Conditions: Arterial Calcification CD73 Deficiency
Keywords: NT5E Bisphosphonates Vascular Calcifications Joint Capsule Calcifications Inherited Disease
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 7
Healthy volunteers: no

Brief summary:
Background:

* Arterial Calcifications due to Deficiency in CD73 (ACDC) is a rare genetic disease. People with ACDC develop calcification in the arteries of the lower extremities as well as calcium deposit in the joints of the fingers, wrists, ankles and feet. The lower extremities calcification causes claudication because of severe ischemia requiring at time revascularization procedures. the calcium deposits in the joints causes severe debilitating pain in the hands and feet. Currently, there are no standard treatments for ACDC.
* Etidronate is a bisphosphonate that interferes with bone metabolism. It is approved to treat Paget's disease, a condition in which the bones are soft and weak and may be deformed, painful, or easily broken. It is also used to treat high blood calcium levels. Researchers want to see if it can be used to treat the calcifications of ACDC and improve pain and blood flow in the lower extremities and arthritic pain of the hands and feet.

Objectives:

\- To see if etidronate is a safe and effective treatment for ACDC.

Eligibility:

\- People between 18 and 80 years of age who have been diagnosed with ACDC.

Design:

* Participants will be screened with a physical exam and medical history. They will also have imaging studies, including CT scan of the lower extremities, x-rays and DEXA bone scans, before starting treatment. Blood and urine samples will be collected. An exercise tolerance test will also be given and ABI (ankle brachial index will be measured.
* Participants will take etidronate by mouth once a day for 14 days every 3 months. They will be assigned an individualized 6- month drug schedule to follow.
* Participants will have regular study visits throughout the treatment period. These visits will involve imaging studies, full dental exams, and blood and urine tests. Participants will also have exercise tolerance tests and ABIs measured.
* Participants may also provide tissue samples for further study.
* Treatment will continue for up to 3 years as long as the side effects are not severe and the condition does not become worse. Participants will have a final follow-up visit after stopping treatment.

Detailed description:
We have recently identified a novel genetic disease affecting nine known adults in whom de novo vascular calcifications develop in the lower extremity arteries and juxta-articular joint capsules of the fingers, wrists, ankles and feet. This rare disease results from bi-allelic mutations in the gene ecto-5-prime-nucleotidase (NT5E), encoding the CD73 protein. CD73, an enzyme involved in the extracellular ATP metabolic pathway, converts extracellular AMP to adenosine and inorganic phosphate. The clinical symptoms of this rare disease, termed ACDC (Arterial Calcifications due to Deficiency in CD73), include claudication of the calves, thighs, and buttocks, chronic ischemic pain of the feet at rest with threat of potential limb loss, and debilitating rheumatoid pain in the wrists and hands. Radiological and histological evaluations do not resemble classic atherosclerotic vascular calcification, since the calcification and dysplasia in ACDC occur in the medial portion of the arterial blood vessel wall. Data from patient-specific cell lines indicate increased activity of tissue non-specific alkaline phosphatase (TNAP), a key mediator of pathological ectopic tissue calcification, and thus reveals a potential therapeutic target.

To date, no effective therapy exists for ACDC patients. However, since bisphosphonates are potent competitive inhibitors of TNAP activity and are widely used to modulate bone metabolism, they may beneficially alter vascular calcification. In addition, our preliminary in vitro studies demonstrate the effectiveness of etidronate, a nitrogen-containing bisphosphonate, in lowering TNAP activity in cells isolated from ACDC patients. Etidronate, and bisphosphonates in general, have proven safe and are well tolerated by most patients.

This protocol provides for the administration of etidronate to ACDC patients, for whom no alternative treatment is available. Patients will be examined at the NIH Clinical Center bi-annually for 3 years. The primary objective of this clinical study is to test the effectiveness of etidronate in attenuating the progression of lower extremity arterial calcification and vascular blood flow based on CT calcium score and Ankle Brachial Index (ABI).

Arms:
arm 1: Participants diagnosed with Arterial Calcifications due to Deficiency in CD73 (ACDC) will receive Etidronate. Etidronate will be administered orally at total dose of 20mg/kg daily x 14 days, followed by 10 weeks off study drug (12 weeks = one cycle). Participants may receive up to 12 cycles of etidronate.

Interventions: intervention 1: Etidronate intervention 2: MRI Scan
Intervention descriptions: intervention 1: Didronel (etidronate disodium)- Didronel tablets contain either 200 mg or 400 mg of etidronate disodium, the disodium salt of (1-hydroxyethylidene) diphosphonic acid, for oral administration. This compound, also known as EHDP. regulates bone metabolism. It is a white powder, highly soluble in water. The 400 mg tablets are white capsule-shaped tablets with ED 400 on one side and G on the other side. intervention 2: Visit on day 0 and visit at 36 month - Exploratory MRI studies may be employed to evaluate the lumen of the artery and degree of collateral vessels, given that the high degree of calcification is precluding that evaluation by CT.

Protocol document:
Prot_SAP_000.pdf: 
Page 1 of 27 
Protocol # 12-H-0110
PI: Alessandra Brofferio, M.D.
Amendment U, December 21, 2021
CLINICAL RESEARCH PROJECT
       Protocol # 12-H-0110
Protocol Title: An Open-label, Non-randomized, Single-arm Pilot Study to Evaluate the Effectiveness of 
Etidronate Treatment for Arterial Calcifications due to Deficiency in CD73 (ACDC)
Short Title: Etidronate for ACDC
Keywords: NT5E, Bisphosphonates, Vascular calcifications, Joint capsule calcifications, inherited disease
Principal Investigator:
Alessandra Brofferio, MD
 301-496-2634 – 10CRC/5-3130 
                 NHLBI/CB(E)
Non-NIH engaged non-enrolling Investigators under FWA coverage:
Cynthia St. Hilaire, PhD              412-648-9441 – U. of Pittsburgh Division of Cardiology                               
Subjects of study:
Number
Sex
      Age range
                               20
             M, F
       18-80
Sponsor: NHLBI Office of the Clinical Director
Sponsor’s Authorized Representative: Alessandra Brofferio, MD
Project involves ionizing radiation: 
 Yes 
Off-site project:
                           No
Multi-Institutional project: 
              No 
DSMB involvement:
                           No 
Tech Transfer: CRADA 
 No
                            MTA                             Yes
Investigational Device Exemption (IDE)
The test article is a non-significant risk medical device eligible for abbreviated IDE requirements of 21 CFR 
812.2(b). No IDE license number is applicable.


Page 2 of 27 
Protocol # 12-H-0110
PI: Alessandra Brofferio, M.D.
Amendment U, December 21, 2021
Table of Contents
1.
Précis......................................................................................................................................................4
2.
Objectives...............................................................................................................................................4
2.1
Primary Objective ........................................................................................................................4
2.2
Secondary Objectives...................................................................................................................4
3.
Background ............................................................................................................................................4
3.1
Pathophysiology...........................................................................................................................5
3.2
Treatment Options........................................................................................................................5
4.
Investigational Plan................................................................................................................................7
4.1
Subject Population Selection and Eligibility................................................................................7
4.2
Inclusion and Exclusion Criteria..................................................................................................7
4.3
Study Design and Methods ..........................................................................................................8
4.4
Pre-Treatment Phase ....................................................................................................................9
4.4.1 Establishing Baseline Rate of Disease Progression......................................................................9
4.4.2 Pre-Treatment Evaluations...........................................................................................................9
4.5
Treatment Phase (Clinical Monitoring and Intervention) ..........................................................10
4.6
Medical and Surgical History and Physical Examination..........................................................11
4.7
Laboratory Tests and Procedures...............................................................................................11
4.8
Study Procedures........................................................................................................................12
4.9
Pharmaceuticals .........................................................................................................................15
4.10
Off-label Use of Drugs...............................................................................................................16
4.11
Off-Treatment............................................................................................................................17
4.12
Follow-up Visits ........................................................................................................................17
5.
Biostatistical Considerations ................................................................................................................17
5.1
Primary Endpoint and Analysis .................................................................................................17
5.2
Sample Size................................................................................................................................18
6.
Sample Collection, Storage, and Data Management ...........................................................................18
7.
Data and Safety Monitoring .................................................................................................................18
7.1
Monitoring .................................................................................................................................18
7.2
Adverse Event Management ......................................................................................................19
7.3
NIH IRB, FDA, and CD reporting.............................................................................................19
7.4
Removal of Subjects from the Study .........................................................................................19
8.
Human Subjects Protection ..................................................................................................................20


Page 3 of 27 
Protocol # 12-H-0110
PI: Alessandra Brofferio, M.D.
Amendment U, December 21, 2021
8.1
Rationale for Subject Selection..................................................................................................20
8.2
Rationale for the Exclusion of Children.....................................................................................20
8.3
Rationale for the Exclusion of Pregnant Women.......................................................................20
8.4
Evaluation of Benefits and Risks/Discomforts ..........................................................................20
8.5
Informed Consent Process and Documentation .........................................................................23
8.6
Subject Advocate .......................................................................................................................24
9.
Conflict of Interest ...............................................................................................................................24
10.
Reimbursement for Travel ...................................................................................................................24
11.
Monetary Compensation ......................................................................................................................24
12.
Agreements ..........................................................................................................................................24
13. References................................................................................................................................................24
Appendix 1 .................................................................................................................................................31
Appendix 2 .................................................................................................................................................32


