Mechanisms Underlying the Bone Modeling Effects of Combined Anabolic/Antiresorptive Administration
Phase: PHASE4
Conditions: Postmenopausal Osteoporosis
Sponsor: Massachusetts General Hospital

 
 
Benjamin Z. Leder - confidential 
 
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Version: 9.0, August 11, 2021 
 
 
 
 
 
Mechanisms Underlying the Bone Modeling Effects of 
Combined Anabolic/Antiresorptive Administration 
 
 
PHRC Protocol Number: 2018P002537 
 
Principal Investigator: Benjamin Z. Leder 
Funded by: National Institute of Arthritis and Musculoskeletal and Skin Diseases 
(NIAMS) 
 
Version Number:  v.9.0 
August 11, 2021 
 
 
 
 

 
 
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Version: 9.0, August 11, 2021 
I. Statement of Compliance  
 
The trial will be conducted in accordance with International Conference on Harmonisation Good Clinical 
Practice (ICH GCP), applicable United States (US) Code of Federal Regulations (CFR), and the NIAMS Terms 
and Conditions of Award. The Principal Investigator will assure that no deviation from, or changes to the 
protocol will take place without prior agreement from the Investigational New Drug (IND) or Investigational 
Device Exemption (IDE) sponsor, funding agency and documented approval from the Institutional Review 
Board (IRB), except where necessary to eliminate an immediate hazard(s) to the trial participants. All 
personnel involved in the conduct of this study have completed Human Subjects Protection and ICH GCP 
Training. 
 
The protocol, informed consent form(s), recruitment materials, and all participant materials will be submitted to 
the IRB for review and approval.  Approval of both the protocol and the consent form must be obtained before 
any participant is enrolled. Any amendment to the protocol will require review and approval by the IRB before 
the changes are implemented to the study. All changes to the consent form will be IRB approved; a 
determination will be made regarding whether a new consent needs to be obtained from participants who 
provided consent, using a previously approved consent form. 
 
II. Background and Significance 
 
A. Historical background 
In the United States, osteoporosis affects over 20 million women and men and 1.5 million Americans 
experience a fragility fracture every year.1 One in five women will experience a hip fracture in their lifetime, a 
devastating consequence that conveys a 24% excess 1-year mortality.2,3 Current osteoporosis therapies 
increase bone mineral density (BMD) modestly and reduce fracture incidence in high-risk populations, but their 
efficacy is limited, especially in the appendicular skeleton where fracture reduction does not exceed 50% for 
any agent.4-7 The real world utility of these drugs is further limited by poor adherence and safety concerns.8-10 
 
Osteoporosis medications can be separated into two categories: (1) antiresorptive medications such as the 
nitrogen-containing bisphosphonates and the receptor activator of nuclear factor kappa-B ligand (RANKL) 
inhibitor, denosumab, and (2) anabolic agents such as teriparatide (parathyroid hormone (PTH)-1-34) and 
abaloparatide. Over the past decade, several groups have investigated the efficacy of combining anabolic 
agents with bisphosphonates but no combination was shown to increase bone mass more than the individual 
monotherapies.11-14 In contrast to results with these anabolic/bisphosphonate combinations, we recently 
reported in the Denosumab And Teriparatide Administration (DATA) study that concurrent denosumab and 
teriparatide increased spine and hip BMD and improved bone microarchitecture more rapidly and to a greater 
extent than either drug alone.15,16 The mechanistic basis for the efficacy of this combination, however, has not 
yet been defined. Based on the changes in biochemical markers of bone turnover observed in DATA, we have 
hypothesized that the combination may derive its efficacy from denosumab’s ability to fully block the pro-
resorptive effects of teriparatide while still allowing for teriparatide to stimulate modeling-based bone formation. 
We now aim to define the mechanisms that form the basis of these novel clinical observations. In so doing, we 
will build a framework for the design of robust clinical trials, including fracture prevention outcomes, with the 
potential to fundamentally advance osteoporosis treatment. 
 
Effects of teriparatide on bone metabolism and histomorphometry: Teriparatide, through its binding to the 
PTH/PTHrP receptor stimulates bone remodeling by increasing the activity of both osteoclastic bone resorption 
and osteoblastic bone formation (with intermittent administration favoring bone formation).17,18 The effects on 
remodeling peak at 3-12 months, after which rates revert towards baseline.7,11,13,15 Additionally, PTH and its 
analogs influence the activity of other bone cells including osteocytes (decreasing the expression of the sost 
gene that encodes the osteoblast inhibitor, sclerostin and increasing the expression of RANKL) and lining cells 
(activating) though the relative physiologic importance of each of these activities is not clear.19,20 In humans, 
teriparatide has been shown to increase BMD at the hip and spine and reduce the risk of both spine and non-

 
 
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spine fractures in postmenopausal women.7,21-26 In iliac crest bone biopsy specimens from teriparatide-treated 
women, there are detectable increases in bone formation rates (BFR) and mineralizing surface/bone surface 
(MS/BS) as early as 1 month after the treatment initiation in all four bone compartments (cancellous, 
endocortical, intracortical, and periosteal).21,27,28 Pro-resorptive effects, including increasing cortical porosity, 
have also been consistently demonstrated both by biopsy and high-resolution imaging.9,16,27,29-32 Notably, the 
relative importance of modeling-based bone formation (MBF) in humans remains controversial.5 In a recently 
reported study by Dempster et al., however, it was reported that in women receiving teriparatide for 3 months, 
both modeling and remodeling-based formation (RBF) increased in the cancellous and endocortical 
compartments whereas only MBF increased in the periosteal envelope.27 While the precise molecular 
mechanisms that underlie the net anabolic activity of PTH and PTH analogs are still being defined, they have 
been attributed to mechanisms both dependent and independent of bone remodeling, including:  
 
1) Stimulating RANKL-mediated bone resorption, leading to the release of osteo-anabolic growth factors 
from the bone matrix.33 
2) Suppressing expression of the Wnt inhibitor, sclerostin.34 
3) Inhibition of osteoblast apoptosis.35 
4) Activation of quiescent osteoblasts into bone forming osteoblasts.20 
5) PTH induction of Wnt10b by activated T lymphocytes.36 
6) Differentiation of bone marrow stromal cells (BMSCs) into cells of the osteoblast lineage.37 
 
Effects of denosumab on bone metabolism and histomorphometry: Denosumab is a monoclonal antibody that 
inhibits the binding of RANKL to its receptor on cells of the osteoclastic lineage (RANK).38 It is a potent 
antiresorptive agent that decreases both osteoclast and osteoblast activity within days of its administration and 
increases BMD while reducing fracture risk.39-44 Biopsies taken from women treated with denosumab for up to 
5 years have consistently demonstrated a decrease in bone resorption (eroded surface (ES)) as well as BFR 
and MS/BS. Moreover, up to 1/3 of patients treated with denosumab demonstrate an absence of either a 
double or single tetracycline label in trabecular and/or cortical compartments and 50% of patients show a 
complete absence of osteoclasts in the biopsy specimens.27,39,41,45 More recently, it was reported that in women 
who received denosumab for only 3-months, all types of bone formation were decreased or unchanged with 
the exception of modeling-based bone formation in the cancellous envelope, which increased significantly.46  
 
Effects of combination anabolic/antiresorptive therapy on bone metabolism and histomorphometry: While the 
RANK-ligand mediated mechanisms of bone resorption have been well studied over the past decade, the 
mechanisms of coupling of this bone resorption to bone formation are less well defined.38,47 Potential coupling 
factors include matrix derived signals such as TGFβ, IGF-1,48-51 secreted factors such as BMP-6 and 
Wnt10b,47,52-54 as well as signals to and from other marrow components, including osteoclasts themselves.55 
Depending on the agents studied, animal models revealed variable findings on the effect of combined anabolic 
+ antiresorptive agents on bone metabolism and histomorphometry.  
 
