our 
prior ZEST 1 Study data supports a level ≥ 25 ng/mL for better performance and 
improved walking (98). Patients can continue on glucocorticoids and anticonvulsants 
because their use is common (116). Women on raloxifene, calcitonin or using protective 
hip pads will be allowed to participate. 
 
Exclusion Criteria #3, #4, and #5 will not apply to participants in the Follow-up Extension 
phase of this study. 
Statistical Considerations 
 
Overview: All statistical analyses will use SAS® version 9 (SAS Institute, Inc., Cary, North 
Carolina) based on intention-to-treat. First, appropriate descriptive statistics will be computed 
and graphical summarizations made for all variables for each treatment group for each time 
point as well as raw and percent change scores from baseline to obtain an overall sense of the 
distributional characteristics, missing values and general data quality. Second, baseline 
measures will be compared across arms using independent samples t- and a Wilcoxon rank 
sum tests, as appropriate, for continuous variables and chi-square and Fisher’s exact tests, as 
appropriate, for categorical variables. Although no significant differences are expected due to 
randomization and large sample size, any variables found to be significantly different will be 
accounted for by including them as covariates in sensitivity analyses. Third, main analyses will 
be performed using a negative binomial regression model. Finally, exploratory analyses will be 
performed. 
 
Main Analyses: First, we will fit a series of negative binomial regression implemented in the 
SAS® GENMOD procedure with each fracture outcome as the dependent variable; duration of 
follow-up as an offset to account for each participant's exposure; treatment arm as the main 
independent factor of interest; and any covariates deemed important as additional independent 
variables in sensitivity analyses. The incident rate ratio and its statistical significance at α=0.05 
level for the primary outcome of all new fractures will serve as a formal test of the main fracture 
reduction hypothesis (H1). Second, we will fit a series of linear mixed models using the SAS® 
MIXED procedure with raw and percent change each from baseline in each of the continuous 
outcomes representing laboratory measures related to safety as the dependent variable; 
treatment arm, follow-up time point (12-/24-/36-month, as available) and their interaction as the 

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fixed effects of interest; and a subject random effect. For some measures, we will fit simpler 
models (due to only one planned post-infusion assessment) using the GLM procedure with raw 
and percent change each as the dependent variable, and treatment arm as the main factor of 
interest. We will appropriately construct sets of contrasts to test various hypotheses of interest 
by comparing the said change over time between the two arms. Third, we will fit a series of 
logistic regression models using the SAS® LOGISTIC procedure with each incident 
dichotomous adverse event during the 36-month follow-up (any and specific types of adverse 
events) as the dependent variable, and treatment arm as the independent factor of interest. The 
resulting odds ratios and levels of significances will be used to test the specific hypotheses for 
Aim 2. We will repeat all main analysis after stratification by institutional setting (assisted 
living/nursing home). 
 
Findings from the proposed trial will be most useful if (a) we can also identify a target sub-
population of the long-term care that is most likely to benefit from zoledronic acid and/or least 
likely to experience side effects; and (b) we can speculate whether fracture reduction or non-
reduction in women, despite improvement in BMD, depends on bone microstructure as 
measured by TBS. Therefore, using several techniques, some with an exploratory data mining 
philosophy, we will identify such sub-populations to address Aim 3(a). First, we will include all 
baseline clinical and laboratory characteristics, one-at-a-time, as an additional fixed effect in the 
models above, along with an interaction term with treatment group. Statistical significance of the 
interaction term will be interpreted as a patient baseline characteristic whose levels exhibit 
potentially differential treatment effects and/or safety profiles. Second, we will stratify analyses 
by such baseline criteria to actually obtain preliminary estimates of subgroup-specific treatment 
effects/safety differences. Third (data mining), using only the active arm, we will fit a series of 
logistic regression models using the SAS® LOGISTIC procedure and classification tree models 
using Salford Predictive Miner® software (Salford Systems, Inc., San Diego, California) with 
incident fracture as the dichotomous response variable; and baseline characteristics as 
predictors. Logistic models are more efficient when associations are linear, and hard-to-discover 
higher order interactions and non-linearity and/or multicollinearities among predictors do not 
exist, while classification tree models are more efficient when they do exist. Thus we will use 
both methods to obtain areas under the receiver operator characteristic curve (AUROCC) to 
quantify predictive accuracy, and use the results from the method with a greater AUROCC. For 
logistic regression we will use the backward elimination and for classification trees minimum 
misclassification-complexity cost tree to guard against overfitting. We will use the method of 
DeLong to obtain statistical significance of the differences in predictive accuracy between the 
models. For predicting improvements in continuous BMD change outcomes, we will employ a 
similar approach but with standard multiple linear regression and regression tree models and 
proportion of explained variance (R2) to quantify predictive accuracy. 
 
For Aim 3(b), the association between zoledronic acid, BMD, TBS and fracture reduction will be 
explored in two ways. First, we proposed above to examine the effect of the study drug on TBS 
with standard linear models. Second, using only the active arm of the trial, we will fit a negative 
binomial model with number of fractures within 3 years as the dependent variable; and baseline 
BMD, 3-year change in BMD, baseline TBS, and 3-year change in TBS as independent 
variables. The statistical significance of regression coefficients for TBS measures will be 
interpreted as evidence supporting that fractures over a 3-year period depend on TBS and its 
changes over the same period independent of BMD. 
 
Exploratory Analyses: We will perform exploratory analyses to assess the robustness of results 
and make more refined conclusions. For example, we will stratify adverse event and fracture 

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analyses to identify any differences between short- and long-term effects and/or within organ 
systems.