Clinical trial NCT00578643
Conditions: Chronic Granulomatous Disease
Keywords: Stem Cell Transplant Chronic Granulomatous Disease Fludarabine Busulfan Cyclophosphamide
Sponsor: Baylor College of Medicine
Start date: 2004-03-01; Completion date: 2017-11-24
Enrollment: 15 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
This study is for patients with chronic granulomatous disease (CGD), which is a disorder of the immune system that puts them at risk for severe infections. CGD is caused by a genetic defect that stops or prevents the white blood cells from killing certain bacteria and fungi. This condition cannot presently be cured by standard treatment with drugs or surgery. Medicine including antibiotics, antifungals, and interferon gamma, may help some patients with CGD; however even with continuous treatment, most patients with CGD will have chronic and recurrent infections. Transfusion of white blood cells may help overcome infection, but white cell transfusions lead to allergic reactions and fever and the benefit of transfusion lasts only a matter of hours. Ultimately, chronic infections can damage or injure the body organs. Injury to the lung or liver can lead to lung or liver failure and death. Medicines used to treat infection can damage body organs too. Infections may become resistant to antibiotic or antifungal treatment, and infections not responding to treatment can be deadly.

It is now known that under specific conditions and with special treatment, blood stem cells (the cells that make blood) can be transplanted from one person to another. Stem cell transplantation has been done for patients with CGD who have a healthy sibling and who share the same immune type (HLA type) as the patient. Stem cell transplantation allows healthy or normal white cells from the stem cell donor to grow or develop in the patient's bone marrow. These healthy white cells can fight infection and prevent future infections for a patient with CGD.

Patients on this study will receive stem cells from a related or unrelated donor. The donor will be closely matched to the patient's immune type but the donor is not a sibling. The reason this treatment is investigational is that we do not know the likelihood of benefit that the patient will receive. It is possible that they will have great benefit, like some of the patients who have been transplanted from a brother or sister. It is possible that the side-effects of treatment may be too severe so that the transplant won't work.

The purpose of this research study is to evaluate whether or not patients with CGD treated with a stem cell transplant from a non-matched and/or non-related donor can have a good outcome from the procedure with an acceptable number of side-effects.

Detailed description:
In order to transplant stem cells we will need to give the patient drugs or high-dose chemotherapy to kill or destroy most of the blood forming and immune cells in the bone marrow. This is necessary to allow the donor stem cells to live and grow (engraft) in the bone marrow space. After the drug treatment is completed, the patient will be given the stem cells from the donor. The drug treatment is as follows:

Day -9 Busulfan

Day -8 Busulfan

Day -7 Busulfan

Day -6 Busulfan

Day -5 Alemtuzumab, Fludarabine, Cyclophosphamide

Day -4 Alemtuzumab, Fludarabine, Cyclophosphamide

Day -3 Alemtuzumab, Fludarabine, Cyclophosphamide

Day -2 Alemtuzumab, Fludarabine, Cyclosporine, Cyclophosphamide

Day -1 REST

Day 0 Stem cell infusion

The day after the chemotherapy treatment is completed, the patient will receive the healthy stem cells by vein, like a blood transfusion. Once in the bloodstream, the marrow cells will go to the bone marrow and grow.

It is also possible that if the marrow takes, it will cause a disease known as graft-versus-host disease (GVHD). To prevent GVHD, we will give the patient cyclosporine and Methotrexate. Methotrexate will be administered on Days 1, 3, 6 and 11 after the transplant. The cyclosporine therapy will continue for a longer period of time, however if the patient does not develop GVHD, it will be discontinued by 6 months after the stem cell transplant.

Arms: arm 1: Conditioning from Day -9 to Day -1. Stem cells given on Day 0. Busulfan, alemtuzumab, cyclophosphamide, fludarabine, cyclosporine, stem cell infusion.
Interventions: intervention 1: Busulfan intervention 2: Alemtuzumab intervention 3: Cyclophosphamide intervention 4: Fludarabine intervention 5: Cyclosporine intervention 6: Stem Cell Infusion
Intervention descriptions: intervention 1: Days -9 through -6

1 mg/kg initially (based on weight) intervention 2: Day -5 through Day -2

Dose is based on weight:

Less than 15 kg: 3 mg

More than 15 kg to 30 kg: 5 mg

More than 30 kg: 15 mg intervention 3: Days -5 through -2

50 mg/kg intervention 4: Day -5 through Day -2

30 mg/m\^2 intervention 5: Cyclosporine will be administered beginning Day -2. Initial dose will 5 mg/kg infused over 24 hours. intervention 6: Stem Cell: Either bone marrow, cord blood, or peripheral blood stem cells may be used for stem cell transplantation. It is desired to infuse: for bone marrow, nucleated cells ≥ 4 X 10\^8/kg recipient weight; for cord blood ≥ 3 X 10\^7/kg nucleated cells; for peripheral blood stem cells ≥ 1 X 10\^/kg CD34+ cells.

Protocol documents:
Prot_SAP_000.pdf: 
1 
Version 1.0 dated 11-25-03 
Version 6.0 dated 8-30-17 
Version 1.1 dated 2-9-04 
 
 
 
 
Version 7.0 dated 3-13-18 
Version 2.0 dated 12-9-08 
Version 3.0 dated 10-29-09 
Version 4.0 dated 05-30-13 
Version 5.0 dated 11-04-13 
 
HLA Matched Unrelated or Non-genotype Identical Related Donor 
Transplantation for Chronic Granulomatous Disease 
 
Principal Investigator 
Robert Krance, MD 
 
Co-Investigators 
Malcolm Brenner, MD 
Helen Heslop, MD 
Bambi Grilley, RPh 
Caridad Martinez, MD 
 
 
 
 
 
 
Texas Children’s Hospital 
6621 Fannin Street 
Houston, TX  77030-2399 
 
 


2 
Table of Contents 
Objectives ......................................................................................................................... 4 
Background ...................................................................................................................... 4 
Stem Cell Transplantation - Rationale ....................................................................... 5 
Drug Profiles...................................................................................................................... 8 
Eligibility ............................................................................................................................ 8 
Exclusion Criteria ............................................................................................................. 9 
Treatment Plan.................................................................................................................. 9 
Supportive Care ............................................................................................................. 11 
Studies to be Obtained Prior to Transplantation ................................................... 11 
Studies to be Obtained Day 8 to day 100 .................................................................. 11 
Evaluation of Toxicity..................................................................................................... 11 
Risks and Potential Hazards ....................................................................................... 12 
Statistical Considerations ............................................................................................ 13 
Informed Consent ............................................................................................................ 15 
Reference List ................................................................................................................ 16 
 


3 
ELIGIBILITY CHECKLIST 
 
PATIENT ID  
 
PATIENT NAME  
 
 
The following must be answered “YES” for a patient to be eligible to participate in this 
study: 
 
YES 
NO 
 
 
____  ____ Chronic Granulomatous Disease (CGD) patients as documented by an 
abnormal NBT assay in a male patient and/or abnormal NADPH enzyme 
mutation confirmed by genetic analysis with abnormal NBT.  
____  ____ Patient must not have an HLA genotype identical donor. 
____  ____ Patient has a 5/6 or 6/6 HLA matched unrelated donor or a 5/6 or 6/6 
HLA phenotype matched related donor. 
____  ____ Patient has had at least one serious infection characteristic of those 
manifested in patients with CGD.  
____    
____     ____ Patients must not have active infection. An active infection may include the 
following:  1) clinical findings consistent with an infection such as fever, 
cavitary organ lesions, osteomyelitis 2) progression of presumed infection 
based upon findings of diagnostic imaging [two or more studies at least 1 
month a part] 
____ 
____ Patient has echocardiogram shortening fraction ≥ 28%. 
____ 
____ Patient has DLCO ≥ 50% predicted or FEV1 ≥ 50% predicted. 
____  ____ Patient has no cumulative organ dysfunction that in the estimation of the 
treating physicians will diminish the patient’s likelihood to survive this 
procedure. 
____ 
____ Negative pregnancy test for post-pubertal female patients. 
 
The following must be answered NO for a patient to be eligible to enroll in this study: 
 
_____ _____ Active or uncontrolled infection (e.g. lung infection, cavitary organ lesions, 
osteomyelitis). 
_____ _____ Markedly elevated C reactive protein or sedimentation rate relative to  
 
 
patient’s baseline.  
_____ _____Invasive bone or bone marrow disease. 
_____ _____Lack of potential hematologic blood product donors in the past (related to 
McLeod phenotype).   
 
