The Use of Novel Therapies to Reconstitute Blood Cell Production and Promote Organ Performance Using Bone Marrow Failure as a Model: a Pilot, Phase I/II Study of the Amino Acid Leucine in the Treatment of Patients With Transfusion-Dependent Diamond Blackfan Anemia
Phase: PHASE1
Conditions: Diamond Blackfan Anemia, Blackfan Diamond Syndrome, DBA, Congenital Hypoplastic Anemia, Pure Red Cell Aplasia
Sponsor: Northwell Health

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The Use of Novel Therapies to Reconstitute Blood Cell Production and Promote 
Organ Performance, using Bone Marrow Failure as a Model: A Pilot, Phase I/II 
Study of the Amino Acid Leucine in the Treatment of Patients with Transfusion-
Dependent Diamond Blackfan Anemia 
     
Proposal 09196004 
Lead Principal Investigator 
Adrianna Vlachos, MD 
Associate Professor of Pediatrics  
Hofstra North Shore-LIJ School of Medicine   
Cohen Children’s Medical Center of New York 
Hematology/Oncology and Stem Cell Transplantation 
The Feinstein Institute for Medical Research 
269-01 76th Avenue, Rm. 255 
New Hyde Park, NY 
Phone: (516) 562-1506 Fax: (516) 562-1599 
E-mail: avlachos@nshs.edu 
 
Study Team Members 
Monica Bessler, MD, PhD 
George Buchanan, MD  
Steven R Ellis, PhD 
Bertil Glader, MD, PhD 
Helge Hartung, MD 
Jeffrey M. Lipton, MD, PhD 
Zora Rogers, MD 
Colin Sieff, MD 
 
Central Study Coordinator 
Eva Atsidaftos, MA, CCRA  
The Feinstein Institute for Medical Research 
350 Community Drive, Rm. 3146 
Manhasset, NY 11030 
 
 
 
 
 
 
Phone:   
516-562-1505 
 
 
 
 
 
 
Fax:   
516-562-1599 
 
 
 
 
 
 
E-mail:  
eatsidaf@nshs.edu 
 
Protocol revised dates: v.2, 12/3/12; v.3, 6/7/13; v.4 8/27/13, v. 5.0 10/03/2013, v. 6.0 10/1/14 
 
This study is being sponsored by the Department of Defense (DOD) Telemedicine and Advanced Technology 
Research Center (TATRC). The protocol will be conducted in accordance with the protocol submitted to and 
approved by the USAMRMC ORP HRPO and will not be initiated until written notification of approval of 
the research project is issued by the USAMRMC ORP HRPO.   
 
This document contains confidential information. It is provided for the sole use of the research team, IRB or 
independent ethics committee, and regulatory authorities. By accepting this document, you agree to maintain the 
information as confidential and to use it only for the purpose of conducting the study. 

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The Use of Novel Therapies to Reconstitute Blood Cell Production and Promote Organ 
Performance, using Bone Marrow Failure as a Model: 
 
A Pilot, Phase I/II Study of the Amino Acid Leucine in the Treatment of Patients with 
Transfusion-Dependent Diamond Blackfan Anemia 
 
 
 
Signature Page 
 
Medical Coordinating Center Principal Investigator Approval. 
 
Signature: ______________________  Date: _____________________ 
 
 
 
Adrianna Vlachos, MD 
 
 
Sub Site Principal Investigator Approval: 
 
Signature: _______________________Date: _____________________ 
 
 
 
 
Please fax this page back to Primary Coordinating Site, prior to site initiation. 
Attn: Eva Atsidaftos 
F) 516-562-1599 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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TABLE OF CONTENTS                                                                                      PAGE  
      
1.0 STUDY TEAM    ………………………………………………………………    6 
 
2.0 STUDY TEAM ROLES AND RESPONSIBILITIES ………………………...    8 
    
 
3.0  STUDY ABSTRACT  ………………………………………………………....  9 
 
4.0  OBJECTIVES  …………………………………………………………………  9 
4.1 Primary.…….………………………………………………….………..   9 
4.2 Secondary ……………………………………………………………..   10 
 
5.0 BACKGROUND ………………………………………………………………   10 
5.1 Diamond Blackfan anemia (DBA) …………………………………… 
 10 
5.2 Review of Experimental Treatment Modalities ……………………… 
 10 
 
 
6.0 DRUG INFORMATION ………………………………………………….….. 
11 
6.1   Leucine Preparation …………………………………………………. 12 
6.2   Pharmacokinetics …………………………………………………… 
12 
6.3   Mechanism of Action/Pharmacology ……………………………..…  12 
6.4   Contraindications …………………………………………………….. 13 
6.5   Adverse Effects ………………………………………………………   13 
6.6   Drug Interactions ……………………………………………………  13 
6.7   Pregnancy/Lactation ………………………………………………… 13 
6.8   Overdosage …………………………………………………………. 
13 
6.9   Leucine Levels ……………………………………………………… 
14 
 
7.0 SCIENTIFIC AND CLINICAL RATIONALE AND SIGNIFICANCE ……. 
14 
 
8.0 STUDY DESIGN …………………………………………………………….. 
14 
 
8.1 Primary Endpoints ………………………………………………….. 
14 
 
8.2  Secondary Endpoint ………………………………………………… 
14 
 
9.0 PATIENT ELIGIBILITY ……………………………………………………… 15 
 
9.1 Inclusion Criteria …………………………………………………….. 15 
 
9.2 Exclusion Criteria …………………………………………………… 17 
 
10.0 SUBJECT RECRUITMENT ………………………………………………… 17 
 
11.0 TREATMENT OF SUBJECTS ……………………………………………… 18  

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 11.1 Informed Consent, Eligibility Screening and Subject Enrollment…….  18  
11.2 IRB Approval ……………………………………………………….. 
20 
11.3  Diamond Blackfan Anemia Registry (DBAR) …..…………………... 20 
 
12.0 DATA COLLECTION ……………………………………………………... 
20 
12.1 Enrollment Form ….………………………………………………… 
20 
12.2 Case Report Form …………………………………………………… 20 
    
12.3 During Treatment …………………………………………………… 
21  
12.4 Data Submission ……………………………………………………. 
21 
12.5 Data Storage ………………………………………………………… 
22 
 
13.0 STUDY DRUG ADMINISTRATION ……………………………………... 
22 
 
13.1 Study Drug – Leucine ……………………………………………….. 23 
 
13.2 Transfusion Therapy during Study Drug Administration …………… 23 
 
13.3 Schedule of Evaluations ……………………………………………… 23 
 
13.4 Grading of Adverse Events …………………………………………... 24 
 
13.5  Dose Modifications for Adverse Events ……………………………… 24  
 
14.0 EVALUATION CRITERIA ………………………………………………… 
24 
 
14.1  On-Study Response Criteria …………………………………………. 24 
 
15.0 WITHDRAWAL AND OFF STUDY CRITERIA …………………………. 
24 
 
15.1 Subject’s Choice ……………………………………………………… 24 
 
15.2 PI Decision …………………………………………………………… 25 
 
15.3 Discontinuation for Toxicity ………………………………………… 25 
 
15.4 Discontinuation for Non-Adherence ………………………………… 25 
 
16.0 BIOSTATISTICAL CONSIDERATIONS …………………………………. 
25 
16.1 General Study Objectives …………………………………………….. 25 
16.2 Specific Statistical Aims of the Study (for the Data Analysis)...……... 25 
16.3 Endpoint Variables …………………………………………………… 25 
16.4 Statistical Methods …………………………………………………… 26  
16.5 Sample Size Considerations ………………………………………….. 26 
 
17.0 Research MONITOR REQUIREMENT …………………………………. 
27 
 
18.0 SAFETY MONITORING …………………………………………………... 
27 
18.1 Reporting of Adverse Events and Unanticipated Problems ………… 
27 
18.2 Clinical Research Monitoring Services …………………………….. 
27 
 
   18.3 Reporting to the U.S. Army Medical Research and Materiel Command 28 
 
   18.4 SAE Definitions ……………………………………………………… 28  
18.5 Assessment Terms for SAE and Causal Relationship to Study Drug … 28 
 
   18.6 Grades of Severity …………………………………………………… 29  
18.7 Non-Serious Adverse Event (AE) Reporting ……………………….. 
29 
 
19.0 HUMAN SUBJECT PROTECTION ………………………………………... 30  

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 19.1 Rationale for Subject Selection ………………………………………. 30 
 19.2 Participation of Children ……………………………………………... 30   
 19.3 Risks and Discomforts ……………………………………………….. 30 
 19.4 Risks in Relation to Benefit for Adult and Pediatric Subjects ……….. 30   
 
20.0 COMPLIANCE ……………………………………………………………… 31 
 
21.0 REFERENCES ……………………………………………………………… 
31  
 
APPENDIX A - Diagnosis of Diamond Blackfan Anemia 
APPENDIX B - Informed Consent/Assent  
APPENDIX C- Eligibility Screening Checklist  
APPENDIX D - Enrollment Form  
APPENDIX E - Leucine Dosage Calculation Table  
APPENDIX F-  Leucine Dose Assignment Form and Supply Request 
APPENDIX G-  Case Report Form  
APPENDIX H - Amino Acid Levels – Collection and Shipping Instructions (to be determined) 
APPENDIX I-  Severe Adverse Event (SAE) Reporting Form  
 

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1.0 STUDY TEAM 
Lead Principal Investigator 
Adrianna Vlachos, MD  
 
Hofstra North Shore-LIJ School of Medicine 
Cohen Children’s Medical Center of New York 
Hematology/Oncology and Stem Cell Transplantation 
The Feinstein Institute for Medical Research 
269-01 76th Avenue 
New Hyde Park, NY 
Phone:  
516-562-1504 
Fax:  
516-562-1599 
E-mail:  
avlachos@nshs.edu 
Study Team Members 
Monica Bessler, MD, PhD 
Children’s Hospital of Philadelphia 
3615 Civic Center Blvd. 
Philadelphia, PA 19104 
Phone: 267-426-8782 
E-mail: besslerm@email.chop.edu 
 
George Buchanan, MD  
University of Texas Southwestern Medical Center 
Dept. of Pediatrics 
5323 Harry Hines Blvd. 
Dallas, TX 75235-9063 
Phone: 
214-648-8594 
Fax:  
214-648-3122 
E-mail:  
George.buchanan@utsouthwestern.edu 
Steven R Ellis, PhD 
University of Louisville 
Dept. of Biochemistry and Molecular Biology 
Phone: 
502-852-5222 
Fax:  
502-852-6222 
E-mail: 
srellis@louisville.edu 
Bertil Glader, MD, PhD 
Stanford University 
1000 Welch Rd. Ste. 300 
Palo Alto, CA 94305 
Phone: 
650-723-5535  
Fax#: 
650-723-5231 
E-mail:  
bglader@stanford.edu 
Helge Hartung, MD 
 
