Conditions: Autosomal Dominant Osteopetrosis Type 2
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 12
Healthy volunteers: no

Brief summary:
This study is an open label use of ACTIMMUNE for patients with Autosomal Dominant Osteopetrosis Type 2(ADO2). Effects of treatment will be evaluated after 14 weeks on ACTIMMUNE by bone resorption markers. This study will treat 12 patients with ADO2 recruited from Indiana University and Riley Hospital for Children at Indiana University Health.

Detailed description:
This is a single center, open-label, dose-escalation study evaluating the efficacy, as defined by biochemical endpoints, and safety profiles of ACTIMMUNE in ADO2 subject.

The investigators will treat 12 ADO2 subjects (children or adults age 3-65) with Actimmune® via a dose escalation protocol to a dose of 50 µg/m2 subcutaneously three times per week (TIW) for 8 weeks. If serum CTX does not increase by more than 25% by week 8, the dose will be increased to 100 µg/m2 subcutaneously TIW.

Individual subjects in whom ACTIMMUNE administration increases bone resorption markers during the 14 weeks of this trial will be eligible for a 1 year extension trial.

Arms:
arm 1: ACTIMMUNE 50 µg/m2 subcutaneously three times per week (TIW) for 8 weeks

Interventions: intervention 1: ACTIMMUNE
Intervention descriptions: intervention 1: None

Protocol document:
Prot_SAP_000.pdf: 
Protocol Version 3 
 8 August 2016 
1 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Phase 2a Study of Interferon Gamma-1b for the 
Treatment of Autosomal Dominant Type 2 
Osteopetrosis 
 
Michael Econs, MD 
Indiana University School of Medicine Department of Endocrinology and Metabolism 
1120 West Michigan Street Room 459 
Indianapolis, IN 46202 


Protocol Version 3 
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Phase 2a Study of Interferon Gamma-1b for the 
Treatment of Autosomal Dominant Type 2 Osteopetrosis 
 
Protocol #1510340687 
Version 3 (8 August 2016)  
IND # 128455 
NCT# 02584608 
 
 
 
 
Protocol Physician (Sponsor-investigator) 
 
Michael J. Econs, MD 
Glenn W. Irwin, Jr., Professor of Endocrinology and Metabolism 
Director: Division of Endocrinology and Metabolism 
Professor of Medicine and Medical and Molecular Genetics 
Indiana University School of Medicine 
Endocrinology Service Line Leaders, Indiana University Health Physicians 
1120 W. Michigan Street 
Gatch Clinical Building CL 459 
Indianapolis, IN 46202 
Tel:  317-274-1339 
Fax: 317-278-0658 
Email: mecons@iu.edu 
 
 
 
 
This document is confidential.  It is provided for review only to investigators, study staff, safety 
monitoring boards, and the institutional review board. 


Protocol Version 3 
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PROTOCOL SIGNATURE PAGE 
 
The signature below constitutes the approval of this protocol and the attachments, and 
provides the necessary assurances that this trial will be conducted according to all stipulations 
of the protocol, including all statements regarding confidentiality, and according to local legal 
and regulatory requirements and applicable U.S. federal regulations and ICH guidelines. 
 
 
 
The Lead Principal Investigator should sign below. 
 
Principal Investigator:    
Michael J Econs, M.D.   
 
 
__________________________________________________________________ Date __________________________ 
Name / Title  
The original signature pages should be filed with Indiana University. A copy of this signature 
page should be maintained at each site in the regulatory binder. 
 
 


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Contents 
1.0 – OVERVIEW AND BACKGROUND .................................................................................................................................. 7 
1.1 
BACKGROUND ............................................................................................................................................................ 7 
1.2 
PRECLINICAL AND CLINICAL EXPERIENCE ................................................................................................... 8 
1.3 
SCIENTIFIC RATIONALE ......................................................................................................................................... 8 
2.0 – OBJECTIVES .......................................................................................................................................................................... 9 
2.1 
PRIMARY OBJECTIVE ............................................................................................................................................... 9 
2.2 
SECONDARY OBJECTIVES ...................................................................................................................................... 9 
2.3 SAFETY OBJECTIVE ......................................................................................................................................................... 9 
3.0 – STUDY DESIGN..................................................................................................................................................................... 9 
3.1 
STUDY DESCRIPTION .............................................................................................................................................. 9 
3.2 
STUDY DURATION ................................................................................................................................................. 10 
3.3       ENROLLMENT .......................................................................................................................................................... 10 
3.4       STUDY ENDPOINTS ................................................................................................................................................ 10 
Primary Efficacy Endpoint ........................................................................................................................................... 10 
Secondary Efficacy Endpoints ..................................................................................................................................... 10 
Safety Endpoints ............................................................................................................................................................... 10 
3.5   ONGOING SAFETY REVIEW .................................................................................................................................... 11 
3.6    STUDY RISKS ................................................................................................................................................................ 11 
Risks of Taking Actimmune ......................................................................................................................................... 11 
Radiation .............................................................................................................................................................................. 12 
Risk of Blood Draws ........................................................................................................................................................ 12 
Loss of Confidentiality .................................................................................................................................................... 12 
3.7 
STUDY BENEFITS ................................................................................................................................................... 12 
3.8 
INCLUSION OF CHILDREN .................................................................................................................................. 12 
4.0 – STUDY MEDICATION ...................................................................................................................................................... 13 
4.1 
STRUCTURE AND NOMENCLATURE .............................................................................................................. 13 
4.2 
PHYSICAL PROPERTIES ....................................................................................................................................... 13 
4.3 
MECHANISM OF ACTION ..................................................................................................................................... 13 
Pharmacokinetics ............................................................................................................................................................. 13 
4.4 
STUDY RELATED INFORMATION .................................................................................................................... 14 
Contraindications ............................................................................................................................................................. 14 
Precautions ......................................................................................................................................................................... 14 
5.0 – ELIGIBILITY ....................................................................................................................................................................... 14 
5.1 
INCLUSION CRITERIA ........................................................................................................................................... 14 
5.2 
EXCLUSION CRITERIA .......................................................................................................................................... 14 


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5.3 
EARLY TERMINATION OF A STUDY PARTICIPANT ................................................................................. 15 
6.0 – STUDY PROCEDURES ..................................................................................................................................................... 17 
6.1 INFORMED CONSENT .................................................................................................................................................. 17 
6.2 PHYSICAL EXAM ............................................................................................................................................................ 17 
6.3 VITAL SIGNS .................................................................................................................................................................... 17 
6.4 ADVERSE EVENTS ......................................................................................................................................................... 17 
6.5 CONCOMITANT MEDICATIONS ............................................................................................................................... 18 
6.6 RENAL ULTRASOUND ................................................................................................................................................. 18 
6.7 HEIGHT, WEIGHT AND BSA ...................................................................................................................................... 18 
6.8 STUDY DRUG ................................................................................................................................................................... 18 
Study medication diary .................................................................................................................................................. 19 
Compliance ......................................................................................................................................................................... 20 
6.9       LAB COLLECTION.................................................................................................................................................... 20 
6.10     RADIOGRAPHS ......................................................................................................................................................... 20 
Skeletal survey radiographs ........................................................................................................................................ 20 
Duel Energy absorptiometry (DXA) ......................................................................................................................... 20 
Spine QCT with Volumetric Spine BMD .................................................................................................................. 20 
pQCT of Radius and Tibia ............................................................................................................................................. 20 
6.11 STUDY VISIT SCHEMATIC ....................................................................................................................................... 22 
6.12 VISIT DETAILS .............................................................................................................................................................. 24 
Screening (-30 to -1) ....................................................................................................................................................... 24 
Visit 1 Baseline (day 0) .................................................................................................................................................. 24 
Visit 2 week 2/ day 14 (±3 day) ................................................................................................................................. 24 
Visits 3 week4(±3 day) and visit 5 week 11 (±5 day) ....................................................................................... 25 
Visit 4 week 8 (±5 day) .................................................................................................................................................. 25 
Visit 6 week 14 or early termination (±5 day) ..................................................................................................... 25 
Visit 7 week 18 Follow up (±3 day) .......................................................................................................................... 25 
6.13      RETENTION PLAN ................................................................................................................................................. 25 
7. 0 - REPORTING OF ADVERSE EVENTS OR UNANTICIPATED PROBLEMS INVOLVING RISK TO 
PARTICIPANTS OR OTHERS .................................................................................................................................................. 26 
7.1       UNANTICIPATED PROBLEMS ............................................................................................................................ 26 
7.2 
DATA SAFETY MONITORING BOARD............................................................................................................. 26 
7.3 
ADVERSE EVENTS .................................................................................................................................................. 26 
8. 0 – STATISTICAL CONSIDERATIONS ............................................................................................................................. 28 
9. 0 – PRIVACY/CONFIDENTIALITY ISSUES .................................................................................................................... 29 
10.0 FOLLOW-UP AND RECORD RETENTION ............................................................................................................ 29 


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10.1 
SUBJECT COMPENSATION .................................................................................................................................. 29 
10.2 
RECORD RETENTION............................................................................................................................................ 29 
11.0 – REFERENCES CITED .................................................................................................................................................... 29 
 