 


4 
1. Objectives 
1.1. 
PRIMARY: To estimate the engraftment rate for patients with CGD using 
busulfan, cyclophosphamide, fludarabine and Alemtuzumab (Campath 1H) as 
conditioning therapy for SCT from 5/6 or 6/6 HLA matched unrelated or 5/6 or 
6/6 HLA phenotype matched related donors. 
1.2. 
SECONDARY: 
1.2.1. To estimate the likelihood of complete donor chimerism for patients with 
CGD using busulfan, cyclophosphamide, fludarabine and Alemtuzumab 
(Campath 1H) as conditioning therapy for SCT from 5/6 or 6/6 HLA matched 
unrelated or 5/6 or 6/6 HLA phenotype matched related donors. 
1.2.2. To estimate the risk for acute GVHD and regimen related 
morbidity/mortality for patients with CGD following SCT from 5/6 or 6/6 HLA 
matched unrelated or 5/6 or 6/6 HLA phenotype matched related donors. 
1.2.3. To estimate the risk for chronic GVHD and regimen related 
morbidity/mortality for patients with CGD following SCT from 5/6 or 6/6 HLA 
matched unrelated or 5/6 or 6/6 HLA phenotype matched related donors. 
1.3. 
EXPLORATORY:  
1.3.1. To explore the ability to treat patients with Chronic Granulomatous 
Disease (CGD) refractory to conventional therapy and without a HLA matched 
sibling donor by performing stem cell transplantation (SCT) from 5/6 or 6/6 
HLA matched unrelated or 5/6 or 6/6 HLA phenotype matched related donors. 
1.3.2. To examine the potential for reversal of organ toxicity (e.g. lung, liver, 
intestine) following engraftment and stable normal neutrophil function. 
 
2. Background  
2.1. 
Chronic Granulomatous Disease (CGD) is a life threatening primary 
immunodeficiency caused by the abnormal function of any of four components of the 
nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system present in the 
phagocitic cells.  The estimated incidence of CGD in the United States is between 
1/200,000 – 1 /255,000 live births.(1) 
 
2.2. 
The most common (accounting for 70% of CGD occurrences) and severe form of 
CGD is caused by an X-linked recessive mutation of the NADPH oxidase protein 
subunit gp91phox.  This mutation as well as 3 additional mutations affecting other 
component proteins of the NADPH oxidase system result in marked susceptibility to 
life-threatening infections, primarily severe recurrent bacterial and fungal infections.  
These infections are the direct result of an inability of phagocytic leukocytes (e.g. 
neutrophils, eosinophils, monocytes), to generate superoxide and its metabolites, 
hydrogen peroxide and hypochlorous acid, necessary for intracellular killing and 
activation of anti-microbicidal enzymes.(2) CGD patients have exaggerated 
susceptibility to infection with catalase-positive pathogens (e.g. Staphylococcus aureus, 
Aspergillus species, Nocardia species, Candida species, Gram-negative enteric bacilli).  
These infections present primarily as pneumonia (75-80%), abscess formation (64-70%, 
[subcutaneous, liver, lung, perirectal, and brain]), adenitis (32-59%) or osteomyelitis (14 
– 27%), among others.(1) Absent effective intracellular killing necessitates the 
prolonged use of antibiotics and ultimately the appearance of resistant strains. This 


5 
illustrates the complicated task of providing adequate antimicrobial prophylaxis and 
treatment to CGD patients.(3) 
 
2.3. 
In spite of our efforts, the morbidity and mortality for this patient population 
continues to be extremely high, primarily among patients with the X-linked form of the 
disease who appear to have a more severe clinical phenotype.   These patients often die 
in the first or second decade of life.     Morbidity and mortality often follow bouts of 
severe pneumonia with attendant complications (e.g. dissemination to chest wall, 
vertebrae and central nervous system). 
 
2.4. 
  Continuous prophylactic antibiotic therapy with trimethropin-
sulfamethoxasole or itraconazole is not sufficient to prevent recurrent infections in this 
patient population.  In our experience, prophylactic administration of antifungal 
agents has failed to prevent infection. Once infected these agents are often inadequate 
to the task. As eradication of invasive fungal infections relies primarily on aggressive 
antibiotic therapy, CGD affected children repeatedly spend weeks or months in the 
hospital receiving therapy with multiple antibiotics.  
 
2.5. 
A landmark in the medical care of CGD patients was the introduction during 
the last decade of interferon gamma therapy as standard of care for this patient 
population. The mechanism by which interferon therapy decreases the number of 
infections is unclear but in spite of this therapy about 50% of children still experience 
severe recurrent infections. (1;4;5) 
2.6. 
Morbidity that accompanies prolonged hospitalizations and aggressive 
antibiotic therapy may be worsened by the need for surgical resection of necrotic and 
infected tissue. Granuloma of the lung or liver, abscess, empyema, osteomyelitis, etc. 
complicate the management of infections.  Due to the frequency and severity of lung 
infections and the need for surgical intervention, patients are often left with severe 
pulmonary insufficiency, oxygen dependency or inability to participate in activities of 
daily life (e.g. school).  In addition, this aggressive care can lead to irreparable 
secondary organ damage (e.g. renal failure, deafness, oxygen dependency, 
pancreatitis), further compromising the already poor quality of life, and perpetuating 
an endless need for chronic care. 
 
3. Stem Cell Transplantation – Rationale  
3.1. 
Therapy for CGD has been largely symptomatic as noted above. While 
symptomatic treatment may adequately control specific infections, over time CGD 
patients invariably develop progressive tissue injury, such as to the lung or liver, from 
chronic infections. The treatment itself can be injurious, for example persistent 
administration of renal toxic antibiotics may compromise renal function. More than 
half of CGD patients die before the end of the third decade of life and for most of the 
others the quality of life is diminished by the chronic disease manifestations.(6) 
 
3.2. 
Neutrophil dysfunction that arises from the defect that underlies CGD can be 
compensated by granulocyte transfusions. These have been reported as life-saving in 
circumstances of severe infections, poorly responsive to drug therapy and 


6 
unapproachable surgically. Clearly the benefit of this approach is due to the 
administration of functioning neutrophils capable of effective microbial phagocytosis. 
Unfortunately, granulocyte transfusions are complicated by fever and respiratory 
distress, and with the invariable development of leukoagglutinins poor granulocyte 
recovery. White blood cell transfusions are never an effective maintenance strategy.(1)  
 
3.3. 
Based upon patterns of X-inactivation in X-linked carriers of CGD, both 
affected and unaffected neutrophils are produced. Unaffected or normal neutrophil 
production does not show a proliferative advantage over abnormal neutrophils. 
Typically, CGD carriers with as few as 10% normal phagocytes do not develop serious 
infections.(1)This suggests that susceptibility to infection in CGD can be prevented by a 
relatively small portion of normal cells admixed with a larger number of abnormal 
cells. 
3.4. 
Clinical manifestations of CGD are limited to the consequences of abnormal 
neutrophil function. Implicit therefore is that CGD patients should benefit 
substantially, and perhaps be cured, if normal neutrophil production can be 
established. This is the premise underlying the role for stem cell transplantation in 
CGD.(1)  
 
3.5. 
There are now adequate data to substantiate stem cell transplantation as best 
practice for CGD patients with HLA matched related donors. 
 
 
3.5.1. The European Group for Blood and Marrow Transplantation (EBMT) and 
the European Society for Immunodeficiencies (ESID) reported on the outcome for 
27 patients with CGD undergoing allogeneic stem cell transplantation between 
1985 and 2000.(7) Patients (23 males) ranged from 0.8 to 38.7 years of age, median 
age 7 years, at transplantation. All patients had at least one prior episode of 
invasive infection; 9 patients had therapy-refractory infection (n=8 fungus; n=1 
mycobacterium) at transplantation. Of the remaining 18 patients, 7 evinced signs 
of active inflammation or organ sequelae from prior illness. Donors were HLA 
matched siblings for 25 patients (5/25 CGD carriers) and matched unrelated 
donors for 2 patients. Busulfan (16 – 20 mg/kg total dose) combined with 
cyclophosphamide, fludarabine, or melphalan comprised the conditioning 
regimen for 23 patients; 4 patients received less than fully myeloablative therapy. 
All patients received cyclosporine; some received either methotrexate or 
prednisone in addition, as GVHD preventive therapy. 
 
Twenty-two/23 patients transplanted after myeloablative therapy engrafted, 
while 2/4 patients transplanted using submyeloablative therapy failed to 
engraft. The median time to granulocyte recovery was 18 days (9 to 40 days). For 
the 24 engrafted patients, NBT assay reached donor levels (5 pts transplanted 
from CGD carriers exhibited a mosaic pattern of NBT positivity).  
 
Four/27 patients have died and deaths were exclusive to the group of 9 patients 
who underwent transplant with therapy-refractory infection (n=2 from 
progressive infection; n=1 carotid hemorrhage; n=1 inflammatory pulmonary 


7 
reaction and skin GVHD). It is noteworthy, however, among this same 
therapy-refractory cohort, 4 patients actively infected with Aspergillus survived 
and eradicated their infection after engraftment. Overall 81%, 22/27, patients are 
alive, 21/22 maintain complete donor myeloid chimerism, and all are without 
clinical or laboratory findings of CGD. Seven/27 patients experienced grade II-IV 
GVHD, and 3 patients have developed chronic GVHD. Two patients who 
received stem cells from HLA matched but unrelated donors (MUD) are alive 
and well.  
 