In the DATA study, the combination of denosumab and teriparatide resulted in less suppression of bone 
formation markers than seen with denosumab alone whereas bone resorption markers were potently and 
equally suppressed in both the denosumab alone and combination groups. Additionally, combining teriparatide 
and denosumab suppressed bone resorption markers more completely than has been reported in studies of 
combined teriparatide and bisphosphonates.11,13,56 This likely contributes to the relatively greater increases in 
BMD with combined denosumab/teriparatide (discussed in detail below). 
 
Quadruple Label Bone Biopsy in Humans:  Paired iliac crest bone biopsies have classically been used to 
assess longitudinal changes in bone histomorphometry.57 Quadruple labeling, however, with 2 pairs of 
tetracycline administrations separated by a period of weeks or months, permits longitudinal assessment with a 
single biopsy procedure.58 Specifically, the mineral apposition rate and mineralizing perimeter can be 
measured for the two separate labeling periods, and changes in bone formation rates can thus be calculated at 
2 separate time points. A further advantage of this technique is that each patient is able to serve as her own 

 
 
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pre-treatment control, thus reducing variability in histomorphometric parameters and increasing statistical 
power at any given sample size. Indeed, quadruple-labeling has been used successfully to assess the effects 
of teriparatide and other osteoporosis medications in prior studies.27 Moreover, this technique has proven 
especially useful in assessing the extent of modeling versus remodeling-based bone formation in the different 
bone envelopes,27,58 and thus is a key tool to directly address the fundamental question regarding combination 
therapy that we are addressing in this proposal.  
 
B. Preliminary Data:  
The rationale and premise for this protocol comes directly from the data reviewed in the 
Background/Significance section above and the results of the DATA study. In DATA, postmenopausal women 
with osteoporosis were randomized to one of the following 2-year treatment groups: teriparatide 20-μg daily 
(n=31), denosumab 60-mg every 6 months (n=33), or the combination of both medications (n=30). Shown 
below are the 2-year changes in BMD in hip and spine *(P<0.05 vs other groups). 
 
    Lumbar Spine BMD      Total Hip BMD            
 
            months 
              months 
             
 
The changes in a biochemical markers of bone resorption (serum C-telopeptide, CTX) and bone formation 
(P1NP, osteocalcin) during the first year of treatment, the time during which the advantage of combination 
therapy is observed, are shown below.  
 
 
These results demonstrate that unlike the combination of teriparatide and bisphosphonates, the combination of 
teriparatide and denosumab has additive effects on BMD. The mechanisms underlying these observations are 
the focus of this protocol.  
 
Based on the results above, we hypothesize that that denosumab is able to fully block teriparatide’s pro-
resorptive effects (as indicated by the identical changes in CTX between the denosumab monotherapy and 
combined groups) while permitting ongoing modeling-based bone formation (as indicated by the early 
differences in osteocalcin and P1NP between the denosumab and combined groups). Confirmation of this 
hypothesis rerquires histomorphometric analysis as proposed in the current studies. 
 
a p<0·001 versus 
denosumab and versus 
combination; b p<0·01 
versus denosumab; and 
*p<0·05 versus baseline. 
Bars show standard error 
of the mean. (Data for 
teriparatide group and 
other groups graphed 
separately for figure 
clarity). 
 

 
 
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III. Specific Aims 
The Specific Aim of this protocol is to test the following hypothesis: 
Hypothesis: This study described below will tests the hypothesis that in postmenopausal osteoporotic women, 
combined PTH-receptor stimulation (teriparatide) and RANKL inhibition (denosumab) will result in 
histomorphometrically-demonstrated inhibition of teriparatide-induced skeletal remodeling while allowing for 
continued stimulation of modeling-based bone formation in all bone compartments (cancellous, endocortical, 
intra-cortical, and periosteal).  
 
IV. Subject Selection 
Study population: 36 female volunteers will be recruited in accord with institutional guidelines for clinical 
studies.  
Participation will be based on meeting the following entry criteria: 
Inclusion Criteria: 
Must satisfy A and B and C below: 
A. women aged > 45 
B. postmenopausal by either of the following criteria: 
 
> 36 months since last spontaneous menses or 
 
> 36 months since hysterectomy, plus serum FSH > 40 units / liter if < 60 years 
C. osteoporosis with high risk of fracture by one or more of the following criteria: 
• 
DXA spine or hip T-score ≤ - 2.5;  
• 
DXA spine or hip T-score ≤ - 2.0 plus > 1 of the following BMD-independent risk factors for fracture: 
fracture after age 50, parental hip fracture after age 50, prior hyperthyroidism, inability to rise from a chair 
with one's arms elevated, current tobacco smoker. 
• 
DXA spine, hip, or forearm T-score ≤ - 1.0 plus > 1 adult low-trauma fracture (low-trauma fracture = 
fracture after no trauma; or fracture after falling < 6 inches when stationary or moving slower than a run). 
 
Exclusion Criteria: Exclusion criteria similar to those used for our prior osteoporosis studies using these 
medications will apply11,13 (also see ClinicalTrials.gov #NCT00926380). 
• 
confirmed serum alkaline phosphatase above upper normal limit with no explanation 
• 
liver disease (AST or ALT > 2 x upper normal limit) 
• 
renal disease (eGFR < 30mL/min) 
• 
hypocalcemia or hypercalcemia (Ca <8.5 mg/dL or >10.5 mg/dL) 
• 
abnormal blood PTH (intact PTH < 10 pg/mL or > 65 pg/mL) 
• 
serum 25-OH vitamin D < 20 ng/ml or >60 ng/ml  
• 
hematocrit < 32% 
• 
history of malignancy (except non-melanoma skin carcinoma), radiation therapy, or gouty arthritis 
• 
history of urolithiasis within the last one year  
• 
significant cardiopulmonary disease including unstable coronary artery disease, stage D ACC/AHA 
heart failure or any other condition that the investigator deems may preclude the subject from 
participating safely or completing the protocol procedures 
• 
major psychiatric disease that in the opinion of the investigator would preclude the subject from 
providing adequate informed consent or completing the protocol procedures 
• 
excessive alcohol use or substance abuse that in the opinion of the investigator would preclude the 
subject from providing adequate informed consent or completing the protocol procedures 
• 
known congenital or acquired bone disease other than osteoporosis (including osteomalacia, 
hyperparathyroidism, Paget’s disease) 
• 
history of intravenous bisphosphonate, strontium, denosumab, abaloparatide or teriparatide use 

 
 
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• 
history of oral bisphosphonate use within the last 5 years or if use >5 years ago, cumulative use > 1 
year  
• 
use within the past 3 months of estrogens, SERMs, or calcitonin 
• 
use of oral or parenteral glucocorticoids for more than 14 days within the past 6 months 
• 
known sensitivity to mammalian cell-derived drug products 
• 
known sensitivity to teriparatide, denosumab, or any of their excipients 
• 
tooth extraction or dental implant within the past 2 months or planned in the upcoming 2 months 
• 
history of osteonecrosis of the jaw 
• 
known sensitivity to tetracycline, demeclocycline or other antibiotics in this drug class 
• 
continuous use of tetracycline for >1-month duration within the last 10 years  
 
Source of subjects and recruitment methods 
Recruitment flyers will be posted in approved locations throughout the MGH (including the MGH Bone Mineral 
Density Center) and email announcements will be sent through the Partners Clinical Research Program 
Network.  Letters will be sent to subjects identified through RSVP for Health. Additionally, a mailing will be sent 
to targeted populations, including subjects who have expressed an interest in one of our research group’s 
previous studies. 
 
Subjects may be recruited from the clinical practice (MGH Endocrine Associates) of the principal investigator 
and co-investigator(s). To avoid potential coercion, after the investigator has briefly presented the study, the 
participant will be asked if they may be re-contacted for a more in-depth phone call from another study 
physician. Additionally, informed consent for screening will be completed by a research coordinator/study 
physician and informed consent for the main study will be completed by a study physician who is not a provider 
for each of the patients/subjects of this recruitment method. 
 
NOTE: Entry criteria were established with input from the FDA (Reproductive Section). These criteria are 
similar to those used in our most recent human studies for which we have been able to meet all enrollment 
goals. Dr. Leder currently holds IND #122883 for the use of teriparatide in combination with denosumab. This 
protocol will be submitted to the FDA as an amendment. 
 