Page 4 of 27 
Protocol # 12-H-0110
PI: Alessandra Brofferio, M.D.
Amendment U, December 21, 2021
1. Précis
We have recently identified a novel genetic disease affecting nine known adults in whom de novo vascular 
calcifications develop in the lower extremity arteries and juxta-articular joint capsules of the fingers, wrists, 
ankles and feet. This rare disease results from bi-allelic mutations in the gene ecto-5-prime-nucleotidase 
(NT5E), encoding the CD73 protein. CD73, an enzyme involved in the extracellular ATP metabolic pathway, 
converts extracellular AMP to adenosine and inorganic phosphate. The clinical symptoms of this rare 
disease, termed ACDC (Arterial Calcifications due to Deficiency in CD73), include claudication of the calves, 
thighs, and buttocks, chronic ischemic pain of the feet at rest with threat of potential limb loss, and debilitating 
rheumatoid pain in the wrists and hands. Radiological and histological evaluations do not resemble classic 
atherosclerotic vascular calcification, since the calcification and dysplasia in ACDC occur in the medial 
portion of the arterial blood vessel wall. Data from patient-specific cell lines indicate increased activity of 
tissue non-specific alkaline phosphatase (TNAP), a key mediator of pathological ectopic tissue calcification, 
and thus reveals a potential therapeutic target.
To date, no effective therapy exists for ACDC patients. However, since bisphosphonates are potent 
competitive inhibitors of TNAP activity and are widely used to modulate bone metabolism, they may 
beneficially alter vascular calcification. In addition, our preliminary in vitro studies demonstrate the 
effectiveness of etidronate, a nitrogen-containing bisphosphonate, in lowering TNAP activity in cells isolated 
from ACDC patients. Etidronate, and bisphophonates in general, have proven safe and well tolerated by most 
patients.
This protocol provides for the administration of etidronate to ACDC patients, for whom no alternative 
treatment is available. Patients will be examined at the NIH Clinical Center bi-annually for 3 years. The 
primary objective of this clinical study is to test the effectiveness of etidronate in attenuating the progression 
of lower extremity arterial calcification and vascular blood flow based on CT calcium score and Ankle–
brachial index (ABI).
2. Objectives
2.1 Primary Objective
This study will examine the effectiveness of an oral bisphosphonate, etidronate, in attenuating the progression 
of lower extremity arterial calcification and improved vascular blood flow in subjects diagnosed with the rare 
disease, ACDC, over a three-year period, based on CT calcium score and Ankle- brachial index (ABI).
2.2 Secondary Objectives
The secondary objectives include:

Functional improvement in treadmill test results

Decrease in hand pain based on Rheumatoid Arthritis assessment tool

Changes in hand joint calcification based on hand x-ray 

Analysis of the composition of collected surgical tissue with calcification/inflammation
3. Background


Page 5 of 27 
Protocol # 12-H-0110
PI: Alessandra Brofferio, M.D.
Amendment U, December 21, 2021
3.1 Pathophysiology
We recently identified the molecular and enzymatic basis of a novel disease, ACDC, in nine patients with 
three different mutations in the NT5E gene.1  Individuals with ACDC form de novo vascular calcifications 
with chondro-osseous histology in the lower extremity arteries. The affected arteries are tortuous, and 
calcification appears to be localized to the medial layer (Fig. 2). Interestingly, other vascular beds appear 
unaffected. Definitive diagnosis of ACDC is based on genetic studies demonstrating mutations in NT5E. 
Preliminary data based on quantitative CT scans conducted at the NIH Clinical Center under NHLBI 
protocols 10-H-0126 Cardiovascular Disease Discovery Protocol or 02-H-0050 Technical Development of 
Cardiovascular Magnetic Resonance Imaging, or the NHGRI 76-HG-0238 Undiagnosed Disease Protocol 
from 3 subjects shows a rate of lower extremity calcium progression averaging 9% per year (range 6.5–
13.8%) (unpublished observation).
Deficiency of CD73 impairs production of adenosine, and the lack of extracellular adenosine signaling in 
patients with CD73 deficiency fosters calcification in lower extremity arteries. We found that lack of 
adenosine signaling in ACDC patient fibroblasts resulted in increased expression and activity of tissue non-
specific alkaline phosphatase (TNAP). TNAP is a key enzyme in bone mineralization and is highly expressed 
by osteoblasts. CD73-deficient fibroblasts exhibit increased TNAP mRNA expression both at baseline and 
upon exposure to medium promoting osteogenic differentiation. Genetic rescue or addition of adenosine 
reduced TNAP expression in CD73-deficient cells to levels found in control cells, suggesting that adenosine 
elicits its effects via binding to cognate cell surface receptors and not by acting directly on the TNAP enzyme. 
Adenosine receptor agonists are not approved for use in human patients, mitigating against their potential 
therapeutic use. However, bisphosphonates, a class of drugs known to inhibit the activity of the enzyme 
TNAP, are widely used to treat disorders of bone metabolism.
3.2 Treatment Options
Due to their profound and powerful effects in altering bone physiology, bisphosphonates represent a major 
class of drugs used for the treatment of bone diseases. Bisphosphonates inhibit bone resorption by selectively 
adhering to mineral surfaces and interfering with bone-resorbing osteoclasts.2 They are chemically stable 
analogs of a naturally occurring pyrophosphate characterized by the presence of a P-C- P bond. The P-C-P 
structure facilitates conformational variation, either by changing the two lateral chains (R1 or R2) or by 
esterifying the phosphate groups. The ability of bisphosphonates to bind hydroxyapatite crystals and prevent 
crystal growth and dissolution is enhanced when the R1 side change comprises a hydroxyl group,3 thereby 
increasing its affinity for calcium and, therefore, bone mineral.4  For maximal potency, the nitrogen atom in 
the R2 side chain must be a critical distance away from the P-C-P group and in a specific configuration. 
Bisphosphonates can be classified into 2 major groups: non-nitrogen- containing and nitrogen-containing. 
The former group, such as clodronate and etidronate (Didronel), can be metabolically incorporated into 
nonhydrolyzable forms of ATP by inhibiting ATP-dependent intracellular enzymes. The more potent, 
nitrogen-containing type of bisphosphonates, such as pamidronate (Aredia), alendronate (Fosamax), 
risedronate (Actonel), ibandronate (Boniva), and zolendronate (Zometa), inhibit farnesyl pyrophosphate 
synthase, thereby preventing the biosynthesis of isoprenoid compounds essential for the posttranslational 
modification of small guanosine trisphosphate (GTP)-binding proteins such as Rabs, Rhos, and Racs.5


Page 6 of 27 
Protocol # 12-H-0110
PI: Alessandra Brofferio, M.D.
Amendment U, December 21, 2021
In animals, bisphosphonates, administered orally or parenterally, efficiently inhibit ectopic calcification in 
vivo by preventing experimentally induced calcification of soft tissues. They decrease not only mineral 
deposits but also the accumulation of cholesterol, elastin, and collagen in the arteries. Etidronate 
administered subcutaneously inhibits calcification of aortic valves when implanted subcutaneously in rats.6  
The vascular calcification produced in rats with adenine-induced renal failure by feeding high phosphate 
chow was prevented by daily treatment with either etidronate or pamidronate, the latter being 100-fold 
more potent. In addition, aortic calcification and bone formation were reduced, and the calcification of pre-
calcified vessels was arrested, but not reversed.7
Bisphosphonates are used to treat several bone disorders, including osteoporosis in adults, Paget’s disease, 
bone metastases, postmenopausal women with vertebral compression fractures, elderly men with non-
traumatic fractures, and pediatric bone diseases, such as osteogenesis imperfecta. In humans, etidronate 
was the initial bisphosphonate to be used for fibrodysplasia ossificans progressiva and Paget’s disease, 
the first clinical disorder in which a dose-dependent inhibition of bone resorption could be demonstrated. 
In pediatrics, pamidronate has proven remarkably effective in increasing bone in children with the 
inherited brittle bone disorder, osteogenesis imperfecta. The continued improvement in bisphosphonate 
formulations fosters enhanced suppression of disease progression. Bisphosphonates accumulate in bone, 
making it important to evaluate the effects of long-term treatment. From a clinical standpoint, it is 
promising that the inhibition of bone resorption reaches a steady-state level and is then progressively 
lowered when bisphosphonates are given continuously. There seems to be no progression of 
antiresorptive effects with time, suggesting that a bisphosphonate remains active as long as it stays buried 
in bone.8 Within the therapeutic dose range, there appears little risk of a continuous and progressive 
decrease in bone turnover that would lead to an increase in bone fragility. In vitro, bisphosphonates can, 
in a dose-dependent fashion, inhibit the formation of osteoclast-like cells in long-term cultures of human 
bone marrow cells, suggesting that long-term use of bisphosphonates could reduce the production of bone-
resorbing cells.2
In a large longitudinal cohort study (MESA; the Multi-Ethnic Study of Atherosclerosis, 3,710 women 
assessed), investigators evaluated the relationship between bisphosphonates and cardiovascular 
calcifications.9 This study reported a decreased prevalence of cardiovascular calcification in older subjects 
(>65 years of age) and more prevalent cardiovascular calcification in younger ones (<65 years of age). 
However, results from younger patients are surprising. It is still unclear the extent to which 
bisphosphonate treatment in younger patients correlates with more advanced osteoporosis, which is linked 
to an increased risk of developing cardiovascular diseases. Although the vascular calcifications in ACDC 
are clearly distinct, there may be some degree of commonality between the two.
In a recent study the effect of a combination therapy with atorvastatin and etidronate on aortic 
atherosclerotic plaque formation and composition was evaluated by MRI.10 A total of 87 patients with 
hypercholesterolemia were treated either with atorvastatin alone or in combination with etidronate. The 
combination treatment significantly reduces both thoracic and abdominal aortic plaques whereas 
treatment with atorvastatin alone was less effective.
There are several smaller clinical studies evaluating the impact of bisphosphonates (mostly etidronate) on 
vascular calcifications in long-term hemodialysis patients. Vascular calcifications in these patients are 
more complex than calcifications in atherosclerotic lesions and involve intimal calcification 