Beyond the excellent survival and the ability to eradicate infection in 
therapy-refractory patients, improvements were documented in lung function 
(lessened restrictive pulmonary function, discontinued supplemental oxygen, 
resolution of clubbing), resolution of colitis, and increased growth rate. 
3.5.2. In a study conducted at the NIH, 10 patients with CGD underwent 
peripheral blood stem cell transplantation (PBSCT) from matched siblings.(8) All 
patients were male, from 5 – 36 y/o, median 15 years; CGD in 8 of these was 
caused by the X-linked mutation. Seeking to avoid transplant related 
morbidity/mortality, nonmyeloablative transplant conditioning therapy was 
employed: fludarabine 25 mg/m2 days –5 to –1 and cyclophosphamide 60 
mg/kg days –7 and -6. GVHD preventive therapy included cyclosporine and 
T-cell depletion. Donors underwent leukapharesis after 4 days G-CSF priming 
and were collected on days 5 and 6. Manipulation of these products included 
CD34+ selection using the Isolex 300i system and treatment with T-cell 
monoclonal antibodies (CD2, CD6, and CD7).  Other manipulations included 
T-cell ‘add-back’ to provide the recipient with 1 X 105/kg CD3+ T-cells, and 
periodic infusions of donor T-cells post PBSCT to promote full donor 
engraftment. 
 
As anticipated conditioning regimen related toxicity was negligible. Neutrophil 
recovery occurred at the median d+10 (range 6-22 days) and thrombocytopenia 
(< 20,000/mm3) occurred in only 4 patients, resolving by d+11 at the latest. 
Unfortunately, 1 patient failed to engraft and died from complications related to 
a second transplant; and 1 patient developed late graft failure. At 17 months 
median follow up post PBSCT, 6/8 patients maintain complete (n=4) or almost 
complete myeloid and lymphoid chimerism, while 2 patients are stable mixed 
chimeras. Two of these 8 patients died, 1 from complications of GVHD (3 
patients developed > grade II acute GVHD) and 1 from bacterial sepsis. It is 
noteworthy that the onset of acute GVHD followed donor T-cell infusions. 
 
Although hematopoietic recovery ensued rapidly post PBSCT, complete donor 
chimerism, when it occurred, required weeks to months to establish. 
Furthermore it was necessary to administer multiple donor T-cell infusions to 
promote donor chimerism and for several patients this process instigated GVHD. 
Others have observed incomplete chimerism following subablative conditioning. 
Thus the benefit of this approach is not compelling when compared to the 
EBMT/ESID report.  
 


8 
3.5.3. While fewer patients with CGD have been transplanted from donors 
other than HLA matched siblings, reports indicate that the outcome is no less 
satisfactory.(7;9-11)  Seven of eight such patients were reported to have engrafted 
following fully ablative conditioning, and all engrafted patients remained alive.  
 
3.6. 
A number of reports find that the overall survival is comparable between 
matched sibling and matched unrelated donor transplants.(12;13)Comparison between 
patient groups is problematic; however our results for transplantation of children with 
aplastic anemia find the 4-year event free survival very similar for patients 
transplanted from matched sibling donors (92%) verus patients transplanted form 
matched unrelated donor (88%). There is no reason to believe that results should be 
inferior in the setting of matched unrelated donor transplantation for CGD. 
 
3.7. 
 Subablative or nonmyeloablative transplant conditioning therapy may permit 
more rapid hematopoietic recovery with lessened immediate transplant morbidity. 
This approach has been advantageous when transplantation has been performed in 
older patients or patients with significant pretransplant morbidity; engraftment has 
been satisfactory and is regularly accompanied by complete donor chimerism. 
However these results have been limited to patients with hematopoietic malignancy, 
for whom prior treatment has induced immunosuppression and reduced marrow 
reserve. These circumstances are not characteristic for patients with CGD. Indeed 
several reports document the failure to engraft or maintain donor engraftment for 
CGD patients treated with subablative conditioning regimens.(7;8)  
 
3.8. 
Busulfan combined with cyclophosphamide has been used most widely and 
with considerable success to condition CGD patients for SCT.(7) The regimen that is 
proposed in this study, which adds fludarabine to busulfan and cyclophosphamide, 
has been used effectively to treat patients with a variety of malignant and 
nonmalignant diseases.(14)(personal communication Lawson, Amrolia) In the past, 
busulfan has presented problems related to unpredictable absorption. With the 
availability of an intravenous formulation of busulfan and the ability to assay plasma 
busulfan concentration, problems related to under or over dosing have been overcome. 
  
3.9. 
Additionally, patients will be treated with Campath (Alemtuzumab) an 
antibody directed against the CD52 antigen, which is present on most lymphocytes. 
This agent has shown efficacy as an immunosuppressive promoting donor 
engraftment and prevention of GVHD.(15)  
 
4. Drug profiles (see Appendix 1)  
4.1. 
Busulfan 
4.2. 
Fludarabine 
4.3. 
Cyclophosphamide 
4.4. 
Alemtuzumab 
4.5. 
Cyclosporine 
4.6. 
Methotrexate 
 
5. Eligibility  


9 
5.1. 
CGD patients as documented by an abnormal NBT assay in a male patient 
and/or abnormal NADPH enzyme mutation confirmed by genetic analysis with 
abnormal NBT.  
5.2. 
Patients must not have an HLA genotype identical donor 
5.3. 
Patients must have a 5/6 or 6/6 HLA matched unrelated donor or a 5/6 or 6/6 
HLA phenotype matched related donor. 
5.4. 
Patients must have had at least one serious infection characteristic of those 
manifested in patients with CGD.  
5.5. 
Patients must not have active infection. An active infection may include the 
following:  1) clinical findings consistent with an infection such as fever, cavitary organ 
lesions, osteomyelitis 2) progression of presumed infection based upon findings of 
diagnostic imaging [two or more studies at least 1 month a part] 
5.6. 
No cumulative organ dysfunction that in the estimation of the treating 
physicians will diminish the patient’s likelihood to survive this procedure. 
5.7. 
Negative pregnancy test for post-pubertal female patients. 
5.8. 
Echocardiogram shortening fraction ≥ 28%. 
5.9. 
DLCO ≥ 50% predicted or FEV1 ≥ 50% predicted. 
 
6. Exclusion criteria  
6.1. 
 Active or uncontrolled infection (e.g. lung infection, cavitary organ lesions, 
osteomyelitis). 
6.2. 
 Markedly elevated C reactive protein or sedimentation rate relative to patient’s 
baseline.  
6.3. 
 Invasive bone or bone marrow disease. 
6.4. 
 Lack of potential hematologic blood product donors in the past (related to 
McLeod phenotype). 
 
 
7. Treatment Plan  
 
Protocol day  
Treatment 
-10  
 
 
 
Anti-Convulsant therapy begins 
-9  
 
 
 
Busulfan (see below regarding dosing) 
-8  
 
 
 
Busulfan 
 
-7  
 
 
 
Busulfan 
 
 
-6  
 
 
 
Busulfan 
 
 
-5  
 
 
 
Alemtuzumab  (see below regarding dosing) 
 
 
 
 
 
Fludarabine 30 mg/m2  
 
 
 
 
 
Cyclophosphamide 50mg/kg +MESNA 
-4  
 
 
 
Alemtuzumab 
 
 
 
 
 
Fludarabine 30 mg/m2   
 
 
 
 
 
Cyclophosphamide 50mg/kg+MESNA 
 
 
 
 
 
Anti-Convulsant therapy ends 
-3  
 
 
 
Alemtuzumab  
 
 
 
 
 
Fludarabine 30 mg/m2 
 
 
 
 
 
Cyclophosphamide 50 mg/kg + MESNA  
-2  
 
 
 
Alemtuzumab 


10 
 
 
 
 
 
Fludarabine 30 mg/m2 
 
 
 
 
 
Cyclosporine  
 
 
 
 
 
Cyclophosphamide 50mg/kg +MESNA  
-1  
       
 
REST 
 0  
 
 
 
Stem cell infusion 
+1  
 
 
 
Mtx 
+3  
 
 
 
Mtx 
+6  
 
 
 
Mtx 
+11  
 
 
 