 
 
 
 
 
 
 
 
 
         
Children’s Hospital of Philadelphia 
34th Street and Civic Center Blvd 

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Philadelphia, PA 19104 
Phone: 267-425-2126 
Fax: 215-590-3992 
E-mail: hartungh1@email.chop.edu 
 
Jeffrey M. Lipton, MD, PhD 
Director, Hematology/Oncology and Stem Cell Transplantation  
Cohen Children’s Medical Center of NY 
269-01 76th Avenue, Room 255 
New Hyde Park, NY  11040 
Phone:   
718-470-3703 
Fax:   
718-343-4642  
E-mail:  
jlipton@nshs.edu 
 
Zora Rogers, MD  
University of Texas Southwestern Medical Center 
Dept. of Pediatrics 
5323 Harry Hines Blvd. 
Dallas, TX 75235-9063 
Phone: 
214-648-8594 
Fax:  
214-648-3122 
E-mail:  
zora.rogers@utsouthwestern.edu 
Colin A. Sieff, MB.BCH., FRCPath, 
Director, Bone Marrow Failure Program,  
Attending in Hematology, 
Boston Children’s Hospital, 
300 Longwood Ave., 
Boston, MA 02115 
Phone:  
617-919-4241 or 617- 355-8246 
Fax: 
 
617-730-0934 
E-mail:  
 c.sieff@childrens.harvard.edu 
 
Study Coordinator 
Eva Atsidaftos, MA, CCRA 
The Feinstein Institute for Medical Research 
350 Community Drive, Rm. 3146 
Manhasset, NY 11030 
Phone:  
516-562-1505  
Fax: 
 
516-562-1599 
E-mail:  
eatsidaf@nshs.edu 
 
 
 
 
 
 

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2.0 STUDY TEAM ROLES AND RESPONSIBILITIES 
The Study Team consists of the Lead Principal Investigator (Adrianna Vlachos, MD - Cohen 
Children’s Medical Center of New York, New Hyde Park, NY and The Feinstein Institute for 
Medical Research, Manhasset, NY) and five Co-Investigators from the four Diamond Blackfan 
Anemia Resource Centers (Jeffrey M. Lipton, MD, PhD - Cohen Children’s Medical Center of 
New York, New Hyde Park, NY; George Buchanan, MD and Zora Rogers, MD - University of 
Texas Southwestern Medical Center, Dallas, TX; Bertil Glader, MD, PhD - Stanford University, 
Palo Alto, CA; Colin Sieff, MD - Dana Farber Cancer Institute, Boston, MA) established in 
cooperation with the Centers for Disease Control and Prevention.  This protocol was initially 
discussed at the annual Diamond Blackfan Anemia International Consensus Conference and 
three more Co-Investigators were added to the Study Team at that time Steven Ellis, PhD is also 
a Study Team member.  He is a researcher in ribosome biology and will be providing assistance 
in the scientific nature of this study.  He has been involved in providing the background for the 
study design. 
 
The lead PI will be responsible for submitting documents to the Institutional Review Board of 
the primary institution and the USAMRMC ORP HRPO, verifying IRB approvals at all sub-
sites, recruiting DBA patients, verifying eligibility, consenting patients to the study, overseeing 
adherence to the protocol specifics, monitoring for drug toxicity and safety, evaluating feasibility 
and outcome and analyzing data and reporting results after the study is complete.  
The Coordinating Center will be at The Feinstein Institute for Medical Research (FIMR) and the 
Central Study Coordinator will also be at the lead PI’s institution. The Study Coordinator will be 
responsible for assisting the lead PI in the above-mentioned delegated roles.  In addition the 
Central Study Coordinator will be responsible for assisting participating institutions in IRB 
submission of the protocol, drug shipment to the participating physicians/institutions, collecting 
Case Report Forms (CRFs) from all sites and assisting with their completion, as well as tracking 
protocol compliance at the participating sites. She will also assist in the analysis of the data. 
All Study Team members, except for Steven Ellis, PhD, will be recruiting patients on to the 
study from their surrounding areas and, as such, will be engaged in human subjects’ research.  
They will serve as local site PIs.  Thus, they will be responsible for obtaining IRB approval and 
the conduct of the study at their institutions. They will also be responsible for submission of the 
required CRFs to the Central Study Coordinator.  The Study Team will also review the data at 
completion of the study and assist in the analysis.  They will also serve as authors in reporting of 
the data. 
 
The funding for this study allows for up to 17 institutions to enroll 50 subjects. Aside for the PI’s 
and Co-I’s institutions, other collaborating institutions will be added once they are identified by 
their request to the Central Study Coordinator to enroll subjects. A designee at these participating 
institutions will be added as Co-Investigator and will be responsible for subject recruitment, 
obtaining IRB approval and completion and submission of CRFs to the Central Study 
Coordinator.   
 
Site Information: The Feinstein Institute for Medical Research will be the main location of 
research activity.  Subjects will also be enrolled at the participating institutions and data will be 
submitted to The FIMR. 

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Type of Research: Drug 
 
3.0 STUDY ABSTRACT 
Diamond Blackfan anemia (DBA) is a rare inherited pure red cell aplasia.  Over the past ten 
years mutations have been described in genes encoding both the small and large ribosome 
associated proteins. There is wide variability in clinical and biologic features, familial history, 
and therapeutic responses. Currently standard therapy includes corticosteroids, red cell 
transfusions or stem cell transplantation.  Approximately 80% of patients have an initial response 
to corticosteroids.  Of these patients approximately half will fail to achieve a taper to a tolerable 
steroid dose and will need to discontinue steroid therapy.  These patients and the 20% who do 
not respond initially to steroid therapy must be maintained on a chronic red cell transfusion/iron 
chelation program.  Chronic transfusion/chelation therapy has considerable treatment-related 
morbidity, most notably iron overload related organ dysfunction. 
 
The branched chain amino acids (BCAA) are leucine, isoleucine, and valine. These amino acids 
are "essential" as they are not stored and must be supplied by a dietary source.  These amino 
acids are utilized by muscle for energy and are the only amino acids that are not metabolized by 
the liver. Branched-chain amino acid therapy has been used in patients with hepatic 
encephalopathy, pre-operative care and post-operative recovery, burns, post-trauma, cirrhosis, 
phenylketonuria, renal insufficiency, sepsis, and spinocerebellar degeneration; however their role 
is not clear.  Leucine is one of three BCAA that have been studied extensively over the years for 
the ability to promote muscle mass when given orally.  Dietary supplementation with branched 
chain amino acids has been used by athletes to stimulate muscle mass and increase endurance 
and, in clinical settings, to reverse muscle deterioration.  Leucine is sold over-the-counter as a 
nutritional supplement and L-Leucine has been Generally Recognized as Safe (GRAS), 
consistent with Section 201(s) of the Federal Food, Drug, and Cosmetic Act.  
 
Leucine has been administered successfully in one patient reported in the literature with DBA. A 
complete response was associated with its administration.  In unpublished data 5 more patients 
have been placed on a leucine trial with partial responses in 4 of the 5 patients without any 
toxicity.  The hypothesis for the molecular basis for the possible use of the amino acid leucine as 
a therapeutic agent for DBA is based upon the observations in skeletal muscle that leucine 
supplementation can upregulate components of the translational machinery.  These components 
include ribosomal proteins, whose haploinsufficiency appears to be the underlying cause of 
DBA. This pilot study will test the feasibility of administration of leucine in a larger group of 
patients with DBA.  Subjects will also be monitored for clinical hematologic response. Leucine 
levels and total amino acid profiles will be ascertained in an attempt to correlate response with 
baseline levels.  Responders will be evaluated with regard to phenotypic characteristics that 
might distinguish them prospectively from other patients. 
 
4.0 OBJECTIVES 
4.1    Primary  
4.1.1 To determine the feasibility of administering the amino acid leucine and to determine the 
pharmacokinetics of leucine administration in patients with Diamond Blackfan anemia 
(DBA) who are red cell transfusion-dependent.  

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4.1.2 To determine the efficacy of leucine to produce a hematologic response in transfusion-
dependent DBA patients.  
4.1.3 To determine the safety of leucine administration in transfusion-dependent DBA patients. 
 
4.2  Secondary  
4.2.1 To determine if patients with DBA are deficient in branched chain amino acids. 
 
5.0 BACKGROUND   
5.1 Diamond Blackfan anemia (DBA) 
Diamond Blackfan anemia is a rare inherited pure red cell aplasia.  Over the past ten years 
mutations have been described in genes encoding both the small and large ribosome associated 
proteins (1-6). There is wide variability in clinical and biologic features, familial history, and 
therapeutic responses. Currently standard therapy includes corticosteroids, red cell transfusions 
or stem cell transplantation (7, 8).  Approximately 80% of patients have an initial response to 
corticosteroids. Often patients require chronic, potentially life-long treatment, which is fraught 
with a variety of short-term and long-term complications including osteoporosis, Cushingoid 
features, diabetes, hypertension, severe growth retardation, aseptic necrosis, glaucoma, and 
cataracts.  Twenty percent of patients are initially unresponsive to corticosteroid treatment and 
about half of the initial responders cannot be tapered to a safe and effective dose.  Other patients 
may become refractory to corticosteroids after a period of responsiveness. These patients must be 
maintained on a chronic red cell transfusion/iron chelation program.  Long-term red cell 
transfusions will result in iron overload and iron chelation therapy is required.  With inadequate 
chelation, iron overload will inevitably lead to organ damage, especially of the heart and liver, 
and may result in death.   
 
 
 
The alternative to corticosteroids or red blood cell transfusions and chelation is hematopoietic 
stem cell transplantation. Hematopoietic allogeneic stem cell transplant with a histocompatible 
donor has been proven to be effective therapy and has been performed successfully for a select 
group of patients. The DBAR experience has been recently updated (9, 10).  However, because 
of remissions (sustained physiologically acceptable erythropoiesis for > 6 months) in up to 20% 
of DBA patients, stem cell transplantation, regardless of donor type, is not consistently offered to 
corticosteroid-dependent or transfusion-dependent DBA patients. For many patients, the lack of 
a suitable donor does not allow stem cell transplantation as a therapeutic option.  In addition, 
HLA-matched unrelated donor transplants have been associated with mortality and morbidity.   
 
