PEDS024, Phase I/II Feasibility Study of Busulfan Fludarabine and Thiotepa Conditioning Regimen for Allogeneic Hematopoietic Stem-Cell Transplantation (HSCT) for Children With Non-Malignant Disorders
Phase: PHASE1
Conditions: Bone Marrow Failure Syndrome, Thalassemia, Sickle Cell Disease, Diamond Blackfan Anemia, Acquired Neutropenia in Newborn, Acquired Anemia Hemolytic, Acquired Thrombocytopenia, Hemophagocytic Lymphohistiocytoses, Wiskott-Aldrich Syndrome, Chronic Granulomatous Disease, Common Variable Immunodeficiency, X-linked Lymphoproliferative Disease, Severe Combined Immunodeficiency, Hurler Syndrome, Mannosidosis, Adrenoleukodystrophy
Sponsor: University of Florida

 Phase I/II Feasibility Study of Busulfan Fludarabine and Thiotepa Conditioning 
Regimen for Allogeneic Hematopoietic Stem-Cell Transplantation (HSCT) for 
Children and Young Adults with Non-Malignant Disorders 
NCT03513328 
Protocol Version 19JUL2020 

PEDS024 
            
 
 
 
2020-7-19 
 
 
 
Phase I/II Feasibility Study of Busulfan Fludarabine and Thiotepa Conditioning Regimen for 
Allogeneic Hematopoietic Stem-Cell Transplantation (HSCT) for Children and Young Adults 
with Non-Malignant Disorders  
 
Protocol Number: 
PEDS024  
 
IRB #:  
 
 
 
201800798 
 
 
 
 
 
 
Coordinating Center: 
 
University of Florida 
 
Principal Investigator:  
 
Biljana Horn, MD 
University of Florida 
1600 SW Archer Road 
Gainesville, FL 32608 
Telephone: (352) 273-9120 
Mobile: (415) 215 6930 
Fax: (352) 294-8091 
Email: biljana.horn@ufl.edu  
 
 
Pharmacogenomics  Investigator:           Jatinder Lamba Ph.D. 
                                                                    University of Florida, Department of 
                                                                    Pharmacotherapy and Translational  Research                    
                                                                    PG-25 HSC Building 
                                                                    1345 Center Drive  
                                                                    PO box# 100486 
                                                                    Gainesville, FL-32610 
                                                                    Phone: (352)273-6425 
                                                                    Fax: (352) 273-6121 
                                                                    Email: jlamba@cop.ufl.edu 
                                                                    Email: jatinderklamba@ufl.edu 
 
Neuropsychology:        
 
Erika Cascio, Psy.D. 
                                                            University of Florida 
1600 SW Archer Road 
Gainesville, FL 32608 
Telephone: (352)265-0680 
Email: emellott@phhp.ufl.edu  
 
Statistician:  
 
 
 
 
 
 
Co-Investigators:                              
                              
                        Paul Castillo, MD 
University of Florida 
1600 SW Archer Road 

PEDS024 
 
 
2020-7-15 
 
2 | P a g e  
 
Gainesville, FL 32608 
Telephone: (352) 273-9122 
Fax: (352) 294-8759 
Email: castillopa@ufl.edu  
 
   
 
 
                        Lucien Black, MD 
                                                            University of Florida 
1600 SW Archer Road 
Gainesville, FL 32608 
Telephone: (885)476-6722 
Email: vblack@ufl.edu 
  
 
                                     
                                                            William Slayton, MD 
                                                            University of Florida 
1600 SW Archer Road 
Gainesville, FL 32608 
Telephone: (352) 273-9120 
Email: slaytwb@ufl.edu 
 
                                                            John Fort, MD 
                                                            University of Florida 
1600 SW Archer Road 
Gainesville, FL 32608 
Telephone: (352) 392-0672 
Email: john.fort@ufl.edu  
                                                                   Nosha Farhadfar, MD 
                                                                   University of Florida 
1600 SW Archer Road 
Gainesville, FL 32610-0278 
P.O. Box 100278 
Telephone: (352) 265-0111 Ex:48506 
E-mail: nosha.farhadfar@medicine.ufl.edu 
 
Coy Heldermon, MD, PhD 
UF Center for Pediatric Neuromuscular and Rare Diseases 
2000 SW Archer Road, 2nd floor 
Gainesville, FL 32608 
Telephone: (877) 543-7783 
E-mail: coy.heldermon@medicine.ufl.edu 
 
Gustavo Maegawa, MD, PhD 
University of Florida 
1600 SW Archer Road 
Gainesville, FL 32610 
Telephone: (352)273-9731 
E-mail: gmaegawa@ufl.edu 

PEDS024 
 
 
2020-7-15 
 
3 | P a g e  
 
 
   
Protocol Manager:  
 
TBA 
 
Clinical Phase:         
Phase I/II 
 
 
IND Status: 
Not applicable 
 
Protocol Agent(s): 
busulfan, fludarabine, thiotepa, anti-thymocyte globulin, 
tacrolimus, methotrexate – IV only, mycophenolate mofetil  
 
Date of Original Protocol:  
April 4, 2018 
 
Date of Version 1.1 Protocol : 
September 1, 2018  
 
Date of Current Protocol                 July 15, 2020 
 
Version of Current Protocol: 
 1.2  
 
PROTOCOL REVISION RECORD 
 
 
Keywords: 
allogeneic, hematopoietic stem cell transplantation, 
pediatric, non-malignant disorders 
 
CONFIDENTIAL 
This protocol was designed and developed by the University of Florida College of Medicine information 
contained in this document is regarded as confidential and, except to the extent necessary to obtain informed 
consent, may not be disclosed to another party unless law or regulations require such disclosure.  Persons to 
whom the information is disclosed must be informed that the information is confidential and may not be 
further disclosed by them. 
 

PEDS024 
 
 
2020-7-15 
 
4 | P a g e  
 
Table of Contents 
1. 
ABBREVIATIONS ................................................................................................................................. 7 
2. 
PROTOCOL SIGNATURE PAGE .................................................................................................... 11 
3. 
STUDY SCHEMA I:  Phase I/II Thiotepa DOSE ESCALATION........................................... 12 
 ................................................................................................................................................................. 12 
4. 
PROTOCOL SYNOPSIS ..................................................................................................................... 13 
5. 
BACKGROUND ................................................................................................................................... 22 
6. 
OBJECTIVES ........................................................................................................................................ 27 
6.1  Primary ............................................................................................................................................. 27 
6.2  Secondary ........................................................................................................................................ 27 
6.3  Exploratory ..................................................................................................................................... 27 
7. 
STUDY DESIGN ............................................................................................................................... 27 
7.1 Study Overview............................................................................................................................... 27 
7.2 Subject Selection ............................................................................................................................ 28 
7.2.1  Number of Subjects .............................................................................................................. 28 
7.2.2  Inclusion Criteria .................................................................................................................. 28 
7.3 Exclusion Criteria ........................................................................................................................... 30 
7.4   Inclusion of women, children and minorities ................................................................... 31 
8.0 Study procedures .................................................................................................................................. 31 
8.1 Registration ...................................................................................................................................... 31 
8.2. Screening Procedures ................................................................................................................. 32 
8.3  Study Procedures  ......................................................................................................................... 32 
8.4. Pharmacogenomic Testing ........................................................................................................ 33 
8.5   Data Collection .............................................................................................................................. 34 
9.0 STUDY TREATMENT ...................................................................................................................... 34 
9.1 Conditioning Regimen and GVHD Prophylaxis .................................................................. 34 
9.2 Treatment Schedule/Administration..................................................................................... 36 
9.2.1 Thiotepa .................................................................................................................................... 36 
9.2.2 Fludarabine .............................................................................................................................. 36 
9.2.3 Busulfan (including PK) ...................................................................................................... 36 
9.2.4 Thymoglobulin® ..................................................................................................................... 38 
9.2.5 GVHD Prophylaxis ................................................................................................................. 38 
9.3 Stem Cell Infusion .......................................................................................................................... 39 
 

PEDS024 
 
 
2020-7-15 
 
5 | P a g e  
 
9.4 G-CSF ................................................................................................................................................... 39 
9.5 Allowed Concomitant Therapy ................................................................................................. 39 
9.6 Thiotepa Escalation based on graft failure .......................................................................... 39 
9.7 Supportive Care Guidelines ....................................................................................................... 42 
10. 
TREATMENT DISCONTINUATION .................................................................................... 42 
10.1 Removal of Subjects From Study ........................................................................................... 42 
10.2 Criteria For Study Treatment Discontinuation ................................................................ 42 
10.3 Replacement of Subjects ........................................................................................................... 42 
11. 
BIOLOGICAL SPECIMENS AND CORRELATIVES ..................................................... 43 
11.1 Source of Specimens .................................................................................................................. 43 
11.2 Preparation, Shipment and Storage of Specimens ......................................................... 43 
12. 
STUDY DRUG INFORMATION ............................................................................................. 43 
13.      ADVERSE EVENT REPORTING REQUIREMENTS ..................................................... 43 
13.1 Purpose ............................................................................................................................................. 43 
13.2 Determination of Reporting Requirements ............................................................................ 43 
14.      STATISTICAL METHODS ....................................................................................................... 45 
14.1  Sample Size Determination .................................................................................................... 45 
14.2  Analysis of Primary Endpoint ............................................................................................... 46 
14.4  Analysis of Exploratory Endpoints ...................................................................................... 48 
14.5  Analysis of Safety Data ............................................................................................................. 48 
14.6  Interim Analysis .......................................................................................................................... 48 
15.       DATA AND SAFETY MONITORING ................................................................................. 48 
15.1  Data Safety  Monitoring Board (DSMB) ............................................................................. 48 
15.2  Principal Investigator Responsibilities .............................................................................. 48 
16.     EMERGENCY PROCEDURES ................................................................................................. 49 
16.1  Emergency Contact .................................................................................................................... 49 
16.2  Emergency Treatment .............................................................................................................. 49 
17.      ADMINISTRATIVE, ETHICAL, and REGULATORY CONSIDERATIONS ......... 49 
17.1  Good Clinical Practice ............................................................................................................... 49 
17.2  Institutional Review Board .................................................................................................... 49 
17.3  Compliance with Laws and Regulations............................................................................ 50 
17.4  Delegation of Investigator Responsibilities ..................................................................... 50 
17.5  Subject Information and Informed Consent .................................................................... 50 

PEDS024 
 
 
2020-7-15 
 
6 | P a g e  
 
17.6  Confidentiality ............................................................................................................................. 51 
17.7  Protocol Amendments .............................................................................................................. 51 
17.8  Case Report Forms .................................................................................................................... 51 
17.9  Record Retention........................................................................................................................ 51 
18.   REFERENCES ...................................................................................................................................... 52 
19.  APPENDICES ...................................................................................................................................... 55 
APPENDIX A: SCHEDULE OF ASSESSMENTS ............................................................................. 55 
APPENDIX B: MODIFIED GLUCKSBERG STAGING CRITERIA FOR ACUTE GRAFT 
VERSUS HOST DISEASE ...................................................................................................................... 57 
APPENDIX B2: CGVHD GRADING AS MIDLE, MODERATE AND SEVERE BY NIH 2014 
CONSENSUS CONFERENCE CRITERIA .......................................................................................... 58 
APPENDIX C: STUDY REQUIRED SPECIMENS, VOLUMES, HANDLING AND SHIPPING 
INSTRUCTIONS ...................................................................................................................................... 59 
APPENDIX D: PROTOCOL DRUGS ................................................................................................... 61 
1. 
Busulfan Source and Pharmacology (Busulfan-Injection (07/01/2015) 
(Busulfex® NSC #750) ......................................................................................................................... 61 
2. 
Fludarabine Source and Pharmacology (Fludarabine (05/09/2011) (Fludara®, 
fludarabine phosphate, (2-fluror-ara-AMP) NSC# 3121887) ............................................. 63 
3. 
Thiotepa Source and Pharmacology (Thiotepa 11/15/2016) (Tepadina, Tespa, 
Thiophosphamide, Triethylenethiophosphoramide Tspa, WR-45312) NSC #6396 .. 67 
4. 
Anti-thymocyte Source and Pharmacology (Anti-thymocyte Globulin (Rabbit) 
(05/06/11) (Rabbit ATG, RATG – Rabbit, Antithymocyte Globulin, Thymoglobulin®) 
NSC #720095) ........................................................................................................................................ 70 
5. 
Tacrolimus Source and Pharmacology (Tacrolimus (05/10/2011) (FK-506, 
Prograf®) NSC #717865 ..................................................................................................................... 72 
6. 
Methotrexate Source and Pharmacology (Methotrexate – IV only 
(02/29/2012) (MTX, amethopterin) NCS #000740 ............................................................... 75 
7. 
Mycophenolate Mofetil Source and Pharmacology (Mycophenolate Mofetil – 
(12/05/16)) CellCept®, MMF, RS-61443) NSC# 724229 ..................................................... 77 
     APPENDIX E.  STANDARD OF CARE NEUROPYSCHOLOGY TESTING BY AGE .............. 81 
 
 
 
 
 
 
 
 
 

PEDS024 
 
 
2020-7-15 
 
7 | P a g e  
 
1. ABBREVIATIONS  
ABO 
blood group system 
ADL 
activities of daily living 
AE 
adverse event 
ANC 
absolute neutrophil count 
araA 
L-arabinose isomerase  
ARDS 
acute respiratory distress syndrome 
ATN 
acute nephrotic toxicity 
AUC 
area under curve 
BM 
bone marrow 
BMT 
bone marrow transplant 
Bu 
busulfan 
BUN 
blood urea nitrogen 
C 
centigrade 
CBC 
complete blood count 
cGVHD 
chronic graft vs host disease 
CI 
confidence interval 
CIBMTR 
Center for International Blood & Marrow Transplant Research 
CMV 
cytomegalovirus 
CNS 
central nervous system 
CRF/eCRF 
case report form/electronic case report form 
CSF 
cerebrospinal fluid 
CSTD 
closed-system transfer device 
CTEP-AERS 
Cancer Therapy Evaluation Program – Adverse Event Reporting System 
CTCAE 
Common Terminology Criteria for Adverse Events 
CTMS 
clinical trials management system 
CTO 
Clinical Trials Office 
D5W 
dextrose 5% in water 
DFS 
disease-free survival 
DL 
deciliter 
DLCO 
diffusing capacity of the lungs for carbon monoxide  
DLT 
dose-limiting toxicity 
DMA 
dynamic mechanical analysis 
DNA 
deoxyribonucleic acid 
DSMB 
Data Safety Monitoring Board 
EBV 
Epstein-Barr virus 
EDTA 
ethylenediaminetetraacetic acid 
e.g. 
example 
F 
fahrenheit 
FACT 
Foundation for Accreditation of Cellular Therapy 

PEDS024 
 
 
2020-7-15 
 
8 | P a g e  
 
FDA 
Food and Drug Administration 
FDAAA 
Food and Drug Administration Amendments Act  
FEV1 
forced expiratory volume in one second 
FLU 
fludarabine 
FSH 
follicle stimulating hormone 
GCP 
Good Clinical Practice 
G-CSF 
Granulocyte colony stimulating factor 
GI 
gastrointestinal 
GVHD 
Graft vs Host Disease 
HBV 
hepatitis B virus  
HCO-60 
polyoxyethylene castor oil derivative 
HCV 
hepatitis C virus 
HHV-6 
human herpes virus 6  
HIV 
Human immunodeficiency virus 
HLA 
human leukocyte antigen 
HRT 
hormone replacement therapy 
HSCT 
hematopoietic stem cell transplant 
IATA 
International Air Transport Association 
IBW 
ideal body weight 
ICAO 
International Civil Aviation Organization  
ICF 
informed consent form 
ICH 
International Conference on Harmonization 
IgA 
immunoglobulin A 
IgG 
immunoglobulin G 
IgM 
Immunoglobin M 
IMPDH 
Inosine-5′-monophosphate dehydrogenase 
IND 
investigational new drug application  
IRB 
Institutional Review Board 
ITP 
idiopathic thrombocytopenic 
IU 
international unit 
IV 
intravenous 
JC 
John Cunningham 
kg  
kilogram(s) 
KM 
Kaplan-Meier 
L 
liter 
LFT 
liver function test 
m 
meter 
MED 
minimum effective dose 
MF1 
Mean fluorescence intensity 
mg 
milligram 

PEDS024 
 
 
2020-7-15 
 
9 | P a g e  
 
MMF 
mycophenolate mofetil 
MPA 
mycophenolic acid 
MPAG 
7-O-MPA-Beta-Glucuronide (metabolized mycophenolic acid) 
MRD 
minimal residual disease 
MRI 
magnetic resonance imaging 
MTD 
maximum tolerated dose 
MTX 
methotrexate 
NaCL 
sodium chloride 
NK-SCID 
natural killer cell-positive severe combined immunodeficiency disease 
NSAIDS 
non-steroidal anti-inflammatory drugs 
NS 
normal saline 
NSAE 
non-serious adverse event 
NSC 
identifying number assigned an agent or product  
NTPR 
National Transplant Pregnancy Registry 
OS 
overall survival 
PB 
peripheral blood 
PBSC 
peripheral blood stem cell 
PCR 
polymerase chain reaction 
PFT 
pulmonary function test 
pH 
potential of hydrogen 
PHI 
protected health information 
PI 
principal investigator 
PK 
pharmacokinetics 
PML 
progressive multifocal leukoencephalopathy 
PO 
by mouth 
rATG 
active rabbit antithymocyte globulin  
SAE/SE 
serious adverse event 
SD 
standard deviation 
SOC 
standard of care 
SOS 
sinusoidal obstruction syndrome 
SNP 
single nucleotide polymorphisms 
TBI 
total body irradiation 
TID 
three times a day 
TNC 
total nucleated cell 
UF 
University of Florida 
ULN 
upper limit of normal 
μ 
micron 
μm 
micrometer 
US 
United States 
USP 
United States Pharmacopeia 

PEDS024 
 
 
2020-7-15 
 
10 | P a g e  
 
VOD 
veno-occlusive disease 
Vss 
volume steady state 
WOCBP 
women of childbearing potential 
 

PEDS024 
 
 
2020-7-15 
 
11 | P a g e  
 
2. PROTOCOL SIGNATURE PAGE 
 
Feasibility Phase I/II Radiation-Free Allogeneic Hematopoietic Stem-Cell Transplantation 
(HSCT) for Children with High-Risk Hematologic Malignancies 
 
Principal Investigator: 
 
 
 
 
 
 
Signature of Investigator 
 
Biljana Horn, MD 
 
Date 
 
 
 
Printed Name of Investigator 
 
University of Florida 
 
 
 
Name of Facility 
 
Gainesville, Florida 
 
 
 
Location of Facility (City/State) 
 
 
 
By my signature, I agree to personally supervise the 
conduct of this study and to ensure its conduct in 
compliance with the protocol, informed consent, IRB 
procedures, the Declaration of Helsinki, ICH Good 
Clinical Practices guidelines, and the applicable parts of 
the United States Code of Federal Regulations or local 
regulations governing the conduct of clinical studies. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

PEDS024 
 
 
2020-7-15 
 
12 | P a g e  
 
3. STUDY SCHEMA I:  PHASE I/II THIOTEPA DOSE 
ESCALATION        
 
 
 
                                                
                                    
 
 
 
 
 
 
 
 
 
 
 
 
 
                                                                                                                                                           
                                                                                                                                                           
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
1 failure of 
engraftment by day 
42,  escalate 
Thiotepa to twice 
daily in both 
Group A and B 
enroll 10 subjects, 
first 5 one at a time 
All subjects engrafted 
by day 42, enroll 
additional 5 subjects   
Enter 5 subjects on Bu/Flu/rATG single 
Thiotepa dose, one at a time 
1 failure of 
engraftment by day 
42, escalate Thiotepa  
to twice daily in 
Group B only, enter 
10 subjects, first 5 
one at a time
>1  failure of 
engrafment, 
close entire 
study 
≤1 failure of 
engraftment, 
enroll 
additional 5 
subjects in 
once a day 
Thiotepa arm 
Enter 5 subjects on Bu/Flu/rATG single 
Thiotepa dose, one at a time 
All subjects 
engrafted by day 
42 enroll 
additional 5 
subjects   
≤ 1 failure of 
engraftment 
among 10 
subjects, enroll 
additional 5 
subjects in 
twice a day 
Thiotepa arm 
 >1  failure 
of 
engraftment
close group 
B only 
≤ 1 failure of 
engraftment, 
enroll 
additional 5 
subjects in 
once a day 
Thiotepa,  
≤ 1 failure of 
engraftment, 
enroll 
additional 5 
subjects in 
twice a day 
Thiotepa arm 
Group A 
Fully matched (10/10) BM 
or PBSC sources only 
Group B 
Mismatched BM or PBSC, all 
cords, T-cell depleted 

PEDS024 
 
 
2020-7-15 
 
13 | P a g e  
 
 
 
4. PROTOCOL SYNOPSIS 
 
Title: 
Phase I/II  Feasibility Study of Busulfan Fludarabine and 
Thiotepa Conditioning Regimen for Allogeneic Hematopoietic 
Stem-Cell Transplantation (HSCT) for Children with Non-
Malignant Disorders  
 
Funding 
Organization: 
TBD 
Rationale: 
Hematopoietic stem cell transplantation is the only curative choice 
for a number of inherited bone marrow failure syndromes, 
hemoglobinopathies, metabolic disorders and primary immune 
deficiencies. While survival of these patients is typically better than 
survival of patients with malignancies, toxicities of conditioning 
regimens and failure of engraftment remain challenges. Most 
children with non-malignant disorders present with normocellular or 
even hypercellular bone marrow, posing a barrier to engraftment and 
requiring intensive conditioning. Commonly used backbone of 
busulfan and fludarabine, although well tolerated, results in variable 
engraftment, in particular with mismatched unrelated donors and 
cord blood recipients. In this study we test 2 dose levels of thiotepa 
(5 mg/kg and 10 mg/kg) added to the backbone of targeted reduced 
dose IV busulfan, fludarabine and rabbit anti-thymocyte globulin 
(rATG) in order to determine the minimum effective dose required 
for reliable engraftment. Subjects are stratified in groups A and B 
based the risk of graft failure.  
Objectives: 
Primary: 
• Determine the minimum effective dose (MED) of thiotepa in 
combination with reduced-dose busulfan, fludarabine and rATG 
required to achieve engraftment in >90% subjects undergoing 
hematopoietic stem cell transplantation for non-malignant 
disorders. The MED will be determined for subjects stratified in 
group A (lower risk of graft failure) and group B (high risk of 
graft failure). The 2 groups will be merged if group A requires 
escalation to the higher dose level. 
Secondary: 
• Evaluate the risk of early (by day 42) and late (by day 365) graft 
failure/rejection. 
• Evaluate 12 and 24 month disease free survival (DFS) and 
overall survival (OS) of children undergoing HSCT using 
busulfan/fludarabine/rATG/thiotepa conditioning regimen. 