Mtx 
 
7.1. 
Busulfan: The initial dose of busulfan is 1 mg/kg. Dosing is based upon actual 
weight unless actual weight exceeds ideal weight by 30% for which adjusted weight 
will be calculated as the ideal body weight plus 25%. Busulfan is administered 
intravenously every 6 hours for 16 doses. Blood samples will be obtained with the first 
and ninth dose and busulfan plasma levels will be determined. Using appropriate 
pharmacokinetic modeling, the busulfan AUC will be estimated. The dose of busulfan 
will be modified to maintain a busulfan AUC 900 – 1200 µmol/min/L. 
7.2. 
All patients require anticonvulsant therapy while receiving busulfan. The clinical 
pharmacist must review orders for anticonvulsants. Blood anticonvulsant levels should 
dictate dosing. 
7.3. 
Fludarabine: The dose of fludarabine is 30 mg/m2.  
7.4. 
Cyclophosphamide: The planned dose of cyclophosphamide is 50 mg/kg but the 
dose will be based upon the ideal body weight or the actual body weight, whichever is 
less. For patients whose actual body weight exceeds the ideal body weight by more 
than 25%, dose adjustment will be required. These adjustments will be made in 
conjunction with the clinical pharmacist. Hemorrhagic cystitis may develop at this 
dose level of cyclophosphamide.  Adequate urine flow is essential and the exact 
hydration and MESNA doses should be followed. 
7.5. 
Alemtuzumab: The dose of Alemtuzumab will be based upon recipient actual 
weight. Alemtuzumab 3 mg will be administered to recipients less than 15 kg; 
Alemtuzumab 5 mg to recipients > 15 kg to 30 kg; Alemtuzumab 10mg to recipients > 
30 kg.  Alemtuzumab is administered on four successive days.  Prior to Alemtuzumab 
patients will receive a premedication as appropriate to prevent allergic reaction. 
7.6. 
Stem Cell: Either bone marrow, cord blood, or peripheral blood stem cells may 
be used for stem cell transplantation. It is desired to infuse: for bone marrow, nucleated 
cells ≥ 4 X 108/kg recipient weight; for cord blood ≥ 3 X 107/kg nucleated cells; for 
peripheral blood stem cells ≥ 1 X 107/kg CD34+ cells. 
7.7. 
Graft versus Host Disease Preventive Therapy: Cyclosporine will be 
administered beginning day -2.  Initial dose will 5 mg/kg infused over 24 hours.  
Cyclosporine dose is based upon the actual body weight unless the actual body weight 
exceeds the ideal body weight by 30%; then the ideal body weight will be used. 
Modifications in cyclosporine dose will be determined by the measured blood 
concentration (cyclosporine levels will be maintained within the acceptable therapeutic 
range).  For patients without GVHD, cyclosporine will be discontinued by 6 months. 
Methotrexate will be administered on day +1, +3, +6, and +11 at 5 mg/m2 IV. 


11 
 
8. Supportive care  
8.1. 
Standard operating procedures will be followed regarding the prevention or 
management of CMV infection, EBV infection, fever and neutropenia, fungus infection, 
GVHD therapy, blood transfusion support  
 
9. Studies to be obtained prior to transplantation  
9.1. 
 Serologic panel: hepatitis A, B, C; HIV 1 & 2, HTLV 1 & 2, CMV, herpes simplex, 
herpes zoster, syphilis,  
9.2. 
 Diagnostic imaging studies as appropriate to evaluate sites of previous or 
current infection 
9.3. 
ECHO and EKG 
9.4. 
24 hour creatinine clearance or glomerular filtration rate 
9.5. 
Liver function tests: ALT, AST, alkaline phosphatase, GGT, total, direct and 
indirect bilirubin, albumin, triglycerides 
9.6. 
CBC, sedimentation rate, prothrombin time, partial thromboplastin time, 
fibrinogen, blood type  
9.7. 
Electrolytes, BUN, creatinine, magnesium, calcium, phosphorus, uric acid 
9.8. 
Urinalyis 
9.9. 
Amylase and lipase 
9.10. Quantitative immunoglobulins 
9.11. Pulmonary function tests where age appropriate 
 
10. Studies to be obtained day –8 to day +100   
10.1. At least weekly from day -8 to day +28 and thereafter as clinically indicated: 
CBC, AST, ALT, alkaline phosphatase, GGT, total and direct and indirect bilirubin, 
albumin, electrolytes, calcium, magnesium, phosphorus, uric acid, triglycerides, 
urinanalysis. 
10.2. CMV antigenemia beginning day +14 and weekly after the ANC has reached 500 
or as indicated until day +120 or cyclosporine and other GVHD therapy is 
discontinued. Thereafter CMV antigenemia test is performed as clinically indicated. 
CMV PCR may used in place of CMV antigenemia if ANC < 500 or as indicated. 
10.3.  Cyclosporine assay weekly until dose is tapered and then perform assay as 
indicated. 
10.4. Chimerism estimation by FISH for sex chromosome or short tandem repeat (STR) 
for sex match between donor and recipient is to be initiated between day +10 and +21 
and repeated as clinically indicated. Either blood or bone marrow are suitable for 
assay. Chimerism determination should be performed 4 weeks, 8 weeks, and 12 weeks  
post transplant and as clinically indicated.  
10.5. NBT assay or other assay for neutrophil function will be performed at 
approximately day+28 and then as clinically indicated. 
10.6. Beyond day +120 studies obtained are as clinically indicated. 
 
 
11. Evaluation of toxicity  


12 
11.1. Definition of a Toxic Event. A toxic event is defined as grade III or grade IV toxicity 
by the NCI Common Toxicity Criteria Version 3.0 
(http://ctep.info.nih.gov/reporting/ctc.html). Fever and hematologic toxicity are excluded 
as these are expected side effects of this therapy. Severe hematologic toxicity resulting 
from cytopenias will be managed in the same way as that which may result from 
conventional conditioning regimen.  
 
12. Risks and potential hazards  
12.1. 
Nonengraftment. A major risk of this protocol is nonengraftment. Since the 
preparative regimen is ablative, we would anticipate the risk for non-engraftment 
would be 5%. This estimate is based upon extensive experience with the busulfan, 
cyclophosphamide, Alemtuzumab regiment used to treat patients with various 
hematolgic diseases. To reduce concern for the risks of engraftment failure, we will 
obtain, when possible, ‘back-up’ marrow from the patient for cryopreservation before 
conditioning is initiated.   
12.2. 
Incomplete engraftment. Even if engraftment occurs it may be incomplete, 
producing a state of mixed chimerism. Complete donor chimerism may become 
established progressively or it may be favored and accelerated by infusion of 
additional donor T lymphocytes. While mixed chimerism would likely be an 
acceptable outcome for treatment of the CGD, we will monitor chimerism and 
functional neutrophil assays to determine the impact of mixed chimerism on clinical 
course of CGD. Thus all patients will be monitored for engraftment at regular intervals 
post transplant. If chimerism is less than complete and the clinical course 
unsatisfactory, e.g. persistent or recurrent infections, patients will be afforded the 
option of Donor lymphocyte Infusion (DLI) infusion as ‘per’ the CAGT SOP, in which 
increasing doses of lymphocytes are administered at predetermined intervals if mixed 
chimerism persists. It is also possible that patients may also be given additional CD34+ 
selected donor stem cells. 
12.3. 
GVHD. Acute GVHD leads to a skin rash, liver dysfunction, and enteritis.  
GVHD prophylaxis in most types of patients reduces the incidence of grade II-IV 
GVHD to about 35% with commonly used regimens of today. Chronic GVHD is a 
multiorgan autoimmune disease usually requiring therapy with steroids and other 
immunosuppressives.  It is seen in about 30% of adults undergoing transplant and a 
lower percentage of children.  Chronic GVHD usually may end after several months or 
may lead to organ failure (liver failure is common) or infectious death.  GVHD will be 
prevented in this study by the use of Alemtuzumab, which should persist over the 
transplant period to produce depletion of infused donor T, cells and by administration 
of cyclosporine and methotrexate. If GVHD occurs it will be treated by steroid 
administration. Patients whose GvHD is steroid resistant will be offered entry to Phase 
I/II protocols for treatment of steroid resistant GvHD. 
12.4. 
Additional Risks. The degree of immune suppression of marrow transplant 
patients leads to an increased risk of opportunistic infections, especially those due to 
CMV, HSV, EBV, Pneumocystis, and other viruses and fungi.  Prophylaxis is given 
where possible (Acyclovir, Bactrim, antifungal and antibacterial mouth rinses, etc.), 
and treatment for suspected agents is initiated very promptly, usually before the 
infection is confirmed. 
 


13 
CGD clinical course may recur. The administration of blood products carries the risk 
of blood born infections such as hepatitis viruses, CMV, and HTLV-III. Blood products 
are screened for these agents but testing is not totally effective in excluding 
contaminated blood products. 
 
Other complications of an unexpected nature may be seen.  In fact, most every patient 
presents with some new or very rare complication during his transplant. Patients are 
made aware of this possibility. 
 
Rarely, patients with CGD have experienced acute inflammatory reactions 
accompanied by severe skin rash at the time of donor engraftment. These reactions 
have been associated with administration of white cell growth factors (GCSF and 
GMCSF), and for this reason, these agents will not be used routinely to promote rapid 
engraftment. 
 