 
 
 
 
 


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1.0 – OVERVIEW AND BACKGROUND 
1.1 BACKGROUND 
Autosomal dominant Osteopetrosis type 2 (ADO2) is a heritable osteosclerotic disorder that 
usually results from heterozygous missense mutations in the CLCN7 gene [1, 2], and is 
characterized by a wide range of symptoms and severity including multiple fractures, 
impaired vision and hearing, pancytopenia and osteomyelitis [3-5].  ADO2 is presently 
incurable, although anecdotal evidence and recent studies in a murine model suggest that 
interferon gamma-1b may have beneficial effects [6]. 
ADO2 (also known as Albers-Schönberg disease and “marble bone disease”) is an autosomal 
dominant disorder with an incidence of 5.5 in 100,000 people and penetrance of 
approximately 66% [2, 4]. We have kept the designation ADO2 for the disease we are 
studying because the disease caused by activating mutations in the LRP5 gene, while not 
technically an osteopetrosis, is inappropriately still referred to as ADO1 in the literature. 
Because of the incomplete penetrance and a mild phenotype in some individuals with 
CLCN7 mutations, ADO2 is sometimes referred to as the "benign" form of osteopetrosis to 
distinguish it from the severe "malignant" recessive form, which is life-threatening in 
infancy. However, referring to ADO2 as “benign” is a misnomer because most ADO2 patients 
suffer complications from their disease. In a previous study of 62 ADO2 patients, hip or 
femur fractures occurred in 16% of ADO2 children and 49% of ADO2 adults, and all 4 of the 
most severely affected patients have died from complications of osteopetrosis [4]. In the ADO2 
population at Indiana University, there was a high rate (19%) of severe visual loss (thought 
to be due to lack of bone resorption to widen the optic canal during growth).  Additionally, 
16% of the patients had osteonecrosis or osteomyelitis, and bone marrow failure occurred 
in 3% of patients [4]. Similar to these study results, Benichou et al [5] studied 42 ADO2 
patients and found that 78% of patients had fractures and over half had orthopedic 
procedures with half of those having at least 3 procedures. They also documented a high 
rate of mandibular osteomyelitis (12.9%). In contrast to our study [4], only 5% had visual 
loss due to optic nerve compression [5]. Thus, in the two largest studies to date, ADO2 
patients manifest significant, and in some cases, severe disease. 
Although very high dose calcitriol (up to 18 mcg per day) has been used anecdotally (L. Key, 
personal communication), this approach has not been shown to be efficacious in ADO2. The 
only reference [7] cited on the clinical management of ADO2 in OMIM 
(http://www.omim.org/entry/166600, accessed May 31, 2015) is a case report of a child 
with severe recessive osteopetrosis, who had a partial response to calcitriol.  Indeed, Dr. 
Econs’ experience treating ADO2 patients with high dose calcitriol has not resulted in 
improvements in bone mineral density or biochemical markers of bone turnover, while 
having risk of nephrolithiasis and nephrocalcinosis. Moreover, the calcitriol studies in the 
ADO2 mouse model indicated no benefit and a possible worsening of the bone phenotype 
(see preliminary data). As a result, Dr. Econs has abandoned the practice of treating ADO2 
patients with calcitriol and is currently following these patients off therapy, essentially 
observing the natural progression of the disease.  
 


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1.2 PRECLINICAL AND CLINICAL EXPERIENCE 
The first evidence of a potentially effective therapy comes from studies of interferon gamma 
in a novel ADO2 “knock-in” mouse with a G213R missense mutation in the Clcn7 gene [8], 
which is syntenic to the human G215R mutation identified in several ADO2 patient families 
[2]. Mice homozygous for the G213R mutation have severe osteopetrosis, absent tooth 
eruption, and die by 4 weeks of age, which is similar to the human recessive disease caused 
by mutations in CLCN7 and several other genes [8]. Mice heterozygous for the G213R 
mutation have high whole body aBMD and bone volume per total volume (BV/TV) at distal 
femur and increased numbers of poorly resorbing osteoclasts, resembling human ADO2 [8]. 
Although the ADO2 mouse does not display spontaneous fractures as seen in the adult 
human disease, the fact that these mice have a 2-fold increase in BV/TV at 12 weeks of age 
and have been successfully used to test two potential therapies, one of which results in a 
partial correction of the phenotype (see below), indicates that this model is an excellent 
model of osteoclast dysfunction.  In concert with human disease, the phenotype of our ADO2 
mice gets more pronounced with age.  
1.3 SCIENTIFIC RATIONALE 
Interferon gamma-1b increases bone resorption and returns the bone phenotype towards 
normal in the ADO2 mouse:  
Interferon gamma-1b (IFN-G) is used in 
patients with recessive forms of osteopetrosis 
to control disease until a donor can be found 
for BMT, which has been shown beneficial in 
this very severe disorder [9].  IFN-G is not 
approved for use in ADO2, as it has never been 
tested in this disease. Moreover, cell culture 
data is inconclusive regarding whether IFN-G 
increases or decreases normal osteoclast 
development, number, and/or activity [10-17]. 
Therefore, we sought to use our newly 
developed ADO2 mouse model to test whether 
murine interferon gamma and/or high dose 
calcitriol would decrease bone mass in ADO2 mice. 
Six-week-old male and female ADO2 mice (N=10/group) were treated with vehicle, or 
calcitriol (low 0.1 µg/kg, medium 0.5 
µg/kg, or high 1 µg/kg doses) or 
interferon gamma (low 22.5 µg/kg, 
medium 37.5 µg/kg, or high 100 µg/kg 
dose) 5 times per week for 8 weeks. Bone 
was phenotyped by DXA and micro-
computed tomography (µCT).  Serum and 
urine biochemistries and bone resorption 
markers were also analyzed. Mice treated 
with low and medium doses of calcitriol 
showed a trend of higher aBMD and 
BV/TV whereas high dose calcitriol 
significantly (p<0.05) increased bone 


Protocol Version 3 
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mass compared to vehicle, indicating that calcitriol actually worsened the bone phenotype 
of ADO2 mice [6].  ADO2 mice treated with all doses of IFN-G significantly (p<0.05) 
attenuated the increase of whole body aBMD [6] and BV/TV at the distal femur in both male 
and female mice as compared to those treated with vehicle (Figure 2A). Importantly, the 
reduction in BV/TV due to IFN-G treatment was to a level approximately half way between 
WT mice and the ADO2 mutant mice (the therapeutic midpoint), indicating that there was 
an incomplete, but meaningful reduction in the abnormal bone phenotype with only 8 
weeks of therapy.  Additionally, serum CTX and the CTX/TRAP ratio were increased in the 
IFN-G groups, but not in the calcitriol treated groups, indicating that there was an increase 
in bone resorption with IFN-G treatment ([6] and Figure 2B).  This study demonstrates that 
IFN-G has potential to treat human ADO2 and these results have led us to propose a short-
term human trial with human Interferon gamma-1b (Actimmune®).  
2.0 – OBJECTIVES 
2.1 PRIMARY OBJECTIVE 
• 
To evaluate the effect of treatment with ACTIMMUNE at week 14 on bone resorption 
markers (i.e. C-telopeptide [CTX], and N-telopeptide (NTX)/creatinine ratio) in 
subjects, age 3-65, diagnosed with Autosomal Dominant Osteopetrosis Type 2 (ADO2) 
due to heterozygous mutations in the chloride channel 7 (CLCN7) gene. 
2.2 SECONDARY OBJECTIVES 
• 
To evaluate the effect of ACTIMMUNE at a stable dose over a 6 to 12 week treatment 
period on bone turnover markers (tartrate-resistant acid phosphatase type 5b 
[TRAP5b], N-telopeptide (NTX), and CTX/TRAP5b ratio).  
• 
To evaluate the change in CTX level from the end of treatment (Week 14 visit) to 
1 month after discontinuation of ACTIMMUNE treatment (Week 18). 
• 
To evaluate changes in bone formation markers (P1NP and bone specific alkaline 
phosphatase) at Weeks 8 and 14. 
2.3 SAFETY OBJECTIVE 
• 
To evaluate the safety and tolerability of ACTIMMUNE in ADO2 adults. 
3.0 – STUDY DESIGN 
3.1 STUDY DESCRIPTION  
This is a single center, open-label, dose-escalation study evaluating the efficacy, as defined 
by biochemical endpoints, and safety profiles of ACTIMMUNE in ADO2 subject.   
We will treat 12 ADO2 subjects (children or adults age 3-65) with Actimmune® via a dose 
escalation protocol to a dose of 50 µg/m2 subcutaneously three times per week (TIW) for 8 
weeks.  If serum CTX does not increase by more than 25% by week 8, the dose will be 
increased to 100 µg/m2 subcutaneously TIW.   
Individual subjects in whom ACTIMMUNE administration increases bone resorption 
markers during the 14 weeks of this trial will be eligible for a 1 year extension trial. 