5.2 Review of Experimental Treatment Modalities  
Historically the lack of truly effective and safe therapy has prompted the investigation of a 
number of other agents. These modalities have achieved only anecdotal success. 
5.2.1 High-dose Corticosteroids  
Several small published trials have evaluated the efficacy of high-dose corticosteroids in those 
patients failing conventional therapy (11-13). In total, 10 of 29 patients have become transfusion 
independent after either intravenous methylprednisolone or oral prednisone/intravenous 
methylprednisolone.  One patient had a partial response and 5 patients had a transient response.  
Varied responses at different doses have been reported, and the cumulative data suggest that 
extremely high doses may be necessary. Responses were not always durable upon 

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discontinuation of therapy and death from infection and the long-term side effects of high-dose 
steroids have justifiably led to the abandonment of this approach. 
 
5.2.2 Cyclosporine A and Antithymocyte Globulin  
Cyclosporine A (CSA) has been studied in DBA patients, but has demonstrated limited success. 
By combining several reports (14-21), a total of 30 patients tried CSA alone (10 patients) or in 
combination with steroids (20 patients).  Only two of the CSA alone patients had a sustained 
response.  Half of the patients treated with CSA plus steroid had a response but it was transient 
and failed once steroids were discontinued.  Antithymocyte globulin (ATG) has also had very 
limited use in DBA. A few case reports of patients with acquired pure red cell aplasia have 
demonstrated mixed responses (22-24). A Phase II NIH-sponsored protocol [98-H-0144] 
combining CSA and ATG closed due to poor responses. 
   
5.2.3 Intravenous Immunoglobulin  
Intravenous immunoglobulin (IVIG) has been used in very few cases and has had minimal 
success in patients who were refractory to conventional-dose corticosteroids. In one case, a 
patient was able to achieve transfusion independence, although continued to require monthly 
IVIG infusions in order to sustain a remission (25). In a few other cases, only limited therapeutic 
benefit was observed (26-28).  This modality is not recommended.   
 
5.2.4 Interleukin-3   
Interleukin-3 (IL-3) has been studied in a number of trials. An increase in erythroid burst 
forming unit colonies and an improved response to erythropoietin with the addition of IL-3 to in 
vitro cultures of bone marrow from DBA patients (29) prompted the investigation of IL-3 in 
clinical trials.  Over 100 patients have been treated with an approximate 10% response (30-35). 
In one report two of the patients who responded, required discontinuation of therapy due to deep 
venous thrombosis.  Others required discontinuation of therapy because of side effects (urticaria, 
wheezing, local reactions). The reason for the disparity between the laboratory results and the 
clinical trials is unclear. IL-3 is no longer available for clinical use. 
 
5.2.5 Metoclopramide   
Metoclopramide has also shown to be effective in DBA.  Abkowitz et al. (36), described a 33% 
hematologic response rate in a small group of patients with DBA using metoclopramide, an 
inexpensive, commonly used dopamine antagonist that induces the release of prolactin from the 
pituitary gland, thereby increasing prolactin levels.  It was proposed that prolactin likely 
improves erythropoiesis by stimulating cells in the microenvironment of erythroblasts.  
Unfortunately other studies in the US and Europe did not confirm these responses but showed a 
10% response rate (37, 38). 
 
In summary none of the agents described have been shown to be unequivocally effective and 
some carry significant treatment related toxicity.  Thus a search for effective agents based upon 
the emergence of new scientific data is ongoing. 
 
6.0 DRUG INFORMATION 
The branched chain amino acids (BCAA) are leucine, isoleucine, and valine (39, 40). These 
amino acids are utilized by muscle for energy and are the only amino acids that are not 

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metabolized by the liver. Branched chain amino acid therapy has been used in patients with 
hepatic encephalopathy, bronchitis, preoperative care, cirrhosis, phenylketonuria, renal 
insufficiency, sepsis, sleep disorders, and spinocerebellar degeneration; however their role is not 
clear.  Leucine is one of three BCAA that have been studied extensively over the years for the 
ability to promote muscle mass when given orally.  Dietary supplementation with branched chain 
amino acids has been used by athletes to stimulate muscle mass and increase endurance and in 
clinical settings to reverse muscle deterioration (41).  These latter clinical settings include 
patients undergoing post-operative recovery, in burn wards, post-trauma and cancer therapy.  
The potential benefit of BCAA enrichment, especially leucine, is still not clear however some 
small controlled studies have shown improved nitrogen balance and decreases in postoperative 
morbidity (42).  Research on BCAA supplementation as an adjunct therapy in these clinical 
settings continues.     
 
Branched chain amino acid products vary widely in their composition, ranging from 200-300mg 
of each BCAA daily to 2-5 grams of each daily.  The total BCAA requirement for young adults 
and children 6-10 years of age is 147 mg/kg/day with increased doses for neonates and infants 
(46).  While there is little indication that supplementation with BCAA produce significant results 
in healthy individuals, there are a number of targeted clinical uses. BCAA may support liver 
health in patients with liver disease such as cirrhosis and hepatic encephalopathy (47, 48).  Also, 
patients suffering from amyotrophic lateral sclerosis may show improvement after using BCAA 
(49).  BCAA may help support health and recovery in patients who have experienced trauma, 
extreme physical stress, kidney failure, and burns (50).  BCAA may aid in recovery after surgery 
(51).  
 
6.1 Leucine Preparation 
Manufacturer:  Ajinomoto Aminoscience LLC  
Leucine is an amino acid available in many over-the-counter nutritional supplements. Leucine is 
a white or almost white, odorless crystalline powder or shiny flakes. Leucine can be administered 
orally or intravenously and can be supplied as tablet, capsule, powder, or solution.  This pilot 
study will only utilize commercially available, pharmaceutical grade L-leucine from Ajinomoto 
Aminoscience LLC, Raleigh, NC.  The leucine will be supplied in capsular form as 250 
milligrams per capsule with excipient approved for use being vegetable cellulose.   
 
6.2 Pharmacokinetics 
6.2.1 Distribution Sites   
Amino acids are widely distributed throughout the tissues of the body (54). 
 
6.2.2 Metabolism Sites   
Metabolism sites include all body tissues. Factors that influence the metabolism of amino acid 
include injury or stress which increases metabolism and liver dysfunction which decreases 
metabolism. Sepsis increases amino acid metabolism, while renal dysfunction decreases 
metabolism (55).  
 
6.3 Mechanism of Action/Pharmacology  
Various mechanisms of action for branched chain amino acids have been proposed. During 
exercise, supplementation appears to exert an anabolic and protein-sparing response. The 

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branched chain amino acids also appear to be a preferred energy substrate for muscles, at least in 
cirrhotic patients. The administration of branched chain amino acid resulted in an anabolic effect 
on protein metabolism during recovery following exercise in human subjects. 
 
6.4 Contraindications 
Hypersensitivity to branched chain amino acids, anuria, and inborn errors of amino acid 
metabolism, especially those that involve branched chain amino acid metabolism, such as maple 
syrup urine disease and isovaleric acidemia are contraindications.  
 
6.5 Adverse Effects 
Toxicity from orally administered leucine is extremely low. Ingestion of even large quantities 
produces only minor gastrointestinal symptoms such as nausea and/or vomiting.  No consistent 
evidence of toxicity has been linked to leucine supplements. In healthy subjects, no adverse 
events have been reported by subjects consuming oral leucine for a 6-week period (56). In 
another study, healthy elderly men were supplemented with 2.5 g of leucine at each main meal 
for a period of 3 months without any reported adverse effects (44). Scarna et al. (45) has reported 
that no adverse effects are associated with repeated oral administration of up to 60 g/day of 
BCAAs (providing 24 g/day of leucine) in patients with bipolar disorder. The body of evidence 
on oral administration of leucine in humans indicates that it can be consumed in considerable 
amounts without adverse effects. There are no Dose-Limiting Toxicities known with oral 
Leucine. There is also no defined Maximum Tolerated Dose and no Recommended Phase II 
Dose reported for this supplement. 
 
Thrombophlebitis, thrombocytopenia, hyperammonemia, nausea, flushing, and fever have been 
reported with administration of intravenous amino acids, but not with oral supplementation. 
Leucine aggravates a rare genetic condition called Maple Syrup Urine Disease and can cause 
delirium and life-threatening neurologic symptoms in these patients. It has been thought that 
higher levels of leucine taken over a prolonged period of time may contribute to pellagra. 
Pellagra is defined as a disease that results from a lack of niacin (vitamin B3) and tryptophan 
(another essential amino acid) but also rarely from an excess of leucine. Its symptoms include 
diarrhea, dermatitis (dry skin rash), dementia and even death in its extreme form. There have 
been reports of severe neurologic damage and death when given intravenously (IV) in high 
doses, which will not be done in this study.   
 
6.6 Drug Interactions  
No drug interaction data are available. 
 
6.7 Pregnancy/Lactation 
Excessive leucine administration interferes with embryo development in rats. Scientific evidence 
for the safe use of branched chain amino acids during pregnancy and lactation is not available as 
there are no human data.  
 
6.8 Overdosage 
Toxicity from orally administered amino acids is extremely low. A minimum toxic dose has not 
been established.  
 

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6.9 Leucine Levels 
Normal reference blood levels of leucine (57) by age are:  
Premature neonate, 1 day: 0.92 ± 0.33 mg/dL (70 ± 25 micromol/L)  
Neonate, 1 day: 0.62-1.43 mg/dL (47-109 micromol/L) 
Infants 1 to 3 months: 1.36 ± 0.39 mg/dL (104 ± 30 micromol/L)  
Infants 9 to 24 months: 0.59-2.03 mg/dL (45-155 micromol/L)  
Children 3 to 10 years: 0.73-2.33 mg/dL (56-178 micromol/L)  
Children 6 to 18 years: 1.03-2.28 mg/dL (79-174 micromol/L)  
Adults: 0.98-2.29 mg/dL (75-175 micromol/L) 
 
7.0 SCIENTIFIC AND CLINICAL RATIONALE AND SIGNIFICANCE 
The molecular basis for the use of the amino acid leucine as a therapeutic agent for Diamond 
Blackfan anemia is based upon the observations in skeletal muscle that leucine supplementation 
can upregulate components of the translational machinery (53).  These components include 
ribosomal proteins, whose haploinsufficiency appears to be the underlying cause of DBA (5). 
Successful administration of leucine has been reported in the literature in one patient with DBA 
(52). A complete response was observed after approximately 6 months of use.  In unpublished 
data reported by the same investigator at conferences 5 more patients were placed on the 
proposed dose of leucine for this trial (2100 mg/M2/day in 3 divided doses, well below the 
highest reported safe dose in humans reported in the GRAS Exemption Claim) with partial 
responses in 4 of the 5 patients – 2 patients were able to reduce their steroid dose and 2 patients 
were able to lengthen their time between transfusions; 1 patient did not have a demonstrated 
difference in transfusion frequency.  No toxicity was reported in these patients (personal 
communication, Pospisilova).  This pilot study will test the feasibility of administration of 
leucine to DBA patients who are transfusion-dependent. The patients will be monitored for 
hematologic response.  Although there is no pathophysiologic reason to expect incremental 
toxicity to leucine in patients with DBA, patients will be carefully monitored for side effects.  
Leucine levels and total amino acid profiles will be ascertained in an attempt to correlate 
response with baseline levels. Where available, responders will also be evaluated with regard to 
phenotypic and genotypic characteristics that might distinguish them prospectively from other 
patients.  
 