Page 7 of 27 
Protocol # 12-H-0110
PI: Alessandra Brofferio, M.D.
Amendment U, December 21, 2021
(atherosclerotic type) and medial calcifications (Monkeberg sclerosis type). In these trials on uremic 
patients, bisphosphonate treatment is associated with a decrease in vascular calcifications.11,12
Calciphylaxis is a rare and often lethal complication in patients with end-stage kidney disease. It is 
characterized by rapid progressive systemic medial calcification in arteries, and leads to vascular 
thrombosis and skin necrosis. There are several case reports indicating that bisphosphonates were 
successful in treating calciphylaxis.13
Fahr’s syndrome or Idiopathic Basal Ganglia Calcification is a rare inherited neurological disorder 
characterized by calcifications of the basal ganglia, and cerebral cortex. This progressive neurological 
disorder results in disability and death. Histological evaluations showed severe medial calcifications of 
midsize to small arteries. There have been case reports where bisphosphonate treatment improved 
neurological dysfunctions.14
Generalized Arterial Calcification of Infancy (GACI) is a severe form of vascular calcification localized 
within the medial vessel wall in all major arteries throughout the body.15 GACI is caused by inactivating 
mutations in the ENPP1 gene that is directly upstream of CD73 in the extracellular ATP metabolism 
pathway (Fig. 1). GACI is associated with a high mortality rate owing to the development of arterial 
calcification with subsequent detrimental complications of the cardiovascular system in early infancy.
Inactivating mutations of ENPP1 lead to decreased levels of inorganic pyrophosphate that can be 
hydrolyzed by tissue non-specific alkaline phosphatase (TNAP), a key enzyme whose activity is 
dysregulated in ACDC. Bisphosphonates decrease mortality in GACI. One infant was started on 
bisphosphonates on the seventh day of life, and the arterial calcifications completely resolved by 3 months 
of age. The mechanisms by which bisphosphonates influence GACI are uncertain but they might act at 
various levels to alter calcium balance16,17. The mechanistic relationship and histopathological appearance 
suggest that GACI and ACDC involve overlapping signaling pathways.
4. Investigational Plan
4.1 Subject Population Selection and Eligibility
Study subjects must meet diagnostic criteria for ACDC. Enrolled subjects must be able to travel to the 
NIH for visits. Although only 9 patients have been diagnosed with ACDC so far, the accrual ceiling is set 
at 20 participants to allow for the enrollment of newly diagnosed ACDC patients. Additional subjects may 
be recruited with the help of referring physicians who report newly diagnosed cases of ACDC. 
Information about this study will be posted on ClinicalTrials.gov and the NHLBI recruitment website.
Currently, six subjects from the U.S. and one subject from Italy are available for screening and enrollment 
in the trial, which will take place in the United States at the NIH Clinical Center in Bethesda, Maryland 
for all subjects. This is proposed as a ten-year study but may be extended with IRB approval due to 
recruitment of newly diagnosed ACDC patients.
4.2 Inclusion and Exclusion Criteria
Inclusion Criteria
Inclusion and exclusion criteria are to be assessed at Screening and Baseline prior to starting study drug. 
Each subject must meet the following criteria to be enrolled in this study: Subjects must be diagnosed with 
ACDC based on genetic tests confirming mutation(s) in NT5E and evidence of lower extremity arterial 


Page 8 of 27 
Protocol # 12-H-0110
PI: Alessandra Brofferio, M.D.
Amendment U, December 21, 2021
calcifications.

Either gender and any ethnic background or race

Age 18-80 years

Willingness and legal ability to give and sign informed study consent

Willingness to travel to NIH and local sites for scheduled protocol studies and treatment
Exclusion Criteria
Subjects who meet any of the following criteria will be excluded from the study:

Subjects not diagnosed with ACDC

Subjects <18 or >80

Subjects who are unable or unwilling to sign an informed consent

Severe renal impairment [estimated creatinine clearance/eGFR of < 30ml/min (calculated using CKD-EPI 
equation)]

Longstanding diabetes mellitus (more than 10 years)

Known abnormality of the esophagus that would interfere with the passage of the drug

Known sensitivity to etidronate

Pregnancy

Any other medical or social condition that, in the opinion of the Principal Investigator, might put the 
subject at risk of harm during the study or might adversely affect the interpretation of the study data.
4.3 Study Design and Methods
A total of nine patients have been diagnosed with the newly described, rare disease, ACDC.1 The small patient 
population precludes us from conducting a larger scale clinical trial. There are currently no treatment options 
available for ACDC patients. However, our preliminary in vitro studies using patient fibroblasts suggest that 
bisphosphonate administration is a potential therapeutic approach for stopping the progression of lower extremity 
vascular calcification, because of its potent effects in inhibiting TNAP activity.
This is an open-label, non-randomized, single-arm pilot study to evaluate the effectiveness of etidronate treatment 
in attenuating the progression of lower extremity arterial calcification in patients diagnosed with ACDC. Currently, 
there are seven patients identified (five patients from one family; two patients from two separate families). Patients 
newly diagnosed with ACDC may also be enrolled if all study criteria are met. Subjects living in the U.S. will be 
followed and evaluated over a 3-year treatment period, with scheduled visits to the clinic approximately every 6 
months (see table 2. Schedule of Events).
Subjects living abroad will be followed and evaluated with scheduled visits every year, to mitigate the travel burden 
in this sick population. Please see marked sections of the schedule of events that applies for those subjects being 
seen yearly. The study will be conducted in three phases described in Section 4.4 through 4.6.
With Amendment T, the last and only study subject still on treatment will only receive 11 out of 12 cycles of 
etidronate due to drug discontinuation by the manufacturer, Mylan/Teva Pharmaceuticals and last lot of study drug 
expiring on December 31, 2019. Follow-up and testing will be performed otherwise as per protocol schedule of 
events.
At this time, as we will not have access to additional drug, this study is now in follow-up only.
Potential and enrolled participants may have had evaluation procedures conducted for clinical purposes outside of 
the NIH or at NIH under the following NHLBI and NHGRI protocols:


Page 9 of 27 
Protocol # 12-H-0110
PI: Alessandra Brofferio, M.D.
Amendment U, December 21, 2021

10-H-0126 Cardiovascular Disease Discovery Protocol

 02-H-0050 Technical Development of Cardiovascular Magnetic Resonance Imaging

18-H-0108 Vascular Disease Discovery 

95-H-0047 Diagnosis and Treatment of Patients with Heart and Vascular Disease (Training Protocol)

76-HG-0238 Undiagnosed Disease Protocol 
Prior, concurrent and subsequent results from procedures performed under these protocols or outside the NIH for clinical 
purposes may be used to supplement the clinical evaluations and research data analysis performed under this protocol to 
address primary and secondary objectives.
All tests will follow NIH Clinical Center policies. Clinical consent forms for individual tests will be obtained in 
addition to the study protocol consent. All the tests are performed to monitor disease progression and drug effects.
4.4 Pre-Treatment Phase
Note: Due to the nature of the disease being studied, and the potentially lengthy pre-treatment phase of this study, it 
is possible that patients already enrolled in the pre-treatment phase of the study may need to undergo clinically 
indicated interventions related to their disease course. In these situations, these clinical procedures and laboratory 
tests may be used in the data analysis for this study. Any cost resulting from clinically indicated procedures or 
preventative action taken prior to beginning the study treatment will be the responsibility of the subject and/or the 
subject’s insurance. The pre-treatment phase can take between one and five years to complete.
4.4.1
Establishing Baseline Rate of Disease Progression
Rate of disease progression will be established using two CT calcium score studies and two ABI studies. CT 
calcium scores will be observed at least 1 year (but no more than 5 years) apart, and ABI studies will be observed 
at least 6 months (but no more than 5 years) apart. Potential participants may have had CT calcium score and 
ABI conducted at NIH under the protocols listed in section 4.3 above or, for clinical purposes outside of the 
NIH. If these CT calcium scores and/ or ABI are comparable to those conducted for this protocol, these tests can 
be used to establish rate of disease progression. The quality of this CT scans will be established by Marcus Y. 
Chen, M.D. In these cases, the first studies for the CT calcium score and ABI to establish rate of disease 
progression may not need to be repeated during the pre-treatment phase. If these studies have not been done on 
other NIH or NHLBI protocols, the first studies for both the CT and the ABI may be collected during a separate 
pre-treatment evaluation visit to occur within one year prior to the initiation of treatment. To ensure consistent 
measurement of calcification for this population, all CT calcium scoring for ACDC patients and potential ACDC 
patients is done by Marcus Chen, M.D., an associate investigator on this protocol.
4.4.2
Pre-Treatment Evaluations