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3.2 STUDY DURATION 
Subject involvement from screening to completion of study will be approximately 157 days 
or approximately 5 months.  Screening procedures can be done over a 30 day period prior 
to baseline (day 0).  Study drug will be started on Day 0 and continued through 14 weeks.  
Subjects will be seen at the completion of the 14 weeks for end of treatment and then 4 
weeks later for a safety follow-up visit.  There will be 8 visits during the trial.  Six of these 
visits will occur at Indiana University Hospital.  Visit 3 and 5 can occur by phone with the 
coordinator and have labs collected at a local lab collection site.   
3.3       ENROLLMENT 
There will be 12 subjects enrolled into the treatment phase of this study.  Additional 
subjects may be required to allow for screen failures and if a subject drops out before visit 5 
they will be replaced with a new subject.  If a subject drops out after visit 5 they will not be 
replaced.   Enrollment is not expected to be difficult due to the extensive experience of the 
investigator with this patient population and the strong Osteopetrosis Support group.   
Screening will focus on patients from known ADO2 kindreds.  Individuals should have either 
been diagnosed with Osteopetrosis and have a clinical phenotype and/or family history that 
is consistent with ADO2, have been told that they have an abnormally high bone density 
(>3SD about mean for age and sex), or a clinical presentation consistent with ADO2.    
3.4       STUDY ENDPOINTS 
Primary Efficacy Endpoint 
 
The primary efficacy endpoint is the difference in the subject’s observed change in bone 
markers (serum CTX and fasting urine NTX) over the treatment period (from Baseline to 
Week 14).  
Secondary Efficacy Endpoints 
• 
Secondary endpoints will include analysis of CTX and NTX over time while on a stable 
dose of Actimmune® and analysis of these markers after discontinuing drug to 
determine if patients rebound off drug.   
• 
In addition, we will measure markers of bone formation (P1NP and bone specific 
alkaline phosphatase) to test whether increased bone resorption from interferon 
gamma-1b therapy is coupled to increased bone formation.   
• 
We will also obtain baseline measurements of bone density via DXA, spine QCT, and 
pQCT of the distal radius and tibia, as well as retrospectively assessing the fracture 
rate in the 5 years prior to initiation of therapy (via a questionnaire with 
confirmation by review of radiographs films and/or reports) to facilitate a future 
longer term extension trial that would test whether long term treatment with 
Actimmune® decreases BMD and reduces fractures in ADO2 patients. 
Please see statistical section (section 8.0) for the statistical analysis plan.   
Safety Endpoints 
Adverse event (AE) data will be presented by subject. Clinical laboratory safety data and 
vital sign data will be summarized by subject with descriptive statistics for Baseline, post-
dose, and change from Baseline to post-dose values. Physical examination findings will be 
listed by subject. AEs will be summarized by categories and grades. Counts and 95% 


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Clinical Toxicity 
ACTIMMUNE 
Placebo 
Fever 
52 
28 
Headache 
33 
9 
Rash 
17 
6 
Chills 
14 
0 
Injection site erythema or tenderness 
14 
2 
Fatigue 
14 
11 
Diarrhea 
14 
12 
Vomiting 
13 
5 
Nausea 
10 
2 
Myalgia 
6 
0 
Arthralgia 
2 
0 
Injection site pain 
0 
2 
Agresti-Coull confidence intervals will be calculated. 
The percentage of subjects with baseline nephrocalcinosis will be summarized using 
descriptive statistics along with degree of nephrocalcinosis.  Individual subjects 
development of new nephrocalcinosis or worsening of existing nephrocalcinosis (identified 
by increasing grade) will be listed as an AE. 
3.5   ONGOING SAFETY REVIEW 
All collected safety data including adverse events, safety test results and changes in 
concomitant medications will be reviewed by the principal investigator or designated 
physician sub investigator in a timely fashion. Study nurses will review the results for 
abnormal results as soon as received and if necessary contact the physician immediately.   
A data and safety monitoring board (DSMB) will be established to monitor the study.  This 
DSMB will be approved by the IRB prior to enrollment in the study is started.  The DSMB 
will review data twice a year.  The DSMB will have authority to stop the trial for safety 
reason or to modify the trial if necessary for safety issues.  Due to the small study population 
and short study duration the DSMB will not be doing interim analysis for effectiveness.   
3.6    STUDY RISKS 
Risks of Taking Actimmune 
The following data on adverse reactions are based on the subcutaneous administration of 
ACTIMMUNE at a dose of 50 mcg/m2, three times weekly, in patients with Chronic 
Granulomatous Disease (CGD) during an investigational trial in the United States and 
Europe. 
The most common adverse events observed in patients with CGD are shown in the following 
table (from product insert): 
Percent of Patients 
 
 
 
 
 
 
 
 
 
 
 
 
 
Miscellaneous adverse events, which occurred infrequently in patients with CGD and may 
have been related to underlying disease included back pain (2 percent versus 0 percent), 
abdominal pain (8 percent versus 3 percent) and depression (3 percent versus 0 percent) 


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for ACTIMMUNE and placebo treated patients, respectively (product label). 
Similar safety data were observed in 34 patients with severe malignant Osteopetrosis. 
Radiation  
Subjects will be exposed to radiation as part of the protocol testing.  The protocol will be 
approved by Indiana University Radiation Safety Committee prior to enrollment.  The 
informed consent will provide subjects information about the risks of additional radiation 
exposure above what would be considered standard of care for ADO2 subjects.   
Risk of Blood Draws 
The risks that can occur with venipuncture include discomfort, bruising, feeling faint or 
passing out, and infection. To reduce the likelihood of complications from blood draws, only 
trained phlebotomists or nurses will draw blood at the study visits using clean technique. 
Loss of Confidentiality 
There is the unlikely chance that health information is viewed by someone outside the 
research team who is not authorized to see health information. However, standard 
procedures to ensure that this does not happen will be followed. 
3.7 STUDY BENEFITS  
 
 
The major potential benefit to the subject is that the treatment may result in increased bone 
resorption, which may reduce disease burden by reducing bone fragility and, thereby, 
fracture risk.  Additionally, treatment may reduce the risk of infection, particularly 
osteomyelitis, and may improve hematopoietic marrow space, resulting in less anemia, 
pancytopenia, and/or extra medullary hematopoiesis.   
ADO2 is an osteosclerotic disorder due to defective bone resorption.  There is wide 
variation in disease severity, but most affected individuals experience fractures with almost 
half of affected adults experiencing hip or femur fractures.  Other manifestations include 
visual loss, osteonecrosis and/or osteomyelitis, and bone marrow failure.  Currently, there 
is no therapy for the disease; however, studies in our ADO2 mouse model indicate that 
interferon gamma-1b may be effective in treating the human disease.  Successful completion 
of the proposed study will provide evidence that administration of Actimmune® induces 
osteoclastic bone resorption in ADO2 patients, a critical first step to determining whether 
the drug is a useful treatment for the disorder.  This study will provide critical data on 
feasibility and dosing and determine if a long-term study is indicated.  Interferon gamma-1b 
could be the first successful therapy for ADO2.  
3.8 INCLUSION OF CHILDREN 
ADO2 affects children as well as adults.  The study medication has been previously tested in 
children for other indications.  It is felt that the earlier the intervention can occur in disease 
process the increased chance of improved outcomes.   
 


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4.0 – STUDY MEDICATION 
4.1 STRUCTURE AND NOMENCLATURE 
ACTIMMUNE® (Interferon gamma-1b) is a single-chain polypeptide containing 140 amino 
acids. Production of ACTIMMUNE is achieved by fermentation of a genetically engineered 
Escherichia   coli containing   the   DNA, which   encodes for the human protein. 
ACTIMMUNE is a highly purified sterile solution consisting of non-covalent dimers of two 
identical 16,465 dalton monomers; with a specific activity of 20 million International Units 
(IU)/mg (2x106 IU per 0.5 mL) which is equivalent to 30 million units/mg (product label). 
4.2 PHYSICAL PROPERTIES 
ACTIMMUNE is a sterile, clear, colorless solution filled in a single-use vial for 
subcutaneous injection.  Each 0.5 mL of ACTIMMUNE contains: 100 mcg (2 
million IU) of Interferon gamma-1b formulated in 20 mg mannitol, 0.37 mg disodium 
succinate hexahydrate, 0.14 mg succinic acid, and 0.05 mg polysorbate 20 and Sterile 
Water for Injection. Note that the above activity is expressed in International Units (1 million 
IU/50mcg). This is equivalent to what was previously expressed as units (1.5 million 
U/50mcg) (product label). 
4.3 MECHANISM OF ACTION 
Interferons bind to specific cell surface receptors and initiate a sequence of intracellular 
events that lead to the transcription of interferon-stimulated genes. The three major groups 
of interferons (alpha, beta, and gamma) have partially overlapping biological activities that 
include immunoregulation such as increased resistance to microbial pathogens and 
inhibition of cell proliferation. Type 1 interferons (alpha and beta) bind to the alpha/beta 
receptor. Interferon-gamma binds to a different cell surface receptor and is classified as 
Type 2 interferon. Specific effects of interferon-gamma include the enhancement of the 
oxidative metabolism of macrophages, antibody dependent cellular cytotoxicity (ADCC), 
activation of natural killer (NK) cells, and the expression of Fc receptors and major 
histocompatibility antigens (product label). 
In recessive osteopetrosis, the exact mechanism(s) by which ACTIMMUNE has a treatment 
effect has not been established. Changes in superoxide levels during ACTIMMUNE therapy 
do not predict efficacy.  
Pharmacokinetics 
The intravenous, intramuscular, and subcutaneous pharmacokinetics of ACTIMMUNE have 
been investigated in 24 healthy male subjects following single-dose administration of 100 
mcg/m2. ACTIMMUNE is rapidly cleared after intravenous administration (1.4 liters/minute) 
and slowly absorbed after intramuscular or subcutaneous injection. After intramuscular or 
subcutaneous injection, the apparent fraction of dose absorbed was greater than 89%. The 
mean elimination half-life after intravenous administration of 100 mcg/m2   in healthy 
male subjects was 38 minutes. The mean elimination half-lives for subcutaneous 
dosing with 100 mcg/m2  was 5.9 hours.  Peak plasma concentrations, determined by 
ELISA, occurred approximately 7 hours (0.6 ng/mL) after subcutaneous dosing.  Multiple 
dose subcutaneous   pharmacokinetic studies were conducted in 38 healthy male subjects. 
There was no accumulation of ACTIMMUNE after 12 consecutive daily injections of 100   