PEDS024 
 
 
2020-7-15 
 
14 | P a g e  
 
• Evaluate transplant-related mortality at 100 days and 12 months 
post-transplant. 
• Evaluate rate of acute (Grade II-IV) graft-versus-host disease 
(GVHD) by 12 months post-transplant 
• Evaluate rate of chronic GVHD by 24 months post-transplant 
• Evaluate transplant-related complications at 12 and 24 months 
post-transplant 
 
Exploratory objectives: 
• Describe changes in neurocognitive functioning compared to pre-
transplant 
• Describe association between pharmacogenomics of genes 
involved in metabolism of chemotherapy agents and toxicity 
• Describe immune reconstitution following this conditioning 
regimen 
 
 
Study Design: 
This is a Phase I/II feasibility study of allogeneic stem cell 
transplantation using a conditioning regimen which tests 2 different 
doses of thiotepa added to the backbone of reduced-dose busulfan, 
fludarabine and rATG for children with non-malignant disorders in 
order to achieve reliable engraftment.  
Following donor identification and standard of care pre-transplant 
evaluation, subjects will start conditioning regimen.  
The study builds in rules for escalation of thiotepa dose based on 
graft failure by day 42. Patients are stratified in two groups A (lower 
risk of graft failure) and B (higher risk of graft failure). Patients 
undergoing 10/10 HLA matched bone marrow and peripheral blood 
transplants are assigned to group A, and patients receiving <10/10 
matched bone marrow or peripheral blood, or receiving cord blood, 
even if fully matched, as well as T-cell depleted transplants, are 
assigned to group B.  
If criteria, based on graft failure rate, for thiotepa dose escalation are 
met first in group A, which has a lower risk of graft failure, thiotepa 
dose will be escalated for all subjects (group A and B). If criteria for 
thiotepa dose escalation are met first in group B, dose will be 
escalated only in group B, and group A will continue enrolling 
patients at a lower dose level. 
Treatment will start in each group with addition of a single dose of 
thiotepa (5mg/kg/day) for the first 5 subjects.  
If failure of engraftment has not been observed by day 42, 5 
additional subjects will be enrolled in the same dose level. If 0 or 1 
failure of engraftment is observed among the first 10 subjects of each 

PEDS024 
 
 
2020-7-15 
 
15 | P a g e  
 
dose level, 5 additional subjects will be enrolled at the same dose 
level. 
If a failure of engraftment is observed among the first 5 subjects, the 
dose of thiotepa will be increased to twice a day and 10 subjects 
enrolled at that dose level.  If there is 0 or 1 graft failure among the 
first 10 subjects, additional 5 subjects will be enrolled in twice a day 
thiotepa arm.  If there are 2 or more graft failures among the 10 
subjects at twice a day thiotepa dose level in group A, the entire 
study will be closed to further accrual. If there are 2 or more graft 
failures in group B, only that group will be closed to further accrual.  
 
With this design, a minimum of 15 subjects will be enrolled at each 
effective dose level. The study may confirm a different MED of 
thiotepa for group A and group B. 
 
Exploratory objectives include evaluating neurocognitive functioning 
following this conditioning regimen and assessment of immune 
reconstitution. We will also gather additional information on 
pharmacogenomics and possible associations between genes 
involved in metabolism of busulfan, fludarabine and thiotepa and 
toxicity and efficacy. 
Accrual Goal: 
The study will enroll a minimum of 15 patients at each effective 
thiotepa dose level. The maximum number of patients will be 35. 
Inclusion 
Criteria: 
Individuals eligible for Phase I/II study participation must meet the 
following criteria:  
• Subjects with any non-malignant disorder known to benefit 
from allogeneic transplantation for cure or amelioration of 
symptoms of the underlying disease. The following groups of 
diagnoses are eligible: 
• Hemoglobinopathies (e.g. thalassemia or sickle cell 
disease),  
• Cytopenias (e.g.Diamond-Blackfan anemia, congenital or 
acquired neutropenia, congenital or acquired 
thrombocytopenia, congenital or acquired anemia, and 
others, regardless clonality),  
• Hemophagocytic lymphohystiocytosis, 
• Primary immunodeficiencies (e.g. Wiscott Aldrich 
Syndrome, chronic granulomatous disease, common 
variable immune deficiency, X-linked 
lymphoproliferative disease, NK+ severe combined 
immune deficiencies), 

PEDS024 
 
 
2020-7-15 
 
16 | P a g e  
 
• Metabolic disorders (Hurler’s syndrome, mannosidosis, 
adrenal leuko-dystrophy)  
• Other non-malignant disorders for which there is 
      published evidence that HSCT is a curative therapy. 
• Donor Requirements 
• Related or unrelated donor who is suitable and willing 
to donate bone marrow or peripheral blood stem cells. 
HLA typing should be done by high-resolution typing 
at A, B, C, DrB1 and DQ loci and the donor should be 
a ≥8/10 match (with one antigen/allele mismatch 
allowed at A, B, or C-loci and other at DQ loci). 
• Cord blood units must be matched at a minimum of 
6/8 antigens/alleles at A, B, C and DrB1 loci. High 
resolution typing at all loci is required. The minimum 
TNC dose pre-cryopreservation must be ≥3.7 
x10^7/kg of recipient’s weight, if a single cord blood 
unit is used, or at least 2x10^7/kg per unit, if two cord 
blood units are used. The mismatches cannot be at the 
same loci (e.g. double A mismatch).  
• Haploidentical related stem cell donor who is suitable 
and willing to donate peripheral blood stem cells. T-
cell depletion is required if haploidentical donors are 
used.   
 
•  Adequate organ function defined as: 
• Cardiac: ejection fraction ≥55% or shortening fraction 
≥30% 
• creatinine clearance ≥70 ml/min/1.73m2 
• Pulse oximetry >95% on room air or FEV1/DLCO 
>60% 
• LFTs < 3 x ULN, Total bilirubin <3 mg/dl (unless due 
to non-hepatic cause (e.g. Gilbert’s syndrome or 
hemolysis) 
• Lansky/Karnofsky score ≥60%                          
• Age: ≥3 months and ≤39 years of age at the time of signing 
informed consent 
• Written informed consent obtained from the subject or 
parental/guardian permission ± child’s assent per institutional 
guidelines  

PEDS024 
 
 
2020-7-15 
 
17 | P a g e  
 
• Women of childbearing potential (WOCBP) must be using an 
adequate method of contraception to avoid pregnancy for at 
least 1 month after completion of conditioning. 
WOCBP include any woman who has experienced menarche 
and who has not undergone successful surgical sterilization 
(hysterectomy, bilateral tubal ligation, or bilateral 
oophorectomy) or who is not post-menopausal. Post-
menopause is defined as: 
• Amenorrhea that has lasted for ≥ 12 consecutive 
months without another cause, or 
• For women with irregular menstrual periods who are 
taking hormone replacement therapy (HRT), a 
documented serum follicle-stimulating hormone 
(FSH) level of greater than 35 mIU/mL. 
• Males with female partners of childbearing potential must 
agree to use physician-approved contraceptive methods (e.g., 
abstinence, condoms, or vasectomy) for at least one month 
after completion of conditioning. 
Exclusion 
Criteria: 
Subjects with any of the following will not be eligible for study 
participation:  
• Diagnoses that do not require myeloablative transplant for 
cure (e.g. NK- SCID patients), unless they previously did not 
engraft with non-myeloablative or reduced intensity 
conditioning transplant. 
• Known or suspected sensitivity to chemotherapy or radiation 
(e.g  Fanconi’s anemia, Dyskeratosis congenita, Ligase IV 
deficiency, etc).   
• Cytopenias with increased blasts (>5%) 
• Patients with fast-progressing neurodegenerative disorders 
(e.g. Krabbe disease or adrenal leukodystrophy with Loes 
score of ≥10) 
• Presence of anti-donor HLA antibodies (positive anti-donor 
HLA antibody is defined as a positive cross-match test of any 
titer (by complement-dependent cytotoxicity or flow 
cytometric testing) or the presence of anti-donor HLA 
antibody to the high expression loci HLA-A, B, C, DRB1 with 
mean fluorescence intensity (MFI)>3000 by solid phase 
immunoassay. This will be measured before the final donor 
selection.  

PEDS024 
 
 
2020-7-15 
 
18 | P a g e  
 
• Prior allogeneic stem cell transplant, except for patients with 
immune 
deficiencies 
who 
underwent 
previous 
non-
myeloablative or reduced intensity transplants. 
• Haploidentical donor using in vivo T-cell depletion (e.g. post 
transplant cyclophosphamide). 
• Uncontrolled bacterial, viral, or fungal infection at the time of 
enrollment. Uncontrolled is defined as currently taking 
medication and with progression or no clinical improvement 
on adequate medical treatment. 
• Seropositive for HIV 
• Active Hepatitis B or C determined by a detectable viral load 
of HBV or HCV by PCR 
• Bridging fibrosis or liver cirrhosis 
• Females or males of childbearing potential who are unwilling 
or unable to use an acceptable method to avoid pregnancy for 
the entire study period and for at least 1 month after the end of 
conditioning 
• Females who are pregnant or breastfeeding 
• History of any other disease, metabolic dysfunction, physical 
examination finding, or clinical laboratory finding giving 
reasonable suspicion of a disease or condition that 
contraindicates the use of protocol therapy or that might affect 
the interpretation of the results of the study or that puts the 
subject at high risk for treatment complications, in the opinion 
of the treating physician. 
• Subjects demonstrating an inability to understand the study 
and comply with the study and/or follow-up procedures. 
 
Efficacy 
Assessments: 
 
Primary Endpoint: 
• Determine the minimum effective dose (MED) of thiotepa in 
combination with reduced-dose busulfan, fludarabine and rATG 
required to achieve engraftment in >90% subjects undergoing 
hematopoietic stem cell transplantation for non-malignant 
disorders. The MED will be determined for subjects stratified in 
group A (lower risk of graft failure) and group B (high risk of 
graft failure). The two groups will be merged if the group A 
requires escalation to the higher dose level. 

PEDS024 
 
 
2020-7-15 
 
19 | P a g e  
 
Effective dose of Thiotepa in combination with busulfan/fludarabine 
and rATG is defined as a dose that results in engraftment of  >90% 
study subjects by day 42 with acceptable toxicity, defined as  ≤20% 
grade 5 toxicities and ≤40% combined grade 4 and 5 toxicities across 
all subjects treated with the same dose level. 
Definition of engraftment failure used for dose escalation in this 
study includes:  
• Failure to achieve sustained ANC recovery of >0.5x10^9/L 
with evidence of donor chimerism by day 42, in light of 
absence of myelosupressive medications (e.g. ganciclovir), or   
• Engraftment with subsequent loss of donor chimerism to <5% 
by day 42, or 
• Need for the 2nd conditioned transplant by day 42 to correct 
manifestations of disease or cytopenia.  
 
Secondary Endpoints: 
Evaluate the risk of early (by day 42) and late (by day 365) graft 
rejection/failure 
Proportion and associated confidence interval (CI) of all subjects 
who initiated conditioning regimen and have sustained engraftment 
failure (as outlined above) at 42 days and at 1 year post transplant.  
. 
  
 
Proportion and associated CI of subjects who initiated conditioning 
regimen and who die due to a cause unrelated to the underlying 
disease. Kaplan-Meier plots will also be generated. 
• Evaluate 12 and 24 month disease free survival 
(DFS)/event-free-survival  and overall survival (OS) of 
children undergoing HSCT using 
busulfan/fludarabine/rATG/thiotepa conditioning 
regimen 
Proportion and associated confidence interval (CI) of all 
subjects who initiated conditioning regimen and are alive 
(OS) and without evidence of underlying disease (DFS) at 12 
months post-transplant will be calculated. KM estimates of  
OS and DFS will also be used. OS is defined as time from 

PEDS024 
 
 
2020-7-15 
 
20 | P a g e  
 
transplant (day 0) until date of death or date of last contact 
for those who have not died. DFS/event-free survival is 
defined as time from transplant (day 0) until  disease 
recurrence/progression, or date of death from any cause, or 
date of last contact for those who are disease-free. Disease 
progression will follow-standard of care definitions for each 
disorder. In general, disease-free-survival implies 
improvement of reversible manifestations of a disorder  and 
arrest in further progression of symptoms in light of presence 
of donor engraftment and immune reconstitution. 
• Evaluate transplant-related mortality at 100 days and 12 
months post-transplant 
Proportion and associated CI of subjects who initiated 
conditioning regimen and who die due to a cause unrelated to 
the underlying disease. Time to transplant-related mortality is 
defined as time from transplant (day 0) until date of transplant 
related mortality. Those who die from other causes will be 
censored at date of death; those who were alive at last contact 
will be censored at their date of last contact. 
 
Rate of acute (grade II-IV) graft-versus-host disease by 12 
months post-transplant: 
Proportion and associated CI of subjects who reached engraftment 
(sustained ANC>500 x 3 days by day 42) and developed acute 
GVHD grade II-IV (by modified Glucksberg criteria) (Appendix B) 
 
Rate of chronic graft-versus host disease by 24 months post-
transplant: 
Proportion and associated CI of subjects who reached engraftment 
and developed moderate or severe GVHD by 2014 NIH Consensus 
Criteria (Appendix B) 
 
Transplant-related complications at 12 and 24 months post-
transplant:  
Complications gathered via CIBMTR post-transplant form will be 
tabulated and described by treatment received 
 
Exploratory endpoints: 

PEDS024 
 
 
2020-7-15 
 
21 | P a g e  
 
 
 
 
Analyses of exploratory objectives (neurocognitive testing, 
pharmacogenomics, and immune reconstitution) will utilize 
descriptive statistics, calculations of predictive values, homogeneity 
testing, regression analyses and others.                                                   
Statistical 
Considerations: 
This is a Phase I/II study feasibility study of a new HSCT 
conditioning regimen for pediatric high-risk non-hematologic 
malignancies. The study is designed so that the minimum 
informative number of subjects is enrolled at each dose level. 
See statistical section for details.                                                             
Estimated 
Enrollment 
Period: 
3 years 
                                                                                              
Estimated Study 
Duration: 
4 years 
                                                                                               

PEDS024 
 
 
2020-7-15 
 
22 | P a g e  
 
5. BACKGROUND  
 
Hematopoietic Stem Cell Transplant for Pediatric Non-malignant Disorders   
    
 
Hematopoietic stem cell transplant is the treatment of choice and the only curative therapy for a 
number of non-malignant disorders, including hemoglobinopathies, inherited bone marrow 
failure syndromes, primary immunodeficiency disorders and metabolic disorders. These patients 
present different challenges than patients with malignant disorders. They are typically younger 
and often with infections or organ failures related to the underlying disease (Slatter et al, 2015).  
Most children with non-malignant disorders have normocellular or even hypercellular bone 
marrow which poses a barrier to engraftment. As a result, these children may require more 
aggressive conditioning to establish sustained donor engraftment. Due to a variety of disorders 
and small number of patients in each disease category, disease-specific regimens are rare and  
designed for disorders such as Fanconi’s anemia, or dyskeratosis congenita that require 
significant reduction in conditioning regimens due to known sensitivity to 
radiation/chemotherapy. Also, patients with severe combined immune deficiencies and those 
with severe aplastic anemia typically do not require myeloablative conditioning. Patients 
requiring specialized conditioning regimens or those for whom a disease-specific alternative 
protocol is available, are excluded from this study. This study will determine the dose of thiotepa 
(5 mg/kg/day or 10 mg/kg/day), added to the backbone of reduced dose of targeted intravenous 
busulfan, fludarabine and thymoglobulin, required to achieve sustained engraftment in children 
with non-malignant disorders.  
 
Non-TBI Conditioning Regimens  
 
Due to known age-related effects of total body radiation on neurocognitive functioning and 
growth (Willard et al, 2014; Kelly et al, 2017), children with non-malignant disorders have 
historically been conditioned with non-TBI regimens, originally designed for patients with 
myeloid malignancies.  
 
The first well-described and widely used non-TBI regimen was oral busulfan and 
cyclophophamide (Santos et al, 1983; Tutschka et al, 1987). Veno-occlusive liver disease and 
mucositis were the major toxicities of busulfan/cyclophosphamide conditioning. This was 
ameliorated by introduction of intravenous busulfan which avoids hepatic first-pass effect and 
high busulfan concentrations in portal-hepatic venous system. Use of intravenous busulfan 
resulted in reduction in VOD (8% vs 33%) and 100-day mortality (Kashyap et al, 2002). As oral 
busulfan was changed to intravenous formulation, fludarabine was introduced as a replacement 
for cyclophosphamide in combination with busulfan. 

PEDS024 
 
 
2020-7-15 
 
23 | P a g e  
 
In 1998 Slavin introduced the combination of fludarabine and low-dose busulfan (8mg/kg) as a 
non-myeloablative conditioning for malignant and non-malignant disorders (Slavin et al, 1998). 
Subsequently a number of studies tested IV busulfan and fludarabine combination mainly for 
myeloid malignancies, and confirmed that busulfan and fludarabine compare favorably to 
busulfan and cyclophosphamide (Bronhauser et al, 2003; Andersson et al, 2008). In 2002 Russell 
introduced single daily busulfan dosing of 3.2 mg/kg for 4 days  infused over 3 hours on days -5 
to -2, fludarabine, 50 mg/m2 for 5 days on days -6 to -2, and rATG 4.5 mg/kg for 3 days prior to 
transplant (Russell et al, 2002, 2013). Daily busulfan and fludarabine has been previously studied 
in pediatric patients, including infants with nonmalignant disorders. In a study of 33 infants with 
primary immune deficiencies and metabolic disorders only 2 daily busulfan doses were given, 
targeting the 2 exposure levels  (AUC  of 4000 and 5000 µM/L*min/day), in combination with 
fludarabine 30 mg/sqm x 5 and rabbit ATG (2 mg/kg/day x 4). Primary or secondary graft failure 
occurred in 6 out of 12 (50%) of patients targeted at 4000 µM/min but only in 1/21 (5%) who 
had busulfan target increased to 5000 µM/min. However, 4 patients receiving umbilical cord 
blood in the higher busulfan AUC also received 5 mg/kg of Thiotepa a day prior to 
busulfan/fludarabine (Ward et al, 2015). Additional pediatric data on use of daily busulfan 
(targeted 5400 µM/L*min/day) fludarabine (160 mg/m2) and serotherapy (10 mg/kg of ATG for 
unrelated and cord blood recipients) comes from Netherlands (Bartelink et al, 2014). They 
enrolled 64 pediatric patients with the median age of 4.8 years (range 0.16-18.6), 37 of whom 
had nonmalignant disorders (19 had primary immunodeficiencies and 16 metabolic disorders). 
Outcomes of these patients were compared with 50 patients receiving busulfan 
cyclophosphamide with or without melphalan, also receiving rATG at 10 mg/kg, if unrelated 
donors were used. A large number of patients received umbilical cord blood transplant in both 
groups (total of 75).  Probability of engraftment was 98%. Busulfan/fludarabine/rATG had 
similar survival but improved toxicity profile when compared to busulfan cyclophosphamide ± 
melphalan group, in particular the incidence of lung injury, veno-occlusive liver disease, 
cGVHD, adenovirus infection and HHV6 reactivation was lower in busulfan/fludarabine group. 
Also, duration of neutropenia was reduced (11 vs 22 days) and fewer transfusions were required 
in busulfan/fludarabine group. The authors conclude that their excellent engraftment rate with 
busulfan/fludarabine was related to targeting higher AUC (~5400 µM/L*min/day) and use of 
ATG (Bartelink et al, 2014). 
 
Two other pediatric studies using targeted busulfan/fludarabine and rATG or alemtuzumab, 
targeting lower AUC in pediatric non-malignant disorders (equivalent of 3500µM/L*min/day) 
encountered high rejection rates in (21% in rATG and 9% in alemtuzumab group). Patients with 
non-malignant disorders receiving unrelated donor transplant, in particular mismatched unrelated 
donor transplant were at a particularly high risk of rejection (Law et al, 2012; Horn et al, 2006). 
Similarly, Horowitz described 10 patients with myeloid malignancies who received daily 
busulfan and fludarabine without ATG, followed by double cord blood transplantation. Graft 
failure rate was 80% and study was discontinued prematurely (Horowitz et al, 2008). 

PEDS024 
 
 
2020-7-15 
 
24 | P a g e  
 
A large study of pediatric reduced-toxicity regimens using lower doses of busulfan, fludarabine 
and rATG or alemtuzumab also confirmed high risk of graft failure in pediatric patients receiving 
unrelated cord blood transplants and patients with non-malignant disorders who did not receive 
chemotherapy previously (Satwani et al, 2013) 
 
Role of Thiotepa in Conditioning for Pediatric Non-Malignant Disorders 
Thiotepa has been introduced in allogeneic bone marrow transplant conditioning regimens in 
early 1990s (Aversa et al, 1991; Przepiorka et al, 1994), in combination with busulfan and 
cyclophosphamide, with a goal of enhancing engraftment. Animal studies suggested that, in 
addition to myeloablation, thiotepa has immune suppressive properties. Thiotepa is toxic to 
committed progenitor population, while primitive stem cells remained relatively resistant to this 
drug (Down et al, 1998).  In previously described study of 33 infants with primary immune 
deficiencies and metabolic disorders that used only 2 daily busulfan doses, fludarabine and rabbit 
ATG; targeting of busulfan to the AUC of 5000 µM/L*min/day and addition of a single dose of 
Thiotepa for umbilical cord blood recipients (5mg/kg) reduced failure of engraftment from 50% 
to 5% (Ward et al, 2015). 
 
Addition of two doses of Thiotepa (5 mg/kg/day x 2) to daily Busulfan (3.2 mg/kg/day x 3), 
fludarabine 150 mg/m2 over 3 days and rATG 8 mg/kg, resulted in 94% cumulative engraftment 
rate in a 88 adult and pediatric patients with hematologic malignancies receiving single-umbilical 
cord blood transplantation from unrelated donors (Sanz J et al, 2012). The study concluded that 
the addition of thiotepa and ATG were crucial for the improvement of engraftment. 
 