8.0 STUDY DESIGN   
This study will be conducted as a pilot, phase I/II, multi-center clinical trial.   
8.1 Primary Endpoints  
1)  To determine the feasibility of administering the amino acid leucine and to determine the 
pharmacokinetics of leucine administration in patients with Diamond Blackfan anemia 
(DBA) who are red cell transfusion-dependent.  
2)  To determine the safety of leucine administration in transfusion-dependent DBA patients. 
3)  To determine the efficacy of leucine to produce a hematologic response in transfusion-
dependent DBA patients. 
 
8.2 Secondary Endpoint  
4)  To determine if patients with DBA are deficient in branched chain amino acids. 
 

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Since the previous reported patients experienced their responses at 6 months, the total length of 
this study will be 12- 15 months.  The patients will have a screening visit and follow-up visits. 
Open-label, pharmaceutical-grade leucine capsules will be used and supplied to the subjects for 
consistency in leucine dosing and preparation. Thus, oral leucine will be given for 9 months at 
700 milligrams/M2/dose three times daily. 
 
9.0 PATIENT ELIGIBILITY 
All clinical and laboratory studies to determine patient eligibility must be performed within 30 
days prior to study enrollment unless otherwise indicated. Laboratory values used to assess 
eligibility must be no older than thirty days at the start of therapy. Laboratory tests need not be 
repeated if therapy starts within thirty days of obtaining labs to assess eligibility. If a post-
enrollment lab value is outside the limits of eligibility, or laboratory values are >30 days old, 
then the following laboratory evaluations must be re-checked prior to initiating therapy: CBC 
with differential, bilirubin, ALT (SGPT) and serum creatinine. If the recheck is outside the limits 
of eligibility, the patient may not receive protocol therapy and will be considered off protocol.  
Important note: The eligibility criteria listed below are interpreted literally and cannot be 
waived.  All clinical and laboratory data required for determining eligibility of a subject enrolled 
on this trial must be available in the patient’s medical or research record which will serve as the 
source document for verification at the time of audit. 
9.1   Inclusion Criteria    
 
9.1.1  Transfusion dependent DBA age 2 years and older. 
9.1.1.1 Transfusion dependence is defined as >10 cc/kg or if over 60 kg 2 units of RBC per 28 
days averaged over 84 days (3 months) prior to study entry. 
9.1.1.2 Diagnostic and supporting criteria for the diagnosis of DBA as published in British 
Journal of Haematology (8) and described in table below.   
 
Table. Diagnosis of Diamond Blackfan Anemia 
Diagnostic criteria 
Age less than 1 year 
Macrocytic or normocytic anemia with no other significant cytopenias 
Reticulocytopenia 
Normal marrow cellularity with a paucity of erythroid precursors 
Supporting criteria 
Major 
Ribosomal protein (RP) gene or GATA1 mutation identified 
Positive family history 
Minor 
Elevated erythrocyte adenosine deaminase activity 
Presence of congenital anomaly(ies) 
Elevated fetal hemoglobin 
No evidence of another inherited bone marrow failure syndrome 
 
A diagnosis of “classical” DBA is made if all the diagnostic criteria are met.  When there is a 
positive family history, an otherwise normal individual should be considered as having “non-
classical” DBA if a mutation shared by affected family members is present.  Anyone suspected 
of having DBA, but with insufficient diagnostic criteria, should be considered as having 

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sporadic, non-classical DBA if a reported mutation is present.  A patient can be assigned as 
having a “probable” diagnosis, with a decreasing degree of certitude if; 3 diagnostic criteria are 
present along with a positive family history; 2 diagnostic criteria and 3 minor supporting criteria 
are present; or, a positive family history and 3 minor supporting criteria are evident, even in the 
absence of diagnostic criteria.  Steroid responsiveness is not considered a diagnostic criterion and 
corticosteroids should not be administered until a diagnosis is made.   
 
If the first three diagnostic criteria are present, but there is no paucity of red cell precursors in the 
bone marrow and no supporting criteria, the diagnosis of DBA cannot be made.  A bone marrow 
evaluation should be repeated at a later date as red cell marrow hypoplasia may develop after 
anemia and reticulocytopenia are observed.  
 
The criteria used for diagnosis must be delineated (Appendix A).  Prior to enrollment the 
principal investigator will determine if the patient meets the diagnostic criteria for inclusion in 
the study. 
 
9.1.2  The study is restricted to patients who are receiving no DBA specific therapy other than 
red blood cell transfusions.  Patient will remain on his/her transfusion schedule for the duration 
of this study without any other DBA medications such as therapeutic corticosteroids, anabolic 
steroids, cyclosporine, etc. to be taken simultaneously or added during the course of this study.  
Patients must be off of therapeutic corticosteroids or any immunosuppressive therapy for 30 days 
prior to study enrollment.  Hydrocortisone for treatment of adrenal insufficiency will be 
permitted. Iron chelation medications will be allowed as standard of care for transfused patients. 
9.1.3  Patient should have adequate renal function defined as: 
 
- Creatinine clearance or radioisotope GFR ≥ 70ml/min/1.73 m2 OR 
 
- A serum creatinine based on age/gender as follows: 
Age  
Maximum Serum Creatinine 
(mg/dL)  
 
Male  
Female 
1 to < 2 years  
0.6  
0.6  
2 to < 6 years  
0.8  
0.8  
6 to < 10 years  
1  
1  
10 to < 13 years  
1.2  
1.2  
13 to < 16 years  
1.5  
1.4  
≥ 16 years  
1.7  
1.4  
The threshold creatinine values in this table were derived from the Schwartz 
formula for estimating GFR utilizing child length and stature data published 
by the CDC.35 
9.1.4 Patient should have adequate liver function defined as: 
 
- Total Bilirubin ≤ 1.5 x upper limit of normal (ULN) for age, and 
 
- SGPT (ALT) < 5 x upper limit of normal (ULN) for age 
 
- Serum albumin ≥ 2 g/dL 
 
9.1.5 Negative pregnancy test if subject is a menstruating female and documentation of  
agreement to use adequate contraception for at least 28 days before starting the study, while 
taking the leucine, and for at least 3 months after the last dose of study medication .  

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9.1.6 Signed informed consent.  
9.1.7 Registration in the Diamond Blackfan Anemia Registry (DBAR) may be offered but is 
not required for study enrollment. 
9.2   Exclusion Criteria  
9.2.1 Known hypersensitivity to branched chain amino acids 
9.2.2 Diagnosis of an inborn error of amino acid metabolism disorder  
9.2.3 Prior hematopoietic stem cell transplantation 
9.2.4 Pregnancy, or plans to become pregnant during duration of trial   
 
10.0 SUBJECT RECRUITMENT 
Patients with Diamond Blackfan anemia are generally cared for at larger academic centers 
because of the rarity of the disease and the need for transfusion therapy in about half of the 
patients. The PI of this protocol is a known clinical researcher in the DBA field. Dr. Vlachos 
directs the Comprehensive Diamond Blackfan Anemia Clinical Care Center and the Diamond 
Blackfan Anemia Surveillance and Awareness Program housed at the Steven and Alexandra 
Cohen Children’s Medical Center of New York. Through these efforts many physicians and 
patients/parents of patients often contact Dr. Vlachos and the DBA Study Coordinator Eva 
Atsidaftos for information about DBA. Contact is made via telephone, fax and/or e-mail. 
Recruitment for this study can occur via three methods: 
 
1) The PI and Co-Investigators’ institutions will be recruiting patients by word of mouth as they 
have multiple DBA patients whom they care for that are eligible for enrollment.  Many of these 
patients have already shown interest in this study.  
 
2) Other physicians caring for DBA patients throughout the country that are interested in the 
study may directly contact the PI and/or the Study Coordinator. The protocol and consent will be 
sent to these physicians.  IRB approval will be obtained at these institutions. The patient will be 
enrolled onto the study if they meet the eligibility requirements. 
 
3) Subjects/Parents of subjects may contact the PI or Study Coordinator directly.  The patient 
will be referred to a local institution that has an IRB-approved protocol.  If one does not exist, 
the patient will be instructed to have his/her physician contact the PI and/or Study Coordinator to 
obtain the protocol.  The PI and/or Study Coordinator will assist the physician to obtain IRB 
approval and enroll the patient if the patient is eligible. 
 
This study will be announced via the Diamond Blackfan Anemia Foundation website 
(www.dbafoundation.org), the DBA Yahoo Group chat room, the Daniella Maria Arturi 
Foundation website (www.DMAF.org), the DBAR website (www.dbar.org), and the Diamond 
Blackfan Anemia Surveillance and Awareness Program Facebook site. As the DBA community 
is a close-knit one with an excellent network, recruitment is expected to be accomplished rapidly 
through word of mouth.   
 
This study is funded for 50 subjects. We expect to recruit 50-60 subjects of which the first 50 
eligible patients will be enrolled. Our target population is the DBA patient community, aged 2 

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years and older, males and females alike. This disease is noted in all racial and ethnic groups so 
all are included.  The consent will be available in English only at this time. For non-English 
speaking subjects, a certified oral interpreter service will be used for obtaining informed consent.   
 
11.0 TREATMENT OF SUBJECTS   
This study will be a multi-institution study.  Institutions must enroll patients directly on this 
protocol. Patients will also be offered enrollment on the Diamond Blackfan Anemia Registry 
(DBAR - see section 11.3).     
 