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mcg/m2 (package insert).    
4.4 STUDY RELATED INFORMATION 
Contraindications 
ACTIMMUNE is contraindicated in patients who develop or have known hypersensitivity 
to interferon-gamma, E. coli derived products, or any component of the product. 
Precautions  
Isolated cases of acute serious hypersensitivity reactions have been observed in patients 
receiving ACTIMMUNE. If such an acute reaction develops the drug should be 
discontinued immediately and appropriate medical therapy instituted. Transient 
cutaneous rashes have occurred in some patients following injection but have rarely 
necessitated treatment interruption (product label). 
Actimmune is a category C drug in pregnancy, therefore, female subjects who are pregnant or 
anticipating pregnancy will not be enrolled and pregnancy tests will be done for females of 
reproductive potential. 
 
5.0 – ELIGIBILITY 
5.1 INCLUSION CRITERIA 
Subjects must meet all of the following criteria: 
1. Subject is diagnosed with clinically significant ADO2 as determined by the 
investigator. 
Individuals will be screened who have either been diagnosed with osteopetrosis and have a 
clinical phenotype and/or family history that is consistent with ADO2, have been told that 
they have an abnormally high bone density (>3SD above mean for age and sex), or a clinical 
presentation consistent with ADO2.  Initial contact will be with members of ADO2 kindreds 
who have known disease.   
2. Provide written informed consent for competent adults and for minors provide 
written assent (if appropriate) and written informed consent by a legally 
authorized representative after the nature of the study has been explained, and 
prior to any research-related procedures 
3. Ages 3 to 65 years inclusive. 
 
4. Willing to use reliable method of contraception [i.e. oral or patch hormonal 
contraceptives, intrauterine device, physical barrier methods, tubal ligation or 
hysterectomy, vasectomy (partner) or abstinence] throughout the study and for 
30 days after the last dose of study drug. 
5.2 EXCLUSION CRITERIA 
Subjects will be ineligible for study participation if they meet any of the following criteria.   
1.  Any unstable illness that in the investigator’s opinion precludes participation in 
the study. 
2. Serum calcium >10.6 mg/dl at screening. 


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3. eGFR using the MDRD equation in adults (or the modified Schwartz equation for 
children) of < 35 ml/min/1.73m2. 
4. Nephrocalcinosis on screening ultrasound Grade 3 or higher [18].  Subjects with 
grade 3 or higher nephrocalcinosis will be excluded because we anticipate that 
use of study drug will increase bone resorption, resulting in increased urinary 
calcium excretion, which could, potentially, lead to worsening nephrocalcinosis.  
The grading scale is listed below: 
0 = Normal  
1 = Faint hyperechogenic rim around the sides and tip of the medullary pyramids  
2 = More intense echogenic rim with echoes faintly filling the entire medullary pyramid  
3 = Intense echoes throughout the medullary pyramid  
4 = Solitary focus of echoes at the tip of the medullary pyramid/nephrolithiasis  
5. Use of any investigational product (drug or device) within 30 days prior to 
randomization. 
6. Subject reported history of hepatitis C.  
7. A recent (past 5 years) history of alcoholism or intravenous drug abuse. 
8. History of hypersensitivity to IFN-ɣ or E. coli-derived products. 
9. History of liver disease as evidenced by laboratory results at Screening (aspartate 
aminotransferase [AST] or alanine aminotransferase [ALT] >2x the upper limit of 
normal), except when in the opinion of the investigator the liver disease is caused 
by extra medullary hematopoiesis. 
10. Pregnant or nursing women or those who plan on becoming pregnant during the 
study.  
11. Preexisting cardiac conditions including ischemia, congestive heart failure, or 
arrhythmia.   
5.3 EARLY TERMINATION OF A STUDY PARTICIPANT 
Treatment will stop and the subject will return for an exit visit if any of the following 
conditions occur: 
1. withdrawal of consent 
2. pregnancy (female participants) 
3. anaphylaxis requiring hemodynamic support (i.e. epinephrine and/or blood pressure 
medications) or mechanical ventilation 
4. any medically important event such as a concurrent illness or complications judged to 
significantly increase the risk of study therapy. 
5. any investigator judgment 
6. If any of the following dose-limiting toxicities occur: 
a. Increase in liver transaminases more than 5 times upper end of normal, total 
bilirubin ≥2.5 mg/d. 
b. Grade 3 or higher AE except as mentioned below. 
c. Develops Grade 2 or higher nephrocalcinosis, or symptomatic nephrolithiasis. 
 
Special circumstance: Grade 3 and 4 toxicity of absolute neutrophil count (ANC, includes 
neutrophils and bands) or platelet count:  
1. Marrow failure due to crowding from lack of bone resorption is part of the underlying 
disease of ADO, and there is no current effective approved therapy for ADO to prevent or 
treat this.  Because of this, some subjects may have baseline values of ANC or platelets 
that are near the Grade 3 toxicity level, prior to study drug.   However, since there is no 


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effective therapy for ADO, and there is potential for interferon gamma 1b to improve 
marrow space and function if it proves effective to increase osteoclastic activity, subjects 
will be allowed to continue in study under the following conditions: 
a. If there is a decrease in absolute neutrophil count (ANC, includes neutrophils and 
bands) or platelet count to a Grade 4 toxicity (e.g. ANC < 500 or Platelets < 25k), 
we will hold study drug and monitor CBC with differential weekly until recovery 
to a grade 3 toxicity level (ANC 500 – 1,000; or platelets 25k-50k) and, after 
consultation with the patient and/or family (if a minor), restart at half the 
previous dose of interferon gamma 1b and continue to monitor CBC with 
differential weekly.   
b. If the patient remains above the target ANC after 2 weeks the investigators may 
consider re-challenge with the previous dose of interferon gamma 1b, with 
weekly CBC with differential monitoring. 
c. If toxicity reoccurs at the lowered dose we will again hold study drug and monitor 
CBC with differential weekly until recovery to a grade 3 toxicity level (ANC 500 – 
1,000; or platelets 25k-50k), and then can restart again with further lowering the 
dose by half again, or discontinue therapy, in consultation with the patient and/or 
family.   
d. If the patient remains above the target ANC after 4 weeks  the investigators may 
consider re-challenge with the previous dose of interferon gamma 1b, with 
weekly CBC with differential monitoring.  
 
Additional Considerations: 
 
1. There are potential for SAE during the study related to the underlying disease. These may 
include hospitalization or surgery for a fracture, or complications of anemia, bone marrow 
failure or infection, or vision loss.   Patients will discontinue study drug during hospitalizations 
or surgeries, but will not be eliminated from the study except as described below, or at the 
investigator discretion based on severity of the incident and likelihood of relationship to study 
drug.  
2. Severe vision loss, which may possibly occur during the study period, is a part of the underling 
disease, usually presenting in children, for which there is not currently any effective medical 
therapy to prevent. In fact the severe vision loss that occurs in a percentage of patients with 
this disorder is only one of the reasons that this study is needed, because it may be possible 
with an effective treatment to prevent severe vision loss, but that cannot be determined except 
in future larger and longer trials, because the mechanism of vision loss is thought to be optic 
nerve compression during skull growth. Although unlikely to occur during the short time 
frame of the study, if severe vision loss is detected clinically during this study, the individual 
patient will be stopped in the trial for the purposes of facilitating potential surgical treatment 
if deemed necessary by the treating clinician.  
3. A threshold of 25% of subjects having unexpected SAE’s would result in an expedited review 
by the DSMB for whether the study should be modified or completely discontinued, except 
when the SAE is death or pregnancy having birth defect or for other SAE considered by the 
investigator (or DSMB) to represent a significant medical hazard. In the unlikely event of a 
subject death or the latter two events occur during the study, the study will be stopped and 
reviewed by the DSMB for whether the study should be discontinued.  Because there is some 


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risk of death with the underlying disorder, this will need to be assessed by the DSMB on a case 
by case basis.  
 