V. Subject Enrollment 
This outpatient study will either be conducted at one or more of the following sites:  the General Clinical 
Research Center (GCRC), the Osteoporosis Research Center or the Endocrine Unit Clinical Space (all within 
the Massachusetts General Hospital.   
 
The general design is a randomized, 3-arm, open label study. 
 
Procedure for informed consent: 
Screening only informed consent will be obtained by a member of the study staff at the Screen visit. Main 
study informed consent will be obtained by a study physician at the beginning of the first study visit.  
 
Subjects will first be screened and those who appear eligible will have a medical history and interview with a 
study investigator at the first study visit and those who remain eligible will be randomized. 
 
Groups Assignment and Randomization: 
Those who remain eligible will be formally accepted into the study by a study investigator and assigned to 
one of the 3 interventions by computer-generated assignment using a randomly varying blocking scheme 
(block sizes of 3 or 6) to minimize the predictability of treatment assignments (1:1:1 ratio). To account for 
imbalance in the dropout rate as of August 11, 2021, group B will be closed. Subjects will be assigned to one 
of the 2 remaining interventions by computer-generated assignment using a randomly varying blocking 
schema (block size 2 or 4) to minimize the predictability of treatment assignments. Please see the 
Biostatistical analysis for re-analysis of power calculations. 

 
 
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Group A: 
daily subcutaneous teriparatide 20-µg (self-administered) 
Group B: 
one dose of subcutaneous denosumab 60-mg (administered by the study physician).  
Group C: 
daily subcutaneous teriparatide 20-µg + one dose of subcutaneous denosumab 60-mg  
 
There will be 12 subjects in each group. 
 
VI. Study Procedures 
 
Telephone Screening and screening labs: 
Members of research staff will screen all interested subjects over the telephone.  If subjects meet initial criteria 
and are interested in the study, a member of the study staff will schedule a screening visit wherein the subject 
will sign a separate “screening only” informed consent form (ICF).  This ICF will allow for a blood draw and 
bone mineral density testing. 
 
Prior to signing this screening only ICF, subjects will be offered the option to speak with a physician 
investigator if they wish or if they have questions that can't be answered by the research assistant. Research 
assistants will document this conversation on study records.  
 
At this screen visit, bone mineral density of the spine and hip will be obtained and the following will be 
measured in serum or blood: 
• 
PTH, 25-OH vitamin D, uric acid, CBC, and comprehensive chemistry panel (serum glucose, BUN, Cr, 
eGFR, Na, K, Cl, Ca, total protein, albumin, bilirubin, ALP, ALT, AST) 
• 
FSH (if necessary due to prior hysterectomy) 
 
Participants who have serum uric acid levels > 6.5 mg/dL and are randomized to receive teriparatide will be 
treated with urate lowering therapy prior to receiving teriparatide. 
 
If a subject’s screen eligibility labs contain an abnormal result/s that, in the opinion of the investigator, should 
be repeated, the subject will be invited to have the abnormal test/s repeated with study staff or study staff will 
recommend that the subject have the test/s repeated with the subject’s clinical care team. 
 
If greater than 3 months have lapsed between the subject’s screen labs and their first main study visit, study 
staff may request the subject to have all or some of their screening labs repeated for safety purposes.   
 
If greater than 12 months have lapsed between the subject’s screening visit and their first main study visit, 
study staff will invite subjects to undergo a re-screen for accuracy and safety purposes.   
 
The study period lasts 3 months.  
 
Visit 1: 
If the subject meets the above BMD and laboratory criteria, she will then be scheduled for Visit #1.  At this visit, 
a study physician will obtain written informed consent for participation in the complete study.  If subjects require 
more time to consider participating after reading the main study ICF the study visit will not continue.  If the 
subject is ready to sign the main study ICF, the study physician will then perform a history and physical on her. 
If all inclusion/exclusion criteria are met, subjects will then proceed with the rest of visit 1 and the subsequent 
visits.  
 
Visit 1-4: 
These visits will occur at the following times: 
Visit 1 (pre-treatment, +/- 7 days) 

 
 
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Visit 2 (day 1, +/- 7 days) 
Visit 3 (month 1, +/- 7 days)  
Visit 4 (month 3, +/- 7 days) 
 
Subjects will be seen at MGH prior to 10 a.m. and will be instructed to be fasting. The following blood/serum 
tests will be measured based on the schema and visit schedule below: 
 
• 
Comprehensive chemistry panel (serum glucose, BUN, Cr, eGFR, Na, K, Cl, Ca, total protein, albumin, 
bilirubin, ALP, ALT, AST) 
• 
Serum CTX (pooled* assay at end of study) 
• 
Serum osteocalcin (pooled* assay at end of study) 
• 
Serum PINP (pooled* assay at end of study) 
 
*Samples for CTX, PINP and osteocalcin will be run in a batch assay at the conclusion of the study. 
 
If a subject’s visit labs contain an abnormal result/s that, in the opinion of the investigator, should be repeated, 
the subject will be invited to have the abnormal test/s repeated with study staff or study staff will recommend 
that the subject have the test/s repeated with the subject’s clinical care team. 
 
After which the subjects will be given breakfast, and then have the following procedures per table below: 
 
Schema and visit schedule: 
 
Screen  
 
 
Visit 1 
 
Visit 2 
+7 days 
Visit 3 
+/- 7 days 
Visit 4 
+/- 7 days 
 
 
Pre-treatment 
Day 1 
Month 1 
Month 3 
Informed Consent 
X 
X 
 
 
 
BMD by DXA (spine, hip)* 
X 
 
 
 
 
Comprehensive chemistry 
panel 
X 
 
X 
X 
X 
Uric acid, PTH, 25-OH 
vitamin D, CBC 
X 
 
 
 
History and Physical Exam 
 
X 
 
 
 
Randomization assignment 
 
X 
 
 
Dispense labeling 
antibiotics with schedule 
 
X 
X 
Dispense teriparatide  
 
 
X 
 
 
Administer denosumab 
 
 
X 
Bone Turnover Markers 
(serum CTX, P1NP, 
osteocalcin) 
 
 
X 
X 
X 
 
*May use historic DXA performed for clinical indications or undergone as part of another protocol if the DXA 
scan is performed within 6 months of the Screen Visit. 
 
At visit 2, denosumab (sold under the brand name Prolia) will be administered to subjects assigned to the 
appropriate group after all blood sampling is complete. Also at visit 2, subjects will be trained in the use of the 
teriparatide injection pen and will give themselves the first injection while observed by a member of the study 
staff. A sufficient supply of teriparatide (if assigned) will be given to each subject at the conclusion of visit 2. 
Fluorochrome labels will be administered as described in the section below. 

 
 
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All subjects who report dietary intake <1200mg of calcium daily will be given supplementation (600mg 
elemental calcium + 400 IU Vitamin D). Total calcium intake (dietary and supplemental) should be at most 
1200mg/day. Subjects with a calcium dietary intake > 1200 mg will receive Vitamin D only. Participants who 
have a screen 25-OH vitamin D level > 50 ng/mL will refrain from vitamin D supplements for the first month and 
then start at 800 units/day. 
 
Note:  Any remaining serum samples will be saved and may be used to measure other variables related to 
bone metabolism at a future time. Samples will be stored with code number and date of sampling in a locked –
80-degree freezer and only approved study personnel will have access to the samples. It is understood that 
any future use of samples must be reviewed and approved separately by the Partners Human Research 
Committee (PHRC) prior to their use.  Personal-Identifying information will be removed before any sample is 
sent out for analysis to a non-Partners affiliated institution. 
 
Note: The Principal Investigator may end the subject's participation without the subject's consent in order to 
protect the health of the subject, if the subject is unable to attend to study visits, if the sponsor decides to stop 
the study, or due to other administrative reasons.  
 
Biochemical markers of bone turnovers of serum procollagen type I N-terminal propeptide (P1NP), serum 
osteocalcin, and serum β-c-terminal telopeptide of type one collagen (CTX) will be performed on fasting 
morning blood samples (24 hours after last injection if taking teriparatide). Samples will be run in batch assay 
at the conclusion of the study. Samples will be stored at -80C.   
 