Treosulfan in Pediatric Conditioning Regimens 

PEDS024 
 
 
2020-7-15 
 
25 | P a g e  
 
Since 2004, treosulfan has increasingly replaced busulfan in pediatric transplantation for 
malignant and non-malignant disorders in Europe, due to its good myeloablative and 
immunoablative properties and toxicity profile. Treosulfan is believed to have less CNS 
toxicities and hepatic toxicities than busulfan; however comparative study has not been done yet. 
Slatter described a large cohort of 316 pediatric patients with non-malignant disorders who 
recieved treosulfan as a part of conditioning regimen in Europe between 2005 and 2010 (Slatter 
et al, 2015). Thirty percent of patients received transplant from matched related donor and others 
from matched or mismatched unrelated donors. The median total dose of treosulfan was 42 g/m2 
divided in 3 doses. In addition to treosulfan, 106 patients in this study received fludarabine 150 
mg/m2, 98 received cyclophosphamide 200 mg/kg, and 104 received fludarabine and thiotepa 
(median dose 8mg/kg). The cohort receiving fludarabine and thiotepa in addition to treosulfan 
had the best 3-year overall survival (88%, SE 4%), followed by patients receiving fludarabine 
(85%, SE 4%), and those receiving cyclophosphamide/treosulfan combination (3-year OS 79%, 
SE 4%). Recent US experience using treosulfan in combination with fludarabine and rabbit anti-
thymocyte globulin indicated excellent engraftment among 14 patients with bone marrow failure 
disorders who received bone marrow or peripheral blood stem cell transplants (Burroughs et al, 
2017). 
 
Rationale for selected conditioning regimen 
 
Since treosulfan is not an FDA approved drug and is not available for studies in the US, 
medications with the best track record for pediatric non-malignant disorders include carefully 
targeted intravenous busulfan, fludarabine and rATG. This combination provides sufficient 
engraftment in children receiving fully matched related or unrelated donor transplants. However, 
in patients receiving mismatched unrelated, and in particular mismatched cord blood 
transplantation, additional immune and myeloablation is needed.  As previously described, 
thiotepa  in combination with reduced dose of busulfan and fludarabine, or treosulfan and 
fludarabine, has a good track record of facilitating engraftment in patients receiving cord blood 
transplant for malignant and non-malignant disorders (Sanz J, et al, 2012, Ward et al, 2015, 
Slatter et al, 2015).    
 

PEDS024 
 
 
2020-7-15 
 
26 | P a g e  
 
Literature indicates importance of optimal exposure to conditioning agents such as busulfan and 
fludarabine, because exposure predicts survival and toxicity. Individualized model-based 
algorithms for busuflan  and fludarabine clearance that incorporate body size, age and creatinine 
level (such as Insight Rx software), provide improved targeted therapy when compared to 
stratified weight or age-based regimens alone (Bartelink IH, et al, 2016, Ivaturi V, et al, 
2017).This protocol will be offered to infants diagnosed with metabolic disorders during 
newborn screening for whom there are scarce data on standard dosing of chemotherapy agents. 
In similar situation (CSIDE multi-institutional study for infants undergoing transplant for severe 
combined immune deficiencies), the Pediatric Blood and Marrow Transplant Consortium  has 
endorsed using Insight Rx platform which provides individualized dose for fludarabine, busulfan 
and rATG based on desired drug exposure (area under the curve).  
 
This study adds thiotepa to busulfan, fludarabine and rabbit ATG in order to reduce the risk of 
graft failure while minimizing toxicities. In addition to determining the minimum effective dose 
of thiotepa in combination with reduced dose of busulfan and fludarabine for groups with low 
and high risk of graft failure, this study will provide additional safety and efficacy data, 
including study of effects on neurocognitive development and immune reconstitutions in infants 
and children with a variety of non-malignant disorders.      
 
Pharmacogenomics 
  
Inter-patient variation in the pharmacokinetics of drugs can result in unpredictable variability in 
occurrence of adverse events and toxicity as well as variation in therapeutic efficacy. Although a 
multitude of factors contribute to this observed variation, inherited variation in genes involved in 
drug metabolism and transport has been shown to play a significant role. Thiotepa undergoes 
metabolism by cytochrome P450s (CYP2B6, CYP3A) and glutathione S transferases (GSTA1, 
GST-P1). Busulfan, the second drug in this study, undergoes significant metabolism by GSTs. 
Fludarabine pathway genes include deoxycytidine kinase (DCK) and ribonucleotide reductase 
catalytic subunit M1/2 (RRM 1/2).  Genetic polymorphisms can influence the gene expression 
and/or activity, thereby impacting drug pharmacokinetics and causing inter-individual variation 
in drug levels which can alter risk of toxicity or therapeutic efficacy. One of the exploratory 
goals of this study is to identify pharmacogenomic biomarkers predicting drug response and 
toxicity. Identification of such biomarkers, followed by further validation in a larger cohort will 
establish clinically relevant biomarkers allowing for personalizing therapy to achieve maximum 
benefit and minimal side-effects in future studies. 
                                                          
 
                

PEDS024 
 
 
2020-7-15 
 
27 | P a g e  
 
6. OBJECTIVES 
6.1 
Primary 
 
• Determine the minimum effective dose (MED) of thiotepa in combination with 
reduced-dose busulfan, fludarabine and rATG required to achieve engraftment in 
>90% subjects undergoing hematopoietic stem cell transplantation for non-
malignant disorders. The MED will be determined for subjects stratified in group 
A (lower risk of graft failure) and group B (high risk of graft failure). The two 
groups will be merged if group A requires escalation to the higher dose level. 
 
6.2    Secondary  
 
• Evaluate the risk of early (by day 42) and late (by day 365) graft failure/rejection. 
• Evaluate 12 and 24 month disease free survival (DFS) and overall survival (OS) 
of children undergoing HSCT using busulfan/fludarabine/rATG/thiotepa 
conditioning regimen. 
• Evaluate transplant-related mortality at 100 days and 12 months post-transplant. 
• Evaluate rate of acute (Grade II-IV) graft-versus-host disease (GVHD) by 12 
months post-transplant 
• Evaluate rate of chronic GVHD by 24 months post-transplant 
• Evaluate transplant-related complications at 12 and 24 months post-transplant 
[Comments][Comments]  
 
6.3    Exploratory 
 
• Describe change in neurocognitive functioning compared to pre-transplant 
• Describe association between pharmacogenomics of genes involved in metabolism of 
chemotherapy agents and toxicity 
• Describe immune reconstitution following this conditioning regimen 
 
[Subject]      
7. STUDY DESIGN 
 
7.1 Study Overview  
 
This is a feasibility Phase I/II multi-institutional study of allogeneic stem cell transplant using a 
conditioning regimen which tests 2 different doses of thiotepa added to the backbone of reduced-
dose busulfan, fludarabine and rATG for children with non-malignant disorders in order to 
achieve reliable engraftment. Following donor identification and standard of care pre-transplant 
evaluation, subjects will start conditioning regimen.  
 

PEDS024 
 
 
2020-7-15 
 
28 | P a g e  
 
The study builds in rules for escalation of thiotepa dose based on graft failure by day 42. Patients 
are stratified in two groups A (lower risk of graft failure) and B (higher risk of graft failure). 
Patients undergoing 10/10 HLA matched bone marrow and peripheral blood transplants are 
assigned to group A, and patients receiving <10/10 matched bone marrow or peripheral blood, or 
receiving cord blood, even if fully matched, or T-cell depleted transplants, are assigned to group 
B.  
 
If criteria for thiotepa dose escalation are met first in group A, which has a lower risk of graft 
failure, thiotepa dose will be escalated for all subjects (group A and B). If criteria for thiotepa dose 
escalation are met first in group B, dose will be escalated only in group B and group A will continue 
enrolling patients at a lower dose level. 
 
Exploratory objectives include evaluating neurocognitive functioning following this conditioning 
regimen and assessment of immune reconstitution. We will also gather additional information 
onpharmacogenomics and possible associations between genes involved in metabolism of 
busulfan, fludarabine and thiotepa and toxicity and efficacy. 
 
If failure of engraftment has not been observed by day 42, 5 additional subjects will be enrolled in 
the same dose level. If 0 or 1 failure of engraftment is observed among the first 10 subjects of each 
dose level, 5 additional subjects will be enrolled at the same dose level. If a failure of engraftment 
is observed among the first 5 subjects, the dose of thiotepa will be increased to twice a day and 10 
subjects enrolled at that dose level.  If there is 0 or 1 graft failure among the first 10 subjects, 
additional 5 subjects will be enrolled in twice a day thiotepa arm.  If there are 2 or more graft 
failures among the 10 subjects at twice a day thiotepa dose level in group A, the entire study will 
be closed. If there are 2 or more graft failures in group B, only that group will be closed to further 
accrual.  
 
With this design a minimum of 15 subjects will be enrolled at each effective dose level. The 
study may confirm a different MED of thiotepa for group A and group B. 
 
7.2 
Subject Selection  
7.2.1  Number of Subjects 
 
Up to 35 subjects will be enrolled in this study. The study aims to enroll a minimum of 15 
patients at the dose of thiotepa which is determined to be effective. A minimum of 16 
subjects and maximum of 35 subjects will be enrolled in the entire study. The minimum 
number will be enrolled if the first patient in the group with the low risk of graft failure 
sustains a graft failure, in which case the dose of thiotepa will be escalated in both group A 
and group B. 
 
7.2.2  Inclusion Criteria   
• Diagnoses: 

PEDS024 
 
 
2020-7-15 
 
29 | P a g e  
 
Subjects with any non-malignant disorder known to benefit from allogeneic 
transplantation for cure or amelioration of symptoms of the underlying 
disease.The following groups of diagnoses are eligible: 
• Hemoglobinopathies (e.g. thalassemia or sickle cell disease),  
• Cytopenias (e.g.Diamond-Blackfan anemia, congenital or acquired 
neutropenia, congenital or acquired thrombocytopenia, congenital or acquired 
anemia, and others, regardless clonality),  
• Hemophagocytic lymphohystiocytosis, 
• Primary immunodeficiencies (e.g. Wiscott Aldrich Syndrome, chronic 
granulomatous disease, common variable immune deficiency, X-linked 
lymphoproliferative disease, NK+ severe combined immune deficiencies), 
• Metabolic disorders (Hurler’s syndrome, mannosidosis, adrenal leuko-
dystrophy)  
• Other non-malignant disorders for which there is 
            published evidence that HSCT is a curative therapy. 
• Donor Requirements 
• Related or unrelated donor who is suitable and willing to donate bone marrow or 
peripheral blood stem cells. HLA typing should be done by high-resolution typing 
at A, B, C, DrB1 and DQ loci and the donor should be at a minimum ≥8/10 match 
(with one antigen/allele mismatch allowed at A, B, or C-loci and other at DQ 
loci). 
• Cord blood units must be matched at a minimum of 6/8 antigens/alleles at A, B, C 
and DrB1 loci. High resolution typing at all loci is required. The minimum TNC 
dose pre-cryopreservation must be ≥3.7 x10^7/kg of recipient’s weight, if a single 
cord blood unit is used, or at least 2x10^7/kg per unit, if two cord blood units are 
used. The mismatches cannot be at the same loci (e.g. double A mismatch).  
• Haploidentical related stem cell donor who is suitable and willing to donate peripheral 
blood stem cells. T-cell depletion is required if haploidentical donors are used.  Adequate 
organ function defined as: 
• Cardiac: ejection fraction ≥55% or shortening fraction ≥30% 
• creatinine clearance ≥70 ml/min/1.73m2 
• Pulse oximetry >95% on room air or FEV1/DLCO >60% 
• LFTs < 3 x ULN, Total bilirubin <3 mg/dl (unless due to non-hepatic cause (e.g. 
Gilbert’s syndrome or hemolysis) 
• Lansky/Karnofsky score ≥60% 
• Age: ≥6 months and ≤39 years of age at the time of signing informed consent 
• Written informed consent obtained from the subject or parental/guardian permission ± 
child’s assent per institutional guidelines  

PEDS024 
 
 
2020-7-15 
 
30 | P a g e  
 
• Males with female partners of childbearing potential must agree to use physician-
approved contraceptive methods (e.g., abstinence, condoms, or vasectomy) for at least 
one month after completion of conditioning. 
• Women of childbearing potential (WOCBP) must be using an adequate method of 
contraception to avoid pregnancy for at least 1 month after completion of conditioning. 
WOCBP include any woman who has experienced menarche and who has not undergone 
successful surgical sterilization (hysterectomy, bilateral tubal ligation, or bilateral 
oophorectomy) or who is not post-menopausal. Post-menopause is defined as: 
• Amenorrhea that has lasted for ≥ 12 consecutive months without another cause, or 
• For women with irregular menstrual periods who are taking hormone replacement 
therapy (HRT), a documented serum follicle-stimulating hormone (FSH) level of 
greater than 35 mIU/mL. 
7.3 Exclusion Criteria  
• Subjects with any of the following will not be eligible for study participation:  
• Diagnoses that do not require myeloablative transplant for cure (e.g. NK- SCID 
patients), unless they previously did not engraft with non-myeloablative or reduced 
intensity conditioning transplant. 
• Known or suspected sensitivity to chemotherapy or radiation (e.g  Fanconi’s anemia, 
Dyskeratosis congenita, Ligase IV deficiency, etc).   
•  Subjects with fast-progressing neurodegenerative disorders (e.g. Krabbe disease or 
adrenal leukodystrophy with Loes score of ≥10) 
• Cytopenias with increased blasts (>5%) 
• Presence of anti-donor HLA antibodies (positive anti-donor HLA antibody is defined 
as a positive cross-match test of any titer (by complement-dependent cytotoxicity or 
flow cytometric testing) or the presence of anti-donor HLA antibody to the high 
expression loci HLA-A, B, C, DRB1 with mean fluorescence intensity (MFI)>3000 
by solid phase 

PEDS024 
 
 
2020-7-15 
 
31 | P a g e  
 
 
7.4   Inclusion of women, children and minorities  
Both men and women and members of all races and ethnic groups are eligible for this trial as 
long as they meet inclusion criteria. 
8.0 STUDY PROCEDURES 
 
8.1 Registration 
All subjects must be registered with the UF Health system ion into the trial. The participating site 
must fax or email the completed study specific eligibility checklist and registration forms, 
supporting documents and signed informed consent to the Coordinating Center to be entered by a 
delegated UF Health stem cell transplant study team member.  Clarification will be requested for 
unsigned or incomplete forms. Once documents are received, the designated Research 
Coordinator will review the documents to confirm eligibility and to complete the registration 
process. If eligibility cannot be confirmed, the research coordinator will query the site for 
clarification or additional documents as needed. Subjects failing to meet all study eligibility 
requirements will not be registered and will be unable to participate in the trial. 
 
• Prior allogeneic stem cell transplant, except for patients with immune deficiencies who 
underwent previous non-myeloablative or reduced intensity transplants. 
• Haploidentical donor using in vivo T-cell depletion (e.g. postransplant cyclophosphamide). 
• Uncontrolled bacterial, viral, or fungal infection at the time of enrollment. Uncontrolled is 
defined as currently taking medication and with progression or no clinical improvement 
on adequate medical treatment. 
• Seropositive for HIV 
• Active Hepatitis B or C determined by a detectable viral load of HBV or HCV by PCR 
• Bridging fibrosis or liver cirrhosis 
• Females or males of childbearing potential who are unwilling or unable to use an 
acceptable method to avoid pregnancy for the entire study period and for at least 1 
months after the end of conditioning 
• Females who are pregnant or breastfeeding 
• History of any other disease, metabolic dysfunction, physical examination finding, or 
clinical laboratory finding giving reasonable suspicion of a disease or condition that 
contraindicates the use of protocol therapy or that might affect the interpretation of the 
results of the study or that puts the subject at high risk for treatment complications, in 
the opinion of the treating physician. 
• Subjects demonstrating an inability to understand the study and comply with the study 
and/or follow-up procedures. 

PEDS024 
 
 
2020-7-15 
 
32 | P a g e  
 
8.2. Screening Procedures     
Written informed consent must be obtained at least 24 hours prior to starting conditioning 
regimen.  
All pre-transplant evaluations performed as standard of care within 30 days of initiation of 
conditioning regimen but prior to informed consent, will be accepted for this study and need not 
be repeated. Neurocognitive testing done up to 90 days prior to transplant will be acceptable as 
baseline. 
Pre-transplant donor and recipient evaluation will follow established standard-of care, FACT 
recommended guidelines. At a minimum pre-transplant evaluation will include: 
• Demographic data, including date of birth, gender, and race 
• Vital signs including height, weight, and blood pressure 
• Medical history, including major diseases and/or surgeries; 
• List of active problems 
• Lansky/Karnofsky score 
• Pre-transplant  disease evaluation will be done per SOC and will be  specific for 
the underlying disease (e.g. liver MRI with quantification of iron for subjects who 
received > 20 PRBC  transfusions previously, brain MRI for subjects with adrenal 
leukodystrophy, etc.) 
• Serum pregnancy test within 7 days of conditioning start for females of child 
bearing potential 
• Echocardiogram 
• Pulse oximetry or pulmonary function tests for patients able to cooperate with the 
test 
• Calculated creatinine clearance  
• Liver function tests  
• Infectious disease testing as per institutional guidelines 
• Neurocognitive testing:  may be done between 3 months prior to transplant and 
day -5 – see neurocognitive testing details (section 6.3). 
•  3mL of peripheral blood collected in EDTA tubes for pharmacogenomics testing 
should be obtained prior to initiation of conditioning regimen 
     
 8.3   Study Procedures (See Appendix A, schedule of assessments for additional details.) 
     
Weekly until day 60, then with every clinic visit until day 100: 
• Assessment for AE and SAEs 
• Assessment for aGVHD (grade II-IV acute), and moderate and severe 
cGVHD once patient engrafted 
• Assessment for graft failure (blood counts, or as clinically indicated) 
 
Immune Reconstitution Tests: At 100 days and every 3 months subsequently until 
normal  
• Immunoglobulin levels (IgG, IgM, IgA) 

PEDS024 
 
 
2020-7-15 
 
33 | P a g e  
 
• Enumeration of lymphocytes (CD4, CD8, CD19 at a minimum) 
• Lymphocyte proliferation to mitogens (start once CD4 > 200/µL) 
• Post-immunization titers (obtain at the end of study, if immunization started) 
 
Donor chimerism: 
• Obtain at day +30 if ANC has recovered 
• Obtain at day +42 or any time that graft failure is suspected 
• Obtain at 12 months  
• Obtain at 24 months or at the end of study 
 
Neurocognitive testing (Appendix 4) 
• Prior to transplant (day -90 to -5) 
• At 12 months post-transplant 
• At the end of study (if at least 12 months have elapsed since the previous testing) 
 
Any time during follow up: 
• Assess for graft failure (blood counts, chimerism if needed) 
• Assess for toxicities (AE and SAE) felt to be related to conditioning regimen  
• Assess for GVHD (grade II-IV acute and moderate and severe cGVHD) 
 
8.4. Pharmacogenomic Testing  
Genomic DNA will be isolated from a blood sample obtained pre-transplant. 15 SNPs listed in 
Table 8.1 will be genotyped using Quant studio in patients enrolled in the study. Additionally, 
GSTM1 gene deletion will be tested in these patients. These SNPs have been selected based on the 
evidence in literature for any functional impact and if they occur with the minimum allele 
frequency of greater than 5% in a population. SNPs or GSTM1 deletion will be evaluated for 
association with efficacy and observed toxicities in transplant patients receiving drugs of interest.  
 
Table 8.1  Key candidate genes and SNPs of significance in pharmacokinetics of 
the drugs used for conditioning 
PK genes 
Genetic polymorphisms 
Drugs impacted 
CYP2B6 
rs2279343, rs3211371,rs3745274 
Thipotepa 
CYP3A4 
 rs35599367 
Thipotepa 
CYP3A5 
 rs776746 
Thipotepa 
GSTA1 
rs3957357, rs3957356 
Thipotepa, Busulfan 
GSTP1 
rs1695 and rs1138272 
Thipotepa, Busulfan 
GSTM1 
Deletion 
Thipotepa, Busulfan 
CYP39A1  
rs2277119  
Busulfan 
DCK 
rs2306744, rs11544786, rs9997790 
Fludarabine 
RRM1/2 
rs1042919, rs1561876 
Fludarabine 

PEDS024 
 
 
2020-7-15 
 
34 | P a g e  
 
 
 
8.5   Data Collection  
 
Other than AEs and SAEs up to day 100 reporting and eligibility documentation, this study will 
use  the following CRFs for data collection. 
 
Table 8.2 – Forms for Data Collection 
Time of assessment 
Enrollment form 
Pre-transplant Evaluation 
Post-Transplant Evaluation 
Day 100, 180, 1 and 2 years post-transplant 
 
9.0 STUDY TREATMENT 
All subjects entering the screening phase will receive a unique subject number. This number will 
be used to identify the subject throughout the study. Subjects withdrawn from the study will retain 
their subject number.  
 
9.1 Conditioning Regimen and GVHD Prophylaxis 
 
Dose adjustments of medications used for conditioning will be made for obesity only in children 
> 2 years of age. Obesity isdefined as BMI  ≥95% based on CDC BMI caluculator (see Appendix 
F for details) This includes thiotepa fludarabine, and the first dose of busulfan. For obese 
subjects, the first busulfan dose will be given based on adjusted weight until pharmacokinetic 
(PK) results are available. Once PK values are available, busulfan dose will be modified to target 
AUC of 20mgxh/L per day for the total AUC of 60 mgxh/L over 3 days. Fludarabin will be 
targeted to  4.5 mgxh/L/day or 18 mgxh/L over 4 days. Thymoglobulin will not be adjusted 
for weight; however, capping of thymoglobulin dose in severely obese patients is 
acceptable at a dose of 150 mg/day. 
 
Starting dose for busulfan and fludarabine will be based on InsightRx platform, whenever 
feasible, given paucity of dosing for young infants. 
 
Tacrolimus should be adjusted based on levels. Post-transplant Methotrexate and MMF may be 
adjusted in obese subjects per institutional guidelines. 
 
In children < 12 kg, all drug doses will be calculated per kg rather than per sqm. 
 