11.1 Informed Consent, Subject Enrollment and Eligibility Screening  
The process of informed consent, eligibility screening and enrollment is as follows: 
a. The local PI or his/her designee calls the Central Study Coordinator to verify that the 
study is still accruing subjects. 
b. The local PI will obtain informed consent from the subject or his/her parents/legal 
guardian for enrollment into the study. 
c. Once informed consent is signed, the local PI will call/e-mail the Coordinating Center 
and a participant identification number will be assigned by the Central Study 
Coordinator. 
d. Screening of the patient for eligibility will proceed with completion of the Eligibility 
Screening Checklist which will be forwarded to the Coordinating Center. 
e. The local PI will be informed of the patient’s eligibility by fax and/or e-mail within 24 
hours of receipt of the Eligibility Screening Checklist or by the next business day.  
f. Participant enrollment will then proceed with assignment of the leucine dose. The 
Enrollment Form will be submitted by local PI as required. 
g. This process will be repeated until 50 subjects are enrolled. Patients found to be not 
eligible after screening will be deemed ‘screen failures’.   
 
11.1.1 Current Study Status 
Local investigators or their designee must contact the Primary Institution (1-516-562-1505) to 
determine if the study is currently open for accrual. This is to ensure that no more than 50 
subjects are enrolled on the study. Once 50 subjects are enrolled, excluding the ‘screen failures’, 
a memo will be sent out to all participating institutions that accrual has been reached. 
 
11.1.2 Informed Consent/Assent (Appendix B) 
A signed informed consent document will be obtained prior to entry onto this study.  The consent 
documents may be mailed or e-mailed for potential subjects to review, however the actual 
consent process will take place in person.  The principal investigator and/or sub investigator will 
obtain consent after the patient and/or patient’s parent/legal guardian are informed of the 
investigational nature and research objectives of the trial, the procedures and treatments involved 
and their attendant risks and discomforts as well as potential alternative therapies. The local PI 
and his/her designees will be well versed in this study. There is no urgency in the start of this 
medication for DBA patients.  Patients and their guardians will be allotted ample time to review 
the study risks/benefits before signing consent. It will be allowable to have those patients not 
treated at the consenting institution to take the consent home to discuss with their treating 
hematologist, only after discussing the study and going through the consent process with 
appropriate documentation. If they decide to enroll, they may mail the consent back to the 

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coordinating center. It will not be uncommon to discuss the study with the patient and consent 
them at their next transfusion visit 3-4 weeks later.  Any and all questions will be answered 
regarding the study and the required tasks.  The informed consent and assent will be signed 
according to institutional guidelines.  Documentation of the informed consent will be maintained 
in the subject’s research chart.  
11.1.2.1 Consent will be obtained from the subject himself/herself, or, in the case of a minor, 
from the parent or legal guardian.  Subjects ages 7-11 and 12-17 (or as determined by the local 
IRB) who are able to read and understand an assent will be asked to sign a simplified document, 
along with their parent or guardian signing the standard consent form. Minors who turn 18 while 
still active in the study will be consented as adults. These may be mailed, as a face to face 
reconsent may not be feasible for those treated at outside facilities. A copy will be given to the 
subject/subject’s legal guardian by the person obtaining the consent. 
11.1.2.2 The original consent form will be kept in the subject’s chart at the subject’s local 
institution and will be available for review if needed by a monitoring body.   
11.1.2.3 If at any time during study participation new information becomes available relating to 
risks, adverse events, or toxicities, this information will be provided orally or in writing to each 
enrolled subject and re-consent obtained as appropriate. Documentation will be provided to the 
IRB and if necessary, the informed consent amended to reflect relevant information.   
 
11.1.3 Subject Identification 
Once the subject/parent of patient/legal guardian has signed consent, the local institution will call 
the Coordinating Center (1-516-562-1505) to obtain participant identification (ID) number from 
the Central Study Coordinator.  A confidential copy will be sent to the local institution with the 
subject’s ID for confirmation. A participant identification number will be assigned to identify the 
subject to that investigator and institution and will be used in all future communications with the 
site.  This is to assure that no two possible subjects are enrolled simultaneously. 
11.1.3.1 Participant Identification Number (PIN) 
Each subject registered will be assigned a unique participant identification number. This PIN will 
consist of two parts: the first two digits will identify the local institution who is registering the 
subject, and the three last digits will number the subjects from that institution consecutively.  The 
funding allows for a total of 17 possible institutions so the first two digits will range from 01 (the 
Coordinating Center) to 17.  For example, PIN 01-001 will indicate the first subject on study, 
which in this case would have been enrolled by the Coordinating Center. Each potential patient 
will be assigned an ID once the consent has been signed. If the subject does not meet the 
eligibility criteria once screening occurs then that number will be skipped.   
 
11.1.4 Eligibility Screening    
Diagnostic or laboratory studies performed exclusively to determine eligibility for this study 
must be done only after obtaining written informed consent. Eligibility is then verified by 
meeting the criteria listed in the Eligibility Screening Checklist (Appendix C). Studies or 
procedures that were performed for clinical indications (not exclusively for eligibility for this 
study) may be used for baseline values if the studies were done within thirty days of when the 
informed consent was obtained.  Before the subject can be enrolled, the responsible institutional 
investigator must sign and date the completed Eligibility Screening Checklist. A signed copy of 
the Eligibility Screening Checklist will be faxed to the Coordinating Center (1-516-562-1599).  
The fax should be sent to the attention of the Central Study Coordinator and must include the 

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associated study number and participant identification number. The lead PI and Central Study 
Coordinator will review the Eligibility Screening Checklist. After review, the local investigator 
will be informed of eligibility status via a fax and/or e-mail within 24 hours, if received Monday 
through Thursday. If the form is received on Friday, eligibility will be confirmed by the next 
business day. 
 
11.1.5 Study Enrollment 
Subjects may be enrolled on the study once all eligibility requirements for the study have been 
met and confirmed. Subjects who sign informed consent for the protocol and undergo 
screening for eligibility should not be started on the study drug until the screening is 
completed and they are deemed to meet all eligibility criteria. Study enrollment is 
accomplished by completing the Enrollment Form (Appendix D). The Enrollment Form will be 
completed and faxed to the Coordinating Center (1-516-562-1599).  The fax should be sent to the 
attention of the Central Study Coordinator and must include the associated study number and 
participant identification number.   
 
11.1.6 Dose Assignment 
The dose will be assigned by the local PI and Study Coordinator at the time of study enrollment 
based on the subject’s body surface area. The prescribed amount of leucine will be rounded up to 
the nearest dosage tablet as indicated in the Leucine Dosage Calculation Table (Appendix E). A 
Leucine Dose Assignment Form (Appendix F) will be faxed to the participating institution from 
the Coordinating Center.    
 
11.2 IRB Approval 
Local IRB approval must be obtained at all participating sites in order to begin enrollment onto 
this protocol.  Sites must submit copies of IRB approval to the Coordinating Center. IRB 
approval documents should be faxed to the Central Study Coordinator along with a cover sheet 
(Eva Atsidaftos at 1-516-562-1599), e-mailed (eatsidaf@nshs.edu) or mailed to the Coordinating 
Center.   
 
11.3 Diamond Blackfan Anemia Registry (DBAR) 
The Diamond Blackfan Anemia Registry is a demographic, laboratory and clinical database of 
patients with DBA in North America.  Subjects screened for this study may be offered 
enrollment in the DBAR (through a separate consent and questionnaire) by their local 
investigator(s).  Enrollment in the DBAR is not required for participation in this study.   
 
12.0 DATA COLLECTION (Appendices D and G)   
12.1 Enrollment Data (Appendix D) 
12.1.1   Demographics required for shipping refills   
12.1.2   Sex, race, date of birth, date of diagnosis (if available) (birth date to be collected in order 
to reduce error of mistaking one subject for another with the same age and initials) 
12.1.3   Current and past medications   
12.1.4   Transfusion history   
12.1.5   Completion of DBAR consent and questionnaire – if the subject (or his/her 
parent/guardian) wants to enroll in the DBA Registry – which is not part of this protocol but will 
be offered to the subject under its own protocol/consent  

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12.2 Case Report Form Data (Appendix G)  
The Case Report Form data is to be collected at baseline, at Weeks 2 and 4 and at Months 2, 3, 4, 
5, 6, 7, 8, and 9 (or at time of transfusion, except where indicated otherwise). Each visit can be 
+/- 1 week of the requested time except week 2. Blood drawings can also be +/- 1 week of the 
requested time except week 2. 
 
12.2.1 Physical Examination   
12.2.1.1 Temperature, Pulse and Blood Pressure  
12.2.1.2 Height (cm) at baseline, month 4 and month 9, weight (kg), and body surface area (M2) 
 
12.2.2 Laboratory Examinations    
12.2.2.1 CBC with platelet count, differential, and red cell indices   
12.2.2.2 Reticulocyte count  
12.2.2.3 Percent Hemoglobin F at baseline and then every 3 months and at study completion 
 
12.2.2.4 Hepatic transaminases, serum bilirubin, creatinine, and blood urea nitrogen 
12.3.2.5 Serum ferritin at baseline and then every three months and at study completion 
12.2.2.6 If female and of child-bearing potential, pregnancy test at baseline and every 3 months 
12.2.2.7 Plasma leucine level as well as levels of isoleucine and valine and an amino acid profile 
at baseline. Plasma leucine levels are obtained for research only and will not be used for clinical 
decision making. This extra blood will be drawn at the same time as standard routine clinical 
blood draws.  A standard 10cc’s (2 teaspoons) will be taken as part of clinical care, with an 
additional 5 cc’s (1 teaspoon) taken after Leucine treatment begins to monitor leucine levels in 
the blood.  Plasma leucine levels (5 cc’s) will also be drawn at 2 weeks, 4 weeks, then months 3, 
6, and 9 months (These are the extra tests for this study protocol.  All the others are standard of 
care for the enrolled subjects.) Subjects will fast for 4 hours and hold the morning dose of 
Leucine for evaluation of the Leucine level at these time points. Blood drawings can be + 7 days 
of the requested time, except week 2.  
  
Laboratory examinations 12.2.2.1, 12.2.2.2, 12.2.2.4 and 12.2.2.5 are standard of care for 
transfused DBA subjects who are on chelation medication – which the chronically transfused 
subjects eligible for this protocol will be receiving; 12.2.2.3, 12.2.2.6 and 12.2.2.7 are extra tests 
that are specific to the study protocol. The instructions for sample collection and shipping of 
blood for amino acid levels are delineated in Appendix H.  The blood samples will be batched 
and shipped directly from the participating institution to Case Western Reserve University, Ohio 
where the tests will be performed. The costs for the mailing will be provided by the Coordinating 
Center through a FedEx account. 
 