Any available Hand X-rays
Within 2 years of start of treatment:

Coronary CT
Within 1 year of start of treatment:

DEXA scan
Within 3 months of start of treatment:


Page 10 of 27 
Protocol # 12-H-0110
PI: Alessandra Brofferio, M.D.
Amendment U, December 21, 2021

Treadmill test

MRI

CAVI (optional)
Within 1 month of start of treatment:

Complete history and physical examination

Laboratory tests listed in Table 1 (except for the pregnancy test)

Screening 12-lead EKG

Dental Consult

Cardiovascular Consult

Questionnaires (Appendices 1 and 2)
No more than 48 hours prior to the start of treatment:

Women of childbearing potential will have a urine or serum hCG pregnancy test
In the event of a treatment delay for clinically indicated interventions resulting in testing falling out of the 
specified window, the tests will be repeated. As a consequence, the pre-treatment phase may result in multiple 
visits and will only be completed with the initiation of drug. The first day the patient receives drug will be 
considered Day 0 of the Treatment Phase (see Section 4.5)
4.5 Treatment Phase (Clinical Monitoring and Intervention)
The Treatment Phase consists of twelve Drug Cycles [approximately 3 months each, ± 2 weeks (with exception for 
acceptable drug interruptions described below)]. Participants living in the U.S will have one scheduled visit to the 
NIH Clinical Center every 6 months. Participants living abroad will have one scheduled visit to the NIH once a year.
Etidronate will be administered at 20mg/kg daily x 14 days, followed by 10 weeks off study drug (12 weeks = one 
cycle). Participants living in the U.S will return to the NIH Clinical Center for evaluation every 6 months. Participants 
living abroad will return for evaluation every 12 months. Subjects may receive up to 12 cycles of etidronate.
In instances where subjects experience gastrointestinal discomfort (i.e. diarrhea, nausea), etidronate may be 
administered as a divided daily dose (10mg/kg twice daily x 14 days). Dividing the daily dose often alleviates 
gastrointestinal complaints that occur in 2 to 3 out of 10 patients taking a 10 to 20mg/kg/day dose of Etidronate.21
Drug interruptions: Unscheduled, clinically indicated treatment interruptions will be allowed for up to 1 
year. Subjects will continue to be monitored during the drug interruption. All visits for the treatment phase 
will be completed regardless of drug interruption; therefore, the treatment phase may take longer than  3 
years to complete if drug interruption occurs.
Participants who have a drug interruption longer than 1 year will require re-establishment of baseline rate of 
progression to begin the treatment phase again.
If a subject misses a dose of the study drug, the subject will be instructed to add a day to the 14- d a y  drug 
cycle for each missed day to take the missed dose(s) of study drug. At least a 7-week drug-free interval must 
be maintained between the drug cycles. If a subject is not able to take all missed doses 7 weeks prior to the 
next drug cycle, he/she will be instructed not to take the missed doses past the 7-week cutoff and to resume 
the drug schedule at the next schedule drug cycle.


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Amendment U, December 21, 2021
Unscheduled visits may occur during the treatment phase. During these unscheduled visits, other appropriate clinical 
tests and procedures will be utilized as indicated to manage complications related to the primary disease and to 
stabilize the clinical condition. Additional visits or extended course visits to the NIH Clinical Center may be 
necessary to manage disease related complications. 
If a subject undergoes lower extremity bypass, amputation, or other clinical intervention, during the study, the 
bypass/intervention limb, or remaining limb in the case of an amputation, will still be monitored.
However, only the data collected on the remaining/non-intervention limb will be used to assess the effectiveness of 
the study drug.
Continued communication will be maintained with subjects’ primary physician throughout their participation in the 
study.
4.6 Medical and Surgical History and Physical Examination
Medical history (including medication history involving over-the-counter and herbal medications), physical 
examination, and laboratory values will be completed on each subject for every admission while on the study. Vital 
signs and biophysical data (including blood pressure, heart rate, respiratory rate, temperature, height, and weight) 
will also be collected at each visit. At each study visit participants will complete a Rheumatoid Arthritis questionnaire 
(Appendix 1) to assess hand pain; and, the Duke Activity Index (Appendix 2) to measure functional capacity.  These 
questionnaires will not be collected at follow-up visits.
Research test results and clinical data including, but not limited to, medical history, physical examination, ABI data, 
and MRI data will be evaluated (i.e. Cardiovascular Consultation) to determine if any clinically indicated 
interventions are necessary prior to the start of the treatment phase) and throughout the study.
The principal investigator and medical advisory investigator will review the results of the cardiovascular 
consultations to determine if there are any significant issues that will need to be closely monitored throughout 
treatment.
Subjects will also be sent for a dental consult where an oral exam will be performed and a panoramic x- ray will be 
taken to document his/her oral health prior to the first dose of the study drug, at Visit 3 and at the end of the treatment 
phase (Visit 6). The purpose of this consult is to monitor the participants’ oral health, as etidronate has been 
associated with jaw osteonecrosis. The incidence of jaw osteonecrosis is associated with long-term (i.e. greater than 
3 years) use of oral bisphosphonates18. It is not anticipated that participants will development jaw osteonecrosis 
within the treatment period of 3 years for this study.
However, the principal investigator and medical advisory investigator will review the results of the dental exam to 
determine if there are any significant dental issues that will need to be closely monitored throughout treatment. Prior 
to treatment with the study drug, incidental findings from the dental consultation requiring intervention will be sent 
to the subject’s designated dentist for treatment prior to beginning the study drug. Any cost resulting from clinically 
indicated procedures or preventative action taken prior to beginning the study treatment, will be the responsibility of 
the subject and/or the subjects insurance. Any dental problems that arise during the study treatment phase that are 
considered likely attributable to the study drug and will be paid for by NIH. We may request that participants provide 
NIH with a record of dental examinations or treatments from the subjects’ designated dentist during the course of 
this protocol.
4.7 Laboratory Tests and Procedures
Blood and urine specimens for the measurement and evaluation of serum chemistries will be collected at each site 


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Amendment U, December 21, 2021
visit. Clinical laboratory procedures may include those presented in Table 1.
We may also perform 24-hour urine collection to determine calcium and phosphate content of urine. Any laboratory 
test result that the Investigator considers clinically significant will be repeated at the discretion of the investigator to 
rule out laboratory error. For tests where a persistent abnormality is considered to be drug related, repeat analyses 
will be performed until the cause is determined and either a return to normality occurs or the investigator deems the 
abnormality to be of no clinical significance. Any values judged by the investigator to represent a clinically 
significant, abnormal change from baseline during the treatment phase will be recorded as an adverse event. Per 
Clinical Center policy, no more than 550 mL of blood will be collected over an 8-week period.
Table 1. List of Laboratory Tests
Hematology:

Complete CBC and Differential
Urine for:

Phosphorus - Inorganic

Calcium

Urinalysis
Serum Chemistry:
Chem 14
•  Acute Care Panel
•  Mineral Panel
•  Hepatic Panel
•  Total Protein
•  CK
•  Uric Acid
•  LD
• Alkaline phosphatase (bone specific/heat specific)
• Lipid panel
• C-reactive protein (CRP)
• Fibrinogen
• Human chorionic gonadotropin (hCG) (for females of 
childbearing potential)
• activated Partial thromboplastin time (aPTT)
• PT/INR
• Research blood, for storage for studies related to the 
subject’s disorder
• Parathyroid hormone (PTH)
• 25-hydroxyvitamin D levels (Vitamin D) level in serum
Research Blood: Serum and plasma will be collected per visit (keeping blood draw volume limits as per NIH 
CC policy), stored at - 80C and analyzed when sufficient samples are accumulated. We will screen research 
blood for known marker associated with vascular calcification like, MGP, Osteopontin, RankL, BMPs, 
Fetuin among others.
4.8 Study Procedures
Note: Study procedures will be performed following the Schedule of Events Table 2 at the end of this section.
Surface Electrocardiogram (EKG): A surface EKG will be recorded to help exclude cardiac abnormalities 
in subjects with vascular calcifications. The EKG will take approximately 5 to 10 minutes.
Treadmill Test: A symptom-limited treadmill progressive test (Gardner Protocol) with gradual increase in 
grade will be performed in accordance with standard practice to evaluation exercise capacity and pain on 
exertion in subjects with decreased peripheral arterial perfusion. Briefly, the test will use a constant