 
 
 
 
 

PEDS024 
 
 
2020-7-15 
 
35 | P a g e  
 
Table 9.1 Conditioning Schedule 
Day -8 
Admit, start hydration at least 12 hours prior to Thiotepa, 
Start anti-seizure prophylaxis (Levetiracetam or Ativan)  
Day -7 
Thiotepa 5 mg/kg x1 (first dose level) 
Thiotepa 5 mg/kg q 12 hours x 2 (2nd dose level) 
Skin care per institutional guidelines 
Day -6 to -3 
Fludarabine 40 mg/m2/dose daily iv over 30 min or 1.3 mg/kg/dose IV if 
<12 kg or InsightRx dose targeting 4.5 mgxh/L/day 18 mgxh/L total dose 
Day -6  
Day -4, -3 
Busulfan  iv over 3 hours, given following Fludarabine. Use weight-based 
dosing or InsightRx platform targeting 20 mgxh/L/day 60 mgxh/L total 
dose 
PK on day -6 after the first dose. NOTE Busulfan is skipped on day -5 
Day -3 to -1 
rATG (Thymoglobulin®) 2mg/kg x 3 for subjects  receiving bone marrow, 
or  peripheral blood stem cells as stem cell source (total 6 mg/kg).  
Subjects receiving cord blood or T-cell depleted product will receive 2 
mg/kg x 2 on days -2 and -1 only (total 4 mg/kg) 
May cap daily dose to 150 mg for severely obese patients 
Day -2 
Begin Tacrolimus (0.03 mg/kg/day, initial maximum dose 1 mg/day) IV 
continuous infusion, adjust based on levels 
Day 0 
Stem Cell Infusion 
Day +1 
Methotrexate 5 mg/m2/dose IV (0.15 mg/kg) 
Day +3 
Methotrexate 5 mg/m2/dose IV 
Day +6 
Methotrexate 5 mg/m2/dose IV 
Day +11 
Methotrexate 5 mg/m2/dose IV   
Cord blood transplant recipients will not receive post-transplant Methotrexate; their GVHD 
prophylaxis will consist of Tacrolimus as described in the table and Mycophenolate Mofetil 
(MMF) 15 mg/kg PO TID up to 1 gm TID (or iv equivalent), starting on day -2 and continuing 
until day +35. 
Subjects receiving T-cell depleted transplant (using CliniMACS® or similar T-cell depletion) will 
not receive any pharmacologic GVHD prophylaxis. The infused T-cell dose (CD3+) in these 

PEDS024 
 
 
2020-7-15 
 
36 | P a g e  
 
transplants should not exceed 3x104 CD3+ cells/kg of recipient’s weight if CD34+ selection is 
done Patients receiving αβ T-cell depletion will receive CD3+ and CD19+ doses and/or Rituxan as 
prescribed in the αβ  T-cell depletion protocol. 
9.2 Treatment Schedule/Administration 
                                                                                                                                                     
9.2.1 Thiotepa 
 
The initial Thiotepa dose is 5 mg/kg. It will be administered IV over 2 hours on Day -7.  In 
case of dose escalation, Thiotepa will be administered IV twice 12 hours apart on Day -7.  
Hydration and skin care prior to and during Thiotepa will follow institutional guidelines.  
 
9.2.2 Fludarabine 
 
Fludarabine dose 40 mg/m2, or 1.3 mg/kg in children <12 kg, or dose targeting the AUC of 
4.5 mgxh/L/day for the total AUC of 18 mgxh/L over 4 days. InsightRx  platform will be a 
preferred way of determining the dose for infants. AFludarabine will be administered over 60 
minutes intravenously on days -6 to -3 immediately prior to busulfan.  Potential study 
participants who have an estimated or measured creatinine clearance <70 mL/min/1.73 m2 are 
excluded from enrollment, thus there will be no adjustment in the fludarabine dose for renal 
insufficiency; however InsightRx platform will adjust the dose based on patient’s creatinine 
level. 
 
Pentamidine should be avoided with fludarabine. 
 
9.2.3 Busulfan (including PK) 
 
First dose will be based on weight following European Medicines Agency IV busulfan dose 
guidelines (Palmer et al, 2016), or based on InsightRx platform individualized 
recommendation. Once PK results are available dose will be based on PK results to target 
AUC of  20 mgxh/L±10% / day for the total AUC of 60 mgxh/L over 3 doses.). Note that 
there are only 3 doses of busulfan in this study..  
 
Table 9.2 – European Medicines Agency IV Busulfan Dose Guidelines 
Weight: 
Dose: 
<9kg 
4 mg/kg/dose 
≥9 to <16 kg 
4.8 mg/kg/dose 
≥16 to<23 kg 
4.4 mg/kg/dose 
≥23 to <34 kg 
3.8 mg/kg/dose 
≥34 kg 
3.2 mg/kg dose 
 

PEDS024 
 
 
2020-7-15 
 
37 | P a g e  
 
Administration: Busulfan is to be administered intravenously via a central venous catheter as 
a 3-hour infusion once daily on days -6, -4 and -3 for a total of 3 doses. Use of alternative (ie, 
divided) dosing schedules is NOT permitted on this study. Please note: the timing of busulfan 
PK sample collection is based upon this 3-hour infusion. Busulfan is administered at a final 
concentration of approximately 0.5 mg/mL in D5W or 0.9% NaCl over 3 hours via central 
line. Flush line with D5W or 0.9% NaCl before and after busulfan administration. Do not use 
polycarbonate syringes or filters. 
 
Busulfan PK studies are required, with first dose pharmacokinetics performed to achieve an 
average daily area under the curve (AUC) for busulfan of 20 mgxh/L/day for the total AUC of 
60 mgxh/L over 3 doses. 
 
For the first dose, busulfan should be started early enough to allow that pharmacokinetic 
studies can be sent out the same day so that the doses on days -4 and -3 may be adjusted. 
Busulfan should be adjusted if first dose PK results indicate that the level is  >10% outside of 
targeted AUC.. Decision about adjustment will be made in discussion with the pharmacologist 
and institutional PI. In general, the busulfan dose should not be increased by more than 50% 
of the starting dose. When adjusting busulfan, the goal is to achieve the outlined AUC for 
future doses (e.g. do not try to make up for already given busulfan dose). 
 
Anti-seizure prophylaxis should start on admission and continue for a minimum of 48 hours 
after completion of Busulfan. Subjects at a high risk for seizures may continue with anti-
seizure prophylaxis during therapy with calcineurin inhibitors. Institutions may use lorazepam 
or levetiracetam for seizure prophylaxis, phenytoin is not allowed. For subjects who are 
already on anti-seizure medications, consult neurologist and pharmacologist regarding 
continuation of patient’s anti-seizure medications. This is allowed, assuming there are no 
interactions with the conditioning regimen medications. 
 
The following medications should be avoided with busulfan:  
• phenytoin or fosphenytoin (increased clearance of busulfan) 
• acetaminophen (decreased clearance of busulfan) 
• itraconazole – reduced clearance of busulfan (fluconazole and voriconazole had no 
effect on the clearance of busulfan) 
• metronidazole – reduced clearance of busulfan. 
 
Busulfan PK Samples 

PEDS024 
 
 
2020-7-15 
 
38 | P a g e  
 
Samples should not be drawn from the lumen used to infuse busulfan. In case infusion runs 
more or less than 3 hours, draw one sample immediately when infusion ends. Then draw the 
next sample 15 minutes later and continue to draw 4, 5, 6, and 8 hours samples by counting 
from beginning of the infusion. Busulfan PKs is sent to central laboratory as standard of care 
test (Philadelphia or Seattle). It can also be done at any other laboratory that performs 
busulfan PK, assuming the above guidelines are followed. 
 
Table 9.3 – Busulfan PK Sample Timing 
PK Sample timing from START of 1st Busulfan dose 
3 hours (end of infusion) 
3 hours 15 minutes 
4 hours 
5 hours 
6 hours 
8 hours 
 
9.2.4 Thymoglobulin® 
 
Thymoglobulin® (rATG) will be given in this study at 2 different dose levels (4 mg/kg and 6 
mg/kg, see conditioning regimen). Thymoglobulin will be infused through a 0.22-micron 
filter over 4-6 hours with pre-medications to include: steroids, oral or intravenous (IV) 
acetaminophen and diphenhydramine. It is recommended that the first day infusion be 6 
hours. An anaphylaxis kit will be kept at bedside during Thymoglobulin administration. 
Steroids should be administered per institutional standard to prevent infusion toxicity 
(suggested steroid dose is 1 mg/kg of Solumedrol prior to infusion with repeating the dose of 
1 mg/kg 4-6 hours later only if required by patient’s symptoms). 
 
9.2.5 GVHD Prophylaxis 
 
1. Mycophenolate Moefetil for CORD BLOOD recipients  
MMF will be given at a dose of 15 mg/kg PO TID with the maximum daily dose not 
to exceed 3 grams (1 gram PO TID). MMF prophylaxis will be used only for CORD 
BLOOD recipients. It will begin on day -2 and continue until day +35, unless active 
GVHD is present. At day 35 MMF can be discontinued a once. 
 
2. Tacrolimus 
Tacrolimus IV or PO should be started on Day -2 and administered per institutional 
standards.  The recommended IV starting dose is 0.03 mg/kg/day with PO equivalent 
acceptable. May change to oral dosing once therapeutic levels are achieved or per 
institutional standards. Young children may require more frequent (e.g. three times a 
day) oral dosing to maintain appropriate levels. If Tacrolimus is not tolerated, 

PEDS024 
 
 
2020-7-15 
 
39 | P a g e  
 
cyclosporine may be substituted IV or PO. Immunosuppression will be withdrawn as 
per institutional guidelines. 
 
3. Methotrexate  
Methotrexate IV push at 5 mg/m2 (0.15 mg/kg for children < 10 kg) will be given on 
Day 1 (a minimum of 24 hours after stem cell infusion) and repeated on Day +3, +6 
and +11. Institutional guidelines will be used for adjustment of methotrexate for 
toxicities. 
 
9.3 Stem Cell Infusion   
 
Stem cell dose and infusion will follow institutional guidelines. The following are general 
guidelines for stem cell doses. If cord blood is used, a minimum of 3.7x107/kg of cryopreserved 
nucleated cells should be available for transplant or at least 2x10^7/kg per unit, if two cord blood 
units are used. The mismatches cannot be at the same loci (e.g. double A mismatch).  Use cord 
blood bank or institutional guidelines for instructions about the washing, dilution and infusion of 
cord blood products. There are no limits to cord blood dose. If in vitro T-cell depletion is used, 
infuse >5 and <20x106/kg of CD34+ cells and a fixed dose of 3x104/kg of CD3+ cells.  For 
peripheral blood stem cells, infuse a minimum of 2 and the maximum of 8x106/kg of CD34+ 
cells. For bone marrow infusion, the target is 5x108 /kg, ( acceptable range 2-8) of total viable 
nucleated cells (TVNC). Follow institutional guidelines for ABO incompatible stem cell 
products.  
 
9.4 G-CSF                                                                                                                                             
 
G-CSF may be started at day +15 if there is no evidence of ANC recovery, or it can be used per 
institutional guidelines.  
 
9.5 Allowed Concomitant Therapy 
 
All standard of care supportive care treatments (e.g. anti-emetics, seizure prophylaxis, pain 
medications, antimicrobials and transfusions) are allowed. All standard of care supportive care 
treatments (e.g. anti-emetics, seizure prophylaxis, pain medications, antimicrobials and 
transfusions) are allowed.  
Patients on this study are allowed to be on concomitant trials for GVHD therapy, donor source 
selection, T-cell depletion, and others, assuming that the outlined conditioning regimen is 
permitted on those trials. 
 
 
9.6 Thiotepa Escalation based on graft failure 
 
 

PEDS024 
 
 
2020-7-15 
 
40 | P a g e  
 
 
STUDY SCHEMA I:  PHASE I/II THIOTEPA DOSE ESCALATION        
 
 
 
                                                
                                    
 
 
 
 
 
 
 
 
 
 
 
 
 
                                                                                                                                                           
                                                                                                                                                           
 
 
 
 
  
 
 
 
 
The above study schema will be used for thiotepa dose escalation based on failure of engraftment 
by day 42. 
 
 
 
 
1 failure of 
engraftment by day 
42,  escalate 
Thiotepa to twice 
daily in both 
Group A and B 
enroll 10 subjects, 
first 5 one at a time 
All subjects engrafted 
by day 42, enroll 
additional 5 subjects   
Enter 5 subjects on Bu/Flu/rATG single 
Thiotepa dose, one at a time 
1 failure of 
engraftment by day 
42, escalate Thiotepa  
to twice daily in 
Group B only, enter 
10 subjects, first 5 
one at a time
>1  failure of 
engrafment, 
close entire 
study 
≤1 failure of 
engraftment, 
enroll 
additional 5 
subjects in 
once a day 
Thiotepa arm 
Enter 5 subjects on Bu/Flu/rATG single 
Thiotepa dose, one at a time 
All subjects 
engrafted by day 
42 enroll 
additional 5 
subjects   
≤ 1 failure of 
engraftment 
among 10 
subjects, enroll 
additional 5 
subjects in 
twice a day 
Thiotepa arm 
 >1  failure 
of 
engraftment
close group 
B only 
≤ 1 failure of 
engraftment, 
enroll 
additional 5 
subjects in 
once a day 
Thiotepa,  
≤ 1 failure of 
engraftment, 
enroll 
additional 5 
subjects in 
twice a day 
Thiotepa arm 
Group A 
Fully matched (10/10) BM 
or PBSC sources only 
Group B 
Mismatched BM or PBSC, all 
cords, T-cell depleted 

PEDS024 
 
 
2020-7-15 
 
41 | P a g e  
 
Definition of engraftment failure used for dose escalation in this study includes:  
• Failure to achieve sustained ANC recovery of >0.5x10^9/L with evidence of donor 
chimerism by day 42, in light of absence of myelosupressive medications (e.g. 
ganciclovir), or   
• Engraftment with subsequent loss of donor chimerism to <5% by day 42, or 
• Need for the 2n  conditioned transplant by day 42 to correct manifestations of disease or 
cytopenia.  
9.7 Toxicity based stopping rules 
Table  9.1 – Toxicity based stopping rules  
 
N of  enrolled Subjects 
≤10 
11-13 
14-16 
17-19 
20 
N of subjects with Grade 4 
or 5 toxicities that will 
trigger stopping rules 
≥4  
≥5 
 ≥6 
 ≥7 
 ≥8 
N of subjects with grade 5 
toxicities that will trigger 
stopping rules 
≥2 
 ≥3 
 ≥3 
≥4 
≥4 
Toxicities up to 100 days post-transplant will be used as triggers for stopping the study for 
excessive toxicities.  If toxicity stopping rules have been met, the DSMB will review data and 
make recommendations regarding study modifications and conditions for study continuation.  
Graft failure is not counted in these grade 4 and 5 toxicities as study modifications for graft failure 
already exist. 
The rate of acceptable combined Grade 4 and 5 toxicities is ≤40%, or ≤20% Grade 5 toxicities. 
DLTs will be followed cumulatively for each dose level (5 mg/kg of thiotepa or 10 mg/kg of 
thiotepa). 
DLTs (CTCAE version 5.0 criteria) are defined as:  
Grade 5 toxicities: any death that is in the opinion of study investigator possibly, probably or 
definitely related to toxicities of the conditioning regimen. 
Grade 4 toxicities: any grade 4 neurologic, cardiac, GI, lung, infections or any other system 
toxicity, that is in the opinion of investigator possibly, probably or definitely related to 
conditioning regimen. 
Severe VOD will also be considered Grade 4 toxicity (defined as VOD with renal or pulmonary 
dysfunction).  
Acute GVHD of grade IV will also be considered as grade 4 toxicity. 

PEDS024 
 
 
2020-7-15 
 
42 | P a g e  
 
Post-transplant expected hematopoietic and immunologic toxicities will not be considered DLTs 
and do not require reporting.  
If the enrollment is brisk following the first 5 subjects enrollment in each dose level and each 
group the enrollment will be held so that at any given time 5 or fewer subjects are <100 days 
post-transplant 
 
 
 
 
  
9.7 Supportive Care Guidelines 
 
Subjects should receive full supportive care, including transfusions of blood products, 
antibiotics, anti-emetics, antidiarrheals, analgesics, etc., when appropriate, according to 
institutional standard of care guidelines.  Available disease-specific supportive care guidelines 
should be followed (e.g. for sickle cell disease including reduction of Hemoglobin S to <35% 
prior to start of conditioning, strict control of blood pressure, continuation of anti-seizure 
prophylaxis during calcineurin inhibitor use and maintenance of normal magnesium levels 
throughout transplant). 
 
9.8  Infectious Disease Monitoring 
 
Infectious disease monitoring should follow institutional guidelines. In patients receiving 
mismatched transplants, follow-up for reactivation of viruses (CMV, EBV, HHV6, adenovirus, 
etc.) should continue at a minimum until the absolute  CD4 lymphocyte count reaches 
200/microL. 
 
10. TREATMENT DISCONTINUATION 
10.1 Removal of Subjects From Study 
Any patients who initiate conditioning regimen will be considered a study subject. 
Subjects who are not initiated on study drug, but have signed an informed consent will be 
considered screening failures. A record of these subjects will be maintained by the study site. 
10.2 Criteria For Study Treatment Discontinuation 
 
The Investigator will make every reasonable effort to keep each subject in the study unless it is 
in the subject’s best interests to discontinue participation.  If a subject is removed from the study 
or declines further participation, a description of the reason(s) for withdrawal from the study 
must be recorded on the case report form (CRF). The Investigator should also ensure that all 
subjects are followed up for survival status until 24 months post-transplant. 
 
10.3 Replacement of Subjects 
 
There will be no replacement of subjects on this study. 

PEDS024 
 
 
2020-7-15 
 
43 | P a g e  
 
11. BIOLOGICAL SPECIMENS AND CORRELATIVES 
11.1 Source of Specimens 
 
Peripheral blood specimens for pharmacogenomics will be obtained for the purposes of this study 
and sent to Dr. Jatinder’s laboratory. All other blood tests will be done as SOC. 
11.2 Preparation, Shipment and Storage of Specimens 
 
See Appendix C for collection, processing and shipping instructions for all tissue and blood 
specimens. 
 
12.  STUDY DRUG INFORMATION 
 
This study uses commercial agents. The current known toxicities for each commercial agent is 
provided in the drug package insert. 
 
See Appendix D for current study drug information. Always refer to the drug insert for the most 
up to date information. 
 
13.  ADVERSE EVENT REPORTING REQUIREMENTS  
 
13.1 Purpose  
 
Adverse event data collection and reporting, which are required as part of every clinical trial, are 
done to ensure the safety of subjects enrolled in the studies as well as those who will enroll in 
future studies using similar agents.  
 
This study uses commercial agents and AE reporting will follow Cancer Therapy Evaluation 
Program (CTEP) guidelines for reporting of AE that occur during therapy with commercial 
agents. 
  
13.2 Determination of Reporting Requirements 
 
Reporting requirements should include the following considerations: 1) the characteristics of the 
adverse event including the grade (severity); 2) the relationship to the study therapy 
(attribution); and 3) the prior experience (expectedness) of the adverse event.  
 
Commercial agents are those agents not provided under an IND but obtained instead from a 
commercial source. In some cases, an agent obtained commercially may be used for indications 
not included in the package label. In addition, NCI may on some occasions distribute commercial 
supplies for a trial. Even in these cases, the agent is still considered to be a commercial agent and 
the procedures described below should be followed.  
 
Determine the prior experience: Expected events are those that have been previously identified 
as resulting from administration of the agent. An adverse event is considered unexpected, for 
reporting purposes only, when either the type of event or the severity of the event is not listed in: 
 

PEDS024 
 
 
2020-7-15 
 
44 | P a g e  
 
• the current known toxicities for each commercial agent as provided described in 
Appendix D, or  
• the drug package inserts.  
 
The following AE will be reported within 5 calendar days of learning about them for 
participants in the interventional trial: 
 
• Grade 3 and 4 toxicities that are unexpected and related (possibly, probably or 
definitely related) to study treatment and occur within 100 days of transplant, will be 
reported. 
• Use the assigned protocol number and the protocol-specific subject ID provided during 
trial registration on all reports.  
 
CTCAE term (AE description) and grade: The descriptions and grading scales found in the 
NCI Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 will be utilized for 
AE reporting and are located on the CTEP website at:  
 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm.   
 
All serious adverse events (SAE) that occur within 100 days of transplant will be reported 
within 5 days of learning about them. A serious adverse events is one that: 
• Results in death 
• Is life-threatening (defined as an event in which the subject was at risk of death at the 
time of the event; it does not refer to an event which hypothetically might have caused 
death if it were more severe) 
• Requires inpatient hospitalization or causes prolongation of existing hospitalization (see 
note below for exceptions) 
• Results in persistent or significant disability/incapacity 
• Is a congenital anomaly/birth defect 
• Is an important medical event, defined as a medical event that may not be immediately 
life-threatening or result in death or hospitalization but, based on appropriate medical and 
scientific judgment, may jeopardize the subject or may require intervention (e.g., 
medical, surgical) to prevent one of the other serious outcomes listed above. Examples of 
such events include but are not limited to intensive treatment in an emergency department 
or at home for allergic bronchospasm; blood dyscrasias or convulsions that do not result 
in hospitalization. “Medically important” should be marked only if no other serious 
criteria are met. 
 
In addition to the above, the study also requires reporting of following Adverse Events of Special 
Interest within 5 days of learning of the event throughout study participation: 
 
• Graft failure 
• New malignancy that occurs any time following treatment  
• Grade 4 toxicity of any organ that occurs at any time during study follow-up and is felt to 
be related to conditioning regimen (e.g. late pulmonary toxicity) 
• GVHD (acute grade 2-4) and cGVHD of moderate and severe grade should be reported 
throughout participation in this study 

PEDS024 
 
 
2020-7-15 
 
45 | P a g e  
 
• Grade 5 toxicity (death) should be reported throughout participation in the study 
• Severe VOD with multiorgan involvement 
 
Secondary Malignancy  
 
A secondary malignancy is a cancer caused by treatment for a previous malignancy (eg, 
treatment with investigational agent/intervention, radiation or chemotherapy). A metastasis of the 
initial neoplasm is not considered a secondary malignancy. 
 
All secondary malignancies that occur following treatment need to be reported to this study. 
  
Three options are available to describe the event:  
• Leukemia secondary to oncology chemotherapy  
• Myelodysplastic syndrome  
• Treatment related secondary malignancy  
14.  STATISTICAL METHODS 
14.1  Sample Size Determination 
 
This is a feasibility Phase I/II study of a new HSCT conditioning regimen for pediatric non-
malignant disorders. Engraftment is the primary endpoint. The study is designed to examine two 
dose levels of thiotepa and determine the minimum effective dose in two groups: (A)  low risk of 
graft failure group, and (B) high risk of graft failure group.  Initially, 5 subjects will be enrolled 
in each group and receive once daily thiotepa.  If all 5 engraft by day 42, 5 more subjects will be 
enrolled at this dose.  Then, if there are 0 or 1 failures out of 10, 5 more subjects will be enrolled 
at this dose in each cohort.  For 1 or more failures among the first 5 subjects in a cohort, the dose 
will be increased to twice daily. If the graft failure is encountered among the first 5 subjects from 
group A, the dose will be increased to twice daily for both groups. If graft failure is encountered 
among the first 5 subjects in group B, the dose of thiotepa will be increased for this group only.   
Ten additional subjects will be enrolled in twice a day thiotepa dose level. If there are 0 or 1 graft 
failure among 10 subjects, additional 5 subjects will be enrolled in twice a day thiotepa dose 
level. For 2 or more failures among the first 10 subjects at twice a day dose level, the group will 
be closed if this occurs in group B (high risk of graft failure), or the entire study will be closed if 
this occurs in group A. This scheme results in a minimum of 15 subjects at each MED. We 
would like to determine the dose such that engraftment is ≥90%.  With 5 subjects enrolled, if the 
true engraftment rate is 90%, there is a 59% chance all will engraft and the dose will not be 
increased.  If the true engraftment rate is less, 80% (or 70%), there is only a 33% (or 17%) 
chance that the dose will not be increased.  Similarly, after 10 subjects are enrolled, if the true 
engraftment rate is 90%, there is a 73% chance that the dose will not be increased.  And, if the 
true engraftment rate is less, 80% (or 70%), there is only a 37% (or 15%) chance that the dose 
will not be increased. 
 