 
Subjects who stop study will be asked to return for a final early termination visit.  
 
6.0 – STUDY PROCEDURES 
6.1 INFORMED CONSENT  
Consent/assents will be completed prior to any study related procedure.  Consents/assents 
can be obtained by designated staff as long as there is a physician investigator available for 
questions.   
The procedures planned will be fully explained to the subject and/or family.  All 
consent/assent discussions will occur in a private room.  The investigational nature and 
objectives of the trial, the procedures involved, and their attendant risks and discomforts 
will be carefully explained to the potential subject and their questions answered. Potential 
subjects (or parents/guardians) will be informed that they have the right to decline 
participation in the study and that this decision will not impact their medical care. Subjects 
will also be notified of their right to withdraw from the study at their (or their 
parent/guardian’s) choice, though recommendations for monitoring for subject safety will 
also be provided when appropriate. 
Assents will be obtained from all children age 7 to 18 if appropriate.  If a child is not 
appropriate for assenting then justification of this must be documented by a physician 
investigator.  If a child turns 7 during the conduct of the study then assent must be obtained 
at next visit.  It is also expected if a child turns 18 while on trial they will be asked to sign a 
full consent at next visit.  
6.2 PHYSICAL EXAM 
Physical exam will be completed by a physician investigator.  Complete physicals will be 
done at screening and visit 1 (unless within 10 days of screening) 4 and 6. A brief physical 
exam can occur at visit 2 and 7.   
6.3 VITAL SIGNS  
Measured at all clinic visits after subject has had a chance to rest for approx. 5 mins.  Vital 
signs should be taken with subject sitting with legs uncrossed.  Attempt should be made to 
use same arm and cuff size for each visit.  Abnormal results should be rechecked and 
physician notified if vitals remain abnormal.   
6.4 ADVERSE EVENTS 
Subjects will be asked about any changes in his/her health status at each site visit and 
phone call.  Adverse events that occur within four weeks prior to Day 0 and prior to dosing 
on Day 0 will be considered baseline signs/symptoms. Adverse events or a worsening of a 
condition listed as medical history that occur after the dose on Day 0 through the end of the 


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study will be considered treatment-emergent adverse events (TEAEs). All SAEs that occur 
from the signing of informed consent through two weeks after study discontinuation will be 
recorded. 
All AE and SAE information will be reviewed by physician investigator for assessment.   
6.5 CONCOMITANT MEDICATIONS 
Concomitant medication data will be collected from the subject, for all medication taken in 
the last 30 days.  This will include prescription, over the counter medications and 
vitamins/supplements.  Subjects will be asked about any medication changes at each site 
visit and phone call, with a particular focus on pain medications.  Changes in the use of pain 
medicines will be analyzed to determine if subjects experience change in pain with therapy.  
This analysis may be complicated by the use of some pain medications as antipyretics 
(NSAIDs, acetaminophen).  Efforts should be made to determine the exact indication for use 
of pain medications such as NSAID and Acetaminophen.   
6.6 RENAL ULTRASOUND 
Standard renal ultrasound will be completed according to visit schedule.  Ultrasounds 
should be read locally and nephrocalcinosis graded according to the following grading scale:  
0 = Normal  
1 = Faint hyperechogenic rim around the sides and tip of the medullary pyramids  
2 = More intense echogenic rim with echoes faintly filling the entire medullary pyramid  
3 = Intense echoes throughout the medullary pyramid  
4 = Solitary focus of echoes at the tip of the medullary pyramid/nephrolithiasis 
6.7 HEIGHT, WEIGHT AND BSA  
Height will be measured using a stadiometer that has routine calibration.  Weight will be 
measured on scales that are routinely calibrated.  All measurements should occur without 
shoes, pockets emptied and minimal clothing.  (i.e. coats, heavy sweaters removed)  BSA will 
be calculated using the equation below 
𝐵𝑜𝑑𝑦 𝑆𝑢𝑟𝑓𝑎𝑐𝑒 𝐴𝑟𝑒𝑎= √height (cm)x weight (kg) ÷ 3600 
6.8 STUDY DRUG 
Subjects will receive subcutaneous doses of ACTIMMUNE TIW for a total of 14 weeks. The 
dose of study drug will be determined using the subject’s body surface area (BSA) calculated 
at each clinic visit as: 
𝐵𝑜𝑑𝑦 𝑆𝑢𝑟𝑓𝑎𝑐𝑒 𝐴𝑟𝑒𝑎= √height (cm)x weight (kg) ÷ 3600 
To minimize side effects of the medication and improve tolerance we will start with a low 
dose and increase the dose according to a protocol developed by Devane et al [19].  Dose 
escalation will be titrated as follows: 
• 
Day 0 to day 6: Subjects will receive  15 µg/m2 SC TIW 
• 
Day 7 to 14 : Subjects will receive 30 µg/m2 SC TIW  
• 
Day 15-55: Subjects will receive 50 µg/m2 SC TIW  
• 
Day 56 (end of week 8): Serum CTX will be assessed. Titration will occur by week 9 
based upon serum CTX 


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• 
Week 9 (approx. day 63):  
o If CTX has increased by ≥25% above the mean of screening and baseline visits 
CTX values, the ACTIMMUNE dose stays at 50 µg/m2 TIW through week 14. 
o If CTX is <25% above the mean of screening and baseline visits CTX values, 
then the ACTIMMUNE dose is increased to 100 µg/m2 TIW through week 14. 
These doses are based on the current approved dose of Actimmune® for severe, malignant 
recessive osteopetrosis and animal studies [6] demonstrating that relatively high doses of 
mouse interferon gamma 1b increased bone resorption in the ADO2 mouse.  Data in 
melanoma patients showed that doses of 100 µg /m2 TIW subcutaneously (SC) were well 
tolerated (N=5), however daily dosing of either 100 µg /m2 SC (N=5), 100 µg /m2 IM (N=10), 
or 250 µg /m2 IM (N=5) resulted in suppressed absolute neutrophil count, and at the highest 
dose increased liver enzymes [20].  Their data also suggested that monocyte activation 
returned to normal after 72 hours of dosing, supporting the need for TIW dosing.   
Dose reductions will be allowed on a case-by-case basis to manage subsequent drug-related 
adverse events (AEs).  If, in the opinion of the investigator, severe reactions occur during 
dose escalation, the dose will be reduced to the previous dose level or will be interrupted 
until the adverse event resolves and then restarted at the previous or a lower dose level.  
Additionally, the dose can be reduced based on laboratory values.  For example, we 
anticipate that the calcium/creatinine ratio will increase on drug due to increased bone 
resorption (the goal of therapy).  Subjects will be encouraged to have high fluid 
consumption (>2 L/day for adults, >30ml/kg/day for children) and those subjects with 
urine calcium/creatinine ratios above 0.25 will be encouraged to consume a low calcium 
diet of  less than half the age appropriate RDA.  However, if either fasting random or 24 hour 
urine calcium/creatinine ratio increases above 0.5 the investigator can reduce the dose of 
study drug and/or put the subjects on a lower calcium diet.  
Attempts should be made to keep dosing on a Monday, Wednesday, Friday dosing schedule.  
Missed doses can be taken the next day but two doses should not be taken in the same day.  
If a dose is missed and it is time for the next dose do not give 2 injections in one day.  If two 
days have passed since the last dose the dose will be skipped. 
Subjects should be instructed on how to give subcutaneous injections including drawing up 
the medication.  The optimal sites for injection are right and left posterior arm and upper 
leg.   
Study medication and supplies will be provided to subjects at clinic visits.  Vials of 
ACTIMMUNE must be placed in a 2 – 8 °C (36 – 46 °F) refrigerator immediately upon receipt 
to ensure optimal retention of physical and biochemical integrity.  DO NOT FREEZE. Avoid 
excessive or vigorous agitation. DO  NOT SHAKE. An unentered vial of ACTIMMUNE 
should not be left at room temperature for a total time exceeding 12 hours prior to use. 
Vials exceeding this time period should not be returned to the refrigerator; such vials 
should be marked do not use. Vials are single dose vials and should be marked used after 
one use.   
Vials should be provided to subject with cooler and gel packs sufficient to keep 
refrigerated during transit home.  Subjects should also be provided with syringes, needles, 
alcohol wipes and sharps containers.   
Study medication diary 
Subjects will be provided a dosing diary.  This should be used to note all doses given 