13. Statistical considerations  
 
13.1. 
Patients with CGD who do not have a HLA genotype identical sibling donor are eligible 
for this  nonrandomized phase II study to examine the efficacy of allogeneic stem cell 
transplantation from unrelated 5/6 or 6/6 HLA matched or phenotype 5/6 or 6/6 HLA matched 
donors. The preparatory regimen is busulfan, cyclophosphamide, fludarabine, and Alemtuzumab 
IH.  We expect to enroll 10 to 15 patients over a period of 3 to 4 years.  Safety monitoring will be 
conducted using Wald's sequential probability ratio test to monitor the rate of engraftment.  The 
primary endpoint, or neutrophil engraftment is determined at or before day 28 and defined as the 
first day of 3 consecutive days for which the absolute neutrophil count exceeds 500/uL.  Donor 
chimerism will be assessed for each patient at 120 days post transplant (+120).  Complete donor 
is defined as >90% donor cells.  Acute GVHD is assessed between day 0 and day 100 while 
chronic GVHD is assessed between day 100 and day 365.  We expect 90% of the patients to 
engraft with an incidence of acute GVHD of 15%-20% grade I, and 5%-10% grade II or greater. 
 
13.2. 
The table, below, lists the total number of engraftment failures (including deaths 
or lost grafts prior to day +120) observed on day +120 necessary to trigger a suspension 
of accrual until a thorough reevaluation of the protocol has been completed.  
Additionally, if at any time two deaths prior to day +120 are observed accrual will be 
suspended and a thorough analysis conducted.  These estimates provide a 10% chance 
of erroneously concluding that the true rate of engraftment is 90% or greater when in 
fact it is less than 80%.  They also allow for a 10% chance of mistakenly concluding that 
the true engraftment rate is less than 80% when in fact it is at least 90%. 
 
 
 
 
 
 
 
 
 
 


14 
 
Engraftment Failure  
 
Safety Monitoring Guidelines 
 
Patients Entered (n) 
 
Engraftment Failures 
 
1 
 
- 
 
2 
 
- 
 
3 
 
- 
 
4 
 
4 
 
5 
 
4 
 
6 
 
4 
 
7 
 
4 
 
8 
 
4 
 
9 
 
5 
 
10 
 
5 
 
11 
 
5 
 
12 
 
5 
 
13 
 
5 
 
14 
 
5 
 
15 
 
5 
 
13.3. 
GVHD will be monitored so that patient entry will be suspended until the study 
investigators have thoroughly reevaluated the protocol when either:  (a) the sum of the 
overall grades assigned to each entered patient exceeds 21, or (b) two patients develop 
grade III or IV GVHD.  Although the literature suggests the incidence of grade I or II 
GVHD to be 25%, it is not uncommon for all patients in a series to exhibit grade I 
GVHD.   By using a sum of 21 as the reevaluation trigger; a sample acceptable 
distribution of GVHD might include 10 grade I, 4 grade II, and 1 grade 3 GVHD patients.  
These criteria also monitor against unacceptable levels of extreme GVHD which exceed 
the expected 3% incidence. 
 
13.4. 
Kaplan-Meier estimates and associated confidence intervals of disease free 
survival, time to engraftment failure, and survival will be developed for all patients 
registered on the study. 
 
13.5. 
The other objective of the protocol, to evaluate the reversal of organ dysfunction 
is strictly observational in nature.  The patient group is expected to exhibit a variety of 
organ dysfunctionality at the time of transplant making it nearly impossible to predict the 
types of dysfunction much less develop criteria for reversal of the dysfunction.  
Improvement can only be determined in relationship to the pre-transplant findings (e.g. 
lung function as assayed by pulmonary function tests).  Results will be reported 
descriptively, and no statistical analyses will be undertaken. 


15 
 
13.6. 
One year post transplant (day 365) should be considered the off-study date. 
 
14. Data Collection and Adverse Event Reporting: 
 
14.1. 
Any questions regarding patients on this study should be addressed to Dr. Krance 
(Ph. 832-824-4693). 
 
14.2. 
Register all patients with the research coordinator at 832-824-4881. 
 
14.3. 
Drug Toxicity and/or adverse reactions 
Adverse events will be collected according to SOP J02.05.XX, J02.06.XX and 
J02.78.XX.     
 
14.4 
Relapse and survival data will be collected until 1 year post transplant. 
 
14.5 
Data Collection: 
 
The following forms will be used to collect data 
 
 
Prestudy  
 
 
Adverse events 
 
 
Response 
 
 
Relapse / progression 
 
 
Off study 
 
 
Death. 
 
 
15. Informed consent  
15.1. 
All patents and/or their legal guardian must sign a document of informed consent 
consistent with local institutional and Federal guidelines stating that they are aware of the 
investigational nature of this protocol and of the possible side effects of treatment.  
Further, patients must be informed that no efficacy of this therapy is guaranteed, and that 
unforeseen toxicities may occur.  Patients have the right to withdraw from this protocol at 
any time.  No patient will be accepted for treatment without such a document signed by 
him or his legal guardian. Full confidentiality of patients and patient records will be 
provided according to institutional guidelines. The costs associated with the transplant 
will be billed to the third party payors. The patients or their families will not receive any 
payment for participation in this study.  
  


16 
 
Reference List  
 
 
(1)  Segal BH, Leto TL, Gallin JI, Malech HL, Holland SM. Genetic, biochemical, and 
clinical features of chronic granulomatous disease. Medicine 2000; 79(3):170-200. 
 
 
(2)  Holland SM. Update on phagocytic defects. Pediatr Infect Dis J 2003; 22(1):87-88. 
 
 
(3)  Guide SV, Stock F, Gill VJ, Anderson VL, Malech HL, Gallin JI et al. Reinfection, 
rather than persistent infection, in patients with chronic granulomatous disease. 
 
  J Infect Dis 2003; 187(5):845-853. 
 
 
(4)  Jackson SH, Miller GF, Segal BH, Mardiney M, III, Domachowske JB, Gallin JI et 
al. IFN-gamma is effective in reducing infections in the mouse model of chronic 
granulomatous disease (CGD). J Interferon Cytokine Res 2001; 21(8):567-573. 
 
 
(5)  Gallin JI. Interferon-gamma in the treatment of the chronic granulomatous 
diseases of childhood. Clin Immunol Immunopathol 1991; 61(2 Pt 2):S100-S105. 
 
 
(6)  Winkelstein JA, Marino MC, Johnston RB, Jr., Boyle J, Curnutte J, Gallin JI et al. 
Chronic granulomatous disease. Report on a national registry of 368 patients. 
Medicine (Baltimore) 2000; 79(3):155-169. 
 
 
(7)  Seger RA, Gungor T, Belohradsky BH, Blanche S, Bordigoni P, di Bartolomeo P et 
al. Treatment of chronic granulomatous disease with myeloablative conditioning 
and an unmodified hemopoietic allograft: a survey of the European experience, 
1985-2000. Blood 2002; 100(13):4344-4350. 
 
 
(8)  Horwitz ME, Barrett AJ, Brown MR, Carter CS, Childs R, Gallin JI et al. 
Treatment of chronic granulomatous disease with nonmyeloablative 
conditioning and a T-cell-depleted hematopoietic allograft. N Engl J Med 2001; 
344(12):881-888. 
 
 
(9)  Leung T, Chik K, Li C, Shing M, Yuen P. Bone marrow transplantation for 
chronic granulomatous disease: long-term follow-up and review of literature. 
Bone Marrow Transplant 1999; 24(5):567-570. 
 
 (10)  Del G, I, Iori AP, Mengarelli A, Testi AM, Romano A, Cerretti R et al. Allogeneic 
stem cell transplant from HLA-identical sibling for chronic granulomatous 
disease and review of the literature. Ann Hematol 2003; 82(3):189-192. 
 
 (11)  Watanabe C, Yajima S, Taguchi T, Toya K, Fujii Y, Hongo T et al. Successful 
unrelated bone marrow transplantation for a patient with chronic granulomatous 
disease and associated resistant pneumonitis and Aspergillus osteomyelitis. Bone 
Marrow Transplant 2001; 28(1):83-87. 
 


17 
 (12)  Hongeng S, Krance RA, Bowman LC, Srivastava DK, Cunningham JM, Horwitz 
EM et al. Outcomes of transplantation with matched-sibling and unrelated-donor 
bone marrow in children with leukaemia. Lancet 1997; 350(9080):767-771. 
 (13)  Deeg H, Leisenring W, Storb R, Nims J, Flowers M, Witherspoon R et al. Long-
term outcome after marrow transplantation for severe aplastic anemia. Blood 
1998; 91(10):3637-3645. 
 (14)  Lawson SE, Roberts IA, Amrolia P, Dokal I, Szydlo R, Darbyshire PJ. Bone 
marrow transplantation for beta-thalassaemia major: the UK experience in two 
paediatric centres. Br J Haematol 2003; 120(2):289-295. 
 
 (15)  Oakhill A, Pamphilon DH, Potter MN, Steward CG, Goodman S, Green A et al. 
Unrelated donor bone marrow transplantation for children with relapsed acute 
lymphoblastic leukaemia in second complete remission. Br J Haematol 1996; 
94(3):574-578. 
 