PEDS024 
 
 
2020-7-15 
 
46 | P a g e  
 
This feasibility study is primarily descriptive and the width of a 95% confidence interval can be 
used to determine how precisely the parameters can be estimated for a specified outcome and a 
given sample size.   By group, the sample size will be 15 or 10.  The table below illustrates the 
precision for estimating engraftment rate by day 42 for the combined sample and by group 
assuming the engraftment rate is approximately 90%. 
 
 
N 
# with 
engraftment 
% with 
engraftment 
95% confidence 
interval 
width of 
confidence 
interval 
combined 
30 
27 
90% 
(0.73-0.98) 
0.25 
25 
21 
84% 
(0.64-0.95) 
0.31 
20 
18 
90% 
(0.68-0.99) 
0.31 
by group 
15 
13 
87% 
(0.60-0.98) 
0.38 
10 
9 
90% 
(0.55-01.00) 
0.45 
14.2  Analysis of Primary Endpoint 
 
Determine the minimum effective dose (MED) of thiotepa in combination with reduced-dose 
busulfan, fludarabine and rATG required to achieve engraftment in >90% subjects undergoing 
hematopoietic stem cell transplantation for non-malignant disorders. The MED will be 
determined for subjects stratified in group A (lower risk of graft failure) and group B (high risk 
of graft failure). The two groups will be merged if the group A requires escalation to the higher 
dose. 
Effective dose of Thiotepa in combination with busulfan/fludarabine and rATG is defined as a 
dose that results in engraftment of >90% study subjects by day 42 with acceptable toxicity, 
defined as ≤10% grade 5 toxicities and ≤40% combined grade 4 and 5 toxicities across all 
subjects treated with the same dose level. 
Definition of engraftment failure used for dose escalation in this study includes:  
• Failure to achieve sustained ANC recovery of >0.5x10^9/L with evidence of donor 
chimerism by day 42, in light of absence of myelosupressive medications (e.g. 
ganciclovir), or   
• Engraftment with subsequent loss of donor chimerism to <5% by day 42, or 
• Need for the 2nd conditioned transplant by day 42 to correct manifestations of disease or 
cytopenia.  
 

PEDS024 
 
 
2020-7-15 
 
47 | P a g e  
 
14.3  Analysis of Secondary Endpoints  
• Evaluate the risk of early (by day 42) and late (by day 365) graft rejection/failure 
Proportion and associated confidence interval (CI) of all subjects who initiated 
conditioning regimen and have sustained engraftment failure (as outlined above) at 42 
days and at 1 year post transplant.  
• Evaluate 12 and 24 month disease free survival (DFS)/event-free-survival and 
overall survival (OS) of children undergoing HSCT using 
busulfan/fludarabine/rATG/thiotepa conditioning regimen 
Proportion and associated confidence interval (CI) of all subjects who initiated 
conditioning regimen and are alive (OS) and without evidence of underlying disease 
(DFS) at 12 months post-transplant will be calculated. KM estimates of OS and DFS will 
also be used. OS is defined as time from transplant (day 0) until date of death or date of 
last contact for those who have not died. DFS/event-free survival is defined as time from 
transplant (day 0) until disease recurrence/progression, or date of death from any cause, 
or date of last contact for those who are disease-free. Disease progression will follow-
standard of care definitions for each disorder. In general, disease-free-survival implies 
improvement of reversible manifestations of a disorder and arrest in further progression 
of symptoms in light of presence of donor engraftment and immune reconstitution. 
• Evaluate transplant-related mortality at 100 days and 12 months post-transplant 
Proportion and associated CI of subjects who initiated conditioning regimen and who die 
due to a cause unrelated to the underlying disease. Time to transplant-related mortality is 
defined as time from transplant (day 0) until date of transplant related mortality. Those 
who die from other causes will be censored at date of death; those who were alive at last 
contact will be censored at their date of last contact. 
• Rate of acute (grade II-IV) graft-versus-host disease by 12 months post-transplant 
Proportion and associated CI of subjects who reached engraftment (sustained ANC>500 x 
3 days by day 42) and developed acute GVHD grade II-IV (by modified Glucksberg 
criteria) (Appendix B). 
• Rate of chronic graft-versus host disease by 24 months post-transplant 
Proportion and associated CI of subjects who reached engraftment and developed 
moderate or severe GVHD by 2014 NIH Consensus Criteria (Appendix B) 
• Transplant-related complications at 12 and 24 months post-transplant  
Complications gathered via CIBMTR post-transplant form will be tabulated and 
described by treatment received. 
 
 
 
 
 
  

PEDS024 
 
 
2020-7-15 
 
48 | P a g e  
 
14.4  Analysis of Exploratory Endpoints                                                                                                                 
          
Statistical analysis of pharmacogenomics, immune reconstitution, and neurocognitive 
exploratory endpoints may include descriptive statistics, analyses of variance, regression 
analyses and other appropriate statistical tests.   
 
14.5  Analysis of Safety Data 
            All reported AEs and SAEs will be tabulated and described. 
 
14.6  Interim Analysis 
No formal interim analysis is planned. Refer to toxicity based stopping rules section ( 
9.7) for description of ongoing analyses  for toxicity and graft failure and plan for 
temporary suspension of enrollment while assessing for toxicities.  
 
15.     DATA AND SAFETY MONITORING 
15.1  Data Safety  Monitoring Board (DSMB) 
 
This protocol will be reviewed and monitored by an independent DSMB chartered for 
this study in accordance with FDA guidelines, policies, and procedures.  The committee 
will review and monitor study progress, toxicity, safety and other data from this trial. The 
review will be done at a minimum twice a year, and more frequently if requested by 
investigators or DSMB members. Questions about subject safety or protocol performance 
will be addressed with the investigator, statistician and study team members. Should any 
major concerns arise; the DSMB will offer recommendations regarding whether or not to 
suspend the trial. DSMB Charter contains full description of DSMB and sponsor’s 
responsibilities related to safety. 
 
Briefly, the independent chartered DSMB activities will include:  
• Review of clinical trial conducted for progress and safety  
• Review of all adverse events as defined in the protocol Notification of the 
investigators of recommended action  
15.2  Principal Investigator Responsibilities 
 
As part of the responsibilities assumed by conducting this study, the Principal 
Investigator (PI) agrees to maintain and have available for monitoring adequate case 
records (accurate source documents and CRFs) for the subjects treated under this 
protocol.  
 
The PI will be primarily responsible for monitoring of adverse events, protocol 
violations, and other immediate protocol issues. The study coordinator will collect 
information on subjects enrolled through the use of electronic or paper adverse event 
(AE) forms, CRFs, and Informed Consent forms.  

PEDS024 
 
 
2020-7-15 
 
49 | P a g e  
 
16.  EMERGENCY PROCEDURES 
16.1  Emergency Contact 
In emergency situations, the treating physician should contact the Principal Investigator by 
telephone at the number listed on the title page of the protocol. 
16.2  Emergency Treatment 
 
During and following a subject’s participation in the study, the treating physician and/or 
institution should ensure that adequate medical care is provided to a subject for any adverse 
events, including clinically significant laboratory values, related to the study. 
17.  ADMINISTRATIVE, ETHICAL, AND REGULATORY CONSIDERATIONS 
 
17.1  Good Clinical Practice 
 
The procedures set out in this study protocol pertaining to the conduct, evaluation, and 
documentation of this study are designed to ensure that the Principal Investigator and Co-
Investigators abide by Good Clinical Practice (GCP), as described in International Conference on 
Harmonization (ICH) Guideline E6 and in accordance with the general ethical principles outlined 
in the Declaration of Helsinki.  
 
The study will be conducted in compliance with the protocol. The protocol, any amendments, 
and the subject informed consent will receive Institutional Review Board (IRB) approval before 
initiation of the study.  
 
The Principal Investigator will conduct all aspects of this study in accordance with applicable 
national, state, and local laws of the pertinent regulatory authorities. 
 
All potential serious breaches must be reported immediately to the independent DSMB and IRB 
of record, if applicable. A serious breach is a breach of the conditions and principles of GCP in 
connection with the study or the protocol, which is likely to affect, to a significant degree, the 
safety or physical or mental integrity of the subjects of the study or the scientific value of the 
study. 
17.2  Institutional Review Board 
 
Before study initiation, the investigator must have written and dated approval from the IRB for 
the protocol, consent form, subject recruitment materials/process (e.g., advertisements), and any 
other written information to be provided to subjects. The investigator should also provide the 
IRB with a copy of the Investigator Brochure or product labeling, information to be provided to 
subjects, and any updates. The investigator should provide the IRB with reports, updates, and 
other information (e.g., amendments, and administrative letters) according to regulatory 
requirements or institution procedures. 
 

PEDS024 
 
 
2020-7-15 
 
50 | P a g e  
 
17.3  Compliance with Laws and Regulations 
 
It is intended that the proposed study be conducted according to the International Conference on 
Harmonization E6 Guideline for Good Clinical Practice (GCP) and the Declaration of Helsinki.  
Please refer to the International Conference on Harmonization and GCP:  
http://www.fda.gov/oc/gcp/guidance.html; Declaration of Helsinki:   
http://www.fda.gov/oc/health/helsinki89.html; Code of Federal Regulations, Title 21: 
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch. cfm]  
 
The study will be registered with ClinicalTrials.gov by the Protocol Development Officer or 
assigned designee.  All studies must be registered no later than 21 days after enrollment of the first 
participant.  The Protocol Development Officer will maintain the responsibility of updating trials 
registered with ClinicalTrials.gov; per the FDA’s updating requirements, information must be 
updated at least every twelve months and the registry must be updated within thirty days of any 
changes in recruitment status or completion of the study. 
17.4  Delegation of Investigator Responsibilities 
 
The Principal Investigator will ensure that all persons assisting with the study are adequately 
informed about the protocol, any amendments to the protocol, the study treatments, and their 
study-related duties and functions. The Principal Investigator will maintain a list of Co-
Investigators and other appropriately qualified persons to whom he/she has delegated significant 
study-related duties.  
 
Study personnel involved in conducting this study will be qualified by education, training, and 
experience to perform their respective tasks. This study will not use the services of study 
personnel where sanctions have been invoked or where there has been scientific misconduct or 
fraud (e.g., loss of medical licensure; debarment).  
 
17.5  Subject Information and Informed Consent 
 
Before being enrolled in this clinical trial, the subject must consent to participate after the nature, 
scope, and possible consequences of the clinical study have been explained in a form 
understandable to him or her. An informed consent document that includes both information 
about the study and the consent form will be prepared and given to the subject. This document 
will contain all ICH, GCP, and locally required regulatory elements. The document must be in a 
language understandable to the subject and must specify the person who obtained informed 
consent. 
After reading the informed consent document, the subject must give consent in writing. The 
written informed consent will be obtained prior to conducting any study-related procedures or 
tests. The subject’s consent must be confirmed at the time of consent by the personally dated 
signature of the person conducting the informed consent discussions. A copy of the signed 
consent document must be given to the subject. 
The PI will retain the original signed consent document. The PI will not undertake any measures 
specifically required only for the clinical study until valid consent has been obtained. 

PEDS024 
 
 
2020-7-15 
 
51 | P a g e  
 
17.6  Confidentiality 
All records identifying the subject will be kept confidential and, to the extent permitted by the 
applicable laws and/or regulations, will not be made publicly available. 
Should direct access to medical records require a waiver or authorization separate from the 
subject’s statement of informed consent, it is the responsibility of the Investigator to obtain such 
permission in writing from the appropriate individual.   
Subjects will be told that the IRB, the independent DSMB, or regulatory authorities may inspect 
their medical records to verify the information collected, and that all personal information made 
available for inspection will be handled in strictest confidence and in accordance with local data 
protection law. 
17.7  Protocol Amendments 
Once the study has started, amendments should be made only in exceptional cases.  Protocol 
amendments cannot be implemented without prior written IRB approval except as necessary to 
eliminate immediate safety hazards to subjects.  A protocol amendment intended to eliminate an 
apparent immediate hazard to subjects may be implemented immediately, provided the IRB is 
notified within five business days.   All amendments will be submitted to the IRB and written 
verification that the amendment was submitted and subsequently approved is to be obtained.  
Study projected dose escalations will be communicated to sites as a memo. 
 
17.8  Case Report Forms 
The Principal Investigator, or his/her designee, will prepare and maintain adequate and accurate 
participant case histories with observations and data pertinent to the study.  Study-specific Case 
Report Forms (CRFs) will document safety and treatment outcomes for safety monitoring and 
data analysis.   
An electronic case report form (eCRF) is required and must be completed for each included 
subject. The completed dataset is the sole property of the University of Florida and should not be 
made available in any form to third parties, except for authorized representatives of appropriate 
Health/Regulatory Authorities, without written permission from the University of Florida. 
17.9  Record Retention 
Study documentation includes all eCRFs, data correction forms or queries, source documents, 
Sponsor-Investigator correspondence, monitoring logs/letters, and regulatory documents (e.g., 
protocol and amendments, IRB correspondence and approval, signed subject consent forms). 
Source documents include all recordings of observations or notations of clinical activities and all 
reports and records necessary for the evaluation and reconstruction of the clinical research study. 
Government agency regulations and directives require that all study documentation pertaining to 
the conduct of a clinical trial must be retained by the study investigator. In the case of a study 
with a drug seeking regulatory approval and marketing, these documents shall be retained for at 
least two years after the last approval of marketing application in an International Conference on 
Harmonization (ICH) region.  In all other cases, study documents should be kept on file until 
three years after the completion and final study report of this investigational study. 

PEDS024 
 
 
2020-7-15 
 
52 | P a g e  
 
All study documentation will be maintained for at least 6 years from the date of final study 
publication.  No study records may be destroyed without prior authorization from UF. 
 
 
18.   REFERENCES 
 
Andersson BS, deLima M, Thall PF, et al. Once daily i.v. busulfan and fludarbine (i.v. Bu-Flu) 
compares favorably with i.v. busulfan and cyclophosphamide (i.v. BuCy2) as pretransplant 
conditioning therapy in AML/MDS. Biol Blood Marrow Transplant 2008;6:672-84. 
 
Aversa F, Pelicci PG, Terenzi A et al. Results of T-depleted BMT in chronic myelogeneous 
leukemia after a conditioning regimen than included thiotepa. Bone Marrow Transplant 1991;7 
(suppl 2):24. 
 
Bartelink IH, van Reij EML, Gerhardt CE, et al. Fludarabine and Exposure-Targeted Busulfan 
Compares Favorably with Busulfan/Cyclophosphamide-Based regimens in Pediatric 
Hematopoietic Cell Transplantation: maintaining Efficacy with Less Toxicity. Biol Blood 
Marrow Transplant 2014;20:345-353. 
 
Bartelink IH, Lalmohamed A, van Reij E, et al. A new harmonized approach to estimated 
busulfan exposure predicts survival and toxicity after hematopoietic cell transplantation in 
children and young adults: a multicenter retrospective cohort analysis. Lancet Haematol 
2016;3(11):e526-e536. Doi:10.1016/S2352-3026(16)30114-4. 
 
Burroughs L, Shimamura A, Talano J-A, et al. Allogeneic hematopoietic cell transplantation 
using treosulfan-based conditioning for treatment of marrow failure disorders. Biol Blood 
Marrow Transplant 2017;23:1669-1677. 
 
Bronhauser M, Storer B, Slattery JT, et al. Conditioning with fludarabine and targeted busulfan 
for transplantation of allogeneic hematopoietic stem cells. Blood 2003;102:820-6. 
 
Down JD, Westerhof GR, Boudewijn A, et al. Thiotepa improves allogeneic bone marrow 
engraftment without enhancing stem cell deplation in irradiated mice. BMT 1998;21:327-330. 
 
Horn B, Baxter-Lowe L-A, Englert L et al. Reduced intensity conditioning using intravenous 
busulfan, fludarabine and rabbit ATG for children with nonmalignant disorders and CML. BMT 
2006;37:263-269. 
 
Horowitz ME, Morris A, Gasparetto C, et al. Myeloablative intravenous busulfan/fludarabine 
conditioning does not facilitate reliable engraftment of dual umbilical cord blood grafts in adult 
recipients. Biol Blood Marrow Transplant 2008;14:591-4. 

PEDS024 
 
 
2020-7-15 
 
53 | P a g e  
 
 
Ivaturi V, Dvorak C, Chan D, et al. Pharmacokinetics and model-based dosing to optimize 
fludarabine therapy in pediatric hematopoietic cell transplant recipients. Biol Blood Marrow 
Transplant 2017;23(10):1701-1713. 
 
Jagasia M, Greinix H, Arora M, et al. National Institutes of Health Consensus Development 
Project on Criteria for Clilnical Trials in Chronic Graft-versus-Host Disease: 1. The 2014 
Diagnosis and Staging Working Group Report. Biol Blood Marrow transplant 2015:21:389-401. 
 
Kashyap A, Wingard J, Cagnoni P, et al. Intravenous versus oral busulfan as part of a 
busulfan/cyclophosphamide preparative regimen for allogeneic hematopoietic stem cell 
transplantation: decreased incidence of hepatic venoocclusive disease (HVOD), HVOD-related 
mortality, and overall 100-day mortality. Biol Blood Marrow Transplant. 2002;8:493-500. 
 
Kelly DL, Duchbinder D, Duarte R, et al. Neurocognitive Dysfunction in Hematopoietic Cell 
Transplant Recipients: Expert Review from the Late Effects and Quality of Life Working 
Committee of the Center for International Blood and Marrow Transplant Research and 
Complications and Quality of Life Working Party of the European Society for Blood and 
Marrow Transplantation. Biol Blood Marrow Transplant 2017. 
https://doi.org/10.1016/j.bbmt.2017.09.004. 
 
Law J, Cowan M, Dvorak C, et al. Busulfan, Fludarabine and Alemtuzumab As a Reduced 
Toxicity Regimen for Children with Malignant and Nonmalignant Diseases Improves 
Engraftment and Graft-versus-Host Disease without Delaying Immune Reconstitution. Biol 
Blood Marrow Transplant 2012;18:1656-1663. 
 
Palmer J, McCune J, Miguel-Angel P, et al. Personalizing Busulfan-based conditioning: 
Considerations from the American Society for blood and marrow transplantation practice 
guidelines committee. Biol Blood Marrow Transplant 2016;22:1915-25. 
 
Przepiorka D, Dimopoulus M, Smith T, et al. Thiotepa, busulfan, and cyclophosphamide as a 
preparative regimen for marrow transplantation: risk factors for early regimen-reltaed toxicity. 
Ann Hematol 1994;68:183-188. 
 
Russell JA, Tran HT, Quinlan D, et al. Once-daily intravenous busulfan given with fludarabine 
as conditioning for allogeneic stem cell transplantation: study of pharmacokinetics and early 
clinical outcomes. Biol Blood Marrow Transplant 2002;8:468-477. 
 

PEDS024 
 
 
2020-7-15 
 
54 | P a g e  
 
Russell JA, Kangarloo SB, Williamson T, et al. Establishing a target exposure for once-daily 
intravenous busulfan given with fludarabine and thymoglobulin before allogeneic 
transplantation. Biol Blood Marrow Transplant 2013:19:1381-6. 
 
Sanz J, Boluda JCH, Martin C, et al. Single-unit umbilical cord blood transplantation from 
unrelated donors in patients with hematological malignancy using busulfan, thiotepa, fludarabine 
and ATG as myeloablative conditioning regimen. BMT 2012;47:1287-93. 
 
Santos GW, Tutschka PJ, Brookmeyer R, et al. Marrow transplantation for acute nonlymphocytic 
leukemia after treatment with busulfan and cyclophosphamide. N Engl J Med. 1983;309:1247-
53. 
Satwani P, Jin Z, Duffy D, et al. Transplantation-related mortality, graft failure, and survival 
after reduced-toxicity conditioning and allogeneic hematopoietic stem cell transplantation in 100 
consecutive pediatric recipients. Biol Blood Marrow Transplant 2013;19:552-61. 
 
Slavin S, Nagler A, Naperstek E, et al. Nonmyeloablative stem cell transplantation and cell 
therapy as an alternative to conventional bone marrow transplantation with lethal cytoreduction 
for the treatment of malignant and nonmalignant diseases. Blood. 1998;91:756-763. 
 
Slatter MA, Boztug H, Potschger U, et al. Treosulfan-based conditioning regimen for allogeneic 
hematopoietic stem cell transplantation in children with non-malignant diseases. Bone Marrow 
Transplantation 2015;50:1536-41. 
 
Tutschka PJ, Copelan EA, Klein JP. Bone marrow transplantation for acute leukemia following a 
new busulfan and cyclophosphamide regimen. Blood 1987;70:1382-1388 
 
Ward J, Ketzel M, Duerst R, et al. Single Daily Busulfan Dosing for Infants with Nonmalignant 
Diseases Undergoing Reduced-Intensity Conditioning for Allogeneic Hematopoietic Progenitor 
Cell Transplantation. Biol Blood Marrow Transplant 2015;21:1612-21. 
 
 
Willard V, Leung W, Huang Q, et al. Cognitive Outcome After pediatric Stem-Cell 
Transplantation: Impact of Age and Total Body Irradiation. J Clin Oncol 2014;32:3982-3988. 
 