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including the dose, date and time.  This should also be used to record missing doses.   
Compliance 
 All vials used and unused should be returned with dosing diary at each study site visit.  
Subject compliance will be assessed based on dosing diary.  Compliance will be calculated: 
(Actual doses taken ÷ expected dose)*100= percent compliant  
6.9       LAB COLLECTION 
Venous blood samples will be collected by the experienced study staff from each subject 
using clean technique. Aliquots of blood will be sent for laboratory tests described in 
schedule of events.  
Blood should be collected with the subjects in a fasting state with nothing but water 8 hrs 
prior to the draw. 
Urine NTX should be from the second morning urine.  Subject should void upon rising from 
bed and discard.  The sample should then be collected from the next void.   
6.10     RADIOGRAPHS  
Skeletal survey radiographs 
This should include anterior/posterior (A/P) and lateral cervical, thoracic and lumbar 
spines, swimmers view, A/P upper and lower ribs, pelvis, A/P femurs, A/P humeri, lateral 
skull. 
Duel Energy absorptiometry (DXA) 
This will measure lumbar spine, total body (total body minus head for children <18), total 
hip and femoral neck. Bone density testing is being done to document skeletal phenotype 
and to provide baseline if subjects continue in possible extension.   
Spine QCT with Volumetric Spine BMD 
This will be done per standard procedure for the radiology department.  This will only be 
done in subjects 10 and older.   
pQCT of Radius and Tibia 
 This will be done according to standard procedure.  This will only be done on subjects 5y/o 
and over.  pQCT will be used to assess cortical and trabecular bone sites at the radius and 
tibia to provide baseline evidence of the disease phenotype and to follow into possible 
extension study.  Tomographic images of the distal (4% of bone length from distal end) and 
diaphyseal (66% of bone length from distal end) radius and tibia will be acquired with a 
Stratec XCT-3000 pQCT machine [21-24]. Standardized segmenting and thresholding 
algorithms will be used to acquire cortical and trabecular bone properties at the diaphyseal 
and distal sites, respectively. Measures will include vBMD, BMC, total area, cortical area, 
average cortical thickness, and periosteal and endosteal perimeters. The polar strength-
strain index (SSI) and bone strength index (BSI) will be determined at the diaphyseal and 
distal sites, respectively [25]. 
Osteopetrosis is a disease of incredibly dense and thickened cortical bone, thus the 
quanititative assessment of trabecular bone density by QCT is a more accurate method for 
identifying the subtle or early changes of treatment which is paramount for this study.   


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22 
 
 
6.11 STUDY VISIT SCHEMATIC  
Study Phase 
Screening  
Treatment Period 
Follow-up 
Visit # 
SV1 
12 
2 
3 
4 
5 
6 
73 
Week 
 
Baseline 
2 
4 
8 
11 
14/ET 
18 
Study Days (± visit window) 
-30 to -1 days 
Day 0 
14 (± 3)  
days 
28 (± 3)  
days 
56 (± 5)  
days 
77 (± 5)  
days 
98 (± 5) days 
126 (± 3) days 
In person visit to IU4 
X 
X 
X 
 
X 
 
X 
X 
Informed consent 
X 
 
 
 
 
 
 
 
Review of inclusion/exclusion criteria 
X 
X 
 
 
 
 
 
 
Demographics and  medical history 
X 
 
 
 
 
 
 
 
SF-36 
 
X 
 
 
 
 
X 
 
Safety Assessments 
 
 
 
 
 
 
 
 
Physical examination 
X 
X 
X 
 
X 
 
X 
X10 
Vital Signs: blood pressure, pulse, temperature 
X 
X 
X 
 
X 
 
X 
X 
Baseline sign/symptom9 
X 
X 
 
 
 
 
 
 
Adverse Events9 
 
X 
X 
X 
X 
X 
X 
X 
Prior/concomitant medications 
X 
X 
X 
X 
X 
X 
X 
X 
Renal ultrasound 
X 
 
 
 
 
 
 
X11 
Study Medication 
 
 
 
 
 
 
 
 
Weight/height, BSA5 
X 
X 
X 
 
X 
 
X 
 
Educate/review of dosing technique and dose 
level 
 
X 
X 
 
X 
 
 
 
Study medication dose given at study visit6 
 
X 
X 
 
X 
 
 
 
Dispense study drug7 
 
X 
X 
 
X 
 
 
 
Drug compliance assessed by diary 
 
 
X 
X 
X 
X 
X 
 
Dose escalation decision  
 
 
 
 
X 
 
 
 
Laboratory Testing 
 
 
 
 
 
 
 
 
Serum pregnancy test 14 
X 
 
 
 
 
 
 
 
Urine pregnancy test 14 
 
X 
X 
X 
X 
X 
X 
X 
Genotype (not required for enrollment) 
 
X 
 
 
 
 
 
 
Efficacy assessments:  
Resorption marker testing  -C-telopeptide 
[CTX], NTX, Tartrate resistant acid 
phosphatase type5b [TRAP5b]) 
X 
X 
X 
X 
X 
X 
X 
X 
  Formation marker Testing (bone specific 
alkaline phosphatase [BAP], type 1 
procollagen [P1NP]) 
X 
X 
X 
X 
X 
X 
X 
X 
  RANKL, OPG, CK-BB (all batched and 
measured at the end of the study) 
 
X 
 
 
X 
 
X 
 
Clinical laboratory evaluation  
(hematology, chemistry, PTH, Vit D 
1,25(OH)2, urinalysis) 
X 
X 
X 
X 
X 
X 
X 
X 
Fasting Urine: Calcium  & Creatinine 
X 
X 
X 
X 
X 
X 
X 
X 
IFN γ level prior to dose Day1; 6 hours post 
dose V1, V2 and V4 
 
X 
X 
 
X 
 
X 
 
IFN γ immunogenicity testing (prior to dosing 
except safety visit)15 
 
X 
X 
X 
X 
 
X 
X 


23 
 
 
Radiology Testing 
 
 
 
 
 
 
 
 
Skeletal survey (radiographs) 
X 
 
 
 
 
 
 
 
  Dual energy X-ray absorptiometry    
  (DXA) lumbar spine, total body,  
  total hip and femoral neck8 
X 
 
 
 
 
 
 
 
Spine QCT13 
X 
 
 
 
 
 
 
 
pQCT of radius and tibia 12 
X 
 
 
 
 
 
 
 
spine volumetric BMD by QCT13 
x 
 
 
 
 
 
 
 
1  Screening procedures can take place over more than one day/clinic visit provided consent/assent is obtained first and all assessments are completed within the designated window. 
2 On Day 0 (Baseline), subjects will receive the first dose of study drug while in the clinic; however, Baseline assessments will be performed prior to dosing.  
3 Eligible subjects completing 14 weeks of treatment may enroll in a long-term extension study at week 14 visit. 
4   Subjects and/or caregivers will be contacted by email or phone to monitor safety and dosing logistics on a weekly basis through Week 14. 
5 Height and weight will be measured at Screening and then at all treatment visits to determine BSA and dose. 
6 Dosing on Day 0 and Weeks 2, 8 and 14 will be performed at the clinic. Starting dose on Day 0 will be 15 mcg/m2 for 7 days (Week 1), then escalated to 30 mcg/m2 for 7 days (Week 2). At the 
Week 2 visit, subjects will be dosed at 50 mcg/m2 in the clinic.  If tolerated, subjects will continue on this dose until Week 8. At the Week 8 visit the dose may be escalated to 100 mcg/m2 until Week 
14. All other doses will be administered at home before bedtime on a three day a week (TIW) schedule. 
7 Subjects will be given a 2 week supply of study drug on Day 0.  At Week 2 visit, subjects will be given six week supply. At Week 8 visit, subjects will be given a 6 week supply of study drug.  
8    Baseline values will be used to measure efficacy in subjects rolling over into the long-term extension study.  
9   Any change in health status including any new signs and symptoms that occur after consent but prior to Day 0 and prior to dosing on Day 0 will be considered baseline signs/symptoms. Adverse 
events occurring or worsening after the dose on Day 0 through the end of the study will be considered treatment-emergent adverse events (TEAEs). All SAEs that occur from the signing of informed 
consent through two weeks after study discontinuation will be recorded. 
10 Brief physical examinations will be performed at the Week 2 and Follow-Up Safety Visits; all other examinations will be complete physical examinations.  
11 If a subject undergoes early termination from the study after week 6, then a renal ultrasound will be included during an early termination visit. 
12 competed only if subject ≥5 years old 
13 completed  only if subject is ≥ 10 years old. 
14 for women of child bearing potential. 
15.  If a subject tests positive for anti-drug antibodies (ADA), he/she will be followed until ADA levels revert to baseline.   
 


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6.12 VISIT DETAILS 
Screening (-30 to -1) 
Screening can occur over more than one day as long as the consent processes is completed 
first.   
Screening will include all of the following: 
 
Consent 
 
Review of inclusion/exclusion 
Collection of demographics, medical history, prior and current medications and 
baseline signs and symptoms. 
 