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Amendment U, December 21, 2021
speed of 2 mph and gradual increase in grade of 2% every 2 minutes beginning at 0% grade. Distance to 
onset of pain and to maximal pain will be recorded. Lower extremity arterial Doppler pressure will be 
measured immediately post exercise.
Ankle-brachial index measurements: The ABI is performed to investigate the functional consequences of 
abnormalities of vascular anatomy. Systolic blood pressure is measured using Doppler ultrasound on both 
brachial, posterior tibial, and dorsalis pedis arteries using a blood pressure cuff inflated over the brachial or 
gastrocnemius muscles. Measurements are obtained during resting steady-state conditions in both limbs, 
and immediately post exercise for 26 minutes or until lower extremity arterial pressure returns to baseline. 
The ABI for each limb represents the ratio of the highest arterial pulse pressure for each limb to the highest 
brachial pressure of the two limbs.
Cardiovascular and Lower Extremity CT: Calcium scores will be obtained from non-contrast computed 
tomography (CT) scans to evaluate peripheral arterial calcifications. Imaging procedures may include scout 
images of the body in the posterior-anterior and lateral directions, non-contrast images of the heart, and non-
contrast images of the lower extremity.
After the CT data have been acquired and stored, FDA-approved software will be used to reconstruct images 
from the acquired data. This reconstruction could be performed on the scanner itself or off-line on a separate 
computer system. The reconstructed images may be further processed to extract quantitative data with respect 
to the pathophysiology involved and/or to evaluate the images themselves. Data and images may be stored 
for future evaluation and additional processing. Coronary and lower extremity calcium scores will be 
determined according to Agatston19.
MRI: Exploratory MRI studies may be employed to evaluate the lumen of the artery and degree of collateral 
vessels, given that the high degree of calcification is precluding that evaluation by CT. Study- specific MRI 
scans will be performed using an FDA-approved MRI scanner. Imaging procedures will include localizer 
scans, two-dimensional studies of cardiovascular anatomy and function, contrast- enhanced cardiovascular 
MRI studies, and three-dimensional angiograms. The exact examination performed depends on the 
individual patient requirements or clinical findings.
Gadolinium Contrast (optional): Subjects may receive an intravenous injection of gadolinium (Gd) 
chelate not to exceed 0.2 mmol/kg of Gd per bolus injection and 0.4 mmol/kg of Gd per examination. The 
injection will be made by hand or using an MRI compatible power injector. Gadolinium contrast is an 
extra-cellular agent, so the distribution volume is a measure of the fractional volume occupied by the 
vascular and interstitial space.
DEXA Scan: A dual x-ray absorptiometry (DEXA) scan will be performed to evaluate mineral bone density 
before initiation of bisphosphonates. The standard sites for DEXA for bone density determination are PA 
and lateral lumbar spine, Proximal Femur (Hip), and Forearm (1/3 radius). DEXA is a low radiation x-ray 
procedure. The radiation exposure is less than 1 mRem for the 3 tests combined, which is much less than 
daily background radiation.
Hand X-Ray: Standard x-rays of the hands for assessment of metacarpal joint calcification will be 
performed. The estimated radiation exposure is less than 1 mRem.
Cardio-Ankle Vascular Index (CAVI) (optional): CAVI is a novel indicator of arterial stiffness. This 
measurement will be obtained to assess initial stiffness of the arteries and changes during the treatment phase. 
The test is not invasive and consists of measurement of BP in the four extremities while monitoring ECG by 


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Amendment U, December 21, 2021
single lead and the heart sound.
Tissue otherwise discarded at the time of medically indicated surgery: Subjects who enter the protocol may 
require medically indicated surgery where diseased or redundant tissue at the surgical site would be 
extracted. Portions of the tissue/s may be submitted to the pathology laboratory at the request of the surgeon. 
If residual tissue is available that would otherwise be discarded as ‘surgical-waste’, the investigator, with 
fully informed consent from the subject, may request the tissue/s to study to enhance the understanding of 
the disease pathophysiology. Participants who undergo medically indicated surgical procedures will be 
asked to sign a separate Tissue Collection informed consent document.
Table 2. Schedule of Events
Treatment Evaluations
Evaluation
Pre-
Treatment 
Evaluations*
/Time 0
Visit
1/6mo 
(±1mo)
Visit 
2/12mo 
(±1mo)*
Visit 
3/18mo 
(±1mo)
Visit 
4/24mo 
(±1mo)*
Visit 
5/30mo 
(±1mo)
Visit
6/36mo 
(±1mo)*
Follow-up 
Evaluations8,#
(non-
mandatory)
Informed Consent
X
I/E Criteria
X
Demographics
X
Dental Consult
X
X#
X
Cardiovascular ConsultX
X
X
X
X
X
X
X
Medical History2
X
X
X
X
X
X
X
X
Questionnaires
X
X
X
X
X
X
X
Vital Signs3
X
X
X
X
X
X
X
X
Physical Exam
X
X
X
X
X
X
X
X
Laboratory Tests
X
X#
X
X#
X
X#
X
X
Research Blood
X
X
X
X
X
X
X
X
Lipid Panel
X
X
X
X
X
CRP
X
X
X
X
X
Urine Test7
X
X#
X
X#
X
X#
X
X
Pregnancy Test3
X
X#
X
X#
X
X#
X
X
EKG
X
X
X
X
X
X
X
X
ABI
X1
X#
X
X#
X
X#
X
X
CAVI (optional)
X
X
X
X
X
X
X
X
Treadmill Test
X
X
X
X
X
X
X
X
MRI
X
X
X
CT lower extremities
X1
X
X
X
X
CT coronaries
X4
X
X
Hand X-Ray
X9
X
X
X
X
DEXA scan
X
X
X
X
X
Drug Accountability
X6
X
X
X
X
X
X
AE Assessment5
X6
X
X
X
X
X
X
1.   Subjects’ must have two CT scans of the lower extremities (at least 1 year apart, but no more than 5 years apart) and two ABI tests (at least 6
months apart, but no more than 5 years apart) to establish baseline rate of disease progression (see Section 4.4.1)
2.   Include a review of previous/ongoing medications
3.   Only females who are not diagnosed as postmenopausal or not of childbearing potential
4.   If not performed within two years prior to involvement
5.   Adverse Events will also be assessed during follow-up contact.
6.  Drug Accountability and AE Assessment will only be necessary for the visit at which the subject in administered the study drug (Day 0).
7. Urine test may include 24-hour urine collection
8. Optional follow-up evaluations may take periodically as needed. All follow-up visits and procedure are optional.
9. Any available during life time Hand X-rays may be collected for comparison
#   Follow-up Evaluations/Procedures/Visits for subjects living abroad may be conducted in their county of residence
* Scheduled visits to the NIH Clinical Center for subjects living abroad.


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Amendment U, December 21, 2021
4.9 Pharmaceuticals
Study Treatment
Product Name: Didronel (etidronate disodium)
Supply: Dispense in a tight, light-resistant container as defined in the USP using a child-resistant closure. 
Commercial supply will be received through the NIH Pharmacy. Drug will be supplied to subjects by 
the NIH Pharmacy every 3 to 6 months.
Product Description: Didronel tablets contain either 200 mg or 400 mg of etidronate disodium, the 
disodium salt of (1-hydroxyethylidene) diphosphonic acid, for oral administration. This compound, also 
known as EHDP, regulates bone metabolism. It is a white powder, highly soluble in water. The 400 mg 
tablets are white capsule-shaped tablets with ED 400 on one side and G on the other side.
Storage: Store at 25°C (77°F), excursions permitted to 15-30°C (59-86°F), in a dry place.
Route of Administration: Oral
Dose: 20mg/kg daily x 14 days, or 10mg/kg twice daily x 14 days, followed by 10 weeks off study drug 
(rounded to the nearest tablet size)
Toxicities: see section 8.4, risks of etidronate
Drug interactions: There have been isolated reports of patients experiencing increases in their 
prothrombin times when etidronate was added to warfarin therapy. Increased INR monitoring will be 
highly recommended during the initiation and all subsequent re-initiations of study drug throughout the 
study. If a patient is on warfarin during the treatment phase of the study, INR should be monitored at 
least weekly during the initiation of etidronate treatment until a stable INR is achieved. While the patient 
is on etidronate and warfarin, INR should be monitored at least monthly. If etidronate therapy is 
discontinued, INR should continue to be monitored weekly until a stable value is reached. Continued 
communication will be maintained with subjects’ primary physician throughout etidronate therapy to 
ensure that he/she is monitored between visits to the NIH Clinical Center.
Results for INR monitoring will be requested from the primary physician but will not be used for research 
analysis.
Stability: Stable under normal conditions
Packaging and Labeling: See attached package insert20.
Selection and Timing of Dose: When all entry criteria have been met and scheduled tests have been 
completed, the first dose of study drug (20mg/kg) will be taken by the subject under supervision of the study 
personnel while at the NIH Clinical Center. Dose will be rounded to nearest tablet size. All subjects will be 
assigned a 6-month drug schedule prior to their departure. Participants living abroad will be assigned a 12-
month drug schedule prior to their departure.
Etidronate is prescribed on a cyclical drug schedule. For the purposes of this study, participants will take 
etidronate daily for two weeks (14 days), followed by 10 weeks off study drug (with exception for acceptable 
drug interruption described in section 4.5).10 This drug schedule will occur four times yearly during the 6-


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Amendment U, December 21, 2021
course treatment phase.
Dosing Instructions: To maximize absorption and clinical benefit, etidronate should be taken at least one 
hour prior to first food or drink (other than water) of the day. To facilitate delivery to the stomach and thus 
reduce potential for esophageal irritation, etidronate tablets should be swallowed whole with a full glass of 
plain water (6-8 oz) while the subject is standing or sitting in an upright position. Subjects should not lie 
down for one hour after taking etidronate. To maximize absorption, patients should avoid taking the following 
items within two hours of dosing:

Food, especially food high in calcium, such as milk or milk products.