 
 
 
 
 
 
 
 
 
 
 

PEDS024 
            
 
 
 
2020-7-19 
 
 
 
19.  APPENDICES 
 
APPENDIX A: SCHEDULE OF ASSESSMENTS   
 
                                      Visit: 
 
 
Procedure: 
SCREENING 
(PRE-TRANSPLANT 
EVALUATION1) 
 
TREATMENT 
(DAY -7 to   
DAY 11) 
 
(WEEKLY UNTIL 
DAY 60 then with 
every visit until 100 
daysPOST 
TRANSPLANT) 
ON STUDY  
(DAY 100 AND EVERY 3 
MONTHS UNTIL NORMAL) 
12 and 24 months post- 
transplant (or end of 
study)12 
 
Informed Consent2 
X 
 
 
 
 
Demographics3 
X 
 
 
 
 
Vital Signs4 
X 
 
 
 
 
Complete Medical History 
X 
 
 
 
 
Neurocognitive assessment (up to age 
16 years at the time of testing) 
X 
 
 
 
X 
Lansky/Karnofsky Score 
X 
 
 
 
X 
Pre-Transplant Disease Evaluation5 
X 
 
 
 
 
Pregnancy Test (Serum)6 
X 
 
 
 
 
Echocardiogram 
X 
 
 
 
 
Calculated Creatinine Clearance 
X 
 
 
 
 
Liver Function Tests 
X 
 
 
 
 
PFTs or O2 saturation 
X 
 
 
 
 
Peripheral blood specimen for 
Pharmacogenomic studies 
X 
 
 
 
 
Protocol Treatment: 
 
 
 
 
 
Busulfan 
 
X7 
 
 
 
Fludarabine 
 
X7 
 
 
 
Thiotepa 
 
X7 
 
 
 
Anti-thymocyte globulin 
 
X7 
 
 
 

PEDS024 
 
 
2020-7-15 
 
56 | P a g e  
 
 
 
 
 
Tacrolimus 
 
X7 
 
 
 
Methotrexate – IV only 
 
X7 
 
 
 
Mycophenolate mofetil 
 
X7 
 
 
 
PK Sampling (Busulfan only) 
 
X8 
 
 
 
Immune Reconstitutions Studies 
 
 
 
X 
X 
Chimerism testing9 
 
 
            X9 
X 
SAE Reporting10 
 
X10 
GVHD Assessment10 
 
 
                                               X10 
 
CIBMTR forms reporting11 
X 
 
X11 
X 
1 All pre-transplant evaluations performed as SOC within 30 days of initiation of conditioning regimen but prior to informed consent, will be accepted for this study and 
need not be repeated. Neurocognitive testing up to 90 days prior to start of conditioning will be accepted. 
2 Written informed consent must be obtained at least 24 hours prior to subject starting conditioning regimen.   
3 Demographics include date of birth, gender,  rac and ethnicity. 
4 Vital signs include height, weight, blood pressure, and pulse oximetry. 
5 Pre-transplant disease evaluation will be determined by treating physician, it is disease specific and will follow SOC guidelines 
6 Pregnancy test to be completed within 7 days of starting conditioning for WOCBP. 
7 Refer to section 7.1 for treatment schedule and administration. 
8 Busulfan PK collection obtained at the following time points from the start of first busulfan dose: 3 hours (end of infusion), 3 hours 15 minutes, 4 hours, 5 hours, 6 
hours, and 8 hours. 
9Obtain at day 30 if ANC has recovered, or at day 42 if there is failure of engraftment, obtain any time there is suspected failure of engraftment and then at 12 months 
and end of study 
10Assess for SAEs and GVHD weekly until day 60, then at every clinic visit 
 11See section 6.3 for CIBMTR forms requirements, post transplant forms are due on day 100, 180 and 12 and 24 months 
12Survival status will also be collected at year 3 and 4, if the study stays open. 

PEDS024 
            
 
 
 
2020-7-19 
 
 
 
 
APPENDIX B: MODIFIED GLUCKSBERG STAGING CRITERIA FOR ACUTE 
GRAFT VERSUS HOST DISEASE 
 
 
 
For GI staging: The “adult” stool output values should be used for patients > 50 kg in weight. Use 
3 day averages for GI staging based on stool output. If stool and urine are mixed, stool output is 
presumed to be 50% of total stool/urine mix. 
For Stage 4 GI: the term “severe abdominal pain” will be defined as: 
(a) Pain control requiring institution of opioid use, or an increase in on-going opioid use, PLUS 
(b) Pain that significantly impacts performance status, as determined by the treating MD. 
If colon or rectal biopsy is +, but stool output is < 500 mL/day (< 10 mL/kg/day), then consider as 
GI Stage 0. 
 
There is no modification of liver staging for other causes of hyperbilirubinemia 
Overall Clinical Grade (based on the highest stage obtained): 
Grade 0: No Stage 1-4 of any organ 
Grade I: Stage 1-2 skin and no liver or gut involvement 
Grade II: Stage 3 skin, or Stage 1 liver involvement, or Stage 1 GI 
Grade III: Stage 0-3 skin, with Stage 2-3 liver, or Stage 2-3 GI 
Grade IV: Stage 4 skin, liver or GI involvement 
 
 
 
 
 

PEDS024 
 
 
2020-7-15 
 
58 | P a g e  
 
APPENDIX B2: CGVHD GRADING AS MIDLE, MODERATE AND SEVERE BY NIH 
2014 CONSENSUS CONFERENCE CRITERIA 
 
Refer to published details on diagnostic signs and symptoms and scores by involved organs 
(Jagasia et al, 2015)  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

PEDS024 
 
 
2020-7-15 
 
59 | P a g e  
 
APPENDIX C: STUDY REQUIRED SPECIMENS, VOLUMES, HANDLING AND 
SHIPPING INSTRUCTIONS 
 
 
Specimen description 
Diagnosis 
 
Pharmacogenomic studies – 3 mL in 
EDTA tube  
 
 
 
X 
 
 
After being obtained, blood specimens in EDTA tubes should be kept in the refrigerator until 
shipping. The specimens should be shipped on ice package overnight. Specific collection, 
packaging and shipping instructions are as follows: 
Procedure: 
1. Draw sample into labeled purple top (EDTA) tube 
a. Peripheral blood: 3mL 
2. Samples should be slowly inverted 8 to 10 times to ensure the mixing of the sample 
and the anti-coagulant liquid inside the tube 
3. Label tube with: 
a. Subject identifier (see below) 
b. Specimen type (peripheral blood) 
c. Date of collection 
4. Secure and tape the top of the tube by wrapping the cap with parafilm to prevent 
spilling during shipment. 
5. Individually wrap each tube with paper towels and rubber band to prevent freezing 
of sample and absorb any leaks. 
6. Seal each wrapped tube in biohazard bag/secondary receptacle to prevent sample 
from leaking. 
7. Place samples in the rigid outer container along with frozen gel packs. 
8. Secure the outer container with packing tape and attach the provided shipping label. 
Notes:  
• Samples should be sent as soon as they are obtained. If sample cannot be sent 
immediately, it can be stored at 4C for up to 24 hours. 
• Do not provide any protected health information (PHI) on the sample or in the 
shipment if possible. 
• Include a marking on the package that properly identifies the shipment as "Exempt 
Human Specimen" as appropriate to comply with current IATA and ICAO 
regulations.   
 
Samples are to be labeled using the following format: 
 
Study Center Identifier –Subject ID- Specimen Number – Sample Source 
 
• Subject IDs to start at 01 and increase sequentially with each newly consented subject. 

PEDS024 
 
 
2020-7-15 
 
60 | P a g e  
 
• Specimen numbers to start at 01 for the diagnostic/baseline specimen and increase 
sequentially with each newly collected sample. 
• Sample source: 
o Peripheral Blood (PB) 
• For this study specimens will be labeled: e.g. UFP-01(first subject)-01-PB (all 
subjects will be having only one specimen and peripheral blood will be the only 
source). 
 
• Contact Dr. Jatinder Lamba Ph.D, prior to shipping. Ship to the following address: 
 
                                                                   University of Florida, Department of 
                                                                    Pharmacotherapy and Translational Research                    
                                                                    Office PG-25 HSC Building 
                                                        Lab PG-06 HSC Building 
                                                                    1345 Center Drive  
                                                                    PO box# 100486 
                                                                    Gainesville, FL-32610 
                                                                    Office Phone: (352) 273-6425 
                                                        Lab Phone: (352) 294 5745 
                                                                    Fax: (352) 273-6121 
                                                                    Email: jlamba@cop.ufl.edu 
                                                                                Email: jatinderklamba@ufl.edu 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

PEDS024 
 
 
2020-7-15 
 
61 | P a g e  
 
APPENDIX D: PROTOCOL DRUGS 
 
1. Busulfan Source and Pharmacology (Busulfan-Injection (07/01/2015) (Busulfex® NSC 
#750) 
 
Busulfan is a non-cell-cycle-specific bi-functional alkylating agent. In aqueous media, busulfan 
hydrolyzes to release methanesulfonate groups. This produces reactive carbonium ions that 
interact with cellular thiol groups and nucleic acids to form DNA cross-links. Busulfan injection 
is 100% bioavailable by definition of intravenous administration. The elimination of busulfan 
appears to be independent of renal function, presumably reflecting the extensive metabolism of 
the drug in the liver, since less than 2% of the administered dose is excreted in the urine 
unchanged within 24 hours. The drug is metabolized by enzymatic activity to at least 12 
metabolites, among which tetrahydrothiophene, tetrahydrothiophene 12-oxide, sulfolane, and 3-
hydroxysulfolane were identified. These metabolites do not have cytotoxic activity. Irreversible 
binding to plasma proteins (primarily albumin) is approximately 32.4%. Busulfan has a plasma 
terminal elimination half-life (t1/2) of about 2.6 hours and demonstrates linear kinetics. It is 
rapidly distributed into tissue and crosses the blood-brain and the placental barriers. CSF 
concentrations are approximately equal to those in plasma. Itraconazole reduced busulfan 
clearance by up to 25% in patients receiving itraconazole compared to patients who did not 
receive itraconazole. Higher busulfan exposure due to concomitant itraconazole could lead to 
toxic plasma levels in some patients. Fluconazole had no effect on the clearance of busulfan. 
 
Busulfan Formulation and Stability 
 
Each ampoule or vial of busulfan injection contains 60 mg (6 mg/mL) of busulfan, N,N-
dimethylacetamide (DMA) 33% vol/vol and Polyethylene Glycol 400, 67% vol/vol. Store 
refrigerated at 2°- 8°C, (36°- 46°F).  
 
Busulfan Guidelines for Administration 
 
Dilute busulfan injection to a final concentration of approximately 0.5 mg/mL with NS or D5W. 
The drug should not be infused with any other drug or IV solution other than NS or D5W. 
Always add the busulfan to the diluent, not the diluent to the busulfan injection. Mix thoroughly 
by inverting several times. 
 

PEDS024 
 
 
2020-7-15 
 
62 | P a g e  
 
Do not use polycarbonate syringes or filter needles with busulfan injection. Busulfan injection 
diluted in NS or D5W is stable at room temperature (25°C) for up to 8 hours but the infusion 
must be completed within that time. Busulfan injection diluted in NS is stable at refrigerated 
conditions 2°- 8°C (36°-46°F) for up to 12 hours but the infusion must be completed within that 
time. Busulfan injection contains N,N-dimethylacetamide, which is incompatible with many 
closed-system transfer devices (CSTDs); the plastic components of CSTDs may dissolve and 
result in subsequent leakage and potential infusion of dissolved plastic into the subject. 
 
Busulfan injection should be administered by IV infusion through a central venous catheter. 
Subjects receiving busulfan in a conditioning regimen for bone marrow transplant must receive 
seizure prophylaxis. If phenytoin is used, it should be given 12 hours prior to the start of 
busulfan, then daily during busulfan administration and for 48 hours after completion of 
busulfan. In dose-finding studies of busulfan where patients received concomitant busulfan and 
phenytoin, phenytoin reduced busulfan plasma AUC by approximately 15%. Use of other 
anticonvulsants may result in higher busulfan plasma AUCs, and an increased risk of sinusoidal 
obstruction syndrome, (SOS, formerly VOD) or seizures. After an initial dose of busulfan 
injection, blood levels are monitored with bone marrow transplant patients in order to achieve a 
target area-under-the-curve (AUC) plasma concentration.  
 
Busulfan Supplier 
 
Busulfan is commercially available from various manufacturers. Reference the package insert for 
further information 
 
Busulfan Toxicities 
 
Please refer to Table 19.1 for busulfan toxicities. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

PEDS024 
 
 
2020-7-15 
 
63 | P a g e  
 
 
Table 19.1- Busulfan Toxicities: 
 
Common 
(Occurs in 21-100 children out of 
every 100) 
Occasional 
(Occurs in 5-10 children out of every 
100) 
Rare 
(Occurs in < 5 children out of every 
100) 
Immediate: 
(Within 1-2 days of 
receiving drug) 
Nausea, vomiting, fever, 
electrolyte changes 
(hypokalemia, hypomagnesemia, 
hypocalcemia, 
hypophosphatemia, and 
hyponatremia), hyperglycemia, 
dizziness, rash, pruritus, urticaria, 
injection site pain & 
inflammation, back pain, 
tachycardia, chest pain, edema, 
insomnia, anxiety depression, 
headache, abdominal pain, 
diarrhea, (L) or constipation, 
anorexia, rectal discomfort, 
dyspnea, epistaxis 
Weight gain, confusion 
Seizures (rare with anti-seizure 
prophylaxis), hematemesis, 
hyperuricemia, arrhythmias other 
than tachycardia, pleural effusion, 
alveolar hemorrhage 
Prompt: 
(Within 2-3 weeks) 
Myelosuppression, asthenia, 
immunosuppression (L), 
mucositis, hyperbilirubinemia 
Hepatotoxicity, sinusoidal 
obstruction syndrome (SOS, 
formerly VOD) (L), mild 
alopecia (L), arthralgia, myalgia, 
hemorrhagic cystitis, 
hyperpigmentation (L), elevated 
creatinine, and BUN 
Reduced adrenal function (L), 
esophagitis, radiation recall 
reactions 
Late: 
(Any time after 
completion of 
treatment) 
Infertility, testicular atrophy & 
azoospermia, amenorrhea, 
ovarian failure 
 
Secondary malignancy, breast 
enlargement, cataracts, idiopathic 
pulmonary syndrome (cough, 
dyspnea, pleural effusion, 
infiltrates, & hypoxemia), 
bronchopulmonary dysplasia with 
interstitial pulmonary fibrosis & 
pneumonitis, myocardial fibrosis, 
osteonecrosis 
Unknown 
Frequency & 
Timing: 
Fetal toxicities and teratogenic effects of busulfan and its solvent have been noted in animals.  Toxicities 
include multiple anomalies and low birth weight.  It is unknown whether the drug or its solvent is excreted 
in breast milk. 
(L) Toxicity may also occur later 
 
2. Fludarabine Source and Pharmacology (Fludarabine (05/09/2011) (Fludara®, 
fludarabine phosphate, (2-fluror-ara-AMP) NSC# 3121887) 
 
Fludarabine phosphate is a synthetic purine nucleoside. It differs from the physiologic 
nucleosides, adenosine, in that the sugar moiety is arabinose instead of ribose, and by the 
addition of a fluorine atom to the purine base adenine. Fludarabine is also a fluorinated 
nucleotide analog the antiviral agent vidarabine, (ara-A).  The addition of fluorine results in 
increased aqueous solubility and resistance to enzymatic degradation by adenosine deaminase. 
Fludarabine (2-fluoro-ara-A) is commercially available as the monophosphate salt (2-fluoro-ara-

PEDS024 
 
 
2020-7-15 
 
64 | P a g e  
 
AMP). The monophosphorylation increases the drug's aqueous solubility while maintaining 
pharmacologic activity. The chemical name for fludarabine phosphate is 9HPurin-6-amine, 2-
fluoro-9-(5-0-phosphono β-D-arabino-furanosyl) (2-fluoro-ara-AMP) and the molecular weight 
is 365.2. Fludarabine is a purine antagonist antimetabolite. In vivo, fludarabine phosphate is 
rapidly dephosphorylated to 2-fluoro-ara-A and then it is phosphorylated intracellularly by 
deoxycytidine kinase to the active triphosphate, 2-fluoro-ara-ATP. This metabolite appears to act 
by inhibiting DNA polymerase alpha, ribonucleotide reductase and DNA primase, thus 
inhibitingDNA synthesis. The mechanism of action of this antimetabolite is not completely 
characterized and may be multi-faceted.  Phase I studies in humans have demonstrated that 
within several minutes after IV infusion, fludarabine phosphate is converted to the active 
metabolite 2-fluror-ara-A and becomes undetectable.  Therefore, PK studies have focused on 2-
fluror-ara-A.  Fludarabine phosphate 25 mg/m2 infused intravenously over 30 minutes to adult 
cancer patients, showed a moderate accumulation of 2-fluoro-ara-A. During a 5-day treatment 
schedule, 2-fluoro-ara-A plasma trough levels increased by a factor of about 2. Fludarabine is 
widely distributed. The volume of distribution at steady state (Vss) reported after daily 
administration of 25 mg/m2 for 5 days to adults averaged at 96-98 L/m2. Tissue distribution 
studies in animals indicate that the highest concentrations of the drug are in liver, kidney, and 
spleen. Although the extent to which fludarabine and/or its metabolites distribute into the CNS in 
humans has not been determined to date, severe neurologic toxicity (e.g., blindness, coma) has 
been reported in patients receiving the drug, particularly in high dosages. There is evidence from 
animal studies that fludarabine distributes into the CNS and that a toxic metabolite (2-
fluoroadenine, possibly formed by bacteria in the GI tract), can be absorbed systematically via 
enterohepatic circulation and distributed into CSF. According to in vitro data, about 19-29% of 
fludarabine is bound to plasma proteins. Following IV administration, fludarabine phosphate is 
dephosphorylated rapidly to fludarabine. Plasma concentrations of fludarabine decline in a 
linear, dose-independent manner. The elimination profile of fludarabine also has been reported to 
be either biphasic or triphasic; however, reported terminal elimination half-lives have been 
similar. In adult cancer patients receiving fludarabine 25 mg/m2 as a 30-minute IV infusion daily 
for 5 days, a terminal half-life of about 20 hours was reported. In a limited number of pediatric 
patients, the plasma concentration profile of fludarabine exhibited both monoexponential and 
biexponential decay, with a mean t1/2 of 10.5 hours in patients with monoexponential 
elimination and a t1/2 of 1.2-1.4 and 12.4-19 hours, respectively, in patients with biexponential 
elimination. Renal clearance accounts for about 40% of the total body clearance of fludarabine. 
Renal elimination appears to become more important at high dosages of the drug. The dose of 
fludarabine needs to be adjusted in patients with moderate renal impairment.  The use of 
fludarabine in combination with pentostatin is not recommended due to the risk of severe 
pulmonary toxicity. 
 
 
 

PEDS024 
 
 
2020-7-15 
 
65 | P a g e  
 
Fludarabine Formulation and Stability 
 
Fludarabine phosphate injection is available as sterile lyophilized powder and in solution. Each 
single-dose vial of powder contains 50 mg of the active ingredient fludarabine phosphate, 50 mg 
of mannitol, and sodium hydroxide to adjust the pH to 7.7. After reconstitution, the pH range for 
the final product is 7.2-8.2. The single-dose solution vial contains 25 mg/mL, 2 mL of 
fludarabine phosphate. It may contain mannitol and is preservative-free. Fludarabine phosphate 
vials should be stored refrigerated at 2-8ºC (36-46ºF).  
 
Fludarabine Guidelines for Administration 
 
Prior to administration fludarabine phosphate powder should be reconstituted with Sterile Water 
for Injection and further diluted in D5W or NS to a concentration of 10–25 mg/mL. Fludarabine 
25 mg/mL solution should be further diluted in the same manner. Fludarabine phosphate powder 
should be reconstituted with 2 mL of Sterile Water for Injection. The solid cake should fully 
dissolve in 15 seconds or less. The resulting concentration is 25 mg/mL. When reconstituted to a 
final concentration of 25 mg/mL, the drug is stable for at least 16 days at room temperature and 
normal light conditions. The manufacturer recommends that the solution be used within 8 hours 
after reconstitution. Prior to administration, fludarabine 25 mg/mL solution or the reconstituted 
25 mg/mL solution should be further diluted in 100 mL or 125 mL of D5W or NS. When diluted 
to a final concentration of 1 mg/mL, fludarabine is stable for at least 16 days at room temperature 
and normal light conditions. The  manufacturer recommends that the diluted solution be used 
within 8 hours after preparation. Parenteral drug products should be inspected visually for 
particulate matter and discoloration prior to administration. 
 
Fludarabine Supplier 
 
Fludarabine is commercially available from various manufacturers. Reference the package insert 
for further information. 
 
Fludarabine Toxicities 
 
Please refer to Table 19.2 for fludarabine toxicities. 
 
 
 
 
 
 
 
 
 
 

PEDS024 
 
 
2020-7-15 
 
66 | P a g e  
 
Table 19.2 - Fludarabine Toxicities: 
 
Common 
(Occurs in 21-100 subjects out of 
every 100) 
Occasional 
(Occurs in 5-20 subjects out of every 
100) 
Rare 
(Occurs in < 5 subjects out of every 
100) 
Immediate: 
(Within 1-2 days of 
receiving drug) 
Fever, fatigue, weakness, pain, 
nausea, vomiting, anorexia, 
cough, dyspnea 
Edema including peripheral 
edema, chills, rash, diarrhea, 
rhinitis, diaphoresis, malaise, 
abdominal pain, headache, back 
pain, myalgia, stomatitis, flu-like 
syndrome 
Anaphylaxis, tumor lysis 
syndrome, dehydration* 
Prompt: 
(Within 2-3 weeks, 
prior to next course) 
Myelosuppression (anemia, 
neutropenia, thrombocytopenia) 
infection (urinary tract infection, 
herpes simplex infection, 
pneumonia upper respiratory) 
Weight loss, gastrointestinal 
bleeding, hemoptysis, 
paresthesia, allergic pneumonitis, 
bronchitis, pharyngitis, visual 
disturbance, hearing loss, 
hyperglycemia 
Sinusitis, dysuria, opportunistic 
infections and reactivation of 
latent viral infections like 
Epstein-Barr virus (EBV), herpes 
zoster and John Cunningham (JC) 
virus (progressive multifocal 
leukoencephalopathy [PML])L, 
EBV associated 
lymphoproliferative disorder, 
pancytopenia (can be prolonged), 
pulmonary hypersensitivity a 
(dyspnea, cough, hypoxia, 
interstitial pulmonary infiltrate), 
pulmonary toxicity (acute 
respiratory distress syndrome) 
[ARDS], pulmonary fibrosis, 
pulmonary hemorrhage, 
respiratory distress, respiratory 
failure), pericardial effusion, skin 
toxicity (erythema multiforme, 
Stevens-Johnson syndrome, toxic 
epidermal necrolysis, 
pemphigus), liver failure, renal 
failure, hemorrhage, transfusion-
associated GVHD has occurred 
following transfusion of non-
irradiated blood products, 
phlebitis*, sleep disorder*, 
cerebellar syndrome*, 
depression*, mentation 
impaired*, alopecia*, pruritus*, 
seborrhea*, esophagitis*, 
constipation*, mucositis*, 
dysphagia* hesitancy*, 
cholelithiasis*, abnormal liver 
function tests*, osteoporosis*, 
arthralgia*, abnormal renal 
function test*, proteinuria*, 
epistaxis*, hemorrhagic cystitis* 
eosinophilia* 

PEDS024 
 
 
2020-7-15 
 
67 | P a g e  
 
Delayed: 
(Any time later 
during therapy, 
excluding the above 
conditions) 
 
 
Neurotoxicity (increased with 
high doses): seizures, agitation, 
confusion, weakness, visual 
disturbances, optic neuritis, optic 
neuropathy, photophobia, 
blindness, paralysis, coma, death, 
peripheral neuropathy a); 
autoimmune phenomena: 
thrombocytopenia/idiopathic 
thrombocytopenic purpura (ITP), 
Evans syndrome, hemolytic 
anemia, acquired hemophilia 
Late: 
(Any time after 
completion of 
treatment) 
 
 
Myelodysplastic syndrome/acute 
myeloid leukemia (mainly 
associated with prior or 
concomitant or subsequent 
treatment with other anticancer 
treatments), skin cancer (new 
onset or exacerbation) 
Unknown 
Frequency & 
Timing: 
Pregnancy Category D 
Based on its mechanism of action, fludarabine phosphate can cause fetal harm when administered to a 
pregnant woman.  Fludarabine phosphate was embryo-lethal and teratogenic in both rates and rabbits. 
(L) Toxicity may also occur later. 
* Reported in ≤ 3% of subjects.  Since these are not considered life threatening, they are not 
included in the consent form. 
a These effects were not reported in children. 
 