12.2.3 Other Specifics   
12.3.3.1 Transfusion history (date and amount in ml and/or units q 3-4 weeks based on 
transfusions) 
12.3.3.2 Adverse side effects  
12.3.3.3 Hospitalizations (cause, dates) 
  
12.4 Data Submission  

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Case Report Forms (Appendix G) will be completed by the local PI and/or his/her designee at 
each subject visit. Case Report forms will be faxed to the Coordinating Center (1-516-562-1599) 
where data will be stored on a secure database.  These forms will be required to be submitted 
every 3 months, including at study completion (three times in total). The central Study 
Coordinator will perform data integrity monitoring visits of a minimum of 20% of the entered 
data through cross-checking and source documentation.  Data will be analyzed with biostatistical 
support after completion of the study. 
 
12.5 Data Storage 
An institution or, when appropriate, an IRB shall prepare and maintain adequate documentation 
of IRB activities, including the following: 
(1) Copies of all research proposals reviewed; scientific evaluations, if any, that accompany the 
proposals; approved sample consent documents; progress reports submitted by investigators; and 
reports of injuries to subjects. 
(2) Copies of all correspondence between the IRB and the investigators. 
 
With regard to the consent document, all sites must specify consent document storage and length 
of time, as well as destroyed/disposed specifics.  Also research records and data storage and time 
destroyed/disposed, hard copies vs. electronic storage, research specimens storage and time 
destroyed/disposed, and any special storage conditions must be specified according to federal 
and institutional requirements.   
 
Electronic and paper clinical research records will be retained for at least 7 years for adult 
subjects and 10 years for pediatric subjects after completion of the research study or at least 2 
years after marketing approval by the FDA (or if not approved, until 2 years after shipment and 
delivery for investigational use is discontinued), whichever is longer. Electronic or paperless data 
sets should be kept on a secure server with adequate backup to assure record retention. Paper 
research records should be maintained in a secure office, in a secure file cabinet for the duration 
of the study and then archived. 
  
All data collected from all health system sources will be maintained on a secure and password 
protected server and backed up every 24 hours.  Study staff should have restricted access to 
directories and files on the server, according to project responsibilities. “Login/password 
security” should be used to control access to the network. Individuals with data entry 
permissions are able to add records only to the databases to which they have access.     
 
A unique participant identification number will be assigned to each subject at the time of 
enrollment.  In an effort to correlate information every unique PIN will be linked (coded) with 
the subject’s medical record information.  The coded link between this unique identifier and the 
subject’s identifying information will be maintained in locked files.  When information is shared 
internally or externally, investigators will only be provided with the subject’s unique PIN.  
 
Accurate and complete study records will be maintained and made available to representatives of 
the U.S. Army Medical Research and Materiel Command, the North Shore-LIJ Health System, 
and other federal and state authorities as a part of their responsibility to protect human subjects in 

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research.  Research records will be stored in a confidential manner so as to protect the 
confidentiality of subject information.   
 
13.0 STUDY DRUG ADMINISTRATION   
Each subject will receive standard dose Leucine therapy for 9 months:          
 
 
● 700 mg/m2/dose orally three times daily for a total of 2100 mg/m2/day                  
 
 
 
13.1 Study Drug - Leucine 
This clinical trial will only utilize commercially available Leucine from Ajinomoto 
Aminoscience LLC, Raleigh, NC.  The Leucine will be supplied in capsule form in 250 
mg/capsule concentration.  The Coordinating Center will purchase the Leucine supply.  
13.1.1 Dispensing of Study Drug Once the subject has signed consent and is enrolled on the 
study, the local PI will be faxed the Dose Assignment Form from the Coordinating Center. The 
participating center will complete the Supply Request (Appendix F) and fax it to the 
Coordinating Center at 516-562-1599. Once received the Coordinating Center’s Study Pharmacy 
will be informed of the subject enrollment and will dispense a 3-month supply of the Leucine to 
the participating subject. The Leucine will be dispensed in 250 mg capsules, in child-proof 
capped containers, to be stored at room temperature. The shipment will be sent directly to the 
subject. Upon request, local investigators may dispense the study drug after showing they are 
able to store drug in a locked area and maintain a log of receiving and dispensing the study 
drug13.1.2 Dispensing of Study Drug to Subject 
The drug will be shipped directly to the subject from the coordinating site.  The amount sent will 
be enough drug for a 3 month period. 
13.1.3 Medication Instructions   
The Leucine Dose Assignment Form (Appendix F) will be faxed/e-mailed to the local PI to be 
reviewed with the subject. Instructions for medication usage will be distributed to the subject 
and/or parent/guardian of the subject along with the dose. May be taken with or without food.  
Capsules can be mixed into food or dissolved in liquids.  Total daily dose will be divided into 3 
doses to be given AM, afternoon and PM. 
13.1.4 Concomitant Medications 
Concomitant medications will be documented on the Case Report Form (Appendix G).  
 
13.2 Transfusion Therapy During Study Drug Administration 
Subjects will receive 10-15 ml/kg/transfusion, with all volumes recorded on the Case Report 
Forms.  
13.2.1 If the hemoglobin (Hb) is less than 8 gm/dL and there is no evidence of reticulocyte 
response (retic count < 0.5%), the subject will be transfused as usual. 
13.2.2 If the Hb is greater than 8 gm/dL, the reticulocyte count is greater than 0.5% (or 
baseline) and the subject is clinically stable (without fever, respiratory illness, etc.), the 
transfusion should be held. The subject should be followed again in 1-2 weeks and 
reassessment will be made to see if a response is occurring.  
13.2.3 If the Hb is between 7-8 gm/dL, the reticulocyte count is greater than 0.5% (or baseline), 
and the subject is clinically stable, then the subject can opt out of transfusion, based upon clinical 
considerations, for one week, or receive 10 ml/kg packed red blood cell transfusion so as not to 

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abort a possible response. If the subject continues to show a response then Hb can be checked 
every 2 weeks and transfusion held. If not, the subject will resume the transfusion schedule. 
 
13.3 Schedule of Evaluations  
Oral Leucine x 9 months  
VISIT * 
0  
2wk 
4wk  
2mo 
3mo 
4mo 
5mo 
6mo 
7mo 
8mo 
9mo 
PE ** 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
BP  
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Height  
X 
 
 
 
 
X 
 
 
 
 
X 
Weight  
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
CBC with 
Retic *** 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
%Hb F 
X 
 
 
 
X 
 
 
X 
 
 
X 
Chemistry**** 
Panel  
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Ferritin  
X 
 
 
 
X 
 
 
X 
 
 
X 
Pregnancy test 
X 
 
 
 
X 
 
 
X 
 
 
 
Leucine level 
X    
X 
X 
 
X 
 
 
X 
 
 
X 
*Each visit can be + 1 week of the requested time – except visit 2. 
** PE includes Temperature, Pulse and Blood Pressure complete physical exam as 
clinically necessary *** CBC to include WBC, RBC, Hb, Hct, Plts, differential and 
reticulocyte count (%) 
**** Chemistry Panel to includeUrea Nitrogen, Creatinine, AST, ALT, Bilirubin, total and 
direct,  
 
13.4 Grading of Adverse Events 
Adverse events (toxicities) will be graded according to the NCI Common Terminology Criteria 
for Adverse Events v4.0 (CTCAE) (http://ctep.cancer.gov/reporting/ctc.html). Any suspected or 
confirmed dose-limiting toxicity should be reported immediately (within 24 hours) to the Study 
PI. 
 
13.5 Dose Modifications for Adverse Events 
13.5.1 Dose Reduction 
There will be no drug reduction for the study drug. 
13.5.2 Dose Escalation 
There will be no drug escalation for the study drug. 
13.5.3 Dose Discontinuation  
In the event of any serious adverse event (SAE), the study drug will be discontinued until all 
symptoms resolve.  If the SAE is deemed possibly related to the protocol therapy, the subject 
will be removed from the study and leucine will be permanently discontinued. 
 
14.0 EVALUATION CRITERIA 
14.1 On-Study Response Criteria - evaluated at 6 and 9 months of treatment 
14.1.1 Complete Response (CR) – Hb > 9 gm/dL and weaned off of transfusion therapy  
 
1a- Complete response lasting > 6 months 

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14.1.2 Partial Response (PR) - Hb < 9 gm/dL with an increase in reticulocyte count to 1% and 
any transfusion interval increase 
14.1.3 No Response (NR) - No change in transfusion needs, no change in Hb or reticulocyte 
count 
14.1.4 Progression - An increased transfusion requirement with increased volume or increased 
interval 
 
15.0 WITHDRAWAL AND OFF STUDY CRITERIA 
15.1 Subject’s choice: A subject may withdraw from the study at any time.  The risk of 
withdrawing will be discussed, as will alternative treatment options. Once off study, subjects will 
be referred back to his or her referring physician.  Alternative treatment will then be discussed 
with the primary hematologist.  
15.2 PI Decision:  Alternative treatment options will be discussed with the subject, who will 
then be referred back to his or her referring physician.  
15.3 Discontinuation for Toxicity: Subjects will be discontinued from study for any Grade 
III/IV toxicity related to the study drug as determined by PI.  
15.4 Discontinuation for Non-Adherence: Subjects will be discontinued from study if non-
adherent to protocol.   
 
16.0 BIOSTATISTICAL CONSIDERATIONS 
Response to treatment (Complete, Partial, None, Progression) will be evaluated at 6 and 9 
months.  Time-to-achieve a complete or partial response will be examined as well. 
 
16.1 General Study Objectives 
16.1.1 To determine the feasibility of administering the amino acid leucine and to determine the 
pharmacokinetics of leucine administration in subjects with Diamond Blackfan anemia (DBA) 
who are red cell transfusion-dependent.  
16.1.2 To determine the efficacy of leucine to produce a hematologic response in subjects with 
DBA. 
16.1.3 To determine the side effects of leucine administration in DBA subjects (monthly). 
16.1.4 To determine if subjects with DBA are deficient in branched chain amino acids (baseline). 
 