Iliac crest bone biopsy procedure: 
Iliac crest bone biopsies will be performed at the 3-month time point, corresponding to the period of 
teriparatide’s maximal stimulation of osteoblasts and anabolism.11 The biopsies will be performed in the MGH 
Clinical Research Center by either Dr. Joseph Schwab, Dr. Stuart Hershman, Dr Daniel Tobert or Dr. Harold 
Fogel of MGH Orthopedic Surgery. A trained staff physician or nurse anesthetist will supervise anesthesia. 
Before proceeding, the procedure will be discussed with the participant in detail, including the risks involved 
(see human subjects section).  A model (synthetic pelvic bone model) will be used to help explain the 
procedure with particular attention to the location of the biopsy. The use of short-acting oral 
(lorazepam/alprazolam) or parenteral sedation (midazolam and/or fentanyl, in titrated doses) will also be 
discussed with the participant and its use is optional depending upon the participant’s comfort level. The iliac 
biopsy is obtained using a Bordier trephine, 7 mm in internal diameter, to avoid fracture and minimize sampling 
error. The biopsy site is 1 inch posterior to the antero-superior iliac spine and 1 inch inferior to the summit of 
the iliac crest. The skin and subcutaneous tissue will be injected with 1% xylocaine with epinephrine 
(1:2100,000) anesthetic (it is often necessary to re-dose during the procedure). The periosteum is then injected 
(both the superficial and the deep surface to prevent pain during biopsy). After the local anesthetic has been 
allowed to achieve its desired effect, the incision is made at the pre-marked site. The superficial surface of the 
ileum is identified and the trephine cutting surface is directly applied to the bone (the trephine will be marked 
with the desired depth of penetration in order to limit the depth of penetration into the iliac crest). The trephine 
is then turned in a clockwise fashion through the superficial cortical surface of bone and the cancellous bone 
(perpendicular with the iliac crest from the outer table to the inner table. The trephine is then removed and the 
biopsy specimen (core, including cancellous bone and a single cortical layer) is placed in 70% ethanol in a 
sterile container labeled with the subject’s unique identifier.  After similar local anesthesia, a second smaller 
core biopsy (3.5 mm x 2 cm) will be obtained from just posterior to the first biopsy. If the participant has 
provided consent, a bone marrow aspirate of 10 cc maximum will be obtained from the same site as the 
second biopsy. Adequacy of the aspirate will be confirmed by the presence of bony spicules. The 3.5 mm core 
will be homogenized in Qiazol and the aspirate will be filtered (100 micron mesh) and spun to separate cells 
from the marrow supernatant, the latter of which will be banked for future analyses of marrow 
cytokines/adipokines which is beyond the scope of the current proposal.  Pressure is applied to ensure 
hemostasis and the biopsy site will be closed in layers using 3.0 monocryl sutures and the skin closed with 

 
 
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absorbable sutures. The procedure lasts 30-40 minutes. After the bone biopsy procedure, subjects will be 
instructed to lie flat for 2 hours in the Clinical Research Center before going home in order to minimize swelling 
and hematoma accumulation within the periosteum or adjacent muscles. Subjects will be instructed on strict 
bed rest for 24 hours after the procedure (apart from necessary activities such as eating/using the restroom) 
and then reduced activity for 14 days (no hiking/biking/exercise classes and walking less than 1 mile). As done 
by our collaborators at the Mayo Clinic, subjects will be instructed to use acetaminophen for analgesia. Tools 
used for the bone biopsy procedure will be sharpened and sterilized after each use.  
 
Outcomes: Note that the analysis of the biopsy specimens and other endpoints will be performed in a 
blinded fashion (i.e. the interpretation will be done without knowledge of group assignment). 
 
Bone Biopsy Microarchitectural and Histomorphometric Analyses will address the hypothesis that 
combined therapy with teriparatide and denosumab will inhibit teriparatide-induced skeletal remodeling while 
allowing for continued stimulation of modeling-based bone formation. Fluorochrome labels will be administered 
as reported by Dr. Dempster (see figure below, DEM=demeclocycline, TET=tetracycline).58,59 The subject will 
receive 150 mg DEM QID for three days, no label for 12 days and DEM again for three days (3:12:3), ending 2-
5 days prior to visit 1(baseline labeling). Tetracycline (TET, 250 mg) will be administered in a similar fashion 
prior to visit 3. The biopsy is performed 5–8 days following the last tetracycline dose. 
 
Core biopsy samples will be fixed immediately in 70% ETOH and high-resolution micro-CT (µCT, µCT40, 
Scanco Medical AG) will be performed in the P30 Core facility to evaluate trabecular and cortical bone 
microarchitecture as previously published.32,60-63. The long axis of the biopsy will be aligned with the rotational 
axis of the scanner.  Scans will be acquired with 70 kVP, 160 µAs with an integration time of 200 ms and 
nominal voxel size of 10 µm. Images will be reconstructed, subjected to Gaussian filtration and the mineralized 
tissue segmented using a global threshold.  Trabecular and cortical morphometric indices will be assessed 
using direct 3D approaches.64 Trabecular bone outcomes will include volumetric density, bone volume fraction, 
thickness, and separation, connectivity density and trabecular bone pattern factor. Cortical outcomes will 
include volumetric density, tissue mineral density, thickness and porosity. 
 
The 8mm core biopsy specimen will then be embedded in methylmethacrylate. Dynamic parameters will be 
evaluated on unstained 10m sections and static parameters will be evaluated on 5 micron sections stained 
with Massons Trichrome. Analyses will be performed on at least two nonconsecutive sections of each sample 
using Osteomeasure software.  Mineralizing surface/bone surface will be calculated using the length of all 
double labels and the length of half of the single labels. Since demeclocycline can overestimate the length of 
the label relative to tetracycline, the length of the tetracycline labels will be multiplied by a correction factor of 
1.34 as was done by our consultant Dr. Dempster.59 Because each bone compartment has distinct remodeling 
characteristics, four compartments will be evaluated: cancellous (bone surround by bone marrow), endocortical 
(bone in contact with the inner boundary of the cortical bone), intracortical (Haversian canal surface), and 
periosteal (outer boundary of cortex). All parameters will be expressed according to the most recent guidelines 
from the ASBMR.8 Static Parameters include osteoid surface, osteoid volume, osteoid thickness, osteoblast 
surface, osteoclast surface and eroded surface. In addition, the area occupied by marrow adipocytes 
(adipocyte volume/tissue volume) will be quantitated as will the number of osteoblasts, lining cells and 
osteoclasts (Nob/perimeter, NLc/ perimeter and NOcl/perimeter).  Dynamic parameters include mineral 
apposition rate, mineralization surface, mineralization lag time, resorption period, resorption rate, and 
activation frequency. Structural parameters include trabecular bone area, trabecular thickness, trabecular 
number, trabecular separation, cortical width, osteocyte lacunar area and cortical porosity. Analyses will be 
performed according to Frost’s method for tetracycline-labeled trabecular bone and the results will be 
interpreted with regard to Frost’s “basic Multicellular unit” (BMU) theory.65,66 One exception to BMU theory will 

 
 
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be made, however, since there is evidence that bone formation can occur without preceding resorption 
(modeling-based formation). A bone-forming unit will be identified by the presence of single or double 
tetracycline labels. Remodeling-based formation units (RmF.U) and modeling-based formation units (MF.U) in 
the unlabeled sections will be identified by examining the underlying reversal and cement lines in adjacent 
trichrome-stained sections.  A scalloped reversal line indicates that the forming unit is remodeling-based, 
whereas a smooth cement line indicates that it is modeling-based. Furthermore, we will term a RmF.U with 
tetracycline label(s) extending beyond the extent of the scalloped reversal line unit as an overfilled remodeling 
unit (oRmF.U). Collagen fiber alignment will be evaluated under polarized microscopy to identify interrupted vs. 
uniformly oriented fibers, characteristic of remodeling and modeling-based formation respectively. 
 
