Trial Information

Pharmacodynamics, Pharmacogenomics, and Preliminary Safety and Efficacy of Alemtuzumab Induction and Tacrolimus in Pediatric Intestinal Transplantation (IND # 100496)

Intraoperatively, it is our impression that Alemtuzumab may predispose to mild coagulopathy.
Therefore, we have elected to administer steroids, and withhold Alemtuzumab in the operating
room for all small bowel transplant recipients.

1. Premedication with acetaminophen 10 mg/kg orally + diphenhydramine 1 mg/kg
intravenously + methylprednisolone 2 mg/kg, intravenously will occur 30 minutes before
Alemtuzumab administration.

2. Steroids up to 10 mg/kg as bolus will be administered intravenously in the operating
room prior to reperfusion of the allograft followed by decreasing amounts of low-dose
steroids post-transplant.

Day 1 post-transplant: up to 5 mg/kg can be given Day 2 post-transplant: up to 4 mg/kg
can be given Day 3 post-transplant: up to 3 mg/kg can be given Day 4 post-transplant:
up to 2 mg/kg can be given Day 5 post-transplant: up to 1 mg/kg can be given

Maintenance steroids are tapered thereafter by switching to equivalent amounts of oral
prednisone, as soon as ileus results and oral intake intake is possible. By the end of
the first month, all patients receive no more than 2.5-5 mg/day of prednisone. This
amount is exceeded only if rejection occurs. Long-term prednisone usage may be elected
in patients re-transplanted for chronic rejection.

3. A single dose of Alemtuzumab 0.4-0.5 mg/kg will be given intravenously slowly
post-operatively. Total dose will not exceed 30 mg.

Procedures:

1. Tacrolimus will be initiated at 0.01 mg per kg body weight orally. If biopsy-proven
rejection does not occur, Tacrolimus will be titrated to whole blood concentrations:

Month 1: 15-20 ng/ml Months 2-3: 10-15 ng/ml Month 4-6: 8-10 ng/ml Months 6-12: 5-10
ng/ml

2. This minimization protocol will be delayed by 3 months if biopsy proven acute cellular
rejection occurs. At the event of rejection, Tacrolimus minimization and steroids
sparing will be discontinued and standard immunosuppression will be instituted. For
example, if rejection occurs, Tacrolimus is increased to month 1 levels of 15-20 ng/ml,
and steroids added to the regimen. Steroids will be reduced or eliminated within 3
months of rejection, and tacrolimus minimization resumed as described above. These
levels of Tacrolimus are our standards of care in the event of rejection. Current
immunosuppressive protocols at our center include rabbit anti-human thymocyte globulin
(rATG) with Tacrolimus monotherapy, or Tacrolimus + steroid without induction.

3. Laboratory tests per clinical protocol. The clinical standard of care will be followed
in performing post-Tx monitoring labs consisting of complete blood count with
differential count, serum sodium, potassium, chloride, bicarbonate, BUN, creatinine and
glucose, and liver function tests consisting of Total bilirubin, aspartate alanine
transaminase (ALT), aspartate glutamine transaminase (AST), and glutamyl galactosyl
transaminase (GGT), and TAC whole blood concentrations. These laboratory tests are
performed at least weekly in the first and second months, twice monthly in month 3, and
monthly thereafter to the sixth month. The extra 1 to 11 ml needed for pharmacodynamic
and pharmacogenomic studies is discussed further in items 5 and in Table 1.

4. Pharmacogenomic studies will consist of characterizing 550,000 SNPs using the Infineum
chip (Illumina, Dan Diego, CA) in DNA extracted from 3 ml of whole blood obtained
pre-Tx, from each of 75 children.

5. Table 1 (Table appended to this protocol application). Blood sampling strategy for:
pharmacodynamic studies which will measure the lymphocyte subsets CD3+, CD4+, CD8+,
CD19+, CD16/56+, αβ and γδ subsets as well as dendritic cells types 1 and 2 are shown
in the following Table (Table 1). Concentration of alemtuzumab will also be measured by
ELISA in 0.1 ml human plasma obtained from the same blood sample. Concentration of
Alemtuzumab associated with half-maximal depletion of each subset will be determined by
Emax pharmacodynamic models based on Hill equations using the WINNONLIN software
(Pharsight Inc, Palo Alto, CA.) The time points a) for immunological studies and mRNA
sampling are chosen based on our documentation of significant changes in donor-specific
alloreactivity in prior studies (12), and b) for pharmacodynamics/pharmacogenomics are
chosen based on mean half-life of Alemtuzumab (24 hours) and terminal half-life (14
days). The 2-month and 8-month samples are added to assess more closely, reconstitution
of lymphocyte subphenotypes.

Drug administration will occur in Children's Hospital of Pittsburgh. Once discharged,
subjects will take the drugs on a daily basis based on the standard of care.

Assays designed will be carried out in the laboratories of Dr. Rakesh Sindhi at the
John G. Rangos Sr. Research Center, 530 45th St, Pittsburgh, PA 15201. Specimens will
be stored in the transplant laboratory indefinitely, and will be under the control of
the principal investigator, Dr. Rakesh Sindhi. The specimens will be stored using
assigned code numbers and the information linking these code numbers to the
corresponding subjects' identities will be kept in a separate, secure location. The
specimens may be shared with secondary investigators without identifiers.

6. Surveillance intestinal allograft biopsies will be performed per clinical surveillance
protocol-twice weekly in the first month, weekly in the second month, two-weekly in the
third month, and at least monthly thereafter until the end of the 6th month, and at
least annually thereafter. Surveillance biopsies are done because no blood test exists
to indicate intestinal allograft dysfunction.