 


18 
 
 
 
 
 
 
 
 
 
 
Appendix 1 
Drug Profiles 
 


1 9  
 
 
F L U D A R A BI N E  
 
T h er a p e uti c Cl a s sifi c ati o n :  P uri n e a nti m et a b olit e. 
P h ar m a c e uti c al D at a :  I n vi al s of 5 0 m g. 
S ol uti o n Pr e p ar ati o n : R e c o n stit ut e d wit h 2 ml of st eril e w at er f or i nj e cti o n.  T h e r e s ulti n g 
s ol uti o n will c o nt ai n 2 5 m g/ ml.  F or i nf u si o n ( m a xi m u m c o n c e ntr ati o n of 1 0 m g/ ml) 
i ntr a v e n o u sl y i n 1 0 0 ml of st a n d ar d i ntr a v e n o u s pi g g y b a c k fl ui d ( d e xtr o s e 5 %, or 
n or m al s ali n e) o v er 3 0 mi n ut e s.  
St a bilit y a n d St or a g e R e q uir e m e nt s : Pri or t o mi xi n g: St or e u n d er r efri g er ati o n 2 t o 8 
d e gr e e s C el si u s. Aft er mi xi n g:  St a bl e f or 1 6 d a y s at r o o m t e m p er at ur e, b ut n e e d s 
t o b e u s e d i n 8 h o ur s b e c a u s e of t h e l a c k of a nti b a ct eri al pr e s er v ati v e s. 
R o ut e s of A d mi ni str ati o n : I V i nf u si o n. 
U s u al D o s a g e R a n g e :   2 5-3 0 m g/ m 2  i n a si n gl e d o s e e a c h d a y f or 5 d a y s e v er y 4 w e e k s. 
Si d e e ff e ct s:  M y el o s u p pr e s si o n, e x a c er b ati o n of h e m ol yti c a n e mi a Pr ol o n g e d 
i m m u n o s u p pr e s si o n, o p p ort u ni sti c i nf e cti o n a n d r ar e N e ur ot o xi cit y. 
S p e ci al Pr e c a uti o n s : I n cr e a s e d m y el o s u p pr e s si o n i n p ati e nt s wit h  
Cr e ati ni n e cl e ar a n c e s of l e s s t h a n 5 0 ml/ mi n.   
M e c h a n i s m of A cti o n:  A p uri n e a nti m et a b olit e m o difi e d wit h fl u ori n e a n d m o n o p h o s p h at e 
t o r e si st d e a mi n ati o n b y a d e n o si n e d e a mi n a s e a n d t o i n cr e a s e d s ol u bilit y.  
D e p h o s p h or yl ati o n f oll o w e d b y c ell ul ar i n c or p or ati o n a n d c o n v er si o n t o a cti v e 
tri p h o s p a ht e, w hi c h i s a c o m p etiti v e i n hi bit or of D N A s y nt h e si s.  
A ntit u m or D at a :  T h e dr u g h a s gr e at er a cti vit y t o T-c ell s t h a n B -c ell s, b ut cli ni c al a cti vit y i s 
o b s er v e d i n B -c ell m ali g n a n ci e s.  
H u m a n P h ar m a c ol o g y :  Fl u d ar a bi n e c a n o nl y b e gi v e n vi a i ntr a v e n o u s r o ut e.  R e n al 
e x cr eti o n a c c o u nt s f or 2 3 %.  H alf -lif e i s 1 0 h o ur s.   
 
B U S U L F A N  
T h er a p e uti c Cl a s sifi c ati o n:   Bif u n cti o n al al k yl ati n g a g e nt  
 
P h ar m a c e uti c al  D at a:    B u s ulf a n  ( B u s ulf e x  I nj e cti o n ®  Or p h a n  M e di c al)  i s  s u p pli e d  a s  a 
st eril e  s ol uti o n  i n  si n gl e -u s e  a m p ul e s  c o nt ai ni n g  6 0  m g  at  a  c o n c e ntr ati o n  of 
6 m g/ ml.  It i s pr o vi d e d a s a mi xt ur e of d e m et h yl a c et a mi d e ( D M A) a n d p ol y et h yl e n e 
gl y c ol 4 0 0 ( P E G 4 0 0).   
S ol uti o n  Pr e p ar ati o n:  B u s ulf a n  s ol uti o n  f or  i nj e cti o n  m u st  b e  dil ut e d  wit h  eit h er  0. 9 % 
S o di u m  C hl ori d e  I nj e cti o n  ( N S)  or  5 %  D e xtr o s e  I nj e cti o n  ( D 5 W)  pri or  t o 
a d mi ni str ati o n.    T h e  dil u e nt  q u a ntit y  m u st  b e  1 0  ti m e s  t h e v ol u m e  of  b u s ulf a n, 
e n s uri n g t h at t h e fi n al c o n c e ntr ati o n i s > 0. 5 m g/ ml.  S a m pl e c al c ul ati o n f or a 5 0 k g 
p ati e nt:  ( 5 0 k g) x ( 0. 8 m g/ k g of b u s ulf a n) = 4 0 m g = 6. 7 ml.  6. 7 ml of b u s ulf a n + 
6 7 ml of N S = 7 4 ml t ot al v ol u m e.  Fi n al c o n c e ntr ati o n: 0. 5 4 m g/ ml.  
St a bilit y a n d St or a g e R e q uir e m e nt s:   Aft er dil uti o n wit h N S or D 5 W, b u s ulf a n i s st a bl e at 
r o o m  t e m p er at ur e  ( 2 5  d e gr e e s  C el si u s)  f or  8  h o ur s.    T h e  i nf u si o n  m u st b e 
c o m pl et e d  wit hi n  t h at  ti m e.    Pri or  t o  mi xi n g:  St or e  u n d er  r efri g er ati o n  ( 2  t o  8 
d e gr e e s C el si u s). B u s ulf a n f or i nj e cti o n i s st a bl e at 4 ° f or at l e a st 1 2 m o nt h s.   
R o ut e  of  A d mi ni str ati o n:   B u s ulf a n  s h o ul d  b e  a d mi ni st er e d  i ntr a v e n o u sl y  vi a  a  c e ntr al 
v e n o u s c at h et er a s a t w o -h o ur i nf u si o n.  
U s u al D o s a g e R a n g e :  0. 8-1 m g/ k g/ d o s e gi v e n e v er y 6 h o ur s f or a t ot al of 1 6 d o s e s.  F or 
p ati e nt s  l e s s  t h a n  4  y e ar s  of  a g e  a  d o s e  of  1  m g/ k g/ d o s e  will  b e  u s e d  a n d  f or 
p ati e nt s > 4  y e ar s t h e st arti n g d o s e will b e 0. 8 m g/ k g/ d o s e.  D o s e s ar e b a s e d o n 
a ct u al  b o d y  w ei g ht,  u nl e s s  t h e  p ati e nt’ s  w ei g ht  i s  gr e at er  t h a n  3 0 %  of  i d e al  b o d y 
w ei g ht, t h e n d o si n g will b e b a s e d o n a dj u st e d w ei g ht of i d e al pl u s 2 5 %.  B u s ulf a n 
p h ar m a c o ki n eti c s  will  b e  p erf or m e d  o n  all  p ati e nt s  wit h  d o s e  a dj u st m e nt  a s 
a p pr o pri at e.  
P h ar m a c o ki n eti c s:   D o s e s will b e a dj u st e d t o a c hi e v e t h e d e sir e d pl a s m a ar e a u n d er t h e 
c ur v e ( A U C) of 9 0 0 – 1 2 0 0 m ol-mi n/ L.  D o s e s will b e a dj u st e d a s n e c e s s ar y 
p e n di n g t h e r e s ult s of t h e fir st d o s e p h ar m a c o ki n eti c s.  F or p ati e nt s w h o s e A U C 