16.2 Specific Statistical Aims of the Study (for the Data Analysis) 
16.2.1 The primary objective of the study is to estimate the proportion of complete responders, 
partial responders or non-responders at 9 months. These proportions will also be estimated at 6 
months. 
16.2.2 To determine the time-to-first occurrence of a CR or PR.  Subjects who do not achieve 
CR or PR by the end of the study at 9 months will be considered ‘censored for CR (PR)’.   
16.2.3 To catalogue the phenotypic characteristics of the responders and non-responders at 6 and 
9 months.   
16.2.4 To examine the frequency and severity of adverse events (AEs) that may arise from the 
study and to determine if these AEs are related to the study drug.   
 
16.3 Endpoint Variables 
The primary outcome is the type of response observed at 9 months (and 6 months).  Response to 
treatment can be one of the following: 

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a. Complete response (CR): Hb > 9 gm/dL and transfusion-independence as defined in 
DBA 
b. Partial response (PR): Hb < 9 gm/dL and increased reticulocyte count to greater than 
1% and any increase in transfusion interval from baseline. (Baseline reticulocytes 
range from 0.1 to 0.5 and transfusions are usually performed every 3 weeks.  An 
increase of reticulocyte counts to over 1 to 1.5% and any increase in transfusion 
interval will be considered a PR.)  
 
c. No response (NR): no change in transfusion requirements and no significant change 
in Hb or reticulocytes (or any response that does not satisfy the conditions of either a 
PR or CR) 
d. Progression:  worsening of disease as defined by the need for more frequent 
transfusions 
 
Secondary outcomes include time-to-occurrence of either a PR or CR, and safety parameters 
such as type, frequency, and severity of adverse events and relationship to leucine (according to 
the NCI CTCAE version 4.0). 
 
16.4 Statistical Methods 
This is a standard Phase I/II study with the goal of determining the feasibility, safety and efficacy 
of the use of leucine in transfusion-dependent DBA subjects.  A maximum of 50 subjects will be 
enrolled into the study.  The statistical approach will be primarily descriptive in nature.   
 
For specific aim 1, the proportion of subjects achieving a specific response at 6 and 9 months 
(CR, PR, NR or progression) along with their corresponding 95% confidence intervals, will be 
calculated using standard methods of estimating proportions.  
 
For specific aim 2, time-to-response (separately for CR and PR) will be analyzed using survival 
analysis techniques. Subjects who do not achieve CR or PR by the end of the study at 9 months 
will be considered ‘censored for CR (PR)’. Kaplan-Meier curves will be generated and the log-
rank test will be used for any comparisons with respect to time-to-CR (PR), if feasible. 
 
For specific aim 3, the approach will be primarily descriptive.  However, we may carry out some 
analyses if feasible.  First, response status at 9 months (separately for CR and PR) will be cross-
tabulated with certain subject phenotypes; a chi-square test or Fisher’s exact test, as appropriate, 
will be used to determine if there is an association between response status and the certain 
phenotype.  Second, time-to-response will be analyzed.  Kaplan-Meier curves will be generated 
and the log-rank test will be used to compare phenotypes with respect to time-to-CR (PR), if 
feasible. Subjects who do not achieve CR or PR by the end of the study at 9 months will be 
considered ‘censored for CR (PR)’.   
 
For specific aim 4, the proportion of adverse events will be tabulated and their corresponding 
95% confidence interval will be calculated. 
 
16.5 Sample Size Considerations  
The sample size for the Phase I/II study (i.e. a maximum of 50 subjects) is a sample size of 
convenience. The number of subjects in the proposed study is based on logistics/funding 

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provided by the Department of Defense Telemedicine and Advanced Technology Research 
Center (TATRC); it is not based on any formal power calculations. Results from this study may 
provide valuable information for quantifying the effects of leucine in this group of transfusion-
dependent DBA subjects and may be useful in estimating the effect size necessary for 
appropriately designing future larger-scale Phase III clinical trials. However, in order to have 
some idea as to the precision of estimates that can be obtained from this study, when the sample 
size is 50, a two-sided 95% confidence interval for a single proportion using the large sample 
normal approximation will be no wider than 50%±13.9% i.e. the 95% confidence interval will 
range from at most, 36.1% to 63.9%. 
 
17.0 RESEARCH MONITOR REQUIREMENT 
17.1 Per DOD Directive 3216.2, all GREATER THAN MINIMAL RISK STUDIES require a 
Research Monitor.  This individual should be a qualified physician, other than the Principal 
Investigator, not associated with the protocol, able to provide medical care to research volunteers 
for conditions that may arise during the conduct of the study, and who will monitor the 
volunteers during the conduct of the study.  The research monitor plays a role in reviewing 
monitoring plans and unanticipated problems. 
 
17.2 The research monitor is required to review all unanticipated problems involving risk to 
subjects or others associated with the protocol and provide an unbiased written report of the 
event.  At a minimum, the research monitor will comment on the outcomes of the event or 
problem.   The research monitor will also indicate whether he/she concurs with the details of the 
report provided by the site principal investigator.   
17.3 The Research Monitor has been identified as: 
 
Dr. Lawrence Wolfe,  
at The Steven and Alexandra Cohen Children’s Medical Center of New York 
269-01 76th Avenue, Rm. 255 
New Hyde Park, NY 11040 
Phone: 718-470-3460 
Fax: 718-343-4642 
E-mail: lwolfe2@nshs.edu 
 
18.0 SAFETY MONITORING 
18.1 Reporting of Adverse Events and Unanticipated Problems 
Information about all serious adverse events, which may be noted through history, investigator 
questioning, by physical examination, or by subject volunteering, will be reported to the 
Coordinating Center by fax (516-562-1599) on the Severe Adverse Event (SAE) Reporting Form 
(Appendix I) and the local IRB within 24 hours of learning of the occurrence of the event.    
  
18.2 Clinical Research Monitoring Services 
The Office of Research Compliance (ORC), located at The Feinstein Institute of Medical 
Research (FIMR) and part of the Coordinating Center will provide clinical research monitoring 
services.   
18.2.1 These services include good clinical practice regulatory review, subject case review of 
critical study data and processes including: 

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- Case report form completion and source document verification 
 
- Protocol and informed consent compliance 
 
- Protocol drug accountability 
18.2.2 The ORC agrees to provide primarily remote monitoring services for all performance sites 
according to the risk-based monitoring plan including the following: 
 
- Site initiation: Prior to study start-up for good clinical practice (GCP) and protocol 
overview 
 
-Follow-up monitoring: Within 2 weeks after the first subject is enrolled and then after 
every fifth subject is enrolled (for a minimum of 11 monitoring visits) in addition to targeted 
reviews as required 
 
- Final monitoring: Study close out 
18.2.3 Reporting: Regular monitoring reports will be generated by the ORC after each review 
and will be provided to the respective performance site and Study PI.  Resolution of queries and 
outstanding issues or concerns will be the responsibility of the individual performance site and 
local PI. The lead PI will be responsible for reporting incidents of IRB non-compliance to the 
North Shore-LIJ Health System IRB and for securing compliance at all trial sites. 
 
18.3 Reporting to the U.S. Army Medical Research and Materiel Command  
18.3.1 The protocol will be conducted in accordance with the protocol submitted to and approved 
by the ORP HRPO and will not be initiated until written notification of approval of the research 
project is issued by the ORP HRPO.  
18.3.2 Accurate and complete study records will be maintained and made available to 
representatives of the U.S. Army Medical Research and Materiel Command as a part of their 
responsibility to protect human subjects in research.  Research records will be stored in a 
confidential manner so as to protect the confidentiality of subject information. 
18.3.3 Any deviation to the protocol that may have an effect on the safety or rights of the subject 
or the integrity of the study must be reported to the USAMRMC ORP HRPO as soon as the 
deviation is identified.   
18.3.4 Major modifications to the research protocol and any modifications that could potentially 
increase risk to subjects must be submitted to the USAMRMC ORP HRPO for approval prior to 
implementation.  All other amendments will be submitted with the continuing review report to 
the USAMRMC ORP HRPO for acceptance. 
18.3.5 A copy of the approved continuing review report and the IRB approval notification will 
be submitted to the USAMRMC ORP HRPO as soon as these documents become available.  A 
copy of the approved final study report and IRB approval notification will be submitted to the 
USAMRMC ORP HRPO as soon as these documents become available.   
18.3.6 The knowledge of any pending compliance inspection/visit by the FDA, DHHS-OHRP, or 
other government agency concerning clinical investigation or research, the issuance of Inspection 
Reports, FDA Form 483, warning letters or actions taken by any Regulatory Agencies including 
legal or medical actions and any instances of serious or continuing noncompliance with the 
regulations or requirements will be reported immediately to USAMRMC ORP HRPO.  
 
18.4 SAE Definitions   An SAE is any untoward medical occurrence that:  
18.4.1 Results in death 

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18.4.2 Is life-threatening (defined as an event in which the subject is at risk of death at the time 
of SAE; this does not refer to an event which hypothetically might have caused death if more 
severe)  
18.4.3 Requires inpatient hospitalization or prolongation of existing hospitalization  
18.4.4 Results in more frequent need for erythrocyte transfusions (more than pre-study) 
18.4.5 Results in persistence or significant disability or incapacity 
18.4.6 Results in overdose (defined as the intentional or accidental ingestion of study drug at any 
dose which is considered medically relevant and excessive)   
18.4.7 Is medically significant (defined as the event may jeopardize the subject and may require 
medical or surgical intervention to prevent one of the outcomes listed above)  
18.4.8 Any SAE occurring after the subject has signed the informed consent form, and until 12 
weeks after the subject has stopped study participation, must be reported.   
 
18.5 Assessment Terms for SAE and Causal Relationship to Study Drug 
18. 5.1 DEFINITE means there is a reasonable causal relationship between study drug and the 
SAE. The event responds to withdrawal of drug and recurs with re-administration of the study 
drug (if clinically feasible).    
18.5.2 PROBABLE means there is a reasonable causal relationship between study drug and the 
SAE. The SAE tapers or diminishes once study drug has been discontinued.  Re-administration 
of the study drug not required. Relationship between study drug and SAE cannot be reasonably 
explained by the known characteristics of subject’s clinical state.   
18.5.3 POSSIBLE means there is a reasonable causal relationship between study drug and the 
SAE but the SAE could have been produced by subject’s clinical state or by other means of 
treatment or medications concurrently administered to the subject.   
18.5.4 UNLIKELY means there is a temporal relationship to study drug administration but no 
reasonable causal relationship exists between study drug and SAE.  The SAE does not follow a 
reasonable sequence from administration of study drug.  SAE could have been produced by 
subject’s clinical state or by other means of treatment or medications concurrently administered 
to the subject.   
18.5.5 UNRELATED means there is no temporal relationship between SAE and study drug 
administration (e.g., the SAE occurred too early, too late, or study drug not taken) or there is 
reasonable causal relationship between SAE and another drug, concurrent disease, or 
circumstance and the SAE.  Subjects experiencing AEs that cause an interruption or 
discontinuation of study drug or those who experience AEs that are present at the end of study 
should receive appropriate follow-up care.  Sites should report the outcome of any AE that 
caused permanent discontinuation of the study drug or that was present at the end of the study.   
 