Vitamins with mineral supplements or antacids which are high in metals such as calcium, iron, 
magnesium, or aluminum.
In instances where subjects experience gastrointestinal discomfort (i.e. diarrhea, nausea), etidronate may be 
administered as a divided daily dose (10mg/kg twice daily x 14 days). Dividing the daily dose often alleviates 
gastrointestinal complaints that occur in 2 to 3 out of 10 patients taking a 10 to 20mg/kg/day dose of 
etidronate.21
Once the study has ended, subjects who wish to continue taking etidronate will be responsible for the cost of 
the medication, and treatment will be the responsibility of their primary physician.
Treatment Compliance: Investigators carefully selected the study drug for optimal safety, compliance, and 
efficacy. Subjects will be prescribed and given the drug initially at the NIH Clinical Center. Subjects will be 
presented with a pre-determined dosing schedule and will be called to remind and/or verify that the drug has 
been taken. Follow-up phone contact may be made the week before the first dose day, again after the first 
week of the study drug, and the week following the end for the drug cycle to ensure subject compliance and 
to assess any adverse reactions to the study drug. At least two phone calls will be made per drug cycle to 
assess compliance with the drug schedule and monitor for adverse reactions. Records of follow-up contact 
to verify subject compliance with drug intake will also be documented in the individual research chart.
If a subject misses a dose of the study drug, the subject will be instructed to add a day to the 14-day drug 
cycle for each missed day to take the missed dose(s) of study drug. At least a 7 week drug-free interval must 
be maintained between the drug cycles. If a subject is not able to take all missed doses 7 weeks prior to the 
next drug cycle, he/she will be instructed not to take the missed doses past the 7-week cutoff and to resume 
the drug schedule at the next schedule drug cycle.
Follow-up phone calls may be placed as often as daily while the subject is on study drug if a subject needs 
assistance to remain compliant with the drug schedule. If subjects become unable or unwilling to continue 
treatment they will be removed from the study. The subjects will be instructed to return remaining drug, in 
original drug containers, to NIH at each applicable study visit for a drug count. Any missed doses of the study 
drug will be disposed of in a medical waste box. The study pharmacist or designee will be responsible for 
drug accountability and record of drug interruptions.
4.10
Off-label Use of Drugs
Medications will be used beyond the indications specified in the Prescriber Information. In addition to the 
study drug, the off-label uses apply to beta-blockers and calcium-channel blockers for slowing the resting 


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Protocol # 12-H-0110
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Amendment U, December 21, 2021
heart rate, and gadolinium-based contrast agent for imaging the heart. These off-label uses will comply with 
prevailing community standards. An IND is not required since the objective of this study is not to develop 
information about the product's safety or efficacy and these uses meet the requirements for exemption.
The use of these drugs for this protocol meets the requirements for exemption from the Investigational New 
Drug regulations, 21 CFR 312, specifically:
1.
The investigational drug is lawfully marketed in the United States
2.
The investigation is not intended to be reported to the FDA as a well-controlled study in support of 
a new indication for use of the drug product
3.
The investigation is not intended to support a significant change in advertising to an existing lawfully 
marketed prescription drug product
4.
The investigation does not involve a route of administration or dosage level or other factor that 
significantly increases the risks (or decreases the acceptability of the risks) associated with the use 
of the drug product.
5.
The investigation will be conducted in compliance with the requirements for institutional review set 
forth in FDA regulations 21 CFR 56, and requirements for informed consent as set forth in FDA 
regulations 21 CFR 50
6.
The investigation will be conducted in compliance with FDA regulations 21 CFR 312.7: Promotion 
and charging for investigational drugs.
4.11
Off-Treatment
After completing all 6 courses of a treatment subjects will be considered off treatment. Off-treatment date 
is the last day of the last dose of the study medication taken.
4.12
Follow-up Visits
Once subjects complete the full course of treatment, they may be asked to return to NIH periodically for 
non-mandatory follow-up visits based on patient availability During these visits, patients may receive any 
of the tests listed on the schedule of events, tailored on the basis of their symptoms and disease 
progression.
5. Biostatistical Considerations
5.1 Primary Endpoint and Analysis
This trial is designed to evaluate the effectiveness of etidronate in subjects diagnosed with ACDC. The 
primary endpoints will be CT calcium score and ankle-brachial Index (ABI). The planned analyses will 
include descriptive statistics and longitudinal trends in the CT calcium score and ABI. Percentage changes 
in CT calcium score and ABI will be compared between the pre-treatment phase baseline and treatment 
phase. The significance of the changes will be reported with the use of t-test or rank-based test as appropriate. 
Secondary endpoints include, treadmill test results (section 4.7), functional capacity measured by the Duke 
Activity Index (Appendix 2), and changes in hand pain based on Rheumatoid Arthritis questionnaire 
(Appendix 1).
Data analysis will include all data collected on 12 drug cycles and follow-up for the first 6 patients accrued, 
as well as the 11 drug cycles and follow-up for patient #7. 


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PI: Alessandra Brofferio, M.D.
Amendment U, December 21, 2021
5.2 Sample Size
There are no CT calcium score data or ABI data on patients with ACDC in the published literature on which 
to base sample size calculations. The study will accrue up to 20 subjects (9 patients have been diagnosed so 
far). Per subject off study criteria has been discussed on section 7.4. No formal study level stopping rule 
will be imposed due to lack of effectiveness profile of the treatment and limited sample size.
6. Sample Collection, Storage, and Data Management
Samples will be de-identified prior to storage on the 5th floor of building 10 in laboratory for the principal 
investigator following current NIH sample storage guidelines. Access to research samples will be limited, 
using a locked freezer. Samples and data will be stored, using codes assigned by the investigators or their 
designee(s). Data will be kept on the NHLBI P:drive, accessible through password-protected computers. 
Only the members of the research team will have access to the samples and data.
All human subjects’ personally identifiable information (PII), eligibility and consent verification will be 
recorded. Primary data obtained during the conduct of the protocol will be kept in secure network drives that 
comply with NIH security standards. Primary and final analyzed data will have identifiers so that research 
data can be attributed to an individual participant.
At the completion of the protocol (termination), samples and data will either be destroyed, or after IRB 
approval, transferred to another existing protocol or a repository. Participants are provided the option 
(Standard informed consent document, section 14) to be contacted in the future should information be 
obtained about their diagnosis, treatment, related symptoms, or associated risks of their disorder from future 
research using their samples. Participants who agree to be contacted in the future will be asked to maintain 
communication with the research team to provide up-to-date contact information following the completion 
of this study.
End of study procedures: Data will be stored in locked cabinets and in password protected network servers 
until no longer of scientific value.
Loss or destruction of data: Should we become aware that a major breech in our plan to protect subject 
confidentiality and trial data has occurred, the IRB will be notified.
Data will not be sent outside NIH without IRB notification and an executed agreement. However, incidental 
findings requiring intervention from the dental consultation will be sent to the subject’s designated dentist.
7. Data and Safety Monitoring
7.1 Monitoring
Accrual and safety data will be monitored by the Sponsor/Principal Investigator. The protocol will be 
continuously evaluated for any unusual or unpredicted complications with the aim of detecting and 
preventing unacceptable increase in morbidity and mortality over and above that anticipated from etidronate. 
Literature searches will be conducted at least annually to seek safety information (21 CFR 312.32(b)).
Prior to implementation of this study, the protocol and the proposed subject informed consent document will 
be reviewed and approved by a properly constituted IRB operating according to the 21 CFR 56. This 


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Amendment U, December 21, 2021
committee must approve all amendments to the protocol or informed consent document, and conduct 
continuing annual review so long as the protocol is open to accrual or follow up of subjects.
Quality assurance and control monitoring will be consistent with the NHLBI Division of Intramural Research 
Clinical Research Quality Assurance and Quality Control Policy.
Please refer to policy 801 for definitions.
7.2 Adverse Event Management
The principal investigator or designee will be responsible for assessing adverse events. Information on 
adverse events will be solicited from subjects through questions from study personnel and information 
volunteered by the subject. All AEs, regardless of severity, will be recorded, graded, assigned an attribution, 
verified, and followed until satisfactory resolution. Adverse events will be captured from the start of the first 
etidronate administration (day 0) until 30 days following the last dose of treatment.
Following this period, any observed or volunteered adverse events will NOT be reported or recorded unless 
they are greater than Grade 1 and deemed possibly, probably or definitely related to a procedure, test or 
screening examination that was administered as part of this protocol. 
Adverse events will be attributed (unrelated, unlikely, possibly, probably or definitively) to study medication 
and/ or disease and/or procedure/testing. AEs will be graded by severity utilizing CTCAE version 4.0. A 
copy 
of 
the 
criteria 
can 
be 
downloaded 
at 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm.
7.3 NIH IRB, FDA, and CD reporting
Reports to the IRB: The PI or designee will refer to HRPP Policy 801 “Reporting Research Events” to 
determine IRB reporting requirements and timelines.
Reports to the CD: The PI or designee will refer to NHLBI DIR guidelines to determine CD reporting 
requirements and timelines.
Expedited Reporting: events requiring expedite reporting will be submitted to the IRB per Policy 801, 
”Reporting Research Events.”
Reports to the IRB at the time of Continuing Review: The PI or designee will refer to HRPP Policy 801 '' 
Reporting Research Events 'to determine IRB reporting requirements.
Reports to the FDA: The sponsor-representative or designee will refer to abbreviated IDE requirements of 
21 CFR 812.2(b), to determine FDA reporting requirements and timelines.
7.4 Removal of Subjects from the Study
The investigator may withdraw a subject from the study for any of the following reasons:

A serious or intolerable adverse event related to etidronate occurs


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Protocol # 12-H-0110
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Amendment U, December 21, 2021

A clinically significant change in a laboratory parameter related to etidronate occurs requiring drug 
discontinuation

The sponsor investigator terminates the study

The subject requests to be discontinued from the study

Subject non-compliance

A clinically significant change that requires a drug interruption longer than 1 year (2 courses).
8. Human Subjects Protection
8.1 Rationale for Subject Selection
Subjects of both genders will be considered for inclusion in this study. There will be no racial, ethnic, or 
gender discrimination. Cognitively impaired and institutionalized persons will not participate in this study. 
Criteria for exclusion or withdrawal from the study are based on the presence of other disease processes that 
may be harmful to the health of subjects.
8.2 Rationale for the Exclusion of Children
Subjects under 18 years of age will not be considered for inclusion in this protocol because the disease has 
not been described in children, and the safety and effectiveness of etidronate has not been established in 
pediatric subjects.
8.3 Rationale for the Exclusion of Pregnant Women
Pregnant women will not be eligible for this study. There are no adequate and well controlled studies of 
etidronate in pregnant women. Females of childbearing potential must not be pregnant or actively seeking 
pregnancy in order to participate in this study. Some form of contraception must be used by female subjects 
of childbearing potential while enrolled. Contraception use will be confirmed at time of enrollment and at 
subsequent visits.
8.4 Evaluation of Benefits and Risks/Discomforts
The research involves greater than minimal risk to subjects, with the prospect of direct benefit (45 CFR 
46.102).
Subjects will receive close monitoring for disease progression. There is potential benefit on disease 
progression and reversal through drug treatment, and benefit secondary to treatment with decrease in lower 
extremity pain and increased mobility and exercise tolerance.
Etidronate: Etidronate may cause local irritation of the upper gastrointestinal mucosa. Subjects will be 
monitored after the first dose given at CC and at every course visit for dysphagia, odynophagia, retrosternal 
pain, new or worsening heartburn, new thigh or groin pain for atypical femoral fracture risk, and 
hypersensitivity reactions. Hypocalcemia, musculoskeletal pain, and osteonecrosis of the jaw have been 
reported. Most common adverse reactions reported include back pain, dyspepsia, pain in extremity, diarrhea, 
headache, alopecia, nausea, and myalgia. Subjects will be instructed to contact the research team to report 
problems with the listed side effects of etidronate.
CT Scans: Non-contrast CT scans of the heart and lower extremities to assess calcification are safe 
procedures; however, it is associated with a small amount of ionizing radiation. The radiation exposure for 


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the coronary CT calcium score is estimated to be less than 0.5 rem (5.0 mSv) and the exposure for the lower 
extremity CT calcium score is estimated to be less than 1.4 rem (14 mSv). Risks of radiation exposure are 
detailed in the consent section using wording provided by the NIH Radiation Safety Committee. In some 
subjects, lying on the table may result in some discomfort due to back pain or claustrophobia.
Although uncommon, if a subject has resting tachycardia, beta-blocker may be used to achieve adequate 
image quality. Heart rates of less than 80 beats per minute are desired for coronary calcium scoring CT exam. 
If a contraindication to metoprolol use is present (asthma with regular use of inhaler, chronic severe lung 
disease, or active decompensated heart failure, Mobitz type 2 second degree or 3rd degree atrio-ventricular 
heart block), metoprolol will not be administered. Diltiazem may be administered in subjects with asthma 
or chronic severe lung disease to help slow the resting heart rate.
Prior to administering metoprolol or diltiazem, a baseline heart rate (HR) and blood pressure (BP) will be 
obtained. Since the approach to pre-scan resting heart rate reduction is rapidly changing, we will generally 
adhere to the following guidelines but recognize that the supervising physician may customize the doses on 
an individual patient:

A net dose of metoprolol 50-200 mg or diltiazem 90-360 mg will be administered orally over a 
period of approximately 1-2 hours titrated to achieve a heart rate less than 60 beats per minute. In 
addition, metoprolol 5-30 mg intravenously or diltiazem 5-30 mg intravenously may be administered 
at the judgment of the supervising physician if heart rate is not adequately controlled and the patient 
appears capable of tolerating the additional dose.

If baseline HR is <60 bpm, generally no additional metoprolol or diltiazem is administered; however, 
the supervising physician may elect to use a small dose to blunt heart rate increases during the scan.

The physician covering the cardiac CT may adjust the dose of metoprolol or diltiazem on an 
individual basis.
Cardiac vital signs will be obtained and documented again approximately thirty minutes following 
metoprolol or diltiazem administration. If at any time the heart rate is <50 bpm and/or the systolic BP is
<100, the supervising physician will be notified.
MRI: MRI uses no ionizing radiation and is quite safe when performed on a properly screened population. 
Potential risks relate to the magnetic field’s effect on subjects with implanted metal objects (i.e. cerebral 
aneurysm clips, cochlear implants, etc.). The magnetic field can cause twisting or movement of these objects, 
thus causing harm to the subject. Also, the radiofrequency deposition of MRI can potentially cause burns in 
subjects with pacemakers or other implanted coiled wires. Subjects will be screened for these objects and 
excluded as indicated.
Although unlikely with our imaging parameters, peripheral nerve stimulation may cause discomfort. 
Peripheral nerve stimulation occurs when magnetic field gradient switching occurs fast enough to cause 
peripheral nerve depolarization. Typically, the patient may feel a twitch such as in the buttock, leg, or across 
the bridge of the nose. There is no physical danger from peripheral nerve stimulation. However, as gradient 
switching rates increase, smaller peripheral nerves (such as those that cause pain) can be excited. The 
switched gradients also generate noise in the scanner. Peak sound power produced in the magnet will be less 
than 140 dB and 119 dBA. The FDA limits constant noise exposure to 140 dB and 99 dBA for two hours. 
To stay within FDA guidelines on dBA limits, all subjects will wear hearing protection in the form of 


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earplugs and/or headphones.
All our scans are within FDA guidelines for radiofrequency power levels. However, in cases of a damaged 
or dysfunctional surface coil, there is a small chance of local warming in the body. For safety purposes, we 
ask patients to report this sensation so we can modify scans or verify integrity of hardware.
There is no data that shows any significant adverse effects of exposure to static magnetic fields. There are 
well known minor adverse effects associated with high magnetic fields. These include nausea, metallic taste, 
and detection of flashes of light. All of these are associated with moving too rapidly in the magnetic field.
Non-significant Risk IDE: The use of the MRI scanner constitutes a non-significant risk (NSR) device 
(IDE). With regard to radiofrequency power levels (SAR limits), we operate the MRI scanner at the FDA 
limitsestablished for MRI in general applications. We utilize the following limits for the body coil: SAR 4 
watts/kg average over 15 minutes. The limits for the head transmit/receive coil are: SAR 8 watts/kg average 
over 5 minutes. The scanner gradient performance (dB/dt limit) is set on an individual basis to avoid painful 
peripheral nerve stimulation.
Investigational or research MRI coils may be used in the protocol. The coils are noninvasive devices external 
to the body. The coils act as antennae to receive small radiofrequency signals out of the body. Coils of the 
type we use are used daily in clinical MRI practice.
The use of the MRI research coils, research pulse sequencing, and research image processing constitute the 
research component of this device which are not FDA approved. All research coils undergo safety testing at 
the NIH which is supervised by the NMR Safety Committee. There is no potential for serious risk to the 
health, safety, or welfare of the subjects using these research modalities.
Currently, none of these devices are undergoing FDA Premarket Notification (510(k)) submission. Thus, 
there is no IDE for the protocol. Research image acquisition programs, research analysis software, and 
research radiofrequency (RF) receive coils may be used in these MRI studies. Participants will not receive 
sedation or anesthesia for the MRI scan.
Gadolinium-based contrast agents (GBCAs) are injected medications used to improve MRI images. Most 
patients experience a metallic taste when gadolinium contrast is injected. Some (2%) report mild symptoms 
such as headache, nausea or vomiting, or a rash near the injection site. Rarely (<0.1%) patients experience 
severe symptoms such as wheezing, shortness of breath, and low blood pressure as part of an allergic 
(anaphylactoid) reaction that may require emergency medical treatment.
In a few cases per million, usually in patients with severe kidney disease, gadolinium contrast can cause a 
rare, debilitating or even fatal, skin disease called Nephrogenic Systemic Fibrosis (NSF) that cause thickening 
of the skin and other organs. Since physicians became aware of the disease, began screening patients at risk 
of kidney disease, and switched to safer (“macrocyclic”) forms of gadolinium contrast, new reports of NSF 
are much rarer.
There are also reports of gadolinium retained in the brain, bone, and skin. It is not known whether this is 
important to health. We use “macrocyclic” forms of gadolinium contrast, such as gadolinium contrast, such 
as gadobutrol, that are thought to reduce this risk.