2 0  
v al u e s ar e gr e at er t h a n 5 % o ut si d e t h e a c c e pt a bl e A U C r a n g e, t h e  d o s e will b e 
a dj u st e d t o a c hi e v e a t ar g et A U C of 1 1 2 5 m ol-mi n/ L ( mi d p oi nt of a c c e pt a bl e 
r a n g e) n ot t o e x c e e d a m a xi m u m d o s e of 1. 6 m g/ k g p er d o s e of b u s ulf a n. 
Si d e eff e ct s:   M y el o s u p pr e s si o n, n e ur ot o xi cit y ( m a nif e sti n g a s s ei z ur e s), mil d t o m o d er at e 
n a u s e a a n d v o miti n g, mil d t o m o d er at e t a c h y c ar di a, s ki n h y p er pi g m e nt ati o n, 
st erilit y, a n d r ar el y h e p at ot o xi cit y ( h e p ati c v e n o -o c cl u si v e di s e a s e) a n d p ul m o n ar y 
t o xi cit y (i nt er stiti al fi br o si s). 
S p e ci al Pr e c a uti o n s:   I n cr e a s e d t o xi cit y i n o b e s e p ati e nt s u nl e s s d o s e i s a dj u st e d 
a p pr o pri at el y. G e n er ali z e d s ei z ur e s h a v e b e e n r e p ort e d aft er u s e of hi g h d o s e 
b u s ulf a n.  All p ati e nt s will b e tr e at e d wit h p h e n yt oi n 5 m g/ k g/ d o s e (I V or P O) q 6 hr 
b e gi n ni n g o n d a y -1 0 f or 2 4 hr u ntil c o m pl eti o n of b u s ulf a n.  A tr o u g h p h e n y t oi n 
l e v el s h o ul d b e o bt ai n e d 6 h o ur s aft er t h e f o urt h d o s e.  T h e t h er a p e uti c r a n g e f or 
t hi s dr u g i s 1 0-2 0 µ g/ ml.  If t h e l e v el i s b el o w 1 0 µ g/ ml, t w o a d diti o n al d o s e s of 5 
m g/ k g/ d o s e s h o ul d b e a d mi ni st er e d q 6 h o ur s.  If t h e l e v el i s b el o w 5 µ g/ ml, a n 
a d d iti o n al f o ur d o s e s of p h e n yt oi n s h o ul d b e a d mi ni st er e d q 6 h o ur s o v er t h e n e xt 
2 4 h o ur s.  If t h e p h e n yt oi n l e v el i s wit hi n t h e t h er a p e uti c r a n g e, m ai nt e n a n c e 
t h er a p y s h o ul d b e gi n at a d o s e of 5 m g/ k g/ d a y (I V or p. o.) i n t w o di vi d e d d o s e s 
u ntil d a y -4.  P ati e nt s e x p eri e n ci n g s ei z ur e a cti vit y s h o ul d b e e v al u at e d 
n e ur ol o gi c all y a n d tr e at e d a s cli ni c all y i n di c at e d.  
M e c h a ni s m of A cti o n :  B u s ulf a n i s a bif u n cti o n al al k yl ati n g a g e nt i n w hi c h t w o l a bil e 
m et h a n e s ulf o n at e gr o u p s ar e att a c h e d t o o p p o sit e e n d s of a f o u r-c ar b o n al k yl 
c h ai n.  I n a q u e o u s m e di a, b u s ulf a n h y dr ol y z e s t o r el e a s e t h e m et h a n e s ulf o n at e 
gr o u p s.  T hi s pr o d u c e s r e a cti v e c ar b o ni u m i o n s t h at c a n al k yl at e D N A.  D N A 
d a m a g e i s t h o u g ht t o b e r e s p o n si bl e f or m u c h of t h e c yt ot o xi cit y of b u s ulf a n.  
H u m a n P h ar m a c ol o g y:   B u s ulf a n c a n b e a d mi ni st er e d or all y or i ntr a v e n o u sl y.  B u s ulf a n 
a c hi e v e s l e v el s i n c er e br o s pi n al fl ui d si mil ar t o pl a s m a l e v el s.  B u s ulf a n i s 
pr e d o mi n a ntl y m et a b oli z e d b y c o nj u g ati o n wit h gl ut at hi o n e, b ot h s p o nt a n e o u sl y 
a n d b y gl ut at hi o n e S -tr a nsf er a s e ( G S T) c at al y si s.  T hi s c o nj u g at e u n d er g o e s 
f urt h er e xt e n si v e o xi d ati v e m et a b oli s m i n t h e li v er.  A p pr o xi m at el y 3 0 % of b u s ulf a n 
a n d m et a b olit e s c a n b e r e c o v er e d i n t h e uri n e wit hi n 4 8 h o ur s aft er a d mi ni str ati o n.  
 
 
C A M P A T H 1 H  
S o ur c e a n d P h ar m a c ol o g y:  C A M P A T H -1 H i s a h u m a ni z e d a ntil y m p h o c yt e m o n o cl o n al 
a nti b o d y.  T h e C a m p at h -1 a nti g e n i n h u m a n s ( C D 5 2) i s pr e d o mi n a ntl y e x pr e s s e d 
o n p eri p h er al bl o o d l y m p h o c yt e s, m o n o c yt e s, a n d m a cr o p h a g e s.  C A M P A T H -1 H  
c a u s e s l y si s of l y m p h o c yt e s b y fi xi n g t o C D 5 2, a hi g hl y e x pr e s s e d, n o n -m o d ul ati n g 
a nti g e n o n t h e s urf a c e of l y m p h o c yt e s.  It m e di at e s t h e l y si s of l y m p h o c yt e s vi a 
c o m pl e m e nt a n d a nti b o d y d e p e n d e nt c ell m e di at e d c yt ot o xi cit y m e c h a ni s m s.  
C A M P A T H -1 H r a pi dl y r e a c h e s a p e a k c o n c e ntr ati o n aft er I V a d mi ni str ati o n.  T h e 
h alf -lif e h a s b e e n t h o u g ht t o b e a p pr o xi m at el y 6 0 h o ur s, alt h o u g h r e c e nt d at a 
s u g g e st a m or e pr ol o n g e d p er si st e n c e.  T h e r o ut e of eli mi n ati o n i s n ot k n o w n, b ut 
i s pr o b a bl y vi a u pt a k e a n d m et a b oli s m b y t h e r eti c ul o-e n d ot h eli al s y st e m of t h e 
li v er a n d s pl e e n.  
D o s e: T h e d o s e  h a s b e e n c h o s e n b a s e d o n t h e pl a s m a c o n c e ntr ati o n s r e q uir e d f or 
a cti vit y, a n d o n t h e pr e vi o u s e xt e n si v e p u bli s h e d  h u m a n e x p eri e n c e u si n g t hi s 
d o s e f or l y m p h o c yt e d e pl eti o n  
F or m ul ati o n a n d St a bilit y :  C A M P A T H-1 H i s s u p pli e d a s a p urifi e d pr e p ar ati o n dil ut e d i n 
p h o s p h at e b uff er e d s ali n e ( P B S) wit h 0. 0 5 m m ol E D T A.  T w e e n 8 0 i s a d d e d t o a 
c o n c e ntr ati o n of 0. 0 1 %.  T h e fi n al pr o d u ct i s cl e ar, c ol orl e s s i s ot o ni c s ol uti o n fr e e 
of vi si bl e p arti c ul at e m att er.  T h e gl a s s a m p ul e s will c o nt ai n 3 0 m g of a nti b o d y i n 3 
m L of stril e P B S at a c o n c e ntr ati o n of 1 0 m g/ m L.   It s h o ul d b e st or e d, pr ot e ct e d 
fr o m li g ht, i n a r efri g er at or at b et w e e n 2° a n d 8 ° C. Aft er dil uti o n i n D 5 W or N S, t h e 
r e s ulti n g s ol uti o n i s st a bl e f or 2 4 h o ur s at r o o m t e m p er at ur e. Ho w e v er, t h e pr o d u ct 
c o nt ai n s n o pr e s er v ati v e a n d m u st t h er ef or e b e u s e d wit hi n 8 h o ur s.  I ntr a v e n o u s 
C A M P A T H -1 H will b e dil ut e d i n 1 0 0 c c of 0. 9 % n or m al s ali n e or 5 % D e xtr o s e a n d 


21 
administered intravenously over 2 hours.  CAMPATH-1H must be filtered with a 
sterile, low-protein binding, 5 m filter prior to dilution. 
Supplier:  Investigational.  L&I Partners, LP 
Toxicity: CAMPATH-1H is a potent lymphocyte depleting agent. WHO Grade 3 and 4 
neutropenia and thrombocytopenia emerge on treatment in approximately 10-20% 
of patients.  Infections resulting from CAMPATH-1H induced immunosuppression 
are the major type of adverse event occurring outside the CAMPATH-1H 
administration period.  The most common infections being mucocutaneous herpes 
simplex and candidiasis.   
The majority of other adverse events seen in CAMPATH-1H trials can be 
categorized as being administration-related and of short duration.  There is usually 
a first dose effect consisting of cytokine release type phenomena including 
hypotension, rigors, fever, shortness of breath, chills, rashes, etc.  These side 
effects can be significantly ameliorated or avoided by premedicating the patients 
with methylprednisolone or hydrocortisone.  
Route of Administration:  Intravenous  
 