VII. Biostatistical Analysis 
 
This study tests the hypothesis that in postmenopausal osteoporotic women, combined PTH-receptor 
stimulation (teriparatide) and RANKL inhibition (denosumab) will result in inhibition of teriparatide-induced 
skeletal remodeling while allowing for continued vigorous stimulation of modeling-based bone formation.  
 
The primary endpoint of this study is the change from baseline to month 3 in the cancellous bone formation 
rate (BFR/BS) in order to test the hypothesis that modelling based bone formation is occurring in the 
combination therapy group.  
 
Key secondary endpoints include MS/BS in the cancellous, endocortical, intracortical, and periosteal 
compartment separately as well as separate calculations of the proportion of the rates of remodeling-based 
bone formation (RBF/BS), modeling based bone formation (MBF/BS), and overflow MBF (oMBF) in the 
cancellous, endocortical, periosteal compartments.  
 
Between-group differences in endpoints will be assessed by comparing each treatment group’s 3-month mean 
change by one-way ANOVA followed by a post-hoc test.   Normality of the outcome data will be examined by 
Shapiro-Wilk’s test. If the outcomes are not normally distributed, a proper transformation will be performed 
before applying the ANOVA or the use of the Kruskal-Wallis test followed by Tukey Kramer parametric or 
Dunn’s non-parametric post-hoc test. 
 
All study participants who have had a bone biopsy at month 3 will be included in the primary outcome analysis. 
For the primary outcome analysis, we will adopt a Bonferroni corrected overall Type-1 error rate of 5% for the 
three-group comparisons (i.e., individual test’s alpha = 0.017). For the secondary outcomes, the Type-1 error 
control for the multiple comparisons will not be considered (i.e., individual test’s alpha = 0.05).   
 
Additional secondary analyses will include an examination of the within-group mean change of each endpoint 
by paired samples t-test or Wilcoxon’s signed rank test depending on the distribution of the outcome. The 
micro-CT analyses of human bone biopsy specimens are exploratory. 
 
Effect size estimates: The power analyses were conducted using the estimated standard deviations 
reported in the AVA study (reference 46). This study utilized identical quadruple-labeling biopsy methodology 
in a short-term comparison of 2 different interventions (denosumab versus teriparatide). We conservatively 
predict that 10 subjects in each group will complete the study (83% completion rate). With this group size, the 
table below shows our effect size estimates for a 3-group multiple comparisons at a Bonferroni’s corrected 
overall Type-1 error rate of 5% with 80% power. This detectable difference is much smaller than that reported 
in published studies comparing short term interventions in similar populations, including the AVA study in 
which BFR/BS increased from 0.0096 to 0.0366 mm3/mm2/y (greater than 3-fold increase) in women treated 
with teriparatide and decreased from 0.0091 to 0.0014 mm3/mm2/y (greater than 6-fold decrease) in women 
treated with denosumab. Similar differences were reported in endocortical MS/BS (increase from 9% to 38 
with teriparatide and decrease from 9% to 7% with denosumab, between group difference >30%), 
intracortical MS/BS (increase from 9% to 21% with teriparatide and decrease from 10% to 3% with 

 
 
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denosumab, between group difference >19%), and periosteal MS/BS (increase from 1% to 4% with 
teriparatide and decrease from 1% to 0% with denosumab, between group difference >4%). Study 
participants that fall outside the month 3 study window will be included in the primary outcome analysis if the 
bone biopsy is performed within 21 days from the last tetracycline labeling dose AND if they continue to 
receive study drug throughout this time.  
 
To estimate our ability to detect between-group differences in the changes in RBF/BS, MBF/BS, and 
oMBF/BS in cancellous, endocortical, and periosteal bone, we use the standard deviation from an 
unpublished analysis of the AVA study (Dempster, personal communication). The minimal detectable 
differences shown below are significantly smaller than the reported between group differences in the change 
of each parameter between denosumab and teriparatide treated women in the AVA study. For example, the 
difference between the 3-month change in cancellous MBF between teriparatide and denosumab treated 
women was 1.5% whereas we will be able to detect a difference of 0.15%. While differences in MBF between 
the combination group and either monotherapy group will likely be lower than those observed between 
teriparatide and denosumab, the study will still have sufficient power to detect small between-group 
differences. Thus, we will have adequate power to test our hypothesis that in postmenopausal osteoporotic 
women, treatment with combined teriparatide and denosumab will result in increased bone formation 
(compared to denosumab alone), and specifically modeling-based bone formation in all bone compartments. 
 
The study will not have sufficient power to detect any differences in any safety endpoints between the three 
groups.  
 
Parameter  
Est. SD of the 3-month ∆  
min detectable difference (5% sig, 80% power) 
Cancellous BFR/BS 
(mm3/mm2/y) 
0.005 (mm3/mm2/y) 
0.007 (mm3/mm2/y) 
Cancellous MS/BS (%) 
2.60% 
4.06% 
Endocortical MS/BS(%) 
5.11% 
6.77% 
Intracortical MS/BS(%) 
7.57% 
10.03% 
Periosteal MS/BS(%) 
1.32% 
1.74% 
Cancellous RBF (%) 
3.06% 
4.05% 
Cancellous MBF (%) 
0.11% 
0.15% 
Cancellous oMBF (%) 
0.28% 
0.37% 
Endocortical RBF (%) 
6.28% 
8.32% 
Endocortical MBF (%) 
1.11% 
1.47% 
Endocortical oMBF (%) 
0.89% 
1.18% 
Periosteal RBF (%) 
0.44% 
0.58% 
Periosteal MBF (%) 
3.11% 
4.12% 
Periosteal oMBF (%) 
0.17% 
0.23% 
 
To account for imbalance in the dropout rate as of August 11, 2021, the following power calculation analysis is 
for an outcome of predicted 8 subjects in each group. Effect sizes were estimated for a 3-group multiple 
comparisons at a Bonferroni’s corrected overall Type-I error rate of 5% (alpha 0.017) with 80% power. For the 
8-8-8 outcomes, the detectable differences for our primary endpoint as well as a number of key secondary 
endpoints is much smaller than that reported in published studies comparing short term interventions in similar 
populations, in particular the AVA study (reference 46). While between-group differences between the 
combination group and either monotherapy group will be smaller than the between-group differences between 
teriparatide and denosumab, the study will have sufficient power to detect small between-group differences.  
  
Primary Endpoint:  

 
 
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1. Cancellous BFR/BS, minimum detectable difference for a 8-8-8 outcome is 0.009 mm3/mm2/y. 
Between group difference reported in the AVA study (denosumab vs. teriparatide) was 0.0347 
mm3/mm2/y. 
  
Secondary Endpoints:  
1. Cancellous MS/BS = 4.65 for a 8-8-8 outcome, between group difference in AVA study = 17.33  
2. Endocortical MS/BS = 9.14 for a 8-8-8 outcome, between group difference in AVA study = 33.89 
3. Intracortical  MS/BS = 13.54 for a 8-8-8 outcome, between group difference in AVA study = 19.81 
4. Periosteal MS/BS = 2.36 for a 8-8-8 outcome, between group difference in AVA study = 4.44 
5. Cancellous RBF = 5.47 for a 8-8-8 outcome, between group difference in AVA study = 12.718 
6. Cancellous MBF = 0.20 for a 8-8-8 outcome, between group difference in AVA study = 1.244 
 
 
VIII. Risk and Discomforts 
Risks of these medications are summarized below: 
 
Denosumab 
DMAB is an FDA approved medication used to treat osteoporosis in various populations, including 
postmenopausal women. More than 13,500 patients have been treated with denosumab in clinical studies, and 
it is generally well tolerated.6,43,67 In clinical studies, it has been reported that DMAB may produce the following 
side-effects: hypersensitivity including anaphylactic reaction, hypocalcemia, osteonecrosis of the jaw, atypical 
femoral fractures, multiple vertebral fractures following denosumab discontinuation, serious infections including 
skin infections and cystitis, dermatitis, rashes, eczema, musculoskeletal pain, and hypercholesterolemia. 
Pancreatitis has been reported in clinical trials. To reduce the likelihood of adverse events, subjects with any 
contraindications to denosumab, including dental procedures, will be excluded. Subjects will be monitored for 
report of thigh or groin pain and would be evaluated to rule out an atypical femur fracture. Subjects who 
withdraw consent during the study will be counseled on the increased risk of fracture when denosumab is 
discontinued and not followed by subsequent osteoporosis treatment and offered a referral to a specialist if 
desired. 
 