18.6 Grades of Severity  
18.6.1 MILD An event that is easily tolerated by the subject, causing minimal discomfort and 
not interfering with everyday activities.   
18.6.2 MODERATE An event that is sufficiently discomforting to interfere with normal 
everyday activities.    
18.6.3 SEVERE An event that prevents normal everyday activities.  
 
18.7 Non-Serious Adverse Event (AE) Reporting  

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18.7.1 An Adverse Event (AE) is any unexpected, non-serious event that is NOT an SAE.  An 
AE is any untoward medical occurrence or worsening of a pre-existing medical condition in a 
subject who receives a study drug and which does not have a necessarily causal relationship with 
study treatment.     
18.7.2 A “study drug” is defined as a pharmaceutical form of an active ingredient being tested in    
this study.     
18.7.3 An AE, therefore, can be any unfavorable and unintentional sign (including a clinically 
significant abnormal laboratory finding, for example), symptom or disease associated with the 
use of the medicinal product, whether or not considered related to the medicinal product.  The 
collection of Non-Serious AE information should begin at Day 1, initiation of study drug.   
18.7.4 All identified AEs must be recorded on the data capture forms, including description of 
AE, date and time of onset and resolution; duration of AE if less than 24 hours, severity of event, 
investigator’s opinion of the relationship of the study drug, study treatment action (if any), and 
outcome of AE (if known).     
 
19.0 HUMAN SUBJECT PROTECTION  
19.1 Rationale for Subject Selection  
19.1.1 This study will be open to all subjects who fully meet the inclusion and exclusion criteria 
as described in section 9.0.   Epidemiologic data suggests that the gender will be approximately 
evenly split between males and females as the DBAR has reported the male: female ratio to be 
1.03:1.00.  DBA has been reported in nearly all ethnicities.   
19.1.2 This study will be listed on:    
● www.clinicaltrials.gov (NHLBI patient recruitment site)     
● www.dbar.org (Diamond Blackfan Anemia Registry site)  
● www.dmaf.org (Daniella Maria Arturi Foundation site)  
● www.dbafoundation.org (Diamond Blackfan Anemia Foundation site)   
 
19.2 Participation of Children    
DBA is an extremely rare disorder with approximately 1,000 cases in the United States.  An 
estimated 90% are diagnosed before one year of age.  Consequently, it is vital to include children 
in this protocol because this age group is most likely to benefit from future intervention. The 
majority of subjects expected to be treated will be children as  
a) this is a congenital disorder 
b) 90% of DBA patients are anemic by 1 year of age 
c) the mean age of the patients in the DBAR is 18 years, and 
d) it is desirable to avoid the complications of chronic transfusion therapy as soon as possible. 
 
19.3 Risks and Discomforts 
19.3.1 Related to Leucine (See Section 6.0)   
19.3.2 Related to blood draws  
Blood samples will be obtained during the screening and periodic visits for diagnostic tests to 
monitor the effectiveness of the therapy, detect possible side effects, and for research purposes. 
No major risk is associated with blood drawing. Minor complications may include bleeding, 
pain, and hematoma formation at the site.   
 
19.4 Risks in Relation to Benefit for Adult and Pediatric Subjects   

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19.4.1 Benefits to the patients themselves could be a reduction or even absence of transfusion 
requirements.  This could in the long run prevent morbidity and mortality from transfusion-
associated viral agents and more importantly, from iron overload.  The need for chronic chelation 
could also be avoided in responding subjects.  Therefore, this research involves more than a 
minor increase over minimal risk to subjects with the prospect of direct benefit.  
19.4.2 Risks in Relation to Benefit for Pediatric Subjects Specifically  This study meets the 
DHHS regulation §46.405 as follows:   a)  the risk is justified by the anticipated benefit to the 
subjects Pediatric subjects with a serious, chronic hematologic disease will be offered a possible 
alternative to transfusion therapy.  b)  the relation of the anticipated benefit to the risk is at least 
as favorable to the subjects as that presented by available alternative approaches    The benefits 
to the patients could be a reduction or even a discontinuation of transfusion requirements and/or 
improvement of anemia, resulting in improved quality of life.  It may offer decreased morbidity 
and mortality from transfusion-associated viral agents and the complications of iron overload. 
The need for chronic iron chelation could also be avoided in responding subjects.   
Adequate provisions are made for soliciting the assent of the children and permission of their 
parents or legal guardians.  Therefore, for pediatric subjects, this research involves greater than 
minimal risk but presents the prospect of direct benefit to the individual subjects.  
 
20.0 COMPLIANCE 
The knowledge of any pending compliance inspection/visit by the FDA, DHHS-OHRP, or other 
government agency concerning clinical investigation or research, the issuance of Inspection 
Records, FDA Form 483, warning letters or actions taken by any Regulatory Agencies including 
legal or medical actions and any instances of serious or continuing noncompliance with the 
regulations or requirements will be reported immediately to ORP HRPO and according to 
institutional policy.  
 
21.0 REFERENCES  
1.  
 Draptchinskaia N, Gustavsson P, Andersson B, Pettersson M, Willig TN, Dianzani I, Ball 
S, Tchernia G, Klar J, Matsson H, Tentler D, Mohandas N, Carlsson B, Dahl N. The gene 
encoding ribosomal protein S19 is mutated in Diamond-Blackfan anaemia. Nat Genet 1999; 
21(2):169-75. 
2.  
 Gazda HT, Grabowska A, Merida-Long LB, Latawiec E, Schneider HE, Lipton JM, 
Vlachos A, Atsidaftos E, Ball SE, Orfali KA, Niewiadomska E, Da Costa L, Tchernia G, 
Niemeyer C, Meerpohl JJ, Stahl J, Schratt G, Glader B, Backer K, Wong C, Nathan DG, Beggs 
AH, Sieff CA. Ribosomal protein S24 gene is mutated in Diamond-Blackfan anemia. Am J Hum 
Genet 2006; 79(6):1110-8. 
3.  
 Cmejla R, Cmejlova J, Handrkova H, Petrak J, Pospisilova D. Ribosomal protein S17 
gene (RPS17) is mutated in Diamond-Blackfan anemia. Hum Mutat 2007; 28(12):1178-82. 
4.  
 Farrar JE, Nater M, Caywood E, McDevitt MA, Kowalski J, Takemoto CM, Talbot CC 
Jr, Meltzer P, Esposito D, Beggs AH, Schneider HE, Grabowska A, Ball SE, Niewiadomska E, 
Sieff CA, Vlachos A, Atsidaftos E, Ellis SR, Lipton JM, Gazda HT, Arceci RJ. Abnormalities of 
the large ribosomal subunit protein, Rpl35a, in Diamond-Blackfan anemia. Blood 2008; 
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5.  
 Gazda HT, Sheen MR, Vlachos A, Choesmel V, O'Donohue MF, Schneider H, Darras N, 
Hasman C, Sieff CA, Newburger PE, Ball SE, Niewiadomska E, Matysiak M, Zaucha JM, 
Glader B, Niemeyer C, Meerpohl JJ, Atsidaftos E, Lipton JM, Gleizes PE, Beggs AH. 

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Ribosomal protein L5 and L11 mutations are associated with cleft palate and abnormal thumbs in 
Diamond-Blackfan anemia patients. Am J Hum Genet 2008; 83(6):769-80. 
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 Doherty L, Sheen MR, Vlachos A, Choesmel V, O'Donohue M, Clinton C, Schneider H, 
Sieff CA, Newburger PE, Ball SE, Niewiadomska E, Matysiak M, Glader B, Atsidaftos E, 
Lipton JM, Gleizes P-E, Gazda HT. Ribosomal protein genes S10 and S26 are commonly 
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Jan 28. Erratum in: Am J Hum Genet 2010 Apr 9;86(4):655. 
7.  
 Vlachos A, Klein GW, Lipton JM.The Diamond Blackfan Anemia Registry: tool for 
investigating the epidemiology and biology of Diamond-Blackfan anemia. J Pediatr Hematol 
Oncol 2001; 23(6):377-82. 
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 Vlachos A, Ball S, Dahl N, Alter BP, Sheth S, Ramenghi U, Meerpohl J, Karlsson S, Liu 
JM, Leblanc T, Paley C, Kang EM, Leder EJ, Atsidaftos E, Shimamura A, Bessler M, Glader B, 
Lipton JM; Participants of Sixth Annual Daniella Maria Arturi International Consensus 
Conference. Diagnosing and treating Diamond Blackfan anaemia: results of an international 
clinical consensus conference. Br J Haematol 2008; 142:859-76. 
9.  
 Vlachos A, Federman N, Reyes-Haley C, Abramson J, Lipton JM. Hematopoietic stem 
cell transplantation for Diamond Blackfan anemia: a report from the Diamond Blackfan Anemia 
Registry. Bone Marrow Transplant 2001; 27(4):381-86. 
10.   Lipton JM, Atsidaftos E, Zyskind I, Vlachos A.  Improving clinical care and elucidating 
the pathophysiology of Diamond Blackfan anemia: an update from the Diamond Blackfan 
Anemia Registry. Pediatr Blood Cancer 2006; 46(5):558-64. 
11.   Ozsoylu S. Oral megadose methylprednisolone for the treatment of Diamond-Blackfan 
anemia. Pediatr Hematol Oncol 1994; 11:561-562, 567-568. 
12. 
 Bernini JC, Carrillo JM, Buchanan GR. High-dose intravenous methylprednisolone 
therapy for patients with Diamond-Blackfan anemia refractory to conventional doses of 
prednisone. J Pediatr 1995; 127(4):654-59.  
13.   Buchanan GR. Oral megadose methylprednisolone therapy for refractory Diamond-
Blackfan anemia. International Diamond-Blackfan Anemia Study Group. J Pediatr Hematol 
Oncol 2001; 23:353-56. 
14.   Totterman TH, Nisell J, Killander A, Gahrton G, Lonnqvist B. Successful treatment of 
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