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DEXA Scan: Although DEXA is a safe procedure it is associated with a small amount of ionizing radiation. 
The radiation exposure is less than 1 mRem per study. In some subjects, lying on the table may result in 
some discomfort due to back pain or claustrophobia.
Hand X-Ray: Hand X-ray is a safe procedure but is associated with a very small amount of ionizing radiation 
exposure less than 1 mRem per study.
Treadmill Test: Shortness of breath and fatigue are often felt at the end of the exercise, and occasionally 
subjects may experience chest discomfort. Abnormal heart rhythms can be triggered by exercise, but these 
are rarely persistent or severe.
Ankle-brachial index (ABI): Inflation of blood pressure cuffs may cause transient discomfort. Subjects with 
fragile skin may suffer minor trauma. There is felt to be no other risk.
Surface EKG: There are no clinically significant discomforts associated with a routine EKG. Shaving local 
body hair with a sterile hair razor may be necessary to improve the quality of the EKG recording. Skin 
irritation may occur at the site of application for the adhesive electrode patches; however, the EKG will take 
approximately 5 to 10 minutes only and any skin irritation should be minimal.
Cardio-Ankle Vascular Index (CAVI) (optional): Inflation of blood pressure cuffs may cause transient 
discomfort. Subjects with fragile skin may suffer minor trauma (as per ABIs, related to usual BP 
measurements). This procedure involves the use of a VaSera VS-1500N system; an FDA approved vascular 
screening system.
Phlebotomy: Standard precautions for obtaining human blood samples will be taken. Transient discomfort 
and minor bruising may occur at the phlebotomy site. Vasovagal symptoms can occur during blood drawing. 
Intravenous catheters will be inserted by a nurse/technician who is well trained in that procedure.
8.5 Informed Consent Process and Documentation
Each participant will receive an oral and written explanation of the goals, procedures, and risks of this study. 
The Principal Investigator and those Associate Investigators who are listed on the Key Study Personnel  page 
of the protocol with consenting rights may obtain informed consent from research participants.
Consent will be obtained at the NIH Clinical Center. The original, signed informed consent document will 
be placed in the medical record, and the subject will receive a signed copy of the informed consent document. 
The informed consent document is translated into Italian.
In instances where a subject has medically indicated surgery outside of the NIH Clinical Center, informed 
consent will be obtained via technology and/or electronic processes rather than in person per the Clinical 
Center Policy and Communication Bulletin M77-2, using the Tissue Collection informed consent document. 
Subjects will be contacted by telephone and the investigational nature and research objectives of the tissue 
collection for this study. The subject will sign and date the informed consent. A witness will sign to confirm 
the consent process. The informed consent document will be faxed or mailed back to the consenting 
investigator, who will sign and date and send back a fully executed copy for the subject’s records. The 
informed consent process will be documented on a progress note and the signed informed consent document 
will be placed in the medical record. Subjects will be provided a pre-paid envelop to send the signed consent 
document back to the investigator.


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Should a potential non-English speaking subject be referred for participation in this study, the full informed 
consent documents will be translated into the subject’s language. The use of short forms will not be 
requested, as this is a longitudinal study.
8.6 Subject Advocate
A subject’s rights representative is available to subjects on this protocol. The representative can be reached 
at 301-496-2626 and is located in Building 10. Subjects may ask any questions about the study and may 
withdraw their consent at any time.
9. Conflict of Interest
The National Institutes of Health reviews NIH staff researchers at least yearly for conflicts of interest. The 
following link contains details on this process http://ethics.od.nih.gov/forms/Protocol-Review-  Guide.pdf.
None of the members of the research team reported a potential conflict of interest.
10.Reimbursement for Travel
Reimbursement for local travel, U.S. travel, food, and lodging will be in accordance with NIH and NHLBI 
guidelines.
11.Monetary Compensation
Subjects will not be offered compensation for time and inconvenience while participating in this study.
12.Agreements
FWA: Dr. Hilaire is now in University of Pittsburg and no longer a volunteer in NIH, will be analyzing 
identifiable data as a Non-NIH engaged non-enrolling Investigator in this protocol. Dr. Hilaire’s role in 
the research will be limited to data analysis. An FWA coverage agreement to cover this activity will be 
executed. 
MTA: between NHLBI and Ann Rosenthal, MD and Jeffrey Wesson, MD, PhD of Medical College of 
Wisconsin. These recipients will receive de-identified patient and/or control tissues and will help us 
identify the composition of calcification and minerals found in the samples we send. We may link 
analyzed data to identifiers here in NIH. 
13. References
1.  St. Hilaire C, Ziegler SG, Markello TC, et al. NT5E mutations and arterial calcifications. The New 
England journal of medicine 2011; 364:432-42.
2.
Russell RG. Bisphosphonates: mode of action and pharmacology. Pediatrics 2007;119 Suppl 2: S150- 
62.
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Fleisch H. Bisphosphonates: mechanisms of action. Endocrine reviews 1998; 19:80-100.
4.
Itoh F, Kojima M, Furihata-Komatsu H, et al. Reductions in bone mass, structure, and strength in axial 
and appendicular skeletons associated with increased turnover after ovariectomy in mature cynomolgus 
monkeys and preventive effects of clodronate. Journal of bone and mineral research: the official journal 
of the American Society for Bone and Mineral Research 2002; 17:534-43.
5.
Russell RG. Bisphosphonates: from bench to bedside. Annals of the New York Academy of Sciences 


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2006; 1068:367-401.
6.
Hashiba H, Aizawa S, Tamura K, Kogo H. Inhibition of the progression of aortic calcification by 
etidronate treatment in hemodialysis patients: long-term effects. Therapeutic apheresis and dialysis: 
official peer-reviewed journal of the International Society for Apheresis, the Japanese Society for 
Apheresis, the Japanese Society for Dialysis Therapy 2006;10:59-64.
7.
Lomashvili KA, Monier-Faugere MC, Wang X, Malluche HH, O'Neill WC. Effect of bisphosphonates 
on vascular calcification and bone metabolism in experimental renal failure. Kidney international 2009; 
75:617-25.
8.
Russell RG, Watts NB, Ebetino FH, Rogers MJ. Mechanisms of action of bisphosphonates: similarities 
and differences and their potential influence on clinical efficacy. Osteoporosis international: a journal 
established as result of cooperation between the European Foundation for Osteoporosis and the National 
Osteoporosis Foundation of the USA 2008; 19:733-59.
9.
Elmariah S, Delaney JA, O'Brien KD, et al. Bisphosphonate Use and Prevalence of Valvular and 
Vascular Calcification in Women MESA (The Multi-Ethnic Study of Atherosclerosis). Journal of the 
American College of Cardiology 2010; 56:1752-9.
10. Kawahara T, Nishikawa M, Furusawa T, Inazu T, Suzuki G. Effect of atorvastatin and etidronate 
combination therapy on regression of aortic atherosclerotic plaques evaluated by magnetic resonance 
imaging. Journal of atherosclerosis and thrombosis 2011; 18:384-95.
11. Ariyoshi T, Eishi K, Sakamoto I, Matsukuma S, Odate T. Effect of etidronic acid on arterial 
calcification in dialysis patients. Clinical drug investigation 2006; 26:215-22.
12. Nitta K, Akiba T, Suzuki K, et al. Effects of cyclic intermittent etidronate therapy on coronary artery 
calcification in patients receiving long-term hemodialysis. American journal of kidney diseases: the 
official journal of the National Kidney Foundation 2004; 44:680-8.
13. Monney P, Nguyen QV, Perroud H, Descombes E. Rapid improvement of calciphylaxis after 
intravenous pamidronate therapy in a patient with chronic renal failure. Nephrology, dialysis, 
transplantation: official publication of the European Dialysis and Transplant Association - European 
Renal Association 2004;19:2130-2.
14. Loeb JA, Sohrab SA, Huq M, Fuerst DR. Brain calcifications induce neurological dysfunction that 
can be reversed by a bone drug. Journal of the neurological sciences 2006; 243:77-81.
15. Rutsch F, Ruf N, Vaingankar S, et al. Mutations in ENPP1 are associated with 'idiopathic' infantile 
arterial calcification. Nature genetics 2003; 34:379-81.
16. Ramjan KA, Roscioli T, Rutsch F, Sillence D, Munns CF. Generalized arterial calcification of 
infancy: treatment with bisphosphonates. Nature clinical practice Endocrinology & metabolism 
2009; 5:167-72.
17. Rutsch F, Boyer P, Nitschke Y, et al. Hypophosphatemia, hyperphosphaturia, and bisphosphonate 
treatment are associated with survival beyond infancy in generalized arterial calcification of infancy. 
Circulation Cardiovascular genetics 2008; 1:133-40.
18. Ruggiero SL, Dodson TB, Assael LA, Landesberg R, Marx RE, Mehrotra B. American Association of 
Oral and Maxillofacial Surgeons position paper on bisphosphonate-related osteonecrosis of the jaw
- 2009 update. Aust Endod J 2009; 35:119-30.
19. Agatston AS, Janowitz WR, Hildner FJ, Zusmer NR, Viamonte M, Jr., Detrano R. Quantification of 
coronary artery calcium using ultrafast computed tomography. Journal of the American College of 
Cardiology 1990; 15:827-32.
20. Didronel [package insert]. In: Proctor & Gamble Pharmaceuticals I, ed. Cincinnati, OH; 2010.
21. Etidronate [prescribing information]. Mylan Pharmaceuticals, Morgantown, WV; Aug 2010.


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Appendix 


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Appendix