Teriparatide 
TPTD is an FDA approved medication used to treat postmenopausal women with osteoporosis who are at high 
risk of fracture. The most commonly reported side effects of TPTD are overall pain, asthenia, nausea, 
headache, leg cramps, sinus tachycardia, arthralgia, rhinitis, and dizziness. TPTD can cause transient 
hypercalcemia, which is generally mild.7 Repeated daily administration of TPTD in high doses to rodents 
causes dose-dependent osteosclerosis, bony obliteration of the marrow space, and extra-medullary 
hematopoiesis, followed by osteosarcomas.68 The relevance of these findings to humans is unclear and will 
remain so for many years, because osteosarcoma is rare in adults (4 cases/million/year).69 Eli Lilly and the 
FDA are monitoring the incidence of osteosarcoma in TPTD treated patients and have not reported a linkage 
thus far. Hypercalcemia is a well-described complication of TPTD treatment.  Elevated blood calcium levels will 
be handled in the manner recommended by Antoniucci et al.5 (Figure 1) 
 
Antibiotic Labels: Short-term administrations of the tetracycline and demeclocycline are generally very well 
tolerated with the most common side effect being gastrointestinal symptoms such as anorexia, nausea, and 
vomiting. Rarely, maculopapular and erythematous rashes, erythema multiforme and Stevens-Johnson 
syndrome have been reported. Hypersensitivity reactions including urticaria, angioneurotic edema, 
polyarthralgia, and anaphylaxis have also been rarely reported. 
 
Sedation with Lorazepam/Alprazolam/Midazolam/Fentanyl (optional):  
Subjects may opt to have sedation during the bone biopsy procedure. Sedation is generally very safe but there 
is still a risk of side effects. If these side effects do occur, the majority of them are temporary, although long-
term complications may occur. All reasonable precautions will be applied to avoid any complications. Common 

 
 
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side effects include nausea and vomiting, headache, sore or dry throat, dizziness or feeling faint, fall in blood 
pressure or a mild allergic reaction such as itching or a rash. If the level of sedation is too deep, subjects may 
experience some difficulty breathing that may require breathing support until the effects of the sedation wear 
off. In rare cases, there can be serious allergic reactions or cardiac arrest (stopping of the heart).   
 
Ondansetron (optional): 
If subjects experience nausea or vomiting from the sedation during the bone biopsy procedure, they will be 
offered ondansetron to help minimize these side effects. Common side effects of ondansetron include 
headache, dizziness, drowsiness, constipation or diarrhea. Additional rare side effects include serious 
arrythmias and hypoxia.   
 
Risks of Bone Biopsy Procedure: 
Bone biopsies of the iliac crest are associated with discomfort but pain can be largely eliminated or reduced 
when the skin and periosteum are adequately treated with local anesthetic prior to the procedure. Serious 
complications are very rare but include pain, hematoma, and wound infection. Neuropathy (transient) is an 
extremely unusual complication of bone biopsies. In the most comprehensive study of bone biopsy 
complications observed in 9131 trans-iliac biopsies, the incidence of overall complications was 0.6%; 0.2% 
experienced prolonged pain, 0.2% developed a hematoma, 0.12% experienced transient neuropathy, and 
0.07% developed a wound infection. Two patients (0.02%) experienced hip fracture, and one patient (0.01%) 
developed osteomyelitis. No patient died or experienced permanent disability.70 The short courses of 
tetracycline and demeclocycline antibiotics are generally well tolerated but can cause gastrointestinal 
discomfort and sun-sensitivity as discussed above. 
 
Minimizing biopsy risks: 
As stated above, the common risks of the procedure are discussed in detail with the participant. The signs and 
symptoms of infection (fever above 101.5, a draining wound, expanding redness around the incision, 
worsening pain and chills) are described and the subject is instructed to contact study staff immediately should 
any of these occur.  Post-procedural pain is a risk of the procedure although not technically a complication. 
The subject is instructed that ice be applied to the biopsy site using a cloth barrier between the ice pack and 
the subject’s skin. The subject is also instructed that over the counter pain medications should also be used to 
help manage pain (acetaminophen and non-steroidal anti-inflammatory medicine). Thigh numbness and pain 
are uncommon complications of this procedure caused by damage to the lateral branch of the lateral femoral 
cutaneous nerve (LFCN).  The biopsy is taken 3 cm lateral to the anteriorsuperior iliac spine to avoid the lateral 
branch of the LFCN. Some bleeding after the biopsy is expected.  It is explained to the subject that while some 
bloody staining of the dressing applied at the end of the procedure is to be expected; bloody drainage from the 
incision should be reported to the investigator immediately. Due to the anesthesia, subjects will be instructed to 
arrange for transportation from the office to their home. Subjects will be asked to limit physical activity and hard 
physical work 3-4 days after the biopsy and not to exercise for 7 days after the biopsy.  
 
All adverse events will be reported to the IRB either immediately (in the case of definitely related or 
probably/possibly related serious adverse events) or at the time of the scheduled renewal process (for all other 
adverse events).  The study physicians and the study staff will meet on a weekly basis to discuss any issues 
relating to adverse events, safety data and outcome data. The risks of bone biopsy will be minimized by careful 
attention to proper technique and will be performed by a board-certified orthopedic surgeon.  
 
Imaging studies 
Over the 3-month study period, subjects will receive only a single DXA of the spine and hip, with consequent 
radiation of <1 mrem. This amount of radiation is equal to the background radiation one is exposed from the 
earth and sky under one month. 
 
IX. Potential Benefits 
The medications given in this study have been shown to reduce fracture risk in postmenopausal women with 

 
 
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osteoporosis when used according to their FDA approved label; however, it is unknown if subjects’ 
osteoporosis will get better, worse, or stay the same in this study. It is hoped that the knowledge gained from 
this study will improve osteoporosis care. Given the short-term nature of the study, participants will be 
encouraged to discuss continuing osteoporosis care with their providers at the end of the study.  
 
Subjects will be compensated for their time with $25 after each visit and $400 for completing the bone biopsy, 
for a total of $500. Subjects will not be compensated for the screen visit and will be mailed a copy of their 
screen visit bone density results.  
 
Parking vouchers will be provided to subjects at each visit, including the screening visit. 
 
X. Monitoring and Quality Assurance 
The Institutional Review Board (IRB) will review and approve the study protocol, ICF and any other applicable 
subject material before any study-specific activities or procedures begin. Before a subject can participate in 
any study-specific activities/procedures, she will sign, or have a legally authorized representative sign and date 
the IRB-approved ICF. 
 
A study physician will review all laboratory test results. The study physicians and the study staff meet on a 
weekly basis to discuss any issues relating to adverse events, safety data and outcome data. Subjects will be 
discontinued from the study if they have any finding that in the opinion of the study physician requires 
withdrawal from the study. Disqualifying findings will be communicated to both the subject and her primary care 
physician. If the subject does not have a primary care physician, the principal investigator will coordinate 
appropriate follow-up with a physician of the subject's choosing.  
 