3. Thiotepa Source and Pharmacology (Thiotepa 11/15/2016) (Tepadina, Tespa, 
Thiophosphamide, Triethylenethiophosphoramide Tspa, WR-45312) NSC #6396  
 
Thiotepa is a cytotoxic agent of the polyfunctional type, related chemically and 
pharmacologically to nitrogen mustard. The radiomimetic action of thiotepa is believed to occur 
through the release of ethylenimine radicals, which, like irradiation, disrupt the bonds of DNA. 
One of the principal bond disruptions is initiated by alkylation of guanine at the N-7 position, 
which severs the linkage between the purine base and the sugar and liberates alkylated guanines. 
Thiotepa is desulfurated by cytochrome P-450 enzymes such as 2B1 and 2C11 which catalyze 
the conversion of thiotepa to tepa. Tepa is less toxic than thiotepa and has been demonstrated to 
produce alkali-labile sites in DNA, rather than cross-links. These findings indicate that tepa 
reacts differently from thiotepa and produces monofunctional alkylation of DNA. A second 
metabolite of thiotepa, a mercapturic acid conjugate, is formed via glutathione conjugation.  
Monochloro tepa is the third metabolite found in the urine. Following short intravenous infusion 
(less than 5 minutes), peak concentrations of thiotepa were measured within 5 minutes. At steady 
state, the volume of distribution was independent of dose and ranged from 0.3 to 1.6 liters per 
kilogram (L/kg). Approximately 4.2% of the original dose is eliminated in the urine within 24 
hours as tepa. The elimination half-life of thiotepa ranges from 2.3 to 2.4 hours. The half-life of 
tepa ranged from 3 to 21.1 hours in one study.  

PEDS024 
 
 
2020-7-15 
 
68 | P a g e  
 
 
Thiotepa Formulation and Stability 
 
Thiotepa for Injection USP, for single use only, is available in vials containing 15 mg of non-
pyrogenic, sterile lyophilized powder. Store in a refrigerator at 2°-8°C (36°-46°F). PROTECT 
FROM LIGHT AT ALL TIMES. Note: FDA is allowing temporary importation of a European 
thiotepa product (Tepadina®). Verify product, storage, and preparation instructions prior to 
dispensation and administration. Refer to specific product labeling for details. Tepadina®: Store 
intact vials under refrigeration at 2°C to 8°C (36°F to 46°F). Protect from light; do not freeze. 
Reconstituted solution (10 mg/mL) is stable for 8 hours when stored at 2°C to 8°C (36°F to 
46°F). Solution further diluted for infusion is stable for 24 hours when stored at 2°C to 8°C 
(36°F to 46°F), or for 4 hours when stored at 25°C (77°F).  
 
Thiotepa Guidelines for Administration 
 
Reconstitute Thiotepa for Injection with 1.5 mL of Sterile Water for Injection resulting in a drug 
concentration of approximately 10 mg/mL. (As per manufacturer’s information: Actual content 
per vial 15.6 mg; withdrawable amount 14.7 mg/1.4 mL; approximate reconstituted 
concentration: 10.4 mg/mL). When reconstituted with Sterile Water for Injection, solutions of 
thiotepa should be stored at refrigerated temperatures 2°-8°C (36°-46°F) protected from light and 
used within 8 hours. The reconstituted solution is hypotonic and should be further diluted with 
Sodium Chloride Injection (0.9% NaCl) prior to use. Thiotepa at a concentration of 1-5 mg/mL 
in 0.9% NaCl is stable for 24 hours at room temperature. At concentration of 0.5mg/mL it is 
stable for only one hour and stability decreases significantly at concentrations of less than 
0.5mg/mL. Therefore, solutions diluted to 0.5mg/mL should be used immediately. In order to 
eliminate haze, filter solutions through a 0.22 micron filter [Polysulfone membrane (Gelman’s 
Sterile Aerodisc®, Single Use) or triton-free mixed ester of cellulose/PVC (Millipore’s 
MILLEX®- GSFilter Unit)] prior to administration. Filtering does not alter solution potency. 
Reconstituted solutions should be clear. Solutions that remain opaque or precipitate after 
filtration should not be used. Tepadina®: Reconstitute each 15 mg vial with 1.5 mL SWFI, or 
each 100 mg vial with 10 mL SWFI, to a concentration of 10 mg/mL. Gently mix by repeated 
inversions. Solution may be clear or opalescent; do not use if particulate matter is present.  
Further dilute reconstituted solution for IV infusion in 500 mL NS (1,000 mL NS if dose > 500 
mg). If dose is < 250 mg, dilute in an appropriate volume of NS to achieve a final concentration 
of 0.5 to 1 mg/mL. When thiotepa is given in bone marrow transplant doses, bath the patient and 
change linen frequently (≥ 2 baths/day) to avoid the contact dermatitis and discoloration of the 
skin that is seen with high dose.  
 
 
 
 

PEDS024 
 
 
2020-7-15 
 
69 | P a g e  
 
 
Thiotepa Supplier 
 
The FDA is allowing temporary importation of Tepadina® (thiotepa), from Adienne Pharma & 
Biotech. Product may be ordered directly through Adienne Pharm & Biotech.  Reference the 
package insert for further information. Please review the letter regarding importation of thiotepa 
on the FDA website: 
http://www.fda.gov/downloads/Drugs/DrugSafety/DrugShortages/UCM251666.pdf 
 
Thiotepa Toxicities 
 
Please refer to Table 19.3 for thiotepa toxicities. 
 
Table 19.3 - Thiotepa Toxicities: 
 
Common 
(Occurs in 21-100 subjects out of 
every 100) 
Occasional 
(Occurs in 5-20 subjects out of 
every 100) 
Rare 
(Occurs in < 5 subjects out of every 100) 
Immediate: 
(Within 1-2 days of 
receiving drug) 
Nausea, vomiting, anorexia, 
fatigue, weakness 
Pain at the injection site, 
dizziness, headache, blurred 
vision, abdominal pain, contact 
dermatitis, rash 
Anaphylaxis, laryngeal edema, 
wheezing, hives 
Prompt: 
(Within 2-3 weeks, 
prior to next course) 
Myelosuppression; at higher 
doses in conditioning regimens 
for BMT: mucositis, esophagitis 
At higher doses in conditioning 
regimens for BMT: 
encephalopathy (inappropriate 
behavior, confusion, 
somnolence), increased liver 
transaminases, increased 
bilirubin, hyperpigmentation of 
the skin (bronzing effect) 
Febrile reaction, conjunctivitis, 
dysuria, urinary retention 
Delayed: 
(Any time later 
during therapy, 
excluding the above 
conditions) 
Gonadal dysfunction/infertility, 
azoospermia, amenorrhea 
 
Alopecia, secondary malignancy 
Unknown 
Frequency & 
Timing: 
Fetal and teratogenic toxicities: Carcinogenic and teratogenic effects of thiotepa have been noted in animal 
models at doses ≤ those used in humans.  It is not known if thiotepa is excreted into human breast milk.  
(L) Toxicity may also occur later. 
 
 
 
 
 
 
 
 

PEDS024 
 
 
2020-7-15 
 
70 | P a g e  
 
4. Anti-thymocyte Source and Pharmacology (Anti-thymocyte Globulin (Rabbit) 
(05/06/11) (Rabbit ATG, RATG – Rabbit, Antithymocyte Globulin, Thymoglobulin®) 
NSC #720095) 
 
Anti-thymocyte globulin (rabbit) is a purified, pasteurized, gamma immune globulin, obtained by 
immunization of rabbits with human thymocytes. This immunosuppressive product contains 
cytotoxic antibodies directed against antigens expressed on human T-lymphocytes. The 
mechanism of action by which polyclonal anti-lymphocyte preparations suppress immune 
responses is not fully understood. Possible mechanisms by which anti-thymocyte globulin 
(rabbit) may induce immunosuppression in vivo include: T-cell clearance from the circulation 
and modulation of T-cell activation, homing, and cytotoxic activities. Anti-thymocyte globulin 
(rabbit) includes antibodies against T-cell markers such as CD2, CD3, CD4, CD8, CD11a, 
CD18, CD25, CD44, CD45, HLA-DR, HLA Class I heavy chains, and (beta) 2 microglobulin. In 
vitro, anti-thymocyte globulin (rabbit) (concentrations > 0.1 mg/mL) mediates T-cell suppressive  
effects via inhibition of proliferative responses to several mitogens. In patients, T-cell depletion 
is usually observed within a day from initiating anti-thymocyte globulin (rabbit) therapy. After 
an intravenous dose of 1.25 to 1.5 mg/kg/day (over 4 hours for 7-11 days) 4-8 hours post-
infusion, anti-thymocyte globulin (rabbit) levels were on average 21.5 mcg/mL (10-40 mcg/mL) 
with a half-life of 2-3 days after the first dose, and 87 mcg/mL (23-170 mcg/mL) after the last  
dose. During an anti-thymocyte globulin (rabbit) Phase III randomized trial, anti-rabbit 
antibodies developed in 68% of the Thymoglobulin-treated patients (108 of 163 patients 
evaluated). The volume of distribution is 0.12 L/kg. Approximately 1% of the dose excreted in 
the urine. 
 
Anti-thymocyte Formulation and Stability 
 
Each vial contains anti-thymocyte globulin (rabbit) 25 mg and inactive ingredients: glycine (50 
mg), mannitol (50 mg), sodium chloride (10 mg). The reconstituted preparation contains 
approximately 5 mg/mL of anti-thymocyte globulin (rabbit), of which > 90% is rabbit gamma 
immune globulin (IgG). The reconstituted solution has a pH of 7 ± 0.4. Each anti-thymocyte 
globulin (rabbit) lot is released following potency testing (lymphocytotoxicity and E-rosette 
inhibition assays), and cross-reactive antibody testing (hemagglutination, platelet agglutination, 
anti-human serum protein antibody, antiglomerular basement membrane antibody, and fibroblast 
toxicity assays) on every fifth lot. 
 
Anti-thymocyte Guidelines for Administration 
 
Reference the Treatment and Dose Modifications sections of the protocol. Reconstitute anti-
thymocyte globulin (rabbit) with the supplied diluent, SWFI, immediately before use. Anti-
thymocyte globulin (rabbit) should be used within 4 hours after reconstitution if kept at room  

PEDS024 
 
 
2020-7-15 
 
71 | P a g e  
 
temperature. Allow Thymoglobulin and SWFI vials to reach room temperature before 
reconstituting the lyophilized product. 1. Reconstitute each vial of anti-thymocyte globulin 
(rabbit) lyophilized powder with 5 mL of SWFI. 2. Inject SWFI slowly into the vial containing 
the lyophilized powder. 3. Rotate vial gently until powder is completely dissolved. Each 
reconstituted vial contains 25 mg or 5 mg/mL of anti-thymocyte globulin (rabbit). 4. Inspect 
solution for particulate matter after reconstitution. Should some particulate matter remain, 
continue to gently rotate the vial until no particulate matter is visible. If particulate matter 
persists, discard this vial. Dilution: 1. Transfer the contents of the calculated number of anti-
thymocyte globulin (rabbit) vials into the bag of infusion solution (saline or dextrose). 
Recommended volume: per one vial of anti-thymocyte globulin (rabbit), use 50 mL of infusion 
solution (total volume usually between 50 to 500 mL). 2. Mix the solution by inverting the bag 
gently only once or twice. Administer by intravenous infusion through a high-flow vein. Anti- 
thymocyte globulin (rabbit) should be infused over a minimum of 6 hours for the first infusion 
and over at least 4 hours on subsequent days of therapy. Administer through an in-line 0.22 µm 
filter. Premedication with corticosteroids, acetaminophen, and/or an antihistamine 1 hour prior to 
the infusion is recommended and may reduce the incidence and intensity of side effects during 
the infusion. Always keep appropriate resuscitation equipment at the patient’s bedside while 
anti-thymocyte globulin (rabbit) is being administered. Observe the patient continuously for 
possible allergic reactions throughout the infusions.  
 
Anti-thymocyte Supplier 
 
Anti-thymocyte is commercially available from various manufacturers. See package insert for 
further information. 
 
Anti-thymocyte Toxicities 
 
Please refer to Table 19.4 for anti-thymocyte toxicities. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

PEDS024 
 
 
2020-7-15 
 
72 | P a g e  
 
Table 19.4 – Anti-thymocyte Toxicities: 
 
 
Common 
(Happens to 21-100 children out 
of every 100) 
Occasional 
(Happens to 5-20 children out of 
every 100) 
Rare 
(Happens to < 5 children out of 
every 100) 
Immediate: 
(Within 1-2 days of receiving 
drug) 
Fever, chills, rash, dyspnea 
w/wo bronchoconstriction, 
hypertension, tachycardia, 
peripheral edema, nausea, 
vomiting, diarrhea, 
abdominal pain, myalgia, 
headache 
Pruritis, dizziness 
Anaphylaxis, swelling, 
redness or phlebitis at the 
infusion site (peripheral 
veins) 
Prompt: 
(Within 2-3 weeks, prior to next 
course) 
Thrombocytopenia, 
leukopenia, pain, 
hyperkalemia, asthenia, 
infection 
Malaise 
Abnormal liver function test 
Delayed: 
(Any time later during therapy, 
excluding the above conditions) 
Antibody development to 
rabbit ATG (may persist for 
1 year) 
Serum sickness (L) which 
can have some or all of the 
following symptoms: 
glomerulonephritis, fever, 
myalgia, arthralgia, and 
periorbital edema (incidence 
reduced with use of 
corticosteroid medication) 
Secondary malignancy 
Unknown Frequency 
and Timing: 
Animal reproduction studies have not been conducted with anti-thymocyte globulin (rabbit).  
It is not known whether anti-thymocyte globulin (rabbit) can cause fetal harm when 
administered to a pregnant woman or affect reproductive capacity. It is unknown whether the 
drug is excreted in breast milk. 
 
(L) Toxicity may also occur later. 
  
5. Tacrolimus Source and Pharmacology (Tacrolimus (05/10/2011) (FK-506, Prograf®) 
NSC #717865 
Tacrolimus is a macrolide immunosuppressant produced by Streptomyces tsukubaensis. 
Tacrolimus is a potent immunosuppressive agent which prolongs the survival of the host and 
transplanted grafts in animal transplant models of liver, kidney, heart, bone marrow, small bowel 
and pancreas, lung and trachea, skin, cornea, and limb. Tacrolimus inhibits T-lymphocyte 
activation, although the exact mechanism of action is not known. Experimental evidence 
suggests that tacrolimus binds to an intracellular protein, FKBP-12. A complex of tacrolimus- 
 

PEDS024 
 
 
2020-7-15 
 
73 | P a g e  
 
FKBP-12, calcium, calmodulin, and calcineurin is then formed and the phosphatase activity of 
calcineurin inhibited. This effect may prevent the dephosphorylation and translocation of nuclear 
factor of activated T-cells (NF-AT), a nuclear component thought to initiate gene transcription 
for the formation of lymphokines (such as interleukin-2, gamma interferon). The net result is the 
inhibition of T-lymphocyte activation (immunosuppression). Additionally, tacrolimus may 
inhibit cellular activities such as nitric oxide synthetase activation and apoptosis, and may 
potentiate the action of corticosteroids in these processes. Tacrolimus activity is primarily due to 
the parent drug. The plasma protein binding of tacrolimus is approximately 99% and is 
independent of concentration over a range of 5-50 ng/mL. Tacrolimus is bound mainly to 
albumin and alpha-1-acid glycoprotein, and has a high level of association with erythrocytes. 
The t½ in adult patients ranges from 11-19 hours. The pharmacokinetics of tacrolimus have been 
studied in pediatric liver transplant patients (0.7 to 13.2 years of age). Following the IV 
administration of a 0.037 mg/kg/day dose to 12 pediatric patients, mean terminal half-life, 
volume of distribution and clearance were 11.5 ± 3.8 hours, 2.6 ± 2.1 L/kg and 0.138 ± 0.071 
L/hr/kg, respectively. Following oral administration to 9 pediatric patients, the absolute 
bioavailability was 31 ± 21%. Whole blood trough concentrations from 31 patients less than 12 
years old showed that pediatric patients needed higher doses than adults to achieve similar 
tacrolimus trough concentrations. Tacrolimus is extensively metabolized by the mixed-function 
oxidase system, primarily the cytochrome P-450 system (CYP3A) in the liver and to a lesser 
extent in the intestinal mucosa. The major metabolite identified in incubations with human liver 
microsomes is 13-demethyl tacrolimus. The main route of elimination is via the biliary tract and 
excretion in faeces. The mean clearance in renal dysfunction and mild hepatic dysfunction is the 
same as normal volunteers. Severe hepatic dysfunction (Pugh score > 10) led to a substantially 
decreased clearance. A retrospective comparison of Black and Caucasian kidney transplant 
patients indicated that Black patients required higher tacrolimus doses to attain similar trough 
 concentrations; there were no gender-based differences. The absorption of tacrolimus from the 
gastrointestinal tract is incomplete and variable exhibiting large intra- and inter-patient 
variability. Administration with food significantly decreases the rate and extent of absorption 
Drugs that stimulate or inhibit hepatic p-450 enzymes will alter clearance of tacrolimus and close 
attention to potential drug interactions is crucial.  
 
Tacrolimus Formulation and Stability 
 
Injection: Tacrolimus is available as a sterile solution (tacrolimus injection) containing the 
equivalent of 5 mg anhydrous tacrolimus per 1 mL. Each mL also contains polyoxyl 60 
hydrogenated castor oil (HCO-60), 200 mg, and dehydrated alcohol, USP, 80% v/v. Store 
between 5°C and 25°C (41°F and 77°F).  

PEDS024 
 
 
2020-7-15 
 
74 | P a g e  
 
Oral: Tacrolimus is available for oral administration as capsules containing the equivalent of 0.5 
mg, 1 mg or 5 mg of anhydrous tacrolimus. Inactive ingredients include lactose, hydroxypropyl 
methylcellulose, croscarmellose sodium, and magnesium stearate. The 0.5 mg capsule shell 
contains gelatin, titanium dioxide and ferric oxide, the 1 mg capsule shell contains gelatin and 
titanium dioxide, and the 5 mg capsule shell contains gelatin, titanium dioxide and ferric oxide. 
Store at 25°C (77°F); excursions permitted to 15°-30°C (59°-86°F).  
 
Tacrolimus Toxicities 
 
Please refer to Table 19.5 for tacrolimus toxicities. 
 
Table 19.5 - Tacrolimus Toxicities: 
 
 
Common 
(Happens to 21-100 children out 
of every 100) 
Occasional 
(Happens to 5-20 children out of 
every 100) 
Rare 
(Happens to < 5 children out of 
every 100) 
Immediate: 
(Within 1-2 days of receiving 
drug) 
Headache (L), hypertension 
(L), nausea, vomiting, 
anorexia, 
immunosuppression (L), 
diarrhea, constipation, fever 
Chest pain 
Anaphylaxis with the 
injection, allergic reaction, 
hypotension, asthma, 
dyspnea, increased cough, 
flu like syndrome, pleural 
effusion, seizure (L), 
tachycardia, angina 
Prompt: 
(Within 2-3 weeks, prior to next 
course) 
Tremor (L), renal 
dysfunction (acute with 
decrease in GFR, impaired 
urinary concentrating ability, 
and sodium retention), 
elevated creatinine/BUN, 
anemia, insomnia, asthenia, 
pain (abdominal, back pain), 
hyperglycemia, 
hypomagnesemia (L), 
hyper/hypokalemia (L), 
hypophosphatemia, 
paresthesia 
Alopecia, dizziness, elevated 
LFTs, UTI, peripheral 
edema, rash, pruritis, 
hyperlipidemia, 
hypercholesteremia 
Dyspepsia, dysphagia, 
gastritis, esophagitis, 
flatulence, CNS 
abnormalities (confusion (L), 
somnolence (L), depression 
(L), anxiety, anxiousness, 
abnormal dreams, emotional 
labiality, hallucinations, 
psychosis, hypertonia, 
incoordination, neuropathy, 
nervousness 
encephalopathy), coagulation 
disorder, leukopenia (L), 
thrombocytopenia, 
polycythemia, anemia, 
leukocytosis, infections 
(bacterial, fungal, viral – 
sepsis, cellulites, fungal 
dermatitis, herpes simplex, 
sinusitis, pharyngitis, 
abscess, pneumonia, 
bronchitis, peritonitis), 
hyperbilirubinemia (L), 
thrombosis, phlebitis, 
arthralgia, myalgia, 
electrolyte abnormalities 

PEDS024 
 
 
2020-7-15 
 
75 | P a g e  
 
Delayed: 
(Any time later during therapy, 
excluding the above conditions) 
 
 
Acne, exfoliative dermatitis, 
skin discoloration, 
photosensitivity reaction, 
skin ulcer, delayed wound 
healing, hirsuitism 
(hypertrichosis) (L), gingival 
hyperplasia, abnormal 
vision, amblyopia, ear pain, 
otitis, tinnitus, GI 
hemorrhage, GI perforation, 
cholelithiasis, cholestatic 
jaundice, chronic renal 
dysfunction, renal failure, 
post-transplant diabetes 
mellitus (L), myocardial 
hypertrophy, elevated liver 
function tests, liver damage, 
ascites 
Late: 
(Any time after the completion 
of treatment) 
 
 
Lymphoproliferative 
disorders, skin malignancies 
Unknown Frequency 
and Timing: 
Fetal toxic effects of tacrolimus have been noted in animals.  Tacrolimus is transported across 
the placenta and its use during pregnancy has been associated with neonatal hyperkalemia and 
renal dysfunction. Tacroimus is excreted in human milk, nursing should be avoided. 
(L) Toxicity may also occur later. 
 