Physical exam, vitals, height and weight 
 
Renal ultrasound 
 
Skeletal survey 
 
DXA 
 
Spine QCT 
 
pQCT 
Blood testing will include: pregnancy, hematology, Chemistry, PTH, Vit D 1,25, CTX, 
TRAP5b, Bone specific alk phos (BAP), P1NP 
Urine tests (fasting) will include: NTX, UA, calcium, creatinine 
Visit 1 Baseline (day 0) 
 
Review of inclusion/exclusions 
 
Physical exam, vitals, height and weight 
 
Review of baseline symptoms and concomitant medication 
 
Educate on dosing technique and dose level 
 
Study medication dose given (1st dose) 
Review of adverse events (acute events as first dose will be given on this day) 
 
SF-36  
 
Dispensing of study medication for 2 weeks 
 
Dispense dosing diary 
Blood testing will include: Genotype, CTX, TRAP5b, BAP, P1NP, RANKL, OPG, CK-BB, 
hematology, Chemistry, PTH, Vit D 1,25(OH)2 ,  IFN γ  pre dose immunogenicity 
IFN γ level predose and 6 hours post dose. 
Urine test will include: pregnancy, NTX, UA, calcium, creatinine 
Visit 2 week 2/ day 14 (±3 day) 
Vitals, height and weight 
 
Review of adverse events and concomitant medication 
 
Review of dosing and injection procedures 
 
Study medication dose given  
 
Dispensing of study medication for 6 weeks 
 
Dispense dosing diary 
Collect all used study medication 
 
Assess study medication compliance after diary review 
Blood testing will include: CTX, TRAP5b, BAP, P1NP, hematology, Chemistry, PTH, Vit 
D 1,25(OH)2, IFN γ  immunogenicity 
IFN γ level predose and 6 hours post dose. 
Urine test will include: pregnancy, NTX, UA, calcium, creatinine 


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Visits 3 week 4 (±3 day) and visit 5 week 11 (±5 day) 
These visits may be completed over the phone after blood work has been at a local central 
lab collection location. 
 
Review of adverse events and concomitant medication 
 
Assess study medication compliance after diary review 
Labs to be collected at local collection site: Blood testing will include: CTX, TRAP5b, 
BAP, P1NP, hematology, Chemistry, PTH, Vit D 1,25(OH)2 
Urine test will include: pregnancy, NTX, UA, calcium, creatinine 
IFN γ  immunogenicity at Week 4 
Visit 4 week 8 (±5 day) 
 
Physical exam, vitals, height and weight 
 
Review of adverse events and concomitant medication 
 
Review of dosing and injection procedures 
Dose escalation decision 
 
Study medication dose given 
Collect all used study medication 
 
Dispensing of study medication for 6 weeks 
 
Dispense dosing diary 
Assess study medication compliance after diary review 
Blood testing will include: CTX, TRAP5b, BAP, P1NP, RANKL, OPG, CK-BB, hematology, 
Chemistry, PTH, Vit D 1,25(OH)2, IFN γ immunogenicity 
IFN γ level predose and 6 hours post dose. 
Urine test will include: pregnancy, NTX, UA, calcium, creatinine 
Visit 6 week 14 or early termination (±5 day) 
  
Physical exam, vitals, height and weight 
 
Review of adverse events and concomitant medication 
SF 36 
 
Assess study medication compliance after diary review 
Collect all study medication supplies 
Blood testing will include: CTX, TRAP5b, BAP, P1NP, RANKL, OPG, CK-BB, hematology, 
Chemistry, PTH, Vit D 1,25(OH)2 , IFN γ  immunogenicity 
IFN γ level predose. 
Urine test will include: pregnancy, NTX, UA, calcium, creatinine 
If early term after 6 weeks renal ultrasound should also be done. 
Visit 7 week 18 Follow up (±3 day) 
  
Physical exam, vitals, height and weight 
 
Review of adverse events and concomitant medication 
 
Renal ultrasound 
Blood testing will include: CTX, TRAP5b, BAP, P1NP, RANKL, OPG, CK-BB, hematology, 
Chemistry, PTH, Vit D 1,25(OH)2 , IFN γ  immunogenicity 
IFN γ level predose. 
Urine test will include: pregnancy, NTX, UA, calcium, creatinine 
6.13      RETENTION PLAN 
Participating subjects will be seen or contacted by phone (or email) at least weekly to query 
regarding any concerns the subject may have regarding participation or adverse events, and 


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to improve retention and compliance. If a subject is having problems with flu like symptoms, 
which may affect participation in the study, dosage adjustments may be made. 
In addition to aid in retention, two study visits may be done over the phone with a local 
collection of blood and urine for laboratory assessments, in order to be more convenient for 
the subject. However, the Screening, Day 0, week 2, week 8, week 14 and week 18 visits 
must occur at the Indiana University School of Medicine 
7. 0 - REPORTING OF ADVERSE EVENTS OR UNANTICIPATED 
PROBLEMS INVOLVING RISK TO PARTICIPANTS OR OTHERS 
7.1       UNANTICIPATED PROBLEMS 
In the event of physical injury resulting from the participation in this research, necessary 
medical treatment will be provided and billed as part of the subjects medical expenses.  
Costs not covered by the subject’s health care insurer will be the subject’s responsibility.  
Also, it is the subject’s responsibility to determine the extent of health care coverage.  There 
is no program in place for other monetary compensation for such injuries. However, the 
subject has not given up any legal rights or benefits to which he/she is otherwise entitled.   
7.2 DATA SAFETY MONITORING BOARD 
An external and independent Data Safety Monitoring Board (DSMB) from the MARCH 
(Midwest Area Research Consortium for Health) will monitor this study and be provided 
with information about subjects’ outcomes and adverse events every six months. 
The study team will review the data for safety and benefits on a continual basis.  The team 
will then generate a summary report based upon that internal review including a decision to 
stop dose escalation. This report will be provided to our primary MARCH DSMB contact, 
who will review at their next full DSMB meeting. All adverse events related to dose limiting 
toxicity will be reported, as they occur, to the MARCH DSMB. 
A threshold of 25% of subjects having unexpected SAE’s would result in an expedited 
review by the DSMB for whether the study should be modified or completely discontinued, 
except when the SAE is death or pregnancy having birth defect or for other SAE considered 
by the investigator (or DSMB) to represent a significant medical hazard. In the unlikely 
event of a subject death or the latter two events occur during the study, the study will be 
stopped and reviewed by the DSMB for whether the study should be discontinued.  Because 
there is some risk of death with the underlying disorder, this will need to be assessed by the 
DSMB on a case by case basis. 
7.3 ADVERSE EVENTS 
An adverse event is defined as any unintended or abnormal clinical observation that is not of 
benefit to the subject.  Either the condition was not present prior to exposure to test 
medication, or it has worsened in intensity or frequency following exposure to test 
medication.  Adverse events will be graded according to the National Cancer Institute (NCI) 
Common Terminology Criteria v4.0 
(CTCAE):http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm 
 


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The Sponsor-Investigator will be responsible for fulfillment of safety reporting obligations 
to authorities, investigators, and Ethic Committees according to applicable regulations, 
including reporting to applicable competent authority. 
  
Principal investigators (PI) must report to the IRB as soon as possible, but in all cases within 
5 working days from notification any event that appears on the List of Events that Require 
Prompt Reporting to the IUPUI IRB. 
 
List of Events that Require Prompt Reporting to the IUPUI IRB: Any of the following: 
❖ Event (including adverse events, injuries, side effects during the research study), 
which in the opinion of the PI 
• 
caused harm to one or more subjects or others, or placed one or more subjects 
or others at increased risk of harm; AND 
• 
was unexpected; AND 
• 
was related to the research procedures or study medications 
 
Note:  After the study is closed with the IRB, these events should only be reported if they are 
profound or they demonstrate long-term risks that would necessitate notifying subjects. 
❖ Protocol deviation/violation (as defined under this policy and on-site only) 
❖ Change to the protocol taken without prior IRB review to eliminate apparent 
immediate hazard to a research subject (e.g. purposeful and for subject safety) (on-
site only) 
❖ Complaint of a subject that indicates unexpected risks, or complaint that cannot be 
resolved by the research team (on-site only) 
❖ Interim findings and safety monitoring reports that indicate an unexpected change to 
the risks or potential benefits of the research, in terms of severity or frequency 
❖ Publication in the literature that indicates an unexpected change to the risks or 
potential benefits of the research 
❖ Change in FDA labeling or withdrawal from marketing of a drug, device, or biologic 
used in a research study 
❖ Noncompliance (as defined in this policy and on-site only) 
 
Unanticipated problems involving risk to volunteers or others, serious adverse events 
related to participation in the study, and all volunteer deaths related to participation in the 
study should be promptly reported to study staff by phone (317-948-8346), by email 
(marihart@iu.edu), or by facsimile (317-948-2254). 
 
The principal investigator must complete the FDA MedWatch 3500a form and assess the 
relationship to study treatment and send the initial completed MedWatch form within 24 
hours. The investigator must then ensure that the form is accurately and fully completed 
with follow-up information within 2 to 3 calendar days for deaths or life-threatening events 
and 5 calendar days for other serious adverse events. The original and the duplicate copies 
of the FDA MedWatch form and the fax confirmation sheet must be kept with the case report 
forms at the study site. 