Study Completion and Closeout Procedures  
Dr. Leder and Dr. Tsai will verify that:  
- study interventions are complete and that all data have been collected 
- correspondence and study files are accessible for audits 
- study records are maintained  
- NIAMS and the IRB are notified of the study’s completion and store a copy of the notifications 
- a clinicaltrials.gov report is completed 
- subjects are notified of study completion and asked if they would like to be informed of the results and/or 
receive a copy of the publication of the trial results 
 
At their third study visit, all study participants will meet with a study physician. The study physician will review 
the experience of the participant during the study and explain to the participant that it is imperative that they 
arrange follow up with their primary care physician within 3 months of finishing the study, due to the increased 
risk of multiple vertebral fractures when denosumab is discontinued and not followed by subsequent 
osteoporosis treatment. Subjects will be encouraged to see a physician with some expertise in the treatment of 
osteoporosis and offered the opportunity to schedule an appointment in the Endocrine Associates outpatient 
clinic to discuss therapy beyond the study period. With the subject’s permission, a letter will be sent to their 
primary care physician, at completion of the study, including this information and the results of all relevant 
investigations from the study period. Study participants will be reminded of this information at their final study 
visit and a follow-up phone call will be made 3 months after exit from the study to ensure that they have 
received appropriate follow-up. 
 
The Data Safety Monitoring Board (DSMB) will monitor this study. This DSMB is comprised of experienced 
clinical investigators with expertise in osteoporosis: Dr. Kristine Ensrud, Dr. Susan Ott, and Dr. George 
Howard.  
 
Definition of Adverse Events  
An adverse event (AE) is any unfavorable and unintended diagnosis, sign (including an abnormal laboratory 

 
 
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finding), symptom, or disease temporarily associated with the study intervention, which may or may not be 
related to the intervention. AEs include any new events not present during the pre-intervention period or events 
that were present during the pre-intervention period which increased in severity. 
 
Definition of Serious Adverse Event 
A serious adverse event (SAE) is any untoward medical occurrence that results in death, is life-threatening, 
requires or prolongs hospitalization, causes persistent or significant disability/incapacity, results in congenital 
anomalies/birth defects, or, in the opinion of the investigators, represents other significant hazards or 
potentially serious harm to research participants or others. 
 
Definition of Unanticipated Problems 
Unanticipated problems (UPs) are defined as being unexpected in terms of nature, severity, or frequency 
based on the IRB-approved research protocol, informed consent documents and the participant population 
being studied; being related or possibly related to participation in the research (“possibly related” means there 
is a reasonable possibility that the UP may have been caused by the procedures involved in the research); 
AND suggesting that the research places participants or others at a greater risk of harm (including physical, 
psychological, economic, or social harm) than was previously known or recognized. 
 
Classification of an Adverse Event  
All AEs will be assessed by the study clinician and assigned to the following categories: 
 
Mild: Events require minimal or no treatment and do not interfere with the participant’s daily activities.  
 
Moderate: Events result in a low level of inconvenience or concern with the therapeutic measures. Moderate 
events may cause some interference with functioning. 
 
Severe: Events interrupt a participant’s usual daily activity and may require systemic drug therapy or other 
treatment. Severe events are usually potentially life-threatening or incapacitating.  Of note, the term “severe” 
does not necessarily equate to “serious”. 
 
Relationship to Study Intervention  
All AEs will have their relationship to study intervention assessed by the clinician who examines and evaluates 
the participant based on temporal relationship and his/her clinical judgment. The degree of certainty about 
causality will be graded using the categories below:  
 
Definite: There is clear evidence to suggest a causal relationship, and other possible contributing factors can 
be ruled out. The clinical event, including an abnormal laboratory test result, occurs in a plausible time 
relationship to study intervention administration and cannot be explained by concurrent disease or other drugs 
or chemicals. The response to withdrawal of the study intervention (dechallenge) should be clinically plausible. 
The event must be pharmacologically or phenomenologically definitive, with use of a satisfactory rechallenge 
procedure if necessary. 
 
Probable/Possible: There is evidence to suggest a causal relationship (e.g. the event occurred within a 
reasonable time after administration of the trial medication), but other factors are present which may have 
contributed to the event (e.g. the participant’s clinical condition, other concomitant events). However, there is 
greater than 50% certainty that the event is related to study intervention. The event follows a clinically 
reasonable response on withdrawal (dechallenge). Rechallenge information is not required to fulfill this 
definition. Although an AE may rate as “probable/possible” upon discovery, it can be flagged as requiring more 
information and later be upgraded to “definite,” as appropriate. 
 
Unrelated/Unlikely related: The AE is completely independent of study intervention administration, and/or 
evidence exists that the event is definitely or likely related to another etiology. 

 
 
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Expectedness 
The study investigator will be responsible for determining whether an AE is expected or unexpected. An AE will 
be considered unexpected if the nature, severity, or frequency of the event is not consistent with the risk 
information previously described for the study intervention.  
 
Time Period and Frequency for Event Assessment and Follow-Up 
The occurrence of an AE or SAE may come to the attention of study personnel during study visits and 
interviews of a study participant presenting for medical care, or upon review by a study monitor. 
 
All AEs including local and systemic reactions not meeting the criteria for SAEs will be captured on the 
appropriate case report form. Information to be collected includes event description, time of onset, clinician’s 
assessment of severity, relationship to study product (assessed only by those with the training and authority to 
make a diagnosis), and time of resolution/stabilization of the event. All AEs occurring while on study must be 
documented appropriately regardless of relationship. All AEs will be followed to adequate resolution. 
 
Any medical condition that is present at the time that the participant is screened will be considered as baseline 
and not reported as an AE. However, if the study participant’s condition deteriorates at any time during the 
study, it will be recorded as an AE.  
 
Changes in the severity of an AE will be documented to allow an assessment of the duration of the event at 
each level of severity to be performed. AEs characterized as intermittent require documentation of onset and 
duration of each episode. 
 
A Study Team member will record all reportable events with start dates occurring any time after informed 
consent is obtained until 7 (for non-serious AEs) or 30 days (for SAEs) after the last day of study participation, 
unless reporting is deemed necessary in the opinion of the study investigator.  At each study visit, the 
investigator will inquire about the occurrence of AE/SAEs since the last visit.  Events will be followed for 
outcome information until resolution or stabilization. 
 
Reporting of Adverse Events 
Adverse events and serious adverse events will be reported to all appropriate regulatory bodies as required by 
current regulations. Study staff will record all adverse events in the Adverse Event Log upon learning of such 
events. The Adverse Event Log will be shared annually with the IRB at the time of Continuing Review and with 
KAI at the time of the annual meeting. Study staff will also complete a Serious Adverse Event Form for all 
serious adverse events, to be filed in the subject’s study file. After identification of any adverse event, the PI 
will advise the study team regarding screening, enrollment, and ongoing participation and initiatives to prevent 
further AEs if relevant. The principal investigator or Dr. Demay or Dr. Tsai will evaluate the adverse event and 
determine whether the adverse event affects the Risk/Benefit ratio of the study and whether modifications to 
the protocol or consent form are required. The PI will be responsible for determining if the study protocol may 
be continued, terminated, or modified based on the observed beneficial or adverse effects of the treatment 
under study. 
 
Reports submitted to Amgen will include an annual and final safety report plus any other aggregate analyses 
(of any report containing safety data generated during the course of the study) submitted to the Partners IRB. 
 
Adverse Events Requiring Immediate Reporting 
Serious adverse events 
- Study staff will notify KAI of all serious adverse events within 48 hours of learning of such events. KAI will 
notify the NIAMS and the DSMB. This will be followed by a written report as soon as possible. 
- Study staff will notify the FDA of all serious adverse events within 15 calendar days. The sponsor of the IND 
(Dr. Leder) will adjudicate all adverse events in determining whether reporting to the FDA is necessary. In the 

 
 
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case that such an event causes death or is life-threatening, this will be reported to the FDA within 7 calendar 
days. An event is considered life-threatening, as defined by the FDA, if “in the view of either the investigator or 
sponsor, its occurrence places the patient or subject at immediate risk of death” (21 CFR 312.32). Non-serious 
adverse events do not require reporting to the FDA. 
- If a serious adverse event is determined to be definitely or probably/possibly related to denosumab, a report 
of the event will also be submitted to Amgen at the same time as the IRB report. 
 
Unexpected adverse events (serious or non-serious) that are definitely related or probably/possibly 
related  
- Study staff will report the event to the IRB within 5 business days or 7 calendar days 
 
Reporting of Unanticipated Problems 
The following procedures will be followed for all Unanticipated Problems (UPs) that do not meet the definition 
of an adverse event. 
 