6. Methotrexate Source and Pharmacology (Methotrexate – IV only (02/29/2012) (MTX, 
amethopterin) NCS #000740 
 
A folate analogue which reversibly inhibits dihydrofolate reductase, the enzyme that reduces 
folic acid to tetrahydrofolic acid. Inhibition of tetrahydrofolate formation limits the availability 
of one carbon fragments necessary for the synthesis of purines and the conversion of 
deoxyuridylate to thymidylate in the synthesis of DNA and cell reproduction. The 
polyglutamated metabolites of MTX also contribute to the cytotoxic effect of MTX on DNA 
repair and/or strand breaks. MTX cytotoxicity is highly dependent on the absolute drug 
concentration and the duration of drug exposure. MTX is actively transported across cell 
membranes. At serum methotrexate concentrations exceeding 0.1 µmol/mL, passive diffusion  
becomes a major means of intracellular transport of MTX. The drug is widely distributed  

PEDS024 
 
 
2020-7-15 
 
76 | P a g e  
 
throughout the body with the highest concentration in the kidney, liver, spleen, gallbladder and 
skin. Plasma concentrations following high dose IV MTX decline in a biphasic manner with an 
initial half-life of 1.5-3.5 hours, and a terminal half life of 8-15 hours. About 50% is bound to 
protein. MTX is excreted primarily by the kidneys via glomerular filtration and active secretion 
into the proximal tubules. Renal clearance usually equals or exceeds creatinine clearance. Small 
amounts are excreted in the feces. There is significant entero-hepatic circulation of MTX. The 
distribution of MTX into third-space fluid collections, such as pleural effusions and ascitic fluid, 
can substantially alter MTX pharmacokinetics. The slow release of accumulated MTX from 
these third spaces over time prolongs the terminal half-life of the drug, leading to potentially 
increased clinical toxicity. 
 
Methotrexate Formulation and Stability 
 
Methotrexate for Injection is available as a lyophilized powder for injection in 1000 mg vials. 
The powder for injection contains approximately 7 mEq sodium in the 1000 mg vial. 
Methotrexate for Injection is also available as a 25 mg/mL solution in 2, 4, 8, 10, and 40 mL 
preservative free vials and 2 and 10 mL vials with preservative. The 2, 4, 8, 10, and 40 mL 
solutions contain approximately 0.43 0.86, 1.72, 2.15, and 8.6 mEq sodium per vial, respectively. 
The preserved vials contain 0.9% benzyl alcohol as a preservative. Sterile methotrexate powder 
or solution is stable at 20º-25ºC (68º-77ºF); excursions permitted to 15º-30ºC (59º- 86 Fº). 
Protect from light.  
 
Methotrexate Guidelines for Administration 
 
Reference the Treatment and Dose Modifications sections of protocol. Leucovorin rescue may be 
necessary with certain doses of methotrexate. For IV use: Powder for injection: Dilute 1000 mg 
vial with 19.4 mL of preservative free SWFI, D5W or NS to a 50 mg/mL concentration. The 
powder for injection may be further diluted in NS or dextrose containing solutions to a 
concentration of ≤ 25 mg/mL for IV use. 
 
Do not use the preserved solution for high dose methotrexate administration due to risk of benzyl 
alcohol toxicity. Methotrexate dilutions are chemically stable for at least 7 days at room 
temperature but contain no preservative and should be used within 24 hours. Diluted solutions 
especially those containing bicarbonate exposed to direct sunlight for periods exceeding 4 hours 
should be protected from light. High dose Methotrexate requires alkalinization of the urine, 
adequate hydration and leucovorin rescue. Avoid probenecid, penicillins, cephalosporins, aspirin, 
proton pump inhibitors, and NSAIDS as renal excretion of MTX is inhibited by these agents.  
 
Methotrexate Supplier 
 
Methotrexate is commercially available from various manufacturers. Reference the package 
insert for further information. 

PEDS024 
 
 
2020-7-15 
 
77 | P a g e  
 
 
Methotrexate Toxicities 
 
Please refer to Table 19.6 for methotrexate toxicities. 
 
Table 19.6 - Methotrexate Toxicities: 
 
Common 
(Happens to 21-100 children out 
of every 100) 
Occasional 
(Happens to 5-20 children out of 
every 100) 
Rare 
(Happens to < 5 children out of 
every 100) 
Immediate: 
(Within 1-2 days of receiving 
drug) 
Transaminase elevations 
Nausea, vomiting, anorexia 
Anaphylaxis, chills, fever, 
dizziness, malaise, 
drowsiness, blurred vision, 
acral erythema, urticaria, 
pruritis, toxic epidermal 
necrolysis, Stevens-Johnson 
Syndrome, tumor lysis 
syndrome, seizures1, 
photosensitivity 
Prompt: 
(Within 2-3 weeks, prior to next 
course) 
 
Myelosuppression, 
stomatitis, gingivitis, 
photosensitivity, fatigue 
Alopecia, folliculitis, acne, 
renal toxicity (ATN, 
increased creatinine/BUN, 
hematuria), enteritis, GI 
ulceration and bleeding, 
acute neurotoxicity1 
(headache, drowsiness, 
aphasia, paresis, blurred 
vision, transient blindness, 
dysarthria, hemiparesis, 
decreased reflexes), diarrhea, 
conjuntivitis 
Delayed: 
(Any time later during therapy, 
excluding the above conditions) 
 
Learning disability1 (L) 
Pneumonitis, pulmonary 
fibrosis (L), hepatic fibrosis 
(L), osteonecrosis (L), 
leukoencephalophthy1 (L), 
pericarditis, pericardial 
effusions, hyperpigmentation 
of the nails 
Late: 
(Any time after the completion 
of treatment) 
 
 
Progressive CNS 
deterioration1 
Unknown Frequency 
and Timing: 
Methotrexate crosses the placenta. Fetal toxicities and teratogenic effects of methotrexate 
have been noted in humans. The toxicities include: congenital defects, chromosomal 
abnormalities, severe newborn myelosuppression, low birth weight, abortion, and fetal death. 
Methotrexate is excreted into breast milk in low concentrations. 
1 May be enhanced by HDMTX and/or cranial irradiation 
(L) Toxicity may also occur later. 
 
7. Mycophenolate Mofetil Source and Pharmacology (Mycophenolate Mofetil – 
(12/05/16)) CellCept®, MMF, RS-61443) NSC# 724229 
  
Mycophenolate (MMF) is the morpholinoethyl ester of mycophenolic acid (MPA), an antibiotic 
with immunosuppressant properties isolated from Penicillium spp. The chemical name for oral 

PEDS024 
 
 
2020-7-15 
 
78 | P a g e  
 
mycophenolate mofetil is 2-morpholinoethyl (E)-6-(1,3-dihydro-4-hydroxy-6-methoxy-7-
methyl-3-oxo-5-isobenzofuranyl)-4-methyl-4-hexenoate. It has an empirical formula of 
C23H31NO7 and a molecular weight of 433.50. Mycophenolate mofetil is a white to off-white 
crystalline powder which is slightly soluble in water (43 μg/mL at pH 7.4); the solubility 
increases in acidic medium (4.27 mg/mL at pH 31 3.6). The intravenous product is the 
hydrochloride salt of mycophenolate mofetil. The chemical name for the hydrochloride salt of 
mycophenolate mofetil is 2-morpholinoethyl (E)-6-(1,3-dihydro-4-hydroxy-6-methoxy-7-
methyl-3-oxo-5-isobenzofuranyl)-4-methyl-4-hexenoate hydrochloride. It has an empirical 
formula of C23H31NO7 HCl and a molecular weight of 469.96. 
 
MMF has been used in a variety of solid organ and hematopoietic stem cell transplant settings 
for the prevention of acute rejection. MMF is a prodrug which, after oral administration, is 
rapidly and primarily hydrolyzed by the liver to the biologically active metabolite mycophenolic 
acid. MPA is metabolized principally by glucuronyl transferase to form the pharmacologically 
inactive phenolic glucuronide of MPA (MPAG). In vivo, MPAG is converted to MPA via 
enterohepatic recirculation. Mycophenolic acid inhibits nucleic acid synthesis and produces a 
potent, noncompetitive, and reversible inhibition of inosine monophosphate dehydrogenase 
(IMPDH), blocking the de novo synthesis of guanosine nucleotides without being incorporated 
into DNA. Both T and B lymphocytes rely on this de novo pathway for purine synthesis. As a 
result, the proliferative responses of T and B lymphocytes to both mitogenic and allospecific 
stimulation are inhibited. Other rapidly dividing cell lines are capable of recycling purine 
nucleotides via the "salvage" pathway, which is not blocked by mycophenolic acid. 
 
In vitro and in vivo studies have demonstrated the ability of mycophenolic acid to block 
proliferative responses of T and B lymphocytes, inhibit antibody formation and the generation of 
cytotoxic T-cells, and suppress antibody formation by B lymphocytes. Mycophenolic acid 
prevents the glycosylation of lymphocyte and monocyte glycoproteins that are involved in 
intercellular adhesion of these cells to endothelial cells, and may inhibit recruitment of 
leukocytes into sites of inflammation and graft rejection. Antirejection effects have been 
attributed to decreased recruitment of activated lymphocytes to the graft site. 
 
The mean absolute bioavailability of oral mycophenolate mofetil relative to intravenous 
mycophenolate mofetil (based on MPA AUC) was 94% in a small sample of healthy, adult 
volunteers. In this group the mean (±SD) apparent volume of distribution of MPA was 
approximately 3.6 (±1.5) and 4.0 (±1.2) L/kg following intravenous and oral administration, 
respectively. At clinically relevant concentrations, MPA is 97% bound to plasma albumin. 
MPAG is 82% bound to plasma albumin at MPAG concentration ranges that are normally seen 
in stable renal transplant patients; however, at higher MPAG concentrations (e.g., patients with 
renal impairment), the binding of MPA may be reduced as a result of competition between 
MPAG and MPA for protein binding. A negligible amount of the agent (< 1% of dose) is 
excreted as MPA in the urine. Most of the administered dose (~87%) is excreted in the urine as 
MPAG. Bile acid sequestrants (e.g., cholestyramine) reduce the AUC of MPA by interfering 
with the enterohepatic circulation of the drug. 
 
Mycophenolate mofetil can cause fetal harm when administered to a pregnant woman. Use of 
MMF during pregnancy is associated with an increased risk of first trimester pregnancy loss and 
an increased risk of congenital malformations, especially external ear and other facial 
abnormalities including cleft lip and palate, and anomalies of the distal limbs, heart, esophagus, 

PEDS024 
 
 
2020-7-15 
 
79 | P a g e  
 
and kidney. According to the package labeling, the National Transplantation Pregnancy Registry 
(NTPR) presents data on 33 MMF-exposed pregnancies in 24 transplant patients. Of these, there 
were 15 spontaneous abortions and 18 live-born infants. Four of the 18 infants had structural 
malformations (22%). In postmarketing data (collected 1995-2007) of 77 women exposed to 
systemic MMF during pregnancy, 25 had spontaneous abortions and 14 had a malformed infant 
or fetus. Six of 14 malformed offspring had ear abnormalities. Because these postmarketing data 
are reported voluntarily, it is not always possible to reliably estimate the frequency of particular 
adverse outcomes. These malformations seen in offspring were similar to findings in animal 
reproductive toxicology studies. In animal reproductive toxicology studies, there were increased 
rates of fetal resorptions and malformations in the absence of maternal toxicity. Female rats and 
rabbits received MMF doses equivalent to 0.02 to 0.9 times the recommended human dose for 
renal and cardiac transplant patients, based on body surface area conversions. In rat offspring, 
malformations included anophthalmia, agnathia, and hydrocephaly. In rabbit offspring, 
malformations included ectopia cordis, ectopic kidneys, diaphragmatic hernia, and umbilical 
hernia. If this drug is used during pregnancy, or if the patient becomes pregnant while taking this 
drug, the patient should be apprised of the potential hazard to the fetus. 
 
WOCBP should have a negative serum or urine pregnancy test with a sensitivity of at least 25 
mIU/mL within 1 week prior to beginning therapy (manufacturer’s recommendation). MMF 
should not be initiated until a negative pregnancy test report is obtained. WOCBP taking MMF 
must receive contraceptive counseling and use effective contraception. It is recommended by the 
manufacturer that the patient begin using two chosen methods of contraception 4 weeks prior to 
starting MMF, unless abstinence is the chosen method. She should continue contraceptive use 
during therapy and for 6 weeks after stopping MMF.  Subjects should be aware that MMF 
reduces blood levels of the hormones in the oral contraceptive pill and could theoretically reduce 
its effectiveness. 
 
An approved medication guide (www.fda.gov/downloads/Drugs/DrugSafety/UCM170919.pdf) 
must be dispensed with each refill of mycophenolate mofetil. 
 
Mycophenolate Mofetil Formulation and Stability 
Mycophenolate mofetil is available in the following preparations: 
• Capsule: 250 mg 
• Tablet: 500 mg 
• Powder for suspension, oral: 200 mg/mL (following reconstitution) 
• Powder for reconstitution, injection: 500 mg per vial 
 
A delayed release tablet (mycophenolic acid, Myfortic) is also commercially available. This 
preparation is not interchangeable with mycophenolate mofetil (Cellcept®, MMF) due to 
differences in absorption. This tablet is not discussed in this monograph and should not be used 
by subjects treated on this protocol. 
 
Inactive ingredients in the 250 mg capsules include the following: croscarmellose sodium, 
magnesium stearate, povidone (K-90) and pregelatinized starch. The capsule shells contain black 
iron oxide, FD&C blue #2, gelatin, red iron oxide, silicon dioxide, sodium lauryl sulfate, 
titanium dioxide, and yellow iron oxide. 
 
Inactive ingredients in the 500 mg tablets include black iron oxide, croscarmellose sodium, 
FD&C blue #2 aluminum lake, hydroxypropyl cellulose, hydroxypropyl methylcellulose, 

PEDS024 
 
 
2020-7-15 
 
80 | P a g e  
 
magnesium stearate, microcrystalline cellulose, polyethylene glycol 400, povidone (K-90), red 
iron oxide, talc, and titanium dioxide; may also contain ammonium hydroxide, ethyl alcohol, 
methyl alcohol, n-butyl alcohol, propylene glycol, and shellac. 
 
Inactive ingredients in the powder for oral suspension include aspartame, citric acid anhydrous, 
colloidal silicon dioxide, methylparaben, mixed fruit flavor, sodium citrate dihydrate, sorbitol, 
soybean lecithin, and xanthan gum.  
 
The injectable product is available as a sterile, white to off-white, lyophilized powder in vials 
containing mycophenolate mofetil hydrochloride for administration by intravenous infusion only. 
Each vial contains the equivalent of 500 mg mycophenolate mofetil as the hydrochloride salt. 
Inactive ingredients include polysorbate 80 (25 mg), citric acid (5 mg), and sodium hydroxide to 
adjust the pH. Reconstitution and dilution with D5W yields a final solution of mycophenolate 
mofetil that is slightly yellow in color. 
 
Mycophenolate Mofetil Toxicities 
 
Please refer to Table 19.7 for mycophenolate mofetil toxicities. 
 
Table 19.7 – Mycophenolate Mofetil Toxicities: 
 
Incidence 
Toxicities 
Common 
(> 20% of patients) 
Hypertension, edema (face, limbs, trunk), rash maculo-papular, cholesterol high, 
hyperglycemia, hyperkalemia, hypocalcemia, hypokalemia, hypomagnesemia, abdominal pain, 
constipation, diarrhea, nausea, vomiting, anorexia, dyspepsia, anemia, white blood cell 
decreased, platelet count decreased, back pain, anxiety, generalized muscle weakness, 
dizziness, headache, insomnia, tremor, creatinine increased, dyspnea, cough, fever, pleural 
effusion, alanine aminotransferase increased, alkaline phosphatase increased, aspartate 
aminotransferase increased, blood bilirubin increased, GGT increased, pain, paresthesia, 
infection1 
Occasional  
(4-20% of patients) 
Sepsis, paresthesia, urinary tract pain, urinary frequency, phlebitis (IV only), thrombosis (IV 
only) 
Rare 
(≤ 3% of patients) 
Neoplasms benign, malignant, and unspecified (including cysts and polyps) – Other, 
[Malignant epithelial neoplasm of skin, non-melanoma; lymphoproliferative disease or 
lymphoma], gastric ulcer, gastrointestinal hemorrhage, gastric perforation, mucositis oral, 
thromboembolic event, infective endocarditis, renal calculi, pulmonary fibrosis, pneumonitis, 
neutrophil count decreased, leukoencephalopathy, colitis, pancreatitis, pure red cell aplasia 
Pregnancy & Lactation 
Pregnancy Category D 
Mycophenolate is associated with an increased risk of congenital malformations and 
spontaneous abortions when used during pregnancy.  Adverse events have been reported in 
animal studies at doses less than the equivalent recommended human dose.  Data from the 
National Transplantation Pregnancy Registry (NTPR) have observed an increase in structural 
malformations (including ear malformations) in infants born to mothers taking mycophenolate 
during pregnancy.  Spontaneous abortions have also been noted.  Females of childbearing 
potential should have a negative pregnancy test within 1 week prior to beginning therapy.  Two 
reliable forms of contraception should be used beginning 4 weeks prior to, during, and for 6 
weeks after therapy.  The effectiveness of hormonal contraceptive agents may be affected by 
mycophenolate. 
 
It is unknown if mycophenolate is excreted in human milk.  Due to potentially serious adverse 
reactions, the decision to discontinue the drug or discontinue breast-feeding should be 
considered.  Breast-feeding is not recommended during therapy or for 6 weeks after treatment 
is complete. 
1 Infection includes all 75 sites of infection under the INFECTIONS AND INFESTATIONS SOC. 
 
 

PEDS024 
 
 
2020-7-15 
 
81 | P a g e  
 
APPENDIX E.  STANDARD OF CARE NEUROPYSCHOLOGY TESTING BY AGE 
Neurocognitive testing will be obtained prior to transplantation and then at 1 year post-transplant 
and at study exit (assuming that at least 12 months has elapsed since the previous testing). 
Due to complexity of scheduling neurocognitive testing, the study allows that testing be 
completed up to 90 days prior to transplant for baseline. Neurocognitive testing is not a part of 
eligibility criteria and patients who cannot obtain the test may be enrolled, but lack of testing of 
eligible patients will be considered protocol violation. 
The following standardized measures will be used to assess broad neurocognitive/developmental 
functioning: 
For ages 6–35 months: Bayley Scales of Infant & Toddler Development-3rd Edition (Bayley-3), 
which assesses development across cognitive, language (expressive/receptive) and motor (fine & 
gross) domains; Bayley Social Emotional and Adaptive Questionnaire (parent rating scale). 
For ages 3-6 years: 
• Intelligence- Wechsler Preschool & Primary Scale of Intelligence, 4th Edition (WPPSI-
IV)  
• Memory- Ages 3-4: NEPSY-II, Narrative Memory; Ages 5-6: Children's Memory Scale 
(CMS), Dot Locations & Stories 
• Visuoconstruction/graphomotor- Beery Buktenica Test of Visuo-motor Integration (VMI-
6) 
• Attention- Ages 4-6: Conners’ Kiddie Continuous Performance Test-2nd Edition 
• Receptive language/school readiness- Bracken Basic Concept Scale-3rd Edition-
Receptive 
• Emotional, behavioral & adaptive functioning- Behavior Assessment Scale for Children-
3rd Edition, parent rating scales 
For ages 7-15 years:  
 
• Intelligence- Wechsler Intelligence Scale for Children, 5th Edition (WISC-V)  
• Memory- Children's Memory Scale (CMS), Dot Locations & Stories 
• Visuoconstruction/graphomotor- Beery Buktenica Test of Visuo-motor Integration (VMI-
6) 
• Attention- Conners’ Continuous Performance Test-3rd Edition 
• Executive Functioning- Ages 8+: Delis Kaplan Executive Function System (DKEFS), 
Trail Making Test & Verbal Fluency   
• Emotional, behavioral & adaptive functioning- Behavior Assessment Scale for Children-
3rd Edition, parent rating scales & self-report form (ages 8+) 
 
 
 
 

PEDS024 
 
 
2020-7-15 
 
82 | P a g e  
 
Appendix E 
 
 
Dosing Weight Adjustments in Pediatric BMT Patients 
 
Actual Weight (BW) = ______kg taken on ______________ (Must be within 2 
weeks of admission for patients > 12kg, within one week for ≤ 12 kg) 
 
Height (Ht): _________ cm = _________inches taken on ______________ 
 
BSA  ___________m2 
 
Age ____________ 
 
BMI % __________ 
(CDC BMI calculator for children and teens   
https://www.cdc.gov/healthyweight/bmi/calculator.html:) 
 
 
Ideal Body Weight (IBW) Calculations 
 <2yo or <12 kg 
Use actual body weight, calculate all chemotherapy using weight 
rather than BSA (divide BSA with 30 to get dose per kg) 
 2-17.99 years old AND < 5ft 
 
IBW= (height in cm2 x 1.65)  
                 1000 
 
 2-17.99 years old AND > 5ft 
 
 IBW (male) = 39 + (2.27 x height in inches over 5 feet) 
 
 IBW (female) = 42.2 + (2.27 x height in inches over 5 feet) 
 ≥ 18 years old 
 
 IBW (male) = 50 + (2.3 x height in inches over 5 feet) 
 IBW (male) = 50 – (2.3 x height in inches UNDER 5 feet) 
 
 IBW (female) = 45.5 + (2.3 x height in inches over 5 feet) 
 IBW (female)= 45.5 – (2.3 x height in inches UNDER 5 feet) 
 
 
 
 
 Do not need to weight adjust, all children <12 kg 
 
 Weight adjust according to calculation below: 
 
Adjusted Body Weight (Adj Wt) =  
 
IBW (____) + [(BW _____ – IBW _____) x 0.4] = 
 
 
Adjusted Body Surface Area =  
 
Ht in cm divided by 2.54 = Ht in inches 
 
5ft = 60inches 
______kg 
If the calculated BMI using CDC calculator is ≥95%, use adjusted body weight for calculation of 
chemotherapy but not serotherapy. Serotherapy may be adjusted in severely obese patients at 
MD discretion.
Pt Name:  
 
MR# 
______m2