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Follow-up information should describe whether the event has resolved or continues, if and 
how it was treated, whether the blind was broken or not, and whether the subject continued 
or discontinued study participation. The MedWatch form sheet must be retained at Indiana 
University. Pregnancy follow-up should describe the outcome of the pregnancy, including 
any voluntary or spontaneous termination, details of the birth, and the presence or absence 
of any congenital abnormalities or birth defects. 
8. 0 – STATISTICAL CONSIDERATIONS 
The sample size N=12 is justified based on the following hypothesis testing: H0: ΔCTX(%)=0 
versus Ha: ΔCTX(%)≥25% using one-sample Student’s t-test. Consequently, we will have a 
power of 80% at type I error level 0.05 if the corresponding standard deviation is no bigger 
than 28.1%, which is considered as obtainable from our previous experiences.    
Percent changes in bone markers (serum CTX and fasting NTX)  from baseline, denoted as 
Δ’s, will be summarized as means and standard deviations (SDs) and be tested as H0: Δ=0 
versus Ha: Δ≠0. This endpoint will be assessed on all subjects tolerating at least 30 ug/m2 
TIW. 
Longitudinal profiles of bone turnover markers will be evaluated by linear mixed effects 
models. Time will be included as both a continuous variable and a categorical variable to 
account for potentially nonlinear effects. Graphs of individual subject curves of CTX and 
NTX/creatinine ratios and other bone turnover markers over time will also be generated. 
CTX and NTX/creatinine concentrations during dose escalation, at peak dose, and at study 
end will be analyzed by repeated measure analysis of variance to evaluate dose response 
and to determine whether bone resorption markers are stable once final dose therapy is 
achieved. 
Fasting urine calcium/creatinine ratio between baseline and while on drug, which are 
anticipated to increase with therapy, will be analyzed by linear or nonlinear mixed effects 
models, depending the distribution of the ratio.   
Baseline fracture rates (fractures per year in the 2 years prior to the start of the study) and 
the fracture rates in the year while on therapy for subjects enrolled in the long-term 
extension study will be summarized by descriptive statistics. We do not expect that there 
will be enough statistical power to conduct a formal hypothesis test for these 12 subjects 
alone. 
Change from baseline in DXA in subjects enrolling in the long-term extension study will be 
measured at the end of one year of therapy in the extension study (primary endpoint for the 
extension study). DXA changes will be summarized by mean (SD) and 95% confidence 
interval. 
The proportions of subjects obtaining ≥25% bone marker changes from baseline will be 
estimated with their 95% Agresti-Coull confidence intervals. 
Baseline characteristics will be summarized by descriptive statistics. 
Subjects may drop out for a variety of reasons including expected or unexpected AEs of the 
therapy.  If a subject drops out before visit 5 they will be replaced with a new subject.  This 
decision is based on having adequate data on the primary outcome of bone turnover 


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markers to interpret the study results.  If a subject drops out after visit 5, they will not be 
replaced. 
9. 0 – PRIVACY/CONFIDENTIALITY ISSUES 
Data sources will include paper and electronic medical records and test results. Data will be 
recorded on paper, desktop computers, and laptop computers. The Principal Investigators, 
Research Coordinators, Co- Investigators, Biostatistics, and Governmental Agencies will 
have access to the individually identifiable data. This data will be safeguarded by locking 
storage cabinets and office doors, keeping information in areas with limited public access, 
password protecting computers and files, and regular back-ups of electronic data. Data will 
be retained indefinitely. When confidential data needs to be discarded, paper records will be 
shredded, CD’s and diskettes will be destroyed, and data will be permanently deleted from 
computers. When possible, only de-identified data will be shared. If identifiable data will be 
shared, it will be done by fax in a secured area, on a shared drive with password protection, 
or by personal delivery by authorized research personnel. 
10.0 FOLLOW-UP AND RECORD RETENTION 
10.1 SUBJECT COMPENSATION 
Subjects will be reimbursed for travel (driving) and parking to study visits and will receive an 
honoraria for each on site visit as follows: $100 for the first visit; $40 for subsequent visits; and 
$25 for local sample collection.  
10.2 RECORD RETENTION 
All records will be kept for a minimum of 15 years. 
11.0 – REFERENCES CITED 
1. 
Cleiren, E., et al., Albers-Schonberg disease (autosomal dominant osteopetrosis, type II) 
results from mutations in the ClCN7 chloride channel gene. Hum Mol Genet, 2001. 
10(25): p. 2861-7. 
2. 
Waguespack, S.G., et al., Chloride channel 7 (ClCN7) gene mutations and autosomal 
dominant osteopetrosis, type II. J Bone Miner Res, 2003. 18(8): p. 1513-8. 
3. 
Johnston, C.C., Jr., et al., In vivo measurement of bone mass in the radius. Metabolism, 
1968. 17(12): p. 1140-53. 
4. 
Waguespack, S.G., et al., Autosomal dominant osteopetrosis: clinical severity and 
natural history of 94 subjects with a chloride channel 7 gene mutation. J Clin Endocrinol 
Metab, 2007. 92(3): p. 771-8. 
5. 
Benichou, O.D., J.D. Laredo, and M.C. de Vernejoul, Type II autosomal dominant 
osteopetrosis (Albers-Schonberg disease): clinical and radiological manifestations in 42 
patients. Bone, 2000. 26(1): p. 87-93. 
6. 
Alam, I., et al., Interferon Gamma, but not Calcitriol Improves the Osteopetrotic 
Phenotypes in ADO2 Mice. J Bone Miner Res, 2015. 
7. 
Key, L., et al., Treatment of congenital osteopetrosis with high-dose calcitriol. N Engl J 


Protocol Version 3 
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30 
 
 
Med, 1984. 310(7): p. 409-15. 
8. 
Alam, I., et al., Generation of the first autosomal dominant osteopetrosis type II (ADO2) 
disease models. Bone, 2014. 59: p. 66-75. 
9. 
Key, L.L., Jr., et al., Long-term treatment of osteopetrosis with recombinant human 
interferon gamma. N Engl J Med, 1995. 332(24): p. 1594-9. 
10. 
Madyastha, P.R., et al., IFN-gamma enhances osteoclast generation in cultures of 
peripheral blood from osteopetrotic patients and normalizes superoxide production. J 
Interferon Cytokine Res, 2000. 20(7): p. 645-52. 
11. 
Kamolmatyakul, S., W. Chen, and Y.P. Li, Interferon-gamma down-regulates gene 
expression of cathepsin K in osteoclasts and inhibits osteoclast formation. J Dent Res, 
2001. 80(1): p. 351-5. 
12. 
Fox, S.W. and T.J. Chambers, Interferon-gamma directly inhibits TRANCE-induced 
osteoclastogenesis. Biochem Biophys Res Commun, 2000. 276(3): p. 868-72. 
13. 
Cheng, J., et al., Molecular mechanisms of the biphasic effects of interferon-gamma on 
osteoclastogenesis. J Interferon Cytokine Res, 2012. 32(1): p. 34-45. 
14. 
Darden, A.G., et al., Osteoclastic superoxide production and bone resorption: stimulation 
and inhibition by modulators of NADPH oxidase. J Bone Miner Res, 1996. 11(5): p. 671-
5. 
15. 
Rodriguiz, R.M., L.L. Key, Jr., and W.L. Ries, Combination macrophage-colony 
stimulating factor and interferon-gamma administration ameliorates the osteopetrotic 
condition in microphthalmic (mi/mi) mice. Pediatr Res, 1993. 33(4 Pt 1): p. 384-9. 
16. 
Gao, Y., et al., IFN-gamma stimulates osteoclast formation and bone loss in vivo via 
antigen-driven T cell activation. J Clin Invest, 2007. 117(1): p. 122-32. 
17. 
Kim, J.W., et al., Effect of interferon-gamma on the fusion of mononuclear osteoclasts 
into bone-resorbing osteoclasts. BMB Rep, 2012. 45(5): p. 281-6. 
18. 
Patriquin, H. and P. Robitaille, Renal calcium deposition in children: sonographic 
demonstration of the Anderson-Carr progression. AJR Am J Roentgenol, 1986. 146(6): 
p. 1253-6. 
19. 
Devane, J.G., M.L. Martin, and M.A. Matson, A short 2 week dose titration regimen 
reduces the severity of flu-like symptoms with initial interferon gamma-1b treatment. 
Curr Med Res Opin, 2014. 30(6): p. 1179-87. 
20. 
Maluish, A.E., et al., The determination of an immunologically active dose of interferon-
gamma in patients with melanoma. J Clin Oncol, 1988. 6(3): p. 434-45. 
21. 
Moyer-Mileur, L.J., J.L. Quick, and M.A. Murray, Peripheral Quantitative Computed 
Tomography of the Tibia: Pediatric Reference Values. Journal of Clinical Densitometry, 
2008. 11(2): p. 283-294. 
22. 
Ashby, R.L., et al., A reference database for the Stratec XCT-2000 peripheral 
quantitative computed tomography (pQCT) scanner in healthy children and young 
adults aged 6–19 years. Osteoporosis International, 2009. 20(8): p. 1337-1346. 
23. 
Swinford, R.R. and S.J. Warden, Factors affecting short-term precision of 
musculoskeletal measures using peripheral quantitative computed tomography (pQCT). 
Osteoporos Int, 2010. 21(11): p. 1863-70. 
24. 
Warden, S.J., et al., Racial differences in cortical bone and their relationship to 
biochemical variables in Black and White children in the early stages of puberty. 


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Osteoporos Int, 2013. 24(6): p. 1869-79. 
25. 
Kroger, H., et al., Bone densitometry of the spine and femur in children by dual-energy x-
ray absorptiometry. Bone Miner, 1992. 17(1): p. 75-85.