Unanticipated problems will be reported within 48 hours to KAI (followed by a written report) and within 7 
calendar days to the IRB. Unanticipated problems that do not meet the definition of an adverse event will be 
reported to the IRB as an ‘Other Event’ submission on Insight, and must include the following information: a 
detailed description of the unanticipated problem; the basis for determining that the problem is unexpected; the 
basis for determining that the problem indicates that the research places subjects at an increased risk of harm; 
and whether any changes to the research or other corrective action are warranted. The PI will advise the study 
team regarding screening, enrollment, and ongoing participation after identification and resolution of these 
events. 
 
Further clarification on which events require immediate reporting can be found in the Partners Healthcare 
policy on Reporting Unanticipated Problems including Adverse Events (for reporting to the IRB) and 21 CFR 
312.32 (for reporting to the FDA). 
 
All subjects will be informed of their rights under the Health Insurance and Portability and Accountability Act of 
1996 per federal law and hospital policy. The screening only and main study ICFs contain an IRB-approved 
template that describes what protected health information from the research study may be used or shared with 
others. Data collected strictly for research purposes are stored in locked files and on computers with 
passwords required for access. When the data are published, no names or other materials that allow 
identification of an individual will be used. 
 
The study database will be secured with password protection and only IRB-approved study staff will have 
access. The study biostatistician will receive only de-identified coded information. Electronic or written 
communication with any outside collaborators will involve only unidentifiable information. Adverse event reports 
and annual continuing reviews will not include subject identifiable material. Data will be entered into REDCap, 
a free, secure, web-based application designed to support data entry for research studies. REDCap was 
developed by a multi-institutional consortium initiated at Vanderbilt University. Data collection will be 
customized for this project by the research team with guidance from Harvard Catalyst staff. The REDCap built-
in system for data verification to safeguard against incorrect data entry will be used (e.g. data entry to be 
performed twice and verified within REDCap). REDCap is designed to comply with HIPAA regulations. Data 
within REDCap will be categorized as verified or unverified. Once all verified data are acquired and entered, 
the final dataset will be locked within REDCap. De-identified information sets may be extracted from REDCap 
to be shared with outside collaborators as needed, which would be performed only in accordance with the 
Partners IRB approval and the ICF. Collaborators will be provided with de-identified, secure data to be used 
solely for research. Collaborators will be requested to return or destroy data once data analysis is complete. 
 
XI. Publication Policy 
Publication of complete data from the study is planned. It is anticipated that the results of 

 
 
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this study will be published in a peer reviewed scientific or medical journal and may be presented at scientific 
meetings. Investigators will publish results from the study in compliance with their agreement with Amgen. 
 
 
 

 
 
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XII. Abbreviations  
 
25-OH vitamin D  
25-Hydroxy Vitamin D 
ACC 
American College of Cardiology  
AE 
Adverse Event 
AHA 
American Heart Association  
ALT 
Alanine Transaminase 
ANOVA 
Analysis of Variance 
ASBMR 
American Society for Bone and Mineral Research 
AST 
Aspartate Aminotransferase  
BFR 
Bone Formation Rate 
BMD 
Bone Mineral Density  
BMP6 
Bone Morphogenetic Protein 6 
BMSCs 
Bone Marrow Stromal Cells  
BS 
Bone Surface  
BUN 
Blood Urea Nitrogen 
Ca 
Calcium 
CBC 
Complete Blood Count 
CFR 
Code of Federal Regulations 
Cr 
Creatinine  
CT 
Computed Tomography  
CTX 
C-Telopeptide 
DATA 
Denosumab and Teriparatide Administration 
DEM  
Demeclocycline 
DSMB 
Data Safety Monitoring Board 
DXA 
Dual-Energy X-Ray Absorptiometry 
ES 
Eroded Surface 
ETOH 
Ethanol 
FDA 
Food and Drug Administration 
FSH  
Follicle Stimulating Hormone 
GCP 
Good Clinical Practice 
GCRC 
General Clinical Research Centre 
PHRC 
Partners Human Research Committee 
ICF 
Informed Consent Form  
ICH 
International Conference on Harmonisation  
IDE 
Investigational Device Exemption 
IGF-1 
Insulin Like Growth Factor 1 
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
LFCN 
Lateral Femoral Cutaneous Nerve 
MBF 
Modeling-Based Bone Formation 
MF.U 
Modeling-Based Formation Units 
MGH  
Massachusetts General Hospital  
MS 
Mineralizing Surface  
NIAMS 
National Institute of Arthritis and Musculoskeletal and Skin Diseases 
oMBF 
Overflow Modeling Based Bone Formation 
oRmF.U 
Overfilled Remodeling Unit  
P1NP 
Procollagen Type 1 N-Terminal Propeptide  
Phos 
Phosphate 
PTH  
Parathyroid Hormone 

 
 
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PTHrP 
Parathyroid Hormone-Related Peptide 
RANKL 
Receptor Activator of Nuclear Factor Kappa-B Ligand 
RBF 
Remodeling-Based Formation 
RmF.U 
Remodeling-Based Formation Units 
SAE 
Serious Adverse Event 
SD 
Standard Deviation 
SERMs 
Selective Estrogen Receptor Modulators 
TET 
Tetracycline  
TGF-ß 
Transforming Growth Factor-Beta 
TPTD 
Teriparatide  
UPs 
Unanticipated Problems 
US 
United States 
 

 
 
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Version: 9.0, August 11, 2021 
XIII. Protocol Amendment History  
 
Version 
Date 
Description of Change  
Brief Rationale 
2.0 
08 Feb  
2019 
Page 7: blood samples will be 
stored by code number and date 
only, not participant name 
To ensure de-identification of study 
participants 
3.0 
27 June 
2019 
Changes made in response to KAI 
and DSMB document review and 
teleconference on 04/18/2019 and 
06/27/2019. 
Changes made in response to KAI 
and DSMB document review and 
teleconference on 04/18/2019 and 
06/27/2019. 
4.0 
16 July 
2019 
Changes made in respond to KAI 
and DSMB document review 
following 6/27/19 teleconference. 
Changes made in respond to KAI 
and DSMB document review 
following 6/27/19 teleconference. 
5.0 
26 
November 
2019 
FSH testing only required if women 
are <60 years; change of oral BP 
exclusion criteria; addition of 3 
other orthopedic staff who will 
perform the bone biopsies; 
addition of short-acting oral 
sedation to list of sedative meds 
that can be given during the bone 
biopsy. 
 
 
6.0 
05 May 
2020 
Modifications to safety reporting 
procedures. 
Language was clarified to match IRB 
and FDA policies. 
7.0 
01 April 
2021; 
revised 
June 01 
2021  
Changes made to allow for 
labs/DXA to be repeated under 
certain circumstances and to allow 
for DXA’s done for clinical 
indication or under other study 
protocols to be used for this study 
if done within 6 months of screen 
date. Adverse event reporting 
language clarified. Risk of sedation 
and ondansetron (optional meds) 
previously included in main ICF 
have been included in the protocol. 
 
Changes made on June 01, 2021 to 
reflect feedback received from 
expert external reviewer, Dr. Paul 
Miller, in context of adverse events 
(OE 11 and OE 12).  
Changes made in response to QI audit, 
and recent subject adverse event.  
8.0 
08 July 
2021 
Change made to collect 10cc bone 
marrow instead of maximum 40cc 
Change made in response to 
suggestion made by our NIH-
appointed DSMB. 
9.0 
11 August 
2021 
Change made to close assignment 
to the denosumab only group and 
to state clearly how the change in 
randomization assignment will 
occur moving forward.  
Change made in response to an 
imbalance in the study dropout rate 
amongst the 3 study groups and in 
response to the research pharmacy’s 
request for a revision in detailed 
protocol.   

 
 
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Figure 1: Hypercalcemia workflow 
 
 
 
If the serum calcium concentration rises above 11.2 mg/dl, TPTD will be stopped and serum calcium measured 
at least daily until normal.  Thereafter TPTD will be resumed every other day and the above algorithm followed, 
starting at "Reduce PTH to every other day". 
 
 
 
 
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