Methotrexate 
Therapeutic classification:  An antimetabolite and antifolate agent with antineoplastic and 
Immunosuppressant activities.  
Pharmaceutical Data:  Methotrexate injection is a sterile isotonic liquid, preservative free, 
supplied in a single dose vial containing 25 mg/mL of methotrexate in the following 
packaging strengths:  2 mL (50 mg), 4 mL (100mg), 8 mL (200 mg), and 10 mL(250 
mg).  Methotrexate injection, preservative free, is also supplied in a sterile 1 gram 
single dose vial of lyophilized powder containing 1 gram methotrexate as the base.   
Solution Preparation:  Single dose vial containing 25 mg/mL:  If desired the solution may 
be further diluted immediately prior to use with an appropriate sterile preservative 
free medium such as 5% dextrose solution or sodium chloride injection.     
Single dose vial of 1 gram lyophilized powder:  Reconstitution should be with an 
appropriate sterile preservative free medium such as 5% dextrose solution, or 
sodium chloride injection.  The 1 gram vial should be reconstituted with 19.4 mL to 
a concentration of 50 mg/mL.  When high doses of methotrexate are administered 
by IV infusion, the total dose is diluted in 5% dextrose solution.  
Stability and Storage:  Store at room temperature 20°-25°C (68°-77°F); protect from light.  
Use immediately after diluting or reconstituting drug.   
Route of Administration:  Intravenous (IV). 
Usual dosage range:  Methotrexate may be given by IV on Days 1, 3, 6 and 11. Dosing is 
either  5mg/m2/day for all four doses or 15mg/m2 on day 1 and 10mg/m2 for three 
remaining doses. Consider reducing dose by 50% or holding dose for Grade 3 
mucositis or weight gain > 5 kg (>5% baseline for children); consider leucovorin 
rescue. Hold dose for Grade 4 mucositis, doubling of serum creatinine from 
baseline, or weight gain >10 kg (>10% baseline for children) or as clinically 
indicated. 
Side Effects:  Methotrexate is a medication used to try to prevent GVHD. It causes 
damage to cells, and therefore can affect many different tissues of your body. It 
may cause or can worsen the mouth sores or inflammation of the mouth.  
Methotrexate may slow down the recovery of blood cells after transplantation. 
Methotrexate can cause kidney damage or worsen kidney function.  If kidney 
damage does occur, the methotrexate dose may be reduced or the medication may 
not be given at all.  Methotrexate can interfere with the body’s defense system (the 
immune system) causing more infections (especially viral infections and 
pneumonia) for several months after stem cell transplant. 
Mechanism of action:  Methotrexate binds to and inhibits the enzyme dihydrofolate 
reductase, resulting in inhibition of purine nucleotide and thymidylate synthesis 
and, subsequently, inhibition of DNA and RNA syntheses. Methotrexate also 


22 
exhibits potent immunosuppressant activity although the mechanism(s) of actions 
is unclear.  
 
CYCLOPHOSPHAMIDE (Cytoxan)  
Source and Pharmacology: Cyclophosphamide is an alkylating agent related to nitrogen 
mustard. 
Cyclophosphamide is inactive until it is metabolized by P-450 isoenzymes 
(CYP2B6, CYP2C9 and CYP3A4) in the liver to active compounds. The initial 
product is 4-hydroxycyclophosphamide (4-HC) which is in equilibrium with 
aldophosphamide which spontaneously releases acrolein to produce 
phosphoramide mustard. Phosphoramide mustard, which is an active bifunctional 
alkylating species, is 10 times more potent in vitro than is 4-HC and has been 
shown to produce interstrand DNA cross-link analogous to those produced by 
mechlorethamine. Approximately 70% of a dose of cyclophosphamide is 
excreted in the urine as the inactive carboxyphosphamide and 5-25% as 
unchanged drug. The plasma halflife ranges from 4.1 to 16 hours after IV 
administration. 
 
 
Formulation and Stability: 
Cyclophosphamide for Injection is available as powder for injection or lyophilized powder for 
injection in 500 mg, 1 gram and 2 gram vials. The powder for injection contains 82 mg sodium 
bicarbonate/100 mg cyclophosphamide and the lyophilized powder for injection contains 75 mg 
mannitol/100 mg cyclophosphamide. Storage at or below 25ºC (77ºF) is recommended. The 
product will withstand brief exposures to temperatures up to 30º C (86ºF). 
Guidelines for Administration:  
Cyclophosphamide for Injection: Reconstitute with sterile water or Bacteriostatic water for 
injection (paraben preserved only) to a concentration of 20 mg/ml. Solutions reconstituted with 
preservative should be used within 24 hours if stored at room temperature or within 6 days if 
stored under refrigeration. If administered as undiluted drug at the 20 mg/ml concentration, 
reconstitute with normal saline (NS) only to avoid a hypotonic solution. 
Cyclophosphamide may be further diluted in dextrose or saline containing solutions for IV use. 
Supplier: Commercially available from various manufacturers.  
 


23 
CYCLOSPORINE A (cyclosporine, CYA, Sandimmune®, Neoral®, Gengraf®): 
 
Source and Pharmacology: Cyclosporine (CSA) is a lipophilic fungal peptide consisting of 11 
amino acids. CSA is a potent immunosuppressive agent which prolongs survival of allogeneic 
transplants involving skin, heart, kidney, pancreas, bone marrow, small intestine, and lung. 
Current evidence suggests that cyclosporine selectively inhibits the transcription of IL-2; the 
action of which stimulates the proliferation of activated T-lymphocytes. CSA has been shown in 
vitro to be a potent inhibitor of P-glycoprotein, which has been postulated to be a factor in multi-
drug resistance to various antineoplastic agents. The terminal half-life of CSA is approximately 19 
hours (range 10-27 hours). Ninety-nine percent of CSA is metabolized. Elimination is primarily 
biliary with approximately 6% excreted in the urine. In the circulation, CSA is mainly bound to 
high, low, or very low density lipoproteins and to chylomicrons. Only a small fraction circulates 
unbound. The volume of distribution varies from 3.5 L/kg to 13 L/kg with higher concentrations of 
drug found in the liver, lymphocytes, kidney, heart, lung, pancreas, fat, neural and muscle cells. 
CSA clearance rates have been shown to be higher in pediatric patients and for patients < 25 
years old. 
 
The absorption of cyclosporine from the gastrointestinal tract is incomplete and variable, 
exhibiting large intra- and inter-patient variability. Drugs that stimulate or inhibit hepatic p-450 
enzymes will alter clearance of CSA and close attention to potential drug interactions is crucial. 
 
 
 
Formulation and Stability: 
IV formulation: 
Cyclosporine (Sandimmune®) is available as a (50 mg/mL) 5mL ampule use containing 650 mg 
polyoxyethylated castor oil (cremophor) and 32.9% alcohol. Store at temperatures below 30°C 
(86°F) and protected from light. 
 
Oral formulations: 
Cyclosporine (Sandimmune®) 25 mg, and 100 mg capsule, 100 mg/mL oral solution. Inactive 
ingredients include: Sandimmune® Capsule = Dehydrated alcohol, sorbitol, glycerol, corn oil, 
gelatin, polyoxyethylated glycolysed glycerides; Sandimmune® Solution = Olive oil, dehydrated 
alcohol, polyoxyethylated glycolysed glycerides. 


24 
Cyclosporine USP modified (microemulsion) (Neoral®, Gengraf®) 25 mg, 100 mg capsule, 100 
mg/mL oral solution. Inactive ingredients include: Neoral® capsule and solution = dehydrated 
alcohol, corn oil, polyoxyl 40 hydrogenated castor oil, tocopherol, gelatin, and propylene glycol. 
Gengraf® Capsule and solution = dehydrated alcohol, gelatin, polyethylene glycol, polyoxyl 35 
castor oil, polysorbate 80, propylene glycol, and sorbitan monooleate. 
Store capsules in the original unit-dose container at controlled room temperature 15°-30°C (59°-
86°F). 
 
Store Oral solutions in the original container at controlled room temperature 68°-77°F (20°-25°C). 
Do not store in the refrigerator. Once opened, the contents must be used within two months. At 
temperatures below 68°F (20°C) the solution may gel; light flocculation or the formation of a light 
sediment may also occur. There is no impact on product performance or dosing using the syringe 
provided. Allow to warm to room temperature 77°F (25°C) to reverse these changes. 
NOTE: Sandimmune®, Neoral® and Gengraf® ARE NOT BIOEQUIVALENT. Liquid formulations 
of each trade name are equivalent to capsules of that same trade name. Conversion from one 
trade name product to another is generally done at a 1:1 ratio, but requires close monitoring. 
Conversions from IV to PO are usually done at a 1:3 ratio, but should be monitored closely. 
Adjusting emulsion products to the same trough concentration as other oral products results in 
greater total exposure to the drug. 
 
Dilute I.V. concentrate 1 mL (50 mg) of cyclosporine injection in 20 mL-100 mL 0.9% Sodium 
Chloride Injection or 5% Dextrose Injection (0.5- 2.5 mg/mL). Diluted infusion solutions are stable 
for 24 hours at room temperature under fluorescent light. 
 
The Cremophor®EL (polyoxyethylated castor oil) contained in the concentrate for intravenous 
infusion can cause phthalate stripping from PVC. It is strongly recommended that glass bottles 
and non-PVC tubing be used to minimize patient exposure to DEHP. 
Monitor closely for an acute allergic reaction for the first 30 minutes and at frequent intervals 
thereafter. Note: cyclosporine absorbs into plastics and can give falsely high serum or blood 
concentrations if blood samples are collected from the same line through which cyclosporine was 
administered. 
 
Oral: Administer at a consistent time of day and at consistent intervals with regard to meals. Do 
not use plastic or styrofoam cups. If diluted with juice or milk, use a glass container and rinse with 
additional diluent, and then consume to ensure that complete dose has been taken. Do not use 
water or cleaning agents on the dosing syringe. To improve palatability, mix Sandimmune® with 
milk, chocolate milk or orange juice; mix Neoral® or Gengraf® with orange juice or apple juice but 
NOT milk. After mixed, have patient consume immediately. DO NOT MIX GRAPEFRUIT JUICE 
with any CSA product. 
 
Supplier: Commercially available from various manufacturers.